Acute exposure to organophosphorus pesticide metabolites compromises buffalo sperm function and impairs fertility.

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.

Buffalo sperm membrane glycan-binding proteins reveal precise and preferential binding signatures with specific glycans targets on oviduct epithelium and zona pellucida-an implication in fertilization.

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3'-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 µg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3'-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(ß1-4)GlcNAc further supports the abundance of 3'-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3'-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.

Epsilon poly-lysine in buffalo semen extender: A step towards reducing the development of antibiotic resistance.

The use of antibiotics in semen extenders can contribute to the development of antibiotic resistance. The objective of the study was to evaluate epsilon-polylysine (Ɛ-PL) as a substitute for antibiotics in the buffalo semen extender. For this, 20 semen ejaculates were collected from four Murrah buffalo bulls. Each ejaculate was divided into three equal aliquots and extended into an egg yolk-based semen extender containing either antibiotics (strepto-penicillin) or different concentrations of Ɛ-PL (0.64 and 1.28 g/L) to make the final concentration 80 million sperm/mL and cryopreserved as per the standard procedure. The antibiogram sensitivity test confirmed that Ɛ-PL is an effective antimicrobial against microbes present in buffalo semen ejaculates. Furthermore, the addition of Ɛ-PL in the semen extender significantly reduces the colony forming unit (CFU)/mL in cryopreserved semen equivalent to strepto-penicillin. The sperm motility and kinematic parameters assessed by a computer-assisted sperm analyser showed that Ɛ-PL did not inhibit either sperm motility not kinematic parameters of cryopreserved sperm. The flow-cytometric evaluation of frozen-thawed sperm revealed interesting results. The extender supplemented with Ɛ-PL protected sperm acrosome and mitochondrial membrane potential greater than the extender supplemented with strepto-penicillin. Further, Ɛ-PL reduced significantly the production of superoxide anions from mitochondria during the cryopreservation process. In this way, Ɛ-PL may be a suitable alternative to antibiotics in semen extenders. In conclusion, Ɛ-PL at a concentration of 0.64 g/L acts as an effective antimicrobial as well as antioxidant in semen extender for cryopreservation of buffalo sperm.

Establishment of a repertoire of fertility associated sperm proteins and their differential abundance in buffalo bulls (Bubalus bubalis) with contrasting fertility.

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E-08) besides metabolic (p = 4.42E-31) and reactive oxygen species (p = 1.81E-30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E-07) and flagellated sperm motility (p = 7.02E-05) processes besides oxidative phosphorylation pathway (p = 6.61E-15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility.

miR-1246 is implicated as a possible candidate for endometrium remodelling facilitating implantation in buffalo (Bubalus bubalis).

BACKGROUND: The 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. OBJECTIVES: This study was undertaken to elucidate the function of TE secreted miRNAs in the maternal-foetal cross-talk during implantation/placentation in buffalo. METHODS: The 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. RESULTS: The 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). CONCLUSIONS: The 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.

Beta-defensins as marker for male fertility: a comprehensive review†.

Bovine male fertility in animals has a direct impact on the productivity of dairy herds. The epididymal sperm maturations involve extensive sperm surface modifications to gain the fertilizing ability, especially by absorptions of the plethora of biomolecules, including glycoprotein beta-defensins (BDs), enzymes, organic ions, protein, and phospholipids. Defensins are broad-range nonspecific antimicrobial peptides that exhibit strong relations with innate and adaptive immunity, but their roles in male fertility are relatively recently identified. In the course of evolution, BD genes give rise to different clusters with specific functions, especially reproductive functions, by undergoing duplications and nonsynonymous mutations. BD polymorphisms have been reported with milk compositions, disease resistance, and antimicrobial activities. However, in recent decades, the link of BD polymorphisms with fertility has emerged as an appealing improvement of reproductive performance such as sperm motility, membrane integrity, cervical mucus penetration, evading of uterus immunosurveillance, oviduct cell attachment, and egg recognition. The reproductive-specific glycosylated BD class-A BDs (CA-BDs) have shown age- and sex-specific expressions in male reproductive organs, signifying their physiological pleiotropism, especially in the sperm maturation and sperm transport in the female reproductive tract. By considering adult male reproductive organ-specific BD expressions, importance in sperm functionalities, and bioinformatic analysis, we have selected two bovine BBD126 and BBD129 genes as novel potential biomarkers of bovine male fertility. Despite the importance of BDs, however, genomic characterization of most BD genes across most livestock and nonmodel organisms remains predictive/incomplete. The current review discusses our understanding of BD pleiotropic functions, polymorphism, and genomic structural attributes concerning the fertilizability of the male gamete in dairy animals.

Transient suppression of Wnt signaling in poor-quality buffalo oocytes improves their developmental competence.

Introduction: One 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. Methods: The 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. Results: The 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 (ß-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. Conclusion: These 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 cryopreservation process induces alterations in proteins associated with bull sperm quality: The equilibration process could be a probable critical control point.

The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography-mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3',5'-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

Analysis of amplification and association polymorphisms in the bovine beta-defensin 129 (BBD129) gene revealed its function in bull fertility.

ß-defensins are adsorbable on the sperm surface in the male reproductive tract (MRT) and enhance sperm functional characteristics. The beta-defensin 129 (DEFB129) antimicrobial peptide is involved in sperm maturation, motility, and fertilization. However, its role in bovine fertility has not been well investigated. This study examines the relationship between the bovine BBD129 gene and Bos indicus x Bos taurus bull fertility. The complete coding sequence of BBD129 mRNA was identified by RNA Ligase Mediated-Rapid Amplification of cDNA End (RLM-RACE) and Sanger sequencing methodologies. It consisted of 582 nucleotides (nts) including 5' untranslated region (UTR) (46nts) and 3'UTR (23nts). It conserves all beta-defensin-like features. The expression level of BBD129 was checked by RT-qPCR and maximal expression was detected in the corpus-epididymis region compared to other parts of MRT. Polymorphism in BBD129 was also confirmed by Sanger sequencing of 254 clones from 5 high fertile (HF) and 6 low fertile (LF) bulls at two positions, 169 T > G and 329A > G, which change the S57A and N110S in the protein sequence respectively. These two mutations give rise to four types of BBD129 haplotypes. The non-mutated TA-BBD129 (169 T/329A) haplotype was substantially more prevalent among high-fertile bulls (P < 0.005), while the double-site mutated GG-BBD129 (169 T > G/329A > G) haplotype was significantly more prevalent among low-fertile bulls (P < 0.005). The in silico analysis confirmed that the polymorphism in BBD129 results in changes in mRNA secondary structure, protein conformations, protein stability, extracellular-surface availability, post-translational modifications (O-glycosylation and phosphorylation), and affects antibacterial and immunomodulatory capabilities. In conclusion, the mRNA expression of BBD129 in the MRT indicates its region-specific dynamics in sperm maturation. BBD129 polymorphisms were identified as the deciding elements accountable for the changed proteins with impaired functionality, contributing to cross-bred bulls' poor fertility.

Cryopreservation process alters the expression of genes involved in pathways associated with the fertility of bull spermatozoa.

In bovines, cryopreserved semen is used for artificial insemination; however, the fertility of cryopreserved semen is far lower than that of fresh semen. Although cryopreservation alters sperm phenotypic characteristics, its effect on sperm molecular health is not thoroughly understood. The present study applied next-generation sequencing to investigate the effect of cryopreservation on the sperm transcriptomic composition of bull spermatozoa. While freshly ejaculated bull spermatozoa showed 14,280 transcripts, cryopreserved spermatozoa showed only 12,375 transcripts. Comparative analysis revealed that 241 genes were upregulated, 662 genes were downregulated, and 215 genes showed neutral expression in cryopreserved spermatozoa compared to fresh spermatozoa. Gene ontology analysis indicated that the dysregulated transcripts were involved in nucleic acid binding, transcription-specific activity, and protein kinase binding involving protein autophosphorylation, ventricular septum morphogenesis, and organ development. Moreover, the dysregulated genes in cryopreserved spermatozoa were involved in pathways associated with glycogen metabolism, MAPK signalling, embryonic organ morphogenesis, ectodermal placode formation, and regulation of protein auto-phosphorylation. These findings suggest that the cryopreservation process induced alterations in the abundance of sperm transcripts related to potential fertility-associated functions and pathways, which might partly explain the reduced fertility observed with cryopreserved bull spermatozoa.

Codelivery of Gemcitabine and MUC1 Inhibitor Using PEG-PCL Nanoparticles for Breast Cancer Therapy.

In breast cancer therapy, Gemcitabine (Gem) is an antineoplastic antimetabolite with greater anticancer efficacy and tolerability. However, effectiveness of Gem is limited by its off-target effects. The synergistic potential of MUC1 (mucin 1) inhibitors and Gem-loaded polymeric nanoparticles (NPs) was discussed in this work in order to reduce dose-related toxicities and enhance the therapeutic efficacy. The double emulsion solvent evaporation method was used to prepare poly(ethylene glycol) methyl ether-block-poly-caprolactone (PEG-PCL)-loaded Gem and MUC 1 inhibitor NPs. The average size of Gem and MUC 1 inhibitor-loaded NPs was 128 nm, with a spherical shape. Twin-loaded NPs containing Gem and the MUC1 inhibitor decreased IC50 and behaved synergistically. Furthermore, in vitro mechanistic studies, that is, loss of MMP, clonogenic assay, Annexin V FITC assay, and Western blotting to confirm apoptosis with simultaneous induction of autophagy using acridine orange (AO) staining were performed in this study. Furthermore, the investigated NPs upon combination exhibited greater loss of MMP and decreased clonogenic potential with simultaneous induction of autophagy in MCF-7 cells. Annexin V FITC clearly showed the percentage of apoptosis while Western blotting protein expression analysis revealed an increase in caspase-3 activity with simultaneous decrease in Bcl-2 protein expression, a hallmark of apoptosis. The effectiveness of the Ehrlich ascites solid (EAT) mice treated with Gem-MUC1 inhibitor NPs was higher than that of the animals treated alone. Overall, the combined administration of Gem and MUC1 inhibitor-loaded NPs was found to be more efficacious than Gem and MUC1 inhibitor delivered separately.

Whole-Genome-Based Web Genomic Resource for Water Buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis), belonging to the Bovidae family, is an economically important animal as it is the major source of milk, meat, and drought in numerous countries. It is mainly distributed in tropical and subtropical regions with a global population of approximately 202 million. The advent of low cost and rapid sequencing technologies has opened a new vista for global buffalo researchers. In this study, we utilized the genomic data of five commercially important buffalo breeds, distributed globally, namely, Mediterranean, Egyptian, Bangladesh, Jaffrarabadi, and Murrah. Since there is no whole-genome sequence analysis of these five distinct buffalo breeds, which represent a highly diverse ecosystem, we made an attempt for the same. We report the first comprehensive, holistic, and user-friendly web genomic resource of buffalo (BuffGR) accessible at http://backlin.cabgrid.res.in/buffgr/, that catalogues 6028881 SNPs and 613403 InDels extracted from a set of 31 buffalo tissues. We found a total of 7727122 SNPs and 634124 InDels distributed in four breeds of buffalo (Murrah, Bangladesh, Jaffarabadi, and Egyptian) with reference to the Mediterranean breed. It also houses 4504691 SSR markers from all the breeds along with 1458 unique circRNAs, 37712 lncRNAs, and 938 miRNAs. This comprehensive web resource can be widely used by buffalo researchers across the globe for use of markers in marker trait association, genetic diversity among the different breeds of buffalo, use of ncRNAs as regulatory molecules, post-transcriptional regulations, and role in various diseases/stresses. These SNPs and InDelscan also be used as biomarkers to address adulteration and traceability. This resource can also be useful in buffalo improvement programs and disease/breed management.

Comparative high-throughput analysis of sperm membrane proteins from crossbred bulls with contrasting fertility.

The aim of the present study was to identify fertility associated sperm membrane proteins in crossbred bulls. Sperm membrane proteins from high- and low-fertile Holstein Friesian crossbred bulls (n = 3 each) were subjected to high-throughput liquid chromatography-mass spectrometry (LC-MS/MS) for comparative proteomic analysis. Proteomic profiling identified a total of 456 proteins in crossbred bull spermatozoa; it was found that 108 proteins were up regulated while 26 proteins were down regulated (>1.5-folds) in spermatozoa from low- compared to high-fertile bulls. Gene ontology classification revealed that upregulated proteins in low-fertile bulls were involved in biological process such as oxidation-reduction process (p = 3.14E-06), fusion of sperm to egg plasma membrane (p = 7.51E-04), sperm motility (p = 0.03), and capacitation (p = 0.09), while down regulated proteins were associated with transport (p = 6.94E-04), superoxide metabolic process (p = 0.02), and tricarboxylic acid cycle (p = 0.04). KEGG pathway analysis revealed that oxidative phosphorylation and tricarboxylic acid cycle pathways are the most significantly affected pathway in low-fertile bulls. It was concluded that expression of proteins associated with oxidative phosphorylation and tricarboxylic acid cycle pathways were altered in low-fertile crossbred bulls, and expression levels of SPATA19, ELSPBP1, ACRBP, CLU, SUCLA2, and SPATC1 could aid in assessing potential fertility of crossbred bulls.

Sperm Transcripts Associated With Odorant Binding and Olfactory Transduction Pathways Are Altered in Breeding Bulls Producing Poor-Quality Semen.

Spermatozoa carries a reservoir of mRNAs regulating sperm functions and fertilizing potential. Although it is well recognized that a considerable proportion of high genetic merit breeding bulls produce poor-quality semen, the transcriptomic alterations in spermatozoa from such bulls are not understood. In the present study, comparative high-throughput transcriptomic profiling of spermatozoa from good and poor-quality semen-producing bulls was carried out to identify the transcripts associated with semen quality. Using next-generation sequencing (NGS), we identified 11,632 transcripts in Holstein Friesian bull spermatozoa; after total hit normalization, a total of 544 transcripts were detected, of which 185 transcripts were common to both good and poor-quality semen, while 181 sperm transcripts were unique to good quality semen, and 178 transcripts were unique to poor-quality semen. Among the co-expressed transcripts, 31 were upregulated, while 108 were downregulated, and 46 were neutrally expressed in poor-quality semen. Bioinformatics analysis revealed that the dysregulated transcripts were predominantly involved in molecular function, such as olfactory receptor activity and odor binding, and in biological process, such as detection of chemical stimulus involved in sensory perception, sensory perception of smell, signal transduction, and signal synaptic transmission. Since a majority of the dysregulated transcripts were involved in the olfactory pathway (85% of enriched dysregulated genes were involved in this pathway), the expression of selected five transcripts associated with this pathway (OR2T11, OR10S1, ORIL3, OR5M11, and PRRX1) were validated using real-time qPCR, and it was found that their transcriptional abundance followed the same trend as observed in NGS; the sperm transcriptional abundance of OR2T11 and OR10S1 differed significantly (p < 0.05) between good and poor-quality semen. It is concluded that poor-quality semen showed altered expression of transcripts associated with olfactory receptors and pathways indicating the relationship between olfactory pathway and semen quality in bulls.

Recombinant expression and molecular characterization of buffalo sperm lysozyme-like protein 1.

Several sperm lysozyme-like genes evolved from lysozyme by successive duplications and mutations; however their functional role in the reproduction of farm animals is not well understood. To understand the function and molecular properties of buffalo sperm lysozyme-like protein 1 (buSLLP1), it was expressed in E. coli; however, it partitioned to inclusion bodies. Lowering of temperature and inducer concentration did not help in the recovery of the expressed protein in the biologically active form. Therefore, buSLLP1 was cloned and expressed in Pichiapink system based on auxotrophic Pichia pastoris in a labscale fermenter. The expressed protein was obtained in flow-through by using a 30 kDa ultrafiltration membrane followed by MonoQ anion exchange chromatography, resulting in a homogenous preparation of 40 mg recombinant buSLLP1 per liter of initial spent culture-supernatant. Circular dichroism spectroscopy showed that recombinant buSLLP1 possessed a native-like secondary structure. The recombinant buSLLP1 also showed thermal denaturation profile typical of folded globular proteins; however, the thermal stability was lower than the hen egg white lysozyme. Binding of buSLLP1 to chitin and zona pellucida of buffalo oocytes showed that the recombinant buSLLP1 possessed a competent binding pocket, therefore, the produced protein could be used to study its functional role in the reproduction of farm animals.

Genome Wide Prediction, Mapping and Development of Genomic Resources of Mastitis Associated Genes in Water Buffalo.

Water buffalo (Bubalus bubalis) are an important animal resource that contributes milk, meat, leather, dairy products, and power for plowing and transport. However, mastitis, a bacterial disease affecting milk production and reproduction efficiency, is most prevalent in populations having intensive selection for higher milk yield, especially where the inbreeding level is also high. Climate change and poor hygiene management practices further complicate the issue. The management of this disease faces major challenges, like antibiotic resistance, maximum residue level, horizontal gene transfer, and limited success in resistance breeding. Bovine mastitis genome wide association studies have had limited success due to breed differences, sample sizes, and minor allele frequency, lowering the power to detect the diseases associated with SNPs. In this work, we focused on the application of targeted gene panels (TGPs) in screening for candidate gene association analysis, and how this approach overcomes the limitation of genome wide association studies. This work will facilitate the targeted sequencing of buffalo genomic regions with high depth coverage required to mine the extremely rare variants potentially associated with buffalo mastitis. Although the whole genome assembly of water buffalo is available, neither mastitis genes are predicted nor TGP in the form of web-genomic resources are available for future variant mining and association studies. Out of the 129 mastitis associated genes of cattle, 101 were completely mapped on the buffalo genome to make TGP. This further helped in identifying rare variants in water buffalo. Eighty-five genes were validated in the buffalo gene expression atlas, with the RNA-Seq data of 50 tissues. The functions of 97 genes were predicted, revealing 225 pathways. The mastitis proteins were used for protein-protein interaction network analysis to obtain additional cross-talking proteins. A total of 1,306 SNPs and 152 indels were identified from 101 genes. Water Buffalo-MSTdb was developed with 3-tier architecture to retrieve mastitis associated genes having genomic coordinates with chromosomal details for TGP sequencing for mining of minor alleles for further association studies. Lastly, a web-genomic resource was made available to mine variants of targeted gene panels in buffalo for mastitis resistance breeding in an endeavor to ensure improved productivity and the reproductive efficiency of water buffalo.

Cellular and Molecular Insights Into the Etiology of Subfertility/Infertility in Crossbred Bulls (Bos taurus × Bos indicus): A Review.

Crossbreeding of indigenous cattle (Bos indicus) with improved (Bos taurus) breeds gained momentum and economic relevance in several countries to increase milk production. While production performance of the crossbred offspring is high due to hybrid vigor, they suffer from a high incidence of reproductive problems. Specifically, the crossbred males suffer from serious forms of subfertility/infertility, which can have a significant effect because semen from a single male is used to breed several thousand females. During the last two decades, attempts have been made to understand the probable reasons for infertility in crossbred bulls. Published evidence indicates that testicular cytology indices, hormonal concentrations, sperm phenotypic characteristics and seminal plasma composition were altered in crossbred compared to purebred males. A few recent studies compared crossbred bull semen with purebred bull semen using genomics, transcriptomics, proteomics and metabolomics; molecules potentially associated with subfertility/infertility in crossbred bulls were identified. Nevertheless, the precise reason behind the poor quality of semen and high incidence of sub-fertility/infertility in crossbred bulls are not yet well defined. To identify the underlying etiology for infertility in crossbred bulls, a thorough understanding of the magnitude of the problem and an overview of the prior art is needed; however, such systematically reviewed information is not available. Therefore, the primary focus of this review is to compile and analyze earlier findings on crossbred bull fertility/infertility. In addition, the differences between purebred and crossbred males in terms of testicular composition, sperm phenotypic characteristics, molecular composition, environmental influence and other details are described; future prospects for research on crossbred males are also outlined.

Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction.

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.

A novel lincRNA identified in buffalo oocytes with protein binding characteristics could hold the key for oocyte competence.

Studying the maternal oocyte-specific genes, in farm animals is a significant step towards delineating the underlying mechanisms that regulate oocyte quality, early embryonic development and survival. With the creation of buffalo oocyte-specific subtracted cDNA library, it has raised new questions which need to be answered. The present study has characterized one of the ESTs selected from the library and highlighted its importance in the oocyte quality. The selected EST was made full length by RLM-RACE and four transcript variants were identified. Bioinformatics analysis indicated the novelty of full-length transcript along with conserved intergenic nature. The largest transcript was identified as long intergenic noncoding RNA based upon coding potential calculator output. The expression analysis at different hours of oocyte maturation showed a significant variation in developmentally competent oocytes to that of incompetent ones. Along with this, the transcript was also found to have protein binding ability which was confirmed by RNA electrophoretic mobility shift assay. The protein used in the experiment was isolated from oocyte and cumulus cells via sonication. A novel lincRNA has been reported here that might have an important role in maturation of oocytes, inferred from its relative gene expression study and protein binding characteristics.

Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction.

Crossbred bulls produced by crossing Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation (biological process) and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.