317 SEX DETERMINATION OF EQUINE EMBRYOS BY PCR USING BLASTOCOELE FLUID

Abstract

Pre-implantation genetic diagnosis (PGD) is used in different species to determine specific genetic traits of early stage embryos. The first part of this technique involves obtaining one or more cells from the embryo. Then, the DNA in the sampled cells is amplified by PCR using specific primers. Recently Palini et al. (2013) performed PGD of human blastocysts using the DNA in blastocoele fluid. The aim of our work was to study if the sex of equine in vivo produced blastocysts could be determined by PGD using blastocoele fluid as a sample for PCR. Thirteen equine blastocysts produced by artificial insemination and uterine flush were used for this study. Once obtained each embryo was placed on a 50-µL droplet of D-PBS without calcium and magnesium supplemented with 10% FBS and 50 µg mL^–1 of gentamicin (working medium) under mineral oil, on an inverted microscope equipped with a micromanipulation system. The blastocoele fluid was aspirated using a beveled micropipette (9 µm ID) and then discharged on a 1-µL microdroplet of working medium. The microdroplet containing the blastocoele fluid was transferred to a 0.2-mL DNAse-free tube containing 4 µL of DNAse-free water. A duplex PCR was performed to amplify the Y-encoded testis-specific protein (TSPY) and amelogenin (AMEL) genes. The primer sequences used for this study were: AMEL-F 5′-CCAACCCAACACCACCAGCCAAACCTCCCT-3′, AMEL-R 5′-AGCATAGGGGGCAAGGGCTGCAAGGGGAAT-3′, TSPY-F 5′-GAAGTCAGGCACACCAGTGA-3′ and TSPY-R 5′-TAAGGCTGCAGTTGTCATGC-3′. The PCR was performed twice, using the same primers and cycling protocol. Embryonic cells from embryos which have been previously diagnosed as male and female were used as positive controls for PCR. As a negative control, one tube was included containing all the components for PCR, except the DNA sample was replaced with the same volume of DNAse-free water. The amplification products were electrophoresed on a 2% agarose gel stained with ethidium bromide and visualised under UV light. Embryo viability post-blastocoele fluid aspiration was studied by individually incubating seven aspirated embryos in 50-mL microdroplets of SOFm with 19 mM D-glucose with 10% FBS under mineral oil at 38°C, in 7% O₂ and 5% CO₂ and observed at 24, 48, and 72 h. The diameter of embryos was registered immediately before blastocoele fluid aspiration and every 24 h during in vitro culture. The sex of 11 out of 13 embryos (84.6%) was determined and they were diagnosed as 8 males and 3 females. Six of the 7 embryos cultured in vitro expanded at 24 h and increased their diameter at 48 and 72 h. Our results demonstrate that PGD of equine embryos can be performed using blastocoele fluid only, avoiding the need of extracting cells from the embryo. The in vitro embryo survival after blastocoele fluid aspiration was high, showing that this technique does not impair the embryo's viability. Further studies are being undertaken using a larger number of embryos to demonstrate if this approach can be used with high efficiency.