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THE EFFECTS OF EGG WASHING SOLUTIONS ON HATCHABILITY
AND BACTERIAL CONTENT OF INCUBATED DUCK EGGS

By
Mounira Naguib Ismail

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

198”

ABSTRACT

THE EFFECTS OF EGG WASHING SOLUTIONS ON HATCHABILITY
AND BACTERIAL CONTENT OF INCUBATED DUCK EGGS

By
Mounira Naguib Ismail

White Pekin Duck hatching eggs from three Michigan
commerical breeder farms were used in this study. Eggs were
randomly divided into four groups and were dipped into one
of three commercially available egg washing products (Amway;
Bioguard; Roccal II) or warm water, prior to incubation.
Hatchability varied from 79% to 8H1. The differences were
not statistically significant (p>0.05). At ten days of age,
the weight gain of ducklings hatched from eggs dipped in
Roccal II solution was significantly (p<0.01) greater than
the weight gain of ducklings hatched from the other
treatment groups.

Periodic bacterial isolation from dead duck embryos
during incubation indicated that Escherichia coli,
§tggptococcus and Proteus species were the principal
contaminants. These isolates did not show a direct
relationship to hatchability. There was however, both an
increase in Proteus species isolation and a significant

(p<0.01) increase in embryonic mortality near the end of

Mounira Naguib Ismail

incubation. This result suggests the possibility that

Proteus species might have influenced embryonic mortality.

ACKNOWLEDGEMENTS

The author wishes to express appreciation to
Dr. Timothy S. Chang, my major advisor, for his guidance and
suggestions during the course of my graduate study and in
planning the research and preparation of this thesis.

My gratitude is also expressed to Dr. Theo H. Coleman
and Dr. Cal Flegal for their helpful suggestions, for
serving on the graduate committee, and for reviewing the
thesis, and to Dr. John L. Gill for his assistance in the
analysis of the data.

I wish to acknowledge the assistance of and to express
appreciation to Ms. Bridget Gregus for her help in the
incubator room, Ms. Yvonne Rumminger for her help in the
Avian microbiology laboratory, Ms. Marina Garza for her help
in the animal room, and Mr. Mike McKinney for his help in
collecting the eggs.

- My special thanks go to my mother for her constant
support and encouragement during the course of my graduate
study. It is a good chance to thank my sisters, Hoda Fadly
and Laila Naguib; my brothers, Tarek Effat and Aly Fadly; my
friends, Mona Boules and Eglall Mandily for their moral

support, advice, prayers and great help.

ii

Mounira Naguib Ismail

Finally, and most importantly, I wish to thank my
husband, Nader; daughter, Deena; and son, Ameer; for their
patience, assistance and encouragement during the period of

graduate study and research.

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . .

INTRODUCTION 0 O O O O O O O O O O O O O O 0

REVIEW OF LITERATURE . . . . . . . . . . . .

I.
II.

III.

IV.

E88
E88
A.
B.

D.
Eas
A.
B.
c.
D.

Defense . . . . . . . . . . . .
Microbiology. . . . . . . . . .
Egg Microbial Content . . . . .
Infection . . . . . . . . . . .
1. Microorganism Strength. . .
2. Characters of Rot-Producing
3. Dominant Microorganisms . .
Factors Affecting Contamination
1. Egg Strength. . . . . . . .
2. Organisms Strength. . . . .
3. Temperature . . . . . . . .
u. Flock-age . . . . . . . . .
Source of Egg Contamination . .
Washing . . . . . . . . . . . .
Washing . . . . . . . . . . . .
Fumigation. . . . . . . . . . .
Disinfection. . . . . . . . . .

Methods of Disinfection . . . .

Farm and Hatchery Hygiene . . . . .

iv

0
\O (D co ‘1 N) '4 4 ‘1 0‘ U1 U1 U1 01 J: $7

. 1O
. 10

V.

Hatchability. . . . . . . . .

A. Relation of Washing and Hatchability. . . .

B. Relation of Contamination

MATERIALS AND METHODS. . . . . . . . .

I.

II.

Hatchability of Duck Eggs . .
Source of Eggs. . . . . . . .
Egg Washing Products. . . . .
Egg Handling Procedures . . .
1. Before Incubation . . . .
2. Incubation. . . . . . . .
3. During Incubation . . . .
u. After Incubation. . . . .
Bacteriological Examination .
Sampling Procedures . . . . .
Culture Media and Biochemical

Isolation and Identification.

RESULTS AND DISCUSSION . . . . . . . .

I.

II.

Hatchability of Duck Eggs . .
Hatchability Studies. . . . .
Embryonic Mortality Studies .
Duckling Performance Studies.

Bacteriological Examination .

and Hatchability.

Examination of the Wash Solutions

Examination of the Dead Embryos .

Page
12

13
13
15
15
15
15
16
16
17
19
21
22
22
23
28
33
33
33
36
111
115
115
145

SUMMARY AND CONCLUSIONS.

I.
II.
REFERENCES .

vi

Hatchability of Duck Eggs .

Bacteriological Examination

50
50
51
53

 

LIST OF TABLES

Page

Hatchability Rate of Fertile Duck Eggs Dipped
in Four Wash Solutions . . . . . . . . . . . . . . 3n

Two-Way Analysis of Variance for the Hatchability
Rate Of Duck Eggs. 0 O O O O O O O O O O O O O O I 35

The Average Embryonic Mortality Rate of Fertile
Duck Eggs at Three Different Stages of Incubation. 37

Two-Way Analysis of Variance for the Arcsin
Transformed Embryonic Mortality Rate of
DuCR Eggs. 0 O O O O O O O O O O O O O O O 0 0 0 O 38

Transformed and Rearranged Embryonic Mortality Rate
of Duck Eggs in Two Periods. . . . . . . . . . . . 39

Statistical Analysis of the Embryonic Mortality
Rate in Two PeriOdSo O O O O O O O O O O O O O O 0 no

The Ten-Day Average Weight Gain in Grams per
Duckling for Wash Treatment and Farm . . . . . . . uz

Two-Way Analysis of Variance for the Weight Gain
of Ducklings . . . . . . . . . . . . . . . . . . . nu

Percentages of Various Bacteria Isolated from Dead
Duck Embryos During Incubation . . . . . . . . . . u7

vii

INTRODUCTION

Poor hatchability of fertile duck eggs is a problem for
several large commercial hatcheries. An increase of as
little as 1% in hatchability would represent a difference of
many dollars to these hatcheries. It is therefore important
to study factors that can affect hatchability, including
genetics, egg shell condition, egg size, environmental
conditions, and egg shell contamination. Changing any of-
the environmental conditions in a commercial duck hatchery,
such as egg washing solutions or washing techniques, may
help to improve the rate of hatchability.

Eggs which rot during storage have been the subject of
many investigations (Brooks and Taylor, 1955; Board, 1965;
Board and Board, 1968; Board, 1969; Seviour and Board,
1972), while incubated chicken eggs (Harry, 1963; Hodgetts,
et al., 1976; Bruce and Johnson, 1978; Furuta and Maruyama,
1981; Hacking, 1982) and duck eggs (Seviour et al., 1972;
Moudgal et al., 1976; Joyce and Chaplin, 1978) seem to
attract little attention.

Microbiological studies have shown that Gram-negative
bacteria are the principal contaminants of rotten eggs.
Examples include Pseudomonas species from rotten table eggs

(Board and Board, 1968; Board, 1969) and Escherichia coli

from duck eggs that became rotten during incubation (Seviour
et al., 1972). However, Gram-positive organisms have been
reported as the predominant contaminants of unhatched
chicken eggs (Bruce and Johnson, 1978).

In a study on the effects of washing hatching eggs, it
was reported that unwashed dirty eggs have a higher surface
bacterial count (Brooks and Taylor, 1955) and lower
hatchability than clean eggs (Furuta and Watanabe, 1978;
Joyce and Chaplin, 1978). However, Bruce and Johnson (1978)
failed to prove a direct relationship between any specific,
isolated, microorganism and hatchability. Furuta and Sato
(1977) reported that dipping eggs in disinfectant solutions,
such as phenolic derivatives or iodophore, reduced bacterial
counts on the eggs, but did not increase hatchability. It
was also demonstrated that the hatchability of the
disinfected eggs was better when the eggs were dipped for a
shorter period of time (A minutes) as compared to those eggs
which were dipped for a longer period of time (8 minutes)
(Furuta and Watanabe, 1978).

Improving farm and hatchery sanitation programs have
resulted in both increased hatchability (Hodgetts et al.,
1976; Joyce and Chaplin, 1978; Hacking, 1982) and higher
quality chicks and ducklings (Chute and Gershman, 1961;
Gentry et al., 1962; Hodgetts et al., 1976; Joyce and
Chaplin, 1978; Shane, 1981).

Based on the assumption that a good sanitizer will

reduce bacterial contamination on eggs and indirectly

increase hatchability, commercially available egg washing
products were compared and bacterial contents of incubated
duck-eggs were identified in this experiment. The
objectives of this experiment were to (1) determine the
effects of washing duck eggs prior to incubation in three
different detergents and/or detergent sanitizers, on both
hatchability and the early performance of the hatched
ducklings; (2) identify bacteria which were isolated from
the contents of the duck embryos that died during
incubation; and (3) seek answers in regard to the
relationship between bacterial content of duck eggs and

hatchability.

REVIEW OF LITERATURE

I. ‘gggDefense

The egg_can be viewed as an ecosystem with a need only
for exchange of respiratory gases, a source of heat, and
regular movement by turning (Board, 1969). The egg
structure is a complex physiochemical system in which
enzyme-mediated energy transfer and chemical transformation
to the cells of the blastoderm takes place (Brooks and
Taylor, 1955).

The shell provides the prebrooding and prenatal stages
with protection against damage by impact or crushing, as
well as allowing the exchange of respiratory gases. The
shell is perforated with 7.17 x 103 pores having an average '
diameter of 9-35,” (Board, 1969). Although the pore size is
large enough to allow the passage of microbial organisms,
both the shell membrane and cuticle help to act as a barrier
against this invasion.

The structure of the yolk and white are important in
the egg's defense. The albumen can be viewed as a medium
that is unsuitable for microbial growth, since it contains

the following:

1. Lysozyme, with its lysis action, destroys bacterial
cell walls by causing flocculation of the bacterial cells
and the hydrolysis of 1-u p glycosidic bond (Board, 1969).

2. Conalbumen (ovotransferrin) chelates iron, copper,
and zinc and inhibits microbial growth in the presence of
low hydrogen ion concentration (Board, 1969).

The viscosity of the albumen and the gelatinous nature
of the albuminous sac are important components of the egg's
defense, since actual rotting does not occur until organisms
reach the yolk (Board, 1969). For this reason, it seems
that regular turning of the eggs during incubation maintains
tension in the chalazae, thereby ensuring that the yolk is

retained in a central position.

II. 'EgggMicrobiolggy
A. .Egg:Microbial Content
Brooks and Taylor (1955) and Board (1968) have
concluded that microorganisms are absent from the majority
of eggs laid by healthy hens. Even when contamination does
occur, the type of organisms in the egg at oviposition
differ markedly from those in rotten eggs at or those which

failed to hatch (Harry, 1963).

B. Infection

1. Microorganism Strength

The structure of the cell wall and the cytoplasmic
membrane of the eubacterial cell is considered to have a

role similar to that of the egg shell (Board, 1969). It

gives the cell a characteristic shape and protection against
damage that could ensue from sudden changes in osmotic
pressure. The outer layers of the cell wall of Gram-
negative bacteria provide a permiability barrier to egg
lysozyme. Different organisms showed different degrees of
resistance to egg conalbumen, i.e. Micrococcus is more
sensitive to conalbumen than Bacillus species (spp.) and the
latter are more sensitive than Gram-negative bacteria.

(Board, 1969).

2. Characteristics of Rot-producing Organisms

Rot-producing organisms are characterized by one or
more of the following (Board, 1969):
a. Production of pigment, e.g. Pseudomonas fluorescent
caused "pink-rot", and Pseudomonas putida produced
"fluorescent green-rot" (Board and Board, 1968);
b. Digestion of protein with or without the production of
hydrogen sulfide, e.g., Aromonas and Proteus sppg.have been
isolated from rotten eggs and they produced "black-rot" type
I and II, respectively. Enterobacter with its proteolytic
activity caused "custard-rot" in-vitro (Board and Board,
1968).
c. An ability to attack lecithin.

Since Gram-positive bacteria such as Bacillus,
Micrococcug_and.§tggptococcus failed to change the
appearance of the yolk or white, when inoculated in fresh

eggs (Board and Board, 1968), they are not considered rot-

producing organisms.

3. Dominant Microorganisms

Gram-negative bacteria were the principal contaminants
of rotten eggs from commercial duck hatcheries, incubated
eggs (Seviour et al., 1972) and from hen eggs which have
been designated for human consumption (Board and Board,
1968; Board, 1969). They were identified as Pseudomonas,

spp. from the rotten table eggs, and as Escherichia coli

 

from the rotten incubated duck eggs. However, Gram-positive
bacteria were the principal contaminants of unhatched
chicken eggs, and they were identified as Micrococcus spg.

(Bruce and Johnson, 1978).

C. Factors Affecting Contamination

1. Egg Strength

Since the egg shell membrane imposes a barrier to
microbial movement following invasion of the shell, the
infection remains confined to the shell membrane for up to

15-20 days (Board, 1959)-

2. 'Qgganism Strength,

The dominance of Gram-negative bacteria in rotten eggs
might be due to their simple nutritional requirements and
their ability to grow in the presence of conalbumen (Seviour

et al., 1972).

3. Temperature

The dominance of certain microorganisms occurs while

they are retained in the shell membrane. Temperature is an

important factor for their selected growth. Thus,

Pseudomonas and related organisms were dominant in eggs held
at room temperature, while Coliforms were predominantly
found in eggs held at 37°C (Seviour and Board, 1972). '
It has been reported that eggs laid on the floor tend
to cool rapidly (The Shaver Technical Bulletin, 1982), as do
eggs that are dipped in a cold bacterial suspension (Board,
1969). This causes the liquid contents of the egg to
contract, thus aiding in the penetration of organisms. For
this reason, floor-laid eggs should not be used for hatching
(Joyce and Chaplin, 1978), and hatching eggs should be
washed in a solution warmer than the temperature of the eggs

(Moats, 1978).

A. Flock-age

It was noted by Bruce and Johnson (1978) that there was
a significant increase in the incidence of contamination of
incubated hens eggs as the flock aged. The Shaver Technical
Bulletin (1982) further explained that when breeder flocks
are young, the nest litter is relatively clean, and eggs
have strong, thick shells, so bacteria have difficulty
penetrating. However, in the older breeder flock, there is
a build up of droppings in the litter, and the flock lays
larger eggs with thinner egg shells. Thus, organisms can

penetrate the shells more rapidly.

D. Source of Egg Contamination
The Shaver Technical Bulletin (1982) reported that eggs

from healthy birds, when laid, were clean and free of

organisms. However, the egg is an ideal host for organisms
because it is warm and moist. The egg shell can be infected
when passing through the hen's vent by feces or by contact
with other dirty surfaces (Board, 1969). Therefore, poor
hygiene in the nesting areas provided ample opportunity for
the shell to acquire organisms of fecal origin (Seviour et
al., 1972). It is believed that Coliforms are the dominant
contaminating organisms during the egg incubation. However,
Burce and Johnson (1978) reported that Enterobacteriaggag_
and Streptococcus spa; were isolated more frequently. It is
commonly agreed that the organisms on the shells originated
from a common source, namely feces, and that the bacteria,
ever present in the pens, were carried by the hen's feet and

feathers to contaminate nest litter and eggs.

III. Egg Washing
A. Washing

Although washing eggs was widely condemned because it
resulted in increasing spoilage losses during long term
storage (Board, 1969), it is now a common practice and is
required in plants operating under Federal Grading Services
(Moats, 1978). However, the importance of washing eggs in
water warmer than the eggs to reduce bacterial contamination
is well established (Moats, 1978). Furuta and Maruyama
(1981) showed that unwashed dirty eggs from floor pens have

significantly higher bacterial counts than do clean eggs.

10

When the dirty eggs were washed with running tap water at
AO°C, a significant decrease in the viable bacterial count

was observed.

B. Fumigation

Since washing hatching eggs may introduce
microorganisms into the eggs, it is not always recommended
in the poultry hatching egg industry. Instead, almost all
hatching eggs are fumigated with formaldehyde (Furuta and
Sato, 1977; Nesheim et al., 1979). Furuta and Maruyama
(1981), using the agar plate culture method, recovered no
bacteria from clean eggs after fumigation with
formaldehyde. However, they isolated a small number of
bacteria from three out of five dirty eggs even though they
were fumigated. Recent laboratory tests at the Shaver
Company (1982) illustrated that many antibiotics and most
disinfectants are not effective against Pseudomonas sppg.but
when formalin was used as an antimicrobial agent, it proved

to be most effective.

C. .Qisinfection

Treating the eubacterial cell with an alkaline solution
such as Ethylenediaminetetraacetic acid will disrupt the
surface architecture of the cell wall and increase the
sensitivity of the bacterium to the egg's lysozyme (Board,
1969). Many investigations have been concerned with the
disinfection of egg shells. Furuta and Sato (1977) found

that disinfectant solutions, such as phenolic derivatives or

11

iodophore, have a complete bacteriocidal effect on bacteria
in broth culture within two minutes. However, organisms
still survived on egg shells even after the eggs were dipped
in the same solutions for eight minutes. Therefore, they
concluded that the efectiveness of a disinfectant solution
on bacterially contaminated egg shells could not be assessed
by its effect on bacteria in culture broth. This conclusion
agreed with the result reported by Moats (1978), who
believed that treatment of hen eggs with a sanitizing
chemical does not necessarily destroy bacteria that are

embedded in the egg shells.

D. Methods of Disinfection

According to Furuta and Sato (1977), the results of
using disinfectant on contaminated egg shells applied by
spraying were not satisfactory. A better degree of
disinfection was observed when shell contaminated eggs were
dipped in the disinfectant solution. However, a complete
disinfection of the egg shells was not achieved by either
spraying or dipping methods.

IV. Farm and Hatcherngygiene
Improvement of sanitary measures, both on the farms and
at the hatchery, together with improvement of egg handling
before and during incubation has led to an overall
substantial increase of 101 in hatchability and also an
improvement in chick quality (Hodgetts, et al.,1976). Joyce
and Chaplin (1978) showed that clean duck-eggs, collected

12

from nest boxes, have an approximately 20% higher _
hatchability than floor eggs. First quality ducklings,
produced from clean nest eggs, were found to be 6.2$ heavier
than those from dirty eggs. Therefore, the importance of a
good sanitation program in both the poultry house and
hatchery in achieving a high level of hatchability (Hodgetts
et al., 1976; Joyce and Chaplin, 1978; Hacking, 1982) and
ensuring the production of high quality chicks (Chute and
Gershman, 1961; Gentry et al., 1962; Hodgetts et al., 1976;
Shane, 1981), and ducklings (Joyce and Chapplin, 1978) has
been firmly established.

Poultrymen may also aid egg shell bacterial penetration
by damaging the cuticle which covers the shell pores, by
rough handling, wet litter, improper washing, improper
dipping or by using an improper dry-cleaning practice. The
shells might be damaged during collection, shipping,
cleaning or traying (The Shaver Technical Bulletin, 1982).
Thus, no aspect of cleanliness or proper handling should be

ignored.

V. Hatchability
Hatchability can be affected by many factors such as

fertility, storage conditions (Moudgal et al., 1976), egg
size, nutrition of the dam, conditions of the egg shell,
genetic constitution of the embryo, incubation temperature,
humidity, gaseous environment, and disease (Nesheim et al.,

1979).

13

A. Relation of Washingand Hatchability

Furuta and Watanabe (1978) studied the hatchability of
chicken eggs disinfected by formaldehyde or certain other
disinfectants. Formaldehyde, phenolic derivatives,
iodophore and soap were used to wash eggs by dipping for
variable lengths or time (A, 6 and 8 minutes). Hatchability
rates were not significantly different among those
disinfectant groups. However, hatchability rate varied
according to the length of dipping time. The hatchability
was lower when eggs were dipped in the disinfectant solution
for 6 or 8 minutes, as compared to the hatchability of eggs
dipped in the disinfectant for A minutes.

B. .figlation of Contamination and Hatchability

It has been anticipated that as the level of
contamination increased, the level of hatchability
decreased. Bruce and Johnson (1978) found that the
relationship between egg contamination and hatchability was
not clear. However, there was a reduction in hatchability
where multiple contamination occured and as the flock
aged. Likewise, Joyce and Chaplin (1978) showed that dirty
floor eggs resulted in significantly lower hatchability than
clean floor eggs, which in turn were significantly lower
than the hatchability of nest eggs. Furthermore, the
presence of Pseudomonas spp. showed a significant
correlation with decreased hatchability (Bruce and Johnson,

1978). Pseudomonas organisms were believed to be the major

1”

cause of the exploding egg problem that occurs in all
hatcheries from time to time (Shaver Technical Bulletin,
1982).

According to Furuta and Watanabe (1978), hatchability
of fumigated eggs obtained from a Specific Pathogen Free
(SPF) chicken flock was significantly higher than that
obtained from eggs from a commercial chicken flock. In
another investigation, Furuta and Maruyama (1981) used clean
fumigated SPF eggs for hatching and found that, at the end
of hatching, eggs which contained dead embryos were highly
contaminated. These results indicated thst even when they
used fumigated SPF eggs for hatching, contaminating bacteria

were still present.

MATERIALS AND METHODS

I. Hatchability of Duck Eggs
Source of Egg§_

A total of 1A40 White Pekin duck eggs were acquired
from three commercial breeder flocks in Michigan. Since
ducks habitually lay their eggs in the early hours of the
morning (Joyce and Chaplin, 1978), #80 of these eggs were
collected early on February 15, 1983 from each farm. They
were randomly divided into four equal groups. Each group
consisted of 120 eggs from each farm, for a total of 360

eggs per group.

‘EgggWashing Products
Each group of eggs were dipped in one of the
commercially available egg washing solutions. Each product
was used according to the manufacturer's directions as
follows:

1. L00 organic cleaner, an Amway1

product which contains
no phosphorus compound, was used at a concentration of 57.8
millilitres (ml) in 68.3 litres (1.) of water. Eggs were

dipped in this solution at “2°C for 2 minutes (min).

 

1Amway Corporation, Ada, Michigan H9355

15

16

2. HEDS detergent sanitizerz, a Bio-Lab product which
contains Sodium carbonate 29%; Sodium metasilicate 251;
Alkyle (C1u 901, C12 51, C16 51) dimethyl benzyl ammonium
chloride 5.961; Alkyle (C1u 901, C12 51, C16 51) dimethyl
ethyl ammonium bromide 0.961; Ethylenediaminetetraacetic
acid, tetra sodium salt 0.381; Alkenyle (C18 751, C15 251)
dimethyl ammonium acetate 0.19%:Bis (2 hydroxyethyl) alkyle
(as in fatty acids of coconut oil) ammonium acetate 0.191;
Dodecyl benzyl alkyle (C12 701, C1“ 301) and dimethyl
ammonium chloride 0.191. It was used at a concentration of
28.u grams (gr.) in 13.7 1. of water. Eggs were dipped in
this solution at uz—u5°c for 3 min.

3. Roccal II 101 sanitizer and germicidal, a Lehn and
Fink3 product which contains alkyl (C1u 501, C12 “01, C16
101) dimethyl benzyl ammonium chloride 101, ethylalcohol
1.261 and water 88.731. It was used at a concentration of
7.1 ml/4.6 l. of water. Eggs were dipped in this solution'
at ”2°C for approximately 30 seconds.

Egg Handling Procedures
1. Before Incubation
The eggs were handled with care and clean hands.

Soiled and cracked eggs were discarded. Equipment such as

 

2Bio-Lab, Inc., P.0. Box 1u89, Decatur, Georgia 30031
3National Laboratories, Lehn & Fink Industrial Products

Division of Sterling Drug, Inc., Montvale, New Jersy 076H5

17

the egg setting and hatching trays, incubators, and washing
containers, was cleaned, disinfected and/or fumigated with
formaldehyde prior to use.

Eggs from the three different farms were washed in
separate containers. The egg dipping solutions were
prepared by dissolving the recommended amount (which was 19
ml. LOC, H8 gr. HEDS, or 36 m1. Roccal II) in 22.8 1. of tap
water. The first group of eggs was dipped into tap water,
without adding any chemical at ”2°C, as a negative
control. The second group of eggs was dipped into L00
solution, as a positive control. The third group was dipped
into HEDS solution. The fourth group was dipped into Roccal
II 101 which has been used in some research laboratories.
After dipping, a sample from each dip solution was collected

for bacteriological examination.

2. Incubation

The eggs were air-dried and set, with their blunt ends
up, on the egg trays and placed in incubators. Four
incubators (Jamesway model 252) were used for the four
groups of washed eggs. Eggs from each farm were set on two
trays for replicas of 60 eggs each. Each tray was marked
with the date, the wash product, the source of eggs (Farm A,
B or C), and the replica number (1 or 2).

This experiment is a factorial experiment with two
factors and balanced data. The first factor was the wash

solution (Fixed) and the second factor was the source of

duck eggs (Random). The experiment consisted of 12

18

wash treatment combinations with two replicas each. The

experimental design can be summarized as follows:

Summary of the Experimental Design

 

 

 

 

 

 

 

I II II
: I No. of Eggs--Treatment Group :
Farm (Replica I Water LOC HEDS Roccal II I Total Eggs
I
A l 1 | 60 60 60 60 |
: 2 : 60 60 60 60 : 480
I I |
B I 1 l 60 60 60 60 I
i 2 : 60 60 60 60 : N80
. I 5r |
C I 1 I 60 60 - 6O 60 I
: 2 i 60 60 60 60 : ueo
I I I
TotalI 6 I 360 360 360 360 I 1"”0
Eggs l Replicasl I
_Jl J |

 

The incubators were checked for temperature, humidity
and ventillation before incubation began and repeatedly
throughout the incubation period. They were adjusted as
scheduled by Jamesway manufacturing company for incubating
duck eggs as follows: I
1. from the 1st to the 3rd day, the temperature (temp) was
37.6° centigrade (C), relative humidity (RH) was 39%, and
the exhaust vent (exh) was closed;

2. from the 3rd to the 16th day, RH was 2H1 to 171,
respectively, temp. was 37.5°C, and exh. vent was Opened to

0.6 centimeter (cm.);

19

3. from the 16th to 23rd day (transfer to hatcher), RH was
231 to 131, respectively, temp. was 37.2°C, and exh. vent
was opened 0.6 cm.;

A. from the 23rd to 28th day hatching, RH was 381 to 551.
respectively, temp. was 36.8°C, and the exh. vent was opened
1.3 cm.

5. relative humidity was allowed to increase from 381 to
551 as hatching progressed and the exhaust was fully opened

to control RH and temp.

3. DuringIncubatigg_

During incubation, all the eggs were fumigated with
Russel Incubator Fumigant“, 12 ml. per incubator. The eggs
were also sprayed with Bioshield 902 solution (a Bio-Lab
product) at a concentration of 30 ml. Bioshield per u.6 l.
of water according to a commercially recomended procedure.
The schedule for fumigation, spraying and transferring the
duck eggs during the incubation period is summarized as

follows:

 

uL.D. Russel Co., Kansas City, Missouri 6H1u1

20

HatchingSchedule as Follwed by a Commercial Hatchery

 

 

Serial ,
Number Date Instruction

Set 2-16 Set eggs, fumigate (12 ml. Russell)
1 2-17 Spray eggs with Bioshield 90$ (30 ml./H.6 l.)
2 2-18 Spray " " " "
3 2-19 Spray " " " "
A 2-20 Spray " " " "
5 2-21

6 2-22

7 2-23 Spray " " " "
8 2-2u Spray and Candle " "
9 2-25 Spray " "
10 2-26 Spray " "
11 2-27

12 2-28

13 3-1

1“ 3-2 Fumigate (12 ml. Russell)

15 3-3 Spray eggs with Bioshield 902
16 3-H Spray " " " "
17 3_5 Spray I! N N N
18 3-6 Spray " " " "
19 3-7

20 3-8

21 3-9 Spray ” " " "

22 3-10 Spray " " " "

23 3-11 Transfer and Candle

2“ 3-12

25 3-13

26 3-1A Fumigate (A ml. Russell)

27 3-15

28 3-16

29 3-17 Hatch

21

The duck eggs were candled during incubation three
times as follows:

1. at the 8th day of incubation to remove infertile eggs,
which showed no embryonic development, and the early dead
embryos.

2. at the 23rd day of incubation (transfer day) to remove
any spoiled eggs and late dead embryos.

3. at the 29th day of incubation (hatching day) to remove
pipped eggs and unhatched eggs.

Early and late dead embryos, and unhatched duck-eggs
were kept for bacteriological examination in part II of this
experiment. Infertile eggs were discarded and recorded.
Pipped eggs (cracked with live or dead embryos) were not
used in bacteriological examination because of external
bacterial contamination (Bruce and Johnson, 1978). Pipped
eggs with live or dead embryos were recorded as unhatched.
Pipped eggs with dead embryos were considered as embryonic

mortality.

A. After Incubation

Newly hatched ducklings were wingbanded and raised for
ten days on a commercially available duckling diet
(Purina). Ducklilngs from each group and farm were placed
in separate sections of a battery brooder (Petersime). Body
weights of individual ducklings were recorded on the first
and tenth days.

Results of hatchability, embryonic mortality during

incubation, and the weight gain of ducklings were subjected

22

to statistical analysis (Gill, 1978). The variances were
analyzed as in two-factor models with balanced data.
Fisher's variance ratio (F-distribution) was applied to test
the variation among treatments or farms. Dunnett's test
method was followed to compare the mean of each experimental

group with the mean of the control group (LOC group).

II. Bacteriological Examination
Sampling Procedures

Wash solution samples were collected from all twelve
wash treatments in 12 sterilized half-gallon bottles and
were labeled accordingly. These samples were subjected to
bacteriological examinations immediately after washing the
eggs. All organisms isolated were identified and recorded.

Early and late dead embryos, and unhatched eggs found
during the incubation were examined for bacterial
contents. Cracked eggs were excluded from the samples to
minimize the incidence of external contamination (Bruce and
Johnson, 1978). All dead embryos from each treatment
combination (e.g. LOC treatment X farm B) were pooled as one
sample for a total of 12 samples each time the eggs were
candled.

All eggs were swabbed with alcohol and iodine solution
and allowed to dry, then cracked at the equator with a sharp
sterile knife. The egg contents including embryos at all
stages of embryonic development, were poured into a sterile

blender jar, mixed and ground at a high speed as suggested

by Hitchner et a1. (1980).

23

Culture Media and Biochemical Tests

Different kinds of media and biochemical tests were
used in isolation and identification of bacteria from the
wash solutions and egg samples. The media, the biochemical
test and the purpose of using it (Schoenhard, 1979; Carter,
[1978) are summarized as follows:

1. Brain heart infusion agar (BHI):

This is a general purpose medium for the growth of
fastidious bacteria.

2. ,Bgain heart infusion broth (BHI broth):

A useful general purpose broth.

3. Sglenite broth (SB):

This is an enrichment medium for the isolation of
Salmonella and Shigella. Proteus and Pseudomonas aeruginosa
are not completely inhibited.

A. Ethelyne violet agide broth (EVA):

This is a-sefective medium for the isolation of
Streptococcus spp.. Gram-negative organisms are usually
inhibited by this media.

5. Brilliant green agar (BC):

This is a highly selective medium recommended for the
isolation of Salmonellae directly from feces. Salmonella
colonies appear as slightly pink-white, opaque colonies
surrounded by a brilliant red zone. Lactose or sucrose
fermenting organisms produce colonies that are yellow-green

and surrounded by an intensive yellowish green zone.

 

2H

6. Eosin methylene blue agar (EMB):

This differential medium is used for direct plating and
also subculturing from Selenite and Tetrathionate broths.
Colonies have the following apearances: E. Coli, metalic
sheen; Aerobacter and Elabsiella, brownish; Salmonella and
Shigella. transparent, amber to colorless.

7. Enterococcus agar (EA):

This is a modified selective medium for the isolation
of Streptococci which appear as pink or colorless pinpoint
colonies.

8. Ppeudosalagar (PA) or Centrimide aga_:

This is a selective medium for the isolation of
Pseudomonag, The medium favors the production of pyocyanin
and fluorescin pigments.

9. 93am staining (Gr):

On a clean greaseless glass slide, a smear of bacteria
in a drop of distilled water was prepared, air-dried and
heat fixed. It was covered with crystal violet for one
minute, then washed with tap water. It was then flooded
with Gram's iodine for 2 minutes and again washed. It was
decolorized with acetone alcohol for a second then washed,
and counterstained with Safranin for one minute. It was
finally dried with bibulous paper. This slide was checked
under the microscope. The shape and grouping of the organ-
ism was recorded as coccus (c) or bacillus (b) single, in
groups or chains. Its color was recorded as purple for

Gram-positive (G+) or pink for Gram-negative (G-).

25

10. MacConkey agar (Mac):

Lactose fermenting enteric bacteria produce red

 

colonies while non-lactose fermenters do not. Included
among those producing pale colorless colonies are
Salmonella, Proteus spp., and Alcaligenes fecalis. Some
organisms cannot grow on MacConkey agar. .

11. Fermentation test (with the production of acid and
gas):

Five kinds of carbohydrate (CHO) media, glucose (gluc),
lactose (lact), maltose (malt), manitol (manit), and
saccharose (sacc), were prepared in Durham fermentation
tubes containing phenol red as an indicator. The tubes were
inoculated with the suspected colony, and incubated at 37°C
for 2H hours. CHO fermentation patterns were identified
when the media turned yellow due to the presence of acid end
products. Aerogenic organisms were identified when gas was
trapped in the inverted Durham tube.

12. Catalase test (Cate) (for the production of
peroxidase):

On a clean glass slide, a small amount of the suspected
colony was mixed with two drops of hydrogen peroxide. A
positive test was indicated by the production of oxygen gas.

13. Oxidase test (Oxid) (for the production of cytochrome
oxidase):

Two drops of 0.51 N, N, N', N' - tetra dimethyl aniline
monohydrochloride were added to part of the pure plate
culture. The positive colonies produced a dark purple

color.

26

1A. Indole test (Ind) (indole is a product of tryptophane
hydrolysis):

One ml. of chloroform was added to the BHI broth
culture, then a few drops of Kovac's reagent were layered on
top. A red line of precipitate was formed in the chloroform
layer if the culture was positive for indole production.

15. Methyl red test (MR) (indicates acidic condition):

A few drops of methyl red indicator were added to the
suspected MR-VP broth culture which had been incubated at
37°C for 2” hours. A positive reaction was indicated by a
distinct red color while a yellow color indicated a negative

reaction.

16. V0 us-Proskauer test (VP) (to test for 2, 3
butanediol):

A few drops of Barritt's reagent solution A and

 

solution B were added to the suspected MR-VP broth culture
which had been incubated for 2H hours at 37°C. The tube was
vigorously agitated for 30 seconds and allowed to stand for
2-2u hours. A positive reaction was indicated by the
development of a pink color. -

17. [Qippate test (Cit) (for utilization of citrate):

Simons citrate agar slant was inoculated with the
suspected colony. The slant was streaked and the butt
stabbed. Then, it was incubated at 37°C for 2H hours. A
blue color with colony growth indicated an alkaline pH, due

to the utilization of citrate as the sole source of carbon.

27

18. Nitrite Test (Nip) (to test nitrate reduction):

The inoculated nitrate broth culture, incubated at 37°C
for 2a hours, was tested for nitrite by adding three drops
if nitrite reagents solutions A and B, respectively. A
cherry red color was developed immediately if nitrite was
present. If the test was negative, nitrate was either not
reduced or reduced beyond nitrite. With a toothpick a few
grains of zinc dust were added to the tube indicating
negative results. If there was no change, then nitrate was
reduced beyond nitrite (positive); if there was a change to
cherry red, then nitrate was not reduced by the organism
(negative).

19. Hydrogen Sulphide testngas) (for the production of
H S):
2

Triple sugar iron agar slants (TSI) were inoculated
with the suspected colonies. The slant was streaked and the
butt stabbed, then it was incubated for 2n hours at 37°C. A
black color in the slant indicated the productin of H23 from
cysteine.

20. Motility test (Mot):

Tubes of SIM medium were inoculated with a straight
stab, to a depth of about two inches. Motility was
indicated by diffuse growth producing turbidity throughout
the medium after 2A hours of incubation at 37°C.

21. Urea hydrolysis test (for the production of urease):

A tube of filtered urea broth was inoculated with the

suspected colony and incubated at 37°C for an hours. A

28

cerise red color indicated an alkaline pH due to the
production of ammonia from the hydrolysis of urea.
22. Oxidation fermentation test (O-F):

This test demonstrated whether the breakdown of sugars
was by oxidation or fermentation. Two tubes of Hugh and
Leifson's O-F medium were heated for five minutes in boiling
water prior to inoculation. One of the tubes was covered
after inoculation with "Vaspar" seal (one part petrolatum
and one part paraffin). Those bacteria that oxidize show
acid production in the open tube only, while those that
ferment produce acid in both tubes.

23. Spore stain:

A smear was fixed and flooded with malachite green
(51); steamed gently over a flame for 30 seconds; washed
with water and stained with safranin for 30 seconds; washed
with water, dried and examined for the presence of green

endospore inside of and free of red vegatative cell.

Isolation and Identification
The microbiological work of isolation and
identification was performed under aseptic conditions. From
the wash-solutin samples, microbial isolation and
identification procedures were carried out as follows:
1. 0.1 ml. sample was spread onto each BHI, BG, EMB, and
PA agar plate. They were incubated at 37°C for 29 hours;

29

2. Different types of single colonies, i.e. shape, size,
and color from the BHI agar culture were picked up with a
sterile platinum loop and transferred onto fresh BHI agar
plates;

3. The suspected single colonies, such as Salmonella
(pink), E. coli (metalic sheen), Streptococcus (red or
colorless), or Ppppdomonas spp. (green or yellow) from the
BG, EMB, EA, or PA agar culture-plates, respectively, were
transferred separately onto fresh BHI agar plates for
purification, and were incubated at 37°C for 23 hours;

A. The purity of the replated-isolates was tested by a
second replating on BHI agar plates, which were then
incubated at 37°C for 2h hours.

5. Each of the pure colonies was subjected to different

biochemical tests for identification.

From the egg samples, the isolation and identification
were carried out as follows:
a. A one ml. sample was inoculated into five ml. of BHI
broth, SB, and EVA broth. This step was done to help the
growth of certain specific bacteria if present in the sample
and, at the same time, to dilute the suspended egg
particles. The three tubes from each sample were incubated
at 37°C for 2H hours.
b. One ml. of the incubated BHI broth culture was spread
onto the BHI, BG, EMB, EA, and PA agar plates. Also, one

ml. of the SB culture was inoculated onto the BG agar plate,

30

and one ml. of EVA culture onto the EA agar plate. The
plates were all incubated at 37°C for 24 hours.
c.- The same procedures for isolation and identification
were used as in steps 2 to 5 for the wash solution protocol.
All observations of the biochemical tests were recorded
and used as criteria for microbial identification. The
modified King's key for identification of aerobic bacteria
has been listed by Carter (1978). It was useful in
identifying the isolated bacteria of this experiment. In
addition a scheme outlined by Schoenhard (1979) was also
helpful in identification. Primary and secondary
identification was determined according to the following

schemes:

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32

Primary Identification Scheme

I . Gram Negative

A. Fermenters

 

 

 

 

 

If 'I
Mac+ Mac-
, I __J
(I I If I
Oxid-n- Oxid- Oxid-o- Oxid- .
I Enterobacteriaceae Pasteurella- Bacillus ssp.
l 121M
II I
Mot Non-Mot

Aeranonas Pasteurella hemolytica

 

 

 

 

 

 

 

B. Oxidizers
, J_
1'5 - I
Mac+ tho-
Oxid+ Oxid-
Pseudanonas aer_uginosa Pseudanonas mallei
Pseudanonas Montana
C. Non-Oxidizers _
non-Fermenters
Al
I If
Mao-1- Mac-
Oxidt .
T I Bacillus spp.
Oxid+ Oxid-
Alcaligpnes Pseudomonas-
mltopgilia
II. Gram Positive
___L—__
I I
Mac-13 Mac-
Cata-
r I Streptococcus (group D)
Cata+ Cata-
I Streptococcus
I T
rod Cocci

Bacillus Sta lococcus (fermenter)
Micro-coccus (Oxidizer)

many bacillus grow very lightly and very weakly

RESULTS AND DISCUSSION

I. HATCHABILITY 0F DUCK EGGS
Hatchability Studies

Healthy, culled and dead ducklings were considered
”hatched". Pipped and not pipped eggs containing dead
embryos were considered as "unhatched" eggs. Results of
hatchability from the four wash groups and the three farms
(A, B and C) are summarized in Table 1.

Two-way analysis of variance (Gill, 1978) for the
hatchability of fertile duck eggs is presented in Table 2.
The hatchability did not vary significantly (p>0.05) among
the different wash groups; water (801), LOC (83$), HEDS
(831), and Roccal II (8H1). The differences in the
hatchability between the three farms, A (801), B (861), and
C (801), were also not significantly (p<0.05) different.

These results indicated that washing duck eggs prior to
incubation with warm tap water, LOC, HEDS, or Roccal II
produced no significant changes in hatchability. A similar
result was reported by Furuta and Watanabe (1978) when they
used phenolic derivatives, iodophore, and soap for washing

chicken eggs.

33

3“

Table 1. Hatchability Rate of Fertile Duck EggsDipped in
our wash Solutions

 

Hatchability (1)
Treatment Grou
Farm Replica Water LOC HEDS Roccal II Av. Farm

 

79 83 79 77

A 1

2 85 86 77 77 80a
13 1 81 88 83 87

2 83 87 88 92 86"
c 1 75 7o 79 86

2 711 81 90 85 80131

 

Av. Treatment 80A 83A 83A 84A

 

A Not significantly different (P > 0.05)

a,b Different letter superscripts in the farms are
significantly different (P < 0.05)

35

 

Table 2. Two-Wa Anal sis of Variance for the Hatchability
Rate of Duck Eggs

 

 

Source of d.f. SS MS F c.v.
variation

Wash solution 3 72.85 2“.28 0.579 > “.76
Farm 2 183.59 91.8 6.63' > 3.89
Interaction 6 251.59 “1.93

Error 12 166.05 13.8“

 

d.f. degree of freedom

SS sum of squares
MS mean square
F F-distribution test

c.v. critical value

p < 0.05 (significantly)

36

Embryonic Mortality Studies

The early and late dead embryos, the unhatched duck
eggs and the dead pipped eggs were combined to give the
embryonic mortality of the fertile eggs. Embryonic
mortality was recorded at the 8th, 23rd, and 29th days of
incubation. The results from the four wash treatments and
the three farms are summarized in Table 3.

Embryonic mortality data was transformed from binomial
percentages to angles of equal information in degrees
(arcsin) for statistical calculation (Bliss, 1937). Two way
analysis of variance of the arcsin transformed embryonic
mortality (Table “) showed that it did not vary
significantly (p>0.05) among the treatments. However,
embryonic mortality significantly (p<0.01) varied between
the farms. In order to compare embryonic mortality during
the first eight days to embryonic mortality during the last
six days of incubation, the data was rearranged as shown in
Table 5. The differences between the two incubation periods
were analyzed statistically and are presented in Table 6.
The results indicate that embryonic mortality of the last
six days of incubation (29th day embryonic mortality) was
significantly (p<0.01) higher than that of the first eight
days of incubation (8th day embryonic mortality). This
observation may be in agreement with the findings of Board
(1969), who stated that following invasion of the shell the

infection remains confined to the shell membranes for up to

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38

Table H. Two-Wax Analysis of Variance for the Arcsin
Transformed Embryonic Mortalitx Rate of Duck-Eggs.

 

Source of d.f. SS MS F c.v.
Variation

6n.26 21.n2 0.5359 >u.76

 

Nash solution 3

Farms 2 55.29 27.65 17.28" >6.93
Interaction 6 239.83 395.97 '

Error 12 19.36 1.6

d.f. Degrees of freedom

SS . Sum of squares

MS Mean square

F F-distribution test

c.v. Critical value

** p<0.01 (significant)

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Table 6. Statistical Analysis of the Embryonic Mortalityy
Rate in Two Periods

 

32D Variance of the difference 5.96
SD Standard deviation 2.uu
t-Test MD / (SD / r) 8.87"

c.v. Critical value 2.71

 

"The difference is significant (p<0.01)

N1

15-20 days, and that actual rotting does not occur until
organisms made contact with the yolk because of the
following reasons:
1. the egg shell membrane acts as a barrier to microbial
movement;
2. the viscosity of the albumen delays the microbial
movement;
3. Regular turning of the egg during incubation prevents
the precipitation of the microorganisms on the yolk surface.
Those findings of Board (1969) could explain why the
highest rate of embryonic mortality occurred near the end of
the incubation period of this trial. Furuta and Maruyama
(1981) also reported that dead embryos found at the end of
the hatching period were heavily contaminated with bacteria
even though the eggs were originally considered clean at the

beginning of the period.

Duckling Performance Studies

The weight gains of the ten-day old ducklings were
recorded according to each farm and by each wash-treatment
as shown in Table 7. The ten-day average weight gains of
ducklings hatched from eggs dipped in the different wash
solutions were 151 gr., 153 gr., 1H6 gr., and 161 gr. for
the water, LOC, HEDS and Roccal II groups, respectively.
The ten-day weight gains of ducklings hatched from eggs from
the different farms were 156 gr., 1H1 gr., and 161 gr. for

farms A, B, and C, respectively. These results were

subjected to two-way analysis of variance (Gill, 1978) and

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are presented in Table 8. Statistical analysis showed that
there is a significant difference in the average weight gain
among the four wash treatments and among the three farms.
Further analysis was carried out by using Dunnett's test
(TD) to compare the result of LOC (as a positive control) to
results of each of the water, HEDS, and the Roccal II
treatment groups. The weight gain of ducklings hatched from
eggs dipped in water did not vary significantly (p>0.05)
from that of ducklings hatched from eggs dipped in LOC
solution. The weight gain of ducklings from the HEDS group
was significantly lower (p<0.01) than that of ducklings from
the LOC group. Ducklings hatched from eggs treated with
Roccal II solution had the highest (p<0.01) ten day weight
gain compared to the other treatment groups. As per
manufacture's recommendations, eggs that were dipped in
Roccal II had the shortest dipping period (V2 min.), as
compared to LOC (2 min.) and HEDS (3 min.). It could be
speculated that the length of the dipping period is
important to the health of the hatched ducklings. This
speculation would agree with the findings of Furuta and
Watanabe (1978) being that eggs dipped in the disinfectant
solution for six or eight minutes (long period) had lower
hatchability than those dipped for four minutes (short
period). They attributed that reuslt to the persistence of
disinfectant solutin on the egg shell. Therefore, they
suggested that hatching eggs should be dipped in a diluted

11 of phenolic derivative solution at ”0°C for four minutes,

nu

Table 8. Two-Way Analysis of Variance for the Weight
Gain of Ducklings.

 

Source of d.f. SS MS F c.v.
Variation

3 683.6h 227.88 un.77** >9.78
Farms 2 2019.09 1009.55 8.55" >6.93
Interaction 6 30.58 5.09

2 1316.56 118.05

Nash solution

Error 1

 

d.f. Degrees of freedom
SS Sum of squares

MS Mean square

F F-distribution test
c.v. Critical value

an p<0.01 (significant)

HS

then eggs Should be washed with water, after dipping to

remove any remaining disinfectant solution.

II. Bacteriological Examination
Examination of the Wash Solutions:

.Several different colonies grew on the BHI agar plates
that were streaked from the samples of LOC wash solutions
and the wash water. These colonies were identified as
Staphylococcus, Micrococcus, Streptococcus, Aeromonas
species and Enterobacteriaceae from both the wash water and
the LOC solution. In addition, Pseudomonas and Bacillus
species were isolated from the wash water and Alcaligenes
was isolated from the LOC solution. These results indicate
that LOC cleanser did not kill or reduce the diversity of
bacteria in the wash solutions. The eggs that were dipped
in LOC could therefore have a similar degree of bacterial
contamination as the eggs dipped into warm tap water.

0n the other hand, samples of HEDS and Roccal II wash
solutions showed no bacterial growth on the BHI agar or any
other culture plates. This indicates that HEDS and Roccal
II sanitizers killed all of the bacteria in the wash
solutions. However, washing or treating eggs with
sanitizing chemical does not necessarily destroy bacteria

embedded in shells (Moats, 1978; Furuta and Sata, 1977).

Examination of the Dead Embryos
During the incubation period, a variety of

microorganisms were isolated at three different stages of

R6

incubation from dead embryos. By following the normal
microbiological identification procedures, they were
identified according to their morphology, gram stain
appearance and biochemical results, such as fermentation
patter, EMB, BG, EA, PA, Mac, O-F, Oxid, Cata, Ind, MR, VP,
Cit, Nit, H2, Mot, Urea hydrolysis, and spore stains.

According to the results of this study, a total of 123
different types of bacteria were isolated, of which 6H1 of
the organisms were Cram-negative and 36$ were Gram-
positive. These results were in agreement with the
classification of bacteria isolated from rotten chicken
eggs by Board (1965) and Board and Board (1968) and from
duck eggs by Seviour et al. (1972). However, Bruce and
Johnson (1978) reported that Gram-positive bacteria were the
principal contaminants of unhatched chicken eggs.

The percentage of microorganisms isolated from dead
duck embryos at three different stages of incubation are
summarized in Table 9. The major isolates were E. coli,
Streptococcus, and Proteus spp. being 30%, 27$, and 17%,
respectively, of the total isolates. The isolation of
Proteus spp. increased from 121 to 1u$ then to 25% at the
8th, 23rd, and 29th days of incubation while the percentage
of Streptococcus spp. increased from 18% to 35$ then
decreased to 27%. The percentage of E. coli isolated
decreased slightly at the 29th day of incubation. These
results seemed to suggest that the presence of Proteus spp.

might have influenced the embryonic mortality near the end

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of the incubation period. Perhaps due to a synergistic
relationship may have been demonstrated between
Streptococcus and Proteus, causing the increase of embryonic
mortality near the end of incubation. Harry (1957) reported
that Streptococcus fecalis and Bacillus cereus have a
synergistic effect to enhance their individual spoilage
potential of egg yolk when studied in vitro. Board and
Board (1968) have isolated Proteus species from rotten

eggs. It was proven to be the cause of "black rot" in

eggs. It is believed that Proteus species gained entrance
to the egg before incubation, but its fatal action was
delayed until the end of the incubation period, when the
organism was able to contact the yolk and cause the actual
rot (Board, 1969).

It is difficult to pin point the relationship between
bacteria isolated from egg content and hatchability of duck
eggs since it was proven that there is no statistical
difference in hatchability. Even when hatchability varied,
Bruce and Johnson (1978) failed to show the relationship
between them.

The frequent isolation of E. coli, Streptococcus, and
Proteus species during the incubation period supported the
explanation that feces is the main source of egg
contamination. Salmonella spp. was not isolated in this

study. Although Pseudomonas spp; was numerically a minor

n9

contaminant in this study compared to E. coli and Proteus,
it has been frequently isolated from rotten hen-eggs (Board,
1965).

Bruce and Johnson (1978) believed that Pseudomonas
isolated from unhatched hen eggs were correlated with
decreased hatchability. Shaver Technical Bulletin (1982)
reported that Pseudomonas was a frequent cause of exploding
egg problems in many hatcheries. The importance of
Pseudomonas was also demonstrated in our laboratory from a
related research experiment to show its effect on
hatchability. When hatching eggs were immersed in a culture
of Pseudomonas solution before being placed in the

incubator, the rate of hatchability was severly reduced.

SUMMARY AND CONCLUSIONS

This experiment was conducted to determine the effect
of several preincubation washing treatments on the
hatchability of fertile duck eggs, on the early ten-day
performance of the hatched ducklings, and on the bacterial
content of dead embryos and to find whether a relationship
betrween the organisms isolated and embryonic mortality
could be established. Hatching eggs were dipped in one of
three commercially available detergents or detergent
sanitizers [LOC (Amway); HEDS (Bio-Lab); Roccal II 10% (Lehn
& Fink)] or warm tap water. The wash products were used
according to the manufacturers' directions.

The results of this experiment can be summarized as

follows:

I. Hatchability of Duck Eggs
1. HEDS and Roccal II have better sanitizing action on the
egg contaminating bacteria than that of warm water and LOC.
2. Embryonic mortality in all treatment groups
significantly increased during the last six days of
incubation as compared to embryonic mortality during the

first eight days of incubation.

50

51

3. Percent of hatchability achieved in this experiment
appeared to be normal compared to other investigations and
to commerical duck hatcheries.

H. Dipping the duck eggs in warm wawter, LOC, HEDS, and
Roccal II produced no significant differences in
hatchability.

5. Ducklings hatched from eggs treated with Roccal II did.
however, have a statistically higher initial ten-day weight

gain over ducklings hatched from other treatment groups.

II. Bacteriological Examination
1. The isolatin of Proteus and Streptococcus app;
increased with a significant increase in embryonic
mortality, near the end of incubation. This reslt indicates
that either Proteus spp. alone is important in causing
embryonic mortality, or that a synergistic action between
Proteus and §tggptococcus causes embryonic mortality. In
conclusion, further research should be directed toward the
role of Streptococcus and/or Proteus in influencing
embryonic mortality.
2. E. coli, Streptococcus, and Proteus spp;_were
frequently isolated during incubation. This result
indicates that feces are the major source of egg
contamination. It is suggested that the sanitary programs
on the farm and at the hatchery should be improved and

concentrated on reducing Proteus, Streptococcus, and E.coli.

52

3. The major contaminant isolated during incubation was
E. coli. However, it showed no relationship to the rate of
hatchability or embryonic mortality.

n. A direct relationship between the organisms isolated,
during incubation, and hatchability or embronic mortality
could not be established. The fact that an organism is
present in an egg which has failed to hatch does not
necessarily implicate the organism in the arrest of
embryonic development. Factors other than the presence of
contaminating organisms can influence hatchability. Some
organisms are known to reduce hatchability and cause embryo
mortality, while the importance of others is this respect in

uncertain.

REFERENCES

REFERENCES

Bliss, 0.1., 1937. Plant Protection (Linengrad), 12: 67.

Board, P.A. and Board, R.G., 1968. A Diagnostic Key for
Identifying Organisms Recovered from Rotten Eggs. Br.
Poult. Sci. 9: 111-120.

Board, R.G., 1965. The Properties and Classification of the
Predominant Bacteria in Rotten Eggs. J. Appl. Bact.

Board, R.G., 1968. In "Egg Quality: A Study of Hen's
Egg." T.C. Carter, ed. Oliver and Boyd, Edinburgh.
p. 133.

Board, R.G., 1969. The Microbiology of the Hen's Egg. Adv.
in Appl. Micro. 11: 295-81.

Brooks, J. and Taylor, D.J., 1955. Eggs and Egg-Product.
Rept. Food Invest. Bd. No. 60 HMSO, London.‘

Bruce, J. and Johnson, A.L., 1978. The Bacterial Flora of
Unhatched Eggs. Br. Poult. Sci. 19: 681-89.

Carter, G.R., 1978. Diagnostic Procedures in Veterinary
Microbiology, 2nd Ed., Chapters A, 5, 7, 15, 16, and
19. Charles C. Thomas, Publisher, Springfield,
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Chute, H.L. and Gershman, M., 1961. A New Approach to
Hatchery Sanitation. Poult. Sci. “0: 568-71.

Furuta, K. and Sato, S., 1977. Studies on the Disinfection
of Hatching Eggs. II the Effect of Disinfection by
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Furuta, KI. and Watanabe, KI., 1978. Studies on the
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53

5H

Furuta, K. and Maruyama, S., 1981. Bacterial Contamination
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25 .

Gentry, R.P., Mitrovic, M. and Bubash, G.E., 1962.
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Gill, J.L., 1978. Design and Analysis of Experiments in the
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Hacking, B., 1982. Sanitation for Hatchery and Farm.
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Harry, E.G., 1963. British Poultry Science u: 63-70.

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Pathologists, Texas A and M University, College
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Hodgetts, B., Chaplin, N.R.C., Jones, G.E. and Joyce, D.A.,
1976. Hatchery Problems in a Field Report. Vet.
Record. 98: 70-1.

Joyce, D.A. and Chaplin, N.R.C., 1978. Hygiene and
Hatchability of Duck Eggs--a Field Study. Vet. Rec.
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Moats, W.A., 1978. Egg Washing--A Review. J. of Food
Protection #1: 919-25.

Moudgal, R.P., Pandey, J.N. and Razdan, M.N., 1976. The
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Chicgen and Duck Eggs. Indian J. of Poult. Sci. 12:
178- 1c

Nesheim, M.C., Austic, R.E.'and Card, L.E., 1979. Poultry
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Schoenhard, D.E., 1979. Basic Laboratory Technique and
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55

Seviour, E.M. and Board, R.G., 1972. The Behavior of Mixed
Bacterial Infections in the Shell Membranes of the
Hen's E880 Brio POUlto $010 13: 33-”3.

Seviour, E.M., Sykes, F.R. and Board, R.G., 1972. A
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Shane, S.M., 1981. Broiler Chick Mortality Linked to
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