CA2150892C - A ruminant feed composition and a method for improving feed efficiency - Google Patents
A ruminant feed composition and a method for improving feed efficiency Download PDFInfo
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- CA2150892C CA2150892C CA002150892A CA2150892A CA2150892C CA 2150892 C CA2150892 C CA 2150892C CA 002150892 A CA002150892 A CA 002150892A CA 2150892 A CA2150892 A CA 2150892A CA 2150892 C CA2150892 C CA 2150892C
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- ruminant
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
- A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S426/00—Food or edible material: processes, compositions, and products
- Y10S426/807—Poultry or ruminant feed
Abstract
A ruminant feed composition for depressing rumen methanogenesis and improving feed efficiency, which is safe and does not adversely affect on a ruminant, and a method for feeding the composition to a ruminant. As an effective component of the feed composition, cysteine and/or its salts are contained.
Cysteine and/or its salts are fed to a ruminant in an amount of 0.02 to 0.21 g sulfur equivalentkg body weight- 75 per day.
Cysteine and/or its salts are fed to a ruminant in an amount of 0.02 to 0.21 g sulfur equivalentkg body weight- 75 per day.
Description
-,-, A RUMINANT FEED COMPOSITION AND
A METHOD FOR IMPROVING FEED EFFICIENCY
BAC~(G:ROUND OF T'HE INVENTION
This invention relates to a composition which is fed to a ruminant so that t:he methanogenesis in the rumen thereof is depressed and the feed c~f.ficiency is improved. The present invention also relates to a method for depressing the methanogenesis in 'the rtunen of a ruminant and improving feed efficiency by the use of i;he composition.
Methane gas is generated in the rumen of a ruminant, such as cattle, sheep, goal: and the like, and emitted in the air in a large amount, and :>uch met;hanogenesis (methane generation) has become a serious problem because methane is one of potent greenhouse gases contributing global warming. The methanogenesis means energy loss of feed which are taken by a ruminant. From this standpoint also, the rumen methanogenesis is not preferable. In a rumen, extremely various kinds of microorganisms exist and contribute to fermentation, resulting in the production of various metabolites. Methane is one of the metabolites, produced by methane-producing bacteria. On the other hand, propionic acid is also produced by rumen fermentation as a result of microbial fermentation of carbohydrates, and it is thought that the energy metabolism depends upon the amount of propionic acid produced. In detail, it is thought that energy utilization efficiency depends upon the final position, namely, propionic acid or methane, to which electron geneoated by the oxidation of energy source in a rumen is transferred. Conventionally, it has been known that an ionophore antibiotic:, such as monensin and salinomycin, coordinates the flow of electrons so that the rumen methane 1 production is depressed and the propionic acid production is increased, and recently, the ionophore antibiotic is widely used for growing beef cattle. It is reported that the use of an ionophore antibiotic brings about 10%
!5 increase of the feed efficiency of a ruminant ("FEED, Revised", 1985, edited by Hiroshi Morimoto, published by Youkendo, Japan, pp.254-255; Ryoji Onodera et al., "Livestock Nutrition", 1989, published by Kawasima Shoten, p.184).
:LO However, since the ionophore such as monensin and salinomycin is an antibiotic, it has an adverse effect such that it depresses the activities of not only methane-producing bacteria but also the other rumen microorganisms. There=_fore, attention should be drawn to 1.5 the dosage so that essential rumen microorganisms may not be damaged. Further, there is a problem that even though an ionophore ant:ibiot:ic is initially effective, the methane-producing baci:eria in a rumen attain resistance to the ionophore~ antibiotic with the lapse of time.
20 Cross-resistance between the antibiotics is another problem in the practice as feed additives (C. J. Newbold et al., Journal of Applied Bacteriology, 1993, 75:129-134) .
SLJMMARY OF THE INVENTION
25 The present inventor has made extensive and 1 intensive studies wii:h a view to depressing the rumen methanogenesis effecl:ively without the above-mentioned problems. To this end, the inventor has examined various chemical compounds as a feed additive. As a result, the present inventor has surprisingly found that when a specific amino acid, cysteine, is fed to a ruminant, not only rumen methanogenesis, but also oxygen consumption, carbon dioxide gas production and propionic acid production, one of volatile fatty acids (VFA), are :10 markedly depressed, contributing the increase of feed efficiency. The present invention has been made based on such a novel finding.
Therefore, it is an object of the present invention to provide a novel ruminant feed composition which is :L5 excellent in de~~ressing the rumen methanogenesis and increasing feed efficiency.
It is another object of the present invention to provide a method for effectively depressing rumen methanogenesis and improving feed efficiency of a 20 ruminant.
The foregoing and other objects are achieved by providing a ruminant feed composition for feeding a ruminant to depress methanogenesis in the rumen thereof and improving feed efficiency, which comprises at least 25 one substance selected from cysteine and its salts, and also achieved by a method which comprises feeding at least one substance selected from cysteine and its salts to ruminant in an amount of 0.02 to 0.21 g sulfur equivalent'kg body weight-'~Sper day as a total of said at least one substance.
In another aspect, the present invention provid~s a method for depressing methanogenesis in the rumen of a ruminant and improving feed efficiency, which comprises feeding at least one substance selected from the group consisting of cysteine and its salts to a ruminant in a total amount of 0.02 to 0.1 g sulfur equivalent/kg of metabolic body weight per day.
In another aspect, the present invention provides use of a ruminant feeding composition comprising at least one substance selected from cysteine and its salts for depressing methanogenesis in the rumen of a ruminant and improving feed efficiency.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG.1 is a graph showing the effect of the ruminant feed composition of the present invention on the depression of rumen methanogenesis;
FIG.2 is a graph showing the effect of the ruminant feed composition of the present invention of the depression of carbon dioxide production in a rumen; and - 4a -FIG.3 is a graph showing the effect of the ruminant feed composition of the present invention on the depression of oxygen consumption in a rumen.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there is provided a ruminant feed composition for feeding a ruminant to depress methanogenesis in the rumen thereof and improving feed efficiency, which comprises at least one substance selected from cysteine and its salts.
The ruminant feed composition of the present invention comprises at least one substance selected from cysteine and its salts. As the cysteine and its salts, any optical isomers, that is, L-, D- and DL-isomers, may _2150892 1 be mentioned in the ;present invention.
As the salts of cysteine, there may be mentioned any salts which can be fed to a ruminant. For example, there may be mentioncad cysteine hydrochloride, but not restricted thereto. There is no difference in the effect of methanogene::is depression and feed efficiency improvement between c:ysteine and its salts. However, cysteine is easily oxidized to cystine that is less effective than cysteine. Therefore, a salt of cysteine is more advantageous as compared to cysteine from the standpoint of protection from oxidation, namely, stabi lity and storage .
With respect to the amount of cysteine and its salts, when the content in the feed composition of the present invention is high, the feeding amount can be decreased. On the other hand, when the content is low, the feeding amount can be increased. Therefore, the content of cyst~eine and its salts in the present invention is not limited. However, for feeding cysteine :>.0 and its salts to a ruminant in an appropriate amount uniformly, it i:~ preferred that the feed composition of the present invcantion be _250892 _ 6 _ 1 fed to a ruminant, in an amount of about 50 g to about 1 kg daily.
On the other hand, the daily amount of cysteine and its salts to be fed to a rumic,ant is 10 to 100 g, preferably 25 to 50 g per animal in germs c~f cysteine weight. Accordingly, the content of cysteine and its salts in the feed composition of the present invention i.s 1 to 100 '~ in terms of cysteine content.
The cysteine and it;s salts contained in the feed composition of the present invention in an amount as mentioned above may be identified, for example, by the Folin-Chiocalteu method, mercury method and the like. The analytical method is described in, for example, "Handbook of Nn<~lytical Chemistry" (Bunsekikagaku Benran), edited by Japanese Society of Analytical Chemistry, published by Maruzen Co., Ltd., Japan, pp.1281-1288.
As the component of the feed composition of the present invention other than cysi~eine and its salts, there may be mentioned substrate materials which are generally used as a feed composition of ruminant feeds. As the representative examples of such substrate materials, there may be mentioned wheat bran, rice bran, corn meal, cereal grains such as barley, wheat, rye and oat, soybean meal, alf;~lfa meal, wheat powder, and the like. The shape of feed composition of the present invention is not restricted and may be in any form of a conventional feed composition, such as a powder and a pellet. The feed composition of the present invention may be produced according to the generally employed method for producing a compound feed and a feed supplement. For example, the present feed composition may be produced by a method comprising a premixing step in which cysteine and its salts are mixed with a small amount, of a substrate material to prepare a premix; a mixing step in which the premix is combined with the remaining large amount of the substrate material; and a finishing _2150892 _, _ step comprising 2, process of supplementing liquid materials, a granulating process and a pelleting process. Such a method is described in, for example, "Lecture on Compound Feed"(Haigoshiryou Koza), the second volume, published by Chikusan Shuppansha, Japan, pp.l5-27.
The feed composition of the present invention may also be formulated in the same form as a pharmaceutical composition for livestock. For example, it may be in the form of granules, tablets, pellets, capsules and the like. As components other than cysteine and its salts, in the case of granules, there may be mentioned carriers such as lactose, sodium hydrogen carbonate, sodium chloride, sucrose., mannitol, starch, crystalline cellulose, calcium sulfate, precipitated calcium carbonate and calcium phosphate; collapsing agents such as starch and crystalline ~5 cellulose; and binders such as an aqueous 10-20~ gum arabic solution, an aqueous 5-10 ~ starch solution, an aqueous or alcoholic 5-10 ~ PVP solution and an aqueous or alcoholic 1-2 ro cellulose derivative solution. In the case of tablets, there may be mentioned, for example, carriers such as lactose, sucrose, 20 glucose, starch and crysi:alline cellulose; binders such as an aqueous 5-10 ~ starch sol~.ution, an aqueous t>,vdroxypropyl cellulose solution, an aqueous carboxymetl>,yl cellulose solution, an aqueous gum arabic solution, an aqueous tragacanth solution, an aqueous sucrose solution, an aqueous glucose solution and sodium alginate;
25 collapsing agents such as starch and calcium carboxymetlLyl cellulose; and luhricant:~ such as refined tare, stearic acid, magnesium stearate and calcium stearate. In the case of pellets and capsules, there may be mentioned, in addition to the above mentioned carriers and binders, coating agents such as sucrose, 30 starch, tarc and 'the like for pellets, and gelatin, glycerol, _210892 _ 8_ sorbitol and the like for capsules. The above-mentioned compositions may be produced according to the method generally employed for producing a pharmaceutical composition for livestock well known in the art. According to need, a stabilizing agent such as an antioxidant may be combined in the present feed composition.
By feeding t'.ne present feed composition to a ruminant, the rumen methanogene;sis can be depressed and the feed efficiency can be improved. Accordingly., it is another object of the present invention to provide a method for depressing methanogenesis in the rumen of a ruminant and :improving feed efficiency, which comprises feeding at least one substance selected from cysteine and its salts to a ruminant in an amount of 0.02 to 0.21 g sulfur equivalent~kg body weighi~w 'S per day as a total of said at least one substance. In the specification of the present application, "kg body weightw 'S" means 1 kg of metabolic body weight.
The feeding :~rnount, is not restricted as long as the methanogenesis is efficiently depressed while the nutrient balance is not adversely :~ffect,ed. From the standpoint of cost and feed efficiency, it is preferred that the feeding amount of the total 20 of cysteine and its salts be in the range of 0.02 to 0.21 g sulfur equivalent~kg body weight:-w 'S per day, more preferably 0.05 to 0.10 g sulfur equivalent°kg body weight'' 'S per day. The upper limit of the feeding amount is the daily sulfur demand according to National Research Council (NRC) 1985, U. S. A. Therefore, even if 25 cysteine and its salts are fed in an amount larger than the upper limit, the effect is not increased as compared to the cost increase. On the other hand, when the feeding amount is lower than the above-mentioned lower limit, it is possible that the rumen methanogenesis depression and feed efficiency become 30 insufficient. In detail, in the case of, for example, a cattle _ 9 _ 1 having a body weight of 700 kg, the feeding amount of cysteine and its salts may be preferably 10 to 100 g, more preferably 25 to 50 g per animal per day in terms of cysteine weight. The manner of feeding is not restricted, and the feed composition of the present invention maJ- be fed by top-dressing over the compound feed, or fed after t:he present feed composition is mixed with the compound feed.
Since the ruminant feed composition of the present invention comprises cysteine which is found in nature as a component of a protein, andjor salts thereof, it can be effectively and safely fed to a ruminant as compared to the ionophore antibiotics, such as monensin and salinomyc:in.
The present invention will now be described in more detail with reference to the following Example, which should in no way be ~5 construed to be limiting the scope of the present invention.
Example 1 Two rumen-fi;~turat:ed, adult sheep ISuffolkl wethers (No.l and No.2) were individually caged in a controlled em~ironment at 18 °C
and 45 ~ RH (relative ;:umidity). Chopped grass hay of cocksfoot (Dactylis glomera~a) was daily offered once a day at 8:00 a.m. in an amount of 55 g!kg body weight''''. Simultaneously, cysteine was fed in an amount of 0.105 g sulfur equivalent~'kg body weight'~'S per da~r. lhat.er was freely available. On the eighth (8th) day from the initiation of administration, methane production, carbon dioxide production, oxygen consumption in rumen were determined at the time of administration of cysteine and thereafter continually at intervals of 15 minutes, and pH and UFA
(volatile fatty acids) contents in rumen juice were determined 4 hours after the administration. The determination of methane _210892 -i0 -1 production, carbon dioxide production, oxygen consumption in rumen was conducted in the following manner. Respiratory gas exchanges were monitored by means of an open-circuit respiratory system using a hood and face ma:;k, by compulsorily ventilating the expiration of an animal by means of a blower and detecting the expiration volume by means of a dry gas meter. The pressure and temperature of the expiration, and the atmospheric pressure and temperature were measw~ecl at intervals of 30 minutes. The carbon dioxide concentration and methane concentration in the expiration were measured by ;means oj° an infrared spectrometer model VIA-500 (manufactured by HORIf3:4 Co. , Ltd. , Japan) and oxygen concentration was measured by means of a magnetic oxygen analyzer model MAG-2 (manufactured by Shimadzu Corp., Japan). Each concentration mea;~ured was automatically input in a computer (PC-9801UM, manufactured by NEC, Japan) and calculated in terms of a value at 0 °C; at 1 atom. The pH was measured by means of a pH
meter model E-13 (manufac;tured by Hitachi-Horiba, Japan). The volatile fatty acids were measured according to the following method. 1.5 ml of a rumen juice sample was taken from the fistura, and centrifuged air 8,000: g for 5 minutes, thereby to obtain a supernatant.. To 0.8 ml of the supernatant was added 0.2 ml of 25 ~ methaphosphoric acid, followed by freezing overnight. Then, the resultant mixture was fused and centrifuged at 8,000 X g for 5 minutes to obtain a supernatant. To 0.5 ml of the supernatant was added 1 ml of 10 n~M 2-ethylbutyTic acid as an internal standard (sample solution). A calibration agent was prepared in substantially the same manner as mentioned above except that a mixture of 50 mM acetic acid, 50 m.~1 propionic acid, 10 mM
isobut~Tic acid, G.0 mM butyric acid, 10 mM isovalerianic acid, 10 ,~ valerianic acid, 5 mM isocaproic acid and 5 mM caproic acid was _210892 _11 _ used instead of a. rumen juice sample. The UFA concentration was analyzed by means of a gas chromatography model CG-14A
(manufactured by Shimadzu Corp., Japan) and calculated by means of an automatically calculating program (Chromato-Pack, manufactured by Shimadzu Corp., Japan).
On the other hand, as a control, No.2 animal was fed in substantially the same manner as mentioned above except that cysteine is not fed.
From the ninth (9th) day of the initiation, the feed was exchanged between the No.l and No.2 sheeps, followed by the measurement in the same rnanner as mentioned above. This exchange was further repeated so i~hat the measurement was conducted in 3 repetitions in total. The average and standard deviation were calculated with respect t:o each item. The results are shown in Tables l, 2, 3 an~j 4 and Figs. 1, 2 and 3.
As apparent from t:he results shown in the Tables and Figs, the rumen methanogenesis was markedly depressed by the feeding of cysteine. This exhibits that the energy loss resulting from methanogenesis was depre~;sed. Further, the carbon dioxide production and ox;~gen consumption were also depressed. These results exhibit that tr:e heat increment (the sum of maintenance energy increment <~nd prod.uction energy increment) was depressed, that is, the feed efficiency (energy efficiency of feed that a ruminant has taken) has been increased.
The pH value was not changed by the feeding of cysteine. On the other hand, with respect to the UFA content, the propionic acid content was decreased while the acetic acid content was increased. It is known that the conventionally used ionophore antibiotic, such as monensin and salinomycin, increases the propionic acid content. Therefore, it seems that cysteine affects '12-the rumen methanogenesis in a manner different from the case of ionophore antibiotic.
Table 1 Met=hane gas production Time ~ ml/min/kg 0.75 (hour) Examp le 1 Control 0 0.58!5 t 0.394 0.697 t 0.133 0.95'7 t 0.331 1.183 ~ 0.145 1.29~~ t 0.028 1.417 ~- 0.297 1.05! t 0.314 1.283 t 0.085 1 1.153 t 0.419 1.418 t 0.176 0.99~a '~ 0.374 1.382 ~ 0.162 1.04~i t 0.453 1.206 _ 0.193 1.190 t 0.361 1.205 ~- 0.353 2 1.217. t 0.260 1.325 0.166 0.83~~ t 0.305 1.340 t 0.312 0.91() t 0.196 1.140 r 0.053 0.897 t 0.275 1.240 t 0.016 3 1.17~~ t 0.370 1.493 _ 0.058 0.76? t 0.131 1.102 0.066 1.04, ~- 0.192 1.213 0.350 1.037 0.259 0.968 0.085 4 0.907 a 0.326 1.194 ~ 0.140 0.825 ~ 0.364 1.377 t 0.328 0.698 0.364 0.917 = 0.151 0.849 t 0.344 1.013 0.076 0.909 ~ 0.203 1.461 -f- 0.436 0.744 t 0.160 1.070 ~ 0.090 0.935 t 0.562 0.923 ~ 0.148 0.788 t 0.494 _- 0.865 _ 0.024 -, _210892 Table 2 Carbon dioxide gas production Time ml/min/kg 0.75 (hour) Example 1 Co ntrol 0 9.87 t 2.312 12.88 t 3.127 13.29 t 2.509 19.10 t 6.248 21.59 .t 2.162 25.86 t 1.342 19.70 t 3.910 24.27 t 1.756 1 19.53 a 4.398 24.96 1.866 15.96 a 3.829 23.35 f 2.895 13.85 .t 3.655 19.09 ~- 3.171 13.36 t 3.553 16.94 -~- 4.466 2 16.12 a 2.523 16.78 t 0.818 12.59 _~ 2.766 17.54 t 3.359 12.60 ~ 2.372 17.48 3.150 12:05 ~ 3.222 15.73 2.307 3 14.31 t- 3.775 16.43 t 1.970 11.37 t 2.389 16.38 t 2.013 12.99 ~ 2.558 15.23 2.332 12.69 t 1.090 12.86 t 1.908 4 12.27 2.629 13.19 t 1.302 11.93 t 2.140 15.13 3.040 13.68 t 2.542 13.75 t 1.878 12.43 2.782 12.69 t 1.982 11.88 t 2.388 14.09 ~ 0.673 10.15 ~ 1.896 14.35 t 2.222 11.51 t 3.099 13.43 ~ 1.936 12.11 t 4.658 12.39 t 1.688 _2150892 Table 3 Oxygen consumption Time ml/min/kg 0.75 (hour? Example Control 0 8.20 t 2.014 10.28 t 1.591 11.25 1.403 16.12 t 4.860 17.5;2 t 0.940 21.36 1.246 15.6'7 t 2.348 19.78 t 2.311 1 15.3! t 2.423 19.86 f 1.895 12.7:L 2.143 16.32 t 3.368 10.95 ~ 2.101 15.26 t 2.564 10.85 t 2.891 13.96 t 3.762 2 13.7E> 1.341 13.78 f 0.474 11.1.. t 2.421 13.99 2.212 11.0~~ t 2.305 14.32 t 1.929 10.93 t 2.204 13.55 t 1.970 3 12.6~~ t 2.459 14.79 t 2.473 10.28 ~ 0.533 14.65 t 2.364 11.77 a 0.952 12.74 1.071 11.49 a 0.771 10.90 t 1.292 4 11.49 t 2.782 11.23 0.514 10.70 0.948 13.21 1.903 12.89 0.926 12.17 t 0.803 12.08 a 1.844 11.42 t 1.619 11.15 ~ 1.120 13.08 t 0.254 9.80 != 1.050 13.25 1.944 11.02 t 2.166 12.81 t 1.546 11.55 ~ 3.635 11.60 t 1.560 Table 4 VFA content (mol %) and pH in a rumen juice Acetic acid F>ropionicacid Butyric acid pH
Control 71.7 0.12 20.07 0.56 8.23 0.59 6.46 0.15 t E~anple 1 75.12 0.7E 16.55 f 0.57 8.33 0.31 6.31 0.06 t t
A METHOD FOR IMPROVING FEED EFFICIENCY
BAC~(G:ROUND OF T'HE INVENTION
This invention relates to a composition which is fed to a ruminant so that t:he methanogenesis in the rumen thereof is depressed and the feed c~f.ficiency is improved. The present invention also relates to a method for depressing the methanogenesis in 'the rtunen of a ruminant and improving feed efficiency by the use of i;he composition.
Methane gas is generated in the rumen of a ruminant, such as cattle, sheep, goal: and the like, and emitted in the air in a large amount, and :>uch met;hanogenesis (methane generation) has become a serious problem because methane is one of potent greenhouse gases contributing global warming. The methanogenesis means energy loss of feed which are taken by a ruminant. From this standpoint also, the rumen methanogenesis is not preferable. In a rumen, extremely various kinds of microorganisms exist and contribute to fermentation, resulting in the production of various metabolites. Methane is one of the metabolites, produced by methane-producing bacteria. On the other hand, propionic acid is also produced by rumen fermentation as a result of microbial fermentation of carbohydrates, and it is thought that the energy metabolism depends upon the amount of propionic acid produced. In detail, it is thought that energy utilization efficiency depends upon the final position, namely, propionic acid or methane, to which electron geneoated by the oxidation of energy source in a rumen is transferred. Conventionally, it has been known that an ionophore antibiotic:, such as monensin and salinomycin, coordinates the flow of electrons so that the rumen methane 1 production is depressed and the propionic acid production is increased, and recently, the ionophore antibiotic is widely used for growing beef cattle. It is reported that the use of an ionophore antibiotic brings about 10%
!5 increase of the feed efficiency of a ruminant ("FEED, Revised", 1985, edited by Hiroshi Morimoto, published by Youkendo, Japan, pp.254-255; Ryoji Onodera et al., "Livestock Nutrition", 1989, published by Kawasima Shoten, p.184).
:LO However, since the ionophore such as monensin and salinomycin is an antibiotic, it has an adverse effect such that it depresses the activities of not only methane-producing bacteria but also the other rumen microorganisms. There=_fore, attention should be drawn to 1.5 the dosage so that essential rumen microorganisms may not be damaged. Further, there is a problem that even though an ionophore ant:ibiot:ic is initially effective, the methane-producing baci:eria in a rumen attain resistance to the ionophore~ antibiotic with the lapse of time.
20 Cross-resistance between the antibiotics is another problem in the practice as feed additives (C. J. Newbold et al., Journal of Applied Bacteriology, 1993, 75:129-134) .
SLJMMARY OF THE INVENTION
25 The present inventor has made extensive and 1 intensive studies wii:h a view to depressing the rumen methanogenesis effecl:ively without the above-mentioned problems. To this end, the inventor has examined various chemical compounds as a feed additive. As a result, the present inventor has surprisingly found that when a specific amino acid, cysteine, is fed to a ruminant, not only rumen methanogenesis, but also oxygen consumption, carbon dioxide gas production and propionic acid production, one of volatile fatty acids (VFA), are :10 markedly depressed, contributing the increase of feed efficiency. The present invention has been made based on such a novel finding.
Therefore, it is an object of the present invention to provide a novel ruminant feed composition which is :L5 excellent in de~~ressing the rumen methanogenesis and increasing feed efficiency.
It is another object of the present invention to provide a method for effectively depressing rumen methanogenesis and improving feed efficiency of a 20 ruminant.
The foregoing and other objects are achieved by providing a ruminant feed composition for feeding a ruminant to depress methanogenesis in the rumen thereof and improving feed efficiency, which comprises at least 25 one substance selected from cysteine and its salts, and also achieved by a method which comprises feeding at least one substance selected from cysteine and its salts to ruminant in an amount of 0.02 to 0.21 g sulfur equivalent'kg body weight-'~Sper day as a total of said at least one substance.
In another aspect, the present invention provid~s a method for depressing methanogenesis in the rumen of a ruminant and improving feed efficiency, which comprises feeding at least one substance selected from the group consisting of cysteine and its salts to a ruminant in a total amount of 0.02 to 0.1 g sulfur equivalent/kg of metabolic body weight per day.
In another aspect, the present invention provides use of a ruminant feeding composition comprising at least one substance selected from cysteine and its salts for depressing methanogenesis in the rumen of a ruminant and improving feed efficiency.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG.1 is a graph showing the effect of the ruminant feed composition of the present invention on the depression of rumen methanogenesis;
FIG.2 is a graph showing the effect of the ruminant feed composition of the present invention of the depression of carbon dioxide production in a rumen; and - 4a -FIG.3 is a graph showing the effect of the ruminant feed composition of the present invention on the depression of oxygen consumption in a rumen.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there is provided a ruminant feed composition for feeding a ruminant to depress methanogenesis in the rumen thereof and improving feed efficiency, which comprises at least one substance selected from cysteine and its salts.
The ruminant feed composition of the present invention comprises at least one substance selected from cysteine and its salts. As the cysteine and its salts, any optical isomers, that is, L-, D- and DL-isomers, may _2150892 1 be mentioned in the ;present invention.
As the salts of cysteine, there may be mentioned any salts which can be fed to a ruminant. For example, there may be mentioncad cysteine hydrochloride, but not restricted thereto. There is no difference in the effect of methanogene::is depression and feed efficiency improvement between c:ysteine and its salts. However, cysteine is easily oxidized to cystine that is less effective than cysteine. Therefore, a salt of cysteine is more advantageous as compared to cysteine from the standpoint of protection from oxidation, namely, stabi lity and storage .
With respect to the amount of cysteine and its salts, when the content in the feed composition of the present invention is high, the feeding amount can be decreased. On the other hand, when the content is low, the feeding amount can be increased. Therefore, the content of cyst~eine and its salts in the present invention is not limited. However, for feeding cysteine :>.0 and its salts to a ruminant in an appropriate amount uniformly, it i:~ preferred that the feed composition of the present invcantion be _250892 _ 6 _ 1 fed to a ruminant, in an amount of about 50 g to about 1 kg daily.
On the other hand, the daily amount of cysteine and its salts to be fed to a rumic,ant is 10 to 100 g, preferably 25 to 50 g per animal in germs c~f cysteine weight. Accordingly, the content of cysteine and its salts in the feed composition of the present invention i.s 1 to 100 '~ in terms of cysteine content.
The cysteine and it;s salts contained in the feed composition of the present invention in an amount as mentioned above may be identified, for example, by the Folin-Chiocalteu method, mercury method and the like. The analytical method is described in, for example, "Handbook of Nn<~lytical Chemistry" (Bunsekikagaku Benran), edited by Japanese Society of Analytical Chemistry, published by Maruzen Co., Ltd., Japan, pp.1281-1288.
As the component of the feed composition of the present invention other than cysi~eine and its salts, there may be mentioned substrate materials which are generally used as a feed composition of ruminant feeds. As the representative examples of such substrate materials, there may be mentioned wheat bran, rice bran, corn meal, cereal grains such as barley, wheat, rye and oat, soybean meal, alf;~lfa meal, wheat powder, and the like. The shape of feed composition of the present invention is not restricted and may be in any form of a conventional feed composition, such as a powder and a pellet. The feed composition of the present invention may be produced according to the generally employed method for producing a compound feed and a feed supplement. For example, the present feed composition may be produced by a method comprising a premixing step in which cysteine and its salts are mixed with a small amount, of a substrate material to prepare a premix; a mixing step in which the premix is combined with the remaining large amount of the substrate material; and a finishing _2150892 _, _ step comprising 2, process of supplementing liquid materials, a granulating process and a pelleting process. Such a method is described in, for example, "Lecture on Compound Feed"(Haigoshiryou Koza), the second volume, published by Chikusan Shuppansha, Japan, pp.l5-27.
The feed composition of the present invention may also be formulated in the same form as a pharmaceutical composition for livestock. For example, it may be in the form of granules, tablets, pellets, capsules and the like. As components other than cysteine and its salts, in the case of granules, there may be mentioned carriers such as lactose, sodium hydrogen carbonate, sodium chloride, sucrose., mannitol, starch, crystalline cellulose, calcium sulfate, precipitated calcium carbonate and calcium phosphate; collapsing agents such as starch and crystalline ~5 cellulose; and binders such as an aqueous 10-20~ gum arabic solution, an aqueous 5-10 ~ starch solution, an aqueous or alcoholic 5-10 ~ PVP solution and an aqueous or alcoholic 1-2 ro cellulose derivative solution. In the case of tablets, there may be mentioned, for example, carriers such as lactose, sucrose, 20 glucose, starch and crysi:alline cellulose; binders such as an aqueous 5-10 ~ starch sol~.ution, an aqueous t>,vdroxypropyl cellulose solution, an aqueous carboxymetl>,yl cellulose solution, an aqueous gum arabic solution, an aqueous tragacanth solution, an aqueous sucrose solution, an aqueous glucose solution and sodium alginate;
25 collapsing agents such as starch and calcium carboxymetlLyl cellulose; and luhricant:~ such as refined tare, stearic acid, magnesium stearate and calcium stearate. In the case of pellets and capsules, there may be mentioned, in addition to the above mentioned carriers and binders, coating agents such as sucrose, 30 starch, tarc and 'the like for pellets, and gelatin, glycerol, _210892 _ 8_ sorbitol and the like for capsules. The above-mentioned compositions may be produced according to the method generally employed for producing a pharmaceutical composition for livestock well known in the art. According to need, a stabilizing agent such as an antioxidant may be combined in the present feed composition.
By feeding t'.ne present feed composition to a ruminant, the rumen methanogene;sis can be depressed and the feed efficiency can be improved. Accordingly., it is another object of the present invention to provide a method for depressing methanogenesis in the rumen of a ruminant and :improving feed efficiency, which comprises feeding at least one substance selected from cysteine and its salts to a ruminant in an amount of 0.02 to 0.21 g sulfur equivalent~kg body weighi~w 'S per day as a total of said at least one substance. In the specification of the present application, "kg body weightw 'S" means 1 kg of metabolic body weight.
The feeding :~rnount, is not restricted as long as the methanogenesis is efficiently depressed while the nutrient balance is not adversely :~ffect,ed. From the standpoint of cost and feed efficiency, it is preferred that the feeding amount of the total 20 of cysteine and its salts be in the range of 0.02 to 0.21 g sulfur equivalent~kg body weight:-w 'S per day, more preferably 0.05 to 0.10 g sulfur equivalent°kg body weight'' 'S per day. The upper limit of the feeding amount is the daily sulfur demand according to National Research Council (NRC) 1985, U. S. A. Therefore, even if 25 cysteine and its salts are fed in an amount larger than the upper limit, the effect is not increased as compared to the cost increase. On the other hand, when the feeding amount is lower than the above-mentioned lower limit, it is possible that the rumen methanogenesis depression and feed efficiency become 30 insufficient. In detail, in the case of, for example, a cattle _ 9 _ 1 having a body weight of 700 kg, the feeding amount of cysteine and its salts may be preferably 10 to 100 g, more preferably 25 to 50 g per animal per day in terms of cysteine weight. The manner of feeding is not restricted, and the feed composition of the present invention maJ- be fed by top-dressing over the compound feed, or fed after t:he present feed composition is mixed with the compound feed.
Since the ruminant feed composition of the present invention comprises cysteine which is found in nature as a component of a protein, andjor salts thereof, it can be effectively and safely fed to a ruminant as compared to the ionophore antibiotics, such as monensin and salinomyc:in.
The present invention will now be described in more detail with reference to the following Example, which should in no way be ~5 construed to be limiting the scope of the present invention.
Example 1 Two rumen-fi;~turat:ed, adult sheep ISuffolkl wethers (No.l and No.2) were individually caged in a controlled em~ironment at 18 °C
and 45 ~ RH (relative ;:umidity). Chopped grass hay of cocksfoot (Dactylis glomera~a) was daily offered once a day at 8:00 a.m. in an amount of 55 g!kg body weight''''. Simultaneously, cysteine was fed in an amount of 0.105 g sulfur equivalent~'kg body weight'~'S per da~r. lhat.er was freely available. On the eighth (8th) day from the initiation of administration, methane production, carbon dioxide production, oxygen consumption in rumen were determined at the time of administration of cysteine and thereafter continually at intervals of 15 minutes, and pH and UFA
(volatile fatty acids) contents in rumen juice were determined 4 hours after the administration. The determination of methane _210892 -i0 -1 production, carbon dioxide production, oxygen consumption in rumen was conducted in the following manner. Respiratory gas exchanges were monitored by means of an open-circuit respiratory system using a hood and face ma:;k, by compulsorily ventilating the expiration of an animal by means of a blower and detecting the expiration volume by means of a dry gas meter. The pressure and temperature of the expiration, and the atmospheric pressure and temperature were measw~ecl at intervals of 30 minutes. The carbon dioxide concentration and methane concentration in the expiration were measured by ;means oj° an infrared spectrometer model VIA-500 (manufactured by HORIf3:4 Co. , Ltd. , Japan) and oxygen concentration was measured by means of a magnetic oxygen analyzer model MAG-2 (manufactured by Shimadzu Corp., Japan). Each concentration mea;~ured was automatically input in a computer (PC-9801UM, manufactured by NEC, Japan) and calculated in terms of a value at 0 °C; at 1 atom. The pH was measured by means of a pH
meter model E-13 (manufac;tured by Hitachi-Horiba, Japan). The volatile fatty acids were measured according to the following method. 1.5 ml of a rumen juice sample was taken from the fistura, and centrifuged air 8,000: g for 5 minutes, thereby to obtain a supernatant.. To 0.8 ml of the supernatant was added 0.2 ml of 25 ~ methaphosphoric acid, followed by freezing overnight. Then, the resultant mixture was fused and centrifuged at 8,000 X g for 5 minutes to obtain a supernatant. To 0.5 ml of the supernatant was added 1 ml of 10 n~M 2-ethylbutyTic acid as an internal standard (sample solution). A calibration agent was prepared in substantially the same manner as mentioned above except that a mixture of 50 mM acetic acid, 50 m.~1 propionic acid, 10 mM
isobut~Tic acid, G.0 mM butyric acid, 10 mM isovalerianic acid, 10 ,~ valerianic acid, 5 mM isocaproic acid and 5 mM caproic acid was _210892 _11 _ used instead of a. rumen juice sample. The UFA concentration was analyzed by means of a gas chromatography model CG-14A
(manufactured by Shimadzu Corp., Japan) and calculated by means of an automatically calculating program (Chromato-Pack, manufactured by Shimadzu Corp., Japan).
On the other hand, as a control, No.2 animal was fed in substantially the same manner as mentioned above except that cysteine is not fed.
From the ninth (9th) day of the initiation, the feed was exchanged between the No.l and No.2 sheeps, followed by the measurement in the same rnanner as mentioned above. This exchange was further repeated so i~hat the measurement was conducted in 3 repetitions in total. The average and standard deviation were calculated with respect t:o each item. The results are shown in Tables l, 2, 3 an~j 4 and Figs. 1, 2 and 3.
As apparent from t:he results shown in the Tables and Figs, the rumen methanogenesis was markedly depressed by the feeding of cysteine. This exhibits that the energy loss resulting from methanogenesis was depre~;sed. Further, the carbon dioxide production and ox;~gen consumption were also depressed. These results exhibit that tr:e heat increment (the sum of maintenance energy increment <~nd prod.uction energy increment) was depressed, that is, the feed efficiency (energy efficiency of feed that a ruminant has taken) has been increased.
The pH value was not changed by the feeding of cysteine. On the other hand, with respect to the UFA content, the propionic acid content was decreased while the acetic acid content was increased. It is known that the conventionally used ionophore antibiotic, such as monensin and salinomycin, increases the propionic acid content. Therefore, it seems that cysteine affects '12-the rumen methanogenesis in a manner different from the case of ionophore antibiotic.
Table 1 Met=hane gas production Time ~ ml/min/kg 0.75 (hour) Examp le 1 Control 0 0.58!5 t 0.394 0.697 t 0.133 0.95'7 t 0.331 1.183 ~ 0.145 1.29~~ t 0.028 1.417 ~- 0.297 1.05! t 0.314 1.283 t 0.085 1 1.153 t 0.419 1.418 t 0.176 0.99~a '~ 0.374 1.382 ~ 0.162 1.04~i t 0.453 1.206 _ 0.193 1.190 t 0.361 1.205 ~- 0.353 2 1.217. t 0.260 1.325 0.166 0.83~~ t 0.305 1.340 t 0.312 0.91() t 0.196 1.140 r 0.053 0.897 t 0.275 1.240 t 0.016 3 1.17~~ t 0.370 1.493 _ 0.058 0.76? t 0.131 1.102 0.066 1.04, ~- 0.192 1.213 0.350 1.037 0.259 0.968 0.085 4 0.907 a 0.326 1.194 ~ 0.140 0.825 ~ 0.364 1.377 t 0.328 0.698 0.364 0.917 = 0.151 0.849 t 0.344 1.013 0.076 0.909 ~ 0.203 1.461 -f- 0.436 0.744 t 0.160 1.070 ~ 0.090 0.935 t 0.562 0.923 ~ 0.148 0.788 t 0.494 _- 0.865 _ 0.024 -, _210892 Table 2 Carbon dioxide gas production Time ml/min/kg 0.75 (hour) Example 1 Co ntrol 0 9.87 t 2.312 12.88 t 3.127 13.29 t 2.509 19.10 t 6.248 21.59 .t 2.162 25.86 t 1.342 19.70 t 3.910 24.27 t 1.756 1 19.53 a 4.398 24.96 1.866 15.96 a 3.829 23.35 f 2.895 13.85 .t 3.655 19.09 ~- 3.171 13.36 t 3.553 16.94 -~- 4.466 2 16.12 a 2.523 16.78 t 0.818 12.59 _~ 2.766 17.54 t 3.359 12.60 ~ 2.372 17.48 3.150 12:05 ~ 3.222 15.73 2.307 3 14.31 t- 3.775 16.43 t 1.970 11.37 t 2.389 16.38 t 2.013 12.99 ~ 2.558 15.23 2.332 12.69 t 1.090 12.86 t 1.908 4 12.27 2.629 13.19 t 1.302 11.93 t 2.140 15.13 3.040 13.68 t 2.542 13.75 t 1.878 12.43 2.782 12.69 t 1.982 11.88 t 2.388 14.09 ~ 0.673 10.15 ~ 1.896 14.35 t 2.222 11.51 t 3.099 13.43 ~ 1.936 12.11 t 4.658 12.39 t 1.688 _2150892 Table 3 Oxygen consumption Time ml/min/kg 0.75 (hour? Example Control 0 8.20 t 2.014 10.28 t 1.591 11.25 1.403 16.12 t 4.860 17.5;2 t 0.940 21.36 1.246 15.6'7 t 2.348 19.78 t 2.311 1 15.3! t 2.423 19.86 f 1.895 12.7:L 2.143 16.32 t 3.368 10.95 ~ 2.101 15.26 t 2.564 10.85 t 2.891 13.96 t 3.762 2 13.7E> 1.341 13.78 f 0.474 11.1.. t 2.421 13.99 2.212 11.0~~ t 2.305 14.32 t 1.929 10.93 t 2.204 13.55 t 1.970 3 12.6~~ t 2.459 14.79 t 2.473 10.28 ~ 0.533 14.65 t 2.364 11.77 a 0.952 12.74 1.071 11.49 a 0.771 10.90 t 1.292 4 11.49 t 2.782 11.23 0.514 10.70 0.948 13.21 1.903 12.89 0.926 12.17 t 0.803 12.08 a 1.844 11.42 t 1.619 11.15 ~ 1.120 13.08 t 0.254 9.80 != 1.050 13.25 1.944 11.02 t 2.166 12.81 t 1.546 11.55 ~ 3.635 11.60 t 1.560 Table 4 VFA content (mol %) and pH in a rumen juice Acetic acid F>ropionicacid Butyric acid pH
Control 71.7 0.12 20.07 0.56 8.23 0.59 6.46 0.15 t E~anple 1 75.12 0.7E 16.55 f 0.57 8.33 0.31 6.31 0.06 t t
Claims (4)
1. A ruminant feed composition for feeding a ruminant to depress methanogenesis in the rumen thereof and improving feed efficiency, characterized in that the composition comprises at least one substance selected from cysteine and it salts.
2. A method for depressing methanogenesis in the rumen of a ruminant and improving feed efficiency, characterized by feeding at least one substance selected from cysteine and its salts to a ruminant in an amount of 0.02 to 0.21 g sulfur equivalent/kg of metabolic body weight per day as a total of said at least one substance.
3. A method for depressing methanogenesis in the rumen of a ruminant and improving feed efficiency, which comprises feeding at least one substance selected from the group consisting of cysteine and its salts to a ruminant in a total amount of 0.02 to 0.1 g sulfur equivalent/kg of metabolic body weight per day.
4. Use of a ruminant feeding composition comprising at least one substance selected from cysteine and its salts for depressing methanogenesis in the rumen of a ruminant and improving feed efficiency.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12087794A JP3232193B2 (en) | 1994-06-02 | 1994-06-02 | Ruminant feeding composition and method for improving ruminant feed efficiency |
| JP120877/1994 | 1994-06-02 |
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| Publication Number | Publication Date |
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| CA2150892A1 CA2150892A1 (en) | 1995-12-03 |
| CA2150892C true CA2150892C (en) | 2003-08-26 |
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| CA002150892A Expired - Lifetime CA2150892C (en) | 1994-06-02 | 1995-06-02 | A ruminant feed composition and a method for improving feed efficiency |
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| US (1) | US5843498A (en) |
| EP (1) | EP0685169B1 (en) |
| JP (1) | JP3232193B2 (en) |
| AU (1) | AU701845B2 (en) |
| CA (1) | CA2150892C (en) |
| DE (1) | DE69520304T2 (en) |
| DK (1) | DK0685169T3 (en) |
| NZ (1) | NZ272268A (en) |
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| US6440447B1 (en) | 1999-06-22 | 2002-08-27 | Land O'lakes, Inc. | Method and composition for enhancing milk production |
| JP2001008640A (en) * | 1999-06-29 | 2001-01-16 | Kyodo Shiryo Kk | Gel-like formula feed and method for feeding the same |
| US6681717B2 (en) * | 2000-12-15 | 2004-01-27 | Can Technologies, Inc. | Computer system for determining a customized animal feed |
| JP4783512B2 (en) * | 2001-03-27 | 2011-09-28 | 社団法人農林水産先端技術産業振興センター | A feed composition that suppresses methane production in ruminants |
| WO2003038109A2 (en) * | 2001-09-18 | 2003-05-08 | The Board Of Regents Of The University Of Nebraska | Method for the inhibition of methanogenesis |
| KR20020062910A (en) * | 2002-07-16 | 2002-07-31 | 옥임호 | Liver function ameliorant for animal feed additive |
| US7348037B2 (en) * | 2002-08-16 | 2008-03-25 | Evonik Degussa Gmbh | Sulfur-containing animal-feed additives |
| US6805074B2 (en) * | 2002-12-23 | 2004-10-19 | Cargill, Incorporated | System and method of customizing an animal feed based on heat increment |
| US8519008B2 (en) | 2003-01-22 | 2013-08-27 | Purina Animal Nutrition Llc | Method and composition for improving the health of young monogastric mammals |
| US8110214B2 (en) | 2003-12-23 | 2012-02-07 | Land O'lakes Purina Feed Llc | Method and composition for enhancing milk production and milk component concentrations |
| US20080189085A1 (en) * | 2004-07-29 | 2008-08-07 | Can Technologies, Inc. | System and method for optimizing animal production |
| US20060036419A1 (en) * | 2004-07-29 | 2006-02-16 | Can Technologies, Inc. | System and method for animal production optimization |
| US20070209599A1 (en) * | 2006-03-10 | 2007-09-13 | Archer-Daniels-Midland Company | Methods and compositions for increased productivity in animals |
| JP2008051787A (en) * | 2006-08-28 | 2008-03-06 | Meiji Milk Prod Co Ltd | Method of evaluating effect of physiological active material |
| UY32802A (en) | 2009-07-23 | 2011-01-31 | Provimi Holding B V | COMPOSITIONS TO REDUCE GASTROINTESTINAL METANOGENESIS IN RUMINANTS |
| AU2011256017B2 (en) | 2010-05-18 | 2013-11-28 | Able Corporation | Method for inhibition of methane production |
| US8673219B2 (en) | 2010-11-10 | 2014-03-18 | Invention Science Fund I | Nasal passage insertion device for treatment of ruminant exhalations |
| NZ610031A (en) * | 2010-12-20 | 2015-01-30 | Dsm Ip Assets Bv | Use of nitrooxy organic molecules in feed for reducing methane emission in ruminants, and/or to improve ruminant performance |
| BRPI1102284A2 (en) * | 2011-05-23 | 2013-11-05 | Grasp Ind E Com Ltda | ADDITIVE FOR ANIMAL NUTRITION BASED ON NITRATES AND SULFATES ENCAPSULATED TO REDUCE METHANE EMISSION FROM RUMINAL FERMENTATION |
| JP6202445B2 (en) * | 2011-05-26 | 2017-09-27 | ディーエスエム アイピー アセッツ ビー．ブイ． | Use of feed compositions to reduce ruminant methane emissions and / or to improve ruminant performance |
| US8658199B2 (en) | 2012-02-01 | 2014-02-25 | Purina Animal Nutrition Llc | Systems and methods for feeding sugar alcohol to ruminants during periods of heat stress |
| US20160183564A1 (en) * | 2013-08-07 | 2016-06-30 | Dsm Ip Assets B.V. | Use of a feed composition for reducing methane emission in ruminants, and or to improve ruminant performance |
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| US3949090A (en) * | 1974-08-26 | 1976-04-06 | Smithkline Corporation | Methods and compositions for improving feed efficiency in ruminants using dithiooxamide |
| JPS56164749A (en) * | 1980-05-22 | 1981-12-17 | Chubu Shiryo Kk | Breeding method of livestock |
| JP2800901B2 (en) * | 1990-01-27 | 1998-09-21 | 高橋 潤一 | Nitrate adverse effects reduction method |
| RU2002426C1 (en) * | 1991-05-16 | 1993-11-15 | Иван Абаксимович Долгов | Method of fodder quality evaluation |
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1994
- 1994-06-02 JP JP12087794A patent/JP3232193B2/en not_active Expired - Lifetime
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- 1995-06-01 DE DE69520304T patent/DE69520304T2/en not_active Expired - Lifetime
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- 1995-06-01 NZ NZ272268A patent/NZ272268A/en not_active IP Right Cessation
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| DK0685169T3 (en) | 2001-05-14 |
| DE69520304D1 (en) | 2001-04-19 |
| JP3232193B2 (en) | 2001-11-26 |
| AU2044395A (en) | 1995-12-14 |
| EP0685169A1 (en) | 1995-12-06 |
| CA2150892A1 (en) | 1995-12-03 |
| US5843498A (en) | 1998-12-01 |
| DE69520304T2 (en) | 2001-09-20 |
| AU701845B2 (en) | 1999-02-04 |
| JPH07322828A (en) | 1995-12-12 |
| EP0685169B1 (en) | 2001-03-14 |
| NZ272268A (en) | 1996-06-25 |
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