Ewa Kulczykowska

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.

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.

Responses of circulating arginine vasotocin, isotocin, and melatonin to osmotic and disturbance stress in rainbow trout (Oncorhynchus mykiss)

In teleost fish exposed to stressors AVT has been implicated in activation of the hypothalamo-pituitary-interrenal axis. Mel has been shown to counteract several behavioural and endocrine consequences of stress in mammals. These studies were undertaken to investigate the effects of either disturbance or osmotic stress, applied either separately or simultaneously, on plasma AVT, IT and melatonin in rainbow trout (Oncorhynchus mykiss). Hormones were determined in plasma by high-performance liquid chromatography preceded by solid-phase extraction. The results showed that both forms of stress caused significant increase in plasma AVT concentration, although more pronounced elevation was observed in physically disturbed fish. Conversely, neither osmotic nor disturbance stress affected plasma IT concentration. The apparent difference in response to stress by the two close related neurohypophysial nonapeptides suggests independent mechanisms controlling their synthesis and/or release and supports the idea that only AVT plays a role in physiological response to stress. Plasma Mel level was depressed in fish subjected to disturbance stress and to both stresses applied simultaneously, an effect possibly associated with the elevation of plasma AVT concentration. Results are discussed in relation to physiological interactions between hormones.

A review of the multifunctional hormone melatonin and a new hypothesis involving osmoregulation

The pineal hormone melatonin is a potentregulator of seasonal and circadian rhythms invertebrates, among them fish. Melatoninsynthesis shows a diurnal rhythm with higherlevels at night. In recent years, the pinealgland and its major product gained a number ofattributes suggesting their role in integrationof various neural and endocrine functions. Besides the well-established physiologicaleffects mediated via high-affinity cellmembrane receptors belonging to the superfamilyof G-protein – coupled receptors, melatoninreveals direct intracellular actions.This paper attempts to synthesise thephysiological roles of this multifacetedhormone in fish. The use of higher vertebratesparadigms (considering every limit ininterpretation) was essential due to lack ofsatisfactory data on fish. The actions ofmelatonin in major organs responsible forosmoregulation in fish are discussed. Theinfluence of melatonin on water/ion excretionby affecting the circulatory blood hemodynamicand by interrelations with other hormonessystems engaged in water/ion homeostasis areconsidered. New data providing the firstevidence for the presence of melatonin bindingsites in fish gills and kidneys are presented. The paper suggests a new approach that may leadto an improved understanding of osmoregulationprocesses.

Day–night variations in plasma melatonin and arginine vasotocin concentrations in chronically cannulated flounder (Platichthys flesus)

Chronically catheterised, free swimming flounder (Platichthys flesus) have been used in experiments examining the day-night variations in circulating levels of melatonin (Mel) and arginine vasotocin (AVT). Under normal photoperiod (16 h light/8 h dark) serial blood samples taken from individual fish demonstrated a Mel rhythm with daytime levels at 09.00 and 15.00 h (238+/-14 and 179+/-12 fmol x ml(-1), respectively) lower than those at 23.00 h (1920+/-128 fmol x ml(-1)). Maintenance of fish in 24-h light abolished the light/dark Mel rhythm and circulating levels were comparable to those measured during the day in fish under normal photoperiod illumination. In fish maintained under 24 h dark, although a daily rhythm was still apparent, at the time when it would be normally dark, plasma Mel concentration was reduced and at times when it would be normally light, levels were higher than in fish maintained under normal light/dark illumination. Plasma AVT concentrations were higher in fish during the day (4.4+/-0.8 fmol x ml(-1)) than those at night (1.5+/-0.4 fmol x ml(-1)), the opposite to that seen with Mel. During acute study infusion of AVT resulted in reduced levels of plasma Mel, although this did not achieve statistical significance. Infusion of Mel did not alter circulating AVT concentration.

Diel changes in plasma arginine vasotocin, isotocin, and melatonin in rainbow trout (Oncorhynchus mykiss)

The diel changes in plasma AVT, IT and Mel in rainbow trout (Oncorhynchus mykiss) were studied to assess potential relationships. Blood was sampled at 05:00, 11:00, 16:00, 22:30 and 05:00 in freshwater-adapted fish and at 22:30 in brackish water-adapted fish maintained under natural photoperiod. A few of the FW-acclimated fish were assigned to one of two experimental groups and adapted to DD or LL lighting regimes. Blood samples were taken at 11:00 and 22:30. Hormones were extracted from plasma by solid phase extraction and determined by high-performance liquid chromatography. Marked diel variations in AVT and Mel were detected in fish maintained under natural photoperiod. Plasma AVT (fmol ml−1) increased during the light to reach the maximal level at the end of that phase (261.7±23.1). Thereafter, AVT concentration decreased and became minimal at 05:00 (68.9±11.5) 3 h before the sunrise. Plasma Mel (pmol ml−1) increased between 16:00 and 22:30 when a peak value was reached (1204.0±55.5). Thereafter, Mel levels decreased and were minimal after the onset of the light phase (242.8±37.0). IT levels displayed no significant diel changes. Linear regression analysis indicated the negative correlation between plasma Mel and AVT for five collecting times of the daily 24 h cycle in freshwater fish and at 22:30 in brackish water fish. A similar correlation occurred at 11:00 in the DD group and at 22:30 in the LL group. To elucidate the character of the Mel-AVT relationships further studies are required.

RESPONSE OF CIRCULATING ARGININE VASOTOCIN AND ISOTOCIN TO RAPID OSMOTIC CHALLENGE IN RAINBOW TROUT

Abstract The plasma levels of arginine vasotocin (AVT) and isotocin (IT) in rainbow trout ( Oncorhynchus mykiss ) were studied to demonstrate changes in neurohypophysial nonapeptides following rapid osmotic challenge. Freshwater-adapted fish were transferred to brackish Baltic water (BW) and brackishwater-adapted fish were transferred to fresh water (FW). In both experiments blood samples for AVT, IT and osmolality were taken 30 min, 1, 2, 24 hr, and 10 days after transfer. Hormones were determined by gradient high-performance liquid chromatography preceded by solid-phase extraction. The pattern of AVT and IT response to external salinity changes was similar during the first hours and showed transient increase or decrease after transfer to brackish or fresh water, respectively. The AVT plasma concentration after 10 days in FW was significantly higher than that in BW, whereas the IT plasma concentrations in both salinities did not differ. These data suggest that the synthesis and or release of AVT and IT are controlled independently, and the roles of hormones in long-term osmotic adaptation are different.

Arginine vasotocin-melatonin interactions in fish: a hypothesis

Arginine vasotocin (AVT) and isotocin (ichthyotocin) are two nonapeptides produced, according to osmotic stimuli, in separate hypothalamic neurosecretory neurons in teleost fish. The neurosecretory cells concentrated in the preoptic nucleus (NPO) give rise to separate vasotocinergic and isotocinergic axons, which end in the neurohypophysis, where the hormones are stored and released (Goossens et al., 1977; Van den Dungen et al., 1982). In fish, AVT plays a role in osmoregulation, cardiovascular activity, endocrine secretion and reproductive processes (Bentley, 1971; Henderson and Wales, 1974; Babiker and Rankin, 1978, 1979, 1980; Fryer and Leung, 1982; Pang et al., 1983; Rodriquez and Specker, 1991). Furthermore, the widespread distribution of AVTimmunoreactive fibres and AVT binding sites in various regions of the brain suggests that AVT may act as a neurotransmitter and/or neuromodulator in the central nervous system in fish (Goossens et al., 1977; Van den Dungen et al., 1982). Arginine vasotocin and isotocin are closely related with the mammalian vasopressin (AVP), and vasotocin is regarded as the original parent neurohypophysial hormone in vertebrates (Fig. 1, upper). Several lines of evidence indicate that AVT and AVP are synthesized de novo also in the mammalian pineal (Binkley, 1988; Olcese et al., 1993) and AVT has been detected in the fish pineal (Holder et al., 1979; Binkley, 1988). Melatonin is a product of tryptophan metabolism by the pineal gland and retina in vertebrates. In all species examined, melatonin synthesis is photoperiod dependent and shows a diurnal rhythm with high levels during the night-time and low levels during daytime (Reiter, 1991; Skene et al., 1991). In teleosts, the pineal containing photoreceptor cells is considered to be the major source of melatonin synthesis (Kezuka et al., 1992; Zachmann et al., 1992b) and is involved in the control of rhythmic adaptations to daily and seasonal cycles (Morton and Forbes, 1988; Falcon and Collin, 1989; Underwood, 1989; Iigo et al., 1991; Zachmann etal. , 1991, 1992a,b; Max and Menaker, 1992).

Vasotocinergic and isotocinergic systems in the gilthead sea bream (Sparus aurata): An osmoregulatory story

To investigate the physiological roles of arginine vasotocin (AVT) and isotocin (IT) in osmoregulatory process in gilthead sea bream (Sparus aurata), a time course study (0, 12h, and 1, 3, 7 and 14 days) has been performed in specimens submitted to hypoosmotic (from 40‰ salinity to 5‰ salinity) or hyperosmotic (from 40‰ salinity to 55‰ salinity) challenges. Plasma and liver osmoregulatory and metabolic parameters, as well as AVT and IT pituitary contents were determined concomitantly with hypothalamic pro-vasotocin (pro-VT) and pro-isotocin (pro-IT) mRNA expression levels. Previously, sequences coding for pro-VT and pro-IT cDNAs were cloned. Two osmoregulatory periods related to plasma osmolality and metabolic parameter variations could be distinguished: i) an adaptative period, from 12h to 3 days after transfer, and ii) a chronic regulatory period, starting at day 3 after transfer. Higher values in hypothalamic pro-VT and pro-IT mRNA expression as well as in pituitary AVT and IT storage levels in both hypo- and/or hyper-osmotic transfers have been distinguished. These increase correlated with changes in plasma cortisol levels, suggesting an interaction between this hormone and pro-VT expression. Furthermore, pro-IT expression enhancement also suggests a role of the isotocinergic system as a modulator in the acute stress response induced by hyper-osmotic challenge in S. aurata.

Changes in hematological parameters in free-living pigeons (Columba livia f. urbana) during the breeding cycle

The blood parameters—red blood cell (RBC) count, hematocrit (Ht), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)—were studied for the first time in free ranging pigeons Columba livia f. urbana. The aim of the study was to establish a set of reference values for this ubiquitous urban species and to examine a potential influence of the factors such as sex and phase of the breeding cycle on the hematological parameters. Blood was sampled from the adult individuals of both sexes during the breeding cycle. Significant variations in Ht and Hb were observed, while RBC count remained unchanged. The lower hematocrit value and hemoglobin concentration found in both males and females during molt probably resulted from the increase of plasma volume not accompanied by an increase in RBC. On the other hand, a significant physiological stress of nutritional deficiency during this energy-consuming stage may manifest itself in the decreased Ht and Hb values. The higher values of Ht and Hb during the laying phase could be related to the dehydration status. There were no overall differences in MCV, MCH, and MCHC values in either of the breeding stage. A lack of sex dimorphism in the measured and calculated blood parameters was apparent. Data collected herein contribute to the better understanding of general physiology of the ubiquitous urban species. The knowledge of variation in the blood indexes with respect to sex and breeding stage is crucial before blood parameters can provide an useful information on physical condition of the bird.