152 Effect of biphasic in vitro maturation with C-type natriuretic peptide on meiosis arrest and in vitro embryo production of oocytes from prepubertal and adult sheep

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Biphasic maturation with meiotic inhibitors has shown to increase developmental competence of prepubertal goat oocytes (Soto et al. 2019 PLoS One 14, e0221663). The aim of this study was to assess the effect of C-type natriuretic peptide (CNP) and 17β-oestradiol (E₂) in lamb and ewe oocytes. Ovaries from lamb (1–5 months old) and ewe (end of productive life) were recovered at a local slaughterhouse. Cumulus–oocyte complexes (COCs) were collected by slicing (lamb) or aspiration (ewe) and selected using HEPES-buffered (25 mM) TCM-199 medium with the meiotic inhibitor 3-isobutyl-1-methylxanthine (500 µM) and heparin. COCs were cultured in a prematuration (preIVM) medium with 200 nM CNP and 10 nM E₂, or in a control medium without meiotic inhibitors, for 6 h at 38.5°C with 5% CO₂. After that, a group of COCs were denuded and stained with Hoechst 33258 to assess meiotic arrest as the rate of germinal vesicles (GV). Remaining COCs were further cultured in our conventional maturation (IVM) medium (TCM-199 with FSH, LH, E₂, and epidermal growth factor). After 24 h, oocytes were in vitro fertilised and embryo cultured for 8 days. Data were statistically analysed by chi-squared test with Yates correction test. In ewe oocytes, we observed a higher (P < 0.05) meiotic arrest in CNP group (85.5% GV; n = 83) than in the control group (28.1% GV; n = 64) but embryo and blastocyst production decreased (P < 0.05) in the CNP group (48.8% and 25.6%, respectively; n = 43) compared with the control group (75.8% and 53.2%, respectively; n = 62). In oocytes recovered from lambs, there was no significant difference in meiotic arrest (GV) at 6 h, embryo production, or blastocyst rate between CNP (87.6%, n = 89; 29.6% and 4.0%, n = 125; respectively) and control (78.4%, n = 74; 19.0% and 5.8%, n = 121; respectively) groups. However, we found a tendency (P = 0.053) of CNP to increase the embryo rate. Regarding the results obtained, preIVM with CNP + E₂ for 6 h seems to compromise the competence of ewe oocytes to produce embryos but we have observed a tendency to improve developmental competence in lamb oocytes. Further experiments are required.

This study was funded by the Spanish Ministry of Science, Innovation and Universities (AGL2017-85837-R).