265 SEXING OF BOVINE EMBRYOS BASED ON PCR TECHNIQUE

Abstract

Turkish cattle breeds are well adapted to harsh environmental and poor feeding conditions. However, their productivity is low. Increasing fertility rate and obtaining a high number of progeny from high-quality animals are important parameters in animal husbandry. The objective of the present project is to produce sexed embryos and cryopreserve them for subsequent transfer. The birth of the calves produced from the transferred embryos with sex determined prior to transfer by PCR are additional objectives of the study. To develop and optimize the PCR method, DNA was first isolated by using standard phenol-chloroform extraction from blood samples of cows and bulls to use as positive control. Then two multiplex PCR methods were developed using one autosomal (bovine 1.715 satellite locus which produces 216 bp long PCR product), and two Y-chromosome specific loci BRY4.a (300 bp) and BRY1 (300 bp). Both multiplexes include bovine 1.715 satellite locus, and they either include BRY4.a or BRY.1 as the second locus. Female individuals produce one PCR band, whereas male individuals produce two PCR bands. Bovine parthenogenetic blastocysts were used to test these two multiplex PCR methods. Immature bovine oocytes were aspirated from slaughterhouse material and in vitro matured in tissue culture medium-199 (TCM-199) supplemented with 10% FCS, sodium pyruvate, EGF, bLH, bFSH and penicillin/streptomycin for 18 h at 39°C and 5% CO₂ in humidified air. After removing the cumulus cells of matured oocytes (MII), meiotic spindles and first polar bodies were removed. Oocyte-cell complexes were fused by one 30 μs pulse of 133V/500 μm. All fusion units were subjected to chemical activation. Afterwards, parthenogenetic oocytes were cultured in Sage cleavage^® medium supplemented with 8 mg mL^–1 BSA for 72 h and then developing embryos were cultured in Sage blastocyst^® media supplemented with 4 mg mL^–1 BSA + 5% FCS for 4 additional days. Then they were stored at –20°C until DNA extraction. For DNA extraction two methods (Park et al. 2001 Theriogenology 55, 1843–1853; Tshimangadzo et al. 2004 Biol. Reprod. 71, 1671–1676) were employed to test their efficiency in our laboratory conditions, and we got better results with the former method. Repeated PCR tests of these parthenogenetic blastocysts were carried out and tests revealed only one PCR product of 216 bp corresponding to the 1.715 satellite locus as expected. The multiplex PCR methods will also be employed for Nuclear Transfer and IVF embryos.

This project is supported by Turkish Scientific and Technical Research Council-TOVAG (project no: KAMAG 107G027).