Polymorphism in inhibin alpha gene and its association with semen quality traits in Murrah bulls

Murrah buffaloes are well known for milk production and used as a dairy animal in India. Its home tract is spread over Rohtak, Hisar, Jind and Gurugram districts of Haryana, India. However, this breed is also available in other parts of the country and is being bred either in pure form or is being used as improver breed for grading-up of non-descript buffaloes. Inhibin is a glycoprotein hormone belonging to the transforming growth factor-β superfamily, which suppresses synthesis and secretion of follicle-stimulating hormone (Rivier et al., 1985). Inhibin gene encoding inhibin alpha and inhibin beta A are potential candidate gene for fertility analysis and are located on chromosome 2 and 4, respectively. INHA and INHBA are 2.97 kb and 11.50 kb long respectively, with 2 exons each for both genes. Review of published scientific literature revealed that two inhibin hormones viz.,inhibin A and inhibin B, sharing a common α-subunit but different β-subunits (βA or βB) were identified (Mason et al., 1985). The INHA was detected in spermatogonia, spermatocytes and early spermatids of Human (Toulis et al., 2010). In boar, allele substitution in INHA had a significant effect on plasma droplets rate and abnormal sperm rate (Lin et al., 2006). In Chinese Holstein bulls, polymorphism in INHA gene was significantly associated with acrosomal integrity rate (Sang et al., 2011). Environment, management, physiological status and genetic factors affect semen quality traits and it is difficult to perform direct selection for semen quality because of their low heritability (Mathevon et al., 1998). With the development of molecular biological techniques, the candidate gene approach might offer the specific markers to predict sperm quality traits in bulls. The hormones and hormone receptors are good candidate genes for bull semen quality traits due to their modulating roles in many male reproductive pathways (Giesecke et al., 2010). Hence, the present study aimed to identify the single nucleotide polymorphisms (SNPs) in the INHA gene and to associate these polymorphisms with semen quality traits in Murrah bull.

Experimental animals and their management
 
All procedures involving animals were approved by the Institutional Animal Ethics Committee (IAEC) of ICAR-NDRI, SRS, Bengaluru, India. A total of 103 Murrah bulls maintained at Frozen Semen Bull Station, Nandyal, Kurnool, Andhra Pradesh and Frozen Semen Bull Station, Karimnagar, Telangana under standard managemental conditions were used in the study. The semen was collected using an artificial vagina method. Immediately after collection, the ejaculates were transferred to water bath maintained at 37°C temperature. Fresh semen quality traits including semen volume per ejaculate and sperm concentration and percent sperm motility, percent live spermatozoa, functional membrane integrity and acrosomal integrity were evaluated using standard techniques.
 
Isolation of genomic DNA
 
Genomic DNA was isolated from blood samples (N=103) by the high salt method (Miller et al., 1988) and the working solution was prepared by diluting the stock to 100 ng/μL for utilising as DNA template in PCR.
 
PCR conditions
 
Three sets of primers (Table 1) were designed for INHA gene based on reference sequence for Bubalus bubalis (NCBI Reference Sequence: NC_037546.1) by using Primer 3 (http://primer3.ut.ee/) online software and were procured from Sigma-Aldrich, Bengaluru, India.
 

       
The Polymerase Chain Reaction (PCR) was carried out on about 100 ng of genomic DNA in 25 μl per reaction volume. The PCR reaction mixture consisted of 200 μM of each dNTPs, 10X Taq assay buffer, 1U Taq polymerase enzyme and 20 pM of each primer. The thermo cycler conditions included an initial denaturation at 94°C for 2 min, followed by 35 cycles with denaturation at 94°C for 30 sec with varying annealing temperatures based on primer set (Table 1), extension at 72°C for 1 min followed by a final extension at 72°C for 10 min. The PCR products were electrophoresed at 100 V in 1.5% agarose gel in 1X TBE buffer containing 0.5 μg/ml ethidium bromide along with a 100 bp DNA marker. The gels were visualised and documented using Gel documentation system (Gel Doc 1000, Bio-Rad, USA).
 
Single Strand Conformation Polymorphism analysis and SNP identification
 
The amplified PCR products were further subjected to polyacrylamide gel electrophoresis (PAGE) as described by Barani et al., (2017) and visualised by silver staining method (Sambrook and Russell, 2001). Band patterns were characterised by the number of bands, mobility shifts and scored manually. Representative PCR products giving unique SSCP patterns were custom sequenced using automated ABI DNA Sequencer (Medauxin Pvt. Ltd., Bengaluru., India) to confirm the mobility shift in each pattern. Sequence data were analysed using DNA Baser and Bio edit software Clustal omega multiple alignments for detecting single nucleotide polymorphisms (SNPs) by comparing the observed sequence with the Bubalus bubalis INHA gene refecrence sequence.
 
Statistical analysis
 
The association study of SSCP variants and semen quality parameters (semen volume per ejaculate and sperm concentration) were done by General Linear Model and compared by Duncan’s Multiple Range Test (SPSS 16.0) as shown below.

 
                        Y_ijklm= µ + G_i + S_j + A_k+H_l + e_ijklm

 
Where,
y_ijklm is the phenotypic value of traits; µ was the population mean; Gi was fixed effect of genotypes; S_j was fixed effect of season of collection; A_k was fixed effect of age; H_l was fixed effect of the herd; e_ijklm was a random residual error which is NID (0, σe²).

In this investigation, PCR-SSCP analysis of amplicons of INHA exon 2 of INHA gene exhibited polymorphism while the INHA exon 1 revealed monomorphic pattern. The analysis of the first fragment of INHA exon 2  gene revealed three distinct band patterns viz., pattern 1 (P1), pattern 2 (P2) and pattern 3 (P3) (Fig 1). The frequency of patterns 1, 2 and 3 were 0.36, 0.31 and 0.33, respectively. The amplicon comprising the second fragment of INHA exon 2 gene (INHA exon 2 FR2) revealed three different band patterns viz., pattern 1(P1), pattern 2 (P2) and pattern 3 (P3) (Fig 2). The frequencies of pattern1, 2 and 3 were 0.56, 0.35 and 0.09, respectively.
 

 

       
Representative samples were custom sequenced to confirm the mobility shift in each pattern. The retrieved sequences representing each of the unique PCR-SSCP patterns were further analyzed by comparing these sequences with the bovine INHA gene reference sequence for Bubalus Bubalis using Clustal omega multiple sequence alignment tool and DNA Baser for detecting Single Nucleotide Polymorphism (SNP) and their respective deduced amino acid variations (Table 2). Three mutations were identified in INHA gene, two in the first fragment of INHA exon 2 (C267119T and G267033T) and one in the second fragment of INHA exon 2 (T266510C). The transversion at the G267033T position was present in the coding region and predicted to change amino acid from Valine to Leucine. The transition at T266510C position was present in the coding region and predicted to change amino acid from Leucine to Proline in translated product. Clustal omega and Chromatograph analysis of the first and second fragment of INHA exon 2 are presented in Fig 3, 4, 5 and 6. Based on the previous study on INHA gene with semen quality traits, it is evident that INHA gene played a pivotal role in historical maturation for the development of testes and acquisition of fertility in male (Seok et al., 2004). Though there is no report available on polymorphism in INHA gene in buffaloes, very few reports are available in cattle. The polymorphism of INHA gene in Chinese Holstein bulls using PCR-RFLP method was observed (Sang et al., 2011). The SNPs, A192G (rs41257116) reported by them was in the exonic region of INHA gene. The polymorphism in the coding region (47727773) of INHA in Brahman bulls was also observed (Fortes et al., 2013).
 

 

 

 



       
The analysis of association of polymorphic band patterns with semen quality parameters revealed a significant association. SSCP patterns of the first fragment of INHA exon 2 gene (INHA exon 2 FR1) were significantly associated with semen volume per ejaculate (P<0.05) and sperm concentration (P<0.01) (Table 3). The bulls with pattern 2 (TT) had higher semen volume per ejaculate (3.69±0.15 ml) in comparison to pattern 1 (GG) (3.19±0.13 ml) and pattern 3 (GT) (3.34±0.14). The sperm concentration of bulls with pattern 1 (GG) (1308.97±24.21×10⁶) was found to be highest followed by pattern 3 (GT) bulls (1095.52±25.52×10⁶) and lowest in pattern 2 (TT) bulls (898.79±26.04×10⁶). The SSCP patterns of the second fragment of INHA exon 2 gene (INHA exon 2 FR2) were significantly associated with sperm concentration (P<0.01) and percent functional membrane integrity (P<0.01) (Table 4). The bulls with pattern 3 (TT) had high semen concentration (1252.01±57.78×10⁶) in comparison to pattern 1(CC) (1123.73±22.75×10⁶) and pattern 2(CT) (1045.69±29.33×10⁶). The percent functional membrane integrity of bulls with pattern 3 (TT) (75.78±2.33) was highest followed by pattern 2 (CT) bulls (70.45±1.18) and bull with pattern 1 (CC) (69.33±0.91). INHA gene polymorphism was significantly associated with acrosomal integrity rate in Chinese Holstein bulls (Sang et al., 2011). Hence, in this study, it is revealed that INHA gene and sperm quality traits exhibited that both the fragments of INHA exon 2 might have a favourable positive effect on sperm concentration.
 

 

       
SSCP variants were observed in both first fragment of INHA exon 2 and second fragment of INHA exon 2 genes of Murrah bulls. SSCP variants of the first fragment INHA of exon 2 gene had an association with semen volume per ejaculate and sperm concentration and the second fragment of INHA exon 2 genes was associated with sperm concentration and functional membrane integrity. The present study indicated that there is a possibility for application of genetic variants of inhibin alpha gene and its association with semen quality traits towards using it as bull fertility marker for achieving early selection of superior animals with high fertility after validating in a large number of the animals.

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. It is also certified that there is no conflict of interest among the authors.