Selecting oocyte donors based on anti-Müllerian hormone (AMH) concentrations: A critical analysis of using cutoff values as exclusion criterion for an in vitro embryo production program in Gir cattle.

The aims of this study were to determine anti-müllerian hormone (AMH) cutoff values for selecting Gir (Bos taurus indicus) oocyte donors and estimate the impact of using AMH concentrations as a selection criterion. In Exp. 1, Gir heifers (n=120) were sampled for AMH analysis and submitted to ovum pick-up and in vitro embryo production (OPU-IVEP). AMH cutoff values were calculated using ROC analysis or, alternatively, by the successive exclusion of heifers with the lowest AMH values. The correlations between AMH and OPU-IVEP outcomes were significant (P<0.001), though low or moderate (r= 0.34-0.52). We estimated an improvement (P<0.05) after the use of AMH cutoff values to select donors of +15.3% for total oocyes, +19.4% for viable COC, and +23.4% for blastocysts. This selection pressure, however, led to the exclusion of 32.8%, 37.9%, and 50.0% of the initial potential donors, respectively. In Exp. 2, we analyzed data from OPU-IVEP sessions of 658 Gir donors with known genomic values for predicted transmitting ability for milk (GPTAm) and age at first calving (GPTAafc). The selection based on the number of oocytes recovered had no effect (P>0.05) on the average GPTAm nor GPTAafc values of the remaining donors. In summary, plasma AMH ≥700 pg/mL is a cutoff value that can be used to select Gir heifers with a greater potential as oocyte donors. Nevertheless, this selection leads to the exclusion of up to 50% of potential donors. Finally, exclusion of poor responders had no effect on mean genomic estimates for milk production or age at first calving in the selected subset of donors.

RA33, an analogue of resveratrol, improves the development of in vitro-fertilized bovine embryos.

Oxidative stress is an undesirable effect of in vitro culture, which requires antioxidant supplementation. This study investigated the analogue of resveratrol (RA33) as an alternative to resveratrol, an antioxidant molecule, for the in vitro culture of in vitro-fertilized bovine embryos. The effect of different concentrations of RA33 on embryo development was evaluated and a comparison between RA33 and resveratrol was performed. The cleavage rate was higher (P < 0.05) with 2.5 µM (69.0 ± 4.4%) than at 0, 0.1 or 0.5 µM RA33 (62.1 ± 2.0%, 60.7 ± 5.9% and 56.7 ± 5.8%, respectively). The blastocyst rates on days 7 and 8 post-fertilization with 2.5 µM RA33 (19.4 ± 3.3% and 24.6 ± 3.3%, respectively) were higher (P < 0.05) than for 0 µM (12.4 ± 2.5% and 15.2±2.5%, respectively). When 2.5 µM RA33 was compared with 0.5 µM resveratrol, similar (P > 0.05) cleavage and blastocyst rates were found between them, but the cleavage rate was higher (P < 0.05) in the control (80.8 ± 3.4%) than for the resveratrol treatment (76.4 ± 3.6%). The numbers of apoptotic cells and the apoptotic index were lower (P < 0.05) with RA33 (6.5 ± 0.6 cells and 6.4 ± 0.7%, respectively) and resveratrol (5 ± 0.8 cells and 5.5 ± 1.0%, respectively) than in the control group (9.8 ± 1.2 cells and 8.9 ± 1.1%, respectively). In conclusion, RA33 can enhance the preimplantation development of in vitro-fertilized bovine embryos and be an alternative to resveratrol in embryo culture medium.

Improvement in early antral follicle development and gene expression modulation prior to follicle aspiration in bovine cumulus-oocyte complexes by equine chorionic gonadotropin.

We aimed to evaluate the morphological ovarian response to equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) and its effects on the molecular phenotype of immature cumulus-oocyte complexes (COCs) from Nelore cow (Bos indicus) donors. To this end, 20 Nelore cows were distributed randomly into the synchronized-OPU (Sync-OPU) and synchronized plus stimulated-OPU (Sync + eCG-OPU) groups using a cross-over experimental design, as each cow was used in both treatments. On a random day of the estrus cycle (Day 0), all cows received an intravaginal implant with 1.0 g of progesterone and 2 mg IM of estradiol benzoate. On the morning of Day 3, only the Sync + eCG-OPU group received 400 IU of eCG IM. On the morning of Day 5, the P4 device was removed and OPU was conducted in both groups. Before OPU management, ultrasonography was used to identify and measure the follicles. The aspirated COCs were morphologically classified based on their cumulus cells (CC) layers and the texture of the ooplasm. The COCs classified as Grade 1, Grade 2, and Grade 3 were considered viable and used for the assessment of quality markers. Oocytes and CC were mechanically separated from pools of 25 immature COCs of the Sync-OPU and Sync + eCG-OPU groups immediately after the follicular aspiration and stored at -80 °C until RNA extraction. Relative quantification of several markers for oocyte quality was assessed by RT-qPCR. The eCG treatment increased the number of follicles sized 3.0-5.0 mm and >5.0 mm compared to that in Sync-OPU group. Moreover, the protocol with eCG improved the total number of oocytes and the number of viable oocytes, which is related to a high number of oocytes in Grade 3. Regarding the impact on transcriptional regulation in immature oocytes, the mRNA encoding BMP15, SMAD1, SMAD2, SMAD3, ACACA, and CPT1A was upregulated in Sync + eCG-OPU compared with the Sync-OPU group. Moreover, the relative mRNA abundance of CTSZ, a member of the cathepsins family functionally related to reduced oocyte competence, was lower in the Sync + eCG-OPU group than in the Sync-OPU group. In addition, CC CTSB, CTSS, and CTSK mRNA abundances were lower in the Sync + eCG-OPU group than in the Sync-OPU group. However, the relative abundance of AREG and EREG mRNA was higher in CC recovered from cows stimulated with eCG. In conclusion, the eCG approach addressing follicular stimulation in Nelore cows had a positive impact on early antral follicle development, followed by a positive morphological and molecular phenotype in bovine COCs.

Timing of early resynchronization protocols affects subsequent pregnancy outcome in dairy cows.

The aim of this study was to evaluate the outcomes of an early resynchronization protocol (Resynch) initiated at different timepoints after timed artificial insemination (TAI) and with unknown pregnancy status. Holstein cows (n = 164) were submitted to the following TAI protocol: D0, insertion of an intravaginal progesterone (P4) device and 2 mg im estradiol benzoate (EB); D8, removal of P4 device and treatment with 0.5 mg im sodium cloprostenol (PGF); D9, 0.1 mg im Lecirelin (LEC); and D10, TAI1. Cows were then randomly assigned to Resynch protocols starting either on day 20 (Resynch20D, n = 82) or 25 after TAI1 (Resynch25D, n = 82) with the insertion of a new P4 device and EB treatment. In both groups, P4 device was removed on day 8 after the beginning of Resynch, the same day of pregnancy diagnosis by ultrasonography. In pregnant cows there was no further action. Non-pregnant cows were treated with 0.5 mg im PGF, had a blood sample collected for serum P4 analysis and we measured and recorded the size of the largest follicle and the presence or absence of a corpus luteum (CL). One day later, cows were treated with 0.1 mg im LEC and TAI2 occurred 12-14 h later. The diameter of the largest follicle and serum P4 were compared between groups by ANOVA for the main effects of treatment, presence of a CL, and their interaction, whereas pregnancy per artificial insemination (P/AI) and the percentage of cows with a CL on the day of ultrasonography were analyzed using the Chi-square test. Follicle diameter on day 8 of Resynch was greater for cows in the Resynch20D group compared with Resynch25D (15.9 ± 3.9 vs 12.2 ± 2.5 mm, respectively; P = 0.046). The Resynch25D group had a greater percentage of cows with a CL (51.9 vs 18.9%, respectively; P = 0.0008) and higher serum P4 (2.8 ± 1.1 vs 1.7 ± 0.8 ng/mL; P = 0.041) at the end of the protocol compared with Resynch20D. P/AI at TAI1 was 35.4 and 36.6% (P > 0.10) for cows enrolled in Resynch20D and Resynch25D groups, respectively. P/AI to TAI2, after Resynch protocols, was greater in Resynch25D than Resynch20D (44.2 vs 22.6%, respectively; P < 0.05). In conclusion, starting an early resynchronization protocol 25 days after TAI increases P/AI compared with starting 20 days after TAI, and this was associated with a presumed greater proportion of cows with a functional CL at the moment of P4 device removal.

Weight gain potential affects pregnancy rates in bovine embryo recipients raised under pasture conditions.

The aim of the present study was to evaluate the effect of differences in body weight gain after embryo transfer on the pregnancy rates of crossbred heifers used as recipients and raised under a grazing system. The study was performed during the dry (April to September) and the rainy (October to March) seasons. The embryos transferred were produced by in vitro fertilization. The body weight of each recipient was measured immediately before the embryo transfer and 23 to 25 days later, when the diagnosis of pregnancy was performed by ultrasonography. The associations among initial body weight (IBW), daily body weight gain (DWG), season, and pregnancy rate were evaluated using a logistic procedure that included the effect of the IBW, season, and linear and quadratic effects of the DWG. Altogether, there was no effect of season and pregnancy rates did not change between the dry and rainy seasons (42.3 vs. 45.8%, respectively; P > 0.05). However, the pregnancy rate was greater in the recipients with daily body weight gains over 250 g/day, regardless of the season. In addition, the pregnancy rate of the recipients was better (P < 0.04) explained by a logistic regression model that included the linear and quadratic effects of the DWG. The probability of each heifer to become pregnant according to DWG is explained by the follow equation: P(y = 1) = (Exp((-1.06703 + 0.0108 * DWG - 0.00002 * DWG ^ 2)))/(1 + Exp((-1.6703 + 0.0108 * DWG - 0.00002 * DWG ^ 2))). In conclusion, body weight gain potential is a critical factor for the pregnancy rates of in vitro embryo recipients managed under grazing systems.

Sincronização folicular e vascularização do folículo dominante em novilhas mestiças tratadas com estradiol / Follicle synchronization and changes in vascularization of the dominantfollicle in crossbred heifers after estradiol treatment

Avaliou-se a sincronização folicular e vascularização na parede do folículo dominante (FD) após o tratamento com Cipionato (CE) ou Benzoato de estradiol (BE). No Experimento 1, 45 novilhas receberam implante de P4 (1,0g) e foram distribuídas em 3 grupos de acordo com éster de estradiol administrado pela via intramuscular: 1mL salina; 0,5mg CE ou 2,0mg BE. O diâmetro do FD e a emergência folicular (EF) foram monitorados por 5 dias. No experimento 2, D0 (dia 0): 30 novilhas receberam implante de P4 e 2,0mg BE; D8, retirada do implante e 0,5mg de prostaglandina (cloprostenol); D9: tratamentos: 1mL salina; 0,5mg CE ou 1,0mg BE. O diâmetro e a vascularização do FD foram monitorados do D8 ao D10. Os dados foram analisados por ANOVA ou Kruskal-Wallis, e as médias comparadas por Tukey (5%). No experimento 1, a EF ocorreu mais tarde (P 0,0013) no grupo BE (4,3±0,8 vs. 3,5±0,8d). No experimento 2, o fluxo sanguíneo do FD aumentou (P 0,001) 24h após o tratamento com BE. A taxa de ovulação foi semelhante (P>0,05) entre os grupos (80,0, 70,0 e 55,5%, BE, CE e salina). Conclui-se que nas concentrações utilizadas, o BE, no início do protocolo, retardou a EF e, no final do protocolo, promoveu uma melhor sincronia do FD demonstrada pela elevação do fluxo sanguíneo.

The study aimed to evaluate synchronization and blood flow of the dominant follicle (DF) after estradiol cypionate (EC) or benzoate(EB) treatment. In experiment 1, progesterone (P4) implants (1.0g) were inserted in 45 heifers, which were given 1mL saline,0.5mg EC or 2.0mg EB. The diameter of the DF and follicle emergence (FE) were recorded for 5 days. For experiment 2, 30 heiferswere given: D0 (day 0): P4 implant and 2.0mg EB; D8: implant removal and 0.5mg of prostaglandin (cloprostenol). On D9, theywere assigned in 1mL saline, 0.5mg EC or 1.0mg EB. From D8 to D10, the diameter and blood flow of the DF was monitored.Data were analyzed by ANOVA or Kruskal-Wallis test, and compared by Tukey (5%). In experiment 1, the FE occurred later(P0.05) between groups (80.0, 70.0 and 55.5%, EB, EC and saline). The interpretation was that,in these concentrations, the EB, at the beginning, delayed FE and, at the end of the protocol, promoted a better synchrony of theDF, confirmed by the increase of the blood flow.