Abstract 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.

REFERENCES

[1]. Alevra, A. I., Exadactylos, A., Mente, E., & Papadopoulos, S. (2022). The protective role of melatonin in sperm cryopreservation of farm animals and human: Lessons for male fish cryopreservation. Animals, 12(6), 791. https://doi.org/10.3390/ani12060791

[2]. Aramli, M. S., Moghanlou, K. S., & Anzabi, M. P. (2024). A brief review of the methodology and cryoprotectants in selected fish and mammalian species. Reproduction in Domestic Animals, 59(5), e14575. https://doi.org/10.1111/rda.14575

[3]. Bibi, S., Awan, M. A., Rakha, B. A., Ejaz, R., Qadeer, S., Arshad, J., Anjum, M. Z., & Akhter, S. (2023). Evaluation of carboxylated poly-l-lysine for cryopreservation of Labeo rohita sperm. Reproduction in Domestic Animals, 58(7), 990– 996. https://doi.org/10.1111/rda.14391

[4]. Chelewani, A. P., Takahashi, E., Nishimura, T., & Fujimoto, T. (2024). Optimizing the post-thaw quality of cryopreserved masu salmon (Oncorhynchus masou) sperm: Evaluating the effects of antioxidant-supplemented extender. Aquaculture, 593, 741332. https://doi.org/10.1016/j.aquaculture.2024.741332

[5]. da Costa, B. B., Lassen, P. G., & Streit, D. P., Jr. (2024). Cryopreservation-induced morphological changes in freshwater fish sperm: A systematic review. Biopreservation and Biobanking, 22(5), 416–427. https://doi.org/10.1089/bio.2023.0008

[6]. Gholami, D., Sharafi, M., Esmaeili, V., Nadri, T., Alaei, L., Riazi, G., & Shahverdi, A. (2023). Beneficial effects of trehalose and gentiobiose on human sperm cryopreservation. PLOS ONE, 18(4), e0271210. https://doi.org/10.1371/journal.pone.0271210

[7]. Jia, B., Memon, S., Liang, J., Lv, C., Hong, Q., Wu, G., & Quan, G. (2021). Trehalose modifies the protein profile of ram spermatozoa during cryopreservation. Theriogenology, 171, 21–29. https://doi.org/10.1016/j.theriogenology.2021.05.004

[8]. Kumar, S., Kumar, A., Sah, R. S., Singh, A. K., Yadav, A., Verma, A., Negi, D., Tamut, H., & Lal, K. K. (2023). Optimisation of cryovials and milt dilution ratios to upscale carp sperm cryopreservation for use in hatchery seed production. Indian Journal of Fisheries, 70(3), 90–97. https://doi.org/10.21077/ijf.2023.70.3.134401-12

[9]. Motta, N. C., Egger, R. C., Monteiro, K. S., Oliveira, A. V., & Murgas, L. D. S. (2022). Effects of melatonin supplementation on the quality of cryopreserved sperm in the neotropical fish Prochilodus lineatus. Theriogenology, 179, 14–21. https://doi.org/10.1016/j.theriogenology.2021.11.012

[10]. Neyrão, I. M., Santos, F. B. P., Rodrigues, R. B., Streit, D. P., Jr., & Godoy, L. (2024). Use of powdered milk in semen cryopreservation protocols for fish: A systematic review. Biopreservation and Biobanking, 22(1), 4–20. https://doi.org/10.1089/bio.2022.0091

[11]. Palhares, P. C., Assis, I. L., Machado, G. J., Freitas, R. M. P., Freitas, M. B. D., Paula, D. A. J., Carneiro, W. F., Motta, N. C., & Murgas, L. D. S. (2021). Sperm characteristics, peroxidation lipid and antioxidant enzyme activity changes in milt of Brycon orbignyanus cryopreserved with melatonin in different freezing curves. Theriogenology, 176, 18–25. https://doi.org/10.1016/j.theriogenology.2021.09.013

[12]. Rezaei, A., Bahmani, H. R., Mafakheri, S., Farshad, A., Nazari, P., & Masoudi, R. (2023). Protective effects of different doses of MitoQ separately and combined with trehalose on oxidative stress and sperm function of cryopreserved Markhoz goat semen. Cryobiology, 110, 36–43. https://doi.org/10.1016/j.cryobiol.2022.12.019

[13]. Sandoval-Vargas, L., Silva Jiménez, M., Risopatrón González, J., Villalobos, E. F., Cabrita, E., & Valdebenito Isler, I. (2021). Oxidative stress and use of antioxidants in fish semen cryopreservation. Reviews in Aquaculture, 13(1), 365–387. https://doi.org/10.1111/raq.12479

[14]. Seki, S., Yano, M., Higashiya, M., Oikawa, T., Yamazaki, W., & Yoshizaki, G. (2024). Vitrification of medaka whole testis with a trehalose-containing solution and production of medaka individuals derived from the vitrified cells. Cryobiology, 116, 104936. https://doi.org/10.1016/j.cryobiol.2024.104936

[15]. Shazada, N. E., Alavi, S. M. H., Siddique, M. A. M., Cheng, Y., Zhang, S., Rodina, M., Kocour, M., & Linhart, O. (2024). Short-term storage of sperm in common carp from laboratory research to commercial production—A review. Reviews in Aquaculture, 16(1), 174–189. https://doi.org/10.1111/raq.12827

[16]. Tamut, H., Singh, R., Kumar, S., Kumar, A., Gupta, M., Singh, A., Nishad, C. S., Sah, R. S., Singh, A. K., Kumar, A., & Lal, K. K. (2025). Semen characteristics and optimisation of sperm cryopreservation protocol of a commercially important minor carp Labeo gonius (Hamilton, 1822). Indian Journal of Fisheries, 72(1). https://doi.org/10.21077/ijf.2025.72.1.152786-15

[17]. Varela Junior, A. S., Jardim, R. D., Streit, D. P., Jr., Cardoso, T. F., Silva, E. F., Lucia, T., Jr., Figueiredo, M. R. C., & Corcini, C. D. (2022). Trehalose in extenders for cryopreservation of Tambaqui (Colossoma macropomum) sperm. CryoLetters, 43(5), 264–268. https://doi.org/10.54680/fr22510110312

[18]. Wood, K. R., Bosworth, B., Liyanage, S. S. N., Nowicki, A. E., Martin, K. A., Dunham, R. A., Beck, B. H., Abernathy, J. W., Wang, X., Roy, L. A., & Butts, I. A. E. (2025). Cryopreservation techniques for blue catfish, Ictalurus furcatus, sperm using 2.5 mL straws. Cryobiology, 121, 105335. https://doi.org/10.1016/j.cryobiol.2025.105335

[19]. Zhang, Y., Liu, D., Wang, Q., Ruan, Q., Hua, S., Zhang, W., Yang, S., & Meng, Z. (2024). Addition of cryoprotectant DMSO reduces damage to spermatozoa of yellow catfish (Pelteobagrus fulvidraco) during cryopreservation: Ultrastructural damage, oxidative damage and DNA damage. Animals, 14(18), 2652. https://doi.org/10.3390/ani14182652

[20]. Zidni, I., Lee, H. B., Yoon, J. H., Park, J. Y., Oh, Y. D., Jang, H. S., Cho, Y. S., Lee, I. Y., & Lim, H. K. (2022). Effect of antioxidants in cryopreservation media on spotted halibut (Verasfer variegatus) sperm quality during cryopreservation. Aquaculture, 557, 738351. https://doi.org/10.1016/j.aquaculture.2022.738351