| An experiment was conducted to study the sperm of Crassostrea hongkongensis using cold-store technology.Different diluents, cryoprotectants, equilibrium time, dilution ratio, freezing, freezing procedure and method of recovery volume were screened to establish a set of mature sperm freezing Crassostrea hongkongensis preservation technology. Through determination and observation on the survival rate of sperm before and after freezing, vitality, fertilization rate and sperm ultrastructure, We evaluate the effect of the technique to the sperm cryopreservation and to provide a scientific basis and technical support for Crassostrea hongkongensis breeding and genetic breeding. The main results of this study are as follows:1. At4℃is more conducive than25℃to Crassostrea hongkongensis sperm preservation, and Low temperature can effectively prolong the survival time and maintain the vitality of sperm in vitro.2. With the highest sperm survival Citric acid sodium glycine solution and Physiological saline turn out to be the best spermatozoa cryopreserved dilution for Crassostrea hongkongensis sperm preservation, and the optimal solution are82.90±0.19%and77.57±0.49%. We choose DMSO as the cryoprotectant, as the protect effect of glycerol and methanol is inferior to that of DMSO. And the optimum concentration of cryoprotectant and trehalose corresponding to different dilutions is different, the optimum concentration of DMSO and trehalose is14%and0.25mol/L when Physiological saline is kept as diluent, while it is12%and0.15mol/L when Citric acid sodium glycine solution is kept as diluent.3. Different programmed cooling method do have significant difference in cryopreservation, the research shows that the proper program of temperature control is program Ⅳ with the highest sperm survival(Sperm motility was81.31±6.61%when physiological saline was kept as diluent, while citric acid sodium glycine was78.23±5.17%.), followed by program Ⅱ and program Ⅲ.4. The effect of different dilution ratio on sperm cryopreservation always varies, and the appropriate dilution ratio is1:40, of which the survival rate and vitality of sperm is the highest. There isn’t significant difference in sperm cryopreservation between different freezing volumes. The difference in the survival rate of different equilibrium time frozen semen after resuscitation is not significant, but the frozen sperm survival rate is the highest when equilibrium time is30min.5. The survival rate of frozen sperm in the two frozen semen dilution reaches the highest value when the resuscitation temperature is35℃(The survival rate in physiological saline group and Citric acid sodium glycine group is81.76±1.97%and78.57±3.28%respectively.), while there isn’t significant difference between25～30℃and40～50℃. The fertilization rate is similar to that of fresh sperm when the frozen semen insemination sperm egg ratio is40:1, and sperm should be activated with0.3125%o ammonia water rather than natural seawater.6. Before and after the Crassostrea hongkongensis sperm ultrastructure observation shows, Crassostrea hongkongensis sperm is mainly composed of head and tail, its head is approximate circular, Sperm midpiece consists of4mitochondria surrounding the centriole. The top of the head with an acrosome, The elongated tail flagellum, The axoneme has a typical "9+2" structure; Effects of cryopreserved sperm are significant, leading the main injury to the acrosome totally or partly disappeared, nuclear top sag, head of the emergence of various shades of depression, nuclear surface uneven, nuclear deformation, increased nuclear vacuoles, and even the whole decomposition, Mitochondrial structure loose spherical or even falling off, the contents of mitochondrial deletion stripping, what’s more cristae structure loose some sperm flagella from the middle fracture, some from the base of the sperm tail midpiece fracture; swelling, severe bending deformation. |
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