Effects of dihydrotestosterone administration with and without estradiol pretreatment on gonadotropin secretion in ovariectomized pony mares

Twenty ovariectomized pony mares were used to determine if dihydrotestosterone propionate (DHTP) administration, with or without estradiol benzoate (EB) pretreatment, would have the same effects on follicle stimulating hormone (FSH) and luteinizing hormone (LH) secretion as testosterone propionate (TP) administration. All mares were given an initial injection of gonadotropin releasing hormone (GnRH) to characterize their LH and FSH response, and then two groups of mares (n = 4/group) were administered EB (22 micrograms/kg of body weight), two groups were administered vehicle (safflower oil) and a fifth group was administered TP (175 micrograms/kg of body weight) daily for 10 days. Following a second injection of GnRH, one group of EB-treated mares and one group of oil-treated mares were administered DHTP (175 micrograms/kg of body weight) daily for 10 days; the other EB- and oil-treated mares were administered oil and the TP-treated mares were continued on the same dose of TP for 10 days. A final injection of GnRH was then given. Treatment with EB increased (P less than .01) concentrations of LH in daily blood samples and increased (P less than .05) the LH response to exogenous GnRH. Administration of TP or DHTP reduced (P less than .05) both daily LH concentrations and the LH response to exogenous GnRH. Concentrations of FSH in daily blood samples were reduced (P less than .05) and the FSH response to exogenous GnRH was increased (P less than .05) by administration of EB alone, DHTP alone or TP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 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.     

Androgen and estradiol effects on gonadotropin secretion and response to GnRH in ovariectomized pony mares

In Exp. 1, 16 long-term ovariectomized pony mares were used to determine the effects of treatment with estradiol benzoate (EB) and dihydrotestosterone (DHT) benzoate alone, and in combination, on secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in daily blood samples and after three consecutive injections of gonadotropin releasing hormone (GnRH). Administration of EB alone, or in combination with DHT, every other day for 11 d reduced (P less than .05) concentrations of FSH and increased (P less than .05) concentrations of LH in daily blood samples, and increased (P less than .05) the secretion of both gonadotropins after administration of GnRH. Treatment with DHT alone had no effect (P greater than .10) on LH or FSH concentrations in daily blood samples and no effect on the LH response to exogenous GnRH. There was no interaction (P greater than .10) between DHT and EB treatment for any hormonal characteristic. In Exp. 2, the control mares and mares treated with DHT in Exp. 1 were equally allotted to treatment with vehicle or testosterone propionate (TP) every other day for six injections, and then GnRH was administered as in Exp. 1. Treatment with TP had no effect (P greater than .10) on LH or FSH concentrations in daily blood samples but increased (P less than .05) the FSH response to exogenous GnRH, confirming our findings in previous experiments. It is concluded that the TP-induced stimulation of FSH secretion after exogenous GnRH in ovariectomized mares may involve estrogens produced from aromatization of the injected androgen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma concentrations of cortisol, prolactin, luteinizing hormone, and follicle-stimulating hormone in stallions after physical exercise and injection of secretagogue before and after sulpiride treatment in winter

Ten lighthorse stallions were used to determine 1) whether prolactin (PRL) and cortisol responses previously observed after acute exercise in summer would occur in winter when PRL secretion is normally low, 2) whether subsequent treatment with a dopamine receptor antagonist, sulpiride, for 14 d would increase PRL secretion and response to thyrotropin-releasing hormone (TRH) and exercise, and 3) whether secretion of LH, FSH, and cortisol would be affected by sulpiride treatment. On January 11, blood samples were drawn from all stallions before and after a 5-min period of strenuous running. On January 12, blood samples were drawn before and after an i.v. injection of GnRH plus TRH. From January 13 through 26, five stallions were injected s.c. daily with 500 mg of sulpiride; the remaining five stallions received vehicle. The exercise and secretagogue regimens were repeated on January 27 and 28, respectively. Before sulpiride injection, concentrations of both cortisol and PRL increased (P less than .05) 40 to 80% in response to exercise; concentrations of LH and FSH also increased (P less than .05) approximately 5 to 10%. Sulpiride treatment resulted in (P less than .05) a six- to eightfold increase in daily PRL secretion. The PRL response to TRH increased (P less than .05) fourfold in stallions treated with sulpiride but was unchanged in control stallions. Sulpiride treatment did not affect (P greater than .05) the LH or FSH response to exogenous GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testosterone propionate effects on gonadotropin secretion in steers after administration of GnRH

Two experiments with steers were performed to determine the effects of 10 days of treatment with testosterone propionate (TP) on secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) after the administration of gonadotropin releasing hormone (GnRH). In Experiment 1, eight 15-month-old Holstein steers castrated at 12 months of age were used. In Experiment 2, eight beef-type steers between the ages of 12–18 months old and which had been castrated for 8–15 months were used. An initial injection of GnRH was followed by 10 daily injections of TP (175 ug/ kg of body weight) or safflower oil, and on the eleventh day a second injection of GnRH was given. Hormonal endpoints in long-term castrated steers (Experiment 2) were not altered by TP treatment. In contrast, TP treatment significantly suppressed FSH and LH concentrations in daily blood samples from Experiment 1 steers and significantly increased the response to GnRH. The lack of response in the long-term castrated steers (Experiment 2) to TP treatment is similar to the response observed in previous experiments with long-term ovariectomized cows. However, from the results of Experiment 1, it appears that the timing of castration influences the response to TP treatment.     

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.     

Gonadotropin secretion in ovariectomized pony mares treated with dexamethasone or progesterone and subsequently with dihydrotestosterone

Twelve long-term ovariectomized (OVX) pony mares were used to determine the effects of dexamethasone (DEX) or progesterone (PR) on concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in daily blood samples and after administration of gonadotropin releasing hormone (GnRH). All mares were subsequently administered dihydrotestosterone (DHT) to determine if DEX or PR treatment altered the FSH or LH response to this androgen. Daily blood sampling was started on day 1. After a pretreatment injection of GnRH on day 5, four mares were administered DEX at 125 micrograms/kg of body weight (BW), four mares were administered PR at 500 micrograms/kg of BW and four mares were administered vehicle. Injections were given subcutaneously in vegetable shortening daily through day 14. After a second injection of GnRH on day 15, all mares were administered DHT in shortening at 150 micrograms/kg of BW. Injections of DHT were given daily through day 24. A final injection of GnRH was given on day 25. Treatment of mares with DEX 1) reduced (P less than .01) daily LH secretion and briefly increased (P less than .05) daily FSH secretion and 2) increased (P less than .01) the FSH response to exogenous GnRH. Treatment of mares with PR had no effect on daily LH secretion but increased (P less than .05) daily FSH secretion and increased (P less than .01) the FSH response to exogenous GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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)     

Effect of daily injections of growth hormone-releasing factor and thyrotropin-releasing hormone on growth and endocrine parameters in chickens

The control of growth is a complex mechanism regulated by several metabolic hormones including growth hormone (GH) and thyroid hormones. In avian species, as well as in mammals, GH secretion is regulated by hypothalamic hypophysiotropic hormones. Since thyrotropin-releasing hormone (TRH) and growth hormone-releasing factor (GRF) are potent GH secretagogues in poultry, we were interested in determining the influence of daily intravenous administration of either peptide or both simultaneously on circulating GH and IGF-I concentrations and whether an improvement in growth rate or efficiency would be obtained.

Male broiler chicks were injected once daily for a period of 21 days with either GRF (10 μg/kg), TRH (1 μg/kg) or both GRF and TRH (10 and 1 μg/kg respectively) between four and seven weeks of age. On the last day of the experiment, following intravenous injection of TRH, GRF or a combination of GRF and TRH, plasma GH levels were significantly (P<.05) increased to a similar extent in control chicks and in those which had received daily peptide injections for the previous 21 days. Circulating GH levels between 10 and 90 min post-injection were significantly (P<.05) greater and more than additive than GH levels in chicks injected with both GRF and TRH when compared to those injected with either peptide alone. Mean plasma T₃ concentrations during that same time period were significantly elevated (P<.05) above saline-injected control chick levels in birds treated with TRH or GRF and TRH respectively, regardless of whether the chicks had received peptide injections for the previous 21 days. There was no evidence of pituitary refractoriness to chronic administration of either TRH or GRF injection in terms of growth or thyroid hormone secretion.

Despite the large elevation in GH concentration each day, growth rate, feed efficiency and circulating IGF-I concentrations were not enhanced. Thus the quantity or secretory pattern of GH secretion induced by TRH or GRF administration was not sufficient to increase plasma IGF-I concentration or growth.     

The effect of pulsatile gonadotropin-releasing hormone and estradiol administration on luteinizing hormone and follicle-stimulating hormone concentrations in pituitary stalk-sectioned ovariectomized pony mares

Hourly pulses of gonadotropin-releasing hormone (GnRH) or bi-daily injections of estradiol (E2) can increase luteinizing hormone (LH) secretion in ovariectomized, anestrous pony mares. However, the site (pituitary versus hypothalamus) of positive feedback of estradiol on gonadotropin secretion has not been described in mares. Thus, one of our objectives involved investigating the feedback of estradiol on the pituitary. The second objective consisted of determining if hourly pulses of GnRH could re-establish physiological LH and FSH concentrations after pituitary stalk-section (PSS), and the third objective was to describe the declining time trends of LH and FSH secretion after PSS. During summer months, ovariectomized pony mares were divided into three groups: Group 1 (control, n = 2), Group 2 (pulsatile GnRH (25 μg/hr), n = 3), and Group 3 (estradiol (5 mg/12 hr), n = 3). All mares were stalk-sectioned and treatment begun immediately after stalk-section. Blood samples were collected every 30 min for 8 h on the day before surgery (DO) and 5 d post surgery (D5) to facilitate the comparison of gonadotropin levels before and after pituitary stalk-section. Additionally, jugular blood samples were collected every 12 hr beginning the evening of surgery, allowing for evaluation of the gonadotropin secretory time trends over the 10 d of treatment. On Day 10, animals were euthanized to confirm pituitary stalk-section and to submit tissue for messenger RNA analysis (parallel study). Plasma samples were assayed for LH and FSH by RIA. Mean LH secretion decreased from Day 0 to Day 5 in Groups 1 and 3, whereas LH secretion tended (P < 0.08) to decrease in Group 2 mares. On Day 5, LH was higher (P < 0.01) in Group 2 (17.26 ± 3.68 ng/ml; LSMEANS ± SEM), than either Group 1 (2.65 ± 4.64 ng/ml) or group 3 (4.28 ± 3.68 ng/ml). Group 1 did not differ from Group 3 on Day 5 (P < 0.40). Similarly, mean FSH levels decreased in all groups after surgery, yet Group 2 mares had significantly (P < 0.001) higher FSH concentrations (17.66 ± 1.53 ng/ml) than Group 1 or Group 3 (8.34 ± 1.84 and 7.69 ± 1. 63 ng/ml, respectively). Regression analysis of bi-daily LH and FSH levels indicated that the time trends were not parallel. These findings indicate: 1) Pituitary stalk-section lowered LH and FSH to undetectable levels within 5 d after surgery, 2) pulsatile administration of GnRH (25 μg/hr) maintained LH and FSH secretion, although concentrations tended to be lower than on Day 0, and 3) E2 did not stimulate LH or FSH secretion.     

Responses of seasonally anovulatory mares to daily administration of thyrotropin-releasing hormone and(or) gonadotropin-releasing hormone analog

Seventeen seasonally anovulatory light horse mares were treated daily, starting January 5 (d 1), for 28 d with GnRH analog (GnRH-A; 50 ng/kg BW) and(or) thyrotropin-releasing hormone (TRH; 5 microg/kg BW) in a 2 x 2 factorial arrangement of treatments to test the hypothesis that combined treatment may stimulate follicular growth and development. Ovaries were examined via ultrasonography and jugular blood samples were collected every 3 d. Frequent blood samples were collected after treatment injections on d 1, 2, 4, 7, 11, 16, and 22; on d 29, all mares received an i.v. mixture of GnRH, TRH, sulpiride, and EP51389 (a growth hormone secretagogue) to assess pituitary responsiveness. No consistent effects (P > 0.1) of treatment were observed for plasma LH, FSH, prolactin, or thyroxine concentrations in samples collected every 3 d. The only effect on ovarian follicle numbers was a reduction in number of follicles 11 to 19 mm in diameter due to TRH treatment (P = 0.029). No mare ovulated during treatment. On the days of frequent sampling, mean LH (P = 0.0001) and FSH (P = 0.001) concentrations were higher in mares receiving GnRH-A and tended to increase from d 1 through 7. In contrast, mean prolactin (P = 0.001) and thyroid-stimulating hormone (P = 0.0001) concentrations were high in mares receiving TRH on d 1 but rapidly decreased thereafter. When mares were administered the secretagogue mixture on d 29, the LH response was greater (P = 0.0002) in mares that had previously received GnRH-A but the FSH response was not affected (P > 0.1); the prolactin response was greater (P = 0.014) and the TSH response was smaller (P = 0.0005) in mares that had previously received TRH. Surprisingly, an immediate growth hormone response to EP51389 was absent in all mares. In conclusion, daily GnRH-A treatment stimulated plasma LH and FSH concentrations immediately after injection; although no long-term elevation in preinjection concentrations was achieved, the responses gradually increased over time, indicating a stimulation of gonadotropin production and storage. Daily treatment with TRH stimulated plasma TSH and prolactin concentrations, but the response diminished rapidly and was minimal within a few days, indicating a depletion of pituitary stores and little or no stimulation of production. There was no beneficial effect of adding TRH treatment to the daily GnRH-A regimen.     

Production of immunoactive inhibin by bovine granulosa cells in serum-free culture: Effects of exogenous steroids and FSH

Granulosa cells from pooled bovine follicles were cultured under chemically-defined (serum-free) conditions to study the effects of exogenous steroids and FSH on production of immunoactive (ia) inhibin, oestradiol and progesterone. Levels of ia-inhibin in media samples and cell lysates were measured by radioimmunoassay (RIA) using an antiserum raised against a synthetic fragment of human inhibin -subunit [hI(1–32)].

Cells secreted measurable amounts of ia-inhibin, oestradiol and progesterone for at least 7 d of culture, although intracellular levels of inhibin were very low, indicating that newly-synthesized ia-inhibin is rapidly released from the cells. Treatment with androstenedione (0.2μmol/l) or testosterone (0.2μmol/l) increased ia-inhibin secretion markedly; levels on Day 5 of culture were approximately 6-fold (P<0.005) higher than control values. In contrast, treatment with the non-aromatizable androgen dihydrotestosterone (DHT; 0.2μmol/l) resulted in only a one- to two-fold increase (P<0.05) over control values (Day 5). Addition of exogenous oestradiol (8nmol/l) markedly increased ia-inhibin secretion (8–9 fold on Day 5; P<0.05) compared with basal levels, whereas progesterone had no effect. Secretion of oestradiol, undetectable in the absence of exogenous androgens, rose daily in the presence of either androstenedione or testosterone, levels rising approximately 6-fold and 9-fold respectively over a 4-d treatment period. Progesterone secretion increased 2-fold over the culture period and was unaffected by any steroid treatment.

Treatment with ovine FSH (10ng/ml) alone stimulated secretion of progesterone over basal levels (3-fold higher on Day 6; P<0.005), but did not affect output of either ia-inhibin or oestradiol. However, exposure to FSH in the presence of androstenedione not only promoted a further 4-fold increase in progesterone output but also led to a dose-dependent suppression of both ia-inhibin (90% lower on Day 6; P<0.001) and oestradiol (80% lower on Day 6; P<0.001) secretion compared to cells treated with androstenedione alone.

These observations indicate that the secretion of ia-inhibin by bovine granulosa cells in culture is positively regulated by oestradiol, implying an autocrine/paracrine role for this hormone in control of ovarian inhibin production. The ability of aromatizable androgens to stimulate secretion of inhibin, coupled with the inability of the non-aromatizable androgen DHT to elicit such an effect, suggests that inhibin output is largely unaffected by androgens prior to their conversion to oestradiol. The absence of any change in output of ia-inhibin or oestradiol following treatment with exogenous progesterone argues against a local role for this steroid hormone in regulation of inhibin or oestradiol production in the bovine follicle. Finally, the observation that co-treatment with FSH and andostenedione not only stimulated progesterone output but also suppressed secretion of ia-inhibin and oestradiol, indicates a synergistic positive effect of FSH and androgens on cellular luteinization.     

Effect of deslorelin acetate on gonadotropin secretion and ovarian follicle development in cycling mares

OBJECTIVE: To evaluate gonadotropin secretion and ovarian function after administration of deslorelin acetate to induce ovulation in mares. DESIGN: Randomized controlled trial. ANIMALS: 16 healthy mares with normal estrous cycles. PROCEDURE: 8 control mares were allowed to ovulate spontaneously, whereas 8 study mares received deslorelin to induce ovulation when an ovarian follicle > 35 mm in diameter was detected. Follicle development and serum concentrations of gonadotropins were monitored daily during 1 estrous cycle. Pituitary responsiveness to administration of gonadotropin-releasing hormone (GnRH) was evaluated 10 days after initial ovulation. RESULTS: Interovulatory intervals of mares treated with deslorelin (mean +/- SD, 25.6 +/- 2.6 days) were longer than those of control mares (22.9 +/- 1.8 days). Diameter of the largest follicle was significantly smaller during 2 days of the diestrous period after ovulation in deslorelin-treated mares than in control mares. Concentrations of follicle-stimulating hormone (FSH) were lower in deslorelin-treated mares on days 5 through 14 than in control mares. Concentrations of luteinizing hormone were not different between groups during most of the cycle. Gonadotropin release in response to administration of GnRH was lower in mares treated with deslorelin acetate than in control mares. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of deslorelin was associated with reduction in circulating concentrations of FSH and gonadotropin response to administration of GnRH during the estrous cycle. Low concentration of FSH in treated mares may lead to delayed follicular development and an increased interovulatory interval.     

Leptin secretion in horses: effects of dexamethasone, gender, and testosterone

Five experiments were performed to evaluate the effects of dexamethasone (DEX), gender, and testosterone on plasma leptin concentrations in horses. In experiment 1, plasma leptin, insulin, glucose, and IGF-1 concentrations were increased (P < 0.01) in stallions following five daily injections of DEX (125 microg/kg BW). In experiment 2, leptin concentrations increased (P < 0.01) in mares, geldings, and stallions following a single injection of DEX, and the response was greater (P < 0.01) in mares and geldings than in stallions. The gender effect was confounded by differences in body condition scores and diet; however, based on stepwise regression analysis, both BCS and gender were significant sources of variation in the best fit model for pre-DEX leptin concentrations (R(2) = 0.65) and for maximum leptin response to DEX (R(2) = 0.75). In experiment 3, in which mares and stallions were pair-matched based on age and body condition and fed similar diets, mares again had higher (P < 0.01) leptin concentrations than stallions after DEX treatment as used in experiment 2. In experiment 4, there was no difference (P > 0.1) in plasma leptin response in mares following four single-injection doses of DEX from 15.6 to 125 microg/kg BW. In experiment 5, treatment of mares with testosterone propionate every other day for 5 days did not alter (P > 0.1) plasma leptin concentrations or the leptin response to DEX. In conclusion, multiple injections of DEX increase leptin concentrations in stallions, as does a single injection in mares (as low as 15.6 microg/kg BW), geldings and stallions. The greater leptin levels observed in mares and geldings relative to stallions were due partially to their greater body condition and partially to the presence of hyperleptinemic individuals; however, even after accounting for body condition and diet, mares still had greater leptin concentrations than stallions after DEX administration. Elevation of testosterone levels in mares for approximately 10 days did not alter leptin concentrations in mares.     

Opioid modulation of gonadotropin releasing hormone release from the hypothalamic preoptic area in the pig

Two experiments (Exp) were conducted to examine in vitro the release of gonadotropin releasing hormone (GnRH) from the hypothalamus after treatment with naloxone (NAL) or morphine (MOR). In Exp 1, hypothalamic-preoptic area (HYP-POA) collected from 3 market weight gilts at sacrifice and sagitally halved were perifused for 90 min prior to a 10 min pulse of morphine (MOR; 4.5 × 10⁻⁶ M) followed by NAL (3.1 × 10⁻⁵ M) during the last 5 min of MOR (MOR + NAL; N=3). The other half of the explants (n=3) were exposed to NAL for 5 min. Fragments were exposed to KCl (60 mM) at 175 min to assess residual GnRH releasability. In Exp 2, nine gilts were ovariectomized and received either oil vehicle im (V; n=3); 10 μg estradiol-17β/kg BW im 42 hr before sacrifice (E; n=3); .85 mg progesterone/kg BW im twice daily for 6 d prior to sacrifice (P₄; n=3). Blood was collected to assess pituitary sensitivity to GnRH (.2 μg/kg BW) on the day prior to sacrifice. On the day of sacrifice HYP-POA explants were collected and treated as described in Exp 1 except tissue received only NAL. In Exp 1, NAL increased (P<.05) GnRH release. This response to NAL was attenuated (P<.05) by coadministration of MOR. Cumulative GnRH release after NAL was greater (P<.05) than after MOR + NAL. All tissues responded similarly to KCl with an increase (P<.05) in GnRH release. In Exp 2, pretreatment luteinizing hormone (LH) concentrations were lower (P<.05) in E gilts compared to V and P₄ animals with P₄ being lower (P<.05) than V gilts. LH response to GnRH was lower (P<.05) in E pigs than in V and P₄ animals, while the responses was similar between V and P₄ gilts. NAL increased GnRH release in all explants, whereas, KCl increased GnRH release in 6 of 9 explants. These results indicate that endogenous opioid peptides may modulate in vitro GnRH release from the hypothalamus in the gilt.     

Effects of pituitary stalk-transection and type of barrier on pituitary and luteal function during the estrous cycle of the ewe

Effects of pituitary stalk-transection on plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) prolactin (PRL) and progesterone were investigated during the estrous cycle of ewes. Pituitary stalk (SS) or sham (SH) transection was performed on day 1 (estrus = day 0) of the estrous cycle. A Teflon or Silastic barrier was placed between the cut ends of the stalk to prevent reorganization of the portal vasculature. Immediately following surgery, pulsatile administration of gonadotropin releasing hormone (GnRH, 200 ng/hr) or .9% NaCl was initiated and continued for the duration of the experiment. Estradiol benzoate (EB, 50 μg im) was administered to all ewes on day 3. Mean concentrations of LH were greater in SS ewes than in SH ewes (P<.05). There was a trend (P=.06) for the concentration of LH to be higher in ewes with Teflon compared with Silastic barriers between the cut ends of the stalk. Infusion of GnRH elevated concentrations of LH in both SS and SH ewes (P<.05). Concentrations of progesterone were reduced (P<.01) in saline-infused SS ewes while infusion of GnRH in SS ewes maintained concentrations of progesterone similar to saline-infused SH ewes. The concentrations of FSH or PRL were unaffected by SS, type of barrier or treatment with GnRH. Administration of EB failed to induce a surge of LH except in a SH ewe infused with GnRH. Ewes were more responsive to infusion of GnRH following SS than after SH as reflected by increased plasma concentrations of LH and progesterone.     

Circulating concentrations of immunoreactive inhibin and FSH during the estrous cycle of mares

A heterologous radioimmunoassay developed to measure inhibin in rat plasma was validated and used to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to follicle stimulating hormone (FSH) concentrations during the estrous cycle of mares. The primary antiserum used in the assay was developed against a synthetic porcine inhibin -subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin -subunit standard curve and porcine follicular fluid dose-response curve. Twelve mares were bled once daily beginning when diameter of the largest follicle reached ≤25 mm and continuing until 3 days after the end of an interovulatory interval (ovulation=Day 0). Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26). There was an effect of day for concentrations of ir-inhibin (P<0.001) and FSH (P<0.006). Significant mean changes were as follows: 1) ir-inhibin decreased between Days 0 and 1, whereas FSH increased between Days 0 and 5; and 2) ir-inhibin increased between Days 7 and 12, whereas FSH decreased between Days 11 and 14. Mean concentrations of ir-inhibin and FSH were negatively correlated (r=-0.548; P<0.002). In conclusion, mean peripheral concentrations of ir-inhibin and FSH were inversely related during the estrous cycle of mares.