Apoptosis And Oxidative Damage In Parthenogenetic Bovine Embryos

Parthenogenesis (PA) of the oocyte is essential to a number of oocyte- or embryo-related technologies such as intracytoplasmic sperm injection and cloning by nuclear transfer. The cleaved rate for the PA embryos was lower, compared with in-vitro fertilized (IVF) embryos, and so was the rate of development to the blastocyte stage, because of activation method, in-vitro culture medium, and oxygen concentration. The present study investigated apoptosis and oxidative damage of bovine PA embryos, aiming to improve perthenogenetic activation as a technical base of cloning.1. Bovine oocytes were parthenogenetic activated by Ionomycin+6-DMAP (ID) and Ionomycin + Cycloheximide (IX) respevtively, and the onset and frequency of apoptosis in PA embryos and the morphological changes that conform to the general criteria of apoptotic cell death by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay were investigated. IX group had a higher cleavage rate (86% vs 79%), and a lower development rate to the blastocyst stage (20% vs 25%), relative to ID group. The earliest positive TUNEL signal in IX group was detected on Day 6, 1 day later than that in ID group. Apoptosis in ID group increased from 8% of the embryos on Day 6 to 11% on Day 8. There was a similar result in IX group, the percentage of apoptosis increased from 13% on Day 6 to 19% on Day 8. The mean cell number in IX group was signicantly lower than that of ID group, whereas the percentage of apoptosis in IX group was signicantly higher than that of ID group. As results showed, bovine PA embryos did undergo apoptosis. It was better for the development of embryos by using Ionomycin combined with 6-DMAP to activate bovine oocytes.2. The effects ofβ-mercaptoethanol (β-ME) on bovine oocytes nucear maturation and the development of parthenogenetic embryo were investigated. Bovine oocytes were matured in the medium containing 0.05, 0.1, 0.15 mmol/Lβ-ME, and the nuclear maturation rate was determined 22～24 h later. Activeated oocytes were cultured in SOF containing 0.05, 0.1, 0.15 mmol/Lβ-ME, or addition of 0.1 mmol/Lβ-ME to SOF at 1-cell stage, 8- to 16-cell stage and morula stage, and the blastocytes rate was checked at 168～192. As results, nucear maturation did not differ in the absence or presence ofβ-ME(P﹥0.05). 0.05 and 0.1 mmol/Lβ-ME improved cleaved rates and blastocytes development significantly, but the latter group was better. Before bovine parthenogenetic embryo progressed to 8～16 cell-stage,β-ME could significantly improved blastocyte development(P﹤0.05),rate and the number of blastocytes were significantly different between 1-cell stage group and 8～16 cell-stage group,therefore, it was the best time for addition ofβ-ME to embryo culture medium at 1-cell stage.