Bull fertility is essential since bull DNA is the primary mechanism through which genetic improvements can be efficiently accomplished. Although 20 to 40 per cent of bulls may have reduced fertility, few are completely sterile (Kastelic and Coulter, 1999). Sub fertile bulls delay conception, prolong the calving season, reduce calf weaning weights and increase the number of females culled, thereby resulting in economic losses and threatening sustainability of a livestock population. Bull semen quality is a quantitative trait controlled by multiple genes. By identifying various genes that have effects on fertility, the genetic cause behind sub fertility can be explored.

The process of spermatogenesis is regulated by reproductive hormones in gonadotropin axis and controlled by large number of genes. Therefore, hormone and their receptors are presumed to be good candidate genes for reproductive traits (Vincent et al., 1998). Follicle Stimulating Hormone (FSH) is a glycoprotein hormone secreted by the anterior pituitary gland primarily in response to gonadotropin releasing hormone (GnRH) and plays an important role in the growth, development and function of the ovaries and testes and it is also important for regulating reproduction in mammals (Ulloa-Aguirre et al., 1995). The biological protein consists of an alpha (α) and beta (β) subunit; however the β subunit is responsible for the specific biological actions of FSH and its interactions with the FSH-receptor (Bernard et al., 2010). In males, FSH promotes spermatogenesis, the process of sperm development, in the seminiferous tubules primarily via actions on sertoli cells (Sairam and Krishnamurthy, 2001). Follicle stimulating hormone plays a critical role in the hormonal control of spermatogenesis by binding to FSH-receptors on sertoli cells and stimulating primary spermatocytes to undergo the first meiotic division to form secondary spermatocytes. Since each sertoli cell can only support a limited number of developing spermatozoa at a time, it is important to understand how polymorphisms of the FSHβ subunit gene can affect male fertility (Sairam and Krishnamurthy, 2001).

In males, FSH in combination with testosterone is the most important tropic hormone regulating sertoli cell function initiate and maintain the quality and quantity in spermatogenesis (Ohta et al., 2007). The published sequence for bovine FSHβ gene (Lin et al., 2006), (genbank No: M83753) comprises 1 non coding exon and 2 translated exons that encode the 129 amino acid pre- protein. Bovine FSHβ gene is located on chromosome 15 that comprises two introns and three exons with a length of 6601 bp (Hediger et al., 1991). Variants of this gene were three alleles, namely A, B and C. (Dai et al., 2011) found 9 SNPs mutations, i.e. 4 mutations in promoter section (‘5 URR), 3 in intron 2, and 2 in exon 3. Polymorphism of FSHβ subunit gene in exon 3 on the study significantly influenced the fresh and frozen semen quality. Polymorphism of FSHβ subunit affects the production and sperm quality in Simmental, Charolais and Limousin (Dai et al., 2009). The diversity of FSHβ subunit genes with litter size in sow (Yaofeng et al., 1998) and (Liu et al., 2012), sperm quality in boar (Lin et al., 2006), litter size in ewes (Xiaopeng et al., 2010) and equine sperm quality (Samper and Plough, 2010) was co-related. Considering the paucity of research in this area the present research work was planned to study the polymorphism of FSH beta subunit on testicular and seminal attributes in Holstein Friesian and Jersey crossbred bulls.

Materials and Methods

The research work was carried on twenty five HF (75%) crossbred as well as six Jersey (50%) crossbred bulls from Frozen Semen Laboratory Harsul, Aurangabad and Nagpur. The testicular length (TL), testicular width (TW) and testicular thickness (TT) of each testis along with scrotal circumference (SC) were measured with a metric tape in the widest region of the scrotum after adequate ventrocaudal traction of the gonads. The semen was collected by artificial vagina method twice weekly for analyzing various semen quality traits and six replicates of samples were studied. The semen volume, concentration, initial motility, post thaw motility, acrosome integrity, hypo osmotic swelling test and abnormal spermatozoa rate were studied. Genomic DNA was extracted from the collected blood samples using DNA extraction Mini kit and the quality and the purity of the DNA were checked and quantification was done by agarose gel electrophoresis.

Fig. 1: DNA in agarose gel electrophoresis

The DNA samples were diluted at different dilutions 1:40, 1:50, 1:80 and 1:100 (20 µl of DNA in 980 µl nuclease free water) and subjected to agarose gel electrophoresis for quality check. FSH beta subunit gene specific primers were custom synthesized and utilized in the study is given below in Table 1.

Table 1: Primer sequence for FSH beta subunit gene used to evaluate the presence of SNP

Primers supplied in freeze dried powder form were reconstituted in nuclease free water to the volume 291 (in µl) and 347 (in µl) as in the case of Forward and Reverse primers, respectively. PCR was carried out in a final reaction volume of 27 µl Table 2.

Table 2: Components used for each PCR reaction for amplification of FSH beta subunit gene

All reaction was carried out in 0.2 ml thin wall PCR tubes. PCR tubes containing mixture were taped gently and quickly spinned @ 10,000 rpm for few seconds. PCR amplification was confirmed by running 9 µl of PCR product from each tube plus 1 µl of DNA loading dye on 2% agarose in 1X TAE buffer (depending on the expected size of amplified product) at a voltage of 120 V and 100 V for 10 min and 30 minutes, respectively. Ethidium bromide was incorporated @ 2 µl of 1% solution/40 gel solution in the gel itself. The amplified product was visualized as a single compact fluorescent band of expected size under UV light. The PCR product for FSH beta subunit gene was digested by restriction enzyme Pst 1 using above protocol and kept in water bath for 1 hour at 37^OC. Enzyme digestion was analyzed by running 9 µl of digested PCR product from each tube plus 1 µl of DNA loading dye on 2% agarose in 1X TAE buffer at a voltage of 120 V and 100 V for 10 min and 30 minutes, respectively. Statistical analysis was carried out by using ANOVA (Analysis of variance) using statistically Web Based Agricultural Statistics Software Package (WASP).

Fig. 2: Genotypes observed after RE digested product in agarose gel electrophoresis

Results and Discussion

In the present study, three different genotypes BB, AB and AA having amplification size 313 bp; 313 bp,202 bp,99 bp and 202 bp, 99 bp, respectively were identified. The results of present study for the three genotypes are in concurrence with Dai et al. (2009), Ghasemi and Ghorbani (2014) and Kadam et al. (2015). Genotypic frequencies were 0.38, 0.54 and 0.08 for AA, AB and BB genotype while gene frequency of A allele was 0.65 and B allele was 0.35. Kadam (2015) found the gene frequency for A and B allele to be 0.702 and 0.307, respectively while the genotype frequency for BB and AB genotypes were observed to be 0.404 and 0.595, respectively. Ghasemi and Ghorbani (2014) observed the A allele was more frequent than B allele (0.675 vs 0.325) which is in relation with result of the present study. However, Dai et al. (2011) reported that bulls with B allele were more frequent than A and C alleles.

Co-relation of FSH beta Subunit Gene with Testicular and Seminal Attributes

Testicular Parameters

The scrotal circumference, testicular length, testicular width and testicular thickness (mean ± S.E) in BB, AB and AA genotypes are depicted in Table 3.

Table 3: Testicular parameters (mean ± S.E) of BB, AB and AA genotypes in H.F and Jersey crossbred bulls

In the present study, all the testicular parameters are statistically non significant (P<0.05) between the three genotypes. The results of the present study are in correlation with Kadam et al. (2015) observed 40.174 ± 0.88 and 39.148± 0.636 cm average scrotal circumference in BB and AB genotypes, respectively. There was no statistically difference (P<0.01) among the scrotal circumference among BB and AB genotypes. The mean age, mean body weight, scrotal volume and scrotal weight were statistically non significant among the genotypes.

Seminal Attributes

The semen volume, sperm concentration, initial motility, post thaw motility, HOST, ASR and AIT(mean ± S.E) in BB, AB and AA genotypes are depicted in Table 4. In the present study, all the seminal attributes differed non significantly (P<0.05) between the BB, AB and AA genotypes. The results of present study for semen volume (4.181±0.288 and 4.996±0.185 ml), sperm concentration (1119.779±76.86 and 1041.212±44.47 millions/ml), initial motility (66.82±1.301 and 67.88±0.655 %), post thaw motility(50.47±0.15 and 50.40±0.11), acrsome integrity (84.504±.0.504 and 84.95±0.321 %) and per cent intact acrosome (77.89±0.405 and 78.77±0.148) are in accordance with Kadam et al. (2015) who reported non significant difference for these seminal attributes between BB and AB genotypes, respectively.

Table 4: Seminal attributes (mean ± S.E) of BB, AB and AA genotypes in H.F and Jersey crossbred bulls

However, hypo osmotic swelling test (60.02±.0.355 and 61.44±.0.261%) and abnormal sperm major defect (3.27±.0.646 and 3.044±.0.158) were significant at (p<0.01) in BB and AB genotypes in HF bulls. The results of present study are not in agreement with Dai et al., (2009) reported bulls with mutation in exon 3 gene of FSHβ exhibited a significantly lower sperm concentration in fresh semen and a lower percentage of acrosome integrity in both fresh and frozen semen (P<0.05), higher percentage of sperm deformity in fresh semen (P<0.05), which was more pronounced in frozen semen (P<0.01), and a significantly lower sperm motility in frozen semen (P<0.05). For fertility evaluation, the non return rates obtained from 14,416 inseminations revealed that bulls with this genotype showed significantly lower non return rates (P<0.05). Ghasemi and Ghorbani (2014) reported significant association of AA and AB genotype on total sperm, fresh semen motility and number of produced pay out but there was non significant association with semen volume, concentration, post thaw motility and sperm in each milt ejaculation in 83 bulls.

Davis et al. (2012) reported percent motile, progressive sperm and rapid sperm were statistically significant (P<0.01) while per cent major, minor and total abnormalities in sperm were non significant   in GG and GA genotypes of SNP 485 A for FSH beta subunit in Angus and Balancer bulls. Ishak et al. (2009) reported that Bali cattle was monomorphic and the other cattle’s in study i.e. Simmental, Limousin, Brahman were polymorphic for FSH beta subunit gene polymorphism with sperm quality traits in 470 samples and the higher incidence of percentage of sperm abnormalities in Simmental, Limousin, Brahman compared to Bali were reported.

Conclusion

In the present study, three different genotypes BB, AB and AA having amplification size 313 bp; 313 bp,202 bp,99 bp and 202 bp, 99 bp, respectively were identified and which has non significant (P<0.05) effect on testicular parameters as well as seminal attributes in H.F and Jersey crossbred bulls.

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