Studies On Structure Characteristics Of Spermatozoa Of Acipenser Sinensis And Its Sperm Cryopreservation

The Chinese sturgeon (Acipenser sinensis Gray) is one among the large anadromousfish that involve riverine spawning migration. It lives mainly in the East China Sea,Yellow Sea, Yangtze and Pearl Rivers. Their nature populations were damaged severelyby long-term over-exploitation and increasing pollution in habitats where they thrive. Andby the construction of Gezhouba Dam, the operation of Three Gorges Dam and otherhuman activities in the near future, the risk of losing most of the populations and bringingthem close to extinction is rapidly increasing. So we need immediately developconservation measures for the population, which of them sperm cryopreservationpossesses important values on scientific reseach and application. On the base ofcomparison of biology characteristics of Acipenseriformes and other teleost, spermcryopreservation was studied. And from the study using computer-assisted sperm analysis(CASA) systems conducted on prefreeze sperm, post-thaw sperm and sperm underdifferent active medium, the most sperm motility parameters were compared. Cometassay was also used to detect DNA integrity of paddlefish sperm followingcryopreservation. Conclusions from the studies are as follows:1. The spermatozoa of Chinese sturgeon consistes of an elongated head with adistinct acrosome and nucleus region, a midpiece and a flagellum. The mean length of thehead and midpiece, the flagellum and total length of spermatozoon were 4.48, 33.3 and37.8μm, respectively. The acrosome contains posterolateral projections, subacrosome andgranular material and actin filament found within the anterior acrosome end etc. Threeendonuclear canals were found in the center of nucleus in a helical arrangement. Themidpiece behind the head comprises proximal centriole, distal centriole, mitochondrialsand cytoplasmic sheath. The flagellum unthreads the cytoplasmic canal and its mainstructure is axoneme, around which there are lateral fins.2. Using E5 (30 mM Sucrose, 20 mM Tris, 5 mM KCl, pH 8.1, MeOH 8%) ascryopreservation dilution for Chinese sturgeon sperm, fertilization rate was the highestafter thawing (6.98%), however, it had significant difference comparing with control(32.74-33.04%) (P＜0.05). No significant difference was found on different equilibriumtime in cryopreserved samples (P＜0.05). It was confirmed that the percentage of spermmotility, VSL, VAP, VCL, the percentage of A grade motility sperm and ALH wereimportant parameters for estimating fish sperm quality and there were highly positivecorrelations (r＞0.8, P＜0.05) between the parameters and fertilization rate. We concludedthat sperm motility was also high when fertilization rate was high. However, fertilizationrate was not aways high when sperm motility was high. The study was planned to providesperm cryopreservation of Chinese sturgeon with theory bases. 3. We detected significant differences (P＜0.05) in the degree of DNA damage incryopreserved sperm of paddlefish (Polyodon spathula) using different extenders.According to osmolality of the extenders, DNA damages of Sb (20 mM Tris, 75 mMSucrose, 0.5 mM KCl, pH 8.5) sperm was the least, which showed that the percentage oftail DNA of Sb (17.87%-35.28%) was lower than those of Sa (20 mM Tris, 50 mMSucrose, 0.5 mM KCl, pH 8.5) and Sc (20 mM Tris, 100 mM Sucrose, 0.5 mM KCl, pH8.5), moreover, A and B class sperm cells provided absolutely the most of the Sb sperm(＞50%). However, in light of concentration of methanol, DNA damages of M8 (methanolconcentration was 8%) sperm was the lightest, including a lower percentage of the tailDNA (21.56%-30.86%), C and D class sperm cells (＜30%), regardless of the osmolalityof the extenders. In conclusion, when the dilution was 20 mM Tris, 75 mM Sucrose, 0.5mM KCl, pH 8.5 and the concentration of methanol was 8%, the extenders was the bestfor cryopreservation of paddlefish sperm. In addition, our results indicated that the extentof damage caused by freeze-thawing to sperm motility was correlated with DNAbreakage (|r|＞0.8). This implied that cryopreservation could damage sperm DNA ofpaddlefish, when the osmolality and the concentrations of the cryoprotectants of theextender were inappropriate. Under such circumstances, we suggested thatcryopreservation could also damage intensely sperm DNA of Chinese sturgeon, whichmay be another reason for getting a terribly low fertilization rate using cryopreservedsperm, by evolutionary linkage between Chinese sturgeon and paddlefish.