IT HAS been well established that

The Effect of Different Holding Temperatures on the Hatchability of Hens' Eggs M. W. OLSEN AND S. K. HAYNES Agricultural Research Center, Beltsville, Maryland IT HAS been well established that storage temperature plays an important role in preserving the viability of the unincubated chick embryo. Research has demonstrated that eggs stored at too high or too low a temperature for prolonged periods hatch poorly. Several workers have studied the effect of temperature on hatchability but most of the reports in the literature are based either upon relatively small numbers of eggs, a few isolated temperatures, or poorly controlled temperature conditions. Phillips (1909) was among the first investigators who were interested primarily in storage temperature as a factor affecting hatchability. In his experiments, three groups of 50 eggs each were stored for 14 days prior to incubation. One group was stored in a room in a heating plant where the temperature averaged 80 F.; another group was stored in a laboratory room where the air temperature averaged 65 F.; and the third group was held in a refrigerator at 50 F. The percentage hatch of fertile eggs set was 0.0%, 43.1% and 75% for each of the respective groups. Mauro (1923) stored two lots of hatching eggs for 10 days; one lot was held at an average temperature of 32.9 F. and the other at 57.2 F. He found that the embryos in the eggs stored at 32.9 F. did not develop when the eggs were removed from storage and placed in the incubator (Received for publication November 24, 1947) but he obtained a 60% hatch of fertile eggs which had been stored at 57.2 F. for 10 days. Mussehl and Bancroft (1924-25) investigated the cause of lower fertility and hatchability of eggs produced during and shortly after storms. They subjected a series of different groups of eggs to a temperature of 32 F. for periods ranging from 2 to 36 hours. Other groups of eggs were subjected to temperatures of 38 F. for 6, 12, and 18 hours. The eggs used for these tests were less than 8 days old and during the tests were exposed to the temperature indicated by placing them on open wire trays for the periods indicated. On removal from storage, all eggs in each series were kept at room temperature from 12 to 24 hours before placing them in the in-, cubator. These investigators found that hatchability of the eggs so treated was not adversely affected under these conditions and concluded "that the lowered fertility and hatching power of eggs produced during and shortly after storms is due to the lowered vitality of the breeding stock rather than a direct influence of low temperature on the eggs produced." Dougherty (1926) stored a group of 300 hatching eggs, less than 3 days old, in a refrigerator at 40 F. for 6 days. A second group of 300 eggs was held at 50 F. for the same length of time. He obtained a hatch of 55.25% of fertile eggs from the 420

EFFECT OF HOLDING TEMPERATURES ON HATCHABILITY 421 group held at 40 F. and a hatch of 60.46% from those held at 50%F. Scott (1933) stored two groups of White Leghorn eggs and two groups of Bronze turkey eggs at average temperatures of 36.3 F. and 54.2 F. for periods of 0 to 34 days. These eggs were less than 24 hours old and were stored in egg cases which were 'turned twice daily during the holding periods. He found that "the hatching power of Leghorn eggs was not materially affected adversely when held from 0 to 6 days at a mean temperature of 36.3 +.2 F. The viability of the Leghorn embryos was markedly reduced when eggs were held at this temperature for longer periods. No Leghorn embryos survived the incubation period after the eggs had been held for 8 days at a mean temperature of 36.3 ±.2 F. Leghorn embryos from eggs held at a mean temperature of 54.2 +.26 F. from 0 to 6 days hatched 69.35%, from 7 to 13 days 66.66%, 14 to 20 days 67.95%, from 21 to 27 days 44.3%; and 28 to 34 days 32.05%." The turkey embryos appeared to be more resistant to low temperatures than did the White Leghorn embryos, and the break in hatchability was not as sharp as that observed with Leghorn eggs. In turkey eggs stored at 36.3 F. for 0 to 6 days before incubation, the hatch was 65.63% of fertile eggs set; in those stored for 7 to 13 days, 52.38%; in those held for 14 to 20 days, 26.79%; and in those stored for 21 to 27 days, 5.18%. No turkey eggs hatched after being stored at 36.3 F. for 28 days or longer. The average hatch of fertile turkey eggs for those groups stored from 0 to 34 days at 54.3 F. was 68.47%. Funk (1934) chilled chicken eggs in an electric household refrigerator at 32 to 38 F. for periods ranging from 6 to 192 hours. He found that chilling eggs for 48 hours or less under these conditions was not harmful to hatchability. Holding eggs under these conditions for 96 hours or longer decreased the hatchability of the eggs until the zero point was reached at 168 hours. Phillips (1945) subjected three groups of New Hampshire hatching eggs to temperatures of 32 F., 38 F., and 52 F. for 1 to 7 days. The eggs used were less than 1 day old and were stored for the first 24 hours in open trays, and for the remaining portion of each test the eggs were kept in egg cases. During the course of five incubation periods, 4,545 eggs were set. The resulting hatches based on number of fertile eggs set were 85.0%, 83.5%, and 84.6% respectively. Phillips concluded "that 32 F. and 38 F. holding temperatures had no influence upon hatchability when compared with comparable eggs stored at the usual recommended temperature of 52 F." He suggested the possibility of differences between breeds of chickens in the ability of their embryos to withstand low temperatures. Recently, Jull and co-workers (1947) subjected several groups of chicken and turkey eggs to temperatures of 1 to 3 F. and 20 F. The eggs used for these tests were less than 6 days old and were stored in egg cases. These investigators found that hen eggs (where shells did not break) could be held as long as 10 hours at 1 to 3 F. without reducing hatchability. Turkey eggs were also held at this temperature for as long as 4 hours without reducing hatchability. Other groups of hen and turkey eggs were stored for periods ranging from 1 to 4 days at 20 F. The percentage hatch of fertile hen eggs was as follows: 65.2%, 46.1% and 6.7% for 1, 2 and 3 days' storage periods, respectively. The percentage hatch of corresponding control groups was 85.7%, 72.0% and 74.1% of fertile eggs set. Turkey eggs stored at 20 F. for 2, 3 and 4 days gave a

422 M. W. OLSEN AND S. K. HAYNES hatchability of 71.4%, 40.0% and 0.0% respectively as compared with 87% and 80% for two control groups. MATERIAL AND METHODS The eggs used in this study were obtained from mass-mated flocks of Single- Comb White Leghorn and Single-Comb Rhode Island Red pullets at the "Agricultural Research Center, Beltsville, Md. The eggs were collected twice daily during the experimental period and placed in an egg holding room at 55 F. until ready for use. No effort was made to segregate the eggs from these two breeds during the holding or hatching period. In the first part of this study, eggs 1 to 5 days old were subjected to different storage temperatures. The eggs were divided at random into 5 groups and placed in open, wire-bottomed trays. Each group was stored in a different refrigerator or room. Four refrigerators were operated at average temperatures of 30, 40, 50, and 60 F., respectively. The remaining group of eggs was stored in an insulated room in which the average air temperature was 70 F. The eggs were not turned during the storage period and no attempt was made to control humidity in the storage rooms. At the end of the second day of storage, one-fourth of the eggs of each group were removed and placed in an electric forced draft incubator which was operated at 99f F. and at 55% relative humidity. At the end of the fourth, sixth, and eighth days of storage, equal numbers of eggs were removed from the various storage rooms and placed in the incubator previously mentioned. The eggs were candled on the seventh day of incubation, at which time infertile eggs and those containing dead germs were removed, broken and examined macroscopically for fertility. At the close of each incubation period all the eggs which failed to hatch were broken and the embryos examined and classified as to time of death. A record was kept of the number and type of terata found among the embryos of each of the different groups of eggs. Four separate incubations were made for each holding period and each temperature. The second part of this study was designed to test the efficiency of holding temperatures of 50 and 55 F. in preserving the hatchability of hen eggs over extended periods. For this phase of the experiment, hatching eggs from mass-mated flocks of Single-Comb White Leghorn and Single- Comb Rhode Island Red pullets were used. Each day, for a period of 18 days, a group of eggs less than 24 hours old was divided into two lots, the division being made so that each lot would contain approximately the same number of eggs from each breed. Each day, two lots of eggs were racked, large and uppermost, in wire-bottomed incubator trays and then placed in holding rooms, one at 50 F. and the other at 55 F. The trays were placed in racks which were so designed that the eggs could be turned twice daily during the storage period. At the end of the 7th, 14th, 21st, 28th, 35th, and 42nd days of storage, one lot of eggs was removed from each of the two holding rooms and placed in a small, forced-draft incubator which was operated at 99f F. and at a wet bulb reading of 86 F. The eggs were candled on the 7th day of incubation and eggs appearing before the candler as infertile or with dead germs were removed, broken and examined macroscopically for indications of development and time of death of the embryos. Three separate incubations were made for each holding period and each temperature. RESULTS AND DISCUSSION Data are presented in table 1 showing the effects of different holding tempera-

EFFECT OF HOLDING TEMPERATURES ON HATCHABILITY 423 TABLE 1. The effect of holding eggs at different temperatures for period of 2-4 days and 6-8 days prior to incubation Item 2 to 4 day holding period 30 F. 40 F. 50 F. 60 F. 70 F. Eggs put in incubator 952 964 963 956 738 Percent of eggs fertile 87.6 89.2 89.2 89.2 91.6 Percent of eggs with dead germs: 1st week 30.9 6.4 7.6 8.0 7.4 2ndweek 1.2 1.4 1.1 1.2.7 3rd week 11.2 11.9 9.9 11.9 12.1 Percent hatch of total eggs 50.7 71.7 72.7 71.2 73.1 Percent hatch of fertile eggs 57.9 80.4 81.5 79.8 79.7 6 to 8 day holding period 30 F. 40 F. 50 F. 60 F. 70 F. Eggs put in incubator 949 955 935 941 740 Percent of eggs fertile 90.4 92.9 90.8 91.3 93.6 Percent of eggs with dead germs: 1st week 97.2 15.4 10.6 9.0 10.8 2ndweek.0 1.1 1.4 1.5 1.2 3rd week.6 12.3 9.4 12.8 14.3 Percent hatch of total eggs 2.0 66.1 71.3 70.0 69.1 Percent hatch of fertile eggs 2.2 71.1 78.6 76.7 73.7 tures for periods of 2 to 4 and 6 to 8 days on the subsequent hatchability of eggs. Of the five temperatures tested, the 50 F. temperature proved to be the most desirable for storage of hatching eggs. The data presented in table 1 show that a temperature of 30 F. for 2 to 4 days is definitely more harmful to the unincubated blastoderm than are temperatures well above the 50 F. optimum. Fifty-eight percent of the fertile eggs hatched after storage at 30 F. for 2 to 4 days; only 2.2% hatched after storage at 30 F. for 6 to 8 days. In contrast, eggs stored at 50 F. for 2 to 4 days hatched at the rate of 81.5% of fertile eggs, and eggs at 50 F. for 6 to 8 days at the rate of 78.6% of fertile eggs. These results are not in accord with findings reported by Phillips (1945), who found no detrimental effect on hatchability due to storage at low temperatures. He reported that 85.0 and 83.5% of fertile eggs hatched after the eggs had been stored at 32 and 38 for 1 to 7 days. Phillips used New Hampshire eggs which were less than one day old when stored, whereas in this study eggs from mass-mated flocks of White Leghorn and Rhode Island Reds were used, and these eggs were 1 to 5 days old when stored at the different temperatures. In this present study there was a very high embryonic mortality during the first few days of incubation in eggs stored at 30 and 40 F. Most of the blastoderms in eggs stored at low temperatures failed to develop after the eggs were placed in the incubator. These eggs appeared to be infertile when candled at 7 days' incubation, but when they were broken and examined, almost all the eggs were fertile but had failed to develop. This latter condition was also noted by Mauro (1923), in eggs stored at 32.9 F. for 10 days. The data in table 1 also show that 40 F. is too low a temperature for storing hatching eggs except for short periods of time. Fertile eggs, 1 to 5 days old, stored at 40 F. for 6 to 8 days prior to incubation hatched at the rate of 71.1%. This was

424 M. W. OLSEN AND S. K. HAYNES TABLE 2. Hatchability of eggs as affected by storage at 50 and 55 F. for extended periods Days of storage No. eggs Percent fertile 1 Percent eggs containing dead germs 1st wk. 2nd wk. 3rd wk. of fertile eggs Percent hatch of total eggs 7 14 21 28 35 42 50 539 409 270 50 538 409 409 408 270 55 50 84.4 83.8 88.0 88.9 91.6 93.9 87.4 92.1 81.0 79.7 51.9 53.7 8.1 10.2 7.0 7.0 14.1 14.6 25.9 56.0 57.7 86.4 86.2 99.2 1.3 1.3 1.4 2.0 2.2 3.4 4.3 3.8 3.7 1.9 1.4 0.0 9.5 8.4 14.6 18.9 23.6 21.6 19.9 13.9 16.4 5.9 5.8 0.8 80.4 80.0 77.0 72.1 60.1 60.4 50.0 26.3 22.2 5.9 6.5 0.0 50-67.9 67.2 54.5 43.2 17.8 3.3 55 67.1 63.9 56.7 24.2 4.7 0.0 1 Many of the eggs in the lots stored for 35 and 42 days failed to start development upon incubation and consequently the blastoderms after seven days in the incubator could not be distinguished from infertile eggs. 7.5% less than for corresponding groups of eggs which had been stored for the same length of time at 50 F. In eggs 1 to 5 days old, stored at 60 and 70 F. for 6 to 8 days, the percentage hatchability of fertile eggs was 76.7 and 73.7 respectively. Hatchability data on two groups of eggs stored at 50 and 55 F. for 7 to 42 days in 7-day periods are presented in table 2. As stated earlier these eggs were less than 24 hours old when placed in storage. These data show that hatching eggs may be stored for 7 to 21 days at either 50 or 55 F. without materially affecting the hatchability. However, longer holding periods showed that 50 was the most desirable holding temperature since the hatchability of some eggs was preserved as long as 42 days whereas no chicks were hatched from eggs held at 55 F. for the same length of time. Furthermore, after 28 to 42 days in the holding rooms, the blastoderms of many fertile eggs failed to resume development after the eggs were placed in the incubator. Such fertile eggs, at the 7th (day) candling period appeared to be infertile but when these eggs were broken out and examined, they could not be classified accurately as to fertility or infertility. This condition was noted in eggs held at both 50 and 55 F. but as indicated in table 2, was more pronounced in the group of eggs held at the higher temperature. This accounts for the apparent increase in the percentage of eggs listed as infertiles in groups held for 28 days or longer. Data are presented in table 3 showing the number and types of embryonic terata found among the unhatched eggs after the third day of incubation. Data published by Hutt and Greenwood (1928 1929) and Byerly (1930) are also included for purposes of comparison. The data of Hutt and Greenwood (1928-1929) represents the number and type of terata one TABLE 3. Data on embryonic terata after the 3rd day of incubation Author Twins No. of t ^. Time of un- Hyperen- Micro- v t Exen- Dupliihfre stora «e hatched cephaly phthalmia act0 P la cephaly citas embryos Miscellarleous Total P««t- Olsen Olsen Olsen Hutt & Greenwood (hatchery flocks) Byerly (Belts. Res. Center) 30+40 F. 2~8davs 50 F. 2-8 days 60+7O F. 2-8 days Not given Not given 11,797 Not given 1-7 days 445 213 495 2,104.45.47 i:s2.38 11 8.45 7.47.81 3.52.45 1.21.05 1.62.90.20.84.43 1.80 1.41.20.07.47.45.47? 0 0 0.37 2.95 15.06 10.79 9.09 3.67 9.36 *

EFFECT OF HOLDING TEMPERATURES ON HATCHABILITY 425 would expect to find in eggs produced under field conditions since their data were obtained by breaking unhatched eggs from several farm flocks which supplied eggs for a local hatchery. The data published by Byerly (1930) on the other hand, were obtained by breaking unhatched eggs which had been produced by the experimental flocks at the Beltsville Research Center. No definite data were given by these investigators as to the temperature at which eggs were held previous to incubation. The data gathered for this present study indicates that the low temperatures used for holding eggs prior to incubation caused an increase in the number of embryonic terata. Twelve cases or 1.04 percent of duplicitates and 3 cases or 0.26 percent of twins (two embryos on the same yolk) were found on breaking the 1,153 unhatched eggs. There were 8 or 1.80 percent duplicitates in 445 unhatched eggs which had been stored previous to incubation at temperatures of 30 and 40 F.; 3 or 1.41 percent in 213 unhatched eggs stored at 50 C F.; and one or.20 percent in 496 unhatched eggs stored at 60 F. Of the 12 cases, 11 were classified as posterior duplicitates. The incidence of duplicitates where eggs were stored at 30 F., 40 F. and 50 F. was over two times as great as that reported by Byerly and 15 times more than reported by Hutt and Greenwood. Other types of terata found during this study with their percentage of incidence are given in table 3. In general, the colder the storage temperature previous to incubation the greater was the incidence of most types of embryonic terata. SUMMARY Data are presented showing the effect of holding temperatures prior to incubation on the subsequent hatchability of White Leghorn and Rhode Island Red eggs. Groups of approximately 950 hatching eggs 1 to 5 days old were stored for 2 to 4 days and 6 to 8 days prior to incubation at temperatures of 30, 40, 50, 60 and 70 F. The highest hatchability (78.6%) was obtained in eggs stored at 50 F. and the lowest (2.2%) in eggs stored at 30 F. for 6 to 8 days. Subsequent tests were conducted on the efficiency of temperatures of 50 and 55 F. in preserving the hatchability of eggs for periods ranging from 7 to 42 days in 7 day intervals. There was little difference in the hatchability of eggs stored at these two temperatures for the first 21 days. The hatchability of fertile eggs stored at 50 F. for 28, 35 and 42 days was 50.0, 22.2 and 6.5%; whereas hatchability of fertile eggs stored at 55 F., for the same periods was only 26.3, 5.9 and 0.0%. Data are also presented giving the relative frequency of various types of terata found among the unhatched embryos. In general, there was an increase in the incidence of micropthalmia and duplicitas in the embryos when eggs were stored at temperatures of 30 and 40 F. as compared with the incidence at higher temperatures previous to incubation. Twelve cases of duplicitas and 3 sets of twins (two embryos on the same yolk) were found among the unhatched embryos, 14 of which were in eggs which had been stored at temperatures ranging from 30 to 50 F. REFERENCES Byerly, T. C, 1930. Time of occurrence and probable causes of mortality in chick embryos. Proc. 4th World's Poultry Cong. London: pp. 178 183. Doughtery, J. M., 1926. Studies in incubation. I. The effect of low temperatures previous to incubation on hatchability of eggs set. Amer. Jour. Physiol. 79: pp. 39-43. Funk, E. M., 1934. Factors influencing hatchability in the domestic fowl. Mo. Agr. Exp. Sta. Bui. 341: pp. 1-22.

426 M. W. OLSEN AND S. K. HAYNES Hutt, F. B., and A. W. Greenwood, 1928-29. Studies in embryonic mortality in the fowl. I. The frequencies of various malpositions of the chick embryo and their significance. Chondrodystrophy in the chick. III. Chick monsters in relation to embryonic mortality. Proc. Roy. Soc. of Edinburgh. XLIX: pp. 118-155. Jull, M. A., M. G. McCartney, and H. M. El- Ibiary, 1947. Hatchability of chicken and turkey eggs held in freezing temperatures. Poultry Sci. (in press). Mauro, M. F., 1923. II trattamento firgarifice delle uova (di-gallina) e la sua influenza sulla capacita di sviluppos della macula germinative. Atti della Soc. Ital., de Scienz Naturali 62: pp. 239-246. Mussehl, Frank E., and Paul Bancroft, 1924-25. Effect of low temperatures on hatching power of hens' eggs. Poul. Sci. 4: pp. 79-81. Phillips, Allen C, 1909. Keeping eggs for hatching. Kans. Farmer 47(6): pp. 3-7. Phillips, R. E., 1945. Hatchability as influenced by environmental and different storage temperatures. Poul. Sci. 24: pp. 25-28. Scott, H. M., 1933. The effect of age and holding temperatures on hatchability of turkey and chicken eggs. Poul. Sci. 12: pp. 49-54.