NUTRITIONAL EVALUATION OF AZOLLA...ii nutritional evaluation of azolla (azolla pinnata) and its...

i

NUTRITIONAL EVALUATION OF AZOLLA

(Azolla pinnata) AND ITS SUPPLEMENTARY EFFECT ON

IN VITRO DIGESTIBILITY OF CROP RESIDUES AND

TOTAL MIXED RATION

KAVYA, K.

DEPARTMENT OF ANIMAL NUTRITION

VETERINARY COLLEGE, BANGALORE

KARNATAKA VETERINARY, ANIMAL AND FISHERIES

SCIENCES UNIVERSITY, NANDINAGAR, BIDAR

JUNE, 2014

ii

NUTRITIONAL EVALUATION OF AZOLLA

(Azolla pinnata) AND ITS SUPPLEMENTARY EFFECT ON

IN VITRO DIGESTIBILITY OF CROP RESIDUES AND

TOTAL MIXED RATION

Thesis submitted to the


SCIENCES UNIVERSITY, BIDAR

In partial fulfillment of the requirements

for the award of the degree of

MASTER OF VETERINARY SCIENCE

in

ANIMAL NUTRITION

By

KAVYA, K.


VETERINARY COLLEGE, BANGALORE


SCIENCES UNIVERSITY, NANDINAGAR, BIDAR

JUNE, 2014

iii


SCIENCES UNIVERSITY, BIDAR


VETERINARY COLLEGE, HEBBAL, BANGALORE

CERTIFICATE

This is to certify that the thesis entitled “NUTRITIONAL EVALUATION OF

AZOLLA (Azolla pinnata) AND ITS SUPPLEMENTARY EFFECT ON IN VITRO

DIGESTIBILITY OF CROP RESIDUES AND TOTAL MIXED RATION’’

submitted by Ms. KAVYA, K., ID No. MVHK 1204 in partial fulfillment of the

requirements for the award of MASTER OF VETERINARY SCIENCE in ANIMAL

NUTRITION of the Karnataka Veterinary, Animal and Fisheries Sciences University,

Bidar is a record of bonafide research work carried out by her during the period of her

study in this University under my guidance and supervision and the thesis has not

previously formed the basis for the award of any degree, diploma, associateship,

fellowship or other similar titles.

Place: Bangalore (Dr. T. M. PRABHU)

Date: June, 2014 Associate Professor

Major advisor

Approved by:

Chairman : ______________________________

(Dr. T.M. PRABHU)

Members : 1. ______________________________

(Dr. R. GIDEON GLORIDOSS)

2. ______________________________

(Dr. K. CHANDRAPAL SINGH)

3. ______________________________

(Dr. Y. B. RAJESHWARI)

4. ______________________________

(Dr. SIDDARAMANNA)

iv

Affectionately Dedicated To

My Beloved Parents

v

ACKNOWLEDGEMENT

I wish to express my deep sense of gratitude to Dr. T. M. Prabhu, Associate

Professor, Department of Animal Nutrition, Veterinary College, Bangalore and the

Chairman of my Advisory Committee. My sincere thanks are due to his constant

supervision, valuable suggestions, continuous encouragement and his efforts for

providing me the atmosphere and facilities that were needed for completing this work

successfully.

I wish to express my profound gratitude and sincere respect to

Dr. R. Gideon Gloridoss, Professor and Head, Department of Animal Nutrition,

Veterinary College, Bangalore and member of my Advisory Committee for his inspiring

guidance, valuable suggestions, constant encouragement throughout my study.

I express my gratitude to my Advisory Committee members

Dr. K. Chandrapal Singh, Head, Division of Animal and Poultry Science, KVAFSU,

Bidar, Dr. Y. B. Rajeshwari, Professor, Department of Livestock Production and

Mangement and Dr. Siddaramanna, Senior Technical Officer, NDRI, Adugodi,

Bangalore for their kind assistance, co-operation and suggestions

I am extremely thankful to Dr. U. Krishnamoorthy, Professor and Head,

Department of Livestock Production and Management, Veterinary College, Bangalore for

providing me the laboratory facilities for in vitro studies.

My sincere thanks to Dr. N. K. Shivakumar Gowda, Principal Scientist (Animal

Nurtrition), NIANP, Adugodi, Bangalore for providing me the Laboratory facilities to

analyse mineral profile of samples.

My sincere thanks to Mr. Hemanth Pandey, Research Scientist, Alltech

Ruminant Nutrition Laboratory, KVAFSU, Bangalore for providing me the Laboratory

facilities for in vitro studies.

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My sincere thanks to Dr. Kiran Doranalli, Regional Technical Manager (Asia

South), Health and Nutrition –Feed Additives, Evonik (SEA) Private Limited, Singapore

for his help to analyze the amino acid composition of Azolla at Evonik laboratory,

Singapore.

I express my heartfelt thanks and gratitude to my seniors Dr. Shivasharanappa

Biradar, Dr. Vikram, Dr. Venkatesh B. S, Dr. Anjaneya and Dr. Basvanth Kumar

for their constant help, encouragement and support.

It is my privilege to extend sincere thanks to my friends Dr. Thahir Ahamed,

Dr. Kavya. P. S, Dr. Sharadha and Dr. Pradeep for their cordial help and cooperation

during my research work.

My special thanks to the supporting staff such as Sri Rangaswami and

Smt. Shakuntala, Department of Animal Nutrition for their constant help during the

period of study.

It is family members without whose support and encouragement, I would not be

in this position today. At this explicable moment, I wish to express my gratitude to my

father Sri. R. Kalidas, mother Smt. G. Lakshmi and sister Soumya. K for their moral

support and encouragement which enabled me to achieve this today.

June, 2014

Bangalore (KAVYA, K.)

vii

CONTENTS

CHAPTER TITLE PAGE No.

I INTRODUCTION 1-4

II REVIEW OF LITERATURE 5-25

III MATERIALS AND METHODS 26-37

IV RESULTS 38-66

V DISCUSSION 67-76

VI SUMMARY 77-80

VII BIBLIOGRAPHY 81-89

VIII ABSTRACT 90

IX APPENDICES 91-106

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LIST OF TABLES

Table

No. Title of Table

Page

No.

2.1 Per cent chemical composition (DMB) of aquatic plants and alfalfa. 6

2.4.1 Per cent chemical composition of different species of Azolla (Dry matter

basis). 14

2.4.2 Chemical composition (on per cent dry matter basis) of Azolla pinnata. 16

2.4.3 Chemical composition of Azolla varieties 18

2.4.4 Amino acid composition of azolla. 20

2.5.1 Chemical composition of varieties of paddy straw. 22

2.5.2 Digestibility of OM and ME content of different varieties of paddy straw. 22

2.5.3 Chemical composition of rice straw in early, mid and late growing period. 23

4.1.1 Proximate principles (on per cent dry matter basis) of Azolla pinnata,

Crop residues and CFM. 39

4.1.2 Fiber fractions, total silica, biogenic silica and sand silica (on per cent dry

matter basis) of Azolla pinnata, crop residues and CFM. 41

4.1.3 Mineral profile of Azolla pinnata. 43

4.1.4 Amino acid profile of Azolla pinnata 44

4.2.1

Chemical composition (Percentage on DMB) of various diets comprising

paddy straw supplemented with different levels of azolla from 0 to 9 per

cent and the TMR comprising paddy straw and CFM supplemented with

different levels of azolla from 0 to 9 per cent.

45

4.2.2


ragi straw supplemented with different levels of azolla from 0 to 9 per

cent and the TMR comprising ragi straw and CFM supplemented with


46

4.2.3

Chemical composition (Percentage on DMB) of various diets containing

maize stover supplemented with different levels of azolla from 0 to 9 per

cent and the TMR comprising maize stover and CFM supplemented with


47

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Table

No. Title of Table

Page

No.

4.2.4


maize husk supplemented with different levels of azolla from 0 to 9 per

cent and the TMR comprising maize husk and CFM supplemented with


49

4.2.5


sorghum stover supplemented with different levels of azolla from 0 to 9

per cent and the TMR comprising sorghum stover and CFM supplemented

with different levels of azolla from 0 to 9 per cent.

50

4.2.6


Bengal gram husk supplemented with different levels of azolla from 0 to 9

per cent.

51

4.3

Rumen in vitro net gas production at 24 hours (IVGP-24h) and predicted

metabolizable energy (ME) content of Azolla pinnata, Crop residues and

CFM.

52

4.4.1 Rumen in vitro net gas production of diets comprising roughage replaced

with different levels of azolla from 0 to 9 per cent (Type I diet). 54

4.4.2

Rumen in vitro net gas production of diets (TMR) comprising roughage

(60 per cent kept constant) and CFM (31 to 40 per cent) replaced with

different levels of azolla from 0 to 9 per cent (Type II diet).

55

4.4.3

Rumen in vitro net gas production of diets (TMR) comprising CFM (40

per cent kept constant) and roughage (51 to 60 per cent) replaced with

different levels of azolla from 0 to 9 per cent (Type III diet).

56

4.5.1 Metabolizable energy (ME) content of diets comprising roughage

replaced with different levels of azolla from 0 to 9 per cent (Type I diet). 57


replaced with different levels of azolla from 0 to 9 per cent (Type II diet). 59


replaced with different levels of azolla from 0 to 9 per cent (Type III diet). 61

4.6.1

Apparent digestible dry matter (ADDM %) and True digestible dry matter

(TDDM %) of Azolla pinnata and the diets comprising roughage replaced

with azolla at 0 per cent and 9 per cent (Type diet).

63

4.6.2

Apparent digestible dry matter (ADDM %) and Total digestible dry

matter (TDDM %) of the diets (TMR) comprising CFM (40 per cent kept

constant) and roughage (51 to 60 per cent) replaced with azolla at 0 per

cent and 9 per cent (Type III diet).

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x

LIST OF PLATES

Plate No. Title of Plate Page No.

3.1 Cultivation of Azolla (Azolla pinnata) in cement tanks 30

xi

LIST OF APPENDICES

Appendix No. Title Page

No.

Appendix I Gas production data of Hay standard and Concentrate standard

of 4 trials at different time intervals over 24 hours 91-92

Appendix II Gas production data of Azolla at different time interval over 24

hours 93

Appendix III Gas production data of Crop residues at different time interval

over 24 hours 94

Appendix IV

Gas production data of various diets comprising paddy straw

supplemented with different levels of azolla from 0 to 9 per cent

and the TMR comprising paddy straw and CFM supplemented


95-97

Appendix V

Gas production data of various diets comprising Ragi straw




98-99

Appendix VI

Gas production data of various diets comprising Maize stover




100-101

Appendix VII

Gas production data of various diets comprising Maize husk




102-103

Appendix VIII

Gas production data of various diets comprising Sorghum stover




104-105

Appendix IX

Gas production data of various diets comprising Bengal gram

husk supplemented with different levels of azolla from 0 to 9

per cent

106

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LIST OF ABBREVIATIONS

ADDM -

Apparent digestible dry matter

ADF - Acid detergent fiber

ADL -

Acid detergent lignin

ANOVA -

Analysis of Variance

AOAC -

Association of Official Analytical Chemists

BS - Biogenic silica

CF - Crude fiber

CFM -

Compounded feed mixture

CP - Crude protein

DM -

Dry matter

DMB - Dry matter basis

DMD

Dry matter digestibility

EE - Ether extract

g - Gram

GP -

Gas production

ICP-AES -

Inductively coupled plasma-atomic emission spectrophotometer

Kg - Kilogram

ME - Metabolizable energy

NDF -

Neutral detergent fiber

NFE -

Nitrogen free extractives

OM -

Organic matter

RIVGP -

Rumen in vitro gas production

SS -

Sand silica

TA -

Total ash

TDDM -

True digestible dry matter

TMR -

Total mixed ration

IInnttrroodduuccttiioonn

I. INTRODUCTION

India has the largest livestock population in the world. The livestock population is

expected to grow at the rate of 0.55 per cent in the coming years, and the population is

likely to be around 781 million by 2050. Though India is among the leading producers of

milk, meat and eggs; productivity of our animals is 20-60 per cent lower than the global

average due to improper nutrition, inadequate health-care and management. Half of the

total losses in livestock productivity are contributed by the inadequacy in supply of feed

and fodder (ICAR, 2012).

The demand for milk and meat has been increasing and animal husbandry as a

profitable occupation, is expanding. However, there is a substantial decline in fodder

production, owing to the decreasing area under forest and grasslands. The fodder

availability from various crops has also decreased largely due to the introduction of high

yielding dwarf varieties. The shortage of fodder is therefore, being compensated with

commercial feed, resulting in increased cost of production of meat and milk. (Pillai et al.,

2002).

A major constraint to livestock production in developing countries is the scarcity

and fluctuating quantity and quality of the year-round feed supply. Providing adequate

good quality feed to livestock to raise and maintain their productivity is a major challenge

to agricultural scientists all over the world. The increase in population and rapid growth

in world economies will lead to an enormous increase in demand for animal products, a

large part of which will be from developing countries. Future hopes of feeding the

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millions and safeguarding their food security will depend on the enhanced and efficient

utilization of alternative feed resources (FAO report, 2006).

Forage-based economical feeding strategies are required to reduce production cost

of quality livestock products; as feed alone constitutes 60-70% of production cost. At

present, the country faces a net deficit of 35.6 per cent of green fodder, 26 per cent of

dry-crop residues and 41 per cent of concentrate feed ingredients(ICAR, 2012).

In India, Karnataka is second state next to Rajasthan in terms of total geographical

area which is drought prone. Out of the 29 districts of Karnataka, 6 districts belonged to

adequate dry matter (DM) available category with the mean availability of 87.51 %. Five

districts belonged to moderately adequate (60 -79%) and 10 districts belonged to

deficient DM available categories (40-59%). However, one in every four districts

belonged to severely DM deficient category (below 40%). The mean DM availability for

the state was 56.46 %. Total contribution of crop residues to DM in the state was 72.59

%, with coarse straw contribution being one third of it. The contribution of green fodder

to the total DM availability in the state was 23.6 % and concentrates was 3.81%

(Nagaratna and Vinod, 2013).

The estimates by different group of workers have consistently pointed out the

deficit of the feed resources for livestock in terms of dry roughages, greens and

concentrates. Conventional sources of feeds are not enough to mitigate the shortage of

feeds and fodder and to make animal production viable and profitable. The gap between

the demand and supply is also increasing. In order to bridge this gap and to ensure

optimum production of livestock throughout the year use of non-conventional feed

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resources as supplement or replacement of conventional feed without compromising the

quality is the area of focus in recent years. (Chatterjee et al, 2013).

There are varieties of unconventional feedstuffs available which find use as

protein and energy source for livestock. The supplementary resources in India include

aquatic macrophytes which have rich nutrient and mineral profile. Throughout the world,

and particularly in Asia, farmers have harvested naturally produced aquatic plants for

number of purposes which includes animal feed, green manure and for their family food.

The best known of these include the free floating plants; water lettuce (Pistia), water

hyacinth (Eichhcornia), duckweed (Lemna) and azolla and some bottom growing plants.

In recent years commonly occurring aquatic plant, azollahas become prominent and has

attracted the attention of scientists because of its apparent high potential as a feed

resource for livestock. Therefore, some workers also called it as Green gold mine due to

its high nutritive value and Super plant due to its fast growth (Wagner, 1997). Azolla is

considered as the most economic and efficient feed substitute and a sustainable feed for

livestock. It is a potential source of nitrogen and thereby a potential feed ingredient for

livestock (Lumpkin, 1984; Pannaerker, 1988).

Azolla is a free floating aquatic fern belonging to family Azolaceae, order

Pteridophyta distributed throughout the tropical, subtropical and temperate fresh water

ecosystems. It is used as nitrogen source for paddy cropping systems. Azolla can supply

around 25-30 kg of nitrogen per hectare (Pillai et al., 2002). In India Azolla is found

floating on the water in the shallow ditches and in channels. Azolla can fix atmospheric

nitrogen with help of blue green algae, Anabaena azollae, found in cavities of dorsal part

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of leaves. This fact makes the azolla tend to contain relatively high levels of nitrogen and

can be a protein source for animal feeding.

The farmers, particularly in South East Asia and probably elsewhere had

developed the use of azolla as a source of nutrients for livestock. There are some reports

on the use of azollaas feed supplement for poultry and livestock, in which normal feed

protein sources have been replaced by azolla on an iso-nitrogenous basis (Chatterjee,

2013). An attempt has been made to evaluate azolla as a nutrient supplement by invitro

studies to assess its utility as a ruminant feed.

The present study was conducted with the following objectives:

1. To evaluate azolla in terms of its chemical composition, digestibility and

metabolizability by in vitro techniques.

2. To study the supplementary effect of azolla on in vitro digestibility and

metabolizability of crop residues and total mixed ration.

RReevviieeww ooff LLiitteerraattuurree

II. REVIEW OF LITERATURE

Inadequate availability of good quality feed is regarded as a major constraint to

the prevalent livestock production system. The conventional feed ingredients, particularly

protein supplement are expensive and are not always available at affordable prices. Since

the cost of feeding is a significant factor dictating the economic viability of livestock

industry, it must be reduced by adopting new measures in the ration formulation. Hence

to make livestock production a profitable enterprise, there is a great need to use alternate

feedstuffs replacing the traditional sources. This leads to a search for cheap and easily

available protein sources. Aquatic plants have long been used in many developing

countries as feed source for livestock and poultry.

The aquatic plants such as water hyacinth and hydrilla species (Boyed, 1969);

Duck weed (Culley and Epps, 1973) are approximately equal to alfalfa in crude protein

and crude fiber contents (Spencer et al., 1969). The lower level of fiber content in some

of the aquatic species depends on location and stage of growth. However, the ash and

xanthophylls contents are fairly high, when compared to the conventional forages.

Although, high ash content is being one of the apparent limitation in nutritional value of

such aquatic plants, but high xanthophylls contents possibly offset the limitation and thus,

makes these aquatic plants superior to other plants.

2.1 Nutritive value of Aquatic plants

Mutzar et al. (1976) studied the nutritional value of five different species of dried

ground aquatic plants, Azolla pinnata, caladophora, pond weed, milofoil and vallisneria

duck weed. These species of aquatic plants revealed (Table 2.1) that, the water weeds had

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extremely variable levels of ash (18.4-62.5 per cent) and crude protein (5.6-15.5 per cent)

but contained relatively less crude fiber and ether extract than dehydrated alfalfa meal.

The calcium content varied from 3.0 to 16.7 per cent as compared to 1.9 per cent in

dehydrated alfalfa meal. Lipstein and Hurwitz (1980) reported 574 g of total protein, 22 g

fiber, 89 g moisture, 11.7 g phosphorus and 2.0 g calcium per kg of algae meal.

Table 2.1: Per cent chemical composition (DMB) of aquatic plants and alfalfa

Plant species Crude protein Crude fiber Ether extract Ash Ca P

Azolla pinnata 21.4 12.7 2.7 16.2 1.16 1.29

Cladophora 12.93 14.5 1.22 59.83 4.93 0.28

Duck weed 15.53 16.64 3.14 18.43 3.03 0.39

Milofoil 5.61 17.04 0.94 62.53 16.72 0.10

Pond weed 10.54 20.0 2.90 40.44 11.90 0.19

Alfalfa meal 17.7 24.2 3.65 9.9 1.9 0.27

2.2 Azolla

Azolla (mosquito fern, duckweed fern, fairy mass and water fern) is a floating

fern in shallow water. It floats on the surface of water by means of numerous, small,

closely overlapping scale like leaves with their roots hanging in the water. They are

extremely reduced in form and specialized, looking nothing like conventional fern but

more resembling duckweed. It can readily colonize areas of fresh water and grows at

great speed doubling its biomass every two to three days (Van Hove and Lejeune, 2002).

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2.2.1 Morphology

Azolla is an aquatic fern consisting of a short, branched, floating stem, bearing

roots which hang down in the water. The leaves are alternately arranged and each

consists of a thick aerial dorsal lobe containing green chlorophyll and a thin floating

ventral lobe of slightly larger size that is colorless. Under certain conditions, an

anthocyanin pigment gives the fern a reddish-brown color. Plant diameter ranges from 1-

2.5 cm for small species, such as Azolla pinnata, to 15 or more cm like Azolla nilotica.

Azolla plants are triangular or polygonal in shape, and float on the water surface

individually or in mats. They give the appearance of a dark green to reddish carpet,

except Azolla nilotica which does not produce the red anthocyanin pigment. The most

remarkable characteristic of azolla is its symbiotic relationship with the nitrogen-fixing

blue-green algae (cyanobacterium) Anabaena azollae. The fern provides nutrients and a

protective cavity in each leaf to Anabaena colonies in exchange for fixed atmospheric

nitrogen and possibly other growth-promoting substances (Lumpkin, 1984).

2.2.2 Taxonomy and biogeography

Azolla is the only genus in the family Azollaceae and has a worldwide

distribution from temperate to tropical climates. There are seven species of azolla, which

are widely distributed in different parts of the world as Azolla caroliniana (North

America and the Caribbean), Azolla filiculoides (South America), Azolla microphylla

(Tropical and subtropical America), Azolla mexicana (North and South America), Azolla

nilotica (upper reaches of the Nile to Sudan), Azolla rubra (Japan, Korea, Australia and

New Zealand) and Azolla Pinnata commonly found in India (Van Hove and Lejeune,

2002).

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2.2.3 Symbiotic interaction with Anabaena azollae

Azolla hosts symbiotic blue green algae Anaebaena azollae, which is responsible

for the fixation and assimilation of atmospheric nitrogen. Azolla, in turn, provides the

carbon source and favorable environment for the growth and development of the algae. It

is the unique symbiotic relationship that makes azolla, a wonderful plant with high

protein content (Pillai et al., 2002).This symbiotic relationship gives azolla a competitive

advantage over other floating hydrophytes in environments such as rice fields, which are

relatively low in available nitrogen. The symbiosis is of interest to agriculturists, since

incorporation and decomposition of azolla crop in rice field soil can result in, increased

amount of available nitrogen to a companion or succeeding rice crop. Azolla can

accumulate more than 10 kg N / ha per day. Some strains of azolla can fix as much as 1-3

kg of nitrogen / ha / day and its annual yield is 730 tonnes / ha as a green azolla for

feeding animals (Lumpkin and Plucknett, 1982).

2.2.4 Azolla in agriculture

Azolla can be used as a food, mosquito inhibitor, green manure, herbicide, water

saver, water purifier, nitrogen fertilizer saver, drug and for reclaiming saline soils (Van

Hove and Lejeune, 2002). It has long been used by farmers, mainly in Asia, as feed for

the animals and as green manure. A number of laboratory and field studies have shown

an unquestionable beneficial effect of azolla as an organic nitrogen fertilizer, mainly in

terms of increasing rice grain yield. In addition, presence of an azolla mat on the surface

of the water body has been shown to significantly reduce weed development, reduce

volatilization of applied N fertilizer and purify water.

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Azolla is used as a nitrogenous crop in Vietnam, China, Taiwan and other South

East Asian countries. Azolla is capable of assimilating atmospheric nitrogen efficiently,

due to presence of an algal symbiont in the leaves (Moore, 1969).

Fresh azolla is used in the preparation of compost. Since, the fern has an excellent

carbon nitrogen ratio, it decomposes rapidly and accelerates the decomposition of other

organic residues inside the compost pit and used as a biofertilizer in coffee plantations

(Anandand Geetha., 2007).

2.2.5 Yield

The plant multiplies rapidly and gives good dry matter yield in spite of its high

water content. Singh (1982) reported that under conditions in Cuttack regions of East

coast, an annual production of fresh azolla was 347 tonnes / ha in field and 321 tonnes /

ha in concrete tank.

Pillai et al. (2002) reported that 500-600g of fresh azolla can be harvested daily

from a pit of 2x 2 x 0.2 m size after 10-15 days of adding culture.

2.2.6 Azolla production and multiplication

The cultivation of azolla is a continuous process requiring a certain amount of

attention throughout the year (Lumpkin and Plucknett, 1980). Usually a certain area of

field within a production unit is set aside for such cultivation.

Azolla nurseries of small plot (50-100 m2) area are prepared to avoid wind action.

A standing water of 3 to 5 cm depth is essential. A pH of 5.0 to 7.0 is ideal and acidic

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soils with pH 4-6 are not suitable unless lime is used to correct pH. Winter temperature in

the range of 14oC to 35

oC is preferable but the optimum range is 20

oC to 30

oC. The azolla

inoculums at 0.1 to 0.4 kg per hectare is most desirable for production of 8 to 10 tonnes

per hectare green manure in 20 days. Application of super phosphate at 75 kg per hectare

is essential for rapid growth of azolla. Use of carbofuran/ furadon at one to two kg per

hectare or 0.625 g per m2 prevents the rapid spread of insects, parasites and the

consequent destruction of azolla nurseries (Pillai et al., 2002). At the good growth period

the azolla mat should be separated once in a week. Azolla doubles its weight in 3-5 days.

From a start of 1 tonne / ha, it can reach a fresh weight of 15-20 tonnes / ha in about 20

days (Khan, 1983).

2.3 Azolla as a livestock feed

Azolla is rich in protein. On a dry weight basis, it contains 25 - 35 per cent

protein, 10 - 15 per cent minerals and 7 – 10 per cent of amino acids, bio-active

substances and bio-polymers. The carbohydrate and fat content of azolla is very low. Its

nutrient composition makes it a highly efficient and effective feed for livestock. Azolla

can be used in animal feed and it is a potential feed ingredient for broilers; it is an income

generating crop (Reddy, 2007). Azolla is one of the most nutritive aquatic plant, owing to

its high protein and carotenoid contents and of generally good amino acid profile. It can

be incorporated into the feed of ruminants (Nik Khah and Motaghi, 1992), small

ruminants (Tamang et al, 1993; Ali and Leeson, 1995), pigs (Bacerra et al., 1995), broiler

chickens (Basak et al., 2002) and rabbits (Sreemannarayana et al., 1993).

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Feeding trials with azolla in dairy cows and buffaloes, growing buffaloes, sheep

and goats have been carried out in India. Since the 2000s, azolla is being promoted in

India for dairy production (Pillai et al., 2002). Azolla meal (dried azolla) can be included

upto 15 per cent of total concentrate requirement of growing Osmanabadi goats (Ghodake

et al., 2012).

Azolla is used as a feed ingredient for ruminants and non-ruminants (Singh and

Subudhi, 1978). Azolla has long been used as green manure and as a feed for poultry

(Basak et al., 2002). The use of azolla as a green feed in cattle, swine, poultry and fish

has been tested with favourable results (Alalade and Iyayi, 2006). Azolla can serve as a

potential alternative nutrient supplement for the crossbred cattle for the improvement of

productivity in terms of growth, milk, meat etc (Chatterjee et al., 2013).

Azolla can be fed to the livestock either in a fresh or dried form. It can be given

directly or mixed with concentrates to cattle, poultry, sheep, goat, pigs and rabbits.

(Giridhar et al., 2012). Nik Khah and Motaghi (1992) reported that, the azolla can be

incorporated in the concentrate mixture at the level of up to 35 per cent in lactating cows

without any deleterious effect.

Murrah buffalo bulls fed with sun dried azolla at 225 g / day replaced 25% of

protein in concentrate feed (Kumar et al., 2012). Tamang et al. (1993) indicated that the

sun dried azolla can be incorporated up to 20 per cent in the concentrate mixture without

any deleterious effects on the performance, digestibility of various nutrients, carcass

characteristics of Black Bengal goats. Duran (1994) reported that the aquatic azolla can

replace up to 20 per cent of the soya bean protein with no adverse effect on the

performance of growing and fattening pigs.

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Replacement of groundnut cake nitrogen with azolla at 50 per cent level for 90

days period improved the digestibility of major nutrients and resulted weight gain in

buffalo calves (Indira et al., 2009).

Five feeds (mixture of gram straw and concentrate in 60:40 ratio) were evaluated

for methane emission and digestibility with goat rumen liquor as inoculums in an in vitro

gas production test. These five feeds were formulated by replacement of oil cake by

azolla meal at 0%, 25%, 50%, 75% and 100% in the concentrate mixture. There was

significant difference (P<0.05) in the net methane production per g DM among feeds.

With the replacement of oil cake with azolla meal, a decreasing trend of dry matter and

organic matter digestibility was reported. It was concluded that 50% replacement of oil

cake with azolla meal reduces the methane production in in vitro condition with no

satisfactory significant effect on digestibility (Kumar et al., 2013).

Utilization of Sun-dried azolla at 10 and 20% in the diets of Marwari and

Patanwadi weaner lambs for 4 months resulted in lower DM and protein digestibility but

higher DM and protein intake and no effect on carcass traits (Wadhwani et al., 2010).

Different diets were formulated and subjected for in vitro gas production kinetic

studies by using cow rumen liquor. The paddy straw and ragi straw were supplemented

with 0%, 2%, 4% and 8% azolla. There was no significant difference in gas production

and dry matter digestibility in different feeds. Addition of azolla at different levels in two

different straws has failed to improve the gas production and it was similar to

unsupplemented straw (Ramesh, 2008).

13

13

2.4 Nutritional assessment of azolla

2.4.1 Chemical composition

Chemical composition of different species of azolla are reported by various

workers (Table 2.4.1). As cited by various authors the crude protein content of azolla

varies from 15.4 to 27.93, crude fibre content between 9.07 and 22.25%, ether extract

value varies between 1.60 and 5.05 % while total ash was in the range of 10.15-36.10%

and NFE values were found to vary between 30.08 and 52.46%. Van Hove (1989) noted

that the crude protein content of azolla might vary from 13.0 to 34.5%. These variations

in the nutrient composition of azolla meal is due to differences in the response of azolla

strains to environmental conditions such as temperature, light intensity and soil nutrients

which consequently affect their growth morphology and chemical composition.

Furthermore, contamination with epiphytic algae could also be important to such a degree

as to affect the results of chemical composition (Sanginga and Van Hove, 1989). The cell

wall composition of azolla is highly variable depending upon the species and the season

of cultivation of azolla. NDF content of azolla was found to be in the range of 36.88-70%

(Chatterjee et al., 2013) while ADF was reported to be in range of 25.24-47.08%.

Cellulose and hemicelluloses content was found to be in the range of 6.8 to 36.7% and

10.09 to 17.8, respectively. Lignin was reported to vary between 9.27 and 28.24% and

silica content varies between 4.8 and 16%. Chemical composition indicated that it was a

fair source of plant protein (210.7-296.7 g kg−1 DM). The mean concentration (per cent)

of organic matter, crude protein, crude fibre, ether extract, total ash, NFE, NDF, ADF and

ADL in Azolla microphylla meal were 80.53±0.59, 24.06±0.35, 13.44±1.20, 3.27±0.18,

19.47±0.59, 37.71±1.83,45.52±1.93, 30.16±1.12 and 8.96±0.56, respectively.

14

Table 2.4.1: Per cent chemical composition of different species of Azolla (Dry matter basis)

Proximate principles Azolla filiculoides Azolla pinnata Azolla microphylla Azolla pinnata

Beckingham et al., 1978 Parnerker et al., 1986 Taklimi, 1990 Alalade and Iyayi, 2006

Dry matter - 90

Crude Protein 23-42 15.63 25.33 21.4

Ether Extract 5.05 2.84 3.01 2.7

Crude Fiber - 13.01 11.60 12.7

Total Ash 15.54 23.59 16.2

Nitrogen free extract - 52.70 16.91 47.0

Neutral detergent fiber 39.16 52.76 40.36 36.88

Acid detergent fiber 26.58 25.24 47.08

Calcium 0.937 1.64 1.70 1.16

Phosphorus 0.487 0.56 1.05 1.29

15

15

In general azolla was reported to be rich in mineral profile, the fern was found to

be a rich source of calcium, phosphorous, potassium, ferrous, copper, magnesium and

zinc. Calcium content of azolla varies from 0.8- 4.99 %, while phosphorus between 0.3

and 1.3%. Querubin et al. (1986) reported the following mineral composition, Ca-2.07%,

P-0.77%, Mn-0.27%, Fe-0.25%, Mg-0.17%, Na-0.49%, K-4.93%, Cu-17.6 ppm, Zinc-

71.8 ppm in A. pinnata and the carotene content ranged from 206 to 619 mg / kg on a dry

matter basis and differed significantly between strains. Carotene content was maximal

during the linear phase of growth and minimal during the stationary phase for the all

strains (Lejeunea et al., 2000).

Variations in the chemical composition of Azolla pinnata as been reported by

different authors Singh and Subudhi (1978), Tamang et al. (1993), Anand Titus and

Geetha Pereira (2007), Ali and Leeson (1995), Khatun et al., (1999), Parthasarathy et al.

(2001), Alalade and Iyayi (2006) and Balaji et al. (2009) is presented in the Table 2.4.2.

Singh and Subudhi (1978) reported that azolla contained 24 to 34 per cent crude

protein, 3.18 per cent ether extract, 9.1 per cent crude fiber and 10.5 per cent total ash on

dry matter basis. The calcium and phosphorus contents were found to be in the range of

0.4 to 1.0 per cent and 0.5 to 0.9 per cent, respectively. They also reported that azolla is

rich in vitamins, minerals, xanthophylls and carotenoids.

The freshly harvested azolla contains 85 to 99 per cent of moisture and 0.4 to 6.0

per cent nitrogen (Moore, 1969). The moisture content varied with the stage of maturity

of the aquatic plant harvested and the time allowed for the drain/ drip, post harvesting.

16

Table 2.4.2: Chemical Composition (on per cent dry matter basis) of Azolla pinnata

Source

Singh and

Subudhi

(1978)

Tamang

et al.,

(1993)

Ali and

Leeson

(1995)

Khatun

et al.

(1999)

Parthasarathy

et al. (2001)

Alalade

and Iyayi

(2006)

Anand Titus and

Geetha Pereira

(2007)

Balaji

et al.

(2009)

Proximate Principles

Dry Matter 90.12 92 90.59 89.70

Crude Protein 24.3 15.37 16.5 28.54 26.02 21.4 20-25 24.5

Crude fiber 9.1 14.13 12.5 12.38 13.6 12.7 - 14.9

Ether extract D3-3.6 2.73 1.6 - 2.37 2.7 3-3.5 3.7

Total ash 10.5 20.35 36.1 - 12.3 16.2 17

NFE - 47.42 33.2 - 45.71 47 - 39.9

Cell wall fractions

Neutral detergent fiber - 67.54 47.8 44.57 - 36.88 - -

Acid detergent fiber - 51.96 46.7 33.41 - 47.08 - -

Cellulose - 15.61 - - - 12.76 - -

Lignin - 17.48 - - - 28.24 - -

Mineral

Calcium 0.4-1.0 1.54 1.43 - 1.24 1.16 0.45-1.25 2.14

Phosphorus 0.5-0.9 0.35 0.31 - 0.72 1.29 0.15-11 0.44

Potassium 2-4.5 - - - - 1.25 0.25-5.5 -

Mananesium 0.5-0.65 - - - - 0.35 0.25-0.5 -

Sulphur - - - - - - 0.2-0.75 -

Sodium - - - - - 23.79 - -

Manganese,ppm - - - - - 174.42 60-2500 -

Zinc.ppm - - - - - 87.59 25-750 -

Copper, ppm - - - - - 16.74 2-250 -

Iron, ppm 60-260 - - - - 755.73 40-500 -

Silica, ppm - 15.98 - - - 3.82 0.15-3.5 -

17

17

Basak et al. (2002) reported that azolla meal contains 25.78 per cent crude

protein, 3.47 per cent ether extract, 15.76 per cent total ash and 30.08 per cent NFE. The

gross energy was found to be 4243 kcal per kg. The NDF and ADF values were 47.85

and 42.16 per cent respectively. Azolla microphylla found to contain 80.53% OM, 24.06

% CP, 13.44% CF, 3.27% EE, 19.47% TA, 37.71% NFE, 45.52% NDF, 30.16% ADF

and 8.96% ADL (Chatterjee et al., 2013).

Parnerkar et al. (1986) analyzed the proximate principles of azolla and reported

15.63 per cent CP, 13.01 per cent CF, 2.84 per cent EE, 1.624 per cent calcium, 0.56 per

cent phosphorus on dry matter basis. Bacerra et al. (1995) analysed the chemical

composition of azolla and reported 26.7 per cent CP, 15.1 per cent TA, 94.4 per cent

moisture, 4.6 per cent EE, 11.2 per cent CF and 0.4 per cent phosphorus and 0.8 per cent

calcium.

Ramesh, (2008) has reported that azolla contains 4.1 % DM, 28.52 % CP, 4.13%

EE, 14.43 % TA, 57.71 % NDF and 26.22 % ADL. The in vitro gas production was

found to be 29.3 ml / 200mg DM / 24 hour and ME of 7.1 MJ/kg DM. The chemical

composition of azolla varieties reported by Bolka (2011) is presented in Table 2.4.4

Pillai et al. (2002) reported that azolla is very rich in proteins, essential amino

acid, vitamins (vitamin A, vitamin B12 and Beta-carotene), growth promoter

intermediaries and minerals like calcium, phosphorus, potassium, iron, copper,

magnesium. Sujatha et al. (2013) reported that azolla contains 21.17 per cent CP, 14.6

per cent CF, 3.39 per cent EE, 19.91 per cent of TA, 1.05 per cent Calcium, 0.52 per cent

Phosphorus and 0.49 per cent Iron.

18

18

Table 2.4.3: Chemical composition of Azolla varieties (Bolka, 2011)

Constituent

Azolla

pinnata

(KVAFSU

sample)

Azolla

pinnata

(Raichur

sample)

Azolla

pinnatae

(Hessaraghatta

sample)

Azolla

pinnata

(TRRI

sample)

Azolla

microlophylla

(GKVK sample)

Organic matter 81.25 80.75 81.02 70.83 75.83

Total ash 18.75 19.25 18.98 29.17 24.17

Crude protein 25.82 24.15 24.55 24.12 24.56

Crude fiber 17.53 19.75 19.85 16.25 15.17

Ether extract 4.85 3.95 4.12 3.80 3.38

NFE 33.05 32.90 32.50 26.66 32.72

Calcium - - - 1.95 -

Phosphorus - - - 0.40 -

Buckingham et al. (1978) has reported that azolla contains 31.36% CP, 5.08 %

EE, 12.2 % CF, 13.3 % TA, in vitro gas production was found to be 13.6 ml / 200 mg

DM / 24 h, digestibility of organic matter was 49.9% and ME was around 5.91 MJ / kg

DM. Khan et al. (2002) reported that the rate of gas production was highest in azolla and

lowest in water-hyacinth.

Alalade and Iyayi (2006) reported the gross energy and ME content of Azolla

pinnata as 8.53 MJ/kg DM and 6.99 MJ/kg DM, respectively. Balaji et al. (2009)

recorded the gross energy of azolla as 1807 Kcal/kg DM. Parthasarathy et al. (2001)

reported that, the apparent and true metabolisable energy values of Azolla pinnata as

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19

1529 and 1855 Kcal/kg DM, respectively. Khatun et al. (1999) has reported the ME

content of azolla as 7.59 MJ / kg DM.

Among different aquatic plants, the in-vitro dry matter digestibility (52.2%) was

found to be highest for Azolla (Becerra et al., 1995). It was reported that replacement of

50% of oil cake with azolla meal in concentrate portion of TMR (Total Mixed Ration)

reduced the methane production under in vitro condition (Kumar et al., 2013).

On the basis of in vitro analysis (Rumen Simulation Technique, RUSITEC)

Ahirwar et al. (2009) reported that the Azolla pinnata could be used as protein

supplement by replacing 30 per cent of the conventional nitrogen source in complete

ration of ruminants. Khan et al. (2002) reported that, the rate of gas production was

highest in azolla as compared to water-hyacinth. A similar trend was observed with in

situ DM degradability.

Parashuramulu and Nagalakshmi (2013) reported that, azolla is a good protein

supplement with 21.37% crude protein, crude fibre - 12.5%, ether extract - 2.3%. The

average in vitro dry matter digestibility was 79.5 per cent, organic matter digestibility

was 63.8 mg / 200 mg DM and ME of 1759 kcal ME / kg or 7.36 MJ / kg DM.

2.4.4 Amino acid composition

The amino acid profile of azolla reported by different authors is presented in the

Table 2.4.2. Sanginga and Van Hove (1989) compared the total nitrogen and amino acid

composition of seven azolla strains at four different growth phases. Azolla microphylla

strain was found to be the best source of amino acids and A. filiculoides strain found to be

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20

the poorest under the cultural conditions used for green manure production. Data on the

amino acid analysis reported by Alalade and Lyayi (2006) indicated that lysine, arginine,

isoleucine, leucine, phenylalanine, glycine and valine were predominant. However, the

sulphur-containing amino acids did not meet the recommended value of 3.5g / 100g

protein. Mandal et al. (2012) also reported azolla as a rich source of protein (21.6%) with

all essential amino acids, including lysine, arginine and methionine.

Table 2.4.4 Amino acid composition of azolla

Amino acids Ali and Leeson (1995)

Alalade and Iyayi

(2006)

Buckingham

et al., (1978)

% DM g /100 g CP %DM g/100g CP g/100g CP

Lysine

Methionine

Cystine

Threonine

Tryptophan

Arginine

Isoleucine

Leucine

Phenylalanine

Tyrosine

Glysine

Serine

Valine

Alanine

Histidine

Proline

Aspartic acid

Glutamic acid

0.62

0.25

0.15

0.66

0.08

0.82

0.69

1.28

0.77

0.49

0.86

0.66

0.84

0.95

0.26

0.67

1.37

1.56

3.80

1.50

0.90

4.00

0.50

5.00

4.20

7.70

4.60

4.00

5.20

4.00

5.10

5.80

1.60

4.00

8.30

9.60

0.98

0.34

0.18

0.87

0.39

1.15

0.93

1.65

1.01

0.68

1.00

0.90

1.18

4.58

1.59

0.84

4.07

1.82

5.37

4.35

7.71

4.72

3.18

4.60

4.21

5.51

6.45

1.88

2.26

4.70

4.10

6.62

5.38

9.05

5.64

4.10

5.72

4.10

6.75

6.45

2.31

4.48

9.39

12.72

2.5 Chemical composition of different crop residues

Chemical composition and digestibility of organic matter and ME content of

different varieties of Paddy straw as reported by Rahaman et al. (2010) are presented in

21

21

Table 2.5.1 and 2.5.2, respectively. Chemical composition of different stages of rice

straw Table as reported by Sarnklong et al. (2010) is presented in Table 2.5.3.

Preston et al. (2000)reported that rice straw contains 90 per cent DM, 88.3 per

cent OM, 8.4 per cent CP, 62.5 per cent NDF, 36.6 per cent ADF, 11.7 per cent TA, in

vitro organic matter digestibility was found to be 27.7 per cent.

Akinfemi and Ogunwole (2012) reported that rice straw contains 93 per cent DM,

CP 4.69 per cent, CF 32.89 per cent, EE 1.66 per cent, TA 11.95 per cent, NFE 48.81 per

cent, NDF 69.96 per cent, ADF 56.28 per cent, ADL 12.54 per cent, cellulose 43.74 per

cent, hemicellulose 13.68, invitro gas production found to be 30 ml/ 24 h, ME 6.49 MJ/kg

DM and OMD 51.17%.Kumar et al. (1999) reported that rice straw contains 88.9% DM,

83.3% OM, 3.33% CP, 36.5% CF, 1.25% EE, 42.45% NFE.

Bhatta et al. (2000) reported that finger millet straw contains 91.38% OM, 3.33%

CP, 0.51% EE, 36.68% CF, 8.62% TA, NDF 80.73%, ADF 51.41 %, ADL 5.38%, ME -

6.02 MJ/Kg DM. Ramachandra (1997) has reported that ragi straw contains 89.69 % DM,

91.55 % OM, 13.68 % CP, 2.32 % EE, 32.58 % CF, 8.45 %TA, 6.55 AIA, 52.31 %NFE,

66.69 %NDF, 46.83% ADF and 19.84% hemicellulose.

Madibela and Modiakgotla (2004) had reported that, finger millet straw contains

16.9 per cent TA, 7.45 per cent CP, 69.7 per cent NDF, 40.9%ADF, 4.76 per cent ADL,

40.9 per cent NFE, Ca of 0.88 per cent, P of 0.03 per cent and in vitro dry matter

digestibility found to be 50.1 per cent.

22

22

Finger millet straw found to contain 92.7% OM, 5% CP, 0.8% EE, 75.1% NDF,

46.2% ADF, 6% ADL, in vitro gas production of 27.3 ± 0.45 ml / 200 mg DM / 24 h and

ME of 6.5MJ/kg DM (Sreerangaraju et al., 2000).

Table 2.5.1 Chemical composition of varieties of paddy straw (Rahaman et al.,

2010)

Varieties DM OM CP NDF ADF ADL Ca P

BRRI 29 93.05 84.49 5.1 72.53 41.38 6.97 0.165 0.146

BINA 92.81 81.21 4.64 72.16 43.62 4 0.10 0.08

Pajam 92.84 82.56 4.39 74.2 42.83 4.34 0.2450 0.074

Kablabadam 92.84 85.08 3.49 74.95 44.22 5.94 0.14 0.06

Nijerhail 92.8 86.24 3.61 74.86 46.32 4.3 0.17 0.068

BR 11 92.2 84.91 4.52 477.57 43.64 4.3 0.245 0.046

Table 2.5.2 Digestibility of OM and ME content of different varieties of paddy

straw (Rahaman et al., 2010)

Different varieties of Paddy

straw

Digestibility of OM

(%)

ME content (MJ/kg DM) ±

SE

BRRI 29 44.58a ± 0.565 6.68

a ± 0.021

Nijershail 39.94b

± 0.047 5.99b

± 0.031

BINA 5 39.87b

± 0.243 5.97b

± 0.070

Pajam 38.30c ± 0.093 5.73

c ± 0.010

Kablabadam 37.67c ± 0.055 5.65

c ± 0.015

BR 11 37.53c ± 0.104 5.61

c ± 0.021

23

23

Table 2.5.3: Chemical composition of rice straw in early, mid and late growing

period (Sarnklong et al., 2011)

Cultivation season DM NDF ADF Hemicellulose Cellulose ADL

Early 96.40 72.53 43.52 29.01 35.81 4.90

Mid 96.20 70.03 41.09 29.08 32.80 4.66

Late 96.87 71.97 39.83 32.24 31.96 4.63

Akinfemi et al. (2009) had reported that, maize husk contains 88.85 per cent DM,

7.44 per cent CP, 1.27 per cent EE, 3.32 per cent TA, 30.45 per cent CF, 42.48 per cent

NFE, 49.15 per cent ADF, 71.14 per cent NDF, 14.87 per cent ADL, 34.25 per cent

cellulose, 21.99 per cent hemicellulose, in vitro gas production found to be 20.33 ml /

200mg DM / 24 h and ME of 5.45 MJ / kg DM and Organic matter digestibility (OMD)

found to be 38.28%.

Kiangi et al. (1981) reported the maize stover contains 93.4 per cent DM, 12.1 per

cent TA, 2.3 per cent CP, 45.3 per cent CF, 1.8 per cent of EE and 38.4 per cent NFE and

the in vitro DMD was found to be 51.7%, while Ojiet al. (1997) recorded 3.5 per cent CP,

39.4 per cent CF, 0.6 per cent EE, 48.4 per cent NFE, 80.7 per cent NDF and 56.7 % of in

vitro DMD.

Sreerangaraju et al.(2000) had reported that, Bengal gram husk contains 5.1 per

cent CP, 1.1 per cent EE, 76 per cent NDF, 65.2 per cent ADF, 6.1 per cent ADL, in vitro

gas production found to be 45±0.39 ml / 200 mg DM / 24 h, ME 8.6 MJ / kg DM.

24

24

Sorghum stover contains 91.23% DM, 2.54 CP, 31.65% CF, 6.19% EE, 6.28%

TA, 53.34% NFE, 70.23% NDF, 46.69% ADF, 15.21% ADL, 31.48% Cellulose, 23.54%

Hemicellulose, in vitro gas production was 26 ml/200 mg DM/24 h, ME of about

5.97MJ/kg DM and OMD was about 42.99% (Akinfemi and Doherty, 2010).

2.6 Fiber degradation in ruminants

In ruminants forages are necessary for normal rumen function. However, high

proportions of structural carbohydrate found in forages limit the voluntary intake and

digestion of feedstuffs by ruminants because they slow down the rates of microbial

fermentation. Abundance and diversity of rumen microorganisms, particularly of

cellulolytic bacteria and anaerobic fungi, are necessary to degrade the fibrous dietary

components (Wilson, 2008). Thus, a method for promoting the growth of fiber-degrading

microorganisms would be beneficial in increasing the degradation of cellulose and

hemicellulose in the rumen. Most ruminal cellulolytic microorganisms, such as

Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, and

Butyrivibio fibrisolvents, require branched-chain volatile fatty acids (BCVFA, i.e.,

isobutyric, isovaleric, valeric, and 2-methylbutyric acids) for growth. Ruminal branched-

chain volatile fatty acids primarily originates from dietary protein or recycling of

bacterial protein by ruminal oxidative deamination and decarboxylation of valine,

leucine, and isoleucine (Argyl and Baldwin, 1989). Studies have shown that branched

chain fatty acids can improve apparent dry matter digestibility and microbial growth, and

enhance microbial functions and enzyme activities in the rumen (Moharrery, 2004).

Dietary supplementation with branched-chain volatile fatty acids improves rumen

fermentation and enhances digestion in cattle (Liu et al., 2009). Branched-chain volatile

http://ajas.info/journal/view.php?number=4677#b26-ajas-26-4-523-10



25

25

fatty acids are used to synthesize branched-chain amino acids (BCAA, i.e., valine,

leucine, and isoleucine) by ruminal microorganisms. Several researchers have

demonstrated that amino acids are often stimulatory for ruminal microorganisms, even

when ammonia and carbohydrates exceed the requirements. These results indicate that

ruminal microorganisms could benefit more by direct provision of branched chain amino

acids rather than the corresponding branched-chain volatile fatty acids (Yang, 2002).

Low concentration of valine, leucine or isoleucine supplementation resulted in

higher VFA production during in vitro rumen fermentation of wheat straw. So branched

chain amino acids could be used as additives in ruminants to improve forage utilization

(Zhang et al., 2013).

In general, it can be inferred that the azolla appears to be a valuable non

conventional feedstuff for livestock and it may improve the utilization of crop residues.

The positive and in some respect controversial data of relevant literature have prompted

us to study supplementary effect of azolla on utilization of crop residues and total mixed

ration.


MMaatteerriiaallss aanndd MMeetthhooddss

III. MATERIALS AND METHODS

A brief account of experimental procedures and analytical techniques adopted

during the course of study are presented in this chapter.

3.1 Location

The experiment was conducted at the Department of Animal Nutrition and

Livestock Production and Management, Veterinary College, KVAFSU, Bangalore and

Alltech Ruminant Nutrition Laboratory, Veterinary College, KVAFSU, Bangalore.

3.2 Feed samples used

Crop residues:

1. Finger millet (Eleusine coracana) straw

2. Paddy (Oryza sativa) straw

3. Maize (Zea mays) stover

4. Sorghum (Sorghum bicolor) stover

5. Maize (Zea mays) husk

6. Bengal (Cicer arietinum) gram husk

Crop residues commonly used for dairy cattle feeding in Karnataka were selected.

The crop residues were dried at 65oC for 2 days and ground to pass through a one mm

sieve and preserved in air tight bottles at room temperature while using for chemical

analysis and in vitro studies.

Azolla pinnata was harvested from azolla tanks maintained at College of

Agriculture, University of Agricultural and Horticultural Sciences, Shivamogga. Fresh

27

27

samples of azolla was dried in hot air oven at 50oC for 48 h and dried samples was

ground to pass through a one mm sieve and preserved in airtight bottles at room

temperature.

3.3 Dietary treatments

Three types of diets were formulated by using azolla, crop residues and

compound feed mixture. The first type of diet consisted only roughage which was

supplemented with different levels of azolla that is 0%, 3%, 6%, and 9%. The second

type of diet consisted Total mixed ration (TMR) prepared using roughage and

concentrate in the ratio of 60:40 and the portion of concentrate in the TMR was replaced

with 0%, 3%, 6% and 9% azolla. The third type of diet consisted TMR, prepared using

roughage and concentrate in the ratio of 60:40 and the portion of roughage in the TMR

was replaced with 0%, 3%, 6% and 9% azolla.

The compounded feed mixture (CFM) or concentrate feed was formulated with

18% CP and 70% TDN by using following ingredients

Ingredients Percent

Maize 43%

Wheat bran 31%

Groundnut cake 23%

Salt 1%

Mineral mixture 2%

28

28

The treatment design is as follows

Type I diet (Crop residue + Azolla)

Crop residues Levels of azolla supplementation

0% (T1-control) 3% (T2) 6% (T3) 9% (T4)

Paddy Straw (R1) T1R1 T2R1 T2R1 T4R1

Ragi straw (R2) T1R2 T2R2 T3R2 T4R2

Maize stover (R3) T1R3 T2R3 T3R3 T4R2

Maize husk (R4) T1R4 T2R4 T3R4 T4R4

Sorghum stover (R5) T1R5 T2R5 T3R5 T4R5

Bengal gram husk (R6) T1R6 T2R6 T3R6 T4R6

Where, T1=Treatment with 0% azolla (Control), T2= Treatment with 3% azolla, T3= Treatment

with 6% azolla, T4= Treatment with 9% azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize

Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.

Type II diet (TMR)

TMR Levels of azolla supplementation

0% (T5- control) 3% (T6) 6% (T7) 9% (T8)

Paddy straw(60% kept constant) +

Concentrate feed+ Azolla T5R1 T6R1 T7R1 T8R1

Ragi straw(60% kept constant) +

Concentrate feed +Azolla T5R2 T6R2 T7R2 T8R2

Maize Stover(60% kept constant) +

Concentrate feed+Azolla T5R3 T6R3 T7R3 T8R3

Maize husk(60% kept constant) +


Sorghum stover(60% kept constant) +


Where, T5= Treatment with 0% Azolla (Control), T6= Treatment with 3% Azolla, T7= Treatment

with 6% Azolla, T8 = Treatment with 6% Azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize

Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.

29

29

Here roughage and concentrate taken in the ratio of 60:40. The portion of

concentrate in the TMR was replaced with azolla at 0%, 3%, 6% and 9% and the

roughage portion was kept constant (60%).

Type III diet (TMR)

TMR Levels of azolla supplementation

0%(T9- control) 3%(T10) 6% (T11) 9% (T12)

Paddy straw + Concentrate feed (40

% kept constant) T9R1 T10R1 T11R1 T12R1

Ragi straw+ Concentrate feed (40 %

kept constant) +Azolla T9R2 T10R2 T11R2 T12R2

Maize Stover+Concentrate feed(40

% kept constant) +Azolla T9R3 T10R3 T11R3 T12R3

Maize husk + Concentrate feed(40

% kept constant) +Azolla T9R4 T10R4 T11R4 T12R4

Sorghum Stover + Concentrate

feed(40 % kept constant) +Azolla T9R5 T10R5 T11R5 T12R5

Where, T9 = Treatment with 0% azolla (Control), T10 = Treatment with 3% azolla, T11= Treatment

with 6% azolla, T12 = Treatment with 6% Azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize

stover, R4 = Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk

Here roughage and concentrate taken in the ratio of 60:40. The portion of

roughage in the TMR was replaced with azolla at 0%, 3%, 6% and 9% and the

concentrate portion was kept constant (40%).

30

Plate 3.1: Cultivation of Azolla (Azolla pinnata) in cement tanks

31

31

3.4 Chemical Analysis

The DM content of feed samples were analyzed by drying to a constant weight in

a forced hot air oven at 105oC. The ash content in the samples was estimated as residue

after incineration of samples at 600oC for 3 hours. Crude protein (N X 6.25) was

analyzed using Gerhardt digestion and distillation unit (AOAC, 2005). The Ether extract

(EE) content in the sample was analyzed after extraction with petroleum ether using the

procedure of AOAC (2005). The fiber fractions were determined according to the

methods described by Van Soest et al. (1991).

3.5 Donor cow and collection of rumen fluid

A crossbred (Holstein Friesian x Bos indicus) lactating dairy cow, weighing 400

kgs, producing 9 kg milk per day, fitted with a flexible rumen cannula of large diameter

(Bar Diamond, Inc. USA), served as the donor of the rumen inoculum. It was fed with

basal diet consisting of finger millet straw at the rate of 6.5 to 7 kg per day and CFM

(Maize- 54%, Wheat bran - 41%, mineral mixture - 2%, salt -1%, urea – 2%) of 4.0

kg/day in two equal portions at the time of milking at 6.00 A.M and 1.30 P.M. The rumen

fluid was collected between 9 A.M and 9.30 A.M before offering finger millet straw.

3.6 Rumen fluid collection

The rumen inoculum collected from rumen‐cannulated cow was brought in a

closed plastic container (Thermos) pre‐warmed with hot water. Completely filled

container with rumen fluid content brought to the laboratory, filtered through four layers

of cheese cloth and then mixed with the medium prepared (Menke and Steingas, 1988).

32

32

The buffered rumen fluid was supplied with CO2 to minimize changes in microbial

populations and to avoid O2 contamination while handling at 39oC at all the time.

3.7 Preparation of Medium for Rumen in vitro gas production kinetic study

(Incubation of 102 in vitro glass syringes)

Micro mineral solution – 0.28 ml

Macro mineral solution – 542.5 ml

Buffer solution – 542.5 ml

Resazurin – 2.8 ml

All these solution were mixed with 1085 ml of distilled water to get 2 liters of

buffer solution.

3.8 Estimation of Metabolizable energy (ME) content (Menke and Steingass, 1988)

1. The medium was prepared on the previous day of incubation in a water bath set at

39oC and carbon dioxide was supplied to the medium for 15 to 20 minutes and the

medium was continuously stirred with a magnetic stirrer.

2. On the day of incubation rumen fluid was collected from rumen fistulated animal

and was filtered through four layers of cheese cloth.

3. Reducing solution was added to the medium prepared. The medium turns pink to

colorless when the CO2 was supplied to the medium.

4. About 1137.5 ml of rumen fluid was added to the medium and mixed. CO2

wassupplied continuously until the completion of filling 102 syringes with rumen

fluid to maintain the anaerobic environment.

33

33

5. Air equilibrated samples of crop residues and TMR (200 ± 5mg) were incubated

in 100 ml calibrated glass syringes supplemented with azolla at 0, 3, 6, 9 percent

and azolla as such in triplicates with 30 ml of buffered rumen fluid with three

blank incubations and the reference standards of roughage and concentrates.

6. The incubation was done in a water bath maintained at 39oC

7. The readings of displaced syringes were recorded at different time intervals over

24 hours that is at 2 hours, 5 hours and 24 hours. Whenever the syringe readings

exceed 90 ml, the readings were reset to 30 ml. The cumulative gas production

was calculated.

8. For determination of ME content, 24 hours net cumulative gas production was

corrected for reference standards.

9. Using chemical composition and net gas production (corrected for blank and

reference standard) at 24 hr of incubation, ME was calculated by using the

following equations.

Roughages:

ME = 2.2 + 0.1357 GP + 0.0057 CP + 0.0002859 EE2

For Azolla and compounded feed or TMR

ME= 1.06 + 0.1570 GP + 0.0084 CP + 0.022 EE - 0.0081 TA

Where, ME = Metabolizable energy (MJ / Kg DM), GP = gas production for 24 h in

ml/200mg DM, CP = Crude protein (g / kg DM), EE = ether extract (g / kg DM), TA=

total ash in g / kg DM.

34

34

3.9 Estimation of ADDM and TDDM by Modified in vitro two stage digestion

technique (Goering and Van Soest, 1970)

1. The medium was prepared on the previous day of incubation by dissolving 3.35

liters of buffer solution with 250 ml of reducing solution and the CO2 was

dispensed untill the medium turns blue to colourless and the medium was stored

at 39oC incubator overnight.

2. On the day of incubation rumen fluid was collected from rumen fistulated animal

and was filtered through four layers of cheese cloth.

3. Air equilibrated samples in duplicates of about 400 ± 5mg was weighed in pre-

labeled and weighed filter bags and sealed with thermo sealer and transferred into

respective Erlenmeyer flask and 80 ml of buffered rumen fluid was added to the

each flask

4. Incubation was done in the water bath at 39oC for 48 hours, blank bags was also

kept for incubation.

5. Anaerobic condition of the inoculums was maintained by supplying carbon

dioxide.

6. After incubation for 48 hours, the bags were taken out and gently squeezed to

remove excess liquid, washed with distilled water and dried at 55oC for minimum

of 5 hours.

7. After cooling weight of bag was recorded for calculation of ADDM (Apparent

digestible dry matter).

35

35

8. The dried bags were refluxed with neutral detergent solution (NDS) for 1 hour, 15

minutes and the bags washed with hot distilled water and dried at 105oC for 2 to 3

hours.

9. After cooling weight of bag was taken for calculation of TDDM (True digestible

dry matter).

Calculation

NDF residue = [W3 - (W1xC1)] / W2 x 100

Where, W 1= Empty bag weight, W2 = Sample weight, W3 = Dried weight of bag with

fiber after extraction process, C1 = Blank bag correction (final oven dried weight divided

by original blank bag weight)

TDDM % =100 - NDF residue

ADDM% =100 - weight of residue before NDS refluxing

3.10 Estimation of mineral profile of Azolla

Preparation of sample

1. About 2 g of azolla samples was taken into the silica crucible

2. The sample in the crucible was kept in the muffle furnace at 550oC for 3 hrs to

make it ash

3. Crucible was taken out from muffle furnace after cooling

4. The ash was transferred quantitatively to 250 ml beaker using little quantity of

distilled water.

36

36

5. To the beaker containing ash 25 ml of dilute HCl (1:2) was added and boiled for

10 minutes.

6. The cooled solution was filtered through whatman No. 1 filter paper into a

volumetric flask of around 250 ml capacity and the volume was made upto the

mark with distilled water and mixed thoroughly.

Analysis of azolla for minerals by Inductively coupled plasma-atomic emission

spectrophotometer (ICP-AES).

The following steps were performed while working with Atomic Emission Spectrometry

1. The computer, printer and main power of the ICP was turned on.

2. The valve on argon tank was opened and the tank was filled upto 500 lbs and the gas

regulator on the argon tank was set at 80 psi.

3. The inlet and outlet tubing on peristaltic pump was set and the pump was switched on

to the low setting.

4. Plasma was ignited and the instrument was allowed to warm up to 20 minutes to

stabilize the wavelength.

5. The method file from the operating software was verified for all the elements of

interest.

6. The right wavelength for each element and the concentrations of the calibration

standards were set.

7. Analyses was done in the order of blanks, standards and azolla sample.

37

37

8. After analyses, the azolla sample was run with 5% HNO3 through the ICP for 5

minutes, followed by distilled water for 10 minutes.

9. The peristaltic pump and the main power was turned off.

10. The valve of argon tank was closed.

11. The ICP analysis program on the computer was closed.

12. The concentration of the liquid digest sample was reported in milligrams/liter (mg/L).

Calculation

Concentration of mineral in sample solution (mg / L ) x Volume made (ml)

µg / g = --------------------------------------------------------------------------------------------------

Weight of sample (g)

3.10 Estimation of amino acid profile of Azolla

The amino acid composition of azolla was analyzed at Evonik (SEA) Laboratory,

Singapore using the fast and reliable Near Infrared Reflectance Spectroscopy (AMINO

NIR).

3.11 Statistical Analysis

The mean values of ME content of the various diets were subjected to statistical

analysis using the software Graph pad prism version 5.0. One way ANOVA was used to

test the hypothesis according to the procedures described by Snedecor and Cochran

(1994). Individual differences between the mean values of ME were tested using

Bonferoni ‘t’ test when the treatment effect was significant. Individual differences

between the mean values of ADDM and TDDM were tested using Unpaired ‘t’ test.

RReessuullttss

IV. RESULTS

The results of the present study are presented in the form of Tables at the end of

this chapter. Detailed experimental results and data are provided in the appendices.

4.1 Chemical composition of Azolla, Crop residues and CFM

4.1.1 Proximate priciples of Azolla, Crop residues and CFM

The proximate principles of Azolla pinnata, Paddy straw, Finger millet straw,

Maize stover, Maize husk, Sorghum stover, Bengal gram husk and Compound feed

mixture (CFM) are presented in Table 4.1.1. The moisture content of fresh azolla was

found to be 95.63 per cent (4.37 per cent DM). The Dry matter content of dried sample of

azollawas found to be 96.52 per cent. The DM content was found to be 95.63 per cent in

paddy straw, 84.64 per cent in ragi straw, 94.46 per cent in maize stover, 89.72 per cent

in maize husk, 87.61 per cent in sorghum stover, 87.44 per cent in bengal gram husk and

86.74 per cent in compound feed mixture. The organic matter content (DMB) was found

to be 82.16 per cent in azolla, 84.21 per cent in paddy straw, 89.25 per cent in ragi straw,

92.80 per cent in maize stover, 97.45 per cent in maize husk, 93.48 per cent in sorghum

stover, 96.38 per cent in bengal gram husk and 94.00 per cent in compound feed mixture

(CFM). The crude protein (DMB) content was found highest in azolla (21.66 per cent)

followed by 17.89 per cent in compound feed mixture , 4.27 per cent in bengal gram

husk, 3.55 per cent in ragi straw, 3.25 per cent in sorghum stover, 2.92 in maize stover,

2.51 per cent in paddy straw and 1.84 per cent in maize husk. The ether extract (DMB)

content was found to be 4.41 per cent in azolla, 0.84 per cent in paddy straw, 0.98 per

cent in ragi straw, 0.87 per cent in maize stover, 0.61 per cent in maize husk, 1.08 per

39

Table 4.1.1: Proximate principles1 (on per cent dry matter basis) of Azolla pinnata, Crop residues and CFM

Samples Proximate principles

DM % OM CP EE CF TA NFE

Azolla pinnata 4.371 82.16 21.66 4.41 15.15 17.84 40.94

Paddy straw 95.63 84.21 2.513 0.841 35.31 15.79 45.55

Ragi straw 84.64 89.25 3.558 0.983 32.49 10.75 52.23

Maize stover 94.46 92.80 2.921 0.873 30.51 7.217 58.49

Maize husk 89.72 97.45 1.844 0.611 31.83 2.551 63.17

Sorghum stover 87.61 93.48 3.253 1.084 30.64 6.522 58.51

Bengal gram husk 87.44 96.38 4.272 0.793 46.72 3.624 44.60

Compound feed mixture 86.74 94.00 17.89 4.081 8.351 6.00 63.68

1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean

40

40

cent in sorghum stover, 0.79 per cent in bengal gram husk and 4.08 per cent in compound

feed mixture. The Total ash (DMB) content was found highest in azolla (17.84 per cent)

followed by 15.79 per cent in paddy straw, 10.75 per cent in ragi straw, 7.2 per cent in

maize stover, 6.52 per cent in sorghum stover, 6.0 per cent in compound feed mixture,

3.62 per cent in bengal gram husk and 2.55 per cent in maize husk. The crude fiber

(DMB) content was found to be 15.15 per cent in azolla, 35.31 per cent in paddy straw,

32.49 per cent in ragi straw, 30.51 per cent in maize stover, 31.83 per cent in maize husk,

30.64 per cent in sorghum stover, 46.72 per cent in bengal gram husk and 8.35 per cent in

CFM.

4.1.2 Fiber fractions of Azolla, Crop residues and CFM

The fiber fractions of Azolla, paddy straw, ragi straw, maize stover, maize husk,

sorghum stover, bengal gram husk and compound feed mixture are presented in Table

4.1.2. The NDF (DMB) content was found to be 54.86 per cent in azolla, 71.50 per cent

in paddy straw, 66.26 per cent in ragi straw, 70.71 per cent in maize stover, 75.71 per

cent in maize husk, 66.72 per cent in sorghum stover, 71.09 per cent in bengal gram husk

and 19.80 per cent in CFM. The ADF (DMB) content was found to be 36.57 per cent in

azolla, 50.0 per cent in paddy straw, 37.33 per cent in ragi straw, 49.71 per cent in maize

stover, 42.18 per cent in maize husk, 38.21 per cent in sorghum stover, 58.2 per cent in

bengal gram husk and 10.11 per cent in compound feed mixture. The ADL (DMB)

content was found to be highest in azolla (24.03 per cent) followed by, 6.26 per cent in

maize stover, 5.14 per cent in ragi straw, 4.93 per cent in sorghum stover, 4.76 per cent in

bengal gram husk, 4.78 per cent in paddy straw, 2.16 per cent in maize husk and 1.13 per

cent in CFM.

41

Table 4.1.2: Fiber fractions1, total silica, biogenic silica and sand silica (on per cent dry matter basis) of Azolla pinnata, Crop

residues and CFM

Samples Azolla

pinnata

Paddy

straw Ragi straw Maize stover Maize husk Sorghum stover

Bengal

gram husk CFM

NDF 54.86 71.50 66.26 70.71 75.71 66.72 71.09 19.89

ADF 36.57 50.00 37.33 49.71 42.18 38.21 58.20 10.113

ADL 24.03 4.78 5.141 6.262 2.166 4.931 4.764 1.132

Hemicellulose 18.29 21.50 28.93 21.00 33.53 28.51 12.89 9.692

Cellulose 12.54 45.22 32.19 43.45 40.02 33.28 51.44 8.981

Total silica(ADF ash) 5.61 10.96 5.381 7.968 3.744 3.662 2.892 2.776

Biogenic silica(ADF

ash-NDF ash) 3.341 6.754 3.138 4.289 2.081 2.481 1.294 1.984

Sand silica

(NDF ash) 2.272 4.217 2.251 3.684 1.664 1.188 1.613 0.791


42

42

4.1.3 Mineral profile of Azolla

The mineral profile of Azolla pinnata is presented in Table 4.1.3. Azolla pinnata

was found to contain 1.64 per cent Calcium, 0.34 per cent magnesium, 2.71 per cent

potassium, 9.1 ppm of copper, 325 ppm of Zinc, 1569 ppm of Iron, 8.11 ppm of cobalt,

5.06 ppm of chromium, 2418 ppm of manganese, 31 ppm of boron, 5.33 ppm nickel, 8.1

ppm of lead, 1.2 ppm of cadmium.

4.1.4 Amino acid profile of Azolla

The amino acid profile of azolla is presented in Table 4.1.4. Azolla found to be

rich in all essential amino acids including branched chain amino acids like valine, leucine

and isoleucine.

4.2 Chemical composition of various diets

The chemical composition of various diets comprising paddy straw supplemented

with different levels of azolla from 0 to 9 per cent and the TMR comprising paddy straw

and CFM supplemented with different levels of azolla from 0 to 9 per cent are presented

in Table 4.2.1. The chemical composition of various diets comprising ragi straw

supplemented with different levels of azolla from 0 to 9 per cent and the TMR

comprising ragi straw and CFM supplemented with different levels of azolla from 0 to 9

per cent are presented in Table 4.2.2. The chemical composition of various diets

comprising maize stover supplemented with different levels of azolla from 0 to 9 per cent

and the TMR comprising maize stover and CFM supplemented with different levels of

azolla from 0 to 9 per cent are presented in Table 4.2.3. The chemical composition of

43

43

Table 4.1.3: Mineral profile of Azolla pinnata (on per cent DMB)

Minerals Percentage ppm

Calcium 1.64 -

Magnesium 0.34 -

Pottassium 2.71 -

Copper - 9.1

Manganese - 2418

Zinc - 325

Iron - 1569

Cobalt - 8.11

Chromium - 5.06

Boron - 31

Nickel - 5.33

Lead - 8.1

Cadmium - 1.2

44

44

Table 4.1.4: Amino acid profile of Azolla pinnata

Amino acids (AA) AA % DMB AA% in CP

Lysine 1.231 4.940

Methionine 0.413 1.657

Cystine 0.194 0.778

Threonine 1.164 4.671

Arginine 1.414 5.674

Isoleucine 1.160 4.655

Leucine 2.072 8.315

Phenylalanine 1.377 5.526

Glycine 1.341 5.381

Serine 1.124 4.510

Valine 1.445 5.799

Alanine 1.539 6.176

Histidine 0.488 1.956

Proline 1.032 4.141

Aspartic acid 2.303 9.242

Glutamic acid 2.740 10.995

Total (without NH3) 21.037 84.418

Ammonia 0.509 2.043

Total 21.546 86.461

45

Table 4.2.1: Chemical composition1 (Percentage on DMB) of various diets comprising Paddy straw supplemented with

different levels of azolla (0 to 9 per cent) and the TMR comprising paddy straw and CFM supplemented with

different levels of azolla (0 to 9 per cent).

Samples Chemical composition (% DMB)

DM % CP EE TA CF NFE NDF ADF ADL

T1R1(Control)* 0.982 2.5 0.84 15.5 35.3 44.6 71.5 50 4.7

T2R1 0.9815 3.08 0.95 15.6 34.7 44.4 71.0 49.5 5.28

T3R1 0.9809 3.64 1.05 15.6 34.1 44.3 70.5 49.1 5.87

T4R1 0.9805 4.22 1.16 15.7 33.4 44.2 70.0 48.7 6.46

T5R1(Control)** 0.973 7.72 2.14 11.7 24.5 53.1 50.8 34.0 3.40

T6R1 0.9732 7.90 2.15 12.0 24.7 52.4 51.8 34.8 4.09

T7R1 0.9733 8.9 2.16 12.4 24.9 51.6 52.9 35.6 4.7

T8R1 0.9735 8.27 2.17 12.7 25.13 50.89 53.97 36.42 5.46

T9R1 (Control)*** 0.973 7.72 2.14 11.71 24.5 53.1 50.8 34.04 3.4

T10R1 0.9725 8.29 2.24 11.93 23.9 53.0 50.3 33.6 3.9

T11R1 0.972 8.87 2.35 11.99 23.3 52.9 49.8 33.2 4.5

T12R1 0.9754 9.44 2.46 12.05 22.7 52.8 49.3 32.8 5.1

1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean,

* Type I diets

** Type II diet where CFM portion of TMR is replaced with azolla (0 to 9%)

*** Type III diet where roughage portion of TMR is replaced with azolla (0 to 9%)

46

Table 4.2.2: Chemical composition1 (Percentage on DMB) of various diets comprising Ragi straw supplemented with different

levels of azolla (0 to 9 per cent) and the TMR comprising ragi straw and CFM supplemented with different levels

of azolla (0 to 9 per cent).


DM% CP EE TA CF NFE NDF ADF ADL

T1R2(Control)* 0.9647 3.55 0.98 10.7 32.4 52.0 66.36 37.33 5.1

T2R2 0.9647 4.09 1.08 10.9 31.9 51.6 66.0 37.30 5.67

T3R2 0.9642 4.63 1.19 11.1 31.4 51.3 65.67 37.28 6.25

T4R2 0.964 5.17 1.29 11.3 30.9 50.9 65.3 37.26 6.82

T5R2(Control)** 0.9626 8.35 2.22 8.84 22.8 57.6 47.7 26.4 3.64

T6R2 0.9626 8.89 2.23 9.2 23.0 56.8 48.7 27.23 4.33

T7R2 0.9627 9.44 2.24 9.5 23.2 56.0 49.83 28.26 5.01

T8R2 0.9627 9.98 2.25 9.61 23.44 55.33 50.89 28.82 5.70

T9R1 (Control)*** 0.9626 8.5 2.22 8.84 22.8 57.6 47.7 26.4 3.64

T10R2 0.9626 8.89 2.32 9.06 22.3 57.2 47.39 26.41 4.22

T11R2 0.9627 9.44 2.43 9.27 21.7 56.9 47.04 26.39 4.8

T12R2 0.9633 9.98 2.53 9.49 21.2 56.5 46.7 26.36 5.37

1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean,

* Type I diet



47

Table 4.2.3: Chemical composition1 (Percentage on DMB) of various diets containing Maize stover supplemented with

different levels of azolla (0 to 9 per cent) and the TMR comprising maize stover and CFM supplemented with




T1R3(Control)* 0.9897 2.9 0.87 10.1 30.5 57.5 75.7 41.9 5.5

T2R3 0.9889 3.46 0.98 10.3 30.0 57.0 75.0 41.82 6.0

T3R3 0.9882 4.02 1.08 10.5 29 56.5 74.4 41.66 6.6

T4R3 0.9874 4.58 1.19 10.8 29.1 56.0 73.8 41.50 7.1

T5R3(Control)** 0.9776 7.96 2.15 8.4 21.6 60.9 50.3 33.8 4.3

T6R3 0.9778 8.14 2.16 8.8 21.8 60.1 51.3 34.66 4.9

T7R3 0.978 8.33 2.17 9.1 22.0 59.4 52.4 35.4 5.6

T8R3 0.9781 8.51 2.18 9.5 22.25 58.66 53.50 36.24 6.36

T9R3 (Control)*** 0.9776 7.96 2.15 8.4 21.6 60.9 50.3 33.8 4.3

T10R3 0.9769 8.52 2.26 8.7 21.1 60.4 49.8 33.4 4.8

T11R3 0.9762 9.09 2.37 8.8 20.7 59.9 49.3 33.0 5.3

T12R3 0.9808 9.1 2.47 8.9 20.2 594 48.9 32.6 5.93


* Type I diet



48

48

various diets comprising maize husk supplemented with different levels of azolla from 0

to 9 per cent and the TMR comprising maize husk and CFM supplemented with different

levels of azolla from 0 to 9 per cent are presented in Table 4.2.4. The chemical

composition of various diets comprising sorghum stover supplemented with different

levels of azolla from 0 to 9 per cent and the TMR comprising sorghum stover and CFM

supplemented with different levels of azolla from 0 to 9 per cent are presented in Table

4.2.5 and the chemical composition of various diets comprising bengal gram husk

supplemented with different levels of azolla from 0 to 9 per cent are presented in Table

4.2.6.

4.3 Rumen in vitro net gas production and ME content of Azolla pinnata, Crop

residues and CFM

The rumen in vitro net gas production at 24 hours and predicted ME content of

Azolla pinnata, crop residues and CFM are presented in Table 4.3. The net gas

production (ml/200 mg DM) for 24 hours observed was highest in compound feed

mixture (48.73 ml) followed by 46.03 ml in maize husk, 45.44 ml in bengal gram husk,

41.51 ml in sorghum stover, 34.39 ml in ragi straw, 30.21 ml in maize stover, 22.37 ml in

paddy straw and 19.45 ml in Azolla pinnata. The ME (MJ / kg DM) content was found

highest for compound feed mixture (9.62 MJ / kg DM) followed by 8.57 MJ/kg DM in

bengal gram husk, 8.56 MJ / kg DM in maize husk, 8.13 MJ / kg DM in sorghum stover,

7.11 MJ / kg DM in ragi straw, 6.56 MJ / kg DM in maize stover, 5.47 MJ/kg DM in

paddy straw and 5.44 MJ/kg DM in Azolla pinnata. The gas production data of Azolla

and crop residues at different time intervals (2h, 5h) over 24 h period is presented in

Appendix II and III respectively.

49

Table 4.2.4: Chemical composition1 (Percentage on DMB) of various diets containing Maize husk supplemented with different

levels of azolla (0 to 9 per cent) and the TMR comprising maize husk and CFM supplemented with different

levels of azolla (0 to 9 per cent)



T1R4(Control)* 0.9904 1.8 0.61 2.55 31.8 63.5 75.7 41.9 5.5

T2R4 0.9896 2.39 0.72 3.01 31.3 62.5 75.0 41.8 6.06

T3R4 0.9888 2.99 0.83 3.47 30.8 61.8 74.4 41.6 6.63

T4R4 0.9881 3.58 0.95 3.93 30.3 61.1 73.8 41.5 7.19

T5R4(Control)** 0.978 7.30 1.9 3.93 22.4 64.3 50.3 33.8 4.31

T6R4 0.9782 7.48 2.00 4.29 22.6 63.5 51.4 34.6 4.99

T7R4 0.9784 7.67 2.01 4.64 22.8 62.8 52.4 35.4 5.68

T8R4 0.9786 7.85 2.02 5.0 22.25 62.0 53.5 36.2 6.36

T9R4 (Control)*** 0.9776 7.30 1.9 3.93 22.4 64.3 50.3 33.8 4.31

T10R4 0.9769 7.9 2.11 4.39 21.9 63.6 49.8 33.4 4.85

T11R4 0.9762 8.49 2.22 4.85 21.4 62.9 49.4 33.08 5.39

T12R4 0.9808 9.09 2.34 5.3 20.9 62 48.9 32.6 5.94


* Type I diet



50

Table 4.2.5: Chemical composition1 (Percentage on DMB) of various diets containing Sorghum stover supplemented with

different levels of azolla (0 to 9 per cent) and the TMR comprising sorghum stover and CFM supplemented with




T1R5(Control)* 0.9824 3.25 1.08 6.54 30.6 57.6 66.72 38.21 4.9

T2R5 0.9818 3.80 1.18 6.88 30.1 57.0 66.36 38.16 5.48

T3R5 0.9813 4.35 1.28 7.23 29.7 56 66.01 38.11 6.06

T4R5 0.9808 4.90 1.38 7.57 29.2 56.0 65.65 38.06 6.65

T5R5(Control)** 0.9819 8.17 2.28 6.32 21.7 60.9 47.95 26.97 3.53

T6R5 0.9814 8.35 2.29 6.68 21.9 60.2 49 27.76 4.21

T7R5 0.981 8.54 2.3 7.04 22.1 59.4 50.06 28.55 4.9

T8R5 0.9805 8.72 2.31 7.4 22.3 58.6 51.11 29.35 5.58

T9R4 (Control)*** 0.9819 8.17 2.28 6.32 21.7 60.9 47.95 26.97 3.53

T10R5 0.9814 8.72 2.38 6.67 21.2 60.4 47.6 26.92 4.11

T11R5 0.9809 92.7 2.48 7.01 20.7 59.9 47.24 26.87 4.69

T12R4 0.982 9.83 2.58 7.35 20.3 59.4 46.88 26.82 5.27


* Type I diet



51

Table 4.2.6: Chemical composition1 (Percentage on DMB) of various diets comprising Bengal gram husk supplemented with


Samples

Chemical composition (% DMB)


T1R6(Control)* 0.9857 4.27 0.79 3.63 46.7 44.6 71.09 58.2 4.76

T2R6 0.985 4.79 0.9 4.06 45.7 44.5 70.6 57.34 5.29

T3R6 0.9844 5.31 1.01 4.49 44.8 44.3 70.12 56.48 5.81

T4R6 0.9838 5.83 1.12 4.92 43.8 44.2 69.63 55.62 6.33

1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean.

* Type I diet

52

Table 4.3: Rumen in vitro net gas production1 at 24 hours (IVGP-24h) and predicted metabolizable energy

2 (ME) content of

Azolla pinnata, Crop residues and CFM.

Samples Rumen in vitro net gas production

(ml/ 200 mg/ 24h) ME(MJ/kg DM)

Azolla pinnata 19.45 5.44

Paddy straw 22.37 5.47

Ragi straw 34.39 7.11

Maize stover 30.21 6.56

Maize husk 46.03 8.56

Sorghum stover 41.51 8.13

Bengal gram husk 45.44 8.57

Compound feed mixture 48.73 9.62

1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.


53

53

4.4 Rumen in vitro gas production of various diets

The rumen in vitro gas production of type I diet, type II diet and type III diet are

presented in the Table 4.4.1., Table 4.4.2 and Table 4.4.3, respectively. The detailed gas

production data of various diets at different time intervals (2h, 5h) over 24 h period is

presented in appendix I to IX.

4.5 Metabolizable energy content of various diets

4.5.1 Metabolizable energy content of Type I diet

The ME content of diets comprising crop residues supplemented with different

levels of azolla from 0 to 9 per cent are presented in Table 4.5.1. The ME content of

paddy straw (T1R1-control) was 5.47 MJ / kg DM. The ME content of the diet comprising

paddy straw supplemented with 3 % (T2R1), 6% (T3R1) and 9% (T4R1) azolla was 5.39MJ

/ kg DM, 5.28 MJ / kg DM and 5.43 MJ/ kg DM respectively. There was no significant

difference (P>0.05) in the ME content of the diets supplemented with different levels of

azolla compared with the control. The ME content of ragi straw (T1R2-control group) was

7.11 MJ/ kg DM. The ME (MJ/kg DM) content of the diet comprising ragi straw

supplemented with 3 % (T2R2), 6% (T3R2)and 9% (T4R2) azolla was 6.91, 6.28 and 6.35,

respectively. There was significant difference (P<0.05) in the ME content of the diets

supplemented with 3%, 6% and 9% of azolla compared with control. The ME content of

maize stover (T1R3- control group) was 6.56 MJ/ kg DM. The ME (MJ/kg DM) content of

the diet comprising maize stover supplemented with 3% (T2R3), 6% (T3R3) and 9%

(T4R3) azolla was 6.17, 6.13 and 6.12, respectively. There was significant difference

(P<0.05) in the ME content of the diets supplemented with different levels of azolla

54

Table 4.4.1: Rumen in vitro net gas production1 of diets comprising roughage replaced with different levels of azolla from 0 to

9 per cent (Type I diet).

Samples

Rumen in vitro net gas production(ml/200 mg DM/24 h)

0% Azolla (T1) 3% Azolla (T2) 6% Azolla (T3) 9% Azolla (T4)

Paddy straw (R1) 22.37 21.49 20.42 21.23

Ragi straw (R2) 34.39 34.13 28.11 27.99

Maize stover (R3) 30.21 27.08 26.42 26.09

Maize husk (R4) 46.03 45.03 43.96 40.69

Sorghum stover (R5) 41.51 38.97 39.59 38.56

Bengal gram husk (R6) 45.44 44.98 44.67 44.05


T1=Treatment with 0% azolla (Control group), T2= Treatment with 3% azolla, T3= Treatment with 6% azolla, T4= Treatment with 9%

azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk

55

Table 4.4.2: Rumen in vitro net gas production1 of diets (TMR) comprising roughage (60 per cent kept constant) and CFM (31

to 40 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type II diet).

Samples



Paddy straw + CFM+ azolla (R1) 41.6 39.56 38.01 36.31

Ragi straw + CFM+ azolla (R2) 43.36 42.16 41.49 40.67

Maize stover +CFM + azolla (R3) 40.26 39.57 39.00 38.4

Maize husk+ CFM+ azolla (R3) 46.65 46.49 46.14 45.91

Sorghum stover +CFM + azolla (R4) 47.3 46.9 45.84 44.15


T5= Treatment with 0% Azolla (Control), T6= Treatment with 3% Azolla, T7= Treatment with 6% Azolla, T8 = Treatment with 6%

Azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.

56

Table 4.4.3: Rumen in vitro net gas production1 of diets (TMR) comprising CFM (40 per cent kept constant) and roughage (51

to 60 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type III diet)

Samples



Paddy straw + CFM + azolla (R1) 41.6 40.88 37.91 37

Ragi straw + CFM+ azolla (R2) 43.36 39.73 37.84 36.84

Maize stover +CFM + azolla (R3) 40.26 38.23 37.5 36.18

Maize husk+ CFM+ azolla (R4) 46.65 46.22 46.13 45.42

Sorghum stover CFM + azolla (R5) 47.3 46.72 46.1 45.6


T9 = Treatment with 0% azolla (Control), T10 = Treatment with 3% azolla, T11= Treatment with 6% azolla, T12 = Treatment with 6%

Azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 = Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk

57

Table 4.5.1: Metabolizable energy1 (ME) content of diets comprising roughage replaced with different levels of azolla from 0

to 9 per cent (Type I diet).

Samples

ME (MJ / kg DM)(Mean ± SE)


Paddy straw (R1) 5.47 ± 0.04 5.39 ± 0.12 5.28 ± 0.09 5.43 ± 0.04

Ragi straw (R2) 7.11a ± 0.05

6.91

b ± 0.10

6.28

b ± 0.05

6.19

b ± 0.04

Maize stover (R3) 6.56a ± 0.06

6.17

b ± 0.06

6.13

b ± 0.04

6.12

b ± 0.04

Maize husk (R4) 8.56a ± 0.07

8.46

b ± 0.09

8.16

b ± 0.03

7.88

b ± 0.10

Sorghum stover (R5) 8.13 ± 0.04 7.80 ± 0.04 7.95 ± 0.05 7.85 ± 0.14

Bengal gram husk (R6) 8.57 ± 0.06 8.46 ± 0.14 8.58 ± 0.07 8.53 ± 0.07

Means bearing different superscripts in a row differ significantly (P<0.05). 1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.

T1=Treatment with 0% azolla (Control group), T2= Treatment with 3% azolla, T3= Treatment with 6% azolla, T4= Treatment with 9%

azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram hus

58

58

compared with the control. There was decreasing trend of ME content as the percentage

of supplementation of azolla was increased. The ME content of maize husk (T1R4-

control group) was 8.56 MJ/ kg DM. The ME (MJ/kg DM) content of the diet comprising

maize husk supplemented with 3% (T2R4), 6% (T3R4) and 9% (T4R4) azolla was 8.46,

8.16 and 7.88, respectively. There was significant (P<0.05) difference between the ME

content of diet supplemented with different levels of azolla compared to the control.

There was decreased ME content in the supplemented diet compared to control. The ME

content of sorghum stover (T1R5-control group) was 8.13 MJ / kg DM. The ME (MJ / kg

DM) content of the diet comprising sorghum stover supplemented with 3 % (T2R5), 6%

(T3R5) and 9% (T4R5) azolla was 7.80, 7.95 and 7.85, respectively. There was no

significant (P>0.05) difference between three supplemented diets and control. The ME

content of bengal gram husk (T1R6- control group) was 8.57 MJ/ kg DM. The ME

(MJ/kg DM) content of the diet comprising bengal gram husk supplemented with 3%

(T2R6), 6% (T3R6) and 9% (T4R6) azolla was 8.46, 8.58 and 8.53, respectively. The

difference among the three different levels of supplementation was not statistically

significant (P>0.05) compared with the control.

4.5.2 Metabolizable energy (ME) content of Type II diet

The ME content of diets comprising crop residue (60 per cent kept constant) and

CFM which was replaced with different levels of azolla from 0 to 9 per cent are presented

in Table 4.5.2. The ME content of Paddy straw based TMR: (T5R1- control) was 7.57 MJ

/ kg DM. The ME (MJ / kg DM) content of the supplemented diets T6R1, T7R1 and T8R1

was 7.26, 7.03 and 6.78, respectively. There was significant difference (P<0.05) in the

ME content of the diets supplemented with different levels of azolla compared with the

59

Table 4.5.2: Metabolizable energy1 (ME) content of diets (TMR) comprising roughage (60 per cent kept constant) and CFM

(31 to 40 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type II diet).

Samples

ME (MJ / kg DM) (Mean ± SE)


Paddy straw + CFM+ azolla (R1) 7.57a ± 0.03

7.26

b ± 0.10

7.03

b ± 0.09

6.78

b ± 0.02

Ragi straw + CFM+ azolla (R2) 8.05 ± 0.29 7.91 ± 0.26 7.86 ± 0.21 7.78 ± 0.13

Maize stover +CFM + azolla (R3) 7.65 ± 0.17 7.55 ± 0.09 7.48 ± 0.07 7.40 ± 0.10

Maize husk+ CFM+ azolla (R3) 8.80 ± 0.02 8.78 ± 0.08 8.74 ± 0.1 8.89 ± 0.13

Sorghum stover +CFM + azolla (R4) 8.96 ± 0.16 8.91 ± 0.12 8.76 ± 0.19 8.51 ± 0.12

Means bearing different superscripts in a row differ significantly(P<0.05).


T5= Treatment with 0% Azolla (Control), T6= Treatment with 3% Azolla, T7= Treatment with 6% Azolla, T8 = Treatment with 6%

Azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.

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60

control. There was decreased ME content in the supplemented compared to the control.

The ME (MJ / kg DM) content of ragi straw based TMR: T5R2- control group, T6R2, T7R2

and T8R2 (supplemented diets) was 8.05, 7.91, 7.86 and 7.78, respectively. There was no

significant difference (P>0.05) in the ME content of the three different levels of

supplementation compared to the control. The ME (MJ / kg DM) content of maize stover

based TMR: T5R3- control, T6R3, T7R3 and T8R3 (supplemented diets) was 7.65, 7.55,

7.48, and 7.40, respectively. There was no significant difference (P>0.05) in the ME

content of the three different levels of supplementation compared to the control. The ME

(MJ / kg DM) content of maize husk based TMR: T5R4- control group, T6R4, T7R4 and

T8R4 (supplemented) was 8.8, 8.78, 8.74 and 8.89, respectively. There was no significant

difference (P>0.05) in the ME content of azolla supplemented diet compared to the

control. The ME (MJ / kg DM) content of Sorghum stover based TMR: T5R5- control,

T654, T7R5 and T8R5 (supplemented) was 8.96, 8.91, 8.76 and 8.51 respectively. The

difference among the three supplemented diet was not statistically significant (P>0.05)

compared to the control.

4.5.3 Metabolizable energy (ME) content of Type III diet

The ME content of diets (TMR) comprising CFM (40 per cent kept constant) and

roughage which was replaced with different levels of azolla from 0 to 9 per cent are

presented in Table 4.5.3. The ME content of Paddy straw based TMR: T9R1 (control) was

7.57 MJ/ kg DM. The ME (MJ/ kg DM) content of the diets T10R1, T11R1 and T12R1

(supplemented) was 7.52, 7.45 and 7.36, respectively. There was significant difference

(P<0.05) in the ME content of the diets (TMR) supplemented with different levels of

azolla compared with the control group. However there was no significant difference

61

Table 4.5.3: Metabolizable energy1 (ME) content of diets (TMR) comprising CFM (40 per cent kept constant) and roughage

(51 to 60 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type III diet).

Samples

ME (MJ / kg DM) (Mean ± SE)


Paddy straw + CFM+ azolla (R1) 7.57a ± 0.03

7.24

b ± 0.24

7.11

b ± 0.19

7.05

b ± 0.26

Ragi straw + CFM+ azolla (R2) 8.05a ± 0.29

7.53

b ± 0.08

7.28

b ± 0.21

7.18

b ± 0.12

Maize stover +CFM + azolla (R3) 7.65 ± 0.17 7.57 ± 0.15 7.39 ± 0.12 7.40 ± 0.07

Maize husk+ CFM+ azolla (R3) 8.80 ± 0.02 8.77 ± 0.08 8.8 ± 0.12 8.72 ± 0.07

Sorghum stover +CFM + azolla (R4) 8.96 ± 0.16 8.91 ± 0.08 8.85 ± 0.13 8.80 ± 0.06

Means bearing different superscripts in a row differ significantly (P<0.05).


T9 = Treatment with 0% azolla (Control), T10 = Treatment with 3% azolla, T11= Treatment with 6% azolla, T12 = Treatment with 6%

Azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 = Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk

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62

among the supplemented diets. The ME (MJ/kg DM) content of the diets T9R2 (control),

T10R2, T11R2 and T12R2 (supplemented) was 8.05, 7.79, 7.80 and 7.69, respectively. There

was significant difference (P<0.05) in the ME content of the three supplemented diets

compared to the control but there was no significant difference among the diets

supplemented with different levels of azolla. The ME (MJ / kg DM) content of maize

stover based TMR: T9R3- control group, T10R3, T11R3 and T12R3 (supplemented) was

7.65, 7.57, 7.39 and 7.40, respectively. There was no significant difference (P>0.05) in

the ME content of the three groups of diet compared to the control. The ME (MJ / kg

DM) content of maize husk based TMR: T9R4- control group, T10R4, T11R4 and T12R4

(supplemented) was 8.8, 8.77, 8.8 and 8.72, respectively. There was no significant

(P>0.05) difference between supplemented and control diets. The ME (MJ / kg DM)

content of sorghum stover based TMR: T9R5- control group, T10R5, T11R5 and T12R5

(supplemented) was 8.96, 8.91, 8.85 and 8.80, respectively. The difference among the

three treatment diets was not statistically significant (P>0.05) compared with the control.

4.6 Apparent digestible dry matter (ADDM, per cent DMB) and True digestible

dry matter (TDDM, per cent DMB)

4.6.1 Apparent digestible dry matter (ADDM) and True digestible dry matter

(TDDM) of Type I diet

The Apparent digestible dry matter (ADDM) and True digestible dry matter

(TDDM) of Azolla pinnata and the diets comprising roughage supplemented with azolla

at 0 and 9 per cent is presented in Table 4.6.1. The ADDM and TDDM of Azolla was

found to be 29.59 % and 54.49 %, respectively. The ADDM of diet comprising paddy

straw supplemented with 0% (control) and 9% azolla was 30.27 % and 23.06 per cent,

63

Table 4.6.1: Apparent digestible dry matter (ADDM %) and True digestible dry matter (TDDM %) of Azolla pinnata and the

diets comprising roughage replaced with azolla at 0 per cent and 9 per cent (Type I diet).

Samples

ADDM %(Mean ± SE) TDDM %(Mean ± SE)


Azolla pinnata 29.59 ± 0.31 - 54.49 ± 0.43 -

Paddy straw with azolla (R1) 30.27a ± 1.34

23.07

b ± 2.08

51.18

c ± 1.24

46.29

d ± 0.94

Ragi straw with azolla (R2) 46.12a ± 0.25

35.49

b ± 0.73

62.19

c ± 0.49

53.96

d ± 0.26

Maize stover with azolla( R3) 31.01 ± 4.77 28.66 ± 0.22 49.50 ± 4.6 49.27 ± 0.22

Maize husk with azolla( R4) 32.89 ± 0.78 28.71 ± 0.81 50.14 ± 0.56 48.62 ± 0.52

Sorghum stover with azolla( R5) 30.16 ± 0.061 29.34 ± 0.40 50.70 ± 0.82 48.45 ± 0.35

Means bearing different superscripts in a row differ significantly (P<0.05).

T1= Treatment with 0% azolla (Control), T4 = Treatment with 9% azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 =

Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk

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64

respectively. The TDDM of diet comprising paddy straw supplemented with 0% (control)

and 9% azolla was 51.18 % and 46.28 per cent, respectively. There was significant

(P>0.05) difference in the ADDM and TDDM between the control and diet supplemented

with 9% azolla. There was decreased ADDM and TDDM % in 9% azolla supplemented

diet. The ADDM and TDDM of ragi straw based diets T1R2 (control) andT4R2 (9% azolla

treatment)were 46.12%, 35.49% and 62.19%, 53.96%, respectively. There was

significant (P<0.05) difference in ADDM and TDDM content in 9% treated diet

compared to control. There was decreased ADDM and TDDM % in 9% azolla

supplemented diet. The ADDM and TDDM of maize stover based diets T1R3 (control)

andT4R3 (9% azolla treatment)were 31.01%, 28.66% and 49.50, 49.27%, respectively.

There was no significant (P>0.05) difference between control and treatment diets. The

ADDM and TDDM content of maize husk based diets T1R4 (control) andT4R4 (9% azolla

treatment)were 32.89%, 28.71% and 50.14%, 48.62%, respectively. There was no

significant (P>0.05) difference between control and treatment diets. The ADDM and

TDDM of sorghum stover based diets T1R5 (control),T4R5 (9% azolla treatment)were

30.16%, 29.34% and 50.70%, 48.45% respectively. The difference among the control and

treatment diet was not statistically significant (P>0.05).

4.6.2 Apparent digestible dry matter (ADDM) and True digestible dry matter

(TDDM) content of Type III diet

The Apparent digestible dry matter (ADDM) and True digestible dry matter

(TDDM) of the mixed diets comprising CFM (40 per cent kept constant) and roughage

supplemented with azolla at 0 and 9 per cent is presented in Table 4.6.2. The ADDM

content of paddy straw based TMR: T9R1 and T12R1 were 48.18% and 40.81%,

65

Table 4.6.2: Apparent digestible dry matter (ADDM %) and Total digestible dry matter (TDDM %) of the diets (TMR)

comprising CFM (40 per cent kept constant) and roughage (51 to 60 per cent) replaced with azolla at 0 per cent

and 9 per cent (Type III diet).

Samples

ADDM %(Mean ± SE) TDDM %(Mean ± SE)

0 %Azolla (T1) 9% Azolla (T12) 0% Azolla (T1) 9% Azolla (T12)

Paddy straw + CFM + Azolla (R1) 48.18a ± 1.96

40.81

b ± 0.740

67.58

c ± 2.13

60.76

d ± 2.8

Ragi straw + CFM + Azolla (R2) 49.84a ± 1.35

44.62

b ± 2.6

67.35

c ± 2.74

63.89

d ± 2.2

Maize stover + CFM + Azolla (R3) 42.66 ± 0.52 41.29 ± 2.72 61.07 ± 0.31 60.70 ± 1.86

Maize husk + CFM + Azolla (R4) 43.56 ± 0.57 42.31 ± 0.34 60.09 ± 0.54 58.9 ± 0.95

Sorghum stover + CFM + Azolla (R5) 41.2 ± 0.64

38.3 ± 0.71

62.36 ± 0.35

58.64 ± 0.64

Means bearing different superscripts in a row differ significantly (P <0.05).

T1= Treatment with 0% azolla (Control), T12 = Treatment with 9% azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 =

Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk

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66

respectively. The TDDM content of paddy sraw based TMR: T9R1 and T12R1 were

67.58% and 60.76%, respectively. There was significant (P>0.05) difference between

control and the diet supplemented with 9% azolla. There was decreased ADDM and

TDDM % in 9% azolla supplemented diet. The ADDM and TDDM of the ragi straw

based TMR: T9R2 (control) andT12R2 (9% azolla treatment)were 49.84%, 44.62 % and

67.35%, 63.89%, respectively. There was significant (P<0.05) difference between control

and supplemented diet. There was decreased ADDM and TDDM % in 9% azolla

supplemented diet. The ADDM and TDDM of the maize stover based TMR: T9R3

(control) and T12R3 (9% azolla treatment) were 42.66%, 41.29% and 61.07%, 60.70%,

respectively. There was no significant (P>0.05) difference between control and treatment

diets. The ADDM and TDDM of the maize husk based TMR: T9R4 (control) and T12R4

(9% azolla treatment) were 43.56%, 42.31% and 60.09%, 58.9%, respectively. There was

no significant (P>0.05) difference between control and treatment diets. The ADDM and

TDDM of the sorghum based TMR: T9R5 (control) and T12R5 (9% azolla treatment) were

41.2%, 38.3% and 62.36%, 58.64 %, respectively. The difference between the control

and treatment diet was not statistically significant (P>0.05).

DDiissccuussssiioonn

V. DISCUSSION

The present study was conducted to determine the supplementary effect of Azolla

pinnata on the metabolizable energy (ME) and digestibility of the crop residues and total

mixed ration (TMR) by rumen in vitro gas production technique and modified in vitro

two stage digestion technique, respectively. As azolla is a good source of protein, amino

acids, it could be a supplementary source in enhancing the ME content of the diet. Hence,

the present study was conducted by formulating the different diets comprising crop

residues which were supplemented with different levels of azolla ranging from 0 to 9 per

cent and the TMR which were supplemented with azolla at different levels ranging from

0 to 9 per cent by replacing either roughage portion or concentrate portion and subjected

to rumen in vitro gas production study (Hohenheim gas test) and modified in vitro two

stage digestion study to know the supplementary effect of azolla. As the dry matter

content of azolla is very less, it cannot be considered at high proportion, it can only be

tried at supplementary levels. Hence, the present study considered azolla supplemention

at a maximum level of 9 per cent of the diet.

5.1 Chemical composition of Azolla pinnata

The results of the proximate analyses of azolla obtained in the present study are in

close agreement with the values reported by Bolka (2011). The crude protein content of

azolla obtained in present study is similar to the values reported by Alalade and Iyayi

(2006) and Mutzar et al. (1976). The total ash content of azolla obtained in the present

study is lower than the values reported by Tamang et al. (1993) and Ali and Leeson

(1995) but higher than the value reported by Singh et al. (1978) and Parthasarathy et al.

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68

(2002). The ether extract content obtained in present study is higher than the values

reported by Parthasarathy et al. (2001), Alalade and Iyayi (2006), Tamang et al. (1993)

and Balaji et al. (2009). The crude fiber content of azolla obtained in present study is

slightly higher than the observations of Alalade and Iyayi (2006) and Singh and Subudhi

(1978). The NFE content of azolla is in close agreement with the results of Balaji et al.

(2009) but lower than the values reported by Alalade and Iyayi (2006) and Tamang et al.

(1993). The NDF content of azolla is in close agreement with the value reported by

Parnerkar et al. (1986) but higher than the values reported by Alalade and Iyayi (2006),

Buckingham et al. (1978), Ali and Leeson (1995) and Taklimi (1990). The ADF content

of azolla is almost similar to the value reported by Khatun et al. (1996). The ADL content

of azolla obtained in present study is almost similar to the value reported by Ramesh

(2008) but higher than the value reported by Tamang et al. (1993). The variations in the

nutrient composition of azolla may be due to differences in the response of azolla strains

to environmental conditions such as temperature, light intensity and soil nutrients which

consequently affect their growth morphology and chemical composition.

5.2 Mineral profile of Azolla pinnata

The mineral profile of azolla obtained in the present study is almost similar to the

values reported by Anand and Geetha (2007). Calcium content of azollais similar to the

reports of Tamang et al. (1993). Magnesium content of azollaobtained in the present

study is similar to the value reported by Alalade and Iyayi (2006). Higher level of heavy

metals like nickel, lead, cadmium was obtained in the sample of azolla used for the

present study indicating biooccumilation of heavy metals by azolla. Jain, et al.

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(2007)studied the absorption of iron, copper, cadmium, nickel, lead, zinc, manganese,

and cobalt by Azolla pinnata indicating bioaccumilation of heavy metals by azolla.

5.3 Amino acid profile of Azolla

The data on amino acid profile indicate that lysine, isoleucine, leucine,

phenylealanine, glycine, arginine and valine were predominant. However, the

concentration of sulphur containing amino acids like methionine and cystine is

comparatively less in Azolla pinnata. Amino acid values obtained in this study were in

corroboration with the findings of Alalade and Iyayi (2006). Beckingham et al. (1978)

reported higher concentration of essential amino acids in Azolla filiculoides.

5.4 Chemical composition of crop residues

The chemical composition of paddy straw obtained in the present study is in close

agreement with the reports of Rahaman et al. (2010). The DM content of paddy straw is

almost similar to the reports of Sarnklong et al. (2012) but little higher than the values

reported by Kumar et al. (1999). The crude protein content of paddy straw is lower than

the values reported by Preston et al. (2000) and Akinfemi and Ogunwole (2012). The

ether extract content of paddy straw is little higher than the values recorded by Kumar et

al. (1999). The CF, NDF and the total ash contents of paddy straw obtained in the present

study are little higher than the values recorded by Preston et al. (2000) and Akinfemi and

Ogunwole (2012).The lignin content of paddy straw obtained in the present study is

almost similar to the value recorded by Sarnklong et al. (2011) but lesser than the value

reported by Rahaman et al. (2009).

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The crude protein content of ragi straw obtained in the present study is similar to

the value reported by Bhatta et al. (2000) but lower than the values reported by Madibela

and Modiakgotla (2004) and Sreerangaraju et al. (2000). The ether extract content of ragi

straw is little higher than the values recorded by Bhatta et al. (2000) and Sreerangaraju et

al. (2000). The NDF, ADF and ADL content of of ragi straw is almost similar to the

values reported by Madibella et al. (2004).

The DM, TA, CF, NFE, ADF, NDF content of the maize husk obtained in the

present is in close agreement with the values reported by Akinfemi et al. (2009) but it

contained lower content of CP, EE and ADL.

The CP, CF and TA content of sorghum stover is in close agreement with the

values reported by Akinfemi et al. (2010) but it contained lower content of NDF, ADF

and ADL.

The CP, EE, NDF, ADF and ADL content of bengal gram husk obtained in the

present study is in close agreement with the values reported by Sreerangraju et al. (2000).

The limitation of crop residues includes high fibre, low protein and less of

fermentable carbohydrates.

5.5 Metabolizable energy content and in vitro dry matter digestibility of Azolla

pinnata

The in vitro net gas production and metabolizable energy density of azolla

obtained in the study is almost similar to the value reported by Buckingham et al. (1978)

but lower than the values reported by Alalade and Iyayi (2006), Parashuramulu and

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Nagalakshmi (2013), Khatun et al. (1999) and Ramesh (2008). The in vitro dry matter

digestibility of azolla obtained in the present study is almost similar to the value reported

by Becerra et al. (1995). In comparison with crop residues, azolla contains low level of

organic matter and higher levels of Acid detergent lignin and biogenic silica which limits

the digestibility and ME content to the lower extent.

5.6 Metabolizable energy content and in vitro dry matter digestibility of crop

residues

The ME content of paddy straw obtained in the present study is lesser than the

value reported by Akinfemi and Ogunwole (2012). The in vitro gas production and ME

content of ragi straw obtained in the present study is more than the values reported by

Sreerangaraju et al. (2000). The in vitro gas production and ME content of maize husk

obtained in the present study is higher than the values reported by Akinfemi et al. (2009).

The in vitro dry matter digestibility of maize stover obtained in the present study is

almost similar to the value reported by Kiangi et al. (1981) but lower than the value

reported by Oji et al. (1997). The in vitro gas production and ME content of sorghum

stover obtained in the present study is higher than the values reported by Akinfemi et al.

(2010). The in vitro gas production and ME content of bengal gram husk obtained in the

present study is almost similar to the value reported by Sreerangaraju et al. (2000).

Among the crop residues, the high crude fibre content and sand silica limits the

digestibility.

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5.7 Metabolizable energy content of Type I diet

In case of the diet comprising paddy straw supplemented with azolla at different

levels there was no significant difference between the control and supplemented diets.

Similar observation was made in case of sorghum stover and bengal gram husk

supplemented with azolla at different levels indicating no supplementary effect of azolla

on crop residues. The results are similar to those of Ramesh (2008) who has reported that,

there was no significant difference in gas production and dry matter digestibility of the

paddy straw supplemented with azolla at different levels and the addition of azolla has no

supplementary effect on paddy straw. In case of diet comprising ragi straw supplemented

with azolla at different levels, there was significant (P≤0.05) difference between the

control and the diet supplemented with 6% and 9% per cent azolla. In the diet comprising

maize stover supplemented with azolla at different levels there was significant (P ≤ 0.05)

difference between the control and the diet supplemented with 3%, 6% and 9% per cent

azolla. Similar observations were made in case of diet comprising maize husk

supplemented with azolla at different levels. There was decrease in ME content of all the

treatments compared to the control. The lower ME content of the treatment diet

supplemented with different levels of azolla may be due to the presence of high lignin

content (24.30%) and high total ash content (17.84 %) of the azolla sample. This

indicates that there is negative effect of azolla supplementation on utilization of crop

residues. There is a negative correlation between lignin content and digestibility of forage

materials in ruminant. Lignin is considered as an antinutritive component of forages as it

cannot be readily fermented by rumen microbes. It has a negative impact on

metabolisable energy (ME) values of forages (Van Soest, 1994). Lignin was identified as

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a dominant factor limiting the feed value in perennial grasses, maize stems and tropical

forages. Apart from high lignin content, higher level of biogenic silica and presence of

heavy metals in azolla would also have been the reason for lower ME content and

digestibility of the diets. It has been reported that elevated concentration of heavy metals

like nickel, cadmium reduces the in vitro dry matter digestibility of fiber (cellulose) upto

50 to 60 % (Martinez and Church, 1990). In general we cannot replace any of the crop

residues with azolla.

5.8 Metabolizable energy content of Type II diet

In the diet (TMR) comprising paddy straw and CFM which was replaced with

azolla at different levels, there was significant (P ≤ 0.05) difference between the control

diet and the diet supplemented with 3%, 6% and 9% azolla. There was decrease in ME

content of all the treatment diets compared to the control. The results were similar to

those of Kumar et al. (2013) who has reported that 50% replacement of oil cake with

azolla meal reduced the gas production in in vitro condition with no satisfactory

significant effect on digestibility. In the diet comprising ragi straw and CFM which was

replaced with azolla at different levels there was no significant difference between the

control and the treatment diets. Similar observations were made in case of all other diets

(TMR) supplemented with azolla at different levels. This indicates that there is no

supplementary effect of azolla on TMR, where concentrate portion was substituted with

azolla at different levels. Compared to CFM, azolla contains good amount of protein,

fibre, lignin and biogenic silica but it has low energy content because of low fermentable

organic matter. So replacement of CFM with azolla reduces ME content and digestibility.

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5.9 Metabolizable energy content of Type III diet

In the diet comprising CFM and paddy straw which was replaced with azolla at

different levels, there was significant (P ≤ 0.05) difference between the control and diet

supplemented with 3%, 6% and 9% azolla. There was decrease in ME content of all the

treatment diets compared to the basal control diet. The lower ME content of the diet

incorporated with different levels of azolla may be due higher lignin content (24.30%)

and higher total ash content (17.84 %) of azolla. This indicates that there is no

supplementary effect of azolla on TMR where roughage portion was substituted with

azolla at different levels ranging from 0 to 9%.

5.10 Apparent digestible dry matter (ADDM) and true digestible dry matter

(TDDM) content of Type I diet

In case of the diet comprising either paddy straw as such and the paddy straw

supplemented with azolla at 9% there was significant difference between the

supplemented and control diet. In case of the diet comprising ragi straw supplemented

with azolla at 9% there was significant (P ≤ 0.05) decrease in digestibility. The decreased

ADDM and TDDM in the diet supplemented with 9% azolla might be due to high lignin

content of azolla sample. In case of the diet comprising maize stover supplemented with

azolla at 9% there was no significant difference between the treatment and control diet.

Similar observations were made in case of diet comprising maize husk and sorghum

stover. This indicates that there is no satisfactory effect on the digestibility by

supplementing azolla to various crop residues.

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5.11 Apparent digestible dry matter (ADDM) and true digestible dry matter

(TDDM) content of type III diet

In the diet comprising CFM and paddy straw which was replaced with azolla at

different levels, there was significant difference between the control and diet

supplemented with 9% azolla. In the diet comprising CFM and ragi straw which was

replaced with azolla at different levels, there was significant (P≤0.05) difference between

the control and diet supplemented with 9% azolla. There was decrease in ADDM and

TDDM content of supplemented diet. The cause for the lower dry matter digestibility of

the diet incorporated with 9% azolla might be high lignin and total ash content of the

azolla sample, indicating no effect of azolla supplementation on digestibility of TMR as

well. In the diet comprising CFM and maize stover which was replaced with azolla at

different levels, there was no significant difference between the control and diet

supplemented with 9% azolla. Similar observations were made in all other diet

supplemented with azolla (0 to 9%) indicating there is is no satisfactory effect on the

digestibility of TMR by supplementing azolla at different levels (0 to 9%).

The greatest problem of the Indian livestock farmers facing today, is the shortage

of fodder. The arrival of high yielding varieties, shifted the emphasis from the bio-mass

oriented agriculture to grain yield oriented agriculture. This in turn highly reduced the

sources of fodder from agricultural crops. The rapid shrinkage of common lands of the

villages and expansion of urban areas also led to disappearance of grazing lands and

pastures. Traditional sources of cattle feed like oil cakes and coarse grains also declined

as other cash crops occupied their areas. Such varied factors have contributed in an inter-

linked way to aggravate the fodder crisis. So to substitute the cattle feeds with natural

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feeds that are rich in useful nutrients, azolla has gained importance and is being used as a

protein substitute in the diet of cattle due to its good content of proteins, essential amino

acids, vitamins (vitamin A, vitamin B12, Beta Carotene), growth promoter intermediaries

and minerals, its ability to proliferate without inorganic nitrogen fertilization and its high

rate of growth in water without the need to displace existing crops or natural ecological

systems. It has been used for many years throughout Asia to feed the livestock. One of

the advantages of growing azolla, is that it not only requires less water, but is also cost-

effective for the dairy farmers. With azolla, there is no need to clear the weeds and level

the soil. Even the water left after the second rinsing of the plant could be used. Besides,

the field claims states that it helps in increasing milk yield by 15 to 20 per cent and

reduction in feed cost by 20-30 per cent (Pillai et al., 2002).

The results of the present study however, confirm that azolla can only be

considered as a source of protein, amino acids, major and trace mineral elements, since it

did not improve the digestibility or metabolizability of crop residues or total mixed ration

even at higher level of supplementation.

As the biomass yield of azolla is extremely low, it is practically difficult to

include even 3% of azolla in the diet of ruminants, therefore it could only be a

supplementary source of protein, amino acids and mineral elements.

http://timesofindia.indiatimes.com/topic/Weeds

SSuummmmaarryy

VI. SUMMARY

The present study was conducted to determine the supplementary effect of Azolla

pinnata on the metabolizable energy (ME) and digestibility of the crop residues and Total

mixed ration (TMR) by rumen in vitro gas production study (Hohenheim gas test) and

modified two stage digestion technique, respectively. As azolla is a good source of

protein, amino acids and mineral elements, it could be a supplementary source in

enhancing the ME content of the diet. Hence, the present study was conducted by

formulating the diets comprising crop residues which were supplemented with different

levels of azolla from 0 to 9 per cent and the TMR which were supplemented with azolla

at different levels from 0 to 9 per cent by replacing either roughage portion or concentrate

portion in the mixed diets and subjected to rumen in vitro gas production and digestibility

studies to verify the supplementary effect of azolla on utilization of different types of

diet.

The study included chemical analyses, in vitro gas production and digestibility

studies. The study was conducted at the Department of Animal Nutrition, Livestock

Production and Management, Veterinary College, KVAFSU, Bangalore and Alltech

Ruminant Nutrition laboratory, Veterinary College, Bangalore.

Crop residues commonly used for dairy cattle feeding in Karnataka were selected

for evaluation. The crop residues were ground to pass through a one mm sieve and

preserved in airtight bottles at room temperature while using for chemical analysis and in

vitro studies. Azolla pinnata was harvested from azolla tanks maintained at University of

Agricultural and Horticultural Sciences, College of Agriculture, Shivamogga. Fresh

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samples of Azolla was dried in hot air oven at 50oC for 48 h and the dried samples was

ground to pass through a one mm sieve and preserved in airtight bottles at room

temperature.

Three types of diets were formulated by using azolla, crop residues and

compound feed mixture. The first type of diet comprised only roughage which was

supplemented with different levels of azolla that is 0%, 3%, 6%, and 9%. The second

type of diet comprised of Total mixed ration (TMR) with roughage and CFM taken in

the ratio of 60:40 and the CFM portion of TMR was replaced with 0%, 3%, 6% and 9%

azolla. The third type of diet also comprised of TMR, roughage and concentrate taken in

the ratio of 60:40 and the roughage portion of TMR was substituted with 0%, 3%, 6%

and 9% azolla.

The samples of azolla and different types of diets were subjected to rumen in vitro

gas production study to know the supplementary effect of azolla on ME content of

various diets.

A crossbred (Holstein Friesian x Bos indicus) lactating dairy cow fitted with a

flexible rumen cannula of large diameter served as the donor of the rumen inoculum. The

ME content of the various diet formulated was obtained by in vitro gas test (Menke and

Steingass, 1988). The per cent ADDM and TDDM contents of the diets was obtained by

modified in vitro two stage digestion technique (Goering and Van Soest, 1970).

The chemical analyses of the azolla revealed that it is a good source of protein,

amino acids and minerals. Estimation of ME content of azolla revealed that it is a low

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energy feed resource compared to all other crop residues and TMR. Estimation of mineral

profile of azolla revealed that, it is a good source of major and trace mineral elements.

Reduction in the ME content was observed in the diet comprising ragi straw

supplemented with azolla at different levels (0 to 9%) as compared to the control diet.

Similar observations were made in case of diets comprising maize husk and maize straw.

This indicates negative effect of azolla supplementation on the ME values of the crop

residues. In the diet comprising paddy straw supplemented with azolla at different levels,

there was no significant difference between the control and all other supplemented diets.

Similar observations were made in case of the diet comprising sorghum stover and bengal

gram husk indicating that there is no supplementary effect of azolla on ME content.

Similarly there was no significant difference in the in vitro dry matter digestibility

between the treatment diet and control diet indicating no beneficial effect of azolla

supplementation on digestibility of crop residues.

In the type II diet there was no significant difference in the ME content between

the control and all other supplemented diets except for the one comprising paddy straw

and CFM, where ME content was reduced in the azolla supplemented diet compared to

the control diet. This indicates that, there is no beneficial effect of azolla supplementation

on ME value of TMR in which concentrate portion is replaced with different levels of

azolla ranging from 3 to 9 %.As ME value of azolla is less, it caused reduction of ME

value of other diets which was supplemented with azolla.

Identical observations were made even with type III diet preparation in which the

roughage portion was substituted with different levels of azolla ranging from 0 to 9%.

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There was no significant difference either in ME density or digestibility of DM with

respect to most of the type III diet preparations, except the mixed diet comprising paddy

straw and CFM and ragi straw and CFM, where in the ME content was reduced with

azolla supplementation.

Based on the results of the present study, following conclusions can be drawn.

1. The chemical composition analyses indicated that, the azolla is a good source of

protein, amino acids and mineral elements.

2. Estimation of ME content in azolla revealed that, it is a low energy feed resource

compared to all other crop residues and TMR preparations.

3. By considering the ME content of various diets supplemented with azolla, it was

concluded that, there is no difference between the control diet and the one

supplemented with different levels of azolla ranging from 0 to 9 per cent.

4. By considering the in vitro dry matter digestibility percentage of various diets

supplemented with azolla, it was concluded that, there was decrease in the in vitro dry

matter digestibility of the diet supplemented with 9% azolla as compared to basal diet

indicating that there is negative effect of azolla on the digestibility of various diets.

BBiibblliiooggrraapphhyy

VII. BIBLIOGRAPHY

A.O.A.C., 2005. Official methods of analysis, 18th

Edition. Association of Official

Analytical Chemists, Washington, D.C.

AHIRWAR, M. K., LEELA, V. and BALAKRISHNAN, V., 2009. In-vitro fermentation

pattern of Azolla pinnata as protein supplement in forage based diet for

ruminants. Indian Vet. J., 86 (1): 60-62.

AKINFEMI, A., BABAYEMI, O. J. and JONATHAN, S. G., 2009. Bioconversion of

maize husk into value added ruminant feed by using white-rot fungus. Revista

UDO Agricola., 9(4): 972-978.

AKINFEMI, A., ADU, O. A. and DOHERTY, F., 2010. Conversion of sorghum stover

into animal feed with white-rot fungi: Pleurotus ostreatus and Pleurotus

pulmonarius. African J. Biotech., 9(11): 1706-1712.

AKINFEMI, A. and OGUNWOLE, O. A., 2012. Chemical composition and in vitro

digestibility of rice straw treated with Pleurotus ostreatus, Pleurotus pulmonarius

and Pleurotus tuber-regium. Slovak J. Anim. Sci., 45(1): 14-20.

ALALADE, M. and IYAYI, E. A., 2006. Chemical composition and the feeding value of

Azolla (Azolla pinnata) meal for egg-type chicks. Int. J. Poult. Sci., 5:137-141.

ALI, M. A. and LEESON, S., 1995. Nutritional value and aquatic weeds in the diet of

poultry. World’s Poultry Science Journal., 50: 239-251.

ANAND, T. and GEETHA, N. P., 2007. Azolla as a Biofertilizer in Coffee Plantation. I

need Coffee. Htm.,

ARGYLE, J. L. and BALDWIN, R. L., 1989. Effects of amino acids and peptides on

rumen microbial growth yields. J. Dairy Sci., 72: 2017–2027.

http://www.cabdirect.org/search.html?q=au%3A%22Ahirwar%2C+M.+K.%22

82

82

BACERRA, M., PRESTON, T. R. and OGLE, B., 1995. Effect of replacing whole boiled

soya beans with azolla in the diets of growing ducks. Livest. Res. Rural Dev.,7: 1-

11.

BALAJI, K., JALALUDEEN, A., CHURCHIL, R. R. and PEETHAMBARAN, P. A and

SENTHIL KUMAR, S., 2009. Effect of dietary inclusion of Azolla (Azolla

pinnata) on production performance of broiler chicken. Indian J. poult. Sci.,

44(2): 195-198

BASAK, B., PRAMANIK, A. H., RAHAMAN, M. S and ROY, B. C., 2002. Azolla

(Azolla pinnata) as a feed ingredient in broiler ration. Int. J. Poult. Sci., 1: 29-32.

BHATTA, R., KRISHNAMOORTHY, U. and MOHAMMED, F., 2000. Effect of

feeding tamarind (Tamarindus indica) seed husk as a source of tannin on dry

matter intake, digestibility of nutrients and production performance of crossbred

dairy cows in mid-lactation. Ani. Feed Sci.Tech.,83: 67-74.

BOLKA, P. C., 2011. Nutritional evaluation of Azolla (Azolla pinnata) in broilers and

layers. Ph.D. Thesis, Karnataka Veterinary, Animal and Fisheries Sciences

University, Bidar.

BOYED, C. E., 1969. The nutritive value of three species of water weeds.Econ. Bot.,

23:123-127.

BECKINGHAM, M. K. W., ELA, S. W., MORIS, J. G. and GOLDMAN, C. R., 1978.

Nutritive value of the Nitrogen-fixing aquatic fern Azolla filiculoides. J. Agri.

Food Chem., 26: 1230-1234.

CHATTERJEE, SHARMA, P., GHOSH, M. K., MANDAL, M. and ROY, P. K., 2013.

Utilization of Azolla Microphylla as Feed Supplement for Crossbred Cattle. Int. J.

Agri. Food Sci. Tech.,4(3): 207-214.

CULLEY, D. D. and EPPS, E. A., 1973. Use of duck weed for waste water treatment and

animal feed water pollution control. Fed. J., 45: 337-347.

83

83

DURAN, A. O., 1994. Raw palm oil as the energy source in pig fattening diets and

Azolla filiculoides as a substitute for soya bean meal. Livestock Research for

Rural Development., 6(1): 34-35.

FAO Report, 2006. Improving Animal Productivity by Supplementary Feeding of

Multinutrient Blocks, Controlling Internal Parasites and Enhancing Utilization of

Alternate Feed Resources.

GHODAKE, S. S., FERNANDES, A. P., DARADE, R. V. and ZAGADE, B. G., 2012.

Effect of different levels of Azolla meal on growth performance of Osmanabadi

kids. Res. J. Anim. Husbandry and Dairy Sci., 3(1): 13-16.

GIRIDHAR, K., ELANGOVAN, A. V., KHANDEKAR, P., SHARANGOUDA and

SAMPATH, K. T. 2012. Cultivation and use of Azolla as nutritive feed

supplement for the livestock. Indian Farming., 62(2): 20-22.

GOERING, H. K. and VAN SAOEST, P. J., 1970. Forage fibre analyses. Agriculture

Handbook No. 379, USDA, Washington, DC.

ICAR Report, 2012. Department of Agriculture Research and Education, Ministry of

Agriculture, Government of India, New Delhi.

INDIRA, D., SARJAN, R. K., SURESH J., VENUGOPAL, N. K. and RAVI, A. 2009.

Azolla (Azolla pinnata) as Feed Supplement in Buffalo Calves on Growth

Performance. Indian J. Ani.Nutr., 26(4): 345-348.

JAIN, H., PRABHA, K. and PADMAVATHIAMMA. 2007. Phytoremediation

Technology: Hyper accumulation Metals in Plants. Water Air Soil Pollut., 184:

105–126

KHAN, M. J., STEINGASS, H and DROCHNER, W., 2002.Evaluation of Some Aquatic

Plants from Bangladesh through Mineral Composition, In Vitro Gas Production

and In Situ Degradation Measurements. Asian-Aust. J. Anim. Sci., 15(4): 537-542.

84

84

KHAN, M. M., 1983. A primer on Azolla Production and Utilization in Agriculture,

UPLB, PCARRD and SEARCA, Los Banos.

KHATUN, A., ALI, M. A. and DINGLE, J. G., 1999. Utilization of Azolla (Azolla

pinnata) in the diets of laying hen. Anim. Feed Sci. Technol., 8(1) 43-56.

KIANGI, E. M., KATEGILE, J.A. and SUNDSTAL, F., 1981. Different sources of

ammonia for improving the nutritive value of low quality roughage. Anim. Feed

Sci. Technol., 6: 377-386.

KUMAR, D. S, PRASAD, R. V, KISHORE, K. R. and RAO, E. R., 2012. Effect of

azolla (Azolla pinnata) based concentrate mixture on nutrient utilization in buffalo

bulls. Indian Journal of Animal Research., 46: 268-271.

KUMAR, R., TRIPATHI, P., CHAUDHARY, U. B., GUPTA, B., SHARMA, H., 2013.

Effect of replacement of oil cake with azolla meal on in vitro gas, methane

production and digestibility with rumen liquor of goat. XX Annual Conference of

IAAVR, KVAFSU, 16-17 April, Bangalore, India.

KUMAR, S., GUPTA, B. S. and YADAV, K. R., 1999. Effect of different roughage

protein and energy sources on in vitro rumen fermentation using rumen liquor

inoculant. Indian J. Ani. Nutr.16: 112-116.

LEJEUNEA, A., PENGA, J., BOULENGE, L., LARONDELLEC, Y. and VAN HOVE,

C., 2000. Carotene content of Azolla and its variations during drying and storage

treatments. Animal Feed Science and Technology.,84: 295-301.

LIPSTEIN, B. and HURWITZ, S., 1980. The nutritional value of algae for poultry. Brit.

Poult. Sci.,21: 9-21.

LIU, J. X., SUSENBETH, A. and SÜDEKUM, K. H., 2002. In vitro gas production

measurements to evaluate interactions between untreated and chemically treated

rice straws, grass hay, and mulberry leaves. J. Anim. Sci. 80: 517-524.

85

85

LIU, Q., WANG, C., HUANG, Y., DONG, K. H., YANG, W. Z. and ZHANG, S. L.

2009. Effects of isovalerate on ruminal fermentation, urinary excretion of purine

derivatives and digestibility in steers. J. Anim. Physiol. Anim. Nutr., 93: 716–725.

LUMPKIN, T. A. and PLUCKNETT, D. L., 1980. Azolla: botany, physiology, and use as

a green manure. Econ. Bot., 34(2): 111-153.

LUMPKIN, T. A. and PLUCKNETT, D. L., 1982. Azolla as a green manure: use and

management in crop production. West view Trap. Agric. Ser., 5: 15, pp. 230

LUMPKIN, T. A. and PLUCKNETT, D. L., 1982. Azolla as a green manure: use and

management in crop production. Int. Rice Commi. News., 34: 23-24-25.

LUMPKIN, T. A., 1984. Assessing the potential for Azolla use in the humid tropics. Int.

Rice Commi. News., 33: 30- 33.

MADIBELA, O. R. and MODIAKGOTLA, E., 2004. Chemical composition and in

vitro dry matter digestibility of indigenous finger millet (Eleusine coracana) in

Botswana. Livestock Research for Rural Development, 16(4): 14

MANDAL, R. N., PANDEY, B. K., CHATTOPADHYAY, D. N. and

MUKHOPADHYAY, P. K., 2012. Azolla – an aquatic fern of significance to

small-scale aquaculture. Aquaculture Asia., 17 (1): 35-36

MARTINEZ,A. and CHURCH, D. C., 1990.Effect of mineral elements on in vitro rumen

cellulose digestion. J. Anim. Sci., 31: 982-990.

MENKE, K. H and STEINGASS, H., 1988. Estimation of energetic feed value obtained

from chemical analysis and in vitro gas production using rumen fluid. Anim. Res.

Devpt., 28: 7-55

MOORE, A. W., 1969. Azolla: Biology and agronomic significance. Bot. Rev., 35: 17-34.

http://www.lrrd.org/lrrd16/4/cont1604.htm

86

86

MUTZAR, A. J., SLINGER, S. J. and BURTON, J. H., 1976.Nutritive value of aquatic

plants for chicks. J. Poult. Sci., 55: 1917-1992.

NAGARATNA, B. and VINOD, K., 2013. Analysis of fodder status in Karnataka. Indian

J. Anim. Sci., 83(10): 1078-1083.

NIK-KHAH, A and MOTAGHI, M., 1992.The use of Azolla in lactating cows. Iranian J.

Agric. Sci., 23(1): 47-56.

NWANNA, L. C. and FALAYE, A. E., 1997. Substitution of Azolla meal for groundnut

cake diets for Nile tilapia, Oreochromis niloticus. Appl. Trop. Agri., 2: 139-143.

PANNAERKER, S., 1988. Azolla as a livestock and poultry feed. Livestock Adviser., 13:

22-26.

PARASHURAMULU, P. S. and NAGALAKSHMI, D., 2013. Protein fractionation and

in vitro digestibility of Azolla in ruminants. J. Anim. Feed Res., 3(3): 129-132.

PARNERKAR, S., PATEL, J. M., BHAGOJI, G. P., PATEL, A. P. and DAVE, F. D.,

1986. Voluntary intake and nutritive value of the sun dried water fern Azolla

pinnata in sheep. Indian J. Anim. Nutr., 3910: 37-39.

PARTHASARATHY, R., KADRIVEL, R. and KATHAPERUMAL, V., 2001. Chemical

evaluation of Azolla as a poultry feed ingredient. Indian Vet. J., 79: 144-146.

PILLAI, K. P., PREMALATHA, S. and RAJAMONY, S., 2002. Azolla: A sustainable

feed substitute for livestock. LEISA Magazine, India, 4: 15-17.

PRESTON, T. R., LAI, N. V. and RODRIGUEZ, L., 2000. In vitro gas production and

washing losses of tropical crop residues for ruminants and pigs. Livestock

Research for Rural Development.

87

87

QUERUBIN, L. J., ALCANTARA, P. F. and PRINCESA, A. O., 1986. Chemical

composition of three Azolla species (A. caroliniana, A.microphylla and

A. pinnata) and feeding value of azolla meal in broiler ration. Philippines J. Vet.

Anim. Sci., 12:65.

RAHAMAN, M. M., ALAM, M. R., AMIN, M. R. and DAS, N. G., 2010.Comparative

Study of the Nutritive Values of the different Varieties of Rice Straw. Bang. J.

Anim. Sci., 39(1&2): 75-82.

RAMACHANDRA, B., 1997. Effect of feeding urea ammoniated ragi straw on body

weight gain of crossbred heifers. Karnataka J. Agri. Sci., 10(1): 197-201.

RAMESH. H. 2008. Nutritional evaluation of Azolla (Azolla pinnata) and its

supplementary effect on in vitro digestibility of crop residues. M.V.Sc. Thesis,

Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar.

REDDY, G. R., 2007.Azolla- a sustainable feed substitute for livestock, Rural

Development Organisation, Andhra Pradesh, India.

SANGINGA, N and VAN HOVE, C., 1989. Amino acid Composition of Azolla as

affected by strains and population density. Plant and Soil.117: 263-267.

SARNKLONG, C., CONE, J. W., PELLIKAAN, W. and HENDRIKS, W. H.,.

Utilization of Rice Straw and Different Treatments to Improve its Feed Value for

Ruminants: A Review. Asian-Aust. J. Anim. Sci., 23(5): 680-692.

SINGH, P. K. and SUBUDHI, B. P. R., 1978. Utilization of Azolla in poultry feed.

Indian Farming.27: 37-39.

SINGH, P. K., 1982. The use of Azolla pinnata as a green manure for rice. Indian

Farming, 27: 19-20.

SNEDECOR, G. W and COCHRAN, W. G. 1994.Statistical methods. Oxford and IBH

Publishing Co., New Delhi.

88

88

SPENCER, R. R., BICKOFF, E. M., KOHLER, G. O. and MOTTLLA, A., 1969. Alfalfa

products by wet fractionation. ASAE paper., 69-179.

SREEMANNARAYANA, O., RAMACHAMDRAIAH, K., SUDARSHAN, K. M.,

RAMANAIAH, N. V. and RAMAPRASAD, J., 1993. Utilization of azolla as

rabbit feed. Indian. Vet. J., 70(3): 285-286.

SREERANGARAJUA, G., KRISHNAMOORTHY, U. and KAILAS, M. M.,

2000.Evaluation of Bengal gram (Cicer arietinum) husk as a source of tannin and

its interference in rumen and post-rumen nutrient digestion in sheep. Anim. Feed

Sci. Technol., 85: 131-138.

SRINIVAS, K. D., PRASAD, R. M. V., RAJA, K. K. and RAGHAVA, R. E., 2012.

Effect of Azolla (Azolla pinnata) based concentrate mixture on nutrient utilization

in buffalo bulls. Indian J. Ani. Nutr., 46(3): 268-271.

SUJATHA, T., KUNDU, A., JEYAKUMAR, S. and KUNDU, M. S., 2013. Azolla

supplementation: Feed cost benefit in duck ration in Andaman Islands. Tamilnadu

J. Vet. Ani. Sci.,9(2): 130-136.

TAKLIMI S., 1990. Utilization of Azolla microphylla in broiler feeding. MSc. Thesis,

University of Agricultural Sciences, Bangalore (India).

TAMANG, Y., SAMANTA, G., CHAKRABORTY, N. and MONDAL, L., 1993.

Nutritive value of Azolla (Azolla pinnata) and its potentiality of feeding in goats.

Environment and Ecology., 10: 755-756.

VAN HOVE, C. 1989. Azolla and its multiple uses with emphasis on Africa. FAO,

Rome. pp. 53.

VAN HOVE, C. and LEJEUNE, A., 2002. The Azolla-anabaena symbiosis. Biology and

Environment: Proceedings of the Royal Irish Academy., 102 B(1): 23-26.

89

89

VAN SOEST, P. J. and ROBERTTSON, J. B., LEWIS, B. A., 1991. Methods of dietary

fibre, neutral detergent fibre and non starch polysaccharides in relation to animal

nutrition. J. Dairy Sci., 74: 3583-3597.

VAN SOEST, P. J., 1994. Nutritional Ecology of the Ruminants. 2nd

Ed. Cornell

University Press, Ithaca, New York, USA.

WADHWANI, K. N., PARNERKAR, S., SAIYED, L. H. and PATEL, A. M., 2010.

Feedlot Performance of Weaner Lambs on Conventional and Non Conventional

Total Mixed Ration. Indian J. Anim. Res., 44(1): 16-21.

WAGNER, G. M., 1997. Azolla: A review of its biology and utilization. The Botanical

Review., 63(1): 1-26.

WILSON., 2008. Isolation and characterization of several cellulolytic bacteria from in

vitro rumen fermentations. J. Dairy Sci., 43: 217–222.

YANG, C. M. 2002. Response of forage fiber degradation by ruminal microorganisms to

branched-chain volatile fatty acids, amino acids, and dipeptides. J. Dairy Sci. 85:

1183-1190.

ZHANG, H., 2013.Effects of Branched-chain Amino Acids on In vitro Ruminal

Fermentation of Wheat Straw. Asian Australaus. J. Anim. Sci., 36: 264

AAbbssttrraacctt

90

90

VIII. ABSTRACT

The objective of the study was nutritional evaluation of Azolla pinnata and its

supplementary effect on the metabolizable energy (ME) and digestibility of the crop

residues and total mixed ration (TMR) by rumen in vitro gas production technique and

modified in vitro two stage digestion technique, respectively. Three different types of

diets were formulated using either crop residues alone or combination of crop residue and

Compounded feed mixture (CFM). The first type of diet comprised of only roughage

which was supplemented with 0%, 3%, 6%, and 9% azolla. The second type of diet

consisted of TMR, where roughage and CFM taken in the ratio of 60:40 and the CFM

portion was replaced with 0%, 3%, 6% and 9% azolla. The third type of diet consisted of

TMR, where roughage and CFM taken in the ratio of 60:40 and the roughage portion was

replaced with 0%, 3%, 6% and 9% azolla. The CP, CF, EE, TA, NFE (on %DMB)

contents of azolla were at a level of 21.66, 15.15, 4.41, 17.84 and 40.79, respectively.

The NDF, ADF, ADL and biogenic silica (on %DMB) content of azolla were found to be

54.86, 36.5, 24.03 and 3.34, respectively. Azolla was found to contain 1.64 % Ca, 0.34 %

Mg, 2.71 % K, 9.1 ppm Cu, 325 ppm of Zn, 1569 ppm of Fe, 8.11 ppm of Co, 5.06 ppm

of Cr, 2418 ppm of Mn, 31 ppm of Bo, 5.33 ppm Ni, 8.1 ppm of Pb, 1.2 ppm of Cd and

also rich in all essential amino acids. The in vitro dry matter digestibility (IVDMD) of

azolla was found to be 54.49 per cent and the ME content was found to be 5.44 MJ/kg

DM. There was no significant difference in the ME content and IVDMD in the diet

comprising crop residues (paddy straw, sorghum stover and bengal gram husk)

supplemented with azolla at different levels (0 to 9%) indicating no significant effect of

azolla supplementation on utilization of crop residues. There was significant decrease in

the ME content of the diet comprising crop residues (ragi straw, maize stover, maize

husk) supplemented with azolla at various levels (0 to 9%). There was decrease in the

IVDMD of the diet comprising ragi straw supplemented with azolla at 9% compared to

the control diet or TMR comprising CFM and ragi straw supplemented with azolla at 9%

compared to control diet indicating negative effect of supplementing azolla on

digestibility of crop residues and TMR. It is concluded that, azolla can only serve as a

source of protein, amino acids and minerals with limited or no supplementary effect on

digestibility or metabolizability of crop residues or total mixed ration.

AAppppeennddiicceess

91

APPENDIX I: Gas production data of Hay standard and Concentrate standard of 4 trials at different time intervals over 24 hours

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank Net gas in ml

(Total gas -Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Blank

28 39 2 4 11 11.66

TRIAL I

29 41 2 4.5 12

29 41 3 5 12

Hay

Standard

0.2016 0.9116 29 82 7 15 53 11.66 41.34 44.98 1.07

0.201 0.9116 29.5 81 6.5 15.5 51.5 11.66 39.84 43.48

0.204 0.9116 29.5 79.5 6.5 15.5 50 11.66 38.34 41.23

0 0 0 0

43.23

Concentrate

Standard

0.2006 0.9014 29.5 91.5 11.5 35.5 62 11.66 50.34 55.67

0.2037 0.9014 29 90 11.5 38 61 11.66 49.34 53.74

0.2055 0.9014 29 92 12 36 63 11.66 51.34 55.43

54.95

1

Blank

29.5 42 3 5 12.5 15.83

TRIAL II

28.5 46 3 7.5 17.5

29 46.5 4 8 17.5

3

Hay

Standard

0.2021 0.9116 29.5 87 6 15.5 57.5 15.83 41.67 45.23 1.07

0.2048 0.9116 29.5 87 6.5 15 57.5 15.83 41.67 44.63

0.2028 0.9116 29.5 85.5 5 14.5 56 15.83 40.17 43.45

0 0 0

44.44

Concentrate

Standard

0.2016 0.9014 30 95 12 34 65 15.83 49.17 54.11

0.2016 0.9014 29 95 12 35 66 15.83 50.17 55.21

0.2045 0.9014 29.5 94 10.5 35 64.5 15.83 48.67 52.8

54.04

Contd…

92

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200 mg

DM)

Correction

factor

Blank

30 48 3 5 18 17.83

TRIAL III

29 47 3 7.5 18

29.5 47 4 8 17.5

1

Hay

Standard

0.2011 0.9116 29 87 5.5 14 58 17.83 40.17 43.82 1.06

0.2008 0.9116 30 87.5 4.5 14 57.5 17.83 39.67 43.3

0.2006 0.9116 29.5 88 3.5 15.5 58.5 17.83 40.67 44.48

0 0 0

43.88

Concentrate

Standard

0.2003 0.9014 29.5 96 13.5 34.5 66.5 17.83 48.67 53.91

0.2007 0.9014 29.5 97.5 12.5 33.5 68 17.83 50.17 55.46

0.2005 0.9014 29.5 98 13.5 34 68.5 17.83 50.67 56.07

55.14

Blank

29 41 3.5 6 12 12.33

TRIAL IV

28.5 41 2 5.5 12.5

27.5 40 3 5 12.5

1

Hay

Standard

0.2004 0.9116 29.5 81 6 12 51.5 12.33 39.17 42.88 1.07

0.201 0.9116 30 81.5 5.5 11.5 51.5 12.33 39.17 42.75

0.201 0.9116 29.5 80.5 5 11 51 12.33 38.67 42.2

42.6

1

Concentrate

Standard

0.2009 0.9014 29 90.5 11 33.5 61.5 12.33 49.17 54.30

0.2003 0.9014 29.5 92 11.5 33.5 62.5 12.33 50.17 55.5

0.2002 0.9014 28.5 92 11 34 63.5 12.33 51.17 56.71

55.52

93

Appendix II: Gas production data of Azolla at different time interval over 24 hours

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

Production

(2hour)

Gas

Production

(5hour)

Gas

Production

(24hour)

Blank

Net gas in ml

(Total gas -

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

AZOLLA 0.2005 0.9652 31 63 3.5 8 32 12.33 19.67 20.32 1.07 21.75

0.2008 0.9652 30 60.5 3 7 31.5 12.33 19.17 19.78 1.07 21.16

0.201 0.9652 30.5 60 3.5 8 29.5 12.33 17.17 17.70 1.07 18.93

0.2004 0.9652 30 62 3.5 8.5 32.5 12.33 20.17 20.85 1.07 22.31

0.2009 0.9652 31 59 2.5 8 30.5 12.33 18.17 18.74 1.07 20.05

0.2012 0.9652 31 60 3 8.5 30 12.33 17.67 18.19 1.07 19.47

0.2002 0.9652 30 58 3.5 7.5 28 11.66 16.34 16.91 1.07 18.09

0.2038 0.9652 30.5 57 3 7.5 26.5 11.66 14.84 15.08 1.07 16.14

0.2030 0.9652 31 58.5 3.5 7.5 27.5 11.66 15.84 16.16 1.07 17.30

19.45

Calculation of Corrected net gas production of Azolla

Sample weight = 0.2005

Gas production for 24 h (ml) = 32 ml

Blank =12.33

Net gas (ml) = Total gas –Blank

= 32-12.33

= 19.67ml

Net gas / 200 mg DM = (19.67x 0.2)/0.2005

= 19.62ml

DM = 0.9652

Net gas / 200 mg DM = 19.62 / 0.9652

= 20.32

Correction factor = 1.07

Corrected net gas (ml) = 20.32 x 1.07

= 21.75ml

94

APPENDIX III: Gas production data of Crop residues at different time interval over 24 hours

Samples Weight of

Sample DM

Initial reading

Final reading

GP*

(2hour) GP

*

(5hour) GP

*

(24hour) Blank

Net gas in ml (Total gas Blank)

Net gas (ml/200 mg DM)

Correction factor

Corrected net gas

Paddy Straw

0.207 0.982 33 67 5.5 10 34 11.66 22.34 21.98 1.07 23.52 0.2072 0.982 29 63 5 8.5 34 11.66 22.34 21.96 1.07 23.5

0.2024 0.982 29 61 5 9 32 11.66 20.34 20.47 1.07 21.9

0.2015 0.982 29.5 66 4.5 9.5 36.5 15.83 20.67 20.89 1.07 22.35

0.205 0.982 29 66 6 13 37 15.83 21.17 21.03 1.07 22.5

0.2039 0.982 29 65 5 11 36 15.83 20.17 20.15 1.06 21.36

0.2026 0.982 29 67 3.5 7.5 38 17.83 20.17 20.28 1.06 21.49

22.37

Ragi Straw

0.2064 0.9647 30 73 6.5 18.5 43 11.66 31.34 31.48 1.07 33.68 0.2067 0.9647 29.5 74 8.5 15.5 44.5 11.66 32.84 32.94 1.07 35.24

0.2015 0.9647 30 74 7 14 44 11.66 32.34 33.27 1.07 35.6

0.2023 0.9647 28 77 10 20 49 17.83 31.17 31.94 1.06 33.86

0.2007 0.9647 29.5 78 10.5 20.5 48.5 17.83 30.67 31.68 1.06 33.58

34.39

Maize 0.2042 0.9897 28 67 4 8.5 39 11.66 27.34 27.06 1.07 28.95 Stover 0.2039 0.9897 28 68 4 9 40 11.66 28.34 28.09 1.07 30.05

0.2007 0.9897 29.5 69 4.5 9.5 39.5 11.66 27.84 28.03 1.07 29.99

0.2019 0.9897 29 77 6 11 48 17.83 30.17 30.2 1.06 32.01

0.2006 0.9897 28.5 74.5 5.5 11.5 46 17.83 28.17 28.38 1.06 30.08

30.22

Maize Husk

0.2002 0.9904 29 86 3 10 57 15.83 41.17 41.52 1.07 44.43 0.2024 0.9904 29 89 4 13 60 15.83 44.17 44.07 1.07 47.15

0.203 0.9904 29 88 4.5 13.5 59 15.83 43.17 42.94 1.07 45.95 0.2011 0.9904 29 90 3 11 61 17.83 43.17 43.35 1.06 47.21 0.2003 0.9904 30 91 2.5 10 61 17.83 43.17 43.52 1.06 45.46 46.04

Sorghum Stover

0.2029 0.9824 29.5 85 9 19 55.5 15.83 39.67 39.8 1.07 42.59 0.2044 0.9824 30 85 9 19.5 55 15.83 39.17 39.01 1.07 41.74

0.2081 0.9824 29.5 84 10.5 22 54.5 15.83 38.67 37.83 1.07 40.48 0.2028 0.9824 28.5 85 10.5 21.5 56.5 17.83 38.67 38.82 1.06 41.15 0.2032 0.9824 29 86 10 21.5 57 17.83 39.17 39.24 1.06 41.6 41.51

Bengal Gram Husk

0.2009 0.9857 28.5 85 6.5 15 56.5 15.83 40.67 41.08 1.07 43.95 0.2004 0.9857 28.5 87 5.5 14 58.5 15.83 42.67 43.2 1.07 46.23 0.2005 0.9857 29 88 6 14 59 17.83 41.17 41.66 1.06 45.7

0.201 0.9857 29 89 5.5 13 60 17.83 42.17 42.57 1.06 45.91 45.45

*GP = Gas production

95

Appendix IV: Gas production data of various diets comprising paddy straw supplemented with different levels of azolla from 0 to 9 per

cent and the TMR comprising paddy straw and CFM supplemented with different levels of azolla from 0 to 9 per cent.

Samples

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas

Blank)

Net gas

(ml/200 mg

DM)

Correction

factor

Corrected

net gas

T1R1 0.207 0.982 33 67 5.5 10 34 11.66 22.34 21.98 1.07 23.5

0.2072 0.982 29 63 5 8.5 34 11.66 22.34 21.96 1.07 23.5

0.2024 0.982 29 61 5 9 32 11.66 20.34 20.47 1.07 21.9

0.2015 0.982 29.5 66 4.5 9.5 36.5 15.83 20.67 20.89 1.07 22.4

0.205 0.982 29 66 6 13 37 15.83 21.17 21.03 1.07 22.5

0.2039 0.982 29 65 5 11 36 15.83 20.17 20.15 1.06 21.4

0.2026 0.982 29 67 3.5 7.5 38 17.83 20.17 20.28 1.06 21.5

0 0

22.4

T2R1 0.2059 0.9814 24 56 3 6.5 32 11.66 20.34 20.13 1.07 21.5

0.2089 0.9814 29.5 61 4.5 8.5 31.5 11.66 19.84 19.35 1.07 20.7

0.2035 0.9814 29 60 5 10 31 11.66 19.34 19.37 1.07 20.7

0.2009 0.9814 29 61 5 13.5 32 15.83 16.17 16.4 1.07 17.6

0.204 0.9814 27 67 6 13.5 40 15.83 24.17 24.15 1.07 25.8

0.2038 0.9814 29.5 68 3 9.5 38.5 17.83 20.67 20.67 1.06 21.9

0.2013 0.9814 29.5 68 4.5 10 38.5 17.83 20.67 20.93 1.06 22.2

0 0

21.5

T3R1 0.2012 0.9809 30 61 5 9.5 31 11.66 19.34 19.6 1.07 21

0.2078 0.9809 29 62 5 9 30 11.66 18.34 18 1.07 19.3

0.2096 0.9809 29 60 5 9.5 31 11.66 19.34 18.81 1.07 20.1

0.2012 0.9809 29.5 67 6.5 14 35.5 17.83 17.67 17.91 1.06 19

0.201 0.9809 30 69 6 13.5 39 17.83 21.17 21.47 1.06 22.8

0 0

20.4

T4R1 0.201 0.9804 29.5 60 4.5 8.5 30.5 11.66 18.84 19.12 1.07 20.5

0.2052 0.9804 29 61 5.5 10 32 11.66 20.34 20.22 1.07 21.6

0.2066 0.9804 30 61 4.5 10 31 11.66 19.34 19.1 1.07 20.4

0.2033 0.9804 28 64 6 13 36 15.83 20.17 20.24 1.07 21.7

0.2004 0.9804 29 65 5 12 36 15.83 20.17 20.53 1.07 22

1.07 21.2

Contd….

96

Samples

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas

Blank)

Net gas

(ml/200 mg

DM)

Correction

factor

Corrected

net gas

T5R1 0.2078 0.9816 29 80 10 20 51 11.66 39.34 38.57 1.07 41.3

0.2037 0.9816 29 80 9 19.5 51 11.66 39.34 39.35 1.07 42.1

0.2035 0.9816 27.5 78.5 8.5 18 51 11.66 39.34 39.39 1.07 42.1

0.2016 0.9816 29 85 11 23.5 56 17.83 38.17 38.58 1.06 40.9

0.2008 0.9816 29.5 86 11 23 56.5 17.83 38.67 39.24 1.06 41.6

41.6

T6R1 0.2023 0.9812 29 78 10 20 49 11.66 37.34 37.62 1.07 40.3

0.2025 0.9812 29 75 8.5 18 46 11.66 34.34 34.57 1.07 37

0.2056 0.9812 29 79 9 18 50 11.66 38.34 38.01 1.07 40.7

0.2005 0.9812 29.5 84 11 23 54.5 17.83 36.67 37.28 1.06 39.5

0.2041 0.9812 29 85 10.5 23.5 56 17.83 38.17 38.12 1.06 40.4

39.6

T7R1 0.2037 0.9807 29.5 76 8.5 18 46.5 11.66 34.84 34.88 1.07 37.32

0.2063 0.9807 29 76 10 20 47 11.66 35.34 34.94 1.07 37.38

0.2028 0.9807 29 77 9.5 19 48 11.66 36.34 36.54 1.07 39.1

0.2048 0.9807 29.5 82 10.5 23 52.5 17.83 34.67 34.52 1.06 36.6

0.2052 0.9807 29.5 85 11 24 55.5 17.83 37.67 37.44 1.06 39.68

38.02

T8R1 0.2077 0.9802 28.5 74 8.5 18.5 45.5 11.66 33.84 33.24 1.07 35.57

0.2046 0.9802 29.5 74 9 18.5 44.5 11.66 32.84 32.75 1.07 35.04

0.2012 0.9802 29 76 8.5 17 47 11.66 35.34 35.84 1.07 38.35

0.2045 0.9802 29.5 82 10.5 22.5 52.5 17.83 34.67 34.59 1.06 36.67

0.2026 0.9802 29.5 81 10.5 22 51.5 17.83 33.67 33.91 1.06 35.94

36.31

T9R1 0.2078 0.9816 29 80 5 10 51 11.66 39.34 38.57 1.07 41.27

0.2037 0.9816 29 80 5 10.5 51 11.66 39.34 39.35 1.07 42.1

0.2035 0.9816 27.5 78.5 6 10.5 51 11.66 39.34 39.39 1.07 42.15

0.2016 0.9816 29 85 4 9 56 17.83 38.17 38.58 1.06 40.89

0.2008 0.9816 29.5 86 4 9 56.5 17.83 38.67 39.24 1.06 41.59

Contd….

97

Samples

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas

Blank)

Net gas

(ml/200 mg

DM)

Correction

factor

Corrected

net gas

T10R1 0.2005 0.982 29.5 79 7 16.5 49.5 12.33 37.17 37.76 1.07 40.4

0.2011 0.982 29.5 81.5 6.5 16 52 12.33 39.67 40.18 1.07 42.99

0.2008 0.982 29 77.5 7.5 17.5 48.5 12.33 36.17 36.69 1.07 39.25

40.88

T11R1 0.2011 0.982 29.5 77.5 6.5 15.5 48 12.33 35.67 36.13 1.07 38.65

0.201 0.982 29.5 76 7.5 16.5 46.5 12.33 34.17 34.62 1.07 37.05

0.2015 0.982 29.5 77 8 16 47.5 12.33 35.17 35.55 1.07 38.04

37.91

T12R1 0.2008 0.982 29.5 79 6 16 49.5 12.33 37.17 37.7 1.07 40.34

0.2008 0.982 29.5 78.5 8 18 49 12.33 36.67 37.19 1.07 39.8

0.2005 0.982 29.5 76 6.5 16 46.5 12.33 34.17 34.71 1.07 37.14

37.01

98

Appendix V: Gas production data of various diets comprising Ragi straw supplemented with different levels of azolla from 0 to 9 per cent

and the TMR comprising paddy straw and CFM supplemented with different levels of azolla from 0 to 9 per cent.

Samples

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

Factor

Corrected

net gas

T1R2 0.2064 0.9647 30 73 6.5 18.5 43 11.66 31.34 31.48 1.07 33.7

0.2067 0.9647 29.5 74 8.5 15.5 44.5 11.66 32.84 32.94 1.07 35.2

0.2015 0.9647 30 74 7 14 44 11.66 32.34 33.27 1.07 35.6

0.2023 0.9647 28 77 10 20 49 17.83 31.17 31.94 1.06 33.9

0.2007 0.9647 29.5 78 10.5 20.5 48.5 17.83 30.67 31.68 1.06 33.6

34.4

T2R2 0.2072 0.9647 29.5 72 8.5 17 42.5 11.66 30.84 30.86 1.07 33

0.2041 0.9647 28 72 10 18.5 44 11.66 32.34 32.85 1.07 35.1

0.2026 0.9647 28.5 73 10.5 20.5 44.5 11.66 32.84 33.6 1.07 36

0.2027 0.9647 30 80 8.5 18.5 50 17.83 32.17 32.9 1.06 34.9

0.2024 0.9647 29.5 76.5 9.5 19.5 47 17.83 29.17 29.88 1.06 31.7

34.1

T3R2 0.2004 0.9647 28 65 8 16 37 11.66 25.34 26.21 1.07 28

0.2026 0.9647 27.5 64 4.5 10.5 36.5 11.66 24.84 25.42 1.07 27.2

0.2095 0.9647 29 68 4 9 39 11.66 27.34 27.06 1.07 28.9

0.2018 0.9647 29 72 9.5 18.5 43 17.83 25.17 25.86 1.06 27.4

0.2022 0.9647 29.5 74 10.5 20.5 44.5 17.83 26.67 27.35 1.06 29

28.1

T4R2 0.2091 0.9647 28.5 66 6 0.5 37.5 11.66 25.84 25.62 1.07 27.4

0.2055 0.9647 28 66 6 11 38 11.66 26.34 26.57 1.07 28.4

0.2075 0.9647 30 75 5 12 45 17.83 27.17 27.15 1.06 28.8

0.2021 0.9647 30 73 9 19 43 17.83 25.17 25.82 1.06 27.4

28

T5R2 0.2044 0.9713 29.5 85 11 14.5 55.5 17.83 37.67 37.95 1.06 40.2

0.2042 0.9713 28.5 87 9.5 20.5 58.5 17.83 40.67 41.01 1.06 43.5

0.2089 0.9713 25 78 14 25 53 11.66 41.34 40.75 1.07 43.6

0.2025 0.9713 28 81 11 28 53 11.66 41.34 42.04 1.07 45

0.2044 0.9713 28.5 81.5 12 27.5 53 11.66 41.34 41.65 1.07 44.6

43.4

Contd….

99

Samples

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

Factor

Corrected

net gas

T6R2 0.205 0.9708 29.5 85 7.5 18.5 55.5 17.83 37.67 37.86 1.06 40.1

0.2059 0.9708 30 87 10 21.5 57 17.83 39.17 39.19 1.06 41.5

0.2023 0.9708 28 79 9 24 51 11.66 39.34 40.06 1.07 42.9

0.2066 0.9708 29.5 82.5 13 23.5 53 11.66 41.34 41.22 1.07 44.1

42.2

T7R2 0.2037 0.9703 29.5 88 8.5 19 58.5 17.83 40.67 41.15 1.06 43.6

0.2048 0.9703 29 86 5.5 17 57 17.83 39.17 39.42 1.06 41.8

0.2033 0.9703 28 77 10 20 49 11.66 37.34 37.86 1.07 40.5

0.2003 0.9703 25 73 17 31.5 48 11.66 36.34 37.4 1.07 40

0.2088 0.9703 28 79 13.5 28 51 11.66 39.34 38.84 1.07 41.6

41.5

T8R2 0.202 0.9698 29 82 8.5 20 53 17.83 35.17 35.91 1.06 38.1

0.2016 0.9698 28 82.5 10 21 54.5 17.83 36.67 37.51 1.06 39.8

0.2023 0.9698 28.5 79.5 10 19 51 11.66 39.34 40.1 1.07 42.9

0.2092 0.9698 28.5 80 11 23.5 51.5 11.66 39.84 39.27 1.07 42

0.2055 0.9698 28.5 78 9.5 21.5 49.5 11.66 37.84 37.97 1.07 40.6

40.7

T9R2 0.2044 0.9713 29.5 85 5.5 13 55.5 17.83 37.67 37.95 1.06 40.2

0.2042 0.9713 28.5 69 7.5 15 58.5 17.83 40.67 41.01 1.06 43.5

0.2089 0.9713 25 78 10 17.5 53 11.66 41.34 40.75 1.07 43.6

0.2025 0.9713 28 81 6.4 15.5 53 11.66 41.34 42.04 1.07 45

0.2044 0.9713 28.5 81.5 6 14.5 53 11.66 41.34 41.65 1.07 44.6

43.4

T10R2 0.2007 0.9708 29.5 76 8.5 21 46.5 12.33 34.17 35.08 1.07 37.5

0.2007 0.9708 28.5 78 9.5 21 49.5 12.33 37.17 38.15 1.07 40.8

0.2006 0.9708 29 78.5 8 19.5 49.5 12.33 37.17 38.17 1.07 40.8

39.7

T11R2 0.2006 0.9703 28.5 76 7 18.5 47.5 12.33 35.17 36.14 1.07 38.7

0.2006 0.9703 28.5 79.5 8.5 21 46 12.33 33.67 34.6 1.07 37

37.8

T12R2 0.2005 0.9698 28.5 77.5 8.5 20.5 47 12.33 34.67 35.66 1.07 38.2

0.2009 0.9698 28.5 76 7.5 19 44.5 12.33 32.17 33.02 1.07 35.3

0.2006 0.9698 29 79 7.5 19 46 12.33 33.67 34.61 1.07 37

0 0 36.8

100

Appendix VI: Gas production data of various diets comprising Maize stover supplemented with different levels of azolla from 0 to 9 per

cent and the TMR comprising Maize stover and CFM supplemented with different levels of azolla from 0 to 9 per cent.

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

T1R3 0.2039 0.9897 28 68 4 9 40 11.66 28.34 28.1 1.07 30.05

0.2007 0.9897 29.5 69 4.5 9.5 39.5 11.66 27.84 28 1.07 29.99

0.2019 0.9897 29 77 6 11 48 17.83 30.17 30.2 1.06 32.01

0.2006 0.9897 28.5 74.5 5.5 11.5 46 17.83 28.17 28.4 1.06 30.08

30.22

T2R3 0.2062 0.9889 27 64 5 10 37 11.66 25.34 24.9 1.07 26.59

0.202 0.9889 28.5 65.5 3.5 8 37 11.66 25.34 25.4 1.07 27.15

0.2049 0.9889 28.5 64.5 2.5 7 36 11.66 24.34 24 1.07 25.71

0.2003 0.9889 29 72.5 3 8 43.5 17.83 25.67 25.9 1.06 27.47

0.2005 0.9889 28.5 73 4.5 9 44.5 17.83 26.67 26.9 1.06 28.52

27.09

T3R3 0.2038 0.9882 28.5 64 4.5 9 35.5 11.66 23.84 23.7 1.07 25.33

0.2078 0.9882 29.5 67 5 10 37.5 11.66 25.84 25.2 1.07 26.93

0.2023 0.9882 29 65.5 3 8 36.5 11.66 24.84 24.9 1.07 26.59

0.2009 0.9882 28.5 70.5 3.5 9.5 42 17.83 24.17 24.3 1.06 25.81

0.2005 0.9882 28 71.5 5 9 43.5 17.83 25.67 25.9 1.06 27.47

26.43

T4R3 0.2014 0.9874 29 64.5 5 11 35.5 11.66 23.84 24 1.07 25.65

0.2017 0.9874 29 65.5 3 8.5 36.5 11.66 24.84 24.9 1.07 26.69

0.2005 0.9874 29.5 65 4 8.5 35.5 11.66 23.84 24.1 1.07 25.77

0.2003 0.9874 29 70.5 5 11 41.5 17.83 23.67 23.9 1.06 25.37

0.2002 0.9874 29 72 5.5 11 43 17.83 25.17 25.5 1.06 26.99

26.1

T5R3 0.2079 0.9863 29 82 9.5 21 53 11.66 41.34 40.3 1.07 43.14

0.2048 0.9863 28.5 80.5 8.5 19.5 52 11.66 40.34 39.9 1.07 42.74

0.2015 0.9863 28 75 8 19 47 11.66 35.34 35.6 1.07 38.05

0.2045 0.9863 27.5 82.5 9.5 21 55 17.83 37.17 36.9 1.06 39.07

0.2056 0.9863 29.5 84 7.5 19 54.5 17.83 36.67 36.2 1.06 38.34

40.27

Contd….

101

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

T6R3 0.2038 0.9858 28.5 76 8.5 18.5 47.5 11.66 35.84 35.7 1.07 38.18 0.2026 0.9858 29.5 78.5 8.5 20.5 49 11.66 37.34 37.4 1.07 40.01 0.2033 0.9858 29 76.5 7.5 17.5 47.5 11.66 35.84 35.8 1.07 38.27 0.2 0.9858 28.5 84.5 10 19.5 56 17.83 38.17 38.7 1.06 41.04 0.2007 0.9858 28.5 84 9.5 20 55.5 17.83 37.67 38.1 1.06 40.36 39.57

T7R3 0.2009 0.9853 30 77 7 17.5 47 11.66 35.34 35.7 1.07 38.21 0.2036 0.9853 29.5 78 8.5 19.5 48.5 11.66 36.84 36.7 1.07 39.3 0.2005 0.9853 28.5 75.5 8.5 18.5 47 11.66 35.34 35.8 1.07 38.28 0.2001 0.9853 28.5 82 8.5 18 53.5 17.83 35.67 36.2 1.06 38.36 0.2009 0.9853 28.5 84.5 7.5 18 56 17.83 38.17 38.6 1.06 40.88 39

T8R3 0.2004 0.9848 30 75 7 16.5 45 11.66 33.34 33.8 1.07 36.15 0.2014 0.9848 29 76 8 18.5 47 11.66 35.34 35.6 1.07 38.13 0.2004 0.9848 30 78.5 7 16 48.5 11.66 36.84 37.3 1.07 39.95 0.2028 0.9848 28.5 82.5 7.5 16.5 54 17.83 36.17 36.2 1.06 38.39 0.203 0.9848 28.5 83.5 7.5 18 55 17.83 37.17 37.2 1.06 39.42 38.41

T9R3 0.2079 0.9863 29 82 4.5 17 53 11.66 41.34 40.3 1.07 43.14 0.2048 0.9863 28.5 80.5 7.5 18.5 52 11.66 40.34 39.9 1.07 42.74 0.2015 0.9863 28 75 9 20.5 47 11.66 35.34 35.6 1.07 38.05 0.2045 0.9863 27.5 82.5 9.5 21 55 17.83 37.17 36.9 1.06 39.07 0.2056 0.9863 29.5 84 6.5 18 54.5 17.83 36.67 36.2 1.06 38.34 40.27

T10R3 0.2005 0.9858 28.5 75 6 16 46.5 12.33 34.17 34.6 1.07 37 0.2005 0.9858 28 77 6.5 16.5 49 12.33 36.67 37.1 1.07 39.7 0.2009 0.9858 28.5 76 6 16.5 47.5 12.33 35.17 35.5 1.07 38 38.23

T11R3 0.2008 0.9853 28.5 75.5 7.5 18.5 45 12.33 32.67 33 1.07 35.34 0.2007 0.9853 29.5 76 7.5 19 47 12.33 34.67 35.1 1.07 37.52 0.2008 0.9853 28.5 77.5 7 17.5 49 12.33 36.67 37.1 1.07 39.66 37.51

T12R3 0.201 0.9848 28 76 7 18 48 12.33 35.67 36 1.07 38.56 0.2011 0.9848 29 74 6 15.5 45 12.33 32.67 33 1.07 35.3 0.2016 0.9848 28.5 76 7 18 44.5 12.33 32.17 32.4 1.07 34.68

0 0 36.18

102

Appendix VI1: Gas production data of various diets comprising Maize husk supplemented with different levels of azolla from 0 to 9 per

cent and the TMR comprising paddy straw and CFM supplemented with different levels of azolla from 0 to 9 per cent.

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

T1R4 0.2002 0.9904 29 86 3 10 57 15.83 41.17 41.52 1.07 44.43

0.2024 0.9904 29 89 4 13 60 15.83 44.17 44.07 1.07 47.15

0.203 0.9904 29 88 4.5 13.5 59 15.83 43.17 42.94 1.07 45.95

0.2011 0.9904 29 90 3 11 61 17.83 43.17 43.35 1.06 47.21

0.2003 0.9904 30 91 2.5 10 61 17.83 43.17 43.52 1.06 45.46

46.04

T2R4 0.2001 0.9896 28.5 89.5 4.5 13 61 17.83 43.17 43.6 1.06 46.22

0.2003 0.9896 29 89.5 3 11 60.5 17.83 42.67 43.05 1.06 45.64

0.2091 0.9896 29.5 90 2.5 10.5 60.5 15.83 44.67 43.17 1.07 46.2

0.207 0.9896 29 87.5 2 12 58.5 15.83 42.67 41.66 1.07 44.58

0.2016 0.9896 28.5 84 3 13 55.5 15.83 39.67 39.77 1.07 42.55

45.04

T3R4 0.2072 0.9888 28 86.5 4 11.5 58.5 15.83 42.67 41.65 1.07 44.57

0.2096 0.9888 29.5 87.5 3.5 10.5 58 15.83 42.17 40.69 1.07 43.54

0.2076 0.9888 29 87.5 3 11 58.5 15.83 42.67 41.57 1.06 44.07

0.2003 0.9888 28.5 86.5 2.5 12.5 58 17.83 40.17 40.56 1.06 43

0.2002 0.9888 28.5 88 3 12.5 59.5 17.83 41.67 42.1 1.06 44.63

43.96

T4R4 0.204 0.9881 28.5 85.5 4 10 57 17.83 39.17 38.86 1.06 41.2

0.2051 0.9881 28.5 87 3 9 58.5 17.83 40.67 40.14 1.06 42.54

0.2054 0.9881 29.5 82.5 3 10 53 15.83 37.17 36.63 1.07 39.19

0.2021 0.9881 29 82 2 9 53 15.83 37.17 37.23 1.07 39.83

T5R4 0.2073 0.9867 29 89.5 8 21.5 60.5 15.83 44.67 43.68 1.07 46.74

0.2008 0.9867 29 88 7.5 26 59 15.83 43.17 43.58 1.07 46.63

0.2027 0.9867 28.5 87.5 6.5 19 59 15.83 43.17 43.17 1.07 46.19

0.2062 0.9867 28.5 91.5 7.5 20.5 63 17.83 45.17 44.4 1.06 47.07

46.66

Contd….

103

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

T6R4 0.2002 0.9862 28 89.5 7 20.5 61.5 17.83 43.67 44.24 1.06 46.89

0.2009 0.9862 29 91.5 6 19.5 62.5 17.83 44.67 45.09 1.06 47.8

0.2057 0.9862 27 86.5 7 20.5 59.5 15.83 43.67 43.05 1.07 46.07

0.2048 0.9862 28.5 87 6 18.5 58.5 15.83 42.67 42.25 1.07 45.21

46.49

T7R4 0.2072 0.9857 29.5 91 9.5 24.5 61.5 15.83 45.67 44.72 1.07 47.85

0.2015 0.9857 29.5 88.5 11.5 27.5 59 15.83 43.17 43.47 1.07 46.51

0.2034 0.9857 29 89 8 20 60 17.83 42.17 42.07 1.06 44.59

0.2059 0.9857 29 90.5 10 28 61.5 17.83 43.67 43.03 1.06 45.62

1.06 46.14

T8R4 0.2038 0.9852 28 91 9 23.5 63 17.83 45.17 44.99 1.06 47.69

0.2026 0.9852 28.5 89.5 9 23.5 61 17.83 43.17 43.26 1.06 45.85

0.2073 0.9852 29.5 87.5 8.5 20.5 58 15.83 42.17 41.3 1.07 44.19

0.2041 0.9852 29.5 90.5 9 22 61 15.83 45.17 44.93 1.07 48.07

0.2019 0.9852 29 90.5 9 21.5 61.5 15.83 45.67 45.92 1.07 49.13

45.91

T9R4 0.2073 0.9867 29 89.5 8 21.5 60.5 15.83 44.67 43.68 1.07 46.74

0.2008 0.9867 29 88 7.5 26 59 15.83 43.17 43.58 1.07 46.63

0.2027 0.9867 28.5 87.5 6.5 19 59 15.83 43.17 43.17 1.07 46.19

0.2062 0.9867 28.5 91.5 7.5 20.5 63 17.83 45.17 44.4 1.06 47.07

46.66

T10R4 0.2006 0.9862 29 85 6 15 56 12.33 43.67 44.15 1.06 46.8

0.2009 0.9862 29 84 7 18 55 12.33 42.67 43.07 1.06 45.66

46.23

T11R4 0.2005 0.9857 28.5 83 6.5 18.5 54.5 12.33 42.17 42.68 1.06 45.24

0.2007 0.9857 29 86 5.5 16 57 12.33 44.67 45.16 1.06 47.87

0.2005 0.9857 29 85 6 18 56 12.33 43.67 44.19 1.06 46.84

46.13

T12R4 0.2005 0.9852 28.5 83 7.5 20 54.5 12.33 42.17 42.7 1.06 45.26

0.2006 0.9852 29 84.5 6.5 17 55.5 12.33 43.17 43.69 1.06 46.31

0.2006 0.9852 29 83 7 18.5 54 12.33 41.67 42.17 1.06 44.7

45.42

104

Appendix VIII: Gas production data of various diets comprising Sorghum stover supplemented with different levels of azolla

from 0 to 9 per cent and the TMR comprising Sorghum stover and CFM supplemented with different levels

of azolla from 0 to 9 per cent

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

T1R5 0.2029 0.9824 29.5 85 9 19 55.5 15.83 39.67 39.8 1.07 42.59

0.2044 0.9824 30 85 9 19.5 55 15.83 39.17 39.01 1.07 41.74

0.2081 0.9824 29.5 84 10.5 22 54.5 15.83 38.67 37.83 1.07 40.48

0.2028 0.9824 28.5 85 10.5 21.5 56.5 17.83 38.67 38.82 1.06 41.15

0.2032 0.9824 29 86 10 21.5 57 17.83 39.17 39.24 1.06 41.6

41.51

T2R5 0.2082 0.9818 29 82.5 9 19.5 53.5 15.83 37.67 36.86 1.07 39.44

0.2086 0.9818 29.5 82 8.5 18.5 52.5 15.83 36.67 35.81 1.07 38.32

0.2072 0.9818 30 83.5 8 18.5 53.5 15.83 37.67 37.04 1.07 39.63

0.2025 0.9818 29 82.5 8 18 53.5 17.83 35.67 35.88 1.06 38.04

0.2024 0.9818 29.5 83.5 8 17.5 54 17.83 36.17 36.4 1.06 38.59

1.06 38.8

T3R5 0.2005 0.9813 29.5 83.5 8.5 19.5 54 17.83 36.17 36.77 1.06 38.97

0.2033 0.9813 30 85 7.5 17.5 55 17.83 37.17 37.26 1.06 39.5

0.2064 0.9813 29.5 82 8.5 19 52.5 15.83 36.67 36.21 1.07 38.74

0.2021 0.9813 29 81.5 8 20 52.5 15.83 36.67 36.98 1.07 39.57

0.202 0.9813 29.5 83.5 7.5 19.5 54 15.83 38.17 38.51 1.07 41.21

1.07 39.6

T4R5 0.2002 0.9808 30 79.5 8 18.5 49.5 15.83 33.67 34.29 1.07 36.7

0.2012 0.9808 29.5 81.5 8 17.5 52 15.83 36.17 36.66 1.07 39.22

0.2005 0.9808 30 84.5 8 18 54.5 15.83 38.67 39.33 1.07 42.08

0.2031 0.9808 29.5 83.5 7.5 17.5 54 17.83 36.17 36.32 1.06 38.49

T5R5

(Control)

0.2022 0.9819 29.5 90.5 11 25 61 17.83 43.17 43.49 1.06 46.1

0.2038 0.9819 29 89.5 11.5 26 60.5 17.83 42.67 42.65 1.06 45.21

0.2008 0.9819 29.5 89.5 10 26.5 60 15.83 44.17 44.8 1.07 47.94

0.2079 0.9819 29 92.5 9 25 63.5 15.83 47.67 46.7 1.07 49.97

47.3

Contd….

105

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

T6R5 0.2003 0.9814 29.5 88.5 10 23.5 59 15.83 43.17 43.92 1.07 47

0.2058 0.9814 29.5 91.5 11 24.5 62 15.83 46.17 45.72 1.07 48.92

0.2033 0.9814 29.5 90.5 10 24.5 61 15.83 45.17 45.28 1.07 48.45

0.2027 0.9814 29.5 89.5 8.5 25 60 17.83 42.17 42.4 1.06 44.94

0.2015 0.9814 29.5 89.5 8.5 25 60 17.83 42.17 42.65 1.06 45.21

46.9

T7R5 0.2015 0.9809 28.5 88.5 10.5 25.5 60 17.83 42.17 42.67 1.06 45.23

0.2031 0.9809 28.5 87 10.5 24.5 58.5 17.83 40.67 40.83 1.06 43.28

0.2081 0.9809 28 89 11.5 24 61 15.83 45.17 44.26 1.07 47.36

0.2065 0.9809 30 93 10.5 21.5 63 15.83 47.17 46.57 1.07 49.84

0.2063 0.9809 29.5 86.5 10.5 22 57 15.83 41.17 40.69 1.07 43.54

0 0 45.85

T8R5 0.2045 0.9804 30 89 10 23.5 59 15.83 43.17 43.06 1.07 46.08

0.2003 0.9804 29.5 86.5 11 24.5 57 15.83 41.17 41.93 1.07 44.87

0.2035 0.9804 30 88 10 23 58 15.83 42.17 42.27 1.07 45.23

0.2002 0.9804 29.5 86.5 11.5 23.5 57 17.83 39.17 39.91 1.06 42.31

0.2002 0.9804 29 86 11 23 57 17.83 39.17 39.91 1.06 42.31

44.16

T9R5 0.2022 0.9819 29.5 90 11.5 22.5 60.5 17.83 42.67 42.98 1.06 45.56

0.2038 0.9819 29 89 12.5 24 60 17.83 42.17 42.15 1.06 44.68

0.2008 0.9819 29.5 90 12 23.5 60.5 15.83 44.67 45.31 1.07 48.48

0.2079 0.9819 29 92.5 -29 -29 63.5 15.83 47.67 46.7 1.07 49.97

92.5 0 0 47.17

T10R5 0.2023 0.9814 29.5 87.5 11.5 20.5 58 15.83 42.17 42.48 1.07 45.45

0.2032 0.9814 29.5 90 11 21 60.5 15.83 44.67 44.8 1.07 47.94

0.2012 0.9814 29.5 88.5 11 17.5 59 15.83 43.17 43.73 1.07 46.79

46.73

T11R5 0.2043 0.9809 28.5 91 11.5 22.5 62.5 17.83 44.67 44.58 1.06 47.26

0.2011 0.9809 28.5 88.5 11 21 60 17.83 42.17 42.76 1.06 45.32

0.2012 0.9809 28 86 10.5 21 58 15.83 42.17 42.73 1.07 45.73

46.1

T12R5 0.2031 0.9804 30 86.5 10 18.5 56.5 15.83 40.67 40.85 1.07 43.71

0.2002 0.9804 29.5 87.5 8.5 18.5 58 15.83 42.17 42.97 1.07 45.98

0.2023 0.9804 30 89.5 8 19 59.5 15.83 43.67 44.04 1.07 47.12

45.6

106

Appendix IX: Gas production data of various diets comprising Bengal gram husk supplemented with different levels of azolla

from 0 to 9 per cent.

Sample

Weight

of

Sample

DM Initial

reading

Final

reading

Gas

production

(2hour)

Gas

production

(5hour)

Gas

production

(24hour)

Blank

Net gas in ml

(Total gas –

Blank)

Net gas

(ml/200

mg DM)

Correction

factor

Corrected

net gas

T1R6

0.2009 0.9857 28.5 85 6.5 15 56.5 15.83 40.67 41.08 1.07 43.95

0.2004 0.9857 28.5 87 5.5 14 58.5 15.83 42.67 43.2 1.07 46.23

0.2005 0.9857 29 88 6 14 59 17.83 41.17 41.66 1.06 45.7

0.201 0.9857 29 89 5.5 13 60 17.83 42.17 42.57 1.06 45.91

45.45

T2R6 0.2004 0.985 29 86 6 13.5 57 17.83 39.17 39.69 1.06 42.07

0.2005 0.985 29 85 5 12.5 56 17.83 38.17 38.65 1.06 40.97

0.2012 0.985 29 87 3.5 11 58 15.83 42.17 42.56 1.07 45.54

0.2004 0.985 29 88 5 11.5 59 15.83 43.17 43.74 1.07 46.8

0.2007 0.985 28.5 85.5 5.5 12 57 15.83 41.17 41.65 1.07 44.57

44.99

T3R6 0.2012 0.9844 28.5 85 5.5 12 56.5 15.83 40.67 41.07 1.07 43.94

0.2029 0.9844 28 84 7 15.5 56 15.83 40.17 40.22 1.07 43.04

0.2025 0.9844 28.5 86.5 5.5 13.5 58 15.83 42.17 42.31 1.07 45.27

0.2007 0.9844 29.5 89.5 3.5 11.5 60 17.83 42.17 42.69 1.06 45.25

0.2003 0.9844 29.5 90 5.6 13 60.5 17.83 42.67 43.28 1.06 45.88

44.68

T4R6 0.2005 0.9838 28 85.5 7 15.5 57.5 17.83 39.67 40.22 1.06 42.64

0.2009 0.9838 28 87 5 13 59 17.83 41.17 41.66 1.06 44.16

0.2000 0.9838 29 84.5 7 15.5 55.5 15.83 39.67 40.32 1.07 43.15

0.2009 0.9838 27.5 85 6.5 15.5 57.5 15.83 41.67 42.17 1.07 45.12

0.2004 0.9838 29.5 87 5 14 57.5 15.83 41.67 42.27 1.07 45.23

44.06

NUTRITIONAL EVALUATION OF AZOLLA...ii nutritional evaluation of azolla (azolla pinnata) and its...

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