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.

Luteinizing hormone beta gene polymorphism and its effect on semen quality traits and luteinizing hormone concentrations in Murrah buffalo bulls.

OBJECTIVE: Present investigation was aimed to study the Single Nucleotide Variants of the luteinizing hormone beta (LHß) gene and to analyze their association with the semen quality (fresh and post-thawed frozen semen) and luteinizing hormone (LH) concentrations in Murrah buffalo bulls. METHODS: Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and Sanger sequencing method is used to study genetic variability in LHß gene. LH assay was carried out using enzyme-linked immunosorbent assay method. A fixed general linear model was used to analyze association of single nucleotide polymorphism (SNP) of LHß gene with semen quality in 109 and LH concentrations in 80 Murrah bulls. RESULTS: LHß gene was found to be polymorphic. Total six SNPs were identified in LHß gene g C356090A, g C356113T, g A356701G, g G355869A, g G356330C, and g G356606T. Single Stranded Conformational Polymorphism variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on sperm concentration (million/mL), percent mass motility, acrosome integrity and membrane integrity in fresh and frozen semen whereas significant (p<0.05) effect was observed on percent live spermatozoa. SSCP variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on luteinizing hormone concentrations too. CONCLUSION: The observed association between SSCP variants of LHß gene with semen quality parameters and LH concentrations indicated the possibilities of using LHß as a candidate gene for identification of markers for semen quality traits and LH concentrations in Murrah buffaloes.

Optimizing age of bull at first use in relation to fertility of Murrah breeding bulls.

AIM: The aim of the present investigation was to optimize the age at first use (AAFU) of semen of Murrah breeding bulls, which will help in early selection of bulls under progeny testing program for improving the reproductive performance in the herd. MATERIALS AND METHODS: The data on AAFU, conception rate based on first A.I. (CRFAI), overall conception rate (OCR), and birth weight (B.WT) of 57 Murrah bulls during 1993-2014 at NDRI center pertaining to 14 sets of Network Project on Buffalo Improvement at ICAR-National Dairy Research Institute, Karnal, Haryana, India were adjusted for significant environmental influences and subsequently analyzed. Simple and multiple regression models were used for prediction of CRFAI and OCR of Murrah breeding bulls. Comparative evaluation of three developed models (I-III) showed that Model III, having AAFU and B.WT, fulfill the accuracy of model as revealed by high coefficient of determination, low mean sum of squares due to error, low conceptual predictive value, and low Bayesian information criterion. RESULTS: The results revealed that the average predicted CRFAI was highest (39.95%) at <3.5 years and lowest (34.87%) at >4.5 years of age at first A.I/use. Similarly, average predicted OCR was highest (41.05%) at <3.5 years and lowest (39.42%) at >4.5 years of age at first A.I/use of Murrah bulls. CONCLUSION: In organized herd under progeny testing program, Murrah bulls should be used at young age, i.e. prior to 3.5 years, which is expected to result in 5.08% better CRFAI and 1.63% better OCR in comparison to Murrah bulls used after 4.5 years of age.