EDUCATION AND PRODUCTION

Effect of a Quaternary Ammonium Sanitizer for

Hatching Eggs on Their Contamination, Permeability,
Water Loss, and Hatchability 1

J. BRAKE2 and B. W. SHELDON3

North Carolina State University,

Raleigh, North Carolina 27695-7608

(Received for publication March 20, 1989)

ABSTRACT The present study tested the biocidal effectiveness of the quaternary ammonium Hatching Egg
Sanitizer Spray® (HES Spray®) in disinfecting broiler hatching eggs and in terms of its effects on eggshell
permeability, water loss, and hatchability. The application of HES at a 1.5% or a 3.0% concentration resulted
in significant reductions in the total aerobic counts on the egg surface of 98.1% and 99.9%, respectively,
within 30 min of application. Molds and yeasts were significantly reduced by 3.0% HES at 14 days of
incubation.
Significant reductions in total aerobic counts on the egg surface due to 1.5% and 3.0% HES were also
observed on eggs that were allowed to "sweat." The 3.0% HES concentration also reduced coliforms on egg
surfaces.
Hatchability of fertile eggs from a 32-wk-old flock was significantly increased, over 6.0%, by spraying
1.5% or 3.0% HES in comparison to controls that were not sprayed, with no significant difference in
hatchability due to treatment observed in eggs from flocks that were 36,42,46, or 62 wk of age. This change in
hatchability associated with spraying HES may be due to a change in eggshell permeability (respiration)
caused by an interaction of HES with the eggshell cuticle.
(Key words: disinfection, hatching eggs, hatching egg spray, hatchability, contamination)

1990 Poultry Science 69:517-525

INTRODUCTION Aspergillus fumigatus. All of these microorga-

The importance of an effective sanitation
program in the hatchery to achieve a high level
of hatchability and to ensure the production of
high-quality chicks has been firmly established
(Wright and Epps, 1958; Avens et al., 1975;
Mowry et al., 1980). The environment of a
chick hatchery and its surroundings may
become contaminated with microorganisms
from various sources. Microorganisms on or in
a few hatching eggs can be easily distributed
throughout the hatchery by air movements
during hatching, thus contaminating or infect-
ing all other chicks in the hatcher (Magwood,
1964). Included in the list of such microorga-
nisms are several pathogens such as Escheri-
chia coli, Staphylocci sp., Streptococci sp., and
'Paper Number 12088 of the Journal Series, North
Carolina Agricultural Research Service, Raleigh, NC
27695-7643. Trade names used in this publication do not
imply endorsement of the products mentioned nor criticism
of similar products not mentioned. Presented at the 77th
Annual Meeting of the Poultry Science Association, Baton
Rouge, Louisiana, July 25-29, 1988.
Department of Poultry Science.
department of Food Science.
nisms are suspected of being associated with
early chick mortality. Chute and Gershman
(1961) concluded that it was more desirable to
incubate and hatch eggs in a clean environ-
ment, regardless of whether or not the patho-
genicity of the microorganisms present within
the hatchery has been confirmed.
Effective cleaning and sanitation programs
are vitally needed in the poultry hatchery.
Included in any hatchery sanitation program is
the application of effective disinfectants. His-
torically, formaldehyde was the recommended
fumigant used in hatcheries due to its bacterial
effectiveness and ease of application (Funk and
Irwin, 1955). However, several disadvantages
are associated with using this sanitizer, such as
the disinfectant vapors being inhaled by
hatchery workers, the lingering unpleasant
odor left by the disinfectant following decon-
tamination, the difficulty in venting (Walker,
1944; Ernst et at., 1974), and most importantly
recent actions by the Environmental Protection
Agency that regulate the use of formaldehyde
under the Toxic Substances Control Act due to
its suspected carcinogenicity (Chemical and
Engineering News, 1984). Furthermore, virtu-
ally all disinfectants are not registered for use

517
518 BRAKE AND SHELDON
as a sanitizer for hatching eggs. This limits the
legal options available to a hatchery manager
apart from washing the eggs.
The eggshell to which sanitizers are applied
is part of the respiratory structure of the
embryo and consists of an overlying cuticle, a
crystalline calcium-carbonate layer with two
underlying proteinaceous membranes (Taylor,
1970). The cuticle has been described as an
uneven organic layer composed primarily of
protein with some polysaccharide and lipid
material (Baker and Balch, 1962; Simons,
1971). The cuticle may either bridge the pore
openings or extend down into them to form a
plug (Board, 1982). The literature contains
reports of cuticle providing resistance to
microbial invasion and contamination (Fromm,
1963; Mayes and Takeballi, 1983).
The cuticle may have a role in regulating
the gaseous exchange (conductance) between
the egg and the embryo. The cuticle has been
described as a barrier to the loss of water vapor
(Simons, 1971; Board and Halls, 1973; Meir et
at, 1984) and to vital gas exchange (Roman-
off, 1943; Fromm, 1963). Conversely, other
reports discuss enhanced water-vapor loss via
the cuticle (Bryant and Sharp, 1934; Marshall
and Cruickshank, 1938). These reported differ-
ences may be due to environmental or age-
related influences. Deeming (1987) showed a
significant increase in conductance due to
cuticle removal in a dessicated environment;
but Sparks and Board (1984) failed to show
differences under similar circumstances. These
studies did not take into account the age-
related changes resulting from cuticle removal
in broiler hatching eggs, as reported by Peebles
and Brake (1986). It was apparent that the
cuticle may be affected by the application of
sanitizers so as to alter eggshell permeability
and embryonic development.
The three primary objectives of the present
study were: 1) to determine the effect of a
registered, quaternary ammonium hatching-egg
sanitizer (HES) on eggshell microbial counts;
2) to assess the effects of egg-sweating on the
microbial counts of HES on sanitized eggs;
and 3) to assess the effects of HES on eggshell
permeability. Hatchability and water loss were
assessed as an aid in quantitating these effects.
4
BioLab, Inc., Decatur, GA 30031-1489.
5
ModeI F-80 Spray Pal, Delta Industries, Philadelphia,
PA 19146).
"Difco Laboratories, Detroit, MI.
7
Natureform, Jacksonville, FL 32202.
MATERIALS AND METHODS
Experiment 1
The objective of this investigation was to
evaluate the biocidal effectiveness of Hatching
Egg Sanitizer Spray® (HES Spray®)4 in water
on eggs at an application dosage of either 15.6
mL (1.5%) or 31.2 mL (3.0%) per L. Freshly laid
hatching eggs (300) from a commercial strain of
broiler-breeder hens (Arbor Acres) fed a stan-
dard breeder ration (16% CP, 2.89 Kcal of ME
per g, 3.2% calcium, .45% available phospho-
rus) were collected from the North Carolina
State University research farm on the day of lay
and randomly assigned to one of three equally
sized groups. Fecal-contaminated eggshells or
eggshells with visible checks were discarded,
and any remaining nesting debris was gendy
removed by hand from the eggshell surface.
Two hundred eggs were immediately placed
on sanitized plastic egg flats. For 100 eggs, each
one was sprayed with a 1.5% or a 3.0% per-liter
aqueous solution (25 C) of HES using a hand-
sprayer.5 A room temperature of 25 C was
selected, since this disinfectant does not need to
penetrate the eggshell and contains no tempera-
ture-dependent ingredients. All eggs were com-
pletely wetted with the disinfectant and were
allowed to air dry at room temperature. The third
group of 100 eggs was not treated, and served as
controls.
A whole-egg washing technique was used to
recover the shell-associated microorganisms for
estimating the total aerobic plate count, pre-
sumptive coliforms, and fungal counts of 5 eggs
per treatment. Individual eggs from each treat-
ment were placed in sterile plastic bags and were
rinsed with 18 mL of sterile .1% peptone water.
The rinse from each egg was serially diluted in
.1% peptone water. The total aerobes (37 C, 48
h), presumptive coliforms (37 C, 18 h), and yeast
and molds (25 C, 72 h) were enumerated using
the plate count, violet red bile, or potato dextrose
agars,6 respectively. All counts were reported
per egg by multiplying the counts per mL of
rinse by 18. Eggs were sampled at 30 min post
spray application (designated as 0 time), at
placement in incubators (4 days post disinfec-
tion) and at 7 and 14 days of incubation. Prior to
incubation, the eggs were stored at 18.3 C (60%
relative humidity) in an egg cooler for 4 days.
All eggs were placed in a common minisetter-
hatcher7 and were incubated at 37.5 C and at
53% relative humidity. The experiment was
 DISINFECTING HATCHING EGGS 519

repeated a second time using similar procedures, area was calculated from the following algo-
except that the eggs were sampled at 24 h post rithm: SA = 4.835 w662; where w = fresh egg
disinfection instead of at 4 days post disinfec- weight (grams) (Paganelli etai, 1974). The eggs
tion. The eggs were held in the egg cooler for were then sprayed as described in Experiment 1.
only 1 day. All other sampling times remained After air-drying, the eggs were again placed in
the same. dessicators and were reweighed at 24 and
at 96 h. Eggshell permeability was calculated
Experiment 2 and was compared with the previous determina-
tions. This experiment was replicated with eggs
The objective of this experiment was to from the same flock at 44 wk of age.
assess the effects of "egg-sweating" on the
lethality of HES as a disinfectant for egg Experiment 4
surfaces. Freshly laid hatching eggs (300) were
collected from the same research facility as for The objective of this investigation was
Experiment 1 and were randomly grouped into similar to that of Experiment 3, except that
four treatments (75 eggs per treatment). Treat- eggshell permeability was determined only after
ments 1 and 2 (controls) were not sanitized with spraying. Fresh eggs from the same broiler-
HES. Eggs from Treatment 2 were refrigerated breeder flock were used for Replication 1 (48 wk
at 4.4 C for 24 h while those from Treatment 1 of age), Replication 2 (51 wk of age), and
were held at room temperature for 24 h. Eggs Replication 3 (55 wk of age). Eighty eggs per
assigned to the remaining two treatments were replicate were randomly divided into three
sanitized, as described in Experiment 1, with a treatment groups of 16 eggs each, as described
1.5% or 3.0% HES application dosage, respec- for Experiment 3; then, the eggs were sprayed,
tively, and were refrigerated for 24 h at 4.4 C. No air-dried, placed in dessicators, and weighed at
precautions were taken to cover the eggs during 24 and 96 h of dessication, respectively. Shell
this 24-h storage period. Following refrigera- permeability was measured, calculated in the
tion, the eggs were left uncovered so they could same manner as with the previous experiments.
equilibrate at room temperature to promote
"egg-sweating," and were then air-dried. Aero- Experiment 5
bic plate counts and coliforms were enumerated
after drying using the same methods as in The objective of this experiment was to
Experiment 1 (approximately 24 h after treat- evaluate the effect of spraying HES in water at
ment). 1.5% or at 3.0% on hatchability and water loss.
One hundred seventy-six eggs (two replicate
Experiment 3 trays) from each of two broiler-breeder flocks,
32 and 62 wk of age, were sprayed immediately
The objective of this study was to evaluate prior to setting for each HES treatment. The eggs
the effect of spraying HES in water at 1.5% or were incubated (as described in Experiment 1).
3.0% on eggshell permeability. Forty, fresh, Random samples of 15 eggs per treatment were
hatching eggs were obtained from a weighed before spraying and at 6 days of
38-wk-old, broiler-breeder flock. Eight eggs incubation, respectively, in order to calculate
were randomly assigned to each of the following water loss, expressed as milligrams of weight
treatments: Control, not sprayed; sprayed with loss per day per cubic centimeter of eggshell
1.5% HES in water; and sprayed with 3.0% HES surface area. At hatch, all of the unhatched eggs
in water. The eggs were weighed initially, were opened and were examined for evidence of
placed in dessicators containing fresh dessicant embryonic development. Eggs were character-
(CaSC^), and maintained at 25 C. These eggs ized as infertile, early dead (1 to 7 days), late
were individually weighed at 24 and 96 h to dead (8 to 20 days), or pipped (the beak had
determine the initial eggshell permeability. penetrated the shell).
Eggshell permeability was expressed as the
milligrams of water lost per day between 24 and
Experiment 6
96 h per torr per square centimeter of eggshell
surface area (Paganelli etai, 1974). Weight loss This study was conducted in a similar fashion
was corrected to standard temperature and to Experiment 5, with the exception that 352
pressure (Ar et al., 1974). The eggshell surface eggs (two replicate trays) from a 36-wk-old
520 BRAKE AND SHELDON
broiler-breeder flock were set per treatment
(identical treatments as in Experiment 5) with 30
eggs per treatment weighed and the weight loss
calculated. All unhatched eggs were opened and
were examined for evidence of embryonic
development.
Experiment 7
In this investigation, 528 eggs (three replicate
trays) from a 42-wk-old, broiler-breeder flock
were set for each of three treatments. Weight
loss for the eggs was not determined. Fertility,
hatchability, and embryonic mortality were
determined, as described for Experiment 5.
Experiment 8
In this experiment, 704 eggs (four replicate
trays) per treatment frcm a 46-wk-old broiler-
breeder flock were set as the controls using 1.5%
HES in water treatments. Fertility, hatchability,
and embryonic mortality were determined, as
described for Experiment 5. Incubation condi-
tions were altered in that the relative humidity
was increased to 58% during the first 18 days.
Statistical Analyses
The data from Experiments 1 and 2 were
subjected to ANOVA using a randomized
complete block design with the Statistical
Analysis System-ANOVA procedure (SAS In-
stitute, 1982). Differences among means were
partitioned by the Waller-Duncan K-ratio t test
(SAS Institute, 1982). The model was tested for
treatment and for sampling-day sources of
variation. The remaining model variation (treat-
ment-by-sampling day interaction) was pooled
and was used as the error term. The data from
Experiments 3 through 8 were subjected to a
one-way ANOVA using the variation within
treatment as the error term with the general
linear model procedure of the Statistical Analy-
sis System (SAS Institute, 1982). Differences
among means were partitioned using the Duncan
option. Statements of statistical significance
were based on P<.05 unless otherwise noted.
Salmonella sp. and E. coll are classified as coliforms.
RESULTS AND DISCUSSION
Experiment 1
This experiment was replicated twice; but
since sampling times varied between replicates,
the data from the two replicates were not pooled
and averaged. At either application dosage, HES
significantly reduced the aerobic plate count
(APC) within 30 min of application in both
experimental replications (Tables 1 and 2).
Compared to the untreated control eggs, the
eggshells of the HES-sanitized eggs had micro-
bial populations that were over 98% lower,
representing log reductions ranging from 1.73 to
2.95. The higher application dose of HES (3.0%)
exhibited the greater kill, although the differ-
ences between the 1.5% and 3.0% rates were
small. Similar findings were evident at each
sampling time. Furthermore, significant reduc-
tions in the APC were detected at each
subsequent sampling time, including the control
eggs.
Several factors may have contributed to the
microbial reductions seen in the control eggs at 7
and at 14 days of incubation, respectively. A
possible explanation may be associated with the
environmental conditions surrounding the set-
ters. The gradual drying of the cuticle during
incubation probably led to the destruction of
some of the surface microflora through dessica-
tion. Furthermore, the lack of available nutrients
for the surface microflora might explain these
reductions. Similar microbial reductions in the
microbial contaminants on eggshells during
incubation were reported by Gentry and Quarles
(1972) and by Furata and Maruyama (1981).
Also, residual disinfectant activity may have
been present. The HES was effective in signifi-
candy reducing the APC compared to the control
eggs.
The effects of HES on presumptive coliforms
are summarized in Tables 1 and 2. In general,
coliforms comprised less than 1 log or only
about .01% of the APC. However, the cleanli-
ness of the eggshell surface usually dictates the
level of coliforms present.8 The amount of
organic matter present on the egg surface at the
time of sanitation will directly influence the
effectiveness of the disinfectant. One or more
eggs in all treatments at each sampling time had
no coliform contamination.
Of the three classes of microorganisms
examined in the present study, HES appeared to
have the least effect on molds and yeast. Molds
comprised the majority of such microorganisms
cultured. Compared with the controls, the level
of reduction resulting from the application of
 DISINFECTING HATCHING EGGS 521
TABLE 1. Experiment 1: Effectiveness of hatching-egg sanitizer spray (HES) as a disinfectant for eggs.
Replicate 1: Geometric means logio ± standard deviation (range)

Sampling time1
2
Treatment 30 min Set 7 days 14 days
(Aerobic plate count , cfu per egg)
Control 5.17 ± .es"* 5.32 ± . 3 2 " 4.26 ± .30ay 3.47 ± .53**
(4.37 - 6.13) (4.77 - 5.59) (3.89 - 4.65) (3.05 - 4.28)
1.5% HES 3.44 ± .74 bx 2.84 ± .73bxy 2.43 ± .52by 2.47 ± .22bxy
(2.52 - 4.46) (2.09 - 3.88) (1.90 - 2.95) (2.13 - 2.73)
3.0% HES 2.22 ±1.17c 2.05 ± .45 b 2.13 ± .38 b 2.10 ± .37b
( .95 - 3.62) (1.43 - 2.66) (1.73 - 2.73) (1.79 - 2.69)
(Coliforms, cfu
Control .50 ± .72 .64 ±1.44 0 .73 ± .74
(0 - 1.56) (0 - 3.21) (0 1.73)
1.5% HES .98 ±1.35 .44 ± .61 0 0
(0 - 2.69) (0 - 1.25)
3.0% HES 0 .19 ± .42 0 .25 ± .56
(0 - 0.95) (0 - 1.25)
cfu per egg)
Control 1.99 ± .31 2.62 ± .85 a 2.06 ± .16 2.09 ± .42"
(1.73 - 2.46) (1.73 - 3.09) (1.91 - 2.33) (1.43 - 2.48)
1.5% HES 2.41 ± .61 1.90 ±1.16 ab 1.65 ± .54 1.36 ± .81 a b
(1.73 - 3.09) (0 - 2.97) (0.95 - 2.38) (0 - 2.13)
b
3.0% HES 1.47 ± .85 .98 ± .99 1.50 ± .43 .93 ± .93 b
(0 - 2.18) (0 - 2.13) ( .95 - 1.95) (0 - 2.16)
a,b c
' Means in the same column and classification with no common superscripts are significantly different (P<.05). N = 5
eggs per treatment per sampling time.
x,y
Means in the same row with no common superscripts are significantly different (P<.05).
30 min = 30 min after disinfection; set = time at placement in setter; 7 and 14 days = days of incubation.
2
Control = no treatment; 1.5% HES = 15.6 mLofHES per L of water; 3.0% HES = 31.2 mL of HES per L of water. HES
was obtained from BioLab, Inc., Decatur, GA.

HES ranged from 61.1% (.41 log) for the 1.5%
application dose at 7 days in Replication 1 to
97.7% (1.64 logs) for the 3.0% HES application
(Day 4, Replication 1). The higher the HES
application concentration, the greater the kill
(similar to the findings observed for the APC).
This was evident in 50% of the eggs sampled.
These findings indicate that HES was effective
immediately in sanitizing the eggshell surface
following application. In most cases, the higher
the disinfectant dosage, the greater the microbial
destruction.
Experiment 2
The findings of the "egg-sweating" experi-
ment are presented in Table 3. Both Controls 1
and 2 had significandy higher APC by a factor of
2.04 to 4.09 logs than the HES-sanitized eggs.
These reductions were similar to the findings
from Replication 2 of Experiment 1, demon-
strating that "egg-sweating" did not influence
the effectiveness of the HES. Eggs sanitized
with 3.0% HES had significantly lower coliform
counts than Control 1 and 2. Eggs exposed to the
surrounding environment for 24 h exhibited
significantly higher coliform counts than the
refrigerated controls (Control 2), suggesting that
refrigeration reduced microbial growth on the
eggshell surface.
Experiments 3 and 4
The ability of HES to influence water loss
was determined. With each egg serving as its
own control (via a permeability measurement
before spraying), only the 3.0% HES treatment
significandy decreased eggshell permeability in
Replication 1; whereas both dosages of HES
significantly decreased eggshell permeability in
Replication 2 (Table 4). Decreased permeability
was also observed with HES in Replication 1 of
Experiment 4 (Table 5). However, lower per-
meability was observed in control eggs in
Replication 2 and in Replication 3 of Experi-
ment 4.
522 BRAKE AND SHELDON

TABLE 2. Experiment 1: Effectiveness of hatching-egg sanitizer spray (HES) as a disinfectant for eggs.
Replicate 2: Geometric means logjg ± standard deviation (range)

Sampling time'
Treatment2 30 min Set 7 days 14 days
(Aerobic plate count , cfu per egg)
Control 5.31 ± .39" 5.11 ± .41axy 4.61 ± .23axy 4.27 ± .30 av
(4.64 - 5.65) (4.46 - 5.55) (4.28 - 4.90) (3.84 - 4.58)
1.5% HES 3.57 ±1.73 2.67 ± .32 b 2.45 ± .28 b 2.79 ± .77b
(1.55 - 5.59) (2.38 - 3.20) (2.13 - 2.85) (2.13 - 3.95)
3.0% HES 3.52 ±1.18X 2.20 ± .65bxy 1.73 ± .82 cv 1.22 ± .82°y
(2.28 - 4.83) (1.43 - 3.09) (0.95 - 2.91) (0 - 2.10)
(Coliforms, cfu per egg)
Control .91 ± .91 1.10 ±1.50 .84 ± .86 0
(0 - 2.03) (0 - 2.84) (0 - 2.00)
1.5% HES .95 ±1.65 0 .38 ± .52 .25 ± .56
(0 - 3.81) (0 - 0.95) (0 - 1.26)
3.0% HES .19 ± .42 .19 ± .42 .25 ± .56 0
(0 - 0.95) (0 - 0.95) (0 - 1.26)

Control 1.94 ± .34 2.25 ± .81 1.95 ± .46a 1.70 ± .53 a

(1.55 - 2.30) (1.26 - 3.42) (1.43 - 2.56) (0.95 - 2.30)
1.5% HES 1.68 ±1.39 1.42 ± .93 1.42 ± .86a 1.28 ± .87 ab
(0 - 3.86) (0 - 2.50) (0 - 2.30) (0 - 2.33)
3.0% HES 1.04 ±1.07 1.33 ± .85 .35 ± .77b .44 ± .62b
(0 - 2.30) (0 - 1.95) (0 - 1.73) (0 - 1.26)
abc
' ' Means in the same column and classification with no common superscripts are significantly different (P<05). N = 5
eggs per treatment per sampling time.
x,v
Means in the same row with no common superscripts are significantly different (P<.05).
30 min = 30 min after disinfection; set = time at placement in setter; 7 and 14 days = days of incubation.
2
Control = no treatment; 1.5% HES = 15.6 mL of HES per L of water; 3.0% HES = 31.2 mL of HES per L of water. HES
was obtained from BioLab, Inc., Decatur, GA.

TABLE 3. Experiment 2: Effect of "egg-sweating" on the effectiveness of hatching-egg sanitizer spray (HES)
as a disinfectant: Geometric means logio ± standard deviation (range)

Temperature
treatment' HES 2 APC (cfu per egg) Coliforms (cfu per egg)
Control 1 5.66 ± .49 A 2.98 ± .35 A
25 to 25 C (4.84 - 5.86) (2.57 - 3.41)
Control 2 4.86 ± .47 A .96 ± .96 B
4.4 to 25 C (4.27 - 5.46) (0 - 2.0)
4.4 to 25 C 1.5% 2.82 ± ,70B .35 ± .77 Bc
(1.91 - 3.80) (0 - 1.73)
4.4 to 25 C 3.0% 1.57 ± .97 C 0C
(0 - 2.57)
A,B,c
Means in the same column with no common superscripts are significantly different (P<.01). N = 5 eggs per
treatment.
'Control 1 = no HES, no refrigeration (25 C), no sweating of eggs collected the day before. Control 2 = refrigerated (4.4
C) overnight to promote sweating. Remaining eggs refrigerated overnight as Control 2.
2
1.5% HES = 15.6 mL of HES per L of water-application dosage, refrigerated (4.4 C), sweating; 3.0% HES = 31.2 mL of
HES per L of water-application dosage, refrigerated (4.4 C), sweating. Control 1 and Control 2 received no HES. HES was
obtained from BioLab, Inc., Decatur, GA.
J
APC = aerobic plate count.
 DISINFECTING HATCHING EGGS 523
TABLE 4. Experiment 3: Eggshell permeability before and after the application
of hatching-egg sanitizer spray (HES)

Eggshell permeability
(mg of H2O per day per per torr per cm2)
Replicate l 1 Replicate 2 2
3
HES treatment Before After Difference Before After Difference
Control .079* .104* .024* .088* .126* .038*
1.5% .101* .107* .005* .103* .103* .000b
3.0% .107* .071 b -.036 b .100b .106* .006b
a,b c
' Means in the same column with no common superscripts are significantly different (P<.05).
'Replicate 1 = 38 wk of age.
2
Replicate 2 = 44 wk of age.
3
HES in water sprayed on each egg after an initial determination of eggshell permeability. 1.5% = 15.6 mL of HES per
liter; 3.0% = 31.2 mL of HES per liter. HES was obtained from BioLab, Inc., Decatur, GA.

The outermost surface material of the egg-
shell is the cuticle, which must be contacted by
HES to cause the observed effects. The func-
tional properties of the cuticle are not static. The
cuticle of the eggshell has been reported to differ
in its functional properties as a broiler-breeder
flock ages; in untreated eggshells the cuticle has
been shown to contribute to lower permeability
early in lay and to higher permeability late in lay
(Peebles and Brake, 1986). The period between
45 and 50 wk of age may be transitional with
regard to permeability.
The control data shown in Tables 4 and 5 tend
to support this concept. The data suggested that
HES further decreased permeability in the
young flock and increased permeability in the
older flock, measured at a very low humidity.
This can only be explained by a chemical
interaction with the proteinaceous cuticle (Baker
and Balch, 1962), since a simple blocking of the
pores or removal of the cuticle would not be
consistent with the data. The data from Experi-
ment 3 also suggested that the resistance of the
cuticle to water-vapor diffusion during a long
period at low humidity declines and that HES
ameliorates this decline.
Experiments 5 to 8
The effects of HES on hatchability and on
water loss are shown in Table 6. Applications of
1.5% or of 3.0% HES increased the hatchability
of fertile eggs in the 32-wk-old flock but did not
have a significant effect at other flock ages. This
improved hatchability in the youngest flock
appeared to be due primarily to a reduction in
early embryonic mortality. As has been demon-
strated, increased water loss improves the
hatchability of eggs by reducing early embry-
onic mortality in eggs from young, but not from
old, broiler-breeder flocks (Vick and Brake,
1986). In contrast with the data from Experi-
ments 3 and 4, which were conducted at a very
low humidity, the data from Experiments 5 and 6
show average water losses at all ages of 4.24,
4.53, and 4.41 mg per day per cm2 for control,
eggs sprayed with 1.5% HES, and eggs sprayed
with 3.0% HES, respectively, for the first six
days of incubation at 53% relative humidity. The
significantly increased hatchability for eggs
sprayed with HES in the youngest flock only
might be associated with a slightly increased
water loss for these eggs. The pooled hatchabil-
ity of fertile eggs from the 32-, 36-, and
42-wk-old flocks for the controls, eggs sprayed
TABLE 5. Experiment 4: Eggshell permeability after
an application of hatching-egg sanitizer spray (HES)
Eggshell permeability
HES (mg of H2O per day per torr per cm )
Treatment1 Replicate l 2 Replicate 2 3 Replicate 3 4
Control .116* .092b .087b
1.5% .109*b .109* .114*
3.0% .101 b .104* .110*
*'bMeans in the same column with no common super-
scripts are significantly different (P<.05).
'HES in water sprayed on each egg prior to the
determination of eggshell permeability. 1.5% = 15.6 mL of
HES per L; 3.0% = 31.2 mL of HES per L. HES was obtained
from BioLab, Inc., Decatur, GA.
2
Replicate 1 = 48 wk of age.
3
Replicate 2 = 51 wk of age.
4
Replicate 3 = 55 wk of age.
524 BRAKE AND SHELDON

TABLE 6. Experiments 5 to 8: Effect of applying hatching-egg sanitizer spray (HES)

on the hatchability and water loss of eggs from young and old flocks

Flock Water loss

age Experiment HES Hatch of Early Late (mg per day
(wk) number treatment1 Fertile fertile eggs dead dead Pipped per CHIT
._ (%\
32 5 Control 98.9 90.2b 6.8 2.3 .6 4.38
1.5% 97.2 96.5a 2.3 .6 .6 4.81
3.0% 97.7 96.4a 2.3 2.9 .6 4.46
36 6 Control 96.3 95.9 1.1 2.3 .0 4.43
1.5% 96.3 93.2 3.7 2.3 .6 4.58
3.0% 95.4 96.4 1.4 1.7 .3 4.53
42 7 Control 90.8 93.0 1.1 2.3 .0 ND
1.5% 89.8 94.2 3.6 1.3 .4 ND
3.0% 89.5 92.3 4.0 2.4 .6 ND
46 8 Control 89.6 93.3 3.6 1.7 .7 ND
1.5% 85.4 92.7 3.6 2.6 .0 ND
62 5 Control 94.9 86.2 8.0 5.1 .0 3.92
1.5% 82.4 85.5 8.5 1.7 1.7 4.21
3.0% 89.7 84.1 7.4 .4 2.9 4.23
a
' Means in the same column and age with no common superscripts are significantly different (P<.05).
'HES in water sprayed on each egg immediately prior to placement in the incubator. 1.5% = 15.6 mL of HES per L; 3.(
31.2 mL of HES per L. HES was obtained from BioLab, Inc., Decatur, GA.
Determined as the weight loss from setting to 6 days of incubation.
3
ND = not determined.

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midity would be appropriate in order to balance
the effect (Peebles et al, 1987).
In summary, spraying hatching eggs with
HES significantly reduced bacterial contamina-
tion on the eggshell surface but affected the
functional properties of the eggshell with respect
to water loss and gas exchange. The latter result
complicates the situation regarding any uniform
application of an HES sanitation program. The
effects of HES on eggshell permeability must be
considered when using HES as a sanitizer for
eggs.
ACKNOWLEDGMENTS
The authors express their appreciation to
BioLab Inc., Decatur, Georgia for its partial
financial support of the present research. The
excellent technical assistance of Susan Hale,
Susan Creech, and Grace Brockman is also
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