366 SEXING OF GOAT BLASTOCYSTS PRODUCED IN VITRO BY FISH USING CHROMOSOME X AND Y OVINE SPECIFIC PROBES

The aim of this study was to test a FISH approach using ovine-painted probes specific for the chromosomes X and Y, on goat interphase and metaphase nuclei of blastocyst cells. Oocytes of prepubertal goats were recovered at a slaughterhouse and selected by morphological criteria. Oocytes were matured in TCM-199 supplemented with hormones, serum and cysteine at 38.5°C for 27 h. IVM-oocytes were fertilized in vitro and the presumptive zygotes were cultured for 10 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. The blastocyst nuclei were spread using a modified Tarkowski method (1966). Briefly, individual embryos were immersed into hypotonic solution for 5 min, followed by fixative solution of methanol/acetic acid (Carnoy’s solution) until the embryos acquired a transparent appearance. Next, the embryos were transferred to a Superfrost plus Slide (Menzel Gläser, Braunschweig, Germany) in a small droplet mixture of distilled water and Carnoy The zona pellucida and the blastomere cytoplasm dissolved gradually and Carnoy solution was added dropwise to the slide before the nuclei dried out. The morphology and total number of nuclei in each embryo were analyzed under phase contrast microscope and stored at -18°C. Embryos with appropriate fixation were then subjected to hybridization with ovine-painted probes specific to chromosomes X (Fluorocrom green-FITC) and Y (Fluorocrom orange-TAMRA) (Chrombios-Molecular Cytogenetics GmbH, Raubling, Germany) according to the manufacturers protocol and adjusted for the caprine species. Briefly, slides were then incubated at 60°C for 1 h. The chromosomal DNA was then denatured by immersing slides in 70% formamide/30% 2 × SSC at 70°C for 1.5 min, and immediately dehydrated in an ethanol series (70%, 90%, and 100%), 4 min duration per solution and air dried. In parallel, X- and Y-probes were added to the hybridization solution (50% deionized formamide, 10% dextran sulfate, 2 × SSC) and denatured at 75°C for 10 min. Aliquots (0.5 to 1.5 μL) of this solution were placed on each slide and sealed with a coverslip and glue prior to incubation at 37°C (Hybrite; Vysis Inc, Dowers Grove, IL, USA). After 22 to 24 h the coverslip was removed and the slides were washed three times. The first and third washes were performed with 2 × SSC at room temperature while the second wash was in a 0.4 × SSC/0.1% Tween at 73°C for 3 min. Nuclear DNA was counterstained with diamino-phenyl-indole solution (DAPI) and examined with a fluorescence microscope (Olympus BX61, Olympus America Inc., Melville, NY, USA) equipped with appropriate filters. From a total of 69 blastocysts, 11 355 blastomeres were analyzed and 7,825 were correctly hybridized (68.9%). The results of the embryo sexing were: 24 embryos XX, 11 XY, 22 polyploid embryos (of which 13 presented more than 80% of cells XX), 3 haploid embryos (X0), 2 tetraploid embryos, and 5 no result. In summary, goat blastocysts were successfully sexed using FISH with painted ovine X- and Y-specific probes. Grant sponsor Spanish Ministery of Science and Innovation.Code: AGL2007-60227-CO2-01.