Embryo

Handmade Somatic Cell Cloning in Cattle: Analysis of Factors Contributing to High Efficiency In Vitro

Abstract

Widespread application of somatic cell cloning is hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (ie. hand-made) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour, were among the highest described so far. Cattle serum used in our experiments was found to be superior to other protein sources for in vitro embryo developmental rates. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum when compared to commercially available fetal calf serum. Morphological analysis of produced blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved hand-made somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive and efficient alternative to traditional somatic cell nuclear transfer.