LPS-Induced Mitochondrial Dysfunction Reduces Oocyte Maturation and Developmental Competence of Buffalo Embryos via ROS Mediated TLR4 Signalling.

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.

Enhancing the quality of inferior oocytes of buffalo for in vitro embryo production: The impact of melatonin on maturation, SCNT, and epigenetic modifications.

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.

Design and evaluation of fluorescent chitosan-starch hydrogel for drug delivery and sensing applications.

Composite bio-based hydrogels have been obtaining a significant attention in recent years as one of the most promising drug delivery systems. In the present study, the preparation of composite chitosan-starch hydrogel using maleic acid as a cross-linker was optimized with the help of response surface methodology. The synthesized hydrogel was fluorescent owing to clustering of large number of functional groups. Different analytical techniques, including XRD, FTIR, SEM, XPS, fluorescence and BET were utilized to characterize the prepared hydrogel. XRD analysis confirmed the formation of non-crystalline hydrogel with random arrangement of macromolecular chains. The composite hydrogel exhibited good swelling percentage with pH sensitivity, hemocompatibility and degradability. BET analysis confirmed that the variation in concentration of crosslinker significantly influences the pore volume of the hydrogel. The synthesized composite chitosan-starch hydrogel was utilized as a prospective candidate for controlling drug release. Cefixime as a model drug was loaded onto the synthesized hydrogel utilizing the swelling diffusion method. SEM micrographs showed uniform distribution of drug molecules in the drug loaded hydrogel. In vitro drug release experiments indicated the swelling dependent drug release behaviour of chitosan-starch hydrogel with higher drug release at pH 7.4 (93.08 %) compared to pH 1.2 (67.85 %). The composite chitosan-starch hydrogel was able to prolong and control the drug release up to 12 h. The drug release from the hydrogel followed Korsmeyer-Peppas and Makoid-Banakar model with Fickian diffusion mechanism. Further, the composite hydrogel displayed excitation dependent fluorescence emission with most intense blue emission band at 425 nm with an excitation wavelength of 350 nm. The inclusion of cefixime drug in the hydrogel matrix significantly reduced the fluorescence intensity; the decrease was linearly correlated to the concentration of the drug. Moreover, the fluorescence emission the chitosan-starch hydrogel was found to be dependent upon pH. The synthesized hydrogel is expected to be a potential candidate for controlled drug release as well as for fluorescent sensing applications.

Deoxyfluorinated amidation and esterification of carboxylic acid by pyridinesulfonyl fluoride.

Amide bond synthesis is one of the most used reactions in medicinal chemistry. We report an amide bond formation reaction through deoxyfluorinated carboxylic acids under mild conditions using 2-pyridinesulfonyl fluoride. The reaction procedure has been used in a one-pot synthesis of amides and esters via in situ generation of acyl fluoride. This one-pot synthetic method provides easy access to amides and esters. Using this method, we have sequentially synthesized a tetrapeptide and calceolarioside-B glycoside derivative with good yields.

Groundwater quality assessment for drinking purpose using traditional and fuzzy-GIS-based water quality index in Gurugram District of Haryana, India.

Primarily groundwater is consumed for the drinking and irrigation purpose. However, due to increasing anthropogenic activities, its quality and quantity have substantially declined over time. The focus of this study is to evaluate the pre-monsoonal groundwater quality and its spatial variability for drinking purposes in the Gurugram, Haryana, India. Ground Water quality index (GWQI) developed on the basis of the Geographical Information System is effective in the assessment of groundwater quality and its spatial variability, but it is unable to account for uncertainties related to environmental problems. Thus, a Hybrid Fuzzy-GIS-based Water Quality Index (FGQI) has been proposed for the assessment of groundwater quality. The study conducted factor analysis to identify the prime factors responsible for groundwater contamination and collected pre-monsoonal groundwater samples through primary sampling. The groundwater quality was assessed based on eight hydro geochemical parameters (pH, TDS, Calcium, Chloride, Sulfate, Fluoride, Potassium, and Sodium). The spatial interpolation of each parameter was performed using appropriate techniques, selected based on a normality test. The guidelines of the World Health Organization and Bureau of Indian Standard were incorporated in the development of GWQI and FGQI, respectively. Correlation analysis was performed to determine the best fuzzy overlay technique for FGQI, and the Fuzzy GAMMA technique with gamma equal to 0.9 was selected. Finally, the GWQI and FGQI were classified into three classes: unsuitable, moderate suitable, and suitable using "natural break". A higher index indicates a higher water quality. The results show that the groundwater in the central part of Gurugram is suitable for drinking, while it is not suitable in the extreme north, south-east, and western regions. In conclusion, this study finds that FGQI effectively assesses the groundwater quality in the region better than GWQI.

Mito-Q supplementation of in vitro maturation or in vitro culture medium improves maturation of buffalo oocytes and developmental competence of cloned embryos by reducing ROS production.

Mito-Q is a well-known mitochondria-specific superoxide scavenger. To our knowledge, the effect of Mito-Q on buffalo oocyte maturation and developmental competency of cloned embryos has not been examined. To investigate the effects of Mito-Q on the in vitro maturation (IVM) of buffalo oocytes and the developmental competence of cloned embryos, different concentration of Mito-Q were supplemented with IVM (0, 0.1, 0.5, 1, 2 µM) and in vitro culture (IVC) medium (0, 0.1 µM). Supplementation of IVM medium with 0.1 µM Mito-Q significantly (P ≤ 0.05) increased the cumulus expansion, nuclear maturation, mitochondrial membrane potential (MMP) and antioxidants genes (GPX1 and SOD2) expression and effectively reduced ROS production leading to a significant improvement in the maturation rate of buffalo oocytes. Further, the supplementation of 0.1 µM Mito-Q in IVC medium promotes the cleavage and blastocyst rate significantly over the control. Mito-Q supplementation improves (P ≤ 0.05) MMP, antioxidant gene (GPX1) expression and reduced the ROS level and apoptosis related genes (caspase 9) expression in cloned blastocysts. In conclusion, the present study demonstrated that the supplementation of 0.1 µM Mito-Q in IVM and IVC media exerts a protective role against oxidative stress by reducing ROS production and improving MMP, fostering improved maturation of buffalo oocytes and enhanced developmental competence of cloned embryos. These findings contribute valuable insights into the optimization of assisted reproductive technologies protocols for buffalo breeding and potentially offer novel strategies to enhance reproductive outcomes in livestock species.

Stress distribution in bone around an implant-supported three-unit fixed dental prosthesis using two different computer-aided designing/computer-aided milling provisional crown materials: Milled polymethylmethacrylate and milled polyetheretherketone - A finite element analysis.

Background: Occlusal loading of osseointegrated implants is believed to be an essential determining factor in the long-term success of an implant treatment. Numerous studies have been conducted on the evaluation of stress distribution by definitive restoration materials for Implant-supported fixed prosthesis, but very few have evaluated provisional restoration materials for the same. This study aims to evaluate the influence of provisional restoration material - Milled Polymethylmethacrylate (PMMA) and Milled Polyetheretherketone (PEEK), over stress distribution on the peri-implant bone around an implant-supported three-unit, fixed dental prosthesis using finite element analysis method. Materials and Methods: Three-dimensional models of a pair of bone-level implant system and titanium base abutments were created using the standard tessellation language data of original implant components. A bone block representing the mandibular posterior area was created, and the implants were placed in the bone block with 100% osseointegration in the 2nd premolar to 2nd molar region. A superstructure of an implant-supported 3-unit bridge was modeled on top of the abutments, each crown to be 8 mm in height and with an outer diameter of 6 mm in 2nd premolar region and 10 mm in 1st molar and 2nd molar region. Two different models were created according to combinations of provisional restoration materials, namely, Milled PMMA and Milled PEEK based on. In each model, the implants were loaded vertically (300 N) and obliquely (150 N at 30°). The stress distribution in the cortical bone, cancellous bone, and implant was evaluated through the von Mises stress analysis. Results: The results showed no difference in stress distribution due to the different provisional restorations - Milled PMMA and Milled PEEK. In addition, the vertical load resulted in higher stress values in the implant components, cortical bone, and cancellous bone in both PEEK and PMMA models as compared to oblique loading. Conclusion: The new polymer, PEEK was seen to provide comparable stress generation in the current study without exceeding the physiological limits of peri-implant bone. Thus, it can be considered as a good alternative to PMMA resin as a provisional crown material since it provides certain additional benefits.

Effects of Heterologous Bovine Seminal Plasma-Supplemented Egg Yolk-Based Extender on Cryosurvivability of Pantja Buck Semen.

The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the extender before cryopreservation. In a preliminary experiment, different levels of BSP were supplemented (1, 3, 5, 7, and 9% v/v) in egg yolk (7.5% egg yolk)-tris (EYT) extender and used for cryopreservation of Pantja buck semen. Results in terms of motility, viability, plasma membrane integrity, acrosome integrity, and lipid peroxidation showed that 5% BSP was suitable for maintaining Pantja buck semen quality during cryopreservation. In the final experiment, pooled semen from four Pantja bucks was split into three aliquots (I, II, and III). Aliquot I was directly diluted in EYT extender and grouped as the control (C); aliquot II and III were washed separately with TALP solution and diluted as D1 (Washed semen with EYT extender) and D2 (Washed semen with EYT extender containing 5% BSP), respectively. Seminal attributes (sperm individual motility, viability, plasma membrane integrity, acrosome integrity, and total morphological abnormalities) were assessed at the postdilution, postequilibration, and post-thawing stages. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) concentration, and glutathione peroxidase (GSH-Px) activity were measured at post-thaw. Washed semen significantly improved (p < 0.05) seminal parameters at post-thaw compared with unwashed semen (control). A significant difference (p < 0.05) was observed in seminal attributes between freezing stages and between dilution groups. Significantly higher (p < 0.05) post-thaw sperm motility, viability, plasma membrane integrity, acrosome integrity, and GSH-Px activity, and significantly lower (p < 0.05) MDA concentration and extracellular release of enzymes (ALT, AST) were observed in group D2 compared with control and D1. The results of the present study demonstrated that cryopreservation of washed Pantja buck semen diluted with 5% BSP-supplemented EYT extender can improve post-thaw semen quality.

Attitude, Opinions, and Working Preferences Survey among Pet Practitioners Relating to Antimicrobials in India.

The indiscriminate usage and overuse of antimicrobials in pets or companion animals are underlying causes of antimicrobial resistance (AMR). Despite the multi-faceted global challenge presented by antimicrobial resistance, very few studies have appraised pet practitioners' factors, such as written policy on antimicrobials, dose rate prescribed, use of critically important antimicrobials, and antimicrobial prescription in clean surgical procedures, which can contribute to AMR. In the present study, an online cross-sectional survey among randomly selected pet practitioners (n = 104) of various Indian provinces and union territories was conducted using a questionnaire comprising 33 closed-ended questions on different parameters, viz., the dosage regimen and level of compliance towards guidelines of the World Health Organization (WHO), other relevant veterinary associations, and their opinion while prescribing antimicrobials. Almost every practitioner of the 104 respondents had revealed the difficulties with owner compliance; i.e., incomplete course of the antibiotics, inappropriate follow-ups, and improper care of the sick animals. The majority of practitioners (95%) reported self-prescription of antimicrobials by the owner before presenting the pet(s) to the veterinary clinic, whereas more than half of the respondents (64%) revealed unavailability of antibiogram facilities. Furthermore, a large number (76%) of practitioners stated empirical treatment based on their experience as the main criteria for antimicrobial choice in the absence of timely results from the laboratory. Although non-necessitated use of antimicrobials in clean surgical procedures has been claimed, surprisingly, the majority of pet practitioners (97%) reported their use to reduce the post-operative complications. The use of the highest priority, critically important antimicrobials (HPCIA) listed by the WHO for humans, particularly quinolones and third-generation cephalosporin, also has been reported for different infections. The treatment durations were nearly as per the recommended guidelines issued by the Danish Small Animal Veterinary Association (DSAVA) for different ailments. Analysis using chi-square tests exhibited a significant correlation between less experienced veterinarians (less than 5 years) and prescription of antimicrobials restricted for critically important infections in human medicine. However, there seems to be no association between the experience of the practitioner and the further studied parameters, namely, antimicrobial regimen prescription, weighing the animals before prescription, dose rate calculation, and antimicrobial selection and use after clean surgical operations. The findings suggest periodic awareness campaigns among practitioners regarding the implementation of the official guidelines, the need for systematic surveillance of AMR, awareness among pet owners about antimicrobial resistance, and the importance of rational use of antimicrobials on their pets.

COVID-19's lockdown effect on air quality in Indian cities using air quality zonal modeling.

The complete lockdown due to COVID-19 pandemic has contributed to the improvement of air quality across the countries particularly in developing countries including India. This study aims to assess the air quality by monitoring major atmospheric pollutants such as AOD, CO, PM2.5, NO2, O3 and SO2 in 15 major cities of India using Air Quality Zonal Modeling. The study is based on two different data sources; (a) grid data (MODIS- Terra, MERRA-2, OMI and AIRS, Global Modeling and Assimilation Office, NASA) and (b) ground monitoring station data provided by Central Pollution Control Board (CPCB) / State Pollution Control Board (SPCB). The remotely sensed data demonstrated that the concentration of PM2.5 has declined by 14%, about 30% of NO2 in million-plus cities, 2.06% CO, SO2 within the range of 5 to 60%, whereas the concentration of O3 has increased by 1 to 3% in majority of cities compared with pre lockdown. On the other hand, CPCB/SPCB data showed more than 40% decrease in PM2.5 and 47% decrease in PM10 in north Indian cities, more than 35% decrease in NO2 in metropolitan cities, more than 85% decrease in SO2 in Chennai and Nagpur and more than 17% increase in O3 in five cities amid 43 days pandemic lockdown. The restrictions of anthropogenic activities have substantial effect on the emission of primary atmospheric pollutants.

Effect of strontium substitution on the cytocompatibility and 3-D scaffold structure for the xSrO-(10-x) MgO-60SiO2-20CaO-10 P2O5 (2 ≤ x ≤ 8) sol-gel glasses.

In the present study, novel glasses xSrO-(10-x) MgO-60SiO2-20CaO-10 P2O5 (2 ≤ x ≤ 8, in steps of 2) are synthesized via sol-gel method. The current work focusses on the evaluation of mechanical, physical and biocompatible properties for sol-gel glasses. The pore size and surface area of these glasses were studied using BET analysis. The structural aspect of the glasses/glass ceramics was studied by XRD and Raman spectroscopy. The cytotoxicity assays were conducted for MG63 human osteosarcoma cell line. Furthermore, the as prepared glasses were used for the fabrication of 3-D porous scaffolds via polymer replication method. The loaded green bodies have been sintered at 700, 800 and 900 °C and were kept for 6 h to densify the glass network. The effect of sintering temperature on the structure and properties of as prepared scaffolds were analyzed via scanning electron microscopy (SEM) and porosity calculations.

Antibacterial and anticancerous drug loading kinetics for (10-x)CuO-xZnO-20CaO-60SiO2-10P2O5 (2 ≤ x ≤ 8) mesoporous bioactive glasses.

In the present study, antibacterial and anticancerous drug loading kinetics for the (10-x)CuO-xZnO-20CaO-60SiO2-10P2O5 (2≤x≤8, varying in steps of 2) mesoporous bioactive glasses (MBGs) have been studied. XRD analysis of the as prepared glass samples proved its amorphous nature. Scanning electron microscopy (SEM) revealed the apatite layer formation on the surface of the MBGs after soaking for 15 days in SBF. Ion dissolution studies of calcium, phosphorous and silicon have been performed using inductively coupled plasma (ICP). FTIR and Raman analysis depicted about the presence of various bonds and groups present in the glasses. The pore size of MBGs lies in the range of 4.2-9.7 nm. Apart from this, specific surface area of the MBGs varied from 263 to 402 cm2/g. The MBGs were loaded with Doxorubicin (DOX), Vancomycin (VANCO) and Tetracycline (TETRA) drugs among which, the decreasing copper content influenced the loading properties of doxorubicin and tetracycline drugs. Vancomycin was fully loaded almost in all the MBGs, whereas other drugs depicted varying loading with respect to the copper content.