Optimization of dissolved oxygen levels in extender prevents development of cryocapacitation like changes, oxidative stress and augments zona binding capacity of crossbred bull spermatozoa.

The present study was undertaken to determine the efficacy of partial deoxygenation of extender at constant temperature (35°C) in freezability of crossbred bull semen. The dissolved oxygen (DO) levels were reduced by the use of newly developed technique of nitrogen effervescence at a flow rate of 2-3 bubbles per second. Four different levels of oxygen in semen extender, that is 11.7, 2, 4 and 8 ppm as control (Group-I), Group-II, Group-III and Group-IV, respectively, were used to assess the effect of partial deoxygenation on semen quality parameters. The 4 ppm level of DO resulted in higher (p < 0.05) progressive motility in comparison with non-treated group at post-thaw stage, whereas reduction up to 2 ppm resulted in drastic fall in motility. Oxidative stress status revealed low superoxide dismutase (SOD) and total antioxidant capacity (TAC) in Group-II, whereas higher (p < 0.05) SOD and TAC activities were observed in Group-III in comparison with non-treated group at pre-freeze and post-thaw stages. The sperm-zona binding at 4 ppm level of DO was significantly higher than control group, 2 and 8 ppm levels of DO. In conclusion, reduction of DO in the extender up to 4 ppm reduced oxidative stress and improved in vitro fertility of crossbred bull spermatozoa.

Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis).

Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein-30 (SMP-30), is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation. In Drosophila, RGN is reportedly a putative cold-tolerance gene and a cytoprotective role for RGN against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN has anticapacitatory effect and abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 µg/ml), as a supplement for Tris-egg yolk-based semen extender. Post-thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 µg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post-thaw progressive motility of spermatozoa (50.6 ± 3.5% vs 40.6 ± 2.6%; p < 0.01), acrosome integrity (53.3 ± 7.4 vs 75.6 ± 6.8; p < 0.05), and zona pellucida binding (31.6 ± 14.0 vs 191.9 ± 12.3 bound spermatozoa; p < 0.01) compared to control conditions without RGN. Thus, ∼1 µM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender.

Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei.

Blackleg, an economically important and highly fatal disease of ruminants, is caused by anaerobic bacillus, Clostridium chauvoei. Identification and differentiation of the causative agent is crucial for implementation of therapeutic and control measures in real time. Most of the diagnostic tests available for blackleg are PCR based, and only a couple of serological tests have been reported. In this study, we targeted flagellin, an important immunogenic protein of C. chauvoei, to develop a sandwich ELISA for detection of C. chauvoei. Sequence analysis of flagellin gene of related Clostridium species showed that central region of flagellin gene is unique to C. chauvoei. Hence, we cloned and expressed central region of flagellin in a prokaryotic expression system. Antiserum against recombinant flagellin was generated in rabbits and chickens. A sandwich ELISA was developed, in which rabbit anti-flagellin antibodies were used as capture antibodies and chicken anti-flagellin antibodies as detecting antibodies. The test was specific and sensitive in detection of up to 10(4) CFU/ml of C. chauvoei. This study shows that assay developed can be used for detection of C. chauvoei in suspected samples.