This study reports the first evaluation of sperm hyaluronan binding assay (HBA) for predicting the fertility of Nili-Ravi buffalo bulls in relation to standard parameters of sperm quality. Cryopreserved semen doses of low (
n = 6), medium (
n = 3) and high fertility (
n = 8) bulls based on their respective return rates were used. Significantly, more spermatozoa bound to hyaluronan from the most fertile bulls (57.15% ± 1.44) compared with medium (42.46% ± 1.08) and low fertility bulls (29.70% ± 0.78). A strongly positive correlation (
r = .824,
p < .01) was found between HBA and fertility that predicts a 67.9% variability (
r2 = .679,
p < .01) in fertility. HBA was also strongly positively correlated with sperm viability (
r = .679,
p < .01) followed by their live/dead ratio (
r = .637,
p < .01), uncapacitated spermatozoa (
r = .631,
p < .01), normal apical ridge (
r = .459,
p < .01), motility (
r = .434,
p < .01), mature spermatozoa with low residual histones (
r = .364,
p < .01), high plasma membrane integrity (
r = .316,
p < .01) and nonfragmented DNA levels (
r = .236,
p < .05). It was negatively correlated with spermatozoa having reacted acrosome (
r = −.654,
p < .01). A fertility model built using a combination of sperm HBA and either sperm livability or viability predicts, respectively, 86.1% (
r2 = .861,
p < .01) and 85.9% (
r2 = .859,
p < .01) variability in buffalo bull fertility. In conclusion, sperm HBA may prove to be a single robust predictor of Nili-Ravi buffalo bull fertility.
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