Effects of broiler breeder age and length of egg storage on albumen characteristics and hatchability

Effects of Broiler Breeder Age and Length of Egg Storage on Albumen Characteristics and Hatchability ˜ O,* L. T. GAMA,† and M. CHAVEIRO SOARES†,1 C. LAPA *Faculdade de Medicina Veterina´ria, 1100 Lisboa Codex, Portugal, and †Instituto Superior de Agronomia, 1399 Lisboa Codex, Portugal ABSTRACT Two experiments were conducted with the purpose of determining the influence of broiler breeder age and storage time on egg albumen characteristics, embryonic mortality, and hatchability. Eggs from four commercial flocks of the same strain (Peterson × Minibro Shaver), under the same management and nutritional regimen, were incubated after storage at 16 C and 78% relative humidity, for periods of 0 (fresh), 1, 4, or 8 d. Albumen height and albumen pH were recorded immediately prior to each setting in Experiment 1 (eggs collected from 32- and 54-wk-old flocks) and at 0, 12, 24, 38, and 60 h of incubation in Experiment 2 (eggs from 42- and 59-wk-old flocks). Overall, albumen pH was 0.95 higher in eggs stored for 8 d than in fresh eggs, but most of this increase occurred during the first 4 d of storage. At 0 d of

storage, pH increased (P < 0.05) with flock age, but age differences were negligible at 8 d of storage. Albumen height decreased with hen age and storage time (P < 0.05). Embryo viability was affected by the storage length by flock age interaction, such that longer periods of storage decreased viability in all flock ages. Decreased viability was pronounced in older flocks, with regression coefficients of viability on days of storage being –0.82 and –1.92% at 32 and 59 wk of age, respectively. The detrimental effects of storage time on viability in older flocks were mostly due to an increased incidence of culls and embryonic losses at all stages. Present results suggest that declines in hatchability with presetting storage start 1 d after lay, possibly due to deterioration in egg albumen quality.

(Key words: broiler breeder, hatching egg storage, albumen height, albumen pH, hatchability) 1999 Poultry Science 78:640–645

derm and malformations in the embryo (Arora and Kosin, 1966a; Mather and Laughlin, 1979) with increased cell necrosis (Arora and Kosin, 1966b). On the other hand, after oviposition, carbon dioxide is released from the egg, resulting in an increase in albumen pH from about 7.6 to 9.5 within a short period of time, whereas the yolk remains slightly acid, at a pH around 6.5. Therefore, a 1,000-fold hydrogen ion concentration gradient (3 pH units) may exist across the blastoderm (Stern, 1991), in its intermediary position between albumen and yolk. This pH gradient means that the blastoderm faces a very alkaline environment on the dorsal side of the epiblast, whereas its basal surface faces a slightly acid environment. Meijerhof (1994a) suggested that a specific pH gradient over the blastoderm, attained in association with an albumen pH of approximately 8.2 (Sauveur et al., 1967; Gillespie and McHanwell, 1987), is optimal for early embryo development and subsequent hatching. The rise in albumen pH with storage time and hen age is associated with a decrease in albumen height and viscosity. Albumen liquefaction probably facilitates the movement of nutrients from the albumen to the blastoderm (Brake et al., 1997) and may reduce resistance to gaseous diffusion (Meuer and Baumann, 1988). The

INTRODUCTION Storage of hatching eggs is a necessary part of commercial incubation, even though storage length and conditions may influence postovipositional mortality of embryos. The mechanisms behind this influence are not completely clear; however, it is well known that a dramatic decline in hatchability is observed in broiler breeder eggs stored for extended periods. Furthermore, conflicting results have been reported regarding the effects of short-term storage on hatching performance (Oluyemi and George, 1972; Mayes and Takeballi, 1984). The effects of preincubational egg storage on embryonic viability depend on storage time, environmental conditions, hen age, and strain (Brake et al., 1997). It has been suggested that the decrease in viability of the embryo may be caused by changes in the embryo or by changes in certain physical aspects of the egg, namely albumen pH (Meijerhof, 1994a). Preincubational egg storage leads to morphological changes in the blasto-

Received for publication June 23, 1998. Accepted for publication December 22, 1998. 1To whom correspondence should be addressed: mcsoares@ bigfoot.com

640

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EGG STORAGE AND BROILER HATCHABILITY TABLE 1. Outline of experiments Egg collection time

Number of eggs used at different storage periods

Experiment

Flock age

Date

0 d

1 d

4 d

8 d

11 22 1 2

(wk) 32 42 54 59

11/30/96 1/12/97 11/30/96 1/12/97

460 500 460 500

460 500 460 500

460 500 460 500

460 500 310 500

1In Experiment 1, 10 eggs were used per age-storage period combination to record albumen characteristics prior to incubation, for a total of 80 eggs utilized to measure albumen height and pH of the thick albumen; remaining eggs were set, such that a total of 3,450 eggs were used to determine incubation performance. 2In Experiment 2, 10 eggs were used at each age-storage period-incubation time combination to record albumen characteristics at 0, 12, 24, 38, and 60 h of incubation, such that a total of 400 eggs was utilized to measure albumin height and pH of the thick albumen; remaining eggs were maintained in the setters, for a total of 3,600 eggs used to determine incubation performance.

above cited effect of storage and hen age upon albumen viscosity would explain why short-term storage may have beneficial effects on hatchability of eggs from young flocks (Brake, 1996a), as these flocks generally lay eggs that have albumens of good quality (viscosity) and that are fairly resistant to degradation. However, extended periods of egg storage allow the albumen to degrade excessively. This degradation causes the blastoderm to move into close proximity to the eggshell, so that early embryonic mortality results from dehydration during the early stages of incubation (Brake et al., 1993). The detrimental effect of long-term storage is more pronounced in eggs from old breeder flocks (Kirk et al., 1980; Meijerhof, 1994a), which is a result of lower albumen quality at oviposition and a subsequent increased rate of decline during storage (Hurnik et al., 1978). The objectives of the experiments described herein were to determine the influences of storage time and broiler breeder age on albumen height and pH, hatchability, and embryonic mortality at different stages of incubation.

MATERIALS AND METHODS

Broiler Hatching Eggs Two experiments were carried out over two periods, from October 30 to November 29, 1996 (Experiment 1), and from January 12 to February 11, 1997 (Experiment 2), using eggs from four commercial flocks of Peterson × Minibro Shaver broiler breeding stocks of different ages, and housed in different farms at a density of 0.22 m2 per bird. The flocks were reared in floor pens and kept during lay under standard management conditions. At bird placement (20 wk of age) the male: female ratio was 1:10. All birds received the same mash broiler breeder laying ration (16.50% CP, 2,800 kcal MEn/kg, 3.10% calcium,

2Petersime

n.v., Zulte, Belgium.

0.35% available phosphorus), formulated to meet or exceed NRC (1994) requirements. Separate-sex feeding was carried out and birds were feed-restricted in accordance with the management guide of the primary breeders (Peterson, 1993; Shaver, 1995). Water was available for ad libitum consumption and natural daylight was supplemented with artificial light to give a 17-h photoperiod. Temperature recordings showed that low and high in-house air temperatures at egg collection day ranged from 13 to 23, 16 to 20, 17 to 22, and 15 to 19 C at ages 32, 42, 54, and 59 wk, respectively. All flocks laid eggs at a normally expected rate. Eggs were collected at 32 and 54 wk of age (Experiment 1), and at 42 and 59 wk of age (Experiment 2). For both experiments, eggs were collected on a single day, as outlined in Table 1. Eggs laid before 0800 h were removed from the nest boxes and discarded from the experiments. Eggs were collected from nests between 0830 and 1230 h, placed in setter trays (150 eggs per tray), and transported in a closed vehicle to the hatchery located near the poultry houses. A total of 3,530 and 4,000 hatching eggs was used in Experiments 1 and 2, respectively (Table 1). At approximately 1400 h, eggs were culled to exclude from the experiments those that were cracked, visibly dirty, misshapen, or of extreme size (those weighing less than 51 g or more than 79 g). After removing culled eggs, eggs from each flock were randomized into four groups (Table 1) and placed on incubator trollies, to allow air circulation around the eggs. Thereafter, eggs were fumigated for 20 min with formaldehyde gas, and one group was set in the incubator on the same day (collection day), whereas the other three groups were stored for 1, 4, or 8 d in the hatching egg cooler at 16 C and 78% relative humidity. Number of eggs used for albumen analysis and for setting are provided in Table 1.

Incubation and Hatching Both experiments were conducted using electronically controlled, single-stage incubators (Model 576). 2 Four identical machines were used per experiment, and eggs from the same experiment and presetting storage period

642

˜ O ET AL. LAPA

were placed in the same incubator. Previous results obtained in the same hatchery (Reis and Soares, 1993) indicated that machines did not represent a significant source of variation for hatchability of total eggs set and viability of fertile eggs. The experimental egg trays were randomly placed in one quadrant (front right) on the same trolley, which was then filled with other eggs. Quadrant and trolley positions were the same in all eight settings to reduce possible position effects. The eggs were prewarmed in the incubator for 8 h at around 24 C and 65% relative humidity, just before the incubation period, and fumigated in the incubator on the day of setting. The eggs were turned hourly through 90° and incubated according to conventional temperature and humidity conditions (Reis et al., 1997), which were automatically monitored. On the 18th d of incubation, eggs were individually candled in the transfer-room (around 24 C and 60% relative humidity), using a hand candling lamp. “Clear” eggs were removed and broken out for macroscopic examination, in order to determine early-dead embryo mortalities (< 7 d) and those that were infertile, as outlined in Brake (1996b). Remaining eggs with apparently living embryos were transferred to hatching baskets and randomly distributed in the front part of the same trolley. The hatcher (Model 192) operated under conventional conditions (Reis et al., 1997). All chicks were removed at 21.5 d of incubation. The numbers of saleable chicks and culls were determined as practiced in commercial operations (Guillou, 1996). Unhatched eggs were opened, examined macroscopically, and assigned to one of the following categories: mid-dead (8 to 18 d), late-dead (after 19 d), pips (i.e., pipped shell but not emerged), and contaminated eggs (i.e., rots). From the data, both hatchability (number of saleable chicks hatched per all eggs set × 100), and viability (number of saleable chicks hatched per number of fertile eggs set × 100) were calculated. The latter estimate alone lacks accuracy due to the difficulty in distinguishing between infertile blastodiscs and very early dead blastoderms in eggs stored for some days before incubation (Mather and Laughlin, 1976). In fact, some very early deads will likely be classified as “infertile” using macroscopic examination (Walsh et al., 1995; Novo et al., 1997).

Albumen Analysis In both experiments, albumen quality was determined. In Experiment 1, 10 eggs from each flock-age (32 and 54 wk) by storage period (0, 1, 4, and 8 d) combination, or a total of 80 eggs were analyzed just prior to each setting (Table 1). In Experiment 2, 10 eggs from each flock-age (42 and 54 wk) by storage period combination, or a total of 400

3Roch Luneville, Avenue de Jolivet, B.P. 45, 54303 Luneville Cedex, France. 4Hanna Instruments Srl, 35010 Ronchi di Villa Franca, Italy.

eggs were analyzed at 0, 12, 24, 38, and 60 h of incubation. Eggs were opened in a laboratory at approximately 17 C to record the albumen height and pH of the thick albumen. The height of the thick albumen was measured to the nearest 0.01 mm using a Roch tripod micrometer.3 Albumen height was measured in the middle of the thick albumen equidistant from the outer edge of the albumen and the yolk (Benton and Brake, 1996). Albumen pH was measured with a Model HI 8424 combination pH microelectrode FC 200 B4 to the nearest 0.01 units. The pH electrode was standardized using buffer solutions of pH 7.01 and 9.01.

Statistical Analysis Data were analyzed by least squares procedures, using the General Linear Models procedure (PROC GLM) of SAS (SAS Institute, 1989). Albumen height and pH were analyzed with a linear model including the effects of flock age, length of storage, and the interaction among these factors. Hatchability, viability of fertile eggs, early-, middead, late-dead, and pipped embryonic mortalities, contaminated eggs, and culled chicks were analyzed with a linear model including the effects of flock age (dummy variable) and days of storage (continuous variable), as well as their interaction. For a given egg, a specific type of mortality was coded as 1 if it was the cause of embryonic death and 0 otherwise, and these codes were then submitted to analysis of covariance, as suggested by Harvey (1982), with the abovementioned model and the General Linear Models procedure (PROC GLM) of SAS (SAS Institute, 1989). A preliminary analysis including the quadratic effect of length of storage showed that this effect was not statistically significant (P > 0.10) for any of the traits analyzed, and thus only the linear effect was kept in the model.

RESULTS AND DISCUSSION

Albumen Characteristics Upon combining the results of Experiments 1 and 2, the effects of flock age and presetting storage of broiler breeder eggs on thick albumen pH and height just prior to setting are presented in Table 2. A highly significant (P < 0.001) interaction between flock age and storage period was observed for albumen pH but not for height. At all ages, albumen pH increased with storage time. This effect was pronounced in eggs from younger hens. Overall, albumen pH increased from 8.20 to 9.15 in eggs stored between 0 and 8 d, but most of this increase occurred during the first 4 d of storage. This rapid increase was followed by a progressively slower rate of increase throughout the remainder of the storage period. At 0 d of storage, pH increased significantly (P < 0.05) with flock age, but differences between ages were smaller at 1 d of storage, and were not significant (P > 0.05) after 4 and 8 d of storage. There are several possible reasons that flock age could affect albumen pH. Firstly, this may be

643

EGG STORAGE AND BROILER HATCHABILITY TABLE 2. Least squares means and tests of significance for albumen pH and height, for age class by storage period, Experiments 1 and 2 Significance1

Storage period Variable pH

Height, mm

Flock age

0 d

(wk) 32 42 54 59 Pooled 32 42 54 59 Pooled

8.08 8.19 8.23 8.30 8.20 7.74 7.44 7.06 6.28 7.13

1 d ± ± ± ± ± ± ± ± ± ±

0.02a 0.02e 0.02c 0.02h 0.01z 0.29 0.29 0.29 0.29 0.14x

8.59 8.66 8.76 8.81 8.71 7.14 7.23 6.64 6.16 6.79

4 d ± ± ± ± ± ± ± ± ± ±

0.02b 0.02f 0.02g 0.02g 0.01y 0.29 0.29 0.29 0.29 0.14y

9.00 9.03 9.04 9.04 9.03 6.18 5.59 5.34 5.37 5.62

8 d ± ± ± ± ± ± ± ± ± ±

0.02c 0.02c 0.02c 0.02c 0.01x 0.29 0.29 0.29 0.29 0.14z

9.12 9.15 9.16 9.17 9.15 5.67 5.23 5.17 4.93 5.25

± ± ± ± ± ± ± ± ± ±

0.02d 0.02d 0.02d 0.02d 0.01x 0.29 0.29 0.29 0.29 0.14z

n

Pooled

40 40 40 40

8.70 8.76 8.80 8.83

± ± ± ±

40 40 40 40

6.68 6.37 6.05 5.68

± ± ± ±

A

S

A × S

0.01z 0.01y 0.01x 0.01x

***

***

***

0.14x 0.14xy 0.14yz 0.14z

***

***

NS2

a–hMeans

for the same trait and effect, or combination of effects, with no common superscript differ significantly (P < 0.05). means with no common superscript differ significantly (P < 0.05). 1A = flock age; S = storage period. 2P > 0.10. ***P < 0.001. x–zPooled

explained by an increased incidence of first and last sequence eggs laid by older hens, as these eggs contain more advanced embryos at oviposition than intermediate eggs (Butler, 1991). Increased metabolic activity of the embryo is associated with increased outward diffusion of carbon dioxide in those eggs (Kucera and Raddatz, 1980) and subsequent increases in pH of the embryonic tissues. Another possible explanation of the observed differences is that a higher eggshell conductance in eggs from older hens would allow for a more rapid release of carbon dioxide from the eggs (Meijerhof, 1994a). Regarding the evolution of pH with storage, the buffering capacity of albumen is weakest between pH 7.5 and 8.5 (Cotterill et al., 1959), which explains the pattern of pH change observed in the present study. Our findings

are in agreement with the work of Brake (1995), who reported that albumen pH approaches 9 at 4 d of storage and does not increase much more thereafter. An alkaline plateau is reached at a pH of 9.0 (at 4 C) to 9.5 (at 38 C) according to Goodrum et al. (1989), and Stern (1991). Albumen height (Table 2) was significantly higher (P < 0.05) in younger (mean of 6.68 mm) than in older hens (5.68 mm), and was also higher (P < 0.05) in fresh (mean of 7.13 mm) than in stored eggs (5.25 mm). No significant interaction (P > 0.05) was observed between age and storage time. The degradation of albumen quality during storage occurred according to normal expectations for broiler breeder eggs. Thick albumen pH increased with length of storage and incubation time, as evidenced by a highly significant

TABLE 3. Least squares means and tests of significance for albumen pH and height, for incubation time by storage period, Experiment 2

Variable pH

Height, mm

a–kMeans

Significance1

Storage period

Incubation time

0 d

(h) 12 24 38 60 Pooled 12 24 38 60 Pooled

8.65 8.95 9.05 9.15 8.95 5.70 4.98 4.45 4.05 4.79

1 d ± ± ± ± ± ± ± ± ± ±

0.01a 0.01c 0.01d 0.01e 0.01z 0.20 0.20 0.20 0.20 0.10x

8.79 8.98 9.08 9.19 9.01 5.29 4.86 4.31 3.77 4.56

4 d ± ± ± ± ± ± ± ± ± ±

0.01b 0.01c 0.01df 0.01eg 0.01y 0.20 0.20 0.20 0.20 0.10x

9.10 9.18 9.31 9.36 9.24 4.53 4.27 3.87 3.34 4.00

8 d ± ± ± ± ± ± ± ± ± ±

0.01f 0.01eh 0.01i 0.01j 0.01x 0.20 0.20 0.20 0.20 0.10y

9.20 9.23 9.32 9.36 9.28 4.48 4.36 4.06 3.19 4.02

± ± ± ± ± ± ± ± ± ±

0.01gh 0.01g 0.01ik 0.01jk 0.01x 0.20 0.20 0.20 0.20 0.10y

n

Pooled

40 40 40 40

8.94 9.08 9.19 9.26

± ± ± ±

40 40 40 40

5.00 4.62 4.17 3.59

± ± ± ±

I

S

I × S

0.01z 0.01y 0.01x 0.01x

***

***

***

0.10x 0.10y 0.10z 0.10z

***

***

NS2

for the same trait and effect, or combination of effects, with no common superscript differ significantly (P < 0.05). means with no common superscript differ significantly (P < 0.05). 1I = incubation time; S = storage period. 2P > 0.10. ***P < 0.001. x–zPooled

˜ O ET AL. LAPA

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TABLE 4. Regression coefficients on storage period, by flock age Significance1

Flock age Variable

32 wk

42 wk

Hatchability Viability Embryonic mortality Days 0 to 7 Days 8 to 18 Days 19 to 21 Pipped Culls Contaminated

–0.890 ± 0.278 –0.819 ± 0.265

–1.042 ± 0.278 –0.964 ± 0.266

0.117 0.163 –0.023 –0.024 0.586 –0.000

± ± ± ± ± ±

0.169 0.111 0.056 0.069 0.173 0.026

0.277 –0.098 0.069 –0.010 0.698 0.028

54 wk

± ± ± ± ± ±

0.170 0.112 0.057 0.069 0.174 0.026

(%) –1.185 ± 0.313 –1.165 ± 0.300 –0.084 0.423 0.078 0.042 0.706 0.000

± ± ± ± ± ±

0.192 0.126 0.064 0.078 0.196 0.029

59 wk

Pooled

A

S

A × S

–1.867 ± 0.278 –1.919 ± 0.271

–1.249 ± 0.142 –1.212 ± 0.137

** **

** **

*

± ± ± ± ± ±

** NS NS NS NS **

* ** * * ** NS

NS2 ** NS ** NS **

0.366 0.383 0.079 0.277 0.927 –0.114

± ± ± ± ± ±

0.173 0.114 0.058 0.070 0.177 0.027

0.182 0.204 0.049 0.071 0.728 –0.022

0.088 0.057 0.029 0.036 0.090 0.013

†

1A

= flock age; S = storage period. > 0.10. *P < 0.05. **P < 0.01. †P < 0.10. 2P

interaction (P < 0.001) between days of egg storage and hours of incubation (Table 3). For all storage periods, albumen pH increased through 60 h of incubation, but the rate of increase declined with storage time. Average changes in albumen pH between 12 and 60 h for eggs stored 0, 1, 4, and 8 d were 0.5, 0.4, 0.26, and 0.16 units, respectively. As a result, differences due to egg storage time on albumen pH at 60 h of incubation were minor. This result is in agreement with those of Benton and Brake (1996), who concluded that stored eggs, for which pH has risen during storage, begin incubation near a pH of 9 with little change afterwards, which is due to the strong buffering capacity of the egg albumen at that pH. The effect of presetting storage of eggs and incubation time on albumen height is illustrated in Table 3. No significant interaction (P > 0.05) was observed between days of egg storage and hours of incubation. The mean initial albumen height (Table 3) was 5.00 mm at 12 h of incubation. Albumen height decreased (P < 0.05) to 3.59 mm by 60 h, whereas at 0 h of incubation (Table 2) eggs from the same flock ages (42 and 59 wk) had average albumen heights of 6.37 and 5.68 mm, respectively. These results are in agreement with those of Benton and Brake (1996), who reported that most of the albumen height decline occurred during the first 24 h of incubation.

Hatching Performance The effects of length of egg storage prior to incubation (expressed as regression coefficients per day of storage), and their interaction with flock age, on hatchability, embryo viability, culls, and embryonic mortality at different stages are shown in Table 4. Viability was significantly (P < 0.05) affected by an interaction between flock age and length of egg storage. Longer storage periods decreased viability in all flock ages, with more pronounced decreases occurring in older flocks. Decreases in viability per additional day of storage were 0.82 at 32 wk of age, and 1.92% at 59 wk of age, with intermediate

regression coefficients at 42 and 54 wk of age (Table 4). The pattern was similar for hatchability, even though the interaction between length of storage and breeder age was of questionable significance (P < 0.10). These findings are in general agreement with the results reported by other researchers (Kirk et al., 1980; Meijerhof, 1994b; Reis et al., 1997), who suggested that, where there is an option, eggs from younger flocks should be stored rather than those from older flocks. Decreased viability with increased storage length in older flocks was mainly due to an increased incidence of culls and embryonic losses at all stages (Table 4). The results of several workers (Bohren et al., 1961; Byng and Nash, 1962; Tandron et al., 1983) have indicated that declines in hatchability start 2 to 3 d after lay. However, it is often suggested that hatchability starts to decline only after 7 d of storage (Mayes and Takeballi, 1984; Meijerhof, 1994a). In contrast, Oluyemi and George (1972) reported that storage for 4 to 6 d tended to improve hatchability of fertile eggs. The above-cited discrepancies may be attributable to differences in presetting egg storage conditions, flock age and strain. Our results, based upon commercial hatchery conditions, strongly indicate a steady decline in hatchability starting as early as 1 d of storage, with an additional day of storage causing a decline in viability by as much as two percentage points in older flocks. Because albumen pH was similar for all flock ages after 4 d of storage, and embryo viability declined to a greater extend in older flocks, no clear relationship could be established between egg albumen pH and hatchability. However, albumen height did decrease significantly with increased flock age. This deterioration in albumen quality could affect embryonic survival. Our results suggest that, in the poultry industry, if an extended preincubational egg storage is necessary, eggs from younger flocks should be stored rather than those from older flocks. More research with different commercial strains of broiler breeders and younger flocks is necessary before general recommendations for commercial hatcheries can be made.

EGG STORAGE AND BROILER HATCHABILITY

ACKNOWLEDGMENTS This project was supported by the Valouro Group, Portugal, and a Program PRAXIS XXI (JNICT) grant to C. Lapa˜o.

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