Accelerating Embryonic Growth During Incubation ...

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were kept in the same egg storage room for the final 2 d and were .... 641a. 648a. 3. Control. 617. 11.6. 638. 21.8. 638. High. 616. 14.5. 639. 22.3. 636. Mean.
Accelerating Embryonic Growth During Incubation Following Prolonged Egg Storage 1. Embryonic Livability1 V. L. Christensen,2 J. L. Grimes, M. J. Wineland, and G. S. Davis Department of Poultry Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, North Carolina 27695-7608 ABSTRACT The hypothesis was proposed that shorter incubation periods and faster growth rates for long-stored eggs would improve embryonic survival and poult hatchling quality. Increased incubation temperatures were tested for their efficacy in improving embryonic livability in fertilized eggs stored for 15 d prior to setting in the incubator compared to controls stored for only 3 d. Two temperature treatments were applied. In experiment 1, a 37.8°C set point for dry bulb temperature was used to accelerate development for the initial 2 wk compared to the controls at 37.5°C. Following treatment, the accelerated embryos were returned to the same machine as the controls. In experiment 2, higher temperature exposure was only for the initial week of incubation. The temperature and storage treatments were in a completely

random 2 × 2 factorial arrangement of treatments. At the completion of 28 d of incubation, survival rates of all treatments were determined by opening all nonhatching eggs to differentiate truly fertilized eggs from unfertilized. Hatchability was determined by dividing the total number of poults on a hatching tray by the number of fertilized eggs on a tray. Incubator trays were the experimental unit. Tissues were sampled in both experiments to verify treatment effects on growth and metabolism. Hatching times were observed at 4-h intervals during the actual hatching process beginning at 25 d of incubation. It was concluded that delayed growth and depressed metabolism of fertilized turkey eggs stored for 15 d can be compensated for by exposure to higher incubation temperatures for the initial 1 or 2 wk of incubation.

(Key words: egg storage, embryonic growth, hatchability, incubation) 2003 Poultry Science 82:1863–1868

INTRODUCTION Storage of fertilized poultry eggs depresses embryonic survival in direct proportion to the length of the storage period (Meijerhof, 1992; Fasenko, 1996). Egg storage is a necessity in modern poultry production because of the demand for large numbers of hatchlings within a constrained period. Hot weather decreases egg production rates so even longer storage periods are required in hot climates or hot weather to accumulate sufficient numbers of eggs prior to setting in incubators. Recent data indicate that pre-incubation of eggs stored for greater than 14 d and increased embryonic development of stored eggs prior to setting improved livability of embryos (Fasenko et al., 2001 a,b). Prolonged incubation periods and reduced embryonic growth accompanied by reduced carbohydrate metabolism have been noted as well (Fasenko,

2003 Poultry Science Association, Inc. Received for publication July 29, 2002. Accepted for publication July 31, 2003. 1 The mention of trade names in this publication does not imply endorsement of the products mentioned or criticism of similar products not mentioned. 2 To whom correspondence should be addressed: vern_christensen @ncsu.edu.

1996). Different incubation temperatures for turkey eggs from hens of different ages were shown recently to change incubation periods and affect embryonic livability (Christensen et al., 2001). The hypothesis was proposed that increasing incubation temperatures and thereby accelerating development during the incubation period rather than prior to incubation (Fasenko et al., 2001a,b) would improve embryonic survival. Hatchling survival was measured by observing hatchability of eggs incubated at higher temperatures for the initial 1 or 2 wk of development.

MATERIALS AND METHODS Three preliminary experiments were conducted to examine incubation treatments to stimulate embryonic growth and affect embryonic livability. Based on these preliminary results, it was decided to use high temperature treatments during wk 1 and 2 of incubation to accelerate the growth and shorten hatching times of turkey embryos from eggs stored for 15 d prior to setting.

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Abbreviation Key: k = conductance constant.

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All eggs in both experiments were weighed 3 times (nearest 0.01 g). Eggs were weighed at the beginning of the storage period, at setting, and at the time of transfer to the hatcher. Egg weight loss data were used to compute conductance (Tullett, 1981). The hatching time of each egg was noted at 4-h intervals, and the time of development from internal to external pipping was noted on 25 eggs per treatment at the same intervals. Increased incubation temperatures determined from the preliminary trials were tested for their efficacy in improving embryonic mortality in fertilized eggs stored for 15 d prior to setting in the incubator compared to controls stored for only 3 d. In each of 2 identical replicate trials of experiment 1, fertilized turkey eggs were collected from a commercial flock 2 times, 12 d apart in each trial. Eggs from the first collection were placed on paper flats on shelves in a storage room and kept at 12.8°C and 75% relative humidity for 14 d. This group of 1,000 eggs was designated as the stored treatment group. At the beginning of the 15th d of storage, the eggs were removed from the cold room and allowed to warm overnight to room temperature. Eggs at the second collection time were kept in the same egg storage room for the final 2 d and were designated as the control group. The control group was also removed from the cold storage room 24 h before setting and allowed to warm to room temperature prior to setting in the incubators. On the day of setting, eggs were placed into 2 incubation cabinets described in our previous study (Christensen et al., 1996). One cabinet operated at 37.8 ± 0.1°C and 50% relative humidity, and the other operated at 37.5 ± 0.1°C and 54% relative humidity. In experiment 2, 2 additional trials were conducted. Identical experimental procedures were used in experiment 2 with the following exceptions. First, each trial included 2,000 fertilized turkey eggs, and second, at the completion of 14 d of incubation in experiment 1 and 7 d of development in experiment 2, eggs were moved to the same cabinet operated at 37.5°C. Thus, the higher temperature for accelerating development was applied for wk 1 and 2 in the first experiment and only wk 1 in the second. The incubators were reversed in the second trial of each experiment to account for machine effects. At the beginning of the 25th day of incubation, all eggs in both treatments and experiments were transferred to a second machine for hatching. The hatching machines operated at 36.8°C and 75% relative humidity. Hatchability data in both trials were collected following 28 d of incubation. All nonhatching eggs remaining on the hatching trays were broken, fertility was determined, and the age of the embryo at death was estimated. Embryo mortality data were pooled on a weekly basis, and percentages were determined by dividing the number of embryos by the total number of fertilized eggs per experimental unit for analysis. Hatchability was calculated based on trays of 25 eggs as experimental units. The number of poults and nonhatching eggs on trays containing eggs were counted. Hatchability was computed by dividing the number of poults hatching by the number of fertilized eggs. Thus, the sample size for each trial of

experiment 1 was approximately 90 (ca. 20 per treatment combination), whereas that for each trial of experiment 2 was 180 (ca. 40 per treatment combination). Statistical analyses were performed using the general linear models procedure of SAS software (SAS Institute, 1998). Data were in a completely random design with 2 egg storage treatments (15 d and 3 d) and 2 incubation treatments (high and control) arranged in a 2 × 2 factorial. Trial was included as a fixed factor in both experiments, but no significant trial effects were noted so the data were pooled across trial for final analysis. All possible main effects and interactions were tested. Probability of significance was based on P < 0.05 unless otherwise noted.

RESULTS Experiment 1 Fertility was identical in all four treatment combinations (15 d High = 97.6%; 15 d control = 97.4%; 3 d high = 96.5% and 3 d control = 97.2%). Thus, the statistical analysis for hatchability analysis based on total or fertilized eggs was identical so only hatchability of fertilized eggs will be reported. Hatchability of the fertilized eggs was improved by 1.5% when exposed to the high incubation temperature compared with controls regardless of egg storage period prior to setting (Table 1). Eggs stored for 15 d exhibited depressed hatchability by approximately 7% compared to control eggs stored for 3 d prior to setting. No storage-by-incubation period interaction occurred. Higher incubation temperatures decreased embryonic mortality during wk 1 and 4 but had no effect on the percentage of embryos dying during pipping. Again, no interaction of storage treatment by incubation period occurred for times of embryonic mortality. The time to attain a developmental stage, the time the embryo remained at a stage, and the time of hatching are given in Table 2. Embryos from 15-d stored eggs reached internal pipping and external pipping approximately 4 to 5 h after 3 d of storage, and embryos treated with high incubation temperature similarly attained internal pipping 4 h before controls. High incubation temperature treatment eggs required an additional 4.1 h at internal pipping compared with controls. Hatching times between 15- and 3-d storage groups differed by approximately 11 h. Eggshell conductance constant (k) measurements from all eggs are given in Table 3. Egg weights at setting differed between the 15- and 3-d groups because additional time in the egg storage room allowed more weight loss over 15 than 3 d of storage. Weights at the beginning of the respective storage periods did not differ. Eggshell conductance values exhibited an interaction of storageby-incubation temperature. Eggs stored 15 d in the control temperature had lower conductance than those in the high, but no such difference occurred among eggs stored 3 d. Hatching times again reflected the 9-h difference between the 15- and 3-d storage groups with no significant differences due to temperature treatments. Combin-

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EGG STORAGE TABLE 1. Hatchability (%) and mortality (%) of fertilized turkey eggs during incubation when exposed to 2 storage periods and 2 incubation temperatures in experiment 1 Storage (d)1 15

3

Incubation temperature2

Hatchability

Week 1 mortality

Control High Mean Control High Mean Control mean High mean Mean ± SEM Storage (S) Incubation temperature (T) S × T

77.9 83.9 80.9b 86.1 88.8 87.5a 82.0b 86.3a 84.2 ± 0.8 0.0003 0.01 NS

9.0 6.8 7.9a 4.6 3.1 3.8b 6.8a 4.9b 5.8 ± 0.4 0.0001 0.05 NS

Week 4 mortality

Pipping mortality

4.3 2.2

8.3 6.7

3.7 1.9

5.4 5.7

4.0a 2.0b 3.0 ± 0.3 NS 0.006 NS

6.4 ± 0.6 NS NS NS

Columnar means with different superscripts differ significantly (P < 0.05). Eggs were stored at 12.8°C and 75% relative humidity for 15 or 3 d prior to setting in the incubator. 2 Control = eggs were incubated at 37.5°C for the entire 28-d incubation period; High = eggs were incubated at 37.8°C for the initial 2 wk after which they were placed into the same machine as the controls. a,b 1

ing all 3 of the previous measurements and computing the conductance constant also revealed a significant storage-by-temperature interaction. Greater k values were noted in the eggs stored 15 d and exposed to high temperature than in any other interaction mean combination (Table 3).

Experiment 2 Embryos were exposed to the higher temperature (37.8°C) in experiment 2 for only the initial 7 d of incubation. There were similarly no significant differences noted in fertility (15-d high = 96.2%; 15-d control = 96.3%; 3d high = 97.4% and 3-d control = 96.7%). Analyses for hatchability of total eggs or fertilized eggs were similar so only hatchability of fertilized eggs is reported. Hatchability showed a similar pattern to that observed for ex-

periment 1 (Table 4). High temperature improved embryonic survival of 15- and 3-d stored eggs, and the 15-d storage period depressed survival of embryos compared with the 3-d period. Week one embryonic mortality was increased in the 15-d compared to the 3-d embryos but exposing them to the higher temperature during the initial week of development did not affect the amount of mortality. Week four mortality was nearly 5% greater in 15- than 3-d stored eggs, and higher incubation temperatures reduced wk 4 mortality by approximately 6%. Time to attain a stage of development as well as time spent at a stage of development revealed interactions of storage-by-incubation temperature (Table 5). The time to attain internal and external pipping was shortened in the 15-d eggs by approximately 16 h when eggs were exposed to the higher incubation temperature. The eggs stored 3 d had only a 9- to 12-h shorter incubation period. Time

TABLE 2. Time of hatching (h) of fertilized turkey eggs when exposed to 2 storage periods and 2 incubation temperatures in Experiment 1 Internal pip Storage (d)1 15

3

Incubation temperature2 Control High Mean Control High Mean Control mean High mean Mean ± SEM Storage (S) Incubation temperature (T) S × T

Time to attain stage 621 622 621a 617 616 616b

Time at stage 11.4 16.6 11.6 14.5

External pip Time to attain stage 640 641 641a 638 639 638b

Hatched

Time at stage

Time to attain stage

19.4 19.2

650 647 648a 638 636 637b

21.8 22.3

11.5b 15.6a 619 ± 1 0.02 NS NS

13.5 ± 0.7 NS 0.01 NS

639 ± 2 0.05 NS NS

20.7 ± 0.9 NS NS NS

642 ± 0.1 0.0001 NS NS

Columnar means with different superscripts differ significantly (P < 0.05). Eggs were stored at 12.8°C and 75% relative humidity for 15 or 3 d prior to setting in the incubator. 2 Control = eggs were incubated at 37.5°C for the entire 28-d incubation period; High = eggs were incubated at 37.8°C for the initial 2 wk after which they were placed into the same machine as the controls. a,b 1

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CHRISTENSEN ET AL. TABLE 3. Egg weights (g), eggshell conductance (mg of H2O/d per mm of Hg), hatching times (h), and conductance constants of fertilized turkey eggs when exposed to 2 storage periods and 2 incubation temperatures in experiment 1 Incubation temperature2

Storage (d)1

Egg weight at setting

Eggshell conductance

Hatching time

Conductance constant

15

Control High Mean

86.0 86.4 86.2b

17.4b 18.6a

648 647 648a

5.44b 5.85a

3

Control High Mean

87.8 89.5 88.6a

18.3a 18.1ab

637 637 637b

5.59b 5.40b

Mean ± SEM Storage (S) Incubation temperature (T) S × T

87.4 ± 0.4 0.007 NS NS

18.1 ± 0.2 NS NS 0.05

642 ± 1 0.0001 NS NS

5.57 ± 0.05 NS NS 0.005

Columnar means with different superscripts differ significantly (P < 0.05). Eggs were stored at 12.8°C and 75% relative humidity for 15 or 3 d prior to setting in the incubator. 2 Control = eggs were incubated at 37.5°C for the entire 28-d incubation period; High = eggs were incubated at 37.8°C for the initial 2 wk after which they were placed into the same machine as the controls. a,b 1

TABLE 4. Hatchability (%) of fertilized turkey eggs and embryonic mortality (%) at times of development when exposed to 2 storage periods and 2 incubation periods in experiment 2 Incubation temperature2

Hatchability

Control High Mean Control High Mean Control mean High mean Mean ± SEM Storage (S) Incubation temperature (T) S × T

74.2 83.1 78.6b 87.3 92.9 90.1a 80.7b 88.0a 84.4 ± 0.8 0.0001 0.0001 NS

Storage (h)1 15

3

Week 1 mortality

Week 4 mortality

9.9 8.7 9.3a 3.8 2.9 3.4b

15.0 6.9 11.0a 8.1 4.1 6.1b 11.6a 5.5b 8.5 ± 0.6 0.0004 0.0001 NS

6.2 ± 0.5 0.0001 NS NS

Columnar means with different superscripts differ significantly (P < 0.05). Eggs were stored at 12.8°d and 75% relative humidity for 15 or 3 d prior to setting in the incubator. 2 Control = eggs were incubated at 37.5°C for the entire 28-d incubation period; High = eggs were incubated at 37.8°C for the initial wk after which they were placed into the same machine as the controls. a,b 1

TABLE 5. Time of hatching (h) of fertilized turkey eggs when exposed to 2 storage periods and 2 incubation temperatures in experiment 2 Prepipping

Storage (d)1 15

3

Incubation temperature2

Time to attain

Time at stage

Internal pipping Time to attain

Time at stage

External pipping Time to attain

Time at stage

Hatch Time to attain

Control High Mean

593 584 588a

22.2a 14.9b

615a 598c

19.2 19.8

634a 618b

29.1a 19.5c

659a 637c

Control High Mean Control mean High mean Mean SEM Storage (S) Incubation temperature (T) S × T

588 580 584b 590a 582b 587 0.5 0.006 0.0001 NS

17.1b 16.0b

603b 591d

16.2 18.4

618b 609c

25.4a 21.5b

644b 631d

17.6 0.5 NS 0.0007 0.01

601 0.8 0.0001 0.0001 0.05

18.4 0.9 NS NS NS

619 0.9 0.0001 0.0001 0.03

23.5 0.7 NS 0.0001 0.05

642 0.8 0.0001 0.0001 0.007

Columnar means with different superscripts differ significantly (P < 0.05). Eggs were stored at 12.8°C and 75% relative humidity for 15 or 3 d prior to setting in the incubator. 2 Control = eggs were incubated at 37.5°C for the entire 28-d incubation period; High = eggs were incubated at 37.8°C for the initial wk after which they were placed into the same machine as the controls. a,b 1

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EGG STORAGE TABLE 6. Egg weights (g), eggshell conductance (mg of H2O/d per mm of Hg), hatching times (h), and conductance constants of fertilized turkey eggs when exposed to 2 storage periods and 2 incubation temperatures in experiment 2 Storage (d)1 15

3

Incubation temperature2 Control High Mean Control High Mean

Egg weight at setting 86.9 87.1 87.0b 89.6 89.4 89.0a

Control mean High mean Mean ± SEM Storage (S) Incubation temperature (T) S × T

88.2 ± 0.1 0.0001 NS NS

Eggshell conductance

Hatching time

Conductance constant

17.5 18.3 17.9b

652 637 644b

5.42 5.53

17.9 18.7 18.3a

642 629 636b

5.39 5.47

17.7b 18.4a

647a 633b

5.40b 5.50a

18.1 ± 0.1 0.006 0.0001 NS

640 ± 1 0.0001 0.0001 NS

5.45 ± 0.02 NS 0.03 NS

Columnar means with different superscripts differ significantly (P < 0.05). Eggs were stored at 12.8°C and 75% relative humidity for 15 or 3 d prior to setting in the incubator. 2 Control = eggs were incubated at 37.5°C for the entire 28-d incubation period; High = eggs were incubated at 37.8°C for the initial wk after which they were placed into the same machine as the controls. a,b 1

spent at internal pipping did not differ among the treatment groups, but time spent at external pipping displayed storage-by-incubation period interaction. Eggs stored 15 d remained about 10 h longer at external pipping in the control than in the high incubation period, whereas 3-d stored eggs had their external pipping time shortened by about 4 h. Time to hatching displayed storage-byincubation temperature interaction as well. Eggs stored 15 d hatched 22 h earlier when exposed to the high treatment than did controls, but 3-d stored eggs hatched only 13 h earlier in high than controls. As was observed in experiment 1, initial egg mass differed among treatments at setting, but this was confirmed to be due to additional time spent in the egg storage room and not in the incubator (Table 6). Eggshell conductance was greater in the 3-d than the 15-d stored eggs, and higher incubation temperatures increased eggshell conductance compared with controls. Hatching times were 8 h earlier in 3-d stored embryos than in the 15-d group, and higher incubation temperature during the initial 7 d of incubation shortened the incubation period by 14 h compared with controls. Eggshell conductance constants differed only between incubation temperature treatments in experiment 2 with high incubation temperatures having greater k than controls.

DISCUSSION Storage of eggs prior to incubation leads to morphological changes in the embryo (Byng and Nash, 1962; Bakst and Gupta, 1997), slower growth rate following extended storage (Singal and Kosin, 1969), shrinkage of the blastoderm, and delays in initiation of development following storage (Mather and Laughlin, 1979). Embryonic survival rates are similarly depressed following extended storage of eggs prior to setting (Meijerhof, 1992). More recently, pre-incubation warming or interruption of the storage period with incubation have been utilized to improve the

growth and embryonic livability of eggs stored for longer than 14 d prior to setting (Fasenko et al., 2001a,b). The hypothesis tested by the current study was that increased temperatures during incubation could accelerate the slowed turkey embryonic development and improve the livability of poult embryos from stored eggs. The results obtained in this experiment suggest that increased incubation temperature during the initial stages of development of eggs stored 15 d can improve livability of turkey embryos. An additional observation suggests that such incubator profiles can improve livability of eggs stored 3 d as well. In experiment 1, higher temperatures were utilized for d 1 through 14 of incubation and resulted in 4.3% greater livability among eggs stored 15- and 3-d. In experiment 2, higher temperatures were utilized for d 1 through 7 of development, and a 6% increase in embryonic livability was noted. These data show clearly that turkey embryos residing in eggs stored for extended periods of time can survive better when their incubation period is shortened by increased incubation temperature during the initial period of development. The data also suggest that increased incubation temperatures and accelerating growth during the initial 7 d of development may be as effective as during the initial 14 d. The egg k of each treatment group best validated the effect of the treatments on development. The 15-d eggs, the treatment that exhibited a lower k value, hatched nearly 7 to 12% less than did the 3-d group. When this situation was rectified by increased incubator temperature, k values increased and survival improved. This situation may be similar to that observed for eggs from hens of different ages that also have different k values and incubation periods (Christensen et al., 1996, 2001). Differences observed in conductance values in the stored eggs for each temperature treatment of experiment 1 did not repeat in experiment 2 and were consequently thought to be spurious. Thus, no inference can be made about the conductance requirement of such eggs.

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The current study demonstrates clearly that the delayed growth and metabolism of fertilized turkey eggs stored for 15 d can be compensated for by exposing the eggs to higher incubation temperatures for the initial 1 or 2 wk of incubation. Thus, increased temperature may cause embryos in stored eggs to compensate for delayed growth rates and better utilize carbohydrate metabolism to enable survival during pipping and hatching as reported by Fasenko et al. (2001b). Thus, higher incubation temperatures during the initial stages of incubation may favor survival of turkey embryos developing in eggs stored for greater than 15 d prior to setting in the incubator.

REFERENCES Bakst, M. R., and S. K. Gupta. 1997. Preincubation storage of turkey eggs: Impact on rate of early embryonic development. Br. Poult. Sci. 38:374–377. Byng, A. L., and D. Nash. 1962. The effects of egg storage on hatchability. Br. Poult. Sci. 3:81–87. Christensen, V. L., W. E. Donaldson, and J. P. McMurtry. 1996. Physiological differences in late embryos from turkey breeders at different ages. Poult. Sci. 75:172–178. Christensen, V. L., J. L. Grimes, M. J. Wineland, and L. G. Bagley. 2001. Effects of turkey breeder hen age, strain, and length of

the incubation period on survival of embryos and hatchlings. J. Appl. Poult. Res. 10:5–15. Fasenko, G. M. 1996. Factors influencing embryo and poult viability and growth in stored turkey eggs. Ph.D. Dissertation, North Carolina State Univ., Raleigh, NC. Fasenko, G. M., V. L. Christensen, M. J. Wineland, and J. N. Petitte. 2001a. Examining the effects of prestorage incubation of turkey breeder eggs on embryonic development and hatchability of eggs stored for four or fourteen days. Poult. Sci. 80:132–138. Fasenko, G. M., F. E. Robinson, A. I Whelan, K. M. Kremeniuk, and J. A. Walker. 2001b. Prestorage incubation of long-term stored broiler breeder eggs: 1. Effect on hatchability. Poult. Sci. 80:1406–1411. Mather, C. M., and K. F. Laughlin. 1979. Storage of hatching eggs: The interaction between parental age and early embryonic development. Br. Poult. Sci. 20:595–604. Meijerhof, R. 1992. Pre-incubation holding of hatching eggs. World’s Poult. Sci. J. 48:57–68. SAS Institute. 1998. A User’s Guide to SAS 98. Sparks Press, Cary, NC. Singal, D. P., and I. L. Kosin. 1969. Induced preincubation of the avian egg and subsequent development of the embryo, as revealed by the DNA, RNA and protein level of its spleen. Proc. Soc. Exp. Biol. Med. 132:871–877. Tullett, S. G. 1981. Theoretical and practical aspects of eggshell porosity. Turkeys 29:24–28.