The present invention relates to the use of a feed comprising a compound selected from guanidinoacetic acid and/or creatine and/or their salts in aquaculture, in particular for improving the growth of aquatic animals and/or for improving the meat quality of aquatic animals.

Aquaculture is a form of agriculture that involves the propagation and cultivation of aquatic animals in a controlled environment and subsequent use of the meat as obtained as food for human beings. Typically, the feed for rearing the aquatic animals comprises fishmeal as an essential ingredient. Fishmeal is normally derived from wild caught species of small pelagic fish like anchovy, blue whiting, capelin, jack, mackerel, menhaden or sardine. Due to its processing, the fishmeal is usually a pelleted or flaked product.

Due to the increase of aquaculture in the course of the last years and the parallel stagnancy or even stepwise decrease in supply of fishmeal, there is a need to find substitute ingredients which are useful for improving the growth of the aquatic animals kept in aquaculture and/or for improving the meat quality of such aquatic animals and which further ideally might be used as a coequal replacement for fishmeal.

Surprisingly, it has been found in accordance with the invention that guanidinoacetic acid has a positive effect on the growth of aquatic animals in aquaculture, in particular on the growth of tilapia. Indeed, guanidinoacetic acid results in a significantly increased weight gain of aquatic animals, in particular tilapia.

Furthermore, it has been found that feeding guanidinoacetic acid to the aquatic animals also results in further improved characteristics, in particular in an increase of the survival rate, the feed intake, the feed conversion rate, the nutrient retention, the meat elasticity and the meat hardness of the aquatic animals, in particular tilapia.

Furthermore, it has been found out that guanidinoacetic acid qualifies as a suitable substitute for fishmeal in the diet of aquatic animals in aquaculture, in particular in combination with plant meals and/or in combination with meal of terrestrial animals.

Guanidinoacetic acid is a bodily substance which occurs in animals and also in humans and which plays a central role in creatine biosynthesis. Creatine can both be taken up with the food and be formed endogenously. The biosynthesis starts from glycine and L- arginine. In mammals, the guanidine group of L-arginine is cleaved by the enzyme amidinotransferase mainly in the kidneys, but also in the liver and in the pancreas, and an N-C-N group is transferred to the glycine. In this process, L-arginine is converted into L- ornithine. In the next step, the guanidinoacetic acid thus formed is converted into creatine with the aid of the enzyme transmethylase, a process which, in vertebrates, exclusively takes place in the liver. In this process, S-adenosylmethionine acts as methyl group donor. The creatine subsequently diffuses into the circulation, where it is transported to the target organs. The transport across the cell membrane into the cells is effected by a specific creatine transporter.

Guanidinoacetic acid is furthermore known to have antibacterial activity and has been applied successfully in animal experiments against bacterial infections (Staphylococcus aureus) (Preparation for protecting mammals against infection (Stanley Drug Products Inc., USA). Neth. Appl (1976), 7 pp. NL 741 1216).

Creatine plays an important role in the cell’s energy metabolism, where, besides adenosine triphosphate (ATP), it forms an essential energy reserve of the muscle in the form of energy-rich phosphocreatine. When the muscle is at rest, ATP can transfer a phosphate group to creatine, giving rise to phosphocreatine, which is then in direct equilibrium with ATP. When the muscle works, it is of crucial importance to replenish the ATP reserves as quickly as possible. Phosphocreatine becomes available for this purpose during the first seconds of maximum muscle work. This phosphocreatine is capable of transferring, through the enzyme creatine kinase, a phosphate group to adenosine diphosphate in a very rapid reaction and therefore of regenerating ATP. This is also called the Lohmann reaction.

Creatine has long been known as a suitable food supplement and animal feed for terrestrial animals. Upon hard and prolonged muscle work, the creatine reserves which are naturally present in the body are rapidly depleted. This is why targeted administrations of creatine have positive effects on stamina and performance, in particular in competitive sportsmen and -women, and undesirable accumulation processes in the body or disadvantageous degradation products are unknown in this context. The reason herefor is that creatine, when supplied in unduly high quantities, is excreted by the body in the form of creatinine.

Besides creatine itself, in other words creatine monohydrate, a large number of other creatine salts, such as creatine ascorbate, creatine citrate, creatine pyruvate and others, have in the meantime also proved to be suitable food supplements. European patent EP 894 083 and the German Offenlegungsschrift DE 197 07 694 A1 may be mentioned at this point as representatives of the prior art. Creatine and guanidinoacetic acid do not only have proven positive effects in humans, but also in terrestrial animals, which is why their use in animal feeds has likewise already been described. Thus, it is known from international patent applications WO 00/67 590 and WO 05/120246 to use creatine and/or guanidinoacetic acid and their salts as feed additive for breeding terrestrial animals and fatteners, for increasing fertility and reproductive performance, as a replacement for meat meal, fish meal and/or antimicrobial growth promoters, growth hormones and anabolic steroids.

GB 2 300 103 teaches the use of creatine in the form of a dog biscuit, for which purpose the creatine monohydrate together with meat is fed as an extruded substance. Owing to its poor solubility, creatine monohydrate’s bioavailability is only limited, and so it is recommended that it be used together with other physiologically active compounds, preferably in salt form. German Offenlegungsschrift DE 198 36 450 A1 relates to the use of stable pyruvic acid salts, in particular creatine pyruvate, in formulations which are suitable for animal nutrition.

DE 100 03 835 A1 relates to formulations for use in states of dehydration as are generally observed in older people, in particular those with limited mobility. In this case, creatine acts as transport medium for water so as to supply moisture to those tissues which are most affected by dehydration symptoms.

Publications concerning the use of creatine or guanidinoacetic acid (GAA) for feeding aquatic animals are rare, as due to the differences in the metabolism of aquatic animals in comparison to terrestrial animals, for which positive effects of GAA have been clearly proven, it is still controversial whether these compounds are beneficial in the diets of aquatic animals, as well.

There is one early publication, disclosing in general the use of GAA for feeding salmonids and natania in predominantly vegetarian diets, i.e. in diets which are essentially free of ingredients deriving from animals (EP1758463).

Qin et al. (Journal of the Chinese Cereals and Oils Association (2015), 30 (3), 85-90) disclose a method of rearing carp, wherein the carp is fed a diet which comprises GAA in concentrations of 0.025 to 0.1 wt.-% and wherein the diet comprises 10 wt.-% fishmeal. Qin et al. observed that the supplementation with GAA did not effect the final weight, specific growth rate, weight gain rate, average daily feed intake or survival rate of the fish.

No state of the art was found disclosing positive effects of GAA on the feeding of tilapia or other cichlids. Further, no state of the art was found, disclosing positive effects of GAA on the survival rate and/or the weight gain and/or the feed intake and/or the nutrient retention and/or the meat quality, in particular the meat elasticity and/or the meat hardness, of the aquatic animals. Furthermore no state of the art was found disclosing the use of GAA in combination with meal of terrestrial animals, in general.

A first subject-matter of the present invention is, therefore, the use of a feed comprising a compound selected from guanidinoacetic acid, creatine and salts of these molecules and mixtures thereof for feeding aquatic animals in aquaculture, in particular for increasing the survival rate and/or the weight gain and/or the feed intake and/or the feed conversion rate and/or the nutrient retention and/or the meat quality, in particular the meat elasticity and/or the meat hardness, of the aquatic animals.

Preferred subject-matter in this context is the use of guanidinoacetic acid and/or its salts for feeding aquatic animals in aquaculture, in particular for increasing the survival rate and/or the weight gain and/or the feed intake and/or the feed conversion rate and/or the nutrient retention and/or the meat quality, in particular the meat elasticity and/or the meat hardness, of the aquatic animals.

A further subject-matter of the present invention is therefore likewise a method for feeding aquatic animals in aquaculture, in particular for increasing the survival rate and/or the weight gain and/or the feed intake and/or the feed conversion rate and/or the nutrient retention and/or the meat quality, in particular the meat elasticity and/or the meat hardness, of aquatic animals, preferably finfish, above all tilapia, in aquaculture, in which method the aquatic animals are fed a feed which comprises a compound, selected from guanidinoacetic acid, creatine and their salts and mixtures thereof.

Preferred subject-matter in this context is a method for feeding aquatic animals in aquaculture, in particular for increasing the survival rate and/or the weight gain and/or the feed intake and/or the feed conversion rate and/or the nutrient retention and/or the meat quality, in particular the meat elasticity and/or the meat hardness, of aquatic animals, preferably finfish, above all tilapia, in aquaculture, in which method the aquatic animals are fed a feed which comprises guanidinoacetic acid or its salts.

According to the invention,“improving growth” is understood in particular as meaning an improvement of the weight gain of aquatic animals.

According to the invention,“nutrient retention” is understood in particular as meaning “energy retention” and/or“protein retention”.

The terms“feed” and“feedstuff” are used interchangeably according to the invention. Uses and methods according to the invention are implemented in a preferred embodiment for non-therapeutic purposes.

The aquatic animals according to the invention are preferably finfish, in particular of the class Actinopterygii, or crustaceans. Actinopterygii include, in particular, tilapia and other cichlids, carps and other cyprinids, salmons and other salmonids, catfish, in particular African catfish and pangasius, tuna, perch, cod, smelt, milkfish, gourami, seabass, in particular barramundi, seabream, grouper and snakehead fish.

Preferred types of salmon and salmonids in this context are the Atlantic salmon, red salmon (sockeye salmon), masu salmon (cherry salmon), king salmon (Chinook salmon), keta salmon (chum salmon), coho salmon, Danube salmon, Pacific salmon, pink salmon and trout. The aquatic animals according to the invention are very preferred tilapia.

Crustaceans include in particular shrimps, lobster, crabs, prawns and crayfish.

The aquatic animals may in particular also be fish which is subsequently processed into fish meal or fish oil. In this connection, the fish are preferably herring, pollack, cod or small pelagic fish like anchovy, blue whiting, capelin, driftfish, jack, mackerel, menhaden, sardine or scad fish. The fish meal or fish oil thus obtained, in turn, can be used in aquaculture for farming edible fish or crustaceans.

The aquatic animals may further be oysters, clams, cockles, arkshells, mussels or scallops.

However, the aquatic animals may also be small organisms which are used as feedstuff in aquaculture. These small organisms may take the form of, for example, nematodes, crustaceans or rotifers.

The farming of aquatic animals may take place in ponds, tanks, basins or else in segregated areas in the sea or in lakes or in rivers, in particular in this case in cages or net pens. Farming may be used for farming the finished edible fish, but also may be used for farming fry which are subsequently released so as to restock the wild fish stocks.

In salmon farming, the fish are preferably first grown into smolts in freshwater tanks or artificial watercourses and then grown on in cages or net pens which float in the sea, ponds or rivers and which are preferably anchored in bays or fjords.

Accordingly, the feedstuff according to the invention is preferably a feedstuff for use in the farming of the above-mentioned animals. In accordance with the invention, guanidinoacetic acid, creatine and their salts can be employed in a wide dosage range. Daily doses are, for example, in the range of between approximately 5 mg and approximately 200 mg, in particular in the range of from approximately 10 mg to approximately 100 mg, preferably in the range of from

approximately 20 to approximately 60 mg, more preferably in the range from

approximately 35 to 50 mg, per kilogram live weight, in particular in the case of finfish, more particular in the case of tilapia.

The aquatic animals are preferably fed a feedstuff which contains guanidinoacetic acid and/or creatine and/or their salts or combinations thereof, preferably guanidinoacetic acid and/or its salts, in an amount of 0.02 to 0.20 wt.-%, more preferably in an amount of 0.05 to 0.16 wt.-%, in particular in an amount of 0.08 to 0.15 wt.-%, above all in an amount of 0.1 1 to 0.14 wt.-%.

Preferably the feedstuff is provided to the aquatic animals, in particular in the case of finfish, in an amount of between 2 and 4% of the actual body weight of the aquatic animals per day, more preferably in an amount of between 2.5 and 3.5% of the body weight of the aquatic animals per day, wherein the feedstuff is preferably fed to the aquatic animals in 2 to 4 rations per day.

The uses and methods according to the invention are preferably applied in aquaculture in a commercial scale, i.e. in particular for rearing aquatic animals, in particular finfish, preferably tilapia, in parallel to a final total weight of aquatic animals of at least 100 kg, preferably to a final total weight of at least 200, 500, 1000 or 1500 kg per aquaculture approach.

Owing to feeding feed comprising guanidinoacetic acid, very good results were especially observed for tilapia.

A further subject-matter of the present invention is therefore a method for feeding tilapia, characterized in that tilapia is fed a feed which comprises a compound selected from guanidinoacetic acid, creatine and their salts and mixtures thereof, preferably

guanidinoacetic acid and/or its salts, in particular in an amount as mentioned before.

The aquatic animals may be fed guanidinoacetic acid and/or creatine and/or their salts over the entire lifetime of the aquatic animals in order to achieve the effects according to the invention. Alternatively they may be fed those compounds only during certain dietary cycles of the rearing. In accordance with the invention, the guanidinoacetic acid and/or the creatine and/or their salts can be provided, for example, as a powder, as granules, as lozenges, as capsules, as pellets or as jelly (hydrocolloidal) products for incorporation into the feedstuff.

Depending on the respective specific intended use, it is preferred to employ the guanidinoacetic acid and/or the creatine and/or their salts as feed additive in combination with other physiologically active substances, in particular with carbohydrates, fats, amino acids, proteins, vitamins, minerals, trace elements and their derivatives and any mixtures thereof.

A further subject of the current invention is a feedstuff as employed for feeding aquatic animals, in particular finfish, preferably Actinopterygii, more preferably tilapia and other cichlids, comprising guanidinoacetic acid and/or creatine and/or their salts or

combinations thereof, preferably guanidinoacetic acid and/or its salts, preferably in an amount of 0.02 to 0.20 wt.-%, more preferably in an amount of 0.05 to 0.16 wt.-%, in particular in an amount of 0.08 to 0.15 wt.-%, above all in an amount of 0.11 to 0.14 wt- %.

This feedstuff comprises further feedstuff ingredients which are preferably selected from protein sources, carbohydrate sources, fat sources and furthermore from other additives such as minerals, vitamins, pigments and amino acids.

Further, besides nutrients, structurants may also be present, for example to improve the texture or the appearance of the feedstuff. Furthermore, it is also possible to employ binders or coatings so as to influence the consistency of the feedstuff. A component which is preferably employed and which constitutes both a nutrient and a structurant is starch.

As a coating may serve fats or polymers like ethylcellulose.

Fats are typically provided as marine oils or vegetable oils or combinations thereof.

Examples for vegetable oils are soybean oil, rapeseed oil, sunflower seed oil, canola oil, cottonseed oil and flax seed oil. Besides the isolated oils the defatted biomass itself, if used as feed ingredient, also provides a certain amount of oil, in particular the fish meal, meal from other marine animals, or plant meal like soybean meal, rapeseed meal, sunflower meal, canola meal, cottonseed meal or flax seed meal.

Plant meals like soybean meal and sunflower meal primarily serve as a source for carbohydrates, in particular besides corn and wheat.

Due to their protein content, plant meals like soybean meal, sunflower meal, corn gluten meal, cottonseed meal and canola meal are also a source for proteins besides wheat gluten and rice. Protein sources of animal origin are animal and/or animal by-product protein sources as known to those skilled in the state of the art, in particular meals of marine animals as well as meals of terrestrial animals, wherein the meals of terrestrial animals are preferably selected from meat meal, meat and bone meal, blood meal, liver meal, poultry meal and silkworm pupae meal. Further protein sources of animal origin are whey powder and egg powder.

A feedstuff according to the invention preferably has a total protein content of 20 to 50% by weight, preferably 25 to 45% by weight, in particular 27 to 40% by weight.

Furthermore, a feedstuff according to the invention preferably has a total fat content of 1 to 15% by weight, preferably 2 to 10% by weight, in particular 3 to 8% by weight.

Furthermore, a feedstuff according to the invention preferably has a total carbohydrate content of 20 to 60% by weight, preferably 30 to 50% by weight, more preferably 35 to 45% by weight.

Furthermore, a feedstuff according to the invention preferably contains meals of marine animals, in particular fish meal, in an amount of less than 10 wt.-%, preferably in an amount of less than 5 wt.-%, more preferably in an amount of less than 2, 1 or 0.5 wt.-%.

Furthermore, a feedstuff according to the invention preferably contains plant meal, in particular selected from soybean meal, rapeseed meal, canola meal, cottonseed meal and combinations thereof, more preferably soybean meal, preferably in an amount of from 10 to 60% by weight, more preferably in an amount from 15 to 50% by weight, in particular in an amount from 20 to 40% by weight;

Furthermore, a feedstuff according to the invention preferably contains animal and/or animal by-product protein sources of terrestrial animals, in particular meal of terrestrial animals, in an amount of at least 2 wt.-%, in particular in an amount of 2 to 30 wt.-%, preferably in an amount of at least 5 or 10 wt.-%, more preferably in an amount of 5 to 30 or 10 to 25 wt.-%, in particular in an amount of 14 to 20 wt.-%.

The feedstuffs according to the invention preferably comprise methionine, betaine and/or choline and/or other physiologically effective methyl group donors. Betaine and choline can be converted into methionine in the body in the presence of homocysteine, which plays a role in particular in the synthesis of creatine starting from guanidinoacetic acid. Methyl groups, which are transferred with formation of homocysteine from S- adenosylmethionine are required for this purpose. If not enough betaine or choline is available, methionine is consumed, which may result in a methionine deficit in the metabolism. In a method which is preferred in accordance with the invention, the feed employed for feeding the aquatic animals, preferably finfish, in particular tilapia, comprises at least one, preferably at least two, three or four, in particular at least five, six or seven, especially preferably all, of the following components: a) plant meal, in particular selected from soybean meal, rapeseed meal,

canola meal, cottonseed meal and combinations thereof, more preferably soybean meal, preferably in an amount of from 10 to 60% by weight, more preferably in an amount from 15 to 50% by weight, in particular in an amount from 20 to 40% by weight;

b) an oil or fat component, in particular a marine oil, corn oil or combinations thereof, preferably in an amount of from 1 to 10% by weight, in particular 3 to 8% by weight;

c) wheat or wheat flour, preferably in an amount of 10 to 50% by weight, more preferably in an amount of 15 to 45% by weight, in particular in an amount of 20 or 40% by weight;

d) meal of terrestrial animals, in particular poultry meal, meat&bone meal or combinations thereof, preferably in an amount of 5 to 30% by weight, more preferably 10 to 25% by weight, in particular 14 to 20% by weight;

e) phosphate, preferably in an amount of from 0.5 to 5% by weight, in

particular 2 to 5% by weight;

f) cassava, preferably in an amount of 1 to 5% by weight;

g) amino acids, preferably selected from lysine, methionine, threonine, valine, histidine, tryptophan and mixtures thereof;

h) vitamins.

This feedstuff preferably comprises fishmeal, more preferably meal of marine animals, in general, in an amount of less than 10 wt.-%, more preferably in an amount of less than 5 wt.-%, in particular in an amount of less than 2, 1 or 0.5 wt.-%, and above all, does not contain any meal of marine animals, at all. According to the invention, "meal from marine organisms" is to be understood in general to mean the processed product of marine organisms in particular the processed product of marine animals. Besides fish meal, which is preferably used according to the invention, this is to be understood according to the invention to mean in particular also krill meal, bivalve meal, squid meal or shrimp shells, which are classic replacement products for fish meal. However, the meal from marine organisms is especially preferably fish meal. According to the invention, "oil from marine animals" or“marine oil” is to be understood in general to mean an oil obtained from a marine organism, preferably from a marine animal. Besides fish oil, which is preferred according to the invention, it also refers to oils isolated from other marine organisms, in particular from marine animals, for example from krill, bivalves, squids or shrimps. Preferably, the marine oil to be used according to the invention is fish oil, in particular a fatty oil from fish, especially preferably a fatty oil from fish of the families Engraulidae, Carangidae, Clupeidae, Osmeridae, Scombridae and/or Ammodytidae.

According to the invention,“meal of terrestrial animals” is to be understood in general to mean a meal which is the processed product of terrestrial animals and preferably refers to meat meal, meat and bone meal, blood meal, liver meal, poultry meal, silkworm pupae meal and combinations thereof.

In one embodiment of the the invention, the guanidinoacetic acid, the creatine and/or their salts are employed as feed additive in predominantly vegetarian rations. The expression“predominantly vegetarian ration” used herein describes a ration which, preferably in agreement with the legal guidelines of the European Union, does not comprise any animal components. The exception of this context is only a possible addition of fish meal. Furthermore, a“predominantly vegetarian ration” in accordance with the present invention is also understood as meaning a partial substitution of fish meal or meat meal by guanidinoacetic acid, creatine and/or their salts. Alternatively, however, it is also possible to employ guanidinoacetic acid, creatine and/or their salts in addition to animal components, in particular in combination with fish meal and/or meat meal.

A feedstuff comprising the components which follow has proved to be especially advantageous in accordance with the invention: a) crude protein in an amount of from 25 to 45% by weight, in particular of from 27 to 40% by weight;

b) calcium in an amount of from 1 to 5% by weight;

c) phosphorus in an amount of from 0.2 to 1.5% by weight;

d) methionine and/or cysteine in an amount of from 0.25 to 1.0% by weight;

e) lysine in an amount of from 0.5 to 2.5% by weight;

f) preferably valine and threonine each in an amount from 0.5 to 2.0% by weight; g) guanidinoacetic acid and/or creatine and/or a salt thereof in an amount of from 0.02 to 0.20% by weight, preferably 0.05 to 0.16% by weight, especially preferably 0.08 to 0.15% by weight, above all 0.11 to 0.14% by weight. Therefore, this feedstuff, too, is subject-matter of the present invention, as are methods for feeding aquatic animals, in particular finfish, above all tilapia, in particular in aquaculture, in which this feed is employed.

In accordance with the invention, it is preferred to employ in particular a feedstuff which comprises the following components: a) plant meal, in particular selected from soybean meal, rapeseed meal, canola meal, cottonseed meal and combinations thereof, more preferably soybean meal, preferably in an amount of from 10 to 60% by weight, more preferably in an amount from 15 to 50% by weight, in particular in an amount from 20 to 40% by weight;

b) an oil or fat component, in particular fish oil, corn oil or combinations

thereof, preferably in an amount of from 1 to 10% by weight, in particular 3 to 8% by weight;

c) wheat or wheat flour, preferably in an amount of 10 to 50% by weight, more preferably in an amount of 15 to 45% by weight, in particular in an amount of 20 or 40% by weight;

d) meal of terrestrial animals, in particular poultry meal, meat&bone meal or combinations thereof, preferably in an amount of 5 to 30% by weight, more preferably 10 to 25% by weight, in particular 14 to 20% by weight;

e) phosphate, preferably in an amount of from 0.5 to 5% by weight, in

particular 2 to 5% by weight;

f) preferably cassava, in particular in an amount of 1 to 5% by weight;

g) amino acids, preferably selected from lysine, methionine, threonine, valine, histidine, tryptophan and mixtures thereof;

h) vitamins.

This feedstuff is therefore also a subject of the present invention, as is a method in which this feedstuff is employed for feeding aquatic animals, in particular in aquaculture.

Guanidinoacetic acid can be prepared in a simple and economical fashion, for example by processes like reacting glycine and cyanamide in aqueous solutions (Production of guanidino fatty acids (Vassel, Bruno; Janssens, Walter D.) (1952), US 2,620,354 ; Method of preparation of guanidino fatty acids (Vassel, Bruno; Garst, Roger) (1953), 5pp. US 2,654,779). In contrast to creatine and creatine monohydrate, guanidinoacetic acid and its salts are, moreover, markedly more stable in the acidic aqueous solution, and they are converted into creatine only under physiological conditions. In this context, guanidinoacetic acid is only converted into creatine after its absorption, above all in the liver. In contrast to creatine, therefore, the predominant fraction of the administered, or fed, compounds, guanidinoacetic acid and/or salts of guanidinoacetic acid, is not degraded by instability reactions, for example in the stomach, and secreted before the absorption, but is indeed available when the corresponding physiological metabolic reaction takes place.

Suitable for the purposes of the present invention are, in principle, all those

guanidinoacetic acid salts and creatine salts which are nutritionally acceptable.

Compounds which have proven to be particularly advantageous for the use according to the invention are guanidinoacetic acid salts and creatine salts which are obtained with hydrochloric acid, hydrobromic acid and phosphoric acid. It is also possible to employ mixtures of guanidinoacetic acid and/or creatine together with one or more of these salts, or else mixtures of the salts with each other.

In total, the present invention serves to provide guanidinoacetic acid, creatine and their salts for novel uses as feedstuff or feed additive in aquaculture. The examples which follow illustrate the present invention further.

Working examples

Materials and methods

A total number a 300 red tilapia ( Oreochromis sp.) with an average initial weight of 5±0.02g were randomly distributed to 175L aquariums. Each aquarium contains 15 fish (equivalent to 85 fish/m ³ ) with temperature ranging from 29-30°C, pH 6.51-7.02, salinity 0 ppt, dissolved oxygen 6.2-7.01 ppm, total ammonia 0.16-0.254 ppm, hardness 38-54 ppm and nitrate 0.5-1.01 ppm. For a period of 60 days, the fish were fed on the 4 experimental treatments 3 times per day (08:00, 12:00 & 16:00) to apparent satiation. Each of the treatment was quintuplicate with all diets containing 0% fish meal, but containing the main animal protein sources poultry meal as well as meat and bone meal (Table 1 ). All experimental diets originated from the same batch with the only difference being the different inclusion levels of GAA (CreAMINO ^® from Evonik, Germany). Control diets had no inclusion of CreAMINO ^® and the supplementation gradually increased in treatments GAA0.5, GAA1.0 and GAA1.5 from 644 via 1373 to 1861 mg of GAA per kg of feed, respectively (Table 2). Survival rate (%), feed intake (g/tank), average body weight (g/fish), biomass gain (g/aquarium), feed conversion ratio (g fed/g biomass gain), and specific growth rate (%/d) were determined. The data were analysed with SPSS 16.0 using ANOVA and Tukey test (p<0.05). Further, retention of energy, lipid and protein was determined.

Texture profile analysis was carried out by using a TA-XT2i texture analyser with probe size 75 to determine the meat texture of the fish products. Compression was carried out on fillet samples 5mm thick and 2x2 cm of size. Samples were compressed twice to a degree of 50% at 1 mm/sec crosshead speed at room temperature for 5 seconds. The following parameters were determined:‘Hardness’, defined as the maximum force (N) or peak first compression;‘Elasticity’, defined as the base line (mm) second compression; ‘Cohesion’, defined as the ratio the area under the second peak to the area under the first peak.

Table 1 : Ingredient and nutrient composition of the experimental diets

Table 2: Analysed levels (mq/kq) of GAA and CreAMINO ^® in the experimental diets

Results and discussions: Feed Intake, Weight Gain, Feed Conversion Ratio, Specific Growth Rate

The tilapia fed the 0% FM diets show a clear response to the graded lower levels of GAA supplemented in the feeds (Table 3). During the 2-month experiment there were no mortalities recorded. The fish exposed to the control diet without any supplementation of GAA show significantly lower growth performance compared to the fish fed with diets supplemented with 0.06 or 0.14 wt.-% GAA. The tilapia fed on diet GAA1.0 (with supplementation of 1 37kg GAA per MT) had significantly higher biomass gain per individual fish and per aquarium compared to all the other treatments. The same trend was observed for the other performance parameters. While a stepwise increase of the different performance parameters was observed from the control via diet GAA0.5 to diet GAA1.0, there was no significant improvement of the performance parameters for the fish fed the diet GAA1.5 in comparison to the negative control. Thus, this study suggests that there is an optimal concentration of GAA in the feed which leads to a clear improvement of the performance of the fish, while transgressing the optimal concentration levels out the positive effects of GAA. Thus, the hypothesis is supported that tilapia can metabolise GAA well and GAA can improve significantly the growth performance of the fish at least at low creatine (and/or low to no fishmeal) levels in the feed.

Table 3: Growth performance (mean ± 1 SD) of tilapia fed the four different experimental diets.

Meat Quality

Table 4 shows the elasticity and hardness of tilapia meat before and after freezing Table 4: Tilapia Meat elasticity (%) and hardness (qf/mm ² )

elasticity and meat hardness, in comparison to fish of the control group without GAA. In contrast to the growth performance parameters, where transgressing the optimal concentration of GAA led to a levelling out of the positive effects of GAA, increasing the level of GAA in feed leads to a continuous increase of the meat quality parameters at least for the fish before freezing.

Nutrient Retention

Table 5 shows the values of energy, lipid and protein retention by the tilapia after rearing for 60 days. Table 5: Retention of energy, lipid and protein of tilapia fed experimental diets

The results for the energy and protein retention are similar to the results for the growth performance of the fish. The highest energy and protein retention was observed in fish fed diet GAA1.0, while transgressing this concentration leads to a significant decrease in energy and protein retention values.