BOVINE EMBRYO SEXING USING THE FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Abstract

The purpose of this study was to elaborate a Y-chromosome specific DNA probe that can be used in order to efficiently sex the bovine embryos using biopsied blastomeres. Another objective was to evaluate the pregnancy rate in recipient cows in which the biopsied and sexed embryos were transferred. A number of 76 bovine embryos have been non-surgically collected from a batch of 8 Simmental donor cows, following superovulation and artificial insemination. The embryos have been morphologically evaluated and biopsy has been performed in order to collect a small number of blastomeres from the inner cell mass. After the biopsy, the embryos have been immediately transferred to recipient cows, whose estrous cycle had previously been synchronized with the donor cows. The biopsied blastomeres were treated with 1 µg/ml vinblastine sulphate for 6 hours in order to induce the chromosomal metaphases, fixed on a slide using methanol and acetic acid and then kept in the freezer until use. The DNA probe has been synthesized as follows: the sequence of the Y-chromosome specific BtY2 gene has been used in order to obtain male specific primers; the primers were used in order to amplify a male specific DNA sequence obtained from a bull’s blood; after amplification and electrophoresis the specific bands were cut and DNA has been extracted from the gel; the male specific probe has subsequently been biotinylated by nick translation. Using this probe FISH has been performed and fluorescent signal has been amplified using the tyramide signal amplification kit. Slides have been examined using a fluorescence microscope (the presence of fluorescence signifying a male embryo while its absence a female embryo). The pregnancy rate has been evaluated by rectal palpation, 60 days after the transfer of biopsied embryos and was of 51.3%, while the accuracy of embryo sexing was evaluated at birth, when the FISH predicted sex has been compared with the morphological sex of the offspring and was of 92%. In conclusion, the fluorescence in situ hybridization method of embryo sexing turned out to be quite difficult but yielded good results and can be used for bovine embryo sexing.