Tryptophan supplementation reduces body weight but does not reduce anxiety-like behavior in zebrafish

Tryptophan supplementation reduces body weight but does not reduce anxiety-like behavior in zebrafish | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tryptophan supplementation reduces body weight but does not reduce anxiety-like behavior in zebrafish Pêssi Socorro Lima de Sousa, Jhon Buenhombre, Erika Alexandra Daza-Cardona, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4151627/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Under certain circumstances, some nutrients can affect the synthesis of central neurotransmitters and potentially behavior. The aim of this study was to characterize the influence of tryptophan supplementation on potential anxiety-like behaviors in zebrafish ( Danio rerio ), as well as to assess the suitability of a new standardized diet formulation for the better maintenance and performance of this species. To achieve this, the proportion of the amino acid tryptophan supplementation was experimentally modified at 5%, 50%, and 100% levels, individually, in the zebrafish diet. The investigation was conducted through the Y-maze test, light-dark preference test, and inhibitory avoidance test. The results indicated that tryptophan supplementation did not lead to the expected reduction in anxiety-like effects in the Y-maze and light-dark tests. However, a significant difference in avoidance times was observed in the inhibitory avoidance test between the groups with 5% and 50% supplemented diets, but a significant difference in avoidance times was observed in the inhibitory avoidance test between the groups with 5% and 50% supplemented diets. Changing the diet did not alter the animal's learning or natural anxiety. However, in most animals, tryptophan supplementation led to a decrease in body mass. Anxiety like behaviours tryptophan serotonin zebrafish memory Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION The increase in brain serotonin levels has been associated with social behaviors, such as affiliation and cooperation (Steenbergen et al 2016 ). Theories that link serotonin to animal behavior provide a framework for understanding how serotonin influences behavior across various species (Lucki 1998 ). The zebrafish ( Danio rerio Hamilton 1822) popularly known as the zebrafish, belongs to the Cyprinidae family (Mansur, Rodrigues and Gouveia Jr 2014 ). As a low-cost and easily maintainable animal species (Aoki, Tsuboi and Okamoto 2015 ; Maximino et al 2010a ), it is investigated in numerous disciplines, including neuroscience research (Cachat et al 2010 ; Serra, Medalha and Mattioli 1999 ; Walsh-Monteiro et al 2016 ). Despite its success as a model for biomedical studies, diets for Danio rerio still largely consist of nutrients whose composition is unknown for their physiology, potentially including carcinogenic diet components. Most formulated fish feeds prioritize growth rate over health and, consequently, longevity (Penglase, Moren and Hamre 2012 ; Watts et al 2016 ). Nevertheless, the use of zebrafish as a model for nutritional research is still limited due to a lack of data (Kaushik, Georga and Koumoundouros, 2011 ; Lawrence 2007 ; Smith Jr et al 2013 ). Additionally, the manipulation of tryptophan in emotional processing is of interest for the treatment of social behavior in animals and humans (Attenburrow et al 2003 ). This work aims to address the development needs of a diet that optimizes the behavior and maintenance of zebrafish, an animal whose learning and memory capabilities have already been effectively demonstrated (Blank et al 2009 ; Sison and Gerlai 2010 ). In this context, behavioral studies are crucial to increase our understanding of pathological models that affect the central nervous system, allowing for the evaluation of toxic or neuroprotective agents on cognition (Cognato 2012). Specifically, the conservation of the serotonergic system across the evolutionary scale of vertebrates provides a bridge between assessing treatment efficacy in fish and its potential therapeutic effects consistent with small mammals and even humans (Mcdonald 2017 ). However, despite this conservation, the role of serotonin in social behavior cannot be simply established in terms of pure inhibition or facilitation of aggression (Teles 2013) Perhaps because of this, the behavioral effects of tryptophan supplementation may be inconclusive (Meunier-Salaün 1991). In this species, to date, there is no consensus regarding the predominance of sex versus aggression (Dahlbom et al 2012 ) and anxiety-like behavior (Bozi et al 2021 ; Egan et al 2009 ; Magno et al 2015 ). Therefore, fish of both sexes are used. In this study, we examined the biobehavioral effects of serotonergic manipulation in zebrafish (Teixeira et al 2021 ) induced by controlled tryptophan supplementation at different treatment times, using unconditioned exploration models that yield rapid results without painful stimuli. 2. MATERIALS AND METHODS 2.1 Subjects Eighty adult wild-type Danio rerio fish of undetermined sex and age, obtained from local suppliers, were used in this study. They were housed in a technical reservoir connected to a microenvironmental control unit called the “autonomous zebtec active blue – zebrafish housing system,” manufactured by TECNIPLAST S.p.. Fish were kept in 3.5-liter aquariums with the water maintained at a pH of 7 ± 1, with a temperature of 28º C. The laboratory maintained a climate-controlled environment with a temperature set at 24 ± 1º C. The lighting cycle was programmed at 10/14 hours, light/dark, starting at 6:00 AM. This research was approved by the Ethics Committee on Animal Use at the Federal University of Pará (CEUA/UFPA) under protocol number 2903141118 (ID 001143). The period from its introduction to the individual aquarium until the start of the feeding test was considered an acclimatization period. All behavioral experiments were conducted between 8:00 AM and 1:00 PM, with a minimum one-day interval between tests (Maximino et al 2010b ). All animals underwent the tests in the following order: first, the Y-maze test, then the light-dark test, and finally the inhibitory avoidance test, always with a minimum interval of one day between tests. The water in the apparatuses was changed after each individual test. 2.2 Feeding and Treatment The fish were kept in individual aquariums, and their food was fractionated and packaged in separate samples, using pre-weighed aliquots. Feeding occurred once daily, between 09:30 and 11:30 AM, using pre-weighed aliquots. The amount of food was calculated at 4% of the average biomass of 60 animals, with an average of 0.47g, ranging between 0.0020 and 0.0025g (Dammski et al 2011 ). On test days, the animals were fed at least 30 minutes after returning to their original aquarium. We used standard commercial fish food (Tetra Min®) for feeding. The test diet in this study was modified by adding 280 mg of Tryptophan VITALAB® to the standard food as follows: For the 5% supplemented diet, 50g of food was weighed, and 0.025 mg of tryptophan was added to it. For the 50% supplemented diet, another 50g portion of food had 0.127 mg added, and for the 100% tryptophan supplemented diet, 0.255 mg of this amino acid was added to 50g of food. An electronic analytical balance (BIOPRECISA® model FA2104N) was used for quantification. The quantities of the amino acid added to the modified diets were based on the specifications (%) found in a standard diet for the species of fish in question: 0.50% tryptophan (Smith Jr et al 2013 ). 2.3 Biomass Evaluation The animals, which had been previously identified, were weighed before starting controlled feeding and after the final test using a pocket scale ® model MH-500. 2.4 Procedures The fish were divided into 8 groups, each consisting of 10 individuals, based on their nutritional treatment (standard diet or supplemented) and the duration of diet use, which included 4 and 8 weeks. One group received the standard diet (control group), while the remaining groups were treated with the same diet supplemented with 5%, 50%, or 100% tryptophan. After completing the designated duration of dietary treatment, all fish were subjected to the experimental tests described below. The water in the apparatuses was changed after each individual test. The procedures were recorded using a digital camera throughout the period (JVC® camera model GZ-EX210), and the files were subsequently transferred to the computer for evaluation using the "Movies & TV" program (Microsoft®). 2.4.1 Y-Maze Test In this experiment, adapted from Cognato (2012), a transparent glass aquarium with three compartments ("arms") was used, each measuring 25 cm in length x 8 cm in width x 15 cm in height. Visual cues in the form of paper geometric shapes, including vertical stripes, horizontal stripes, and squares, were affixed to the external walls of the apparatus. Black adhesive plastic covered the remaining areas of the walls, while the floor was covered in white. The initial compartment for the maze was determined randomly. During the first phase of the test, the fish were allowed to freely explore two of the arms for 5 minutes. After a 1-hour interval, they were released into the same arm where they had started the previous test and could explore all three arms. We analyzed various parameters, including the time spent in each arm and the number of crossings. 2.4.2 Light/Dark Preference Test The test (Maximino et al 2010c ) was conducted in an aquarium with alternating white and black compartments separated by a central area, which served as the starting compartment for the test. After a 5-minute habituation period, the sliding doors were removed, granting access to the compartments and allowing the animal to explore the environment for 15 minutes. The parameters evaluated included the time spent in each compartment of the tank, the latency to enter the white compartment, and the number of entries into the white compartment. 2.4.3 Inhibitory Avoidance Test The inhibitory avoidance test, also known as passive avoidance, is a form of aversive learning. For this test, an aquarium was used, which had a guillotine door dividing it into a larger black compartment and a white compartment (Santos et al 2019 ). After a 5-minute acclimation period, the guillotine door was opened, and the subject's latency to enter the white compartment was measured. At the end of 5 minutes interval, the fish was gently removed from the aquarium using a specialized net and reintroduced into the smaller compartment, with the guillotine door closed. The test consisted of a total of five episodes. 3. RESULTS 3.1 Evaluation of Pre and Post-Treatment Biomass Most of the animals decreased their mass after Tryptophan supplementation. There was a significant difference (Z = -3.999, p = 0.000) between the pre-treatment (mean = 0.4108) and post-treatment (mean = 0.3514) mass after 4-week supplementation. Likewise, there was a significant difference (Z = -4.385, p = 0.000) for pre-treatment (mean = 0.4359) and post-treatment (mean = 0.3462) for 8 weeks of supplementation (Fig. 1 ). 3.2 Y-Maze Test In both the tests conducted after 4 weeks and 8 weeks of treatment, The Kolmogorov-Smirnov test and Levene’s test were used. For group comparisons, we employed a one-way ANOVA followed by Tukey's post hoc test. There was no significant effect of the level of tryptophan on the start arm of the maze (p = 0.335), the other arm (p = 0.498), and the novel arm of the maze (p = 0.891) (Fig. 2 ). 3.3 Light-Dark Preference Test 3.4 Inhibitory Avoidance Test The data were compared using the Kolmogorov-Smirnov test, the Friedman test, and a Kruskal-Wallis test followed by pairwise comparison with Bonferroni correction. After 4 weeks, the control group showed a significant difference in the time between L1 and L4 (p = 0.041) and L1 and L5 (p = 0.041). However, contrary to expectations, there was a decrease in latency to enter the white compartment. This also occurred with all groups combined between L1 and L3 (, L1 and L4 (p = 0.012), and L1 and L5 (p = 0.07). Among the groups treated for 8 weeks, not even the control group showed a significant difference between the trials (p = 0.406). Despite the results of both treatment doses not showing significant effects, there was a noticeable decrease in latency time to the white compartment across trials, suggesting excitement/anxiety/stress (Maximino et al 2011 ). Learning deficits can also be interpreted as impulsivity (Chirinéa and Gouveia Jr 2019 ) or even habituation to the white compartment. Interestingly, in experiments conducted by Santos et al ( 2019 ), treatment with fluoxetine in animals did not prevent the acquisition of inhibitory avoidance (Fig. 4 ). Inhibitory avoidance 8 weeks for all groups All groups 8 weeks Here, the consumption of individual doses of food caused social animals to remain isolated during the equivalent treatment period, which likely influenced them in the form of stress. It was not possible to quantify brain serotonin levels in this study. However, it was possible to investigate commonly used behavioral parameters after the oral administration of precise levels of tryptophan in zebrafish. 4. DISCUSSION The evaluation of pre- and post-treatment biomass results presents the greatest evidence. Since tryptophan is a precursor of serotonin, the serotonergic system has long been associated with satiety (Halford et al 2011 ), which could explain the mass loss. Another possibility is that tryptophan supplementation may lead to a decrease in body weight in rats by oxidizing fatty acids in the body for lipid synthesis (Ruan et al 2014 ). On the first day, we tested the fish’s memory: In the y-maze test, the expected outcome would be animals showing a preference for the unexplored arm (Cognato et al 2012 ). In our experiments, there was no preference for any arm, making it inconclusive whether there was recognition of the arms explored earlier in the treatment groups with different concentrations at both proposed treatment durations. Similar results were obtained after the use of fluoxetine. Despite being known for its anxiolytic effects, its use did not alter memory acquisition in the y-maze test but reduced the activity of the zebrafish at the bottom of the tank (also indicative of anxiety-like effects) in the novel tank test, similar to diazepam (Giacomini et al 2016 ). In rats, there has been recognized difficulty with the y-maze, related to avoidance of the novel arm stemming from anxiety-like behavior (Hughes 2004 ). Findings suggest that vertebrate species such as zebrafish, rodents, and humans perform exploration in similar behavioral models (Cleal et al 2021 ). In the second stage, the light-dark preference test results suggest some predominance of anxiety-like behavioral responses and/or attention deficits (Chirinéa and Gouveia Jr 2019 ). Maximino et al ( 2013 ) also observed this type of behavior in zebrafish in the light-dark test after the administration of 2.5 mg/kg of fluoxetine intraperitoneally, while the dose of 10 mg/kg led to an increase in the number of entries. In the novel tank test, zebrafish exhibited a tendency to prevent anxiety-like effects after immersion in 300 mg/L of tryptophan (Giacomini et al 2020 ). In the next test, the inhibitory avoidance test, despite the results of both treatment doses not showing significant effects, there was a noticeable decrease in latency time to the white compartment across trials, suggesting excitement/anxiety/stress (Maximino et al 2011 ). Learning deficits can also be interpreted as impulsivity (Chirinéa and Gouveia Jr 2019 ) or even habituation to the white compartment. Interestingly, in experiments conducted by Santos et al ( 2019 ), treatment with fluoxetine in animals did not prevent the acquisition of inhibitory avoidance. Evidence indicates that the alteration of the metabolic process determined by the nutritional condition of these animals cold lead to behavioral responses (Lieberman 2003 ; Gomez-Requeni 2010). Here, the consumption of individual doses of food caused social animals to remain isolated during the equivalent treatment period, which likely influenced them in the form of stress. It was not possible to quantify brain serotonin levels in this study. However, it was possible to investigate commonly used behavioral parameters after the oral administration of precise levels of tryptophan in zebrafish. 5. CONCLUSION It is known that the induction of an increase in cerebral serotonin levels can be achieved through dietary supplementation with tryptophan (Teixeira et al 2021 ). This study had the advantage of individually examining potential anxiolytic and cognitive effects in different models without the need for long training periods. It tested oral tryptophan supplementation at different concentrations and after two time periods, one of which (8 weeks) was considered prolonged use for the Danio rerio species. Studies have observed that affective states influence attention, memory and even decision- making in various species (Buenhombre et al, 2021 ; Buenhombre et al, 2023 ). The results were integrated and tryptophan couldn’t affect the results on better learning and memory in zebrafish. Tryptophan was not enough to neutralize the animals' anxiety and therefore was unable to affect the results in better learning and memory in zebrafish; However, this indicates the safety of its use for breeding and suggests potential differences from what occurs in mammals, reinforcing that the relationship between tryptophan and its behavioral paradigm is still not well understood. Declarations Ethical Approval: This research was approved by the Ethics Committee on Animal Use at the Federal University of Pará (CEUA/UFPA) under protocol number 2903141118 (ID 001143). Conflicts of Interest: The authors have no conflict of interest to declare. Funding: This study was financed in part by the Coordination for the Improvement of Higher Education Personnel - Brazil - CAPES. 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Behav Brain Res 253:17–24. https://doi.org/10.1016/j.bbr.2013.07.012 Walsh-Monteiro A, Pessoa RDS, Sanches ÉM et al (2016) A new anxiety test for zebrafish: Plus maze with ramp. Psychol Neurosci 9:457–464. https://doi.org/10.1037/pne0000067 Watts SA, Lawrence C, Powell M et al (2016) The vital relationship between nutrition and health in zebrafish. Zebrafish 13:S72–S76. https://doi.org/10.1089/zeb.2016.1299 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 26 Mar, 2024 Submission checks completed at journal 26 Mar, 2024 First submitted to journal 22 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4151627","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284218993,"identity":"d2564e9c-a07d-4daa-ad0f-6222a59e5d8d","order_by":0,"name":"Pêssi Socorro Lima de Sousa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFADZuYDQFJChhQtbAkgLTykWMNjACYJqpNv7zH+8HOHjb18O8/nVzdqLHgY2A8f3YBPi8GZM2aSvWfSEjcc5t1mnXMM6DCetLQbeLVI5Jgx8LYdTjBg5t1mnMMG1CLBY4ZXi/yMHOOPf9v+28s38zwzzvlHhBaGGzkG0rxtBxgbDvMwP85tI0KLwZljZdKyZ5KBfmEzY87tk+BhI+QX+fbmzR/f7rCzl+8//Phzzrc6OX72w8fwOwwEGBvAFJsEmCSoHEkL8weiVI+CUTAKRsGIAwDv7EQxO7yHkQAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Para","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Pêssi","middleName":"Socorro Lima","lastName":"de Sousa","suffix":""},{"id":284219001,"identity":"b9aac44e-c775-4c33-b043-30ac54088f14","order_by":1,"name":"Jhon Buenhombre","email":"","orcid":"","institution":"Faculty of Veterinary Medicine, Fundación Universitaria Agraria de Colombia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jhon","middleName":"","lastName":"Buenhombre","suffix":""},{"id":284219005,"identity":"99a01388-0358-4661-9eaa-f348892be0fa","order_by":2,"name":"Erika Alexandra Daza-Cardona","email":"","orcid":"","institution":"Faculty of Veterinary Medicine, Fundación Universitaria Agraria de Colombia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Erika","middleName":"Alexandra","lastName":"Daza-Cardona","suffix":""},{"id":284219006,"identity":"d8cd6cea-6ac1-4983-bab9-f8ec64ad4429","order_by":3,"name":"Yuri Richard","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuri","middleName":"","lastName":"Richard","suffix":""},{"id":284219008,"identity":"5942839b-6c42-43e2-bbb2-83af847728c3","order_by":4,"name":"Amauri Gouveia Jr","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amauri","middleName":"","lastName":"Gouveia","suffix":"Jr"}],"badges":[],"createdAt":"2024-03-22 19:08:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4151627/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4151627/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53774229,"identity":"1f96bd38-dee8-47d5-b41b-6caed4386778","added_by":"auto","created_at":"2024-03-30 07:36:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31579,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Comparison of body masses before and after 4 weeks of treatment, (B) After 8 weeks of treatment.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-4151627/v1/d04a6d952592ec1626cd0c6e.png"},{"id":53773953,"identity":"f0eb43fc-e20f-4cd3-b8f0-109c66d07087","added_by":"auto","created_at":"2024-03-30 07:28:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48801,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Analysis of the percentage of time spent in each arm of the maze after 4 weeks and (B) after 8 weeks.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-4151627/v1/fd28ad07c18ab70cb6bc162c.png"},{"id":53773950,"identity":"4a0c3843-0b8a-4589-84f6-5d88b728218e","added_by":"auto","created_at":"2024-03-30 07:28:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99678,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the light-dark test. After 4 weeks: (A) Time spent by each group in the light compartment, (B) Number of entries into the light compartment, (C) Latency to enter the white compartment. After 8 weeks: (D) Duration in the light compartment, (E) Entries into the light compartment, (F) Latency to enter the light compartment.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-4151627/v1/2c6b9eaf479500173013a419.png"},{"id":53773951,"identity":"3b4f41db-3aed-4baf-a549-33d8945ceaae","added_by":"auto","created_at":"2024-03-30 07:28:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74563,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the Inhibitory Avoidance Test. After 4 weeks: (A) Differences in latency to enter the white compartment with all groups, (B) Latency to enter the white compartment for each group. After 8 weeks: (C) Differences in latency to enter the white compartment with all groups, (D) Latency to enter the white compartment for each group.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-4151627/v1/fe3bfc94a0049e0a2d87d59f.png"},{"id":53774480,"identity":"bceb3a62-636e-4f52-85e3-bf4400e25809","added_by":"auto","created_at":"2024-03-30 07:44:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":528769,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4151627/v1/4838e088-726f-44dc-9055-580162851984.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tryptophan supplementation reduces body weight but does not reduce anxiety-like behavior in zebrafish","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe increase in brain serotonin levels has been associated with social behaviors, such as affiliation and cooperation (Steenbergen et al \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Theories that link serotonin to animal behavior provide a framework for understanding how serotonin influences behavior across various species (Lucki \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e Hamilton 1822) popularly known as the zebrafish, belongs to the Cyprinidae family (Mansur, Rodrigues and Gouveia Jr \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). As a low-cost and easily maintainable animal species (Aoki, Tsuboi and Okamoto \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Maximino et al \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e), it is investigated in numerous disciplines, including neuroscience research (Cachat et al \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Serra, Medalha and Mattioli \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Walsh-Monteiro et al \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite its success as a model for biomedical studies, diets for \u003cem\u003eDanio rerio\u003c/em\u003e still largely consist of nutrients whose composition is unknown for their physiology, potentially including carcinogenic diet components. Most formulated fish feeds prioritize growth rate over health and, consequently, longevity (Penglase, Moren and Hamre \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Watts et al \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Nevertheless, the use of zebrafish as a model for nutritional research is still limited due to a lack of data (Kaushik, Georga and Koumoundouros, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lawrence \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Smith Jr et al \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Additionally, the manipulation of tryptophan in emotional processing is of interest for the treatment of social behavior in animals and humans (Attenburrow et al \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis work aims to address the development needs of a diet that optimizes the behavior and maintenance of zebrafish, an animal whose learning and memory capabilities have already been effectively demonstrated (Blank et al \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sison and Gerlai \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this context, behavioral studies are crucial to increase our understanding of pathological models that affect the central nervous system, allowing for the evaluation of toxic or neuroprotective agents on cognition (Cognato 2012). Specifically, the conservation of the serotonergic system across the evolutionary scale of vertebrates provides a bridge between assessing treatment efficacy in fish and its potential therapeutic effects consistent with small mammals and even humans (Mcdonald \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, despite this conservation, the role of serotonin in social behavior cannot be simply established in terms of pure inhibition or facilitation of aggression (Teles 2013) Perhaps because of this, the behavioral effects of tryptophan supplementation may be inconclusive (Meunier-Sala\u0026uuml;n 1991). In this species, to date, there is no consensus regarding the predominance of sex versus aggression (Dahlbom et al \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and anxiety-like behavior (Bozi et al \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Egan et al \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Magno et al \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, fish of both sexes are used. In this study, we examined the biobehavioral effects of serotonergic manipulation in zebrafish (Teixeira et al \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) induced by controlled tryptophan supplementation at different treatment times, using unconditioned exploration models that yield rapid results without painful stimuli.\u003c/p\u003e "},{"header":"2. MATERIALS AND METHODS","content":" \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Subjects\u003c/h2\u003e \u003cp\u003eEighty adult wild-type \u003cem\u003eDanio rerio\u003c/em\u003e fish of undetermined sex and age, obtained from local suppliers, were used in this study. They were housed in a technical reservoir connected to a microenvironmental control unit called the \u0026ldquo;autonomous zebtec active blue \u0026ndash; zebrafish housing system,\u0026rdquo; manufactured by TECNIPLAST S.p.. Fish were kept in 3.5-liter aquariums with the water maintained at a pH of 7\u0026thinsp;\u0026plusmn;\u0026thinsp;1, with a temperature of 28\u0026ordm; C. The laboratory maintained a climate-controlled environment with a temperature set at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026ordm; C. The lighting cycle was programmed at 10/14 hours, light/dark, starting at 6:00 AM.\u003c/p\u003e \u003cp\u003e This research was approved by the Ethics Committee on Animal Use at the Federal University of Par\u0026aacute; (CEUA/UFPA) under protocol number 2903141118 (ID 001143). The period from its introduction to the individual aquarium until the start of the feeding test was considered an acclimatization period.\u003c/p\u003e \u003cp\u003eAll behavioral experiments were conducted between 8:00 AM and 1:00 PM, with a minimum one-day interval between tests (Maximino et al \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e). All animals underwent the tests in the following order: first, the Y-maze test, then the light-dark test, and finally the inhibitory avoidance test, always with a minimum interval of one day between tests. The water in the apparatuses was changed after each individual test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Feeding and Treatment\u003c/h2\u003e \u003cp\u003eThe fish were kept in individual aquariums, and their food was fractionated and packaged in separate samples, using pre-weighed aliquots. Feeding occurred once daily, between 09:30 and 11:30 AM, using pre-weighed aliquots. The amount of food was calculated at 4% of the average biomass of 60 animals, with an average of 0.47g, ranging between 0.0020 and 0.0025g (Dammski et al \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). On test days, the animals were fed at least 30 minutes after returning to their original aquarium.\u003c/p\u003e \u003cp\u003eWe used standard commercial fish food (Tetra Min\u0026reg;) for feeding. The test diet in this study was modified by adding 280 mg of Tryptophan VITALAB\u0026reg; to the standard food as follows: For the 5% supplemented diet, 50g of food was weighed, and 0.025 mg of tryptophan was added to it. For the 50% supplemented diet, another 50g portion of food had 0.127 mg added, and for the 100% tryptophan supplemented diet, 0.255 mg of this amino acid was added to 50g of food. An electronic analytical balance (BIOPRECISA\u0026reg; model FA2104N) was used for quantification. The quantities of the amino acid added to the modified diets were based on the specifications (%) found in a standard diet for the species of fish in question: 0.50% tryptophan (Smith Jr et al \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Biomass Evaluation\u003c/h2\u003e \u003cp\u003eThe animals, which had been previously identified, were weighed before starting controlled feeding and after the final test using a pocket scale \u0026reg; model MH-500.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Procedures\u003c/h2\u003e \u003cp\u003eThe fish were divided into 8 groups, each consisting of 10 individuals, based on their nutritional treatment (standard diet or supplemented) and the duration of diet use, which included 4 and 8 weeks. One group received the standard diet (control group), while the remaining groups were treated with the same diet supplemented with 5%, 50%, or 100% tryptophan.\u003c/p\u003e \u003cp\u003eAfter completing the designated duration of dietary treatment, all fish were subjected to the experimental tests described below. The water in the apparatuses was changed after each individual test. The procedures were recorded using a digital camera throughout the period (JVC\u0026reg; camera model GZ-EX210), and the files were subsequently transferred to the computer for evaluation using the \"Movies \u0026amp; TV\" program (Microsoft\u0026reg;).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Y-Maze Test\u003c/h2\u003e \u003cp\u003eIn this experiment, adapted from Cognato (2012), a transparent glass aquarium with three compartments (\"arms\") was used, each measuring 25 cm in length x 8 cm in width x 15 cm in height. Visual cues in the form of paper geometric shapes, including vertical stripes, horizontal stripes, and squares, were affixed to the external walls of the apparatus. Black adhesive plastic covered the remaining areas of the walls, while the floor was covered in white. The initial compartment for the maze was determined randomly. During the first phase of the test, the fish were allowed to freely explore two of the arms for 5 minutes. After a 1-hour interval, they were released into the same arm where they had started the previous test and could explore all three arms. We analyzed various parameters, including the time spent in each arm and the number of crossings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Light/Dark Preference Test\u003c/h2\u003e \u003cp\u003eThe test (Maximino et al \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010c\u003c/span\u003e) was conducted in an aquarium with alternating white and black compartments separated by a central area, which served as the starting compartment for the test. After a 5-minute habituation period, the sliding doors were removed, granting access to the compartments and allowing the animal to explore the environment for 15 minutes. The parameters evaluated included the time spent in each compartment of the tank, the latency to enter the white compartment, and the number of entries into the white compartment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Inhibitory Avoidance Test\u003c/h2\u003e \u003cp\u003eThe inhibitory avoidance test, also known as passive avoidance, is a form of aversive learning. For this test, an aquarium was used, which had a guillotine door dividing it into a larger black compartment and a white compartment (Santos et al \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After a 5-minute acclimation period, the guillotine door was opened, and the subject's latency to enter the white compartment was measured. At the end of 5 minutes interval, the fish was gently removed from the aquarium using a specialized net and reintroduced into the smaller compartment, with the guillotine door closed. The test consisted of a total of five episodes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Evaluation of Pre and Post-Treatment Biomass\u003c/h2\u003e \u003cp\u003eMost of the animals decreased their mass after Tryptophan supplementation. There was a significant difference (Z = -3.999, p\u0026thinsp;=\u0026thinsp;0.000) between the pre-treatment (mean\u0026thinsp;=\u0026thinsp;0.4108) and post-treatment (mean\u0026thinsp;=\u0026thinsp;0.3514) mass after 4-week supplementation. Likewise, there was a significant difference (Z = -4.385, p\u0026thinsp;=\u0026thinsp;0.000) for pre-treatment (mean\u0026thinsp;=\u0026thinsp;0.4359) and post-treatment (mean\u0026thinsp;=\u0026thinsp;0.3462) for 8 weeks of supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Y-Maze Test\u003c/h2\u003e \u003cp\u003eIn both the tests conducted after 4 weeks and 8 weeks of treatment, The Kolmogorov-Smirnov test and Levene\u0026rsquo;s test were used. For group comparisons, we employed a one-way ANOVA followed by Tukey's post hoc test. There was no significant effect of the level of tryptophan on the start arm of the maze (p\u0026thinsp;=\u0026thinsp;0.335), the other arm (p\u0026thinsp;=\u0026thinsp;0.498), and the novel arm of the maze (p\u0026thinsp;=\u0026thinsp;0.891) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Light-Dark Preference Test\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Inhibitory Avoidance Test\u003c/h2\u003e \u003cp\u003eThe data were compared using the Kolmogorov-Smirnov test, the Friedman test, and a Kruskal-Wallis test followed by pairwise comparison with Bonferroni correction.\u003c/p\u003e \u003cp\u003eAfter 4 weeks, the control group showed a significant difference in the time between L1 and L4 (p\u0026thinsp;=\u0026thinsp;0.041) and L1 and L5 (p\u0026thinsp;=\u0026thinsp;0.041). However, contrary to expectations, there was a decrease in latency to enter the white compartment. This also occurred with all groups combined between L1 and L3 (, L1 and L4 (p\u0026thinsp;=\u0026thinsp;0.012), and L1 and L5 (p\u0026thinsp;=\u0026thinsp;0.07). Among the groups treated for 8 weeks, not even the control group showed a significant difference between the trials (p\u0026thinsp;=\u0026thinsp;0.406). Despite the results of both treatment doses not showing significant effects, there was a noticeable decrease in latency time to the white compartment across trials, suggesting excitement/anxiety/stress (Maximino et al \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Learning deficits can also be interpreted as impulsivity (Chirin\u0026eacute;a and Gouveia Jr \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or even habituation to the white compartment. Interestingly, in experiments conducted by Santos et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), treatment with fluoxetine in animals did not prevent the acquisition of inhibitory avoidance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibitory avoidance 8 weeks for all groups All groups 8 weeks\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHere, the consumption of individual doses of food caused social animals to remain isolated during the equivalent treatment period, which likely influenced them in the form of stress. It was not possible to quantify brain serotonin levels in this study. However, it was possible to investigate commonly used behavioral parameters after the oral administration of precise levels of tryptophan in zebrafish.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe evaluation of pre- and post-treatment biomass results presents the greatest evidence. Since tryptophan is a precursor of serotonin, the serotonergic system has long been associated with satiety (Halford et al \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which could explain the mass loss. Another possibility is that tryptophan supplementation may lead to a decrease in body weight in rats by oxidizing fatty acids in the body for lipid synthesis (Ruan et al \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the first day, we tested the fish\u0026rsquo;s memory: In the y-maze test, the expected outcome would be animals showing a preference for the unexplored arm (Cognato et al \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In our experiments, there was no preference for any arm, making it inconclusive whether there was recognition of the arms explored earlier in the treatment groups with different concentrations at both proposed treatment durations. Similar results were obtained after the use of fluoxetine. Despite being known for its anxiolytic effects, its use did not alter memory acquisition in the y-maze test but reduced the activity of the zebrafish at the bottom of the tank (also indicative of anxiety-like effects) in the novel tank test, similar to diazepam (Giacomini et al \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn rats, there has been recognized difficulty with the y-maze, related to avoidance of the novel arm stemming from anxiety-like behavior (Hughes \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Findings suggest that vertebrate species such as zebrafish, rodents, and humans perform exploration in similar behavioral models (Cleal et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the second stage, the light-dark preference test results suggest some predominance of anxiety-like behavioral responses and/or attention deficits (Chirin\u0026eacute;a and Gouveia Jr \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Maximino et al (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) also observed this type of behavior in zebrafish in the light-dark test after the administration of 2.5 mg/kg of fluoxetine intraperitoneally, while the dose of 10 mg/kg led to an increase in the number of entries. In the novel tank test, zebrafish exhibited a tendency to prevent anxiety-like effects after immersion in 300 mg/L of tryptophan (Giacomini et al \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the next test, the inhibitory avoidance test, despite the results of both treatment doses not showing significant effects, there was a noticeable decrease in latency time to the white compartment across trials, suggesting excitement/anxiety/stress (Maximino et al \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Learning deficits can also be interpreted as impulsivity (Chirin\u0026eacute;a and Gouveia Jr \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or even habituation to the white compartment. Interestingly, in experiments conducted by Santos et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), treatment with fluoxetine in animals did not prevent the acquisition of inhibitory avoidance.\u003c/p\u003e \u003cp\u003eEvidence indicates that the alteration of the metabolic process determined by the nutritional condition of these animals cold lead to behavioral responses (Lieberman \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Gomez-Requeni 2010).\u003c/p\u003e \u003cp\u003eHere, the consumption of individual doses of food caused social animals to remain isolated during the equivalent treatment period, which likely influenced them in the form of stress. It was not possible to quantify brain serotonin levels in this study. However, it was possible to investigate commonly used behavioral parameters after the oral administration of precise levels of tryptophan in zebrafish.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIt is known that the induction of an increase in cerebral serotonin levels can be achieved through dietary supplementation with tryptophan (Teixeira et al \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This study had the advantage of individually examining potential anxiolytic and cognitive effects in different models without the need for long training periods. It tested oral tryptophan supplementation at different concentrations and after two time periods, one of which (8 weeks) was considered prolonged use for the \u003cem\u003eDanio rerio\u003c/em\u003e species. Studies have observed that affective states influence attention, memory and even decision- making in various species (Buenhombre et al, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Buenhombre et al, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The results were integrated and tryptophan couldn\u0026rsquo;t affect the results on better learning and memory in zebrafish. Tryptophan was not enough to neutralize the animals' anxiety and therefore was unable to affect the results in better learning and memory in zebrafish; However, this indicates the safety of its use for breeding and suggests potential differences from what occurs in mammals, reinforcing that the relationship between tryptophan and its behavioral paradigm is still not well understood.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e This research was approved by the Ethics Committee on Animal Use at the Federal University of Par\u0026aacute; (CEUA/UFPA) under protocol number 2903141118 (ID 001143).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was financed in part by the Coordination for the Improvement of Higher Education Personnel - Brazil - CAPES.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP\u0026ecirc;ssi Sousa - Wrote the main manuscript text; Jhon Buenhombre - Interpretation of data for the work; Prepared figuresErika Alexandra Daza-Cardona - Reviewed the manuscriptYuri Richard - Reviewed the manuscriptAmauri Gouveia Jr - Design of the work; Supervision\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAoki R, Tsuboi T, Okamoto H (2015) Y-maze avoidance: An automated and rapid associative learning paradigm in zebrafish. 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Zebrafish 13:S72\u0026ndash;S76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/zeb.2016.1299\u003c/span\u003e\u003cspan address=\"10.1089/zeb.2016.1299\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"animal-cognition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anco","sideBox":"Learn more about [Animal Cognition](http://link.springer.com/journal/10071)","snPcode":"10071","submissionUrl":"https://submission.nature.com/new-submission/10071/3","title":"Animal Cognition","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Anxiety like behaviours, tryptophan, serotonin, zebrafish, memory","lastPublishedDoi":"10.21203/rs.3.rs-4151627/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4151627/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnder certain circumstances, some nutrients can affect the synthesis of central neurotransmitters and potentially behavior. The aim of this study was to characterize the influence of tryptophan supplementation on potential anxiety-like behaviors in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e), as well as to assess the suitability of a new standardized diet formulation for the better maintenance and performance of this species. To achieve this, the proportion of the amino acid tryptophan supplementation was experimentally modified at 5%, 50%, and 100% levels, individually, in the zebrafish diet. The investigation was conducted through the Y-maze test, light-dark preference test, and inhibitory avoidance test. The results indicated that tryptophan supplementation did not lead to the expected reduction in anxiety-like effects in the Y-maze and light-dark tests. However, a significant difference in avoidance times was observed in the inhibitory avoidance test between the groups with 5% and 50% supplemented diets, but a significant difference in avoidance times was observed in the inhibitory avoidance test between the groups with 5% and 50% supplemented diets. Changing the diet did not alter the animal's learning or natural anxiety. However, in most animals, tryptophan supplementation led to a decrease in body mass.\u003c/p\u003e","manuscriptTitle":"Tryptophan supplementation reduces body weight but does not reduce anxiety-like behavior in zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-30 07:28:45","doi":"10.21203/rs.3.rs-4151627/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-03-26T17:57:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-26T07:36:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Animal Cognition","date":"2024-03-22T19:07:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"animal-cognition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anco","sideBox":"Learn more about [Animal Cognition](http://link.springer.com/journal/10071)","snPcode":"10071","submissionUrl":"https://submission.nature.com/new-submission/10071/3","title":"Animal Cognition","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fa59b48d-d2f1-464e-85af-06a6cb36844b","owner":[],"postedDate":"March 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-30T07:28:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-30 07:28:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4151627","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4151627","identity":"rs-4151627","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}