Background of the invention Periodic feeding restrictions are frequently utilised as a management strategy in fish aquaculture, e. g. to control the rate of production, to affect sexual maturation in the rearing population, or to obtain fish of a certain size at predetermined time intervals.

However, feeding restrictions entail higher levels of competition for feed and may result in disproportionate feed acquisition and heterogeneous growth of fish (McCarthy et al., 1992; Jobling and Koskela, 1996; Damsgård et al., 1997).

The formation of dominance hierarchies due to intraspecific competition for limited resources is particularly evident in salmonid species, of which several are of considerable economic importance. Examples are rainbow trout (Oncorhynchus mykiss) which is reared for food production and for put-and-take fishing, Atlantic salmon (Salmo salar) and Arctic charr (Salvelinus alpinus) which are reared for food production, as well as for restocking of natural populations (compensation for spawning sites destroyed by hydroelectric power plants). These species are all highly aggressive, especially at life stages when they are territorial in nature, and develop strong dominance hierarchies. The stress effect of social subordination is probably enhanced under conditions of artificial rearing, where opportunities for social signaling and retreat are limited. Stress-induced appetite reduction in subordinates (Overli et al., 1998), along with direct feeding competition from dominants, are among the proposed mechanisms causing differential food acquisition of fish in a rearing unit.

It is also likely that elevated levels of stress hormones, mainly cortisol, will negatively influence the defence system, which in turn can be expected to cause health problems and contribute to reduced growth rate. Metabolic effects of social stress, which might further contribute to retarded growth of subordinate fish, also have been demonstrated (Jobling, 1994).

However, it is reasonable to believe that the stress exerted upon individual fish from dominant members of a group diminish as group size and rearing density increases.

Observations of aggressive behaviour as well as recordings of inter-individual variation in growth rate and food intake indeed suggest that the intensity of social hierarchy formation decrease with increasing stocking densities. Growth depensation and differential feed intake are however common also at higher rearing densities, and often constitute a disadvantage to aquaculture production. In addition, elevated heterogeneity observed under restricted feeding conditions (Jobling and Koskela, 1996; Damsgard et al., 1997) present a major problem, making production control in aquaculture difficult.

Summary of the invention The invention relates to use of L-tryptophan, and tryptophan derivatives, to control aggressive behaviour and stress reactions in fish. The use is especially intended for fin fish, such as salmonids, sea bass, tilapia, sea bream, channel catfish, turbot, halibut, yellowtail, barrimundi, stripped bass, hirame flounder and eel.

The amino acid L-tryptophan (TRP) is the precursor of the monoaminergic neurotransmitter, serotonin (5-HT) (Boadle-Biber, 1982). The serotonergic system of the brain is believed to be involved in the regulation of agonistic behaviour among diverse animal groups, and in most vertebrate species increased 5-HT activity appears to have an inhibitory effect on aggressive behaviour (reviewed by Winberg and Nilsson, 1993). We have recently obtained results suggesting that supplementation with dietary TRP increases brain 5-HT activity, and thereby decreases aggressive behaviour in juvenile rainbow trout (Oncorhynchus mykiss).

The rate of 5-HT synthesis is normally restricted by TRP availability (Boadle-Biber, 1982) and the local concentration of TRP is an important factor governing the rate of 5-HT synthesis in the mammalian brain (Fernstrom, 1983).

The regulation of brain 5-HT synthesis has been extensively studied in mammals (reviewed by Boadle-Biber, 1993). The first and rate-limiting step in the biosynthesis of 5-HT is the hydroxylation of TRP to 5-hydoroxytryptophan, a reaction catalysed by the enzyme tryptophanhydroxylase. This enzyme is not saturated by its substrate, TRP, in vivo. In addition, tryptophanhydroxyalse does not appear to be subjected to any inhibition by the end product of the reaction pathway, 5-HT. Consequently, an elevation of brain TRP levels results in an increase in the rate of 5-HT synthesis.

Further, it appears that brain TRP levels are remarkably sensitive to the supply of the amino acid from the circulation. The major factor regulating TRP uptake to the mammalian brain is a transport carrier located at the blood-brain barrier, a carrier that transports not only TRP, but also several other large, neutral amino acids (LNAA), including tyrosine, phenylalanine, leucine, isoleucine, and valine, into brain (Fernstrom, 1983). Thus, the uptake of TRP depends not only on the level of TRP in the blood, but also on the blood concentrations of other LNAA. Hence, the amino acid composition of the diet will affect brain TRP concentrations and, thus, 5-HT synthesis. Elevating dietary TRP and/or lowering dietary intake of other LNAA could raise brain 5-HT synthesis. On the other hand, reducing dietary TRP, and/or increasing dietary intake of other LNAA, will decrease brain 5-HT synthesis (Fernstrom, 1983). A meal rich in protein will reduce brain TRP levels since it provides large amounts of other large LNAA competing with TRP for uptake to the brain.

Our knowledge on the regulation of 5-HT biosynthesis in teleost fish and other non- mammalian vertebrates is still very restricted. However, recent results suggest that the rate of 5-HT biosynthesis in fish is regulated by mechanisms very similar to those observed in mammals, and brain 5-HT synthesis appear to be restricted by TRP availability also in teleosts (Johnston et al., 1990; Aldegunde et al., 1998).

Therefore, the suppression of aggressive behaviour observed in rainbow trout fed with TRP supplemented feed could be mediated by the brain 5-HT system.

Supplementation with dietary TRP has been reported to decrease aggressive behaviour in broiler breeder males, an effect that seems to be mediated by the brain 5-HT system (Shea-Moore et al., 1996).

Providing feed with increased TRP might be an interesting aquaculture management strategy, especially during periods of feed restriction. The effect of dietary TRP will be most pronounced in dominant individuals, who consume the larger part of the feed offered. Therefore the tendency to develop strong dominance hierarchies will be diminished.

Detailed description of the invention The object of the present invention was to provide an alternative strategy for production control in aquaculture, reducing the level of intra-specific aggressive behaviour and stress.

Providing feed enriched in TRP will very effectively diminish the tendency to develop dominance hierarchies, an acknowledged problem in aquaculture operations especially during periods of feed restriction. Further, since increased brain serotonergic activity not only inhibits aggressive behaviour, but probably also appetite and feed intake, TRP supplemented feed would provide the desired effects of restricted feeding (i. e. reduced growth, delayed sexual maturation, etc), in the absence of the undesired side-effects (i. e. increased competition for feed, disproportionate feed acquisition, stress, and heterogeneous growth). Interestingly, the effects of elevated dietary TRP will be most pronounced in socially dominant fish, which consume the larger part of the feed offered, and are most aggressive.

Results from experimental work Juvenile rainbow trout (Oncorhynchus mykiss) were kept visually isolated and fed commercial trout pellets during a one week acclimation period. The fish were fed ad libitum and the individual feed intake was quantified continuously during the experiment by counting the number of pellets consumed. Following this week of acclimation the fish were subjected to an aggression test after which the feed was exchanged for experimental feed, with or without (control) supplemented TRP. Two levels of TRP supplementation was used, 1.5 % and 0.15% TRP (as wet weight).

The aggression test was repeated twice, after receiving TRP supplemented feed for 3 and 7 days.

The aggression test applied was a residence-intruder test. In this test a small (50% in body mass compared to the resident fish) conspecific was introduced to the isolated fish. The fish were video recorded for one hour after which the intruder was removed.

The latency to first attack and the frequency of aggressive acts were quantified from the video recordings. Following the final aggression test (after being fed TRP supplemented feed for 7 days) the fish were sacrificed, and blood and brain tissue were collected. Blood plasma was analysed for TRP and cortisol concentrations.

Levels of TRP, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA, the major 5-HT metabolite) were quantified in brain tissue, and the ratio of 5-HIAA to 5-HT was calculated as a measure of brain 5-HT activity.

Blood samples and brain tissue were also collected from fish that were held visually isolated and fed TRP supplemented (1.5 or 0.15%) or control feed for 7 days, but not subjected to any aggression test.

Fish fed TRP supplemented feed for 7 days showed significantly lower levels of aggression than fish fed control feed (Fig. 1). However, there was no significant effect on aggression following 3 days of feeding with TRP supplemented feed (Fig.

1). The two different levels of TRP supplementation had almost identical behavioural effects, and there was no significant difference in aggression between fish receiving 1.5% TRP and 0.15% TRP (Fig. 1).

Plasma TRP levels were drastically increased in fish fed the highest level of TRP whereas fish fed 0.15% TRP showed only modest, but still significant, elevations of plasma TRP levels (Fig. 2). Similarly, brain TRP concentrations were strikingly elevated in fish receiving feed supplemented by 1.5% TRP, whereas fish receiving the lower TRP dose showed a modest, but significant, elevation of brain TRP concentrations (Fig. 2).

Brain 5-HT activity, as indicated by the brain 5-HIAA/5-HT ratio, was elevated in fish receiving TRP supplemented feed (Fig. 3).

Fish subjected to the aggression test showed weakly but significantly elevated plasma cortisol levels, as compared to undisturbed fish (Fig. 4). In fish subjected to the aggression test there was no significant difference in plasma cortisol concentrations between fish receiving different feeds (Fig. 4). However, in undisturbed fish receiving TRP supplemented feed had a significant effect on plasma cortisol levels, fish receiving feed supplemented by 1.5% TRP displaying elevated plasma cortisol concentrations whereas fish receiving feed supplemented by 0.15% TRP showing decreased plasma cortisol concentrations, as compared to controls (Fig. 4).

Figure Legends Fig. 1. The number of aggressive acts performed during three 30 min intruder- resident tests in isolated rainbow trout (Oncorhynchus mykiss) fed control feed or feed supplemented with L-tryptophan (0.15 or 1. 5 % by wet weight), Intruder resident tests were performed at three occasions, before feeding the experimental fish supplemental TRP (Day 0), and after 3 (Day 3) and 7 (Day 7) days of TRP feeding.

Values are mean and SEM from 12 individuals. On day 3 fish fed TRP supplemented, feed, at the level 0.15% as well as 1.5%, were significantly less aggressive than fish fed control feed (p<0-05).

Fig. 2. Concentrations of free L-tryptophan (TRP) in blood plasma (A) and brain tissue (hypothalamus) (B) of intruder tested and undisturbed rainbow trout (Oncorhynchus mykiss). The fish were given feed supplemented with 0. 15% TRP, 1. 5% TRP or feed not supplemented with TRP (control feed) for 7 days.

Fig. 3. Brain serotonergic activity (as indexed by hypothalamic 5-HIAAIS-HT ratios) of intruder tested and undisturbed (isolated controls) rainbow trout (Oncorhynchus mykiss). The fish were given feed supplemented with 0.15% TRP, 1,5% TRP or feed not supplemented with TRP (control feed) for 7 days, Fig. 4. Concentrations of cortisol in blood plasma of intruder tested and undisturbed (isolated controls) rainbow trout (Oncorhynchus mykiss). The fish were given feed supplemented with 0.15% TRP, 1. 5% TRP or feed not supplemented with TRP (control feed) for 7 days.

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