Introduction
Bull infertility or subfertility critically regulates the herd reproductive performance and consequently, the economics of a dairy farm (Amann and DeJarnette, Reference Amann and DeJarnette2012, Taylor et al., Reference Taylor, Schnabel and Sutovsky2018, Butler et al., Reference Butler, Bormann, Weaber, Grieger and Rolf2020). It has a significant effect on the major performance traits including days open (Hagiya et al., Reference Hagiya, Hanamure, Hayakawa, Abe, Baba, Muranishi and Terawaki2018), daughter pregnancy rate (Raheja et al., Reference Raheja, Nadarajah and Burnside1989), average daily gain (Raidan et al., Reference Raidan, Tineo, de Moraes, Escarce, de Araújo, Gomes, Ventura and Toral2017), in vitro seminal parameters (Oliveira et al., Reference Oliveira, de Arruda, de Andrade, Celeghini, dos Santos, Beletti, Peres, Oliveira and Hossepian de Lima2012, Mapel et al., Reference Mapel, Hiltpold, Kadri, Witschi and Pausch2022) and sperm defects (Leite et al., Reference Leite, de Losano, Kawai, Rui, Nagai, Castiglioni, Siqueira, D’Avila Assumpção, Baruselli and Nichi2022). The ‘omics’ revolution in animal breeding has brought new insights into the genetic make-up of sperm and its variability in individuals (Taylor et al., Reference Taylor, Schnabel and Sutovsky2018), thus bringing more clarity into the cases of bull infertility or subfertility (Han and Peñagaricano, Reference Han and Peñagaricano2016, Rezende et al., Reference Rezende, Dietsch and Peñagaricano2018, Das et al., Reference Das, Begum, Choudhury, Medhi, Paul and Das2020, Kumaresan et al., Reference Kumaresan, Elango, Datta and Morrell2021). It has been found that sperm infertility is modulated by the immunological milieu in the male reproductive tract.
Spermatogenesis (and the consequent sperm fertility) is crucially regulated by the inflammatory and non-inflammatory responses induced by the immune cells (Ye et al., Reference Ye, Huang, Liu, Cai, Hong, Xiao, Thiele, Zeng, Song and Diao2021). Physiological and pathological injuries activate the immune regulatory molecules which, though, offer protection to the sperm against these attacks but end up having a detrimental effect on its fertility (Archana et al., Reference Archana, Selvaraju, Binsila, Arangasamy and Krawetz2019). Oxidative stress produced as a result of microbial and viral attack on the sperm can also cause anatomical changes in the male reproductive tract including testicular damage, reduction in Leydig cell mass and atrophy of seminiferous tubules and Sertoli cells (Akhigbe et al., Reference Akhigbe, Dutta, Hamed, Ajayi, Sengupta and Ahmad2022, Das et al., Reference Das, Roychoudhury, Dey, Jha, Kumar, Roychoudhury, Slama and Kesari2022a). Immune response-related genes like IL6, IL8, IL1A (Robertson and Sharkey, Reference Robertson and Sharkey2016), IFN (Hansen, Reference Hansen2007), HLA-DRA, HLA-DRB1, TNFRSF14 and VRK1 (Salvi et al., Reference Salvi, Gawde, Idicula-Thomas and Biswas2022, Cerván-Martín et al., Reference Cerván-Martín, Tüttelmann, Lopes, Bossini-Castillo, Rivera-Egea, Garrido, Lujan, Romeu, Santos-Ribeiro, Castilla, Carmen Gonzalvo, Clavero, Maldonado, Vicente, González-Muñoz, Guzmán-Jiménez, Burgos, Jiménez, Pacheco, González, Gómez, Amorós, Aguilar, Quintana, Calhaz-Jorge, Aguiar, Nunes, Sousa, Pereira, Pinto, Correia, Sánchez-Curbelo, López-Rodrigo, Martín, Pereira-Caetano, Marques, Carvalho, Barros, Gromoll, Bassas, Seixas, Gonçalves, Larriba, Kliesch, Palomino-Morales and Carmona2022) have been usually implicated in sperm infertility or subfertility resulting from immunological causes.
Yak (Bos grunniens) is a seasonal breeder owing to the pastoral management system that offers rich forages and nutrition for breeding in the high altitudes during summers, whereas scarcity of vegetation renders them reproductively anestrous during winters (Prakash et al., Reference Prakash, Sarkar, Paul, Mishra, Mishra and Meyer2005, Das et al., Reference Das, Kour, Deori, Begum, Pukhrambam, Maiti, Sivalingam, Paul and Sarkar2022b). This production system renders the species highly vulnerable to the dangers of extinction (Das et al., Reference Das, Begum, Choudhury, Medhi, Paul and Das2020, Kour et al., Reference Kour, Niranjan, Malayaperumal, Surati, Pukhrambam, Sivalingam, Kumar and Sarkar2022). In this scenario, culling of infertile or subfertile bulls is crucial for continued economic viability and conservation of this unique germplasm. Infertility studies in yaks have been majorly directed at improving the understanding of hybrid male sterility seen in cattle yak (Tumennasan et al., Reference Tumennasan, Tuya, Hotta, Takase, Speed and Chandley1997; Wang et al., Reference Wang, Pan, Zhang, Xie, Liu and Li2012). Most of these studies have been conducted by comparing mRNA expression between hybrids and their parents (Wang et al., Reference Wang, Pan, Zhang, Xie, Liu and Li2012; Cai et al., Reference Cai, Yu, Mipam, Yang, Zhao, Liu, Cao, Shen, Zhao, Sun, Xu and Wu2017; Wu et al., Reference Wu, Zhang, Zuo and Zhang2019; Zhao et al., Reference Zhao, Sun, Chen, Li, Wang, Lai and Jia2022), while others have focused on DNA methylome of the male hybrids to reveal the probable epigenetic roles (Liu et al., Reference Liu, Li, Pan, Qu, Zhang and Xie2011; Luo et al., Reference Luo, Mipam, Wu, Xu, Yi, Zhao, Chai, Chen, Wu, Wang, Wang, Wang, Zhong and Cai2022). Some of the workers have pursued a candidate gene approach targeting Y-chromosome linked genes, namely, MSY, TSPY, TSPY2, PRAMEY, UTY, OFD1Y, USP9Y and SCYP3 to identify their roles in regulating yak bull fertility (Wang et al., Reference Wang, Pan, Zhang, Xie, Liu and Li2012; Zhang et al., Reference Zhang, Wu, Luo, Guan, Wang, Luo and Zuo2019; Wu et al., Reference Wu, Zhang, Zuo and Zhang2019). Das and co-workers (2020) deduced the involvement of small non-coding RNAs like miRNA19a, miRNA142 and miRNA143 in determining subfertility in Arunachali yak bulls.
This study particularly aimed at understanding the omic backgrounds of infertile or subfertile yak bulls. Therefore, infertile or subfertile yak sperm was subjected to genomic and transcriptomic evaluations to unveil the genes and biological processes regulating infertility or subfertility in yak bulls. This study provided initial leads for further detailed investigations in the area.
Materials and methods
Sample collection
Three adult healthy Arunachali yak bulls with true to the breed characters (Das et al., Reference Das, Kour, Deori, Begum, Pukhrambam, Maiti, Sivalingam, Paul and Sarkar2022b) and showing infertility or subfertility were considered for the study. Animals were kept in the bull shed of the experimental yak farm of the Indian Council of Agricultural Research (ICAR)-National Research Centre on Yak at Nyukmadung, Arunachal Pradesh, at an elevation of 9000 ft above mean sea level (Figure 1). Animals were housed in open shelters (CGI roof) with concrete floors and were fed concentrate feed at 2–3% of their body weight. This was supplemented with green grass and paddy straw (roughages) and an ad libitum supply of drinking water. All the bulls were apparently healthy and were regularly vaccinated against major diseases. Semen was generally collected at fortnightly intervals using a teaser bull and was microscopically evaluated for its quality before being used for insemination. A bull was considered infertile when its conception rate was zero after being successively used for breeding for 2 years, and a bull was considered subfertile when its conception rate was less than 5% after being used successively for 5 years in the herd.
Figure 1. Arunachali yak bull housed at the ICAR-National Research Centre on Yak.
Fresh ejaculates were collected from three infertile or subfertile yak bulls by artificial vagina method using the Missouri model (Das et al. Reference Das, McCarthy, Vishnoi, Paria, Gresham, Li, Kachroo, Sudderth, Teague, Love, Varner, Chowdhary and Raudsepp2013). Semen samples were collected in accordance with the approval of the Institute Animal Ethics Committee of the ICAR-National Research Centre on Yak, Dirang, India, and the approved animal use protocol number was 4(17)/NRCY/IAEC-02. Subsequently, semen samples were processed and purified (Das et al. Reference Das, McCarthy, Vishnoi, Paria, Gresham, Li, Kachroo, Sudderth, Teague, Love, Varner, Chowdhary and Raudsepp2013) and stored at –80oC until further use. DNA was isolated using a protocol developed by Wu et al. (Reference Wu, de Gannes, Luchetti and Richard Pilsner2015) with slight modification. RNA from sperm was isolated using a published protocol with the help of a 27 gauge needle and Trizol (Das et al. Reference Das, Paria, Gustafson-Seabury, Vishnoi, Chaki, Love, Varner, Chowdhary and Raudsepp2010).
Whole-genome and transcriptome sequencing
1 ng of sperm DNA concentration was used to prepare libraries using Illumina’s Nextera XT DNA (Catalog no: FC-131-1024) and Nextera DNA Flex (Catalog no: 20018704) library preparation kits. Each pool of libraries with raw cluster densities of 202 and 189 for Nextera XT and Nextera DNA Flex, respectively, were loaded and sequenced separately on a NextSeq 500 System. Finally, paired-end reads of 2 × 151 bp were generated for further analysis.
Total RNA from the sperm of three infertile or subfertile yak bulls was pooled together and used for next-generation sequencing on the Roche platform. RNA concentration was measured using Nanodrop and the integrity of RNA was checked by Bioanalyzer. A cDNA library was prepared from the sperm RNA involving the following steps: fragmentation of nucleic acids to 300–400 bp lengths followed by end repair, ligation of adapters to ends of target sequences, library amplification and quantification, selection of appropriate fragments and removal of adapter dimers. Roche single-end RNA sequencing was performed using library fragments constructed according to the Roche protocol.
Data analysis
DNA sequencing data
The generated data were demultiplexed using bcl2fastq (version v2.17.1.14; Illumina), and the final sequence reads were trimmed using trimmomatic (version 0.38) (Bolger et al., Reference Bolger, Lohse and Usadel2014) using default parameters (illuminaclip :2:30:10). The sequenced contigs data were processed in mpiBLAST (Darling et al., Reference Darling, Carey and Wu2003) to generate an xml file. This file was subsequently used to search for similarity in BLAST2GO software (Götz et al., Reference Götz, García-Gómez, Terol, Williams, Nagaraj, Nueda, Robles, Talón, Dopazo and Conesa2008), and contigs with similarity mean >80% were considered for further mapping and annotation. Gene ontology (GO) IDs associated with the data were analysed in PANTHER (Mi et al., Reference Mi, Dong, Muruganujan, Gaudet, Lewis and Thomas2010) to find out the statistical overrepresentation of particular biological processes in yak sperm DNA at False Discovery Rate (FDR) ≤ 0.01.
RNA-sequencing data
The raw reads obtained by RNA sequencing were converted to fastq format, and the quality check was performed. The quality of the reads was evaluated using FASTQC (Andrews, Reference Andrews2010), and PRINSEQ lite v0.20.4 (Schmieder and Edwards, Reference Schmieder and Edwards2011) was used to trim the adaptor tags from the single-end reads. Thereafter, Bos taurus (assembly ARS-UCD1.2), as well as Bos grunniens (GCA_005887515.2 BosGru v3.0) reference genome, was downloaded and indexed, and reads were aligned to both the genomes using Hisat2 (Kim et al., Reference Kim, Langmead and Salzberg2015). The .sam file containing the mapped reads was sorted with the help of SAMtools (Li et al, Reference Li, Handsaker, Wysoker, Fennell, Ruan, Homer, Marth, Abecasis and Durbin2009) and, subsequently assembled using StringTie (Pertea et al., Reference Pertea, Pertea, Antonescu, Chang, Mendell and Salzberg2015). StringTie was also used to estimate the gene abundance of the transcripts assembled from both Bos taurus and Bos grunniens reference genomes. The transcripts with gene coverage ≥1 were selected for further gene annotation and ontology. The retrieved gene list was fed into PANTHER (Mi et al., Reference Mi, Dong, Muruganujan, Gaudet, Lewis and Thomas2010) to identify statistically overrepresented GO terms (FDR ≤ 0.01).
Results
The total 36,542 contigs obtained through sperm genome sequencing were blasted, mapped and annotated to reveal GO IDs corresponding to >80% similarity mean. These GO IDs were fed into Ensembl Biomart to retrieve a list of 8,337 protein-coding genes (Supplementary Table 1). Gene ontology analysis highlighted 1,089 statistically overrepresented biological processes (Figure 2). The complete list of statistically significant GO terms has been presented in Supplementary Table 2. The significant biological processes could be majorly categorized into parent groups, namely, biological regulation (GO: 0044848), cellular process (GO: 0009987), localization (GO: 0051179), metabolic process (GO: 0008152), response to stimulus (GO: 0050896) and signalling (GO: 0023052). Significant biological processes related to immunity included immune system development (GO:0002520), immune system process (GO:0002376), regulation of immune response (GO:0050776), regulation of T cell (GO:0050863) and lymphocyte activation (GO:0051249), natural killer cell activation involved in immune response (GO:0002323), regulation of cytokine production involved in immune response (GO:0002718) etc.
Figure 2. Network map of gene ontology terms related to biological processes in sperm DNA.
A cDNA library was prepared from the sperm samples of Arunachali yak and raw reads were generated. After alignment of the reads with Bos grunniens and Bos taurus genomes, assembled transcripts from both genomes were evaluated for gene abundance, and those with gene coverage ≥1 were highlighted. However, transcripts assembled from cattle genome corresponded to a number of protein-coded genes including VAMP7, MYLK, ARAP2 and MARCH6. All the protein-coding genes were subjected to gene ontology analysis for reflecting statistically overrepresented biological processes (FDR ≤ 0.01). The GO terms for significant biological processes were all related to immune responses and have been presented in Table 1. Since we did not perform a comparative transcriptomic study in fertile bulls, it may be possible that bulls considered in this study may be suffering from some underlying infection or inflammation at the time of sampling due to which immune response was overrepresented. Although there was no clinical history of any disease or infection at the time of sampling, we cannot ignore this reason. This is also possible since biological processes related to immune responses were only found to be significantly overrepresented.
Table 1. Significant gene ontology (GO) terms for biological processes in yak sperm cDNA
Discussion
The infertile yak sperm genome is comprised of all the cellular components, molecular functions and biological processes necessary for the growth, development, survival, immunity and multiplication of an organism. This is quite obvious given the fact that sperm, being a gamete, possesses one complete set of genes that combines with the other set from the ovum to produce a diploid individual with normal bodily functions and development.
The transcriptome of infertile or subfertile yak sperm highlighted that the GO terms related to immune-related processes were highly significant. Though this study suffered from a limitation that differential expression and transcriptome comparison with fertile yak sperm was not carried out, nonetheless, it provided insights into the genomic and transcriptomic landscape of infertile or subfertile yak sperm. These findings will definitely help in designing further detailed downstream studies in the future.
Based on RNA Seq results, granulocytes-mediated immune response was found to be significant in infertile yak sperm granulocytes or polymorphonuclear leukocytes (PMN), namely, eosinophils, neutrophils and basophils, constituting the most prevalent white blood cells in semen followed by macrophages and T-lymphocytes (Wolff, Reference Wolff1995). Additionally, granulocytes in semen are negatively correlated with normal sperm morphology and positively with mid-piece abnormalities, thus highlighting their role in male infertility or subfertility (Thomas et al., Reference Thomas, Fishel, Hall, Green, Newton and Thornton1997). Specifically, neutrophil activation results in the production of reactive oxygen species, which severely dents the sperm motility (Kovalski et al., Reference Kovalski, De Lamirande and Gagnon1992). Being the first-line innate immune defense system, PMNs release bactericidal enzymes by degranulation to form neutrophil extracellular traps (NETs) and, hence, phagocytosis (Brinkmann et al., Reference Brinkmann, Reichard, Goosmann, Fauler, Uhlemann, Weiss, Weinrauch and Zychlinsky2004; Borregaard, Reference Borregaard2010). When the blood-testis barrier is breached, antigens on the sperm surface induce PMN activation, thus extruding their DNA and resulting in the trapping of sperm in NETs, thus hindering sperm motility. However, neutrophil activation does not necessarily indicate deterioration of sperm function as DNAse activity of seminal plasma proteins helps in the digestion of extruded DNA and frees the entangled spermatozoa, greatly boosting the chances of conception in the female reproductive tract (Alghamdi and Foster, Reference Alghamdi and Foster2005). Also, recent evidence suggests that the phagocytic activity executed by PMNs is crucial for the therapeutic activity of the sperm head in the female reproductive tract (Pakravan et al., Reference Pakravan, Hassan and Abbasi2021).
Eosinophils also play an important role in the bodily immune response by protecting against allergens and parasitic infestation (Shamri et al., Reference Shamri, Xenakis and Spencer2011). Eosinophilic degranulation is exacerbated in response to infections and inflammation, and an RNAse-mediated positive feedback loop is established between eosinophils and mast cells in which the former induces mast cell activation by secretion of RNase and mast cells, in turn, secrete cytokines like IL-5, which further stimulate RNase release from eosinophils (Bystrom et al., Reference Bystrom, Amin and Bishop-Bailey2011). Subsequently, eosinophilic activation is mediated by these RNases through the toll-like receptor signalling pathway and results in the release of cytokines and various immune regulatory molecules (Rudd et al., Reference Rudd, Burstein, Duckett, Li and Lukacs2005; Phipps et al., Reference Phipps, En Lam, Mahalingam, Newhouse, Ramirez, Rosenberg, Foster and Matthaei2007). It has been reported that eosinophilic degranulation is observed in female cervical mucosa on exposure to IgA antibodies, thus evoking an allergenic reaction and disrupting immune tolerance (Brazdova et al., Reference Brazdova, Senechal, Peltre and Poncet2016). This may be an important mechanism underpinning sperm infertility or subfertility in the female reproductive tract.
Another probable proposition for the increased expression of immune response in infertile or subfertile yak spermatozoa could be the presence of antisperm antibodies (ASAs). Autoimmunity to spermatozoa or the presence of ASAs is increasingly being implicated as a major cause underlying human male infertility or subfertility (Archana et al., Reference Archana, Selvaraju, Binsila, Arangasamy and Krawetz2019). Though the testis is an immune-privileged site and protects the spermatozoa against autoimmune attack, the blood-testis barrier provided by the organ is not invincible (Wang and Holstein, Reference Wang and Holstein1983) as ASAs have been reported in around 18% of the infertile males (Bozhedomov and Teodorovich, Reference Bozhedomov and Teodorovich2005). Antigenic interaction between microorganisms and sperm (Bozhedomov and Teodorovich, Reference Bozhedomov and Teodorovich2005), inflammatory conditions (Marconi et al., Reference Marconi, Pilatz, Wagenlehner, Diemer and Weidner2009), tumors (Bronson et al., Reference Bronson, O’Connor, Wilson, Bronson, Chasalow and Droesch1992) and reduced levels of cellular and humoral immunomodulatory factors in seminal plasma (Cooley et al., Reference Cooley, El Shikh, Li, Keim, Zhang, Strauss, Zhang and Conrad2016) pose a grave challenge to the immunosuppressive environment provided by the testis. These insults lead to the production of cytokines, leukocytes and T-cell activators, all of which subsequently impact the fertilizing ability of sperm (Bohring and Krause, Reference Bohring and Krause2003). This has been further validated by improvements seen in sperm motility, sperm concentration and overall conception rate on the administration of immune-suppressive corticosteroid therapy in infertile human males (Skau and Folstad, Reference Skau and Folstad2005). Although there is a paucity of research in this area concerning farm animals, a significant association has been reported between ASAs in serum or seminal plasma and fertility in cattle bulls (Zodinsanga et al., Reference Zodinsanga, Cheema and Mavi2015; Ferrer et al., Reference Ferrer, Laflin, Anderson, Miesner, Wilkerson, George, Miller, Larson and Garcia Flores2015). Sperm-bound ASAs were reported to be associated with poor post-thaw motility and breeding soundness in stallions (Ferrer and Miller, Reference Ferrer and Miller2018; Ferrer et al., Reference Ferrer, Canisso, Podico, Ellerbrock, Hurley and Palomares2021). Though we did not test for the presence of ASAs in the sperm of infertile or subfertile bulls considered in the study, this cause cannot be ruled out completely.
Genes like VAMP7, MYLK, ARAP2 and MARCH6 showed increased expression in infertile or subfertile yak bulls. Over-expression of VAMP7 has been associated with increased transcription of oestrogen receptors resulting in reduced sperm motility and spermatogenic failure (Tannour-Louet et al., Reference Tannour-Louet, Han, Louet, Zhang, Romero, Addai, Sahin, Cheung and Lamb2014). MYLK gene plays a crucial role in upregulation of AGBL4 gene which subsequently, results in teratozoospermia in males (Wu et al., Reference Wu, Rong, Correia, Min and Morgan2015, Han et al., Reference Han, Wang, Yu, Ge, Li, Jiang, Shen and Sun2021). CENTD1 gene (analogue of human ARAP2 gene) is significantly downregulated in the presence of miR-10a which causes acute myeloid leukaemia in humans (Bryant et al., Reference Bryant, Palma, Jayaswal, Yang, Lutherborrow and Ma2012). MARCH6 was identified as a differentially methylated gene in infertile sperm samples and could be postulated as a novel biomarker gene for male infertility (Cassuto et al., Reference Cassuto, Piquemal, Boitrelle, Larue, Ledee, Hatem, Ruoso, Bouret, Siffroi, Rouen and Assou2021). This indicates that the reduction in DNA methylation in immune-related genes can lead to increased transcript expression of these genes in infertile males (Schütte et al., Reference Schütte, El Hajj, Kuhtz, Nanda, Gromoll, Hahn, Dittrich, Schorsch, Müller and Haaf2013). Though this study suffered from some limitations, it explored the ‘omic’ landscape of infertile or subfertile yak sperm. Further functional studies can be carried out to validate our findings and to bring out interesting insights.
Conclusions
‘Omic’ analysis of infertility or subfertility in yak sperm highlighted that genes including VAMP7, MYLK, ARAP2 and MARCH6 were regulating the phenotype. Furthermore, immune-related biological processes (GO:0043308, GO:0002447, GO:0002278, GO:0043307, GO:0043312, GO:0002283, GO:0043299 and GO:0002446) were significantly overrepresented (FDR ≤ 0.01). These findings may be indicative of a crucial role being played by genotype-environment interactions in determining infertility or subfertility in male yaks. However, further downstream studies in this direction can validate our findings and propositions.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0967199424000194.
Data availability
The data generated as a part of this study have been successfully submitted to Sequence Read Archive with Bioproject ID PRJNA931839.
Acknowledgements
The authors are highly grateful to the ICAR for providing financial resources to carry out this study. Special thanks are due to ICAR-Indian Agricultural Statistics Research Institute, New Delhi, for providing unconditional help and support.
Author contributions
PJD, AK and MS conceptualized and designed the study; AK, JB and DCM carried out the data analysis; AK and PJD wrote the paper; and DCM, JB and MS critically revised the manuscript.
Funding
This study received no external funding
Competing interests
All the authors declare that the research was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest.
Ethical Standards
This study was approved by the Institute Animal Ethics Committee of the ICAR-National Research Centre on Yak, Dirang, India, vide approval number 4(17)/NRCY/IAEC-02.
