Introduction
In aquaculture, fish undergo a series of events and practices that can compromise their welfare and health. One of the most frequently used stress indicators in fish is the quantification of cortisol in conventional matrices, such as plasma. Nevertheless, blood sampling can be extremely invasive, becoming itself a stressor and cortisol quick increase after stress may lead to unreliable results. This is why non-conventional matrices such as muscle, fin and skin mucus have already been tested as valuable alternatives to plasma (Bertotto et al., 2010; Sadoul & Geffroy, 2019). Among them, scales have also been recently taken into consideration as less invasive matrices and as novel and reliable marker for chronic stress in European sea bass and other species (Samaras et al., 2021). Cortisol in scales is accumulated slowly, due to their bony nature, making them unsuitable for acute stress studies but essential to evaluate fish stress response over the mid-long term. This study aims to evaluate the stress status of European seabass subjected to hypoxia by quantifying cortisol levels in a suite of matrices using specific radioimmunoassays (RIAs). For a deeper insight into the stress response, measurements of hematocrit and oxidative stress were also taken.
Materials and methods
European sea bass (n=60; 158±10 g) were acclimatized for 4 weeks at 22.5±0.5°C and 8.5±0.5 mg/L of oxygen. Fish were then randomly distributed in 6X1000L tanks with a daily renewal rate of approximately 5%. The experiment was performed at the Department of Veterinary Medical Sciences (University of Bologna, Italy) and during the whole period temperature was kept at 26±1°C and the photoperiod was set at 12L:12D. Three tanks were used as control and had normal oxygen levels (8.7±0.4 mg/L), whereas the other three had low oxygen levels (4±0.2 mg/L) to establish hypoxic stress. Fish were sampled at the beginning of the trial, before exposure to hypoxia, and at the end of the trial, which lasted 7 days. During the samplings, fish were sacrificed by excess of anesthetic (MS222 Sigma-Aldrich) and subsequent cut of the spinal cord. Blood was collected from the caudal vein using heparinized tubes and samples of skin mucus and scales were collected by scraping the side of the fish. Immediately after collection, two aliquots from each blood sample were taken for hematocrit measurements, while the remaining part was centrifuged to obtain plasma samples. Small portions of lateral muscle (1cm3) and caudal fin (1cm2) were also collected. All the matrices were subsequently stored at -20°C until the analysis.
Cortisol was measured in all the matrices by a previously validated specific RIA protocol for use in European seas bass (Bertotto et al., 2010). For the scales, different extraction methods were tested and a specific RIA protocol was developed and validated. Plasma was also used for the assessment of lipid peroxidation, i.e. malondialdehyde (MDA), by measuring thiobarbituric acid-reactive substances (TBARS). All the data are expressed as mean ± standard error and were previously evaluated for normality distribution. Differences between treatments were analyzed using a general linear model (GLM) using time and hypoxia stress as main factors. Correlations in cortisol concentrations between plasma and the various matrices were performed using Pearson’s correlation coefficient. The level of statistical significance was set at p < 0.05.
Results and discussion
The RIA protocol developed to assess cortisol in the scales showed good parallelism and reproducibility (linear regression curve coefficient R2: 0.99; CV% intra-assay = 5.14; CV% inter-assay = 4.39) and the recovery test with value higher than 90% confirmed the efficiency of the method for steroid extraction.
Results showed that cortisol concentration in plasma, fin and skin mucus increased over time, although not significantly (p=0.21, p=0.27 and p=0.07, respectively). Neither the exposure to hypoxia affected the hormone levels in the same matrices (p=0.92, p=0.90 and p=0.18, respectively). On the other hand, a significant increase in cortisol concentration in muscle and scales was observed in fish over time (p<0.01 and p=0.001, respectively), but no differences were found in fish exposed to hypoxia stress compared to controls (p=0.83 and p=0.88). As scales are composed of bone tissue and slowly accumulate cortisol, they are little influenced by the acute effect of the sampling and allow to quantify the level of stress in the mid-long term. However, the low oxygen conditions used in this trial were too mild to trigger a strong stress response and a high increase of blood cortisol, whereas the scales probably seized the low increase of cortisol during the prolonged mild stress response or the increase of cortisol during the first sampling. For the same reason, hematocrit values did not differ (p=0.1) in relation to oxygen conditions, whereas the hematocrit decrease (p<0.05) over time could be a sign of anemia. The correlation of cortisol levels in plasma and in the alternative matrices, i.e. muscle, fin and mucus, was evident and significant (p<0.001, r=0.68; p<0.001, r=0.72 and p<0.001, r=0.60, respectively). Correlation between plasma and scales cortisol levels was also positive, even if less marked due to the different meaning of the hormone in these matrices in term of acute and long stress (p<0.05, r=0.36). Finally, plasma MDA concentrations were not significantly affected neither by the time nor by hypoxia and were similar to those already reported for European sea bass (Vinagre et al., 2012), highlighting the absence of oxidative stress.
Conclusions
The present study confirmed that scales could represent one of the most effective matrices to quantify cortisol levels and to evaluate the effects of sub-chronic and chronic stress, even in mild conditions. The hypoxia conditions applied in this study did not strongly impact European sea bass stress response, indicating that this species might have a high tolerance to poor water oxygenation. Nevertheless, these results need to be confirmed in future trials evaluating a further decrease of oxygen concentration and longer-term effects (beyond 7 days) both in terms of stress response and overall performance of fish.
Acknowledgements
The authors wish to thank Elena Negrato and Carlo Poltronieri for their technical assistance.
References
Bertotto, D. et al. (2010). Alternative matrices for cortisol measurement in fish. Aquaculture Research, 41, 1261-1267.
Samaras, A. et al. (2021). Cortisol concentration in scales is a valid indicator for the assessment of chronic stress in European sea bass, Dicentrarchus labrax L. Aquaculture, 545, 737257.
Sadoul, B & Geffroy, B. (2019). Measuring cortisol, the major stress hormone in fishes. Journal of Fish Biology, 94, 540-555.
Vinagre, C. et al. (2012). Effect of temperature on oxidative stress in fish: Lipid peroxidation and catalase activity in the muscle of juvenile seabass, Dicentrarchus labrax. Ecological Indicators, 23, 274-279.