CRYOPRESERVATI ON OF ERIOCHEIR SINENSIS SPER M S (IN VITRO)W ITH D IFFERENT CRYOPROTECTIVE SOLUTI ONS AND PREFREEZING TIM E

As for genetic breeding and conservation of biotic germplasm resources, the cryopreservation of sperms serves asan effective protection. However, the process of cryopreservation and cryoprotective solutions both enable to cause damageof sperms to some extent, includingDNA damage of sperms. Anyhow, serving as vectors of genetic information, the integrityof DNA is necessary for the normal development of offspring. Therefore, the extent of DNA damage caused during cryopr2eservation can be used as an important index to evaluate the cryopreservative effects. In this study, sperms of Eriocheirsinensis cryopreserved in glycerol and dimethyl sulfoxide (DMSO) (in vitro) were detected for the cryopreservation effectswith different cryoprotective solutions and prefreezing times. Viability rate and DNA damage of sperms were betaken asevaluation indices. Sperms of Eriocheir sinensiswere acquired by trypsinase digestion and cryopreserved in the liquefied ni2trogen for over 8 hourswith sperm density at 107sperm/mL. Meanwhile, viability rate of spermswas assessed by eosin stainand DNA damagewas detected by single cell gel electrophoresis(SCGE). Ten experimental groups and three control groupswere set. Those ten groupswere four glycerol and DMSO scalar solutions(for each) (5%, 10%, 1215%, 15%) and twocomposite solutions with equivalent glycerol and DMSO (5%, 10%). Results revealed that cryoprotective solution with1215% glycerolwas optimum for cryopreservation of Eriocheir sinensis sperms(in vitro), where viability rate reached to62160%. Furthermore, cryoprotective solution with 10% glycerolwas used to study its cryopreservation effectswith differ2ent prefreezing times and five time gradients(5, 10, 20, 30 and 40 min) were set. It revealed that prefreezing time had aconspicuous influence on cryopreservation effects, i. e. sperms were thorough dead with overwhelming DNA damage afterless than 20 minπprefreezing, however, viability rate of sperms ascend obviously with DNA damage decreasing distinctlywhen prefreezing time was over 30min.