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Problem: Lipopolysaccharide (LPS) from gram‐negative bacteria has reportedly been associated with infectious diseases like metritis, which has a substantial adverse effect on animal reproductive performance and causes serious financial losses for the dairy sector. The current work aimed to establish the impact of LPS on in vitro oocyte maturation and subsequent in vitro developmental competence of oocytes, as well as to investigate the explanatory molecular mechanism underlying this effect. Method of Study: Buffalo cumulus‐oocyte complexes (COCs) were challenged with 0, 5, 10 and 20 µg/mL LPS during IVM followed by IVF and IVC. Cytoplasmic and nuclear maturation, cleavage and blastocyst rate, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP, ΔΨm) and transcript abundance of genes related to inflammation, antioxidation and apoptosis were evaluated. Results: The maturation and subsequent embryonic development competency were found to be significantly (p ≤ 0.05) reduced with the addition of 10 and 20 µg/mL LPS to IVM media. ROS production accompanied by a decreased ΔΨm was recorded in LPS‐treated oocytes in comparison to the control group (p ≤ 0.05). Our results were further supported by the transcriptional expression of proinflammatory (TLR4, CD14 and RPS27A) and apoptotic gene (Caspase 3) which were found to be significantly increased while antioxidant genes (SOD2 and GPX1) were decreased significantly in matured oocytes and blastocyst after LPS exposure. Conclusions: The deleterious effects of LPS are mediated through ROS generation, which triggers inflammatory processes via the TLR4 pathway and impairs oocyte maturation and subsequent embryonic development. [ABSTRACT FROM AUTHOR]

Electroporation is a widely used method for delivering CRISPR components into cells; however, it presents challenges when applied to difficult-to-transfect cells like adult buffalo fibroblasts. In this study, the ITGB2 gene (encoding the CD18 protein), plays vital for cellular adhesion and immune responses, was selected for editing experiments. To optimize electroporation conditions, we investigated parameters such as electric field strength, pulse duration, plasmid DNA amount, cuvette type, and cell type. The best transfection rates were obtained in a 4 mm gap cuvette with a single 20-millisecond pulse of 300 V using a 10 μg of all-in-one CRISPR plasmid for 106 cells in 100 μL of electroporation buffer. Increasing DNA quantity enhanced transfection rates but compromised cell viability. The 4 mm cuvette gap had high transfection rates than the 2 mm gap, and newborn cells exhibited higher transfection rates than adult cells. We achieved transfection rates of 10–12% with a cell viability of 25–30% for adult fibroblast cells. Subsequently, successfully edited the ITGB2 gene with a 30% editing efficiency, confirmed through various analysis methods, including T7E1 assay, TIDE and ICE analysis, and TA cloning. In conclusion, electroporation conditions reported here can edit buffalo gene(s) for various biotechnological research applications. [ABSTRACT FROM AUTHOR]

Semen ejaculates were collected from Murrah buffalo (Bubalus bubalis) bulls and were diluted with low density lipoprotein (LDL) based extender into 20 million sperm/0.25 mL and 2 million sperm/0.25 mL. Dinitrophenol (DNP) was added (@ 0, 1, 10 and 50 µM) to 20 million and 2 million sperm concentration which was followed by cryopreservation. After thawing, the parameters studied were plasma membrane integrity by HOST, sperm motility and kinetics by CASA, the thermal resistance of sperm by incubation test, mitochondrial superoxide status by MitoSOX through flow cytometry, mitochondrial membrane potential (MMP) evaluation by JC-1 through flow cytometry. There was no significant (P>0.05) change in plasma membrane integrity, sperm motility and kinematics; thermal resistance of sperm, mitochondrial superoxide status and hMMP in comparison to control within the 2 and 20 million sperm doses. Two million sperm doses resulted in low plasma membrane integrity, low thermal resistance of sperm, high mitochondrial superoxide status and decreased high MMP and no significant (P>0.05) change in sperm motility and kinematics, but reduced total motility, beat cross frequency and no change in progressive motility, straight linear velocity, average path velocity, curvilinear velocity, amplitude of lateral head displacement, straightness, linearity, and wobble in comparison to 20 million sperm doses. Supplementation of DNP (0, 1, 10 and 50 µM) in extender failed to improve semen quality in both 2 and 20 million sperm doses in buffalo. [ABSTRACT FROM AUTHOR]

India owns the best buffalo breeds, particularly Murrah which is famous all over the world for high milk production. India's white and pink revolution cannot be imagined without the contribution of buffalo and to achieve these, the best productive animals need to be produced through scientific interventions. Animal cloning is a technique used to produce multiple copies of the best animals without normal reproduction. In India, buffalo cloning has already happened and India's first cloned buffalo was produced in 2009. Later, several buffalo clones were produced and attempts are ongoing to produce stock of more elite animals. Buffalo cloning has made its way from scientific curiosity to farmer's farm. In this viewpoint article, we provide an overview of the progress of buffalo cloning and we discuss some of the public perceptions of animal cloning such as aging and food safety of cloned animal products. [ABSTRACT FROM AUTHOR]

Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)