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"Pal, Ankit"
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Acute exposure to organophosphorus pesticide metabolites compromises buffalo sperm function and impairs fertility
by
Datta, Tirtha Kumar
,
Kumaresan, Arumugam
,
Chhillar, Shivani
in
631/80/2373
,
631/92/609
,
Agrochemicals
2023
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.
Journal Article
Circulatory extracellular vesicle derived miR-195-5p promotes cellular apoptosis and suppresses cell proliferation in the buffalo endometrial primary cell culture
2023
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.
Journal Article
Response surface optimization of CMCase from Staphylococcus saprophyticus and Klebsiella quasipneumoniae subsp. Similipneumoniae originated from termitarium of Odontotermes obesus
by
Kumar, Krishan
,
Shaikh, Mohammedfaizan
,
Bhut, Vishruti
in
Alternative energy
,
Alternative energy sources
,
Bacteria
2025
The continuous depletion of fossil fuel reserves and the rising demand for energy have led researchers to look into alternative renewable energy sources. Bioethanol derived from the lignocellulosic biomass (LCB) could be a cost-effective and viable alternative source. As cellulose is a major component of LCB, therefore, in this study, two potential CMCase producing bacterial strains were screened out from the termitarium of Odontotermes obesus. Based on morphological, biochemical, and molecular sequencing, isolates T12 and T13 were identified as Staphylococcus saprophyticus T12 and Klebsiella quasipneumoniae subsp. Similipneumoniae, respectively. Upon optimization through method like one factor at a time (OFAT) and response surface methodology (RSM), bacterial isolate T12 produced 17.47 U/mL of crude CMCase under optimal conditions of pH 7.0, 35 °C, and 48 h of incubation, representing a 1.25-fold increase from 13.87 U/mL in unoptimized conditions. Similarly, isolate T13 produced 18.18 U/mL of crude CMCase at pH 6.0, 41 °C, and 62 h of incubation, achieving a 1.50-fold increase from its unoptimized activity of 12.07 U/mL. Crude enzyme of both bacterial isolates exhibited high CMCase activity at mesophilic ranges, which makes these bacterial strains suitable for industrial processes such as bioethanol production, biopolishing and biostoning processes in textile industries.Article highlightsIsolation of CMCase-producing bacteria from termitarium of O. obesus.Characterization of CMCase-producing bacterial strains.RSM-CCD based CMCase production optimization.
Journal Article
Extracellular vesicles in seminal plasma of Sahiwal cattle bulls carry a differential abundance of sperm fertility-associated proteins for augmenting the functional quality of low-fertile bull spermatozoa
2025
Poor male fertility significantly affects dairy production, primarily due to low conception rates (CR) in bulls, even when cows are inseminated with morphologically normal sperm. Seminal plasma is a key factor in evaluating the fertilizing ability of bull semen. The extracellular vesicles (EVs) in seminal plasma contain fertility-associated proteins like SPAM1, ADAM7, and SP10, which influence sperm function and fertilizing potential. This study aimed to assess EV-associated fertility proteins in bulls with varying fertility levels and to investigate the effects of seminal plasma EVs (SPEVs) from high-fertility (HF) bulls on the spermatozoa of low-fertility (LF)
Sahiwal
bulls. Seminal plasma was isolated from the fresh semen of
Sahiwal
bulls, with four bulls classified as high fertility and three as low fertility. Fertility-associated proteins such as SP10, ADAM7, and SPAM 1 were highly expressed in SPEVs of high-fertility (HF) bulls. PKH26 dye-labeled SPEVs demonstrated significant uptake by spermatozoa at pH 6.8 with ≥ 4 h of co-incubation. Exposure of HF SPEVs to low-fertility (LF) spermatozoa reduced acrosome response, capacitation, and reactive oxygen species (ROS) formation. Our findings suggest that protein repertoires in SPEVs influence sperm activities such as motility, acrosome response, and ROS production. Supplementing LF spermatozoa with HF SPEVs could enhance their functional characteristics, highlighting these proteins as potential resources to modulate cattle bull sperm fertilizing ability.
Journal Article
Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction
by
Kumar, Rakesh
,
Solanki, Subhash
,
Bhushan, Vanya
in
African buffalo
,
Animal Genetics and Genomics
,
Antiinfectives and antibacterials
2021
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.
Journal Article
Identification of protein candidates in spermatozoa of water buffalo (Bubalus bubalis) bulls helps in predicting their fertility status
by
Datta, Tirtha K.
,
Kumar, Rakesh
,
Karanwal, Seema
in
Artificial insemination
,
Bubalus bubalis
,
Cell and Developmental Biology
2023
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.
Journal Article
Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls
2024
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 × 10 11 and 3.57 to 7.74 × 10 11 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.
Journal Article
Differential abundance of microRNAs in seminal plasma extracellular vesicles (EVs) in Sahiwal cattle bull related to male fertility
by
Chauhan, Vitika
,
Kumar, Rakesh
,
Solanki, Subhash
in
Artificial insemination
,
bull fertility
,
Cattle
2024
Sahiwal cattle, known for their high milk yield, are propagated through artificial insemination (AI) using male germplasm, largely contingent on semen quality. Spermatozoa, produced in the testes, carry genetic information and molecular signals essential for successful fertilization. Seminal plasma, in addition to sperm, contains nano-sized lipid-bound extracellular vesicles (SP-EVs) that carry key biomolecules, including fertility-related miRNAs, which are essential for bull fertility. The current study focused on miRNA profiling of SP-EVs from high-fertile (HF) and low-fertile (LF) Sahiwal bulls. SP-EVs were isolated using size exclusion chromatography (SEC) and characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Western blotting detected the EV-specific protein markers TSG101 and CD63. The DLS analysis showed SP-EV sizes of 170–180 nm in HF and 130–140 nm in LF samples. The NTA revealed particle concentrations of 5.76 × 10 10 to 5.86 × 10 11 particles/mL in HF and 5.31 × 10 10 to 2.70 × 10 11 particles/mL in LF groups, with no significant differences in size and concentration between HF and LF. High-throughput miRNA sequencing identified 310 miRNAs in SP-EVs from both groups, with 61 upregulated and 119 downregulated in HF bull. Further analysis identified 41 miRNAs with significant fold changes and p-values, including bta-miR-1246, bta-miR-195, bta-miR-339b, and bta-miR-199b, which were analyzed for target gene prediction. Gene Ontology (GO) and KEGG pathway analyses indicated that these miRNAs target genes involved in transcription regulation, ubiquitin-dependent endoplasmic reticulum-associated degradation (ERAD) pathways, and signalling pathways. Functional exploration revealed that these genes play roles in spermatogenesis, motility, acrosome reactions, and inflammatory responses. qPCR analysis showed that bta-miR-195 had 80% higher expression in HF spermatozoa compared to LF, suggesting its association with fertility status ( p < 0.05). In conclusion, this study elucidates the miRNA cargoes in SP-EVs as indicators of Sahiwal bull fertility, highlighting bta-miR-195 as a potential fertility factor among the various miRNAs identified.
Journal Article
Higher abundance of DLD protein in buffalo bull spermatozoa causes elevated ROS production leading to early sperm capacitation and reduction in fertilizing ability
2024
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.
Journal Article
Euler-type integrals for the generalized hypergeometric matrix function
2023
The special matrix functions have received significant attention in many fields, such as theoretical physics, number theory, probability theory, and the theory of group representations. In 2017, Dwivedi and Sahai introduced the generalized hypergeometric matrix function using matrix parameters and proved the convergence on
. Recently, hypergeometric matrix functions and their potential applications have played a major role in mathematical physics and engineering. Motivated by aforesaid works and in order to enrich this flourishing field, we investigate the Euler-type integral representations for the generalized hypergeometric matrix function and determine various transformations in terms of hypergeometric matrix functions. Furthermore, unit and half arguments have been provided for several particular cases.
Journal Article