Optimization of Extender–cryoprotectant Combinations for Sperm Cryopreservation of Rohu Labeo rohita (Hamilton, 1822)
S. Gokul *
Department of Aquaculture, College of Fisheries, Acharya Narendra Deva University of Agriculture & Technology (ANDUA&T), Kumarganj, Ayodhya, Uttar Pradesh – 224 229, India.
Dinesh Kumar
Department of Aquaculture, College of Fisheries, Acharya Narendra Deva University of Agriculture & Technology (ANDUA&T), Kumarganj, Ayodhya, Uttar Pradesh – 224 229, India.
Santosh Kumar
ICAR – National Bureau of Fish Genetic Resources (NBFGR), Canal Ring Road, P.O. Dilkusha, Lucknow - 226 002, India.
Omdevsinh Wala
Department of Aquaculture, College of Fisheries, Kerala University of Fisheries and Ocean Studies (KUFOS), Panangad, Kochi, Kerala – 682 506, India.
Irtiqa Hamdani
Department of Aquaculture, College of Fisheries, Kerala University of Fisheries and Ocean Studies (KUFOS), Panangad, Kochi, Kerala – 682 506, India.
Shashank Singh
Department of Aquaculture, College of Fisheries, Acharya Narendra Deva University of Agriculture & Technology (ANDUA&T), Kumarganj, Ayodhya, Uttar Pradesh – 224 229, India.
*Author to whom correspondence should be addressed.
Abstract
Sperm cryopreservation is an essential reproductive biotechnology for the long-term conservation of genetic resources and the year-round production of quality seed in aquaculture. The present study aimed to optimise suitable combinations of extenders (7, 9B, and 9C) and cryoprotectants [dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and methanol] for the cryopreservation of rohu (Labeo rohita) sperm. Fresh milt exhibited excellent quality, with sperm concentrations ranging from 9.58 × 10¹⁰ to 1.68 × 10¹¹ cells mL⁻¹ in Lot 1 and from 1.03 × 10¹¹ to 2.25 × 10¹¹ cells mL⁻¹ in Lot 2, and motility ranging from 90–95%. Milt samples were diluted at a ratio of 1:6 (milt:diluent) using different extender–cryoprotectant combinations and cryopreserved in liquid nitrogen (−196°C). Post-thaw fertilisation and hatching trials were conducted to evaluate sperm functionality. Extender 9C produced the highest hatching percentage among the extenders (51.08%), while DMSO produced the highest hatching percentage among the cryoprotectants (51.26%); both values remained lower than the fresh-milt control (79.00%). The results indicate that extender 9C and DMSO were the best-performing extender and cryoprotectant, respectively, under the evaluated conditions. This optimised protocol has considerable potential for large-scale hatchery operations, selective breeding programmes, and long-term germplasm conservation of L. rohita.
Keywords: Labeo rohita, sperm cryopreservation, extender optimization, cryoprotectant, hatching rate, aquaculture biotechnology.