The Effect of Low Ambient Temperatures on Sperm Production, Epididymal Sperm Reserves, and Semen Characteristics of Roars

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1 BIOLOGY OF REPRODUCTION 2, (197) The Effect of Ambient Temperatures on Sperm Production, Epididymal Sperm Reserves, and Semen Characteristics of Roars ERNEST E. SWIERSTRA Research Station, Canada Department of Agriculture, Brando,,, Manitoba, Canada Received April 1, 1969 The effect of low s on reproduction was determined by housing one group of 14 boars (av age 8.5 months) in a controlled research piggery (17#{176}C) and another group of 14 boars (av age 8.7 months) in outside pens. Semen was collected at 72-hr intervals for an 11-week period (January through March). The mean daily s for January, February, and March were -18, -2, and -15#{176}C. respectively. The low did not interfere with testicular development, sperm production, or semen quality. Daily sperm production (DSP), as determined by quantitative testicular histology, was 16.5 X 1 for the control boars and 18.6 X l for the low boars (p <.5). This difference was due to a significant difference in testis size (365 versus 419 g), because the DSP/g of testis was similar for the two groups. The ratio of sperm output to production was 99% for the control boars and 84% for the low bears. Although it is well recognized that high s can affect spermatogenesis and semen quality, knowledge of the effect of low s on reproduction remains limited. Harris and Harrison (1955) showed, by surrounding the exposed rat testis with a mixture of ice and salt, that a of -5#{176}Cfor 1 hr caused some destruction of the germinal epithelium. Severe damage to the germinal epithelium was observed when the was lowered to -11#{176}C.Perrault and Dugal (1962) concluded, from their studies with the rat, that cold per se produced inhibition of the endocrine function of the testes as demonstrated by a regression of the accessory sex glands. Chang (1944) applied ice to the scrotum of rabbits and found that this resulted in disintegration of spermatozoa in the tail of the epididymis. The present research was designed to determine the effect of low s on semen characteristics, sperm production, and epididymal sperm reserves of boars managed under practical conditions. MATERIALS AND METHODS Animals and Design of Experiment Thirteen Yorkshire (6.9 to 8.1 months) and 15 Lacombe boars (6.9 to 8.1 months) were selected for this experiment on the basis of semen quality. All boars produced good quality semen as determined by collecting five to eight ejaculates per boar prior to the start of the experiment. The boars were paired within breeds on the basis of the number of sperm per ejaculate and body weight. One member of each pair was randomly assigned to a control or a low (LAT) group. One pair was made up of the odd boar from each breed. All boars were kept in individual pens. The control boars were housed in a windowless piggery with a mean of 17#{176}C± only a few degrees and with light supplied from 8. to 17. hr. They had free access to water and were handfed 1.36 kg of pelleted sow ration (16% protein) twice daily. The LAT boars were housed in outside pens with single-boarded cabins as shelter. The cabins had plank floors and were bedded with straw. The LAT boars 23

2 24 SWIERSTRA were fed the pelleted sow ration ad libitum and were watered twice daily. The experiment was conducted during a 15-week period from 5 December 1966 to 2 March The mean daily s (mean of daily maximum and minimum s) for December, January, February, and March were -16, -18, -2, and -15#{176}C, respectively (Fig. 1). Corresponding average minimum s for the 4 months were -22, -24, -27, and -21#{176}C. On 23 days, the minimum was lower than -3#{176}C, and the maximum exceeded #{176}Cby a few degrees only on 7 days. The first 4 weeks were used to acclimatize the bears to their respective environments and no semen was collected. Semen was collected by means of an artificial vagina (Swierstra and Rahnefeld, 1967) and each boar was ejaculated every 72 hours. Both groups were ejaculated on the same dates and all collections were made by the same technician. The LAT boars were brought inside the research piggery for the semen collections. After collection the percentage of motile sperm was estimated from to 1 in increments of 1. The gelatinous material (gel) was removed by straining the ejaculate through four layers of cheesecloth (grade 4). Sperm concentration was determined by optical density (Young et al., 196). The bears were killed 24 hr after the last semen collection. The testes and epididymides were weighed and retained for further analyses. Two damaged epididymides were discarded. Epididymal Sperm Reserves Each epididymis was divided into the caput, corpus, and cauda, and each part was processed separately. After mincing the tissue with a pair of scissors, it was transferred quantitatively to a Waring Blendor using 15 ml of homogenizing solution. This solution consisted of physiological saline with.5% Triton X-1 added (Amann and Lambiase, 1969). The tissue was homogenized for 1 mm, and the homogenates were transferred quantitatively to glass jars. Additional homogenizing solution was added to dilute sperm concentrations to convenient levels for accurate hemacytometer counting (4-6 sperm per chamber). Sperm concentrations were determined by counting + o ADAPTATIO sperm present in ten large squares of each of eight hemacytometer chambers. Each chamber was filled with a different pipette, and the homogenates were mixed for 2 mm with a magnetic stirrer just prior to filling pipettes. Daily Sperm Production (DSP) by the Testes The DSP was determined from quantitative testicular histology by Swierstra s modification (1968) of a method proposed by Kennelly and Foote (1964). Tissue samples were taken from three specified locations (Swierstra, 1968) in the testes, fixed in Allen s fixative (Gray, 1958) for 3 days, sectioned serially (8 t), and stained by the Feulgen technique using fast green as a counterstain. The volume percent of round spermatid nuclei in the testes was determined by Chalkley s method (1943). Two sections from each location were scanned at random and 42 hits recorded per testis. Sperm production was compared with the average sperm output in the last 1 ejaculates, after sperm losses in the collection equipment and gel fraction of the ejaculate had been taken into account. Swierstra and Rahnefeld (1967) calculated that these losses equaled 3.28% of the number of spermatozoa present in the semen collection bottle. RESULTS Feed Consumption, Body Weights, and Organ Weights The control boars consumed 2.72 kg of feed per day. The average feed consumption of the LAT Yorkshire and Lacombe boars was 4.47 and 4.4 kg, respectively. Body weight gains as well as the shape of the growth curves were similar for the control and LAT boars. The testicular weight of the control boars was 365 g and that of the LAT boars 419 g. This difference between treatment means was significant when tested by covariance analysis to adjust testis weight for body weight. Neither capita nor corpora epididymides weights differed significantly between control z -2 4Ii. -3 U -4-1 NCOLLECTIONPERIOD DECEMBER JANUARY FEBRUARY MARCH FIG. 1. Mean daily during the experimental period.

3 COLD STRESS AND REPRODUCTION 25 TABLE 1 BoDY, TESTIS, AND EPIDIDYMAL WEIGHTS OF CONTROL AND AMBIENT TEMPERATURE BOARSa Treatment Characteristic Yorkshire Breed Lacombe Control Age at start of semen collection period, 8.6 ± ± ± ±.1 months Body weight at start of semen collection 131.Ob ± #{176}± ± ± 2.6 period, kg Body weight at end of experiment, kg 17.9 ± ± ± ± 3.8 Testis weight, g ± ± b ± #{176}± 11.9 Epididymal weight, g Caput 22.5 ± ± ± ±.9 Corpus 1.8 ± ± ± ±.6 Cauda 3.9 ± ± b ± #{176}± 1.2 Total 64.2 ± ± Ob ± #{176} ± 2. a Values in the table are means and standard errors. b,c Means on the same line under the same major nificantly (p <.5). and LAT boars (Table 1). However, the caudae epididymides of the LAT boars were heavier (p <.1) than those of the control boars. Total epididymal weights of the LAT boars also were heavier (p <.5) than total epididymal weights of the control boars (Table 1). Semen Characteristics The two breeds did not differ in the semen characteristics measured (Table 2). The LAT boars produced larger (.1> p>.5) total and strained semen volumes than the control boars (Fig. 2A). However, sperm concentrations were less (p <.5), so that total and motile sperm/ejaculate did not differ between gups(fig.2 B and C). There were 5ignificant differences among ejaculates (collection dates) and among boars with respect to total volume, strained volume, sperm concentrations, total sperm/ejaculate, and motile sperm/ejaculate. E/ididymal Sperm Reserves Sperm reserves in the capita and in the corpora were not affected by low s. However, sperm reserves in the caudae, and in total, were greater (p <.1) for the LAT heading with different superscript letters differ sig- boars (Table 3). The epididymal sperm reserves (ESR) were significantly correlated with epididymal weights. For the control boars the correlation coefficient was +.76 (p <.1) and for the LAT boars +.72 (p < TABLE 2 EFFECT OF AMBIENT TEMPERATURES ON Characteristic SEMEN CHARAcTERISTICS Control Treatmenta Number of boars Number of ejaculates Total volume, ml 229 ± ± 15 Strained volume, ml 182 ± ± 12 Gel volume, ml 47 ± 3 5 ± 3 Sperm ml, 16 concentration/ 279 ± 15#{176} 231 ± 12 Percent motile sperm 77 ± 1 75 ± 1 Total Motile ejaculate, sperm! ejaculate 2, sperm! io 1O a Mean ± standard error ± ± ± ± 1.6 c,,l Means on the same line with different superscript letters differ significantly (p <.5). Sperm in the collection bottle, not corrected for sperm losses in the collection equipment and gel.

4 26 SWIERSTRA U -J > Ii I-. -j lii z I- I- z lii U z a. U I- -j U U a. UI a j JANUARY - FEBRUARY MARCH EJACULATES FIG. 2. Changes in semen charactristics of control and low bears ejaculated at 72-hr intervals for 11 weeks. A, ejaculate volumes; B, sperm concentrations; C, total sperm per ejaculate. TABLE 3 TIlE EFFECT OF AMBIENT TEMPERATURES ON Part epididymis EPIDIDYMAL SPERM RESERVESa of Control Treatment Caput, ± ± 1.6 Corpus, ± ± 1.7 Cauda, #{176}± ± 2.7 Total per side, 1 8.6#{176} ± d ± 3.9 Total per boar, #{176}± s ± 1.1 a Values in the table represent sperm reserves 24 hr after last semen collection (means and standard errors). Two control Lacombe boars with damaged epididymis excluded. e,d Means on the same line with different superscript letters differ significantly ( <.5)..1). Regression analyses revealed that for each gram increase in epididymal weight there was an increase in ESR of 1.43 X 1#{176} for control and 1.41 X 1#{176} for the LAT boars. Sperm Production and Sperm Output Neither breeds nor treatments differed significantly with respect to the percentage of round spermatid nuclei or the DSP/g of testis (Table 4). However, the total DSP of the control boars was less (p.(.5) than that of the LAT boars (16.5 X 1 versus 18.6 X 1#{176}). Based on the last 1 ejaculates from each boar, sperm output per 72 hr was 48.8 X 1#{176} for the control boars and 46.9 X 1 for the LAT boars. Sperm output equaled 99% of the DSP for the control boars and 84% of the DSP for the LAT boars. Sperm output was the

5 COLD STRESS AND REPRODUCTION 27 TABLE 4 SPERM PRODUCTION AND OUTPUT OF CONTROL AND AMBIENT TEMPERATURE BOARS Treatment Characteristic Yorkshire Breed Lacombe S Control. S Round spermatid nuclei, % DSPB, DSP/g of testis, DSOt, DSO/DSP x a Daily sperm producti on as de termined from quantitative testi cular histology. Average number of sperm in the last 1 ejaculates divided by the semen c;llecti:n interval in days. significantly correlated (r.59, p <.1) with sperm production. DISCUSSION Because Hauser et al. (1952) found that testicular development was more closely associated with body weight than with age, it was decided to use two feeding regimes to maintain a similar rate of gain for the control and LAT boars. Testes and epididymides of the LAT boars were heavier than those of the control boars. Epididymal weights were to some extent influenced by testis weights. Covariance analysis revealed that treatment differences with respect to epididymal weight were non-significant when these weights were adjusted for testis weight. It is postulated that differences in testis size between the LAT and control boars represented either a response to the cold or to the extra energy consumed. However, chance in random sampling cannot be excluded, because no testis measurements were taken prior to assigning the boars to the respective treatments. Larger (p <.1) semen volumes of the LAT boars were associated with lower (p <.5) sperm concentrations, so that sperm output was similar for both groups. These larger volumes represented either a direct response to cold or an association with the higher degree of libido exhibited by the LAT boars. No definite conclusion can be reached with respect to libido, since environmental effects were confounded with differences in boar handling prior to semen collection. The negative association observed between semen volume and sperm concentration agrees with results reported by Ito et al. (1948) and Swierstra and Rahnefeld (1967). However, McKenzie et al. (1938) and Glover (1955) found no relationship between semen volume and sperm concentration. The high sperm concentrations and the large number of sperm in the first few ejaculates (Fig. 2, B and C) resulted from an accumulation of sperm in the epididymides during the 4 weeks of sexual rest prior to semen collections. The sperm output of the control boars was 48.8 X 1 /72 hr. In another experiment, involving 22 Yorkshire and 22 Lacombe boars of similar age and managed under identical conditions, it was found that the sperm output was 28.3 X lo /ejaculate when semen was collected at 48-hr intervals (Swierstra and Rahnefeld, 1967). This suggests that sperm output per unit time is similar for 48- and 72-hr collection intervals s had no significant effect on sperm numbers in the capita and corpora epididymides. However, sperm numbers in the caudae epididymides as well as total ESR of the LAT boars were significantly larger than those of the control boars. The average number of sperm per epididymis.4

6 28 SWIERSTRA was comparable to the 95.4 X 1 reported by Singh (1962). Differences in testis size between treatments accounted for the difference in DSP, because the DSP/g of testis was similar for the control and LAT boars (Table 4). The sperm output of the control boars was close to 1% of the DSP (Table 4). This suggests that nearly all the spermatozoa produced by the testes were ejaculated by the control boars, and that few, if any, spermatozoa were absorbed from the epididymis or passed out by masturbation and micturition. The data for the LAT boars suggest that spermatozoa were resorbed from excurrent ducts or passed out by masturbation and micturition. ACKNOWLEDGMENTS The author gratefully acknowledges the technical assistance of Messrs. H. H. S. Bell, W. D. Sutherland, and D. E. Kitz. Acknowledgment is also made to Rohm and Haas Co. of Canada, Ltd., for supplying Triton X-1. REFERENCES AMANN, R. P. AND LAMBIASE, J. T. (1969). The male rabbit. III. Determination of daily sperm production by means of testicular homogenates. J. Animal Sci. 28, CIIALKLEY, H. W. (1943). Method for quantitative morphologic analysis of tissues. NatI. Cancer Inst. 4, CHANG, M. C. (1944). Disintegration of epididymal spermatozoa by application of ice to the scrotal testis. J. Exptl. Biol. 2, GLOVER, T. D. (1955). The semen of the pig. Vet. Rec. 67, GRAY, P. (1958). Handbook of basic microtechnique. 2nd ed. p. 74. McGraw-Hill, New York. HARRIS, R. AND HARRISON, R. G. (1955). The effect of low on the guinea pig testis. In Studies on Fertility, R. G. Harrison, ed., Vol. 7, p. 23. Charles C Thomas, Springfield, Illinois. HAUSER, E. R., DICKERSON, G. E., AND MAVEN, D. T. (1952). Reproductive development and performance of inbred and crossbred boars. University of Mi.,souri. Agr. Expt. Sta. Bull. 53. ITO, S., NIWA, T., KUDO, A., AND MIzUH, A. (1948). Studies on the artificial insemination in swine. II. Observations on the semen and its storage trial. In Summaries of Research Reports on Reproductive Physiology and Artificial Insemination in Swine, Natl. Inst. Agr. Sci., Chiba-Shi, Japan. KENNELLY, J. J. AND FOOTE, R. H. (1964). Sampling boar testes to study spermatogenesis quantitatively and to predict sperm production. J. Animal Sci. 23, MCKENZIE, F. F., MILLER, 3. C., AND BAUCUESS, L. C. (1938). The reproductive organs and semen of the boar. University of Missouri. Agr. Expt. Sta. Bull PERRAULT, M. J. AND DUGAL, L. P. (1962). Effect of severe cold (-5#{176}C)on the endocrine function of the testis in the albino rat. Can. J. Biochem. Physiol. 4, SINGH, G. (1962). Reserves spermatiques #{233}pididymaires chez le verrat. Ann. Biol. Animate Biochim. Biophys. 2, 47-SO. SWIERSTRA, E. E. (1968). A comparison of spermatozoa production and spermatozoa output of Yorkshire and Lacombe boars. J. Re prod. Fertility 17, SWIERSTRA, E. E. RAHNEFELD, G. W. (1967). Semen and testis characteristics in young Yorkshire and Lacombe boars. J. Animal Sci. 26, YOUNG, D. C., FOOTE, R. H., TURKHEIMER, A. R., AD HAFS, H. D. (196). A photoelectric method for estimating the concentration of sperm in boar semen. I. Animal Sci. 19, 2-25.