CA1225273A - Method for quantitating non-structural carbohydrates in feedstuffs and ration formulations based thereon - Google Patents
Method for quantitating non-structural carbohydrates in feedstuffs and ration formulations based thereonInfo
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Abstract
Application Of: James E. Nocek, et al For: Method For Quantitating Non-Structural Carbohydrates in Feedstuffs and Ration Formulations Based Thereon Abstract Of The Disclosure A novel method of determining the portion of a given dairy cow feedstuff which is attributable to non-structural carbohy-drates is disclosed. The method includes the steps of analy-tically determining the percentages of crude protein, lipid and ash in the dry matter concentration of a first feed sample, and in the neutral detergent fiber which has been separated from the soluble matter of a second sample of the same feed, and subtracting the amounts of crude protein, lipid and ash in the neutral detergent fiber from the respective amounts of the same substances in the original sample. The three difference figures are totaled and subtracted from the percentage of the second sample represented by neutral detergent solubles, i.e., 100-neutral detergent fiber, thereby providing an accurate indication of the percentage of non-structural carbohydrate in the feedstuff. The method is applied to both forages and grains, and is employed in the formulation of dairy cow rations wherein ration is adjusted to optimize milk production, the preferred percentage being from about 30% to about 45% depending on forage type.
Description
'73 Background Of The Invention The present invention relates to dairy cow nutrition and r more specifically, to methods of determining the proportion of non-structural carbohydrates in foodstuffs and formulating rations for optimized milk production based thereon.
US. Patent No. 4,118,513, assigned to applicant's assignee, describes a method of formulating dairy rations based on adjustment of the proportions of soluble and insoluble protein in the total dietary protein to obtain a positive response in milk production. The present invention is the result of research directed toward determining the optimum balance of structural and non-structural carbohydrates in dairy cow rations for increased milk production and feed efficiency.
It is well known, of course, that carbohydrates are the principal energy source for many animals, as well as humans.
Plant carbohydrates may be categorized in two major groups, namely, structural and nonstructural The structural carbon hydrates, found in the cell wall, are essentially rigid in nature, providing strength to the cell walls and thus to the plant itself. The fibrous materials which make up the cell walls, i.e., the structural carbohydrates, also known as neutral detergent fiber, are mainly cellulose, hemicellulose, and lignin. Non-structural carbohydrates, found mainly within the lumen of the cell, include sugars and stanch.
The major source of structural carbohydrates in dairy cattle rations is forage. The quality and degree of structural carbohydrate availability (digestibility) to Ruben microorganisms in different forages are quite variable, being
US. Patent No. 4,118,513, assigned to applicant's assignee, describes a method of formulating dairy rations based on adjustment of the proportions of soluble and insoluble protein in the total dietary protein to obtain a positive response in milk production. The present invention is the result of research directed toward determining the optimum balance of structural and non-structural carbohydrates in dairy cow rations for increased milk production and feed efficiency.
It is well known, of course, that carbohydrates are the principal energy source for many animals, as well as humans.
Plant carbohydrates may be categorized in two major groups, namely, structural and nonstructural The structural carbon hydrates, found in the cell wall, are essentially rigid in nature, providing strength to the cell walls and thus to the plant itself. The fibrous materials which make up the cell walls, i.e., the structural carbohydrates, also known as neutral detergent fiber, are mainly cellulose, hemicellulose, and lignin. Non-structural carbohydrates, found mainly within the lumen of the cell, include sugars and stanch.
The major source of structural carbohydrates in dairy cattle rations is forage. The quality and degree of structural carbohydrate availability (digestibility) to Ruben microorganisms in different forages are quite variable, being
- 2 -ye LO
~.~22~Z'73 influenced by such factors as plant variety, maturity at harvest and storage conditions. Grains generally have more non-structural carbohydrates and are generally less variable in carbohydrate content than forage.
It has been generally accepted that structural carbon hydrate is a negative indicator ration energy concentration.
That is, the more structural carbohydrate a given ration contains, the less energy value it provides, and vice versa.
Present energy-based feeding systems for ruminants are based on this relationship.
In order to develop a feeding system which includes regulation of carbohydrates in the total daily ration, it is necessary, of course, to have available an accurate and repeatable method of quantitating both structural and non-structural carbohydrates in each constituent of the ration.
A procedure which has been used in the past to separate structural and non-structural carbohydrates involves refluxing - a given sample in a neutral detergent solution and filtering out the insoluble portion, termed "neutral detergent fiber."
It has been generally assumed that, for practical purposes, the neutral detergent fiber could be equated to the structural carbohydrate portion of the sample and, conversely, the neutral detergent solubles were essentially the non-structural carbohydrate portion. However, the fractions of non-carbohydrate substances present in both the neutral detergent fiber and solubles are significant to a degree that a reliable regulated carbohydrate feeding program cannot be based upon such generalizations.
Yams ~2~"S~73 Another chemical procedure which has been used to quantitate non-structural carbohydrate utilizes an enzyme called Taka-Diastase. (see Smith, D. "Removing and Analyzing Total Non-Structural Carbohydrates From Plant Tissue."
Wisconsin Air. Exp. Stay Rest Rep. 41, 1969). The procedure has also been modified, utilizing Bacillus subtilis type III
A aimless (see Madrid, JAMS Thesis, Cornell Univ., pp. 76, 77, 1981). These procedures basically involve the chemical action of enzymes on the plant material under consideration.
Although theoretically measuring the total amount of non-structural carbohydrate in a sample, the enzyme dictates what is being digested and therefore what is being quantitated.
- The Taka-Diastase enzyme is an aimless derived from Aspergillus ours, an organism grown on sterilized wheat bran or rice hulls. This extracted enzyme represents more than 30 different enzymatic functions, and is not only amylolytic, but digests proteins and fats as well, according to Merck Index, Thea Ed., p. 81, #633 (1976). Bacillus subtilis type III A enzyme has specific properties to hydrolyze, 1-4 glucosidic linkages of polysaccharides, also according to Merck Index, swooper. The activity of the latter enzyme may therefore be too "specific" to estimate all non-structural carbohydrates in a foodstuff. Therefore, non-structural carbohydrate measurements based on these enzymatic procedures are subject to variability and inconsistencies.
In the most general sense, the object of the present invention is to provide methods of formulating dairy cow rations which have a positive influence on milk production without an offsetting increase in the cost of the feeding program.
Yo-yo Lo . I.
A further object is to provide a novel method of measuring the amounts of structural and non-structural carbohydrates in a given plant material in order to permit reliable ration formulations based on nonstructural carbohydrate content.
Another object is to provide methods of balancing carbohydrate and protein types in dairy cow feeding programs which maximize efficiency of microorganism growth and yield, fiber digestion and overall feed efficiency, and milk production.
Other objects will in part be obvious and will in part appear hereinafter.
Summary Of The Invention -The method of measuring the carbohydrate content of feed-stuffs according to the present invention is based upon quantitation of non-structural carbohydrates in the dry matter of a sample by a "difference" method. The dry matter concentration of the foodstuff sample is determined by drying a portion thereof. Soluble ingredients are removed from one of the samples by refluxing with neutral detergent solution and filtering. The portion which is not dissolved, termed "neutral detergent fiber," consists essentially of structural carbohydrates, as well as neutral detergent insoluble crude proteins, lipid and ash. The material which is dissolved ("neutral detergent solubles") contains non-structural carbohydrates, consisting primarily of yo-yo;
I o starch, various sugar moieties, and pectin, as well as neutral detergent soluble crude protein, lipid and ash.
The other dry matter sample and the neutral detergent fiber are separately analyzed to determine the respective percentages thereof constituted by crude protein, lipid and ash. Crude protein content is calculated based upon the nitrogen content, livid is extracted with ether and ash is measured by weight after exposure of the samples to 500 C for 3 hours. The non-structural carbohydrate content may then be calculated by sub-treating from the neutral detergent solubles (i.e., 100-neutral detergent fiber) the sums of the crude protein, lipid and ash in the feed sample, each reduced by the respective crude pro-loin, lipid and ash in the neutral detergent fiber of the other sample.
Experiments were conducted to test the value of regulating the type and amount of carbohydrates in dairy cow feeding pro-grams which had previously been based upon regulating protein in accordance with previously mentioned patent No. 4,118,513.
Results of the initial research were analyzed to develop conical-sons an recommendations which were implemented in field studies, conducted on a total of 13 commercial dairy herds under actual operating conditions. Results of the field studies supported the findings of the controlled research results.
AL 'I
I,.
In one aspect the invention provides the method of formulating dairy cow rations for optimized milk production comprising: a) determining the percentage, on a dry matter basis, of non-structural carbohydrates Of each of a plurality of components, including at least one grain and one forage, collectively comprising a total daily ration;
and b) adjusting the proportion and composition of at least one of the components to a level wherein the portion of the total daily ration consisting of non-structural carbohydrates is between about 30~ and 45~.
DETAILED DESCRIPTION
Quantitation of non-structural carbohydrates according to the method of the present invention involves a determine-lion of dry matter concentration in a feed sample, ground to pass through - pa -. . , .
~2~5~7~
a 1 mm screen, the separation of neutral detergent fiber and solubles in a dry matter sample, and the calculation of protein, lipid and ash content. Known quantities (e.g., 2 to 5 grams) of two substantially chemically identical sub samples of a given foodstuff are allowed to dry in a forced air oven at 55 C for 48 hours. The difference in - weight of the sub samples before and after drying provides the original moisture content and dry matter concentration.
One of the samples is then reflexed with neutral detergent solution to quantify and separate the neutral detergent fiber and neutral detergent soluble portions. If desired, moisture content may be determined from a sub sample taken from the same sample as the first two, since dry matter concentration would be the same throughout the sample.
The basic procedure used to quantitate neutral detergent fiber was developed some years ago and is set forth in Agriculture Handbook No. 379 (Agric. Rest Serv., USDA, Washington, DO It has been employed with various modifications, the preferred procedure for purposes of the present invention being as follows:
Weigh 0.5 to 1.0g sample and place in a 600 ml Berzelius beaker. Add 50 ml cold neutral-detergent solution, prepared according to Ago Handbook 379. Place on hot plate and heat to boiling. Adjust heat to reduce foaming but boiling sufficiently to keep feed particles suspended. Thirty minutes from the onset of boiling, remove beaker and add 50 ml cold neutral-detergent solution and 2 ml of an enzyme (Aimless) solution.
dissolve 2 g Bacillus Subtilis type III A aimless in 90 ml of water, yo-yo 5~'73 filter through Whatmar.*X54 paper and add 10 ml Ethoxyethanol).
Return beaker to hot plate without adjusting the thermostat and allow to return to boiling. One hour after the initial onset of boiling, filter on a prepared sintered-glass crucible or Whitman X54 paper. Wash twice with boiling water to remove the detergent and twice with acetone. Dry overnight at 105 C
and weigh. Ash at 500C for three hours and weigh. The loss in weight in asking is an estimate of the plant cell wall constituents.
- Crude protein, lipid and ash content of the whole dry matter sample, and of the neutral detergent fiber separated from the other sample are separately determined. The total protein content, * trade mark 52~
expressed as 6.25 times the nitrogen content of the two materials, is determined according to Official Methods of Analysis of the Association of Official Agricultural Chemists (22.053), Thea Ed., 1965. Lipid analysis (Eat concentration) is performed by extracting the fat material in the foodstuff with ether. The materials are then exposed to a 500 C oven for 3 hours, the remaining materials being weighed and constituting the ash portion.
Although it is the non-structural carbohydrate fraction of the sample which is to be quantitated, it is impractical to do so directly since reagents associated with the neutral detergent solution may interfere with quantitation of the crude protein, lipid and ash which are dissolved together with the soluble carbohydrates. In addition, no single method has been developed to permit direct quantitation of non-structural carbohydrates which include starches, sugars and pectins. Therefore, quantitation of crude protein, lipid and ash in the original foodstuff, and in the neutral detergent fiber portion thereof as described above allows the amount of non-structural carbohydrates to be determined by differences.
The following equation and example will demonstrate the calculation of non-structural carbohydrate for a specific feed ingredient.
Equation: % Non-Structural Carbohydrate [Neutral Detergent Solubles Crude Protein in original - Crude Protein in Neutral Detergent Fiber Lipid in original - lipid in - Neutral Detergent Fiber Ash in original-Ash in Neutral Detergent Fiber fractional Dry Matter concentration, where:
ye/ I-~22S~73 Neutral Detergent Solubles=100-Neutral Detergent Fiber (NDF) Example of actual calculation for 48% soybean meal:
chemical Analysis Original Sample Neutral Detergent Fiber As fed Dry Matter As fed Dry Matter basis basis basis basis Moisture (~)10.60.0 10.6 0.0 NDF (%) 9.1 10.2 89.4 100 Crude protein (~)48.954.7 .8 .89 - 10 Fat (~) 1.0 1.1 .6 .67 Ash (~) 6-0 6.7 .4 .45 Calculation using all Dry Matter values:
nonstructural Carbohydrates = [(100-10.2)-[(54.7-.89)+(1.1-.67)+(6.7-.45)]
(Dry matter basis) = [(89.8) -[(53.8) + (.43) +(6.25)]]
- = [(89.8) - [(60.5)]]
= 29.3 Calculation using "As Fed" analysis, with corrections for Dry Matter:
nonstructural Carbohydrates = [(100-10.6-9.1)-[(48.9-.8)+(1.0-.6)+
(6.0-.4)]]/.894 (Dry matter basis) = 29.3 the foregoing method was used to determine the structural and non-structural carbohydrate content of a number of different forages and grains, the results being tabulated in the following table, wherein N is 30 the number of samples tested, NSC the percentage of dry matter constituted by non-structural carbohydrates, and SUM the standard deviation of the mean (SD):
'ho yo-yo. - -Table I
NSC
. N Mean SUM
Ingredient (% Dry Matter Basis) Corn silage 3 36.3 + 5.4 Hay crop silage 3 27.6 + 4.8 Soybean meal 4 29.5 + 1.0 Distillers grains 3 21.8 + 3.5 Dried brewers grains 4 13.2 + 1.8 lo Corn gluten feed 3 34.1 + 3.5 Corn gluten meal 3 25.5 + 7.4 Homing 3 57.6 + .9 Whole corn 3 75.1 + 2.8 Wheat middlings 3 34.0 + 3.9 Soybean hulls 3 20.8 + 2.3 Beet pulp 4 39.5 + 2.9 Based on the foregoing values, three experimental grain mixes were formulated to contain relatively low, medium and high levels of non-structural carbohydrates. The percentages of each ingredient used in the three grain mixes is as follows:
~2~5~ 3 TABLE II
In~redientsLow-NSC Med-NSCHigh-NSC
(us fed basis) Brewers grains 23.5 18.9 8.6 Soybean Molly 2.0 8.9 omen 45.0 49.4 4.0 Corn meal 2.6 4.0 65.7 Soybean Halsey 10.3 ---Wheat middlings 2.0 7.5 7.6 Corn gluten weed 2.0 5.2 2.0 Salt 1.0 1.0 1.0 Dicalcium phosphate .35 .5 .9 Ground limestone .8 1.1 1.2 Dynamite* .1 I .1 Dairy TRA-MIN-MX* .05 .05 .05 VITA ADIEUX .017 .017 The non-structural carbohydrate portions of the composite mixes given above are, low NSC 39.8%, medium NSC 51.2% and high NSC 64.6%, on a dry matter basis.
A trial was conducted in the dairy herd at the Away Cooperative Research Farm in Tulle, New York under controlled conditions. Forty-five multiparous, early lactation cows were balanced according Jo previous lactation 305-day mature equivalent production and parity, assigned to three groups of 15 cows each and fed three respective -total mixed ration treatments starting one day postpartum. The total daily ration consisted of I forage (dry matter basis) which included 22.5~ non-structural carbohydrates, and 60~ of the respective grain mix. This resulted in the non-structural carbohydrate (NSC) content of the total daily ration of the three groups being: low NSC 32.9~, medium NSC 39.7~ and high NSC 47.8~.
*trade mark - 12 -, I '73 All rations were isocaloric (Nil), isonitrogenous and the soluble protein levels were similar between treatments.
The total ration contained a minimum of I crude protein.
Individual feed consumption and milk production were recorded daily.
The forage portion of the ration consisted of hay crop silage, samples of which were collected weekly and composite monthly. Samples of the grain mixes were taken at the mill, at the time each new batch of feed was mixed. All samples were analyzed for percent dry matter, crude protein, soluble protein, acid detergent fiber, neutral detergent fiber, acid detergent bound nitrogen, non-structural carbohydrate, calcium and phosphorus.
Starting with day 6 and 7 postpartum and throughout the trial period, milk samples were collected weekly during your consecutive milkings the same 2 days of each week, composite and analyzed for percent fat and protein. Individual body weights were taken on two consecutive days immediately prior to trial initiation and again upon termination at lo weeks.
results are tabulated as follows:
ye ....
-` ~Z~2~3 TABLE III
Variable Low-NSC Med-NSC Hi-NSC
Castrate 15 15 15 . Dry matter intake (kg/cow/d) 18.7+.7 19.5+.7 18.8+.7 Milk yield (kg/cow/d) 33.8+4.5 35.1+4.3 30.9+3.4 Week of peak 5 8 5 Fat (%) 3.61+.6 3.49+.5 3.67+.4 Fat yield (kg/cow/d) 1.19+.05 1.19+.05 1.12+.04 Protein (%) 3.02+.2 2.99+.2 3.11~.2 protein yield (kg/cow/d) 1.01~.1 1.04+.1 .95+.1 4% FCM 31.4+~1 31.9+2.0 ~9.1~1.9 Body wit (kg) Initial 596 605 581 10 wok 583 592 564 Change -13 -13 -17 The difference in milk yield between the groups on the medium NSC and high NSC rations was statistically significant at a probability of less than 0.01, i.e., the probability was at least 99% that the higher yield was due to the difference in non-structural carbohydrate levels and not to chance. The higher milk yield for the group on the low NSC ration versus that on the high NSC was statistically significant at a probability of less than .10 (greater than 90%), and the 4% FCM (fat corrected milk) differences were statistically significant at a probability of less than .05.
As previously mentioned, the only forage used in the foregoing trial was hay crop silage, which contains higher levels of soluble protein and lower levels of non-structural carbohydrates than forages such as corn silage. Therefore, total rations could be formulated with a wider variation ,; ye/ -:
in the percentage of non-structural carbohydrates by adjustment of -the individual ingredients in the grain portion of the ration. Thus, when hay crop silage or hay comprises the sole forage source, it may be concluded, based upon the results of carefully controlled tests, that a positive response in milk yield is achieved by regulating the total daily ration to contain medium (39.7%) levels of non-structural carbohydrates, as compared to higher ~47.8%) levels. The optimum response was obtained at the medium level and, moreover r the group receiving the lower non-structural carbohydrate levels experienced a greater incidence of health-related problems. Therefore, when feeding hay or hay crop silage as forage, it has been demonstrated that regulating the total daily ration to contain in the neighborhood of 40% (e.g., within a range of 30% to 45%) non-structural carbohydrates provides optimum milk yield and feed efficiency with no deleterious side effects.
Recognizing that most forage rations do not consist entirely of hay or hay crop silage, additional testing was conducted to determine if regulating the total ration non-structural carbohydrate content is beneficial in other forage programs. A second trial was conducted under controlled conditions to evaluate the effect of regulating the non-structural carbohydrate content of dairy rations wherein the corn silage was the only forage. Since corn silage contains higher levels of non-structural carbohydrates than hay or hay crop silage, the percentage of non-structural carbohydrates in the total ration cannot be varied as widely by adjusting the composition of the grain mix in feeding ye/ I, :.
~5~'73 programs based on corn silage. As a practical matter, dairy rations consisting of 50% corn silage and 50%
grain mix, balanced to provide all necessary nutritional factors, may be varied by only about 15 percentage units in the amount of non-structural carbohydrate content.
In the test conducted to determine the effect of non-structural carbohydrate content of total daily ration on milk yield, 45 cows, in their second lactation or beyond, were equally balanced into two test groups and a control group. All cows were fed a total mixed ration consisting of 50% corn silage and 50% treatment grain mix, on a dry matter basis, free choice, over a 15 week period. As in the previously described test, all rations had the same amount of crude protein, soluble protein energy, vitamins and minerals. The only difference between rations was the amount of non-structural carbohydrates, which were balanced to levels of approximately 30~ to 40% for the two test groups. The control group was fed a commercial grain mix with regulated protein volubility which had previously been in use for a number of years. Although no account had been taken of the non-structural carbohydrate content when formulating the standard (control) grain mix, analysis showed that the total daily ration consisting of 50% of this mix and 50% corn silage contained 35.5% non-structural carbohydrates.
Tune compositions of the control and the two test grain mixes, by percentage of individual ingredients, were as follows:
yo-yo -~2~S~;~'73 TABLE IV
Grain Mix Treatments Control 35 5~-NSC 30 0%-NSC 40 0%-NSC
Ingredient (A) (B) (C) Jo (As Fed Basis) __________________________________________ Corn Meal 20.1 -- 36.45 Brewers grains 17.0 17.0 13.0 Distillers grains 9.0 15.0 7.75 Gluten feed -- 8.6 --Homing weed -- 8.75 --Wheat minds 20.5 -- 9.35 Soybean meal 23.0 21.0 26.7 Soybean hulls -- 25.7 2.0 Molasses 5.0 -- --Pellet binder 1.25 -- --Salt 1.0 1.0 1.0 Dicalcium phosphate .25 -- .40 Ground limestone 2.80 2.65 3.0 Dynamite* .25 .25 .25 Dairy TRUMAN* .05 .05 .05 VITA-MX AYE* .034 .034 ,034 * trade mark ,, I.
,", I 3 isle Results of the test are tabulated in the following table:
TABLE V
Treatments Control 35.5%-NSC 30.0%-NSC 40.0%-NSC
Variables (A) (B) (C) Castrate 15 15 15 Dry matter intake 19.2 + .7 20.4 _ .819.3 + .7 ilk yield, kg/day 32.7 _ 1.3 33.7 _ 1.3 34.6 _ 1.2 lo Actual fat, % 3.55 + .14 3.48 + lo 3.28 + .10 Covariantly adjusted fat, % (3.33 + .11) (3.57 + .05) (3.42 + .08) Fat yield, kg/day1.15 _ .05 1.17 _ .041.13 + .04 Protein, % 3.18 + .06 3.13 _ .05 3.13 + .05 - Protein yield, kg/day 1.04 + .03 1.05 _ .04 1.08 _ .03 4% fat-corrected milk, kg/day 30.3 + 1.1 . 31.0 + 1.0 30.8 _ 1.0 Efficiency kg milk/kgDM 1.70 + .09 1.65 + .08 1.79 + .07 kg 4% FC~/kgD~I1.60 + .07 1.54 + .07 1.62 + .07 Avg. gross milk income 8.71 8.88 3.87 ($/cow/day) Body weight Initial, kg 598 + 20 605 + 15582 _ 15 Final, kg 632 + 22 637 + 15614 + 11 Change, kg - 35 + 8 32 + lo 32 + 7 Milk yield was significantly higher in cows receiving the test ration containing 40% non-structural carbohydrate, as compared to the control group, the difference being l.9kg/cow/day, or 5.8% more milk, the average daily production being over 34. 5 kg per cow. The higher level of milk yield was statistically significant at a probability of less than .10, using accepted statistical procedures.
That is, since non-structural carbohydrate content was the only formulated variable, the probability was at least 90 that the higher milk yield was due to the difference in non-structural carbohydrate content in the rations and not to chance. Milk yield from the group receiving the 30%
non-structural carbohydrate test ration fell between those on the 40% and control (35.5%) rations and cannot be found to be different from either in a statistically significant sense.
The test also demonstrated that cows fed total rations with the optimum (40%) non-structural carbohydrate level peaked higher and consistently maintained a higher milk production level over the experimental period. efficiency of milk production was 5.3% higher for cows fed the optimum non-structural carbohydrate level. There was no significant difference in average daily feed intake between groups.
Thus, in each of thought trials described herein, a total ration non-structural carbohydrate level of approximately 40% gave significant positive responses in milk production.
In each trial an economic advantage in gross milk income was realized at the 40% non-structural carbohydrate level.
Although this level has been demonstrated to be effective with different forages, the range of values is wider for forages containing lower percentages of non-structural carbohydrates (e.g., hay and hay crop silage) than for those containing higher percentages (e.g., corn silage).
That is, while the total daily ration in a feeding program wherein the forage is hay or hay crop silage may range from US% to 45~ in non-structural carbohydrate content, ye/
~,S;~'73 the level in a corn silage based ration should be kept between 38~ and 42~.
Following the two previously described controlled tests, a field study was conducted to determine milk production responses when commercial dairy herds, under normal operating conditions, were changed from their normal grain rations to a regulated non-structural carbohydrate grain mix and balanced with the forage portion of the ration. The test included 13 dairy herds using predominately hay crop silage in a forage program in effect for at least two weeks before introduction of the regulated carbohydrate feeding program. Milk production was considered only from cows that were fed the rations regulated for non-structural carbohydrate content for a full 30-day period. A sample of each foodstuff used in the respective herds was obtained and analyzed for percent crude protein, soluble protein, non-structural carbohydrate, acid detergent fiber, neutral detergent fiber, calcium, phosphorus and dry matter. Non-structural carbohydrate and soluble protein of the total ration was calculated for each herd.
Again, the results of the field study confirmed that, in the majority of commercial herds, milk production is improved by a feeding program based upon regulated non-structural carbohydrates.
ye/ -
~.~22~Z'73 influenced by such factors as plant variety, maturity at harvest and storage conditions. Grains generally have more non-structural carbohydrates and are generally less variable in carbohydrate content than forage.
It has been generally accepted that structural carbon hydrate is a negative indicator ration energy concentration.
That is, the more structural carbohydrate a given ration contains, the less energy value it provides, and vice versa.
Present energy-based feeding systems for ruminants are based on this relationship.
In order to develop a feeding system which includes regulation of carbohydrates in the total daily ration, it is necessary, of course, to have available an accurate and repeatable method of quantitating both structural and non-structural carbohydrates in each constituent of the ration.
A procedure which has been used in the past to separate structural and non-structural carbohydrates involves refluxing - a given sample in a neutral detergent solution and filtering out the insoluble portion, termed "neutral detergent fiber."
It has been generally assumed that, for practical purposes, the neutral detergent fiber could be equated to the structural carbohydrate portion of the sample and, conversely, the neutral detergent solubles were essentially the non-structural carbohydrate portion. However, the fractions of non-carbohydrate substances present in both the neutral detergent fiber and solubles are significant to a degree that a reliable regulated carbohydrate feeding program cannot be based upon such generalizations.
Yams ~2~"S~73 Another chemical procedure which has been used to quantitate non-structural carbohydrate utilizes an enzyme called Taka-Diastase. (see Smith, D. "Removing and Analyzing Total Non-Structural Carbohydrates From Plant Tissue."
Wisconsin Air. Exp. Stay Rest Rep. 41, 1969). The procedure has also been modified, utilizing Bacillus subtilis type III
A aimless (see Madrid, JAMS Thesis, Cornell Univ., pp. 76, 77, 1981). These procedures basically involve the chemical action of enzymes on the plant material under consideration.
Although theoretically measuring the total amount of non-structural carbohydrate in a sample, the enzyme dictates what is being digested and therefore what is being quantitated.
- The Taka-Diastase enzyme is an aimless derived from Aspergillus ours, an organism grown on sterilized wheat bran or rice hulls. This extracted enzyme represents more than 30 different enzymatic functions, and is not only amylolytic, but digests proteins and fats as well, according to Merck Index, Thea Ed., p. 81, #633 (1976). Bacillus subtilis type III A enzyme has specific properties to hydrolyze, 1-4 glucosidic linkages of polysaccharides, also according to Merck Index, swooper. The activity of the latter enzyme may therefore be too "specific" to estimate all non-structural carbohydrates in a foodstuff. Therefore, non-structural carbohydrate measurements based on these enzymatic procedures are subject to variability and inconsistencies.
In the most general sense, the object of the present invention is to provide methods of formulating dairy cow rations which have a positive influence on milk production without an offsetting increase in the cost of the feeding program.
Yo-yo Lo . I.
A further object is to provide a novel method of measuring the amounts of structural and non-structural carbohydrates in a given plant material in order to permit reliable ration formulations based on nonstructural carbohydrate content.
Another object is to provide methods of balancing carbohydrate and protein types in dairy cow feeding programs which maximize efficiency of microorganism growth and yield, fiber digestion and overall feed efficiency, and milk production.
Other objects will in part be obvious and will in part appear hereinafter.
Summary Of The Invention -The method of measuring the carbohydrate content of feed-stuffs according to the present invention is based upon quantitation of non-structural carbohydrates in the dry matter of a sample by a "difference" method. The dry matter concentration of the foodstuff sample is determined by drying a portion thereof. Soluble ingredients are removed from one of the samples by refluxing with neutral detergent solution and filtering. The portion which is not dissolved, termed "neutral detergent fiber," consists essentially of structural carbohydrates, as well as neutral detergent insoluble crude proteins, lipid and ash. The material which is dissolved ("neutral detergent solubles") contains non-structural carbohydrates, consisting primarily of yo-yo;
I o starch, various sugar moieties, and pectin, as well as neutral detergent soluble crude protein, lipid and ash.
The other dry matter sample and the neutral detergent fiber are separately analyzed to determine the respective percentages thereof constituted by crude protein, lipid and ash. Crude protein content is calculated based upon the nitrogen content, livid is extracted with ether and ash is measured by weight after exposure of the samples to 500 C for 3 hours. The non-structural carbohydrate content may then be calculated by sub-treating from the neutral detergent solubles (i.e., 100-neutral detergent fiber) the sums of the crude protein, lipid and ash in the feed sample, each reduced by the respective crude pro-loin, lipid and ash in the neutral detergent fiber of the other sample.
Experiments were conducted to test the value of regulating the type and amount of carbohydrates in dairy cow feeding pro-grams which had previously been based upon regulating protein in accordance with previously mentioned patent No. 4,118,513.
Results of the initial research were analyzed to develop conical-sons an recommendations which were implemented in field studies, conducted on a total of 13 commercial dairy herds under actual operating conditions. Results of the field studies supported the findings of the controlled research results.
AL 'I
I,.
In one aspect the invention provides the method of formulating dairy cow rations for optimized milk production comprising: a) determining the percentage, on a dry matter basis, of non-structural carbohydrates Of each of a plurality of components, including at least one grain and one forage, collectively comprising a total daily ration;
and b) adjusting the proportion and composition of at least one of the components to a level wherein the portion of the total daily ration consisting of non-structural carbohydrates is between about 30~ and 45~.
DETAILED DESCRIPTION
Quantitation of non-structural carbohydrates according to the method of the present invention involves a determine-lion of dry matter concentration in a feed sample, ground to pass through - pa -. . , .
~2~5~7~
a 1 mm screen, the separation of neutral detergent fiber and solubles in a dry matter sample, and the calculation of protein, lipid and ash content. Known quantities (e.g., 2 to 5 grams) of two substantially chemically identical sub samples of a given foodstuff are allowed to dry in a forced air oven at 55 C for 48 hours. The difference in - weight of the sub samples before and after drying provides the original moisture content and dry matter concentration.
One of the samples is then reflexed with neutral detergent solution to quantify and separate the neutral detergent fiber and neutral detergent soluble portions. If desired, moisture content may be determined from a sub sample taken from the same sample as the first two, since dry matter concentration would be the same throughout the sample.
The basic procedure used to quantitate neutral detergent fiber was developed some years ago and is set forth in Agriculture Handbook No. 379 (Agric. Rest Serv., USDA, Washington, DO It has been employed with various modifications, the preferred procedure for purposes of the present invention being as follows:
Weigh 0.5 to 1.0g sample and place in a 600 ml Berzelius beaker. Add 50 ml cold neutral-detergent solution, prepared according to Ago Handbook 379. Place on hot plate and heat to boiling. Adjust heat to reduce foaming but boiling sufficiently to keep feed particles suspended. Thirty minutes from the onset of boiling, remove beaker and add 50 ml cold neutral-detergent solution and 2 ml of an enzyme (Aimless) solution.
dissolve 2 g Bacillus Subtilis type III A aimless in 90 ml of water, yo-yo 5~'73 filter through Whatmar.*X54 paper and add 10 ml Ethoxyethanol).
Return beaker to hot plate without adjusting the thermostat and allow to return to boiling. One hour after the initial onset of boiling, filter on a prepared sintered-glass crucible or Whitman X54 paper. Wash twice with boiling water to remove the detergent and twice with acetone. Dry overnight at 105 C
and weigh. Ash at 500C for three hours and weigh. The loss in weight in asking is an estimate of the plant cell wall constituents.
- Crude protein, lipid and ash content of the whole dry matter sample, and of the neutral detergent fiber separated from the other sample are separately determined. The total protein content, * trade mark 52~
expressed as 6.25 times the nitrogen content of the two materials, is determined according to Official Methods of Analysis of the Association of Official Agricultural Chemists (22.053), Thea Ed., 1965. Lipid analysis (Eat concentration) is performed by extracting the fat material in the foodstuff with ether. The materials are then exposed to a 500 C oven for 3 hours, the remaining materials being weighed and constituting the ash portion.
Although it is the non-structural carbohydrate fraction of the sample which is to be quantitated, it is impractical to do so directly since reagents associated with the neutral detergent solution may interfere with quantitation of the crude protein, lipid and ash which are dissolved together with the soluble carbohydrates. In addition, no single method has been developed to permit direct quantitation of non-structural carbohydrates which include starches, sugars and pectins. Therefore, quantitation of crude protein, lipid and ash in the original foodstuff, and in the neutral detergent fiber portion thereof as described above allows the amount of non-structural carbohydrates to be determined by differences.
The following equation and example will demonstrate the calculation of non-structural carbohydrate for a specific feed ingredient.
Equation: % Non-Structural Carbohydrate [Neutral Detergent Solubles Crude Protein in original - Crude Protein in Neutral Detergent Fiber Lipid in original - lipid in - Neutral Detergent Fiber Ash in original-Ash in Neutral Detergent Fiber fractional Dry Matter concentration, where:
ye/ I-~22S~73 Neutral Detergent Solubles=100-Neutral Detergent Fiber (NDF) Example of actual calculation for 48% soybean meal:
chemical Analysis Original Sample Neutral Detergent Fiber As fed Dry Matter As fed Dry Matter basis basis basis basis Moisture (~)10.60.0 10.6 0.0 NDF (%) 9.1 10.2 89.4 100 Crude protein (~)48.954.7 .8 .89 - 10 Fat (~) 1.0 1.1 .6 .67 Ash (~) 6-0 6.7 .4 .45 Calculation using all Dry Matter values:
nonstructural Carbohydrates = [(100-10.2)-[(54.7-.89)+(1.1-.67)+(6.7-.45)]
(Dry matter basis) = [(89.8) -[(53.8) + (.43) +(6.25)]]
- = [(89.8) - [(60.5)]]
= 29.3 Calculation using "As Fed" analysis, with corrections for Dry Matter:
nonstructural Carbohydrates = [(100-10.6-9.1)-[(48.9-.8)+(1.0-.6)+
(6.0-.4)]]/.894 (Dry matter basis) = 29.3 the foregoing method was used to determine the structural and non-structural carbohydrate content of a number of different forages and grains, the results being tabulated in the following table, wherein N is 30 the number of samples tested, NSC the percentage of dry matter constituted by non-structural carbohydrates, and SUM the standard deviation of the mean (SD):
'ho yo-yo. - -Table I
NSC
. N Mean SUM
Ingredient (% Dry Matter Basis) Corn silage 3 36.3 + 5.4 Hay crop silage 3 27.6 + 4.8 Soybean meal 4 29.5 + 1.0 Distillers grains 3 21.8 + 3.5 Dried brewers grains 4 13.2 + 1.8 lo Corn gluten feed 3 34.1 + 3.5 Corn gluten meal 3 25.5 + 7.4 Homing 3 57.6 + .9 Whole corn 3 75.1 + 2.8 Wheat middlings 3 34.0 + 3.9 Soybean hulls 3 20.8 + 2.3 Beet pulp 4 39.5 + 2.9 Based on the foregoing values, three experimental grain mixes were formulated to contain relatively low, medium and high levels of non-structural carbohydrates. The percentages of each ingredient used in the three grain mixes is as follows:
~2~5~ 3 TABLE II
In~redientsLow-NSC Med-NSCHigh-NSC
(us fed basis) Brewers grains 23.5 18.9 8.6 Soybean Molly 2.0 8.9 omen 45.0 49.4 4.0 Corn meal 2.6 4.0 65.7 Soybean Halsey 10.3 ---Wheat middlings 2.0 7.5 7.6 Corn gluten weed 2.0 5.2 2.0 Salt 1.0 1.0 1.0 Dicalcium phosphate .35 .5 .9 Ground limestone .8 1.1 1.2 Dynamite* .1 I .1 Dairy TRA-MIN-MX* .05 .05 .05 VITA ADIEUX .017 .017 The non-structural carbohydrate portions of the composite mixes given above are, low NSC 39.8%, medium NSC 51.2% and high NSC 64.6%, on a dry matter basis.
A trial was conducted in the dairy herd at the Away Cooperative Research Farm in Tulle, New York under controlled conditions. Forty-five multiparous, early lactation cows were balanced according Jo previous lactation 305-day mature equivalent production and parity, assigned to three groups of 15 cows each and fed three respective -total mixed ration treatments starting one day postpartum. The total daily ration consisted of I forage (dry matter basis) which included 22.5~ non-structural carbohydrates, and 60~ of the respective grain mix. This resulted in the non-structural carbohydrate (NSC) content of the total daily ration of the three groups being: low NSC 32.9~, medium NSC 39.7~ and high NSC 47.8~.
*trade mark - 12 -, I '73 All rations were isocaloric (Nil), isonitrogenous and the soluble protein levels were similar between treatments.
The total ration contained a minimum of I crude protein.
Individual feed consumption and milk production were recorded daily.
The forage portion of the ration consisted of hay crop silage, samples of which were collected weekly and composite monthly. Samples of the grain mixes were taken at the mill, at the time each new batch of feed was mixed. All samples were analyzed for percent dry matter, crude protein, soluble protein, acid detergent fiber, neutral detergent fiber, acid detergent bound nitrogen, non-structural carbohydrate, calcium and phosphorus.
Starting with day 6 and 7 postpartum and throughout the trial period, milk samples were collected weekly during your consecutive milkings the same 2 days of each week, composite and analyzed for percent fat and protein. Individual body weights were taken on two consecutive days immediately prior to trial initiation and again upon termination at lo weeks.
results are tabulated as follows:
ye ....
-` ~Z~2~3 TABLE III
Variable Low-NSC Med-NSC Hi-NSC
Castrate 15 15 15 . Dry matter intake (kg/cow/d) 18.7+.7 19.5+.7 18.8+.7 Milk yield (kg/cow/d) 33.8+4.5 35.1+4.3 30.9+3.4 Week of peak 5 8 5 Fat (%) 3.61+.6 3.49+.5 3.67+.4 Fat yield (kg/cow/d) 1.19+.05 1.19+.05 1.12+.04 Protein (%) 3.02+.2 2.99+.2 3.11~.2 protein yield (kg/cow/d) 1.01~.1 1.04+.1 .95+.1 4% FCM 31.4+~1 31.9+2.0 ~9.1~1.9 Body wit (kg) Initial 596 605 581 10 wok 583 592 564 Change -13 -13 -17 The difference in milk yield between the groups on the medium NSC and high NSC rations was statistically significant at a probability of less than 0.01, i.e., the probability was at least 99% that the higher yield was due to the difference in non-structural carbohydrate levels and not to chance. The higher milk yield for the group on the low NSC ration versus that on the high NSC was statistically significant at a probability of less than .10 (greater than 90%), and the 4% FCM (fat corrected milk) differences were statistically significant at a probability of less than .05.
As previously mentioned, the only forage used in the foregoing trial was hay crop silage, which contains higher levels of soluble protein and lower levels of non-structural carbohydrates than forages such as corn silage. Therefore, total rations could be formulated with a wider variation ,; ye/ -:
in the percentage of non-structural carbohydrates by adjustment of -the individual ingredients in the grain portion of the ration. Thus, when hay crop silage or hay comprises the sole forage source, it may be concluded, based upon the results of carefully controlled tests, that a positive response in milk yield is achieved by regulating the total daily ration to contain medium (39.7%) levels of non-structural carbohydrates, as compared to higher ~47.8%) levels. The optimum response was obtained at the medium level and, moreover r the group receiving the lower non-structural carbohydrate levels experienced a greater incidence of health-related problems. Therefore, when feeding hay or hay crop silage as forage, it has been demonstrated that regulating the total daily ration to contain in the neighborhood of 40% (e.g., within a range of 30% to 45%) non-structural carbohydrates provides optimum milk yield and feed efficiency with no deleterious side effects.
Recognizing that most forage rations do not consist entirely of hay or hay crop silage, additional testing was conducted to determine if regulating the total ration non-structural carbohydrate content is beneficial in other forage programs. A second trial was conducted under controlled conditions to evaluate the effect of regulating the non-structural carbohydrate content of dairy rations wherein the corn silage was the only forage. Since corn silage contains higher levels of non-structural carbohydrates than hay or hay crop silage, the percentage of non-structural carbohydrates in the total ration cannot be varied as widely by adjusting the composition of the grain mix in feeding ye/ I, :.
~5~'73 programs based on corn silage. As a practical matter, dairy rations consisting of 50% corn silage and 50%
grain mix, balanced to provide all necessary nutritional factors, may be varied by only about 15 percentage units in the amount of non-structural carbohydrate content.
In the test conducted to determine the effect of non-structural carbohydrate content of total daily ration on milk yield, 45 cows, in their second lactation or beyond, were equally balanced into two test groups and a control group. All cows were fed a total mixed ration consisting of 50% corn silage and 50% treatment grain mix, on a dry matter basis, free choice, over a 15 week period. As in the previously described test, all rations had the same amount of crude protein, soluble protein energy, vitamins and minerals. The only difference between rations was the amount of non-structural carbohydrates, which were balanced to levels of approximately 30~ to 40% for the two test groups. The control group was fed a commercial grain mix with regulated protein volubility which had previously been in use for a number of years. Although no account had been taken of the non-structural carbohydrate content when formulating the standard (control) grain mix, analysis showed that the total daily ration consisting of 50% of this mix and 50% corn silage contained 35.5% non-structural carbohydrates.
Tune compositions of the control and the two test grain mixes, by percentage of individual ingredients, were as follows:
yo-yo -~2~S~;~'73 TABLE IV
Grain Mix Treatments Control 35 5~-NSC 30 0%-NSC 40 0%-NSC
Ingredient (A) (B) (C) Jo (As Fed Basis) __________________________________________ Corn Meal 20.1 -- 36.45 Brewers grains 17.0 17.0 13.0 Distillers grains 9.0 15.0 7.75 Gluten feed -- 8.6 --Homing weed -- 8.75 --Wheat minds 20.5 -- 9.35 Soybean meal 23.0 21.0 26.7 Soybean hulls -- 25.7 2.0 Molasses 5.0 -- --Pellet binder 1.25 -- --Salt 1.0 1.0 1.0 Dicalcium phosphate .25 -- .40 Ground limestone 2.80 2.65 3.0 Dynamite* .25 .25 .25 Dairy TRUMAN* .05 .05 .05 VITA-MX AYE* .034 .034 ,034 * trade mark ,, I.
,", I 3 isle Results of the test are tabulated in the following table:
TABLE V
Treatments Control 35.5%-NSC 30.0%-NSC 40.0%-NSC
Variables (A) (B) (C) Castrate 15 15 15 Dry matter intake 19.2 + .7 20.4 _ .819.3 + .7 ilk yield, kg/day 32.7 _ 1.3 33.7 _ 1.3 34.6 _ 1.2 lo Actual fat, % 3.55 + .14 3.48 + lo 3.28 + .10 Covariantly adjusted fat, % (3.33 + .11) (3.57 + .05) (3.42 + .08) Fat yield, kg/day1.15 _ .05 1.17 _ .041.13 + .04 Protein, % 3.18 + .06 3.13 _ .05 3.13 + .05 - Protein yield, kg/day 1.04 + .03 1.05 _ .04 1.08 _ .03 4% fat-corrected milk, kg/day 30.3 + 1.1 . 31.0 + 1.0 30.8 _ 1.0 Efficiency kg milk/kgDM 1.70 + .09 1.65 + .08 1.79 + .07 kg 4% FC~/kgD~I1.60 + .07 1.54 + .07 1.62 + .07 Avg. gross milk income 8.71 8.88 3.87 ($/cow/day) Body weight Initial, kg 598 + 20 605 + 15582 _ 15 Final, kg 632 + 22 637 + 15614 + 11 Change, kg - 35 + 8 32 + lo 32 + 7 Milk yield was significantly higher in cows receiving the test ration containing 40% non-structural carbohydrate, as compared to the control group, the difference being l.9kg/cow/day, or 5.8% more milk, the average daily production being over 34. 5 kg per cow. The higher level of milk yield was statistically significant at a probability of less than .10, using accepted statistical procedures.
That is, since non-structural carbohydrate content was the only formulated variable, the probability was at least 90 that the higher milk yield was due to the difference in non-structural carbohydrate content in the rations and not to chance. Milk yield from the group receiving the 30%
non-structural carbohydrate test ration fell between those on the 40% and control (35.5%) rations and cannot be found to be different from either in a statistically significant sense.
The test also demonstrated that cows fed total rations with the optimum (40%) non-structural carbohydrate level peaked higher and consistently maintained a higher milk production level over the experimental period. efficiency of milk production was 5.3% higher for cows fed the optimum non-structural carbohydrate level. There was no significant difference in average daily feed intake between groups.
Thus, in each of thought trials described herein, a total ration non-structural carbohydrate level of approximately 40% gave significant positive responses in milk production.
In each trial an economic advantage in gross milk income was realized at the 40% non-structural carbohydrate level.
Although this level has been demonstrated to be effective with different forages, the range of values is wider for forages containing lower percentages of non-structural carbohydrates (e.g., hay and hay crop silage) than for those containing higher percentages (e.g., corn silage).
That is, while the total daily ration in a feeding program wherein the forage is hay or hay crop silage may range from US% to 45~ in non-structural carbohydrate content, ye/
~,S;~'73 the level in a corn silage based ration should be kept between 38~ and 42~.
Following the two previously described controlled tests, a field study was conducted to determine milk production responses when commercial dairy herds, under normal operating conditions, were changed from their normal grain rations to a regulated non-structural carbohydrate grain mix and balanced with the forage portion of the ration. The test included 13 dairy herds using predominately hay crop silage in a forage program in effect for at least two weeks before introduction of the regulated carbohydrate feeding program. Milk production was considered only from cows that were fed the rations regulated for non-structural carbohydrate content for a full 30-day period. A sample of each foodstuff used in the respective herds was obtained and analyzed for percent crude protein, soluble protein, non-structural carbohydrate, acid detergent fiber, neutral detergent fiber, calcium, phosphorus and dry matter. Non-structural carbohydrate and soluble protein of the total ration was calculated for each herd.
Again, the results of the field study confirmed that, in the majority of commercial herds, milk production is improved by a feeding program based upon regulated non-structural carbohydrates.
ye/ -
Claims (5)
- THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OF PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
l. The method of formulating dairy cow rations for optimized milk production comprising:
a) determining the percentage, on a dry matter basis, of non-structural carbohydrates of each of a plurality of components, including at least one grain and one forage, collectively comprising a total daily ration; and b) adjusting the proportion and composition of at least one of said components to a level wherein the portion of said total daily ration consisting of non-structural carbohydrates is between 30% and 45%. - 2. The method of claim l wherein said forage comprises between about 40% and 50% of said total daily ration, on a dry matter basis.
- 3. The method of claim 1 wherein said forage is corn silage and said percentage of non-structural carbohydrates is about 40%.
- 4. The method of claim 1 wherein said forage is hay or hay crop silage.
- 5. The method of claim 1 and further including determining the percentage, on a dry matter basis, of total protein, and the rumen-soluble and insoluble proportions thereof, of each of said components, and adjusting the proportion of at least one of said components to provide a total daily ration meeting the total protein required and wherein at least 15% but not more than 25% of said total protein is rumen-soluble.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000520226A CA1233098A (en) | 1985-01-23 | 1986-10-09 | Method for quantitating non-structural carbohydrates in feedstuffs |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US694,105 | 1976-06-09 | ||
| US69410585A | 1985-01-23 | 1985-01-23 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000520226A Division CA1233098A (en) | 1985-01-23 | 1986-10-09 | Method for quantitating non-structural carbohydrates in feedstuffs |
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| Publication Number | Publication Date |
|---|---|
| CA1225273A true CA1225273A (en) | 1987-08-11 |
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ID=24787417
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000476909A Expired CA1225273A (en) | 1985-01-23 | 1985-03-19 | Method for quantitating non-structural carbohydrates in feedstuffs and ration formulations based thereon |
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| Country | Link |
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| CA (1) | CA1225273A (en) |
-
1985
- 1985-03-19 CA CA000476909A patent/CA1225273A/en not_active Expired
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