| Coreius guichenoti,belonging to Cytcriniformes,Cyprinidae,Gotiongiae and Coreius,is a migratory fish habitually inhabited by turbulent rivers.It often appears in groups in deep pools with numerous rocks and reefs.It was once the dominant species and important economic fish in the upper valley of the Yangtze River in China.However,in recent years,due to the construction of hydropower facilities and ecological environment changes and overfishing by human beings in the Yangtze River basin have destroyed the living environment of Coreius guichenoti,the amount of resources has declined rapiddly,and the proportion of young and middle-aged fish in the catches has increased year by year.It has been listed as an indicator species of rare and endemic fish reserves in the upper valley of the Yangtze River,and it's protection problems need to be solved urgently.Artificial domestication and large-scale breeding of parent fish are the sufficient conditions for stock enhancement which is key important for the protection of rare fish.At present,artificial domestication and the key technical problems of reproduction of Coreius guichenoti has been overcomed.However,many problems restrict the largescale breeding of Coreius guichenoti,such as the difficulty of catching parent fish,small number of breeding parent fish,asynchrony of male,female gamete development,and the small amount of male semen.The purpose of this study was to explore the factors affecting sperm viability and the suitable methods for sperm preservation of Coreius guichenoti,in order to provide a basis data for artificial breeding and cryopreservation of Coreius guichenoti,through the study of sperm viability under different osmotic pressure and storage at 4?cryopreservation and cryopreservation under different antifreeze agents.The main results are as follows:1 In order to investigate the relationship between sperm motility and osmotic pressure,The osmolality of Coreius guichenoti semen and the activity of spermatozoa in 7 osmotic pressure gradients(0?50?100?150?200?250?300 mOsm/kg)in two solutions(NaCl and KCl)were studied.The results showed that the average osmotic pressure of spermatozoa was 252.48±4.20 mOsm/kg;within 0 ~ 150 mOsm/kg osmotic pressure gradient.The fast movement time and lifetime of spermatozoa were prolonged with the increase of osmotic pressure.The fast movement time and lifetime were 35.2 s?30.8 s and 103.13 s?105.13 s,respectively,and within the osmotic pressure gradient of 150 mOsm/kg NaCl and KCl solution.Within the osmotic pressure gradient of 150-300 mOsm/kg,the sperm fast movement time and lifetime of Coreius guichenoti spermatozoa were shortened gradually,even 0.The results of this study can provide reference for semen preservation techniques.2.Results of experiments on the Coreius guichenoti sperm storage at 4?.When sperm was separately stored in modified Kurokura1,D15,L1,L2,L3,L4 six dilutions respectively,the results indicated that the sperm viability,lifetime and movement speed decreased with the prolongation of storage time.Fresh sperm without any treatment survived almost without sperm only after 72 hours of storage.The best preservation was D15 dilution,when the preservation time was 72 hours,the sperm viability rate was(91.65±6.77)%,and the rapid movement time and lifetime were(10.00±1.67)s and(41.33±7.42)s,respectively.The sperm viability could still reach at(75.28±42.56)% after 120 hours of observation.3.In order to establish Coreius guichenoti sperm cryopreservation method,a computer assisted sperm motion analysis system(CASA)was used to find a suitable sperm cryopreservation method by comparing the effects of four extenders,(D15,D20,L1 and D1),and three cryoprotectants including dimethylformamide(DMF),dimethyl sulfoxide(DMSO),methanol(METH)at three concentrations(7.5%,10% and 12.5%,V/V)on sperm motility after thawing.In this study,the best cryopreservation protocol for Coreius guichenoti sperm was 10% METH as antifreeze protective agent in D1,and showed no significant difference compared to fresh sperm group(P>0.05).Following the two-step method to cool the mixture and thawing the frozen semen in 37 ? water bath for 12~15 s,motility of the sperm reached(78.11 ±14.74 %).The 10% METH group performed best in average velocity(50.28± 12.46 ?m/s),average linear velocity(35.06±10.82 ?m/s)and average path velocity(39.44±12.46 ?m/s),which showed significant differences compared to fresh sperm group(P<0.05).The fast movement time and lifetime of sperm were 8.67±1.15 s,33.3±5 s,respectively.Therefore,10% METH was the best cryoprotectant for Coreius guichenoti sperm and could be applied in practice. |
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