The cryopreservation of fish sperm offers a versatile technique for enhancing reproduction, breeding, and conservation efforts. Although sperm cryopreservation technology for Eurasian perch (Perca fluviatilis ) can be successfully used at a commercial-scale in aquaculture, there is still a need for finding suitable markers of cryopreservation effectiveness and fertilizing capacity of frozen-thawed sperm (FTS) . Such markers may be used as selection criteria in cultured fish and for optimization of reproductive protocols.
In this study , we have performed extensive analysis of sperm collected using highly standardized protocols from 20 males and subjected them to thorough evaluation for various quality parameters. A longside classic sperm motility parameters (using computer-assisted sperm assessment [CASA] system), we also checked for various other parameters characterizing crucial cellular components, such as , ROS+ production, mitochondrial membrane potential, ATP levels, integrity and fluidity of cell membranes in Eurasian perch sperm. Later, these analyses were also performed on the same sperm subjected to standardized cryopreservation protocol. Next, we tested fertilizing capacity of the post-thawed sperm samples during controlled in vitro fertilization assay . Briefly, for this purpose we have used sperm-to-egg ratio of 10000:1 and fertilizing ~1g of eggs (~500 eggs) from 3 females for each male (n=20; in triplicates) . In total 180 egg samples were fertilized and later monitored for fertilization and embryonic developmental rates.
We observed positive correlations between fertilizing ability of FTS and average path velocity (VAP, µm s-1) and straight-line velocity (VSL µm s-1) . This aligns with the observations made in previous studies where sperm atozoa’s kinematic parameters were key factors in the fertilization success. Interestingly , we observed negative correlations between cellular membrane integrity, both in fresh and FTS with fertilization rates. This contradicts the well proven fact that sperm membrane intactness is one of the most important quality indicators. However, since cryopreservation has a detrimental effect on spermatozoa and resulted in decrease of sperm motility of about 20%, we can speculate that there is a possibility for leakage of intracellular components from these non-alive spermatozoa which could influence on those that survive/fertilize . One of the possibilities can be leakage of ATP from spermatozoa , since we found its level to be positively correlated with fertilization in our study. It should be emphasized that extracellular ATP has already been reported to improve fertilization rates in other taxa , but not studied so far in any fish species . In conclusion, along with this study we bring first set of validated markers which can be used as predictors of FTS fertilizing capacity in Eurasian perch and may serve as inspiration for other aquaculture species.
This work was funded by National Science Center of Poland (SONATA BIS project, number UMO-2020/38/E/NZ9/00394).