Existing methods of storing fish semen are not perfect. Researchers at our institute, Dr. Radosław Kowalski and Agnieszka Brzyszcz, MSc., have developed special diluents that will significantly improve the process while protecting the sperm.

Climate change has led to the desynchronization of male and female spawning. It is now common for males to mature much earlier than females, hence the need for methods of storing the semen used for fertilization. Under the direction of Radosław Kowalski, Ph.D., researchers from our Institute have created special buffers for carp and trout that can improve this process. The project „Method of storing fish semen under production conditions” was carried out under the Innovation Incubator 4.0 program.

We asked Dr. Radoslaw Kowalski about the significance of his research and what it could change among fish farmers.

How is fish semen currently stored?

Dr. Radosław Kowalski: In hatchery practice, semen taken directly from the fish is stored in a container or bag with oxygen – such methods work well within hours of collection. Keeping undiluted semen risks declining quality immediately, as urine contamination is dangerous. Fish semen has the property that it is activated by low osmotic pressure so that it can be accidentally activated by urine. Long-term storage of semen with oxygen is also not good – our studies have shown that oxidative changes bring more harm than benefit.

What can be done to improve existing methods?

RK: We have developed special immobilizing liquids for storage. If, during sperm retrieval, it becomes contaminated with urine, sperm movement will not be activated after dilution in the immobilizing fluid. In the favorable environment created by the buffer, sperm „recharge their batteries,” that is, rebuild their energy reserve in the form of ATP, which positively affects their motility. Thanks to diluents, we can regenerate any sperm – from 0 to even 90% of mobile sperm.

Does such a method also work in the long run?

RK: When we store sperm in a buffer for a long time, about 5 percent of the resulting offspring develop into embryos with severe deformities, such as heart defects or other physiological changes… It turned out that sperm stored for a long time had oxidative changes in DNA (at the level of the epigenome) that caused permanent genetic changes. Old sperm can be responsible for lower-quality offspring, as we see throughout the natural world.

Can this cellular aging be stopped?

RK: As part of the Innovation Incubator 4.0 project, we used antioxidant additives to protect sperm from oxidative changes within the genetic material. We used three different antioxidants and observed how they worked – vitamin E was the best. We studied two species of the most popular fish in Poland – carp and rainbow trout. These are two extreme examples – trout is a cold-loving fish, and carp is warm-loving. Trout have spermatozoa resistant to oxidative changes in the genome due to the presence of protamines as chromatin-stabilizing proteins. Carp spermatozoa are more sensitive to these changes because their genome is stabilized by histones, which are less efficient at packing DNA.

Our study compared the differences in sperm quality and motility after a designated storage period – in trout for a month and carp two weeks after harvesting. We also analyzed the quality of offspring obtained from fertilization using semen stored in this way. In both cases, we used mixtures of antioxidants, and vitamin E performed best.

What else is in the buffers?

RK: The composition of the buffers was patented back in 2014. I can’t reveal the concentrations of antioxidants, but the basic composition of the buffers itself is public. The most critical substances are sodium and potassium. There is also magnesium, calcium, and a pH stabilizer. In addition, we were the first in the world to enrich the composition of buffers with thickening polysaccharides (alginates, carrageenans, and xanthans), which prevents gravitational sedimentation of sperm and their consequent sinking to the bottom of the containers. Sedimentation is a process by which, after just 48 hours, more than 90% of sperm can be permanently damaged.

Can the composition of buffers be further improved?

RK: Antioxidants of natural origin change their color under different pH. My research goal is to create additives that indicate changes in pH by color. I want to develop a buffer that, on the one hand, would protect sperm and, on the other hand, through its color, would show how advanced the aging changes of sperm are. Such a solution would make life much easier for fish breeders, who could tell from the color changes whether or not to use a given sperm.