Higher abundance of DLD protein in buffalo bull spermatozoa causes elevated ROS production leading to early sperm capacitation and reduction in fertilizing ability.

BACKGROUD: Before fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm's ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction. RESULTS: In this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa. CONCLUSIONS: The present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize.

Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls.

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7 nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity.

Circulatory extracellular vesicle derived miR-195-5p promotes cellular apoptosis and suppresses cell proliferation in the buffalo endometrial primary cell culture.

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.

Identification of protein candidates in spermatozoa of water buffalo (Bubalus bubalis) bulls helps in predicting their fertility status.

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.

PU.1 is involved in the transcriptional up-regulation of RNA and DNA sensing pathway genes in buffalo fibroblasts.

PU.1, CEBPA, and CEBPB are Lineage Determining Transcription Factors (LDTFs) that play roles in biological processes such as cell differentiation and the immune system regulation including the innate immune pathways. The roles of these LDTFs in the innate RNA and DNA sensing pathways have received little attention. We show that in buffalo fibroblasts, PU.1 causes the mRNA up-regulation of the RNA and DNA sensors such as RIG-I (65.1 fold), MDA5 (20.4 fold), IFI16-l (8.0 fold), and cGAS (60.5 fold) while CEBPA does the same but to a lesser extent (RIG-I-26.4 fold, MDA5-10.8 fold, IFI16-l- 3.3 fold and cGAS-8.6 fold). CEBPB does not appear to have a role in the up-regulation of these genes. PU.1 expression also primes the cells to develop a strong immune response against the dsRNA virus mimic polyinosinic:polycytidylic acid (poly I:C) by significantly up-regulating Interferon-ß (14.9 fold change with p-value <0.0001). CEBPA up-regulates Interferon-ß to a lower level than PU.1 (4.7 fold change with p-value 0.0024), whereas CEBPB exhibits non-significant up-regulation (2.1 fold with p-value of 0.1449). As PU.1 robustly up-regulates the nucleic acid sensing pathways, it can prove to be useful in improving the defence against viruses that can cause losses to animal husbandry.

Antibiotic resistance: A cross-sectional study on knowledge, attitude, and practices among veterinarians of Haryana state in India.

AIM: The current study aimed to assess the knowledge, attitude, and practices pertaining to antibiotic usage among the field veterinarians who serve as nodal officers playing a crucial role in disseminating knowledge to the farmers regarding livestock management practices in India. MATERIALS AND METHODS: A pilot study was conducted in which 106 of the 173 field veterinarians of Haryana, India, agreed to contribute through their valuable participation in the study. The collected data were critically analyzed by simple descriptive statistics, and the responses were ranked using Garrett's ranking method. RESULTS: Our study found that most of the clinicians were aware of the fundamental clinical aspects of antibiotic resistance (AR), i.e., the general causes and transmission of resistance, response during treatment failure, and safe disposal of hospital waste. Further, implementation of "antibiotic stewardship" (rational/responsible use of antibiotics) and interruption of AR transmission by means of cross-kingdom pathogens are two ways to restrict the spread of resistant pathogens which were not in the clinical purview of majority of the clinicians. This highlights a lack of awareness and scope of improving clinician's knowledge pertaining to AR. Moreover, we got to know the methodology adopted by farmers for disposal of infected milk from diseased udders as well as their attitude toward diseased and unproductive animals. CONCLUSION: This study provides snippets of the current animal husbandry practices prevalent at the field level which would assist to plug in the gaps of knowledge regarding AR among the veterinarians as well as the general public and serve to reduce its deleterious impacts in Indian animal farming as well as in the world through the concept of "One World, One Health."

Nature of selection varies on different domains of IFI16-like PYHIN genes in ruminants.

BACKGROUND: ALRs (AIM2-like Receptors) are germline encoded PRRs that belong to PYHIN gene family of cytokines, which are having signature N-terminal PYD (Pyrin, PAAD or DAPIN) domain and C-terminal HIN-200 (hematopoietic, interferon-inducible nuclear protein with 200 amino acid repeat) domain joined by a linker region. The positively charged HIN-200 domain senses and binds with negatively charged phosphate groups of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) purely through electrostatic attractions. On the other hand, PYD domain interacts homotypically with a PYD domain of other mediators to pass the signals to effector molecules downwards the pathways for inflammatory responses. There is remarkable inter-specific diversity in the numbers of functional PYHIN genes e.g. one in cow, five in human, thirteen in mice etc., while there is a unique loss of PYHIN genes in the bat genomes which was revealed by Ahn et al. (2016) by studying genomes of ten different bat species belonging to sub-orders yinpterochiroptera and yangochiroptera. The conflicts between host and pathogen interfaces are compared with "Red queen's arms race" which is also described as binding seeking dynamics and binding avoidance dynamics. As a result of this never-ending rivalry, eukaryotes developed PRRs as antiviral mechanism while viruses developed counter mechanisms to evade host immune defense. The PYHIN receptors are directly engaged with pathogenic molecules, so these should have evolved under the influence of selection pressures. In the current study, we investigated the nature of selection pressure on different domain types of IFI16-like (IFI16-L) PYHIN genes in ruminants. RESULTS: Three transcript variants of the IFI16-like gene were found in PBMCs of ruminant animals-water buffalo, zebu cattle, goat, and sheep. The IFI16-like gene has one N-terminal PYD domain and one C-terminal HIN-200 domain, separated by an inter-domain linker region. HIN domain and inter-domain region are positively selected while the PYD domain is under the influence of purifying selection. CONCLUSION: Herein, we conclude that the nature of selection pressure varies on different parts (PYD domain, HIN domain, and inter-domain linker region) of IFI16-like PYHIN genes in the ruminants. This data can be useful to predict the molecular determinants of pathogen interactions.