Cryopreservation of Penaeid Shrimp Embryos: Development of a Germplasm Cryo-Bank for Preservation of High Health and Genetically Improved Stocks

The objectives of the project were to develop a successful protocol for cryopreservation of penaeid germ plasm in order to preserve a pathogen-free broodstock nucleus for commercial exploitation of marine shrimp in aquaculture. The critical parameters to be characterized in the project were: 1. Determination of chill sensitivity and chill tolerant embryonic stages, including a full description and time course study of embryonic developmental stages. 2. Development of protocols for loading and removal of cryoprotectant agents (CPAs) from embryos; determination of optimal concentrations and duration of loading. 3. Characterization of the toxicity of the selected CP As and 4. Establishing optimal cooling and thawing procedures. Studies were performed on two penaeid species: Litopenaeus vannamei (in the USA) and P. semisulcatus (in Israel). The effect of incubation temperature on embryonic development rate and hatching success was studied in L. vannamei, showing that spawns maybe maintained at temperatures ranging from 24°C to 30°C, without compromising hatchability. Embryonic development extends from 12 hr to 19 hr at 30°C and 24°C, respectively. Studies showed that advanced embryonic developmental stages were chill tolerant in the two studied species, but P. semisulcatus could better endure lower temperatures than L. vannamei. A large number of experiments were performed to determine the optimal CP As, their concentration and duration of loading. Permeating (e.g. glycerol, methanol, DMSO, 1,2- propanediol, ethylene glycol, glucose) and non-permeating CPAs (sucrose, PVP, polyethylene glycol) were tested and several combinations of permeating and non-permeating CP As, on fertilized eggs (embryos), nauplii and protozoeae. In general, nauplii tolerated higher CPA concentrations than eggs and nauplii were also more permeable to radiolabeled methanol. Chlorine treatment intended to remove the chitinous envelop from eggs, did not increase dramatically the permeation of radiolabled methanol into eggs. Cooling eggs, nauplii or protozoeae to cryogenic temperatures, by either vitrification or slow cooling protocols, did not result in full survival of thawed samples, despite exhaustive attempts testing various protocols and CP As. Results seemed more encouraging in freezing of nauplii in comparison to eggs or protozoeae. Successful preliminary results in cryopreservation of spermatozoa of P. vannamei, will facilitate preservation of genetic specific to some extent.