Introduction
Pre-slaughter stunning is vital for animal welfare. F or rainbow trout (Oncorhynchus mykiss), thermal and electrical shock are commonly used methods. These techniques trigger stress and tissue damage, raising cortisol levels and affecting energy metabolism, which are important indicators of the physiological impact of stunning (EFSA, 2009; Noble et al., 2020). This study aims to evaluate the effects of three stunning methods under two seasonal conditions (winter and summer), considering the trout poikilothermic nature and the impact of global warming on water temperatures.
Materials and methods
A total of 180 pan-size rainbow trout (349 ± 4.11 g ) were assigned to one of three stunning methods: Thermal shock (TS) through 32-minute ice-water immersion, dry electrical stunning at 200mA for 2 seconds (ES2), and dry electrical stunning at 400mA for 0.5 seconds, followed by 200mA for 1.5 seconds . Both electrical stunning treatments were followed by a 32-minute ice- water immersion . After stunning, blood samples were analyzed for cortisol, osmolality, and energy metabolism markers (glucose, LDH and CPK enzymatic activity, triglycerides, and NEFAs). The trout were then slaughtered, and muscle samples were collected for histological analysis . The trial was conducted in winter (8.67 ± 0.04°C ) and summer (22.3 ± 0.04°C). Data were analyzed using GraphPad Prism 10.3.1, with normality and homogeneity assessed via the Shapiro-Wilk and Bartlett’s tests. A two-way ANOVA was conducted, followed by post -hoc Tukey’s test (p < 0.05).
Results and discussion
In summer, cortisol levels were higher across all stunning methods, with the TS group in winter showing similar levels. However, the ES4 group in winter exhibited the lowest cortisol levels among all groups. Higher cortisol levels generally indicate a stronger stress response (Noble et al., 2020), which in summer could be attributed to the elevated water temperatures and the rainbow trout cold-water nature. In contrast, the TS group in winter showed elevated cortisol levels, likely due to the reduced effectiveness of the ice-water mixture, as the temperature difference between the rearing water and the ice-water mixture was smaller. This method is typically more effective for warm-water species (Ineno et al., 2005; Bordignon et al., 2024). Energy mobilization was notably influenced by season. Blood glucose levels were lower in summer compared to winter, while LDH and CPK enzyme activities were higher during this period, with the highest levels observed in the ES4 group. Lipid reserves also followed a seasonal pattern , with triglycerides decreasing and NEFA levels increasing in summer. These changes can be explained by the higher metabolic rate of poikilothermic organisms at elevated temperatures (Alfonso et al., 2021). Elevated LDH and CPK enzyme activity, along with changes in blood osmolality, are known biomarkers of muscle damage (Yousaf and Powell, 2012). The increased enzyme activity in the ES4 group and the elevated LDH levels in the TS group suggest more significant muscle damage, possibly due to the stress situation and scape responses to TS or the higher electrical current intensity in ES4 (Skjervold et al., 2001; Anders et al., 2019). Histological analysis revealed less extensive rhabdomyolysis in the ES2 group compared to the ES4 group. The higher osmolality observed in the ES4 group during winter further supports this, potentially indicating muscle fiber rupture and the release of intracellular contents (Stožer et al., 2020). Seasonal differences in osmolality may be less noticeable in summer due to osmotic adjustments made by freshwater organisms.
Conclusions
Both the stunning method and season influence stress, metabolism, and muscle integrity. ES2 stands out as a method that reduces stress and muscle damage, making it a more welfare-friendly choice that helps preserve product quality, striking a balance between animal welfare and the final product integrity.
References
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