THEOBROMINE AS AN EFFECTIVE PREVENTION AGAINST CRYODAMAGE OF BOVINE SPERMATOZOA

This research examined a possible protective effects of three selected theobromine (TBR) doses (12.5, 25 and 50 μM) on the cryopreserved bovine spermatozoa following sperm motility, oxidative profile and the expression patterns of heat shock proteins (HSPs) 70 and 90 as well as apoptosis-associated BAX (pro-apoptotic Bcl2-associated X) and Bcl-2 (anti-apoptotic B-cell lymphoma 2) proteins. Cryopreserved samples supplemented with TBR were compared with a native control (CtrlN) and a cryopreserved control (CtrlC) without TBR treatment. Our results demonstrate that the administration with TBR led to a significant improvement of sperm progressive motility (p<0.0001) when compared to CtrlC. The highest levels of protection for sperm DNA (p<0.01), lipids (p<0.0001) and proteins (p<0.001) were detected in samples treated with 25 and 50 μM of TBR against CtrlC. Administration of 25 and 50 μM of TBR significantly preserve HSP90 protein expression (p<0.0001) while HSP70 protein expression stay without changes when compared with the unsupplemented frozen control. No significant differences were observed in the expression patterns of Bcl-2 protein, however a significant decline of BAX protein was observed in the frozen samples enriched with 25 (p<0.05) and 50 (p<0.001) μM of TBR after comparison with the CtrlC. In summary, we may consider that TBR can be effective agent in stabilizing the sperm membranes by preventing reactive oxygen species (ROS) induced lipid peroxidation, protein oxidation and subsequent oxidative damage to molecules critical for a proper sperm function. These protective properties of TBR may lead to higher post-thaw quality and viability of bovine spermatozoa.