| 1, Comparison of two methods for acquiring free sperm of Eriocheir sinensisThe purpose of this paper was to compare different methods of acquiring free sperm cells from spermatophores of the Chinese mitten-handed crab (Eriocheir sinensis), and to find a suitable method of acquiring free sperms from spermatophores of Chinese mitten-handed crab under three factors with five levels including a series of temperature (27,32,37,42 and 47 ℃), different concentrations in Trypsinase sotution(0.05%,0.10%,0.15%,0.20% and 0.25%) and duration (5,10,15,20 and 25 min).The orthogonal experiment results of digesting spermatophore showed it would acquire the largest amount of sperms in per gram spermatophore (7.41×10~8/g) by using 0.2% Trypsinase solution at 37℃ and incubating 5 min. Basing on this, we compared the digestion method with homogenization method. The results showed that digestion method was much better than homogenization method in both the amount of sperm and living rate of sperms.2, Assay the sperm viability of Eriocheir sinensis by two supravital staining techniqueTwo dyes: esion and trypan blue were dissolved in Ca2+-free saline water. And by using them the sperm viability of Eriocheir sinensis was evaluated. They appeared remarkable difference between the dead sperms and live sperms, The dead sperm was stained by both of dyes, especially the acrosome; The size of dead sperm was bigger than the lives; The radial arms and border of death were indistinct. Then two experiments were done to determine a fit dye time and concentration of dyes. In 0.1%and 0.5% esion solution, death rate of sperm appeared significantly higher than in 0.25%, 0.125% (P<0.05) . But there was no significant difference between 1% and0.5% trypan blue solutions. And there was also no significant difference among 0.5% , 0.25% esion solution and 1%, 0.5% trypan blue solution (P>0.05) . So (he lit concentrations of dyes were 0.5% 0.25% esion solution and l%> 0.5% trypan blue solution . The death rate of sperm was increased with the time, because dyes did harm to sperms also. In 0.1%and 0.5% esion solution, death rate of sperm showed significant difference among at 30min, 5 min and 15min. In 0.25%and 0.125% esion solution, death rate of sperm showed significant difference between at 30min, 5min and 15min, but there was no significant difference between at 5 and 15 min. In 0.1%and solution, there was no significant difference in sperm death rate among at 5, 15 and 30min. So the best condition was that 0.5% trypan blue or 0.25% esion under staining 15min. The reliability of esion was compared with the trypan blue staining technique. The sperm samples contained known proportions of fresh and killed cells. The results show that the values determined by esion and trypan blue both agreed closely with the theoretical values(R=0.99, PO.05). And the values determined by esion agreed closely with trypan blue method(R=0.98, P<0.05).3, Assessment of Sperm Viability in Eriocheir sinensis by using a Fluorometric Ethidium Bromide Exclusion ProcedureThe ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on Chinese mitten-handed crab (Eriocheir sinensis). The fluorescence intensities of stained fresh and stained tritonX-100-permeabilized samples were corrected, respectively. The nonspecific fluorescence measures of fresh and tritonX-100-permeabilized samples were both unstained. The contribution of the dye and dye+ tritonX-100 was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for Chinese mitten-handed crab (8-32 xl06cell/ml). The reliability of the fluorometric method was compared with the traditional eosin (E) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defined working cell ranges.4, The use of flow cytometry in the evaluation of sperm cell viability of Eriocheir sinensisThe propidium iodide (PI) exclusion procedure, a flow cytometry method for measuring sperm cell viability, was studied on Chinese mitten-handed crab {Eriocheir sinensis). The sperm samples contained known proportions of fresh and killed cells(sperm dead rate: 0%, 20%, 40%, 60%, 80%). The results showed that the values determined by flow cytometry agreed closely with the theoretical values(R=0.99, P<0.05). These results showed that flow cytometry was a valuable tool to evaluate sperm cell viability in decapod species and it was mote sensitive technique than optical microscopy.
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