Chimeric Blastocyst Production Using Ivf Bovine Embryos (Holstein-Japanese Red} Without Zona Pellucida

Abstract

The objective of this study was to produce chimeric blastocysts by the aggregation of IVF bovine embryos (Holstein- Japanese Red}, without zonae pellucidae. The embryos were produced by the in vitro maturation and fertilization of oocytes were collected from a local slaughterhouse. Oocytes were taken from follicles of 2-5 mm diameter and those with multiple cumulus - cell layers, were matured in TCM-199 supplemented with 5 % superovulated cows serum (SCS, collected on day-7} and 0.01 mg/ml FSH containing 0.58% luteinizing hormone (Denka, Pharmacheutical Co., Japan}. Oocytes were cultured for 21 hours at 38.5°C under 5 % C01 in air, and then fertilized in vitro using frozenthawed sperm. Forty- eight hours after IVF, 8-cell stage embryos were used to produce aggregation chimeras. The zonae pellucidae were microsurgically removed using a microblade and a Narishige micromanipulator unit. Holstein embryos were aggregated with Japanese Red embryos after zona removal by hand manipulation in TCM-199, supplemented with 5 % SCS, then transferred into culture medium (TCM-199 supplemented with 5 % SCS and 5 Ug/ml insulin} at 38.5°C under 5 % COa in air. Embryos were evaluated 4 days after aggregation. Viability of aggregated embryos that developed into blastocysts is shown in Tab.l.