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.