Amplication, Copy Number Variation, Expression and Association of Non-synonymous SNP of Bovine Beta-defensin 129 Gene With Distinct Fertility of Cattle Bull

Background The male reproductive specic class-A β-defensins are adsorbed on sperm surface and enrich sperm functioning thus considered vital for maintaining male fertility. The primate DEFB129 play role in sperm maturation, motility, and fertilization but its contribution to bovine fertility is still unexplored. Method RLM-RACE and RT-qPCR approaches were used to characterize and expression analysis of Indian cattle BBD129 gene. The polymorphism analysis of the BBD129 gene was done by PCR, sequencing, and absolute RT-qPCR on sperm gDNA from distinct fertility cattle bulls. Bioinformatic analysis was performed to understand the structural and functional implications of SNP on BBD129 protein. Results The complete coding sequence of the BBD129 gene consists of 582 bp mRNA including UTRs and conserves all beta-defensin-like characteristics. Sequencing results revealed two conserved non-synonymous T169G (rs378737321, S57A) and A329G (rs383285978, N110S) SNPs in the functional protein-coding exon. Based on SNP position and linkage, BBD129 gene haplotypes were categorized into four groups: TA haplotype (169T & 329A), GA haplotype (T169G polymorphism), TG haplotype (A329G polymorphism), and GG haplotype (when T169G & A329G polymorphisms present together). The frequencies distributions of BBD129 haplotypes in the high fertile group (n=105 clones) were: TA (71.42%), GA (1.90%), TG (2.8%), and GG (24.76%), while in the low fertile group of bulls, the frequencies distributions of observed BBD129 haplotypes (n=149 clones) were: TA (36.24%), GA (0%), TG (2.68%), and GG (61.07%). The distributions of TA haplotype were majorly distributed in bulls with a high conception rate (P=0.5256) while double mutated GG haplotype was signicantly more abundant in bulls with a lower conception rate (P=0.0001). BBD129 exist as a single-copy gene in the bovine genome and found higher expression in the corpus-epididymis region. Bioinformatic analyses found nsSNPs as neutral and non-deleterious but their structural-distorter could result in altered mRNA secondary structure, protein conformations decreased protein stability, and compromised biological functionalities. The polymorphisms resulted in altered O-glycosylations (deletion S57A and insertion N110S) and an increase in phosphorylations (52T-Threonine and 110S-Serine) post-translational-modications. BBD129 gene polymorphism could be associated with the fertility performance of cattle bulls. gene-specic primers, Taq DNA polymerase, reaction buffer, and Nuclease-free water in a 25µl reaction volume. The PCR thermal prole was: Initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing-variable between 45-55°C C for 30 seconds, extension at 72°C for 15 seconds and a nal extension for 5 minutes at 72°C. The selection of best primer concentration was done by RT-qPCR assay with the following reaction components: SYBR (5 µl), forward primer (0.25µl/0.5µl/1µl/1.5µl), reverse primer (0.25µl/0.5µl/1µl/1.5µl), nuclease-free water, and cDNA template (2µl of 10ng/µl). The following RT-qPCR thermal prole was used: initial denaturation (95°C for 3 min), denaturation (95°C for 10 sec), annealing (49°C for 15 sec, extension (72°C for 15 min), and 35 cycles were run on Bio-Rad CFX96 Maestro qPCR machine by using SYBR Green Master Mix (Bio-Rad, USA). After the amplication, a melting peak analysis with a temperature gradient of 0.5°C/sec from 65°C to 95°C was performed to ensure specicity and primer-dimer formation. RT-qPCR reaction eciency done multiple sequence alignment (MSA) and analyses BBD129 twenty-nine indicate a strong evolutionary relationship between the Indian BBD129 gene and the Bovidae family BBD129 Equidae families relationships next Bovidae found Indian cattle is structurally similar to Bos taurus except


Introduction
The molecular mechanism of reproduction is a complex process involving thousands of genes encode for proteins or glycoproteins and any defect or mutation in these genes directly or indirectly in uences the process of gametogenesis, transportation of sperm to the egg, fertilization, and embryo formation [1][2][3]. In the last decade, advances in molecular-omics (genomics, transcriptomics, and proteomics) technologies has exceedingly improved the identi cation of fertility associated markers for selecting the best breeding domestic animals [4][5][6][7]. The testicular spermatozoa are non-motile and do not have the fertilizing ability, therefore, the complete sperm maturation takes place in the epididymis where they are exposed to a consortium of molecules such as lipids, organic ions, energy components, enzymes, and glycoproteins secretions from the epididymis epithelial cells in order to attain maturity for fertilization [8][9]. These uptakes of epididymal glycoproteins on sperm surface protect sperm from premature capacitation, acrosomal reaction and help sperm to cross the hostile female reproductive tract (FRT) barriers including cervical mucus passage, uterine immune evasion, OECs binding, and zona interactions [2,8,[10][11][12][13]. The presence of an essential class of glycosylated beta-defensin proteins is one of the crucial modi cations of sperm surface during epididymal maturation [9]. β-defensin (BD) is a most ancient class of defensin, it contains C1-C5, C2-C4, C3-C6 disul de linkages [14][15][16] that give rises to the typical BD motif with defensin fold containing three antiparallel beta-strands [17][18][19]. Most of the reported BD genes are encoded by two exons and show a conserved signal sequence. The signal peptide encoded by the rst exon and cleaved off to give mature functional peptide which encoded by second exon. Amino acids exposed on the surface of functional proteins are conserved at cysteine positions suggesting the stabilization of core con guration of protein structure for their conserved antimicrobial functions in the species-speci c adaptation during the evolution [20][21][22]. Although originally defensins were intact, during the course of evolution, β-defensin genes have undergone duplications and non-synonymous mutations giving rise to different clusters with region-speci c functions and also have gained their reproductive speci c functions [14,23]. Out of many, class-A BDs (including DEFB126 and DEFB129) have shown agespeci c and sex-speci c expressions signifying their pleiotropic physiological relevance in humans [24], macaque [10][11]25], rat [26][27], bovine [28], ovine [18], porcine [29], and equine [30]. In primates, the heavily glycosylated DEFB126 and rat DEFB22 (orthologs) are coated on the sperm surface which facilitates sperm functioning, and releasing of these BDs during capacitation events in the FRT are essential for the sperm-zona interactions [25]. The polymorphisms in the BDs gene lead to decreased milk contents [31][32], increased round cells in semen [33], compromised sperm ability to cross mucus penetration ability [2, Secondary structure prediction was done by PSIPRED and SOPMA server [42][43]. Signal peptide prediction was done by Signal IP 5.0 server [44]. Biological function prediction was done by Argot2 server [45]. The complete sequence of cattle BBD129 gene was submitted on NCBI-BankIt [46].
Evolutionary phylogenetic tree analysis: The protein sequences of the DEFB129 gene from ruminants and non-ruminants mammalian species were retrieved from the NCBI database by using "beta-defensin 129" keyword (supplementary le 2). To nd the evolutionary relationships between cattle BBD129 (Bovidae family) identi ed by RLM-RACE and its neighbor a family, a phylogenetic evolutionary analysis was performed by the Maximum Likelihood method and Tamura-Nei model by using MEGA 10 bioinformatics tool [47]. This analysis involved 29 protein sequences of DEFB129 genes. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed.
Ampli cation of BBD129 gene from sperm gDNA Collection of frozen semen and sperm genomic DNA extraction Sample collection: Eleven distinct fertility cattle bulls were selected on the basis of their rst and second arti cial inseminations conception rate (CR) data available at the Arti cial Breeding Research Center, NDRI, India. Cattle bulls with CR less than 31% were classi ed in the low fertile group and bulls with CR with more than 50% were classi ed in the high fertile group (Table 1). Frozen semen samples of selected cattle bulls of distinct fertility (5 bulls from high and six from low) were collected into liquid nitrogen. Genomic DNA isolation: Frozen semen samples were thawed by immersing them into the pre-warmed 37˚C distills water for 30 sec and contents were collected into the 15ml falcon tubes containing 8ml of washing buffer-A (150mM NaCl and 10mM EDTA) and centrifuged at 800g for 10 min. Pellets were dissolved into the 300 µl of lysis buffer-B (100mM TRIS-Cl, 10mM EDTA, 0.5M NaCl and 1% SDS) and 100µl of 1M DTT (Dithiothreitol) followed by incubation for 15 min at RT. After that, 100µl of Proteinase-K enzyme (0.2 mg/ml) was added to the tube followed by overnight incubation at 55ºC in the dry bath. The sperm gDNA was isolated by Phenol:Chloroform:Isoamylalcohol (PCI) method [48]. The quality and quantity of gDNA were measured by NanoDrop ND1000 spectrophotometer and 1% AGE.
Absolute quanti cation of BBD129 gene copy number The protocols and procedures used for the optimization of primer concentration, gDNA template, and generation of the standard curve are provided in supplementary le 1. To determine the absolute copy number of BBD129 gene in genomic DNA of distinct fertility cattle bulls, the RT-qPCR reaction was set up with the following components and thermal pro le: SYBR (5 µl), forward primer (1.5µl), reverse primer (1.5 µl), nuclease-free water, and gDNA template (2µl of 1.25ng/µl). The thermal pro le was: initial denaturation (95°C for 3 min), denaturation (95°C for 10 sec), annealing (49°C for 15 sec), extension (72°C for 15 min), and repeated run for 35 cycles. After the ampli cation, a melting peak analysis with a temperature gradient of 0.5°C/sec from 65°C to 95°C was performed.
The expressional analysis of Indian cattle BBD129 by RT-qPCR assay Reverse transcription and Primer designing Total RNA extraction from frozen cattle MRT tissues and cDNA synthesis was done as mentioned above (RNA extraction & DNase I treatment and RLM-RACE primer designing strategy, cDNA synthesis and RACE PCR sections). The primers were designed from our RLM-RACE BBD129 product (supplementary table  1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and eEF-2 (eukaryotic elongation factor 2) reference genes were chosen based on the literature [18,49]. RT-qPCR optimization and validation of reference genes are provide in supplementary le 1.
Annealing temperature (AT) optimization was done by the conventional gradient PCR. The best AT was selected based on the brightest and sharpest band obtained on 2% agarose gel. PCR chemical composition involves cDNA template, dNTPs, gene-speci c primers, Taq DNA polymerase, reaction buffer, and Nuclease-free water in a 25µl reaction volume. The PCR thermal pro le was: Initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing-variable between 45-55°C C for 30 seconds, extension at 72°C for 15 seconds and a nal extension for 5 minutes at 72°C.
The selection of best primer concentration was done by RT-qPCR assay with the following reaction components: SYBR (5 µl), forward primer (0.25µl/0.5µl/1µl/1.5µl), reverse primer (0.25µl/0.5µl/1µl/1.5µl), nuclease-free water, and cDNA template (2µl of 10ng/µl). The following RT-qPCR thermal pro le was used: initial denaturation (95°C for 3 min), denaturation (95°C for 10 sec), annealing (49°C for 15 sec, extension (72°C for 15 min), and 35 cycles were run on Bio-Rad CFX96 Maestro qPCR machine by using SYBR Green Master Mix (Bio-Rad, USA). After the ampli cation, a melting peak analysis with a temperature gradient of 0.5°C/sec from 65°C to 95°C was performed to ensure speci city and primer-dimer formation. RT-qPCR reaction e ciency was done by using a serial dilution by using BBD129 gene-speci c primers for the MRT tissues. RT-qPCR e ciency was calculated according to the E = 10(-1/slope) [50].

Cattle BBD129 RT-qPCR assay
The relative expression of the cattle BBD129 gene was done as duplicate in a 10 µl reaction volume with RT-qPCR thermal pro le: initial denaturation (95°C for 3 min), denaturation (95°C for 10 sec), annealing (49°C for 15 sec, extension (72°C for 15 min), and 35 cycles were run. After the ampli cation, a melting peak analysis with a temperature gradient of 0.5°C/sec from 65°C to 95°C was performed to ensure speci city and primer-dimer formation. A non-template control was run to cross-con rmation of reaction component nucleic acid contamination.
Bioinformatics analysis of cattle BBD129 polymorphism BLASTn and BLASTp alignment analysis was used for the nucleotide and protein search alignment, respectively [51].
Prediction of functional impacts of nsSNPs on mRNA secondary structure To predict mRNA secondary structure, FASTA formats of native and mutated mRNA were submitted to the RNAfold server [52]. It provides three structural information i) conventional secondary structure graph, ii) Dot plot, iii) Mountain plot. A mountain plot is an x-y graph and shows minimum free energy structural curve, centroid structural curve, partition function curve and positional entropy resulting from the pair probabilities. . PSIPRED and SOPMA servers were used for the prediction of the impact of nsSNPs on protein secondary structure. Biological function prediction was done by Argot2 server [45]. The translated amino acid sequences of native and mutated BBD129 gene were submitted above servers.  4). BBD129 was predicted for conserved three beta-sheets secondary structure, immune defensive roles, e ciency to bind with CCR6 chemokine receptor and lipopolysaccharides binding, and extensive post-translation modi cations especially O-glycosylations and phosphorylations ( g 1 and supplementary table 1).
The multiple sequence alignment (MSA) and phylogenetic analyses of the BBD129 gene across the twenty-nine mammalian species indicate a strong evolutionary relationship between the Indian cattle BBD129 gene and the Bovidae family BBD129 ( g 2; supplementary g 5). The Camelidae and Equidae families show relationships next to the Bovidae family. MSA analysis found Indian cattle is structurally similar to Bos taurus except for few amino acids.

Association of cattle BBD129 sequence variations with bull fertility
The BBD129 gene was ampli ed from sperm gDNA of distinct fertility bulls and the full length of exon rst (507bp) and exon second (607bp) were achieved by ampli cation of intronic anking regions of exons (Fig 3). To determine the polymorphisms in the BBD129 gene in the distinct fertility bulls, we sequenced 254 clones from 11 bulls. There was no polymorphism observed in exon rst. However, in exon second, we observed two missense or non-synonymous SNPs at positions 169th (T169G cDNA position, rs378737321) and 329th (A329G cDNA position, rs383285978) in the gDNA from distinct fertility bulls (Fig 4; Supplementary g 6A). After translating nucleotide to amino acid sequence, we have also observed substitutions at the amino acids level. In the BBD129 protein, the serine amino acid at the 57th position is replaced by alanine (S57A/srs378737321) while asparagine amino acid is replaced by serine at the 110th position (N110S/rs383285978) (Fig 4; supplementary g 6B Table 1). There was no signi cant difference in TA haplotype distribution in the high fertile and low fertile groups (P=0.5256) but there was a 35.15% difference in BBD129 TA haplotype in both the group of high fertile and low fertile bulls. There was a signi cant difference in GG haplotypes frequency distribution in the low fertile bulls as compared to high fertile bulls (P=0.0002). The distribution of double mutated GG haplotype was found an association with bull's low conception rate (table 1).

Copy number variation in BBD129 gene in distinct fertility cattle bulls
In order to investigate the fertility related association in the copy number of the bovine beta-defensin 129 (BBD129) gene, we examined the genomic copy number of the BBD129 gene in two groups of distinct fertility cross-bred bulls (n=11). Standard curve-based quanti cation of the BBD129 gene was carried out by absolute real-time quantitative PCR. The known concentration (copy number) of the plasmids carrying the gene of BBD129 fragment was used to construct the standard curve for the BBD129 gene by plotting the log concentration values against the crossing point (Cq) values (Supplementary g 7 & 8).
Crossing point (Cq) values were determined from the RT-qPCR run of equal concentration of genomic DNA (1.25 ng/ul) of distinct fertility categorized bulls. The universal bovine CSN2 (casein beta) gene was used as a single-copy number reference gene. The absolute Cq values and their log transformation for BBD129 gene and CSN2 control are provided in supplementary table 1. It was observed that the mean copy numbers of CSN2 and BBD129 genes did not vary signi cantly between bulls of distinct fertility (Fig 6) suggesting a single copy of BBD129 gene in bovine genome and gure 10B suggesting no signi cant difference of BBD129 gene CNV in high fertile and low fertile bulls.
Expression of beta-defensin BBD129 gene in male reproductive tract The relative distribution of BBD129 expression was analyzed in adult cattle MRT. The expression dynamics of BBD129 were slightly different from the other mammalian species reported earlier, the higher expression of BBD129 was observed in the corpus segment of the epididymis as compared to others MRT tissues (viz. ST, RT, caput, cauda and VD) (Fig 7). The corpus regions show 14.2 fold higher expression of the BBD129 gene than the normalizer rete testis region. The order of reducing expression of BBD129 expression was corpus, cauda, VD, RT, and caput region. The details of mean difference and P-value have  (supplementary g 10). Similarly, SIFT, SNAP, and MAPP predicted S57A mutation as deleterious or disease-causing polymorphism and N110S mutation as neutral. I-Mutant2.0 server predicted both nsSNPs as structure distorters. The result details of the above bioinformatics servers are provided in table 2.
Possible impact of nsSNPs on physiochemical properties of BBD129 protein Comparative ProtParam results between native BBD129 protein sequence and double mutated protein sequence have shown major alterations in the molecular weight, polar and non-polar amino acids, instability index, aliphatic index, and GRAVY (supplementary table 1). PSI-PRED and SOPMA servers predicted alterations in the protein secondary structure especially in the helix and coils (Fig 4). SOPMA predicted native protein sequence with 33.77 % alphahelix, 1.32 % beta-turn, 10.60 % extended strand, and 54.30 % random coil, however, in the mutated BBD129 protein the alfa helix increase by 5.3 % while the beta-turn and random coil regions decrease about 1.32 % and 3.97 %, respectively. Alterations in BBD129 protein stability of missense variants were examined by MUpro software. MUpro predicted that both the substitutions negatively affect the protein stability of BBD129 protein. The substitution of serine to alanine amino acid at 57th position (S57A_rs378737321) decreased the protein stability by con dence score -0.83258649 and -0.998569279497182 in Support Vector Machine and Neural Network Machine, respectively. The substitution of asparagine to serine amino acid at 110th position (N110S_rs378737321) decreased the protein stability by con dence score -0.  (Fig 4; supplementary table 1). SNPs have no effect on N-glycosylation. NetPhos 3.1 predicted fteen sites for phosphorylations in the BBD129 TA haplotype protein (supplementary table 1) while two possible additional phosphorylation sites were predicted in the double mutated BBD129 GG haplotype protein. The motif CKKKTCCIR (52nd amino acid) predicted as a potential candidate for threonine phosphorylation with 0.545/0.503 scores for Phosphokinase-G/Phosphokinase-A enzymes. Another motif IKSASAFAK (110th amino acid) was predicted as a new potential candidate for serine phosphorylation for the PKC enzyme with a 0.617 score. These new phosphorylation sites were the result of S57A and N110S substitutions, respectively (Fig 4 and supplementary table 1).

Possible impact of nsSNPs on BBD129 protein biological functions
As predicted above that nsSNPs decrease protein stability and in uence the protein secondary structure and post-translation modi cations, here we have predicted possible impacts observed nsSNPs on the biological functioning of BBD129 protein by Argot 2 server. The prediction found double mutated BBD129 protein has decreased scores for all predicted biological functions compared to native BBD129 protein suggesting nsSNPs negatively impact the protein biological functioning ( Table 3).
The summarized results of bioinformatics tools used for the predictions of various physiochemical and structural changes in the mutated BBD129 are provided in the table 4.

Discussion
The reproductive-speci c class-A BDs have got many attractions because of their importance in the fertilization events, however, insu ciency of their complete information and characterizations limit their usages. In the present work, we have characterized the bovine β-defensin 129 gene in Indian cattle and showed its association with bull fertility performances. The complete coding sequence of BBD129 mRNA was ampli ed from the corpus-testicular region and expression analysis was performed to explore its region-speci c roles. The sequence variations analyses were performed by PCR ampli cation and absolute quanti cation of BBD129 from sperm genomic DNA of distinct fertility characterized cattle bulls. The bio-computational analyses of non-synonymous polymorphism on the biological functioning of BBD129 protein found an association with cattle bull conception rate percentage. The testicular sperm are not able to fertilize an oocyte as they need post-gonadal maturations to gain fertilizing ability [69,70]. The complete maturation of testicular spermatozoa takes place in the epididymal environment where they are exposed to a plethora of molecules including highly negatively charged cysteine-rich glycosylated beta-defensin antimicrobial peptides secreted from the epididymis epithelial cells and coating of these glycoproteins or exogenously addition of epididymal proteins (e.g. BDs) improve fertilization potential of sperm [71][72][73]. The addition of prokaryotically expressed bovine r-BBD126 protein to non-motile caput-epididymal spermatozoa improves their motility and mucus penetration ability but lacks the ability to improve fertility [74][75]. This failure to improve sperm fertilizing ability could be due to prokaryotic expression of bovine BBD126 because prokaryotes lack post-translational modi cation system and DEFB126 or its orthologs are known for their post-translational modi cations (glycosylations) [23,71]. Another reason could be the absence of an uncharacterized complete coding sequences or predicted sequences of BDs available on the public databases [76]. RACE methodology has been used to amplify capped and tailed mRNAs [77][78], therefore accurately determining the 5' capped end and 3' polyadenylation ends [79][80]. Bos taurus BBD129 gene was characterized as two exons and with 50 bp UTR region at both the end of the gene [81]. Herein, we ampli ed 5' and 3' end of cattle BBD129 mRNA and revealed strong conservation in the coding region (UOA_Brahman_1 GCF_003369695.1). A Bioinformatic analysis revealed its location on chromosome 13 and was observed to retain all the essential characteristics as reported in beta-defensins. The MSA and phylogenetic analysis found its strong conservation in the Bovidae family and other mammalian families. The MSA of DEFB129 across the mammalian species found structural variations in the protein-coding region.
The protein structure of BBD129 is almost similar in the Bovidae family excepts very fewer places and the variations increase as phylogenetic distance increase.
In the bovine, studies are largely focused on genomic SNPs associated with milk production, susceptibility to immune function, and parasite resistance [82].
The non-synonymous mutations (CC/AA and CT/AC) genotypes of cattle BNBD4 and DEFB103 are associated with higher milk, fat, proteins, lactose, dry matter contents, somatic cell counts, and resistance to mastitis [72,75,[83][84]. The genotypic analysis found that the human DEFB126 gene is highly polymorphic bearing 77 known SNPs, out of these, rs140685149 and rs11467497 are associated with human infertility affecting sperm surface glycans, motility (rs11467497), higher round cells in semen, cervical mucus penetration ability [10-11, 33-34, 74]. In Bos taurus, the BBD129 gene has been reported for the twelve missense or non-synonymous SNPs in the genome [81]. In this study, we observed two conserved non-synonymous SNPs (T169G and A329G) in the Indian cattle genome suggesting the conservation nature of snSNPs. These non-synonymous nucleotide polymorphisms also substitute non-synonymous amino acids (S57A and N110S, respectively) in translated BBD129 protein. In Indian bovine species, publically available predicted BBD129 gene has conserved 57A and 110S, however, in our study, RLM-RACE BBD129 ampli cation found 57S and 110N as reported in Bos taurus [81]. The sequencing revealed Indian cattle bulls are producing a heterogeneous population of spermatozoa bearing different haplotypes of the BBD129 gene. The BBD129 native TA haplotype (169T & 329A) and double mutated GG haplotype (169G & 329G) were found majorly distributed in the Indian cattle genome. The TA haplotype is primarily distributed in the group of high fertile bulls with a difference of 35.18 % to low fertile bulls while the major percentage of heterogeneous sperm population from the low fertile bulls found with double mutated GG haplotype and this was signi cantly associated with bull's of low conception rate [87] and in our study, it was used as single copy reference gene. The standard curve analysis has found that there was no duplication in the BBD129 gene and it has been retained as a single-copy gene in the genome of contrasting fertility population of Indian cattle bulls.
The distributions of the beta-defensins in the MRT across the species are similar suggesting their conserved expression pattern and their traditional antimicrobial activity in the male reproductive organs depicting their importance in the reproductive processes [14,18,29,[88][89]. Bos taurus and Bubalus bubalis, newly emerged primate DEFB126 ortholog viz. BBD129 shows region-speci c and sex-speci c expression in the healthy MRT suggests its pleiotropic activities, in addition to traditional antimicrobial importance [23,81]. The DEFB129 is anticipated to play its vital physiological role in sperm maturation via membrane modulations and providing motility [14,27,[90][91]. Human epididymal DEF129 protein bind to the sperm surface chemokine receptor CCR6 and in uence the calcium ion in ux causing sperm hypermotility. The exogenous additions of different concentrations of recombinant-BDs to de cient spermatozoa signi cantly increase their motility, viability, and antimicrobial activity [36, 75,92]. To date, there is no published report on the expression of the Indian cattle BBD129 gene in the male reproductive tract. The expression pattern of BBD129 mRNA in Indian cattle MRT show higher expression in the corpusepididymis region with a region-speci c expression as found in other mammalians [23,81,91,93]. Interestingly, expression of BBD129 gene initiates from the rete testes in Indian cattle and had maximum expression in the middle corpus region of the epididymis, thereafter the expression decreases suggests that as the sperm acquire its BBD129 coat largely in the corpus region of the epididymis. The MRT expression pattern of the BBD129 gene suggests its region-speci c roles in sperm maturation, protection and makes sperm fertilizable. The cross-bred cattle-yak epididymal transcriptomics study reported an association of BBD129 gene down-expression with male sterility [94]. It is well-understood phenomenon that sperm epididymal coat proteins follow the last-in, rst-out sequence in the female reproductive tract in order to execute proper sperm function. The current nding of intense expression of cattle BBD129 gene in healthy matured MRT tissues clearly indicates the region-speci c abundance of BBD129 protein destined for uptake on the sperm surface.
The emerging bio-computational analyses have shortened the issues of expensive in-vitro experimentations and high throughput technologies cost. In this study, bioinformatic analyses underpinned the possible causes of nsSNPs which could alter the structure and functions of BBD129 protein. The polymorphisms could lead to abrupt mRNA synthesis and conformational distorter protein or non-functional proteins. The bovine BNBD-4 SNPs (CT2239) effect gene functioning by incorrect intron splicing results in a premature stop codon and unstable/aberrant mRNA production [95]. The non-synonymous genomic variation (C483A, rs378652941) in TMEM95 gene introduces a premature stop codon leads to mRNA decay and also, reported that this altered codon resides within the transmembrane domain of TMEM95 most likely resulting in a disturbed anchorage of the truncated protein on the sperm plasma membrane [96]. In this study, the mountain curve plot analysis of minimum free energy, centroid secondary structure, partition function, thermodynamic ensemble, and entropy is an easy view of effects of nsSNP on BBD129 mRNA. The possible impact of observed BBD129 polymorphism on mRNA found that they are making more complex BBD129 mRNA secondary structure by altering the base pairing which could affect the transcription or translation processes and may effect BBD129 protein abundance.
The mutation Q47R in PATE1 protein cause structural damages in protein leads to less bio-availability to the sperm membrane and results in reduced sperm motility associated with phosphorylations. [97][98][99]. Bovine SPAG11 gene polymorphisms are associated with higher ejaculate volumes, higher sperm concentrations, higher fresh sperm motility, and higher post-thaw cryopreserved sperm motility [100]. The serine-to-alanine S191A in progesterone receptors cause de-phosphorylation leads to reduced female fertility (litters size) and uterine growth [101][102] Capacitation and acrosome reactions are associated with increased serine, tyrosine, and threonine amino-acids phosphorylations and kinase activities [103][104]. The epididymal secretions and seminal plasma proteins are potent de-capacitational factors that maintain the premature capacitational and acrosomal integrity of the sperm in the epididymis and FRT before the conference with the zona pellucida membrane [104][105][106]. The polymorphism in BBD129 increases threonine and serine phosphorylations which may trigger the sperm hyperactive motility, early capacitation, early acrosomal reaction or may attract immune cells [107][108]. The adsorption of primate DEFB126 on the sperm surface provides a thick glycocalyx which facilitates numbers of sperm functions and polymorphisms in the DEFB126 cause abnormal glycosylations leads to sub-fertility [10-11, 34, 71]. In buffalo, the abundance of glycans on the spermatozoa from distinct fertility bull affect the neutrophil phagocytosis and NETosis process [109]. Similarly in our study, glycosylation prediction revealed nsSNPs causing positional alterations in the O-glycosylations pattern on BBD129 protein. All prediction results suggest snSNPs could result in a substantial change in functional attributes of BBD129 protein or sperm functioning [11,22,34] and their abundance in the group of low fertilizing bulls could be a possible marker for bovine fertility.

Conclusion
The present study clearly suggests a close association between polymorphisms of BBD129 gene with bull's conception rate. Maximum expression of the BBD129 gene in the corpus epididymis region might, however, be of interest for its uptake on the sperm surface. Full-length of BBD129 in Indian cattle was absolute in consonant with Bos taurus sequence re ecting the conserved functionality of protein. A detailed insight into the SPNs proves the non-synonymous polymorphisms in the BBD129 gene are convincingly related to the low conception rate of Indian cattle bulls. Further, the change in functional characteristics of BBD129 protein due to SNPs indicating towards the altered structural and conformational topology of BBD129 protein could be responsible for the distinct fertilizing potential of sperm. However, further study on large sample size and in-vitro validation assay needs to be taken up for assessing the altered fertilization of sperm and other reproductive pleiotropic functions that emerged due to SNPs in BBD129 of Indian cattle.        Distribution of BBD129 TA haplotype and GG haplotype in distinct fertility cattle bulls.

Figure 6
Absolute RT-qPCR analysis: distribution of BBD129 gene copy number in distinct fertility cattle bulls. RT-qPCR expression analysis was done to know the abundance of BBD129 mRNA in different parts of healthy matured cattle bull.

Figure 8
RNAfold server to predict possible impact of nsSNPs on mRNA secondary structure. Mountain plot has shown double mutated BBD129 mRNA get more complex than native BBD129 mRNA. SNPs are in uencing the base pairing between single stranded mRNA.

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