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Biology of Reproduction 60, 1360-1366 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Effect of Long-Term Immunization against Inhibin on Sperm Output in Bulls1

J.H. Bamea, J.C. Daltona, S.D. Degelosa, T.E.M. Goodb, J.L.H. Irelandb, F. Jimenez-Krasselb, T. Sweeneyc, R.G. Saacke2,a, and J.J. Irelandb

a Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 b Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824 c Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To determine the effect of neutralization of inhibin on sperm output, 12 Holstein bulls were paired by birth date and weight on Day 1 of age. Each bull was actively immunized against bovine inhibin {alpha}1–26 gly-tyr (bINH) conjugated to human alpha globulin (HAG, n = 6 bulls) or HAG alone (controls, n = 6) at 60 days of age; booster immunizations were administered at 90, 104, 124, 270, and 395 days of age. Body weights and scrotal circumferences were measured at the time of primary immunization and at 10 days after each booster. In addition, jugular blood was obtained at 60, 70, 100, 114, 134, 280, and 405 days of age, during the 3-wk sperm collection period, and during a 6-h blood-sampling period after sperm collection to determine bINH antibody titer and concentrations of FSH, LH, testosterone, and estradiol. Beginning at 405 days of age, sperm output was measured 3 days/wk for 3 wk with two successive ejaculates collected each day for a total of 18 ejaculates per bull. During Days 60–405 of age, the increase in titer of bINH antibodies, scrotal circumference, and serum concentration of FSH was greater (p < 0.01) for the bINH-immunized compared with control bulls. There were significant (p < 0.01) pair x treatment interactions for sperm output and serum FSH and LH concentrations. Specifically, bINH-immunized bulls for four of the six pairs had nearly 50% greater serum FSH concentrations and sperm output. For the remaining two pairs, sperm output was lower and FSH was either lower or only marginally higher in the bINH-immunized bulls compared with controls. Also, the control bulls for the two remaining pairs produced more sperm than all but one bINH-immunized bull, and had markedly higher serum LH concentrations than all other bulls. To summarize, enhancement of sperm output after immunization against inhibin depends on the subsequent increment in FSH concentrations. We conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FSH is one of the main hormones stimulating spermatogenesis [1]. Inhibin is a 31- to 34-kDa heterodimeric glycoprotein ({alpha}-ßA is inhibin-A, {alpha}-ßB is inhibin-B) produced primarily in gonads with physiological roles important for regulation of testicular function (see reviews in [2–5]). In general, the best-established role for inhibin is as an endocrine negative feedback hormone that decreases secretion of FSH [6–9]. Recent evidence also supports an autocrine or paracrine role for inhibin in regulation of testicular function [2, 3, 5]. Specifically, binding sites/receptors for inhibin [10–12] and receptors for activins [11, 13–15] that contain two inhibin ß subunits [2] are in testicular cells. Paracrine regulation has been demonstrated in vivo: intratesticular injections of inhibin reduce the number of spermatogonia in the treated testis of adult mice and hamsters with no effect on the contralateral testis [16]. In vitro inhibin treatment of rat seminiferous tubule segments reduces DNA synthesis in spermatogonia [17]. The aforementioned results, coupled with studies showing that deletion of the inhibin {alpha} subunit gene in mice results in testicular tumors shortly after birth [18, 19], strongly imply that inhibin and/or inhibin {alpha} subunits have endocrine and local inhibitory roles fundamentally important for regulation of testicular function. In support of the overall suppressive effect of inhibin on pituitary and testicular function, immunization against inhibin increases secretion of FSH in a variety of species, including rats [20, 21], rams [22, 23], bulls [24–26], and monkeys [27]. In addition, immunization of prepubertal animals against inhibin enhances daily sperm output in urine of 23-mo-old rams [22] and testicular spermatid numbers for 9- to 13-mo-old bulls [24, 25]. Taken together, these findings imply that inhibin is a general "suppressor" of spermatogenesis. However, a recent report demonstrates that immunization of adult rams does not alter sperm output, despite an increase in serum FSH [28]. The reason for the absence of a positive effect of inhibin immunization on sperm production in adult rams is unknown, but it may be related to differences in responsiveness of prepubertal and adult animals. Alternatively, a single indirect measure of sperm output, as in the previous studies showing a positive effect of inhibin immunization on sperm production [24, 25], may not accurately reflect sperm output. Consequently, the primary objective of the present study was to determine whether long-term immunization of prepubertal bulls against inhibin increased number of sperm ejaculated in yearling postpubertal bulls.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Immunogen

Bovine inhibin {alpha}1-26 gly-tyr (bINH, 30 mg; [24]) was conjugated to 16.4 mg human alpha globulins (HAG; Sigma Chemical Co., St. Louis, MO) using bisdiazotized benzidine [29] as previously described [24]. A previous study [30] showed that antibodies generated against bINH:HAG cross-react with at least eight different molecular variants of bovine inhibin dimers and inhibin {alpha} subunits, but not with activins or the inhibin/activin serum-binding proteins, follistatin and {alpha}-macroglobulins.

Animals and Treatments

Twelve Holstein bull calves paired by birth date and weight (45.5 ± 0.5 kg, mean ± SEM) on Day 1 of age were injected from 60 to 395 days of age with bINH:HAG or HAG (control). At 60 days of age, each bull was given a primary immunization (s.c., four sites on the neck) of 500 µg bINH: 250 µg HAG or 250 µg HAG dissolved in 0.5-ml sterile deionized water and emulsified in 2 ml of Freund's complete adjuvant. Booster injections emulsified in Freund's incomplete adjuvant were given at 90, 104, and 124 days of age, and boosters in Freund's complete adjuvant were given at 270 and 395 days of age. Jugular blood (10 ml) was collected at the time of primary immunization and at 10 days after each injection to determine bINH antibody titer and quantify serum concentrations of FSH, LH, testosterone, and estradiol. Body weight and scrotal circumference were measured at 60, 70, 100, 114, 134, 186, 220, 240, 280, 365, and 405 days of age. Body weights for Days 60 and 70 were measured for five pairs only. All animal experimentation was undertaken using guidelines set forth by the animal use committee for Virginia Polytechnic Institute and State University.

Analysis of Sperm Output and Quality

At 285 days of age, bulls were moved into individual pens and trained for semen collection into an artificial vagina on a weekly basis. Extragonadal sperm reserves were stabilized by collecting semen from bulls six times a week—2 ejaculates in succession on Mondays, Wednesdays, and Fridays—for 3 wk prior to the sperm output study. To determine sperm output, semen was collected from 405-day-old bulls for 3 wk at the same frequency and in the same manner as for stabilizing extragonadal sperm reserves. Sexual preparation of each bull consisted of two false mounts on a "teaser" bull separated by 2 min of active restraint preceding each ejaculate. This procedure maximizes sperm numbers in each ejaculate [31]. Immediately after collection, semen was evaluated for volume and sperm concentration to calculate sperm output. To examine sperm movement, progressive motility was estimated to the nearest 10% by using a phase-contrast microscope (x250 magnification) equipped with a heated (37°C) stage. The two ejaculates for each bull were pooled after motility determinations, and a 100-µl aliquot of each pool was added to 1-ml glutaraldehyde fixative [32] for evaluation of sperm morphology for each day's collections. Sperm cells were morphologically classified by differentially counting 100 sperm cells from each of two wet smears prepared from the fixed sperm samples, and results were recorded as the average [33]. A single blood sample was taken from each bull immediately after completion of each day's semen collection. On the final day of the sperm output study (Day 423 of age), blood was sampled at 1-h intervals for 6 h to determine serum concentrations of FSH, LH, testosterone, and estradiol.

Inhibin Antibody Titer Determination

Bovine INH was radioiodinated [24], and 20 000 cpm [125I]bINH was incubated with duplicate samples of each bull's serum diluted from 1:10 to 1:2000 in PBS (pH 7.0). Since [125I]bINH binding to the various dilutions of serum was linear (data not shown), percentage [125I]bINH bound to serum diluted 1:100 was used as the index for titer of bINH antibodies, as described and validated previously [24].

RIAs

Concentrations of FSH in serum were determined using a previously validated heterologous RIA [34, 35] that was recently shown to correlate with in vitro bioassay of FSH bioactivity [36]. Ovine FSH (USDA oFSH-19-SIAFP-I-2) was used as radioiodinated tracer, bFSH (USDA bFSH-I-2) as standard, and NIDDK anti-oFSH-1 (AFP-C5288113; Rockville, MD) as antiserum. Intraassay coefficient of variation (CV) was 5.2%, and sensitivity of the assay was 0.03 ng/ml.

Estradiol-17ß concentrations were determined in duplicate 200-µl serum samples previously extracted with ether using a modified version [37] of the commercial Estradiol MAIA Kit (Polymedco Inc., Courtland Manor, NY). Standard curves ranged from 0.195 to 50 pg/ml (or 0.039–10 pg/tube), and the mean (± SEM) ED50 for standard curves (n = 4) averaged 2.39 (± 0.13) pg/ml. Limit of sensitivity, defined as amount of estradiol detected in serum samples from an ovariectomized heifer, averaged 0.04 (± 0.01, n = 4 samples) pg/ml. Intraassay (n = 4 assays) CV was 35.4% for serum samples (n = 4) that averaged 0.04 pg/ml estradiol, 10.5% for ED50 values (n = 4) that averaged 2.39 pg/ml, and 6.6% for samples (n = 4) that averaged 3.2 pg/ml.

Concentrations of LH were determined using USDA bovine (b)LH-I-2 as radioiodinated tracer and standard, and a monoclonal antibody against bLH B-518B7 [38, 39]. Intraassay CV was 5.8%, and sensitivity of the assay was 0.03 ng/ml.

Testosterone concentrations were determined using a nonextraction commercial kit (Diagnostic Products, Los Angeles, CA). Intraassay CV was 6.1%, and sensitivity of the assay was 0.04 ng/ml.

Statistical Analyses

All analyses were computed using the General Linear Models (GLM) procedures of the Statistical Analysis System [40]. Means ± SEM for body weights were determined. Body weights at birth and at 100, 114, 134, 186, 220, 240, 280, 365, and 405 days of age were analyzed using the repeated option of GLM. Scrotal circumference was analyzed with measurement at 60 days of age as the covariate using a model that also included pair, treatment, age, pair x age, and treatment x age. Inhibin titer and concentrations of FSH, LH, testosterone, and estradiol during the immunization period (Days 60–405 of age) were analyzed using a model that included pair of bulls, treatment, age, and the interactions pair x treatment, pair x age, treatment x age. The same model, with week replacing age and with the additional interaction, pair x treatment x week, was used to analyze sperm output, sperm output corrected for body weight at Day 405, sperm output corrected for scrotal circumference at Day 405, inhibin titer, and concentrations of FSH, LH, testosterone, and estradiol during the 3-wk sperm output period (starting at 405 days of age). Inhibin titer and concentrations of FSH, LH, testosterone, and estradiol for the hourly blood-sampling period on Day 423 were analyzed using the same model with hour replacing age. The data for percentages of normal sperm and sperm motility were analyzed using GLM for repeated measures with a model that included pair and treatment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibin Titer

The overall mean titers of bINH antibodies during Days 60–405 of age and during the sperm output and hourly blood-sampling periods were greater (p < 0.01) in bINH-immunized bulls compared with controls (Fig. 1). During Days 60–405 of age, a significant (p < 0.01) treatment x age interaction existed for bINH antibody titers, as shown by the increase in bINH antibody titers in bINH-immunized bulls compared with relatively low and stable titers for controls (Fig. 1).



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  		FIG. 1. Effect of immunization of bulls against a synthetic fragment of inhibin on titer of inhibin antibodies. Bulls were immunized with bINH conjugated to HAG (Treated) or HAG alone (Control) on Days 60, 90, 104, 124, 270, and 395 of age (shown by arrows). The y-axis depicts titer of inhibin antibodies. Titer is defined as percentage of 20 000 cpm of [125I]bINH bound to 10 µl of serum. The three x-axes represent: left, days of age of bulls when blood samples were collected; center, weeks of the sperm output study and range in days of age of bulls; right, number of blood samples taken at hourly intervals on Day 423 of age of bulls. In the left panel, each bar represents the mean (± SEM) for a single daily determination of titer from each of six bulls. In the center, each bar represents the weekly mean (± SEM) for determination of titer on 3 different days per week from each of the six bulls for a grand total of 18 titer determinations for six bulls. On the right, each bar represents the hourly mean (± SEM) for a single determination of titer from each of six bulls.

Effects of Immunizations on Body Weight and Scrotal Circumference

A significant treatment x age interaction (p < 0.01) existed for scrotal circumference, indicating that the increase in scrotal size during Days 60–423 of age was greater in immunized bulls compared with controls (Fig. 2). The average body weight of immunized bulls exceeded that of controls beginning at 100 days of age but was significant (p < 0.05) only for Days 220 and 240 (Fig. 2). Much of the observed difference was attributable to one control bull with slow growth.



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  		FIG. 2. Effect of immunization of bulls against inhibin on scrotal circumference and body weight. Bulls were immunized and measurements made on 60, 70, 100, 114, 134, 186, 220, 240, 280, 365, and 405 days of age as explained in Materials and Methods. Each symbol represents mean (+SEM) weight for six bINH-immunized (Treated) or six control bulls. Day 1 = day of birth.

Effects of Immunizations on Serum Concentrations of FSH, Estradiol, LH, and Testosterone and on Sperm Quality

There was a significant (p < 0.01) treatment x age interaction for serum FSH concentrations during Days 60–405 of age, with immunized bulls having the greater serum FSH concentrations on Days 280 and 405 of age as compared with control bulls (Fig. 3). In addition, there was a significant (p < 0.01) treatment x week interaction during the sperm output period, as shown by the higher but declining serum FSH concentrations in bINH-immunized bulls from Weeks 1 to 3 as compared with controls. Estradiol concentrations in bINH-immunized bulls increased (p < 0.01) markedly from Days 134 to 280 and remained high in relation to controls until the hourly sampling period of Day 423. However, estradiol concentrations tended to be higher (p < 0.10) in bINH-immunized bulls compared with controls during the sperm output period. Serum LH and testosterone concentrations increased (p < 0.01) in immunized and control bulls during Days 60–405 (Fig. 4). However, the significant (p < 0.05) treatment x age interaction indicated that the age effects on testosterone concentrations were not the same in bINH-immunized and control bulls. The large LH increase for control bulls at Days 280 and 405 was attributable primarily to two of the six control bulls. This difference owing to these two control bulls was also observed in the sperm output period (Fig. 5).



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  		FIG. 3. Effect of immunization of bulls against inhibin on serum concentrations of FSH and estradiol. Bulls were immunized and measurements made as explained in Figure 1 legend. The x-axes for each panel and bars are defined in Figure 1 legend.



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  		FIG. 4. Effect of immunization of bulls against inhibin on serum concentrations of LH and testosterone. The high LH concentration for control bulls was due primarily to 2 of the 6 control bulls. Bulls were immunized and measurements made as explained in Figure 1 legend; the x-axes and bars are defined in Figure 1 legend.



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  		FIG. 5. Effect of immunization against inhibin on sperm output, antibody titer, and serum concentrations of FSH, estradiol, LH, and testosterone in individual pairs of bINH-immunized (Treated) and control bulls. Bulls were immunized and sperm collected during Days 405–423 of age as explained in Figure 1 legend. Each bar in the panel for total sperm represents the total number of sperm in 18 ejaculates combined per bull. Each bar in the remaining panels represents the overall mean (± SEM) for nine determinations (3 per week x 3 wk) per bull. Titer is defined in Figure 1 legend.

The least-squares means for normal sperm morphology (59.4 ± 3.8% vs. 68.4 ± 3.8%) and motility (76.6 ± 0.6% vs. 76.9 ± 0.6%) were not different for bINH-immunized and control bulls, respectively.

Effects of Immunizations on Each Pair of Bulls (Treated and Control) during the Sperm Output Period (Days 405–423) and on Day 425

The effect of immunization on parameters measured during the sperm output period is best described by the responses of the six pairs of bulls (Fig. 5). Despite a highly significant bINH antibody titer in treated bulls, immunization did not significantly affect any other main effect measured. However, there were significant pair x treatment interactions (p < 0.01) for sperm output and serum concentrations of FSH, estradiol, and LH during the sperm output period (Fig. 5). Sperm output corrected for differences in scrotal size and body weight did not alter these interactions. In four of the six pairs, the bINH-immunized bull had nearly 50% greater total sperm output after 18 ejaculates compared with his control (pairs 2, 3, 5, and 6; Fig. 5). Associated with the higher total sperm output in the immunized bulls for pairs 2, 3, 5, and 6 were nearly 50% higher serum FSH concentrations compared with the control for each pair (Fig. 5). The two bINH-immunized bulls (in pairs 1 and 4) that had lower total sperm output compared with their controls also had lower serum concentrations of FSH compared with all other bINH-immunized bulls. The high variability among bulls for sperm output was caused primarily by two controls, those in pairs 1 and 4 (Fig. 5). This rendered the main effect of immunization on mean sperm output during the 3-wk period insignificant (p > 0.05) with means ± SEM of 20.7 ± 1.6 and 27.2 ± 1.5 for control and immunized bulls, respectively. Specifically, total sperm output in controls for pairs 1 and 4 was nearly double that for each of the remaining controls, and as high as or higher than total sperm output for all but one of the bINH-immunized bulls. This high sperm output for the two controls could not be explained by corresponding alterations in body weight (data not shown), scrotal circumference (data not shown), titers of inhibin antibodies (Fig. 5), or serum concentrations of FSH, testosterone, and estradiol during the sperm output period (Fig. 5). However, serum concentrations of LH were at least 3- to 5-fold higher in the controls for pairs 1 and 4 compared with all other bulls (Fig. 5). In addition, LH was also at least 2- to 6-fold higher for the controls in pairs 1 and 4 compared with all other bulls at 280 days of age (data not shown). Despite the LH variation, testosterone concentration did not follow this pattern.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study extend previous inhibin studies in males [16, 17, 20, 21, 23–27] by demonstrating that long-term active immunization of prepubertal bulls against inhibin enhances sperm output in some reproductively maturing bulls. That inhibin immunization did not increase sperm output in all bulls in our study may be best explained by the significant pair x treatment interactions for FSH and sperm output, which imply that enhanced sperm output depends on the subsequent increment in serum FSH concentrations after inhibin immunizations. The results of this experiment are in contrast, however, to the recent observation that immunization of adult rams against inhibin did not increase sperm output, despite marked increases in serum FSH concentrations and scrotal circumference [28]. In addition, testicular size was not increased in previous studies in beef bulls despite increased FSH and/or number of spermatids [24, 25]. Perhaps a combination of different breeds of cattle and immunization protocols, coupled with a diminished FSH response in the previous studies compared with our present study and that of McKeown et al. [28], explain the discrepancy among laboratories. Certainly in the present study an increase in scrotal circumference was observed in immunized bulls. Considering the close relationship between scrotal circumference and daily sperm production, some of the effects of immunization could be due to testes size. However, for the bulls in this study, scrotal circumference was not related to sperm output (r = 0.55, p > 0.05). While dissimilarities in sperm output may be due to species differences, or to the different lengths of the sperm collection periods for each study, we speculate that spermatogenic capacity of the postpubertal testis is more easily up-regulated by immunizing against inhibin before puberty than during adulthood.

Determining the mechanisms explaining the multiple, diverse effects of inhibin immunization on gonadotropin and estradiol levels, body weight, and scrotal size was beyond the scope of our study. Nevertheless, in contrast to the actions of inhibin, activin stimulates FSH secretion [2, 5, 6] and proliferation of sperm [17, 41] and Sertoli cells [42]. In addition, "knockout" of the activin type II receptor in mice decreases FSH secretion, volume of the seminiferous tubules, and fertility [9]. Since inhibin antagonizes activin production [43] and action [2, 5, 6], it is possible that increases in the local ratio of activin:inhibin in the pituitary and testes could explain most of the diverse effects of inhibin immunization on bulls in our study. In support of alterations in ratio of activin:inhibin, activin levels are increased in follicles following active immunization of sheep against inhibin [44], and in serum after knockout of the inhibin {alpha} subunit gene in mice [45]. Although a previous report indicates that inhibin antibodies are not detected in rete testes fluid after long-term active immunization of bulls [25], this finding does not exclude the possibility that inhibin antibodies located in other testicular compartments diminish local inhibin action that in turn enhances activin action.

Despite similar weights (at 1 day of age), ages, and breed, sperm output was highly variable among bulls in our study, as previously reported [46, 47]. Since serum FSH concentrations were lower in the bINH-immunized bulls for pairs 1 and 4 as compared with the other bINH-immunized bulls, this may explain why their sperm output was also lower than that of their paired controls. However, sperm output among all the bINH-immunized bulls was similar and was markedly enhanced compared with that of all other controls except those for pairs 1 and 4. Thus, in addition to the relatively low serum FSH concentrations, sperm output was relatively lower in two bINH-immunized bulls probably as the result of an unusually high sperm output for pair 1 and 4 controls. Although precise reasons for this variability are unknown, results from several studies imply that differences in number of Sertoli cells may contribute to variability in sperm output in bulls [47, 48]. Nevertheless, the two high sperm-producing controls in our study, which were primarily responsible for the marked variability in sperm output among bulls, did not have the expected higher serum levels of FSH compared with other bulls. This finding indicates that mechanisms other than those mentioned may increase sperm production, as suggested in previous studies [47, 48]. Interestingly, the two high sperm-producing controls had remarkably higher LH levels throughout different development periods and the hourly blood-sampling regimen compared with all other bulls. This surprising result implies a potentially important positive role for LH in sperm production, as previously shown after immunoneutralization of LH in prepubertal rats [49] and after treatment of bull calves with LHRH [50].

In contrast to the consistent finding among laboratories that serum FSH concentrations increase following immunization against inhibin in males, alterations in basal or episodic secretion of LH and testosterone are inconsistent [20, 23–28]. In bulls, the pattern of secretion of LH and testosterone is highly episodic and dramatically altered during pubertal development, while FSH secretion is relatively unaltered [51]. Thus, we suspect that the aforementioned inconsistencies among laboratories can best be explained by blood-sampling protocols that were inadequate to accurately assess the effects of inhibin immunizations on LH or testosterone secretion during pubertal development. For example, although overall testosterone concentrations were unaltered by immunization against inhibin in the present study, in contrast with previous results [24], testosterone levels were higher in immunized animals at 9 mo of age compared with controls both in our present and in previous studies [24]. In vitro studies examining the effects of inhibin on LH are also inconsistent. For example, while inhibin is generally considered a selective negative regulator of FSH secretion [52], reports also show that inhibin enhances GnRH-induced LH release during culture of sheep pituitary cells [30, 53] but decreases basal [54] and GnRH-induced [55] LH release during culture of pituitary cells from rats. Species and seasonal differences, or differences in response of males to the acute effects of passive [20, 21, 26, 27] vs. long-term active immunizations beginning in prepubertal [24, 25] or adult males [23, 28], could also explain the inconsistencies among laboratories. Because of these caveats, it is necessary to evaluate further the physiological significance of our findings that serum LH concentrations were remarkably greater in the two high sperm output control bulls compared with all other control and bINH-immunized bulls, and that serum testosterone concentrations were unaltered during the sperm output period despite marked alterations in serum levels of LH.

In summary, long-term active immunization of bulls against inhibin during their pre- and early postpubertal development resulted in 1) increased titer of bINH antibodies, scrotal circumference, body size, and serum concentrations of FSH and estradiol during Days 60–405 of age; 2) increased serum FSH and sperm output in most bulls during the sperm output period; and 3) no effect on sperm motility and morphology. This study also indicated that enhancement of sperm output depends on the subsequent increment in serum FSH concentrations after immunization against inhibin. On the basis of these results, we conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls.


   ACKNOWLEDGMENTS
 
The authors wish to thank the graduate and undergraduate students Sher Nadir, Billy Walker, and Christy Carroll for their assistance with the immunization, bleeding, and semen collection. Sincere thanks to Dr. Ronald Pearson for his advice on statistical analysis.


   FOOTNOTES
 
1 Research supported by grants to R.G.S. from Select Sires and NAAB and to J.J.I. from Select Sires, USDA (90-27240-5508) and Research Excellence Funds from Michigan State University. Back

2 Correspondence: R.G. Saacke, Department of Dairy Science, Virginia Polytechnic Institute and State University, 2020 Litton Reaves Hall, Blacksburg, VA 24061. FAX: 540 231 5014; saacke{at}vt.edu Back

Accepted: January 21, 1999.

Received: October 19, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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