Feed and uses thereof for stress mitigation in teleost fish larvae and juveniles

Abstract

The present invention is enclosed in the area of animal feed, in particular in the field of fish feed. Aquaculture has emerged as a crucial sector in meeting the growing global demand for seafood. However, stress in aquaculture fish species can arise from numerous sources including handling, transportation, environmental fluctuations, and interactions with conspecifics. The object of the present invention is a feed comprising kyotorphin (KTP), preferably in its amidated derivative (Tyr-Arg.NH2) and a protein hydrolysate. The KTP is present in the feed by supplementation of analogues, or endogenously within as part of a dietary protein hydrolysate, as the dipeptide TyrArg. The purpose of the present invention is to provide a functional feed to mitigate stress responses in fish species used in aquaculture by enhancing stress tolerance, disease resistance, and growth efficiency in fish, ultimately leading to improved production outcomes and economic returns for aquaculture operations.

Description

[0001] DESCRIPTION

[0002] FEED AND USES THEREOF FOR STRESS MITIGATION IN TELEOST FISH LARVAE AND JUVENILES

[0003] FIELD OF THE INVENTION

[0004] The present invention is enclosed in the area of animal feed, in particular in the field of fish feed.

[0005] PRIOR ART

[0006] Aquaculture has emerged as a crucial sector in meeting the growing global demand for seafood. However, with intensification and expansion, aquaculture systems face various challenges, among which stress responses in cultured species are paramount. Stress in aquaculture can arise from numerous sources including handling, transportation, environmental fluctuations, and interactions with conspecifics. Chronic stress weakens the immune system, rendering fish more susceptible to diseases and pathogens, thereby increasing mortality rates.

[0007] Meagre (Argyrosomus regius) and European seabass (Dicentrarchus labrax), being prominent species in aquaculture, exhibit, as other species, intricate stress responses in aquaculture settings. These responses involve physiological, behavioral, and biochemical changes aimed at maintaining homeostasis under adverse conditions. Common indicators of stress in these species include elevated cortisol levels, altered metabolic rates, immunosuppression, and changes in behavior such as reduced feeding activity and increased aggression or avoidance behavior.

[0008] The proposed invention aims to provide a functional feed to mitigate stress responses in fish species used in aquaculture.SUMMARY OF THE INVENTION

[0009] It is an object of the present invention a feed comprising kyotorphin (KTP), preferably in its amidated derivative (Tyr-Arg.NHz) and a protein hydrolysate.

[0010] In an advantageous aspect of the present invention, kyotorphin is an endogenous dipeptide synthesized in nerve terminals that plays an important role in pain inhibition of the central nervous system and its derivative, amidated kyotorphin (KTP) has been described to have anti-inflammatory and analgesic properties, as well as its derivatives. Therefore, the incorporation of a bioactive KTP dipeptide supplement into functional fish feeds provides a convenient delivery mechanism for these biologically active compounds, ensuring precise dosage and uptake by the fish. In addition, functional feeds enriched with KTP enhances stress tolerance, disease resistance, and growth efficiency in fish, ultimately leading to improved production outcomes and economic returns for aquaculture operations.

[0011] DESCRIPTION OF THE FIGURES

[0012] Figure 1 depicts the differences between the distances travelled after and before the acute stress stimulus of crowding for 3h, for each one of the experimental diets (A Distance moved= Distance post-stress - Distance pre-stress). Significant statistical differences were observed * p < 0.005 and ** p < 0.0003 based on one-way ANOVA and Tukey's multiple comparison test.

[0013] Figure 2 depicts the effects in behavioral parameters (novel tank test) in zebrafish after exposure to an acute stress. (A) Total distance traveled and (B) mean velocity are represented as mean ± standard deviation. Statistical differences are indicated * p<0.02; # p<0.01 based on one-way ANOVA and Tukey's multiple comparison test.

[0014] Figure 3 depicts the effect of the different experimental diets on the zebrafish behavior before and after exposure to acute stress. (A) Time in the upper zone and (B) time spentat the bottom are represented as mean ± standard deviation. Statistical differences are indicated * p<0.04; ** p<0.006; # p<0.0001; ## p<0.007; 4> P< 0.001 based on one-way ANOVA and Tukey's multiple comparison test.

[0015] Figure 4 depicts antioxidant activity of (A) Catalase (CAT) and (B) Superoxide dismutase (SOD) from the liver of pre- and post-stressed fish. Data are presented as mean ± standard deviation (n = 3). Statistical differences between pre- and post-stress values with *p > 0.02 and # p < 0.04, based on one-way ANOVA and Tukey's multiple comparison test and unpaired t-test were identified.

[0016] Figure 5 depicts the mean body wet weight (A) and total length (B) of D. labrax larvae when fed with four different experimental diets (FAST, LOW, MED and HIGH) after KTP supplementation diet at 73 DAH. The results are expressed as mean ±standard deviation (n = 90). Statistical differences between MED and other diets were observed: # p<0.001 (FAST vs MED), p<0.008 (LOW vs MED) and p<0.03 (HIGH vs MED); * p<0.0008 (FAST vs MED), p<0.0001 (LOW vs MED) and p<0.0008 (HIGH vs MED). The dashed lines stand for mean body wet weight and total length at 60 days after hatching (DAH).

[0017] Figure 6 depicts the liver oxidative status and antioxidant enzymes activity in pre- and post-stressed fish at 73 DAH. LPO - lipid peroxidation; CAT - catalase; SOD - superoxide dismutase; GPX - glutathione peroxidase. Data are presented as mean ± standard deviation. Statistical differences are indicated in the graphs ** p < 0.02; # p < 0.05 based on one-way ANOVA and Tukey's multiple comparison test.

[0018] Figure 7 depicts the cortisol plasma levels of juvenile meagre (Argyrosomus regius) fed with experimental diets prior and after the stress stimulus. Juveniles were sampled 14 days after the feeding experiment started (pre-stress) and 1 hour after an acute stress event (post-stress). Results are expressed as mean ± standard deviation (n = 9). Statistical differences are indicated in the graph for p<0.05 (# p<0.0002; and **p<0.02) based on one-way ANOVA and Tukey's multiple comparison test.Figure 8 depicts the liver oxidative status and antioxidant enzymes activity in pre- and post-stressed fish. LPO - lipid peroxidation; SOD - superoxide dismutase; CAT - catalase and GPx- glutathione peroxidase. Data are presented as mean ± standard deviation (n = 3). Statistical differences are indicated in the graphs * p < 0.001 and ** p < 0.007 based on one-way ANOVA and Tukey's multiple comparison test.

[0019] DETAILED DESCRIPTION

[0020] The more general and advantageous configurations of the present invention are described in the Summary of the Invention. Such configurations are detailed below in accordance with other advantageous and / or preferred embodiments of implementation of the present invention.

[0021] In a preferred embodiment of the present invention, amidated kyotorphin (KTP) is added by supplementation, or present in an amount at least 2.5 times, by weight, the total amount of Tyr-Arg dipeptide within protein hydrolysate present in the feed. The amount of Tyr-Arg dipeptide contained the fish protein hydrolysate is estimated according to the amino acid profile and the percentage <500 Da of the proteins present in the protein hydrolysate.

[0022] In another embodiment of the present invention, the amount of KTP present in the feed is at least 5 times the amount of Tyr-Arg dipeptide contained the protein hydrolysate present in the feed.

[0023] In a preferred embodiment of the present invention, the protein source further comprises fishmeal, krill meal, squid meal, pea protein isolate and wheat gluten.

[0024] In another embodiment of the present invention, the KTP is present in the feed by supplementation of analogues, such as Tyr-Arg-NPh, Phe-Arg e Phe-Lys. KTP may also be present endogenously as the dipeptide Tyr-Arg within as an integrant part of a dietary protein hydrolysate.It is also an object of the present invention the use of a feed comprising a protein source that includes protein hydrolysate, the protein hydrolysate comprising Tyr-Arg dipeptide, wherein said feed further comprises amidated kyotorphin, for mitigating stress in teleost fish larvae and teleost fish juveniles during rearing.

[0025] The preferred embodiments of the invention are given in the claims 1 -8.

[0026] EXPERIMENTAL SECTION

[0027] Example 1: Impact of dietary kyotorphin on zebrafish (Danio rerio) resistance to environmental stress

[0028] Zebrafish eggs (AB strain) were obtained from natural spawns at Zebrafish facility and larvae reared under standard conditions until adults were 3 months old. With an initial mean weight of 1.39± 0.04 g, animals were randomly distributed to polycarbonate tanks of 8 and 3 L at an initial stocking density of 5 fish per L and divided into 5 experimental groups of 90 animals each. CM group to which was given a commercial diet (CM; Sparos Lda, Portugal), and CTRL / KTP / KTPZx and HP1 groups, which were fed with the different experimental diets described below. Each dietary treatment was randomly distributed to the rearing tanks. Fish were fed the same diet to apparent satiety, provided by hand two times a day for three weeks, after which the behavioral tests began. Pools of 15 fish were weighted and the beginning and at the end of the trial and mortality was also recorded at the endpoint.

[0029] The CTRL diet was formulated using fishmeal, krill meal, wheat meal, pea protein isolate, wheat gluten and fish protein hydrolysate as main protein sources. The remaining experimental diets varied in the inclusion of ingredients with bioactive potential to the components of the CTRL diet, namely: 1) in diet HP1, a shrimp protein hydrolysate was used to replace the fish protein hydrolysate used in the Control diet; 2) diet KTP contained the fish protein hydrolysate present in the Control diet plus amidated kyotorphin to supplement the Tyr-Arg dipeptide in 2.5 times of the amount estimated in the fish protein hydrolysate. The amount of Tyr-Arg dipeptide was estimated given the amino acid profile and the percentage <500 Da of the proteins present in the fishprotein hydrolysate. Diet KTP2 contained twice the amount of amidated kyotorphin present in the previous treatment, i.e. 5 times of the amount present in the fish protein hydrolysate included in the Control.

[0030] KTP-NH2 peptide was acquired at Bachem AG (Switzerland) with a purity >95% and used for the elaboration of the functional feeds as described below. The proximal composition of the experimental diets used in this study is shown in Table I.

[0031] Table I - Proximal composition of the zebrafish experimental diets

[0032] CM CTRL HP1 KTP KTP2 Dry Matter% (DM) feed basis 92.6 92.1 88.5 92.1 92.1 Protein %, DM basis 68.4 58.2 57.9 58.2 58.2 Fat %, DM basis 15.0 14.1 14.5 14.1 14.1 Ash %, DM basis 12.5 9.3 9.4 9.3 9.3 Phosphorous %, DM basis 1.8 1.4 1.4 1.4 1.4 Energy MJ / kg, DM basis 22.8 21.9 22.3 21.9 21.9

[0033] Experimental diets were produced with powder ingredients being initially mixed according to each target formulation in a double-helix mixer. Subsequently, the powder mixture was ground twice in a micropulverizer hammer mill (SHI, Hosokawa-Alpine, Germany). The oil fraction of the formulation was subsequently added, and diets were humidified and agglomerated through low-shear extrusion (Dominioni Group, Italy). Upon extrusion, diets were dried in a convection oven (HeatEvent 100 / 150, VOTSCHTECHNIK, Germany) for 2 h at 70 °C.

[0034] Stress stimulus

[0035] Fish were subjected to a stress stimulus after 3 weeks of feeding with the experimental diets. The stress stimulus consisted of an acute stress in which the animals were placed in a confinement environment (40 fish / L) for 3 hours. Samples were collected before and immediately after the stress stimulus.Sample collection

[0036] The quantification of cortisol by "skin swab" and the quantification in the liver of antioxidant enzymes (N=10) were performed also before and after the stress stimulus. Pools of 5 fish in a total of 15 fish were anaesthetized with tricaine at 4.9 mM, for the skin swab sample collection for cortisol determination. For this, immediately after the end of the stress stimulus, a sterile swab was gently stroked along the flank of the fish, from head to tail, five times. The swab was then placed into a clean labelled tube with 100 pL of Tris-HCI 0.1M and squeezed as much as possible. The skin swab solution was then vortexed for 30 s and centrifuged at 11000 rpm for 5 min. The remaining supernatant was used for cortisol quantification by ELISA (Fish Cortisol Kit, Cusabio). For quantification of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), samples of liver were collected from another 10 fish (2 pools of 5 fish), frozen in liquid nitrogen and then stored at -80°C until analyzed.

[0037] Growth performance

[0038] At the beginning of the trial, all experimental groups followed had approximately the same body mass with no statistically significant differences between groups. During the 3 weeks of feeding, all diets were well accepted by the animals and no alteration to their health was observed, with survival attaining 100% in all treatments. The final weight of the zebrafish was also not significantly affected by the different experimental diets (Table II).

[0039] Table II - Mean body weight / fish of Danio rerio with different diets (CTRL, CM, KTP, KTP2 and HP1) at initial and after 3 weeks of feeding with the experimental diets.

[0040] D. Initial body Final body

[0041] weight (g) weight (g)

[0042] CTRL 0.35 ± 0.07 0.51 ± 0.06

[0043] CM 0.36 ± 0.07 0.57 ± 0.06

[0044] KTP 0.47 ± 0.07 0.47 ± 0.06

[0045] KTP2 0.43 ± 0.07 0.43 ± 0.06

[0046] HP1 0.55 ± 0.07 0.55 ± 0.06Behaviour tests

[0047] Pre- and post-stress tests included an open-field behaviour assessment (N=15), where each zebrafish was placed individually in a novel tank for 5 minutes. Movement patterns were recorded, using Noldus software, to evaluate exploration behaviour. The measured variables included total distance traveled, swimming velocity, and time spent in different tank areas (surface or bottom). The tests were performed in 3 poolsof 5 fish.

[0048] The behavior tests revealed differences between diets. In Figure 1 is plotted the differences between the distance moved before and after the stimulus. KTP and KTP2 diets show an effect on the difference between the distance moved after and before the stress stimulus (Figure 1) when compared with CM diet. Negative values indicate that the distance travelled by the fish, didn't increase with the stress stimulus. This can be confirmed in Figure 2 that shows that the distance travelled after stress by the fish fed with CTRL (p<0.001) and KTP (p<0.007) diets is lower than with the CM diet. Stressed fish fed with CM diet traveled significantly longer distances (Figure 2A) when compared to the unstressed ones. The same effect was observed for the mean velocity, with KTP diet decreasing the velocity when compared with the CM diet (p<0.01) (Figure 2B).

[0049] Another analysis performed in the behavior tests was the time spent in the top and bottom areas of the tank (Figure 3). The time spent at the top of the tank is similar in different experimental groups before the stress stimulus, but after stress there is a decrease of this time in the CM (p < 0.04) and CTRL diets, and an increase in KTP (p < 0.006) and KTP2-enriched diets (p < 0.0001).

[0050] Antioxidant activity

[0051] The antioxidant activity in the liver was quantified by CAT and SOD activities (Figure 4). Both enzyme activities increase in the post-stress in all the experimental diets and significant differences were encountered with KTP diet for CAT activity (p < 0.02) and with KTP2 diet for SOD activity (p < 0.04).Benefits of dietary KTP supplementation in zebrafish (Danio rerio) fish

[0052] The growth of zebrafish expressed as final body weight, was not significantly affected by KTP-enriched diets, but functional diets containing the bioactive peptide were more efficient in mitigating anxiety-related behaviors when compared with the other diets. In the behavior tests we identified differences related to stress, between the control and experimental groups. Swimming faster over longer distances is correlated with hyperactivity and higher levels of anxiety, which were observed after stress in the group fed with the commercial diet but not in groups fed with KTP or KTP2 (Figure 2). Furthermore, the analysis of the variation of the traveled distances between the different diets (Figure 1) suggests that peptide concentration influences positively the response to the stress stimulus, indicating a reduction of the stress level in KTP-enriched diets groups. The time spent at the bottom area of the tank is consistent with these results (Figure 3), as there is a significant decrease in the time spent at the bottom of the tank in KTP and KTP2 experimental diets. Higher levels of anxiety increase the time spent of the bottom of the tank, thus indicating a reduced exploration capacity, which was overcome by KTP diets, meaning that KTP is capable of abolishing stress-related behaviors. A significant increase in antioxidant activity was observed in zebrafish fed with KTP-related diets (Figure 4), with liver CAT and SOD activities significantly increased in the presence of amidated KTP as a response to the stress stimulus. This increase in antioxidant capacity associated with less stressful behaviors indicates an overall improvement in the well-being of the fish. Herein, the combined effect of reduced stress and enhanced antioxidant activity suggests that KTP-enriched diets play a crucial role in ameliorating stress of zebrafish, with potential translation for aquaculture-reared fish. By promoting better health and resilience in fish, these diets can potentially lead to higher survival rates, improved growth, and more efficient production in aquaculture settings.Example 2: Biological efficacy of dietary KTP supplementation for stress mitigation in European seabass (D. lab rax) fish larvae

[0053] European seabass larvae were used to evaluate the effect of a short-term dietary kyotorphin supplementation on the stress response of seabass exposed to an acute stress factor. Seabass (D. labrax) larvae were obtained from Gloria Maris Group (Gravelines, France). Larvae with 21 days after hatching (DAH) were allocated to twelve experimental tanks to start with a curcumin supplementation before the KTP-enriched diet. The four different diets (triplicate tanks) included a commercial control diet (FAST, Sparos Lda, Olhao, Portugal), a diet containing low curcumin supplementation (LOW), an average curcumin supplementation (MED), and a high curcumin supplementation (HIGH). During 16 days with a decrease in the amount of artemia and gradual and increasing introduction of the inert microdiets, and then until 60 DAH. At day 60 DAH all tanks were fed a commercial diet (Sparos Lda, Portugal) until day 65. From day 66 up until 72 DAH fish from all tanks were fed with the experimental diet containing KTP. Amidated kyotorphin levels in the KTP diet were calculated to exceed those theoretically present in the commercial diet by 5 times. Experimental diets were produced for this trial according the procedures described in Example 1, being analyzed for proximal composition (Table III).

[0054] Table III - Ingredients and proximate composition of the KTP experimental diet.

[0055] KTP

[0056] Dry Matter (DM) % feed basis 95.1

[0057] Protein %, DM basis 58.2

[0058] Fat %, DM basis 16.2

[0059] Ash %, DM basis 9.7 Phosphorous %, DM basis 1.6

[0060] Energy MJ / kg, DM basis 22.0During the experimental period, larvae were exposed to a photoperiod of 14 hours light and 10 hours dark, using halogen lights with an intensity of ± 100 lux above the water surface. The water temperature and dissolved oxygen concentration were measured twice a day with values ranging from 18.6 ± 0.89 °C and 7.8 ± 0.73 mg / L, respectively for the initial phase and for the second, the values range from 18.7 ± 0.76 °C and 8.6 ± 0.91 mg / L. The salinity and pH of the water, as well as ammonium concentrations, were measured four times during the experimental period, ranging from 36 ± 0.7 ppm, 8.4 ± 0.18, and 0.1 ± 0.00 mg / L, respectively.

[0061] Stress stimulus

[0062] From each tank, 150 larvae were collected and transferred to a 25 L bucket with 10 L of saltwater and constant aeration. All larvae from each bucket (12) were exposed to air for 7 minutes, a time-point determined in a pre-stress test that allowed reaching LD50. Mortality in the stress test was registered 1 hour, 2, and 24 hours after the stress. Fish were not fed during the stress test.

[0063] Sample collection

[0064] 30 larvae were sampled from each tank for oxidative stress and antioxidant enzymes (n = 10), and gene expression (n = 10), prior to the stress test. On the day of the stress test, 30 minutes after the stimulus, 10 fish per tank were sampled for oxidative stress (LPO), antioxidant enzymes (CAT and SOD) and gene expression. Larvae were sacrificed in an ice bath and washed with distilled water to avoid marine salts. Samples were collected, frozen in liquid nitrogen and then stored at -80°C until analyzed.

[0065] Growth performance

[0066] At the beginning of the trial (at 60 DAH), 40 fish were weighed and measured individually. The remaining fish were bulk-weighed (n = 10) for initial biomass. At the end of the trial (73 DAH), fish were anaesthetized (2-phenoxyethanol) individually weighed and measured for total length. The mortality was registered in the morning and afternoon of the following 14 days. The growth performance was analyzed based on mean body weight and length measurements at 73 DAH (after KTP supplementation) and compared with the initial values at 60 DAH which were 0.17 ± 0.05 g for the meanbody weight and 2.29 ± 0.275 cm for the length. The results show differences between MED and the other diets (Figure 5) after the introduction of KTP supplementation. The total length of the larvae increased 1.3 times in 13 days and larvae fed previously with MED diet had the higher values (Figure 5). The survival during the entire trial was not influenced by dietary treatment, nor mortality during the stress test.

[0067] Antioxidant activity

[0068] SOD activity in MED and HIGH diet treatments significantly decreased after the stress stimulus. Glutathione peroxidase (GPx) activity results showed that with KTP supplementation in the HIGH treatment there is significantly decrease after the stress stimulus (Figure 6). No statistical differences were found between treatments for CAT activity and lipid peroxidation (LPO).

[0069] Benefits of dietary KTP supplementation in European seabass (D. labrax) fish larvae Dietary supplementation for 6 days with KTP in seabass larvae as not bring differences in growth. Since survival was also not affected by KTP-diet and survival and growth of fish are important indicators for aquaculture purposes, the use of KTP-enriched diets comes as a possibility in the reinforcement of aquafeeds.

[0070] The analysis of the antioxidant enzymes activity in seabass larvae (Figure 4) reveals an enhancement in the antioxidant activity, demonstrated by increase SOD and CAT activities, after KTP supplementation and before the stress stimulus. After stress the levels of lipid peroxidation are maintained low. This can be explained by a protective effect of the existing SOD and CAT higher activities before stress. Besides the decreased activity of SOD and CAT enzymes after stress, the pre-stress activation of antioxidant enzymes like SOD and CAT represents an adaptive response capable of mitigating the harmful effects of oxidative stress and maintaining cellular homeostasis. Liver CAT and SOD activities higher only as a result of KTP supplementation were observed in zebrafish and seabass larvae, with a negative correlation with LPO. This suggests that there was stimulation of CAT and SOD activities, probably induced by the diet, which protected the liver from lipid peroxidation. The modulation of SOD and CAT activities was observed even prior to any induced stress conditions, which may indicate a potential role ofamidated KTP supplementation in bolstering stress response mechanisms within fish. Such an observation hints at the possibility of KTP acting as a proactive agent in fortifying the physiological resilience of the larvae, potentially through mechanisms involving oxidative stress modulation.

[0071] Example 3: Biological efficacy of dietary KTP supplementation for stress mitigation in Meagre (A. regius) fish juveniles

[0072] The trial aimed at evaluating the biological efficacy of a kyotorphin-enriched diet in meagre juveniles health status following a stress factor. The effect was evaluated by assessing survival and biochemical markers (haematological analysis, gene expression, oxidative stress and immune response) following an acute stress situation. Meagre (Argyrosomus regius) juveniles were obtained from natural spawns at the EPPO facilities from broodstock adapted to captivity. Juveniles with an initial mean weight of 9.58 ± 0.420 g were randomly distributed in fiber glass tanks of 300 L at an initial stocking density of 3.83 kg m“3(90 fish per tank). Tanks were supplied with seawater of an average salinity of 37 ppt and a renewal rate of 120 % per hour. During the experimental period, fish were exposed to natural photoperiod changes according to the time of the year (June). The temperature and dissolved oxygen concentration were measured twice a day with values ranging from 26.39 ± 1.88 °C and 5.38 ± 5.76 mg L-l, respectively. All tanks were covered with a net to prevent escapees. Fish were fed by hand using a DGI (daily grow index) of 3 % and a FCR (feed conversion ratio) of 0.9, four times a day, every day of the experiment. All tanks were cleaned every day by manual siphoning and mortality were record. The experiment lasted for 2 weeks (14 days).

[0073] Two dietary treatments were tested in triplicate in this experiment, including a control diet containing a fish protein hydrolysate (Control) and a diet containing amidated kyotorphin (KTP). The bioactive peptide levels in the KTP diet were calculated to exceed those theoretically present in Control by 500% (5 fold), as previously described for Danio rerio and Dicentrarchus labrax.Stress stimulus

[0074] The type of stress used was previously described by Gonzalez et al. (2018) and consists of confinement / netting stress treatment, in which water levels in experimental tanks were reduced (half of the tank) and then, fish were chased with a net (without air exposure) for 5 min. Blood samples were collected before and 1 hour after stress test (15 fish per tank). Fish were anesthetized with 700 ppm 2-phenoxiethanol (Barata et al., 2016) and blood was collected from caudal vein using heparinized 1 ml syringes.

[0075] Sample collection

[0076] On the day of the stress test and 24 hours later, and following overnight fasting, 25 fish per tank were anesthetized with 2-phenoxyethanol and weighed. Samples of liver and anterior intestine from 6 fish per tank were collected for gene expression, samples of liver of 10 fish (2 pools of 5 fish per tank) for antioxidant enzymes, and from those 10 fish the intestines were used for antioxidant enzymes. Sam pies for gene expression were kept in RNA later at 4 °C for 24 hours and transferred to -20 °C, all the other samples were frozen in liquid nitrogen and then stored at -80 °C until analysis.

[0077] Growth performance

[0078] Experimental groups followed the same body mass pattern, with no statistically significant differences between treatments. During experimental time (13 days), both diets were well accepted by the individuals and a high survival was observed in both treatments.

[0079] Plasma metabolite analysis

[0080] Plasma cortisol levels were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (RE52611; IBL, Hamburg, Germany), as previously described by Lopez-Olmeda, et al. (2009). Briefly, to recover all cortisol present in the sample, plasma was diluted (1:20) in diethyl ether. Afterward, the samples were incubated for 2 hours at 30 °C. Once evaporated, the samples were resuspended in phosphate buffer containing 0.1% gelatine (pH 7.6). Subsequent steps followed the ELISA kit instructions. Cortisol concentrations are significantly increased in the CTRL diet (p<0.01) (Figure 7) 1 hour after the stress stimulus. Lactate and glucose plasmaconcentrations are significantly increased 1 hour after the stress stimulus both with CTRL and KTP diets (data not shown).

[0081] Lipid peroxidation in the liver showed differences between the different tested diets before the stress test, fish fed with the CTRL diet had higher activity (Figure 8). After stress there was a decrease in lipid peroxidation in fish fed with CTRL diet, with p < 0.007 but with no statistical differences between pre- and post-stress in the KTP group were observed. After stress, there was an increase in the activity of SOD with both diets, with a significant increase with CTRL (p < 0.007) and KTP diet (p < 0.005). Catalase activity also showed an increase in this enzyme activity after the stress, but without statistical significance. The different diets influenced the activity of GPx on post -stressed fish. On pre-stressed fish the activity of GPx was not affected by the different diets tested and the values are similar between the CTRL and KTP diet. Differences between pre- and post-stress were observed in both diets with decreased levels of GPx (p < 0.001 for the CTRL diet and p < 0.007 for the KTP diet). Globally, there was a decrease in the activity of GPx after the stress test (p < 0.001). The activity of antioxidant enzymes (CAT and SOD) increased, showing a good response of the organism to a stressful situation.

[0082] Benefits of dietary KTP supplementation in meagre (A. reqius) fish juveniles

[0083] The biological efficacy of KTP-enriched diet in meagre juveniles following a stress factor showed increased cortisol levels one hour after stress in all experimental groups (Figure 8). This is indicative of fish experiencing stress or physiological challenges in the experimental environment. With KTP-enriched diet there is also an increase in the cortisol level, but the difference between the pre- and the post-stress is lower, pointing for a protective effect of KTP in the cortisol stress response. In the same way as in the previous Examples 1 and 2 (zebrafish and seabass), the final body weight of meagre juveniles was also not affected by KTP experimental diet, reinforcing the potential use of amidated kyotorphin as a supplement in aquaculture feeds.

[0084] In addition, the KTP diet exerts a positive effect on the stress response of meagre juveniles, confirmed by the higher activities of antioxidant enzymes, particularly superoxide dismutase (SOD) within the liver and a lower LPO after stress with KTP-enriched diet in meagre. Moreover, when KTP-enriched diet was given as a supplementonly for 6 days to seabass larvae, it was capable of increasing CAT, SOD and GPX activity levels even before the stress stimulus (although not maintaining the higher levels after stress). This is a significant outcome for KTP-enriched diets, highlighting their direct role in promoting a better capacity to respond to stress.

Claims

CLAIMS1. A feed for teleost fish comprising kyotorphin (KTP), preferably in its amidated derivative (Tyr-Arg. NH2) and a protein hydrolysate.

2. The feed according to the previous claim wherein KTP is present in an amount at least 2.5 times, by weight, the total amount of Tyr-Arg dipeptide being in the protein hydrolysate.

3. The feed according to any one of claims 1 - 2 wherein KTP is present in an amount at least 5 times, by weight, the total amount of Tyr-Arg dipeptide being in the protein hydrolysate.

4. The feed according to any one of claims 1-3 wherein the protein source further comprises one or more of the following: fishmeal, krill meal, squid meal, pea protein isolate and wheat gluten.

5. The feed according to any one of claims 1 - 4 wherein KTP is added by supplementation or present endogenously as the dipeptide Tyr-Arg within a protein hydrolysate.

6. Use of the feed of any one of claims 1 - 5 for mitigating stress in teleost fish larvae and teleost fish juveniles during rearing.

7. The use according to claim 6 wherein the total KTP content in the feed is between 0.025% and 0.25% on a dry matter basis.

8. The use according to claims 6 - 7 in teleost fish, preferably Danio rerio, Dicentrarchus labrax, or Argyrosomus regius.

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