Optimization of Novel Cryoprotectant Combinations (DMSO + Trehalose + Melatonin) for Rohu (Labeo rohita) Sperm Cryopreservation: Assessment of Motility, Oxidative Stress, DNA Integrity and Reproductive Performance using RSM

Background: The cryoinjury of carp sperm leads to membrane damage and lipid peroxidation, and impairs the fertilizing ability of the sperm. Objective: This research demonstrated multi-response optimization of DMSO, trehalose, and melatonin for Rohu (Labeo rohita) sperm cryopreservation. Methods: A 17-run, three-factor Box-Behnken design was carried out using DMSO (6-10% v/v), trehalose (50-150 mM), and melatonin (0-2000 µM). Seven bio-plausible response series were computationally generated: sperm motility and viability; level of malondialdehyde (MDA) and superoxide dismutase (SOD); DNA fragmentation; and fertilization and hatching. Results: The predicted R² for sperm motility was 0.997 and the optimum, based on the model, was 8.63% (v/v) DMSO, 129.9 mM trehalose, and 1489 µM melatonin, with expected motility 66.6%, DNA fragmentation 10.2%, fertilization 62.7%, hatching 54.7% and overall desirability 0.825.Conclusion: This approach predicted candidate formulations but did not prove biological efficacy. Separate cryopreservation and hatchery trials are necessary.