RESUMO
Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 °C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 °C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans.
Assuntos
Sêmen , Motilidade dos Espermatozoides , Masculino , Humanos , Criopreservação/métodos , Crioprotetores , EspermatozoidesRESUMO
The spermatogenesis of crustaceans includes nuclear deformation and acrosome formation. The mechanism of acrosome formation is one focus of reproductive biology. In this study, Macrobrachium rosenbergii was selected as the research object to explore the mechanism of acrosome formation. The acrosome contains a large number of acrosomal enzymes for the hydrolysis of the egg envelope. How these acrosomal enzymes are transported to the acrosomal site after synthesis is the key scientific question of this study. The acroframosome (AFS) structure of caridean sperm has been reported. We hypothesized that acrosomal enzymes may be transported along the AFS framework to the acrosome by motor proteins. To study this hypothesis, we obtained the full-length cDNA sequences of Mr-kifc1 and Mr-Acrosin from the testis of M. rosenbergii. The Mr-kifc1 and Mr-Acrosin mRNA expression levels were highest in testis. We detected the distribution of Mr-KIFC1 and its colocalization with Mr-Acrosin during spermatogenesis by immunofluorescence. The colocalization of Mr-KIFC1 and microtubule indicated that Mr-KIFC1 may participate in sperm acrosome formation and nucleus maturation. The colocalization of Mr-KIFC1 and Mr-Acrosin indicated that Mr-KIFC1 may be involved in Acrosin transport during spermiogenesis of M. rosenbergii. These results suggest that Mr-KIFC1 may be involved in acrosomal enzymes transport during spermiogenesis of M. rosenbergii.
RESUMO
Dynein is a motor protein with multiple transport functions. However, dynein's role in crustacean testis is still unknown. We cloned the full-length cDNA of cytoplasmic dynein heavy chain (Pt-dhc) gene and its structure was analyzed. Its expression level was highest in testis. We injected the dynein inhibitor sodium orthovanadate (SOV) into the crab. The distribution of Portunus trituberculatus dynein heavy chain (Pt-DHC) in mature sperm was detected by immunofluorescence. The apoptosis of spermatids was detected using a TUNEL kit; gene expression in testis was detected by fluorescence quantitative PCR (qPCR). The expression of immune-related factors in the testis were detected by an enzyme activity kit. The results showed that the distribution of Pt-DHC was abnormal after SOV injection, indicating that the function of dynein was successfully inhibited. Apoptosis-related genes p53 and caspase-3, and antioxidant stress genes HSP70 and NOS were significantly decreased, and anti-apoptosis gene bcl-2 was significantly increased. The activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) were significantly decreased. The results showed that there was no apoptosis in testicular cells after dynein function was inhibited, but the cell function was disordered. This study laid a theoretical foundation for the further study of apoptosis in testis and the function of dynein in testis and breeding of P. trituberculatus.
RESUMO
In this study, the bacteria from the mud in tidal-flat Sinonovacula constricta aquaculture area were isolated each month from March to December, 2002, and the temporal and spatial distribution of heterotrophic bacteria, ammonifying bacteria, denitrifying bacteria, and sulphate reducing bacteria were analyzed. The results showed that all the 515 isolated bacteria mainly belonged to 1 family and 13 genera. The bacterial flora in different layers of the mud was almost consistent, while the composition was different. The predominant genera were Clostridium, Bacillus, Corynebacterium, Photobacterium, and some Enterobacteriaceae. The number of heterotrophic bacteria in the surface layer and the bottom fluctuated in 7.6 x 10(3) cfu x g(-1) - 2.0 x 10(5) and 1.6 x 10(3) - 1.0 x 10(5) cfu x g(-1), ammonifying bacteria fluctuated in 1.5 x 10(6) - 9.0 x 10(7) and 9.0 x 10(5) - 1.0 x 10(7) cfu x g(-1), denitrifying bacteria fluctuated in 9.0 x 10(3) - 4.0 x 10(6) and 5.0 x 10(2) -1.9 x 10(6) cfu x g(-1), and sulphate reducing bacteria fluctuated in 5.0 x 10(4) - 5.0 x 10(6) and 1.9 x 10(4) - 2.0 x 10(6) cfu x g(-1), respectively. The detection rates of ammonifying bacteria, denitrifying bacteria and sulphate reducing bacteria in the mud were all 100%, and these bacteria increased significantly in the second half of the year, indicating that the environment of the Sinonovacula constricta aquaculture area was deteriorated due to the accumulation of NH3, nitrite and H2S, and it is important to regulate the breed capacity and redistribute the breeding environment.
