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Immunocastration using gonadotropin-releasing hormone (GnRH)-based vaccines has been investigated in rams to reduce aggressive and sexual behaviour and to control meat quality. Despite considerable efforts, a practical GnRH vaccine has yet to be developed for rams. In the present study, a A GnRH-lipopeptide vaccine (GnRH-LP) including two copies of GnRH, 2-amino-d,l-hexadecanoic acid (C16), and a unique T helper epitope, was examined in rams. Rams received a primary and secondary vaccination of GnRH-LP without additional adjuvant (Group 1) or with the adjuvant AdjuVac™ (Group 2). In both Group 1 and 2 anti-GnRH antibody titres increased after secondary vaccination, however, the antibody titres were higher (p<0.01) for rams in Group 2. The latter rams showed a marked decrease in testicular size. The marked and sustained reduction in testicular size in rams treated with GnRH-LP+AdjuVac™ provides the basis for an effective immunocastration vaccine in rams., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim was to elucidate the role of LH and FSH in testicular development and growth in the neonatal boar. On Day 10 after birth (Day 0 of study), animals were assigned to one of nine groups (n=6): Group 1, control, no treatment; Group 2, hemicastrated (H); Group 3, H and implanted with GnRH agonist (H+GnRH); Group 4, H+GnRH+FSH 200μg/kg daily from Days 0 to 14 (D0-14); Group 5, H+GnRH+FSH 400μg/kg D0-14; Group 6, H+GnRH+FSH 400μg/kg in PVP D0-14; Group 7, H+GnRH+LH 200μg/kg D0-14; Group 8, H+GnRH+LH 400μg/kg D0-14; Group 9, H+GnRH+LH and FSH 200μg/kg D0-14. The right testis in control and hemicastrated boars was removed on Day 15. Hemicastrated boars had greater (P<0.05) testicular growth than control boars and testicular growth was prevented in boars treated with GnRH agonist. FSH induced Sertoli cell proliferation but not testicular growth whilst LH induced Leydig cell proliferation and testicular growth was similar to control boars but less than hemicastrated boars. LH+FSH induced similar testicular growth as LH alone and neither LH and/or FSH supported testicular hypertrophy in hemicastrated boars. The findings show conclusively for the first time that LH and FSH respectively induce Leydig cell and Sertoli cell proliferation in the neonatal boar. LH additionally supports a normal increase in testicular size in the neonatal boar., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The objective was to ascertain fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), and transforming growth factor-alpha (TGFalpha) mRNA expression and testis morphology during accelerated testicular growth after hemicastration in the neonatal boar. On Day 10 after birth (Day 0), boars were assigned to control (n = 28), no treatment; hemicastrated (n = 28), left testis removed. The right testis in both groups (n = 7) was removed on Days 5, 10, 15, and 20. Expression of mRNA for FGF2, EGF, and TGFalpha was determined by qRT-PCR using TaqMan. Testicular morphology was determined on Day 15. On Day 10, hemicastrated boars had a greater (P = 0.01) testis weight (6.2 +/- 0.8 g; mean +/- SEM) than controls (4.3 +/- 0.4 g) and on Day 15 testis weight in hemicastrated boars (8.8 +/- 0.8 g) was twice (P < 0.01) that of control boars (4.2 +/- 0.3 g). Seminiferous tubule volume was approximately doubled in hemicastrated boars (P < 0.01) and was associated with an increase (P < 0.01) in Sertoli cell number. Interstitial compartment volume was greater (P < 0.01) in hemicastrated boars. Leydig cell numbers were similar (P = 0.14) but volume was greater (P < 0.01) for hemicastrates. There were no differences (P > 0.05) between control and hemicastrated boars in TGFalpha or FGF2 expression on Day 5 or Day 10, and EGF was not detected. It was concluded that upregulation of TGFalpha or FGF2 expression is not a pre-requisite for enhanced testicular growth and increased Sertoli cell proliferation that occurs subsequent to hemicastration in the neonatal boar.

The objective was to compare testis characteristics of Zebu bulls treated with the GnRH agonist, deslorelin, at different times and for different durations during their development. An additional objective was to determine the usefulness of a stain for the transcription factor GATA-binding protein 4 (GATA-4) as a specific marker for Sertoli cell nuclei in cattle. Bulls (54) were allocated to nine groups (n = 6) and received s.c. deslorelin implants as follows: G1 = from birth to 3 mo of age; G2 = from 3 to 6 mo; G3 = from 6 to 9 mo; G4 = from 9 to 12 mo; G5 = from birth to 15 mo; G6 = from 3 to 15 mo; G7 = from 6 to 15 mo; G8 = from 12 to 15 mo; and G9 (control) = no implant. Bulls were castrated at 19 mo of age. Paraffin sections (10 microm) were subjected to quantitative morphometry and GATA-4 immunohistochemistry. At castration, all bulls in the control group (6/6) had attained puberty (scrotal circumference > or = 28 cm), whereas a smaller proportion (P < 0.05) had reached puberty in G2 (2/5) and G6 (1/6). Bulls in G2 and G6 also had a lesser (P < 0.05) testis weight compared with the control group. Total volume of seminiferous epithelium and total daily sperm production in G2 and G6 were only half that observed in the control group. Spermatids were observed in less than 50% of seminiferous tubules in G2, G6, and G7 compared with 82% in the control group (P < 0.05). Staining for GATA-4 was specific for and abundant in the Sertoli cell nucleus in both pre- and postpubertal bulls, and no other cell nucleus inside the seminiferous tubule was positive for GATA-4. Total number of Sertoli cells was not affected by treatment (P = 0.45), but nuclear volume was smaller in G2 and G6 (P < 0.05) compared with the control group. In conclusion, treatment of Zebu bulls with deslorelin had no apparent beneficial effect on testis development and delayed puberty when treatment was initiated at 3 mo of age. Staining for GATA-4 was a useful method for identifying and quantifying Sertoli cell nuclei in both pre- and postpubertal bulls.

The objectives were to determine whether active immunization against gonadotrophin releasing hormone (GnRH) induced a long-term suppression of testicular function in bulls, and to ascertain the effects of immunization against GnRH on carcase and meat quality characteristics. In experiment 1, 6-month-old Zebu bulls were assigned to: control (n=25), no treatment; immunized (n=31), immunized against GnRH at 0 and 4 months (anti-GnRH(2)), with a sub-set of bulls (n=17) immunized again at 10 months (anti-GnRH(3)). After the second immunization, testicular growth ceased for 2 months in 14/31 (45%) bulls and for at least 6 months in 17/31 (55%) bulls. Among the latter bulls (anti-GnRH(3)) the testes did not grow for >1 year after the third immunization in 5/17 (30%) bulls. In experiment 2, 22-month-old Zebu bulls were assigned to: control (n=14), no treatment; immunized (n=17), immunized against GnRH at 0, 2 and 4 weeks. The testes decreased (P<0.05) in size for 2 months after immunization in 11/17 (65%) bulls and then re-initiated growth, whilst in 6/17 (35%) bulls the testes continued to decrease in size for 4 months and did not re-initiate growth for 1 year. At slaughter, the latter immunocastrated bulls had carcase and meat quality characteristics the same as contemporary bulls that had been castrated before puberty. The findings demonstrated that active immunization against GnRH can induce a long-term suppression of testicular function in a proportion of bulls. Also, when bulls are immunocastrated after puberty, carcase and meat quality traits change from those typical of entire bulls to traits that are characteristic of long-term castrated bulls.

Testosterone secretion and the expression and relative contents of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450SCC), 3beta-hydroxysteroid dehydrogenase/delta(5)-->delta(4)-isomerase (3 beta-HSD), and (17)alpha-hydroxylase cytochrome P450/C17-20 lyase (P450(17)alpha) were determined in testicular tissues of bulls treated with a LHRH agonist. Testis morphology and spermatogenesis were also examined. In Experiment 1, bulls (30-mo-old) received no treatment (control, n = 7) or were implanted for 10 days with the LHRH agonist deslorelin (n = 7). Bulls were castrated on Day 10 and testis tissues prepared for Western and Northern blotting. At castration, bulls implanted with deslorelin had greater plasma testosterone (5-fold) and testis content of testosterone (10-fold) compared with control bulls. Relative content (per micrograms total testis protein or RNA) of StAR protein, 3beta-HSD, P450SCC, and mRNA for P450(17)alpha in bulls treated with deslorelin ranged from 3- to 6-fold that of control bulls. In Experiment 2, bulls (20-mo-old) were left untreated (control, n = 6) or implanted with deslorelin (n = 12) for 120 days. On Day 120, bulls were castrated and right testis tissues prepared for morphology. Testis volume and weight were increased (P < 0.01) in bulls treated with deslorelin compared with control bulls. Stereological analysis revealed that this increase occurred in all compartments (seminiferous epithelium, lumen and interstitium) studied, but was significant (P < 0.01) only for the seminiferous epithelium. Absolute numbers of round spermatids per testis were increased (P < 0.05) in bulls treated with deslorelin compared with control bulls. Increased testosterone secretion in bulls treated with deslorelin was associated with increased testicular StAR protein and steroidogenic enzymes. Bulls treated long-term with deslorelin had a faster rate of testis growth and increased daily sperm production at the end of the experiment.

The possibility that estradiol-17 beta may directly influence testicular steroidogenesis in bulls was investigated in vitro. Testicular tissues were incubated for 4 h and regression coefficients (b, ng X ml-1 X h-1) based on the increase in testosterone in the medium were used to describe testosterone secretion rates. In the first experiment, testicular tissues from control bulls and bulls chronically implanted with estradiol were incubated in the absence (basal conditions) or presence of 10 mIU/ml human chorionic gonadotropin (hCG). Under basal conditions, testosterone secretion rates were similar for tissues from control (b = 24.1 +/- 6.0) and implanted (b = 34.7 +/- 6.0) bulls. Testosterone secretion rates were increased approximately fourfold during incubation with hCG; tissues from implanted animals secreted testosterone at a higher rate (P less than .05) than control tissues (b = 120.7 +/- 6.0 vs 81.6 +/- 6.0). In a second experiment, testicular tissues were obtained from control bulls and incubated both with and without hCG (10 mIU/ml) and with and without estradiol (concentrations ranging from 3.7 nM to 37 microM). In this experiment, basal secretion rate (b = 12.6 +/- 3.6) was increased threefold during incubation with hCG (b = 37.8 +/- 3.6). Estradiol failed to inhibit hCG-stimulated testosterone secretion when bovine testicular tissue was incubated with 0, 3.7 and 370 nM estradiol. In contrast, 37 microM estradiol suppressed (P less than .05) testosterone secretion, perhaps reflecting a pharmacologic effect. The results suggest that neither chronic nor acute exposure to estradiol directly affects testosterone secretion by the bovine testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone (LH) secretory profiles have been determined for the male bovine following castration and steroid replacement therapy. Serum LH concentrations increased approximately threefold during the first week following castration and thereafter remained elevated (6.6 +/- .7 ng/ml). Castrates not receiving steroid replacement showed a rhythmic pattern of LH release that was of high frequency (mean pulse interval; 85 +/- 5 min) and high amplitude (mean peak concentration, 11.2 +/- 1.4 ng/ml). Chronic administration of estradiol-17 beta via subdermal Silastic implants reduced mean serum LH concentrations (2.1 +/- .3 ng/ml) and blocked the pulsatile pattern of LH release in all steers. Similar administration of testosterone suppressed mean serum LH and blocked pulsatile LH release in two of four animals. The number of implants used in this study provided physiological concentrations of estradiol (9.8 +/- 1.5 pg/ml) and testosterone (4.1 +/- .2 ng/ml) in systemic blood for the two respective treatment groups. Differences in the LH secretory profiles among testosterone-implanted steers may have been related, in part, to differences in the amounts of steroid not bound to serum proteins. These findings demonstrate that estradiol is a particularly potent inhibitor of pulsatile LH secretion in the male bovine and suggest that gonadal steroid feedback on LH secretion may, in part, be imposed at the level of the hypothalamus. The mechanism for pulsatile LH release is discussed relative to a centrally-located luteinizing hormone releasing hormone pulse generator.

The capacity of the anterior pituitary gland and testes in mature bulls (705 +/- 9 (S.E.M.) kg body wt, n = 4) to respond to graded doses of LH-releasing hormone (LHRH) was assessed relative to endogenous profiles of LH and testosterone secretion. Endogenous hormone profiles were determined by bleeding bulls at 20-min intervals for 12 h. Responses to LHRH were assessed on successive days after single intravenous injections of 1, 5, 10, 50 or 100 ng LHRH/kg body wt. Blood samples were taken at -40, -20, 0, 10, 20, 30, 40, 60 and 120 min relative to LHRH injection. During a 12-h bleed bulls showed spontaneous pulses of LH and testosterone which had peak amplitudes of 2.6 +/- 0.5 micrograms/l and 44.5 +/- 7.1 nmol/l respectively. Respective peak LH (micrograms/l) and testosterone (nmol/l) responses to LHRH were as follows: 1 ng LHRH (3.0 +/- 0.7; 47.3 +/- 4.1); 5 ng LHRH (8.0 +/- 1.2; 52.8 +/- 6.2); 10 ng LHRH (11.1 +/- 2.3; 57.7 +/- 9.1); 50 ng LHRH (19.2 +/- 2.8; 47.9 +/- 8.6); 100 ng LHRH (19.1 +/- 4.7; 43.9 +/- 6.4). A dose of 1 ng LHRH/kg produced LH and testosterone responses which were comparable in amplitude to spontaneous peaks in the respective hormone. There was a linear (y = 0.28 X + 5.72; r = 0.81) increase in the LH response to doses of LHRH between 1 and 50 ng/kg; corresponding testosterone responses showed no relationship with the dose of LHRH. The capacity of the anterior pituitary gland to release amounts of LH eight to ten times in excess of those secreted during spontaneous peaks suggests that (1) there exists a large releasable store of LH in the anterior pituitary gland and (2) hypothalamic LHRH is a limiting factor in gonadotrophin secretion. In contrast to LH release, the androgenic response of the testes to acute gonadotrophic stimulation is determined largely by prevailing steroidogenic activity.

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.

Prepubertal (16 weeks old) ram lambs were used to investigate the effects of hyperthyroidism and hypothyroidism on development of reproductive endocrine function. Over a period of 8 weeks, ram lambs were made hypothyroid (serum T4 less than or equal to 3 ng/ml compared with controls congruent to 30 ng/ml) by daily oral administration of methyl thiouracil or hyperthyroid (serum T4 congruent to 135 ng/ml) by daily sc injection of T4. Hyperthyroidism was associated with decreases in LH pulse frequency (2.25 +/- 0.75/12 h compared with controls 5.75 +/- 0.48/12 h), basal LH, and mean LH concentrations, together with arrested testicular growth and aspermatogenesis. Hypothyroid rams showed normal pubertal development. After iv injection of LHRH, hyperthyroid ram lambs showed similar LH responses to control and hypothyroid rams. Basal testosterone production (5.6 +/- 1.0 ng/min) and plasma testosterone concentrations after human CG (2.0 +/- 0.7 ng/ml) in hyperthyroid rams were significantly lower than in controls (67.5 +/- 24.0 ng/min and 5.2 +/- 1.0 ng/ml, respectively). It is concluded that retarded testicular development in hyperthyroid ram lambs results from changes in hypothalamo-pituitary activity manifested in a decreased LH pulse frequency.

Testicular growth and secretory profiles of LH and testosterone were monitored in three bull calves implanted with oestradiol-17 beta and three bull calves implanted with oestradiol-17 beta and infused intravenously with LH releasing hormone (LH-RH; 500 ng/pulse per h, 30-s pulse) continuously between 34 and 42 weeks of age. Oestradiol-17 beta implants restricted testicular growth and spermatogenesis by interfering with the hypothalamo-pituitary-testicular endocrine axis. Initiation of pulsatile LH release by LH-RH pulse infusion was accompanied by a twofold increase in mean circulating levels of LH (3.4 v. 1.8 microgram/l) and a marked increase in serum testosterone (13.0 v. 0.4 microgram/l). Testicular diameter was enhanced significantly by week 4 of infusion and increased in a linear fashion up to and including week 8. Testicular weight (g) and total daily sperm production (X 10(9)) at 42 weeks of age were decreased in calves implanted with oestradiol-17 beta (105 +/- 14 (S.E.M.); 0.0) when compared with calves implanted with oestradiol-17 beta and infused with LH-RH (254 +/- 12; 1.2 +/- 0.3). Differences in testicular size and sperm production rates between LH-RH-infused and control bulls without implants (352 +/- 26; 3.3 +/- 0.9) were attributed to the 2-month delay between oestradiol-17 beta treatment and the initiation of LH-RH treatment. These results (1) confirm our earlier conclusion that oestradiol-17 beta can interfere with normal pubertal development in beef bulls and (2) provide additional support that pulsatile LH secretion is important for the initiation of testicular growth and spermatogenesis in pubertal bulls.