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: g.bekhet@kfu.edu.sa (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.

References

Acklund, N.R., Hinton, M.R., Denmeade, K.R., 1980. Controlled

formaldehyde fumigation system. Appl. Environ. Microb. 39, 480–

487.

Arhienbuwa, F.E., Adler, E., Wiggins, A.D., 1980. A method of

surveillance for bacteria on the shell of turkey eggs. Poult. Sci. 59,

28–33.

Baydar, H., Sagdic, O., Ozkan, G., Karadogan, T., 2004. Antibacterial

activity and composition of essential oils from Origanum, Thymbra

and Satureja species with commercial importance in Turkey. Food

Control 15, 169–172.

Bouchra, C., Achouri, M., Idrissi Hassani, L.M., Hmamouchi, M.,

2003. Chemical composition and antifungal activity of essential oils

of seven Moroccan Labiatae against Botrytis cinerea Pers. Fr. J.

Ethnopharmacol. 89, 165–169.

Brake, J., Sheldon, B.W., 1990. Effect of a quaternary ammonium

sanitizer for hatching eggs on their contamination, permeability,

water loss and hatchability. Poult. Sci. 69, 517–525.

Brant, A.W., Starr, P.B., 1962. Some physical factors related to egg

spoilage. Poult. Sci. 41, 1468–1473.

Cadirci, S., 2009. Disinfection of hatching eggs by formaldehyde

fumigation – a review. Arch. Geflugelk. 73, 116–123.

Chemical and Engineering News, 1984. Formaldehyde may face

regulation. Chem. Eng. News 62, 8.

Copur, G., Arslan, M., Duru, M., Baylan, M., Canogullari, S., Aksan,

E., 2010. Use of oregano (Origanum onites L.) essential oil as

hatching egg disinfectant. Afr. J. Biotechnol. 8, 2531–2538.

Cortes, C.R., Tellez, I.G., Lopez, C.C., Villaseca, F.J.M., Eslava, C.C.,

2004. Bacterial isolation rate from fertile eggs, hatching eggs and

neonatal broilers with yolk sac infection. Rev. Latinoam. Micro-

biol. 46, 12–16.

Coufal, C.D., Chavez, C., Knape, D., Carey, J.B., 2003. Evaluation of

a method of ultraviolet light sanitation of broiler hatching eggs.

Poult. Sci. 82, 754–759.

Dondua, A.K., Efremov, V.I., Krichinskaya, E.B., Nikolaeva, I.P.,

1966. Mitotic index, duration of mitosis and proliferation activity

in the early phases of the development of the chick embryo. Acta

Biol. Hung. 17, 127–143.

Duncan, D.B., 1955. Multiple range and multiple F-test. Biometrics 11,

1–42.

Dvorak, G., 2005. Disinfection 101. <www.cfsph.iastate.edu>.

El-Zayat, M.A., 1974. Cell Reproduction Kinetics of Entodermal

Derivatives and Methodical Characteristics of Its Analysis in the

Chick Embryo at 4th to 6th day of Incubation (Ph.D. thesis).

Leningrad.

Essawi, T., Srour, M., 2000. Screening of some Palestinian medicinal

plants for antibacterial activity. J. Ethnopharmacol. 70, 343–349.

Ewart, S.L., 2001. Disinfectants and control of environmental

contamination. In: Smith, B.L. (Ed.), Large Animal Medicine:

Diseases of Horses, Cattle, Sheep and Goats, third ed. Mosby, St.

Louis, pp. 1371–1380.

Fueng-Lin, W.O., Carey, J.B., Ricke, S.C., Ha, S.D., 1996. Peroxidase

catalyzes chemical dip, egg shell surface contamination, and

hatching. J. Appl. Poult. Res. 5, 6–13.

Fujita, S., 1967. Quantitative analysis of cell proliferation and

differentiation in the cortex of the postnatal mouse cerebellum. J.

Cell Biol. 32, 277–287.

Gabe, M., 1976. Histological Techniques. Masson, Paris.

Gouladis, M., Tzakoy, O., Verykokidoy, E., Harvala, C., 2003.

Screening of some Greek aromatic plants for antioxidant activity.

Phytother. Res. 17, 194–195.

Grooms, D., 2003. Biosecurity guide for livestock farm visits.

Michigan State University Extension Bulletin, E-2842.

Hamburger, V., Hamilton, H.L., 1951. A series of normal stages in the

development of the chick embryo. J. Morphol. 88, 49–92.

Ibrahim, Ledia L., 1999. Cell proliferation kinetics in the head sense

organs of the 3-days chick embryo; Gallus gallus (Gemma strain). J.

Egypt. Ger. Soc. Zool. 12, 17–32.

Kahn, Z.A., 1974. Humoral response to sheep red blood cells in C57L/

J mice during early and chronic stages of infection with Echinococ-

cus multilocularis1 cysts. Parasitol. Res., 47–54.

Lorenz, F.W., Starr, P.B., 1952. Spoilage of washed eggs: 1. Effect of

sprayed versus static water under different washing conditions.

Poult. Sci. 31, 204–213.

Mansour, A.J., 2001. Hydrogen peroxide a versatile disinfectant.

Poult. Int. 8, 44–45.

McDonnell, G., Russell, A.D., 1999. Antiseptics and disinfectants:

activity, action, and resistance. Clin. Microbiol. Rev. 12, 147–179.

Meijerhof, R., 2000. Embryo temperature as a tool in the incubation

process. Incubation and Fertility Research Group (WPSAWorking

Group 6 (Reproduction)), St. Edmund’s Hall, Oxford, UK.

Michael, M.I., EL-Zayat, M.A., Ibrahim, L.L., 1991. Changes in cell

proliferation kinetics during organogenesis and differentiation of

the brain and optic cup in the chick embryo. Funct. Dev. Morph. J.

Egypt. Ger. Soc. Zool. 2, 43–48.

Modak, S.P., Morris, G., Yamada, T., 1968. DNA synthesis and

mitotic activity during early development of chick lens. Dev. Biol.

17, 544–561.

Muller, R.F., Berger, B., Yegen, O., 1995. Chemical composition and

fungitoxic properties to phytopathogenic fungi of essential oils of

selected aromatic plants growing wild in Turkey. J. Agric. Food

Chem. 43, 2262–2266.

Nychas, G.J.E., 1995. Natural antimicrobials from plants. In: Gould,

G.W. (Ed.), NewMethods of Food Preservation. Blackie Academic

Professional, London.

Oka, Y., Nacar, S., Potievsky, E., Ravid, R., Yaniv, Z., Spiegel, Y.,

2000. Nematicidal activity of essential oils and their components

against the root-knot nematode. Nematology 90, 710–715.

Overton, J.O., 1958. Effect of colchicine on the early chick blastoderm.

J. Exp. Zool. 139, 329–347.

Padron, M., 1990. Salmonella typhimurium penetration through the

eggshell of hatching eggs. Avian Dis. 34, 463–465.

Padron, M., 1995. Egg dipping in hydrogen peroxide solution to

eliminate Salmonella typhimurium from eggshell membranes. Avian

Dis. 39, 627–630.

Quinn, P.J., 2001. Disinfection and disease prevention in veterinary

medicine. In: Block, S.S. (Ed.), Disinfection, Sterilization and

Preservation, fourth ed. Lea and Febiger, Philadelphia, pp. 1069–

1103.

Ralph, A., 2003. Hatching egg production, storage and sanitation.

Rev. Poult. Calif.

Reid, W.M., Maag, R.A., Boyd, F.M., Kleckner, A.L., Schmittle,

S.C., 1961. Embryo and baby chick mortality and morbidity

induced by a strain of Escherichia coli. Poult. Sci. 40, 1497–1502.

Rizk, R.E., 1979. The Main Organisms in Eggs and Their Relation

with Its Spoilage (M.Sc. thesis). High Institute of Public Health of

Alexandria University, Egypt.

Rizk, R.E., 1994. Influence of chicken egg abnormalities on embryonic

development and hatchability. Egypt. Poult. Sci. 14, 317–337.

Russell, A.D., 1976. Inactivation of non-sporing bacteria by gases. In:

Skinner, F.A., Hugo, W.B. (Eds.), Inhibition and Inactivation of

Vegetative Microbes. Society for Applied Bacteriology Symposium

Series (5). Academic Press, London, pp. 61–68.

Sacco, R.E., Renner, P.A., Nestor, K.E., Saif, Y.M., Dearth, R.N.,

1989. Effect of hatching egg sanitizers on embryonic survival and

hatchability of turkey eggs from different lines on egg shell

bacterial populations. Poult. Sci. 68, 1179–1184.

Sarma, D.R.L., Char, N.L., Rao, M.R.K., Kahn, D.I., Narayana, G.,

1985. A comprehensive study on bacterial flora isolated from yolk

Effect of egg disinfectants against bacteria and mitotic indices of chick embryos 15

sac infection (omphalitis) in chicks. Indian J. Poult. Sci. 20, 262–

266.

SAS Institute, 1998. SAS Users Guide: Statistics. SAS Institute Inc.,

Cary, NC, USA.

Scott, T.A., Swetnam, C., 1993a. Screening sanitizing agents and

methods of application for hatching eggs, 1. Environmental and

user friendliness. J. Appl. Poult. Res. 2, 1–6.

Scott, T.A., Swetnam, C., 1993b. Screening sanitizing agents and

methods of application for hatching eggs, 2. Effectiveness against

microorganisms on the egg shell. J. Appl. Poult. Res. 2, 7–11.

Sheldon, B.W., Brake, J., 1991. Hydrogen peroxide as an alternative

hatching egg disinfectant. Poult. Sci. 70, 1092–1098.

Singh, A., Singh, R.K., Bhunia, A.K., Simmon, J.E., 2001. Use of

plant essential oils as antimicrobial agents against Listeria mono-

cytogenes in hotdogs. In: Program listing, The 2001 IFT Annual

Meeting, New Orleans.

Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S.,

Lanaras, T., Arsenakis, M., 1996. Antimicrobial and cytotoxic

activities of Origanum essential oils. J. Agric. Food Chem. 44,

1202–1205.

Sparks, N.H.C., Board, R.G., 1985. Bacterial penetration of the

recently oviposited shell of hens’ eggs. Aust. Vet. J. 62, 169–170.

Sparks, N.H.C., Burgess, D., 1993. Effect of spray sanitising on

hatching egg cuticle efficacy and hatchability. Br. Poult. Sci. 34,

655–662.

Sullivan, J., Krieger, G., 1992. Hazardous Materials Toxicology:

Clinical Principles of Environmental Health. Williams & Wilkins,

Baltimore, MD.

Tepe, B., Daferera, D., Sokmen, M., Polissiou, M., Sokmen, A., 2004.

The in vitro antioxidant and antimicrobial activities of the essential

oil and various extracts of Origanum syriacum L. var. bevanii. J. Sci.

Food Agric. 84, 1389–1396.

Tepe, B., Daferera, D., Sokmen, A., Sokmen, M., Polissiou, M., 2005.

Antimicrobial and antioxidative activities of essential oils and

various extracts of Salvia tomentosa Miller. (Lamiaceae). Food

Chem. 90, 333–340.

Traboulsi, A.F., Taoubi, K., El-Haj, S., Bessiere, J.M., Rammal, S.,

2002. Insecticidal properties of essential plant oils against the

mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Manag.

Sci. 58, 491–495.

Turpin, K., 2013. Ethanol vs. isopropyl alcohol to disinfect. <http://

www.ehow.com/about_6540795_ethanol-vs_-isopropyl-alcohol-

disinfect.html>.

USDA, 1985. National Poultry Improvement Plan and Auxiliary

Provisions. Animal and Plant Health Inspection Service. Veterinary

Service, Miscellaneous Publication. USDA, Hyattsville, MD.

Vagi, E., Simandi, B., Suhajda, A., Hethelyi, E., 2005. Essential oil

composition and antimicrobial activity of Origanum majorana L.

extracts obtained with ethyl alcohol and supercritical carbon

dioxide. Food Res. Int. 38, 51–57.

Wells, J.B., Coufal, C.D., Parker, H.M., McDaniel, C.D., 2010.

Disinfection of eggshells using ultraviolet light and hydrogen

peroxide independently and in combination. Poult. Sci. 89, 2499–

2505.

Wells, J.B., Coufal, C.D., Parker, H.M., Kiess, A.S., Young, K.M.,

McDaniel, C.D., 2011. Hatchablility of broiler breeder eggs

sanitized with a combination of ultraviolet light and hydrogen

peroxide. Int. J. Poult. Sci. 10, 320–324.

Whistler, P.E., Sheldon, B.W., 1989a. Biocidal activity of ozone versus

formaldehyde against poultry pathogens inoculated in a prototype

setter. Poult. Sci. 68, 1068–1073.

Whistler, P.E., Sheldon, B.W., 1989b. Comparison of ozone and

formaldehyde as poultry hatchery disinfectants as an alternative

hatching egg disinfectant. Poult. Sci. 68, 1345–1350.

Williams, J.E., 1969. Effect of high-level formaldehyde fumigation on

bacterial population on the surface of chicken hatching eggs. Avian

Dis. 14, 386–392.

Wilson, C.L., Solar, J.M., El Ghaouth, A., Wisniewski, M.E., 1997.

Rapid evaluation of plant extract and essential oils for antifungal

activity against Botrytis cinerea. Plant Dis. 81, 204–210.

Wilson, H.R., 1991. Effect of egg size on hatchability, chick size and

post-hatching growth. In: Tullet, S.G. (Ed.), Avian Incubation.

Butterworth-Heinemann Ltd., Surrey, UK, pp. 279–283.

Yildirim, I., Yetisir, R., 1999. Effects of disinfecting of hatching eggs

with disinfectants having different active compounds on egg shell

antibacterial activity, embryo development, hatchability of fertile

eggs and first two weeks chick mortality in broiler. Selcuk Univ. J.

Agric. Fac. 19, 78–91.

Yildirim, I., Ozcan, M., 2001. Use of oregano and cumin essential oils

as disinfectant on hatching quail eggs. J. Anim. Res. Inst. 11,

61–63.

Yildirim, I., Ozcan, M., Yetisir, R., 2003. The use of oregano

(Origanum vulgare L.) essential oil as alternative hatching egg

disinfectant versus formaldehyde fumigation in quails (Coturnix

japonica) eggs. Food Control 15, 169–172.