Abstract

Roscovitine (RO) is a reversible inhibitor of MPF. Incubation of immature COCs with RO followed by IVM enhances oocyte competence. Additionally, it is know that SCNT is a complex procedure in which only a limited number of viable embryos can be generated. The reversible delay on oocyte maturation generated by RO might enhances the cytoplasmic maturation of domestic cat oocytes. This could be useful in the generation of embryos by SCNT, giving the possibility of reconstruct a higher number of embryos. However, it has been described that RO reduces the developmental competence of domestic cat embryos generated by IVF (Sananmuang et al. 2010). To prove the effectiveness of RO, the hypothesis of this research proposes that the incubation of domestic cat oocytes with RO allows the generation of embryos without affecting their developmental competence. The objective was to evaluate the effects of RO in the developmental competence of domestic cat embryos generated by different methods: parthenogenetic activation (PA), IVF and SCNT. Six groups were done, in which COCs were subjected to IVM (IVM-IVF, IVM-PA, and IVM-SCNT) or incubation with RO followed by IVM (RO-IVF, RO-PA and RO-SCNT). IVM was done in TCM-199 with 0.3% BSA, 0.36 mM sodium pyruvate, 2 mM glutamine, 2.2 mM calcium lactate, 0.1 IU/mL FSH-LH, 1 μg/mL 17β-estradiol, 20 ng/mL EGF and 50 μg/mL gentamycin, in 5% CO 2 at 38.5°C for 24 h. RO incubation was done similarly, without hormones and adding 12.5 µM of RO, this for 24 h and then followed by IVM for 24 h. Matured oocytes were used for IVF, PA or SCNT. Embryos were cultured in SOF medium, in 5% O 2 , 5% CO 2 and 90% N 2 at 38.5°C for 8 days. Cleavage and blastocyst rates were evaluated between IVM and RO groups using the Kruskal-Wallis non-parametric test (P < 0.05). Additionally, the blastocysts were stained with Hoechst, the total cell number of the blastocysts was estimated and a t-test was used to evaluate statistical differences (P < 0.05). Three replicates were done in RO-IVF, RO-PA and RO-SCNT, two in IVM-SCNT and five in the IVM-IVF and IVM-PA. At day-2 of IVC, the cleavage rates were estimated in the RO groups (RO-PA: 43/62 [69.4%], RO-IVF: 63/141 [44.7%] and RO-SCNT: 43/54 [79.6%]) and the IVM groups (IVM-PA: 81/92 [88.0%], IVM-IVF: 76/183 [41.5%] and IVM-SCNT: 29/37 [78.4%]). At day-8 of IVC, the blastocyst rates were estimated in the RO groups (RO-PA: 22/43 [51.1%], RO-IVF: 19/63 [30.2%] and RO-SCNT: 11/43 [25.6%]) and in the IVM groups (IVM-PA: 33/81 [40.7%], IVM-IVF: 26/76 [34.2%] and IVM-SCNT: 6/29 [20.7%]). No statistical differences were observed in the cleavage and blastocyst rates between the RO groups in their respective counterparts from the IVM groups. Additionally, no differences were observed in the blastocyst total cell number (Mean ± SD) between groups: RO-PA (234.4 ± 110) and IVM-PA (210.6 ± 100.1); RO-IVF (270.1 ± 163.1) and IVM-IVF (329.4 ± 88.7); RO-SCNT (239.4 ± 115.8) and IVM-SCNT (367.0 ± 118.2). These are preliminary results and additional experiments are needed to validation. In conclusion, the incubation of domestic cat COCs with RO before IVM did not negatively affect the developmental competence of embryos. Supported by ANID FONDECYT 3200352