The association between sub-optimal paternal diet and offspring well-being is becoming established. However, the underlying mechanisms are yet to be fully defined. The aim of this study was to establish the impact of over- and under-nutrition, with or without macronutrient supplementation, on male reproductive fitness and post-fertilisation development. Male C57BL/6J mice were fed either control diet (CD), isocaloric low-protein diet (LPD), high-fat/sugar ‘Western’ diet (WD), or LPD or WD supplemented with methyl donors and carriers (MD-LPD or MD-WD, respectively) for 8 weeks before mating with virgin C57/BL6J females. Placental tissue was collected at embryonic day (E)8.5 to assess early placental (ectoplacental cone) morphology and metabolism and E17.5 for sex-specific transcriptomic profiling. Post-mating, stud male tissues were harvested for the assessment of testicular morphology and gene expression, gut microbiota composition, and metabolic status. WD and MD-WD males displayed increased adiposity, hepatic cholesterol and free fatty acids, and gut microbiota dysbiosis when compared to CD-fed males. In the testes, WD and MD-WD perturbed the expression of genes associated with metabolism and transcription regulation. Additionally, we observed differential expression of multiple genes within the Wnt signalling pathway, central in the regulation of cellular proliferation, migration, survival, and cell fate determination during development. Despite no impact on fundamental male fertility, significant changes in ectoplacental cone metabolism, fetal growth, and placental gene expression were observed in response to specific dietary regimens. Interestingly, while CD male and female placentas displayed 301 genome-wide, sexually dimorphic genes, LPD, MD-LPD, WD, and MD-WD male and female placentas possessed only 13, 0, 14, and 15 sexually dimorphic genes, respectively. Our data show that while sub-optimal paternal diet has minimal impact on male fertility, fetal and placental development are perturbed in a sex-specific manner.

In brief:Male reproductive tract extracellular vesicles play a critical role in regulating sperm quality and male fertility. This study shows that extracellular vesicles from distinct regions of the male reproductive tract differ in their size, abundance and composition. Abstract:As sperm transit the male reproductive tract, they undergo a series of dynamic changes, gaining motility, modifying lipid and protein content and refining their epigenetic composition. Extracellular vesicles are central to this post-testicular maturation and changes in their composition could directly impact male reproductive health, sperm quality and post-fertilisation development. This study aimed to characterise and compare extracellular vesicles isolated from distinct regions of the male reproductive tract. Extracellular vesicles were isolated from adult, male C57BL/6J cauda and caput epididymis (epididymosomes) and seminal vesicle fluid by precipitation and size exclusion chromatography. Isolated vesicles were characterised using nanoparticle tracking analysis, transmission electron microscopy, Western blotting and imaging flow cytometry. Epididymosomes and seminal fluid vesicles ranged from 110.26 to 121.26 nm in diameter, had a concentration of 109 to 1010 particles/cm3 and had a typical round, cup-shaped morphology. The size and concentration of extracellular vesicles from the caput were significantly larger than those from the cauda and seminal fluid. Imaging flow cytometry revealed that all isolated extracellular vesicles expressed CD81 and CD9 tetraspanins; however, CD63 was detected only in caput epididymosomes. Furthermore, there were significantly fewer CD9+ vesicles in seminal fluid EVs compared to epididymosomes. Using a range of bulk- and single-vesicle analytical approaches, we show that different regions of the male reproductive tract display distinct vesicle compositional phenotypes. However, additional studies are warranted to define the significance of this heterogeneity, their roles in regulating male reproductive health and the development of their offspring.

In men, the reproductive system has the central purpose of producing spermatozoa capable of fertilising the oocyte within the female reproductive tract. In addition, the male reproductive system must also secrete a series of hormones that stimulate the continuous production of spermatozoa which, once initiated at puberty, will continue throughout a man's life. Finally, the anatomy of the male reproductive system must provide an environment suitable to ensuring the processes of spermatogenesis and spermiation can occur in a continuous and highly coordinated manner. Spermatogenesis involves the dramatic remodelling of relatively undifferentiated diploid, spermatogonial stem cells into highly specialised haploid spermatozoa. Within the seminiferous tubules of the testes, the spermatogonium undergo mitosis, maintaining the stem cell pool while also generating primary spermatocytes. Through subsequent rounds of meiosis and cell remodelling, spermatids are generated, each with unique genetic identity. Subsequently, through the process of spermiogenesis, the spermatids differentiate further into spermatozoa with fully condensed chromatin. Finally, under spermiation, the mature spermatids are released into the lumen of the seminiferous tubule prior to their final maturation within the epididymis. This highly co-ordinated process is maintained and supported by a series of extrinsic and intrinsic factors and supporting cells, all critical in supporting male reproduction and fertility.

Pregnancy represents a stage during which maternal physiology and homeostatic regulation undergo dramatic change and adaptation. The fundamental purpose of these adaptations is to ensure the survival of her offspring through adequate nutrient provision and an environment that is tolerant to the semi-allogenic foetus. While poor maternal diet during pregnancy is associated with perturbed maternal adaptations during pregnancy, the influence of paternal diet on maternal well-being is less clearly defined. We fed C57BL/6 male mice either a control (CD), low protein diet (LPD), a high fat/sugar Western diet (WD) or the LPD or WD supplemented with methyl donors (MD-LPD and MD-WD, respectively) for a minimum of 8 weeks prior to mating with C57BL/6 females. Mated females were culled at day 17 of gestation for the analysis of maternal metabolic, gut, cardiac and bone health. Paternal diet had minimal influences on maternal serum and hepatic metabolite levels or gut microbiota diversity. However, analysis of the maternal hepatic transcriptome revealed distinct profiles of differential gene expression in response to the diet of the father. Paternal LPD and MD-LPD resulted in differential expression of genes associated with lipid metabolism, transcription, ubiquitin conjugation and immunity in dams, while paternal WD and MD-WD modified the expression of genes associated with ubiquitin conjugation and cardiac morphology. Finally, we observed changes in maternal femur length, volume of trabecular bone, trabecular connectivity, volume of the cortical medullar cavity and thickness of the cortical bone in response to the father’s diets. Our current study demonstrates that poor paternal diet at the time of mating can influence the patterns of maternal metabolism and gestation-associated adaptations to her physiology.

Buffalo is a dominant dairy animal in many agriculture-based economies. However, the poor reproductive efficiency (low conception rate) of the buffalo bulls constrains the realization of its full production potential. This in turn leads to economic and welfare issues, especially for the marginal farmers in such economies. The mammalian sperm surface proteins have been implicated in the regulation of survival and function of the spermatozoa in the female reproductive tract (FRT). Nonetheless, the lack of specific studies on buffalo sperm surface makes it difficult for researchers to explore and investigate the role of these proteins in the regulation of mechanisms associated with sperm protection, survival, and function. This study aimed to generate a buffalo sperm surface-specific proteomic fingerprint (LC-MS/MS) and to predict the functional roles of the identified proteins. The three treatments used to remove sperm surface protein viz. Elevated salt, phosphoinositide phospholipase C (PI-PLC) and in vitro capacitation led to the identification of N = 1,695 proteins (≥1 high-quality peptide-spectrum matches (PSMs), p < 0.05, and FDR<0.01). Almost half of these proteins (N = 873) were found to be involved in crucial processes relevant in the context of male fertility, e.g., spermatogenesis, sperm maturation and protection in the FRT, and gamete interaction or fertilization, amongst others. The extensive sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth of identification of reproduction-specific cell-surface proteins and can provide a potential framework for further studies on the functional aspects of buffalo spermatozoa.

IntroductionOne of the most evolutionary conserved communication systems, the Wnt signaling pathway is a major gene regulatory pathway that affects the developmental competence of oocytes and regulates most embryonic developmental processes. The present study was undertaken to modulate the canonical Wnt (Wingless/integration) signaling pathway in the poor-quality (colorless cytoplasm after Brilliant Cresyl Blue staining, BCB-) buffalo cumulus-oocyte complexes (COCs) to improve their in vitro maturation (IVM) and embryo production (IVEP) rates.MethodsThe expression of key Wnt pathway genes was initially assessed in the good (blue cytoplasm after Brilliant Cresyl Blue staining, BCB+) and poor quality (BCB-) buffalo COCs to establish a differential activity of the Wnt pathway. The BCB- COCs were supplemented with the Wnt pathway inhibitor, Dickkopf-related protein 1 (DKK1) and later subjected to IVM and IVEP along with the BCB+ and BCB- controls. The cumulus expansion index (CEI), rate of nuclear maturation (mean percentage of oocytes in the MII stage) and embryo production, and the expression of developmentally important genes were evaluated to assess the effect of Wnt pathway inhibition on the development competence of these poor-quality oocytes.ResultsThe Wnt pathway genes exhibited a significantly higher expression (p < 0.05) in the poor-quality BCB- oocytes compared to the good-quality BCB+ oocytes during the early maturation stages. The supplementation of BCB- COCs with 100 ng/mL DKK1 effectively inhibited the expression of the key mediators of the Wnt pathway (β-catenin and dishevelled homolog 1, DVL1). DKK1 supplemented BCB- COCs exhibited significantly improved cytoplasmic and nuclear maturation indices, development rates and significantly elevated expression (p < 0.05) of genes implicated in germinal vesicle breakdown (GVBD) and embryonic genome activation (EGA) vis-à-vis BCB- control COCs.ConclusionThese data indicate that inhibition of the Wnt pathway during the initial course of oocyte maturation can improve the development competence of poor-quality buffalo oocytes.

The water buffalo (Bubalus bubalis) is an indispensable part of the Indian dairy sector and in several instances, the farmers incur economic losses due to failed pregnancy after artificial insemination (AI). One of the key factors for the failure of conception is the use of semen from the bulls of low fertilizing potential and hence, it becomes important to predict the fertility status before performing AI. In this study, the global proteomic profile of high fertile (HF) and low fertile (LF) buffalo bull spermatozoa was established using a high-throughput LC-MS/MS technique. A total of 1,385 proteins (≥1 high-quality PSM/s, ≥1 unique peptides, p < 0.05, FDR < 0.01) were identified out of which, 1,002 were common between both the HF and LF groups while 288 and 95 proteins were unique to HF and LF groups respectively. We observed 211 and 342 proteins were significantly high (log Fc ≥ 2) and low abundant (log Fc ≤ 0.5) in HF spermatozoa (p < 0.05). Gene ontology analysis revealed that the fertility associated high abundant proteins in HF were involved in spermatogenesis, sperm motility, acrosome integrity, zona pellucida binding and other associated sperm functions. Besides this, the low abundant proteins in HF were involved in glycolysis, fatty acid degradation and inflammation. Furthermore, fertility related differentially abundant proteins (DAPs) on sperm viz., AKAP3, Sp17, and DLD were validated through Western blotting and immunocytochemistry which was in coherence with the LC-MS/MS data. The DAPs identified in this study may be used as potential protein candidates for predicting fertility in buffaloes. Our findings provide an opportunity in mitigating the economic losses that farmers incur due to male infertility.

In pregnant animals, communication between the mother and conceptus occurs via extracellular vesicles (EVs) that carry several biomolecules such as nucleic acids (miRNAs, mRNAs), proteins, and lipids. At the time of implantation, the endometrium undergoes several morphological and physiological changes, such as angiogenesis, apoptosis, and cell proliferation regulation at the implantation site, to attain a receptive state. This study was conducted to detect pregnancy-specific miRNAs derived from extracellular vesicles in the systemic circulation of Bubalus bubalis (water buffalo) and to assess their functional significance in the modulation of endometrial primary cells. The extracellular vesicles were isolated from the blood plasma using a precipitation-based method and further characterized by various methods such as Differential light scattering, Nanoparticle tracking assay, Western blot, and transmission electron microscopy. The relative expression of the selected extracellular vesicles associated miRNAs (EV-miRNA) at different intervals (days 15, 19, 25, and 30) post artificial insemination (AI) was analyzed using RT-qPCR, and expression of miR-195-5p was found to be significantly higher (P < 0.01) in pregnant animals on day 19 post AI (implantation window) as compared to day 15 post AI. The elevated expression might indicate the involvement of this miRNA in the maternal-conceptus cross-talk occurring during the implantation period. The KEGG pathway enrichment and Gene Ontology analyses of the miR-195-5p target genes revealed that these were mostly involved in the PI3-Akt, MAPK, cell cycle, ubiquitin-mediated proteolysis, and mTOR signaling pathways, which are related to the regulation of cell proliferation. Transfecting the in vitro cultured cells with miR-195-5p mimic significantly suppressed (P < 0.05) the expression of its target genes such as YWHAQ, CDC27, AKT-3, FGF-7, MAPK8, SGK1, VEGFA, CACAND1, CUL2, MKNK1, and CACAN2D1. Furthermore, the downregulation of the miR-195-5p target genes was positively correlated with a significant increase in the apoptotic rate and a decrease in the proliferation. In conclusion, the current findings provide vital information on the presence of EV miR-195-5p in maternal circulation during the implantation window indicating its important role in the modulation of buffalo endometrium epithelial cells via promoting cell death. Altogether, the milieu of miR-195-5p may serve as a novel and potential molecular factor facilitating the implantation of the early embryo during the establishment of pregnancy in buffaloes. Thus, miR-195-5p may be identified as a unique circulatory EV biomarker related to establishing pregnancy in buffaloes as early as day 19 post-AI.

Agrichemicals such as organophosphorus pesticides’ metabolites (OPPMs) are more hazardous and pervasive than their parent pesticides. Parental germline exposure to such xenobiotics leads to an elevated susceptibility towards reproductive failures e.g. sub- or in-fertility. This study sought to examine the effects of low-dose, acute OPPM exposure on mammalian sperm function using buffalo as the model organism. The buffalo spermatozoa were briefly (2 h) exposed to metabolites of the three most prevalent organophosphorus pesticides (OPPs) viz. Omethoate (from Dimethoate), paraoxon-methyl (from methyl/ethyl parathion) and 3, 5, 6-trichloro-2-pyridinol (from chlorpyrifos). Exposure to OPPMs resulted in compromised structural and functional integrity (dose-dependent) of the buffalo spermatozoa typified by elevated membrane damage, increased lipid peroxidation, precocious capacitation and tyrosine phosphorylation, perturbed mitochondrial activity and function and (P < 0.05). This led to a decline in the in vitro fertilizing ability (P < 0.01) of the exposed spermatozoa, as indicated by reduced cleavage and blastocyst formation rates. Preliminary data indicate that acute exposure to OPPMs, akin to their parent pesticides, induces biomolecular and physiological changes in spermatozoa that compromise their health and function ultimately affecting their fertility. This is the first study demonstrating the in vitro spermatotoxic effects of multiple OPPMs on male gamete functional integrity.

AbstractBackgroundThe microRNAs (miRs) secreted by the trophectoderm (TE) cells have recently been implicated in the conceptus‐endometrial cross talk during implantation and placentation. These miRs modulate various cellular processes during conception and throughout the pregnancy by regulating the gene expression in the foetal and maternal tissues.ObjectivesThis study was undertaken to elucidate the function of TE secreted miRNAs in the maternal‐foetal cross‐talk during implantation/placentation in buffalo.MethodsThe in vitro produced blastocysts were cultured on a cumulus feeder layer for 21 days. The relative expression profiles of a selected panel of miRs was generated using the spent media collected on Days 0, 7, 12, 16, and 21. A custom‐designed mirVana™ miRNA mimic was used to transfect the endometrial epithelial cells (EECs) in order to determine the role of miRNA exhibiting highest expression on Days 21 and 21.ResultsThe expression of miR‐1246 (p < 0.001) and let‐7b (p < 0.01) was found to be significantly higher on Day 21 of TE culture in comparison to the control (Day 0). This elevated expression indicated the involvement of these miRs in the maternal‐foetal cross‐talk. Interestingly, after the transfection of EECs with miRNA mimic for miR‐1246 (a novel molecule vis‐à‐vis implantation), the expression of beta‐catenin and mucin1 in these cells was found to be significantly (p < 0.05) downregulated vis‐à‐vis the control, that is, the IFN‐τ primed EECs (before transfection).ConclusionsThe TE secreted miR‐1246 appeared to lower the expression of the endometrial receptivity genes (mucin1 and beta‐catenin) which apparently assists the endometrium in preparing for placentation.

As adults, our health can be influenced by a range of lifestyle and environmental factors, increasing the risk for developing a series of non-communicable diseases such as type 2 diabetes, heart disease and obesity. Over the past few decades, our understanding of how our adult health can be shaped by events occurring before birth has developed into a well-supported concept, the Developmental Origins of Health and Disease (DOHaD). Supported by epidemiological data and experimental studies, specific mechanisms have been defined linking environmental perturbations, disrupted fetal and neonatal development and adult ill-health. Originally, such studies focused on the significance of poor maternal health during pregnancy. However, the role of the father in directing the development and well-being of his offspring has come into recent focus. Whereas these studies identify the individual role of each parent in shaping the long-term health of their offspring, few studies have explored the combined influences of both parents on offspring well-being. Such understanding is necessary as parental influences on offspring development extend beyond the direct genetic contributions from the sperm and oocyte. This article reviews our current understanding of the parental contribution to offspring health, exploring some of the mechanisms linking parental well-being with gamete quality, embryo development and offspring health.

AbstractThe mammalian sperm surface is radically modified in its biomolecular composition and structure during the transit of sperm through the male and female reproductive tract (MRT, FRT). Numerous distinct secretagogues such as (glyco)proteins and many GPI-anchored proteins (GPI-APs) are applied as peripheral coats on the sperm surface in the MRT, which is critical for the maturation, survival and function of the spermatozoa. This study aimed to characterize the buffalo sperm surface proteins by extracting them either by using elevated salt (NaCl) or Phosphatidylinositol-specific phospholipase-C (for GPI-APs) or by inducing in vitro capacitation. A buffalo sperm surface-specific proteomic fingerprint was subsequently generated using the protein informatics platform, Proteome Discoverer (v2.2) for protein identification. Overall, 1695 unique proteins isoforms (proteoforms) (≥ 1 high-quality PSM/s, minimum 2 peptides, P < 0.05, FDR < 0.01) that exhibited remarkable heterogeneity in their molecular weight, pI, distribution of expression from the genome and their functional roles in the MRT and the FRT were identified. We characterized the identified including 200 orphan proteins by extrapolation of function from their sequence orthologs using BLAST2GO software. The extensive, yet unexplored, reproduction-specific buffalo sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth identification of fertility-related and reproduction-specific cell-surface proteins.

Abstract Agrichemicals such as metabolites (OPPMs) of the commonly-used organophosphorus pesticides (OPPs) are considered more hazardous and pervasive than their parent pesticides which. This is because parental germline exposure to such xenobiotics has been demonstrated to cause an elevated susceptibility towards various reproductive failures e.g. sub- or in-fertility Buffalo is a vital livestock resource, however, has numerous reproductive restraints e.g. male subfertility (low conception) that negatively impact its lifetime productivity. This study sought to examine the effects of low-dose, acute OPPM exposure on the mammalian sperm survival and its fertilizing ability. The buffalo spermatozoa were briefly exposed to the three most prevalent organophosphorus pesticides metabolites viz. Omethoate (Dimethoate), paraoxon-methyl (methyl parathion) and 3, 5, 6-trichloro-2-pyridinol, (chlorpyrifos). The structural and functional integrity of the OPPM-exposed and control sperm were evaluated by various Cytomics and Biomimetics experiments including assessment of capacitation, lipid-peroxidation, mitochondrial activity and kinematic parameters (CASA), and in vitro fertilization (IVF) apart from their gene-expression profiling (RT-qPCR). Preliminary data indicated that a perturbed phosphoproteomic endowment (PTP) and a dysregulated ROS-homeostasis in the OPPM-exposed spermatozoa (P<0.05) negatively affected their fertilizing ability (P<0.01) vis-à-vis control sperm. This is the first study demonstrating the spermatotoxic effects of OPPMs on gamete survival and function.

The mammalian spermatozoon is a consequence of extensive and intricate biochemical, physiological, and morphological cellular differentiation events which take place in the testes during a spermatozoon’s development. However, the testicular still requires distinct post-gonadal modification events in the various sperm surface biomolecules to become competent to fertilize the oocyte. The chronological interactions of the sperm occurring sequentially with the surrounding medium of the three distinct epididymal regions are believed to conclude the final steps of spermatogenesis. A number of secretagogues, e.g., the highly glycosylated and negatively charged, cysteine-rich antimicrobial peptides, β-defensins (BDs), are applied onto sperm surface which assist the spermatozoa in survival during its tortuous journey in the female reproductive tract (FRT) and are added on spermatozoa surface. Hitherto thought of as effectors of the innate immune system, recent research has revealed multiple and potentially epistatic roles of these proteins which possess the widest taxonomical distribution. Their role in reproduction especially in male fertility has gained considerable attention in the last two decades. The BDs have crucial roles to play in sperm cervical mucus penetration, capacitation, and zona-recognition. Furthermore, these multifunctional molecules “cross talk” with the adaptive immune system and can modulate the host’s immune-competent cell responses, e.g., those mounted against the spermatozoa in the FRT. Overall, these AMPs constitute an important, nonspecific component of the innate immunity in animals and humans with potential reproductive immunobiological roles in multiple mammalian species including the ruminants.

Abstract Background Numerous distinct secretagogues such as (glyco) proteins including the GPI-anchored proteins are added to the sperm surface during their transit through the male reproductive tract (MRT). This remodelling of the sperm surface is critical for sperm maturation, survival and function in the female reproductive tract (FRT). This study aimed to characterize the proteins present on the buffalo sperm surface. Results A buffalo sperm surface-specific proteomic fingerprint was generated using shotgun proteomics (LC-MS/MS). The protein informatics platform, Proteome Discoverer (v2.2) identified 1342, 678, and 982 distinct proteins and isoforms (P < 0.05, FDR < 0.01) in the salt-extracted, PI-PLC treated and capacitated samples, respectively. Overall, 1695 unique proteins (minimum 2 peptides) with ≥ 1 high-quality PSM/s and their isoforms (proteoforms) were identified. Descriptive statistical analysis indicated that these buffalo-specific proteoforms exhibit remarkable heterogeneity in their molecular weight, pI, distribution of expression from the genome and their functional roles in the MRT and the FRT. Subsequent analysis and a thorough literature search revealed that the fertility-related, reproduction-specific proteoforms constituted more than 50% (873) of the identified sperm-surface proteome (1695). Discussion These identified proteoforms are unique to buffalo since a buffalo-specific database, NCBI reference proteome (translated from the latest chromosome level genome assembly, (UOA_WB_1) was used as the search space. These proteoforms were mapped to 252 buffalo-specific proteins implicated in the regulation of various aspects of male reproductive physiology across multiple species. Besides, more than 200 orphan, buffalo-specific proteins and their isoforms (undefined locus, uncharacterized, P < 0.05, FDR < 0.01) were also identified using our computational strategy. This allowed us to consider these novel proteins for considered for mapping their ontology terms. This led to the elucidation of the biological functions of these hitherto unreported, buffalo-specific proteins by extrapolation of function from their sequence orthologs in more several ruminant and non-ruminant (e.g. Primates and Rodents) mammalian. These uncharacterized proteins constitute an extensive, yet unexplored, reproduction-specific sperm-surface proteome repertoire. Conclusions The proteomic signature driving the buffalo sperm production, maturation, survival and function discovered in this study is unparalleled vis-à-vis the depth identification of fertility-related and reproduction-specific cell-surface proteins. These results would facilitate advances in understanding the functional roles of proteins implicated in mammalian sperm function.

BACKGROUND:Low conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins.RESULTS:The raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments.CONCLUSIONS:The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

The glycans on the plasma membrane of cells manifest as the glycocalyx, which serves as an information-rich frontier that is directly in contact with its immediate milieu. The glycoconjugates (GCs) that adorn most of the mammalian cells are also abundant in gametes, especially the spermatozoa where they perform unique reproduction-specific functions e.g., inter-cellular recognition and communication. This study aimed to implicate the sperm glycosylation pattern as one of the factors responsible for low conception rates observed in buffalo bulls. We hypothesized that a differential abundance of glycans exists on the spermatozoa from bulls of contrasting fertilizing abilities endowing them with differential immune evasion abilities. Therefore, we investigated the role of glycan abundance in the phagocytosis and NETosis rates exhibited by female neutrophils (PMNs) upon exposure to such spermatozoa. Our results indicated that the spermatozoa from high fertile (HF) bulls possessed a higher abundance of O-linked glycans e.g., galactosyl (β-1,3)N-acetylgalactosamine and N-linked glycans like [GlcNAc]1-3, N-acetylglucosamine than the low fertile (LF) bull spermatozoa. This differential glycomic endowment appeared to affect the spermiophagy and NETosis rates exhibited by the female neutrophil cells (PMNs). The mean percentage of phagocytizing PMNs was significantly different (P < 0.0001) for HF and LF bulls, 28.44 and 59.59%, respectively. Furthermore, any introduced perturbations in the inherent sperm glycan arrangements promoted phagocytosis by PMNs. For example, after in vitro capacitation the mean phagocytosis rate (MPR) rate in spermatozoa from HF bulls significantly increased to 66.49% (P < 0.01). Likewise, the MPR increased to 70.63% (p < 0.01) after O-glycosidase & α2-3,6,8,9 Neuraminidase A treatment of spermatozoa from HF bulls. Moreover, the percentage of PMNs forming neutrophil extracellular traps (NETs) was significantly higher, 41.47% when exposed to spermatozoa from LF bulls vis-à-vis the spermatozoa from HF bulls, 15.46% (P < 0.0001). This is a pioneer report specifically demonstrating the role of O-linked glycans in the immune responses mounted against spermatozoa. Nevertheless, further studies are warranted to provide the measures to diagnose the sub-fertile phenotype thus preventing the losses incurred by incorrect selection of morphologically normal sperm in the AI/IVF reproduction techniques.