Salinity is an important ecological factor affecting the osmolality of aquatic animals. Solenaia oleivora is an endemic and economically important freshwater shellfish in China. However, its osmotic response and osmoregulatory mechanisms under high salinity stress are still unclear. In this study, S. oleivora was exposed to saline water (salinity: 2.2‰) for 3 h, 6 h, 12 h, 24 h, and 48 h, and then the changes in osmolality, ion concentrations, free amino acid (FAA) content, Na⁺/K⁺-ATPase (NKA) activity, and gill histology were analyzed. The hemolymph osmolality increased from 3 h after salinity stress and stabilized between 24–48 h. Na⁺ in the hemolymph increased from 24 h after salinity stress, and Cl⁻ increased from 3 h. The content of total FAAs in the hemolymph increased after salinity stress. The content of alanine, glycine, glutamine, proline, and other FAAs increased after salinity stress. NKA activity in the gill, hepatopancreases, adductor muscle, and axe foot decreased during 3–48 h of salinity stress. The gill filament space increased and the number of gill cilia decreased after salinity stress. Principal component analysis (PCA) showed that the first two principal components (PC1 and PC2) cumulatively explained 77.6% of the total variation. The NKA activity was positively associated with PC1, while the ion concentration and most FAAs were negatively associated with PC1. Thus, these results indicated that S. oleivora is an osmoconformer, and inorganic ions, FAA, NKA, and gill structure changes play an important role in its osmoregulation.

Sperm cryopreservation is helpful for maintaining the genetic diversity of fish species. This study was aimed at developing efficient methods to cryopreserve the sperm of three fish species, including koi carp (Cyprinus carpio L.), Ya fish (Schizothorax prenanti), and Glyptosternum maculatum. Firstly, based on the analysis of sperm viability, the cryomedium, dilution ratio, volume, and cooling procedure were assessed and optimized in koi carp. The results showed that the highest sperm viability was up to 63.23 ± 1.36% after a 14-day cryopreservation using the optimal method, briefly, sperm frozen with a volume of 50 μL (Vol.sperm:Vol.cryomedium = 1:9) of cryomedium containing 10% DMSO and 3% sucrose in D17 through ultrarapid cooling. Secondly, both the mitochondrial membrane potential and the DNA fragmentation index of sperm were examined and found to be significantly damaged after the cryopreservation. Intriguingly, the fertilization rate of sperm after 14-day cryopreservation is up to 63.03 ± 1.36% and the elongation of cryopreservation time (210 days) just slightly affected the fertilization rate (55.09 ± 4.70%) in koi carp. Thirdly, the optimal cryopreservation method was applied to cryopreserve Glyptosternum maculatum sperm; the cell viability was 45.39 ± 4.70%. And then this method, after a minor modification (3% sucrose of cryomedium replaced with 3% SMP) was adopted to cryopreserve Ya fish sperm, the cell viability was up to 70.45 ± 2.23%. Lastly, the ultrastructure and morphology of sperm was observed by SEM, and it was found that the cryopreservation prominently caused sperm head swelling and tail shortening in three fish species. In conclusion, this study established effective methods for cryopreserving sperm in three fish species and elaborated the injuries on sperm caused by cryopreservation. And the findings facilitate developing more protocols with practical value to cryopreserve sperm in different fish species.