| Cryopreservation of fish gametes plays an important role in aquaculture, genetics breeding and in the preservation of genetic resource. Cryopreservation is an effective method for the long-term storage of viable sperm, and can provide a gear-round supply of male gametes. the technique is useful for the cryopreservation of sperm from genetically superior males for later use, in the transportation of sperm, and for use in breeding programs such as cross breeding, genetic studies or conservation of endangered species. The research on fish sperm cryopreservation, with the focus on cryopreservation protocols, has achieved great advances since the first successful cryopreservation of the herring sperm fifty years ago. To date, sperm from about 200 freshwater and more than 40 marine species has been successfully cryopreserved. However techniques for the routine sperm cryopreservation for all fish species remain to be developed.The present study focused on the red seabream Pagrosomus major, a marine fish species of an important value for aquaculture in China, Japan and Korea. Because of over fishing, marine pollution and natural habitat disturbtion, the wild stock red seabream has markedly declined in recent years. Therefore, the method for the sperm cryopreservation is eagerly demanded.The Sperm collected from red seabream was cryopreserved in 2-mL cryovials by using a programmable freezer. Sperm motility was quantitatively analyzed through computer-assisted sperm motion analyzer. The motility and fertility of both fresh and post-thaw sperm were investigated in order to optimize the spermatozoa cryopreservation protocols for red seabream. Six extenders (Cortland, Hank's balanced salt solution, Modified Mounib's medium, Mounib's medium, Mounib's NaCl medium, Ringer), six cryoprotectants (dimethyl sulfoxide, dimethyl acetamide, propylene glycol, ethylene glycol, methanol, glycerol) with the concentrations between 6% and 20% byvolume, four cooling rates (10, 20, 30, 40 °C/min) from 0 °C to- 150 °C as well as three thawing temperatures (35, 40, 45 °C) were designed and tested in the sperm cryopreservation, and their effect on post-thaw sperm motility and fertility were studied. The ratio of sperm to egg for post-thaw sperm fertilization trials was experimentally standardized and it was optimal at 500:1. Optimal post-thaw motility (79.4 ± 4.7% to 88.6 ± 8.0%), fertilization rates (89.6 ± 2.9% to 95.5 ± 2.0%), and hatching rates (85.3 ± 5.1% to 91.4 ± 4.3%) were achieved when using Cortland extender supplemented with 10% to 20% DMSO, 9%, 12% EG in combination with 20 °C/min cooling rate, and thawing in 40 °C water bath. Moreover, no significant influence of cryopreserved sperm on embryonic development was observed compared with the fresh sperm during incubation process. The results indicated that these methods for red seabream sperm cryopreservation could be suitable for routine aquaculture application and preservation of genetic resources.Alternation both in physiology and morphology of the red seabream after cryopreservation were investigated. Objective evaluation of sperm motility by computer assisted sperm analysis was used for red seabream before and after cryopreservation. It is found that the cryopreservation have no significant influence on sperm motility pattern and swimming velocity of motile sperm cryopreserved with DMSO. But the viability of cryoprserved sperm maintained was remarkably reduced. About 150 s after activation, the total motility of cryopreserved sperm with 15% DMSO was markedly reduced from 80.4% ± 5.8% to 10.7 ± 8.6% compared with fresh sperm from 86.7 ± 4.9% to 60.7 ± 8.6%.The red seabream sperm cryopreserved with 15% DMSO was observed by electron microscopy, about 50%—70% of the sperm showed normal morphology, 10% —20% were damaged to various degrees such as swelling or rupture of head and midpiece region and tail region, about 10%—20% were severely damaged, the plasma were completely ruptured, and only nuclei, mitochondria and flagellum were found. Flow flow cytometic analysis combined with a double staining procedure with Rhodamine 123 (Rh 123) and Propidium iodide (PI). 74.8% of cryopreserved spermatozoa exhibited an intact membrane and a functional mitochondrion which ascertained the high quality of the cryopreserved sperm with 15% DMSO.In addition, the effect of storage time on post-thaw sperm quality was primarily investigated. Long-term storage significantly influenced the quality of post-thaw spenn. The effect of DMSO concentrations on sperm fertilization and hatching rate was significant. The effect of long-term storage on sperm quality is eagerly needed further investigation.
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