Long‐term cryopreservation of the giant freshwater prawn, Macrobrachium rosenbergii, spermatophores using glycerol (Gly) and ethylene glycol (EG) as cryoprotective agents (CPAs) was studied. The tolerance of sperm to cryopreservation was evaluated on the basis of sperm survival and fertilizing ability. The survival of the sperm was determined by trypan blue staining, while the fertilizing ability was assessed from artificial insemination of the cryopreserved spermatophores. The rates of embryo survival on day 5 after spawning and of spermatophores capable of producing embryos survived to hatching were determined. Storage of spermatophores at −20°C without CPA for a short period of up of 1–5 days decreased the sperm survival significantly and did not preserve fertilizing ability. Preservation at −20°C in the presence of 10% or 20% Gly or of 10% or 20% EG offered a simple and efficient short‐term storage up to 10 days. For a long‐term storage, cryopreservation in the presence of 20% EG at −196°C was more efficient than at −20°C. High sperm survival rates and high fertilizing ability were recorded from those cryopreserved at −196°C for up to 150 days. High sperm survival rates with moderate levels of fertilizing ability were obtained from those cryopreserved at −20°C for not more than 30 days. The results indicate that preservation at −196°C with 20% EG is a suitable procedure for long‐term storage of the giant freshwater prawn spermatophores.

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