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Bull fertility is an important factor for improving the economic value of the herd as the single bull can be used to inseminate many cows using artificial insemination. The bull fertility is measured in terms of semen quality traits like sperm motility and plasma membrane integrity of semen. The scrotal circumference of the bulls influences the semen quality in terms of sperm concentration and percentage of normal sperms of bulls ejaculate. These traits were influenced by both genetic and non-genetic factors. The genes MAP3K1, SPEF2 and PLCZ1 influencing the semen quality traits like scrotal circumference, sperm motility and plasma membrane integrity of semen were identified based on GWAS through Chilibot and Connected paper web-based bioinformatic tools. The SNPs rs463712269 (MAP3K1) and rs722354121 (SPEF2) were identified by screening these genes for missense mutations using Ensembl database and their RFLP pattern was verified using NEBcutterV2.0 software. For PLCZ1 gene, the perusal of literature revealed the presence of rs208019489 affecting the plasma membrane integrity of semen. A total of five frozen semen straws per bull and the relevant data were collected from 239 bulls of different breeds residing in FSBS of Andhra Pradesh. A suitable DNA isolation protocol viz., modified high salt method was selected through comparison of five different DNA isolation procedures in frozen semen. PCR-RFLP polymorphisms were examined in each group by using CviQI, HpyCH4V, and AvaII restriction enzymes for digestion of PCR products in MAP3K1, SPEF2 and PLCZ1 genes respectively. Association of MAP3K1, SPEF2 and PLCZ1 genotypes on scrotal circumference, sperm motility and plasma membrane integrity respectively were worked out using General linear model of SPSS Statistics base 26. The MAP3K1/CviQ1 PCR-RFLP assay revealed that the T allele frequency was more in Ongole population while in other cattle breeds C allele frequency was more. The highest homozygosity values were observed in Jersey and a reversal of this was observed in Ongole and HF population. Negative FIS values in the studied population are indicative of heterozygous excess due to outcrossing. In Murrah, the T allele frequency was more wiaaaaaaaaath effective number of allele of 1.13 and heterozygosity excess at this locus. Fixation of T allele was observed in both the exotic pure breeds (HF and Jersey) and presence of C allele was almost insignificant in cattle SPEF2/ HpyCH4V assay. The low selective pressure on SPEF2 locus was observed and the negative FIS values were observed in the cattle groups studied. In Murrah, the abundance of heterozygotes was observed indicating the selective advantage of C allele in this species and they deviated from the Hardy Weinberg equilibrium suggesting a high selection pressure on this locus. The GG (0.64) genotype frequency was higher than the CG (0.22) in Ongole cattle in PLCZ1/AvaII assay. Except in HF crossbred none off the other cattle genetic groups were with CC genotype which could be an inadvertent result of crossbreeding in HF crossbred. The HF crossbred and indigenous Ongole cattle (P<0.01) were not in agreement with the Hardy Weinberg equilibrium. In Murrah buffaloes, the near fixation of G allele (0.98) was observed accounting for the selection. The presence of low genetic diversity and heterozygosity excess in the Murrah population at this locus was found. Association of MAP3K1/CviQ1 and SPEF2/HpyCH4V polymorphisms had no significant influence on scrotal circumference and pre-thaw and post-thaw sperm motility respectively in both cattle and buffaloes. The PLCZ1/AvaII genotypes have significant influence on the plasma membrane integrity of semen in Holstein Freisiean, Jersey (P<0.05) and Ongole bulls (P<0.01). The plasma membrane integrity of semen was reported to be high in heterozygotes (CG) of Jersey and Ongole cattle. In Murrah, the genotypes have no significant effect on plasma membrane integrity of semen. The present study, conclude the importance of PLCZ1 gene as a marker for semen quality assessment and selection in bulls, owing to the association of PLCZ1 polymorphisms with plasma membrane integrity of bulls. Identification of few more SNPs in the vicinity of the verified SNPs suggests the importance of investigations aiming at screening for further casual mutation responsible for the variation of these traits with higher number of bulls.