The most apparent aquaculture benefit of the reproduction of external fertilizers, such as fish, is that their gametes which are naturally released into the environment where fertilization occurs, could be collected before fertilization and used in artificial breeding measures. Thus the gametes handling becomes almost an indispensable part of any artificial reproduction in most fish species. It allows optimization of fertilization rates , producing large numbers of healthy offspring , and applying selective breeding programs , increasing the quality and efficiency of artificial reproduction.
Compared to artificial reproduction programs in other species, including mammals, which generally focus on acquiring a relatively small number of individuals, fishes produce a huge quantity of gametes in both sexes and rely on a large number of offspring. In such a situation, preserving the high quality of maximal numbers of eggs and spermatozoa before fertilization is essential. At the same time, it is important to keep enough high genetic variability to avoid inbreeding and potential homogeneity of progeny.
In this report, we are summarising our recent results on sperm handling and suggesting the most promising steps for optimizing and improving sperm handling methods in the future .
Sperm cryopreservation . Currently, cryopreservation protocols cover the vast majority of commercially important fish species with high preservation of sperm motility and fertilisability after thawing, using different cryoprotectants and freezing techniques. Moreover, sperm cryopreservation still requires the next steps in technology development , probably by cost-cutting and simplifying the methods, to become widely used in aquaculture.
In the past few years, we (1) optimized the protocols for sturgeon and carp sperm cryopreservation by ad justment of sperm concentration before freezing (Nascimento et al., 2021; Sotnikov et al., 2023) ; (2) tested using hypertonic cryopreservation media for cryopreservation of large volumes of carp and sterlet spermatozoa required for fisheries practice (unpublished data) ; (3) optimized two most common methods of uncontrolled sperm cooling by floating raft in Styrofoam box and dry shipper container (Horokhovatskyi et al., 2017) .
Short-term sperm storage . Similarly to cryopreservation , many protocols for short-term storage exist for fish spermatozoa. In contrast to cryopreservation, short-term storage accumulates damage in the spermatozoa slowly , and the last storage stages are commonly associated with increased bacterial growth, suggesting using antibacterial storage media . Therefore understanding the changes in the spermatozoa during storage is the critical element for further improvement of this handling method.
Recently we tested the effect of hypothermic storage: ( 1) on the epigenetics of sperm and the resulting embryos (Cheng et al., 2023) ; ( 2) on spermatozoa’s ability to tolerate temperature (Zhang et al., 2023) and osmotic shock (unpublished data ); (3) on spermatozoa metabolome (unpublished data) and (4) bacterial contamination and apply polyphenolic extracts as an alternative to antibiotics (unpublished data).
Sperm separation . Unavoidable damage (due to ice crystals growing in cryopreservation or sperm aging during short-term storage) results in the appearance of a nonvaluable and semi-valuable population of spermatozoa (which still could be involved in fertilization). Thus, developing spermatozoa separation methods is the most promising next step for improving fertilization outcomes by eliminating damaged sperm populations. At the same time, these methods may allow us to study the separated populations more deeply to understand their specific physiological differences and possibly resistance to different types of damage .
Currently, we are developing and testing methods for spermatozoa separation in different fish species by ultrasound (unpublished data) and Percoll gradient (Horokhovatskyi et al., 2018). At the same time, we are searching for the metabolic markers of individual sperm samples which can predict their storage and freezing capacity to maximize the outcomes.
Cheng, Y., Waghmare, S. G., Zhang, S., Vechtová , P., Schumacher, F., Kleuser , B., Samarin , A. M., Samarin , A. M., Linhartová, Z., Dey, A., Dietrich, M., Sterba, J., Alavi, S. M. H., Labbé, C., & Linhart, O. (2023). Aging of common carp (Cyprinus carpio L.) sperm induced by short-term storage does not alter global DNA methylation and specific histone modifications in offspring. Aquaculture , 571, 739484. https://doi.org/10.1016/j.aquaculture.2023.739484
Horokhovatskyi , Y., Dietrich, M. A., Lebeda, I., Fedorov, P., Rodina, M., & Dzyuba, B. (2018). Cryopreservation effects on a viable sperm sterlet (Acipenser ruthenus ) subpopulation obtained by a Percoll density gradient method. PLOS ONE , 13(8), e0202514. https://doi.org/10.1371/journal.pone.0202514
Horokhovatskyi , Y., Rodina, M., Asyabar, H. D., Boryshpolets, S., & Dzyuba, B. (2017). Consequences of uncontrolled cooling during sterlet (Acipenser ruthenus ) sperm cryopreservation on post-thaw motility and fertilizing ability. Theriogenology , 95, 89–95. https://doi.org/10.1016/j.theriogenology.2017.03.007
Nascimento, J. P., Horokhovatskyi , Y., Kholodnyy, V., Rodina, M., Dzyuba, V., Stechkina , T., Boryshpolets, S., & Dzyuba, B. (2021). Optimization of sterlet sperm concentration for cryopreservation. Aquaculture , 540 (March). https://doi.org/10.1016/j.aquaculture.2021.736682
Sotnikov, A., Rodina, M., Stechkina , T., Benevente , C. F., Gela, D., Boryshpolets, S., Kholodnyy, V., Linhart, O., & Dzyuba, B. (2023). High sperm concentration during cryopreservation decreases post-thaw motility percentage without compromising in vitro fertilization outcomes in common carp. Aquaculture , 562, 738746. https://doi.org/10.1016/j.aquaculture.2022.738746
Zhang, S., Cheng, Y., Alavi, S. M. H., Shazada, N. E., Linhartová, Z., Rodinová, V., & Linhart, O. (2023). Elevated temperature promotes spermatozoa motility kinetics and fertilizing ability following short-term storage: An implication for artificial reproduction of common carp Cyprinus carpio in a hatchery. Aquaculture , 565, 739126. https://doi.org/10.1016/j.aquaculture.2022.739126