WO2020014741A1

WO2020014741A1 - Animal feed supplements and methods thereof

Info

Publication number: WO2020014741A1
Application number: PCT/AU2019/050748
Authority: WO
Inventors: Murray ISMAN; Rod Bradbury; Michael O'shea
Original assignee: Aquaculture Supplements Pty Ltd
Priority date: 2018-07-17
Filing date: 2019-07-17
Publication date: 2020-01-23

Abstract

The present invention relates to a feed supplement comprising an oil that comprises at least one triterpenoid and an oil that comprises 1,8-cineole. The feed supplement can assist in the growth of a subject.

Description

TITLE

Animal feed supplements and methods thereof

FIELD OF THE INVENTION

[0001 ] The present invention relates to the field of supplements. More particularly, the invention relates to a feed supplement. Most particularly, the invention relates to a supplement for fish feed.

BACKGROUND TO THE INVENTION

[0002] Any reference to background art herein is not to be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere.

[0003] Manufactured feeds form an integral part of commercial farming. In particular, commercial aquaculture requires feed to provide the adequate nutritional needs for farmed fish. The feed enables fish to grow to a commercial size and standard. However, a constant issue in aquaculture is the marketability of fish being hindered by pests, diseases and/or infection. Furthermore, it would be appreciated that uptake of feed is an important commercial factor.

[0004] Pests can lead to significant production and economic losses. For example, fish are susceptible to ectoparasite infections. In particular, Atlantic salmon ( Salmo salar ) are prone to sea louse infestations. Left untreated, ectoparasite infections can cause disease and developmental problems. Furthermore, the marketability of a fish can be adversely affected by ectoparasitic infections/infestations which can leave marks on the skin of a fish.

[0005] A typical solution in the art is to apply parasiticide directly to the water, commonly known as bath treatment. Bath treatment requires the administration of parasiticide (for example, organophosphates, hydrogen peroxide and/or pyrethroids) directly to the water, and the fish are treated through complete immersion for a pre-determined period. One major disadvantage, especially when administered using well boats, is that all the parasites are exposed to the same concentration of the parasiticide. Other disadvantages of bath treatments include being very labour intensive, loss of livestock during transfer of fish to well boats, and parasiticides being toxic and detrimental to the environment. Additionally, bath treatment requires a large amount of the parasiticide and can be expensive and inefficient.

[0006] There are also stresses and accidental fish mortalities associated with parasiticide treatments. In this regard, some fish cannot grow to their full size due to fasting before and/or after treatments. Furthermore, there is a risk that residual parasiticide may remain in the flesh of the fish. [0007] Similar disadvantages are faced in relation to disease and infection. In this regard, disease and infection can also lead to significant production and economic loss. Disease and infection can inhibit the growth of fish, and the amount of feed required may be increased due to the fish being diseased or infected. In general, it would be advantageous to have a composition that reduces the amount of feed required for fish growth, and/or can alleviate or ameliorate the problem of pasts, diseases and/or infection.

[0008] It would be advantageous to provide a composition or method which can alleviate or reduce some of the abovementioned problems, and/or at least provide a commercial alternative.

SUMMARY OF THE INVENTION

[0009] In one form, although it need not be the only or indeed the broadest form, the invention resides in a feed supplement comprising an oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole.

[0010] In one embodiment, the triterpenoid can be a tetranortriterpenoid. For instance, an example of a tetranortriterpenoid is azadirachtin.

[0011 ] In one embodiment, the triterpenoid is present as neem oil. Suitably, the neem oil is cold pressed neem oil. Preferably, the cold pressed neem oil is pharmaceutical grade cold pressed neem oil.

[0012] The % by weight amount of the neem oil is between about 30% and about 99%, or between about 50% and about 95%, or between about 60% and about 90%, or between about 75% and about 85%, or about 50%, or about 83%, of the feed supplement.

[0013] In an embodiment, 1 ,8-cineole forms greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80% or greater than about 85% of the oil.

[0014] In one embodiment, the oil that comprises 1 ,8-cineole is selected from the group consisting of eucalyptus oil and rosemary oil. Preferably, the oil is eucalyptus oil.

[0015] The % by weight of the eucalyptus oil is between about 1 % and about 40%, or between about 5% and about 30%, or between about 10% and about 25%, or between about 15% and about 25%, or about 10%, or about 17%, of the feed supplement.

[0016] In another embodiment, the feed supplement further comprises vitamin E and/or carotenoids. The vitamin E and carotenoids is present as palm oil. [0017] The % by weight of the palm oil is between about 0.1% and about 80%, or between about 10% and about 70%, or between about 20% and about 60%, or between about 30% and about 50%, or about 40%, of the feed supplement. Preferably, the palm oil is red palm oil.

[0018] The feed supplement is suitably in the form of a solid or a liquid. In one embodiment, the feed supplement is a solid feed supplement. Alternatively, the feed supplement is a liquid feed supplement.

[0019] In the embodiment that the feed supplement is a solid feed supplement, the feed supplement further comprises a solid substrate. The solid substrate is selected from the group consisting of caseinates and polysaccharides. Suitably, the solid substrate is selected from the group consisting of sodium caseinate and maltodextrin.

[0020] In another form, the invention resides in a fish feed composition comprising the feed supplement.

[0021 ] The % by weight amount of the neem oil is between about 0.1 % and about 20%, or between about 2% and about 10%, or between about 3% and about 7%, or between about 4% and about 6%, or about 5%, of the fish feed composition.

[0022] The % by weight of the eucalyptus oil is between about 0.1 % and about 10%, or between about 0.2% and about 5%, or between about 0.5% and about 4%, or between about 0.5% and about 2%, or about 1 %, of the fish feed composition.

[0023] The % by weight of the palm oil is between about 0.1% and about 20%, or between about 1 % and about 10%, or between about 2% and about 6%, or between about 3% and about 5%, or about 4%, of the fish feed composition.

[0024] In an embodiment, the fish feed composition further comprises a coating. The coating comprises further red palm oil and/or stearin and fish oil and/or fish hydrolase.

[0025] The % by weight of the further amount of palm oil and/or stearin is between about 20% and about 80%, or between about 30% and about 70%, or between about 40% and about 60%, or between about 45% and about 55%, or about 50%, of the coating.

[0026] The % by weight of the fish oil and/or fish hydrolase is between about 20% and about 80%, or between about 30% and about 70%, or about 40% and about 60%, or between about 45% and about 55%, or about 50%, of the coating.

[0027] The % by weight of the further amount of palm oil and/or stearin is between about 0.1 % and about 20%, or between about 0.5% and about 10%, or between about 1 % and about 5%, or between about 1 % and about 3%, or about 2%, of the fish feed composition. [0028] The % by weight of the further amount of fish oil and/or fish hydrolase is between about 0.1 % and about 20%, or between about 0.5% and about 10%, or between about 1 % and about 5%, or between about 1 % and about 3%, or about 2%, of the fish feed composition.

[0029] In another form, the invention resides in a method of treating or preventing pests, diseases and/or infections in a fish including the steps of:

administering a therapeutically effective amount of a fish feed composition comprising a feed supplement to the fish;

wherein the feed supplement comprises an oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole,

to thereby treat or prevent pests, diseases and/or infestations in the fish.

[0030] The feed supplement, fish feed composition, and components thereof, are as substantially described herein.

[0031 ] In another aspect, the invention resides in a method of preparing a feed supplement including the steps of:

mixing an oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole,

to thereby prepare a feed supplement.

[0032] The feed supplement and components thereof are as substantially described herein.

[0033] In another aspect, the invention resides in a method of incorporating a feed supplement into fish feed including the steps of:

providing fish feed;

contacting the fish feed with a feed supplement comprising an oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole.

to thereby form a fish feed composition comprising the feed supplement.

[0034] The fish supplement and components thereof are as substantially described herein.

[0035] In an embodiment, the fish feed composition further comprise a coating. The coating and components thereof are as substantially described herein. In one embodiment, the method further include the step of:

providing a coating; and

coating the fish feed composition with the coating.

[0036] In one aspect, the invention resides in a method of assisting and/or improving weight gain in a fish including the step of: feeding a fish feed composition comprising a feed supplement that comprises an oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole; to thereby assist or improve weight gain in the fish.

[0037] The various features and embodiments of the present invention referred to in the individual sections above and in the description which follows apply, as appropriate, to other sections, mutatis mutandis. Consequently, features specified in one section may be combined with features specified in other sections as appropriate.

[0038] Further features and advantages of the present invention will become apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] To assist in understanding the invention, the invention will be described by way of example only with reference to the accompanying figures, in which:

Figure 1 is a graph of total intake vs theoretical potential uptake of azadirachtin from fish tissue steeped in ethanol;

Figure 2 is a graph of total intake vs theoretical potential uptake of azadirachtin from fish tissue homogenised in ethanol;

Figure 3 shows the droplet temperature during traditional spray drying;

Figure 4 shows the droplet temperature during drying when utilizing an electrostatic effect;

Figure 5 shows a graph of average daily feed intake;

Figure 6 shows a graph of average weight gain as a function of diet;

Figure 7 shows a graph of specific growth rate as a function of diet; and

Figure 8 shows a graph of food conversion ratio (as a function of diet).

DETAILED DESCRIPTION OF THE INVENTION

[0040] Embodiments of the present invention reside primarily in a feed supplement. Accordingly, the methods and compositions have been described with only the specific details that are necessary for understanding the present invention, so as to not obscure the disclosure with excessive detail that will be readily apparent to those of ordinary skill in the art having the benefit of the present description.

[0041 ] In this specification, adjectives such as first, second, further and the like may be used solely to distinguish one element or action from another element or action without necessarily requiring or implying any actual such relationship or order. [0042] As used herein, the terms“comprises” and“includes” are intended to define a non-exclusive inclusion, such that a method or composition that comprises a list of elements does not include only those elements but may include other elements not expressly listed, including elements that are inherent to such a method or composition.

[0043] As used herein, the term‘about’ means the amount is nominally the number following the term ‘about’ but the actual amount may vary from this precise number to an unimportant degree.

[0044] As used herein, the % by weight amounts refer to the % by weight of the constituent relative to the respective composition (wt/wt) unless otherwise state.

[0045] It is postulated that the present invention assists in the growth of a subject. It is believed that this is achieved by increasing the uptake of nutrients of a feed and/or by treating and/or preventing pests, diseases and/or infection. The present invention is predicated, at least in part, on the finding that a feed supplement can be utilized to treat and/or prevent pests, diseases and/or infection in a subject. The subject is suitably any wild, domestic or farmed animal. For instance, the subject may suitably be a fish. The invention has been described in relation to fish for ease of description. However, it will be appreciated that the feed supplement can be used with other animals such as prawns and/or poultry. The present feed supplement may be viewed as a medicated feed supplement or a veterinary medicine or simply as a supplement for feed.

[0046] It is postulated that the feed supplement enhances the immune system of the fish, and this results in the fish being more capable of overcoming, and/or less susceptible to, pests, diseases and infection. The feed supplement improves the immune response of fish to allow them to better adapt to environmental challenges. As such, the fish is able to grow more efficiently. In this regard, the improved immune system results in an improved feed conversion ratio, and thus increases the total biomass with the same quantity of feed.

[0047] In a first aspect, although it need not be the only or indeed the broadest aspect, the invention resides in a feed supplement comprising an oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole. This feed supplement is suitably incorporated into fish feed. In other words, the present invention also resides in a fish feed composition comprising the feed supplement.

[0048] In one embodiment, the triterpenoid is a tetranortriterpenoid. One example of a tetranortriterpenoid is azadirachtin. Triterpenoids, such as azadirachtin, can act as an antifeedant and/or a growth disruptor in pests. Triterpenoids appear to be particularly useful as an antifeedant and growth disruptor in aquatic pests. As such, the consumption of the triterpenoids can eradicate or ameliorate the presence of a pest, disease and/or infection. The triterpenoid may be present in the oil in an amount of at least 300 ppm. Suitably, the triterpenoid may be present in the oil in an amount of greater than 2500 ppm. In a preferred embodiment, the triterpenoid may be present in the oil in an amount of at least 3000 ppm. In one embodiment, the oil that comprises the at least one triterpenoid is selected from the group consisting of chinaberry oil ( Melia azedarach ), melia oil ( Melia volkensii), and neem oil ( Azadirachta indica) Preferably, the oil that comprises the at least one triterpenoid is neem oil.

[0049] The term‘neem oil’ as used herein refers to any oil that is derived from the fruits and seeds, leaves and bark of neem trees. Neem oil may be derived from Azadirachta indica, Azadirachta Siamensis and Aradirachta Excelsa and any of its cultivars or from another species of Azadirachta. Preferably, the neem oil is derived from Azadirachta indica. The neem oil can be extracted in a number of methods known to a person skilled in the art, such as steam distillation, mechanical or chemical means, or cold press. Without being bound to any particular constituent, neem oil may include one or more known chemical compounds such as, for example, linoleic acid, oleic acid, hexadecanoic acid, octadecanoic acid, alpha-linolenic acid, 9-hexadecenoic acid and/or azadirachtin. These chemical compounds can also be produced synthetically or may be derived from other sources including essential oils.

[0050] Other compounds, related to azadirachtin, are present in the seeds, leaves and bark of the neem tree which also show strong biological activity against various pests. These bioactive compounds can be affected by temperature and so the neem oil is preferably obtained by cold pressing. Cold pressed neem oil is preferred as the lower temperatures of production alleviate the problem of beneficial components being degraded. In one embodiment, the neem oil is cold pressed neem oil. It has been found that cold pressed neem oil of a pharmaceutical grade has a sufficient amount of azadirachtin. As such, in one embodiment, the neem oil is pharmaceutical grade cold pressed neem oil.

[0051 ] There are many active compounds in the oil that comprises the at least one triterpenoid that may have a synergistic effect with the active ingredients in the eucalyptus oil (oil that comprises 1 ,8-cineole; mentioned in more detail hereinafter). Non-limiting examples of these actives are gedunin or similar compounds, which are believed to have antifeedant and/or bactericidal properties; nimbin or similar compounds, which are believed to have antifeedant, bactericidal and/or cytotoxic properties; salannin or similar compounds, which are believed to have growth disrupting and/or antifeedant properties; and vilasinn or similar compounds which are believed to have antifeedant and/or sensory disruption properties. It will be noted that the oil that comprises the at least one triterpenoid may comprise many other compounds that have properties that are particular useful as a feed supplement.

[0052] The % by weight amount of the neem oil is suitably between about 30% and about 99%, more suitably between about 50% and about 95%, preferably between about 60% and about 90%, more preferably between about 75% and about 85%, and most preferably about 50%, about 83% or about 86% of the feed supplement.

[0053] The 1 ,8-cineole forms greater than about 30%, suitably greater than 50%, more suitably greater than about 60%, preferably more suitably greater than about 70%, preferably greater than about 80% and most preferably greater than about 85% of the oil.

[0054] The oil that comprises 1 ,8-cineole is selected from the group consisting of eucalyptus oil, litsea oil and rosemary oil. Preferably, the oil that comprises 1 ,8-cineole is eucalyptus oil. The eucalyptus oil is preferred for its high 1 ,8-cineole content.

[0055] The term‘eucalyptus oil’ as used herein refers to oil that is derived from eucalyptus leaves including purified and/or refined derivatives of eucalyptus oil. Eucalyptus oil may be extracted from the leaves of Eucalyptus and any of its cultivars or from another species of Myrtaceae. The eucalyptus oil can be extracted in a number of methods known to a person skilled in the art, such as steam distillation, mechanical or chemical means, or cold press. Without being bound to any particular constituent, eucalyptus oil may include one or more known chemical compounds such as, 1 ,8-cineole, pinene, phellandrene, limonene, terpineol, cymene, terpenen-4-ol, camphor and/or sabinene. These chemical compounds can also be produced synthetically or may be derived from other sources including essential oils.

[0056] The % by weight of the eucalyptus oil is suitably between about 1% and about 40%, more suitably between about 5% and about 30%, preferably between about 10% and about 25%, more preferably between about 15% and about 25%, and most preferably about 10%, 14% or about 17% of the feed supplement.

[0057] The inventors believe that 1 ,8-cineole, and other components of the oil that comprises 1 ,8-cineole, activates triterpenoids. The inventors postulate that this activation results in an improved immune response in a fish. As a result, the combination of an oil that comprises 1 ,8-cineole and an oil that comprises at least one triterpenoid results in a synergist effect over the use of either oil alone.

[0058] Furthermore, the bioactive compounds in the cold pressed neem oil and eucalyptus oil are lipid modulators. It is believed that the eucalyptus oil increases the penetration of the lipophilic gut membrane. This increased penetration increases uptake of the fatty acids in the feed supplement and feed. The feed supplement is therefore postulated to assist in the growth of the subject and not necessarily through pest, diseases and/or infection treatment and prevention. As such, the present feed supplement may be viewed as a supplement that assists and/or improves weight gain and/or growth. In this regard, it is postulated that a subject that consumes the feed supplement with feed will more readily uptake the nutrients therein.

[0059] The eucalyptus oil is also believed to facilitate movement of neem oil and/or palm oil (mentioned hereinafter) across the gut membrane to increase absorption of product in the blood stream, and thus deposition in the body and organs of the fish. This increase in uptake of bioactive compounds allows the fish to exhibit strong biological activity against various pests. The mixture of eucalyptus oil and cold pressed neem oil passes through the integument more rapidly than either ingredient individually. It is postulated that this action results in the improved immune response in the fish. Tissue samples demonstrated cold pressed neem oil content above expectations when compared to cold pressed neem oil being used on its own. In one embodiment, the feed supplement is for use in treating or preventing pests, diseases and/or infections in the subject. In another embodiment, the feed supplement is limited to when it is used in treating or preventing, pests, diseases and/or infections in a subject. In another embodiment, the feed supplement is for use in assisting/improving the uptake of nutrients in fish feed. In yet another embodiment, the feed supplement is limited to when it is used in assisting/improving the uptake of nutrients in fish feed.

[0060] In one embodiment, the feed supplement comprises an oil selected from the group consisting of chinaberry oil, melia oil, and neem oil; and an oil selected from the group consisting of eucalyptus oil, litsea oil and rosemary oil. Preferably, the feed supplement comprises neem oil and eucalyptus oil.

[0061 ] In one embodiment, the feed supplement comprises eucalyptus oil in an amount of between about 15% and 25% by weight of the feed supplement and cold pressed neem oil in an amount of between about 75% and about 85% by weight of the feed supplement. In a specific embodiment, the feed supplement comprises eucalyptus oil in an amount of about 17% by weight of the feed supplement and cold pressed neem oil in an amount of about 83% by weight of the feed supplement. In another embodiment, the feed supplement comprises eucalyptus oil in an amount of about 14% by weight of the feed supplement and cold pressed neem oil in an amount of about 86% by weight of the feed supplement. It will be appreciated that the feed supplement may comprise additional components.

[0062] The feed supplement may further comprise carotenoids. Carotenoids, such as carotene, improve the colour and marketability of the tissue of a fish. The feed supplement may further comprise vitamin E. Vitamin E is a known antioxidant and its addition to the feed supplement stabilizes the biological active ingredients (e.g., the carotenoids and triterpenoids). Carotenoids and triterpenoids can oxidize and degrade, and so the presence of vitamin E alleviates this problem.

[0063] Therefore, vitamin E extends the shelf life of the feed supplement. Vitamin E may comprise tocopherol and tocotrienol. In this regard, tocopherol and tocotrienol further assists in maintaining and improving fish health. The anti inflammatory properties of the tocotrienol and tocopherol have been shown to have health benefits in mammals and invertebrates. The vitamin E, carotene and coenzyme Q10 content can counteract oxidative stress that leads to reduced feed conversion ratio and mortality.

[0064] The vitamin E and carotenoids may be present in the form of palm oil. It has been found that the inclusion of red palm oil advantageously results in the biological actives {e.g., carotenoids and triterpenoids) remaining stable for at least 12 months. In one embodiment, the feed supplement is stable for at least 6 months, more suitably at least 9 months, and most suitably at least 12 months. Additionally, it is postulated that the feed supplement is more stable when nitrogen blanketed (e.g., under a nitrogen atmosphere).

[0065] The term‘palm oil’ as used herein refers to any oil that is derived from the mesocarp of the fruit of oil palms. Palm oil may be derived from Elaeis guineensis and any of its cultivars or from another species of Elaeis. The palm oil can be extracted in a number of methods known to a person skilled in the art, such as steam distillation, mechanical or chemical means, or cold press. Without being bound to any particular constituent, palm oil may include one or more known chemical compounds such as, carotenoids (e.g., alpha and beta carotene and lycopene), sterols, vitamin E (tocopherol and tocotrienol) and/or antioxidants. These chemical compounds can also be produced synthetically or may be derived from other sources including essential oils.

[0066] The combination of neem oil and palm oil creates a beneficial free acid balance. Manipulation of the beneficial free acid balance has been shown to affect fish resistance to pathogens. When cold pressed neem oil is combined with red palm oil, the fatty acid balance is enhanced and produces an immune effect. As such, it is postulated that this immune effect treats and/or prevents pests, diseases and/or infections in a fish. The phospholipid content of cold pressed neem oil is high (>1 ,800 ppm). The phospholipids provide an additional benefit as dietary phospholipids increase the efficiency of transport of dietary fatty acids and lipids from the gut to the rest of the body. In this regard, it is postulated that the dietary phospholipids enhance lipoprotein synthesis. Furthermore, phospholipids play an important role in the metabolism of a feed.

[0067] The combination of cold pressed neem oil, eucalyptus oil and red palm oil leads to increased uptake of all components. As such, less cold pressed neem oil, eucalyptus oil and red palm oil is secreted into the environment. As a result, the environmental impact of using the feed supplement is minimal/reduced.

[0068] The % by weight of the palm oil is suitably between about 0.1 % and about 80%, more suitably between about 10% and about 70%, preferably between about 20% and about 60%, more preferably between about 30% and about 50%, and most preferably about 40% of the feed supplement. In one embodiment, the palm oil is suitably red palm oil. In this regard, red palm oil is rich in carotenoids. It will be appreciated that other oils can be utilized in the feed supplement. An alternative to palm oil may be rice bran oil.

[0069] In one embodiment, the feed supplement comprises:

cold pressed neem oil in an amount between about 40% and about 60% by weight of the feed supplement;

eucalyptus oil in an amount between about 1 % and about 20% by weight of the feed supplement; and

red palm oil in an amount between about 30% and about 50% by weight of the feed supplement.

[0070] In an embodiment, the feed supplement comprises cold pressed neem oil at about 50%, eucalyptus oil at about 10%, and red palm oil at about 40%, by weight of the feed supplement.

[0071 ] The feed supplement may exhibit one or more of: increased penetration, the accumulated compound shows a higher level activation, and less of the active compound is detoxified.

[0072] As previously mentioned, the feed supplement may be incorporated into feed. Non-limiting examples of the feed is aquatic feed or fish feed. The feed supplement may be incorporated into fish feed as a liquid or a solid. In other words, the feed supplement is a liquid feed supplement or a solid feed supplement. The liquid feed supplement is as substantially described hereinabove.

[0073] The solid feed supplement is as described for the feed supplement hereinabove except for the addition of a solid substrate. In other words, the solid feed supplement further comprises a solid substrate. The addition of the solid substrate allows the feed supplement to be formed as a solid. The solid substrate may be selected from the group consisting of starch, starch derivatives, proteins, gums, lipids caseinates and polysaccharides. In one embodiment, the caseinate is selected from the group consisting of sodium caseinate, calcium caseinate and potassium caseinate. In an embodiment, the polysaccharide is maltodextrin.

[0074] The % by weight of the solid substrate is suitably between about 20% and about 80%, more suitably between about 30% and about 70%, preferably between about 40% and about 60%, and most preferably about 50%, by weight of the solid feed supplement. It will be appreciated that the remainder of the solid feed supplement is made up the liquid components (e.g., the components mentioned hereinabove, such as the oil comprising at least one triterpenoid, the oil comprising 1 ,8-cineole and palm oil). In other words, the liquid component may be viewed as the liquid feed supplement mentioned hereinabove.

[0075] In one embodiment, the solid substrate comprises sodium caseinate and maltodextrin. In a further embodiment, the solid substrate comprises:

sodium caseinate in an amount of about 0.1% to about 50%, or about 5% to about 20%, or about 10% to about 20%, or about 15% by weight of the solid substrate; and

maltodextrin in an amount of about 50% to about 99.9%, or about 60% to about 95%, or about 70% to about 95%, or about 80% to about 90%, or about 85% by weight of the solid substrate.

[0076] In an embodiment, the solid substrate comprises sodium caseinate and maltodextrin. In a further embodiment, the solid substrate comprises sodium caseinate in an amount of about 15% by weight of the solid substrate and maltodextrin in an amount of about 85% by weight of the solid substrate.

[0077] In one embodiment, the solid feed supplement is in the form of a powder. In one embodiment, the solid feed supplement is in the form of a microfine. However, it is postulated that the feed supplement may be in the form of a granulated product.

[0078] In one embodiment, the solid feed supplement comprises the solid substrate in an amount of about 50% by weight of the solid feed supplement, and the liquid components in an amount of about 50% by weight of the solid feed supplement. It is postulated that the solid feed supplement can comprise the solid substrate in an amount of about 35% by weight of the solid feed supplement, and the liquid components in an amount of about 65% by weight of the solid feed supplement. Furthermore, the inventors believe that the solid feed supplement can comprise the solid substrate in an amount of about 30% by weight of the solid feed supplement, and the liquid components in an amount of about 70% by weight of the solid feed supplement.

[0079] In one embodiment, the solid feed supplement comprises solid substrate in an amount between about 20% and about 80% by weight of the solid feed supplement; neem oil in an amount between about 30% and about 60% by weight of the solid feed supplement; and eucalyptus oil in an amount between about 5% and about 15% by weight of the solid feed supplement. In yet another embodiment, the solid feed supplement comprises a solid substrate at about 50% by weight of the solid feed supplement; neem oil at about 42% by weight of the solid feed supplement; and eucalyptus oil at about 8% by weight of the solid feed supplement. In one embodiment, the solid feed supplement comprises a solid substrate at about 50% by weight of the solid feed supplement; neem oil at about 43% by weight of the solid feed supplement; and eucalyptus oil at about 7% by weight of the solid feed supplement.

[0080] In an embodiment, fish feed may be treated with the feed supplement to incorporate the components therein to form a fish feed composition. The fish feed is suitably selected from the group consisting of pellets and flakes. In other words, the present invention is in the form of a feed comprising the feed supplement. In one aspect, the present invention resides in a fish feed composition comprising the feed supplement. In one embodiment, the feed supplement is used for supplementing fish feed. The feed supplement may be a solid feed supplement or a liquid feed supplement.

[0081 ] The fish feed is any feed that is edible to fish. The fish feed may or may not provide a source of nutrition to the fish. As such, the fish feed encompasses feeds which are either nutritional feeds and feeds which are non- nutritional. In this regard, the feed may suitably be a substrate for delivering the feed supplement to the fish. Preferably, the fish feed is solid fish feed. The fish feed is preferably porous. The porosity of the fish feed allows the feed supplement to penetrate into the fish feed. Fish consuming the fish feed composition can benefit from an improved immune system and as such are more able to fight off pests, diseases and/or infection. The fish feed composition and thus feed supplement alleviates the problem of pests, disease and/or infection. The present fish feed composition may be viewed as a pesticide or a veterinary medicine as the consumption of the fish feed composition can act as a prophylactic against pests, diseases and infection and/or eradicate pests, diseases and/or infection present in the fish. Furthermore, the fish feed composition may also be considered as an efficient fish feed when compared to the fish feed alone. In this regard, the fish supplement assists/improves uptake of the nutrients of the fish feed by a fish. This advantageously leads to a greater feed conversion ratio, greater weight gain in the fish and improved growth rate.

[0082] The fish may be any fish. The fish may be wild fish and/or farmed fish. The fish may be a fresh water fish or a salt water fish. The fish may suitably be a domestic fish. In one embodiment, the fish is selected from the species Salmo, Lates calcarifer, Carangidae, Cichilid and/or Oncorhynchus. Suitably, the fish is selected from the group consisting of salmon, trout, bass, yellowtail kingfish, tilapia, bream and gold fish. Preferably, the fish is selected from the group consisting of salmon, barramundi and trout. It will be appreciated that the list provided only exemplifies some of the fish that can be treated, and that the person skilled in the art will appreciate that other fish not expressly listed can also be treated. The fish may also include juvenile fish. [0083] The pest, disease and/or infection may be any organism which is detrimental to the health of the fish. The pest, disease and/or infection may be an ecto-parasite and/or an endo-parasite. The pest, disease and/or infection may be selected from the group consisting of worms, helminths, flukes, mites, ticks, crustaceans, lice, copepods, amoeba and louse. The copepod suitably belongs to the Lepeophtheirus genus and/or Caligus genus. Preferably, the pest, disease and/or infestation is selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensi, Caligus rogercreseyi, salmon lice, Monogenean flatworm parasites, myxozoan parasites, kudoa thyrsites parasite in salmon, amoebic gill disease Neoparamoeba perurans and salmon louse. It will be appreciated that the list provided only exemplifies some of the pests, diseases and/or infestations that can be treated, and that the person skilled in the art will appreciate that others not expressly listed can be addressed by using the feed supplement and thus fish feed composition. In this regard, the postulated increase in immune response can assist in combating any pest, disease and/or infection. It is also postulated that the present feed supplement can be used to treat and/or prevent greenhouse pests, such as chewing insects.

[0084] It will be appreciated that the amount of each constituent in the liquid feed supplement will be different to the amount of each of these constituents in the fish feed composition. The liquid feed supplement may be added to fish feed in ratio of suitably between about 1 :5 and about 1 :20, more suitably between about 1 :7 and about 1 :15, preferably between about 1 :8 and about 1 :12, and most preferably about 1 :9 (liquid feed supplement: fish feed).

[0085] The % by weight amount of the neem oil is suitably between about 0.1 % and about 20%, more suitably between about 2% and about 10%, preferably between about 3% and about 7%, more preferably between about 4% and about 6%, and most preferably about 5% of the fish feed composition.

[0086] The % by weight of the eucalyptus oil is suitably between about 0.1 % and about 10%, more suitably between about 0.2% and about 5%, preferably between about 0.5% and about 4%, more preferably between about 0.5% and about 2%, and most preferably about 1 % of the fish feed composition.

[0087] The % by weight of the palm oil is suitably between about 0.1 % and about 20%, more suitably between about 1 % and about 10%, preferably between about 2% and about 6%, more preferably between about 3% and about 5%, and most preferably about 4% of the fish feed composition.

[0088] In one embodiment, the fish feed composition comprises:

cold pressed neem oil in an amount between about 0.1 % and about 1 % by weight of the fish feed composition, preferably about 0.83%; eucalyptus oil in an amount between about 0.1 % and about 0.5% by weight of the fish feed composition, preferably about 0.17%; and solid substrate in an amount between about 0.5% and about 1.5% by weight of the fish feed composition, preferably about 1 %; with the remainder made up with fish feed.

[0089] In one embodiment, the fish feed composition comprises:

cold pressed neem oil in an amount between about 0.1 % and about 0.5% by weight of the fish feed composition, preferably about 0.21%;

eucalyptus oil in an amount between about 0.1 % and about 0.5% by weight of the fish feed composition, preferably about 0.04%; and solid substrate in an amount between about 0.1 % and about 1.0% by weight of the fish feed composition, preferably about 0.25%; with the remainder made up with fish feed.

[0090] In one embodiment, the fish feed composition comprises:

cold pressed neem oil in an amount between about 4% and about 6% by weight of the fish feed composition;

eucalyptus oil in an amount between about 0.1 % and about 2% by weight of the fish feed composition; and

red palm oil in an amount between about 3% and about 5% by weight of the fish feed composition.

[0091 ] In a preferred embodiment, the fish feed composition comprises cold pressed neem oil at about 5%, eucalyptus oil at about 1%, and red palm oil at about 4%, by weight of the fish feed composition.

[0092] The fish feed composition may further comprise a coating. The coating comprising a further amount of palm oil and/or stearin; and fish oil and/or fish hydrolase. Preferably, the palm oil is red palm oil. The palm oil is as described hereinabove.

[0093] The % by weight of the further amount of palm oil and/or stearin is suitably between about 20% and about 80%, more suitably between about 30% and about 70%, preferably between about 40% and about 60%, more preferably between about 45% and about 55%, and most preferably about 50% of the coating.

[0094] The % by weight of the fish oil and/or fish hydrolase is suitably between about 20% and about 80%, more suitably between about 30% and about 70%, preferably between about 40% and about 60%, more preferably between about 45% and about 55%, and most preferably about 50% of the coating. [0095] It will be appreciated that the amount of each component in the coating will be different to the amount of each of these component in the fish feed composition.

[0096] The % by weight of the further amount of palm oil and/or stearin is suitably between about 0.1 % and about 20%, more suitably between about 0.5% and about 10%, preferably between about 1 % and about 5%, more preferably between about 1 % and about 3%, and most preferably about 2% of the fish feed composition.

[0097] The % by weight of the fish oil and/or fish hydrolase is suitably between about 0.1 % and about 20%, more suitably between about 0.5% and about 10%, preferably between about 1 % and about 5%, more preferably between about 1 % and about 3%, and most preferably about 2% of the fish feed composition.

[0098] The term‘fish oil’ as used herein refers to oil that is derived from the tissue of fish. The fish oil can be extracted from the tissue of fish in a number of methods known to the person skilled in the art. Without being bound to any particular constituent, fish oil may include one or more known chemical compounds such as, oleic acid and/or omega-3 fatty acids (e.g., eicosapentaenoic acid and docosahexaenoic acid). These chemical compounds may be produced synthetically or may be derived from other sources.

[0099] The coating acts as a shield to reduce oxidation of the feed supplement in the fish feed composition. Furthermore, the coating shields the bitter taste of neem oil and eucalyptus oil to increase palatability of the fish feed composition. Furthermore, fish oil is known to be a fish supplement due to their omega-3 fatty acid content, such as docosahexaenoic acid and eicosapentaeonic acid. It has been found that a coating comprising a 1 :1 mixture of red palm oil and fish oil results in a similar weight gain to fish where only fish oil is utilized. In this regard, fish oil is more expensive due to the falling supply of the oil. As such, the substitution of part of the fish oil with red palm oil is advantageous. For example, salmon require an energy dense diet that has high lipid content, and this coating can assist in providing provide the required nutritional content to the salmon. The person skilled in the art will appreciate that the composition of the coating can be altered so that it is can be tailored to the dietary requirements of any species of fish.

[00100] Preferably, the coating has a higher melting point than the feed supplement. The higher melting point of the coating advantageously prevents or ameliorates the problem of the feed supplement seeping into the water. As such, the structural integrity of the fish feed composition is advantageously maintained for longer, and the stability of the fish feed composition in the water column is improved. The red palm oil can be exposed to a fractionation process to include partial crystallization. This fractionation can be applied as a top coat.

[00101 ] It will be appreciated that the amount of each constituent in the solid feed supplement will be different to the amount of each of these constituents in the fish feed composition. The solid feed supplement may be added to fish feed in a ratio of suitably between about 1 :100 and about 1 :10, more suitably between about 1 :80 and about 1 :20, preferably between about 1 :60 and about 1 :40, and most preferably about 1 :50 (solid feed supplement: fish feed). In one embodiment, the solid feed supplement is present in the fish feed composition at 2% by weight. In another embodiment, the solid feed supplement is present in the fish feed composition at 0.5% by weight.

[00102] In another aspect, the invention resides in a method of preparing a feed supplement including the steps of:

to thereby prepare a feed supplement.

[00103] The feed supplement and components thereof are as substantially described hereinabove. As mentioned hereinabove, the oil that comprises triterpenoid may suitably be cold pressed neem oil. The oil that comprises 1 ,8- cineole may be selected from the group consisting of eucalyptus oil and rosemary oil.

[00104] The method may further include the step of adding and mixing vitamin E. The method may further include the step of adding and mixing carotenoids. As previously mentioned, the vitamin E and the carotenoids may be present as palm oil. The method may further include the step of adding and mixing palm oil. The palm oil is suitably red palm oil.

[00105] In forming the solid feed supplement, the method may further include the step of adding a solid substrate. The method may further include the step of drying the mixture.

[00106] Traditional spray techniques can utilize heat and an evaporative airstream to dry a composition, and this can lead to the bioactive compounds being degraded due to the higher temperatures. Furthermore, the evaporative airstream can have a further oxidising effect on the bioactive compounds.

[00107] In traditional spray drying, there are two distinct phases of drying: a constant-rate drying phase and a falling-rate drying phase. During the constant- rate phase, the majority of heat transferred to the droplet is latent and used to drive the evaporation of the solvent. The solvent evaporation cools the surrounding drying gas and the droplet temperature remains largely constant. As more of the solvent evaporates from the droplet, the solid content of the outer layer of the droplet increases to a point where a shell is formed. At this point, a particle with a solid shell but wet core is formed, and the drying phase switches to the falling-rate phase. During this phase, heat is transferred to the particle from the drying gas as sensible heat. The temperature of the particle is raised to fully evaporate the remaining solvent from the core of the particle. This increase in temperature can cause the bioactive compounds to degrade. The temperature profile of this process is shown in Figure 3.

[00108] In one embodiment, in the method of forming a solid feed supplement, the method may utilize electrostatic technology. It will be appreciated that electrostatic technology utilizes electric charges or fields. In this regard, each component of the feed supplement has different electrostatic properties. The components that have a greater charge density will repel each other which forces said components to migrate to the outer surface of a droplet. In contrast, the components that have a lower charge density will migrate to the centre of a droplet. The electrostatic effect is used to stratify the components of the feed component during atomization. This is based on the relative polarities of the components. The electrostatic charge applied to the feed allows for control over the agglomerate droplets as they are being dried.

[00109] The oils of the feed supplement are mixed and blended with the solid substrate and solvent to form a sprayable emulsion. This blend is sprayed into a drying chamber where it is subjected to an electrostatic charge. The components of this blend are solvent (water and/or organic solvent), the solid substrate, and the oils (neem oil, eucalyptus oil and/or red palm oil). The solvent will have the greatest charge density, and the solid substrate being less conductive has a lower charge density. The solvent having the greatest charge density is forced to the surface of the droplet. In contrast, the oils migrate to the centre of the droplet and are encapsulated by the solid substrate. This action eliminates oils from the surface and thus the palatability issues. The movement of the solvent to the surface of the droplet also creates excellent drying conditions and leads to excellent encapsulation of the oils without the use of high evaporation temperatures. The temperature profile of the process is shown in Figure 4.

[00110] It is postulated that varying the electrostatic charge, or charge pulse, can lead to the solid feed supplement having different properties. For instance, it is believed that varying the electrostatic charge pulse can lead to the formation of the solid feed supplement in the form of a microfine or a granulated product.

[0011 1 ] In one embodiment, the method of preparing a feed supplement further includes one or more of the following steps: mixing the oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole with a solid substrate and solvent to form a mixture; spraying the mixture into a drying chamber; subjecting the mixture to an electrostatic charge; and drying the mixture. In an embodiment, the drying step is completed at a temperature of below about 60^QC, about 55^QC, about 53^QC or about 50^QC.

[00112] In one embodiment, the drying process was completed using a substantially inert gas as the drying medium. Non-limiting example of the substantially inert gas are nitrogen gas, helium gas, argon gas, krypton gas and xenon gas. Preferably, the inert gas is nitrogen gas. The exclusion of reactive gases reduces oxidation loss. Furthermore, the reduction in oxidation loss alleviates the need for a coating. However, it will be appreciated that a coating can still be applied to the fish feed composition to further extend the shelf life and/or improve palatability.

[00113] In another aspect, the invention resides in a method of incorporating a feed supplement into fish feed including the steps of:

providing fish feed;

contacting the fish feed with a feed supplement comprising an oil that comprises triterpenoid and an oil that comprises 1 ,8-cineole. to thereby form a fish feed composition comprising the feed supplement.

[00114] The fish feed, feed supplement and components thereof are as substantially described hereinabove.

[00115] High temperatures can cause the oils and the bioactive components thereof to degrade and so it is preferable that the temperature is maintained at lower temperatures. Preferably, the step of contacting the fish feed with the fish supplement occurs at a temperature suitably below 60^QC or 55^QC, more suitably below 53^QC and most preferably below 50^QC It will be appreciated that the lower the temperature the slower the evaporation rate of water. As such, it will be appreciated that the optimal temperature at which this occurs is about 50^QC - 53^QC. Testing suggests that greater than 98% of the bioactive compounds are retained in the feed supplement when the temperature is maintained at or below 60^QC.

[00116] The step of contacting the fish feed with a feed supplement may occur after the fish feed has been produced or during the cooling process when temperatures are below 55^QC or 60^QC of the production process of the fish feed. As such, the fish feed may suitably be pre-formed. Alternatively, the feed supplement may be applied during the preparation of the fish feed as long as the feed supplement is not subjected to temperatures of greater than 55^QC or 60^QC. [00117] The contacting step can occur in many different methods known to the person skilled in the art. A non-limiting example includes using a rotating drum. The method may further include the steps of: placing the fish feed into a rotating drum; and spraying the fish feed with the feed supplement. In a further preferred embodiment, the method further includes one or more steps of: placing the fish feed into a rotating drum; subjecting the fish feed to a vacuum; contacting the fish feed with the feed supplement under vacuum; and releasing the vacuum. This vacuum advantageously allows the feed supplement to penetrate into the fish feed. In this regard, upon release of the vacuum, the feed supplement is forced into the fish feed. It will be appreciated that this method is applicable to both the liquid feed supplement and the solid feed supplement.

[00118] As previously mentioned, the fish feed composition may further comprise a coating. However, the fish feed composition need not necessarily require a coating. The coating and components thereof are as substantially described hereinabove. The method may further include the step of:

providing a coating; and

coating the fish feed composition with the coating.

[00119] The coating may simply be coated on the fish feed composition after the feed supplement has been applied thereto. Alternatively, the coating may be advantageously coated on the fish feed composition under vacuum after the feed supplement has been applied thereto. Upon releasing the vacuum, the coating covers the fish feed composition and previously incorporated feed supplement. The release of the vacuum forces the coating into the fish feed and into contact with the feed supplement.

[00120] The coating advantageously alleviates the problem of oxidation of the feed supplement. Furthermore, the coating masks any aroma arising from the feed supplement. As such, the coating increases the shelf life of the fish feed composition, and improves the palatability of the fish feed composition.

[00121 ] The invention also resides in a method of treating or preventing pests, diseases and/or infections in a fish including the steps of:

wherein the feed supplement comprises an oil that comprises triterpenoid and an oil that comprises 1 ,8-cineole,

to thereby treat or prevent pests, diseases and/or infestations in the fish.

[00122] As used herein, the term‘therapeutically effective amount” describes a quantity of the fish feed composition and thus feed supplement to achieve a desired effect in a fish being treated. For example, this can be the amount of a fish feed composition and thus feed supplement necessary to treat and/or prevent pests, diseases and/or infections. In some embodiments, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of diseases and/or infections and/or the presence of a pest. In other embodiments, a“therapeutically effective amount” is an amount sufficient to achieve a desired biological effect. The“therapeutically effective amount” will vary, in a manner which would be understood by a person of skill in the art, with species, age, sex, weight etc.

[00123] The fish feed composition, feed supplement, subject, pests, diseases and/or infection, and components thereof, are as substantially described hereinabove. The effective amount of fish feed composition may suitably be determined from the weight and species of the fish.

[00124] The administration may suitably be over a predetermined period. The predetermined period is suitably greater than 7 days, greater than 14 days, greater than 21 days, or greater than 28 days. Alternatively, the administration may be a continuous administration. In this regard, the amount of the feed supplement may be reduced to a lower amount if continuous feeding of the fish feed composition is administered. The lower amount of feed supplement maintains an active content in the tissue for the control of pests, diseases and/or infection.

[00125] In one embodiment, the administration of a therapeutically effective amount of fish feed composition comprising a feed supplement to a fish is about 1 % body weight/day for a duration of 7 days of a 28 day cycle. In another embodiment, the administration of a therapeutically effective amount of fish feed composition comprising a feed supplement to a fish may be altered at stages of treatment. For instance, in a 28 day treatment cycle, the first 10-14 days may include administering a therapeutically effective amount of fish feed composition comprising a feed supplement to the fish at about 2% body weight/day, and subsequently reduced to a lower amount in subsequent days.

[00126] In another embodiment, when the administering is continuous administration, the cold pressed neem oil is in an amount of about 1.5%, the red palm oil is in an amount of about 1.5%, and the eucalyptus oil is in an amount of about 0.5%, by weight of the fish feed composition. In this embodiment, the total internal lipid content of the supplement is about 3.5%.

[00127] It will be appreciated that the present fish feed composition provides a commercial alternative to those known in the art.

Examples

Liquid Feed Supplement Example [00128] A liquid feed supplement was prepared containing the relative amounts of ingredients shown in Table 1 below. The composition was prepared by the process described in Example 1.

Table 1 - Sample Liquid Feed Supplement

Example 1

[00129] The following working steps were taken to provide a liquid feed supplement with the relative amounts as set out in Table 1 :

a. adding cold pressed neem oil to a suitably sized vessel; b. adding eucalyptus oil;

c. stirring for about 15 minutes;

d. adding red palm oil; and

e. stirring for about 15 minutes.

[00130] The vessel may be made of stainless steel, mild steel, high density polypropylene or has a chemically resistant lining.

[00131 ] A fish feed composition was prepared using the liquid feed supplement described in Table 1 containing the relative amounts of ingredients shown in Table 2 below. The composition was prepared by the process described in Example 2.

Table 2 - Sample Fish Feed Composition

Example 2

[00132] The following working steps were taken to provide a fish feed composition with the relative amounts as set out in Table 2:

a. adding the fish feed to a rotating drum;

b. subjecting the fish feed to a vacuum;

c. spraying the liquid feed supplement onto the fish feed whilst rotating; and

d. releasing the vacuum.

[00133] A coating was prepared containing the relative amounts of the ingredients shown in Table 3 below. The components were added and mixed in a suitably sized vessel.

Table 3 - Sample Coating Composition

[00134] The fish feed composition described in Table 2 was coated with the coating described in Table 3. The following working steps were taken continuing from the working steps of Example 2 to provide a fish feed composition comprising a coating:

a. rotating the rotating drum in which the fish feed composition resides;

b. subjecting the fish feed composition to a vacuum;

c. spraying the coating onto the fish feed composition whilst rotating; and

d. releasing the vacuum.

Solid Feed Supplement Example

[00135] A solid feed supplement was prepared containing the relative amounts of ingredients shown in Table 1 a below. The composition was prepared by the process described in Example 1 a.

Table 1 a - Sample Solid Feed Supplement

Example 1a

[00136] A The following working steps were taken to provide a solid feed supplement with the relative amounts as set out in Table 1 a:

c. stirring for about 15 minutes;

d. adding solid substrate and solvent;

e. stirring for about 15 minutes;

f. spraying the mixture into a dry chamber;

g. subjecting the mixture to an electrostatic charge; and h. drying the mixture in a nitrogen atmosphere and at a temperature below 60^QC.

[00137] The vessel may be made of stainless steel, mild steel, high density polypropylene or has a chemically resistant lining. [00138] A fish feed composition was prepared using the solid feed supplement described in Table 1 a containing the relative amounts of ingredients shown in Table 2a below. The composition was prepared by the process described in Example 2a.

Table 2a - Sample Fish Feed Composition

Example 2a

[00139] The following working steps were taken to provide a fish feed composition with the relative amounts as set out in Table 2:

a. adding the fish feed to a rotating drum;

b. subjecting the fish feed to a vacuum;

c. adding the solid feed supplement to the fish feed whilst rotating; and

d. releasing the vacuum.

Testing Bioactive transfer

[00140] The testing of the above fish feed composition described in Table 2 indicates that the % bioactive compounds contained in the fish feed composition transfers up to 56% of total bioactive compounds to the tissue.

Analysis of Neem residue in fish sample

Method

[00141 ] Aradirachtin was used as a standard on the basis that azadirachtin decomposition or degradation could be used as a measure of degradation of all bioactive compounds in the oil. A comparative analysis was completed based upon an assay of azadirachtin content as a measure of the amount of bioactive compounds in the tissue. Specimens of fish tissue steeped in ethanol were provided for determination of bioactive compounds.

[00142] It appeared that much of the triterpene in the tissue sample was likely to have been extracted from the fish tissue into the steeping ethanol. In this regard, small aliquots of the ethanol were sampled for specific assay with the remaining fish tissue homogenised in the remaining ethanol. The ethanol results were labeled‘ethanol’ (Figure 1 ) and the homogenized fish tissue in ethanol was labelled‘Fish and Ethanol’ (Figure 2).

[00143] Figure 1 illustrates the actual uptake of azadirachtin from three different diets compared to the potential amount available from the various diets, using azadircahtin levels in the ethanol used for extracting the fish tissue. This comparison indicates that azadirachtin content starts to diminish with increasing neem oil content.

[00144] Figure 2 illustrates the actual uptake of azadirachtin from the three different diets compared to the potential amount available from the various diets, using azadirachtin levels present in the homogenate of fish tissue and ethanol. This comparison indicates that azadirachtin content is saturated after 5% neem oil, indicating that higher levels of Neem oil in the diet are not readily absorbed by the fish.

[00145] In both Figure 1 and Figure 2, the x axis is neem oil % and the y axis is total intake/theoretical uptake (ppm). The results from both Figures 1 and 2 indicate that absorption of the neem oil at around 2.5% is achieved, and that no further absorption appears to occur after 5%. At less than 2.5%, good transfer of bioactive compounds into the blood stream and thus tissue of the fish is achieved. The azadirachtin content in the neem oil of these samples was 300ppm. In this regard, the residual amount of azadirachtin content in the fish tissue was about 4 ppm. It is postulated that if the azadirachtin content of the neem oil was higher then the residual content of azadirachtin in the fish tissue would also be higher.

Testing of Electrostatic Method

[00146] Three different solid feed supplements were trialed to confirm that the use of electrostatic effect was suitable.

[00147] Firstly, a trial using 50% eucalyptus oil and 50% solid substrate (sodium caseinate and maltodextrin) was completed to determine whether the volatility of eucalyptus oil would disrupt the encapsulation and allow the oil to migrate to the surface and inhibit the free-flowing properties of the bioactive compounds in the centre of the droplet. The inventors found that the oil was not allowed to migrate to the surface and confirmed the integrity of the barrier encapsulated.

[00148] In the second trial, 50% neem oil and 50% solid substrate was utilized to determine whether the process could run at <60^QC drying temperature and if greater than 95% of the bioactive would transfer to the solid substrate.

[00149] In the third trial, a feed composition utilizing a 50% eucalyptus oil : neem oil mixture (1 :6 ratio) and 50% solid substrate was trialed at <60^QC drying temperature.

[00150] These trials showed that the temperature of drying could be achieved whilst maintaining a temperature of about 53^QC to about 55^QC. As such, the problem of the bioactive components degrading had been alleviated. Furthermore, the trials indicated that a 98% transfer of bioactive compounds could be achieved (determined using LCMS). The tested oil was 2400ppm and the power comprising 41.5% neem oil was 10OOppm.

[00151 ] The resultant solid feed supplement was extremely fine (microfine). The microfine feed supplement formed a suspension in water, and stayed in suspension for at least 4-5 days without phase separation. This demonstrates the stability of the solid feed supplement in an aqueous environment.

Testing of the fish supplement on Barramundi

[00152] A trial was run to show the impact of the feed supplement on the growth of a fish (Barramundi). A solid feed supplement comprising 50% oils (5:1 neem oil: eucalyptus oil) and 50% solid substrate (10-20% sodium caseinate : 80-90% maltodextrin) was prepared. The combination of the oil into the solid substrate powder was achieved through a micro encapsulation process. The Barramundi was not diseased, infected or infested with pests. These results demonstrate the supplementary effects of the feed supplement.

[00153] To show the effect of the feed supplement, three fish feed compositions were compared:

- C1 - control diet (fish feed without the solid feed supplement)

- D2 - the fish feed of C1 with 0.5 wt% solid feed supplement

- D3 - the fish feed of C1 with 2 wt% solid feed supplement

[00154] The average daily feed intake, weight gain by fish, growth rate and food conversion ratio were monitored. These tests were completed in duplicates of five (5). These results should demonstrate the impact of the feed supplement on fish growth.

[00155] The C1 control trial was conducted for 25 days whereby the only diet provided to the fish was the C1 composition. The D2 trial was conducted for 25 days whereby the only diet provided to the fish was the D2 composition. The D3 trial was conducted for 25 days, however, the C1 composition was provided to the fish for the first 20 days and then provided with the D3 composition for 5 further days.

Average Daily Feed Intake

[00156] The average daily feed intake was monitored. In this regard, a table showing the results is provided below and a graph showing the average daily feed intake is presented in Figure 5.

[00157] The average daily feed intake of the D2 composition is about 10.87% lower than the C1 composition. This result suggests that the Barramundi were consuming less of the D2 composition. However, this reduced feed intake is significant when combined with the feed-conversation-ratio (mentioned hereinafter). The D3 composition had an increased intake of 4.6% indicating that there are no palatability issues with the feed supplement.

Weight Gain (g/fish)

[00158] The fish weight gain was also monitored. A table showing the result is provided below and a graph showing the average weight gain as a function of diet is presented in Figure 6.

[00159] As mentioned above, the D2 composition feed intake was 10.87% lower than that of the C1 composition. However, the weight gain of the fish consuming the D2 composition was comparable and slightly greater than the fish consuming the C1 composition. This result suggests that the D2 composition assisted in weight gain of the Barramundi. That is, less fish feed was consumed but a similar weight gain was achieved. The D3 composition resulted in a 12.76% increase in weight gained/fish (5-day treatment) when compared to the C1 composition. These results suggest that the feed supplement assists in absorption of nutrients or at least assists in weight gain of fish.

Specific Growth Rate (harvest weight-starting weight/production time x100)

[00160] A table showing the results of the specific growth rate is provided below and a graph showing the average weight gain as a function of diet is presented in Figure 7.

[00161 ] Firstly, it should be noted that the D2 composition was more consistent as the error bars were smaller than those of the C1 and D3 compositions. Furthermore, the D3 composition had a 7.9% which is greater than both C1 and D2. Food Conversation Ratio (as a function of diet)

[00162] A table showing the results is provided below and a graph showing the food conversation ratio is presented in Figure 8.

[00163] Firstly, it should be noted that the lower the ratio, the more efficient the conversion from feed to tissue. A ratio of 1.3 is a good result for both the D2 and D3 compositions as this represents a more efficient conversion from feed to fish tissue. The D3 composition results in excellent conversion for only 5 days with a specific growth rate of 107.9%.

[00164] These results suggest that the feed supplement assists in the growth of fish. In particular, the D2 and D3 compositions resulted in greater feed conversion ratio which indicates an increased uptake of the nutrients in fish feed. This in turn leads to greater weight gain in fish and improved growth rate.

[00165] The above description of various embodiments of the present invention is provided for purposes of description to one of ordinary skill in the related art. It is not intended to be exhaustive or to limit the invention to a single disclosed embodiment. As mentioned above, numerous alternatives and variations to the present invention will be apparent to those skilled in the art of the above teaching. Accordingly, while some alternative embodiments have been discussed specifically, other embodiments will be apparent or relatively easily developed by those of ordinary skill in the art. Accordingly, this invention is intended to embrace all alternatives, modifications and variations of the present invention that have been discussed herein, and other embodiments that fall within the spirit and scope of the above described invention.

Claims

1. A feed supplement comprising an oil that comprises at least one

triterpenoid and an oil that comprises 1 ,8-cineole.

2. The feed supplement of claim 1 , wherein the oil that comprises at least one triterpenoid is selected from the group consisting of chinaberry oil, melia oil, and neem oil

3. The feed supplement of claim 1 or claim 2, wherein the oil that

comprises at least one triterpenoid is neem oil or cold pressed neem oil.

4. The feed supplement of any one of the preceding claims, wherein the oil that comprises 1 ,8-cineole is selected from the group consisting of eucalyptus oil, litsea oil and rosemary oil.

5. The feed supplement of any one of the preceding claims, wherein the oil that comprises 1 ,8-cineole is eucalyptus oil.

6. The feed supplement of any one of the preceding claims, further

comprising vitamin E and/or carotenoids.

7. The feed supplement of claim 5, wherein the vitamin E and

carotenoids is present as red palm oil.

8. The feed supplement of any one of the previous claims, further

comprising a solid substrate.

9. The feed supplement of any one of the previous claims comprising neem oil, eucalyptus oil and a solid substrate.

10. The feed supplement of claim 8 or claim 9, wherein the solid substrate is present at an amount between about 20% and about 80%, or between about 30% and about 70%, or between about 40% and about 60%, or about 50% by weight.

1 1. The feed supplement of any one of the preceding claims, wherein the oil that comprises at least one triterpenoid is present at an amount between about 30% and about 99%, or between about 50% and about 95%, or between about 60% and about 90%, or between about 75% and about 85% by weight.

12. The feed supplement of any one of the preceding claims, wherein the oil that comprises 1 ,8-cineole is present at an amount between about 1 % and about 40%, or between about 5% and about 30%, or between about 10% and about 25%, or between about 15% and about 25%.

13. A fish feeding composition comprising the feed supplement of any one of the preceding claims.

14. The fish feed composition of claim 12, wherein the feed supplement is present as a solid feed supplement.

15. The fish feed composition of claim 13, wherein the solid feed

supplement is present at an amount between about 0.1 % to about 10% by weight, or between about 0.1 % and about 5% by weight, or between about 0.5% and about 2% by weight, or about 0.5% by weight, or about 2% by weight.

16. The fish feed composition of any one of claims 13-15 further

comprising a coating.

17. The fish feed composition of claim 16, wherein the coating comprises a further amount of palm oil and/or stearin, or fish oil and/or fish

hydrolase.

18. A method of treating or preventing pests, diseases and/or infections in a fish including the steps of:

to thereby treat or prevent pests, diseases and/or infestations in the fish.

19. A method of assisting and/or improving weight gain in a fish including the step of:

feeding a fish feed composition comprising a feed supplement that comprises an oil that comprises at least one triterpenoid and an oil that comprises 1 ,8-cineole;

to thereby assist or improve weight gain in the fish.