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EFFECT OF DIETARY INCLUSION OF SODIUM BICARBONATE ON PRODUCTION PERFORMANCE,
NUTRIENT DIGESTIBILITY AND BLOOD PROFILE OF CAGED LAYERS DURING SUMMER
By
Ghulam Abbas2000-ag-1523
M. Sc. (Hons.) Poultry Science
A thesis submitted in partial fulfillment of the requirement for the degree of
DOCTOROFPHILOSOPHY
IN
POULTRY SCIENCE
INSTITUTE OF ANIMAL AND DAIRY SCIENCESFACULTY OF ANIMAL HUSBANDRY,
UNIVERSITY OF AGRICULTURE,FAISALABAD,
PAKISTAN2017
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THIS HUMBLE EFFORT, FRUIT OF MY LIFE IS
DEDICATED TO MY RESPECTED BROTHER
MUHAMMAD AHMAD SHAHEEDThe Son of My Supervisor
DR. SULTAN MAHMOOD
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AcknowledgementsI offer my humblest thanks from the deepest core of my heart to “ALMIGHTY
ALLAH” who created the universe and bestowed the mankind with knowledge and wisdom
to research for its secrets. I bow before his compassionate endowments. I pay homage to
Holy Prophet Muhammad (Peace be upon Him and His Holy Descendants), the most
perfect and exalted among us who are forever a source of knowledge and guidance for
humanity as a whole.
It is my utmost pleasure to avail this opportunity to extend my heartiest gratitude to
my worthy supervisor Prof. Dr. Sultan Mahmood, professor, Institute of Animal Sciences,
University of Agriculture, Faisalabad (UAF) for providing and facilitating an encouraging
environment for competitive research. I feel highly privileged to pay cordial gratitude to my
reverend, zealot and distinguish supervisor for his keen personal interest, dynamic
supervision, immense cooperation, valuable suggestions, unfailing patience, constructive and
thoughtful criticism and, moral and economical support during my study.
I am also thankful to the members of my supervisory committee, Dr. AhsanulHaq,
Director of Institute of Animal Sciences at UAF and Prof.Dr. Haq Nawaz, Director
Graduate Studies, UAF for their constant support and help throughout the course of this
study.
I also extend my sincere words of admiration and appreciation to my family members
who supported me morally throughout my research. Special thanks are extended to my
brother Sajjad Hussain Hashmi and my wife Razia Abdual Majeed Qureshi for their
prayers and support. I further offer passionate thanks to especially Brother
SajjadHussainHashmifor his extended cooperation.
I extend my exorbitant and hearty thanks to my affectionate and generous parents
who taught me the first word to speak, the first alphabet to write and the first step to take.
They are up and above of all the blessings of ALMIGHTY ALLAH, I enjoyed during my
temporary stay in the world. I also pay sincere thanks to all of my sisters and brothers who
supported me during my study. May ALLAH give them a long, happy and healthy life
(Ameen)
Ghulam Abbas
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LIST OF CONTENTS
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2.14.8 Various egg quality parameters 35
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2.14.9 Body/rectal temperature and respiration rate 372.14.10 Mortality 382.14.11 Hematological profile 402.14.12 Serum metabolites and serum proteins 402.14.13 Plasma electrolytes and minerals 412.14.14 Serum lipids, hormones and enzymatic profile 442.14.15 Immune response 452.14.16 Digestibility of nutrients 46
CHAPTER 3 MATERIALS AND METHODS 483.1 Performance Trial 48
3.1.1 Experimental birds 483.1.2 Allocation of the birds to the cages 483.1.3 Management of the experimental birds 493.1.4 Experimental diets, groups and their feeding plans 49
3.2 Data Collection 493.2.1 Initial body weight of birds 493.2.2 Weight gain 493.2.3 Feed consumption 523.2.4 Egg production 523.2.5 Egg mass 523.2.6 Feed conversion ratio 523.2.7 Water consumption 523.2.8 Rectal temperature and respiration rate 523.2.9 Ambient temperature and Humidity index 52
3.3 Egg quality characteristics 533.3.1 Egg Weight 533.3.2 Shell thickness 533.3.3 Specific Gravity of Eggs 533.3.4 Albumen height 553.3.5 Yolk Height 553.3.6 Yolk Diameter 553.3.7 Blood and meat spots 553.3.8 Yolk index 553.3.9 Haugh unit score 553.3.10 Yolk pH and pH of albumen 56
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3.3.11 Egg yolk cholesterol 563.4 Mortality 563.5 Hematological profi le 57
3.5.1 Glucose 573.2.5 Hemoglobin 583.5.3 ESR 583.5.4 Packed Cell Volume (PCV) 583.5.5 Total leucocyte count 583.5.6 Red blood cells (RBCs) count 59
3.6 Serum proteins 603.6.1 Total serum protein 603.6.2 Serum albumen concentration 613.6.3 Serum globulin concentration 61
3.7 Serum lipids profile 613.7.1 Serum cholesterol concentration 613.7.2 Serum triglycerides 623.7.3 HDL cholesterol 64
3.8 Plasma electrolytes (i.e. Na+, K+, Cl-, HCO3-) and mineral (Ca
and P) profile66
3.8.1 Estimation of Blood pH 663.8.2 Estimation of Sodium (Na+) and potasium 663.8.3 Estimation of chloride 673.8.4 Calcium 673.8.5 Phosphorus 683.8.6 Estimation of HCO3
- 693.9 Hormono-enzymic performance 70
3.9.1 Assay procedure for Triiodothyronine (T3) 703.9.2 Assay procedure for Thyroxin (T4) 713.9.3 Assay procedure for cortisol 723.9.4 Assay procedure for estrogen 723.9.5 Assay procedure for progesterone 733.9.6 Serum Glutamic pyruvic transaminase (SGPT) 743.9.7 Serum Glutamic-Oxaloacetic Transaminase (SGOT)
Principle75
3.10 Serum metabolites 75
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3.10.1 Urea 753.10.2 Serum creatinine concentration 763.10.3 Uric acid 773.10.4 Alkaline Phosphatase (ALP) 78
3.11 Determination of Antibody titre against Newcastle disease virus in birds
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3.12 Digestibility trial 813.13 Proximate composition 82
3.13.1 Dry matter 823.13.2 Crude protein 823.13.3 Ether extract 833.13.4 Crude fiber 833.13.5 Acid Insoluble Ash (AIA) 84
3.14 Mineral analysis 853.14.1 Wet digestion 853.14.2 Determination of calcium 853.14.3 Determination of Phosphorus 863.14.4 Determination of Sodium and potassium by flame
photometer87
CHAPTER 4 RESULTS AND DISCUSSION 88Results 88
4.1 Production performance 884.1.1 Live body weight 884.1.2 Feed consumption 884.1.3 Egg Production 904.1.4 Egg weight 904.1.5 Egg mass 904.1.6 Feed efficiency 92
4.2 Egg quality 934.2.1 Specific gravity 934.2.2 Shell thickness 934.2.3 Albumen height 954.2.4 Haugh unit 954.2.5 Yolk diameter 954.2.6 Yolk height 96
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4.2.7 Yolk Index 964.2.8 Egg yolk cholesterol 964.2.9 Yolk pH 974.2.10 Albumen pH 974.2.11 Meat and blood spots 98
4.3 Rectal temperature, respiration rate and water consumption 984.3.1 Rectal temperature 984.3.2 Respiration rate 984.3.3 Water intake 100
4.4 Mortality 1004.5 Hematological profile 100
4.5.1 Serum glucose 1024.5.2 Packed cell volume 1024.5.3 Blood hemoglobin 1024.5.4 Erythrocytes sedimentation rate 1034.5.5 Red blood cells count 1034.5.6 White blood cell count (WBCs) 103
4.6 Serum metabolites 1034.6.1 Serum urea 1044.6.2 Serum uric acid 1044.6.3 Serum creatinine 1064.6.4 Serum alkaline phosphatase 106
4.7 Serum proteins analysis 1064.7.1 Total proteins 1064.7.2 Albumen 1084.7.3 Globulin 108
4.8 Plasma electrolytes, minerals and serum pH 1084.8.1 Plasma sodium 1084.8.2 Plasma Potassium 1104.8.3 Plasma chloride 1104.8.4 Plasma bicarbonate 1104.8.5 Plasma calcium 1114.8.6 Plasma phosphorus 1114.8.7 Serum pH 111
4.9 Serum lipids profile 112
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4.9.1 Serum cholesterol 1124.9.2 Serum triglyceride 1124.9.3 Serum high density lipoprotein 1124.9.4 Serum low density lipoprotein 114
4.10 Hormones and enzymes 1144.10.1 Tri-iodothyronine (T3) and Thyroxin (T4) 1144.10.2 Oestrogen 1164.10.3 Progesterone 1164.10.4 Corticosterone 1174.10.5 Serum Glutamic-Oxaloacetic Transaminase (SGOT) and
Serum Glutamic-PyruvicTransaminase (SGPT)117
4.11 Immune response 1184.12 Economic Appraisal 120
Discussion 1234.13 Performance 123
4.13.1 Live body weight 1234.13.2 Feed consumption 1244.13.3 Egg Production 1254.13.4 Egg weight 1264.13.5 Egg mass 1274.13.6 Feed efficiency 129
4.14 Egg quality 1304.14.1 Specific gravity 1304.14.2 Shell thickness 1314.14.3 Albumen height 1324.14.4 Haugh unit 1334.14.5 Yolk diameter 1344.14.6 Yolk height 1354.14.7 Yolk Index 1354.14.8 Egg yolk cholesterol 1364.14.9 Yolk pH 1364.14.10 Albumen pH 1364.14.11 Meat and blood spots 137
4.15 Mortality 1374.16 Rectal temperature, respiration rate and water consumption 138
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4.16.1 Rectal temperature 1384.16.2 Respiration rate 1394.16.3 Water intake 140
4.17 Hematological profile 1414.17.1 Serum glucose 1414.17.2 Packed cell volume, ESR and Red blood cells count 1414.17.3 Blood hemoglobin 1424.17.4 White blood cell count (WBCs) 143
4.18 Serum metabolites 1444.18.1 Serum urea 1444.18.2 Serum uric acid 1444.18.3 Serum creatinine 1454.18.4 Serum alkaline phosphatase 145
4.19 Serum proteins analysis 1454.19.1 Total proteins 1454.19.2 Albumin 1454.19.3 Globulin 146
4.20 Plasma electrolytes, minerals and serum pH 1464.20.1 Plasma sodium 1464.20.2 Plasma Potassium 1474.20.3 Plasma chloride 1484.20.4 Plasma bicarbonate 1494.20.5 Plasma calcium and phosphorus 1494.20.6 Serum pH 150
4.21 Serum lipids profile 1514.22 Hormones and enzymes 151
4.22.1 Tri-iodothyronine (T3) and Thyroxin (T4) 1514.22.2 Oestrogen 1524.22.3 Progesterone 1534.22.4 Corticosterone 1534.22.5 Serum Glutamic-Oxaloacetic Transaminase (SGOT) and
Serum Glutamic Pyruvic Transaminase (SGPT)154
4.23 Immune response 155CHAPTER 5 DIGESTIBILITY TRIAL 157
5.1 Introduction 157
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5.2 Materials and methods 1575.2.1 Experimental diets 158
5.2.2 Chemical analysis of excreta 1585.2.3 Statistical analysis 158
5.3 Results 1585.3.1 Dry matter 1605.3.2 Crude protein 1605.3.3 Crude fiber 1605.3.4 Ether extract 161
5.4 Minerals 1615.4.1 Calcium 1615.4.2 Phosphorous 1635.4.3 Iron 1635.4.4 Sodium 1645.4.5 Potassium 164
5.5 Discussion 1655.5.1 Dry matter 1655.5.2 Protein 1665.5.3 Crude fiber 1675.5.4 Ether extract 169
5.6 Minerals 169CHAPTER 6 SUMMARY 173
LITERATURE CITED 177
TABLE OF CONTENTS
No. Title Page
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1 INTRODUCTION 1
2 LITERATURE REVIEW 04
3 MATERIALS AND METHODS 48
4 RESULTS AND DISCUSSION 88
5 DIGESTIBILITY TRAIL 157
7 SUMMARY 173
LITERATURE CITED 177
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LIST OF TABLES
Table # TITLE Page#
3.1 Proportion of ingredients used in experimental diets 50
3.2 Chemical composition of the experimental diets 51
4.1Effect of dietary inclusion of sodium bicarbonate on weight gain and
feed consumption of caged layers89
4.2Effect of dietary inclusion of sodium bicarbonate on production
performance of caged layers91
4.3Effect of dietary inclusion of sodium bicarbonate on egg quality
characteristics of caged layers94
4.4Effect of dietary inclusion of sodium bicarbonate on rectal
temperature, respiration rate and water consumption of caged layers99
4.5Effect of dietary inclusion of sodium bicarbonate on hematological
profile of caged layers101
4.6Effect of dietary inclusion of sodium bicarbonate on serum
metabolites of caged layers105
4.7Effect of dietary inclusion of sodium bicarbonate on serum proteins
concentration of caged layers107
4.8Effect of dietary inclusion of sodium bicarbonate supplementation
on plasma electrolytes and serum pH of caged layers109
4.9Effect of dietary inclusion of sodium bicarbonate on serum lipids
profile of caged layers113
4.10Effect of dietary inclusion of sodium bicarbonate on serum
hormones and liver enzymes of caged layers115
4.11Effect of dietary inclusion of sodium bicarbonate on immune
response of caged layers119
4.12
Economics of production of the layers fed different levels of sodium
bicarbonate, calculated for 12 weeks of production (27th -38th week
of age)
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5.1 Effects of dietary inclusion of sodium bicarbonate on nutrient 159
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digestibility coefficient in layers
5.2Effect of dietary inclusion of sodium bicarbonate on absorbability of
minerals in layers162
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ABSTRACTThe intent of this study was to investigate the effect of dietary inclusion of NaHCO 3 on production performance, nutrient digestibility and blood profile of caged layers during summer. One hundred sixty commercial layers of 24 weeks old were bought from a commercial layer farm and were raised in a group for one week i.e. adaptation period. At the beginning of 26th week of age, these layers were further divided into 20 experimental units/replicates (8 layers/replicate). These 20 replicates/units were further allotted/distributed to five treatment groups (4 replicate/treatment). All the birds were offered diets containing 17% CP and 2700 Kcal/Kg ME with or without supplementation of NaHCO 3 for a period of twelve weeks. Group A served as control, which was provided layer ration without any supplementation, while group B, C, D, and E were offered ration supplemented with 0.5, 1, 1.5 and 2% sodium bicarbonate, respectively. All the diets were iso-nitrogenous (having same protein contents, CP, 17%) and iso-caloric (having same energy level, ME, 2700 Kcal/Kg). these diets were fed to the experimental birds ad libitum, for 12 weeks (26-37 weeks of age). Data on feed consumption, number of eggs produced, egg weight and egg mass laid by the birds were recorded. These data were used for the calculation of feed conversion ratios (FCR) on the basis of per dozen eggs and FCR on the basis of per kg egg mass produced. Five eggs from each replicate were checked weekly for their shell thickness (ST), yolk index (YI), albumen index (AI), Haugh unit (HU) score, yolk pH, albumen pH, specific gravity (SG) and yolk cholesterol. Results revealed that dietary inclusion of sodium bicarbonate significantly (P<0.05) increased feed consumption, weight gain, feed efficiency, egg production, egg weight, egg shell thickness, specific gravity, albumen height, Haugh unit, yolk height and yolk diameter of eggs produced by the birds. Yolk cholesterol was found to be minimum in the eggs laid by the birds fed rations containing 1% NaHCO 3
(group C). Whilst pH of yolk, egg albumen and Serum uric acid concentration were found to be higher in group E. Dietary inclusion of sodium bicarbonate significantly (P<0.05) decreased the rectal temperature and respiration rate of layers, whilst it increased the water intake of the birds significantly (P<0.05). Blood samples were collected from two birds selected randomly from each replicate 10 days post vaccination of 1st, 2nd, and 3rd vaccination to check antibody titer against ND virus. Blood samples were collected from two birds from each replicate at the last day of 37 th week for the analysis of blood profile. Serum glucose, white blood cells count, serum urea, plasma chlorides, serum cortisol and serum glutamic-oxaloacetic transaminase concentration were (SGOT) found to be significantly (P<0.05) higher in control group, whereas, blood hemoglobin concentration, red blood cells count, plasma sodium, potassium, bicarbonate, serum total protein and serum albumen concentration were found to be significantly (P<0.05) higher in birds of group C. However, yolk index, packed cell volume, erythrocyte sedimentation rate, serum creatinine, alkaline phosphatase, plasma calcium, plasma phosphorus, serum globulin and serum glutamic pyruvate transaminase concentration were not affected significantly (P>0.05) due to the dietary treatments. Serum cholesterol, triglycerides and low density lipo-proteincon centration were significantly (P<0.05) decreased, whereas, serum high density lipo-protein concentration was found to be significantly (P<0.05) increased by dietary inclusion of sodium bicarbonate. Birds of group C showed maximum concentration of estrogen, progesterone, T3, T4 and antibody titer against Newcastle disease. For digestibility trial thirty layers having similar body weight were obtained from the same batch which was used for the performance trial. These birds were maintained in individual metabolic cages and were randomly allotted to five experimental diets (same as in performance trial) in such a way that each ration was offered to 6 layer birds and each bird served as a replicate. Feces samples were collected at the end of 38th week of age for two days at the time interval of three hours. Results revealed that digestibility of dry matter , protein, ether extract and crude fiber as well as mineral absorption was found to be better in the birds fed diets containing 1% sodium bicarbonate.
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CHAPTER-1
INTRODUCTION
Pakistan is situated in the subtropical zone of Northern Hemisphere of the world
where temperature usually remains well above the higher side of thermo-neutral zone (25-37
°C) for the greater part of the year (Anjum, 2000). The environmental temperature of some
parts in the region reaches up to 52 °C (Vidal and Walsh, 2010). The optimum temperature
for efficient performance is 19-22°C for laying birds, however, ambient temperature
especially on the higher side is very disruptive and may reduce survival rate and production
(Charles, 2002). Heat stress during summer is a major problem in most parts of Pakistan, and
this has pronounced effects on production performance of layers (Mashaly et al., 2004). Egg
production declines drastically, thereby adversely affecting the economics of poultry
production, which may lead to increase in the number of culled birds.
High laying house temperature causes detrimental effects not only on egg production,
size of egg and egg quality (Farnell et al., 2001) but also adversely affects physiology of the
birds (Sahota et al., 1990) resulting in high mortality. The birds increase panting up to 10
times if ambient temperature is higher than thermo-neutral zone (Nillipour and Melog, 1999).
Ambient temperature and circadian rhythm influence liver glycogen and plasma carbohydrate
levels with distinct changes in blood glucose (Ahmad et al., 2005).
As ambient temperature shoots up, respiratory rate of birds increases resulting in
higher losses of CO2 that causes increase in blood pH and disturbs acid-base balance
(Toyomizu et al., 2005). Any change in acid-base balance does cause alkalosis or acidosis,
diverting the metabolic machinery used for homeostatic regulation rather than used for
production (Carlson, 1997). Alteration in levels of CO2 can cause disruption in blood pH and
deterioration in eggshell quality (Jones, 2006).
Different techniques are being used in poultry production to combat heat stress. These
techniques include nutritional manipulations such as dietary addition of oils (Ghazalah et al.,
2008), reduction in protein level of feed, supplementation of feed with limiting amino acids
(Daghir, 1995) and management practices like intermittent feeding, feeding the birds in
cool hours of the day, time limit feeding (Yalcin et al.,2001; Macleod et al., 1993),
sprinkling of water, evaporative cooling (Donald, 2000), improved ventilation (Nilipour,
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2000) and supplementation of electrolytes (Ahmad et al., 2005). These techniques are
considered helpful in reducing heat stress. Time limit feeding during cool hours of the day is
a common practice for combating/manipulating heat stress. It has also been recommended
that birds should not be fed during hot hour periods (Mahmood et al., 2005) because it only
adds to body heat due to heat increment, which the birds has to dissipate. Whereas time limit
feeding during the cooler part of the day would increase the feed consumption at a time much
suited for its efficient utilization with minimum chances of heat stress. Although, such feed
practice is not likely to increase the overall daily feed intake, yet it is expected to improve the
feed efficiency and production performance.
The practice of feed withdrawal and intermittent feeding during the hottest hours of
the day is being practiced in many broiler producing areas for the prevention of heat stress
and to control mortality (Ahmad et al., 2006). Short term feed withdrawal has been shown to
lower the body temperature of birds and increase the ability to stay alive in acute stress
conditions (Siegel and Jordan, 1997). Practice of supplementation of ascorbic acid in
commercial feed has also been considered a useful and effective tool for heat stress
amelioration (Whitehead and Keller, Khattak et al., 2012).
Among the electrolytes, NaHCO3 may be used to maintain a correct plasma acid-base
balance to combat heat stress. Sodium bicarbonate is a cheap salt (electrolyte) and is also
used as a buffering agent, source of carbon dioxide, an antacid and for the production of
sodium carbonate (Whiting et al., 1991). It provides sodium and positively affects blood pH
supplying bicarbonate ions (Ahmad et al., 2006). Many studies have reported beneficial
effects of supplementing drinking water of broilers with sodium bicarbonate as a sodium
source (Hassan et al. , 2009). However, scientific research information regarding the use of
NaHCO3 in layer diet is still scarce.
Sodium bicarbonate is a good source of providing sodium and bicarbonate ions.
However, the potential benefits of including sodium bicarbonate as a source of sodium in
poultry diets are uncertain (Teeter et al . , 1985). Although it is adjusted with great care in
poultry diets, however, any imbalance of the sodium in poultry diet may lead to depress their
performance (Murakami et al., 2000). A fall in plasma sodium may cause an increase in
aldosterone secretion and increased re-absorption of sodium from the renal tubules (Rector et
al., 2004).
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Supplementation of sodium bicarbonate resulted in better performance in broilers
subjected to high temperature and humidity stress (Khattaket al., 2012). During heat stress
(Gorman and Balnave, 1995) blood values for bicarbonate concentration are drastically
reduced due to excessive panting. Under such circumstances supplementing diets of birds
with bicarbonate may be useful. As feed additive, sodium bicarbonate helps to maintain
proper pH balance, eliminates acidosis and facilitates metabolic process, ensuring maximum
growth and productivity (Danny, 1995). Moreover, rise in blood bicarbonate concentration
favors increase in performance of chicken (Keskin and Durgan, 1997).
Keeping in view the information above, supplementation of NaHCO3 may be a useful
technique to combat heat stress, which still needs to be addressed in layers. Therefore, the
present study was planned to investigate the effect of dietary inclusion of NaHCO3 on
production performance, nutrient digestibility and blood profile of caged layers during
summer under the environmental conditions of Pakistan.
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CHAPTER-2
REVIEW OF LITERATURE
Birds are able to maintain their body temperature within narrow limits (Khattak et al.
2012). An increase in body temperature due to higher ambient temperature or excessive
metabolic activities may cause irreversible thermoregulatory events that could be harmful for
the existence of birds (North and Bell, 1990). However, various techniques are being used in
birds such as addition/supplementation of various products in poultry rations to ameliorate
the effects of heat stress. Supplementation of sodium bicarbonate (NaHCO3) in water or feed
is one of these practices, which are used as an effort to combat the heat stress in broilers
(Ahmad et al., 1997; Mushtaq et al., 2005). However, scientific information regarding its use
in layers during summer is scanty, especially in the areas of Asiatic region.
The following review will address the effects of heat stress on poultry and
possibility of dietary use of NaHCO3 to reduce/mitigate the adverse effects of heat
stress in poultry.
2.1 What is stress?Any physical or physiological change in birds due to internal or external deviation in
birds caused by an unusual process is called stress (Reddy and Dinesh, 2004). It is an
additional burden on the birds, which possibly tends to produce a disharmony in various
physiological systems. Stress can arise from any of a number of internal or external factors
and can cause hormonal changes in the body though pituitary and adrenal glands. General
adaptation syndromes described by Selye (1973a) has become the basis of studies for many
scientists working on the subject of stress in animals. According to him, “stress describes an
animal's defense mechanisms, and thus stressor is any situation that elicits defensive
responses”.
The surrounding environment of the bird is amalgamated of interacting stress factors,
thus bird's success to compete with it depends on the intensity of stressor and bird's
physiological mechanism to react to the stressor (Chrrousos and Gold, 1992). Generally the
environment of the bird is of two types 1) external i.e. temperature, light, etc. and internal i.e.
disease organisms, parasites etc. Any changes in the environment of birds activate regulatory
mechanism in attempt to maintain homeostasis. There are 2 types of regulatory mechanisms:
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1) specific, 2) nonspecific (Carrasco and Van de Kar, 2003). Any particular change in the
environment of the bird will educe/elicit a specific response. For example, when ambient
temperature is increased, it causes: body temperature of bird to rise, vasodilation for
rapid/quick heat dissipation, and feathers are rearranged to ease insulation. To regulate
adaptation process against stress, endocrine and nervous system work together.
2.1.1 Heat Stress mechanism
Stimulus of heat stress is received by the central nervous system, which excites
hypothalamus to activate pituitary glands (Selye, 1956) and in turn releases Adreno
corticotropic hormone (ACTH). Secretion of this hormone is regulated by the secretion of
corticotrophin releasing factor from hypothalamus. Adreno corticotropic hormone stimulates
adrenal gland to produce catecholamine (via adrenal medulla) and corticosteron (via adrenal
cortex). Catecholamine (epinephrine and nor-epinephrine) perform non-essential functions
i.e. increase glucose metabolism (Selye, 1973), blood flow through skeletal muscles (Sahin et
al., 2002), oxygen consumption, heart rate, elevates blood pressure and constricts arterioles
and venules (Grandin, 1998; Siegel, 1980). Adrenal cortex hormones (cortisol and
aldosteron) perform essential functions i.e. promote synthesis of glucose from fat and protein
(gluconeogenesis), maintain heat loss mechanism, blood flow rate and body temperature
(Maxwell, 1993). These hormones also maintain osmotic pressure, regulates sodium
retention and potassium loss through kidney, water balance and mediate response to stress
(Olanrewaju et al., 2006).
Stressors activate a compound array of responses i.e. endocrine system, nervous
system, immune system etc. and the process is called stress response (Carrasco and Van de
Kar, 2003). It causes a number of behavioral and physiological modifications that get better
animal existence when there are homeostatic challenges (Habib et al., 2001). Behavioral
adaptations for a stress response may involve increased awareness, euphoria, improved
cognition (Chrousos and Gold, 1992) and enhanced analgesia (Charmandari et al., 2005).
Physiological effects due to stress include: increase in cardiac activity, increased respiration,
increased intermediate metabolism and restraining of general vegetative functions such as
feeding, digestive, growth, reproductive and immune response (Sapolsky et al., 2000).
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2.2 Thermoregulation mechanism in birdsThe birds being homoeothermic can regulate their deep body temperature within
certain limits of ambient temperature (Pickering, 2000). Birds are also endothermic and can
increase the body temperature by creating considerable quantity of heat within their cells and
tissue. Birds use plumage, fat insulation and salt glands to regulate their body temperature
(Sturkie, 1976). When ambient temperature of the birds goes beyond thermo-neutral zone,
chemical reaction speeds up in body, heat is generated and their body temperature rises
(North and Bell, 1990). In order to keep body temperature normal the birds make effort to
dissipate excessive heat via conduction, convection, radiation and evaporation. During humid
and hot weather evaporative cooling (panting) is a main source of heat loss from the body
which speeds up water to evaporate through respiratory tract resulting in removal of heat
from the body (Angiletta et al., 2010). This mechanism is also affected by ambient relative
humidity. Evaporative cooling, fogging and misting of birds are useful even in hot and humid
climates. As there are no sweat glands in birds hence they excrete heat through respiration
(Nillipour and Melog, 1999). Normally, when atmospheric humidity is increased, the humid
environment slows down the evaporation of water from the respiratory tract. In this situation
birds require more energy to lose heat from body which may cause exhaustion, resulting in
heat prostration (Remus, 2001).
Poultry birds physiologically perform most competently within a narrow comfort
zone of 25-37°C (Sturkie, 1976). This range of ambient temperature depends upon body
weight of birds, amount of plumage, shape of feathers along with their amount and
distribution in body, acclimatization and dehydration status of birds. The higher and lower
temperatures are known as upper and lower critical temperatures. A disturbance in ambient
temperature may cause a change in physiological and metabolic parameters such as rectal
temperature, diseases, metabolic disorders and production losses (Ahmad et al., 2007;
Anjum, 2000; Dibartola, 1992; Carlson, 1997).
2.3 Effect of heat stress on various physiological norms of birds2.3.1 Growth
Adverse effects of high ambient temperature (heat stress) on the production
performance of laying pullets and broiler is well known (Anjum, 2000; Yahav, 2000; Star
et al., 2008; Deng et al., 2012; Sohail et al., 2012). It is also well documented that growth
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rate of birds varies with the fluctuations in environmental temperatures. Impact of different
environmental temperature on weight gain of birds may also vary depending on the age and
breed of birds (Hussan et al., 2011; Diarra and Tabuciri, 2014).
At high ambient temperature and humidity, efficiency of chickens to maintain their
normal body weight is adversely affected. High ambient temperature ranging 34 to 37.8 °C
has shown to reduce body weight gain in chickens (Geraert et al . , 1996) and quails
(Keskin and Durgun, 1997). Similarly adverse effect of heat stress on body weight gain in
birds has also been reported by Njoya and Picard (1994) and Njoya (1995). Macleod and
Hocking (1993) observed a significant loss in the weight of chicken with gradual increase
in ambient temperature. The study of Smith (1992) indicated that layer birds lost significant
body weight during higher ambient temperature. They also reported that the seasonal
variations affected the rate of daily protein requirements.
2.3.2 Feed consumption
Heat stress is known to cause noteworthy decrease in feed consumption in broilers
(Ahmad et al., 2006). At higher ambient temperatures body activities of birds are reduced
and the bird’s needs for energy to keep their body temperature normal are increased which
adversely affect the production performance and feed efficiency of the layers (Anjum, 2000).
Heat stress is reported to depress the feed intake of chickens (Tadtiyanant et al., 1991).
Balnave and Gormen (1993) observed a decrease in feed consumption of layers as the
ambient temperature increased. Roussan et al. (2007) studied the significant adverse effects
of thermal stress on feed consumption of turkeys. Balnave and Muheereza (1997) observed
the effects of high environment temperature (30 °C and 35 °C) on performance of laying
birds. They found significantly decreased feed consumption in birds exposed to higher
temperature.
Mack et al. (2013) observed that the birds kept under high environment temperature
spent more time in drinking, resting, wings elevating and panting whereas spent less time in
feed consuming, moving and walking activities. Feed consumption was reported to be
depressed at high ambient temperature (Geraet et al., 1996; Nakamura et al., 1992; Mikec et
al., 1992; Muiruri and Harrison, 1991a). Similarly, laying hens trained for two meals per day
showed decreased feed intake at higher temperature (Li et al . , 1992).
7
2.3.3 Feed efficiency
It is generally agreed that heat stress adversely affects feed efficiency of poultry birds
(Tanor et al., 1984; Anjum, 2000; Balnave and Gormen, 1993; 2005; Ahmad et al., 2007;
Star et al., 2008; Deng et al., 2012; Yahav, 2000; Sohail et al., 2012). Poor feed efficiency of
the poultry birds may perhaps be ascribed to the decrease in feed consumption of the birds
exposed to heat stress. Under these circumstances a major part of feed consumed is utilized
to fulfill the maintenance requirements of the birds. Moreover, a substantial part of energy is
utilized to dissipate heat from their body. Anjum (2000) reported poor feed conversion
efficiency in White Leghorn layers exposed to heat stress. Similarly, Li et al . (1986)
reported poor efficiency of feed utilization in birds exposed to heat stress. However, Muiruri
and Harrison (1991a) did not find any effect of environmental temperature on feed efficiency
in layers.
2.3.4 Egg production
Negative effects on egg production of layers due to high environmental temperature
conditions have been reported by many scientists (Muiruri and Harrison, 1991a; Bell and
Adams, 1992). Similarly, Melesse, (2011); Mashaly et al. (2004) and Peguri and Coon,
(1991) also observed a significant decrease in egg production in layer birds exposed to
heat stress.
Anjum (2000) reported that production performance of White Leghorn birds reduced
severely when they were exposed to an abrupt increase in their environmental temperature. It
was suggested that functioning of reproductive system might have been depressed under high
environmental temperatures. Findings of Odom et al . (1985) have also revealed a drastic
decrease in egg production of layers subjected to a high environmental temperature.
Peguri and Coon (1991) tested the effects of diverse ambient temperature on the
performance of layers. The birds were reared in rooms, which were constantly maintained at
six environmental temperatures i .e. 16.1, 18.9, 22.2, 25.0, 27.8 and 31°C. The results
revealed significant differences in egg production of the birds during 20 to 36 weeks of age,
which indicated specific effects of warm or cold environments. Deaton et al . (1982)
studied the effect of higher environment temperature i.e. heat stress, on egg production of
layer birds acclimated (adapted) to cyclic versus constant environmental temperature. The
birds were kept (acclimated) to twenty four hours of a linear thermal cycle of either 15.6 °C
8
to 3 °C to 15.6 °C and/or a continuous temperature of 25 °C. Heat stress temperature of 39
°C was maintained with a relative humidity (RH) of 26%. The results showed non-significant
variation in hen day egg production of layers kept (acclimated) to twenty four hours of linear
thermal cycles of either 15.6 °C to 3 °C to 15.6 °C and/or 25 °C. The decline in egg
production percentage was obviously higher for layer birds kept at even temperature of 25 °C
as compared to birds kept at 15.6 °C to 35 °C to 15.6 °C thermal cycle. Results revealed a
higher reduction in production at 39 °C.
In contrast to the findings discussed above, no effect due to heat stress has been
observed on egg production. Roland et al . (1996) checked the dietary manipulation of
calcium and environment temperature on first cycle egg production of commercial Leghorn
layers. Based upon the results of research experiment, warm and cool environment
temperatures did not exert any effect on egg production.
Heat stress has shown a noteworthy effect on the size of eggs in laying birds and has
been observed to decrease with increase in ambient temperature. Heat stress (Hyperthermia)
depressed egg weights in hens (Odom et al . , 1985). However, low ambient temperature has
been reported to increase egg weight of commercial Leghorn pullets housed at 15.6 °C to
23.3 °C (Ronald et al . , 1996).
2.3.5 Egg characteristics
It has been observed that birds exposed to high environmental temperature produced
eggs with poor shell thickness (El-Boushy and Raternick, 1993). Higher environmental
temperature has been shown to decrease shell thickness of eggs because of low feed intake
(Balnave and Muheereza, 1997), which probably caused reduction in calcium intake, an
important element required for shell formation (Karimian et al., 2004). Another probable
reason advocated regarding this aspect is decrease in serum calcium level due to high house
temperature (Hassan et al., 2003). Anjum (2000) has also reported adverse effects of high
environment temperature (heat stress) on egg shell thickness of the eggs produced by White
Leghorn layers and reported that birds exposed to heat stress laid thin shelled eggs.
However, Li et al . (1986) did not observe any effect on egg shell thickness as a
result of high environmental temperature. Slinger (1985) stated that egg shell thickness
decreased when body temperature rose above normal. Deaton et al . (1982) determined the
effects of high environmental temperature (heat stress) on layer birds acclimated (adapted) to
9
cyclic thermal environment versus constant ambient temperature. They conducted two trials
of eight weeks duration; each on 231 Dekalb layers acclimated (adapted) to twenty four
hours linear increase of ambient temperature of either, 15.6 to 35 °C and/or a constant
ambient temperature of 39 °C with a relative humidity of 26%. They observed insignificant
effects on shell thickness of the eggs of layer birds exposed to cyclic vs. (versus) constant
temperatures. Akram et al . (1999) reported that surface wetting of layers by water spraying
can improve thickness of the eggs produced by the birds.
Daniel and Balnave (1981) conducted a study to check the effect of high house
temperature on eggs quality characteristics of White Leghorn x Australorps layers of 32, 70
and 80 weeks old. Hens were exposed to high environmental temperature (35 °C) abruptly
and gradually, between the ranges of 60-80% relative humidity during summer months. No
detrimental effect of heat stress on the Haugh unit score was observed. However, North and
Bell (1990) stated that quality of albumen and yolk deteriorates with the increase in storage
time and temperature.
One of the serious problems in egg production is blood and meat spots in the eggs
which lowers quality as well as age of the eggs (shelf life). Higher protein percentage in diet
is positively correlated with the blood spot occurrence in laying hens (Bearse et al . , 1962).
Nair and Elizabeth (1983) reported that the percentage of eggs with blood spots was 4.5-9.5
in the various seasons and that of meat spots was 9.5-15.5; the differences between seasons
being significant.
Trail (1963) compared blood and meat spot problem in local chicken breeds of
Uganda with five imported ones. Indigenous breed was found to have less (0.7%) eggs
having blood and meat spots. Whereas, imported breeds were found to have more (2.1% to
9.1%) eggs experiencing blood and meat spots. This difference in the rate of eggs meat and
blood spots was attributed to less stress and good adaptation of hot environment rate
experienced by the birds. Akram et al . (1999) found less blood spots in restricted feeding of
hens under normal laying process.
2.4 PhysiologyAmong the factors affecting body temperature, important ones are humidity, heat
loss rate from the body, feed intake, nature of feed, water intake feeding schedules, vitamins,
minerals, acids-base balance, electrolytes, breed, strain, housing conditions and cooling
10
methods. Body temperature of adult chickens also differs with change in the environmental
temperature (Reddy and Dinesh, 2004). When the environmental temperature is equal to the
body temperature, heat cannot be lost from the bird by non-evaporative means. However,
excess heat losts through respiratory lining by evaporation of moisture (Mustaf et al., 2009).
Birds have air sacs for exchange of heat between the body and external environment. These
air sacs contribute to increase the surface area for exchange of gases and evaporative heat
loss (Fedde et al., 1998).
Environmental temperature causes a significant impact on physiological phenomena
in birds. Mack et al. (2013) studied the physiological responses of fowl reared at higher
environmental temperature and reported that birds exposed to higher ambient temperature
(heat stress) conditions spent more time for panting. Heat stress disrupts reproductive
hormones of layer birds secreted by the hypothalamus and ovary (Elnagar et al., 2010). A
decrease in volume of semen fluid, sperm cells concentration and live sperm cells count was
observed in males broiler breeders subjected to high temperature environment (McDaniel et
al., 2004).
Prieto et al. (2010) observed significant less circulating lymphocytes count but an
increase in the number of heterophils count of birds reared under heat stress. Moreover,
broilers subjected to high ambient temperature showed regressed lymphoid organs weights
(Niu et al., 2009), ultimately leading to a poor immune response in laying birds. Birds kept
under heat stress showed depressed systemic humoral immune response, less number of
intraepithelial lymphocytes and Immuno globulin A-secreting cells in their digestive tract
(Niu et al., 2009). The reduction in immune response of the birds was attributed to reduced
antibody response and phagocytic ability of macrophages in birds because of high ambient
temperature.
Teeter and Belay (1996) recommended fasting and starvation as a method of lowering
the rectal temperature in broilers reared under heat stress environment. Sahota et al . (1996)
observed effect of Vitamin C supplemented in diet during summer on rectal temperature of
LSB (Lyallpur Silver Black) and WLH (White Leghorn) layers at the age of 27 weeks when
exposed to heat stress for an experimental period of 12 week. The results of research
exhibited high rectal temperature (42.02 °C) in the control birds which were exposed to heat
stress than those kept under treated groups.
11
2.5 Hematological profile It is generally agreed that environmental temperature, age, season, feed, diurnal
effect, and fasting are related to hematological responses in poultry birds. Thermal
environment has shown a significant effect on white blood cells count (Maxwell et al., 1992).
Maxwell et al. (1992) examined the effects of hot weather environment on differential
leucocyte (DLC) responses in broilers, turkeys and ducks exposed to various degrees of feed
restriction. It was also reported that a mild to moderate heat stress may result in increase in
leucocyte count. An increased white blood cells count in laying birds at high environmental
temperature has also been reported by Anjum (2000).
Packed cell volume value of birds has shown an inverse relationship with high
ambient temperature (Parker et al . , 1982). Hypothermia (8 °C) caused an increase in the
hematocrits, whilst hyperthermia (30 °C) caused a decrease in hematocrit value.
Hyperthermia exhibited a reduction in packed cell volume in chicken and turkeys (Andrade
et al . , 1976; Parker and Boone, 1976). However, supplementation of anti-stress compound
like vitamin C and sodium bicarbonate have been reported to be effective in improving the
packed cell volume in layers exposed to heat stress. Sahota and Gilani (1995) determined the
effects of supplementation Vitamin C in the diet at level of 0, 50 and 100 ppm on the
hematocrit values of LSB and WLH layers kept under high ambient temperature of 31-45 °C
for 12 weeks. It was concluded that influence of ascorbic acid on packed cell volume might
be due to the rise in erythrocyte number. Similarly, Sahota et al .(1993) reported an
improvement in packed cell volume of LSB and WLH breeds of chicken with dietary
ascorbic acid supplementation during heat stress period.
Ekanayake et al. (2004) and Mubarak and Sharkawy, (1999) have reported an
increase in RBCs count in birds fed diets containing sodium bicarbonate. Whereas, Khattak
et al. (2012) reported an increase in WBCs count in the birds kept under heat stress condition
than those fed sodium bicarbonate containing diet at the same temperature. A significant
decrease in packed cell volume has been observed by Oladele et al. (2001) in birds exposed
to high environmental temperature. This increase was attributed to high ambient temperature
which might impaired the synthesis of blood cells in these birds. Heat stress increased blood
glucose and decreased liver glycogen levels in pigeons (Chakraborty and Sadhu, 1983).
Khmilyar (1983) concluded that an increase in ambient temperature above the optimum level
12
caused a considerable drop in serum glucose level in birds. Whereas, opposite to it, Yang et
al . (1992) observed highest blood sugar contents at 23 and 28 °C (223.6 and
221.7mg/100ml) at the exposure of broilers to 12, 18, 23, 28 and 32 °C temperatures.
Sahota and Gilani (1994) observed the effect of heat stress in Lyallpur Silver Black
and White Leghorn layer birds. The birds were exposed to 30 °C and 39 °C, respectively. An
increase in blood glucose in birds of control group was observed as ambient temperature was
raised. They observed a blood glucose level of 196 and 179 mg/dl in 5 weeks old Lyallpur
Silver Black and White Leghorn chicks at 17.5 °C, respectively, which increased to 206 and
195 mg/dl at 27.5 °C at 12 weeks of age.
Mubarak et al. (1999); Al-Hassani et al. (2001) and Ahmad et al. (2005) have
observed an increase in hematocrit values in birds treated with sodium bicarbonate. They
attributed this increase to high house temperature that might impair the synthesis of blood
cells in these birds. Similarly, results of a study executed by Oladele et al. (2001) has
reported a significant decrease in blood packed cell volume in birds exposed to high
environmental temperature when fed sodium bicarbonate supplemented diet.
2.6 Serum metabolites and serum proteinExposure of poultry birds to heat stress has been found to affect their serum
metabolites and the effect may be ameliorated by fortifying their diets with different levels of
sodium bicarbonate (Hassan et al., 2011). An increase in serum urea concentration in layers
kept at higher ambient temperature when compared to those reared under heat combating
systems has been observed by Anjum et al. (2000). Whereas, Yang et al . (1992) has
observed significantly higher serum urea concentration in birds kept at low temperature (12
°C) as compared to those kept at relatively higher ambient temperatures (23 and 28 °C). On
the other hand, Kurtoglu et al. (2007) investigated non-significant (P>0.05) effect due to
dietary inclusion of sodium bicarbonate on uric acid concentration in Brown-Nick layers.
Moreover, Koelkebeck and Odom (1995) did not observed significant effect of higher
ambient temperature on serum uric acid and creatinine concentration in laying birds.
Blood plasma proteins are of two types; albumen and globulin. A
marked decrease in plasma protein concentration has been observed in birds reared under
high ambient temperature (Anjum, 2000) and the decrease was ascribed to heat stress, which
hampered the synthesis of plasma proteins in liver. Higher ambient temperature causes a
13
decrease in serum protein contents in birds (Geraert et al . , 1996). Similarly, findings of
Yang et al . (1992) also revealed higher contents of serum total protein in broilers reared at
12 °C than those reared at 23 °C and 28 °C.
Three studies related to hemodynamic changes in blood protein of fowl kept under
high environmental temperature were conducted by Yahav et al . (1997) in which Cobb
broilers were raised for 4 weeks of age, in battery brooder at 26 °C. Thereafter, for the 1st
trial these birds were acclimated to constant environmental temperatures of 10, 20 and 30 °C
or to diurnal temperatures of low (10 °C) and high (30 °C) up to 8 weeks of age. In their 2nd
trial ambient temperatures were maintained at 15, 25 and 35 °C constantly, whereas in 3rd
trial, non-acclimatized 8 week (W) old birds were exposed to 35 °C. Results of these studies
revealed a decrease in plasma protein concentration at higher temperatures.
There is paucity of information about the response of birds with respect to the serum
uric acid fractions during hot summer conditions. However, Yang et al . (1992) reported
higher serum uric acid level in birds kept at 12 °C than those exposed to 23 °C and 28 °C.
Scientific information regarding the effects of inclusion of NaHCO3 in the diet on serum
alkaline phosphatase in the birds exposed to heat stress is scanty. However, findings of Bogin
et al. (1981) have depicted that broilers subjected to heat stress for two hours showed non-
significant effect on their blood serum alkaline phosphatase level. Findings of Koelkebeck
and Odom (1995) have also revealed non-significant effect of acute heat stress on alkaline
phosphatase enzyme in layers.
2.7 Plasma electrolyte and mineral profileAs ambient temperature exceeds 30ºC, it causes increase in respiration rate of birds,
which might go up to about 10 times more than normal rate (Nillipour and Melog, 1999). It
has been observed that birds started panting along with increase in blood pH during high
ambient temperature. A mild alkalosis (pH 7.55) develops at a temperature of 35 °C, with no
raise in body temperature. Further increase in temperature from 35 °C to 38 °C induces
moderate alkalosis, whereas, severe alkalosis (blood pH 7.65) occurs at higher ambient
temperature i.e. 41 °C (Leeson and Summer, 2001). Findings of Teeter et al. (1985) reported
that pH values higher than 7.25, lowered the performance of poultry birds. This increase in
blood pH can be prevented by reducing panting and rising water intake in birds (Belay and
Teeter, 1993). DEB and blood pH are directly related to each other; at 0 DEB blood pH is
14
always acidic, whilst at 350 DEB it shifts to basic (Ahmad et al., 2009). Acid base balance is
also considered to be important in controlling blood pH in fowls, which in turns improves
efficiency of enzymes and ultimately physiological functions (Patience, 1990).
An increase in blood pH has been shown to depress feed intake which ultimately
reduced the production performance of birds (Yahav et al., 2004). Probably bird performance
is directly associated to blood pH, which shows bird’s physiological and biochemical
condition. Production performance of broiler is higher when blood pH is optimum (7.28),
whereas a reduction in performance is exhibited when pH value is greater than 7.30 or lesser
than 7.20. In verity this narrow range of blood pH establishes the physiology of body enzymes
which is required in expressions of good general health and optimum production of animals
(Lehninger, 1970).
Scientific information regarding the consequence of dietary inclusion of sodium
bicarbonate (NaHCO3) on serum alkaline phosphatase in the birds exposed to heat stress is
scanty. However, findings of Bogin et al. (1981) have shown that broilers subjected to heat
stress for two hours showed non-significant effect on their blood serum alkaline phosphatase
level. Findings of Koelkebeck and Odom (1995) have also revealed acute heat stress had no
effect on alkaline phosphatase enzyme in laying hens. There is paucity of information about
the response of birds with respect to the serum uric acid fractions during hot summer
conditions. However, Yang et al . (1992) reported higher serum uric acid level in birds kept
at 12 °C as compared to those kept at 23 °C and 28 °C. Heat stress decreases the plasma
protein concentration in poultry birds with increase in the environmental temperature
(Anjum, 2000; Geraert et al . , 1996; Yang et al . , 1992). A decrease in blood Na+ level in
broilers kept under heat stress has been observed by Borges et al. (2004) and Takahashi and
Akiba (2002). Plasma potassium (K+) concentration/level was found to be decreased in birds
kept in heat stress (Harper et al., 1977). The decrease in K+ has been reported to be due to
either its increased excretion (Berne and Levy, 1993) or an increase in its uptake by the cells,
or both. Increases in excretion of K+ emerge to preponderate in chronic heat stress, whereas
its increase in uptake by the cells is manifested during acute high temperature environment.
The adverse effects of high environmental temperature/heat stress on blood K+ concentration
has also been found to be similar in different species of birds such as in broilers (Mushtaq et
al., 2005), layers (Ghorbani and Fayazi, 2009) and quails (Keskin and Durgan, 1997).
15
2.8 Serum lipids, hormones and enzymatic profileCholesterol is present in the cells of liver and aortic tissues as well as in the fluids of
animal body. Important factors which affect cholesterol level in the blood are: sex, age,
ration, hyperthermia and starvation. Hevia and Vinsek (1979) reported that fasting increased
blood cholesterol level by mobilizing fat through gluconeogenesis which ultimately increased
blood cholesterol level. High serum cholesterol concentration in birds kept at high
temperature has also been reported by Haazele et al . (1991); Takahashi et al . (1991);
Sahota et al . (1993) and Sahota and Gilani, (1994).
Heat stress affects performance as well as various biochemical processes including
hormone and enzymatic status of birds (Anjum, 2000). Adrenal gland has a central role in the
General Adaption Syndrome (Selye, 1973a) and hormones produced by this gland are
strongly correlated to the heat stress. Thyroid hormone is essential for the development and
normal growth of birds and its secretion rate accurately determines thyroid gland activity
(May et al . , 1974). Thyroid activity had been found to be adversely affected by high
ambient temperature and it was lowest in chickens reared under heat stress, which might
have been due to variations in photoperiod, seasonal reproduction behavior in species and
age of the birds (Bowen and Washburn, 1985).
Hypothalamus and pituitary respond to high environmental temperature by decreasing
the secretion of thyroid gland. Cogburn and Harrison (1980) observed low T3 values in birds
exposed to hot environment. Furthermore, El-Gendy et al . (1995) reported a lower plasma
T3 level in heat stressed broilers at 6 weeks of age. Similar responses on the secretion of T3,
due to heat stress have also been observed by Brigmon et al . (1992) in commercial layers.
Moreover, changes in environmental temperature and level of serum T3 have been found to
be negatively correlated with each other (Brigmon et al . , 1992).
Heat stress is known to influence reproduction performance in pullets (Tojo and
Huston, 1980) and estrogen is a key hormone for efficient reproductive performance in
layers. Kohne and Jones (1976), observed a continuous decrease in plasma levels of estrogen
during heat stress. Progesterone is also a vital hormone, which is related to ovulation process
of birds. Novero et al . (1991) reported that stoppage of progesterone because of malfunction
of ovary due to heat stress, may cause malfunction of positive feedback mechanism to the
hypothalamus resulting in a decrease in secretion of luteinizing hormone in birds.
16
2.9 Immune responseEnvironmental stressors affect immunity and innate resistance of the host directly or
indirectly. Layer birds kept under heat stress experienced a reduction in lymphocytes and a
rise in heterophil concentration (Anjum, 2000). Thaxton and Siegel (1972) reported that high
ambient temperature mediated immune depression. Heat stress caused decrease in total
leucocytes count (Ben Nathan et al., 1977) and thus affected immune response.
Environmental factors other than temperature have also shown to influence immunity in
birds, i.e microbial toxins (Michael et al., 1973), hypoxia (Tengerdy, 1970), non-ionic
radiation (McRee et al., 1977), social connections (Siegel and Latimer, 1975), heavy metals
(Morgan et al., 1975), pesticides (Glick, 1972), nest strain (Thaxton and Briggs, 1972) and
amount of nutrients intake (Tengerdy and Brown, 1977) etc.
The control of antibody mediated immunity at various environmental temperatures
had been studied by many investigators (El-Gendy et al., 1995). Birds exposed to a
temperature of 32.2 °C and higher than 32.2 °C reduced (P<0.05) the agglutinin level in their
blood. A petite exposure (2 or 4 times) to cold and subsequent antigen injection increased the
agglutinin and hemolytic response in birds. Whereas, 30 minutes cold contact for 2 or 4 times
considerably (P<0.05) augmented the IgM antibody concentration and markedly
abridged/decreased the IgG (Suba-Rao and Glick, 1977). El-Gendy et al. (1995) observed
that serum antibodies concentration against Newcastle disease vaccine (NDV) was lesser in
heat stressed broilers as compared to those kept under normal temperatures.
Anti-sheep erythrocyte values have been known to be affected due to high ambient
temperature. Savic et al. (1993) exposed broiler chicks to heat stress at different intervals of
time. The control (maintained at thermo-neutral zone) and heat stressed birds were
vaccinated at 12 days of age using Lasota strain vaccine against Newcastle disease virus. At
the time of vaccination, HI titer of both the groups was < 1:23 and was found to be 1:24 at 18
days post vaccination in control and 1:23 in heat stressed group. Environmental stressors have
been known to affect immunity and innate resistance of the host directly or indirectly
(Robertson, 1998). Bains et al. (1996) investigated a significant (P<0.05) lower immune
response in turkeys due to high ambient temperature Similarly, Tuekam et al. (1994)
observed a negative correlation between serum corticosterone concentration and antibody
titers in heat stressed birds.
17
2.10 DigestibilityHigh ambient temperature may exert a significant influence on digestion and
absorption of nutrients and their metabolism (Macleod, 2004). Increaese in ambient
teperature has been shown to reduce feed consumption in birds to prevent thermogenic effect
(heat increment) associated with nutrient utilization, absorption and assimilation (Koh and
Macleod, 1999). Ambient temperature above 30 °C has shown to cause a decrease in blood
flow towards digestive tract (Wolfenson, 1986). Consequently it may reduce hydrolytic
activities of the respective enzymes in upper part of digestive system (Haiet al.,2000) and
hence may lead to decrease in digestibility of protein.
Brake et al. (1998) reported that at high ambient temperatures (32 °C), arginine
uptake was reduced in birds. Puvadolpirod and Thaxton (2000) observed significantly lower
protein and carbohydrate digestibility in broilers in which ACTH was dispensed to induce
stress. Zuprizal et al. (1993) noted a reduction in the digestibility of rapeseed meal and soya
bean meal protein, at high ambient temperature. However, Virden et al. (2007) investigated
that physiological stress had no effect on amino acid digestibility.
Factors, which may influence digestibility of nutrients include, ambient temperature
(Macleod, 2004; Hai et al., 2000 ; Puvadolpirod and Thaxton, 2000), level of feed intake and
passage rate of digesta (Ravindran et al., 2008; Ahmad et al., 2007), anti-nutritional factors
present in feed ingredients (Hughes and Choct, 1999), age and physiological status of the
bird (Batal and Parsons, 2002; Huang et al., 2007; Garcia et al., 2007) and nutritional
composition of the diet (Leeson and Summer, 2001a).To improve digestibility of feed
ingredients different nutritional manipulations have been used such as adding enzymes in
feed (Selle et al., 2010; Bryden et al.,2009), heat treatment and processing of feed
ingredients and (Friedman, 1996; Amerah et al., 2007) addition of electrolytes in feed/water
during hot weather (Ravindran et al., 2008).
2.11 Significance of electrolytes in combating heat stress Several methods have been proposed to ameliorate high ambient temperature in the
poultry house and to decrease body temperature of birds for successful poultry production
(Daghir, 1995). Dietary electrolyte balance (DEB) in poultry birds plays a significant role
for the better performance. An optimum dietary electrolyte balance is thus required for
efficient performance, proper bone development and good litter quality (Oliveira et al.,
18
2010). However, if DEB is not maintained in normal limits, the performance of the birds is
adversely affected. Maiorka et al. (2004) recommended a dietary DEB of 174mEq/kg for
better feed intake and 163mEq/kg for the best weight gain as compared to 250mEq/kg of
DEB. It has also been observed that a DEB of 175mEq/kg may improve performance in
broilers until 21 days of age (Szabó et al., 2011), but DEB should be 250mEq/kg during the
grower and the finisher phases in broilers. However, role of DEB on performance of layer
birds has not been much studied. Ghasemi et al. (2014) have reported that, under tropical
conditions, using a DEB of 250mEq/Kg achieved a correction of the lay-induced metabolic
acidosis and results in a positive effect on performance of layers.
Electrolytes maintain ionic and water balance in living systems. It is important to note
that requirements of electrolytes cannot be considered individually because there must be an
overall balance among these to achieve homeostasis. Maintaining acid-base balance is a key
strategy to avoid harmful effects of heat stress. Acid base balance is mainly affected by
environmental and nutritional status of the birds. High anions (negative charged ions i.e. Cl-)
may cause acidemia in chickens, whilst high cation contents (positive charged ions i.e. Na+,
K+) in diet cause alkalemia. Both these adverse situations, therefore, may affect performance
of fowls. Dietary electrolyte balance may be calculated using equations developed by various
scientists. However, for the calculation of DEB, it be concerned that oncentration of sodium
(Na+), K+ and chloride (Cl-) should be within adequate range (Mongin, 1981). Physiological
stress, however, tends to cause deviation in electrolyte balance of poultry birds (Yalcin et al.,
2004; Sandercock et al., 2001; 2003; Borges et al., 2003, 2004).
In young birds, Cl− at high levels i.e. 160-240mEq/kg, significantly decreased blood
H+ concentration (Ruiz-Lopez and Austic, 1993). Sodium sulphate has been found to be
relatively more acidic as compared to calcium sulphate and potassium sulphate (Ahmad et
al., 2005). Gorman and Balnave (1994) investigated that heat stress can cause an increased
metabolic need for HCO3− ions. Patience (1990) observed that acid base and electrolytes
balance effect the growth, appetite, thermal stress response and the metabolism of different
nutrients in birds. Borges (2001) viewed that a complete electrolyte equation would be (Na+
+K++Ca+2+ Mg+2) - (Cl− + SO4−2 + 2PO4
−2) and also reported that maximum feed intake was
noted at DEB 264mEq/kg. Rondon et al. (2000) reported 250mEq/kg DEB when Na+ level
were different and 319mEq/kg when K+ level manipulate.
19
Murakami et al. (2001) recommended optimal DEB between 246 and 315mEq/kg for
broilers during starter phase and for the growers between 249 and 257mEq/kg to achieve
efficient performance. Borges et al. (2002) investigated that ideal DEB was found to be
between 246 and 277mEq/kg. Borges et al. (2003a) observed that dietary electrolyte balance
of 240mEq/ kg influenced beneficial effect on body weight and feed efficiency versus dietary
electrolyte balance of 0, 120, and 360mEq/kg, in chicken reared under heat stress. They
concluded that an optimum DEB range of 220 mEq/kg to 240mEq/kg be maintained for
adequate performance. Barbosa et al. (2014) revealed that electrolyte balance may affect
intestinal length, water intake and heart and liver relative weights. They concluded that
electrolyte balance of 120mEq/kg in feed and 30mEq/L in drinking water may cause an
increase in water intake of European quails reared under hot temperature.
Johnson and Karunajeewa (1985) investigated the dietary effect of mineral inclusion
i.e. calcium and available phosphorus and electrolytes i.e. sodium, potassium and chloride on
physiological response of chickens. They did not observe any change in plasma ions
concentration (Ca, inorganic P, Mg, Na, K and Cl) of birds due to treatments.
2.12 Buffering action of sodium bicarbonateFor normal metabolic events such as maintaining the normal structure and functions
of proteins etc., blood pH of poultry birds must be very near to narrow physiological range of
7.35 to 7.45 (Carlson, 1997). Moreover, blood pH is closely related to HCO3– buffering
system, which is the major buffering system for maintaining blood pH and can be described
with the following equation.
CO2 + H2O ⇔ H2CO3⇔ H+ + HCO3–
Blood bicarbonate concentration is primarily under the control of kidneys and to a
less extent, the lungs. Kidneys organize the concentration/level of HCO3– by adjusting its re-
absorption from the renal tubules. Increased breathing under heat stress decreases pCO2
which in turn causes an increase in pH that induces respiratory alkalosis (Belay and Teeter,
1993). The bicarbonate buffer system functions works with double regulatory control of the
lungs and kidneys. In HCO3– buffering system blood pH is represented by the expression
(Berney and Levy, 1993) called Henderson Hasselbalch equation as follows.
pH = 6.1 + log [HCO3–] / 0.03 pCO2
Where;
20
pCO2 = partial pressure of CO2
In normal physiological phenomenon the ratio of HCO3− to pCO2 is 20:1. In an
attempt to keep the body temperature normal, respiration rate of birds increases which lowers
the pCO2, hence increasing the Log term in the Henderson Hasselbalch equation. This causes
an increase in the pH (respiratory alkalosis). In such conditions sodium bicarbonate might be
used as a buffering agent to nullify the problem (Whiting et al., 1991).
2.13 Attempts to improve feed efficiency during hot weatherHigh ambient temperature has shown to cause significant adverse effect on efficiency
of feed utilization in birds. Anjum (2000) reported a poor feed conversion ratio in White
Leghorn layers exposed to heat stress whereas; Muiruri and Harrison (1991) found that
environmental temperature had no effect of on feed efficiency in layers. These types of
contradictory findings observed by various scientists are still causing confusion regarding the
effect of hot weather on feed efficiency of birds reared under different climatic temperatures,
which are direly needed to be addressed.
Several nutritional and managemental manipulations have been used to combat heat
stress. These practices include provision of maximum insulation and improving ventilation of
the poultry house/shed (Nilipour, 2000), use of evaporative cooling systems (Donald, 2000),
thermal conditioning (Yahav, 2000), use of ventilating fans, reducing bird density in the
house (Lott, 1991), provision of adequate cool drinking water, feed withdrawal for certain
periods of time or fasting prior to beginning of heat stress, feeding during cool hours of the
day and acclimation (Yamauchi et al., 1995; Yahav and Hurwitz, 1996).
Al-Zujajy et al . (1978) examined the effect of use of air coolers in poultry house of
birds/broilers kept under the subtropical conditions (Iraq). They reared chicks for 56 days in
two broiler houses in such a manner that one house was provided with two air coolers to
provide cool environment (21.2-29.5 °C), whilst environmental conditions in the other house
were hot and dry (30.1- 39.9 °C). They observed that efficiency of feed utilization was
significantly better in chicks kept under cool housing conditions.
Srivastava et al . (1980) observed the effect of cooling on feed utilization of broiler
chicks in two trials. In 1st trial (hot and dry atmosphere), feed conversion ratios were found
to be 2.34, 2.56, 2.45 and 2.31 in birds kept in house provided either the exhaust fan, a
fogging system plus ceiling fan, an evaporative cooling system or with no cooling system,
21
respectively, at 8 weeks of age. Whereas, corresponding results for trial 2 (hot and humid
atmosphere) were found to be 2.67, 2.78, 2.74 and 2.62, in the respective groups. Wang
(1995) reported that broilers subjected to time limited feeding during cooler hours showed
better feed efficiency than those exposed to heat stress. Moreover, use of air cooler improved
feed conversion efficiency of the birds by 4.3 to 9.7 percent. Therefore, time limit feeding
during cool hours can be a useful practice in poultry birds for combating heat stress. It has
also been recommended that birds should not be fed during hot hour periods (Mahmood et
al., 2005) because it only adds to body heat due to heat increment, which the birds has to
dissipate. Moreover, time limit feeding during the cooler part of the day would increase feed
consumption at a time much suited for its efficient utilization with minimum chances of heat
prostration. Although, this feed practice is not likely to increase the overall daily feed intake,
yet it is expected to improve the feed efficiency and production performance of birds.
Johnson and Karunajeewa (1985) investigated the effect supplementation of calcium
and available phosphorus (minerals) and the sodium (Na), potassium (K) and chloride
(electrolytes) in feed on growth and physiological response of broiler. Results exhibited that
dietary electrolyte balance (DEB) does not define the growth rate of chicken. Lower (<
180mEq/kg) or higher (> 300mEq/kg) electrolyte balance (DEB) in the feed depressed live
weight of the birds at the age of 42 days. Growth rate of chicken fed diets with DEB (dietary
electrolyte balance) higher than 300mEq/kg depends on the type of cat-ions (Na or K). The
range of Na: K ratio for proper growth was found to be 0.5-1.8. Barton (1998) found that
dissolved bicarbonate in drinking water improved feed conversion in turkeys.
2.14 Effect of dietary inclusion of NaHCO3 on various physiological
norms of birdsIn the following text, a brief review regarding the effects of including sodium
bicarbonate in the diets or/and in drinking water of different species of poultry on various
parameters related to their production performance have been presented.
2.14.1 Growth
High ambient temperature is generally known to reduce the average body weight in
layers as compared to those kept under a cool environment (Anjum, 2000). To improve
weight gain under different heat stress conditions, different feeding manipulations have been
practiced (see section 2.8), which have shown positive effects. However, use of feed
22
manipulation alone is not enough to mitigate adverse/devastating effect of heat stress on
body weight in birds completely (Spinu and Degen, 1993; Zakia et al . , 1995). Under such
conditions, dietary inclusion of sodium bicarbonate has shown significant (P<0.05) effect on
growth rate and efficiency of feed efficiency in birds exposed to heat stress (Ramezani et al.,
2011).
Inter-relationship between levels of NaCl, NaHCO3, phosphorus and calcium in the
diet of layers was studied by Junqueira et al. (1984). Three experiments were performed on
Hy-line layer birds kept in individual wire cages. The layers were given a diet based on
yellow maize and soybean meal with or without addition of sodium chloride, calcium
phosphate or sodium bicarbonate. Sodium chloride did not affect body weight gain of the
birds. However, addition of NaHCO3 in the diets of the birds produced a noteworthy increase
(P<0.05) on body weight of layer birds. Moreover, the diets high in sodium and low in
chloride caused increase in mortality of the hens. Supplementation of diet with 0.5% sodium
bicarbonate resulted in 9% increase in body weight gain in broilers suffering with chronic
hyperthermia (Teeter et al., 1985). Barton (1998) investigated the impact of water quality on
the performance of turkeys and found that dissolved bicarbonates in water were positively
correlated with the weight of broilers.
Harms (1982) replaced sodium chloride with sodium bicarbonate in the diet of
turkeys during starting phase. He observed maximum body weight and feed utilization when
NaHCO3 was added to the diet which already had 0.056% Na+ provided by NaCl. It was
concluded that mutual replacement of NaCl with NaHCO3 for the adjustment of sodium and
chloride might have resulted in better growth rate.
Bonsembiante et al . (1988) performed a trial to test the effect of supplementation of
sodium bicarbonate and ammonium chloride on the performance of chicken during hot
weather. Addition of sodium bicarbonate in broiler feed improved the growth rate and
enhanced efficiency of feed utilization of birds than birds of control group. However, chicks
receiving 0.5% sodium bicarbonate plus 1% ammonium chloride did not perform better than
that of controls. Effect of adding NaHCO3 (0.5%) and KCl (0.5%) in water was investigated
on the production performance and carcass parameters of chicken reared in thermo-neutral or
cyclic heat stress (Whiting et al . , 1991b). The results revealed no appreciable improvement
in weight gain of broilers due to the supplementations when compared to those offered water
23
without any supplementation.
Influence of dietary supplementation sodium bicarbonate, ascorbic acid and acetyl
salicylic acid was observed on broiler’s performance kept under the hot weather conditions
(Puron et al., 1994). For this 3 research trials were conducted, in experiment 1 and 2, sodium
bicarbonate was added at the level of 0.5% and 0.6% in the diets of broilers. Whereas, in
experiment 3, the experimental rations for broiler were prepared by dietary inclusion of 0.6%
bicarbonate, 200 ppm ascorbic acid and 250 ppm acetyl salicylic acid. However, control diet
was kept without any supplementation. These diets, when fed to the experimental groups did
not exhibit any significant difference on performance of the broilers, when compared
between control and treatment groups.
Teeter et al., (1985) observed meaningful (P<0.05) increase in body weight gain of
chicken fed diet containing NH4CI (1%) and sodium bicarbonate (0.5%) during hot weather
conditions. Fox et al . (1997) carried out a research experiment to explore the effects of
dietary addition of sodium bicarbonate (NaHCO3) on production performance and intestinal
pH of chicks infested with Eimeria acervulina (Coccidia) . Outcomes of the trial proved
that dietary supplementation of sodium bicarbonate significantly (P<0.05) increased the
growth and efficiency of feed utilization of the birds. Moreover, dietary inclusion of sodium
bicarbonate significantly increased body weight gain in birds infested with coccidiosis.
Hooge et al. (2000) evaluated the influence of dietary NaHCO3 (0 or 0.25%), monensin (0 or
99 ppm), or coccidial inoculation (0 or 2 Eimeria species), singly and in various
combinations on weight gain in broilers. Dietary sodium bicarbonate significantly (P<0.05)
improved the body weight and feed efficiency of the broiler than those fed diets without the
addition of sodium bicarbonate (control).
Kidd et al. (2003) evaluated the impact of dietary addition of NaHCO3 in high and
modest (moderate) temperature conditions on performance of broilers. Results revealed that
dietary treatments had minimum effect on body weight gain and breast meat synthesis of the
birds. Effects of supplementation of Vitamin C (62.5 mg/liter water), acetylsalicylic acid
(62.5 mg/liter water), NaHCO3 (75 mg/ liter water), and KCl (125 mg/L) in water was tested
on broilers exposed to heat stress (Roussan et al., 2007). However, better growth rate was
noted in the birds fed sodium bicarbonate.
Ramezani et al. (2011) conducted an experiment using 216 male Ross chicken to
24
assess the effect of organic selenium and supplementation of NaHCO3 on blood parameters,
body weight gain and carcass weight of broilers kept in heat stress condition. Results of the
study depicted that sodium bicarbonate significantly increased growth performance, along
with their breast and thigh weight. However, selenium supplementation significantly
(P<0.05) reduced the abdominal fat contents and liver weight of the birds.
Effect of dietary inclusion of betaine, vitamin C, vitamin E and sodium bicarbonate
was studied on the production performance of chicken during heat stress (Khattack et al.,
2012). Results showed a significant role of sodium bicarbonate in improving the performance
of broilers reared under heat stress. Supplementation of betaine and sodium bicarbonate were
also found to be useful in protection against heat stress related adverse effects. Balnave and
Gorman (1993) reported an improved weight gain in broilers fed diet added with sodium
bicarbonate. Genedi (2000) also reported that adding anti-stressors like NaHCO3 in to
drinking water (DL) of White Leghorn (WLH) and Matrouh layers increased their weight
gain under heat stressed condition.
In contrast to the findings of various studies mentioned above, Junqueira et al. (2003)
and Osman et al. (2015) found no effect (P>0.05) on growth rate of chicken due to dietary
supplementation of different levels of sodium bicarbonate. Similarly, findings of Hayat et al.
(1999) Wideman et al. (2003) and Saedi and KhajaliI (2010) did not find any effect (P>0.05)
of supplementation of NaHCO3 on body weight gain of birds. Whereas, Squires and Julian,
(2001) and Peng et al. (2013) reported a significant decrease in body weight gain in broilers
fed NaHCO3. Similarly, Wideman et al. (2003) observed 7% reduction in growth rate in
broilers fed diet containing 1% NaHCO3.
2.14.2 Feed consumption
Several nutritional manipulations have been recommended to minimize depression in
feed intake due to heat stress (Baghel and Pradhan, 1989; Fethiere et al., 1994; Anjum, 2000;
Khattak et al., 2012) in species of fowls. Different levels of electrolytes are reported to be
beneficial for broiler birds kept under heat stress conditions (Mushtaq et al., 2007).
Managing acid base balance by dietary supplementation of various electrolytes salts such as
sodium bicarbonate, KCl, ammonium chloride (NH4Cl) and calcium chloride is one of the
preeminent methods used to combat hot weather stress (Teeter et al., 1985; Borges, 1997;
Borges et al., 2003a, b; Ahmad et al., 2005).
25
Dietary addition of NaHCO3 in laying hens has shown a significant improvement in
their performance during heat stress (Ghorbani and Fayazi 2009). Teeter et al. (1986) and
Marinez et al. (1993) also found that dietary supplementation of sodium bicarbonate in
broilers reared under heat stress conditions has shown a significant improvement in their feed
consumption.
Bonsembiante and Chiericato (1990) offered feed to meat type turkeys without or
with 0.50% sodium bicarbonate and observed an average final weight of 8817 and 9046g
with an average daily gain of 63.4 and 65.9g, respectively. However, feed intake or feed
efficiency and levels of electrolytes among treatment groups were non-significantly affected
by the dietary treatments. Fethiere et al. (1994) executed an experiment to check efficacy of
dietary addition of sodium present in sodium zeoilte-A in broilers. Corn soybean meal basal
rations (iso-caloric and iso-nitrogenous) were formulated containing sodium from either
sodium zeolite-A (SZA) or sodium chloride. The addition of sodium in the diet resulted in an
improvement in feed consumption.
Junqueira et al. (1984) examined the effect of NaCl, sodium bicarbonate, calcium and
phosphorus supplemented in diet on performance of Hy-line layers reared in cages
maintained in open house. The layers when fed diet containing sodium bicarbonate at the
level of1 or 6 % showed significantly better performance as compared to those fed sodium
chloride free diets. However, performance of the hens remained unaffected due to the
addition of sodium chloride at the rate of 0.37 and 1.11 % in their diets.
Branton et al . (1986) conducted an experiment in which broilers were exposed to
acute heat stress and were provided water containing sodium bicarbonate and ammonium
chloride. The results indicated 20% increase in water intake by the birds using water
containing sodium chloride (6.25 g/L). However, both water and feed intakes were limited
when consumed sodium chloride at the rate of 31g/L of water. Cooke and Raine (1986) used
bicarbonate as sodium source rather than chloride in the diets of broilers. The results showed
that diets containing 0.13% and 0.19% sodium content decreased water intake (0.2L/bird)
and also exhibited improvement in the quality of litter by about 20%.
Darmon et al . (1986) investigated the effect of NaHCO3 and NaCl supplementation
in broilers on their water intake. They observed increased water consumption due to
increasing level of sodium chloride. Based upon the results it was concluded that sodium
26
from sodium bicarbonate might have been used in a way similar to that of from NaCl.
Balnave and Gorman (1993) studied the efficacy of supplementation of sodium bicarbonate
in broiler birds and observed increase in feed consumption and production performance
(growth rate) of the birds. The response was credited to the bicarbonate ions.
Effect of dietary NaHCO3 supplementation on feed consumption during late laying
period was studied by Yoruk et al. (2004). Hisex Brown layers were randomly divided in to
four groups to obtain one of the four diets (0, or 0.1, or 0.2, or 0.4% level of NaHCO 3) for a
period of 75 days. The results revealed that feed consumption was higher (P<0.05) in hens
fed diets supplemented with NaHCO3. It was also observed that there was a gradual raise in
feed intake of birds with raise in the levels of NaHCO3. Balnave and Gormen (1993) brought
it to fact that feed intake of chicken kept under heat stress can be increased by adding their
diet or water with NaHCO3. The significant beneficial effect might be due to the HCO3 ion or
raising the water intake. However, measuring dietary or retained values for dietary electrolyte
balance (DEB) were found to be inadequate for calculating the feed conversion (FCR) of
chickens.
Puron et al. (1997a) and McDowell (1992) have reported an increase in feed
consumption of birds due to more sodium ions concentration in rations containing sodium
bicarbonate. Ahmad et al. (2006) and Balnave and Gorman (1993) observed a significant
increase in feed consumption in broilers fed diet added with sodium bicarbonate during high
ambient temperature. Gongruttananun and Ratana et al. (2004) determined the response of
dietary addition of NaHCO3 on feed consumption of laying birds. Three experimental diets
were formulated as; control layer diet, diet with 1% inclusion of NaHCO3 and diet with 1.5%
inclusion of NaHCO3.The findings of the study did not reveal any significant effect on feed
consumption in the birds.
Mandal et al. (2010) investigated the effect of adding Vitamin C (ascorbic acid) and
sodium bicarbonate in layer diets, on egg production of the layers exposed to heat stress.
Daily feed consumption was lowered (P<0.02) in ascorbic acid supplemented group in
comparison to those of non-supplemented group (control). Khattak et al. (2012) have also
observed a reduction in feed intake in broilers fed sodium bicarbonate supplemented diets
during heat stress conditions.
However, in contrast to the results of various studies discussed above, findings of
27
research trial conducted by Whiting et al. (1991) did not (P>0.05) reveal any beneficial result
of dietary addition of NaHCO3 production performance of poultry birds reared under hot
weather conditions. Similarly, Puron et al. (1994) found that supplementation of NaHCO3 did
not affect the performance of chicken. They attributed these findings to the lack of response
of NaHCO3, which might be due to climatic conditions and environment of the experiment
which might produced only mild heat stress. Bonsembiante and Chiericato (1990) observed
no (P>0.05) distinction in feed consumption of birds using rations with or without
supplemented sodium bicarbonate. Similarly, Senkoylu et al. (2005) who tested the effects of
inclusion of various levels of NaCl, NaHCO3 and K2CO3 in poultry diets, on feed
consumption of layers during peak production did not observe any effect due to the dietary
inclusion of these compounds on feed intake of the layers. Findings of Balnave and
Muheereza (1997) and Waldroup et al. (2005) have also shown that feed intake of broilers
fed diets with or without sodium bicarbonate remained unaffected. Fuentes et al. (1997)
found no effect of adding different levels of sodium bicarbonate (0.6, 1.2, 1.8 and 2.4%) in
diets on feed consumption of guinea fowls reared at high ambient temperature.
2.14.3 Feed efficiency
Different levels of sodium bicarbonate have been proved beneficial for broilers reared
under different hot climatic conditions but with varying results. Differences in the levels of
sodium bicarbonate used, climatic conditions, species of birds and differences in precision
and accuracy in measurements among different experiments might be a cause of
disagreements among different studies reported. However, dietary supplementation of
sodium bicarbonate in broilers might improve their body weight gain and decrease losses
caused by heat stress (Mirsalimi and Julian, 1993).
It is generally agreed that beneficial effects of NaHCO3 can be only achieved when its
recommended/optimum levels are administered in the diet. Moreover, use of excessive levels
of this chemical compound in the diet has been reported as nephro-toxic and toxic in White
Leghorn layers (Davison and Wideman, 1992), which indicates that there is a dire need to
define the proper dietary level of sodium bicarbonate having proper dietary electrolyte
balance (DEB) for optimum performance of laying hens kept under environmental conditions
of Pakistan. In the following paragraphs a concise review regarding scientific findings has
been reported about the effects of different levels of NaHCO3 used in poultry birds.
28
Drinah et al . (1990) used sodium bicarbonate (0.25%, 0.5%, and 0.75%) to detoxify
the tannins present in the sorghum fed to starting broiler chicks. Findings of the study
revealed that a dietary addition of 0.25% sodium bicarbonate may overcome anti-nutrient
effects of tannins present in sorghums ultimately can cause better body weight and efficiency
of feed utilization. Findings of Keskin and Durgan (1997) have also reported an improved
FCR in quails fed diet supplemented with NaHCO3 (1%), KCl (1%), CaCl2 (1%), NH4Cl (1%)
and CaSO4 (1%).
Hooge et al. (1999) tested incorporation of sodium bicarbonate in the diet of broilers
at level of 0 to 0.4%. Dietary inclusion of NaHCO3 at level of 0.2 to 0.4% acquiesced
significant (P<0.05) increase in growth, feed efficiency/utilization and survivability of the
birds, whereas the level of 0.1% sodium bicarbonate did not exert such beneficial effects. A
range of 0.2 to 0.3% of sodium bicarbonate in the diet of broilers from day-old to market age,
was recommended.
Atlan et al. (2000) investigated the outcome of dietary addition of NaHCO3 on feed
consumption, feed efficiency and rectal temperatures in two strains of layers (brown and
white) reared during summer. Findings of the study indicated that dietary inclusion of sodium
bicarbonate at a level of 0.3% significantly improved feed conversion in both strains of the
layers. Rectal temperatures recorded during the hottest hours of the day were found to be
significantly higher in brown layers than in white layers. However, rectal temperature of the
birds remained unaffected due to NaHCO3 supplementation in their diets. Yoruk et al. (2004)
find out the dietetic effect of NaHCO3 on feed efficiency in layers during their late laying
period. Hisex Brown layers were indiscriminately allocated to obtain one of four treatments
diets having either, 0 or 0.1or 0.2, or 0.4% sodium bicarbonate for 75 days. The results of the
study depicted improved feed conversion ratios in the layers with increase in the level of
NaHCO3 in their diets.
Naseem et al. (2005) surveyed the outcomes of dietary supplementation of KCl and
NaHCO3 on body weight and feed conversion ratio (FCR) in the birds exposed to heat stress.
Hyperthermia in these birds led to a significant (P<0.05) reduce in their weight gain and poor
FCR. Balnave and Gormen (1993) reported that feed consumption and weight gain of
chicken reared under high temperature can be enhanced by adding their diet or drinking
water with NaHCO3. This might be due to an increase in HCO3 ions and may also be linked
29
with boost in water intake. Senkoylu et al. (2005) accounted no (P>0.05) effect of dietary
inclusion of varying levels of NaCl, NaHCO3 and K2CO3 on FCR (calculated on the basis of
gram of feed/grams of egg produced by layers) during peak production. Fuentes et al. (1998)
also observed a no effect (P>0.05) of sodium bicarbonate on FCR values calculated on the
basis of per kg egg mass produced in guinea fowls raised under high ambient temperatures.
2.14.4 Egg production
In order to improve egg production during summer, scientists have used diets
supplemented with varying levels of NaHCO3 and depicted different results. A percentage of
1.5% NaHCO3 was reported to be ineffective in laying hens (Grizzle et al., 1992), whereas
dietary addition of 0.3% to 2% increased the shell quality (Davison and Wideman, 1992) of
the eggs produced by the birds using these diets. Moreover, Davison and Wideman (1992)
noted that dietary supplementation of 3% NaHCO3 led to eggs without shells. In adding
together inclusion of macro minerals, micro minerals, salts and vitamin D, adjustment of
acid-base balance (DEB) by supplementation sodium bicarbonate (Grizzle et al., 1992;
Davison and Wideman, 1992) are existing emergent to get better layers performance.
Harms et al . (1995) conducted a study to access dietary sodium requirements of
Arbor Acres hens. In this study, NaCl was mixed to a corn-soybean diet to endow intakes of
either, 35, 65, 95, 125 or 150mg per hen daily. Egg production was markedly decreased
during 4th and 7th week of the experimental period, when the hens had an intake of 35mg
sodium/day and 65mg sodium/day, respectively. Daily requirements for maximum
production were found to be 113.8 and 96mg and the requisite for egg mass was 105mg and
100mg/day, during the respective production periods.
Dai et al. (2009) observed an improved egg production of the layers fed diet
containing sodium bicarbonate supplemented diet. Ghorbani and Fayazi (2009) studied the
consequence of dietary addition of NaHCO3 on egg production of layers kept under persistent
heat stress and found significant increase in egg production due to dietary inclusion of
sodium bicarbonate. Findings of Hassan et al. (2011) also discovered that addition of sodium
bicarbonate at a level of 0.25 and 0.50% in poultry diet may improve egg production of
layers exposed to heat stress. An increase in egg production has also been observed by
Balnave and Muheereza (1997) because of dietary addition of NaHCO3 (1%). Similarly,
Yoruk et al. (2004) scrutinized the effect of different levels of NaHCO3 (0.1%-0.4%) on
30
production performance of layers. Inclusion of different levels of sodium bicarbonate (0.1%-
0.4%) in laying hens diet improved their egg production significantly.
Makled and Charles (1987) compared the effects of adding calcium source, sodium
bicarbonate in the diets and photoperiod on 240 Hy-line Leghorn hens of 25 weeks age.
Photoperiod and dietary addition of these substances showed a noticeable effect on
production performance of the layers. Gongruttananun and Ratana (2005) also found non-
significant effect of adding varying level of sodium bicarbonate on production performance
in laying birds. They fed diets added with 1.0-1.5% sodium bicarbonate to Thai native hens
and observed that differences in egg production between treated and non-treated birds were
non-significant, even due to the different levels of sodium bicarbonate used. Similar results
regarding egg production are also observed by Grizzle et al. (1992); Gongruttananun et al.
(1999) and Waldroup et al. (2005). Egg production has also been found to remain unaffected
due to the inclusion of different levels of NaCl, NaHCO3 and K2CO3 in the diets of layers
(Senkoylu et al., 2005), during their peak production period. Mandal et al. (2010)
investigated the effect of adding ascorbic acid and sodium bicarbonate in layer diets on egg
production under heat stress. Two additives i.e. ascorbic acid (300mg/kg) and sodium
bicarbonate (1%) in diets were used but egg production was not affected by the dietary
treatments.
2.14.5 Egg weight/size
In order to improve egg weights during summer various scientists have used diets
supplemented with varying levels of sodium bicarbonate and have observed varying results.
Makled and Charles (1987) reported significantly (P<0.05) better weight of eggs in layers,
when fed diet containing 0.5% NaHCO3. Balnave and Muheereza (1997) fed either basal
diet or treated diet containing either 1% sodium bicarbonate or treated diet containing 0.05%
Zinc methionine or treated diet containing 0.04% vitamin C, to layers kept under high
ambient temperature and found significantly higher weight of eggs in the birds fed diet
supplemented with 1% sodium bicarbonate. Similar effects of sodium bicarbonate on egg
weight have also been reported by Ghorbani and Fayazi (2009) in layers. They studied the
significance of feeding various level of sodium bicarbonate (0.5%-1.5%) and rearing systems
on egg weight of layers kept under chronic heat stress and found that dietary levels of sodium
bicarbonate (0.5%-1.5%) in laying hens diet improved their egg weight whereas hens fed diet
31
supplemented with 1.5% sodium bicarbonate produced heavier eggs than its counterparts.
Yoruk et al. (2004) conduct a trial to find out the effects of dietary NaHCO3
supplementation on egg weight during late laying period of hens. Hisex Brown layers were
randomly distributed to get one of four diets having 0, or 0.1, or 0.2 or 0.4% NaHCO 3,for 75
days. Egg weight was more for hens fed diets having NaHCO3. Raising the level of NaHCO3
caused a linear raise in egg weight of the hens. Yin et al. (2001) observed that adding 0.3%
sodium bicarbonate as water supplement caused better egg laying rate and egg mass in laying
hens.
In contrast to the findings discussed above, no effect due to dietary addition of
NaHCO3 has been observed on egg weight of the poultry birds. Ernst et al. (1975) fed diets to
commercial Leghorn layers, with or without addition of sodium bicarbonate to improve their
egg weight. They adjusted Na+ level to 0.23% and Cl- level to 0.18% of the diets. However,
they did not discover any disparity in egg weight of layers due to these treatments. Senkoylu
et al. (2005) tested the effect of inclusion of diverse levels of NaCl, NaHCO3 and K2CO3 in
diet, on egg weight in layers during their peak production period and found no effect due to
dietary inclusion of these compounds on egg weight of the layers. Comparable results have
also been observed by Gongruttananun and Ratana (2005) who found no effect (P>0.05) on
egg weight of Thai native hens due to dietary addition of varying levels of NaHCO3 (1.0-
1.5%). Stevenson (2006) also observed that sodium bicarbonate containing diets did not exert
any effect on egg weight in layers. Similarly, egg weight of layers using diet containing 1%
sodium bicarbonate remained unaffected (Waldroup et al., 2005).
Higher egg mass production of the eggs produced by the birds using sodium
bicarbonate treated rations was reported by Dai et al. (2009). Yoruk et al. (2004) found
beneficial result of dietary addition of NaHCO3 on egg mass of turkeys. Balnave and
Muheereza (1997) fed either basal diet or diets containing 1% sodium bicarbonate, 0.05%
Zinc methionine or 0.04% Vitamin C to layers kept under high ambient temperature. Results
showed a significant improvement in egg mass produced by the bird fed diet supplemented
with 1% sodium bicarbonate. However, in contrast to the findings of various studies
mentioned above, Senkoylu et al. (2005) reported no effect (P>0.05) on egg mass production
due to inclusion of varying levels of NaHCO3 in the diet of layers during their peak
production period.
32
2.14.6 Egg shell thickness
Poor egg shell quality is the main trouble in birds during the summer/high ambient
temperature. Environmental temperature, seasonal changes, bicarbonates, calcium, ascorbic
acid, light intensity and duration, nutrition, genetics and age of hens are possibly the
important factors, which are related to egg shell quality of the birds. Wideman and Buss
(1985) studied the bicarbonate, CO2 and pH values in layers to observe egg shell quality.
Results of the study showed that the hens producing thin egg shell suffered with metabolic
acidosis during the first six hours of oviposition also had markedly lower blood pH and
bicarbonate contents than those producing eggs with thick shell. Ergun (1992) reported a
decline in blood pH and bicarbonate level at 22nd hour of laying cycle.
Austle and Keshavarz (1988) conducted two experiments to determine the results of
supplementation of Na and Cl on egg shell quality of Leghorn layers. In the first experiment,
they observed that presence of chloride in the diets reduced feed intake, egg shell thickness
and strength in the hens receiving 2 % calcium in their diet. In the second experiment,
influence of the dietary addition of sodium and Cl on egg shells of the hens was determined.
Increase in the concentration of sodium contents relative to those of chlorides, reduced feed
intake of the birds but significantly improved egg shell strength and thickness, bicarbonate
concentration, blood pH and base excess.
Balnave and Muheereza (1997) executed two trials to explore the effects of sodium
bicarbonate during heat stress. Layer birds were reared at higher house temperatures (30 and
35 °C) and fed sodium bicarbonate supplemented diet, at the end of lay. Shell thickness was
improved due to dietary addition of sodium bicarbonate. A significant improvement in egg
shell thickness in layers fed NaHCO3 added diet was observed by Hayat et al. (1999). Similar
beneficial effect of including NaHCO3 in poultry diets have also been reported on egg shell
thickness by Davison and Wideman (1992). An improvement in eggshell thickness was also
observed in hens fed diets added with 0.5% NaHCO3 (Makled and Charles, 1987).
Different levels of dietary inclusion of NaHCO3 have shown considerable effects on
egg shell quality of layers. Atlan et al. (2000) investigated the effect of dietary
supplementation of NaHCO3 on egg quality parameters during summer, in two strains of
layers (brown and white). Results of the trial showed that addition of NaHCO3 at the level of
0.3% significantly improved egg shell quality in both strains. Gongruttananun and Ratana et
33
al. (2004) observed the effect of different levels of dietary NaHCO3 (1 to 1.5%)
supplementation on shell thickness of eggs produced by Thai native hens. Three
experimental diets were formulated as; control layer diet, diet with 1% inclusion of NaHCO3
and diet with 1.5% inclusion of NaHCO3.Layers using diets added with NaHCO3 produced
eggs with higher shell thickness (P<0.05) than those of control group. It was concluded that
at moderate temperatures, eggshell quality of the hens could be improved by dietary
supplementation with 1.5% NaHCO3.
Kaya et al. (2004) tested the effect of dietary inclusion of NaHCO3 on blood gases,
blood pH and egg shell thickness in geese birds. Fourteen geese of two year age were
distributed into 2 groups as; 1) control, 2) 0.5% NaHCO3 supplemented group. Unlike the
results of various studies discussed above, in this dietary addition of NaHCO3 revealed no
significant improvement in egg shell thickness of the eggs produced by the birds. Yoruk et
al. (2004) have also observed non-significant effect on egg shell thickness of laying hens due
to dietary addition of different levels of NaHCO3 (0.1%, o.2%, 0.4%).
A significant decrease in shell thickness of eggs laid by the laying hens exposed to
heat stress and fed diets containing different levels of NaHCO3 (0, 0.5, 1 and 1.5%) was
observed by Ghorbani and Fayazi (2009). Findings of Mandal et al. (2010) who investigated
the effect of adding ascorbic acid and sodium bicarbonate in layer diets, on shell thickness of
eggs produced by laying birds kept under heat stress, have revealed that egg shell thickness
was higher in layers maintained at low energy level or ascorbic acid group. EL-Sheikh and
Salama (2010) investigated that adding 100 mg sodium bicarbonate in drinking water did not
significantly affect egg shell weight of layers but albumen weight and yolk weight were
significantly affected as compared to the control group during summer season. Whereas, Dai
and Bessei (2007) found that egg shell thickness was higher (P>0.05) and eggs having shell
deformities were lower in the birds treated with potassium chloride supplementation through
drinking water.
2.14.7 Specific gravity of intact egg
The effect of different dietary levels of sodium bicarbonate (0.1%-0.4%) on specific
gravity of eggs in layers was studied by Yoruk et al. (2004). The results revealed that
inclusion of different levels of sodium bicarbonate in laying hens diet markedly increased
specific gravity of the eggs produced by the hens. It was also reported that higher levels of
34
sodium bicarbonate (0.2%-0.4%) in the diet reduced the specific gravity of eggs laid by the
birds. However, Grizzle et al. (1992) research results do not support the opinion of such
effect on specific gravity of eggs due to dietary supplementation of 1% sodium bicarbonate
in layers. Similarly, Makled and Charles (1987) found no change in specific gravity of eggs
laid by hens fed diet supplemented with 0.5% sodium bicarbonate during their peak
production period. Connor and Arnold(2004) fed pullets (Australorp X White Leghorn) with
diets containing sodium bicarbonate and did not observe any effect due to dietary addition of
sodium bicarbonate on specific gravity of the eggs.
2.14.8 Various egg quality parameters
There are many factors, which may influence egg quality characteristics in birds but
heat stress is the most important factor which has shown pronounced effect on most of the
important egg quality parameters (Anjum, 2000). Although, many scientists have used
sodium bicarbonate in the diet of layers in order to improve egg quality characteristics during
hot weather, yet there is some difference of opinion among the investigators. In the following
paragraphs, scientific information concerning the effects of adding sodium bicarbonate in the
diets of layers, on variables like, Haugh unit values, albumen index and yolk index are
reviewed.
Change in Haugh unit score is affected by many factors and heat stress is one of them
(Anjum, 2000). As the laying birds turns old, Haugh unit score of their eggs decrease in
value (Coutts et al., 1990). In such conditions inclusion of sodium bicarbonate in diet may
prove beneficial. An improved absorption of mono-saccharides and amino acid (Johnson and
Karunajeewa, 1985; Ravindran et al., 2008) due to sodium bicarbonate may cause an
increase in protein content of eggs hence Haugh unit and albumen quality of eggs produced
by these birds.
Hussan et al. (2011) observed that sodium bicarbonate when added in layer diets at
the rate of 0.25%, 0.50% and KCl at 0.2%, 0.3%, alone or in amalgamation exhibited
beneficial effect on egg quality characteristics as compared to those provided diets without
supplementation. Additives (NaHCO3 and KCl) and their arrangement/combination
improved (P>0.05) the various traits of egg characteristics in Golden Montazah hens reared
in hot weather conditions. Yoruk et al. (2004) studied the effect of different levels of sodium
bicarbonate (0.1%-0.4%) on Haugh unit score of layers during late laying period. They found
35
an increase in albumen height of eggs produced by the birds fed sodium bicarbonate added diets.
In contrast to the findings of various studies mentioned above, Haugh unit score of
the eggs produced by the layers kept under chronic heat stress was found to be not affected
due to dietary inclusion of sodium bicarbonate and rearing system. Moreover, neither
addition of sodium bicarbonate nor its various levels (0.5%-1.5%) exhibited any effect on
Haugh unit score in layers (Ghorbani and Fayazi, 2009). Findings of Gongruttananum and
Ratana (2005) have also revealed that different dietary levels of sodium bicarbonate (1%-
1.5%) did not improve Haugh unit score of the egg laid by hens.
Information regarding the effect of sodium bicarbonate on yolk index of eggs is
scanty. Yolk index of eggs was found to be significantly improved by dietary inclusion of
sodium bicarbonate at 0.1% level (Yoruk et al., 2004) when different levels of this chemical
compound (0.1%-0.4%) were used in the ration of layers during late laying period. In
contrast to the findings of the previous study, Ghorbani and Fayazi (2009) observed that
neither dietary inclusion of sodium bicarbonate nor its levels (0.5%-1.5%) exhibited any
effect on yolk index of the eggs produced by the hens. Similarly, Gongruttananum and
Ratana (2005) have also reported that dietary levels of sodium bicarbonate (1%-1.5%) in
laying hens did not improve yolk quality of the eggs produced by the birds.
Presence of sodium is necessary for absorption/uptake of amino acids and
carbohydrates from gastrointestinal tract (Leeson and Summers, 2001a), thus deficiency of
this essential element may render the digested protein and carbohydrates unavailable to the
body. Therefore, addition of this beneficial chemical compound in the ration of poultry birds
may tend to affect Haugh unit, albumen index, albumen quality and other egg quality
parameters.
Ghorbani and Fayazi (2009) and Gongruttananum and Ratana (2005) studied the
effects of dietary addition of NaHCO3 on egg quality characteristics in layers kept under
chronic heat stress. They reported that dietary levels of sodium bicarbonate (0.5%-1.5%) in
the diets of laying hens did not show any improvement in the albumen quality of the eggs
produced by these birds. Similarly dietary addition of ascorbic acid and sodium bicarbonate
in layer diets did not exhibit any effect on albumen index of eggs produced by laying birds
kept under heat stress (Mandal et al., 2010).
Results of the various studies discussed in the paragraphs above, are in accord with
36
observations of EL-Sheikh and Salama (2010) who investigated that adding 100mg NaHCO3
or 75mg KCl/L in drinking water did not affect (P>0.05) albumin weight, yolk weight, yolk
index and Haugh units score, but on the other hand, albumin weight and yolk weight were
affected (P<0.05) by NaHCO3 or KCl supplementation.
The manner, in which different factors influence egg quality parameters have not
been fully determined yet. Although, many research workers have studied the effects of these
factors on some parameters of eggs quality, but still there is dearth of information regarding
the effect of sodium bicarbonate on important egg quality variables i.e., Haugh unit values,
albumen index and yolk index. Hence the expected changes in these factors under various
hyperthermic conditions still need to be addressed. Therefore, this project was planned to
check the effect of dietary inclusion of sodium bicarbonate on egg quality characteristics of
caged layers during summer.
2.14.9Body/rectal temperature and respiration rate It has been observed that birds experiencing heat stress have higher body temperature
than those reared under a comfortable zone of temperature and hence spend more time
panting (Mack et al., 2013). Birds utilize multiple ways to maintain body temperature and
homeostasis when subjected to heat stress, which include increased radiation, convection and
evaporation along with the heat loss by vasodilatation and perspiration (Mustaf et al., 2009).
Increase in respiration rate is correlated with high environment temperature along
with increase in moisture contents of the air. When ambient temperature is increased
chemical reactions speed up in the body, heat is generated and body temperature of birds
rises (North and Bell, 1990). Birds have no sweat glands; hence they dissipate their body heat
mainly through respiration (Nillipour and Melog, 1999). When environmental temperature
exceeds the thermo-neutral zone, respiration rate increases up to 10 times, from a normal rate
of 25 breaths/minute (Remus, 2001), which results in respiratory alkalosis. In such
conditions sodium bicarbonate can be used as a buffering agent to ameliorate the problem
(Whiting et al., 1991a). Angiletta et al. (2010) observed increased respiration rate in birds
exposed to hyper-thermal environment.
Ahmad et al. (2005) investigated the influence of addition of baking soda in the feed
on rectal temperature in broilers experiencing heat stress. They noted a reduction (P<0.05) in
body temperature of birds fed diets having sodium bicarbonate. The birds which were
37
supplied sodium bicarbonate containing diets also exhibited lower respiratory rate and thus
produced less heat for this physiological norm. However, Mushtaq et al. (2007) did not find
any correlation between rectal temperature of poultry birds and dietary sodium levels.
Similarly, findings of Junqueira et al. (2000) showed that layer birds exposed to heat stress at
33 °C receiving diets having varying levels of sodium bicarbonate (0.67 to 2.56%), were not
differed in their body temperature. Similar results are also observed by Dai and Bessei
(2007), that body temperature of birds was not affected due to dietary addition of KCl (0.2
and 0.4 %). In addition, the same trend i.e. non-significant effect due to the inclusion of
sodium bicarbonate (0.65 %) in drinking water of layers exposed to heat stress was observed
by Genedi (2000).
2.14.10Mortality Heat stress not merely reduces productive potential, but it also causes high mortality
leading to a reasonably harmful effect on economics of production in poultry birds. Mortality
of birds increases with increase in ambient temperature than that of thermo-neutral zone
(Anjum, 2000). Seley (1973) suggested that if an animal is unable to adjust it into a new set
of environmental conditions, it exhausts and ultimately dies. It was further reported that
response of the animals to higher temperature was different for a different set of
environment. High environmental temperatures have shown to increase mortality rate in
fowls as has been observed by Zakia et al . (1995) and Mandal et al. (2010)
There exists a significant difference in livability of heat resistant and vulnerable lines
of poultry birds. Mortality rate in heat resistant and susceptible lines of WLH, after twenty
hours of heat stress at 40.6 °C were found to be 44.4% in total (Bohren et al . , 1982).
However, the birds of heat resistant bird’s lines showed less (P<0.05) mortality than the birds
of heat susceptible lines. It was further reported that the birds kept in cold climatic conditions
exhibited higher (P<0.05) mortality under subsequent heat stress conditions.
Smith (1992) observed the effects of feed withdrawal and acclimation on livability in
heat stressed broilers. Chicks were exposed to ambient temperature of 38 °C for twenty four
hours from 1st to 9 days of age. In experiment I, chickens were kept under cycling
temperature of 24 °C to 35 °C for the next 44 days. In experiment 2, chicken were reared at
24 °C for 23 days followed by feed withdrawal for 0 to 24 hours and a rapid temperature
increase to 37 °C for the final 4 hours of feed withdrawal. They found that chickens on full
38
feed showed lower livability than those maintained on feed withdrawal program. Zakia et al .
(1995) studied the effect of feeding time and light on mortality in heat stressed birds and
observed that the birds subjected to heat stress with access to food and light showed higher
mortality (10%).
Dietary addition of anti-stress compounds, like sodium bicarbonate have proved to be
beneficial in controlling mortality of birds during heat stress (Khattak et al., 2012). Mushtaq
et al. (2005) observed no mortality in broilers fed diet supplemented with sodium bicarbonate
(0.025% Na+) under subtropical summer conditions. Similarly Owen et al. (1994) reported
that sodium bicarbonate when included into the poultry diet at the level of 1% reduce the
ascites related mortality because of its alkaline nature. Whereas, inclusion of ammonium
chloride at 1% level resulted in an increase (P<0.05) in ascites related disorders, which was
assumed to be due to its acidic nature (inducing acidosis). These results support the findings
of Genedi (2000) who reported that supplementing anti-stressors (NaHCO3, KCl and
NaHCO3+ KCl) decrease (P<0.05) the mortality level in layers kept under heat stress.
In a study, Merkley and Miller (1983) used non-conventional sodium salt sources
(sodium fluoride and sodium silicate) in broiler ration and found that efficiency of feed
utilization and mortality rate is not influenced by Na+ when provided in the form of these
chemical compounds. Lack of response of sodium supplied by non-conventional source can
be attributed to the absence of bicarbonate ions, which are necessary to combat heat stress.
Mandal et al. (2010) investigated the effect of adding ascorbic acid and sodium bicarbonate
in layer diets on mortality in layers kept under heat stress. Results revealed maximum
mortality in the birds fed sodium bicarbonate. Increase in mortality due to sodium
bicarbonate supplementation was attributed to higher levels of this compound. NaHCO3 at
1.45g/L has shown to increase ascites related mortality (Julian et al., 1986) whilst, a level of
7.50g/L resulted as toxic (Mirsalimi et al., 1993).
Puron et al. (1997a) reported slightly higher mortality in birds given NaHCO3 added
diets. They stated that such type of results might be due to higher stocking density rate,
which may probably be the cause of slightly higher mortality in treated group. Shane (1994)
observed that both humidity and temperature determine the level of heat stress in birds. He
pointed out that at temperature above 36 °C only production is adversely affected; whilst,
mortality losses occurs only when ambient temperature exceeds 47 °C.
39
2.14.11Hematological profile
It has been well documented that high environmental temperature depresses
hemoglobin level in birds (Anjum, 2000) and increase in environmental temperature and
blood hemoglobin concentration in birds, have been found to be negatively correlated.
Moreover, factors like age, sex, season, hormones and hypoxia may also influence quantity
of hemoglobin in blood (Sturkie, 1976a). Hemoglobin concentration was found to be
decreased in the male broiler chicks, as ambient temperature rose from 10 to 30 °C or 15 to
35 °C in two different trials conducted by Yahavet al . (1997). It was concluded that
hemodynamic variations are depicted only in the chicken acclimated to constant temperature
or to fast temperature changes e.g. those taking place during diurnal cycle but not happening
in birds reared under acute high or lower temperature. This might be responsible for the
inability of birds to regulate their body temperature.
Decrease in blood hemoglobin concentration in layers reared under high ambient
temperature is also reported by Vecerek et al. (2002). However, Ahmad et al. (2005)
observed increase in hemoglobin concentration in birds due to inclusion of sodium
bicarbonate in their diet. Similarly, findings of Genedi (2000) have also shown that addition
of anti-stressors like NaHCO3 in drinking water of Leghorn and Matrouh layers markedly
increased their hemoglobin concentration, even under heat stress conditions.
Elevation of blood glucose in experimental birds under heat stress is usually
attributed to hydrolysis of glucagon as a result of increased body temperature. Al-Hassani et
al. (2001) have reported a significant decrease in plasma glucose level in Hisex brown layers
subjected to heat stress, when fed diets containing sodium bicarbonate as compared to those
fed diets without any supplementation (control).
Ahmad et al. (2005) examined a decrease (P<0.05) in glucose level in broilers fed
diet containing sodium bicarbonate as compared to those fed diets without its addition
(controls). However, Koelkebeck and Odom (1995) did not observe any effect on glucose
concentration of the layers, due to high ambient temperature. Similarly, Zakia et al. (2009)
reported no effect of dietary addition of sodium bicarbonate on glucose level in chickens.
2.14.12 Serum metabolites and serum proteins
Heat stress has shown to decrease plasma protein concentration in poultry birds with
increase in the environmental temperature (Anjum, 2000; Geraert et al . , 1996; Yang et al . ,
40
1992). However, dietary inclusion of sodium bicarbonate has shown its potential beneficial
effect in improving serum protein concentration in laying birds during high ambient
temperature. Kurtoglu et al. (2007) reported an increase (P<0.05) in serum total protein due
to dietary inclusion of sodium bicarbonate in Brown-Nick layers. These results are in
agreement with Badran (2003) who resulted out that NaHCO3 supplementation at levels of 2,
3 and 4% increased (P<0.05) the blood protein concentration during heat stress. However,
Genedi (2000) reported that addition of NaHCO3 or KCl into drinking water of heat stressed
Leghorn and Matrouh layers did not cause any effects on the total protein and globulin
concentration in the birds. Heat stress has shown to increase serum uric acid level in poultry
(Yang et al . , 1992) and no effect on serum alkaline phosphatase (Bogin et al., 1981;
Koelkebeck and Odom, 1995). However, scientific information regarding the effect of
dietary inclusion of NaHCO3 on serum alkaline phosphatase and uric acid in the birds
exposed to heat stress is scanty.
Based upon scientific information available, it can be envisaged that high
environmental temperature can negatively influence serum protein concentration in poultry
birds. However, dietary addition of sodium bicarbonate during hot weather may improve
bird’s serum protein concentration. Therefore, it was required to initiate a project to check
response of dietary addition of sodium bicarbonate on serum protein concentration in layers
kept under hot weather conditions.
2.14.13Plasma electrolytes and minerals
Inter-relationship among NaCl, NaHCO3, calcium (Ca) and phosphorus (P) in the
diets of laying hens was studied by Junqueira et al. (1984) and for this purpose; 3
experiments were executed. For this Hy-line layers were kept in individual wire cages
installed in open sheds. The birds were given a diet based on yellow maize and soybean meal
with or without additional sodium chloride, calcium phosphate or sodium bicarbonate. The
results revealed a significant increase in blood pH, base excess and bicarbonate concentration
due to the dietary addition of sodium bicarbonate.
Incidence of respiratory alkalosis in broilers during heat stress period has also been
reported by Teeter et al . (1985). They observed a higher blood pH value of 7.39 in heat
stressed (32 °C) panting birds than those of non-panting (pH 7.28) birds, reared at 24 °C.
Induced heat stress from 32 °C to 41°C for 20 minutes, further elevated blood pH to 7.52 in
41
the same birds. Moreover, chronic hyperthermia caused an intermittent respiratory alkalosis
during panting, whereas an acute hyperthermia induced continuous panting and alkalosis in birds.
To investigate the effect of tap water, carbonated water, NaHCO3 and CaCl2 on blood
acid bases balance, an experiment was conducted on twenty Hubbard broilers (Bottje and
Harrison, 1985). A solution of 2 % Sodium chloride or 3.5 % Calcium chloride having pH
8.0 and pH 7.4, correspondingly, was introduced into the crop of birds and blood pH was
noted. Infusion of Sodium bicarbonate increased blood pH whereas, Calcium chloride
infusion reduced (P<0.05) the blood pH. However, high levels of sodium bicarbonate and
calcium chloride may cause a change in the DEB (dietary electrolyte balance). It is also well
documented that inclusion of NaHCO3 in diet may increase blood pH of poultry birds
(Squires and Julian, 2001; Glahn et al., 1988).
Branton et al. (1986) conducted an experiment in which they used NaHCO3 and
NH4Cl in broilers feed exposed to acute heat stress. The results revealed that sodium
bicarbonate did not exert any significant effect on blood pH of broilers. However, in a similar
study, Brenes et al . (1988) detected that sodium bicarbonate supplementation showed a
noteworthy (P<0.05) effect on distribution of calcium in bones of broilers. Turkeys suffering
from gout were observed for their biochemical profile when fed diets supplemented with
sodium bicarbonate (Mert, 1991). Concentration of bicarbonate was increased in the birds
and found to be 27.37and 35.73mEq/L in healthy and sick chickens, respectively.
Infusion of sodium chloride at10g/L into the crop of broilers, exposed to heat stress
for 90 minutes, resulted in metabolic acidosis due to reduction in blood bicarbonate
concentration. Its concentration was also found to be decreased in birds fed sodium chloride
treated diets (Bottje et al . ,1989). Harms (1991) conducted two experiments with Hyline
hens using corn soybean meal basal diet. Four diets; 1) control; 2) diet containing no sodium
chloride; 3) diet containing sodium chloride but sodium supplied as sodium bicarbonate; and
4) diet containing no added sodium and chloride supplied as calcium chloride, were
formulated and fed to the layers for 19 days period. Based upon the results, it
wasrecommended that provision of sodium from sodium bicarbonate may be used as
substitute of sodium chloride to avoid the presence of chloride in layer diets. Moreover, Kaya
et al. (2004) reported that the NaHCO3 supplementation in the diet proved helpful in
maintaining the venous blood pH, DEB, HCO3– and pCO2 levels at the physiological ranges.
42
Deyhim and Teeter (1991) conducted an experiment to assess the effect of iso-molar
KCl (0.5 %) and NaCl (0.39%) for drinking water supplementation on blood pH, HCO3- and
water intake of birds raised in heat stressed and thermo-neutral environments. At 35 °C,
supplementation of sodium chloride in drinking water of the birds decreased HCO3- contents
in their blood. Austle and Keshavarz (1988) launched 2 experiments to find out the effect of
addition of sodium and chloride contents in the diet, on bicarbonate ions concentration in
Leghorn layers. Blood bicarbonates and base excess were increased due to the presence of
chloride contents in the diet.
A decrease in blood sodium level in broilers kept under heat stress has been observed
by Borges et al. (2004) and Takahashi and Akiba (2002).Whereas, Ahmad et al. (2006)
observed an increase in plasma Na+ concentration in birds fed diets supplemented with
sodium bicarbonate. Similarly, Mushtaq et al. (2005) reported an increased serum sodium
concentration due to the addition of different dietary sodium levels. Bonsembiante and
Chiericato (1990), however, did not observe any effect of dietary inclusion of sodium
bicarbonate on sodium ion concentration in meat type turkeys.
Ruiz-Lopez et al. (1993) reported that elevated levels of chlorides in the diet may
lower the pH of the blood and blood HCO3- concentration. It may also lead to Tibial
dyschondroplasia (TD) and cartilage abnormalities (Nelson et al., 1981). Studies in hot
summer conditions have reported a raise in plasma sodium ions concentration along with
hem-dilution (Whithow et al., 1994; Ait-Boulahsen et al., 1989).
Gongruttananun and Ratana et al. (2004) determined the effect of supplementation of
NaHCO3 in the birds’s diet on plasma sodium and pH. Three experimental diets were
formulated as; control (diet without addition of NaHCO3), diet with 1% inclusion of NaHCO3
and diet with 1.5% inclusion of NaHCO3.Layers fed diet added NaHCO3 exhibited a higher
level of plasma sodium concentrations. An increase (P<0.05) in plasma pH was noted in
birds fed diet supplemented with 1.5%NaHCO3. Findings of Wideman and Buss(1985) have
revealed that the layers which produced thin egg shell had significantly lower blood pH and
bicarbonate contents than those producing eggs having thick shell.
Plasma K+ concentrations were found to be decreased in birds exposed to heat stress.
Increase in excretion of K+ appears to be predominated in chronic heat stress, whereas its
increase in uptake by the cells is manifested during acute heat stress (Berne and Levy, 1993).
43
Naseem et al. (2005) observed the effect of sodium bicarbonate on serum potassium and
serum bicarbonate levels in birds. Hyperthermia in these birds led to significant decrease in
serum potassium and serum bicarbonate level. However, dietary inclusion of NaHCO3 at
levels of 0.5% during heat stress has shown to increase serum potassium and bicarbonate
level in blood of the birds.
The effect of heat stress on blood potassium concentration has also been found to be
similar in different species of birds such as in broilers (Mushtaq et al., 2005), layers
(Ghorbani and Fayazi, 2009) and quails (Keskin and Durgan, 1997). Moreover, dietary
supplementation of different levels of sodium bicarbonate has shown to prevent any decrease
in blood potassium level in heat stressed birds (Ghorbani and Fayazi, 2009).
It can be concluded and is also well documented that the level of blood potassium is
reduced (P<0.05) in the heat stress birds (Borges et al., 2004; Takahashi and Akiba, 2002).
However, addition of sodium in the diet of birds may prevent the blood K+ concentration
when reared under hot weather conditions (Ahmad et al., 2006; Mushtaq et al., 2005).
However, Kurtoglu et al. (2007) have found decreased plasma potassium concentration in
layers fed NaHCO3 containing diets as compared to those fed diets containing either NaCl or
KCl.
2.14.14 Serum lipids, hormones and enzymatic profile
Heat stress shows negative impact on production performance also different
biochemical processes including lipids, hormone and enzymatic status of birds
(Anjum, 2000). Important factors which affect cholesterol level in the body are: sex, age,
ration, hyperthermia and starvation. High serum cholesterol concentration in birds kept at
high temperature has also been reported by Haazele et al . (1991); Takahashi et al . (1991);
Sahota et al . (1993) and Sahota and Gilani, (1994).
Response of birds to dietary inclusion of sodium bicarbonate with respect to growth
hormones has not been studied much. However, Hussan et al. (2011) have observed a
noteworthy (P<0.05) decrease in blood (plasma) T3 and T4 hormones concentration in laying
birds supplementing diet with sodium bicarbonate. Similarly Attlla et al. (2002) found that
supplementation of NaHCO3 and KCl in drinking water, decreased concentration of T3
hormone in layer birds kept at higher ambient temperature (34 °C) for the period of fours
constant hours daily followed by a normal temperature (22° to 24 °C) throughout three
44
months experimental period. Genedi (2000) also reported pronounced effect of NaHCO3 on
plasma triiodothyronine hormone (T3) in Matrouh hens reared in heat stress conditions,
however, the effect was non-significant in Leghorn hens. Findings of Badran, (2003) have
also shown that the level of plasma T3 hormone in egg laying birds due to the addition of
different level of sodium bicarbonate (2, 3 and 4%) remained unaffected.
The investigations presented above have reported some changes in lipids, hormones
and enzymatic profile of poultry birds under the influence of various temperatures. However,
there is a dearth of research regarding the effect of dietary inclusion of sodium bicarbonate
on serum lipids, hormones and enzymatic profile of poultry birds exposed to heat stress.
Therefore, it can be envisaged that there is a dire need to explore the behavior of layers
regarding the variation of these parameters as a result of dietary inclusion of sodium
bicarbonate.
2.14.15 Immune response
Heat stress is known to increase lymphocyte and may severely affect the immunity in
poultry birds (Anjum, 2000; El-Gendy et al., 1995; Savic et al. 1993; Thaxton and Siegel,
1972). Dietary inclusion of sodium bicarbonate, however, has been shown to reduce effects
of heat stress (Ahmad, 1997; Ahmad et al., 2007). Deficiency of sodium has been reported to
suppress immune response and reduction in peripheral gustatory function (Guagliardo et al.,
2009). Use of NaHCO3 may be attributed to the partial correction in acid-base balance, which
may play a vital role in increasing immune response against some diseases. An increase in
dietary electrolyte balance has shown to cause a decrease in heterophil to lymphocyte ratio in
blood, leading to increase in antibody titer (Borges et al., 2003).
Santin et al. (2003) reported an increase in immune response against Newcastle
disease (ND) with increase in dietary electrolyte balance (40, 140, 240, 340mEq/kg) in birds.
Use of sodium bicarbonate in the diet of birds during hot weather significantly increased
antibody titters against Newcastle disease and Avian Influenza in poultry birds (Hussan et
al., 2011). These results are compatible to the results of the research conducted by Genedi
(2000) who found that use of anti-stressors (NaHCO3, KCl and NaHCO3 + KCl) may
improve immune response in Leghorn and Matrouh layers.
Hoshi et al . (1995) studied the effect of oral administration of formalin inactivated
virus suspended in buffered containing sodium bicarbonate in chicken. It was observed that
45
this form of antigen when administered orally, stimulate a serum defense response against
Gumboro disease virus (IBDV) in birds.
Fletcher et al . (1993) used a rinse process using NaHCO3 on recovery of pathogens
(bacteria) from the carcass of chicken. They implemented a three step rinsing procedure with
2% sodium bicarbonate and reported that the process was effective in reducing pathogen
recovery after seven days of the treatment. Findings of Khatak et al. (2012) have revealed
higher haemaglutination inhibition titer against NDV in birds consuming diets containing
sodium bicarbonate.
2.14.16 Digestibility of nutrients
High ambient temperature may exert a significant influence on digestion and
absorption of nutrients and their metabolism (Macleod, 2004; Puvadolpirod and Thaxton,
2000). Heat stress decreases blood flow towards digestive tract (Wolfenson, 1986).
Consequently it may reduce proteolytic activities of the respective enzymes in upper part of
digestive system (Haiet al.,2000) and hence may lead toa decrease in digestibility of protein.
Factors, which may influence digestibility of nutrients include; ambient temperature
(Macleod, 2 004; Hai et al., 2000 ; Puvadolpirod and Thaxton, 2000), level of feed intake and
passage rate of digesta (Ravindran et al., 2008; Ahmad et al., 2007), anti-nutritional factors
present in feed ingredients (Hughes and Choct, 1999), age and physiological status of the
bird (Batal and Parsons, 2002; Huang et al., 2007; Garcia et al., 2007) and composition of
the diet (Leeson and Summer, 2001a).To improve digestibility of feed ingredients different
nutritional manipulations have been used such as adding enzymes in feed (Bryden et
al.,2009; Selle et al., 2010), heat treatment and processing of feed ingredients and (Friedman,
1996; Amerah et al., 2007) addition of electrolytes in feed/water during hot weather
(Ravindran et al., 2008).
NaHCO3 in the diet of poultry birds exposed to heat stress may improve nutrient
digestibility by increasing sodium ions concentration (Fethiere et al., 1994); improving
electrolyte balance in the diet (Borges et al., 2003) and decreasing the production losses
caused by heat stress (Gorman and Balnave, 1994). Supplementation of either sodium or
potassium in drinking water may also increase water intake, which is associated with a
reduction in body temperature (Smith and Teeter, 1989) and an increase in nutrients
utilization. Gous (2004) suggested that producers should encourage the birds to drink more
46
water by adding mineral salts in to their drinking water (2 g NaHCO3/L water) or using
NaHCO3 (up to 16 g/kg) in the feed as a sodium source, or both.
Use of saline solutions has been a common practice to stimulate nutrient digestion
and absorption during heat stress (Jeukendrup et al., 2009) in birds. Electrolyte balance can
influence appetite and the metabolism of certain nutrients (Patience, 1990).Dietary
electrolyte balance (DEB) has also been reported to influence the absorption of
monosaccharides and amino acids (Johnson and Karunajeewa, 1985; Ravindran et al., 2008).
Therefore, an optimum level of dietary electrolyte balance can enhance digestion of nutrients
whereas a significant deviation to either side of optimum DEB may decrease digestibility of
nutrients (Ravindran et al., 2008).
Dietary addition of sodium in birds has been shown to improve digestibility by
increasing sodium ions concentration (Fethiere et al., 1994). Sodium containing compounds
such as sodium bentonites have been successfully used in sorghum containing diets to
prevent deleterious effects of tannins present in it, on nutrient digestibility (Pasha et al.,
2008; Banda-Nyirenda and Vohra, 1990). Santurio et al. (1999) and Salari et al. (2006) also
observed an increase in nutrient digestibility due to addition of sodium bentonite in broiler
diets.
Dietary inclusion of sodium containing compound such as NaHCO3 increase the
dietary electrolyte balance, which favors the proper digestibility of nutrients (Drinah et al.,
1990; Borges et al., 2003; Mahmud et al., 2010; Ahmad, 1997). However, there is dearth of
information in the literature where the significance of dietary inclusion of sodium
bicarbonate during hot weather has been evaluated.
It may be concluded that among many factors which influence digestibility of
nutrients in birds, heat stress is the most important factor, which has shown a pronounced
effect on digestibility of most of the key nutrients. Although, many research workers have
used different substances (mentioned above) in the diets of broilers during hot weather to
improve digestibility of nutrients, however, information regarding dietary inclusion of
sodium bicarbonate to see its effect on nutrient digestibility in layers during heat stress is still
scanty and therefore, is required to be explored.
47
CHAPTER-3
MATERIALS AND METHODSPrimary objective of this study was to explore the effect of varying levels of sodium
bicarbonate (NaHCO3) on production performance, immune response, blood profile and
nutrient digestibility in caged layers during summer. Two separate experiments (performance
trial and digestibility trial) were carried out for this study. The first experiment was designed
to probe the question of cause and effect when diets containing different levels of NaHCO3
were fed to caged layers during summer. Whereas, the second experiment was carried out to
investigate the effect of different levels of dietary NaHCO3 on digestibility of crude protein,
crude fiber and ether extract, as well as absorption of some minerals (Na, K, Ca, P and Fe) in
the layers.
3.1 Performance Trial
The details of the experiment regarding the effect of various level of sodium
bicarbonate on production performance, egg quality characteristics, immune response and
blood profile of caged layers are as follows
3.1.1 Experimental birds
One hundred sixty, 24 weeks White Leghorn layers were employed as experimental
birds for the performance trial. These birds were maintained in individual cages in a Poultry
House of the Department of Parasitology, Faculty of Veterinary Sciences, University of
Agriculture, Faisalabad (Pakistan) for a period of 12 weeks.
3.1.2 Allocation of the birds to the cages
Initially the experimental layer birds were kept in a group and were fed layer ration
during adaptation period (25th week of age). After the adaptation period, at the start of 26 th
week, all the birds were separately weighed, leg banded and allocated to the treatments using
a Completely Randomized Design (CRD). The cages were supplied with feeder and drinker
lines. The dimension of each cage was 41, 39 and 37cm, length, width and height
respectively. The birds were indiscriminately (randomly) divided into 20 experimental
units/replicates having 8 birds in each unit. These units (layer birds) were further allotted to
five experimental diets (4 replicates/treatment). The birds were provided experimental diets
48
throughout the study period (26-37 week of age).
3.1.3 Management of the experimental birds
These birds were maintained in a thoroughly cleaned and disinfected poultry house,
where they were given Erythromycin (1 table spoon/8 liter of water) for a week (25 th week of
age) as prophylactic measure to lessen the probability of any disease outbreak. A weighed
amount (110 g/bird) of the experimental diets was fed two times a day (morning and
evening). Fresh and clean water was offered to the birds. Daily, 17 hours light was
maintained to the birds throughout the study. The data on weekly feed consumption and body
weight were recorded during the experimental period.
3.1.4 Experimental diets, groups and their feeding plans
Diets used for the in vivo trials were iso-nitrogenous (CP, 17%) and isocaloric (ME,
2700 Kcal/kg diet). Five diets i.e. A, B, C, D and E were formulated with or without addition
of sodium bicarbonate (Table 3.1). Diet A, was without sodium bicarbonate and considered
as control whereas, diets B, C, D and E contained 0.5, 1.0, 1.5 and 2.0% sodium bicarbonate,
respectively. All the diets were formulated according to the NRC (1994) requirements. Each
experimental diet was fed for 12 weeks (26-37 weeks of age).
Before the start of experiment, all the diets were analyzed for their proximate
composition as shown in table 3.2, according to the technique described by AOAC (2010), in
the Analytical Laboratory of the Institute of Animal Sciences, Faculty of Animal Husbandry
(FAH), University of Agriculture, Faisalabad (Pakistan).
3.2 Data CollectionDuring this study, following data were recorded.
3.2.1 Initial body weight of the birds
The layer birds of all the experimental groups were individually weighed to have their
initial body weight on very first day of the experimental period.
3.2.2 Weight gain
The experimental birds were weighed every week in order to get weekly weight gain
of the birds and the weight records were maintained accordingly.
49
Table 3.1: Proportion of the ingredients used in the experimental diets
Ingredients
(%)
A
Basal diet
B
0.5% NaHCO3
C
1% NaHCO3
D
1.5% NaHCO3
E
2% NaHCO3
Maize 31.50 28.00 29.00 30.60 30.60
Rice broken 30.20 30.00 30.00 30.00 30.00
Fish meal 3.60 5.50 7.00 7.00 7.00
Soybean meal 17.00 1.50 0.00 2.00 4.40
Canola meal 4.50 14.00 13.60 11.60 8.40
Rapeseed meal 3.10 3.00 3.00 3.00 3.00
Guar meal 0.00 2.50 3.00 3.00 3.00
Corn-gluten 60% 0.00 2.00 2.00 2.00 2.00
Rice polishing 0.00 2.20 2.00 0.00 0.00
Dicalcium
phosphate
0.50 0.00 0.00 0.00 0.00
Limestone 9.00 9.00 8.70 8.70 8.70
Mineralsand
vitamin Premix
0.30 0.30 0.30 0.30 0.30
DL-met 0.13 0.08 0.07 0.07 0.09
Lys sulphate 65% 0.00 0.15 0.14 0.13 0.12
Salt 0.23 0.00 0.17 0.17 0.18
Sodium
bicarbonate
0.00 0.50 1.00 1.50 2.00
Allzymea 0.015 0.015 0.015 0.015 0.015
Lincomixb 0.02 0.02 0.02 0.02 0.02
a: a naturally fermented product with multiple enzyme activities including carbohydrase,
protease and phytase.
b:an antibiotic that provides consistent disease control for necrotic enteritis caused by
Clostridium perfringens
50
Table 3.2: Calculated chemical composition of the experimental diets
Nutrients A
Basal diet
B
0.5% NaHCO3
C
1%
NaHCO3
D
1.5% NaHCO3
E
2% NaHCO3
ME Kcal/Kg 2700 2700 2700 2700 2700
Crude protein (%) 17.00 17.00 17.00 17.00 17.00
Crude fiber (%) 3.29 3.80 3.74 3.44 3.39
Crude fat (%) 3.27 3.9 3.9 3.67 3.65
Crude ash (%) 11.97 11.9 12.01 11.85 11.89
Calcium (%) 3.9 3.9 3.9 3.9 3.9
Available Phosphorus (%) 0.37 0.37 0.38 0.38 0.39
Sodium (%) 0.17 0.23 0.43 0.57 0.7
Potassium (%) 0.70 0.53 0.51 0.52 0.52
Chloride (%) 0.22 0.15 0.20 0.20 0.20
Metabolizable Lys (%) 0.80 0.80 0.80 0.80 0.80
Metabolizable Met (%) 0.44 0.41 0.42 0.42 0.47
Metabolizable Thr (%) 0.55 0.56 0.56 0.57 0.57
MetabolizableTrp (%) 0.16 0.16 0.16 0.16 0.16
Dietary Electolyte
Balance (DEB) mEq/kg
210 210 262 325 388
51
3.2.3 Feed consumption
A weighed amount of feed was provided to each experimental unit twice a day,
throughout each week and left-over (remained) feed was weighed back to determine weekly
feed consumption of each replicate.
3.2.4 Egg production
Record of daily egg production of each replicate was used to calculate average
number of eggs produced/bird/week.
3.2.5 Egg mass
Total number of eggs produced by each replicate were weighed daily to calculate
weekly egg mass produced per replicate.
3.2.6 Feed conversion ratio
Feed conversion ratio (FCR) was calculated in two ways;
a) The kilograms of feed consumed to produce one dozen of eggs.
FCR /dozeneggs= Feed consumed(kg)No . of eggs produce
b) The kilograms of feed consumed to
produce one kilogram of egg mass.
FCR /Kg eggs mass= Feed consumed (kg)Egg mass produced (kg)
3.2.7 Water consumption
A measured quantity of fresh water was offered to each group, in the morning and
evening time. At each time, the residual water was again measured and recorded. Then daily
water intake of each replicate was summed up to calculate water consumption/group.
3.2.8 Rectal temperature and respiration rate
On the last day of each experimental week, rectal temperature and respiration rateof
three birds from each replicate were recorded at 6:00 am, 12:00 noon and 6:00 pm and
thereafter average of these observations was calculated to be used in the statistical analysis.
3.2.9 Ambient temperature and humidity index
A dry bulb thermometer was installed in the middle of the house to manually record
daily ambient temperature. Whereas, daily records of relative humidity inside the poultry
house were maintained by using a digital hygrometer.
52
3.3 Egg quality characteristicsFrom the start of production, at weekly interval, five eggs from each replicate were
selected randomly. These eggs were broken down individually (one by one) and their
material (content) was transferred into a separate Petri dish to find out the following
characters in relation to measurement of the quality of the eggs produced.
3.3.1 Egg weight
All the eggs produced by the birds of each experimental unit were numbered and
weighed daily. At the last day of each experimental week average egg weight was
determined for that particular week.
3.3.2 Shell thickness
Egg shell thickness was measured with a Micrometer Screw Gauge, accurately up to
0.01mm from the samples (eggs) used for meat and blood spots. Shell membranes of the egg
shells were removed by hand and then shell thickness of each egg was measured by taking
three readings (one reading each from broader end, narrow end and girth of the egg).
Average of these three values, however, was taken as the final reading.
Shell thickness (mm)=a+b+c3
Where
a = reading (mm) taken at the broader end of the egg
b = reading (mm) taken at the narrow end of the egg
c = reading (mm) taken at the girth of the egg
3.3.3 Specific gravity of eggs
Specific gravity of intact eggs was determined by the method described by Hamilton
(1982). The detail of the method used is as follows:
Procedure1. Nine beakers having 500ml water each; containing different concentration of salt
solution and having a known specific gravity were used in this process. Specific
gravity of different beakers is described below.
53
Beaker
No.
Conc. Of salt for 3litre H2O
(g)
Conc. Of salt for 500ml
H2O (g)
Specific gravity of
beaker
1. 276 276/6=46.00 1.06
2. 298 298/6=49.66 1.065
3. 320 320/6=53.34 1.07
4. 342 342/6=57.00 1.075
5. 365 365/6=60.84 1.08
6. 390 390/6=65.00 1.085
7. 414 414/6=69.00 1.09
8. 438 438/6=73.00 1.095
9. 462 462/6=77.00 1.1
2. The salt was weighed via a digital balance according to the amount of water in each
beaker. The appropriate amount of salt (as mentioned in the above table) was added
to each beaker and was dissolved properly by thorough manual stirring.
3. Firstly, the eggs were lowered one by one, into a beaker having pure water (without
the addition of salt), considered as pre-dip solution.
4. Thereafter, each egg was carefully lowered down one by one into the 1 st beaker, with
the help of a spoon and sliding down slowly, with the wall of the beaker. The egg was
kept in this state for 15-20seconds. The egg was taken out, when it started floating in
the beaker i.e. broke the surface of water.
5. The egg, if did not float in the 1st beaker, was taken out and dipped for few seconds in
the pre-dip solution again; it was then lowered down in the 2nd beaker having different
salt concentration.
6. The process was continued up to the 9th beaker and/or till the egg floated by itself in
some beaker.
54
7. The value of specific gravity of an egg was taken as that of known value of the
solution in the beaker, into which the egg floated.
3.3.4 Albumen Height
A tripod micrometer was used to determine the height (mm) of the albumen. The
tripod stand was positioned in midway between the yolk and edge of the albumen. Probe of
tripod micrometer was then lowered down on the surface of the albumen so that it made just
touch with it. The point when needle made contact with the surface of the albumin, the
reading was noted.
3.3.5 Yolk Height
A tripod micrometer was used to determine the height (mm) of yolk. The tripod stand
was placed on middle of the yolk. The needle of the micrometer was lowered even it
contacted. The reading was then noted.
3.3.6 Yolk Diameter
Yolk diameter was determined by using digital Verniercaliper. The jaws of Vernier-
caliper were placed around the yolk of the egg and thereafter were brought close to the yolk.
When the jaws just contacted the edges of the yolk, the reading was noted.
3.3.7 Blood and meat spots
The eggs, which were broken to check the egg quality parameters, were carefully
checked for the presence of blood or meat spots, if any.
3.3.8 Yolk index
Yolk index of egg was calculated using the following formula:
Yolk index= Yolk heightYolk diameter 3.3.9 Haugh unit score
The observations obtained for albumen height were used to determine Haugh unit
score of the eggs by the following formula.
Haugh unit = 100 log {H+7.6 – 1.7 W0.37}
Here,
55
H = albumen height (mm)
W = weight of eggs (g)
3.3.10 Yolk and albumen pH
The pH values of albumen and yolk of the eggs broken for the determination of egg
quality parameters were determined by digital pH meter.
3.3.11 Egg yolk cholesterol
Principle
The value of cholesterol in egg yolk was determined after enzymatic (esterase)
hydrolysis and oxidation. The end products of the reaction were cholesterol and fatty acids. It
was converted into cholestene-3-one and hydrogen peroxide by the action of oxidase
enzyme. The indicator quinoneimine was formed from H2O2 and 4-aminoantipyrine in the
presence of phenol and peroxides.
Procedure
For the estimation of cholesterol, Enzymic endpoint of Randox Kit was used
following the procedure of Roeschlau et al. (1974) as described below.
Pipetted out, 10µl standard solution in a standard tube and 10µl of egg yolk in other
separate sample test tube. Thereafter, 1000 µl of the reagent was added in a standard blank,
as well as in the sample tubes. The tubes were then gently shaken and incubated at 25°C for
25 minute. Absorbance of the standard and those of the samples was measured by
spectrophotometer model Meterek Sp-851 at 500nm wavelength. Calculations were made as
follows:
Conc .of Cholesterol(mg /egg)=( A ˚ Sample) /(A ˚ Standard) ˟ Conc. of standard
Where,
A0 sample = absorbance of sample
A0 standard = absorbance of standard
3.4 MortalityA complete record of mortality, if any, was maintained throughout the experiment.
Post-mortem examination of the dead birds was conducted immediately after their death to
find out the cause of death, if any.
3.5 Hematological profile
56
At the last day of 37th week blood samples from two birds (randomly selected from
each replicate) were taken separately in 10 ml test tubes. The tubes containing blood samples
were kept in a test tube rack for 2 hours to obtain blood serum of each sample. After that
supernatant of each tube was separated with micropipette and was put into 2 ml plastic
tubes. The tubes containing the supernatant (serum) were stored at -20 °C till further analysis
for the blood profile.
3.5.1 Glucose
Principle
Serum glucose was determined after enzymatic oxidation (in the presence of glucose
oxidase). The hydrogen peroxide thus formed reacts, under the catalysis of peroxidase, with
phenol and 4-amino-phenazone to form a red-violet quinoneirnine dye as indicator.
Following specifications were applied while using the method of Randox kit (Barham and
Trinder, 1972) for the determination of glucose.
Wavelength 500nm, Hg 546nm
Cuvette 1 cm path length
Temperature 15-25 °C
Measurement against reagent blank
Procedure
1. Pipetted out various necessary solutions into test tubes as under:
Macro Semi Micro
Standard or sample Reagent blank Standard
SampleReagentBlank
Standard or sample
Reagent 20µl2000µl
--------2000µl
10µl-------
1000 µl--------1000µl
2. Mixed the contents thoroughly and then incubated the above mentioned solutions for 10
minutes at 37 °C.
3. Measured the absorbance of the sample solution and standard solution and against the reagent
blank within 60 minutes.
57
4. Glucose concentration was calculated by the formula:
Glucose conc .( mgdl )= Absorbancesample
Absorbance standard×Conc .Standard
3.5.2 Hemoglobin
Hemoglobin (Hb) concentration was determined by using the Sahli’s instrument.
Erythrocytes were leaked in dilute hydrochloric acid to form acid hematin with the specific
color appearance and then the color was matched with color standards provided in the Sahli’s
instrument. Finally, the hemoglobin level was read from the scale where it matched with the
standard color.
3.5.3 Erythrocyte Sedimentation Rate (ESR)
For ESR determination, Westergren tube method (mm/hour) was used. Westergen
tube is 2mm in diameter and has graduation from 0 to 200 at one mm gap. The bottom of
the tube was dipped in the blood and drawn into the tube up to the “0” mark by aspiration.
The tube was then set in an upright position in a rack with a soft rubber cushion at the
bottom. The drop/fall (mm) of the red blood cells in the tube was noted after one hour.
3.5.4 Packed Cell Volume (PCV)
PCV was determined by the method described by Benjamin, (1978). One third of the
micro-haematocrit tube was filled with the blood (well mixed heparinized) and the open end
of the tube was sealed. Placed this tube in a centrifuge machine in such a position that its
sealed end remained away from the center, taking care to balance it against another tube of
the same size. The tube was covered with a screw top and centrifuged from 5-7 minutes at
12000 rpm. Similarly, contents of all the sample tubes were centrifuged. After removing
from the machine, percent values of PCV were recorded directly from the graphic
haematocrit tubes such that each tube was held against the liner chart, with the top of the
liquid exactly at the top line and the bottom of the tube against the bottom line.
3.5.5 Total leucocyte count (TLC)
Total leucocyte count was determined using fresh heparin mixed blood by the method
of Benjamin (1978). The White blood cell counting pipette was filled with the blood up to
mark 0.5 and sucked 3% acetic acid up to mark 11 above the bulb of the pipette. Closed the
tip of the pipette and brought it in horizontal position. Rotated the pipette gently, so that
leucocytes were dissolved in diluting solution. Discarded 1/3 contents of the pipette. Placed a
58
drop of diluted blood to the engraved area of the counting chamber and allowed to settle for
one minute. Set the microscope and counted the white blood cells in the respective squares
and calculated total number of leucocytes in 1 cu.mm by the following formula.
Total number of leucocytes in 1 cu.mm = F/4 x 20 x 10
Where:
F = No. of cells in four fields.
20 = Diluting factor
10 = Factor for converting 1/10.
3.5.6 Red blood cells (RBCs) count
Following are the requisites for enumeration of red blood cells;
Counting chamber with cover glasses
A diluting pipette
Diluting fluid
Hand tally counting
Reagents
Gower solution
Sodium sulphate 12.5g
Glacial acetic acid 33.3ml
Distilled water 200ml
Procedure
1. Blood sample was taken and thoroughly mixed for 3-5 minutes on mechanical mixer
or by inverting the tube for at least 20 times.
2. Diluted the blood 1:200 in a solution of 10 ml 40% formalin in a liter of 32g/l
trisodium citerate
3. Filled the counting chamber as discussed in the protocol for determination of WBC.
4. Let the cells settle (for 2-3 minutes) in a wet chamber (Petri dish with a small piece
of damp blotting paper).
5. Then counted the RBC at the 40X of microscope. An ample number of RBCs were
counted to minimize errors due to uneven cell distribution.
Total erythrocytes count ¿X80
× 40×200×10 ¿X1012
59
Where,
X¿Cells counted in 80small squares
80¿ Subdivisions of 5 small squars
400¿Total numbers of small squares
200¿Dilution 1:200
10¿ Depth of chamber (0.1mm)
3.6 Serum proteinsSamples of blood serum were analyzed for their protein composition including:
1) Total serum protein concentration
2) Albumin concentration
3) Globulin concentration
3.6.1 Total serum protein concentration
Principle
Cupric ions interact with protein peptide bonds in an alkaline medium resultantly
forming a color complex.
Procedure
Total protein was assayed by the Burette method (Henry et al., 1974) as described
below:
A volume of 0.02ml of distilled water, standard and serum was taken in reagent
blank, standard and samples tubes, respectively. Then add 1.0ml of reagent solution in all
the tubes. Mixed thoroughly and incubated the above mentioned solutions for 30 minutes at
25 °C. Then read absorbance of the standard (A° standard) and of the samples (A0 samples)
against the reagent blank at wavelength of 500nm within 60 minutes on spectrophotometer.
Finally total protein concentration was calculated by the following expression:
Total protein conc .( gdl )= Absorbancesample
Absorbance standard×Conc . standard
3.6.2 Serum albumen concentration
Principles
60
The determination of serum albumin is based on its quantities binding to the indicator
3, 3, 5, 5 tetra-bromocresol sulphonphthalen (Bromocresol green BCG).
Procedure
Bromocresol green Randox kit method was used following the procedure of Doumes
et al. (1971). The details of the procedure are described below.
Wavelength Hg 578nm
Cuvette 1cm light path
Incubation Temperature 20-25 °C
Measurement against reagent blank
A volume of 0.01 ml of distilled H2O, serum sample and standard was taken into
the test tubes marked as reagent blank, standard and samples, respectively, and then added
3.0 ml BCG reagent in each tube. Mixed thoroughly and then incubated the above
mentioned solutions for 5 minute at 25 °C. Read the absorbance of standard (A0 standard)
and of the sample (A0 sample) against the reagent blank on spectrophotometer.
Albumin concentration was calculated by the following formula:
AlbuminConc .( gdl )= Absorbance sample
Absorbance standard× Conc . standard
3.6.3 Serum globulin concentration
Serum globulins were determined by deducting albumin from the total concentration
of serum proteins.
Serum globulin (g/dl) = Conc. of total serum protein Conc. of albumin
3.7 Serum lipids profile3.7.1Serum cholesterol concentration
Principle
Serum cholesterol level was assayed by enzymatic (esterase) hydrolysis and
oxidation. The end products of the reaction were cholesterol and fatty acids. It was
converted into cholestene-3, 1 and hydrogen peroxide by the action of enzyme oxidase.
61
The indicator quinoneimine was formed from H2O2 and 4-aminoantipyrine in the presence
of phenol and peroxides.
Procedure
For the estimation of cholesterol, Enzymic endpoint of Randox Kit was used,
following the procedure of Roeschlau et al. (1974) as described below.
10µl standard solution was pipetted out into standard tube and 10µl of serum
sample was taken in another sample tube. Thereafter, 1000µl of reagent was added in both
tubes. The tubes were gently shaken and incubated at 25 °C for 25 minute. Using the
spectrophotometer model Meterek Sp-851, absorbance of standard and samples were
measured at 500nm wavelength and calculation was made as under:
Conc .of Cholesterol(mgdl )= Absorbance sample
Absorbance standard×Conc . standard
3.7.2 Serum triglyceridesSerum triglyceride concentration was estimated by enzymatic GOP-Pap method
(Trinder, 1969) by using kit manufactured by Human.
Principal
The principle involved is determination of triglycerides after enzymatic hydrolysis
with lipases. Indicator is quinoneimine formed from H2O2. 4-amino antipyrine and 4-cholro-
phenol under the catalytic action.
Triglycerides Lipases→
Glycero+fatty acid
Glcerol+ ATP GK→
GLycerol 3 Phosphate+ ADP
Glcerol3 Phosphate+O2 GPO→
dihydroxyacetone phosphate+ H 2O 2
H 2O 2+4−aminoantipyrine POD→
quinoneimine+HCl+2 H 2O+4chlorophenol
Reagents composition
1. Buffer solution (500 ml)
62
PIPES buffer (pH 7.5) 40mMol/L
4-chlorophenol 5mMol/L
Magnesium ions 4.7mMol/L
Sodium-azide 0.05 %
2. Buffer solution (15×7 or 10×50ml )
4-aminoantipyrine 0.4mMol/L
Glycerol-3-phosphate oxidase ≥1.5U/ml
ATP 1mMol/1
Lipases ≥0.2U/ml
Peroxidase ≥0.5U/ml
Glycerol Kinase ≥0.4U/ml
3. 3ml standard
Triglyceride 200g/dl or 2.28mMol/1
Reagent stability
The reagents were stable up to the stated expiry date when stored at 2-8°C (if
contamination avoided). The working reagent was stable for 6 weeks at 2-8°C for days at
15-25°C (if protected from light).
Instrument:
Micro lab 300 made by Merck international
Assay procedure
Wave length 500nm, Hg 546nm
Optimal path 1cm
Temperature 20-25 oC or 37oC
Measurement Against reagent blank
Pipetting scheme
Pipette into Cuvettes Sample/standard Working reagent
63
Reagent blank ----- 1000µl
Sample /Standard 10µl 1000µl
Mixed the contents and incubated these for 10 minutes at 20-25°C or for 5 minutes at 37°C
and then measured the absorbance of the standard and sample (∆ Asample).
Concentration of triglycerides were calculated as under;
With factor
Wave length C (mg/dl) C (mMol/L)
546 nm 1048 x ∆ A 11.95 x ∆ A
With standard`
C (mg/dl) = 200 x ∆ A sampe∆ A standard or
C (mMol/L) = 2.28 x ∆ A sampe∆ A standard
3.7.3 HDL cholesterol
High density lipoproteins (HDL) cholesterol was estimated by enzymatic CHOD-
PAP method as described by Schettler and Nussel (1975) by using the kit made by
DiaSys (Diagnostic System International).
Principle
Chylomicrons, VLDL (Very low density lipoproteins) and LDL are precipitated by
adding Phosphotungstic acid and Magnesium ions to the sample, centrifugation process left
only the HDL in the supernatant and the cholesterol contents can be determined
enzymatically.
Reagent preparation and stability
The precipitant for macro assays was stable up to the end of the indicated expiry date
and was prepared to be used. Reagents were kept contamination free and stored at 15-25 °C.
Precipitation for semi micro assays
The100 ml line was filled with distilled water. The reagent was stable up to the end of
the mentioned expiry date. Contaminations were kept away from and stored at 15-25 °C.
Specimen
64
Serum, Heparinized or EDTA plasma
Serum Tooke apart from the blood clot as quickly as possible
Component and concentration in the test
Mono Reagent
Magnesium Chloride 25mMol/L
Phosphotungstic acid 0.55mMol/L
Instruments
Micro Lab-300 made by Merck international
Procedure
Precipitation
Contents Macro Semi macro
Sample 500µl 200µl
HDL reagent undiluted 1000µl ---------
HDL reagent Diluted ----------- 500µl
Reaction components were mixed well and then permitted to stand for 1 minute at
room temperature (25 °C), then centrifuged at 10000rpm for 2 minutes. Clear supernatant
was separated from the precipitate within one hour. HDL concentration was determined
using the DiaSys (Diagnostic systems) cholesterol FS reagent.
Cholesterol determination
Wavelength 500nm, Hg 546nm
Optical path 1cm
Temperature 20-25 °C
Measurement against reagent blank
65
Pipetted scheme
Contents Blank Sample/standard
Dist. Water 100µl ----------
Supernatant ------------ 100µl
Reagent 1000µl 10000µl
Mixed well, incubated five minutes at 37 °C, finally read the absorbance against
reagent blank within one hour.
Calculation of HDL cholesterol
Cholesterol calibrator of DiaSystem (content 200mg/dl or 5.2mMol/L was used
like a sample in the precipitation step
HDL cholesterol (mg/dl) ¿∆ A sample
∆ A standard× Conc . Std( mg
dl)
Calculation of LDL cholesterol
LDL cholesterol was determined by calculation method using Friede-Wald et al.
(1972) formula:
LDL cholesterol (mg/dl) ¿Total cholesterol Triglyceride5
−HDL cholesterol
3. 8 Plasma electrolytes (i.e. Na+, K+, Cl-, HCO3-) and mineral (Ca and P)
profile
3.8.1 Estimation of plasma pH
For the determination of plasma pH,a digital pH meter was used. The bulb of the
electrode was completely dipped in the blood taken in a glass test tube and the final reading
was read out on the pH meter screen. The bulb of the electrode was washed with distilled
water after every consecutive reading.
3.8.2 Estimation of Sodium (Na+) and/or potassium (K+)
Na+and K+ were determined by using clinical flame photometer model 410-C, U.S.A.
66
The sodium concentration was calculated by the following formula.
Na+or K+(mEq/lit) = T/S x CS
Where,
T = Flame photometer displacement reading of test
S = Flame photometer displacement reading of standard.
CS= Concentration of standard
3.8.3 Estimation of chloride
For the estimation of chloride two reagents were used.
Reagent 1 = Mercury 2 mEq/lit
Thiocyanate iron 20 mEq/lit
Nitrate nitric acid 45 m Eq/lit
Reagent 2 = Chloride standard 100 m Eq/lit.
The spectrophotometer tubes were arranged as under.Blank Standard Sample
Reagent 1 1ml 1ml 1mlReagent 2 — 10µlSample — — 10µl
The displacement was read at 480nm by adjusting the spectrophotometer at zero
through running the blank. Concentration of Cl-was calculated as:
Chloride mEqL
= Absorbance sampleAbsorbance standard
× Conc . of standard
3.8.4 CalciumPrinciple
Calcium present in the sample reacts with arsenazo III forming a colored complex
which can be quantized using the spectrophotometer.
Reagent preparation
Reagent and Standard were made ready to use.
Equipments
Analyzer, spectrophotometer able to read at 650 ± 20nm.
Samples
Serum, heparinized plasma was collected by standard procedures. Calcium in plasma
was stable for 10 days at 2-8ºC.
67
Procedure
1. Took the reagent to room temperature.
2. Pipetted out various test solution into labeled spectrophotometer test tubes as detailed
below.
Blank Standard Sample
Calcium standard (S) ----- 15ul -----
Reagent 1ml 1ml 1ml
3. Mixed thoroughly and let these tubes stand at room temperature for 2 minutes.
4. Read the absorbance (A) of the Sample and Standard at 650 nm against the blank.
Calculations
Concentration of calcium (Ca) in the sample was quantized by the following formula:
Calcium conc .(mgdl )= Absorbance sample
Absorbance standard× Conc . std .× sampledilution factor
3.8.5 Phosphorus
Principle
Inorganic phosphorus (P) in the sample reacts with molybdate in acid medium
forming a phosphor-molybdate complex which can be quantized by spectrophotometer.
Contents and composition
A. Reagent: 3 x 40ml. Sulfuric acid 0.36Mol/L, sodium chloride 154mMol/L.
B. Reagent: 1 x 50ml. Sulfuric acid 0.36mmol/L, sodium chloride 154mMol/L,
ammonium molybdate 3.5mMol/L.
C. Phosphorus Standard: 1 x 5mL. Phosphorus 5mg/dl (1.61mMol/L) and aqueous
primary standard. For further warnings and precautions, see the product safety data
sheet (SDS).
Reagent preparation
Standard (S) was provided ready to use. Working Reagent: Mix thoroughly in the
68
proportion: 7mL Reagent A + 3mL Reagent B. It remains stable for 12 months at 15-30ºC.
Equipment
Analyzer spectrophotometer able to read at 340 ± 20nm.
Samples
Serum, heparinized plasma collected by standard procedures. Phosphorus in plasma was
stable at 2-8ºC for 7 days. Centrifuged the samples and diluted 1/10 with distilled water
before measurement.
Procedure
1. Pipetted various solutions into labeled spectrophotometer test tubes as detailed below.
Reagent blank Sampe blank Sample Stardand
Distilled water 10ul ------- ------- -------
Sample ----- 10ul 10ul -------
P standard ------ ---------
1 Mixed carefully and let the tubes to stand for 5 minutes at room temperature.
2 Read the absorbance (A) of the Sample Blanks at 340nm against distilled water.
4 Read the absorbance (A) of the Samples and of the Standard at 340nm against the
Reagent Blank.
Calculations
The phosphorus concentration in the sample was calculated using the following general
formula:
Phos . conc .( mgdl )= Absorbancesample
Absorbance standard×Conc . standard× sample dilution factor
3.8.6 Estimation of HCO3-
Plasma HCO3̶level was determined in a gasometer. For this a known volume of
serum was used to be reacted with liberating reagent (C3H6O3). The liberating reagent
69
converted the HCO3- to CO2. In the presence of an indicator solution column (C6H5COOH),
after running the standard solution of HCO3- (NaHCO3), the displacement of pressure
produced by CO2 was read out. The HCO3- content was calculated as under:
HCO3- mEq/L = T/S x C.S.
Where,
T = Displacement reading of the test (serum)
S = Displacement reading of the standard.
C.S. = Concentration of standard.
3.9 Hormono-enzymatic determination
Concentrations of different hormones were determined by using the Radio-
immunoassay (RIA) kits manufactured by ICN Pharmaceuticals, Inc. California. The analysis
relies upon the capability of antibody to bind its antigen. To quantify the antigen, radioactive
and non-radioactive type of the antigens vies for binding sites on a specific antibody. Since
more non radioactive antigen is added, less radioactive antigen leftovers bound until
equilibrium establishes between the free and antibody bound antigens.
3.9.1 Assay for Triiodothyronine (T3) determination
Principle
Radioimmunoassay based on the capability of an antibody to bind the antigen. To
quantify the antigen, radioactive and non-radioactive type of the antigens vies for binding
sites on a particular antibody. Since more non radioactive antigen is added, less radioactive
antigen leftovers bound until equilibrium establishes between the free and antibody bound
antigens.
Procedure
Blood serum T3 was assayed by the method used by Abraham (1981). A brief
description of the method is given below.
1. Brought all standard, samples, coated tubes and Triiodothyronine 125I at room temperature
before to use.
70
2. All the tubes were labeled accordingly.
3. Placed the required number of anti-Triiodothyronine coated tubes in test tube frams/racks.
Placed the sample labeled coated tubes in stands.
4. Allowed them to stand to attain room temperature.
5. Pipetted 100µl each of T3 standard, control and samples to their respective tubes.
6. Added one ml of T3 125Ito all tubes, vortexed all the test tubes carefully and incubated at 37
°C for 120 minutes.
7. Thereafter decanted the supernatant from the tubes.
8. Finally, subjected the liquid for counting in a gamma counter, already calibrated for 125I. The results were calculated using RIA data reduction system.
3.9.2 Assay procedure for Thyroxin (T4)
Principle
The antibodies used have alike affinity both for standard and the analyte, which is
present in the serum sample. The unlabeled analyte vies with labeled analyte for the
existing antibody binding sites, thus falling the quantity of the labeled analyte attached to
antibody. Hence the level of radioactivity bound is thus, inversely interrelated to the
quantity of analyte in the sample or standard. Following an adequate incubation period,
the bound and free fractions are separated. Blood serum T4 was assayed by following the
method of Abraham (1981). A brief description of it is given as under.
1. Brought all standard, samples coated tubes and thyroxin to room temperature prior
to use. All the tubes were labeled accordingly.
2. Pipetted 100µl each of T4 standard, control and samples to their respective tubes.
Added 1.0 ml of T4125I to all tubes, vortexed all the tubes thoroughly for a minute
and incubated in a water bath at 37 °C±1 for 60 minutes.
3. Ensured the level of water bath being above the level of the reaction mixture in the
tubes.
4. Aspirated liquid from the tubes, and subjected all the empty tubes for counting in a
71
gamma counter already calibrated for 125I.
5. The results were calculated using the RIA reduction system.
3.9.3 Assay procedure for Cortisol
Radioimmunoassay (RIA) depends upon the capability of an antibody to attach its
antigen. To quantize the antigen, the radioactive and nonradioactive types of the antigen
compete for binding sites on its exact antibody. Since more nonradioactive antigen is
appended, less radioactive antigen leftovers bound until/unless equilibrium establishes
between the free and antibody bound antigen. Blood serum Cortisol was assayed by
following the procedure of Oelkers et al. (1992). A brief description of the method is as
under.
1. Brought all standards, samples, coated tubes and Cortisol 125| to room
temperature before use. Placed the requisite number of anti-Cortisol coated test
tubes in a test tube rack/frame.
2. Resealed the unused tubes in the plastic bag along web desiccant and
refrigerated. In the end, added all solutions in the quantity specified directly
from the reagent vials.
3. Pipetted out 25µl each of standard, control and samples into their respective
coated test tubes. Added 1.0 ml of Cortisol 125ǀ to all test tubes and vortexed for
a short while.
4. Incubated all test tubes in a water bath, set at 37 ± 1 °C for 45 minutes.
5. Decanted the contents of the tubes and then counting in a gamma counter,
already calibrated for 125ǀ
3.9.4 Assay procedure for Estrogen
Principle
In the ImmuChem total estrogen Assay, the reaction follows the Law of Mass Action.
The labeled and non-labeled analytes unite to the antibody in quantity to their relative
quantity. The amount of radioactive analyte which binds is inversely proportional to the
concentration of unbound analyte in the sample. In view of the fact of the inverse relationship
between the quantity of the analyte and the counts bound, counts bound decrease whilst
rising concentration of non-labeled analyte. Blood serum Oestrogen was assayed following
72
the method of Buster and Abraham (1975). A brief account of it is as under.
1. Added 0.6 ml of diluent buffer to the tube number 1 and 2, and0.5 ml to tubes 3
and 4, labeled four tubes 1, 2, 3, and 4.
2. Pipetted 0.6 ml of diluent buffer to test tube number 1 and 2 and 6.5 ml to tubes 3
and 4. Placed 0.5 ml of total Oestrogen standard to tubes 5-16 and took 0.5 ml of
reconstituted samples to tube number 17.
3. Treated all the assay tubes, except 1 and 2 with 0.1 ml of anti-total Oestrogen.
Thereafter, put 0.1 ml of 17 β-oestradiol125I to all the assay tubes mixed the
contents and incubated them for 1.5 hour at room temperature.
4. Finally added 0.1 ml of second antibody tr. all the tubes and incubated at room
temperature for 60 minutes.
5. After incubation centrifuged the tubes at 2300-2500rpm for 15 minutes and
aspirated the supernatant.
6. Subjected the residue of tubes for counting in gamma counter. Calculated the
results using the RIA data reduction system.
3.9.5 Assay procedure for progesterone
Principle
Radioimmunoassay based upon the capability of an antibody to bind its antigen.
In order to quantize the antigen, a radioactive antigen and a non-radioactive antigen
struggle for a limited numbers of binding sites on a specific antibody. As more non-
radioactive antigen is introduced into the system, less binding sites are accessible for
the radioactive antigen creating a method for quantitation. Method of Abraham et al.
(1972) was followed for the assay of progesterone. A brief description of the method is
as follow.
1. Brought all standards, samples, coated tubes and progesterone 125l to room
temperature before use.
2. Then placed the required number of anti- progesterone coated tubes in a test
tube rack/frame. Resealed the unused tubes in the plastic bag together with
the desiccant and refrigerated.
73
3. Pipetted 100 µl each of standard, sample and control into the respective
tubes. Added 1.0 ml of progesterone 125I to all the tubes and vortexed briefly.
4. Incubated the tubes in a water bath, Set at 37±1 °C for 120 minutes.
5. It was ensured that the level of water bath was above the level of reaction
mixture in the tubes.
6. Calculated the results using the RIA data reduction system.
3.9.6 Serum Glutamic Pyruvic Transaminase (SGPT)
Serum Glutamic Pyruvic Transaminase (SGPT) was determined by observing the
amount of pyruvate hydrazone formed with 2, 4-dinitro phenylhydrazine (DNP).
Assay Procedure
The Randox Kit method was used for the assay of GPT following the
procedure of Reitman et al. (1957) as described below.
Wavelength Hg 546nm (530-550nm)
Cuvette 1 cm light path
Incubation temperature 37 °C
1. Measured against reagent blank and pippeted into a test tube 0.1ml of sample.
Added 0.5ml of buffer, each in reagent blank and sample tubes but 0.1ml of
distilled water was put in reagent blank tube only.
2. The content of every test tube were thoroughly mixed and incubated for exactly
half an hour at 37 °C. Then added 0.5 ml of 2, 4-DNP in reagent blank and
sample, respectively.
3. Thoroughly mixed and then permitted to stand for exactly 30 minutes at 25 °C.
Then added 5.0ml of sodium hydroxide in reagent blank and sample, respectively.
Mixed and read the absorbance of sample (A° sample) against the reagent blank
after 5 minutes. The activity of GPT in the serum was obtained from the table by
comparing the absorbance of spectrophotometer.
74
3.9.7 Serum Glutamic-Oxaloacetic Transaminase (SGOT) Principle
Glutamic Oxalocetic transaminase was determined by monitoring the concentration
of pyruvate hydrazine formed with 2,4- dinitro phenylhydrazine (DNP).
Assay procedure
The Randox Kit method was used for the assay of GPT following the procedure of
Reitman et al. (1957) as described below.
Wavelength Hg 546nm (530-550nm)
Cuvette 1cm light path
Incubation temperature 37 °C
4. Measured against reagent blank and pippeted into a test tube 0.1ml of sample.
Added 0.5ml of buffer, each in reagent blank and sample tubes but 0.1ml of
distilled water was put in reagent blank tube only.
5. The content of each test tube were thoroughly mixed and incubated for exactly 20
minutes at 25 °C. Then added 0.5 ml of sodium hydroxide each in reagent blank
and sample, respectively.
6. Thoroughly mixed and then permitted to stand for exactly 30 minutes at 25 °C.
Then mixed 5.0ml of sodium hydroxide in reagent blank and sample, respectively.
7. Mixed and read the absorbance of sample (A° sample) against the reagent blank
after 5 minute.
8. The activity of GOT in the serum was found from the table by comparing the
absorbance of spectrophotometer.
3.10 Serum metabolites3.10.1 Urea
Principle
Urea is hydrolyzed in the occurrence of water and urease to produce ammonia and
CO2, the NH4 formed in the first reaction with α-oxoglutarate and NADH in the presence of
glutamate- dehydrogenase to yield glutamate and NAD+.
Procedure
75
Urea in the serum was estimated by enzymic kinetic method described in the Randox
Kits, following the UV method (Kassirer, 1971).
Wavelength 340nm (Hg 334nm or Hg 365nm)
Cuvette 1cm light path
Incubation Temp. 37 °C
Measurement against reagent blank.
Pipetted out 10µlof each of standard solution and serum into test tubes for standard
and sample. Working reagent 1.0ml was added in each tube (Reagent blank, standard,
sample). Shacked and incubated the above mentioned solutions for at least 3 minutes at 37
°C. Added 200µlreagent 2 in reagent blank, standard and sample, respectively. Shackedand
incubated for at least 5 minutes at 37 °C. Measured the standard (A standard) and the
sample (A sample) against the reagent blank within 2 hours.
Concentration of urea in serum sample was calculated by the formula;
UreaConc .(mg /dl)= Absorbance sampleAbsorbancestandard
×Conc . standard
3.10.2 Serum creatinine concentration
Creatinine produces an orange-red colored compound in an alkaline solution
with picric acid. The absorbance of this complex is directly proportional to the amount
of creatinine in the sample.
Creatinine + Picric acid→ Creatinine-picric acid complex
Reagents
Components and Concentrations
Picric acid (Rl): 70mMol/L
NaOH (R2): 1.6mMol/L
Standard (R3): 176.8 pMol/L (2mg/dl)
Assay
Temperature: +25 °C, +30 °C, +37 °C
Wavelength: Hg 492nm (480-520nm)
Optical path: 1cm
Measurement: against the air
76
Procedure
1. Working reagent was prepared by mixing of one part of reagent number 1 and
one part of reagent number 2.
2. Up to 100µl sample was taken and 1000µl working reagent was added and in it
then mixed carefully. Absorbance was noted/read after 20 seconds and again
after 80 seconds for both, test sample and standard.
Creatinine Conc .(mg /dl)= Absorbance sampleAbsorbance standard
× 2
3.10.3 Uric acid
Principle
Uric acid in the sample originates, by means of the coupled reactions described
below, a colored complex which can be quantified by spectrophotometer.
Uric acid + oxygen + 2 H2O →Alantoin + carbon dioxide + hydrogen peroxide
2 H2O2 + 4 – Aminoantipyrine + DCFS →Quinoneimine + 4 H2O
Composition
A. Reagent: Phosphate 100mMol/L, detergent 1.5 g/L, dichlorophenolsulfonate 4mMol/L,
uricase > 0.12 U/mL, ascorbate oxidase > 5 U/mL, peroxidase > 1 U/mL, 4-aminoantipyrine
0.5mMol/L, pH 7.8.
Serum uric acid standard: Uric acid 6mg/dl (357µMol/L). Aqueous primary standard.
Reagent preparation
Reagent and Standard solutions were made ready to utilize.
Equipments
Water bath set at 37ºC, analyzer and spectrophotometer able to read at 520 ± 10nm
Procedure
1. Took the reagent to room temperature and pipetted then into test tubes having labeled.
2. Pipetted into labeled test tubes:
3. Mixed carefully and incubated the contents of test tubes for for 5 minutes at 37ºC.
4. Measured the absorbance of the standard solution and the Sample solution at 520nm
against the blank.
77
The spectrophotometer tubes were arranged as underBlank Standard Sample
Distilled water 25ul -------- --------
Uric acid standard (S) ------- 25ul --------
Sample --------- --------- 25ul
Reagent 1ml 1ml 1ml
CalculationThe concentration/quantity of uric acid in the sample was calculated by the following general
formula:
Uric acid conc .( mgdl )= Absorbance sample
Absorbance standard× Conc . standard ×sample dilution factor
3.10.4 Alkaline Phosphatase Principle2-amino-2-methyl-1 -propanol + p-nitrophenvlophosphate + H2O ↔4-nitrophenol + 2
amino-2-methyl-1 -propanol phosphate
Reagents/chemicalsComponents and Concentrations
Enzyme/Buffer Reagent2-amino-2-methyl-1 -propanol 350mMol/L
Zn+2 1.0mMol/L
Mg+2 2.0mMol/L
Substratep- Nitrophenyl phosphate 16.0mMol/L
EDTA 2.0mMol/L
AnalysisWavelength: 405nm
Optical path: 1cm
Temperature: 30 °C, 37 °C
Measurement: ‘ against air
ProtocolWorking reagent was get ready d by adding 4 parts of 1-ALP reagent and 1 part of 2-
78
ALP reagent. Firstly 20μl serum sample was taken after that 1000μl of buffer was added in it
then after 1 minute and again after 1, 2 and 3 minutes absorbance was read.
ResultFrom the absorbance readings, ∆A/ minute was determined and the result was
multiplied by the factor i.e. 3433.
3.11 Determination of Antibody titer against Newcastle disease virus (NDV).Five ml of whole blood was collected from healthy adult birds (wing-web) in screw-top test
tube having 1mg/ml EDTA as anticoagulant as described by Allan et al. (1978). The test tube
was gently rotated for the mixing of blood and anticoagulant, but great care was taken to
avoid hemolysis.
Washing of Red Blood Cells (RBC)
a. The blood containing anticoagulant was centrifuged at 1500rpm for 5 minutes.
b. The plasma and buffy coat was separated via sterile pasture pipette.
c. Physiological saline was added to the sediment and the cells were re-suspended
by gentle shaking of test tube.
d. Again centrifuged at 1500rpm for 5 minute.
d. The cells were given 3 washing in this way.
e. A 2 % suspension of washed erythrocytes was prepared in Phosphate Buffer
Saline (PBS).Source of Virus
Newcastle disease virus (Mukteswar strain) was obtained in allantoic fluid from
the Department of Veterinary Microbiology University of Agriculture Faisalabad
(UAF), Pakistan.
Micro Haemagglutination Test (for ND virus)Procedurea. A 50Μl of sterilized physiological saline (pH 7.2) was distributed/dispensed in
all wells of rows A to H of micro titer plate with the help of micro diluter.
b. To well one of each row 50 Μl of NDV in allontoic fluid was added and mixed
with physiological saline.
c. Using micro diluter 2-fold serial dilution of each NDV was prepared up to 11th
well of each row.
79
d. A 50ΜL of 1% RBC’s was added to each of the wells of micro titer plates
e. Well number 12 was considered as control since merely the diluents and red
blood cells control suspension was added to this well.
f. The micro titer plate was quaked/shaken lightly to let mixing of contents in the
wells then incubated at room temperature afterwards. The results were
recorded/documented after 15-20 minutes, when the RBC’s in well number 12
were settled down in the form of a button at the bottom.
The results of HA test were interpreted as follows.
Positive: The bottom of the well covered by the thin layer of finally clumped RBC’s.
Negative: A small sharply outlined button of RBC’s (Bead formation) on the bottom of
the well.
Doubtful: A ring formed by the un-agglutinated RBC’s disrupting the thin layer of
clumped cell coating the bottom of the well. The HA titer was the reciprocal of the
highest dilution exhibiting haemagglutination.
Note: The highest dilution of the virus showing HA was considered as one HA unit. The
4 HA titer was calculated by dividing highest dilution showing HA titer by four.
Collection of serum samples
a. Blood was collected through wing vein by inserting 24 gauge needle fitted with 3cc
syringe.
b. After taking 3ml blood in syringe, it was taken in sterilize glass tubes and kept in
slanting position.
c. Upon clotting, it was kept in refrigerator for 2 hours.
d. Serum oozing out was collected in clean dry plastic vials and stored at -20 °C till
use.
e. Serum samples were collected at 10 days post first, second and third
vaccination.
Haemagglutinion Inhibition (HI) Test for determination of serum antibody titer in test
serum
Heamagglutination inhibitions (HI) test for determination of serum antibody titer described
by Beared (1976).
a. Micro titration plates (8 rows and 12 column of well) were used. Sterile tips were
80
used for micro titration dispenser.
b. All the wells in the plate were filled with 50µl of the normal saline solution.
c. Serum sample of 50µl were placed in the first well of all the rows.
d. With the help of multichannel micro-titration dispenser, the mixture in the first
well of each row was mixed properly and 50µl of the mixture was transferred to 2nd
well of the respective rows thus diluting each sample as 2- fold. Dilutions of serum
samples were done up to the well No. 10.
e. With the help of micro dispenser 50µl of the 4 HA unit of NDV was added to each
well upto well number 11. The plates were incubated at room temperature for 30
minutes.
f. Chickens RBC’s suspension (50µl of 1 % RBC’s) was added with the help of
micro-titration dispenser into each well of the plate from well 1 to 12 of each row.
g. The plates were then agitated/disconcerted backward and forward and from side to
side to make sure even suspension of RBC’s.
h. The plates were then set aside undisturbed/serene at room temperature until/unless
a clear pattern of haemagglutinion inhibition button formation was observed.
Maximum dilution of each serum sample which caused haemagglutination inhibition
was the endpoint. The titer of each serum sample expressed as reciprocal of the highest
serum dilution, which gave positive result (Lin, 1997).
3.12 Digestibility trialA digestibility trial was conducted during the experiment at 38 week of age. For this
purpose a separate group of thirty pullets obtained from the same batch as used for
performance trial, was reared in individual metabolism cages to be used in digestibility trial.
These layers were randomly allotted to five treatments (6 birds/treatment) such that each bird
served as a replicate. These pullets were fed rations mixed with cellite (acid insoluble ash) at
1% as a marker.
The birds of these groups were fed their respective rations for five days (start of 38 th
week of age) to assure that the passage of marker (AIA) in the feces of the birds was
stabilized (Sales and Janssens, 2003) and during this period no feces were collected. After
stabilization of the marker in the feces (week 38), all the birds were offered same amount of
their respective rations. The feed offered to the birds was divided in to two equal portions
81
and half of the feed was given at 9:00 am, and the rest at 9:00 pm. The feed not eaten was
removed from the feeders and weighed at the end of the digestibility period.
Excreta collections, which started at the 6th experimental day, were made for a period
of 48 hours (2 consecutive/uninterrupted days) at the interval of 2 hours. Excreta samples
were immediately/instantly frozen after every collection. The sample thus obtained were
dried, finely ground and then analyzed for the determination of digestibility of dry matter
(DM), crude protein (CP), ether extract (EE) and crude fiber (CF) contents using the AO AC
(2010) methods. The samples collected were also analyzed for their mineral contents (Ca, P,
Na, K, Fe and Mg) using atomic absorption spectrophotometer.
Digestibility of the nutrient was calculated by the following formula:
D(% )=100−( Acid insoluble ash (AIA )∈feedAcid insoluble ash( AIA)∈ feces
× Nutrient∈fecesNutrient∈ feed
×100)3.13 Proximate composition
Proximate analysis of feed and excreta samples were carried out for calculation of
nutrients digestibility for the determination of digestibility of DM, CP, EE and CF contents
by the method described by AOAC (2010). The description of these procedures is given
below,
2.13.1Dry matter
Dry matter contents of the samples (feed and/or excreta) were determined by drying
the samples in hot air oven at 65 °C for 48 hours having no further moisture. The dried
samples were transferred into desiccators for thirty minutes to attain constant weight.
Weights of the samples were recorded prior and after drying. The DM was calculated using
the following formula!
W1 - W2
Dry matter % =––––––––––––––––––––––x 100 W1
Where,
W1= weight (g) of sample before drying
W2= weight (g) after drying
3.13.2 Crude protein
Nitrogen contents of the samples (feed and/or excreta) were determined by the
Kjeldahl method. One gram (gm) of dried and ground sample was taken in digestion flask
82
with 5 gram of digestion mixture containing K2SO4, CuSO4 and FeSO4 (90:9:1) and 25 ml of
concentrated H2SO4. Then digestion flask was placed/put on heater until a clear solution was
attained. Thereafter the contents of flask were cooled and diluted with distilled water up to
250ml in a volumetric flask. Ten ml of this solution was transferred/shifted to a micro
Kjeldahl distillation apparatus and distilled with 10ml of 40% sodium hydroxide solution.
The ammonia (NH3), produced, was accumulated in a beaker having 10ml of 2 % boric acid
solution containing two drops of indicator containing methyl red. Distillate so obtained was
titrated against 0.1 N H2SO4 to end point (pink) and the percentage (%age) of nitrogen was
calculated using the formula given below:
Volume used N/10 x 250 x 0.0014 x l00N2 (%) = –––––––––––––––––––––––––––––––––––––
Weight of sample x 10CP (%) = % N2 x 6.25
3.13.3 Ether extract
Crude fat (ether extract) was determined by Soxhlet’s continuous extraction
apparatus using fat solvent. Five grams of moisture free sample (feed and/or excreta)
was taken into the dried extraction thimble. The thimble was placed in the glass jacket
which is fixed under the condenser for the extraction apparatus. About 150 ml diethyl
ether (BP 40-60 °C) was poured in the already weighed receiving flask of the apparatus.
The water and heater were turned on and extraction was continued for 10 hours at the
condensation rate of 90drops/minute. Thereafter, thimble was removed and the diethyl
ether was collected in the glass jacket until the receiving flask contained about 20ml
ether and fat extracted. Then the receiving flask was heated to evaporate the solvent.
The percentage of ether extract was calculated by the following formula:
Ether extract (%)¿Lossof weight of sample
weight of sample∗100
3.13.4 Crude fiber
Two grams of moisture free sample (feed and/or excreta) was made fat free
(ether extracted) and was taken in a 600ml beaker provided with a reflux condenser.
The sample (feed and/or excreta) was digested at simmering temperature ( 80 °C) with
200 ml of 1.25 % H2SO4 solution for 30 minutes, which hydrolyzed the protein and
carbohydrate. The volume of simmering medium was kept constant by frequent addition
83
of hot water. The contents left were filtered immediately/instantly under vacuum and
the residue remaining after filtration was washed with H 2O and transferred to 600ml
glass beaker. The residue was digested with 1.25 % NaOH simmering solution for
exactly 30 minutes. Again the residue left was washed and filtered in similar way. The
contents were shifted to an oven at l00oC for drying to a constant weight. The dried
residue was ignited in a muffle furnace at a temperature of 550 oC for 20 minutes.
Residue left after ignition was weighed and loss of weight of original sample is reported
as crude fiber. Crude fiber calculated by using the following formula.
Weight of residueCrude fiber (%) = ––––––––––––––––––––– x 100
Weight of sample
3.13.5 Acid Insoluble Ash (AIA)
The AIA was determined using the method of Van Keuien and Young (1977) as follows:
Five gram dried ground sample of feed and/orexcreta were washed overnight at 450
°C.
The ash was transferred/shifted into a conical flask and 100ml of 2N HCl was added.
The mixture obtained was thus boiled for 5 minutes on a crude fiber digestion
apparatus. A condenser was attached to the flask to avoid the loss of HCl.
The hot hydrolysate was then filtered and washed free of acid with hot distilled water.
The ash and filter paper were then transferred/shifted back into the crucible and kept
for conversion into ash, overnight at 450oC.
The crucibles, along with its contents were cooled in a desicator to room temperature/
weighed having ash and reweighed instantly after emptying.
The percentage of AIA was calculated using the following formula!
Wf - We AIA% = ––––––––––––––––––––––––– × 100 WsWhere,
Wf = weight of crucible with ash
We = weight of empty crucible
84
Ws = weight of sample:
3.14 Mineral analysisBefore the estimation of minerals (Ca, P, Na, K, Mg and Fe), the fecal and/or feed
samples were dried for 24 hours. The dried samples were ground in Willy mill and sieved
with stainless steel sieve and stored in air tight containers.
3.14.1 Wet digestion
One gram of dried and ground excreta and/or feed sample was taken into 150 ml
Pyrex beaker, and soaked thoroughly with 10 ml of concentrated Nitric acid (HNO3). Then
3ml of 60 percent per chloric acid (HClO4) was added. The contents were heated slowly on a
hot plate, until frothing ceased and then heated gently with another 10ml of HNO3. The
heating continued/persistent until brown fumes of nitric acid came to an end and white fumes
of HClO 4appeared. The beaker was cooled and the contents were dissolved in 10ml of 6 N
HCl and transferred to 100ml volumetric flask quantitatively and volume was made up with
de-ionized water. Concentration of various mineral elements were determined/found as
described by Smith et al. (1979) by atomic absorption spectrophotometer model 170-10
(Hitachi Ltd.) using air acetylene flame at spectral line 213.9nm. While, sodium and
potassium were determined on Flame Photometer using corning (EEL Model) flame
photometer according to the method as described by AOAC (20100).
3.14.2 Determination of calcium
Calcium was estimated by titration method using micro burette (EDTA-verenuate).
Digested eggshell sample was titrated against 0.01 N Ethylinediaminetetraacetate (EDTA)
solution (Allison et al . , 1954). 0.01 N EDTA was prepared by dissolving 2gm of sodium
salt of EDTA in 1 liter of distilled water and added one crystal of MgCl2. 2.5gm of digested
material was taken in a china dish and added 20ml of distilled water along with 8-10 drops of
4N NaOH. Stirred it with glass rod and added 50gm of ammonium purpurate i.e.(4.5gm
ammonium purpurate and 100gm K2SO4 were ground to form homogeneous mixture) stirred
with glass rod. Pink color was obtained. Titrated it against 0.01 N EDTA up to purple color
end point and then recorded the reading of EDTA used for calculations.
Calculation Volume of sample (feed and/or excreta) solution taken = 10 ml
85
Strength of calcium/L = N× Eq. weight, = Bgm/L
1000ml of sample solution contain (Ca ) = Bgm/L
100 ml of calcium solution contain calcium = Bgm/1000 ×100 = A%
3.14.3 Determination of phosphorus
Three test tubes were taken and these solutions were treated with reagents as
illustrated.
86
Pipetted scheme
Reagents Test (ml) Standard (ml) Blank (ml)
Sample solution (Wet
digested)
1.0 ------ -------
Standard solution of P1 ---------- 1.0 --------
Ammonium molibidate 1.0 1.0 1.0
Aminonapthol
sulfonate 3
0.4 0.4 0.4
Distilled water 7.6 7.6 8.6
Standard phosphateThe contents of test tubes were finely quivered/shaken and then permitted to settle for
the minutes, when blue color appeared in them, the absorbance was read on HITACIII U
1100 spectrophotometer at 720 nm after setting the zero with blanks.
The amount of total Phosphorousin feed and/or feces was calculated by using the following
formula:Phosphorusmg /100 ml=A / B× 0.08
Where,
A = absorbance of sample solution
B= absorbance of standard solution
3.14.4 Determination of Sodium and Potassium by Flame PhotometerAfter wet digestion of the sample(feed and/or excreta), a standard solution of sodium
or potassium was prepared for running in flame analyzer. Recorded the readings of the
standard and sample solutions by the flame analyzer and made the calculations accordingly.
Calculation Reading of standardsolution = X
Reading of sample solution = Y
X reading is due to presence of = 10mg/100ml
Y reading is due to presence of = 10X
×Y = A mgof sodium∨potassium
100 ml
500 mg of feed/or feces contain = A mg of sodium or potassium
% of sodium or potassium in feed/or feces = A
500×100
87
CHAPTER-4
RESULTS AND DISCUSSIONEFFECT OF DIETARY INCLUSION OF SODIUM BICARBONATE ON
PRODUCTION PERFORMANCE AND BLOOD PROFILE OF CAGED
LAYERS DURING SUMMER
4 RESULTS
4.1 Production performanceMeans and their standard deviation values of initial body weight, final body weight,
weight gain and feed consumption of the birds fed diets with or without addition of different
levels of sodium bicarbonate are shown in table 4.1.
4.1.1 Live body weight
Mean values for body weight gain of the layers were found to be 166, 187, 199, 177
and 169g for treatment groups A, B, C, D and E, respectively. Weight gain of the layers was
markedly influenced due to dietary addition of sodium bicarbonate in the layer diets.
Statistical analysis of the data showed that birds using diets containing 0.5 and 1% sodium
bicarbonate gained significantly (P<0.05) more weight as compared to those of groups D, E
and control. Disparity in weight gain among the treated groups was also found to be
significant. Birds of group C, which were fed diet having 1% sodium bicarbonate, exhibited
maximum body weight when compared to the birds of other treated groups, followed by
those of group B, D and E. However, difference in weight gain between groups A, D and E
was non-significant. Similarly, differences in weight gain between treated groups B and C
were also found to be non-significant.
4.1.2 Feed consumption
Mean weekly feed consumption values were 743, 770, 780, 766 and 755 g/bird for
treatment A, B, C, D and E, respectively. Feed consumption of layer birds was markedly
influenced due to dietary addition of sodium bicarbonate. Statistical analysis of the data
showed that birds receiving diets containing sodium bicarbonate consumed significantly
(P<0.05) more feed than those of control (group A). Differences in feed consumption among
treated group were also found to be significant. Birds of group C, which were fed diet
containing 1% sodium bicarbonate showed maximum
88
Table: 4.1 Effect of dietary inclusion of sodium bicarbonate (NaHCO3) on weight gain
and feed consumption caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Initial body
weight (g)1328±14.3 1310 ±7.0 1318 ±16.4 1324 ±11.0 1312 ±12.1
Final body weight
(g) 1494±60.9 1497±53.4 1517±36.7 1501±24.6 1481±61.0
Weight gain (g) 166±11.4c 187±6.4ab 199±15.1a 177±9.3bc 169±13.5c
Feed
consumption (g)743±12.90 770±10.45b 780±14.84a 766±10.51b 755±12.16c
Values within the same row having different superscripts are significantly (P<0.05) different.
89
feed intake followed by those of group B and E among treated groups. Whereas, the lowest
feed intake was observed in the layers of control group. On the other hand, difference in feed
consumption was found to be non-significant between treated groups B and D.
4.1.3 Egg production
Means and their standard deviation values of weekly egg production, egg weight, egg
mass, FCR per kg egg mass produced and FCR per dozen eggs produced by the birds fed
diets with or without addition of different levels of sodium bicarbonate are presented in table
4.2.
Mean values of weekly egg production were 5.33, 5.63, 5.86, 5.51 and 5.42 eggs/bird
for treatment A, B, C, D and E, respectively. Egg production of layers was markedly
influenced due to dietary inclusion/addition of sodium bicarbonate in the layer diets.
Statistical analysis of the data showed that birds consuming diets containing sodium
bicarbonate produced more eggs (P<0.05) as compared to those of control group.
Differences in egg production among treated group were also found to be significant. Layer
birds which were fed diet containing 1% sodium bicarbonate, showed maximum egg
production when compared to the birds of other treated groups. However, differences in egg
production between treated groups D and E were non-significant (P>0.05).
4.1.4 Egg weight
Mean values for egg weight were 50.9, 54.0, 58.2, 56.0 and 55.2 g, for treatment A,
B, C, D and E, respectively. Egg weight of layers was significantly influenced due to dietary
inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data showed that
birds consuming diets containing sodium bicarbonate produced significantly (P<0.05)
heavier eggs as compared to those of control group. Birds of group C, which were fed diet
containing 1% sodium bicarbonate, showed maximum egg weight when compared to the
birds of other treated groups. However, the differences in egg weight among treated groups D
and E were found to be non significant.
4.1.5 Egg massMean values for weekly eggs mass produced were found to be 267, 303, 339, 307 and
299 g for treatments A, B, C, D and E, respectively. Statistical analysis regarding egg mass
90
produced by each replicate showed that inclusion of NaHCO3 in the diet of layers has
91
Table 4.2: Effect of dietary inclusion of sodium bicarbonate on production
performance of caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Egg Prod.(Nos./week)
5
.33±0.20
5.63±0.21b 5.8
6±0.36a5.51±.12c 5.4
2±0.51c
Average egg weight
(g) 50.9±4.0 54.0±3.5c58.
2±1.50a 56.0±1.1b 55.2±1.6b
Egg mass produced
(g/week)
267±10.8 303±13.0b 339±12.6a 307±11.2b 299±17.5b
FCR/ dozen eggs 1
.70±0.052
1.6
4±0.050c
1.6
0±0.051d1.67±0.020b 1.6
7±0.021b
FCR/kg egg mass 2.78
±0.012a
2.54
±0.025b
2.3
0±0.015c2.48 ±0.051b 2.5
2±0.051b
Values within the same row bearing dissimilar superscripts are significantly different (P<0.05)
92
significant positive effect (P<0.05) on egg mass produced by the addition of sodium bicarbonate
in the diet. Difference in mean values of egg mass produced between sodium bicarbonate treated
groups was significant. The birds in treatment group C, consuming diet containing sodium
carbonate at 1% level produced more egg mass than the other treatment groups. However, the
difference in egg mass produced among treated groups B, D and E was found to be non-
significant.
4.1.6 Feed efficiency1. FCR/dozen eggs
Mean feed conversion ratio on the basis of per dozen eggs produced was found to
be1.70, 1.64, 1.60, 1.67 and 1.67 for treatments A, B, C, D and E, respectively. Feed
conversion ratio on the basis of per dozen eggs produced was significantly affected due to
dietary inclusion of NaHCO3 in the diets. Statistical analysis of the data showed that birds
consuming diets containing sodium bicarbonate showed significantly (P<0.05) improved feed
conversion ratio on the basis of per dozen eggs produced as compared to those of control
group. Differences in FCR/ dozen eggs produced among treated groups were also found to be
noteworthy (P<0.05). Birds of group C, which were fed diet containing 1% sodium
bicarbonate, showed better FCR/ dozen eggs produced when compared to the birds of other
treated groups. Whereas, the poor FCR was noted in the birds of control group. However,
differences in the FCR values among treated groups D and E were found to be inconsistent
(P>0.05).
2. FCR/Kg egg mass producedMeans feed conversion ratios on the basis of per kg eggs mass produced were found
to be 2.78, 2.54, 2.30, 2.48 and 2.52 for treatments A, B, C, D and E, respectively. Feed
conversion ratio on the basis of per kg egg mass produced by the layers was significantly
influenced by the dietary addition of NaHCO3. Statistical analysis of the data showed that
birds consuming diets containing sodium bicarbonate showed significantly (P<0.05)
93
improved FCR/Kg egg mass produced than those of control group. The birds in treatment
group C (1% sodium bicarbonate added to the diet) utilized their feed more efficiently than
the other treatment groups. Difference in mean values of FCR/Kg egg mass produced
between sodium bicarbonate treated groups was also significant. Whereas, the poorest
FCR/Kg egg mass produced was observed in the layers of group A (control). However, the
differences in FCR/Kg egg mass produced among treated groups B, D and E were found to
be non-significant.
4.2 Egg qualityMeans and their standard deviation values, of specific gravity, shell thickness,
albumen height, Haugh unit score, yolk diameter, yolk height, yolk index, yolk cholesterol,
yolk pH and albumen pH are shown in table 4.3.
4.2.1 Specific gravity
Mean specific gravity values of the eggs produced by the layers were 1.077, 1.081,
1.086, 1086 and 1.079 for treatment A, B, C, D and E, respectively. Specific gravity values
of the eggs produced by the birds were significantly influenced due to dietary inclusion of
sodium bicarbonate in their diets. Statistical analysis of the data showed that birds consuming
diets containing sodium bicarbonate produced eggs, which had more specific gravity values
(P<0.05) than those of control group. However, the differences in specific gravity values
among treated groups C, D; B, E and A, E were found to be non-significant. Specific gravity
of the eggs produced by the birds of group C, which were fed diet containing 1% sodium
bicarbonate, was found to be higher followed by those of treated groups D, E and B.
4.2.2 Shell thickness
Mean values pertaining to shell thickness of the eggs produced by the layers were
found to be 0.307, 0.335, 0.334, 0.336 and 0.34 for treatment groups A, B, C, D and E,
respectively. Shell thickness values of the eggs produced by the birds were markedly
influenced due to the dietary inclusion of NaHCO3 in their diets. Statistical analysis revealed
that shell thickness of the eggs produced by the birds consuming diets containing sodium
bicarbonate increased (P<0.05) when compared to those of control group. However,
differences in shell thickness among treated groups B, C, D and C, D, E were found to be
non-significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,
apparently but non-significantly showed maximum shell thickness when compared to the
94
birds of other treated groups. The eggs produced by the layer of control group showed the
minimum value of shell thickness of their eggs than all those of treated groups.
Table 4.3: Effect of dietary inclusion of NaHCO3 on egg quality characteristics of caged
layers
Variables
Treatment
A
Control diet
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Specific gravity 1.077±0.0301.081±0.012b 1.086±0.020a 1.086±0.020a 1.079±.02
8bc
Shell thickness (mm)
0
.307±0.0132
0.335±0.010
4b0.334±0.011
9ab0.336±.010
9ab0.34±0.011
0a
Albumen height (mm) 4.39±0.1074.71±0.101b 4.97±0.123a 4.6±0.150bc 4.46±0.13
0cd
Haugh unit 66.9±1.25 68.7±0.73 ab 70.2±0.97a 68±1.29bc 67.2±1.15bc
Yolk diameter (mm) 33.4±1.00 34.5±1.14b 36.6±1.55a 33.9±1.20b 33.4±1.02b
Yolk height (mm) 13.3±0.37 14.2±0.26a 14.5±0.28a 13.8±0.20b 13.49±0.1
4bc
Yolk index0.404±0.01380.401±0.0141 0.393±0.0140 0.408±0.0108 0.401±0.010
1
Yolk Cholesterol
mg/egg
243±5.9a 214±13.7b 193±15.0c 191±17.0c 185±6.4c
Yolk pH 6.8±0.22b 6.8±0.56b 7.1±0.18ab 7.3±0.16a 7.4±0.17a
Albumen pH 6.9±0.32c 7.3±0.29bc 7.7±0.26ab 7.6±0.24ab 7.8±0.21a
95
Values within the same row which have different superscripts letter are significantly different (P<0.05)
4.2.3 Albumen height
Mean values pertaining to albumen height were found to be 4.39, 4.71, 4.97, 4.6 and
4.46 mm for treatment groups A, B, C, D and E, respectively. Albumen height of the eggs
produced by the birds was significantly influenced due to dietary inclusion of NaHCO3 in
their diets. Statistical analysis of the data showed that birds using diets containing sodium
bicarbonate produced eggs having significantly (P<0.05) more albumen height as compared
to those of control group.
Differences in albumen height among treated group were also found to be significant.
Birds of group C, which were offered diet containing 1% sodium bicarbonate, showed
maximum albumen height when compared to the eggs produced by the birds of other treated
groups. However, the differences in albumen height among groups A, E; B, D and D, E were
not observed (P>0.05).
4.2.4 Haugh unit
Mean values pertaining to Haugh unit score were found to be 66.9, 68.7, 70.2, 68.0
and 67.2 for the treatment groups A, B, C, D and E, respectively. Haugh unit score of the
eggs produced by the birds was significantly influenced due to dietary addition of sodium
bicarbonate in their diets. Statistical analysis of the data showed that birds using diets
containing sodium bicarbonate produced eggs having significantly (P<0.05) greater Haugh
unit score as compared to those of control group. Differences in Haugh unit score among
treated groups were also found to be significant. Birds of group C, which were fed diet
containing 1% sodium bicarbonate, showed maximum Haugh unit score when compared to
the eggs produced by the birds of other treated groups. However, differences in Haugh unit
score between treated groups, B, C and D, E were found to be inconsistent (P>0.05).
Differences in Haugh unit values among groups A, D and E were also found to be
inconsistent (P>0.05).
4.2.5 Yolk diameter
96
Mean values pertaining to yolk diameter were found to be 33.4, 34.5, 36.6, 33.9 and
33.4 mm for the treatment groups A, B, C, D and E, respectively. Yolk diameter of the eggs
produced by the birds was significantly influenced due to dietary inclusion of NaHCO3 in
their diets. Statistical analysis of the data showed that birds using diets containing sodium
bicarbonate produced eggs having significantly (P<0.05) more yolk diameter as compared to
those of control group. Differences in yolk diameter among treated groups were also found to
be significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,
showed maximum yolk diameter when compared to the eggs produced by the birds of other
treated groups. However, the differences in yolk diameter among treated group, B, D and E
were found to be non-significant (P>0.05).
4.2.6 Yolk height
Mean values pertaining to yolk height were found to be 13.3, 14.2, 14.5, 13.8 and
13.49 mm for the treatment A, B, C, D and E, respectively. Yolk height values of the eggs
produced were significantly affected due to dietary inclusion of sodium bicarbonate in the
layer diets. Statistical analysis of the data showed that birds using diets containing sodium
bicarbonate produced eggs having higher (P<0.05) yolk height values as compared to those
of control group. Differences in yolk height among treated groups were also found to be
significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,
showed maximum yolk height values when compared to the birds of other treated groups.
However, the differences in these values among treated groups D and E were found to be
non-significant (P>0.05). Similarly, differences in yolk heights between group A and E were
found to be non-significant.
4.2.7 Yolk Index
Mean values pertaining to yolk index of the eggs produced were found to be 0.404,
0.401, 0393, 0.408 and 0.401 for the treatments A, B, C, D and E, respectively. Yolk index of
eggs was non-significantly influenced due to dietary inclusion of sodium bicarbonate in the
layer diets. Statistical analysis of the data showed that birds using diets containing sodium
bicarbonate produced eggs having apparently but inconsistently (P>0.05) slightly higher yolk
index as compared to those of control group.
97
4.2.8 Egg yolk cholesterol
Mean values regarding egg yolk cholesterol concentration for treatment groups A, B,
C, D and E, were found to be 243, 214, 193, 192 and 185 mg/egg, respectively.
Concentration of yolk cholesterol in the eggs produced by the birds of treated group was
significantly influenced due to addition of NaHCO3 in their diets. Statistical analysis of the
data showed that birds using diets containing sodium bicarbonate produced eggs having
lower (P<0.05) yolk cholesterol as compared to those of control group. Differences in yolk
cholesterol concentration among treated groups fed diets having various levels of sodium
bicarbonate were also found to be significant. Birds of group E, which were fed, diet
containing 2% sodium bicarbonate, produced eggs having lowest concentration of yolk
cholesterol when compared to those of other treated groups. However, difference in egg yolk
cholesterol between treated groups C, D and E was found to be non-significant.
4.2.9 Yolk pHMean values regarding egg yolk pH for treatment groups A, B, C, D and E, were
found to be 6.8, 6.8, 7.1, 7.3 and 7.4, respectively. Yolk pH of the eggs produced by the birds
in treated groups was significantly influenced due to addition of NHCO3 in their diets.
Statistical analysis of the data showed that birds using diets containing sodium bicarbonate
produced eggs having higher (P<0.05) egg yolk pH as compared to those of control group.
Differences in yolk pH values among treated groups fed diets containing different levels of
sodium bicarbonate were also found to be significant. Birds of group E which were fed diet
containing 2.0% sodium bicarbonate, produced eggs having maximum yolk pH value when
compared to those of other treated groups. The pH value in the treated group showed a linear
increase with increase in the level of sodium bicarbonate used. However, difference in egg
yolk pH value between treated groups B and C and among those of C, D, and E were not
found to be significant (P>0.05). The eggs produced by the birds of control group exhibited
the lowest pH value in their yolks.
98
4.2.10 Albumen pHMean values regarding egg albumen pH for treatment groups A, B, C, D and E, were
found to be 6.9, 7.3, 7.7, 7.6 and 7.8, respectively. Albumen pH of the eggs produced by the
birds in treated groups was significantly influenced due to addition of NaHCO3 in their diets.
Statistical analysis of the data showed that birds using diets containing sodium bicarbonate
produced eggs having higher (P<0.05) albumen pH as compared to those of group A
(control). The pH values in the treated groups showed a linear increase with increase in the
level of sodium bicarbonate used in the diets. However, differences in albumen pH among
treated groups were also found to be significant. Birds of group E, which were fed diet
containing 2% sodium bicarbonate, showed maximum albumen pH values when compared to
the birds of other treated groups. However, the differences in albumen pH values among treated
groups C, D and E and those of B, C and D were found to be non-significant.
99
4.2.11 Meat and blood spotsThe percentage of meat and blood spots observed in the eggs laid by the birds of
control was found to be 2.5% and 1.25%, respectively. Blood and meat spots were found to
be present only in the eggs laid by the birds of control group, which were fed diet without
addition of sodium bicarbonate, whilst treated groups were devoid of these. It has been
observed that higher ambient temperature is positively correlated with the incidence of meat
and blood spots in laying hens (Anjum, 2000). However, results of this study did not exhibit
presence of any blood or meat spots in the birds fed diet containing different levels of sodium
bicarbonate.
4.3 Rectal temperature, respiration rate and water consumption
Means and their standard deviation values of rectal/body temperature,
respiration rate and water consumption of layers are presented in table 4.4.4.3.1 Rectal temperature
Mean values regarding rectal temperature of the birds kept under treatment groups A,
B, C, D and E, were found to be 106.7, 106.39, 106.37, 106.41 and 106.38oC, respectively.
Rectal temperature of the birds kept under treated groups was significantly influenced due to
addition of sodium bicarbonate in their diets. Statistical analysis of the data revealed that
birds using diets containing sodium bicarbonate exhibited significantly (P<0.05) lower rectal
temperature as compared to those of control group. However, difference in rectal temperature
among the birds of treated groups fed diets containing different levels of sodium bicarbonate
was found to be non-significant. Rectal temperature of the birds kept under treated groups
exhibited a linear decrease with increase in the level of sodium bicarbonate used. Birds of
group C which were fed diet containing 1% sodium bicarbonate apparently experienced
lowest rectal temperature when compared to those of other treated groups. However, the
highest rectal temperature was observed in the birds of control group.
4.3.2 Respiration rate
Mean values pertaining to respiration rate of the layers for treatment groups A, B, C, D and E
were found to be 57.04, 53.46, 47.74, 51.3 and 50.5 respiration/minute, respectively.
100
Table 4.4: Effect of dietary inclusion of sodium bicarbonate on rectal temperature,
respiration rate and water consumption of caged layers
Variables
Treatment
A
Control
B
0.5%NaHC
O3
C
1%NaHC
O3
D
1.5%NaHC
O3
E
2%NaHCO3
Rectal
temperature
(0F)106.75±5.81106.39±4.43b
106.20±4.
2b
106.41±3.14
b
106.38±2.10
b
Respiration
rate (per
minute)57.04±1.7 53.46±3.18b 47.74±3.9d 51.30±4.2bc 50.5±4.1c
Water intake
(ml/day)333±9.51d 380±14.6 c 427±9.7 b 446±10 ab 467±8.7a
Values within the same row having different superscripts letter are significantly different (P<0.05)
101
Finding of the study depicted that respiration rate of the birds was significantly
influenced due to dietary inclusion of NaHCO3 in their diets. Statistical analysis of the data
revealed that birds using diets containing sodium bicarbonate exhibited lower respiration rate
(P<0.05) when compared to those of group A (control). Differences in respiration rate among
the birds of treated groups were also found to be significant. Birds of group C, which were
fed diet containing 1% sodium bicarbonate, showed minimum respiration rate values when
compared to the birds of other treated groups. Similarly the differences in respiration rate
among treated groups D and E was also non-significant (P>0.05). Heart rate quality of all the
birds was found to be normal and satisfactory and no abnormal sound was observed.
4.3.3 Water intakeMean values pertaining to daily water consumption were found to be 333, 380, 427,
446 and 467 ml/bird/day for treatment A, B, C, D and E, respectively. Water consumption of
the layers was significantly influenced due to dietary inclusion of NaHCO3 in their diets.
Statistical analysis of the data showed that birds using diets containing sodium bicarbonate
took more water (P<0.05) as compared to those of control group. There was a linear increase
in water consumption of the birds with increase in the level of NaHCO3 in the diets. Birds of
group E, which were fed diet containing (2%) sodium bicarbonate, took maximum water as
compared to the birds of other treated groups. Differences in water intake among sodium
bicarbonate treated groups were also found to be significant. However, differences in water
consumption among treated groups D and E, and those of groups C and D were found to be
non-significant.
4.4 MortalityIncidence of mortality was zero in all groups. It may probably be due to the reason
that the experiment was conducted under the best possible controlled hygienic conditions
except ambient temperature and the birds were kept well observed.
4.5 Hematological profileMeans and their standard deviation values, regarding serum glucose level, packed cell
102
volume (PCV), blood hemoglobin (Hb), erythrocyte sedimentation rate (ESR), red blood
cells count (RBCs) and white blood cells count (WBCs) are given in table 4.5.
Table 4.5: Effect of dietary addition of sodium bicarbonate on hematological profile of
caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Glucose
(mg/dl)
21
2.7±17.84a
200.2±17.4
0b195.3±5.91b 186.6±5.88c
174.7±10.5
1d
PCV (%) 33±3.56 32.5±2.46 32.7±2.99 32±2.16 32.7±2.63
Hb (mg/dl) 9.72±0.84610.53±0.89
6ab 11.37±0.675a10.58±1.16
0ab
10.75±1.320
ab
ESR
(mm/hour)3.93±0.29 3.75±0.44 3.7±1.04 3.73±0.62 3.7±0.52
RBCs
count
(106/mm3)
2.53±0.522.73±0.45 2.81±0.53 2.69±0.67 2.66±0.18
WBCs
count
(103/mm3)
26.5±1.21 22.5±2.80b 24±2.73ab 25±2.51ab 26.2±1.00a
Means within the same row having dissimilar superscripts letter are significantly different (P<0.05)
103
104
4.5.1 Serum glucose
Mean values pertaining to serum glucose level of the layers for treatment groups were
found to be 212.7, 200.2, 195.3, 186.6 and 174.7 mg/dl, respectively. Findings of the study
depicted that glucose level of birds was significantly affected due to dietary
addition/inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data
revealed that birds consuming diets containing sodium bicarbonate had reduced (P<0.05)
serum glucose level when compared to those of control group. Disparity in glucose level due
to the use of different levels of sodium bicarbonate was also found to be significant among
the birds kept under treated groups. The birds of group E, which were fed diet containing 2%
sodium bicarbonate, showed minimum serum glucose level when compared to the birds of
other treated groups. Yet, the difference in glucose level between groups B and C were non-
significant. Similarly the differences in glucose level between groups C and D were also
known to be non-significant. Maximum level of serum glucose was noted in the blood of the
layers kept under control group.
4.5.2 Packed cell volume (PCV)
Mean values of PCV in blood of the layers were found to be 33, 32.5, 32.7, 32.0 and
32.7% for treatment A, B, C, D and E, respectively. Statistical analysis of the data exposed
insignificant difference among the birds of all groups, indicating no effect on packed cell
volume of the layers due to dietary inclusion of NaHCO3 in their diets. However, birds of
group E, which were fed diet containing 2% sodium bicarbonate, apparently but non
significantly exhibited minimum packed cell volume among the birds of all treatment groups.
4.5.3 Blood hemoglobin
Mean values pertaining to blood hemoglobin of the layers were found to be 9.72,
10.53, 11.37, 10.58 and 10.75 mg/dl for treatment A, B, C, D and E, respectively. Statistical
analysis of the data showed significant difference in hemoglobin values among the birds of
all treatment groups, indicating improved effect on blood hemoglobin value of the layers due
to inclusion of NaHCO3 in their diets. Birds of group C, which were offered feed containing
1% sodium bicarbonate, significantly showed maximum concentration of hemoglobin among
the birds of all treatment groups. However, the difference in glucose level between treated
groups B and C, D and E were found to be insignificant (P>0.05). Similarly the differences in
glucose level between groups A, B, D and E were also found to be insignificant.
105
4.5.4 Erythrocytes sedimentation rate
Mean values regarding to erythrocytes sedimentation rate (ESR) of the layers were
found to be 3.93, 3.75, 3.7, 3.73 and 3.7 mm/ hour for treatments A, B, C, D and E,
respectively. Statistical analysis of the data showed non-significant difference in hemoglobin
values among the birds of all treatment groups, indicating no effect on erythrocytes
sedimentation rate of the layers due to dietary inclusion of NaHCO3 (sodium bicarbonate) in
their diets. However, birds of group A (control) apparently but non significantly exhibited
higher erythrocytes sedimentation rate among the birds of all other groups.
4.5.5 Red blood cells count
Mean values pertaining to red blood cells count in blood of the layers were found to
be 2.53, 2.73, 2.81, 2.69 and 2.66 ×106/mm3for treatment A, B, C, D and E, respectively.
Statistical analysis of the data showed insignificant difference among the birds of all groups,
indicating no effect on red blood cells count of the layers due to dietary inclusion of sodium
bicarbonate in their diets. However, layers of group C, which were offered, diet containing
1% sodium bicarbonate, apparently but non significantly showed maximum red blood cells
count among the birds of all treatment groups.
4.5.6 White blood cell count (WBCs)
Mean values for white blood cells count were 26.5, 22.5, 24, 25 and 26.2 ×103/mm3
for treatment A, B, C, D and E, respectively. White blood cells count was affected due to
dietary inclusion of sodium bicarbonate. Statistical analysis of the data showed that birds
using diets containing sodium bicarbonate had significant (P<0.05) effect on white blood
cells WBCs) count as compared to those of group A. Birds of group B, which were fed diet
containing 0.5% sodium bicarbonate, exhibited minimum white blood cells (WBC) count
when compared to the birds of other treated groups. However, the difference in the result of
white blood cells count among groups A, C, D and E were insignificant. Similarly the
differences in white blood cells count among groups B, C and D were also found to be
insignificant.
4.6 Serum metabolitesMeans and their standard deviation values regarding serum urea, serum uric acid, serum
creatinine and serum alkaline phosphatase levels are presented in table 4.6
106
4.6.1 Serum urea
Mean values relevant to serum urea level were 12.33, 8.96, 8.58, 9.57 and 11.35
mg/dl for treatment A, B, C, D and E, respectively. Serum urea level values of layers were
significantly affected due to dietary inclusion of sodium bicarbonate in the diets. Statistical
analysis of the data showed that birds using diets containing NaHCO3 had less (P<0.05)
serum urea level as compared to those of control group. Variations in serum urea level
among treated group were also found to be noteworthy (P<0.05). Birds of group C, which
were fed diet containing 1% sodium bicarbonate, showed minimum serum urea level when
compared to the birds of other treated groups. However, the differences regarding the serum
urea level among groups B, C and D were found to be insignificant. Similarly the disparity in
serum urea level among groups A and E were found to be insignificant (P>0.05).
4.6.2 Serum uric acid
Mean values regarding serum uric acid level were found to be 5.9, 6.52, 6.62, 7.6 and
7.8mg/dl for treatment A, B, C, D and E, respectively. Serum uric acid values of layers were
significantly affected due to dietary addition/inclusion of NaHCO3 in the diets. Statistical
analysis of the data showed that birds using diets containing sodium bicarbonate had
significantly (P<0.05) less serum uric acid level as compared to those of control group.
Difference in serum uric acid level among groups was also found to be noteworthy (P<0.05).
Birds of group E, which were fed diet containing 2% sodium bicarbonate, showed maximum
serum uric acid level when compared to the birds of other treated groups. However, the
differences in serum uric acid level among groups A, B and C were found to be non-
significant. Similarly the differences in serum uric acid level among groups D and E were
also found to be insignificant (P>0.05).
107
Table 4.6: Effect of dietary inclusion of sodium bicarbonate on serum metabolites of
caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Serum urea
mg/dl12.33±.718.96±1.31b 8.58±0.92b 9.57±1.02b 11.35±0.81a
Serum uric
acid
mg/dl
5.9±0.706.52±0.62b 6.62±0.45b 7.6±0.56a 7.8±0.78a
Serum
creatinine
mg/dl
0.77±0.050.76±0.05 0.69±0.09 0.72±0.08 0.75±0.04
Alkaline
Phosphatase
mg/dl
14.0±0.66 13.62±0.47 13.12±0.62 13.17±0.88 13.77±0.26
Mean values within the same row with dissimilar superscripts are significantly different (P<0.05)
108
4.6.3 Serum creatinine
Mean values pertaining to creatinine concentration in blood of the layers were found
to be 0.77, 0.76, 0.69, 0.72 and 0.75mg/dl for treatment A, B, C, D and E, respectively.
Statistical analysis of the data showed insignificant (P>0.05) difference among the birds of
all treatment groups, indicating no effect on creatinine concentration of the layers due to
dietary inclusion of sodium bicarbonate in their diets. However, birds of group C, which
were fed diet containing 1% sodium bicarbonate, apparently but non significantly showed
minimum creatinine concentration among the birds of all treatment groups.
4.6.4 Serum alkaline phosphatase
Mean values pertaining to serum alkaline phosphatase level in blood of the layers
were found to be 14.0, 13.62, 13.12, 12.77 and 14.77mg/dl for treatment A, B, C, D and E,
respectively. Statistical analysis of the data revealed non-significant difference among the
birds of all treatment groups, indicating no effect on alkaline phosphatase level of the layers
due to dietary inclusion of sodium bicarbonate in their diets. However, birds of group C,
which were fed diet containing 1% sodium bicarbonate, apparently but non significantly
showed minimum alkaline phosphatase level among the birds of all treatment groups.
4.7 Serum proteins analysisMeans and their standard deviation values regarding serum total proteins, serum
albumen and serum globulin concentration are presented in table 4.7.
4.7.1 Total proteins
Mean values regarding total protein were found to be 4.97, 6.13, 6.53, 6.02 and
5.05mg/dl for treatment A, B, C, D and E, respectively. Total protein concentration was
affected due to dietary addition/inclusion of sodium bicarbonate in the layer diets. Statistical
analysis of the data showed that birds using diets containing sodium bicarbonate had
significant (P<0.05) effect on total protein as compared to those of control group. Birds of
group C, which were fed diet containing 1% sodium bicarbonate, showed apparently
maximum total protein when compared to the birds of other treated groups. However, the
differences in
109
Table 4.7 Effect of dietary inclusion of sodium bicarbonate on serum proteins
concentration of caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Total protein
(mg/dl) 4.97±0.59 6.13±0.65a 6.53±0.58a 6.02±0.50ab 5.05±.49b
Albumin
(mg/dl)3.35±0.26 3.82±.26ab 3.9±0.53a 3.5±0.15ab
3.41±0.2
9ab
Globulin
(mg/dl)1.62±0.18 2.31±0.32 2.63±0.62 2.52±0.15 1.63±0.13
Values within the same row which have unlike superscripts are different significantly (P<0.05)
110
serum total protein level among the groups B, C and D were found to be insignificant
(P>0.05). Similarly the differences in serum total proteins among treated groups A and E
were also found to be insignificant P>0.05).
4.7.2 Albumin
Mean values pertaining to serum albumin concentration were found to be 3.35, 3.82,
3.9, 3.5 and 3.41 mg/dl for treatment A, B, C, D and E, respectively. Albumin concentration
was affected due to dietary addition/inclusion of sodium bicarbonate in the layer diets.
Statistical analysis of the data showed that birds using diets containing sodium bicarbonate
had significant effect (P<0.05) on albumin as compared to those of group A.
Birds of group C, which were provided diet containing 1% sodium bicarbonate,
showed apparently maximum serum albumin concentration when compared to the birds of
other treated groups. However, the differences in serum albumin protein level among groups
B, C, D and E were found to be non-significant.
4.7.3 Globulin
Mean values pertaining to globulin concentration in blood of the layers were found to
be 1.62, 2.31, 2.63, 2.52 and 1.63 mg/dl for treatment A, B, C, D and E, respectively. Data
obtained when statistically analyzed revealed non-significant difference among the birds of al
groups, indicating no effect on globulin concentration of the layers due to dietary
addition/inclusion of sodium bicarbonate in their diets. However, the birds of group C, which
were provided diet containing 1% sodium bicarbonate, apparently but non significantly
showed maximum globulin concentration among the birds of all treatment group.
4.8 Plasma electrolytes, minerals and serum pHMeans and their standard deviation values of plasma sodium, potassium, chloride,
bicarbonates, calcium, phosphorus and serum pH are given in table 4.8.
4.8.1 Plasma sodium
Mean values for plasma sodium were 132.7, 141.3, 149.6, 155.2 and 154.7mMol/L
for treatment A, B, C, D and E, respectively. Plasma sodium level was affected due to dietary
inclusion of NaHCO3 in the layer diets. Birds receiving diets containing sodium bicarbonate
exhibited significant (P<0.05) effect on plasma sodium concentration than those of group A
(control group). Birds of group E, which were fed diet containing 2% sodium bicarbonate,
111
Table 4.8: Effect of dietary inclusion of sodium bicarbonate supplementation on
plasma electrolytes and serum pH of caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Plasma Sodium
(mMol/L)
132.7±9.3
5d 141.3±8.70149.6±11.6
2b 155.2±9.10154.7±8.6
9a
Plasma potassium
(mMol/L)3.92±0.314.97±0.20a 4.87±0.22a 4.86±0.54a 4.1±0.26b
Plasma chloride
(mMol/L)
13
6.46±1.84a
118.21±5.9
5b
102.11±3.8
4c 94.63±3.26d77.46±3.0
7e
Plasma HCO3
(mMol/L)
2
1.86±1.30d25.83±2.13c
27.58±
1.50b 27.81±1.54b28.9
3±1.43a
Plasma Calcium
(mg/dl)
10.7±0.51 11.10±0.64 11.30±0.35 11.00±0.45 10.70±0.39
Plasma
Phosphorus
(mg/dl)
7.90±0.43 8.10±0.47 7.80±0.35 7.50±0.76 7.40±0.17
Serum pH 7.65±0.247.42±0.15ab 7.38±0.22ab 7.22±0.19b 7.32±0.2
5b
Values within the same row with different superscripts are different significantly (P<0.05)
112
showed maximum plasma sodium concentration when compared to the birds of other treated
groups except those of group D, where the differences in plasma sodium level were found to
be non-significant.
4.8.2 Plasma Potassium
Mean values for plasma potassium were 3.92, 4.97, 4.87, 4.86, and 4.1mMol/L for
treatment A, B, C, D and E, respectively. Plasma potassium was affected (P<0.05) due to
dietary inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data
showed that birds using diets containing sodium bicarbonate had significant (P<0.05) effect
on plasma potassium as compared to those of group A (control group). Birds of group B,
which were fed diet having 0.5% sodium bicarbonate, showed maximum plasma potassium
when compared to the birds of other treated groups. However, the differences in plasma
potassium protein level among groups B, C and D were found to be non-significant.
Similarly the differences in plasma potassium among treated groups A and E were also found
to be insignificant (P>0.05). A probable explanation of increased plasma potassium level in
treated group may be that treated groups were fed diets containing varying levels of
NaHCO3, therefore as the levels of sodium were increased in diets, it increased sodium in
plasma of birds and decreased plasma chloride ions, therefore level of potassium was
increased in plasma of treated groups.
4.8.3 Plasma chloride
Mean values pertaining to plasma chlorides were 136.46, 118.21, 102.11, 94.63 and
77.46mMol/L for treatment A, B, C, D and E, respectively. Plasma chlorides concentration
was affected due to dietary inclusion of sodium bicarbonate in the layer diets. Statistical
analysis of the data showed that birds using diets containing sodium bicarbonate had
significant (P<0.05) effect on plasma chlorides as compared to those of group A (control).
Birds of group E, which were fed diet containing 2% sodium bicarbonate, showed apparently
maximum plasma chlorides concentration when compared to the birds of other treated
groups.
4.8.4 Plasma bicarbonate
Mean values of plasma bicarbonate were 21.86, 25.83, 27.58, 27.81 and
28.93mMol/L for treatment A, B, C, D and E, respectively. Plasma bicarbonate was affected
due to inclusion of sodium bicarbonate in the diets of layers. Statistical analysis of the data
113
showed that birds using diets containing sodium bicarbonate exhibited significant (P<0.05)
effect on plasma bicarbonate compared to those of group A. Birds of group E, which were
fed diet containing 2% sodium bicarbonate, showed apparently maximum plasma
bicarbonate concentration when compared to the birds of other treated groups. However, the
differences in plasma bicarbonate level among groups C and D were found to be non-
significant.
4.8.5 Plasma calcium
Mean values regarding plasma calcium concentration of the layers were found to be
10.7, 11.1, 11.3, 11 and 10.6mg/dl for treatment A, B, C, D and E, respectively. The data
obtained when analyzed statistically showed non-significant difference among the birds of all
treatment groups, indicating no effect on plasma calcium concentration of the layers due to
dietary inclusion of NaHCO3 in their diets. However, birds of group C, which were provided,
diet containing 1% sodium bicarbonate, apparently but non significantly showed maximum
plasma calcium concentration among the birds of all treatment groups.
4.8.6 Plasma phosphorus
Mean values pertaining to plasma phosphorus concentration in blood of the layers
were found to be 7.9, 8.1, 7.8, 7.5 and 7.4mg/dl for treatment A, B, C, D and E, respectively.
Data obtained when statistically analyzed revealed non-significant difference among the
birds of all treatment groups, indicating no effect on plasma phosphorus concentration of the
layers due to dietary inclusion of NaHCO3 in their diets. However, birds of group B, which
were fed diet having 0.5% sodium bicarbonate, apparently but non significantly showed
maximum plasma phosphorus concentration among the birds of all treatment groups.
4.8.7 Serum pH
Mean values of serum pH were found to be 7.65, 7.42, 7.38, 7.22 and 7.32 for
treatment A, B, C, D and E, respectively. Serum pH was affected due to inclusion of sodium
bicarbonate in the layer diets. Statistical analysis of the data showed that birds using diets
containing sodium bicarbonate exhibited significant (P<0.05) effect on serum pH compared
to those of group A. Birds of group A (control) showed maximum serum pH when compared
to the birds of treated groups. However, the differences in serum pH level among treated
groups B, C, D and E were found to be non-significant.
114
4.9 Serum lipids profile
Means and their standard deviation values, of serum cholesterol, serum high density
lipoprotein (HDL) and low density lipoprotein (LDL) are given in table 4.9.
4.9.1 Serum cholesterol
Mean values pertaining to serum cholesterol concentration of the layers were found to
be 161.25, 149.50, 141.63, 158.1 and 163.13mg/dl for treatment A, B, C, D and E,
respectively. Serum cholesterol was affected due to inclusion of sodium bicarbonate in the
layer diets. Statistical analysis of the data showed that birds using diets containing sodium
bicarbonate exerted significant (P<0.05) result on serum cholesterol concentration when
compared to those of group A. Birds of group C showed the lowest serum cholesterol when
compared to the birds of other groups. However, differences in serum cholesterol level
among groups A, B, D and E were noted as non-significant (P>0.05). Similarly the
differences in serum cholesterol between treated groups B and C were also found to be non-
significant.
4.9.2 Serum triglyceride
Mean values regarding serum triglyceride concentration of the layers were found to
be296,226, 163, 203 and 185mg/dl for treatment A, B, C, D and E, respectively. Serum
triglyceride concentration was markedly affected due to inclusion of sodium bicarbonate in the
layer diets. Statistical analysis of the data showed that birds using diets containing sodium
bicarbonate exhibited significant effect (P<0.05) on serum triglyceride as compared to those of
control group. Birds of group C showed the lowest serum triglyceride when compared to the
birds of other groups. However, the differences in serum triglyceride level among groups B, D
and E were found to be insignificant (P>0.05). Similarly the differences in serum triglyceride
among treated groups C, D and E were also found to be non-significant.
4.9.3 Serum high density lipoprotein
Mean values pertaining to serum high density lipoprotein concentration of the layers
were found to be 121, 130, 138, 141 and 154mg/dl for treatment A, B, C, D and E,
respectively. Serum high density lipoprotein concentration was significantly affected due to
inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data revealed
that birds using diets containing sodium bicarbonate exhibited significant effect (P<0.05) on
the serum high density lipoprotein as compared to those of control group. Birds of
115
Table 4.9: Effect of dietary inclusion of sodium bicarbonate on serum lipids profile
of caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Serum
cholesterol
(mg/dl)
161.25±9.2
0a
149.50
±2.71ab
141.63
±5.62b
158.1
±9.20a
163.13
±6.73a
Serum
triglyceride
(mg/dl)
296±27.0 a 226±19.0 b 163±8.0 c 203±20.0 bc 185±18.0 bc
Serum HDL
(mg/dl)121±9.9 b 130±10.3ab 138±13.7a 141±4.5a 154±9.1a
Serum LDL
(mg/dl)62.6±6.4228.2±2.29b 28.6±2.34b 27.1±2.0b 31.3±1.43b
Values within the same row having different superscripts are differed significantly (P<0.05)
116
control group showed the lowest serum high density lipoprotein concentration when
compared to the birds of treated groups. Birds of group E, which were provided diet
containing 2% sodium bicarbonate, showed highest serum high density lipoprotein
concentration when compared to the birds of other treated groups. However, the differences
in Serum high density lipoprotein level among all treated groups were found to be non-
significant.
4.9.4 Serum low density lipoprotein
Mean values of serum low density lipoprotein (LDL) concentration of the layers were
found to be 61.9, 28.3, 28.1, 27.0 and 31.3mg/dl for treatment A, B, C, D and E, respectively.
Serum low density lipoprotein was affected due to inclusion of sodium bicarbonate in the
layer diets. Statistical analysis of the data showed that diets containing sodium bicarbonate
had significant (P<0.05) effect on serum low density lipoprotein. Birds of control group
showed highest serum low density lipoprotein concentration when compared to the birds of
treated groups. Birds of group D, which were fed diet containing 1.5% sodium bicarbonate,
showed highest serum low density lipoprotein concentration when compared to the birds of
other treated groups. The differences in Serum low density lipoprotein level among all
groups were found to be significant.
4.10 Hormones and enzymesMeans and their standard deviation values of serum tri-iodothyronine (T3), thyroxin
(T4), cortisol, estrogen progesterone, serum glutamic-oxaloacetic transaminase (SGOT) and
glutamic pyruvic transaminase (SGPT) are presented in table 4.10.
4.10.1 Tri-iodothyronine (T3) and Thyroxin (T4)
Mean values regarding serum T3 concentration of the layers for treatment groups A,
B, C, D and E, were found to be 2.87, 3.07, 3.27, 3.25 and 2.99 ng/ml, respectively. Findings
of the study depicted that serum T3 concentration of the birds was significantly influenced
due to dietary inclusion of sodium bicarbonate in their diets. The data obtained when
analyzed statistically showed that birds using diets containing sodium bicarbonate exhibited
higher serum T3 concentration (P<0.05) when compared to those of control group. The
differences in serum T3 concentration among the birds of treated group were also found to be
significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,
exhibited maximum serum T3 concentration when compared to those of other treated groups.
117
Table 4.10: Effect of dietary inclusion of sodium bicarbonate on serum hormones and
liver enzymes of caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
T3 (ng/ml) 2.87±0.163.07±0.15ab 3.27±0.20a 3.25±0.12a 2.99±0.14b
T4 (ng/ml) 1.79±0.06 1.90±0.01c 2.08±0.03a 1.79±0.03c 1.93±0.03b
Cortisol
(ng/ml)
7
1.25±2.22a 69.25±2.75ab 65.77±2.03b 66.50±1.91b 70.19±2.84a
Estrogen
(pg/ml)
143.3±8.00
b 168.6.±5.34a 171.3±3.42 168.3±4.82a 166.8±9.80a
Progesterone
(ng/ml) 0.89±0.101.08±0.14a 1.18±0.05a 1.16±0.03a 1.16±.02a
SGOT (U/L)11
5.50±5.65a 101.75±8.80b 93.88±7.31c 99.00±7.03bc98.13±6.9
5bc
SGPT (U/L) 71.70±2.4067.60±2.56 65.70±3.10 69.50±6.80 63.80±5.27
Means within the same row having different superscripts are differed significantly (P<0.05)
118
However, differences in T3 among treated groups B, C, and D were noted to be non-
significant. Similarly, differences in serum T3 concentration among treated groups A, B and
E, were also found to be insignificant (P>0.05).
Mean values of serum T4 concentration of the layers for treatment groups A, B, C, D
and E, were found to be 1.788, 1.898,2.075, 1.79 and 1.925ng/ml, respectively. Findings of
the study depicted that serum T4 concentration of the birds was significantly influenced due
to dietary inclusion of sodium bicarbonate in their diets. Statistical analysis of the data
revealed that birds using diets containing sodium bicarbonate exhibited significantly
(P<0.05) higher serum T4 concentration when compared to those of control group.
Differences in serum T4 concentration among the birds of treated group were also found to be
significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,
showed maximum serum T4 concentration when compared to the birds of other treated
groups. However, the differences in serum T4 concentration among treated groups B, C and
D, were found to be non-significant. Similarly the differences in serum T4 concentration
among treated groups A, B and E, were also found to be non-significant.
4.10.2 OestrogenMean values regarding serum estrogen concentration of the layers for treatment
groups A, B, C, D and E, were found to be 143.3,168.6, 171.3, 168.3 and 166.8 pg/ml,
respectively. Findings of the study depicted that serum estrogen concentration of the birds
was significantly influenced due to dietary inclusion of sodium bicarbonate in their diets. The
data obtained when analyzed statistically showed that birds using diets containing sodium
bicarbonate exhibited significantly higher (P<0.05) serum estrogen concentration when
compared to those of control group. Differences in serum estrogen concentration among the
birds of treated group were also found to be significant. Birds of group C, which were fed
diet containing 1% sodium bicarbonate, showed maximum serum estrogen concentration
when compared to the birds of other treated groups. However, the differences in serum
estrogen concentration among treated groups B, C, D and E, were noted to be non-
significant.
4.10.3 ProgesteroneMean values of serum progesterone concentration of the layers for treatment group A,
group B, group C, group D and group E, were noted to be 0.89, 1.08, 1.18, 1.16 and
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1.16pg/ml, respectively. Findings of the study depicted that serum progesterone
concentration of the birds was significantly influenced due to addition/inclusion of NaHCO3
in their diets. (P<0.05) that birds using diets containing sodium bicarbonate exhibited
significantly higher serum progesterone concentration (P<0.05) when compared to those of c
group A. However, differences in serum progesterone concentration among the birds of
treated group (B, C, D and E) were found to be non-significant. Birds of group C, which
were fed diet containing 1% sodium bicarbonate, showed maximum serum progesterone
concentration when compared to the birds of control group.
4.10.4 CortisolMean values pertaining to serum cortisol concentration of the layers for treatment
groups A, B, C, D and E, were found to be 71.25, 69.25, 65.77, 66.50 and 70.19ng/ml,
respectively. Findings of the study depicted that serum cortisol concentration of the birds was
significantly influenced due to dietary inclusion of sodium bicarbonate in their diets. The
data obtained when analyzed statistically showed that birds using diets containing sodium
bicarbonate exhibited significantly (P<0.05) lower serum cortisol concentration when
compared to those of control group. Differences in serum cortisol concentration among the
birds of treated group were also found to be significant. Birds of group C, which were fed
diet containing 1% sodium bicarbonate, showed minimum serum cortisol concentration when
compared to the birds of other treated groups. However, differences in serum cortisol
concentration among treated group B, group C and group D were found to be non significant.
Similarly the differences in serum cortisol concentration among treated groups A, B and E
were also found to be non significant.
4.10.5 Serum Glutamic-Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT)Mean values pertaining to SGOT concentration of the layers for treatment groups A,
B, C, D and E, were found to be 115.5, 101.75, 93.88, 99.0and 98.13ng/ml, respectively.
Findings of the study depicted that serum SGOT concentration of the birds was significantly
influenced due to dietary inclusion of sodium bicarbonate in their diets. The data obtained,
when analyzed statistically, showed that birds using diets containing sodium bicarbonate
exhibited significantly (P<0.05) lower serum SGOT concentration when compared to those
of control group.
Differences in serum SGOT concentration among the birds of treated group were also
found to be significant. Birds of group C, which were fed diet containing 1% sodium
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bicarbonate, showed minimum serum SGOT concentration when compared to the birds of
other treated groups. However, differences in serum SGOT concentration among treated
groups C, D and E, were non significant. Similarly, differences in serum SGOT
concentration among treated groups B, D and E, were also found to be non-significant
Mean values of SGPT concentration of the layers for treatment groups A, B, C, D and
E, were found to be 71.7, 7, 67.6, 65.7, 69.5 and 63.8U/L, respectively. Findings of the study
depicted that serum SGPT concentration of the birds was not influenced due to inclusion of
sodium bicarbonate in their diets. The data obtained when analyzed statistically showed that
birds using diets containing sodium bicarbonate exhibited non-significantly (P>0.05) lower
serum SGPT concentration when compared to those of control group.
4.11 Immune responseMean values of antibody titer against Newcastle disease virus (NDV) 10 days post
vaccination during 1st, 2nd and 3rd month are presented in table 4.11.
Mean values pertaining to antibody titer against NDV of the layers for treatment groups A,
B, C, D and E, were found to be 43, 86, 167, 145 and 118, respectively. Findings of the study
depicted that serum antibody titer against Newcastle disease virus of the birds was significantly
influenced due to dietary addition/inclusion of sodium bicarbonate in their diets. Statistical
analysis of the data revealed that birds using diets containing sodium bicarbonate exhibited
significantly (P<0.05) higher serum antibody titer against NDV when compared to those of
control group. Differences in serum antibody titer against NDV among the birds of treated
groups were also found to be significant. Birds of group C, which were fed diet containing
1% sodium bicarbonate, showed maximum serum antibody titer against NDV when
compared to the birds of other treated groups. However, the differences in serum antibody
titer against Newcastle disease virus between treated groups B and E, were found to be non-
significant. Similarly, differences in serum antibody titer against Newcastle disease virus
between treated groups C and D were also found to be non-significant.Mean values regarding antibody titer against Newcastle disease virus,10 days post 2 nd
vaccination, of the layers for treatment groups A, B, C, D and E, were found to be 135, 154,
237, 263 and 205, respectively. Findings of the study depicted that serum antibody titer
against NDV of the birds was significantly influenced due to dietary inclusion of sodium
bicarbonate in their diets. Statistical analysis of the data revealed that birds using diets
containing sodium bicarbonate exhibited significantly (P<0.05) higher serum antibody titer
against NDV when compared to those of control group.
121
122
Table 4.11: Effect of dietary inclusion of sodium bicarbonate on immune response of
caged layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
1st
Vaccination43 d 86 cd 167a 145 ab 118 bc
2nd
Vaccination135 b 154 b 237 a 263 a 205 ab
3rd
Vaccination208 b 272 ab 368 a 288 ab 272 ab
Mean values within the same row with unlike superscripts are significantly different (P<0.05)
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Differences in serum antibody titer against NDV among the birds of treated groups
were found to be significant. Birds of group D, which were fed diet containing 1.5% sodium
bicarbonate, showed maximum serum antibody titer against NDV when compared to the
birds of other treated groups. However, the differences in serum antibody titer against NDV
between treated groups B and E were found to be non-significant. Similarly, differences in
serum antibody titer against NDV between treated groups C and D, were also found to be
non-significant.
Mean values relevant to antibody titer against Newcastle disease virus, 10 days post
3rd vaccination, of the layers for treatment groups A, B, C, D and E, were found to be 208,
272, 368, 288 and 272, respectively. Findings of the study depicted that serum antibody titer
of the birds against NDV was significantly influenced due to dietary inclusion of sodium
bicarbonate in their diets. Statistical analysis of the data revealed that birds using diets
containing sodium bicarbonate exhibited significantly (P<0.05) higher serum antibody titer
against NDV when compared to those of control group. Differences in serum antibody titer
against NDV among the birds of treated groups were also found to be significant.
Birds of group C, which were fed diet containing 1% sodium bicarbonate, showed
maximum serum antibody titer against NDV when compared to the birds of other treated groups.
However, the differences in serum antibody titer against NDV among treated groups were
also found to be non-significant.
4.12 Economic AppraisalEconomics of production of the caged layers calculated on the current values of
various commodities has been described in table 4.12. The table showed that net profit
obtained from the layers of groups A, B, C, D and E, was found to be Rs: 40.64, 60.92,
104.57, 77.12 and 73.16, respectively. These findings revealed that profit margin obtained
from the experimental birds was found to be influenced due to dietary inclusion of sodium
bicarbonate in their diets. The birds of group C, which were fed diet containing 1% sodium
bicarbonate, exhibited maximum profit (Rs. 104.57) followed by those of group D (1.5%
NaHCO3) and E (2% NaHCO3).Better profit margin, from the layers of group C was because
of their higher egg production and efficient utilization of feed containing sodium bicarbonate
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Table: 4.12 Economics of production of the layers fed different levels of sodium
bicarbonate, calculated for 12 weeks of production (26th -37th week of age)
Variables
TreatmentA
Control diet
B0.5%NaHC
O3
C1%NaHC
O3
D1.5%NaHC
O3
E2%NaHC
O3Cost of pullets 400 400 400 400 400Feed consumed
(Kgs)/bird 8.91 9.24 9.36 9.19 9.06
Feed cost @36Rs/kg 320.76 332.64 336.99 330.84 326.16
Housing (equipped)@Rs
2/month6.00 6.00 6.00 6.00 6.00
Labor charges@Rs
2/month6.00 6.00 6.00 6.00 6.00
Vaccination and medication Rs3/month
9.00 9.00 9.00 9.00 9.00
Miscellaneous expenses (Rs) 5.00 5.00 5.00 5.00 5.00
Total expenses 746.76 758.64 762.99 756.84 752.16Sales of table eggs @ Rs.
8/egg482.4 514.56 562.56 528.96 520.32
Sales of culled birds 300 300 300 300 300
Sales of dropping as
manure5 5 5 5 5
Total cash received 787.4 819.56 867.56 833.96 825.32
Net profit 40.64 60.92 104.57 77.12 73.16
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as compared to those of other experimental groups. Moreover, reduction in body temperature
due to the use of sodium bicarbonate might have provided comfortable physiological
conditions to the layers, suitable for efficient egg production as compared to those of control
groups.
However, minimum profit (Rs 40.64/hen) was obtained from the layers of control
group. Lower return from control birds may probably be due to their lower production
performance because of high ambient temperature. Anjum (2000) and Ahmad et al. (1993)
while justifying their findings have also attributed lower egg production of layers to high
ambient temperature. They concluded that high ambient temperature was a major
contributing factor affecting the economics of poultry production.
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DISCUSSIONNumerous acid-base imbalances have been studied in broilers kept under heat-
stressed condition, which have resulted in the incidence of respiratory alkalosis in the birds
(Dibartola, 1992; Carlson, 1997; Ahmad et al., 2005). However, dietary supplementation of
certain minerals such as sodium bicarbonate appears to be helpful in alleviating the effect of
heat stress in broilers (Ahmad et al., 2008) by improving their feed consumption and water
intake.
4.13 Performance4.13.1 Live body weight
Body weight gain of the birds consuming diets containing sodium bicarbonate was
found to be significantly affected due to the inclusion of NaHCO3 in their diets. The birds
receiving diets containing sodium bicarbonate exhibited higher weight gain as compared to
those of untreated group (control). A probable explanation of increased live body weight of
the layers in treated groups may be the higher feed intake of the birds receiving sodium
bicarbonate. Similar results have been observed by Balnave and Gorman (1993) who
reported improved weight gain because of inclusion of NaHCO3 in the diets of birds. Genedi
(2000) also reported that adding anti-stressors like NaHCO3 in to drinking water offered to
Leghorn and Matrouh hens increased their weight gain under heat stress condition. Another
possible explanation of these results may be the response of inclusion of NaHCO3, which
depends upon existence or absence of factors influencing acid-base balance of the birds.
Presence of metabolizable anions (Na+) in poultry diets has shown to exhibit a significant
improvement in the body weight gain of broilers (Ruiz-Lopez and Austic, 1993).
Opposing to the results of the present study, Junqueira et al. (2003) and Osman et al.
(2015), found no effect due to dietary addition of different levels of sodium bicarbonate on
weight gain of poultry birds. Comparable, results are also reported by Hayat et al. (1999) and
Wideman et al. (2003), where inclusion of NaHCO3 in the diets of birds did not exhibit any
significant increase in body weight gain of birds. Saedi and Khajali (2010) found that body
weight of broilers remained unaffected because of dietary addition of sodium bicarbonate.
Moreover, Peng et al. (2013) found a significant decrease in final body weight in
broilers fed NaHCO3 added diets under summer conditions of relatively high temperature.
Similarly, a decrease in body weight of the birds due to the addition of sodium bicarbonate in
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their diets has been reported by Squires and Julian, (2001) who observed that addition of
NaHCO3 (0.42%) reduced body weight gain in broilers. Wideman et al. (2003) have also
observed 7% reduction in final body weight of broilers fed a diet containing of 1% NaHCO3.
Differences among the results of different experiments might have been due either to the
difference in genetic makeup (Kassim and Brence, 2001) of the birds or difference in the
levels of sodium bicarbonate used in these studies.
4.13.2 Feed consumption
Birds receiving diets containing sodium bicarbonate exhibited more feed
consumption as compared to those of untreated group (control). Increase in feed consumption
of the treated groups may be due to more sodium ions concentration in the rations containing
sodium bicarbonate (Puron et al., 1997; Mc Dowell, 1992). Similar effect of increased
sodium ions concentration in broilers has been observed by Fethiere et al. (994) who
concluded that feed intake is reliant upon Na+ level in the ration and high sodium level could
cause more feed consumption. The results of presents study supports the findings of Ahmad
et al. (2006), and Balnave and Gorman (1993) who observed a significant (P<0.05) increase
in feed consumption in broilers fed diet added with sodium bicarbonate as compared to
control group. Puron et al. (1997) also observed a significant improvement in feed intake by
adding sodium bicarbonate (0.5%) in the diets of poultry birds.
Feed intake of the birds was also found to be significantly affected, among the birds
of treated groups, due to different levels of sodium bicarbonate (0.5-2.0%) used in their diets.
These results are comparable to those reported by Gongruttananun and Ratana (2005) who
reported a significant increase in feed consumption of Thai native hens due to the addition of
different levels of sodium bicarbonate (1.0-1.5%) in their diets. Similarly, Yoruk et al. (2004)
examined the effect of different levels of inclusion of NaHCO3 (0.1%-.4%) in the diet on
feed consumption of layers during their late laying period and observed a marked
improvement in feed consumption of the birds.
However, Bonsembiante and Chiericato (1990) have observed a non significant
difference in feed intake (P<0.05) of birds receiving rations with or without supplemented
sodium bicarbonate. Parallel results are also reported by Senkoylu et al. (2005) who explored
the effect of inclusion of different levels of NaCl, NaHCO3 and K2CO3 in poultry diets, on
feed consumption of layers during peak production and did not observe any effect due to the
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dietary inclusion of these compounds on feed intake of the birds. Findings of Balnave and
Muheereza (1997) and Waldroup et al. (2005) have also depicted that feed intake of birds fed
diets with or without sodium bicarbonate remained unaffected. Contrary to the outcomes of
current study, Fuentes et al. (1997) found no effect of different levels of sodium bicarbonate
(0.6, 1.2, 1.8 and 2.4%) in diet on feed consumption in guinea fowl reared at high ambient
temperature.
Moreover, Khattak et al. (2012) observed some reduction in feed consumption of
birds fed diet supplemented with sodium bicarbonate during summer. The discrepancy in the
results of these research results may probably be due to the variations in experimental
conditions maintained, strain/species of birds or levels of sodium bicarbonate added in the
diets (Nayak et al., 2015).
4.13.3 Egg Production
Birds receiving diets containing sodium bicarbonate exhibited more egg production
when compared to those of untreated group (control). Increase in egg production of the
treated groups may be due to either more feed consumption (Dai et al., 2009) or increased
sodium ions concentration or both in the rations containing sodium bicarbonate (Puron et al.,
1997a; Mc Dowell, 1992). Observations of the research are in line with the results of Okan,
(1999) who reported an increase in egg production by supplementation of NaHCO3 in layer
diets. Similar effect has also been observed by Ghorbani and Fayazi (2009) who studied the
effect of inclusion of NaHCO3 in the feed of layers on egg production during chronic heat
stress and found considerable increase (P<0.05) in egg production due to dietary inclusion of
sodium bicarbonate. The results of this study are also compatible with the observations of
Hassan et al. (2011) who found that inclusion of sodium bicarbonate used at 0.25% and
0.50% in poultry diet showed a significant increase in egg production of layers during hot
weather. Improved egg production has also been observed by Balnave and Muheereza (1997)
because of inclusion of NaHCO3 (1%) in layers diet.
The difference in egg production of the birds was also found to be significant among
the birds of treated groups due to different levels of sodium bicarbonate (0.5-2.0%) used in
the diets. Similar results were also reported by Ghorbani and Fayazi (2009) who studied the
effect of addition of NaHCO3 and rearing system on production performance of layers kept
under chronic heat stress. They observed that dietary levels of sodium bicarbonate from
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0.5%-1.5% in laying hens diet improved their egg production. Similarly, Yoruk et al. (2004)
studied the effect of different levels of NaHCO3 (0.1%-0.4%) on egg production of layers.
Results showed that inclusion of different levels of sodium bicarbonate (0.1%-0.4%) in
laying hens diet improved their egg production.
Contrary to the results of present study, Gongruttananun and Ratana (2005) found
non-significant effect of adding varying levels of NaHCO3 on feed consumption in layers.
They fed diets supplemented with 1.0-1.5% sodium bicarbonate to Thai native hens and
observed that differences in egg production between treated and non-treated birds, even due
to different levels of sodium bicarbonate were non-significant. Similar results pertaining to
egg production are reported by Grizzle et al. (1992); Gongruttananun et al. (1999) and
Waldroup et al. (2005) due to the inclusion of sodium bicarbonate in layer diets. Egg
production has also been found unaffected due to the inclusion of different levels of NaCl,
NaHCO3 and K2CO3 in the diets of layers (Senkoylu et al., 2005), during peak production
period.
Moreover, significant reduction of egg production in control group due to heat stress
is also in accord with the studies of Melesse, (2011); Mashaly et al. (2004); Peguri and Coon,
(1991). Furthermore, heat stress not merely lessens feed consumption but is also reported to
decrease absorption of various components of the diet (Bonnet et al., 1997). However,
inclusion of sodium bicarbonate may result in increased absorption and availability of
nutrients and hence results in increased egg production.
4.13.4 Egg weight
Birds receiving diets containing sodium bicarbonate had heavier eggs as compared to
untreated group (control). Increase in egg weight of the treated groups may probably be due
to better utilization of digested proteins, amino acids, monosaccharide and energy due to
metabolic effect of sodium present in sodium bicarbonate containing rations (Murkami et al.,
2001). Another possible reason of better egg weight in treated groups may be higher feed
intake of the birds, as has been observed in the results of present study (see section 4.1.2).
The results of this study are in accordance with the findings of Balnave and Muheereza
(1997) who fed either basal diet or diets containing 1% sodium bicarbonate, 0.05% Zinc
methionine or 0.04% vitamin C, to layers kept under high ambient temperature and found
significantly higher weight of eggs produced by the birds fed diet supplemented with 1%
130
sodium bicarbonate. Similar effects of sodium bicarbonate on egg weight have also been
reported by Ghorbani and Fayazi (2009) in layers.
The difference in egg weight was also known to be considerable among the birds fed
diets containing different levels of sodium bicarbonate (0.5-2.0%). These results are in line
with the results of Yoruk et al. (2004) who observed the effect of different levels of sodium
bicarbonate (0.1%-0.4%) on egg weight of layers during late laying period and observed that
inclusion of different levels of sodium bicarbonate (0.1%-0.4%) in the feed of layers
improved their egg weight. Identical results have also been observed by Ghorbani and Fayazi
(2009) who studied the effect of different levels of sodium bicarbonate (0.5%-1.5%) and
rearing systems on egg weight of layers kept under chronic heat stress and found that dietary
levels of sodium bicarbonate (0.5%-1.5%) in laying hens diet improved their egg weight,
where hens fed diet supplemented with 1.5% sodium bicarbonate produced heavier eggs.
Contrary to the results of present study, Senkoylu et al. (2005) who tested the effect
of inclusion of different levels of NaCl, NaHCO3 and K2CO3 in poultry diets, on egg weight
in layers during peak production, and found no effect due to dietary inclusion of these
compounds on egg weight of layers. Akin results have also been observed by
Gongruttananun and Ratana (2005) who found non-significant effect of sodium bicarbonate
(1.0-1.5%) on egg weight of Thai native hens. Similarly, Waldroup et al. (2005) observed a
non-significant effect of adding sodium bicarbonate (1%) in the diet on mean egg weight of
layers. The discrepancy in the results of these studies might be due to varying environmental
conditions of the experiment, strain/species of birds used in these studies (Nayak et al., 2015)
or difference in the levels of sodium bicarbonate added in the experimental diets.
4.13.5 Egg mass
A significant reduction in egg mass/size of control group was observed due to
hyperthermia. High ambient temperature not merely reduces feeding activity but also has
reported to lessen absorption of various nutrients of the feed (Bonnet et al., 1997). Therefore,
it is quite possible that metabolic machinery of the birds may have been used for homeostasis
regulation rather than to be used for production (Carlson, 1997). However, inclusion of
sodium bicarbonate has been known to ameliorate hyperthermia effects due to beneficial
effect of sodium and bicarbonate ions, by increasing absorption of the nutrients present in the
diets along with their availability and hence resulting in the increase of egg mass.
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Birds receiving diets containing sodium bicarbonate exhibited significantly (P<0.05)
more egg mass than those of untreated group. Moreover, the birds receiving sodium
bicarbonate also exhibited higher feed intake and produced more eggs when compared to
those of control group (see table 4.2). Therefore, higher egg mass production of the eggs by
the layers getting treated ration may possibly be due to more feed consumption coupled with
its efficient utilization by these birds as has also been observed by Dai et al. (2009). Another
possible reason of increased egg mass of the treated groups may be the increase in sodium
ions concentration in the diets containing sodium bicarbonate (Kurtoglu et al., 2007). Yoruk
et al. (2004) also found beneficial effect of sodium bicarbonate on egg mass due to its
addition in the diets of turkey. Better feed utilization of layers receiving sodium bicarbonate
added diets and improved electrolyte balance in these diets might have created favorable
physiological conditions for an improvement in egg mass (Drinah et al., 1990). Results of the
present study are in accord with the findings of Balnave and Muheereza (1997) who fed
either basal diet or diets containing sodium bicarbonate (1%), 0.05% Zinc methionine or
0.04% vitamin C to layers kept under high ambient temperature and exhibited a significant
improvement in egg mass produced by the bird fed diet supplemented with 1% sodium
bicarbonate.
The difference in FCR/Kg egg mass produced by the layers was also found to be
significant among the birds of treated groups due to different levels of sodium bicarbonate
(0.5-2.0%) used in the diets. These results coincide with the findings of Ghorbani and Fayazi
(2009) who studied the effect of dietary addition of NaHCO3 and rearing system on the
performance of layers kept under chronic heat stress. They found that dietary levels of
sodium bicarbonate (0.5%-1.5%) in laying hens diet improved their egg weight/egg mass.
Findings of the current research are also in according with those observed by Yoruk et al.
(2004). They studied the effect of different levels of NaHCO3 (0.1%-.4%) on egg weight/egg
mass of layers during late laying period and observed that inclusion of different levels of
sodium bicarbonate in the diets of laying hens improved their egg weight/egg mass.
Contrary to the results of current study, Senkoylu et al. (2005) reported a non
significant effect due to the inclusion of different levels of NaHCO3 in the diets, on egg mass,
in layers during peak production. The discrepancy in results of these experiments may
probably be due to the differences in stage of production of the birds used in these studies.
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4.13.6 Feed efficiency
a) FCR/dozen eggs
Birds fed diets supplemented with sodium bicarbonate utilized their diets more
efficiently when compared to those of untreated group (control). A probable explanation of
better FCR/dozen eggs produced in the birds of treated groups may be more synthesis of
tissue proteins (Borges et al., 2003) as a result of higher feed consumption. Similar results
regarding efficiency of feed utilization have also been reported by Barton (1996) due to the
inclusion of NaHCO3 in turkey feeds. Another probable explanation of better efficiency of
feed utilization in the birds receiving NaHCO3 may be the improved electrolyte balance in
the diets, which might have created some favorable conditions for improvement in the feed
efficiency (Drinah et al., 1990). Better FCR may also be attributed to better digestion and
absorption of nutrients due to incorporation of sodium carbonate, which ultimately may have
resulted in improved egg production; a vital factor involved in the calculation of feed
efficiency.
Efficiency of feed utilization of the layers, calculated on the basis of per dozen eggs
produced was also found to be due affected due to the inclusion of different levels of sodium
bicarbonate (0.5-2.0%) in layer diets. A probable explanation of better utilization of feed
containing different levels of sodium bicarbonate may be either due to improved feed
consumption or improved egg production and/or both, by the birds of treated groups. These
results are in line with the results of Yoruk et al. (2004) who studied the effect of different
levels of sodium bicarbonate (0.1%-0.4%) on feed conversion ratio of layers during late
laying period. Their findings revealed that dietary inclusion of different levels of sodium
bicarbonate improved feed conversion ratio of the birds, calculated on the basis of one dozen
eggs produced.
b) FCR/Kg egg mass produced
The results regarding efficiency of feed utilization calculated on the basis of per kg
egg mass produced, revealed that the birds receiving diets containing sodium bicarbonate
utilized their feeds more efficiently when compared to those fed diet without addition of
sodium bicarbonate (control). Better FCR/kg eggs mass produced in the treated groups may
probably be due to their higher feed consumption, resulting in increased availability of
nutrients after fulfilling the maintenance requirements of the birds and ultimately leading to
133
more egg production and heavier eggs (see table 4.2). Improvement in efficiency of feed
utilization in birds receiving sodium bicarbonate may also be due to the improved electrolyte
balance, better digestion and absorption of nutrients, enzymatic reactions and synthesis of
tissue proteins in the diet by creating favorable conditions for an improvement in feed
efficiency (Drinah et al., 1990; Borges et al., 2003). The results of the present study are in
line with the findings of Keskin and Durgan (1997) who have reported an improved FCR in
quails fed diet supplemented with NaHCO3, KCl, CaCl2, NH4Cl and CaSO4.
The difference in FCR/Kg egg mass produced of the layers was also found to be
significant among the birds of treated groups due to different levels of sodium bicarbonate
(0.5-2.0%) used in the diets. These results are in line with the findings of Yoruk et al. (2004)
who studied the effect of different levels of sodium bicarbonate (0.1%-0.4%) on feed
conversion ratio of layers during late laying period. They reported that inclusion of different
levels of sodium bicarbonate in laying hens diet improved their feed conversion ratio.
Contrary to the results of present study, Senkoylu et al. (2005) reported non-
significant effect of inclusion of different levels of NaCl, NaHCO3 and K2CO3 in the diet on
FCR (g of feed/g of egg) in layers during peak production. Fuentes et al. (1998) have also
observed non-significant effect of adding different levels of sodium bicarbonate (0.6, 1.2,
1.8, and 2.4%) on FCR values calculated on the basis of per kg egg mass in guinea fowls
raised at high ambient temperatures. Contradictions in the findings of these studies may
probably because of difference in the species of poultry birds used in these studies.
4.14 Egg quality4.14.1 Specific gravity
Birds receiving diets containing sodium bicarbonate exhibited more specific gravity
value as compared to those of untreated group (control). Increase in specific gravity (SG) of
the eggs laid by the birds of treated groups may probably be due to better utilization of
calcium because of metabolic effect of sodium/bicarbonate ions, present in sodium
bicarbonate containing (Keskin and Durgan, 1997, Squire and Julian, 2001), and leading to
the production of thick shell eggs. On the other hand, shell thickness of an egg is closely
correlated with SG of a newly laid egg. That’s why SG measurements are used to find out
shell quality.
Findings of this study are in accordance with those stated by Yoruk et al. (2004) who
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studied the effect of different dietary levels of NaHCO3 (0.1%-0.4%) on specific gravity of
eggs in layers, during late laying period and found that inclusion of NaHCO3 in laying hens
diet increased specific gravity of their eggs. They also reported that higher levels of sodium
bicarbonate (0.2%-.4%) in the diet decreased the SG of eggs produced by birds. However,
Grizzle et al. (1992) did not notice any such effect on specific gravity of eggs because of
dietary addition of 1% NaHCO3 in layers. Similarly, Makled and Charles (1987) also found
no change in specific gravity of eggs in hens fed diet supplemented with 0.5% sodium
bicarbonate during peak production period.
4.14.2 Shell thickness
Shell thickness of eggs produced by the birds consuming diets containing sodium
bicarbonate was found to be significantly higher than those of untreated group (control).
Addition of sodium bicarbonate in the diets has been shown to increase calcium retention in
layers (Ferguson et al., 1974). Therefore, increase in egg shell thickness of the treated groups
may probably be due to more utilization of calcium because of some positive metabolic
effects of sodium and bicarbonate ions in sodium bicarbonate containing diets (Keskin and
Durgan, 1997; Squire and Julian, 2001). Better shell thickness (ST) of the eggs may also be
the result of relatively higher bicarbonate level, which ultimately reduced panting in the birds
using diets containing sodium bicarbonate than those of control group. Production of thick
shell eggs due to the dietary inclusion of sodium bicarbonate level also coincides with better
production performance of the birds, as has been observed in the present study.
El-Boushy and Raternick, (1993) observed that the birds exposed to high
environmental temperature produced eggs with poor ST, which is quite in agreement with the
findings of present study where birds fed diet without adding sodium bicarbonate produced
thin shelled eggs. Decrease in ST of eggs produced by the birds exposed to heat stress has
been related either to low feed intake (Balnave and Muheereza, 1997), which may lead to
ultimate reduction in calcium intake, a necessary element required for shell formation
(Karimian et al., 2004) or to decrease in calcium level in blood (Hassan et al., 2003).
However, shell thickness of eggs produced by the birds fed diet supplemented with 1%
sodium bicarbonate was significantly improved (Balnave and Muheereza, 1997). Therefore, a
probable explanation of production of thinner shelled eggs by the birds under the influence of
heat stress and fed diet without addition of sodium bicarbonate might be due to lower feed
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intake and bicarbonate level in their blood as compared to those fed diets containing different
levels of sodium bicarbonate.
The results observed in this study are in line with the findings of Hayat et al. (1999)
who observed a significant improvement in egg shell thickness in layers fed
NaHCO3supplementeddiet. Similar beneficial effects of including sodium bicarbonate in
poultry diets have also been reported on egg shell thickness by Davison and Wideman
(1992). Makled and Charles (1987) observed an improvement in egg shell thickness in hens
fed diets supplemented with 0.5% sodium bicarbonate. Gongruttananum and Ratna (2005)
also reported markedly higher shell thickness of eggs in birds fed diet supplemented with
sodium bicarbonate. Increasing NaHCO3 level in the diets also increased the DEB level of
these diets (see table 3.2), however, birds fed diet containing 1% sodium bicarbonate
(DEB=262) laid thicker shelled eggs when compared to other groups. Ghasemi et al. (2014)
have also reported that, under tropical conditions, using a DEB of 250mEq/Kg achieved a
correction of the lay-induced metabolic acidosis and resulted in thicker shelled eggs.
Therefore, increase in shell thickness of the eggs may be ascribed to the inclusion of sodium
bicarbonate in the diet, which might have led to an improvement in eggshell thickness.
In contrary to the results of the present study, Kaya et al. (2004) observed
insignificant improvement in shell thickness of the eggs produced by geese layers, offered
diet containing 0.5% NaHCO3. Yoruk et al. (2004) also observed non-significant effect of
adding different levels of sodium bicarbonate (0.1%, o.2%, 0.4%) on egg shell thickness of
laying hens. Contradiction in the results/outcomes of these researches may be due either to
varying level of dietary addition/inclusion of sodium bicarbonate or difference in the poultry
species used (layer vs geese) in these trials, or both.
4.14.3 Albumen height
Eggs produced by the birds receiving diets containing sodium bicarbonate exhibited
more albumen height as compared to those of untreated group (control). An increased
albumen height of the eggs produced by the birds of treated groups may probably be due to
higher feed consumption and better absorption of nutrients present in the experimental diets
containing sodium bicarbonate when compared to those of control group. Moreover, the birds
receiving sodium bicarbonate also exhibited higher serum albumen concentration than those
of control group (see table 4.7). Therefore, higher albumen height of the eggs produced by
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the birds consuming treated ration may possibly be due to more serum albumen
concentration. Inclusion of sodium bicarbonate in feed has also shown potential benefits on
egg characteristics (Balnave and Muheereza, 1997; Kaya et al., 2004) in poultry birds during
heat stress period. Similar results are quoted by Yoruk et al. (2004) who studied the effect of
different levels of NaHCO3 (0.1%-0.4%) on albumen index and Haugh unit score of layers
during late laying period.
The albumen height is essential constituents for calculation of Haugh units and is
known to be affected by the temperature during storage. The quality of albumen and yolk
deteriorates with the increasing storage time and temperature (North and Bell, 1990).
However, in the present study the eggs used were fresh. Therefore, probably albumen quality
of the eggs produced by the birds of control group deteriorated only because of high
environmental temperature. However, according to the findings of present study, dietary
supplementation of sodium bicarbonate improved albumen quality of the eggs produced by
the birds.
Contradictory results have been reported by Ghorbani and Fayazi (2009) and
Gongruttananum and Ratna (2005) who studied the effect of dietary dietary and system of
rearing on egg quality characteristics in layers kept under chronic heat stress. They reported
that dietary levels of sodium bicarbonate (0.5%-1.5%) in the diets of laying hens did not
show any improvement in the albumen quality of the eggs produced by these birds.
4.14.4 Haugh unit
Haugh unit score was found to be significantly better in the birds fed diets containing
sodium bicarbonate as compared to those fed diets without its addition. The difference in
Haugh unit score of the birds was also found to be significant among the birds of treated
groups due to different levels of sodium bicarbonate (0.5-2.0%) used in the diets. An
increased Haugh unit score of the treated groups may probably be due to higher feed
consumption and better absorption of nutrients of experimental diets when compared to those
of control group, as has been reflected in the findings of present study. Moreover, the birds
receiving sodium bicarbonate also exhibited higher serum proteins concentration than those
of control group (see table 4.7), which may have caused an increase in Haugh unit value of
eggs produced by these layers. These results are matched with those reported by Kaya et al.
(2004) who observed that inclusion of sodium bicarbonate in feed has shown potential
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benefits on egg characteristics in poultry birds during heat stress period.
Contradictory to the findings of present study, Ghorbani and Fayazi (2009) did not
observe any effect due to dietary inclusion of sodium bicarbonate and rearing system on
Haugh unit score of the eggs produced by the layers kept under chronic heat stress. Their
findings depicted that neither addition of sodium bicarbonate nor its various levels (0.5%-
1.5%) exhibited any effect on Haugh unit score in the layers. These type of findings have
also been quoted by Yoruk et al. (2004) who also did not examine any effect of dietary
inclusion of different levels of sodium bicarbonate (0.1%-0.4%) on Haugh unit score of the
eggs produced by layers, during their late laying period. Findings of Gongruttananum and
Ratna (2005) have also revealed that dietary levels (1%-1.5%) of sodium bicarbonate in diets
did not improve Haugh unit score of the egg laid by the hens. A probable explanation of
these contradictory findings may be the difference in ambient conditions in which the birds
were kept during these experiments.
4.14.5 Yolk diameter
Effect of dietary addition/inclusion of NaHCO3 on yolk diameter of the eggs
produced by the layers was found to be significant. The eggs produced by the birds, which
used diets containing sodium bicarbonate exhibited more yolk diameter as compared to the
controls. Increase in yolk diameter of the eggs of treated groups may probably be due to
higher feed ingestion and better absorption of nutrients present in the experimental diets, as
has been depicted in the results of this study. Inclusion of sodium bicarbonate in feed has
shown potential benefits on egg characteristics (Balnave and Muheereza, 1997; Kaya et al.,
2004) in poultry birds during heat stress period.
However, findings of Ghorbani and Fayazi (2009) did not showed any effect on yolk
diameter of the eggs produced by the hens due to dietary addition of NaHCO3 and rearing
system in which the birds were kept under chronic heat stress. Similarly, Yoruk et al. (2004)
who studied the effect of different levels of NaHCO3 (0.1%-0.4%) on yolk diameter of eggs
produced by layers during late laying period, did not find any improvement in yolk diameter
of the eggs . Findings of Gongruttananum and Ratna (2005) reported that yolk diameter of
eggs due to different dietary levels of sodium bicarbonate (1%-1.5%) in laying hens
remained unaffected. Probably these contradictory findings may be due to the difference in
experimental conditions maintained during these studies
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4.14.6 Yolk height
The eggs produced by the birds, which used diets containing sodium bicarbonate
exhibited more yolk height as compared to those fed untreated diet. Reasons of increase in
yolk height of the eggs produced by the birds in treated groups may probably be the same as
have been advocated in the previous section (yolk diameter). The results pertaining to egg
yolk height in this trial are in line with those observed by Yoruk et al. (2004). They studied
the effects of dietary supplementation of different levels of sodium bicarbonate (0.1%-0.4%)
on yolk quality of the eggs produced by the layers during their late laying period and found a
significant improvement in yolk quality of eggs because of these treatments. Addition of
NaHCO3 in feed has also shown potential benefits on various egg characteristics in poultry
birds during heat stress period (Davison and Wideman, 1992; Kaya et al., 2004)
Contrary to the results of this study, findings ofGongruttananum and Ratna, (2005)
and Ghorbani and Fayazi, (2009) have also revealed that yolk quality remained unaffected in
the eggs laid by the layers fed diets containing different levels of sodium bicarbonate (1-
1.5%). The reasons of these contradictory results may be the same as have been reported in
the previous section (4.15.4).
4.14.7Yolk Index
The difference in yolk index of eggs produced by the birds fed diets with or without
addition of sodium bicarbonate was found to be non-significant. These findings are in
accordance with those observed by Ghorbani and Fayazi (2009) who studied the effect of
dietary NaHCO3 and rearing system, on yolk index of layers kept under chronic heat stress.
The results of their study revealed that neither dietary inclusion of sodium bicarbonate nor its
levels (0.5%-1.5%) exhibited any effect on yolk index of the eggs produced by the hens.
Similarly, Gongruttananum and Ratna (2005) have also observed that dietary levels of
sodium bicarbonate (1%-1.5%) in laying hens diet did not improve their yolk quality.
However, contradictory results are reported by Yoruk et al. (2004) who studied the
effect of different levels of sodium bicarbonate (0.1%-0.4%) on yolk index of layers during
late laying period. They found significant improvement in yolk index of eggs by dietary
inclusion of sodium bicarbonate at 0.1% level. The differentiation in the results of these
experiments might be due to variation in the levels of sodium bicarbonate incorporated in the
diets used in these studies.
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4.14.8 Egg yolk cholesterol
Addition of NaHCO3 in the diets of layers reduced (P<0.05) the concentration of
cholesterol in their eggs as compared to those produced by the control birds. A probable
explanation of decrease in egg cholesterol may be that sodium bicarbonate might have
stimulated the production of bile acids, which utilized cholesterol for their synthesis
(Naviglio et al., 2011); hence ultimately resulting in decreased concentration of yolk
cholesterol in the birds of treated groups. Another possible reason for decrease in egg
cholesterol in layers offered sodium bicarbonate added diet may be the inhibition of enzyme
“squalene epoxidase” which is vital for the production of cholesterol (Angelovicova, 1997).
Reduction in egg cholesterol perhaps may reduce the chances of cardiovascular
diseases due to the use of chicken eggs, in human beings. Recent research has shown that
intake of eggs having low cholesterol, does not increase cholesterol in patients suffering from
cardiovascular diseases (Harman et al., 2008; Spence et al., 2010).However, reduction of
cholesterol in hatch able eggs received from breeder flocks is questionable because
cholesterol is vital for yolk formation and embryo growth (Griffin, 1992). Hence, strict
cholesterol fall in egg yolk can stop of egg production and increase in embryonic deaths.
4.14.9Yolk pH
Diets containing sodium bicarbonate when fed to the layers, exhibited significantly
higher yolk pH (P<0.05) value of the eggs produced by them than those fed control diet. A
probable explanation of significantly higher pH value in the yolk of the eggs produced by the
birds receiving diets containing sodium bicarbonate may be the increase in concentration of
bicarbonate ions in their guts, which might have given rise to yolk pH of eggs produced by
the birds. Addition of NaHCO3 in the diet of layers has also been reported to support
maintenance of venous blood pH (Kaya et al. (2004). Moreover, it has also been observed
that increase in blood bicarbonate ions concentration can compensate the ill effects of
chloride ions contributed by heat stress (Fethiere et al., 1994). Results of the present
experiment are compatible with the findings of Balnave and Gorman, (1993) who observed
increase in blood pH in birds due to increase in HC03- ions concentration in their diets.
4.14.10 Albumen pH
Dietary addition/inclusion of sodium bicarbonate revealed a significant increase in
albumen pH (P<0.05) of the eggs produced by them than those of untreated group (control).
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The findings are compatible to the results of Kaya et al. (2004) who reported that inclusion
of NaHCO3 in the diet of layers may support to maintain the blood pH of birds. Therefore,
increase in the albumen pH may probably be due to increase in blood bicarbonate ions
concentration of the birds. Addition of NaHCO3 in the diet of layers has been reported to
support the maintenance of venous blood pH (Kaya et al., 2004). Similar results have been
observed by Glahn et al., 1988; Squires and Julian, 2001, who found that dietary use of
NaHCO3 increased blood pH of birds. Increase in albumen pH also coincides with the
findings of Balnave and Gorman, (1993) who observed increase in blood pH in birds due to
increase in HC03- ions concentration in their diets.
4.14.11 Blood and meat spots
The blood and meat spots were observed only in the eggs produced by the birds fed
ration without dietary addition of sodium bicarbonate, while those of treated groups were
devoid of these. The exact mechanism of how dietary inclusion of different levels of sodium
bicarbonate prevented the occurrence of blood spots in the eggs produced by the birds of
treated groups is still not known. However, probably reduction in body temperature of the
layers receiving sodium bicarbonate added diets may have resulted in less stress upon the
reproductive tract of the birds of treated groups.
The findings of this study are in accordance with those stated by North and Bell
(1990), that incidence of meat spots are reported to be affected by the ambient temperature
along with other factors such as genetics and age of the birds. As the layers used in this
experiment were of the same age and strain, therefore no genetic variation or age affect can
be expected. Therefore, production of eggs without any blood or meat spots, by the layers fed
diet containing various levels of sodium bicarbonate may be attributed to less physiological
stress on reproductive system of the birds than those fed control diet without containing
sodium bicarbonate (control).
4.15 MortalityIncidence of mortality was zero in all groups. It may probably be due to the reason
that the experiment was conducted under the best possible controlled hygienic conditions,
(except ambient temperature) and the birds were kept well observed. Resultantly, no bird
died from any group (treated or untreated) during the experimental period. The results of the
present study are in conformity with the findings of Mushtaq et al. (2005) who observed no
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mortality in broilers fed diet supplemented with sodium bicarbonate (0.025% Na+). However,
generally high environmental temperature has been ascribed to more mortality of the birds
(Branton et al., 1986).
4.16 Rectal temperature, respiration rate and water consumption4.16.1 Rectal temperature
Birds receiving diets containing sodium bicarbonate exhibited lower rectal
temperature (P<0.05) when compared to those of untreated group (control). A probable
reason of decrease in rectal temperature of the birds in treated groups may be increased
sodium ions concentration in their diets, which might have resulted in increased water
consumption (Ahmad, 1997; Ahmad, 2007). Increased water consumption in birds has shown
to cause an increase in body heat loss through evaporation (Belay and teeter, 1993).
Therefore, birds in positive water balance (treated groups) were capable to sustain their
internal body temperature to optimum level. Results of the study are in agreement with the
findings of Ahmad et al. (2005) who studied the influence of sodium bicarbonate
supplementation on rectal temperature of heat stressed broilers and observed a significant
(P<0.05) reduction in body temperature of the birds fed diet having sodium bicarbonate. The
birds which were supplied sodium bicarbonate containing diets also exhibited lower
respiration rate and thus produced less heat for this physiological norm. This probably could
be the reason, for significantly lower rectal temperature in this case.
Layers fed diet without addition of sodium bicarbonate (control group) manifested a
rise in their rectal temperature in response to increase in ambient temperature during the
experimental period. However, layers of treated groups revealed a decrease in their rectal
temperature with increase in levels of sodium bicarbonate throughout the period of heat
stress. Higher relative humidity in combination with severe temperature was possibly a
contributing factor for upholding the initial rise in rectal temperature.
Mean values of rectal temperature of the layers fed diet containing 1% sodium
bicarbonate were found to be lower than those of its counterparts. The reduction in
temperature may have been, probably due to comfortable physiological environment of the
birds because of sodium/bicarbonate ions, which might have favored lowering of their body
temperature than its other counterparts.
Contrary to the results of current study, Mushtaq et al. (2007) did not find any
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correlation between rectal temperature of poultry birds and dietary sodium levels in broilers.
Dissimilarity in the results of these researches may be either because of difference in the
levels of sodium bicarbonate incorporated in the diets or due to the different species of the
birds (broilers vs layers) used in these studies, or both.
4.16.2 Respiration rateThe birds fed diets without adding sodium bicarbonate (control) exhibited faster
respiration rate when compared to those of treated groups, which is quite in line with the
findings of Angiletta et al. (2010) who noted increased respiration rate in birds exposed to
hyper-thermal environment. The changes in respiration rate per minute, in general are
correlated with the variations in the ambient temperature and relative humidity. In fact, when
ambient temperature goes beyond thermo-neutral zone, coupled with high humidity,
chemical reactions speed up in the body, heat is generated and body temperature of birds
rises (North and Bell, 1990). Moreover, as there are no sweat glands in birds hence they
dissipate their body heat mainly through respiration (Nillipour and Melog, 1999). Therefore,
when environmental temperature goes higher than the thermo-neutral zone, respiration rate
increases up to 10 times, from a normal rate of 25 breaths/minute (Remus, 2001), which in
turn causes a raise in the pH and thus results in respiratory alkalosis. In such conditions
sodium bicarbonate can be used as a buffering agent to ameliorate the problem (Whiting et
al., 1991) hyperthermia.
It has been observed that birds kept under heat stress spent less time for feeding, more
time for drinking water and panting (Mack et al., 2013). However, birds utilize multiple ways
to maintain their body temperature and homeostasis when subjected to heat stress, which
include increased radiation, convection and evaporation along with the heat loss through
vasodilatation (Mustaf et al., 2009). Birds also have an additional mechanism (air sacs) for
exchange of heat between their body and the ambient environment. Air sacs help air
circulation on surfaces adding increase in gaseous exchange with air and evaporative loss of
heat (Fedde et al., 1998).
Dietary inclusion of sodium bicarbonate has shown to decrease respiration rate of the
birds in treated groups as compared to those of untreated group (control). A probable
explanation of decrease in respiration rate of the birds may be the increase in bicarbonate
ions concentration in the blood of the treated birds. The birds which were fed sodium
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bicarbonate containing diets exhibited lower respiratory rate and thus produced less heat for
this physiological norm. This probably could be the reason for significantly lower rectal
temperature in this case.
4.16.3Water intakeResults of the preent study revealed that birds offered diets containing different levels
of sodium bicarbonate exhibited more water consumption as compared to those of untreated
group (control). An increased water consumption of the treated groups may probably be due
to higher dietary electrolyte balance of experimental diets and more feed consumption by
these birds when compared to those of control group. At high environmental temperatures,
the stimulus for more water intake and increased rate of water exchange in the body of birds
can be useful (Borges et al., 2003). A raise in water intake helps in lowering the body
temperature of broilers exposed to higher ambient temperature (Ahmad et al., 2005). Increase
in water consumption of the treated groups was compatible with the findings of Balnave and
Gorman (1993) and Teeter and Belay (1996) who reported that sodium ions induced increase
in water intake which might have been helpful in heat dissipation.
Water consumption of the birds exhibited a linear increase with increase in the level
of addition of sodium bicarbonate in the experimental diets. Borges et al. (2003) reported that
water consumption of birds increased linearly as the dietary electrolyte balance increased.
They observed that birds fed diets supplemented with NaHCO3 (DEB, 360mEq/kg)
consumed more water. Vieites et al. (2005) compared 0 to 350mEq/kg of DEB in diets and
found the lowest litter moisture contents at 138 and 147mEq/kg, whilst Oliveira et al. (2010)
recommended a DEB of 200mEq/kg for the best litter moisture and bone development. A
significant increase in water intake in sodium bicarbonate supplemented groups during
summer was in accordance with the results observed by Sayed and Scott (2008).
Findings of Ahmad (1997) haverevealed maximum water consumption (176
ml/bird/day) in birds kept on 75% supplemented sodium from NaCl. Whereas, minimum
water consumption (201ml/bird/day) was recorded in the birds kept on 75% supplemented
Na+ from NaHCO3. Contradictory to results of the present study, Fowler (1990) reported an
increase in water intake of birds fed ration containing 100% sodium from NaCl. Similar
findings have also been stated by Hooge (1995) in a review paper that complete replacement
of NaCl with NaHCO3 in broiler diets reduced water consumption by 3.04%.
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4.17 Hematological profile4.17.1 Serum glucose
The birds fed diet without addition of sodium bicarbonate (control group), exhibited
the highest glucose level in their blood as compared to those of treated groups (B, C, D and
E). As the ambient temperature was very high during the experimental period, therefore, the
birds experienced a continuous heat stress. In response, to combat this heat stress the birds in
control group might have secreted higher level of hormones like glucocorticoid, adrenaline
and nor-adrenaline, which might have caused gluconeogenesis, ultimately leading to rise in
blood glucose concentration (Yang et al . , 1992). Similar results have also been reported by
Borges (2001) in the birds exposed to heat stress.
On the other hand, the birds which used diets containing sodium bicarbonate
exhibited lower blood glucose level than those of control group. Decrease in blood glucose
level of the birds kept in treated groups may probably be because of decrease in heat stress
upon the birds used in the present study. That’s why the treated birds have exhibited
significantly lower rectal temperature as compared to those of group A. The results of this
study are close to the findings of Ahmad et al. (2005) who observed a decrease (P<0.05) in
glucose level in broilers fed diet containing sodium bicarbonate those of controls. Al-Hassani
et al. (2001) also reported a decrease (P<0.05) in glucose level in Hisex brown layers
subjected to heat stress when fed diet containing sodium bicarbonate.
In contradiction to the findings of present study, Koelkebeck and Odom (1995) did
not observe effect of high ambient temperature on blood glucose concentration in layers.
Similarly, Zakaria et al. (2009) also did not discover any outcome of dietary addition of
sodium bicarbonate on glucose level in chickens. Discrepancy in the results of these research
experiments may be due either to the variation in the levels of sodium bicarbonate
incorporated in the diets used or temperature stress to which the birds were exposed in these
studies, or both.
4.17.2 Packed cell volume (PCV), Erythrocyte sedimentation rate (ESR) and Red blood
cells count (RBCs)
PCV, ESR and RBCs count of all the experimental birds remained unaffected (P>0.05) due
to the dietary addition/inclusion of NaHCO3. Conflicting results have so far been reported
regarding the effect of dietary addition/inclusion of NaHCO3 on packed cell volume of
145
poultry birds. A significant decrease in packed cell volume has been observed by Oladele et
al. (2001) in birds exposed to high environmental temperature. They attributed this increase
to high ambient temperature and nutritional stress, which impaired the production of blood
cells in the birds. Whereas, Mubarak et al. (1999); Al-Hassani et al. (2001) and Ahmad et al.
(2005) have observed an increase in hematocrit values in birds treated with sodium
bicarbonate and these findings are quite in contrast to the findings of present study. Similarly,
contradictory to the findings of this study, Ekanayake et al. (2004); Mubarak and Sharkawy,
(1999) have reported increase in RBCs count in birds fed diets containing sodium
bicarbonate.
In fact, increase in water consumption due to the use of sodium bicarbonate may
result in peripheral vasodilation, which has been known to cause an influx of extracellular
fluid in to vascular space (Boulahsen, 1989), thus causing decrease in packed cell volume of
the birds. However, results of this study did not reflect such effects even water consumption
of the birds receiving sodium bicarbonate has shown a significant increase (see section
4.3.3). However, hematological profile of birds varies with age, sex, environmental factors
including season and stress to which the animals have been exposed (Olayemi and Arowolo,
2009).
4.17.3 Blood hemoglobin
Birds receiving diets containing sodium bicarbonate exhibited significantly higher
hemoglobin concentration in their blood as compared to those of untreated group (control). In
the present study, decline in hemoglobin concentration at higher environmental temperature
in the birds of control group also coincides with the findings of Yahav et al. (1997). Sahota
and Gilani, (1995) and Vecerek et al. (2002) have also observed a decreased hemoglobin
concentration in layers kept at high ambient temperature.
However, findings of Genedi (2000) have shown that addition of anti-stressors
(NaHCO3) in drinking water of Leghorn and Matrouh hens markedly increased their
hemoglobin concentration (%), even under heat stress conditions. Therefore, increased
hemoglobin concentration in sodium bicarbonate treated groups may probably be due to
increased nutrient uptake and reduction in body temperature, which may have led to
improved physiological performance of the layers. Results of the current study are in
accordance with report of Ahmad et al. (2005) who noted an increase in hemoglobin
146
concentration in birds due to inclusion of sodium bicarbonate in their diet.
However, the highest level of sodium bicarbonate (2%) included in the diet of the
experimental birds did not show any significant difference on their hemoglobin concentration
when compared to those of control group. A probable explanation of this result may be the
decrease in feed intake (see section 4.1.2), which might have quenched the thirst due to
increase in sodium intake, probably because of higher blood osmotic pressure of the birds
(Borges et al., 2003) and ultimately resulted in increased dilution of blood/blood contents.
On the other hand, excessive water intake probably improved feed passage rate and diluted
the enzymes of digestive tract resulting in reduced nutrient uptake (Ahmad et al., 2009;
Ravindran et al., 2008). Ultimately it might have reduced hemoglobin concentration in the
birds fed higher level of sodium carbonate.
4.17.4 White blood cell count (WBCs)
Concentration of WBCs was known to be higher in the birds fed diet without
inclusion of sodium bicarbonate (control group)) when compared to those of treated groups.
Higher WBCs count in the control birds may probably be due to high environmental
temperature to which these birds were exposed. Results of the present study are compatible
to those examined by Khattak et al. (2012) who found increase in WBCs count (3.2×104µl)
in the birds exposed to 38-40oC as compared to those fed sodium bicarbonate containing diet
(1.8× 104µl) at the same temperature. An increased white blood cells count at higher
environmental temperature has also been reported by Anjum (2000).
The birds fed diets containing sodium bicarbonate exhibited a decrease (P<0.05) in
WBCs count than those of control group. The reduction in WBCs count coincides with
reduction in bogy temperature of the birds of various groups (see section 4.3.1) due to the
treatments. Therefore, reduction in WBCs count due to the dietary inclusion of sodium
bicarbonate may be credited to the reduction in body temperature of the birds which might
have decreased heat stress and thus resulting in decrease in WBCs count (Maxwell et al.,
1992). The other factors, which may influence this parameter (hematological profile) in birds
include age, sex, season and stress to which the animals have been exposed (Olayemi and
Arowolo, 2009).
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4.18 Serum metabolites4.18.1Serum urea
Concentration of serum urea was discovered to be higher in the birds fed diet without
inclusion of sodium bicarbonate (control group) when compared to those of treated groups.
Higher concentration of serum urea in the control birds may probably be due to high
environmental temperature to which these birds were exposed. These results are matched to
those reported by Anjum et al. (2000) who observed an increase in serum urea concentration
in layers kept at higher ambient temperature as compared to those reared under heat
combating systems. Results of the current research are also in accordance with the findings
of Yang et al . (1992) who found significantly higher serum urea concentration in birds kept
at low temperature (12 °C). However birds kept at relatively high ambient temperatures (23
and 28 °C) showed lower serum urea concentration.
The birds fed diets containing sodium bicarbonate exhibited a decrease (P<0.05) in
their serum urea concentration as compared to those of control group. The reduction in serum
urea concentration also coincides with the reduction in body temperature of the birds of
various groups (see table 4.4) due to the treatments. Therefore, reduction in serum urea
concentration due to the dietary inclusion of sodium bicarbonate may be attributed to the
reduction in body temperature of the birds which may have decreased heat stress and hence
resulting in decrease in serum urea concentration (Maxwell et al. (1992). Olayemi and
Arowolo, (2009) have also reported that environmental factors and stress to which the
animals have been exposed, may affect their hematological profile.
4.18.2 Serum uric acid
The birds fed diets containing sodium bicarbonate exhibited a significant decrease in
their serum uric acid concentration as compared to those of control group. A possible
explanation of these results may be that excessive intake of minerals like sodium may cause
synergistic effect on other minerals i.e. excess of one can reduce the availability of another.
Excessive intake of sodium may cause hypernatremia (Mc Dowell, 1992; Oviedo-Ronden et
al., 2001) and thus may cause excessive water intake, diarrhea and increase in serum uric
acid (Davison and Wideman, 1992) in poultry birds.
However, Kurtoglu et al. (2007) did not observe effect of sodium bicarbonate on uric
acid level in Brown-Nick layer hens. Similarly, Koelkebeck and Odom (1995) also reported
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different results that heat stress had no effect on serum uric acid concentration of laying
birds. Dissimilarity in the results of these studies may probably be because of the differences
in the levels of sodium bicarbonate used.
4.18.3 Serum creatinine
Addition of different levels of sodium bicarbonate in the diets of layer did not give
any significant effect on serum creatinine values of the birds when compared to those fed diet
without inclusion of sodium bicarbonate (control group). These results are in line with the
research results of Koelkebeck and Odom (1995) who found that layers kept under acute heat
stress had no effect on their serum creatinine levels.
4.18.4 Serum alkaline phosphatase
Addition of different levels of sodium bicarbonate in the diets of layer did not affect
(P>0.05) serum alkaline phosphatase level of the birds. These results are in line with those
reported by Bogin et al. (1981) who reported that broilers subjected to heat stress for two
hours showed non-significant effect on their blood serum alkaline phosphatase level. Results
of the present study are also in line to those reported by Koelkebeck and Odom (1995) who
observed that acute heat stress had no effect on alkaline phosphatase enzyme of laying hens.
4.19 Serum proteins analysis4.19.1 Total proteins
Dietary inclusion of different levels of sodium bicarbonate showed a significant
increase in total protein concentration in blood serum of layers when compared to those fed
diet without any addition of sodium bicarbonate (control group). Results of the present study
are in accordance with the findings of Kurtoglu et al. (2007) who reported significant
(P<0.05) effect due to dietary inclusion of sodium bicarbonate on total protein in blood
serum of Brown-Nick layers indicating that increase in sodium ions concentration may
improve synthesis of total proteins in layers.
4.19.2 Albumin
The birds fed control diet exhibited a significantly lower albumin concentration in
their blood than those fed diet containing different levels of sodium bicarbonate (treated
groups). Lower albumen concentration in the birds of control group may probably be due to
decrease in digestion and absorption of protein contents of the diet because of heat stress
(Koh and Macleod, 1 999 ;), which possibly may also have exerted negative effect on protein
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synthesis, ultimately resulting in reduced albumin concentration. Findings of this study are in
accordance with those of Geraert et al. (1996) and Yahav et al. (1997) who observed
decreased protein level in birds reared in high ambient temperature (heat stress).
Inclusion of NaHCO3 in the diets of birds, however, showed an increase in their blood
albumen concentration probably because of lowering down body temperature of the birds,
which also had a positive effect on digestion and absorption of nutrients. Another probable
explanation of his fact may be the increased feed intake of the birds fed sodium bicarbonate
added diets. These findings coincide with those observed by Kurtoglu et al. (2007) who
found an increase (P<0.05) in blood albumen concentration of Brown-Nick layers fed diet
containing sodium bicarbonate.
In contrast to the findings of present study, Anjum (2000) noted increased serum albumin
concentration in heat stressed birds. Contradictions in these results may be due to difference in
environmental factors like season and type of stress to which the birds were exposed. Change
in albumin concentration of birds due to these factors (season and type of stress) has also
been reported by Olayemi and Arowolo, (2009).
4.19.3 Globulin
Addition of different levels of sodium bicarbonate in the diets of layer did not cause
any significant effect (P>0.05) on globulin concentration of the birds when compared to
those fed diet without inclusion of sodium bicarbonate (control group). Information regarding
the effect of inclusion of sodium bicarbonate on blood globulin concentration is very scanty.
However, decline in serum globulin of birds is observed by Yang et al. (1992); Geraert et al.
(1996); Anjum, (2000) due to heat stress. They also reported decreased protein level in the
heat exposed birds. Discrepancy in results of these research studies may perhaps be because
of difference in environmental factors like season and type of stress to which the birds were
affected. Olayemi and Arowolo, (2009) have also observed change in albumen concentration
of birds due to these factors (season and type of stress).
4.20 Plasma electrolytes, minerals and serum pH4.20.1Plasma sodium
Dietary inclusion of different levels of sodium bicarbonate depicted a significant
increase in plasma sodium concentration of layers when compared to those fed diet without
addition of sodium bicarbonate (control group). Increase in plasma sodium concentration in
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may be due to the fact that the treated birds were being fed more sodium in their diets with
sodium bicarbonate. However, birds fed diet without addition of sodium bicarbonate (control
group) showed low plasma sodium concentration. A probable explanation of lower sodium
concentration in blood plasma of heat stressed birds (control) may be due to increase in
urinary excretion of this essential mineral (Gorman et al., 1997). Results of the study are in
line to those observed by Borges et al. (2004) who reported decreased blood sodium level in
broilers kept under heat stress. A decline in blood sodium ions level in birds kept under heat
stress has also been observed by Takahashi and Akiba (2002).
On the other hand, increased plasma sodium ion concentration in the birds of treated
groups may be related to the dietary addition of different levels of sodium bicarbonate,
because a linear increase in sodium ion concentration has been observed with increase in its
level of inclusion. These results are compatible to those observed by Ahmad et al. (2006).
They observed a rise in plasma Na+ concentration in birds due to the supplementation of
sodium bicarbonate in their diets. Similarly, Mushtaq et al. (2005) found an increased serum
sodium concentration because of dietary addition of different levels of sodium.
In contrast to the findings of present study, Bonsembiante and Chiericato (1990) did
not observe effect (P>0.05) of dietary addition/inclusion of sodium bicarbonate on sodium
ion concentration in meat type turkeys. Contradictions in these results may either be due to
the difference in species of the birds (layers vs turkeys) used in these studies or to the degree
of heat stress to which the birds were exposed (Olayemi and Arowolo, 2009; Teeter et al.
1985), or both.
4.20.2Plasma Potassium
Concentration of plasma potassium was found to be lower in the birds fed diet
without inclusion of sodium bicarbonate (control group) when compared to those of treated
groups. A probable explanation of lower potassium ion concentration in blood plasma of heat
stressed birds (control) is increase in urinary excretion of this essential mineral (Gorman et
al., 1997). These results are in line to those observed by Borges et al. (2004) who reported
decreased blood potassium concentration in broilers kept under heat stress. A decline in the
level of potassium ions in birds kept under heat stress has also been observed by Takahashi
and Akiba (2002).
However, birds receiving diets containing sodium bicarbonate exhibited increased
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plasma potassium level as compared to untreated group (control). Results of the present study
supports the findings of Ahmad et al. (2006) who observed that blood K+, was significantly
increased by the addition of sodium sources (sodium carbonate, NaHCO3 or sodium sulfate)
in the diet. Similar effect has also been observed by Ghorbani and Fayazi (2009) who studied
the dietary effect of NaHCO3 on plasma potassium concentration of layers kept under
constant/chronic heat stress and found an increase (P<0.05) in plasma potassium
concentration due to dietary inclusion of sodium bicarbonate. Similarly Mushtaq et al. (2005)
reported that serum K+ level of birds was significantly affected (P<0.05) by inclusion of
varying dietary sodium levels.
Contrary to the results of current research, Kurtoglu et al. (2007) found a decreased
plasma potassium concentration in layers offered diets having NaHCO3 as compared to those
fed diets containing either NaCl or KCl. Similarly, Keskin and Durgan (1997) found
decreased plasma potassium concentration in quails fed diets containing sodium bicarbonate
(1%) kept at high ambient temperature when compared to those kept at thermo-neutral zone.
Dissimilarity in results of these experiments may possibly be due either to difference in
environmental factors like ambient temperature or difference in species of the birds (layers vs
quails) used in these studies.
4.20.3 Plasma chloride
Birds receiving diets containing sodium bicarbonate exhibited decreased plasma
chloride level as compared to those of untreated group (control). Exchange of Na+/H+ takes
place in kidneys of the birds. When level of sodium is increased in blood, the excretion of
chloride in urine is also increased (Pech-Waffenschmidt et al., 1995), ultimately resulting in
reduction of its level in blood plasma. Therefore, decrease in plasma chloride concentration
in the birds fed various levels of sodium bicarbonate may be attributed to increased level of
sodium in their blood, as has been depicted in the results of present study. These findings are
compatible to the findings of Ahmad et al. (2006) who reported that blood Cl-, was decreased
by the supplementation of sodium sources (sodium carbonate, NaHCO3 or sodium sulfate) in
the diet of poultry birds.
Contrary to the results of present research, Ahmad et al. (1997) reported that blood
Cl- concentration was not affected due to the addition of dietary sodium bicarbonate in the
diet of broilers. Contradictory results have also been reported by Bonsembiante and
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Chiericato, (1990) who observed non-significant effect (P>0.05) of dietary addition/inclusion
of NaHCO3, on blood chloride in meat type turkeys. Differences in these results may be due
to the various species of the birds or levels of sodium bicarbonate used in these studies or
both.
4.20.4 Plasma bicarbonate
Concentration of plasma bicarbonate (HCO3-) was known to be higher in the birds fed
diet containing sodium bicarbonate (treated groups) as compared to those of control group.
Increase in plasma HCO3- concentration in treated birds may possibly be due to the inclusion
of sodium bicarbonate in their diets, which led to increased availability of these ions to the
birds. Generally, a decrease in plasma Cl– increases HCO3– re-absorption by the kidneys (Ait-
Boulahsen et al., 1989), leading to higher concentration of HCO3– in blood plasma. Results of
the current study supports the findings of Keskin and Durgan (1997) who found an increase
(P<0.05) in plasma bicarbonate level due to the inclusion of NaHCO3 (1%) in the diet of
birds exposed to heat stress. Similar findings have also been reported by Squire and Julian
(2001) who observed a significant rise in blood HC03- of the layers receiving ration
supplemented with sodium bicarbonate.
Whereas, plasma bicarbonate level was significantly decreased in birds kept under
heat stress (control) probably because of increased respiration rate (Teeter et al.,1985).
Moreover, heat stress can induce increase in metabolic requirements of this essential anion
(Gorman and Balnave, 1994). However, this situation can be improved as has been depicted
in the results of this study, by the dietary inclusion sodium bicarbonate. On the other hand,
excessive level of NaHCO3 in diet may cause increase in water consumption, diarrhea and
urine pH (Mert, 1991; Davison and Wideman, 1992). Therefore, based upon the discussion
above, it may be concluded that sodium bicarbonate if used at an optimum level in the ration,
can increase HC03- and hence may lead to higher blood pH (Austic and Keshavarz, 1988) of
broilers resulting in their better rate of growth.
4.20.5 Plasma calcium and phosphorus
The difference in plasma calcium and phosphorus concentration in birds fed diets
with or without addition of sodium bicarbonate was found to be non-significant. These
findings are in accordance with those observed by Ghorbani and Fayazi (2009) who studied
the effect of varying dietary levels NaHCO3 and rearing system on plasma calcium of layers
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kept under chronic heat stress. Results of their study revealed that neither dietary inclusion of
sodium bicarbonate nor its levels (0.5%-1.5%) exhibited any effect on plasma calcium
concentration in layers. Findings of the present research also coincide with those observed by
Kurtoglu et al. (2007) who found no effect (P>0.05) of NaHCO3 on plasma calcium
concentration in layers. Similarly, Keskin and Durgan (1997) found non-significant
differences in blood calcium concentration between thermo-neutral group and sodium
bicarbonate supplemented (1% NaHCO3 in diet) group of quails exposed to heat stress.
4.20.6 Serum pH
Diets containing sodium bicarbonate when fed to experimental birds, exhibited a
decrease in their serum pH as compared to those of untreated group (control). High ambient
temperature during summer (heat stress) has shown to increase heat production in birds
(Teeter & Belay, 1996; Macleod et al., 1984). To get rid of this excessive body heat, birds
start panting which may increase their respiratory rate, resulting in excessive loss of CO2.
This loss causes an increase in blood pH of birds and may lead to respiratory alkalosis. Under
such conditions sodium bicarbonate can be used as a buffering agent, which is known to
normalize blood pH by supplying bicarbonate ions (Whiting et al., 1991). Therefore, it may
be inferred that the birds fed diets containing sodium bicarbonate may have low serum pH
value (optimum physiological range), as has been investigated in the current study.
Findings of the present research are compatible to the findings of Ahmad et al. (2005)
who observed that blood pH was decreased by the supplementation of sodium sources
(sodium carbonate, sodium bicarbonate or sodium sulfate) in the diets of birds. Similarly
Mushtaq et al. (2005) investigated that blood pH was decreased (P<0.05) by increasing
dietary addition of sodium levels 0.20% vs 0.25%. Similar results are also observed by
Fuentes et al. (1998) who reported a decrease in blood pH in guinea fowl fed diets containing
different levels (0.6-2.4%) of sodium bicarbonate. Khattak et al. (2012) also observed
decreased pH (8.04) in broilers kept at 38-40 0C as compared to those fed sodium bicarbonate
containing diet (8.34 pH) at the same temperature.
However, Keskin and Durgan (1997) found a significant increase in blood pH of
quails exposed to heat stress when fed diet containing 1% NaHCO3. Disparity in results of
these experiments may possibly be due to variation in ambient temperature or difference in
species of the birds (layers vs quails) used in these studies (Olayemi and Arowolo, 2009).
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4.21 Serum lipids profile
Birds receiving diets containing sodium bicarbonate exhibited significantly less
serum lipids profile i.e. low density lipoproteins (LDL) and triglyceride in their blood when
compared to those of control group. A probable explanation of decrease in this parameter
may be that sodium bicarbonate might have stimulated the synthesis of bile acids from
cholesterol, leading to decreased level of blood cholesterol in this group (Naviglio et al.,
2011). Another possible reason for reduction in serum cholesterol may be the inhibition of
“squalene epoxidase” an enzyme which is necessary for production of cholesterol
(Angelovicova, 1997). The reduction of serum low density lipoprotein to some extent may
reduce the chances of cardiovascular diseases.
Birds receiving diets containing sodium bicarbonate exhibited less serum triglyceride
level as compared to untreated group (control). Layers in group C which were fed diet
containing 1% sodium bicarbonate showed the lowest serum triglyceride level in their blood.
A possible explanation of lower level of triglyceride in treated groups may be inhibition of
fatty acid synthesis and production of bile acids from cholesterol (Naviglio et al., 2011),
leading to decreased concentration of serum triglyceride in this group. Moreover, inhibition
of an enzyme called “squalene epoxidase” may also cause reduction in serum triglyceride
(Angelovicova, 1997).
Probable explanation of decreased serum triglycerides level in the birds fed sodium
bicarbonate containing diets may be decrease in cortisol level of the birds, as has been
observed in the results of present study (see section 4.11.4). A similar relationship between
serum triglyceride and cortisol level has been observed by Sahin et al. (2002) who observed a
linear rapport between serum triglycerides and ACTH level, due to its catabolic effect, in
birds kept under high ambient temperature.
4.22 Hormones and enzymes4.22.1Tri-iodothyronine (T3) and Thyroxin (T4)
Addition of different levels of sodium bicarbonate in the diets of layer showed
significant effect on serum T3 and T4 concentration of the birds when compared to those fed
diet without inclusion of sodium bicarbonate (control group). The birds fed diets without
adding sodium bicarbonate, exhibited the lowest level of these growth hormones (T3 and T4)
in their blood. Secretion rate of thyroxin (T3 and T4) is known to be influenced by
155
environmental temperature (Wright et al., 1952) and these hormones are secreted at their best
when the birds are free from all types of stress, particularly heat stress. Moreover, fall in
temperature results in increased thyroid secretion. As incorporation of sodium bicarbonate in
the diets has shown to reduce heat stress in the present study, therefore, higher concentration
of these hormones in the birds of treated groups may probably be due to the heat stress
combating ability of dietary sodium bicarbonate.
Hypothalamus and pituitary receive stimulus of high environmental temperature and
in turn causes a decline in the secretions of growth hormones (Anjum, 2000). However,
dietary inclusion of sodium bicarbonate during hot period was found to be accompanied by
reduction in heat stress (Remus, 2001). Hence, dietary addition of bicarbonate might have
caused an increase in concentration of growth hormones in the blood of treated birds. Higher
concentration of these hormones corresponded to maximum performance as has also been
observed by Anjum, (2000).
Findings of the present research coincide with the research results of Sahin et al.
(2001) who investigated a decrease (P<0.05) in serum T4 and T3 concentration in birds kept
under heat stress. Decrease in concentration of these hormones in birds reared under heat
stress has also been reported by Honog et al . (1995) and Anjum, (2000).
4.22.2 Estrogen
Difference in serum estrogen concentration in birds fed diets with or without addition
of sodium bicarbonate was found to be significant. The birds fed diets without adding sodium
bicarbonate (control) exhibited lower serum estrogen concentration when compared to those
of treated groups. Estrogen plays a pivotal role in the reproductive efficiency of fowls (Lile,
1976), therefore, its decreased concentration in control birds consequently adversely affected
the performance of the birds. In fact, when ambient temperature goes beyond thermo-neutral
zone, chemical reactions speed up in the body, heat is generated and body temperature of
birds rises (North and Bell, 1990) which probably may have been the cause of adverse effect
on normal synthesis of this hormone. It has been observed that under such conditions sodium
bicarbonate can acts as a buffering agent to mitigate the heat stress (Whiting et al., 1991).
Results of this study are compatible to the research results of Anjum (2000) who reported
significant decrease in the level of estrogen in layers kept under heat stress conditions.
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4.22.3 Progesterone
Concentration of progesterone was found to be lower in birds kept at high ambient
temperature when compared to those of treated groups. A possible explanation of this result
may probably be that high ambient temperature may have depressed ovarian function, posing
hindrance against the release of leutinizing hormone, subsequently resulting in decreased
progesterone concentration. These results are in line with the research results observed by
Anjum (2000) who reported a significant decrease in progesterone concentration in layers
exposed to heat stress. Furthermore the results of the current research are also in concord
with the earlier findings of Novero et al . (1991) and Chostesangasa (1992), where higher
environmental temperature has been reported to inhibit growth and sexual maturity,
respectively, as a result of decreased secretion of progesterone in birds.
4.22.4 Corticosterone
Dietary inclusion of different levels of sodium bicarbonate depicted significantly
lower concentration of corticosterone hormone in layers when compared to those fed diet
without sodium bicarbonate. In fact, when ambient temperature goes beyond its thermo-
neutral zone, chemical reactions speed up in the body, heat is generated and body
temperature of birds rises (North and Bell, 1990), which causes heat stress in birds. In such
conditions sodium bicarbonate can be used as an anti-stress agent to ameliorate the effect of
heat stress (Whiting et al., 1991). Therefore, a probable explanation of decrease in cortisol
level of the birds may probably be due to increase in bicarbonate ions concentration in the
blood of the treated birds.
Reduction in serum corticosterone concentration in the layers fed diets containing
sodium bicarbonate also corresponds to decrease in their rectal temperature, as has been
depicted in the results of this study. Whereas, the highest level of this hormone was noted in
the serum of layers of group A (control), which were offered diet without addition of sodium
bicarbonate. An increase in corticosterone level of birds, with increase in environmental
temperature has also been observed by Sahin et al. (2002), Teukam et al. (1996), Bains
(1996) and Yang et al. (1992),.
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4.22.5 Serum Glutamic-Oxaloacetic Transaminase and Serum Glutamic Pyruvic
Transaminase
Birds receiving diet without addition of sodium bicarbonate (control group) exhibited
higher concentration of liver enzyme Serum Glutamic-Oxaloacetic Transaminase (SGOT),
when compared to those of treated groups. Heat stress has shown to increase plasma cortisol
concentration (Sahin, et al., 2002) in birds. This increase may cause an increase in catabolic
effect in liver, which exerts maximum stress on it, leading to a raised level of serum SGOT
of birds exposed to heat stress. Therefore, increase in serum SGOT concentration in the birds
of control group may probably be because of stress on their liver due to higher body
temperature of these birds as compared to those of treated groups. These results are similar to
those reported by Anjum, (2000) who observed an increase in SGOT in layers kept/reared
under heat stress. Reduction in concentration of serum SGOT in layers kept under heat
combating systems was also noted when compared to those kept at high ambient temperature.
Another possible explanation of higher concentration in blood serum of heat stressed
birds may be the deviation in blood pH of birds exposed to heat stress as a result of
respiratory alkalosis. Though pH influences many aspects of cell structure and functions, yet
catalytic activity of the enzymes is specifically sensitive to it (Lehninger, 1970). Each
enzyme has maximum activity at a specific pH, called optimum pH, and the activity reduces
sharply on either side of the optimum pH. Therefore, biological control of pH of cells and
body fluids is of utmost importance for metabolism and cellular functions.
On the other hand, the birds fed diets containing sodium bicarbonate exhibited a
significant reduction in SGOT concentration. A possible explanation of this fact may
probably be the reduction in body temperature of birds, which might have reduced stress on
their liver and hence led to a decrease in SGOT concentration in the birds of treated groups.
Based upon these findings, it can be envisaged that dietary inclusion of sodium bicarbonate
may help alleviate heat stress by decreasing body temperature and concentration of plasma
cortisol. These findings are compatible to those observed by Ahmad et al. (2005) who
reported that dietary inclusion of sodium bicarbonate can decrease body temperature of heat
stressed birds.
In contrast to the results of present study, Ozbey et al. (2004) observed that blood
SGOT concentration was not affected due to heat stress in quails. Dissimilarity in results of
158
these researches, however, may likely be due to variation in ambient temperature maintained
or difference in species of the birds (layers vs. quails ), used in these studies. However,
concentration of Serum Glutamic Pyruvic Transaminase (SGPT) remained unaffected in the
birds due to the inclusion of NaHCO3 in their diets.
4.23 Immune responseDietary inclusion of different levels of sodium bicarbonate depicted an increase
(P<0.05) in antibody titer against NDV in layers when compared to those fed diet without its
addition (control group). Environmental stressors have been known to affect immunity and
innate resistance of the host directly or indirectly (Robertson, 1998). Therefore, increase in
antibody titer against NDV in birds offered diets having varying levels of sodium bicarbonate
may probably be due either to less heat stress upon these birds because of reduction in their
body temperature or lower cortisol concentration as compared to those of group A (control),
or both. Results of the present experiment are compatible to the observations of Khatak et al.
(2012) who investigated higher haemaglutination inhibition titer against NDV in birds
consuming diets containing sodium bicarbonate.
Borges et al. (2003) have investigated that increase in DEB may cause decrease in
heterophil to lymphocyte ratio in blood, leading to increase in antibody titer. Similarly,
Santin et al. (2003) have reported a linear increase (P<0.05) in NDV antibody titers with
rising DEB (40, 140, 240, 340mEq/kg), receiving NaCl, NaHCO3, and NH4Cl as
supplements, Therefore, it may safely be concluded that dietary addition of sodium
bicarbonate may improve antibody titter against NDV in birds.
On the other hand, birds receiving diets without sodium bicarbonate (control)
exhibited reduced antibody titer against NDV comparing to those of treated groups. Control
of antibody mediated immunity at various environmental temperatures hasbeen studied by
El-Gendy et al. (1995) and they found that birds exposed to heat stress had significantly
depressed agglutinin levels. Moreover, exposure of layer birds to stressors like heat stress
have shown to cause a decrease (P<0.05) in lymphocytes and increase (P<0.05) in
heterophils (Borges et al., 2004), resulting in reduced immunity. Therefore, outcomes of
present study correspond with the results of El-Gendy et al. (1995) who observed that
antibody titer against NDV was lower in heat stressed broilers as compared to those kept
under normal temperatures. Similar effects have also been reported by Anjum (2000) who
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observed decrease in antibody titer against Newcastle disease virus, probably because of
increase in total leukocyte count (Maxwell, 1993), in birds exposed to heat stress. Bains et al.
(1996) have observed an effect (P<0.05) on immune system of turkey breeder hens when
exposed to heat stress. Comparable findings were also noted by Savic et al. (1993) who
exposed the birds to heat stress at different intervals and found lower antibody titer against
Lasota strain virus in heat stressed birds.
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CHAPTER-5
DIGESTIBILITY TRIALEFFECT OF DIETARY INCLUSION OFNaHCO3ON NUTRIENT
DIGESTIBILITY IN CAGED LAYERS DURING SUMMER
5.1 IntroductionSodium bicarbonate (NaHCO3) is a white solid crystalline compound soluble in
water, which is commonly used as an antacid to treat acid indigestion (Thomas and
Stone, 1994). It is generally supplemented as a simple solution for reinstate the pH of
water which has a high concentration of chlorine (Teeter et al., 1985; Whiting et al.,
1991). Sodium bicarbonate in feed or water has also shown potential benefits on production
performance (Ahmad et al., 2005; Khatak et al., 2012), egg characteristics (Kaya et al.,
2004), blood profile (Kurtoglu et al., 2007) and nutrient digestibility (Ahmad, 2007), in
poultry birds reared under heat stress.
Sodium bicarbonate in the diet of layers may improve nutrient digestibility by
increasing sodium ions concentration (Fethiere et al., 1994); improving electrolyte balance in
the diet (Borges et al., 2003); meeting the requirements for the HCO3- ions (Gorman and
Balnave, 1994) and decreasing the losses caused by heat stress (Gorman and Balnave, 1994;
Mirsalimi and Julian, 1993; Braton et al., 1989). It is cheap, easily available and easy to
handle, therefore, can be safely incorporated in poultry diets to ameliorate the adverse effects
caused by heat stress.
Therefore, the present trial was conducted to study the effects of dietary inclusion of
sodium bicarbonate on in vivo digestibility of dry matter (DM), crude protein (CP), crude
fibers (CF) and ether extract (EE). The effect of addition of this compound in poultry diets
was also studied on absorption of some minerals i.e. calcium, phosphorus, sodium, potassium
and iron, in caged layers during summer.
5.2 Materials and methodsThe digestibility trial (in vivo) was conducted in 36 weeks old layers. Thirty layers
having similar body weight were obtained from the same batch which was used for
performance trial. All the experimental layer birds were maintained/kept in individual
metabolic cages. These layer birds were randomly allotted/allocated to five experimental
161
diets/rations in such a way that each diet was offered to 6 layers so that each bird served as a
replicate. At the end of 38th week of age, fecal samples were collected for two days at the
interval of 3 hours. The birds in all the groups were fed same amount of feed during the
collection period.
5.2.1 Experimental diets
Five experimental diets i.e. A (control, without Sodium bicarbonate), B (0.5%Sodium
bicarbonate), C (1 % Sodium bicarbonate), D (1.5% Sodium bicarbonate), E (2% Sodium
bicarbonate) used in the performance trial were also used in the digestibility trial, however
acid insoluble ash (1%) was included/incorporated as an indigestible marker in the diets to be
used in the digestibility trial (See table 3). These diets/feeds were formulated according to the
NRC (1994) recommendations for nutrient requirements of layers. All the diets were iso-
nitrogenous (CP 17 %) and iso-caloric (ME 2700 Kcal/Kg diet).
5.2.2 Chemical analysis of feed/excreta
Feed and or excreta samples were analyzed for DM, CP, CF, EE, calcium,
phosphorus, sodium, potassium, iron and AIA marker determination as described by AOAC
(2010). The nutrients digestibilities were calculated receiving the equations outlined in
chapter 3.
5.2.3 Statistical analysis
The data collected were subjected to statistical analysis for interpretation of results
using completely randomized design (CRD). Treatment means were compared by the Least
Significance Differences (Steel et al., 1997) test.
5.3 ResultsMean values regarding digestibility of DM, CP, CF and EE in birds fed diets with or
without dietary inclusion of sodium bicarbonate are shown in table 5.1.
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Table 5.1: Effects of dietary inclusion/addition of NaHCO3 on nutrient
digestibility coefficient in layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Dry matter (%)70.4±
6.09c 73.4±5.89b 77.1 ±5.79a 74.1± 6.34b 71.6±2636c
Crude protein
(%)
68.6±5.7
2c 72.7±4.65a 75±3.27a 72.2±3.38ab69.
2±5.44bc
Crude fibers %)29.
0±2.81b 33.9±2.36ab 40.7 ±2.85a 33.2 ±2.1ab 30.9±2.95b
Ether extract
(%)
82.
0±5.57b 89.4± 4.02a 93.7± 5.46a 84.8±7.78b82.7±
4.67b
Values within the same row with unlike superscripts are significantly different (P<0.05)
163
5.3.1 Dry matter
Mean values of dry matter (DM) digestibility for treatments A, B, C, D and E, were
found to be 70.4, 73.4, 77.1, 74.1 and 71.6%, respectively. The results revealed an effect
(P<0.05) on DM digestibility due to the addition/ inclusion of NaHCO3 in the diets of layer
when compared to those of control group. Statistical analysis of the data depicted that the
birds of treated groups, receiving diets containing sodium bicarbonate showed higher
(P<0.05) DM digestibility than those of group A (control). The differences in DM
digestibility values were also found to be significant among the treated groups. Birds
receiving diet containing 1% sodium bicarbonate exhibited maximum digestibility followed
by those of group D and B whereas, the lowest dry matter digestibility was recorded in
groups A (control) and E. However, differences in the digestibility values of groups B and D,
and those between A and E were found to non-significant (P>0.05).
5.3.2 Crude protein
Mean values of crude protein (CP) digestibility for treatment A, B, C, D and E, were
found to be 68.6, 72.7, 75.0, 72.2 and 69.2%, respectively. The results revealed a significant
effect on CP digestibility due to the inclusion of NaHCO3 in the diets of layer when
compared to those of control group. Statistical analysis of the data depicted that the birds of
treated groups, receiving diets containing sodium bicarbonate showed higher (P<0.05) CP
digestibility than those of group A. The difference in CP digestibility values was also
significant among the treated groups. Birds receiving diet containing 1% sodium bicarbonate
exhibited maximum digestibility followed by those of group B, D and E. whereas, the lowest
CP digestibility was recorded in the control group. However, differences in the digestibility
values of groups B, C and D, and those between A and E and between D and E were found to
be non-significant (P>0.05).
5.3.3 Crude fiber
Mean values of crude fiber (CF) digestibility for treatment A, B, C, D and E, were
found to be 29.0, 33.9, 40.7, 33.2 and 30.9%, respectively. The results revealed a significant
effect on CF digestibility due to the addition/inclusion of NaHCO3 in the diets of layers when
compared to those of control group. Statistical analysis of the data depicted that the birds of
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treated groups, receiving diets containing sodium bicarbonate showed higher CF (P<0.05)
digestibility than those of group A. The difference in CF digestibility values was also
significant among the treated groups. Birds receiving diet containing 1% sodium bicarbonate
exhibited maximum digestibility followed by those of group B, D and E, whereas, the lowest
CF digestibility was recorded in the control group. However, differences in the digestibility
values of groups B, C and D, and those among A, B, D and E were found to non-significant.
5.3.4 Ether extract
Means values of ether extract (EE) digestibility for treatment A, B, C, D and E, were
found to be 82.1, 89.4, 93.7, 82.3 and 82.8%, respectively. The results revealed a significant
effect on EE digestibility due to the inclusion of NaHCO3 in the diets of layer when
compared to those of control group. Statistical analysis of the data depicted that the birds of
treated groups, receiving diets containing sodium bicarbonate showed higher (P<0.05) EE
digestibility than those of control group. The difference in EE digestibility values was also
significant among the treated groups. Birds receiving diet containing 1% sodium bicarbonate
exhibited maximum digestibility followed by those of group B, D and E. whereas, the lowest
EE digestibility was recorded in the group A. However, differences in the digestibility values
of groups B and C, and those among A, D and E were found to non-significant (P>0.05).
5.4 Minerals Mean values pertaining to the absorption of minerals i.e. calcium, phosphorous, iron,
sodium, and potassium in birds fed diets with or without dietary inclusion of sodium
bicarbonate are shown in table 5.2.
5.4.1 Calcium
Means values of regarding digestibility of calcium (Ca), for treatment A, B, C, D and
E, were found to be 56.1, 58.0, 60.3, 58.1 and 54.8, respectively. The results revealed a
significant effect on digestibility of Ca due to the inclusion of NaHCO3 in the diets of layer
when compared to those of control group. Statistical analysis of the data depicted
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Table 5.2: Effect of dietary inclusion of NaHCO3 on absorbability of minerals in
layers
Variables
Treatment
A
Control
B
0.5%NaHCO3
C
1%NaHCO3
D
1.5%NaHCO3
E
2%NaHCO3
Calcium 56.1± 3.29c 58.3±3.10b 60.3±3.20a 58.1± 3.28b 54.8 ±3.19c
Phosphorus 50.8±2.32c 53.5±3.09b 57.6±3.27a 53±3.33b 49.6±2.40c
Iron50.5 ±
2.36d 53.1±1.38c 60.8 ±3.34a 55.6 ±3.2b 50±2.27d
Sodium 51.6± 2.1c 54.1± 1.3b 59.2± 1.1a 53.5±1.11b51.6.±
1.913b
Potassium 51.8±2 c 53.5±2.47 b 56.3±3.64 a 53.5±3.64 b 52±3.47 c
Values within the same row with unlike superscripts are significantly different (P<0.05)
166
that the birds of treated groups, receiving diets containing sodium bicarbonate showed
significantly (P<0.05) higher Ca digestibility as compared to those of control group. The
difference in Ca digestibility values was also significant among the treated groups. Birds
receiving diet containing 1% sodium bicarbonate exhibited maximum digestibility followed
by those of group B, D and E. whereas, the lowest Ca digestibility was recorded in the
control group. However, differences in the digestibility values of groups B and D, and those
between A, and E were found to non-significant (P>0.05).
5.4.2 Phosphorous
Means values of regarding digestibility of phosphorous (P), for treatment A, B, C,
D and E, were found to be 50.8, 53.5, 57.6, 53.0 and 49.6, respectively. The results
revealed a significant effect on digestibility of P due to the inclusion of NaHCO 3 in the
diets of layer when compared to those of control group. Statistical analysis of the data
depicted that the birds of treated groups, receiving diets containing sodium bicarbonate
showed higher (P<0.05) P digestibility than those of control group. The difference in P
digestibility values was also significant among the treated groups. Birds receiving diet
containing 1% sodium bicarbonate exhibited maximum digestibility followed by those of
group B, D and E. Whereas, the lowest P digestibility was recorded in the control group.
However, differences in the digestibility values of groups B and D, and those between A,
and E were found to non-significant (P>0.05).
5.4.3 Iron
Means values of regarding digestibility of Iron, for treatment A, B, C, D and E, were
found to be 50.5, 53.1, 60.8, 55.6 and 50.0, respectively. The results revealed a significant
effect on digestibility of Iron due to the inclusion of sodium bicarbonate in the diets of layer
when compared to those of control group. Statistical analysis of the data depicted that the
birds of treated groups, receiving diets containing sodium bicarbonate showed significantly
(P<0.05) higher Iron digestibility as compared to those of control group. The difference in
the values of digestibility of Iron also found to be significant among the treated groups. Birds
receiving diet containing 1% sodium bicarbonate exhibited maximum digestibility followed
by those of group D, B and E, whereas the lowest Iron digestibility was recorded in the
control group. However, difference in the digestibility values of groups A, and E was found
to non-significant.
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5.4.4 SodiumMeans values of regarding digestibility of sodium (Na), for treatment A, B, C, D and
E, were found to be 51.6, 54.1, 59.2, 53.5 and 51.6, respectively. The results revealed a
significant effect on digestibility of Na due to the inclusion of NaHCO3 in the diets of layer
when compared to those of control group. Statistical analysis of the data depicted that the
birds of treated groups, receiving diets containing sodium bicarbonate showed higher
(P<0.05) Na digestibility than those of control group. The difference in Na digestibility
values was also significant among the treated groups. Birds receiving diet containing 1%
sodium bicarbonate exhibited maximum digestibility followed by those of group B, D and E.
Whereas, the lowest Na digestibility was recorded in the control group. However, differences
among the digestibility values of groups B, D and A were found to non-significant.
5.4.5 PotassiumMeans values of regarding digestibility of potassium (K), for treatment A, B, C, D
and E, were found to be 51.8, 53.5, 56.3, 53.5 and 52.0, respectively. The results revealed a
significant effect on digestibility of K due to the inclusion of NaHCO3 in the diets of layer
when compared to those of control group. Statistical analysis of the data depicted that the
birds of treated groups, receiving diets containing sodium bicarbonate showed higher
(P<0.05) K digestibility than those of control group. The difference in K digestibility values
was also significant among the treated groups. Birds receiving diet containing 1% sodium
bicarbonate exhibited maximum digestibility followed by those of group B, D and E. whilst,
the lowest digestibility of K was recorded in the control group. However, differences in the
digestibility values of group B and group D, and those between group A, and group E were
found to be non-significant (P>0.05).
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5.5 Discussion5.5.1 Dry matter
Diets containing sodium bicarbonate exhibited better digestibility of dry matter in
layers. Increase in digestibility of dry matter of the treated groups may be due to more
sodium ions concentration in rations containing sodium bicarbonate. Similar effect of
increased sodium ions concentration in broilers has been observed by Fethiere et al. (1994).
Dietary inclusion of sodium bicarbonate might have improved the DEB by forming favorable
circumstances for improving the digestibility of nutrient (Borges et al., 2003; Mahmud et al.,
2010). Gorman and Balnave (1994) reported that high ambient temperature could lead to a
metabolic need for the HCO3- ions. The pancreatic juices which involve digestion of most of
the nutrients primarily contain H2O, NaCl, and NaHCO3. The function of NaHCO3 in
pancreatic juice is to neutralize high acidity (pH) of chyme and raise it to be alkaline to
prepare the chyme for digestion and absorption. This process takes place in the small
intestine (Leeson and Summer, 2001). Therefore, decreased digestibility of dry matter in
control group may have been due to lower bicarbonate and sodium levels, which probably
occurred due to increase in respiration rate.
Heat stress may exert a negative influence on digestion and/or absorption of dietary
nutrients and their metabolism (Macleod, 2004; Puvadolpirod and Thaxton, 2000), as have
been observed in the birds of control group. Presence of sodium bicarbonate in the diets of
treated birds might have improved their digestibility and prevented losses caused by heat
stress (Mirsalimi and Julian, 1993; and Braton et al., 1989). However, beneficial effects of
NaHCO3 can be achieved only when its recommended/optimum levels are incorporated in
the diets. An excessive level of this chemical compound in the diet has been reported to be
toxic in White Leghorn layers (Davison and Wideman, 1992). Therefore, it might be the
possible reason of reduced digestibility of dry matter in group E, which were fed a diet
containing 2% sodium bicarbonate. Another reason for decrease in digestibility of dry matter
in group E (2% NaHCO3) might be increased passage rate of digesta (Ravindran et al., 2008).
However, Ahmad (1997) observed that dry matter digestibility in broilers was not influenced
due to dietary inclusion of sodium bicarbonate.
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5.5.2 ProteinThe birds fed diet without inclusion of sodium bicarbonate (control) exhibited the
lowest digestibility of protein. At ambient temperature above 30 °C, thermoregulatory
system is activated and causes an increase in blood flow to upper respiratory tract and other
organs associated in heat excretion i.e. combs and wattles, which causes a decrease in blood
flow to the digestive tract (Wolfenson, 1 986 ). Consequently, activities of proteolytic
enzymes may be decreased in the upper gastrointestinal tract (Hai et al., 2 000 ), ultimately
leading to decrease in protein digestibility.Considering the fact that heat stressed birds use
glucogenic amino acids for glucose production (Nelson et al., 2000) during the process of
gluconeogenesis inbirds, which is metabolically expensive process (Nelson et al., 2000),
provision of sodium bicarbonate in their diets can decrease glucose production from amino
acids, which may lead to improved digestibility of protein during stress.
Addition/inclusion of NaHCO3 in the diet of layers exhibited more digestibility of
protein in these bids as compared to those of control group. Protein ingested by the birds is
broken down by the action certain enzymes to its constituent amino acids (a.a.) in the
gastrointestinal tract prior to absorption, and most of these a.a. require sodium (Leeson and
summer, 2001) for this process. Therefore, increase in digestibility of protein of the treated
groups may probably be due to the presence of more sodium ions concentration in the rations
containing sodium bicarbonate. Sodium containing compounds such as sodium bentonites
have been successfully used in sorghum containing diets to prevent deleterious effects of
tannins present in it, on digestibility of protein (Pasha et al., 2008).
The results of present experiment are in accordance with the investigation of Ahmad
(1997) who found maximum digestibility of protein in birds kept on 75% supplemented
sodium from sodium bicarbonate whereas, minimum digestibility of protein was noted in
birds kept on 100% supplemented sodium from sodium chloride. Banda-Nyirenda and Vohra
(1990) found significant improvement in apparent protein digestibility by treating sorghum
by sodium bicarbonate. The results are also in agreement with the observations of Choi and
Han (1982) who observed improved protein digestibility due to dietary inclusion of sodium
bicarbonate in broiler ration having low crude protein level.
Gorman and Balnave (1994) investigated that heat stress could lead to a metabolic
needs for the bicarbonate ions. Therefore, decreased digestibility of protein in control group
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might be due to lower bicarbonate and sodium levels which probably occurred due to
increased respiration. Sodium bicarbonate in the diet of broilers has shown to improve
digestibility and decrease losses caused by heat stress (Mirsalimi and Julian, 1993; Braton et
al., 1989). An improvement in protein digestibility and nitrogen retention was observed in
broilers offered sorghum grains treated with alkaline solution (Mohammed and Ali., 1988).
Sodium salts and electrolytes may respond differently to amino acid during heat
stress. Chen et al. (2005) investigated that respond to synthetic a.a. is affected by DEB.
Brake et al. (1998) found that increasing the Arginine: Lysine ratio with low levels of NaCl
in the diets of broilers reared at 31 °C, enhanced their body weight and efficiency of feed
utilization. However, Balnave and Brake (2001) observed that NaHCO3 enhanced the
performance with high Arg: Lys ratio in broilers. Gonzales-Esquerra and Leeson (2006)
observed that the Arg: Lys ratio, methionine source and time of contact to heat stress can
disturb the protein utilization in birds reared at higher ambient temperature.
5.5.3 Crude fiber Dietary inclusion of different levels of sodium bicarbonate depicted a significant
increase in digestibility of crude fibers in layers when compared to those fed diet without
addition of sodium bicarbonate. Increase in digestibility of crude fibers of the treated groups
may be due to availability of more sodium ions and bicarbonate ions concentration in rations
containing sodium bicarbonate. Dietary inclusion of sodium bicarbonate has also shown to
improve DEB by creating physiological conditions better for enhancing the digestibility of
nutrients. Pasha et al. (2008) used different levels of sodium bentonite in broiler rations and
found an improvement in nutrient digestibility as compared to the control group (without
sodium bentonit). Similarly, Santurio et al. (1999) and Salari et al. (2006) have also observed
improvement in nutrient digestibility due to addition of sodium bentonite in broiler diets.
High ambient temperature has shown a significant influence on feed consumption,
digestion, nutrients absorption and their metabolism (Macleod, 2004). As ambient teperature
shoots up, feed intake of the birds is reduced (Ain-Baziz et al., 1 996 ) to avoid the
thermogenic effect of heat increment associated with nutrient utilization, absorption and
assimilation (Koh and Macleod, 1999 ). However, at ambient temperature above 30 °C,
thermoregulatory system is activated and exhibits increase in blood flow to upper respiratory
tract and other organs associated in heat excretion i.e. combs and wattles, which intern causes
171
a decrease in digestibility ofcrude fibers.
Glucose is the main energy source for poultry birds. Absorption/assimilation of
glucose from the gastro intestinal tract requires sodium. Galactose also needs sodium for
transport system as glucose. Absorption of glucose, galactose, and amino acids require
transporters in the gastro intestinal tract that require sodium to be transported along with
these, as has been stated by Nelson and Cox, (2000). They have also pointed out that sodium
may remarkably be reduced in birds during high ambient temperature due to its loss via
excretion, which may reduce the process of digestion and absorption. However, addition of
NaHCO3 in such situation can improve digestibility of nutrients.
Sodium bicarbonate is associated in increasing sodium ions concentration (Fethiere et
al., 1994); improving electrolyte balance in the diet (Borges et al., 2003), decreasing the
losses caused by heat stress (Gorman and Balnave, 1994) and reduction in body temperature
(Smith and Teeter, 1989). As all these factors are related to the performance, therefore, it is
expected that inclusion of NaHCO3 in the diet may result in better performance of birds,
including better digestibility of crude fiber. Use of saline solutions has been a common
practice to stimulate nutrient digestion and absorption during heat stress (Jeukendrup et al.,
2009) in birds.
The birds fed diet without inclusion of NaHCO3 (control) exhibited the lowest
digestibility of crude fiber in the present study. Gorman and Balnave, 1994) have reported
that high ambient temperature (heat stress) may lead to an increase in metabolic requirements
for HCO3- ions. Keeping this fact in view, a possible explanation of decreased digestibility of
crude fibers in control birds may be the less availability of bicarbonate ions (HCO3-), which
probably occurred due to increased respiration. Therefore, presence of enough HCO3- ions
because of adding NaHCO3 in the diets of broilers might have improved digestibility and
decreased losses caused by heat stress (Mirsalimi and Julian, 1993; Braton et al., 1989).
On the other hand, presence of excessive levels/addition of NaHCO3 in the diets has
been reported to be toxic in White Leghorn layers (Davison and Wideman, 1992). This may
be the probable reason of decreased digestibility of crude fiber in the birds of group E, which
were fed a diet containing 2% NaHCO3. Another possible explanation of decrease in
digestibility of crude fibers in group E (2% NaHCO3) was because of accelerated passage of
feed (Ravindran et al., 2008).
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5.5.4 Ether extractDigestibility of ether extract was found to be significantly better in the birds receiving
diets containing sodium bicarbonate as compared to those of untreated group. Hyperthermia
seems to be the most possible contributing factor for decreased digestibility andabsorption of
ether extract in the birds fed diet without sodium bicarbonate as have been observed by Koh
and Macleod, ( 1999 ). Leeson and summer, (2001), while discussing the factors affecting
digestibility of fats, have also stated that fat digestibility is negatively affected in the birds
exposed to heat stress. These results are coincided to those reported by Bonnet et al. (1997)
in birds maintained under hyperthermic (heat stress) conditions.
On the other hand, the results have shown an increase in digestibility of ether extract
contents in layers fed diets containing different levels of sodium bicarbonate. Increase in
digestibility of ether extract of the treated groups may probably be due to more Na+ and
HCO3- concentration in the diets containing sodium bicarbonate (Gormanand Balnave, 2004).
Findings of our study have also exhibited that digestibility of ether extract was affected due
to the level of feed intake of birds as has been reported by Ravindran et al.(2008).
Bile salts also reported to have an important role in the digestibility and absorption of
fats (Leeson and summer, 2001)). These are sodium containing salts like sodium
glycocholate and sodium taurocholate, which emulsify fats and thus rendering them
digestible. Therefore, it is quite possible that addition of NaHCO3 might have improved the
production of bile salts in the birds receiving diets containing different levels of NaHCO3,
which increased the digestibility of ether extract of the diets. Moreover, buffers like NaHCO3
when added to diets may increase or stabilize the pH, improving enzymatic activity (Paggi et
al., 1999), hence result in better digestion of the nutrients. Contribution of better electrolyte
balance in the birds fed diets containing different levels of NaHCO3 has already been
discussed in the previous section (5.5.2) and the same is expected in this particular case. In
contrast to the findings of present study, Ahmad (1997) observed that digestibility of ether
extract in broiler was not affected by dietary inclusion of NaHCO3. This contradiction in the
results might because of varying species of the birds used in these studies.
5.6 MineralsDietary inclusion of sodium bicarbonate significantly influenced the absorption of all
minerals (Ca, P, Fe, Na and K) studied in this trial. Birds receiving diets containing sodium
bicarbonate exhibited better absorption of these minerals as compared to those of untreated
group (control). Minerals and trace elements are essential for optimum performance (Leeson
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and summers, 2001) of poultry birds. Therefore, increased mineral absorption/assimilation in
NaHCO3 fed birds may either be due to more accessibility of minerals because of increased
feed intake (Karimian et al., 2004) or improved DEB (Borges et al., 2003), or both.
Another possible reason of increased absorption of the minerals by the birds receiving
sodium bicarbonate in their diets may be increased availability of sodium ions (Fethiere et
al., 1994), leading to more uptake of the minerals. Body temperature record of the
experimental birds of treated groups has also shown a decrease in the body temperature of
birds probably because of enough availability of minerals and HCO3- ions to meet body
requirements (Gorman and Balnave, 1994), thus repairing/decreasing the losses of these
elements caused by heat stress (Mirsalimi and Julian, 1993; Braton et al., 1989).
The birds of control group, which were fed diet without inclusion of sodium
bicarbonate exhibited lower feed intake as compared to their counterparts, resulting in less
availability of bicarbonate ions. This situation may have caused increased respiration rate and
ultimately led to heat stress. Moreover, heat stress in birds may lead to increase in metabolic
requirements for the HCO3 ions (Gorman and Balnave, 1994) and less availability of these
ions to the birds fed control diet might have ultimately resulted in low absorption of the
minerals. Therefore, decreased absorption of minerals in control group may be attributed to
less feed intake of the birds.
Absorption of mineral ions may be accelerated by dietary sodium salts, which can
enhance function of digestive and absorptive enzymes and passive permeability (Nelson and
Cox, 2000). Increased influx of dietary sodium salts may also cause changes in fluid
absorption and/or secretion, and could also stimulate intestinal HCO3- secretion. It has been
reported that phytates and fiber present in plant ingredients reduce the availability of calcium
and other minerals (Champagne, 1989; Sugiura et al., 1998), however, absorbability of these
key minerals can be improved by dietary addition of sodium bicarbonate.
Minerals absorption may decrease under hot conditions (Smith and Teeter, 1987). In
addition to effect on specific nutrients, gastrointestinal size is also reported to be decreased in
heat-exposed chickens (Savory, 1986; Mitchell and Carlisle, 1992). In all of these studies, the
high ambient temperature also caused some reduction in feed intake. Therefore, decreased
mineral absorption in the birds kept under heat exposure may have been somewhat due to
decrease in feed consumption. Reduced minerals absorption in birds exposed to heat stress
174
had also been observed by Smith and Teeter (1987) and Belay et al.(1992). However,
improvement in absorption of minerals in the birds of treated groups may be obtained by
dietary addition of sodium bicarbonate (table 5.2).
In fact, a big proportion of dietary minerals is absorbed/assimilated from the small
intestine receiving a Na+ reliant transport system (Leeson and Summer, 2001), therefore,
transport of mineral ions and sodium must be at the same time. However, presence of an
optimum level of NaHCO3in the diet is important for its maximum utility/efficiency
(Ravindran et al., 2008). Therefore, a possible reason for decrease in absorption of these
minerals in the birds fed diet containing higher level of sodium bicarbonate (2%)when
compared to those of other treated groups, may be its (NaHCO3) less effectiveness/utility
because of increased passage rate of digesta of the birds.
An other possible reason of decreased absorption of minerals in the birds of control
group may be decreased blood flow towards their digestive tracts under the influence of high
ambient temperature. At higher ambient temperature, thermoregulatory system of the birds
might have been activated and caused an increased blood flow to their upper respiratory
tracts and organs associated in heat dissipation (Wolfenson, 1 986 ).Obviously this situation
might have decreased blood flow to the digestive tracts andconsequently resulted in reduced
absorption of minerals in control birds as compared to those of NaHCO3 treated groups.
ConclusionsIn making final assessment of the study, inclusion of NaHCO3 in the diet of layers
during summer improved production performance, egg quality characteristics, blood profile,
immune response, and digestibility of proteins, fats and carbohydrates as well as absorption
of some minerals, in caged White Leghorn layers. Addition of NaHCO3 @1% (DEB= 262) in
the diet of the layers was found to be the most effective in ameliorating/mitigating the effect
of heat stress upon the performance of the caged layers.
RecommendationsDietary inclusion of sodium bicarbonate @ 1% is recommended to be used for better
efficiency of production performance, blood profile, immune response against Newcastle
disease virus, digestibility of nutrients and absorption of some minerals, in the caged layers
during summer.
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Practical Implications1- Sustainable productivity of layers during summer receiving 1% sodium bicarbonate in
diet.
2- Reduction in expenditures to overcome heat stress using mechanical devices.
3- Reduction in economic losses due to mortality and reduced production performance
of layers reared in heat stress conditions.
Future work1- Further research may be conducted for a longer periods to compare production
performance of layers receiving NaHCO3 during first and second production cycles.
2- The effect of NaHCO3 at different ages and seasons can also be probed along with
economics of production.
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CHAPTER-6
SUMMARYPakistan is situated in the subtropical zone of Northern Hemisphere of the world
where temperature usually remains well beyond the higher side of thermo-neutral zone (25-
370C) for greater part of the year. The optimum temperature for efficient performance is 19-
220C for laying birds; however, ambient temperature especially on higher side is very
disruptive and may reduce survival rate and production. Egg production declines drastically,
thereby adversely affecting the economics of poultry production. Hence, low egg production
may lead to excessive culling of layers from a flock.
Different techniques are being used in poultry production to combat heat stress,
which include nutritional manipulations such as dietary addition of oils reduction in protein
level of feed, supplementation of feed with limiting amino acids and management practices
like intermittent feeding, feeding the birds in cool hours of the day, time limit feeding,
sprinkling of water, evaporative cooling, improved ventilation and supplementation of
electrolytes. Many studies have reported beneficial effects of supplementing drinking water
and/or diets of broilers with sodium bicarbonate as sodium source. However, scientific
information regarding the use of NaHCO3 in layer diet is still scarce. Therefore, this study
was planned to investigate the effect of dietary inclusion of NaHCO3 on production
performance, nutrient digestibility and blood profile of caged layers, during summer.
One hundred sixty commercial layers (24 weeks old) were purchased from a poultry
farm and were reared in a group for one week (adaptation period).At the start of 26 th week of
age, these layers were divided into 20 experimental units/replicates (8 layers/replicate),
which were further allotted to five treatment groups (4 replicate/treatment).Five diets (A, B,
C, D and E) were prepared with or without addition of sodium bicarbonate. Diet A, was
without sodium bicarbonate and served as control whereas, diets B, C, D, and E contained
0.5, 1.0, 1.5 and 2.0% sodium bicarbonate, respectively. All the diets were iso-nitrogenous
(CP 17%) and iso-caloric (ME 2700 Kcal/Kg) and were formulated according to the
requirements prescribed by NRC (1994). The diets were fed to the experimental birds ad
libitum, for 12 weeks (26-37 weeks of age).
Data on feed consumption, number of eggs, egg weight and egg mass laid by the
177
birds were recorded. From these observation feed conversion ratios on the basis of per dozen
eggs and per kg egg mass produced, were calculated. Five eggs from each replicate were
checked weekly for their shell thickness, yolk index, albumen index, Haugh unit yolk pH,
albumen pH, specific gravity and yolk cholesterol. At the last day of each week, rectal
temperature and respiration rate of three layers from each replicate were recorded. At the end
of the experiment (37th week of age), blood sample of two birds/replicate were collected for
hematological analysis, hormonal profile (T3, T4, Cortisol, Progesterone and Estrogen), serum
proteins, lipids profile, plasma electrolytes (HCO3–,Cl Na+ and K+), plasma minerals analysis
(Calcium and Phosphorus).Blood samples were also collected from two birds from each
replicate 10 days post vaccination of 1st, 2nd, and 3rd vaccination to the immune response.
Digestibility of dry matter, crude protein, crude fiber, ether extract and absorption of
minerals (calcium, phosphorus, sodium, potassium and iron)was also determined in a
separate trial, during the last week (38th week) of experiment. For this purpose 30 layers
obtained from the same batch, as were used for the performance trial, were put into
individual cages. Each bird acted as a replicate. These birds were allotted to the treatment
groups (A, B, C, D and E) such that each treatment received 6 birds (replicate). The diets
used in this trail were the same, which were used in the performance trial except that 1%acid
insoluble ash was added in these diets, as an indigestible marker. Economics of each
treatment was calculated at the end of experiment. The data thus collected were statistically
analyzed under completely randomized design.
The results of the study revealed that dietary inclusion of sodium bicarbonate
significantly (P<0.05) improved feed intake, weight gain, feed efficiency, egg production and
egg weight of the birds. The birds of Group C which were fed diet containing 1% NaHCO3
performed better than those of its counterparts and exhibited better feed consumption, weight
gain, egg production and egg mass production. Moreover, the birds fed diets containing
sodium bicarbonate utilized their feed more efficiently than those fed diet without any such
addition (control).
Egg quality characteristics such as specific gravity, shell thickness, albumen height,
haugh unit score, yolk height and yolk diameter were also improved by dietary inclusion of
sodium bicarbonate. Yolk cholesterol was found to be the lowest in the eggs produced by the
birds of group C, which were fed diet containing 1% NaHCO3. Whilst, pH values of yolk and
178
albumen were found to be the highest in the birds of group E which were fed diet containing
2% NaHCO3.
Dietary inclusion of sodium bicarbonate significantly reduced rectal temperature and
respiration rate of layers; however, their water intake was significantly increased. Serum
glucose and WBCs count was found to be higher in birds of control group. Birds of group C,
which were fed diet containing 1% sodium bicarbonate, showed maximum concentration of
hemoglobin in their blood. However, red blood cells count, PCV and ESR values were not
affected due to the dietary treatments. Serum urea concentration was found to be the highest
in the layers of control group whereas, serum uric acid concentration was found to be the
highest in birds of group E (2% NaHCO3). However, values of serum creatinine and alkaline
phosphatase were not affected due to the treatments. Dietary inclusion of sodium bicarbonate
significantly increased serum total protein and albumen concentration of the birds. Birds fed
diets containing 1% sodium bicarbonate exhibited higher concentration of these proteins as
compared to those of other groups. However, serum globulin contents of birds remained
unaffected due to the dietary inclusion of sodium bicarbonate.
Plasma sodium level showed a linear increase with increase in the level of dietary
inclusion of sodium bicarbonate. Serum potassium and bicarbonate also showed increasing
trend due to the dietary treatments. Whereas, birds fed diets containing sodium bicarbonate
exhibited decreased levels of serum chlorides and serum pH. However, plasma calcium and
potassium levels remained unaffected due to sodium bicarbonate treatments. Serum
cholesterol, triglycerides and LDL were decreased by the dietary inclusion of sodium
bicarbonate and birds fed diets containing 1% NaHCO3 exhibited lower concentration of
these lipids as compared to those of other treated groups. However, concentration of HDL
was found to be increased in the birds fed diets containing 1% sodium bicarbonate.
The birds of group C (1% NaHCO3) showed a marked increase in the concentrations
of estrogen, progesterone, T3 and T4 hormones, whereas, serum cortisol was higher in birds
exposed to higher temperature and fed diet without NaHCO3).Serum SGPT concentration
was not affected due to the dietary treatments. While, serum SGOT concentration was
significantly decreased due to the use of NaHCO3 in the diets during heat stress. Dietary
inclusion of NaHCO3 has also shown a significant (P<0.05) increase in immune response
against Newcastle disease virus in the layers when compared to those of control group.
179
Digestibility of DM, crude protein, crude fiber and ether extract was found to be
higher in the birds fed diets containing sodium bicarbonate than those of control group. The
best digestibility of these nutrients was observed in the birds fed diet containing 1% sodium
bicarbonate among those of treated groups. Similarly, absorption of the minerals studied
(calcium, phosphorus, sodium, potassium and iron)was also found to higher in the treated
birds as compared to untreated ones.
Findings of this study clearly indicated that dietary inclusion of sodium bicarbonate
exhibited beneficial effect on production performance, digestibility of nutrients and
biochemical profile of the layers during summer, addition of 1% NaHCO3 being the best. It
was, therefore, concluded that dietary inclusion of 1% NaHCO3 may be used for efficient and
economical production performance of layers during summer season. However, effects of
dietary sodium bicarbonate on production performance of birds at different ages and during
different seasons of the years can be addressed, as future research work.
180
LITERATURE CITEDAbraham, G. E. 1981. Radio-assay Systems in Clinical Endocrinology. Marcel Dekker, Inc.,
New York, USA.
Abraham, G. E., W. D. Odell, R. S. Swerdoloff and K. Hopper. 1972. Simultaneous
radioimmunoassay of plasma FSH, LH, progesterone, 17-hydroxyprogesterone and
estradiol-17 B during the menstrual cycle. J. Clin. Endocrinol. Metabol. 34: 312-318.
Aerni, V., H. El-Lethey and B. Wechsler. 2000. Effect of foraging material and food form on
feather pecking in laying hens. Br. Poult. Sci.41: 16-21.
Ahmed, A. 1993. Effect of cage density and ascorbic acid supplementation on the
performance of layers during summer. M.Sc. Thesis. University of Agricuture,
Faisaiabad, Pakistan.
Ahmad, H., H. Rashid, M. F. Ullah and M. Akram. 1993. Influence of ascorbic acid
supplementation on the performance of layers kept in cages during summer season. J.
Anim. Poult. Sci. 3: 99-100.
Ahmad, R. 1997. Growth performance and electrolyte balance of broiler as affected by two
sources of sodium. M. Sc. (Hons.) Thesis, Department of Poultry Science.
Universityof Agriculture, Faisalabad.
Ahmad, T., M. Sarwar, M. Nisa, A. Haq, Z. Hasan. 2005. Influence of varying
sources of dietary electrolytes on the performance of broilers reared in a high
temperature environment. Anim. Feed Sci. Technol. 120: 277–298.Ahmad, F., S. Mahmood, Z. Rehman, M. Ashraf, M. Alam and A. Muzaffar. 2006. Effect
of feeding management on thermoregulation, production performance and
immunological response of broilers during summer. Int. J. Agri. Biol. 4: 550–553.
Ahmad, M. M. and M. Sarwar. 2006. Dietary electrolyte balance implications in heat stressed
broilers. World’s Poult. Sci. J. 62: 638-653.
Ahmad, T., T. Khalil, T. Mushtaq, M. A. Mirza, A. Nadeem, M. E. Barabar and G. Ahmad.
2008. Effect of KCl supplementation in drinking water on broiler performance under
heat stress conditions. Poult. Sci. 87: 1276-1280.
Ahmad, T., T. Mushtaq, M. A. Khan, M. E. Babar, M. Yousaf, Zia-ul-Hassan, Z. Kamran.
2009. Influence of varying dietary electrolyte balance on broiler performance under
tropical summer conditions. J. Anim. Physiol. Anim. Nutr. 93: 613–621.
181
Ain-Baziz, H. A., P. A. Geraert, J. C. F. Padilha and S. Guillaumin. 1996. Chronic heat
exposure enhances fat deposition and modifies muscle and fat partition in broiler
carcasses. Poult. Sci. 75: 505-513.
Ait-Boulahsen, J. D. Garlich, and F. W. Edens. 1989. Effect of fasting and acute heatstress
on body temperature, blood acid-baseand electrolyte status in chickens. Comp.
Biochem. Physiol. 94A:683.
Akram, M., H. Ahmed, M. L. Khan and A. Iqbal. 1999. Egg quality characteristics
influenced by frequency of body wetting of layers during summer. Inter. J. Agri. Biol.
1: 45-47.
Akram, M., H. Ahmed, M. S. Anjum, A. Iqbal, M. Usman and T. Tasneem. 1999. Impact of
surface wetting upon the physiologicalbehavior of commercial layers during summer.
Pak. J. Sci. 51: 1-3.
Ali, M. A. and M. F. Ullah. 1970. Effect of Vitamin C supplementation on egg shell
thickness in layers ration during summer. Pak. J. Agri. Res. 8: 90-95.
Ali, S., C. M. Saleem, and M. S. Anjum. 1995. Effect of ascorbic acid on the growth and
subsequent performance of Fayoumi birds. Pak. J. Sci. 47: 113-115.
Allan, W. H., J. E. Lancester and B. Toth. 1978. A report on the production and use of ND
vaccines. F.A.O. (UN). Rome: pp: 57-62.
Allison, L. E., L. Bernstein and C. A. Bower. 1954. Diagnosis and Improvement of Saline
and Alkali Soils. L. A. Richards (Ed.). Oxford and IBH Publishing Company, New
Delhi, India. pp: 129- 134.
Al-Zujajy, R. J., H. El-Hammady and M. A. Abudulla. 1978. The use of aircoolers in broiler
houses under subtropical conditions in Iraq. Br. Poult. Sci., 19: 731-735.
Amerah, A.M., V.Ravindran, R. G.Lentle and D. G. Thomas. Influence of feed particle size
and feed form on the performance, energy utilization, digestive tract development,
and digesta parameters of broiler starters.Poult. Sci. 86: 2615-2623, 2007.
Anerah, A. M., V. Ravindran, R. G. Lentle. 2007. Feed particle size: implications on
the digestion and performance in poultry. World's Poult. Sci. J. 63: 439-451.Andrade, De. A. N., J. C. Rogler and W. R. Featherston. 1976. Influence of constant elevated
temperature and diet on egg production and shell quality. Poult. Sci. 55: 685-693.
Angelovicova, M. 1997. Reduced cholesterol content in market eggs as influenced by diets
withAmaranthus (Amaranthus hypochondriacus). Achta-Zootechnica. 53: 81-87.
182
Angilletta, M. J., B. S. Cooper, M. S. Schuler, J. G. Boyles. 2010. The evolution of thermal
physiology in endotherms. Frontiers in Bioscience E2: 861–881.
Anjum, M. S. 2000. Productive performance and physiological behavior of White Leghorn
caged layers under different heat combating practices during summer. PhD. Thesis,
Department of Poultry Science. University of Agriculture, Faisalabad.
AOAC, 2010. Official Methods of Analysis. Association of official analytical chemists.
EUA. 19th edition, Washington D. C. USA.
Arad, Z, E. Moskovits and J. Marder. 1975. A preliminary study of egg production and heat
tolerance in a new breed of fowl (Leghorn x Bedouin). Poult. Sci. 54: 780-783.
Arad, Z. and J. Marder. 1984. Strain differences in heat resistance to acute heat stress,
between the Bedouin desert fowl, the White Leghorn and their crossbreeds. Comp.
Biochem. Physiol. 72: 191-193.
Arad, Z., J. Marder and U. Eylath. 1983. Serum electrolyte and enzyme responses to heat
stress and dehydration in the fowl (Gallus domesticus) Comp. Biochem. Physiol. 74:
449-453.
Arieli, A., A. Meltzer and A. Berman. 1980. The thermoneutral temperature zone and
seasonal acclimation in the hens. Br. Poult. Sci., 21: 471-478.
Arima, Y., F. B. Mather and M. M. Ahmad. 1976. Response of egg production and shell
quality to increases in environmental temperature in two age groups of hens. Poult.
Sci. 55: 818- 820.
Astier, H. S. and W. S. Newcomer. 1978. Extrathyroid conversion of thyroxine to
triiodothyronine in a bird: the Pekin duck. Gen. Comp. Endocrinol. 35: 496-499.
Atlan, A., O. Atlan, S. Ozkan, Z. Acikgoz and K. Ozkan. 2000. Effect of dietary sodium
bicarbonate on egg production and egg quality of laying hens during high summer
temperature. Archiv fur Geflugelkunde. 64: 269-272.
Attlla, A.A., M. R. Fatma, S. M. Salwa and M. G.Doaa, 2002. Effect of some electrolytes on
the performance and some physiological parameters of laying hens.Egypt. Poult. Sci.
22: 219-233.
Austle, R. E. and K. Keshavarz. 1988. Interaction of dietary calcium and chloride and the
influence of monovalent minerals on egg shell quality. Poult. Sci. 67: 750.
Badran, A. M. M. 2003. Effect of Sodium bicarbonate supplementation on laying
performance and physiological parameters during hot weather. M.Sc. Thesis Faculty
of Agriculture, Cairo University.
183
Baghel, R.P.S. K. and Pradhan, 1989. Energy, protein and limiting amino acid requirements
of broilers at very high ambient temperature. Br. Poult. Sci., 30: 295-304
Bains, B. S. 1996. The role of vitamin C in stress management. World’s Poult. Misset. 12:
38-41.
Balnave, D. and I. Gorman, 1993. A role for sodium bicarbonate supplements for
growing broilers at high temperatures. World’s Poult. Sci. J. 49: 236- 241.Balnave, D. and J. Brake. 2001. Different responses of broilers at low, high or cyclic
moderate – high temperature to dietary sodium bicarbonate supplementation due to
differences in dietary formulation. Aus. J. Agric. Res. 52: 609-613.
Balnave, D. and S. K. Muheereza. 1997. Improving egg shell quality at high temperatures
with dietary sodium bicarbonate. Poult. Sci. 76: 588-593.
Banda-Nyirenda, D. B. C. and P. Vohra. 1990. Nutritional improvement of tannin containing
sorghums (Sorghum bicolor) by sodium bicarbonate. Cereal Chem. 67: 533-537.
Barbosa L. R., J. H. V. da-Silva, P. E. N. Givisiez, T. D. D. Martins, T. D. D. Saraiva
, F. G. P. Costa and M. Macari. 2014. Influence of environmental temperature
andelectrolyte balance on the performance of quails(Coturnix Coturnix
Coturnix). Brazilian J. Poult. Sci. 16: 249-256.Barham, D. and P. Trinder. 1972. An improved color reagent for thedetermination ofblood
glucose by the oxidase system. Analyst.97: 142-145.
Barton, T. L. 1996. Relevance of water quality to broiler and turkey performance. Poult. Sci.
75: 854-856.
Batal, A. B., C.M. Parsons.2002. Effects of age on nutrient digestibility in chicks fed
different diets. Poult. Sci. 81: 400-407.Bearse, G. E., L. R. Berg and A. H. Massey. 1962. Protein level of the diet and incidence of
blood spots in chicken eggs. Poult. Sci. 41: 1625- 1626.
Belay, T., and R. G. Teeter. 1993. Broiler water balance and thermo-balance during thermo-
neutral and high ambient temperature exposure. Poult. Sci. 72:116.
Belay, T., C. J. Wiernusz, and R. G. Teeter, 1992. Mineralbalance and urinary and fecal
mineral excretion profile ofbroilers housed in thermo-neutral and heat-
distressedenvironments. Poult. Sci. 71:1043–1047.
Bell, D. D. and C. J. Adams. 1992. Performance responses to temperature as affected by age
in table egg flocks. Proceedings of 19thPoultry Congress, Amsterdam, Netherlands,
20-24 Sept. 1992.
184
i
Bell, G. H., J. N. Davidson and D. E. Smith. 1975. Text book of physiology and
biochemistry. 8th Ed. The English Language Book Society and Churchill,
Livingstone, U.K.
Ben Nathan, D., E. D. Heller and M. Perek. 1976. The effect of short heat stress upon
leucocyte count, plasma corticosterone level, plasma and leucocytes ascorbic acid
content of chickens. Br. Poult. Sci. 17: 481-485.
Benjamin, M. M. 1978. Outline of Veterinary Clinical Pathology. 3rd Ed. The Iowa State
University Press, USA.
Ben-Nathan, D., E. D. Heller, and M. Perek. 1977. The effects of starvation on antibody
production of chicks. Poult. Sci. 56:1468-1471.
Benton, C. E., D. Balnave and J. Brake. 1998. The use of dietary minerals during heat stress
in broilers: Review. The Professional Animal Scientist 14: 193-196.
Berne, R. M. and M. N. Levy. 1993. Physiology. R. Ferrall (Ed.). pp 797. Mosby Year
Book, Inc., St. Louis, MO.
Bernier, G., J. B. Phaneuf, R. Filion. 1977. Necrotic enteritis in broiler chickens. Study on
the factors favoring the multiplication of Clostridium perfringens and the
experimental transmission of the disease. Can. J. Comp. Medi. 41: 112-11 6.
Bligh, J. and K. G. Johnson. 1973. Glossary of terms for thermal Physiology. J. Appl.
Physiol. 35: 941-961.
Bobek, S., J. Niegoda, M. Pietras, M. Kacinska and Z. Ewy. 1980. The effect of acute cold
and warm ambient temperature on the thyroid hormone concentration in blood
plasma, blood supply, and oxygen consumption in Japanese quail. Gen. Comp.
Endocrinol. 40: 201-210.
Bogin, E., Y. Weisman and Y. Friedman. 1981. Effect of heat shock on the biochemical
composition of chicken blood. Ref. Vet. 38: 98-104.
Bohren, B. B., J. C. Rogler and J. R. Carson. 1982. Performance at two rearing temperatures
of White Leghorn lines selected for increased and decreased survival under heat
stress. Poult. Sci. 61: 1939-1943.
Bonnet, S., P. A. Geraert, M. Lessire, B. Carre, S. Guillaumin.1997. Effect of high ambient
temperature on feed digestibility in broilers. Poult. Sci. 76: 857-863. Bonsembianate, M., G. M. Cliericato and L. Bailoni. 1990. Use of sodium bicarbonate in
diets for meet turkeys reared at high environmental temperature and humidity. Rev.
Avic. 59: 37-41.
185
Bonsembiante, M. and G. M. Chiericato. 1990. Effect of sodium bicarbonate on blood
chemistry of meat turkeys subjected to stress (high environmental temperature and
humidity) Rivista di Avicoltura. 59: 85-89. (Pool. Abst. 1990; 18: 2044).
Bonsembiante, M., G. M. Chiericato and L. Bailoni. 1988. The effect of Sodium bicarbonate
on the performance of broilers subjected to temperature and humidity stress. Rivista
di avicoltura 57 (6): Poult. Abstracts 1989, 15(1).
Bordas, A. and P. Merat. 1984. Effects of naked-neck gene on traits associated with egg
laying in dwarf stock at two temperatures. Br. Poult. Sci. 25: 195-207.
Borges, S. A. 2001. Balanço eletrolítico e sua inter-relação com o equilíbrio ácido-base em
frangos de corte submetidos a estresse calórico tese. Jaboticabal (SP): Universidade
Estadual Paulista.
Borges, S. A., A. V. Fisher da Silva, J. Ariki, D. M. Hooge, K. R. Cummings. 2003a.
Dietary electrolyte balance for broiler chickens under moderately high ambient
temperatures and relative humidities. Poult Sci. 82: 301–308.
Borges, S. A., A. V. Fisher da Silva, J. Ariki, D. M. Hooge, K. R. Cummings. 2003b.
Dietary electrolyte balance for broiler chickens exposed to thermo-neutral or heat–
stress environments. Poult Sci. 82: 482–435.
Borges, S. A., A. V. Fisher da Silva, A. Majorka, D. M. Hooge, K. R. Cummings. 2004a.
Physiological responses of broiler chickens to heat stress and dietary electrolyte
balance (sodium plus potassium minus chloride, milliequivalents per kilogram). Poult
Sci. 83: 1551–1558.
Borges, S. A., A. V. Fisher da Silva, A. D. A. Meira, T. Moura, A Maiorka, A Ostrensky.
2004 b. Electrolyte balance in broiler growing diets. Int. J. Poult. Sci. 3: 623–628.
Bottje, W. G., P. C. Harrison. 1985. The effect of tap water, carbonated water, sodium
bicarbonate and calcium chloride on blood acid- base balance in cockerels subjected
to heat stress. Poult. Sci. 64: 107-113.
Bottje, W. G., T. J. Kaup, S. Wang. 1989. Effect of ammonium chloride on the bicarbonate
buffer system in heat-stressed broilers. Br. Poult. Sci. 30: 899-905.
Boulahsen, A., J.D. Garlich and F.W. Edens, 1989. Effect of fasting and acute heat stress on
body temperature, blood acid-base and electrolyte status in chickens. Comp.
Biochem. Physiol. 94: 683- 687.
Bowen, S.J. and K.W. Washburn. 1985. Thyroid and adrenal response to heat stress in
chickens and quail differing in heat tolerance. Poult. Sci. 64: 149-154.
186
Brake, J. and M. Baker. 1982. Physiological changes in cage layers during forced molting. 4
Leucocytes and packed cell volume. Poult. Sci.61: 790-795.
Brake, J., D. Balnave and J. J. Dibner. 1998. Optimum dietary arginine: lysine ratio for
broiler chickens is altered during heat stress in association with changes in intestinal
uptake and dietary sodium chloride. Br. Poult. Sci. 39: 639-647.
Branton, S. L., F. N. Reece and J. W. Deaton. 1986. Use of ammonium chloride and sodium
bicarbonate in acute heat exposure of broilers. Poult. Sci. 65: 1659-1963.
Brenes, A., M. V. Diez, P. Yuste, L. A. Rubio. 1988. Effect of salt and sodium bicarbonate
on abdominal fat and bone minerals contents in chicks. Archivos de Zootecnia. 37:
105-113.
Brigmon, R. L., E. L. Besch and F. B. Mather. 1992. Seasonal temperature and its influence
on plasma corticosterone, tri-iodothyronine, thyroxine, plasma protein and packed
cell volume in mature male chickens. Biochem. Physiol. 102: 289-293.
Bryden, W.L., P.H. Selle D.J. Cadogan. 2009. A review of the nutritive value of sorghum for
broilers.RIRDC Publication 09/077. A report for the rural industries research and
development corporation.
Buster, J. E. and G. E. Abraham. 1975. The applications of stored hormones
radioimmunoassays to clinical obstetrics. Obstet. Gynoc. 46, 489.
Calder, W. A., J. R. King. 1974. Thermal and Caloric Relations of Birds. In: Farner DS, King
JR, editors. Avian Biology, Volume IV. New York: Academic Press. pp. 259–413.
Cannon W. B. 1929. Bodily changes in Pain, Fear and rape: An account of recent research
into the function of emotional excitement. 2nd ed. Appleton, NY.
Carlson, G. P. 1997. Fluid, electrolyte and acid-base balance. In: Clinical Biochemistry of
Domestic Animals. Eds.Kaneko J. J., Harvey J. W., Bruss M. L.5th Ed.Academic
Press, Boston. pp: 485–516.
Carrasco, G. A., L. D.Van de Kar .2003. Neuroendocrinem pharmacology of stress. Eur. J.
Pharmacol. 463: 235–272.
Chakraborty, A. K. and D. P. Sudhu. 1983. Effect of acute heat stress and its modifications
by adrenaline and adrenolytic drugs in pigeons. Int. J. Anim. Sci. 53: 575-578.
Charles, D. R. 2002. Responses to the thermal environment. In: Poultry Environment
Problems, A guide to solutions (Charles, D. A. and Walker, A. W. eds.), Nottingham
University Press, Nottingham, United Kingdom, pp: 1-16.
187
Champagne, E.T. 1989. Low gastric hydrochloric acid secretion and mineral bioavailability.
In: Dintzis, F. R.; Laszlo, J. A. (Eds.) Mineral absorption in the monogastric GI tract.
New York: Plenum Press. Pp: 173-184.
Charmandari E., Tsigos C., Chrousos G. Endocrinology of the stress response. Annu Rev.
Physiol. 2005; 67:259–284.
Chaudhry, M. R. and M. B. Sial. 1972. Studies on the effects of lowered temperature during
summer nights on the productive behavior of Leghorn and Lyallpur Silver Black
layers. Pak. J. Agric. Res. 10: 64-69.
Chaudhry, M. R. and M. B. Sial. 1973. Studies on the effect of lowered temperature during
summer nights on the physiological behavior of white Leghorn and Lyallpur Silver
Black Layers. Pak. J. Sci. 25: 179-182.
Cheng, T. K., C. N. Coon and M. L. Hamre. 1990. Effect of environment of laying hens.
Poult. Sci. 69: 774-780.
Ching, C.Y. 1992. Effects of acute heat stress on the blood characteristics of Taiwan country
chickens and broilers. J. Chin. Society Anim. Sci. 21: 57-66.
Choi, J. H. and I. K. Han. 1983. Dietary interaction of phosphorus with sodium from either
chloride or bicarbonate affects laying hen performance. Poult. Sci. 62: 341-344.
Chotesangasa, R. 1992. Effect of feed restriction on basal steroid hormone levels and egg
production in the layer. Kasetsart J. Nat. Sci. 26: 384-392.
Chrousos, G. P., and P. W. Gold. 1992. The Concepts of Stress and Stress System Disorders.
JAMA: The Journal of the American Medical Association 267: 1244-1252.
Connor, J. K., K. F Arnold. 1972. The effects of calcium, fat and sodium bicarbonate on
eggshell strength. Aust J Exp Agric Anim Husb. 121:146–151.
Cooke, B. C. and H. M. Raine. 1986. Nutrients requirements of poultry and nutritional
research. (Editor KN Boorman and C. Fisher). Butterworths.
Cowan, P. J. and W. Michie. 1978. Environmental temperature and broiler performance: The
use of diets containing increased amounts of protein. Br. Poult. Sci. 19: 601-605.
Cowan, P. J. and W. Michie. 1980. Increasing the environmental temperature later in lay and
performance of the fowl. Br. Poult. Sci. 21: 339-343.
Crimes, G. R. and R. E. Moreng. 1965. Body temperature response to breed, environmental
temperature and ascorbic acid. Poult. Sci. 44: 1374.
Daghir, N. J. 1991. Broiler feeding and management in hot climate. Zootecnica International,
10: 32-37.
188
Dai, N. V. and W. Bessei. 2007. Potassium chloride supplementation in drinking water of
laying hens as a means to maintain high productivity under high ambient temperature.
Conference International Agricultural Research for Development, Germany. pp: 1-6.
Dai, N. V., W. Bessei, 2009: The effects of sodium chloride and potassium chloride
supplementation in drinking water on performance of broilers under tropical summer
conditions. Arch.Gefluegelk. 73: 41-48.
Damron, D. L., W. L. Johnson, and L. S. Kelly. 1986. Utilization of sodium from sodium
bicarbonate by broiler chicks. Poult. Sci. 65: 782-785.
Daniel, M. and D. Balnave. 1981. Responses of laying hens to gradual and abrupt increases
in ambient temperature and humidity. Aus. J. Exp. Agric. Anim. Husb. 21: 189-195.
Danny, M. H. 1995. Dietary electrolyte influence on metabolic processes of poultry. Feed
Stuff. 4: 14-18.
Datta, P., P. C. Gangwar, R. K. Srivastava and D. P. Dhingra. 1984. Effect of environmental
cooling on growth attributes in broilers. Int. J. Anim. Sci. 54: 77-88.
Davison, S. and R. F. Wideman. 1992. Excess sodium bicarbonate in diet and its effect on
Leghorn chicken. Brit. Poult. Sci. 33: 859–870.
Deaton, J. W., J. L. McNaughton and B. D. Lott. 1982. Effect of heat stress on laying hens
acclimated to cyclic versus constant temperature. Poult. Sci. 61: 875-878.
De-Basilio, V., Vilarino, M., Yahav, S. and M. Picard. 2001. Early age thermal conditioning
and a dual feeding program for male broilers challenged by heat stress. Poult. Sci.80:
29-36.
Deng, W., X. F. Dong, J. M. Tong, and Q. Zhang. 2012. The probiotic Bacillus licheniformis
ameliorates heat stress-induced impairment of egg production, gut morphology, and
intestinal mucosal immunity in laying hens. Poult. Sci. 91:575–582
Depeters, E. J., A. H. Fredeen, D. L. Bath and N. E. Smith. 1984. Effect of sodium
bicarbonate addition to alfalfa hay-based diets on digestibility of dietary fractions and
rumen characteristics. J. Dairy Sci. 67:2344.
Despotis G. J., A. Alsofier, C. Wltoque, T. N. Zoys, L. T. Goodnough, S. A. Santoro, K. M.
Kater, P. Bames and D. G. Lappas. 1996. Evaluation of complete blood count results
from a new, on site hemocytometer compared with laboratory based hemocytometer.
Crit. Care Med. 24: 1163-1167.
189
Deyhim, F., R. G. Teeter. 1991. Research note: sodium and potassium chloride drinking
water supplementation effects on acid-base balance and plasma corticosterone in
broilers reared in thermoneutral and heat distressed environments. Poult. Sci. 70:
2551-2553.
Deyhim, R. G. Teeter. 1995. Effect of heat stress and drinking water salt supplements on
plasma electrolytes and aldesterone concentration in broiler chickens. Int. J.
Biometerol. 38: 216-217.
Diarra, S.S. and P. Tabuaciri. 2014. Feeding Management of Poultry in High environmental
temperature. Int. J. Poult. Sci. 13: 657-661.
Dibartola, S. P. 1992. Metabolic acidosis. In: Fluid Therapy in Small Animal Practice. W.B.
Saunders Co., Harcourt Brace Javanovich Inc., Philadelphia, USA. pp: 261–243.
Dickson, W. M. 1975. The thyroid glands. In "Dukes, Physiology of; Domestic Animals". 8 th
Edit. Comstok Publishing Associates, London, pp. 1203-1212.
Donald, J. 2000. Getting the most from evaporative cooling systems in tunnel ventilated
broiler houses. World Poult. 16: 34-39.
Doumes, B.T., W.A.Watson, and H.C. Biggs. 1971. Albumin standards and the measurement
of serum albumin with bromocresol green. Clin. Chim. Acta, 31: 87-96.
Drinah, B. C., Banda-Nyirenda and Pranvohra. 1990. Nutritional improvement of tannin
containing sorghums (Sorghum bicolor) by sodium bicarbonate. Cereal Chem. 67:
533-537.
Ekanayake S., S. S. Silva, N. Priyankarage, U. T. Herath, M. U. Jayasekara, N. U.
Horadagoda, P. Abeynayake and S. P. Gunaratne. 2004. Effect of excess sodium in
feed on haematological parameters and plasma sodium level in broiler chickens. Br.
Poult. Sci. 1: 53-54.
Elahi, F., P. Horst and P. K. Mathur. 1985. Body weight, oviduct weight and egg weight
inter-relations under warm and temperate environmental conditions. Proceedings of
the 3rd AAAP Animal Science Congress, May 6-10 1985. Seoul, Korea.
El-Boushy, A. R. and R. Raternick. 1993. Egg shell strength, cases of egg breakage in
relation to nutrition. Management and Environment. Poult. Advis. 26: 47-55.
El-Gammal, A.M. and M.N. Makled, 1977. Incorporation of sodium bicarbonate into laying
rations. I. Effect on egg production and hatchability. Beitr. Trop. Landwirtsch.
Veterinarmed. 15: 173-176.
190
El-Gendy, E.A., A.A. Atallah, F.R. Mohamed, A. M. Atta and SAS Institute. 1997.
SAS/STAT User's Guide: Statistics. N.E. Goher, 1995. Strain variation in young
chicken in response to chronic heat stress conditions. Egyptian J. Anim. Prod. 32:
237-251.
Elnagar, S. A., Scheideler, S. E., Beck, M. M. 2010. Reproductive hormones, hepatic
deiodinase messenger ribonucleic acid, and vaso-active e intestinal polypeptide-
immunoreactive cells in hypothalamus in the heat stress-induced or chemically
induced hypothyroid laying hen. Poult. Sci. 89: 2001–2009.
El-Sheikh, S.E.M. and A. A. Salama. 2010. Effect of sodium bicarbonate and potassium
chloride as water additives on productive performance and egg quality of heat
stressed local laying hen in Siwa oasis. Proc. of the 3rd Animal Wealth Research
Conf. in the Middle East and North Africa. Pp. 108 – 122.
Ernst, R. A., F. R. Frank, F. C. Price, R. E. Burger and H. R. Halloran. 1975. The effect of
feeding low chloride diets with added sodium bicarbonate on egg shell quality and
other economic traits. Poult. Sci. 54: 270-274.
Ergun, A. 1992. The usage of sodium bicarbonate in poultry diets. In: Symposium on Sodium
Bicarbonate in Animal Nutrition, Jointly organized by Ministry of Agricultural and
Rural Afairs, Associa-tion of Feed Manifacturers and Bottle-Glass Factories of
Turkey, 14th May, Klassis Hotel, Silivri-Istan-bul, Turkey, 61–72.
Farnell, M. B., R. W. Moore, A. P. McElroy, B. M Hargis, D. J. Caldwell. 2001. Effect of
prolonged heat stress in single-comb white leghorn hens on progeny resistance to
Salmonella enteritidis organ invasion. Avian Dis. 45: 479–485
Farrell, D. J., S. l. Atmamihardja and R. L. Hood. 1981. A note on the effects of heat stress
on carcass composition and adipose tissue cellularity of ducklings. Br. Poult. Sci. 22:
533-536.
Fedde, M. R. 1998. Relationship of structure and function of the avian respiratory system to
disease susceptibility. Poult. Sci. 77: 1130–1138.
Ferguson, T.M., J.T. Scott, D.H. Miller, J.W. Bradley and C.R. Creger, 1974. Bone strength
of caged layers as affected by Portland cement and sodium bicarbonate. Poult. Sci.,
53: 303-307.
Fethiere, R., R. D. Miles, R. H. Harms. 1994. The utilization of sodium in sodium zeolite-A
by broilers. Poult. Sci. 73: 118-121.
191
Fletcher, D. L., S. M. Russel, J. M. Walker, J. S. Bailey. 1993. An evaluation of a rinse
procedure using sodium bicarbonate and hydrogen peroxide on the recovery of
bacteria from broiler carcasses. Poult. Sci. 72: 2152-2156.
Fowler, N. G. 1990. in Diseases of Poultry by Jordan, F.T.W. (Editor). 3rd Ed. English
Language Book Society/Bailliere, Tindall, London.
Fox, M. C., D. R. Brown, L. L. Southern. 1987. Effect of dietary buffer additions on gain,
efficiency, duodena! pH and copper concentration in liver of infected chicks. Poult.
Sci. 66: 500-504.
Friedewald, W. T., R. I. Levy, D. S. Fredrickson.1972. Estimation of the concentration of
low-density lipoprotein cholesterol in plasma, without use of the preparative
ultracentrifuge. Clin Chem. 18:499-502.
Friedman, M. 1996. Food browning and its prevention: an overview, Journal of Agricultural
and Food Chem.44: 631-653, 1996.
Fuentes, M. F., J. F. Zapata, G. B. Espindola, E. R. Freitas, M. G. Santos and F. M. Sousa.
1998. Research notes: Sodium bicarbonate supplementation in diets for guinea fowl
raised at high environmental temperatures. Poult Sci. 77: 714-717.
Garcia, A.R., A. B. Batal, N. M. A. Dale. 2007. Comparison of methods to determine amino
acid digestibility of feed ingredients for chickens, Poult. Sci. 86: 94-101.
Genedi, D. M. M. 2000.The role of some anti- stressors on layer performance during hot
climate conditions. M.Sc., Thesis Faculty of Agriculture, Cairo University.
Geraert, P. A., J. C. F. Padilha and S. Guillaumin. 1996a. Metabolic and endocrine changes
induced by chronic heat exposure in broiler chickens: growth performance, body
composition and energy retention. Br. J. Nut. 75: 195-204.
Geraert, P. A., J. C. F. Padilha and S. Guillaumin. 1996. Metabolic and endocrine changes
induced by chronic heat exposure in broiler chickens: growth performance, body
composition and energy retention. Br. J. Nut. 75: 195-204.
Ghazalah, A. A., M. O. Abd – Elsamee and A. M. Ali. 2008. Influence of dietary energy and
poultry fat on the response of broiler chicks to heat therm. Int. J. Poult. Sci. 7: 355-
359.
Ghorbani M. R. and J. Fayazi. 2009. Effects of dietary sodium bicarbonate and rearing
system on laying hens performance, egg quality and plasma cations reared under
chronic heat stress. Biol. Sci. Res. J. 4: 562-565.
192
Glahn R. P., R. F. J. Wideman and B. S. Cowen. 1988. Effect of dietary acidification and
alkalinization on urolith formation and renal function in single comb white Leg-horn
laying hens. Poult. Sci. 67: 1694–1701.
Glick, B. 1972. The immunobiological influence of Mirex and DDT. Poult. Sci., 51: 1861.
Glick, B. and S. Whatley. 1966. The effect of o, p-DDD in the chicken. Experientia. 2: 179-
182.
Gongruttananun, N. and C. Ratana. 2005. Effect of dietary sodium bicarbonate
supplementation on egg shell quality and hatchability in Thai native hens. Kasetsart J.
Nat. Sci. 39: 53-63.
Gonzales-Esquerra, R. and S. Leeson. 2006. Physiological and metabolic responses of
broilers to heat stress – implicationsfor protein and amino acid nutrition. World’s
Poult. Sci. J. 62: 282-295.
Gorman, I., D. Balnave. 1994. Effects of dietary mineral supplementation on the performance
and mineral excretions of broilers at high ambient temperatures. Br. Poult Sci. 35:
563–572.
Gous, R. M. 2004. Nutritional interventions in alleviating the effects of high temperatures in
broiler production. Proceedings of the 12th World’s Poultry Congress, Istanbul,
Turkey, June 8th – 13th.
Grandin, T. 1998. Information resources for Livestock and Poultry Handling and Transport.
In AWIC Resources Series No. 4. J. Odriscol, ed. USDA, National Agriculture
Library, Animal Welfare Information Center, Beltsville, MD.
Griffin, H. G. 1992. Manipulation of eggyolk cholesterol: A physiologist’s view. World’s
Poult. Sci. J. 48: 101-102.
Grizzle, J., M. Iheanacho, A. Saxton and J. Broaden.1992. Nutritional and environmental
factors involved in egg shell quality of laying hens. Brit. Poult. Sci. 33: 781-794.
Guagliardo, N. A., West. K. N., McCluskey, L. P. and D.L. Hill. 2009. Attenuation of
peripheral salt taste responses and local immune function contralateral to gustatory
nerve injury: effects of aldosterone. Am. J. Physiol. Regular. Integra. Comp. Physiol.
297: 1103- 1110.
Habib K. E., Gold P. W., Chrousos G. P. 2001. Neuroendocrinology of stress. Endocrinol
Metab. Clin. North. Am. 30: 695–728.
Hai, L., D. R ong, Z.Y. Z hang. 2000. The effect of thermal environment on the digestion of
broilers. J. Anim. Physiol. Anim. Nutr. 83: 57-64.
193
Hamilton, R. M. G. 1982. Methods and factors that affect the measurement of egg shell
quality. Poult. Sci. 61: 2002-2039.
Harms, R. H. 1982. Chloride requirements of young turkeys. Poult. Sci. 61: 247-2449.
Harms, R. H. 1991. Effect of removing salt, sodium or chloride from the diet of commercial
layers. Poult. Sci. 70: 333-336.
Harms, R. H., K. K. Kuchinski, D. R. Sloan, G. B. Russel. 1995. Sodium requirement of
broiler breeder hen. Poult. Sci. 74: 1311- 1316.
Hassan, A. M., H. May, A. Azeem and P. G. Reddy. 2009. Effect of Some Water
Supplements on the Performance and Immune System of Chronically Heat-Stressed
Broiler Chicks. Int. J. Poult. Sci. 8: 432-436.
Hassan, M. S. H., O. A. El-Sayed and M. M. Namera. 2011. Effect of dietary sodium
bicarbonate and potassium chloride supplementation on acid-base balance, plasma
electrolytes and aldosterone hormone of golden montazah hens under hot climate
condition. Egypt. Poult. Sci. 31: 285-303.
Hayat, J., D. Balnave, J. Brake. 1999. Sodium bicarbonate and potassium supplements for
broilers can cause poor performance at high temperature. Br. Poult. Sci. 40: 411–418.
Henry, R. J., Cannon, D. C. and J. W. Winkelman. 1974. "Clinical chemistry, Principles and
Techniques. 2nd Ed. Harper and Row.
Hevia, P. and W. J. Vinsek. 1979. Dietary protein and plasma cholesterol in chickens. J. Nut.
109: 32-38.
Hooge, D. M. 1995. Dietary electrolytes influence metabolic processes of poultry. Feedstuffs
67: 14-15, 17-19, 21.
Hooge, D. M., K. R Cummings, J. L. Mc-Naughton.1999. Evaluation of sodium bicarbonate,
chloride, or sulfate with a coccidiostat in corn-soy or corn-soy-meat diets for broiler
chickens. Poult Sci. 78:1300–1306.
Hooge, D. M., K. R. Cummings, and J. L. McNaughton. 2000. Dietary sodium
bicarbonate, monensin, or coccidial inoculation and productive performance of
market turkeys on built-up litter. J. Appl. Poult. Res. 9: 343-351.
Hooge, D. M. 2003. Practicabilities of using dietary sodium and potassium supplements to
improve poultry performance. Proceedings of Arkansas Nutrition Conference; 2003;
Fayetteville, Arkansas. USA. p.19.
194
Hoshi, S., T. Nakamura; T. Nunoya, S. Ueda. 1995, Induction of protective immunity in
chickens orally immunized with inactivated Infectious Bursa disease virus. Vaccine.
13: 245-252.
Huang, K. H., V. Ravindran, X. LI. 2007. Apparent ileal digestibility of amino acids in feed
ingredients determined with broilers and layers. J. Sci. Food Agri. 87: 47-53.
Hughes R. J. 1988. Inter-relationships between eggshell quality, blood acid-base balance and
dietary electrolytes. World’s Poult. Sci. J. 44: 30–40.
Hughes, R.J. and M. Choct.1999. Chemical and physical characteristics of grains related to
variability in energy and amino acid availability in poultry. Aust. J. Agri. Res.
50:689-701.
Jeukendrup, A. E., K. Currell, J. Clarke, J. Cole, A. K. Blannin. 2009. Effect of beverage
glucose and sodium content on fluid delivery. Nutrition and Metabolism. 6:9-15.
Johnson, R. J, H. Karunajeewa. 1985. The effects of dietary minerals and electrolytes on the
growth and physiology of the young chick. J. Nutr. 115:1680–1690.
Jones, D. R., 2006. Conserving and Monitoring Shell Egg Quality. Proceedings of the 18 th
Annual Australian Poultry Science Symposium, pp. 157-165.
Julian R. J., L. J. Caston, S. Leeson.1992. The effect of dietary sodium on right ventricular
failure-induced ascites, gain and fat deposition in meat-type chickens. Can. J. Vet.
Res. 56: 214–219.
Junqueira, O. M., P. T. Costa, R. D. Miles, R. H. Harms. 1984. Interrelationship between
sodium chloride, calcium and phosphorus in laying hen diets. Poult. Sci. 63: 123-130.
Junqueira, O. M., R. D. Miles, R. H. Harms. 1993. The inability of sodium bicarbonate to
induce an improvement in egg shell quality in the presence of sulfanilamide. Poult.
Sci. 62: 2062-2064.
Junqueira, O. M., F. B de-Comargo, L. F. Araujo, S. C. S. de Araujo and N. K.Sakomura.
2000. Effects of the source and levels of sodium, chlorine and potassium and (Na+
K)/Cl ratio on performance and plasma blood characteristics of laying hens. Revista
Brasileirade Zool. Teccnia. 29: 1110-1116
Junqueira, O. M., L. E. C. Fonseca, L. F. Araujo, K. F. Duarte, cs da S Araujo, EAP
Rodrigues. 2003. Feed restriction on performance and blood parameters of broilers
fed diets with differed sodium levels. Rev. Bras. Cienc. Avic. Vol.2 Campinas
May/Aug.,2003.
195
Karimian, M. R., M. Tohidian and M. Khiyavi. 2004. Effect of replacement of maize by
sorghum on layer quail performances. The jointagriculture and natural resources
Symp. Tabriz-Ganja, Iran
Kassirer, J. P. 1971. Enzymatic Kinetic Method for urea determination New Eng. J. Med.
285: 385.
Kawas, J. R., R. García-castillo, H. Fimbres-durazo, F. Garza-cazares, J. F. G. Hernández-
vidaL, E. Olivares-sáenz, C. D. Lu. 2007. Effects of sodium bicarbonate and yeast on
nutrient intake, digestibility, and ruminal fermentation of light-weight lambs
fedfinishing diets. Small Ruminant Res. 67: 149-156.
Kaya, I., B. Karademir and O. Ukar. 2004. The effects of diet supplemented with sodium
bicarbonate upon blood pH, blood gases and eggshell quality in laying geese.Vet.
Med. Czech. 49: 201–206.
Kekeocha, C. C. 1984. Poultry production handbook. Poultry health and diseases. pp: 110.
Keshavarz, K. 1996. The effects of different levels of vitamin C and cholecalciferol with
adequate or marginal levels of dietary calcium on performance and egg shell quality
of laying hens. Poult. Sci. 75: 1227-1235.
Keskin, E. and Z. Durgan, 1997. Effects of supplemental NaHCo3, KCl, CaCl2, NH4Cl and
CaSo4 on acidbase balance, weight gain and feed intake inJapanese quails exposed to
constant chronic heat stress. Pak. Vet. J. 17: 60-64.
Khattak, F. M., T. Acamovic, N. Sparks, T. N. Pasha, M. H. Hussain, M. Joiya and Z. Ali.
2012. Comparative efficacy of different supplements used to reduce heat stress in
broilers. Pak. J. Zool. 44: 31.
Khmilyar, D. D. 1983. Effect of high air temperature on carbohydrate and nucleotide
metabolism in blood of laying hens. Visnik Sil Skogodar's Koi Nauki 1: 42-43.
Kidd M. T., L. M. Pote and R. W. Keirs. 2003. Lack of interaction between dietary threonine
and Eimeria acervulina in chicks. J. Appl. Poult. Res. 12: 124-129.
Koelkebeck K. W., T. W. Odom. 1995. Laying hen responses to acute heat stress and carbon
dioxide supplementation: II. Changes in plasma enzymes, metabolites and
electrolytes. Comp. Biochem. Physiol. A Physiol. 112:119–122.
Koh, K, M. G. Macleod. 1999. Effects of ambient temperature on heat increment of feeding
and energy retention in growing broilers maintained at different food intakes. Br.
Poult. Sci. 40: 511-516.
196
Kohne, H. J. and J. E. Jones. 1975. Acid-base balance, plasma electrolytes and production
performance of adult turkey hens under condition of increasing ambient temperature.
Poult. Sci. 54: 2038-2045.
Kohne, H. J., J. E. Jones. 1975. Changes in plasma electrolytes, acid base balance and other
physiological parameters of adult female turkeys under conditions of acute
hyperthermia. Poult. Sci. 54: 6: 2034-2038.
Kohne, H. J. and J. E. Jones. 1976. The relationship of circulating levels of estrogens,
corticosterone and calcium to production performance of adult turkey hens conditions
of increasing ambient temperature. Poult. Sci. 55: 277-285.
Kurtoglu, V. , F. Kurtoglu and T. Balevı. 2007. Effects of sodium bicarbonate, potassium
chloride and sodium chloride supplementation on some blood biochemical parameters
in laying hens.World Poultry Science Association, Proceedings of the 16th European
Symposium on Poultry Nutrition, Strasbourg, France, 26-30 August, 2007. pp. 189-
192.
Leeson, S., J. D. Summers. 2001. Scott’s nutrition of the chicken. 4th Ed. University Books;
Guelph, Ontario, Canada.
Leeson, S. and J. D. Summers. 2001a. Determination of digestibility. In: Nutrition of the
Chicken. 4th ed. International Book Distribution Company (Publishing Division)
Charbagh, Lucknow, India. Pp: 533-534.
Lehninger, A. L.1970. Biochemistry. Worth Publishers Inc.; New York: pp. 50–51.
Li, B. M, Y. J. Zhou and Y. N. Cui. 1992. Study and use of tunnel ventilation system for
poultry houses in summer. Transaction of the Chinese Society of Agri. Engineering.
8: 83-89.
Li, C. C., W. H. Bruke and M. G. Hulsey. 1986. Effect of environmental temperature on feed
intake, body weight, rate of egg laying, egg weight, egg shell strength, blood calcium
content and the level of oestradioi in the laying hen. Anim. Husb. Vet. Med. 18: 97-
98.
Lile, N. R. 1976.The role of estrogen and progesterone in the regulation of reproductive
behaviour in female ring doves (Streptopelia risoria) under long vs. short
photoperiods.Can J Zool. 54:1409-22.
Lin, H., Mertens, K., Kemps, B., Govaerts, T., De Ketelaere, B., De Baerdemaeker, J.,
Decuypere, E and J. Buyse. 2004. New approach of testing the effect of heat stress on
eggshell quality: Mechanical and material properties of eggshell and membrane. Br.
197
Poult. Sci. 45: 476–482.
Lott, B. D. 1991. The effect of feed intake on body temperature and water consumption of
male broilers during heat exposure. Poult. Sci. 70: 756–759.
Mack, L. A., J. N. Felver-Gant, R. L. Dennis, H. W. Cheng. 2013. Genetic variation alters
production and behavioral responses following heat stress in 2 strains of laying hens.
Poult. Sci. 92: 285–294.
MacLeod, M. G. 2004. Climate-nutrition interaction in poultry. 1stAnn. Confr. FVM.
Moshtohor, pp 1-24.
MacLeod, M. G. and P. M. Hocking. 1993. Thermoregulation of high temperature of
genetically fat and lean broiler hens fed ad libitum or on a controlled-feeding regime.
Br. Poult. Sci. 34: 589-596.
Macleod, M. G., C. J. Savory, C. C. McCorquodale and A. Boyd. 1993. Effect of long term
food restriction on energy expenditure and thermoregulation in broiler-breeder fowls
(Callus donfesticus). Comp. Biochem. Physiol. 106: 221-225.
MacLeod, M.G. and Jewitt, T.R. 1984. Circadian variation in the heat production rate of the
domestic fowl (Gallus domesticus): effects of limiting feeding to a single daily meal.
Comparative Biochem. and Physiol.78: 687-690.
Maff, A. 1984. Manual of Veterinary Investigation. Volume 2. 3rd ed. Her Majesty
stationary officer, London.
Mahmood, A. 1993. Influence of restricted feeding and ascorbic acid supplementation on the
performance and physiological parameters of broiler chicks. M.Sc. Thesis.
Department. of Poultry Husbandry. University. of Agriculture, Faisalabad.
Mahmood, S., S. Hasan, F. Ahmad, M. Ashraf, M. Alam and A. Muzaffar. 2005. Influence
of feed withdrawal for different durations on theperformance of broilers in summer.
Int. J. Agri. Biol. 6: 975–978.
Mahmud, A., Z. Hayat, M. Z. Khan, A. Khalique and M. Younus. 2010. Comparison of
source and levels of sodium in broilers under low temperature conditions.Pak. J.
Zool.42: 383-388.
Maiorka A., N. Magro, H. A. S. Bartels and A. M. Kessler. 2004. Different sodium levels
and electrolyte balance in pre-starter diets for broilers. Revista Brasileira de Ciência
Avícola. 6: 143-146.
Makled, M. N., O. W. Charles. 1987. Egg shell quality as influenced by sodium bicarbonate,
calcium source and photoperiod. Poult. Sci. 66: 705-712.
198
Mandal A. B., A. V. Elangovan, T. Saroj, K. T. Praveen and K. T. Pramod. 2010. Effect of
ascorbic acid and sodium bicarbonate supplementation in diets with two energy levels
on the performance of laying Japanese quails during extreme dry summer. Int. J.
Poult. Sci. 45: 141-145.
Mariinez, K. K., J. Salazar and G. Vela. 1993. Estudio controlado del efecto del bicarbonato
de sodio en pollos bajo estrts calbrico. Pages 138-141 in: Proc. 18 th Convencibn
Nacional ANECA, Cancun Q. Roo, Mexico.
Mashaly, M. M., G. L. Hendricks, M. A. Kalama, A. E. Gehad, A. O. Abbas, P. H.
Patterson. 2004. Effect of heat stress on production parameters and immune responses
of commercial laying hens. Poult. Sci. 83: 889–894
Maxwell, M. H. 1993. Avian blood leukocyte responses to stress. World’s Poult. Sci. J. 9:
34-43.
Maxwell, M. H., P. M. Hocking and G.W. Robertson. 1992. Differential leucocyte response
to various degrees of food restriction in broilers, turkeys and ducks. Br. J. Poult. Sci.
33: 177-187.
May, J. D., J. W. Deaton, F. N. Reece and S. L. Branton. 1986. Effect of acclimation and
heat stress on thyroid hormone concentration. Poult. Sci. 65:1211-13.
McDaniel, C. D., Hood, J. E., Parker, H. M. 2004. An attempt at alleviating heat stress
infertility in male broiler breeder chickens with dietary ascorbic acid. Int. J. Poult.
Sci. 3: 593–602.
McDonald, P., R.A. Edward, J.F.D. Greenlagh and C.A. Morgan, 1998. Animal Nutrition, 5 th
Edition. Chapter th 10: Evaluation of Foods (A) Digestibility, pp:221-229.
Mckee, J. S., P. C. Harrison and G. L. Rishowski. 1997. Effect of supplemental ascorbic acid
on the energy conversion of broiler chicks during heat stress and feed withdrawal.
Poult. Sci. 76: 1278-1286.
McRee, D. I., P. E. Hamrick, P. Thaxton and C. R. Parkhurst. 1977. Humoral immunity of
Japanese quail subjected to microwave radiation. Health Phys., 33: 23-33.
Melesse A. 2011. Performance and physiological responses of naked-neck chickens and their
F1 crosses with commercial Layer Breeds to long-term high ambient temperature.
Global Veterinaria 6: 272-280.
Mert, N. 1991. A biochemical investigation on chicken gout observed in narmara region in
Turkey. Adv. Exp. Biol. 309A: 251-254.
199
Michael, G. Y., J. P. Thaxton and P. B. Hamilton.1973. Impairment of the reticuloendothelial
system of chickens during aflatoxicosis. Poult. Sci. 52: l206-1207.
Mirsalimi, S. M., P. J. O. Brlen, R. J, Julian. 1992. Changes in erythrocyte deformabillty in
NaCI-induced right sided cardiac failure in broiler chickens. Am. J. Vet. Res. 53:
2359-2363.
Mitchell, M. A., and A. J. Carlisle, 1992. The effects of chronic exposure to elevated
environmental temperature on intestinal morphology and nutrient absorption in the
domestic fowl (Gallus domesticus). Comp. Biochem. Physiol. 101A:137–142.
Mongin, P. 1981. Recent advances in dietary cation anio balance in poultry. In: Recent
Advances in Nutrition, W. Haresign and D. J. A. Cole (Eds.). pp: 109-119.
Butterworths, London, UK.
Moore, D. T., K. Baker and J. D. Firman, 2001. Digestible sulfur amino acid requirements
for male turkeys to five weeks of age. J. Appl. Poult. Res. 10: 363‒370
Moorhead, P. D. and R. F. Cross. 1970. Whole blood ascorbic acid levels in chickens with
aplastic anemia and the effect of supplemental ascorbic acid and minerals on
mortality and pathologic manifestation. Poult. Sci. 49: 1065-1069.
Morgan, G. W., F. W. Edens, P. Thaxton and C. R. Parkhurst. 1975. Toxicity of dietary lead
in Japanese quail. Poult. Sci. 54: 1636-1642.
Mubarak, M., A. A. and Sharkawy. 1999. Toxopathology of gout induced inlaying pullets by
sodium bicarbonate toxicity. Environmental Toxicol. Pharmacol. 7: 227-236.
Muiruri, H. K., P. C. Harrison and H. W. Gonyou. 1991. The use of water cooled roosts by
hens for thermoregulation. Appl. Anim. Behav. Sci. 28: 333-339.
Muiruri, H. K. and P. C. Harrison. 1991a. Effect of roost temperature on performance of
chicken in hot ambient environments. Poult. Sci. 70: 2253-2258.
Murakami, A. E., E. Rondon, E. N. Martins, M. S. Pereira, C . Scapinello. 2001. Sodium and
chloride requirements of growing broiler chickens (twenty-one to forty-two days of
age) fed corn-soybean diets. Poult Sci. 80: 289–294.
Murakami, A. E, E. A Saleh, S. E. Watkins and P. W. Waldroup. 2000. Sodium source and
level in broiler diets with and without high levels of animal protein. J Appl. Poult.
Res. 9:53–61.
200
Mushtaq M. M. H., T. N. Pasha, M. Akram, T. Mushtaq, R. Parvin, H. C. Choi, J. Hwangbo,
J. H. Kim 2013. Growth performance, carcass characteristics and plasma mineral
chemistry as affected by dietary chloride and chloride salts fed to broiler chickens
reared under phase feeding system. Asian Aust. J. Anim. Sci. 26: 845–855.
Mushtaq, T., M. A. Mirza, M. Athar, D. M. Hooge, T. Ahmad, G. Ahmad, M. M. H.
Mushtaq, U. Noreen 2007. Dietary sodium and chloride for twenty-nine to forty-two-
day-old broiler chickens at constant electrolyte balance under subtropical summer
conditions. J Appl. Poult. Res. 16: 161–170.
Mushtaq, T., M. Sarwar, H. Nawaz, M. A. Mirza, T. Ahmad. 2005. Effect and interactions of
dietary sodium and chloride on broiler starter performance (hatching to twenty-eight
days of age) under subtropical summer conditions. Poult. Sci. 84: 1716–1722.
Mustaf, S., Kahraman, N. S.; Firat, M. Z. 2009. Intermittent partial surface wetting and its
effect on body-surface temperatures and egg production of white brown domestic
laying hens in Antalya (Turkey). Br. Poult. Sci. 50: 33–38.
Nair, R. S. and V. K. Elizabeth. 1983. Effect of age and season on quality of chicken eggs.
Int. J. Poult. Sci. 18: 207-210.
Nakamura, Y., Y. Aoyagi, and T. Nakaya. 1992. Effect of dietary ascorbic acid on growth
and ascorbic acid level of chicks exposed to high ambient temperature. Japanese
Poult. Sci., 29: 41-46.
Naseem, M. T., S. Naseem, M. Younus, Z. I. Chauhdary, A. Ghafoor, A. Aslam and S.
Akhter. 2005. Effect of potassium chloride and sodium bicarbonate supplementation
on thermo-tolerance of broilers exposed to heat stress. Int. J. Poult. Sci. 4: 891-895.
Nathen, D. B., E. D. Heller and M. Perek. 1977 The effect of starvation on antibody
production of chicks. Poult. Sci. 56: 1468-1471.
Naviglio, D., M. Gallo, L. L. Grottaglie, C. Scala, L. Ferrara and A. Santini. 2011.
Determination of cholesterol in Italian chicken eggs. J.food Chem.11: 3-7.
Nayak, G. D., N. C. Behura, K. K. Sardar and P. K. Mishra. 2015. Effect of climatic
variables on production and reproduction traits of coloured broiler breeder
poultry.Vet. World 8: 472-477.
Nelson, D. L., and M. M. Cox. 2000. Lehninger Principles of Biochemistry. Worth
Publishers, New York, NY.
Nilipour, A. H. 2000. Modern broilers require optimum ventilation. World Poult. 16: 30-31.
201
Nillipour, A. H. and Melog. 1999. Feeding techniques during heat stress. Poultry Digest 58:
3-34.
Niu, Z. Y., Liu, F. Z., Yan, Q. L., Li, W. C. 2009. Effects of different levels of vitamin E on
growth performance and immune responses of broilers under heat stress. Poult. Sci.
88: 2101–2107.
Njoya, J. 1995. Effect of diet and natural variations in climates on the performance of laying
hens. Poult. Sci. 36: 537-554.
Njoya, J. and M. Picard, 1994. Climatic adaptation of laying hens. Trop. Animal Health and
Production. 26: 180-186.
North, M. O. and D. D. Bell. 1990. "Egg quality" Commercial Chicken Production Manual
4th ed. Van Nostrand Rinolt, New York, NY.
Novero, R. P., M. M. Beck, E. W. Gleaves, A. L. Johnson and J. A. Deshazer. 1991. Plasma
progesterone, luteinizing hormone concentrations and granulosa cell responsiveness
in heat stressed hens. Poult. Sci. 70:2335-2339.
NRC. 1994. Nutrient requirements of poultry. 9th revised Ed. National Academy Press.
Washington D.C. USA.
Odom, T. W., C. R. Creger, J. R. Cain, D. Costello and C. A. Bailey. 1983, The effect of
thermal stress on parathyroid gland weight, duodenal calcium-binding protein activity
and bone mineral n SCVVL. Poult. Sci. 62: 1476.
Odom, T. W., P. C. Harrison and M. J. Darre. 1985. The effects of drinking carbonated water
on the egg shell quality of single Comb White Leghorn hens exposed to high
environmental temperature. Poult. Sci. 64: 594-596.
Oelkers, W., S. Diederich and V. Bähr. 1992. Diagnosis and therapy surveillance in
Addison's disease: rapid adrenocorticotropin (ACTH) test and measurement of
plasma ACTH, renin activity, and aldosterone. J. Clin. Endocrinol. Metab. 75: 259-64
Okan, F. 1999.Effects of dietary supplemental sodium bicarbonate on some egg
characteristics and blood parameters in Japanese Quail reared under high
enviromental temperature. Tr. J. Vet. Anim. Sci. 1: 139-143.
Oladele, S. B., S. Ogundipc, J. O. Ayo and K. A. N.Esievo. 2001. Effects of season and sex
on packed cell volume, haemoglobin and total proteins of indigenous pigeons in
Zaria, Northern Nigeria. Veterinarski Arhiv. 71: 277–286.
Olanrewaju, H. A., S. Wongpichet, J. P. Thaxton, W. A. Dozier and S. L. Branton. 2006.
Stress and Acid-Base Balance in Chickens. Poult. Sci. 85:1266–1274.
202
Oliveira MC, U. M. Arantes and J. Stringhinix. 2010. Efeito do balanço eletrolítico da ração
sobre parâmetros ósseos e da cama de frango. Biotemas. 23: 203-209.
Oluyemi, J. A. and A. Adebanjo. 1979. Measures applied to combat thermal stress in poultry
under practical tropical environment. Poult. Sci. 58: 767-773.
Olayemi, F. O and R. O. A. Arowolo. 2009. Seasonal variation in the Haematological values
of the Nigerian Duck. Int.J. Poult Sci. 8: 813-815.
Osman, A. A. B., H. M. A. Hamed, M. M. Muna and S. M. Mysara. 2015. Effects of sodium
bicarbonate levels on the performance of broiler chickens under Sudan condition.
Asian J. Agri. Food Sci. 3: 89-94.
Oviedo-Rondon, E. O., A. E. Murakami, A. C. Furlan, I. Moreira and M. Macari. 2001.
Sodium and chloride requirements of young broiler chickens fed corn-soybean diets
(one to twenty one days of age). Poult. Sci. 80:592–598.
Ozbey, O., N. Yildiz, M. H. Aysöndü and O. Ozmen. 2004. The effects of high temperature
on blood serum parameters and the egg productivity characteristics of japanese quails
(Coturnix coturnix japonica). Int. J. Poult. Sci. 3: 485-489
Paggi, R. A., J. P. Fay and H. M. Fernandez. 1999. Effect of short-chain acids and glycerol
on the proteolytic activity of rumen fluid. Anim. Feed Sci. Technol. 78: 341-347.
Pardue, S. L. and J. P. Thaxton. 1982. Enhanced livability and improved immunological
responsiveness in ascorbic acid supplemented cockerels during acute heat stress.
Poult. Sci. 61:1522.
Pardue, S. L., J. P. Thaxton and J. Brake. 1985. Role of ascorbic acid in chicks exposed to
high environmental temperature. J. Appl. Physiol. 58: 1511-1516.
Pardue, S. L. and J. B. Thaxton. 1986. Ascorbic acid in poultry: a review. World’s Poult. ci.
42: 107-123.
Parker, J. T. and M. A. Boone, 1976. Thermal stress effects on certain blood characteristics
of adult male turkeys. Poult. Sci. 50: 1287- 1295.
Parker, J., G. C. Haarris, Jr., R. Selers, V. Scogin and K. Sexton, 1982. The effects of heat
stress and supplemental glucose on broiler breeder cockerels. Poult. Sci. 61: 1391.
Pasha, T. N., A. Mahmood, F. M. Khattak, M. A. Jabbar and A. D. Khan. 2008. The effect
of feed supplemented with different sodium bentonite treatments on broiler
performance. Turk. J. Vet. Anim. Sci. 32: 245-248.
203
Patience, J. F., R. E. Austic, R. D. Boyd. 1986. The effect of sodium bicarbonate or
potassium bicarbonate on acid base status and protein and energy digestibility in
swine. Nutr. Res. 6:263–273.
Patience, J. F. 1990. A review of the role of acid base balance in amino acid nutrition. J.
Anim. Sci.68: 398-408.
Pech‐Waffenschmidt, V., E. Bogin, Y. Avidar and P. Horst. 1995. Metabolic and
biochemical changesduring heat stress in relation to the feathering degree of the
domestic hen. Avian Pathology. 24: 33-44.
Peguri, A. and C. Coon. 1991. Effect of temperature and dietary energy on layer
performance. Poult. Sci. 70: 126-138. ,
Peng, Y., Y. Wang, D. Ning and Y. Guo.2013.Estimation of dietary sodium bicarbonate dose
limit in broiler under high ambient temperatures.J. Poult. Sci.50: 346-353.
Pickering, R. E. 2000. Moving through the air. Complete Biology. Oxford University press,
printed by G. Canale and SPA Turin, Italy. pp 126-130.
Prieto, M. T., J. L. Campo. 2010. Effect of heat and several additives related to stress levels
on fluctuating asymmetry, heterophil: lymphocyte ratio, and tonic immobility
duration in White Leghorn chicks. Poult. Sci. 89: 2071–2077.
Puron, D., R. Santamaria and J. C. Segura. 1994. Effects of sodium bicarbonate,
acetylsalicylic and ascorbic acid on broiler performance in a tropical environment. J.
Appl. Poult. Res. 3: 141-145.
Puron, D., R. Santamaria and J. C. Segura. 1997. Sodium bicarbonat and broiler performance
at high stocking densities in a tropical environment. J. Appl. Poult. Res. 6: 443-448.
Puvadolpirod, S., and J. P. Thaxton. 2000. Model of physiological stress in chickens:
Digestion and Metabolism. Poult. Sci. 79: 383–390.
Ramezani, S., A. Riasi, N. Afzali and F. Nasari. 2011. Effect of selenium and sodium
bicarbonate supplementation diets on blood biochemical properties, growth
performance and carcass traits of broilers in heat stress condition.Vet. J. 90: 13-22.
Rauch, W. 1964. Influence of the feed on shell strength of hen's eggs. Arch. Geflugelk. 28:
437-451.
Ravindran, V., A. J.Cowieson and P. H. Selle. 2008. Influence of dietary electrolyte balance
and microbial phytase on growth performance, nutrient utilization, and excreta quality
of broiler chickens. Poult. Sci. 87: 677-688
204
Rector, Floyd C. Brenner, M. Barry. 2004. Brenner and Rector's the kidney. Philadelphia:
Saunders. ISBN 0-7216-0164-2. OCLC 51838812
Reddy , V. R., T. B. Dinesh. 2004. Nutrition management under adverse environment. Hand
book of poultry nutrition. International book distributing company, Chaman studio
building, 2nd floor, Charbagh, Lucknow (India). Pp 228-229.
Rehman, Z. U., J. A. Khan, T. Khaliq and A Ali. 2012. Manual of Physiology-1. 5 thEd,
Department of Physiology and Pharmacology, University of Agriculture, Faisalabad.
pp: 12-55.
Reid, B. L., A. A. Kurnick and B. J. Huliet. 1965. Relationship of protein level, age and a
ambient temperature on laying hen performance. Poult. Sci. 44: 1113-1122.
Reitman, S. and S. Frankel. 1957. A colorimetric method for the determination of serum
glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28: 56–
63.
Remus, J. 2001. Betaine for increased breast meat yield in turkey. World Poultry. Elsevier
17: 14-15.
Remus, J. 2002. Feed stuff. 74: 11-13.
Roeschlau P., E. Bernt and N. J. Gruber. 1974. Serum cholesterol determination procedure.
Clin. Biochem. 12: 403-403
Roland, D. A. Sr., M. M. Bryant and H. W. Rabon. 1996. Influence of calcium and
environmental temperature on performance of first cycle (phase 1) commercial
Leghorns. Poult. Sci. 75: 62-68.
Rondon E., A. E. Murakami, A. C. Furlan, I. Moreira, M. Macari. 2001. Sodium and chloride
requirements of young broiler chickens fed corn-soybean diets (one to twenty–one
days of age). Poult Sci. 80: 592–598.
Roussan, D. A., G.Y. Khwaldeh, R. R.Haddad, I. A. Shaheen, G. Salameh and R. AlRifai.
2012. Effect of ascorbic acid, acetylsalicylic acid, sodium bicarbonate and potassium
chloride supplementation in water on the performance of broiler chickens exposed to
heat stress. J. Appl. Poult. Res. 17: 141-144.
Ruiz–Lopez B., R. E. Austic. 1993. The effects of selected minerals on acid–base balance of
growing chicks. Poult Sci. 72: 1054–1062.
Ruiz-Lopez., M. Rangel-Lugo and R. E. Austic. 1993. Effects of selected minerals on acid-
base balance and tibial dyschondroplasia in broiler chickens. Poult. Sci. 72: 1693-
1704.
205
Sabry, N., A. A. Khalil, S. Hamdy and A. R. A. Akkada. 1978. Effects of high
environmental temperature on feed efficiency and performance of chickens.
Alexandria J. Agri. Res. 26: 63-71.
Sahin, K., O.Kucuk, N.Sahin, M.Sari. 2001. Effect of vitamin C and vitamin E on lipid
peroxidation b status, some serum hormone, metabolite and mineral concentrations of
Japanese quails reared under heat stress (34 °C). Int. J. Vitam. Nutr. Res. 71: 27-31
Sahota, A. W, M. F. Ullah, A. H. Giliani and M. R. Chaudhry. 1990. Effect of ascorbic
acid supplementation on the performance of laying hens exposed to heat stress. Proc.
3rd. Int. Cong. Pak. Vet. Med. Assoc. (November 28-29). pp:322-329.
Sahota, A. W. and A. H. Gilani. 1994. A study on physiological behaviour of Lyallpur Silver
Black and White Leghorn breeds of chicken subjected to high temperature stress. Pak.
J. Sci., 46: 112-115.
Sahota, A. W. and A. H. Gilani. 1995. Effect of dietary ascorbic acid supplementation on
haematology and body temperature of adult layers maintained under high ambient
temperature. Pak. J.Sci. Res. 47: 82-86.
Sahota, A. W., A. H. Gilani and M. F. Ullah. 1993. Effect of ascorbic acid supplementation
on blood composition of chickens exposed to heat stress. Pak. J. Sci. 45: 21-26.
Sahota, A. W., A. H. Gilani and M. F. Ullah. 1994. Comparative hematology of Lyallpur
Silver Black and White Leghorn breeds of chicken affected by heat stress. Pak. J. Sci.
Res. 45: 105-112.
Sahota, A. W., M. F. Ullah and A. H. Gilani. 1993. Blood and tissue ascorbic acid status of
chicken exposed to heat stress. J. Shandong Agri. Univ. 23: 363-367.
Sahota, A. W., M. F. Ullah, A. H. Gilani and M. D. Ahmad. 1993. Effect of ascorbic acid
supplementation on the performance of Lyallpur Silver Black and White Leghron
chicks exposed to heat stress. Pak. Vet. J. 12: 28-31.
Sahota, A. W., M. F. Ullah, A. H. Gilani and M. D. Ahmad. 1993. Effect of ascorbic acid supplementation on the performance of Lyallpur Silver Black and White Leghron chicks exposed to heat stress. Pak. Vet. J. 12: 28-31.
Sahota, A. W., M. F. Ullah. and A. H. Gilani. 1996. Egg quality characters and blood calcium levels of the hens as influenced by ascorbic acid supplementation during hot summer season. Pak, J. Sci. Res. 48: 90-92.
Salari, S., H. Kermanshahi and M.H. Nasiri, 2006. Effect of sodium bentonite and comparison of pellet vs mash on performance of broiler chickens. Int. J. Poult. Sci., 5: 31–34
206
Sales, J. and G. P. J. Janssens. 2003. Methods to determine metabolizable energy and
digestibility of feed ingredients in the domestic pigeon (Columba liviadomestica ).
Poult. Sci. 82: 1457–1461.
Sandercock, D. A., R. R. Hunter, G. R. Nute, M. A. Mitchell, and P. M. Hocking. 2001.
Acute heat stress-induced alterations in acid-base status and skeletal muscle
membrane integrity in broiler chickens at two ages: Implications for meat quality.
Poult. Sci. 80: 418–425.
Santin, E., S. A. Borges, A. V. Fischer da Silva, D. M. Hooge, and K. R. Cummings. 2003.
Effect of dietary electrolyte balance on the immune response (Newcastle disease virus
antibody titers) of broiler chickens at various ages following vaccination and during
heat stress. Int. Poult. Sci. Forum, Atlanta, Georgia. Abstract 74.
Santurio, J. M. 1999. Effect of sodium bentonite on the performance and blood variables of
broiler chickens intoxicated with aflatoxins. Br. Poult. Sci., 40: 115–119
Sapolsky, RM., L. M. Romero and A U.Munck. 2000. How do glucocorticoids influence
stress responses? Integrating permissive, suppressive, stimulatory, and preparative
actions. Endocr Rev. 21: 55–89.
Savic, V., M. Mikec, P. Pavicic and M. Tisljar. 1993. Effect of repeated heat stress on the
humoral immune response and productivity of broiler chicks. Veterinarska Stanica,
24: 195-202.
Savory, C. J. 1986. Influence of ambient temperature on feeding activity parameters and
digestive function in domestic fowls. Physiol. Behav. 38:353–357.
Sayed, M. A. M. and T. A. Scot. 2008. Maintaining electrolyte and water balance to alleviate
heat stress in broiler chickens. In Proceeding of the 19th Australian Poultry Science
Symposium,New South Wales,Australia.
Schettler, G., E. Nussel. 1975. Massnahmen Zur Prevention der Artherosklerose.
Arb.Med.Soz. Med. Prav. Med, 10: 25
Schmidt, R. R., S. Kaplan and J. J. Smith. 1976. Mechanical stress (shaking) and
reticuloendothelial function in the chick embryo. Life Sci. 18: 1273-1278.
Scott, T. A. and D. Balnave. 1988. Influence of dietary energy, nutrient density and
environmental temperature on puliet performance in early lay. Br. Poult. Sci., 29:
155-165.
207
Scott, T. A. and D. Balnave. 1989. Responses of sexually maturing pullets to self-selection
feeding under different temperature and Sighting regimens. Br. Poult. Sci. 30: 135-
150.
Selle, P.H., D.J. Cadogan X. LI. 2010. Implications of sorghum in the nutrition of broiler
chickens. Anim. Feed Sci. and Tech. 156: 57-74.
Selye, H. 1956. Endocrine reactions during stress. Current Res. Aneth. 1: 182-193.
Selye, H. 1973. Homeostasis and heterostasis. Perspectives in Biology and Medicine. 16:
441-445
Selye, H. 1973a. The Evolution of the Stress Concept: The originator of the concept traces its
development from the discovery in 1936 of the alarm reaction to modern therapeutic
applications of syntoxic and catatoxic hormones. Am. Sci. 61: 692-699.
Senkoylu, N., H. Akyurek, H. Ersin-Samli and A. Agma.2005. Assessment the Impacts of
Dietary Electrolyte Balance Levels on Laying Performance of Commercial White
Layers. Pak. J. Nut. 4: 423-427.
Siegel, H. S. and J. W. Latimer. 1975. Social interactions and antibody titers in young male
chickens (Galus domesticus). Anim. Behav. 23: 323-330.
Siegel, P. and F. Jordan. 1997. Improving Poultry Production. Proceedings of 19th World
Poultry Congress. Poult. Int. J. pp: 62-68.
Smith, M. O. 1992. Effects of feed withdrawal and acciimation on weight gain, body
temperature, survival and carcass traits of heat stressed broilers. Tennessee Farm and
Home Science. 156: 4-10.
Smith, M. O. and R. G. Teeter. 1987. Potassium balance of 5 to8-week old broilers exposed
to constant heat or cycling high temperature stress and the effects of
supplementalpotassium chloride on body weight gain and feedefficiency. Poult. Sci.
66:487–492.
Smith, M. O. and R. G. Teeter. 1988. Effects of potassium chloride and fasting on broiler
performance during summer. Anim. Sci. Res. Report. 125: 255-258.
Smith, W.O. and R. G. Teeter. 1989. Effects of sodium and potassium salts on gain, water
consumption and body temperature of 4- to 7-week-old heat stressed broilers. Nutr.
Reports Int. 40: 161-169.
Smith, M. O. 1992. Effects of feed withdrawal and acciimation on weight gain, body
temperature, survival and carcass traits of heat stressed broilers. Tennessee Farm and
Home Science. 156: 4-10.
208
Sohail, M. U., M. E. Hume, J. A. Byrd, D. J. Nisbet, A. Ijaz, A. Sohail, M. Z. Shabbir and H.
Rehman. 2012. Effect of supplementation of prebiotic mannan-oligosaccharides and
probiotic mixture on growth performance of broilers subjected to chronic heat
stress. Poult Sci.91: 2235–2240.
Spence, J. D., D. J. Jenkins, and J. Davignon. 2010. Dietary cholesterol and egg yolks: Not
for patients at risk of vascular disease. Can. J. Card. 26: 336–339.
Spinu, M. and A. A. Degen. 1993. Effect of cold stress on performance and immune
responses of Bedouin and White Leghorn hens. Br. Poult. Sci. 34: 177-185.
Squires, E.J., R.J. Julian. 2001. The effect of dietary chloride and bicarbonate on blood pH,
haematological variables,pulmonary hypertension and ascites in broiler chickens. Br.
Poult. Sci. 42: 207-212.
Srivastava, R. K., D. P. Dhingra, A. K. Sharma and B. K. Shingari. 1980. Effect of cooling
on growth and feed utilization of commercial broiler chicks during summer months.
6th European Poultry Conference, Hamburg, 8-12 Sept., 1980.
Star, L., Decuypere, E., Parmentier, H. K., Kemp, B. 2008. Effect of single or combined
climatic and hygienic stress in four layer lines: 2. Endocrine and oxidative stress
responses. Poult. Sci. 87: 1031–103
Stark, J. M. 1974. Rate of antigen catabolism and immuno-genecity of 1311 B66 in mice.
Immunology.19: 457-468.
Steel, R. G. D, J. H. Torrie and D. A. Dickey. 1997. Principles and Procedures of Statistics, a
biometrical approach. 3rd ed. McGraw Hill Book CO. Inc. New York.
Stevens, B. R., J. D. Kannitz and E. M. Wright. 1984. Intestinal transport of amino acids and
sugars: Advances using membrane vesicles. Annu. Rev. Physiol. 46: 417‒433
Stoimenov, K. 1976. Effect of some trace elements on Heterakis gallinarum invasion. Vet.
Med. Nauki. 13: 5-11.
Sturkie, P. D. 1976. Avian physiology 3rd Ed. Springer Virlag. N. Y. Heidelberg, Berlin
Sturkie, P. D. and P. Griminger. 1976a. Blood: Physical characteristics, formed elements'
haemoglobin and coagulation. In "Avian physiology" 3rd edition, edited by Sturkie,
P. D. Sringerr Verlag, New York, pp 54-75.
Suba-Rao, D. S.V. and B. Click. 1977. Effect of cold exposure on the immune response of
chicken. Poult. Sci., 56: 992-996.
Swenson, M. J. 1970. Duke's physiology of domestic animals. 8th Ed. Comstock Publishing
Associates, London. 753.
209
Szabó J, Vucskits AV, Andrásofszky E, Berta E, Bersényi A, Börzsönyi L, Pálfi V,Hullár I.
2011. Effect of dietary electrolyte balance on production, immune response and
mineral concentrations of the femur in broilers. Acta Veterinaria Hungarica. 59: 295-
310.
Takahashi, K., Y. Akiba and M. Horiguctti. 1991. Effects of supplemental ascorbic acid on
performance, organ weight and plasma cholesterol concentration in broilers treated
with prophyl- thiouracii. Br. Poult. Sci. 32: 545-554.
Takahashi, K. , and Y. Akiba. 2002. Effect of oral administration of Diakur (a glucose and
electrolyte additive) on growth and some physiological responses in broilers reared in
a high temperature environment. Asian-aust. J. Anim. Sci.15:1341–1347.
Tanor, M. A., S. Lesson and J. D. Summers. 1984. Effect of heat stress and diet composition
on performance of White Leghorn hens. Poult. Sci. 63: 304-310.
Teeter, R. G., M. O. Smith, F. N. Owens, S. C. Arp, S. Sangiah and J. E. Breazile. 1985.
Chronic heat stress and respiratory alkalosis: Occurrence and treatment in broiler
chickens. Poult. Sci. 64: 1060-1064.
Teeter, R. G. 1988. Enhancing broiler productivity during chronic and acute heat stress.
Monsanto Nutrition Update, 6: 1-6.
Teeter, R. G., M. O. Smith and C. J. Wiernusz. 1992. Research note: Broiler acclimation to
heat distress and feed intake effects on body temperature in birds exposed to
thermoneutral and high ambient temperatures. Poult. Sci.71: 1101-1104.
Teeter, R. G. and T. Belay. 1996. Broiler management during acute heat stress. Animate
Feed Science and Technology. Oklahoma State University Stillwater, USA. 58: 127-
142.
Tengerdy, R. P. 1970. The immune suppressive effect of hypoxia on chicken embryos.
Experimentia 26: 309-310.
Tengerdy, R. P. and J. C. Brown. 1977. Effect of vitamin E and A on humoral immunity and
phagocytosis in E. coli infected chickens. Poult. Sci. 56: 957-963.
Thaxton, P. and C. R. Parkhurst. 1973. Toxicity of mercury to young chickens, Changes in
immunological responsiveness. Poult. Sci. 52: 761-764.
Thaxton, P. and D. M. Briggs. 1972. Effect of immobilization and formaldehyde on
immunological responsiveness in young chickens. Poult. Sci. 51: 342-344.
Thaxton, P. and H. S. Siegel. 1970. Immuno depression in young chickens by high
environmentaltemperature. Poult. Sci. 49: 202-205.
210
Thomason, D. M., A. T. Leighton. Jr. and J. P. Jr. Mason. 1977. Effect of temperature,
environment and laying cages on the reproductive performance of turkeys. Poult. Sci.
56: 426-434.
Tojo, H. and T. M. Huston. 1980. Effect of environmental temperature on the concentration
of serum estradiol, progesterone and calcium in maturing female domestic fowl.
Poult. Sci. 59: 2797-2802.
Toyomizu, M., M. Ueda, S. Sato, Y. Seki, K. Sato and Y. Akiba. 2005. Cold-induced
mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in
chicken skeletal muscles. Federation of European Biochemical Societies Letters. 529:
313–318.
Trail, J. C. M.1963. Shell and egg interiorquality of the indigenous poultry of Uganda
compared with five imported breeds and crosses. Poult. Sci. 42: 1887-1891.
Trinder, P. 1969. Enzymatic colorimtric determination of triglycerides by GPO-PAP method.
Ann. Clin. Biochm. 6: 24-27.
Tuekam, T. D., R. D. Miles and G. D. Butcher. 1994. Performance and humoral response in heat stressed broilers fed an ascorbic acid supplemented diet. J. Applied Anim. Res. 6: 121-130.
Van Keulen, J. and B. A. Young. 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 44: 282–287.
Vecerek, V., E. Strakova, P. Suchy and E. Voslarova. 2002. Influence of high environmental
temperature on production and haematological and biochemical indexes in broiler
chickens. Czech. J. Anim. Sci. 47: 176182.
Vidal, J. and D. Walsh. 2010. "Temperatures reach record high in Pakistan". guardian.co.uk (London). Retrieved 21July, 2010. Accessed on http://www.theguardian.com/world/2010/jun/01/pakistan-record-temperatures-heatwave.
Vieites, F. M., G. H. K. Moraes, L. F. T. Albino, H. S. Rostagno, A. Atencio, J. G. Jr. Vargas. 2005. Balanço eletrolítico e níveis de proteína bruta sobre o desempenho, o rendimento de carcaça e a umidade da cama de frangos de corte de 1 a 42 dias de idade. Revista Brasileira de Zootecnia. 34: 1990-1999.
Vincek, C. 1967. Vitamin C in nutrition of man and animals. Vet. Glasnik. 21: 351-359.Virden, W. S., M. S. Lilburn, J. P. Thaxton, A. Corzo, D. Hoehler, and M. T. Kidd. 2007.
The effect of corticosterone- induced stress on amino acid digestibility in Ross broilers. Poult. Sci. 86: 338–342.
211
Waldroup, P. W., S. E. Watkins and H. M. Hellwig. 2005. Influence of sodium source and
level on performance of second-cycle hens fed diets with different levels of
nonphytate phosphorus. Int. J. Poult Sci. 4: 399-407.I
Warriss, P. D., Pagazaurtundua, A. Brown, S. N. 2005. Relationship between maximum daily
temperature and mortality of broiler chickens during transport and lairage. Br. Poult.
Sci. 46: 647–651.
Washburn, K. W. and T. M. Huston. 1968. Effect of environmental temperature on iron
deficiency anemia in Athens Canadian randombred. Poult. Sci. 47: 1532-1535.
Whitehead, C. C. and D. W. F. Shannon. 1974. The control of egg production using a low
sodium diet. Br. Poult. Sci. 15: 429-434.
Whiting, T. S., L. D. Andrews, and L. Stamps. 1991. Effects of sodium bicarbonate and
potassium chloride drinking water supplementation. Performance and exterior
carcass quality of broilers grown under thermoneutral or cyclic heat-stress conditions.
Poult. Sci. 70: 53- 59.
Whiting, T. S., L. S. Andrews, M. H. Adams and L. Stamps. 1991a. Effects of sodium
bicarbonate and potassium chloride drinking water supplementation. Meat and
carcass characteristics of broilers grown under thermoneutral and cyclic heat-stress
conditions. Poult. Sci. 70: 60-66.
Wideman, R. F. J. and E. G. Buss. 1985. Arterial blood gas, pH and bicarbonate values in
laying hens selected for thick or thin egg shell production. Poult. Sci. 64: 1015-1019.
Wideman, R. F., D. M. Hooge and K. R. Cummings. 2003. Dietary sodium bicarbonate, cool
temperatures, and feed withdrawal: Impact on arterial and venous blood gas values in
broilers. Poult. Sci. 82: 560–570.
Wolfenson, D.1986. The effect of acclimatization on blood flow and its distribution in
normothermic and hyperthermic domestic fowl. Comp. Biochem. Physiol. 85:739-
742.
Yahav, S. and Hurwitz, S. 1996. Induction of thermotolerance in male broiler chickens by
temperature conditioning at an early age. Poult, Sci. 75: 402–406.
Yahav, S., A. Straschnow, I. Palvnik and S. Hurvitz. 1997. Blood system response of chicken
to changes in environmental temperature. Poult. Sci., 76: 627-633.
Yahav, S. 2000. Domestic fowl-Strategies to confront environmental conditions. Avian and
Poult. Biol. Rev. 11: 81-95.
212
Yahav, S., A. Straschnow, D. Luger, D. Shinder, J. Tanny and S. Cohen. 2004. Ventilation,
sensible heat loss, broiler energy, and water balance under harsh environmental
conditions. Poult. Sci. 83: 253-258.
Yalcin, S., S. Ozkan, G. Oktay, M. Cabuk, Z. Erbayraktar, and S. F. Bilgili. 2004. Age-
Related effects of catching, crating, and transportation at different seasons on core
body temperature and physiological blood parameters in broilers. J. Appl. Poult. Res.
13:549–560.
Yalcin, S., S. Ozkan, L. Turkmut and P. B. Siegel. 2001. Responses to heat stress in
commercial and local broiler stocks.1. Performance traits. Br. Poult. Sci. 42: 149-152.
Yamauchi. 1995. Effect of cool air on the performance of laying hens fed in free cage with
alternate racks in the summer. J. app. Poult. Sci. 32: 350-358.
Yang, Q. M., Q. W. Mu., Z. H. Yu and H. Lin. 1992. A study of influence of environmental
temperature on some biochemical indices in serum of broilers. J. Shandong Agri.
Univ. 23: 363-367.
Yaqoob, M. 1966. Effect of varying levels of heat stress on the physiological behaviours of
Desi and White Leghorn layers. Ph.D Thesis, Department of Poultry Science.
University of Agriculture, Faisalabad.
Yaqoob, M., S. M. N. Haider and M. Z. Siddiqui. 1965. Preliminary studies on the growth
pattern and productive behavior of local chicken in West Pakistan. Pak. J. Agri. Sci.
2: 103-107.
Yin, Z.Z.; Yuan S.R. and Pan G.C. 2001. Effects of adding Sodium compounds to Corn-rape
seed meal type diet on production performance of laying hens. Journal of Zhejiang
University Agriculture and Life Sciences. 27: 99-102.
Yoruk , M. A., M. Gul, A. Hayirli and M. Karaoglu. 2004. Laying performance and egg
quality of hens supplemented with sodium bicarbonate during the late laying period.
Int. J. Poult. Sci. 3: 272-278.
Yousef, M. K. 1985. Stress Physiology in Livestock. Vol. II. Poultry CRC Press, Inc. Boca
Raton, Florida, Pp: 125-127.
Zakaria, H. A., M. J. Tabbaa, K. M. Alshawabkehand K. Altaif. 2009. The effect of dietary
sodium bicarbonate on performance and blood parameters of broiler chickens and
local Balady breed inoculated with Salmonella gallinarum. J. Anim. and Feed Sci. 18:
335–347
213
Zakia, A. M. A., E. El-Khashab, A. M. El-Nabarawi and J. EIjaky. 1995. Effect of light and
food withdrawal on the broiler responses to heat stress and/or Salmonella
typhimurium infection at early growing stage. Vet. Med. J. Giza and Zonoses. 43:
461-468.
Zimmerman, R. A, D. C. Snetsinger and D. E. Green. 1973. Significance of day length and
air circulation for commercial layers under heat stress. Poult. Sci. 52: 2106.
Zimmerman, R. A., D. C. Snetsinger and D. E, Green. 1975. Comparative performance of
laying chickens in constant versus cyclic temperature environments. Poult. Sci. 54:
1831.
Zook, J. and G. R. Sharpless. 1938. Vitamin C nutrition in artificial fever. Proc. Soc. Exp.
Biol. Med. 39: 233-236.
Zuprizal, Larbier, M. Chagneau, A. M. and P.A. Geraert. 1993. Influence of ambient
temperature on true digestibility of protein and amino acids of rapeseed and soybean
meals in broilers. Poult. Sci.72: 289-295.
214