This study examined the metabolic rate (MO2, oxygen consumption) of goldfish (Carassius auratus) under normal management conditions in aquaculture. Using an intermittent respirometry system, we assessed daily variations and the effects of feeding, handling, temperature increase, and anesthetics. MO2 exhibited a daily rhythm, with higher values during day. Feeding to satiety produced a 35% increase in MO2 compared to fasted animals, with a maximum peak after 3 h and returning to baseline after 7 h. Handling stress (5 min) produced a 140% MO2 peak (from 180 to 252 mg O2 kg−1 h−1), returning to the routine MO2 after 2.5 h. An increase in water temperature (+0.1 ◦C min−1) up to 30 ◦C caused MO2 to peak at 200% after 2.5 h from the start of the temperature increase. The use of common anesthetics in aquaculture (MS-222, 2-phenoxyethanol and clove oil in deep anesthesia concentration) affects MO2 during the first few minutes after anesthetic recovery,
but also during the following 4 h. It can be concluded that the metabolic rate is a good indicator of the goldfish’s response to aquaculture practices involving energy expenditure and stress. Thus, intermittent respirometry is a valuable non-invasive tool for understanding and improving fish welfare in aquaculture, This study was funded by PID2022-136288OB-C32 (MCIN/AEI//10.13039/501100011033), Peer reviewed

Experimental data published in the article "Metabolic rate of goldfish (Carassius auratus) in the face of common aquaculture challenges" in the journal "Biology2, Ministerio de Ciencia e Innovación, Peer reviewed

[Data availability]
The datasets generated during and/or analysed during the present study are available in the UCM database DOCTA (https://hdl.handle.net/20.500.14352/134899)., The synchronization of biological rhythms to environmental cues is essential for maintaining temporal homeostasis. However, the specific neuroendocrine and metabolic effects of circadian misalignment in fish remain poorly understood. This study investigated the effects of chronodisruption on energy homeostasis in goldfish (Carassius auratus). Fish were exposed for 53 days to three conditions: Control (12L:12D photoperiod, feeding at ZT 1); Random Feeding (RF; 12L:12D, feeding at random times) and continuous light (LL, feeding at ZT 1). Feed intake, metabolic rate, and locomotor activity were monitored as indicators of energy input and expenditure. Biometric indices (weight and length gain, growth, nutritional and hepatosomatic indices) were calculated. The expression of feeding and growth regulating genes was also analyzed in hypothalamus (hcrt, npy, crh, pomca, cartpt1, and cartpt2), telencephalon (cnr1), liver (igf1, and lepa1), and intestine (cck1 and ghrl). Plasma and liver metabolites (triglycerides, glycogen, lactate, and glucose) and plasma cortisol levels were analyzed to assess metabolic and stress responses. Both RF and LL groups showed elevated metabolic rate and feed intake, accompanied by changes in the expression of feeding regulators that favored orexigenic signals. However, only RF fish showed increased growth, weight, hepatosomatic index, and plasma glucose, suggesting a positive energy balance. LL fish showed growth similar to controls, likely due to a greater increase in energy expenditure. Overall, our results show that disruption of temporal feeding or light cues alters energy balance, with different physiological outcomes depending on the type of disruption. This study highlights the critical role of circadian alignment in maintaining energy homeostasis in goldfish., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by Spanish Ministry of Science and Innovation [PID2022-136288OB-C32 (MCIN/AEI//https://doi.org/10.13039/501100011033)] to N.d.P. and E.I. N.S. and L.H.-C. have been pre-doctoral fellows from the Universidad Complutense de Madrid (CT42/18-CT43/18 and CT63/19-CT64/19) during the experimental part of this work., Peer reviewed

Experimental data of the project FishFedEx, This work was supported by Spanish Ministry of Science and Innovation [PID2022-136288OB-C32 (MCIN/AEI//https://doi.org/10.13039/501100011033)], Peer reviewed

This study evaluates the effectiveness of different anesthetic compounds in the red swamp crayfish, Procambarus clarkii, a species of major commercial importance. The study addresses the growing need for improved welfare standards in crustacean aquaculture by assessing how anesthetics can mitigate the physiological and behavioral stress responses resulting from handling. We first characterized the acute stress response in crayfish after a 5-min air exposure, observing significant changes in hemolymph pH, lactate, chloride, and hemocyanin. Next, we tested the efficacy of several anesthetic/sedative agents, including 2-phenoxyethanol, ethanol, tricaine methanesulfonate (MS-222), clove oil, ketamine, and xylazine. We found that 2-phenoxyethanol, ethanol, and MS-222 were ineffective in inducing deep anesthesia. In contrast, clove oil (0.5 mL L−1), ketamine (50 μg g−1), and xylazine (15 μg g−1) were effective, with each showing distinct induction and recovery times. Physiological analysis one hour after anesthesia revealed that xylazine treatment significantly increased hemolymph lactate, as seen after an acute stress. Similarly, in an open-field test, xylazine-treated crayfish showed reduced time close to the wall, consistent with a putative anxiolytic effect. Ketamine appears to delay full recovery, showing signs of sedation one hour after anesthesia. In summary, our findings suggest that clove oil is a valid anesthetic for P. clarkii, as it induces deep anesthesia with a rapid recovery while effectively mitigating stress-related physiological responses. This study provides a foundational protocol for improving the welfare of decapod crustaceans in aquaculture and research through the use of anesthetics., This work was partially supported by a “Ramón y Cajal” Fellowship (RYC2021-032451-I) and the project PID2022-142638OA-100 (MCIN/AEI/10.13039/50110011033/FEDER, UE), funded by the Spanish Ministry of Science and Innovation, and partially funded by the Recovery, Transformation and Resilience Plan of the European Union granted to IRJ, and by the Spanish Ministry of Science and Innovation though the project PID2022-136288OB-C32 (MCIN/AEI//10.13039/501100011033) granted to N.d.P. and E.I. L.H.C. is beneficiary of a pre-doctoral fellow from the Universidad Complutense de Madrid (CT63/19-CT64/19)., Peer reviewed

The accumulation of microplastics in aquatic ecosystems poses a significant threat to fish physiology and welfare. This study investigated the impact of exposure to virgin polystyrene microplastics (15 µm) on energy balance and welfare in goldfish (Carassius auratus). Fish were exposed for 14 days, and the effects were assessed through an integrated analysis of behavioral, metabolic, neuroendocrine, and physiological parameters. Microplastic exposure significantly reduces feed intake and feed anticipatory activity, indicating a potent anorexigenic effect. This effect was driven by neuroendocrine disruption, characterized by the downregulation of orexigenic neuropeptides (npy, agrp, hcrt) and the upregulation of anorexigenic signaling (pomca, cartpt, lepa). Simultaneously, exposed fish exhibited increased oxygen consumption, suggesting elevated metabolic demands. These factors converged to impaired growth and reduced hepatosomatic index, suggesting altered energy allocation. Furthermore, microplastic exposure induced anxiety-like responses and increased plasma cortisol levels, confirming the activation of the physiological stress response. Overall, these findings demonstrate that microplastics disrupt energy homeostasis and trigger behavioral shifts that ultimately compromise fish welfare and the biological resilience of aquatic species.

The synchronization of biological rhythms to environmental cues is essential for maintaining temporal homeostasis. However, the specific neuroendocrine and metabolic effects of circadian misalignment in fish remain poorly understood. This study investigated the effects of chronodisruption on energy homeostasis in goldfish (Carassius auratus). Fish were exposed for 53 days to three conditions: Control (12L:12D photoperiod, feeding at ZT 1); Random Feeding (RF; 12L:12D, feeding at random times) and continuous light (LL, feeding at ZT 1). Feed intake, metabolic rate, and locomotor activity were monitored as indicators of energy input and expenditure. Biometric indices (weight and length gain, growth, nutritional and hepatosomatic indices) were calculated. The expression of feeding and growth regulating genes was also analyzed in hypothalamus (hcrt, npy, crh, pomca, cartpt1, and cartpt2), telencephalon (cnr1), liver (igf1, and lepa1), and intestine (cck1 and ghrl). Plasma and liver metabolites (triglycerides, glycogen, lactate, and glucose) and plasma cortisol levels were analyzed to assess metabolic and stress responses. Both RF and LL groups showed elevated metabolic rate and feed intake, accompanied by changes in the expression of feeding regulators that favored orexigenic signals. However, only RF fish showed increased growth, weight, hepatosomatic index, and plasma glucose, suggesting a positive energy balance. LL fish showed growth similar to controls, likely due to a greater increase in energy expenditure. Overall, our results show that disruption of temporal feeding or light cues alters energy balance, with different physiological outcomes depending on the type of disruption. This study highlights the critical role of circadian alignment in maintaining energy homeostasis in goldfish.

Social rewards may have evolved in social species to reinforce adaptive social interactions. Yet, evidence for social rewards is still scarce, and the underlying mechanisms remain unclear. A key candidate to regulate the value of social stimuli is oxytocin due to its role in social affiliation, which is traced to its origins in ray-finned fish—but whether it encodes rewards is uncertain. Using a single-trial conditioned place preference test, we found that wild-type zebrafish increased preference for a neutral unpreferred cue associated with a same-sex sibling, while oxytocin receptor (oxtr) mutants did not. These findings demonstrate the necessity of oxtr for social rewards, while the short exposure infers its role in encoding rather than consolidation. Our results provide evidence for an evolutionarily conserved role of oxytocin in social reward encoding given the available evidence for similar effects in rodents.

Background: Melatonin is a key hormone in regulation of circadian rhythms, and involved in many rhythmic functions, such as feeding and locomotor activity. Melatonin reportedly counteracts stress responses in many vertebrates, including fish. However, targets for this action of melatonin and underlying mechanisms remain unknown.

Results: This study reports potential anti-stress properties of melatonin in goldfish (Carassius auratus), with a focus on its effect on plasma cortisol, food intake, and locomotor activity, all of them involved in the responses to stress exposure. Indeed, acute injection of melatonin counteracted stress-induced hypercortisolinemia and reduced food intake. The reduced locomotor activity following melatonin treatment suggests a possible sedative role in fish. To assess whether this anti-stress effects of melatonin involve direct actions on interrenal tissue, in vitro cultures of head kidney (containing the interrenal cortisol-producing tissue) were carried out in presence of ACTH, melatonin, and luzindole, an antagonist of melatonin receptors. Melatonin in vitro reduced ACTH-stimulated cortisol release, an effect attenuated by luzindole; this suggests the presence of specific melatonin receptors in interrenal tissue.

Conclusions: Our data support a role for melatonin as an anti-stress signal in goldfish, and suggest that the interrenal tissue of teleosts may be a plausible target for melatonin action decreasing cortisol production.

Di-2-ethylhexyl phthalate (DEHP), a widely used plasticiser, is a pervasive environmental contaminant with potential detrimental effects on aquatic organisms. The objective of this study was to provide an integrative analysis of how DEHP alters energy balance, temporal homeostasis and fish welfare - interrelated aspects critical to animal survival - to address critical gaps in our understanding of its toxicological effects. Goldfish (Carassius auratus) were chronically (14 days) treated with DEHP. Energy balance was assessed through locomotor activity, metabolic rate, feed intake, and growth indices. Daily of locomotor and metabolic rate rhythms were examined to explore potential circadian disruptions. Anxiety-like behaviours were also examined to assess welfare. DEHP decreased feed intake and food-anticipatory activity (FAA), suggesting an anorexigenic effect, which may have been mediated by increased expression of anorexigenic genes in the hypothalamus and liver, along with decreased expression of orexigenic npy (neuropeptide Y) gene in the hypothalamus. Growth parameters remained unchanged, probably due to compensatory reductions in energy expenditure, as indicated by decreased locomotor activity and metabolic rate. Daily rhythms in these two parameters were preserved, suggesting no disruption in temporal homeostasis. DEHP increased hepatic expression of peroxisome proliferator-activated receptor (PPAR)-related genes, suggesting that PPARs activation is a potential mode of action for DEHP in fish. Anxiety levels were elevated, as evidenced by increased thigmotaxis and scototaxis in behavioural tests, which may be mediated by changes in hypothalamic neuropeptides. These findings highlight the adverse effects of DEHP on energy regulation and animal welfare, providing novel insights into its broader physiological consequences in fish.

Data of the article: Three Non-Invasive Tests Reveal Anxiety-like Responses During Food Anticipation in Rainbow Trout . Anxiety-like behavior in fish is commonly assessed using non-invasive behavioral paradigms such as the Light/Dark preference, Novel Tank, and Open Field tests. In this study, we validated these three assays in rainbow trout (Oncorhynchus mykiss), a species of commercial relevance, to characterize their anxiety-related responses. To explore behavioral changes associated with feeding anticipation and satiety, we implemented a feeding schedule consisting of two daily meals and conducted behavioral tests at specific times before and after feeding. Trout exhibited clear patterns of scototaxis, geotaxis, and thigmotaxis, consistent with anxiety-like behavior described in other teleosts. Our results showed a significant increase in anxiety-like responses before feeding, coinciding with food anticipatory activity observed prior to expected feeding schedules, which diminished after food intake, as evidenced by each test individually. Moreover, multivariate analysis combining parameters from all three tests improved discrimination between anxious and relaxed fish. The behavioral states before and after feeding resembled anxiety-like and anxiolytic conditions reported in other species, supporting that food anticipatory activity reflects an anxious state in rainbow trout as well. These findings endorse using a multi-test behavioral battery to assess anxiety-like states and provide a framework for studying neurobiological mechanisms of emotional regulation related to feeding in teleosts.

Food anticipatory activity (FAA), the increase in locomotor activity prior to a scheduled feeding time, is linked not only to energy demands but also to food reward expectations. However, the mechanisms behind FAA remain unclear. We hypothesize that FAA induces anxiety due to reward anticipation. To test this, anxiety-like behavior was assessed in goldfish using open field and black-and-white preference tests under different feeding conditions and daytimes. Increased thigmotactic and scototactic behavior during FAA, compared to the post-FAA period, was observed, even in animals under constant light. Additionally, an unexpected meal did not reduce anxiety, while more hours of fasting did not increase it, suggesting that FAA is an anxiogenic state driven by a food-entrained oscillator rather than energy status. Ghrelin signaling was further explored using two antagonists, both of which reduced anxiety-like behavior when administered during FAA. Moreover, ghrelin administration induced anxiety-like behavior that was reversed by co-injection with an antagonist, supporting its anxiogenic role. These findings suggest a shared neural mechanism between FAA and anxiety, mediated by ghrelin. The possible involvement of ghrelin in activating dopamine-reward circuits in teleosts should be explored in future studies to gain further insight into the neurobiological basis of food anticipatory activity.

Anxiety-like behavior in fish is commonly assessed using non-invasive behavioral paradigms such as the Light/Dark preference, Novel Tank, and Open Field tests. In this study, we validated these three assays in rainbow trout (Oncorhynchus mykiss), a species of commercial relevance, to characterize their anxiety-related responses. To explore behavioral changes associated with feeding anticipation and satiety, we implemented a feeding schedule consisting of two daily meals and conducted behavioral tests at specific times before and after feeding. Trout exhibited clear patterns of scototaxis, geotaxis, and thigmotaxis, consistent with anxiety-like behavior described in other teleosts. Our results showed a significant increase in anxiety-like responses before feeding, coinciding with food anticipatory activity observed prior to expected feeding schedules, which diminished after food intake, as evidenced by each test individually. Moreover, multivariate analysis combining parameters from all three tests improved discrimination between anxious and relaxed fish. The behavioral states before and after feeding resembled anxiety-like and anxiolytic conditions reported in other species, supporting that food anticipatory activity reflects an anxious state in rainbow trout as well. These findings endorse using a multi-test behavioral battery to assess anxiety-like states and provide a framework for studying neurobiological mechanisms of emotional regulation related to feeding in teleosts.