Comparison of testosterone and insulin-like peptide 3 secretions in response to human chorionic gonadotropin in cultured interstitial cells from scrotal and retained testes in dogs

Levels of testosterone and insulin-like peptide 3 (INSL3) secretions in response to different doses of human chorionic gonadotropin (hCG) in cultured interstitial cells were compared between retained and scrotal testes in dogs. Retained (n=10) and scrotal (n=9) testes were obtained from small-breed dogs. The testicular tissues were dispersed in Dulbecco's Modified Eagle Medium with Ham's nutrient mixture containing 2000 PU/ml dispase II and 10% fetal bovine serum. The cells were plated with differing concentrations (0-10 IU/ml) of hCG for 18 h in multiwell-plates. Testosterone and INSL3 in the same spent medium were measured by enzyme-immunoassays (EIA). A new EIA with a reliable detection range of 0.025-5 ng/ml was developed in order to measure canine INSL3 in culture medium. Dose-dependent stimulation of testosterone by hCG was observed in the cells of both retained and scrotal testes. The incremental rate of testosterone secretion was significantly lower at 0.1, 1 and 10 IU/ml hCG in the cells of retained testes than in scrotal testes, however. INSL3 secretion was significantly stimulated at 10 IU/ml hCG relative to unstimulated controls comprising cells of scrotal testes; no such stimulation was observed in the cells of retained testes. At 10 IU/ml hCG, the incremental rate of INSL3 was significantly lower in the cells of retained testes than scrotal testes. These results suggest that LH-induced secretory testosterone and INSL3 responses are lower in the interstitial cells of retained testes than of scrotal testes. Furthermore, the high concentrations of LH may acutely stimulate INSL3 release in scrotal testes of dogs, but not in retained testes.     

Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1-11 before collection) and during spermatogenesis (average temperature of days 12-65 before collection). Age of bull significantly affected all traits (P<0.01 to P<0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P<0.05 to P<0.001) while collection intervals between 4-9 days and 1-6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P<0.10 to P<0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P<0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5-15 degrees C were found to be optimal for semen production.     

Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden

Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n=8) and Swedish Holstein (SLB, n=4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n=3) or pooling two consecutive ejaculates (n=104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing >10% of morphologically deviating sperm head shapes, a commonly used threshold for young AI bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires.     

Predictors of testosterone in zoo-managed male African elephants (Loxodonta Africana)

Reproductive complications for both male and female zoo-managed African elephants (Loxodonta africana) contribute to the rapidly declining population. In zoo-managed bull elephants, few studies have explored the potential physiological, physical, social, and environmental factors that influence bull fertility, particularly, androgen production. Testosterone is the essential steroid hormone for male sexual maturation and inadequate concentrations can be detrimental for spermatogenesis. Testosterone, fecal glucocorticoid metabolites, leptin, glucose, insulin, and triglycerides were analyzed from weekly fecal and blood serum samples taken over 6 months from six zoo-managed African elephant bulls (10–19 years of age). Testosterone levels were compared to endocrine factors, weekly social and environmental variables, daily musth signs, and body condition scores (BCS). The glucose-to-insulin ratio (G:I) was the only physiological biomarker found to be positively associated with testosterone. Predictive physical variables included Musth Score (+) and Moderate Exercise (+). Bulls with BCS signifying overweight (BCS 4) had lower testosterone (36.6 ± 1.6 ng/g fecal extraction [FE]) than bulls with healthy BCS 3; 51.2 ± 4.9 ng/g FE). Numerous social variables influenced testosterone concentrations, including Total Contact Day (+), Female Interaction Day (+), Indirect Contact Day (+), Indirect Contact Night (+) and Total No Contact (−). Both percentage of Time Outdoor and Time Mixed positively influenced testosterone, whereas testosterone decreased for percentage of Time Indoors. Each additional daily browse opportunity increased testosterone by approximately 7 ng/g FE. In managed care, the emphasis should be placed on optimizing these predictors of testosterone production to promote bull reproductive health.     

Regional differences in the testosterone to dihydrotestosterone ratio in the epididymis and vas deferens of adult mice

The concentrations of testosterone and dihydrotestosterone (DHT) were measured in the testis and in different segments of the epididymis and vas deferens of adult mice. There were marked regional variations in the concentrations of testosterone and DHT from the testis to the caudal part of the vas deferens. In the testis, testosterone was the predominant androgen (364 +/- 90 ng/g) while DHT was weakly represented (8 +/- 2 ng/g). Qualitative and quantitative changes occurred in epididymis: DHT was the main steroid in the caput (29.3 +/- 2.7 ng/g) and corpus (33.1 +/- 4.4 ng/g) while testosterone and DHT were in similar quantities in the cauda (18.6 +/- 2.6 and 19.0 +/- 2.7 ng/g, respectively). The proximal region of the vas deferens contained higher amounts (71.4 +/- 8.0 ng/g) of androgens (testosterone + DHT) than did the caput epididymidis (39.1 +/- 3.3 ng/g). Testosterone was the predominant androgen in each part of the vas deferens and its concentrations decreased from the proximal (64.5 +/- 7.5 ng/g) to the caudal (26.9 +/- 4.3 ng/g) region. Castration and section of the efferent ducts of the testis showed that the epididymis received testosterone essentially via the blood supply and that epididymal DHT was produced locally from circulating testosterone.     

Serum testosterone and musth in captive male African and Asian elephants

Testosterone concentrations in serum samples collected weekly over a 5-year period from a young adult male Asian elephant (Elephas maximus) and a young adult male African forest elephant (Loxodonta africana cyclotis) were measured by radioimmunoassay. Testosterone profiles during this maturational period were compared between the two species and related to the occurrence of musth, a recurring physiological and behavioral condition exhibited by most mature Asian, and some African, bull elephants. Musth is characterized by secretion from the bull's temporal glands, dribbling urine, and increased aggression. Serum testosterone concentrations in the Asian bull were elevated substantially between April and September each year, coincident with the presence of temporal gland secretion, urine dribbling, and aggressive behavior. Testosterone levels from April through September averaged (± SEM) 41.2 ± 2.8 ng/ml, compared to 7.6 ± 1.0 ng/ml during the rest of the year. In contrast, the testosterone profile of the African bull showed greater variation and lower levels overall, the only pattern being a tendency for levels to be lowest from November to February (avg. 6.8 ± 1.5 vs. 10.3 ± 0.8 ng/ml during the rest of the year). Temporal gland secretion and other signs of musth were first observed in this bull in 1988, at age 17. While his testosterone profile did not show a pattern comparable to that in the Asian bull, average testosterone values were significantly greater in 1988 compared to previous years. The Asian bull showed sexual attention to preovulatory (estrous) cows whether in musth or not, and exposure to estrous cows did not appear to alter the highly consistent, annual pattern of musth as evidenced in temporal gland flow.     

In vitro effects of estradiol-17β, monobutyl phthalate and mono-(2-ethylhexyl) phthalate on the secretion of testosterone and insulin-like peptide 3 by interstitial cells of scrotal and retained testes in dogs

The objective was to determine the effects of estradiol-17β, monobutyl phthalate (MBP) and mono-(2-ethylhexyl) phthalate (MEHP) on testosterone and insulin-like peptide 3 (INSL3) secretions in cultured testicular interstitial cells isolated (enzymatic dispersion) from scrotal and retained testes of small-breed dogs. Suspension cultures were treated with estradiol-17β (0, 10, and 100 ng/mL), MBP (0, 0.8, and 8 mmol/L) or MEHP (0, 0.2, and 0.8 mmol/L) for 18 h, in the presence or absence of 0.1 IU/mL hCG. Testosterone (both basal and hCG-induced) and INSL3 (basal) concentrations were measured in spent medium. Effects of estradiol-17β, MBP, and MEHP on testosterone and INSL3 secretions were not affected (P > 0.15) by cell source (scrotal versus retained testis); therefore, data were combined and analyzed, and outcomes reported as percentage relative to the control. In testicular interstitial cells, basal testosterone secretion was increased (P < 0.01) by 100 ng/mL estradiol-17β (130.2 ± 10.6% of control). Among phthalates, 0.2 and 0.8 mmol/L MEHP stimulated (P < 0.01) basal testosterone secretion (135.5 ± 8.3% and 154.6 ± 12.9%, respectively). However, hCG-induced testosterone secretion was inhibited (P < 0.01) by 8 mmol/L MBP (67.7 ± 6.0%), and tended to be inhibited (P = 0.056) by 0.8 mmol/L MEHP (84.5 ± 5.6%). Basal INSL3 secretion was inhibited (P < 0.01) by 8 mmol/L MBP (73.6 ± 6.8%) and 0.8 mmol/L MEHP (76.9 ± 11.3%). In conclusion, we inferred that estradiol-17β and certain phthalate monoesters had direct effects on secretions of testosterone and INSL3 in canine testicular interstitial cells, with no significant difference between scrotal and retained testes.     

Assessment of bull fertility using a mucus penetration test and a human chorionic gonadotrophin stimulation test

The relative predictive abilities of an in vitro mucus penetration test and a human chorionic gonadotrophin (hCG) stimulation test were compared for assessing bull fertility. Cervical mucus was collected from 22 Holstein cows and then stored in liquid nitrogen. Semen was collected from 13 Holstein bulls and evaluated for standard semen parameters. The mucus penetration test was performed with fresh ejaculates, and the results were expressed as the distance traveled by the vanguard spermatozoa during incubation at 38 degrees C for 10 min. Increases in plasma testosterone levels were determined by a ratio of testosterone concentration before and after hCG injection. Sperm motility and mucus penetration were significantly correlated with the conception rate. However, no significant correlation was confirmed between the increased plasma testosterone levels and conception rate. The results indicate that the mucus penetration test is an effective method for predicting the fertility of bulls.     

Testicular growth, hormone concentrations, seminal characteristics and sexual behaviour in stallions

Puberty was studied using 15 colts of Quarter Horse phenotype. Total scrotal width was measured every 8 weeks from 48 to 96 weeks. Blood samples were taken from 8 colts at 8, 16 and 24 weeks and then every 4 weeks until 100 weeks to measure changes in LH, FSH and testosterone concentrations. Seminal collections were attempted monthly from 48 to 64 weeks and every 2 weeks thereafter until puberty resumed every 3rd day from 96 weeks for 15 ejaculates. For all collections, times to erection, mount and ejaculation and seminal characteristics were recorded. Age at puberty was defined as the first ejaculate containing 50 x 10(6) spermatozoa, with greater than or equal to 10% motile. Colts were castrated at 2 years to enable determination of daily sperm production (DSP), epididymal sperm reserves and normality of spermatogenesis. Total scrotal width increased linearly from 48 to 96 weeks. Age at puberty averaged 83 weeks (56-97 weeks). Changes in serum concentrations of LH and FSH were parallel, rising at 36-40 weeks, declining after 40 weeks and rising again at 68-80 weeks. Testosterone was low until 68 weeks after which concentrations rose slowly to 80 weeks and increased rapidly to a plateau at 92 weeks. Sexual behaviour and seminal characteristics differed (P less than 0.05) between puberty and 2 years, except for time to erection, time to mount, and percentage of motile spermatozoa. DSP at 2 years averaged 1.7 x 10(9) and daily sperm output (DSO) averaged 1.1 x 10(9). The correlation between DSP and DSO was 0.83 (P less than 0.01). There were 9.57 x 10(9) spermatozoa/epididymis of which 67% were in the cauda.     

Effect of hCG injection on prostaglandin E concentrations in ram seminal plasma

Ram and bull seminal plasma, respectively, contain 0.5-20 microg PGE/ml and 5-10 ng PGE/ml. To demonstrate that PGE concentrations in the seminal plasma are related to sperm quality and could be affected by hormonal stimulation in vivo, four rams were injected with 500 IU hCG, in and out of season. The rams responded 1 week after hCG with a 1.5- to 4-fold increase in seminal plasma PGE. The PGE peak was temporally separate from the hCG-induced rise in seminal plasma testosterone which was observed after 1 day. Using a simulated cryptochid ram, peaks in seminal fluid PGE were found to be associated with increased sperm velocity and sperm counts. In bulls, PGE concentrations in the seminal plasma of good bulls were significantly higher than that found in poor and cryptorchid bulls.     

Effect of long-term immunization against inhibin on sperm output in bulls.

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 alpha1-26 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.     

Influence of interactions between semen extender and number of spermatozoa on nonreturn rate estimates of fertility for individual Holstein bulls

The aim of the present study was to investigate the possibility of adapting sperm cell concentration per insemination unit to the intrinsic fertility of a given bull. For this purpose, split ejaculates of seven proven Holstein sires of unknown fertility were diluted in TRIS or in Laiciphos extenders. TRIS diluted doses contained either 8.12 or 16 million total sperm cells (T8, T12, T16 respectively), while the routinely-used Laiciphos units contained 16 million sperm (L16). A total of 18.068 first artificial inseminations were performed by three different AI centers. No difference in the average fertility occurred between T16 and L16 (69.2 vs 69%), but the individual bull results varied; L16 displayed lower inter-bull variability. The nonreturn rates (60 to 90 days) obtained with T8 and T12 (65.7 and 66.8%, respectively) were significantly lower than with T16 and L16 (P < 0.02). The effect of AI center was also significant (P < 0.05). Despite a highly significant effect of the sire (P < 0.001), no interaction was found between the bull and cell concentration or the AI center. The bull effect indicates that it may be worthwile especially for the bulls of the highest genetic merit to test for the appropriate lowest number of spermatozoa per unit to allow wider commercial and genetic diffusion without compromising the average nonreturn rate of the sire.     

Electroejaculation, semen characteristics and serum testosterone concentrations of free-ranging african elephants (Loxodonta africana)

A regimented electroejaculation protocol (120 electrical stimulations; 10-30 V) was used to collect semen and characterize ejaculate quality from 9 adult, free-ranging African elephants under anaesthesia. Eight of the 9 ejaculates contained high concentrations of progressively motile spermatozoa. The overall mean ejaculate volume, sperm concentration/ml ejaculate, sperm motility, sperm status and ejaculate pH were 93.3 ml, 2408.6 X 10(6) spermatozoa/ml, 70%, 3.9 and 7.4, respectively. A high percentage (mean 77.5%) of spermatozoa within each ejaculate was morphologically normal. Of the aberrant spermatozoa, 72% had a cytoplasmic droplet defect. When sperm viability was tested in vitro at 37 degrees C, sperm motility rating declined by at least half of the initial assessment within 3.5 h of semen collection. Generally, spermatozoa maintained motility in vitro for less than 6 h. Serum testosterone ranged from 1.4 to 8.2 ng/ml in 4 males evaluated in the morning (07:30-08:00 h). In 4 of the 5 bulls assessed in the afternoon (15:00-18:00 h), testosterone levels were less than 0.9 ng/ml. The remaining bull, evaluated at 16:00 h, had exceptionally high testosterone concentrations (peak 25.6 ng/ml) and a preputial discharge potentially indicative of 'musth'. The present study demonstrates that high quality semen can be collected consistently from the African elephant and that striking differences exist in serum testosterone amongst free-ranging males which may be due, in part, to a diurnal rhythm.     

Regulation of serum testosterone in men with steroid sulfatase deficiency: response to human chorionic gonadotropin

Human chorionic gonadotropin (hCG; 5000 IU) was administered to 6 control men and 6 patients with recessive x-linked ichthyosis (RXLI) with verified 3 beta-hydroxysteroid sulfate sulfatase (3 beta-HSS) deficiency in their skin biopsy samples. Concentrations of steroids and their sulfate conjugates were determined in peripheral serum specimens collected a day before and 4 days after hCG administration. Testosterone concentrations were identical in patients and controls. Baseline serum LH concentrations were also identical in the 2 groups showing that there were no major differences in the regulation of the hypothalamic-pituitary-gonadal axis. The significantly increased (31-82%) serum concentrations of sulfated pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and 5-androstene-3 beta,17 beta-diol in patients compared with controls indicated that their circulating concentrations were regulated by 3 beta-HSS. This is in line with the fact that the baseline concentrations of the same unconjugated steroids were significantly lower (32-90%) in patients with RXLI, suggesting that a proportion of these circulating steroids were derived from the corresponding sulfated precursors. The response patterns and actual concentrations of testosterone, 17-hydroxyprogesterone and estradiol were similar in the patients and the controls after hCG. The decreased concentrations of testosterone sulfated at carbon 17 under baseline conditions and after hCG in patients with RXLI remains enigmatic. In conclusion, testosterone production and the response to hCG seem to be identical in patients with RXLI and controls despite the fact that significant differences were observed in the circulating concentrations of several unconjugated and sulfated testosterone precursors.     

Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture.

Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process. In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome. After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml. At 11 days in culture, samples were removed for morphological characterization and determination of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD). Testosterone concentration was determined by RIA in the culture medium at different intervals. Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions. No mature Leydig cells were present. In basal conditions, the percentages of 3 beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%, respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng.10(6) cells.24 h, respectively. In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng.10(6) cells, respectively). Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired semen quality of AI bulls fed with moldy hay: a case report

The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonretum rate was normal. The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography-mass spectrometry (GC-MS) detected Fusarium mycotoxins HT-2 and T-2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay.     

Reproductive hormone secretion and testicular growth in bull calves actively immunized against testosterone and oestradiol-17 beta

Groups of bull calves received a primary immunization against testosterone (Group T; N = 7) or oestradiol-17 beta (Group E; N = 9) at 3 months of age and booster injections on four occasions at approximately 2 month intervals. Controls (Group C, N = 7) were immunized against human serum albumin alone using the same protocol. Immunity was achieved against both steroids as judged by the secondary antisteroid antibody titres in Group T (730 +/- 231; reciprocal of titre) and Group E (12,205 +/- 4366) bulls; however, peak antibody titres generally declined with successive booster injections. Mean plasma concentrations of LH, FSH and testosterone during the period from 3 to 10 months of age were higher (P less than 0.05) in Group T bulls than in Groups C and E. Group T bulls had larger testes compared with controls from 6 months of age onwards. At castration at 14 months of age, testes of Group T bulls were heavier (P less than 0.05) than those of Groups C and E (179 +/- 13, 145 +/- 8 and 147 +/- 6 g, respectively). At 10 months of age, there were no differences among treatment groups in LH responses to LHRH, but the testosterone responses were greater (P less than 0.05) in bulls in Group T (26.2 +/- 4.9 ng/ml) and Group E (16.6 +/- 1.8 ng/ml) compared with those in Group C (6.9 +/- 0.6 ng/ml). Testosterone responses to hCG determined at 13 months of age were also greater (P less than 0.05) in Groups T and E relative to controls. At 14 months of age daily sperm production rates per bull (X 10(-9)) were higher (P less than 0.10) in Group T bulls (2.2 +/- 0.1) than those in Groups C (1.6 +/- 0.2) and E (1.6 +/- 0.1). These results indicate that early immunity against testosterone is associated with increased gonadotrophin secretion and accelerated growth of the testes in prepubertal bulls. Also, chronic immunity against testosterone or oestradiol-17 beta enhances the steroidogenic response of bull testes to gonadotrophic stimulation. If the above responses observed in young bulls are shown to be sustained, then immunity against gonadal steroids early in life may confer some reproductive advantage in mature animals.     

Effect of vaccination against gonadotropin-releasing factor (GnRF) with Bopriva® in the prepubertal bull calf

The aim of this study was to evaluate the effect of immunization against gonadotropin-releasing factor (GnRF) with Bopriva(?) (Pfizer Animal Health, Parkville, Australia) in prepubertal bull calves. For the study, 6 calves were vaccinated at the age of 3 and 6 weeks with 1 mL Bopriva(?), and 6 animals served as matched controls. Concentrations of GnRF antibodies, testosterone and LH were determined in serum samples out to 30 weeks after the first immunization. Body weight and scrotal circumference were measured for 59 weeks. At slaughter, 65 weeks after the first immunization, the quality of epididymal sperm was evaluated. The results showed that vaccination against GnRF influenced (P<0.05) anti-GnRF titer, LH and testosterone concentrations as well as scrotal circumference. Antibody titers significantly (P<0.05) increased after the booster vaccination and reached peak values 2 weeks later. Compared to control animals, inhibition (P<0.05) of the prepubertal LH secretion was observed in vaccinated calves at weeks 10 and 12-14 after the first vaccination. In vaccinated calves testosterone concentrations decreased after the booster injection to values below 0.5 ng/mL serum and remained for at least 22 weeks at this low level. Animals vaccinated with Bopriva(?) showed a delay in testes growth and smaller scrotal circumference. Puberty occurred at the age between 46 and 55 weeks in vaccinated and between 38 and 52 weeks in control animals and body weight gain was similar in both groups. All vaccinated bulls attained spermatogenic capacity at slaughter when they were 68 weeks old.     

Androgen secretion and characteristics of testicular HCG binding in cryptorchid rats

Response of the cryptorchid testis to gonadotrophic stimulation was assessed by comparison of the androgen production capability in vivo and in vitro with that of the normal scrotal testis. Serum androgen concentrations in cryptorchid rats were similar to those in normal rats, and the incremental increase 60 min after 50 i.u. hCG (i.v.) was about 7-fold for both groups. Basal and hCG-stimulated androgen production in vitro was higher for abdominal testes (557 and 3286 ng/pair) than for scrotal tests (157 and 504 ng/pair). Specific binding of hCG by testicular homogenates was slightly higher (P < 0.05) for cryptorchid testes when expressed per unit weight, but Scatchard analysis indicated that although hCG binding affinities did not differ (Ka = 2 x 10(10) M-1), hCG binding capacity of cryptorchid testes was only 75 ng, compared to 219 ng for scrotal testes. These data indicate that a discrepancy exists between androgen production in vivo and in vitro by cryptorchid testes and that normal serum androgen concentrations are maintained in the presence of decreased numbers of testicular LH/hCG receptors.     

Assessment of reproductive status in male echidnas

This study reports the development and application of techniques to assess the reproductive status of male echidnas. The pattern of testosterone secretion over a 24-h period in five echidnas was documented. Testosterone secretion after injection i.m. of either 1000 IU hCG (n=4) or 4 microg GnRH agonist (n=6) was determined to establish whether this could be used as a practical index of the prevailing steroidogenic capacity of the testes. hCG (1000 IU) was also used to assess seasonal changes in testosterone secretion in six echidnas over a 13-month period. Seasonal changes in testicular volume were examined by transabdominal ultrasonography. Electroejaculation was attempted to monitor seasonal changes in sperm production, which was also determined by spermatorrhea. There was no apparent diurnal pattern of testosterone secretion in echidnas and circulating concentrations of testosterone remained relatively low (maximum 1.2 ng/mL) and stable over 24h. Injection of hCG resulted in an increase (P<0.01; n=4) in testosterone concentration with a peak (2.9+/-0.3 ng/mL) approximately 4h after injection. GnRH also induced an increase (P<0.01; n=6) in circulating testosterone that was apparent after 1h (2.6+/-0.3 ng/mL) and concentrations remained elevated (3.4+/-0.3 ng/mL) for up to 8h after injection. Seasonal changes in testosterone secretion determined after injection of hCG, increased (P=0.03; n=6) from late-autumn, peaked in late-winter, and decreased by early-spring. Testicular volume followed a similar seasonal pattern (P<0.01; n=6) with an increase from late-autumn, peak in winter and a decline in mid-spring. There was no seasonal change in live weight. Electroejaculation was attempted throughout two breeding seasons but no semen was obtained. Spermatorrhoea in the echidna was described for the first time and was subsequently used to assess seasonal sperm production. Spermatozoa were found in the urine from June to September. This study has demonstrated that exogenous hormones can be used to obtain an index of the prevailing steroidogenic capacity of the testes in echidnas, which is not apparent with repetitive non-stimulated samples over 24 h. The assessment of testosterone secretion after injection of trophic hormones provides a valuable and practical procedure for the assessment of reproductive status. Testicular ultrasonography and spermatorrhea are useful in assessing reproductive status and in this study were successfully used to determine seasonal reproduction in captive echidnas.