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The Effect of Broiler Breeder Flock Age and Length of Egg Storage on Egg Albumen During Early Incubation1
EDUCATION AND PRODUCTION The Effect of Broiler Breeder Flock Age and Length of Egg Storage on Egg Albumen During Early Incubation1 C. E. BENTON, JR. and J. BRAKE2 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608 1. Fresh eggs had significantly greater albumen height and significantly lower albumen pH than stored eggs in both experiments. These differences diminished with length of incubation. Because the blastoderm is located adjacent to the albumen, changes in the viscosity or pH of the albumen may play an integral role in determining the viability of the embryo during the very early stages of development. Incubation of fresh eggs without storage appears to expose the developing embryo to an inappropriate trans-vitelline membrane pH gradient and a thick albumen that may slow vital gas diffusion and limit nutrient availability. These conditions may cause an increased incidence of embryonic death.
(Key words: albumen, flock age, incubation, embryo, storage) 1996 Poultry Science 75:1069-1075
INTRODUCTION Egg storage prior to incubation has been reported to have both detrimental as well as beneficial effects (Brake et ah, 1993). Excessively long storage prior to incubation causes a decline in hatchability (Becker, 1964). On the contrary, eggs stored for a few days have been reported to hatch better than those set in an incubator soon after oviposition (Asmundson and Macllriath, 1948). Albumen is known to play two major roles in embryonic development. These roles are protection of the yolk and embryo from pathogenic microbes and provision of a supply of nutrients necessary for proper growth and development to the embryo. At oviposition, albumen's purpose is primarily antimicrobial in nature. Albumen viscosity is maximal at oviposition (Walsh, 1993) with a pH of about 7.6 (Stern, 1991). The chalaza, in combination with the generally high viscosity of the albumen, holds the yolk in a central position away from
Received for publication September 14, 1995. Accepted for publication May 17, 1996. 1 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products mentioned, nor criticism of similar products not mentioned. 2 To whom correspondence should be addressed.
the eggshell, where possible contamination may arise (Board and Fuller, 1974). The role of albumen then expands to include providing water, proteins, and a variety of nutrients to the developing embryo (Burley and Vadehra, 1989). Within a few days of storage, albumen viscosity decreases substantially (Hurnik et ah, 1978; Walsh, 1993) and reaches an alkaline plateau of about 9 (Stern, 1991). This process, known as albumen liquefaction, facilitates the movement of various nutritive substances from the albumen towards the embryo (Burley and Vadehra, 1989). Liquefaction might also reduce any physical barrier to gaseous diffusion of oxygen that the albumen may present (Meuer and Baumann, 1988). This basic pH presents the embryo with a 1,000-fold pH gradient across the layer of epiblast cells and adjacent perivitelline layer (Stern, 1991). In addition to these roles, water from egg albumen also forms the subembryonic fluid, which appears to be essential to proper embryonic development. Albumen quality (viscosity) is also influenced by storage (Walsh, 1993) and flock age (Romanoff and Romanoff, 1949). Albumen quality appears to decrease with flock age. Hurnik et ah (1978) found that the lower the albumen quality at oviposition, the more rapid its decline during storage. Associated with this decline during storage is a loss of weight due to the evaporative loss of water (Romanoff and Romanoff, 1949). These
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Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
ABSTRACT The objective of these two experiments was to determine the temporal changes in albumen during storage and early incubation as a means of understanding some of the effects of egg storage on early embryonic development. Eggs from 30- or 50-wk-old broiler breeder hens were incubated (37.5 C dry bulb, 30 C wet bulb) after storage for 0 (fresh) or 5 d (18 C, 75% RH) in Experiment 1. Albumen height, albumen pH, and egg weight loss were recorded at 2, 24, 48, and 66 h of incubation. The same measurements were taken on another group of eggs from 43-wk-old hens stored for 0 (fresh), 4, 8, or 12 d in Experiment 2. All hens were of the same strain. Egg weight loss during incubation was significantly greater in fresh eggs than in stored eggs in Experiment
1070
BENTON AND BRAKE TABLE 1. Albumen pH during the first 66 h of incubation of fresh eggs or of eggs stored for 5 d from young (30 wk) or old (SO wk) broiler breeder hens, Experiment l 1 Hours of incubation Main effect means Flock age, wk 30 50 Days of egg storage 0 5 Hours of incubation
n
2 h
24 h
48 h
66 h
n
X
24 24 SE
8.29 8.32 0.02
8.92 8.97 0.02
9.14 9.16 0.02
9.11 9.10 0.02
96 96 SE
8.86 8.89 0.01
24 24 SE 48 SE
7.68 8.93 0.02 8.30b 0.01
8.75 9.13 0.02 8.94b 0.01
9.07 9.23 0.02
9.08 9.13 0.02 9.10" 0.01
96 96 SE
8.65B 9.10* 0.01
9.15a 0.01
ab
' Main effect means with no common superscript differ significantly (P <, 0.05). - Main effect means with no common superscript differ significantly (P < 0.01). ^ E for n = 24, n = 48, or n = 96 calculated from flock age by egg storage by hours of incubation analysis.
A B
MATERIALS AND METHODS Experiment 1 Eggs were collected twice within a 5-d period and randomly assigned in a factorial arrangement of treatments consisting of flock age, storage time, and incubation time. Eggs were collected from 30- and 50-wk-old Arbor Acres hens, placed in flats, and stored at 18 C and 75% RH on the 1st d of the experiment. Eggs were again collected 5 d later. All eggs collected on 0 and 5 d were then weighed and incubation was initiated. The eggs were placed in an incubator that was operated at a dry bulb temperature of 37.5 ± 0.2 C and wet bulb temperature of 30.0 ± 0.2 C. At 2, 24, 48, and 66 h of incubation, eggs were randomly removed and analyzed in consecutive groups of four, one from each factorial treatment. An initial incubation time of 2 h was used instead of 0 h to ensure that pH would be recorded at the same egg temperature throughout the experiment. Eggs were quickly weighed and broken open to record the albumen height and pH of the thick albumen. The sensing bulb of a glass micro (5 fit) pH electrode3 connected to a pH meter4 was used to determine pH to the
3
Model MI-410, Microelectronics, Inc., Londonberry, NH 03053-2103. 4 Accumet Mini pH Meter Model 640 and certified buffer solutions, Fisher Scientific, Pittsburgh, PA 15219-4785.
nearest 0.01 unit. Albumen height was measured in the middle of the thick albumen equidistant from the outer edge of the albumen and the yolk. Weight loss during incubation was calculated. Twelve eggs were utilized per treatment at each sample time.
Experiment 2 Eggs were collected four times in 4-d intervals from a single Arbor Acres breeder flock managed as the flocks in Experiment 1, starting when the hens were 43 wk of age. Eggs were collected, weighed, placed in flats, and stored at 18 C and 75% RH. Eggs that had been stored 4,8, and 12 d were again weighed prior to the initiation of incubation with the fresh (0 d) eggs. Incubation conditions and all measurements taken thereafter were identical to those described for Experiment 1. Egg weight loss during storage and incubation was calculated. Twelve eggs were used per treatment for each incubation time.
Statistical Analysis The data of Experiments 1 and 2 were subjected to a multiway ANOVA using the General Linear Models (GLM) procedure of SAS® software (SAS Institute, 1989). The main effects in Experiment 1 were hen age, length of storage, and incubation time. The main effects in Experiment 2 were storage time and incubation time. Error for both experiments was estimated from the variation among eggs. Statements of statistical significance were based on P < 0.05 unless otherwise stated.
RESULTS Experiment 1 The main effects of flock age and storage of eggs prior to incubation on albumen pH during incubation is shown in Table 1. There was a significant interaction (P < 0.0001) between days of egg storage and hours of incubation
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changes that occur within the albumen probably have profound effects upon the developing embryo. The research described herein delineates some aspects of the dynamics of albumen liquefaction during early embryonic development by measuring weight loss, albumen height, and pH of incubated eggs stored for different lengths of time from broiler breeder hens of different ages. The objective of these experiments was to determine the temporal changes in albumen during storage and early incubation as a means of understanding some of the effects of egg storage on early embryonic development.
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HATCHING EGG STORAGE AND ALBUMEN TABLE 2. Albumen height during the first 66 h of incubation of fresh eggs or of eggs stored for 5 d from young (30 wk) or old (50 wk) broiler breeder hens, Experiment l 1 Hours of incubation n
Main effect means
2 h
24 h
48 h
66 h
n
X
(mm) Flock age, wk 30 50 Days of egg storage 0 5 Hours of incubation
24 24 SE
6.9 6.5 0.2
5.0 4.5 0.2
4.6 3.7 0.2
4.1 3.9 0.2
96 96 SE
5.2* 4.7" 0.1
24 24 SE
8.0 5.4 0.2 6.7* 0.1
5.1 4.4 0.2 4.8B 0.1
4.3 4.0 0.2 4.2C 0.1
4.4 3.6 0.2 4.0C 0.1
96 96 SE
5.5* 4.4B 0.1
48 SE
A_c
(Figure 1). Fresh eggs maintained a pH significantly below that of stored eggs through 48 h of incubation. Fresh eggs exhibited a rapid increase in albumen pH during this time. Stored eggs at 2 h of incubation had a pH of 8.93, which increased to 9.13 by 24 h of incubation. Fresh eggs had a lower pH (P < 0.01) of 7.68 at 2 h of incubation but increased dramatically to a pH of 9.07 by 48 h of incubation such that no difference in albumen pH existed between fresh and stored eggs at 66 h of incubation. There were no significant effects on albumen pH due to flock age. The main effects of flock age and storage of eggs prior to incubation on albumen height during incubation is illustrated in Table 2. A general decline in albumen height
X 9$ o. c
^
~ /
E
% V T D
/
3 8 -
/ /
r
0
^
i
= 30 30 50 50
— i
24
• 3 0 wk - 0 V 30 wk - 5 T 50 wk - 0 • 50 wk - 5
^ wk wk wk wk
48
-
during incubation was inconsistent for stored and fresh eggs as evidenced by a highly significant interaction (P < 0.0001) between days of egg storage and hours of incubation (Figure 2). Fresh eggs had an albumen height of 8.0 mm at 2 h of incubation, which decreased to 4.3 mm by 48 h. Stored eggs had an initial albumen height of 5.4 mm at 2 h and decreased substantially less than fresh eggs throughout incubation to a height of 3.6 mm by 66 h incubation. Most of this decline occurred during the first 24 h of incubation. Albumen height was significantly higher in 30-wk-old hens than in 50-wk-old hens (P < 0.01). The effect of flock age and storage of eggs prior to incubation on egg weight loss is shown in Table 3. Fresh eggs lost significantly more weight than stored eggs.
0 5 0 5
day day day day
day day day day
1
72
Hours FIGURE 1. Albumen pH during the first 66 h of incubation of fresh eggs (0 d) or of eggs stored for 5 d from young (30 wk) or old (50 wk) hens in Experiment 1. There was an interaction between day of storage and hour of incubation (P < 0.0001). Vertical bars represent the SE for each mean of 12 eggs.
Hours FIGURE 2. Albumen height during the first 66 h of incubation of fresh eggs (0 d) or of eggs stored for 5 d from young (30 wk) or old (50 wk) hens in Experiment 1. There was an interaction between day of storage and hour of incubation (P < 0.0001). Vertical bars represent the SE for each mean of 12 eggs.
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Main effect means with no common superscripts differ significantly (P S 0.01). ^ E for n = 24, n = 48, or n = 96 calculated from flock age by egg storage by h of incubation analysis.
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BENTON AND BRAKE
DISCUSSION
9.5 i ?. 9.0 0 4 T B D 12
24
48
Days Days Days Days
72
FIGURE 3. Albumen pH during the first 66 h of incubation of eggs from 43-wk-old hens stored 0,4,8, and 12 d in Experiment 2. There was an interaction between day of storage and hour of incubation (P ^ 0.0001). Vertical bars represent the SE for each mean of 12 eggs.
There were no differences in average initial egg weight (data not shown).
Experiment 2 The overall effect of storage of eggs on albumen pH during incubation is illustrated in Table 4. An increase in albumen pH was inconsistent over hours of incubation for different lengths of egg storage as evidenced by a highly significant interaction (P < 0.0001) between days of egg storage and hours of incubation (Figure 3). A small increase of pH in eggs stored for 4,8, or 12 d through 48 h of incubation was observed, but eggs set without storage exhibited a dramatic increase in albumen pH during the first 24 h followed by a slowing rate of increase throughout the remainder of the incubation period (Figure 3). This pattern of pH flux for eggs set without storage resulted in a lower pH overall (Table 4). Eggs stored for 8 and 12 d exhibited a higher average pH than those stored for 0 or 4 d. The overall effect of storage of eggs on albumen height during incubation is illustrated in Table 4. There was a highly significant interaction (P < 0.0002) between egg storage and hour of incubation (Figure 4). Albumen height at 2 h of incubation was much higher in eggs stored for 0 d (fresh) than in eggs stored for 4,8, or 12 d. A large decline during the first 24 h was observed in fresh eggs. Overall, albumen height was greater for eggs stored for 0 d than for eggs stored for 4 d, which was in turn greater than that of eggs stored 8 or 12 d (Table 4). Egg weight loss during incubation and total weight loss during storage and incubation combined is shown in Table 4. A stepwise increase in total weight loss was observed with increasing length of storage and hour of incubation. There were no differences in average initial egg weight (data not shown).
# V T •
0 4 B 12
Day Day Day Day
Hours FIGURE 4. Albumen height during the first 66 h of incubation of eggs from 43-wk-old hens stored 0,4,8, and 12 d in Experiment 2. There was an interaction between day of storage and hour of incubation (P < 0.0002). Vertical bars represent the SE for each mean of 12 eggs.
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Hours
Avian egg albumen undergoes its most rapid changes during the first 24 h of incubation if set fresh or similar changes during the first 4 d of storage at 18 C and 75% RH (Figures 3 and 4) Fresh egg albumen is buffered most weakly between pH 7.0 and 9.0 and most strongly at pH 6 and 10 (Cotterill et al, 1959). Under normal incubation conditions, buffering capacity will be the major determinant of the rate of pH increase at any given time. This phenomenon explains the observed interaction between storage and incubation with respect to albumen pH (Figures 1 and 3). Stored eggs, whose pH has risen during storage, initiate incubation near pH 9 and there is little change in pH due to the strong buffering capacity of the egg albumen at this pH. Fresh eggs initiate incubation at a pH at which buffering capacity is weaker. This fact accounts for the sharp increase in pH of fresh egg albumen during the first 24 h of incubation. As incubation proceeds and albumen pH of the fresh eggs approaches 9, the change in pH slows. Albumen height is a relative measure of albumen viscosity. In theory, the more viscous the albumen the greater the barrier it presents to gaseous diffusion of oxygen to the blastoderm (Meuer and Baumann, 1988). Fresh eggs begin incubation with an albumen height significantly higher than that observed for stored eggs (Tables 2 and 4). Such a high viscosity might prevent an adequate supply of oxygen from reaching the embryo, thereby resulting in mortality prior to any appreciable formation of blood (membrane dead). By 24 h of
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HATCHING EGG STORAGE AND ALBUMEN TABLE 3. Incubation weight loss during the first 66 h of incubation of fresh eggs or of eggs stored for 5 d from young (30 wk) or old (50 wk) broiler breeder hens, Experiment 1 Hours of incubation Variable
n
2 h
24 h
48 h
,m
n
X
s
^ S^
Main effect means Flock age, wk 30 50 Days of iegg storage 0 5 Hours of ncubation
24 24 SE3
57 62 137
487 564 137
985 1,017 137
1,490 1,333 137
96 96 SE*
755 741 68
24 24 SE3 48 SE5
73 47 137
554 497 137 525C 10
1,080 922 137 1,001B 10
1,501 1,322 137 1,412A 10
96 96 SE^
802 697 68
12 12 12 12 SEi
72 42 73 52 193
514 459 593 534 193
1,076 894 1,084 951 193
1,702 1,278 1,301 1,366 193
48 48 48 48 SE2
841 668 763 726 97
60° 10
means 1
Egg storage, d 0 5 0 5
A D
~ Main effect means with no common superscript differ significantly (P < 0.0001). SE for n = 12 calculated from flock age by egg storage by hours of incubation analysis. 2 SE for n = 48 calculated from flock age by egg storage analysis. 3 SE for n = 24 from flock age or days of storage by hours of incubation analysis. 4 SE for n = 68 from flock age, days of storage, or hours of incubation analysis. 5SE for n = 48 from h of incubation analysis. !
incubation, such a diffusive barrier would no longer exist, as albumen height decreases to a height equivalent to that of eggs stored for 4 or more d (Figures 2 and 4). This difference in viscosity might provide one explanation why the incubation of fresh eggs often result in increased "membrane dead" embryos when compared to eggs stored prior to incubation. Sufficient amounts of oxygen to support a basal rate of metabolism during the very early stages of incubation may be essential (Meuer and Baumann, 1988). It is important to make a distinction between diffusion of oxygen into the egg and the diffusion of oxygen to the embryo proper. High quality albumen does not seem to retard the diffusion of water out of the egg. If diffusive water loss is proportional to oxygen diffusion into the egg (Romanoff and Romanoff, 1949), then albumen does not appear to interfere with this exchange. During the first 24 h of incubation, when albumen height decreases the most for fresh set eggs (Table 4), there is only a small increase in weight loss (Table 4). However, thick albumen may prevent oxygen that passes through the eggshell from reaching the cells of the blastoderm. The decrease in albumen height due to storage or incubation can be attributed to the increase in albumen pH. However, this decrease cannot account for the lower albumen height observed in fresh eggs from the 50-wk-old breeder flocks (Table 1). There is no flock age effect on albumen pH. Perhaps albumen undergoes a general decline in protein tertiary structure or integrity
with increasing flock age as a result of an "exhaustion phenomenon" as the hen is unable to cope with the high demands of continuous production (Wilcox and Wilson, 1962). The degradation of albumen viscosity and its associated pH increase enhance the flow of water and solutes across the vitelline membrane or into the yolk for utilization (Burley and Vadehra, 1989; Stern, 1991). Freshly set eggs may endure a delay in the release of these energy sources from the high viscosity albumen. This delay may also partially account for the high incidence of poor chick quality and "membrane deads" associated with freshly set eggs (Walsh, 1993). The inevitable increase in albumen p H may have a unique role in embryogenesis. The asymmetry in pH between yolk and albumen established by the albumen pH increase may be necessary to facilitate certain transport functions across the vitelline membrane. Rymen and Stockx (1974) measured ion potentials across isolated vitelline membranes and concluded that it was an asymmetrical ion-exchange membrane. Conversely, an extended exposure to a basic p H such as that of the eggs stored for 8 and 12 d in Experiment 2 (Table 4) may be detrimental. Evidence of necrosis and regressive changes in the blastoderm have been reported as a result of extended storage (Funk and Biellier, 1944; Arora and Kosin, 1966). Weight loss in these two experiments was inconsistent. There did not appear to be a direct relationship
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:Interaction Flock age, w k 30 30 50 50
66 h
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BENTON AND BRAKE TABLE 4. Albumen pH, albumen height, incubation weight loss, and total weight loss through 66 h of incubation for eggs from 43-wk-old 1broiler breeder hens stored 0, 4, 8 and 12 d, Experiment 2 Hours of incubation E
Variable Albumen p H
gg storage
n
(d) 0 4 8 12
24 h
2 h
48 h
66 h
5c
5c
12 12 12 12 48
7.79 8.92 9.05 9.08 8.71 d
8.82 9.12 9.21 9.19 9.09 '
9.10 9.25 9.28 9.29 9.23»
9.13 9.19 9.22 9.22 9.19b
8.70C 9.12" 9.19* 9.20A SE2 = 0.03
5c
12 12 12 12 48
6.8 4.7 4.7 4.4 5.1*
4.8 4.8 3.9 4.3 4.5B
4.4 4.2 3.7 3.7 4.0^
4.4 3.8 3.6 3.6 4.0C
5.1" 4.4b 4.0c 4.0* SE2 = 0.1
3<
12 12 12 12 48
90 74 61 72 74D
X"
12 12 12 12 48
90 567 895 1,302 714D
Albumen height, mm
Incubation weight loss, mg 0 4 8 12
1,177 1,119 1,182 1,114 1,148"
1,524 1,522 1,458 1,369 1,468*
855 512 810 778 SE2 = 35
1,177 628 1,753 969 2,084 1,358 2,286 1,696 1,163C 1,780B
1,524 1,977 2,260 2,423 2,046*
855 d 1,271c 1,649" 1,927' SE2 = 35
628 534 539 556 564C
Total weight loss, mg 0 4 8 12
"-dMain effect means with no common superscript differ significantly (P 2 0.05). *~DMain effect means with no common superscript differ significantly (P £ 0.01). Significant interactions for Albumen pH: Days of egg storage by hours of incubation (P <. 0.0001), for Albumen height: Days of egg storage by hours of incubation (P <. 0.0002). 2 SE for n = 48 calculated from flock age by egg storage by hours of incubation analysis.
between water loss and albumen p H and height changes. The exact nature of the relationship between egg storage and its influence on water movement warrants further research. This study suggests that there are exact timedependent changes with respect to albumen viscosity and p H that may increase access of the blastoderm to oxygen, establish essential gradients, and support the acquisition of nutrients by the early chick embryo during the first days of incubation. Incubation of eggs prior to these changes may result in insufficient oxygen or metabolite acquisition to meet the metabolic demands of the early chick embryo, which may result in increased mortality, extended incubation, and poor chick quality.
REFERENCES Arora, L. L., and I. L. Kosin, 1966. Developmental response of early turkey and chicken embryos to preincubation holding of eggs: Inter- and intra-species differences. Poultry Sri. 45:958-970. Asmundson, V. S., and J. J. Macllriath, 1948. Preincubation tests with turkey eggs. Poultry Sri. 27:394-401. Becker, W. A., 1964. The storage of white leghorn chicken eggs in plastic bags. Poultry Sri. 43:1109-1112. Board, R. G„ and R. Fuller, 1974. Non-specific antimicrobial defenses of the avian egg, embryo, and neonate. Biol. Rev. 49:15-49.
Brake, J., T. J. Walsh, and S. V. Vick, 1993. Relationship of egg storage time, storage conditions, flock age, eggshell and albumen characteristics, incubation conditions, and machine capacity to broiler hatchability—Review and model synthesis. Zootech. Int. 16(1):30-41. Burley, R. W., and D. V. Vadehra, 1989. Pages 65-145 in: The Avian Egg. Chemistry and Biology. John Wiley and Sons, New York. NY. Cotterill, O. J., F. A. Gardner, F. E. Cunningham, and E. M. Funk, 1959. Titration curves and turbidity of whole egg white. Poultry Sci. 38:836-842. Funk, E. M., and H. V. Biellier, 1944. The minimum temperature for embryonic development in the domestic fowl (Gallus domesticus). Poultry Sci. 23:538-540. Hurnik, G. I., B. S. Reinhart, and J. F. Humik, 1978. Relationship between albumen quality and hatchability in fresh and stored eggs. Poultry Sci. 57:854-857. Meuer, H. J., and R. Baumann, 1988. Oxygen pressure in intraand extraembryonic blood vessels of early chick embryo. Resp. Physiol. 71:331-342. Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. John Wiley and Sons Inc., New York, NY. Rymen, T., and J. Stockx, 1974. The vitelline membrane of the unfertilized hen's egg; electrolyte and water transport. Ann. Biol. Anim. Biochim. Biophys. 14:651-666. SAS Institute, 1989. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Stern, C. D., 1991. The sub-embryonic fluid of the domestic
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
0 4 8 12
HATCHING EGG STORAGE AND ALBUMEN fowl and its relationship to the early development of the embryo. Pages 81-90 in: Avian Incubation. Carfax Publishing Co., London, UK. Walsh, T. J. 1993. The effects of flock age, storage humidity, carbon dioxide, and length of storage on albumen
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characteristics, weight loss and embryonic development of broiler eggs. Masters thesis, North Carolina State University, Raleigh, NC 27695-7608. Wilcox, F. H., and H. R. Wilson, 1962. Changes in albumen quality with time. Poultry Sci. 41:883-886.
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
(Key words: albumen, flock age, incubation, embryo, storage) 1996 Poultry Science 75:1069-1075
INTRODUCTION Egg storage prior to incubation has been reported to have both detrimental as well as beneficial effects (Brake et ah, 1993). Excessively long storage prior to incubation causes a decline in hatchability (Becker, 1964). On the contrary, eggs stored for a few days have been reported to hatch better than those set in an incubator soon after oviposition (Asmundson and Macllriath, 1948). Albumen is known to play two major roles in embryonic development. These roles are protection of the yolk and embryo from pathogenic microbes and provision of a supply of nutrients necessary for proper growth and development to the embryo. At oviposition, albumen's purpose is primarily antimicrobial in nature. Albumen viscosity is maximal at oviposition (Walsh, 1993) with a pH of about 7.6 (Stern, 1991). The chalaza, in combination with the generally high viscosity of the albumen, holds the yolk in a central position away from
Received for publication September 14, 1995. Accepted for publication May 17, 1996. 1 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products mentioned, nor criticism of similar products not mentioned. 2 To whom correspondence should be addressed.
the eggshell, where possible contamination may arise (Board and Fuller, 1974). The role of albumen then expands to include providing water, proteins, and a variety of nutrients to the developing embryo (Burley and Vadehra, 1989). Within a few days of storage, albumen viscosity decreases substantially (Hurnik et ah, 1978; Walsh, 1993) and reaches an alkaline plateau of about 9 (Stern, 1991). This process, known as albumen liquefaction, facilitates the movement of various nutritive substances from the albumen towards the embryo (Burley and Vadehra, 1989). Liquefaction might also reduce any physical barrier to gaseous diffusion of oxygen that the albumen may present (Meuer and Baumann, 1988). This basic pH presents the embryo with a 1,000-fold pH gradient across the layer of epiblast cells and adjacent perivitelline layer (Stern, 1991). In addition to these roles, water from egg albumen also forms the subembryonic fluid, which appears to be essential to proper embryonic development. Albumen quality (viscosity) is also influenced by storage (Walsh, 1993) and flock age (Romanoff and Romanoff, 1949). Albumen quality appears to decrease with flock age. Hurnik et ah (1978) found that the lower the albumen quality at oviposition, the more rapid its decline during storage. Associated with this decline during storage is a loss of weight due to the evaporative loss of water (Romanoff and Romanoff, 1949). These
1069
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
ABSTRACT The objective of these two experiments was to determine the temporal changes in albumen during storage and early incubation as a means of understanding some of the effects of egg storage on early embryonic development. Eggs from 30- or 50-wk-old broiler breeder hens were incubated (37.5 C dry bulb, 30 C wet bulb) after storage for 0 (fresh) or 5 d (18 C, 75% RH) in Experiment 1. Albumen height, albumen pH, and egg weight loss were recorded at 2, 24, 48, and 66 h of incubation. The same measurements were taken on another group of eggs from 43-wk-old hens stored for 0 (fresh), 4, 8, or 12 d in Experiment 2. All hens were of the same strain. Egg weight loss during incubation was significantly greater in fresh eggs than in stored eggs in Experiment
1070
BENTON AND BRAKE TABLE 1. Albumen pH during the first 66 h of incubation of fresh eggs or of eggs stored for 5 d from young (30 wk) or old (SO wk) broiler breeder hens, Experiment l 1 Hours of incubation Main effect means Flock age, wk 30 50 Days of egg storage 0 5 Hours of incubation
n
2 h
24 h
48 h
66 h
n
X
24 24 SE
8.29 8.32 0.02
8.92 8.97 0.02
9.14 9.16 0.02
9.11 9.10 0.02
96 96 SE
8.86 8.89 0.01
24 24 SE 48 SE
7.68 8.93 0.02 8.30b 0.01
8.75 9.13 0.02 8.94b 0.01
9.07 9.23 0.02
9.08 9.13 0.02 9.10" 0.01
96 96 SE
8.65B 9.10* 0.01
9.15a 0.01
ab
' Main effect means with no common superscript differ significantly (P <, 0.05). - Main effect means with no common superscript differ significantly (P < 0.01). ^ E for n = 24, n = 48, or n = 96 calculated from flock age by egg storage by hours of incubation analysis.
A B
MATERIALS AND METHODS Experiment 1 Eggs were collected twice within a 5-d period and randomly assigned in a factorial arrangement of treatments consisting of flock age, storage time, and incubation time. Eggs were collected from 30- and 50-wk-old Arbor Acres hens, placed in flats, and stored at 18 C and 75% RH on the 1st d of the experiment. Eggs were again collected 5 d later. All eggs collected on 0 and 5 d were then weighed and incubation was initiated. The eggs were placed in an incubator that was operated at a dry bulb temperature of 37.5 ± 0.2 C and wet bulb temperature of 30.0 ± 0.2 C. At 2, 24, 48, and 66 h of incubation, eggs were randomly removed and analyzed in consecutive groups of four, one from each factorial treatment. An initial incubation time of 2 h was used instead of 0 h to ensure that pH would be recorded at the same egg temperature throughout the experiment. Eggs were quickly weighed and broken open to record the albumen height and pH of the thick albumen. The sensing bulb of a glass micro (5 fit) pH electrode3 connected to a pH meter4 was used to determine pH to the
3
Model MI-410, Microelectronics, Inc., Londonberry, NH 03053-2103. 4 Accumet Mini pH Meter Model 640 and certified buffer solutions, Fisher Scientific, Pittsburgh, PA 15219-4785.
nearest 0.01 unit. Albumen height was measured in the middle of the thick albumen equidistant from the outer edge of the albumen and the yolk. Weight loss during incubation was calculated. Twelve eggs were utilized per treatment at each sample time.
Experiment 2 Eggs were collected four times in 4-d intervals from a single Arbor Acres breeder flock managed as the flocks in Experiment 1, starting when the hens were 43 wk of age. Eggs were collected, weighed, placed in flats, and stored at 18 C and 75% RH. Eggs that had been stored 4,8, and 12 d were again weighed prior to the initiation of incubation with the fresh (0 d) eggs. Incubation conditions and all measurements taken thereafter were identical to those described for Experiment 1. Egg weight loss during storage and incubation was calculated. Twelve eggs were used per treatment for each incubation time.
Statistical Analysis The data of Experiments 1 and 2 were subjected to a multiway ANOVA using the General Linear Models (GLM) procedure of SAS® software (SAS Institute, 1989). The main effects in Experiment 1 were hen age, length of storage, and incubation time. The main effects in Experiment 2 were storage time and incubation time. Error for both experiments was estimated from the variation among eggs. Statements of statistical significance were based on P < 0.05 unless otherwise stated.
RESULTS Experiment 1 The main effects of flock age and storage of eggs prior to incubation on albumen pH during incubation is shown in Table 1. There was a significant interaction (P < 0.0001) between days of egg storage and hours of incubation
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changes that occur within the albumen probably have profound effects upon the developing embryo. The research described herein delineates some aspects of the dynamics of albumen liquefaction during early embryonic development by measuring weight loss, albumen height, and pH of incubated eggs stored for different lengths of time from broiler breeder hens of different ages. The objective of these experiments was to determine the temporal changes in albumen during storage and early incubation as a means of understanding some of the effects of egg storage on early embryonic development.
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HATCHING EGG STORAGE AND ALBUMEN TABLE 2. Albumen height during the first 66 h of incubation of fresh eggs or of eggs stored for 5 d from young (30 wk) or old (50 wk) broiler breeder hens, Experiment l 1 Hours of incubation n
Main effect means
2 h
24 h
48 h
66 h
n
X
(mm) Flock age, wk 30 50 Days of egg storage 0 5 Hours of incubation
24 24 SE
6.9 6.5 0.2
5.0 4.5 0.2
4.6 3.7 0.2
4.1 3.9 0.2
96 96 SE
5.2* 4.7" 0.1
24 24 SE
8.0 5.4 0.2 6.7* 0.1
5.1 4.4 0.2 4.8B 0.1
4.3 4.0 0.2 4.2C 0.1
4.4 3.6 0.2 4.0C 0.1
96 96 SE
5.5* 4.4B 0.1
48 SE
A_c
(Figure 1). Fresh eggs maintained a pH significantly below that of stored eggs through 48 h of incubation. Fresh eggs exhibited a rapid increase in albumen pH during this time. Stored eggs at 2 h of incubation had a pH of 8.93, which increased to 9.13 by 24 h of incubation. Fresh eggs had a lower pH (P < 0.01) of 7.68 at 2 h of incubation but increased dramatically to a pH of 9.07 by 48 h of incubation such that no difference in albumen pH existed between fresh and stored eggs at 66 h of incubation. There were no significant effects on albumen pH due to flock age. The main effects of flock age and storage of eggs prior to incubation on albumen height during incubation is illustrated in Table 2. A general decline in albumen height
X 9$ o. c
^
~ /
E
% V T D
/
3 8 -
/ /
r
0
^
i
= 30 30 50 50
— i
24
• 3 0 wk - 0 V 30 wk - 5 T 50 wk - 0 • 50 wk - 5
^ wk wk wk wk
48
-
during incubation was inconsistent for stored and fresh eggs as evidenced by a highly significant interaction (P < 0.0001) between days of egg storage and hours of incubation (Figure 2). Fresh eggs had an albumen height of 8.0 mm at 2 h of incubation, which decreased to 4.3 mm by 48 h. Stored eggs had an initial albumen height of 5.4 mm at 2 h and decreased substantially less than fresh eggs throughout incubation to a height of 3.6 mm by 66 h incubation. Most of this decline occurred during the first 24 h of incubation. Albumen height was significantly higher in 30-wk-old hens than in 50-wk-old hens (P < 0.01). The effect of flock age and storage of eggs prior to incubation on egg weight loss is shown in Table 3. Fresh eggs lost significantly more weight than stored eggs.
0 5 0 5
day day day day
day day day day
1
72
Hours FIGURE 1. Albumen pH during the first 66 h of incubation of fresh eggs (0 d) or of eggs stored for 5 d from young (30 wk) or old (50 wk) hens in Experiment 1. There was an interaction between day of storage and hour of incubation (P < 0.0001). Vertical bars represent the SE for each mean of 12 eggs.
Hours FIGURE 2. Albumen height during the first 66 h of incubation of fresh eggs (0 d) or of eggs stored for 5 d from young (30 wk) or old (50 wk) hens in Experiment 1. There was an interaction between day of storage and hour of incubation (P < 0.0001). Vertical bars represent the SE for each mean of 12 eggs.
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Main effect means with no common superscripts differ significantly (P S 0.01). ^ E for n = 24, n = 48, or n = 96 calculated from flock age by egg storage by h of incubation analysis.
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BENTON AND BRAKE
DISCUSSION
9.5 i ?. 9.0 0 4 T B D 12
24
48
Days Days Days Days
72
FIGURE 3. Albumen pH during the first 66 h of incubation of eggs from 43-wk-old hens stored 0,4,8, and 12 d in Experiment 2. There was an interaction between day of storage and hour of incubation (P ^ 0.0001). Vertical bars represent the SE for each mean of 12 eggs.
There were no differences in average initial egg weight (data not shown).
Experiment 2 The overall effect of storage of eggs on albumen pH during incubation is illustrated in Table 4. An increase in albumen pH was inconsistent over hours of incubation for different lengths of egg storage as evidenced by a highly significant interaction (P < 0.0001) between days of egg storage and hours of incubation (Figure 3). A small increase of pH in eggs stored for 4,8, or 12 d through 48 h of incubation was observed, but eggs set without storage exhibited a dramatic increase in albumen pH during the first 24 h followed by a slowing rate of increase throughout the remainder of the incubation period (Figure 3). This pattern of pH flux for eggs set without storage resulted in a lower pH overall (Table 4). Eggs stored for 8 and 12 d exhibited a higher average pH than those stored for 0 or 4 d. The overall effect of storage of eggs on albumen height during incubation is illustrated in Table 4. There was a highly significant interaction (P < 0.0002) between egg storage and hour of incubation (Figure 4). Albumen height at 2 h of incubation was much higher in eggs stored for 0 d (fresh) than in eggs stored for 4,8, or 12 d. A large decline during the first 24 h was observed in fresh eggs. Overall, albumen height was greater for eggs stored for 0 d than for eggs stored for 4 d, which was in turn greater than that of eggs stored 8 or 12 d (Table 4). Egg weight loss during incubation and total weight loss during storage and incubation combined is shown in Table 4. A stepwise increase in total weight loss was observed with increasing length of storage and hour of incubation. There were no differences in average initial egg weight (data not shown).
# V T •
0 4 B 12
Day Day Day Day
Hours FIGURE 4. Albumen height during the first 66 h of incubation of eggs from 43-wk-old hens stored 0,4,8, and 12 d in Experiment 2. There was an interaction between day of storage and hour of incubation (P < 0.0002). Vertical bars represent the SE for each mean of 12 eggs.
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Hours
Avian egg albumen undergoes its most rapid changes during the first 24 h of incubation if set fresh or similar changes during the first 4 d of storage at 18 C and 75% RH (Figures 3 and 4) Fresh egg albumen is buffered most weakly between pH 7.0 and 9.0 and most strongly at pH 6 and 10 (Cotterill et al, 1959). Under normal incubation conditions, buffering capacity will be the major determinant of the rate of pH increase at any given time. This phenomenon explains the observed interaction between storage and incubation with respect to albumen pH (Figures 1 and 3). Stored eggs, whose pH has risen during storage, initiate incubation near pH 9 and there is little change in pH due to the strong buffering capacity of the egg albumen at this pH. Fresh eggs initiate incubation at a pH at which buffering capacity is weaker. This fact accounts for the sharp increase in pH of fresh egg albumen during the first 24 h of incubation. As incubation proceeds and albumen pH of the fresh eggs approaches 9, the change in pH slows. Albumen height is a relative measure of albumen viscosity. In theory, the more viscous the albumen the greater the barrier it presents to gaseous diffusion of oxygen to the blastoderm (Meuer and Baumann, 1988). Fresh eggs begin incubation with an albumen height significantly higher than that observed for stored eggs (Tables 2 and 4). Such a high viscosity might prevent an adequate supply of oxygen from reaching the embryo, thereby resulting in mortality prior to any appreciable formation of blood (membrane dead). By 24 h of
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HATCHING EGG STORAGE AND ALBUMEN TABLE 3. Incubation weight loss during the first 66 h of incubation of fresh eggs or of eggs stored for 5 d from young (30 wk) or old (50 wk) broiler breeder hens, Experiment 1 Hours of incubation Variable
n
2 h
24 h
48 h
,m
n
X
s
^ S^
Main effect means Flock age, wk 30 50 Days of iegg storage 0 5 Hours of ncubation
24 24 SE3
57 62 137
487 564 137
985 1,017 137
1,490 1,333 137
96 96 SE*
755 741 68
24 24 SE3 48 SE5
73 47 137
554 497 137 525C 10
1,080 922 137 1,001B 10
1,501 1,322 137 1,412A 10
96 96 SE^
802 697 68
12 12 12 12 SEi
72 42 73 52 193
514 459 593 534 193
1,076 894 1,084 951 193
1,702 1,278 1,301 1,366 193
48 48 48 48 SE2
841 668 763 726 97
60° 10
means 1
Egg storage, d 0 5 0 5
A D
~ Main effect means with no common superscript differ significantly (P < 0.0001). SE for n = 12 calculated from flock age by egg storage by hours of incubation analysis. 2 SE for n = 48 calculated from flock age by egg storage analysis. 3 SE for n = 24 from flock age or days of storage by hours of incubation analysis. 4 SE for n = 68 from flock age, days of storage, or hours of incubation analysis. 5SE for n = 48 from h of incubation analysis. !
incubation, such a diffusive barrier would no longer exist, as albumen height decreases to a height equivalent to that of eggs stored for 4 or more d (Figures 2 and 4). This difference in viscosity might provide one explanation why the incubation of fresh eggs often result in increased "membrane dead" embryos when compared to eggs stored prior to incubation. Sufficient amounts of oxygen to support a basal rate of metabolism during the very early stages of incubation may be essential (Meuer and Baumann, 1988). It is important to make a distinction between diffusion of oxygen into the egg and the diffusion of oxygen to the embryo proper. High quality albumen does not seem to retard the diffusion of water out of the egg. If diffusive water loss is proportional to oxygen diffusion into the egg (Romanoff and Romanoff, 1949), then albumen does not appear to interfere with this exchange. During the first 24 h of incubation, when albumen height decreases the most for fresh set eggs (Table 4), there is only a small increase in weight loss (Table 4). However, thick albumen may prevent oxygen that passes through the eggshell from reaching the cells of the blastoderm. The decrease in albumen height due to storage or incubation can be attributed to the increase in albumen pH. However, this decrease cannot account for the lower albumen height observed in fresh eggs from the 50-wk-old breeder flocks (Table 1). There is no flock age effect on albumen pH. Perhaps albumen undergoes a general decline in protein tertiary structure or integrity
with increasing flock age as a result of an "exhaustion phenomenon" as the hen is unable to cope with the high demands of continuous production (Wilcox and Wilson, 1962). The degradation of albumen viscosity and its associated pH increase enhance the flow of water and solutes across the vitelline membrane or into the yolk for utilization (Burley and Vadehra, 1989; Stern, 1991). Freshly set eggs may endure a delay in the release of these energy sources from the high viscosity albumen. This delay may also partially account for the high incidence of poor chick quality and "membrane deads" associated with freshly set eggs (Walsh, 1993). The inevitable increase in albumen p H may have a unique role in embryogenesis. The asymmetry in pH between yolk and albumen established by the albumen pH increase may be necessary to facilitate certain transport functions across the vitelline membrane. Rymen and Stockx (1974) measured ion potentials across isolated vitelline membranes and concluded that it was an asymmetrical ion-exchange membrane. Conversely, an extended exposure to a basic p H such as that of the eggs stored for 8 and 12 d in Experiment 2 (Table 4) may be detrimental. Evidence of necrosis and regressive changes in the blastoderm have been reported as a result of extended storage (Funk and Biellier, 1944; Arora and Kosin, 1966). Weight loss in these two experiments was inconsistent. There did not appear to be a direct relationship
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:Interaction Flock age, w k 30 30 50 50
66 h
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BENTON AND BRAKE TABLE 4. Albumen pH, albumen height, incubation weight loss, and total weight loss through 66 h of incubation for eggs from 43-wk-old 1broiler breeder hens stored 0, 4, 8 and 12 d, Experiment 2 Hours of incubation E
Variable Albumen p H
gg storage
n
(d) 0 4 8 12
24 h
2 h
48 h
66 h
5c
5c
12 12 12 12 48
7.79 8.92 9.05 9.08 8.71 d
8.82 9.12 9.21 9.19 9.09 '
9.10 9.25 9.28 9.29 9.23»
9.13 9.19 9.22 9.22 9.19b
8.70C 9.12" 9.19* 9.20A SE2 = 0.03
5c
12 12 12 12 48
6.8 4.7 4.7 4.4 5.1*
4.8 4.8 3.9 4.3 4.5B
4.4 4.2 3.7 3.7 4.0^
4.4 3.8 3.6 3.6 4.0C
5.1" 4.4b 4.0c 4.0* SE2 = 0.1
3<
12 12 12 12 48
90 74 61 72 74D
X"
12 12 12 12 48
90 567 895 1,302 714D
Albumen height, mm
Incubation weight loss, mg 0 4 8 12
1,177 1,119 1,182 1,114 1,148"
1,524 1,522 1,458 1,369 1,468*
855 512 810 778 SE2 = 35
1,177 628 1,753 969 2,084 1,358 2,286 1,696 1,163C 1,780B
1,524 1,977 2,260 2,423 2,046*
855 d 1,271c 1,649" 1,927' SE2 = 35
628 534 539 556 564C
Total weight loss, mg 0 4 8 12
"-dMain effect means with no common superscript differ significantly (P 2 0.05). *~DMain effect means with no common superscript differ significantly (P £ 0.01). Significant interactions for Albumen pH: Days of egg storage by hours of incubation (P <. 0.0001), for Albumen height: Days of egg storage by hours of incubation (P <. 0.0002). 2 SE for n = 48 calculated from flock age by egg storage by hours of incubation analysis.
between water loss and albumen p H and height changes. The exact nature of the relationship between egg storage and its influence on water movement warrants further research. This study suggests that there are exact timedependent changes with respect to albumen viscosity and p H that may increase access of the blastoderm to oxygen, establish essential gradients, and support the acquisition of nutrients by the early chick embryo during the first days of incubation. Incubation of eggs prior to these changes may result in insufficient oxygen or metabolite acquisition to meet the metabolic demands of the early chick embryo, which may result in increased mortality, extended incubation, and poor chick quality.
REFERENCES Arora, L. L., and I. L. Kosin, 1966. Developmental response of early turkey and chicken embryos to preincubation holding of eggs: Inter- and intra-species differences. Poultry Sri. 45:958-970. Asmundson, V. S., and J. J. Macllriath, 1948. Preincubation tests with turkey eggs. Poultry Sri. 27:394-401. Becker, W. A., 1964. The storage of white leghorn chicken eggs in plastic bags. Poultry Sri. 43:1109-1112. Board, R. G„ and R. Fuller, 1974. Non-specific antimicrobial defenses of the avian egg, embryo, and neonate. Biol. Rev. 49:15-49.
Brake, J., T. J. Walsh, and S. V. Vick, 1993. Relationship of egg storage time, storage conditions, flock age, eggshell and albumen characteristics, incubation conditions, and machine capacity to broiler hatchability—Review and model synthesis. Zootech. Int. 16(1):30-41. Burley, R. W., and D. V. Vadehra, 1989. Pages 65-145 in: The Avian Egg. Chemistry and Biology. John Wiley and Sons, New York. NY. Cotterill, O. J., F. A. Gardner, F. E. Cunningham, and E. M. Funk, 1959. Titration curves and turbidity of whole egg white. Poultry Sci. 38:836-842. Funk, E. M., and H. V. Biellier, 1944. The minimum temperature for embryonic development in the domestic fowl (Gallus domesticus). Poultry Sci. 23:538-540. Hurnik, G. I., B. S. Reinhart, and J. F. Humik, 1978. Relationship between albumen quality and hatchability in fresh and stored eggs. Poultry Sci. 57:854-857. Meuer, H. J., and R. Baumann, 1988. Oxygen pressure in intraand extraembryonic blood vessels of early chick embryo. Resp. Physiol. 71:331-342. Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. John Wiley and Sons Inc., New York, NY. Rymen, T., and J. Stockx, 1974. The vitelline membrane of the unfertilized hen's egg; electrolyte and water transport. Ann. Biol. Anim. Biochim. Biophys. 14:651-666. SAS Institute, 1989. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Stern, C. D., 1991. The sub-embryonic fluid of the domestic
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0 4 8 12
HATCHING EGG STORAGE AND ALBUMEN fowl and its relationship to the early development of the embryo. Pages 81-90 in: Avian Incubation. Carfax Publishing Co., London, UK. Walsh, T. J. 1993. The effects of flock age, storage humidity, carbon dioxide, and length of storage on albumen
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characteristics, weight loss and embryonic development of broiler eggs. Masters thesis, North Carolina State University, Raleigh, NC 27695-7608. Wilcox, F. H., and H. R. Wilson, 1962. Changes in albumen quality with time. Poultry Sci. 41:883-886.
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