title: Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender.
abstract: In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.
authors: Reproductive Biochemistry Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal, Haryana, India.
authors: V K
authors: VK
authors: Singh
authors: S K
authors: SK
authors: Atreja
authors: R
authors: R
authors: Kumar
authors: S
authors: S
authors: Chhillar
authors: A K
authors: AK
authors: Singh
mesh_terms: D000818:Animals; D002020:Buffaloes; D002118:Calcium / Q000032:analysis*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D003451:Cryoprotective Agents / Q000008:administration & dosage*; D000242:Cyclic AMP / Q000032:analysis*; D004075:Diglycerides / Q000032:analysis*; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary; D013081:Sperm Motility; D013094:Spermatozoa / Q000737:chemistry* / Q000502:physiology; D013654:Taurine / Q000008:administration & dosage; D014199:Trehalose / Q000008:administration & dosage
chemical_list: C011439:1,2-diacylglycerol; D003451:Cryoprotective Agents; D004075:Diglycerides; D013654:Taurine; D014199:Trehalose; D000242:Cyclic AMP; D002118:Calcium