Introduction
Fish welfare is traditionally assessed by measuring cortisol levels, which is the quintessential stress hormone in these animals. However, the best practice to assess animal welfare is to utilize multiple indicators in order to provide a more comprehensive portrait of the animal condition, especially when assessing long-term stress . Among the physiological indicators, dehydroepiandrosterone (DHEA), a precursory androgen with anti-stress effects, seems to be promising in diagnosing mammalian chronic stress through the cortisol:DHEA ratio [1] besides DHEA has recently been demonstrated to increase in fish exposed to a chronic stress [2] . However, while DHEA i n mammals has been shown to act in opposition to cortisol in many physiological pathways and to exhibit antioxidant, neuroprotective and immuno-protective characteristics [3] , still very little is known about its physiological meaning and possible implication in the stress response in fish . For this reason, the present study aimed to investigate how the levels of this steroid are affected not only b y a common stressful aquaculture practice, but also by sex and degree of sexual maturity in rainbow trout (Oncorhynchus mykiss) scales, a promising medium for multi-h ormone s tress analyses [4].
Materials and methods
The experiment was performed at a commercial farm where four groups of rainbow trout (n=96 ) were sampled: mature females (990 ± 28.5 g) and males (1005 ± 40.7 g), immature females (1350 ± 48.8 g) and males (1394 ± 33.3 g) . Half fish of each group were subjected to 30 minutes confinement stress and the other half were controls. Prior to sampling , fish were sacrificed with an excess of anesthetic (MS222 Sigma-Aldrich) and subsequent cut of the spinal cord. S cales were collected after removing excess mucus by scraping the side of the fish with a small plastic rod and subsequently stored at -20°C until the analysis.
Cortisol and DHEA were quantified in the scal es using a specific microtitre radioimmunoassay (RIA). Different washing protocols were tested and the RIA protocol was adapted and validated. All the data are expressed as mean ± standard error and were previously evaluated for normal distribution. Differences between treatments were analyzed using a general linear model (GLM) using sex , maturity and stress as main factors. The level of statistical significance was set at p < 0.05.
Results and discussion
Among the three factors studied, the degree of sexual maturity was the major factor influencing cortisol concentration , with mature fish having significantly higher cortisol levels than the immature (p<0.01) . This result aligns with previous studies conducted on rainbow trout, in which mature individuals, both males and females, showed higher plasma cortisol levels than immature [5]. Differently, sex affected DHEA concentration, with males showing significantly higher levels than fem ales (p<0.05). To our knowledge, DHEA has not been previously quantified in relation to sex in fish and thus there is currently no information available to compare our findings. However, there is evidence of higher DHEA concentrations in males than females both in humans and other mammals [6,7]. R esults did not show stress-related difference in both cortisol and DHEA levels. T his could be linked to the kind of stress applied in this study , i.e. acute, and to the bony nature of the scales, that slowly accumulate hormones. Nevertheless, the cortisol:DHEA ratio , resulted higher in the stressed fish compared to the controls, although not significantly (p =0.058). At present , knowledge about the involvement of DHEA in fish stress response is scarce. However, considering that it has been shown to counteract cortisol in mammals, this ratio might better describe an animal’s stress status than either hormone alone . Indeed, in humans and other vertebrates, high ratios of cortisol to DHEA have been considered indicative of chronic stress [8] . Finally, the meaning of the interactions between the three factors studied (sex, maturity and stress) is still under investigation .
Conclusions
In the present study, both cortisol and DHEA levels were successfully quantified in the scales of rainbow trout. Furthermore, the obtained levels are perfectly in line with those reported in the only other study currently present on rainbow trout exposed to chronic stress [2], attesting the applicability of the RIA method developed in this study in quantifying these hormones in fish scales. T o our knowledge, this is the first time DHEA has been evaluated with respect to sex and degree of sexual maturity in fish. Nevertheless, further investigation is required to better understand the role of this hormone in fish physiology. Moreover , these results need to be implemented by assessing a longer-term stress in order to test the suitability of DHEA as an alternative physiological indicator to be used alongside cortisol in the assessment of chronic stress in fish.
References
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