Method for the In Vivo Collection of Epididymal Spermatozoa and for Their Comparison With Ejaculated Cells

Transcription

1 Method for the In Vivo Collection of Epididymal Spermatozoa and for Their Comparison With Ejaculated Cells I. G. White, B.Sc., Ph.D., L. H. larsen, B.V.Sc., M.S., and R. G. Wales, B.V.Sc. HAMMOND AND ASDELL have shown that rabbit spermatozoa are fertile after 40 days in the ligated epididymis at a temperature only slightly below that of the body. This is better than has been achieved for ejaculated semen in any diluent at temperatures above freezing point, and along with other morphological and biochemical evidence 9 suggests there may be important differences in the properties of epididymal and ejaculated spermatozoa. Hitherto, one of the difficulties in the study of epididymal semen has been the necessity of killing animals in order to collect material by the methods of Czarnetzky and Herle or Lasley and Bogart. Thus it has been impossible to make repeated observations on the epididymal semen of an individual animal, and difficult to compare epididymal and ejaculated spermatozoa fi'om the one animal. It occurred to the authors tfat the problem might be approached by establishing a fistula into the vas deferens of one testis, so that on electrical stimulation ejaculated spermatozoa could be obtained from the penis and epididymal spermatozoa from the fistula. The ram was chosen as the experimental subject as it is particularly well suited for testicular surgery. From the Department of Veterinary Physiology (J.G.W. and RG.W.), and the Department of Veterinary Surgery (L.H.L.), University of Sydney, Sydney, Australia. This work has been aided by grants from the Nuffield Foundation (RG.W.), the Rural Credits Development Fund of the Commonwealth Bank of Australia, and the Planned Parenthood Federation of America (J.G.W. and R.G.W.). The authors are indebted to Professor C. W. Emmens for his interest and advice. 571

2 572 WHITE ET At. Fertility & Sterility DUCTUS DEFERENS FISTULA Anatomical Considerations The ductus deferens of the ram and of other ruminants is small in caliber and has a thin wall. It pursues a course from the tail of the epididymis along the posteromedial border of the testicle to the inguinal canal in a relatively constant relation to the other structures of the spermatic cord. It is surrounded by a serosal layer of epithelium that is a reflection of the mesorchium. The well-developed muscular fibers constitute the greater part of the duct wall, and except under conditions of active sperm movement, virtually obliterate the lumen. The lumen is lined by simple columnar or pseudostratified epithelium, and while it is capable of considerable dilatation it is very difficult to identify in the normal contracted state. Several tortuous vessels run the entire length of the duct in the subserosallayer. In the ram they form two quite distinct groups on either side of the duct, thus presenting two zones of minimal blood supply where access to the lumen affords least interference in blood flow. Breed differences in the length of the spermatic cord and the amount of proximal scrotal adipose tissue are sufficient to affect the continued patency of the fistulas. The long pendulous scrotum of the Merino, with a minimal accumulation of adipose tissue, permitted an easier fistula development than did that of the Dorset Horn, where the opposite tendency was observed. Surgical Technique Each animal was fistulated in the right ductus deferens, leaving the left duct intact. The site found most satisfactory was approximately midway between the dorsum of the scrotum and the inguinal canal. If the fistulas were established close to the abdominal wall the collection of semen was difficult; fistulas established immediately dorsal to the testes were subject to increased tension due to the marked contraction of the external cremaster muscle during ejaculation. The well developed spermatic cord in this area also increased the difficulty of access at operation. General anesthesia throughout the operation was necessary to control movement and to prevent contraction of the external cremaster muscle during the exposure of the duct. Intermittent intravenous injections of a shortacting barbiturate were preferable to closed-circuit gas techniques with

3 Vol. 10, No.6, 1959 IN VIVO COLLECTION OF EPIDIDYMAL SPERMATOZOA 573 cyclopropane or nitrous oxide, because the barbiturate depressed respiration and provided a more stationary operative field. Strict asepsis was maintained during the operation, because localized Corynebacterium ovis infections had occurred in previous surgical procedures involving cutaneous fistulas in sheep. A half-inch incision was made on the anterior aspect of the neck of the scrotum, immediately lateral to the midline. The anterior approach was preferred to a posterior one since it gave greater protection to the fistula. The spermatic cord was then rotated anterolaterally and held by an assistant in this position, thus locating the vas deferens immediately beneath the original incision. In some rams considerable adipose tissue had to be dissected from the subcutaneous area to expose the spermatic cord. A half-inch incision was then made in the parietal tunica vaginalis immediately below the cutaneous incision. Great care was necessary to avoid damage to the highly vascular spermatic cord immediately below this layer. This incision was then extended at its ventral margin to allow greater mobility of the exteriorized vas deferens, during the marked contraction of the external cremaster muscle following electrical stimulation. With a blunt aneurysm needle the vas deferens was drawn through the incision and temporarily secured by two probes passed through the reflected mesorchium. The establishment of a patent fistula was found to be largely dependent on the next phase, the successful exposure of the duct with a minimum of tissue damage. Several techniques were tried and modified until the following procedure was found to give complete and most efficient exposure. With a fine Bard Parker scalpel, a quarter-inch incision was made in the relatively nonvascular part of the vas deferens, exactly parallel to its course and reaching a depth of approximately half the muscle layer. An assistant then gently massaged the tail and body of the epididymis in a proximal direction, causing a flow of epididymal spermatozoa up the duct. The semen distended the duct at this point and outlined its lumen immediately below the incision. Very gentle stroking with the scalpel at the base of the incision permitted exposure of the lumen. The lumen was then extended proximally, to produce a fistula half an inch in length, by inserting a small ophthalmic probe into the duct and incising down onto it with the scalpel. The seromuscular walls of the fistulated duct were secured to the skin by interrupted "arterial" silk sutures, three or four being inserted on each side, with a round pointed needle. The remainder of the original skin

4 574 WHITE ET AL. Fertility & Sterility incision was then closed. Exposure of half an inch of the duct permitted sufficient sutures to be inserted to distribute any tension on the duct wall. A complete transection of the vas deferens and exposure of the distal end at the cutaneous site was unsatisfactory. Other methods with probes, needles, and injected fluids to identify the contracted duct, were also less successful. After-care consisted of the application of "Chlorocort" (Parke-Davis) ointment twice daily. This is a combination of the broad spectrum antibiotic chloramphenicol and cortisone acetate. The local application of a cortical steroid was made in an effort to reduce the inflammatory response in the tissues around the fistulas. Dusting powders were avoided and any surface protective coverings were found unsatisfactory because of the excessive skin secretions in this area. Despite the application of cortisone, the inflammatory reaction in the area was sufficient to prevent secretion from the fistula during the first four or six days. After ten to fourteen days, patency of the duct was well established in all cases, although earlier collections were made in some cases when the sutures were removed at the seventh to eighth day. The continued patency of the duct was apparent at each collection by the accumulation of coagulated semen around the orifice. In the earlier cases penicillin and streptomycin were given parenterally for the first three days; they were not used in the last seven cases.. COLLECTION AND EVALUATION OF SEMEN The area around the fistula was swabbed clean with ether and allowed to dry. After the application of pressure to the tail of the epididymis and the beginning of the vas deferens in a proximal direction, epididymal semen collected at the fistula and was sucked up into a 0.5 ml. opsonic pipet. A bipolar electrode2 was then inserted into the rectum, and on stimulation ejaculated semen was collected from the penis into a graduated centrifuge tube; at the same time more epididymal semen was obtained from the fistula. Sperm motility was scored, by the system of Emmens, with a warm stage and a low power microscope. Full motility was scored as 4 and complete immotility as O. Since the motility of epididymal spermatozoa falls off under anaerobic conditions in the absence of glycolysable sugar,lo the semen was examined routinely either after mixing with a small volume of a diluent (0.132 M NaCI, M KCI, M KH2P04, M MgS04.- 7H20, M Na2HP04) containing 100 mg./100 cc. fructose, or after it had been well aerated.

5 Vol. 10, No.6, 1959 IN VIVO COLLECTION OF EPIDIDYMAL SPERMATOZOA 575 The total sperm counts were made with a hemocytometer, the semen being diluted with 3 per cent sodium chloride. A mixture of 2 per cent congo red and 5 per cent nigrosin in 2.6 per cent sodium citrate dihydrate was used to assess the percentage of live spermatozoa.a One drop of semen was mixed with two drops of stain, and duplicate smears were made after 5 minutes. One hundred spermatozoa were counted on each smear and the mean percentage of unstained spermatozoa was used as unit observation. The percentage of attached kinoplasmic droplets (A.K.D.) was determined from a mixture of 1 drop of semen with 5 m!. of diluent containing 1 drop of neutral formalin. This suspension was mounted on a slide under a cover slip and examined through the phase-contrast microscope. The percentage of spermatozoa was attached kinoplasmic droplets was estimated by examining 100 spermatozoa. RESULTS AND DISCUSSION Fistulas were established into one testis of 16 mature rams (13 Merinos, 1 Border Leicester, and 2 Southdown crosses). A week after the operation, semen was obtained from the fistula of 13 animals and further collections were made over the period shown in Table 1. The fistulas have remained patent for periods of from 1 month to 2 years (Table 1). Two rams developed an abscess at the site of the fistula; one of them completely recovered after surgery and treatment with antibiotics, but the other developed an acute orchitis and was destroyed. Table 2 shows some of the characteristics of the epididymal and ejaculated semen collected from the rams. Both types of semen are clearly of high quality as judged by motility, total sperm counts, and the percentage of live cells. The sperm count of the ejaculated semen, of course, represents the spermatazoa from one testis only, mixed with the total secretions from the accessory organs. The volume of epididymal semen obtained is small but very concentrated and has a higher percentage of attached kinoplasmic drops than does ejaculated semen. TABLE 1. Period During Which the Vas Deferens Fistulas Have Remained Patent No. of rams Months patent 25 (still patent) 7 (still patent) 3 1

6 576 WHITE ET AL. Fertility & Sterility TABLE 2. Observations of Epididymal and Ejaculated Semen Collected From 13 Rams with Fistulas Into 1 Vas Deferens Ejac. Epid. No. of paired observations Mean S.D. Mean S.D. Volume (ml.) Motility Spenn count (106 Iml.) % alive % attached kinoplasmic droplets The question of whether spermatozoa are motile or not in the epididymis of the living animal seems to be controversia1. 10 When immediately examined under the microscope, spermatozoa from the fistula showed good motility, even when precautions were taken to exclude air by collecting into a capillary tube under paraffin. This suggests that ram spermatozoa may be motile in the epididymis. Further evidence on this point was obtained by anesthetizing 3 rams with thiopentone sodium B.P., isolating the vas deferens, and exposing the duct under several centimeters of gas-free paraffin. Epididymal semen was squeezed out and drawn into a capillary tube that had been sealed with paraffin and plasticine. When examined immediately under the microscope the spermatozoa appeared quite motile. Mter some minutes the motility declined, but was fully restored at the air interface when the capillary tube was broken. The motility of epididymal spermatozoa would seem, therefore, to be very much dependent upon oxygen tension. Although Bishop and Matthews apparently have evidence that the oxygen tension may be low in the vas deferens of some animals, the epididymis of the ram is a highly vascular organ, and one might expect the oxygen tension in the living animal to be sufficient to support motility. After death the oxygen tension would fall rapidly, so that the use of excised organs or postmortem material may account for the widespread belief that spermatozoa are immotile in the epididymis. The technique described in this paper makes a supply of epididymal semen readily available, and would seem to be a promising one for the critical comparison of epididymal and ejaculated spermatozoa. The only previous attempts to obtain semen from the vas deferens in vivo seem to be those of Gunn and Walton and Rowson (unpublished data, quoted by Walton), who also used the ram as the experimental subject. Neither

7 Vol. 10, No.6, 1959 IN VIVO COLLECTION OF EPIDIDYMAL SPERMATOZOA 577 author gives any details of the technique but the latter workers established an artificial fistula of silicone rubber between the vas deferens and the surface of the scrotum. In both cases active spermatozoa were obtained from the orifice of the fistula, but it only remained patent for a few weeks. SUMMARY A technique is described for establishing in the ram a unilateral vas deferens fistula, so that on electrical stimulation, both epididymal and ejaculated spermatozoa can be obtained and compared. REFERENCES l. BISHOP, D. W., and MATTHEWS, H. P. Inhibition of sperm motility by tetrazolium salts. Science 115:208, BLACKSHAW, A. W. A bipolar rectal electrode for the electrical production of ejaculation in sheep. Australian Vet. ]. 30:249, BLACKSHAW, A. W. The effects of glycerol on the supra-vital staining of spermatozoa. Australian Vet. ]. 34:71, CZARNETZKY, E. J., and HERLE, W. A pressure device for the separation of mammalian spermatozoa from the isolated epididymis. Proc. Soc. Exper. Biol. & Med. 38:63, EMMENS, C. W. The motility and viability of rabbit spermatozoa at different hydrogen-ion concentrations. J. Physiol. 106:471, GUNN, R. M. C. Fertility in Sheep. Council for Sc. & Indust. Res. Bull., No. 94, HAMMOND, J., and ASDELL, S. A. The vitality of the spermatozoa in the male and female reproductive tracts. ]. Exper. Biol. 4: 155, LASLEY, J. F., and BOGART, R. A comparative study of epididymal and ejaculated spermatozoa of the boar. ]. Anim. Sci. 3:360, MANN, T. The Biochemistry of Semen. London, Methuen, WALTON, A. The initiation of motility in mammalian spermatozoa. Studies on Fertility 8:53, 1956.