FOR PROMOTING FISH GROWTH PERFORMANCE

TECHNICAL FIELD OF THE INVENTION

The present invention relates to teleost fish larval diets supplemented with natural extracts of curcumin, green tea and grape seed and to a method for improving fish growth performance by providing these diets to fish during the larvae and post-larvae phases.

Growth of fish is directly dependent on feed composition and quality. Inclusion of plants extracts in fish diets can promote multiple functional physiological changes that includes antioxidant, and anti-inflammatory responses, of curcumin (CC) , green tea (GT) and grape seed (GS) as dietary supplements .

The present invention is thus in the field of aquaculture, nutrition, and preparation of marine fish larval diets.

BACKGROUND OF THE INVENTION

Growth of fish is directly dependent on feed composition and quality. Somatic growth is a balance between protein synthesis and degradation that it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term.

Senegalese sole ( Solea senegalensis) is a highly valuable flatfish species for aquaculture diversification in Southern- European countries. However, only recently optimised sole weaning protocols have been developed, with a high impact on larval survival and growth rates.

Gilthead seabream ( Sparus aurata) is one of the most produced species in aquaculture in the Mediterranean Sea and southern European countries. In the 80s the rearing protocols were developed for the species, which allowed the beginning of cultivation in intensive conditions. Nevertheless, despite being an important species in aquaculture, research is still needed to improve larval quality because similarly to sole this species presents low survival during the hatchery phase.

Fish larvae have a tremendous growth potential, displaying simultaneously growth rates that may exceed 30% a day and low survival rates usually between 10-20%. This brings a high cellular metabolism of fish larvae, reflected in much higher oxygen consumption rates in fish larvae than in fish juveniles, giving rise to a much higher formation of reactive oxygen species (ROS) compared to larger fish. Excessive ROS may interact with all types of biomolecules, including proteins, enzymes, and amino acids, causing oxidative damage leading to an increase protein degradation. To maintain homeostasis and prevent oxidative stress, living organisms have evolved antioxidant defence mechanisms, which includes both enzymatic and non-enzymatic components. Several antioxidants, plants and extracts rich in polyphenols, have been proven to improve antioxidant defences, and immune response in fish.

Incorporation of curcumin, green tea and grape seed extracts in fish diets started to be reported recently in juvenile fish. These studies are based on a high range of inclusion levels, as well as highly variable extract origins and compositions. For rainbow trout similar doses of green tea (dried leaves of green tea) were include in the diet at 0.02; 0.1 and 0.5 g/ kg of feed. In grass carp juveniles was reported a 10-fold higher inclusion of green tea at 50 g/kg (green tea purchased in supermarket) . Two different studies tested extracts from grape seed in rainbow trout, with two ranges of supplementation. The former with supplementation of 0.5, 1 and 2 g/kg in feed while the latter with higher inclusions of 10 and 50 g/kg. The inclusion of curcumin in diets for Wuchang bream juveniles were 0.015; 0.03; 0.06; 0.12 and 0.24 g/ Kg feed. In tilapia several supplementation doses were tested, two using higher doses of inclusion 5 and 10 g/ kg and a third supplementing doses between 0.05 - 0.4 g/ kg feed.

However, literature is silent when it concerns the supplementation of these compounds to early stages of development of teleost fish in aquaculture conditions. Larval and post-larval stages of teleost fish, such as Senegalese sole and gilthead seabream, typically require diets very rich in protein, lipid, long-chain n-3 polyunsaturated fatty acids, phospholipids and other nutrients, to sustain growth rates that typically range between 10 and 30% per day. Juveniles teleost fish typically growth at 2-5% per day and have therefore lower nutritional requirements.

Moreover, larval and post-larval stages of teleost fish have poorly developed digestive systems, requiring very high- quality ingredients thereof, which are typically too expensive for juvenile fish feeds. All this is also valid not only for Senegalese sole and gilthead seabream, but also for all currently farmed teleost fish, except for salmonids . Therefore, Senegalese sole and gilthead seabream are good model species, representative of farmed teleost fish in what concerns larval nutrition and digestive physiology.

Therefore, there is a great need of developing teleost fish larval diets to improve fish growth performance in a reliable and efficient way in particular by providing a suitable intake of antioxidant and additives that contribute for increasing the growth performance and survival rate of fish larvae whilst maintaining all the remaining necessary dietary requirements, such as a very high nutrient density in terms of protein, lipid, long-chain n-3 polyunsaturated fatty acids and phospholipids .

The present invention solves this problem by providing a correct amount of antioxidant supplements in marine larviculture diets, with incorporation of curcumin, green tea and grape seed extracts as antioxidant supplements in diets having an additional advantage of being environmentally friend, natural and suitable for Human consumption. DESCRIPTION OF THE FIGURES

Figure 1 represents the dry weight and standard length of fish by the end of the 25 day of feeding with different diets supplemented with curcumin (CC) , green tea (GT) and grape seed (GS) extracts and a commercial diet (CTRL) , without any supplementation of the extracts, in Trial 1 wherein:

Fig. 1A shows the dry weight (mg) of Senegalese sole at 70 days after hatching (DAH) of the different treatments (CTRL; CC; GT and GS) , and

Fig. IB shows the standard length (mm) of Senegalese sole at 70 days after hatching (DAH) of the several treatments (CTRL; CC; GT and GS) . Values are mean ± SD (N = 120 /treatment ) . Different superscript letters at sole age indicate significant differences (One-way ANOVA, P<0.05; followed by Tukey test) .

Table 1 represents the expression of muscle development and antioxidant related genes of Senegalese sole at 70 days after hatching (DAH) of the different treatments (CTRL; CC; GT and GS) . Values are mean ± SD (N = 4/treatment) . Different superscript letters indicate significant differences (One-way ANOVA, P<0.05; followed by Tukey test) .

Figure 2 represents the content of Heat shock protein 70 (HSP70) of Senegalese sole at the end of the growth trial (70 days after hatching, DAH) and after thermal acute stress (72 days after hatching, DAH) of the different treatments (CTRL; CC; GT and GS) . Values are mean ± SD (N = 9/treatment) . Different superscript letters indicate significant differences (Two-way ANOVA, P<0.05; followed by Tukey test) . Figure 3 represents the results of antioxidant biomarkers of gilthead seabream at 62 days after hatching (DAH) of feeding with different diets supplemented with two doses of curcumin extracts (LOW and HIGH) and a commercial diet (CTRL) , without any supplementation of the extracts, in Trial 2, wherein

Fig. 3A shows the total antioxidant capacity (TAC) (mM Trolox equivalents/mg protein) of gilthead seabream at 62 days after hatching (DAH) of the different treatments (CTRL; LOW and HIGH) , and

Fig. 3B shows the protein carbonyl content (PC) (nmol carbonyl/mg protein) of gilthead seabream at 62 days after hatching (DAH) of the several treatments (CTRL; LOW and HIGH) . Values are mean ± SD (N = 12 /treatment ) . Different superscript letters indicate significant differences (One-way ANOVA, P<0.05; followed by Tukey test) .

Figure 4 represents the activity of digestive enzymes of gilthead seabream at 31 days after hatching (DAH) of feeding with different diets supplemented with two doses of curcumin extracts (LOW and HIGH) and a commercial diet (CTRL) , without any supplementation of the extracts, in Trial 3, wherein:

Fig. 4A shows the activity of chymotrypsin (RFU/ mg DW) of gilthead seabream at 31 days after hatching (DAH) of the different treatments (CTRL; LOW and HIGH) , and

Fig. 4B shows the activity of trypsin (RFU/ mg DW) of gilthead seabream at 31 days after hatching (DAH) of the different treatments (CTRL; LOW and HIGH) . Values are mean ± SD (N = 6/treatment) . Different superscript letters indicate significant differences (One-way ANOVA, P<0.05; followed by Tukey test) . DESCRIPTION OF THE INVENTION

The present invention relates to teleost fish larval diets supplemented with natural extracts of curcumin, green tea and grape seed and to a method for improving fish growth performance by providing these diets to fish during the larvae and post-larvae phases.

Curcumin is a yellow compound extracted from the rhizome of turmeric (Curcuma longa) , and commonly used as a spice, and has been part of the traditional Asian medicine for centuries. This lipophilic polyphenol was reported to have strong antioxidant and anti-inflammatory properties, moreover when supplemented in fish diets shown to improve immune response, and digestives enzymes activity.

Green tea ( Camellia sinensis L .) is a source of polyphenolic compounds, secondary plant metabolites that are involved in a wide range of specialized physiological functions. In aquaculture production, the inclusion of green tea fish diets, was reported to have an immunostimulant and antioxidant functions .

Grape seed, from the berries of Vitis vinifera L. ssp sativa, a by-product of the winery and grape juice industry, is a source of numerous bioactive compounds, such as polyphenols. Dietary grape seed extract was reported to have beneficial for effects in meat quality, survival rate and gene expression of antioxidant and innate immunity responses. 1 . Preparation of fish diets

Fish diets according to the present invention comprise natural extracts of curcumin, green tea and grape seed extract in different concentrations to a premium-quality base-diet for fish larvae comprising, in respective to the total amount of dry weight of the diet:

- crude protein: 60 to 67%,

- crude fat: 17 to 22%, wherein from these 2.0 to 4.0% are n-3 polyunsaturated fatty acids, and 5.0 to 10% are phospholipids ,

- fibre: 0.1 to 3%, and

- ash: 5 to 15%.

More preferably, the composition of the base-diet comprises:

- crude protein: 64 to 67%, and

- crude fat: 19 to 21%, and wherein from these, 2.5 to 4.0% are n-3 polyunsaturated fatty acids, and 6.0 to 10% are phospholipids, in respective to the total amount of dry weight of the diet.

Natural extracts of curcumin, green-tea and grape seed extract are added to any of the above-mentioned base-diets in the following concentrations:

- curcumin extract having 95% purity in a range of 0.01% to 1.5%,

- green-tea extract having 50% polyphenols in a range of 0.003% to 0.250%, and

- grape seeds extract having 80% polyphenols in a range of 0.0008% to 0.1875%, also in respective to the total amount of dry weight of the diet. More preferably, the natural extracts are added to any of the base-diets in the following concentrations:

- curcumin extract having 95% purity in a range of 0.020% to 1.0%,

- green tea extract having 50% polyphenols in a range of 0.004 % to 0.125%, and

- grape seeds extract having 80% polyphenols in a range of 0.0012% to 0.012%, also in respective to the total amount of dry weight of the diet.

Even more preferably, the natural extracts are added to any of the base-diets in the following concentrations:

- curcumin extract having 95% purity in a range of 0.020% to 0.75%,

- green-tea extract having 50% polyphenols in a range of 0.005 % to 0.125%, and

- grape seeds extract having 80% polyphenols in a range of 0.0025% to 0.0625%, also in respective to the total amount of dry weight of the diet.

These premium-quality microdiets for fish larvae supplemented with the extracts according to the present invention can be prepared by mixing micronized ingredients such as fishmeal, fish protein hydrolysate, squid-meal, krill-meal, soybean protein concentrate, green mussel extract, porcine liver, wheat gluten, pea protein concentrate, soy lecithin, vitamins, minerals, natural extracts of curcumin, green tea and grape seed and others. A mixture of algae, fish, rapeseed, and linseed oils can be subsequently added.

The obtained mixture is then humidified and agglomerated through low-shear cold-extrusion known methods. The resultant pellets are subsequently dried, crumbled and sieved in different size-ranges preferably of less than <100 pm to 1 mm, preferably of 100 pm to 700pm, more preferably of 100pm to 300pm. Pellets of such sizes may also be obtained by other micro-diet production methods.

2. Method of promoting fish performance

The method of improving marine fish larval growth according to the present invention comprises feeding to fish or larvae of fish a diet supplemented with natural extracts of curcumin, green tea or grape seed as described in any of the claims 1 to 2, wherein the diets are provided to fish for a period 4 to 70 days after hatching (DAH) , preferably for a period of 15 to 45 days (DAH) , preferably 30 days (DAH) .

3. Evaluation of fish performance

Fish performance can be evaluated by growth, muscle cellularity, redox status, digestive capacity, and the expression of muscle growth and redox status related genes.

To assess the effect of these natural extracts supplementation three different trials were performed in Senegalese sole and gilthead seabream at several larvae developmental stages.

First trial - Senegalese sole were supplemented with three doses of different extracts (curcumin, green tea and grape seed) during post-larvae (45 - 70 DAH) growth-phase for 25 days in a recirculation aquaculture system. Second trial - Gilthead seabream were supplemented with 2 different doses of curcumin extract during post-larvae (42 - 62 DAH) growth-phase for 20 days in a recirculation aquaculture system .

Third trial - Gilthead seabream were supplemented with 2 different doses of curcumin extract during larvae (4 - 31 DAH) growth-phase for 27 days in a recirculation aquaculture system.

In addition, the overall analyses of redox status can provide insight on the fitness condition and stress resistance. Moreover, in marine larvae the gut development is not fully achieved after hatching and assessing the activity of digestive enzymes are of particular interest, since the rate of digestion in the intestinal system limits the uptake of nutrients and can potentially limit the growth of the whole organism. For this purpose, several marine fish larval diets were tested.

Trial 1, four marine fish larval diets were tested in Senegalese sole post-larvae, three of them were supplemented with curcumin (CC) , green tea (GT) and grape seed (GS) extracts, and a fourth diet (CTRL), without any supplementation of these extracts was commercial acquired and fed to different group of fish of 45 days after hatching (DAH), for 25 days.

Trial 2, three marine fish larval diets were tested in Gilthead seabream post-larvae, two of them were supplemented with different doses of curcumin (CC) , and a third diet (CTRL) , without any supplementation of these extracts was commercial acquired and fed to different group of fish of 42 days after hatching (DAH), for 20 days. Trial 3, three marine fish larval diets were tested in Gilthead seabream larvae, two of them were supplemented with different doses of curcumin (CC) , and a third diet (CTRL) , without any supplementation of these extracts was commercial acquired and fed to different group of fish of 4 days after hatching (DAH) , for 27 days .

At the beginning and at the end of these experiments, fish were individually sampled for dry weight (DW; mg) and body length (Standard length (SL; mm) determination. Survival was determined at the end of the experiments. (Trials 1,2 and 3) .

In Trial 1, at the beginning and at the end of the experiment sole were collected for morphometric muscle analyses. Standard histological procedures included: fixation, decalcification, dehydration, and inclusion in paraffin. The total transversal cross-section of the muscle [Muscle CSA (mm ² ) ] were measured and photos were taken. The area (pm ² ) of fibres per cross- section and total number of fibres (N) was recorded in each photo and extrapolated to the total muscle cross-section.

For the expression of antioxidant defences and muscle development related genes, sole post-larvae were individually sampled at the end of the growth trial. Samples were homogenized with 1 ml Tri Reagent. The supernatant content was transferred to columns of the Isolate II RNA Mini Kit following the manufacturer's protocols. qPCR assays were performed in a 10 pL volume containing cDNA generated from 10 ng of the original RNA template, 300 nM of each specific forward and reverse primers, and 10 pi of iQ™ SYBR ^® Green Supermix. The qPCR amplification protocol was as follows: 7 min for denaturation and enzyme activation at 95 °C followed by 40 cycles of 30 s at 95 °C and 1 min at 60 °C. Expression data were normalized using the geometric mean of two reference genes, ubiquitin ( ubi ) and glyceraldehyde-3-phosphate dehydrogenase 2 ( gadph2) and the relative mRNA expression calculated using the comparative Ct method. (Trial 1) .

For oxidative status biomarkers analysis, pools of fish were sampled. Samples were homogenized on ice using 1500m1 of ultra- pure water. From each sample, 2 aliquots of the supernatant were taken. One aliquot of 200m1 containing 4m1 of 4% butylated hydroxytoluene (BHT) in methanol was used for the determination of endogenous lipid peroxidation (LPO) . The other aliquot of 500 mΐ was diluted (1:1) 0.2 M K-phosphate buffer, pH 7.4, and centrifuged for 10 min at 10,000 g (4 °C) . The post- mitochondrial supernatant (PMS) was divided into microtubes and kept in -80 °C until further analyses. All biomarkers were determined spectrophotometrically, in micro-assays set up in 96 well flat bottom plates, with the Microplate reader (Trials 1 , 2 and 3 ) .

In Trial 3, gut maturation of gilthead larvae was assessed throughout the growth trial. Samples were homogenised in ultra- pure water and centrifuge for 5 min at 13, 000 rpm, 4°C. The supernatant was used for enzymes analyses, that were expressed as RFU (Relative Fluorescence Units) per mg larvae dry weight.

From the evaluation as above described it is possible to observe that :

- The dietary inclusion of curcumin (CC diet) and grape seed (GS diet) extracts resulted in significantly larger sole post-larvae compared to post-larvae that were fed no supplemented diet (CTRL diet) .

- The dietary inclusion of curcumin (CC diet) extract resulted in significantly larger muscle cross sectional area in these fish when compared to fish that were fed with no supplemented diet (CTRL diet),

- Sole fed the CC diet had the highest number of muscle fibres indicating that the curcumin promoted muscle hyperplastic growth,

- The mean fibre diameter did not differ among treatments, however, the proportion of large-sized (>25 pm) increased, suggesting that inclusion of curcumin (CC diet) extract can also improve hypertrophic growth,

- Differences in the phenotype can be associated with a significant up-regulation of the myogenic differentiation 2 and the myomaker transcripts involved in myocyte differentiation and fusion, respectively, during larval development in fish fed the diet with inclusion of curcumin (CC diet) extract,

- The content of HSP70 was significative higher in sole fed green tea (GT diet) extract after an acute thermal stress. These family of protein chaperons are essential to prevent the denaturation of protein which tend to increase in stressful situations,

- The positively modulatory effect of green tea extract (GT diet) inclusion in the diet in the content of fish HSP70 after an acute stress seems to prevent the increase of protein carbonylation (PC) in long term exposer of thermal stress ,

- In gilthead seabream the inclusion of curcumin increased the total antioxidant capacity (TAC) and decreased the amount of protein carbonylation (PC) of the post-larvae indicating a better oxidative defence system,

- In larvae of gilthead seabream fed high dose of curcumin a significative increase was observed in the activity of chymotrypsin and trypsin, suggesting a higher digestive capacity . In conclusion, these results demonstrate that curcumin and grape seed extracts supplementation improves growth performance of sole. Curcumin supplementation in diet can positively modulate muscle development in sole post-larvae by hyperplasia and hypertrophy of muscle fibres. Green tea improves stress resistance of sole post-larvae through an improvement of HSP70 that prevent protein oxidative damage. In post-larvae of gilthead seabream supplementation of curcumin extract improves oxidative status of the fish. Moreover, at early larvae stage the inclusion of curcumin in the fish diet enhances protein digestion by increasing the activity of protease trypsin and chymotrypsin .

EXAMPLES

Example 1. Preparation of fish diets for Senegalese sole postlarvae

Several marine fish larval diets were prepared supplemented with curcumin (CC) , green tea (GT) and grape seed (GS) extracts with the following compositions.

A premium-quality micro-diet for fish larvae was used as control (CTRL-1) and constituted the basis for the 3 test diets to which the natural extracts were added.

The composition of the CTRL diet comprised 65.3% crude protein, 19.6% crude fat, 2.9% long-chain n-3 polyunsaturated fatty acids, and 7.2% total phospholipids, of dry weight in respective of the total dry weight of the diet (w/w%) . This diet comprised as ingredients krill meal, squid meal, wheat gluten, fish meal, shrimp meal, fish hydrolysate, pea protein concentrate, fish gelatine, fish oil, lecithin and a micronutrient premix comprising vitamins, minerals and other additives .

1.1 supplemented with curcumin (CC)

The diet consisted of 99.95% w/w of the control diet and 0.05% w/w curcumin extract. The nutrient composition was equal to the control diet. Natural extracts were acquired from Denk Ingredients GmbH (Munich, Germany) .

1.2. supplemented with green tea (GT)

The diet consisted of 99.9875% w/w of the control diet and 0.0125% w/w green tea extract. The nutrient composition was equal to the control diet. Natural extracts were acquired from Denk Ingredients GmbH (Munich, Germany) .

1.3 supplemented with grape seed (GS)

The diet consisted of 99.975% w/w of the control diet and 0.025% w/w green tea extract. The nutrient composition was equal to the control diet. The natural extract used was acquired to Parchem (New Rochelle, NY, USA) .

Trial 1, four marine fish larval diets were tested in Senegalese sole post-larvae, three of them were supplemented with curcumin (CC) , green tea (GT) and grape seed (GS) extracts, and a fourth diet (CTRL), without any supplementation of these extracts was a commercial diet. Diets were fed to different group of fish of 45 days after hatching (DAH) , for 25 days. The experiment was done in triplicates tanks and abiotic conditions were maintained at optimum values for maximum sole growth. Inert diet was delivered semi-continuously with automatic feeders for 24 h (cycles of 2 h of feeding followed by 1 h break) .

At two sampling points (45 DAH and 70 DAH) post-larvae were collected to determinate growth performance, muscle cellularity. At the end of the growth trial oxidative status, and expression of candidate genes related to muscle growth, protein catabolism and oxidative status were assessed.

At the end of growth trial, the post-larvae were submitted to a thermal shock for one week. The system water temperature was raised from 21 to 25°C. Fish were sampled at 24h and seven days after the temperature shock for analyses of oxidative status and gene expression.

It is possible to observe that the fish that were fed the diet without supplementation (CTRL diet) showed the lowest results in terms of growth performance (Figure 1) . The final dry weight of the fish in the CTRL group was 163.8 ± 40.2 mg; this was 17% lower than the observed in the CC group (192.2±39.4 mg) . Fish fed with the supplemented diets (CC, GT and GS) had a significant improvement of about 9% in the length compare to the CTRL group.

The muscle growth is a highly controlled process with spatial- temporal expression patterns of several growth-related genes. The differences in the phenotype observed in the sole fed CC diet were accompanied by a concomitant and significant up- regulation of the myod2 and the mymk transcripts. The myod2 is a master regulator of the skeletal muscle for its visible effects in the recruitment of stem cells into the skeletal muscle lineage, as well as, in the proliferation of myoblasts. In turn, mymk is a recently identified gene that encodes muscle-specific proteins that directly govern the fusion process of myoblasts. We hypothesize that the up-regulation of both myod2 and the mymk in fish fed CC diet could activate the differentiation and fusion of myoblasts that in turn would increase both the number and the proportion of large-sized fast-twitch muscle fibres observed in this study.

Our results showed that the sole fed GT diet were able to have a stronger and faster adaptative response under stress caused by the increase of water temperature, compare to the other dietary treatments. The post-larvae of GT treatment were able to significantly increased HSP70 response after 24h of thermal stress exposure.

Example 2. Preparation of fish diets for gilthead larvae and post-larvae

Marine fish larval diets were prepared supplemented with curcumin (CC) extract with the following compositions.

2.1 Diet for gilthead seabream post-larvae supplemented with curcumin (CC)

The diets comprised 99.85% w/w and 99.70% w/w of the control diet, and 0.15% w/w and 0.30% w/w of curcumin extract, respectively. The nutrient composition was equal to the control diet. The natural extract used was acquired to Denk Ingredients GmbH (Munich, Germany) . A premium-quality micro-diet for fish larvae was used as control (CTRL-2) and constituted the basis for the 2 test diets to which the curcumin natural extract was added. The composition of CTRL-2 diet was 66.2% w/w crude protein, 17.4% w/w crude fat, 3.1% w/w long-chain n-3 polyunsaturated fatty acids, and 7.4% w/w of total phospholipids. This diet comprises as ingredients: squid meal, wheat gluten, fish meal, shrimp meal, fish hydrolysate, fish gelatine, fish oil, lecithin and a micronutrient premix comprising vitamins, minerals, and other additives.

In Trial 2, three marine fish larval diets were tested in Gilthead seabream post-larvae, two of them were supplemented with different doses of curcumin (CC) , and a third diet (CTRL- 3), without any supplementation of these extracts was commercial acquired and fed to different group of fish of 42 days after hatching (DAH) , for 20 days.

The experiment was done in quadruplicates tanks and abiotic conditions were maintained at optimum values for maximum seabream growth. Inert diet was delivered by hand to visual satiety during 8h and semi-continuously with automatic feeders for 16 h (cycles of 2 h of feeding followed by 1 h break) .

At the end of growth trial seabream post-larvae were sampled for growth performance and oxidative status.

2.2 Diets for gilthead seabream larvae supplemented with curcumin (CC)

The diets comprised 99.925% w/w and 99.85% w/w of the control diet, and 0.075% w/w and 0.15% w/w curcumin extract, respectively. The nutrient composition was equal to the control diet. The natural extract used was acquired to Denk Ingredients GmbH (Munich, Germany) .

A premium-quality micro-diet for fish larvae was used as control (CTRL-3) and constituted the basis for the 2 test diets to which the curcumin natural extract was added. The composition of CTRL-3 diet was 66.3% w/w crude protein, 17.6% w/w crude fat, 3.0% w/w long-chain n-3 polyunsaturated fatty acids, and 7.5% w/w of total phospholipids. This diet comprises as ingredients: squid meal, wheat gluten, fish meal, shrimp meal, fish hydrolysate, fish gelatine, fish oil, lecithin and a micronutrient premix comprising vitamins, minerals, and other additives.

In Trial 3, three marine fish larval diets were tested in Gilthead seabream larvae, two of them were supplemented with different doses of curcumin (CC) , and a third diet (CTRL-3), without any supplementation of these extracts was commercial acquired and fed to different group of fish of 4 days after hatching (DAH) , for 27 days.

Trial 3 was done in triplicates tanks and abiotic conditions were maintained at optimum values for maximum seabream growth. Total daily amount of inert diet was divided in five meals per day while the total amount of live preys was initially divided in three meals (4-9 DAH) and later reduced to two times per day (10-23 DAH) . Larval rearing system was based on green water technique using Nannochloropsis oculata (4-23 DAH) .

At three sampling points (10 DAH, 24 DAH and 31 DAH) larvae were collected to determinate growth performance, oxidative status, and gut maturation. Larvae represents a transitory period of fish species, where it undergoes several morphological and metabolic changes. They present a tremendous growth potential, displaying growth rates that may exceed 70% a day. These periods are highly demanding in energy and oxygen uptake, thus, may lead to a saturation of antioxidant defences and improve susceptibly to cytotoxic effects of oxidative stress.

The inclusion of curcumin in seabream diet improved the total antioxidant capacity and decrease the protein carbonylation content of the post-larvae in comparation to CTRL diet (Fig 4A and B) . The result corroborates the effect of curcumin as a scavenger of reactive oxygen species (ROS) .

In the long term the post-larvae fed curcumin might increase growth performance by allowing larvae to reduce the costs of endogenous antioxidant defences and by decreasing protein degradation. Therefore, allowing fish to invest more energy for growth improvement .

The diet composition has been recognized to impact the activity of digestive enzymes. The inclusion of curcumin enhances the chymotrypsin and trypsin activity compared to no supplemented diet (CTRL diet) fed fish (Fig 5A and B) .

These enzymes are pivotal for protein digestion so with this supplementation larvae of seabream might have a better protein digestion than CTRL larvae. In a long term fed curcumin larvae might present a better growth performance and feed utilization than CTRL fed fish. This is very important, as fish larvae have an immature digestive system in the first 4 weeks of life.