Effects of active immunization against gonadotropin releasing hormone on gonadotropin secretion after ovariectomy and testosterone propionate administration to mares

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) conjugated to bovine serum albumin (BSA) to study the involvement of GnRH in luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion following ovariectomy (OVX) and after administration of testosterone propionate (TP). Five mares immunized against BSA served as controls. Immunizations were started on November 1, and OVX was performed in June (d 1). All mares were treated with TP from d 50 to 59 after OVX. On the day of OVX, concentrations of LH were lower (P less than .05) in GnRH-immunized mares than in BSA-immunized mares and were generally nondetectable; FSH concentrations were reduced (P less than .05) by 50% in GnRH-immunized mares relative to BSA-immunized mares. In contrast to BSA-immunized mares, plasma concentrations of LH or FSH did not increase after OVX in GnRH-immunized mares. The LH response to GnRH analog (less than .1% cross-reactive with GnRH antibodies) on d 50 was reduced (P less than .05) by 97% in GnRH-immunized mares relative to BSA-immunized mares, whereas the FSH response was similar for both groups. Treatment with TP for 10 d reduced (P less than .01) the LH response and increased (P less than .01) the FSH response to GnRH analog in BSA-immunized mares, but it had no effect (P greater than .1) on the response of either gonadotropin in GnRH-immunized mares.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization of pigs against chicken gonadotropin-releasing hormone-II and lamprey gonadotropin-releasing hormone-III: effects on gonadotropin secretion and testicular function

The objective of this experiment was to evaluate the effects of active immunization against 2 GnRH isoforms on gonadotropin secretion and testicular function in pigs. Synthetic chicken (c) GnRH-II and lamprey (l) GnRH-III peptides, with the common pGlu-His-Trp-Ser sequence at the N-terminal omitted, were conjugated to BSA. Forty-eight male piglets were randomly assigned to 1 of 4 treatments. Pigs on treatment 1 were actively immunized against cGnRH-II, whereas pigs on treatment 2 were actively immunized against lGnRH-III. Control pigs on treatment 3 were actively immunized against the carrier protein (BSA), and pigs on treatment 4 were castrated and actively immunized against BSA. The BSA conjugate was emulsified in Freund's Incomplete Adjuvant and diethylaminoethyldextran. Primary immunization was given at 13 wk of age (WOA) with booster immunizations given at 16 and 19 WOA. Body weight and plasma samples were collected weekly beginning at 11 WOA. Treatments did not affect BW during the experimental period. Antibody titers were increased in animals immunized against cGnRH-II and lGnRH-III (P < 0.001). Cross-reactivity of the antibodies to mammalian GnRH or between cGnRH-II and lGnRH-III was minimal. Concentrations of testosterone were maximal in control boars (treatment 3) and minimal in control barrows (treatment 4) and immunized pigs (treatment x week; P < 0.01). Immunized animals had concentrations of LH (P < 0.001) and FSH (treatment x week; P < 0.03) that were less than control barrows and similar to control boars. At the end of the experiment, intact (noncastrated) pigs were exsanguinated. Testes were removed immediately; Leydig cells were isolated and treated with 0, 1, or 10 ng/mL of LH. There was an LH x GnRH treatment effect on testosterone concentrations (P < 0.03), indicating that Leydig cells were sensitive to the immunization protocol and doses of LH. Taken together, these data suggest that immunization against GnRH isoforms decreased gonadotropin secretion compared with control barrows. Additionally, immunization against cGnRH-II and lGnRH-III reduced the ability of Leydig cells to respond to LH challenges.     

Testosterone effects on gonadotropin response to GNRH: cows and pony mares

Effects of testosterone propionate (TP) treatment on plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) before and after an injection of gonadotropin releasing hormone (GnRH) were studied using ovariectomized cows and pony mares. An initial injection of GnRH (1 microgram/kg of body weight) was followed by either TP treatment or control injections for 10 (cows) or 11 (ponies) d. A second GnRH injection was administered 1 d after the last TP or oil injection. Concentrations of LH and FSH were determined in samples of plasma taken before and after each GnRH injection. Control injections did not alter the response to GnRH (area under curve) nor the pre-GnRH concentrations of LH and FSH in ovariectomized cows or ponies. Testosterone treatment increased (P less than .01) the FSH release in response to GnRH in ovariectomized mares by 4.9-fold; there was no effect in cows, even though average daily testosterone concentrations were 59% higher than in pony mares. Testosterone treatment reduced the LH release in response to GnRH by 26% in ovariectomized mares (P less than .05) and by 17% in ovariectomized cows (P approximately equal to .051). These results are consistent with a model that involves ovarian androgens in the regulation of FSH secretion in the estrous cycle of the mare, but do not support such a model in the cow.     

Feedlot performance of steers and bulls actively immunized against gonadotropin-releasing hormone.

Feedlot performance and testicular and pituitary function were assessed in cattle actively immunized against GnRH. In Trial 1, 50 steers were either unimmunized (n = 10), actively immunized against keyhole limpet hemocyanin (KLH; n = 10), or immunized against a GnRH-KLH conjugate (n = 30). Fifteen of 30 steers immunized against GnRH-KLH received a secondary immunization 8 wk after primary immunization. Antibodies against GnRH were not evident in unimmunized steers or steers actively immunized against KLH. Antibodies against GnRH were noted in all immunized animals (n = 30) within 6 wk of primary immunization and anti-GnRH antibody concentrations became maximal 20 to 24 wk after immunization. The increasing anti-GnRH titer in immunized steers was associated with decreasing serum concentrations of LH. Serum concentrations of LH were depressed (P less than .05) within 8 wk of primary immunization and reached a nadir by wk 20. The patterns of increase in GnRH titer and decrease in serum concentrations of LH did not differ (P greater than .05) in animals receiving primary immunization alone or primary and secondary immunization. Feedlot performance and carcass quality were not affected (P greater than .05) by immunization against KLH or the GnRH-KLH conjugate. In Trial 2, 60 bull calves (mean weight = 325.2 +/- 2.8 kg) were randomly assigned to a 2 x 3 factorial experiment. The two classes (n = 30) were 1) unimplanted and 2) implanted with Synovex-S. The three treatments (n = 20) were 1) intact control, 2) actively immunized against GnRH, and 3) castrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of active immunization against estrogen on gonadotropin response to testosterone propionate treatment in ovariectomized pony mares

An experiment was conducted to determine whether partial neutralization of estrogens via active immunization alters testosterone propionate (TP)-induced increases in FSH secretion after GnRH administration in ovariectomized pony mares. Twenty mares were used in a 2 X 2 factorial arrangement of treatments (n = 5/group). Factor 1 was long-term active immunization against either bovine serum albumin (BSA) or estrone-17-oxime-BSA. Factor 2 was 11-d administration of either vehicle (vegetable oil) or TP (175 micrograms/kg BW). Plasma concentrations of FSH were not affected (P greater than .1) by either factor. As expected, the FSH response to exogenous GnRH was threefold greater (P less than .05) in BSA-immunized mares treated with TP than in BSA-immunized mares receiving oil. However, immunization against estrogens reduced (P less than .05) this TP-induced increase in FSH response by 52%. Plasma concentrations of LH were decreased (P less than .08) by TP; this effect was not altered (P greater than .1) by immunization against estrogen. The LH response to exogenous GnRH was not affected (P greater than .1) by either factor. We conclude that aromatization of testosterone to estrogen is partially responsible for the increased FSH response to exogenous GnRH in TP-treated mares. In contrast, suppression of LH concentrations by TP appears to involve only the androgenic effect of TP.     

Reproductive function and feedlot performance of beef heifers actively immunized against GnRH1

Two trials were conducted to examine reproductive function and feedlot performance by heifers after active immunization against GnRH. In trial 1, heifers were not immunized or were immunized with one of three doses of a GnRH-KLH (keyhole limpet hemocyanin) conjugate in Freund's complete adjuvant. Antibodies against GnRH were not detectable in non-immunized heifers (n = 9). However, antibodies against GnRH were noted in all immunized animals (n = 30) within 8 wk of primary immunization; anti-GnRH antibody concentrations were at a maximum 16 to 20 wk after immunization. This increased anti-GnRH titer was associated with a decreased serum concentration of progesterone. Ovarian and uterine weight and tissue concentrations of LH and GnRH receptor were reduced (P less than .05) by immunoneutralization of GnRH. Similarly, immunization against GnRH reduced (P less than .05) weight gain during feedlot confinement. In trial 2, feedlot performance after insertion of anabolic steroid implants (Synovex H) was evaluated in non-immunized heifers (n = 15), heifers actively immunized against GnRH-KLH (n = 15) or KLH alone (n = 15), or non-immunized heifers treated with melengestrol acetate (MGA; n = 15). Serum concentrations of progesterone were depressed in anti-GnRH and MGA-fed groups, but ovarian and uterine weights were depressed (P less than .05) only in heifers immunized against GnRH. Total weight gain and gain during the final 4 wk of confinement did not differ (P greater than .05) among groups with steroid implants. The GnRH-KLH conjugate is an effective immunogen in heifers, leading to suppression of reproductive activity. The depression of weight gain that attends development of anti-GnRH titers may be reversed by use of implants that contain anabolic steroids.     

Pituitary and testicular responses of beef bulls to active immunization against inhibin alpha

Prepubertal crossbred beef bulls served as controls or were actively immunized against the N-terminal, 30-amino acid synthetic fragment of porcine inhibin alpha, pI alpha (1-30). Antibody titers were detected in sera (greater than 40% B/BO in sera diluted 1,000-fold) but not in rete testis fluid of 390-d-old bulls. Serum FSH and inhibin remained static during a 5-h intensive bleed; inhibin was not acutely affected by a 15-fold LH rise and a threefold FSH rise induced by exogenous GnRH. Serum FSH, but not LH or testosterone, was consistently elevated (P less than .05) in immunized bulls compared with control bulls. Neither pituitary weight, pituitary gonadotropin content nor pituitary FSH/LH ratios were affected (P greater than .10) by pI alpha(1-30) active immunization. Testicular sperm density was greater (60 x 10(6) vs 45 x 10(6) sperm/g testis; P less than .10) in immunized bulls, but testes weight, epididymides weight and total daily sperm production remained unchanged. These results suggest that inhibin is important for regulation of FSH secretion and testicular function. Immunization with suitable inhibin vaccines may improve bull fertility.     

Relationships among LH, FSH and prolactin secretion, storage and response to secretagogue and hypothalamic GnRH content in ovariectomized pony mares administered testosterone, dihydrotestosterone, estradiol, progesterone, dexamethasone or follicular fluid

Thirty-five ovariectomized pony mares were used to study the relationships among luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) concentrations in blood (secretion), in pituitary (storage) and in blood after secretagogue administration, as well as the content of gonadotropin releasing hormone (GnRH) in hypothalamic areas, under various conditions of steroidal and nonsteroidal treatment. Five mares each were treated daily for 21 d with vegetable shortening (controls), testosterone (T; 150 micrograms/kg of body weight, BW), dihydrotestosterone (DHT; 150 micrograms/kg BW), estradiol (E2; 35 micrograms/kg BW), progesterone (P4; 500 micrograms/kg BW), dexamethasone (DEX; 125 micrograms/kg BW) or charcoal-stripped equine follicular fluid (FF; 10 ml). Secretagogue injections (GnRH and thyrotropin releasing hormone, TRH, at 1 and 4 micrograms/kg of BW, respectively) were given one d prior to treatment and again after 15 d of treatment. Relative to controls, treatment with T, DHT and DEX reduced (P less than .05) LH secretion, storage and response to exogenous GnRH, whereas treatment with E2 increased (P less than .05) these same characteristics. Treatment with P4 reduced (P less than .05) only LH secretion. Treatment with T, DHT, E2 and DEX reduced (P less than .05) FSH secretion, whereas treatment with P4 increased (P less than .05) it and FF had no effect (P greater than .1). All treatments increased (P less than .05) FSH storage, whereas only treatment with T and DHT increased (P less than .05) the FSH response to exogenous GnRH. Other than a brief increase (P less than .05) in PRL secretion in mares treated with E2, secretion of PRL did not differ (P greater than .1) among groups. Only treatment with E2 increased (P less than .01) PRL storage, yet treatment with T or DHT (but not E2) increased (P less than .05) the PRL response to exogenous TRH. Content of GnRH in the body and pre-optic area of the hypothalamus was not affected (P greater than .1) by treatment, whereas treatment with T, E2 and DEX increased (P less than .1) GnRH content in the median eminence. For LH, secretion, storage and response to exogenous GnRH were all highly correlated (r greater than or equal to .77; P less than .01). For FSH, only storage and response to exogenous GnRH were related (r = .62; P less than .01). PRL characteristics were not significantly related to one another. Moreover, the amount of GnRH in the median eminence was not related (P greater than .1) to any LH or FSH characteristic.     

Ovarian response to pregnant mare serum gonadotropin and porcine pituitary extract in gilts actively immunized against gonadotropin releasing hormone

Two experiments were conducted to determine the effect of exogenous gonadotropins on follicular development in gilts actively immunized against gonadotropin releasing hormone (GnRH). Four gilts, which had become acyclic after immunization against GnRH, and four control gilts were given 1,000 IU pregnant mare serum gonadotropin (PMSG), while four additional control gilts were given saline. Control animals were prepuberal crossbred gilts averaging 100 kg body weight. Control gilts given saline had ovaries containing antral follicles (4 to 6 mm in diameter). Control gilts given PMSG exhibited estrus and their ovaries contained corpora hemorrhagica and corpora lutea. PMSG failed to stimulate follicular growth in gilts immunized against GnRH, and ovaries contained regressed corpora albicantia and small antral follicles (less than 1 mm in diameter). Concentrations of luteinizing hormone (LH) and estradiol-17 beta (E2) were non-detectable in gilts immunized against GnRH and given PMSG. In the second experiment, five gilts actively immunized against GnRH were given increasing doses of PMSG every third day until unilateral ovariectomy on d 50. PMSG failed to stimulate follicular growth, and concentrations of follicle stimulating hormone (FSH), E2 and LH were not detectable. Six weeks later, gilts were given a booster immunization and then were given 112 micrograms LH and 15 micrograms FSH intravenously every 6 h for 9 d. The remaining ovary was removed on d 10. Although LH and FSH concentrations were elevated, administration of gonadotropins did not stimulate follicular growth or increase E2 concentrations. These results indicate that neither PMSG or exogenous LH and FSH can induce E2 synthesis or sustain follicular development in gilts actively immunized against GnRH.     

Influences of season and artificial photoperiod on stallions: pituitary and testicular responses to exogenous GnRH

Effects of season and photoperiod on the anterior pituitary gland and testes were studied by responses to exogenous GnRH. Stallions were assigned to one of three treatments: 1) control, exposed to natural day length; 2) S-L, 8 h of light and 16 h dark (8:16) for 20 wk beginning July 16, 1982 then 16:8 from December 2, 1982 until March 5, 1984; or 3) S-S, 8:16 from July 16, 1982 until March 5, 1984. Approximately every 8 wk, stallions were administered GnRH (2 micrograms/kg BW) and blood was sampled at 20-min intervals for 2 h before and 8 h after GnRH administration. Concentrations of LH, FSH and testosterone were determined. Baseline concentrations (mean of pre-GnRH samples) of all hormones fluctuated seasonally (P less than .05), but only LH and testosterone displayed seasonal changes (P less than .05) in maximum response to GnRH (highest concentration above baseline after GnRH). The FSH response to GnRH was not affected (P greater than .05) by season, photoperiod or the season X treatment interaction. Exposure of S-L stallions to 16:8 in December resulted in early recrudescence of baseline concentrations of LH, FSH and testosterone. Maximum concentration of testosterone in response to GnRH was stimulated by 16:8, but the increase in baseline LH concentrations in S-L stallions was not associated with an increase in maximum LH response to GnRH. Seasonal patterns of baseline concentrations of FSH and testosterone and maximum LH response to GnRH in S-S stallions were similar to those for control stallions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulsatile administration of gonadotropin-releasing hormone agonist to gilts actively immunized against gonadotropin-releasing hormone

Sexually mature gilts (n = 20) were actively immunized against GnRH. Primary and booster immunizations of GnRH conjugated to bovine serum albumin induced production of antibodies in all gilts. Nineteen of the gilts became acyclic with suppressed concentrations of gonadotropins and estradiol. Intravenous challenges with 100 micrograms GnRH and 5 micrograms D-(Ala6, des-Gly-NH2(10)) ethylamide GnRH (a GnRH agonist that did not cross-react with antibodies produced by the gilts) caused release of LH and FSH, indicating maintenance of secretory capacity of pituitary gonadotropes in the immunized animals. Gilts were given 100 ng GnRH agonist at 2-h intervals for 72 h (n = 4) or 144 h (n = 10) or did not receive agonist (n = 5). Blood samples were taken every 6 h, and detectable concentrations of LH were observed in 42% and 52% of samples taken from gilts treated with or without agonist. In contrast, serum concentrations of FSH and estradiol were undetectable. Reproductive tracts and anterior pituitaries were taken from gilts at the conclusion of pulsatile administration of GnRH agonist or at 144 h for controls. Pituitary concentration of LH and FSH, uterine wet and dry weight, and size of the uterus were similar among groups. Paired ovarian weights for treated gilts pulsed with GnRH agonist for 72 h were heavier (P less than .05); however, ovaries from all immunized gilts were atrophied without follicular structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary receptors for GnRH and estradiol, and pituitary content of gonadotropins in beef cows. I. Changes during the estrous cycle

To further characterize the endocrinological changes in the hypothalamo-hypophyseal axis thoughout the bovine estrous cycle, cycling beef heifers (n = 24) were randomly assigned to six groups. These heifers were slaughtered 6, 12, 18, 19, 20 or 21 days following their previous estrus (day 0). Anterior pituitaries and hypothalami were collected. Hypothalami were divided into the preoptic area and medial basal hypothalamus, and content of gonadotropin-releasing hormone (GnRH) was quantified by radioimmunoassay. Contents of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the anterior pituitary gland were quantified by radioimmunoassay. Membrane receptors for GnRH were quantified by a standard curve technique and receptors for estradiol in anterior pituitary cytosol were quantified by saturation analysis. There was no significant change in content of GnRH in the hypothalamus or content of FSH in the anterior pituitary on any of the days examined; however, content of GnRH in the preoptic area was lower (P less than .1) on day 19 postestrus. Cytosolic receptors for estradiol increased (P less than .05) on day 18 post-estrus and returned to baseline by day 19. Content of LH and the number of receptors for GnRH in the anterior pituitary gland decreased (P less than .01) on day 19 postestrus, and the number of receptors for GnRH remained low through day 21 postestrus. The reduction in anterior pituitary content of LH was transient indicating that synthesis of LH restores pituitary content to preovulatory levels before the number of receptors for GnRH returns to normal.     

Prolactin and Gonadotropin Responses in Geldings to Injections of Estradiol Benzoate in Oil, Estradiol Benzoate in Biodegradable Microspheres, and Estradiol Cypionate

We previously reported success in inducing early ovulation in seasonally anovulatory mares with a combination of estradiol pretreatment followed by daily administration of a dopamine antagonist (sulpiride). Although every-other-day injections of estradiol benzoate (EB) were effective in that experiment, practical application of this technology would require simplification of the treatment regimen. The current experiment was designed to compare, in a gelding model, the biologic responses of two alternative, one-injection regimens for estradiol delivery to the established EB treatment used previously. Fifteen long-term geldings were sampled via jugular venipuncture from November 5 to 7, 2006, and were then administered intramuscular injections of vegetable oil (n = 4); EB, 11 mg in oil (n = 4; controls); EB in biodegradable microspheres (300 mg; n = 3); or estradiol cypionate, 100 mg in oil (n = 4). Injections of EB in oil were repeated every other day for a total of 10 injections, as was done in our previous experiment. Jugular blood samples were drawn from all geldings at 3, 6, 12, 24, 36, and 48 hours relative to injections, and then on the mornings of days 3, 4, 6, 8, 10 to 18, 22, 26, and 30. On days 10 through 13, all geldings received subcutaneous injections of 125 mg sulpiride, a dopamine receptor antagonist, to stimulate prolactin secretion. On day 12, each gelding received an intravenous injection of 30 μg gonadotropin-releasing hormone (GnRH) analog and 3 mg thyrotropin-releasing hormone (TRH); frequent blood samples were drawn to characterize the luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin responses. Relative to geldings receiving oil, all geldings receiving estradiol injections had a rise (P < .05) in estradiol concentrations lasting at least 12 days. Daily LH concentrations increased (P < 0.01) in all treated groups, but the response was delayed approximately 14 days in the geldings receiving EB in microspheres. Daily FSH concentrations decreased (P < .01) in all treated groups, with the greatest response in the geldings receiving EB in microspheres. Prolactin in daily samples increased (P < .01) similarly in all estradiol-treated groups after injection of sulpiride. The LH response to GnRH analog was greatest (P < .05) in geldings receiving EB in oil and estradiol cypionate; the FSH response was not altered by treatment. The prolactin response to TRH was greater (P < .01) in estradiol-treated geldings relative to controls, but did not differ among groups. Compared with the responses to every-other-day EB injections in oil, as we used previously, a single injection of 100 mg estradiol cypionate gave the most similar and consistent responses. Because of these similar responses in this gelding model, it is likely that a single injection of 100 mg estradiol cypionate can be used in lieu of every-other-day injections of EB in oil in the treatment regimen we reported previously for stimulating ovarian activity in seasonally anovulatory mares.     

Effects of deslorelin acetate implants in horses: single implants in stallions and steroid-treated geldings and multiple implants in mares

Three experiments were performed to test the following hypotheses: 1) stallions and/or progesterone-estradiol-treated geldings could serve as models for the effects of a single implant of the GnRH analog, deslorelin acetate, on LH and FSH secretion by mares; and 2) multiple implants of deslorelin acetate could be used as a means of inducing ovarian atrophy in mares for future study of the mechanisms involved in the atrophy observed in some mares after a single implant. In Exp. 1, nine light horse stallions received either a single deslorelin implant (n = 5) or a sham injection (n = 4) on d 0. In Exp. 2, 12 geldings received daily injections of progesterone on d -20 through -4, followed by twice-daily injections of estradiol on d -2 to 0. On the morning of d 0, geldings received either a single deslorelin implant (n = 6) or a sham injection (n = 6). Daily injections of progesterone were resumed on d 2 through 15. In Exp. 1, plasma LH and FSH were elevated (P < 0.05) in the treatment group relative to controls at 4, 8, and 12 h after implant insertion. In the treated stallions, FSH was decreased (P < 0.05) on d 3 to 13, and LH was decreased on d 6 to 13. In Exp. 2, plasma LH and FSH were elevated (P < 0.05) at 4,8, and 12 h after deslorelin implant insertion. Plasma LH was suppressed (P < 0.05) below controls on d 2 to 7, 9, and 11 to 15; plasma FSH was suppressed (P < 0.05) on d 4 to 15. In Exp. 3, 21 mares were used to determine whether multiple doses of deslorelin would cause ovarian atrophy. Mares received one of three treatments: 1) sham injections; 2) three implants on the first day; or 3) one implant per day for 3 d (n = 7 per group). Treatment with multiple implants increased (P < 0.05) the interovulatory interval by 14.8 d and suppressed (P < 0.01) LH and FSH concentrations for approximately 25 d; no mare exhibited ovarian atrophy. In conclusion, after an initial short-term increase in LH and FSH secretion, deslorelin implants caused long-term suppression of both gonadotropins in stallions as well as in geldings treated with progesterone and estradiol to mimic the estrous cycle. It is likely that either of these models may be useful for further study of this suppression in horses. Although multiple implants in mares suppressed gonadotropin secretion longer than a single implant, the lack of ovarian atrophy indicates that the atrophy observed after a single implant in previous experiments was likely due to the susceptibility of individual mares.     

Concentrations of prolactin, luteinizing hormone and follicle stimulating hormone in pituitary and serum of horses: effect of sex, season and reproductive state

Pituitary and serum from 86 male or female horses of various reproductive states were collected in the normal breeding season (summer) and in the nonbreeding season (winter) at a commercial slaughterhouse. Concentrations of prolactin (PRL), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured by radioimmunoassay. Concentrations of pregnant mare serum gonadotropin and reproductive steroids in serum and gross appearance of the reproductive tract and gonads were used to catagorize reproductive state. Concentrations of PRL were higher (P less than .01) in summer than in winter in pituitary and serum of mares, stallions and geldings. In summer, mares had higher (P less than .01) concentrations of PRL in serum than stallions. In mares, concentrations of LH in pituitary were higher (P less than .05) in summer than in winter. Concentrations of LH in serum were higher (P less than .01) in summer than in winter in mares and geldings, higher (P less than .01) in mares than in stallions in summer, higher (P less than .01) in geldings than in stallions in summer and higher (P less than .01) in mares with low serum progesterone (P) concentrations than in mares with high P concentrations in summer. Concentrations of FSH in pituitary and serum did not differ between summer and winter for any type of horse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization of Sheep Against GNRH Early in Life: Effects on Gonadotropins,Follicular Growth and Responsiveness of Granulosa Cells To FSH and IGF-I in Two Breeds of Sheep With Different Prolificacy (Romanov and ILE-DE-France)

The prolific Romanov (R, ovulation rate = 3) and non-prolific Ile-de-France (IF, ovulation rate = 1) breeds were compared for their ovarian sensitivity to gonadotropins and IGF-I before puberty. For this purpose, the effects of in vivo immunization against GnRH on populations of ovarian follicles and in vitro sensitivity of granulosa cells to FSH and IGF-I were studied in prepuberal lambs from both breeds. Seventeen prepuberal lambs of each breed were actively immunized against GnRH between 3 wk and 6 mo of age. Relative to untreated lambs, FSH levels at 4, 5, and 6 mo of age were (respectively) 41%, 25%, and 29% for IF, and 43%, 24%, and 36% for R lambs. In a first experiment, histological analysis of ovaries was performed. Immunization treatment decreased the number of small (100–390 μm in diameter) and large size follicles (<1500 μm) in both breeds at 6 mo of age. In both breeds, gonadotropin (FSH - LH -hCG) treatment increased the number of large size follicles (<1500 μm in diameter) and induced the formation of preovulatory follicles in immunized as well as untreated lambs. The ovulation rate was less in immunized animals, but it was not different between breeds. In a second experiment, the effects of FSH and IGF-I were studied on granulosa cells from follicles between 1000 and 2000 μm in diameter. In both breeds, IGF-I increased granulosa cell proliferation, but enhanced progesterone secretion was observed only in R lambs after FSH and IGF-I stimulation. Granulosa cell response to FSH treatment was lost by immunization, whereas response to IGF-I remained unchanged in both breeds. These results indicate that long-term immunization of prepuberal lambs against GnRH reduced systemic concentrations of FSH, follicular development, and response to gonadotropins in vivo, similarly in the prolific R and the non-prolific IF breed. However, granulosa cells from R lambs had higher steroidogenic capacities and were more responsive to FSH. In addition, these results suggest that IGF-I could play an important role in regulating growth of small follicles both in immunized and non-immunized lambs.     

Immunization of beef heifers against gonadotropin-releasing hormone prevents luteal activity and pregnancy: Effect of conjugation to different proteins and effectiveness of adjuvants

Three experiments were conducted to evaluate methods of immunization against GnRH on antibody titer, luteal activity, and pregnancy in beef heifers. Experiment 1 evaluated the efficacy of adjuvants with 30 heifers. Control heifers were immunized against human serum albumin (HSA) emulsified in Freund's complete adjuvant (FCA). The other 4 treatments contained GnRH conjugated to HSA (HSA-GnRH) emulsified in FCA, Freund's incomplete adjuvant (FIA), DEAE dextran (DD) + mineral oil (MO), or DD+FIA. Treatment was in the mammary gland for all experiments. Titers against GnRH for heifers immunized against HSA-GnRH with FCA, DD+MO, or DD+FIA were greater than titers for HSA-GnRH with FIA or control heifers (P < 0.01). Body weight was reduced (P < 0.05) in control and FCA heifers compared with FIA, DD+MO, and DD+FIA heifers. Heifers immunized with DD+MO and DD+FIA had fewer granulomas in mammary glands than heifers treated with FCA (P < 0.01). In Exp. 2, 36 heifers were used to determine the effect of the protein conjugated to GnRH on titers against GnRH. Heifers (6/treatment) received a primary immunization against GnRH conjugated to HSA (HSA-GnRH), ovalbumin (OA-GnRH), or keyhole limpet hemocyanin (KL-GnRH), or heifers were immunized against each carrier protein. Antigens were emulsified in DD+FIA. Immunization of heifers against OA-GnRH, KL-GnRH, or HSA-GnRH suppressed luteal activity (P < 0.01) for 23, 16, and 12 wk, respectively, and antibody titers against GnRH were greater (P < 0.01) for 19, 5, and 7 wk, respectively, compared with heifers immunized against the carrier proteins. In Exp. 3, 90 heifers were used to determine the effect of immunization against GnRH on ovarian activity and pregnancy rate. Heifers (30/treatment) received a primary and 2 or 3 booster immunizations against GnRH conjugated to OA, and controls received a primary and 2 booster immunizations against OA. All antigens were emulsified in DD+FIA. At 8 wk after primary immunization, heifers were exposed to fertile bulls for 24 wk. Pregnancy rate was less (P < 0.01) for 3-booster heifers (13%) compared with control (83%) and 2-booster (62%) heifers. We conclude that immunization against GnRH, conjugated to OA and emulsified in DD+FIA, does not influence ADG and produces sufficient titers against GnRH to prevent estrous cycles with few mammary granulomas. Immunization against GnRH with 3 booster immunizations prevented luteal activity and pregnancy in most beef heifers for more than 4 mo.     

Central injection of exogenous IL-1β in the control activities of hypothalamic-pituitary-gonadal axis in anestrous ewes

This study was performed to determine the effect of intracerebroventricular (icv) injection of interleukin (IL)-1β on the gene expression, translation and release of gonadotropin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) gene expression in the hypothalamus of anestrous ewes. In the anterior pituitary gland (AP), the expression of genes encoding: GnRHR, β subunits of luteinizing hormone (LH) and folliculotropic hormone (FSH) was determined as well as the effect of IL-1β on pituitary gonadotropins release. The relative mRNA level was determined by real-time PCR, GnRH concentration in the cerebrospinal fluid (CSF) was assayed by ELISA and the plasma concentration of LH and FSH were determined by radioimmunoassay. Our results showed that icv injection of IL-1β (10 or 50 μg/animal) decreased the GnRH mRNA level in the pre-optic area (POA) (35% and 40% respectively; p ≤ 0.01) and median eminence (ME) (75% and 70% respectively; p ≤ 0.01) and GnRHR gene expression in ME (55% and 50% respectively; p ≤ 0.01). A significant decrease in GnRHR mRNA level in the AP in the group treated with the 50 μg (60%; p ≤ 0.01) but not with the 10 μg dose was observed. The centrally administrated IL-1β lowered also GnRH concentration in the CSF (60%; p ≤ 0.01) and reduced the intensity of GnRH translation in the POA (p ≤ 0.01). It was not found any effect of icv IL-1β injection upon the release of LH and FSH. However, the central injection of IL-1β strongly decreased the LHβ mRNA level (41% and 50%; p ≤ 0.01; respectively) and FSHβ mRNA in the case of the 50 μg dose (49%; p ≤ 0.01) in the pituitary of anestrous ewes. These results demonstrate that the central IL-1β is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.     

Effects of dihydrotestosterone benzoate administration on gonadotropin secretion in ovariectomized pony mares

Eight long-term ovariectomized pony mares were treated with either dihydrotestosterone (DHT) benzoate (400 micrograms/kg body weight) in safflower oil or an equivalent amount of oil every other day for 21 d to determine the effects of DHT on follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in blood samples drawn once daily and after administration of three successive injections of gonadotropin releasing hormone (GnRH). The GnRH injections were given at 4-h intervals on the day following the last DHT or oil injection. Treatment with DHT benzoate did not alter (P greater than .10) concentrations of FSH or LH in daily blood samples relative to controls. The FSH and LH response, assessed by areas under the GnRH curves, decreased (P less than .05) from the first to third injection of GnRH when averaged over both groups of mares. There was no effect of DHT treatment on FSH response to GnRH. There was an interaction (P less than .05) between treatment and GnRH injection for LH areas; areas decreased (P less than .05) for DHT-treated mares from the first to third GnRH injection but were unchanged for control mares. It seems that DHT alone cannot mimic the stimulatory effects of testosterone on FSH production and secretion as observed in previous experiments with ovariectomized and intact mares. Moreover, because intact mares have been shown previously to respond to DHT treatment with an increase in GnRH-induced FSH secretion, it appears that some mechanism is lost in long-term ovariectomized mares, making them unresponsive to DHT treatment.     

Influence of dose, frequency, and duration of infused gonadotropin-releasing hormone on secretion of luteinizing hormone and follicle-stimulating hormone in nutritionally anestrous beef cows.

Nutritionally induced anovulatory cows were ovariectomized and used to determine the relationships between dose, frequency, and duration of exogenous gonadotropin-releasing hormone (GnRH) pulses and amplitude, frequency, and concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum. In Experiment 1, cows were given pulses of saline (control) or 2 micrograms of GnRH infused i.v. during a 0.1-, 1.25-, 5-, 10-, or 20-min period. Concentrations of LH and FSH during 35 min after GnRH infusion were greater than in control cows (P < 0.01), and FSH concentrations were greater when GnRH infusions were for 10 min or less compared with 20 min. In Experiment 2, the effect of GnRH pulse frequency and dose on LH and FSH concentrations, pulse frequency, and pulse amplitude were determined. Exogenous GnRH (0, 2, or 4 micrograms) was infused in 5 min at frequencies of once every hour or once every 4th hr for 3 d. There was a dose of GnRH x frequency x day effect on LH and FSH concentrations (P < 0.01), indicating that gonadotropes are sensitive to changes in pulse frequency, dose, and time of exposure to GnRH. There were more LH pulses when GnRH was infused every hour, compared with an infusion every 4th hr (P < 0.04). Amplitudes of LH pulses were greater with increased GnRH dose (P < 0.05), and there was a frequency x dose x day effect on FSH pulse amplitude (P < 0.0006). We conclude that LH and FSH secretion in the bovine is differentially regulated by frequency and dose of GnRH infusions.