Cryopreservation of Spermatophores and Seminal Plasma of the Edible Crab Scylla serrata

¹ Unit of Invertebrate Reproduction, Department of Zoology, University of Madras, Guindy Campus, Madras 600 025, India

This paper describes the development of a suitable biotechnology to cryopreserve the spermatophores of the edible crab Scylla serrata in a viable condition. Three temperatures (–4°C, –79°C, and –196°C) were chosen to preserve the spermatophores and seminal plasma, collected from the middle region of vas deferens of mature male crabs, for 30 days. Sperm viability was determined by the eosin-nigrosin dye exclusion method, as applied to entire spermatophores. Of the three temperatures tested, the maximum percentage of sperm viability was obtained at –196°C, whereas it significantly decreased at –4°C. Biochemical alterations of the major substrates such as proteins, carbohydrates, and lipids, as well as the enzyme Lactate Dehydrogenase (LDH) occurred only at –4°C, reflecting their use in the metabolic activities of the spermatozoa contained within the spermatophores. At –4°C, the TCA-soluble total free sugars increased in correspondence with a decline in the bound sugars, suggesting that the latter may be used rapidly during sperm storage. Our data also suggest that, at –79°C and –196°C, the frozen spermatozoa retain viability but do not exhibit metabolic activity.

To investigate the role of cryoprotectants in preventing damage to the sperm cells/spermatophores during cryopreservation, four cryoprotectants, glycerol, dimethyl sulfoxide (DMSO), trehalose, and DMSO + trehalose combination, were tested. Using the phosphate buffer as the standard diluent, glycerol gave the best result. When used alone, trehalose gave only a low sperm survival, but in combination with DMSO, it gave an increased viability that equalled the result with glycerol. We recommend that glycerol is the best cryoprotectant inasmuch as the biochemical alterations in the glycerol medium is less, compared to that of DMSO + trehalose. When used alone, DMSO may be more toxic to the sperm cells as it gave a low viability value even at –196°C and –79°C.