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Studies on Preservation in Vitro of Spermatozoa and Embryos of Exopalaemon Carinicauda

Author: PuYunHui
Tutor: YanBinLun;LiuZhaoPu
School: Nanjing Agricultural College
Course: Marine biology
Keywords: Exopalaemon carinicauda Spermatozoa Embryo In vitro preservation
CLC: S966.12
Type: Master's thesis
Year: 2013
Downloads: 5
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In recent years, the development of cryobiology has been more and more rapid. As one of the most important economic shrimps, there are few studies on spermatozoa and embryo in Exopalaemon carinicauda. Therefore, we make some relative researches on in vitro preservation of spermatozoa and embryo, select the suitable conditions of the spermatozoa and embryos of cryopreservation (-196℃). The study provides theoretical guidance to cryopreservation of spermatozoa, as well as provide a reference for its metabolism and physiological study. Embryos in vitro preservation can momentarily provide materials for research on breeding and production.These researches have important significance for germplasm, optimization, gynogenesis, establishing good germplasm.when the salinity in20-25%o and pH at8.0, the sperm survival rate and acrosome enzyme activity are high. The quality of spermatozoa is detected using survival rate and acrosin activity, researching on the effect of cryopreservation of spermatozoa based on different dilution solutions, and the effect of cryopreservation of spermatozoa based on different cryoprotectants. The result shows that Ringer’s extender is the best dilution solution of spermatozoa in vitro preservation. Under this condition, after preservation for4d at4℃, survival rate is71.50%, and acrosin activity reduce to1.80μIU/106. The two kinds of index values are higher than the other experimental group. In each group of dilution solution, survival rate significantly correlates with acrosin activity,7=0.846(p<0.05); After preservation for2d in liquid nitrogen, under the condition of Ringer’s extender, survival rate is83.50%, acrosin activity reduces to2.51μIU/10. The two kinds of index values are higher than the other experimental group as well as at4℃, the condition of diluting solution I (Ringer’s extender). There exists a highly significant correlation between survival rate and acrosin activity in each group, y=0.862(p<0.01).15%DMSO is the best cryoprotectant for cryopreservation. Under this condition,2d after preservation, survival rate is81.32%, and acrosin activity reduces to1.39μIU/106. There exists a highly significant correlation between survival rate and acrosin activity in each group, y=0.864(p<0.01).The research studies the effects of cryopreservation (-196℃) on the enzyme activity (SDH, LDH, Na+/K+-ATP, SOD, CAT, GR and Acrosin) in Exopalaemon carinicauda spermatozoa, in order to provide a theoretical basis for cryopreservation effect on Exopalaemon carinicauda spermatozoa, and lay a foundation for artificial propagation of Exopalaemon carinicauda.Set a control group, and three experimental groups, detect the enzyme activities on day0,1,3,5,7,15. The result showed that, after cryopreservation, in addition to GR,the other enzyme activity decrease significantly (p<0.05), and the biggest drop in the control group. GR activity significantly increases in7days, and the control group is significantly higher than the experimental group (p<0.05), declining again in15days. The group of15%DMSO measured enzyme activities are higher than the other groups, indicating that15%DMSO has protective effect on enzyme of spermatozoa. From the decline range to compare, cryopreservation has the biggest influence on SOD, followed by Na+/K+-ATPase.Inspecting the DNA damage level of Exopalaemon carinicauda of spermatozoa after cryopreservation by using the single cell gel electrophoresis assay (SCGE) method. After cryopreservation, the spermatozoas are damaged to different extents. The comet rate of fresh spermatozoa is6.67%. After cryopreservation, the comet rate of control group is significantly different with experimental groups (p<0.05), the comet rate is40.00%. The result show that the cryoprotectant is necessary to the cryopreservation. Under the condition of15%DMSO, the damage of spermatozoas’ DNA is the lowest, lower than other concentrations. The study provides a scientific basis for further study on cryopreservation of Exopalaemon carinicauda of spermatozoa.In the research of embryo’s in vitro preservation of Exopalaemon carinicauda, the optimum conditions are:pH7.0, salinities20-25%o, and the survival rate being the highest. Among selected seven diluting solutions, BS2solution is the best one. Choose DMSO, glycerol,1,2-propylene glycol as cryoprotectant, the toxicity to embryo in descending order is1,2-propylene glycol, DMSO, glycerol, and survival rate increases in concentration of5%-10%, reduces in10%-20%.10%glycerol is the best cryoprotectant for cryopreservation, the survival rate is78.94%after10days. Each of the components in embryos is significantly changed after cryopreservation when researching effects on cryopreservation of Exopalaemon carinicauda. There are29kinds of main fatty acids in embryos, the carbon chain length of is mainly14to24, there are9kinds of saturated fatty acids (SFAs), accounting for37.948%, eight kinds of monounsaturated fatty acids (MUFAs), accounting for30.091%, nine kinds of polyunsaturated fatty acids (PUFAs), accounting for30.763%. After cryopreservation, saturated fatty acids of C15:0and C17:0decrease, showing a significantly difference (p<0.05) compared with the control group. Meanwhile, relative contents of C16:0and C18:0have a significant increase in the fresh embryos and significantly different with the experimental group. Monounsaturated fatty acid of C16:1in experimental group increases significantly after cryopreservation, decreases significantly in the experimental group1,2, showing a significantly difference with fresh embryos and control group (p<0,05). C18:ln9c in embryos increases significantly with significant differences among the groups (p<0.05). After crypreservation there is a significant difference(p<0.05) of polyunsaturated fatty acids of C18:2n6c among groups. The content of DHA decreases significantly and show significant differences (p<0.05), while EPA decreases significantly after crypreservation. Cn-3/Cn-6is on a downward trend. The result shows that the groups with cryoprotectant and the group without cryoprotectant are significantly different.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquaculture technology > Other aquaculture > Crustacean culture > Freshwater shrimp farming
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