Assessment of Intracellular Ca2+, cAMP and 1,2‐Diacylglycerol in Cryopreserved Buffalo (Bubalus bubalis) Spermatozoa on Supplementation of Taurine and Trehalose in the Extender
2012
Singh, VK | Atreja, SK | Kumar, R. | Chhillar, S | Singh, AK
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 A2 (PLA2) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2‐diacylglycerol (DAG) and IP3, 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.
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