202 ASSOCIATION BETWEEN BMP4 GENE POLYMORPHISM AND OVUM PICK-UP IN VITRO PRODUCTION TRAITS IN BRAZILIAN Gyr COWS

Abstract

In Bos taurus taurus, previous studies have reported an association between single nucleotide polymorphism (SNP) in BMP4 gene and the blastocyst rate in cows evaluated for in vitro embryo production efficacy. The aim of this study was to evaluate the degree of association between a polymorphism in the BMP4 gene and characteristics related to in vitro embryo production as well as pregnancies thereof in Bos taurus indicus cattle (Gyr breed). Data from 212 ovum pick-up in vitro (OPU-IPV) sessions were collected of 50 Gyr cows in the Rio de Janeiro state, Brazil. The OPU-IVP procedures were performed and associated with marker SNP. The OPU-IVP traits were number and proportion of viable cumulus-oocyte complexes, number and proportion of cleaved embryos at Day 4 of culture, number and ratio of transferable embryos at Day 7 of culture, number and proportion of pregnancies at Day 30 after transfer, and number and proportion of pregnancies at Day 60 after transfer. DNA was extracted from hair follicles. BMP4 polymorphism, the segment of the BMP4 gene (exon 2, SNP rs109778173) was amplified by PCR with a specific primer. PCR-restriction fragment length polymorphism (RFLP) was used for genotyping. HinfI restriction enzyme that recognises and cleaves the sequence 5′ G ? ANTC 3′ was used. The samples were submitted to capillary electrophoresis Fragment Analyzer™ (Advanced Analytical Technologies Inc., Ames, IA) for allelic discrimination by size and polymorphism identification. Genotypic frequency, allele frequency, Hardy–Weinberg equilibrium probability, gene homozygosity, gene heterozygosity, effective allele number, and polymorphism information content were statistically analysed in the software PowerMarker v.3.25 (North Carolina State University, Raleigh, NC, USA). The association between DNA marker and the OPU-IVP traits and pregnancy rates was make by analysis of variance of repeated data PROC GLIMMIX of SAS 2009 (SAS Institute Inc., Cary, NC, USA). The domain of the protein coding of BMP4 gene is located in exon 2. A fragment of the sequence of DNA in exon 2 was amplified, where the mutation SNP rs109778173 was identified (G > T) in Gyr cows using PCR-RFLP and capillary electrophoresis methods. Three genotypes were identified (GG, TT, and GT) with genotypic frequencies of 0.64, 0.32, and 0.04 respectively; allelic frequency and Hardy–Weinberg equilibrium. The polymorphism was significantly associated with the number of viable oocyte complexes, ratio of viable oocyte complex, and ratio of pregnancies at 30 days (P > 0.01). The GT genotype affects negatively the number and proportion of viable cumulus-oocyte complexes and the proportion of pregnancies at Day 30 after transfer. This finding is an indication of a genetic effect of these characteristics.