Cornell University, Ithaca, New York

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1 T TESTICLAR GROWTH AND RELATED SPERM OTPT IN DAIRY BLLS 1.2 J. HAI-IN, 3 R. H. FOOTE AND G. E. SEIDEL, JR. Cornell niversity, Ithaca, New York HE demand for sperm from outstanding sires has increased with the development of frozen semen and the growth of large artificial breeding organizations. Methods for predicting potential sperm production, and particularly for identifying bulls with high sperm output potential at an early age are important. Several investigators (Boyd and VanDemark, 1957; Willett and Ohms, I957; Almquist and Amann, 1961; Amann and Almquist, 1961; Amann and Almquist, 1962a, b) have reported that testis circumference or testis weight at slaughter were positively correlated with number of sperm obtained by frequent ejaculation and with gonadal reserves. Testicular and body growth of a large number of dairy bulls were measured to develop techniques which would give repeatable results and to determine which ones were most highly correlated with sperm output. This study also was designed to provide the basis for investigation of parameters useful in predicting future reproductive performance and in establishing the extent to which differences in these parameters are inherited. Materials and Methods The bulls used for this study were Holsteins owned by Eastern Artificial Insemination Cooperative, Inc. They ranged in age from young bulls 7 months old, which had not yet been sampled for progeny testing, to proven bulls, 6 to 15 yr. old, with extensive histories on semen quality and fertility. In the first study withers height, circumference of both testes, and length, width and depth of each testicle were estimated independently in succession by two technicians to determine repeatability of these measurements obtained by different persons. The measurements were repeated 4 weeks later to estimate repeatability within technicians after allowing sufficient time to minimize any bias due to knowledge of the 1 Department of AnlmaI Science. 2 Supported in part by a grant from Eastern Artificial Insemination Cooperative, Inc. The assistance of this organization in providing bulls also is gratefully acknowledged. S Present address: Tier~irztliche Hochschule, 3 Hanover, Germany. 41 previous values. All animals were restrained for measurement. Withers height was measured by a metal device built especially for this purpose. Testis circumference was obtained with a flexible cloth or plastic tape around the greatest diameter of the testes and scrotum (Foote, 1969). The measurements of testis length, width and depth, and the combined width of the two testes was taken at the point of maximum dimensions with calipers simpler than some used previously (Podany, 1964). Care was taken to exclude the epididymides in measuring the length. The measurements, which were found in this study to be most accurate and useful, were taken every 6 months to obtain information on the normal variation of testis size within age groups and the changes in testis size with age. During this study 35 bulls were slaughtered, and the relationship between estimated testis size in the live animal and actual testis weight was determined. Four experiments were conducted in which semen was collected from Holstein bulls 17 to 22, 34 to 42, 42 to 53 and 59 to 69 too. of age. There were 10 bulls in each of the groups. Bulls were ejaculated twice each Tuesday and Friday for 4.5 weeks. Data for the first collection period were discarded and sperm output was based on the next 16 ejaculates. Before each semen collection the bulls were intensively prepared sexually over a period of 5 min., utilizing a combination of two to three false mounts and active partial restraint. As soon as the first ejaculate of semen was collected from each bull the sexual preparation procedure was repeated and a second sample was collected approximately 5 min. after the first sample. In addition a group of 15 bulls 72 to 150 too. of age, which were in active service, were placed on a semen collection schedule of two ejaculates per day 2 days per week. Data the first week were discarded to allow for depletion. Some of these bulls were more sluggish than those in the younger groups, and the interval between collection of the first and

2 42 HAHN, FOOTE AND SEIDEL, JR. Measurement a (cm.) TABLE 1. SIMILARITY OF MEASREMENTS OBTAINED ON 120 BLLS Withers height Testis circumference ~ L. testis length L. testis depth R. testis length R. testis depth Total testis width L.=left, R.:right. b Technicians 1 and 2. Means Repeatability 1st series 2nd series between series Tech. 1 b Tech. 2 b Tech. 1 Tech. 2 Tech. 1 Tech " second ejaculates was increased. The time interval varied in this group depending upon the necessity of changing teasers and locations to create the novel stimuli necessary. Semen samples were brought immediately to the laboratory and identified. Semen volume was recorded to the nearest 0.1 milliliter. The percentage of progressively motile sperm was estimated microscopically to the nearest 10% after dilution with physiological saline to make individual sperm visible. Sperm cell concentration was estimated by optical density. Total and motile sperm per ejaculate were calculated from these measurements. Results and Discussion The results shown in table 1 indicate that both technicians obtained the same average values for the different measurements, and that all measurements were highly repeatable (P(.01). Thus, data obtained by different technicians would be expected to be comparable. Younger animals grew some in the 4 weeks between the two series of measurements. This explains the slightly larger means for the second series of measurements. Correlations between the different measurements for the 120 bulls are shown in table 2. Withers height was somewhat more highly TABLE 2. CORRELATIONS BETWEEN VARIOS MEASREMENTS Correlations between measurements a Measurement Age Withers height Circumference Left testis length Left testis depth Right testis length Right testis depth Total testis width... a All correlation coefficients were significant, P(.O1. correlated with the various testicular measurements than was age. As would be expected the various measurements of testis size were highly correlated with each other. More difficulty was encountered in measuring the length and depth of the right testes, and this fact probably explains the slightly lower correlations which were obtained between this and several other variables. All correlation coefficients were significant (P<.01). Circumference of the testes was highly correlated with other testicular measurements (table 2), it was highly repeatable (table 1) and it was a simple measurement to obtain. The correlations among this trait and other testicular measurements on a within-age group basis are not given, but they remained high. However, the correlations of testicular measurements with withers height for the different age groups generally were positive but low (0 to 0.39), indicating that the larger faster gaining bulls did not necessari 'y have larger testes than smaller bulls of a cdmparable age. An exception to this was the selected group of AB-proven bulls over 6 yr. old, in which the correlation between withers height and circumference of the testes was 0.68 (P<.01). The relationship between scrotal circumference (X) and testicular weight (~) was computed for 35 mature bulls slaughtered during the study. Results are shown graphically in figure 1. The relationship is linear and high. The regression equation shown in figure 1 is similar to one calculated on 16 slaughterhouse bulls by Willett and Ohms (1957), in which Yz X, Both equations may require modifications for young bulls, since young bulls capable of producing sperm (Bratton et al., 1959; Macmillan and Hafs, 1968) may have a scrotal circumference of not more than 20 centimeters.

3 TESTICLAR GROWTH IN BLLS A :E L Z uj P~ :[ Q~.-I o ul J r ,f -W I 400 I I I I i I I f I! O WEIGHT OF TESTES (GM.) Figure 1. Relationship between the circumference of the testes plus scrotum in the live bull and the combined weight of left and right testes after slaughter. The equations given would estimate testicular size to be zero in such young bulls. The correlation of 0.92 between scrotal circumference and testis weight is similar to the values of 0.92 to 0.95 reported by several workers (VanDemark, 1956; Boyd and Van- Demark, 1957; Willett and Ohms, 1957; Seidel, 1968) for various comparisons between testicular dimensions and weight. Since circumference of the testes was easy to measure, the measurement had high repeatability and it was highly correlated with testis weight and volume, this measurement subsequently was taken on all bulls examined. The scrotal circumference of 184 bulls was measured in May, 1967, and again in June When these were divided into the following age groups: 7 to 12, 13 to 18, 19 to 24, 25 to 30, 31 to 36, 37 to 48, 49 to 60, 61 to 72, 73 to 96 and 97 to 148 too., the correlations between the 2 yr. were 0.53, 0.76, 0.44, 0.82, 0.81, 0.67, 0.74, 0.90, 0.77, 0.77 and 0.78, respectively. Over all age groups the correlation coefficient was 0.78 (P~.01). These generally represent highly significant correlations, and indicate that the size of testis measured 1 yr. is an indication of size measured approximately 1 yr. later. The lower correlations tended to be in the younger bull groups; these groups had the greatest increase in testis size. Scrotal circumference (testes plus scrotum) in the different age groups is shown in table 3, and the curvilinear relationship to age is displayed in figure 2. The multiple regression equation ~= ~ log X (log X) 2 was calculated, where ~=scrotal circumference in cm. and X=age in months. This equation appeared to fit the data better than one employing only the log transformation, particularly during the first 2 to 3 yr. when growth was rapid. The correlation between age and scrotal circumference transformed by the use of log and (log) 2 was 0.87; whereas, with only the log transformation, it was The linear correlations between age and testis size were 0.85 before 6 yr. of age and 0.12 after that age. The variation in scrotal circumference of Holstein bulls is shown in figure 2, and bulls

4 44 HAHN, FOOTE AND SEIDEL, JR. TABLE 3. SCROTAL CIRCMFERENCE MEASREMENTS ON BLLS AT DIFFERENT AGES Scrotal circumference No. of Mean Standard Age in too. bulls (cm.) deviation below the 80% confidence limits should be viewed with some suspicion that they may be subnormal in development9 These data further indicate that maximum scrotal circumference is reached at 5 to 6 yr. of age and remains relatively stable after that time9 Iljinskaja (1966) reported that testes decrease in size after bulls are 8 to 9 yr. old. Although this is the situation also described by the curvilinear regression shown in figure 2, no valid assessment is possible with present measurements because they represent different bulls9 Table 4 shows the results of testicular measurements and semen characteristics of 55 bulls in five age groups9 Testis weight was calculated by the regression formula given in figure 1. Total sperm output and sperm output per gram of testes are given per ejaculate, per week and per day. The values for sperm output are uncorrected for sperm loss in the semen collection equipment, since they were not measured for each age group9 Most values in the literature are not corrected for this. However, based on the study by Foote and Heath (1963) they may underestimate true output by about 12~c. Sperm output per bull per unit of time was much lower in the " 9 ' t ~ ~,,.-:. 38 ~ :,,.,.r : I ) ~.,".".':'~ -'" "~:: 9 " " "-8o~ "'/".~T/. C " " " " " " ' -95%"..7.,~ : "/1'_ 9..:..... :. 9. ;;,:... '-`~. ~. ~ 32 ~ ao 11:!-7'I ' " i AGE (MO.) Figure 2. Relationship between scrotal circumference and age in bulls. The 80 and 95% confidence limits for the curvilinear regression are shown.

5 TESTICLAR GROWTH IN BLLS 45 TABLE 4. AGE, TESTIS SIZE AND SPERM OTPT '~ Age in too. Variable Over-all Scrotal circumference, cm Estimated testes weight, gin Semen volume, ml Sperm concentration, 10~/ml Total sperm output, 106 per ejaculate per wk per day Sperm output/gin, of testis, 106 per ejaculate per wk per day '~ I0 bulls per group, excepting there were 15 bulls in the oldest group bulls 17 to 22 months of age than in the more mature bulls. In the oldest group of bulls sperm output also was much lower. In the latter group this may be due to a true decline with age, to the greater difficulty in providing proper stimuli for some of the old bulls in poor physical condition, and to differences among bulls which are confounded with age. Sperm output per gram of testis was similar in all groups excepting the oldest one. Kugimoto (1941), Abdel-Raouf (1960) and Macmillan and Hafs (1968) reported that dairy bulls can attain their mature rate of spermatogenesis at about 1 yr. of age. After this age the growth of the testes is primarily responsible for further increases in potential sperm output. Willett and Ohms (1957) suggested that with the approach of senescence the testes diminish in their capacity to produce spermatozoa. In the present study the partial correlation coefficient calculated between age of the bull and total sperm output, holding circumference of the testes constant, was (P<.01). This relationship also indicates a declining spermatogenic capability per unit of testis with age. The figures obtained for total sperm output and sperm output per gin. of testis are m accordance with results of other experiments in which depletion or exhaustion tests were used to achieve maximal sperm output (Boyd and VanDemark, 1957; Willett and Ohms, 1957; Hafs, Hoyt and Bratton, 1959; Almquist and Amann, 1961; Amann and Almquist, 1962a). Amann and Almquist (1962a) compared the potential daily sperm production from histological data and gonadal reserves with daily sperm output when bulls were ejaculated 6X per week. Daily sperm output for young bulls (average age 29 months) was 4.58 X 109 and for mature bulls was 5.26 X 109. This was 36 to 44~o of the estimated sperm production in these bulls. Daily sperm output (table 4) range from 4.02 X 109 to 6.10 X 109, suggesting that the procedure of collecting semen 4X per week with intensive sexual preparation in the present experiments was efficient in removing sperm from the bulls. The bulls appeared to be stabilized in sperm output during the experiment. In the four younger groups, where all bulls were in good physical condition, the average sperm output per bull, in millions, for weeks 1, 2, 3 and 4 was 37,405, 36,894, 37,601 and 38,761. Volume of semen (table 4) tended to increase with age of the bulls (r~0.52, P<.01). Other correlations among semen characteristics generally were low and are not presented. Correlations between testis size and sperm output per week were calculated within each age group. The relationship was high in younger animals, and it decreased with age, as shown by the following correlation coefficients for the groups in order of increasing age: r=0.81, 0.72, 0.64, 0.40 and Willett and Ohms (1957) also reported a similar trend and found a correlation of between sperm output and scrotal circumference in aged bulls. These results indicate that the prediction of sperm output from the measurement of scrotal circumference should be valuable in young bulls, but holds little promise in older bulls. Additional information on this point was obtained by collecting semen from the youngest group of bulls again 1 yr. later. One bull had been sold in the meantime, and a second bull showed pathological changes in one testis. The cor-

6 46 HAHN, FOOTE AND SEIDEL, JR. relation of scrotal circumference with sperm output per week for the remaining eight bulls was 0.69 (P ~.05). Scrotal circumference in consecutive years was highly correlated (r~0.91, P~.01). These preliminary results suggest that bulls with a potential for subnormal testicular development may be detected at an early age. Elimination of such bulls might reduce the incidence of bulls with hereditary factors associated with abnormal spermatogenesis and low fertility (Eriksson, 1952 ; Lagerl/Sf and Settergren, 1953 ; Aehnelt and Hahn, 1965). The analysis of variance of bull differences within age groups for ejaculate volume, sperm concentration and total sperm output showed that the bull effect was significant in all instances (P(.01). The large variation observed among bulls within groups is due, in part, to some variation in age within groups. However, it emphasizes the fact that estimates of changes in testicular development and semen quality can be refined considerably by evaluating the same bulls at different ages to avoid the confounding between bulls and ages. The present study will be continued for several more years to obtain these data and to estimate differences among progeny groups. Summary Scrotal circumference in living Holstein bulls was found to be a better measure of testis size than various linear measurements. This variable was easy to measure, was highly repeatable (r~-0.98) when measured by different investigators and was highly correlated with testis weight (r~0.92). The regression equation for estimating testis weight (~) from scrotal circumference (X) in mature bulls was ~ X. Scrotal circumference (~r) and age of bulls in too. (X) showed a curvilinear relationship described by the multiple regression equation ~ log X (log X) 2. Semen was collected twice each Tuesday and Friday for 4 weeks from groups of Holstein bulls (10 bulls per group) which were 17 to 22, 34 to 42, 42 to 53 and 59 to 69 mo. of age. In addition 15 proven bulls over 72 months of age, varying greatly in physical condition, were placed on the same schedule after an initial period of depletion. Average sperm output per week in order of increasing age group was 28.4, 41.5, 38.0, 42.7 and 28.2 X 109. These values are not corrected for any loss in the collection equipment. Corresponding values for sperm output per week per gram of testis were 61.2, 58.5, 53.6, 52.8 and 34.4 X 106. These data strongly suggest that there is a reduction in spermatogenesis per unit of testis in old bulls, and indicate the desirability of capitalizing upon the large spermatogenic potential of sires 2 to 6 yr. old. Correlations between scrotal circumference and sperm output per week were 0.81, 0.72, 0.64, 0.40 and --.22, respectively. The youngest group of bulls was ejaculated a year later and the correlation between scrotal circumference and sperm output per week was The correlation between scrotal circumference measurements in consecutive years was These results indicate that scrotal circumference is a useful indicator of potential current sperm output and subsequent testicular development in young bulls. However, it appears to be of little value in bulls more than 5 to 6 years of age, where other changes in the testes apparently mask the relationship of testis size to total spermatogenic potential. Literature Cited Abdel-Raouf, M The postnatal development of the reproductive organs in bulls with special reference to puberty. Acta Endocr. 34: (Suppl. 49) 1. Aehnelt, E. and J. Hahn Fruchtbarkeitsbeurteilung bei Bullen und Zfichtung auf Fruchtbarkeit. Ziichtungskde. 37: 71. Almquist, J. O. and R. P. Amann Reproductive capacity of dairy bulls. II. Gonadal and extragonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia. J. Dairy Sci. 44:1668. Amann, R. P. and J. O. Almquist Reproductive capacity of dairy bulls. V. Detection of testicular deficiencies and requirements for experimentally evaluating testis function from semen characteristics. J. Dairy Sci. 44:2283. Amann, R. P. and J. O. Almquist. 1962a. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production. J. Dairy Sci. 45:774. Amann, R. P. and J. O. Almquist. 1962b. Reproductive capacity of dairy bulls. VI. Effect of unilateral vasectomy and ejaculation frequency on sperm reserves; aspects of epididymal physiology. J. Reprod. Fertil. 3:260. Boyd, L. J. and N. L. VanDemark Spermatogenic capacity of the male bovine. I. A measurement technique. J. Dairy Sci. 40:689. Bratton, R. W., S. D. Musgrave, H. O. Dunn and R. H. Foote Causes and prevention of reproductive failures in dairy cattle. II. Influence of underfeeding and overfeeding from birth to 80 weeks of age on growth, sexual development, and semen production of Holstein bulls. Cornell niv. Agr. Exp. Sta. Bul. 940.

7 TESTICLAR GROWTH IN BLLS 47 Eriksson, K Hereditary disturbances of reproduction in cattle. 14th Int. Vet. Congr., London, 3: 216. Foote, R. H Research Techniques to Study Reproductive Physiology in the Male. In Techniques and Procedures in Animal Production Research. American Society of Animal Science. Albany, N.Y. Foote, R. H. and A. Heath Effect of sperm losses in semen collection equipment on estimated sperm output by dairy bulls. J. Dairy Sci. 46:242. Hafs, H. D., R. S. Hoyt and R. W. Bratton Libido, sperm characteristics, sperm output, and fertility of mature dairy bulls ejaculated daily or weekly for thirty-two weeks. J. Dairy Sci. 42:626. Iljinskaja, T. P Changes in the testes of bulls of different ages. Veterinariya 43:67 (In Russian. An. Breed. Abstr. 35:429, 1967). Kugimoto, S Studies on the age of sexual maturity of the bull, with particular reference to the development of the testes and spermatozoa. Imp. Zootech. Exp. Sta. Res. Bul. 46. (In Japanese). LagerlBf, N. and I. Settergrem Results of seventeen years' control of hereditary ovarian hypoplasia in cattle of the Swedish Highland breed. Cornell Vet. 43 : 52. Macmillan, K. L. and H. D. Hafs Gonadal and extra-gonadal sperm numbers during reproductive development of Holstein bulls. J. Animal Sci. 27: 697. Podany, J Testikulare biometrie an bullen. VIth Int. Congr. on An. Reprod., Trento. III:403. Seidel, G. E., Jr Effect of collection interval and artificial vagina length and temperature on the bovine ejaculate, compartmental analysis using biochemical markers, and motion picture analysis of ejaculation. M.S. Thesis, Cornell niversity. VanDemark, N. L Quantitative aspects of semen production in bulls. IInd Int. Congr. on An. Reprod., Cambridge, Plenary Papers:80. Willett, E. L. and J. I. Ohms Measurement of tesficular size and its relation to production of spermatozoa by bulls. J. Dairy Sei. 40:1559.