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The Journal of Basic & Applied Zoology (2015) 70, 1–15
HO ST E D BYThe Egyptian German Society for Zoology
The Journal of Basic & Applied Zoology
www.egsz.orgwww.sciencedirect.com
Comparing the effectiveness of egg disinfectants
against bacteria and mitotic indices of developing
chick embryos
* Corresponding author at: Department of Zoology, Faculty of
Science, University of Alexandria, Alexandria 21511, Egypt.
E-mail address: [email protected] (G.M. Bekhet).1 Current address: Department of Biology, Faculty of Science,
King Faisal University, P.O. Box 1759, Al Hofuf 31982, Al Hasa,
Saudi Arabia. Tel.: +966 3 5800000x1858, mobile: +966 0569323209;
fax: +966 3 5886437, +966 3 5886439.
Peer review under responsibility of The Egyptian German Society for
Zoology.
http://dx.doi.org/10.1016/j.jobaz.2014.12.0052090-9896 ª 2015 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H.S. Zeweila, R.E. Rizk
b, G.M. Bekhet
c,1,*, Mona R. Ahmedb
a Anim. and Fish Prod. Dept., Faculty of Agric., Saba Basha, Alex. Univ., Egyptb Anim. Prod. Res. Inst., Agric. Res. Center, Giza, Egyptc Zoology Dept., Faculty of Sci., Alex. Univ., Egypt
Received 29 March 2014; revised 30 November 2014; accepted 2 December 2014Available online 23 February 2015
KEYWORDS
Chemical disinfectants;
Natural disinfectants;
Antibacterial activity;
Malformation;
Chick embryos;
Mitotic indices
Abstract Total bacterial counts on hatching eggshell surface were significantly (P < 0.05) reduced
as a result of using all disinfectants with different concentrations and formaldehyde fumigation
treatments compared with those for eggs before treatment except for those subjected to water only
which are considered as control with water. Chemical disinfectants significantly reduced the eggshell
total bacterial count from 7.07 Logs to 2.41 Logs with 65.9% reduction and decreased again to
1.96 Logs with 72.3% reduction before setting in the incubator. Also, natural disinfectants sig-
nificantly reduced the total bacterial count from 7.0 Logs to 1.86 Logs with 73.7% reduction and
decreased again to 1.34 Logs with 81% reduction before setting in the incubator. Whereas, treat-
ment with formaldehyde fumigation significantly reduced the bacterial count from 7.07 Logs to
2.53 Log with 64.2% reduction, but the bacterial count had increased numerically again during
storage and before setting in the incubator to 4.20 Logs. Chemical disinfectant effects on developing
chick embryos resulted in retarded growth as reflected by malformed limbs and beaks and muscle
weakness was seen in a few hatched chicks. The mitotic indices of the spinal cord for chicks from
egg treated by cumin 0.2% at 3rd and 4th day of age are slightly higher being 5.5% and 4.8%
respectively, than those for other treatment and control groups. The mitotic index revealed that
there was a significant (P < 0.05) difference between all disinfection and control groups on days
4, 7 and 10 of incubation with respect to skin systems, whereas skin system of newly hatched chicks
did not demonstrate any significant differences between mitotic indices of experimented groups.
2 H.S. Zeweil et al.
Mitotic indices of embryonic dermal system on days 4 and 10 of incubation were slightly higher for
natural disinfectant (being 4.7 and 0.1) compared with those for the chemical disinfectant (being 4
and 0.6), formaldehyde fumigation (being 3 and 0.4) and control group (being 4 and 0.9).
ª 2015 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Disinfectants are an essential part of infection control practicesand aid in the prevention of disease outbreaks on farms(Dvorak, 2005). Currently there are over 5000 antimicrobialproducts registered with the Environmental Protection Agency
as ‘‘antimicrobial pesticides’’, which are substances or mixturesof substances used to destroy or suppress the growth of harm-ful. Many parent compounds have been made more effective,
stable and less irritating by the addition of other chemicalgroups. Therefore, it is not appropriate to generalize the activ-ity of a parent compound such as iodine or phenol, upon the
commercial derivatives available (Ralph, 2003). There aremany disinfectants to choose but they basically fall into afew categories based on the active ingredients and abilities tokill different micro-organisms. Alcohol compounds are fast
acting and highly effective against both Gram positive andGram negative bacteria but have no residual activity such as,ethyl alcohol (ethanol, alcohol), isopropyl alcohol (iso-
propanol, propan-2-01) and n-propanol (McDonnell andRussell, 1999; Dvorak, 2005; Ewart, 2001; Turpin, 2013).Formaldehyde (CH2CO, formalin, formol) is commonly used
as a disinfectant, as it is cheap, not corrosive, and kills mostbacteria and fungi (including their spores) (Acklund et al.,1980; Williams, 1969; Russell, 1976; Cadirci, 2009). Chlorhex-
idine compounds can kill microorganisms by damaging outercell layers (McDonnell et al., 1999; Quinn, 2001). Sodium chlo-ride is reported for wide antibacterial activity and low toxicitytoward man and animal (Grooms, 2003). Virkon-S was essen-
tially ineffective against the inoculated microorganisms (Scottand Swetnam, 1993b). Hydrogen peroxide also, has been usedas a satisfactory disinfectant for inanimate materials (Scott
and Swetnam, 1993a; Sheldon and Brake, 1991; Mansour,2001; Sullivan and Krieger, 1992). Researchers have beeninterested in biologically active compounds isolated from plant
species for the elimination of pathogenic microorganismsbecause of the resistance that microorganisms have built again-st antibiotics (Nychas, 1995; Essawi and Srour, 2000; Singh
et al., 2001). The essential oil of oregano has anti-bacterial(Baydar et al., 2004; Vagi et al., 2005), anti-oxidant(Gouladis et al., 2003; Tepe et al., 2004), anti-fungal (Mulleret al., 1995; Bouchra et al., 2003), cytotoxic (Sivropoulou
et al., 1996; Wilson et al., 1997), insecticidal (Traboulsi et al.,2002) and nematicidal properties (Oka et al., 2000), the cuminseed contains powerful compounds. These natural constituents
possess a remarkable antioxidant, antitoxic, anti-microbial,anti fungal, anti-parasitic, anti-spasmodic and diuretic actions(Tepe et al., 2005). Sanitizers and disinfectants that are most
critical to the normal development of the embryo are thosethat occur before and during incubation and hatchingprocesses (Wilson, 1991; Meijerhof, 2000). The magnitude ofthe mitotic index is a reliable indication of the rate of cell pro-
liferation. As a general rule, when the mitotic index is high,
proliferation is rapid and when it is low, the birth rate of cellsis also low. El-Zayat (1974) and Michael et al. (1991) reported
that during organogenesis the mitotic index is high at the timeof proliferation and drops sharply by the onset of cellular dif-ferentiation. Rizk (1994) also concluded that the cell division
of the nervous system could be affected by the egg abnor-malities and then the survival and development of the embryosand finally hatching power. The present study was carried out
to investigate the effect of chemical and natural egg disinfec-tions against bacteria, embryonic development, embryonicmitotic indices in Bandarah local strain.
Materials and methods
A total of 1442 hatched eggs from Bandarah chicken strainwere used in this experiment. Hatched eggs were divided into
two divisions: firstly, forty two hatched eggs for bacterialcount, secondly, 1400 eggs for embryonic inspection. The eggsfor each division were divided into 14 groups, which represent
the disinfectants used (Tables 1a and 1b), as disinfectants fromchemical sources and others from natural sources with theircombinations, formaldehyde fumigation and control groups.
Each group for studying the development of chick embryoscontains 100 developing and hatching eggs.
Egg from this group was treated with formaldehyde fumi-
gation (triple strength), approximately 1 g potassium perman-ganate (KMnO4) to 2 ml formalin (CH2CO) per 1 m3. Triplestrength formaldehyde gas was produced inside the setter for20 min (USDA, 1985). For embryonic study, three incubated
eggs from Bandarah chicken strain were selected randomlyrepresenting each trial of experiment. Each egg was weighedand opened on days 3, 4, 7, 10, 13 and 21, then the embryos
were separated from the remaining egg contents. Three devel-oping embryos at each day of the preceding days of incubationfor each experimental and control groups were used for deter-
mination of morphological examination and mitotic index askinetic parameter of the cell cycle in two regions of the nervoussystem and skin .The region of the nervous system was thespinal cord. For histological preparation and studying,
embryos were rinsed in saline water and fixed in Bouin’s fluidfor 24 h as described in the method of Gabe (1976). Fixationconsists of the following compositions: saturated aqueous
solution of picric acid 100 parts, formaldehyde solution 25%parts and five parts of glacial acetic acid were added promptlybefore using. This solution acts as a fixative and/or preserva-
tive. After fixation, embryos were thoroughly washed with70% ethyl alcohol. Then they were dehydrated through anascending series of alcohol then cleared in xylene and embed-
ded in paraffin. Paraffin blocks were treated, fixed over theblock holder of the microtome and serially sectioned at 4and 5 u. The obtained paraffin ribbons containing the serialsections were cut into pieces of 5 cm long and mounted over
a slide placed over a hotplate adjusted at 40 �C. The mounted
Table 1a Disinfectant types.
Disinfectants Active ingredient Effect
Hydrogen peroxide (H2O2) 50% Oxygen (O) Killing of aerobic and Gram-positive bacteria,
Gram-negative bacteria, yeasts, and
antibiotic-resistant bacteria
Sodium chloride (NaCl) Chlorine (Cl2) Antimicrobial and a preservative
Betadine Povidone iodine (C6H9I2NO) Killing of aerobic and aerobic Gram-positive
bacteria, Gram-negative bacteria, yeasts, and
antibiotic-resistant bacteria
Virkon S Potassium peroxymonosulfate 0.4% sodium
chloride 1.5%
Viruses, Gram positive and Gram negative
bacteria, fungi (molds and yeasts), and
mycoplasma
Origanum vulgare L. (Oregano) b-Pinene (0.5%) – thymol (0.86%), terpinene and
p-cymene–carvacrol (57.01%)
Killing microorganism, anti-oxidant,
anti-bacterial and anti-fungal
Cuminum cyminum (Cumin) Cumin aldehyde (p-isopropyl-benzaldehyde,
25–35%), perilla aldehyde, cumin alcohol,
a- and b-pinene (21%), dipentene, p-cymene
and b-phellandrene
Antioxidant, antitoxic, anti-microbial,
anti-fungal, anti-parasitic, anti-spasmodic
and diuretic actions
Table 1b Disinfectant concentration.
Treatment used Concentration Method of application
Control untreated No –
Control with water To make sure that there is any synergistic effect or not. Because, H2O2,
NaCl, Virkon betadine have been solved in the water
Dipping
Control with alcohol 125 ml ethanol alcohol per liter taped water to make sure that there is any
synergistic effect or not, because, oil has been solved in the alcohol
Dipping
Sodium chloride 10% per liter taped water Dipping
Hydrogen peroxide 5% per liter taped water Dipping
Betadine 2% per liter taped water Dipping
Virkon S 0.5% per liter taped water Dipping
Oregano 0.2% 125 ml alcohol + 0.2% oregano per liter taped water Dipping
Oregano 0.4% 125 ml alcohol + 0.4% oregano per liter taped water Dipping
Cumin 0.2% 125 ml alcohol + 0.2% cumin per liter taped water Dipping
Cumin 0.4% 125 ml alcohol + 0.4% cumin per liter solution Dipping
Oregano 0.1%+ cumin 0.1% 125 ml alcohol + 0.1% oregano + 0.1% cumin per liter taped water Dipping
Oregano 0.2%+ cumin 0.2% 125 ml alcohol + 0.2 ml oregano + 0.2 ml cumin per liter taped water Dipping
Effect of egg disinfectants against bacteria and mitotic indices of chick embryos 3
sections were allowed to dry completely in an oven adjusted at40 �C for 1 week. After complete dryness and sticking of the
sections over the slides, it was taken off the incubator and sub-jected to the staining process, then dehydrated, cleared,mounted in balsam and glass slip. The mounted stained sec-
tions are placed in oven at 40 �C to ensure complete drynessbefore microscopic examination and determination of themitotic index. At least 1000 cells from the organ under inves-
tigation in each embryo were counted (Overton, 1958). Alsothe mitotic figure within these 1000 cells was counted andthe mitotic index was calculated according the formula ofDondua et al. (1966) which is: the statical analysis conducted
using the SAS program (SAS, 1998) software, the followingmodel was used: Yij = M + Li + eij where Yij = observationrecord, M = the overall mean, Li = is the effect of disinfec-
tion, i= 1–14 and eij = is the random error. Mean differenceswere separated by Duncan New Multiple range test (Duncan,1955).
Results
Effect of hatching egg disinfection on the total bacterial count on
eggshell surface
Data obtained concerning effect of hatching egg disinfectionon total bacterial count of eggshell surface are shown inTable 2. Results demonstrated in this table reveal that totalbacterial counts on hatching eggshell surface were significantly
(P< 0.05) reduced as a result of using all disinfectants withdifferent concentrations and formaldehyde fumigation treat-ments compared with bacterial count before egg treatment
except for eggs subjected with water which are considered ascontrol with water. The same effect of this significant reduc-tion on bacterial count was continued for all groups with
one exception for the formaldehyde fumigation group whichrecorded numerical change in bacterial count after treatmentsand before setting the eggs in the incubator. Also, there were
Table 2 Effect of hatching egg disinfection on total bacterial eggshell count (cfu per egg) (log ± SE).
Treatment Total bacterial count
Before treatment After treatment Before setting in the incubator Significant
Control with water 7.07 ± 1.34 6.83 ± 1.01a 7.33 ± 0.88a N.S
Control with alcohol 7.07 ± 1.34A 2.57 ± 0.18bB 1.96 ± 0.03cB *
Formaldehyde fumigation 7.07 ± 1.34A 2.53 ± 0.29bB 4.20 ± 0.40bAB *
Sodium chloride 10% 7.07 ± 1.34A 2.33 ± 0.14bB 2.00 ± 0.06cB *
Hydrogen peroxide 5% 7.07 ± 1.34A 2.20 ± 0.06bB 2.13 ± 0.13cB *
Betadine 2% 7.07 ± 1.34A 2.73 ± 0.12bB 1.53 ± 0.78cB *
Virkon S 0.5% 7.07 ± 1.34A 2.30 ± 0.3.5bB 2.00 ± 0.58cB *
Oregano 0.2% 7.07 ± 1.34A 1.80 ± 0.15bB 1.70 ± 0.06cB *
Oregano 0.4% 7.07 ± 1.34A 2.00 ± 0.01bB 1.00 ± 0.58cB *
Cumin 0.2% 7.07 ± 1.34A 1.83 ± 0.22bB 1.63 ± 0.09cB *
Cumin 0.4% 7.07 ± 1.34A 1.73 ± 0.18bB 1.30 ± 0.65cB *
Oregano + cumin 0.1% 7.07 ± 1.34A 2.00 ± 0.10bB 1.03 ± 0.51cB *
Oregano + cumin 0.2% 7.07 ± 1.34A 1.80 ± 0.10bB 1.40 ± 0.06cB *
Significant N.S * *
a,b,cMeans within each column for each item with different superscripts are significantly different (P < 0.05).A,BMeans within each row for each item with different superscripts are significantly different (P < 0.05).
N.S: non significant.* Significant (P < 0.05).
Table 3 Effect of hatching egg disinfectants from chemical and natural sources and formaldehyde fumigation on total bacterial
eggshell count (cfu per egg) (log ± SE).
Treatment Total bacterial count
Before treatment After treatment Before setting in the incubation Significant
Average of chemical disinfectants 7.07 ± 1.34A 2.41 ± 0.10aB 1.96 ± 0.19bB *
Average of natural disinfectants 7.07 ± 1.34A 1.86 ± 0.06bB 1.34 ± 0.16bB *
Formaldehyde fumigation 7.07 ± 1.34A 2.53 ± 0.29aB 4.20 ± 0.40aAB *
Significant N.S * *
a,bMeans within each column for each item with different superscripts are significantly different (P< 0.05).A,BMeans within each row for each item with different superscripts are significantly different (P < 0.05).
N.S: non significant.* Significant (P < 0.05).
4 H.S. Zeweil et al.
no significant differences between the total bacterial counts onall egg groups subjected to disinfection, whereas all counts ofegg groups were significantly (P < 0.05) decreased compared
with those for the control with water groups. Also, this obser-vation with the same significant difference was detected for alltreatment groups compared with control plus water for thebacterial count before incubation.
Effects of hatching egg disinfectants from different sourcesand formaldehyde fumigation on total bacterial count for egg-shells are shown in Table 3. Treatment of the eggs with chemi-
cal disinfectant as average, significantly (P < 0.05) reduced thetotal bacterial count from 7.07 Log to 2.41 Log with 65.9% ofreduction and decreased again to 1.96 Log with 72.3% before
setting in the incubator. Also, treatment of the eggs with nat-ural disinfectant as average, significantly (P < 0.05) reducedthe total bacterial count from 7.07 Log to 1.86 Log with73.7% reduction and decreased again to 1.34 Log with 81%
reduction before setting in the incubator. Whereas, treatmentwith formaldehyde fumigation significantly (P < 0.05)reduced the bacterial count from 7.07 Log to 2.53 Log with
64.2% reduction, but the count increased numerically againduring storage at 4.2 Log before setting in the incubator.
Moreover, total bacterial count after treatment was sig-nificantly (P < 0.05) reduced for the natural disinfectantgroup compared with those for chemical and formaldehyde
fumigation groups, while there was no significant differencebetween bacterial count of chemical and formaldehyde fumiga-tion groups. Bacterial count for the formaldehyde fumigationgroup before setting in the incubator was significantly
(P < 0.05) increased (4.20 Log) compared with those for disin-fectants either for chemical (1.96 Log) or natural (1.34 Log),while the significant difference between chemical and natural
disinfectants was not detected. This conclusion confirms theresults of the previous table which demonstrates that formalde-hyde fumigation has not possessed a residual effect, therefore
the bacterial count increased during storage compared withthe residual effect of chemical and natural disinfectants.
Embryonic weight
Table 4a represents the effect of hatching egg disinfectantsfrom natural and chemical sources compared with the
formaldehyde fumigation and control group on absolute andrelative embryonic weights at different ages during incubation.
Table
4a
Effectofhatchingeggdisinfectiononabsolute
andrelativeem
bryonic
weights
atdifferentages
(2nd–5th
days)
duringincubation(X
±E).
Treatm
ent
Embryonic
weight
2day
3day
4day
5day
g%
g%
g%
g%
Controluntreated
0.023±
0.004f
0.048±
0.009de
0.074±
0.006fg
0.136±
0.011fg
0.284±
0.010de
0.553±
0.022cd
0.540±
0.016de
1.094±
0.020cd
Controlwithwater
0.023±
0.001ef
0.048±
0.002de
0.093±
0.007ef
0.179±
0.015ef
0.284±
0.006de
0.514±
0.011de
0.583±
0.003d
1.059±
0.012d
Controlwithalcohol
0.018±
0.002h
0.036±
0.002fg
0.116±
0.005de
0.244±
0.013cd
0.265±
0.001ef
0.481±
0.028ef
0.593±
0.011cd
1.170±
0.020bc
Form
aldehydeFumigation
0.018±
0.001h
0.033±
0.004g
0.063±
0.005g
0.113±
0.009g
0.161±
0.004h
0.335±
0.014g
0.465±
0.037f
0.906±
0.070e
Sodium
chloride
0.011±
0.003i
0.022±
0.006h
0.073±
0.011f
0.155±
0.021fg
0.236±
0.007g
0.449±
0.011f
0.490±
0.010ef
0.948±
0.018e
Hydrogen
peroxide
0.022±
0.002fg
0.043±
0.004ef
0.089±
0.010efg
0.169±
0.018ef
0.276±
0.011def
0.561±
0.029cd
0.564±
0.023d
1.103±
0.036cd
Betadine
0.019±
0.003gh
0.036±
0.007fg
0.112±
0.006de
0.199±
0.008de
0.253±
0.003fg
0.517±
0.004de
0.573±
0.009d
1.179±
0.007cd
VirkonS
0.019±
0.001gh
0.039±
0.003fg
0.117±
0.007de
0.202±
0.012de
0.266±
0.002ef
0.529±
0.007de
0.594±
0.031cd
1.175±
0.057bc
Oregano0.2%
0.028±
0.001c
0.059±
0.006c
0.126±
0.016cd
0.236±
0.031cd
0.298±
0.007cd
0.581±
0.019bc
0.645±
0.014bc
1.232±
0.025b
Oregano0.4%
0.037±
0.002a
0.076±
0.004a
0.172±
0.001a
0.330±
0.007a
0.327±
0.010a
0.625±
0.018ab
0.754±
0.006a
1.480±
0.001a
Cumin
0.2%
0.026±
0.001cde
0.051±
0.001d
0.155±
0.005abc
0.281±
0.011abc
0.320±
0.004abc
0.593±
0.014bc
0.772±
0.017a
1.562±
0.018a
Cumin
0.4%
0.024±
0.007def
0.046±
0.014de
0.136±
0.008bcd
0.259±
0.017bc
0.300±
0.007bcd
0.659±
0.010a
0.650±
0.001b
1.235±
0.004b
Oregano+
Cumin
0.1%
0.027±
0.004cd
0.052±
0.007d
0.127±
0.006cd
0.233±
0.013cd
0.324±
0.009ab
0.625±
0.021ab
0.747±
0.024a
1.486±
0.028a
Oregano+
Cumin
0.2%
0.033±
0.006b
0.067±
0.014b
0.163±
0.020ab
0.310±
0.033ab
0.312±
0.007abc
0.591±
0.008bc
0.772±
0.014a
1.532±
0.021a
Significant
**
**
**
**
a,b,c,d,e,f,g,h,iMeanswithin
each
columnforeach
item
withdifferentsuperscripts
are
significantlydifferent(P
<0.05).
*Significant(P
<0.05).
Effect of egg disinfectants against bacteria and mitotic indices of chick embryos 5
Results reveal that averages for either absolute or relativeembryonic weight produced from egg treated with oregano0.4% aged 2 days were significantly (P < 0.05) greater
than those for other treated groups followed by those fororegano + cumin 0.2% with the same significant difference.The same egg group, which disinfected with oregano 0.4% rep-
resented the highest significant (P < 0.05) increase of absolute(0.172 g) and relative (0.33%) embryonic weights on the thirdday of incubation compared to those for other treated egg
groups except that for cumin 0.2% and oregano + cumin0.2% groups. Generally, the lowest significant (P < 0.05) abso-lute and relative embryonic weights on the third day of incuba-tion were detected in formaldehyde fumigation, sodium
chloride, hydrogen peroxide and control groups for untreatedeggs and those subjected to water compared with other treatedgroups. Nearly, the same trends of increasing or decreasing
absolute and relative embryonic weights on second and thirddays of incubation were recorded in the fourth day of incuba-tion. On the fourth day of incubation, the heaviest significant
(P< 0.05) embryonic weight by gram was recorded for thegroup treated with oregano 0.4% compared with other treatedgroups besides numerical increase compared with those for
cumin 0.2% and oregano + cumin 0.1% and oregano +cumin 0.2%. Generally relative embryonic weight on the sameday of incubation was significantly (P < 0.05) increased forgroup of cumin 0.4% followed by those of oregano 0.4% and
oregano + cumin 0.1% compared with other groups. The low-est significant (P < 0.05) records of absolute and relativeembryonic weights on the fourth day of incubation were for
the formaldehyde fumigation group compared with those forother experimented groups. On the fifth day of incubation, ore-gano 0.4% realized the best significant (P < 0.05) increase of
embryonic weight either by absolute or relative and it sharedgroups of cumin 0.2%, oregano + cumin 0.1% andoregano + cumin 0.2% with the same significant increase
compared to those for other experimented treated groups.Moreover, the least significant (P < 0.05) embryonic weighton the same day of incubation were recorded in egg groupsof formaldehyde fumigation and sodium chloride compared
with other groups.Results in Table 4b reveal that relative embryonic weight
on sixth day of incubation, oregano 0.4% recorded lonely
the best significant (P < 0.05) increase of embryonic weight(2.42%) compared with those for other treated groups fol-lowed by oregano 0.2% (2.15%), cumin 0.2% (2.15%) and
cumin 0.4% (2.21%) with a significant difference with oregano0.4%. Regarding the absolute embryonic weight on the sameday of incubation, the heaviest significant (P < 0.05) increaseof absolute embryonic weight was recorded for groups subject-
ed to oregano 0.4% (1.15 g), cumin 0.2% (1.12 g) andoregano + cumin 0.2% (1.1 g) compared to those for othergroups. On the seventh day of incubation, the highest
significant (P < 0.05) increase of both absolute and relativeembryonic weights was detected for cumin 0.2% (1.67 g and3.11%), respectively compared to those for all other groups.
Moreover, formaldehyde fumigation recorded the least sig-nificant (P < 0.05) relative embryonic weight (2.23%) com-pared to those for all other groups. It can be observed that
oregano 0.4% represented the highest embryonic weight andformaldehyde fumigation represented the least one throughthe early embryonic development during the first seven daysof incubation. On day 8 of incubation, embryos which were
Table
4b
Effectofhatchingeggdisinfectiononabsolute
andrelativeem
bryonic
weights
atdifferentages
(6th–9th
days)
duringincubation(X
±E).
Treatm
ent
Embryonic
weight
6day
7day
8day
9day
g%
g%
g%
g%
Controluntreated
0.83±
0.01efg
1.44±
0.01ef
1.36±
0.02de
2.41±
0.02f
2.28±
0.10def
4.47±
0.17c
2.96±
0.01h
5.90±
0.18d
Controlwithwater
0.83±
0.02efg
1.46±
0.03ef
1.39±
0.03cd
2.56±
0.05de
2.29±
0.05def
4.22±
0.06cd
3.40±
0.04fg
6.42±
0.08c
Controlwithalcohol
0.91±
0.02d
1.88±
0.05d
1.35±
0.03de
2.48±
0.02ef
2.32±
0.05cde
4.34±
0.07c
3.58±
0.06cde
6.90±
0.12b
Form
aldehydefumigation
0.78±
0.01g
1.41±
0.01f
1.25±
0.02f
2.23±
0.02g
2.12±
0.03fg
4.23±
0.04cd
2.66±
0.04i
5.19±
0.13e
Sodium
chloride
0.78±
0.01g
1.50±
0.02ef
1.26±
0.03f
2.52±
0.06de
1.95±
0.02g
3.75±
0.05e
2.71±
0.01i
5.34±
0.04e
Hydrogen
peroxide
0.80±
0.02de
1.54±
0.03e
1.31±
0.01ef
2.60±
0.02d
2.14±
0.04ef
3.93±
0.04de
3.05±
0.09h
6.48±
0.26c
Betadine
0.88±
0.03gf
1.77±
0.06d
1.39±
0.01ef
2.39±
0.01f
2.27±
0.03def
4.31±
0.07c
3.04±
0.03h
5.85±
0.03d
VirkonS
0.85±
0.01ef
1.76±
0.04d
1.31±
0.01ef
2.56±
0.01de
2.32±
0.10cde
4.88±
0.24b
3.29±
0.01g
6.64±
0.01bc
Oregano0.2%
1.08±
0.01b
2.15±
0.02b
1.38±
0.01d
2.75±
0.02c
2.41±
0.05cd
4.47±
0.13c
3.84±
0.12b
6.96±
0.22b
Oregano0.4%
1.15±
0.02a
2.42±
0.05a
1.51±
0.03b
2.88±
0.03b
2.49±
0.01c
4.84±
0.02b
3.72±
0.05bc
6.98±
0.07b
Cumin
0.2%
1.12±
0.03ab
2.15±
0.06b
1.67±
0.02a
3.11±
0.07a
3.17±
0.09a
6.80±
0.21a
4.14±
0.07a
7.78±
0.20a
Cumin
0.4%
1.02±
0.03c
2.21±
0.07b
1.40±
0.03cd
2.39±
0.03bc
2.40±
0.09cd
4.50±
0.14c
3.49±
0.06ef
7.57±
0.07a
Oregano+
Cumin
0.1%
0.91±
0.02d
1.87±
0.05d
1.45±
0.03bc
2.91±
0.01b
2.43±
0.06cd
4.48±
0.07c
3.67±
0.05bcd
7.69±
0.15a
Oregano+
Cumin
0.2%
1.11±
0.02ab
2.01±
0.02c
1.49±
0.01b
2.92±
0.02b
2.68±
0.02b
4.99±
0.08b
3.53±
0.03def
7.45±
0.05a
Significant
**
**
**
**
a,b,c,d,e,f,g,hMeanswithin
each
columnforeach
item
withdifferentsuperscripts
are
significantlydifferent(P
<0.05).
*Significant(P
<0.05).
6 H.S. Zeweil et al.
produced from egg treated with cumin 0.2% (3.17 g) were sig-nificantly (P < 0.05) larger than those produced from eggtreated with other disinfectant and control groups followed
by those of oregano + cumin 0.2% (2.68 g) with the same sig-nificant difference. Also, relative embryonic weight on thesame day of incubation was significantly (P < 0.05) increased
for group of cumin 0.2% (6.80%) followed by oregano +cumin 0.2% (4.99%), oregano 0.4% (4.84%) and Virkon S(4.88%) with the same significant difference. Numerically,
the lowest records of absolute and relative embryonic weightson the 8th day of incubation were recorded for sodium chlo-ride, being 1.95 g and 3.75%, respectively. On day 9 of incuba-tion, the heaviest significant (P < 0.05) embryonic weight by
gram was recorded for the group treated with cumin 0.2%compared with other treated groups. Besides, the highest sig-nificant (P < 0.05) records of relative embryonic weight were
recorded for groups treated with cumin 0.2%, cumin 0.4%,oregano + cumin 0.1% and oregano + cumin 0.2% whichbeing 7.78%, 7.57%, 7.69% and 7.45%, respectively. Relative
embryonic weight on the same day of incubation was recordedin egg formaldehyde fumigation and sodium chloride groupscompared with other groups.
It appears from Table 4c that, on day 10 of incubation,absolute embryonic weight had significantly (P < 0.05)increased for the group of oregano + cumin 0.2% (5.91 g)compared with other treated groups besides numerical increase
compared with those for cumin 0.4%. Generally, relativeembryonic weight on the same day of incubation recordedthe highest significant (P < 0.05) values with cumin for both
concentrations and oregano + cumin 0.2%. On the sameday of incubation the lowest significant (P < 0.05) recordsof absolute and relative embryonic weights were observed in
groups subjected to formaldehyde fumigation and sodiumchloride compared with those for other experimented groups.On day 11, the highest significant (P < 0.05) values of abso-
lute embryonic weight were observed in cumin 0.4% (7.52 g),oregano 0.2% (7.38 g) and cumin 0.2% (7.33 g) compared withthose for other treated groups, while the lowest values wererecorded in formaldehyde fumigation and sodium chloride
groups. Generally, at the same day of incubation the sametrend was observed in relative embryonic weight. The highestsignificant (P < 0.05) records for average of absolute embryo
weight on day12 were noticed for egg groups treated with ore-gano 0.4%, oregano + cumin 0.1% and oregano + cumin0.2% which being 11.34, 11.29 and 11.32 g respectively, com-
pared to those for other treatments. The same egg group whichwas disinfected with oregano 0.4% represented the highest sig-nificant (P < 0.05) increase of relative embryonic weight onthe same day of incubation compared to those for other treat-
ed egg groups. On 13th day of incubation both absolute andrelative embryonic weights from egg treated with cumin0.2% (12.64 g and 27.33%) and oregano + cumin 0.2%
(12.64 g and 26.80%) had significantly (P < 0.05) increasedcompared to those for other groups.
Results in Table 4d show that the group which disinfected
with cumin 0.2% represented the highest significant(P < 0.05) increase of absolute (16.76 g) and relative(37.40%) embryonic weights on 14th day of incubation com-
pared to those for other treated egg groups. Absolute embry-onic weight on 15th day of incubation had significantly(P < 0.05) increased for groups treated with oregano 0.4%(17.98 g) and cumin 0.2% (17.33 g) compared to other groups
Table
4c
Effectofhatchingeggdisinfectiononabsolute
andrelativeem
bryonic
weights
atdifferentages
(10th–13th
days)
duringincubation(X
±E).
Treatm
ent
Embryonic
weight
10day
11day
12day
13day
g%
g%
g%
g%
Controluntreated
3.84±
0.07g
7.71±
0.23f
5.43±
0.03e
11.41±
0.18e
8.88±
0.10c
21.27±
0.44d
9.36±
0.08e
22.55±
0.10fg
Controlwithwater
4.78±
0.13e
9.75±
0.33cd
5.02±
0.15f
10.69±
0.32e
7.82±
0.28ef
19.02±
0.55e
9.28±
0.05e
20.82±
0.11h
Controlwithalcohol
5.10±
0.05d
9.30±
0.07d
5.54±
0.21e
11.25±
0.05e
10.53±
0.08b
23.38±
0.51c
11.07±
0.15cd
23.08±
0.35ef
Form
aldehydefumigation
4.22±
0.11f
8.48±
0.12e
4.37±
0.08g
8.59±
0.21g
6.28±
0.08g
14.77±
0.30g
7.00±
0.11g
19.00±
0.08i
Sodium
chloride
3.31±
0.05h
6.57±
0.05g
4.35±
0.17g
9.57±
0.05f
7.64±
0.07f
16.59±
0.22f
7.73±
0.19f
19.67±
0.31i
Hydrogen
peroxide
4.80±
0.08e
9.74±
0.17cd
5.43±
0.09e
11.31±
0.12e
8.20±
0.04de
19.20±
0.24e
9.03±
0.02e
22.02±
0.14g
Betadine
4.58±
0.06e
9.66±
0.22cd
5.53±
0.07e
11.44±
0.14e
8.43±
0.09d
19.02±
0.33e
9.28±
0.05e
20.96±
0.10h
VirkonS
4.60±
0.10e
9.57±
0.19cd
5.24±
0.06ef
10.63±
0.11e
8.15±
0.03de
18.39±
0.24e
10.89±
0.21d
23.82±
0.54de
Oregano0.2%
5.12±
0.06d
10.09±
0.05bc
7.38±
0.07a
15.71±
0.27a
10.83±
0.15b
23.68±
0.57c
11.91±
0.02b
24.18±
0.25cd
Oregano0.4%
5.47±
0.07bc
10.45±
0.32b
6.18±
0.09d
12.36±
0.05d
11.34±
0.07a
27.13±
0.65a
11.99±
0.14b
25.93±
0.27b
Cumin
0.2%
5.40±
0.05c
11.63±
0.30a
7.33±
0.10ab
15.24±
0.32a
10.73±
0.30b
25.24±
0.57b
12.64±
0.05a
27.33±
0.12a
Cumin
0.4%
5.74±
0.10ab
12.02±
0.16a
7.52±
0.01a
14.89±
0.16ab
10.49±
0.04b
24.23±
0.14bc
10.95±
0.18d
24.63±
0.45cd
Oregano+
Cumin
0.1%
4.69±
0.18e
9.38±
0.28d
7.04±
0.02b
14.27±
0.028bc
11.29±
0.11a
25.00±
0.14b
11.37±
0.10c
24.82±
0.21c
Oregano+
Cumin
0.2%
5.91±
0.14a
11.87±
0.11a
6.57±
0.10c
13.32±
0.11c
11.32±
0.11a
24.90±
0.30b
12.64±
0.05a
26.80±
0.56a
Significant
**
**
**
**
a,b,c,d,e,f,g,hMeanswithin
each
columnforeach
item
withdifferentsuperscripts
are
significantlydifferent(P
<0.05).
*Significant(P
<0.05).
Effect of egg disinfectants against bacteria and mitotic indices of chick embryos 7
except that for oregano + cumin 0.1% (16.91 g). On the sameday of incubation, egg treated with oregano 0.4% (40.77%),cumin 0.2% (39.11%), oregano + cumin 0.1% (39.07%) had
significantly (P < 0.05) surpassed other groups besides numer-ical increase compared with those for oregano + cumin 0.2%(38.60%). Generally the lowest significant (P < 0.05) absolute
embryonic weight on the day 15 of incubation were detected informaldehyde fumigation, sodium chloride, betadine, Virkon Sand control with water compared with other treated groups.
Nearly, the same trend of decreasing relative embryonic weighton 15th days of incubation was recorded for formaldehydefumigation and sodium chloride. On the day 16 of incubation,the heaviest significant (P < 0.05) embryonic weight was
recorded for groups treated with cumin 0.2% (21.78 g) andoregano 0.4% (21.46 g) compared with other treated groups,besides it increased numerically compared with those for ore-
gano + cumin 0.1%. Regarding relative embryonic weight atthe same day, egg treated with cumin 0.2% and oregano0.4% had the best significant (P < 0.05) values than those
for other egg disinfectant and control groups. On the day 16of incubation the lowest significant (P < 0.05) records of abso-lute and relative embryonic weights were recorded for groups
subjected to formaldehyde fumigation and sodium chloride,respectively. Results demonstrated that there was numericalincrease of embryonic weight on 17th and 18th days of incuba-tion from egg treated with oregano 0.4% compared with
cumin 0.2%, besides significantly increased (P < 0.05) com-pared with other treated groups. Also, on the 17th and 18thdays of incubation, oregano 0.4% recorded lonely the best sig-
nificant (P < 0.05) increase of relative embryonic weight com-pared with those for other treated groups, also there wascontinuous effect of disinfectant treatments with cumin
0.4%, oregano + cumin 0.2%. On the other hand, there wasnumerical decrease of absolute and relative embryonic weightsfor formaldehyde fumigation and sodium chloride on the 17th
and18th days of incubation.Results of average absolute and relative embryonic weights
at different incubation ages as affected by different disinfec-tants from different sources and formaldehyde fumigation
are summarized in Tables 5 and 6. It appears from Table 5 thateggs subjected to disinfectants from natural sources had theheaviest (P < 0.05) absolute embryonic weight daily from 2
to 17 days compared to those subjected to other disinfectantsfrom chemical sources, formaldehyde fumigation and controluntreated eggs. Absolute embryonic weight produced from
eggs subjected to disinfectants from natural sources was theheaviest (P < 0.05) than those for other treatments except thatfor control untreated eggs on the day 18 of incubation. Inaddition, the lowest significant (P < 0.05) absolute embryonic
weight was detected in the formaldehyde fumigated groupfrom day 11 of incubation onwards up to day 14 comparedto other treatments. The superiority of the disinfectants from
natural sources in embryonic weight still appeared throughthe days of incubation from 2 to 18 (Table 5). Moreover, onthe same days of incubation, relative embryonic weight record-
ed the same trend of the significant increase with disinfectantfrom natural sources compared to other treatments and con-trol. The highest relative embryonic weight (P < 0.05) was
recorded for the average of the natural disinfectant group com-pared to other treatments through the same days of incuba-tion. On days 16, 17 and 18, relative embryonic weightbetween formaldehyde fumigation and control untreated egg
Table
4d
Effectofhatchingeggdisinfectiononabsolute
andrelativeem
bryonic
weights
atdifferentages
(14th–18th
days)
duringincubation(X
±E).
Treatm
ent
Embryonic
weight
14day
15day
16day
17day
18day
g%
g%
g%
g%
g%
Controluntreated
12.38±
0.08d
29.01±
0.04d
14.69±
0.24f
34.07±
0.12de
19.33±
0.17d
39.94±
0.09g
23.68±
0.54cde
44.68±
1.34e
26.70±
0.54cde
49.72±
1.34e
Controlwithwater
10.92±
0.27e
26.07±
0.94f
13.58±
0.38g
33.38±
1.33e
17.72±
0.33ef
40.43±
0.54g
22.84±
0.87edf
53.15±
0.32abc
25.86±
0.87def
58.20±
2.54abc
Controlwithalcohol
12.36±
0.15d
31.31±
0.05c
15.40±
0.20ef
34.72±
093cde
21.46±
0.12a
49.27±
0.56a
23.83±
0.27cd
46.57±
0.38de
26.85±
0.27de
51.60±
0.38de
Form
aldehydefumigation
8.82±
0.22g
21.37±
0.36h
13.39±
0.16g
29.73±
1.01f
15.63±
0.35h
39.57±
0.59g
21.07±
0.23fg
44.61±
0.78e
24.09±
0.23fg
49.64±
0.78e
Sodium
chloride
10.01±
0.55f
23.82±
1.78g
13.41±
0.11g
30.62±
0.31f
16.66±
0.14g
38.01±
0.20h
20.46±
0.74g
44.35±
1.11e
23.47±
0.71g
49.37±
1.11e
Hydrogen
peroxide
11.82±
0.34d
27.45±
1.14def15.10±
0.21f
34.67±
0.66cde
18.04±
0.23e
42.44±
0.53f
21.93±
0.21bcd
52.27±
0.19bc
27.48±
0.21bcd
57.30±
0.19bc
Betadine
11.55±
0.24de
26.97±
0.24de
13.68±
0.18g
34.39±
0.48de
17.06±
0.28fg
42.91±
0.55f
21.93±
0.65efg
48.27±
1.14de
24.95±
0.65efg
53.31±
1.14de
VirkonS
11.85±
0.09d
28.62±
0.05c
13.40±
0.30g
35.84±
0.28cd
18.04±
0.23e
41.91±
0.48f
21.58±
0.21fg
49.61±
0.33cd
25.60±
0.21fg
54.65±
0.33cd
Oregano0.2%
14.92±
0.43b
31.96±
0.45b
15.51±
0.09def36.79±
0.80bc
19.57±
0.36d
47.09±
0.22de
24.79±
0.17bc
47.06±
0.68de
27.83±
0.17bc
52.14±
0.67ed
Oregano0.4%
15.19±
0.19b
34.76±
0.13a
17.98±
0.54a
4.08±
0.77a
19.99±
0.36cd
47.57±
0.46cd
27.90±
1.25a
57.18±
3.12a
30.93±
1.25a
62.24±
3.12a
Cumin
0.2%
16.76±
0.31a
37.40±
0.71a
17.33±
0.33ab
39.11±
0.09a
21.77±
0.08a
48.63±
0.18bc
26.30±
1.14ab
52.84±
1.13bc
29.32±
1.14ab
57.90±
1.14bc
Cumin
0.4%
14.66±
0.15bc
32.58±
0.21c
15.99±
0.49de
36.40±
1.35cd
18.02±
0.24e
42.83±
0.24f
24.92±
0.28bc
53.76±
0.94abc
27.95±
0.28bc
58.81±
0.93abc
Oregano+
Cumin
0.1%
13.91±
0.34c
32.21±
0.94c
16.91±
0.05bc
39.07±
1.45a
21.03±
0.24ab
52.29±
0.50a
24.99±
0.07bc
54.62±
0.46ab
28.02±
0.06bc
59.68±
0.46ab
Oregano+
Cumin
0.2%
14.54±
0.27bc
32.80±
0.39c
16.26±
0.11cd
38.60±
0.33ab
20.64±
0.39bc
45.90±
0.48e
25.94±
0.44b
54.71±
1.06ab
29.00±
0.44b
57.78±
1.06ab
Significant
**
**
**
**
**
a,b,c,d,e,f,g,hMeanswithin
each
columnforeach
item
withdifferentsuperscripts
are
significantlydifferent(P
<0.05).
*Significant(P
<0.05).
8 H.S. Zeweil et al.
groups did not significantly differ, while embryo weight forchemical disinfectant sources was intermediate among thetreatments.
This explanation highlights the main causes which mayinfluence the growth and weights of embryo at different incu-bation days with some disinfectant from natural sources com-
pared to others.
Chick embryo development
Untreated cases, control cases treated with water and alcoholshowed low-weighed embryos with morphology were delayedthan that of typically 13-day (Fig. 1). Chemically treated cases
showed that morphology of the embryo was typically 13-daywhile in both cases treated with fumigation with formaldehydeand treated cases with sodium chloride (in the upper row casesnumber 3 and 4 from left to right respectively) showed
low-weighing and underdeveloped embryos with malformedtwisting limbs, and on the other hand treated cases withnatural disinfectants showed high-weighing and more
developed embryos as typically 14-day (Fig. 2).
Mitotic index for spinal cord and dermal system
The obtained results of mitotic index as calculated accordingto Dondua et al. (1966) are illustrated in histograms ofFig. 3. Statistical analyses for mitotic indices reveled that thereare significant differences between the different treatments of
egg disinfection and control groups with respect to the spinalcord. From this histogram we can observe that the mitoticindices of the spinal cord for chicks from egg treated with
cumin 0.2% at 3rd and 4th days of age are slightly higherwhich being 5.5 and 4.8 respectively, than those for other treat-ment and control groups. Embryos from egg treated with
formaldehyde fumigation acquired the lowest value of mitoticindex (4) at 3rd day of incubation. Moreover, embryo from eggtreated with alcohol as control acquired nearly the lowest val-
ues of mitotic index of the spinal cord (2.8) at 4th day of incu-bation compared to other treated egg groups.
The obtained results of mitotic index in the spinal cord ofthe embryo from egg treated with averages of natural and arti-
ficial disinfectants and formaldehyde fumigation are illustratedin histogram (Fig. 4). The mean values of mitotic index of thestudied parts taken from embryo chicks for egg treated with
natural disinfectant are slightly higher in the spinal cord whichbeing 5.8 and 4.4 compared to others at days 3 and 4 of incu-bation, respectively. On the other hand, the mitotic index of
the spinal cord of egg treated with formaldehyde fumigationin embryo on day 3 of incubation has a lower value than thoseat the other treated and control group. The control group on
4 th day of incubation had a lower value of mitotic index(3.1) compared to others.
Fig. 5 shows the mean values of mitotic index in one ofstudied part namely the skin system of chick embryos at the
days 4, 7, and 10, and for newly hatched chicks. The mitoticindex revealed that there was a significant (P < 0.05) differ-ence between all disinfection and control groups on days 4, 7
and 10 of incubation with respect to skin systems, whereas skinsystem of newly hatched chicks did not demonstrate any sig-nificant differences between mitotic indices of experimented
groups. From this histogram we can observe that the mitotic
Table 5 Effect of hatching egg disinfectants from chemical and natural sources and formaldehyde fumigation on absolute embryonic
weight of incubation (X ± E).
Treatment Control untreated Average of chemical
disinfectants
Average of natural
disinfectants
Formaldehyde
fumigation
Significant
2nd day 0.023 ± 0.004b 0.018 ± 0.001c 0.029 ± 0.001a 0.018 ± 0.001c *
3rd day 0.074 ± 0.006c 0.098 ± 0.003b 0.147 ± 0.003a 0.063 ± 0.005c *
4th day 0.284 ± 0.010b 0.266 ± 0.004b 0.308 ± 0.004a 0.161 ± 0.004c *
5th day 0.540 ± 0.016b 0.555 ± 0.010b 0.724 ± 0.007a 0.465 ± 0.037c *
6th day 0.83 ± 0.01b 0.83 ± 0.01b 0.07 ± 0.01a 0.78 ± 0.01b *
7th day 1.36 ± 0.02b 1.29 ± 0.01c 1.49 ± 0.01a 1.25 ± 0.02c *
8th day 2.28 ± 0.10b 2.17 ± 0.03b 2.60 ± 0.03a 2.12 ± 0.03b *
9th day 2.96 ± 0.01b 3.02 ± 0.03b 3.73 ± 0.03a 2.66 ± 0.04c *
10th day 3.84 ± 0.07c 4.32 ± 0.07b 5.39 ± 0.05a 4.22 ± 0.11b *
11th day 5.43 ± 0.03b 5.14 ± 0.07c 7.00 ± 0.05a 4.37 ± 0.08d *
12th day 8.88 ± 0.10c 8.70 ± 0.12b 10.60 ± 0.10a 6.28 ± 0.08c *
13th day 9.36 ± 0.08b 9.23 ± 0.13b 11.92 ± 0.06a 7.00 ± 0.11c *
14th day 12.38 ± 0.08b 11.31 ± 0.19c 15.00 ± 0.14a 8.82 ± 0.22d *
15th day 14.69 ± 0.24b 13.90 ± 0.12c 16.66 ± 0.15a 13.39 ± 0.16c *
16th day 19.33 ± 0.17d 17.45 ± 0.12c 20.17 ± 0.15a 15.63 ± 0.35d *
17th day 23.68 ± 0.54b 22.11 ± 0.29c 25.81 ± 0.30a 21.07 ± 0.23c *
18th day 26.70 ± 0.54a 25.13 ± 0.54b 28.03 ± 0.33a 24.09 ± 0.23b *
a,b,c,dMeans within each row for each item with different superscripts are significantly different (P < 0.05).* Significant (P < 0.05).
Table 6 (continued) Effect of hatching egg disinfectants from chemical and natural sources and formaldehyde fumigation on relative
embryonic weight of incubation (X ± E).
Treatment Control untreated Average of chemical
disinfectants
Average of natural
disinfectants
Formaldehyde
fumigation
Significant
2nd day 0.048 ± 0.009b 0.035 ± 0.001c 0.059 ± 0.001a 0.033 ± 0.004c *
3rd day 0.136 ± 0.011c 0.178 ± 0.005b 0.275 ± 0.005a 0.113 ± 0.009c *
4th day 0.553 ± 0.022b 0.527 ± 0.01b 0.596 ± 0.01a 0.335 ± 0.014c *
5th day 1.094 ± 0.020b 1.101 ± 0.019b 1.421 ± 0.019a 0.906 ± 0.070c *
6th day 1.44 ± 0.01c 1.64 ± 0.02b 2.14 ± 0.02a 1.41 ± 0.01c *
7th day 2.41 ± 0.02c 2.52 ± 0.02b 2.90 ± 0.02a 2.23 ± 0.02d *
8th day 4.47 ± 0.17b 4.22 ± 0.08b 5.01 ± 0.08a 4.23 ± 0.04b *
9th day 5.90 ± 0.18c 6.08 ± 0.08b 7.40 ± 0.08a 5.19 ± 0.13c *
10th day 7.71 ± 0.23c 8.8 ± 0.15b 10.91 ± 0.12a 8.48 ± 0.12b *
11th day 11.41 ± 0.18b 10.74 ± 0.16c 14.38 ± 0.13a 8.59 ± 0.21d *
12th day 21.27 ± 0.44b 19.47 ± 0.30c 24.27 ± 0.25a 14.77 ± 0.30d *
13th day 22.55 ± 0.10b 21.62 ± 0.21c 25.61 ± 0.16a 19.00 ± 0.08d *
14th day 29.01 ± 0.04b 26.72 ± 0.44c 33.62 ± 0.26a 21.37 ± 0.36d *
15th day 34.07 ± 0.12b 32.35 ± 0.39c 38.3435 ± 0.31a 29.73 ± 1.01d *
16th day 39.94 ± 0.09c 41.32 ± 0.29b 47.39 ± 0.29a 39.57 ± 0.59c *
17th day 44.68 ± 1.34c 48.62 ± 0.48b 53.36 ± 0.65a 44.61 ± 0.78c *
18th day 49.72 ± 1.34c 53.66 ± 0.48b 56.74 ± 0.68a 49.64 ± 0.78c *
a,b,c,dMeans within each row for each item with different superscripts are significantly different (P < 0.05).* Significant (P < 0.05).
Effect of egg disinfectants against bacteria and mitotic indices of chick embryos 9
index of the skin system of chick embryos at 4 and 7 days ofincubation beside newly hatched chicks for egg treated with
cumin 0.2% is slightly higher (being 5.3 and 3.9), respectivelythan that from all treated groups. Moreover, the mitotic indexof the skin system of oregano + cumin 0.1% or 0.2% chick
embryos at day 10 of incubation is slightly higher (1.3) thanthat from other treated groups. On the other hand, the mitoticindex of the formaldehyde fumigation group at days 4 and 7 of
incubation has acquired the lowest value of M which being 3and 2.4, respectively. The mitotic index of the embryonic skinsystem for eggs treated with Virkon S and formaldehyde fumi-
gation at day 10 of incubation had acquired the lowest meanvalue (0.4) compared to those for other groups. Also, sodium
chloride, Virkon S, formaldehyde fumigation and control withwater groups recorded the lowest values of dermal mitoticindex (0.1) for newly hatched chicks compared with others.
The obtained results of mitotic index in the skin system at days4, 7 and 10, of incubation and newly hatched chicks are illus-trated in Fig. 6. The mitotic indices of embryonic dermal sys-
tem on days 4 and 10 of incubation were slightly higher fornatural disinfectant being 4.7 and 0.1, respectively comparedwith those for THE chemical disinfectant which being 4 and
Figure 1 Photo micrograph showing a 13-day-old embryos control untreated, treated with water, formaldehyde fumigation (A–C
respectively) and chemical disinfectants sodium chloride, hydrogen peroxide, betadine, and Virkon S (D–G respectively).
Figure 2 Photo micrograph showing a 13-day-old embryos (A: control treated with alcohol) and natural disinfectants (B–G respectively)
oregano 0.2 and 0.4%, cumin 0.2 and 0.4% and oregano + cumin 0.1% and 0.2%).
10 H.S. Zeweil et al.
0.6, formaldehyde fumigation (being 3 and 0.4) and controlgroup (4 and 0.9), respectively. Whereas the control untreated
group at day 7 of incubation, recorded the highest value ofmitotic index (3.4) compared to other treatment groups. More-over, formaldehyde fumigation recorded the lowest values of
mitotic indices on days 4, 7, and 10, and newly hatched chicksbeing 3, 2.4, 4 and 0.1, respectively compared to other treatedgroups.
Discussion
It has been demonstrated that if hatching eggs are not sanitized
prior to incubation, excessive bacterial contamination and sub-sequent growth can lead to decreased hatchability, poor chick
quality, growth and performance (Scott and Swetnam, 1993a),and increased mortality (Reid et al., 1961). Results of this
study regarding using cumin as natural disinfectant agree withthe findings of Yildirim and Ozcan (2001) who observed thatturkey eggs treated with cumin and oregano essential oils rep-
resented a significant bacterial decrease of populations. Also,Yildirim et al. (2003) and Copur et al. (2010) reported that ore-gano essential oil eliminates microbial populations naturally
occurring on the egg shell surface. Effect of essential oil onbacteria was attributed to its interaction with the microbial cellmembranes by means of their physiochemical properties and
molecular shapes that influence their enzymes, carriers, ionchannels and receptors. Similarly, Arhienbuwa et al. (1980),Sarma et al. (1985) and Cortes et al. (2004) demonstrated thatEscherichia coli was the predominant bacteria on the surface of
Figure 3 Spinal cord index at different embryonic ages during incubation for eggs subjected to natural and artificial disinfectants
compared with fumigation.
Effect of egg disinfectants against bacteria and mitotic indices of chick embryos 11
the hatching eggs. It is known that pathogenic bacteria presenton the surface of egg may contaminate the egg shell and pene-
trate the egg through shell pores. The increase of bacterialcount in the egg group treated with water as a control in thisexperiment is expected that water is considered as a good envi-ronment to encourage the enrichment of bacteria and this
result is keeping with those reported by Lorenz and Starr(1952), Brant and Starr (1962) and Rizk (1979) who indicatedthat the historical practices of egg washing resulted in an
increase of internal bacterial contamination. Also, it is possiblefor egg contents to be contaminated via the shell, especially ifcontamination occurs before the cuticle has dried (Sparks and
Board, 1985; Padron, 1990, 1995). The data herein in this
Figure 4 Spinal cord mitotic index at different embryonic ages for e
formaldehyde fumigation and control untreated.
experiment regarding the effect of hydrogen peroxide onbacterial count are in harmony with those reported by Wells
et al. (2010) who mentioned that hydrogen peroxide reducedeggshell bacterial counts by 2 Log cfu/egg and it is possiblethat hatchability and chick quality of breeder eggs might beimproved by treatment. Moreover, Wells et al. (2011) reported
the same conclusion of bacterial count reduction on eggshellsurface due to using 1.5% hydrogen peroxide with no effecton hatchability. Sacco et al. (1989) observed that formaldehy-
de fumigation eliminated the majority of bacteria of the egg-shell microorganism population. Also, some researchersmentioned that formaldehyde fumigation has been used suc-
cessfully in the poultry industry to control microbes on the
ggs subjected to artificial and natural disinfectants compared with
Figure 5 Mitotic index means of skin system in chick embryos at different ages produced from eggs treated with disinfection.
Figure 6 Dermal mitotic index at different embryonic ages for eggs subjected to natural and artificial disinfectants compared with
formaldehyde fumigation and control.
12 H.S. Zeweil et al.
eggshell surface (Whistler and Sheldon, 1989a; Brake and
Sheldon, 1990; Yildirim and Yetisir, 1999; Yildirim et al.,2001). It could be concluded from this table that all disinfec-tants used in this experiment had a residual effect on the shell
except that for formaldehyde fumigation treatment in which itsresidual effect was not persisted through the storage periodand its bacterial count increased again before setting in the
incubator.Control of microorganisms on the shell surface of hatching
eggs requires a disinfectant effective in killing the pathogenswithout injury to the live chick embryo (Fueng-Lin et al.,
1996). Fumigationwith formaldehyde has been themethod usedby most producers to achieve that, but the implication of thecontrol of substances hazardous to health (COSHH) legislating
is causingmany procedures sanitizing techniques (Sheldon et al.,1991; Sparks and Burgess, 1993). Moreover, formaldehydefumigation is an irritant for the eyes and the nose and has a lin-
gering noxious odor, venting of its vapors is difficult (Whistler
and Sheldon, 1989a). Most importantly, recent actions by the
environmental protection agency that regulate the use offormaldehyde fumigation under the toxic substances controlact due to its suspected carcinogenicity (Chemical and
Engineering News, 1984). Thus, effective alternative disinfec-tants are needed to replace formaldehyde fumigation in theevent that the environmental protection agency bans its use
(Whistler and Sheldon, 1989b). It is supposed that the increasingof embryonic development and consequently in embryonicweight resulting from using disinfectant from natural sourcescompared to those from chemical source, formaldehyde fumiga-
tion and control is due to that of these disinfectants did notadversely affect the cuticle and shell properties. These resultsare in accordancewith those reported byBrake (1987) whomen-
tioned that the cuticle may be affected by application of sanitiz-ers so as to alter embryonic development. Whereas, Yildirimet al. (2003) andCopur et al. (2010) reported that oregano essen-
tial oil had no detrimental impact on the development embryo.
Effect of egg disinfectants against bacteria and mitotic indices of chick embryos 13
Avian eggs are laid with a finite amount of water depositedin their yolk and albumen. This water is redistributed to theembryonic tissue and extra embryonic compartment within
the egg during incubation. In addition, some of this water islost across the eggshell during incubation and more water isproduced by oxidation of yolk (Ar and Rahn, 1980; Drent,
1975; Paganelli, 1980). Variation in shell conductance or inthe vapor pressure difference across the eggshell from egg toegg will produce variation in the amount of water lost. Consid-
erable variation in the water vapor conductance has beenreported for a number of avian species (Ar et al., 1974; Hoyt,1979). Increased or decreased water loss decreases hatchingsuccess (Lundy, 1969) and may influence growth and develop-
ment of the embryo (Simkiss, 1980; Tullett and Deeming,1987).
Mitotic index is the number of divided cells expressed as
percentage and indicates the proliferation activity. In general,when mitotic index is high consequently the proliferation israpid, and when it is lower than that reported by Hamburger
and Hamilton (1951), it means that the birth-rate of cells isalso low provided that the mitotic index remains constant(Dondua et al., 1966). In embryonic development, differential
cell proliferation, morphogenetic movements and cell death areimportant major processes. Immature cells are not only lack-ing in morphologic characteristics but also change their shapeand localization continuously as they differentiate (Ibrahim,
1999). The appearance of new cell types during embryonicdevelopment is frequently accompanied by changing patternsand rates of cell division (Fujita, 1967). Measurements of cell
population kinetics include, time intervals (duration of mitosis,interkinetic time and cell cycle time) population, size and num-ber of mitotic figures. Therefore, mitotic index (Dondua et al.,
1966) and labeling index (Modak et al., 1968; Kahn, 1974)were used as proliferative indices. As observed herein in theprevious Figs. 34 and 36, the slowing down in the mitotic index
was detected in the spinal cord at days 3 and 4 of incubationand for the dermal system of embryos at days 4, 7, and 10,besides newly hatched chicks for the chemical disinfectant,formaldehyde fumigation and control group. Variation in the
proliferation activity reported in the present investigationmay be due to (1) differentiation process which may changethe rate of cell replication by changing the duration of the cell
cycle phases, (2) cells which may leave the replicating phase orpermanently go into a terminal phase and (3) the number ofcells in the organ compartment which may be influenced by cell
loss (death or migration) or cell addition. These conclusionsare coinciding with those reported by Ibrahim (1999).
In conclusion, all the disinfectants used either from chemi-cal or natural sources, control with alcohol and formaldehyde
fumigation have a bactericidal effect on the eggshell surface asall treatment significantly diminished the bacterial count.Furthermore, all of treatments have a residual effect after
egg storage except that for formaldehyde fumigation.Natural and chemical disinfectants were investigated the
egg during fertilization, egg development in the oviduct of
the hen or immediately after oviposition or laid eggs couldbe contaminated with some infectious organisms passingthrough the eggshell pores upon contact with contaminated
feces or bedding. Therefore, sanitation is essential in successfulhealthy hatchings. Several methods are available for sanitizinghatching eggs as reported by Whistler and Sheldon (1989a) andCoufal et al. (2003). Many embryos infected with E. coli die
late in incubation or shortly after hatching. If hatching eggsare not sanitized prior to incubation, excessive bacterial con-tamination and subsequent growth of bacterial population
can lead to decrease embryonic development (Scott andSwetnam, 1993b) and increased mortality (Reid et al., 1961).Essential oils represent a rich potential source of alternative
and environmentally acceptable control agents for infectiousorganisms due to their antimicrobial properties. In the presentstudy, the effect of fourteen commercially disinfectants has
been determined on the developing chick embryo initiatingfrom the first 24 hour period till 13 days old. No distinctiveabnormalities were grossly visible among treated embryos until13 days of incubation. Formaldehyde fumigation untreated
cases, control cases treated with water and alcohol showedlow-weighing embryos with morphology were delayed thanthat of typically 13-day (Fig. 2). Chemically treated cases
showed that morphology of the embryo was typically 13-daywhile in treated cases with fumigation with formaldehydeshowed low-weighing and underdeveloped embryos, and on
the other hand treated cases with natural disinfectants showedhigh-weighing and more developed embryos as typically14-day.
Disinfectants reduced bacterial contamination of the egg-shell and affected the functional properties of the eggshell withrespect to egg water loss and gas exchange during incubation.These results complicate the situation regarding application of
any new egg disinfectants, therefore eggshell permeabilityshould be taken in our concept in choosing any method ofegg disinfection. The slowing down in mitotic index for the
spinal cord at days 3 and 4 of incubation and for the embryon-ic dermal system at days 4, 7 and 10, besides newly hatchedchicks for groups of chemical disinfectant, formaldehyde fumi-
gation and control compared to eggs treated with natural dis-infection reveal clearly the reason of early hatch of chicks fornatural disinfectant by about 8 h compared to other treat-
ments. Formaldehyde produces teratogenic and toxic effectsin the developing chicken and causing malformations on someembryos such as swelling part below the lower jaw of the beak.Chemical disinfectants are capable of causing slow develop-
ment to the central nervous system of the developing chickembryo. All the disinfectants used either from chemical or nat-ural sources, control with alcohol and formaldehyde fumiga-
tion have a bactericidal effect on the eggshell surface as alltreatments significantly diminished total bacterial count, fur-thermore, all of them have a residual effect after egg storage
except that for formaldehyde fumigation.The data suggested that chemical disinfectants are capable
of causing slow development to the central nervous systemof the developing chick embryo. This was manifested after
hatching by grossly abnormal behavior of chicks, such as tre-mor, non purposeful bodily movement and a total incapabilityof either standing or walking normally. These disinfectants
varied in their toxigenic properties for the chick embryo. Itwas determined that differences in the numbers of deathsoccurring in natural disinfectant-treated groups compared
with untreated and chemically treated control groups of chickembryos with significance at less than the .01 level. The effectof chemical disinfectants on developing chick embryos was
investigated. The embryos were immersed in different disinfec-tants. This resulted in retarded growth, as reflected by lowerembryonic body weight, reduced crown-rump length and billlength. Muscle weakness was seen in a few hatched chicks
14 H.S. Zeweil et al.
and that was evident in a number of chicks which survived toterm but were too weak to break out of the shell. It is conclud-ed that formaldehyde produces teratogenic and toxic effects on
the developing chicken.
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