AU2014257626B2

AU2014257626B2 - Method for improving mineral resorption in farmed fish and crustacean

Info

Publication number: AU2014257626B2
Application number: AU2014257626A
Authority: AU
Inventors: Robert Aebi; Heinrich Bachmann
Original assignee: Herbonis AG
Current assignee: Herbonis AG
Priority date: 2013-04-23
Filing date: 2014-04-22
Publication date: 2017-03-30
Anticipated expiration: 2034-04-22
Also published as: CA2910568C; CL2015003106A1; NO20151589A1; WO2014173862A1; AU2014257626A1; CA2910568A1; GB2550100A; GB201519078D0; GB2550100B

Description

PCT/EP2014/058057 WO 2014/173862

Method for improving mineral resorption in farmed fish and crustacean

Field of the Invention

The invention relates generally to the field of fish and crustacean farming, in particular to specific feed components or additives suitable to feed fishes or crustaceans. More specifically the invention relates to means useful for improving mineral resorption in fishes and crustaceans.

Background of the Invention

Supplying the growing world population with food is highly challenging. One of the most valuable nutritive components is protein which comes typically from meat of land animals but also from fish. Consumption of fish and crustacean is a meat source since prehistoric times. However, in the last decades, ocean fishing came to a critical point in which over-fishing reduced catches of many marine species such as sardines, tuna and herring. Also, modem fishery practices destroy the ocean floor and this impairs hatching ground for many marine animals.

Fish farming is therefore a suitable alternative to ocean fishing. So, for example salmons have the best feed conversion ratio (FCR) of all farmed animals

However, fish and crustacean aquaculture has also its shortcomings, which needs to be overcome in order to make aquaculture sustainable: 1. 2. 3.

Stocking density as a cause of stress and growth anomalies Over fertilization of coastal areas caused by excreta and wasted feed The use of fish meal and fish oil as feed components WO 2014/173862 2 PCT/EP2014/058057

Such draw-backs are recognized by the industry and intensive research is ongoing to improve the situation and make aquaculture more sustainable. One of the proposed strategies consists to replace the use of fish meal and oil by raw material of plant origin and to correct eventual imbalances. Another strategy is to find additives which would induce a better use of other nutrients.

In addition, fishes and crustaceans in aquaculture do not have access to the natural feed components in marine or freshwater environment like essential nutrients e. g. vitamins from plankton. On the other hand freshwater and seawater contain some minerals in sufficient amounts but are deficient in others like e.g. phosphorus.

In contrast to calcium phosphorus is a growth-limiting nutrient for aquatic animals and must be supplemented by feed. Phosphorus is together with calcium a main component of bone and scales in fish, but also in the exoskeleton of crustaceans. In addition, phosphorus is an essential element in muscle function.

It is therefore of high interest to find methods which help to make an efficient use of such nutrients and not to waste it in the environment.

The invention obviates adequately all the obstacles still met in fish and crustacean farming and responds adequately to an obvious unmet need in aquaculture.

The present invention relates, in particular, to a method which improves calcium and phosphorus uptake in teleost fishes and crustaceans and that improves growth and animal body composition and that yields better meat quality. This allows an easy and profitable reduction in such components in the feed whereas it still allows achieving the desired performance.

The invention is defined in the claims appearing here below. WO 2014/173862 3 PCT/EP2014/058057

Summary of the Invention A first objet of the invention is a composition for use in improving or enhancing resorption of minerals in fishes and crustaceans which comprises as an active ingredient Vitamin D metabolites or any source of same.

Another object of the invention is a feed or a feed material useful for improving or enhancing resorption of minerals in fishes and crustaceans which comprises as active ingredient Vitamin D metabolites or any source of same in combination with a source of carbohydrates, a source of lipids and a source of proteins.

Still another object of the invention is a feed additive useful for improving or enhancing resorption of minerals in fishes and crustaceans which comprises as an active ingredient Vitamin D metabolites or any source of same.

Still another object of the invention is a method for reducing phosphorus pollution in aquaculture, especially in fish and crustacean farming which comprises feeding said fishes and crustaceans by means of a feed or a feed material mentioned here above.

Additional objects of the invention shall appear in the specification below.

Detailed description of the Invention

According to the invention the terms “Vitamin D metabolites” mean either 25-Hydroxyvitamin D3 (Calcidiol, CAS number: 19356-17-3) , 25-Hydroxyvitamin D2; 1,25-Dihydroxyvitamin D3 (Calcitriol, CAS 32222-06-3) , 1,25-Dihydroxyvitamin D2; 1-a-Hydroxy-cholecalciferol , Ι-α-Hydroxyergosterol or 1,25-Dihydroxyvitamin D3-glycosides whereas the term “glycosides” defines glucoside, fructoside, galactoside units or any hexose and pentose unit bound to the aglycon. 1,25-Dihydroxyvitamin D3-glycosides represent one of the preferred Vitamin D metabolites used within the frame of the invention.

According to the invention the terms “source of Vitamin D metabolites” comprise preparations or part of plants, and namely leaves, obtained from plants like Solanum WO 2014/173862 4 PCT/EP2014/058057 glaucophyllum, Cestrum diurnum, Trisetum flavescens and Nierembergia veitchii. That list, however, is not limitative.

According to the invention said vitamin D metabolites can be applied alone, i.e. as unique vitamin entity or in addition to regular vitamin D whereby the addition to regular Vitamin D3 represents the preferred application.

According to the invention the term “fish” comprise any species suitable to farming or aquaculture and in particular marine fishes such as Atlantic salmon, milkfish, halibut, Atlantic cod, Pacific salmon, turbot, sole, sea bass, finfish and eels as well as freshwater fishes like trout e.g. rainbow trout, carps, tilapia, catfish, pangasius and bream.

According to the invention the term “crustaceans” comprise any species suitable to farming or aquaculture, ant in particular marine shrimps and prawns like e.g. tiger prawns and fresh water prawns, crabs and crayfish.

According to the invention the term “mineral” includes phosphorus, calcium and magnesium; phosphorus is first of all of concern within the frame of this invention as being a major source of pollution in fish farming.

According to the invention the terms “mineral resorption” means intestinal resorption or resorption through the scales (fishes) or through the shell (crustaceans).

According to the invention Solanum glaucophyllum plant material represents a preferred source of Vitamin D3 metabolites, especially of 1,25-dihydroxyvitamin D3-glycosides. This plant material is provided either from wild or from cultivated plants or hybrids of same like e.g. “HERVIT®” according to Plant Variety Certificate EU 25473.

As plant material one can use either dried leaves of said plant or any water extract of same, any water/alcohol extract of same or any enriched and purified water/alcohol extract of same (see examples below).

According to the invention 1,25-dihydroxyvitamin D3-glycosides are provided to the fish and crustacean in their feed at a daily dose of about 0.1 pg to about 100 pg of 1,25- WO 2014/173862 5 PCT/EP2014/058057 dihydroxyvitamin D3-glycosides per kg fish feed or crustacean feed, a dose which is usually expressed analytically as pg of 1,25-dihydroxyvitamin D3.

One uses preferably from about 0.2 to about 20 pg 1,25-dihydroxyvitamin D3-glycosides per kg of feed material and still more preferably form about 0.5 to about 10 pg 1,25-dihydroxyvitamin D3-glycosides per kg of feed material. The vitamin D metabolites can be incorporated to any feed material or component dedicated to any state of development of fish and crustacean.

Such feed or feed material can further comprise from about 25 pg to about 75 pg regular (synthetic) Vitamin D, e.g. (synthetic) Vitamin D3 per kg feed or feed material.

When applying 1,25-dihydroxyvitamin D3-glycosides in accordance with the provisions here above, and in particular when performing the application of a single dose of 1,25-dihydroxyvitamin D3-glycosides - e.g. provided as water soluble extract of Solanum glaucophyllum according to WO2009/129818) - or of synthetic 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 or of 1 -α-hydroxyvitamin D3 to adult Atlantic salmons one observes increased blood levels of 1,25-dihydroxyvitamin D3. When comparing the above mentioned fishes to untreated control subjects one also observes a substantial increased uptake of calcium and phosphorus from the standard feed provided to the said fishes.

According to another series of test performed with juvenile Atlantic salmon and making use of 1,25-dihydroxyvitamin D3-glycosides in the form of a water soluble extract of Solanum glaucophyllum (see above) on observes, after 30 days of feeding, a definite better growth rate, a better feed conversion and lower bone and scales deformations or distortions. Similar results have been achieved when using Panbonis® 10 as an alternative source of 1,25-dihydroxyvitamin D3-glycosides.

According to another object the invention one can decrease advantageously the phosphorus content in the feed or the feed component used in aquaculture when reducing the initial standard and generally recommended proportion for a given species and stage of development by 10 to 50 % of said initial proportion while adding thereto an efficient amount of Vitamin D3 metabolites referred to here above and while still achieving the same end performance, i.e. growth rate, meat and bone quality. WO 2014/173862 6 PCT/EP2014/058057

For example feeding adult Atlantic salmon with different doses of 1,25-dihydroxyvitamin 03-glycosides - provided as an extract of Solanum glaucophyllum (see above) - to diets with graded reductions of added feed phosphorus over 60 days resulted in a better growth rate, a 5 better feed conversion and less phosphorus in excreta when compared to a non-phosphorus-reduced control group. Similar results have been achieved by using a combination of Panbonis® 10 together with a feed having reduced phosphorus content as compared to a control group where phosphorus content of their diet was not reduced. 10 Thus, by means of the invention, one can advantageously reduce the phosphorus pollution in aquaculture, especially in fish and crustacean farming when feeding said fishes and crustaceans by means of a feed or feed additive mentioned here above.

When feeding adult Atlantic salmon with different doses of 1,25-dihydroxyvitamin D3- 15 glycosides- provided as an extract of Solanum glaucophyllum (see above) - to diets with graded reduction of added fish meal and fish oil and by replacing same with plant proteins and plant oils one observes, nevertheless, the same growth performance than that of the non-reduced control group. 20 Similar results have been achieved when using Panbonis® 10 as an alternative source of 1,25-dihydroxyvitamin D3-glycosides to a feed material in which a fraction of fish meal and of fish oil was replaced by plant proteins and vegetal oils, when compared to non-replaced control feed material. 25 When fishes or crustaceans are fed according to the invention one observes a definite improvement of their well-being which is reflected, in particular, in the quality of their meat and their bone and scales health and, also, in their growth rate and their feed conversion ratio.

One could also improve substantially the growth rate and reduce at the same tome the bone 30 deformation in farmed fishes and crustaceans by means of a feed or feed material or feed additive as referred to here above.

Consequently the invention further provides a method for improving growth rate and wellbeing of farmed fishes and crustaceans when a part of fish meal and fish oil is replaced by WO 2014/173862 7 PCT/EP2014/058057 vegetable proteins and oils by means of a feed or feed additive referred to here above. Following such a way one is today able to correct efficiently potential imbalances which would be caused by these feed substitutions. 5 The invention still further provides a method for improving growth rate and well-being of farmed fishes and crustaceans when a part of fish meal and fish oil is replaced by vegetable proteins and vegetal oils and supplemented at the same time with a feed or feed additive mentioned here above and in combination with the use of phytase enzymes when anti-nutrient phytic acid is present in said vegetable oils and proteins. 10

The incorporation of selected Vitamin D3 metabolites, especially of 1,25-dihydroxyvitamin D3 glycosides, is performed according to the techniques usually applied in this field for producing pellets or crumbles or similar feed material, namely extrusion, coating, spraying, etc. The selected metabolites are primarily mixed with the carbohydrates, a first portion of 15 lipids and the proteins and then subjected to extrusion to afford granule or pellet cores of. A protective coating made of the second portion of selected lipids is then sprayed onto the cores to provide the ready to use pellets. 20 Examples

Preparation of a Solanum glaucophyllum plant extract A selected batch of dry leaves of Solanum glaucophyllum has been macerated under agitation 25 at 40 - 60 °C for about 24 hours with the 5 to 12 liter per kg of a water/ethanol mixture.

Previous analytical assessment of the content in 1,25-dihydroxyvitamin D3-glycosides of various batches of dry leaves material available in the storehouse led to prepare an 85/15 in volume water / ethanol mixture. The use of the proper mixture allows the achievement of the 30 desired content of vitamin D3 derivative - approx. 100 ppm in this example. The liquid fraction was separated and set aside whereas maceration was repeated for a second turn of 24 hours. WO 2014/173862 8 PCT/EP2014/058057

The collected water / ethanol extracts were then combined and added with ascorbic acid in a ratio of 0.25 % by weight and pH of same was adjusted to 5 - 6.5 by means of a food-compatible acid, citric acid in this particular case.

The stabilized extract was then filtered and concentrated to approx. 30 to 50 % of dry matter content by means of vacuum drying technique and the resulting concentrated was poured onto a cationic exchanger resin equilibrated in its H+ form.

The collected effluent was filtered and then subjected to UHT sterilization (125° C for 1 min). After analytically assessment of the 1,25-dihydroxyvitamin D3-glycosides content, a specific amount of excipient, preferable maltodextrin, lactose or com starch, is added the sterilized material in order to standardize the final content of active ingredients in the plant extract -100 ppm of 1,25-dihydroxyvitamin D3-glycosides.

The standardized mixture is eventually spray dried or vacuum dried in a conventional spray drier to afford the desired Solanum glaucophyllum plant extract in powder form.

Alternatively Solanum glaucophyllum plant material can be subjected to an extraction by percolation with the water / ethanol solvent mix referred to above followed by the addition of stabilizing additives like e.g. ascorbic acid and pH adjustment, before subsequent UHT treatment and drying (vacuum drying or spray drying).

Preparation of a feed component (or feed additive): Panbonis® 10

Wheat middling (excipient) is intimately milled with dried leaves of Solanum glaucophyllum and then processed until obtaining a homogeneous powdered material. The inactive excipient is added to the dried leaves in order to obtain a standardized product comprising 0.001 % (weight) of 1,25-dihydroxyvitamin D3-glycosides (measured analytically as “active Vitamin D3” i.e. as 1,25-dihydroxyvitamin D3 or, expressed differently, as l,25(OH)2 D3); Panbonis® 10 is more conveniently characterized here below as comprising 10 pg 1,25-dihydroxyvitamin D3 / kg. WO 2014/173862 9 PCT/EP2014/058057

The analytical content of the excipient may vary according to the following range: 8 - 12 % (weight) of crude fibers, 14-25 % (weight) of starch. The mill processing is set in order to afford a particle size distribution comprised between ca. 50 and ca. 1000 pm. 5

Fish feeding experiments Experiment no 1

To a total of 116 brown trout (Salmo Salar) having each a body weight extending from 250 g 10 to 500 g to which was periodically provided a commercial grower feed (crude protein: 46%; crude fat 12%; carbohydrate 20%; crude fibers 3%; ash 9%; calcium “.0%; P 1.5%; vitamin A 5000IU/kg; vitamin D3 2500 IU/kg) and moving in separate tanks, one gave to each of same one capsule containing 1.75 pg/kg bodyweight of 1,25-Dihydroxyvitamin D3. At different times the fishes were anesthetized and blood was withdrawn from them, serum samples were 15 prepared and submitted to analytical quantification of 1,25-Dihydroxyvitamin D3, calcium and phosphorus.

Table 1: Serum l,25(OH)2D3, calcium and phosphor values

Blood Time Time Time Time Time parameter 0 hrs. 12 hrs. 24 hrs. 48 hrs. 72 hrs. l,25(OH)2D3 [pg/ml] 51 246 1254 391 72 Ca [mmol/L] 3.06 2.53 2.41 2.38 2.59 P [mmol/L] 3.18 3.18 3.70 4.24 4.48

One observes a blood serum increase of l,25(OH)2 D3 with a peak concentration at 24 hours followed by a progressive decrease down to the pretreatment level. A slight increase in calcium and phosphorus levels can only be seen 48 hours after administration. WO 2014/173862 10 PCT/EP2014/058057

Experiment no 2

Thirty-five juvenile rainbow trout (Oncorhynchus mykiss) having an average body weight of 19 gram were kept per tank in 4 replicates per treatment. The duration of the feeding 5 experiment extended over 73 days at a water temperature of 14°C. Fishes were fed once per day to apparent satiation with a diet according to Table 2. Panbonis® 10 was used as the source of 1,25(0H)2 D3, in a ratio of 200 mg/kg diet, respectively of 1000 mg/kg diet.

Table 2: Composition of the diet of Experiment 2

Raw materials (%) Control Panbonis-1 Panbonis-2 Fish meal 10.0 10.0 10.0 Wheat flour 12.0 12.0 12.0 Vegetable protein 67.8 67.8 67.8 Fish meal 10.0 10.0 10.0 Vitamin premix 0.4 0.4 0.4 Mineral premix 6.0 6.0 6.0 Cellulose powder 1.0 1.0 0.9 Panbonis® 10 — 0.020 0.100 Fish oil 3.0 3.0 3.0 Total 100 100 100

Table 3: Result providing by the parameters applied in Experiment 2

Indices Control Panbonis-1 Panbonis-2 Fish meal (%) 10 10 10 Initial fish number 35 35 35 Starting biomass (g) 674 668 666 Average body weight (g) 19.2 ± 1.5 19.1 ± 1.4 19.0 ±1 .3 Final biomass (g) 2,036 2,134 2,179 Average body weight (g) 58.2±12.9 61.0±13.5 62.2±15.6 Survival rate (%) 100 100 100 Weight gain (g) 1,362 1,466 1,512

Scrum analysis Panbonis(K) 10 (%) Control Panbonis-1 0.020 Panbonis-2 0.100 1,25(OH)2 D3 (pg ml)* 89.2±15.0 102.3=2.8 133.1=1=31.2 * Serum samples collected from 3 fishes were mixed to one specimen for the detection of l,25(OH)2 D3. WO 2014/173862 11 PCT/EP2014/058057

Average weight gain (%) 302 319 327

On observes a dose-dependent increase in average weight gain as result of the addition of the 1,25-Dihydroxyvitamin D3-glycosides provided by the various dosages of Panbonis® 10 The increase of serum concentration in 1,25-Dihydroxyvitamin D3-glycosides - measured as 5 l,25(OH)2 D3 - was also dose-dependent. The survival rate of the fishes was 100%.

Experiment no 3 10 Thirty-five juvenile rainbow trout (Oncorhynchus mykiss) of an average body weight of 28 gram were spread over 6 tanks during the whole treatment. Duration of the feeding experiment was 32 days at a water temperature of 14°C. Fish were fed once per day to apparent satiation with a diet as shown in Table 4. Panbonis was used as test article in a concentration of 0.1, 0.2, 1.0, 5.0 and 10 g/kg diet. 15

Table 4: Composition of the diet of Experiment 3

Raw Materials (%) Control Panbonis 0.1 g/kg Panbonis 0.2g/kg Panbonis 1.0g/kg Panbonis 5.0g/kg Panbonis 1 Og/kg Wheat flour 16.64 16.64 16.64 16.64 16.64 16.64 Vegetable protein 44 44 44 44 44 44 Fish meal 25 25 25 25 25 25 Vitamin premix 0.72 0.72 0.72 0.72 0.72 0.72 Mineral premix 1.2 1.2 1.2 1.2 1.2 1.2 Others 4.94 4.94 4.94 4.94 4.94 4.94 Defatted rice bran 1 0.99 0.98 0.9 0.5 0 WO 2014/173862 12 PCT/EP2014/058057

Panbonis® 10 0 0.01 0.02 0.1 0.5 1 Fish oil 6.5 6.5 6.5 6.5 6.5 6.5 Total 100 100 100 100 100 100

Table 5: Results of Experiment 3

Indicators Control Panbonis O.lg/kg Panbonis 0.2g/kg Panbonis l.Og/kg Panbonis 5.0g/kg Panbonis 10g/kg Fish meal(%) 25 25 25 25 25 25 Start biomass (g) 663 658 663 662 662 663 Aver, body weight (g) 28.8 ±2.6 28.6 ±2.3 28.8 ±2.8 28.8 ±2.2 28.8 ±2.6 28.8 ±2.6 Final Biomass (g) 1,260 1,248 1,304 1,494 1,410 1,301 Aver, body weight 0(g) 54.8 ± 6.8a 54.3 ± 7.6a 56.7 ± 5.6a 65.0 ± 7.0b 61.3 ± 5.5b 56.6 ± 6.0a Survival rate (%) 100 100 kkT 100 Too 100 Weight gain (g) 597 590 ~ 642 832 749 638 Aver, weight gain (%) 190 190 197 226 213 198 Ca content (g/fish) 0.42 0.39 0.43 0.54 0.46 0.44 P content (g/fish) 0.42 0.4 0.42 0.56 0.49 0.43 5

As result of this short term experiment it one observes a similar outcome than that of Experiment 2 in terms of growth performance. The trial shows an increased response for calcium & phosphor deposition in the fish. 10 The optimal dose was found at 1 gram Panbonis® 10 per kg of diet, what corresponds to 10 pg 1,25-Dihydroxyvitamin D3-glycosides per kg of fish diet.

Experiment no 4 (fish meal reduction experiment) 15

Forty juvenile rainbow trout (Oncorhynchus mykiss) having an average body weight of 26 grams in average were kept per tank in 6 replicates per treatment. The feeding experiment WO 2014/173862 13 PCT/EP2014/058057 took place over 62 days at a water temperature of 14°C. Fishes were fed once per day to apparent satiation with a diet as shown in Table 6. Panbonis® 10 was used as test article in a concentration of 1.0 g/kg diet.

Table 6: Composition of the diet of Experiment 4

Raw materials (%) Control Control low fish meal Panbonis 1.0 g/kg Fish meal 40 25 25 Wheat flour 18.99 14.64 14.64 Vegetable protein 35 44 44 Fish meal 40 25 25 Vitamin premix 0.35 0.72 0.72 Mineral premix 0.8 1.2 1.2 Fish oil 6.5 8.5 8.5 Others 0.36 4.8 3.3 Panbonis® 10 — — 0.10 Deffatcd rice bran — 1.14 2.54 Total 100 100 100

Table 7: Results of Experiment 4

Indicators Fish meal Low fish Panbonis control meal 1.0 g/kg Initial biomass (g) 1,048 1,034 1,033 Average body weight (g) 26.2±1.9 25.9±2.4 25.8±2.0 Final biomass (g) 3,247 3,058 3,444 Average body weight (g) 81.2±17.0‘' 76.4=19.5'’ 86.1±17. f Survival rate (%) 100 100 100 Weight gain (g) 2,199 2,023 2,411 Average weight gain (%) 310 296 333 WO 2014/173862 14 PCT/EP2014/058057

One observes that a reduction in fishmeal affected the weight performance in rainbow trout. However, the addition of 1 g/kg Panbonis® 10 to the diet having a lower content of fish meal improved the growth performance significantly although fishmeal inclusion was reduced in the feed.

As a conclusion from experiments 2, 3 and 4 shows that: - Calcium and Phosphorus utilization can be improved - Fishmeal replacement is possible - Improved growth performance is real - Improved serum levels of active vitamin D3 are attained. A further experiment was then performed in which the initial proportion of added phosphor in the diet was reduced.

Experiment 5 (Phosphorus reduction)

Thirty juvenile rainbow trout (Oncorhynchus mykiss) of 126 grams in average were kept per tank in 4, respective 3 replicates per treatment. Duration of the feeding experiment was 63 days at a water temperature of 15°C in a recirculating system.

Fish feed to feeding table to ensure no feed wastage and optimal feed conversion ratio (FCR) and fish were fed once per day to apparent satiation with a diet as shown in Table 8. Lighting was a constant 24h light regime. Dissolved oxygen was maintained above 7mg/l.

Positive control diet used commercially used standard P level of 0.90%, whereas the test diets used a P level of 0.70% at 26% below the positive control. Phytase was used at 750FTU/g. Panbonis was included at 0.15 and 0.3 g/kg diet and in conjunction with Phytase.

Table 8: Composition of the basal diet (negative control - NC)

Ingredient g/kg diet Fish meal 220 15

Soy protein concentrate 210 Wheat gluten 86 Corn gluten 60 Wheat meal 170 Lysine 6 Vitamin-mineral premix 20 Phosphate (MCP) - Rapcsccd oil 160 Fish oil 68

Table 9: Composition of the supplemented diet (negative control - NC)

Treatment code Panbonis Phytase, Inorganic P mg/kg U/kg g/kg Replicates Negative control (NC) - 4 NC + Phosphate 2 4 NC + Panbonis® 10 150 3 NC + Panbonis® 10 300 3 NC + Phytase 1750 l- 3 NC + Phytase + Panbonis® 150 750 3

Table 10: Fish weight at end of experiment, (FCR) & bone ash in Experiment 5

Parameters Positive Negative NC + NC + NC + NC + Control Control Phytase Phytase +Panl50 Panl50 Pan300 Fish end weight [g] 454 448 446 448 440 450 FCR 0.87 0.88 0.89 0.88 0.90 0.87 Total bone ash mean 49.8 45.3 50.1 50.3 47.6 45.7 WO 2014/173862 PCT/EP2014/058057

As result, the addition of the 1,25-Dihydroxyvitamin D3-glycosides containing product Panbonis to a diet reduced in phosphorus (from normal 0.9% to 0.7%) resulted in a definite WO 2014/173862 16 PCT/EP2014/058057 improvement of the feed conversion ratio and bone ash when the phosphorus reduced diet was supplemented with either Phytase or Panbonis® 10 or a mixture of both.

Experiment no 6

5 15 Japanese prawn (Marsupenaeus japonica - average body weight around 6.5 gram in 200L polycarbonate resin tank with (4 tanks ?) - water temperature 14°C - feeding once per day in the morning up to apparent satiation - dried Solanum glaucophyllum leaves constitute the source of 1,25-dihydroxyvitamin D3-glycosides. 10 Table 11: Composition of the feed formulation in Experiment 6

Table 11 Control-5 Test-9 Test-10 Test-11 Panbonis® 10 (%) - 0.001 0.01 0.10 l,25(OH)2Vit D3 (pg/kg) - 0.1 1 10 Animal protein 71.00 Fish meal 1.00 Mono Calcium Phosphate 4.00 Vitamin premix 2.00 Others 22.00 22.00 21.00 21.90 Total 100.00 100.00 1100.00 100.00 Level of Vit D3 derived from Vitamin premix and fish oil (pg/kg) 188

Tables 12,13 & 14: Results of Experiment 6

Table 12 Control-5 Test-9 Test-10 Test-11 Panbonis® 10 (%) — 0.001 0.01 0.10 1,25(013)2Vit D3 (pg/kg) — 0.1 1 10 Initial number 45 45 45 45 Average body weight (g) 6.4±0.9 6.5±0.9 6.4±0.8 6.5±0.8 WO 2014/173862 17 PCT/EP2014/058057

Final number 44 45 44 45 Average body weight (g) 11.6±1.6 11.5±1.3 n.3±i.i 11.7Ϊ1.5 Total weight gain (g) 222.0 222.5 210.1 233.2 Average weight gain (%) 181 "ill 111 180

Shell hardness *

Table 13 Control-5 Test-9 Test-10 Test-11 Panbonis® 10 (%) — 0.001 0.01 0.10 1.25(011),Vit Di (g) — "o.\ 1 ~ 10“ Shell hardness (g) 175±39.8a 283±118b ~3Ϊ5±65Λ^" 267±55.4b 1st abdominal segment (g) il6.6±31.5a 178'6±26.3b 219.0±5L8C ”i78’2±49.9’b” 5 * Detected by Rheometer CR300 (Sun Scientific Co. Ltd.).

Calcium content *

Table 14 Control-5 Test-9 Test-10 Test-11 Panbonis® 10 (%) — 0.001 0.01 0.10 l,25(OH)2Vit D3 (g) — 0.1 1 10 Ca content (% in dry matter) 4.42 5.13 5.68 5.19 10 * 6 shrimps were collected from each treatment and mixed to afford one sample for Ca content measurement.

One observes - see Table 13 - a significant improvement of shell concerning the crustaceans fed with the composition according to the invention, hardness as compared to the control 15 population. Table 14 further shows a parallel improvement of the calcium absorption. 2014257626 28 Nov 2016

17A

The discussion of the background to the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was part of the common general knowledge in Australia or elsewhere as at the priority date of this application. 5 Further, and unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense - that is to say, in the sense of “including, but not being limited to” - as opposed to an exclusive or exhaustive sense - that is to say meaning “including this and nothing else”. .0

Claims

1. A method for improving growth rate of farmed fishes and crustaceans fed on fish meal and fish oil wherein part of said fish meal and fish oil is replaced by vegetable proteins and vegetal oils supplemented with an efficient amount of 1,25- dihydroxyvitamin D3-glycosides provided from a source consisting of dried leaves of Solanum glaucophyllum in order to correct imbalances introduced by said replacement.

2. A method for improving growth rate of farmed fishes and crustaceans fed on fish meal and fish oil wherein part of said fish meal and fish oil is replaced by vegetable proteins and vegetal oils supplemented with an efficient amount of 1,25- dihydroxyvitamin D3-glycosides provided from a source consisting of dried leaves of Solanum glaucophyllum and further supplemented by means of phytase enzymes when anti-nutrient phytic acid is present in said vegetable oils and proteins.

3. The method according to any of the preceding claims wherein the fishes are selected from marine fishes such as Atlantic salmon, milkfish, halibut, Atlantic cod, Pacific salmon, turbot, sole, sea bass, finfish and eels as well as freshwater fishes like trout e.g. rainbow trout, carps, tilapia, catfish, pangasius and bream.

4. The method according to any of the preceding claims wherein the crustaceans are selected from marine shrimps and prawns like tiger prawns and fresh water prawns, crabs and crayfish.

5. The method according to any of the preceding claims wherein said 1,25-dihydroxyvitamin D3-glycosides are provided to the fishes or crustaceans in their feed at a daily dose of about 0.1 pg to about 100 pg of 1,25-dihydroxyvitamin D3-glycosides per kg fish or crustacean feed.

6. The method according to any of the preceding claims wherein said 1,25-dihydroxyvitamin D3-glycosides are provided in addition to regular Vitamin D3 originating from other source.