The study does not warrant permission from Institutional Animal Ethics Committee as no experimentation was conducted on the animals but only observational study based on the DNA isolated from blood samples. The blood samples were collected aseptically from 270 bulls (Ongole: 54, Jersey: 30, Jersey crossbred: 56, Holstein Friesian (HF): 20, Holstein Friesian crossbred: 9, Murrah: 101) maintained at various Frozen Semen Bull Stations (FSBS) of Andhra Pradesh Livestock Development Agency (APLDA) located at Visakapatnam, Nandyal, Banavasi and Livestock Research Station (Sri Venkateswara Veterinary University), Lam, Guntur. Genomic DNA was isolated from blood samples as described by Miller et al. (1988) and stored at−20^oC until further use.

Multiple sequence alignment was performed to understand the homology based on the sequence information of DAZL genes in cattle and buffaloes available in NCBI. The variant data of Ensemble was screened for reported variants in DAZL genes and two SNP’s (rs42740178 and rs440858806) were identified. The possibility to differentiate the alleles at polymorphic site using restriction enzyme was verified by using the online tool, NEBcutter V2.0 (Vincze et al., 2003). Primers were designed (Table 1) for the target SNP along with the flanking region using Primer3 and the genomic regions were amplified subjecting to PCR in a reaction volume of 10 μl comprising of PCR mastermix (2x Taq Master Mix Red, Amplicon, Denmark) along with 5 pmol of each primer and 1µl of template DNA. At every PCR setup a negative control was run along with the samples. The conditions for amplification included initial denaturation at 94 °C/3 min, followed by 35 cycles: 94 °C/30s, 55 °C/45s, 72 °C/30s, for 3’UTR region; 94 °C/30s, 60 °C/45s, 72 °C/30s for missense mutation region, and a final extension for each at 72 °C/10 min. Amplicon of each sample (10 mL) was subjected to RFLP with 2 units of DdeI and BstY1 to genotype each animal with respect to the 3’ UTR and missense variants respectively. All the digested samples were electrophoresed in agarose gel and visualized in gel image system (Omega Fluor™ Plus Documentation Systems, BioExpress, USA).

In the present study the regions of DAZL gene spanning rs42740178 (3’UTR) and rs440858806 (missense mutation) were amplified in all the genetic groups. The corresponding PCR products observed were 300 bp and 364 bp respectively.

The restriction fragment pattern of 3’UTR/DedI corresponding to three genotypes AA (243, 39 and 18 bp), AC (243,160, 83, 39, 18 bp) and CC (160, 83, 39 and 18 bp) indicative of presence of polymorphism was observed (Fig. 1). Polymorphism was evident in Murrah bulls and all the studied genetic groups of cattle, except in HF crossbred. All the HF crossbred bulls were monomorphic and were typed to AA genotype. None of the Exotic or crossbred bulls belonged to CC genotype. The observed genotype and allele frequencies are presented in Table 2. In the genetic groups examined, the A allele frequency was almost nearing to fixation in exotic, and crosses with exotic bulls (0.95 to 0.99). Fixation of A allele was observed in HF crossbred bulls. In none of the exotic and crossbred population under study CC genotypes could be observed and only one bull belonging to Ongole breed was genotyped as CC. The 300 bp PCR amplicon of the DAZL gene in Murrah yielded a two different band pattern. Bands of 243, 39 and 18 bp were observed in almost all samples corresponding to the AA genotype. Only one animal was genotyped to heterozygous AC. At the same locus (rs42740178) in 3’ UTR region the frequency of C and A alleles in IRBT (Iranian Bos taurus) population from NextGen project (http://feb2021.archive.ensembl. org/Bos_taurus/Variation/ Population?r=1:153640009153641009;v=rs42740178;vdb=variation;vf=24718109) was 0.31 and 0.69. The A allele frequency in the studied zebu bulls, exotic and crosses involving exotic bulls and in Murrah bulls was higher than that was reported in IRBT population.

The population genetic indices at 3’UTR /DedI of DAZL locus in the examined groups of cattle and buffalo bulls for each genetic group under study and were presented in Table 2 and 3. The expected and observed genotypic frequencies in all the exotic bulls and crosses with exotic bull groups were almost similar. The analysis on 3’UTR /DedI locus revealed that the present populations were consistent with Hardy-Weinberg equilibrium (P > 0.05) except for indigenous cattle i.e. Ongole bulls (P=0.004). The C allele is found to be rare allele in the Murrah population. The distribution of 3’UTR variant (rs42740178) genotypic frequencies in all the cattle populations was almost equal to the expected heterozygosity (Table 3). The observed heterozygosity was less than expected (P< 0.01) in Ongole bull population. The effective number of alleles (Ne) across the studied genetic groups ranged between 1.069 (HFx) to 1.1 (HF). Murrah buffalo bulls were with highest heterozygosity among the studied populations. The estimates of polymorphic information content (PIC) values varied from 0.01 (Murrah, & Jersey crosses) to 0.09 (HF) among the different cattle populations which is considered to be lesser degree of informativeness for a marker. The Ongole bulls were observed to be in HardyWeinberg disequilibrium (P<0.05) and the F_IS values were positive in this breed indicating heterozygote deficiency.

Digestion of DAZL PCR amplicon (364 bp) encompassing missense variant in exon 3 was performed with BstY1 restriction enzyme to identify the polymorphism and the study revealed only CC genotypes (315 bp and 49 bp) in all the animals (Fig. 2). As all the genetic groups were monomorphic further studies could not be conducted.

Some of the earlier studies in human revealed that the SNPs within DAZL have significant influence on male fertility (Zhu et al., 2014) and those with non-obstructive azoospermia showed a declining expression of DAZL in the testis (Hashemi et al., 2018). In studies involving livestock, a significant association between SNPs of DAZL and epididymis weight was reported in rams (Yuan et al., 2020). The biometrical traits of epididymis are affecting the ejaculated semen volume and sperm motility (Ytournel et al., 2014) and sperm abnormality (Waldner et al., 2010). Owing to the highly conserved nature of the gene among species capture of sire fertility phenotypes in dairy herds utilizing DAZL gene polymorphism will contribute for genomic selection for male fertility. Ogoreves et al. (2011) reported nine SNPs in 3’ UTR region of DAZL gene in cattle and opined that it is the most polymorphic candidate with substantial level of variation and affecting male reproductive traits.

The present study revealed a substitution of C→A at position 2519 position in 3’UTR region (rs42740178) in all the bulls except in HF crossbred. HF crossbred bulls were monomorphic and were typed to AA genotype. In Murrah bulls only one was heterozygous (AC) and interestingly none of the other exotic or crossbred bulls belonged to CC genotype. Since, the sequences within the UTRs mediate the translational regulation of mRNAs further studies on polymorphism, differential expression and association may shed light on role in bull fertility.