Use of GnRH agonist implants for long-term suppression of fertility in extensively managed heifers and cows

The ability of gonadotrophin releasing hormone (GnRH) agonist implants to suppress ovarian activity and prevent pregnancies, long-term, was examined in heifers and cows maintained under extensive management. At three cattle stations, heifers (2-year-old) and older cows (3- to 16-year-old) were assigned to a control group that received no treatment, or were treated with high-dose (12 mg, Station A) or low-dose (8 mg, Station B and Station Q GnRH agonist implants. The respective numbers of control and GnRH agonist-treated animals (heifers + cows) at each station were: Station A, 20 and 99; Station B, 19 and 89; Station C, 20 and 76. Animals were maintained with 4% bulls and monitored for pregnancy at 2-monthly intervals for approximately 12 months. Pregnancy rates for control heifers and control cows ranged from 60-90% and 80-100%, respectively, depending on the study site. The respective number of animals (heifers + cows) treated with GnRH agonist that conceived, and days to first conception, were: Station A, 9 (9%) and 336 3 days; Station B, 8 (10%) and 244 +/- 13 days; Station C, 20 (26%) and 231 +/- 3 days. Treatment with high-dose GnRH agonist prevented pregnancies for longer (similar to300 days) than treatment with low-dose GnRH agonist (similar to200 days). In the majority of heifers and cows treated with GnRH agonist, ovarian follicular growth was restricted to early antral follicles (2-4 mm). The findings indicate that GnRH agonist implants have considerable potential as a practical technology to suppress ovarian activity and control reproduction in female cattle maintained in extensive rangelands environments. The technology also has broader applications in diverse cattle production systems. (C) 2002 Elsevier Science B.V. All rights reserved.

Control of ovulation with a GnRH agonist after superstimulation of follicular growth in buffalo: fertilization and embryo recovery

The potential to use a GnRH agonist bioimplant and injection of exogenous LH to control the time of ovulation in a multiple ovulation and embryo transfer (MOET) protocol was examined in buffalo. Mixed-parity buffalo (Bubalus bubalis; 4-15-year-old; 529 13 kg LW) were randomly assigned to one of five groups (n = 6): Group 1, conventional MOET protocol; Group 2, conventional MOET with 12 It delay in injection of PGF(2alpha); Group 3, implanted with GnRH agonist to block the pre-ovulatory surge release of LH; Group 4, implanted with GnRH agonist and injected with exogenous LH (Lutropin(R), 25 mg) 24 h after 4 days of superstimulation with FSH; Group 5, implanted with GnRH agonist and injected with LH 36 h after superstimulation with FSH. Ovarian follicular growth in all buffaloes was stimulated by treatment with FSH (Folltropin-V(R), 200 mg) administered over 4 days, and was monitored by ovarian ultrasonography. At the time of estrus, the number of follicles greater than or equal to8 mm. was greater (P < 0.05) for buffaloes in Group 2 (12.8) than for buffaloes in Groups 1 (8.5), 3 (7.3), 4 (6.1) and 5 (6.8), which did not differ. All buffaloes were mated by AI after spontaneous (Groups 1-3) or induced (Groups 4 and 5) ovulation. The respective number of buffalo that ovulated, number of corpora lutea, ovulation rate (%), and embryos + oocytes recovered were: Group 1 (2, 1.8 +/- 1.6, 18.0 +/- 13.6, 0.2 +/- 0.2); Group 2 (4, 6.1 +/- 2.9, 40.5 +/- 17.5, 3.7 +/- 2.1); Group 3 (0, 0, 0, 0); Group 4 (6, 4.3 +/- 1.2, 69.3 +/- 14.2, 2.0 +/- 0.9); and Group 5 (1, 2.5 +/- 2.5, 15.5 +/- 15.5, 2.1 +/- 2.1). All buffaloes in Group 4 ovulated after injection of LH and had a relatively high ovulation rate (69%) and embryo recovery (46%). It has been shown that the GnRH agonist-LH protocol can be used to improve the efficiency of MOET in buffalo. (C) 2002 Elsevier Science Inc. All rights reserved.

The stability of lipidic analogues of GnRH in plasma and kidney preparations: the stereoselective release of the parent peptide

The conjugation of a lipoamino acid to the N-terminus of Gonadotropin releasing hormone (GnRH) produces a lipophilic peptide from which the parent GnRH peptide is released into solution on treatment with plasma and kidney enzyme preparation. Our findings show that one stereoisomer of the Laa is cleaved very rapidly, providing a bolus dose of the peptide while the opposite stereoisomer is cleaved much more slowly, providing prolonged elevation of peptide concentration. The Laa-Glu linkage appears to act as a two phase prodrug system. © 2005 Elsevier Ltd. All rights reserved.

Use of a GnRH agonist and hCG to obtain an index of testosterone secretory capacity in the koala (Phascolarctos cinereus)

Testosterone secretion in mammals typically occurs in random pulses such that a single blood sample provides limited information on reproductive endocrine status. However, it has been shown in several species that an index of the prevailing testosterone biosynthetic capacity of the testes can be obtained by measuring the increase in circulating testosterone after injection of a GnRH agonist or human chorionic gonadotrophin (hCG). Hence, the aims of the present study were to examine fluctuations in testosterone secretion in the koala (n = 6) over a 24-hour period and then characterise testosterone secretion after injection of the GnRH agonist buserelin (4 mu g) or hCG (1000 IU). The latter was used to establish an index of the prevailing testosterone biosynthetic capacity of the koala testis. Individual koalas showed major changes in blood testosterone concentrations over 24 hours, but there was no apparent diurnal pattern of testosterone secretion (P >.05). Injection of buserelin and hCG resulted in an increase (P

Gonadotrophin and prolactin secretion in castrated male sheep following subcutaneous or intracranial treatment with testicular hormones

Interactions between testosterone, estradiol, and inhibin in the control of gonadotrophin secretion in males are poorly understood. Castrated rams were treated with steroid-free bovine follicular fluid (bFF), testosterone, or estradiol and for 7 d (2 x 2 x 2 factorial design). Given independently, none of the exogenous hormones affected follicle-stimulating hormone (FSH) concentrations, but the combination of one or both steroids with bFF reduced FSH secretion. Testosterone and estradiol reduced luteinizing hormone (LH) pulse frequency (there was no synergism), and bFF had no effect. Plasma prolactin concentrations were not affected by any treatment. To locate the central sites of steroid action, castrated rams were bilaterally implanted in the preoptic area (POA), ventromedial nucleus (VMH), or arcuate nucleus (ARC). These implants did not affect FSH or prolactin concentrations, or LH pulse amplitude. The frequency of the LH pulses was not affected by testosterone in any site. Estradiol located in the ARC, but not the POA or VMH, decreased LH pulse frequency. In summary, FSH secretion is controlled by synergistic interactions between inhibin and estradiol or testosterone, whereas GnRH/LH pulse frequency is controlled by testicular steroids. Estradiol acts partly, at least, in the ARC, but the central site of action, testosterone remains unknown.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 mu mol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l-1 mu mol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 mu mol/l; P<0.05), PACAP also stimulated FSH secretion in cells from some animals although this effect was small, The GH response to PACAP was inhibited by PACAP(6-38), a putative PACAP antagonist; but not by (N-Ac-Tyr(1), D-Arg(2))-GHRH(1-29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1-24 h, VIP (1 mu mol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l-1 mu mol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l-1 mu mol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 mu mol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.

A method for drug infusion into the lateral median eminence and arcuate nucleus of sheep

The role of catecholamines in the control of the GnRH pulse generator is unclear as studies have relied on the use of peripheral or intracerebroventricular injections, which lack specificity in relation to the anatomical site of action. Direct brain site infusions have been used, however, these are limited by the ability to accurately target small brain regions. One such area of interest in the control of GnRH is the median eminence and arcuate nucleus within the medial basal hypothalamus. Here we describe a method of stereotaxically targeting this area in a large animal (sheep) and an infusion system to deliver drugs into unrestrained conscious animals. To test our technique we infused the dopamine agonist, quinpirole or vehicle into the medial basal hypothalamus of ovariectomised ewes. Quinpirole significantly suppressed LH pulsatility only in animals with injectors located close to the lateral median eminence. This in vivo result supports the hypothesis that dopamine inhibits GnRH secretion by presynaptic inhibition in the lateral median eminence. Also infusion of quinpirole into the medial basal hypothalamus suppressed prolactin secretion providing in vivo evidence that is consistent with the hypothesis that there are stimulatory autoreceptors on tubero-infundibular dopamine neurons. (C) 1997 Elsevier Science B.V.

Unusual ovarian activity in a mare preceding the development of an ovarian granulosa cell tumour

An 8-year-old mare, with a foal at foot, was inseminated on foal heat with frozen semen, with the resultant pregnancy lost between days 34 and 41. The right ovary developed a large anovulatory follicle that was non-responsive to multiple doses of ovulating agents. The follicle eventually appeared to luteinise, although plasma progesterone concentrations did not reflect this. Another follicle developed, responded to GnRH and resulted in a pregnancy from frozen semen that went to term with a healthy foal. When the mare was examined after foaling, the structure on the right ovary appeared to be a granulosa cell tumour; the left ovary was smaller than normal and non-functional. Surgical removal of the right ovary before increasing photoperiod resulted in a return to function of the left ovary and a pregnancy to frozen semen on the second cycle following removal. Figures showing concentrations of inhibin, progesterone, androstenedione, oestradiol and testosterone are presented for this entire period. Unusual ovarian activity in the mare might be a prelude to the development of a granulosa cell tumour.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R,) that confers agonist selectivity for Are in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L-296, N-297, and R-298. A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

Embryonic mortality in buffaloes synchronized and mated by AI during the seasonal decline in reproductive function

The aim was to determine the factors that contribute to embryonic mortality in buffaloes mated by AI during a period of increasing day length which corresponds to a natural decline in reproductive activity. Italian Mediterranean buffalo cows (n = 243) showing regular estrous cycles were synchronized using the Ovsynch-TAI program and mated by AI at 16 and 40 h after the second injection of GnRH. Blood samples were collected on Days 10 and 20 after the first AI and assayed for progesterone (P-4). Pregnancy diagnosis was undertaken on Days 26 and 40 after the first AI using rectal ultrasonography. Buffaloes with a conceptus on Day 26 but not on Day 40 were judged to have undergone embryonic mortality and for these animals uterine fluid was recovered by flushing and analysed for common infectious agents. Estrus synchronization was achieved in 86% of buffaloes and the pregnancy rate on Day 40 was 34%. Embryonic mortality between Days 26 and 40 occurred in 45% of buffaloes and was associated with the presence of significant infectious agents in only 10 buffaloes (8%). Concentrations of P-4 on Day 10 after AI were higher (P < 0.05) in buffaloes that established a pregnancy than in buffaloes that showed embryonic mortality that was not associated with infectious agents. Similarly, on Day 20 after AI P-4 concentrations were higher (P < 0.01) in pregnant buffaloes compared with non-pregnant buffaloes and buffaloes that had embryonic mortality. It is concluded that a reduced capacity for P-4 secretion can explain around 50% of embryonic mortalities in buffaloes synchronised and mated by AI during a period of low reproductive activity and that other as yet unidentified factors also have a significant effect on embryonic survival. (c) 2004 Elsevier Inc. All rights reserved.