BackgroundThe buffalo, despite its superior milk-producing ability, suffers from reproductive limitations that constrain its lifetime productivity. Male sub-fertility, manifested as low conception rates (CRs), is a major concern in buffaloes. The epididymal sperm surface-binding proteins which participate in the sperm surface remodelling (SSR) events affect the survival and performance of the spermatozoa in the female reproductive tract (FRT). A mutation in an epididymal secreted protein, beta-defensin 126 (DEFB-126/BD-126), a class-A beta-defensin (CA-BD), resulted in decreased CRs in human cohorts across the globe. To better understand the role of CA-BDs in buffalo reproduction, this study aimed to identify the BD genes for characterization of the selection pressure(s) acting on them, and to identify the most abundant CA-BD transcript in the buffalo male reproductive tract (MRT) for predicting its reproductive functional significance.ResultsDespite the low protein sequence homology with their orthologs, the CA-BDs have maintained the molecular framework and the structural core vital to their biological functions. Their coding-sequences in ruminants revealed evidence of pervasive purifying and episodic diversifying selection pressures. The buffalo CA-BD genes were expressed in the major reproductive and non-reproductive tissues exhibiting spatial variations. The Buffalo BD-129 (BuBD-129) was the most abundant and the longest CA-BD in the distal-MRT segments and was predicted to be heavily O-glycosylated.ConclusionsThe maintenance of the structural core, despite the sequence divergence, indicated the conservation of the molecular functions of the CA-BDs. The expression of the buffalo CA-BDs in both the distal-MRT segments and non-reproductive tissues indicate the retention the primordial microbicidal activity, which was also predicted by in silico sequence analyses. However, the observed spatial variations in their expression across the MRT hint at their region-specific roles. Their comparison across mammalian species revealed a pattern in which the various CA-BDs appeared to follow dissimilar evolutionary paths. This pattern appears to maintain only the highly efficacious CA-BD alleles and diversify their functional repertoire in the ruminants. Our preliminary results and analyses indicated that BuBD-129 could be the functional ortholog of the primate DEFB-126. Further studies are warranted to assess its molecular functions to elucidate its role in immunity, reproduction and fertility.

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.

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.

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 &lt; 0.05, FDR &lt; 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 &lt; 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.

Germinal vesicle breakdown (GVBD) is the first milestone that an oocyte needs to achieve toward completing the maturation and gaining potential to fertilize. Significantly lower in vitro embryo production rate in buffaloes can be attributed to heterogeneity of GVBD occurrence among oocytes obtained from abattoir derived ovaries. Evidence from our earlier work had suggested that different qualities of buffalo oocytes differ significantly in their timing of GVBD. Besides, these oocytes also differ in terms of volume of Akt phosphorylation, which initiates the process of GVBD. With objective of synchronizing the oocytes for GVBD, immature buffalo oocytes were subjected to a two-step culture protocol, initially in the presence of GVBD inhibitors and subsequently, in vitro maturation (IVM) with added SC79 (activates Akt). Expression of developmentally important genes was assessed along with embryo development rate and blastocyst health to interpret the consequences. Oocytes subjected to a short GVBD inhibition period of 6 h followed by IVM with SC79 resulted in improved cleavage and blastocyst rates. Resultant blastocysts also possessed higher ICM: TE ratio. Further, GVBD inhibited oocytes displayed a sustained cytoplasmic maturation status in terms of reorganization of cortical granules (CGs), mitochondrial membrane potential, and glutathione levels during the period of inhibition. We conclude that a temporary GVBD arrest of buffalo oocytes and modulation of Akt improves the in vitro embryo development rate as well as quality of resultant embryos. Besides, our meiotic arrest protocol does not affect the cytoplasmic maturation.

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.

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants.