Bovine Blastocysts Obtained from Reconstructed Cytoplast and Karyoplasts Using a Simple Portable CO 2 Incubator

Abstract

To enable both the multiplication of elite livestock and the engineering of transgenic animals for various agricultural and biochemical purposes, scientists around the world are intensively studying efficient ways of improving developmental competency of bovine embryos reconstructed by somatic cell nuclear transfer. Because it is widely accepted that culture conditions along with many other factors contribute to the developmental competency of reconstructed embryos, this preliminary study was designed to test whether or not bovine reconstructed embryos could develop in vitro using a simple portable CO₂ incubator. CO₂ and O₂ gas tensions and air pressure can be varied using this system. The parameters used in the five conducted trials were low CO₂ (2-5%) and O₂ (8-10%) gas tensions, and negative air pressure (of 300 mm Hg). Chamber temperature was maintained at 38.5°C. Bovine fetal fibroblasts were used as donor karyoplasts and were fused into microsurgically enucleated M II oocytes followed by activation and culture. From the 250 enucleated oocytes, 217 (86.8%) fused, 183 (73.2%) cleaved, and 43 (17.2%) developed to the blastocyst stage. While relatively low developmental rates were achieved, technical proficiency may have been a contributing factor. Further studies using this system are needed to determine optimal levels of O₂, CO₂, and air pressure.