2.1. Social Enrichment

Zebrafish are often described as a highly social species, and are generally found in groups in the wild, although observed group sizes vary widely [41,42,43]. In the laboratory, zebrafish larvae display social behaviours from as young as 10 days post-fertilisation (dpf) [44]. Zebrafish also generally express preferences for social contact, choosing to be with other zebrafish over being on their own or with fish of another species [45,46,47,48] and choosing a larger shoal over a smaller one [49]. Zebrafish even prefer tanks with mirrored paper on the walls over bare tanks [48], and will shoal with computer-animated zebrafish [46], suggesting that simulated social contact may be beneficial if zebrafish cannot be housed in direct or visual contact with other zebrafish. However, preferences may be affected by factors such as the sex ratio of the shoals available [50,51]. Housing and husbandry guides for laboratory zebrafish therefore often emphasise the importance of group-housing for zebrafish [1,11,52].

The natural tendencies and preferences of zebrafish for being in groups might suggest that group-housing is beneficial for zebrafish welfare, and some studies support this (Table 1). For example, housing zebrafish in isolation or in pairs can lead to higher cortisol release rates, increased anxiety and hypersensitivity compared with fish housed in groups [53,54,55]. Another study found that group-housed fish were less anxious than isolated fish, but only when the group-housed fish had additional physical enrichment, suggesting that there are interactions between different forms of enrichment in terms of their impacts on welfare—for example, physical enrichment may help zebrafish avoid unwanted social contact [47]. Isolation can also cause long-term changes in monoamine levels which may modulate reward systems and social behaviour—dopamine, serotonin, and their main metabolites (3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5HIAA), respectively) have all been shown to decrease in response to isolation in zebrafish [56,57]. However, several studies have found opposing results: fish housed in groups may show more anxiety [56,58], and higher cortisol release rates than isolated zebrafish [57,58], or no difference in cortisol between isolated and grouped zebrafish [59,60]. One study found that fish which had been raised in groups but then exposed to short-term (1 h) or longer-term (2 weeks) isolation had lower cortisol release rates than group-housed controls, although fish raised in isolation for the first 6 months did not differ from group-housed fish in cortisol release rates [61].

The presence of other fish may also affect how zebrafish respond to and recover from challenges. For example, the behaviour of zebrafish housed in groups has been shown to return to normal levels after handling more quickly than in paired or isolated fish [54], suggesting that the presence of a group promoted faster recovery from stress. In another study, group-housed fish were bolder than isolated fish in a novel object test, which may suggest that the presence of other individuals led to greater feelings of safety, which in turn could indicate a better welfare state [62]. However, some studies have shown that isolated fish mount a smaller response to a stressor than group-housed fish [60,63], while another found that isolated fish had smaller cortisol responses than group-housed fish to being chased with a net, but bigger responses to predation stress [59].

Despite some apparently conflicting results, it seems likely that housing zebrafish in isolation is detrimental to welfare. Although lower levels of cortisol might be assumed to indicate lower levels of stress, this is not necessarily the case—for example, social isolation may cause chronic stress, leading to lower cortisol levels due to a ‘dampening’ or ‘blocking’ effect on the stress response in isolated fish [58,64,65]. It should also be noted that cortisol levels in teleosts are known to fluctuate for a variety of reasons, including natural diurnal cycles, after feeding, and as a result of excitement or activity [66], and so higher cortisol levels should be interpreted cautiously, and alongside other parameters, as they could be related to a positive as well as a negative welfare state.

Differences in the levels of stress and anxiety in isolated fish compared with grouped fish could also be affected by different aspects of social context. Mechanisms such as ‘emotional contagion’ (where the response of one individual to a stimulus influences the response of nearby individuals) and ‘social buffering’ (where fear or stress responses are decreased when individuals are in a group, or are in visual or olfactory contact with a group can influence the strength of a fish’s response to an aversive stimulus [63,67,68]. This could mean that the strength of response to a stressor may depend on the composition of a group—for example, groups containing lots of very highly reactive individuals might respond differently to a stressor than groups containing few reactive individuals [59]. This could also explain why groups show different responses to different types of stressor, as there may be variation in how different types of stressor are perceived by the fish. A further factor relating to social context is familiarity—zebrafish have been shown to recognise familiar individuals [69] and it is possible that greater familiarity among shoal-mates increases or moderates the impacts of emotional contagion or social buffering.

Another possible explanation for these mixed results is that the systems of group housing commonly used in laboratories may not reflect the best conditions for welfare, even though group housing is likely to be better overall than individual housing. There may be a need to re-examine some conventional housing practices to establish whether they are best for welfare. For example, one study found that wild zebrafish were markedly more aggressive after being housed in laboratory conditions for three months [70]. This could have been due to factors such as inappropriate stocking densities, or because the barren nature of laboratory tanks forces social contact and does not allow individuals to avoid negative social interactions if they choose. Further exploration of optimal stocking densities and the inclusion of structures (see below) might therefore be ways to improve zebrafish welfare. Furthermore, laboratory zebrafish are often housed in mixed-sex groups, but some evidence suggests that there may be benefits to segregating fish by sex zebrafish housed separately according to sex have been found to have higher fecundity, egg viability, breeding success, growth and lower baseline cortisol levels than those housed in mixed groups [71,72]. However, another study found that female zebrafish become more anxious in sexually segregated groups [73], and it should be noted that prolonged sexual segregation can cause female zebrafish to become ‘egg-bound’—where the oviduct becomes blocked with degenerating eggs—so separately housed females would still need regular exposure to males, which might require more handling of fish and therefore be more detrimental to welfare than mixed housing [52].

Stocking density may also affect stress levels and welfare in group-housed fish, which could help explain the conflicting results in the studies mentioned here. Adult zebrafish are commonly kept at densities of between 4 and 10 fish/L [4], although some facilities may use densities as high as 20 fish/L [52]. Zebrafish have been shown to become stressed at high stocking densities—for example, Ramsay et al. [74] found that whole-body cortisol release rates increased fourfold in fish subjected to densities of 40 fish/L compared with uncrowded controls (0.2 fish/L). Zebrafish have also been shown to have significantly lower cortisol release rates when housed at 5 fish/L than 10, 20, or 40 fish/L [75], which may suggest that stocking densities above 5 fish/L are too high and may cause stress. However, this experiment compared different densities housed in the same volume of water (2 L)—when different stocking densities were compared by changing the size of the tank, fish housed at 2 fish/0.5 L (i.e., 4 fish/L) did not have significantly different cortisol levels than fish housed at higher stocking densities. This suggests that both stocking density and space availability matter to zebrafish and may impact stress and welfare. Furthermore, densities between 3 fish/L and 12 fish/L have been shown to have no impact on average clutch size, spawning success or egg viability—indicators which might be affected if fish were stressed [76]. Finally, animal care staff have observed that low stocking densities can lead to increased levels of aggression due to zebrafish having more space available to defend territories (although aggression may also be affected by the presence of physical enrichment; see below) [1,52]. Establishing clearer guidance on the most appropriate stocking densities and tank sizes is likely to be essential for promoting better zebrafish welfare.

Overall, these findings suggest that group-housing is likely to be the better option for zebrafish, but we should not assume that the mere presence of other zebrafish is always enriching—instead, a socially enriched tank requires thought to be given to other factors such as stocking density, sex ratio, and familiarity. Even where this has been done, we also cannot assume that group housing with no other forms of enrichment meets all the behavioural and welfare needs of zebrafish.

2.2. Physical Enrichment

Wild zebrafish are found in India, Nepal, Bangladesh and Pakistan in a range of habitats including small streams, rivers, pools and rice paddies, which may contain aquatic plants, overhanging vegetation, and substrates including mud, gravel or sand [77,78,79,80]. It is perhaps a desire to mimic some of these features that leads to real or artificial plants, substrate and shelter frequently being suggested as enrichment items for zebrafish (Table 2). Plants, shelters and other structures may provide cover or refuge from negative social interactions (e.g., bullying), from disturbances, from water flow or aquarium lights [20,32,81]. Substrate might provide some camouflage when zebrafish are viewed from above, which may contribute to greater feelings of safety and so an improved welfare state [82].

Zebrafish prefer structures over bare tanks [82,83,84,85,86], and female zebrafish have been found to spend most of their time in close proximity to plants rather than in open areas of a tank [87]. Lavery et al. [85] also found that zebrafish showed stronger preferences for more complex enrichment (plants and gravel over plants alone or gravel alone, and four plants over two plants). Notably, zebrafish preferred an image of gravel affixed to the base of a tank over a barren tank almost as much as real gravel [82]—using an image of gravel removes any potential concerns over adding substrate into the tank, and therefore has been adopted in other studies [88] and for commercial use (e.g., Tecniplast Enrichment Runner). Zebrafish have also been found to show stronger preferences for enrichment at night than during the daytime, emphasising the importance of thoroughly assessing preferences over an extended timescale and with multiple observation points [86]. However, some studies have not found any preference for structures [81,89], and one study found that zebrafish preferred a covered area of a tank over an open area, but did not prefer simulated vegetation [90]. Social dynamics within the tanks may explain this lack of preferences—for example, Lee et al. [89] observed that dominant individuals tended to exclude subordinates from enriched areas of the tank, suggesting that zebrafish placed value on the available resources. Another possible explanation is that some study lengths were too short for zebrafish to habituate and for preferences to stabilise—studies which found no preference tended to take place over shorter time periods ([81]—2.5 h; [89,90]—3 days), whereas studies which found preferences tended to allow fish several days to acclimatise to their new environment, then examined preferences over several more days.

Structures have been found to contribute to a reduction in the stress response in zebrafish. For example, the presence of substrate, shelter and plants was found to blunt the cortisol response to acute [60] and chronic stress [91], and may be as effective as the anti-anxiety drugs diazepam and fluoxetine at blunting the cortisol response to stress [60]. However, von Krogh et al. [92] found higher cortisol levels in fish in structured tanks than those in barren tanks—although cortisol release rates in these fish were still significantly lower than in fish exposed to a stressor [92]. It may be that these slightly increased cortisol levels may indicate a state of ‘eustress’ (i.e., a positive response to a mild stressor) [93], rather than being indicative of poorer welfare—although it is also possible that they did represent a poorer welfare state. It is also possible that the time point at which cortisol levels are measured after fish are provided with structures may affect results: pairs of zebrafish provided with plants showed higher cortisol levels than controls after five days, but lower cortisol levels than controls after ten days [94].

Physical enrichment may also contribute to lower levels of anxiety in zebrafish [47,89,91,95,96], and combining group housing with the presence of structures can lead to lower anxiety than either condition on its own [47]. DePasquale et al. [96] found that zebrafish which had been reared with structures, but later experienced barren housing conditions, still showed lower levels of anxiety than controls—this may be useful in laboratory settings, as it may mean that zebrafish in nursery tanks can be provided with enrichment and still benefit, even if other challenges prevent the use of enrichment in adult tanks. Other behavioural indicators of better welfare in the presence of physical structures include increased exploration and decreased inhibitory avoidance in response to electric shock, suggesting that fish were better at coping with aversive experiences [95] and increased social cohesion [97], a behaviour which has been suggested as being an indicator of positive welfare [98]. Zebrafish have also been shown to have lower levels of locomotor activity, measured as the number of turns made, in the presence of physical structures [92]. This was interpreted as a sign of lower stress, as high levels of turning behaviour may indicate a predator-avoidance response, and because turning behaviour decreased in all fish over time, suggesting they were settling into a novel environment.

The additional environmental complexity provided by physical structures can have positive effects on zebrafish cognition and brain development. For example, zebrafish raised in tanks containing structures have been found to have a faster rate of learning in a maze task than those raised in barren tanks [96,99,100], as well as better memories of how to solve the task after a break in training [99,100], and improved ability to discriminate between similar spatial environments [101]. The presence of structures in rearing environments has also been found to lead to increased overall brain size [96] and a greater number of cells in the telencephalon of zebrafish [92]. Although changes in cognition and brain development are not necessarily direct indicators of better welfare, they are both likely to affect behaviours which may have welfare consequences—for example, better cognitive ability may be linked to improved behavioural flexibility, which may improve the ability of fish to respond to stressors or challenges [102]. Furthermore, as wild zebrafish live in structurally complex environments, this level of improved development and cognition is likely to represent ‘normal’ zebrafish development, which is likely to improve the quality of science.

Further physiological effects which may be linked to welfare have been found in zebrafish provided with physical structures. For example, zebrafish provided with plastic grass had higher total egg counts than those in barren tanks or provided with plastic leaves, and an interaction was found whereby either plastic leaves or plastic grass increased the number of fry at 6 dpf compared with barren environments, depending on the age of the spawning pair [103]. Stress, particularly chronic stress, is often associated with lower fertility and fecundity [38], therefore the increased fertility and fecundity found in this study may indicate lower stress in fish provided with structure. Another study found that fry survivorship increased from 54% to 83% at 30 dpf when zebrafish were provided with plants and gravel [89]. Finally, the presence of structures has been found to reduce production of reactive oxygen species in response to unpredictable chronic stress, and so can protect against oxidative stress [91,104].

A concern that is often raised when considering adding physical structures to zebrafish tanks is that structures may lead to an increase in aggressive behaviour, perhaps because zebrafish may try to monopolise high-value resources. Evidence here is mixed: some have found increased aggression in zebrafish housed with structures [35,105] compared to those in barren tanks, and another found that the initial higher levels of aggression seen in newly set up tanks took longer to settle when structures were present [34]. However, Hamilton and Dill [90] found that enrichment did not result in higher levels of aggression, and others have found lower levels of aggression, injury, and mortality in the presence of structures [94,106,107]. In another study in which highly aggressive behaviour was induced by exposing zebrafish to lead, provision of a shelter helped reduce the number of aggressive interactions [108]. These differing results may be due to variation between studies in terms of stocking densities of fish and the number of physical structures provided—if aggression is due to the presence of desirable resources, lower stocking densities and higher numbers of available structures might minimise competition for these resources. It is therefore possible that physical structures can be provided to the benefit of laboratory zebrafish without causing higher aggression, but that more work must first be done to identify appropriate stocking densities and an appropriate amount of structures for a certain number of fish.

2.3. Nutritional Enrichment

The basic diet provided to captive animals should meet all nutritional needs for health, therefore interventions should provide some further welfare benefit to be considered ‘enriching’—furthermore this paper seeks to review the impacts of environmental enrichment (enrichment external to the zebrafish), so possible nutritional benefits are not considered here. For zebrafish, the provision of live food such as Artemia spp. or rotifers may be referred to as enrichment [3,20,52], but studies comparing diets with and without live food have generally been focussed on parameters like growth and survival which are more likely to be indicative of proper biological functioning rather than demonstrating any additional welfare benefit [109,110,111]. Zebrafish users sometimes report better zebrafish welfare when live food is provided (pers. comms.), and this is usually attributed to live food stimulating natural predatory behaviour [3,52]. Indeed, it is possible that zebrafish are highly motivated to perform this behaviour, as wild zebrafish primarily feed on aquatic insects and their larvae, and zooplankton [78], and so may spend much of their time hunting. However, no studies appear to have yet examined the welfare impacts of providing live food to zebrafish in addition to their usual diet.

There may be other potential avenues for providing nutritional enrichment to zebrafish—for example, it may be possible to provide enrichment through altered feeding schedules, through provision of palatable foods as treats, or by providing a more varied diet. However, these have attracted little research attention so far. One study has shown that feeding frequency can affect zebrafish behaviour, with fish fed once a day showing higher anxiety than fish fed twice a day or more [112], but no other studies were found which explored how the timing and frequency of feeding may affect zebrafish welfare. Another area which has not been explored in zebrafish is the potential welfare benefits of demand feeders—in aquaculture, demand feeders can contribute to reduced aggression and injury compared with fixed feeding regimens, and may also allow fish to self-select diets based on individual nutritional requirements [113,114].

2.4. Occupational Enrichment

Occupational enrichments generally are those which encourage the animal to interact with the environment in some way—for example, puzzles or toys, opportunities for exercise, or the opportunity for animals to exert control over their environment [40]. These forms of enrichment may help to promote normal behaviour, and may alleviate boredom or psychological stress, which can be seriously detrimental to good welfare [115,116]. Such interventions are not easy to design, and so far there is little evidence as to whether zebrafish might be interested in such devices, but there is potential to provide some forms of occupational enrichment to zebrafish (Table 3).

Exercise conveys both physiological and psychological benefits in other vertebrates, so may result in similar benefits in zebrafish [117]. Some of these effects may be linked—for example, larval zebrafish exposed to forced swimming training are better at coping with hypoxia as a result of more efficient oxygen consumption, but this may also be related to a better ability to cope with stress [118]. Exercise can promote muscle and bone development, which may protect against the degenerative effects of aging and thus promote a better welfare state as fish get older [119,120]. Zebrafish have also been found to have improved learning ability and lower anxiety levels in environments with water flow [121,122]. However, wild zebrafish living in flowing water appear to exhibit higher levels of aggression, less group cohesion, and more frequent leadership changes than zebrafish found in still water [41,43], and this has been replicated in the laboratory with higher levels of aggression found amongst fish in flowing water [105]. Housing zebrafish in continually flowing water may therefore lead to more anti-social behaviour. However, it may be possible to promote better welfare by providing zebrafish with a choice of whether to exercise: DePasquale and colleagues [84] found that zebrafish showed an aversion to tank compartments containing flowing water, but a preference for compartments containing both water flow and structures (plants and substrate) over a barren compartment or a compartment containing structures but no water flow. This might be because structures can give shelter from flow and thus provide zebrafish with choice over whether to interact with the flow or not, and suggests that zebrafish do value the presence of flowing water, but only when interaction with it is optional.

Providing animals with choice and control over their environments has been recognised as key for good welfare, allowing animals to cope more effectively with stressors and challenges [29,123,124,125]. However, strategies to provide zebrafish with more choice and control in current laboratory settings are not easily identified. One study found that giving zebrafish the opportunity to explore a novel area in their tank resulted in an increase in socio-positive behaviour, a decrease in socio-negative behaviour, and did not increase anxiety [98]. However, the small tanks commonly used in laboratory settings would not easily allow for this type of intervention. Some other possible forms of enrichment which have already been mentioned, such as demand feeders, may also allow opportunities for choice and control, but these avenues have yet to be explored for zebrafish.

2.5. Sensory Enrichment

Natural environments expose animals to a huge variety of sensory stimuli, which is not usually replicated in a laboratory environment. Sensory enrichment, including visual, olfactory, auditory and tactile stimuli may be able to emulate some of the complexity of the natural environment—and importantly, enrichments in several of these modalities need not involve adding anything into the tank itself.

Most of the studies which have addressed sensory enrichment for zebrafish have focussed on visual stimuli—for example, as mentioned above, zebrafish prefer a gravel image over a barren tank [82] (Table 4). This preference for the image was almost as strong as the preference for real gravel, suggesting that in this case, the visual element is more important than the presence of the physical object—perhaps because the broken pattern of the gravel is perceived as providing camouflage from aerial predators and promotes a sense of safety. Another study found that zebrafish housed in tanks with a blue seascape image on the back vertical wall of the tank produced more eggs than fish in barren tanks or those with the image and additional structural enrichment, possibly because the image led to reduced stress levels in the fish [35]. Lavery et al. [85] found that zebrafish showed no preferences between black tank walls and tanks with an underwater image, but it is unclear whether these images were equally appealing or unappealing, or whether the zebrafish were indifferent to these particular images. Zebrafish seem to express colour preferences, but different studies have found different results—one study found that blue and green were preferred to red and yellow [126], whilst others have found preferences for red and green over blue—and possibly even an aversion to blue [127,128]. This may be problematic, given that many commercially available zebrafish tanks are tinted blue to limit algal growth. However, zebrafish have shown lower levels of anxiety and lower cortisol release rates in blue or black tanks over white tanks [129], so possible zebrafish preferences for tank colour or other visual enrichment items are not yet clear.

The use of dawn-dusk phases in the lighting cycles for laboratory zebrafish has been suggested as a form of visual enrichment [3,14,20], and is used in some facilities. Sudden changes in light levels can induce a startle response in zebrafish [130], which is likely to have a high energetic cost and may cause psychological stress. Using dawn and dusk phases would be a simple way to reduce this potential stressor, and would be relatively easy to introduce in many facilities without compromising any other aspects of zebrafish husbandry or welfare. However, the welfare impacts of dawn and dusk lighting phases have not yet been studied in zebrafish.

There have been relatively few studies of enrichment for zebrafish within other sensory modalities. One study looked at the effect of auditory stimulation by exposing zebrafish to two hours of classical music daily for 15 days—zebrafish showed lower anxiety and increased activity than those exposed to no music, although cortisol levels did not differ [131]. However, this is the only study of its kind in zebrafish so far, so it is not known how other forms of music, or other sounds, may affect fish.

It has been suggested that another reason for improved welfare in zebrafish exposed to water currents is that the flow of the water provides tactile stimulation [132]. Zebrafish which were exposed to a chemical alarm cue and then moved into flowing water showed less anxiety than fish moved into still water [132]. This study also found that fish exposed to alarm cue and a water current showed less of a decrease in behavioural indicators of anxiety, such as freezing and remaining near the bottom of a novel tank, when neuromast cells, which are one mechanism by which fish may detect tactile stimulation, were temporarily impaired. This may support the idea that water currents provide tactile stimulation, rather than anxiety being lowered because the fish were occupied by swimming against the current. It has also been suggested that similar tactile stimulation may be caused by the bubbles created by aeration devices, such as airstones (pers. comm.). However, this has not been tested, and one study has found that zebrafish found airstones in tanks aversive compared to barren compartments [82].