Cryoprotective Effects of Fulvic Acid on Buffalo Bull Spermatozoa: A Concentration-Dependent Response

The present study was aimed to evaluate the effect of fulvic acid supplementation in Tris-fructose-yolk-glycerol (TFYG) extender on sperm quality attributes of buffalo bulls at successive stages of cryopreservation. Semen ejaculates (n=12) collected from healthy breeding bulls using an artificial vagina were split into four aliquots and extended with TFYG containing 0.00 (control), 0.50, 1.00 and 1.50 % (w/v) fulvic acid. The samples were processed, equilibrated and cryopreserved. Progressive sperm motility was evaluated at the post-dilution, pre-freeze and post-thaw stages, while viability, morphological abnormalities, plasma membrane integrity and acrosome integrity were assessed at the post-dilution and post-thaw stages. The response to fulvic acid was distinctly biphasic. Post-thaw sperm motility and plasma membrane integrity were significantly (p<0.01) higher with 1.50 and 1.00 % fulvic acid, and viability with 1.50 %, compared to the control, whereas 0.50 % fulvic acid consistently reduced sperm motility, viability and plasma membrane integrity below the control, the reduction being statistically significant for viability (p<0.05). The extender with 1.50 % fulvic acid also maintained significantly better acrosome integrity (p<0.05) and lower sperm abnormalities than the lower concentrations and control. The findings indicate that fulvic acid confers cryoprotection above a threshold concentration, below which it exerts the opposite effect, and that 1.50 % (w/v) is the most effective inclusion level for buffalo semen cryopreservation