GnRH-treated Groups Research Articles

Effects Of Exogenous GnRH Stimulation On Testicular Spermatogenesis And Characteristics Of The West African Dwarf Buck

The testis in male domestic animals has two primary functions: spermatogenesis and steroidogenesis, coordinated by testicular cells under GnRH control. This study examined the features of the testes of the bucks of the West African dwarf breed to assess the effects of additional GnRH-stimulated levels of FSH, LH, T, and estradiol from 3 to 12 months of age. Twelve 3-month-old bucks were divided in a random manner into GnRH-treated (n = 6) and control (n = 6) groups. Monthly, bucks were weighed, their testicular diameter measured, and two blood samples taken (n = 12) at a 1-hour interval to establish peripheral concentrations. The GnRH-treated group received 0.5μg/ kg of GnRH intramuscularly the next day, while the control group received saline subcutaneously. Blood samples were taken 1 and 2 hours later to establish GnRH-stimulated levels. At 12 months, bucks were castrated, and testes were weighed and prepared for histological analysis. Testes diameters showed no significant difference (p<0.05): 24.52±0.43 mm (GnRHtreated group) and 25.92±0.45 mm (control group). However, yearling testicular mass differed significantly (p<0.05): 41.50±5.80g (GnRH-treated group) and 38.80±7.58g (control group). The number and make-up of seminiferous tubular cells (cells of Sertoli, germ cells in the form of spermatogonia, developing into spermatocytes, further maturing into spermatids, and ultimately forming spermatozoa) indicated increased spermatogenesis in the GnRH-treated group. It is concluded that GnRH super-stimulation increased spermatozoa production in the West African dwarf buck.

Age-related changes in hormonal profiles and testicular diameter of West African Dwarf buck treated with gonadotropin-releasing hormone

This study aimed to investigate the impact of age-related peripheral and Gonadotropin Releasing Hormone (GnRH) -stimulated hormonal profiles on testicular size in West African dwarf bucks. Twelve 3-month-old bucks were randomly divided into two groups: the GnRH- treated group (n = 6) and the control group (n = 6). Over the course of 10 months, from month 3 to month 12, the testicular diameter of all bucks was measured monthly, and blood samples were collected monthly for hormone analysis. The results showed that testicular diameter increased with age and higher concentrations of Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Testosterone (T). The GnRHtreated group reached its peak testicular diameter (28.36±1.44 mm) earlier at 8 months compared to the control group (30.50±1.44 mm) at 10 months, aligning with the peak concentrations of FSH, LH, and T. While the GnRH stimulation led to higher FSH, LH, T, and estrogen levels in the treated group, it didn't significantly affect the final testicular diameter (p<0.05). It can be concluded that hormonal levels rise with age and increasing testicular size, and GnRH stimulation can accelerate this process. Testicular size is an important factor in breeding soundness and is influenced by LH, FSH, and testosterone concentrations. These hormones can reliably indicate the reproductive capacity of the buck. Therefore, hormonal profiling should be an integral part of the breeding soundness examination.

Neurokinin 3 receptor–selective agonist, senktide, decreases core temperature in Japanese Black cattle

Heat stress disrupts reproductive function in cattle. In summer, high ambient temperature and humidity elevate core body temperature, which is considered to be detrimental to reproductive abilities in cattle. Neurokinin B (NKB) is a factor that generates pulsatile GnRH and subsequent LH secretion in mammals. Recent studies have reported that NKB-neurokinin 3 receptor (NK3R) signaling is associated with heat-defense responses in rodents. The present study aimed to clarify the role of NKB-NK3R signaling in thermoregulation in cattle. We examined the effects of an NK3R-selective agonist, senktide, on vaginal temperature as an indicator of core body temperature in winter and summer. In both seasons, continuous infusion of senktide for 4 h immediately decreased vaginal temperature, and the mean temperature change in the senktide-treated group was significantly lower than that of both vehicle- and GnRH-treated groups. Administration of GnRH induced LH elevation, but there was no significant difference in vaginal temperature change between GnRH- and vehicle-treated groups. Moreover, we investigated the effects of senktide on ovarian temperature. Senktide treatment seemed to suppress the increase in ovarian temperature from 2 h after the beginning of administration, although the difference between groups was not statistically significant. Taken together, these results suggest that senktide infusion caused a decline in the vaginal temperature of cattle, in both winter and summer seasons, and this effect was not due to the gonadotropin-releasing action of senktide. These findings provide new therapeutic options for senktide to support both heat-defense responses and GnRH/LH pulse generation.

Nerve growth factor from seminal plasma origin (spβ-NGF) increases CL vascularization and level of mRNA expression of steroidogenic enzymes during the early stage of Corpus Luteum development in llamas

The objectives of the study were to determine the effect of seminal plasma β-NGF on Corpus Luteum morphology and function and level of mRNA expression of steroidogenic enzymes. Llamas were assigned (n = 12/per group) to receive an intramuscular dose of: (a) 1 ml phosphate buffered saline (PBS), (b) 5 μg gonadorelin acetate (GnRH), or (c) 1.0 mg of purified llama spβ-NGF. Ovaries were examined by transrectal B-mode ultrasonography from treatment to ovulation (Day 0 = treatment). B mode/Power Doppler ultrasonography and blood samples collection were performed at Days 4, 8 and 10 (n = 3 llamas per treatment group/per time point) to determine CL diameter, vascularization and plasma progesterone concentration respectively. Plasma progesterone concentration was analyzed in all llamas at Day 0. Then females were submitted to ovariectomy at Days 4, 8 and 10 (n = 3 llamas/treatment/time), CL was removed to determine vascular area, proportion of luteal cells and CYP11A1/P450scc and STAR expression by RT-PCR. Ovulation was similar between llamas treated with GnRH or spβ-NGF and CL diameter did not differ between GnRH or spβ-NGF groups by Day 4, 8 or 10. Vascularization area of the CL was higher (P < 0.01) in llamas from the spβ-NGF than GnRH-treated group by Day 4 and 8. Plasma progesterone concentration was higher (P < 0.05) in llamas from the spβ-NGF compared to females of GnRH group by Day 4 and 8. The proportion of small and large luteal cells did not differ between GnRH or spβ-NGF groups by Day 8. CYP11A1/P450scc was upregulated 3 folds at day 4 and 10 by spβ-NGF compared to GnRH. STAR transcription was 3 folds higher at day 4 in females treated with spβ-NGF. In conclusion, the luteotrophic effect of spβ-NGF could be related to an increase of vascularization and up regulation of CYP11A1/P450scc and STAR transcripts enhancing progesterone secretion.

Fixed-time deep uterine insemination in PGF2α-synchronized goats

The aim of this investigation was to optimize fixed-time insemination in goats by clustering ovulations in prostaglandin F2α-synchronized goats either with gonadotropin releasing hormone (GnRH) or human chorionic gonadotropin (hCG). The underlying intention was to reduce the incidence of short cycles by providing a more sustained stimulation of the corpus luteum by substituting the commonly used GnRH with longer-acting hCG. It was conjectured that this might render the corpus luteum less prone to premature regression. Sixty pluriparous does were administered 5 mg of the prostaglandin F2α preparation dinoprost (Dinolytic; Pharmacia and Upjohn, Erlangen, Germany) during the luteal phase of the estrous cycle. Twenty of these does were administered 0.004 mg of the GnRH analog buserelin (Receptal; Intervet, Unterschleissheim, Germany) 48 hours later; another 20 does received 500 IU hCG (Chorulon; Intervet, Unterschleissheim, Germany) instead. Sixteen hours later the does were inseminated with frozen-thawed semen. The remaining 20 does served as controls and were inseminated 16–18 h after the onset of detected estrus. All 60 treated goats displayed estrous symptoms, the time of onset being similar for all groups (42.6, 37.6, and 40.5 hours after treatment for GnRH-treated, hCG-treated, and control does, respectively). The duration of estrus in the GnRH-treated group was 10 h less than in the other groups (45.1 vs. 56.4 and 54.4 h, P < 0.05). The number of ovulations (assessed by ultrasound monitoring) did not differ among groups (2.4, 2.1, and 2.5, P > 0.05). Monitoring of serum progesterone revealed that the incidence of corpus luteum insufficiency was significantly higher in GnRH- and hCG-treated does than in the control group (40% and 35% vs. 5%, P < 0.05). The pregnancy rate was 50% in the GnRH and 35% in the hCG group as compared with 60% in the controls. Corresponding kidding rates were 40%, 35%, and 60% (P > 0.05). When disregarding does with corpus luteum insufficiency, pregnancy rates would have been 83%, 54%, and 63%, and kidding rates 67%, 54%, and 63%, respectively. The average number of kids born was 1.88, 1.71, and 1.83, respectively (P > 0.05). It may be concluded that fixed time insemination of cycling does treated with prostaglandin F2α during the luteal phase, followed by ovulation induction with GnRH or hCG, would be an effective management tool if it were possible to control the high incidence of corpus luteum insufficiency. The attempt to achieve this by substituting GnRH with hCG, was not met with success. Until a solution for the problem has been found, it is advisable to inseminate prostaglandin-synchronized does 16–18 hours after the onset of detected estrus.

Cetrorelix suppresses the preovulatory LH surge and ovulation induced by ovulation-inducing factor (OIF) present in llama seminal plasma

BackgroundThe purpose of the study was to determine if the effect of llama OIF on LH secretion is mediated by stimulation of the hypothalamus or pituitary gland.MethodsUsing a 2-by-2 factorial design to examine the effects of OIF vs GnRH with or without a GnRH antagonist, llamas with a growing ovarian follicle greater than or equal to 8 mm were assigned randomly to four groups (n = 7 per group) and a) pre-treated with 1.5 mg of GnRH antagonist (cetrorelix acetate) followed by 1 mg of purified llama OIF, b) pre-treated with 1.5 mg of cetrorelix followed by 50 micrograms of GnRH, c) pre-treated with a placebo (2 ml of saline) followed by 1 mg of purified llama OIF or d) pre-treated with a placebo (2 ml of saline) followed by 50 micrograms of GnRH. Pre-treatment with cetrorelix or saline was given as a single slow intravenous dose 2 hours before intramuscular administration of either GnRH or OIF. Blood samples for LH measurement were taken every 15 minutes from 1.5 hours before to 8 hours after treatment. The ovaries were examined by ultrasonography to detect ovulation and CL formation. Blood samples for progesterone measurement were taken every-other-day from Day 0 (day of treatment) to Day 16.ResultsOvulation rate was not different (P = 0.89) between placebo+GnRH (86%) and placebo+OIF groups (100%); however, no ovulations were detected in llamas pre-treated with cetrorelix. Plasma LH concentrations surged (P < 0.01) after treatment in both placebo+OIF and placebo+GnRH groups, but not in the cetrorelix groups. Maximum plasma LH concentrations and CL diameter profiles did not differ between the placebo-treated groups, but plasma progesterone concentrations were higher (P < 0.05), on days 6, 8 and 12 after treatment, in the OIF- vs GnRH-treated group.ConclusionCetrorelix (GnRH antagonist) inhibited the preovulatory LH surge induced by OIF in llamas suggesting that LH secretion is modulated by a direct or indirect effect of OIF on GnRH neurons in the hypothalamus.

224 THE EFFECT OF TREATMENT WITH VARIOUS DOSES OF A SYNTHETIC GONADOTROPIN-RELEASING HORMONE ON INDUCTION OF OVULATION IN LLAMAS

Llamas require a copulation stimulus to elicit an LH surge that subsequently leads to induced ovulation. Gonadotropin-releasing hormone has been used to induce ovulation in llamas; however, results have been variable because of the different analogues and doses in previous studies (Bravo PW et al. 1992 Biol. Reprod. 47, 884-888; Ratto M et al. 2006 Anim. Reprod. Sci. 91, 299-306). Additionally, the effect of different doses and types of GnRH analogues on CL formation and function has not been described in detail. The purpose of the present study was to conduct a dose response trial to examine the effect of a synthetic analogue of GnRH, gonadorelin acetate (Ovalyse®, Pfizer, Santiago, Chile) on ovulation and CL formation in llamas. Nonpregnant and nonlactating adult llamas (range 4-8 years) weighing 90 to 130 kg were examined once daily by transrectal ultrasonography using a B mode scanner with a 7.5-MHz linear-array transducer (Aloka® SSD 500, International Clinics, Santiago, Chile). Llamas with a follicle ≥8 mm in diameter that had grown for 3 consecutive days were assigned randomly to be given an i.m. administration of gonadorelin at a dose of 1) 50 μg (n = 5); 2) 25 μg (n = 8); 3) 12.5 μg (n = 9); 4) 6.25 μg (n = 8), or 5) i.m. administration of 0.5 mL of PBS as a negative control group (n = 5). Llamas were examined by ultrasonography every 12 h from treatment (Day 0) to ovulation and thereafter on Day 8 to determine CL formation. Ovulation was defined as the sudden disappearance of a large follicle ≥8 mm that was detected during the previous examination and was confirmed by subsequent CL formation on Day 8. Ovulation rate was analyzed by chi-square analysis. Follicle diameter (at the time of treatment), interval from treatment to ovulation, and CL diameter on Day 8 were analyzed using one-way ANOVA. Mean ± SD diameter of preovulatory follicle did not differ (P = 0.4) among groups (11 ± 1.6, 10.6 ± 1.5, 11.5 ± 1.9, 11.7 ± 1.9, and 9.8 ± 2.9 mm for 50, 25, 12.5, and 6.25 μg of gonadorelin and 0.5 mL PBS, respectively). The proportions of ovulated llamas did not differ (P = 0.1) among GnRH-treated groups (100, 62.5, 77.7, and 62.5% for 50, 25, 12.5, and 6.25 μg of gonadorelin, respectively) but they did differ (P &lt; 0.01) from the control group (0%). Also, the interval from treatment to ovulation in those ovulated females ranged from 36 to 48 h and it did not differ (P = 0.6) among groups. Corpus luteum diameter on Day 8 (12.2 ± 1.8, 10.2 ± 1.1, 11.7 ± 2.2, and 11.3 ± 1.8 for 50, 25, 12.5, and 6.25 μg of gonadorelin, respectively) did not differ (P = 0.3) among groups. In conclusion, the minimal dose of gonadorelin used in this study (1/8 dose) was as equally effective as the highest dose to induce ovulation in llamas without compromising CL formation. The study was supported by Proyecto FONDECYT 11080141, Convenio de Desempeño en Investigacion (2007 DGI-CDA-04), Universidad Catolica de Temuco, and College of Graduate Studies, Faculty of Veterinary Sciences, Universidad Austral de Chile.

297 EFFECT OF GONADOTROPIN-RELEASING HORMONE COMPARED WITH ESTRADIOL-17β AT THE BEGINNING OF A SUPERSTIMULATION PROTOCOL ON SUPEROVULATORY RESPONSE AND EMBRYO QUALITY

Success of a superstimulation protocol is largely dependent on avoiding the effects of the dominant follicle. The objective of this study was to determine if gonadotropin-releasing hormone (GnRH) would serve as a suitable alternative to the traditional estradiol-17β in a superstimulation protocol when using a CIDR. Dairy cattle (n = 411) were superstimulated using one of two treatment protocols. The GnRH-treated animals received a CIDR on random days of the estrous cycle (day 0), and GnRH was injected i.m. (100 micrograms of Cystorelin) on day 3. Porcine FSH (Folltropin-V) was administered twice daily in decreasing doses on days 5 to 8, and on day 8, CIDR were removed and 25 mg of dinoprost (PGF2α) was administered twice. The Estradiol-treated animals received a CIDR and 4 mg estradiol-17β i.m. on random days of the estrous cycle (day 0). Porcine FSH was administered twice daily in decreasing doses on days 4 to 7, and on day 7, CIDR were removed and 25 mg of PGF2α was administered twice. All animals were artificially inseminated at standing estrus and at 6-h intervals during estrus. Animals were inseminated a minimum of twice, and those with extended estrous periods were inseminated three times. Animals not showing estrus were inseminated once 48 h after CIDR removal. Seven days following estrus, embryos were collected. For data analysis, animals were divided into lactating cows, dry cows, and heifers. Dosage of FSH for each animal varied according to previous response rates; therefore, animals were further subclassified as high FSH (≥300 mg in 15 mL) or low FSH (&lt;300 mg in 15 mL). As there were no heifers on high FSH, data analysis was done as a 5 � 2 factorial ANOVA. Total number of ova/embryos, quality, development, and fertilization rates were recorded. Analysis of data showed no statistical differences (P &gt; 0.05) between Estradiol- and GnRH-treated groups for number ova/embryos recovered (9.8 � 0.58 v. 9.7 � 0.65), IETS-grade 1 and 2 embryos (4.7 � 0.37 v. 4.5 � 0.41), percentage fertilized (54.1 � 2.5 v. 53.2 � 2.9), and percentage grade 1 and 2 embryos (49.2 � 2.5 v. 49.3 � 2.8), respectively. No significant interactions were found between treatment and lactation status or treatment and FSH dose. Data suggest that GnRH in combination with a CiDR is an effective means of synchronizing follicle wave emergence in a superstimulation protocol. George Seidel, Josh Walker, John Hassler, and Sam Galphin.

Conception rates and serum progesterone concentration in dairy cattle administered gonadotropin releasing hormone 5 days after artificial insemination

The objectives of this study were to determine the effect of administration of exogenous GnRH 5 days after artificial insemination (AI) on ovarian structures, serum progesterone concentration, and conception rates in lactating dairy cows. In experiment 1, 23 Holstein cows were synchronized using the Ovsynch protocol. Five days after AI (day 0) cows were assigned randomly to receive either saline (saline; n = 11) or 100 μg GnRH (GnRH; n = 12). To examine ovarian structures, ultrasonography was performed on day 1 and every other day beginning on day 5 until day 13. On days 5 and 13 blood samples were obtained to measure serum progesterone concentrations. All cows in the GnRH-treated group developed an accessory corpus luteum (CL), whereas cows in the saline group did not. Mean serum progesterone concentrations did not differ between GnRH and saline groups on day 5 (1.64 ± 0.46 ng/ml versus 2.04 ± 0.48 ng/ml). On day 13 serum progesterone concentrations were greater ( P < 0.05) in the GnRH group compared with saline (5.22 ± 0.46 ng/ml versus 3.36 ± 0.48 ng/ml). In experiment 2, 542 lactating cows, at two different commercial dairies, were used to test the effect of administering GnRH 5 days after AI on conception rates. Cows were synchronized and detected for estrus according to tail chalk removal. Cows detected in estrus received AI within 1 h after detection of estrus. Five days after AI, cows were assigned randomly to receive either GnRH ( n = 266) or saline ( n = 276). Pregnancy status was determined by palpation per rectum of uterine contents approximately 40 days after AI. There was no effect of farm on conception rate. There was no effect of treatment as conception rates did not differ between GnRH and saline groups (26.7% GnRH versus 24.3% saline). Regardless of treatment, days in milk, parity, milk yield, and number of services had no effect on the odds ratio of pregnancy. In summary, the results of this study indicated that GnRH administered 5 days after AI increased serum progesterone by developing an accessory CL but did not improve conception rates in dairy cattle.

Acidic mix of FSH isoforms are better facilitators of ovarian follicular maturation and E2 production than the less acidic.

FSH is secreted as a mix of isoforms with varying biologic attributes. To determine the functional significance of FSH heterogeneity, an acidic (ovine pituitary FSH; C-FSH) and less acidic mix (C-FSH exposed to neuraminidase; N-FSH) were administered to prepubertal lambs. Production of GnRH- induced less acidic FSH was blocked with a competitive GnRH receptor antagonist, Nal-Glu. Beginning 24 h after Nal-Glu, lambs were injected with C-FSH or N-FSH and LH. Controls included untreated, GnRH-treated, and Nal-Glu-treated groups. Blood samples were obtained at 2-h intervals. Plasma FSH levels were similar before treatment and increased over time in the C-FSH but not the N-FSH group (P < 0.001). Three of the six GnRH-treated ewes exhibited an LH surge. Peak E2 concentrations in the GnRH-treated animals were achieved 30-36 h after initiation of treatment. Peak circulating E2 levels tended to be higher in the C-FSH than in the GnRH-treated group. Only two of six N-FSH-treated ewes had a serum E2 rise. The C-FSH ewes had more estrogenic follicles than the GnRH and N-FSH groups (P < 0.05). Our findings show that C-FSH clears more slowly than N-FSH, and C-FSH is a better facilitator of follicular development and maturation than N-FSH.

Effect of LH or GnRH on the dominant follicle of the first follicular wave in beef heifers.

A study was designed to characterise ovarian follicular dynamics in heifers treated with porcine luteinizing hormone (pLH) or gonadotropin releasing hormone (GnRH) on days 3, 6 or 9 (ovulation = day 0), corresponding to the growing, early-static, and late-static phases of the first follicular wave. Following ovulation, 65 beef heifers were assigned, by replicate, to the following seven treatment groups: 25 mg im of pLH on days 3, 6 or 9 (n = 9 per group); 100 microg im of GnRH on days 3, 6 or 9 (n = 9 per group); or controls (no treatment; n = 11). Ovulation occurred within 36 h in 67%, 100% and 67% of heifers treated with pLH and in 89%, 56% and 22% of heifers treated with GnRH on days 3, 6 or 9, respectively (treatment-by-day interaction, P < 0.09). Combined for all treatment days, ovulation rates were 78% and 56% in pLH- and GnRH-treated groups, respectively (P < 0.09). Overall, mean day (+/- SD) of emergence of the second follicular wave in heifers that ovulated was different from that in controls or in heifers that did not ovulate (P < 0.05). Mean (+/- SD) day of emergence of the second wave occurred earlier (day 5.6+/-1.2; P < 0.05) in heifers that ovulated after treatment on day 3 (n = 14) than in controls (day 8.7+/-1.6; n = 11); however, wave emergence in all heifers treated on day 6 (day 8.1+/-0.5; n = 18) did not differ from controls, regardless of whether or not ovulation occurred. In the heifers that ovulated in response to treatment on day 9 (n = 8), the emergence of the second follicular wave was delayed (day 10.9+/-0.4; P < 0.05). The day of emergence of the second wave in the 14 treated heifers that failed to ovulate, irrespective of the day of treatment (day 8.9+/-1.4) did not differ from control heifers. The emergence of the second wave was more synchronous in day 6 heifers (regardless of whether they ovulated) and in day 9 heifers that ovulated compared to control heifers (P < 0.05). Results did not support the hypothesis that the administration of pLH or GnRH at known stages of the follicular wave in cycling heifers would consistently induce ovulation or atresia and, thereby, induce emergence of a new follicular wave at a predictable interval. New wave emergence was induced consistently (1.3 days post-treatment) only in those animals that ovulated in response to treatment. However, 22% of LH-treated heifers and 44% of GnRH-treated heifers failed to ovulate. Treatments did not induce atresia of the dominant follicle or alter the interval to new wave emergence in animals that did not ovulate in response to treatment.

Gonadotropin-releasing hormone increases CD4 + T-lymphocyte numbers in an animal model of immunodeficiency

Background: Gonadotropin-releasing hormone (GnRH) possesses immunostimulatory properties. We have previously demonstrated that GnRH antagonists decrease lymphocyte numbers in an animal model of autoimmune disease. We speculated that the converse might be true, that GnRH administration would increase lymphocyte numbers or alter lymphocyte subsets in an immunodeficiency state. Objective: Our purpose was to test the hypothesis that GnRH agonist would increase IgG and CD4 counts in a rat model of immunodeficiency independently of gonadal steroids. Methods: We used diabetes-prone (DP) BB rats. This model has been characterized to have an AIDS-like lymphocyte profile, with lymphopenia and depressed CD4 counts. Ovariectomized female DP rats were randomized to receive subcutaneous injections with GnRH or vehicle 6 times weekly. DR rats were ovariectomized and treated with vehicle as controls. We performed flow cytometric analysis and complete blood cell counts at baseline, 3.5 weeks, and 7 weeks of treatment. We also measured total serum IgG and luteinizing hormone levels. Results: GnRH administration significantly increased total serum IgG levels in DP rats compared with vehicle. The percentages of CD4 + cells in blood were also significantly increased in the GnRH-treated group compared with the vehicle-treated group and compared with baseline. Similarly, the absolute numbers of CD4 + positive T cells were increased over controls at 7 weeks. The effects of GnRH were specific for the CD4 subset because there were no significant differences in numbers of CD8 + positive cells between the 2 treatment groups. Conclusion: GnRH shows potential utility as an immunostimulatory agent in immunodeficient states manifesting diminished numbers of immunocompetent CD4 + T lymphocytes. (J Allergy Clin Immunol 1999;104:653-8.)

Alteration of follicular dynamics and superovulatory responses by gonadotropin releasing hormone and follicular puncture in cattle: A field trial

A field experiment was conducted to determine the influence of follicular alteration on superovulatory responses. Ultrasonography was performed once daily over 4 d prior to gonadotropin treatment (Day 0), on the day of estrus during superstimulation, and on the day of embryo collection to monitor follicular development. Animals were superstimulated between Days 8 and 12 of the estrous cycle. Follicular status was altered 2 d prior to initiation of superstimulation (Day 0) with GnRH (Cystorelin, 200 μg im) administered with (GnRHpuncture group, n=31) or without (GnRH-no puncture group, n=52) concomitant removal of the largest follicle by follicular aspiration. Responses were compared with those of an untreated control group superovulated 8 to 12 d after estrus (n=102). The proportion of animals with a high number (≥ 2) of large follicles (≥ 7 mm) on Day 0 was lower (P < 0.001) in the 2 GnRHtreated groups than in the control group, while the increase in the number of medium size follicles (4 to 6 mm) on Day 0 was greater (P < 0.02) in the GnRH-puncture group. During superstimulation, the proportion of superovulatory cycles with a high follicular (≥ 10 follicles) response was similar in the control and GnRH-no puncture groups. Within the GnRH-treated animals, follicular and ovulatory responses were greater in the GnRH-puncture than in the GnRH-no puncture group (P < 0.001 to P < 0.02). Despite these changes in follicular and ovulatory responses, however, the mean number of embryos produced did not differ (P > 0.1) among treatments (4.3 ± 0.4, 3.7 ± 0.7, and 5.4 ± 0.8 in control, GnRH-no puncture, and GnRHpuncture groups, respectively). This was due primarily to an increase in the mean numbers of unfertilized ova (P < 0.005) and in degenerated embryos (P < 0.06) in the GnRH-puncture group. Results indicate that the beneficial effects of treatment with GnRH and follicular puncture 2 d prior to superstimulation on follicular and ovulatory responses were limited by an increase in the number of unfertilized ova and degenerated embryos.

Comparison of two dosage regimens of the GNRH agonist deslorelin acetate on inducing ovulation in seasonally anestrous mares

This study investigated the efficacy of two dosage regimens of a potent GnRH analogue (GnRHa), deslorelin acetate, in inducing ovulation in seasonally anestrous mares. Forty-five seasonally anestrous mares were randomly assigned according to follicular size to one of three treatment groups: control, increasing GnRHa dose, and constant GnRHa dose. Treatment began on February 28 and continued until ovulation or for a maximum of seven treatments. Mares were palpated every other day until a 35 mm follicle was detected, then every day until ovulation or regression of the follicle occurred. Blood samples were taken from five randomly chosen mares in each treatment group and analyzed for LH levels. Twenty percent of mares in both deslorelin treatment groups ovulated, while no control mares ovulated during the treatment period. There was no difference in the number of mares that ovulated between treatment groups. Four of the six mares that ovulated were in transitional anestrus at the initiation of treatment, while only two were in deep anestrus. Concentrations of LH were greater (p=0.0008) in both GnRH-treated groups than in the control mares. Concentrations of LH did not differ between the two GnRH-treated groups until day 12 of treatment, when mares treated with a constant dosage had higher (p=0.0358) levels of LH than those treated with an increasing dosage. It is possible that administration of larger amounts of the GnRH agonist lowered the sensitivity of the pituitary to stimulation by GnRH. Deslorelin acetate did stimulate follicular growth and ovulation in a limited number of anestrous mares. Further investigation into the potential of this short-term implant to shorten the onsent of the breeding season is recommended.

Estradiol and gonadotropin-releasing hormone (GnRH) interact to increase GnRH receptor expression in ovariectomized ewes after hypothalamic-pituitary disconnection.

Gonadotropin-releasing hormone (GnRH) receptor expression is regulated by estradiol and GnRH itself. The objective of this experiment was to determine the extent to which low levels of estradiol, similar to those observed during the transition from the luteal to the follicular phase of the estrous cycle, and GnRH interact to regulate expression of GnRH receptors and GnRH receptor mRNA. Ewes were ovariectomized (OVX) at least 2 wk prior to initiation of the experiment, and the pituitary gland was surgically disconnected from the hypothalamus to remove ovarian and hypothalamic inputs to the pituitary. Within 24 h after hypothalamic-pituitary disconnection, ewes received pulses of GnRH (250 ng/pulse) every 2 h for 6 d. At the end of 6 d, ewes were randomly assigned to treatments in a 2 x 2 factorial arrangement as follows: half of the animals received a single estradiol implant and half received an empty implant (placebo). At the same time, animals also received one of the following treatments: (1) saline or (2) GnRH (100 ng/pulse/2 h). Additionally, one group of ewes was ovariectomized, but not subjected to hypothalamic-pituitary disconnection (OVX controls). Blood samples were collected 15 min prior to each pulse of GnRH or saline and at 15-min intervals for 1 h after each pulse until tissues were collected and concentrations of luteinizing hormone (LH) were determined. Anterior pituitaries were collected 24 h after implant insertion to quantitate steady-state amounts of GnRH receptor mRNA and numbers of GnRH receptors. Mean LH was greatest in ovariectomized control ewes compared to all other treatments (p < 0.05). Mean LH and LH pulse amplitude in the placebo and GnRH-treated group most closely mimicked LH secretion in ovariectomized control animals. Mean LH and LH pulse amplitude were similar between both GnRH-treated groups (p < 0.05). Mean LH and LH pulse amplitude were significantly lower in all animals treated with saline compared to OVX controls (p < 0.05). Treatment with an estradiol implant and pulsatile GnRH increased (p < 0.05) relative amounts of GnRH receptor mRNA and the number of GnRH receptors compared to all other treatments. There were no differences in GnRH receptor expression between the remaining treatment groups (p > 0.05). Therefore, in OVX ewes after hypothalamic-pituitary disconnection, low levels of estradiol and GnRH are required to increase GnRH receptor mRNA and GnRH receptor numbers. Since we only observed an increase in GnRH receptor expression in the presence of both estradiol and GnRH, we conclude that there is a synergistic interaction between these two hormones in the regulation of GnRH receptor expression.

Dissociation of increases in plasma insulin-like growth factor I and testosterone during the onset of puberty in bulls.

The present study was conducted to examine the relationship between plasma concentrations of testosterone, insulin-like growth factor I (IGF-I) and IGF-binding proteins (IGFBPs) during puberty, in male calves treated with GnRH or testosterone propionate. Twelve male Holstein calves (10 weeks old) were assigned to the control group (n = 6), the GnRH-treated group (n = 3) or the testosterone-treated group (n = 3). For 8 weeks, the GnRH-treated group received a single i.v. injection of GnRH (0.5 microgram kg-1 body mass) each day while the testosterone-treated group received an i.m. injection of testosterone propionate (0.5 mg kg-1 body mass) twice a day. The calves were studied until they were 200 days old. Hormone treatments were stopped one month after puberty was reached in the control group. Blood samples were collected every 30 min for 8 h every third day. Hormone concentrations were determined by radioimmunoassay. Western ligand blotting and immunoblotting, using monoclonal antibodies against IGFBP-2 and IGFBP-3, were used to characterize the IGF-binding proteins. In the control group, puberty occurred at about 120 days of age and was associated with an increase in concentrations of testosterone, IGF-I and IGFBP-3 and a decrease in concentration of IGFBP-2. In the GnRH-treated group, plasma testosterone remained low until 8 weeks after establishment of puberty in the control group (4 weeks after the end of treatment). In the testosterone-treated group, testosterone was high during the treatment period and then decreased to prepubertal values when treatment was stopped. Testosterone values increased again to reach postpubertal values 5 weeks after the end of hormone treatment. Nevertheless, independent of testosterone status, the profile of IGF-I and the IGFBPs in the GnRH- and testosterone-treated groups were parallel to that reported for the control group with the transition from prepubertal to adult values at about 120 days of age. In conclusion, concentrations of testosterone, IGF-I and IGFBP-3 increase together, but probably independently, during the onset of puberty in male calves.

Evidence that the switch from negative to positive feedback at the level of the pituitary gland is an important timing event for the onset of the preovulatory surge in LH in the ewe.

Experiments were performed to test the hypothesis that there is a negative feedback 'clamp' of ovarian hormones on the hypothalamus and pituitary gland during the follicular phase of the oestrous cycle that limits the secretion of GnRH and LH. GnRH secretion was monitored by sampling the hypophysial portal blood of ewes during the luteal phase of the oestrous cycle and either 24 h or 48 h after the induction of luteolysis by the injection of cloprostenol, a prostaglandin analogue. There was an increase in GnRH pulse frequency in the transition from the luteal to the follicular phase of the cycle. A reduction in the amplitude of GnRH pulses did not occur until 48 h after cloprostenol, suggestive of negative feedback at the level of the hypothalamus that is more profound in the latter part of the follicular phase. The responsivity of the pituitary gland to GnRH was monitored in ewes during the luteal phase of the oestrous cycle and 24 h or 48 h after cloprostenol. Injections of 250 ng or 1000 ng GnRH were given (i.v.) to ewes that had been anaesthetised to suppress endogenous secretion of GnRH and LH. Using the lower dose, the responses 48 h after cloprostenol were not significantly different from those in the luteal phase. With the higher dose of GnRH, a significant (P < 0.05) increase in mean responsivity was seen 48 h after cloprostenol. There was, however, a marked variation in response, with some ewes showing profound increases in LH secretion in response to GnRH and others showing responses that were similar to those obtained during the luteal phase of the cycle. These data are interpreted to mean that the secretion of LH is 'clamped' during the follicular phase of the oestrous cycle and the 'clamp' is only released near the time of the preovulatory LH surge. To test whether or not a rise in GnRH input to the pituitary gland could over-ride the 'clamp' on the pituitary secretion of LH in the late follicular phase of the cycle, sheep were treated 40 h after cloprostenol with either a bolus injection of 500 ng GnRH or four pulses of 125 ng GnRH given at 10-min intervals. These treatments caused small elevations in LH secretion but did not always cause preovulatory LH surges. In some cases, a small rise in LH secretion was induced by GnRH treatments and levels of LH in plasma returned to baseline with the preovulatory LH surge occurring a few hours later.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic GnRH administration in prepubertal male pigs

The effect of chronic pulsatile low-dose GnRH treatment on the juvenile testis and associated structures was evaluated in relation to hormonal parameters in the peripheral blood in the pig. Starting at 8 weeks of age, male pigs (crossbreds of Dutch Landrace and Yorkshire breeds) were injected 6 times daily im with 0, 75 or 250 ng GnRH/kg body weight during 4 weeks. Immediately after the treatment period, a GnRH stimulation test with 750 ng GnRH/kg iv was carried out. Samples for plasma LH, FSH, testosterone and 5 alpha DHT measurement were obtained weekly (basal level) and after GnRH stimulation. The pigs were castrated at 12 weeks of age and the weights and lengths of the testis, epididymis and cremaster muscle were recorded. Intratesticular testosterone and 5 alpha DHT concentrations were determined, and the testis and epididymis were evaluated for histological changes. Basal plasma hormone concentrations, intratesticular androgen concentrations and the response of the pituitary gland to stimulation had not been affected by GnRH treatment. Pigs receiving the higher treatment dose of GnRH showed less increase in testosterone levels in response to the stimulation dose at 12 weeks of age than the other pigs. Morphologically, no changes were observed in the epididymis and cremaster muscle after GnRH treatment and no signs of reactivation of structures that can provoke testicular descent could be seen. The development of the seminiferous epithelium was more advanced in the GnRH-treated groups, apparently in a dose-dependent manner.

Pulsatile administration of gonadotropin-releasing hormone advances ovulation in cycling mares.

Cycling standardbred mares were infused with saline or 20 micrograms gonadotropin-releasing hormone (GnRH) in a pulsatile pattern (one 5-sec pulse/h, 2 h or 4 h) beginning on Day 16 of the estrous cycle. Although serum concentrations of luteinizing hormone (LH) increased significantly earlier in all three GnRH-treated groups (within one day of the initiation of infusion) compared to saline-infused controls, there were no differences in peak periovulatory LH concentrations among treatments (overall mean +/- SEM, 8.98 +/- 0.55 ng/ml). The number of days from the start of treatment to ovulation was significantly less in mares infused with 20 micrograms GnRH/h (mean +/- SEM, 2.9 +/- 0.6 days after the initiation of treatment, or 18.9 days from the previous ovulation; N = 7) compared to mares treated with saline (5.9 +/- 0.3 days, or 21.9 days from previous ovulation; N = 7) or 20 micrograms GnRH per 4 h (5.4 +/- 0.9 days or 21.4 days from previous ovulation; N = 5). Although mares infused with 20 micrograms GnRH/2 h ovulated after 4.3 +/- 0.7 days of treatment (Day 20.3; N = 7), this was not significantly different from either the control or 20 micrograms GnRH/h treatment groups. Neither the duration of the resulting luteal phase nor the length of the estrous cycle was different between any of the treatment groups (combined means, 14.7 +/- 0.2 days and 21.3 +/- 0.4 days, respectively). We conclude that pulsatile infusion of GnRH is effective in advancing the time of ovulation in cycling mares, but that the frequency of pulse infusion is a critical variable.