Nutritional Formulas and Uses Thereof

Abstract

The present invention relates generally to nutritional compositions for the development of aquatic crustacean and fish species, and more particularly to nutritional compositions comprising n-3 and n-6 fatty acids and live Artemia for tropical spiny rock lobsters.

Description

[Technical Field]

[0001]

[0001] The present invention relates generally to nutritional compositions for the development of aquatic crustacean and fish species. [Background technology]

[0002]

[0002] Understanding and providing the key nutritional requirements for the full development of larval aquatic crustacean and fish species presents significant challenges to the development of robust commercial aquaculture. An example of this is the tropical spiny rock lobster, Panulirus ornatus, which has recently commanded high market prices primarily due to increased consumer demand and scarcity of wild stocks (due in part to overfishing). While these crustacean species possess many favorable characteristics with respect to the commercial aquaculture industry, they also present many challenges due in large part to the nutritional demands imposed on species undergoing continuous molting cycles and morphological changes.

[0003]

[0003] Similarly, most decapod crustaceans have complex life cycles characterized by a series of molts and morphological transformations that occur over a pelagic larval stage. Nutrition has a major impact on the timing and success of these complex processes, greatly determining the success of the larvae and their likelihood of reaching the juvenile stage. Because the incubation period represents a critical bottleneck for the establishment of a commercial aquaculture sector for palinurid lobsters, understanding the requirements for molting and complete larval development in cultured crustaceans is an important prerequisite for commercial viability.

[0004]

[0004] Up to 24 morphological stages have been documented for the Japanese shrimp, which are typically divided into 11 distinct stages by the identification of specific morphological milestones. The complete molting cycle is divided into four recurring stages: prometamorphosis (pre-molt), which begins with the process of peeling the epidermis from the cuticle; molt, in which the old cuticle is removed; post-molt, which involves the absorption of water and the accumulation of storage organic materials; and the period leading up to the next molt (inter-molt), which is the major period of tissue growth.

[0005]

[0005] Healthy phyllosoma undergo multiple molts throughout the larval life cycle, enabling growth and regeneration. Conversely, phyllosoma that are poorly nourished are expected to exhibit sporadic and incomplete molting processes, leading to poor growth, susceptibility to disease, failure of metamorphosis, and ultimately death. While some understanding exists of the nutritional requirements of benthic juvenile phyllosoma, this information is scarce regarding the pelagic larval stage, which is critical for developing production in this species' closed life cycle. Successful phyllosoma development and metamorphosis are highly dependent on both the efficient use of stored energy and the accumulation of nutrients necessary to reconstruct viable juveniles. Larval lobsters likely have specific nutritional requirements, but their quantitative requirements and available forms are largely unknown. As important nutritional components, total lipid content, the proportions and composition of individual lipid classes, and their constituent fatty acids can affect phyllosoma health, development, and physiology. Lipids are also thought to have a significant impact on the molting process, and many studies have observed that total lipid content in larval crustaceans follows a cyclical pattern during each molt cycle. This is seen as an accumulation of lipids throughout the inter- and pre-molt stages, followed by a decrease in lipids during the post-molt stage. Although the energetic cost of molting is small compared to that of growth and metabolism, its successful completion is crucial for larval development.

[0006]

[0006] Adequate lipid nutrition with a specific lipid class profile has been shown to accelerate larval development and increase the likelihood of successful metamorphosis. Phospholipids are thought to be essential for cell membrane integrity and cholesterol transport during molting, improved growth, and successful metamorphosis. Deficiencies in the fatty acids eicosapentaenoic acid (20:5n-3, EPA), docosahexaenoic acid (22:6n-3, DHA), and arachidonic acid (20:4n-6, ARA) have been reported to cause poor survival, a longer intermolt period, and a narrower carapace width in crustacean larvae. Understanding the nutritional requirements during larval development is essential for establishing an optimal diet for the phyllosoma of the larval shrimp. Researchers have focused on the chemical composition of the phyllosoma immediately before and after molting (i.e., pre- and post-molt) to gain insight into the natural nutritional trends during the larval molt cycle. However, the Phyllosoma of later spiny rock lobsters also appear to undergo dietary changes.

[0007]

[0007] The temperate spiny lobster, Jasus edwardsii, often undergoes periods of variable larval quality when kept in captivity. Summary of the Invention [Problem to be solved by the invention]

[0008] What is needed in the industry is a complete nutritional formula (or feed) that overcomes one or more of the shortcomings of prior art formulas and achieves a high degree of metamorphosis success, particularly in crustacean species, particularly hatchery-produced crabs and clawed lobsters. [Means for solving the problem]

[0009] The present invention is based on the discovery that a high rate of successful metamorphosis can be achieved by formulations having specific combinations of ingredients. Specifically, the present invention increases juvenile survival and advances development beyond the juvenile molt. The compositions disclosed herein allow for the inclusion of ingredients that can alter the form and balance of nutrients provided.

[0010]

[0010] Thus, in one aspect, the present invention provides a method for manufacturing a semiconductor device comprising: (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source; (ii) live Artemia (non-enriched); (iii) at least one sterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant The present invention provides a nutritional formulation comprising:

[0011] In one embodiment, the live Artemia (unenhanced) is a species at the nauplius developmental stage. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a photograph showing the individual components of an extruder gun used to produce noodle-shaped formulations according to one embodiment of the present invention. [Figure 2] FIG. 2 is a photograph showing an extruder gun used to produce noodle-shaped formulations according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0014] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0015]

[0015] In accordance with the present invention, the first component of the nutritional formula is an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source. The term "n-3 fatty acids," as used herein, are also referred to as omega-3 or omega-3 fatty acids and are characterized as polyunsaturated fatty acids (PUFAs) having a double bond at the third carbon atom from the end of the carbon chain. The term "n-6 fatty acids," as used herein, are also referred to as omega-6 or omega-6 fatty acids and are characterized as polyunsaturated fatty acids (PUFAs) having a double bond at the sixth carbon atom from the end of the carbon chain. Common n-3 and n-6 fatty acids include:

[0016] [Table 1]

[0017]

[0016] In the context of the present invention, the most common n-3 fatty acids are EPA and DHA, with relatively small amounts of n-6 fatty acids present entirely in fish oil. However, in the context of the present invention, the inventors have discovered that esterifying fatty acids in such a high-phospholipid source provides benefits in achieving high survival rates and sustainability of crustaceans during metamorphosis. Specifically, this benefit has been found to be realized using a source of marine fatty acids with a high phospholipid:low triacylglycerol (TAG) ratio. For example, fish oil, a convenient marine source of EPA, DHA, and ARA, incorporates these fatty acids into TAG or ethyl esters. Thus, fish oil generally has a higher TAG content. In contrast, krill oil (an alternative source of n-3 / n-6 fatty acids) has esterified EPA, DHA, and ARA in phospholipids, specifically phosphatidylcholine. Thus, krill oil is a convenient source of esterified n-3 and n-6 fatty acids with a high phospholipid:low TAG ratio. Another possible source is squid skin fat, which has a higher ratio of phospholipids to TAGs.

[0018]

[0017] Thus, as used herein, the term "effective" refers to an amount capable of increasing the viability (survival) of developing crustacean larvae during metamorphosis in nutritional formulations utilizing fish oil as a source of fatty acids. This may also include additional saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), such as n-9 fatty acids, highly unsaturated fatty acids (HUFAs), and other PUFAs (e.g., long-chain PUFAs). These fatty acids may contribute as an important additional energy source and may be provided by additional known animal and plant sources.

[0019] In one embodiment, the nutritional formula of the present invention comprises about 0.5-5% wt / wt krill oil, for example, about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8 or about 4.9% wt / wt krill oil. In one embodiment, the formulation is prepared by first forming an emulsion incorporating krill oil (or other source of desired fatty acids). The krill oil component may comprise about 15-25% of the emulsion. The emulsion may comprise about 6-15% of the final formulation (i.e., in the range of about 0.97-3.3 to 0.97-3.8 wt / wt%).

[0020] The formulations of the present invention also incorporate live (unfortified) Artemia as an ingredient. Artemia is a genus of aquatic brine shrimp. In one embodiment, the nutritional formulations of the present invention utilize Artemia species in the nauplius developmental stage. This invention should be distinguished from other feed compositions that contain adult Artemia that have been fortified with beneficial nutritional products, such as phospholipids, ascorbic acid (AA), and antibiotics, prior to formulation. Artemia species in the nauplius developmental stage are an example of live, unfortified Artemia species. In one embodiment, the nutritional formula of the present invention comprises about 3-60% wt / wt live Artemia (unfortified), for example, about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 to about 59% wt / wt live Artemia (unfortified).

[0021] As a third component, the formulation includes at least one sterol. In one embodiment, the at least one sterol is cholesterol. "Sterol," as used herein, includes known steroid alcohols. In one embodiment, the sterol is an animal sterol. In one embodiment, the nutritional formulation of the present invention includes about 0.05-0.5% wt / wt of a sterol. In one embodiment, a sterol, such as cholesterol, is incorporated into the aforementioned emulsion. Cholesterol may be added at about 1.5% of the emulsion.

[0022]

[0021] As an additional component, the formulation includes at least one binder, which in one embodiment is alginate (alginic acid), an anionic polysaccharide derived from the cell walls of brown algae. One additional benefit of using alginate as a binder is that it also serves as an immunostimulant. In one embodiment, the nutritional formulation of the present invention includes about 0.2-3% wt / wt, e.g., about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or about 2.9% wt / wt of at least one binder. In one embodiment where the binder is alginate, the alginate is provided as a liquid (aqueous) extract at a concentration of about 1-4%. In such an embodiment, the % amount of such extract in the formulation may range from 15-25% wt / wt of the total composition.

[0023] As a further component, the formulation includes at least one emulsifier. The emulsifier, in one embodiment, is lecithin. In another embodiment, the emulsifier is gum arabic. In one embodiment, the at least one emulsifier in the formulation is present, for example, in a range of about 0.2-0.8% wt / wt of the total formulation, or about 0.3, 0.4, 0.5, 0.6, or 0.7% wt / wt.

[0024]

[0023] As a further component, the formulation includes at least one mineral component. Suitable mineral components can be selected from calcium, chloride, phosphorus, potassium, iodine, sodium, zinc, magnesium, sulfur, iron, cobalt, selenium, chromium, manganese, and copper. The mineral component is typically provided as an inorganic salt or inorganic compound. For example, in one embodiment, the formulation includes CaCl2.

[0025]

[0024] Mineral components are typically added to the formulation as a salt component during manufacturing. In one embodiment, at least one mineral component is added to the emulsion at about 0.5% of the emulsion; this about 0.5% is about 0.064% of the total formulation. In this regard, the at least one mineral component in the formulation is present in a range of about 0.04 to 0.1% wt / wt of the total formulation, e.g., about 0.05, 0.06, 0.07, 0.08 to about 0.09% wt / wt.

[0026]

[0025] As a further component, the formulation includes at least one vitamin. Added vitamins include ascorbic acid (vitamin C), vitamin A, vitamin B complex, vitamin D, and / or vitamin K. In one embodiment, at least one vitamin is also added to the emulsion in an amount of about 1.2% of the emulsion. In this regard, the at least one vitamin component is preferably present in the formulation in an amount ranging from about 0.05% to 0.5% wt / wt of the total formulation, e.g., 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, Preferably, the hydroxybenzoate is about 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48 or about 0.49% wt / wt.

[0027] As a further component, the formulation includes at least one antioxidant. A preferred antioxidant is the beta-carotenoid astaxanthin, sold under the trade name Carophyll® Pink (Roche). In one embodiment, Carophyll® Pink is added to the emulsion at about 0.4%. In this regard, the at least one antioxidant is preferably present in the formulation in a range of about 0.01 to 0.1% wt / wt of the total formulation, for example, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or about 0.09% wt / wt. In a further embodiment, the present invention also includes a color pigment, which may also be an antioxidant, such as Carophyll® Pink.

[0028] As an additional component, the formulation includes at least one probiont source. Preferred probiont sources include Sanolife®. In one embodiment, the probiont source is in the form of an activated solution in the formulation, within the range of about 1-2% wt / wt of the total formulation, e.g., 1.1, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or about 1.9% wt / wt. The inventors have found that the inclusion of a probiont source aids in the prevention of white gut disease, a disease of bacterial origin, with the primary site of infection being the midgut of early- to mid-stage Phyllosoma. The disease can be rapidly transmitted between individuals, potentially infecting an entire tank. Once an outbreak occurs, established antibiotic treatment protocols are ineffective in controlling the disease.

[0029] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) at least one sterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes.

[0030] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes.

[0031] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier, including lecithin; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes.

[0032] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral including calcium chloride; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes.

[0033] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral including calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant Includes.

[0034] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral including calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant, including astaxanthin Includes.

[0035] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in the formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes.

[0036] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes:

[0037] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in the formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes:

[0038] In one embodiment, the nutritional formula comprises: (i) an effective amount of n-3 and n-6 fatty acids esterified in a high phospholipid source that is krill oil, present in the formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes:

[0039] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in the formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral including calcium chloride; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Includes:

[0040] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in the formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral including calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant Includes:

[0041] In one embodiment, the nutritional formula comprises: (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in the formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier, including lecithin; (vi) at least one mineral including calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant, including astaxanthin Includes:

[0042] The present invention also contemplates nutritional formulations (or feeds) that include one or more of the following additional ingredients: (i) Antibiotics, which can help control conditions such as white leg. Suitable antibiotics include enrofloxacin and cyprofloxacin (e.g., at 0.5%); (ii) free amino acids, as an additional source of energy; (iii) other proteins and peptides, such as gelatin, bovine serum albumin, and caseinates; (iv) carbohydrates and their derivatives, such as cellulose, maltose, chitosan, and gellan; (v) chemoattractants, such as glycine (also a free amino acid); (vi) additional color pigments / dyes, which may be of assistance in having attractant capabilities; and (vii) pH buffering agents, such as citric acid and sodium bicarbonate.

[0043] The nutritional formula of the present invention is preferably produced in the form of extruded noodles with diameters ranging from about 0.3 to 1.0 mm, e.g., about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and about 1.0 mm. When not utilized as a feed, the formula is preferably maintained at a temperature of about 4°C or less. The present invention contemplates the preparation of a feed (nutritional composition) by first forming an emulsion using many of the ingredients discussed above. The emulsion is then added to the remainder of the ingredients to form a feed, which is then extruded to form the final nutritional formula. The inventors have found that when the emulsion comprises more than about 12% of the total formula and the formula is in the form of noodles, the nutritional formula remains neutrally buoyant in the feed tank, which is advantageous for the survival of the larvae during rearing.

[0044] In a further embodiment, the feed formulation is in the form of a noodle having neutral buoyancy, which means that the feed formulation has a slow falling rate, i.e., a falling rate within the range of about 0.02 to 2 cm / sec, e.g., about 0.02, 0.05, 0.07, 0.09, 0.12, 0.15, 0.17, 0.19, 0.20, 0.22, 0.25, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.4 1.6, 0.48, 0.50, 0.55, 0.58, 0.60, 0.64, 0.66, 0.70, 0.75, 0.78, 0.80, 0.85, 0.87, 0.90, 0.92, 0.94, 0.98, 1.0, 1.1, 1.15, 1.2, 1.3, 1.35, 1.4, 1.5, 1.55, 1.6, 1.7, 1.75, 1.8, 1.85, 1.9, or about 2 cm / sec (or a range created by any two of the above numbers). The hydrodynamics of the water flow in the culture vessel are such that the feed and phyllosoma are evenly dispersed throughout the water column, allowing for feeding by the phyllosoma "bumping" into the feed, as the phyllosoma are not actively feeding on their food source / prey. Thus, another advantage of the feed formulation of the present invention is that it behaves more like feed in its natural environment, increasing the contact generation time, thereby allowing for increased feed uptake and increased generation.

[0045] To maintain palatability and increase the nutritional value of the formula, it is an important feature to maintain the pH of the nutrient formula between about 5 and about 10, for example, about 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or about 10.

[0046]

[0045] Therefore, the present invention also provides (i) an effective amount of esterified n-3 and n-6 fatty acids in a high-phospholipid source; (ii) live Artemia (unenriched); (iii) at least one sterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant Also provided is an extruder gun device for extruding a noodle-shaped nutritional formula comprising:

[0047] In order that the present invention may be readily understood and put into practical effect, certain preferred embodiments will now be described by way of the following non-limiting examples.

[0047] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0048]

[0048] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the application of the present invention.

[0049] Throughout this specification, it is the intent to describe preferred embodiments of the present invention without limiting the invention to any one embodiment or particular group of features. Thus, those skilled in the art will appreciate, in light of this disclosure, that various modifications and changes can be made in the specific embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims. [Example]

[0049]

[0048] Formulation Examples (in noodle form)

[0050] [Table 2]

[0051]

[0049] Preparation Examples Alginate solution 1. To make the alginate solution, add alginate to deionized water. 2. Stir the alginate solution into the water until well mixed. 3. Warm the alginate solution in a water bath.

[0052] Wet whole ingredients Live Artemia nauplii (unenhanced) 1. Decant the required volume of live Artemia nauplii from a bucket onto a clean, disinfected 50 micrometer mesh screen. 2. Rinse the Artemia nauplii with deionized water to rinse the saltwater from the nauplii. 3. Prepare trace mineral mix.

[0053] [Table 3]

[0054] 4. Preparing Mineral Mix TRL M2

[0055] [Table 4]

[0056] Wet Premix The moistened premix can be used as one of the main components of a diet. For single feeds, follow standard formulation procedures. 1. Weigh out the alginate into a mixing bowl. 2. Measure out the choline chloride solution into a mixing bowl. 3. Measure out deionized water into a mixing bowl. 4. Weigh out the autoclaved algae paste into a mixing bowl. 5. Measure out the probiotic solution into a mixing bowl. 6. Mix gently with a spatula until all ingredients are evenly combined.

[0057] Preparation of lecithin-cholesterol paste 1. Measure out the required amount of liquid lecithin into a beaker. 2. Weigh out the required amount of cholesterol powder into the same beaker. 3. Leave the solution on the hot plate. 4. Stir the solution regularly to incorporate the cholesterol into the lecithin.

[0058] Preparation of nutrient solution: 5. Measure out the required amount of 10% deionized water into a clean beaker. 6. Add the required amount of Carophyll Pink to the beaker. 7. Mix in the Carophyll Pink. 8. Add the vitamin mix, mineral mix, and vitamin C to the beaker. 9. Mix the nutrient solution until all ingredients are completely dissolved. 10. Add krill oil to the mix and stir gently. 11. Add the lecithin-cholesterol paste to the beaker.

[0059] Dry Premix

[0060] [Table 5]

[0061] Prepare a trace mineral mix.

[0062] [Table 6]

[0063] Prepare the mineral mix, i.e., TRL M2.

[0064] [Table 7]

[0065] emulsion Krill Oil: Emulsion KOv3 component: Deionized water Calofil® Pink Vitamin Mix (TRL-V2) * No choline chloride Mineral Mix (TRL-M2) Rovimix 35 ("Vitamin C") Krill oil.

[0066] Cholesterol-lecithin paste (1:2 ratio) Preparation of cholesterol-lecithin paste 1. Measure out the required amount of liquid lecithin into a beaker. 2. Weigh out the required amount of cholesterol powder into the same beaker. 3. Leave the solution on a hot plate. 4. Stir the solution regularly to incorporate the cholesterol into the lecithin as it softens.

[0067] Preparation of emulsion KOV3: 1. Measure out deionized water into a beaker. 2. Add the required amount of Carophyll Pink, Vitamin Mix, Mineral Mix and Lovimix 35 to the deionized water and mix. 3. Measure the krill oil into a mixing bowl. 4. Add one portion of the nutrient and water mixture and mix. 5. Add another portion of the nutrient and water mixture and mix further. 6. Add cholesterol:lecithin paste and mix. 7. Once the cholesterol:lecithin mix is fully incorporated into the oil and water solution, gradually add the rest of the water while continuing to mix the emulsion.

[0068] Bath settings Preparation of the bath setup: 1. Add calcium chloride (CaCl2) to a carboy (0.2%). 2. Fill each carboy with 1 μm filtered seawater. 3. Autoclave the carboy using a large autoclave.

[0069] Krill Oil: Emulsion KOv2 component: The total emulsion volume required is multiplied by the percentage content of each ingredient to determine the content. Deionized water Calofil® Pink Vitamin Mix (TRL-V2) * No choline chloride Mineral Mix (TRL-M1) Robimix 35 Krill oil Cholesterol / lecithin paste (1:2 ratio).

[0070] Preparation of lecithin-cholesterol paste 1. Measure out the required amount of liquid lecithin into a beaker. 2. Weigh out the required amount of cholesterol powder into the same beaker. 3. Leave the solution on a hot plate. 4. Stir the solution regularly to incorporate the cholesterol into the lecithin as it softens.

[0071] Preparation of nutrient solution: 1. Measure out the required amount of deionized water into a clean beaker. 2. Add the required amount of Carofil® Pink to the beaker. 3. Mix the carophyll pink until completely dissolved. 4. Add Vitamin Mix, Mineral Mix and Robimix 35 to the beaker. 5. Mix the nutrient solution. 6. Add the lecithin-cholesterol paste to the beaker. 7. Add krill oil to nutrient mix and stir.

[0072] Preparation of emulsion KO: 1. Cover the pre-emulsion with plastic wrap and allow to cool. 2. Place the beaker in an ice-water bath. 3. Sonicate the emulsion three times, vigorously stirring the emulsion between each burst of sonication. 4. Place the completed emulsion in a clean, sterile container.

[0073] NN8V3-PB (compound) Preparatory work: 1. Hydrate the probiotic powder and place it in a 30°C water bath. 2. Place all extruder parts (die, chamber, sleeve, plunger, O-rings) in a 10% chlorine bath for sanitization. 3. Recover emulsion KOV3. 4. Prepare a premix in a container sized appropriately for the total premix volume. Add the alginate, choline chloride solution, and algae paste and stir thoroughly. Once the probiotic is fully activated, add the probiotic solution to the remainder of the premix and stir thoroughly again.

[0074] Preparation of Artemia nauplii: 5. Carefully pour the live Artemia nauplii from the bucket onto a clean, sanitized screen. 6. Rotate the screen to remove most of the seawater. 7. Rinse the Artemia nauplii with deionized water.

[0075] Preparation of LNN8V3 premix: 8. Measure out the probiotic premix solution. 9. Measure out the deionized water. 10. Weigh out the emulsion KOV3. 11. Add the required number of drops of acetic acid to the mixing bowl. 12. Mix.

[0076] Addition of Artemia nauplii to LNN premix: 13. Carefully weigh out the dried Artemia nauplii and place them directly into a stainless steel mixing bowl. 14. Mix. 15. Check the pH of the final food mix. Adjust if necessary.

[0077] Extrusion of the compound: 16. Assemble the plunger by installing the two O-rings. 17. Attach a plunger to one end of the chamber, invert the chamber on the bench, and use the tubing to push the plunger to the other end. 18. Turn the chamber upside down and assemble the throttle using the O-ring and secure with the sleeve. 19. Attach the chamber to the arm. 20. Attach the compressed air line to the throttle assembly. 21. Adjust the regulator. 22. Fill the extruder with the required amount of food mixture. 23. Assemble the die, spray it with ethanol, and fasten it to the sleeve. Place the O-ring and sleeve on the extruder and tighten until the rubber seal exerts pressure on it. 24. Pour the calcium chloride bath water into a large plastic tub. 25. Immerse the head of the large extruder into the calcium chloride bath and press the throttle. 26. While removing the noodle gun from the tank, press the pressure release lever.

[0078] Cutting and rinsing food: 27. Food length is cut to maximize feeding and efficient flushing. Extruder Gun There are two extruder guns (see Figures 1 and 2).

[0079] Compressed air is used to drive the extrusion plunger in the extruder gun, facilitating the extrusion of large batches of noodles. The air pressure is regulated to maintain a constant air pressure at the internal plunger, allowing for a consistent rate of noodle extrusion. This is important for obtaining consistent, well-formed noodles without exerting too much pressure on the live Artemia.

[0080] Follow these instructions for operation: 1. Assemble the plunger by installing the two O-rings. 2. Attach the plunger to the lubricated end of the chamber, invert the chamber on the bench, and use a plastic rod to push the plunger to the other end. Push the plunger back and forth until it slides freely within the chamber. Leave the plunger at the bottom end of the chamber. 3. Turn the chamber upside down and assemble the throttle using the O-ring and secure it with the metal sleeve. 4. Attach the extruder to an arm bracket mounted on the benchtop, in a position that will place it above the calcium seawater when inverted. 5. Attach the compressed air line to the throttle assembly. 6. Adjust the air pressure to approximately 6-10 psi using the air pressure regulator in the laboratory. 7. Fill the extruder with the food mixture until it is full. If the chamber is not full, carefully and slowly press the throttle to push the mixture up near the top of the chamber. Carefully stop before the mixture overflows the extruder. 8. Assemble the die into the die housing and spray it with ethanol. Place a rubber O-ring on the die housing, place it on top of the extruder, and tighten it until the rubber seal exerts pressure on it. 9. Use a large spatula to create a strong circular flow in the tank. 10. In one smooth motion, position the noodle gun at the top left of the container, submerge the noodle gun in the bath, and press the throttle while continuing to stir vigorously. Continue to press down on the lever while stirring throughout the extrusion until all of the noodle mixture has been extruded. 11. When removing the noodle gun from the tank, press the pressure release lever. 12. Repeat the process of filling the gun and extruding until all of the noodle mixture has been extruded. 13. Use single or multiple blade scissors to cut food to the appropriate length. 14. Drain the food onto the screen and rinse with tap water followed by deionized water. 15. Dry the food by placing the screen on a paper towel, then place in a container, then place in the refrigerator.

[0081] Comparison with fish oil formulation products When fish oil was the lipid source, tropical rock lobsters (TRL) phyllosoma grew throughout the larval stage, taking 150 to 180+ days to attempt metamorphosis. Successful transformation from the final phyllosoma larval stage to the puerulus larvae was 0% on the fish oil diet.

[0082] When krill oil was utilized as the lipid source (e.g., in the formulation of the present invention), larval development time was reduced from 90 to 120+ days. Successful transformation of the final phyllosoma larval stage to the puerulus larvae was 30-70%.

Claims

1. (i) an effective amount of esterified n-3 and n-6 fatty acids in a high phospholipid source; (ii) live Artemia (unenriched); (iii) at least one sterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant A nutritional formula comprising:

2. (i) an effective amount of esterified n-3 and n-6 fatty acids in a high phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) at least one sterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 10. The nutritional formula of claim 1, comprising:

3. (i) an effective amount of esterified n-3 and n-6 fatty acids in a high phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 3. The nutritional formula of claim 1 or 2, comprising:

4. (i) an effective amount of esterified n-3 and n-6 fatty acids in a high phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 4. The nutritional formula of claim 1, comprising:

5. (i) an effective amount of esterified n-3 and n-6 fatty acids in a high phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 5. The nutritional formula of any one of claims 1 to 4, comprising:

6. (i) an effective amount of esterified n-3 and n-6 fatty acids in a high phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant 6. The nutritional formula of any one of claims 1 to 5, comprising:

7. (i) an effective amount of esterified n-3 and n-6 fatty acids in a high phospholipid source that is krill oil; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant, including astaxanthin 7. The nutritional formula of any one of claims 1 to 6, comprising:

8. (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unenriched); (iii) cholesterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 8. The nutritional formula of any one of claims 1 to 7, comprising:

9. (i) an effective amount of esterified n-3 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 9. The nutritional formula of any one of claims 1 to 8, comprising:

10. (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 10. The nutritional formula of any one of claims 1 to 9, comprising:

11. (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 11. The nutritional formula of any one of claims 1 to 10, comprising:

12. (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin; (viii) at least one probiont source; and (ix) at least one antioxidant 12. The nutritional formula of any one of claims 1 to 11, comprising:

13. (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant 13. The nutritional formula of any one of claims 1 to 12, comprising:

14. (i) an effective amount of esterified n-3 and n-6 fatty acids in a phospholipid-rich source that is krill oil, present in said formulation at about 0.5-5.0% wt / wt of the total formulation; (ii) live Artemia (unaugmented) present in said formulation at about 30-60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one binder comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin, including vitamin C; (viii) at least one probiont source; and (ix) at least one antioxidant, including astaxanthin 14. The nutritional formula of any one of claims 1 to 13, comprising:

15. A nutritional formula according to any one of claims 1 to 14 in the form of noodles.

16. A nutritional formula according to any one of claims 1 to 14 in the form of noodles having a diameter of 0.3 to 1.0 mm.

17. A tropical spiny rock lobster fed with a composition according to any one of claims 1 to 16.

18. 18. The tropical spiny rock lobster of claim 17, which belongs to the species Panulirus ornatus.

19. 17. A method for achieving successful metamorphosis of a population of crustaceans in a hatchery environment, said method comprising feeding said population an effective amount of a formulation according to any one of claims 1 to 16.

20. 20. The method of claim 19, wherein the crustacean population is tropical spiny rock lobster, preferably belonging to the species Calanidae.

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