Prediction of male fertility is of prime importance in production animals. Follicle-stimulating hormone (FSH) plays a pivotal role in the reproduction of mammals. Single nucleotide polymorphisms (SNPs) in FSHB gene have been reported to affect the semen quality and fertility in cattle. The present study aimed to characterize and analyze the presence of SNPs in the FSHB-3 of HFxG 5043 Indian crossbred bull. Analysis revealed that HFxG FSHB-3 has 99.2% homology at nucleotide level and 100% homology at amino acid level with cattle FSHB-3 sequences. Two SNPs were detected in the FSHB-3 sequence of HFxG (4314A>G and 4489A>C) compared to cattle M83753 sequence. Both SNPs detected in HFxG FSHB-3 sequence were synonymous i.e. they did not result in amino acid change in FSHB-3. Findings of this study may provide basis for future research on genetic mechanisms affecting the semen traits and marker assisted selection of such traits in Indian crossbred cattle.
Fertility constitutes an important stake for dairy farm since reproductive problems lead to economic losses due to lost income from milk sales due to less calves produced per year, increase in semen costs because of an increase in the number of artiﬁcial inseminations per conception, costs for treatment and veterinarian or additional costs due to culling of long time infertile animals (Mahmoud & Nawito, 2012). In general, fertility is more important in an individual bull than an individual cow, as one bull may be used to breed up to 40 females with natural service, or potentially hundreds to thousands via artificial insemination. It was reported that 20 to 40% of bulls may have reduced fertility whereas few are completely sterile (Kastelic, 2013). Sub fertile bulls delay conception, prolong the calving season, reduce calf weaning weights, and increase the numbers of females culled, thereby resulting in economic losses and threatening sustainability of a livestock operation. Infertility may be defined as any reduction in the expected number of live animals born / unit time / unit number of females put to the male, which will vary between species, breed, production systems and individual farms. Generally when an infertility problem is brought to the attention of the veterinarian it has history of one or more of the following- 1) failure either to mate or mate properly, 2) increase number of females returning to service, 3) increase number of abortion, 4) increase incidence of dystokia and still birth (Logue & Greig, 1985). Molecular and genomic defects are important aspects responsible for male infertility. Through analysis of single nucleotide polymorphism (SNP) it is possible to identify regions of the genome influencing the fertility. Such information i.e. candidate gene analyses for sperm quality traits in bulls; could offer a tool to select for improved reproductive performance, thereby resulting in economic benefits. In this regard, hormone and hormone receptors are considered good candidate genes for bull fertility traits because they modulate limiting steps in many reproductive pathways (Giesecke et al., 2010).
Follicle-stimulating hormone (FSH), a pituitary glycoprotein hormone, plays an essential role in mammalian spermatogenesis and follicular development. In males, FSH and testosterone are principal endocrine factors responsible for the regulation of Sertoli cell function. FSH is involved in initiation and maintenance of the quality and quantity in spermatogenesis (McLachlan, et al., 1996; Shi, et al., 2018). FSH is heterodimeric hormone comprising of α -glycoprotein subunit (α GSU) and a β -subunit (FSHβ) (Pierce & Parsons, 1981; De Pascali, et al., 2018). Although both FSH subunits participate in the binding to FSH receptor, the beta-subunit dictates its binding specificity (Fan & Hendrickson, 2005). Previous research in mice has shown that spermatogenesis is not completely normal in FSH-deﬁcient mice. It was observed that FSH-deﬁcient mice are fertile; however epididymal sperm numbers and the number of motile sperm in the FSH-deﬁcient mice were lower compared to that in normal mice (Kumar et al., 1997, Hsueh & He, 2018). In case of bovines, the published sequence for FSHB (GenBank No.: M83753) (Kim et al., 1988) comprises 1 non-coding exon and 2 translated exons that encode the 129-amino acid preprotein. Dai et al. (2009) (Dai, et al., 2009) reported associations between a polymorphism in the follicle stimulating hormone β-subunit gene ( FSHB ) and sperm deformity, acrosome integrity, and nonreturnable rate in pure breed bulls of Canada (Dai et al., 2009). A total of 13 substitutions and 1 insertion was reported in the FSHB gene in pure breed bulls of Canada. In exon 3 (FSHB-3), seven substitutions were reported which signiﬁcantly inﬂuenced quality and fertility traits in fresh and frozen semen.
Recently, one study reported associations between polymorphism in FSHB and different semen quality parameters in Holstein Friesian and Jersey crossbred cattle (Dalvi, et al., 2018). In this investigation, non-significant associations between FSHB polymorphism and semen quality parameters was observed. Thus findings of previous studies in cattle (Dai et al., 2009; Dalvi et al., 2018) indicates that mutations in FSHB gene in bulls only affect the fertility i.e. ability to produce viable offspring. However, to the author’s knowledge the information on FSHB gene polymorphism from Indian crossbred bull is scanty. Due to the lack of information about the SNPs present in FSHB-3 exon from Indian crossbred bulls, we report here characterization of FSHB-3 exon from Indian crossbred bull.
Materials and Methods
Blood sample was collected from Holstein Friesian x Gir crossbred bull (HFxG 5043 Bull number 5043) maintained at Sabarmati Ashram Gaushala, Bidaj Farm, and Gujarat, India. 5 ml of blood was collected from jugular vein of cross bred cattle in polypropylene tube containing 0.5 M Ethylenediaminetetraacetic acid (EDTA) as anticoagulant. After collection of blood, the vials were shaken gently to facilitate through mixing of blood. The vials were then kept immediately in ice box containing ice and gel cool pack and were transport to the laboratory immediately. Genomic DNA was extracted by phenol-chloroform method as described previously (John et al., 1991). A pair of primers for amplification of FSHB-3 exon (250bp) were designed based on published FSHB sequence (GenBank No.: M83753) (Kim et al., 1988). The forward primer was of 18 bp (5′- GACTTGGTGTACAGGGAC-3′) and reverse primer was of 18 bp (5’- GAGCAGCGGATGCTTTGA -3′). Polymerase chain reactions (PCR) was carried out in a final volume of 25 µl reaction mixture containing 100ng of template DNA, 1X PCR assay buffer, 1.5 mM of Mg2+, 200 µM of dNTPs , 1 µM of each primer and 1U of Taq DNA polymerase. Amplification was carried out in Thermal cycler (Eppendorf, USA). PCR condition were: initial denaturation at 94º C for 5 minutes; followed by 94º C for 30sec, 57º C for 30sec , 72º C for 30 sec, and a final extension of 72º C for 5min.
PCR products were purified and quantified according to manufacturer’s instructions (QIAquick PCR Purification Kit; Qiagen Inc). Purified PCR products were submitted to geneOmbio technologies pvt ltd. Sequence analysis was done by comparing 5043 FSHB-3 amplicon sequence to FSHB published sequences available at National Center for Biotechnology Information (NCBI, USA) using DNAstar software (USA).
Results and Discussion
The PCR amplification of HFxG 5043 FSHB-3 is shown in Fig. 1a. PCR amplification of 250 bp was checked using 1.5% agarose gel electrophoresis. PCR product was purified using PCR purification kit and sent for sequencing. The HFxG FSHB-3 sequence and its protein translation obtained by using the ExPASy translate tool (ExPASy software, Swiss Institute of Bioinformatics, Geneva, Switzerland) is shown in Fig. 1b.
Fig. 1a: 1.5% agarose gel electrophoresis of HFxG5043 FSHB-3.
Fig. 1b: Predicted amino acid sequence of HFxG5043 FSHB-3
The nucleotide sequence and the deduced amino acid sequence of HFxG FSHB-3 was aligned using DNAstar software. Alignment was done using ClustalW method Fig. 2a and 2b. FSHB-3 sequences used for alignment were obtained from the NCBI. HFxG FSHB-3 nucleotide sequence showed 99.2% homology with cattle FSHB-3 sequences (M83753 & NM_174060) (Fig. 2a). Homology of nucleotide sequence between FSHB-3 sequence of HFxG and bison, yak, buffalo, sheep, goat, camel and pig was 99.2%, 98.4%, 94.8%, 94.0%, 93.6%, 89.6% and 89.6%, respectively. HFxG FSHB-3 at amino acid level (Fig. 2b) showed 100% identity cattle FSHB-3 sequences (M83753 & NM_174060) whereas it had 98.7% identity with bison and yak sequence. HFxG FSHB-3 showed 94.7%, 94.7%, 90.8%, 90.5% and 89.5% identity with pig, camel, goat, sheep and buffalo sequence, respectively. Phylogenetic tree at nucleotide level (Fig. 3a) revealed that HFxG FSHB-3 falls in the cattle group. Next highest identity was seen with bison & yak sequences. Buffalo, pig, camel, goat and sheep sequences are distantly related with HFxG FSHB-3. Phylogenetic tree at amino acid level revealed (Fig. 3b) that HFxG FSHB-3 falls in the cattle group. Next highest identity was seen with bison & yak sequences. Buffalo, goat, sheep, camel and pig sequences are distantly related with HFxG FSHB-3.
Fig. 2a: Sequence distance of HFxG5043 FSHB-3 at nucleotide level
Fig. 2b: Sequence distance of HFxG5043 FSHB-3 at amino acid level
Fig. 3a: Phylogenetic tree of HFxG5043 FSHB-3 at nucleotide level
Fig. 3b: Phylogenetic tree of HFxG5043 FSHB-3 at amino acid level
Two nucleotide substitutions or SNPs were detected in the FSHB-3 sequence of HFxG (4314A>G and 4489A>C) (Fig. 4) compared to cattle M83753 sequence. Both SNPs (4314A>G and 4489A>C) were synonymous and caused no change in the amino acid sequence. This finding is different from the earlier report on pure breed bulls of Canada (Dai et al., 2009). In that study, they detected 7 SNPs (4338T> C, 4341C> T, 4350G> A, 4452C> T, 4453A> C, 4461C> T, 4489A> C) in FSHB-3 of which 4453A>C led to the putative amino acid replacement Ser103Arg in the predicted protein. Other FSHB-3 gene sequence analysis studies in cattle’s are mostly on 5-upstream regulation region (5-URR) or on parts of FSHB-3 gene other than exon 3 [14-17]. Thus, findings of the current study could not be compared with these studies. Nevertheless, results of this study may provide foundation for further research on FSHB gene in Indian crossbred cattle.
Fig. 4: SNP analysis of HFxG5043 FSHB-3
In the current investigation, the partial sequence FSHB-3 of HFxS 5043 crossbred bull was characterized and analysed for the presence of single nucleotide polymorphisms (SNPs). Analysis of FSHB-3 of HFxG crossbred bull revealed that HFxG FSHB-3 has 99.2% homology at nucleotide level and 100% homology at amino acid level with cattle FSHB-3 sequences. Phylogenetic tree showed close relationship between partial sequence FSHB-3 of HFxS 5041 and cattle congeners. Two synonymous SNPs were detected in the HFxG FSHB-3 (4314A>G and 4489A>C) compared to cattle M83753 sequence. Findings of the current study may provide basis for further research on FSHB gene in Indian crossbred cattle.
Authors are thankful to Associate Dean, KNP College of Veterinary Science Shirwal for supporting the research and necessary facilities