Formation of an antioxidant profile in the sea trout (Salmo trutta m. trutta L.) from the Slupia River

Accepted Manuscript Title: Formation of an antioxidant profile in the sea trout (Salmo trutta m. trutta L.) from the Slupia River Author: Natalia Kurhaluk PII: DOI: Reference:

S0944-2006(18)30141-7 https://doi.org/10.1016/j.zool.2019.02.002 ZOOL 25675

To appear in: Received date: Revised date: Accepted date:

8 August 2018 5 February 2019 11 February 2019

Please cite this article as: Kurhaluk N, Formation of an antioxidant profile in the sea trout (Salmo trutta m. trutta L.) from the Slupia River, Zoology (2019), https://doi.org/10.1016/j.zool.2019.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Formation of an antioxidant profile in the sea trout (Salmo trutta m. trutta L.) from the Slupia River

Short title: Antioxidant profile and life cycle of the sea trout

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Natalia Kurhaluk

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Department of Physiology, Institute of Biology and Environmental Protection, Pomeranian University of Slupsk, Slupsk, Poland

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*Corresponding author:

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Natalia Kurhaluk, Ph.D.

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Department of Physiology,

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Pomeranian University of Slupsk,

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Institute of Biology and Environmental Protection,

Arciszewskiego 22b Str., 76-200 Słupsk, Poland

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Highlights

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E-mail: [email protected]; Tel +48 511 311 112

 The functioning of the pro/antioxidant balance reflects the course of stages of the

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trout.

 The research has shown relationships between the antioxidant defense and sex in the adult stage.  The highest level of toxic oxidation reaction was noted in the kelt form in the liver.

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 Rank order for the antioxidant protection is presented as follows: CAT>SOD>GPx>GR and TBARS>OMP KD> TAC> OMP AD.

Abstract Using a stage- and sex-based multivariate significance tests on the sea trout Salmo trutta m.

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trutta L. model, we show dependencies in the balance between lipid peroxidation processes, levels of carbonyl derivatives, and activity of antioxidant enzymes (superoxide dismutase

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SOD, catalase CAT, glutathione reductase GR, and peroxidase GPx) in the processes of

antioxidant profile formation during the fish growing process. The study was aimed at examination of the relationships between the biomarkers of oxidative stress estimated by the

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total antioxidant status as well as the dependencies between the sex (male, female) and

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developmental stage of the wild sea trout from the Slupia River and its catchment area rivers.

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Functioning of the pro/antioxidant balance of the liver tissue reflected the course of the

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individual developmental stages of the trout and was associated with significant intensification of lipoperoxidation, oxidative modification of proteins, and reduction of the

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total antioxidant capacity of fish along with age. Formation of a holistic model for the

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analysis of the involvement of all parameters of antioxidant protection in all stages of development and sex allowed us to obtain the following rank order for the level of processes,

modified

proteins,

and

antioxidant

enzyme

complex:

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lipoperoxidation

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CAT>SOD>GPx>GR and TBARS>OMP KD> TAC> OMP AD.

Key words: antioxidants, developmental stage, sea trout, oxidative stress, oxidatively modified proteins, sex

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1. INTRODUCTION Sea trout are the anadromous and brown trout are the resident forms of the same species Salmo trutta L., and that any morphological and behavioral differences between them are only differences to a degree (Quinn, 2005; Wells et al., 2007). The sea trout resources in the Baltic Sea are at risk; hence, the ecophysiology of this fish depends on water management

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(Kallio-Nyberg et al., 2007; Degerman et al., 2012). Sea trout populations are affected by a

multiplicity of anthropogenic and environmental influences factors: water pollution (Chitra

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and Sajitha 2014), degree of anthropological transformation of the valley, regulation of

waterfronts and river bottoms (Kallio-Nyberg et al., 2002), deforestation of river valleys and

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hydrotechnical development (Dębowski and Bartel, 1995), smoltification process (Dieperink

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et al., 2001), temperature (Lahnsteiner et al., 2012), and eutrophication of the Baltic Sea

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(Ronnberg and Bonsdorff, 2004; Saikku and Asmala, 2010). Sea trout have an especially high

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social and economic importance as a source for aquaculture, water management, recreational angling in both freshwater and seawater and value as food. The sea trout is a very important

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factor enriching the biodiversity of the Pomeranian region of Poland, and the Slupia River in Northern Poland is best known for the possibility of sea trout catching.

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The evaluation of the physiological condition of fish (health, immunity, fecundity) requires

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the use of different test methods. A good measure of the condition of fish is the assessment of the oxidative stress level (Sinha et al., 2014). Free radicals and related molecules are classified as reactive oxygen species (ROS) for their ability to lead to oxidative changes

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within the cell. A wide variety of ROS are produced in the course of the normal metabolism in biological systems. ROS have several important physiological functions, but their accumulation beyond the needs of the cell can potentially damage lipids, proteins, and nucleic acids (Halliwell and Gutteridge, 2007).

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Oxidative stress describes a condition in which cellular antioxidant defences are insufficient to keep the levels of ROS below a toxic threshold (Kolakowska and Bartosz, 2010). Oxidative stress results as a consequence of an imbalance between the production of ROS, and the ability of biological systems to readily detoxify the reactive intermediates or to repair the resulting damage. Elevated cellular oxidative stress has been noted by numerous studies wild

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fish functioning levels and aquaculture conditions (Kurhalyuk et al., 2009; Tkachenko et al., 2014a; Tkachenko et al., 2015; Birnie-Gauvin et al., 2017a,b). The cells possess an intricate

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network of defence mechanisms to neutralize excessive ROS accumulation, including

antioxidant compounds (e.g., glutathione (GSH), vitamin E, vitamin C, selenium, zinc, vitamin A) and antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione

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reductase, and glutathione peroxidases); therefore, under physiological conditions, cells are

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able to cope with the flux of ROS (Halliwell and Gutteridge, 2007).

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Analysis of changes in oxidative stress provides a very important indicator of the adaptive

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abilities and condition of fish (physiological and biochemistry metabolism). The analysis of

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the dynamics of changes in oxidative stress markers and metabolic changes allows assessment of the degree of the physical condition of fish (Dziewulska et al., 2008). From this point of

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view, the sea trout Salmo trutta m. trutta L. is one of the very useful model for assessment of the course of physiological processes. It is sensitive to environmental changes, which is

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reflected by the presence of oxidative stress in tissues (Sreejai and Jaya, 2010) and to climate changes related to temperature (Passi et al., 2005). The species is also used in assessment of

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the cleanness of river and sea waters by following the migrations of this anadromous fish (Valon et al., 2013) and an important element of aquaculture. In the process of individual fish development, the functioning of immune, reproductive and neuroendocrine systems changes with age (Dziewulska and Domagała, 2006; Dziewulska et al., 2008). These changes depend to a different sex of the vertebrates (Costantini, 2018). The

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functioning of the redox system depends on these basic elements of fish physiology as well (Birnie-Gauvin et al., 2017a,b). Since the balance between lipid peroxidation processes, levels of carbonyl derivatives, and antioxidant enzyme activity (superoxide dismutase, catalase, glutathione reductase, and peroxidase) plays an important role in the processes of antioxidant profile formation during the fish growing process, the aim of this study was to examine the

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relationships between the biomarkers of oxidative stress estimated by the total antioxidant

status as well as the dependencies between the sex (male, female) and developmental stage

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(parr, smolt, adult, spawner) and kelt as a form of survival of the wild sea trout Salmo trutta m. trutta L. from the Baltic Sea and its catchment area rivers. In particular, we characterize the trends in the main effects (i.e. the sex and developmental stage of the sea trout) in

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formation of the antioxidant balance by the fish developmental stage and the interaction of the

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sex and developmental stage effects simultaneously. We also identify important determinants

2. Materials and methods

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of the antioxidant balance from one developmental stage to the other.

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2.1 Animals and experimental design

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2.1.1 Characteristics of the experimental groups We used following terminology and the definition of concepts from the work of the

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authors Pratten and Sherarer (1983). Sea trout are the anadromous and brown trout are the resident forms of the same species Salmo trutta L. The parr developmental stage is

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characterized by the age of 2-3 years and stays in fresh water. This form persists until the individuals come to the sea. A proportion of juvenile sea trout does not smoltify during the course of their first seaward migration and are indistinguishable from brown trout parr (Piggins, 1975; Pemberton, 1976). In the smolt stage, the trout species reach a size of 10-20 cm; this is a two-habitat stage, because the fish head for the sea. The adult sea trout

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developmental stage includes individuals that return to the rivers as adult specimens after living for 2-3 years in the sea. The fish reaches a weight of 2-3 kg, is sexually mature, and is characterized by a silver color of the sides (Birt and Green, 1986; Debowski, 1999). During migration, the adult sea trout do not feed. The previous spawner group of the sea trout includes individuals of both sexes mature for breeding and spawning. Kelt group – a fish

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which had recently spawned but had not recovered condition or commenced new growth (Pratten and Sherarer, 1983). Kelts are fish that have just finished spawning, and which will

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return to sea.The kelt form is regarded as a form of trout survival.

The research material was collected in the years 2008 - 2014 from 365 specimens of the sea

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trout in various developmental stages: parr (n = 113, length to 15 cm and weight to 25 g),

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smolt (n = 121, length 13-25 cm and weight 30-40 g), adult (n = 20, male=8 and female=12,

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length to 50-70 cm and weight 2-5 kg), spawner (n = 87, male=34 and female=53, length 50-

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70 cm and weight 2-4 kg), and kelt as form of survival (n = 24, male=12 and female=12, length to 70 cm and weight 1.5-3 kg). At the adult, spawner, and kelt forms the sexual

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dimorphism is well-expressed, which is why the analysis was carried out according to the sex because the phenotypic males and females had testicular and ovarian structures. We analyze

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those stages of fish development, when the changes in the neurohormonal levels determining

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sex are expressed (testicular and ovarian structures, eggs) and fish show gonadal development and differentiation. The physiological state of sea trout was evaluated using a complex of morphological parameters (mean body mass, mean body length) and aging according (Jensen

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and Johonsen, 1982). This corresponded to our aims - to establish a link between the development of oxidative stress, the stage of development and clearly expressed phenotypic males and females. The trout specimens in the parr and smolt developmental stages were caught in Pomeranian rivers: Glazna, Skotawa, Kamienna, Kwacza near Slupsk (Fig. 1). The trout specimens of the

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adult were caught in the estuary of the Slupia River (Ustka), and the sexually mature spawner and kelt sea trouts of both sexes were caught in the Slupia River (Slupsk, Pomeranian Voivodeship, Northern Poland). The fish was collected using the electric fishing method, with the help of a power generator (Honda) with a DC adapter. Technical data of DC adapter: 230 V, 50 Hz, 10 A, maximum voltage 320 V, maximum current 10 A. The trout specimens were

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caught in close cooperation with the "Dolina Slupia" Landscape Park and the District Board of the Polish Angling Association in Slupsk. Percussion stunning followed by destruction of

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the brain was used for fish killing. The tissues for analysis were then collected. Samples of tissues was taken at the site of capture, frozen (-80° C) and homogenized in the lab.

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2.1.2 Tissue isolation

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Liver tissue for biochemical determinations was taken from each fish. The obtained tissue was

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homogenized in a cooled 0.1 M Tris-HCl buffer (pH 7.2) in a ratio of 1:10. The prepared

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homogenate was the basic research material, subjected to further biochemical analyzes, which

2.1.3 Chemicals

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were carried out with the following methods.

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2-Tiobarbituric acid (TBA), reduced glutathione (GSH), oxidised glutathione (GSSG), serum albumin, NADPH, 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and quercetine were

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purchased from Sigma (USA), Serva, (Germany), POCH (Poland). All chemicals were of analytical grade.

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2.1.4 Biochemical assays 2.1.4.1 Thiobarbituric acid reactive substances (TBARS) assay TBARS were measured by the method of Kamyshnikov (2004). Briefly, to 2 mL of distilled water was added to 0.1 mL homogenate of tissue (1;10), 1 mL of 20% trichloracetic acid (TCA) and 1 mL of 0.8% 2-tiobarbituric acid (TBA) reagent, and boiled in a water bath

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at 95 oC for 10 min. The reaction was stopped by placing the tubes in ice-cold water. Then mixture was centrifuged at 3,000g for 10 min. The absorbance of supernatant was read at 540 nm. The µmol of TBARS was calculated by using 1.56∙105 mM-1∙cm-1 as extinction coefficient. TBARS level was expressed in nmol of malondialdehyde (MDA) per mg protein.

albumin as a standard. Absorbance of proteins was recorded at 595 nm.

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2.1.4.2 Protein carbonyl derivatives assay

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TBARS level was recounted in mg protein by Bradford method (1976) with bovine serum

The oxidatively modified protein (OMP) rate was estimated by the reaction of the resultant carbonyl derivatives of amino acids with 2.4-dinitrophenyl hydrazine (DNFH) as described by

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Levine et al. (1990) in modification by Dubinina et al. (1995). The final solution was

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centrifuged to remove any insoluble material. The carbonyl content was calculated from the

·cm-1. Carbonyl groups were determined spectrophotometrically at 370 nm (aldehyde

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absorbance measurement at 370 nm and 430 nm and an absorption coefficient of 22,000 M-

nmol per mg of protein.

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derivates (AD), OMP370) and 430 nm (ketonic derivates (KD), OMP430), and expressed in

Total

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2.1.4.3 Total antioxidant capacity assay antioxidant

capacity

(TAC)

levels

in

the

liver,

tissue

were

estimated

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spectrophotometrically using the TBARS level and following the method with Tween 80 oxidation described by the authors (Galaktionova et al., 1998). Tissue inhibits

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Fe2+/ascorbate-induced oxidation of Tween 80, resulting in a decrease of TBARS level. The absorbance of the obtained solution was measured at 532 nm. Absorbance of blank was determined as 100%. The level of TAC in sample (%) was calculated in respect to the absorbance of blank. 2.1.4.3 Superoxide dismutase activity assay

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SOD activity of the tissues samples in the supernatant were performed according to Kostiuk et al. (1990). It is a colorimetric method based on the principle of measuring absorbance of the coloured quercetin autoxidation complex in an alkaline medium at 406 nm against water blank. SOD activity was determined in units of SOD per mg of protein. 2.1.4.4 Catalase activity assay.

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CAT activity was determined using the Koroliuk et al. (1988) method. Activity of the enzyme was estimated by the decrease in absorbance of hydrogen peroxide at 410 nm. The reaction

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mixture contained liver homogenate diluted in incubation medium (1:10), H2O2 and ammonium molybdate. One unit of catalase activity was expressed in nmol H2O2 per minute per mg of protein.

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2.1.4.5 Glutathione reductase activity assay

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Liver glutathione reductase (GR) activities was determined metric according to the

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colourimetric method of Glatzle et al. (1974) based on the NADPH oxidation in the presence

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of GSSG in sodium phosphate buffer (pH 6.6). The method based on the measurement of

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changes in absorbance at a wavelength of 340 nm, caused by the oxidation NADPH. A blank without NADPH was used, and the GR activity was expressed in nanomoles of NADPH per

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minute per mg of protein.

2.1.4.6 Glutathione peroxidase activity assay

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GPx activity was evaluated by using the nonenzymatic utilization of GSH after incubation of liver homogenate with 5,5-dithiobis-2-nitrobenzoic acid according by the method of Moin

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(1986). The absorbance was measured at 412 nm. The assay mixture contained 0.1 M Tris– HCl buffer, 6 mM EDTA, 12 mM sodium azide (pH 8.9), 4.8 mM GSH, liver homogenate sample, 20 mM t-butylhydroperoxide, and 0.1 mL of 0.01 M 5, 5-dithiobis-2-nitrobenzoic acid. GPx activity was expressed in nanomoles of GSH per minute per mg of protein. 2.2 Statistical analysis

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The results were expressed as mean ± S.D. All variables were tested for normal distribution using the Kolmogorov-Smirnov test (p>0.05) and homogeneity of variance was assessed using Levene’s test. The significance of differences in the level of antioxidant enzyme activity, lipid peroxidation, amino acid carbonyl derivatives, and between all examined groups was determined using one-way analysis of variance (ANOVA) and multifactorial

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analysis of variance (MANOVA). We used Tukey’s post-test for unequal observations. Statistical analysis was carried out in a double way: the effect of biomarkers of oxidative

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stress was compared with those in each developmental stage of the sea trout, and the combined effect of sex and developmental stages and its significance (main effects) was compared with the data of the lipid peroxidation processes, carbonyl derivatives, total

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antioxidant capacity, and antioxidant enzymes separately. Differences were considered

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significant at p<0.05. In addition, the associations between data of all individuals were

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evaluated using Pearson's correlation analysis. The correlation and regression analysis

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comprised the correlation coefficient (r), regression equation, and significance of these

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dependencies (p). We used the coefficients of multiple correlation analysis (R), the coefficient of determination (R2), and its corrected form reduced by random errors (R2 adjusted) in the

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data analysis for description of the full model. We used the SS test to describe the share of all analyzed biomarkers of oxidative stress for assessment of the antioxidant barrier with the F

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test and its significance. All statistical calculations were performed on separate data from each

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individual with STATISTICA 8.0 software (StatSoft Inc., Poland).

3. Results Oxidative stress associated with the analyzed developmental stages of the sea trout affects different organs and systems and is a marker of transition from one stage of life to another. Measurement of oxidative stress intensity is used for estimation of the concentration

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of malondialdehyde (MDA) as the final product of lipid peroxidation, which reacts with 2thiobarbituric acid to form a trimethyl complex with it (TBARS products). Therefore, in our research, we decided to analyze the level of one of the final products of lipoperoxidation processes, i.e. MDA. During the selected developmental stages, the level of lipoperoxidation processes in the liver tissue differed significantly [F7,359=127.99, p=0.000] with age and

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transition to every next stage of life. Data obtained in this analysis are shown in Fig. 2 (A).

There was a general, stable tendency to increase lipoperoxidation processes in the liver tissue,

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which was particularly pronounced for males and females in the last stage of life, namely the

spawner and kelt as a form of survival. In the first three stages (parr, smolt, and adult) of the trout development, we observed the lowest level of TBARS products that did not differ

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significantly between these groups. In the spawner and kelt forms, we observed five- and six-

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fold higher levels of the final value of lipoperoxidation products.

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The primary products of free radical oxidation processes can damage many important

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cell components, especially proteins. Therefore, the next stage of our analysis was devoted to

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determination of an oxidatively modified protein level (OMP) such as protein carbonyl derivatives OMP AD and OMP KD. Our results show a significant effect of the

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developmental stages on the increase in OMP AD [F7,359=68.44, p=0.000] and OMP KD [F7,359=89.86, p=0.000] in the liver. The results are shown in Fig. 2 (B) and Fig. 2 (C).

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However, we showed a statistically significant increase in the OMP AD and OMP KD in the liver together with the increasing age of the sea trout and with the each stage of development,

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respectively. The lipoperoxidation presented earlier and related to the developmental stages accompanies the increasing level of the oxidative modification of proteins. The whole redox reaction was associated with the total antioxidant capacity (TAC) parameter. Therefore, we decided to examine this sensitive index of oxidative balance in the various stages of trout development. The results of our research are presented in Fig. 3. Our

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results indicate significant dependencies [F7,359=6.19, p=0.000] in the TAC value in the liver tissue. We observed statistically significant changes only in individuals in the male smolt and spawner stage, compared with the first developmental stage, i.e. the parr. It should be noted that the highest value of the TAC parameter was observed for individuals in the male and female adult stage, which actively spend their first year in seawater in search of food

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(foraging); and the lowest value was found for the male kelt individuals, which after spawning go back to sea.

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The antioxidant enzyme activity in the liver tissue in response to developmental

processes was found to be highly specific. Our results showed that the SOD activity [F7,359=9.15, p=0.000] changed statistically significantly during the developmental stages as

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follows: there was a significant increase in the adult stage with subsequent reduction in the

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activity in the spawner and kelt forms (Fig. 4, A). It was observed [F7,359=131.00, p=0.000]

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that, due to the activity of SOD, the activity of catalase (CAT), i.e. an enzyme occurring in the

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second antioxidant defense line and inactivating hydrogen peroxide, is inversely correlated

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with SOD, depending on the developmental stage. In the first stages of trout growth (parr, first-year male and female smolt, and less for the smolt stage), the activity of the CAT

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enzyme was lower than in the spawner stage (male and female) and kelt (male and female) form. The results of this series of tests are shown in Fig. 4 (B).

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In our research, the enzyme activities associated with the metabolism of glutathione as

one of the basic antioxidants, namely glutathione reductase and glutathione peroxidase,

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exhibited the following changes presented in Fig. 5 (A, B). The highest activity of glutathione reductase [GR, F7,359=9.89, p=0.000] was observed for the parr and smolt developmental stages with a subsequent distinct tendency to decrease in a statistically significant manner in the male and female spawner and male kelt forms. It was observed that the other enzyme i.e. glutathione peroxidase [GPx, F7,359=14.29, p=0.000] showed an opposite trend to that found

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for GR, i.e. a tendency to increase with the age of the fish and the advancement of the developmental stages. Namely, the reduced GPx activity in the parr, smolt, and adult developmental stages was statistically significantly increased in the male and female spawner stage. In the present study, we have observed statistically significant dependencies between the

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activity of the investigated antioxidant enzymes and biomarkers of oxidative stress during the analyzed developmental stages in the sea trout. The correlation and regression analysis

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comprised the correlation and determination coefficients, the regression equation, and the

significance of these dependencies. The interdependencies are presented in a bivariant model (Table 1) and the most significant ones - in a three-variant manner (Fig. 6, 7). The parr and

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smolt stages were characterized by statistically significant positive and negative

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interdependencies, which had a lower absolute value than in the adult (male and female) and

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kelt (male and female) forms. The highest value of correlation interdependencies was

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observed in the adult stage between the intensive protein modification process estimated by

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the content of aldehyde and ketone derivatives and the intensity of lipid peroxidation in the liver of the male and female individuals (TBARS-OMP AD and TBARS-OMP KD).

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To verify the hypothesis of the impact of the developmental stage and sex on the formation of the antioxidant balance, we decided to evaluate these assumptions using a

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multivariate analysis of variance for three fish forms, where sexual dependence (adult, spawner, and kelt) was visible. The use of multivariate significance analysis of the main

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effects of the developmental stage and sex parameters in Wilk’s and Hotelling’s tests allowed us to obtain statistically significant relationships only for the developmental stage and the stage-sex interaction parameters, which is presented in Table 2. Determination of the significance of the interaction of the main sex-development effects for the selected tissue required the use of post-hoc tests to identify the most important

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relationships. We used this approach in two ways, i.e. separately for the developmental stage (Table 3) and for different sexes (Table 4). These interactions allow a conclusion that both the developmental stage and sex have a particularly important impact on the antioxidant barrier values. In the present study, we have observed dependencies between the investigated developmental stages in one sex in terms of the TAC, TBARS, OMP KD, SOD, CAT, and

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GPx levels. We also assessed the effect of the sex on the most important interdependencies in oxidative stress parameters such as TAC, TBARS, OMP KD, SOD, and GPx.

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We used the coefficients of multiple correlation analysis (R), the coefficient of

determination (R2), and its corrected form reduced by random errors (R2 adjusted) for the data analysis (Table 5). The SS test used to describe the profile of all analyzed biomarkers of

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oxidative stress for the assessment of the antioxidant barrier in the liver of the sea trout with

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the F test and its significance simultaneously allowed us to draw the following conclusions on

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the role of each of these parameters in the formation of full models. These dependencies are

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presented as follows: CAT>SOD>GPx>GR and TBARS>OMP KD> TAC> OMP AD. Such

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dependencies indicate the role of the CAT and SOD enzymes in the development of the sea trout and the intensity of lipoperoxidation processes such as TBARS generation, which can be

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4. Discussion

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used as biomarkers of oxidative stress in the developmental stages of fish.

The term "oxidative stress" is understood as a disturbance in the pro- and antioxidant

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balance, which may include overproduction of reactive oxygen species (ROS) or a decrease in antioxidant defense activity of the organism (Yadav et al., 2015). ROS react with biomolecules and cause damage to the cell, resulting in damage to proteins and lipids of biologically relevant compounds. Unfavorable changes include an increase in the level of

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lipid peroxidation processes as well as oxidative damage to proteins and DNA (Kolakowska and Bartosz, 2010). The present study demonstrated the impact of sex and developmental stages in the wild sea trout Salmo trutta m. trutta L. in formation of antioxidant protection. To date, there are no conclusive data in the available literature showing the relationship between biomarkers of

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oxidative stress and different developmental stages of these fish. In the available literature,

there are few reports describing changes in the functioning of the pro/antioxidant barrier at the

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level of tissues or organs of wild fish (Greani et al., 2017; Birnie-Gauvin et al., 2017ab).

There are even fewer publications from Poland devoted to the analysis of the antioxidant balance parameters among the wild trout of both sexes combined with the analysis of

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developmental stages. In a thorough analysis of the literature review, we found no papers

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about changes in oxidative stress markers and antioxidant balance at various stages of

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development of wild salmonids with the use of the sex division for description of the entire

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process.

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The destructive effects of radicals promoted by the lack of antioxidant capabilities of the organism can affect virtually all biomolecules present in the body, causing damage at the

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molecular and cellular levels (Sreejai and Jaya, 2010). Free radicals in vivo cause mainly chemical modifications and damage to proteins (aggregation and denaturation), lipids

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(peroxidation), carbohydrates, and nucleotides (Tkachenko et al., 2015). They induce changes in the DNA structure leading to mutations or cytotoxic effects. Their damage can even lead to

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cell death.

The liver is one of the tissues with a high level of metabolism; therefore, that is why the

changes occurring in this organ and others well reflect the markers of oxidative stress and hormonal changes determining the sex in assessment of the course of developmental stages (Godwin, 2010; Piferrer et al., 2012; Maitra and Hasan, 2016; Solomon-Lane et al., 2013;

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Martínez et al., 2014; Maitre et al., 2017). In our earlier publications (Kurhalyuk et al., 2009; Kurhalyuk et al., 2011a; Kurhalyuk et al., 2011b; Tkachenko et al., 2014a; Tkachenko et al., 2014b; Tkachenko et al., 2014c) we considered these processes separately in relation with other factors (diseases, aquaculture, rivers of fish occurrence). In this study, an attempt was made to assess the interesting basic phenomenon of the influence of the sex and

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developmental stages as separate elements and main factors for assessment of the formation of antioxidant balance in the sea trout (Table 2).

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There are several novels findings in the study. Firstly, our study demonstrated a general stable tendency to increase lipoperoxidation processes at five- and six-fold higher levels in liver tissue and particularly pronounced in males and females in the last two forms of survival

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with the increasing level of oxidative modification of proteins, namely in the spawner and kelt

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forms. Our research confirms the concept that the process of organism aging evaluated from

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the parr to kelt forms, is associated with an increase in lipoperoxidation and internal cellular

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damage caused by oxidative modification of proteins and simultaneous reduction in the total

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antioxidant balance. Secondly, our data of the antioxidant capacity level indicated that the highest significance of the TAC parameter in individuals of the male and female adult stages,

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which actively spend their first year in seawater and the lowest level - in the case of male kelt individuals, which after spawning go back to the Baltic sea. Our results showed the highest

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values of TAC, which is an indicator of the pro/antioxidant balance and adaptation reserves as well as changes induced in vivo by oxidative stress, in the parr and adult (male and female)

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stages, and in the spawner (only female) stage. The total antioxidant activity is determined by the redistribution of superoxide dismutase and catalase activity within TAC at different stages of development, which is confirmed by the data on the activities of these enzymes themselves and by the multivariate analysis. Thirdly, the results of our multiple correlation analysis (MANOVA) confirmed the working hypothesis that the level of tissue antioxidant balance

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and degradation of protein structures estimated as the carbonyl derivative content and lipoperoxidation level depends on the interactions between the main effects in our model, i.e. the sex-developmental stage effects. Our results demonstrated increased lipoperoxidation processes in liver tissue during development and aging, which was particularly pronounced for males and females in the last

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two forms of life, namely the spawner and kelt. This is related to the fact that lipid peroxidation is a process of oxidation of unsaturated fatty acids or other lipids, e.g.

SC R

phospholipids, components of cell membranes, resulting in peroxidation of these compounds (Harman, 1986; Sreejai and Jaya, 2010). This is an example of a free radical reaction with a non-radical compound that donates an electron and transforms into a free radical form. The

U

products of these compounds are aldehydes (e.g. malonic dialdehyde - MDA),

N

hydroxyaldehydes, and hydrocarbons. The concentration of MDA increases in the conditions

A

of increased RFT production, resulting in a change in the permeability of cell membrane,

M

which loses its biological properties, and disruption of oxidative phosphorylation in the

ED

mitochondria, which ultimately stimulates cell apoptosis (Nunes et al., 2015). Measurement of its concentration is widely recognized as a measure of free radical damage to lipids

PT

(Halliwell and Gutteridge, 2007). MDA has cytotoxic, genotoxic, mutagenic, and carcinogenic properties (Poćwierz-Kotus et al., 2013). It is a marker of oxidative stress,

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biological age, and cancer risk. Malondialdehyde generates the synthesis of lipofuscinic dyes and affects the phenomenon of cross-linking in proteins; it also causes degradation of cell

A

nuclei and inhibits mitotic divisions (Leliuna, 2010). The most reactive ROS present in biological systems are the hydroxyl radical (OH •)

and the superoxide radical (•O2-), because they can react with all biological macromolecules (proteins, lipids, nucleic acids, and carbohydrates) as shown Kolakowska and Bartosz (2010). The initial reaction generates a second radical that can react with the second macromolecule

17

and induce a chain reaction (Hensley et al., 2000; Halliwell and Gutteridge, 2007). The hydroxyl radical poses the greatest danger to the organism because it reacts with every organic molecule (Kaya and Akbulut, 2015). Free radicals involved in lipid peroxidation processes can react simultaneously with proteins. The modified proteins produced at that time can participate in termination reactions,

IP T

creating mixed protein-lipid combinations (Guéraud et al., 2010). Aldehydes, although less reactive than free radicals, react with thiol groups of proteins and with amino acid residues,

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e.g. lysyl, histidyl, arginyl, and tyrosyl. They can then change the antigenic properties of

proteins with which they bind and inhibit the activity of a number of enzymes (Stadman and Levine, 2000). In our investigations, we noted that the level of oxidatively modified proteins

U

estimated by the OMP AD and OMP KD increased with the age and developmental stages

N

and their interrelationships (Table 3 and 4). Our data confirm the general aging concept

A

(Harman, 1986) and exhaustion of the fish body during spawning due to the maximum

M

accumulation of oxidatively modified proteins (Fig. 2).

ED

The protein damage consisting of modification of amino acid residues, prosthetic groups, and aggregation or fragmentation of the protein molecule, leads to the loss of their

PT

biological activity. Our study provides additional results of the positive and negative correlation between oxidative stress parameters and the level of carbonyl derivatives

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depending on the sex and developmental stage (Table 1, Fig. 6). The mechanisms of formation of carbonyl derivatives during the oxidation of the side

A

chains of amino acids in proteins catalyzed with metal ions deserve attention (Stadman and Levine, 2000). Carbonyl derivatives have become one of the most frequently used markers of oxidative stress, and more specifically - oxidative modification of proteins. An increase in the concentration of carbonyl groups was observed for a large number of pathological states. It has been proven that the amount of protein oxidation products increases with the age.

18

Oxidized proteins can show a change in the biological activity and tend to form aggregates. The aggregates have the ability to inhibit systems responsible for their degradation and promote the accumulation of these altered proteins in cells (Sreejai and Jaya, 2010). In our investigations, we noted one of the highest TAC values in the parr stage of the sea trout. It has been determined that the parr stage is one of the key stages for the existence

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of the trout population, because it acquires biological traits and behaviors that decide about

the success in further stages of the life (Amundsen and Gabler, 2008). After leaving the nest,

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young fish begin to feed themselves. The trout fry remains in Polish rivers for about 2 years.

Parr fish intensely feed on invertebrate bottom fauna and actively defend its territory (Amundsen et al., 2001).

U

We concluded that adult individuals of the sea trout are characterized by stability in

N

maintaining high TAC values (Fig. 3). Young individuals undergo the smoltification process,

A

i.e. physiological and physical changes under the influence of hormones, enabling them to

M

move from fresh water to sea conditions (Björnsson and Bradley, 2007). These are physical

ED

changes in the body of smolts consisting in the disappearance of the colors and stains of the parr stage, occurrence of silver coloration, and body slimming (Dieperink et al., 2001). In

PT

terms of behavioral changes, loss or reduction of territorialism, changes in eating habits, leaving hiding places and feeding grounds, as well as tendencies to gather in groups and

CC E

limiting swimming activity, which may result from physiological changes, are observed. The change in environmental conditions to hyperosmotic in relation to the body fluid content of

A

the fish requires a significant amount of energy, which generates a high level of stress (LaizCarrion et al., 2002). Our results demonstrated that the antioxidant activity in the liver determined by SOD, CAT, GR, and GPx, depending on the stage of development and sex, was associated with the preferential activation of only some of the analyzed enzymes. We concluded that the SOD

19

activity (Fig. 4) was significantly increased in the adult stage, followed by subsequent reduction in this activity in the spawner and kelt forms in contrast to CAT (Fig. 4), whose activity was lower compared to the spawner (male and female) and kelt (male and female) forms. In our investigations, we noted that the activity of glutathione peroxidase (GPx), in contrast to GR, exhibited a tendency to increase with the fish age and along the

IP T

developmental stages (Fig.5).

The migration of fish from fresh water in rivers to the water of the salt sea is associated

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with the development of a number of adaptations, and as shown by our data, a significant

change in the antioxidant enzymatic preferences. It is known that Baltic Sea waters have an ion level similar to a physiological state, i.e. around 0.7%. For the proper exchange of ions

U

that does not cause disturbances in fish health, the chloride cells are rich in mitochondria.

N

These structures are located in the gills and epithelia lining the operculum bones, as well as

A

the intestinal epithelium and the trunk kidney (Maetz and Bornancin, 1975). They are

M

connected with the activity of ATP-ases and the functioning of the sodium-potassium pump

ED

(Schmidt-Nielsen, 1993). Their work is to protect the body from dehydration by the outflow of water outside the body in order to maintain the osmotic balance. The entire regulatory

PT

process is associated with the endocrine control. We concluded higher dependencies of the sex-development stage main effects in

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MANOVA analysis for the antioxidant profile of different structures (Table 2-4). The results of our study are in line with those reported by other authors, i.e. the spawning process is one

A

of the most important reproductive stages that ensure species protection during ontogenesis and phylogenesis (Pedersen et al., 2006). This process consists of several cycles characterized by significant changes in the functioning of neuroendocrine, immune, and reproductive systems (Dziewulska and Domagała, 2006; Dziewulska et al., 2008) and in water and electrolyte balance. These changes are largely caused by deviations in the functioning of

20

elemental homeostasis, redistribution of metabolic pathways for energy needs, significant expenditure of energy for the maturation of eggs and dairy, and implementation of the spawning processes themselves (Repecka, 2003; Svendsen et al., 2004). According to Selye (1960), changes observed in the reproductive period of fish are similar to the general stress metabolic syndrome and are adaptive characteristics (Hochacka and Somero, 1971).

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According to our data, the exhaustion of the fish body during and after spawning due to the

maximum accumulation of modified proteins (Fig.2) is associated with redistribution of

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antioxidant enzymes (Fig.4-5) and a decrease in TAC (Fig.3).

This study has some limitations related to the experimental methodology. Due to a high number of analyses, we resigned from the investigation of the effects exerted by where fish of

U

different stages were caught at different locations, and hence we cannot distinguish between

N

river effects and stage effects. We believe that such evaluations are sufficiently present in the

A

literature. Also, we believe we eliminated these factors by giving a environmental stress

M

condition to all health fish investigated. This approach enabled us to obtain the data on the

ED

influence of sex and development stage on oxidative stress in the liver tissue remaining on pollution and other environmental factors and subjected to the oxidative stress biomarkers

PT

induced by sex and development stages; the data that have not been hitherto reported. The results of this work may be useful for the construction of national fisheries

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management principles in industrially and agriculturally exploited regions. Such an ecological and economic approach is important for the assessment of the proper state of the population

A

of this fish species, the importance of which for the fisheries economy is currently growing. Salmonid fish species are considered to be one of the most valuable natural and economic specimens of the ichthyofauna of the Slupia and its catchment areas. These species are also of great angling importance, thanks to which they significantly influence the development of tourism and income from angling in the Pomeranian Voivodeship. It is important that

21

migratory fish are an important element enriching the biological diversity of the rivers in our province. The results of this study provide evidence that functioning of the pro/antioxidant balance of the liver tissue reflects the course of individual developmental stages of the trout (parr, smolt, adult and spawner) and is associated with significant intensification of

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lipoperoxidation, oxidative modification of proteins, and reduction of the total antioxidant capacity of fish with the age. The results regarding the formation of the basic elements of

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antioxidant defense mechanisms, such as the superoxide dismutase, catalase, glutathione

peroxidase and glutathione reductase, show a compensatory effect of the antioxidant protection components depending on the course of developmental stages and the sex of

U

individuals. The research has shown sex-related relationships between the antioxidant defense

N

and the tissue type in the adult stage as well as modifications of the antioxidant defense

A

induced by long-term environmental stress associated with changing the habitats from

None.

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Founding

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Conflict of interest

ED

M

freshwater to salty water and intense migrations.

The study was supported by Pomeranian University of Slupsk.

A

Acknowledgment

The author would like to thank Dr Halina Tkachenko, Dr Katarzyna Palczynska-Gugula and Miroslawa Jagodzinska for technical assistance and help in collecting samples. The author would like to thank District Board of the "Dolina Slupia" Landscape Park and the District Board of the Polish Angling Association in Slupsk.

22

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IP T SC R U N

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Fig. 1. Maps of Pomeranian region in northern Poland. Marked is Slupsk city (Pomeranian

A

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PT

ED

M

region, Poland) where samples from trout were collected.

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cc e f j

Kelt F Kelt M

cc e ee ff

Spawner F

b bb dd b bb d

Spawner M

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Adult F Adult M

Parr 0

100

200

300

400

500

µmol MDA•ml-1

Spawner F

800

cc e cc e

M

Kelt M

700

N

A

Kelt F

600

U

A OMP AD

SC R

Smolt

bb c bb

ED

Spawner M Adult F

Smolt Parr

10

20

aa b a

30

40

50

60

E370/mg protein

B

A

CC E

0

PT

Adult M

aa b

33

OMP KD cc e f

Kelt F Kelt M

cc e ee ff

Spawner F

bb c bb c

Spawner M aa

Adult F

Smolt

a

Parr 10

20

30

40

50

E 420/mg protein

70

80

90

U

C

60

SC R

0

IP T

Adult M

N

Fig. 2. TBARS products in the liver of the sea trout (Salmo trutta m.trutta L.) (A), protein

A

carbonyl derivates OMP AD (E370/mg protein) (B) and OMP KD (E420/mg protein) (C)

M

content during different developmental stages.

Results are expressed as mean ±S.D. Differences between experimental groups were analysed

ED

by one-way ANOVA and Tukey’s post-hock test for unequal observations. Differences were considered significant at p<0.05.

PT

Legend: development stages – Parr, Smolt, Adult Male and Adult Female, Spawner Male and

CC E

Spawner Female, Kelt Male and Kelt Female as a forms of survival. Significant differences between groups are designated as follows: a - Smolt group vs. Parr group;

A

aa – Adult Male or Adult Female vs. Parr group; b – Adult Male or Adult Female vs. Smolt group; bb - Spawner Male group or Spawner Female group vs. Parr group; c - Spawner Male group or Spawner Female group vs. Smolt group; cc - Kelt Male group or Kelt Female group vs. Smolt group;

34

d - Spawner Male group vs. Adult Male group; dd - Spawner Female group vs. Adult Female group; e - Kelt Male group or Kelt Female group vs. Parr group. ee - Kelt Male group vs. Adult Male; f - Kelt Female group vs. Adult Female group;

IP T

ff - Kelt Male group vs. Spawner Male group;

A

CC E

PT

ED

M

A

N

U

SC R

j- Kelt Female group vs. Spawner Female group.

35

Kelt F

Kelt M

Spawner F

bb

Spawner M

IP T

Adult F

Adult M

Smolt

a

0

10

20

30

40

50

%

SC R

Parr

60

70

80

U

Fig. 3. Total antioxidant capacity (%) in the liver of the sea trout (Salmo trutta m. trutta L.)

N

during different development stages.

A

Results are expressed as mean ±S.D. Differences between experimental groups were analysed

considered significant at p<0.05.

A

CC E

PT

ED

Legend: see Legend to Figure 2.

M

by one-way ANOVA and Tukey’s post-hock test for unequal observations. Differences were

36

Kelt F

f

Kelt M

ee

Spawner F Spawner M

d

Adult F aa

Smolt

IP T

Adult M a

0

50

100

150

200

250

SC R

Parr 300

350

U·mg-1 protein

450

500

U

A

400

efj

N

Kelt F

e ee

A

Kelt M Spawner F

Adult F

bb c d

M

Spawner M

bb c dd

b b

ED

Adult M Smolt

CC E

0

PT

Parr 10

20

a

30

40

50

60

70

μmol·min-1·mg-1 protein

B

Fig. 4. Superoxide dismutase activity (U·mg-1 protein) (A) and catalase activity (μmol·min-

A

1·mg-1 protein) (B) in the liver of the sea trout (Salmo trutta m.trutta L.) during different development stages. Results are expressed as mean ±S.D. Differences between groups were analysed by one-way ANOVA and Tukey’s post-hock test for unequal observations. Differences were considered

37

significant at p<0.05.

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Legend: see Legend to Figure 2.

38

Kelt F Kelt M

e bb c

Spawner F bb c

IP T

Spawner M Adult F

Smolt Parr 0

20

40

60

80

100

120

A

N

U

nmol·min-1·mg-1 protein

A

SC R

Adult M

M

Kelt F Kelt M

bb c dd

ED

Spawner F Spawner M

PT

Adult F

bb d

Adult M

CC E

Smolt Parr

A

0

20

40

60

80

100

120

nmol ·min-1·mg-1 protein

B

Fig. 5. Glutathione reductase activity (nmol NADPH2·min-1·mg-1 protein) (A) and glutathione peroxydase activity (nmol min-1·mg-1 protein) (B) in the liver of the sea trout (Salmo trutta m.trutta L.) during different development stages.

39

Results are expressed as mean ±S.D. Differences between groups were analysed by one-way ANOVA and Tukey’s post-hock test for unequal observations. Differences were considered significant at p<0.05.

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Legend: see Legend to Figure 2.

40

74

34

72

33

70

32 31

SC R

68 66 64 62 60 29

30

31

32

33

34

35

30 29 28

OMP KD, E 420/mg protein

35

IP T

76

U

TBARS µmol MDA·mg-1 protein

OMP AD:TBARS, y = 13.4029 + 1.6665*x; r = 0.8093; p = 0.0149; r2 = 0.6550 OMP AD:OMB KD, y = -2.4536 + 1.0617*x; r = 0.9215; p = 0.0011; r2 = 0.8493

27 36

A

A

N

OMPAD, E 370/mg protein

ED

M

TBARS:OMPAD, y = 12.7204 + 0.3573*x; r = 0.8288; p = 0.0009; r2 = 0.6869 TBARS:OMP KD, y = 12.8367 + 0.2894*x; r = 0.8259; p = 0.0009; r2 = 0.6822

34

32 31 30

A

29

32

28

30

27

28

26 48

50

OMP KD, E 420/mg protein

36

33

PT

38

34

CC E

OMP AD, E370/mg protein

40

26 52

54

56

58

60

62

64

66

68

25 70

TBARS, µmol MDA·mg-1 protein

B Fig.6. Significant relationship between OMP AD, TBARS and OMP KD level in liver tissue in

41

the sea trout male (A) and OMP AD, TBARS and OMP KD female (B) individuals during the adult development stage in liver tissue. The figure shows the regression equation, correlation (r) and determination (r2) coefficients, and

A

CC E

PT

ED

M

A

N

U

SC R

IP T

significance of these relationships (p).

42

65

60

60

55

55

50

50

45

45

40

40

SC R

35 30 25

100

120

140

U

20 15 80

OMP AD, E 370/mg protein

65

IP T

CAT, μmol min-1 mg-1 protein

SOD:CAT y = 69.1343 - 0.2051*x; r = -0.5919; p = 0.0426; r2 = 0.3504 SOD:OMPAD y = 57.9255 - 0.1225*x; r = -0.3694; p = 0.2373; r2 = 0.1365

160

180

200

220

35 30 25 20

15 240

M

A

A

N

SOD, U·mg-1 protein

ED

55 50 45

90 80

PT

60

70

40

60

35

50

30 25

40

A

20

15 300

400

500

600

700

800

900

GR, nmol min-1 mg-1 protein

65

CC E

CAT, μmol min-1 mg-1 protein(L)

TBARS:CAT y = 68.3458 - 0.0406*x; r = -0.6135; p = 0.0339; TBARS:GR y = 72.9432 - 0.0199*x; r = -0.2214; p = 0.4891;

30 1000

TBARS, µmol MDA·mg-1 protein

B Fig.7. Significant relationship between OMP AD, SOD and CAT level (A) and TBARS, GR and

43

CAT level (B) in liver tissue in the sea trout male individuals during the kelt form. The figure shows the regression equation, correlation (r) and determination (r2) coefficients, and

A

CC E

PT

ED

M

A

N

U

SC R

IP T

significance of these relationships (p).

44

Table 1. Correlation intergroup interdependencies between the parameters of oxidative stress and antioxidant enzyme activities in the liver tissue during different developmental

Interdependencies

Correlative coefficient

p

Parr

TBARS-TAC

0.245

0.009

OMP AD-GPx

0.246

TAC-OMP KD

-0.266

TBARS-GR

-0,234

0.01

TAC-CAT

-0.187

0.04

0.922

0.026

0.722

0.043

OMP KD- TAC

0.774

0.024

TBARS- OMP AD

0.829

0.001

TBARS- OMP KD

0.826

0.001

OMP AD-GR

0.645

0.023

OMP KD- GR

0.745

0.002

TBARS-CAT

-0.613

0.033

CAT- OMP AD

0.672

0.017

AD OMP KD- GR

-0.644

0.023

SOD-CAT

-0.592

0.043

OMP AD- TAC

0.775

0.003

GR-GPx

0.639

0.025

CC E

PT

ED

M

OMP AD- TAC

A

Kelt male

Kelt female

0.811

A

TBARS- OMP KD

Adult female

0.004

0.025

TBARS- OMP AD

N

Adult male

0.009

U

Smolt

SC R

Development stage

IP T

stages and forms of the sea trout

45

Table 2. Multivariate significance tests and effective hypothesis decomposition for three stages of the sea trout during three developmental forms (adult, spawner, and kelt).

p

Wilks

0.705

0.686

Hotelling

0.705

Wilks

26.058

Hotelling

32.547

0.686

0.000 0.000

Sex●Development stage

0.048

1.758

0.038

A

CC E

PT

ED

M

Hotelling

1.698

A

Wilks

N

U

Development stage

F

IP T

Sex

Test

SC R

Effect

46

Table 3. Statistically significant dependencies of the main effects of the sexdevelopment stage interactions on oxidative stress parameters in the liver of the sea trout during different developmental stages Parameters

p

Kelt - Adult, male

TAC

0.013

Kelt – spawner, male

TAC

0.039

Kelt – Adult, female

TAC

IP T

Stadium, sex

0.008

TBARS

0.000

Kelt – Spawner, male

TBARS

0.003

Kelt – Adult, female

TBARS

0.000

Spawner - Adult, female

TBARS

N

U

SC R

Kelt - Adult, male

OMP KD

A

Kelt - Adult, male Kelt – Spawner, male

0.000 0.000 0.011

OMP KD

0.001

SOD

0.000

SOD

0.000

SOD

0.000

SOD

0.004

Kelt - Adult, male

CAT

0.000

Spawner - Adult, male

CAT

0.000

Kelt – Adult, female

CAT

0.000

Spawner - Adult, female

CAT

0.000

Spawner - Adult, male

GPx

0.017

Spawner - Adult, female

GPx

0.000

M

OMP KD

Kelt - Adult, male Kelt – Spawner, male

PT

Spawner - Adult, female

ED

Spawner - Adult, female

A

CC E

Kelt – Adult, female

47

Differences between all groups for the main effects of the sex-development stage interactions were analyzed by Multivariate ANOVA and Tukey’s post-hock test for unequal

A

CC E

PT

ED

M

A

N

U

SC R

IP T

observations.

48

Table 4. Statistically significant dependencies of the main effects of the sexdevelopment stage interactions on oxidative stress parameters between sexes (male-female) in the liver of the sea trout during different developmental stages and forms.

Parameters

p

Adult, male - Adult, female

TAC

0.039

Spawner, male - Spawner, female

TAC

IP T

Stadium, sex

0.003

TBARS

0.000

Kelt, male – kelt, female

TBARS

0.000

Kelt, male – kelt, female

OMP KD

0.001

SOD

U

0.028

N

SC R

Adult, male - Adult, female

SOD

0.000

GPx

0.006

Spawner, male – Spawner, female

A

Adult, male - Adult, female

M

Spawner, male - Spawner, female

ED

Differences between all groups for the main effects of the sex-development stage

A

CC E

observations.

PT

interactions were analyzed by Multivariate ANOVA and Tukey’s post-hock test for unequal

49

Table 5. SS test of oxidative stress parameters for a full model of the formation of the antioxidant profile in the liver tissue and SS for residues for the three stages of sea trout development and forms

Multiple R

Multiple R2

Multiple

F

adjusted R2 0.502

0.252

0.223

8.50

TBARS

0.650

0.422

0.400

18.42

OMP AD

0.265

0.070

0.033

OMP KD

0.556

0.309

0.282

SOD

0.663

0.440

0.417

CAT

0.821

0.673

0.661

GR

0.315

0.099

GPx

0.547

0.299

0.000

11.29

0.000

19.76

0.000

N

51.97

0.000

0.063

2.77

0.021

0.272

10.77

0.000

U

0.189

A

1.90

A

CC E

PT

ED

M

0.000

SC R

TAC

p

IP T

Parameters

50