The Effect of A PRID®Delta-Based Estrus Synchronization Protocol On Pregnancy Rates In Cows With No Estrus Symptoms

The present study aimed to determine the effects of progesterone-based gonadotropin-releasing hormone (GnRH), prostaglandin F2α (PGF2α), and equine chorionic gonadotropin (eCG) injections on progesterone profiles and pregnancy rates in cows with no estrus symptoms within 60 days after parturition. A total of 80 cows were included in the study. All animals had the progesterone-releasing device PRID®Delta placed intravaginally for nine days with an injection of GnRH. On the eighth day, PGF2α was injected, and PRID®Delta was removed from the vagina on day nine. Artificial insemination was carried out 60 hours after PRID®Delta removal. In half of the animals (n = 40), 600 IU of eCG was injected when PRID®Delta was removed on the ninth day before artificial insemination 60 hours later. Blood samples were taken from the tail vein on days 0 and 8 to determine progesterone levels. The pregnancy rate in the group that received eCG was 37.5%, while it was 27.5% in those that did not (P = 0.4). While the dominant follicle diameter was 15.5 mm in cows injected with eCG during timed artificial insemination, the follicle diameter was 12.4 mm in cows with no eCG injection (P <0.001). There were no differences in serum progesterone values in blood samples taken until the time of artificial insemination. However, progesterone values in the blood taken during artificial insemination were 0.94 ng/ml in the eCG- group and 0.72 ng/ml in the eCG+ group (P <0.05). As a result, it was determined that eCG injections, in addition to progesterone-based GnRH and PGF2α applications, increased the pregnancy rates in cows without symptoms of estrus. The dominant follicle diameter was larger in cows treated with eCG during artificial insemination; however, follicle size did not increase the pregnancy rate.

Assessment of Biostimulation Methods based on Chemical Communication in Female Doe Reproduction

Biostimulation is an animal management practice that helps improve reproductive parameters by modulating animal sensory systems. Chemical signals, mostly known as pheromones, have a great potential in this regard. This study was conducted to determine the influence of short-term female rabbit exposure to different conditions, mainly pheromone-mediated, on reproductive parameters of inseminated does. Groups of 60 females/each were exposed to 1) female urine, 2) male urine, 3) seminal plasma and 4) female-female interaction, just before artificial insemination, and compared to isolated females controls (female-female separated). The following reproductive parameters were analyzed for each group: receptivity (vulvar color), fertility (calving rate), prolificacy and number of born alive and dead kits &frasl; litter. Our results showed that the biostimulation methods employed in this experiment did not significantly improve any of the analyzed parameters. However, female doe exposure to urine, especially to male urine, slightly increased fertility levels when compared to the rest of the experimental conditions. Female-female interaction before artificial insemination, which is a common practice in rabbit farms, did not have any effect, which suggests its removal to avoid unnecessary animal management and time cost. On the other hand, fertility ranges were lower for animals with pale vulvar color whereas no differences were noticed among the other three colours which measure receptivity (pink, red, purple), thus suggesting that these three colours could be grouped together. Additionally, equine chorionic gonadotropin injection could be replaced with various biostimulation methods, therefore reducing or replacing current hormonal treatments, and contributing to animal welfare and to a natural image of animal production.

The Effects of Ram Exposure, Progestogen and eCG on Reproductive Performance of Fat Tail Ewes in Non-Breeding Season

Sheep are considered short-day breeders. Exposure of rams to anestrus ewes results in an increase in LH secretion. The aims of this study was to determine whether the presence and exposure of rams can effect on reproductive performance such as: pregnancy rate, litter size, sex of lamb, fecundity rate and prolificacy rate of fat tail Iranian Shaal breed ewes in non-breeding season using progesterone (p4) analogue with or without equine chorionic gonadotropin (eCG). Totally, 100 ewes out of 138 non-cyclic ewes with <0.5ng/ml P4 were selected for this study. They were aged 2 to >7 years old. They were stratified to two groups (control and treatment). The ewes in treatment and control groups received 13 days a sponge containing 60 mg medroxyprogesterone acetate (MPA). The treatment group consists of 3 subgroups and each group contains 25 ewes equally. They were included: 1- P4, eCG and Ram exposure. 2- P4 and ram exposure. 3- P4 and eCG. Ewes in control group received only P4. The ewes were located in isolated corrals for 40 days. The ram to ewe ratio was 1 to 5. Twenty healthy rams were housed in an isolated pen from ewes with distance 1500 m during 40 days. Rams exposed three days before sponge withdraw (day 10). They were separated by considerable distance (2 meters) using fence. The related groups received 500 IU eCG on the day of sponge removal (day 13). The rams were released into the ewe flock after removing the sponge in treatment and control groups. Pregnancy diagnosis performed using transabdominal ultrasonography. The obtained data analyzed using SPSS version 16. The conception rate in treatment and control groups were differed significantly (P<0.01). The conception rate in P4+ram exposure+ eCG, P4+ramexposure and P4+ eCG were 96%(no.24), 80%(no.20) and 88%(no.22), respectively. However, the conception rate in control group was 60% (no. 15). The fecundity rates were 136%, 100% and 124% in treatment subgroups, however, it was 84% in control group, respectively. There were no significant differences among genus and weight of lambs in treatments subgroups and control group (P>0.05). It is concluded that ram exposure can increase significantly reproductive performance in non-breeding season fat tailed ewes Shaal breed.

Large-scale DNA demethylation occurs in proliferating ovarian granulosa cells during mouse follicular development

During ovarian follicular development, granulosa cells proliferate and progressively differentiate to support oocyte maturation and ovulation. To determine the underlying links between proliferation and differentiation in granulosa cells, we determined changes in 1) the expression of genes regulating DNA methylation and 2) DNA methylation patterns, histone acetylation levels and genomic DNA structure. In response to equine chorionic gonadotropin (eCG), granulosa cell proliferation increased, DNA methyltransferase (DNMT1) significantly decreased and Tet methylcytosine dioxygenase 2 (TET2) significantly increased in S-phase granulosa cells. Comprehensive MeDIP-seq analyses documented that eCG treatment decreased methylation of promoter regions in approximately 40% of the genes in granulosa cells. The expression of specific demethylated genes was significantly increased in association with specific histone modifications and changes in DNA structure. These epigenetic processes were suppressed by a cell cycle inhibitor. Based on these results, we propose that the timing of sequential epigenetic events is essential for progressive, stepwise changes in granulosa cell differentiation.

Equine Chorionic Gonadotropin as an Effective FSH Replacement for In Vitro Ovine Follicle and Oocyte Development

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.

279 ReBreed21, a Rapid Reinsemination Program: Fertility in Bos Indicus Cattle of Different Parities

This study evaluated efficiency of a reinsemination program allowing timed AI (TAI) every 21 days (ReBreed21; RB21) vs. a traditional Resynch program in Nellore females (n = 2,085) of different parities. First TAI followed estradiol (E2)/progesterone (P4)-based synchronization, and was considered d0. For RB21 (n= 1,357), cows received P4 insert on D12. On D19, P4 was removed, followed by treatment with equine chorionic gonadotropin (eCG) and 0.6 mg E2 Cypionate (RB21+EC, n = 60) or nothing (RB21, n=687). On D21, Doppler ultrasound was used to determine CL blood flow [&lt; 25%=nonpregnant (NP)] and NP cows received TAI immediately together with 25µg licerelin acetate (GnRH). A second RB21 program using the same groups was done in cows that received TAI on D21 to allow a third potential TAI on D42. Pregnancy diagnosis was performed on D33 after each TAI. Cows designated pregnant on D21 but NP on D33, were termed False-Positive (FP). On D33, all control (n = 728) designated NP and FP cows were resynchronized with P4+E2 protocol for the second TAI at D42. Data were analyzed with SAS and shown in Table 1. For all cows, there were no differences in the three groups (Control, RB21, RB21+EC) for pregnancy/AI (P/AI). In nulliparous heifers, cumulative pregnancies were greater for both RB21 groups compared to Controls at D21 or D42 of the breeding season. Primiparous cows had greater cumulative pregnancies at D21 but not D42. Primiparous cows at second TAI had lower P/AI in RB21 (34.2%) vs. Control (51.7%); however, RB21-EC (55.8%) was similar to Control. No other parities had differences between RB21 and RB21+EC. Multiparous cows had high fertility at first TAI [69.3% (418/603)] and no differences between groups in cumulative pregnancies (P = 0.23). Thus, ReBreed21 program increases the reproductive efficiency in Nulliparous and Primiparous but not Multiparous Bos Indicus cattle. Adding EC to RB21 program only improves P/AI in Primiparous cows.

Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

Background Equine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGβ/αΔ56, substitution of Asn56 of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR). Results Both rec-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200–250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α were in the ranges of 40–45, 37–42, and 34–36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5–10 kDa. Rec-eCGβ/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn56 of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities. Conclusions Rec-eCGβ/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn56 of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

EFFECT OF THE ADMINISTRATION OF SERUM AND RECOMBINANT EQUINE CHORIONIC GONADOTROPIN ON OVARIAN DYNAMICS AND CONCEPTION RATE IN ANESTRUS PRIMIPAROUS BRANGUS COWS

The objective was to determine the size of the dominant ovulatory follicle, the ovulation time and the conception rate of cows in anestrus treated with serum equine chorionic gonadotropin (eCG), recombinant chorionic gonadotropin and untreated controls. 57 anestrus primiparous Brangus cows were used with 70 ± 26 days of calving and a body condition of 2.50 ± 0.15. They were synchronized with a FTAI protocol based on estrogen and progesterone devices (DI). Upon removal of DI, 3 groups were made according to the type of eCG applied: serum eCG received 400 IU im of equine Chorionic Gonadotropin, recombinant eCG received 140 IU of recombinant Equine Chorionic Gonadotrophin and Control did without treatment. Follicular size and time of ovulation (normal or abnormal) was determined at DI removal, during FTAI and 7 days later. An ANOVA was performed to determine the effect of the treatment on ovarian dynamics and a chi-square test and correspondence analysis to establish associations. In normal ovulation cows at the FTAI, 94.4% of the serum eCG, 55.5% of the recombinant eCG and 71.4% of the Control cows presented a dominant ovulatory follicle (DOF), which in those treated with serum eCG, it was 1.4 mm greater in relation to the Control (P = 0.0073). The ovulation rate, in normal and abnormal ovulation cows, was 94.4% for serum eCG, and for recombinant eCG and 66.6% for Controls. A significant association was detected between ovulation time and the treated groups (P = 0.0042). Normal ovulation was 38.9% higher in cows with serum eCG in relation to those treated with recombinant eCG and 50.8% in comparison with Control. A significant relationship was observed between the conception rate and the groups treated with the different Gonadotropins (P = 0.0574), being 66.7% in serum eCG, 50% in recombinant eCG and 28.6% in the Control. The use of eCG, either serum or recombinant, in anestrus primiparous cows, stimulates the development of DOF at FTAI, increases ovulation rate and improves conception in relation to untreated cows.