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Rapid Disappearance of Spermatozoa After Vasal Occlusion in the Dog

PHILIP S. LI^*,

PETER N. SCHLEGEL^*,

MARC GOLDSTEIN^*,

From the ^* Center for Male Reproductive Medicine and Microsurgery, the James Buchanan Brady Foundation Department of Urology and the Cornell Institute for Reproductive Medicine, Weill Cornell Medical Center, New York, New York; and Center for Biomedical Research, The Population Council, New York, New York.

Correspondence to: Dr Marc Goldstein, PO Box 580, Center for Male Reproductive Medicine and Microsurgery, Cornell Institute for Reproductive Medicine, 525 E. 68th Street, New York, NY 10021.

Received for publication October 11, 2002; accepted for publication November 18, 2002.

	Abstract

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The reproductive system of the male dog is unusual in that the seminal vesicles and bulbourethral glands are absent. Therefore, we chose the dog as a model to evaluate the effect of seminal vesicles on clearance of spermatozoa from the male reproductive tract after vasal occlusion. Thirty adult male beagle dogs with ejaculates containing at least 500 x 10⁶ sperm with greater than 90% motility were used for this study. The dogs' vasa were occluded percutaneously using a Vasocclude clip-applying device through a small scrotal puncture site. Dogs were ejaculated and semen analysis was performed before and after vas occlusion. The first 24 dogs were completely azoospermic 1 week following vas occlusion. In order to explain these unanticipated results, an additional 6 dogs were evaluated to determine the specific time course of sperm disappearance from the ejaculate at 1, 3, 5, and 7 days after vas occlusion. The results revealed that spermatozoa were almost completely absent within 1 day after vas occlusion (99.9% reduction, X = 1.0 + 1.1 x 10⁶ sperm per milliliter at 1 day, P < .0005 vs prevas occlusion). The rapid elimination of spermatozoa after vas occlusion indicates that sperm transit rapidly through the vas deferens in dogs. Therefore, the delayed clearance of spermatozoa from the ejaculate in humans may be due to sperm storage in the seminal vesicles.

     Key words: Vasectomy, sperm clearance, canine

Postvasectomy studies have been conducted extensively in humans. Although the time to achievement of azoospermia postvasectomy in humans is highly variable, studies have suggested that clearance of spermatozoa from the ejaculate may take 4 weeks to 6 months (Freund and Davies, 1969; Marshall and Lyon, 1972; Jouannent and David, 1978; Smith, et al 1998; Goldstein, 2002). These studies have also suggested that anywhere from 6 to more than 20 ejaculations are needed after vasectomy in order to clear the human reproductive tract of spermatozoa.

The time to achieve spermatozoal clearance from the reproductive tract in dogs after vasal occlusion is unknown. Because dogs lack seminal vesicles, there is not likely to be any reservoir to store sperm between the remaining vas deferens and the ejaculatory duct. The purpose of the present study was to determine the specific time course for development of azoospermia following bilateral vasal occlusion.

	Material and Methods

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Animals

Institutional animal care and use committee approval of this study was obtained before experiments were conducted. All animals were handled in accordance with United States Department of Agriculture standards. Thirty adult male beagle dogs weighing 12–16 kg were obtained from White Eagle Laboratories (Doylestown, PA). All 30 dogs had been successfully ejaculated with the aid of a teaser or manual stimulation on at least 2 occasions before vasal occlusion. Sperm concentrations were at least 500 x 10⁶ sperm per milliliter, with >90% sperm motility, progressive motility of 3 (0–4) and a volume of >3 mL for each ejaculation.

Vasal Occlusion

All surgical procedures were performed under thiamylal sodium i.v. anesthesia (8 mg/pound of body weight). Through a small scrotal puncture site under aseptic conditions, bilateral vasal occlusion was performed by using the Vasocclude (Vastech, Inc, New York NY) clip-applying device. This device is a modified clip-applier that places a straight, noncrushing medium-sized titanium clip across the vas. The vas deferens was not divided.

Semen Collection and Evaluation

Semen was collected 1, 2, 4, 8, 12, 24, and 36 weeks after vasal occlusion. A separate group of 6 dogs were ejaculated 1, 3, 5, and 7 days following vasal occlusion. Whole semen from each dog was centrifuged at 3000 x g for 10 minutes and the pellet was examined using a phase-contrast microscope at 200x for sperm. Sperm concentration was determined using a standard hemocytometer counting chamber. Semen volume, pH, color, and percent and progression of motility were recorded, and 1992 World Health Organization criteria were used for grading purposes.

Statistical Analysis

The Student's paired t-test was employed to determine significance of results (P values). A P value of < .05 was considered significant. All data were expressed as ± SD.

	Results

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Semen analyses were performed before and after vasal occlusion in each of 30 dogs. Average ejaculate volume was 4.0 (±1.4) mL per animal with a pH of 6.5. These values did not change (Table 1) from preocclusion levels at any time point measured during the study. The mean sperm count before vasal occlusion was 513 (±165) million/mL, with 91% (± 7%) motility and 3% (±1%) progression. The first 24 dogs were completely azoospermic by 1 week following vas occlusion (Table 1), and remained so for the duration of our study.

In order to explain these unexpected results, we then studied 6 additional dogs to determine the specific time course of sperm disappearance from the ejaculate. The average ejaculate volume was also 4.0 (±1.4) mL per animal, with 510 (±165) million sperm/mL, 90% (±7%) motility, and 3% (±1%) progression (Table 2). Semen analysis 1 day after vas occlusion revealed that spermatozoa were almost completely absent (99.9% decrease, X 1.0 + 1.1 x 10⁶ sperm/mL at 1 day, P < .0005 vs before vasal occlusion). By 3 days after vasal occlusion and thereafter, all dogs were azoospermic (Table 2).

	Discussion

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Although approximately 500 000 North American men undergo vasectomy each year, the minimum criteria for declaring that a vasectomized man is sterile have been debated for years. The disappearance of sperm from the ejaculate usually requires 6 to 20 ejaculations before complete clearance of the human reproductive tract. Eighty percent to 90% of men will be azoospermic following 12 to 15 ejaculations (Marshall and Lyon, 1972; Jouannent and David 1978). However, 80% of men will be azoospermic 6 weeks after vasectomy regardless of ejaculatory frequency (Goldstein, 2002).

Both the seminal vesicles and the ampulla of the vas may function as sperm reservoirs after vasectomy. Freund and Davies (1969) observed a rapid decrease in the number of spermatozoa during the first 10 ejaculations after vasectomy in 13 men. They assumed that most of the spermatozoa stored above the vasectomized site were released during the first ejaculations following vasectomy (Freund and Davies, 1969; Marshall and Lyon, 1972; Jouannent and David, 1978). Other studies have found that clearance of spermatozoa from the human ejaculate can take up to 6 months after vasectomy (Smith et al, 1998). However, to our knowledge, there are no data on the specific time course of spermatozoa clearance from the male reproductive tract and development of azoospermia following bilateral vasectomy in species other than humans.

The dog, as a common experimental animal, has been widely used for studies of vasal morphology and immunologic alteration after vasectomy. However, little attention has been paid to the time of onset of azoospermia after vasectomy. This study revealed that dogs become azoospermic quite rapidly after vasal occlusion. Initially, we expected that at least 1 week, and likely several weeks, would be required for the onset of azoospermia in our dogs following vas occlusion. Our initial observations indicated that sperm disappearance from the ejaculate of dogs following vas ligation is much faster than we anticipated, with complete azoospermia achieved by 1 week after vas ligation.

Our follow-on study examined vas-occluded dogs at earlier time points in order to more precisely define the time course of the onset of azoospermia in dogs after vas ligation. We found that 1 day after vas ligation, our dogs were severely oligospermic, with counts of 1.1 million per cc, and that by 3 days after the procedure, all dogs were completely azoospermic.

The disappearance of spermatozoa from the dogs' ejaculate after vas occlusion was more rapid than predicted, and contrasts markedly with that of humans. Between the site of vas occlusion and the ejaculatory duct in dogs there are no reservoirs for the storage of sperm. In contrast, human sperm may be stored in the ampulla of the vas deferens as well as the seminal vesicle. Our findings suggest that the absence of seminal vesicles in dogs and the rapid clearance of germ cells through its reproductive tract following vasectomy support the hypothesis that the seminal vesicles are an extraepididymal site for storage of spermatozoa.

However, attempts to mechanically clear sperm from the distal vas deferens and seminal vesicles in humans have been unsuccessful to date. Two recent prospective trials examined whether irrigating the vas distal to the site of vasectomy would speed the clearance of sperm from the postvasectomy ejaculate (Leungwattanakij et al, 2001; Mason et al, 2002). Neither study found a statistically significant advantage to irrigating the distal vas with saline in terms of time to clearance of sperm from the ejaculate. One of the studies examined the postprocedure urine for spermatozoa and found that those subjects who underwent irrigation had significantly more sperm in their spun urine (Leungwattanakij et al, 2001). This finding indicates that irrigation does clear some sperm from the distal vas, but that there must be other reservoirs for the spermatozoa that were not irrigated.

We believe that these observations, taken with those of our study, suggest that the seminal vesicle is the other reservoir for spermatozoa. Irrigation of the distal vas would not effectively clear the seminal vesicle because of the angle of the seminal vesicle off the ejaculatory duct. This acute angle would make resistance to the flow of irrigant greater than simply flowing straight out of the ejaculatory duct.

Therefore, simply irrigating the distal vas will not effectively clear the seminal vesicle of sperm. We believe that only a chemical agent that achieves high levels in the seminal vesicle and is spermatotoxic will help to clear potentially live sperm from the ejaculate after vasectomy. Prior work has demonstrated that methylene blue is highly toxic to sperm almost instantaneously (Sheynkin et al, 1999). Perhaps instillation of a chemical irrigant would hasten clearance of viable sperm from the ejaculate after vasectomy.

	Acknowledgments

 
We thank the technical staff at White Eagle Laboratories (Doylestown, PA) for their assistance in collection and analysis of dog semen as for animal care for this study.

	Footnotes

 
Supported by grant DPE3050-A-00-8059-00 from the U.S. Agency for International Development.

	References

Top Abstract Material and Methods Results Discussion References

 
Freund M, Davies J. Disappearance rate of spermatozoa from the ejaculate following vasectomy. Fertil Steril.1969; 20:163 –165.[Medline]

Goldstein M. Surgical management of male infertility and other scrotal disorders. In: Walsh P, Retik A, Vaughan ED, Wein A, eds. Campbell's Urology. 8th ed. New York: WB Saunders;2002 : 1532–1588.

Hart BL. Male reproductive system. The beagle (dog)—as an experimental dog. Ames, Iowa: The Iowa State University Press;1970 .

Jouannent P, David G. Evolution of the properties of semen immediately following vasectomy. Fertil Steril.1978; 29:435 –428.[Medline]

Leungwattanakij S, Lertsuwannaroj A, Ratana-Olarn K. Irrigation of the distal vas deferens during vasectomy: does it accelerate the post-vasectomy sperm-free rate? Int J Androl.2001; 24:241 –245.[Medline]

Marshall S, Lyon R. Variability of sperm Disappearance from the ejaculate after vasectomy. J Urol.1972; 107:815 –818.[Medline]

Mason R, Dodds L, Swami S. Sterile water irrigation of the distal vas deferens at vasectomy: does it accelerate clearance of sperm? A prospective randomized trial. Urology.2002; 59:424 –427.[Medline]

Sheynkin Y, Starr C, Li P, Goldstein M. Effect of methylene blue, indigo carmine, and renografin on human sperm motility. Urology. 1999;53:214 –217.[Medline]

Smith AG, Crooks J, Singh NP, Scott R, Lloyd SN. Is the timing of postvasectomy seminal analysis important? Br J Urol.1998; 81:458 –461.[Medline]

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