191 Effect of the time of oocyte collection through slicing method on meiosis resumption and in vitro embryo production

Abstract

Oocytes from juvenile goats are collected by slicing the ovary surface because the high percentage of small antral follicles limits follicular aspiration. The time of oocyte collection can impair oocyte developmental competence due to spontaneous resumption of meiosis. The aim of this study was to assess whether the time of slicing period affects oocyte meiosis and embryo development after in vitro fertilization. Ovaries from juvenile goats (1-2 months old) were recovered at a local slaughterhouse. Cumulus-oocyte complexes (COCs) were collected by slicing, selected, and kept in the slicing medium at 38.5°C in humidified air with 5% CO₂ until analysis or culture. The slicing medium was HEPES-buffered (25 mM) TCM-199 with 2.2 mg mL⁻¹ NaHCO₃ and 50 mg mL⁻¹ gentamicin. Two slicing periods were tested: T1 (1 h) and T4 (4 h). After this time, a group of oocytes were stained with 1% orcein in 45% acetic acid solution for assessing meiotic arrest and observed as the rate of germinal vesicle (GV; 61-67 oocytes/group from 5 replicates). The remaining COCs were cultured in our conventional IVM medium (TCM-199 with FSH, LH, oestradiol, sodium pyruvate, glutamine, cysteamine, epidermal growth factor, and fetal bovine serum) at 38.5°C with 5% CO₂. After 24 h, a sample of oocytes were stained for assessing nuclear maturation (28-29 oocytes/group, 3 replicates), and the rest were in vitro fertilized with 4 × 10⁶ sperm mL⁻¹ in BO-IVF medium (IVF Bioscience) for 20 h and embryo cultured in BO-IVC medium for 7 days (70-81 oocytes/group, 3 replicates). Blastocysts were stained with Hoechst 33258 for determining the number of cells. Data were analysed with two-way ANOVA with RStudio version 1.2.1335. The time of slicing was set as a fixed factor and the replicate as random variable. Data presented as percentage did not follow a normal distribution and were square root arcsine transformed before analysis. At the end of slicing periods T1 and T4, oocytes at GV were 100% and 84.7 ± 5.0%, respectively (P < 0.05). After 24 h of IVM, the oocytes at MII were 77.0 ± 7.1% and 88.6 ± 7.3%, respectively, without statistical differences. However, oocytes from T1 produced a higher rate of cleaved oocytes (84.6 ± 0.9%) and expanded blastocysts (11.03 ± 5.2%) than T4 (49.8 ± 7.9%, 0%, respectively; P < 0.05). The total blastocyst rate for T1 and T4 was 25.4 ± 5.8% and 9.4 ± 4.9%, respectively (P = 0.068). No differences were observed in blastocyst cell number (75.9 ± 4.0 and 67.5 ± 10.9, respectively). In conclusion, oocytes resume meiosis before IVM during a long slicing period, even though the slicing medium is not supplemented with hormones or growth factors. The longer slicing period does not affect nuclear maturation but impairs oocyte competence, observed as lower cleavage and blastocyst development. Further experiments are needed to determine whether the use of meiotic inhibitors in the slicing medium can prevent the negative effect of the long slicing period.

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