Compositions and processes for providing amino acids and carbohydrates in ruminant feed

Information

  • Patent Application
  • 20030138524
  • Publication Number
    20030138524
  • Date Filed
    September 19, 2002
    22 years ago
  • Date Published
    July 24, 2003
    21 years ago
Abstract
Methods and compositions for improved ruminant diets are disclosed. The invention relates to the use of metal ion/metal ion salts in ruminant feed, at levels from about 0.25 to about 1 g/kg dry matter, in any ruminant diet, for improvement of bypass protein content, as well as to influence the rate of rumen starch digestion and the flow of starch to the duodenum.
Description


STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0002] Not applicable



REFERENCE TO MICROFICHE APPENDIX/SEQUENCE LISTING/TABLE/COMPUTER PROGRAM LISTING APPENDIX

[0003] Not applicable.



BACKGROUND OF THE INVENTION

[0004] The importance of rumen digestion of protein in the productive efficiency of ruminant diet formulations has been recognized for a considerable time. Feeds entering the rumen environment are digested with variable efficiency, such that the contribution of protein and energy to the rumen fermentation, or alternately to the animal via rumen escape followed by intestinal digestion and absorption, varies widely among feedstuffs. The variability in feeding value amongst feeds and animal classes has led to the development of diet evaluation software incorporating digestion rates relating to a variety of nutritionally important protein and carbohydrate fractions (Dairy NRC 2001; Fox, et al., 1992.).


[0005] As summarized in such computer programs, the specific feed value of a dietary ingredient varies both with animal productivity and diet formulation or composition. As animal productivity increases, so do the nutritional requirements for amino acids, metabolizable protein and energy. At low levels of production, nutrition demands are more readily satisfied by the end products of rumen fermentation, as well as volatile fatty acids as energy sources, and by the use of microbial protein to supply metabolizable protein and amino acids. At elevated production levels the gross efficiency of nutrient digestion decreases, increasing the proportion of nutrients escaping rumen fermentation. The specific efficiency of microbial protein produced in the rumen is somewhat variable and difficult to predict, but does not increase sufficiently, such that rumen fermentation is unable to supply the quantity of metabolizable protein required to meet productive demands. This shortfall of rumen microbial protein production increases the dietary demand for rumen bypass protein. Thus, research efforts aimed at achieving continued increases in ruminant productive level and efficiency have emphasized the importance of the nutrients which escape or bypass rumen fermentation. Therefore, a number of rumen escape proteins are now available in the marketplace.


[0006] A parallel development to rumen escape protein has been an increase in dietary energy density to meet the energetic demands of increased production. For high production situations, this has increased the level of rumen fermentable carbohydrate in diets by raising starch levels. Increased feeding of starchy ingredients has led to increased concerns relating to rumen acidosis and loss of productive efficiency from the rumen.


[0007] A variety of methods have been used to reduce the rumen availability of vegetable protein. U.S. Pat. No. 3,619,200 proposes a rumen-inert coating of vegetable meal for protection against rumen microbial digestion. Treatment of feeds with tannin, formaldehyde, or other aldehydes can denature the protein and reduce ruminal fermentation (see U.S. Pat. No. 4,186,213), and rumen digestion of protein can be reduced by heating (Tagari et al., Brit. J. Nutr. 16:237-243 (1982)).


[0008] Hudson, et al., J. Anim. Sci. 30:609 (1970) presents an experiment comparing evaluating the effect of heating on SBM on the post ruminal nitrogen utilization by lambs. The results indicated slower protein digestion by rumen microflora.


[0009] Endres, et al., 1996, and Heitritter, et al., 1998 (U.S. Pat. Nos. 5,508,058 and 5,824,355, with references) summarize the procedures commonly used for production of heat-treated vegetable meals.


[0010] The patents of Meyer, 1987, 1988, and Endres, et al., 1996 (U.S. Pat. Nos. 4,664,905, 4,664,917, 4,704,287,4,737,365, 5,508,058) disclose the use of zinc salts to protect animal feed protein from rumen degradation.


[0011] The patents of Meyer, 1987 and 1988 (U.S. Pat. Nos. 4,664,905, 4,664,917, 4,704,287, 4,737,365) established the use of relatively high levels of zinc salts to improve protein utilization in beef and dairy cattle and sheep. Incorporation levels of zinc were from 0.25 to 1.3% dry weight or alternately 0.005 to 0.0294 parts zinc ion per unit protein in a protein dry blend.


[0012] Endres, et al., 1996 (U.S. Pat. No. 5,508,058) disclose a method to produce heat treated vegetable protein incorporating zinc at a lower level than previously discovered (0.003 to 0.008 parts zinc per part protein). As discussed in that disclosure, the use of lower zinc levels is beneficial in reducing the excretion of zinc into the environment via animal manure while retaining efficacy of reducing rumen protein digestion of the protein feed.


[0013] 1. Field of the Invention


[0014] The present invention relates to methods and compositions for the improvement of ruminant diets. More specifically, the present invention relates to the use of metal ions and/or their salts in feed to improve productive efficiency where alterations in rumen digestion rates of protein or starch are desirable.


[0015] 2. Background Art


[0016] Not applicable.



BRIEF SUMMARY OF THE INVENTION

[0017] The present invention relates to improved animal feed compositions comprising one or more metal ion(s) or metal salt(s) at a concentration of from about 0.25 gram to about 1 gram per Kilogram of feed dry matter. The present invention further relates to ruminant diet formulations comprising such improved feeds, and the process for making such improved feed compositions. The present invention also further relates to a process for improvement of the productive efficiency of a ruminant diet by providing to a ruminant a diet comprising such an improved animal feed.


[0018] This invention has a primary objective of retaining the efficacy equivalent to previous zinc use while further reducing the levels of zinc needed.


[0019] A further objective of this invention is to utilize zinc singly and in combination with other metals or metal salts to modify the rumen degradation of both protein and carbohydrates.


[0020] Previous work has focused on the ability of dry mixtures of metal salts to protect protein-containing feeds as part of protein supplements or complete feeds. In early efforts, manganese was evaluated for its ability to slow rumen protein digestion and was discounted in favor of zinc. However, the present inventors have found that, surprisingly, the use of zinc salts with manganese salts (manganous sulfate) in art-recognized methods of use leads to an unexpected synergistic reduction of rumen protein digestion, where the decrease in protein degradation is greater than that expected based on the levels of either element singly. Furthermore, a similar synergy has been achieved by adding soluble iron salts to the blend. Unexpectedly, the form of iron is particularly important, with ferrous iron preferred to other electron states. Recent research also demonstrates the ability of zinc salts and metal mixtures to influence the rate of rumen starch digestion and the flow of starch to the duodenum of dairy cattle.


[0021] In addition, the present invention takes advantage of the surprising finding that the effects of metal salts may be generalized to all dietary ingredients contributing protein, including forages, although the magnitude of effect is ingredient specific. Thus, the present invention relates to the use of metals salts, in combination with both amino acid formulation, and the formulation of the entire diet, to influence the amino acid and nutrient profile appearing at the duodenum, allowing increased animal performance.



BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0022] Not applicable.







DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention proposes the use of zinc in combination with heat processing at levels from about 0.25 to about 1 g per kg dry matter (DM) of the feed (meal or forage) being utilized. Further, combinations of water soluble salts (preferably sulfate salts, although it is important to note that all water soluble salts, and combinations of metals or metal salts, may be used in the practice of the invention), of zinc, manganese and iron (preferably the ferrous form of iron) may be provided in animal diets singly or in combination at a total concentration of between about 250 and about 750 ppm of diet DM to increase rumen escape of diet protein, to reduce ruminal ammonia production, and to reduce the fermentation rate of dietary starch. In addition, when combined with dietary formulation of amino acid profiles, incorporation of water soluble zinc, manganese, or iron salts may be utilized to modify the profile of amino acids appearing in the post-ruminal digesta flow.


[0024] The present invention may be practiced in any ruminant diet. To obtain the desired results, diets may be formulated to contain single metal ion forms or combinations of metal ions at a concentrations from about 0.25 to about 1 g/kg of diet dry matter. In practice metal salts may be incorporated directly into the animal diets, or mixed into commercial supplements or liquid feeds. As those of skill in the art will recognize, absolute concentrations of the metal salts incorporated into supplements will be dependent on the dietary inclusion rate of the supplemental feed. For example, and not by way of limitation, to an animal eating 25 kg dry matter per day, a mineral supplement or liquid feed offered at 1 kg dry matter per day may be expected to contain between 6.25 to 25 g metal ion per kg of the supplement. If the supplemental inclusion rate were to increase to 10 kg dry matter per day the corresponding concentrations would be 0.625 to 2.5 g metal ion per kg of supplement dry matter.


[0025] This invention may also be used to improve the bypass protein content of animal feeds in combination with moist heat treatment. For example Heitritter, 1998, discloses the use of a moist heat treatment process. To utilize this invention in combination with heat treatment of protein meals, zinc or metal combinations may be blended into the protein meal entering the process at a rate to obtain from about 0.25 to about 1 g of metal ion per kg of feed dry matter, utilizing either dry mixtures or liquid application of salts. Alternately, the metal blends may be incorporated into the feed ingredient after the initial cooking process but prior to the drying of the final mixture.


[0026] During the experimental work related to development of this invention an interaction between processing method and zinc concentration was determined which allowed for the reduction of zinc concentration to between about 0.25 and about 1 g/kg of feed material, or to a level of about one-third the amount previously reported. Table 1 presents the results of a test addressing the interaction of moist heat treatment of soybean meal and zinc application. The expectation at the time was that the response in rumen undegradable protein (RUP) required zinc levels above 1300 ppm in meal DM. Surprisingly, however, there was a clear relationship between zinc concentration and bypass protein content/RUP even at lower than expected metal concentrations.
1TABLE 1Influence of zinc and heat treatment on the rumen undegradableprotein content (RUP) of soybean mealSampleZinc, ppmRUP, % CP426266.51447267.925101069.136172170.2


[0027] In a second embodiment, experimental work by the present inventors has derived improvements over the state of the art by the use of combinations of zinc and manganese or combinations of zinc, manganese and ferrous iron to influence rumen fermentation and animal performance. These improvements occur when metals are provided at concentrations ranging from about 250 to about 1000 ppm total metal ion in diet DM. Specifically, dietary inclusion of metal combinations has been shown to reduce rumen protein digestion (increase RUP), reduce ammonia release from protein and reduce milk urea nitrogen levels, and slow the rate of rumen starch digestion. Thus, depending on diet formulation methods, metal addition may be used to influence the profile of nutrients appearing at the duodenum for absorption.


[0028] Having provided a general description, the invention will now be more readily understood through reference to the following examples, which are provided by way of illustration, and which are not intended to limit the present invention.



EXAMPLES


Example 1

[0029] In vitro digestion of alfalfa silage, a complete dairy pellet, and dairy total mixed ration TMR) was performed using an artificial rumen system (Ankom Daisy System, Ankom Technology, Fairport, N.Y.) in a partial factorial arrangement of treatments incorporating zinc or manganese, at two levels, singly or in combination, to evaluate effects on rumen protein digestion. Metals were incorporated at 150 or 300 mg/L. As shown in Table 2, zinc has a general effect on protein digestion while the effect of manganese is more moderate. The ‘Additive’ column presents the expected results based on a simple additivity of the manganese and zinc concentrations. The combination of zinc and manganese reduces protein digestion in a manner similar to the zinc only treatment although the level of zinc has been reduced by 50%. These data extended previous findings, demonstrating that zinc and the zinc-manganese addition improved the rumen bypass protein content of alfalfa forage and dairy TMR samples as measured in the artificial rumen system (Table 3). Previous efforts focused on the digestion of high protein feeds such as oilseed meals.
2TABLE 2Average rumen undegraded protein, % of protein,for all samplesMetalinclusionZinc (Zn)Manganese (Mn)‘Additive’Zn/Mn, 50:50 040.2150 mg/L4543.242.744.2300 mg/L4842.545.347.4


[0030]

3





TABLE 3










Effect of dietary metal addition on rumen undegraded protein










Metal inclusion
Zinc (Zn)
Manganese (Mn)
Zn/Mn, 50:50










Dairy Concentrate Feeds










 0
47.8




150 mg/L
61.4
50.9
55.7


300 mg/L
63.5
52.3
67.4







Alfalfa Haylage










 0
24.6




150 mg/L
24.4
26.1
31.3


300 mg/L
28.2
24.8
25.5







Dairy total mixed ration










 0
48.3




150 mg/L
48.9
51.8
46.3


300 mg/L
52.5
51.5
48.3











Example 2

[0031] Samples of soybean meal (SBM), heat treated soybean meal, canola meal, heat treated canola meal, and cottonseed meal were fermented in vitro in combination with zinc sulfate, or ferric or ferrous iron sulfate. In those fermentations containing metal, metal ions were added to obtain a concentration of 150 mg/L. Relative to controls with no metal addition all metals increased RUP content measured after 16 h of fermentation (Table 4). Surprisingly, the ferrous form of iron was substantially more effective than the ferric ion for decreasing rumen protein digestion.
4TABLE 4Mean RUP and Metal Effects for Individual IngredientsTreatment50:50 ZincFerrousandIngredientControlFerric IronIronZincManganeseSBM28.6a29.1a42.9b56.4c59.7Canola46.5a51.6ab57.7b57.3b66.3Cottonseed56.3a58.4b62.1bc64.4c66.3Heated Canola79.7a81.1a83.5b85.2b85.9Heated SBM78.6a81.7ab83.9b85.2b85.6a,b,cMeans within row with different superscripts differ P < .05.



Example 3

[0032] Lactating Holstein dairy cows were randomly divided into two groups based on production, days in milk and parity. Both groups of animals received diets based on alfalfa and corn silage supplemented with a commercial concentrate. The treatment diet was formulated to provide 300 ppm of a 50:50 blend of zinc and mangenous sulfate. Calculated soluble protein supplied was 40% of dietary crude protein (CP). The level of heat treated soybean meal was reduced in the metal containing diet to account for the effects on protein digestion (calculated as two percentage decrease in RUP, % of dietary CP). Both diets were formulated to contain RUP of a similar amino acid profile. There were no differences in milk production or milk component levels. There was a significant decrease in milk urea nitrogen levels with metal inclusion. These data demonstrate the effects of metal ions on performance by lactating cows, and are comparable to prior art in which zinc alone was used. The difference in the present example is that combining metals affords efficiency with lower concentrations of zinc than expected being necessary in the final feed product.
5TABLE 5Effects of Zinc and Manganese on Milk Yield by Lactating CowsItemControlZn/MnSEP =Diet CP19.0518.98Diet Zn, ppm88216Diet Mn, ppm78211Milk, kg/d35.635.6.63.86Milk Fat, %3.493.43.06.52Milk Protein, %2.92.92.02.24MUN1, mg/ml14.914.1.20.021MUN = milk urea nitrogen



Example 4

[0033] Individually fed crossbred wether lambs were fed diets containing 14% CP (as a negative control) or 16% CP with low or high RUP content to examine the feeding of divalant metals singly or in combination. Bypass protein content was increased by feeding higher amounts of heat-treated soybean meal. In the 16%, Low RUP diet, the following metal additions were tested: 500 ppm Zn; 250 ppm Zn:250 ppm Mn; and 170 ppm Zn: 170 ppm Mn: 170 ppm Fe. All metals were added in the sulfate form, and iron was in the form of ferrous sulfate. Feeding the 16% CP: High RUP diet or adding metals to the 16% CP: Low RUP tended to decrease gain and feed efficiency with only small effects on feed intake relative to that obtained with the 16%, low RUP diet (Table 6).
6TABLE 6Performance of lambs receiving zinc, or zinc, manganese, iron combinationsHigh CPHigh CPHigh CPNegativeHigh CPHigh CPLow RUPLow RUP +Low RUP +Diet DescriptionControlLow RUPHigh RUPZnZn/MnZn/Mn/FeCP, %14.016.016.016.016.016.0RUP, %5.05.26.45.25.25.2SEMInitial weight, kg 24.4 23.2 24.7 24.7 24.0 23.91.242 day weight, kg 40.1b 44.2a 43.7a 43.8a 42.7a,b 41.8a,b1.4DMI1, kg/day 1.50b 1.59a,b 1.62a,b 1.66a 1.51b 1.49b0.05Total Gain, kg 15.84b 20.44a 18.96a 18.81a 18.88a 18.30a1.12ADG1, g/day394c486a464a,b451a,b446a,b429b,c17G/F1 × 100 26.5b 30.7a 28.7a,b 27.3b 29.6a 28.9a,b1.1a,b,cMeans in the same row with different superscripts are different (P < 10). 1DMI = dry matter intake, ADG = average daily gain, G/F = ratio of weight gain to feed intake


[0034] Although the effects of additional RUP were negative on animal performance for this model animal system, the data substantiate the use of low amounts of zinc, or combinations of zinc manganese and iron to influence rumen protein digestion. The level of response to metal addition was equivalent to that of the heat treated SBM.



Example 5

[0035] A six week lactation study was conducted using 59 Holstein cows to test the effects of zinc on performance and the interaction with dietary bypass protein content (RUP). Treatments were administered by adjusting the RUP in the 20% CP dairy complete feed from 8.0% (Control) to 9.0% RUP (Control+RUP) and zinc from 245 ppm (Control) to 1020 ppm of zinc (Control+Zinc). This provided 755 ppm zinc from zinc sulfate in a ratio of 0.003 part zinc ion per part protein in the complete feed. The source of RUP was a combination of heated soybean meal, corn gluten meal, and distiller dried grains. Increasing dietary zinc numerically improved milk yield by 1.1 kg/d or 3.4%. Feeding higher amounts of bypass protein did not improve yield. Without intending to be limited by theory, these results suggest that zinc may be affecting the supply of nutrients besides protein that are critical for lactation. Zinc may decrease ruminal digestion of fiber and nonfiber so that more of these components are supplied to the intestines. This phenomena may be detrimental in the case of fiber but potentially beneficial in the case of nonfiber (e.g., starch) because energy supply to the cow may be improved. The potential for zinc to shift the site of carbohydrate digestion from the rumen to the intestines has not been described previously.
7TABLE 7Milk production of cows receiving zinc or RUPItemControlControl + ZnControl + RUPMilk, kg/d32.133.231.8Milk Fat, %3.303.253.33Milk Protein, %3.213.203.25



Example 6

[0036] A study was performed to examine whether increasing dietary zinc and/or RUP content would affect ruminal digestion and the flow of amino acids to the small intestine of lactating dairy cows. Cows were fed a TMR containing low and high amounts of RUP and low or high concentrations of zinc (zero or additional 250 ppm zinc from zinc sulfate). The concentration of RUP in high and low RUP diets were manipulated by changing the proportions of low RUP feeds (soybean meal, canola) and high RUP feeds (heated soybean meal, corn gluten meal). Feeding higher amounts of zinc shifted site of nutrient digestion from the rumen to the small intestines. A surprising observation was the effect of zinc on rumen digestion of starch. In both the low and high RUP diets, feeding higher concentrations of zinc reduced the digestion of starch in the rumen.
8TABLE 8Effects of RUP level and zinc on rumen nutrient digestionLow RUP,Low RUP,High RUP,High RUP,ItemLow ZnHigh ZnLow ZnHigh ZnDry matter intake,22.821.821.522.2kg/dIntestinal Amino Acid flows, g/dLysine187.2183.3182.6200.0Methionine51.649.056.158.2Total Essential1398133114691601Amino Acids Ruminal digestion, % of intakeOrganic Matter54.147.345.545.5Neutral Detergent33.522.024.024.8FiberStarch67.053.664.352.8



Example 7

[0037] In vitro digestions of a complete dairy pellet, and a dairy total mixed ration TMR) were performed using an artificial rumen system (Ankom Daisy system) to evaluate the effects of metal ions on rumen starch digestion. Treatments formed a partial factorial arrangement incorporating zinc or manganese, at 150 or 300 mg/L of metal in the in vitro media, singly or in combination, to evaluate rumen starch digestion. Inclusion of zinc or the zinc and manganese combination increased the rumen undegraded starch measured at 16 h and decreased the calculated kinetic rate for starch digestion. The decrease of in vitro pH over the first 16 hours of fermentation was moderated by the inclusion of zinc or zinc-manganese blend as was the rate of decline. The total decline in pH over the 48 hour fermentations (initial pH—final pH) was not statistically different.
9TABLE 9Mean Effect of Metal Addition on In Vitro Starch Digestion and pH50:50 Zinc +ItemControlZincManganeseManganeseMain Effects1Concentration, mg/L0150300150300150300SEZnMnZn/MnRumen Undegraded8.613.212.05.18.013.215.61.94.0−2.05.8*Starch (16 hours), %Ammonia mg/dL31.430.829.732.134.230.629.91.8−1.11.8−1.2Initial pH6.746.696.606.656.646.686.62.03−.1**−.1**−.09**pH at 16 hours−.37−.35−.28−.28−.34−.32−.32.03.05*.05*.05*pH at 48 hours−.32−.30−.23−.30−.27−.30−.25.03.05.03.05Digestion Rates,% per hourRumen Undegraded−.135−.096−.098−.137−.132−.105−.102.007.038**0.0.031**Starch (16 hours)pH Decline.233.209.205.135.247.221.220.04−.027−.043−.0131P < .1 = *, P < .05 = **.



Example 8

[0038] A study was conducted using four duodenally cannulated Holstein cows to investigate the effect of a zinc:manganese blend on the rumen digestion of starch and soluble fiber. Diets contained 10.9 kg alfalfa silage, 3.6 kg mixed hay, 1.4 kg hay pellets, 9. 1 kg complete supplement and 2.7 kg of com/soyhulls or beet pulp/soyhulls (non-forage fiber, or NFF). Dietary RUP was formulated to 32.5% of CP, and the RUP lysine and methionine was adjusted to a 3:1 ratio. Diets containing added metal contained 400 ppm of metal added as 50:50 zinc and manganese. In this experiment, numerical decreases in rumen starch digestion were associated with greater rumen pH, altered volatile fatty acid (VFA) profile, and significantly increased microbial efficiency (Table 10).
10TABLE 10Effects of non-fiber carbohydrate type and metal addition on rumen nutrient digestionDietP =MetalCornNFFNFCandItemCornNFFaZn/MnZn/MnSEMsourceZn/MnNFCDry Matter Intake, kg/d20.218.817.720.2.09.55.60.10Rumen Digestion, % intakeOrganic Matter31.140.832.540.91.7.01.65.63Neutral Detergent Fiber33.443.027.635.58.0.28.42.90Starch75.966.664.061.29.8.60.56.53Duodenal flowNon-Microbial N, % N intake57.552.350.045.03.4.17.10.99Microbial N, % N flow48.846.556.159.22.9.86.03.32Microbial Efficiency g of N/kg32.424.636.231.81.3.01.02.18organic matter digestedAverage Rumen pH5.775.965.835.81.13.50.76.42Acetate:Propionate Ratio2.602.802.962.720.09.85.12.04aNFF = non-forage fiber (beetpulp and soyhulls) b NFC = non-fiber carbohydrate


[0039] In view of the foregoing description and examples, those skilled in the art will be able to practice the invention, in its various embodiments and equivalents, without undue experimentation, and without departing from the spirit and scope of the invention as defined in the appended claims.


Claims
  • 1. An improved animal feed composition comprising an animal feed containing one or more metal ion(s) or metal salt(s) at a concentration of from about 0.25 gram to about 1 gram per Kilogram of feed dry matter.
  • 2. The animal feed composition of claim 1 wherein the one or more metal ion(s) or metal salt(s) are selected from the group consisting of zinc, manganese, and ferrous iron, or water soluble salts thereof.
  • 3. The animal feed composition of claim 1 wherein the feed dry matter is selected from the group consisting of meal dry matter or forage dry matter.
  • 4. A ruminant diet formulation comprising the animal feed composition of claim 1.
  • 5. A process for the improvement of the productive efficiency of a ruminant diet, the process comprising providing to a ruminant a diet comprising an animal feed comprising one or more metal(s) or metal salt(s) at a concentration of from about 0.25 gram to about 1 gram per Kilogram meal dry matter.
  • 6. The process of claim 5 wherein the one or more metal(s) or metal salt(s) are selected from the group consisting of zinc, manganese, and ferrous iron, or water soluble salts thereof.
  • 7. A process for making an improved animal feed composition, the process comprising adding to the animal feed composition an amount of one or more metal(s) or metal salt(s) to give a final concentration of from about 0.25 gram to about 1 gram per Kilogram meal dry matter.
CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No. 60/324,593, filed Sep. 25, 2001, which application has been incorporated by reference in its entirety.

Provisional Applications (1)
Number Date Country
60324593 Sep 2001 US