THERE is considerable evidence that

Effects of High Temperature on Semen Production and Fertility in the Domestic Fowl M. A. BOONE Poultry Department, Clemson College, Clemson, S.C. AND T. M. HUSTON Poultry Department, University of Georgia, Athens, Ga. THERE is considerable evidence that environmental temperature influences fertility and hatchability of eggs in the domestic fowl. Upp and Thompson (97), Funk (935) and Heywang (9) reported that fertility and hatchability decreased during the hot summer months. Ota, McNally and Wilson (955) found that fertility was reduced in a variable environment and that fertility and hatchability were improved by keeping the hens at a constant temperature of 65 F. Wilson (99) showed that fertility in the female was not seriously affected by high temperatures. It has been demonstrated by others, however, that the fertilizing capacity of the cock declines during hot weather (Parker and McSpadden, 93; Huston and Wheeler, 99). Huston and Carmon (958) found no statistically significant differences in fertility between cocks held at high (9 F.) temperatures and those held at normal variable temperatures. Since pooled semen samples were used, it is possible that two or three superior males in the group fertilized most of the eggs and obscured the lower fertility of the other males. A search of the literature did not reveal what the effects might be on individual chicken and turkey semen production if temperatures over 65 F. were employed. Journal Series Paper No. 65. College Experiment Station, College of Agriculture Experiment Station, University of Georgia, Athens, Georgia. (Received for publication November 5, 96) 67 MATERIALS AND METHODS Three separate trials were conducted. Two equal groups of mature White Plymouth Rock males were used in each trial. One group is referred to as the controls and the other is referred to as the temperature exposed group. Within the trials both groups were treated alike except for the period of time the temperature exposed groups were in the environmental chamber. Individual measurements were made for volume, sperm motility and number of sperm per ejaculate. Motility was determined immediately after collection by diluting a drop of raw semen with approximately ml. of avian Ringer's solution. The sample was then placed on a hanging drop slide and examined under a microscope. Motility was scored on the basis of two different criteria: () the percent of viable sperm as determined by motion, was scored -9, and () the speed (sperm ) at which the sperm traveled across the microscopic field, was scored -5 where equalled no movement and the score of 5 equalled the greatest movement. Hence an individual score of 95 would indicate at least 9 percent of the sperm in the field showed some motility and that they were traveling at a maximum speed of five. These data were treated statistically according to the method of least squares with disproportionate subclass numbers Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 5, 6

HIGH TEMPERATURE AND FERTILITY 67 (Harvey, 96). Adjustments were made for previous production by covariance analysis. Trial. Sixteen males, 3 months of age, were used. They were held in individual laying cages and given a commercial allmash breeder ration and water ad libitum. Thirteen hours of light per day was provided with an average light intensity to approximately five foot candles at the head of the bird. Semen samples were collected from each male two days prior to heat stress and five times within the fourteen day period following heat stress as shown in Table. The one man technique of artificial ejaculation devised by Wheeler (98) was used. At least two days were allowed between ejaculations. Semen was collected from a group of eight birds at a time. On the day of stress one half of the males (eight) were placed in the environmental chamber at room temperature and the ambient temperature was raised at a rate of about degrees per hour until the temperature reached F. and a relative humidity of 65%. The birds were maintained at this temperature for a period of three hours. Only three birds were alive at the end of this period. The other five males became prostrate and were removed prior to completion of the test period. The males were ejaculated immediately after removal from the environmental chamber and the measurements were made on the sperm as previously described. The surviving males from the heat exposure group and four males randomly selected from the variable temperature control group were similarly checked four times within the next two weeks. Trial. In this trial the environmental temperature for all males was held constant at approximately 7 F. A total of 6 males 8 months of age were used. The males were paired on the basis of semen volume prior to heat stress to assure equal distribution between the control and treated groups. On the day of test, eight males were placed in the environmental chamber and the temperature raised as before. The temperature was maintained at 3 F. and a relative humidity of 68%, for three and one half hours. The same measurements were made on each of six surviving males as in the previous trial. TABLE. Adjusted mean values for semen volume, sperm, density and number per ejaculate of Domestic cocks exposed to high environmental temperature {trial ) Overall Mean s after stress 5 5 7 7 Volume (ml.).8.63.39.9.3.3..8.7.35. Motile sprem 7 35 36 5 63 5 5 8 36.6.6.6.3 3..39 3.6.7.3.8.7 per mm. 3.98.75.8.7..86.77.88.9.3.89 per ejaculate,35 63,75 686,88,6 973 6,8,3, Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 5, 6 Note: Data from control cf's and 3 heat stress ct's, age 3 months. Stress consisted of 3 hours at F.

67 ' M. A. BOONE AND T. M. HUSTON TABLE. Adjusted mean values for semen vot domestic cocks exposed to high e, sperm, density and number per ejaculate of ironmental temperature {trial ) s after stress Volume (ml.) Motile sperm per mm. 3 per ejaculate.7.7 53 3..93.3.37 868 7 3 3.9.87 39 3.73.53.7.65 58 6 Overall Mean 6 6 9 9 5 5.6.9.8.93.3.69.7.76. Note: Data from 8 control c? 's and 6 heat stress d*' s, age 8 months. Stress consisted of 3J hours at 3 F. Trial 3. This trial was initiated with 8 pedigreed males months of age. Each of the control males had a full brother in the heat exposed group. The males were placed in individual cages three weeks before being exposed to high temperature. Three to four days before heat exposure a type "T" thermocouple enclosed in a pyrex jacket was embedded in the pectoral muscle as described by Boone (96). The control males were similarly treated with a dummy thermocouple. All males were ejaculated two days prior to heat exposure. Semen of each male was measured for volume, sperm motility, sperm count per ejaculate and. ml. was artifically inseminated into each of three hens. Wilcox buffer (Wilcox, 958) was used as the diluent for determining motility. Eggs were then collected for a period of fourteen days starting with the second day after insemination. Fertility of the eggs was determined on the 8th day of incubation by candling. Eggs were broken and examined macroscopically if embryonic development could not be 6 39 6 59 58 6 83 86 5 3.65.93.5 3.53 3. 3.53.33 3.8 3.3.3.7.89..8.8..85.86, 578 77 8 836 9,3,37 838 detected by candling. On the day of exposure males were placed in the environmental chamber and previously inserted thermocouples attached to a station multipoint recorder. The temperature in the chamber was raised ten degrees per hour until.5 ( +.5 F.) was attained. This temperature was then maintained for two hours. The humidity was maintained at 5 (+ ) percent. Individual body temperature was recorded every 7 seconds while the birds were in the chamber. All birds survived the exposure period but two males died within 5 minutes after removal from the chamber. RESULTS The adjusted mean values obtained in the first trial are summarized in Table. These results indicate that semen volume and number of sperm per ejaculate were significantly reduced on day of exposure. The semen volume and number of sperm per ejaculate had returned to the level of the controls five days later. Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 5, 6

HIGH TEMPERATURE AND FERTILITY 673 In the second trial (Table ) the results also indicate immediately after heat exposure semen volume, sperm concentration and total sperm ejaculate were reduced. Semen quality returned to normal within seven to nine days. The lowered sperm concentration of the heat exposed males was not statistically significant in either Trial or Trial. The fluctuation between days for semen volume and percent of motile sperm was statistically significant in Trial. Heat exposure did cause a significant reduction in sperm concentration. In Trial 3 (Table 3), as in the previous trials, heat exposure caused a non-significant reduction in semen volume, but the differences in sperm concentration and number of sperm per ejaculate was highly significant. This reduction was of short duration and within two days these factors equaled that of the control group. The analysis of co-variance for the three trials is shown in Table. Correlation coefficients (Table 5) were determined between fertility and the following variables; volume, sperm concentration, percent of motile sperm and sperm. There were two statistically significant correlations: () percent of motile sperm with fertility for the heat stress group two days after stress, which was found to be highly significant (r =.6) and () fertility with sperm concentration which was found to be highly significant (r =.55) for the control group of males on the 5th day after stress.. DISCUSSION Observation of the adjusted mean values for the three trials indicate that there was a treatment effect on semen volume, sperm concentration and number of sperm per ejaculate. In all three trials these values were reduced on day of stress. However, the only statistically significant differences found were () Heat exposure caused a significantly reduced number of sperm per ejaculate in the first and third trials. () A significantly lower volume in the first trial, and (3) a significantly reduced sperm concentration in the second trial due to heat stress. In no instance was the percent of motile sperm or sperm affected. The results indicate that an exposure of two or three hours at a temperature of F. to F. had only a temporary detrimental effect on cock semen qualities. Coefficients of correlation were deter- TABLE 3. Adjusted mean values for semen volume, sperm, density and number per ejaculate of domestic cocks exposed to high environmental temperature {trial 3) Overall Mean s after stress 5 5 Volume (ml.).87.73.9.9.6.5.3 Motile sperm 7 67 6 67 6 63 65 3.89.7 3.8 3.6.6.9 3.3 per mm. 3.7.8.67.59.56.5.9 per ejaculate 537 35 58 565,,38,6 Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 5, 6 Note: Data from control cf's and heat stressed cf's, age months. Stress consisted of two hours at.5 F. Internal body temperature (mid-pectoral muscle) 5.6T. before stress and. F. at termination of stress.

67 M. A. BOONE AND T. M. HUSTON mined between fertility and semen volume sperm concentration, percent of motile sperm and sperm. In Trial 3, there was a highly significant correlation between fertility and percent of motile sperm for the heat exposed group on the second day after treatment. concentration was found to be significantly correlated with fertility in only one case. The consistent lack of correlation in this study between fertility and the factors studied agrees with the findings of Parker, McKenzie and Kempster (9). Shaffner and Andrews (98) found that semen volume and sperm concentration were not TABLE. The analysis of co-variance for the five variables of the three trials F., 3'F. and.5 F. respectively X X X X X d.f. Trial M.S. 969.55 8,73.*,6. 5,33.79**,333.9 d.f. correlated with fertility but there was a significant correlation between initial motility and fertility. Allen and Champion (955) found a positive correlation between motility and the number of progeny. Cooper and Rowell (958) found fertility was significantly correlated with percentage of dead sperm and motility. Fertility was not correlated with volume or density. McCartney (956), utilizing turkey semen, did not find any correlation between fertility and volume, percent motility, sperm concentration per mm. s, or number of sperm per ejaculate. Since individual variation for heat toler- Trial M.S. Semen Volume 5 9,873.99* 635.53 5,5.,3.7** 5,975.83 No, pei ' mm. 3,95.78 5 5,98.7 966.35 5,66.* 3,93.3 5 5,593.8,67.8** 8,5.6**,96.69 5 6,655.3 No.,639.8 7,6.8*,37.97,59.97**,5.3 Percent Motile,7.88** 5 6.89 369.88 5.3 5.58 5...5.*.63 per Ejaculate 5,8.37 3,58.5 5 3,5.95* 65,8.8** 5,9.3,8.7*,.95 53.6 3,99.6** 89.9 Vigor 5.35.69 5.36. 5. ' ambient temperature was d.f. 9 9 9 9 9 Trial 3 M.S. 3,883.9** 39.69,33.5,9.67** 93.3,66.** 7,37. 3,976.39 8,7.6**,67. 7,8.75** 5,75.3** 5.87,789.,6.37 79. 8.3 8.3 7,833.** 7. 3.**..8 7.87**.6 Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 5, 6 * Significant at the P.<.5 level. ** Significant at the P.<. level.

HIGH TEMPERATURE AND FERTILITY 675 TABLE 5. Correlation coefficients between fertility and semen, sperm concentration, percent motile sperm and sperm by days and by treatment for trial 3 Time of measure- Fertility vs. semen ment in relation volume to days of stress (days) Heat r stress r Fertility vs. sperm concentration Heat stress r Fertility vs. % motile sperm r Heat stress r Fertility vs. sperm Heat stress r before Oday after 5 after.7.8 -..33..38 -.8 -.9..9.38.5**.7.7 -..8..5 -. -.3.3.5.6**.3.7 -.5 -..8 -.3 -.6.39. ** Significant at the P.<. level. ance is recognized, internal body temperature was considered as an additional criterion for heat stress. No significant correlation was found between the internal body temperature of the birds during heat exposure and subsequent fertility of their sperm collected immediately after removal from the heat chamber or two days later. The mortality rate during the exposure period indicates that the temperatures used approached the lethal range. The data presented show that surviving males exposed to a single short period of high temperature recover rather quickly with relatively little detrimental effect on the factors studied. The findings further suggest that summer decline in fertility of poultry flocks is a gradual process. It is highly probable that seasonal factors have a cumulative effect involving the endocrine system and other physiological responses influencing fertility. Since embryonic development is the usual criterion of fertility, the role of the female and her environment should not be ignored. The frequency of mating may also be reduced because of lethargy during hot weather. Any or all of these factors might account for lower fertility during the summer months rather than spermatogenesis per se. SUMMARY Three trials were conducted in which a total of 8 White Plymouth Rock males were subjected to ambient temperatures of.5 F. to F. for a period of two to three and one half hours. The data consistently showed lower, but non-significant, values for semen volume, sperm concentration and number of sperm per ejaculate immediately following high temperature stress. These values usually returned to normal within five to six days after stress. Correlations between fertility and semen volume, sperm concentration, percent of motile sperm and sperm were not significantly different except in two cases. No significant correlation could be found between body temperature on day of stress, and fertility of sperm collected on day of stress, or two days after stress. ACKNOWLEDGMENTS The authors wish to thank Dr. J. L. Carmon, Experiment Station statistician, for his aid in the analysis of these data. Trial 3 was supported in part by the American Poultry and Hatchery Federation. REFERENCES Allen, C. F., and L. R. Champion, 955. Competitive fertilization in the fowl. Poultry Sci. 3: 33-3. Boone, M. A., 96. The effects of high ambient temperature on semen production in domestic fowl. Ph.D. Thesis. University of Georgia. Cooper, D. M., and J. G. Rowell, 958. Relations be- Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 5, 6

676 M. A. BOONE AND T. M. HUSTON tween fertility, embryonic survival and some semen characteristics in the chicken. Poultry Sci. 37: 699-77. Funk, E. M. 935., Factors influencing hatchability in the domestic fowl. Missouri Agr. Exper. Sta. Bulletin 3. Harvey, W., 96. A least squares method of analysis of variance. U. S. Department of Agriculture. ARS -8. Heywang, B. W., 9. Fertility and hatchability when the environmental temperature of chickens is high. Poultry Sci. 3: 33-339. Huston, T. M., and R. S. Wheeler, 99. Effect of synthetic thyroprotein on seasonal variation in ' volume and concentration of cock semen. Poultry Sci. 8: 6-69. Huston, T. M., and J. L. Carmon, 958. Influence of high environmental temperature on fertility and hatchability of eggs of domestic fowl. Physiol. Zool. 3:3-35. McCartney, M. G., 956. Relation between semen quality and fertilizing ability of White Holand turkeys. Poultry Sci. 35: 37-. Ota, H., E. H. McNally and W.., Wilson, 955. STUDIES on the protein requirements of young Bobwhite quail by Norris (935), Nestler et al. (9), Stadelman et al. (95) and Baldini et al. (95), and of young Ringnecked pheasants by Callenbach and Hiller (933), Norris et al. (936) and Scott et al. (95), showed that the usual corn-soybean oil meal practical Respiration calorimeter studies on White Leghorn hens in individual cages. Poultry Sci. 3:. Parker, J. E., and B. J. McSpadden, 93. Seasonal variation in semen production in domestic fowl. Poultry Sci. :-7. Parker, J. E., F. R. McKenzie and H. L. Kempster, 9. Fertility in the male domestic fowl. Missouri Agr. Exper. Sta. Res. Bull. 37: -5. Shaffner, C. S., and F. N. Andrews, 98. The influence of thiouracil on semen quality in the fowl. Poultry Sci. 7: 9-. Upp, C. W., and R. B. Thompson, 97. Influence of time of hatch on hatchability of the egg, rate of growth of the chicks, and characteristics of the adult female. Oklahoma Agr. Exper. Sta. Res. Bull. 67. Wheeler, R. S., 98. One man technique for collecting cock semen. Poultry Sci. 7: 53-5. Wilcox, F. H., 958. The effect of dilution and concentration of chicken semen on fertility. Poultry Sci. 37: 356-36. Wilson, W. O., 99. High environmental temperatures as affecting the reaction of laying hens to iodized casein. Poultry Sci. 8: 58-59. Studies on the Protein and Methionine Requirements of Young Bobwhite Quail and Young Ringnecked Pheasants M. L. SCOTT Department of Poultry Husbandry, Cornell University, Ithaca, New York E. R. HOLM New York Slate Conservation Department, Albany, New York AND R. E. REYNOLDS Ithaca Game Farm, Ithaca, New York (Received for publication November 7, 96) diets must contain approximately 8 percent protein to support normal growth. Baldini et al. (953) found that the protein requirement of young Bobwhite quail could be reduced to -% when a practical corn-soybean oil meal diet was adequately supplemented with methionine and lysine. Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 5, 6