LEVELS OF SOME NORMAL CONSTITUENTS OF BULL SEMEN DURING REPETITIVE EJACULATION

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1 LEVELS OF SOME NORMAL CONSTITUENTS OF BULL SEMEN DURING REPETITIVE EJACULATION K. T. KIRTON, H. D. HAFS and A. G. HUNTER Department of Dairy, Michigan State University, East Lansing, Michigan, U.S.A. (Received \5th November 1963, revised \2th March 1964) at Summary. Four consecutive were ejaculates collected within 1 hr, weekly intervals for 4 weeks, from each of five mature Holstein bulls to determine the influence of repetitive ejaculation on the levels of some seminal constituents. The ph of the fresh semen and the concentrations of fructose and citric acid in the seminal plasma increased (P<\m=.\01) while the concentration and total amount offree amino nitrogen declined from first to fourth ejaculates (P<0\m=.\01).Repetitive ejaculation did not significantly influence the concentrations of protein and zinc in the seminal plasma (P \m=simeq\ 0\m=.\20) or the total citric acid and fructose per ejaculate (P \m=simeq\ 0\m=.\20).Moving boundary electrophoresis of the proteins and agar-gel-diffusion studies of the antigens in seminal plasma revealed that each protein component observed in first ejaculates was also found in fourth ejaculates. However, the proportion of the protein components in fourth ejaculates differed from that in first ejaculates. The results suggested that the total contribution of the seminal vesicles to the seminal plasma was relatively constant, but their relative contribution increased, from the first to the fourth ejaculate, at least partly at the expense of the contribution of the epididymides. INTRODUCTION Although the sperm output of dairy bulls has been investigated extensively in relation to ejaculation frequency (reviewed by Salisbury & VanDemark, 1961)^ comparatively little information is available regarding resulting alterations in the seminal plasma. Cragle, Salisbury & Muntz (1958) found that the potassium and calcium content of seminal plasma decreased from the first to the tenth consecutive ejaculate, while values for sodium and chloride increased. N. L. VanDemark & L. J. Boyd (unpublished data, 1956) measured some constituents in twenty consecutive ejaculates and found a progressive decrease in fructose, potassium and calcium from the first to the twentieth. Hopwood, Masken & Gassner (1961a) analysed sixteen consecutive ejaculates collected from each of three beef bulls. Seminal fructose, total free amino acids and nitrogen declined * Present address : Department of Dairy Husbandry, University of Minnesota, St. Paul, Minnesota, U.S.A. 157

2 158 K. T. Kirton, H. D. Hafs and A. G. Hunter steadily, while the ph of successive ejaculates increased. This decrease in free amino acid content was primarily due to a lowered alanine and glutamic acid content. De Groot (1961) also reported a decrease in seminal fructose and an increase in seminal ph in successive ejaculates. Many other investigators (reviewed by Salisbury & VanDemark, 1961) have reported an increase in ph of semen from succeeding ejaculates. However, in contrast with the results of Hopwood et al. (1961a) and De Groot (1961), Mann (1948) observed that the seminal fructose content (one bull) increased slightly from the first to the sixth ejaculate and then decreased slightly in two further ejaculates, and thus did not decline in parallel with the spermatozoan concentration. Larson & Salisbury (1954) and Larson, Gray & Salisbury (1954) showed that although bull seminal plasma proteins were electrophoretically similar to those found in blood serum, ultracentrifugai and immunochemical analyses revealed that at least some of the seminal proteins were unlike blood or milk serum proteins The research described below was initiated to determine the influence of repetitive ejaculation on some seminal constituents, particularly the proteins. MATERIALS AND METHODS Five Holstein bulls, from 2 to 5 years of age, that had been in routine use at Michigan Artificial Breeders Co-operative, were available for this experiment. At weekly intervals for 4 weeks, four consecutive ejaculates were collected within 1 hr from each bull. Ten to 15 min of intensive sexual preparation, including two or three false mounts, were imposed before each ejaculation. Consequently, 40 to 60 min elapsed from beginning sexual preparation for the first ejaculate to collection of the fourth. Immediately after each ejaculation, the volume of semen was recorded and a small sample was removed to determine spermatozoan concentration and ph. Within 20 min, the remaining portion of the semen sample was centrifuged at 5 C. The supernatant seminal plasma was sampled to determine protein (Gornall, Bardawill & David, 1949) and free amino nitrogen (Harding & MacLean, 1916). The remainder of the seminal plasma was stored at 20 C for subsequent duplicate determinations of citric acid (Saffran & Denstedt, 1948) and fructose (Roe, 1934, as modified by Mann, 1948). Zinc was deter mined in pooled first and fourth ejaculates from each bull (Johnson, Linden, Brammell & Benne, 1959). The frozen samples were also used for electrophoretic and immunochemical analyses. Seminal plasma samples from the first and fourth ejaculates of the first and fourth weeks were analysed by electrophoresis. These samples were adjusted to 1-75 to 2 0 % protein and a total volume of 7-5 ml by the addition of sodium barbital buffer (ph 8-6, µ 0-1). Each sample = was then dialysed twice for 12 hr against buffer at 5 C, and electrophoresis was carried out at 1 to 2 C in a Tiselius apparatus (American Instrument Co, Ine, Silver Spring, Maryland, U.S.A.). The mobilities and concentrations of electrophoretic components were calculated according to the procedures of Alberty (1948) and of Tiselius & Kabat (1939), respectively.

3 Bull semen after repeated ejaculation 159 Immunochemical analyses were made on pooled seminal plasma from the first and fourth ejaculates from each bull. Each pooled sample was adjusted to 2-0 % protein with 0-85 % saline and then homogenized with an equal quantity of Freund's complete adjuvant. One ml of each sample was injected subcutaneously into each of four Dutch Belted rabbits. One week later, each rabbit was similarly injected with the same sample homogenized with Freund's incomplete adjuvant. The rabbits were bled by cardiac puncture about 3 weeks after the second injection. The serum samples were stored at 20 C, merthiolate (0-001 %) being added to retard possible bacterial growth. Subsequently, the antisera were thawed and the numbers of precipitating antibodies were determined by agar-gel-diffusion (Ouchterlony, 1958). Progress of precipitin line formation was recorded photographically. Initially, one titre plate was prepared for the antiserum from each rabbit to determine the minimal number of precipitating antibodies and to determine the optimal antigen concentration for subsequent diffusions. The centre well was filled with antiserum, and the six peripheral wells with serial dilutions of antigen. Subsequently, plates containing four wells in the -pattern (Fox, 1959) were prepared with pooled antisera to Ejaculate 1 in one well, pooled antisera to Ejaculate 4 in another well, seminal plasma from Ejaculate 1 in a third well, and seminal plasma from Ejaculate 4 in the fourth well. Duplicate plates of this variety were prepared for each bull. The Bjorklund (1952) modification of Ouchterlony's double diffusion method was used to determine whether any precipitating antigens were peculiar either to first or to fourth ejaculates. Antiserum to Ejaculate 1 absorbed with was an equal volume of seminal plasma from Ejaculate 4 at 38 C for 30 min. The absorbed antiserum was then allowed to diffuse against seminal plasma from Ejaculate 1 and Ejaculate 4. The significance of differences among the four consecutive ejaculates tested by analysis of variance for each was criterion of response. RESULTS Average values of the volume of semen, concentration of spermatozoa, spermatozoa per ejaculate and seminal plasma ph, free amino nitrogen, fruc tose, citric acid, protein and zinc in the four ejaculates are listed in Table 1. As expected, seminal volume, and the concentration and number of spermato zoa per ejaculate, decreased from the first to the fourth ejaculate ( <0 01). However, the decreases were not as great as would have been expected had not intensive sexual preparation been imposed before each ejaculation. Weekly total output of spermatozoa was quite uniform for each bull, indicating that such a collection schedule could be used to estimate their spermatozoa-produc ing capacity. The average ph of the fresh semen increased from 6-26 for first ejaculates to 6-92 for fourth ejaculates (P<0-01). The average concentration and total amounts of free amino nitrogen in the seminal plasma declined progressively (P<0-01). Seminal fructose and citric acid concentrations increased ( <0 01),

4 160 K. T. Kirton, H. D. Hafs and A. G. Hunter while the total amounts of fructose and citric acid did not decline significantly ~ ( 0-20). Repetitive ejaculation did not significantly influence the average concentration of protein in the seminal plasma of the four consecutive ejaculates ~ ( 0-20). The average concentration of zinc in the seminal plasma of first and fourth ejaculates did not differ ~ significantly ( 0-50). Table 1 summary of averages and standard errors for each seminal component measured in first, second, third and fourth ejaculates Seminal component Semen (ml/ejaculate) Spermatoza/ml ofsemen ( IO9) Spermatozoa/ejaculate ( X IO9) ph Amino nitrogen (mg/100 ml) Amino nitrogen (mg/ejaculate) Fructose (mg/100 ml) Fructose (mg/ejaculate) Citric acid (mg/100 ml) Citric acid (mg/ejaculate) Protein (g/100 ml) Protein (mg/ejaculate) Zinc ^g/100 ml) Ejaculate First Second Third Fourth 8-51 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±74 Table 2 summary of correlation coefficients for the concentration seminal constituents and volume of semen of some Seminal vol. Spermatozoa/ml Citric acid Fructose Protein Free amino nitrogen Spermatozoa/ ml Citric acid 0-29* * Fructose ** 0-36** Protein 0-32** ** -0-27* Free amino nitrogen 0-41** * 0-16 ph 0-32** -0-81** * *i>/<0-05; **P/<0-01. The average values for each ejaculate, summarized in Table 1, appear to reveal relationships between certain components. For example, the concentra tions of fructose and citric acid appear to have a positive relationship, whereas the concentration of spermatozoa and ph appear to have a negative relation ship. Since such relationships could have value in determining the origin of certain constituents, some correlation coefficients were determined by pairing individual results for each comparison (e.g. seminal volume and spermatozoan concentration) for each ejaculate from each bull in each week. Thus, each correlation was determined from eighty pairs of values. The correlations listed in Table 2 reveal several significant relationships.

5 Bull semen after repeated ejaculation 161 For example, the significant negative correlations between spermatozoan concentration and citric acid ( 0-22) or fructose ( 0-40) are indicative of the fact that the origin of spermatozoa differs from the origin of much ofthe fructose and citric acid. In contrast, the significant negative correlation between spermatozoa per ml and ph ( 0-81) suggests either a common origin for spermatozoa and seminal plasma hydrogen ions or that the hydrogen ions were produced by the spermatozoa. A similar comparison of academic interest may be made for each significant value in Table 2, but most of the values are low for purposes of prediction. Although the correlations for the concentration values (Table 2) have more importance in terms of the welfare of ejaculated spermatozoa, the comparable correlations for the total amounts of each seminal constituent per ejaculate (Table 3) are more meaningful in terms of the origins of the constituents and Table 3 summary of correlation coefficients for the amount of some seminal constituents per ejaculate Total spermatozoa Total citric acid Total fructose Total protein Total citric acid 0-39** Total fructose 0-26* 0-61** Total protein 0-44** 041** 0-41** Total free amino nitrogen 0-53** 0-64** 0-68** 0-94** * Pi< 0-05;**Pi<0-01. in terms of the relative contributions of the accessory sex glands. In contrast to the values in Table 2, the correlations shown in Table 3 are all positive. For example, the concentration of spermatozoa was inversely related to the concentration of citric acid, fructose or protein, but total spermatozoa per ejaculate was positively correlated with total citric acid, total fructose and total protein per ejaculate. Also, most of the values in Table 3 were considerably larger than their counterparts in Table 2. Typical Tiselius electrophoresis patterns for first and fourth ejaculates from each bull are presented in Plate 1, and illustrate the striking similarity in the pattern of all the ejaculates from an individual bull. Enlargement of these patterns revealed a total of eight electrophoretic components, six to eight of which were found in each pattern. The average electrophoretic mobilities of these eight components were 14, 2-2, 3-2, 4-1, 4-8, 54, 6-0 and 6-8 (cm2/volt/ sec) 10-5, respectively. Repetitive ejaculation did not significantly alter the mobility of Components 2, 4, 5, 6 or 7. However, the average mobility of 6-7 for Component 8 in fourth ejaculates was significantly less than the corre sponding average of 6-9 in first ejaculates ( <0 01). No statistical procedure was applied to Components 1 or 3, because they were not found in all samples. Electrophoretic Components 4, 5, 6 and 7 were present in quantities sufficient to allow the estimation of their relative proportions. They comprised respect ively, 22, 40, 35 and 3 % of their combined total in first ejaculates, and 29, 38,

6 162 K. T. Kirton, H. D. Hafs and A. G. Hunter 30 and 2 % in fourth ejaculates. Repetitive ejaculation did not significantly affect the proportion of Components 5 or 7 ~ ( 0-17), but the average of 29 % for Component 4 in fourth ejaculates is significantly greater than the average ~ of 22 % in first ejaculates ( 0-03), and the average of 30 % for Component 6 in fourth ejaculates is significantly lower than the average of 35 % in first ejaculates ( 0 02). Thus, although the total protein ~ content of seminal plasma was not measurably altered by repetitive ejaculation, the proportions of the individual protein constituents were significantly altered. Ouchterlony titre plates representing the first and the fourth ejaculates are shown in Plate 2. Eight or nine precipitation lines (which represented the minimum number of precipitating antibodies) were demonstrated for antisera to seminal plasma from first ejaculates. Six or seven lines were demonstrated for antisera to seminal plasma from fourth ejaculates. The number of antigenic components demonstrated by this technique was in general agreement with the number of electrophoretic components (Plate 1). Study of the antigens in the seminal plasma of first and fourth ejaculates in Ouchterlony ' -pattern' plates revealed that all precipitin lines for first ejaculate seminal plasma appeared to be continuous with those for fourth ejaculate seminal plasma. This indicates that there were no antigens in first ejaculate seminal plasma that were not also present in fourth ejaculate seminal plasma. The Bjorklund inhibition studies substantiated this conclusion. DISCUSSION If, as reported by Hopwood, Masken & Gassner (1961b) and by Hess, Ludwick, Martig & Ely (1960), the epididymides and testes are the main sources of free amino acids in bovine semen, the results of the free amino nitrogen analyses presented above indicate a declining contribution of these of the organs to the seminal plasma of successive ejaculates. The similarity decline in total spermatozoa and total free amino nitrogen per ejaculate (Table 1) seems to support this hypothesis. The relevant correlation coefficient (Table 3) is If, as reported by Mann (1948), the seminal vesicles are the primary source of fructose and of citric acid in bovine semen, the results of the analyses pre sented above indicate an increasing contribution of the seminal vesicles, relative to the other accessory sex organs, to the seminal plasma of four succes sive ejaculates. The data suggest that the total contribution of the seminal vesicles remained relatively constant while the total contributions of the other An increase in accessory glands declined from the first to the fourth ejaculate. concentration of fructose with repetitive ejaculation is in general agreement with the limited results listed by Mann (1948), but conflicts with results recorded by Hopwood et al. (1961a) and by De Groot (1961). These discrepan cies may be due to differing intervals of fructolysis before the spermatozoa were removed from the seminal plasma. They may also be due to differing methods of sexual preparation of the bulls, which could influence the relative contributions of the accessory sex organs to the seminal plasma. The magnitude and similarity of the correlation coefficients between total

7 PLATE 1 Electrophoretic patterns of seminal plasma. Figs. 1,3,5,7 and 9 were from first ejaculates, and Figs. 2, 4, 6, 8 and 10 were from fourth ejaculates. Figs. 1 and 2, 3 and 4, 5 and 6, 7 and 8, and 9 and 10 were from bulls A, B, C, D and E, respectively. The detectable electrophoretic components are enumerated on each photograph. (Facing p. 162)

8 PLATE 2 Agar-gel-double-diffusion titre plates. Final photograph and composite drawing for a first (top) and a fourth (bottom) ejaculate. The centre well in each plate was filled with antiserum and the peripheral wells with a serial dilution of seminal well at 3 o'clock to 1:63 in well at 1 o'clock. plasma from 1:1 in

9 Bull semen after repeated ejaculation 163 fructose and total citric acid, between total fructose and total free amino and total citric acid are nitrogen and between total free amino nitrogen indicative of a common origin for most of each of these three seminal con stituents. However, each of these correlation coefficients accounted for less than half of the total variance associated with them, and this fact is indicative of multiple origins for portions of at least two of these three constituents. The relative uniformity of protein concentrations from first to fourth con secutive ejaculates suggests that the protein concentration of the secretions of the various accessory glands is relatively uniform. The correlation of 0-94 (Table 3) between total protein and total free amino nitrogen indicates that most of both had a common origin. However, since the ninhydrin test measured all free amino nitrogen, including that exposed on seminal proteins, the correla had been tion of 0-94 is somewhat higher than would be expected if the protein eliminated before the nitrogen analyses were performed. Electrophoresis of blood serum proteins from the five bulls in the present experiment revealed eight components, in agreement with comparable results by Larson & Salisbury (1954) and by Larson et al. (1954). However, the number ofelectrophoretic components for seminal plasma listed by those authors ranged from six to ten, somewhat higher than the numbers obtained in the present experiment. In agreement with the data presented by Larson & Salisbury (1954), the present data indicate that three of the electrophoretic components together make up most (95 to 97 %) of the protein in the seminal plasma. The data indicate that two of the major electrophoretic components of seminal plasma exhibit mobilities (4-1 and 5-4) similar to those of the 02- and a3-globulins of blood. The third = major component (mobility 4-8) appeared to have no electrophoretic counterpart in blood serum, an observation which differs from that of Larson & Salisbury (1954). ACKNOWLEDGMENTS Thanks are due to the management and personnel of Michigan Artificial Breeders Co-operative, East Lansing, Michigan, whose facilities were used to obtain the semen samples. Claude Desjardins and Kent R. Stevens provided valuable technical assistance. This is Journal Article No from the Michigan Agricultural Experiment Station. REFERENCES Alberty, R. A. (1948) An introduction to electrophoresis. I. Methods and calculations. J1. chem. Educ. 25, 426. Bjorklund, B. (1952) Specific inhibition of precipitation as an aid in antigen analysis with gel diffusion method. Proc. Soc. exp. Biol., N.T. 79, 319. Cragle, R. G., Salisbury, G. W. & Muntz, J. H. (1958) Distribution of bulk and trace minerals in bull reproductive tract fluids and semen. J. Dairy Sci. 41, De Groot,. (1961) The relative contributions of the sex glands to the successive ejaculates of the bull. (Transi, title). Tijdschr. Diergeneesk, 86, 325. Abstract in Anim. Breed. Abstr. 29, 1403 (1961). Fox, A. S. (1959) Genetic determination of sex-specific antigens. J. nat. Cancer Inst. 23, Gornall, A. G., Bardawill, C. J. & David, M. M. (1949) Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751. Harding, J. U. & Maclean, R. M. (1916) A colorimetrie method for the estimation of amino-acid nitrogen. II. Application to the hydrolysis of proteins by pancreatic enzymes. J. biol. Chem. 24, 503.

10 164 K. T. Kirton, H. D. Hafs and A. G. Hunter Hess,.., Ludwick, T. M., Martig, R. C. & Ely, F. (1960) Influence of seminal vesiculectomy on certain physical and biochemical properties of bovine semen. J. Dairy Sci. 43, 256. Hopwood, M. L., Masken, J. F. & Gassner, F. X. (1961a) The effect of exhaustive ejaculation on quality of bovine semen. J. Anim. Sci. 20, 672. Hopwood, M. L., Masken, J. F. & Gassner, F. X. (1961b) The effect of vasectomy and vasligation on free seminal glutamic acid in the bovine. J. Anim. Sci. 20, 672. Johnson,..., Linden,. I., Brammell, W. S. & Benne, E. J. (1959) Report on zinc in plants. J'. Ass. Off. agrie. Chem. 42, 363. Larson,. L., Gray, R. S. & Salisbury, G. W. (1954) The proteins of bovine seminal plasma. II. Ultracentrifugai and immunological studies and comparison with blood and milk serum. J. biol. Chem. 211, 43. Larson, B. L. & Salisbury, G. W. (1954) The proteins of bovine seminal plasma. I. Preliminary and electrophoretic studies. J. biol. Chem. 206, 741. Mann, T. (1948) Fructose content and fructolysis in semen. Practical application in the evaluation of semen quality. J. agrie. Sci. 38, 323. Ouchterlony, O. (1958) Diffusion-in-gel methods for immunological analysis. Progr. Allergy, 5, 1. Roe, J. H. (1934) A colorimetrie method for the determination of fructose in blood and urine. J. biol. Chem. 107, 15. Saffran, M. & Denstedt, O. F. (1948) A rapid method for the determination of citric acid. J. biol. Chem. 175, 849. Salisbury, G. W. & VanDemark, N. L. (1961) Physiology of reproduction and artificial insemination of cattle. Freeman, San Francisco. Tiselius, A. & Kabat, E. A. (1939) An electrophoretic study of immune sera and purified antibody preparations. J. exp. Med. 69, 119.