Carbon Dioxide During Storage of Chicken and Turkey Hatching Eggs1

METHIONINE SUPPLEMENTATION OF SOYBEAN MEAL

may interfere with sulfur amino acid metabolism. SUMMARY Unheated soybean meal and soybeans, when adequately supplemented with DLmethionine supported normal egg production. The source of carbohydrate, glucose or starch did not greatly alter the response of hens to raw and heated soybean meal when 0.5% DL-methionine was added to the diets. In these experiments, pullets fed diets with the lower levels of methionine lost more weight than those fed higher levels. Hens fed the diet with autoclaved soybean meal gained weight. Pancreas size in hens fed diets containing raw soybean meal or raw soybeans was highly variable, but the results showed evidence of pancreatic enlargement, the magnitude of which was much less than that observed in chicks fed raw soybean meal. REFERENCES Bray, D. J., 1964. Pancreatic hypertrophy in laying pullets induced by unheated soybean meal. Poultry Sci. 43 : 382-384.

251

Carver, J. S., J. McGinnis, C. F. McClary and R. J. Evans, 1964. The utilization of raw and heat treated soybean oil meal for egg production and hatchability. Poultry Sci. 25: 399. Fisher, H., D. Johnson, Jr. and S. Ferdo, 1957. The utilization of raw soybean meal protein for egg production in the chicken. J. Nutrition, 6 1 : 611-621. Harmas, R. H., C. R. Douglas and P. W. Waldroup, 1962. Methionine supplementation of laying hen diets. Poultry Sci. 4 1 : 805-812. Hill, R. W., and R. Renner, 1963. Effects of heat treatment on the metabolizable energy value of soybeans and extracted soybean flakes for the hen. J. Nutrition, 80: 375-380. Rogler, J. E., and C. W. Carrick, 1964. Studies on raw and heated unextracted soybeans for layers. Poultry Sci. 43 : 605-612. Saxena, H. C , L. S. Jensen, J. V. Spencer and J. (McGinnis, 1963. Production, interior egg quality, and some physiological effects of feeding raw soybean meal to laying hens. Poultry Sci. 42: 291-293. Saxena, H. C , 1964. Investigations on the mechanism of growth depression and pancreatic hypertrophy by raw soybean meal in the chick. Ph.D. Thesis, Washington State University. P. 75. Summers, J. D., J. D. McConachie, S. J. Slinger and W. F. Pepper, 1965. The value of raw unextracted soybeans for laying hens. Poultry Sci. 45: 165-168.

Carbon Dioxide During Storage of Chicken and Turkey Hatching Eggs 1 WALTER A. BECKER, JOHN V. SPENCER AND JAMES L. SWARTWOOD2 Department of Animal Sciences, Washington State University, Pullman, Washington 99163 (Received for publication June 6, 1967)

W

HEN chicken and turkey hatching eggs are stored in plastic bags for long periods, their hatchability is higher than eggs not packaged (Becker, 1964; 1

Scientific Paper No. 2757, College of Agriculture, Pullman. Project 1808. Supported in part by a grant from the American Poultry and Hatchery Association. 2 Present address: General Mills, Toronto, Ontario, Canada.

Becker et al, 1964; Proudfoot, 1964a). Comparisons of plastic bags with different permeabilities provided information that the exchange of gases with the ambient atmosphere was influencing hatchability, the higher the permeability, the lower the hatchability (Becker, 1964; Becker et al., 1964; Proudfoot, 1965). Krueger et al. (1965) found that the advantage of storing hatching eggs in plastic bags was reduced

252

W. A. BECKER, J. V. SPENCER AND J. L. SWARTWOOD

when control eggs were held in egg cases rather than in open filler flats, an indication that air circulation around the eggs can be a possible factor. Such a factor offers a possible explanation for the smaller positive results obtained by Warren et al. (1965) when they held control eggs in closed egg cases. Storing eggs in high concentrations of carbon dioxide and oxygen was detrimental while storage in nitrogen increased, in general, hatchability when compared with eggs held in closed environments with no added gases (Proudfoot, 1964b, 1965; Gowe, 1965). Thus, the interchange of gases across the shell and shell membrane probably affects the hatchability of stored eggs. In the following two experiments the relationship of one of the gases, carbon dioxide, was studied to determine its effect upon hatchability. PROCEDURES AND RESULTS Experiment 1: Taylor et al. (1956) found that C0 2 concentrations of 1.1% or higher during the first four days of incubation reduced hatchability. Therefore an experiment was performed to ascertain if a high level of CO, in albumen administered just prior to setting affected hatchability when there was full circulation of air in the incubators. The eggs laid between 4:30 p.m. and 8:00 p.m. from two flocks of White Leghorns were discarded. Eggs were then gathered the next day at 8:30 a.m., 11:30 a.m., 2:30 p.m., and 4:30 p.m. Immediately after each gathering the eggs were placed into egg coolers maintained at 12.8°C. Clean plastic flats were used throughout this study, both on the farm and in the egg storage room. In the late afternoon all the eggs were removed to a large egg cooler (11.7°-13.9°C., and 70-80% relative humidity), where two eggs from each gather-

ing and flock were broken out for pH determinations. The egg was broken and the contents placed in a petri dish. A pH probe was inserted immediately into the thick white albumen and a reading taken to the nearest hundredth of a pH value. The probe was cleaned and then inserted into the center of the yolk and a reading taken. The pH determinations are given in Table 1. There was an increase in pH of the albumen depending upon the time of day and subsequent holding time prior to breakage. The pH of the yolk was relatively steady. A rise in pH reflects a loss of C0 2 from the egg and we infer from these results that the C0 2 content of the albumen is highest soon after laying. The remaining eggs were weighed on the day of collection and on the day of setting for incubation. Eggs were collected every third day for three weeks so the storage periods of eggs were 0, 3, 6, 9, 12, 15, 18, and 21 days. The linear regression coefficient of egg weight loss on days was 0.0394 ±0.00458 i.e., a loss of about 0.04 grams per egg per day. This result was significantly different than zero at the 1% level. On day of setting, ten eggs were broken out from each day's storage and the pH of albumen and yolk determined as before. Eggs from one treatment group were placed in a large 71 X 118.5 cm. polyvinylidene chloride copolymer plastic bag (trade name, Cryovac) and the bag filled with CO, gas. The bag was sealed by twisting the open end, binding it back on itself and tying it TABLE 1.—A verage pH of White Leghorn eggs collected at different times of the day Time of gathering

pHof albumen

pHof yolk

4:30 p.m. 2:30 p.m. 11:30 a.m. 8:30 a.m. Average

7.74 7.77 7.98 8.36 7.96

6.06 6.10 5.94 5.91 6.00

HATCHING EGG STORAGE AND C0 2

with a rubber band. Small preliminary trials had determined that storage of one hour in C0 2 produced a pH level about that of a fresh egg, and therefore, the eggs were held in this C0 2 atmosphere for one hour. Eggs were then removed and two eggs from each storage day broken out for pH determination. The eggs from both treatments were set in an incubator and after 24 hours of incubation five eggs from each treatment storage day were removed and broken out for pH determination. The mean pH values of the albumen obtained at the described observations are given in Table 2. The loss of the C0 2 during storage is reflected in the observation that the pH was 1.25 units higher on the day of setting than on the day laid. The C0 2 treatment of eggs lowered the pH to 7.94, a level almost the same as that when the eggs were measured on the day laid.

253

TABLE 2.—Mean pH of thick albumen of White Leghorn eggs Measurement time Day laid Day of setting After 24 hours of incubation CO2 treated eggs Immediately after treatment After 24 hours of incubation

Mean pH

Number eggs

7.907 9.132 9.296

128 80 40

7.937 9.201

16 40

The pH after 24 hours of incubation was higher than when the eggs were set, and the pH of the treated eggs, while still somewhat lower than the untreated eggs, indicated that the C0 2 gas was lost during incubation. The pH values of the yolks changed very little and are, therefore, not reported. The embryonic viability and hatchability of the eggs in the two treatments by days of storage are presented in Table 3. Viabil-

TABLE 3.—Embryonic viability and hatchability of untreated and CO, treated {before incubation) White Leghorn eggs Embryi Dnic viability (%) Storage time in days

Treatment

No. eggs 0-7 days

8-21 days Pipping

Hatchabi Hty (%) Fertile eggs

Total eggs

0

C0 2 Untreated

89 88

91.0 97.6

96.1 96.3

97.2 96.1

84.7 91.6

80.8 87.5

3

C0 2 Untreated

104 104

97.0 98.0

94.8 96.9

95.7 90.6

87.0 86.7

83.6 81.7

6

CO2 Untreated

105 103

99.0 92.9

91.8 95.7

95.2 97.7

85.7 86.8

80.0 83.4

9

CO, Untreated

103 104

93.9 91.9

96.7 87.9

97.7 96.2

89.7 78.7

85.4 75.0

12

CO2 Untreated

115 114

97.3 91.9

90.9 95.6

93.5 97.6

82.3 85.8

80.8 74.5

IS

CO2 Untreated

104 105

90.9 98.0

80.0 90.8

90.9 92.8

66.7 83.0

63.4 79.0

18

CO2 Untreated

112 113

96.2 92.6

87.0 83.0

80.8 72.3

70.1 60.1

65.1 57.5

21

C0 2 Untreated

90 90

87.8 88.0

84.7 80.8

80.4 82.0

62.1 60.2

56.6 55.5

All days

C02 Untreated

822 821

94.4 94.0

90.3 91.0

92.2 91.6

78.8 79.1

74.7 74.2

254

W. A. BECKER, J. V. SPENCER AND J. L. SWARTWOOD

ity and hatchability dropped the longer eggs were stored. Although there were differences between the two treatments among the different days, these were not consistent and the unweighted average over all days of storage indicated that there was essentially no difference between the two treatments in embryonic viability and hatchability. Thus, it would appear that the level of C0 2 in an egg just prior to incubation had no effect on hatchability. Therefore, if C0 2 gas does affect embryonic viability, it would appear to be the level during storage which is responsible. Experiment 2: Eggs from a random bred flock of Broad Breasted Bronze turkeys were gathered five times a day from 40 single sire pens (ten females per pen). After the eggs were gathered, they were placed in a room where the temperature was approximately that of the outside air (about 10-18.3°C, December, 1963). Late in the afternoon, the eggs were taken to the egg storage room (11.7-13.9°C, 70-80% relative humidity) and were left overnight in a closed cardboard egg case. Clean plastic turkey egg flats previously dipped in a phenol disinfectant and then dried, were used in this study. The next morning the dirty, cracked, double-yolked and other unsuitable eggs were removed. Four eggs were chosen at random to be broken for determination of pH of the thick albumen. The eggs were divided at random into three treatment groups and each group weighed to the nearest gram. The number of eggs varied from day to day with an average of 60-70 eggs per treatment/day. The eggs were then treated as follows: 1. Unpackaged. The eggs were left on flats exposed to the air of the egg storage room. 1. Plastic bags. Eggs were placed in 40 X 62 cm., 0.04 mm. thick plastic

(Cryovac) bags and the open end tied tightly. 3. Plastic bags and C0 2 treatment. Eggs were placed in a plastic bag (same type as above) and the air replaced with C0 2 gas. The bag was sealed for one hour, then opened and allowed to drain for IS minutes. Two eggs were then removed and broken out for determination of pH. The bag was then tightly sealed. The eggs were collected for 17 consecutive days. The average albumen pH of the eggs broken out was 8.24. The eggs broken after one hour of C0 2 treatment had an average pH of 7.06. After storage, the eggs were weighed again and the weight loss per egg in grams by treatment is depicted in Figure 1. The weight loss was greatest for the unpackaged eggs, continuing steadily up to the 17 days of storage. The regression coefficient of unpackaged egg weight loss in days was 0.0466 + 0.0036 indicating a loss of about 0.047 grams a day. This regression coefficient was statistically significantly greater than zero at the 0.001 level. The loss from the eggs in the plastic bags was much less and appeared to stabilize after about nine days of storage. Within each plastic bag a set of ten test

.8 -

•;,;/• /s\ O

Y.*^V 2

^ W Y

/2r-*-V • 5

Plastic Bogt CO, • 10

• 15

IT

Days of Storage

FIG. 1. The weight losses of BBB turkey eggs by treatment and storage time.

HATCHING EGG STORAGE AND

papers prepared in accordance with procedures given by Kvaale and Dalhoff (1963) provided some evidence of relative humidity changes within the plastic bags. Table 4 presents these average relative humidities. The test papers were not adjusted to 11.7-13.9°C. so the relative humidity readings are only approximate. However, it appears that the relative humidity remained steady after seven days of storage, a finding which would explain the stabilization of the egg weight loss after about nine days (Figure 1) when eggs were stored in plastic bags. The embryonic viability and hatchability for every four days of storage (five days in the case of eggs stored for 13-17) by storage treatment are presented in Table 5. Up to eight days there was no effect of storage treatments although there was a slightly higher hatchability for eggs in the C0 2 treated groups stored 1-4 days. After eight days of storage the C0 2 treatment had a lower hatchability than the eggs stored untreated in plastic bags. The data were analy-

TABLE 5.-

TABLE 4.-

255

C0 2

-The average relative humidity within the plastic bags by days of storage

Days of storage

Relative humidity (%)

1 2 3 4 5 6 7

75-79 79-84 84-88 84-88 88-90 90-93 <93

1

<93

1

17

zed by analysis of variance and Table 6 presents the results. The storage periods were significantly different at the 0.1% level while the comparison of the hatchability of eggs showed that eggs held in plastic bags and treated with C0 2 had a significantly (at the 5% level) lower hatchability than eggs held in plastic bags. At hatching, the poults were taken off at eight hour intervals beginning after 630 hours of incubation and ending at 658 hours. Hatching time was increased by length of storage of the eggs. The linear regression coefficients of hatching time (in

-Embryonic viability and hatchability of BBB eggs for various periods of storage and treatments Embryonic viability

Storage in days

Storage treatment

No. eggs

1-4

Unpackaged Cryovac Cryovac+C0 2

5-8

Hatchability

0-10 days

11-28 days

Pipping

Fertile eggs

Total eggs

303 304 303

92.1 92.5 94.3

95.3 95.0 97.0

94.1 96.2 97.8

82.6 84.5 89.4

69.0 66.4 72.6

Unpackaged Cryovac Cryovac+C02

302 300 302

92.0 94.0 95.4

91.8 95.3 92.6

95.3 91.1 91.6

80.5 81.6 81.0

77.9 68.0 64.9

9-12

Unpackaged Cryovac Cryovac+C0 2

274 274 273

89.0 91.5 90.0

86.6 92.8 81.0

93.8 96.1 83.1

72.2 81.6 60.7

55.1 63.1 46.9

13-17

Unpackaged Cryovac Cryovac+CC^

247 243 243

84.1 87.9 84.5

76.5 86.9 84.4

87.2 92.8 87.1

56.0 70.9 62.1

41.3 53.1 44.4

1-17

Unpackaged Cryovac Cryovac+C02

1,126 1,121 1,121

89.7 91.7 91.6

88.7 93.0 89.6

93.3 94.0 90.9

74.2 80.2 74.7

59.0 63.2 58.2

256

W.

A. BECKER, J. V. SPENCER AND J.

TABLE 6.—Analysis of variance of halchabilily of fertile BBB eggs ' Source

d.f.

Storage periods Treatments Unpackaged vs. plastic

+plastic+C0 2

Plastic vs. plastic +CO2 PeriodsX treatments Days/periods—treatments

M.S.

3 2

1,422***

1 1 6 36

98 306** 126 94

** Significantly different at 5% level. *** Significantly different at 0.1% level.

hours) on storage (in days) were: unpackaged, 1.417 ± 0 . 1 2 0 ; plastic, 1.115 ± 0 . 1 3 4 ; and plastic + CO,, 0.996 ± 0.189. Thus, every day of storage added about one hour to the hatching time. All regression coefficients were statistically significantly longer than zero at the 0.001 level although none of them were significantly different from one another at the 0.05 level. The poults hatched between 636-644 hours of incubation were divided into ten poult groups, there being three groups per storage time and treatment. Each group was group-weighed and randomly assigned to a different wire floored battery pen in a temperature controlled room. All poults were treated alike and were fed the same turkey starter ration. There were three pens for each storage period per treatment, each pen's poults coming from a different day's storage within that storage period. When the poults were three weeks of age,

L.

SWARTWOOD

they were again group-weighed by pens. The mean weights by storage periods and treatments for hatching weight and three-week body weight gain are given in Table 7. The data were subjected to analysis of variance (Table 8). For hatching weight the interaction treatments X storage periods were highly significant. Using this interaction term as the error term, none of the comparisons of treatments or storage periods were significantly different. In the three-week gain analysis the between days within treatment-period was the error term. The mean gain of the poults from eggs stored 1-3 days was highly significantly different than poults from eggs of longer storage periods. This phenomenon coincides with the finding of Becker (1963) that the storage of eggs reduced the 8 and 24-week body weights of turkeys. The storage treatment comparisons were not significantly different. DISCUSSION AND CONCLUSIONS

In the storage of hatching eggs there are two main factors to be considered: the embryo and its surrounding environment. The genotype of the embryo affects its ability to resist the influences of storage. The developmental stage of an embryo before it is stored can play an important part in whether it will develop further and live to hatching. Kosin (1956), by preheating eggs before storage found that advancing the embryo to a certain developmental stage better enabled the embryo to hatch after storage.

TABLE 7.—Means (in grams) of /latching weights and 3-week body weight gains of BBB turkeys Hatching weights Storage in days

Unpackaged

1-3 4-6 7-11 12-17 Treatment mean

55.3 53.3 53.5 52.5 53.7

3-week gain Plastic

Plastic bag

bag+C0 2

56.5 53.7 54.0 52.8 54.3

56.2 53.4 53.2 51.0 53.5

Storage means Unpackaged 56.0 53.5 53.6 52.1

301.8 281.1 292.9 267.3 285.8

Plastic Plastic bag bag+C0 2 320.1 295.5 286.1 277.3 294.7

292.1 280.5 270.3 271.3 278.5

Storage means 304.7 285.7 283.1 272.0

HATCHING EGG STORAGE AND

257

CO,

TABLE 8.—A nalysis of variance of hatching weights and 3-week body weight gains ofBBB turkeys Mean squares Source

d.f. Hatching w(right

Storage Periods (l-3d) vs. ( 4 - 6 d + 7 - l l d + 1 2 - l 7 d ) (4-6d) vs. (7-ll + 12-17d) (7-1 Id) vs. (12-17d) Treatments Unpackaged vs. plastic+plastic+C02 Plastic vs. plastic+C02 Treatment X Periods Between days within treatment period

3 1 1 1 2 1 1 6 24

3-week gain

57.9 2.4 9.8

4,031** 399 558

1.9 3.8 13.3** 2.7

6 1,571 171 385

Significantly different at 1% level.

The developmental stage and the genotype are related for it has been found that preheating certain high hatching eggs reduced their hatchability (Kosin, 1960; Milby and Sherwood, 1960). The experiments reported in this paper are several in a series designed to explore the possibility of improving the environment surrounding the embryo. What might be called the "fresh egg" concept has been adopted for storage of hatching eggs in that the methods and procedures normally recommended for maintaining egg quality have been used. Under the assumption that C0 2 during storage affects hatchability, it was determined in the first experiment that the level of C0 2 gas in the egg just prior to incubation was not a major factor in hatchability. As expected, at a temperature of 37.5°C. the gas disappeared very rapidly, so that 24 hours later the pH indicated very little C0 2 remained in the albumen. This evidence leads to the inference that if C0 2 influences embryonic viability, it does it during the storage period and not during normal incubation conditions. In the second experiment too much C0 2 was apparently allowed to enter the albumen, for the average pH after the C0 2 administration was lower than that of the fresh egg, i.e., a pH of 7.6-7.8. Probably

because of this high level of C0 2 during storage, the hatchability was lowered as compared to eggs held in plastic bags without the addition of C0 2 . We suggest that there may be an optimum level of C0 2 and 0 2 for the embryo prior to incubation, a level (yet to be determined) which is different from that normally found in air. SUMMARY

The effect of C0 2 gas, prior to incubation, upon the viability and hatchability of chicken and turkey embryos was investigated. White Leghorn eggs were stored at 11.7-13.9°C. and 75% relative humidity up to 3 weeks, and half were exposed to C0 2 for one hour immediately before incubation. The albumen pH of the CO, treated eggs became similar to that of freshly laid eggs. Thus, it is the level of C0 2 during the storage period and not the level just prior to incubation which would be the major factor if CO, affects viability of embryos. In another experiment, BBB turkey eggs were treated with CO, and scored in Cryovac bags up to 17 days. Hatchability was: unpackaged, 56%; C0 2 treated, stored in Cryovac bags, 62%; untreated, stored in Cryovac bags, 71%. In the C0 2 treated eggs the level of C0 2 became higher

258

W. A. BECKER, J. V. SPENCER AND J. L. SWARTWOOD

than that of a freshly laid egg and, therefore, this may have contributed to the lower hatchability. The regression coefficients of hatching time in hours on days of storage were: unpackaged, 1.417; Cryovac bag, 1.115; and Cryovac + C0 2 , 0.996, indicating that every day of storage added about 1 hour to the hatching time. Hatching weights and 3-week body weight gains of poults hatched from these eggs provided evidence that storage of eggs lowered 3-week gains. The three methods of storage had little or no effect on these two traits. It is suggested that there may be an optimal level of C0 2 and 0 2 for the embryo prior to incubation which differs from that of air. ACKNOWLED GMENTS

The authors express their appreciation to Mrs. Mary Helen George for technical assistance in obtaining the pH values of the eggs. W. R. Grace Company, Cambridge, Massachusetts, provided the large Cryovac plastic bags for a portion of this study. REFERENCES Becker, W. A., 1963. Length of pre-incubation storage of turkey eggs and its effects on body weight. Poultry Sci. 42: 13 56-13 59. Becker, W. A., 1964. The storage of White Leghorn hatching eggs in plastic bags. Poultry Sci. 43: 1109-1112. Becker, W. A., J. V. Spencer, and J. L. Swartwood, 1964. The pre-incubation storage of tur-

key eggs in closed environments. Poultry Sci. 43 : 1526-1534. Gowe, R. S., 1965. On the hatchability of chicken eggs stored in plastic bags flushed with nitrogen gas. Poultry Sci. 44: 492-495. Kosin, I. L., 1956. Studies on pre-incubation warming of chicken and turkey eggs. Poultry Sci. 35: 1384-1392. Kosin, I. L., 1960. Heredity—another factor responsible for variable responses of turkey hatching eggs to pre-incubation warming treatments. Poultry Sci. 39: 1266. Krueger, W. F., J. H. Quisenberry and J. W. Bradley, 1965. Preserving the hatching power of eggs through the use of plastic bags. Poultry Sci. 44: 1392. Kvaale, O., and E. Dalhoff, 1963. Determination of the equilibrium relative humidity of foods. Food Tech. 17: 659-661. Milby, T. T., and D. H. Sherwood, 1960. The influence of pre-incubation treatment on hatchability of chicken and turkey eggs. Poultry Sci. 39: 1118-1121. Proudfoot, F. G., 1964a. The effects of plastic packaging and other treatments on hatching eggs. Canad. J. Anim. Sci. 44: 87-95. Proudfoot, F. G., 1964b. The effect of nitrogen and other gases on the hatchability of eggs stored in plastic bags. Canad. J. Anim. Sci. 44: 120-121. Proudfoot, F. G., 1965. The effect of film permeability and concentration of nitrogen, oxygen and helium gases on hatching eggs stored in polythylene and Cryovac bags. Poultry Sci. 44: 636-644. Taylor, L. W., R. A. Sjodin and C. H. Gunns. 1956. The gaseous environment of the chick embryo in relation to its development and hatchability. Poultry Sci. 35: 1206-1215. Warren, D. C , H. A. Roff and E. Long. 1965. Hatchability of eggs stored in plastic-lined egg cases. Poultry Sci. 44: 1278-1280.

NEWS AND NOTES (continued from page 211) signed primarily for educators and food buyers. prise about 12 percent of all eggs produced. It contains much up-to-date, and previously unThe folder is available from the Poultry and available in one source, information including the Egg National Board, 8 South Michigan Ave., Chicomposition and nutritive values of egg products, cago, Illinois 60603. It is priced at $1.90 in lots conversion factors for use of egg products, and a of 100, and $17 in lots of 1,000. specification guide for buyers and users of such MARYLAND NOTES products. At the present time, egg products used comDr. H. M. DeVolt, Chief of Research in Vet(continued on page 263)