The cutaneous stress response system in three-spined stickleback and European flounder exposed to oxidative stress: Different mode of action.

In fish, the skin is directly exposed to multiple environmental stressors and provides the first line of defense against harmful external factors. It turned out that cortisol and melatonin (Mel) are involved in fish cutaneous stress response system (CSRS) similar to mammalian. This study investigates the mode of action of CSRS in two teleost species of different biology and skin characteristics, the three-spined stickleback and the European flounder, after exposure to oxidative stress induced by a potassium dichromate solution. The cutaneous stress response system presents different ways of action in two studied species: Mel concentration increases in the skin of both species, but cortisol concentration increases in the skin only in sticklebacks. Data suggest that stickleback skin cells can produce cortisol. However, cortisol is not involved in the response to oxidative stress in flounders. In stickleback skin, two genes encoding AANAT and ASMT/HIOMT (enzymes involved in Mel synthesis), aanat1a and asmt2, are expressed, but in flounder skin, only one, asmtl. Because gene expression does not change in stickleback skin after exposure to stress, the source of increased Mel is probably outside the skin. A lack of expression of the gene encoding AANAT in flounder skin strongly suggests that Mel is transported to the skin by the bloodstream from other sites of synthesis. Pigment dispersion in the skin after exposure to oxidative stress is found only in sticklebacks.

Melatonin and cortisol as components of the cutaneous stress response system in fish: Response to oxidative stress.

The skin being a passive biological barrier that defends the organism against harmful external factors is also a site of action of the system responding to stress. It appears that melatonin (Mel) and its biologically active metabolite AFMK (N1-acetyl-N2-formyl-5-methoxykynuramine), both known as effective antioxidants, together with cortisol, set up a local (cutaneous) stress response system (CSRS) of fish, similar to that of mammals. Herein we comment on recent studies on CSRS in fish and show the response of three-spined stickleback skin to oxidative stress induced by potassium dichromate. Our study indicates that exposure of the three-spined stickleback to K2Cr2O7 affects Mel and cortisol levels and pigment dispersion in melanophores in the skin. In our opinion, an increased concentration of Mel and cortisol in the skin may be the strategy to cope with oxidative stress, where both components act locally to prevent damage caused by active oxygen molecules. Furthermore, the pigment dispersion may be a valuable, easy-to-observe mark of oxidative stress, useful in the evaluation of fish welfare.

A study of aanat and asmt expression in the three-spined stickleback eye and skin: Not only "on the way to melatonin".

Melatonin synthesis is controlled by aralkylamine N-acetyltransferase (AANAT: EC 2.3.1.87) acetylating serotonin (5-hydroxytryptamine; 5-HT) to N-acetylserotonin (NAS), and N-acetylserotonin O-methyltransferase (ASMT: EC 2.1.1.4) methylating NAS to melatonin (Mel; N-acetyl-5-methoxytryptamine). We examined the levels of expression of the aanat and asmt genes, Mel concentrations as well as AANAT isozyme activity in the eyeball (with retina) and skin of the three-spined stickleback (Gasterosteus aculeatus), at noon and midnight. We found mRNA of four genes (aanat1a, snat, asmt and asmt2) in the eyeball, and two (aanat1a and asmt2) in the skin. The presence of two transcripts of genes encoding AANAT and two of ASMT in the eyeball at noon and midnight, suggests activity of AANAT and ASMT isozymes in metabolic pathways besides "the way to melatonin", all the more so because day/night changes in Mel concentration do not follow the changes in either the expression of genes or the activity of AANAT. The high effectiveness of noon NAS synthesis in the eyeball at low substrate concentrations, which is not reflected in high Mel production, suggests the function of eye NAS beyond that of a precursor to the biosynthesis of Mel. The inhibition of AANAT isozyme activity by product observed in the eyeball may be one of the mechanisms of 5-HT husbanding in the eye (retina). The presence of transcripts of genes encoding both AANAT and ASMT and the activity of AANAT, at noon and midnight, supports a local Mel synthesis in the sticklebacks' skin.

Social context affects aggression and brain vasotocin and isotocin level in the round goby.

On the wild spawning grounds, the round gobies Neogobius melanostomus are subjected to different social cues, such as sex-separation and high fish density. We designed an experiment to stimulate natural social stress when fish are separated from opposite sex individuals and exposed to close proximity of same-sex conspecifics. We examined the effects of different sex compositions on aggressiveness and brain concentrations of arginine vasotocin (AVT) and isotocin (IT), as AVT and IT are known to be involved in aggressive interactions during reproduction. The round gobies were kept in three experimental groups: same-sex groups broken down into male-only and female-only groups and mixed-sex groups. In this study, males and females from same-sex groups showed overt aggression and competition. Separation stress stimulated aggressive responses in both sexes, but the link between brain AVT and IT concentration and aggressive behavior was evident only in male-only group. In the male-only group, AVT and IT levels were the highest. This study shows that sex composition of the social environment can affect aggressive behavior as well as AVT and IT concentration in the whole brain of the round goby.

Parental care and neuropeptide dynamics in a cichlid fish Neolamprologus caudopunctatus.

The hypothalamic neuropeptides arginine vasopressin (AVP) and oxytocin (OT) modulate social behavior across a wide variety of species. However, the role of arginine vasotocin (AVT) and isotocin (IT, the teleost homologs of AVP and OT) in regulating biparental care especially in the context of monogamy is not well studied. Here, using high-performance liquid chromatography (HPLC), we investigated how bioactive whole brain AVT and IT neuropeptide levels vary in relation to the phase of the breeding cycle and sex, in a monogamous biparental cichlid fish, Neolamprologus caudopunctatus. Since non-caring individuals of this species readily cannibalize eggs, but caring parents never do, we further investigated whether there might be changes in AVT/IT whole brain levels that correspond to the transition from a non-breeding, egg cannibal to an egg caring parent. We found that AVT levels were higher in females than in males and that AVT levels were highest when the need to defend the young was greatest. Breeding pairs that had a strong pair-bond and a higher frequency of nest care had the highest levels of AVT, whereas individuals that spent little time close to their breeding partner, displayed aggression towards their partner and neglected their nest duties (signs of a weak pair bond), had lower whole brain AVT levels. Isotocin (IT) levels did not differ between sexes and we did not detect any variation across the breeding cycle, with pair-bonding scores or with parental behavior. Our results show that whole brain AVT levels are linked to the breeding cycle, nest maintenance and pair-bonding score in this species. Furthermore, our study highlights species and sex-specific nonapeptides patterns in bi-parental caring fish and contributes to the increasing knowledge on neuroendocrinological mechanisms underlying parental care.

Cortisol and melatonin in the cutaneous stress response system of fish.

The stress hormone cortisol, together with antioxidants, melatonin (Mel) and its biologically active metabolites, 5-methoxykynuramines, including AFMK, set up a local stress response system in mammalian skin. Our in vitro study of the European flounder (Platichthys flesus) was designed to examine whether Mel and AFMK would respond to cortisol while a glucocorticoid is added to the incubation medium. The concentrations of cortisol in the incubation medium mimic plasma cortisol levels seen in fish exposed to different types of stresses such as handling, confinement, high density, food-deprivation or air-exposure. We measured Mel and AFMK in skin explants and culture media using high-performance liquid chromatography (HPLC) with fluorescence detection. We also analysed melanosome response (dispersion/aggregation) in the explants subjected to the different treatments. Cortisol stimulated the release of Mel and AFMK from skin explants in a dose-dependent manner. Melanosome dispersion and a darkening of the skin explants were observed after incubation with cortisol. This study is the first to demonstrate the interrelationship between cortisol and Mel/AFMK in fish skin. Our data strongly suggest that the cutaneous stress response system (CSRS) is present in fish. The question remains whether Mel, AFMK or cortisol are synthetized locally in fish skin and/or transported by the bloodstream. The presence of the CSRS should be taken into account during elaboration of new indicators of fish welfare both in aquaculture and in the wild.

The time enzyme in melatonin biosynthesis in fish: Day/night expressions of three aralkylamine N-acetyltransferase genes in three-spined stickleback.

In vertebrates, aralkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is a time-keeping enzyme in melatonin (Mel) biosynthesis. Uniquely in fish, there are several AANAT isozymes belonging to two AANAT subfamilies, AANAT1 and AANAT2, which are encoded by distinct genes. The different substrate preferences, kinetics and spatial expression patterns of isozymes indicate that they may have different functions. In the three-spined stickleback (Gasterosteus aculeatus), there are three genes encoding three AANAT isozymes. In this study, for the first time, the levels of aanat1a, aanat1b and aanat2 mRNAs are measured by absolute RT-qPCR in the brain, eye, skin, stomach, gut, heart and kidney collected at noon and midnight. Melatonin levels are analysed by HPLC with fluorescence detection in homogenates of the brain, eye, skin and kidney. The levels of aanats mRNAs differ significantly within and among organs. In the brain, eye, stomach and gut, there are day/night variations in aanats mRNAs levels. The highest levels of aanat1a and aanat1b mRNAs are in the eye. The extremely high expressions of these genes which are reflected in the highest Mel concentrations at this site at noon and midnight strongly suggest that the eye is an important source of the hormone in the three-spined sticklebacks. A very low level of aanat2 mRNA in all organs may suggest that AANAT1a and/or AANAT1b are principal isozymes in the three-spine sticklebacks. A presence of the isozymes of defined substrate preferences provides opportunity for control of acetylation of amines by modulation of individual aanat expression and permits the fine-tuning of indolethylamines and phenylethylamines metabolism to meet the particular needs of a given organ.

Agonistic interactions elicit rapid changes in brain nonapeptide levels in zebrafish.

The teleost fish nonapeptides, arginine vasotocin (AVT) and isotocin (IT), have been implicated in the regulation of social behavior. These peptides are expected to be involved in acute and transient changes in social context, in order to be efficient in modulating the expression of social behavior according to changes in the social environment. Here we tested the hypothesis that short-term social interactions are related to changes in the level of both nonapeptides across different brain regions. For this purpose we exposed male zebrafish to two types of social interactions: (1) real opponent interactions, from which a Winner and a Loser emerged; and (2) mirror-elicited interactions, that produced individuals that did not experience a change in social status despite expressing similar levels of aggressive behavior to those of participants in real-opponent fights. Non-interacting individuals were used as a reference group. Each social phenotype (i.e. Winners, Losers, Mirror-fighters) presented a specific brain profile of nonapeptides when compared to the reference group. Moreover, the comparison between the different social phenotypes allowed to address the specific aspects of the interaction (e.g. assessment of opponent aggressive behavior vs. self-assessment of expressed aggressive behavior) that are linked with neuropeptide responses. Overall, agonistic interactions seem to be more associated with the changes in brain AVT than IT, which highlights the preferential role of AVT in the regulation of aggressive behavior already described for other species.

Cortisol modulates vasotocinergic and isotocinergic pathways in the gilthead sea bream.

In the present study, we assessed the responses of the vasotocinergic and isotocinergic systems to chronic stress induced by cortisol administration in the gilthead sea bream (Sparus aurata). Pituitary and plasma arginine vasotocin (AVT) and isotocin (IT) levels, as well as hypothalamic pro-vasotocin (pro-VT) and pro-isotocin (pro-IT) mRNA expression levels, were analysed. In addition, the mRNA levels of three receptors, AVTR type V1a2, AVTR type V2 and ITR, were analysed in several target organs associated with the following physiological processes: (i) integration and control (hypothalamus), (ii) metabolism and its control (liver and hypothalamus), (iii) osmoregulation (gills) and (iv) stress response (head kidney). Specimens were injected intraperitoneally with slow-release implants (5 µL g(-1) body mass) containing coconut oil alone (control group) or with cortisol (50 µg g(-1) body mass; cortisol group). Both AVT and IT synthesis and release were correlated with plasma cortisol values, suggesting a potential interaction between both hormonal systems and cortisol administration. Our results suggest that the activation of hepatic metabolism as well as the hypothalamic control of metabolic processes provide the energy necessary to overcome stress, which could be partly mediated by AVTRs and ITR. Upregulation of branchial AVT and IT receptor expression following cortisol treatment suggests an involvement of the vasotocinergic and isotocinergic systems in the regulation of ion channels/transporters during stressful situations. Finally, changes in AVT and IT receptor mRNA expression in the head kidney suggest these nonapeptides participate in feedback mechanisms that regulate the synthesis/release of cortisol. Our results indicate a relationship between cortisol and both the vasotocinergic and isotocinergic systems during simulated chronic stress in S. aurata.

Brain levels of nonapeptides in four labrid fish species with different levels of mutualistic behavior.

There is strong evidence that brain nonapeptides are implicated as modulators of a wide array of social and reproductive behaviors in fishes. However, the question remains, as to whether there is a link between the distribution of active nonapeptides across brain regions and fishes specific behavioral phenotypes. To explore this link we compared the nonapeptides' profile across the brains of fishes representing different degrees of mutualistic behavior (here: cleaning behavior). Herein we studied the quantitative distribution of both nonapeptides, arginine vasotocin (AVT) and isotocin (IT), in the brains of four species of fish belonging to the family Labridae: two are obligatory cleaners throughout their entire life (Labroides dimidiatus and Labroides bicolor), one species is a facultative cleaner (Labropsis australis; juveniles are cleaners and adults are corallivorous), and one is a non-cleaner species, corallivorous throughout its entire life (Labrichthys unilineatus). The biologically available AVT and IT concentrations were measured simultaneously in distinct brain macro-areas: forebrain, optic tectum, cerebellum and brain stem, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We showed that the levels of both AVT and IT varied significantly across species, as measured in the whole brain or in the specific macro-areas. Significantly higher AVT concentrations in the cerebellum which were found in the obligate cleaners seemed to be related to expression of mutualistic behavior. On the other hand, the higher levels of brain IT in the non-cleaner L. unilineatus suggested that these might be linked to the development of sexual dimorphism, which occurs only in this non-cleaner species.

Brain arginine vasotocin and isotocin in breeding female three-spined sticklebacks (Gasterosteus aculeatus): the presence of male and egg deposition.

Arginine vasotocin (AVT) and isotocin (IT) are fish hypothalamic nonapeptides involved in numerous social and reproductive behaviors. Vasotocinergic and isotocinergic fibers project to different brain areas where peptides act as neurotransmitters and/or neuromodulators. In this study, we measured whole brain levels of bioactive AVT and IT in breeding females of three-spined stickleback (Gasterosteus aculeatus) when they were kept with: (i) courting nest-owners, (ii) courting males that did not build the nest, (iii) non-courting males, and (iv) alone. Only some of the females kept with courting nest-owners deposited eggs. The highest and similar brain AVT levels were in those of females that did not deposit eggs, regardless of whether they were kept with non-courting or courting male, having the nest or not. The highest IT levels were in females that did not deposit eggs but only in those kept with courting male. We suggest that production of AVT in females' brain is stimulated by the presence of male in close proximity, irrespective of whether or not it displays courting behavior, but that of IT is stimulated by male courtship proxies. Moreover, presence of courting or non-courting male that stimulate IT or/and AVT producing neurones may be decisive for final oocyte maturation or egg deposition, because brain levels of both nonapeptides decrease after egg deposition. Similar AVT levels in brains of aggressive and non-aggressive individuals and lack of correlation between brain IT levels and aggressive behavior of females suggest that the nonapeptides are not related to females aggressiveness in three-spined sticklebacks.

Stocking density influences brain arginine vasotocin (AVT) and isotocin (IT) levels in males and females of three-spined stickleback (Gasterosteus aculeatus).

Arginine vasotocin (AVT) and isotocin (IT) are fish neurohormones produced in separate parvocellular and magnocellular preoptic neurons of Teleostei. Apart from well-established peripheral action as hormones they are important neurotransmitters in central nervous system in fish. In the present study, we examined an influence of stocking density on whole brain AVT and IT concentrations in males and females of three-spined stickleback (Gasterosteus aculeatus). In males, the highest AVT levels have been found at stocking densities of 10 and 30 individuals per 30-l tank. On the other hand, in females, AVT concentration was significantly higher in those kept alone. Brain IT concentrations significantly increased along with stocking density only in females and did not change in males. The sex-dependent responses indicate a different stimulation of AVT and IT neurons in males and females. Consequently, roles of the neurohormones in males and females exposed to stress of overcrowding must be different.

Changes in brain arginine vasotocin, isotocin, plasma 11-ketotestosterone and cortisol in round goby, Neogobius melanostomus, males subjected to overcrowding stress during the breeding season.

In natural spawning grounds, breeding round goby, Neogobius melanostomus, males are exposed to various social stimuli, including high density of same-sex competitors and separation from females. We hypothesize that breeding males subjected to overcrowding in the wild experience high stress that affects their socio-sexual behavior and their relationships among conspecifics. We designed an experiment to mimic natural stimulation when highly aggregated breeding males are subjected to same-sex opponents. Males were sampled sequentially from experimental tank stocked at decreasing fish densities of 15 fish/m(2), 9 fish/m(2) and 4 fish/m(2). We studied the effects of overcrowding on male behavior and selected hormones, brain arginine vasotocin (AVT) and isotocin (IT) and plasma 11-ketotestosterone (11-KT) and cortisol as these are known to play roles in reproduction and related social interactions. The highest brain AVT and plasma cortisol levels were measured in non-aggressive males kept in the overcrowded group of 15 fish/m(2). IT level was elevated in fish kept at the lower density of 9 fish/m(2), and at which the males began to display territoriality and aggression. The plasma level of 11-KT was similar in all the males. Brain AVT and IT and plasma cortisol along with behavioral observations can be applied as species-specific indicators of the well-being of round goby males.

Neuropeptides isotocin and arginine vasotocin in urophysis of three fish species.

In this study, for the first time, both neuropeptides isotocin (IT) and arginine vasotocin (AVT) have been identified and measured in urophysis, the neurohaemal organ of the caudal neurosecretory system of teleost fish. So far, AVT, but not IT, was quantified by radioimmunoassay (RIA) in urophysis of several fish species. We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species. The efficiency of peptide's SPE extraction was 79-85%. In HPLC-FL method, the limits of detection (LOD) and quantification (LOQ) were estimated as 1.0 and 3.4 pmol/mL for IT and 0.25 and 2.20 pmol/mL for AVT. In LC-MS/MS method, LOD and LOQ were estimated as 0.4 and 1.2 pmol/mL for IT and 0.06 and 0.2 pmol/mL for AVT. The chromatographic methods are good alternative for RIA, because enable to measure both nonapeptides simultaneously in one sample. In round goby (Neogobius melanostomus), three-spined stickleback (Gasterosteus aculeatus) and sea bream (Sparus aurata), urophysial IT concentrations ranged between 0.056 and 0.678 pmol/mg tissue and AVT concentrations ranged between 0.0008 (or even below detection threshold) and 0.084 pmol/mg tissue.

Neurohypophysial and paracrine vasopressinergic signaling regulates aquaporin trafficking to hydrate marine teleost oocytes.

The dual aquaporin (Aqp1ab1/Aqp1ab2)-mediated hydration of marine teleost eggs, which occurs during oocyte meiosis resumption (maturation), is considered a key adaptation underpinning their evolutionary success in the oceans. However, the endocrine signals controlling this mechanism are almost unknown. Here, we investigated whether the nonapeptides arginine vasopressin (Avp, formerly vasotocin) and oxytocin (Oxt, formerly isotocin) are involved in marine teleost oocyte hydration using the gilthead seabream (Sparus aurata) as a model. We show that concomitant with an increased systemic production of Avp and Oxt, the nonapeptides are also produced and accumulated locally in the ovarian follicles during oocyte maturation and hydration. Functional characterization of representative Avp and Oxt receptor subtypes indicates that Avpr1aa and Oxtrb, expressed in the postvitellogenic oocyte, activate phospholipase C and protein kinase C pathways, while Avpr2aa, which is highly expressed in the oocyte and in the follicular theca and granulosa cells, activates the cAMP-protein kinase A (PKA) cascade. Using ex vivo, in vitro and mutagenesis approaches, we determined that Avpr2aa plays a major role in the PKA-mediated phosphorylation of the aquaporin subdomains driving membrane insertion of Aqp1ab2 in the theca and granulosa cells, and of Aqp1ab1 and Aqp1ab2 in the distal and proximal regions of the oocyte microvilli, respectively. The data further indicate that luteinizing hormone, which surges during oocyte maturation, induces the synthesis of Avp in the granulosa cells via progestin production and the nuclear progestin receptor. Collectively, our data suggest that both the neurohypophysial and paracrine vasopressinergic systems integrate to differentially regulate the trafficking of the Aqp1ab-type paralogs via a common Avp-Avpr2aa-PKA pathway to avoid competitive occupancy of the same plasma membrane space and maximize water influx during oocyte hydration.

Brain levels of arginine-vasotocin and isotocin in dominant and subordinate males of a cichlid fish.

The nonapeptides arginine-vasotocin (AVT) and isotocin (IT), which are the teleost homologues of arginine-vasopressin and oxytocin in mammals, have well established peripheral effects on osmoregulation and stress response, and central effects on social behavior. However, all studies that have looked so far into the relationship between these nonapeptides and social behavior have used indirect measures of AVT/IT activity (i.e. immunohistochemistry of AVT/IT immunoreactive neurons, or AVT/IT or their receptors mRNA expression with in situ hybridization or qPCR) and therefore direct measures of peptide levels in relation to social behavior are still lacking. Here we use a recently developed high-performance liquid chromatography analysis with fluorescence detection (HPLC-FL) method to quantify the levels of both AVT and IT in macro-dissected brain areas [i.e. olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and hindbrain (= rhombencephalon minus cerebellum)] and pituitary of dominant and subordinate male cichlid fish (Oreochromis mossambicus). The pituitary shows higher levels of both peptides than any of the brain macroareas, and the olfactory bulbs have the highest AVT among all brain areas. Except for IT in the telencephalon there is a lack of correlations between central levels and pituitary peptide levels, suggesting an independent control of hypophysial and CNS nonapeptide secretion. There were also no correlations between AVT and IT levels either for each brain region or for the pituitary gland, suggesting a decoupled activity of the AVT and IT systems at the CNS level. Subordinate AVT pituitary levels are significantly higher than those of dominants, and dominant hindbrain IT levels are significantly higher than those of subordinates, suggesting a potential involvement of AVT in social stress in subordinate fish and of IT in the regulation of dominant behavior at the level of the hindbrain. Since in this species dominant males use urine to communicate social status and since AVT is known to have an antidiuretic effect, we have also investigated the effect of social status on urine storage. As predicted, dominant males stored significantly more urine than subordinates. Given these results we suggest that AVT/IT play a key role in orchestrating social phenotypes, acting both as central neuromodulators that promote behavioral plasticity and as peripheral hormones that promote integrated physiological changes.

Variation in brain arginine vasotocin (AVT) and isotocin (IT) levels with reproductive stage and social status in males of three-spined stickleback (Gasterosteus aculeatus).

Arginine vasotocin (AVT) and isotocin (IT) are fish nonapeptides synthesized in separate hypothalamic neurons from where they are transported to the neurohypophysis for storage and release into circulation. AVT is known to modulate aggression, courtship and parental care or social communication in many species, including fish, amphibians and birds. In this paper we examined a link between the level of AVT and IT in the brain and particular reproductive behavior in males of three-spined stickleback (Gasterosteus aculeatus). AVT and IT levels in whole brain of males of three-spined stickleback vary depending on specific breeding behavior of the individuals and their social status. These studies have shown the highest AVT levels in aggressive males that took care of the eggs. Brain AVT concentrations are also increased in nuptial colored subordinate males that fight to change their social status. On the other hand, IT is significantly higher in aggressive dominant males that defend their territory. IT may be also involved in courtship in three-spined stickleback. These findings highlight the importance of determination of "free", bioavailable neuropeptides' level in behavioral studies.

Behavioural indicators of welfare in farmed fish.

Behaviour represents a reaction to the environment as fish perceive it and is therefore a key element of fish welfare. This review summarises the main findings on how behavioural changes have been used to assess welfare in farmed fish, using both functional and feeling-based approaches. Changes in foraging behaviour, ventilatory activity, aggression, individual and group swimming behaviour, stereotypic and abnormal behaviour have been linked with acute and chronic stressors in aquaculture and can therefore be regarded as likely indicators of poor welfare. On the contrary, measurements of exploratory behaviour, feed anticipatory activity and reward-related operant behaviour are beginning to be considered as indicators of positive emotions and welfare in fish. Despite the lack of scientific agreement about the existence of sentience in fish, the possibility that they are capable of both positive and negative emotions may contribute to the development of new strategies (e.g. environmental enrichment) to promote good welfare. Numerous studies that use behavioural indicators of welfare show that behavioural changes can be interpreted as either good or poor welfare depending on the fish species. It is therefore essential to understand the species-specific biology before drawing any conclusions in relation to welfare. In addition, different individuals within the same species may exhibit divergent coping strategies towards stressors, and what is tolerated by some individuals may be detrimental to others. Therefore, the assessment of welfare in a few individuals may not represent the average welfare of a group and vice versa. This underlines the need to develop on-farm, operational behavioural welfare indicators that can be easily used to assess not only the individual welfare but also the welfare of the whole group (e.g. spatial distribution). With the ongoing development of video technology and image processing, the on-farm surveillance of behaviour may in the near future represent a low-cost, noninvasive tool to assess the welfare of farmed fish.

Stress Response System in the Fish Skin-Welfare Measures Revisited.

The skin of vertebrates acts as a biological barrier defending the organism against many harmful environmental factors. It is well established that the main stress hormone cortisol, together with antioxidants such as melatonin (Mel) and its biologically active metabolites set up a local stress response system in the mammalian skin. Recently, our research group has shown that in fish there are basic conditions for the functioning of a cutaneous stress response system (CSRS) similar to that in mammals, where Mel with its biologically active metabolite AFMK (N1-acetyl-N2-formyl-5-methoxykynuramine) and cortisol act together to protect organism against unfavorable environment. Since aquaculture is making an increasing contribution to the global economy and new laws are demanding people to respect the welfare requirements of animals there has been increasing interest in indicators of fish well-being in aquaculture. This article addresses the problem of on-farm assessment of fish welfare and proposes the CSRS as a new source of information on the welfare status of farmed fish.

Concentrations of melatonin, thyroxine, 17ß-estradiol and 11-ketotestosterone in round goby (Neogobius melanostomus) in different phases of the reproductive cycle.

The aim of this study was to determine changes in concentrations of melatonin (Mel) and thyroxine (T4) in plasma, and 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) in plasma and gonads of female and male round gobies (Neogobius melanostomus) from the Southern Baltic Sea in four phases of the reproductive cycle classified as pre-spawning, spawning, late spawning and non-spawning periods. The concentrations of Mel, T4 and E2 were determined by radioimmunoassay (RIA) whereas 11-KT was quantified using an enzyme immunoassay (EIA). The maturity stage of gonads was determined using histological analyses. The pattern of changes in Mel concentrations of females and males was similar with the greatest concentrations in the spawning and non-spawning phases. In both sexes, there was a similar tendency of change in concentrations of T4 and E2 with the increase being in the pre-spawning and non-spawning phases. The greatest concentrations of 11-KT were observed in the plasma and gonads of males during the spawning phase. In females, there were no changes in 11-KT concentrations either in plasma or gonads during all phases where quantifications occurred. This is the first study for determination of the pattern of changes in Mel and T4 concentrations as well as gonadal steroids E2 and 11-KT, supported by histological analysis of gonads, in batch spawning fish during the reproductive cycle.