title: Effect of different antioxidant additives in semen diluent on cryopreservability (-196°C) of buffalo semen.
abstract: OBJECTIVE
  The aim of this study was to evaluate the effect of different antioxidant additives in standard tris-fructose-egg yolk-glycerol (TFYG) extender on the cryopreservability of buffalo semen.
  
  
  METHODS
  Semen collection using artificial vagina, twice weekly for 5 weeks from three pedigreed health breeding bulls of Mehsani breed, aged between 6 and 8 years. Immediately after initial evaluation all 30 qualifying ejaculates (10/bull) were split into three aliquots and diluted at 34°C keeping the concentration of 100 million spermatozoa/ml with standard TFYG extender as control and TFYG having two antioxidant additives - Cysteine HCl at 1 mg/ml and ascorbic acid at 0.2 mg/ml to study their comparative performance. Semen filled in French Mini straws using IS-4 system and gradually cooled to 4°C and equilibrated for 4 h in cold handing cabinet. After completion of equilibration, straws were cryopreserved in LN2 by Programmable Bio-freezer. Semen was examined at post-dilution, post-equilibration, and post-thaw stages for sperm quality parameters, and at each stage plasma was separated for enzymatic analysis of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP).
  
  
  RESULTS
  The mean percentage of sperms in TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage in terms of progressive motility (52.83±0.52, 57.83±0.52, 57.83±0.52), livability (78.70±0.21, 82.33±0.23, 81.73±0.22), and abnormality (5.43±0.21, 5.03±0.17, 5.23±0.18) varied significantly (p<0.05) between control TFYG and TFYG having antioxidant additives. The mean U/L activities of AST (78.70±0.47, 72.80±0.48, 73.30±0.54), LDH (172.70±0.41, 155.78±0.42, 156.33±0.41), and AKP (103.61±0.34, 90.20±0.34, 91.03±0.34) in semen diluted with TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage, respectively, which showed significantly (p<0.05) higher leakage of enzymes in control TFYG than TFYG incorporated with additives.
  
  
  CONCLUSIONS
  Incorporation of antioxidant additives such as cysteine HCl and ascorbic acid in standard TFYG diluents improves sperm quality parameters, reduces enzyme leakage, and ultimately advances cryopreservability of buffalo semen.
authors.affiliation: Sabarmati Ashram Gaushala, Ahmedabad, Gujarat, India.
authors.forename: Hardik A
authors.initials: HA
authors.lastname: Patel
authors.affiliation: Department of Gynaecology and Obstetrics, College of Veterinary Sceicne and Animal Husbandry, Sardarkrushinagar Dantiwada Agricultural University, Deesa, Banaskanth, Gujarat, India.
authors.forename: G M
authors.initials: GM
authors.lastname: Siddiquee
authors.affiliation: Pashupalan Sansodhan Kendra, Veterinary College, Anand Agricultural University, Anand, Gujarat, India.
authors.forename: Dinesh V
authors.initials: DV
authors.lastname: Chaudhari
authors.affiliation: Directorate of Research, Kamdhenu University, Gandhinagar, Gujarat, India.
authors.forename: Vishal S
authors.initials: VS
authors.lastname: Suthar
keywords: antioxidants; cryopreservation; diluent; egg yolk; enzyme; semen