Introduction
Global production of common carp increased from 1,205,000 t in 1990 to 4,360,000 t in 2020, with aquaculture accounting for the majority (97.2%). Within the European Union, the Czech Rep., Poland, and Hungary contributed 68% of the total common carp output (Shazada et al., 2025). The Czech Republic has maintained a selective breeding program for common carp for over 80 years. Over the past three decades, the program has adopted short-term sperm storage to enable controlled fertilization and preserve genetic diversity. In this context, sperm was collected individually and used at 0.5 to 5 days intervals depending on the breeding and/or testing protocols (Kocour et al., 2005). However, the use of short-term storage of carp sperm greatly reduced sperm fertility. Therefore, during the last five years, we have focused intensively on optimizing the method of sperm short-term storage. Firstly, to enhance the preservation of sperm motility and fertility, and secondly, to verify the potential effect of sperm storage on epigenetic changes in the offspring.
Materials and methods
Mature males of carp were obtained following fish farm pond harvesting and maintained in a laboratory and hatchery aquatic system. Sperm and egg samples were collected individually after treatment with carp pituitary extract at 21°C. Sperm was diluted with carp extender (Cejko et al., 2018) and stored for 6-14 days at 0-2°C under aerobic conditions. Post-storage, 6 mL sperm suspension was warmed at 20 °C for 2 h, followed by assessment of spermatozoa motility, velocity, and hatchability. To investigate how sperm storage may affect offspring, we integrated DNA methylome, transcriptomic, and proteomic analyses. To standardize the protocol and minimize maternal variation, fertilization for the DNA study was conducted using eggs from a single female.
Results and discussion
Carp sperm is always contaminated to some extent with urine (Billard et al., 1995). Although the level of contamination is low, storing sperm without an extender leads to a significant reduction in sperm motility and fertility within 24h, and sperm become immotile after 3 days post-storage (Cheng et al., 2022, Zhang et al., 2023, 2024). The most effective storage medium is the carp extender (110 mM NaCl, 40 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, pH 7.5, and 310 mOsmol/kg) (Cejko et al., 2018). When sperm is used for fertilization within 3h of sperm collection, it is not diluted and cooled to 0-2 ℃ but instead stored in the incubator at 15-20 ℃. This is due to the high sensitivity of carp sperm to temperature (Zhang et al., 2023). Therefore, fertilization within three hours, cooling is avoided. In contrast, sperm intended for later use is diluted 1:1 with pre-cooled extender (Cheng et al, 2022) and stored at 0-2 °C without antibiotics, for up to 14 days (Zhang et al., 2023, 2024). Notably, dilution even after 3h post-collection can still maintain sperm quality, making this practice suitable for hatchery applications (Shazada et al., 2025). Stored and diluted sperm should always be warmed to 20 °C before insemination. A 2-hour warming period significantly improves motility and hatching rates (Fig. 1). Intriguingly, malformation rates increased, especially in fresh sperm compared to those fertilized with stored sperm without warming (Zhang et al., 2023).
Sperm storage negatively affected sperm motility, viability, and DNA integrity, but did not affect the global DNA methylation of sperm or embryos. However, whole-genome bisulfite sequencing revealed altered methylation patterns in differentially methylated regions (DMRs) associated with genes in embryos fertilized with the 6-day-stored sperm. Results provide strong evidence of epigenetic inheritance influencing offspring’s performance following fertilization with stored sperm (Cheng et al., 2024). Therefore, in the carp breeding program, we aim to minimize the sperm storage duration.
Funding information
This study was supported by the Czech National Agency for Agricultural Research, project QK21010141, and the Czech Science Foundation (23-06426S).
References
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