Effect of cryoprotectants and cooling rates on fertility potential of sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man)

• Effect of cryoprotectants on sperm quality of M. rosenbergii on cryopreservation.
• One-step freezing at cooling rate of −1.5 °C/min between 27 °C and −39 °C before LN2.
• Development of freezing protocol with pond water containing DMSO 10% + PG 10%.
• Comparison of sperm acrosome reaction in fresh and cryopreserved spermatophores.

This study evaluates freezing protocol with suitable cryoprotectants and their effects on the fertility potential of sperm in the cryopreserved spermatophores of Macrobrachium rosenbergii. Spermatophores, collected using electroejaculation, were suspended in dimethyl sulfoxide (DMSO), propylene glycol (PG), methanol, glycerol and ethylene glycol (EG) at different concentrations (10, 15 & 20% v/v), prepared in sterile-filtered pond water. Based on the cryoprotectant toxicity assay, DMSO and PG were used individually as well as in combination with three freezing protocols (i.e. −1.5, −3 and −5 °C/min and to final temperature of −39 °C) and plunged into liquid nitrogen at −196 °C. After 90 days of storage (−196 °C) thawing was done at 35 °C in a water bath for 1 min. Results showed that fresh and cryopreserved spermatophores held for 90 days registered sperm viability of 91.4 ± 2.9% and 50.4 ± 1.9% respectively. Further, fertility potential of sperm was assessed based on acrosome reactivity using calcium ionophore (A23187). Observations indicated that cryopreserved sperm registered 28.3 ± 2.2% of acrosome reactivity compared to freshly collected spermatophores (85.3 ± 2.5%). Thus, one-step slow cooling rate of −1.5 °C/min between 27 °C and −39 °C stored in liquid nitrogen at −196 °C with DMSO (10%) + PG (10%) seems to be amenable for cryopreservation of spermatophores, compared to other cooling rates.