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Cryopreservation and Damage Research of Sperm of Cobia (Rachycentron Canadum) and Crimson Snapper (Lutjanus Erythopterus)

Author: DengPuEn
Tutor: LiuChuWu
School: Guangdong Ocean University
Course: Marine biology
Keywords: Sperm Ultra-low temperature freezer Artificial insemination Single cell gel electrophoresis Hypotonic swelling technique
CLC: S961.2
Type: Master's thesis
Year: 2011
Downloads: 22
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Abstract


The marine aquaculture industry has being developed rapidly in recent years. However, the degradation of germplasm resources has become more and more serious, in that case, germplasm conservation is particularly important. As a method of saving germplasm cells, sperm cryopreservation technology should play its proper role. Because of species and differences exist between individuals sperm frozen after the survival rate, fish sperm cryopreservation research is mainly in the experimental stage, and it is difficult to find a universal sperm cryopreservation way. At the meanwhile, freezing damage mechanism has not been known very much. In this study, we researched the sperm cryopopreservation and damage about Cobia ( Rachycentron canadum ) and crimson snapper ( Lutjanus Lutjanus erythopterus ) , its main contents summary is as follows:1. In the in fresh sperm motility observation of Cobia and crimson snapper, we observed the time sperm motility keeping and sperm surviving at room temperature through activating fresh semen sperm and keeping fresh sperm at room temperature, and also through analyzing the data that the software obtained from the size and concentration of sperm. After activating the fresh semen, time of sperm motility with Cobia was a little longer than crimson snapper, but there was no significant difference (P> 0.05), the best sperm motility were both within 3 min, and fell rapidly 3 min later. Regards to the experiments of preserving fresh sperm at room temperature, both Cobia and crimson snapper sperm can be preserved 30 min at room temperature, and was also a better preservation time at room temperature. In this time, the sperm motility was high that it was suitable for the production practice and experimental study of sperm.2. By picking the conditions of ultra-low temperature freezer, such as base fluid, cryoprotectants, cooling procedures, dilution proportion and freezing volume and screening thaw method, we observed vitality of frozen semen about Cobia and crimson snapper, did artificial insemination, then we got the best method for freezing and resurrection. Besides, there were three key factors included base fluid, type and concentration of cryoprotectants and cooling process assured the success of sperm cryopreservation. They affected larger on sperm motility, fertilization rate and hatching rate, followed by the proportion of sperm dilution, preservation volume and thawing method. Considering the hatching rate, fertilization rate and sperm motility of cryopreservation, the best solution for Cobia sperm cryopreservatioon was: MPRS+12% glycerin or DPE+10% glycerin as dilution, the proportion was 1:50, the frozen volume was 0.5 mL, and the frozen process was: 25℃to -20℃, -10℃/min, balanced at -20℃for 5 min, -20℃to -80℃, -10℃/min, balanced at -80℃for 5 min, then put them directly into liquid nitrogen.After thawed by 37℃water bath, sperm motility went up to 90%, and fertilization rate,gastula rate and hatching rate went up to 50% or more.The best solution for crimson snapper sperm cryopreservatioon was: HBSS+12% glycerin as dilution, the proportion was 1:50, the frozen volume was 0.5 mL, and the frozen process was: 25℃to -20℃, -10℃/min, balanced at -20℃for 5 min, -20℃to -80℃, -10℃/min, balanced at -80℃for 5 min, then put them directly into liquid nitrogen.After thawed by 37℃water bath, sperm motility went up to 85%, and fertilization rate,gastula rate and hatching rate went up to 60% or more.3. Analyzed frozen damage to Cobia and crimson snapper cryopreservation sperm nuclei, which was used single cell gel electrophoresis. After turning electrophoresis profiles of sperm into the data, we concluded that 10% glycerol antifreeze played a better role in protecting Cobia and crimson snapper cryopreservation sperm, and different cooling methods was the biggest factor to sperm DNA damage. In the relationship between comted rate and fertilization rate, there was no siginificant linear correlation. Fertilization rate may be affected by many factors during the fertilization process.4. We studied sperm membrane integrity and sperm tail damage with Cobia and crimson snapper sperm cryopreservation through hypotonic swelling technique, Research was supported by that sperm membrane integrity and sperm tail damage was mainly caused by different cooling methods, crystals in the sperm cells more severe the longer the injury.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquaculture technology > Fish breeding and wintering > Artificial propagation
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