Atul Saxena

Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa

To provide new insights into the mechanisms through which reduced glutathione (GSH) is able to protect spermatozoa, we tested the hypothesis that cryocapacitation and apoptosis like changes can contribute to the negative effect of freezing and thawing on bull spermatozoa, and that GSH prevent this damage. Having known protective effects of GSH in terms of a potent antioxidant, we evaluated capacitation, tyrosine phosphorylation and apoptosis like changes in bull spermatozoa after freezing and thawing in egg yolk tris glycerol extender containing (0.5m M-GSH-T1 & 1mM GSH-T2) and without GSH serving as the control (C). Forty ejaculates were collected from four Hariana bulls and were pooled due to non significant variations among the bull ejaculates for the evaluation of sperm attributes. Capacitation like changes, tyrosine phosphorylation, localization of tyrosine phosphorylated proteins, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA fragmentation after final dilution, 4h of equilibration at 4°C and 24h after freezing and thawing were evaluated. GSH supplementation at 0.5mM showed significant reduction in B- and AR- pattern spermatozoa during all stages of semen freezing and thawing. Immunoblot revealed six proteins which were tyrosine phosphorylated and protein of 30 and 75kDa (p30, p75) were the major tyrosine phosphorylted proteins. On further analysis, the p30 showed differential variation in intensity in all the three groups after freezing and thawing. Positive immune reactivity for tyrosine phosphorylated proteins was found in neck, middle piece and post-acrosomal regions of spermatozoa. Addition of 0.5mM GSH decreased percentage of spermatozoa showing fragmented DNA and increased the percentage of spermatozoa having high transmembrane mitochondrial potential (P<0.05). This study demonstrates that GSH favours survival of bull spermatozoa by interfering with apoptotic and cryocapacitation pathways, and thereby protects the spermatozoa from deleterious effects of cryopreservation. The findings of the study indicated that GSH at 0.5mM can be effectively used as an additive in bull semen extender for freezing and thawing.

Seasonal Variations in Seminal Plasma Proteins of Buffalo

The study was designed to evaluate the influence of season on semen characteristics and seminal plasma protein profile of buffalo bull semen. Thirty-six ejaculates were collected in three seasons (winter, summer and rainy) from six adult Bhadawari bulls, and semen characteristics were evaluated immediately after collection. The seminal plasma was harvested by centrifugation and protein profiling, and percentage protein fractions were analysed by SDS-PAGE. The significant effect of season was observed on ejaculate volume, sperm concentration, progressive motility, percentage live spermatozoa, hypo-osmotic swelling test (HOST) and acrosomal integrity. The electrophoretogram of seminal plasma proteins revealed 20 protein bands in winter, 23 bands in rainy and 25 bands in summer seasons, illustrating the significant effect of seasons on seminal plasma proteins. Among these protein bands, 18 bands were observed common in semen samples of all three seasons while protein bands of 46, 55, 58, 144 and 160 kDa were found in rainy and summer seasons. The protein bands of 48 and 60 kDa were observed only in winter season, whereas 184 and 200 kDa were reported in summer season only. The protein fractions (protein%) of common protein bands observed in three seasons revealed a significant effect of season on protein bands of 24.5, 66, 70, 72, 84 and 86 kDa. From the study, it was pertinent that bull seminal plasma contains specific proteins in particular season, which may be associated with some of the semen characteristics, and these proteins could be used as markers of the semen quality of buffalo bulls.

Effect of supplementation of butylated hydroxytoluene on post-thaw sperm viability, motility and membrane integrity of Hariana bulls.

Aim: This study was aimed to see the beneficial effect of butylated hydroxytoluene (BHT) as a semen additive of Hariana bull semen. Materials and Methods: The study was carried out in Hariana bulls. Twenty-four ejaculates from two bulls were used for this study. Each ejaculate was extended with standard glycerolated egg yolk tris extender and supplemented with BHT at two concentrations as 0.5 mM (T1) and 1.0 mM (T2). After dilution, equilibration and 24 h of cryopreservation, the samples were analyzed for progressive motility, sperm viability and membrane integrity. Results: Progressive motility, sperm viability and sperm membrane integrity were significantly (p<0.05) increased in the samples fortified with BHT as compared to the control during the process of cryopreservation and thawing. The BHT concentration of 1 mM revealed better results as compared to 0.5 mM. Conclusion: Addition of 1.0 mM BHT was found better in cryopreservation of Hariana bull semen compared to 0.5 mM BHT and control samples. The addition of BHT has improved the sperm quality by acting as an antioxidant thereby reducing the lipid peroxidation of the sperms.

Molecular characterization of voltage-gated potassium channel (Kv) and its importance in functional dynamics in bull spermatozoa

Present study was undertaken to characterize the voltage gated potassium channel (Kv 1.1) in bull spermatozoa using sixty four ejaculates collected from four Hariana bulls. Functional characterization was undertaken using a selective blocker of Kv channel, 4-Aminopyridine (4-AP) while molecular presence of Kv on bull spermatozoa by immunoblotting and indirect immunofluorescence. Three sets of 100 μL diluted sperm samples namely-negative control (100 μL of sperm dilution medium (SDM) containing 10 × 106 cells), vehicle control (99 μL of SDM containing 10 × 106 cells, and DMSO- 1  μL) and 4-AP treatment group (99 μL of SDM containing 10 × 106 cells, and drug 1 μL 4-AP) were used in the study. Immunoblotting identified a single band of 56 kDa corresponding to Kv1.1 in Hariana bull spermatozoa. Immunolocalization showed the positive immunoreactivity at head, middle piece and principal piece of the spermatozoa for Kv 1.1. Blocking of Kv using 4-AP resulted in significant (p < 0.05) reduction in sperm progressive motility, per cent capacitated spermatozoa (B-pattern) and acrosome reacted (AR-pattern) spermatozoa, while significant (P < 0.05) increase in per cent swollen spermatozoa. Blocking of Kv channels resulted in significantly (P < 0.05) increased percentage of spermatozoa with lower mitochondrial transmembrane potential. Computer assisted semen analysis (CASA) of motion and kinematic parameters in 4-AP treated spermatozoa indicated reduction in sperm motion parameters like LIN, STR, VSL and VAP and higher ALH, VCL, and BCF indicating hyperactivity of spermatozoa. Based on our findings, it may be concluded that voltage-gated potassium channel (Kv) are present on bull spermatozoa and these are associated with functional dynamics of spermatozoa. However, based on our limited study, it is not possible to deduce that how these channels are associated with induction of hyperactivity. Therefore, further studies are warranted to unravel the mechanistic signaling pathways involved in Kv-mediated alterations in functional dynamics of spermatozoa.

Hydrallantois Associated with Fetal Anasarca in a Non-Descript Doe

The incidence of dystocia in goats has been reported about 7% (Abdul-Rahman et al., 2000) to 8.23 % (Mehta et al., 2002). Fetal causes of dystocia are more common than maternal causes in goat (Abdul-Rahman et al., 2000) and sheep (Taha et al., 2005). Among dropsical conditions, the hydrallantois is more common compared to hydramnios (Hafez, 1993) and is frequently reported in bovines (having twins), rarely in mares (Milton et al., 1989) and less frequently in small ruminants. Hydrallantois is excessive accumulation of fluid in the allantoic sac in uterus, usually occurs within 5 to 20 days in advance pregnancy. Goats suffering from hydrallantois are usually presented in their second stage of labor with a history of sudden enlargement of the abdomen after mid gestation (Purohit, 2006). The present report documents a case of hydrallantois associated with fetal anasarca and its successful management in a non-descript doe.

Effect of Reduced Glutathione Supplementation on cryopreservation induced Sperm cryoinjuries in Murrah Bull semen

The experiment was conducted to study cryopreservation induced sperm cryoinjuries and the protective effect of reduced Glutathione supplementation in Murrah bull semen. A total of 20 semen ejaculates were split into two parts after initial examination and were extended in glycerolated egg yolk TRIS diluter (Control group) and glycerolated egg yolk TRIS diluter + 0.5 mM reduced Glutathione (Treatment Group). The diluted semen samples were loaded into 0.25 ml French mini straw and sealing of straws were done. Thereafter, semen straws were kept for equilibration for 4 h at 5 °C and semen was frozen using a standard cryopreservation protocol in automatic biological freezer. Post-thaw analysis was performed after 24 h of semen storage in liquid nitrogen. Fresh and post-thaw sperm assessments included sperm motility, viability (SYBR-14/PI assay), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (HOST). Cryopreservation of semen in liquid nitrogen induced a marked reduction in post-thaw sperm motility, viability, mitochondrial function and plasma membrane integrity and increased moribund type of sperm (SYBR-14/PI assay) in control group as compared to reduced glutathione treated group. There were significant (P < 0.05) cryo injuries in frozen-thawed spermatozoa following cryopreservation in buffalo bull semen. The supplementation of reduced glutathione in treatment group exhibited significantly (P < 0.05) lower cryoinjuries during cryopreservation and semen storage in liquid nitrogen. From the study it was concluded that, spermatozoa from Murrah bulls are susceptible to injuries due to cryopreservation in liquid nitrogen, but these cryo induced damage can be protected significantly (P < 0.05) by the use of reduced Glutathione.

Successful management of dystocia due to ankylosed foetal monster through fetotomy in a buffalo

Dystocia is the most common sequelae of foetal monstrosities in bovines. Fetal anomalies and monstrosities of various kind causing dystocia in cattle (Arthur et al., 1989; Patel et al., 2015) and buffaloes (Kumaresan et al., 2003 and Shukla et al., 2006) have been documented. The present paper describes about management of dystocia due to ankylosed foetal monster through fetotomy in a Murrah buffalo.

Atypical Schistosoma Reflexus with Multiple Congenital Abnormalities in Hariana Cow

The present manuscript reports an unusual case of schistosomus reflexus in a Hariana cow. The clinical findings include free floating viscera, mandibular prognathism, unilateral Amelia, mild hydrocephalus but lacking the typical ventral curvature of spine.

Schistosomus Reflexus in a Goat-A Case Report

A rare case of dystocia due to schistosomus reflexus in a goat and its successful management.

A critical case of closed cervix pyometra in a bitch.

Pyometra is accumulation of purulent material within the uterus, which manifests in both local and systemic symptoms, and demands costly surgical or medical intervention (Hagman et al. 2011). Pyometra is typically a post-oestral syndrome in adult bitches associated with a variety of clinical and pathological manifestations of genital and multisystemic disease (Chaffaux and Thibier 1978). Parity and age are significant risk factors in the development of pyometra (Niskanen and Thrusfield 1998). This paper communicates with successful management of a critical case of closed cervix pyometra in a German shepherd bitch.