C ONSIDERING THE SPECTRUMS of mammalian species in which semen collection

Transcription

1 The Collection of Primate Semen by Electro-ejaculation STEVEN WEISBROTH, D.V.M., and FRANCIS A. YOUNG, Ph.D. C ONSIDERING THE SPECTRUMS of mammalian species in which semen collection has been done, only a comparatively few technics have emerged. Electro-ejaculation has been proved successful in the bull, dog, goat, sheep, fox, and guinea pig, to mention a few. Masturbation has been used as a routine method in the dog, collection of the vaginal-dismount semen is done routinely with stallions (and has been used in primates), and the use of the artificial vagina employing teaser cows, steers, or wooden dummies, has proved practicable with the bull. For various reasons, mostly involving the handling problem, electro-ejaculation would be the method of choice in the monkey. Mastroianni and Manson described the first account of electro-ejaculation in the monkey.4 Their technic employs the attachment of both electrodes to the skin of the penis. The technic described in this report is modeled after that used in domestic ruminants and employs a rectal probe containing both electrodes. This represents a simpler method and perhaps inflicts less trauma on the subject. APPARATUS AND METHODS The electro-ejaculator is composed of two main components, the power supply and the probe. The power supply used is presented diagrammatically in Figure 1. The voltage peaks could be varied from zero to, in the following maximums: 1.5, 2.,.45, 7.4, and 9.1 by the operator. In the probe shown in Fig. 1, the copper-band contacts 1 and, and 2 and 4 are connected together, each pair separate from the other. K-Y Jelli* or surgical liquid soap may be used for lubrication of the probe. The diameter o~ inch) was useable for animals weighing 7-1 kg., but neither limit was fixed. Operating conditions indicated that the probe should have had all rings grouped within a 2 -in. span. From the Berg Institute, New York University Medical Center, New York, N. Y., and the Primate Research Center, Washington State University, Pullman, Wash. *Johnson & Johnson, New Brunswick, N. J. 229

2 2 WEISBROTH & YOUNG FERTILITY & STERILITY Four Macaca nemestrina, two Macaca mulatta, one Macaca fuscata, and two Cynopithecus niger sexually mature males with varying degrees of sexual experience were used. These monkeys varied in weight between 7 and 1kg. STANCOR P-184- TRANSFORMER 1.5 1~ 1 w IIOVIN ---s; o------=e~k Fig. 1. Electrical supply and probe. At first, ejaculation was accomplished without narcosis in order to establish a base line for success or failure in future sedation trials. Judged by behavioral responses, electro-ejaculation is an unpleasant sensation to the monkey because in all cases where this was done in an unanesthetized animal, it was accompanied by efforts to escape the treatment. As a means of both avoiding the task of physically restraining the animal, and making the experience less meaningful to the subject, the technic of ejaculation under narcosis was used. 2 For the purpose of inducing narcosis, we used Semyl ( Sernylan)"' at a dosage schedule of 1 mg. per pound. Semyl is an intramuscular tranquilizer which, at the dosage employed, was sufficient to render the animal incapable of voluntary motor activity, depressed neither respiration nor cardiac rate, and gave a 45- to 9-min. working period before gradual return to consciousness.6 The monkey was held by two assistants in a dorso-ventral, horizontal plane with the thorax supported on a box (e.g., apple box) resting on a table, and with the hind legs stretched caudad. The probe was suitably lubricated and inserted into the rectum. A third assistant was ready with a *Parke, Davis & Company, Detroit, Mich.

3 VoL. 16,No.2,1965 PRIMATE SEMEN COLLECTION 21 container in which to collect the semen when ejaculation was imminent. The operator selected a low voltage output with which to begin stimulation, and with one hand rotated the autotransformer from to peak in a fairly rapid, rhythmic manner. Contact was noted by spasmodic contractions of the monkey's pelvis and thighs. The operators continued thus at the low setting (we used the 2. v setting as a starting point) for a few moments, then stopped stimulation for about a minute. The voltage was increased to the next setting (.45 v) and the procedure repeated. This stepwise increase in voltage with intervening rest periods seemed to give the most consistent results. In most cases the voltage output was on the 7.4 setting before ejaculation occurred. Ordinarily it took ~1 min., in total, for ejaculation to occur. With the other hand, the operator manipulated the direction of the probe, if necessary, within the rectum. For the various examinations of semen quality, specimens were collected in graduated centrifuge tubes so that volume could be approximated. Neubauer counting chambers were used for sperm counts, determined by the undiluted liquid fraction. The chambers were pre-refrigerated to inhibit sperm motility. The proportion of live versus dead sperm in the ejaculate was computed as a per cent by means of the live-dead staining method of Shaffer and Almquist, as given by Herman and Madden. RESULTS In all cases, some degree of erection was noted prior to ejaculation, and seemed to vary from a feeble attempt to full erection. In most cases, full erection was noted just before ejaculation. Semen volume seemed unrelated to the degree of erection. Well-defined semen fraction sequences such as are observed in semen collection by artificial vagina in other species, i.e., seminal fluid preceding a milky sperm-rich portion, was not noted with this procedure. Coagulum was noted on different occasions to come both before and after the sperm-rich liquid fraction. Once the first ejaculation was obtained, two more could be easily obtained in rapid succession with brief ( 1- min.) intervening rest periods. No attempt was made to determine the maximum possible ejaculations during a given session. In every individual attempted, an ejaculation was obtained. The physical characteristics are as follows: Two fractions are obtained in every ejaculation, a fibrinous coagulum and a sperm-bearing liquid fraction. These two fractions are of variable proportions. Most often the coagulum makes up -5% of the total volume, but can vary from to almost 1%. Sections of the fibrinous coagulum reveal it to be almost free of sperm, and, indeed (Table 1), in all cases where the coagulum formed an extensive proportion

4 22 WEISBROTH & YOUNG FERTILITY & STERILITY of the total volume, sperm were of low density. Varizyme"" (streptokinasehuman plasminogen-streptodoronase preparation) was used in an unsuccessful attempt to liquefy the coagulum. The coagulum forms when the secretion of the cranial lobe of the prostate joins the other seminal fluids and occurs within the urethra, before exposure to ai:r. Three variables were considered on a gross level as screening criteria. For descriptive purposes the proportion of coagulum was roughly quantified as follows: small, -4% of total volume; moderate, 4-6% of total volume; and extensive, 6-1% of total volume. Volume was quantified in a similar manner: small, 1 cc. or less; moderate, 1-2 cc.; and large, 2 cc. or more. Sperm density was screened into two categories: sperm-rich, many sperm per microscope field; and sperm-poor, sperm absent or few per microscope field. The results for the entire group, by these criteria, are summarized in Tables 1 and 2. One of the monkeys, pig-tailed macaque No. 78, was examined in further detail. Ejaculations were obtained 1 times over a period of about 5 weeks. Data were kept on 4 of these, including total volume, proportion of coagulum, sperm count, and live-dead ratio. In all ejaculations, the motility was noted, and there was good forward progression. The per cent of motility usually reflected the live-dead per cent and was considered less reliable since more interpretation was required; hence this information was not kept. Table shows the boost in semen volume and sperm count the week after this monkey was removed from a breeding cage to a solitary cage. DISCUSSION Electro-ejaculation of male monkeys has been shown to be a reliable and practical method for obtaining semen in sufficient quantity for quality evaluation and, potentially, for use in a program of artificial insemination. Nalbandov states that in males of high ability at natural fertilization, a total of 5-15 million sperm are required for insemination. Although the minimum number has not been calculated in monkeys, No. 78, for example, would have no difficulty in frequently providing sperm well in excess of that number. The fibrinous coagulum occurring in ejaculations poses no problem to insemination technics. Histologic section did not reveal sperm in the coagulum. One could solve the problem by simply removing it from the collection vial and discarding it. Several factors influence the extent of coagulum. The operator's technique in procuring ejaculation, including the directional *American Cyanamid Company, Wayne, N. J.

5 VoL. 16,No.2,1965 PRIMATE SEMEN CoLLECTION 2 TABLE 1. Screening Criteria for Individual Subjects Examined Times Amount of Species No. ejaculated coagulum Total volume Sperm density Pig-tailed 78 1 Moderate Large Rich Pig-tailed 851 Small Moderate Rich Pig-tailed 85 Small Small Rich Pig-tailed 85 Extensive Large Poor Rhesus P486 Extensive Large Poor Rhesus 72 Moderate Large Rich Japanese Moderate Small Rich Celibes 51 Extensive Large Poor Celibes 57 Small Large Rich manipulation of the probe within the rectum, was subjectively estimated to have varied the amount of coagulum. No attempt was made, nor can data be presented, to substantiate this hypothesis. Secondly, many workers experienced in collecting bovine ejaculations have observed that semen quality, in all particulars, increases in a bull that is regularly subjected to electroejaculation, whereas bulls subjected to this procedure at very infrequent intervals have semen that is less reliable for use in estimating true semen quality. Freund has reported that the amount of coagulum in guinea pig ejaculations, where the problem is identical, decreases in male pigs from which ejaculations are obtained at biweekly intervals, to a point where coagulum does not appear at all. These observations seem to offer hope of procuring semen samples of high sperm density, large volume, and small amounts of coagulum. The routine ejaculation of the same individual over a period of time may show great increases in quality. A trend in this direction can be noted in pig-tailed monkey No. 78. Individual variation is apparently a third factor influencing the amount of coagulum, since some mon- TABLE 2. Analysis of Ejaculate Variables Sperm density Variable Criter,ia No. o/o No. Rich o/o No. Poor o/o Sperm density Coagulum Total volume Rich 6 Poor Extensive Moderate Small Large 6 Moderate 1 Small

6 24 WEISBROTH & YOUNG FERTILITY & STERILITY TABLE. Summary of Semen Quality, Subject No. 78 Sperm Session Physiologic Total Amount of count Live-dead Pre-treat. No. state volume coagulum mil/cc stain with Sernyl 2 With female 2. Extensive % live (-) With female 2.2 Moderate 8. 98% live (+) 4 With female 1.8 Moderate 6. 97% live (+) 5 Alone 2.6 Moderate 2. 99% live (+) keys seem to have more coagulum per ejaculation than others. Of the three mentioned variables in extent of coagulum, the most promising area for investigation appears to be a means for increasing quality as a function of time and frequency of ejaculation, rather than attempts to find coagulumlow males by screening procedures or efforts to refine technics to avoid coagulum. CONCLUSIONS 1. Electro-ejaculation by means of rectal probe electrodes was demonstrated to be a practical method of semen collection in four species of infrahuman primates. 2. Screening criteria were established for use in evaluating semen quality in large numbers of semen samples.. Sperm counts were shown to be of more than sufficient concentration for use in artificial insemination. 4. The fibrinous coagulum noted in most ejaculations was found to be devoid of sperm. 5. Varizyme was found to be ineffectual as a means of coagulum liquefaction. 6. Sperm density was found to be highest in samples with a small amount of coagulum and least in samples with large amounts of coagulum. 7. Ejaculation under Sernylan narcosis was found to be indistinguishable from. that in the wakeful subject, and, in addition, was accomplished with less physical and emotional trauma to the subject and the operators. Primate Research Center Washington State University Pullman, Wash. REFERENCES l. FREUND, M. Collection and liquification of guinea pig semen. Proc. Soc. Exper. Biol. & Med. 98:58, 1958.

7 ,,I II,, VoL. 16, No.2, 1965 PRIMATE SEMEN CoLLECTION HERRICK, J. B. An aid to collection of semen by electro-ejaculation. Chlorpromazine hydrochloride. Vet. Med. 5:41, HERMAN and MADDEN. Artificial Insemination of Dairy Cattle. Lucas Bros., Columbia, Mo., 1919, p MASTROIANNI, L., }R., and MANSON, W. A. Collection of monkey semen by electroejaculation. Proc. Soc. Exper. Biol. & Med. 112:125, NALBANDOV, A. V. Reproductive Physiology. Comparative Reproductive Physiology of Domestic Animals, Laboratory Animals and Man. Freeman, San Francisco, YouNG, F. A. Refraction of the monkey eye under general anesthesia. Vision Res. :1, 196. Journal of the International Federation of Gynaecology and Obstetrics The International Federation of Gynaecology and Obstetrics, started publication of its own periodical in 196. Editors of the journal are Prof. G. Tesauro, Naples, Italy; Prof. W. C. W. Nixon, London, England, and Prof. H. de Watteville, Geneva, Switzerland. Dr. John I. Brewer, of Chicago, is a member of the Advisory Editorial Committee. Yearly subscription is $2.5. A few copies of Vol. 1 ( 196) are still available at the regular price of $2.5. Correspondence should be addressed to: Prof. G. Tesauro, Editor-in Chief, ViaS. Andrea delle Dame 16, Naples, Italy.