OPINION published: 22 February 2021 doi: 10.3389/fvets.2021.634434 About Welfare and Stress in the Early Stages of Fish Juan Ramos*, Joan Carles Balasch and Lluis Tort Department Cell Biology, Physiology and Immunology, Universitat Autonoma de Barcelona, Bellaterra, Spain Keywords: welfare, early fish stages, zebrafish, anesthesia, stress, fish husbandry INTRODUCTION Fish are the most phylogenetically ancient vertebrates and the most varied group in terms of genetic and morphological diversity. Hence, the considerations about fish welfare and the physiological bases for such welfare have been adopted always later than higher vertebrates and it has been more difficult to generalize protocols and methodologies. In recent years there has been a greater social sensitivity in terms of fish welfare, which has been reflected in an increasingly protective (European) legislation of fish, whether they are for aquarium trade (006/88/EU), production Edited by: (standing committee of the European convention for the protection of animals kept for farming Edward Narayan, purposes: recommendation concerning farmed fish adopted by the standing committee on 5 The University of December 2005) or research, (2010/63/EU https://eur-lex.europa.eu/eli/dir/2010/63/oj). This social Queensland, Australia defendant has been associated to a change in the growing scientific perspective and research Reviewed by: regarding animal welfare. The development of different indicators for evaluating the status of the Jose Fernando Lopez-Olmeda, University of Murcia, Spain fish has resulted in a quantifiable set of parameters, either individually or for a given population. Patrick Prunet, But some questions arise regarding welfare during earlier fish stages: When and how the fish start Institut National de la Recherche to experience stress and pain along development? Are stress and pain experiences in mature fish Agronomique (INRA), France applicable to the earliest stages of development? In this contribution, we review the state of the *Correspondence: art regarding the studies dealing with stress and welfare in eggs, larvae and early stages of fish. Juan Ramos Provided that zebrafish, Danio rerio is, by far, the most used species in biomedical research, we juanramoblas@gmail.com focused this opinion paper in this species, although most of the conclusions can be applied to other species, such as medaka or killifish. By law only welfare of fish with independent feeding, should be Specialty section: considered, as they are protected by European directive, and considered legally as research animals. This article was submitted to But the implications on welfare during the early stages (no independent feeding), can affect to Animal Behavior and Welfare, the adults and further generations. Early stages welfare is not required by law but will affect the a section of the journal normal development and reliability of the research. Our conclusion is that current protocols of egg Frontiers in Veterinary Science transport and larval handling, lack of solid analytical background and therefore there is a need of Received: 27 November 2020 specific studies (1, 2) (Table 1). Accepted: 25 January 2021 Welfare during early stages begins with the paternal welfare. The experiences of the parents Published: 22 February 2021 (nutritional, social, environmental), especially during the gametogenesis period, are of great Citation: importance for the progeny. During the gametogenesis the DNA will be reprogrammed, so this Ramos J, Balasch JC and Tort L (2021) About Welfare and Stress in information will be transmitted to the progeny, thus involving transgenerational effects with a the Early Stages of Fish. direct impact in the quantity, viability, social status, neurogenesis, and adaptation of the further Front. Vet. Sci. 8:634434. generations (3–5). Welfare has to be understood as continuous and intergenerational, linking doi: 10.3389/fvets.2021.634434 progeny adaptation to parents resources. Frontiers in Veterinary Science | www.frontiersin.org 1 February 2021 | Volume 8 | Article 634434 Ramos et al. Early Stage Fish Welfare TABLE 1 | Type of protocol and the variables that should be considered to genetic plasticity. Thus, when the environmental parameters are preserve welfare and normal development. not optimal, they try to adapt to the new conditions. If the Protocol Variables Author adaptation is successful, no welfare problems will arise, but some alterations can often occur. The genetic quality of embryonic eggs Environmental Oxygen Reed et al., is determined by the gametes, which results from the parents plus parameter Aleström et al. any own experience. Assessing the protein and genetic profiles of Temperature Sfakianakis et al., Scott et the eggs, may help to predict their quality and the viability of the al., Long et al. embryos (6). In this way, altered embryos can be identified and Light and photoperiod Villamizar et al., discarded avoiding further welfare implications. Basili et al. During their development, embryonic eggs are very sensitive Nitrogen compounds Lin et al. to environmental influences (7). To ensure its correct embryonic Noise and vibrations Lara et al., development and to avoid future alterations in the juvenile or Bhandiwad et al. adult stages, the environmental conditions must be adjusted pH Reed et al., Aleström et al. to the optimal ranges. But how can we assure it along all the Conductivity Reed et al., Aleström et al. development period? Adult zebrafish are kept under recirculation Hardness/alkalinity Reed et al., Aleström et al. but not when they are mating (1, 2). Zebrafish eggs are usually Incubation of eggs Density obtained under static conditions, so water has low oxygen and Egg and gametes quality Yilmaz et al., high ammonia values and kept in these conditions 4 h affecting Labbé et al. their normal development, but this is not usually taken in account Time to be collected Lin et al. in the protocols (8). Environmental parameters Environmental influences on embryogenesis vary between Transgenerational effects Wang et al., species and individuals, thus modifying the normal development Labbé et al., of fish larvae and, so the animal may not be able to cope with Long et al. some environmental conditions. Regarding zebrafish, no studies % Renovation water Water quality have been done to stablish a proper protocol of incubation. Larval rearing Environmental enrichment Lee et al. Thus, the influence conditions like the use of fungustatics or Maternal social status Best C. et al. disinfectants, density of eggs, % of water removal, type of water Social and schooling Gerlach et al., Biechl et al., or type of incubator (with or without photoperiod control) have parameters Groneber et al. not been established. The influence of environmental conditions Diet and nutrition Monteiro et al., is not a trivial issue and should be investigated before stablishing Martins et al., the protocols in order to assure the normal development and Chang et al. avoid further welfare problems (1, 2) (Table 1). Water flow Oteiza et al. Rearing larval density Ribas et al. Behavior Tudorache et al. Microbiota Davis et al., Basili et al. LARVAL RESPONSES Environmental parameters The alteration of appropriate conditions during development Transport of embryos Acclimation period Dhansari et al. will produce changes in DNA methylation, with the consequent Insulation Barton et al. physiological change (7), that may remain for all live stages and Disinfection method Barton et al. the offspring. In zebrafish larvae, an inadequate density induces a Environmental parameters Barton et al. stress response and can influence sex determination (9). Also like Handling Analgesics Lopez-Luna et al. in previous stages, water quality and light conditions modulates Refinement technique Oteiza et al. gene expression and development (10, 11). Environmental parameters Zebrafish larvae are able to process external stimuli, thanks Anesthesia and Type of anesthesia Felix et al., to the presence of neural centralized circuits, and not by euthanasia Strikowski et al., automatic or ecotaxic processes as it was previously thought Collymore et al. (12). For example, zebrafish larvae adapt their swimming Refinement technique depending on water flow (13, 14) thanks to the integration of Environmental parameters the stimuli perceived by the lateral line. During the early stages of development, the nociceptive pathways are already active, allowing the larvae to escape from painful stimuli. This is possible EGG QUALITY AND DEVELOPMENT: thanks the activation of oxytocin neurons, which produce a HANDLING AND ENVIRONMENTAL locomotor reaction, whose activation can be modulated by EFFECTS analgesics (15–17). So analgesic drugs could be used in zebrafish larvae in order to avoid pain and preserve welfare, but also Fish have been able to colonize many ecological niches, so their impact in the larvae, as bioactive molecules, should be they have developed multiple adaptive strategies, thanks to their studied (18). As fish develop they are able to process more Frontiers in Veterinary Science | www.frontiersin.org 2 February 2021 | Volume 8 | Article 634434 Ramos et al. Early Stage Fish Welfare environmental stimuli and elaborate a strategy to cope with them. for a normal development (33–35) has been widely studied If the environment during early stages is complex they will have in zebrafish, none of these factors are usually considered more strategies and less anxiety. So enrichment and complexity for the shipping protocols of embryos neither the relevance of environmental conditions during early stages should be taken of the acclimation period afterwards. The evaluation and in consideration in order to help their adaptation strategies and standardization of environmental conditions during transport improve welfare. of the early stages of zebrafish should then be revisited (1, Another way for fish larvae to avoid external dangers such 2, 36), as it should be during the standard incubation period as predation is schooling, especially in social fishes such as (Table 1). zebrafish. In order to develop this aggregation mechanism, they Fish experimental facilities usually involve noises need to differentiate their congeners from other fish. Thus, and vibrations produced by water pumps or working the olfactory cues (19–21) are key signals, and these have routines that will affect the normal development been found in zebrafish brain from day 6 of development, of fish (37, 38). Fish husbandry facilities could be allowing them to recognize their siblings and perform the designed in order to minimize noise impact, removing schooling pattern. This process would be impossible without pumps, machines from the facility. The routines have memory, that performs the integration of the stimuli and the to be also reduced to minimum, especially in the identification of the habitual environment (22). The social breeding area. conditions of early stages should be also considered in protocols, Visual techniques are very common in zebrafish research, due as they are developing social patterns: schooling and social to the translucency of embryos. It is useful for developmental avoidance (23). studies, but it usually requires immobilization of the Zebrafish larvae can sometimes adopt different coping larvae or embryos with an anesthetic. Nevertheless, the strategies in front of the same stimulus. The stimuli can use of anesthetics, especially in the early stages, has a be processed as an opportunity, as for proactive fish, or broad implication on the present and future welfare of like a risk, as for reactive ones, so they develop anxiety. the individual. So, it must be carefully considered when These differences may be determined by the paternal carrying out the experiments even if these early phases are genetic load, as well as the experiences of the embryo not protected by welfare laws. For instance, the most common or larva during development (24), The use of substrates anesthetic for zebrafish, Tricaine metanosulphonate (MS- and enrichment makes the ambient more complex and 222), is capable of generating oxidative stress, altering the reduces anxiety, improving the boldness (proactive) (25). normalized development of cartilage and inducing apoptotic Environmental enrichment is not common in zebrafish tanks processes (39–41). because of technical implications but this may result in more In terms of euthanasia, an anesthetic overdose is the usual anxious fish. procedure, but its efficiency in larvae is very limited. Since Nutrition has a direct impact in animal welfare especially oxygen is taken by zebrafish through the skin until day during the growing o development period. The use of life preys 14 for respiration, it makes them resistant to most of the in zebrafish (Artemia, paramecium, and rotifers) allows to display anesthetics, as the muscular contraction is not related to the natural behavior as a predator and also use them as vehicle for respiration. Furthermore, it should be taken into account that different nutrients such as polyunsaturated fatty acids. So new cessation of the heartbeat, usually taken as an indicator of protocols have been developed using life preys and special dry death, could not be considered as such, since heart fibers foods that help zebrafish larvae to grow and develop faster (26– are capable of beating for more than 20 min after death (42, 28). The use of probiotics has been tested in zebrafish larvae as a 43). For these reasons, especial euthanasic protocols and more way to improve welfare by modulating anxiety, immunity, or gut clear death indicators should be addressed for zebrafish early function (29). development stages. WELFARE IN EARLY FISH STAGES AND CONCLUSION THE ANTHROPOGENIC IMPACT In conclusion, the investigations carried out up to now Transport is a highly stressful process for adult fish (30) if demonstrate that during the early stages fish show high sensitivity environmental conditions are not properly controlled. Zebrafish to many types of stressors involving an array of responses eggs are commonly transported between facilities as they are to overcome alterations that could affect the animal and be cheaper and easier to ship than adults. If there is an improper transmitted to the progeny. Welfare in eggs and larvae is a isolation and no heater or chiller inside the box, during the continuous process that involve both parental experience and transport, eggs can be exposed to extreme temperatures (higher development, so environmental parameters have to be controlled or lower than their optimal range). Till the eggs arrive to during all the life time, especially during the gamete and the new facility no water or air is exchanged, as they are in organogenesis stages. Standard protocols should be developed watertight containers, and no light is received. Although the including all environmental parameters by studying not just importance of the photoperiod, for the activation of circadian the zoothecnical indexes but also stress and welfare-related rhythms (11, 31, 32), and the importance of temperature indicators such as behavioral traits, stress hormones, or the Frontiers in Veterinary Science | www.frontiersin.org 3 February 2021 | Volume 8 | Article 634434 Ramos et al. Early Stage Fish Welfare expression of genes associated to them. In our opinion, the AUTHOR CONTRIBUTIONS results of the research on these early stages also points out a lack of adequate standards for reliable welfare results in JR collected data and wrote the first draft. JCB and LT designed relation with the procedures for maintenance, husbandry or the paper structure. All authors completed, revised and finished transport (Table 1). the work, and approved it for publication. REFERENCES 19. Gerlach G, Hodgins-Davis A, Avolio C, Schunter C. Kin recognition in zebrafish: a 24-hour window for olfactory imprinting. Proc R Soc B Biol Sci. 1. Aleström P, D’Angelo L, Midtlyng PJ, Schorderet DF, Schulte-Merker S, (2008) 275:2165–70. doi: 10.1098/rspb.2008.0647 Sohm F,et al. Zebrafish: housing and husbandry recommendations. Lab Anim. 20. Biechl D. 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(2018) 19:741–52. doi: 10.1007/s10162-018-00685-0 43. Collymore C, Banks EK, Turner PV. Lidocaine hydrochloride compared with 38. Ayala R., Ornelas R. Impact of noise exposure on development, physiological MS222 for the euthanasia of zebrafish (Danio rerio). J Am Assoc Lab Anim Sci. stress and behavioural patterns in larval zebrafish. Res Square [Preprint]. (2016) 55:816–20. (2020). doi: 10.21203/rs.3.rs-126894/v1 39. Félix L, Coimbra AM, Valentim AM, Antunes L. Review on the use of zebrafish Conflict of Interest: The authors declare that the research was conducted in the embryos to study the effects of anesthetics during early development. Crit Rev absence of any commercial or financial relationships that could be construed as a Toxicol. (2019) 49:357–70. doi: 10.1080/10408444.2019.1617236 potential conflict of interest. 40. Félix LM, Luzio A, Themudo M, Antunes L, Matos M, Coimbra AM, et al. MS-222 short exposure induces developmental and Copyright © 2021 Ramos, Balasch and Tort. This is an open-access article distributed behavioural alterations in zebrafish embryos. Reprod Toxicol. (2018) under the terms of the Creative Commons Attribution License (CC BY). The use, 81:122–31. doi: 10.1016/j.reprotox.2018.07.086 distribution or reproduction in other forums is permitted, provided the original 41. Félix LM, Luzio A, Santos A, Antunes LM, Coimbra AM, Valentim author(s) and the copyright owner(s) are credited and that the original publication AM. MS-222 induces biochemical and transcriptional changes related in this journal is cited, in accordance with accepted academic practice. No use, to oxidative stress, cell proliferation and apoptosis in zebrafish distribution or reproduction is permitted which does not comply with these terms. Frontiers in Veterinary Science | www.frontiersin.org 5 February 2021 | Volume 8 | Article 634434