ABSTRACT ALTERATION OF SOME SEMINAL PLASMA CONSTITUENTS DURING REPETITIVE EJACULATION OF DAIRY BULLS by Kenneth T. Kirton Five Holstein bulls, ranging from 2 to 5 yr of age, were ejaculated four consecutive times at weekly intervals for a period of 4 wk. Ten to fifteen min of intensive sexual prep- aration was imposed prior to each ejaculation. Seminal vol- ume, sperm concentration and pH of the fresh semen was de- termined for each ejaculate. The concentrations of fructose (Roe), citric acid (Saffran and Densted), protein (biuret), and free amino nitrogen (ninhydrin) were determined for each ejaculate. Zinc content of first and fourth ejaculate sem- inal plasma, and of blood serum for each bull was determined by the dithizone method. Proteins of first and fourth ejaculate seminal plasma of the first and fourth week for each bull were further studied by Tiselius moving boundry electrophoresis and by agar gel double diffusion immunochemistry. As anticipated seminal volume, sperm concentration, and total sperm per ejaculate decreased from ejaculate one to by Kenneth T. Kirton ejaculate four (P < .01). A marked similarity between weeks was noticed for each of the five bulls indicating that such an ejaculation procedure may be useful to estimate a bull's sperm output capacity. The average pH of seminal plasma in- creased uniformly from the first to the fourth ejaculates (P < .01). The fructose and citric acid concentration of the seminal plasma increased from the first to the fourth ejaculate (P < .01), but, the total amounts per ejaculate remained relatively constant (P E .20). Since the seminal vesicles are assumed to be the primary source of seminal fructose and citric acid, these data indicated that the seminal vesicles contributed a relatively constant volume to each of the four ejaculates. The average concentration of zinc in first and fourth ejaculate seminal plasma did not differ significantly (P:> .50). Although the average zinc concentration of sem- inal plasma was considerably higher than the average of blood serum, the seminal average did not approach the com- parable values previously reported for some other species. The average seminal free amino nitrogen decreased steadily with progressive ejaculation. Based upon the re- ported high concentration of amino acids in epididymal secretions and the decreasing sperm concentration in by Kenneth T. Kirton succeeding ejaculates, these results indicate a decreasing epididymal contribution from the first to the fourth ejac- ulates. The average concentration of protein in seminal plasma did not significantly change from first to fourth ejaculate (P E .48). Electrophoretic analysis of seminal plasma revealed a maximum of eight protein components, and six or seven of these were found in each ejaculate. No appreciable differ- ence was found between the number of components in first and fourth ejaculates. Electrophoretic mobilities of the major protein components were not altered by repetitive ejaculation. Based upon the electrophoretic data, the per- centage composition of two of the four major protein com— ponents was altered by repetitive ejaculation (P a .03). One of the four major seminal plasma protein components had no electrophoretic counterpart in blood serum. Immunochemical studies revealed a minimum of eight pre- cipitating antigens in first ejaculate seminal plasma and a minimum of six in fourth ejaculate seminal plasma. How- ever, the evidence indicated that the concentration of some minor constituents in fourth ejaculate seminal plasma was too low to be detected by this immunochemical technique. by Kenneth T. Kirton Correlation of the measured components in seminal plasma revealed that the totals per ejaculate were, without exception, more highly correlated than the comparable con- centration values. The magnitude and similarity of the cor- relations between total fructose and total citric acid (r = 0.61), between total fructose and total free amino ni— trogen (r = 0.68), and between total citric acid and total free amino nitrogen (r = 0.64) indicated a common major origin for these seminal constituents. The correlation (- 0.81) between the concentration of sperm and pH of the seminal plasma indicated that seminal hydrogen ions orig- inated primarily in the ductus deferentia and epididymides. ALTERATION OF SOME SEMINAL PLASMA CONSTITUENTS DURING REPETITIVE EJACULATION OF DAIRY BULLS BY Kenneth T. Kirton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 1963 BIOGRAPHICAL SKETCH Kenneth T. Kirton was born at Kingman, Kansas on June 16, 1934. He received his elementary education at Inde- pendence School and was graduated from Iola High School in June, 1952. In September he entered Kansas State College, and re— ceived a Bachelor of Science degree in Dairy Husbandry in June, 1957. Having obtained an R.O.T.C. commission with the United States Army, he then entered upon two years active duty with a Nike-Ajax Battery in Detroit, Michigan. After discharge from active duty in August, 1959, he operated a dairy farm at LaHarpe, Kansas until accepting a graduate assistantship at Michigan State University in September, 1961. He received the Master of Science degree in the Department of Dairy with a major in Reproductive Physiology in June, 1963. ii ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. Harold D. Hafs for his unselfish interest, inspira- tion, and guidance during the course of this experiment and preparation of the manuscript. The author gratefully acknowledges the interest and assistance of Alan Hunter with the experimental procedures. The assistance of Kent Stevens and Claude Desjardins with the collection of the semen is greatly appreciated. Acknowledgement is made to Michigan Artificial Breeders Cooperative for the bulls used in this experiment. The assistance of Dr. E. J. Benne and Miss E. I. Linden, of the Biochemistry Department, with the zinc assays was appreciated. The author wishes to express his sincere appreciation to his wife, Marlena, for her encouragement and patience during the course of the study, as well as for her assis- tance by typing the manuscript. iii TABLE OF BIOGRAPHICAL SKETCH ACKNOWLEDGEMENTS . . LIST OF TABLES . . . . LIST OF FIGURES . . . LIST OF APPENDICES . . INTRODUCTION . . . . REVIEW OF LITERATURE . Behavioral Factors Volume and Sperm Alteration of Seminal Constituents by Repetative Ejacul Characterization of Seminal Constituents EXPERIMENTAL PROCEDURES Semen Collecting and Handling Free Amino Nitrogen Citric Acid Fructose Zinc Total Protein Electrophoretic Ana Immuno-chemical Ana RESULTS AND DISCUSSION Affecting Output ation CONTENTS Seminal lysis of Proteins lysis of Proteins iv Page ii iii vi vii viii 15 l5 16 16 17 18 19 19 20 23 Page Ejaculate Volume and Sperm Numbers 23 Hydrogen Ion Concentration 26 Free Amino (Ninhydrin) Nitrogen 27 Fructose and Citric Acid 28 Zinc 30 Biuret Protein 31 Electrophoretic Analysis of Proteins 32 Immuno-Chemical Analysis of Proteins 35 Relationship Between Some Seminal Constituents 46 Some Histologic Observations on Remark 49 SUMMARY . . . . . . . . . . . . . . . . . . . . . 52 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . 56 APPENDIX . . . . . . . . . . . . . . . . . . . . . 62 10. ll. 12. l3. 14. LIST OF TABLES Milliliters of semen per ejaculate . . . . . Billions of sperm per ml of semen . . . . . Billions of sperm per ejaculate for each . ejaculate . . . . . . . . . . . . . . . . . Averages of pH values for the fresh semen . Average seminal plasma free amino nitrogen values 0 O O I O O O O O O O O O O O O C O 0 Average seminal plasma fructose and citric acid values . . . . . . . . . . . . . . . . Micrograms of zinc per milliliter of seminal plasma and blood serum . . . . . . . . . . . Average seminal plasma biuret protein values Electrophoretic mobilities of measurable seminal plasma proteins . . . . . . . . . . Average percentage composition of major protein constituents of seminal plasma . . . Number of precipitin lines visible in titer plates 0 O O O O O O O O O O O O O O O O O 0 Summary of averages of each seminal component measured in each ejaculate number Summary of correlation coefficients for the concentration of some seminal constituents and volume of semen . . . . . . . . . . . . Summary of correlation coefficients for the amount of some seminal constituents per ejaculate . . . . . . . . . . . . . . . . . vi Page 23 24 25 27 28 29 31 32 37 38 42 46 48 48 LIST OF FIGURES Electrophoretic patterns of seminal plasma . . . . . . . . . . . . . . . . . . Electrophoretic pattern of bovine blood serum 0 O O O O O O O O O O O O O O O O O O Electrophoretic pattern of seminal vesicular fluid . . . . . . . . . . . . . . Ouchterlony titer plates . . . . . . . . . Typical "H" pattern Ouchterlony plate . . . Cross reaction of bovine seminal plasma, bovine blood serum, and respective anti- sera O O O O O O O O O O O O O O O O O O O Photomicrograph of bovine seminal vesicle . Photomicrograph of bovine ampulla of ductus deferens . . . . . . . . . . . . . . . . . Templates and cutter used to prepare Ouchterlony plates . . . . . . . . . . . . vii Page 36 39 39 41 45 45 50 50 69 Appendix Procedure A. LIST OF APPENDICES Dithizone zinc determination . . Preparation Preparation Computation Composition Preparation of biuret reagent . of sodium veronal buffer of electrophoretic mobilities and use of adjuvant of Ouchterlony plates viii Page 62 65 66 66 67 67 I NTRODUCTI ON Although the importance of seminal plasma to the sperm- atozoa contained therein has been a topic for controversy, most of the evidence indicates seminal plasma or some con- stituents (s) of seminal plasma is (are) responsible for improved sperm survival. The seminal plasma unquestionably serves as a vehicle for the sperm during ejaculation. There is little doubt that efficiency of natural mating would be lower without the provision of seminal plasma to serve as an effective diluent and vehicle for the closely packed epididymal spermatozoa. Seminal plasma exerts a definite stimulatory effect on the non-motile epididymal sperm, and provides an important source of nutrition in the form of fructose (Mann, 1946). In addition, seminal plasma is capable of exerting striking biodynamic effects upon blood pressure and smooth muscles when injected intravenously (Mann, 1949). Additionally, the plasma undoubtedly has some effect upon the functional life expectancy of the sperm even though it is progressively diluted in the luminal fluids of the female genitalia at the time of mating. For example, weil (1962) has shown that ejaculated sperm are rapidly and relatively permanently coated with proteins of seminal plasma origin. Mann (1949), in reviewing the available evidence, alluded to the impor- tance of the buffering capacity of seminal plasma for the sperm. Nwmerous authors have cited evidence for the metabolic utilization of nutrients from seminal plasma by sperm. Dilution of sperm with seminal plasma did not effect fruc- tolysis rate, while dilution with an artificial isotonic solution invariably decreased the rate of fructose utiliza- tion (Mann and Mann, 1951). A further indication of the beneficial effect of seminal plasma on sperm was provided by the increased fertilizing capacity of minimal numbers of sperm in the presence of increased concentrations of seminal plasma (Chang, 1947). Desjardins and Hafs (1962) presented evidence indicating that bull sperm survive deep freezing better when frozen with high sperm cell concentrations. Kinney and VanDemark (1954) showed that sperm from second ejaculates survive freezing better than sperm from first ejaculates. Since the sperm concentration of second ejaculates is leSs than that of first ejaculates, the concentration of seminal plasma during freezing would be greater for second ejac- uLates because bull semen is usually frozen with a constant concentration of sperm. Improved quality of sperm from second and third ejaculates has been reported by many au— thors. Although this improved quality could be attrib- utable to inherent differences between the sperm popula- tions, Desjardins and Hafs's data seem to emphasize the role of the seminal plasma. Alterations of sperm production by repetitive ejacula- tion have been investigated extensively (Hale and Almquist, 1963, VanDemark, 1956, Hafs _§__l,, 1962). In contrast, seminal plasma constituents during repetitive ejaculation have not been extensively investigated. The purpose of this thesis was to determine whether repetitive ejaculation affected the proportions of some seminal plasma constituents of mature dairy bulls. In View of the lack of information on seminal plasma proteins and because of their potential importance to sperm physi- ology, they were emphasized in this thesis. REVIEW OF LITERATURE Behavioral Factors Affecting Seminal Volume and Sperm Output: While artificial insemination was still in its infancy; Lager16f (1934) observed that bulls frequently yield first ejaculates with few or no sperm. Later, Hellstrom (1947) found that restraining bulls for a few minutes before ejac- ulation resulted in larger seminal volumes. Collins gt_§l, (1951) were the first to publish quantitative information concerning pre-ejaculation sexual preparation of bulls. They concluded that restraining a bull in the vicinity of a mount animal and allowing one false mount prior to ejacula- tion increased seminal volume and numbers of motile sperm per ejaculate significantly. Branton et_§1, (1952) obtained similar increases with one false mount. Hale and Almquist (1960) summarized previous data by stating that at collection frequencies of one or two ejacu- Lates per week, one may reasonably anticipate that a single false mount prior to ejaculation will increase sperm output by approximately 50%. Two additional false mounts may in— crease output per ejaculate by another 50%. representing double the output with no preparation. Restraining a bull for about 10 min was estimated to have essentially the same effect as several false mounts (Almquist §t_a1,, 1958, and Cromback, 1958). This work was subsequently substantiated by Hafs _§_a1, (1962) who obtained almost 230% as many total sperm per ejaculate after 10 min of active restraint as was obtained from bulls with no sexual preparation. This increase in sperm output was accompanied by an increase of 153% in seminal volume. The same authors found that the increased sperm output obtained as a result of false mounting was primarily a factor of seminal volume, which was significantly increased by false mounting, whereas the concentration of sperm was not. Alteration of Seminal Constituents by Repetitive Ejaculation: t al. (1958) found a definite change in mineral Cragle content of seminal plasma of ten consecutive ejactulates taken from dairy bulls. Values for potassium and calcium de- creased from 155 and 39 mg/100 ml, respectively, in the first ejaculate to 91 and 13 mg/100 ml, respectively, in the tenth ejaculate. Values for sodium and chloride increased from 289 and 154 mg/100 ml, respectively, in the first ejaculate to 325 and 411 mg/100 ml, resPectively, in the tenth ejaculate. VanDemark and Boyd (1956) determined some chemical components in twenty consecutive ejaculates. They found a progressive decrease in fructose, potassium, and calcium from the first to twentieth ejaculates. Hopwood _£.§L- (1961a) analyzed 16 consecutive ejaculates collected from each of three beef bulls. The semen was analyzed for Nessler nitrogen, fructose, and free amino acids. Sperm count, seminal fructose, and nitrogen steadily declined while the pH rose in successive ejaculations. Total free amino acid contentprogressively decreased from the first to the twentieth ejaculate. This decrease was primarily due to a lowered glutamic acid and alanine con- tent while glycine, serine, and aspartic acid did not change appreciably. De Groot (1961) found a decrease in seminal fructose and sperm concentration with succeeding ejaculates taken from triplet bulls subjected to an exhaustion test. The pH of the semen showed a pronounced increase as ejaculate number increased. Mean sperm motility showed little change with ejaculate number, except in the case of the first ejaculate which tended to be lower. Successive ejaculates tend to increase in pH from an average value of 6.75 for first ejaculates (Sergin, 1936 and Anderson, 1942) to about 7.0 (Davis, 1938, Hopwood §t_a1,, 1961a, VanDemark and Boyd, 1956, and Davis and Williams, 1939). An investigation of the buffering capacity of bull semen disclosed a well buffered system at pH 4.0 to 5.5 and 9.0 to 10.0, while the semen was poorly buffered at physio- logical values of 6.0 to 9.0 ( Smith and Asdell, 1940). Dougherty and Ewalt (1941) found seminal pH increased for about 2 hr and then decreased steadily when fresh semen was incubated at room temperature. Mann (1949) attributed the buffering capacity of seminal plasma to the inorganic phos- phate, bicarbonate, citrate, and protein components of the seminal plasma. In view of the sensitivity of sperm to an acid medium, such as occurs due to the accumulation of lactic acid during metabolism, the buffering effect of seminal plasma is of particular value. Characterization of Seminal Constituents: Seminal plasma is a.ndxture of secretions originating in the testis, epididymis, deferent duct, seminal vesicle, prostate, bulbo-urethral gland, and urethra. The seminal vesicle is the major source of seminal fluids, but the relative contributions of the other sources is not well known. Seminal plasma differs remarkably from other body fluids in several respects. The seminal concentrations of citric acid (Schersten, 1929), fructose (Mann, 1946), and phos- phorylcholine (Lundquist, 1946) are much higher than the amounts found in other body fluids. These three substances appear to originate primarily in the seminal vesicle of most domesticated animals. Mann (1949) has speculated that seminal fructose and citric acid are secreted independently of each other, in the sense that the two components are produced by two dis- tinct types of cells. In support of this contention, he showed (Mann _t__1,, 1948) three distinct cell types in the secretory epithelium of the seminal vesicle upon histological examination. While Rothchild and Barnes (1954) found a significant (P < .01) positive correlation between seminal fructose and citric acid, Mann (1948) found no correlation between the seminal content of these two components. Ehlers ._E._1. (1953) found evidence of a cyclic relationship between fructose and citric acid, in that correlations were negative in some months and significantly positive in others. Evidence for the separate origins of sperm and seminal fructose was provided by Mann (1948) who collected eight ejaculates from a bull in a period of 63 min. As expected, sperm numbers decreased from 1,664 X 106/m1 for the first ejaculate to 98 X 106/m1 for the eighth ejaculate. The seminal fructose content increased from first to sixth ejac- ulate and then decreased in the last two ejaculates. The fructose and citric acid content of semen declined markedly following seminal vesiculectomy (Hess _£__1,, 1960). The average values before seminal vesiculectomy were 255 and 245 mg/100 ml for fructose and citric acid, respectively, where—as comparable values after removal were 10.4 and 21.7 mg/100 ml. These data indicate nearly all of the fructose and citric acid found in bovine semen is produced by the seminal vesicle. Bertand and Vladesco (1921) surmised that zinc was in some way important for reproduction. This assumption was based upon the high zinc content found in semen of the stallion and human. Mawson and Fischer (1953) obtained similar values for human semen, and an exceptionally high zinc content in the dorsolateral prostate of the rat. Dyrendahl (1954) found 2.84 mg/100 ml of zinc in fresh bo- vine semen. Cragle §£_§1, (1958) reported finding only 0.2 mg/100 ml and suggested the zinc concentration of bull semen fluctuates widely. However, it is not clear if this value is for whole semen or seminal plasma. Mann (1945) reported a comparable value of 0.28 mg/100 ml for ram seminal plasma. 10 Keilin and Mann (1940) have shown that zinc is the pros- thetic metal of the enzyme carbonic anhydrase, which catal— yzes the reaction; _—9' + . H2CO3 H20 CO2 Much of the zinc content found in tissue may be explained in this manner. However, only small amounts of the enzyme were found in sperm and seminal plasma (Mawson and Fischer, 1953). HOpwood and Gassner (1962) found glutamic acid, alanine, glycine, serine, and aspartic acid in decreasing order of con- centration in bovine seminal plasma. Evidence that the free amino acids were related to testicular and epididymal func- tion was obtained by causing a drastic alteration in amino acid level by (1) removal of the testes and epididymides, (2) blockage of their products by vasectomy, or (3) inter- ference with their function by hyperthermy (Hopwood gt al,, 1961b). The most pronounced change was reduction of glu- tamic acid to a very low level, indicating its origin to be the testis or epididymis. Analysis of the free amino acid content of ampullar and seminal vesicular fluid revealed alanine to be present in the largest quantaties. Adminis- tration of testosterone propionate in the castrated animals restored the level of alanine to near normal, but the levels of the other amino acids did not increase similarly. 11 These findings are not in complete agreement with those of Hess et_al, (1960) who found glycine to be more concen-‘ trated than alanine in seminal plasma of normal dairy bulls. Mean concentrations of five individual seminal amino acids were not altered significantly by the removal of the sem- inal vesicles. Hess stated these results were similar to the findings of Brown (1952). Huggins gt_al. (1942) found only a small per cent of the proteins of human seminal plasma to be heat-coagulable, and approximately 60% of the proteins were of a proteose nature and dialyzable. Electrophoretic mobilities of the undialyzable proteins corresponded to the d1, d2, and d3 globulins, and serum albumin of blood. As ejaculation fre- quency increased, a definite decrease in seminal plasma protein content was found. Ross g§_§l. (1942), and Ross (1946) found proteoses, blood globulins, and glycoproteins present in quantity, but little serum albumin in human sem- inal plasma. Although the proteins of human seminal plasma have been extensively investigated, few studies have been published on the proteins of bovine seminal plasma. Larson and Salis- bury (1954) found the nitrogen content of the total seminal plasma proteins to be 14.4%. About 90% of the total nitrogen 12 present in seminal plasma was found to be protein in nature and non—dialyzable. Seventy six per cent of these proteins were heat-coagulable. A weak Molish test of exhaustively dialyzed seminal plasma indicated that non-dialyzable carbo- hydrate constituents are present in low concentration. These results are not in complete agreement with those of Sarkar §t_al, (1947) who found only 12.1% nitrogen in dried bovine seminal plasma protein. Electrophoretic examination of bovine seminal plasma protein revealed eleven components, three of which accounted for the majority of the total protein found (Larson and Salis— bury, 1954). The three major components possessed mobil- ities similar to those of blood a globulins. Immune-globulins were found in higher concentration in the seminal plasma of older bulls. The relative proportions of the proteins found in seminal plasma did not resemble the proportions existent in blood serum. According to the authors, the three com- ponents found in largest quantities in seminal plasma had a and a electrophoretic mobilities similar to the d 2, 3 ll globulins of blood. Proteins with mobilities of blood serum albumin were either absent from the seminal plasma or present at a very low concentration in the from of one minor compo- nent. The electrophoretic patterns of the seminal plasma 13 of all bulls studied were remarkably similar. Although the major components possess electrophoretic mobilities similar to blood globulins, they were found to differ by possessing a greater stability to physical treatment as well as lower carbohydrate and lipid contents. Ultracentrifugal analysis of bovine seminal plasma pro- teins by Larson et_al, (1954) revealed five components. The three present in largest quantities accounted for 95% of the total protein. The relative proportion of the proteins found in seminal plasma did not resemble the proportions existent in blood serum. Two components found in seminal plasma possessed sedimentation velocities similar to compo- nents found in blood serum. The authors stated that 30 to 40% of the bovine seminal plasma proteins are unlike any found in blood. Ultracentrifugal analysis of milk serum proteins revealed they resembled those of seminal plasma more closely than did proteins found in blood serum. Immunological studies conducted by Larson et_al. (1954) indicated that proteins found in seminal plasma were much more antigenic than those from milk serum or blood serum. The primary causative factors were found to be heat labile and non-dialyzable. A weak cross reaction between seminal \ plasma protein and milk serum protein, which was known to be 14 high in immune-globulin, indicated a low content of these proteins in seminal plasma. Substantiating evidence was ob- tained by a weak reaction between antisera to a purified preparation of immune-globulins and seminal plasma. A cross reaction obtained between blood serum and seminal plasma and their respective antisera indicated the systems have some antigens in common. The authors suggested that the cross reactions of seminal plasma and blood serum proteins were due to components which were either antigenically weak, or present in low concentra- tion. They further stated that with the exception of one component which appeared to be similar to lactalbumin of milk serum, "the major protein components of bovine seminal plasma are definite chemical entities which are not similar to any of the major protein components of blood or milk serum." EXPERIMENTAL PROCEDURES Semen Collection and Handling: The semen used in these studies was collected from five Holstein bulls ranging from 2 to 5 yr of age. These bulls had been in routine use at Michigan Artificial Breeders Co- operative. Based upon 60— to 90—day non—return percentages of routine artificial inseminations, they had normal fertil- ity. Four consecutive ejaculates were collected from each bull at weekly intervals for a period of 4 wk. Ten to fif- teen min of intensive sexual preparation including two or three false mounts were imposed previous to each ejaculation. Immediately after a semen sample was collected, the vol- ume of semen was recorded and a small sample was removed to, determine sperm concentration with a photelometer, and to determine pH of the fresh semen with a Beckman Zero-matic pH meter equipped with a one-drop electrode. Within 20 min, the remaining portion of the semen sample was centrifuged at 50 C with a centrifugal force of 26,000 x g for 20 min. Following centrifugation, the seminal plasma was decanted from the sperm cells and placed in plastic vials. A sample was re- moved at this time to determine biuret protein and ninhydrin- nitrogen. The rest of the seminal plasma was frozen and 15 l6 stored at -200 C for subsequent determination of citric acid, fructose, and zinc and for electrophoretic and im- mune-chemical analysis of the proteins. Free Amino Nitrogen: The free amino nitrogen content of seminal plasma was determined by the ninhydrin reaction (Harding and Mac Lean, 1916). This test gives positive results with proteins, peptides, amino acids, and other primary amines, including ammonia. One tenth ml of seminal plasma was diluted with 0.9 ml of distilled water, 1.0 m1 of 10% aqueous pyridine, and 1.0 ml of freshly prepared 2.0% solution of aqueous ninhydrin. The samples were then heated at 1000 C for 20 min, cooled to room temperature, and diluted to 200 ml with distilled water. The optical density was determined at 565 mu in a Spectronic 20 spectrophotometer and compared to a standard curve which had been previously constructed using the amino acid alanine. Citric Acid: Citric acid was determined by the procedure of Saffran and Denstedt (1948). Although this method is neither as accurate nor as specific as some other assays, it's rela- tive simplicity motivated its use here. A sample of frozen l7 seminal plasma was thawed and 0.2 ml of the sample was de- proteinized by adding 4.8 m1 of 5% trichloroacetic acid at 50 C. The resultant precipitate was removed by centrifuga- tion. Duplicate l-ml samples of the supernatant were each mixed with 8 ml of acetic anhydride and incubated for 10 min at 600 C. One ml of pyridine was then added and the mixture was incubated at 600 C for an additional 40 min. The samples were then cooled to room temperature and the optical density was determined at 400 mu in a Spectronic 20 spectrophotometer and compared to a previously prepared standard curve. Fructose: Fructose was determined by the method of Roe §t_al,(1934). This reaction is quite specific, the main disadvantage being that the phosphorylated esters of fructose are less reactive than the sugar itself. A sample of frozen seminal plasma was thawed and 0.2 ml of the sample was deproteinized by adding 4.8 ml of 5% trichloroacetic acid at 50 C. The re- sultant precipitate was removed by centrifugation. Duplicate 0.5 m1 samples of the supernatant fluid were diluted with 1.5 m1 of 5% trichlaroacetic acid and mixed with 2.0 ml of 30% resorcinol and 6.0 m1 of 30% HCL. These samples were incubated at 800 C for 8 min, quickly cooled to room l8 temperature, and the optical density was determined at 490 mu in a Spectronic 20 Spectrophotometer and compared with a previously prepared standard curve. The fructose to be used to establish the standard curve was dissolved in a saturated aqueous solution of benzoic acid to prevent deterioration with time. 2129.: Zinc was determined by the dithizone reaction as out- lined by Johnson §t_al, (1959). This method is extremely sensitive, detecting as little as 0.1 u g/ml. Because of this sensitivity, avoidance of contamination from glassware or reagents is of utmost importance. The samples of frozen seminal plasma were thawed and 0.5 ml taken from each of the four first ejaculates from each bull to obtain a 2.0 ml pooled first ejaculate for each bull. A similar procedure was followed for fourth ejaculates. Each 2.0 m1 sample was wet ashed and zinc determinations were performed according to the procedure described by Johnson §t_al, (1959) (Appen- dix, Procedure A). 19 Total Protein: Total protein content of the seminal plasma was deter— mined by the biuret method of Kingsley (1939) as modified by Gornall et_al. (1949). There are practically no sub- stances other than protein in biological fluids which give the biuret reaction, the only exception being bile pigments which are not present in sufficient quantity to cause an appreciable error. One-tenth ml of seminal plasma was diluted with 1.9 ml of distilled water and 4.0 m1 of biuret reagent (Gornall gt_al,, 1949) (Appendix, Procedure B) was added. »The mixture was incubated for 30 min at 380 C and the optical density was determined at 540 mu in a Spectronic 20 Spectro- photometer and compared to a standard curve which had been previously constructed using bovine serum albumin. Electrophoretic Analysis gngroteinsz Electrophoretic analyses were per formed at 10 to 20 C in a Tiselius electrophoresis apparatus (American Instrument Company) equipped with a cylindrical lens and a rotating slit. Seminal plasma from the first and from the fourth ejaculates of the first and fourth weeks for each bull were thawed, adjusted to a biuret protein content of 1.75 to 2.00% and a total volume of 7.5 ml by addition of sodium veronal 20 buffer (Appendix, Procedure C) at pH 8.6 and ionic strength 0.1. Each sample was then dialyzed in Visking cellulose casing with constant agitation for 12 hr at 50 C against each of two changes of buffer, each about 100 times the volume of the sample. The electrophoretic mobilities were calculated according to the procedure of Alberty (1948) which is outlined in the Appendix, Procedure D. The rela- tive concentration of each of the major protein constituents in each sample was estimated by drawing the electrophoretic pattern on graph paper, cutting out the curves, and weigh- ing the resultant paper (Tiselius, 1939). Immuno-chemical Analysis of Proteins: Immuno-chemical analyses were performed by injecting samples of seminal plasma and adjuvant into rabbits and analyzing the resultant antisera by the agar del double dif- fusion method of Ouchterlony (1958). The samples of frozen seminal plasma were thawed and 0.25 ml taken from each of the four first ejaculates from each bull to obtain a 1.0 ml pooled first ejaculate for each bull. A similar procedure was followed for fourth ejaculates. Each sample was adjusted to a biuret protein content of 2.0% with 0.85% saline and then emulsified with an equal 21 quantity of Freund's complete adjuvant (Appendix, Procedure E). The mode of action of adjuvants was discussed by Freund (1947, 1951). One ml of each sample was subcutaneously in- jected into five intrascapular sites in each of four Dutch Belted rabbits. One week later, each rabbit was similarly injected with the same sample homogenized with Freund's incomplete adjuvant (Appendix, Procedure E). The rabbits were bled by cardiac puncture 2 wk after the second injection and the blood was allowed to clot. The serum was decanted and then centrifuged to remove any remaining cells. Merthiolate was added to each serum sample to a concentration of 0.001% and the samples were stored at -200 C. Subsequently, the antisera were thawed and the numbers of precipitating antibodies were determined by dif- fusing the antisera against seminal plasma samples in Ouchterlony plates (Appendix, Procedure F). The progress of precipitin line formation was followed and recorded by photographing the plate against a black background with angularly transmitted light. Initially, one titer plate was prepared for the anti- sera of each immunized rabbit to determine the optimal anti- gen concentration for subsequent diffusions. The center well was filled with antisera, and the six peripheral wells 22 with serial dilutions of antigen. The presence of anti- bodies and the relative titer were determined in this man- ner. Subsequently, plates containing four wells in the H- pattern (Fox, 1959) were prepared with pooled antisera to ejaculate one in one well, pooled antisera to ejaculate four in another well, seminal plasma (antigen) from ejacu- late one in a third well, and seminal plasma from ejaculate four 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 utilized to determine any pre- cipitating antigens peculiar to either the first or to the fourth ejaculates. Antiserum to ejaculate one was absorbed with an equal volume of ejaculate four seminal plasma at 380 C for 0.5 hr. The absorbed antisera were then allowed to diffuse against seminal plasma from ejaculate one and ejaculate four. Cross reactions between the antisera to seminal plasma proteins and bovine blood serum were also determined by the double diffusion in gel method. RESULTS AND DISCUSSION Ejaculate Volume and Sperm Numbers: The average volume of semen for each of the four con- secutive ejaculates for each bull is listed in Table I, where it is apparent that the volume of semen declined pro- gressively from the first to the fourth ejaculate (P < .01). Table 1. Milliliters of semen per ejaculate. Ejaculate Bull l 2 3 4 Remark 8.8 8.5 7.9 7 3 Climax .4 8.9 7.1 5 9 Imperial 8.5 6.8 . 5.5 Explorer 10.4 7.4 Pure Gold 5.4 4.5 . 4.4 Avg 8.5 7.2 6.2 5.7 The average number of sperm per milliliter of semen for each of the four consecutive ejaculates for each bull is listed in Table 2. Although there is considerable variation among the bulls, the average concentration of sperm progres- sively declined from the first to the fourth ejaculate for 23 24 each bull (P < .01). This fact indicates that the epidi— dymal contribution decreases with repetitive ejaculation. Table 2. Billions of sperm per ml of semen. Ejaculate Bull l 2 3 4 Remark 1.390 1.137 0.686 0.571 Climax 1.527 0.945 0.688 0.471 Imperial 2.130 1.469 0.742 0.650 Explorer 2.031 1.050 0.561 0.419 Pure Gold 1.911 1.563 0.789 0.698 Avg 1.798 1.233 0.693 0.562 The total sperm in each ejaculate for each bull is pre- sented in Table 3. As was the case for volume of semen and for concentration of sperm, the average number of total sperm per ejaculate declined progressively from the first to the fourth ejaculates for each bull (P < .01). However, the decline from first to fourth ejaculate was considerably greater for some bulls than for others (P < .01). Total sperm production for each bull was remarkably constant among weeks for each of the four ejaculates and for the total sperm in the four ejaculates. These data agree with previous re— ports providing evidence that the ”exhaustion trial" is an 25 Table 3. Billions of sperm per ejaculate for each ejaculate. Ejaculate Bull Week 1 2 3 4 Sum Remark 1 10.5 9.4 6.8 3.9 30.6 2 14.8 9.3 4.8 3.8 32.8 3 10.6 9.5 5.4 3.7 29.1 4 13.0 9.6 4.6 4.9 32.1 Avg 12.2 9.5 5.4 4.1 31.2 Climax 1 14.0 8.1 5.8 2.8 30.8 2 12.2 7.5 6.5 2.8 28.8 3 12.5 9.0 3.8 3.7 29.1 4 19.3 8.2 3.6 1.1 32.2 Avg 14.5 8.2 4.9 2.6 30.2 Imperial 1 17.3 13.9 5.8 4.7 41.6 2 17.3 7.0 3.7 3.2 31.2 3 18.9 9.9 1.4 4.3 34.5 4 16.6 8.8 5.0 1.3 31.8 Avg 17.5 9.9 4.0 3.4 34.8 Explorer 1 17.0 12.1 4.9 2.0 35.9 2 26.0 5.2 4.3 1.2 36.6 3 18.8 7.8 2.9 3.0 32.6 4 22.6 6.2 2.3 2.6 33.7 Avg 21.1 7.8 3.6 2.2 34.7 Pure Gold 1 12.3 8.9 2.8 1.8 25.7 2 8.5 5.9 2.8 2.5 19.7 3 7.5 7.4 4.5 4.2 23.5 4 13.1 6.4 3.0 3.7 26.2 Avg 10.3 7.1 3.3 3.0 23.8 Avg 15.1 8.5 4.2 3.1 30.9 26 excellent means of estimating the sperm output capacity of bulls (walton and Edwards, 1938, and Hale and Almquist, 1960). Hydrogen Ion Concentration: The average pH of the fresh semen for each ejaculate for each bull is listed in Table 4. These data revealed a uniform increase in the pH from the first to the fourth ejaculate for each bull (P < .01). The bulls were remark— ably similar in this regard (P‘> .75). This progressive increase in pH has been reported by other workers (Rimoldi and Brigatti, 1945, and Davis and Williams, 1939). The average pH of first ejaculates was 6.26, somewhat lower than some reports, but well within the range reported for normal bovine semen (Anderson, 1942). Values obtained for each bull seemed characteristic for what particular animal, with little fluctuation between weeks. Several randomly chosen pH determinations following freezing and thawing of the seminal plasma showed no appreciable change from the fresh semen values previously obtained for the same semen sample. This indicated that bicarbonate probably was not a major factor in the buffering system of seminal plasma. 27 Table 4. Averages of pH values for the fresh semen. Ejaculate Bull l 2 3 4 Remark 6.39 6.52 6.66 6.77 Climax 6.36 6.63 6.82 7.09 Imperial 6.19 6.41 6.68 6.96 Explorer 6.15 6.35 6.74 6.91 Pure Gold 6.21 6.41 6.75 6.85 Avg 6.26 6.46 6.73 6.92 Free Amino (Ninhydrin) Nitrggen: The concentration of free amino nitrogen in the seminal plasma, and the total free amino nitrogen per ejaculate are listed in Table 5. The average concentration of ninhydrin nitrogen declined progressively from the first to the fourth ejaculate (P < .01) a trend which was magnified when presented in terms of total free amino nitrogen per ejaculate (P < .01). If we assume that the epididymides and ductus deferentia are the primary sources of free amino nitrogen (Hess §t_a1,, 1960, and HOpwood gt al,, 1961b), the data in Table 5 in- dicate that the epididymides and ductus deferentia contri- bute declining proportions to the seminal plasma of successive ejaculates. Table 5. Average seminal plasma free amino nitrogen values. Units of Enll Ejaculate measurement 1 2. 3 4 ug/ml Remark 1,023 945 1,038 813 Climax 976 928 856 834 Imperial 574 563 470 375 Explorer 930 850 787 708 Pure Gold 695 697 649 625 Avg 840 797 760 671 mg/ejac Remark 9.02 8.05 8.45 6.30 Climax 9.20 8.13 6.05 4.80 Imperial 4.77 3.73 2.45 1.83 Explorer 9.67 6.35 5.03 3.70 Pure Gold 3.63 3.15 2.77 2.80 Avg 7.26 5.88 4.95 3.88 Fructose and Citric Acid: The concentrations of fructose and citric acid in the seminal plasma and the total amounts per ejaculate are pre— sented in Table 6. The concentration of both of these con- stituents progressively increased with successive ejaculation (P‘< .01). The similarity of the values for concentration of fructose in third and fourth ejaculates indicates that fur- ther ejaculations would not have increased fructose concen- tration appreciably. However, the total amounts of fructose and citric acid per ejaculate were quite constant (P E .20) relative to the comparable values for free amino nitrogen 29 o.©m n.¢m o.mm m.m¢ maw wmm mam omm m>< N.om m.am N.HN m.mm mum mom one hmw UHOO musm m.Hv m.mv m.m¢ 0.65 mom «up moo ems HmHonxm o.nm N.mm m.mm m.a¢ 0mm va mom mow HmwummEH o.mm o.m~ N.ov m.mm Hmv mmq mov na¢ meHHO m.©m o.m¢ N.m¢ m.wv own ume hwm mmm meEmm Uflofi UHHDHO N.Om 5.00 m.¢© m.H© mam mum mum Hmo m>< m.mm m.mm m.mm N.©m 0mm nmm mun mam UHOO whom m.on m.mm w.Nm N.¢oa mmm.a oH¢.H nom.a ooo.H HwHonxm m.vm m.om N.v¢ o.nm mmv mom mmo mqv HMHHmQEH m.m¢ m.mo m.mm m.mh who who qmm mqn meHHO N.no m.mo o.mm o.m© nmm Now vhh mom meEmm mmouuznm w m N H w m N H Adam pamSpHpmnoo mumasomnm manasomhm omhm\mz as ooa\mz .mmsam> Uflum UHHHHU can mmouosnm mammHm HMCHEmm mmmnm>¢ .0 OHQMB 30 presented above. From the standpoint of the welfare of the sperm, the concentration values are probably more important. we may assume that the seminal vesicles are the primary source of fructose and citric acid and that the concentra- tion of these two constituents in the seminal vesicular contribution to the first through the fourth ejaculates was constant (Mann, 1949). Then the data in Table 6 indicate that the total seminal vesicular contribution was quite constant from the first to the fourth ejaculates. However, the relative contribution of the seminal vesicles increased from the first to the fourth ejaculate, presumably because of declining contributions from the other accessory sex organs. This contention is supported by the free amino ni- trogen values presented above and by the zinc values pre- sented below. The average concentrations of zinc in seminal plasma from first and fourth ejaculates and in blood serum from each of the five bulls are listed in Table 7. The average concen— tration of zinc in blood serum was considerably less than that in seminal plasma (P < .05). The average concentrations in ejaculates one and four did not differ greatly (P’) .50). 31 The tissues from one bull (Remark) were available for analysis at the end of the experiment. The prostate tissue had an average zinc concentration of 29 ug/gm of fresh tissue a value only about 10 to 30% of those found in other species. The seminal vesicular secretions had an average zinc concentration of 6.5 ug/ml. These facts in addition to the relatively small size of the bovine prostate. in- dicate that the seminal vesicles were the primary source of seminal plasma zinc, at least in this bull. Table 7. Micrograms of zinc per milliliter of seminal plasma and blood serum. Ejaculate Bull Blood serum 1 4 Remark 6.0 9.0 4.0 Climax 11.3 10.2 5.5 Imperial 8.3 6.6 7.3 Explorer 8.4 7.7 4.5 Pure Gold 13.1 10.5 8.3 Avg 9.4 8.8 5.9 Biuret Protein: The average concentrations of protein in seminal plasma are listed in Table 8. Repetitive ejaculation failed to 32 influence the concentration of protein (P£¥.48). This re- lative uniformity in protein concentration seems indica- tive of a homeostasis that may be important to sperm sur— vival. The total amount of protein per ejaculate declined with ejaculate number (P < .02)- a result which reflects declining seminal volume. Table 8. Average seminal plasma biuret protein values. Unit of B 11 Ejaculate u measurement 1 2 3 4 mg/100 ml Remark 8.29 8.85 8.44 7.97 Climax 8.55 9.16 9.63 8.71 Imperial 8.14 7.89 7.45 7.15 Explorer -5.31 5.74 5.36 4.62 Pure Gold 5.42 5.68 6.47 6.85 Avg 7.14 7.47 7.47 7.06 mg/ejac Remark 0.730 0.747 0.663 0.590 Climax 0.805 0.810 0.687 0.507 Imperial 0.700 0.545 0.373 0.417 Explorer 0.547 0.425 0.345 0.235 Pure Gold 0.283 0.260 0.280 0.303 Avg 0.613 0.557 0.469 0.410 Electrophoretic Analysis of Proteins. Typical Tiseulius electrophoretic patterns for the first and fourth ejaculates from each bull are presented in figure 33 1. Enlargement of these patterns revealed six or seven components in each pattern. The components were classified by electrophoretic mobility and are listed in Table 9. Repetitive ejaculation failed to alter the mobilities for components 2, 4, 5, 6, or 7 (P'> .15). However, the av— erage mobility of 6.70 for component eight in fourth ejac— ulates was significantly less than the corresponding av- erage of 6.88 in first ejaculates (P < .01). The full meaning of this result is not clear. No statistical pro— cedure was applied to components 1 or 3, because they were found only occasionally. Electrophoretic components 4, 5, 6, and 7 were present in sufficient amounts to estimate the relative proportion of these components from the patterns. These proportions are listed in Table 10. Repetitive ejaculation did not affect the proportion of components 5 or 7 (P > .17). How- ever, fourth ejaculates contained an average of 28.8% of component 4, significantly more than the average of 22.0% for first ejaculates (P 25.03) and fourth ejaculates con- tained an average of 30.4% of component 6, significantly less than the 35.3% for first ejaculates (P g .02). Thus, although the total protein content of seminal plasma was not measurably altered by repetitive ejaculation, the proportion 34 of the protein constituents was significantly altered. Electrophoretic analysis of blood serum proteins from the Remark bull revealed eight components (figure 2)- in excellent agreement with comparable results by Larson and Salisbury (1954). The number of electrophoretic components for seminal plasma listed by those authors ranged from six to ten, somewhat higher than the numbers obtained in the present experiment (Table 9). The present author is of the opinion that the patterns presented by Larson and Salisbury do not provide adequate evidence for as many as ten electrophoretic components in seminal plasma. Conse- quently, regarding the number of components, their results were very similar to those presented here. In agreement with the data presented by Larson and Salisbury (1954), the present data indicate that three components comprise the great majority (95 to 97%) of the protein in seminal plasma. Larson and Salisbury claimed that "the major components of the seminal plasma exhibit mobilities similar to those of the dl—, d2-, and d3— glob- ulins of blood.” The data in the present experiment in- dicate that tWO 0f the major components (c0mponents 4 and 6) of seminal plasma exhibited mobilities similar to those of the d2- and d3— globulins of blood. The third major 35 component (component 5) appeared to have no electrophoretic counterpart in blood serum. Figure 3 pictures an electrophoretic pattern of the protein components in seminal vesicular fluid from Remark. Seminal vesicular fluid contained all of the components for seminal plasma displayed in figure 1 and listed in Table 9. The relative proportions of the components in seminal vesic- ular fluid were very similar to those for ejaculated seminal plasma. Immunodchemical Analysis of Proteins: Photographs of Ouchterlony titer plates representing the first and the fourth ejaculates from each of the five bulls are displayed in figure 4. The number of visible precipitin lines obtained when antisera from each rabbit was reacted with its antigen in Ouchterlony titer plate is presented in Table 11. There was considerable variation in the measurable antibody response from rabbit to rabbit with- in a particular seminal plasma sample, probably due to a lower sensitivity of the reticulo-endothelial systems of certain rabbits. The rabbit yielding the maximum number of precipitin lines for a particular seminal plasma sample demonstrated the minimum number of antigens in that sample. Additional antigens may not have been detected due to the Figure l. Electrophoretic Patterns of Seminal Plasma. c '9 Remark 0 ~, 4 7 ,5 4 ml ./"'.‘\_2 1 i- __ ‘_ ‘ Ejaculate 1 Ejaculate 4 Climax 4 G- 7 7 1 Ejaculate l Ejaculate u ° 5 Imperial 6 5 ’1 4 2 6 L, h A ‘3 7 _3 ’7‘ Ejaculate 1 Ejaculate u 5 ’f g. 5 fi/ 2 Ejaculate 1 Ejaculate u Pure Gold 4 a 5 4 7 5 ‘7‘ 2 2'1, 5 7 Allllll... ., .. ;;_ Ejaculate 1 Ejaculate u Numbered components correspond to electrOphoretic components listed in Table 9 and 10. 37 Table 9. Electrophoretic mobilities* of measurable seminal plasma proteins. Electrophoretic component Bull week Ejac l 2 3 4 8 JRemark 1 l 1.8 4.1 6.2 7.1 4 1.8 4.4 6.2 7.0 4 l 1.9 4.1 6.2 7.1 4 1.9 4.3 6.3 6.8 Climax 1 1 2.0 4.2 6.3 . 4 l 5 2.6 4.0 6.0 4 l 1.5 3.2 3.8 5.5 4 1.7 3.0 3.8 5.7 Imperial 1 1 2.3 4.0 6.0 4 1.9 4.0 6.2 4 l 2.3 3.9 5.5 4 1.0 2 3 3.9 5.3 Explorer 1 l 1.8 2 9 4.2 6.2 7.2 4 2.1 4.1 6.1 7.0 4 l l 9 4.0 5.9 6.7 4 1.6 2 7 3.8 5.9 6.5 Pure Gold 1 l . 4.3 6.0 4 3 5 4.1 5.8 .7 4 l 0.4 2 4 4.2 6.1 7.3 4 1.3 4.2 5.7 7.0 Avg 1 l. 4. l. 4. 5. . * Mobilities in (cm2/volt sec) ( x 10-5). Table 10. 38 constituents of seminal plasma. Average* percentage composition of major protein Electrophoretic component Bull Ejac 4 5 6 7 Remark 24.7 38.1 35.1 2.5 4 31.7 35.1 30.1 3.2 Climax 1 31.9 40.7 24.9 2.4 4 42.6 34.5 21.3 Imperial 16.3 40.7 40.2 2.9 4 12.9 44.9 40.3 2.1 Explorer 24.8 43.1 28.7 3.5 4 28.3 42.9 25.7 3.2 Pure Gold 1 11.9 37.7 47.5 2.9 4 28.3 34.4 34.4 2.8 Avg 22.0 40.1 35.3 4 28.8 38.3 30.4 2. * Entries are averages of first and fourth week samples , a- Figure 2. Electrophoretic pattern of bovine blood "crum. mi Figure 3. Electrophoretic pattern of seminal vesicular fluid. Numbered components correspond to similarly numbered components in seminal plasma. ' 40 Figure 4. Ouchterlony titer plates. Each plate was prepared in the following manner: Center well: rabbit anti-bovine seminal plasma immune-serum Outer wells: serial dilutions of bovine seminal plasma, well 1 at three o'clock 1:1, to well 6 at one o'clock 1:63 A. Remark, ejaculate l B. Remark, ejaculate 4 C. Climax, ejaculate l D. Climax, ejaculate 4 E. Imperial, ejaculate F. Imperial, ejaculate G. Explorer, ejaculate a ,. .n +4 H. Explorer, ejaculate I. Pure Gold, ejaculate 1 J. Pure Gold, ejaculate 4 42 critical nature of the concentrations of the reactants necessary to obtain a precipitin line. It is also pos- sible that some antigen—antibody complexes were soluble and consequently undetected. Table 11. Number of precipitin lines visible in titer plates Rabbit Maximum Bull Ejac 1 2 3. 4 no. of lines Remark 1 5 8 3 4 8 4 5 4 5 7 Climax 1 8 7 7 7 8 4 6 5 7 7 7 Imperial 1 6 8 7 - 8 4 5 3 5 5 5 Explorer 1 4 7 6 8 8 4 5 6 - Pure Gold 1 8 5 7 8 4 5 5 — The average maximum of eight precipitin lines for sem- inal plasma from first ejaculates was significantly greater than the comparable average of six for fourth ejaculates 43 (P < .01). The number of protein components demonstrated in the seminal plasma from first ejaculates by this immuno- chemical technique is in good agreement with the number of components demonstrated electrophoretically. However, the concentrations of some of the proteins from fourth ejacu- lates apparently were too low to be detected by the immuno- chemical technique, since seven electrophoretic components were found in seminal plasma from fourth ejaculates. A photograph of a typical Ouchterlony "H—pattern“ plate is displayed in figure 5. A continuous precipitin line such as that marked in figure 5 demonstrated a common pro— tein antigen in the seminal plasma from first and fourth ejaculates. All precipitin lines appeared to be continuous when examined under magnification. These results indicated that there were no antigens in first ejaculate seminal plasma that were not also present in fourth ejaculate sem— inal plasma. However, as concluded above, the concentration of some of the proteins from fourth ejaculates were appar- ently too low to be detected by this technique. The Bjorklund inhibition studies substantiated these results. 44 Figure 5. Typical “H” pattern Ouchterlony plate. Well 1 (upper) Rabbit anti—sera to ejaculate one seminal plasma. Well 2 (right) Ejaculate one seminal plasma. Well 3 (lower) Rabbit anti—sera to ejaculate four seminal plasma. Well 4 (left) Ejaculate four seminal plasma. An antibody formed as the result of an antigen common to first and fourth ejaculate seminal plasma resulted in a continuous precipitin line (e.g., 1). Figure 6. Cross reaction of bovine seminal plasma, blood serum, and their respective anti—sera. Well 1 (upper) Rabbit anti—seminal plasma sera. Well 2 (right) Pooled ejaculate one and ejaculate four seminal plasma. Well 3 (lower) Rabbit anti—blood sera. Well 4 (left) Bovine blood serum. Precipitin line indicating an antigen common to seminal plasma, and blood serum. Precipitin line indicating an antigen present in seminal plasma, but not in blood serum. 46 Relationship Between Some Seminal Constituents: When the average values for each ejaculate number were summarized, as in Table 12, there appeared to be a relation- ship between certain components. For example, the concentra- tions of fructose and citric acid appeared to be quite paral— lel, where-as the concentrations of ninhydrin-nitrogen and fructose appeared'to have a negative relationship. such relationships could have value in determining the ori- gin of certain constituents, correlation coefficients were determined by pairing values obtained for each ejaculate from each bull in each week, resulting in 80 paired values from which to estimate each correlation coefficient. Table 12. Summary of averages of each seminal component measured in each ejaculate number. Seminal Ejaculate Component 1 2 3 4 M1 of semen/ejac 8.51 7.22 6.15 5.68 Sperm/ml of semen (x10 ) 1.80 1.23 0.69 0.56 Sperm/ejac (x109) 15.14 8.51 4.24 3.06 pH 6.26 6.47 6.75 6.92 Ninhydrin-N (us/m1) 840 797 760 671 Ninhydrin-N (mg/ejac) 7.26 5.88 4.78 3.78 Fructose (mg/100 ml) 681 879 973 917 Fructose (mg/ejac) 61.8 64.6 60.8 50.2 Citric acid (mg/100 ml) 520 518 564 619 Citric Acid (mg/ejac) 45.8 38.0 34.7 36.0 Protein (gm/100 ml) 7.1 7.5 7.5 7.1 Protein (mg/ejac) 61.3 55.8 47.0 41.0 47 The correlation coefficients between the concentration of seminal constituents and volume of semen are listed in Table 13 with their significance levels. These values, while demonstrating some significant relationships, generally are too low for predictive purposes, with the single pos- sible exception of the correlation of —0.81 between concen- tration of sperm and pH. Although the correlations for the concentration values listed in Table 13 have more importance in terms of the wel- fare of ejaculated sperm, the comparable correlations for the total amounts of each seminal constituent per ejaculate (Table 14) have more significance in terms of the origin of the constituents and in terms of the relative contribution of the adnexal sex glands. The correlations shown in Table 14 are all considerably larger than their counter- parts in Table 13. The correlation of 0.94 between total protein and total ninhydrin-nitrogen indicated that the majority of each of these components has a common origin. On the other hand, since the ninhydrin test measured all free amino nitrogen, including that exposed on proteins, the correlation of 0.94 is higher than would be expected if the protein had been eliminated before the ninhydrin test was performed. Table 13. 48 Summary of correlation coefficients for the concen— tration of some seminal constituents and volume of semen. Sperm Citric Fructose Protein NHz—N pH conc. acid Seminal vol. 0.29a 0.07 0.02 0.32a 0.41a 0.32a Sperm conc. -0.22b -0.40a —0.14 0.07 -0.81a Citric acid 0.36a -0.33a 0.13 0.02 Fructose -0.27b 0.26b 0.19 Protein 0.16 0.14 NHz-N -0.27b a PI < .01 b PI < .05 Table 14. Summary of correlation coefficients for the amount of some seminal constituents per ejaculate. Total Total _ . Total Total Total sperm Cltrlc fructose protein NH2-N aCld Seminal volume 0.69a 0.68a 0.64a 0.84a 0.82a Total sperm 0.39a 0.26b '0.44a 0.53a Total citric acid 0.61a 0.41a 0.64a Total fructose 0.41a 0.68a Total protein 0.94a a PI < .01 b PI < .05 49 The magnitude and similarity of the correlation coef- ficients between total fructose and total citric acid, be— tween total fructose and total ninhydrin-nitrogen, and be- tween total ninhydrin-nitrogen and total citric seem indica- tive of a common origin for these three seminal constituents. However, each of these correlation coefficients account for less than half of the total variance associated with them and this fact strongly implicates multiple origins for at least two of the three constituents. Some Histologic Observations on Remark: Tissues from the various parts of the reproductive tract of the Remark bull were obtained at the time of slaughter and fixed in Bouin's fixative preparatory to histologic examination. Based upon these examinations, the testes, epididymides, ductus deferentia, prostate, and bulbo—ure- thral glands all closely resembled those repeatedly described in the literature (Bailey, 1958). Micromorphologic examination of the seminal vesicles revealed three cell types corresponding to those described by Mann §£_§1, (1948). The three cell types are identified in the photomicrograph of a seminal vesicle displayed in figure 7. Secretion blebs comparable to those mentioned by Figure 7. Photomicrograph of bovine seminal vesicle (X 736). l. Secretion blob. Figure 8. Phozgmicrograph of ampulla of ductus deferens X 0 . 1. Secretion bleb. 51 Mann _t__l, (1948) were present at the luminal extremity of many of the cells in the seminal vesicle. These blebs are pictured in figure 7 and appeared to represent an apocrine type of secretion. Micromorphologic examination of the ampullae of the ductus deferentia revealed a striking similarity between the epithelium of this organ with that of the seminal ves- icles. The photomicropgraph of an ampulla displayed in figure 8 identifies the same three cell types described for the seminal vesicles. Although this anatomical simi- larity has not been previously reported, Mann §§_a1, (1948) reported that ampullar secretions were quite similar to seminal vesicular secretions. S UMMARY Five Holstein bulls, ranging from 2 to 5 yr of age, were ejaculated four consecutive times at weekly intervals for a period of 4 wk. Seminal volume, pH, and sperm concen— tration were determined and the sperm were removed by cen- trifugation. The concentrations of fructose, citric acid, protein, free amino nitrogen, and zinc in the seminal plas- ma were determined. The proteins were also studied elec- trophoretically and immunochemically. As anticipated, seminal volume, sperm concentration, and total sperm per ejaculate decreased from ejaculate one to ejaculate four. There was a marked similarity in sperm output from week to week for each of the five bulls, indi- cating that such an ejaculation procedure could be used to predict a bull's sperm producing capacity. The average pH of seminal plasma from first, second, third, and fourth ejaculates was 6.26, 6.46, 6.73, and 6.92, respectively (P < .01). Comparable averages for fructose in seminal plasma were 681, 879, 973, and 917 mg/100 ml, re- spectively (P < .01), and for citric acid were 520, 518, 564, and 619 mg/100 ml, respectively (P < .01). Neither the total fructose nor the total citric acid in the first, sec- ond, third, and fourth ejaculates differed significantly 52 53 (P'> .20). These data indicate that the seminal vesicle contributed a relative constant volume to each ejaculate. However, the relative contribution of the seminal vesicle increased from the first to the fourth ejaculates, presum- ably because of declining contributions from the other ac- cessory sex organs. The average concentration of zinc in first and fourth ejaculate seminal plasma did not differ significantly (P'> .50). Although the average zinc concentration of 9.1 u g/ml of seminal plasma was considerably larger than the average of 5.9 u g/ml of blood serum, the seminal average did not approach the comparable values previously reported for some other species. Average seminal free amino nitrogen, in first, second, third, and fourth ejaculates were 840, 797, 760, and 671 u g/ml, respectively (P < .01). In view of the report- edly high concentration of amino acids in epididymal sec- retions, these results indicate that the epididymis con— tributes progressively lower proportions to the seminal plasma of successive ejaculates. The average concentra- tion of protein in seminal plasma did not significantly change from the first to the fourth ejaculates. 54 Electrophoretic analysis of seminal plasma revealed eight protein components and six or seven of these were found in each ejaculate. No appreciable difference was found between the number of components in first and fourth ejaculates. Electrophoretic mobilities of the major pro— tein components were not altered by repetitive ejaculation. Based upon the electrophoretic data, the percentage com— position of two of four major protein components was al- tered by repetitive ejaculation (P E .03). One of the four major seminal plasma protein components had no electro- phoretic counterpart in blood serum. Immunochemical studies revealed a minimum of eight pre- cipitating antigens in first ejaculate seminal plasma and a minimum of six in fourth ejaculate seminal plasma. Hew- ever, the evidence indicated that the concentration of some minor constituents in fourth ejaculate seminal plasma was too low to be detected by this immunochemical technique. Correlation of the measured components in seminal plasma revealed that the totals per ejaculate were, without excep- tion, more highly correlated than the comparable concentra— tion values. The magnitude and similarity of the correla- tions between total fructose and total citric acid (r = 0.61), between total fructose and total free amino nitrogen 55 (r = 0.68), and between total citric acid and total free amino nitrogen (r = 0.64) indicated a common major origin for these seminal constituents. The correlation between the concentration of sperm and pH was -0.81, suggesting that seminal hydrogen ions and sperm have similar origins. BIBLIOGRAPHY Alberty, R. A. 1948 An introduction to electrophoresis. I. Methods and calculations. J. Chem. Ed" 25: 426. Almquist, J. 0., Hale, E. B., and Amann, R. 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Freund, J. 1951 The effect of paraffin oil and mycobacteria on antibody formation and senitization. J. Clin. Path., 21: 645. Gornall, A. G., Bardawill, C. J., and David, M. M. 1949 Determination of serum proteins by means of the biuret reaction. J. Biol. Chem., 177: 751. Hafs, H. D., Knisely, R. C., and Desjardins, C. 1962 Sperm output of dairy bulls with varying degrees of sexual preparation. J. Dairy Sci., 45: 788. Hale, E. B., and Almquist, J. O. 1960 Relation of sexual behavior to germ cell output in farm animals. Supp. J. Dairy Sci., 4;: April. 145. Harding, J. V., and Mac Lean, R. M. 1916 A colorimetric method for the estimation of amino-acid nitrogen. II. Application to the hydrolysis of proteins by pancreatic enzymes. J. Biol. Chem., 24; 503. Hellstrom, P. 1947 Bulls which appear too eager should be checked. (Trans. title.) Lantmannen (Stockh.), 31: 198. Abstr. in Animal Breeding Abstr., 1E5 177. 1947. Hess, E. A., Ludwick, T. M., Martig, R. 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General aspects, occurence and distribution of cyto- chrome, certain enzymes and co—enzymes. Biochem. J. 39: 451. 6O Mann, T. 1946 Studies on the metabolism of semen. III. Fructose as a normal constituent of seminal plasma. Site of formation and function of fructose in semen. Biochem. J., 49: 481. Mann, T. 1948 Fructose content and fructolysis in semen. Practical application in the evaluation of semen quality. J. Agric. Sci., 38: 323. Mann, T. 1949 Metabolism of semen. (In Advances in en— zymology, 2:329, Interscience Pub1., London). Mann, T., Davies, D. V., and Humphrey, G. H. 1948 Fructose and citric acid assay in the secretions of the accessory glands of reproduction as indicator tests of male sex hormone activity. J. Endo., 6: 75. Mann, T., and Lutwak—Mann, C. 1951 Secretory function of male accessory organs of reproduction in mammals. Physiol. Rev., 31: 27. Mawson, C. A., and Fischer, M. I. 1953 Zinc and carbonic anhydrase in human semen. Biochem. J., 55: 696. 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Exptl. Biol. Med., 109: 567. APPENDIX Procedure A. Dithizone Zinc Determination (Johnson gt a1,, 1959): Preparation of reagents: A. Ammonium hydroxide solution: (1) 1N: Prepare a solution slightly stronger than required, then standardize against standard acid and dilute as required. (2) 0.01 N: Dilute 10 ml solution (1) to one liter. B. Ammonium citrate solution: (1) Solution I (0.05 M): Dissolve 226 g ammon- ium citrate in 2,000 ml distilled water. Add concentrated ammonium hydroxide until the pH of the solution is 8.5-8.7 (80 to 85 m1). (2) Solution II: Add 70 m1 of I N ammonium hy- droxide to 500 m1 of Solution I (0.05 M) and dilute to 2,000 m1. C. Carbamate solution: Dissolve 1.25 g sodium diethyldithiocarbamate in distilled water and dilute to 1,000 m1. Store at 50 C. 62 63 D. Diphenylthiocarbazone (dithizone): (A) Concentrated solution: Dissolve 0.0375 g dithizone in 500 m1 CC14.(Prepare fresh daily.) (B) Dilute solution: Add one volume of the con— centrated solution to four volumes of carbon tetrachloride. Zinc determinations: Twenty—five m1 of concentrated HNO3 was added to 2 m1 of seminal plasma in a 150 m1 Pyrex glass beaker. Each beaker was covered with a watch glass and allowed to react at room temperature for 15 min. The beakers were then transferred to a hot plate and heated to approximately 1000 C with caution to prevent excessive frothing. Digestion was con— tinued for about 2 hr until the color was light yellow. Beakers were removed from the heat and cooled to room temperature, before addition of 6 m1 of 70-72% perchloric acid. The samples were then refluxed vigorously until a colorless or pale yellow solution remained. If necessary, additional perchloric acid was added to prevent the samples from going to dryness. Upon completion of digestion, the samples were evaporated to dryness by placing the cover glass slightly to one side. The beakers were then removed 64 from the source of heat, and the cover glass and sides of the beaker washed down with distilled water. The solution was made just basic to litmus with concen— trated ammonium hydroxide, then just acid with concentrated hydrochloric acid, and two drops of acid were added in ex- cess. The solution was transferred to a 100 m1 volumetric flask and the beaker was washed several times with distilled water. One ml of 1.0 N HCl was added and the flask was filled to volume with distilled water. The pH was 1.8 to 2.2. Thirty to thirty-five m1 of the solution from the vol- umetric flask was measured into a 1 X 8 in glass-stoppered Pyrex test tube. Ten m1 of concentrated dithizone was added and the tube stoppered and shaken 1 min. The layers were allowed to separate. If the carbon tetrachloride layer was red or purple it was removed with a pipette and the dithizone extraction was repeated. The following reagents were added to a 125 ml red, glass stoppered, Erlenmeyer flask in the order listed: (1) 50 m1 ammonium citrate solution II (2) 5 m1 carbamate reagent (3) 15 ml of aqueous sample solution from the test tube (4) 25 m1 dithizone solution B. The flask was stoppered, shaken vigorously for l min, the 65 layers were allowed to separate, and then the aqueous phase was decanted and discarded. Fifty m1 of 0.01 N ammonium hy- droxide was added to the flask and the flask was stoppered, shaken 30 sec, and the layers were allowed to separate. The aqueous phase was decanted and discarded. The ammonium hydroxide extraction was repeated and the aqueous phase was decanted just prior to determining the absorption of the organic phase. A cuvette was filled with the organic phase and the absorption was read at 535 mu. Two blanks were processed with the samples and all readings corrected for the average values obtained for the blanks. The corrected values were then compared to a previously constructed standard curve. Procedure B. Preparation of Biuret Reagent (Gornall §£_§l,, 1949): Place 1.5 g copper sulfate and 6.0 g sodium potassium tartrate in a 1,000 m1 flask and add sufficient water to dissolve. Slowly add 300 ml 2.5 N sodium hydroxide. Add 1.0 g potassium iodide and dilute to 1,000 ml. Store anaerobically at 50 C and discard when a dark precipitate forms. 66 Procedure C. Preparation of Sodium Veronal Buffer: Add 5.594 g Barbitol and 41.2 g sodium Barbitol to 800 ml boiling water in a 2,000 ml vol flask and add glass dis— tilled water to volume. Procedure D. Computation of Electrophoretic Mobilities (E): Mobilities of the seminal plasma proteins were calcu- lated by the formula: __3__ H = v c I R I Where: u mobility in (cm2 /volt sec) (x 10_5) v = velocity in cm/sec q = cross section of cell (cmz) c = cell constant I = amperes of current applied R = resistance of protein solution (ohms) Photographs used to compute mobilities were taken with a Polaroid camera. The starting boundry was recorded after compensation but before power was applied to the cell. Patterns were photographed twice during, and upon comple- tion of each run. The negatives were projected with an enlarger, the patterns were traced on paper, and the dis- placement of each component from the reference boundry was measured. Migration of a particular component was determined 67 by the distance from the initial boundry to a point which bi- sected the curve representing the component. Velocity (v) was calculated by determining the migration (cm) per unit of time (sec). Procedure E. Composition and Use of Adjuvant: Immunization was accomplished using Freund's complete adjuvant and incomplete adjuvant (Bacto-Adjuvant, Difco Laboratories). The complete adjuvant contained 1.5 ml mannide monooleate, 8.5 ml paraffin oil, and 5 mg killed and dried Mycobacterium butyricum. The incomplete adjuvant contains the same quantities of mannide monooleate and paraffin oil, but no bacteria. An oil in water emulsion of the antigen and adjuvant was prepared by emulsification in a Serval Omnimixer. Appendix F. Preparation of Ouchterlony Plates: Plates to be used for the double diffusion technique of Ouchterlony (1958) were prepared as follows: 1. Eight strips of filter paper (Whatman No. 1), mea- suring 1.0 by 0.51JL, were placed evenly around the rim of a 90 mm petri dish. A 24 in. 26 ga stainless steel wire was coiled so as to hold the 68 paper strips around the periphery of the petri dish. 2. Thirty m1 of melted 0.85% agara, dissolved in phos- phate-buffered saline, was poured into each plate. The phosphate-buffered saline was prepared by dis- solving 1.4198 g of Na HPO and 0.3233 g of KH PO 2 4 2 4 in 2,475 m1 of 0.85% sodium chloride. 3. "H-pattern" wells were cut with a template and tube cutter (figure 9). Seven—well patterns were cut with a Feinberg No. 1801 agar cutter (figure 9). The bottoms of the wells were sealed with a few drops of molten agar. 4. Antigen and antibody solutions were added to the wells and the plates held at 50 C while the re- actants diffused. Agar Agar, No. 3 (Consolidate Labs.) .mopmao zcoanopnoso mammopz on com: poppso cam mopmHaEme .m mpSUHm an??? huwvi wilfully?) 111’: M! {1 (1(1ij (in) (F