GnRH Treatment Research Articles

373 STRATEGIES TO IMPROVE CONCEPTION RATES IN DAIRY HEIFERS INDENTIFIED IN ESTRUS USING TAIL HEAD PAINTING AND INSEMINATED WITH SEXED SEMEN

Two strategies to improve the conception rates in dairy heifers identified in estrus using tail head painting and inseminated with sexed semen were evaluated. In the first trial, the effect of GnRH injection (100 μg of gonadorelin) at estrus on ovarian responses and conception rates (CR) were verified. A total of 413 virgin Jersey heifers 11 to 12 mo of age were used. All heifers were treated with 25 mg of dinoprost tromethamine at the first day of the AI program. All inseminations were performed 12 h after estrus detection using sexed semen (2.1 million sperm) and the estrus was detected based upon removal of tail-head paint. At estrus detection, heifers were randomly assigned to receive GnRH or not (GnRH = 212 and No GnRH = 201). A subset of heifers (GnRH = 70 and No GnRH = 70), was scanned by ultrasound every 12 h after estrus detection until ovulation. The largest follicle diameter at estrus (LF), ovulatory follicular diameter (OF), ovulation rate (OR), interval between beginning of estrus and ovulation (IEO), and the proportion of heifers presenting late ovulation (LO, ovulation occurred with more than 36 h after the beginning of estrus) were recorded. The GnRH did not influence the LF (13.3 ± 0.9 v. 13.4 ± 0.2 mm; P = 0.87), the OF (13.4 ± 0.3 v. 13.9 ± 0.2 mm; P = 0.17), or OR [98.6% (69/70) v. 97.1% (68/70); P = 0.57]. However, GnRH treatment tended to shorten the IEO (29.2 ± 1.1 h v. 32.0 ± 1.3 h; P = 0.07) and reduced the occurrence of LO (2.9% (2/69) v. 16.2% (11/68); P = 0.02). However, GnRH injection did not influence the CR [GnRH = 47.2% (100/212) v. No GnRH = 51.7% (104/201); P = 0.38]. The second trial evaluated the CRin dairy heifers after the use of single or double insemination dose. A total of 576 Jersey heifers receiving first, second, or third insemination services were used. The heifers were assigned according to sire utilized into one of the three groups (Single 12 h, Double 12 h, or Double 12/24 h). In Single 12 h (n = 193) or Double 12 h (n = 193) groups, heifers received one or two insemination doses (2.1 million sperm) of sexed semen 12 h after estrus detection, respectively. The Double 12/24 h (n = 190) group heifers received two doses 12 h apart from each other (first dose at 12 h and second dose at 24 h after estrus detection). Differences were found between the number of AI services (P = 0.004) on the CR [First = 55.3%a (115/208); Second = 46.1%a (94/204); Third = 34.8% (57/165); P = 0.004]. However, there was no effect of treatment on the CR [Single 12 h = 45.1% (87/193); Double 12 h = 44.0% (85/193); Double 12/24 h = 49.5% (94/190); P = 0.51]. In conclusion, the GnRH injection altered the timing of ovulation, but failed to increase the CR. Furthermore, double insemination doses of sexed semen did not influence the CR in dairy heifers. Dalhart Jersey Ranch-TX, USA and Sexing Technologies-SP, Brazil.

Effect of RF-Amide-Related Peptide-3 on Luteinizing Hormone and Follicle-Stimulating Hormone Synthesis and Secretion in Ovine Pituitary Gonadotropes

GnRH provides the primary stimulus for the reproductive axis, but original work also revealed the existence of a gonadotropin-inhibitory hormone (GnIH) in birds. In mammals, GnIH properties are displayed by a hypothalamic dodecapeptide, which is a member of the RF-amide family, namely RF-amide-related peptide (RFRP)-3. This peptide inhibits GnRH-stimulated gonadotropin secretion from ovine pituitary cells in culture, but it is not known whether there are effects on gonadotropin synthesis. The aim of the present study was to determine the effects of RFRP-3 on the expression of genes for beta-subunits of the gonadotropins in ovine pituitary cells from gonadectomized ewes and rams. Cells in primary culture were given GnRH or vehicle pulses every 8 h for 24 h with and without RFRP-3 treatment. GnRH stimulated LH and FSH secretion, which was reduced by RFRP-3. Quantitative real-time PCR revealed increased expression of LHbeta and FSHbeta subunit genes after GnRH treatment and a specific reduction in expression after RFRP-3 treatment. There was no effect on the expression of GH, proopiomelanocortin, or prolactin genes. Western blotting showed that GnRH stimulated phosphorylation of ERK (phospho-ERK-1/2), and this effect was abolished by RFRP-3. We conclude that RFRP-3 acts on the pituitary gonadotropes to inhibit synthesis of the gonadotropins, and this effect may be mediated by a reduction in the GnRH-stimulated second messenger phospho-ERK-1/2.

Comparison of the Timing of the Second Dose of Prostaglandin F2 Alpha in a Five-Day Progesterone-based Timed AI Synchronization Protocol in Angus Cross Beef Cows

Artificial insemination (AI) is a highly effective reproductive technology for promoting genetic improvement in beef cattle. The most commonly used timed-AI program in beef cattle is the CO-Synch program. Pregnancy rates achieved through this program have ranged from 31 to 66% across a series of published reports, a rate that is acceptable to many beef producers. It has been shown that reducing the interval from the initial GnRH treatment to PGF2a (and withdrawal of a progestin) improves the outcome of timed-AI. With this protocol, termed the 5-day Co-Synch+ CIDR protocol, cows are given GnRH and have a CIDR device placed intravaginally, have the device removed after five days, and are given PGF2a, and then are time inseminated at 72 hours. However, this protocol requires the injection of two doses of PGF2a with an inconvenient 12-hour wait between the two doses. Reducing the interval between the two doses of PGF2a will allow cows to be handled twice with only a single penning and sorting to remove calves. The objective of this study was to compare reproductive performance of Angus cross beef cows synchronized with 5-day Co-Synch+ CIDR protocol and two doses of dinoprost tromethamine (PGF) given at CIDR removal and either at 2 h or 8 h following CIDR withdrawal.

Osmotic stress response is altered in leiomyoma cells

OBJECTIVE: Clinical treatment of leiomyomas with GnRH analogue produces a rapid reduction in leiomyoma volume. Rapid leiomyoma regrowth when GnRH treatment is withdrawn suggests the reduction in volume is due to transport of water, yet little is known about the response of leiomyoma cells to osmotic stress. Nuclear factor of activated T cells 5 (NFAT5) is the key osmo-sensing transcription factor in human cells. Our goal was to characterize osmosensing via NFAT5 in leiomyoma cells. DESIGN: Prospective, human model experiments MATERIALS AND METHODS: Osmotic stress was induced by culturing immortalized human leiomyoma and myometrial cells in DMEM-10% FBS with additional osmolytes of NaCl at 25, 50 or 100 mM for 8 hours. Proliferation assays were performed by the sulforhodamine B method. Apoptosis was assessed by caspase 3/7 activity assay. Expression of NFAT5 in tissue and cells was determined with qRT-PCR and western blot analysis. RESULTS: Leiomyoma cell growth was inhibited 18% at 50 mM NaCl compared to 48% in myometrial cells (p<0.05), consistent with altered osmosensing in leiomyoma cells. This finding was not due to apoptosis, as evidenced by no change in caspase activity. NFAT5 transcripts were increased in leiomyoma 1.5 fold ± 0.19 compared to myometrium in 4 of 7 patient samples (p<0.05). Concentration dependent upregulation of NFAT5 mRNA was observed in osmotically stressed leiomyoma cells. At 50mM NaCl, NFAT5 transcripts were increased 2.5-fold compared to control. CONCLUSIONS: Leiomyoma cells were more resistant to changes in osmotic stress than myometrial cells, suggesting dysregulation in osmotic signaling in leiomyomas. This change was accompanied by differential expression of NFAT5 in leiomyoma tissue. Altered osmotic signaling in leiomyoma cells suggests a central role for water regulation in the rapid change of leiomyoma volume with GnRH analogue treatment.

Effects of GnRH treatment on initiation of pulses of LH, LH release, and subsequent concentrations of progesterone

Progesterone is essential for establishment and maintenance of pregnancy. One proposed method to increase progesterone is administering GnRH at insemination. However, this method has resulted in conflicting results. Therefore, 2 experiments were conducted to evaluate how administering GnRH at insemination affected pulses of luteinizing hormone (LH) and subsequent progesterone. In Experiment 1, cows were allotted to 2 treatments: (1) GnRH (100 μg) given approximately 12 h after initiation of estrus (n = 5); and (2) Control (n = 5). Blood samples were collected at 15-min intervals for 6 h at 12 (blood sampling period 1), 26 (blood sampling period 2), 40 (blood sampling period 3), 54 (blood sampling period 4), and 68 (blood sampling period 5) h after onset of estrus. Daily blood samples were collected for 17 d. In Experiment 2, cows were allotted into 2 treatments: GnRH administered 10 to 11 h (n = 10) or 14 to 15 h (n = 10) after onset of estrus. Daily blood samples were collected for 17 d. Cows treated with GnRH tended ( P ≤ 0.075) to have greater LH release during blood sampling period 1, tended ( P = 0.095) to have fewer pulses during blood sampling period 2, tended ( P = 0.067) to have greater concentrations of progesterone, and had an earlier ( P = 0.05) increase in progesterone than control cows. Cows treated with GnRH 10 to 11 h after onset of estrus had greater ( P = 0.01) progesterone and an earlier ( P = 0.04) increase in progesterone than cows treated 14 to 15 h. In conclusion, timing of GnRH treatment following onset of estrus influenced pulses of LH and subsequent progesterone.

LH pulse frequency and the emergence and growth of ovarian antral follicular waves in the ewe during the luteal phase of the estrous cycle

BackgroundIn the ewe, ovarian antral follicles emerge or grow from a pool of 2–3 mm follicles in a wave like pattern, reaching greater than or equal to 5 mm in diameter before regression or ovulation. There are 3 or 4 such follicular waves during each estrous cycle. Each wave is preceded by a peak in serum FSH concentrations. The role of pulsatile LH in ovarian antral follicular emergence and growth is unclear; therefore, the purpose of the present study was to further define this role.MethodsEwes (n = 7) were given 200 ng of GnRH (IV) every hour for 96 h from Day 7 of the estrous cycle, to increase LH pulse frequency. Controls (n = 6) received saline. In a second study, ewes (n = 6) received subcutaneous progesterone-releasing implants for 10 days starting on Day 4 of the cycle, to decrease LH pulse frequency. Controls (n = 6) underwent sham surgery. Daily transrectal ovarian ultrasonography and blood sampling was performed on all ewes from the day of estrus to the day of ovulation at the end of the cycle of the study. At appropriate times, additional blood samples were taken every 12 minutes for 6 h and 36 min or 6 h in studies 1 and 2 respectively.ResultsThe largest follicle of the follicular wave growing when GnRH treatment started, grew to a larger diameter than the equivalent wave in control ewes (P < 0.05). Mean serum estradiol and progesterone concentrations were higher but mean serum FSH concentrations were lower during GnRH treatment compared to control ewes (P < 0.05). The increased serum concentrations of estradiol and progesterone, in GnRH treated ewes, suppressed a peak in serum concentrations of FSH, causing a follicular wave to be missed. Treatment with progesterone decreased the frequency of LH pulses but did not have any influence on serum FSH concentrations or follicular waves.ConclusionWe concluded that waves of ovarian follicular growth can occur at LH pulse frequencies lower than those seen in the luteal phase of the estrous cycle but frequencies seen in the follicular phase, when applied during the mid-luteal phase, in the presence of progesterone, do enhance follicular growth to resemble an ovulatory follicle, blocking the emergence of the next wave.

Gonadotropin-Releasing Hormone-Mediated Phosphorylation of Estrogen Receptor-α Contributes to fosB Expression in Mouse Gonadotrophs

Estrogen receptors (ERs) are activated by their ligands as well as signaling pathways that alter ER phosphorylation in response to peptide hormones and growth factors. In pituitary gonadotrophs, GnRHs act via the type I GnRH receptor (GnRHR). Both GnRH subtypes (GnRH-I and -II) activate an estrogen response element (ERE)-driven luciferase reporter gene in LbetaT2 mouse pituitary cells, and GnRH-I is most potent in this regard. Moreover, antide (a GnRH antagonist) and a GnRHR small interfering RNA (siRNA) abrogate this effect, whereas an ERalpha antagonist (ICI 182,780) does not. The ERalpha in LbetaT2 cells is phosphorylated at Ser(118) in the nucleus and at Ser(167) in both nucleus and cytoplasm after GnRH treatments and coincided with increased ERalpha binding to its coactivator, the p300/cAMP response element-binding protein-associated factor (PCAF). Moreover, siRNA-mediated knockdown of PCAF levels attenuated GnRH-induced ERE-luciferase transactivation in these cells. Most importantly, both GnRH subtypes robustly up-regulated expression of the immediate early response gene, fosB, whereas cotreatment with ERalpha siRNA or PCAF siRNA attenuated this effect. This appears to occur at the transcriptional level because corecruitment of ERalpha and PCAF to an ERE within the endogenous fosB promoter was increased by GnRH treatments, as shown by chromatin immunoprecipitation assays. These data demonstrate that GnRH-mediated phosphorylation of ERalpha in mouse LbetaT2 pituitary cells results in its rapid association with PCAF and the transcriptional activation of fosB, and we demonstrate that this in turn likely activates other genes in pituitary cells including the FSH beta-subunit gene.

Identification of Amnionless Gene as an Estrogen/ER Alpha-Regulated Gene in the Mouse Pituitary.

Estrogen plays a key role in regulating gonadotropin secretion from the pituitary through both negative and positive feedback pathways. However, the direct mechanisms of this regulation have yet to be defined. We hypothesize that the positive feedback effect of estrogen is achieved by ERα-mediated transcriptional regulation of cellular components needed for LH vesicle transport and secretion of its components into the bloodstream, thus equipping cells with the secretory machinery required for the LH surge. To identify these ERα-regulated components of LH secretion, total RNA was extracted from the pituitaries of cycling mice in metestrus or proestrus (low and high levels of circulating estrogen, respectively), ovariectomized (OVX) WT mice treated with vehicle or estrogen, and OVX ERαKO mice treated with vehicle or estrogen, and subjected to microarray analysis. Overall, 183 genes were found to be differentially expressed in an estrogen/ERα-dependent manner. In this study, we characterized the expression pattern of a gene (amn) that codes for Amnionless (Amn), a type I transmembrane protein known to be involved in vitamin B12 absorption by distal intestinal epithelia and proximal tubules of the kidney. The amn gene was selected as one gene of interest due to significant effect of estrogen treatment on transcription levels and the novelty of its characterization in the pituitary. The microarray analysis revealed a 30-fold increase of amn transcription in proestrus compared to metestrus, and a fold change of 25 in OVX WT mice with estrogen compared to vehicle treatment. ERαKO mice showed no increase in transcription of amn with estrogen treatment. This pattern of amn expression was confirmed using RT-PCR, showing a 9 fold increase in transcription levels in proestrus versus metestrus, and 16-fold increase in the OVX WT estrogen treated compared to vehicle treated groups. Immunohistochemistry confirmed that protein levels increased concurrently with mRNA levels in proestrus versus metestrus, but not in ERαKO mice. In silico analysis of the amn proximal promoter reveals 8 putative EREs within 2 kilo bases upstream of the start codon, evidence of a possible direct regulation of amn expression by the classical ERα pathway, which will be determined by ChIP assay. Currently, we are determining the functional significance of ERα-regulated amn expression in LH secretion using in vitro models. Both primary pituitary cells and isolated gonadotroph cells are treated with anti-Amnionless antibody, in order to block the functional capacity of the protein, followed by GnRH treatment. This experiment in the primary pituitary cells will allow us to determine if Amn in the pituitary plays a critical role in facilitating LH secretion, and the experiment in isolated gonadotroph cells will clarify if the effect on LH secretion is due to Amn in gonadotrophs themselves, or secondarily due to Amn expression in other cell types of the pituitary. For isolation of gonadotroph cells, we have generated ROSA-GFP CgaCre mice, who express Cre, and thus GFP, specifically in gonadotroph. Using flow cytometry, live gonadotrophs can be segregated from other cell types of the pituitary. Future directions will be aimed at elucidation of the role of Amn in preparing the gonadotroph for LH secretion in relation to the well-known function of Amn in facilitating vitamin B12 absorption. In summary, the gene encoding the Amnionless protein has been identified as an estrogen/ERα-regulated gene in the pituitary. The findings we may make from the study of Amn function in the pituitary will add to the understanding of the molecular mechanisms of estrogen priming of the pituitary in preparation for the LH surge. Supported by a COBRE grant from NIH NCRR: P20 RR15592. (poster)

Lutropin alpha, recombinant human luteinizing hormone, for the stimulation of follicular development in profoundly LH-deficient hypogonadotropic hypogonadal women: a review

Lutropin alpha, recombinant human luteinizing hormone, for the stimulation of follicular development in profoundly LH-deficient hypogonadotropic hypogonadal women: a review Bernd Th Krause1, Ralf Ohlinger2, Annette Haase31Center for Endocrinology and Reproductive Medicine, MVZ Uhlandstr, Berlin, Germany; 2Ernst-Moritz-Arndt-University, Department of Gynecology and Obstetrics, Greifswald, Germany; 3Uhlandstr. 162, 10719 BerlinAbstract: Hypogonadotropic hypogonadism is defined as a medical condition with low or undetectable gonadotropin secretion, associated with a complete arrest of follicular growth and very low estradiol. The main cause can be traced back to an irregular or absent hypothalamic GnRH secretion, whereas only a minority suffers from a pituitary disorder. The choice of treatment to reverse this situation is a pulsatile GnRH application or a direct ovarian stimulation using gonadotropin injections. The goal is to achieve a proper ovarian function in these cases for a short time to allow ovulation and chance of pregnancy. Since the pulsatile GnRH treatment lost its former importance, several gonadotropins are in use to stimulate follicular growth, such as urine-derived human menopausal gonadotropin, highly purified follicle stimulating hormone (FSH) or recombinant FSH, all with different success. The introduction of recombinant luteinizing hormone (LH) and FSH provided an opportunity to investigate the distinct influences of LH and FSH alone and in combination on follicular growth in monofollicular ovulation induction cycles, and additionally on oocyte maturation, fertilization competence of the oocyte and embryo quality in downregulated IVF patients. Whereas FSH was known to be indispensable for normal follicular growth, the role of LH remained questionable. Downregulated IVF patients with this short-term gonadotropin depletion displayed no advance in stimulation success with the use of recombinant LH. Patients with hypogonadotropic hypogonadism undergoing monofollicular stimulation for ovulation induction showed clearly a specific role and need for both hormones in normal follicular growth. Therefore, a combined stimulation with FSH and LH seems to be the best treatment choice. In the first half of the stimulation cycle the FSH dosage should exceed that of LH by 2:1, with an inverse ratio for the second half.Keywords: hypogonadotropic hypogonadism, ovarian stimulation, ovulation induction, recombinantluteinizing hormone

Lutropin alpha, recombinant human luteinizing hormone, for the stimulation of follicular development in profoundly LH-deficient hypogonadotropic hypogonadal women: a review.

Hypogonadotropic hypogonadism is defined as a medical condition with low or undetectable gonadotropin secretion, associated with a complete arrest of follicular growth and very low estradiol. The main cause can be traced back to an irregular or absent hypothalamic GnRH secretion, whereas only a minority suffers from a pituitary disorder. The choice of treatment to reverse this situation is a pulsatile GnRH application or a direct ovarian stimulation using gonadotropin injections. The goal is to achieve a proper ovarian function in these cases for a short time to allow ovulation and chance of pregnancy. Since the pulsatile GnRH treatment lost its former importance, several gonadotropins are in use to stimulate follicular growth, such as urine-derived human menopausal gonadotropin, highly purified follicle stimulating hormone (FSH) or recombinant FSH, all with different success. The introduction of recombinant luteinizing hormone (LH) and FSH provided an opportunity to investigate the distinct influences of LH and FSH alone and in combination on follicular growth in monofollicular ovulation induction cycles, and additionally on oocyte maturation, fertilization competence of the oocyte and embryo quality in downregulated IVF patients. Whereas FSH was known to be indispensable for normal follicular growth, the role of LH remained questionable. Downregulated IVF patients with this short-term gonadotropin depletion displayed no advance in stimulation success with the use of recombinant LH. Patients with hypogonadotropic hypogonadism undergoing monofollicular stimulation for ovulation induction showed clearly a specific role and need for both hormones in normal follicular growth. Therefore, a combined stimulation with FSH and LH seems to be the best treatment choice. In the first half of the stimulation cycle the FSH dosage should exceed that of LH by 2:1, with an inverse ratio for the second half.

Cisto ovariano em vacas de leite: incidência, resposta à aplicação de GnRH e desempenho reprodutivo

A incidência de cistos ovarianos, a resposta ao tratamento com GnRH e os efeitos da ocorrência de cisto no desempenho reprodutivo e na taxa de descarte foram determinados em vacas lactantes da raça Holandesa. Vacas lactantes (n=333) foram avaliadas semanalmente por ultrassonografia a partir da quarta semana pós-parto, visando à detecção de corpos lúteos (CL) e de folículos ovarianos maiores que 10mm. Na sétima semana pós-parto, as vacas foram classificadas: em ciclando (n=248; presença de CL em um dos exames ultrassonográficos); em anestro (n=54; ausência de CL e de folículos &gt;25mm) e com cisto (n=31; ausência de CL e presença de estruturas &gt;25mm), quando foram distribuídas em: grupo-controle (n=16; sem tratamento) e grupo-tratamento (n=15; vacas que receberam uma aplicação de GnRH). A taxa de cura foi de 60,0% no grupo das vacas tratadas e de 87,5% no grupo-controle. As vacas com cistos apresentaram maior intervalo parto-primeira inseminação artificial (P&lt;0,05; 91,4±8,3 vs. 77,8±2,5), maior número de serviços por concepção (P&lt;0,05; 4,4±1,2 vs. 3,6±1,5), maior intervalo parto-concepção (P&lt;0,05; 214,8±25,9 vs. 174,9±7,7) e maior taxa de descarte (P&lt;0,05; 41,2 vs. 21,8%) do que as vacas ciclando. Vacas diagnosticadas com cisto na sétima semana pós-parto apresentaram recuperação espontânea, embora o intervalo parto-concepção e a taxa de descarte tenham sido maiores para essas vacas.

Strategies to improve fertility in postpartum multiparous Bos indicus cows submitted to a fixed-time insemination protocol with gonadotropin-releasing hormone and prostaglandin F2α1

In Exp. 1, we evaluated the effects of 2 lengths of progesterone exposure [CIDR (controlled intravaginal drug release); 7 vs. 14 d] before a modified CO-Synch protocol [50.0-microg injection of GnRH 6.5 d before a 25.0-mg injection of PGF(2alpha) followed by another injection of GnRH and fixed-time AI (TAI) 2 d after PGF(2alpha)], with or without temporary weaning (TW) before GnRH treatments, on fertility of suckled multiparous Bos indicus cows (n = 283) and on calf performance. Timed AI pregnancy rates for cows receiving 7 d CIDR + TW, 7 d CIDR, 14 d CIDR + TW, and 14 d CIDR were 53, 47, 46, and 41%, respectively (P > 0.10). Calves submitted to two 48-h TW 6 d apart had decreased mean BW at 240 d (187.9 +/- 2.7 vs. 195.5 +/- 2.7 kg; P < 0.05), but BW at 420 d was not affected by TW (240.1 +/- 5.1 kg). In Exp. 2, we evaluated the effect of no treatment and treatment with or without a CIDR insert between GnRH and PGF(2alpha) treatments of a modified CO-Synch protocol on pregnancy rate to TAI, and throughout a 90-d breeding season in suckled multiparous Bos indicus cows (n = 453). The inclusion of a CIDR between first GnRH and PGF(2alpha) treatments of a modified CO-Synch protocol did not improve pregnancy rate (29 and 33% for cows receiving CO-Synch + CIDR and CO-Synch protocol, respectively), and cycling cows had poorer TAI pregnancy rates than anestrous cows treated with either synchronization protocol (21.7 vs. 40.7%; P < 0.05). However, regardless of treatment with CIDR, cows submitted to TAI protocol had greater (P < 0.05) pregnancy rates at 30 (54.8 vs. 11.2%), 60 (72.1 vs. 38.8%), and 90 d (82.0 vs. 57.9%) of breeding season than untreated cows.

The effect of addition of equine chorionic gonadotropin to a progesterone-based estrous synchronization protocol in buffaloes ( Bubalus bubalis) under tropical conditions

Poor estrus expression and anestrus decrease the reproductive efficiency of buffaloes. The objective of this study was to determine whether the addition of equine chorionic gonadotropin (eCG) to an estrous synchronization protocol and timed insemination could improve ovulation and pregnancy rates of anestrous buffalo cows under tropical conditions. The study population comprised 65 lactating Murrah buffalo cows which were assigned to CIDR ( n = 33) or CIDR + eCG ( n = 32) treatment groups. Cows in the CIDR group were fitted for 8 d with a controlled intravaginal drug release (CIDR) device containing 1.38 g progesterone, received GnRH (10 μg i.m.) on D 0, PGF 2α (750 μg i.m.) on D 7, and GnRH (10 μg i.m.) on D 9; whereas cows in the CIDR + eCG group received the same treatment plus eCG (500 IU, i.m.) at the time of PGF 2α treatment. All cows were inseminated 16–20 h after the second GnRH treatment. Blood samples were obtained 10 d before the start of synchronization treatment (Day –10) and at the onset of treatment (Day 0). Cows with plasma progesterone concentrations <1 ng/mL recorded in both samples (Low–Low levels of P4) were classified as non-cyclic cows. Similarly, when either one or both of the sample pair contained concentrations of serum progesterone ≥1 ng/mL (High–High, Low–High, or High–Low levels of P4), the buffaloes were classified as cyclic cows. Ovulation rate, defined as the number of buffaloes with at least one corpus luteum 10 days after insemination, was significantly higher ( P = 0.018) in the CIDR + eCG (84.4%) cows than in the CIDR cows (57.6%). Pregnancy rate was numerically lower in CIDR (27.3%) than CIDR + eCG (40.6%) cows, though differences were not significant ( P = 0.25). Pregnancy rates for CIDR + eCG cows were similar to that of cows inseminated after natural estrus (40.9%; 29/71). In the non-cyclic animals, higher ovulation rates ( P = 0.026) were recorded for the CIDR + eCG (81%) than for the CIDR cows (47.4%). Our results indicate that the addition of eCG to a progesterone-based estrous synchronization regimen substantially improves the ovulation rate in non-cyclic buffaloes. When this treatment is followed by timed AI, pregnancy rates achieved in anestrous buffaloes, whether cyclic and non-cyclic, may approach the rates observed in cows inseminated at natural estrus.

Remoção temporária de bezerros em dois momentos do protocolo de sincronização da ovulação GnRH-PGF2α-BE em vacas Nelore pós-parto

Os efeitos da remoção temporária de bezerros (RB) sobre características foliculares e taxas de ovulação e de prenhez de vacas Nelore foram avaliados em dois experimentos quando da inseminação artificial em tempo fixo (IATF), utilizando-se um protocolo à base de GnRH-PGF2α-BE. No experimento 1, 139 vacas Nelore, lactantes, cíclicas ou acíclicas, receberam um protocolo hormonal envolvendo a aplicação de 100±g de análogo GnRH no dia zero (D0-GnRH), 25mg de PGF2α no dia 7 (D7-PGF2α) e 1,0mg de benzoato de estradiol no dia 8 (D8-BE). Os animais foram aleatoriamente distribuídos para serem submetidas ou não à RB (48 horas) antes da aplicação de GnRH (RB1) ou após a aplicação de PGF2α (RB2). Avaliaram-se o diâmetro folicular (DF) nos dias 0 (D0) e 9 (D9) do protocolo e as taxas de ovulação (T0), advindas dos tratamentos. As vacas acíclicas que receberam RB1 apresentaram maiores DF no D0 (P&lt;0,05) e taxa de ovulação (P&lt;0,05) quando da aplicação de GnRH, em relação às que não receberam RB1. Nas vacas cíclicas, não houve efeito da RB1 sobre as variáveis analisadas. As vacas que receberam RB2 apresentaram maiores DF no D9 (P&lt;0,05) e taxa de ovulação (P&lt;0,01) quando da aplicação de BE, em relação às que não receberam a RB2. No experimento 2, 376 vacas da raça Nelore, lactantes, foram submetidas ao mesmo protocolo hormonal e aos mesmos tratamentos do experimento 1, mas foram inseminadas 24 a 30 horas após a aplicação de BE no D8, visando avaliar a taxa de prenhez após a IATF. A taxa de prenhez foi maior nos animais que receberam as duas RB em relação às que não receberam RB (29,8% vs 10,6%; P&lt;0,05). A remoção de bezerros associada ao protocolo hormonal aplicado pode ter aumentado as taxas de prenhez devido às maiores taxas de ovulação, em resposta ao GnRH ou ao benzoato de estradiol.

Synchronization of estrus with short- and long-term progestagen treatments and the use of GnRH prior to short-term progestagen treatment in ewes

The objective of the present study was to determine the efficacy of the synchronization of estrus using short- and long-term progestagen treatments in ewes at the onset of the breeding season, and to evaluate the effect of the exogenous GnRH administration immediately prior to short-term progestagen treatment on the reproductive performance. A total of 240 Tahirova cross-bred ewes, aged 18–24 months, and 40 rams, aged 2–4 years-old, were used in the trial. Ewes were divided equally into 3 groups ( n = 80 per group). Intravaginal progestagen sponges containing FGA (30 mg) were inserted in the ewes for 7 d in the FGA1 (short-term) and GnRH treatment groups, and for 12 d in the FGA2 group (long-term). The ewes in the GnRH group received 10.5 μg busereline acetate i.m. at the time of sponge insertion. Tiaprost tromethamol (PGF 2α; 0.294 mg) and eCG (400 IU) were injected i.m. on the 6th day of progestagen treatment in the GnRH and FGA1 groups, and on the 11th day in the FGA2 group following sponge insertion. All ewes were hand-mated once at the detection of estrus. The estrous response, fertility rate, multiple birth rate and litter size recorded was 88.7, 87.3, 51.6% and 1.6 in the FGA1 group, 92.5, 71.6, 50.9% and 1.5 in the FGA2 group, and 96.2, 89.6, 71.0% and 1.8 in the GnRH group, respectively. No significant difference in estrous response between the groups was recorded, but the fertility rate in the FGA1 and GnRH groups was significantly ( P < 0.05) higher than in the FGA2 group. The occurrence of multiple births and litter sizes were significantly ( P < 0.05) higher in the GnRH group, compared to both the FGA1 and FGA2 groups, with the number of single lambs being significantly ( P < 0.05) higher in the FGA1 (48.4%) and FGA2 (49.0%) groups than in the GnRH (29.0%) group. However, the differences recorded between any of the groups in terms of the number of twin and triplet lambs were insignificant. In conclusion, it can be said that estrous synchronization using the 12-d-FGA-eCG-PGF 2α regimen could be replaced with the 7-d-FGA-eCG-PGF 2α regime in sheep at the onset of the breeding season. However, the combination of GnRH with the latter regimen (7-d-GnRH-FGA-eCG-PGF 2α) increased the multiple birth rate and litter size in the ewes.

Plasma luteinizing hormone concentrations in cows given repeated treatments or three different doses of gonadotropin releasing hormone

We hypothesized that: (i) repeated GnRH treatments would increase the magnitude and duration of the LH surge and would increase progesterone (P4) concentrations after ovulation; and (ii) the release of pituitary LH would be greater in response to larger doses of GnRH. In Experiment 1, ovary-intact cows were given an intravaginal P4 (1.9 g) insert (CIDR) for 10 d and 500 μg cloprostenol (PGF) at CIDR removal to synchronize estrus. On Days 7 or 8 after estrus, cows received two PGF treatments (12 h apart) and 100 μg GnRH at 36 (Control), 36 and 38 (GnRH38), or 36 and 40 h (GnRH40) after the first PGF. Mean plasma LH concentration (ng/mL) was greater ( P < 0.05) in GnRH38 (8.8 ± 1.1) than in Control (5.1 ± 1.3), with that in GnRH40 (5.8 ± 1.3) being intermediate. Although the duration (h) of the LH surge was longer in GnRH40 (8.0 ± 0.4) than in either GnRH38 ( P < 0.05; 7.0 ± 0.3) or Control ( P < 0.09; 7.1 ± 0.4), mean postovulatory P4 (ng/mL) was greater ( P < 0.01) in Control (4.2 ± 0.7) than in GnRH38 (2.9 ± 0.6) or GnRH40 (3.0 ± 0.7) cows. In Experiment 2, ovariectomized cows were given a CIDR for 10 d and 2 mg of estradiol cypionate im at CIDR insertion. Thirty-six hours after CIDR removal, cows received, 50, 100, or 250 μg of GnRH. Cows given 250 μg GnRH released more LH (9.4 ± 1.4 ng/mL) than those given 50 or 100 μg (6.1 ± 1.3 and 5.4 ± 1.4 ng/mL, respectively), and had an LH surge of longer duration than those given 50 μg (6.8 ± 0.4 vs. 5.1 ± 0.3 h). In summary, ovary-intact cows in the GnRH38 group had greater mean and peak LH concentrations, but subsequent plasma P4 concentrations were lower than in Control cows. Ovariectomized cows given 250 μg GnRH had a greater pituitary release of LH.

Effect of time interval between prostaglandin F 2α and GnRH treatments on occurrence of short estrous cycles in cyclic dairy heifers and cows

Prostaglandin F 2α (PGF 2α) and GnRH treatments, when administered 24 h apart during early diestrus, cause short estrous cycles in some dairy cows and heifers [J. Taponen, M. Kulcsar, T. Katila, L. Katai, G. Huszenicza, H. Rodriguez-Martinez, Short estrous cycles and estrous signs after premature ovulations induced with cloprostenol and gonadotropin-releasing hormone in cyclic dairy cows, Theriogenology 2002; 58, 1291–1302]. We investigated the effect of a time interval between PGF 2α and GnRH administration on the appearance of short cycles. Estrus was induced in heifers with dexcloprostenol. A second luteolysis was induced similarly on day 7 after ovulation, and either 0 (T0) or 24 h (T24) later an injection of GnRH (0.1 mg of gonadorelin) was administered. We monitored ovarian activity with progesterone analyses from blood plasma samples and with ultrasonography. Fourteen cases (12 in T0 and 2 in T24) were excluded due to either incomplete luteolysis (2 cases) or unresponsiveness to GnRH (10 in T0 and 2 in T24). Short estrous cycles (7 to 8 d) were detected in 11/11 and 8/17 heifers in groups T0 and T24, respectively, with a significant difference in the incidence of short cycles ( P < 0.01). In Experiment 2, estrus was induced in cows on day 8 (D8, n = 18), 9 (D9, n = 5), or 10 (D10, n = 3) with cloprostenol and gonadorelin administered simultaneously. Daily milk samples were collected for progesterone analysis until subsequent estrus was detected and ovarian ultrasound examinations were performed. Eight cases had to be excluded due to unresponsiveness to GnRH, leaving 18 cases eligible for the study. Short estrous cycles (7–12 d) were detected in 14/18 cows. In conclusion, shortening the time interval between PGF 2α and GnRH treatments increased the incidence of short estrous cycles and appeared to increase the proportion of females unresponsive to GnRH treatment.

Pregnancy patterns during the early fetal period in high producing dairy cows treated with GnRH or progesterone

In order to explore pregnancy patterns in high producing dairy cows treated with GnRH or progesterone at pregnancy diagnosis (Days 28–34), two consecutive experiments were designed. In Experiment 1, cows bearing a single embryo were randomly assigned to a PRID ( n = 40; cows fitted with a progesterone releasing intra-vaginal device for 28 days), GnRH ( n = 40; cows receiving GnRH) or Control ( n = 26; untreated cows) group. PRID treatment led to a rise in plasma progesterone concentrations in the 7 days following the onset of treatment compared to the other two groups. In Experiment 2, in which we also examined twin pregnancies, animals were randomly assigned to PRID ( n = 312) or GnRH ( n = 294) treatment groups. Treatments were the same as described for Experiment 1. Logistic regression procedures revealed that in cows with a single corpus luteum, the probability of pregnancy loss between the first (Days 28–34) and second (Days 65–62) pregnancy diagnosis decreased by a factor of 0.51 in the PRID group compared to the GnRH group. However, in cows with two or more corpora lutea, PRID treatment increased the likelihood of pregnancy loss by a factor of three, compared to GnRH treatment. In cows carrying twins, the conceptus reduction rate was higher ( P = 0.02) for the GnRH (36%) than for the PRID (16.4%) group. Formation of a new corpus luteum was recorded in 17.7% of cows in the GnRH group. Our results indicate that compared to GnRH treatment, progesterone treatment given at pregnancy diagnosis in high producing dairy cows, reduced by a factor of 0.51 and increased by a factor of 3 the probability of pregnancy loss in cows with a single or with two or more corpora lutea, respectively, and reduced the conceptus reduction rate in cows carrying twins. The practical implications of our findings are that in herds with a high incidence of early fetal loss of a non-infectious nature, treatment at the time of pregnancy diagnosis with PRID in cows with one corpus luteum and with GnRH in cows with two or more corpora lutea should offer considerable benefits.

Proteasome Regulation of Dynamic Transcription Factor Occupancy on the GnRH-Stimulated Luteinizing Hormone β-Subunit Promoter

GnRH is the main modulator of LH secretion and transcription of the LH subunit genes in pituitary gonadotropes. The LHbeta gene is preferentially transcribed during pulsatile GnRH stimuli of one pulse/30 min and is thus carefully controlled by specific signaling pathways and transcription factors. We now show that GnRH-stimulated LHbeta transcription is also influenced by the ubiquitin-proteasome system. GnRH-stimulated activity of an LHbeta reporter gene was prevented by proteasome inhibitors MG-132 and lactacystin. Inhibition was not rescued by overexpression of two key transcription factors for LHbeta, early growth response-1 (Egr-1) and steroidogenic factor-1 (SF-1). Increased endogenous LHbeta transcription after GnRH treatment was also prevented by MG-132, as measured by primary transcript assays. To investigate possible mechanisms of LHbeta transcriptional inhibition by proteasome blockade, we employed chromatin immunoprecipitation to measure LHbeta promoter occupancy by transcription factors. Without GnRH, binding was low and unorganized. With GnRH, Egr-1 and SF-1 associations were stimulated, cyclic, and coincidental, with a period of approximately 30 min. MG-132 disrupted GnRH-induced Egr-1 and SF-1 binding and prevented phosphorylated RNA polymerase II association with the LHbeta promoter. Egr-1, but not SF-1, protein was induced by GnRH and accumulated with MG-132. Egr-1 and SF-1 were ubiquitinated in gonadotropes and ubiquitinated forms of these factors associated with the LHbeta promoter, suggesting their degradation may be key for LHbeta proteasome-dependent transcription. Together, these results demonstrate that degradation via the proteasome is vital to GnRH-stimulated LHbeta expression, and this occurs in part by allowing proper transcription factor associations with the LHbeta promoter.

171 ADMINISTRATION OF GnRH ON DAY 6 ALTERS THE NUMBER OF FOLLICULAR WAVES FROM 2 TO 3 DURING THE ESTROUS CYCLE OF HOLSTEIN COWS

It is suggested that pregnancy rate is greater in lactating cows inseminated following ovulation of a third-wave follicle compared with a second-wave follicle. The number of follicular waves is not apparent during the estrous cycle. However, GnRH injection on Day 6 is supposed to initiate a new follicular wave earlier; as a result, the number of cows with 3 follicular waves will be increased. This study was done to change the 2-follicular-wave cycles to 3 follicular waves during the estrous cycle. The estrous cycles of 10 cows were synchronized with 2 i.m. injections of prostaglandin F2α given 11 days apart. The cows were randomly assigned to 1 of 2 treatments. Cows in the control treatment received no treatment, whereas GnRH6 cows received a GnRH injection on Day 6 of the estrous cycle (estrus = Day 0). Ovaries of all cows were scanned daily, from estrus (Day 0) to the subsequent estrus (Day 23). Profiles of the mean number of follicles (i.e. 4- to 6-mm and ≥7 mm), the diameter of the largest follicle (F1), and comparison of 2- and 3-wave cows were compared by least squares analysis of variance using the general linear model (GLM) procedure of SAS. The number of 4 to 6 mm follicles had a tendency to decline until Day 6 (3.0 ± 2.1 v. 0.8 ± 1.6). In response to an injection of GnRH, the number of small follicles in group GnRH6 on Day 6 (1.5 ± 1.6 follicles) increased (P &lt; 0.05) on Day 8 (5.0 ± 1.7 follicles) and the number of medium and large follicles (≥7 mm) on Day 6 (1.8 ± 0.5 follicles) decreased (P &lt; 0.05) on Day 9 (0.8 ± 0.6 follicles). Over the 4-day period before GnRH treatment, the diameter of F1 was increasing (from 4.7 ± 0.9 mm to 10.0 ± 0.6 mm) in cows treated with GnRH on Day 6 of the estrous cycle; thereafter, the diameter of the F1 decreased (P &lt; 0.05) within 2 days of GnRH treatment. In the control group the diameter of the F1 increased over the 4-day period before GnRH treatment (from 5.0 ± 0.9 mm to 11.2 ± 0.6 mm), whereas it remained constant (11.2 ± 1.2 mm to 11.2 ± 1.2 mm) between Days 6 and 9 of the estrous cycle (P &lt; 0.003). Results showed that 4 out of 5 cows in control group had 2 follicular waves, whereas all of the cows (n = 5) in GnRH6 group had 3 follicular waves. The first follicular wave begins at Day 1.1 ± 0.50 and 0.70 ± 0.50 in the control and GnRH groups, respectively. The second follicular waves appeared at Day 11.00 ± 0.80 and 8.30 ± 1.50 (P ≤ 0.05) for the 2- and 3-wave animals, respectively. The third follicular waves (n = 6) initiated at Day 16.30 ± 1.50. Cows with 2 follicular waves had shorter estrous cycles (P ≤ 0.05) than 3-wave cows (20.50 ± 0.60 v. 22.40 ± 0.90). The ovulatory follicle in 2-wave cows grew larger (14.00 ± 1.80 v. 12.50 ± 0.90 mm; P ≤ 0.05), and maintained for a longer period of time (9.50 ± 1.80 v. 6.1 ± 0.90; P ≤ 0.05) than in 3-wave cows. In conclusion, an injection of GnRH on Day 6 of the estrous cycle could promote the emergence of a new follicular wave earlier in cows and alter the estrous cycles with 2 waves into 3 follicular waves.