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The effects of stanozolol and boldenone undecylenate on scrotal width, testis weight, and sperm production in pony stallions
THERIOGENOLOCY THE EFFECTS OF STANOZOLOL AND BOLDENONE UNDECYLENATE ON SCROTAL WIDTH, TESTIS WEIGHT, AND SPERM PRODUCTION IN PONY STALLIONS T. L. Blanchard (a), R. G. Elmore (a), R. S. Youngquist (a), W. E. Loch (b), D. K. Hardin (a), C. J. Bierschwal (a), V. K. Ganjam (c), J. M. Balke (a), M. R. Ellersieck (d), L. J. Dawson (a), and W. S. Miner (e)
(a) (b) (c) (d)
Department of Veterinary Medicine and Surgery. Department of Animal Science. Department of Veterinary Anatomy and Physiology. Agriculture Experiment Station.
University of Missouri-Columbia,
Columbia, MO
65211
(e) Sterling-Winthrop Research Institute, Rensselaer, NY 12144, and Sterling Animal Health Products, New York, NY 10016. Received for publication: March 8, 1983 Accepted: May 12, 1983 ABSTRACT Fifty mature pony stallions were randomly assigned to one of five treatment groups: Group 1 - controls (no treatment), Group 2 - 0.55 mg/kg stanozolol weekly for 13 treatments, Group 3- 1.1 mg/kg stanozolol every 3 weeks for 5 treatments, Group 4 - 1.1 mg/kg boldenone undecylenate every 3 weeks for 5 treatments, and Group 5 - 0.55 mg/kg boldenone undecylenate weekly Scrotal widths (SW), combined testis weights for 13 treatments. (CTW), and daily sperm productions (DSP) were not different between Groups 1 and 2. Ponies in Group 5 had smaller SW (P < O.Ol), smaller CTW and decreased DSP compared to controls (P < 0.05). Although SW for ponies in Groups 3 and 4 were less than for controls (P < O.Ol), CTW and DSP were not different. The only treatment regime that did not alter SW, CTW, and DSP was Group 2 (0.55 mg/kg stanozolol weekly for 13 treatments).
____________________--____ Acknowledgements: This study was supported by a grant from the Winthrop Veterinary, Sterling Animal Health Products, Division of Sterling Drug Inc. The authors wish to express thanks to S. Mellskog, S. Rigby, J. Travers, L. Brockschmidt and N. Sights for technical assistance.
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THERIOGENOLOGY INTRODUCTION Anabolic steroids are derivatives of testosterone which are altered to provide maximal anabolic action and minimal androgenic side effects (1,2). The administration of anabolic steroids causes a retention of body water, nitrogen, potassium, sodium, calcium, chloride, sulfate, and phosphorus ions. Muscle mass increases due to promotion of positive nitrogen balance by stimulating protein production and reducing protein destruction (1). Anabolic steroids also stimulate production and/or release of erythropoietin from the kidneys and stimulate undifferentiated stem cells in bone marrow to enter an erythroid critical stage (3). Therapeutic uses for anabolic steroids are to correct tissue-depleting processes following systemic disease, malnutrition, parasitism, or wasting disease of old age; to speed tissue repair following surgery or trauma; and to promote nonspecific stimulation of erythropoiesis (4). They also are used to reverse some of the effects of glucocorticoids, specifically negative nitrogen and calcium balance and catabolic effects on muscle and connective tissue (2). Many human athletes, physicians, veterinarians, and trainers attest to gains in physical strength, stamina, and mental attitude following use of anabolic steroids (1, 5-7). It is not yet feasible to produce an anabolic steroid without androgenic activity (2). This androgenic activity accounts for most of the undesirable side effects of these drugs. If given early in life in large quantities, anabolic steroids may result in premature closure of the epiphyseal plates and prevent further growth in bone length (7). Administration of norethandrolone for 8-25 weeks results in loss of libido, decreased potency, diminished testicular size, and azoospermia in man (8). Administration of boldenone undecylenate and nandrolone decanoate at three-week intervals for 15 weeks decreases testis size, sperm production, sperm output, and semen quality in stallions (9). These effects may stem from feedback inhibition of gonadotropin release from the pituitary (8-10). Suppressed pituitary luteinizing hormone (LH) levels eventually lead to markedly decreased endogenous testosterone production, resulting in lowered intratesticular testosterone levels and cessation of spermatogenesis (11). Chemical alteration in structure of androgenic steroids can result in compounds that differ significantly in their relative peripheral effects on androgenic and anabolic parameters, even though separation between anabolic and antigonadotrophic activities is difficult to achieve (10).
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Testicular weight provides an accurate estimate of the amount of sperm-producing parenchyma in the testes of bulls (12,13), rams (14), and stallions (15). Testicular weights cannot be measured directly in breeding animals, so indirect measurements have been developed for live animals. Scrotal circumference is an accurate predictor of both testicular weight and sperm output in growing bulls (16) and rams (14). Scrotal width correlates significantly with testicular weight and sperm output in the stallion, although the measurement is less repeatable than is the measurement of scrotal circumference in bulls (17). At a certain point during spetmiogenesis, the nuclei of developing spermatids become extremely resistant to physical destruction. Testicular spermatid reserves can be estimated by homogenization of testicular tissue and subsequent enumeration of the elongated spermatids, which are resistant to homogenization, by hemacytometry. Daily sperm production (DSP) can be calculated by dividing the number of spermatid nuclei in a testicular homogenate by a time divisor which is the number of days'production these reserves represent (18). This has been done in rams (19), rabbits (20, 21), monkeys (22), bulls (12,13), and stallions (15,23). The technique can be used for comparing spermatozoa1 production rates among members of the same species in the absence of procedures for identification of stages of the cycle (24). Daily sperm production, as determined by testis homogenization, is correlated with testicular weight and daily sperm output in stallions ejaculated twice every four days (25). Daily sperm output, when corrected for sperm losses in collection equipment and gel, averaged 87 percent of DSP in horses 2-16 years of age (23). Both stanozolol and boldenone undecylenate are anabolic steroids currently approved by FDA for use in horses, although neither are approved for stallions or pregnant mares. The purpose of this study was to evaluate the effects of stanozolol and boldenone undecylenate on scrotal width, testis weight, and daily sperm production in mature pony stallions. MATERIALS AND METHODS Fifty mature (age 2-16 years) pony stallions were randomly assigned to one of five treatment groups, with 10 ponies per group. This randomization produced five treatment groups that were statistically equal with respect to age and weight. The treatment groups were as follows: Group 1 - controls, no treatment;
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THERIOGENOLOGY Group
-
mg/kg
1.1 mg/kg boldenone undecylenate (b) by deep intramuscular injection every 3 weeks for five treatments; Group 5 - 0.55 mg/kg boldenone undecylenate by deep intramuscular injection weekly for thirteen treatments. The treatments were given during May, June, and July. The ponies were weighed at the start of the experiment and once monthly to adjust the drug dosages for weight changes. Each pony's total scrotal width was measured with calipers (c) weekly. All ponies were castrated one week after the last treatment. At castration, the parietal tunics were dissected from the testes and epididymides, the epididymides were cut from the testes, and each testis was weighed. A section of testicular tissue was homogenized in a Waring blender in an aqueous solution containing approximately 100 ml 150 mM NaCl, 3.8 mM NaN3 and 0.05% (v/v) Triton X-100 per gram of parenchyma according to Amann, et al. (22,26). The homogenates were stored 22-26 hours at 5 degrees C before counting the spermatid and spermatozoa1 nuclei resistant to homogenization. Five additional untreated pony stallions were castrated. Immediately after castration , each testis was prepared as before and weighed. The tunica albuginea was stripped, cleaned, and weighed, and percent contribution of albuginea to the total testis weight was determined. The average tunica albuginea contribution to testis weight was used to determine testis parenchyma weight in the fifty experimental pony stallions. The number of spermatids and spermatozoa in the total mass of testicular parenchyma was calculated in the experimental Since a time divisor has not been determined for animals. pony stallion testicular spermatids, DSP was calculated by dividing the number of spermatids per testis by six days, which is the calculated time divisor for horse testicular spermatids under these conditions (23).
(a) "Winstrol-V" - Winthrop Veterinary, Sterling Animal Health Products, New York, NY 10016. (b) "Equipoise" - E. R. Squibb and Sons, Inc., Princeton, NJ
08540. (c) "Scrotal Caliper" - Lane Manufacturing,
Inc.,
Denver, CO
80222.
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THERIOGENOLOGY The variable scrotal width was analyzed as a repeated measurement design according to Gill and Hafs (18). The analysis of variance model contained the main effects of treatment (treatment groups l-5), time (weekly intervals), and the interaction of treatment and time. The least significant difference (LSD) statistic was used for mean separation. The variables weight (left testis weight, right testis weight, and combined testes weight) and daily sperm production were measured only at one time; thus a simple one-way analysis of variance was performed. The LSD statistic was used for mean separation. RESULTS AND DISCUSSION The correlation coefficient for final scrotal widths and combined testis weights at castration were highly significant Others have (0.93; P < 0.01) in this group of 50 pony stallions. reported the number of spermatozoa that a horse stallion is capable of producing is related to the size of the testes, providing the testes are normal, and mean scrotal width is correlated with the size of the testes (17). They reported a coefficient of correlation between combined testis weight and in vivo total testis width of 0.83 (P < 0.01). However, they caution that testicular weight accounted for only 60 percent of the variation in daily sperm output in horse stallions, and thus scrotal widths cannot be expected to predict daily sperm output accurately. The effects of stanozolol and boldenone undecylenate on scrotal width are shown in Figure 1. Scrotal widths (SW) among the groups did not differ at the start of the study. The mean scrotal widths of Groups 1 and 2 did not differ throughout the study except for weeks 1 and 6. For the last six weeks of the study (weeks 7-12) Group 1 and 2 did not differ, but were different from Groups 3-5 (P < 0.01). Also computed for each treatment group was the overall change from the initial scrotal width to the final scrotal width. The overall change in mean scrotal width was not different for Group 1 (+.49 cm) and Group 2 (t.24 cm). There was a significant difference (P z 0.01) in overall change in mean scrotal width between the control ponies (Group 1) and Groups 3-5 (-.26 cm, -.64 cm, and -1.30 cm, respectively).
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1983 VOL. 20 NO. 1
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6.5
I
1
OF ANABOLIC STEROIDS ON WIDTH IN PONY STALLIONS
1
I 2
3
I
4
I
5
I
WEEK
6
I
7
I
8
I
9
I
0 Controls (n=lO) 0.55 mg/kg stanozolol weekly (n=lO) : 1.1 mg/kg stanozolol every 3 weeks (n=lO) 0 1.1 mg/kg boldenone undecylenate every 3 weeks (n=lO) I 0.55 mg/kg boldenone undecylenate weekly (n=lO)
EFFECT SCROTAL
FIGURE
\
I
IO
I
11
/
t
I 12
THERIOGENOLOGY Mean scrotal widths for pony stallions injected with both boldenone undecylenate treatment regimens were significantly less than that of control pony stallions by five weeks after initiation of treatment, and continued to decrease and remained lower than controls throughout the rest of the treatment period. Other workers have stated that treatment of horse stallions with 1.1 mg and 4.4 mg/kg boldenone undecylenate once every three weeks resulted in scrotal widths significantly less than those in control stallions by five weeks after start of treatment, and scrotal widths remained so throughout a treatment period lasting fifteen weeks (19). Mean scrotal width for pony stallions injected with 1.1 mg/kg stanozolol every three weeks was significantly less than that of control pony stallions by seven weeks after start of treatment, and continued to decrease and remained lower than controls throughout the treatment period. Means for testicular weights and daily sperm production (DSP) are presented in Table 1. The average percent contribution of albuginea to total testis weight of 11.48%, derived from 5 additional ponies, was used to calculate DSP of testicular parenchyma. For both combined testis weights (CTW) and DSP, Group 2 was no different from Group 1, but was heavier and produced more sperm than all other groups (P < 0.05), and Group 5 was not different from Groups 3 and 4, but was lighter and produced fewer sperm than Groups 1 and 2 (P < 0.05). There was no significant difference in final mean scrotal width (SW), combined testis weight (CTW), and daily sperm production (OSP) between control pony stallions and pony stallions injected with 0.55 mg/kg stanozolol.weekly. Stanozolol, at this dose, had no detrimental effect on the pony testis parameters measured in this study. There was a significant decrease, compared to control ponies, in SW, CTW, and DSP in pony stallions injected with 0.55 mg/kg boldenone undecylenate weekly. That dosage of boldenone undecylenate had a marked detrimental effect on pony testis size and sperm production. Although there was a significant decrease, compared to control ponies, in SW in pony stallions injected with 1.1 mg/kg stanozolol and 1.1 mg/kg boldenone undecylenate every 3 weeks, there were no differences in CTW and DSP (P < This may have been due to the 0.15; P < 0.21 respectively). large variation in testicular size among individuals within each group. There may be differences in mechanism of action or in cumulative effect between stanozolol and boldenone undecylenate.
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5 c
*N = 10 ponies per group. **Values are corrected for average of 11.48% albuginea weight.
0.55 mg/kg weekly for 13 injections
1.208ab
2 * 304,
1.841,,
Daily Sperm Production (LISP) by Combined Testes, log**
Within columns, values with different subscripts are significantly different (P < 0.05).
92. 70ab
49. 3gab
43. 30ab
1.1 mg/kg every 3 weeks
boldenone undecylenate
boldenone undecylenate
93. 07ab
50. 48ab
42. 5gab
1.1 mg/kg every 3 weeks for 5 injections
stanozolol
3
for 5 injections
141.77,
72,52,
69.25,
0.55 mg/kg weekly for 13 injections
stanozolol
126.97,,
Combined
2
70.42,
56.54,,
-_
_Left
Right
Dose
none-controls
Treatment
1
Group*
TESTIS WEIGHT (gm)
EFFECTS OF 12 WEEKS OF ANABOLIC STEROID ON MEAN TESTIS WEIGHT AND DAILY SPERM PRODUCTION IN PONY STALLIONS
TABLE 1
2
6
3
3
THERIOGENOLOGY REFERENCES 1.
Beroza, G.A. Anabolic steroids in the horse, J. Am. Vet. Med. Assoc. 179:278-280 (1981).
2.
Barraguy, T.B. Anabolic steroids: J. 28_:28-33 (1974).
3.
Low, D.G. Medical Management of Polymic Renal Failure: Anabolic Agents, in Kirk, R.W. Current Veterinary Therapy, ed 7. W.B. Saunders Co., Philadelphia, 1980, p. 1102.
4.
McDonald, L.E. Hormones Affecting Reproduction, in Jones, L.M., Booth, N.H. and McDonald, L.E. Veterinary Pharmacology and Therapeutics, ed 4. Iowa State Univ. Press, 1977, pp. 635-637.
5.
Frasier, S.D. Androgens and athletes, Am. 3. Dis. Child. 125:479-480 (1973).
6.
Wade, N. Anabolic steroids: Doctors denounce them, but athletes aren't listening, Science, June 30, 1972, pp. 1399-1403.
7.
Tobin, T. Pharmacology review: The anabolic steroid androgen group of drugs, J. Equ ine Med. Surg. 2:163-165 (1978).
8.
Heller, H.G. Effects of progesterone and synthetic progestins on the reproductive physiology of normal men, Fed. Proc. -18:1057-1065 (1959).
9.
Squires, E.L. The Effect of Steroids, Testosterones on Reproduction, Harness Horseman International, Sept., 1980, pp. 29-32.
A review, Irish Vet.
10.
Boris, A., Stevenson, R.H. and Trmal, T. Comparative androgenic, myotropic and antigonadotrophic properties of some anabolic steroids, Steroids -15:61-71 (1970).
11.
Steinberger, E. Current status of research on hormonal contraception in the male, Research Frontiers in Fertility Regulation L:l-12 (1980).
12.
Almquist, J.O. and Amann, R.P. Reproductive capacity of dairy bulls. II. Gonadal and extragonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia, J. Dairy Sci. -44:16681678 (1961).
JULY 1983 VOL.20NO. 1
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THERIOGENOLOGY 13.
Amann, R.P. and Almquist, J.O. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production, J. Dairy Sci. 4!j:774-781 (1962).
14.
Lino, B.F. The output of spermatozoa in rams. II. Relationsh ip to scrotal c ircumference, testis weight, and the number of spermatozoa in different parts of the urogenital tract, Aust. J . Biol. Sci. 25:359-366 (1972).
15.
Gebauer, M.R., Pickett, B.W. and Swierstra, E.E. Reproductive physiology of the stallion. II. Daily production and output of sperm, J. Anim. Sci. -39:732-736 (1974).
16.
Coulter, G.H. and Foote, R.H. Bovine testicular measurements and indicators of reproductive performance and their relationship to productive traits in cattle: A review, Theriogenology -11:297-311 (1979).
17.
Gebauer, M.R., Pickett, B.W. and Swierstra, E.E. Reproductive physiology of the stallion: Daily sperm output and testicular measurements, J. Am. Vet. !4ed. Assoc. 165: 711-713 (1974).
18.
Amann, R.P. Sperm Production Rates, in Johnson, A.D. and Gomes, W.R. (eds.), The Testis, Vol. 1, Academic Press, New York, 1970, pp. 438-441.
19.
Ortavant, R. Le Cycle Spermatogenetique Chez le Belier. D. SC. Thesis, University of Paris, Paris (as cited by Amann, 1970) in Sperm Production Rates, in Johnson, A.D. and Gomes, W.R. (eds.), The Testis, Vol. 1, Academic Press, New York, 1970, pp. 438-441.
20.
Orgebin-Christ, M.C. Gonadal and epididymal sperm reserves in the rabbit: Estimation of the daily sperm production. J. Reprod. Fertil. -15:15-25 (1968).
21.
Amann, R.P. and Lambiase, J-T., Jr. The male rabbit. III. Determination of daily sperm production by means of J. Anim. Sci. -28:369-374 (1969). testicular homogenates.
22.
Amann, R.P., Johnson, L., Thompson, D.L., Jr. and Pickett, B.W. Daily spermatozoa1 production, epididymal spermatozoa1 reserves and transit time of spermatozoa through the epididymis of the rhesus monkey. Biol. Reprod. -15:586-592 (1976).
130
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THERIOGENOLOGY 23.
Thompson, D.L., Jr., Picket-ti,B.W., Squires, E.L. and Amann, R.P. Testicular measurements and reproductive characteristics in stallions. J. Reprod. Fert., Suppl. 27:13-17 (1979).
24.
Berndtson, W.E. spermatogenesis:
833 (1977).
Methods for quantifying mammalian J. Anim. Sci. -44:818A review.
R.P., Thompson, D.L., Jr., Squires, E.L. and Pickett, B.W. Effects of age and frequency of ejaculation on sperm production and extragonadal sperm reserves in stallions. J. Reprod. Fert., Suppl. -27:1-6 (1979).
25.
Amann,
26.
Amann, R.P. and Almquist, J.O. Reproductive capacity of dairy bulls. I. Technique for direct measurement of gonadal and extragonadal sperm reserves. J. Dairy Sci. 44:1537-1543 (1961).
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(a) (b) (c) (d)
Department of Veterinary Medicine and Surgery. Department of Animal Science. Department of Veterinary Anatomy and Physiology. Agriculture Experiment Station.
University of Missouri-Columbia,
Columbia, MO
65211
(e) Sterling-Winthrop Research Institute, Rensselaer, NY 12144, and Sterling Animal Health Products, New York, NY 10016. Received for publication: March 8, 1983 Accepted: May 12, 1983 ABSTRACT Fifty mature pony stallions were randomly assigned to one of five treatment groups: Group 1 - controls (no treatment), Group 2 - 0.55 mg/kg stanozolol weekly for 13 treatments, Group 3- 1.1 mg/kg stanozolol every 3 weeks for 5 treatments, Group 4 - 1.1 mg/kg boldenone undecylenate every 3 weeks for 5 treatments, and Group 5 - 0.55 mg/kg boldenone undecylenate weekly Scrotal widths (SW), combined testis weights for 13 treatments. (CTW), and daily sperm productions (DSP) were not different between Groups 1 and 2. Ponies in Group 5 had smaller SW (P < O.Ol), smaller CTW and decreased DSP compared to controls (P < 0.05). Although SW for ponies in Groups 3 and 4 were less than for controls (P < O.Ol), CTW and DSP were not different. The only treatment regime that did not alter SW, CTW, and DSP was Group 2 (0.55 mg/kg stanozolol weekly for 13 treatments).
____________________--____ Acknowledgements: This study was supported by a grant from the Winthrop Veterinary, Sterling Animal Health Products, Division of Sterling Drug Inc. The authors wish to express thanks to S. Mellskog, S. Rigby, J. Travers, L. Brockschmidt and N. Sights for technical assistance.
JULY 1983 VOL. 20 NO. 1
121
THERIOGENOLOGY INTRODUCTION Anabolic steroids are derivatives of testosterone which are altered to provide maximal anabolic action and minimal androgenic side effects (1,2). The administration of anabolic steroids causes a retention of body water, nitrogen, potassium, sodium, calcium, chloride, sulfate, and phosphorus ions. Muscle mass increases due to promotion of positive nitrogen balance by stimulating protein production and reducing protein destruction (1). Anabolic steroids also stimulate production and/or release of erythropoietin from the kidneys and stimulate undifferentiated stem cells in bone marrow to enter an erythroid critical stage (3). Therapeutic uses for anabolic steroids are to correct tissue-depleting processes following systemic disease, malnutrition, parasitism, or wasting disease of old age; to speed tissue repair following surgery or trauma; and to promote nonspecific stimulation of erythropoiesis (4). They also are used to reverse some of the effects of glucocorticoids, specifically negative nitrogen and calcium balance and catabolic effects on muscle and connective tissue (2). Many human athletes, physicians, veterinarians, and trainers attest to gains in physical strength, stamina, and mental attitude following use of anabolic steroids (1, 5-7). It is not yet feasible to produce an anabolic steroid without androgenic activity (2). This androgenic activity accounts for most of the undesirable side effects of these drugs. If given early in life in large quantities, anabolic steroids may result in premature closure of the epiphyseal plates and prevent further growth in bone length (7). Administration of norethandrolone for 8-25 weeks results in loss of libido, decreased potency, diminished testicular size, and azoospermia in man (8). Administration of boldenone undecylenate and nandrolone decanoate at three-week intervals for 15 weeks decreases testis size, sperm production, sperm output, and semen quality in stallions (9). These effects may stem from feedback inhibition of gonadotropin release from the pituitary (8-10). Suppressed pituitary luteinizing hormone (LH) levels eventually lead to markedly decreased endogenous testosterone production, resulting in lowered intratesticular testosterone levels and cessation of spermatogenesis (11). Chemical alteration in structure of androgenic steroids can result in compounds that differ significantly in their relative peripheral effects on androgenic and anabolic parameters, even though separation between anabolic and antigonadotrophic activities is difficult to achieve (10).
122
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THERIOGENOLOGY
Testicular weight provides an accurate estimate of the amount of sperm-producing parenchyma in the testes of bulls (12,13), rams (14), and stallions (15). Testicular weights cannot be measured directly in breeding animals, so indirect measurements have been developed for live animals. Scrotal circumference is an accurate predictor of both testicular weight and sperm output in growing bulls (16) and rams (14). Scrotal width correlates significantly with testicular weight and sperm output in the stallion, although the measurement is less repeatable than is the measurement of scrotal circumference in bulls (17). At a certain point during spetmiogenesis, the nuclei of developing spermatids become extremely resistant to physical destruction. Testicular spermatid reserves can be estimated by homogenization of testicular tissue and subsequent enumeration of the elongated spermatids, which are resistant to homogenization, by hemacytometry. Daily sperm production (DSP) can be calculated by dividing the number of spermatid nuclei in a testicular homogenate by a time divisor which is the number of days'production these reserves represent (18). This has been done in rams (19), rabbits (20, 21), monkeys (22), bulls (12,13), and stallions (15,23). The technique can be used for comparing spermatozoa1 production rates among members of the same species in the absence of procedures for identification of stages of the cycle (24). Daily sperm production, as determined by testis homogenization, is correlated with testicular weight and daily sperm output in stallions ejaculated twice every four days (25). Daily sperm output, when corrected for sperm losses in collection equipment and gel, averaged 87 percent of DSP in horses 2-16 years of age (23). Both stanozolol and boldenone undecylenate are anabolic steroids currently approved by FDA for use in horses, although neither are approved for stallions or pregnant mares. The purpose of this study was to evaluate the effects of stanozolol and boldenone undecylenate on scrotal width, testis weight, and daily sperm production in mature pony stallions. MATERIALS AND METHODS Fifty mature (age 2-16 years) pony stallions were randomly assigned to one of five treatment groups, with 10 ponies per group. This randomization produced five treatment groups that were statistically equal with respect to age and weight. The treatment groups were as follows: Group 1 - controls, no treatment;
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THERIOGENOLOGY Group
-
mg/kg
1.1 mg/kg boldenone undecylenate (b) by deep intramuscular injection every 3 weeks for five treatments; Group 5 - 0.55 mg/kg boldenone undecylenate by deep intramuscular injection weekly for thirteen treatments. The treatments were given during May, June, and July. The ponies were weighed at the start of the experiment and once monthly to adjust the drug dosages for weight changes. Each pony's total scrotal width was measured with calipers (c) weekly. All ponies were castrated one week after the last treatment. At castration, the parietal tunics were dissected from the testes and epididymides, the epididymides were cut from the testes, and each testis was weighed. A section of testicular tissue was homogenized in a Waring blender in an aqueous solution containing approximately 100 ml 150 mM NaCl, 3.8 mM NaN3 and 0.05% (v/v) Triton X-100 per gram of parenchyma according to Amann, et al. (22,26). The homogenates were stored 22-26 hours at 5 degrees C before counting the spermatid and spermatozoa1 nuclei resistant to homogenization. Five additional untreated pony stallions were castrated. Immediately after castration , each testis was prepared as before and weighed. The tunica albuginea was stripped, cleaned, and weighed, and percent contribution of albuginea to the total testis weight was determined. The average tunica albuginea contribution to testis weight was used to determine testis parenchyma weight in the fifty experimental pony stallions. The number of spermatids and spermatozoa in the total mass of testicular parenchyma was calculated in the experimental Since a time divisor has not been determined for animals. pony stallion testicular spermatids, DSP was calculated by dividing the number of spermatids per testis by six days, which is the calculated time divisor for horse testicular spermatids under these conditions (23).
(a) "Winstrol-V" - Winthrop Veterinary, Sterling Animal Health Products, New York, NY 10016. (b) "Equipoise" - E. R. Squibb and Sons, Inc., Princeton, NJ
08540. (c) "Scrotal Caliper" - Lane Manufacturing,
Inc.,
Denver, CO
80222.
124
JULY
1983 VOL. 20 NO. 1
THERIOGENOLOGY The variable scrotal width was analyzed as a repeated measurement design according to Gill and Hafs (18). The analysis of variance model contained the main effects of treatment (treatment groups l-5), time (weekly intervals), and the interaction of treatment and time. The least significant difference (LSD) statistic was used for mean separation. The variables weight (left testis weight, right testis weight, and combined testes weight) and daily sperm production were measured only at one time; thus a simple one-way analysis of variance was performed. The LSD statistic was used for mean separation. RESULTS AND DISCUSSION The correlation coefficient for final scrotal widths and combined testis weights at castration were highly significant Others have (0.93; P < 0.01) in this group of 50 pony stallions. reported the number of spermatozoa that a horse stallion is capable of producing is related to the size of the testes, providing the testes are normal, and mean scrotal width is correlated with the size of the testes (17). They reported a coefficient of correlation between combined testis weight and in vivo total testis width of 0.83 (P < 0.01). However, they caution that testicular weight accounted for only 60 percent of the variation in daily sperm output in horse stallions, and thus scrotal widths cannot be expected to predict daily sperm output accurately. The effects of stanozolol and boldenone undecylenate on scrotal width are shown in Figure 1. Scrotal widths (SW) among the groups did not differ at the start of the study. The mean scrotal widths of Groups 1 and 2 did not differ throughout the study except for weeks 1 and 6. For the last six weeks of the study (weeks 7-12) Group 1 and 2 did not differ, but were different from Groups 3-5 (P < 0.01). Also computed for each treatment group was the overall change from the initial scrotal width to the final scrotal width. The overall change in mean scrotal width was not different for Group 1 (+.49 cm) and Group 2 (t.24 cm). There was a significant difference (P z 0.01) in overall change in mean scrotal width between the control ponies (Group 1) and Groups 3-5 (-.26 cm, -.64 cm, and -1.30 cm, respectively).
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1983 VOL. 20 NO. 1
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6.5
I
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OF ANABOLIC STEROIDS ON WIDTH IN PONY STALLIONS
1
I 2
3
I
4
I
5
I
WEEK
6
I
7
I
8
I
9
I
0 Controls (n=lO) 0.55 mg/kg stanozolol weekly (n=lO) : 1.1 mg/kg stanozolol every 3 weeks (n=lO) 0 1.1 mg/kg boldenone undecylenate every 3 weeks (n=lO) I 0.55 mg/kg boldenone undecylenate weekly (n=lO)
EFFECT SCROTAL
FIGURE
\
I
IO
I
11
/
t
I 12
THERIOGENOLOGY Mean scrotal widths for pony stallions injected with both boldenone undecylenate treatment regimens were significantly less than that of control pony stallions by five weeks after initiation of treatment, and continued to decrease and remained lower than controls throughout the rest of the treatment period. Other workers have stated that treatment of horse stallions with 1.1 mg and 4.4 mg/kg boldenone undecylenate once every three weeks resulted in scrotal widths significantly less than those in control stallions by five weeks after start of treatment, and scrotal widths remained so throughout a treatment period lasting fifteen weeks (19). Mean scrotal width for pony stallions injected with 1.1 mg/kg stanozolol every three weeks was significantly less than that of control pony stallions by seven weeks after start of treatment, and continued to decrease and remained lower than controls throughout the treatment period. Means for testicular weights and daily sperm production (DSP) are presented in Table 1. The average percent contribution of albuginea to total testis weight of 11.48%, derived from 5 additional ponies, was used to calculate DSP of testicular parenchyma. For both combined testis weights (CTW) and DSP, Group 2 was no different from Group 1, but was heavier and produced more sperm than all other groups (P < 0.05), and Group 5 was not different from Groups 3 and 4, but was lighter and produced fewer sperm than Groups 1 and 2 (P < 0.05). There was no significant difference in final mean scrotal width (SW), combined testis weight (CTW), and daily sperm production (OSP) between control pony stallions and pony stallions injected with 0.55 mg/kg stanozolol.weekly. Stanozolol, at this dose, had no detrimental effect on the pony testis parameters measured in this study. There was a significant decrease, compared to control ponies, in SW, CTW, and DSP in pony stallions injected with 0.55 mg/kg boldenone undecylenate weekly. That dosage of boldenone undecylenate had a marked detrimental effect on pony testis size and sperm production. Although there was a significant decrease, compared to control ponies, in SW in pony stallions injected with 1.1 mg/kg stanozolol and 1.1 mg/kg boldenone undecylenate every 3 weeks, there were no differences in CTW and DSP (P < This may have been due to the 0.15; P < 0.21 respectively). large variation in testicular size among individuals within each group. There may be differences in mechanism of action or in cumulative effect between stanozolol and boldenone undecylenate.
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1983 VOL. 20 NO. 1
127
5 c
*N = 10 ponies per group. **Values are corrected for average of 11.48% albuginea weight.
0.55 mg/kg weekly for 13 injections
1.208ab
2 * 304,
1.841,,
Daily Sperm Production (LISP) by Combined Testes, log**
Within columns, values with different subscripts are significantly different (P < 0.05).
92. 70ab
49. 3gab
43. 30ab
1.1 mg/kg every 3 weeks
boldenone undecylenate
boldenone undecylenate
93. 07ab
50. 48ab
42. 5gab
1.1 mg/kg every 3 weeks for 5 injections
stanozolol
3
for 5 injections
141.77,
72,52,
69.25,
0.55 mg/kg weekly for 13 injections
stanozolol
126.97,,
Combined
2
70.42,
56.54,,
-_
_Left
Right
Dose
none-controls
Treatment
1
Group*
TESTIS WEIGHT (gm)
EFFECTS OF 12 WEEKS OF ANABOLIC STEROID ON MEAN TESTIS WEIGHT AND DAILY SPERM PRODUCTION IN PONY STALLIONS
TABLE 1
2
6
3
3
THERIOGENOLOGY REFERENCES 1.
Beroza, G.A. Anabolic steroids in the horse, J. Am. Vet. Med. Assoc. 179:278-280 (1981).
2.
Barraguy, T.B. Anabolic steroids: J. 28_:28-33 (1974).
3.
Low, D.G. Medical Management of Polymic Renal Failure: Anabolic Agents, in Kirk, R.W. Current Veterinary Therapy, ed 7. W.B. Saunders Co., Philadelphia, 1980, p. 1102.
4.
McDonald, L.E. Hormones Affecting Reproduction, in Jones, L.M., Booth, N.H. and McDonald, L.E. Veterinary Pharmacology and Therapeutics, ed 4. Iowa State Univ. Press, 1977, pp. 635-637.
5.
Frasier, S.D. Androgens and athletes, Am. 3. Dis. Child. 125:479-480 (1973).
6.
Wade, N. Anabolic steroids: Doctors denounce them, but athletes aren't listening, Science, June 30, 1972, pp. 1399-1403.
7.
Tobin, T. Pharmacology review: The anabolic steroid androgen group of drugs, J. Equ ine Med. Surg. 2:163-165 (1978).
8.
Heller, H.G. Effects of progesterone and synthetic progestins on the reproductive physiology of normal men, Fed. Proc. -18:1057-1065 (1959).
9.
Squires, E.L. The Effect of Steroids, Testosterones on Reproduction, Harness Horseman International, Sept., 1980, pp. 29-32.
A review, Irish Vet.
10.
Boris, A., Stevenson, R.H. and Trmal, T. Comparative androgenic, myotropic and antigonadotrophic properties of some anabolic steroids, Steroids -15:61-71 (1970).
11.
Steinberger, E. Current status of research on hormonal contraception in the male, Research Frontiers in Fertility Regulation L:l-12 (1980).
12.
Almquist, J.O. and Amann, R.P. Reproductive capacity of dairy bulls. II. Gonadal and extragonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia, J. Dairy Sci. -44:16681678 (1961).
JULY 1983 VOL.20NO. 1
129
THERIOGENOLOGY 13.
Amann, R.P. and Almquist, J.O. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production, J. Dairy Sci. 4!j:774-781 (1962).
14.
Lino, B.F. The output of spermatozoa in rams. II. Relationsh ip to scrotal c ircumference, testis weight, and the number of spermatozoa in different parts of the urogenital tract, Aust. J . Biol. Sci. 25:359-366 (1972).
15.
Gebauer, M.R., Pickett, B.W. and Swierstra, E.E. Reproductive physiology of the stallion. II. Daily production and output of sperm, J. Anim. Sci. -39:732-736 (1974).
16.
Coulter, G.H. and Foote, R.H. Bovine testicular measurements and indicators of reproductive performance and their relationship to productive traits in cattle: A review, Theriogenology -11:297-311 (1979).
17.
Gebauer, M.R., Pickett, B.W. and Swierstra, E.E. Reproductive physiology of the stallion: Daily sperm output and testicular measurements, J. Am. Vet. !4ed. Assoc. 165: 711-713 (1974).
18.
Amann, R.P. Sperm Production Rates, in Johnson, A.D. and Gomes, W.R. (eds.), The Testis, Vol. 1, Academic Press, New York, 1970, pp. 438-441.
19.
Ortavant, R. Le Cycle Spermatogenetique Chez le Belier. D. SC. Thesis, University of Paris, Paris (as cited by Amann, 1970) in Sperm Production Rates, in Johnson, A.D. and Gomes, W.R. (eds.), The Testis, Vol. 1, Academic Press, New York, 1970, pp. 438-441.
20.
Orgebin-Christ, M.C. Gonadal and epididymal sperm reserves in the rabbit: Estimation of the daily sperm production. J. Reprod. Fertil. -15:15-25 (1968).
21.
Amann, R.P. and Lambiase, J-T., Jr. The male rabbit. III. Determination of daily sperm production by means of J. Anim. Sci. -28:369-374 (1969). testicular homogenates.
22.
Amann, R.P., Johnson, L., Thompson, D.L., Jr. and Pickett, B.W. Daily spermatozoa1 production, epididymal spermatozoa1 reserves and transit time of spermatozoa through the epididymis of the rhesus monkey. Biol. Reprod. -15:586-592 (1976).
130
JULY
1983 VOL. 20 NO. 1
THERIOGENOLOGY 23.
Thompson, D.L., Jr., Picket-ti,B.W., Squires, E.L. and Amann, R.P. Testicular measurements and reproductive characteristics in stallions. J. Reprod. Fert., Suppl. 27:13-17 (1979).
24.
Berndtson, W.E. spermatogenesis:
833 (1977).
Methods for quantifying mammalian J. Anim. Sci. -44:818A review.
R.P., Thompson, D.L., Jr., Squires, E.L. and Pickett, B.W. Effects of age and frequency of ejaculation on sperm production and extragonadal sperm reserves in stallions. J. Reprod. Fert., Suppl. -27:1-6 (1979).
25.
Amann,
26.
Amann, R.P. and Almquist, J.O. Reproductive capacity of dairy bulls. I. Technique for direct measurement of gonadal and extragonadal sperm reserves. J. Dairy Sci. 44:1537-1543 (1961).
JULY 1983 VOL. 20 NO. 1
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