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Mating behavior, serum testosterone and semen characteristics in vasectomized and short scrotum rams
THERIOGENOLOGY
MATING BEHAVIOR, SERUM T~S?~STER~NF AND SEMEN CHA~~T~R?ST~CS IN VASECTOMIZED AN? SHORT SCROTUM RAMS a,b L. A. Tierney and D. M. Hallford
Department of Animal and Range Sciences College of Agriculture ard Home Economics New Mexico State University Las Cruces, New Mexico 88003 Received for publication: September Accepted: December
1, 1984 27, 1984
ABSTRACT Ter similar rams were either vasectomized (VAS) or altered by securing the testes near the abdomen using elastrator bands (short scrotum, SS). Semen characteristics (electroejaculation) were similar (P>O.lO) between groups before treatment and ejaculate volumes remained relatively constant (P>O.?o) throughout the trial. Remaining sperm cells were nonmotile and 95% abnormal by 6 days after vasectomy. By 12 days after surgery, ejaculates contained no cells. Two SS rams had detectable sperm numbers at 12 days after treatment, but 95% were abnormal and all but 1% were nonmotile. Ejaculates collected 3 months after surgery were similar between treatments, in that all existing cells were nonmotile and abnormal. Partial spermatngenic function was regained in SS rsms 15 months after treatment as indicated by the presence of low concentrations of motile sperm. Three and 15 months after treatment , sexual activity was estinated hy exposing rams to a group of four estrous ewes. During the 15-minute exposure, VAS males exhibited more (P
vasectomy, short scrntum, semen, libido, testosterone
iJournal Article IORO of the New Mexico Agricultural Experiment Station. The authors appreciate the help of C. Crusberg, D. Morrical, R. Hudgens, M. Scboenemann, S. Mischler, D. Sxnson, M. Neave and P. Ross. We also thank Dr. G. D. Niswender (Dept. of Physiol. and Biophys., Colorado State Univ., Fort Collins, CO) for the supply of testosterone-11 BSA antiserum (GDN 250) used in testosterone determinations. Direct reprint requests to D. M. Hallford.
MARCH
1985VOL. 23N0.3
535
THERIOGENOLOGY
INTRODUCTION In most domestic species, reliable estrous detection records are difficult to obtain without using intact or sterile males or andrcgenized females. In addition, Restall (1) observed an increased lambing response in artificially inseminated ewes placed with vasectomized rams for 6 hours. In contrast, Hawk (2) demonstrated that exposing artificially inseminated or naturally mated ewes to vasectomized rams did not improve sperm numbers in the uterus, oviducts or zona pellucidae. Schinckel (3,4) showed that joining rams with Merino ewes during transition from the nonbreeding to the breeding season stimulated ovulation without estrus in most ewes that had not already cycled. The Mor-Lean method of male sterilization (short scrotum, patent number 3,915,15O) was developed at New Mexico State University (5) as a means of increasing lean carcass yield. Ruttle et al. (6) demonstrated that this procedure resulted in atrophy of the germinal epithelium within 10 days in ram lambs and that all sperm cells in ejaculates from short scrotum animals were dead 8 days after treatment. Data relative to effects of this methnd on libido, however, are lacking. This study compared long-term influences of vasectomy and shortening the scrotum on semen characteristics, libido and serum testosterone in rams.
MATERIALS AND METHODS Ten fine-wool ram lambs of similar ages and breeding were weaned at approximately 80 days of age and subsequently allowed ad libitum access to a pelleted diet having the following composition (dry matter basis): 93.4% dry matter, 18.5% crude protein, 5.7% ether extract, 18.8% acid detergent fiber and 3.9 kcal/g gross energy. Animals were maintained in a single 6.2 x 9.1 m pen with continuous access to water, salt and mineral blocks. At approximately 6 months of age, rams were vaccinated against tetanus and randomly allotted to one of two treatments. Animals were weighed at 3-week intervals beginning 1 week before treatment and continuing for 10 weeks. Five lambs that had been fasted for 24 hcurs were lightly sedated (Rompun, Haver-Lockhart; Shawnee, KS) and prepared for surgery by shaving and disinfecting the proximal portion of the scrotum and adjacent areas and administering a local anesthetic (Lidocaine, Sussex; Edison, NJ). Each spermatic cord was exteriorized by making a midventral incision (4 cm) near the dorsal pole of each testicle. A 2.5 cm segment of each vas deferens was then ligated and removed. The peritoneum and skin incisions were subsequently closed. Skin sutures were removed after 10 days. Five ram lambs were sterilized by the short scrotum technique described by Ray and Felling (5). Testes were elevated and secured near the body cavity using elastrator bands. After treatment, all animals (both groups) received antibiotic therapy (Combiotic, Pfizer; New York, NY) for 3 consecutive days. Semen samples were collected by electroe!aculation 1 week before
536
MARCH
1985 VOL. 23 NO. 3
THERIOGENOLOGY
treatment and every third day after treatment for 15 days. Additional samples were collected 1 week before conducting each libido trial. Ejaculates were examined for volume, motility, concentration and percentage abnormal cells using procedures outlined by Sorensen (7). Mating behavior (libido) of both vasectomized and short scrotum rams was assessed in September of 2 consecutive years. Trials were conducted in a similar fashion during both years except ewes used in the first trial were synchronized with intravaginal pessaries while ewes in the second trial underwent an injection scheme to induce estrus. During the first year, 10 fine-wool ewe lambs (9 months of age) received intravaginal pessaries containing 20 mg flurogestone acetate (Syncro-mate, Searle; Skokie, IL). After pessaries had been in place for 4 days and while ewes were not sexually receptive, treated rams (9 months of age) were joined with the females for 6 days to allow animals to become accustomed to each other and to the pen facility. Pessaries were removed 10 days after insertion at which time rams were moved to a separate facility. Ewes synchronized during the second year received 10 mg of progesterone (Reprogest, Burns Biotec; Oakland, CA) daily (intramuscular injection) for 14 days. Progesterone treatments were followed by intramuscular injections containing 0.5 mg estradiol cypionate (Upjohn; Kalamazoo, MI) for 2 consecutive days. On the day after the second estradiol cypionate injection, eight ewes were determined to be in estrus by a mature, sexually aggressive vasectomized ram. Ewes were then randomized into one of two groups containing four estrous ewes and one nonreceptive ewe. Rams were subsequently ordered in a random sequence within treatment and exposed to one of the two groups of ewes for a 15-minute period. The test facility was constructed to eliminate outside distractions and to prohibit the ram being tested from seeing any sheep other than his group of ewes. Libido was assessed by observing displays of sexual behavior and recording the number and type of responses each ram exhibited toward a ewe. Nonmating sexual approaches included pawing, flehmen, licking the flank, nudging and rubbing along the ewe's side. Incomplete mating was described as an attempted or successful mount with no ejaculation. A completed mating was recorded when both intromission and ejaculation occurred. At the end of the 15-minute period, each ram was also assigned an overall sexual activity score (1 = low, 5 = high). Numerous blood samples were obtained during libido trials. Bach ram was bled before entering the enclosure where the libido trial was conducted, at first sight of receptive ewes and again midway (7.5 min) through the libido trial. Additional samples were obtained at completion of the trial and 30 minutes, 24 hours and 48 hours after the libido trial. Blood was collected by jugular venipuncture into serum separation tubes (Corvac, Sherwood; St. Louis, MO) and was allowed to coagulate at room temperature for 30 minutes before centrifugation (2,300 x g, 15 minutes at 4" C). Serum was harvested and stored at -20" C until serum testosterone was quantified. Serum testosterone was quantified by radioimmunoassay using anti-
MARCH
1985 VOL. 23 NO. 3
537
THERIOGENOLOGY
sera which has been described previously (8). Aliquots of serum (usually 0.1 ml) were extracted by vigorous inversion (5 minutes) using 3 ml of hexane:benzene (2:l; v:v). Solvent extracts were transferred to 12 x 75 mm culture tubes and evaporated to dryness under nitrogen gas. A standard solution was prepared by diluting 1 pg testosterone (17B-hydroxy-4-androsten-3-one, Steraloids; Wilton, NH) in 100 ml of 0.01 M phosphate buffered saline containing 0.1% gelatin (PBS-Gel) such that each ml contained 10 ng testosterone. This stock standard solution was dispensed by l-ml aliquots into polyethylene culture tubes (12 x 75 mm) and frozen at -80" C. For each assay, 1 ml of the stock standard solution was thawed in cool water and an appropriate amount was pipetted to yield duplicate sets of seven standards containing 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ng of testosterone. AI1 standard and solvent extract tubes were then normalized to 0.1 ml using PBS-Gel. Anti-testosterone (GDN no. 250) diluted 1:80,000 using PBS-Gel was subsequently added to each assay tube (0.2 ml) after which tubes were vortexed and allowed to incubate at room temperature for 15 minutes. Then30.2 ml of a solution containing approximately 30,000 cpm of 1, 2, 6, 7- H- testosterone (New England Nuclear; Boston, MA) in PBS-Gel was pipetted into each tube and the assay was incubated at 4" C for 2 hours. Separation of free and bound hormone was accomplished by addition of 1 ml dextran-coated charcoal (0.25 g dextran T 150 and 2.5 g neutral norit charcoal/ liter of water) followed by centrifugation (1,600 x g) at 4" C for 15 minutes. Radioactivity was determined in the supernatant fraction and concentration of hormone in each serum sample was calculated after corrections were made for procedural losses. Testosterone concentrations in eight benzene:hexane extracts were quantified before and after chromatographic isolation using Sephadex LH-20 column chromatography (benzene:methanol:9:1, v:v). Testosterone concentrations (mean + SE) in extracts (5.3 + 1.7 ng/ml) before chromatography did not differ (P>O.lO) from those determined after column chromatography (6.5 + 0.7 ng/ml; r = 0.96). The within assay coefficient of variation for nine determinations of an ovine serum pool was 7.3%, while the between assay coefficient of variation for 34 determinations of the same pool was 17.1%. When 5 ng testosterone were added to 1 ml of wether serum, 5.1 + 0.5 ng/ml (97%) were recovered. Analyses of variance for completely randomized designs were used to evaluate treatment influences on animal weight, semen volume, sperm motility, sperm concentration, abnormal cells and mating activity (9). Rate of forward movement, libido rating and other nonparametric data were subjected to chi-square procedures (9). Treatment effects on serum testosterone profiles were examined by split-plot analysis of variance for repeated measurements of animals (10).
RESULTS AND DISCUSSION Animal weights did not differ (P>O.lO) before surgery in the two groups of rams (46.9 and 49.2 kg for vasectomized and short scrotum rams, respectively), indicating that randomization of animals into the respective treatments resulted in an even distribution of weights
538
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1985VOL.23N0.3
THERIOGENOLOGY between groups. This similarity in weight response remained apparent in vasectomized (VAS) and short scrotum (SS) rams at 3 (50.8 and 49.6 kg, respectively), 6 (55.2 and 57.0 kg, respectively) and IO (61.4 and 65.4 kg, respectively) weeks after treatment. Thus, shortening the scrotum of 6-month-old ram lambs did not adversely influence animal weight responses any more than did the more conventional method of rendering males infertile (i.e., vasectomy). Semen
@ualitv
As mentioned previously, semen samples were collected frequently during the 15-day period immediately following treatment. Realizing the variable nature of samples obtained by electroejaculation, these data were used only to allow estimates of when rams became sterile. Semen quality is, therefore, discussed in descriptive terms related to differences between treatments rather than presenting actual numbers in tabular form. Cells were nonmotile 6 and I? days after treatment in VAS and SS animals, respectively. Although VAS males had a higher (PcO.10) percentage of abnormal cells than did SS rams, over 70% of remaining cells in both treatments were considered abnormal by 6 days after surgery. By 12 days following treatment, no cells were detected in ejaculates from VAS rams at which time these males were considered sterile. These results compare favorably with those of Dunlop et al. (11). who found rams to be sterile 1 week after vasectomy when ejaculated daily. Thwaites (I?) demonstrated that three ejaculations were required following vasectomy to completely sterilize rams. Semen quality of SS rams I? days after treatment was inferior and males at that time were consjdered sterile. Only two SS rams had detectable numbers of cells in their ejaculates and of these cells, 95% were abnormal and all but 1% were non-motile. Tn a similar study (6), 8-month-old SS rams had no motile cells and 58% abnormals 8 days after treatment. Semen quality of VAS rams remained unchanged past 12 days after treatment. Ko cells were detected in ejaculates collected at 15 and 18 days7or at 3 and 15 months after surgery. One SS ram had a few cells (1 x 10 cells/ml) present 18 days after imposing treatments; however, none of the cells were motile and all were considered abnormal. The SS technique appeared to be effective in rendering males sterile by 15 days after treatment. Additional samples collected from SS and VAS rams 1 week before the first libido trial 13 months after surgery (Table 1); were similar to the previous sample obtained at 18 days in that an almost undetectable concentration was evident with all cells being abnormal and nonmotile. The presence of motile spermatozoa in ejaculates of three SS rams indicated that at least partial testicular function was regained by 15 mccths after treatment (Table 1). Although overall concentration and motility were low (73 x 10 and 38.3%, respectively), concentration in the ejaculate of one SS ram approyched those values normally seen in mature, unaltered males (185 x 10 cells/ml with 95% motility). Shortening the scrotum by the method of Ray and Belling (5),
MARCH
1985 VOL. 23 NO. 3
539
THERIOGENOLOGY
” r
8
,
1
I 4 I 4 , ,
, )
c
540
MARCH
1985 VOL. 23 NO. 3
therefore, apparently resulted in temporary cessation of spermatogenesis. These data indicate that as SS rams became mature, testicular function was no longer totally impaired and partial spermatogenesis was regained. The reason for this resumption of sperm production is not clear but the rate and degree to which spermatogenesis is regained in an individual may be dependent upon the extent of tubular damage or the age at which the animal was altered (13). Since the Mor-Lean method was primarily developed for use in feedlot animals destined for slaughter before maturity, semen quality has not been examined in older animals. If scrotal growth and repositioning of the testes occurred after treatment such that portions of the testicle were not exposed to abdominal heat, spermatogenesis might occur if at least some spermatogonia were still present. Mating Behavior Vasectomized rams exhibited a higher degree (PCO.10) of courtship behavior than did SS rams (12.0 t 1.7 and 7.4 2 1.7 approaches/animal, respectively) during the first year of the trial (Table 2). Much of this difference was due to the lack of interest in receptive females expressed by one SS ram. Rams were sexually inexperienced before their first exposure to receptive ewes and several rams showed little interest until the latter portion of the trial. Nonmating approaches during the second libido test were similar (P>O.lO) among treatment groups. As might be expected, both SS and VAS males exhibited responses such as flehmen, pawing, sniffing the vulva or rubbing along the ewe's side more frequently during the second libido trial. Furthermore, most males responded immediately when introduced to the ewe group during the second year, which was not the case during year one. During a 15-minute exposure to a group of receptive ewes, SS males performed similarly (P>O.lO) to VAS rams with regard to the number of incomplete and completed matings. Overall mating activity was somewhat higher for both treatment groups during the second libido trial when compared with the first, which is likely attributable to the fact that all rams were sexually experienced and responded immediately upon exposure to estrous females. The overall degree of sexual activity was also comparable (P>O.lO) as indicated by the same percentage of males in each treatment having high and low libido ratings (Table 2) during year one. Libido ratings of VAS rams during the second year were similar to those obtained for year one, whereas more animals in the SS group had higher libido scores in year two than in year one. In any event, distributions of libido ratings were similar (P>O.lO) between treatments during both years. These data indicate that shortening the scrotum of &month-old ram lambs by the method of Ray and Belling (5) has little effect on mating behavior or sexual activity when compared with VAS rams. These data apply to rams that are 6 and 9 months of age at the time of surgery and first exposure to females, respectively. Whether younger or older animals would respond in the same manner to shortening the scrotum is yet to be determined.
MARCH
1985 VOL. 23 NO. 3
541
N
P
ul
60.0
Animals having a 1:~ libido rating, % 60.0
40.0
5.4
3.8
16.8 7.4b
'Row means within year do not differ (P>O.lO).
bRow means for year 1 differ (P
pi esponses during a 15 min exposure to receptive ewes.
40.0
7.4
Completed matingaC
Animals having a h:gh libido rating, %
4.6
Incomplete matingac
14.4 12.0b
Vasectomized
Year 1 Short Scrotum
2.6
I.2
3.4 1.7
SE
40.0
60.0
9.4
5.4
17.6 16.4'
Vasectomized
20.0
80.0
13.0
5.4
19.6 21.8'
Year 2 Short Scrotum
3.0
1.4
3.1 2.9
SE
MATING BEHAVIOR OF VASECTOMIZED AND SHORT SCROTUM RAMS DURING LIBIDO TRAILS CONDUCTED IN THE FALL OF TWO CONSECUTIVE YEARS.
Non-mating approachesa Sniffing vulvaC Courting behavior
Item
TABLE 2.
5 0 I(
g
$
g s
THERIOGENOLOGY
MARCH
1985 VOL. 23
3
THERIOGENOLOCY Serum Testosterone Serum testosterone was examined at numerous intervals during and after libido trials (Table 3). No treatment differences (P>O.90) were observed before, during or after each trial and a split-plot analysis revealed no treatment x period interactions within year (P>O.20). Overall, testosterone levels throughout the libido trial of year one were low when compared with serum levels observed during the second year. Somewhat elevated levels of testosterone in both treatment groups during the second year of the libido trial may be a reflection of physical maturity. These data indicate that shortening the scrotum of 6-month-old rams has little effect on serum testosterone when compared with VAS rams. Results obtained in this study concur with those of Schanbacher and Ford (13) who reported that serum testosterone levels in SS males were similar to values observed in intact rams. Several authors (14,15,16) were unable to demonstrate relationships between copulation or mating behavior and serum levels of testosterone; however, Kamel et al. (17) observed elevated testosterone levels in rats approximately 1 hour after mating activity. In the same study, testosterone levels of sexually naive rats were unaffected by the mating stimulus. Similar trends were observed in both SS and VAS rams during libido trials. Mean testosterone levels of sexually naive rams were similar during all periods of the libido trial conducted in year one, but were noticeably higher 30 minutes and 24 hours following the second trial (year two). Although hormonal responses could not be directly correlated with mating activity, data reported herein demonstrate that SS males respond similarly to VAS rams when exposed to receptive ewes.
REFERENCES 1.
Restall, B. J. Artificial insemination of sheep. VI. The effect of post-inseminal coitus on percentage of ewes lambing to a 37:70-72 (1961). single insemination. Australian Vet. J. -
2.
Hawk, H. W. Failure of vasectomized rams to improve sperm transport in inseminated ewes. J. Anim. Sci. -35:69-72 (1972).
3.
Schinckel, P. G. The effect of the ram on the incidence and occurrence of oestrus in ewes. Australian Vet. .I. -30:189-195 (1954).
4.
Schinckel, P. G. The effect of the presence of the ram on the ovarian activity of the ewe. Australian J. Agr. Res. 2:465-469 (1954).
5.
Ray, E. E. and Belling, T. H. The effects of shortening the scrotum on growth rate in lambs. Growth -31:39-42 (1967).
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1985VOL.23 NO.3
THERIOGENOLOGY
6.
Ruttle, J. L., McFadden, W. D. and Mata, F. The effect of short scrotum on histology of the testis and semen production in rams. New Mexico Agr. Exp. Sta. Res. Rep. 304 (1975).
7.
Sorensen, A. M. Repro Lab: A Laboratory Manual for Animal Reproduction (3rd Ed.). Kendall/Hunt Publishing Co., Dubuque, IA, 1975.
8.
Gay, V. L. and Kerlan, J. T. Serum LH and FSH following passive immunization against circulating testosterone in the intact male rat and in orchidectomized rats bearing subcutaneous silastic implants of testosterone. Arch. Androl. -1:257-266 (1978).
9.
Statistical Methods (6th Snedecor, G. W. and Cochran, W. G. Ed.). Iowa State Univ. Press, Ames, IA, 1967.
10.
Gill, J. L. and Hafs, H. D. Analysis of repeated measurements of animals. J. Anim. Sci. -33:331-336 (1971).
11.
Dunlop, A. A., Moule, G. R. and Southcott, W. H. Spermatozoa in ejaculates of vasectomized rams. Australian Vet. J. -39:46-48 (1963).
12.
Thwaites, C. J. Semen quality after vasectomy in the ram. Livestock Prod. Sci. 8:529-534 (1982).
13.
Schanbacher, B. D. and Ford, J. J. Luteinizing hormone, testosterone, growth and carcass responses to sexual alteration in the ram. J. Anim. Sci. -43:638-643 (1976).
14.
Purvis, K. and Haynes, N. B. Short-term effects of copulation, human chorionic gonadotrophin injection and non-tactile association with a female on testosterone levels in the male rat. J. Endocrinol. 60:429-439 (1974).
15.
Illius, A. W., Haynes, N. B. and Lamming, G. E. Effects of ewe proximity on peripheral plasma testosterone levels and behavior in the ram. J. Reprod. Fertil. -48:25-32 (1976).
16.
Illius, A. W., Haynes, N. B., Purvis, K. and Lamming, G. E. Plasma concentration of testosterone in the developing ram in different social environments. J. Reprod. Ferti.1. 48:17-24 (1976).
17.
Kamel, F., Wright, W., Mock, E. and Frankel, A. The influence of mating and related stimuli on plasma levels of luteinizing hormone, follicle stimulating hormone, prolactin and testosterone in the male rat. Endocrinology -101:421-429 (1977).
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1985 VOL. 23 NO. 3
545
MATING BEHAVIOR, SERUM T~S?~STER~NF AND SEMEN CHA~~T~R?ST~CS IN VASECTOMIZED AN? SHORT SCROTUM RAMS a,b L. A. Tierney and D. M. Hallford
Department of Animal and Range Sciences College of Agriculture ard Home Economics New Mexico State University Las Cruces, New Mexico 88003 Received for publication: September Accepted: December
1, 1984 27, 1984
ABSTRACT Ter similar rams were either vasectomized (VAS) or altered by securing the testes near the abdomen using elastrator bands (short scrotum, SS). Semen characteristics (electroejaculation) were similar (P>O.lO) between groups before treatment and ejaculate volumes remained relatively constant (P>O.?o) throughout the trial. Remaining sperm cells were nonmotile and 95% abnormal by 6 days after vasectomy. By 12 days after surgery, ejaculates contained no cells. Two SS rams had detectable sperm numbers at 12 days after treatment, but 95% were abnormal and all but 1% were nonmotile. Ejaculates collected 3 months after surgery were similar between treatments, in that all existing cells were nonmotile and abnormal. Partial spermatngenic function was regained in SS rsms 15 months after treatment as indicated by the presence of low concentrations of motile sperm. Three and 15 months after treatment , sexual activity was estinated hy exposing rams to a group of four estrous ewes. During the 15-minute exposure, VAS males exhibited more (P
vasectomy, short scrntum, semen, libido, testosterone
iJournal Article IORO of the New Mexico Agricultural Experiment Station. The authors appreciate the help of C. Crusberg, D. Morrical, R. Hudgens, M. Scboenemann, S. Mischler, D. Sxnson, M. Neave and P. Ross. We also thank Dr. G. D. Niswender (Dept. of Physiol. and Biophys., Colorado State Univ., Fort Collins, CO) for the supply of testosterone-11 BSA antiserum (GDN 250) used in testosterone determinations. Direct reprint requests to D. M. Hallford.
MARCH
1985VOL. 23N0.3
535
THERIOGENOLOGY
INTRODUCTION In most domestic species, reliable estrous detection records are difficult to obtain without using intact or sterile males or andrcgenized females. In addition, Restall (1) observed an increased lambing response in artificially inseminated ewes placed with vasectomized rams for 6 hours. In contrast, Hawk (2) demonstrated that exposing artificially inseminated or naturally mated ewes to vasectomized rams did not improve sperm numbers in the uterus, oviducts or zona pellucidae. Schinckel (3,4) showed that joining rams with Merino ewes during transition from the nonbreeding to the breeding season stimulated ovulation without estrus in most ewes that had not already cycled. The Mor-Lean method of male sterilization (short scrotum, patent number 3,915,15O) was developed at New Mexico State University (5) as a means of increasing lean carcass yield. Ruttle et al. (6) demonstrated that this procedure resulted in atrophy of the germinal epithelium within 10 days in ram lambs and that all sperm cells in ejaculates from short scrotum animals were dead 8 days after treatment. Data relative to effects of this methnd on libido, however, are lacking. This study compared long-term influences of vasectomy and shortening the scrotum on semen characteristics, libido and serum testosterone in rams.
MATERIALS AND METHODS Ten fine-wool ram lambs of similar ages and breeding were weaned at approximately 80 days of age and subsequently allowed ad libitum access to a pelleted diet having the following composition (dry matter basis): 93.4% dry matter, 18.5% crude protein, 5.7% ether extract, 18.8% acid detergent fiber and 3.9 kcal/g gross energy. Animals were maintained in a single 6.2 x 9.1 m pen with continuous access to water, salt and mineral blocks. At approximately 6 months of age, rams were vaccinated against tetanus and randomly allotted to one of two treatments. Animals were weighed at 3-week intervals beginning 1 week before treatment and continuing for 10 weeks. Five lambs that had been fasted for 24 hcurs were lightly sedated (Rompun, Haver-Lockhart; Shawnee, KS) and prepared for surgery by shaving and disinfecting the proximal portion of the scrotum and adjacent areas and administering a local anesthetic (Lidocaine, Sussex; Edison, NJ). Each spermatic cord was exteriorized by making a midventral incision (4 cm) near the dorsal pole of each testicle. A 2.5 cm segment of each vas deferens was then ligated and removed. The peritoneum and skin incisions were subsequently closed. Skin sutures were removed after 10 days. Five ram lambs were sterilized by the short scrotum technique described by Ray and Felling (5). Testes were elevated and secured near the body cavity using elastrator bands. After treatment, all animals (both groups) received antibiotic therapy (Combiotic, Pfizer; New York, NY) for 3 consecutive days. Semen samples were collected by electroe!aculation 1 week before
536
MARCH
1985 VOL. 23 NO. 3
THERIOGENOLOGY
treatment and every third day after treatment for 15 days. Additional samples were collected 1 week before conducting each libido trial. Ejaculates were examined for volume, motility, concentration and percentage abnormal cells using procedures outlined by Sorensen (7). Mating behavior (libido) of both vasectomized and short scrotum rams was assessed in September of 2 consecutive years. Trials were conducted in a similar fashion during both years except ewes used in the first trial were synchronized with intravaginal pessaries while ewes in the second trial underwent an injection scheme to induce estrus. During the first year, 10 fine-wool ewe lambs (9 months of age) received intravaginal pessaries containing 20 mg flurogestone acetate (Syncro-mate, Searle; Skokie, IL). After pessaries had been in place for 4 days and while ewes were not sexually receptive, treated rams (9 months of age) were joined with the females for 6 days to allow animals to become accustomed to each other and to the pen facility. Pessaries were removed 10 days after insertion at which time rams were moved to a separate facility. Ewes synchronized during the second year received 10 mg of progesterone (Reprogest, Burns Biotec; Oakland, CA) daily (intramuscular injection) for 14 days. Progesterone treatments were followed by intramuscular injections containing 0.5 mg estradiol cypionate (Upjohn; Kalamazoo, MI) for 2 consecutive days. On the day after the second estradiol cypionate injection, eight ewes were determined to be in estrus by a mature, sexually aggressive vasectomized ram. Ewes were then randomized into one of two groups containing four estrous ewes and one nonreceptive ewe. Rams were subsequently ordered in a random sequence within treatment and exposed to one of the two groups of ewes for a 15-minute period. The test facility was constructed to eliminate outside distractions and to prohibit the ram being tested from seeing any sheep other than his group of ewes. Libido was assessed by observing displays of sexual behavior and recording the number and type of responses each ram exhibited toward a ewe. Nonmating sexual approaches included pawing, flehmen, licking the flank, nudging and rubbing along the ewe's side. Incomplete mating was described as an attempted or successful mount with no ejaculation. A completed mating was recorded when both intromission and ejaculation occurred. At the end of the 15-minute period, each ram was also assigned an overall sexual activity score (1 = low, 5 = high). Numerous blood samples were obtained during libido trials. Bach ram was bled before entering the enclosure where the libido trial was conducted, at first sight of receptive ewes and again midway (7.5 min) through the libido trial. Additional samples were obtained at completion of the trial and 30 minutes, 24 hours and 48 hours after the libido trial. Blood was collected by jugular venipuncture into serum separation tubes (Corvac, Sherwood; St. Louis, MO) and was allowed to coagulate at room temperature for 30 minutes before centrifugation (2,300 x g, 15 minutes at 4" C). Serum was harvested and stored at -20" C until serum testosterone was quantified. Serum testosterone was quantified by radioimmunoassay using anti-
MARCH
1985 VOL. 23 NO. 3
537
THERIOGENOLOGY
sera which has been described previously (8). Aliquots of serum (usually 0.1 ml) were extracted by vigorous inversion (5 minutes) using 3 ml of hexane:benzene (2:l; v:v). Solvent extracts were transferred to 12 x 75 mm culture tubes and evaporated to dryness under nitrogen gas. A standard solution was prepared by diluting 1 pg testosterone (17B-hydroxy-4-androsten-3-one, Steraloids; Wilton, NH) in 100 ml of 0.01 M phosphate buffered saline containing 0.1% gelatin (PBS-Gel) such that each ml contained 10 ng testosterone. This stock standard solution was dispensed by l-ml aliquots into polyethylene culture tubes (12 x 75 mm) and frozen at -80" C. For each assay, 1 ml of the stock standard solution was thawed in cool water and an appropriate amount was pipetted to yield duplicate sets of seven standards containing 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ng of testosterone. AI1 standard and solvent extract tubes were then normalized to 0.1 ml using PBS-Gel. Anti-testosterone (GDN no. 250) diluted 1:80,000 using PBS-Gel was subsequently added to each assay tube (0.2 ml) after which tubes were vortexed and allowed to incubate at room temperature for 15 minutes. Then30.2 ml of a solution containing approximately 30,000 cpm of 1, 2, 6, 7- H- testosterone (New England Nuclear; Boston, MA) in PBS-Gel was pipetted into each tube and the assay was incubated at 4" C for 2 hours. Separation of free and bound hormone was accomplished by addition of 1 ml dextran-coated charcoal (0.25 g dextran T 150 and 2.5 g neutral norit charcoal/ liter of water) followed by centrifugation (1,600 x g) at 4" C for 15 minutes. Radioactivity was determined in the supernatant fraction and concentration of hormone in each serum sample was calculated after corrections were made for procedural losses. Testosterone concentrations in eight benzene:hexane extracts were quantified before and after chromatographic isolation using Sephadex LH-20 column chromatography (benzene:methanol:9:1, v:v). Testosterone concentrations (mean + SE) in extracts (5.3 + 1.7 ng/ml) before chromatography did not differ (P>O.lO) from those determined after column chromatography (6.5 + 0.7 ng/ml; r = 0.96). The within assay coefficient of variation for nine determinations of an ovine serum pool was 7.3%, while the between assay coefficient of variation for 34 determinations of the same pool was 17.1%. When 5 ng testosterone were added to 1 ml of wether serum, 5.1 + 0.5 ng/ml (97%) were recovered. Analyses of variance for completely randomized designs were used to evaluate treatment influences on animal weight, semen volume, sperm motility, sperm concentration, abnormal cells and mating activity (9). Rate of forward movement, libido rating and other nonparametric data were subjected to chi-square procedures (9). Treatment effects on serum testosterone profiles were examined by split-plot analysis of variance for repeated measurements of animals (10).
RESULTS AND DISCUSSION Animal weights did not differ (P>O.lO) before surgery in the two groups of rams (46.9 and 49.2 kg for vasectomized and short scrotum rams, respectively), indicating that randomization of animals into the respective treatments resulted in an even distribution of weights
538
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1985VOL.23N0.3
THERIOGENOLOGY between groups. This similarity in weight response remained apparent in vasectomized (VAS) and short scrotum (SS) rams at 3 (50.8 and 49.6 kg, respectively), 6 (55.2 and 57.0 kg, respectively) and IO (61.4 and 65.4 kg, respectively) weeks after treatment. Thus, shortening the scrotum of 6-month-old ram lambs did not adversely influence animal weight responses any more than did the more conventional method of rendering males infertile (i.e., vasectomy). Semen
@ualitv
As mentioned previously, semen samples were collected frequently during the 15-day period immediately following treatment. Realizing the variable nature of samples obtained by electroejaculation, these data were used only to allow estimates of when rams became sterile. Semen quality is, therefore, discussed in descriptive terms related to differences between treatments rather than presenting actual numbers in tabular form. Cells were nonmotile 6 and I? days after treatment in VAS and SS animals, respectively. Although VAS males had a higher (PcO.10) percentage of abnormal cells than did SS rams, over 70% of remaining cells in both treatments were considered abnormal by 6 days after surgery. By 12 days following treatment, no cells were detected in ejaculates from VAS rams at which time these males were considered sterile. These results compare favorably with those of Dunlop et al. (11). who found rams to be sterile 1 week after vasectomy when ejaculated daily. Thwaites (I?) demonstrated that three ejaculations were required following vasectomy to completely sterilize rams. Semen quality of SS rams I? days after treatment was inferior and males at that time were consjdered sterile. Only two SS rams had detectable numbers of cells in their ejaculates and of these cells, 95% were abnormal and all but 1% were non-motile. Tn a similar study (6), 8-month-old SS rams had no motile cells and 58% abnormals 8 days after treatment. Semen quality of VAS rams remained unchanged past 12 days after treatment. Ko cells were detected in ejaculates collected at 15 and 18 days7or at 3 and 15 months after surgery. One SS ram had a few cells (1 x 10 cells/ml) present 18 days after imposing treatments; however, none of the cells were motile and all were considered abnormal. The SS technique appeared to be effective in rendering males sterile by 15 days after treatment. Additional samples collected from SS and VAS rams 1 week before the first libido trial 13 months after surgery (Table 1); were similar to the previous sample obtained at 18 days in that an almost undetectable concentration was evident with all cells being abnormal and nonmotile. The presence of motile spermatozoa in ejaculates of three SS rams indicated that at least partial testicular function was regained by 15 mccths after treatment (Table 1). Although overall concentration and motility were low (73 x 10 and 38.3%, respectively), concentration in the ejaculate of one SS ram approyched those values normally seen in mature, unaltered males (185 x 10 cells/ml with 95% motility). Shortening the scrotum by the method of Ray and Belling (5),
MARCH
1985 VOL. 23 NO. 3
539
THERIOGENOLOGY
” r
8
,
1
I 4 I 4 , ,
, )
c
540
MARCH
1985 VOL. 23 NO. 3
therefore, apparently resulted in temporary cessation of spermatogenesis. These data indicate that as SS rams became mature, testicular function was no longer totally impaired and partial spermatogenesis was regained. The reason for this resumption of sperm production is not clear but the rate and degree to which spermatogenesis is regained in an individual may be dependent upon the extent of tubular damage or the age at which the animal was altered (13). Since the Mor-Lean method was primarily developed for use in feedlot animals destined for slaughter before maturity, semen quality has not been examined in older animals. If scrotal growth and repositioning of the testes occurred after treatment such that portions of the testicle were not exposed to abdominal heat, spermatogenesis might occur if at least some spermatogonia were still present. Mating Behavior Vasectomized rams exhibited a higher degree (PCO.10) of courtship behavior than did SS rams (12.0 t 1.7 and 7.4 2 1.7 approaches/animal, respectively) during the first year of the trial (Table 2). Much of this difference was due to the lack of interest in receptive females expressed by one SS ram. Rams were sexually inexperienced before their first exposure to receptive ewes and several rams showed little interest until the latter portion of the trial. Nonmating approaches during the second libido test were similar (P>O.lO) among treatment groups. As might be expected, both SS and VAS males exhibited responses such as flehmen, pawing, sniffing the vulva or rubbing along the ewe's side more frequently during the second libido trial. Furthermore, most males responded immediately when introduced to the ewe group during the second year, which was not the case during year one. During a 15-minute exposure to a group of receptive ewes, SS males performed similarly (P>O.lO) to VAS rams with regard to the number of incomplete and completed matings. Overall mating activity was somewhat higher for both treatment groups during the second libido trial when compared with the first, which is likely attributable to the fact that all rams were sexually experienced and responded immediately upon exposure to estrous females. The overall degree of sexual activity was also comparable (P>O.lO) as indicated by the same percentage of males in each treatment having high and low libido ratings (Table 2) during year one. Libido ratings of VAS rams during the second year were similar to those obtained for year one, whereas more animals in the SS group had higher libido scores in year two than in year one. In any event, distributions of libido ratings were similar (P>O.lO) between treatments during both years. These data indicate that shortening the scrotum of &month-old ram lambs by the method of Ray and Belling (5) has little effect on mating behavior or sexual activity when compared with VAS rams. These data apply to rams that are 6 and 9 months of age at the time of surgery and first exposure to females, respectively. Whether younger or older animals would respond in the same manner to shortening the scrotum is yet to be determined.
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1985 VOL. 23 NO. 3
541
N
P
ul
60.0
Animals having a 1:~ libido rating, % 60.0
40.0
5.4
3.8
16.8 7.4b
'Row means within year do not differ (P>O.lO).
bRow means for year 1 differ (P
pi esponses during a 15 min exposure to receptive ewes.
40.0
7.4
Completed matingaC
Animals having a h:gh libido rating, %
4.6
Incomplete matingac
14.4 12.0b
Vasectomized
Year 1 Short Scrotum
2.6
I.2
3.4 1.7
SE
40.0
60.0
9.4
5.4
17.6 16.4'
Vasectomized
20.0
80.0
13.0
5.4
19.6 21.8'
Year 2 Short Scrotum
3.0
1.4
3.1 2.9
SE
MATING BEHAVIOR OF VASECTOMIZED AND SHORT SCROTUM RAMS DURING LIBIDO TRAILS CONDUCTED IN THE FALL OF TWO CONSECUTIVE YEARS.
Non-mating approachesa Sniffing vulvaC Courting behavior
Item
TABLE 2.
5 0 I(
g
$
g s
THERIOGENOLOGY
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1985 VOL. 23
3
THERIOGENOLOCY Serum Testosterone Serum testosterone was examined at numerous intervals during and after libido trials (Table 3). No treatment differences (P>O.90) were observed before, during or after each trial and a split-plot analysis revealed no treatment x period interactions within year (P>O.20). Overall, testosterone levels throughout the libido trial of year one were low when compared with serum levels observed during the second year. Somewhat elevated levels of testosterone in both treatment groups during the second year of the libido trial may be a reflection of physical maturity. These data indicate that shortening the scrotum of 6-month-old rams has little effect on serum testosterone when compared with VAS rams. Results obtained in this study concur with those of Schanbacher and Ford (13) who reported that serum testosterone levels in SS males were similar to values observed in intact rams. Several authors (14,15,16) were unable to demonstrate relationships between copulation or mating behavior and serum levels of testosterone; however, Kamel et al. (17) observed elevated testosterone levels in rats approximately 1 hour after mating activity. In the same study, testosterone levels of sexually naive rats were unaffected by the mating stimulus. Similar trends were observed in both SS and VAS rams during libido trials. Mean testosterone levels of sexually naive rams were similar during all periods of the libido trial conducted in year one, but were noticeably higher 30 minutes and 24 hours following the second trial (year two). Although hormonal responses could not be directly correlated with mating activity, data reported herein demonstrate that SS males respond similarly to VAS rams when exposed to receptive ewes.
REFERENCES 1.
Restall, B. J. Artificial insemination of sheep. VI. The effect of post-inseminal coitus on percentage of ewes lambing to a 37:70-72 (1961). single insemination. Australian Vet. J. -
2.
Hawk, H. W. Failure of vasectomized rams to improve sperm transport in inseminated ewes. J. Anim. Sci. -35:69-72 (1972).
3.
Schinckel, P. G. The effect of the ram on the incidence and occurrence of oestrus in ewes. Australian Vet. .I. -30:189-195 (1954).
4.
Schinckel, P. G. The effect of the presence of the ram on the ovarian activity of the ewe. Australian J. Agr. Res. 2:465-469 (1954).
5.
Ray, E. E. and Belling, T. H. The effects of shortening the scrotum on growth rate in lambs. Growth -31:39-42 (1967).
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THERIOGENOLOGY
6.
Ruttle, J. L., McFadden, W. D. and Mata, F. The effect of short scrotum on histology of the testis and semen production in rams. New Mexico Agr. Exp. Sta. Res. Rep. 304 (1975).
7.
Sorensen, A. M. Repro Lab: A Laboratory Manual for Animal Reproduction (3rd Ed.). Kendall/Hunt Publishing Co., Dubuque, IA, 1975.
8.
Gay, V. L. and Kerlan, J. T. Serum LH and FSH following passive immunization against circulating testosterone in the intact male rat and in orchidectomized rats bearing subcutaneous silastic implants of testosterone. Arch. Androl. -1:257-266 (1978).
9.
Statistical Methods (6th Snedecor, G. W. and Cochran, W. G. Ed.). Iowa State Univ. Press, Ames, IA, 1967.
10.
Gill, J. L. and Hafs, H. D. Analysis of repeated measurements of animals. J. Anim. Sci. -33:331-336 (1971).
11.
Dunlop, A. A., Moule, G. R. and Southcott, W. H. Spermatozoa in ejaculates of vasectomized rams. Australian Vet. J. -39:46-48 (1963).
12.
Thwaites, C. J. Semen quality after vasectomy in the ram. Livestock Prod. Sci. 8:529-534 (1982).
13.
Schanbacher, B. D. and Ford, J. J. Luteinizing hormone, testosterone, growth and carcass responses to sexual alteration in the ram. J. Anim. Sci. -43:638-643 (1976).
14.
Purvis, K. and Haynes, N. B. Short-term effects of copulation, human chorionic gonadotrophin injection and non-tactile association with a female on testosterone levels in the male rat. J. Endocrinol. 60:429-439 (1974).
15.
Illius, A. W., Haynes, N. B. and Lamming, G. E. Effects of ewe proximity on peripheral plasma testosterone levels and behavior in the ram. J. Reprod. Fertil. -48:25-32 (1976).
16.
Illius, A. W., Haynes, N. B., Purvis, K. and Lamming, G. E. Plasma concentration of testosterone in the developing ram in different social environments. J. Reprod. Ferti.1. 48:17-24 (1976).
17.
Kamel, F., Wright, W., Mock, E. and Frankel, A. The influence of mating and related stimuli on plasma levels of luteinizing hormone, follicle stimulating hormone, prolactin and testosterone in the male rat. Endocrinology -101:421-429 (1977).
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