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The present study comprehensively evaluates the postnatal growth, hematology, telomere length, and semen attributes of multiple clones and re-clone derived from superior buffalo breeding bulls. To the best of our knowledge, we successfully produced multiple clones and a re-clone of an earlier cloned buffalo bull from an embryo developed from an adult bull's skin-derived cell for the first time. The cloned bulls' growth, blood hematology, plasma biochemistry, and telomere length were all shown to be normal at various stages of development. The bulls were used for semen production after being screened for testicular growth and training. Semen characteristics such as volume, concentration, and initial motility of fresh sperm as well as motility and kinetics characteristics such as straightness (STR), average lateral head displacement (ALH), and beat cross frequency (BCF) of frozen-thawed sperms of the cloned bulls were found to be similar to those of non-cloned bulls, including the donor bulls. Additionally, it was found that cloned bulls' functional sperm attributes, including acrosome intactness, mitochondrial membrane potential, and superoxide anion status, were comparable to those of non-cloned bulls. These characteristics are necessary for sperm to pass through the female reproductive system, penetrate the oocyte, and efficiently fertilize. Finally, this study adds to our understanding of the postnatal development, hematology, telomere length, and sperm characteristics of superior buffalo breeding bulls that have been cloned and re-cloned. The findings provide the groundwork for improving cloning practices, refining reproductive procedures, and optimizing the use of cloned genetic material in animal breeding and conservation. • Using skin-derived cells, the study produced multiple clones and effectively re-cloned a buffalo bull, achieving major scientific breakthrough. • Clones of buffalo bulls demonstrated normal postnatal growth, indicating that the cloning procedure had no negative impact on their overall health and development. • The cloned bulls' telomere length, remained within normal ranges, indicating that their cells maintained genetic stability over time. • Multiple clones produce high-quality sperm, a key aspect of their reproductive potential. [ABSTRACT FROM AUTHOR]

Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10−9 M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10−9 M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10−9 M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10−9 M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning. • Approximately 30–40 % of cattle and buffalo COCs collected are classified as C or D grade which are usually discarded. • We explored the potential of melatonin to improve the quality of substandard oocytes. • Melatonin acts by scavenging ROS thus protecting oocytes and embryos against oxidative stress. [ABSTRACT FROM AUTHOR]

The objective of this study was to optimise the electroporation conditions for efficient integration of Venus construct in buffalo fetal fibroblasts using Sleeping Beauty (SB) based transposition and to produce Venus expressing transgenic cloned embryos through handmade cloning (HMC) approach. Primary culture of buffalo fetal fibroblast cells was established and subsequently cultured cells were co-transfected with Venus and helper plasmid at different combinations of electroporation condition. In different combinations of voltage, time and plasmid dose, we observed that 300 V, single pulse for 10 ms in 2 mm cuvette and 1.5–2.0 μg transposons with 200–300 ng transposase dose was optimum for expressing Venus fluorescence in cells via electroporation. After electroporation, the cells were cultured for 2–3 days and then Venus expressing cells were picked with the help of a Pasteur pipette under the fluorescence microscope to enrich them through single cell culture method before using as donor cells for HMC. In vitro matured oocytes were reconstructed with either transfected or non-transfected buffalo somatic cells by electric fusion followed by activation. The reconstructed, activated embryos were cultured in 400 μL of Research Vitro Cleave medium supplemented with 1% fatty acid-free BSA in 4-well dish, covered with mineral oil and incubated in an incubator (5% CO 2 in air) at 38.5 °C for 8 days and the developmental competence was observed. The percentage of cleaved, 4–8 and 8–16 cells stage embryos generated through Venus expressing cells were comparable with control, whereas, the morula (21.0 vs 53.0%) and blastocysts (10.5 vs 30.6%) produced through Venus expressing cells was found low as compared to control. These results indicate that fetal fibroblasts transfected with Venus could be used as donor cells for buffalo cloning and that Venus gene can be safely used as a marker of foreign gene in buffalo transgenesis. [ABSTRACT FROM AUTHOR]

The somatic cells having higher levels of DNA methylation and reducing it in donor cells before nuclear transfer (NT) by treating them with chemicals, may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Therefore, the present study was planned to understand mechanism of epigenetic changes in donor cells (buffalo fibroblasts) treated with different concentration of sodium butyrate (NaBu)-a chromatin remodeling agent. The cultured fibroblasts purity and lineage were confirmed by fibroblast specific protein and gene markers (Vimentin, Tubulin and Cytokeratin) at different passages using immuno-staining and q PCR respectively. The buffalo fibroblast cells were treated with 1, 3 and 5 mM of NaBu and observations were taken on their morphological changes, population doubling time and cell proliferation after 48 h of treatment. The epigenetic changes were observed using acetylation (H3K9ac) and methylation (H3K27me3) markers expression. The fibroblast cells derived from new born ear tissue started emerging and anchoring to cell culture flasks within 24 h and showed spindle-shaped morphology. The confluent culture was cryopreserved at different time interval. The post thaw culture behavior of the cryopreserved cells was also observed at different time interval and passages. The morphology of NaBu treated cells were changed with increase of dosages of NaBu treatment. The increase population doubling times and decreases the proliferation rate in the dose dependent manner. The NaBu treatment showed that the significantly increased the acetylation (H3K9ac) and methylation (H3K27me3) level over the control. Based on the observations of fibroblast characterization as well as epigenetic modifications of these cells after treatment with NaBu, results suggest that the cells may provide a useful approach for better epigenetic reprogramming in SCNT embryos. [ABSTRACT FROM AUTHOR]

Cryopreservation commonly decreases the cellular functionality and post-thaw viability of cells. Reactive oxygen species (ROS) generated during cryopreservation degrade mitochondrial activity and promote the release of cytochrome C which activates caspases required for apoptosis. Antioxidants have the potential to improve the recovery efficiency of cells by reducing ROS production and maintaining mitochondrial membrane potential (MMP). The present study was conducted to explore the role of MitoQ, a derivative of coenzyme Q10 on cryopreserved fibroblasts derived from buffalo skin. To achieve our goal, buffalo skin fibroblasts were treated with varying concentrations of MitoQ (0, 0.1, 0.5, 1, 2, and 10 μM) for 24, 48, and 72 h. The MMP, ROS generation, cell viability was measured by flow cytometry. Furthermore, expression of genes related to mitochondrial oxidative stress (NRF2, GPX, and SOD), apoptosis (BAK and caspase 3) and cell proliferation (AKT) were also assessed. The results showed that over a period of 72 h lower concentrations of MitoQ (0.1–0.5 μM) decrease the ROS production, improves MMP and cell viability whilst the high concentration of MitoQ (2–10 μM) increased the oxidative damage to the cells. Taken together, our study provide important insights into the novel role of MitoQ in cryopreserved buffalo skin fibroblasts. In conclusion, we demonstrated the dose-dependent functional role of MitoQ on cryopreserved fibroblasts for improving post-thaw cell viability and cellular function. • Cryopreservation hampers viability and cellular functions of somatic cells due to ROS productions which limit their usages. • MitoQ showed the ability to improve the cells recovery by reducing ROS production and maintaining MMP. • MitoQ has antioxidant action, but it has also been seen that with high dose of MitoQ concentration, its activity turns prooxidant. • MitoQ possesses antioxidant properties, but at high concentrations, its activity becomes prooxidant. Due to its antioxidant properties, MitoQ must be added in the optimum quantity to various tissues and cells. • In cryopreserved buffalo fibroblast cells, the effect of MitoQ and the mechanism underlying its protective role were examined. [ABSTRACT FROM AUTHOR]

Abstract: X-ray in-line phase-contrast imaging is an emerging technique made practical by high brilliance synchrotron X-ray sources and high-performance X-ray detectors. This technique is capable of providing improved contrast along with edge enhancement for weakly absorbing samples and hence it is of immense value in study of materials like carbon fiber, carbon composite, etc. This technique is also very useful for determination of coating thickness, uniformity of pyrocarbon coating on the fuel kernel particles. The applications of such fuel kernel particles demand a high-quality of uniformity and precise thickness. The thickness and uniformity of such coatings depends upon several parameters of the system during their preparation. In this paper, we have explored the use of X-ray phase-contrast techniques for the measurement of coating thickness and its uniformity. We present the results of such experiments on SiC and pyrocarbon (PyC)-coated zirconia micro-spheres. This information may be of importance in optimization of the various parameters during the deposition process. The results bring out the importance of the X-ray phase-contrast technique in materials research. [Copyright &y& Elsevier]

Abstract: In-line X-ray phase-contrast imaging technique is an emerging method for the study of materials such as carbon fibers, carbon composite materials, polymers, etc. Similarly this technique is also well suited for the imaging of soft materials such as tissues, distinguishing between tumor and normal tissue. These represent the class of materials for which X-ray attenuation cross-section is very small. Thus this method promises a far better contrast for low X-ray absorbing substances than the conventional radiography method. We have set up an experimental facility using a combination of X-ray CCD detector and a microfocus X-ray source. This facility is dedicated to micro-imaging experiments such as microtomography and high-resolution phase-contrast experiments. In this paper, the results of X-ray phase-contrast imaging experiments are described. [Copyright &y& Elsevier]

Abstract: A theoretical study of physical and electronic properties of silicon has been made by using pseudopotential method within density functional theory (DFT) in generalized gradient approximation (GGA). The effects caused by the expansion of the lattice not studied so far have been investigated. The energy gap for the experimental lattice constant calculated in GW approximation is seen to be in agreement with the experimental value. We observe a crossover from an indirect gap to direct gap at a lattice constant of 5.918Å corresponding to a lattice expansion of about 8.39%. Silicon is found to be metallic for a lattice constant of 7.61Å in the tetrahedral structure. [Copyright &y& Elsevier]

Abstract: We perform an ab initio study of the stability, structural and vibrational properties of small silicon–carbon binary microclusters after using a self-consistent pseudopotential method within density functional theory in the generalized gradient approximation considering spin-polarization. The binding energies, various bond lengths and vibrational frequencies have been computed for the various microclusters. The computed values of different Si–Si, Si–C and C–C bond lengths are in very good agreement with the available experimental data. Also, the calculated vibrational frequencies for different clusters are in reasonable agreement with the available experimental values. The clusters containing the maximum number of C–C and Si–C bonds are found to be most stable. [Copyright &y& Elsevier]

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance. • Genome editing is a well-known method for introducing targeted genetic alterations into livestock genomes, however its progress in buffaloes has lagged behind. • In the present study, we aimed to generate MSTN-edited buffaloes using SCNT and Zygotic electroporation. • The best transfection method was identified for introduction of RNP complex into buffalo fibroblast which was further used for single cell isolation and generation of MSTN edited embryos via SCNT. • The embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation were compared. [ABSTRACT FROM AUTHOR]

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 μM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 μM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process. • Study disclosed the new role of H89. • H89 reduced cholesterol efflux from spermatozoa. • H89 restricted the influx of calcium in spermatozoa. • H89 prevented phosphorylation of tyrosine-containing proteins. • Effects of H89 were reversed in vitro capacitation media. [ABSTRACT FROM AUTHOR]

Exotic properties provided by the adsorption of non-magnetic Si atoms on a two-dimensional–GaN (2D–GaN) monolayer have been predicted using a generalized gradient approximation within density functional theory. This includes the occurrence of spin filtering, high magnetism and the continuous emission of electromagnetic radiation. A comprehensive ab-initio study of the structural, electronic, magnetic and optical properties of a 2D–GaN monolayer possessing several Si atoms as adsorbents has been performed. The semiconducting pure GaN monolayer was converted into its metallic analogue in several Si-adsorbed GaN monolayer systems. Along with the main strong absorption lying in the deep ultraviolet region near 5.5 eV observed in all of the Si-adsorbed 2D-GaN monolayer systems, a strong continuous absorption from the far infrared to visible light region has been observed in some of the systems studied. These systems may be useful for the development of light-emitting devices, which will emit in the various regions of the electromagnetic spectrum, including the visible light region. The emission in the deep ultraviolet light region may be used for sterilization, water purification etc. • The adsorption of Si atom on 2D-GaN monolayer makes it magnetic. • The induced magnetic behaviour is site independent. • The binding energies per adatom of Si-atoms adsorbed above N are larger than those of Si-atoms adsorbed above Ga. • Spin filtering has been observed in many of the studied systems. • The occurrence of the absorption in the ultraviolet light region can be utilized in various applications e.g. optoelectronic devices, sterilization, water filtering etc. [ABSTRACT FROM AUTHOR]