Introduction
The Atlantic salmon is an emblematic species of Western Europe rivers that flow into the North Atlantic Ocean, yet its populations have been in decline throughout the 20th century. Several European programs in Western Europe (ie, such as those in Denmark, Germany, Wales or Belgium), aim to sustainably restore salmon habitats and populations by implementing restocking actions-including the release of emerging yolk fry and parr, removal of migration barriers, and habitat restoration measures. Nevertheless, adult salmon returns remain relatively low and show considerable interannual variability. The use of cryopreserved sperm from genetically valuable males in aquaculture production provides a promising tool for conservation. It allows for long-term storage of genetic material and helps maintaining sperm motility, ultimately impacting reproductive success (Erraud et al. 2024). Although no significant effects of cryopreservation was observed on fertilization rates, growth, survival nor pathogen resistance during the embryo stage, Nusbaumer et al. (2019) found that fertilization by cryopreserved sperm significantly reduced larval growth after hatching.
In addition, salmon released through restocking programs will be increasingly exposed to the impacts of climate change. It is now well established that rivers are experiencing accelerated warming during the summer months, along with a higher frequency of extreme temperature events. These changes are particularly concerning for cold-water species such as Atlantic salmon, which are highly sensitive to elevated water temperatures (Buisson et al., 2008).
Our experiment aimed at evaluating the effect of using cryopreserved sperm from the Meuse River strain (Belgian) on fry development and their resistance to environmental stressors such as river warming and bacterial infection. The effects of two stressors experienced by hatchery-reared juveniles upon release were tested: daily thermal fluctuations specific on the recipient river and an infection with a bacterial pathogen. The environmentally realistic thermal conditions tested in this study were set based on temperature records from three Belgian rivers (Ry de Ferron (constant 10°C), Berwinne (6.5–12.3°C), and Ry d’Oxhe (4.5–16°C) rivers).
Material and Methods
Artificial reproductions were performed using anadromous male breeders (using both fresh and cryopreserved sperm) and 2+ female breeders. Eggs were collected, pooled and divided into batches of 300 eggs. They were then fertilized with fresh and cryopreserved sperm to obtain a standard sperm-to-egg ratio of 10⁶ and transferred into individual vertical micro-incubators supplied with recirculated freshwater at 9.0 ± 0.4°C throughout the incubation period. Fifteen days post-hatching, salmon fry (N = 360) from fertilization with fresh and cryopreserved sperm were subjected to thermal stress as such: a constant temperature of 9 °C, a daily fluctuation between 7-12°C, or a fluctuation between 3-15°C. Biometric variables such as size, weight, and yolk sac size were measured at the start of the experiment, after 24 h and 11 days of thermal stress exposure. Nine individuals per condition were euthanized using a final concentration of 200 mg L⁻¹ buffered MS-222 to collect biological material for molecular analyses via qPCR.
Following thermal stress, half of the remaining organisms were exposed through a bath infection to the bacterial pathogen Aeromonas salmonicida achromogenes at 1.41 × 109 CFU/mL for 6 hours at 9°C; the other half was kept as a control group uninfected. After 6 h of bacterial challenge, half of the exposed fry and half of the control fry were euthanized and sampled for molecular analyses using qPCR analyses.
Results and discussion
This study represents the first investigation assessing the impact of sperm cryopreservation on the development of yolk-sac fry intended for restocking, as well as their resistance to two successive environmental stressors: thermal stress and bacterial challenge. Particularly, the higher daily thermal fluctuations (3–15°C) resulted in reduced growth rates and yolk reserves. These phenotypic observations were sustained by transcriptomic analyses, which revealed that progeny derived from cryopreserved sperm exhibited upregulated expression of genes associated with antioxidant defences and energy metabolism. These high fluctuated temperatures also induced an increase in intracellular reactive oxygen species (ROS), leading to heightened energy expenditure to counteract oxidative stress. Consequently, energy allocated to stress responses was unavailable for somatic growth. It is therefore likely that fry originating from cryopreserved sperm possess lower critical thermal minima and maxima compared to those produced with fresh sperm.
Moreover, the bacterial challenge demonstrated that fry from the cryopreserved group were able to face the bacterial infection with an effective immune response only when they had not been previously exposed to thermal stress (i.e., 3-15°C). Our results suggest that sperm cryopreservation could have reduced the capacity of progeny to cope with multiple environmental stressors. While the use of cryopreserved sperm remains a viable strategy for restocking salmon populations, caution is warranted when releasing these individuals into rivers subject to substantial daily temperature fluctuations, such as the Ry d’Oxhe in Belgium.
References
Buisson, L., Blanc, L. & Grenouillet, G. (2008) Modelling stream fish species distribution in a river network: the relative effects of temperature versus physical factors. Ecology of Freshwater Fish, 17, 244–257.
Erraud, A., Cornet, V., D’Halluin, F., Lambert, J., Neus, Y., & Kestemont, P. (2024). Sperm cryotolerance in mature male parr of Atlantic salmon (Salmo salar L. 1758): Effects of dietary lipid content and n-3 essential fatty acids composition. Aquaculture, 593.
Long, A. P., Vaughan, L., Tray, E., Thomas, K., Maoileidigh, N., Poole, R., Cotter, D., Doogan, A., & Brophy, D. (2023). Recent marine growth declines in wild and ranched Atlantic salmon Salmo salar from a western European catchment discovered using a 62-year time series. ICES Journal of Marine Science, 80(6), 1697–1709.
C. Magnotti, V. Cerqueira, M. Lee-Estevez, J.G. Farias, I. Valdebenito, E. Figueroa. (2018). Cryopreservation and vitrification of fish semen: a review with special emphasis on marine species. Rev. Aquacult., 10 (1), pp. 15-25
Nusbaumer, D., da Cunha, L. M., & Wedekind, C. (2019). Sperm cryopreservation reduces offspring growth. Proceedings of the Royal Society B: Biological Sciences, 286(1911).