Note: Descriptions are shown in the official language in which they were submitted.
~2~41
Means for enhancing the protein synthesis of animals
This invention relates to feedstuff additives for
enhancing the protein synthesis of animals.
It is known that the biological element ~inc is
indispensable for the growth of juvenile animals be-
cause it is a structural component of ribonucleicacid polymerases and deoxyribonucleic acid polymerases
(W.S. Wegener and A. H. Romano; Science, 142 (1963),
1669 - J.E. Coleman; Biochem. Biophys. Res. Comm., 60
~1974), 641 - M. Fujioka and I. Liebermann, J. Biol~
Chem., 239 (1964), 1164 - J. P. Slater, A.S. Mildvan and
L.A. Loeb; Biochem. Biophys. Res. Comm., 44 (1971), 37),
and because it is an activator of nu~erous enzyyme systems
~W. Seeling and I. Seeling; ZinkstoE~wechsel- Bedeutung
fur Klinik und Praxis; TM-Verlag, Bad Oeynhausen, 1979).
Conversely, a scarcity of zinc in animals causes a dis~
ruption of protein synthesis, growth retardation, loss
of hair, reduction of endochondral bone growth and other
pathophysioloqical disruptions of metabolism ~R.C. Thever
and W.G. Hiekstra, J. of Nutrit.f 89 (1966), 448 - H.G. Day
and E.V. McCollum; Proc. ~oc. Exp. Biol. Med., 45 (1940),
282 - R.H. Follis, H.G. Day and E.V. McCollum; J. of
Nutrit., 22 (1941), 223 - E. Hove, C.A. Elvehjem and
E.G. Hart; A~. J. Physiol., 113 ~1937), 768 - F.E. Stirn,
C.A. Elvehje~ and R.B. Hart; J~ Biol. Chem., 109 (1935),
347).
841
~ ontinuing from this, N. Danzig (West German
Offenlegungsschrift 2,704,746) proved that zinc methionate
(1:2) exerts a growth enhancing effect on calves, cattle
and sheep when simultaneously reducing the feed during
intensive fattening, wherein the optimum dosage lies in
the region of 250 to 1500 ppm zinc methionate (1:2).
It is also known that a deficiency of the biological
element copper in young animals, which have a high copper
requirement, also produces a stunting of growth, particu-
larly in association with the bones, the reproductivesystem and the central nervous system (Th. Bersin:
Biochemie der Mineral- and Spurenelemente, Akad
Verlagsges., Frankfurt-Main, 1963~. Under human medical
conditions, it has been observed that upon intravenous
hyperalimentation with amino acid solutions, as well as
upon the acceleration of wound healing with zinc, mere
zinc substitution does not always lead to a reproduc-
ible result and that, on the contrary, Erequént Eailures
occur. Such controversial reactions can be explained by
a prevailing deficit of copper r which can even increase
during an ongoing zinc application. Zinc and copper have
the same orbital occupation and therefore at irst operate
antagonistically in metabolism, but the two elements can
also work to advantage in the sense of having a synergistic
~5 ef~ect (M. D~vid, M.D. Ota, V. Bruce, M.D. MacFadyen,
T. Elisabeth, B.S. Gum, J. Stanley and M.D. Dudrick; in:
zinc and Copper in Clinical Medicine, SP Medical and
Scientific Books, New York, 1976~. Deficiency of copper
limits the complete utilization of food, as can be
in~erred from a study carried out by A. Cordano (in:
Zinc and Copper in Clinical Medicine; loco cit.).
The growth enhancing effect of copper sulfate com-
monly used when fattening nogs at a dose of 250 ppm/feed
has been attributed to the germicidal and fungicidal pro-
perties of copper, but these antibacterial effects haverecently been cast in doubt as the result of detailed
~2~41
3 -
research, whereby priority has been ascribed to the
pharmacodyn~mic effects of the nutritive properties
of copper (M.G. Beyer; Landw. Forschung, 29 (1970),
53 - M. Kirchgessner and E. Grassman; Z. Tierphysiol.,
Tierernahr. and Futtermittelkde., 26 (1970), 3 -
H. Meyer and H. Kroger; Ubers. Tierern., 1 (1973), 9).
In this sense, N~ Danzig has confirmed that copper
(II)-methionate in doses of 250-900 mg is suitable as a
growth enhancing supplement for the feeding of hogs (DP
2546051).
We have found that the chelate of asparaginic acid
(aspartic acid) with zinc and with copper is an especially
effective agent for the improvement of protein synthesis
in the liver, and is considerably superior to other
compounds used for the same purpose, e.g. copper sulphate,
copper (II)-methionate and zinc methionate.
According ~o one aspect of the invention there is
provided a feedstuff additive for improving protein
synthesis during the fattening of animals, comprising
zinc aspartate at a dose of about 10 to about 100 mg.
zinc as zinc aspartate per kg. of feedstuff.
According to another aspect of the invention there
is provided a feedstuff additive Eor improving protein
synthesis during the fattening of animals, comprising
copper aspartate at a dose of about 10 to about 100 mg.
copper as copper aspartate per kg. of feedstuEf.
According to yet another aspect oE the invention
there is provided a feedstuff additive for improving
protein synth~sis during the fattening of animals,
comprising a mixture oE zinc aspartate and copper
aspartate with about 3 to about 20 mg. zinc as zinc
aspartate per kg. of feedstuff and about 25 to about
100 mg. copper as copper aspartate per kg. of Eeedstuff.
The invention and its preferred forms are described
in more detail below with reference to the accompanying
drawings, in which:
Fig. l is graphs showing the protein synthesis rates
in the livers of male rats after feeding for two weeks
with feedstuÇfs having various additives; and
Fig. 2 is a graph showing the protein synthesis rates
of liver cell microsomes of rats after feeding with
various copper salts over two weeks, the doses being given
in mg Cu2 /kg. feed.
The following description illustrates the methods by
which the invention was evaluated and tested.
Methods
Groups of six male Wistar rats having a starting
weight of ca. 609. were held in Makrolon cages on
sprinkled wood shavings. The ambient temperature was
24 ~ 1C. Water and feed formulated according to Drepper
and Weik (1972) was provided for each group of 6 rats.
The feed formulation is given in Table 3. According to
the experimental plan itemized in Table l, zinc aspartate
was added to the feed for the rats of groups 2 and 3,
copper aspartate was added to the Eeed for the rats of
groups 4 and 5 and a mixture of zinc aspartate and copper
aspartate was added to the feed for the rats of groups 6
and 7 in the doses given in the table.
The eEfect of the additions of copper aspartate to
the feed in the doses given in Table 2 was compared with
the efEect oE the additions of copper sulfate or copper
methiorlate to the Eeeds of the rat groups according to
experimental plan B (Table 2).
Table 1: Experimental Plan A
Dose Duration of
~roup n Substance (ppm Metal) Feedin~
6 Control
2 6 Zn-aspartate 3,5
3 6 Zn-aspartate 7,0
4 6 Cu-aspartate 50,0 2 weeks
6 Cu-aspartate 100,0
6 6 Zn-& Cu-aspartate 3,5 -~ 50,0
7 6 Zn--& Cu-aspartate 7,0 * 50,0
Table 2 : Experimental Plan B
Dose Duration of
Substance ppm Metal ppm Substan~e Feeding
1 6 Control
2 6 Cu~O4 2562,~
3 6 CuSO4 50125,6
6 CuSO4 1~0251,3
6 Cu-aspartate 25132r3 2 weelcs
6 Cu-aspartate 50264,6
7 6 Cu-aspartate 100529,1
8 6 Cu-methionate25119,0
9 6 Cu-methionate50238,1
6 Cu-metilLonate100476,2
4~
~able 3 ; Formulation of Mixed Feed ~ccording
to Drepper and Weik (1972)
Formulation of the feed:
18 % Fish meal (64 ~ raw protein)
13 % Soya meal extract (43 ~ raw protein)
30 ~ Corn meal
28 % Wheat bran
2 ~ Saccharose
4 ~ Soya oil
3 % Mineral mixture
2 % Vitamin mixture
100 %
Raw Food Contents ~Weender Analysis)
raw protein 18,3 %
raw fat 6,3
raw fibre 9,8
raw ash 8,3 ~
Minerals and trace elements Vitamins ( /kg feed)
Ca10,0 9 ~ Vi~. A15000 I.E.
P 7,5 g Vit. D3 500 I.
Mg3,5 9 Vit. E 150 I.E.
Na3,5 ~ Vit. ~ 10 mg
K14,0 g ~ HCl 20 mg
Fe170,0 mg Vit. B2 20 mg
Mn90,0 mg Vit. ~6HC115 mg
Zn20,0 mg ~ /kg feed Vit. B1220 ~g
Cu5,0 mg Ca-Pantothenate 50 mg
I0,4 mg Nicotinic acid 50 mg
Mo0,25 mg Choline 1 g
F3,6 mg Folic acid10 mg
Se0,22 mg Biotin 200 ~g
Co 0,12 mg ~ Inositol100 mg
p-Aminobenzoin 100 mg
Vit. C. 20 mg
Methionine 1.5 g
- 7 -
Measurement of Protein Synthesis Rate in the Liver ~ccording
to Kaemmerer and Dey-Hazra (Vet. Med. Nachr. 2 (1980) 99).
At the end of the feed period, the animals were
killed at 8 a.m. MEZ (Middle European Time) by cervical
dislocation.
The working up of the liver tests was carried out
according to the directions of Appel (Diss. Hanover, 1973):
3g of liver was homogenized in an ice cooled TKM-
buffer-saccharose-solution, the solution was then
centrifuged for 20 minutes in an ultra centrifuge at
lOOOOg and the upper layer was drawn off as "microsomal
cell fluid" for protein synthesis~ After the determination
of the protein content in the microsomal cell fluid by
means of the Biuret-method, the protein concentration in
lS the mix~ure was adjusted to 1.0 mg/ml with TKM-bu~fer
(tris potassium chloride magnesium chloride). Tilereafter,
0.15 mL of the reaction mixture according to Siler and
Fried (Biochem. J., 109 (196~), 185), 5~ pivruvatekinase-
solution and ~.1 ml 14C_amino acid mixture were added to
0.25 ml of the resulting microsomal cell fluid (~ 0.25 mg
protein), and incubated at 37C for 35 minutes. The
residue was washed 8 to 12 times with trichloracetic acid
(10%) and, upon reachin~ the reEerence value, measured in
a PRI~S (Trade Mark)-flui~ity-scintillation counter PL
(Paclcard) after the addition oE Di~estin ~ (~erck) ~or
dissolvin~ the protein and the addition o~ scintillation
fluid (6 ml Lipoluma ~t Baker). For further details
of the method see Kaemmerer and Dey-Hazra (loco cit.).
Results:
The results oE the comparison tests of zinc aspartate,
copper sulfate, copper aspartate and copper methionate,
as well as the com~ination of zinc aspartate and copper
aspartate, in rats are shown in Table 4 below and Fig. 1.
~2~
Table 4: Results of the Determination of the Rate of Protein Syn-
thesis in the Livers of ~ats After Feeding for 2 Weeks
Dose CPM % re.
Substance (ppm Metal) x s ControL Significance
Control - 345 42
Zinc aspartate 3,5 345 24 0
zinc-aspartate 7,0 382 44 + 10,7 not signif.
Copper-aspartate 50,0 412 43 + 19,4 significant
Copper-aspartate 100,0 41~ 50 + 21,4 significant
Zinc- & copper-aspartate 3,5+50 443 26 + 28,4 highly signif.
Zinc- ~ copper-aspartate 7,0+50 420 24 ~ 21,7 highly signi~.
Control - 390 48
Zinc-methionatè 3,5 381 24 - 2,3 not signif.
Zinc-methionate 7tO 356 38 - 8,7 not signif~
Zinc-me~hionate 14,0 391 59 + 0t3 not signif.
Table 5: Number of Rats Having an Increase of Protein
Synthesis After Copper ~ddition to the Feed
DoseAnimals Showin~ an Increase o~ Protein Synthesis
(ppm Cu )(6 Animals per Group)
CuSO~ Copper-aspartate
1 2
4 6
100 5 6
34:1
Table 6: Optimal Value of the Protein Synthesis in Rat Livers
ppm Cu/Feed ppm Cu/FeedStatistical
Substance~Substance) (Element)Safeguard
CuS~4 125,7 50
CUSO4 251,3 100 x
Copper-aspartate 264,6 50 xx
Copper-aspartate 529,1 100 xxx
Copper-methionate238,1 50 x
Co,oper-methionate476,2 100
Discussion of Tables 4-6 and of Fig. 2:
COPPERSULFATE:
Coppersulfate in this experimental procedure casued a
slight reduction oE the protein synthesis in the liver of
6.6% at a dose of 25 mg Cu/Kg feed. This effect was not
statistically sound. In contrast, a dose oE 100 mg Cu/Kg
feed resulted in an increase oE 31DO~ of the liver protein
synthesis, which had a signi~icant status in comparison
with the control group.
COPPER ASPARTAT~:
The protein synthesis was increased by 3~3~r 22.7~
and 33 . 5% respectively upon the use of 25, 50 or 100 mg
copper~kg feed. The measured values are significant for
50 mg and llighly significant for 100 mg copper/kg Eeed.
COPPER METHIONATE:
Upon the feeding of 25 mg COQper methionate~kg feed,
the rate of protein synthesis in the liver was increased
by 5.6%, at 50 mg copper methionate by 22.0~, which is
stati~tically signiEicant, and at 100 mg merely by 16.7~.
According to the Eeedstuff formulation for hogs, the
feed may contain 125 mg copper/kg feed, and for piglets up
to 16 weeks it may contain 200 mg copper/kg feed. Accord-
ing to Kirchgessner (Bayer. Landw. Jb., 50 (1973), 79) a
copper dose outside the requirements creates no improved
i
~2~8~
-- 10 --
result and no be~ter health. Tlle current view requires
a minimum dose of from 100 mg copper/kg feed for the
trigg~ring of a nutritive effect. Quantities in the
region of from 62-63 mg/kg feed are ineffective, as shown
by Meyer and Kroger (Ubers. Tierern., 1 (1973), 44).
~hese conclusions confirm the findings of Kirchengessner
and Friesecke (Wirkstoffe in der Praktischen Tierernahrung;
Bayer. Land. Verlg., Munchen 1966), that anabolic e~fects
fail to appear, if the amount of copper in the feed is
reduced to about 50 mg/kg feed. Kaemmerer and Kietzmann
(D. tierartzl. Wochenschr., in print) show that the
increase of the liver protein synthesis upon the addition
of 100 mg copper sulphate/kg feed is larger than at low
concentrations. In contrast, at 50 mg copper~kg feed as
aspartate or methionate, just as many animals reactl
as can be seen from Fig. 2, which also shows the large
superiority of copper aspartate.
ZINC ASPARTATE:
Zinc aspartate increases the protein synthesis in the
liver by 10~7% upon feeding at 7 mg zinc/kg feed.
ZINC METHIONATE:
Zinc methionate in doses o~ 3.5 or 7.0 or 14.0 mg
zinc/kg Eeed causes no increase in the protein synthesis
in the livers of rats; in comparison with the control.
At the lower doses even a slight ciecrease oE the albumin
synthesis was measured.
THE COMBINATION OF ZINC AND COPPER ASPARTATES
The combined feeding of 50 mg copper aspartate/kg feed
and 3.5 mg or 7.0 mg zinc aspartate/kg feed, caused highly
signiEicalt increases of 28.4% and 21.7% of protein syn-
thesis in the liver in comparison with the control group.
