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The Effects of Oral Altrenogest on Hormonal, Testicular, and Behavioral Profiles of Two-Year-Old Stallions12
TheThe Effects of Altrenogest on Two-Year-Old Stallions Professional Animal Scientist 17:75–80
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Effects of Oral Altrenogest on TheHormonal, Testicular, and Behavioral Profiles of Two-Year-Old Stallions 1,2 N. L. HENINGER*, H. A. BRADY*,3, PAS, S. D. PRIEN†, A. D. HERRING*,2, PAS, K. A. GUAY*, and L. A. JANECKA* *Department of Animal Science and Food Technology, Texas Tech University, Lubbock, TX 79409 and †Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center, Lubbock, TX 79430
Abstract Altrenogest is frequently administered to young stallions in the equine industry as an off-label use to suppress sexual/ aggressive behavior. In this study, 2-yrold Quarter Horse stallions in earlyperformance training were administered altrenogest (0.044 mg/kg BW daily) to determine the effects on sexual/aggressive behavior, testicular parameters, and steroid hormone profiles. Horses were randomly assigned to treatment (n = 5) and control (n = 5) groups. The treatment group was administered a daily oral dose of 0.044 mg/kg BW daily for 67 d; the control group was given a daily
1Funding
for this project was provided by the Houston Livestock Show and Rodeo Association. Research was conducted in accordance with TTU ACUC # 9872. TTU CASNR manuscript T-R-404. 2The authors would like to acknowledge C. Guay, D. Johnson, and H. Vartorella for their assistance with the project.
oral sham dose of corn oil for 67 d. No significant differences were found between treatment groups in BW or body condition scores. Altrenogest had no effect on any behavioral parameters or seminal parameters measured. Averages of total scrotal width (TSW) were lower (P<0.03) for treatment animals at the end of treatment period (d 67). By d 157 mean TSW for both treatment and control stallions were not different. Both were significantly higher (P<0.03) than pretrial values at d 157. Comparisons of testicular weight (d 157) between control and treatment groups were not different. Histological analysis of testicular tissue revealed no significant difference for the average number of spermatids per seminiferous tubule. Altrenogest treatment reduced serum estrogen levels (estradiol 17-b ) by d 67 (P<0.02); however, estrogen levels returned to pretreatment values after a 90-d recovery. Serum testosterone values were not affected by treatment. Further research is needed to determine dose and age effects of altrenogest in the stallion.
3To whom correspondence should be ad- (Key Words: Stallion, Altrenogest, dressed: [email protected] Behavior, Hormone.)
Introduction Androgens are essential to maintain the normal sexual physiology of the male (1). Previous research exists in many species examining the use of progesterone in an effort to biochemically interrupt normal androgen production in the male. Studies involving rodents (5, 7, 8), boars (11, 12), rams (4, 6), and humans (13) have shown that progesterone reduces testosterone-dependent male sexual behavior. Altrenogest (ReguMate®; Intervet, Millsboro, DE), an oral synthetic progestin, is labeled for the suppression of estrus in mares. Altrenogest has also been used in a variety of male domestic farm animals for the reduction of sexual behavior with varying results depending on species, age, and dose. In the stallion, reported effects of altrenogest treatment have varied depending on length of treatment, dose, and age of stallion (3, 10, 14, 16). Squires et al. (16) examined the effects of 0.088 mg/kg BW daily in mature stallions. Stallions fed altrenogest for 150 or 240 d had
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decreased serum concentrations of testosterone and luteinizing hormone. Total scrotal width (TSW), daily sperm output (DSO), and libido were also significantly decreased by treatment (16). Miller et al. (14) examined the effects of a short-term administration (30 d) of altrenogest (0.044 mg/kg BW daily) in mature stallions. Only minor effects on stallion sexual/ aggressive behavior and semen quality were reported. Previous studies in our lab (10, 3) examined the suppressive effects of altrenogest (0.088 mg/kg BW daily) for 56 d on 2- to 4-yr-old stallions. Altrenogest decreased serum testosterone, estrogen, and LH (3). Decreases were found in DSO and scrotal circumference in stallions within the treatment group (10). Abnormalities in sperm morphology were significantly increased in the treatment group between the pre-trial collection period and the 8-wk collection period (10). The purpose of this study was to determine the effects of a daily oral dose (0.044 mg/kg BW) of altrenogest administered for 67 d to 2-yr-old stallions in training. This dose represents the label dose for suppression of estrus in mares and is referred to in the industry as the “single mare dose.” This study examined behavioral, hormonal, testicular, and seminal parameters to fully evaluate the potential of the drug as a temporary alternative to castration.
Materials and Methods General. Ten untrained 2-yr-old Quarter Horse stallions leased from four different ranches located in the Texas Panhandle and eastern New Mexico were used in the study. All horses were foaled in large pastures, and the exact age in months was not known; however, a mean age of 26 mo was estimated from ranch records. Stallions were housed together in a single 100' by 100' drylot paddock at the Texas Tech University Ranch Horse Center in New Deal, Texas. Upon arrival,
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horses were dewormed (Safeguardâ; Intervet) and vaccinated (Cephalovacâ VEWT; Boehringer Ingelheim Vetmedica Inc., St. Josephs, MO). Horses were fed highquality round-baled alfalfa hay (21% CP). Horses had ad libitum access to hay, salt blocks, and water. Three weeks prior to the commencement of the study, horses were halter-broken and trained under saddle in a manner typical for the young ranch horse. In this training process, horses were ridden on a schedule of walking and trotting for 1 hr with brief (10 min) loping patterns and rail work interspersed. Care was taken to equalize the workloads as much as possible by timing exercise schedules. During this 3-wk period, horses were also trained to use an artificial vagina for semen collection. In this training regimen, horses were teased every 48 h with an estrous mare and encouraged to mount for semen collection using an artificial vagina. A time limit of 20 min was set for each training session. Measurements of BW were determined monthly by individually weighing each animal with a Mettler-Toledo Digital Livestock Pen Scale (MettlerToledo, Inc., Columbus, OH). On d –7, 67, 157 of the trial, horses were assigned a body condition score (BCS) by two trained observers according to the equine system of Henneke et al. (9). Stallions were stratified across treatments according to pre-trial test breeding performance (measured by libido and mounting attempts) and separation of same sire pairs, and then randomly assigned to treatment (n = 5) and control (n = 5) groups. On d 1, treatment horses were orally administered altrenogest (ReguMateâ; Intervet) at a daily dose of 0.044 mg/kg BW for a period of 67 d (9.5 wk) during June, July, and the first week in August. Control horses were orally administered a daily sham dose of corn oil for the same time period (67 d). A 90-d recovery period followed the treatment period to examine physiological or behavioral modifications. Recovery period lasted
from the end of treatment until the first week in November. One control animal had very mild laminitis (not requiring medication) and was separated for 1 wk. One treatment animal was injured in the hock and was separated for 1 wk; treatment was continued in this animal. Sexual/Aggressive Behavior. Sexual/aggressive behavior was analyzed using a 3-min Standardized Teasing Test (10) (0 to 180 s) (d –7, 67, 157). Stallions were presented to a hand-held estrous mare over a solid partition and video-recorded in real time (30 frames/s). The positioning of the mare (front vs rear presentation) was held constant from stallion to stallion to avoid handler influence on the teasing sessions. Tapes were analyzed for frequency, duration, and latency to the following behaviors: sniffing/licking, biting/chewing, vocalization, Flehman’s response dropping of penis, erections, and aggressive behavior. Aggressive behavior was designated as any behavior directed at the handlers or mares (striking, rearing, or kicking). Duration was expressed in seconds of response within the 180-s observation period. Latency was expressed as seconds until first response of parameter is achieved. Frequency was expressed as mean number of times that the response occurred within the 180-s observation period. Mounting behavior, analyzed separately (d –7, 67, 157), examined the number of mounts or attempted mounts during the semen collection periods. Testicular and Seminal Parameters. Seminal parameters were analyzed three times beginning on d –7, 67, and 157. Stallions were collected using a hand-held mare in estrus and a Missouri artificial vagina. Daily sperm output was determined using a 7-d sequential collection period. Stallions were collected each day for 7 d to deplete sperm reserves; semen collection on d 7 was used for evaluation of concentration (Model 534A mod 1 Densimeter; Animal Reproduction Systems, Chino, CA). Motility was determined manually using light micros-
The Effects of Altrenogest on Two-Year-Old Stallions
TABLE 1. Effects of altrenogest on duration, latency, and frequency of behavioral parameters using a standardized teasing test (0 to 180 s). Overall comparisons
Duration
Latency
Frequency
Item
Control
Biting/chewing Sniffing/licking Flehman’s Vocalization Dropping penis Complete erection Aggressive behavior Biting/chewing Sniffing/licking Flehman’s Vocalization Dropping penis Complete erection Aggressive behavior Biting/chewing Sniffing/licking Flehman’s Vocalization Dropping penis Complete erection Aggressive behavior
18.3 101.6 10.3 22.6 73.3 36.6 8.0 107.0 16.3 124.0 103.3 77.3 139.6 143.6 2.0 5.4 1.1 2.3 1.5 0.7 0.9
copy by two trained evaluators. Total sperm and total progressive sperm were determined by standard calculations. Samples for morphology (d 7 semen ejaculate) were fixed in formalin (Formalin 10; Animal Reproduction Systems) for microscopic evaluation. Dry, stained slides were prepared using Hemotoxylin and Eosin stain (H&E). One hundred sperm cells per stallion were counted and classified as normal or abnormal (head, midpiece, or tail). Values for TSW were determined on d –7, 67, and 157 using a scrotal width caliper (Model 549A Stallion Scrotal Caliper; Animal Reproduction Systems). Measurements were taken three times by one trained operator and averaged to determine TSW for each period. Stallions were castrated at the end of the recovery period (d 157). After removal of the epididymus, testicles were weighed for comparison to control group. Tissue samples for histology were excised from the middle one-third of the left testicle of each stallion and fixed in Bouin’s
Treatment 22.3 92.6 5.0 29.3 114.0 70.6 6.0 95.0 5.0 130.0 82.0 61.3 95.3 144.6 2.3 5.4 1.1 1.9 1.1 1.1 0.7
P
SEM
0.76 0.77 0.38 0.74 0.34 0.21 0.78 0.75 0.51 0.38 0.68 0.74 0.18 0.97 0.89 0.61 0.84 0.60 0.82 0.17 0.28
8.9 12.9 3.4 13.7 23.9 28.6 4.7 28.2 6.9 23.57 23.6 22.3 29.9 24.0 1.2 1.0 0.6 1.0 0.5 0.4 0.5
solution for paraffin sectioning and H&E staining (Pathology Laboratory Texas Tech Health Sciences Center, Lubbock, TX). The mean number of elongated spermatids within 25 round seminiferous tubules was calculated by three trained evaluators using a Nikon Alphaphot Microscope (Nikon Inc., Melville, NY) with brightfield optics at 100x magnification. Spermatid counts were averaged across evaluators to determine values for statistical analyses. Hormonal Analysis. A blood sample was obtained from each stallion by jugular veni-puncture on 7-d intervals in the morning prior to feeding. Samples were allowed to clot at ambient temperature for 30 min and refrigerated at 5°C for 24 h. Samples were spun to isolate serum which was frozen at –20°C until all samples had been collected. Serum concentrations of testosterone and estrogen (estradiol 17-ß) were determined using radioimmunoassay kits (Diagnostic Products Corp., Los Angeles, CA), which have been validated in our lab (Dept. of Obstet-
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rics and Gynecology, Texas Tech Health Sciences Center, Lubbock, TX) for a number of species, including the horse. The samples were assayed without extraction, and all samples were analyzed for a single hormone within a single run. Sensitivities of the assays were 0.1 ng/mL for testosterone (with an interassay CV of 4.5%) and 8 pg/mL for estrogen (with an interassay CV of 3.8%). Statistical Analysis. Weight, body condition scores, testicular, and seminal and hormonal data were analyzed by ANOVA using general linear models procedures (15). Sexual/aggressive behavior as measured in the standardized teasing test including frequencies (n), latency (0 to 180 s) and duration (0 to 180 s), were also analyzed by ANOVA (15). For all data, treatment, time, horse within treatment, and treatment by time were included in the statistical model. The “horse within treatment” term was used as the error term to determine treatment differences. Means for all analysis were separated using Fisher’s protected LSD procedure.
Results and Discussion No differences in BW or body condition scores were found between control and treatment groups. Body weights did not differ between control and treatment stallions at d –7 (424.6 ± 15.2 vs 425.9 ± 6.8 kg, respectively), d 67 (430.5 ± 15.4 vs 424.1 ± 8.5 kg), or at the end of the recovery period, d 167 (455.0 ± 18.4 vs 450.9 ± 7.9 kg). Similarly, no differences were found between control and treatment animals in body condition scores at d-7 (4.7 ± 0.3 vs 5.2 ± 0.4, respectively), d 67 (5.1 ± 0.2 vs 5.2 ± 0.1) or at d 167 (5.4 ± 0.1 vs 5.6 ± 0.2). These data suggest that the use of altrenogest (Regu-Mate®) at the dose given did not suppress or augment overall growth or condition of these young stallions. There were no significant effects for any measured behavioral parameter throughout pretreatment, treatment, and recovery periods.
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from this animal on d 67; however, treatment was continued. On d 67, three of five control and two of five Pretrial (d 0) Control SEM Treatment SEM treatment stallions were able to be collected. For the recovery period (d 157), four of five controls and three (n = 3) (n = 3) of five treatment animals were Concentration (1 x 106/mL) 93.6 41.5 113.6 37.2 collected. No differences between Volume (mL) 15.2 9.3 28.0 8.4 treatments were observed for any Motility (%) 27.2 19.9 21.6 17.8 6 measured seminal parameter (Table Total sperm (1 x 10 ) 1,019.9 1,074.3 2,567.3 960.9 2). Very large differences were seen End of Treatment (d 67) Control SEM Treatment SEM across individual horses. This is in contrast to Johnson et al. (10) and b Squires et al. (16), who reported (n = 3) (n = 2) 6 significant decreases in DSO in Concentration (1 x 10 /mL) 142.3 41.5 161.0 49.1 Volume (mL) 5.8 9.3 17.6 11.0 altrenogest-treated horses using a Motility (%) 51.2 19.9 25.6 24.5 regimen of 0.088 mg/kg BW, suggestTotal sperm (1 x 106) 854.9 1,074.3 1,774.2 1,271.1 ing a dose effect of altrenogest on seminal quality in stallions. The End of Recovery (d 157) Control SEM Treatment SEM seminal effects appeared to be partially reversible in mature stallions as (n = 4) (n = 3) DSO was reported to increase after a Concentration (1 x 106/mL) 137.0 32.2 109.3 37.1 recovery period (16), although DSO Volume (mL) 9.5 7.2 14.0 8.4 remained suppressed 8 wk after Motility (%) 66.3 15.4 37.6 17.8 cessation of altrenogest treatment in Total sperm (1 x 106) 1,026.3 832.1 1,276.0 960.9 young stallions (10). Miller and coworkers (14) found only minor effects aNumbers are based on stallions collected as described in text. in semen quality, which returned to bOne treatment stallion was not included because of lameness. pretreatment levels by a period of 30 d following cessation of altrenogest treatment. We found that treatment with altrenogest had no significant Analysis of the standardized teasing short-term administration of effects on sperm morphology (pertest revealed no differences between altrenogest for 30 d at 0.044 mg/kg centage of normal sperm, head, midtreatment groups in duration, latency BW resulted in only minor behavpiece, and tail abnormalities) after 67 or frequency of behavioral paramioral modifications. Stallion–mare d (Table 3). This is in contrast to eters including biting/chewing, interactions in the breeding shed studies by Johnson et al. (10) using a sniffing/licking, Flehman’s response, such as time to erection frequency, higher dose (0.088 mg/kg BW) of vocalization, dropping of the penis, mounting ability, and ejaculation complete erections, or aggressive were not reduced during the course of altrenogest, finding that sperm head and tail abnormalities were signifibehavior (Table 1). Large differences the treatment (14). It appears that cantly increased in the treatment were observed between individual there are large dose and age effects group between the pretrial period horses. Additionally, no significant associated with behavioral modificaand the end of the 8-wk treatment differences were noticed for mounttions in the stallion. period. ing behavior/attempted mounts for After the 3-wk training period, a Significant differences were found any 7-d sequential collection period total of three control and three (P<0.03) in TSW as a treatment by (P>0.05). Previous research has treatment stallions were able to be demonstrated that feeding collected due to differences in altrenogest at 0.088 mg/kg BW baseline libido of these young staldecreased libido, Flehman’s response, lions (Table 2). As a result, three of vocalization, and erections in 2- to 4- five stallions within each group were yr-old treated stallions (10). Squires able to be collected at the pre-trial et al. (16) also found significant period (d –7 to d 0). The same three sexual behavioral suppression in animals in each group were collected mature stallions (3 to 18 yr) adminis- at each time point with the exception Figure 1. Scrotal widths for pretrial, end of tered 0.088 mg/kg BW for 150 or 240 of d 67. One treatment animal was treatment, and end of recovery periods. a,b,cDifferent superscripts indicate significant d. This contrasts with the study by injured in the hock on d 65; no Miller (14) in mature horses in which attempt was made to collect semen differences. Standard error = 1.5.
TABLE 2. Effects of altrenogest on mean seminal parameters pretrial, end of treatment, and end of recovery after 7-d sequential collection perioda.
The Effects of Altrenogest on Two-Year-Old Stallions
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TABLE 3. Effects of altrenogest on head, mid-piece, and tail abnormalities in 2-yr-old stallions after 67 d (end of treatment)a. Overall comparison Item Normal Head Mid-piece Tail
Control (%)
SEM
Treatment
SEM
63.7 3.5 12.8 20.0
7.1 1.7 1.4 7.3
58.1 7.0 11.6 23.3
8.7 2.1 1.7 8.9
Figure 2. Estradiol values (17-b) for pretrial, end of treatment, and end of recovery period. a,b,cDifferent superscripts indicate significant differences. Standard error = 1.5.
in response to a 0.088 mg/kg BW regimen of altrenogest was reported are based on stallions collected. Recovery period data not available for by Brady et al. (3). Work in the boar morphology. found that altrenogest decreased levels of testosterone (11) as well as time interaction (Figure 1). Averages animals at the end of the recovery estrogen (12). Kluber et al. (11) of TSW across control and treatment period (18.3 vs 11.3, respectively, hypothesized that there may be a groups were lower for treatment spermatids per seminiferous tubule). threshold concentration of testosteranimals at the end of the treatment Large differences were seen between one in growing boars that is responperiod. Values were not significant individual horses. Johnson et al. (10) sible for sexual behavior, libido, and by the end of the recovery period. reported a significant decrease in maintenance of spermatogenesis. Both groups were significantly higher spermatids per seminiferous tubule. This may hold true across most than pretrial values at the end of the Kluber et al. (11) also found that domestic farm animal species, includrecovery period (P<0.03). In agreetotal sperm per testis, concentration ing the young stallion. ment with Squires et al. (16) the of sperm/gram of tissue, total seminIt is acknowledged that season authors found a significant decrease iferous epithelium, and total intersti- and increasing age in months may in TSW in altrenogest-treated staltium per testis were less for boars fed affect different parameters of this lions. The present study documented altrenogest. trial. Treatment data were in coma return to TSW pretreatment values Estrogen concentrations of treatparison to control animal data. The after a 90-d recovery period. Simiment stallions were lower (P<0.02) age at which a colt reaches puberty larly, Squires et al. (16) reported that than control stallions by the end of also must be considered. Puberty, in mature stallions, the effect apthe treatment period (Figure 2). No depending on the definition, can peared to be partially reversible differences were found between range from 14 to 24 mo of age or resulting from an increase in TSW groups 90-d post-cessation of treatmore (2). The colts used in this study after cessation of progestin treatment. A significant reduction in may have been in various early stages ment. However, Johnson et al. (10) estrogen levels in young stallions of post-puberty. This may be due in found that mean scrotal circumferfollowing 8 wk of altrenogest treatpart to previous environmental ence for young altrenogest-treated ment was found by Brady et al. (3); conditions and late birth dates. stallions was less than control anihowever, estrogen levels returned to These effects may have contributed to mals at cessation of treatment. This pretreatment levels after an 8-wk lower levels of libido demonstrated difference remained after an 8-wk recovery period. Thompson et al. by some individuals within each recovery period (10). Research with (17) demonstrated that variations in treatment as well as the large variathe boar (11) also has supported a libido are mediated by estrogen levels, tion between horses. reduction in testicular size associated which are high in the stallion. No with altrenogest. Kluber et al. (11) significant differences were observed found boar testicular weight and in serum testosterone values throughvolume declined linearly as duration out the duration of the trial. It has Little information exists regarding of altrenogest feeding increased. been demonstrated that testosterone the extra-label use of altrenogest in No significant differences were secretion is dependent upon the young stallions; however, it is used observed in mean testicular weights secretion of anterior pituitary gonawidely in the performance horse at castration (d 157) between control dotrophins. Squires et al. (16) industry. In this study, altrenogest at and treatment groups (123.9 g vs documented a decrease in serum the dose given had little effect on 124.1 g, respectively). Data from testosterone in mature stallions in behavioral and seminal parameters in histological analysis showed no response to a 0.088 mg/kg BW regicontrast to studies using higher differences between groups in sperma- men of altrenogest. A suppression of doses; however, mean TSW was tids per seminiferous tubule in treated testosterone levels in young stallions reduced after altrenogest treatment. aNumbers
Implications
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Our data have shown that altrenogest decreased levels of estrogen after 60 d of treatment. This finding is important in that estrogen is related to libido in the stallion. In reviewing existing studies, there are many factors that may affect both short- and long-term physiological effects of altrenogest in the stallion, including dose, length of treatment, age of stallion, and stallion-tostallion variation. It is premature to be able to give clear recommendations to horse owners regarding the safe use of altrenogest in the stallion. Further work must be done to elucidate the risks and benefits involved.
2. Amann, R. P. 1993. Physiology and endocrinology. In: Equine Reproduction. A.O. McKinnon and J.L. Voss (Eds.). p. 658. Lea and Febiger, Philadelphia, PA.
11. Kluber, E. F., D. S. Pollman, D. L. Davis, and J. S. Stevenson. 1985. Body growth and testicular characteristics of boars fed a progestin, altrenogest. J. Anim. Sci. 66: 470.
3. Brady, H. A., N. N. Johnson, C. S. Whisnant, S. D. Prien, and J. M. Hellman. 1997. Effects of oral altrenogest on testicular parameters, steroidal profiles, and seminal characteristics in young stallions. Proc. Am. Assoc. Eq. Pract. 43:195.
12. Kluber E. F., J. E. Minton, J. S. Stevenson, M. C. Hunt, D. L. Davis, T. L. Hoagland, and J. L. Nelssen. 1987. Growth, carcass traits, boar odor, and testicular endocrine functions of male pigs fed a progestagen, altrenogest. J. Anim. Sci. 66:470.
4. Bolt, D. J. 1971. Changes in the concentration of leutinizing hormone in plasma of rams following administration of oestradiol, progesterone, and testosterone. J. Reprod. Fertil. 24:435.
13. Meyer, W. J., C. Cole, and E. Emory. 1992. Depo Provera treatment for sex offending behavior: an evaluation of outcome. Bull. Am. Acad. Psy. and Law. 20:249.
5. Diamond, M. 1966. Progestogen inhibition of normal sexual behavior in the male guinea pig. Nature 209:1322. 6. Eckternkamp, S. E., and D. D. Lunstra. 1984. Relationship between LH and testicular development in progesterone-implanted prepubertal ram lambs. J. Anim. Sci. 59:441. 7. Erpino, M. 1973. Temporary inhibition by progesterone of sexual behavior in intact male mice. Horm. Behav. 4:355.
Literature Cited 1. Allrich, R. D., R. K. Christenson, J. J. Ford, and D. R. Zimmerman. 1983. Pubertal development in the boar: Age-related changes in testicular morphology and in vitro production of testosterone and estradiol. Biol. Reprod. 28:902.
8. Griffo, W., and C. Lee. 1973. Progesterone antagonism of androgen-dependent marking in gerbils. Horm. Behav. 4:351. 9. Henneke, D. R., G. D. Potter, J. L. Kreider, and B. F. Yeates. 1983. Relationship between body condition score, physical measurements, and body fat percentage in mares. Eq. Vet. J. 15:371. 10. Johnson, N. N., H. A. Brady, C. S. Whisnant, and P. A. LaCasha. 1998. Effects of altrenogest on sexual/aggressive behavior, hormonal profiles, and testicular parameters in young stallions. J. Eq. Vet. Sci. 18:249.
14. Miller, C. D., D. V. Dickson, T. L. Blanchard, J. A. Thompson, and L. Johnson. 1997. Effects of a commonly used progestagen on behavior and reproductive function of stallions. Proc. Fourth Ann. Race Horse Conf. Austin, TX. 15. SAS. 1998. SAS/STAT® User’s Guide (Release 6.03 Ed.). SAS Inst. Inc., Cary, NC. 16. Squires, E. L., S. L. Badzinski, R. P. Amann, P. M. McCue, and T. M. Nett. 1997. Effects of altrenogest on total scrotal width, seminal characteristics, concentrations of LH and testosterone, and sexual behavior of stallions. Theriogenology. 48:313. 17. Thompson, D. L. Jr., B. W. Pickett, E. L. Squires, and T. M. Nett. 1980. Sexual behavior, seminal ph and accessory sex gland weights in geldings administered testosterone and (or) estradiol-17b. J. Anim. Sci. 51:1358.
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Effects of Oral Altrenogest on TheHormonal, Testicular, and Behavioral Profiles of Two-Year-Old Stallions 1,2 N. L. HENINGER*, H. A. BRADY*,3, PAS, S. D. PRIEN†, A. D. HERRING*,2, PAS, K. A. GUAY*, and L. A. JANECKA* *Department of Animal Science and Food Technology, Texas Tech University, Lubbock, TX 79409 and †Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center, Lubbock, TX 79430
Abstract Altrenogest is frequently administered to young stallions in the equine industry as an off-label use to suppress sexual/ aggressive behavior. In this study, 2-yrold Quarter Horse stallions in earlyperformance training were administered altrenogest (0.044 mg/kg BW daily) to determine the effects on sexual/aggressive behavior, testicular parameters, and steroid hormone profiles. Horses were randomly assigned to treatment (n = 5) and control (n = 5) groups. The treatment group was administered a daily oral dose of 0.044 mg/kg BW daily for 67 d; the control group was given a daily
1Funding
for this project was provided by the Houston Livestock Show and Rodeo Association. Research was conducted in accordance with TTU ACUC # 9872. TTU CASNR manuscript T-R-404. 2The authors would like to acknowledge C. Guay, D. Johnson, and H. Vartorella for their assistance with the project.
oral sham dose of corn oil for 67 d. No significant differences were found between treatment groups in BW or body condition scores. Altrenogest had no effect on any behavioral parameters or seminal parameters measured. Averages of total scrotal width (TSW) were lower (P<0.03) for treatment animals at the end of treatment period (d 67). By d 157 mean TSW for both treatment and control stallions were not different. Both were significantly higher (P<0.03) than pretrial values at d 157. Comparisons of testicular weight (d 157) between control and treatment groups were not different. Histological analysis of testicular tissue revealed no significant difference for the average number of spermatids per seminiferous tubule. Altrenogest treatment reduced serum estrogen levels (estradiol 17-b ) by d 67 (P<0.02); however, estrogen levels returned to pretreatment values after a 90-d recovery. Serum testosterone values were not affected by treatment. Further research is needed to determine dose and age effects of altrenogest in the stallion.
3To whom correspondence should be ad- (Key Words: Stallion, Altrenogest, dressed: [email protected] Behavior, Hormone.)
Introduction Androgens are essential to maintain the normal sexual physiology of the male (1). Previous research exists in many species examining the use of progesterone in an effort to biochemically interrupt normal androgen production in the male. Studies involving rodents (5, 7, 8), boars (11, 12), rams (4, 6), and humans (13) have shown that progesterone reduces testosterone-dependent male sexual behavior. Altrenogest (ReguMate®; Intervet, Millsboro, DE), an oral synthetic progestin, is labeled for the suppression of estrus in mares. Altrenogest has also been used in a variety of male domestic farm animals for the reduction of sexual behavior with varying results depending on species, age, and dose. In the stallion, reported effects of altrenogest treatment have varied depending on length of treatment, dose, and age of stallion (3, 10, 14, 16). Squires et al. (16) examined the effects of 0.088 mg/kg BW daily in mature stallions. Stallions fed altrenogest for 150 or 240 d had
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decreased serum concentrations of testosterone and luteinizing hormone. Total scrotal width (TSW), daily sperm output (DSO), and libido were also significantly decreased by treatment (16). Miller et al. (14) examined the effects of a short-term administration (30 d) of altrenogest (0.044 mg/kg BW daily) in mature stallions. Only minor effects on stallion sexual/ aggressive behavior and semen quality were reported. Previous studies in our lab (10, 3) examined the suppressive effects of altrenogest (0.088 mg/kg BW daily) for 56 d on 2- to 4-yr-old stallions. Altrenogest decreased serum testosterone, estrogen, and LH (3). Decreases were found in DSO and scrotal circumference in stallions within the treatment group (10). Abnormalities in sperm morphology were significantly increased in the treatment group between the pre-trial collection period and the 8-wk collection period (10). The purpose of this study was to determine the effects of a daily oral dose (0.044 mg/kg BW) of altrenogest administered for 67 d to 2-yr-old stallions in training. This dose represents the label dose for suppression of estrus in mares and is referred to in the industry as the “single mare dose.” This study examined behavioral, hormonal, testicular, and seminal parameters to fully evaluate the potential of the drug as a temporary alternative to castration.
Materials and Methods General. Ten untrained 2-yr-old Quarter Horse stallions leased from four different ranches located in the Texas Panhandle and eastern New Mexico were used in the study. All horses were foaled in large pastures, and the exact age in months was not known; however, a mean age of 26 mo was estimated from ranch records. Stallions were housed together in a single 100' by 100' drylot paddock at the Texas Tech University Ranch Horse Center in New Deal, Texas. Upon arrival,
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horses were dewormed (Safeguardâ; Intervet) and vaccinated (Cephalovacâ VEWT; Boehringer Ingelheim Vetmedica Inc., St. Josephs, MO). Horses were fed highquality round-baled alfalfa hay (21% CP). Horses had ad libitum access to hay, salt blocks, and water. Three weeks prior to the commencement of the study, horses were halter-broken and trained under saddle in a manner typical for the young ranch horse. In this training process, horses were ridden on a schedule of walking and trotting for 1 hr with brief (10 min) loping patterns and rail work interspersed. Care was taken to equalize the workloads as much as possible by timing exercise schedules. During this 3-wk period, horses were also trained to use an artificial vagina for semen collection. In this training regimen, horses were teased every 48 h with an estrous mare and encouraged to mount for semen collection using an artificial vagina. A time limit of 20 min was set for each training session. Measurements of BW were determined monthly by individually weighing each animal with a Mettler-Toledo Digital Livestock Pen Scale (MettlerToledo, Inc., Columbus, OH). On d –7, 67, 157 of the trial, horses were assigned a body condition score (BCS) by two trained observers according to the equine system of Henneke et al. (9). Stallions were stratified across treatments according to pre-trial test breeding performance (measured by libido and mounting attempts) and separation of same sire pairs, and then randomly assigned to treatment (n = 5) and control (n = 5) groups. On d 1, treatment horses were orally administered altrenogest (ReguMateâ; Intervet) at a daily dose of 0.044 mg/kg BW for a period of 67 d (9.5 wk) during June, July, and the first week in August. Control horses were orally administered a daily sham dose of corn oil for the same time period (67 d). A 90-d recovery period followed the treatment period to examine physiological or behavioral modifications. Recovery period lasted
from the end of treatment until the first week in November. One control animal had very mild laminitis (not requiring medication) and was separated for 1 wk. One treatment animal was injured in the hock and was separated for 1 wk; treatment was continued in this animal. Sexual/Aggressive Behavior. Sexual/aggressive behavior was analyzed using a 3-min Standardized Teasing Test (10) (0 to 180 s) (d –7, 67, 157). Stallions were presented to a hand-held estrous mare over a solid partition and video-recorded in real time (30 frames/s). The positioning of the mare (front vs rear presentation) was held constant from stallion to stallion to avoid handler influence on the teasing sessions. Tapes were analyzed for frequency, duration, and latency to the following behaviors: sniffing/licking, biting/chewing, vocalization, Flehman’s response dropping of penis, erections, and aggressive behavior. Aggressive behavior was designated as any behavior directed at the handlers or mares (striking, rearing, or kicking). Duration was expressed in seconds of response within the 180-s observation period. Latency was expressed as seconds until first response of parameter is achieved. Frequency was expressed as mean number of times that the response occurred within the 180-s observation period. Mounting behavior, analyzed separately (d –7, 67, 157), examined the number of mounts or attempted mounts during the semen collection periods. Testicular and Seminal Parameters. Seminal parameters were analyzed three times beginning on d –7, 67, and 157. Stallions were collected using a hand-held mare in estrus and a Missouri artificial vagina. Daily sperm output was determined using a 7-d sequential collection period. Stallions were collected each day for 7 d to deplete sperm reserves; semen collection on d 7 was used for evaluation of concentration (Model 534A mod 1 Densimeter; Animal Reproduction Systems, Chino, CA). Motility was determined manually using light micros-
The Effects of Altrenogest on Two-Year-Old Stallions
TABLE 1. Effects of altrenogest on duration, latency, and frequency of behavioral parameters using a standardized teasing test (0 to 180 s). Overall comparisons
Duration
Latency
Frequency
Item
Control
Biting/chewing Sniffing/licking Flehman’s Vocalization Dropping penis Complete erection Aggressive behavior Biting/chewing Sniffing/licking Flehman’s Vocalization Dropping penis Complete erection Aggressive behavior Biting/chewing Sniffing/licking Flehman’s Vocalization Dropping penis Complete erection Aggressive behavior
18.3 101.6 10.3 22.6 73.3 36.6 8.0 107.0 16.3 124.0 103.3 77.3 139.6 143.6 2.0 5.4 1.1 2.3 1.5 0.7 0.9
copy by two trained evaluators. Total sperm and total progressive sperm were determined by standard calculations. Samples for morphology (d 7 semen ejaculate) were fixed in formalin (Formalin 10; Animal Reproduction Systems) for microscopic evaluation. Dry, stained slides were prepared using Hemotoxylin and Eosin stain (H&E). One hundred sperm cells per stallion were counted and classified as normal or abnormal (head, midpiece, or tail). Values for TSW were determined on d –7, 67, and 157 using a scrotal width caliper (Model 549A Stallion Scrotal Caliper; Animal Reproduction Systems). Measurements were taken three times by one trained operator and averaged to determine TSW for each period. Stallions were castrated at the end of the recovery period (d 157). After removal of the epididymus, testicles were weighed for comparison to control group. Tissue samples for histology were excised from the middle one-third of the left testicle of each stallion and fixed in Bouin’s
Treatment 22.3 92.6 5.0 29.3 114.0 70.6 6.0 95.0 5.0 130.0 82.0 61.3 95.3 144.6 2.3 5.4 1.1 1.9 1.1 1.1 0.7
P
SEM
0.76 0.77 0.38 0.74 0.34 0.21 0.78 0.75 0.51 0.38 0.68 0.74 0.18 0.97 0.89 0.61 0.84 0.60 0.82 0.17 0.28
8.9 12.9 3.4 13.7 23.9 28.6 4.7 28.2 6.9 23.57 23.6 22.3 29.9 24.0 1.2 1.0 0.6 1.0 0.5 0.4 0.5
solution for paraffin sectioning and H&E staining (Pathology Laboratory Texas Tech Health Sciences Center, Lubbock, TX). The mean number of elongated spermatids within 25 round seminiferous tubules was calculated by three trained evaluators using a Nikon Alphaphot Microscope (Nikon Inc., Melville, NY) with brightfield optics at 100x magnification. Spermatid counts were averaged across evaluators to determine values for statistical analyses. Hormonal Analysis. A blood sample was obtained from each stallion by jugular veni-puncture on 7-d intervals in the morning prior to feeding. Samples were allowed to clot at ambient temperature for 30 min and refrigerated at 5°C for 24 h. Samples were spun to isolate serum which was frozen at –20°C until all samples had been collected. Serum concentrations of testosterone and estrogen (estradiol 17-ß) were determined using radioimmunoassay kits (Diagnostic Products Corp., Los Angeles, CA), which have been validated in our lab (Dept. of Obstet-
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rics and Gynecology, Texas Tech Health Sciences Center, Lubbock, TX) for a number of species, including the horse. The samples were assayed without extraction, and all samples were analyzed for a single hormone within a single run. Sensitivities of the assays were 0.1 ng/mL for testosterone (with an interassay CV of 4.5%) and 8 pg/mL for estrogen (with an interassay CV of 3.8%). Statistical Analysis. Weight, body condition scores, testicular, and seminal and hormonal data were analyzed by ANOVA using general linear models procedures (15). Sexual/aggressive behavior as measured in the standardized teasing test including frequencies (n), latency (0 to 180 s) and duration (0 to 180 s), were also analyzed by ANOVA (15). For all data, treatment, time, horse within treatment, and treatment by time were included in the statistical model. The “horse within treatment” term was used as the error term to determine treatment differences. Means for all analysis were separated using Fisher’s protected LSD procedure.
Results and Discussion No differences in BW or body condition scores were found between control and treatment groups. Body weights did not differ between control and treatment stallions at d –7 (424.6 ± 15.2 vs 425.9 ± 6.8 kg, respectively), d 67 (430.5 ± 15.4 vs 424.1 ± 8.5 kg), or at the end of the recovery period, d 167 (455.0 ± 18.4 vs 450.9 ± 7.9 kg). Similarly, no differences were found between control and treatment animals in body condition scores at d-7 (4.7 ± 0.3 vs 5.2 ± 0.4, respectively), d 67 (5.1 ± 0.2 vs 5.2 ± 0.1) or at d 167 (5.4 ± 0.1 vs 5.6 ± 0.2). These data suggest that the use of altrenogest (Regu-Mate®) at the dose given did not suppress or augment overall growth or condition of these young stallions. There were no significant effects for any measured behavioral parameter throughout pretreatment, treatment, and recovery periods.
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from this animal on d 67; however, treatment was continued. On d 67, three of five control and two of five Pretrial (d 0) Control SEM Treatment SEM treatment stallions were able to be collected. For the recovery period (d 157), four of five controls and three (n = 3) (n = 3) of five treatment animals were Concentration (1 x 106/mL) 93.6 41.5 113.6 37.2 collected. No differences between Volume (mL) 15.2 9.3 28.0 8.4 treatments were observed for any Motility (%) 27.2 19.9 21.6 17.8 6 measured seminal parameter (Table Total sperm (1 x 10 ) 1,019.9 1,074.3 2,567.3 960.9 2). Very large differences were seen End of Treatment (d 67) Control SEM Treatment SEM across individual horses. This is in contrast to Johnson et al. (10) and b Squires et al. (16), who reported (n = 3) (n = 2) 6 significant decreases in DSO in Concentration (1 x 10 /mL) 142.3 41.5 161.0 49.1 Volume (mL) 5.8 9.3 17.6 11.0 altrenogest-treated horses using a Motility (%) 51.2 19.9 25.6 24.5 regimen of 0.088 mg/kg BW, suggestTotal sperm (1 x 106) 854.9 1,074.3 1,774.2 1,271.1 ing a dose effect of altrenogest on seminal quality in stallions. The End of Recovery (d 157) Control SEM Treatment SEM seminal effects appeared to be partially reversible in mature stallions as (n = 4) (n = 3) DSO was reported to increase after a Concentration (1 x 106/mL) 137.0 32.2 109.3 37.1 recovery period (16), although DSO Volume (mL) 9.5 7.2 14.0 8.4 remained suppressed 8 wk after Motility (%) 66.3 15.4 37.6 17.8 cessation of altrenogest treatment in Total sperm (1 x 106) 1,026.3 832.1 1,276.0 960.9 young stallions (10). Miller and coworkers (14) found only minor effects aNumbers are based on stallions collected as described in text. in semen quality, which returned to bOne treatment stallion was not included because of lameness. pretreatment levels by a period of 30 d following cessation of altrenogest treatment. We found that treatment with altrenogest had no significant Analysis of the standardized teasing short-term administration of effects on sperm morphology (pertest revealed no differences between altrenogest for 30 d at 0.044 mg/kg centage of normal sperm, head, midtreatment groups in duration, latency BW resulted in only minor behavpiece, and tail abnormalities) after 67 or frequency of behavioral paramioral modifications. Stallion–mare d (Table 3). This is in contrast to eters including biting/chewing, interactions in the breeding shed studies by Johnson et al. (10) using a sniffing/licking, Flehman’s response, such as time to erection frequency, higher dose (0.088 mg/kg BW) of vocalization, dropping of the penis, mounting ability, and ejaculation complete erections, or aggressive were not reduced during the course of altrenogest, finding that sperm head and tail abnormalities were signifibehavior (Table 1). Large differences the treatment (14). It appears that cantly increased in the treatment were observed between individual there are large dose and age effects group between the pretrial period horses. Additionally, no significant associated with behavioral modificaand the end of the 8-wk treatment differences were noticed for mounttions in the stallion. period. ing behavior/attempted mounts for After the 3-wk training period, a Significant differences were found any 7-d sequential collection period total of three control and three (P<0.03) in TSW as a treatment by (P>0.05). Previous research has treatment stallions were able to be demonstrated that feeding collected due to differences in altrenogest at 0.088 mg/kg BW baseline libido of these young staldecreased libido, Flehman’s response, lions (Table 2). As a result, three of vocalization, and erections in 2- to 4- five stallions within each group were yr-old treated stallions (10). Squires able to be collected at the pre-trial et al. (16) also found significant period (d –7 to d 0). The same three sexual behavioral suppression in animals in each group were collected mature stallions (3 to 18 yr) adminis- at each time point with the exception Figure 1. Scrotal widths for pretrial, end of tered 0.088 mg/kg BW for 150 or 240 of d 67. One treatment animal was treatment, and end of recovery periods. a,b,cDifferent superscripts indicate significant d. This contrasts with the study by injured in the hock on d 65; no Miller (14) in mature horses in which attempt was made to collect semen differences. Standard error = 1.5.
TABLE 2. Effects of altrenogest on mean seminal parameters pretrial, end of treatment, and end of recovery after 7-d sequential collection perioda.
The Effects of Altrenogest on Two-Year-Old Stallions
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TABLE 3. Effects of altrenogest on head, mid-piece, and tail abnormalities in 2-yr-old stallions after 67 d (end of treatment)a. Overall comparison Item Normal Head Mid-piece Tail
Control (%)
SEM
Treatment
SEM
63.7 3.5 12.8 20.0
7.1 1.7 1.4 7.3
58.1 7.0 11.6 23.3
8.7 2.1 1.7 8.9
Figure 2. Estradiol values (17-b) for pretrial, end of treatment, and end of recovery period. a,b,cDifferent superscripts indicate significant differences. Standard error = 1.5.
in response to a 0.088 mg/kg BW regimen of altrenogest was reported are based on stallions collected. Recovery period data not available for by Brady et al. (3). Work in the boar morphology. found that altrenogest decreased levels of testosterone (11) as well as time interaction (Figure 1). Averages animals at the end of the recovery estrogen (12). Kluber et al. (11) of TSW across control and treatment period (18.3 vs 11.3, respectively, hypothesized that there may be a groups were lower for treatment spermatids per seminiferous tubule). threshold concentration of testosteranimals at the end of the treatment Large differences were seen between one in growing boars that is responperiod. Values were not significant individual horses. Johnson et al. (10) sible for sexual behavior, libido, and by the end of the recovery period. reported a significant decrease in maintenance of spermatogenesis. Both groups were significantly higher spermatids per seminiferous tubule. This may hold true across most than pretrial values at the end of the Kluber et al. (11) also found that domestic farm animal species, includrecovery period (P<0.03). In agreetotal sperm per testis, concentration ing the young stallion. ment with Squires et al. (16) the of sperm/gram of tissue, total seminIt is acknowledged that season authors found a significant decrease iferous epithelium, and total intersti- and increasing age in months may in TSW in altrenogest-treated staltium per testis were less for boars fed affect different parameters of this lions. The present study documented altrenogest. trial. Treatment data were in coma return to TSW pretreatment values Estrogen concentrations of treatparison to control animal data. The after a 90-d recovery period. Simiment stallions were lower (P<0.02) age at which a colt reaches puberty larly, Squires et al. (16) reported that than control stallions by the end of also must be considered. Puberty, in mature stallions, the effect apthe treatment period (Figure 2). No depending on the definition, can peared to be partially reversible differences were found between range from 14 to 24 mo of age or resulting from an increase in TSW groups 90-d post-cessation of treatmore (2). The colts used in this study after cessation of progestin treatment. A significant reduction in may have been in various early stages ment. However, Johnson et al. (10) estrogen levels in young stallions of post-puberty. This may be due in found that mean scrotal circumferfollowing 8 wk of altrenogest treatpart to previous environmental ence for young altrenogest-treated ment was found by Brady et al. (3); conditions and late birth dates. stallions was less than control anihowever, estrogen levels returned to These effects may have contributed to mals at cessation of treatment. This pretreatment levels after an 8-wk lower levels of libido demonstrated difference remained after an 8-wk recovery period. Thompson et al. by some individuals within each recovery period (10). Research with (17) demonstrated that variations in treatment as well as the large variathe boar (11) also has supported a libido are mediated by estrogen levels, tion between horses. reduction in testicular size associated which are high in the stallion. No with altrenogest. Kluber et al. (11) significant differences were observed found boar testicular weight and in serum testosterone values throughvolume declined linearly as duration out the duration of the trial. It has Little information exists regarding of altrenogest feeding increased. been demonstrated that testosterone the extra-label use of altrenogest in No significant differences were secretion is dependent upon the young stallions; however, it is used observed in mean testicular weights secretion of anterior pituitary gonawidely in the performance horse at castration (d 157) between control dotrophins. Squires et al. (16) industry. In this study, altrenogest at and treatment groups (123.9 g vs documented a decrease in serum the dose given had little effect on 124.1 g, respectively). Data from testosterone in mature stallions in behavioral and seminal parameters in histological analysis showed no response to a 0.088 mg/kg BW regicontrast to studies using higher differences between groups in sperma- men of altrenogest. A suppression of doses; however, mean TSW was tids per seminiferous tubule in treated testosterone levels in young stallions reduced after altrenogest treatment. aNumbers
Implications
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Our data have shown that altrenogest decreased levels of estrogen after 60 d of treatment. This finding is important in that estrogen is related to libido in the stallion. In reviewing existing studies, there are many factors that may affect both short- and long-term physiological effects of altrenogest in the stallion, including dose, length of treatment, age of stallion, and stallion-tostallion variation. It is premature to be able to give clear recommendations to horse owners regarding the safe use of altrenogest in the stallion. Further work must be done to elucidate the risks and benefits involved.
2. Amann, R. P. 1993. Physiology and endocrinology. In: Equine Reproduction. A.O. McKinnon and J.L. Voss (Eds.). p. 658. Lea and Febiger, Philadelphia, PA.
11. Kluber, E. F., D. S. Pollman, D. L. Davis, and J. S. Stevenson. 1985. Body growth and testicular characteristics of boars fed a progestin, altrenogest. J. Anim. Sci. 66: 470.
3. Brady, H. A., N. N. Johnson, C. S. Whisnant, S. D. Prien, and J. M. Hellman. 1997. Effects of oral altrenogest on testicular parameters, steroidal profiles, and seminal characteristics in young stallions. Proc. Am. Assoc. Eq. Pract. 43:195.
12. Kluber E. F., J. E. Minton, J. S. Stevenson, M. C. Hunt, D. L. Davis, T. L. Hoagland, and J. L. Nelssen. 1987. Growth, carcass traits, boar odor, and testicular endocrine functions of male pigs fed a progestagen, altrenogest. J. Anim. Sci. 66:470.
4. Bolt, D. J. 1971. Changes in the concentration of leutinizing hormone in plasma of rams following administration of oestradiol, progesterone, and testosterone. J. Reprod. Fertil. 24:435.
13. Meyer, W. J., C. Cole, and E. Emory. 1992. Depo Provera treatment for sex offending behavior: an evaluation of outcome. Bull. Am. Acad. Psy. and Law. 20:249.
5. Diamond, M. 1966. Progestogen inhibition of normal sexual behavior in the male guinea pig. Nature 209:1322. 6. Eckternkamp, S. E., and D. D. Lunstra. 1984. Relationship between LH and testicular development in progesterone-implanted prepubertal ram lambs. J. Anim. Sci. 59:441. 7. Erpino, M. 1973. Temporary inhibition by progesterone of sexual behavior in intact male mice. Horm. Behav. 4:355.
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14. Miller, C. D., D. V. Dickson, T. L. Blanchard, J. A. Thompson, and L. Johnson. 1997. Effects of a commonly used progestagen on behavior and reproductive function of stallions. Proc. Fourth Ann. Race Horse Conf. Austin, TX. 15. SAS. 1998. SAS/STAT® User’s Guide (Release 6.03 Ed.). SAS Inst. Inc., Cary, NC. 16. Squires, E. L., S. L. Badzinski, R. P. Amann, P. M. McCue, and T. M. Nett. 1997. Effects of altrenogest on total scrotal width, seminal characteristics, concentrations of LH and testosterone, and sexual behavior of stallions. Theriogenology. 48:313. 17. Thompson, D. L. Jr., B. W. Pickett, E. L. Squires, and T. M. Nett. 1980. Sexual behavior, seminal ph and accessory sex gland weights in geldings administered testosterone and (or) estradiol-17b. J. Anim. Sci. 51:1358.