Bovine embryo genotyping and freezability after biopsy

The reproduction process in its various forms is the instrument that allows the transmission of genes to the next generation. Reproduction science and in particular embryo technologies applied to animal breeding have the important role in increasing the impact of superior genotypes in the population. With the introduction of multiple ovulation, embryo recovery and transfer techniques plus embryo freeze-thaw methods in the early 1980s (in Lithuania these processes started in 1985), the breeding industry has the tools to increase the number of calves from donors of high genetic merit. However, embryo technologies are used on a limited scale in Lithuania. Embryo genotyping, which is required for continuous scientific progress, is one of the tools that could be used in selection programmes [3]. The latest development and application of polymerase chain reaction (PCR) for amplification of sex-specific DNA sequences, opened new ways for bovine embryo sex determination [2]. Our previous studies indicated that successful sex determination of bovine embryos can be achieved using several blastomeres, by detection of male-specific Y-chromosomal DNA using the PCR and by PCR-amplified ZFX/ZFY loci. The first method resulted in 92.6% and the second in 82.2% successful sex determinations [7]. Besides embryo sex determination, approaches have already being used for the identification of embryos carries of genetic diseases such as BLAD (bovine leukocyte adhesion deficiency), citrulinemia, and will be extended to CVM (complex vertebral malformation) and others conditions [1, 5, 6]. Another possibility being explored at present is to screen embryos for loci of breeding value [4]. In our sex determination experiment, k-casein loci were additionally PCR-amplified. This amplification was less successful, and k-casein loci were determined in 74.1% of analysed samples [7]. The method of biopsy had a direct influence on the amplification of the k-casein gene loci: efficiency was 37.5% after suction and after cutting - 86.7%. In routine PCR, 50-100 ng DNA is used, it has been established that 10 cells contain about 20 pg of DNA. Therefore, one of the main problems in embryo genotyping is very small amount of DNA available, i.e. 1 or 2 cells (the lowest level of reaction sensitivity), because only a few blastomeres can be collected from the embryo. It is necessary to have a reliable and highly accurate total DNA amplification procedure and then to screen for the loci of interest. A larger embryo biopsy reduces its viability and, hence embryo genotyping becomes meaningless. The aim of the study was to determine the effect of biopsy on bovine embryo freezability and viability depending on the method and magnitude of biopsy.