| Metabolism of sperm is expected to be in a suspended state and metabolic activity of sperm almost stops when sperm were cryopreserved in liquid nitrogen. It has been proposed that the storage time would not affect the sperm quality. While, with the application of cryopreservation technique and the improvement of sperm quality detection, some literatures reported that there were changes in the quality of sperm when cryopreserved in liquid nitrogen.In this paper, the effect of long-term cryopreservation on structural properties, physiological function, biochemical and genetic material properties of red seabream (Pagrus major) sperm which were cryopreserved from 1 month to 84 months were systematically studied, by observing the longevity, motility, fertilization rate and ultrastructure of post-thaw sperm, by analyzing the activities of superoxide dismutase (SOD), catalase (CAT), lactate dehydrogenase(LDH) and succinodehydrogenase(SDH), and by examining DNA damage of post-thaw sperm, to show the change law of structural and function properties of sperm during long-term cryopreservation; to reveal the effect of long-term cryopresrvation on sperm quality and the mechanism of it. The results are as follows:1. For 2, 12, 25, 60 and 84 months storage, the motility, fertilization rate and hatching rate decreased with the storage time significantly(P<0.05). The highest motility, fertilization rate and hatching rate were obtained in sperm cyropreserved for2 months, and they were 85.00%±2.00%, 79.01%±12.86% and 71.61%±3.26%, respectively; The motility, fertilization rate and hatching rate of sperm cryopreserved for 12, 25, 37 months have no significant differences with those of sperm cryopreserved for 2 months; The motility, fertilization rate and hatching rate of sperm cryopreserved for 60 months were significantly lower than those of sperm cryopreserved for 2, 12, 25 and 37months; (P<0.05)When the storage time increased to 84 months, the motility, fertilization rate and hatching rate decreased to 45.00%±2.00%, 16.74%±3.00% and 11.22%±2.71%, respectively.2. For 1, 13, 26, 48 and 73 months storage, the duration time of post-thaw sperm was 180 s, 180 s, 150 s, 140 s and 100 s, respectively;At 4℃, the longevity of post-thaw sperm was 120h, 38h, 38h, 28h and 20h, respectively. In addition, the motility of sperm cryopreserved for 1, 13 and 26 months were up to the highest value immediately after addition of seawater. However, the motility of sperm cryopreserved for 48 and 73 months was about only 10% for the first 10-20 s, then, the number of motile sperm increased lowly and up to the highest value after 50-60 s of addition of seawater.3. The ultrastructure of post-thaw sperm was studied and the results showed the head region of intact sperm was similar circular and strongly connected mid-piece region and tail region. The diameter of intact sperm was 1.3-1.4μm; the damage of head, mitochondria and plasma and nuclear membranes is the main ultrastructure damage of post-thaw sperm. The membrane damage is the most serious one, and 80%-90% damaged sperm were with membrane damage.4. Enzymatic activities of red seabream were analysed. The results showed there were no significant differences in SOD activities of sperm cryopreserved for 1 and 13 months; SOD activities of sperm cryopreserved for 48 and 73 months were were significantly lower than those for 1 and 13months(P<0.05), SOD activities of sperm cryopreserved for 26 months had no significant difference with those for 1, 13 , 48 and 73 months; the highest CAT activity was obtained in sperm for 1month storage, and it is significantly higher than those of sperm for 13, 26, 48 and 73months storage (P<0.05).There were no significant differences were observed in CAT activities of sperm cryopresevered for 13, 26, 48 and 73 months; LDH activities was obtained the highest value in sperm for 13 months storage (1817.22±431.41 u/mgprot), and it is significantly higher than that of post-thaw sperm of other groups(P<0.05); The highest value of SDH activities was obtained in sperm for 73 months storage (18.83±1.34 u/mgprot), and the lowest value in sperm for 1months storge.5. The red seabream sperm cryopreserved for 11, 23, 36, 58, 83 months were studied by using single-cell gel electropheresis assay. The results showed the comet rates of post-thaw sperm were among 34.00%±4.00%～42.67%±9.45%, and the comet rate of fresh sperm was 30.67%±6.43%. There were no significant differences were obtained among post-thaw sperm and fresh sperm, so there were no significant differences in the genetic material stability of sperm cryopreserved for 11, 23, 36, 58, 83 months and fresh sperm (P>0.05).This study showed there were significant effects of the cryopresrvation on sperm quality, the long-term cryopresrvation mainly affect the longevity, motility, fertilization rate, hatching rate and antioxidant enzyme activities of poth-thaw sperm in red seabream, and all of those parameters decreased with the storage time prolonged. |
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