<![CDATA[Cryopreservation is a crucial technique for preserving fish spermatozoa; however, its success heavily relies on the efficacy of extenders in mitigating cellular damage during the freeze-thaw process. Honey, rich in antioxidants and capable of modulating extracellular osmotic pressure, presents a promising natural cryoprotectant. This study aimed to determine the optimal concentration of honey as a natural extender for the cryopreservation of kissing gourami (Helostoma temminckii) spermatozoa. The experimental study utilized a Completely Randomized Design (CRD) with three biological replicates to evaluate extender formulations by combining honey and Ringer's lactate solution at concentrations of 0% (P0), 0.25% (P1), 0.5% (P2), and 0.75% (P3) honey, supplemented with 10% dimethyl sulfoxide (DMSO) as an intracellular cryoprotectant and 10% egg yolk. Initial evaluations of fresh sperm demonstrated an optimal physiological baseline: a volume of 0.6 mL, pH 7.0, milky white coloration, a concentration of 1.11 × 10⁹ cells mL⁻¹, a mass movement score of 5, and 93.2% viability. After 14 days of cryopreservation in liquid nitrogen (-196°C), Analysis of Variance (ANOVA), followed by the Least Significant Difference (LSD) test, revealed that the addition of 0.5% honey (P2) exhibited the most effective and statistically significant (p<0.05) protective efficacy. This optimal dose successfully maintained a mass movement score of 4, prolonged motility duration to 112 seconds, and preserved the highest spermatozoa viability at 76.18%. In conclusion, supplementing with 0.5% honey serves as an effective natural extender as it significantly minimizes cold-shock damage while preserving the physiological viability and motility dynamics of H. temminckii spermatozoa during low-temperature storage.]]>
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