Gel structure for live feed replacement for promoting early stage of life of aquatic organisms

Abstract

Disclosed are nutritional supplements suitable for ingestion by shrimp larvae or fish larvae. The nutritional supplement comprises a granular gel structure, wherein the granular gel structure comprises agar, an attractant and water; and an aqueous medium comprising at least one salt. The particulate gel structure may be made in the form of gel particles, such as particles, and the gel particles may be added to the aqueous medium. The invention also relates to a method for preparing the nutritional supplement and a method for co-feeding the nutritional supplement to shrimp larvae or fish larvae with at least one wet feed or dry feed, such as a fully wet feed or fully dry feed.

Description

[0001] The present invention relates to a nutritional supplement suitable for ingestion by shrimp larvae or fish larvae. The nutritional supplement comprises a particulate gel structure, which may be in the form of gel particles, such as granules. The gel structure is dispersed in an aqueous medium comprising at least one salt, such as sodium chloride. The present invention also relates to a method for preparing the nutritional supplement. The present invention also relates to a method for feeding shrimp larvae or fish larvae with the nutritional supplement and at least one wet feed or dry feed, such as a complete wet feed or a complete dry feed.

[0002] abbreviation

[0003] ALF artificial live feed

[0004] ALF(P),ALFP pasteurized live feed

[0005] ALFF Feeding artificial live feeds on a fixed feeding regimen

[0006] ALFV Feeding artificial live feeds on a variable feeding schedule

[0007] ART Artemia

[0008] DD Dry Diet

[0009] M1, M2, M3 Mysid stages 1, 2, 3

[0010] N5 Nauplius stage 5

[0011] Z1, Z2, Z3 Zoa stage 1, 2, 3

[0012] PLn Postlarval stage

[0013] Background and Problem Description

[0014] Despite the continuous improvement in the physical and nutritional properties of dry diets used for larval culture of marine finfish and shrimp species, it remains essential to provide them with live feeds during the critical early developmental stages. Live feeds mainly consist of rotifers and Artemia, but sometimes also include wild zooplankton harvested from nature or produced in hatcheries.

[0015] Due to the constant biohazard risk of disease transmission through live feed production on the one hand and the insufficient and variable nutritional status of these wet feeds on the other, many sterilization and enrichment procedures have been developed. All these responses and treatments ultimately add several additional operational costs but are essential to upgrade live feeds to an acceptable wet feed state.

[0016] Many attempts have been made to fully integrate the nutritional content of live feeds into so-called dry replacement diets or wet replacement diets, but none have been completely satisfactory. Complex dry diets, such as micro-bound particles, micro-coated particles, protein-walled microcapsules, etc., usually produced on pharmaceutical equipment, have been developed containing all possible nutrients and chemical characteristics to meet the needs of small aquatic animals, but often fail to achieve the right balance between water stability, particle digestibility and buoyancy.

[0017] Due to their higher water content, wet diets generally result in more buoyant formulations but encounter higher filtration and preservation challenges when retaining peptides, amino acids, vitamins, and essential lipophilic and hydrophilic compounds of high oxidative potential in one matrix.

[0018] The best compromise to solve the problem of live feed replacement may be to combine the best features of wet and dry diets by co-feeding them. Co-feeding dry and live feeds before weaning is a well-established procedure that can be further explored in the direction of dry and wet diets by mimicking the live feed feeding strategies currently used. For shrimp and fish, this means that during the critical stages before molting to the early PL stage (post larval stage; for shrimp) and before metamorphosis (for fish), the live feed can be reduced to its minimum essential functions and elements, which are lacking in dry diets. It is an object of the present invention to remedy or alleviate at least one of the disadvantages of the prior art, or at least to provide a useful alternative to the prior art.

[0019] Live diets, which have been subject to analysis for decades, hold no secrets in terms of the nutrients that make up the bulk of their composition. Functional nutrients are well known and can be incorporated into dry diets without having to be repeated in wet diet formulations. In this regard, wet diets may be better used as a supplement to chemically incompatible nutrients in dry diets, but may also be used to compensate for temporary specific enhancements during critical stages. Highly sensory attractive molecules and excellent physical buoyancy properties should further stimulate interest in the on-demand ingestion of these new generation wet diets by larvae.

[0020] The life cycle of shrimp includes egg, nauplii (1-6), zoea (1-3), mysid (1-3), postlarval stage (PLn), juvenile stage, subadult stage and adult stage. The life cycle of fish depends on the fish species but usually includes egg, larvae, juveniles and adults, although it may be different for some fish species.

[0021] Artemia is used in most shrimp hatcheries, but its use is limited to the larval rearing period, when it forms a small part of the total formulated (dry) diet provided. Average Artemia cyst consumption is usually limited to 1-5 kg ​​of cysts per million shrimp produced.

[0022] There are many reasons why Artemia usage is limited, but the most important reasons are cost, biohazard issues, sustainability, chemical contamination, variable hatching, and quality issues. Despite all these issues, Artemia is still used because of its unique role in overcoming the critical stages of early shrimp development from mysids to early postlarvae (PL).

[0023] The additional benefits of Artemia factors to shrimp larvae have been studied for many years but have never been fully elucidated. The hypotheses are many: buoyancy and motility, digestibility of readily autolytic organisms, accompanying bacterial flora, micronutrients, enzymes, hormones, etc.

[0024] Most likely, the Artemia factor is the result of a complex multifactorial combination of physical, microbiological and nutritional packages that are difficult to replicate due to their high variability, but are nonetheless complementary, such as but not limited to:

[0025] - has digestion-enhancing properties due to the simple hydration of dry feed by live food, which contains about 90% water;

[0026] - Recolonization of the larval gut by different microbial communities released from rapid autolysosomes, and release of bacterial gut flora from live feeds after ingestion by fish or shrimp;

[0027] - protection of the intestine by the soft matrix formed by live feed and the harder dry diet;

[0028] —The viscosity and movement of live prey promotes intestinal motility.

[0029] Dry feed and wet feed

[0030] Depending on the size and development stage of shrimps and fish, dry feed particles are provided in sizes of, for example, 50 μm to 100 μm, 100 μm to 200 μm, 200 μm to 300 μm, 300 μm to 400 μm and 400 μm to 500 μm.

[0031] Wet feeds generally follow a similar size hierarchy as they attempt to completely replace dry feeds and / or Artemia. These feeds consist of a mixture of: a gelling substance / binder, water and a complete list of nutrients, i.e. a fat source, a protein source, minerals, vitamins, etc. Other wet feeds have been designed for use with growing fish or shrimp in aquaculture, long after they have been weaned off a live diet. Some wet feeds are only for use by hobbyists who keep fish tanks, where the animals are not in the food chain.

[0032] WO 02 / 078463 discloses formulated micro-bound dietary products in dry or wet form for culturing larval fish and crustaceans, containing proteins such as fish protein hydrolysate, casein and egg yolk, and binders such as soya lecithin, wheat gluten and alginates.

[0033] WO02 / 071867 discloses a solid non-liquid granular food composition that effectively meets the nutritional needs of aquatic organisms. The food of the invention comprises oil-coated nutrients and moistened nutrient feed particles, which are embedded in a gel or an ingestible polymer mixture. The gel of alginate and gelatin as a gel is cross-linked by a water-soluble multivalent antimicrobial salt such as calcium chloride.

[0034] EP 1006807 discloses a feed for aquatic animals in the form of a viscous gel used as a substitute for frozen feed, comprising 0.001 to 50% of a gelling agent, 0.1-90% of a natural nutrient, a water content of 20-99%, and a viscosity of 1 to 2.10 6 mpa.s.

[0035] It is an object of the present invention to remedy or alleviate at least one disadvantage of the prior art, or at least to provide a useful alternative to the prior art. More specifically, it is an object of the present invention to improve the feeding of fish and / or shrimp larvae, preferably without the use of live feeds, and / or to improve the growth and / or survival of fish and / or shrimp larvae.

[0036] This object is achieved by the features specified in the following description and in the following claims.

[0037] General Description of the Invention

[0038] The gist of the present invention is to move away from either a completely dry diet or a completely wet diet (i.e. a nutritionally complete diet) and to focus on the drawbacks of either dry or wet diets, particularly during periods of difficulty encountered by juvenile shrimp and juvenile fish during specific molting periods, weaning or dietary transitions. During this period, the animals are undergoing a huge transformation and require additional dietary and digestive needs, but their digestive systems are still underdeveloped and rely on the interaction of live feed and bacteria to partially digest the ingested feed. Without wishing to be bound by theory, it is assumed that the gist of the present invention is to replace the specific functions of live feed by forming a nutritional supplement, i.e. a physical nutritional granular gel structure dispersed in an aqueous medium (e.g., saline) containing at least one salt, to promote the intake and digestion of a co-fed dry diet. The granular gel structure is buoyant, rich in water, and can be used for bacterial adhesion and electrolyte exchange.

[0039] The present disclosure relates to a nutritional supplement comprising a particulate gel structure preserved in an aqueous medium containing at least one salt, the nutritional supplement replacing the function of live feeds. The nutritional supplement is preferably co-fed with conventional and / or commercial formulated dry feeds during the critical transformation stages of larval shrimp and larval fish.

[0040] Surprisingly, it was found that feeding such a nutritionally incomplete nutritional supplement, i.e. one that does not contain all the nutrients required for the growth of fish and / or shrimp larvae, together with a formulated dry feed that is itself nutritionally complete, increased the survival of shrimp larvae from the mysid to the early PL stage to the same extent as the co-feeding of live feeds.

[0041] According to one aspect of the present disclosure, a granular wet gel structure may be preformed by gel coagulation based on a mixture of a gelling agent, an attractant and water, and optionally a colorant, omega-3 fatty acids and phospholipids.

[0042] As used herein, the term "weight percent" or "wt%" refers to the weight percent on a wet weight basis, which is also referred to as an "as-is basis", i.e., water or moisture is part of the composition and the inclusion level of each specified ingredient is calculated relative to the composition. This is different from a dry basis, which is expressed as the weight of the specified ingredient as a percentage of completely dry solids, i.e., excluding water or moisture content.

[0043] After gel coagulation, the nutritional supplement can be obtained by wet sieving the granular gel structure and diluting the granular gel structure in an aqueous medium comprising at least one salt. The aqueous medium comprising at least one salt can contain about 100-200 ppt of NaCl. The nutritional supplement can be stored in a tank.

[0044] The invention is defined by the independent patent claims. The dependent claims define advantageous embodiments of the invention.

[0045] In a first aspect, the present disclosure relates to a nutritional supplement suitable for ingestion by shrimp larvae or fish larvae, the nutritional supplement comprising:

[0046] I) A granular gel structure comprising:

[0047] a) agar;

[0048] b) attractants; and

[0049] c) water;

[0050] and

[0051] II) an aqueous medium comprising at least one salt.

[0052] The nutritional supplement may further comprise an ingredient selected from a colorant, omega-3 fatty acids and phospholipids, or any combination thereof. In an alternative embodiment, the nutritional supplement further comprises a colorant, omega-3 fatty acids and phospholipids.

[0053] The aqueous medium may contain at least 80 ppt, such as at least 100 ppt, at least 125 ppt, at least 150 ppt, at least 175 ppt, or at least 200 ppt of salt. The aqueous medium may contain up to 500 ppt, preferably up to 400 ppt, more preferably up to 300 ppt, even more preferably up to 250 ppt of salt. The salt may be any salt. Those skilled in the art will be able to select a suitable salt. The salt may include, but is not limited to, sodium chloride, magnesium chloride, calcium chloride, potassium chloride, sodium carbonate, magnesium carbonate, calcium carbonate, potassium carbonate, sodium sulfate, magnesium sulfate, calcium sulfate and potassium sulfate, or any combination thereof. In a preferred embodiment, the aqueous medium comprises sodium chloride, preferably at least 80 ppt, such as at least 100 ppt, at least 125 ppt, at least 150 ppt, at least 175 ppt or at least 200 ppt of sodium chloride, and at most 400 ppt, such as at most 350 ppt, at most 325 ppt, at most 300 ppt or at most 250 ppt of sodium chloride. The advantage of sodium chloride is that it can act as a preservative to prevent the nutritional supplement taught herein from deteriorating. However, other salts can also be used for this purpose. The aqueous medium can also contain other salts taught herein.

[0054] The granular gel structure may be in the form of individual gel particles, such as granules, preferably granules having a particle size of 1 μm to 2 mm. The granules may be 5 μm to 800 μm, such as 20 μm to 600 μm, such as 50 μm to 300 μm, such as 50 μm to 200 μm. The granular gel structure may be coagulated into individual particles having such a particle size. The granular gel structure may also be referred to as a granular wet coagulated gel structure to emphasize that the gel structure may be prepared by coagulation and is therefore in a solid (albeit gelled) state and that the particles are not dried after coagulation or gelling.

[0055] The particulate gel structure, for example in the form of gelled particles, such as granules, can be buoyant, slowly sinking, or preferably neutrally buoyant, i.e. it remains in the water column without sinking or rising. For example, its density can be 0.995 to 1.200 g / ml. Such a density allows for suitable buoyancy, such as neutral buoyancy, in water tanks typically used to cultivate fish and / or shrimp larvae.

[0056] The gelled particles, e.g. granules, may be obtained by compression, spraying, coagulation, flocculation or other techniques. The use of agar keeps the various ingredients together. The gelled particles, e.g. granules, may themselves also contain smaller particles. The gelled particles, e.g. granules, may have any form, e.g. they may be round, oval, square or elongated, like grains.

[0057] A granular gel structure or nutritional supplement as taught herein may comprise 0.01 wt % to 10 wt %, e.g., 0.05 wt % to 7.5 wt %, 0.1 wt % to 6 wt %, 0.5 wt % to 5 wt %, 1 wt % to 4 wt %, 1.5 wt % to 3 wt %, or 1.5 wt % to 2.5 wt % agar, by wet weight.

[0058] The attractant can be an amino acid, an amino acid mixture, a dipeptide, a polypeptide, a dipeptide mixture, a polypeptide mixture, a mixture of a dipeptide and a polypeptide, a hydrolyzed protein source (wherein the protein source can be a plant source or an animal source, wherein the protein source can be an aquatic or marine source, such as zooplankton and fish), and a mixture of any of amino acids, dipeptides and polypeptides and hydrolyzed protein sources. The attractant can be provided in the form of a dry powder, a wet paste or a solution. The attractant can be present in the granular gel structure or nutritional supplement taught herein in the following amounts: 0.0001% to 20% by weight, such as 0.0005% to 18% by weight, 0.001% to 16% by weight, 0.005% to 14% by weight, 0.01% to 12% by weight, 0.05% to 10% by weight, 0.1% to 8% by weight, or 0.5% to 6% by weight.

[0059] The ω-3 fatty acids can preferably be selected from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and preferably DHA. Fish meal, fish oil, seaweed and phytoplankton are the main sources of EPA and DHA, and DHA and EPA accumulate in larval fish and / or larval shrimp that eat these algae and phytoplankton. However, ω-3 fatty acids can also be obtained from (optionally genetically modified) plant sources, such as Brassica plants, at present. In one embodiment, the ω-3 fatty acids can be from an animal source. In an alternative embodiment, the ω-3 fatty acid source can be from a plant source. The ω-3 fatty acid source can include DHA from an animal source. As an alternative or supplement, the ω-3 fatty acid source can include DHA from a plant source. Omega-3 fatty acids, such as DHA, can be present in the granular gel structures or nutritional supplements taught herein in amounts ranging from 0.001% to 20% by weight, such as from 0.005% to 18% by weight, from 0.01% to 16% by weight, from 0.05% to 14% by weight, from 0.1% to 10% by weight, or from 0.5% to 6% by weight.

[0060] The phospholipids can include lecithin. Lecithin is a mixture of glycerophospholipids, which includes phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid. Therefore, they are a good source of various phospholipids. Lecithin can come from any available source, including but not limited to egg yolk, marine origin, soybean, milk, rapeseed, cottonseed and sunflower oil. In one embodiment, the lecithin can be soybean lecithin. The phospholipids, such as lecithin, can be present in the granular gel structure or nutritional supplement taught herein in the following amounts: 0.01% by weight to 20% by weight, such as 0.05% by weight to 15% by weight, 0.1% by weight to 10% by weight, or 0.5% by weight to 6% by weight.

[0061] The colorant may be any colorant that is used to make the granular gel structure visible in a water tank that is typically used to culture shrimp larvae and / or fish larvae. The colorant may be a pigment, a dye, or an ionized solution. In one embodiment, the colorant may be selected from carotenoids such as carotene or xanthophylls, carbon, clay, minerals, anthocyanins, colored algae such as red algae or green algae, etc. In one embodiment, the colorant may be a carotenoid, preferably lutein, and even more preferably astaxanthin.

[0062] The coloring agent can be present in the granular gel structure or nutritional supplement taught herein in the following amounts: 0.0001 wt % to 20 wt %, such as 0.0005 wt % to 18 wt %, 0.001 wt % to 16 wt %, 0.005 wt % to 14 wt %, 0.01 wt % to 12 wt %, 0.015 wt % to 10 wt %, 0.02 wt % to 8 wt %, 0.1 wt % to 6 wt %, or 0.5 wt % to 5 wt %, depending on the type of coloring agent used. One skilled in the art will be able to select the correct amount of coloring agent needed to achieve the desired effect.

[0063] The granular gel structure may, for example, contain, by wet weight, 0.01 to 10 wt % agar, 0.01 to 10 wt % attractant, 0.1 to 3 wt % DHA, 0.01 to 10 wt % phospholipids, 0.01 to 20 wt % colorant and 40 to 95 wt % water.

[0064] The weight ratio between the particulate gel structure and the aqueous medium may be 0.2:1 to 1:0.2, preferably 0.5:1 to 1:0.5, more preferably 0.75:1 to 1:0.75.

[0065] In a second aspect, the present disclosure more particularly relates to a method of preparing a nutritional supplement suitable for co-feeding shrimp or fish larvae as taught herein, wherein the method comprises the following steps:

[0066] i) coagulating a gelling mixture comprising agar, water and an attractant, and optionally omega-3 fatty acids, phospholipids and / or a colorant, to form a particulate gel structure, such as granules;

[0067] ii) optionally, wet sieving the particulate gel structure, such as granules, to obtain a particulate gel structure of appropriate size; and

[0068] iii) adding the particulate gel structure to an aqueous medium comprising at least one salt to obtain a nutritional supplement suitable for ingestion by shrimp larvae or fish larvae.

[0069] The gelling mixture is preferably solidified by heating the gelling mixture to a temperature that melts the agar in the gelling mixture, and then solidifying the gelling mixture, for example, by forming suitable particles and solidifying them. A person skilled in the art knows how to determine the appropriate temperature to melt the agar in the gelling mixture, and a person skilled in the art also knows how to determine the appropriate temperature to solidify the gelling mixture.

[0070] In one embodiment, the gelling mixture can be prepared as follows:

[0071] a) mixing water, an attractant, and optionally omega-3 fatty acids, phospholipids and / or a colorant;

[0072] b) heating the mixture of a); and

[0073] c) adding agar to the mixture during heating to form the gelled mixture.

[0074] In another embodiment, the gelling mixture can be prepared as follows:

[0075] a1) mixing water, agar, an attractant, and optionally omega-3 fatty acids, phospholipids and / or a colorant; and

[0076] b1) heating the mixture of a1) to form the gelled mixture.

[0077] In an alternative embodiment, the gelling mixture may be prepared as follows:

[0078] a2) mixing water and agar;

[0079] b2) adding an attractant and, optionally, omega-3 fatty acids, phospholipids and / or a colorant to the mixture of a2); and

[0080] c2) heating the mixture of b2) to form said gelled mixture.

[0081] In an alternative embodiment, the gelling mixture may be prepared as follows:

[0082] a3) mixing water and agar;

[0083] b3) heating the mixture of a3);

[0084] c3) adding an attractant, and optionally omega-3 fatty acids, phospholipids and / or colorants to the mixture of b3) to form the gelled mixture.

[0085] The present disclosure also provides nutritional supplements obtainable by any of the preparation methods taught herein.

[0086] In a third aspect, the present disclosure more specifically relates to a method for feeding shrimp larvae or fish larvae, comprising feeding the shrimp larvae or fish larvae with a nutritional supplement as taught herein and at least one wet feed or dry feed, such as a complete wet feed or a complete dry feed. As used herein, a wet feed contains more than 10% by weight of water based on wet weight. A dry feed contains 10% or less by weight of water based on wet weight. Complete wet feeds and complete dry feeds are diets that cover all the nutrients required for healthy growth of shrimp or fish at a specific life stage. Suitable wet feeds and dry feeds are commercially available. Those skilled in the art know how to select suitable wet feeds or dry feeds to be fed with the nutritional supplements taught herein.

[0087] In a fourth aspect, the present disclosure relates to a method of increasing the survival rate of shrimp larvae or fish larvae, wherein the method comprises the step of co-feeding a nutritional supplement as taught herein with at least one wet feed or dry feed.

[0088] In a fifth aspect, the present disclosure more specifically relates to a method for increasing the total biomass of shrimp larvae or fish larvae, the method comprising the step of feeding the nutritional supplement taught herein with at least one wet feed or dry feed. During the hatching period, the nutritional supplement can be fed with at least one wet feed or dry feed.

[0089] In a sixth aspect, the present disclosure more specifically relates to a method of increasing the growth of shrimp or fish larvae, wherein the method comprises the step of feeding a nutritional supplement as taught herein with at least one wet feed or dry feed. During the hatching period, the nutritional supplement may be fed with at least one wet feed or dry feed. Increased growth may be observed during the nursery period because the nutritional supplement as taught herein is fed with at least one wet feed or dry feed during the hatching period. Therefore, when the larvae emerge from the hatching period, they may have better health conditions that continue into the next developmental stage when fed the nutritional supplement as taught herein and at least one wet feed or dry feed.

[0090] In a seventh aspect, the present invention more specifically relates to a method for reducing microorganisms (such as harmful microorganisms, fungi and viruses) in fish or shrimp larvae culture, the method comprising the step of feeding the nutritional supplement taught herein with at least one wet feed or dry feed. The reduction in microorganisms is compared to the use of live feed. Without wishing to be bound by theory, it is assumed that the reduction in the number of microorganisms observed during the culture process is due to the absence of live feed known to carry microorganisms during the culture process.

[0091] In an eighth aspect, the present disclosure more particularly relates to a method of reducing waste in fish or shrimp larvae culture, the method comprising the step of co-feeding a nutritional supplement as taught herein with at least one wet feed or dry feed.

[0092] In a ninth aspect, the present disclosure more particularly relates to a nutritional supplement as taught herein for improving the survival rate of shrimp larvae or fish larvae.

[0093] In another aspect, the present disclosure provides use of a nutritional supplement as taught herein for preparing a composition for improving the survival rate of shrimp larvae or fish larvae.

[0094] The nutritional supplement and the at least one wet feed or dry feed may be fed to the shrimp or fish larvae at the same frequency or at different frequencies, for example, such that the nutritional supplement is fed to the shrimp or fish larvae at a lower frequency than the at least one dry feed or wet feed. The amount of the nutritional supplement that can be fed per day depends on the larval stage of the fish or shrimp larvae. The same is true for the amount of the at least one wet feed or dry feed. During the hatching period, the nutritional supplement may be fed together with the at least one wet feed or dry feed.

[0095] BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1A -B shows the survival rate of shrimp larvae at stage PL12 (%; Figure 1A ) and average weight

[0097] (mg; Figure 1B ), the shrimp larvae were fed dry diet alone (DD), or dry diet with Artemia (DD-ART), pasteurized ALF (DD-ALFP), or non-pasteurized ALF (DD ALF). Four bar graphs for each treatment describe the results of each replicate;

[0098] Figure 2A -C shows the survival rate of shrimp larvae at stage PL12 (%; Figure 2A ) and average weight

[0099] (mg; Figure 2B ), the shrimp larvae were fed dry feed (DD) alone or in combination with Artemia (DD ART), or ALF (DD ALFF and DD ALFV, respectively; ALFF was fed a fixed daily ration,

[0100] whereas ALFV was fed with a variable daily ration from Z3 / M1 to PL4). Figure 2C

[0101] The relationship between survival rate and larval weight is depicted;

[0102] Figure 3A -B shows the survival rate of shrimp in the nursery fed with DD ART or DD ALF during hatchery (%; Figure 3A ) and average weight (g; Figure 3B ). All shrimp received a commercial nursery diet during the nursery period;

[0103] Figure 4A -B shows the molting distribution of shrimp two days after the mysid stage ( Figure 4A ) and the corresponding survival rate ( Figure 4B );and

[0104] Figure 5A -B shows the difference in Vibrio colony formation in the ALF treatment group ( Figure 5A ) and the difference between the waste obtained after siphoning ( Figure 5B ). Example

[0105] In the examples, it was investigated whether some physical characteristics of Artemia can be mimicked by a simplified artificial live feed (ALF) as taught herein as a nutritional supplement. In addition, it was investigated whether ALF can completely replace Artemia in the larval diet. It was further investigated whether the combined use of commercial dry feed (DD) and the nutritional supplement (ALF) taught herein can serve as an alternative solution to the existing shrimp and / or fish larvae weaning strategy using Artemia as a co-feed with commercial dry feed.

[0106] Important beneficial features can be added to overcome some of the bottlenecks in live feed use. These beneficial features include, but are not limited to:

[0107] -- ALF uses a wider range of particle sizes than live feeds;

[0108] - Use hygienic and controlled production processes to reduce biohazards of live feeds in clinical settings;

[0109] - Use products that are available off the shelf, as opposed to live feeds that need to be scaled up, maintained sterile and enriched;

[0110] - Products that are naturally buoyant and uniform in the water column make dosing and control easier than live feeds that attract light, concentrate, grow (Artemia), or even multiply (rotifers) and are propagated in larval shrimp or fish tanks.

[0111] Since ALF is not a complete feed, but more of a physical imitation of a live feed, it is preferably used within a limited time frame. Since it is fed with a dry diet that provides all the nutrients required by the larvae, replacing Artemia with ALF does not result in underfeeding. In the example of P. vannamei, ALF was used during the critical molting period M3-PL5 at a dose of 0.5-3 kg per million shrimp produced. For fish and other shrimp, ALF can be used at the same or different stages, with similar or different dosages.

[0112] diet

[0113] The dry feed (DD) used in Examples 1-6 below was a commercially available dry feed for feeding shrimp M3-PL5 during the molting period.

[0114] ALF

[0115] To obtain the artificial live feed (ALF) / nutritional supplement taught herein, a gel mixture comprising agar, water, marine protein hydrolysate, astaxanthin, DHA and lecithin is spray-coagulated into granules, which are then wet-sieved to obtain a particle size of 50-200 μm. The spray-coagulated granules comprise 2 wt% agar, 5 wt% attractant (on a dry matter basis; in the form of marine protein hydrolysate), 0.2 wt% DHA, 0.5 wt% lecithin, 0.2 wt% astaxanthin and 92.1 wt% water. The granules are stored in a saline solution containing NaCl (salinity of 150 ppt) to form a nutritional supplement. The weight ratio of granules to saline solution is 1:1. The nutritional supplement is thus provided to shrimp larvae. The nutritional supplement (comprising granules and saline solution in a weight ratio of 1:1) contains 78% water, 14% ash, 4% fat, 3% carbohydrates and about 1% protein. The granules were pasteurized at 60°C for 60 minutes to obtain ALFP.

[0116] Feeding

[0117] Shrimp trials were conducted in 60-liter tanks in four replicates. For the hatchery period, shrimp were stocked at the nauplius stage at a density of 130 nauplii per liter.

[0118] From Z3 / M1 to PL6, Artemia or ALF were fed 6 times a day.

[0119] The water temperature was maintained at 29°C ± 1°C; the oxygen was 4.5 mg / l ± 0.5 mg / l, and the pH was 8.2 ± 0.2. The water was full concentration seawater (35 ppt).

[0120] Example 1

[0121] The trial included 4 treatments:

[0122] DD ART: Dry Diet and Artemia

[0123] DD: Dry Diet

[0124] DD ALFP: Dry Diet + Pasteurized ALF

[0125] DD ALF: Dry diet + ALF

[0126] Compared with feeding the dry diet alone, feeding the dry diet with Artemia, pasteurized ALF, or ALF resulted in a higher survival rate ( Figure 1A ) and average body weight ( Figure 1B ) was improved. No statistically significant differences were found between DD ART and DD ALFP and DD ALF feeding methods. Therefore, ALFP and ALF are suitable alternatives to ART.

[0127] Example 2

[0128] It was tested whether co-feeding a dry diet with a fixed daily feed ration (ALFF) or a variable daily feed ration (ALFV) would affect performance. In ALFF, the same dose was given from M2 to PL4. In ALFV, feeding started with a lower dose at Z3 / M1, reached a maximum at M3 / Pl1, and was gradually reduced until PL4. After PL4, larvae were fed only dry diet. The two ALF treatments were also compared with a treatment where Artemia and dry diet were co-fed (DD ART) and a treatment where dry diet was fed only (DD). The results are shown in Figure 2. Figure 2A -C. The ALF treatments (DD ALFF and DD ALFV) had the highest survival, followed by the Artemia treatment (DD ART). The treatment that received only dry diet (DD) performed poorly ( Figure 2A The weights of shrimp in the different treatments were relatively equal ( Figure 2B ). When survival and body weight are combined to understand the total biomass produced ( Figure 2C ), it is clear that the overall biomass production of the ALF treatment was better than that of the Artemia treatment and the dry diet only treatment.

[0129] Example 3

[0130] At the end of the hatchery (PL12), the shrimp were followed for 8 weeks in the nursery facility. They were reared with 350 postlarvae in two replicates, all of which received the same commercial diet containing 25% protein and 9% fat according to a fixed feeding regimen based on shrimp body weight.

[0131] Table 1 Relationship between daily feed ratio and shrimp body weight

[0132]

[0133] No Artemia or ALF were fed during this period. This period was used to evaluate the effects of hatchery dietary treatments (DD ART or DD ALF) on shrimp survival and biomass increase during the nursery or grow-out phases.

[0134] At the end of the nursery period, the survival rate of shrimp fed DD ALF during hatchery was significantly higher than that of shrimp fed DD ART during hatchery ( Figure 3A ) and average body weight ( Figure 3B This indicates that shrimp can be reared without any live feed and show excellent survival and growth performance in further development in nursery and grower ponds.

[0135] Example 4

[0136] The hatching experiment described in Example 2 was repeated. The experiment included four treatments (A, B, C and D) with four replicates for each treatment. All treatments received the same amount of dry diet and were supplemented with Artemia, except for treatment C where ALF replaced Artemia.

[0137] All treatments had good survival until the end of the mysid stage. Surprisingly, animals that received Artemia in addition to the complete diet experienced problems molting to the postlarval stage, while those that received the ALF treatment instead of Artemia experienced fewer molting problems ( Figure 4A Molting problems resulted in a gradual and rapid death in treatments A, B, and D ( Figure 4B ). This ultimately led to total mortality in these treatments at PL5, while all replicates in treatment C survived. Furthermore, animals that received ALF in addition to the complete diet reached the PL1 / PL2 stage earlier than animals that received Artemia in addition to the complete diet. The experiment ended at PL12, with approximately the same survival rates in all replicates of treatment C, while there were no shrimp in all other treatments. This suggests that ALF not only has properties that help shrimp overcome difficult developmental stages, but also provides a more hygienic and controlled production process, reducing the biohazards of live feeds.

[0138] Example 5

[0139] The incubation trial described in Example 4 was repeated in time with the addition of UV water filtration and daily tank siphoning. Each of the four treatments (A, B, C, and D) was again run in 4 replicates. Treatments A and B received the same dry diet as in Example 4, and treatments C and D also received the same dry diet, but from different batches.

[0140] All treatments received the same amount of dry diet and supplemented with Artemia except for treatments B and C where ALF replaced Artemia. Dietary consumption per million shrimp produced was calculated to be 3.5 kg dry diet, 2 kg Artemia eggs, and 1.3 kg ALF. During the transition from M3 to PL1, shrimp experienced difficulties in molting, but survival was not affected, with all treatments producing PL12 with survival rates between 40-45%. During incubation periods M3-PL5, the presence of Vibrio spp. was determined by placing incubation water samples on TCBS medium. Figure 5AIt was shown that the presence of Vibrio on the plates was more evident in the Artemia treatments compared to the ALF treatment. It was also observed that more Artemia debris was found during siphoning in treatments A and D compared to the treatment that received ALF ( Figure 5B ). This again suggests that live feeds such as Artemia can be replaced by ALF with the advantage of generating lower bacterial loads and less waste during larval rearing.

[0141] Example 6

[0142] To find the source of Vibrio contamination, a simplified test was conducted using beakers filled with seawater without shrimp. The test was conducted with 3 replicates and 5 treatments. The treatments included:

[0143] ——Control group (no feed)

[0144] - Dry diet

[0145] ——Dry diet and ALF

[0146] - Dry diet vs. cooked Artemia

[0147] - Dry diet vs. live Artemia

[0148] The concentration of each diet was equivalent to one day's feeding amount. The feed was kept suspended by ventilation. After one day, the precipitate was filtered out and analyzed by inoculating the contents of the filter on TCBS medium plates in triplicate. Vibrio was counted after incubation of the plates. No Vibrio was detected in all treatments except the treatment containing live Artemia (all three plates turned yellow due to the presence of Vibrio).

[0149] deal with Vibrio counts on TCBS plates Control group (no feed) --- Dry diet --- Dry Diet with ALF --- Dry Diet vs. Cooked Artemia --- Dry Diet vs. Live Artemia +++

[0150] Example 7

[0151] ALF was also co-fed with a commercial diet (GemmaMicro, manufactured by Skretting) to sea bass larvae and zebrafish larvae. ALF was ingested by fish larvae (sea bass and zebrafish) as observed by imaging (presence of ALF in the larval gut is visible in red, while presence of GemmaMicro in the larval gut is visible in green). Fish larvae were apparently attracted to ALF. Based on excreta, ALF was observed to be ingested and digested.

[0152] It should be noted that the above embodiments illustrate rather than limit the present invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference symbols between brackets shall not be construed as limiting the claim. The use of the verb "comprise" and its variations does not exclude the presence of elements or steps other than those described in the claims. The English article "a" or "an" before an element does not exclude the presence of a plurality of such elements.

[0153] The mere fact that certain measures are recited separately in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

Claims

1. A nutritional supplement suitable for shrimp larvae or fish larvae, the nutritional supplement comprising: I) A granular gel structure comprising: a) agar; b) attractants; and c) water; and II) an aqueous medium comprising at least one salt.

2. The nutritional supplement of claim 1, wherein the nutritional supplement further comprises an ingredient selected from the group consisting of a colorant, omega-3 fatty acids, and phospholipids, or any combination thereof.

3. The nutritional supplement of claim 2, wherein the nutritional supplement comprises a colorant, omega-3 fatty acids, and phospholipids.

4. The nutritional supplement according to any one of the preceding claims, wherein the aqueous medium comprises at least 80 ppt of salt, preferably at least 80 ppt of sodium chloride.

5. A nutritional supplement according to any one of the preceding claims, wherein the particulate gel structure is in the form of gel particles, such as granules, preferably granules having a particle size of 1 μm to 2 mm, preferably 5 μm to 800 μm.

6. A nutritional supplement according to any one of the preceding claims, wherein the particulate gel structure, for example in the form of granules, has a density of 0.995 g / ml to 1.200 g / ml.

7. A nutritional supplement according to any one of the preceding claims, wherein the gel structure comprises 0.01 to 10 wt% agar on a wet weight basis.

8. The nutritional supplement of any one of claims 2 to 7, wherein the omega-3 fatty acid comprises docosahexaenoic acid (DHA).

9. The nutritional supplement of any one of claims 2 to 8, wherein the phospholipid comprises lecithin.

10. A nutritional supplement according to any one of claims 2 to 9, wherein the colorant is selected from carotenoids such as carotene or preferably lutein, carbon, clay, minerals, anthocyanins or colored algae such as red or green algae.

11. The nutritional supplement of claim 10, wherein the colorant is carotene.

12. The nutritional supplement of claim 10, wherein the colorant is astaxanthin.

13. Nutritional supplement according to any one of the preceding claims, wherein the aqueous medium comprises sodium chloride (NaCl) as salt, preferably 100 to 300 ppt of NaCl.

14. The nutritional supplement according to any one of the preceding claims, wherein the weight ratio between the particulate gel structure and the aqueous medium is from 0.2:1 to 1:0.

2.

15. A method for preparing a nutritional supplement suitable for co-feeding to shrimp larvae or fish larvae, wherein the method comprises the following steps: i) coagulating a gelling mixture comprising agar, water and an attractant, and optionally omega-3 fatty acids, phospholipids and a colorant, to form a particulate gel structure, such as granules; ii) optionally, wet sieving the particulate gel structure, such as granules, to obtain particulate gel structures, such as granules, of appropriate size; as well as iii) adding the particulate gel structure, such as particles, to an aqueous medium comprising at least one salt to obtain a nutritional supplement suitable for ingestion by shrimp larvae or fish larvae.

16. A nutritional supplement obtainable by the method of claim 15.

17. A method for feeding shrimp or fish larvae, increasing the growth of shrimp or fish larvae, increasing the total biomass of shrimp or fish larvae, reducing microorganisms in fish or shrimp larvae culture, and / or reducing waste in fish or shrimp larvae culture, wherein the method comprises feeding the shrimp or fish larvae with a nutritional supplement as described in any one of claims 1 to 14 or 16 and at least one wet feed or dry feed (e.g., a complete wet feed or a complete dry feed).

18. The method of claim 17, wherein the nutritional supplement and the at least one wet feed or dry feed are fed to the shrimp or fish larvae at different frequencies, such that the nutritional supplement is fed to the shrimp or fish larvae less frequently than the at least one wet feed or dry feed.

19. The method according to claim 17 or 18, wherein the nutritional supplement is co-fed with the at least one wet feed or dry feed during the incubation period.

20. The nutritional supplement of any one of claims 1 to 14 or claim 16, for use in improving the survival rate of shrimp larvae or fish larvae.

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