151 Effect of biphasic in vitro maturation with C-type natriuretic peptide on meiosis arrest and in vitro embryo production of prepubertal and adult goats

Biphasic maturation with meiotic inhibitors has shown to increase developmental competence of prepubertal goat oocytes (Soto et al. 2019 PLoS One 14, e0221663). Oocyte developmental competence is related to the follicle diameter from which they come from, and biphasic maturation has been suggested to specially affect poorly developed oocytes. Our aim was to study its effect on three types of oocytes of different competences: oocytes recovered from follicles (1) < 3 mm and (2) > mm of prepubertal goats and from follicles (3) >3 mm of adult goats. Ovaries from juvenile (1–2 months old) and adult goats were recovered at a local slaughterhouse. Cumulus–oocyte complexes (COCs) were collected either by follicle aspiration (large follicles) or slicing (small follicles) and selected in a medium composed of HEPES-buffered (25 mM) TCM-199 supplemented with the meiotic inhibitor 3-isobutyl-1-methylxanthine at 500 µM. COCs were cultured in a prematuration (preIVM) medium with 200 nM C-type natriuretic peptide (CNP) and 10 nM 17β-oestradiol, or in a control medium without meiotic inhibitors, at 38.5°C with 5% CO₂. After 6 h, a group of COCs were denuded and stained with Hoechst 33258 for assessing 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, oestradiol, and epidermal growth factor). After 24 h, a sample of oocytes were stained for assessing nuclear maturation and the rest were in vitro fertilised and embryo cultured for 8 days. Data were statistically analysed by chi-squared test. Oocytes recovered from small prepubertal follicles showed a GV rate of 67.1% (n = 231) and 28.6% (n = 220) after CNP and control preIVM, respectively (P < 0.01). After biphasic and conventional IVM, MII was 80.0% (n = 70) and 73.1% (n = 78); and percentage of total embryos was 42.3% (n = 163) and 52.7% (n = 167), respectively. For oocytes recovered from large prepubertal follicles, GV was 64.4% (n = 174) and 26.6% (n = 169) (P < 0.01), MII was 67.0% (n = 91) and 70.5% (n = 105), and total embryo percentage was 41.2% (n = 170) and 38.2% (n = 102), for CNP and control groups, respectively. In oocytes from adult goats treated with CNP and control group, we observed percentages of GV 64.6% (n = 158) vs. 47.8% (n = 157) (P < 0.01), an MII of 85.1% (n = 107) vs. 90.1% (n = 121), and total embryos of 55.0% (n = 40) vs. 32.3% (n = 65) (P < 0.05), respectively. As seen, biphasic maturation affects meiotic arrest in all oocyte types but does not impair its maturation rate after IVM. Regarding oocyte competence, biphasic IVM significantly increases embryo production in oocytes from adult goats but it does not affect it in any other group. Further experiments are required to expand embryo sample size and to generate blastocyst production data.

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