| Abstract |
Τhe last decade, animal welfare is of increasing concern to scientists, consumers and producers. Although there is not a generally accepted definition of welfare, it refers to an animal’s physical and mental state with regard to its environment. Stress can be an important factor of welfare disturbance and it is considered to be a condition in which homeostasis is threatened or disturbed. Stress is not necessarily detrimental for the organism. On the contrary, the response to stress is considered an adaptive mechanism that allows the fish to cope with harmful or threatening situations. When fish are exposed to severe or persistent stressors and they are not capable of regaining homeostasis, then the responses themselves may become maladaptive and threaten the fish’s health and well-being. The allostasis concept has been introduced recently. Allostasis, i.e. stability through change, is a fundamental process through which organisms actively adjust to both predictable and unpredictable events.
Physiological stress responses are divided into primary (endocrine level: release of cortisol and catecholamines), secondary (level of metabolism and osmoregulation) and tertiary (level of the organism). Increased plasma cortisol is a reliable and easily detected indicator but it is influenced by husbandry conditions, capture and bleeding, type of stress (acute or chronic), physical factors and the physiological state of the fish..
Recently, research has focused on the development of non-invasive methods for estimating stress response, such as water cortisol determination. Free cortisol release into the water represents a passive “leakage” through the gills, due to concentration differences between plasma and water. There are several advantages of the non-invasive procedure compared to blood sampling: (i) water sampling does not disturb the fish; (ii) repeated samples can be taken from the same population in time-course experiments reducing the numbers of tanks and animals required, and (iii) stress experiments can be carried out on fishes that are too small to be bled. In addition, behavioral and physiological monitoring can be performed at the same time. The disadvantages of the non-invasive procedure are that it effectively integrates the cortisol release of all members of a population and it is likely to depend not only upon the plasma cortisol concentration, but also on the rate of diffusion of cortisol through the gills. Diffusion of cortisol through the gills is influenced by a variety of factors including gill surface area, permeability, branchial blood flow and ventilatory water flow and it may vary with species, fish size and environmental factors, such as water temperature.
The overall aim of the present study was to investigate the stress response and to develop and evaluate a non-invasive method for the determination of free cortisol release into seawater in several Mediterranean fish species, with emphasis on the European sea bass, Dicentrarchus labrax.
The study is divided in two sections. The first section mainly concerns methodological items and basic experimentation, and in particular the:
(i) Development and evaluation of a non-invasive method for the determination of free cortisol released into seawater for European sea bass (Chapter 2),
(ii) Determination of the effect of sampling on the stress response of six fish species (European sea bass, gilthead seabream, Sparus aurata, common Pandora, Pagellus erythrinus, sharpsnout seabream, Diplodus puntazzo, dusky grouper, Epinephelus marginatus and meager, Argyrosomus regius) of 4 Mediterranean families (Chapter 3), and
(iii) Determination of the species-specificity in the acute stress response of seven Mediterranean fish species (European sea bass, gilthead seabream, common pandora, sharpsnout seabream, dusky grouper, meager and common dentex, Dentex dentex) (Chapter 4).
Results showed that the non-invasive method is reliable for the assessment of stress response for European sea bass maintained in sea water, although the cortisol release rate (ng g-1 h-1) was lower than expected based on the high plasma cortisol concentrations observed in this species. In addition, it was shown that there is species-specificity in capture tolerance and that capture with hook and line generally induced a lower stress response. Finally, for the first time in these species, results clearly showed species-specificity of the primary and secondary stress response both in the magnitude and the pattern of changes. Cortisol release rates into the water were lower than those of fresh water species with regard to the plasma cortisol concentrations.
The second section of the study is focused on the investigation of significant parameters of the European sea bass stress response and in particular the:
(i) Peripheral regulation of cortisol (comparative approach: European sea bass and dusky grouper, species with high and low cortisol stress response, respectively) (Chapter 5),
(ii) Consistency of the cortisol stress response: investigation into the presence of high (HR) and low (LR) stress-responsive fish (Chapter 6),
(iii) Ontogeny of the cortisol stress response during early development (Chapter 7),
(iv) Effect of fish size on the cortisol release rate (Chapter 8), and
(v) Effect of sex and sexual maturation in plasma cortisol and cortisol release rate (Chapter 9).
Results showed that European sea bass generally presented higher cortisol concentrations in the tissues studied, but only liver and spleen were statistically significantly different compared to the dusky grouper. In regard to the consistency of the stress response, results did not confirm the existence of consistently divergent plasma cortisol and glucose responses in European sea bass exposed to acute stress. Ontogenesis of cortisol in European sea bass under intensive farming conditions displayed a similar pattern to that of other fish species studied so far, but with higher whole-body cortisol concentrations, especially after the stage of flexion (27 Days Post Hatching, DPH). Whole-body cortisol content was decreased during the embryonic development to reach a minimum at hatching and then started to increase due to de novo cortisol synthesis (11 DPH). Stress response was activated at first feeding stage (11 DPH), when the Hypothalamus-Pituitary-Interrenal (HPI) axis became functional, though at a lower extent than subsequent stages. Transport simulation of juvenile European sea bass in two different densities (20 kg m-3 and 50 kg m-3 ) showed elevated cortisol release rates. Results indicate that there is a difference in cortisol release rate with size, although the two sizes studied had similar plasma cortisol concentrations. Larger individuals presented a 3-fold higher cortisol release rate than smaller fish, in accordance to their body mass difference. Finally, results confirmed that plasma cortisol concentrations are increased in mature individuals of both sexes. In addition, plasma cortisol was higher in males reared isolated from females than in males in the mixed group (1:1 sex ratio), suggesting possible stress due to altered social relationships or due to the absence of female hormones
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