Mitigation of Stress in Zebrafish (Danio rerio) induced in a Commonly Employed Housing Method for Research Adam Grierson, Charles R. Tyler and Gregory C. Paull Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, EX4 4QD  The development of innovative racking systems for maintaining large numbers of zebrafish in a relatively small space and novel tanks for the mass production of embryos have been fundamental for research establishments using this fish species. However, we don’t know how some of these systems/practices impinge on certain aspects of the health and wellbeing of the fish kept within them. In this study, we investigate ‘pair-wise mating systems’, the most commonly employed practice for breeding zebrafish. Given that we know zebrafish can become very aggressive when kept at low densities, it is surprising that little is described on how to best optimise pairings that enables good egg delivery whilst maintaining the best health and wellbeing of the animals. Here, we look at fairly simple measures of behaviour to monitor stress in pair-wise mating systems and investigate whether manipulating differential size between the two sexes and/or the complexity of the environment can mitigate against stress imposed by this practice. Introduction Results Size and Dominance 4/6 males became dominant against size matched female fish (< 5% differential in body weight between sexes). 6/6 males became dominant against females when males were > 10% larger in body weight than females. 5/6 females became dominant over males when females were > 10% larger in body weight than males. Dominant females were on average 16% larger (body weight) than males. Dominant males were on average 6% larger (body weight) than females. Experimental Approach Adult WIK (Wild Indian Karyotype) zebrafish were removed from large (>100L) mixed-sexed holding tanks, weighed and randomly assigned to their experimental tanks. The experimental breeding tanks (commonly used for pair-wise breeding and genotyping/screening ) were approximately 2L in capacity. Experiment 1: Size and Dominance – 36 fish (18 males and 18 females) were assigned to 18 breeding tanks. 6 pairs were of approximately equal weight (<5 % differential in body weight between males and females); 6 pairs manipulated to have larger males (>10% of the females body weight) and 6 pairs with larger females (>10% of the males body weight). Experiment 2: Enrichment – 16 fish (8 males and 8 females) were assigned to 8 experimental tanks. 4 tanks contained an artificial grass plant, whilst the remaining 4 tanks were left bare. The fish were size matched to be within <10% of each others body weight. This experiment was replicated 5 times (80 fish; 40 males and 40 females). Behavioural Observations: Behavioural observations were conducted for 6 minutes per tank (3 minutes for chase and repel: 3 minutes for bite and spar), twice daily at 11am and 4pm for 4 days. Observations were made manually and by video capture. Four main types of behaviour were assessed for both male and female fish, see below. Fig.1. Average fish weights in female dominated and male dominated tanks Fig.2. Average male and female weights from tanks where females were removed and tanks which completed the 4-day study Behaviour as an Indicator of Stress Stress imposed (and a requirement to remove fish from the experiments) was correlated with elevated levels of chasing and biting by dominant males (p<0.001). 5 subordinate females had to be removed before the end of the 4-day study to avoid fatalities. Average Chase in Seconds Average Bite in Counts Fig.3. Average chase duration (per 3 minute observation period) by dominant males towards subordinate females in tanks where females completed the 4-day study versus females that had to be removed. Fig.4. Average number of bites(per 3 minute observation period) by dominant males towards subordinate females in tanks where females completed the 4-day study period versus females that had to be removed. Aggression Because males showed no ill effect of female aggression we concentrated on male behaviour only. Male aggression was positively correlated with body size, with an increase in the duration of chases towards subordinate females observed as male size increased. Chasing by males also increased as females became larger in comparison to male size. Measuring Zebrafish Behaviour Average time male spent chasing Subordinate female (seconds) Average time male spent chasing Subordinate female (seconds) Definitions used to Score Aggressive Behaviours for Zebrafish Behaviour Description Chase: Fish swims directly towards another fish causing it to increase its speed (and possibly change direction) and actively pursues the fish. Chase begins when the other fish is forced to retreat and ends when pursuing fish turns away or loses 2 body lengths of distance from retreating fish. Repel: When approached by another fish, the fish makes a sudden movement that causes the approaching fish to change direction, or the fish swims a short distance toward another fish causing it to suddenly change direction but then stops and immediately returns to its previous position (i.e., does not actively pursue it). Bite: Fish clearly opens its mouth and bites other fish, often on the ventral fins. Spar: Fish slowly approaches another fish with its fins flared and offers his/her body flank to the other fish gently oscillating its body with the effect of making it appear a larger size. The opposing fish either moves off or reciprocates the behaviour, sometimes resulting in an elaborate circling motion between the two fish, which can last from a few seconds through to several minutes. Percentage Weight Difference of Females compared to Males Male weight (grams) Fig.5. Average time males spent chasing subordinate females with increasing male size Fig.6. Average time males spent chasing subordinate females with increasing female body weight relative to males Enrichment Average aggression levels were lower in tanks containing plastic plants (p<0.001) 4 females were removed from bare tanks and only 1 female from an enriched tank due to stress. Males from tanks where females had to be removed were on average around 0.56 grams, whilst average male weight from tanks which did not require removal were 0.5 grams. Conclusions Pair-wise mating can invoke aggressive behaviour in zebrafish, invoked particularly by larger males . Unlike for females, subordinate males never showed signs of excessive stress or physical damage even when females were dominant. Enrichment mitigated levels of aggression. Pairing larger females with smaller males with enrichment may help reduce stress and avoid physical damage to subordinates in this commonly employed breeding practice. Fig.7. Average chase per 3 minute observations (seconds) in enriched and bare tanks. Fig.8. Predicting safety margins for pair matches based on size differential between sexes and the presence or absence of enrichment. Future Work To investigate the effects of zebrafish strain and tank size on levels of aggression. Include biochemical markers of physiological stress in then fish studied (e.g. Plasma cortisol). Measure egg output to measure breeding success in the different scenarios