Note: Descriptions are shown in the official language in which they were submitted.
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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,
modern 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. Stocking density as a cause of stress and growth anomalies
2. Over fertilization of coastal areas caused by excreta and wasted feed
3. The use of fish meal and fish oil as feed components
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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.
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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-
Hydro xy- cholecalciferol , 1 -a-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
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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. "HERVITO" 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 iug to about 100 iug of
1,25-
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dihydroxyvitamin D3-glycosides per kg fish feed or crustacean feed, a dose
which is usually
expressed analytically as iug of 1,25-dihydroxyvitamin D3.
One uses preferably from about 0.2 to about 20 iug 1,25-dihydroxyvitamin D3-
glycosides per
kg of feed material and still more preferably form about 0.5 to about 10 iug
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 g to about 75
iug 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 W02009/129818) - or of synthetic 1,25-
dihydroxyvitamin D3,
25-hydroxyvitamin D3 or of 1-a-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 Panbonis0 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.
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For example feeding adult Atlantic salmon with different doses of 1,25-
dihydroxyvitamin D3-
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
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
Panbonis0 10 together with a feed having reduced phosphorus content as
compared to a
control group where phosphorus content of their diet was not reduced.
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-
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.
Similar results have been achieved when using Panbonis0 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.
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
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 well-
being of farmed fishes and crustaceans when a part of fish meal and fish oil
is replaced by
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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.
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.
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
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.
Examples
Preparation of a Solanum glaucophyllum plant extract
A selected batch of dry leaves of Solanum glaucophyllum has been macerated
under agitation
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
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.
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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 corn 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): Panbonis0 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 1,25(OH)2
D3);
Panbonis0 10 is more conveniently characterized here below as comprising 10
iLig 1,25-
dihydroxyvitamin D3 / kg.
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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 gm.
Fish feeding experiments
Experiment no 1
To a total of 116 brown trout (Salmo Salar) having each a body weight
extending from 250 g
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
50001U/kg; vitamin D3 2500 IU/kg) and moving in separate tanks, one gave to
each of same
one capsule containing 1.75 gg/kg bodyweight of 1,25-Dihydroxyvitamin D3. At
different
times the fishes were anesthetized and blood was withdrawn from them, serum
samples were
prepared and submitted to analytical quantification of 1,25-Dihydroxyvitamin
D3, calcium
and phosphorus.
Table 1: Serum 1,25(OH)2D3, calcium and phosphor values
Blood Time Time Time Time Time
parameter 0 hrs. 12 hrs. 24 hrs. 48 hrs. 72 hrs.
1,25(OH)2D3 [pg/m1] 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 1,25(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.
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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
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. Panbonis0 10 was
used as the
source of 1,25(OH)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 1
7 H
Vitamin premix 0.40.4 0.4
1-. H
Mineral premix 6.0 6.0 6.0
1- H
Cellulose powder 1.0 1.0 0.9
-t-- H
Panbonis0 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
1
i Indices Control Panbonis-1 Panbonis-2
I Fish meal (%) 10 10 10
1
! Initial fish number 35 35 35
I Starting biomass (g) 674 668 666
! Average body weight (g) 19.2 1.5 19.1 1.4 19.0 1 .3
i 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
i
I Weight gain (g) 1,362 1,466 1,512
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Average weight gain (%) 302 319 327
!Serum analysis - Control Panbonis-1 Panbonis-i-1
Panbonis 10 (%) 0.020 0.100
1,25(OH)2 D3 (m/m1)* 89.2 15.0 102 .3 2 .8 133 .1
31.2
* Serum samples collected from 3 fishes were mixed to one specimen
for the detection of 1,25(OH)2 D3.
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 in1,25-Dihydroxyvitamin D3-glycosides -
measured as
1,25(OH)2 D3 - was also dose-dependent. The survival rate of the fishes was
100%.
Experiment no 3
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.
Table 4: Composition of the diet of Experiment 3
Raw Materials (%) Control Panbonis Panbonis Panbonis Panbonis
Panbonis
0.1g/kg 0.2g/kg 1.0g/kg 5.0g/kg 10g/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
1Others 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
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Panbonis 10 0 0.01 0.02 0.1 0.5 1
1Fish oil 6.5 6.5 6.5 6.5 6.5
6.5
Total 100 100 100 100 100
100
L - _ _
Table 5: Results of Experiment 3
Indicators Control Panbonis Panbonis Panbonis Panbonis
Panbonis
0.1g/kg 0.2g/kg 1.0g/kg 5.0g/kg 10g/kg
I-
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 56.7 65.0 61.3
56.6
7.6a 5.6a 7.0b 5.5b
Survival rate (%) 100 100 100 100 100 100
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
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.
The optimal dose was found at 1 gram Panbonis 10 per kg of diet, what
corresponds to 10
iug 1,25-Dihydroxyvitamin D3-glycosides per kg of fish diet.
Experiment no 4 (fish meal reduction experiment)
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
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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
IRaw materials (%) Control Control low Panbonis i
I fish meal 1.0 g/kg
Fish meal 40 25 25
1
1Wheat flour 18.99 14.64 14.64
II Vegetable protein 35 44 44
1Fish meal 40 25 25
liVitamin premix 0.35 0.72 0.72
1Mineral premix 0.8 1.2 1.2
1Fish oil 6.5 8.5 8.5
1Others 0.36 4.8 3.3
1
1Panbonis 10 ---- ---- 0.10
1Deffated rice bran ---- 1.14 2.54
1Total 100 100 100
Table 7: Results of Experiment 4
I Indicators Fish meal Low fish Panbonis
I control meal 1.0 g/kg
Initial biomass (g) 1,048 1,034 1,033
,
,
lAverage body weight (g) 26.2 1.9 25.9 2.4 25.8 2.0
1Final biomass (g) 3,247 3,058 3,444
I Average body weight (g) 81.2 17.0a 76.4 19.5b 86.1 17.1a
1Survival rate (%) 100 100 100
,
1
1Weight gain (g) 2,199 2,023 2,411
I Average weight gain (%) 310 296 333
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One observes that a reduction in fishmeal affected the weight performance in
rainbow trout.
However, the addition of 1 g/kg Panbonis0 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
1Fish meal 220
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Soy protein concentrate 210
1Wheat gluten 86
Corn gluten 60
Wheat meal 170
Lysine 6
IVitamin-mineral premix 20
I Phosphate (MCP)
Rapeseed oil 160
1Fish oil 68
Table 9: Composition of the supplemented diet (negative control ¨ NC)
Treatment code Panbonis Phytase, Inorganic P Replicates
mg/kg U/kg g/kg
Negative control (NC) 4
NC + Phosphate 2 4
NC + Panbonis 10 150 3
! NC + Panbonis 10 300 3
I NC + Phytase 750 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 Pan150 Pan300
+Pan150
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
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
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improvement of the feed conversion ratio and bone ash when the phosphorus
reduced diet was
supplemented with either Phytase or Panbonis0 10 or a mixture of both.
Experiment no 6
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.
Table 11: Composition of the feed formulation in Experiment 6
Table 11 Control-5 Test-9 Test-10 Test-11
Panbonis0 10 (%) - 0.001 0.01 0.10
1,25(OH)2Vit D3 (m/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 188
fish oil (m/kg)
Tables 12, 13 & 14: Results of Experiment 6
Table 12 Control-5 Test-9 Test-10 Test-11
Panbonis0 10 (%) ---- 0.001 0.01 0.10
1,25(OH)2Vit D3 (m/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
CA 02910568 2015-10-22
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 11.3 1.1 11.7 1.5
Total weight gain (g) 222.0 222.5 210.1 233.2
Average weight gain (%) 181 177 177 180
Shell hardness *
Table 13 Control-5 Test-9 Test-10 Test-11
Panbonis0 10 (%) ---- 0.001 0.01 0.10
1,25(OH)2Vit D3 (g) ---- 0.1 1 10
Shell hardness (g) 175 39.8a 283 118b 315 65.1b 267 55.4b
1st abdominal segment (g) 116.6 31.5a 178.6 26.3b 219.0 51.8c 178.2 49.9b
* Detected by Rheometer CR300 (Sun Scientific Co. Ltd.).
Calcium content *
Table 14 Control-5 Test-9 Test-10 Test-11
Panbonis0 10 (%) ---- 0.001 0.01 0.10
1,25(OH)2Vit D3 (g) ---- 0.1 1 10
Ca content (% in dry
4.42 5.13 5.68 5.19
matter)
* 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
population. Table 14 further shows a parallel improvement of the calcium
absorption.
