SNP Identification in Sperm Associated Antigen 11B Gene and Its Association with Sperm Quality Traits in Murrah Bulls

摘要

Background: The beta-defensin family antigens present on sperm surface suggestively play pivotal role in sperm fertility by aiding in various steps of sperm maturation, motility, capacitation, immune recognition and sperm-oocyte interaction in female tract. It is imperative to explore genetic polymorphisms to build a better understanding of individual variation in male fertility. The experiment was designed to identify DNA polymorphism in sperm associated antigen 11B (SPAG11B) gene and to analyze association between genetic variants with sperm quality traits in buffalo bulls (Murrah) in ICAR-National Dairy Research Institute, Karnal. Methods: Genomic DNA was extracted from hundred and thirty Murrah bulls. A 395 base pair region covering partial Intron 2, Exon 3 and Partial Intron 3 of bovine SPAG11 gene was amplified and genotyped using PCR-RFLP method. The PCR amplified product was purified, sequenced and further ClustalW analysis was done to align edited sequence with reported Bos taurus sequence (AC_000158.1). Gene and genotype frequencies, effective number of alleles, level of heterozygosity and polymorphic information content of various genotypes were estimated by standard procedure. Seminal parameters (Post thaw sperm motility, sperm viability, acrosomal integrity, HOST and abnormality) were estimated and statistical analysis was carried out. Result: A novel SNP (G>A substitution) at 2266 base of the SPAG11B gene was identified by sequencing. The fragment of SPAG11 gene containing 395 base pairs was amplified using PCR and digested with restriction enzyme i.e. MunI which showed three distinct genotypes viz., AA (266 bp and 107 bp fragment), AG (307 bp, 266 bp and 107 bp) and GG (373 bp fragment). Least squares means of seminal parameters for the SNP was estimated and compared after correction for non-genetic factors. Between genotypes, significant differences were found only for acrosomal integrity and was highest in AG (74.22 +/- 0.72) followed by AA (72.6 +/- 0.89) and GG (71.12 +/- 0.97), respectively. The identified novel SNP of SPAG11 gene showed significant association with acrosome integrity with genotype AG being superior to other genotypes. However, an association cannot be established with other seminal parameters with this SNP, further studies are required in order to validate the impact of g.2266G>A on sperm quality traits in a large population.