GnRH Treatment Research Articles

Effect of intrauterine administration of gonadotropin releasing hormone on serum LH concentrations in lactating dairy cows

The objectives were to compare: (1) preovulatory serum LH concentrations, and (2) synchronization of ovulation, after im or iu administration of the second GnRH treatment of Ovsynch in lactating dairy cows. Lactating cows (N = 23) were presynchronized with two injections of PGF2α given 14 days apart (starting at 34 ± 3 days in milk), followed by Ovsynch (GnRH-7 d-PGF2α-56 h-GnRH) 12 days later. At the time of the second GnRH of Ovsynch (Hour 0), cows were blocked by parity and randomly assigned to 1 of 3 groups: (1) control group (CON; N = 7) were given 2 mL sterile water im; (2) intramuscular group (IM; N = 8) received 100 μg of GnRH im; and (3) intrauterine group (IU; N = 8) had 100 μg GnRH infused in the uterus (2 mL). Blood samples for serum LH concentrations were collected at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Furthermore, ultrasonography was performed twice daily (12-h intervals) from Hours 0 to 60 to confirm ovulation. The LH concentrations were greater (P < 0.05) in the IM than IU and CON groups at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Although LH concentrations were numerically higher in the IU group, LH concentrations within the IU and CON groups did not change over time. More cows ovulated in the IM (8/8) and IU (7/8) groups within 60 h after the second GnRH administration compared with the CON (2/7) group. In summary, serum LH concentrations were lower in the IU versus IM group, but the proportion of cows that ovulated within 60 h was similar between these two groups. Therefore, iu administration of GnRH may be an alternative route of delivery to synchronize ovulation in beef and dairy cattle.

Study of Testosterone Concentrations during Breeding Season of Two Breeds of Goat Bucks and their Crossbred Under Exogenous GnRH Treatments

Study of Testosterone Concentrations during Breeding Season of Two Breeds of Goat Bucks and their Crossbred Under Exogenous GnRH Treatments

Effect of progesterone on magnitude of the luteinizing hormone surge induced by two different doses of gonadotropin-releasing hormone in lactating dairy cows

Ovulation to the first GnRH injection of Ovsynch-type protocols is lower in cows with high progesterone (P4) concentrations compared with cows with low P4 concentrations, suggesting that P4 may suppress the release of LH from the anterior pituitary after GnRH treatment. The objectives of this study were to determine the effect of 1) circulating P4 concentrations at the time of GnRH treatment on GnRH-induced LH secretion in lactating dairy cows and 2) increasing the dose of GnRH from 100 to 200μg on LH secretion in a high- and low-P4 environment. A Double-Ovsynch (Pre-Ovsynch: GnRH, PGF2α 7d later, GnRH 3d later, and Breeding-Ovsynch 7d later: GnRH, PGF2α 7d later, and GnRH 48h later) synchronization protocol was used to create the high- and low-P4 environments. At the first GnRH injection of Breeding-Ovsynch (high P4), all cows with a corpus luteum ≥20mm were randomly assigned to receive 100 or 200μg of GnRH. At the second GnRH injection of Breeding-Ovsynch (low P4) cows were again randomized to receive 100 or 200μg of GnRH. Blood samples were collected every 15min from −15 to 180min after GnRH treatment, and then hourly until 6h after GnRH treatment. As expected, mean P4 concentrations were greater for cows in the high- than the low-P4 environment. For cows receiving 100μg of GnRH, the LH peak and area under the curve (AUC) were greater in the low- than in the high-P4 environment. Similarly, for cows receiving 200μg of GnRH, the LH peak and AUC were greater in the low- than the high-P4 environment. Cows receiving 100 or 200μg of GnRH had greater mean LH concentration in the low- than the high-P4 environment from 1 to 6h after GnRH treatment. On the other hand, when comparing the effect of the 2 GnRH doses in the high- and low-P4 environments, cows receiving 200μg of GnRH had a greater LH peak and AUC than cows treated with 100μg of GnRH both in the high- and low-P4 environments. For the high-P4 environment, mean LH was greater from 1.5 to 5h after GnRH treatment for cows receiving 200μg of GnRH than for those receiving 100μg of GnRH. In the low-P4 environment, mean LH was greater for cows receiving 200μg of GnRH than for those receiving 100μg of GnRH from 1 to 2.5h after GnRH treatment. We conclude that the P4 environment at GnRH treatment dramatically affects GnRH-induced LH secretion, and that a 200-μg dose of GnRH can increase LH secretion in either a high- or a low-P4 environment.

Ovulation-inducing factor (OIF) induces LH secretion from pituitary cells

A substance in the seminal plasma of llamas and alpacas has been discovered that induces ovulation and growth of the corpus luteum (CL) in the female of the same species. The ovarian effects of the ovulation-inducing factor (OIF) are associated with a surge release of LH into circulation. We hypothesize that OIF stimulates LH release from gonadotroph cells in the anterior pituitary gland. Four experiments were done to determine if purified OIF isolated from llama seminal plasma stimulates LH secretion in pituitary cells using tissue from an induced ovulator (llama) and spontaneous ovulator (cattle). Anterior pituitary cells were cultured in vitro for two days, and on the third day, wells were incubated for 2h with media containing no treatment (control), GnRH or OIF. Concentrations of LH in the culture medium were measured using radioimmunoassay and compared among groups by analysis of variance. In all experiments, GnRH and OIF treatments induced more LH secretion than untreated controls (P<0.05). A dose-related effect was evident in the llama pituitary cell cultures in that mean LH concentrations were greater (P<0.05) in wells treated with a higher dose of OIF (5.41±0.28ng/mL) compared to wells treated with a lower dose (2.70±0.50ng/mL), both of which were higher (P<0.05) than in wells with no treatment (0.87±0.18ng/mL). Although OIF stimulated LH release in bovine cell cultures, a dose-related effect was not detected. We conclude that OIF stimulates LH secretion from pituitary gonadotrophs in vitro.

Fertility after ovarian follicular wave synchronization and fixed-time natural mating compared to random natural mating in dromedary camels (Camelus dromedarius)

The objective of the study was to compare the efficiency of two ovarian follicular wave synchronization protocols coupled with fixed-time natural mating with that of random mating in dromedary camels. Dromedaries were assigned randomly to one of the three treatment groups. Group 1 animals (RM; n=46) were mated randomly. Group 2 camels (1×GnRH-FTM; n=46) were given a GnRH analog (Buserelin, 20μg/animal, i.v.; Receptal, Intervet, Holland) at random, then were mated 14 days later. In Group 3 animals (2×GnRH-FTM; n=41), random GnRH analog was followed by repeated GnRH injection 14 days later and fixed-time natural mating on Day 28. Transrectal examination and ultrasonography were performed at weekly intervals to evaluate ovarian follicular status, diagnose ovulation and pregnancy. Blood samples were collected for progesterone determination by ELISA to confirm ovulation and pregnancy. All female dromedaries were assigned randomly to one of thirteen fertile bulls and were bred once on Days 1, 14 and 28 in Groups 1–3, respectively. Ovarian follicular status and ovulation rate was similar among groups at the start of the study. Seventy-five of the 133 dromedaries (56.4%) ovulated after random natural mating or random GnRH treatment. Mean length of mating was 386±17.8 (±SEM) seconds. There was no significant difference in mating time among groups and in pregnancy rate among dromedary bulls. In Group 3 (2×GnRH-FTM), ovarian follicular status before mating (P<0.05), ovulation rate (n=37, 90.2%, P<0.001) and pregnancy rate at 21 and 60 days (PR 21 days n=22, 53.7% and PR 60 days n=19, 46.3%, P<0.05) were greater compared to random natural mating (Group 1: OR n=25, 54.3%, PR 21 days n=13, 28.3% and PR 60 days n=12, 26.1%). In Group 2 dromedaries (1×GnRH-FTM), treatment tended to improve follicular status before mating, ovulation rate (n=34, 73.9%) and pregnancy rate at 21 and 60 days (PR 21 days n=21, 45.7% and PR 60 days n=16, 34.8%), but the effect was not significant compared to random natural mating. In conclusion, this is the first study demonstrating that favorable pregnancy rate can be achieved following ovarian follicular wave synchronization with repeated GnRH analog and fixed-time natural mating at 14 days intervals in dromedary camels.

Effects of eCG injection combined with FSH and GnRH treatment on the lambing rate in synchronized Afshari ewes

Due to the substantial contribution of lambing rate to profitability of sheep holders, the present trial was investigated the effects of eCG injection combined with FSH and GnRH administrations on ovarian follicular development and the lambing rate in Afshari ewes during breeding season. The estrous cycle of 64 cycling multiparous Iranian Afshari ewes, weighing 65±2.5kg, was synchronized using CIDR's, inserted for 14 days. eCG (300IU) was injected to ewes a day before CIDR removal. The ewes were allocated randomly to 1 of 4 groups (n=16/group). Control group did not receive FSH treatment. In FSH/0h, FSH/12h and FSH/24h groups, ewes received 10mg FSH at CIDR removal, 12 or 24h later, respectively. GnRH (50μg) was administered 48h following CIDR removal to half of each group of ewes. Ewes were mated with the proven rams. Ovarian follicular activity was monitored by transrectal ultrasonography at CIDR removal (day 0) and a day later (day 1). The repeated data analyzed using the mixed procedure of SAS (9.1). Effects of weight and age of ewes were added as a covariate to the model. The reproductive parameters (%) were analyzed using the chi-square test. The results have been showed that FSH treatment did not increase the mean number of small and medium sized follicles. Following FSH treatment, the mean number of large follicles had a tendency to increase (P<0.1) in FSH/12h group of ewes. Lambing rate in control, FSH/0h, FSH/12h and FSH/24h were 137.50%, 131.25%, 150.00% and 181.25%, respectively. The lambing rate and litter size increased to ≥150% (P<0.05) in groups of ewes which received GnRH. FSH/24h group of ewes had a significantly higher lambing rate, litter size and multiple birth rate (P<0.05) compared to other treatment groups. In conclusion, GnRH injection 48h after CIDR removal and eCG injection increase the lambing rate, litter size and multiple birth rates in ewes. The lambing rate (>180%) was greater in ewes which eCG injection was combined with FSH treatment 24h after CIDR removal with or without GnRH injection. These results indicate that lambing rate and litter size after CIDR and eCG treatment can be increased when GnRH is administered 48h after CIDR removal or when FSH is given 24h after CIDR removal.

Enhancing follicular growth as a prerequisite for GnRH treatment of true anestrum in buffalo

A total of 140 true anestrous buffalo were divided on the basis of receiving short-term (20 days) nutritional supplementation (N, n=80) or not (WN, n=60). The animals in N group were subdivided into NQ (n=40) where the quantity of the offered diet was increased by 20% and NF (n=40) where the offered diet was supplemented by 3% of dry protected fat. Buffaloes in either NQ or NF were equally allotted on the following treatment regimens: Insulin/GnRH (NQi or NFi, n=10 for each); rbST/GnRH (NQr or NFr, n=10 for each); GnRH alone treated (NQG or NFG, n=10 for each) and saline-treated control (NQc or NFc, n=10 for each). Insulin-treated subgroups (NQi or NFi) received s/c injection of insulin at a dose of 0.25I.U./kg on Days 21, 22 and 23 while rbST–treated subgroups (NQr or NFr) received single IM injection of rbST (500mg Sometribove) on Day 21. GnRH was injected at a dose of 0.020mg buserelin (5ml Receptal®) on Day 24 in all subgroups except NQCand NFC where Day 1 was the first day of the short-term nutritional improvement. Buffalo in the WN (n=60) were equally allotted on the same treatment regimens applied in the first group insulin/GnRH (WNi, n=15), rbST/GnRH (WNr, n=15); GnRH alone treated (WNG, n=15) and saline-treated control (WNC, n=15).Ultrasonic scanning of ovaries was conducted on Day 24 to measure largest follicle diameter (LFD). The results showed increases (P<0.05) in the LFD following nutritional supplementation with insulin or rbST. The recorded EIRs for GnRH pre-treated with nutritional improvement – metabolic hormones combinations (9/10 and 8/10 for NQi and NFi or 8/10 for NQr) were greater (P<0.05) than those pre-treated with either metabolic hormone alone (7/15 for WNi and/or WNr) or nutritional improvement alone (6/10 for NQG and/or NFG) and control as well. The greatest CR was recorded in the NQi group.It could be concluded that pre-GnRH nutritional improvement plus administration of insulin or rbST increases LFD in true anestrous buffalo having LFD<8.5mm thereby increasing their fertility response to GnRH in terms of EIRs and CRs.

The endocrine changes, the timing of ovulation and the efficacy of the Doublesynch protocol in the Murrah buffalo (Bubalus bubalis)

Experiments were conducted to investigate (a) the timing of ovulation and the associated endocrine changes (progesterone, estrogen and LH) during estrous cycle and (b) the efficacy, with respect to the pregnancy rate, in cycling and anestrus in Murrah buffaloes subjected to the Doublesynch protocol during the low breeding season. In experiment 1, 10 cycling buffaloes were administered PGF2α on day 0 (without regard to the estrous cycle stage), GnRH on day 2, a second PGF2α injection on day 9, and a second GnRH injection on day 11. Transrectal palpation was performed at 2-h intervals after the first and second GnRH treatments until ovulation was detected or for upto 96h. The plasma progesterone and total estrogen concentrations were determined in blood samples collected at daily intervals starting 2days before the onset of the protocol and continued until the day of the second detected ovulation. The plasma LH and total estrogen concentrations were measured in blood samples collected at 30-min intervals for 8h following the first and second GnRH injections and thereafter at 2-h intervals until 2h after the detection of ovulation. Ovulation occurred in 9/10 buffaloes (90%) at 22.2±1.2h (mean±S.E.M.; range 18.0–26.0h) and 10/10 buffaloes (100%) at 23.2±1.0h (mean±S.E.M.; range 20.0–28.0h) after the first and second GnRH treatments, respectively. The peak LH concentrations of 99.8±28.5ng/ml (range 37.8–320.0ng/ml) and 62.3±11.9ng/ml (range 20.9–143.9ng/ml) occurred 2.1±0.3h (range 1.0–3.5h) and 2.3±0.3h (range 0.5–3.0h) after the first and second GnRH treatments, respectively. The total estrogen concentration gradually increased from the day of both the first and second PGF2α administrations until the LH peak (with great variability) and then gradually declined to the basal level, which was reached at the time ovulation was detected. In experiment 2, 10 cycling and 11 non-lactating anestrus buffaloes were subjected to the Doublesynch protocol with timed artificial insemination (TAI) 16 and 24h after the second GnRH treatment, and 55 cycling buffaloes were inseminated after spontaneous estrus was detected (control group). The pregnancy rates were 60% using TAI on cycling buffaloes (experiments 1 and 2), 55% for anestrus buffaloes (experiment 2), and 27.3% for cycling buffaloes inseminated following spontaneous estrus. The overall pregnancy success rates after the Doublesynch protocol in both cycling and anestrus buffaloes increased by 30.8% compared to spontaneous estrus (58.1% vs. 27.3%).In conclusion, the Doublesynch protocol effectively synchronized ovulation twice (after the first and second GnRH treatments) irrespective of the stage of estrous cycle in Murrah buffaloes. The study also demonstrated that the Doublesynch protocol followed by TAI significantly (P<0.005) enhanced the pregnancy rate in cycling and anestrus buffaloes in comparison to untreated controls during the low breeding season.

Secretion of luteinizing hormone into pituitary venous effluent of the follicular and luteal phase mare: novel acceleration of episodic release during constant infusion of gonadotropin-releasing hormone

We tested the hypothesis that continuous infusion of native GnRH into mares during the estrous cycle, at a dose of 100 μg/h, would elevate circulating concentrations of LH without disrupting the endogenous, episodic pattern of LH release. Ten cyclic mares were assigned to one of two groups (n = 5/group): (1) Control (saline) and (2) GnRH in saline (100 μg/h). On experimental day 0 (3 to 6 d after ovulation), osmotic pumps containing saline or GnRH were placed subcutaneously and connected to a jugular infusion catheter. Blood samples were collected from jugular catheters daily and at 5-min intervals from catheters placed in the intercavernous sinus (ICS) for 8 h on experimental day 4 (luteal phase; 7 to 10 d after ovulation), followed by an additional 6-h intensive sampling period 36 h after PGF2α-induced luteal regression (experimental day 6; follicular phase). Treatment with GnRH increased (P < 0.001) concentrations of LH by 3- to 4-fold in the peripheral circulation and 4- to 5-fold in the ICS. Continuous GnRH treatment accelerated (P < 0.01) the frequency of LH release and decreased the interepisodic interval during both luteal and follicular phases. Treatment with GnRH during the luteal phase eliminated the low-frequency, long-duration pattern of episodic LH release and converted it to a high-frequency, short-duration pattern reminiscent of the follicular phase. These observations appear to be unique to the horse. Further studies that exploit this experimental model are likely to reveal novel mechanisms regulating the control of gonadotrope function in this species.

Effect of synchronization protocols and GnRH treatment on the reproductive performance in goats

Experiment 1 was designed to determine if the estrous response and pregnancy rates differ between two synchronization protocols (Ovsynch vs two PGF2α injections, 10 days apart), in Beetal and Dwarf does. The Ovsynch group of goats (n=14) received an intramuscular injection of a GnRH analogue (12.5μg lecirelin) on day 0 and a treatment of a PGF2α analogue (37.5μg d-cloprostenol), on day 7 – followed by a second injection of the GnRH analogue 48h later (day 9). The double PGF-treated goats (n=14) received two intramuscular injections of a PGF2α analogue 10 days apart. The estrous response did not differ significantly between the regimes (Ovsynch 71%; double PGF 100%). Further, the interval from standing estrus to ovulation (24±3.7h vs 30±2.7h), ovulatory diameter (7.1±0.2mm vs 7.0±0.2mm), pregnancy rate (60% vs 78%) and fecundity (1.6±0.5 vs 1.6±0.7) was not significantly different between the Ovsynch and PGF treatment groups. The objective of Experiment 2 was to determine if the administration of GnRH at the time of natural breeding increases the pregnancy rate in Beetal goats. Goats were randomly allocated to two groups (GnRH, n=11 and Control, n=14). The GnRH-treated does received 12.5μg lecirelin on the day of natural breeding, while the control does received no treatment. The interval from standing estrus to ovulation (31.2±2.9h vs 36.0±5.3h; P>0.05), ovulatory follicle diameter (6.7±0.1mm vs 7.2±0.5mm; P<0.05), pregnancy rate (54% vs 64%; P>0.05) and fecundity (1.5±0.5 vs 1.7±0.5; P>0.05) were evaluated for the GnRH and control does, respectively. It could be concluded that the Ovsynch treatment appeared to be similar to the double PGF protocol in terms of the reproductive response. However, this needs to be tested on a larger herd size. Furthermore, it would seem that the use of GnRH at the time of breeding also did not significantly improve the reproductive rate in goats.

Reproductive performance of dairy cows with luteal or follicular ovarian cysts after treatment with buserelin

In dairy farm management economic losses resulting from cystic ovarian degeneration are well known. In spite of this, neither the definition nor the aetiopathology of ovarian cysts are clear and agreed upon. Also the usual classification in luteal and follicular cysts, requiring ultrasound examination together with assessment of P4 to be accurate, is not very helpful in field conditions. Consequently a single treatment is often provided for both types of cysts, and since the 1970s treatments with GnRH and its analogues have been considered very useful. Nevertheless differences in recovery rates after GnRH treatment in animals with either luteal or follicular cysts are reported. Thus, the aim of this study was to evaluate recovery rate, recovery time and conception rate after treatment with buserelin (GnRH-analogue) in cows with ovarian luteal or follicular cysts. In a 5 years period, 150 cows with cysts out of a total of 990 animals, were detected and treated intravenously between 45 and 60 days PP with 20 μg buserelin. No statistically significant differences were found in recovery rates and in conception rates between the two types of cysts. Comparison of recovery times showed significantly shorter recovery for cows with luteal cysts. The results emphasise the usefulness of GnRH to treat ovarian cysts regardless of their type, in relation to both recovery and conception rates. Intervals from treatment to resumption of ovarian activity were affected by the characteristics of ovarian cysts, with a faster recovery for the luteal type.

Effects of GnRH or Progesterone Treatment on Day 5 Post‐AI on Plasma Progesterone, Luteal Blood Flow and Leucocyte Counts During the Luteal Phase in Dairy Cows

This study was designed to test the effects of progesterone or GnRH treatment on day 5 post-AI on fertility and luteal function in dairy cows and heifers. Five days after AI, 32 animals were randomly assigned to a control, intravaginal progesterone for 14 days progesterone releasing intravaginal device (PRID) or GnRH treatment group. On days 5, 7, 12, 14, 17 and 19 post-AI, each animal underwent colour Doppler ultrasonography of the corpus luteum and blood samples were collected for cell counts and plasma progesterone determination. Through general linear model repeated measures analysis of variance, significant effects were observed of treatment, parity, inseminating bull, reduced vascularization of the CL and pregnancy on plasma progesterone concentrations, whereas mean plasma progesterone and time luteal phase day, and treatment and plasma progesterone concentration on day 5 post-AI were found to, respectively, affect neutrophil and lymphocyte counts throughout the luteal phase. Moreover, two binary logistic regression analyses were performed. Based on the odds ratio, the likelihood of pregnancy by days 26-32 post-AI was 23.4 times higher in animals with high mean progesterone levels throughout the study period, compared with animals with low mean progesterone. The likelihood of reduced CL vascularization was 14 times higher in animals treated with PRID, compared with control and GnRH-treated animals. In conclusion, our results indicate that treatment on day 5 post-AI with PRID reduced subsequent CL vascularization, whereas GnRH treatment increased plasma progesterone concentrations on day 12 post-AI, although an effect was identified of the inseminating bull on plasma progesterone levels. Pregnant animals showed higher mean plasma progesterone concentrations than in nonpregnant ones and heifers higher than in lactating cows, whereas blood cell counts differed depending on the treatment and on the mean plasma progesterone concentration on day 5 post-AI.

Presynchronization with GnRH 7 days prior to resynchronization with CO-Synch did not improve pregnancy rate in lactating dairy cows

The objective was to determine the effect of presynchronization with GnRH 7 d prior to the initiation of resynchronization with CO-Synch on pregnancy/AI (P/AI) of resynchronization in lactating dairy cows, and the effect of GnRH on P/AI from previous breeding. All parity Holstein cows (n = 3287) from four dairy farms were enrolled. Cows not detected in estrus by 28 ± 3 d (Day -7) after a previous breeding were assigned to receive either GnRH (100 μg, im; n = 1636) or no GnRH (Control; n = 1651). Cows not detected in estrus during the 7 d after GnRH underwent pregnancy diagnosis (35 ± 3 d after previous breeding, Day 0); non-pregnant cows (n = 1232) in the Control (n = 645) and GnRH (n = 587) groups were resynchronized with a CO-Synch protocol. Briefly, cows received 100 μg GnRH on Day 0, 25 mg PGF2α on Day 7, and 72 h later (Day 10) were given 100 μg GnRH and concurrently inseminated. Serum progesterone concentrations (n = 55 cows) were elevated in 47.3, 70.9, and 74.5% of cows on Days -7, 0, and 7, respectively. The proportion of cows with high progesterone concentrations on Day -7 and Day 0 were 44.1% and 88.2% (P < 0.003), and 55.2% and 33.2% (P > 0.1), for GnRH and Control groups, respectively. Accounting for significant variables such as locations (P < 0.0001) and parity categories (P < 0.05), the P/AI (35 ± 3 d after AI) for resynchronization was not different between GnRH and Control groups [26.7% (95% CI: 23.2, 30.5; (157/587) vs 28.4% (95% CI: 25.0, 31.9; (183/645); P > 0.1]. There were no significant location by treatment or parity by treatment interactions. Accounting for significant variables such as location (P < 0.0001) and parity categories (P < 0.001), the P/AI was not different between GnRH and Control groups for the previous service [60.2%; 95% CI: 57.9, 62.6; (986/1636) vs 59.1%; 95% CI: 56.7, 61.5; (976/1651); P > 0.1)]. There were no significant location by treatment or parity by treatment interactions. In conclusion, more cows presynchronized with GnRH 7 d prior to resynchronization with CO-Synch had elevated progesterone concentrations at initiation of resynchronization than those not presynchronized. The GnRH treatment 7 d prior to resynchronization with CO-Synch, when given 28 ± 3 d after a previous breeding, did not improve P/AI in lactating dairy cows; furthermore, compared to the control, it did not significantly affect pregnancy rate from the previous breeding.

Neither duration of progesterone insert nor initial GnRH treatment affected pregnancy per timed-insemination in dairy heifers subjected to a Co-synch protocol

Our objectives were to: 1) compare response to cloprostenol, synchrony of ovulation, and pregnancy per timed-AI (P/TAI) in a 5 d versus a 7 d Co-synch + PRID protocol (Experiment 1); and 2) investigate whether the initial GnRH is necessary to achieve acceptable P/TAI in a 5 d Co-synch + PRID protocol (Experiment 2) in dairy heifers. In Experiment 1, 64 Holstein heifers, 15 to 17 mo, were assigned by age to receive 100 μg of GnRH and a PRID for 5 or 7 d (PRID5 and PRID7, respectively). At PRID removal 500 μg of cloprostenol (PGF) was given i.m. Heifers received the second GnRH treatment concurrently with TAI at 72 (PRID5) or 56 (PRID7) h after PRID removal. Transrectal ultrasonography monitored ovarian dynamics, ovulation synchrony, and pregnancy status (28 and 45 d after TAI). Plasma progesterone concentrations were determined at PRID removal and TAI. Five of seven heifers that ovulated before TAI became pregnant, and only two heifers did not respond to PGF treatment in the PRID5 group. Five PRID5 and 2 PRID7 heifers failed to ovulate after the second GnRH. However, P/TAI did not differ between PRID5 (59.4%) and PRID7 (58.1%). Overall ovulation response to first GnRH treatment was only 31.7%, and a larger proportion of heifers that did not ovulate became pregnant (65.1 versus 45.0%). In Experiment 2, 56 Holstein heifers, assigned as in Experiment 1, were subjected to a PRID5 protocol with (PRID5G) or without (PRID5NoG) GnRH at PRID insertion; all heifers were TAI 72 h after PRID removal. Transrectal ultrasonography and progesterone determinations were performed as in Experiment 1. Pregnancy per TAI did not differ whether or not heifers received GnRH at PRID insertion (67.9 versus 71.4%). Consistent with our previous findings, seven of nine heifers that ovulated before TAI became pregnant, and only two heifers did not respond to PGF treatment. Combining both experiments, length of proestrus but not ovulatory follicle diameter was identified as a significant predictor of probability of pregnancy 28 d after TAI, with a maximum predicted probability of 80.1% when the length of proestrus was 3 d. In summary, a PRID5 protocol resulted in comparable P/TAI to a PRID7 protocol. Most of the heifers that ovulated before TAI in the PRID5, PRID5G, and PRID5NoG protocols became pregnant. More than one PGF or a GnRH treatment at PRID insertion in a 5 d Co-synch + PRID protocol was not required to achieve acceptable P/TAI in dairy heifers.

Importance of estrus on pregnancy per insemination in suckled Bos indicus cows submitted to estradiol/progesterone-based timed insemination protocols

The objective was to evaluate the effect of estrus occurrence (based on removal of tail-head marks) on ovarian responses and pregnancy per AI (P/AI; 30 d after AI) in suckled Bos indicus beef cows submitted to timed AI (TAI) protocols. Cows received an intravaginal device containing 1.0 g progesterone, and 2.0 mg estradiol benzoate im; 8 d later, the intravaginal device was removed, and they were given PGF2α (0.25 mg of cloprostenol sodium) and 300 IU of eCG, with TAI 48 to 52 h later. In Experiment 1, cows were assigned to receive one of three treatments: 1 mg of estradiol cypionate (ECP) im at progesterone (P4) device removal (N = 178); 10 μg of GnRH im at TAI (N = 190); or both treatments (N = 172). In cows given estradiol (ECP or ECP + GnRH), more displayed estrus (P = 0.002) and became pregnant (P < 0.0001) compared with those receiving only GnRH. In Experiment 2, the effect of the occurrence of estrus on ovarian responses was evaluated in cows (N = 53) synchronized using ECP at device removal. Cows that displayed estrus had a greater diameter of the largest follicle (LF) at device removal (P < 0.0001), a greater diameter at TAI (P < 0.0001), a greater ovulation rate (P = 0.02), a larger CL (P = 0.02), and a greater P4 concentration (P < 0.0001) than cows that did not display estrus. In Experiment 3, the effect of GnRH treatment on P/AI at TAI was evaluated in cows that received ECP at device removal, and either displayed, or did not display, estrus (N = 726). There was no estrus by GnRH interaction (P = 0.22); the P/AI was greater (P < 0.0001) in cows that displayed estrus (61.9%) than cows that did not display estrus (41.4%). However, GnRH did not improve (P = 0.81) P/AI (GnRH = 53.7% vs. no GnRH = 52.6%). In conclusion, exogenous estradiol at device removal increased both the proportion of suckled Bos indicus cows that displayed estrus and P/AI. Cows that displayed estrus had better ovarian responses (i.e., larger follicles at TAI, a greater ovulation rate, larger CL, and greater P4 concentrations) following an estradiol/P4-based synchronization protocol. Although occurrence of estrus improved pregnancy outcomes, GnRH at TAI did not improve P/AI in suckled Bos indicus cows treated with ECP, regardless of estrus occurrence.

Regulation of pituitary inhibin/activin subunits and follistatin gene expression by GnRH in female rats

Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homodimeric molecule (β(B)-β(B)), while inhibin B contains an α and a β(B) subunit. The regulation of gene expression of α, β(B), and follistatin by local and endocrine hormones was examined in pituitaries from female rats and in perifused pituitary cells by RT-PCR. Ovariectomy (OVX) induced an elevation in the mRNA level of α and β(B) subunits and follistatin. Short-term (4 h) treatment of pituitary cells with GnRH decreased both the inhibin α and the inhibin/activin β(B) subunit mRNA levels, while long-term treatment (20 h) with 100 nM GnRH stimulated the expression of both subunits. In contrast, the mRNA level of follistatin was elevated after the short-term GnRH treatment. Long-term exposure of pituitary cells to estradiol and inhibin B suppressed the mRNA expression of β(B) and had no effect on the expression of α subunit and follistatin. Our results demonstrate that the increased expressions of inhibin/activin subunits and follistatin in the post-OVX period can be induced by the lack of gonadal negative feedback, resulting in a high GnRH environment in the pituitary. This study reports for the first time that GnRH administered in high doses and for a long period stimulates the gene expression of inhibin/activin subunits and thereby may contribute to the stimulatory effect of OVX on the expression of these genes.

Comparison of responses to Ovsynch between Holstein-Friesian and Swedish Red cows

The Ovsynch protocol was designed to synchronize ovulation, thereby allowing timed artificial insemination (TAI) of all cows without detection of estrus. However, the effectiveness of Ovsynch in different breeds of dairy cows has not been previously compared. The aim of this study was to compare the response to Ovsynch in cycling lactating Holstein-Friesian (HF) and Swedish Red (SR) dairy cows. A total of 495 cyclic cows (n=347 HF, n=148 SR) were housed together and treated with Ovsynch (GnRH – 7 d – PGF2α – 56h – GnRH – 16 to 18h – TAI). Ovulatory responses, synchronization rate, maximal follicle size at the time of AI, and percentage of pregnant cows per AI (P/AI at 31 and 62 d after AI) were compared between breeds. Ultrasonography was performed during Ovsynch at first GnRH, PGF2α, at time of AI, and 7 d after AI. Ovulatory response and synchronization rate were similar in HF versus SR cows (60.2 vs. 62.2%; 88.4 vs. 88.5%, respectively). Cows that ovulated to the first GnRH of Ovsynch had smaller follicle size at AI (15.9±0.1 vs. 16.4±0.2mm). Maximal follicle size at AI was greater for HF (16.4±2.2mm) than SR (15.5±2.3mm) cows. The P/AI was greater for SR than HF cows at the 62-d pregnancy diagnosis (56.1 vs. 46.1%). In addition, pregnancy loss between 31 and 62 d of pregnancy was greater in HF (10.1%) than SR (3.5%) cows. Fertility was less in HF cows during the hot season (57.7 in cold vs. 38.1% in the hot season), whereas such a decrease was not observed in SR (60.0 in cold vs. 53.5% in the hot season) cows. Thus, although the GnRH treatments of Ovsynch were equally effective in SR and HF cows, pregnancy outcomes (P/AI at d 62 and pregnancy survival) were greater in SR than HF cows, and P/AI in SR cows was not compromised during the hot season as was found for HF cows.

Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors

The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi ( Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH ( n = 24), which consisted of eight pFSH injections, or FSH/eCG ( n = 24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2 mg estradiol benzoate and the insertion of an intravaginal progesterone-releasing device on Day 0. On Day 4, superstimulatory treatments were initiated and 100 mg pFSH was divided into twice daily decreasing doses over a 4-day period. In the FSH/eCG group, the last two doses of pFSH were replaced by two doses of eCG (150 IU eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg PGF 2α was injected i.m. The intravaginal progesterone-releasing device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg GnRH 18 h later. All females were artificially inseminated with frozen–thawed semen from the same bull 6 and 18 h after GnRH treatment. Seven days after GnRH treatment, embryos/ova were recovered and classified. Follicular superstimulatory (number of follicles ≥6 mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means ± standard error. The FSH/eCG group had higher superstimulatory (33.8 ± 3.9 compared to 23.8 ± 2.6 follicles; P = 0.03) and SOV (16.8 ± 2.9 compared to 10.8 ± 2.1 CL; P = 0.10) responses. Although the number of total ova/embryos was not different between groups (8.2 ± 1.8 compared to 5.9 ± 1.4 for FSH/eCG and FSH groups, respectively; P = 0.25), the number (5.8 ± 1.3 compared to 2.6 ± 0.7; P = 0.02) and percentage (75.6 ± 5.7 compared to 53.2 ± 9.7%; P = 0.05) of transferable embryos was greater for the FSH/eCG females. Therefore, there was improvement in follicular superstimulatory and SOV responses and embryo quality in FSH/eCG-treated females.

Gonadotropin-Releasing Hormone Induces miR-132 and miR-212 to Regulate Cellular Morphology and Migration in Immortalized LβT2 Pituitary Gonadotrope Cells

GnRH is central to the regulation of reproductive function. It acts on pituitary gonadotropes to stimulate LH and FSH synthesis and secretion. We had previously presented evidence for translational control of LHβ synthesis; therefore we investigated whether micro-RNAs might play a role in GnRH regulation in LβT2 cells. We show here that GnRH strongly induces the AK006051 gene transcript that encodes two micro-RNAs, miR-132 and miR-212, within the first intron. We show furthermore that the AK006051 promoter region is highly GnRH responsive. We verify that the p250Rho GTPase activating protein (GAP) is a target of miR-132/212 and show that GnRH treatment leads to a decrease in mRNA and protein expression. This reduction is blocked by an anti-miR to miR-132/212 and mimicked by a pre-miR-132. GnRH inhibits p250RhoGAP expression through a miR-132/212 response element within the 3'-untranslated region. The loss of p250RhoGAP expression leads to activation of Rac and marked increases in both the number and length of neurite-like processes extending from the cell. Knockdown of p250RhoGAP by small interfering RNA induces the same morphological changes observed with GnRH treatment. In addition, loss of p250RhoGAP causes an increase in cellular motility. Our findings suggest a novel pathway regulating long-term changes in cellular motility and process formation via the GnRH induction of miR-132/212 with the subsequent down-regulation of p250RhoGAP.

Comparison of long-term progestin-based protocols to synchronize estrus before fixed-time artificial insemination in beef heifers1

Two experiments were conducted to compare pregnancy rates resulting from fixed-time AI (FTAI) after administration of 1 of 2 long-term controlled internal drug release (CIDR)-based protocols. Heifers were assigned to treatment by age, BW, and pubertal status. The CIDR Select-treated heifers (Exp. 1, n = 37; Exp. 2, n = 192) received a CIDR (1.38 g of progesterone) from d 0 to 14, followed by 100 µg of GnRH, intramuscularly (i.m.) 9 d after CIDR removal (d 23) and PGF(2α) (25 mg, i.m.) 7 d after GnRH treatment (d 30). Heifers assigned to the Show-Me-Synch protocol (Exp. 1, n = 40; Exp. 2, n = 200) received a CIDR from d 0 to 14, followed by PGF(2α) 16 d later (d 30). Artificial insemination was performed at 72 or 66 h after PGF(2α) treatment for the CIDR Select- and Show-Me-Synch-treated heifers, respectively, and each heifer was given GnRH (100 µg, i.m.) at the time of AI. In Exp. 1, ovaries of each heifer were examined by transrectal ultrasonography on d 23 and 30 to characterize follicular dynamics. Follicles ≥5 mm and the presence of corpora lutea were recorded. On d 25, ovaries of each heifer were examined to characterize the status of dominant follicles recorded on d 23. Heifers were fitted with HeatWatch (DDx Inc., Denver, CO) estrus-detection transmitters at PGF(2α) to characterize estrus distribution up to FTAI. The diameter of dominant follicles on d 23 at PGF(2α) and on d 30, and the estrous response after PGF(2α) treatment up to the point of FTAI did not differ between CIDR Select- and Show-Me-Synch-treated heifers. Concentrations of progesterone in serum at PGF(2α) were greater (P = 0.07) in Show-Me-Synch- than CIDR Select-treated heifers (6.0 vs. 4.8 ng/mL, respectively). Pregnancy rates of heifers resulting from FTAI did not differ (P = 0.33) between CIDR Select- and Show-Me-Synch-treated heifers (CIDR Select, 59%; Show-Me-Synch, 70%). In Exp. 2, FTAI pregnancy rates tended (P = 0.07) to be greater in Show-Me-Synch-treated (62%) than in CIDR Select-treated (51%) heifers. Pregnancy rates at the end of the breeding season did not differ (P = 0.72; CIDR Select, 85%; Show-Me-Synch, 83%) between treatments. In summary, pregnancy rates resulting from FTAI were comparable for heifers assigned to each of the 2 long-term progestin-based protocols. The reduced treatment cost and animal handling associated with administration of the Show-Me-Synch protocol offer distinct advantages over the CIDR Select protocol despite similarities in pregnancy rates resulting from FTAI.