Protocols have been developed for sperm cryopreservation of major carps more or less simultaneously at the National Bureau of Fish Genetic Resource (NBFGR) and The Central Institute of Freshwater Aquaculture (CIFA) in 1988. The optimum requirements have been developed in respect to extender, activation media, dilution rate, activation period, sperm and egg ratio difference between species. Stocks of L.rohita milt cryopreserved in 1988 are being maintained along with other species in a mini gene bank at NBFGR. Viable hatchlings have been produced from five-year old rohu cryopreserved milt (Ponniah, 1997).
The ionic composition of seminal and variation fluid of rohu and mrigal have been studied at the Bureau with the objective of providing basic information that can be used to fashion new extenders and spermatozoa motility inducing media. Gupta and Rath (1993) preserved the milt of C. catla, L. rohita and Cirrhinus mrigala under cryogenic condition for three years. The quality of milt samples were analysed when preserved and were expressed in terms of spermatocrit value and sperm cell count, which in catla, rohu and mrigal ranged from 65–75, 75–85 and 65–75 respectively while sperm cell counts ranged between 2–2.5 X 107, 3.0–3.25 × 107 and 2.0–2.5 X 107 respectively. The most effective diluent was found to be an electrolyte medium when Dimethyl Sulfoxide (DMSO) acted as cryoprotectant and the ratio of milt and diluent was 1:4. Quick Thawing procedure (38+2°C).
Gupta and Rath (1995) have also studied the efficacy of cryopreserved milt in fertilizing the eggs compared with normal milt. The survival and growth rates from larvae to fry and to fingerling produced from eggs fertilized with cryopreserved and fresh milt were not found statistically different.
The fertilization efficiency of cryopreserved spermatozoa thawed at different temperatures of catla, rohu and mrigal over a period of four years was assessed at different time intervals after thawing, (Gupta et al., 1995). Milt thawed at 39 + 1C and 30 + 1C had fertilization rates of 56.7% and 12.5% during 900 and 1500 seconds. The motility and fertility were reduced drastically at the next 60 seconds and 300 seconds.
Cryopreservation of embryos of Cyprinus carpio and L. rohita have been attempted (John et al., 1993). It has been reported that C. carpio at tail bud stage was more tolerant to cryoprotectants than morula stage as revealed by the hatching rates of treated embryos. This may be due to some ontogenic differences in the tolerance to handling, membrane permeability or osmoregulation potential, rather than cryoprotectant susceptibility. Rohu tail-bud embryos treated in methanol and DMSO (Cryoprotectents) in different concentrations and in combination, showed high hatching rates in all the treatments but no pattern was evident. When embryos from all treatments were kept immersed in liquid nitrogen for about 3 hours and thawed, no survival was observed. This was felt due to defective thawing (John et al., 1993).
Cryoprotectant toxicities have been also worked out in two embryonic stages of prawns viz. Penaeus monodon and P. indicus. Success has been achieved in obtaining viable larvae of P. indicus frozen to -40C and of P. monodon frozen to -30C. The frozen P. monodon larvae developed further without any abnormalities. Also, safe levels of cryoprotectants have been worked out for the embryos of L. rohita and common carp. Further research to develop, vitrification and cryopreservation of carp embryos is in progress (Ponniah, 1997).
The development of effective protocols for cryopreservation of sperm and embryos will go a long way in support of sustainability in aquaculture. It will also facilitate the establishment of a national facility for gene banking of fish gametes, embryos and DNA material. The existence of such gene bank facilities will be also highly helpful for the obvious reasons that it reduces the maintenance problems and costs involved in raising brood fish in in situ condition, provides a wider choice of genetic material for selective breeding, and enables the process of dissemination of genetically improved varieties of fish with ease. Conservation of germplasm of endangered and wild populations would become easier. As mentioned earlier the mini gene bank at NBFGR can be upgraded into a National Fish Germplasm Bank for wild and endangered species. Similar gene banking facilities can be also developed at CIFA for preservation of genetically improved carp and other domesticated culture species.
