**2.2 Redox balance**

*Redox*

studies in fish.

Like to the studies in mammals' species, the most of fish approaches on redox aquaculture subjects were focused in liver as the main physiological organ. Liver shows powerful enzymatic antioxidant machinery, it is involved on glutathione synthesis and it is a main target of reactive oxygen species (ROS). However, digestive tract, white and red muscle even plasma are of a novel targets for redox

Despite the bibliography reporting redox markers in the last years are increasing, there are only few works tackling on the redox balance and the consequences of its alteration in fish welfare. This chapter aims to evaluate the most relevant and recent studies, and the advances of oxidative status, antioxidant defences and global welfare in fish. Along the chapter we will be focused on the studies which report

Oxygen Free Radicals are highly reactive species which are known to be the major factor in oxidative cell injury via the oxidation and subsequent functional impairment of lipids, carbohydrates, proteins and DNA. In the 1950s, free radicals were first identified in biological systems and were proposed to be involved in pathological processes [3]. The major source of intracellular free radicals is mitochondria due to the presence of an electron transport chain [4, 5], which consumes 85–90% of the oxygen utilized by cell [4, 6]. While passing through the mitochondrial electron transport chain, up to 2% of the total oxygen consumed undergoes one-electron reduction to generate superoxide anion radicals (O2•<sup>−</sup>) and hydrogen peroxide (H2O2). This hydrogen peroxide may lead to hydroxyl radical (OH•

most reactive free radical produced in biological systems, with the participation of transition metals in the Haber-Weiss reaction [7]. In addition, different stressors, particularly those induced by environmental physical and chemical factors were reported to increase levels of free radicals. As research on free radicals focused on oxygen radicals, with some other forms of non-radical active oxygen, they are col-

As the formation of reactive oxygen species (ROS) is a part of natural cellular oxidative metabolism, the question if living organisms possess regulated enzymatic systems to defend against ROS, suddenly arises. This was first confirmed by McCord and Fridovich [8] who discovered the enzyme superoxide dismutase (SOD) and demonstrated that living organisms have developed protective mechanisms against ROS. Over time, this was supported by continuing discoveries of several mechanisms by which ROS can be neutralized: antioxidant enzymes and low molecular mass antioxidants. During normal oxidative metabolism ROS are produced continually, but they are scavenged by superoxide dismutase (SOD), glutathione peroxidase and catalase [9]. Other small molecular antioxidants: glutathione, ascorbic acid and a-tocopherol are also involved in the detoxification of free radicals. Those reported evidences lead to Helmut Sies [10] to first propose the following definition of oxidative stress as 'imbalance between oxidants and antioxidants in favour of the oxidants, potentially leading to damage'. Oxidative stress was considered to be harmful, while antioxidants provided defence and prevention of tissue damage. However, ROS were recently found to play signalling roles not only in ROS-related processes, but in many

basic functions such as fertilization, growth, and differentiation [11–14].

), the

dietary effects and environmental challenges on fish culture.

**2. Redox reactions on ectotherms versus endotherms**

lectively referred to as reactive oxygen species (ROS).

**2.1 Definition of oxidative stress**

**94**

We have described how reactive oxygen species (ROS) can be both harmful and beneficial. Consequently, under physiological conditions, the cellular redox equilibrium is tightly regulated on the one hand by pro-oxidants and on the other by enzymatic and non-enzymatic antioxidants (**Figure 1**). Due to the central role of ROS in many pathologies, restoring the redox balance forms an innovative target in the development of new strategies for treating several conditions. For example, Coenzyme-Q and its redox status -that was mostly found in the reduced form- have been proposed as an adaptation to different thermal environments in Antarctic fishes [15], and to reflects species-specific ecological habits and physiological constraint associated with oxygen demand represent an adaptation to environmental oxygen availability in coral reef fishes [16]. Recently, the determination of the redox balance of liver Coenzyme-Q from fish has been observed to have a potential, based on physiological principles, to be used as a practical biomarker for polycyclic aromatic hydrocarbon (PAH) contamination in aquatic biotopes [17].

The main marker of oxidative damage in fish tissues have been considered as lipid peroxidation (LPO, usually measured as ThioBarbituric Reactive Substances, TBARS) [18–20]. However, the application of mammals' techniques to measure the direct oxidation of the amino acid side chains, so-called the Advanced Oxidation Protein Products, (AOPPs) or/and the tissue accumulation of 4-hydroxinonenal (4-HNE) cross-linked proteins are novel approach in the study of oxidative insult in fish species [21].

To cope with the oxidative damage resulting from metabolism, animals use non-enzymatic defences, such as thiol groups and glutathione, and enzymes with antioxidant activity [1, 20, 22]. Thus, in fish as in mammals, antioxidant liver capacity has been classically measured via total glutathione, (tGSH) and its oxidized and reduced forms (GSH and GSSG, respectively), and via the main antioxidant enzyme activities: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR).

### **2.3 Comparative studies: ectotherms versus endotherms**

The generic term 'fish' comprises an extremely diverse group of vertebrates, which represents 40% of the world's vertebrates. Fish are adapted to aquatic environment, showing a wide type of adaptations to different environmental conditions of temperature, salinity, oxygen level and water chemistry. To survive in such diverse environments, fish need high adaptive potential. If it is overwhelmed, organisms may enter stress conditions. The most studied environmental stress conditions in aquatic environments include changes in salinity, ion composition, and temperature and oxygen availability. Recently, they also included pollutants exposure due to human activity.

If the previous studies of comparative physiology were focused on the fish availability of O2, the generation of ROS and its harmful consequences are currently taken into account. In fishes, the main physiological source of ROS is also the mitochondria, and the mechanism of ROS production similar to that of mammal. However, the fraction of total mitochondrial electron flux that generate H2O2 (the fractional electron leak), was far lower in rat than in all the ectothermic fishes assayed [23]. Results previously published concluded that mitochondria of true endotherms (such as birds and mammals) produce lower rates of ROS generation compared to fish and showed higher levels of antioxidant enzymes compared to fish [24].

Despite similarities in mitochondrial coupling mechanisms and proton leakage, differences in mitochondrial function between mammals and fish have been reported. A greater phospholipid unsaturation, the presence of cardiolipin and the absence of cholesterol in the mitochondrial membranes of fish can establish different aspects of mitochondrial functionality in fish. In fact, the presence of the phospholipid cardiolipine and the absence of cholesterol may confer a high structural flexibility in fish mitochondria. Moreover, differences in membrane phospholipid composition involving polyunsaturated fatty acids (PUFA) may play a role in the proton leak across the mitochondrial membranes (which can explain greater proton leak in endoherms when compared to ectotherms) and in the lipid peroxidation process. It has been previously demonstrated the correlation between the consumption of polyunsaturated fatty acid diet and the enhancement of endogenous lipid peroxidation, in rats [1, 25, 26]. As the increased unsaturation of fatty acids leads to increased parameters of oxidative stress damage, this increases the oxidative challenge to fish tissues. However, some important differences in the lipid composition of membranes have been reported for phylogenetic distinct marine fish species [27]. In the referred work, the elasmobranch *Raja erinacea* showed lower percentage of polyunsaturated fatty acids when compared to non-elasmobranch species. Fishes of the Antarctic seas showed relatively high proportion of polyunsaturated fatty acids in mitochondria [28], which make them prone to lipid peroxidation. Indeed, reported TBARS levels are much higher in fish liver then in mammals [24].

As reported above, all aerobic forms of life developed antioxidant defences. Both, low-molecular weight antioxidants and antioxidant enzymes like catalase, superoxide dismutase and glutathione- dependent enzymes, have been detected in different fish species [1]. Many studies confirm that enzymatic antioxidant activities in fish were lower than in endotherms. As fish refers to different evolved species, some studies have been performed to determine whether antioxidants correlate with phylogenetic position [29]. Marine fishes showed high levels of the antioxidant vitamin E [30] and elasmobranchs compensate limited antioxidant enzymes with high levels of glutathione and urea [31]. It seems that low molecular weight antioxidants appeared early in evolution to later develop enzymatic antioxidant systems.

**97**

*+*

*\**

**Table 1.**

*Redox Balance Affects Fish Welfare*

**Diet effect** *Dicentrarchus labrax*

*DOI: http://dx.doi.org/10.5772/intechopen.89842*

**3.1 Redox balance in feeding strategies**

Mediterranean fish related to redox balance.

*Sparus aurata* Liver Supplemented diet Met

Liver, intestine

Liver, intestine

Mucus, gut, skin

Liver, intestine

Liver, intestine

Liver, intestine

white muscle

Liver, intestine

plasma

*Gene expression of antioxidant enzymes.*

*To enhance the response to handling stress. \*\*To enhance the response to thermal stress.*

*Dentex dentex* Liver,

**Diet to enhance welfare**

*Sparus aurata* Liver,

*Dicentrarchus labrax*

*Scophthalmus maximus*

*Solea senegalensis*

**3. Advances in redox balance of temperate fish species**

Substitution diets and carbohydrates reduction [32]

Palm fruit extract and

[43]

Soybean and wheat with glutamine [38]

Substitution diets and carbohydrates reduction [34]

supplemented with Met and

Different carbohydrates and carbohydrates amount [35]

Substitution diets and carbohydrates reduction\*

Soybean and wheat with arginine

and Tea [41]

probiotics+

[39]

Liver Soybean replacement

*Last decade works related to feeding strategies in Mediterranean teleost fish.*

phosphate [37]

Usually, the main aim of the diet trials in fish pursue to improve growth performance as well as food conversion and efficiency, guaranteeing a high-quality product, reducing fish mortality and with economic positive gains. Recently, feeding studies include redox balance markers as additional indicators of the fish physiological status. **Table 1** represents the most relevant feeding strategies in

**Tissue Diet strategy Oxidants Antioxidants**

Soy based diets and Taurine [40] LPO SOD, CAT

Intestine Sprayed diet porcine protein [42] TBARS CAT, GR, GST, GPX

Liver Soybean replacement [36] SOD, CAT, GR, GPX, GST

[33]

Liver Reduced dietary proteins\*\* [19] LPO SOD, CAT, GR, GPX,

Liver Prebiotics\*\* [53] LPO SOD, CAT, GR, GPX,

LPO, carbonylated proteins

Lipid reduction\*\* [21] LPO, AOPP SOD, CAT, GR, GPX, GSH

LPO SOD, CAT, GR, GPX,

LPO SOD, CAT, GR, GPX,

LPO SOD, CAT, GR, GPX, G6PDH

LPO SOD, CAT, GR, GPX, G6PDH

LPO SOD, CAT, GR, GPX,

G6PDH

LPO SOD, CAT, GR, GPX, G6PDH

G6PDH

and GSSG

G6PDH

LPO, H2O2 SOD, CAT, GR

G6PDH, GSH and GSSG

G6PDH, GSH and GSSG

G6PDH, GSH and GSSG

SOD, CAT, GR, GPX, GST

SOD, CAT, GR, GPX,

*Redox*

**2.3 Comparative studies: ectotherms versus endotherms**

exposure due to human activity.

The generic term 'fish' comprises an extremely diverse group of vertebrates, which represents 40% of the world's vertebrates. Fish are adapted to aquatic environment, showing a wide type of adaptations to different environmental conditions of temperature, salinity, oxygen level and water chemistry. To survive in such diverse environments, fish need high adaptive potential. If it is overwhelmed, organisms may enter stress conditions. The most studied environmental stress conditions in aquatic environments include changes in salinity, ion composition, and temperature and oxygen availability. Recently, they also included pollutants

If the previous studies of comparative physiology were focused on the fish availability of O2, the generation of ROS and its harmful consequences are currently taken into account. In fishes, the main physiological source of ROS is also the mitochondria, and the mechanism of ROS production similar to that of mammal. However, the fraction of total mitochondrial electron flux that generate H2O2 (the fractional electron leak), was far lower in rat than in all the ectothermic fishes assayed [23]. Results previously published concluded that mitochondria of true endotherms (such as birds and mammals) produce lower rates of ROS generation compared to fish and

Despite similarities in mitochondrial coupling mechanisms and proton leakage, differences in mitochondrial function between mammals and fish have been reported. A greater phospholipid unsaturation, the presence of cardiolipin and the absence of cholesterol in the mitochondrial membranes of fish can establish different aspects of mitochondrial functionality in fish. In fact, the presence of the phospholipid cardiolipine and the absence of cholesterol may confer a high structural flexibility in fish mitochondria. Moreover, differences in membrane phospholipid composition involving polyunsaturated fatty acids (PUFA) may play a role in the proton leak across the mitochondrial membranes (which can explain greater proton leak in endoherms when compared to ectotherms) and in the lipid peroxidation process. It has been previously demonstrated the correlation between the consumption of polyunsaturated fatty acid diet and the enhancement of endogenous lipid peroxidation, in rats [1, 25, 26]. As the increased unsaturation of fatty acids leads to increased parameters of oxidative stress damage, this increases the oxidative challenge to fish tissues. However, some important differences in the lipid composition of membranes have been reported for phylogenetic distinct marine fish species [27]. In the referred work, the elasmobranch *Raja erinacea* showed lower percentage of polyunsaturated fatty acids when compared to non-elasmobranch species. Fishes of the Antarctic seas showed relatively high proportion of polyunsaturated fatty acids in mitochondria [28], which make them prone to lipid peroxidation. Indeed,

reported TBARS levels are much higher in fish liver then in mammals [24].

As reported above, all aerobic forms of life developed antioxidant defences. Both, low-molecular weight antioxidants and antioxidant enzymes like catalase, superoxide dismutase and glutathione- dependent enzymes, have been detected in different fish species [1]. Many studies confirm that enzymatic antioxidant activities in fish were lower than in endotherms. As fish refers to different evolved species, some studies have been performed to determine whether antioxidants correlate with phylogenetic position [29]. Marine fishes showed high levels of the antioxidant vitamin E [30] and elasmobranchs compensate limited antioxidant enzymes with high levels of glutathione and urea [31]. It seems that low molecular weight antioxidants appeared early in evolution to later develop enzymatic antioxi-

showed higher levels of antioxidant enzymes compared to fish [24].

**96**

dant systems.
