The effect of L-tryptophan on aggressive interactions in barramundi (Lates calcarifer), and food intake of Atlantic salmon (Salmo salar) during seawater transfer

Aggressive behaviour between fish is common in both freshwater and seawater environments, at all stages of development and both between species and within species groups. In monoculture fish farms intracohort aggressive behaviours result in the establishment of dominance and feeding hierarchies, growth depensation (the increase in size variability within a population over time due to differences in growth rates), stress, injury, increased pathogen susceptibility and death. Given the material impact of aggressive behaviours on stock value, Australian fish farmers of both Atlantic salmon and barramundi, carry out specific husbandry practices and provide culture environments that aim to reduce these interactions. Tryptophan (TRP), an essential amino acid, is the precursor of serotonin (5-HT), a monoamine neurotransmitter implicated in mood modulation and behavioural change. This study examined whether supplementary dietary tryptophan (TRP) reduces the rate of aggressive interactions between barramundi. It also examined whether supplementary dietary tryptophan causes a quicker resumption of feeding in Atlantic salmon smolt post-seawater transfer (SWT). These research questions were considered of sufficient commercial interest to warrant sponsorship of this study by an Australian aqua feed manufacturer. This study involved 5 experiments: (1) the effect of supplementary dietary TRP on the behavioural response of a resident barramundi to a smaller intruder; (2) the behavioural response of isolated barramundi to their reflection, a foreign object , and a smaller intruder, with measurement of physiological stress responses and brain serotonergic activity; (3) the effect of supplementary dietary TRP on growth performance, cannibalism, physiological stress response and brain serotonergic activity in groups of barramundi fed to satiation; (4) the effect of supplementary dietary TRP on growth performance, cannibalism, physiological stress response and brain serotonergic activity in groups of barramundi fed a restricted ration; (5) the effect of supplementary dietary TRP on resumption of feeding post-SWT, growth performance, physiological stress response and brain serotonergic activity in groups of Atlantic salmon smolt fed to satiation. In experiment 1, aggressive behaviour of a resident barramundi, fed either a reference feed or one supplemented with TRP at 19.4 mg.g\\(^{-1}\\) total inclusion, toward a 50% smaller (length) conspecific intruder was examined. Intruder fish were confined with residents for 24 hours after the residents had been fed the experimental feeds for 1, 2, 3, 4, 5, 6, 7, 8 or 14 days. Video data were analysed for 5 minutes of each hour over the 24 hour period and behaviours were presented for the first 5 minutes of confinement, the first 6 hours of confinement, and the full 24 hours of confinement. Data for the full 24 hours of confinement are presented for only days 1 and 14. No differences in behavior were found between these treatments during the first 5 minutes of confinement for any day. In relation to the 6 hours of confinement no differences in behavior were observed on all but 2 days tested. On day 14 more attacks were perpetrated on the intruder by fish fed the reference feed. On day 6 a greater latency to chasing by resident fish was observed in the treatment fed the reference diet compared to those fed the TRP supplemented feed and a greater latency to eyeballing by resident fish was observed for fish fed TRP supplemented feed compared to those fed the reference feed. Across 24 hours of confinement on day 14 more attacks were perpetrated on the intruder by fish fed the reference feed. A greater latency to eyeballing by resident fish was observed on day 1 over 24 hours of confinement for fish fed TRP supplemented feed compared to those fed the reference feed. No differences in survival of intruders were observed at either initial confinement, or after 6 or 24 hours of confinement between feed treatments. Experiment 2 aimed to identify behavioural types in barramundi by comparing individual responses to 3 different stimuli with serotonergic and physiological stress responses. Stepwise regression was used to identify predictor variables of behavioural response. Solitary resident barramundi were acclimated to test chambers and exposed to each of the 3 stimuli (reflection, foreign object and intruder) for 1 hour, with a 72 hour gap between tests to reduce the possible behavioural effect of one test upon the next. After the final test (the intruder test) resident fish were sampled for blood and brain tissue. Eyeballing by residents was found to be the strongest predictor of attack by residents on intruders. Eyeballing the foreign object was the strongest predictor for chasing the intruder. Blood glucose was elevated in resident fish chased by intruders, however was not significantly elevated in residents that chased the intruder. Due to a processing error data for serotonergic activity were not available. In experiments 3 and 4 growth performance, rates of cannibalism, stress physiology and serotonergic activity were examined. Across the two experiments fish were offered feed supplemented with TRP at 4.6, 19, 21.4, 23.8, 28.0, 31.0, 33.6, 40.9 mg.g\\(^{-1}\\) (Exp 3); and 4.6, 14.8, 15.9 and 19.0 mg.g\\(^{-1}\\) (Exp 4). Fish were fed to satiation in Exp 3, and to either satiation or 50% of satiation in Exp 4. Supplementary dietary TRP was found to inhibit food intake in barramundi in a dose dependent manner, and specific growth rate was negatively correlated with increased dose. When barramundi were offered a range of TRP supplemented feeds to satiation, fish fed the reference feed, and the feed with the least supplementary TRP had more efficient feed conversion ratios. No differences in feed conversion ratio were observed between barramundi offered either a satiation or 50% of satiation ration at any of the TRP inclusions, despite specific growth rate being strongly affected by ration. Neither ration size nor TRP content affected survival or physiological stress response measured as whole body cortisol, and whole blood glucose and lactate. Supplementary dietary TRP (4.9, 10.9, 21.8, and 46.3 mg.g\\(^{-1}\\) of feed) was found to inhibit food intake in Atlantic salmon smolt in a dose dependent manner prior to seawater transfer in Exp 5. Seawater transfer of Atlantic salmon smolt reduced food intake for both high (period of low water level followed by immersion in seawater ) and low (slow flooding of tanks with seawater ) stress transfer types and for all feed types (4.9, 10.9, 21.8, and 46.3 mg.g\\(^{-1}\\) of feed). Supplementation with TRP did not increase the level of food intake or reduce the period of reduced food intake post-seawater transfer. Neither supplementation of feed with TRP, nor seawater transfer type affected growth or the feed conversion ratio. Serum cortisol was increased by seawater transfer however no differences in serum cortisol response were observed between seawater transfer type. Serum osmolality values were higher for Atlantic salmon subjected to high stress seawater transfer compared to low stress seawater transfer. Brain TRP concentration was higher post-seawater transfer in fish fed the reference feed and subjected to high stress transfer compared to those subjected to low stress transfer. Experiments show that supplementary dietary TRP, at the inclusion levels used in the current study, in general do not significantly inhibit behavioural interactions (measured either directly by video or by the proxy of relative growth performance) between barramundi or Atlantic salmon. The non-feeding period observed in Atlantic salmon smolt post-seawater transfer was not alleviated by supplementing food with TRP. Furthermore the strong hypophagic effect of TRP further reduces its potential as a functional ingredient for practical application in fin fish food. This thesis discusses possible pathways for the observed hypophagic action of dietary TRP, as well as the apparent lack of behavioural modulation. The attribution of TRP, serotonin (5-HT), 5-hydroxyindoleacetic acid (5-OHIAA) or the ratio 5-HT : 5-OHIAA, to food intake and behavioural observations are discussed in context with sometimes contrary findings of other studies. Associations between serotonergic activity and HPI axis activation are examined. All aspects of the study conformed to the requirements of the NHMRC Australian code of practice for the care and use of animals for scientific purposes 8th edition 2013, and more specifically to the UTAS Animal Ethics Committee approved project AOO14598.