25 Effect of roscovitine on the cumulus cells expansion, oocyte maturation and in vitro development of domestic cat embryos generated by in vitro fertilisation

The domestic cat is a valuable model for development of assisted reproductive techniques in endangered felids. Roscovitine (RO) is a reversible inhibitor of M-phase promoting factor (MPF) that generates a delay in maturation, thus improving oocyte competence. However, prematuration with RO negatively affects the viability of cumulus cells and in vitro development of domestic cat embryos. More studies are needed to understand the effects of RO on oocyte competence in domestic cats. The objective of this study was to evaluate the effect of prematuration with RO on the oocyte competence and in vitro development of domestic cat embryos. Two experimental groups were generated: (1) cumulus-oocyte complexes (COCs) subjected to IVM (IVM-control group), and (2) COCs subjected to prematuration with RO followed by IVM (RO-IVM group). Prematuration was performed in M-199 with 0.3% bovine serum albumin, 0.36 mM sodium pyruvate, 2 mM glutamine, 2.2 mM calcium lactate, 50 μg mL⁻¹ gentamicin and 12.5 µM RO. IVM was performed similarly, without RO and adding 0.1 IU mL⁻¹ FSH-LH, 1 μg mL⁻¹ 17β-oestradiol, 20 ng mL⁻¹ epidermal growth factor, and 10 µL mL⁻¹ insulin-transferrin-selenium (ITS). Prematuration and IVM were conducted in 5% CO₂ at 38.5°C for 24 h. After IVM, COCs were subjected to IVF for 24 h. Zygotes were cultured in synthetic oviductal fluid (SOF), in 5% O₂, 5% CO₂ and 90% N₂ at 38.5°C for 8 days. First, the expansion of cumulus cells from each COC was measured after prematuration with RO and IVM using Micrometrics SE (Accu-Scope Inc.). Subsequently, a proportion of oocytes was stained with Hoechst to estimate the MII rate. Finally, the cleavage, morula, and blastocyst rates were assessed on Days 2, 5 and 8 of IVC. The expansion of cumulus cells between groups was evaluated by ANOVA (Infostat 2020, UNC). The Wilcoxon nonparametric test was used to evaluate the proportion of MII oocytes and in vitro embryo development (Infostat 2020, UNC) (P < 0.05). Regarding cumulus expansion in the RO-IVM group, when the COCs exposed to RO were subjected to IVM, the expansion of their cumulus cells significantly increased (mean ± s.d.) (323.3 ± 144.8 vs. 474.6 ± 310.0 µm; P < 0.001). After IVM, the cumulus expansion of the COCs from the RO-IVM group (474.6 ± 310.0 µm) was significantly higher than that of their counterparts from the IVM-control group (388.5 ± 221.7 µm; P = 0.005). No differences were observed after IVM in the proportion of oocytes in MII (mean ± s.d.) between the RO-IVM (58.5 ± 2.9%) and the IVM-control group (61.5 ± 13.7%; P > 0.05). After IVC, no differences were observed between groups (P > 0.05), as follows. In the RO-IVM group: cleavage: 131/264 (49.6%), morula: 66/131 (50.4%), and blastocyst rate: 31/131 (23.7%). In the IVM-control group: cleavage: 126/264 (47.7%), morula: 69/126 (54.8%), and blastocyst rate: 35/126 (27.8%). In conclusion, prematuration with RO followed by IVM increased the expansion of cumulus cells and did not affect resumption of meiosis in oocytes. In this study, prematuration with RO did not affect the in vitro development of domestic cat embryos generated by IVF.

This research was supported by ANID FONDECYT 3200352.