Influence of Seminal Vesiculectomy on Bovine Semen

Influence of Seminal Vesiculectomy on Bovine Semen

Influence of Seminal Vesiculectomy on Bovine Semen G. J. KING and J. W. MACPHERSON Department of Animal Science, University of Guelph, Guelph, Ontario...

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Influence of Seminal Vesiculectomy on Bovine Semen G. J. KING and J. W. MACPHERSON Department of Animal Science, University of Guelph, Guelph, Ontario, Canada Abstract

Therefore, to avoid these effects, it might prove advantageous to collect ejaculates which are free of vesicular secretion for use in artificial insemination. I n the bull, seminal vesicular secretions contribute the largest portion of the seminal plasma. Removal of these glands could eliminate or reduce the concentration of certain constituents which might have some effect on reproductive efficiency. This study was designed to examine certain physical and chemical properties, freczability and fertility of bovine semen in the absence of seminal vesicular secretion.

A study was designed to investigate some physical and chemical responses resulting from elimination of seminal vesicular contribution to the bovine ejaculate and to determine the effect on freezability and fertility of the semen. Seminal vesiculectomy resulted in a reduction in protein and reducing sugar and an increase in the chloride levels of seminal plasma. Glycerylphosphorylcholine was increased in three of four bulls. The operation almost completely eliminated sugar from ejaculates of two bulls; therefore, the concept o£ fructose production by the ampulla is questioned. Ejaculate volume was reduced and sperm concentration increased in all but one bull. The change in total spermatozoa per ejaculate was not significant, but the results suggest individual bulls may vary in their response. The operation did not appear to have any effect on initial semen quality. However, vital staining demonstrated a greater loss of spermatozoa during the freezing process after surgery. Removal of the seminal vesicles may have eliminated or reduced the concentration of some factor which protect cells during freezing. Seminal vesiculeetomy did not alter respiratory activity or fertilizing capacity of spermatozoa which survived the freezing process. Introduction

Dott (3) has observed that fertility levels obtained with ram epididymal spermatozoa incubated for 4 hr in a continuous flow dialysis apparatus are superior to fertility levels obtained with ejaculated semen treated in the same way. Fulka, Sulcova, and Va]enta (5) reported that the cold-shock resistance of bovine epididymal cells is markedly reduced by incubation with seminal vesicular secretions. Shannon (19) has detected a toxic protein fraction in bovine semen which depresses the livability of extended semen during storage. Antigens have been demonstrated in bovine seminal plasma by H u n t e r and H a f s (10) and these could be involved in antifertility reaction. Received for publication June 10, 1969.

Experlmental Procedures Six ejaculates were collected by artificial vagina from each of four bulls before surgery. Seminal vesiculectomy was performed as previously described (11) and after a six-week recovery period, six additional ejaculates p e r bull were obtained. Bulls were collected two or three times weekly following intensive sexual preparation, as suggested by Hale and Almquist (8). A portion of each ejaculate was extended in tris-buffered yolk extender (22) and cooled to 5 C in 2.5 hr. Additional extender containing glycerol was then added slowly to give a final concentration of 25 X 106 live cells per milliliter and 6.8 per cent glycerol ( v / v ) . The extended semen was packaged in 1-ml glass ampoules and frozen in an automatic, alcohol bath freezing device after 3 hr of equilibration. All frozen semen was stored in liquid nitrogen. The percentage of motile spermatozoa at collection time was determined microscopically, the per cent live by vital staining with t r y p a n blue (7) and the concentration by haemocytometer. The same percentages were determined on frozen samples after three weeks of storage and the survival p e r cent calculated by the formulas: % motile post-freezing % Motile sur× 100 viving freezing ---% motile at collection

% Live sur-

% live post-freezing

riving freezing = % llve at collection

x 100

A f t e r glycerolization, the concentration of a portion of extended semen was adjusted to give 50 × 106 live spermatozoa in 3 ml of extender.

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Three-milliliter volumes were incubated at 37 C for 4 hr in a Warburg apparatus and the prefreezing oxygen uptake by 50 × 10 ~ live spermatozoa recorded. Post-freezing oxygen uptake was determined by placing 3 ml of thawed semen into the reaction vessels and incubating for 4 hr. The number of live spermatozoa in this 3-ml volume was determined and the 4-hr oxygen uptake was corrected to 50 )< 1@ live cells. Portions of unprocessed ejaculates not required for motility, metabolic, or freezing studies were frozen immediately after collection and stored in liquid nitrogen for subsequent analysis. This was done to prevent metabolic changes during the interval between collection and laboratory determinations. These samples were subsequently thawed and used for the physical and chemical analysis. A Radiometer p H meter with micro electrode was used for p H deternlinations and measurements of osmotic pressure were made with an Advanced Instruments Osmometer. Samples were then centrifuged at 3,360 X g for 20 rain to obtain cell-free seminal plasma. Individual ejaculates were analyzed for total protein using the Biuret method (24) and total reducing sugar by the method of Somogyi (20). Presurgical ejaculates from individual bulls were pooled and likewise post-surgical ejaculates pooled for determination of glycerolphosphorylcholine (GPC) by the method of Renkonen (14) and chloride levels by the technique of Scbales and Sehales (16). The pooled samples were subjected to electrophoresis on Sepraphore I I I , cellulose polyacetate strips, using the Gehnan high resolution buffer. Eleetrophoresis was performed for 40 min at 5 C with a constant current of 1.5 ma per strip. Strips from each sample were stained with periodic acid-Schiff stain to demonstrate glycoprotcins (6). A fertility trial was conducted with four presurgical and four post-surgical ejaculates from each of two bulls. The pre- and postsurgical semen was distributed so that each inseminator received equal numbers of ampules from each group. Trial inseminations were restricted to first-service cows only and the 60- to 90-day nonreturn rate calculated. Unfortunately, it was not possible to perform sufficient numbers of inseminations with the two other bulls. Data were analyzed according to procedures described by Steel and Torrie (21). Arcsin transformations were used with all percentage data. J . DAII~Y SCIENCE VOL. 52, NO. 11

Results and Discussion

Seminal vesiculectomy had no apparent effect on libido. All experimental bulls mounted teasers one or two days after surgery and semen was collected from all except Bull A at this time. Traces of blood and inflammatory exudate were present in semen collected for five weeks following surgery. After five weeks of attempted collections and several unproductive sessions with the electro-ejaculator, Bull A regained his ejaculation reflex and produced semen with no visible trace of inflammatory material. Rectal palpation and post-surgical levels of sugar, protein, and chloride showed that the seminal vesicles were almost completely removed from Bulls A and D. Some functional tissue remained in Bull B and a trace was probably present in Bull C. Table ] presents the means of pre- and post-surgical data for all physical and biochemical characteristics measured. Analyses of these data indicated significant bull × surgery interaction for certain characteristics; therefore, the individual bull means are included in this table. The concentration of spermatozoa in ejaculates from Bull A was lower after the operation. No obvious explanation can be offered for this except that perhaps the operation resulted in occlusion of the ampullae or affected the neural supply. I f only partial function was restored to this area, a significant number of spermatozoa may have remained in the ampullae and pelvic urethra during postsurgical ejaculations. A procedure such as seminal vesiculectomy, which alters ejaculate volume and concentration, must be evahmted in terms of the combined effect of these changes. Therefore, the most important aspect is the total number of spermatozoa which can be harvested before and after the treatment. Analysis of this data indicated no significant difference between the total sperm before and after surgery but the bull × surgery interaction was significant. The first three bulls showed a reduction in total sperm following the operation, whereas Bull D exhibited a considerable increase. It is possible that individual bulls may vary in their total sperm response, but additional replication would be required before conclusions could be meaningful. Secretions from the seminal vesicles have a lower p H (23) and osmitie pressure (15) than fuids from the ampullae and epididymis. Therefore, elimination of part or all of the vesicular portion of the ejaculate should be accompanied by an increase in the alkalinity

TABLE 1. Some physical and chemical characteristics of bovine semen before and after seminal vesiculectomy,a Bull A Criteria

50

Q



Pre-S

Bull B

Post-S

Pre-S

Bull C Post-S

Pre-S

Bull D

Post-S

Pre-S 4.8

Means

Post-S 5.1

Pre-S 6.1

Post-S

Ejaculate volume (ml) b

7.1

4.8

6.7

5.2

5.9

3.9

4.7 ¢

Concentration (106/ml) b

1,768

1,402

1,733

1,917

1,702

2,077

722

1,765

1,481

Total sperm (109/ejac.) b

13.28

6.74

11.76

10.12

10.07

7.74

3.58

8139

9.67

8.25

Osmotic pressure b (mosmol/kg)

292

361

301

292

292

325

297

326

296

326 c

Semen p H

6.33

6.64

6.51

6.55

6.38

6.64

6.61

6.60

6.46

6.61 c

Total protein b (g/100 ml plasma)

5.0

1.4

5.3

2.3

5.0

2.0

5.3

1.1

5.2

1.7 e

Total reducing sugar b (rag/100 ml plasma)

338

11

450

136

405

32

524

9

429

GPC (rag/100 ml plasma) d

976

810

469

606

999

1,231

485

1,365

732

1,003

Chloride (rag/100 ml plasma) d

138

316

110

284

e

220

217

298

155

280

1,790 c

47 c

a Individual bull values are means of six ejaculates. Bull × surgery interaction significant, P ~ 0.05. c Difference between pre- and post-surgical means significant, P--~ 0.05. a Criteria determined on pooled seminal plasma. e Very low value.

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and osmotic pressure of ejaculated semen. An increase in osmotic pressure was demonstrated with semen collected from three of the bulls. A significant rise in p H was also observed, in agreement with results reported by Hess et al. (9). However, Shah, Hopwood, and Faulkner (18) did not observe a rise in semen p H following seminal vesiculectomy in Hereford bulls. Szumowski and Theret (23) compared fluids from all regions of the male reproductive tract and found that secretions of the seminal vesicles contain the highest levels of protein and fructose. Aalbers ( l ) found only ]9.5 ~ 5 rag/100 ml fructose in a bull born without seminal vesicles. Removal of these glands should result in considerable reduction in the plasma level of both protein and sugar. Postsurgical total reducing sugar for Bulls A and D was below the level reported by AMbers. Bull B had a considerably higher sugar level, but a remnant of the right gland could be palpated in this animal. The level of seminal plasma protein was reduced considerably in all bulls after surgery. The significant interaction obtained in analysis of the data for both protein and sugar levels was due to the response of Bull D. Samples collected from this animal yielded the highest presurgical and the lowest post-surgical levels for both components. Several researchers (13, 23) have analyzed fluids collected from reproductive tracts after slaughter and reported that the bovine ampullae are also a source of fructose. Total reducing sugar was almost completely eliminated by seminal vesiculectomy from two of the bulls in this study. It would, therefore, appear that the ampullae could not have been

contributing significant amounts of fructose in these bulls. It is possible that vesicular secretion is expressed at slaughter and may flow back into the ampullae. This could amount for the relatively high levels of fructose detected in ampullae fluid from slaughterhouse specinmns. Glycerylphosphorylcho]ine is secreted by the epididymis (13) and the major source of chloride is the urethral glands (1). These components should both increase when the vesicular secretion is eliminated from the ejaculate. The chloride levels in pooled seminal plasma did show the expected increase above normal leveIs following surgery, whereas the GPC level in post-surgical ejaculates was increased in all animals except Bull A. However, the total sperm output of this bull was also reduced following the operation and it has been shown that total sperm numbers are positively correlated with GPC content of the bovine ejaculate (12). The GPC values reported here are higher than most previously published levels but in close agreement with several recent reports (17, ]8). The ratio of GPC to spermatozoa in this study was 5.1 mg GPC/109 sperm before seminal vesiculectonly and 5.8 mg GPC/109 sperm after. This is in very close agreement with the mean of 5.5 reported by Seidel and Foote (17). Observations on the percentages of motile and live spermatozoa at collection and surviving the freezing process before and after seminal vesiculectomy are presented in Table 2. Data analyses of the per cent motile cells at collection indicated that post-surgical ejaculates

TABLE 2. Motile and live spermatozoa at collection and surviving freezing, oxygen uptake, and fertility levels before and after seminal vesiculectomy. Criterion

Pre-surgery

Post-surgery

S~

Motile spermatozoa a At collection Survival ( % )

56.28 b 39.77

52.68 38.69

1.07 1.33

Live spermatozoa a At collection Survival (%)

57.77 40.725

57.79 35.32

1.22 1.00

02 Uptake ~liters/50 × 106 cells/4 hr

26.8

27.2

0.83

Fertility levelc 60/90 day nonreturns

71.5

71.0

1.9

a Aresin values. b Difference between pre- and post-surgery means significant, P--~ 0.05. e Frozen semen from Bulls A and B; 200 pre- and 200 post-surgical inseminations from each bull. J'. DAIRY SCIENCE VOL. 52, NO. I I

SEMEN P R O P E R T I E S

contained significantly fewer motile spermatozoa. This may not be meaningful, since large reductions occurred in only two bulls. Hess et al. (9) observed a significant reduction in the per cent of motile cells after senfinal vesiculectomy but concluded this was due to the poor quality of the first few post-surgical ejaculates. Bulls may differ in their response to this operation, or low levels of reducing sugar in postsurgical ejaculates could inhibit motility in some samples. A more critical evaluation is the per cent of live spermatozoa revealed by vital staining. Analyses of these data revealed no significant difference in semen collected before or after removal of the glands. However, in a similar study, Faulkner, Hopwood, and Wiltbank (4) found a significant reduction in the per cent of live cells in two of six bulls following semihal vesieuleetomy. Again, this indicates the vesicular secretion may be useful or essential in certain bulls but not in others. Hess et al. (9) found that removal of the seminal vesicles did not affect the per cent of spermatozoa surviving the freezing process when progressive motility was used as the criterion for evaluation. The findings in this study support this observation. However, when vital staining was employed, elimination of seminal vesicular secretions resulted in a reduction of spermatozoa which survived freezing. This indicates components in seminal vesicular fluid may have some protective function. Table 2 also shows the spermatozoa in post-surgical ejaculates that do survive are capable of normal respiration and fertility. The protective action of vesicular secretion is perhaps an all-or-none effect which allows survival of higher numbers of cells but does not benefit those capable of surviving without this protection. Oxygen uptake was measured to determine what effect seminal vesiculeetomy might have on the metabolism of spermatozoa. The analyses of variance on these data indicated neither freezing nor surgery affected the uptake by comparable numbers of live cells. Therefore, spernmtozoa which survive the freezing process, with or without exposure to vesicular secretion, were capable of respiring at their prefreeze rate. The fertility trial carried out with frozen semen from Bulls A and B resulted in approximately 200 services before and after surgery for each bull. The combined 60/90-day nonreturn rate was 71.5 before and 71.0% after seminal vesiculectomy. This indicates that even though cell losses during freezing were in-

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creased after seminal vesiculectomy, the vitality of surviving spermatozoa was not affected and adequate numbers survived to maintain normal fertility levels. Seminal plasma apparently has different effects on various aspects of semen processing. Exposure to vesicular secretion causes epididymal spermatozoa to become more susceptible to cold shock (5). I n this study, these fluids afforded some degree of protection during freezing. A glycoprotein coat has been described surrounding various cells and this m a y function to protect surface membranes and maintain cellular mieroenvironment (2). This protective action may be very useful during cooling and freezing semen for artificial insemination. Visual evaluation of periodic acidSehiff stained eleetrophoresis strips indicated glycoprotein in seminal plasma was practically eliminated after seminal vesieulectomy. The reduction in level of this protein component may have contributed to the lower percentage of live cells after freezing. A sudden temperature drop might cause a rapid contraction of cellular contents and membranes which may be important features of cold shock. The plasma membrane of epididymal spermatozoa may be able to maintain its structural integrity during this sudden contraction. At ejaculation tile macromoleeules, including the glycoprotein in seminal vesicular fluid, become quickly and firmly adhered to the surface of the spermatozoa. Adhering forces formed between these macromoleeules and the fibrous protein layer of ejaculated spermatozoa may impair the ability of the surface membrane to contract rapidly and uniformly. Secondary and tertiary bonds which stabilize the structure of these membrane proteins could be broken and t h e integrity of the cell membrane disrupted. This would then allow the leakage of intracellular enzymes and the increased permeability to vital stains which accompanies cold shock. The slow cooling carried out in semen processing may allow the cell surface to compensate for these adhering forces and prevent membrane damage. During the actual freezing of semen, the coating of macromolecules from vesicular secretion may be acting as a protective shield and, therefore, minimizing membrane damage which would be caused by increased salt concentration. This would reduce the incidence of spermatozoal death while the medium is undergoing the change from liquid to solid state. These results suggest seminal vesieulectomy will reduce the total sperm output in individual bulls. Also, the per cent of live spermatozoa J. DAIRY SCIElqCE YOL. 52, NO. ] 1

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surviving f r e e z i n g was reduced. Therefore, elhnination o f seminal vesiculnr secretion would not be beneficial f o r most bulls in commercial artificial insemination service.

Acknowledgments The authors t h a n k !VIrs. J e a n Claxton for her capable assistance in performance of laboratory tests and Dr. R. H. Foote, Cornell University, for his assistance and critical reading of the manuscript. Appreciation is extended to the ~ a m i t t o n and Waterloo Cattle Breeding Associations for providing experimental animals and conducting field trials. Financial support was provided by the National Research Council of Canada and the Ontario Association of Animal Breeders.

References (1) Aalbers, J. C. ]966. The contributions of the epididymides and accessory glands to bull ejaculates a f t e r two-sided vasectomy or lacking glandulae vesicu]aries. Tijdschr. Diergeneesk, 91: 1745. (2) ]gennett, ]:I. S. 1963. Morphological aspects of extracellular polysaccharides. J. Histochem. Cytochem., 11: 14. (3) Dott, H. 1~[. 1961. The f e r t i l i t y of ram semen t r e a t e d in the continuous flow dialysis apparatus. Proc. I V t h Int. Congr. Anita. Reprod. (The Hague), IV: 873. (4) Faulkner, L. C., M. L. Hopwood, and J. N. Wiltbank. 1968. Seminal veslculeetomy in bulls. II. Seminal characteristics and breeding trials. J. Reprod. Fertility, 16: 179. (5) Fulka, J., H. Sulcova, and M. Valenta. 1964. P r o t e i n polymorphism of the seminal vesicles of bulls and the sensitivity of bull spermatozoa to cold shock. Blood groups of animals. Proe. 9th European Anim. Blood Group Conf., (Prague), 381. (6) Gelman. 1966. Advanced electrophoresis techniques for Scpraphore I2I cellulose polyacetate. Gelman I n s t r u m e n t Co., Ann Arbor, ~Iichigan. (7) Hackett, A. J., and J. W. Macpherson. 1965. A method for differential staining of bovine spermatozoa a f t e r extension in sterile milk. Canadian Vet. J., 6: 117. (8) Hale, E. ]9., and J. O. Ahnquist. 1960. Relation of sexual behavior to germ cell output in f a r m animals. J. Dairy Sci., 43 (Suppl.}: 145. (9) Hess, E. A., T. M. Ludwick, R. C. Martrig, and F. Ely. 1960. Influence of seminal vesiculectomy on certain physical and biochemical properties of bovine semen. J. Dairy Sci., 43: 256.

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(10) Hunter, A. G., and H. D. Hafs. 1964. Antigenicity and cross-reactions of bovine spermatozoa. J. Reprod. Fertility, 7: 357. (11) King, G. J., and J. W. Macpherson. 1968. The effects of seminal vesiculectomy on f e r t i l i t y of frozen bovine semen. Proc. V I t h Cong. Anita. Reprod. (Paris). I I : 1259. (12) MacMillan, K. L., C. Desjardins, K. T. Kirton and It. D. Hafs. 1967. Relationship of glycerylphosphorylcho]ine to other constituents of bull semen. J. Dairy Sci., 50: 1310. (13) Mann, T. 1964. The Biochemistry of Semen and of the Male Reproductive Tract. J o h n Wiley and Sons, Inc., New York. (14) Renkonen, O. 1962. Determination of glycerol in phosphatides. ]giochim. ]giophys. Acta, 56: 367. (15) Salisbury, G. W., and R. G. Cragle. 1956. Freezing point depression and mineral levels of fluids of the ruminant male reproductive tract. Proc. II][rd Int. Congr. Anita. Reprod. (Cambridge), I : 25. (16) Schales, O., and S. S. Schales. 1941. Simple and accurate method for determination of chloride in biological fluids. J. ]giol. Chem., 140 : 879. (17) Seidel, G. E., Jr., and R. H. Foote. 1969. Influence of semen collection techniques on composition of bull seminal plasma. J. Dairy Sci., 52: 1080. (18) Shah, ]9. A., M. L. Hopwood, and L. C. Fau]kner. 1968. Seminal vesiculeetomy in bulls. I. Seminal biochemistry. J. Reprod. Fertility, 16: 171. (19) Shamlon, P. 1965. Presence of a heat-labile toxic protein in bovine seminal plasma. J. Dairy Sci., 48: ]362. (20) Somogyi, M. 1945. Determination of blood sugar. J. Biol. Chem., 160: 69. (21) Steel, R. G. D., a~ld J. H. Torrie. 1960. The Princip]es and Procedures of Statistics. McGraw-Hill, Inc., Toronto, Canada. (22) Steinbach, J., and R. H. Foote. 1964. Post-thaw survival of bovine spermatozoa frozen by different methods in buffered-yolk and skimmilk extenders J. Dairy Sci., 47 : 909. (23) Szumowski, P., and M. Theret. 1967. Contribution a, ]'Etude biochimique des ]ecretions des differentes Glandes sexuelles du Tractus genital du Taureau. Ree. Mcd. Vet. Ec. Alfort, 143: 97. (24) Weichselbaum, T. E. 1946. An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Amer. J. Clin. Pathol., ]6: 40.