31 DEVELOPMENT AND APOPTOSIS IN BOVINE CLONED EMBRYOS RECONSTRUCTED WITH OOCYTES COLLECTED BY REPEATED OVUM PICKUP SESSIONS OR FROM SLAUGHTERED COW OVARIES

Abstract

The oocyte has important components for nuclear reprogramming and its cytoplasmic background may influence the somatic cell nuclear transfer success. The current study attempted to evaluate the competence of cytoplasm from oocytes recovered by repeated ovum pickup (OPU) in living cows (OPU group) or obtained from ovaries collected at slaughterhouse from unknown source crossbred cows (SH group) to produce nuclear-transferred bovine embryos. For the OPU group, oocytes were recovered from 4 Bos indicus × Bos taurus crossbred cows in 4 repeated OPU sessions. Oocytes of OPU and SH groups were matured in vitro for 17 to 18 h, denuded and exposed to Hoechst 33342 (Sigma, St. Louis, MO, USA) and cytochalasin (Sigma) before enucleation. Embryos of OPU (n = 100) and SH (n = 105) groups were reconstructed with somatic cells from adult Gyr (Bos indicus) cow, fused with double electric pulse of 2.4 kV cm^–1 for 30 μs and activated with ionomycin (Sigma) and 6-DMAP (Sigma). Embryos were cultured in CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C. Cleavage and blastocyst rates were evaluated at 72 h and 168 h post-activation, respectively. Blastocysts at 168 h post-activation were fixed and permeabilized for TUNEL assay (DeadEnd™ Fluorimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. IVF bovine blastocysts (IVF group; n = 245) obtained with oocytes of slaughtered cows were used as control group. Fusion, cleavage, and blastocyst rates were analysed by chi-square test and total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analysed by ANOVA. There were no differences (P > 0.05) in fusion (71.0% and 61.0%), cleavage (74.6% and 78.1%) or blastocyst (32.3% and 31.2%) rates between OPU and SH groups, respectively, but both groups presented greater (P < 0.05) blastocyst rates than the IVF group (15.1%). Total cell number (80.66 ± 5.36 and 82.10 ± 4.79), apoptotic cell number (12.66 ± 3.20 and 15.60 ± 3.04), and apoptotic cell index (0.15 ± 0.03 and 0.20 ± 0.04) were also similar (P > 0.05) between OPU and SH groups, respectively. However, apoptotic cell number (7.40 ± 0.93) and apoptotic cell index (0.07 ± 0.01) were lower (P < 0.05) in the IVF group than the SH group and similar (P > 0.05) to the OPU group. In conclusion, oocytes cytoplasm from both groups (OPU and SH) have the same potential to produce nuclear-transferred bovine embryos but only blastocysts from the OPU group present apoptosis levels similar to its in vitro-fertilized counterpart.