The objectives of this study were to develop a two-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in gilt oocytes using chromosome-specific DNA probes, and to establish baseline frequencies of aneuploidy in pig oocytes matured in vitro. The ovaries were collected from gilts at the local slaughterhouse. Immature oocytes were isolated by slicing the cortex of the ovaries. The oocytes were matured in microplate wells using TCM-199 medium supplemented with 10% estrous cow serum, sodium pyruvate, antibiotics, and gonadotrophins. After 44 h of maturation the oocytes were incubated with hyaluronidase and the cumulus cells were removed by vortexing. Single oocytes were transferred into 1 microL drops of a lysing buffer (0.01 N HCl/0.1% Tween 20) on clean microscopic slides. Two-color FISH was performed using probes specific for Chromosomes 1 and 10. The probe for Chromosome 1 was labeled with Cy3-dUTP and a probe labeled with fluorescein-11-dUTP was used for Chromosome 10. Only oocytes in which a complementary first polar body was found were confirmed as aneuploid. The final assessment of aneuploidy was based on results of 1189 haploid oocytes. Thirty-four (3%) of the examined oocytes were aneuploid. Disomy of Chromosome 1 and Chromosome 10 was found in 12 of 34 and 8 of 34 of the aneuploid oocytes, respectively. Nullisomy of Chromosome 1 and Chromosome 10 was found in 8 of 34 and 6 of 34 of the aneuploid oocytes. No significant differences were found in the frequencies of disomies and nullisomies of oocytes or in the frequencies of aneuploidies of Chromosomes 1 and 10. The frequency of aneuploid oocytes determined by FISH seems to be higher than that determined by conventional methods in other laboratories.

Veterinary Research Institute Brno Czech Republic

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