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The effects of stanozolol and boldenone undecylenate on plasma testosterone and gonadotropins and on testis histology in pony stallions
THERIOGENOLOGY
THE EFFECTS OF STANOZOLOL AND BOLDENONE UNDECYLENATE ON PLASMA TESTOSTERONE AND GONADOTROPINS AND ON TESTIS HISTOLOGY IN PONY STALLIONS M.C. Garcia’, V.K. Ganjam2 T.L.Blanchard3 E.Brown2, K.Hfrdin3, R.S.Youngqt&, W.E.Loch ‘, M.R.Ellersieck4, and J.M.Balke
R.G.Elmore3,
University of Pennsylvania, New Bolton Center, 1 School of Veterinary Medicin Kennett Square, PA 19348. College of V?terinary Medicine, University of College4 of Veterinary Medicine, Missouri-Columbia, Columbia, MO 65211. College of Agriculture, Texas A&M University, College Station, TX 72847. RR 1, Box 27, Seadog, Nanoosc University of Missouri, Columbia, MO 65211. Bay, Brit. Col., Canada VOR 2R0. Received for publication: Accepted:
December
27,
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day 19, 19~7
ABSTRACT Fifty 2- to 16- yr old pony stallions were randomly assigned to one of five treatments: Group 1, controls (no treatment); Group 2, 0.55 mg/k stanozolol weekly for 13 treatments; Group 3, 1.1 mg/kg stanozolol every ‘5 wk for 5 treatments; Group 4, 1.1 mg/kg boldenone undecylenate every 3 wk for 5 treatments; and Group 5, 0.55 boldenone undecylenate weekly for 13 treatments. Mean lasma testosterone levels for Groups 2,4, and 5 were elevated over controls (P < 0.11) at 2, 8, and 9 wk, respectively. Testosterone levels for ponies in Group 3 did not differ from controls (P > 0.05). There were no differences in mean plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels among groups (P > 0.05). Daily spermatid production per gram of testicular parenchyma (DSP/gm) in Group 5 was lower than in controis (P < 0.05) whereas DSP/gm was not different among groups 1 to 4 (P > 0.05). There were no differences among groups (P > 0.05) in the percentage of Stage 8 tubules or relative number of Leydig cells. The mean diameter of Leydig cells was less for Group 5 than for controls (P < O.OS),but was not different for Groups 1 to 3 (P > 0.05). Key words:
stallion, anabolic gonadotropins
steroid,
spermatogencsis,
testosterone.
INTRODUCTION Anabolic steroids are derivatives of testosterone that are altered to provide maximal anabolic action and minimal androgenic side effects (1,2). It is not yet feasible to produce an anabolic steroid without androgenic activit (2). This androgenic activity accounts for most of the undesirable srde effects o tythese drugs. Administration of norethandrolone for 8 to 25 wk results in loss of libido, decreased potency, diminished testicular size, and azoospermia in men (3). Acknowledgments: Portions of this study were supported by the Winthrop Sterling Animal Health Products, Division of Sterling Drug, Inc., and in part by BRSG 520559 awarded by NIH. The gifts of anti-human FSH from the National Hormone and Pituitary Program, NIH, and of RIA materials from Dr. O.J. Ginther are apDrs. D. Kinden, E. Swierstra, and R.M. Kenney, and M. preciated. We thank Floyd, S. Mellskog, S. Rigby, J. Travers, L. Brockschmidt, and N. Sights for technical assistance.
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Administration of boldenone undecylenate and nandrolone decanoate at 3wk intervals for 15 wk decreases testis size, sperm production, sperm output, and semen quality in stallions (4). These effects may stem from feedback inhibition of gonadotropin release from the pituitary (3-5). Suppressed pituitary LH levels eventually lead to markedly decreased endogenous testosterone production, resulting in lowered intratesticular testosterone levels and cessation of spermatogenesis (6). 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 se aration between anabolic and antigonadotrophic activities is difficult to achieve P5). Currently there are only two anabolic steroids (stanozolol and boldenone undecylenate) approved by the Federal Drug Administration for use in horses, although neither is approved for stallions or pregnant mares. In a previous paper WC reported that boldenone undecylenate (0.55 mg/kg weekly for 13 wk) resulted in smaller scrotal width, decreased testis weight, and depressed daily sperm production comnared with untreated control nonv stallions (7). Stanozolol (1.1 mg/kg weekly for 13 wk) and boldenone undecyjenate (1.1 mg/kg every 3 wk for five-inicctions) did not depress daily sperm production. The purpose of this paper is to report the effects of these drugs on plasma testosterone and gonadotropins and on testis histology in those ponies. MATERIALS
AND METHODS
Fifty 2- to 16-year-old pony stallions of mixed breeding were randomly assigned to one of five treatment groups, with 10 ponies per group. This randomization produced five treatment groups that were statistically comparable with respect to age and weight. The treatment groups were as follows: Group 1, controls, no treatment; Group 2, 0.55 mg/kg stanozolola by deep i.m. injection weekly for 13 treatments; Group 3, 1.1 m@g stanozolol by deep i.m. injegtion every 3 wk for five treatments; Group 4, 1.1 mg&g boldenone undecylenate by deep i.m. injection every 3 wk for five treatments; Group 5, 0.55 m@g boldenone undecylenate by deep i.m. injection weekly for 13 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. Blood samples from each pony were collected in heparinized tubes weekly prior to treatment. Immediately after collection, each tube of blood was placed on ice and maintained upright until centrifuged prior to collection of plasma. Plasmas were frozen until assayed for hormones. Total plasma unconjugated testosterone was quantified using a double antibody radioimmunoassay (RIA; RSL testosterone assay No. 1102-D).’ The first antibody was raised against testosterone 19-carboxymethylether-bovine serum albumin in rabbits. The second antibody was goat antirabbit gammaglobulin. The sensitivity of the assay was about 0.1 ng/ml at 85% binding. The cross reactivities of the other steroids (testosterone = 100% at 50% displacement) are: 5 alpha dihydrotestosterone (DHT), 3.4%; androstenedione, 0.56%; other 17keto_androgens, ~1%; estrogens and progestagens, ~0.05%. Neither stanozolol nor boldenone undecylenate specifically bound to this antibody. aWinstrol-V
NY.
-Winthron
Veterinary.<. Sterling Animal Health Products,
New York,
‘“Equipoise” - E.R. Squibb and Sons, Inc., Princeton, NJ. ‘Radioassay Systems Laboratories, Inc., Carson, CA.
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Plasma concentrations of LH and FSH were determined by double antibody RIAs(8). The LH assay used antibody against equine chorionic gonadotropin raised in a rabbit for the first antibody and sheep antirabbit gammaglobulin for the second antibody. The sensitivity of the assay at 85% binding was 0.1 rig/ml. Mean intraand inter-assay coefficients of variation were 4.0 and 9.5%, respectively, for triplicate tubes of pooled estrus plasma run in the last three assays. The FSH RTA is a heterologous system which used antihuman FSH for the first antibody and sheep antirabbit ammaglobulin for the second antibody. The cross reactivity of equine LH in the F .!H assay was less than 1%. The assay sensitivity was about 0.2 ng/ml at 85% binding. The mean intra- and inter-assay coefficients of variation were 8 and 12%, respectively, for triplicate samples of pooled diestrus plasma run in the last three assays. All ponies were castrated following blood sampling 1 wk after the last trcatment. The parietal tunics were dissected from the testes and epididymides, the epididymides were separated from the testes, and each testis was weighed. Each testis was sliced and a center section was stored at -5’C for determination of spcrmatid reserves (7). The average daily sperm produced per gram of testicular parenchyma was calculated with the following formula: (DSP/gm of left testis x left testis parenchyma weight + DSP/gm of right testis x right testis parenchyma weight)/total The remainder of the testis was fixed in mercuric weight of testicular parenchyma. formol for 3 d, washed in running water for 24 h. and stored in 70% alcohol until being trimmed. A section from the middle plane (which was adjacent to the section taken for determination of spermatid reserves) of each testis was embedded in paraffin, sectioned, and stained with periodic-acid-Schiff-hematoxylin. These sections were examined histologically for determination of the pcrcentage of seminiferous tubules in Stage 8 (Figure 3) according to the method of Swierstra et al (9). Calculation of DSP by testicular homogenization assumes that at some stage elongated spermatids become resistant to homogenization. Howcvcr. the technique does not establish whether spcrmatogenesis is being complctcd. Therefore, histologic assessment was used to confirm completion of spermatogenesis. Because Leydig cells appeared to be less prominent in the testes of stallions in some treatment groups, the number and size of Lcydig cells were dctcrmined retrospectively. The number of Leydig cells was determined by counting the number of hits of 25 randomly selected dots in a Chalkley eyepiece (10) on the nuclei of Lcydig cells in 100 fields using a Zeiss Universal microscope.” Lcydig ccl1 diameters were measured using an ocular micrometer by obtaining two mcasurcmcnts perpendicular to each other on 25 Leydig cells per testis, each in a different field. The variables testosterone, LH, and FSH were analyzed as a rcpcatctl measures analysis of variance according to Gill and Hafs (11). The analysis ol variance model contained the main effects of treatment (Groups 1 to S), time (weekly intervals), and the interaction of treatment and time. Animal within trcament was used as an error term (Error A) for determining significant treatment effect. The animal by time within treatment (Error B) was used to test time and cffects of treatment by time. The least significant difference statistic was used for mean separation. The variable percentage of Stage 8 tubules was transformed to square roots and a one-way analysis of variance was performed. The variables Leydig cell number and size were analyzed by one-way analysis of variance, and least significant difference was used for mean separation. ’ Carl Zeiss, Germany (Seller Instrument
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Mfg. Co., St. Louis, MO).
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rHERIOGENOLOGY
RESULTS Initial testosterone levels did not differ among groups (Figure 1; P > 0.05). Testosterone levels for Group 2 did not differ from those for controls (P > 0.05) except during Weeks 1 and 3, when those of Group 2 were higher (P < 0.01). Testosterone levels for Group 3 did not differ from controls (P > 0.05). Testosterone levels for Group 4 were higher than those for controls during Weeks 1,3 to 5,7,9 to 11,and 13 (P c 0.01).
FIGURE
?
600 500
400 300
__-
0
1
Group Group Group Group Group
Figure 1.
2
1 2 3 4 5
l
0 A A n
3
4
Controls 0.55 mg/kg 1 .I0 mg/kg 1.10 mg/kg 0.55 mg/kg
5 WfL
stanozolol stanozolol boldenone boldenone
7
6
9
10
weekly every 3 weeks undecylenate undecylenate
Effects of stanozolol and boldenone terone in pony stallions.
11
12
13
every 3 weeks weekly
undecylenate
on plasma testos-
Testosterone levels for Group 5 were higher (P < 0.01) than those for controls during Weeks 1. 3. 5 to 7. 9. 10. 12. and 13. The correlations between testis measurem&ts (testisweight, DSP’ per gram of testis, daily sperm production), and testosterone values at castration; 0.20, -0.02, and 0.13, respectively, were not significant (P > 0.05). There were no differences in mean levels of gonadotropins among the treatment groups (Figure 2; P > 0.05). There was a significant effect of time on mean plasma LH concentration (P < 0.05).
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-1
1
3
5
7
9
11
13
WEEK
Figure 2.
Bi-weekly mean plasma LH and FSH concentration
in pony stallions.
Due to a significant age effect on average DSP/gm of testicular parenchyma, an analysis of covariance was performed to adjust for the effect of age. Average DSP/gm of testicular parenchyma was lower in Group 5 ponies (Table 1; P < 0.05) than in control ponies. There were no differences in average DSP/gm of testicular parenchyma in ponies in Groups 1 to 4 (P > 0.05). There also were no differences in percentage of Stage 8 tubules among treatment groups (Table 1; P > 0.05). Mean number of Leydig cells did not differ among groups (Table 1; P > 0.05). However, the mean diameter of Leydig cells was less for ponies in Group 5 (P < 0.05) than for control ponies. The mean diameter of Leydig cells was not different for ponies in Groups 1 to 4 (P > 0.05). Marked cytoplasmic vacuolization, shrunken tubules and Leydig cells, and multinucleated giant cells containing phagocytosed spermatids indicative of tcstitular degeneration (Figure 4) occurred in the testes of some ponies. This was most often observed in testes of Group 5 stallions and was more pronounced in younger animals. Correlation coefficients (r) for the histologic parameters measured were as follows: 0.74 (P c 0.01) for percentage of Sta e 8 tubules and DSP/gm of testicular parenchyma, 0.35 (P < 0.01) for percentage of s tage 8 tubules and number of Leydig cells, 0.67 (P < 0.01) for percentage of Stage 8 tubules and diameter of Leydig cells,
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0.25 (P < 0.09) for number of Leydig cells and DSP/gm of testicular parenchyma, and 0.52 (P < 0.01) for diameter of Leydig cells and DSP/gm of testicular parenchyma. The simple correlation coefficients and probability values between LH and testosterone were Group 1, r = 0.22, P c 0.06; Group 2, r = 0.20, P c 0.08; Group 3, r = 0.24, P -Z0.05; Group 4, r = 0.15, P < 0.23; Group 5, r = -0.06, P < 0.63. No significant correlations between FSH and testosterone were observed for any of the five treatment groups.
Table 1.
Group
Effects of stanozolol and boldenone undecylenate on daily spermatid production per gram (DSP/gm) of testis and on testis histology in pony stallions.
Treatment
n
DSPigm of testis
Stage 8 tubules (%)
Relative number of Leydig cells
Mean diameter of Leydig cells
X+SEM
X+SEM
X+SEM
XzSEM
1
control
10
16.22+1.30
9.61k1.12
40.39+5.77
14.75+0.81
2
stanozolol 0.55 mg!kg weekly
10
17.51+1.95
8.35+1.65
45.3027.26
15.43+0.60
3
stanozolol 1.1 mg/kg every 3 wk
10
15.6022.07
8.45~1.42
39.8OL5.38
14.36+0.81
4
boldenone undecylenate 1.1 mg/kg every 3 wk
10
10.52t2.64
6.6021.92
50.30+7.13
12.97+1.20
5
boldenone undecylenate 0.55 mg/kg weekly
10
9.56+2.46*
4.8Ot1.99
44.5529.71
11.27+0.72*
* Differs from controls (P -Z0.05).
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Figure 3.
Photomicrograph of testis histology for Pony 13 (Group 1, control). Left testis, middle section with seminiferous tubule in Sta e 8 of spermatogenesis. dig cells (L). Elongated spermatids (8 p) lmc the tubule lumen . 520x.
Figure 4.
Photomicrograph of testis histology for Pony 54 (Group 5~0.55 mg’kg boldenone undecylenate once weekly). Left testis, middle section. Few small Leydig cells (L) are present. Lumen (LU) of seminiferous tubule has no elongating spermatids but has desquamatcd spcrmatocytcs (D). 520x.
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DISCUSSION Adverse effects of exogenous testosterone are thought to result from feedback inhibition of gonadotropm release from the pituitary (12-15). Pelletier suggested that testosterone may depress gonadotropin releasing hormone synthesis in rams (16). Suppressed pituitary LH levels eventually lead to markedly decreased endogenous testosterone production, thus resulting in lowered intratesticular testosterone levels: some workers feel that these lowered levels result in cessation of spermatogenesis (6). However, critical examinations of the necessity of high concentrations of testosterone in the milieu surrounding the developing germ cells for the maintenance of normal spermatogenesis have not been performed. Qualitatively normal spermatogenesis, in spite of lower than normal intratesticular testosterone concentration, has been reported in intact (17) and hypophysectomized (18) rats administered testosterone. However, while testosterone treatment reduces the percentage of tubules with differentiating spermatozoa, considerable reduction in testis wei ht may occur even though spermatogenesis may still occur in some tubules (18 ). Mean plasma LH concentration at the start of the experiment (3.7 @ml) was similar to that reported for adult ponies >3 yr old (3.2 rig/ml) and higher than that reported for yearling pony colts (about 1 rig/ml) using the same assay system 20). Similarly, the mean plasma FSH concentration at the start of the experiment 11.5 rig/ml) was comparable to that reported in adult ponies (7.0 rig/ml) using the same assay system. The finding that administering boldenone undecylenate did not affect plasma onadotropins but increased plasma testosterone and decreased testis function is dif. Brcult to explain. However, the correlation between LH and testosterone for Group 1 was positive and tended to be significant while that for Group 5 was negative. The failure to find decreased concentrations of plasma gonadotropins as a result of boldenone undecylenate treatment in this study may be due to breed differences or may be due to the infrequent (biweekly) sampling regimen. There is evidence for episodic fluctuation in the secretion of androgens in the stallion (21), and a secretory attern of LH secretion has been described (22). Administration of anabolic steroi .d”s to men reduces plasma concentrations of both testosterone and gonadotropins and reduces spermatogenesis (23-27). Likewise, administration of 1.1 and 4.4 mg/kg boldenone undecylenate and 1.1 mg/kg nandrolone decanoate at 3 wk intervals for 15 wk decreased serum LH concentration, testis size, sperm production, sperm output, and semen quality in horse stallions (28). The mean percentage of Stage 8 tubules in testes of control ponies (9.61%) was lower than that reported for horse stallions (15.7%) (9), possibly due to breed difference or to technician error. Only Stage 8 tubules were classified in our study, whereas all tubules were classified into one of eight stages in the horse testes studied. Not having to place all tubules into a category could also account for the lower percentage of Stage 8 tubules found in the ony stallions. Regardless, because of the high correlation between average D !$P/gm of testicular parenchyma and percentage of Stage 8 tubules (0.75; P < O.OOOl),percentage of Stage 8 tubules is a valid comparison of relative treatment effects between groups. We had previously found that treating ponies with 0.55 mgkg boldenone undecylenate once weekly significantly reduced testis weight and daily sperm production (7). This dosage of boldenone undecylenate apparently decreases the efficiency of spermatogenesis as well as the amount of testicular parenchyma available to produce spermatozoa. Others have demonstrated that testosterone (12,13,29) and anabolic steroid administration (28) arrests spermatogenesis at the spermatogonia or spermatocyte stages.
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It is not known if there are any adverse long-term effects of anabolic steroids on reproductive capacity of stallions. The adverse effects of anabolic steroids are thought, in general, to be reversible in man (25). Adverse effects of administration of anabolic steroids for 1 yr to growing fillies persisted for at least 6 mo after trcatment ceased (30). Adverse effects of anabolic steroids appeared to be more pronounced in the younger ponies. Testicular degeneration may preclude return to a normal level of spermatogenesis. Despite the failure to demonstrate decreased concentrations of plasma gonadotropins, Leydig cell diameter diminished in the testes of ponies treated with 0.55 mg/kg boldenone undecylenate weekly. The failure to detect changes in gonadotropin levels may have been due to a change in the pattern or magnitude of pituitary gonadotropin release which could not be discriminated using the present sampling protocol. Testicular hormones have been shown to be necessary to maintain the normal secretory pattern of LH secretion in the stallion (22). The possible need for elevated intratesticular testosterone concentrations for normal spermatogenesis was supported by the finding that Leydig cell number and diameter were positively correlated with the average DSP/gm of testicular parenchyma and the percentage of Stage 8 tubules. This would agree with the finding that the onset of the breeding season was associated with increases in the diameter of seminiferous tubules, the testicular concentration of testosterone, and the rate of spcrmatogenesis in the stallion (31). The shrunken Leydig cells were perhaps associated with decreased intratesticular testosterone levels with resultant suppression of spermatogenesis. It is difficult to explain the elevated plasma testosterone in the ponies treated with boldenone undecylenate. Others have reported elevated plasma testosterone in horses treated with boldenone undecylenate (32). Neither stanozolol nor boldenone undecylenate (up to 10 @ml) bound to the antibody used in the testosterone assay. Perhaps once boldenone undecylenate is administered, its metabolite is recognized by the antibody in the RIA, resulting in elevated testosterone levels. The failure of either dose of stanozolol to alter concentrations of plasma testosterone or gonadotropins, DSP/gm of testicular parenchyma, percentage of Stage 8 tubules, or number and diameter of Leydig cells would suggest that stanozolol is less androgenic in pony stallions than is boldenone undecylenate. No adverse effects of stanozolol were found on the testis parameters measured in this study. REFERENCES 1.
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THE EFFECTS OF STANOZOLOL AND BOLDENONE UNDECYLENATE ON PLASMA TESTOSTERONE AND GONADOTROPINS AND ON TESTIS HISTOLOGY IN PONY STALLIONS M.C. Garcia’, V.K. Ganjam2 T.L.Blanchard3 E.Brown2, K.Hfrdin3, R.S.Youngqt&, W.E.Loch ‘, M.R.Ellersieck4, and J.M.Balke
R.G.Elmore3,
University of Pennsylvania, New Bolton Center, 1 School of Veterinary Medicin Kennett Square, PA 19348. College of V?terinary Medicine, University of College4 of Veterinary Medicine, Missouri-Columbia, Columbia, MO 65211. College of Agriculture, Texas A&M University, College Station, TX 72847. RR 1, Box 27, Seadog, Nanoosc University of Missouri, Columbia, MO 65211. Bay, Brit. Col., Canada VOR 2R0. Received for publication: Accepted:
December
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;984
day 19, 19~7
ABSTRACT Fifty 2- to 16- yr old pony stallions were randomly assigned to one of five treatments: Group 1, controls (no treatment); Group 2, 0.55 mg/k stanozolol weekly for 13 treatments; Group 3, 1.1 mg/kg stanozolol every ‘5 wk for 5 treatments; Group 4, 1.1 mg/kg boldenone undecylenate every 3 wk for 5 treatments; and Group 5, 0.55 boldenone undecylenate weekly for 13 treatments. Mean lasma testosterone levels for Groups 2,4, and 5 were elevated over controls (P < 0.11) at 2, 8, and 9 wk, respectively. Testosterone levels for ponies in Group 3 did not differ from controls (P > 0.05). There were no differences in mean plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels among groups (P > 0.05). Daily spermatid production per gram of testicular parenchyma (DSP/gm) in Group 5 was lower than in controis (P < 0.05) whereas DSP/gm was not different among groups 1 to 4 (P > 0.05). There were no differences among groups (P > 0.05) in the percentage of Stage 8 tubules or relative number of Leydig cells. The mean diameter of Leydig cells was less for Group 5 than for controls (P < O.OS),but was not different for Groups 1 to 3 (P > 0.05). Key words:
stallion, anabolic gonadotropins
steroid,
spermatogencsis,
testosterone.
INTRODUCTION Anabolic steroids are derivatives of testosterone that are altered to provide maximal anabolic action and minimal androgenic side effects (1,2). It is not yet feasible to produce an anabolic steroid without androgenic activit (2). This androgenic activity accounts for most of the undesirable srde effects o tythese drugs. Administration of norethandrolone for 8 to 25 wk results in loss of libido, decreased potency, diminished testicular size, and azoospermia in men (3). Acknowledgments: Portions of this study were supported by the Winthrop Sterling Animal Health Products, Division of Sterling Drug, Inc., and in part by BRSG 520559 awarded by NIH. The gifts of anti-human FSH from the National Hormone and Pituitary Program, NIH, and of RIA materials from Dr. O.J. Ginther are apDrs. D. Kinden, E. Swierstra, and R.M. Kenney, and M. preciated. We thank Floyd, S. Mellskog, S. Rigby, J. Travers, L. Brockschmidt, and N. Sights for technical assistance.
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Administration of boldenone undecylenate and nandrolone decanoate at 3wk intervals for 15 wk decreases testis size, sperm production, sperm output, and semen quality in stallions (4). These effects may stem from feedback inhibition of gonadotropin release from the pituitary (3-5). Suppressed pituitary LH levels eventually lead to markedly decreased endogenous testosterone production, resulting in lowered intratesticular testosterone levels and cessation of spermatogenesis (6). 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 se aration between anabolic and antigonadotrophic activities is difficult to achieve P5). Currently there are only two anabolic steroids (stanozolol and boldenone undecylenate) approved by the Federal Drug Administration for use in horses, although neither is approved for stallions or pregnant mares. In a previous paper WC reported that boldenone undecylenate (0.55 mg/kg weekly for 13 wk) resulted in smaller scrotal width, decreased testis weight, and depressed daily sperm production comnared with untreated control nonv stallions (7). Stanozolol (1.1 mg/kg weekly for 13 wk) and boldenone undecyjenate (1.1 mg/kg every 3 wk for five-inicctions) did not depress daily sperm production. The purpose of this paper is to report the effects of these drugs on plasma testosterone and gonadotropins and on testis histology in those ponies. MATERIALS
AND METHODS
Fifty 2- to 16-year-old pony stallions of mixed breeding were randomly assigned to one of five treatment groups, with 10 ponies per group. This randomization produced five treatment groups that were statistically comparable with respect to age and weight. The treatment groups were as follows: Group 1, controls, no treatment; Group 2, 0.55 mg/kg stanozolola by deep i.m. injection weekly for 13 treatments; Group 3, 1.1 m@g stanozolol by deep i.m. injegtion every 3 wk for five treatments; Group 4, 1.1 mg&g boldenone undecylenate by deep i.m. injection every 3 wk for five treatments; Group 5, 0.55 m@g boldenone undecylenate by deep i.m. injection weekly for 13 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. Blood samples from each pony were collected in heparinized tubes weekly prior to treatment. Immediately after collection, each tube of blood was placed on ice and maintained upright until centrifuged prior to collection of plasma. Plasmas were frozen until assayed for hormones. Total plasma unconjugated testosterone was quantified using a double antibody radioimmunoassay (RIA; RSL testosterone assay No. 1102-D).’ The first antibody was raised against testosterone 19-carboxymethylether-bovine serum albumin in rabbits. The second antibody was goat antirabbit gammaglobulin. The sensitivity of the assay was about 0.1 ng/ml at 85% binding. The cross reactivities of the other steroids (testosterone = 100% at 50% displacement) are: 5 alpha dihydrotestosterone (DHT), 3.4%; androstenedione, 0.56%; other 17keto_androgens, ~1%; estrogens and progestagens, ~0.05%. Neither stanozolol nor boldenone undecylenate specifically bound to this antibody. aWinstrol-V
NY.
-Winthron
Veterinary.<. Sterling Animal Health Products,
New York,
‘“Equipoise” - E.R. Squibb and Sons, Inc., Princeton, NJ. ‘Radioassay Systems Laboratories, Inc., Carson, CA.
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Plasma concentrations of LH and FSH were determined by double antibody RIAs(8). The LH assay used antibody against equine chorionic gonadotropin raised in a rabbit for the first antibody and sheep antirabbit gammaglobulin for the second antibody. The sensitivity of the assay at 85% binding was 0.1 rig/ml. Mean intraand inter-assay coefficients of variation were 4.0 and 9.5%, respectively, for triplicate tubes of pooled estrus plasma run in the last three assays. The FSH RTA is a heterologous system which used antihuman FSH for the first antibody and sheep antirabbit ammaglobulin for the second antibody. The cross reactivity of equine LH in the F .!H assay was less than 1%. The assay sensitivity was about 0.2 ng/ml at 85% binding. The mean intra- and inter-assay coefficients of variation were 8 and 12%, respectively, for triplicate samples of pooled diestrus plasma run in the last three assays. All ponies were castrated following blood sampling 1 wk after the last trcatment. The parietal tunics were dissected from the testes and epididymides, the epididymides were separated from the testes, and each testis was weighed. Each testis was sliced and a center section was stored at -5’C for determination of spcrmatid reserves (7). The average daily sperm produced per gram of testicular parenchyma was calculated with the following formula: (DSP/gm of left testis x left testis parenchyma weight + DSP/gm of right testis x right testis parenchyma weight)/total The remainder of the testis was fixed in mercuric weight of testicular parenchyma. formol for 3 d, washed in running water for 24 h. and stored in 70% alcohol until being trimmed. A section from the middle plane (which was adjacent to the section taken for determination of spermatid reserves) of each testis was embedded in paraffin, sectioned, and stained with periodic-acid-Schiff-hematoxylin. These sections were examined histologically for determination of the pcrcentage of seminiferous tubules in Stage 8 (Figure 3) according to the method of Swierstra et al (9). Calculation of DSP by testicular homogenization assumes that at some stage elongated spermatids become resistant to homogenization. Howcvcr. the technique does not establish whether spcrmatogenesis is being complctcd. Therefore, histologic assessment was used to confirm completion of spermatogenesis. Because Leydig cells appeared to be less prominent in the testes of stallions in some treatment groups, the number and size of Lcydig cells were dctcrmined retrospectively. The number of Leydig cells was determined by counting the number of hits of 25 randomly selected dots in a Chalkley eyepiece (10) on the nuclei of Lcydig cells in 100 fields using a Zeiss Universal microscope.” Lcydig ccl1 diameters were measured using an ocular micrometer by obtaining two mcasurcmcnts perpendicular to each other on 25 Leydig cells per testis, each in a different field. The variables testosterone, LH, and FSH were analyzed as a rcpcatctl measures analysis of variance according to Gill and Hafs (11). The analysis ol variance model contained the main effects of treatment (Groups 1 to S), time (weekly intervals), and the interaction of treatment and time. Animal within trcament was used as an error term (Error A) for determining significant treatment effect. The animal by time within treatment (Error B) was used to test time and cffects of treatment by time. The least significant difference statistic was used for mean separation. The variable percentage of Stage 8 tubules was transformed to square roots and a one-way analysis of variance was performed. The variables Leydig cell number and size were analyzed by one-way analysis of variance, and least significant difference was used for mean separation. ’ Carl Zeiss, Germany (Seller Instrument
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Mfg. Co., St. Louis, MO).
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rHERIOGENOLOGY
RESULTS Initial testosterone levels did not differ among groups (Figure 1; P > 0.05). Testosterone levels for Group 2 did not differ from those for controls (P > 0.05) except during Weeks 1 and 3, when those of Group 2 were higher (P < 0.01). Testosterone levels for Group 3 did not differ from controls (P > 0.05). Testosterone levels for Group 4 were higher than those for controls during Weeks 1,3 to 5,7,9 to 11,and 13 (P c 0.01).
FIGURE
?
600 500
400 300
__-
0
1
Group Group Group Group Group
Figure 1.
2
1 2 3 4 5
l
0 A A n
3
4
Controls 0.55 mg/kg 1 .I0 mg/kg 1.10 mg/kg 0.55 mg/kg
5 WfL
stanozolol stanozolol boldenone boldenone
7
6
9
10
weekly every 3 weeks undecylenate undecylenate
Effects of stanozolol and boldenone terone in pony stallions.
11
12
13
every 3 weeks weekly
undecylenate
on plasma testos-
Testosterone levels for Group 5 were higher (P < 0.01) than those for controls during Weeks 1. 3. 5 to 7. 9. 10. 12. and 13. The correlations between testis measurem&ts (testisweight, DSP’ per gram of testis, daily sperm production), and testosterone values at castration; 0.20, -0.02, and 0.13, respectively, were not significant (P > 0.05). There were no differences in mean levels of gonadotropins among the treatment groups (Figure 2; P > 0.05). There was a significant effect of time on mean plasma LH concentration (P < 0.05).
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-1
1
3
5
7
9
11
13
WEEK
Figure 2.
Bi-weekly mean plasma LH and FSH concentration
in pony stallions.
Due to a significant age effect on average DSP/gm of testicular parenchyma, an analysis of covariance was performed to adjust for the effect of age. Average DSP/gm of testicular parenchyma was lower in Group 5 ponies (Table 1; P < 0.05) than in control ponies. There were no differences in average DSP/gm of testicular parenchyma in ponies in Groups 1 to 4 (P > 0.05). There also were no differences in percentage of Stage 8 tubules among treatment groups (Table 1; P > 0.05). Mean number of Leydig cells did not differ among groups (Table 1; P > 0.05). However, the mean diameter of Leydig cells was less for ponies in Group 5 (P < 0.05) than for control ponies. The mean diameter of Leydig cells was not different for ponies in Groups 1 to 4 (P > 0.05). Marked cytoplasmic vacuolization, shrunken tubules and Leydig cells, and multinucleated giant cells containing phagocytosed spermatids indicative of tcstitular degeneration (Figure 4) occurred in the testes of some ponies. This was most often observed in testes of Group 5 stallions and was more pronounced in younger animals. Correlation coefficients (r) for the histologic parameters measured were as follows: 0.74 (P c 0.01) for percentage of Sta e 8 tubules and DSP/gm of testicular parenchyma, 0.35 (P < 0.01) for percentage of s tage 8 tubules and number of Leydig cells, 0.67 (P < 0.01) for percentage of Stage 8 tubules and diameter of Leydig cells,
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THERIOGENOLOGY
0.25 (P < 0.09) for number of Leydig cells and DSP/gm of testicular parenchyma, and 0.52 (P < 0.01) for diameter of Leydig cells and DSP/gm of testicular parenchyma. The simple correlation coefficients and probability values between LH and testosterone were Group 1, r = 0.22, P c 0.06; Group 2, r = 0.20, P c 0.08; Group 3, r = 0.24, P -Z0.05; Group 4, r = 0.15, P < 0.23; Group 5, r = -0.06, P < 0.63. No significant correlations between FSH and testosterone were observed for any of the five treatment groups.
Table 1.
Group
Effects of stanozolol and boldenone undecylenate on daily spermatid production per gram (DSP/gm) of testis and on testis histology in pony stallions.
Treatment
n
DSPigm of testis
Stage 8 tubules (%)
Relative number of Leydig cells
Mean diameter of Leydig cells
X+SEM
X+SEM
X+SEM
XzSEM
1
control
10
16.22+1.30
9.61k1.12
40.39+5.77
14.75+0.81
2
stanozolol 0.55 mg!kg weekly
10
17.51+1.95
8.35+1.65
45.3027.26
15.43+0.60
3
stanozolol 1.1 mg/kg every 3 wk
10
15.6022.07
8.45~1.42
39.8OL5.38
14.36+0.81
4
boldenone undecylenate 1.1 mg/kg every 3 wk
10
10.52t2.64
6.6021.92
50.30+7.13
12.97+1.20
5
boldenone undecylenate 0.55 mg/kg weekly
10
9.56+2.46*
4.8Ot1.99
44.5529.71
11.27+0.72*
* Differs from controls (P -Z0.05).
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Figure 3.
Photomicrograph of testis histology for Pony 13 (Group 1, control). Left testis, middle section with seminiferous tubule in Sta e 8 of spermatogenesis. dig cells (L). Elongated spermatids (8 p) lmc the tubule lumen . 520x.
Figure 4.
Photomicrograph of testis histology for Pony 54 (Group 5~0.55 mg’kg boldenone undecylenate once weekly). Left testis, middle section. Few small Leydig cells (L) are present. Lumen (LU) of seminiferous tubule has no elongating spermatids but has desquamatcd spcrmatocytcs (D). 520x.
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THERIOGENOLOGY
DISCUSSION Adverse effects of exogenous testosterone are thought to result from feedback inhibition of gonadotropm release from the pituitary (12-15). Pelletier suggested that testosterone may depress gonadotropin releasing hormone synthesis in rams (16). Suppressed pituitary LH levels eventually lead to markedly decreased endogenous testosterone production, thus resulting in lowered intratesticular testosterone levels: some workers feel that these lowered levels result in cessation of spermatogenesis (6). However, critical examinations of the necessity of high concentrations of testosterone in the milieu surrounding the developing germ cells for the maintenance of normal spermatogenesis have not been performed. Qualitatively normal spermatogenesis, in spite of lower than normal intratesticular testosterone concentration, has been reported in intact (17) and hypophysectomized (18) rats administered testosterone. However, while testosterone treatment reduces the percentage of tubules with differentiating spermatozoa, considerable reduction in testis wei ht may occur even though spermatogenesis may still occur in some tubules (18 ). Mean plasma LH concentration at the start of the experiment (3.7 @ml) was similar to that reported for adult ponies >3 yr old (3.2 rig/ml) and higher than that reported for yearling pony colts (about 1 rig/ml) using the same assay system 20). Similarly, the mean plasma FSH concentration at the start of the experiment 11.5 rig/ml) was comparable to that reported in adult ponies (7.0 rig/ml) using the same assay system. The finding that administering boldenone undecylenate did not affect plasma onadotropins but increased plasma testosterone and decreased testis function is dif. Brcult to explain. However, the correlation between LH and testosterone for Group 1 was positive and tended to be significant while that for Group 5 was negative. The failure to find decreased concentrations of plasma gonadotropins as a result of boldenone undecylenate treatment in this study may be due to breed differences or may be due to the infrequent (biweekly) sampling regimen. There is evidence for episodic fluctuation in the secretion of androgens in the stallion (21), and a secretory attern of LH secretion has been described (22). Administration of anabolic steroi .d”s to men reduces plasma concentrations of both testosterone and gonadotropins and reduces spermatogenesis (23-27). Likewise, administration of 1.1 and 4.4 mg/kg boldenone undecylenate and 1.1 mg/kg nandrolone decanoate at 3 wk intervals for 15 wk decreased serum LH concentration, testis size, sperm production, sperm output, and semen quality in horse stallions (28). The mean percentage of Stage 8 tubules in testes of control ponies (9.61%) was lower than that reported for horse stallions (15.7%) (9), possibly due to breed difference or to technician error. Only Stage 8 tubules were classified in our study, whereas all tubules were classified into one of eight stages in the horse testes studied. Not having to place all tubules into a category could also account for the lower percentage of Stage 8 tubules found in the ony stallions. Regardless, because of the high correlation between average D !$P/gm of testicular parenchyma and percentage of Stage 8 tubules (0.75; P < O.OOOl),percentage of Stage 8 tubules is a valid comparison of relative treatment effects between groups. We had previously found that treating ponies with 0.55 mgkg boldenone undecylenate once weekly significantly reduced testis weight and daily sperm production (7). This dosage of boldenone undecylenate apparently decreases the efficiency of spermatogenesis as well as the amount of testicular parenchyma available to produce spermatozoa. Others have demonstrated that testosterone (12,13,29) and anabolic steroid administration (28) arrests spermatogenesis at the spermatogonia or spermatocyte stages.
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It is not known if there are any adverse long-term effects of anabolic steroids on reproductive capacity of stallions. The adverse effects of anabolic steroids are thought, in general, to be reversible in man (25). Adverse effects of administration of anabolic steroids for 1 yr to growing fillies persisted for at least 6 mo after trcatment ceased (30). Adverse effects of anabolic steroids appeared to be more pronounced in the younger ponies. Testicular degeneration may preclude return to a normal level of spermatogenesis. Despite the failure to demonstrate decreased concentrations of plasma gonadotropins, Leydig cell diameter diminished in the testes of ponies treated with 0.55 mg/kg boldenone undecylenate weekly. The failure to detect changes in gonadotropin levels may have been due to a change in the pattern or magnitude of pituitary gonadotropin release which could not be discriminated using the present sampling protocol. Testicular hormones have been shown to be necessary to maintain the normal secretory pattern of LH secretion in the stallion (22). The possible need for elevated intratesticular testosterone concentrations for normal spermatogenesis was supported by the finding that Leydig cell number and diameter were positively correlated with the average DSP/gm of testicular parenchyma and the percentage of Stage 8 tubules. This would agree with the finding that the onset of the breeding season was associated with increases in the diameter of seminiferous tubules, the testicular concentration of testosterone, and the rate of spcrmatogenesis in the stallion (31). The shrunken Leydig cells were perhaps associated with decreased intratesticular testosterone levels with resultant suppression of spermatogenesis. It is difficult to explain the elevated plasma testosterone in the ponies treated with boldenone undecylenate. Others have reported elevated plasma testosterone in horses treated with boldenone undecylenate (32). Neither stanozolol nor boldenone undecylenate (up to 10 @ml) bound to the antibody used in the testosterone assay. Perhaps once boldenone undecylenate is administered, its metabolite is recognized by the antibody in the RIA, resulting in elevated testosterone levels. The failure of either dose of stanozolol to alter concentrations of plasma testosterone or gonadotropins, DSP/gm of testicular parenchyma, percentage of Stage 8 tubules, or number and diameter of Leydig cells would suggest that stanozolol is less androgenic in pony stallions than is boldenone undecylenate. No adverse effects of stanozolol were found on the testis parameters measured in this study. REFERENCES 1.
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