The coasting time affects the quality of cumulus-oocyte complexes in superstimulated ewes.

We hypothesized that the coasting time may be beneficial to the quality of cumulus-oocyte complexes recovered from live ewes, as reported in cattle. The present study assessed the effect of coasting times on the quantity and quality of cumulus-oocyte complexes (COCs) in sheep. All ewes were subjected to the "Day 0 protocol", followed by an ovarian stimulation (80 mg of pFSH in three decreasing doses), varying only the coasting time [12 (G12), 36 (G36), or 60 h (G60]. In Experiment 1, data regarding follicular population was assessed. In Experiment 2, the COC quality was checked by their morphology, brilliant cresyl blue (BCB) test, evaluation of chromatin condensation pattern, and oocyte diameter. In Experiment 3, genes related to oocyte developmental competence were evaluated in BCB + COCs. The oocytes in the G60 group had more (P < 0.05) large follicles than the other groups and oocytes with a greater diameter than the G12. Oocyte morphology was similar (P > 0.05) among groups, as well as the BCB + COCs quantity. The G60-oocytes presented a better (P < 0.05) configuration of chromatin condensation compared with the other groups and a greater (P < 0.05) gene expression of BMP15, MATER, ZAR1, and PTGS2 compared with G12, and PTGS2 and HAS2 compared with G36 group. In conclusion, 60 h of coasting time positively affects the quality of COCs recovered after subjecting ewes to the "Day 0 protocol" and ovarian superstimulation. Implementing the appropriate coasting time to a given protocol can positively impact the in vitro embryo production outcomes in sheep.

Gene expression patterns of in vivo-derived sheep blastocysts is more affected by vitrification than slow freezing technique.

Transfer of fresh sheep embryos frequently results in higher pregnancy rate compared to cryopreserved ones, possibly due to a failure in the communication between the cryopreserved embryo and the endometrium during pre-implantation and pregnancy establishment. Thus, this study assessed the effect of sheep embryo cryopreservation (slow freezing or vitrification) on embryo survival rate and expression of genes related to trophectoderm differentiation (CDX2), pluripotency maintenance (NANOG), cell proliferation (TGFB1), mitochondrial activity (NRF1) and apoptosis (BAX and BCL2). Superovulation (n = 32 ewes) was performed and embryos were transcervically collected. One hundred good quality (Grade I and II) embryos were allocated into three groups: fresh embryos (CTL; n = 15), slow freezing (SF; n = 42) or vitrification (VT; n = 43). After thawing/warming, three pools of five blastocysts per group were used for RT-qPCR; the remaining 55 embryos were cultured in vitro in SOFaa medium at 38.5 °C and 5% CO2 (SF: n = 27 and VT: n = 28). Survival rate of SF and VT were, respectively, 29.6% (8/27) and 14.2% (4/28) at 24 h; and 48.1% (13/27) and 32.1% (9/28) at 48 h (P > 0.05). Only CDX2 was affected (up-regulated, P < 0.05) in both groups compared to CTL. The BAX transcript was upregulated in VT, compared to SF group. The VT increased (P < 0.05) the expression of all genes, except for NANOG and NRF1, when compared to the CTL. In conclusion, although in vitro survival was similar between techniques, VT led to increased changes in blastocyst gene expression compared to CTL and SF.