Illllllllll!Illllllllllllllllllllllllllllllllllll* a it 3 1293 10533 7749 9'7“"? ': an? THESlS as“. ”-4433; '2 ..-. .- ‘f'a.’ I > ' '. . , w _‘ ,..1 u»; 3" c, '1‘“, ‘D ~ This is to certify that the thesis entitled Evaluation of Markers for Determining Site and Extent of Digestion and Digesta, Flow Patterns in Steers Fed Corn Silage Diets . presented by Gary Michael Weber has been accepted towards fulfillment of the requirements for Ph.D. degree in Animal Science Major professor [MNe January 19, 1984 0-7639 MSU is an Affirmative Action/Equal Opportunity Institution 1 v *‘m- -—-vv—-o‘o . , “m... ‘1 . r .I M. ‘i‘LAVJ‘J' '# X' ' I' - MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record.. FINES will be charged if book is returned after the date stamped below. Evaluation of Markers for Determining Site and Extent of Digestion and Digesta Flow Patterns in Steers Fed Corn Silage Diets By Gary Michael Weber BOSO, MOS. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Animal Science 1983 ABSTRACT Evaluation of Markers for Determining Site and Extent of Digestion and Digests Flow Patterns in Steers Fed Corn Silage Diets By Gary Michael Weber The effect of marker choice Zlignin, ytterbium, lanthanum, chromium EDTA) upon estimates of site and extent of digestion of corn silage based diets was investigated. An abomasal infusion of polyethylene glycol (PEG) was utilized to evaluate digests flow patterns and marker recovery at the duodenum. Superimposed upon these investigations was an evaluation of the effect of several supplemental protein sources (urea, soybean, .corn gluten meal, wet distillers grains) upon digestion of major dietary constituents in steers fed corn silage based diets. Studies utilized Latin square, crossover designs with Holstein steers having duodenal (T) cannulae, or in addition, abomasal infusion cannulae. Estimates of apparent rumen digestibility of dietary constituents was significantly affected by marker selected. Lignin levels were elevated in duodenal samples, perhaps due to elevated acid detergent fiber levels. Estimates of total tract digestibility of dietary constituents do not vary significantly due to marker choice. Fecal lignin recovery tended to be elevated (average, 110% of intake) and produced slightly higher digestibility estimates than other markers. Fecal chromium levels, when corrected for an estimated 5% absorption, provided estimates of digestibility similar to other markers. Ytterbium satisfies the requirements of an ideal marker in more respects than any other marker evaluated. Digests flow patterns, estimated by PEG infusion, indicated the presence of a phasic pattern of duodenal flow of digesta. The pattern appeared to vary with supplemental protein source. Corn silage diets supplemented with urea, soybean and corn gluten meal exhibited nearly equal ruminal and total tract digestion of nitrogen, organic matter and acid detergent fiber. Corn silage diets supplemented with wet distillers grains exhibited reduced ruminal and total tract nitrogen availability as compared to diets supplemented with urea ans soybean meal. DEDICATION This thesis is dedicated in loving memory of my father, Alan Weber. My father's commitment to excellence in all aspects of life set a prece- dent I am proud to follow. I will always remember..."like sparks from flint and steel". ii ACKNOWLEDGEMENTS I am indebted to so many people for their council, friendship and assistance during my graduate program. The outstanding members of my advisory committee, Dr. John Waller, Dr. Werner Bergen, Dr. Dave Hawkins and Dr. J. Roy Black, were a pleasure to work with. I feel privileged to have had such an excellent committee. Their contributions will always be appreciated. I am also very thankful for having the Opportunity to work with people like Kris Johnson, Patty Dickerson, Brad Knudsen, Matt Parsons, Doug Bates, Ken Geuns, Steve Baertsche and Farabee McCarthy. Their friendship and assistance will always be remembered. My most sincere thanks to Dr. Ron Nelson and Dr. Harlan Ritchie for their valuable advice on career opportunities. The appropriate words do not exist to express my appreciation and respect for fellow graduate students, Bill Rumpler, Don Banner and Scott Barao. They have been friends and colleagues for which there are no equals. The persbn I am most indebted to is my wife, Roberta. As a friend and colleague, her infinite patience, love and assistance in my life are greatly appreciated. iii TABLE OF CONTENTS LIST OF TABLES .............. LIST OF FIGURES ............. INTRODUCTION ................ LITERATURE REVIEW ........... Markers in Ruminant Nutrition Marker Systems Marker Ratio Techniques Nonabsorption of Markers Inert, Indigestible Markers Marker and Digests Flow Nontoxic Markers Analytical Quantification of Marker Marker Effect Upon Digestion of Dietary Constituents Marker Association and Movement Estimation of Feed Component Rate of Passage 0......00...00.. In-situ Dacron Bag Digestibility Studies Integration of Rate of Passage and Degradability Calculations Double Marker Method Two Marker Method Effects of Cannulation Upon Digestion of Diets in Ruminants Digestibility of Corn Silage and Supple- mental Protein Sources by Ruminants Factors Affecting the Digestibility of Supplemental Protein Sources Protein Chemistry Protein Bypass Estimates Level of Intake Effects on Site and Extent of Digestion Effects of Shifting the Site and Extent of Digestion SUMMARY PRELIMINARY EVALUATION MARKER BINDING BEHAVIOR IntrOduction .0.....0..................0 Materials and Methods Results and Discussion iv 15 17 18 21 23 25 26 28 29 33 34 35 36 39 4O 4O PRELIMINARY EVALUATION: Introduction .................... Materials and Methods ........... Results and Discussion .......... MATERIALS AND METHODS Experiment One Experimental Design ............. Cannula Design and Insertion .... Diet Formulation ................ Markers ......................... Housing, Adaptation, Feeding .... Sampling, Compositing, Processing Feed .......................... Duodenal ...................... Fecal ......................... Analytical Techniques Nitrogen ...................... Ammonia NitrOgen .............. Acid Detergent Fiber, Lignin, Nitrogen ...................... Dry Matter, Ash, Organic Matter Activation Analysis: Ytterbium, Chromium ........... Calculations: Flow and Digestibility Experiment Two Experimental Design ............. Cannula Design and Insertion .... Diet Formulation ................ Markers ......................... Housing, Feeding, Adaptation .... Sampling, Compositing, Processing Feed .......................... Duodenal .....................: Fecal ......................... Analytical Techniques Nitrogen ...................... Ammonia Nitrogen .............. Dry Matter, Ash, Organic Matter Acid Detergent Fiber, Lignin .. Activation AnalySis 0.0.00.0000000 Polyethylene Glycol Analysis ..... Calculations: Flow and Digestibility FECAL EXCRETION OF MARKERS 42 42 43 45 47 48 54 57 6O 62 65 67 67 69 71 71 75 78 79 80 83 85 86 86 87 87 87 88 88 88 89 91 RESULTS AND DISCUSSION Experiment One The Effect of Supplemental Protein Source Upon Site and Extent of Digestion of Corn Silage Based Diets Rumen Disappearance and Flow ........... 94 Lower Tract Digestion .................. 99 Total Tract Digestion ..................lOO ADIN Flow: Total Tract .................102 Lignin Flow: Total Tract ...............104 Comparison of Digests Markers: Rumen and Total Tract Estimates ..................110 Evaluation of Markers: Selection Based on magnitude 0f the SEM .....000.000.000.000000000000116 RESULTS AND DISCUSSION Experiment Two The Effect of Soybean and Corn Gluten Meal Supplementation Upon Site and Extent of Digestion of Corn Silage Diets by Steers Rumen Disappearance and Flow ...........120 Lower Tract Digestion ..................122 Total Tract Digestion ..................122 Lignin Flow: Total Tract ......----.----129 Comparison of Digests Markers: Rumen and Total Tract Estimates ............................129 Evaluation of Markers: Selection Based on magnitude Of the SEM 00......000000000000...00.... 132 Evaluation of Digests Flow Patterns in Steers Fed Corn Silage Based Diets ......................136 SUMMARY 0.00..0.00....000.0...0....0......0.00.00.00.00156 APPENDIX .0......0.0..00..0.......0....0..0...00..0.00.159 BIBLIOGRAPHY .0...00.......00000.000.00.000...0.0.0000.184 vi E a d—h—b—b—b—b—b—bd-fi N O '21 22 23 24 25 26 27 28 29 LIST OF TABLES Digestibility and Rate of Passage Markers .... Minimal Analytical Levels For Markers ........ Protein Content of Cereal Grains ............. Protein Bypass Estimates ..................... Diet Formulation: Experiment One ............. Supplement Composition: Experiment One ....... Source of Nitrogen in Corn Silage Based Diets Diet Analysis: Experiment One ................ Diet Formulation: Experiment Two ............. Supplement Composition: Experiment Two ....... Diet Analysis: Experiment Two ................ Experiment One: Intake of Dietary Constituents. Experiment One: Lignin as a Marker ........... Experiment One: Ytterbium as s‘Marker ........ Experiment One: Chromium as s Marker ......... Experiment One: Digestibility of Nitrogen .... Experiment One: Corn Silage-Soybean Meal ..... Experiment One: Corn Silage- Urea ............ Experiment One: Corn Silage- Wet Distillers Grains ........................ Experiment One: Corn Silage- Wet Distillers Grains-Urea ................... Experiment One: Comparison of Marker SEM Estimates ........... Experiment Two: Lignin as a Marker ........... Experiment Two: Ytterbium as a Marker ........ Experiment Two: Lanthanum as a Marker ........ Experiment Two: Chromium as a Marker ......... Experiment Two: PEG Infusion ................. Experiment Two: Corn Silage-Soybean Meal ..... Experiment Two: Corn Silage-Corn Gluten Meal . Experiment Two: Comparison of Marker SEM Estimates ........... vii PAGE 13 30 34 50 51 52 S3 81 82 82 105 106 107 108 109 112 113 114 115 119 124 125 126 127 128 130 131 135 Appen Table 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 '52 53 dix Experiment Two: Whole Digests Flow, % of Total Daily 00.000.000.00.00.0.0000...0.0...000.000.0000 Experiment Two: Dry Matter Flow, Experiment Two: Nitrogen Flow, Experiment Two: Organic Matter Flow, % of Total Daily. % of Total Daily .. % of Total Daily ............................................ Experiment Two: ADF Flow, % of Total Daily ....... Experiment Two: Lignin Flow, % of Total Daily .... Experiment Two: Yb Flow, % of Total Daily ........ Experiment Two: Cr Flow, % of Total Daily ........ Experiment Two: La Flow, % of Total Daily ........ Feed Analysis Data: Experiment One ... Duodenal Analysis Data: Experiment One Fecal Analysis Data: Experiment One .. Experiment Two: Cr Data ...... Experiment Two: Lignin Data .. Experiment Two: La Data ...... Experiment Two: Yb Data ...... Experiment Two: Infusion Data, 813, Soybean Meal Diet ....... Experiment Two: Infusion Data, 819, Soybean Meal Diet ....... . Experiment Two: Infusion Data, 820, Corn Gluten Meal Diet ... Experiment Two: Infusion Data, 550, Corn Gluten Meal Diet ... Experiment Two: Infusion Data, 813, Corn Gluten Meal Diet ... Experiment Two: Infusion Data, 819, Corn Gluten Meal Diet ... Experiment Two: Infusion Data, 820, Soybean Meal Diet ....... Experiment Two: Infusion Data, 550, Soybean Meal Diet ....... viii Period Period Period Period Period Period N0 N0 N o , Animal , Animal , Animal , Animal Page 159 160 161 162 163 164 165 166 167 169 170 171 172 173 174 175 176 177 178 179 180 181 182 FIGURE u-h—hd-L-J-h—b—b O‘D Fecal Excretion of Digests Markers LIST OF FIGURES Experiment One Design ........... Experiment Two Design Adaptation and Sampling Protocol Feed Processing System .......... Duodenal Sample Processing Fecal Sample Processing ......... PEG Analysis System Flow Flow Flow Flow Flow Flow Flow Flow Flow Flow Patterns Patterns Patterns Patterns Patterns Patterns Patterns Patterns Patterns Patterns OM, N. TD, L. ADF, OM, N. Yb, L. TD, N, ADF Soybean Meal Diets . Yb, L Soybean Meal Diets ... Yb, Cr Soybean Meal Diets . TD, La Soybean Meal Diets .. ix L Soybean Meal Diets N, ADF Corn Gluten Meal Diets L, Yb Corn Gluten Meal Diets Cr, TD Corn Gluten Meal Diets TD, La Corn Gluten Meal Diets DM All Diets Averaged PAGE 44 46 78 58 61 64 66 90 146 147 148 149 '150 151 152 153 154 155 INTRODUCTION The ruminant occupies a unique niche in animal agriculture. Ruminants harness the cellulolytic and protein synthesis capabilities of the rumen microbes. They are therefore capable of utilizing diets composed of fibrous materials and low biological value proteins. As a result of the symbiotic relationship between rumen microbes and host, the ruminant animal can produce meat, milk, fiber and serve as beasts of burden without competing with humans for high quality dietary components. No other group of livestock occupy a position of such importance in animal agriculture. Realistically, it is currently not possible to maximize the production of meat, milk and fiber without the incorporation of some high quality dietary components in ruminant diets. However, a thorough understanding of factors influencing the efficient utilization of dietary components by ruminants will allow maximum productivity with a minimum of competition for human food resources. Efforts to evaluate the efficiency of production in ruminants typically involves an evaluation of feed input versus product output; i.e. feed efficiency. It has long been understood that certain combinations of dietary components result in optimal animal performance whereas other combinations result in poorer performance than expected. The phenomena of negative 1 2 associative effects, where performance on a particular diet or digestibility of a diet are not as expected, does exist. Intuitively, situations must exist where combinations of dietary ingredients act synergistically and result in positive associative effects. The exact nature of negative or positive associative effects, with respect to digestibility, are most commonly attributed to changes in site and extent of digestion of dietary constituents. Systems to estimate digestibility and nutrient flow through the digestive tract must be utilized to evaluate the interactions of dietary components upon the site and extent of digestion of the total diet. Markers which are indigestible, nonsbsorbable, do not influence digestibility of dietary components and can be analytically quantitated, are used to evaluate the site and extent of digestion as well as nutrient flow through the digestive tract. The purpose of this research was to evaluate the use of various markers to estimate the site and extent of digestion of corn silage diets supplemented with several common protein sources. The specific goal was to more explicitly define and develop appropriate marker systems for the determination of site and extent of digestion in ruminants. LITURATURE REVIEW Markers in Ruminant Nutrition There are literally hundreds of substances which can be used in ruminant nutrition as markers for flow and digestibility of dietary constituents. Kotb and Luckey (1972) provided an outstanding review of markers used in nutrition research. Table 1 lists a partial summary of several markers used in ruminant nutrition and the principal researchers involved with the markers. In recent years, rare earth lanthsnide series elements have been very popular for nutrition research. Kyker (1961) discussed the chemistry of rare earth elements. The chemistry of the rare earth elements dictates their acceptibility as markers for specific types of nutritional research. The intent of this literature review is to discuss and emphasize the principles of marker studies. It is not the author's intent to evaluate the efficacy of every available element or compound for marker studies. An understanding of the principles pertaining to marker use is essential. The principles represent the foundation for decision making regarding the choice of markers used in nutrition research. Marker Systems Markers can be used to evaluate three general areas of interest to ruminant nutritionists. The three areas of marker application require one to. make three sets of assumptions regarding specific criteria which are critical for marker derived data to be valid and reliable: ‘ Three areas of marker application include; 1) Digestibility and nutrient intake estimates based on marker ratio techniques (Bergeim, 1926; Stanley and Cheng, 1957; Kleiber, 1961), 2) Estimation of the rate of passage of specific dietary components, particles or phases (Blaxter et al., 1956; Grovum and Williams, 1973b; Ellis et al., 1979), 3) Estimation of the contribution of microbial components to digests flow (Siddens et al., 1982). These three areas provide researchers with detailed information. However, as stated earlier, each technique requires the establishment and adherence to a specific group of assumptions and analytical techniques. The uSe of markers of microbial flow were reviewed by Siddens et al. (1982). The use of microbial markers is not discussed in the literature review, although the general principles of digests markers do apply to the use of microbial markers. Marker Ratio Techniques The selection of a suitable marker for use in digestibility studies requires an evaluation of the proposed marker in relation to several important criteria. Table 1 is a brief list of the more commonly used markers. Specific criteria for marker ratio determination of digestibility have been discussed by Alvarez (1950), Engelhardt (1974) and Kotb and Luckey‘(1972). The important criteria for marker ratio digestibility studies requires that markers be: 1) Nonabsorbable 2) Inert, Indigestible 3) Flowing with the total digests: steady state 4) Nontoxic to gut tissues or microbes 5) Noninterfering with respect to digestibility of dietary components 6) Analytically quantifiable by specific and sensitive means 7) Noninterfering with analysis of other dietary components Few, if any, of the available substances can fulfill all of these criteria in all respects. An evaluation of the importance of each assumption and the resultant effect on data when an assumption is violated is necessary to completely understand the role of markers in ruminant nutrition. Table 1 Digestibility and Rate of Passage Markers Marker Phase Absorption Reference PEG 4000 liquid slight CrEDTA liquid 10% 2% 5% Cr-51 EDTA liquid 22% CoEDTA liquid 3-28% Phenol red liquid slight Chromic oxide particles ? Mg ferrite particles ? Lignin solids variable .IADF solids ? (Indigestible acid detergent fiber) AIA solids ? (Acid insoluble ash) Chromium mordanted fiber solids Ru-103 Phenanthroline particles Cerium particles/solids Dysprosium particles/solids Lanthanum particles/solids Samarium particles/solids Ytterbium particles/solid Winne and Gorig, 1982 Clemens, 1982 Neudoerffer et al.,1982 Ulystt, 1964 Kay, 1969 Corbet, 1981 Goodall and Kay, 1973 Faichney, 1975 Downes,McDonald, 1964 Uden et al., 1980 Uden et al., 1980 Schedl et al., 1966 Miller,Schedl, 1970 Kunihara,Teruhiko, 1981 Drennan et al., 1970 Wilkinson,Prescott, 1970 Prigge et al., 1981 Purser,Mori, 1966 Neumark et al., 1975 Neumark et al., 1981 Thonney et al., 1979 Balch, 1957 Galyesn et al., 1979 Fahey and Jung, 1983 Waller et al., 1980 Penning,Johnson, 1983 Frape et al., 1982 Van Keulen,Young, 1977 Thonney et al., 1979 Furuichi,Takahashi, 1981 Uden et al., 1980 Tan et al., 1971 Warner, 1981 Huston and Ellis, 1968 Ellis and Huston, 1967 Ellis, 1968 Young et al., 1976 Crooker et al., 1982 Stern et al., 1983 Crooker et al., 1982 Teeter et al., 1979 Prigge et al., 1981 Sklsn et al., 1975 Nonabsorption of Markers Utilization of the marker ratio technique for calculating digestibility of any dietary component requires that the marker utilized be nonsbsorbable. This is a very important criteria. Absorption of marker from the digestive tract results in an increased estimate of nutrient flow and a concurrent underestimation of component digestibility. Equation 1 illustrates the role of marker concentration of digests samples (digestas feces, ileal, duodenal or abomasal) and feed.samples in calculation of nutrient flow through the digestive tract and consequently digestion of nutrients. (1) A B ' C G (Nutrient Conc. /Marker Conc. ) Ratio in Digests Sample A B D (Nutrient Conc. /Marker Conc. ) Ratio in Feed Sample G 1. ( ) x 100 = % Nutrient flow of Intake G 2. 1 - ( ) x 100 - % Nutrient Digestibility G 3. ( ) x Nutrient Intake - Nutrient Flow 4. (Nutrient Intake - Nutrient Flow) A x 100 = Digestibility Nutrient Intake A) Nutrient concentration expressed as grams/kg of dry matter or other suitable measure B) Marker concentration can be expressed as a percentage of dry matter or g, mg/kg of dry matter or other suitable measure C) Digests can be of abomasal, duodenal, ileal or fecal material D) Feed could be replaced in this ratio by rumen, abomasal, duodenal or ileal data and nutrient flow and digestibility calculated from this point of origin to any site further down the digestive tract, i.e. duodenal to fecal, etc. G) A ratio of ratios It is clear a decline in the concentration of a marker in the digests sample, in Equation 1, will result in elevated flow and concurrent depression in digestibility estimates for all components of interest. Absorption of markers from the digestive tract is well documented. Absorption of low molecular weight components of PEG 4000 has been reported (Winne and Gorig, 1982). Chromium absorption from CrEDTA complexes has been observed by Corbett (1981), Goodall and Kay (1973) and Faichney (1975). Cobalt absorption from CoEDTA complexes has been reported by Uden et a1. (1980). Many researchers have adjusted marker concentration in digests samples to account for absorption. Although this can theoretically be done, book values for marker absorption cannot be adapted to apply to all situations. Therefore, correction of marker concentration can only be done, with any significant degree of accuracy, by analysis of urinary excretion of marker and tissue retention. This would significantly increase the complexity of studies. It is obvious that markers which are not absorbable are to be used whenever possible. Inert, Indigestible Markers It should be noted that a marker need not be absorbed to create problems of over estimation of dietary nutrient flow. Alterations of marker concentration due to digestive processes or analytical problems with detection of markers in digests can create errors in the estimation of nutrient flow and digestion. The problem with alteration of marker concentration due to digestive processes may be of special significance when a marker such as lignin is used. Apparent digestibility, absorption and/or failure of gravimetric methodology to accurately detect lignin residues has been observed by Thonney et a1. (1979) and Muntifering et al. (1981). Marker and Digests Flow As indicated, an important assumption is that markers move with the total digests. As a result, compositing of digests from numerous samples will provide a representative sample of total digests with true concentration of markers and nutrients. Unfortunately, numerous problems arise when digests composites do not represent the true digests which has moved through the digestive tract during the sampling period. Many researchers use a sampling sequence similar to the one described by Faichney (1980). With this technique, 10 twelve samples of digests (abomasal, duodenal, ileal or fecal) are taken over a three day period, with 4 to 6 hours between sampling. These samples are then composited on an equal wet weight basis. This assumes that each sample represents an equal contribution, with respect to volume and content, to total daily digests flow. This is the point at which many digests passage and digestibility studies confront severe data evaluation problems. The existance of steady state digests flow may not be valid under many conditions. It does appear. that, with respect to fecal and perhaps ileal sampling, the assumption of steady state flow and therefore equal weight compositing may be adequate. It has been well documented that repeated sampling and compositing of fecal samples is equal to total collection schemes for many species of livestock and nonfarm animals: Beef steers; Young et al., (1976), Thonney et al., (1979), Beef cows; Prigge et al., (1981), Sheep; VanKeulen and Young, (1977), Horses; Sutton et al., (1977), Rabbits; Furuichi and Takshashi, (1981). Numerous studies have shown that abomasal or duodenal samples will not produce the same degree of accuracy when compared with total collection schemes. Corbett and Pickering (1983) have shown, in grazing sheep, marker and nutrient concentrations in abomasal samples varied with time of sampling . They observed a variation of plus or minus 30% from values calculated from a 24 hour mean concentration of marker. These data illustrate the presence of nonstesdy flows in animals eating several meals each day while grazing. 11 Uden et al.,.(1980) has illustrated the different rates of passage of the liquid phase marker CoEDTA and a solid phase marker, chromium mordanted fiber. These data illustrate the existance of differential flow rates. Therefore, material moving with the liquid phase leaves the rumen at s more rapid rate than those materials moving with the solid phase. As a result of these flow differences, the estimation of digestibility or flow can be grossly affected by marker choice. It would appear that compositing of duodenal or abomasal samples on an equal weight basis will not correct for 'the rapid movement of liquid phase markers and slower movement of solid phase markers. These observations coupled with the difficulty to collect representative samples of digests passing abomasal or duodenal cannula led Faichney (1980s) to propose the two marker and double marker systems. Faichney (1980) discussed the work of Hogan and Weston (1967) who observed differences between samples collected from a simple cannula and digests flowing past the cannula. This failure to acquire a representative sample is a problem, however, careful sampling may minimize the differences between samples and true digests. Nontoxic Markers There is not a significant amount of information regarding the toxicity of markers to gut tissues, microflora or the whole animal. Toxicity of rare earth elements to 12 bacteria has been evaluated by Johnson and Kyker (1966). Toxic effects of ruthenium phenanthroline on rumen microbes is reported by Evans et al. (1977) and Beaver et al. (1978). 1 Analytical Quantification of Markers Evaluation of marker concentrations in digests samples is a very important consideration. Table 2 illustrates the detection limits of several elements used in marker studies as summarized by Ellis et a1. (1980). Due to the nonspecific nature of gravimetric procedures for estimation of markers such as lignin, indigestible acid detergent fiber (Penning and Johnson, 1983) and acid- insoluble ash (Van Keulen and Young, 1977) analysis of each animal's feed, digests and fecal samples must be conducted within an animal set, under the same laboratory conditions. With gravimetric methodology, laboratory conditions must be standardized. This includes standardization of reagents, lab temperature, reaction times and sample dry matter corrections. Evidence of the need for strict control of gravimetric analysis is discussed by Mueller (1956) with respect to lignin analysis and by Van Keulen and Young (1977) with respect to acid-insoluble ash analysis. 13 Table 2 Minimal Analytical Levels for Markers Procedure Element Activation Analysis Atomic Absorption a b ug/ssmple ug/ml “8/8 Cobalt .5 1 10 Chromium 1.5 2 20 Dysprosium - 4 40 Lanthanum .06 - - Ytterbium .07 2 20 a) ug/ml aspirated or extracted b) assumes 2g sample ashed, extracted by 20ml, analyzed in nitrous oxide supported flame Marker Effect upon Digestibility of Dietary Constituents When a marker is used to calculate the rate of passage of a particular feed component through the digestive tract, its effect on digestion must be considered. Theoretically, feed particles small enough to pass through the omasal orifice prior to digestion will not be affected by reduction in rate of digestion due to marker inhibition. Teeter et a1. (1979) report no effect of marker upon dacron bag degradability of feedstuffs. However, Teeter et a1. (1981) report that ytterbium did reduce digestibility (disappearance from dacron bags). Due to the role of digestion upon rate of passage of fibrous fed particles, reduction in digestibility 14 due to markers is more critical than with most protein sources. Uden et al. (1980) report increasing chromium content in mordanting fiber decreased digestibility of the material. Marker Association and Movement In order to calculate the rate of passage of specific fed components the marker must be closely associated with the feed components. Hartnell and Satter (1979) discuss the binding of several rare earth elements and marker movement to other feed components. The nature of rare earth binding characteristics has been discussed by Kyker (1961), Ellis and ‘Huston (1968) and Ellis et al. (1979). In general, the marker should not move independently of the feed which it is to mark. Teeter et a1. (1979) indicate that the method of marking feeds will affect the proportion of marker bound tenaciously to feedstuffs. There appear to be numerous sites on feed particles which bind rare earth elements. These sites have differing binding affinities. Ellis et al. (1983) discussed the presence of acid resistant binding sites on feedstuffs for ytterbium. These sites have very high affinities for ytterbium. When utilizing a marker for the calculation of rate of passage of a particular feed component, studies must be conducted or evaluation of existing data must be done to insure that the marker is actually moving with the particular feed component. 15 Estimation of Feed Component, Rate of Passage It is well known that the retention time of a feed component in the rumen is a key factor in determining the extent of digestion which can occur. Retention time is a function of liquid turnover for soluble and small feed components. Digestion rate is a key factor in determining the retention time of feed particles too large to leave the rumen through the omasal orifice. Marker technology has been applied to the quantification of rate of passage of specific feed comonents. Calculation of the rate of passage of specific markers has been discussed in detail by Grovum and Phillips (1973), Grovum and Williams (1973b) and Grovum and Williams (1977). Briefly, the rate of passage of a marked feed component or marked digests phase can be calculated from pulse dose techniques. Assuming rules of kinetics apply to the passage of digests through the digestive tract, mathematical analysis of flow as described by Shippley and Clark (1972) can be accomplished. A dose of marker placed in the rumen will move from the rumen at a rate proportional to the amount remaining. Samples of duodenal digests can be acquired or feed samples can be taken sequentially over a period of several days to calculate the rate of decline of marker concentration. Multiple pool analysis using curve peeling techniques has been discussed by Grovum and Williams (1973a)- 16 However for most marker studies a single _pool model is suitable to reach conclusions regarding the flow of a marked feed particle or phase. The rate constant (k) is the fraction of marker or percent excreted per unit of time based on analysis of the semilog plot of the log of marker concentration in sequential samples versus time. Equation 2 can be used to calculate numerous attributes of digests flow. (2) -kt Y a Y . e O Y - concentration of marker in a sample at time t YO= concentration of marker in a sample at t=0 Equation 2 can be used to solve for rate constant (k) if only the time between two sampling periods and marker concentrations are known. Turnover time (T) for a marker can be calculated from Equation 3; this is also the mean retention time value. (3) r: 1/k The half life of the marker in the digestive tract is calculated from Equation 4. (4) .693 1/2 k 17 Researchers have utilized marker technology to calculate the specific rate of passage of particular feed components. Uden et al. (1980) bound chromium to fiber components and followed their passage through the digestive tract. It is possible to couple rate of passage of marked feed components with dacron bag, in-situ, degradability studies described by Orskov et al. (1980) and Mehrez and Orskov (1976). with estimates of rate of feed component passage and rate of degradation within the rumen, estimates of feed components escaping rumen digestion can be acquired. Several assumptions must be evaluated before data derived from marker rate of passage and dacron bag degradability studies are utilized. These assumptions include the following: 1) Markers must be closely associated with the particular component of interest. 2) Markers must not interfere with digestion of feed components, if digestion is required for passage through the omasal orifice. 3) Dacron bag degradability estimates must represent actual degradation of free floating components. In-situ Dacron Bag Digestibility Studies A key factor involved with coupling rate of passage data with digestion rate is the effect the dacron bag has on digestibility rates. Factors affecting the accuracy of dacron .bag data have been evaluated by Mehrez and Orskov (1977). In general, a key problem exists in reference to coupling dacron bag data to passage data. One must assume 18 for purposes of calculation that disappearance of nitrogen and dry matter from nylon bags is synonymous with digestion. In reality, small particles may leave the dacron bag and are not' necessarily digested and may pass to (the lower tract intact. An important consideration, which has not been discussed in detail in the literature, is the effect of diet upon digestibility of dietary ingredients. It appears protein source digestibility within the rumen may be a function of the other dietary components being fed. This raises questions with regard to digestibility estimates derived from dacron bag studies which may have been conducted in cattle fed diets which could alter protein degradability within the dacron bags. Research indicating the effect of diet upon dacron bag degradability of protein sources has been conducted by Loerch et a1. (1983). In-situ degradability estimates may need to be studied with particular feed components evaluated in animals fed the feed components. Further research needs to be conducted to establish the nature of these problems. Integration of Rate of Passage and Degradability Calculations Orskov and McDonald (1979) and Orskov (1982) discuss the calculation of the amount of material remaining in dacron bags after a unit of time. Equation 5 illustrates the calculation for a feed component which has little or no rapid 19 release of water soluble or rapidly digestible material. (5) . -ct P= 100(1 - e ) Where P is the amount degraded after time "t" and c is the degradation rate for the component of interest. Many feed components exhibit a complex digestion pattern. In essence, there are water soluble and rapidly degradable portions, slowly degraded portions and components which may never be degraded during their normal residence time in the rumen. This more complex type of digestion was discussed by Orskov and McDonald (1979). Equation 6 is used to describe digestion of a more complex nature. (6) -ct P: a + b (1 - e ) In this equation, P is the amount degraded at time "t". I. N N b fl '0 N n I. In the equation, a , and c are constants where a represents the percent of feed rapidly degraded and "b" the remaining material which is degraded at a rate described by "c". In the equation, the sum of a and "b" cannot exceed 100% but the value of "a" + "b" is normally the percent digested when time nears infinity. As discussed earlier, the rate of passage of labeled feeds can be coupled to estimates of rumen degradability and estimates of rumen outflow calculated. This technique is subject to the assumptions evaluated previously. As described by Gsnev et a1. (1979) and Orskov (1982), Equation 7 can be used to calculate the amount of a feed component degraded in the rumen after a unit of time. .20 (7) (b i c) -(c+k)t P: a + ...-I— (1 ' e (c + k) In this equation, as. in 5 and 6, P is the percent of the feed component degraded after a unit of time "t". The unit "a" is the percent of feed components degraded rapidly, ”b" the percentage of feed components degraded at rate 0 after time "t" and "k" is the fractional outflow rate of the feed component calculated from the pulse dose decay curve of marked feed. Essentially, after any significant length of time, the equation is most easily represented by Equation 8. (8) The rate constant "k" can be calculated from pulse dose, sampling systems of marked feed components as discussed earlier. Broderick (1978) also discused the application of feed component movement through the digestive tract coupled with the rate of degradation within the rumen, to estimate a percent of feed component escaping rumen degradation. The following equation for estimated feed component escape from the rumen is illustrated in Equation 9. (9) % escape = [Kr/(Kr + Kd)] x 100 In the equation "Kr" is the rumen flow rate constant for the feed component and "Kd" is the rate constant for degradation in units per hour. This calculation assumes a constant degradation rate constant for the feed components. Evaluation of the amount of a feed component leaving the rumen must be done with care. 21 One should consider the importance of the major assumptions listed earlier. Double Marker Method Faichney (1980a,b) described the calculation procedure for the double marker method as follows for samples separated into filtrate"and filtrand by straining through Terylene cloth and utilizing a constant 'rumen infusion of both solid and liquid phase markers. Equation 10 illustrates Faichney's reconstitution factor calculation. Marker concentrations are expressed as a fraction of marker infused each day per kg of digests or filtrate, dry matter (DM) concentrations are expressed as a fraction of the original sample and nitrogen (N) concentrations were expressed as g/kg of digests or filtrate. Concentration of markers, such as CrEDTA were corrected for absorption based on data of Faichney (1975). The reconstitution factor (R) is the number of units of filtrate which must be added to or removed from one unit of digests to obtain true digests. Assuming the liquid and solid phase markers are in equilibrium within the rumen, by virtue of constant infusion, the reconstitution factor would be equal to 1 if the digests samples (abomasal, duodenal, ileal or fecal) are representative of true digests. 22 (10) R= (Conc. Digests Solid Marker - Conc. Digests Liquid Marker) g (Conc. Filtrate Liquid Mkr.- Conc. Filtrate Solid Mkr.) (11) True Digests Marker Concentration = Conc. Digests Liquid Mkr. + (R x Filtrate Liquid Mkr. Conc.) 1 + R (12) True Digests Flow = 1/True Digests Marker Conc. The flow of any dietary constituent is calculated by replacing marker concentrations in Equation 11 by the Vconcentration of the constituent of interest. This value is then multiplied by the flow calculated with Equation 12. The double marker method, in summary, corrects for difference in marker ratio (digests solid phase marker to digests liquid phase marker ratio should be equal to the infused ratios or ratios in the feed). This correction is necessary if a nonrepresentative sample of digests is acquired due to simple cannula characteristics. The technique assumes that steady state conditions exist within the rumen as a result of feeding multiple meals through the day. In the discussion of the double marker method, Faichney does not evaluate the role of discrete intervals of water consumption which could easily disrupt steady state. 23 ’Two Marker Method Faichney (1980) discussed a two marker method for the calculation of digests flow. This method differs from the double marker method in that it is designed for studies where animals are fed only once or twice daily. In essence, steady state is not exhibited by Faichney's definition, however, both solid and liquid phase markers are constantly infused into the rumen. This method requires marker concentrations be adjusted for absorption anterior to the sampling site. Digests samples may be composited on an equal weight basis to represent feeding cycles. The composited samples are filtered through Terylene cloth. The samples must not be centrifuged as can be done with the double marker method. The sample, after filtration, is divided into a liquid phase filtrate and a solid phase filtrand. It is proposed, by virtue of the differential movement of the filtrate and filtrand, that dietary constituents move with one phase or the other. Estimates of each phase rate of flow coupled with the content of dietary constituents within each phase could predict digests flow accurately. The two marker method (Faichney, 1980a) includes the following calculations. Digests Sample Filtrate Fraction (FF) FF= (1—Digesta DM) / (1-Filtrate DM) 24 Effective Infusion Fraction for Liquid Marker Liquid Mkr.-(Filtrate Liquid Mkr.)/(Digesta Liquid Mkr.) x FF Corrected Liquid Marker Concentration Filtrate Flow 8 FF/Digesta Liquid Marker Effective Infusion Fraction for Solid Marker I=~1- (Filtrate Solid Marker x Filtrate Flow) Filtrand Solid Marker = [Digests Solid Marker - (FF x Filtrate Solid Marker)] (1 - FF) x I Filtrand Flow= 1/Filtrand Solid Marker Digests Flow= Filtrate Flow + Filtrand Flow Calculation of Nutrient Flow Nitrogen Flow= (Filtrate N x Filtrate Flow) + [Digests N - (FF x Filtrate N)] A X Filtrate Flow 1 - FF The two marker method assumes that dietary constituents associated with the solid and liquid markers move with the marker fractions. Ellis and Huston (1968) and Faichney (1980b) have shown that digests flow represents a complex mixture of different flow rates for different constituents. Liquid markers move the most rapidly followed by particulate 25 markers such as chromic oxide and ruthenium phenanthroline and the. slowest movement is for lignin or other fiber associated markers. Mudgal et sl.(1982) reported the retention time of lignin in the rumen was 2.1 to 2.3 times longer than ruthenium phenanthroline. Therefore, as indicated by the two marker method, materials moving with ruthenium phenanthroline would move more rapidly than those associated with lignin. Effects of Cannulation Upon Digestion of Diets in Ruminants It is a common practice to use cannulated animals in digestibility studies with ruminants: Several types of cannula are available for use with ruminants. An innovative re-entrant cannula design has been discussed by Ivan and Johnson (1981). A modified T-type cannula design has been developed and tested by Komarek (1981a and 1981b). Investigations of the effects of cannulation on intestinal motility and digests flow in ruminants have been conducted by Wenham and Wyburn (1980), Sissons and Smith (1982), Hayes et a1. (1964) and Poncet et a1. (1982). In general, surgical modification of the digestive tract alters flow of digests. This is especially true of the re-entrant cannula as discussed by Wenham and Wyburn (1980). The re- entrant cannula appears to be the most troublesome of all cannulation procedures. Hayes et a1. (1967) report that rumenal and abomasal fistulation did not alter digestibility 26 coefficients of their diets. Poncet et al. (1982) report the Y reeentrant cannula created marked reductions in the rate of digests flow through the duodenum. The effect of slowing digests movement might result in longer digests retention throughout the digestive tract and alter digestibility estimates. Digestibility of Corn Silage and Supplemental Protein Sources by Ruminants The digestibility and utilization of components present in ruminant diets involves a complex interaction of level of intake, concentrate level, particle size, roughage source, nitrogen level and source and many other variables. The rumen environment represents the result of its microbial population interacting with and being selected by dietary ingredients. Within the rumen, the optimal situation would include prolific microbial growth resulting in maximal digestion of dietary roughage with a minimum digestion of high quality dietary proteins. Data indicate that there is a range of rumen ammonia levels which maintain optimal growth of the microbial population. Pilgrim et al. (1970) indicated that so to 75 percent of the nitrogen present in rumen bacteria originated as ammonia. Satter and Slyter (1974) have reported optimal microbial growth occured at a theoretical rumen ammonia level of 5 mg/dl. Hume et al. (1970) observed maximum microbial growth in sheep occuring at ammonia concentrations of 9 27 mg/dl. In contrast to these data, Mehrez et al. (1977) report optimal microbial activity, not necessarily. growth, occured at 23.5 mg/dl of rumen fluid. Recent interest in bypass proteins has raised concerns that low rumen degradability of dietary proteins could result in levels of rumen ammonia too low for optimal digestion of dietary roughages. Wohlt et a1. ((1976) has provided data indicating that rumen solubility of dietary proteins, is directly correlated to degradability. Wohlt et al. (1976) formulated a series of low and high solubility diets. When these diets were fed to sheep, the low solubility diets produced rumen ammonia levels of from 5 to 6.8 mg/dl. These levels of ammonia are near the minimum required for optimal microbial growth and certainly well below the level indicated by Mehrez et al. (1977) to provide optimal digestion. Data presented by Spears et al. (1980) and Sharma et al. (1972) indicate chemical treatment of protein sources can result in drastically low levels of rumen ammonia. Sharma et al. (1972) also reported calves fed formaldehyde treated rapeseed had reduced dry matter digestion. There is limited data that selection of a low rumen degradable protein source for corn silage diets will reduce digestibility of the roughage components. Cottrill et a1. (1982) report the inclusion of fish meal (a protein source of low rumen degradability) for urea in corn silage diets tended to reduce rumen organic matter digestion. As discussed by Bergen et al. (1974), some of the nitrogenous constituents of corn silage may not be readily available to rumen microbial 28 digestion and assimilation into microbial. protein. These observations indicate there may be a need to provide some soluble proteins in corn silage diets to maintain adequate microbiar digestion of dietary constituents.‘ Soluble or readily degradable protein sources may also provide the alpha keto acids; phenylacetic, isobutyric, isovaleric and two methyl butyric acids which have been shown to be required by many cellulolytic bacteria for protein synthesis (Hume et al., 1970; Bryant and Doetsch, 1955 and Cline et al., 1966). These alpha keto acids are carboxylated and aminated to form phenylalanine, valine,‘ leucine and isoleucine respectively (Allison et al., 1966 and Allison and Bryant, 1963). Factors Affecting the Digestibility of Supplemental Protein Sources As discussed earlier, the digestibility of supplemental protein sources may affect the extent of digestion of dietary roughage sources. Protein sources which are resistant to microbial proteolytic activity have been labeled bypass protein sources. Many researchers regret the popularity of the phrase "bypass protein" since these protein sources are more appropriately "microbial protease resistant proteins" since they do not actually bypass the rumen. The importance of bypass protein sources in ruminant diets has been discussed by Chalupa (1975) and Clark (1975). Many researchers have studied animal performance in relation to bypass protein supplementation. Klopfenstein et al. 29 (1976, 1978) have conducted numerous experiments and reported data evaluating protein sources and animal performance. Rock at al. (1983) reported steers fed corn gluten meal, as the only supplemental protein source, gained slower than steers fed dehydrated alfalfa. Ruminants fed bypass protein sources, such as corn gluten meal, benefit from the addition of urea as a source of supplemental, readily degradable nitrogen for microbial protein synthesis and growth. This response is referred to as a "complementary" effect_ by Klopfenstein et a1. (1978). This effect may also be described as a positive associative effect. These observations are also supported by Cottrill et al. (1982) where increasing level of fish meal, a "microbial protease resistant protein" replacing urea resulted in a decrease in digestibility of organic matter in the rumen. It is clear that one must formulate a diet to provide nitrogen for maximum microbial growth and sufficient total nitrogen flow to to the lower tract to support optimal animal performance. Protein Chemistry The availability of nitrogen from supplemental protein sources for microbial growth is directly related to the chemistry of proteins, including their secondary and tertiary structures. The chemistry of cereal proteins determines the potential for degradation by microbial proteases. Brohult and Sandegren (1954) have discussed the chemical nature of cereal proteins. 3O Cereal proteins are made up of combinations of four general classes of proteins. These classes include: albumin, globulin, prolamin and glutelin fractions. Table 3 illustrates the composition of several cereal proteins. 1 Table 3 Protein Content of Cereal Grains ‘ ---‘-------—-------------------------------_----------------- Protein Fractions, % of Total Protein % Crude Protein ------------------------------------- Item (dry basis) Albumin Globulin Prolamin Glutelin Corn 7-13 +/- 5-6 50-55 30-45 Barley 10-16 ‘1'/- 10-20 35-40 35-45 Wheat 10-15 3-5 6-10 40-50 30-40 Oats 8-14 1 80 10-15 5 2 SBM 30-50 +/- 85-95 - - 1. Brohult and Sandegren (1954) 2. Bailey et al. (1935) As discussed by Osborne (1924), the albumins are soluble in water and the globulins are soluble in water and. dilute saline solutions. Protein sources high in albumins and globulins, such as soybean meal, are very soluble in the rumen and as discussed by Wohlt (1973), this is highly correlated to rumen to rumen degradability. As a result, soybean meal is very degradable in the rumen. The glutelins are soluble in very dilute acid or alkaline solutions, insoluble in water, saline or solutions of alcohol. Prolamins are only soluble in solutions of alcohol. Proteins high in prolamin and glutelin fractions, such as corn gluten meal, are relatively insoluble in rumen fluid and resistant to microbial protease attack. Solubility, however, is not 31 the only factor affecting protein degradability in the rumen. Mahadevan et al. (1980) reports the protein, bovine serum albumin, is highly soluble in the rumen. Studies show this protein is very resistant to microbial protease activity. Mahadevau states the resistance may be due to the proteins 16 disulfide linkages which may inhibit microbial protease activity. Casein is very soluble in rumen fluid, lacks disulfide linkages and is readily degraded in the rumen. Many researchers have attempted to modify the rumen degradability ' of high quality dietary protein sources. Methods have included: formaldehyde (Spears et al., 1980), aldehyde treatments (Peter at al., 1971), tannic acid treatment (Driedger and Hatfield, 1972) and heat treatments (Nishimutu et al., 1973). Formaldehyde has been used extensively to alter rumen degradation of dietary proteins. Formaldehyde treatment results in the formation of methyl groups on terminal alpha amino groups of lysine, followed by condensation of these groups with primary amide groups of asparagine, glutamine and guanidyl groups of srginine. These condensations form intermolecular and intramolecular methylene bridges. The treated proteins are stable and resistant to microbial protease. activity. The methylene .bridges may prevent degradation by altering tertiary structure similar to the action of disulfide bonds. The methylene bridges are broken in the acid environment of the abomasum. Excessive formaldehyde treatment may render the proteins indigestible throughout the total tract. Formaldehyde treatment of high 32 quality dietary proteins has increased nitrogen retention under several feeding regimes (Faichney, 1971; Tamminga, 1979). There are factors other than the direct chemistry of proteins.which affect solubility and degradability of cereal proteins in the rumen. However, these other factors are the result of the rumen environment interacting with the chemistry of the cereal proteins. Smith et al. (1959) and leases and Owens (1972) evaluated the solubility of protein sources at varying pH and salt concentrations. In general, soybean meal is least soluble at a pH range of 4 to 5. Solubility of soybean meal plateaus at a pH of 8. Corn proteins are more soluble at the lower pH ranges. Smith et al. (1959) reports an increase in salt concentration of their solvent solution, in vitro, from O to .1M depress solubility of soybean meal from 90% at 0 to 50% soluble at .1M. Salt concentrations above .25M increased soybean meal solubility. Loerch et al. (1983) have illustrated the effects of in- vivo pH on in-situ dacron bag degradability of supplemetnal protein sources. Feeding a diet to the fistulated steers, in which the in-situ study was conducted, of 80% corn, as compared to 20%, produced a lower rumen pH. The rate of soybean meal nitrogen disappearance was 2.9% per hour in the 80% corn diet and 7.8% per hour in the 20% corn diet. This was not observed for blood meal, meat and bone meal or corn gluten meal. These studies also indicate the supplemental protein source fed the fistulated steers also affected nitrogen disappearance from the dacron bags. 33 Protein Bypass Estimates It is obvious that estimates of' rumen degradability protein sources are a function of several factors. The primary factors include: microbial protease activity, protein chemistry, secondary and tertiary structure, rumen pH, ionic strength of rumen fluid and perhaps Some unkonwn dietary interactions. Specific rates of protein outflow from the rumen are as important, under many circumstances, as protein chemistry. Calculation of protein outflow rates an estimates of total degradability have been discussed by Orskov and McDonald (1970) and Orskov and McDonald (1977). Table 4 provides a summary of current estimates of degradability of supplemental protein sources. The range of estimates for protein bypass are quite large. It is interesting to note the difference in bypass value of soybean and corn gluten meal with only a small increase in level of intake (Zinn et al. (1981). Estimates of protein bypass are only significant and unique for a given level of intake relative to rumen/total tract volume and nature of the total diet, especially with respect to the level of concentrates in the diet due to their affect on rumen pH. 34 Table 4 Protein Bypass Estimates Feedstuff Bypass % ,Reference Soybean meal 61 Hume, 1974 Soybean meal 20 Kropp et al., 1976 Soybean meal 19-24 Orskov and McDonald, 1970 Soybean meal 35 Stern and Sstter, 1982 Soybean meal 15 (1) Zinn et al., 1981 Soybean meal 18 (2) Zinn et al., 1981 Corn gluten meal 57 Stern et al., 1983 Corn gluten meal 46 (1) Zinn et al., 1981 Corn gluten meal 61 (2) Zinn et al., 1981 Dried distillers grains 32 Stern and Sstter, 1982 Dried distillers grains with solubles 55 Santos and Sstter, 1980 1) Steers, intake 1.2 x maintenance 2) Steers, intake 1.6 x maintenance Level of Intake: Effects on Site and Extent of Digestion Several authors have discussed the role of level of intake and its effects on site and extent of digestion of dietary constituents (Zinn et al., 1983; Zinn et al., 1981; Miller, 1973; Tamminga et al., 1979 and Mudgal et al., 1982). Evans, 1981a and 1981b, has reviewed the relationships between dietary parameters and liquid, solids turnover rates. In brief; liquid turnover rates increase as feed intake increases, liquid turnover decreases as digestible energy content of the diet increases, liquid turnover decreases as forage intake decreases, solid turnover increases as feed intake increases, solid turnover declines as digestible energy content of the diet increases (for sheep, not related in cattle), solids turnover increases as forage percentage of 35 the diet increases. In general, increasing the level of intake increases the turnover of both liquids and solids from the rumen. This reduces rumen retention time, reduces digestion of dietary constituents and produces a shift in the site of digestion. Bypass of dietary protein as a result of increasing turnover is beneficial; bypassing fiber to the lower tract may not be beneficial. Independent of level of intake, compounds such as monensin reduce liquid turnover as described by Lemenager et a1. (1978). The result is a longer ruminal retention time. Despite longer rumen retention in animals fed monensin, there is less rumen degradation of dietary constituents due to a depression in microbial growth. Muntifering et a1. (1981) report a shift in nitrogen and starch digestion to the lower tract in steers fed monensin. Effects of Shifting the Site and Extent of Digestion As discussed earlier, increasing the rate of passage of feeds from the rumen increases dietary protein flow to the lower tract. This is a preferred situation since it is more efficient to have high quality dietary proteins absorbed by the animal intact and provide nonprotein nitrogen to rumen microbes. A shift in starch digestion to the lower tract is an efficiency promoting event. Glucose absorption from starch in the small intestine is energetically more efficient than conversion to volatile fatty acids and their absorption 36 from the rumen. Black (1971) has discussed the theoretical basis for improved efficiency of energy and protein utilization when there is increased rumen bypass of the diet. Fiber components bypassing digestion in the rumen pass through the small intestine without significant digestion. Fiber components may be digested in the cecum and proximal colon and partially compensate for reduced ruminal digestion of fiber due to increased rate of passage from the rumen. Data indicate a significant digestion of dietary fiber can occur post ruminally. Putnam and Davis (1965) indicate that alfalfa and wood cellulose fiber had a digestion coefficient of 29 percent when placed directly into the abomasum as compared to 43 and 63 percent respectively when fed per os. Dixon and Nolan (1982) report significant fermentation and absorption of dietary constituents bypassing the small intestine does occur in the cecum and proximal colon and to a much less extent in more distal portions of the large intestine. SUMMARY It is apparent that factors affecting the amount of passage of dietary proteins depends on a large number of variables. These variables include; protein chemistry, rumen environment, level of intake and addition of antibiotics such as monensin to the diet. Maximizstion of dietary protein bypass must not be accomplished at the expense of providing for microbial 37 growth. Microbial growth can be improved .when readily available nitrogen sources are included in diets containing "microbial protease resistant proteins". There is also potential improvements in ruminal fiber digestion when microbes are properly supplemented with nitrogen and sources of iso-acids. The cecum and proximal large intestine do provide significant contributions to total tract digestion of dietary fiber and may play a major role when bypass of dietary constituents occurs to any great extent. Extreme care must be taken when selecting a system of markers to evaluate site and extent of digestion. This thesis evaluate5\ several aspects of marker function which affect estimates of site and extent of digestion. The diets studied contain corn silage and several common supplemental protein sources. The effects of protein source and marker Selection upon the site and extent of digestion of corn silage based diets are studied and evaluated in detail. It is evident that the level of intake in these studies is quite low. This will most likely bias the digestibility estimated toward the the upper limits of digestibility of corn silage based diets. This is most likely the case due to longer residence times of dietary constituents allowing for maximum microbial attack and consequently Optimal rumen digestion of dietary constituents. Care must be taken to evaluate the level of intake relative to the total capacity of the rumen and total tract not merely relative to percent of body weight. The rumen and total digestive tract reaches 38 its maximum capacity much sooner than body weight reaches a maximum. Evaluation of digestibility estimates for use in models of animal performance must take into account level of intake, source of dietary constituents and markers utilized to provide the parameter estimates. Hopefully this thesis will provide some insight into these considerations. PRELIMINARY EVALUATION: MARKER BINDING BEHAVIOR Introduction At the onset of this research program, little data was available regarding the binding behavior of the rare earth elements Yb and La. This evaluation procedure was conducted to establish potential rare earth elements binding affinity with corn silage, soybean and corn gluten meals and wet distillers grains. This was required to estimate the amount of marked feed reqmired to conduct these research programs. Uden (1978) discussed the relationships between rare earth molecular weight and ionic radius to binding with feedstuffs. Rare earth elements of high molecular weight and small ionic radius bind more tenaciously to feed components than lower molecular weight high ionic radius rare earths. For this reason, ytterbium (Yb) appears to be a better marker than lanthanum (La), assuming binding affinity is an important criteria. It has been discussed by Allen et al. (1982) that rare earth elements may be released from their binding sites by an acid environment. Both corn silage and wet distillers grains have pH values of 4.2 or less and it is of interested as to study their affinity to bind Yb and La. 39 40 Materials and Methods Corn silage, soybean meal and wet distillers grains were soaked in either ytterbium or lanthanum chloride solutions containing 50 mg of rare earth per gram of feedstuff dry matter. Feeds (50 grams of dry matter) were soaked for 24 hours under refrigeration in 1,000 ml beakers. After the 24 hour soaking period, the solution was drained off and a sample of each feedstuff was taken for evaluation of initial marker concentration. The feeds were soaked for 6 hours with distilled water, liquid was drained off, material was rinsed once and feed samples were taken for analysis of marker concentrations. This cycle of soaking for 6 hours in distilled water and one rinse was conducted for three cycles. Concentrations of rare earths on feed samples were evaluated by neutron activation analysis as discussed in experiment one materials and methods. Results and Discussion Corn silage marked with Yb lost 29.5% of the associated Yb after the first soaking-washing cycle. When La was utilized as a marker, 64.8% of the initial marker concentration was removed by the first soaking-rinsing cycle. Soybean meal marked with Yb lost 22.5% of the initial Yb content after the first washing-rinsing cycle and 8.4% of the 41 La. Wet distillers grains lost 64% of the Yb and 67.3% of the La upon the first soaking-rinsing cycle. Corn silage, soybean meal and wet distillers grains contained 10.5, 9.9; 22.0, 7.4; 11.5, 7.5 mg of Yb and La per gram of dry matter respectively after the 3 cycles of soaking and washing. The 24 hour soaking and 3 cycle soaking-rinsing scheme was applied to 45 kg batches of feed. Utilizing soaking solutions contributing twice the potential rare earth concentration present after 3 soaking-rinsing cycles, the bound marker levels were slightly higher than those found in the laboratory. qun silage contained 15.1 to 24.2 mg Yb/gram of dry matter. Soybean meal contained 14.5 to 40.4 mg La/gram, wet distillers grains contained 14.8 mg La/gram and corn gluten meal contained 17.5 mg La/gram. These levels were higher than values found when small amounts of feeds were processed. Likely this represents an inability to rinse the feeds as effectiyely under large scale settings. The higher levels of rare earths bound to feedstuffs provided the basis for determining the dose size to achieve 2 to 3 grams of marker intake per day during periods of adaptation (5 days) and digests sampling (3 days) for experiments one and two. PRELIMINARY EVALUATION: FECAL EXCRETION OF MARKERS Introduction An important consideration involved in any passage study is the period of time required to reach stable marker concentrations in fecal samples. This is critical to achieve valid total tract digestibility estimates, which require steady state flow of markers from the total tract. This requirement must be balanced against time constraints and the cost of markers required for the marker adaptation period. Materials and Methods In this study, two duodenally cannuulated steers from experiment one were fed 323 grams of Yb marked corn silage (31% DM, 15.1 'mg Yb/gram) and 250 mls of, CrEDTA (9.2 mg Cr/ml) mixed with 6.8 kg of diet dry matter. Fecal samples were taken from each steer at 10, 20, 50, 80, 100 and 105 hours after the initial dose. Steers were fed the same level of markers for each subsequent day of the adaptation period. Fecal samples were freeze dried and analyzed for marker concentrations as discussed in experiment one materials and methods. 42 43. Results and Discussion Figure 1 illustrates the pattern of marker concentration in feces averaged across the two steers. The concentration of Yb in the feces reached a peak at the 100 hour sample while Cr level reached a peak at so hours with some slight variation after this point. Teeter et al. (1981) indicated ’peak fecal Yb concentrations, associated with long hay, occured 31.8 hours after dosing. Welch and Smith (1978) found polypropylene ribbon excretion peaked by 48 hours in steers and 72 hours in sheep. Uden (1978) reported peak excretion of several markers occured 40 and 50 post dosing in large and small heifers respectively. These data indicate that peak marker concentrations occur at 48 to 72 hours after dosing. In our research, peak concentrations of Yb occured at 100 hours after dosing and Cr peak was reached essentially 50 hours post dosing. It was concluded from this data and literature sources that a minimum 72 hour marker feeding period would be required for liquid markers and a 96 hour period for solid markers to reach suitably stable levels. We chose to feed marker for a minimum of 5 days (120 hours) prior to initiation of duodenal or fecal sampling. 44 cent. FECAL EXCRETION OF MARKERS PPM aso - sOO - 750 - 7oo .. - . 650 - w A/ \ 600 H- 550 " 500 450 400 350 - 300 -A- Cr. .250; _,_ N 200 - iso - 100 so I T 4 l L l l L L l I l L L 10 20 30 4O 50 60 70 80 90100110 time-hours Figure l Fecal Excretion of Digests Markers MATERIALS AND METHODS Experiment One Experimental Design This study was designed as a 4 x 4 Latin square. It utilized four, duodenally cannulated, 600 kg, Holstein steers. The experiment consisted of four periods within which each Of the steers received one of fOur corn silage based diets. Figure 2a shows the arrangement of animal, periods and dietary allocation. Within each diet, three digests markers were evaluated to study the consequences Of marker choice upon estimates of site and extent Of digestion. Statistical evaluation was conducted, as with standard Latin square crossover experiments, utilizing blocking for animal and period effects. Markers were evaluated, as to their .effects upon digestibility, within diets with four Observations for each marker. With this arrangement, animal and periods are confounded and cannot be separated 'from the error term. Means were compared utilizing the all pair-wise comparisons of the Tukey's test (Gill, 1973; Gill, 1978) as Opposed to orthogonal comparisons which would not provide logical comparisons but would be more sensitive to differences between treatment means. 45 46 Period Animal # 1 2 3 4 788 B A C , D 789 ""c """ 13 """" I. """ 13‘" 790 ___D.__._-C """ I3 ----- I" 791”}; """ é ''''' 13 """" C--- Diets Composition A Corn Silage-Soybean Meal B Corn Silage—Urea C Corn Silage-Wet Distillers Grains D Corn Silage-Wet Distillers Grains with Urea Figure 2a Experiment One Design 47 Cannula Design and Insertion A T-type cannula was surgically placed in the duodenum approximately 10 cm from the phylorus, anterior to the bile duct. The cannulae initially were made from“ viscous plastisol (Norton's Plastic and Synthetics Div., Akron, Ohio and M & R Plastics and Coatings, Maryland Heights, Missouri). The mold utilized to make the cannulae was a copy of one acquired from Dr. L.D. Sstter, University of Wisconsin. These cannulae were not durable. Portions Of the cannulae in contact with digests became brittle and resulted in cannula losses due to cracking off Of the T portion. Cannulae were replaced with cannulae made from 15.9 mm ID X 22.22 OD Silastic tubing (Silastic Medical Grade Tubing, catalog #601- 685, Dow Corning Corp. Medical Products, Midland, Michigan). The 20 cm barrel section was placed through a hole in the center Of the 15 cm T portion of the cannula. The T section was prepared by longitudinaIly cutting, in half, a 15 cm piece of 15.99 mm ID x 22.22 OD tubing, A 22.22 diameter hole was cut in the center Of the T section for barrel insertion. The inner and outer surface of the junction of barrel and T section were joined by placing a liberal bead of Silastic medical adhesive, silicone type, at the junction (adhesive #891, Dow Corning Corp.). The cannula was allowed to cure for 24 hours at which time excess adhesive can be removed and rough edges smoothed with a razor blade. This type of cannula can be sterilized by autoclave or ethylene 48 oxide techniques prior to surgical insertion. Diet Formulation In this study, four corn silage based diets were fed. Diets were mixed daily using fresh corn silage from a 1000 metric ton capacity upright silo. Silage averaged just under 8% crude protein and had a large amount of corn grain, equivalent to approximately 7.0 bushels per ton of 33% dry matter silage. Four diets were designed to be isonitro- genous. A mineral or mineral-urea supplement was designed for each diet. Table 5 lists the average composition Of each diet on a dry matter basis. Table 6 lists the supplement composition. The protein sources were soybean meal, urea, wet distillers grains and wet distillers grains plus a mineral- urea supplement. Table 7 lists the source Of nitrogen for each diet. Diets supplemented with wet distillers grains plus urea have 50% of the supplemental nitrogen from the :wet grains and urea ‘respectively. The diets were formulated to contain 13% crude protein with calcium and phosphorus levels set for those required by 225 kg steers. Table 8 lists the average analysis of the diets. The wet distillers grains were generated from ethanol production at the MSU pilot scale ethanol plant located at the Beef Cattle Research Center. The wet distillers grains were collected from whole stillage immediately after distillation of the mash. The grains were separated from 49 whole stillage on a 24 x 24 mesh, inclined, vibrating screen. The wet grains averaged 22% dry matter, 24% crude protein and contained less than 5% starch. After collection, the wet grains were mixed in a horizontal mixer to insure homogeniety and packaged individually into 10 kg packages. The grains were frozen for storage. Wet distillers grains were thawed at room temperature overnight prior to feeding. 50 Table 5 Diet Formulation: Experiment One ‘ Component 1 Corn Silage 2 Soybean Meal 3 WDG 4 Urea Supplement W ‘ CS-Soy CSeUrea cs-wnc CS-WDG,Urea --------- % Of Total Diet Dry Matter--------- 85.6 95.4 66.7. 79.7 12.5 - - - - - 31.8 16.5 '- 1.8 - .9 1.9 6.8 1.5 3.8 1) Corn silage ensiled, well eared. 33% dry matter, 7.9% CP. 2) Soybean meal, 92% dry matter, 50% CP. 3) Wet distillers grains, 22% dry matter, 24% crude protein, collected from whole stillage by 24x24 mesh vibrating, inclined screen, frozen, stored. 4) Urea was contained in the supplement. 51 Table 6 Supplement Composition: Experiment One ‘ TREATMENT . Component ' CS-Soy CS-Urea CS-WDG CS-WDG,Urea ---------- % of Total Dry Matter---------- Ground Corn 15-7 44-7 16.7 37-7 Limestone 25-6 7.4 29.3 11.7 Calcium Sulfate ‘ 25-5 7-3 ‘ 29.3 11.6 Monosodium Phosphate 19-0 8-8 8-1 9.1 TM Salt1 13-1 3.8 16.6 6.5 Urea -- 28.0 -- 23.4 1 Trace mineral salt contained a minimum of: 0.35% Zn, 0.20% Fe, 0.03% Cu, 0.005% Co, 0.007% I, 96% NaCl. 52 Table 7 Source Of Nitrogen in Corn Silage Based Diets 2 Component Diet CS-Soy CS-U CS-WDG CS-WDG-U Corn Silage N ‘ 61.5 61.5 61.5 61.5 Urea N -“ 3805 "" 1902 Natural Protein N 38.5 -- 38.5 19.3 3 a a b c ADIN % Of Total N 3.6 4.4 10.2 8.0 L; 1 Corn silage based diet, 13% crude protein 2 Expressed as a % Of total nitrogen in the diet 3 ‘ ADIN-Nitrogen bound to acid detergent fiber, means with different superscripts differ (P<.05) 53 Table 8 Diet Analysis: Experiment One‘ Treatment 1 , CS-Soy CS-Urea CS-WDG CS-WDG-Urea Component Unit Dry Matter g 56.06 54.54 26.65 29.88 Crude Protein z 14.04 15.15 15.68 15.55 Organic Matter % 93.94 94-38 94.79 94.73 ADF % 20.48 22.47 21.52 22.49 Lignin z 2.56 2.56 2.81 2.92 Caz z .48 .48 .48 .48 P2 % .34 .34 .34 .34 K2 % 1.08 .90 .66 .79 Salt 1 .25 .25 .25 .25 1 Average value four periods. 2 8, based upon 4 cOmposited samples from the Values calculated based upon similar feed ingredients. 54 Markers In experiment one, three markers (lignin, ytterbium, CrEDTA) were used to evaluate the site and extent Of digestion of major feed constituents; nitrogen, organic matter and acid detergent fiber (ADF). Lignin represents an internal marker and was analyzed in feed, duodenal and fecal samples using the 72% sulfuric acid, gravimetric procedure to determine acid detergent fiber lignin (Van Soest, 1963 and Van Soest and Wine, 1967). Ytterbium (Yb) was utilized as a marker for the solid phase Of digests. It was bound to corn silage by a soaking procedure. Teeter et al., (1979) discussed the binding of ytterbium to feeds by soaking Or spraying methods. Teeter et al., (1981) reported the presence Of binding sites on feeds which had varying degrees of affinity for ytterbium. Preliminary studies on corn silage used in our research have shown tenacious binding- of ytterbium at a level Of 10 mg Yb/kg of corn- silage dry matter. This is the level of Yb remaining after soaking corn silage for 24 hours in a solution providing the potential Of 50 mg Yb/kg of corn silage, followed by 3 cycles of washing and soaking in dis- tilled water for 6 hours. In an effort to efficiently utilize Yb, marker solutions were prepared to provide the potential Yb concentration on the marked corn silage of 20 mg Yb/kg of corn silage dry matter. It was assumed 3 cycles of washing and soaking for 6 55 hours with water would remove the loosely bound Yb. Ytterbium chloride (Research Chemicals, P.0. Box 14588, Phoenix, Arizona) contains a variable number of water molecules Of hydration with 6 being the average value and averages 44.64% Yb. Corn silage was soaked in the ytterbium chloride solution for 24 hours, in large plastic containers, under refrigeration. The corn silage was soaked with enough solution to suspend the material and allow for easy mixing. Following 24 hours of soaking, the plastic containers were covered with 2 layers Of cheesecloth and a layer of screen, which was wired in place. The containers were inverted, allowing the soaking solution to drain Off. The containers were refilled with water, agitated and drained. The con- tainers were refilled with water and soaked for 6 hours followed by draining. This washing, soaking procedure was repeated 3 times. When the final rinse solution was removed, the corn silage was spread out in a thin layer on plastic and allowed to air dry to 30% dry matter. At this time, the marked corn silage was mixed in a horizontal mixer tO insure homo- geneity, packaged in plastic bags with 323 grams per bag, subssmpled for marker concentration and dry matter analyzed. Corn silage marked with Yb for this study averaged 31% dry matter and 15.1 mg Yb/g of silage dry matter. The chromium complex Of ethylenediamine tetracetic acid was prepared by adapting the procedure of Downes and McDonald (1964) for use of the free acid form of ethylenediamine 56 tetracetic acid (EDTA) rather than the disodium salt. The chromium complex (CrEDTA) can be prepared in the following manner: 1. Dissolve 400 grams of. free acid ethylenediamine tetracetic acid dissolved in 4 liters of distilled water. Add 100grams of NaOH to facilitate solubilization Of the EDTA. Stir constantly on a hot plate, heat to a gentle boil. 2. Dissolve 284 grams Of chromium chloride in 2 liters of ~distilled water. 3. Combine solutions 1 and 2. Heat to a gentle boil while stirring. Hold at a gentle boil (100 degrees C), for 1 hour. 4. Add 80 mls of 1 N calcium chloride to the EDTA solution, stir well. The added Ca binds to unoccupied Cr binding sites on the EDTA molecule. 5. Adjust the pH of the CrEDTA solution with NaOH. Final pH should be 5.0 to 5.5. CrEDTA prepared in this manner will contain 9 to 10 mg of Cr per ml of solution. 57 Housing, Adaptation, Feeding This reSearch project was initiated October 1, 1981 and was completed April 7, 1982. .The four duodenally cannulated Holstein steers were confined during the adaptation and collection phases Of the project in 1.8 x 2.4 m pens in an environmentally controlled facility. They had free access to water and there was no bedding as the pens have slatted floors. Four diets were fed to each of the four Holstein steers during four periods. Steers were adapted to each diet for 21 days prior to any duodenal or fecal sampling. Figure 3 illustrates a typical adaptation and sampling schedule. As illustrated by Figure 3, after the 21 days of adaptation, the steers were fed marker for 5 days followed by 3 days Of marker feeding with duodenal and fecal sampling. The slatted floor facility creates severe feet and leg problems, especially with large steers as utilized in this research project. The steers were allowed to rest in bedded pens, outdoors for 14 days between collection periods. During the rest periods, the steers received the corn silage, soybean meal diet. 58 Typical Adaptation and Sampling Schedule Animal Diet Diet Diet Diet Diet + Markers + Markers + markers \ + markers + markers NO markers NO markers No markers NO markers Figure 3 Day Day Day Day Day 1 to 21, adaptation to new diet 22-25 26- Feed sample 27- Feed sample, doudenal and fecal samples: 12 am, 4 am, 12 pm, 6 pm 28— Feed sample, duodenal and fecal samples: 6 am, 10 am, 2 pm, 8 pm 29- Duodenal and fecal samples: 2 am, 8 am, 4 pm, 10 pm 30- Duodenal samples: 8 am, 12 pm, 3 31- Duodenal samples: 8 am, 12 pm, 3 32- Duodenal samples: 8 am, 12 pm, 3 33- Duodenal samples: 8 am, 12 pm, 3 Rest period, outdoors: 14 days Adaptation and Sampling Protocol pm pm pm pm 59 Diets were mixed fresh daily. Corn silages, protein source and the appropriate mineral supplement were weighed on an electronic balance to the nearest tenth of a kg. The diets were mixed in a horizontal mixer. During periods of marker feeding, marked corn silage replaced fresh corn silage on an equal weight basis and was mixed in the horizontal mixer. At the time Of mixing, 250 mls Of CrEDTA was added slowly during the mixing process. Each diet was allowed to mix for several minutes. As the diet was run out into its feed container, a .25 kg subssmple was taken to make up a diet composite sample. This type Of mixing and sampling procedure was conducted for each diet in each period. Three .5 kg feed samples from each diet within a period were taken for future analysis and frozen until analyzed. The steers were fed once each day at 7:30 am. Each steer received 6.8 kg Of diet dry matter each day during the experimental periods. This level Of intake represented a feeding regime Of 1.1 x maintenance calculated based on a requirement of 100 kcal ME/kg.75 body weight. It was assumed each steer initially weighed 500 kg and the diets contained 1.56 mcal ME/kg of diet dry matter. 60 Sampling, Compositing, Processing Feeds As illustrated in Figure 3, three 250 gram feed samples were taken from the mixed diets, one sample the day before duodenal and fecal samples were taken and one sample each Of the first two days of the collection periods. It was assumed the samples were representative of the complete mixed- diet. The three feed samples from each diet, within a period, were composited on an equal wet weight basis. This resulted in a total Of 16 composite feed samples for the entire experimen- tal period. A subssmple of each composite was oven dried at 105 degrees C for 24 hours to estimate original sample dry dry matter. The remaining feed samples were freeze dried for a period of 3 days in a Virtis model 25,SRC freeze dryer (Virtis Co., Gardiner NY). The freeze dried samples were ground in a Wiley mill with a 1 mm Screen and stored in sealed plastic containers. Figure 4 shows the feed processing scheme. Feed Sample Processing and Analysis Individual Feed Samples (3/diet/period) Composited (equal wet weight basis) Freeze Dried Ground (1 mm screen) ‘. ~> Stored in Plastic Containers Nitrogen 105 degree C dried 105 degree C dry matter ‘Pelleted (Hydraulic Press) Ash, Organic Matter Neutron Activation q} Analysis, Cr, Yb Acid Detergent Fiber ADF Lignin ADF-Nitrogen Figure 4 Feed Processing System 62 Duodenal Figure 3 illustrates a typical collection protocol used in this experiment. Twelve duodenal samples were taken from each steer, each period of the experiment. The duodenal samples were collected in individually labled, 400 ml capacity, square plastic containers with lids. An attempt was made to collect at least 300 mls Of duodenal digests from each steer at each sampling time. Care was taken to discard the first 100 mls of duodenal digests leaving the cannula after the plug is removed. This material is often Of very high solids content and should not be used. After the initial surge Of duodenal contents leaves the cannula, Often there is a period Of several minutes where no additional flow is occuring. It has been Observed by the author that replacing the plug and moving to the next steer, purging his cannula and replacing the plug and then going back to sample the first steer, is a system which works well. Surges of 100 mls Of duodenal contents are not uncommon, however, under no circumstances should collections be made by leaving the digests dribble from the cannula. It is much more efficient and intuitively more correct to replace the plug and collect surges Of digests which can only help maintain a correct collection Of duodenal solids and liquids. Samples of duodenal digests were frozen immediately after collection. 63 An alternative to immediate freezing is homogenization in a Waring blender, subssmpling and analysis Of digests dry matter in each sample, then freezing the remainder. At the time Of homogenization, a subssmple can be separated, (50-60 m1) and frozen in a separate container for future analysis of duodenal ammonia nitrogen. In experiment one, the twelve duodenal samples from each steer were homogenized separately in a Waring blender. , At this time, 100 g of each homogenized sample are combined in a large beaker and placed on a stirring plate. The 1200 gram duodenal composite is placed in four plastic containers, being careful to have equal representation of digests in each lcontainer., Three Of the four containers are freeze dried (Virtis freeze dryer) as discussed earIier regarding the feed samples. The fourth container is stored in a freezer as a backup in case a sample is lost or other problems are encountered. After freeze drying, ' the three containers of dry duodenal digests (94-98% DM) are ground in a Wiley mill with a 1 mm screen. At this time, the Contents can be placed in one container and stored for future analysis. Storing feed and duodenal samples prior to analysis should be done under freezing or at least refrigeration. Samples of duodenal digests Of 94 to 98% dry matter content will begin to support a type Of fungal growth in a few months after storage if not kept refrigerated. Figure 5 summarizes the steps involved with processing Of duodenal samples. , 64 Duodenal Samples (12, 300 g each)/steer/period / Frozen ———————+ Thaw fir Homogenize 105 degree C dry matter ‘ Composite (12, 100 gram portions) \, Duodenal Sample (100 grams) Frozen for backup (200 grams) Frozen (900 grams) Prepare 95% Duodenal Fluid 5% TCA Freeze Dried L \\V Spin 20,000 x G 20 minutes Ground (1 mm screen) k(// Supernatent Nitrogen Ammonia Nitrogen Acid Detergent Fiber 105 degree C dry matter ADF-Lignin ADF—Nitrogen Ash, Organic Matter «k 105 degree C Dried i 1 g pellet (hydraulic press) Jr Neutron Activation Analysis (Cr, Yb) Figure 5 Duodenal Sample Processing 65 Fecal A total of twelve fecal samples were taken from each steer,. each period, at the same sampling intervals as the duodenal sample collections as illustrated in Figure 3. The fecal samples were taken by rectal palpation and grab sampling of fecal contents. It is desirable to acquire approximately 200 grams of feces at each sampling interval. The fecal samples were frozen and stored prior tO compositing. Fecal samples were composited on an equal wet weight basis. The frozen samples are thawed and 100 grams from each individual sample is placed in a large container. The 1200 grams of fecal material is mixed well, 300 grams were placed in each of four containers. Three of the containers, (900 grams), containing the fecal composite, were freeze dried. Freeze dried fecal material was ground in a Wiley mill through a 1 mm screen, placed in 2 plastic containers and stored either frozen or in a refrigerator for future analysis. Figure 6 illustrates procedures for fecal processing. 66 Fecal Samples (12, 200 gram samples/steer/period) Composited (100 grams/sample=1200 grams composite) Freeze Dried, 900 grams Ground (1 mm screen) . Stored either frozen or refrigerated Nitrogen 105 degree C Dried 105 degree C dry matter Ash, Organic Matter Pelleted (Hydraulic press) Acid Detergent Fiber 1' l l Neutron Activation Analysis, Cr, Yb ADF-Lignin ADF-Nitrogen Figure 6 Fecal Sample Processing and Analysis 67 Analytical Techniques Nitrogen The total nitrogen present in feeds, duodenal and fecal samples was determined by sulfuric acid digestion in the presence of a copper sulfate based catalyst (Pope Kjeldahl mixtures, P.O. Box 903, Dallas, Texas 75221). The analysis of free nitrogen was conducted on a Technicon Auto Kjeldahl System. Nitrogen determinations were conducted on samples having dry matter of 92-98 % and adjusted to a 100% dry matter basis. Ammonia Nitrogen There are several methods available to determine ammonia nitrogen content Of rumen and duodenal fluid samples. The most common methods include; steam distillation of ammonia from samples made alkaline, (Dixon and Nolan, 1982), ammonia electrodes such as model 95-10 Orion (Orion Research Corp., Cambridge, MA), Nesslers reagent system (Sigma Technical Bulletin No. 14), hypochlorite-alkaline phenol method Of Berthelot (Ngo et al., 1982) and Technicon Auto Kjeldahl system. Review of these systems shows a number of limitations exist for each analytical technique. 68 Due to the large number of duodenal ammonia determinations required for experiments one and two, the automated Kjeldahl system was chosen. The Technicon auto analyzer Operations manual lists modifications required to analyze ammonia nitrogen in liquid samples. The modifications were designed to increase samples size when analyzing samples with very low ammonia levels such as blood plasma. Duodenal ammonia nitrogen concentrations range from a low Of 15 to above 80 mg/1OO mls. These levels were within the detection range for analysis without sample flow modifications. The analysis Of ammonia nitrogen in duodenal samples require the following sample processing: 1. Pipet carefully 9 mls Of duodenal fluid. Let thawed sample solids settle before sampling. 2. Place 9 mls Of duodenal fluid and 1 ml of 50% trichloro- acetic acid (TCA) in 12 ml centrifuge tube. ,3. Centrifuge at 20,000 x g for 20 minutes. 4. Place supernatant in a clean test tube, seal and refri- gerate until analyzed. 5. Analyze in duplicate on Technicon Auto analyzer. 100 and 200 ppm nitrogen standards (from ammomiun sulfate). Utilize ammonia free duodenal fluid prepared as dis- cussed in this section, as the base line, rather than distilled water. Several attributes Of duodenal fluid affect analysis Of ammonia nitrogen. In general, duodenal sample pH was sufficiently acid (pH less than 4) to provide a long storage life (frozen) and adequate hydrogen ion concentration to Prevent ammonia loss. Precipitations Of proteins by TCA and centrifugation will not remove free amino acids from the 6_9 dilution Of duodenal fluid by TCA and other additions, of 2 to 3 ppm of ammonia nitrogen equivalent. The 100 and 200 ppm duodenal samples produced readings equivalent to 1-2 ppm ammonia nitrogen above the 100 and 200 ppm standards. The 1 to 2% difference between duodenal based standards and normal standards is considered not significant enough to warrant using ammonia free duodenal samples spiked with ammonia- nitrogen as standards. However, it would appear advantageous to utilize the ammonia free duodenal fluid (0 ppm ammonia- nitrogen) to produce the baseline readings for the Technicon autoanalyzer system. Acid Detergent Fiber, Lignin, Nitrogen The analysis of acid detergent fiber was conducted in quadruplicate when both lignin and acid detergent fiber nitrogen data were required. Acid detergent fiber analysis was conducted by the technique discussed by Goering and Van Soest (1970). Analysis of lignin utilized the 72% sulfuric acid method as described (by Van Soest (1963) and Van Soest and Wine (1967). In order to reduce error in use of lignin as a marker, each animal's feed, doudenal and fecal composite samples were analyzed as a set to reduce variation due to changes in environmental temperature and chemical reagents. Acid detergent fiber nitrogen, also known as acid detergent insoluble nitrogen, was analyzed by micro Kjeldahl, digestion in concentrated sulfuric acid and with POpe 70 solutions. It is known certain amino acids react with automated Kjeldahl reagents and are measured as ammonia complexes by the analyzer. Final sample dilution on the analyzer. is 3,333:1 and as a result, amino acids can only contribute a small amount Of "ammonia like" activity with the Kjeldahl reagents used in the Technicon system. In an attempt to evaluate the potential contribution of non-ammonia nitrogen components to the total quantification of ammonia nitrogen, the following analysis was conducted. A 180 ml sample Of duodenal fluid, pooled from several samples, was centrifuged at 20,000 x.g for 20 minutes with 20 ml Of 50% TCA. The sample supernatant pH was elevated to 11 with sodium hydroxide and it was heated to 100 degrees C. The solution was held at 100 degrees C for 1 hour. This was conducted to drive off all Of the ammonia nitrogen. The original sample pH (3 to 4) and volume was restored by addition Of sulfuric acid and distilled water. Aliquates Of ammonia free duodenal fluid were prepared to -contain 0, 100 and 200 ppm of ammonia nitrogen by the addition Of ammonium sulfate. These samples were run on the Technicon autosnalyzer and compared to distilled water as a baseline and 100 or 200 ppm ammonia nitrogen standards. The 100 and 200 ppm standards were prepared in the standard Technicon manner; ammonium sulfate as the nitrogen source, sulfuric acid, distilled water, POpe Kjeldahl salts and nitrogen free filter paper. The results indicate the 0 ppm ammonia-nitrogen free duodenal fluid produced a chart reading, corrected for 71 Kjeldahl catalyst. . The samples used were the acid detergent fiber residue remaining after the normal acid detergent fiber analysis. All data were corrected to s 100% dry matter basis, based on 105 degrees C oven dry matter determinations. Dry Matter, Ash, Organic Matter All feed, duodenal and fecal samples were freeze dried. The freeze dried samples were 92 to 98% dry matter. All feed samples and composite duodenal and fecal samples were analyzed for dry matter, ash and organic matter. Dry matter analysis ”of each sample is conducted in duplicate by weighing 1 to 2 grams Of sample into ashing crucibles. The samples were dried at 105 degrees C for 24 hours. The samples were allowed to cool, are re-weighed and then ashed at 500 degrees C for a minimum Of 2 hours at the ashing temperature. This technique Of incorporating several analysis steps into one was very efficient. This dry matter value was then used to correct all other analysis data to a 100% dry matter basis. Activation Analysis; Ytterbium and Chromium Activation analysis represents one of the most sensitive analytical techniques for evaluation Of elements, both quantitatively and qualitatively. With nuclear reactors, the large neutron flux can produce radioisotopes as the result of neutron bombardment. When the neutron flux and period of 72 bombardment are known, the isotopes are produced in proportion to the masses Of elements present. Activation Of a known mess Of an element and an unknown, if exposed to the same flux and time period, will allow for quantification of the mass of the element in the unknown. Activation analysis is a non-destructive method of analysis which works well for detection and quantification Of elements, especially the rare earths and chromium. In experiment one, analysis of ytterbium and chromium, in asamples Of feed, duodenal and fecal material, were conducted in duplicate. A total of 96 samples (48 samples duplicated) and 6 standards were analyzed as a set. Feed, duodenal and fecal samples from each animal were analyzed together, within an activation analysis run. This prevented variation due to standard differences or other factors. Four grams Of feed, duodenal and fecal samples for each animal and period were placed in small glass bottles. These samples were oven dried at 105 degrees C for 24 hours and sealed with screw-on caps while hot. Standards were prepared by drying corn silage collected during the experiment, grinding in a Wiley mill through a 1 mm screen and oven dried at 105 degrees C for 24 hours. A 100 gram sample Of this 100% dry amtter corn silage was placed in a 500 ml beaker. A solution containing 2 ml of a certified 10 mg/ml ytterbium standard (20 mg total Yb), 40 ml of a certified 1 mg/ml chromium standard (40 mg total Cr) and 158 ml of distilled water was prepared. This solution was 73 mixed with the dry corn silage in the 500 ml beaker. The mixture was oven dried at 105 degrees C for 48 hours, reground in a Wiley mill with a 1 mm screen, redried at 105 Idegrees C for 24 hours and placed in an oven dried container and sealed. These standards contained 200 ppm of ytterbium and 400 ppm Of chromium. Preliminary evaluation of neutron activation Of ytterbium, lanthanum and chromium was conducted at Michigan State University using the nuclear reactor in the Chemical Engineering department. Small, 2 dram, polyethylene vials were used to contain samples for irradiation in the nuclear reactor. After irradiation, the samples were allowed to decay for a week to ten days to reduce the amount of Na-24 in the sample. The samples were then counted with a germanium (lithium) [Ge(Li)] detector. At this time, two problems were discovered. One problem was the 100% dry matter samples were coating the sides and top of the polyethylene vials. The other problem was that if two identical samples had different total sample weights and thus filled the vials to different levels, the amount Of element detected per unit Of samples mass was different. It was verified by the reactor operator that sample geometry will affect the efficiency of sample counting on Ge(Li) detectors. In order to prevent sample geometry from affecting element analysis, the sample geometry must be standardized. A pellet formed by a hydraulic press, as used for bomb calorimetry work, seemed to provide a solution to the geometry problem and associated static charge related coating 74 Of vial walls with sample materials. All feed, duodenal, fecal and standard samples were formed into pellets with a hydraulic press. The pellet ranged in mass from .8 to 1.4 grams but due to specific gravity differences, size ,of the pellets did not vary to any significant degree. The pellets were 100 mm in diameter and varied from 50 to 75 mm in height. The pellets were placed in 4 dram polyethylene vials (Olympic Plastics CO. INc., 5800 W. Jefferson Blvd., Los Angeles, CA 90016) and were held in place with 2 cotton balls. The standards ranged in mass from 1.0040 to 1.4317 grams and contained 200 ug/g Yb and 400 ug/g Cr. Six pellets of exactly the same mass (plus or minus .007 grams) were used for each activation analysis run. A group Of 40 to 50 standard pellets were prepared at one time from which, 6 Of equal mass were chosen. The remaining pellets were placed in numbered vials, with their mass recorded and were used in subsequent analysis runs. Sets Of 48 duplicated samples and 6 standards were sent to the University Of Wisconsin Research Reactor for activation analysis (Reactor Lab, 130 Mechanical Eng. Bldg., Univ. Of Wisc., Madison WI 53706). (The four dram polyethylene vials were heat-sealed and \ irradiated in the nuclear reactor for 3 hours at a thermal 11 -2 -1 flux Of approximately 5 x 10 neutrons cm sec. In order to reduce the presence Of interfering, short lived isotopes such as Na-24, the samples and standards were allowed to decay for 10 to 14 days. The samples and 75 standards were counted for 900' sec. of live time. The elements, Cr-51 and Yb4169 were counted at 319.8 Kev and 198.1 Kev respectively. The counting of the gamma radiation emmitted from the samples and standards was accomplished by a Ge(Li) detector coupled to a Tracor Northern TN-11 pulse height analyzer. The effects of interfering energy peaks were removed and corrections for different decay, times were accomplished by a computer program developed by the Nuclear Engineering department at the University of Wisconsin. Calculations: Flow and Digestibility Calculations Of flow and digestibility of dietary constituents can be accomplished without an accurate estimate of feed intake. This can be accomplished when a suitable marker is homogeniously distributed throughout the diet. It was Observed that animals, housed in the slotted floor metabolism room, would lose feed from their mouth while masticating. For some reason, several steers preferred to masticate feed with their heads outside of the feedbunk. Feed loss into the manure pit below the steers was evident. An estimate of feed 1088 was not possible. As a result, actual ingested feed intake is unknown, although feed provided is measured. The calculation of flow and digestibility Of dietary constituents was calculated, as a percent of intake, by assuming the ratio of feed constituents and markers present was not altered by feed waste. The ratio of feed nutrient 76 concentrations and feed marker concentrations can be compared to their ratio at other sampling points to determine nutrient flow and digestibility. Equations 13, 14, 15 and 16 illustrate the calculation site of nutrient flow and digestibility at various points along the digestive tract. Equation 13 was used to calculate the percentage Of nutrient intake which has flowed to the duodenum (duo), grams of nutrient flow per day and percent apparent ruminal digestion. (13) (Nutrient Conc. Duo. DM/Marker Conc. Duo. DM) (Nutrient Conc. Feed DM7Marker Conc. Feed DM) x 100= % Nutrient Flow of Nutrient Intake x - Nutrient Intake (g/day)= Nutrient Flow to Duodenum (g/day) (100 - % Flow Of Nutrient)= % Apparent Ruminal Digestion Total tract nutrient flow and digestion was calculated in a similar manner as illustrated by equation 14. (14) (Nutrient Conc. Fecal DM/Marker Conc. Fecal DM) 7(Nutrient Conc. Feed DM/Marker Conc. Feed DM) x 100= % Nutrient Flow of Nutrient Intake to Feces x - Nutrient Intake (g/day)= Nutrient Flow to Feces (g/day) (100 - % Flow of Nutrients to Feces)= % Apparent Total Tract Digestion 77 (15) % Apparent Total Tract Dig. - % Apparent Ruminal Dig.= % Apparent Lower Tract Digestion The percent apparent digestion Of nutrients entering the lower tract was estimated by equation 16. (16) % Apparent Lower Tract Digestion of Nutrient % Apparent Flow Of Nutrient from Rumen x 1008 % Apparent nutrient digestion of nutrient entering duodenum and lower tract. MATERIALS AND METHODS Experiment Two Experiment Design The design Of this study was a replicated 2 x 2 latin square, crossover experiment. It utilized four duodenally and abomasally cannulated, 350 kg, Holstein steers. The experiment consisted Of two periods within which each of the steers received one Of two corn silage based diets. Figure 2b shows the arrangement Of animals, periods and dietary allocation. Within each diet, four digests markers were evaluated and an infusion of PEG into the abomasum was con- ducted to establish the patterns of digests passage. . Statistical evaluation was conducted as with standard latin square, crossover experiments utilizing anlaysis Of square and treatment effects. Markers were evaluated, as to their effect upon digestibility, within diets, with four Observations for each marker. With this arrangement, animals, periods and squares were confounded and could not be separated in the analysis of variance. Means were compared utilizing the all pair-wise comparisons of the Tukey's test (Gill, 1973: Gill, 1978). 78 79 Square 1 Period Animal 1 2 813 A B 820 B A Square 2 Period Animal 1 2 819 A B 550 B A Diets Composition A Corn Silage-Soybean Meal B Corn Silage-Corn Gluten Meal Figure 2b Experiment Two Design Cannula Design and Insertion A T-type cannula prepared and inserted in the same manner as in experiment one was utilized for sampling of duodenal contents. In addition, each steer had a surgically inserted abomasal infusion cannula. This small cannula was placed in the non-glandular region of the abomasum, near the junction with the omasum. The cannula was prepared with a 25 cm barrel portion of 9.53 mm ID x 12.7 mm OD Silastic brand tubing (Catalog #601- 525, Silastic medical grade tubing, Dow Croning Corp. Medical Products, Midland MI). The T portion was prepared with an 8 80 cm, 1/3 section of 15.99 mm ID x 22.22 mm OD Silastic medical grade tubing (catalog #601-685). The barrel portion was placed through a 12.7 mm hole in the center of the T portion. Five cm of the barrel projected from one side of the cannula, 20 cm on the other. The two portions were bonded together by a liberal bead, on both sides of the T portion, around the junction Of barrel and T portions (Silastic brand Medical Adhesive, catalog #891). The infusion cannula was held in the abomasum with a purse string suture of nonabsorbable material. Both the duodenal and abomasal cannulae were inserted at the same time through a single incision site. The steers require at least a one month recovery period before the experiment was conducted. Diet Formulation In this study, two corn silage diets were fed. One was supplemented with soybean meal (SOY), the other with corn gluten meal (CGM). Diets were mixed daily using fresh corn silage ensiled in an upright silo. The silage averaged nearly 8% crude protein and was not as high in grain content as the corn silage utilized in experiment one. The silage was estimated to contain 6 bushels Of corn equivalent per ton of silage at 33% dry matter. Table 9 lists the composition of each diet on a dry matter basis. The diets were formulated to contain 13% crude protein with calcium and phosphorus levels set for those 81 required by 225 kg steers. Table 10 lists the average analysis of the diets. Table 11 lists the supplement composition. Table 9 Diet Formulation: Experiment Two Treatment Component CS-Soy CS-CGM % of Dry Matter 1 ................................ Corn Silage 85.6 89.7 Soybean Meal2 12.5 -- Corn Gluten Meal3 -- 9.0 Supplement 1.9 1.3 1 Corn silage 6 bu/ton at 33% DM, 7.9% crude protein 2 Soybean meal 92% dry matter, 50% crude protein 3 Corn gluten meal, 90% dry matter, 66% crude protein 82 Table 10 Supplement Formulation: Experiment Two Component Treatment CS-Soy CS-CGM % of Dry Matter Ground Corn 16.7 15.46 Monosodium phosphate 19.0 34.28 Calcium sulfate 25.6 16.75 Limestone 25.6 16.75 Salt-TM1 13.1 16.75 Trace mineral salt contained a minimum level of: 0.35% Zn, 0.20% Fe, 0.03% Cu,0.005% CO, 0.007% I and 96% NaCl Table 11 Diet Analysis, Experiment Two Component Unit Treatment CS-Soy CS-CGM Dry Matter 33.34 31.85 Crude Protein 13.07 12.84 Organic Matter 93.72 94.30 ADF 22.16 24.56 Lignin 2.52 2.59 2 Ca .48 .47 2 P _ ~34 .35 2 K 1.08 .85 Salt .25 .25 1) Average values based upon 4 composite samples, 2 periods 2) Values calculated based upon similar feed ingredients 83 Markers In experiment two, four markers, (lignin, ytterbium, lanthanum, CrEDTA) were utilized to evaluate their effects upon estimates of site and extent of digestion Of dietary constituents. Lignin served as an internal, naturally occuring marker. The lignin evaluated was 72% sulfuric acid lignin as described in experiment one. Ytterbium was bound to corn silage by the soaking and washing procedure as discussed in experiment one materials and methods. The corn silage was individually packaged. Each package contained 1362 grams of marked corn silage at 18.1% dry matter. The marked silage was frozen and stored until required. Prior to mixing into the diets, the corn silage was thawed overnight. As illustrated by Figure 3, markers were placed in each animals' diet for a total Of 8 days. A package, containing 1362 grams of marked corn silage, replaced 1362 grams of normal corn silage at the time of diet preparation. The marked corn silage was 18.1% dry matter and contained an average of 24.17 mg Yb/gram of silage dry matter. ' After removing a subssmple Of the mixed diets for diet analysis, each steer received an average Of 5.76 grams of Yb per day. Lanthanum was bound to both soybean meal and corn gluten meal by the soaking procedure utilized to mark corn silage. 84 A soaking solution was prepared to contain an estimated 40 mg La/gram Of feed to be marked. Lanthanum chloride (7.40% La, Research Chemicals P.O. Box 14588, Phoenix, Arizona 85063) was utilized as a source Of Lanthanum which is highly soluble Soybean and corn gluten meal were oven dried at 105 degrees C for 48 hours. The marked feeds were then ground in a burr mill because after drying, the material had formed hard clumps Of feed. The marked feeds were individually packaged. Packages contained 150 grams of soybean meal at 81.4% dry matter and 40.37 mg La/gram Of dry matter. Corn gluten meal was packaged in 170 gram units at 83.8% dry matter and 17.47 mg La/gram of dry matter. During periods of marker feeding the 150 grams Of marked soybean meal or 170 grams of corn gluten meal replaced the same amount of soybean meal or corn gluten meal at the time of diet mixing. Steers receiving the soybean meal and corn gluten meal diets consumed an average Of 4.83 grams and 2.24 grams of lanthanum respectively during periods of marked feed addition. ‘During periods Of marker feeding, 250 mls of CrEDTA were added to each diet at the time of mixing. The CrEDTA complex was prepared as discussed in experiment one ,msterials and methods. Steers daily Cr intake averaged 2.25 grams per day during periods of marker feeding. The CrEDTA complex averaged 9.2 mg Cr/ml. A polyethylene glycol solution (PEG 4000) was infused into the abomasum. The solution contained 135 mg PEG/ml and was infused at a rate of 2.7 to 2.9 mls/minute. Infusion was 85 conducted by using a Harvard type peristalic pump. The infusion was initiated 12 hours prior to duodenal sampling. Measurement Of infusion rate was conducted by evaluation of weight change Of flasks containing the infusate. Flasks were weighed prior to and immediately after each duodenal sampling period. This provided 12 measures of infusion rate for calculation Of duodenal flow at each sampling time. The steers were connected to 7.6 meters Of 9.5 mm Tygon tubing. The tubing ran through a series Of pulleys and was weighted to keep the tubing tight and yet allowing some freedom of movement of the steers. Steers had to be secured with a halter to prevent their damaging the tubing. Housing, Adaptation, Feeding This research project was initiated January 7, 1982 and completed April 5, 1983. The four Holstein steers were confined during the adaptation and collection phases in an environmentally controlled metabolism room. Figure 3 illustrates the typical adaptation and sampling schedule. The only differences are the steers were infused with polyethylene glycol, into the abomasum, beginning 12 hours prior to duodenal sampling and terminated after the last duodenal samples were taken. Diets were mixed fresh daily. During periods Of marked feed addition, feeds bound with Yb and La replaced equal amounts Of respective unmarked feed ingredients. While the diets were mixing in a horizontal mixer, 250 ml Of CrEDTA 86 were added slowly. Steers were fed once daily at 90% Of ad lib intake of the -steer consuming the lowest average amount Of dry matter per day. Steers averaged 350 kg and their average dry matter intake was 7.2 kg for both diets and periods. Three .5 kg feed samples were taken from each steer's mixed diets over 3 days as indicated in Figure 3. The samples were frozen for future compositing and analysis. Sampling, Compositing, Processing Feeds The three .5 kg feed samples, Obtained as illustrated in Figure 3, were analyzed in the same manner as described in the materials and methods in experiment one, Figure 4. Due to analytical difficulties experienced with determination of acid detergent fiber nitrogen, this analysis was not conducted in experiment two. Lanthanum in feed samples was analyzed as Yb and Cr were in experiment one , Figure 4. Duodenal Figure 3 illustrates a typical collection protocol, as used in experiment one and two. The duodenal samples were processed as described in experiment one, Figure 5, but were not composited. Duodenal samples were analyzed 1 ndividually for all components and markers. Acid detergent insoluble 87 nitrogen was not analyzed in this experiment. Lanthanum was analyzed by activation analysis as indicated for Yb and Cr in Figure 5. Fecal Fecal samples were taken following the schedule illustrated in Figure 3. Figure 6 summarizes the compositing and processing sequence conducted for fecal samples Obtained in experiment two. Acid detergent fiber insoluble nitrogen was not analyzed in fecal samples. Lanthanum was anlayzed by activation analysis as described in Figure 6 for Yb and Cr. Analytical Techniques Nitrogen The total nitrogen in feeds, duodenal and fecal samples was analyzed as samples in experiment one analytical techniques. Ammonia Nitrogen Ammonia nitrogen in duodenal samples was analyzed as described in experiment one analytical techniques. The modifications include that each of the 96 duodenal samples were analyzed individually for ammonia nitrogen. 88 Dry Matter, Ash, Organic Matter Feed, duodenal and fecal samples were analyzed for dry matter, ash and organic matter as discussed in experiment one, analytical techniques. The modifications include that after homogenization of duodenal samples, individual duodenal digests dry matter was analyzed in duplicate for each of the 96 samples. This was conducted by placing 20 to 30 mls of homogenized duodenal digests into small aluminum dry matter pans and dried at 105 degrees C for 24 hours. 1 Analysis of ash, organic matter and freeze dried sample dry matter was conducted on each duodenal samples in duplicate. Acid Detergent Fiber, Lignin The analysis Of acid detergent fiber and lignin were conducted in duplicate on composite samples Of feeds and feces as discussed in experiment one, analytical techniques. All 96 duodenal samples were analyzed in duplicate for acid detergent fiber and lignin. Activation Analysis Activation analysis on samples collected in experiment two was conducted as discussed in experiment one, analytical techniques. All 96 duodenal samples and feed, fecal 89 composites were analyzed in duplicate. Lanthanum 140 radioisotope was counted at 487 Kev. Standards contained 300 ppm La and were prepared as discussed in experiment one. Polyethylene Glycol Analysis Figure 7 illustrates the processing sequence, conducted on each of the 12 duodenal samples from each steer, for PEG analysis. The technique represents only slight modificaiton to an improved, turbidimetric, PEG analysis developed by Malawar and Powell, (1967). Standards for this analysis should be prepared with centrifuged duodenal fluid from cattle fed diets similar to those used in the experiment. The presence of interfering materials, especially tannins, can be corrected for in this manner. (The standards utilized in this study contained 0, 300, 500 and 700 mg/100 mls Of PEG and were processed in the same sequence and at the same time as the duodenal samples. Analysis Of a 1:25 dilution of the individual infusion solutions was also conducted within the same set as the respective duodenal samples. 90 Homogenized Duodenal Sample Centrifuge (20,000 x G, 20 min.) 9 mls + 1 ml 50% TCA Spin 20,000xG, 20 min. Duodenal Analysis Ammonia Nitrogen 1 ml duodenal fluid, standards 1:25 dilution of infusion solution + 10 ml water (distilled, deionized) + 1 ml 10% (w/v) anhydrous barium chloride + 2 ml .3N barium hydroxide + 2 ml 5% (w/v) zinc sulfate (swirl after each addition , after last step cap with parafilm-- shake) Let stand 10 min.-—-————+ Filter, double thickness Whatman 42 filter paper 1 ml filiate 16 x 150 mm test tube + 3 ml gum arabic solution (6-12 mg/l conc.) .L mix gently / + 4 ml, 30% (w/v) TCA with 5% Barium chloride, anhydrous 1 cap, invert 5 times wait 60-90 minutes read on Spectrophotometer at 650 mu, slit width .04 mm Figure 7 PEG Analysis System 91 Calculations: Flow and Digestibility In this experiment, digestibility of dietary constituents was estimated by marker ratio techniques based on lignin, Yb, Cr and La concentrations in feeds and fecal composites and duodenal concentrations based on averaging the levels in the 12 duodenal samples on the equal whole digests basis. Marker concentrations were also estimated in duodenal samples based upon weighted values resultant from abomasal PEG infusion and PEG concentration in duodenal samples. These marker concentrations were compared with those based on averages derived from equal whole digests compositing. Digests flow at each sampling time was calculated as illustrated in equation 17. Percent Of dietary constituents flowing to the duodenum at each sampling time was calculated as illustrated in equation 18. The following calculations were conducted for each duodenal sample within a steer/diet set of data. (17) Digests Flow per Hour Infusate Conc. (mg PEG/ml) x Infusion rate (ml/min) PEG conc./ml whole digests (mg/ml) ml digests flow/min x 60 min/hour Digests flow/hour x % DM Digests dry matter flow/hour at each sampling point 92 (18) Percent of Total Flow at each Sampling Time Digests flow/hr x 100 12 f (Dry matter flow/hr) i=1 i s % of total daily digests flow at sampling time (Digests flow/hr x nutrient or marker conc. in digests DM) (Digests flow/hr x nutrient or marker conc. in digests DM) x 100 s z of total nutrient or marker flow at sampling time Duodenal samples were mathematically composited based on an equal whole digests weight basis and based on percent of total daily flow occuring at the sampling time based on PEG infusion. Ruminal digestion was calculated based on an equal amount dry matter compositing system and the marker ratio techniques as well as PEG determined nutrient flow versus total intake of each nutrient. PEG contributed to apparent organic matter content Of duodenal and fecal samples. The amount of PEG infused per day was used to correct the duodenal and fecal samples to actual organic matter contents prior to determination of organic matter digestion. 'Based upon the percent of total daily flow occuring at each Of the 12 duodenal sampling times, the data can be examined by Fourier analysis. The Fourier transform 93 can help describe the circadian variation in flow. This analysis technique was not utilized to evaluate the infusion system studied in this research, how ever we intend to evaluate the techniques at a later date. The Fourier analysis system was used by Corbett and Pickering (1983). A similar evaluation Of rumination patterns was conducted by Murphy et al. (1983) using cosinar analysis and similar circadian rhythms was described but not mathematically predicted by Gordon and McAllister (1970). RESULTS AND DISCUSSION Experiment One The Effect of Supplemented Protein Source Upon Site and Extent Of Digestion Of Corn Silage Based Diets by Steers. Rumen Disappearance and Flow There were no statistically significant effects of protein source upon apparent rumen disappearance of nitrogen (N), organic matter (OM), acid detergent fiber (ADF) or non- ammonia nitrogen flow (NAN flow) to the lower tract (duodenum). However, specific trends exist which are evident regardless Of the marker (lignin, Yb or Cr) selected to predict digestibility and flow (see tables 13,14,15). The absolute numerical values Of digestiblity vary based on the marker selected. The qualitative differences between dietary constituent digestibility and flow are likely actual occurances, not representations of artifacts Of protein source or marker choice. The NAN, flow as a percent Of nitrogen intake was the least for the soybean meal (SOY) supplemented diet and highest for the wet distillers grains with urea combination (WDG-Urea). The SOY diet NAN flow as a percent Of N intake was 44.9, 65.7 and 73.2 while WDG-Urea averaged 54.3, 70.3 and 94 95 82.4 for lignin, Yb and Cr markers respectively. Crickenberger et al. (1979) reported NAN flow Of 50.9% Of N intake on a corn silage-soy diet at 13% crude protein, based on lignin as a marker. The WDG-Urea diet NAN flow exceeded the SOY supplemented diets by 20.8, 7 and 12.4% based on lignin, Yb and Cr as markers respectively. Diets supplemented with WDG-Urea exhibited less apparent rumen disappearance of organic matter than SOY supplemented diets. The organic matter disappearance was 22, 32.3 and 57.7 % less for WDG-Urea diets compared to SOY diets, based on lignin, Yb and Cr(EDTA) markers respectively. Merchen et al. (1979) reported a brewers dried grains and urea combination, in a corn cob based diet, had 5.5% less apparent rumen organic matter disappearance when compared to a similar diet supplemented with urea alone. They also report 9.8% less microbial nitrogen incorporated per kg Of apparent organic matter digestion for diets supplemented with brewers dried grains with urea versus urea alone. The quantity of apparent organic matter fermented or 'digested in the rumen is directly related to microbial growth and the ‘amount Of dietary nitrogen incorporated per kg of apparent rumen organic matter fermented. Estimates Of grams of microbial nitrogen incorporated per kg Of apparent fermentable organic matter range from 14.6 for low nitrogen diets to 21.3 for high nitrogen diets (Hume et al., 1970, baSed upon grams Of microbial protein divided by 6.25). Merchen et al. (1979) reported 13.3, 14.5 and 12.0 g microbial N/kg OM fermented in diets supplemented with urea, 96 soybean meal and urea, dried brewers grains and urea respectively. The brewers grains and urea diet provided the lowest total microbial nitrogen contribution to the lower tract. Hembry et al. (1975) report 9.6, 13.0, 6.2 and 11.9 grams of microbial N/kg fermented OM for diets supplemented with soybean meal, casein, zein and urea respectively. They also report the addition of isovaleric and valeric acids tO diets containing urea improved microbial growth. Diets supplemented with corn based proteins, (wet distillers grains, dried distillers grains, and zein) or other proteins resistant to degradation, may not support optimal microbial growth. The addition of urea to diets supplemented with corn based proteins may not improve the efficiency Of microbial growth as expected. It has been shown that the alpha keto acids; phenylacetic, isobutyric, isovaleric and two methylbutyric are required by many cellulolytic bacteria for protein synthesis (Hume et al., 1970; Bryant and Doetsch, 1955 and Cline et al., 1966). Perhaps, due to the resistant nature of corn based proteins to digestion within the rumen, there is a deficiency of both nitrogen and specific alpha keto acids for microbial growth. The potential benefits Of alpha keto acids and urea to these diets has not been evaluated. Apparent rumen disappearance of ADF in WDG-Urea supplemented diets was the lowest compared to urea supplemented diets, except when Yb was used as a marker. In this instance, the soybean meal supplemented diets had a slightly higher ADF disappearance than the urea supplemneted 97 diet. The WDG-Urea diets' had 22, 52.5 and 57.7% less apparent ADF disappearance from the rumen compared to the urea diets with lignin, Yb and Cr as markers, respectively. The trend for low rumen digestion Of nitrogen, organic matter and acid detergent fiber in diets containing wet distillers grains with urea is interesting. Corn based proteins, such as wet distillers grains, have low rumen degradability as illustrated by Zinn et al. (1978) and Stern et al. (1983). As described by Pilgrim et al. (1970), 50 to 75% of the microbial nitrogen originates as ammonia. Hembry et al. (1975) has shown in sheep that diets supplemented with the corn protein zein supported the lowest microbial growth. The low microbial growth on diets supplemented with corn based proteins may be related to low rumen ammonia levels generated in these diets. Rumen ammonia levels Of 5 mg/100 ml (Satter and Slyter, 1974) and 9 mg/100 ml (Hume et al., 1970) have been reported to support Optimal microbial growth. Mehrez et al. (1977) report that optimal digestion within the rumen requires rumen ammonia levels of 23.5 mg/100 ml. Klopfenstein et al. (1976) has reported peak rumen ammonia levels Of 2 mg/100 ml in the rumen Of sheep fed dry distillers grains and by 6 hours, values of 0. In comparison, sheep fed urea supplemented diets had peak rumen ammonia levels of 35 mg/100 ml and .6 at 6 hours. Clearly, rumen ammonia levels in sheep fed distillers grains are below those shown Optimal for either microbial growth or digestion Of dietary constituents. 98 Urea is Often added to diets containing corn based proteins or other proteins resistant to rumen degradation. Klopfenstein et al. (1978) reports a "complementary" effect of urea in these diets, where animal performance is signif- icantly improved relative to the diets containing the resistant proteins alone. Aderibigbe and Church (1983) report increased in vitrO dry matter digestion when urea was added to diets containing feather meals. Church et al. (1982) report increased utilization of feather meal when diets are supplemented with urea. Despite the beneficial effects Of urea upon ruminal digestion of dietary constituents, (Church at al. 1982) in diets supplemented with some resistant proteins, this has not been Observed in diets containing dried brewers grains (Merchen et al., 1979) or in our studies. Apparent rumen disappearance of ADF was 8.4, 2.3 and 16.9% less in WDG-Urea diets compared to WDG diets with lignin, Yb and Cr markers respectively. Based upOn the "complementary" effects of urea addiiton to these diets, as reported by Klopfenstein et al. (1978), one must contemplate the sOurce of these performance advantages. The advantages may be unrelated to urea improving ruminal microbial growth. It appears the urea resulted in an increased amount Of ruminal flow of undegraded dietary constituents. As reported by Utley et al. (1970), increased urinary output as a result of urea supplementation may increase water intake. This may increase the flow Of 99 dietary constituents to the lower tract. Black (1971) has indicated that utilization Of nutrients post-ruminally eliminates the energy losses which are assoc- iated with fermentation and conversion of dietary protein to microbial protein. Perhaps containing corn based proteins such as distillers grains, distillers grains, result in a shift in the site of theory, this may be the source Of Of urea described by Klopfenstein Of urea producing a shift in the been elucidated. As discussed isoacids as well as nitrogen in corn based proteins, may be corn gluten meal the addition of urea to diets wet and some brewers grains, digestion. Based on Black's the "complementary" effects et al. (1978). The role site Of digestion has not earlier, a deficiency of diets supplemented with responsible for low rumen microbial growth and high bypass of dietary constituents. Lower Tract Digestion The percent tract In addition, of total digestion occuring in \(duodenum to rectum) was evaluated (Tables the percent digestion Of dietary the lower 13.14.15)- constituents occuring in the lower tract was evaluated. The apparent digestion of duodenum, with Cr(EDTA) as a marker, was nitrogen occuring at the significantly less for WDG supplemented diets compared to urea diets; 57.41 versus 67.47% (PO.10). Table 16 illustrates an estimate of the digestion of protein from corn silage, soybean meal and wet distillers grains.‘ The calculation represent the pooled means Of total tract nitrogen digestibility as predicted by lignin, Yb and Cr as markers. The calculation assumes a 100% digestion of urea and no associative effect of protein source on Corn silage digestibility. It appears the corn silage nitrogen is 55.9% digestible in the total tract. Soybean meal and wet distillers grains nitrogen digestibility were 95.9% and two estimates of 76.9 and 69.0% for wet distillers grains. Diets supplemented with WDG alone exhibited higher total tract 0M digestion than diets supplemented with WDG- Urea (77.3 vs. 68.7%) with lignin as a marker (P<0.05) and (72.5 vs. 68.0%) with Or as a marker (PO.10) (73.8 vs. 71.1%) for SOY vs. WDG-Urea and (73.8 vs. 68.5%) for SOY‘vs. WDG. Total tract ADF digestion was significantly greater for diets supplemented with SOY versus WDG (60.2 vs. 53.4%) for lignin as a marker (P<0.05). When Yb was utilized as a marker, differences were not significant (P>0.10) but similar trends existed (54.0 vs. 51.4% for soy vs. WDG). When Or was utilized as a marker, the differences also were not significant, however, the urea diets had numerically greater 102 ADF digestion than the WDG supplemented diet (56.0 vs. 51.4%). ° Despite a trend for a large amount Of rumen bypass Of dietary constituents in the WDG-Urea diet, the data illustrate the potential contribution, to total tract digestion, supplied by the lower tract. However, in general, diets containing WDG and WDG-Urea had less total tract digestion Of dietary constituents as compared to diets containing soybean meal or urea. These results are similar to data presented by Waller et al. (1980). Diets supplemented with distillers grains and urea for lambs, tended to have lower total tract OM digestion than diets supplemented with urea. Klopfenstein et al. (1976) show higher total tract digestion of N and DM in diets containing soybean meal and urea than for diets containing either corn distillers dried grains or grains with solubles. ADIN Flow-Total Tract Tables 11 and 12 list the percent of total dietary nitrogeh component of acid detergent insoluble nitrogen (ADIN) (Van Soest, 1965) and total intake per day of ADIN respectively. ADIN is often a significant portion Of the total nitrogen present in forages. Goering et al. (1974) report ADIN in dehydrated alfalfa ranging from 6.6 to 20.4% of the total nitrogen. Yu (1976) has reported the role of heat and moisture in the formation Of ADIN in alfalfa stored in several 103 ways. Sutton and Vetter (1971) have shown the decreased nitrogen retention in lambs fed haylage with high ADIN. In our research, corn silage appeared to average 4.4% and wet distillers grains averaged 15% ADIN as a percent Of the total N. Tables 13,14 and 15 illustrate the ADIN total tract flow. It is evident that ADIN present in WDG diets was digestible. ADIN in soybean meal and urea diets flowed from the tract at between 79.1 and 100.5% Of intake depending upon protein source or marker selected. This indicated only marginal digestibility, if any, for ADIN in the SOY and urea supplemented diets. ADIN flow was significantly less (P<0.05) in WDG diets as compared to the soy diet, (79.1 vs. 42.6%), 90.1 vs. 45.2%) and (100.5 vs. 46.0%) for lignin, Yb and Cr markers respectively. It is reasonable to assume that ADIN produced during the ensiling and storage Of forages is virtually indigestible. However, ADIN present in wet distillers grains did appear to be quite digestible in the total tract. Rumen flow of ADIN was not measured. ADF residue in duodenal samples could not be effectively removed from the Gooch crucibles without damaging the fritted glass filter portion. The material adhered very tanaciously and could not be remove without washing and subsequent ashing of the crucibles. 104 Lignin Flow-Total Tract Total tract flow Of lignin was evaluated utilizing Yb and Cr as markers. Tables 14 and 15 illustrate the lignin flow as a percent of intake. Although there were no significant differences between diets for lignin flow, there does appear to be a trend toward lower lignin recovery in WDG supplemented diets than in the soy and urea diets. , Muntifering et al. (1981) has reported the relationship between dietary source of lignin, lignin recovery and gravimetric analysis methodology. It is reported that utilizing the acid lignin method, (72% sulfuric acid treatment of ADF residue, Goering and Van Soest, 1970), lignin recovery Often exceeds 100% when lignin is forage related (fescue in Muntifering's research). When lignin in the diet is related to seed cost and related materials, lignin digestion is extensive (seed related i.e. corn cobs, cottonseed hulls in Muntifering's research). In our research, corn silage diets supplemented with wet distillers grains and wet distillers grains with urea, contained 33 and 16% of these feedstuffs on a dry matter basis (see Table 5). Despite the high levels Of wet grains dietary lignin levels were equal to or higher than in diets supplemented with soy and urea (refer to Tables 11 and 12). Therefore, wet distillers grains were contributing a signif- icant amount of lignin to the diet. Table 12 Experiment One: 105 Intake of Dietary Constituents Component Diet CS-Soy CS-Urea CS-WDG CS-WDG,Urea SEM Intake/Day1 Dry Matter kg 6.80 7.00 6.60 6.80 Nitrogen g 152.80 147.00 144.50 145.20 a a b c ADIN g 5.50 6.58 14.70 11.60 .55 Organic Matter kg 6.40 6.60 6.20 6.40 ADF kg 1.40 1.50 1.40 1.50 Lignin g 160.50 179.20 185.50 198.60 Ytterbium g 2.61 2.72 2.55 2.56 Chromium g 3.76 4.10 3.37 3.86 1 Means within the same row with different superscripts differ (P<0.05). 106 Table 13 Experiment One: Lignin as a Marker Component Diet CS-Soy CS-Urea CS-UDG CS-WDG,Urea SEM Rumen (Apparent,% intake) .litrogen Loss 52.02 47.62 48.10 42.44 5.16 HAN Plow 44.94 49.13 51-35 54-32 4.90 0H Digestion 50.56 51.78 47.60 43.83 3.13 ADF Digestion 49.16 50.74 47.84 43.83 3.13 Lower Tract (Apparent Digestion) N x of Intake 22.45 27.60 17.25 24.92 5.32 N S of Arrived 45.88 50.42 33.20 36.98 5.36 0H 5 of Intake 26.71 24.58 21.71 29.38 3.90 on i of Arrived 52.17 49.88 44.70 46.50 5.57 ADF 1 of Intake 11.04 9.07 5.62 10.66 2.26 ADP z of Arrived 19.94 15.60 9.60 15.49 3.52 1 Total Tract (Apparent Digestion) a Nitrogen S of Intake 74.48 75.22 65.35 67.36 1.46 s OH I of Intake 77.28 76.36 69.32 68.66 1.98 a ADF ‘ of Intake 60.20 59.81 53.46 54.52 1-39 a ADIN Plow g Intake 79.06 66.76 42.63 54.30 5.00 1 Means within the same row with different superscripts differ (P<0.05) 107 Table 14 Experiment One: Ytterbium as a Marker Component Diet CS-Soy CS-Urea CS-WDG CS-WDG,Urea SEM Rumen (Apparent,% intake) Nitrogen Loss 30.07 26.63 27.62 25.36 7.07 NAN Plow 65.70 68.72 68.42 70.30 7.03 OH Digestion 30.52 32.28 27.22 20.67 6.83 ADP Digestion 27.85 33.77 28.74 28.09 2.66 Lower Tract (Apparent Digestion) x x of Intake 40.44 44.65 36.28 41.44 5.32 N S of Arrived 57.83 60.86 50.12 55.51 3.20 on 1 of Intake 43.26 39.63 41.29 50.40 3.82 OH I of Arrived 62.28 58.52 56.73 63.53 2.06 ADP 1 of Intake 26.18 19.19 22.64 24.63 6.14 ADF 1 of Arrived 36.28 28.97 31.77 34.24 7.58 Total Tract (Apparent Digestion) Nitrogen % of Intake 70.50 71.28 63.90 66.80 3.24 on 1 of Intake 73.79 71.92 68.51 71.08 2.88 AD? 1 of Intake 54.00 52.96 51.38 52.72 5.75 a ab b so ADIN Flow 1 Intake 90.12 78.46 43.16 55.53 8.87 Lignin 110w 1 of Intake 116.18 115.53 106.44 98.42 14.93 1 Means within the same row with different superscripts differ (P<0.05) 108 Table 15 Experiment One: Chromium as a Marker Component Diet CS-Soy CS-Urea CS-VDG CS-VDG,Urea SEM Rumen (Apparent,% intake) Nitrogen Loss 20.52 14.26 14.21 12.61 5.62 NAN flow 73.25 79.66 81.10 82.37 5.65 on Digestion 19.52 19.89 15.12 8.26 4.80 ADF Digestion 15.44 22.53 15.54 12.92 3.81 Lower Tract (Apparent Digestion)1 n S of Intake 48.47 57.85 48.68 53.09 4.70 N Z of Arrived 60.97xy 67.47 57.41y 60.75 2.09 on 1 of Intake 53.00 52.75 55.44 59.75 4.96 on x of Arrived 65.85 65.85 65.28 65.12 1.96 AD? S of Intake 36.92 33.50 35.84 38.54 5.35 AD? 1 of Arrived 43.66 43.23 42.42 44.26 5.51 Total Tract (Apparent Digestion)1 Nitrogen I of Intake 68.98ab 72.11 62.89 65.70 1.78 OH 5 of Intake 72.52x 72.64 70.56 68.00y 1.18 ADF % of Intake 52.36 56.02 51.37 51.46 3.43 ADIN Flow 1 Intake 100.48‘ 80.48 46.04 58.59 6.23 Lignin Flow 1 of Intake 125.40 116.00 110.40 105.20 8.30 1 Heans within rows with different superscripts (x,y) differ (Pi:al tract digestibility of N, OM and ADF as the result of marker choice. However, as discussed earlier, Tables 14 and 15 indicate that there may be a difference in lignin recovery 9‘16 to dietary lignin source. The data indicate that lignin from forages tends to produce small increases in total tract 1ignin recovery based on the acid lignin procedure. In contrast, lignin from seed related products tends to be more digestible resulting in lower lignin recovery, especially in the WDG and WDG-Urea diets. This results in (11011; 112 13ab1e 17 Experiment One: Corn Silage- Soybean Neal Site and Component firker Lignin Yb Cr SEN 1’2 a sh b Eiumen Nitrogen Plow 47.0 69.9 79.5 6.30 flPotal Tract Nitrogen Digestion 74.5 70.5 68.9 3.50 a ab b Elumen 0! Flow 49.4 69.5 80.4 6.20 flfotal Tract OM Digestion 77.3 73.8 72.5 6.80 Iiumen ADF Digestion 49.2 27.8 15.4 7.80 Ifotal Tract ADF Digestion 60.2 54.0 52.4 5.10 N ) Expressed as a S of intake 2!) Means within the same row with different superscripts differ (P<0.05) 113 'Table 18 Experiment One: Corn Silage-Urea .1ite and Component Marker Lignin Yb Cr SEM Rumen Nitrogen Plowhz 52.4“ 73.4ab 85.7b 8.20 flflotal Tract Nitrogen Digestion 75.2 71.3 72.1 2.50 a ab Eiumen ON Plow 48.2 67.6 80.1 8.20 fifotal Tract OM Digestion 76.4 71.9 72.6 2.30 Iiumen ADF Digestion 50.7 33.8 22.5 7.80 130ta1 Tract ADF Digestion 59.8 53.0 56.0 4.10 1 5 Expressed as a 1 of intake 22) Means within the same row with different superscripts differ (P<0.05) 114 Table 19 Experiment One: Corn Silage-Net Distillers Grains '—ite and Component iarker Lignin Ib Cr SEN 1'2 a sh b Rumen Nitrogen Plow 51.9 72.4 85.8 5.59 Total Tract Nitrogen Digestion 67.4 66.8 65.7 1.50 a sh b IBumen OH Flow 52.4 72.8 84.9 5.97 'Iotal Tract OM Digestion 69.3 68.5 70.6 3.85 a ab b iBumen ADP Digestion 47.7 28.7 15.5 6.40 {total Tract ADF Digestion 53.5 51.4 51.4 5.70 1) Expressed as a % of intake 2) Means within the same row with different (P<0.05) superscripts differ 115 Table 20 Experiment One: Corn Silage-Vet Distillers Grains-Urea -Tte and Component Marker Lignin Yb Cr SEN 1'2 a sh b Rumen Nitrogen Plow 57.6 74.6 87.4 5.60 Total Tract Nitrogen Digestion 67.4 66.8 65.7 1.50 a ab b Rumen OH Plow 60.7 79.3 91.7 5.30 Total Tract 0M Digestion 68.7 71.1 68.0 2.20 a ab b Rumen ADP Digestion 43.8 28.1 12.9 6.50 Total Tract ADF Digestion 54.5 52.7 51.5 3.30 1) Expressed as a S of intake 2) Means within the same row with different (P<0.05) superscripts differ 116 significant) slightly higher, digestion coefficients for soy and urea diets compared to the WDG and WDG-Urea diets. Evaluation of Markers: Selection Based on Magnitude of the SEM The magnitude of the standard error of the mean (SEM) is an important component of the data set. The SEM value provides information regarding the size of the error or residual components in the analysis of variance. This term indicates, based on its magnitude, the efficiency of the experimental design, blocking and indicates the variation inherent in the sample population. The lower the SEM value, the easier it is to show significant .differences between means. Therefore, experimental designs which reduce the error or residual term, and consequently the SEM, are preferred to other designs. In digests passage studies, it is a common practice to utilize Latin square designs. These designs partition variation into treatment effects, the contribution of nuisance variables such as periods of time and animals and error or the residual variation. 'Table 21 summarizes the SEM values for estimates of flow and digestibility of major dietary constituents based on lignin, Yb and Cr as markers. In general, SEM values are lowest for lignin, followed by Cr and highest for Yb. The SEM values are similar to values found in the literature for simi- lar designs. Crickenberger et sl.(1979). utilizing lignin as a marker, reports an SEM value of 9.2 for nitrogen passage, 117 % of N intake. Galyesn et al.(1979) report a.SEM for rumen 0M digestion of 3.2 with lignin as a marker. Waller et a1. (1980) reported dry matter and nitrogen total tract digestibility, utilizing sheep, estimates resulting in SEM [values of 1.09 and 1.17 respectively. These values are somewhat less, but very similar to values for total tract digestion of organic matter and nitrogen based on lignin and Cr. These values are lower than those reported for Yb. The data would indicate, based on the magnitude of the SEM values, that lignin is an excellent marker for digesiton and flow research. However, as discussed earlier, there are problems related to differential recovery of lignin. These problems with lignin recovery are related to the dietary source of lignin (forage or seed related) and the somewhat qualitative nature of the gravimetric method of analysis. The CrEDTA complex is an excellent marker based on its low SEM values. However, it is well documented there is substantial absorption of Cr from the digestive tract (Goodall and Kay, 1973). It also tends to overestimate flow of dietary constituents, particularly acid detergent fiber, from the rumen. Consequently, suspicious estimates of lower tract ADF digestion are common when Cr(EDTA) is used as a marker. Despite high SEM values for Yb estimates of digestion and flow, there are significant reasons it is superior to lignin and Or as a marker. Yb is not absorbed from the digestive tract as CrEDTA and analysis is much more sensitive than the gravimetric lignin procedure. A low SEM value should 118 not be the sole criteria utilized for marker selection. The nonabsorption and accurate quantification criteria are of equal importance. 119 Table 21 Experiment One: Comparison of Marker SEM Estimates Component Rumen (Apparent, % Intake) N Loss NAN Flow OM Digestion ADF Digestion Lower Tract (Apparent Digestion) N, of Intake N, of N Arriving OM, of Intake OR, of OM Arriving ADF, of Intake ADF, of ADP Arriving Total Tract (Apparent Digestion) Nitrogen Organic Matter ADF ADIN Flow Marker Lignin Yb ............. SEM 5.16 7.07 4.90 7.03 4.40 6.83 3.13 2.66 5032 5.32 5036 3'20 3090 3'82 5.57 2.06 2.26 6.14 3.52 7.58 1046 3‘24 1.39 2.88 1.98 5.75 5.00 8.87 5.62 5.65 4.80 3.81 4.70 2.09 4.96 1.96 ~35 5.51 1.78 1.18 3-43 6.23 RESULTS AND DISCUSSION Experiment Two The Effect of Soybean and Corn Gluten Meal Supplementa- tion Upon Site and Extent of Digestion 0f Corn Silage Based Diets by Steers. Rumen Disappearance and Flow Based on marker estimates (Tables 22, 23, 24, 25) there were no significant effects of protein source upon apparent rumen disappearance of nitrogen (N), organic matter (OM), acid detergent fiber (ADF) or flow of non-ammonia nitrogen (NAN) to the lower tract. Calculations of abomasal flow based on PEG infusion is shown in Table 26. Based on the dilution of PEG and subsequent flow calculations, diets supplemented with corn gluten meal exhibited greater total dietary nitrogen flow to the lower tract (P< 0.10). Estimates of flow were 102.83 and 126.02 percent of dietary nitrogen intake for soybean and corn gluten meal respectively. In these diets, both soybean (SOY) and corn gluten meal (CGM) proteins supplied approximately 38.5% of the dietary N. Stern et al. (1983) report CGM bypass of 57%; other available estimates include 38% (Waller, 1978), 46% at 1.2 x maintain- ance, 61% at 1.6 x maintenance (Zinn et al., 1981). 120 121 Estimates of soybean meal bypass include 20% (Krop et al., 1976) and 35% (Stern and Sstter, 1982). Based upon soybean protein bypass of 20%, CGM bypass of 61%, 38.5% of dietary N from these sources, assuming equal corn silage N degradability, the CGM diet would theoretically have 15.8 % higher flow of dietary N than SOY diets. Assuming literature values of bypass being equally valid, using SOY bypass of 35% and CGM of 38%, CGM diets would have 1.5% more flow of dietary N, as a percent of intake. Estimates of dietary N flow are 1.5, 7 and 23% higher for the CGM diets based on lignin, chromium and PEG infusion as markers respectively. Estimates of dietary N flow based on Ytterbium and lanthanum, respectively, were 1 and 9% less for CGM diets compared to SOY diets. Bypass of nitrogen from soybean meal and corn gluten meal, although not directly measured, appear to be very similar. This would be in agreement with literature values for bypass of nitrogen. The differences between the two protein sources bypass potential is essentially to small to detect with any degree of statistical significance. Data reported by Loerch et al. (1983) illustrate how decreased rumen pH will reduce the degradation rate of soybean meal to a much greater extent than corn gluten meal. These observations would lend more evidence to support finding of very little difference between N flow to the lower tract when diets contain soybean meal or corn gluten meal. Rumen disappearance of 0M and ADF showed no trends toward differences between the diets. This observation is 122 not surprising given the very similar amount of nitrogen available in the rumen, regardless of protein source, based on the flow data. Lower Tract Digestion The potential contribution of lower tract digestion to total digestion of dietary constituents is illustrated in tables 22, 23, 24 and 25. 'There were no significant differences in digestibility of N, 0M or ADF as a % of intake or as a % of arrived. Digestion of ADF arriving at the lower tract averaged 10% when diets and marker estimates were pooled together. These data represent lower estimates of lower tract ADF digestion potential than observed in experiment two. Putman and Davis (1965) and Dixon and Nolan (1982) have illustrated the importance of the lower tract for digestion and absorption of both fermentation endproducts from ADF and OM fermentation and nitrogenous constituents. The lower tract is plays a significant role in the digestive processes of the ruminant. Total Tract Digestion The total tract digestion of N is reported in Tables 22- 25. Apparent nitrogen digestion was 2.9, 4.7, 6.4 and 3.6% greater for the CGM suplemented diets in relation to the SOY supplemented diets based on lignin, La, Yb and Cr as markers 123 respectively. Differences were significant based on Yb and Cr markers (P< 0.05). Klopfenstein et a1. (1976) reported lower total tract N digestion for diets supplemented with corn distillers products as compared to SOY or urea supplemented diets. Data in experiment one illustrated these differences as well, where diets supplemented with wet distillers grains had lower total tract nitrogen digestion than diets containing soybean meal. Reiners and Watson (1975) have evaluated the variation in nutrient content of the high protein (60%) corn gluten meal products from wet milling of corn. DeMuelenaere et a1. (1967) reported virtually 100% of the lysine in CGM was available in their studies with rats. Misra and Potter, (1970) report 92 to 96% total tract digestion of nitrogen in turkeys fed CGM. CGM proteins are very available to monogastric species and as observed in this experiment, very available to the ruminant. The nitrogen availability appears to be equal to or slightly greater than soybean meal by our evaluation. The total tract digestion of 0M did not differ (P) 0.10) due to source of supplemental protein. There is a trend for a somewhat greater total tract OM digestion in diets containing CGM. The trend was evident regardless of the marker selected. Total tract ADF digestion did not differ significantly (P>O.10) with either protein supplement. A trend toward slightly greater total tract ADF digestion in diets supplemented with CGM was evident regardless of digests 124 Table 22 Experiment Two: Lignin as a Marker Component Rumen (Apparent,% intake) Nitrogen Flow NAN Plow OM Digestion ADF Digestion Lower Tract (Apparent Digestion) N S of Intake N S of Arrived OH % of Intake OM % of Arrived ADF S of Intake ADF % of Arrived Total Tract (Apparent Digestion) Nitrogen % of Intake OH % of Intake ADP % of Intake CS-Soy 88.58 85.02 56.12 52.16 58.11 65.60 14-73 35-57 -1.77 -3.70 69-53 70.85 50-39 CS-CGM 90.03 82.51 57.63 52.97 61.46 68.27 14.33 33.82 .18 .38 71.43 72.30 54.08 SEM 4.38 6.53 1090 ~30 4.52 2.07 1.62 3.71 .65 1.34 .23 .18 .45 125 Table 23 Experiment Two: Ytterbium as a Marker Component Rumen (Apparent,% intake) Nitrogen Plow NAN Plow OM Digestion ADP Digestion Lignin Flow Lower Tract (Apparent Digestion) N % of Intake N % of Arrived OM % of Intake OM % of Arrived ADF % of Intake ADF % of Arrived Total Tract (Apparent Digestion) Nitrogen % of Intake on 5 of Intake ADF % of Intake Lignin Plow CS-Soy CS-CGM SEM 111.78 110.90 4.12 108.23 103.22 6.19 41.62 46.64 1.96 38.15 41.55 1.62 127.70 128.40 3.65 78.86 81.17 4.16 70.55 72.82 1.13 26.86 23.76 1.76 46.00 44.56 1.23 9.12 9.24 .73 14076 15082 '19 1 67008 70026 914 68.48 70.41 .18 47.28 50.80 .89 108.38 107.62 .58 1 Means within the same row with different superscripts differ (P<0.05) 126 Table 24 Experiment Two: Lanthanum as a Marker Component CS-Soy Rumen (Apparent,% intake) Nitrogen Flow 99-50 NAN Flow 95-96 OM Digestion 49.12 ADF Digestion 45.90 Lignin Plow 113.02 Lower Tract (Apparent Digestion) N % of Intake 67.50 I z of Arrived 67.40 on 1 of Intake 20.11 OM % of Arrived 38.26 ADF S of Intake 2.84 ADF % of Arrived 6.18 Total Tract (Apparent Digestion) Nitrogen % of Intake 68.00 OM X of Intake . 69.23 ADF % of Intake 48.57 Lignin Flow 107.30 CS-CGM 90.30 82.66 55-25 51.83 106.14 62.69 69.27 15.66 36.99 2.42 5-33 72.39 73.44 54.25 100.82 SEM 2.07 4.14 2.70 2.21 5.30 5.40 3076 4.63 8.05 4.00 5-73 3-33 3.66 6.09 12.42 127 Table 25 Experiment Two: Chromium as a Marker Component Diet CS-Soy CS-CGM SEM Rumen (Apparent,% intake) nitrogen Plow 117.70' 124.87 9.40 NAN Flow 114.15 117.13 11.40 OM Digestion 37.96 39.00 4.96 ADF Digestion 34.72 39.00 .89 Lignin Flow 134.27 142.89 1.26 Lower Tract (Apparent Digestion) N z of Intake 85.28 94.86 9.30 N % of Arrived 72.45 75.97 1.42 OM % of Intake 30.64 31.49 4.36 0H % of Arrived 49.38 51.60 3.18 ADF 1 of Intake 12.69 12.08 1.17 ADF % of Arrived 19.45 19.80 .83 1 Total Tract (Apparent Digestion) Nitrogen % of Intake OM % of Intake ADF % of Intake Lignin Plow 67.58 68.60 47.42 107.71 69-99 70.48 51.08 107.03 .12 .60 .28 .42 1 Means within the same with different superscripts differ (P<0.05) 128 Table 26 Experiment Two: PEG Infusion Component Diet CS-Soy CS-CGM SEM Rumen (Apparent,% intake)1 Nitrogen Plow 102.83x 126.02y 1.46 NAN Flow 99.30 118.37 3.52 OM Digestion 35.05 30.23 .84 ADP Digestion 40.38 31.94 3.71 Lignin Plow 123.83 149.75 11.45 Yb Flow 94.88 110.73 3.99 La Flow 107.61a 145.01b .99 Cr Plow 87.70 99.04 6.34 1 Means within the same row with different superscripts (P0m .mo< .z .20 .mcnouuma zo_a m on:m_a US.» 02.4m14m ...-m see new new. .10. sac saw a: can cam. ago. new 1 1 4 d :1 a q q u q q «I A J. 0 j v . a ._ 0 0.0000... 1 o 0 e e. / see .. h o v e I III” 00 \- I‘ III/ 1 1 Q J] 0000.00... A a t o. ... . N. i n- uoom macro .moz cmon>0m .4 .n> .z .mcnmuuma 30_a m ousm_m u}... 02.4324m new set new so“. s...o. sac saw eat saw 1...»... sec. can n V H A . a, . a r I, a! . o as "4 lllte. “ I ’H’sr eeoe‘efirss I e. h x .7411. 40.11. / track...“ 74 \\ or /. r «l_\\4~W . hes-H\\: 1‘4 .. o so A \ \ I \III m1\ ‘ I \ rseess/e/I \\ \\\se»-e/.I \\eereeee§e¢h”\\ \4\ L m ’\ est |||x\ so. se’l \ as 4. \ 4 .” so 0 I \ so / es s. I \ os\ \ sssfle x N / e O. on \ I \ gyro-e \ I \ I \ / \ .. .. ’\ a 4 L N. . n. n— -4- a» not»... . c. 2 11.1... 304m>4.0m .nu .a> .a» .mcnmuuma zo_a o. on:m_a u 2 . a. o z . .. a .2 a m “1.1m a... new low. e...o. sac saw ....v can sau. ...-o. ...-c C I d d q I d c 1 q 1 q A w . g c o e.. . n F. eseeeee a. .:...: .. . f. 0.0... .000 u “CFODOOODI0050Ieeeeee ee 1 R see e..e so .... “6000‘s.". —. .IllIIIhlqebLJhll-ll'... sense. .0. \ 1 Q A; v . so see...- a? secrete a... Ilell .238 >om 4.. 304... >4.om .mm .nh .4 .mcuouuma zo_a —— onsm_a m2; nines; low so? low ecu. 1.0. can (so as? saw (an. 1.0. sea I d 1 u q u d d d d 4 d 0 1V c . 4 v . 4’ +0 .I \ \ “a, \elll. . o 0 \ \ .:.:...r . a o m o. .. a N. .. n- . ... ...-.:.: able..- . c. 41.1 304..— xido .:...: go 5 «has >om 150 muo_o .moz cmoa>0m .4 .ao< manouuma 30.; ~_ on:m_m mo...» 02.4m24m new set new new. and. saw saw (at can cam. coo. sea a q q q 1 u r H A . W .o 0’ 5.0-see m m. In". . a W fl 4. 1 h 4. n— -4- L O < .008 so. .M“‘\e Jr MXW, \% . m Jw .LW. Mw \fim ; o. A. mm x” 3%. [KM Mflm ... .. .... . N. . n. . e. 204... >343 4.72:. no 3 mhm.o >om 15.1 muo_o .moz sous—o cLOu .ao< .z .zo mcaouuma 30_a m_ oasm_m ma: 623.3; (so see sea son. (.0. sac saw (at law saw. too. can . . 4! . . . . r 1 9‘ d I d1 e- 00-. l__ V t a a O. 1.. k a. ...—0‘ scenes. . n. z DUI-l--. . v. 201.1. 304...»...48 442.0»...0 4. «ZS .:Bz 2:23.330 152 new 1 was .000 \\a as 9000: 60.090960 pesos \ \ " \ 'n. I“.". a» n. nflfluanflflu z '9‘-‘ muo_o .moz nous—c cnou .n> .4 .z menouuma zo_a a. on:m_m warp 02.4a3 mcnouuma zo_a m. on:m_a 153 ma... oz...a_z4.m .zo .ak .mcnmuuma 3o_a m_ onsm_n u}; 02.4aaam. ...-u ...-v ...-u com. .:...: ego sac lav .2; saw. ...-0. ...-o 1 1 I I J u I d s s I I o 1’ . . a v a .. a n n m . a. .. o a m . o. ... .- .. N. . n. :0 scene: coo-seed ....o 304... >.:do 445.0» no I SUMMARY Data in this thesis illustrate factors involved with digestive tract processing of dietary constituents and the importance of sound evaluation of data based on typical markers available to nutritionists. The following conclu- sions can be drawn from the data. 1. Estimates of apparent rumen digestibility of dietary constituents can be affected by the marker selected. Lignin may commonly be too high in duodenal samples due to high ADF levels. This results in high estimates of digestibility of dietary constituents in the rumen. Yb recovery may not be far from 100% with equal sample 'compositing; however, elevated ADF in samples contributes to high OM levels in duodenal samples and as a result, apparent ruminal digeston estimates of ADF and 0M are too low. Due to variable Cr absorption from the digestive tract, its role as a suitable marker is very suspect. Variability of La content in duodenal samples results in high SEM values when it is used as a marker and duodenal recovery is usually high based upon PEG infusion data. 2. Estimates of total tract digestibility of dietary constituents do not vary significantly due to the markers selected. Fecal Cr levels, corrected for 5% total tract 156 157 absorption, provide adequate total tract digestibility estimates. In corn silage based diets, total tract recovery of lignin averaged, across all other digests marker estimates, diets and studies, 110% of intake, resulting in slightly elevated total tract digestibility estimates. PEG based flow patterns of digests passing the duodenal cannula in steers indicates the presence of a phasic pattern. The pattern varies with protein source and dietary constituent of interest. The circadian rhythms of digests flow present may be related to rumination and or water intake patterns. The patterns of digests flow may provide the potential to improve the efficiency of production of meat,' milk and fiber.~ Feeding high quality dietary constituents prior to peak flow periods may .maximize bypass and improve efficiency. Further analysis of flow patterns may prove beneficial for improving marker studies. Based on our studies, Yb appears to represent the best marker for passage studies as compared to CrEDTA, lignin or lanthanum. It is easily quantified by specific and sensitive means. Flow patterns are not excessively variable as those for CrEDTA, lignin, or lanthanum. In general, standard errors of digestibility means are no larger and often smaller when Yb is used as a marker as compared to the other markers studied. Ytterbium has been shown to not be absorbed or toxic to gut tissues or to microbial flora to any great extent. 6. 158 Ruminal and total tract digestion of corn silage diets supplemented with soybean, corn gluten meal and urea indicate equal nitrogen, organic matter and ADF digestion regardless of protein source. Nitrogen contained in wet distillers grains is not as available ruminally as other protein sources evaluated. Nor is total tract digestion as complete as with the other protein sources evaluated. Addition of urea to diets containing wet distillers grains resulted in a shift in site of digestion of a substantial portion of the organic matter, ADF and nitrogen to the lower tract. This may be due to more rapid rumen turnover of digests due to urea addition stimulating water intake as a result of greater urinary output as described by Utley et al. (1970). APPENDIX 159 Table 30 Experiment Two: Whole Digesta Plow, % of Total Daily Soybean Meal Corn Gluten Meal Animals Periods 1 2 1 813 819 820 550 813 819 820 550 Sampling Times 12 AM 6.66 7.80 7.80 8.10 7.80 8.00 . 8.96 8.36 2 AM 7.60 6.72 6.92 9.44 7.92 7.76 7.62 8.26 4 AM 8.80 7.26 8.10 8.54 8.30 8.38 14.82 8.88 6 AM 6.06 7.26 6.84 8.76 7.48 7.72 7.36 9.08 8 AM 8.00 7.20 7.68 8.68 7.28 7.78 9.34 7.58 10 AM 10.18 8.58 7.55 7.48 7.68 7.10 7.28 7.68 12 PM 11.22 9.70 8.80 9.28 10.38 8.68 7.3 8.56 2 PM 8.92 8.92 9.92 7.92 8.02 12.40 8.34 9.94 4 PM 9.46 13.28 9.76 6.48 8.84 8.32 6.68 7.26 6 PM 7.62 7.50 8.68 .8.74 8.98 8.40 8.04 7.56 8 PM 8.04 8.22 8.62 7.96 7.48 7.44 7.28 8.66 10 PM 7.44 7.74 9.08 8.68 9.72 8.02 6.98 8.18 _Total, Kg/day: 86.56 88.50 104.60 105.80 89.77 86.20 106.66 85.63 160 Table 31 Experiment Two: Dry Matter Flow, % of Total Daily Soybean Meal Corn Gluten Meal Animals Periods 1 2 2 1 813 819 820 550 813 819 820 550 Sampling Times 12 AM ' 5.66 6.22 8.34 9.60 7.68 7.54 8.84 8.82 2 AM 7.26 5.64 7.00 9.86 9.56 6.24 7.34 8.02 4 AM 10.92 8.28 6.88 7.40 11.44 7.36 14.16 7.22 6 AM 4.68 5.16 6.52 8.58 7.52 8.98 6.16 10.24 8 AM 8.26 6.46 5.90 9.08 5.52 7.44 8.82 7.12 10 AM 9.30 8.40 8.90 7.42 7.52 6.40 7.04 7.74 12 PM 10.34 9.61 6.34 9.02 18.04 6.60 6.32 9.38 2 PM 7.88 9.00 11.46 8.02 7.42 11.52 8.22 8.48 4 PM 10.06 14.70 10.42 5.98 8.30 7.72 8.26 6.48 6 PM 7.86 7.00 10.04 9.32 8.22 9.5 8.66 7.72 8 PM 8.84 10.96 9.68 7.34 8.92 10.88 9.74 9.30 10 PM 8.92 8.56 8.28 8.46 9.56 9.78 8.34 9.48 Total, Kg/day: 4.60 5.10 4.70 5.16 5.50 5.95 4.85 5.10 Intake/day, Kg: 7.15 '7.06 6.98 7.00 6.93 6.90 7.02 7.00 Table 161 32 Experiment Two: Nitrogen Plow, % of Total Daily Sampling Times 10 Total e/dar Intake, g/day: flow, Periods Soybean Meal Corn Gluten Meal ' Animals W 1 813 819 820 550 813 819 820 550 7.06 7.86 9.60 9.48 8.02 9.1 .50 10.08 7.90 6.70 7.78 11.28 10.32 7.44 7.88 8.98 8.76 7.48 7.46 8.86 11.30 8.34 14.38 7.42 4.60 6.30 6.90 8.16 7.70 8.32 5.98 11.90 7.36 6.90 6.36 8.88. 5.44 7.86 9.12 6.70 8.72 8.20 7.92 7.22 7.04 5.88 6.38 6.60 9.44 8.62 6.86 7.68 19.06 6.32 6.04 8.06 8.66 8.84 19.80 7.66 7.70 11.48 7.72 7.44 10.28 11.72 10.64 5.44 8.40 7.18 5.80 5.70 8.24 7.12 19.20 8.22 8.12 9.98 8.12 7.94 9.28 9-34 7.08 9.62 8.46 9.06 9.12 10.04 140.36 155.42 149.97 166.53 184.82 198.77 172.34 165.65 145.72 153.38 149.54 146.84 145.70 147.38 143.31 135.68 162 Table 33 Experiment Two: Organic Matter Flow, 1 of Total Daily Soybean Meal Corn Gluten Meal Animals Periods 1 2 2 1 813 819 820 550 813 819 820 550 Sampling Times 12 AM 5.44 6.08 8.50 9.82 7.72 7.58 8.58 8.90 2 AM 7.14 5.58 7.02 9.84 9.90 5.98 7.22 7.82 4 AM 11.66 8.64 6.66 7.24 11.94 7.30 15.10 6.88 6 AM 4.56 4.96 6.58 8.54 7.54 8.22 6.02 10.36 8 AM 8.14 6.50 5.64 9.16 5.32 7.82 8.68 7.12 10 AM 8.34 8.58 9.16 7.42 7.26 6.10 7.08 7.82 12 PM 10.26 9.62 6.00 9.08 7.88 6.80 6.28 9.54 2 PM 7.67 8.82 11.68 8.02 7.38 11.44 8.00 8.36 4 PM 10.06 14.68 10.60 5.96 8.44 7.68 6.26 6.30 6 PM 7.84 6.96 10.18 9.44 8.10 9.38 8.32 7.83 8 PM 8.86 10.96 9.86 7.18 8.98 11.82 9.7 9.44 10 PM 9.00 8.66 8.16 8.30 9.56 9.90 8.40 9.60 Total Plow, kg/day : 3.94 4.37 3.86 4.34 4.71 5.00 4.15 4.46 Intake, g/day clulculated : .6070 6060 6055 6057 6053 6053 6063 6058 163 Table 34 Experiment Two: ADP Flow, 1 of Total Daily Soybean Meal Corn Gluten Meal Animals Periods 1 2 2 1 813 819 820 550 813 819- 820 550 Sampling Times 12 AM 3.64 4.48 8.50 10.32 7.02 7.08 8.46 10.64 2 AM 6.66 4.54 6.68 9.34 10.36 5.36 6.16 7.48 4 AM 14.26 9.28 5.30 6.80 13.48 5.92 15.16 5.42 6 AM 2.06 3.52 5.36 8.46 7.72 10.78 5.20 10.64 8 AM 8.32 6.14 3.92 10.16 3.80 6.54 8.90 6.60 10 AM 11.76 9.60 10.96 7.22 8.10 6.64 6.72 7.66 12 PM 12.30 11.20 3.06 10.36 7.00 6.14 5.24 9.58 2 PM 6.26 9.52 15.62 7.56 6.60 12.68 9.18 7.98 4 PM 9.82 15.88 12.78 4.42 9.64 7.54 .38 6.36 6 PM 7.60 6.14 11.36 9.92 5.90 10.72 8.48 9.04 8 PM 7.86 11.68 10.04 6.22 11.00 10.04 11.84 9.3 10 PM 9.48 8.02 6.44 8.22 9.34 10.56 7.56 10.20 Total Plow, kg/day .98 1.09 .79 .86 1.14 1.47 .98 1.02 Intake, kg 1.54 1.64 1.59 1.48 1.85 1.56 1.68 1.75 1611 Table 35 Experiment Two: Lignin Plow, % of Total Daily Soybean Meal Corn Gluten Meal Animals Periods 1 2 2 1 813 819 820 550 813 819 820 550 Sampling Times 12 AM 2.70 5.22 8.48 11.56 7.06 6.14 8.16 9.80 2 AM 6.04 5.82 6.98 9.88 10.24 4.72 6.3 8.32 4 AM 12.84 8.98 5.22 6.72 10.70 6.60 15.44 5.96 6 AM 3.14 3.60 5.60 8.40 8.08 14.16 6.18 11.92 8 AM 7.98 6.28 4.02 9.36 4.60 4.42 8.02 6.20 10 AM 11.68 10.84 10.34 7.40 7.82 7.56 6.44 7.68 12 PM 11.84 11.00 3.06 9.38 8.02 4.36 5.38 9.08 2 PM 7.00 9.80 14.20 7.90 4.42 12.12 9.92 7.48 4 PM 9.36 15.52 13.18 4.78 10.08 7.80 6.18 6.16 6 PM 7.86 6.88 11.72 8.92 6.46 12.16 9.02 9.16 8 PM 9.34 8.70 9.94 6.66 13.54 9.94 11.48 8.52 10 PM 10.22 7.38 6.26 9.08 9.96 10.04 7.44 9.70 Total Plow, g/day : 235.29 248.52 171.75 206.73 192.55 347.49 244.94 268.01 Intake, 3 Calculated : 205.88 202.00 157.75 147.01 153.80 152.50 200.92 213.75 165 Table 36 Experiment Two: Yb Plow, % of Total Daily Soybean Meal Corn Gluten Meal Animals Periods 1 2 ' 2 1 813 819 820 550 813 '819 820 550 Sampling Times 12 AM 6.14 7.56 9.00 9.88 8.12 8.02 9.02 8.26 2 AM 7.80 6.26 7.34 12.48 9.86 6.84 7.76 8.04 4 AM 8.16 7.22 6.18 8.44 8.54 9.36 13.72 8.50 6 AM 3.82 5.44 6.30 8.18 8.04 9.08 6.48 9.34 8 AM 8.60 6.34 5.66 8.28 4.28 7.72 9.10 6.32 10 AM 8.40 8.17 6.66 8.74 8.74 6.00 6.74 7.3 12 PM 9.90 8.48 5.36 7.64 9.12 6.74 6.68 8.16 2 PM 8.44 9.40 8.98 8.01 7.92 11.46 7.80 8.26 6 PM 8.28 7.62 11.50 7.88 7.76 9.52 9.24 9.06 8 PM 9.28 11.74 11.54 6.98 9.28 9.36 8.80 9.66 10 PM 9.66 8.92 11.26 7.58 10.16 8.18 9.30 10.16 Total Plow, g/day : 4.92 6.03 5.3 5.60 5.66 7.04 6.42 5.96 Intake, g/day Calculated : 5.98 5.68 4.95 6.79 5.25 5.61 6.20 5.62 166 Table 37 Experiment Two: Cr Flow, 1 of Total Daily Soybean Meal Corn Gluten Meal - Animals Periods 1 2 _ 2 1 813 819 820 550 813 819 820 550 Sampling Times 12 AM 6.10 8.00 7.44 10.68 10.78 7.86 9.80 8.24 2 AM 6.24 5.58 5.96 12.22 9.52 7.10 7.32 8.90 4 AM 5.32 5.96 6.10 8.52 8.28 8.28 14.26 9.50 6 AM 3.54 5.18 5.08 8.14 6.16 7.28 5.78 8.94 8 AM 5.46 4.06 5.10 6.58 3.74 5.80 8.08 5.88 10 AM 9.26 6.94 5.92 7.00 7.72 6.20 6.18 5.60 12 PM 12.78 10.50 8.04 6.80 7.26 7.64 7.02 5.94 2 PM 10.70 10.24 10.48 8.60 8.7 13.12 8.48 7.84 4 PM 11.94 13.20 10.18 6.40 8.30 9.26 6.28 6.80 6 PM 9.06 9.48 11.76 8.02 6.78 8.80 9.74 8.86 8 PM 10.22 10.82 11.68 7.56 9.86 9.62 8.46 11.38 10 PM 9.28 10.04 12.24 9.50 11.86 8.96 8.48 12.08 Total Plow, g/day : 1.74 2.02 2.04 2.20 2.10 2.64 2.16 2.14 Intake, g/day Calculated . : 2.28 2.25 2.02 2.67 2.18 2.22 2.53 2.24 167 Table 38 Experiment Two: La Flow. 1 of Total Daily Soybean Meal Corn Gluten Meal Animals Periods 1 2 2 1 813 819 820 550 813 819 820 550 Sampling Times 12 AM 5.40 6.28 9.56 12.94 8.14 8.16 9.08 10.08 2 AM 6.90 6.30 7.82 11.62 10.66 6.82 7.24 7.88 4 AM 11.64 11.06 6.90 10.08 14.02 8.82 19.62 5.40 6 AM 3.22 4.84 6.06 10.09 7.70 10.14 6.12 15.06 8 AM 6.54 6.50 5.00 9.36 3.68 7.88 9.34 6.32 10 AM 6.74 7.96 6.22 8.08 7.52 6.60 6.38 8.88 12 PM 8.86 11.92 4.46 8.28 6.92 5.60 5.28 8.50 2 PM 7.80 7.66 8.50 6.62 6.40 10.30 6.68 7.56 4 PM 12.02 10.78 10.42 4.44 7.70 5.88 4.90 4.98 6 PM 9.78 5.70 12.76 7.96 6.48 8.66 7.76 7.40 8 PM 10.38 9.80 11.54 .5.08 9.44 10.76 9.64 7.68 10 PM 10.68 11.20 11.76 6.32 11.32 10.38 7.96 10.24 Total Plow, s/day 4.57 5.32 5.04 5.50 2.71 3.78 2.95 3.35 Intake. g/day Calculated 4.25 4.01 5.25 5.83 2.32 2.76 1.80 2.07 168 Abbreviations Code for Tables_39, 40 and 41 ADF ADIN WDG-U DAN DM DMI Lgn 0M Yb animal numbers acid detergent fiber, Z, dry matter acid detergent insoluble nitrogen, mg/gr dry matter crude protein, %, dry matter -ppm chromium, dry matter diets soybean meal urea wet distillers grains wet distillers grains and urea duodenal ammonia nitrogen, Z of total nitrogen dry matter of original material dry matter intake lignin, %, dry matter organic matter, %, dry matter experimental periods ppm ytterbium, dry basis 169 Table 39 Peed Analysis Data: Experiment One A P D DMI CP 0M ADP ADIN Lgn Yb Cr 788 1 U 15.76 12.64 94.99 23.67 .85 2.81 340.8 573.0 7879 1 11111: 14.68 13.46 94.89 19.94 1.94 2.72 335.92 489.0 790 1 WDG-U 15.17 13.08 94.89 22.24 1.08 3.14 335.84 553.4 791 1 8 14.55 13.25 94.45 20.34 .74 2.55 307.2 500.4 788 2 3 16.26 13.85 94.41 19.66 .79 2.20 312.7 538.5 789 2 UDG-U 15.7 13.18 94.45 23.80 1.13 2.84 327.1 528.2 790 2 WDG 15.20 13.47 94.90 22.64 1.38 3.10 307.1 474.9 791 2 U 16.24 12.34 94.57 22.52 .82 2.57 362.5 540.0 788 3 WDG 14.27 13.61 94.46 21.65 2.77 2.78 392.0 652.4 789 3 3 15.14 13.42 93.51 21.58 .93 2.15 410.4 604.7 790 3 U 15.09 ,13.54 94.10 22.92 1.09 2.65 347.0 596.9 791 3 WDG—U 14.70 13.32 94.92 22.20 2.77 2.86 337.9 595.5 788 4 UDG-U 14.35 13.82 94.66 21.72 1.92 3.37 369.1 595.3 789 4 U 14.29 13.99 93.88 20.78 .90 2.19 363.8 639.4 790 4- S 14.31 15.64 93.39 20.36 .76 2.53 365.7 571.3 791 <4 WDG 13.88 14.18 94.92 21.05 2.82 2.58 ' 406.6 425.7 170 Table 40 Duodenal Analysis Data: Experiment One 4 p 0 us or 043 on 407 Lgn Yb 0: 788 1 u 7.18 11.25 2.86 84.98 19.07 4.45 429.5 399.3 789 1 700 6.16 14.42 3.51 85.02 20.65 5.41 506.4 456.5 790 1 WDG-U 7.96 11.39 3.90 83.68 21.60 44.26 418.4 437.1 791 1 s 6.51 12.18 5.05 84.80 23.05 5.00 468.8 459.3 788 2 s 6.17- 12.40 7.64 85.94 15.08 3.52 398.4 542.8 789 2 700.0 4.90 13.25 6.79 84.18 23.15 6.31 444.8 609.3 790 2 700 4.89 10.55 8.53 83.24 21.20 4.40 420.8 608.8 791 2 u 5.68 13.69 6.71 85.23 18.3 4.19 427.2 632.3 788 3 700 5.59 13.35 5.90 84.84 18.84 5.17 424.0 712.4 789 3 s 4.69 13.62 4.36 83.36 21.38 5.45 499.2 872.6 790 3 0 4.95 12.79 8.31 84.16 19.71 4.76 445.2 850.5 791 3 900-0 5.52 10.98 8.00 86.13 22.44 4.98 421.6 744.9 788 4 1100-11 7.65 10.65 4.87 88.45 11.68 2.94 320.8 531.7 789 14 0 4.26 12.65 7.53 81.32 22.13 6.06 595.6 1057.4 790 14 s 6.12 11.67 7.63 85.11 18.14 3.85 448.4 727.0 791 .4 900. 6.38 10.93 5.83 87.69 15.52 4.46 443.8 423.2 Fecal Analysis Data: Experiment One 171 Table 41 A P 0 or on ADF ADIN Lgn Tb Cr 788 1 U 12.85 87.25 34.19 2.74 10.49 1182.8 1375.5 \789 1 vnc 16.40 87.32 29.37 3.48 9.81 991.2 1094.1 790 1 700.0 12.96 86.80 32.92 2.55 9.90 818.4 1235.8 791 1 8 15.38 86.51 32.41 2.84 9.92 1230.8 1238.0 788 2 8 14.04 88.90 33.35 2.27 9.49 1088.0 1704.5 789 2 700.0 14.16 88.38 33.08 2.40 8.89 1116.4 1893.7 790 2 800 13.12 88.75 29.99 1.96 7.63 1194.8 1953.0 791 2 U 11.99 88.74 35.97 1.90 9.49 1015.2 1917.3 788 3 800 13.39 89.30 32.29 2.50 7.96 977.6 1764.0 789 3 8 14.47 87.99 35.72 2.93 9.31 1131.6 2006.6 ' 790 3 U 11.82 88.68 36.84 2.38 9.46 1037.6 1975.6 791 3 800.0 13.08 89.39 34.68 2.41 8.65 1103.2 1659.0 788 4 WDG-U 13.94 88.20 28.50 2.34 8.89 1218.0 1871.6 789 4 0 14.96 86.16 32.71 2.58 11.08 1598.8 3094.4 790 4 8 14.41 85.83 32.00 2.33 9.77 1622.4 2627.6 791 4 WDG 15.61 88.84 29.59 3.15 9.42 1159.0 1254.2 172 -.-..0.---.-..---. .-----...--..----..-.--..---....-----.-.--..l....:.--..-‘0--'-.-...-------..---.-U.-1II1 1 1 'lll 1 1.----- 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 88 8 8.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.888 . 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.88 88.8 . 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8. 888 888 8 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88888.. 88.88888 . 88.88888 88888888 88888888 88.88888 88.88888 88.88888 8888 888888 888888 888-_8 8-88 8-888 8.888 8.8 8.88 8.88 888888 888888 88888 88 8888 8888 8888 8888 8888 8888 8888 888 .3388. 88.88.83 88.88 .3... 8.832888 88 323382.888 38.. :...: .3 8.883233 8... 888.2 .:...... .883 .6 8882.888. .283 .:..::. 8.... 88.83 8.8 88 3883.. 82.88.88... 983882.883 88...... 8.8.83 88382383 .828... a 8. .5 05... «use 808039 ucmsfiummxm mv magma 1773 'II 1" II 1 | -- . ‘ oo.o8osw8 88.8 >om nu 88.88888 88.8 88.8 88.8 88.8 88.8 8 88.88888 88.8 . 88.8 88.8 88.8 88.8 88.888888 88.8 888 888 8 88.88888 88.8 88.8 88.8 88.8 88.8 88.888888 88.8 888 888 8 88.88888 88. 88.8 88.8 88.8 88.8 88.888888 88.8 888 888 8 88.8 888 88.8 88.8 88.8 88.8 88.8 88.888888 88.8 . 888 88 8 88.88888 88.8 88.8 88.8 88.8 88.8 88.888888 88.8 888 88 8 88.8888. 88.8 . 88.8 88.8 88.8 88.8 88.888888 88.8 888 888 8 88.88888 88.8 .88.8 88.8 88.8 88.8 88.888888 88.8 888 888 8 8888888 8.888888 88.88888 88.88888 88.88888 88.88888 88.88888 88.88888 8888 888888 888888 888.888848 8.88 8-888 8-888 8-8 . 8.88 8.888888 888888 888888 88888 88 8888 8888 8888 8888 8888 8888 8888 888 .888888 8888888 888 888 88888888. 88 8888888888888 888 3888 .8 888888888888 .88888 88888: 888 88:78 88 88 8888883 88888888 88888888888 :9 88888 888888889888 .8888a: a 88 888788 mama c8cm8a8039 8:0588wmxm me wanna 171+ 0. .- .-.. .-- .-.-.-----'.-....-.--.--..------..------------:--.---. .--..-. 8cm 88.8888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 8 88.8888 ., 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 . 8 . 88.888 88.8 88.8 88.8 88.8 88.8 . 88.8888 88.8 888 888 8 88.888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.8888 88.8 88.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 88.888 8.8 8.8 88.8 88.8 88.8 88.8888 88.8 888 888 8 8888888 88888888 88888888 88888888 88888888 8888888. .88888888 88888888 8888 888888 888888 888-88 8-88 8-888 8-888 8-8 8-88 8-88 888888 888888 88888 88 8888 8888 8888 8888 8888 8888 8888 888 .88888: 8888888 888 89¢ .88888888 88 8888888888888 888 3888 88 888888888888 .88888 88888: >83 88:88 :8 =8 admwawc “acmaoaa 98888888zou 898: numan 888888—8888u .8mg8oz a 88 a; name 888039 acm288mmxm 8v manna 0-.---------.-.-l ll ||Ib Al 'I || II |----'- l- ’--"----- wu.nmod Kn.m n~.° “q." m_.° co.“ 0°.ophm do.° ”cm on“ . w m~.u«~_ nu.w m~.° “a." n_.° mm.¢ °°.°nm« mm.° ram on” u nn.np- an.” n_.° on.“ m_.° oo.o °°.°_mn a¢.° sou adm u m~.an, mm.¢ n_.o n¢._ mfi.o no.¢ oo.onmn an. to” "do u 5 . nu .~.nu._ an.¢ -.° an." *_.° 0°.“ °°.°~¢m on.° tug an _ .n.¢un_ no.¢ on.° “a.“ ._.o No.“ 0°.ocmq nm.o :99 on“ _ mn._ou~ 9‘.” °~.° .o.fi m~.° ac.“ ‘ 0°.oocn cm.° Pam «flu ~ .0..ho_ ou.m u.° .n._ nfi.o n~.~ oo.°a.n Nn.° pom ”fin d ,agxa‘. .>.u\94. .,~u\o4. .’.u\ax. .>~u\og. .>~a\ag. .>~s\o:. .,.a\ag. hm“: «mags: ammgzz 939.4» _.=o _.4g¢ _-.a¢ ~-= .-ga _-.» ugcpzfl 4¢=~z¢ 2°94“ ta gong ”wag puHQ bum. hu_a hum: hm_= um“ ..aagmz adamago egg ua¢ .gugOgadg ho xawfidaddmuadu can soak be mgoqam.=u~mg .mdmua godgaz >gu ~m=go cm co ~.m~o~u .mgmuo=u ocddfiuoagcu goaa u.m~a necqgm~=u~mu .go;g.x a ma nu . mama awnoze ucoefluomxm mv «Hams 176 «9.,un oo.nao. .4: “um um. u “n.0m a“.¢ 0,.9" .6.” cg." oo..~° om.a~o ao.a~m .o..~°_ on.” “N." _...nfl ma¢¢u9¢ ~_.uu on..~ om.” ~h.flw om.» N_.n .a.u~ on.ac- ao.m¢n on.~n_“ hm.w an.“ °~.nnh :q°°u°~ u¢.uu cu." a..n .°.¢_ .m.n . an.” a..°~ oo.m‘~d oo...« on.-~. .m.n cm.“ co..m¢ .¢°°ua .m.uu o..nm ._.m cm. u. “a." on.” h¢.a« on.nn~« °~.onq oo.n~._ a..n “a.“ °~.@- .qoonq an. a an.“ uh.. ~“.°N “a.” "g.” on.°~ on.hm_~ ofi.ow. °°.n-« .fi.n nu." om._fin ¢;oou. wa.u~ c..nn fln.q °°.~_ ” on.” an." ...oa °¢.nmo co.n.n oo.n«.. ON.” “a." om.o~4 .Qoonu no.0“ 0°.na we.“ . .mu "m.“ as." .«.n~ “. mm o..mfl. om.nflo_ o... “4." oq.nn¢ giooufl mu.uu o_.on n..w a“..~ on.°_ on.“ oa.n~ o“.owh an.ukn oo.n¢a mm.q .Q.n °o.~nn ..ocuo_ no. u o..«u .o.. an._~ oo.¢ “N.“ °°.h~ °°.hm~ ON.°n~ oo...~_ c..n mg.“ 0“...» :.ooum a .uu oq.na .,.n we.“ 0°.o 0°.” N~”m_ °°.nm% oq.¢mn oq.o~a °~.. ”a." afi.fiam ..ooua om.o~ o..n. No.‘ uu..~ ~_.¢ n«.~ .n.n~ oo.°w°~ oo.n°~ oo.om~ mn.¢ “n." 00.0““ ..oau. .n.~u ow.qm a~.. ~m.¢, co.“ Na.” -.°~ on...o °°.n~n on.a._~ so.” mm.“ °~.¢_n ..oouu a .uu o .na ..." u~.n~ fin.“ ca." q~.n~ °~.h.¢ ca.h°. on.on__ “n.. . mm.“ om.aoo =~o°"~_ ..guaw .aoofixo. .oood\a. .,°°~\a. .~;o°_\a;. .m°°~\a. .acofixo. .a\o=. .o\a=. .axm.. g; .=«z\~.. .~:°°~\mg. u‘__ .Jk a“; 9:9 2: uzu 4a¢ ”z” u=¢ z-n== 029 ._2 ago ‘9 ”=9 «4 uzu a” 920 .» 9°39 .h¢.m2_ uzu am“ an 9““ ..Na. qw.on~ ._=_.u=u ”flu. uo_o .moz cmun>0m .m—m .mE_c< ._ vo_gca .aumo;co_m:mc_ nose ucwsfiummxm we mHnme 177 wq.cu °¢.~nam .«a gum mm.ou ~n.mm oa.. mm.°~ mg.4 mg." q¢.mmo_ e‘.on°_ am..on mn.~°ufi on.” gfl.m °~.~.a mum“ 9¢ un.~u am.om a... ”a... _..n an.” “5.0“ °°._on. °°.ng¢ on.m-_ an.“ ~h.N cfl..4¢ :..onofi “a. N "g.nm ‘5.” ‘«.N~ am.“ no.” m..o~ °°.~m, , o~.~an on.n¢~_ OK.“ as.“ oo.~n¢ géooum u . u o~.mo h.. a¢.a_ __.n “a.” nn.a~ «.a.m °~.~nn on.,mw. an.“ “N." °¢.5Nq :aooum “h.am “ .n ...n a .un . ‘._.n n..~ m~.m_ co.¢¢h °~.¢nn on.un°~ m..« “w.“ oo.mwm :‘oou. an. n “0..“ .n.n .n.~« as.“ ea.” -.a~ °°.omm om..n. °°.¢n~_ _¢.m “N.“ °¢.0Nn .aoouu m~.m. ~«.wa “a.” ca..~ «a. "N.“ «0.5" co.~«m~ ov.nn« °°.n¢°_ uh.“ ...“ om.u~. .;oo“~ mg. n on.Na -.a an..~ .u... “a." nu.o_ a“..a« °¢.¢un co...__ ”a.” 0‘.” o“.dmn ..oouofi m .“u ”u.nm uh.. u_.ou ac.“ “a." mm.ow on.n«°~ o..o.~ on.~n__ mg.“ mu." o_.mn9 ..ocum an. N um.~a 0*.” an... nn.. “5.” m~.nm °~.oma on.aon om.n,~_ .°.¢ “a.“ o..n~w ..ooufl .r.°~ cq.ma on.“ «m.n~ am.“ ”5.“ -.- u.amm_ °°.¢¢~ oo.on°_ an.” _o." on.ua‘ ta°°“* h". u «m.«m no.” .~.K« oa.§ N‘.n N .um oo.m¢~_ °°.n,n oo.ofln~ ¢a.. ON." om.N.~ ..oouu nu. N uu.na «0.. “n.n~ «a. we.” uc..~. oo._m°~ oc.oon on.nn.~ o‘.q _~.n o..n.¢ .QOOuN ..;“a. .moo~\o. .moo—\a. .aoo_\m. .~=°°_\ag. .aoo~\o. .a°°_\m. .axoa. .a\o=. .a\3.. =9 .adg\~:. .~=°o~\m=. u:__ :4“ gum uzu go 929 Jae uzu ua¢ z-n=z 929 pm: 929 mu uzu «a .920 a“ uzu .h aoaa .hm.m=~ uzu cum m4¢=«m . ‘ o o . 3:. .3: 52:8 .ms .255 :8“ .:...: 3°: .5 a: :2 . c .me mama ¢o_m:mc_ nose ucwswummxm 5v oHnwe 178 2.8 2.5. E é ..n.~ .~.na .5.¢ "a... .~.n .n.n .N.oam .~.¢mn ~«.o.. .n.¢~n~ ~m.. _m.~ ~«._¢n mumgw3¢ h:_u one“ .n;. «na_ _1a .95 ”4;" 6.45“ .figaq ona§z "ta owa onaam .a2:°~ h. u and . new an;« .na "95 “ea“ Osage ougan oeawz we; on“ 2x23 530; no.un o..nw an.“ au.o~ ‘ .¢.. an." ~m.om °~.n.n °~.~°n ow.~_¢_ °°.. _Q.N ¢©.°¢m .aooum .w“u and” me; “96“ _fis owe cad“ oqsa. oqga+ oqan: ow; at“ ¢¢gs¢ sag: no.~u °¢.na °_.a an.- ow.¢ .n.n NG.°~ cc.m¢. °°.~‘. oo.mwu. a¢.. .~.~ °~.o_m .Qoonu no.nu 0°.“ cm.. -.*. as.. on.” a~._~ 09.non on.n«. on.¢um~ .9.” .9.“ °¢.‘dfi gioo"~_ ~.~u °~.oa "g.* KN.._ a~.. NW." ~_.°~ ou.~wn ca._,n oo.c¢- ow... ah.“ aq.~mn ..°°u°~ “A” 3.5 3... 3.2 $5 $.n 8.8 2......» 3.8.. 852 2.. a.“ 8.3 583 on.un oa.na £°.n w°.- «a.» w..n “n._u co..°m ow.m,. o .mon~ gm." “a." °«.c°n .aooua un..u o..~¢ fin.“ no.~u u~.¢ .g.n an.“ 0....0 on.o.. on.nm~_ .n.. m5." °~.mm~ ‘moou. o..~u ou.¢a an.. na.o_ mm.. “a.” .~.n~ oa.°°m ow.... on.~,n~ an.. ”N.” on.°mw .ncoun nm..u on.u¢ d¢.. n«.u~ fin.” on.” .¢.nn °¢.m,¢ °¢.oc. 0°.Nnn_ ”n.. ca.“ om.°a. ..ocuu ..gua. ..°°_\a. “aoodua. .ooo_\a. ._:oo_\oz. .mocdua. .acovxa. ..\a=. .,\a=. .a\m,. ta .=Mg\~‘. .~:°°~\a:. u:__ 3. 3“. ea .3 ea 3... ea é 2-22 ea :2 ea ,3 ea .3 ea 5 ea 2. as .22.: as E was.” 9““ ._Ka. @¢.°m_ ._=~.u=u owe. HQ . _o —002 COuJ—U CLOU .CNQ —mE_c< ¢ nose unmsfiumaxm av mflnme 179 oo..°n ca.fimfl. »¢a “a“ no._u °~.~a Nu.” “a... .n.~ "a.” .w.... .0...“ "a..~. .~.n~_~ 5..” ¢a.~ “n._na ug¢¢w>¢ IMMMMm «I guglflnfllfluflglflflauIlaIINNufllfllumumnu wauuflflllmwmufl ulflduom ll: 4llaluuuflmll q Mum mn.°" am.aa Kn.» nn._~ 0°.“ ...n ..._N on..°~ cm.~nn on.~n- ~0.q ~m.w °¢._na :¢°°"°_ ¢°.- .«.am .a.. co..~ «a. ,_.n «u.o_ on.~.n °Q.__n on.°«- o..¢ “a.“ °~.w°¢ gaoona “h. N N..am -.~ ~..n« as.“ an." .a.°~ ck.m~m o“..~. 6°.chn" ¢°.m no." omwwam .acoum a...~_ n_.nm 0..» 5%.." an.~ .m.~ aa.- an.non °¢.un. on.nu- ~n.n ...“ oo.m- .;°°". co.°u .~.¢o ne.. a..‘~ .«.m ”a.“ ~..K« ow..mn on.~an on.an_~ co.“ om.“ ow...” tgaouu no.m“ uu.a. co.“ ...oa fin.“ .5." ¢..~_ °~.n¢n oc..¢~ an..dofi .n.o ¢_.n °¢.-o .aoo"~_ o..°~ .°.ao "a.” «a... ¢n.. “N." on.- on.nnu oa.~°n on..¢°~ _°.Q om.“ o~.oam ..ocuo_ mo.°~ a .hg an.. an.m_ .a.¢ ‘°.n ~_.o~ 00.nan on.a.n o..om°~ on.” as.“ o"..mo :.ooum 0*.au ¢N.ac ._.. ~a.¢a .¢.N NR." on.nw °~.n¢, °~.n¢n 0°.nqofi ~h.¢ om.“ oo.~¢n ..ocnw _«.°~ on.m¢ .n.. ~n.n~ an.“ an." no.0“ °~._.. ca.°nm oo.n~n_ n .4 “a.“ o...~n :.oou. .m.°“ "n.na n..u .m.o. .c.‘ N*.n .m.- °@.n.g on.n¢. oo.-- an.“ ow.“ °N.w~m .woonu .°._~ u..o¢ ...“ Nd..~ ~_.~ ”h." °~.n~ °¢._n~ ow._.n on.n.°_ 0“.“ am." on.¢~§ ..o°"- ..;N,. .aoodxa. ..°°,\,. .,°°.\m. ..:°°_\,g. .,°°~N,. .,°°u\a. .,\o=. .a\... .a\a=. =9 .gd:\~.. .~;°°.\o;. un__ :4; gm“ 929 so 929 49¢ can u=¢ z-n== uzu _~= 929 mg 929 «4 029 go uzu .» =o== .h¢.uz~ 929 um“ m4t=¢n um“ .~\,. , .¢._N~ .mz_.uzu °,._ u 0 ”0 C n. . .... _o _ : mus—g CLOu 0mm .ms_c< .p uo_gom .Mumo co_m:m:_ nose ucmsflummxm me wanna “n.oun °¢.flaan .49 “a“ mm..u n..n¢ 5.." "n.°« °_.a .n.n 5°.na. ~°.no. ma..an n~.nn°~ «d.a KN." ao..~q mo¢mmp¢ mm.~u ow.nc an." -.°~ -.g «n.» a~.°~ ow..~n on..~. 0°.nao. ~°.a gm.“ °°.~nn :aoono. ”w.~“ kn.wm an.” «n.n~ ¢..¢ co.“ «n.h~ om.-m og.-. an.~k°_ on.” an.“ a .ncfi gnoona u .uu on..m an." on... ow.” Na.” -.°m °~.,an o..n,n on.°- .‘.n ma.“ °°.~am :.ocum 0“.“N «a..a d..¢ .~.n« an.“ “a.” kn.°~ o..~.. °~.‘¢. °_.u~, mm.“ ”a." .han .Aoou. __.- .a~.n¢ ,b.~ ...,“ ¢n.~ on." na..~ an.c~. 9°.dm. oo..od_ ac.” No.“ °°._ng .:ooum _ .uu uu.nm on.n. .9.._ _d._~ ON.” °~.n~ °¢.m~. oo.¢.n 0°.mfida .N.. “a." o..m.. .aoouu MW _ .uu fin.~a .‘.n ¢~.- oo.¢ ...” .¢.,_ °~.~o. o..~@n on.n«__ °°.q “a." oo._am =.°°"°_ 1. mn.°« as.” no.“ om.§_ .d._. .n.n 65.0" °~.°nn om.an~ °¢.mu~ .¢.. 0‘.“ o~.¢nq :.ocua .n.- ca.na as." q~._~ .a.a ..." ow.«~ o“..°n co.n.n °o..o__ o_.fi no.“ oo.,°~ .~°°"Q ou.°~ .a.,g an.” a...“ "w.“ "a." “8.0“ °~.noo ou.¢- o..o¢~ ...a we.“ °~.hog .:.00“. “h. u o‘.¢m uh." "..na an.¢ “a." ...«n on...“ ou.°¢n °~._.°« °¢.h “a.” °~.~na ..ccuu “h.°~ ou.fia «u. g.nu. ho.” «n.n. N..- on.n~n on.~nn °°.mm°_ ~°.¢ ac.“ 05.0”. ..ccnu ..4ua. .,°°_\,. .aoo_\m. ..°°"\o. .~:°°,\ag. .aoodxm. .mcofixo. “0‘03. .m\a=. .a\a,. ta .=M‘\_g. .~=°°~\m;. Hg”— ad. um“ uzu x: gag 45¢ 92” “a“ =-n== ”a“ h“: 929 mg 929 «A 020 cu uzu .» aoaa ._“.u=_ uzu gum ”Aggam ugh .~\a. w¢.°n~ ._=_.uzu m_m~ uo_a .mm: cou=_u show .m.» _ae_=< .N vo_go¢ .32. :23»... 63. ucmfiummxm on Same 181 ,_.~¢. o...no. »¢= “um .°.°~ .~.na "N.“ nn..~ N...“ . Kn." -.an§ k~.ana so.~.. nfi.n¢__ no.” on." -._~¢ unamubc uuuuum 1 gunfiunufifiufi»: gm 0 11 Out flu“ ul uuuuuuuau .m.o~ ...ma ...” an..~ -.~n .°.n ~n.o_ o...~£ °~.¢°. °~.,ao ~..a Qa.~ o..~.‘ .¢°°"°~ no.9. a".fig .n.n ¢~.- an.wn “N.“ .n.~_ ou..~‘ oo.~,n on.-o_ ¢°.°~ .~.~ °¢.°¢¢ :.oaum N“... ¢n.~a n..~ NQ.- ‘°._m ..." .~._N om.n~n c..o°. oc.oo_~ .w.~ as.“ 0°.aon .goouo oo.ow on.na .o..n ‘9..~ ,-.n¢ _~.n a..." oc..c. o..~nn ow.nafifi a..§ as." °°.n.m :.OO“. 9a.._ Nn.ng .—.g .~.~« Kn..h ma.” ...on °~.~¢n °°.n°n oo.-__ d..q KN." °¢.o¢n gacou~ n .0“ g..¢¢ ¢°.n c..- «a._. on." ...." °~.¢nn 09.n_n on.¢o~_ .~.n “a." °~.hgm :.°°"~_ 9~.o~ N...“ .a.a na.na __._~ N..n ¢_.u~ °~.wng °~.a~. on.m°_~ n~.§ Q~.~ ca..a¢ .~°¢"°, “N.°~ .~.om ‘..n N‘._~ .o.a an." .°.- o..-‘ on.n.n on.o-~ o‘.fl o..~ o~.nco :.ooua no. a n..‘N °~., .m..~ °~.. .°.n «n.._ °¢.¢_~ oo.¢nn on.m.~_ \no.m om." c..nnq .aoou‘ fin.°~ ¢~.n¢ .~.n ”.... aa.. .5.” a‘.n~ °~.n¢~ 09.non oo.con_ n°.Q .a.m o..m.m :.oou. me. u ...oa d... ._..~ ._.s ...n N..." 0....” 9°.now on.~,~_ .0." om." o...n« =.°°”~ ma.o~ an... “5.. ._.n~ -.‘ ”9.. .¢.n~ °~.¢ofl o..~o. °°.¢m~_ ow.c cc." °°.-g :.°°“~_ ..g\.. .,°°_\a. ..°°_\.. .ao._\a. ._;°°~\.‘. .a°°~\.. .aoon\.. ..\a=. .oxaa. .a\,.. :9 .gflgxd.. ._=°°~\a:. mg.— ... cum uzu :9 929 Age 029 $94 =-n== ox” __= 929 mu use «4 uzu “u can .» =o=a .h¢.kz_ 929 out udmx¢m ugh .daa. on..- ..=_.u,u Oucfl uo_o .moz cops—u chu .m_m .me_c< .~ uo_go¢ .muma co_m:mc_ “039 newsflummxm Hm magma 182 a.“ u ......n ... “u. . .uu 0.... on." a“... .n.. . ”a.” .....°_ .....°. ...... n~.~._. a... wk." ”....” “...... a..._ a...“ .5.” cc... ...n "K." ~°.m. on._.m. ...... on..mn_ a... n..~ .~.a.. taoouo_ .u.“u .5... as.” ...N. ...n a... ....N ou.~h~. ca.~“n on...n. ...“ mm.“ on...“ :...“. n..mu an... .n.. ...._ .... ~..N .N... on...n. ...... 0...... a... a... ...... ...... L .uu an... n... an..~ mm.» ”a. ...ou ...“... ...NN. oo.°~.. a... a5.“ ...... ...... n.- ...n. ~n.. m..." .... "N.” .0... ...... ...”.n on.... ...n ...N ...... :3...“ . .uu ....“ .... .... ...“ ...” ...." c..~nN 0....” ...... ...n ...u ...... ...."N. a .«u ".... .N.. a...” .... ma." .5... .....N ...... 0°.NW. an.” a... ow...” :.oouo_ u .uu on... ...u ..... .... .... a...“ ou..°. ...... n.huo_ n... ...“ .....n ...... ...Nfl an... ...n .n... h~.. mu." ....m ...... :...". ecu...“ .... an.“ ...“... ...... . .uu a...“ “h." ...“. ”a.“ ..." nu..~ °~.-°. oo.~¢m °~.m~.. N... an." as...” :...". n .uu ...”. ...n .0... .... .... ...Nm 0...... ...... ...“... n... ...N ...... ..coua m..uu .B.no n... .9... .... 5..» ...NN on.m-_ on...” oo.n.~_ a... om.“ °~.han :....“ ...... ......o. ......o. ..oo_\.. ..z°._\.=. ......o. ..°°.\o. ....a. ....3. ..x... ta ....\.g. ..toa.\.=. u... 3.. um“ 02. go .2. .a. age “a. z-n== .2. ..z .29 a. 9:. .4 9:. a. 929 .. 9.2. .....x. ”2. a“. “A..." a“. ..... ...... ._=_.u.. one“ uovc:_mut.cmon>0m .omm .mE_c< .N vo_go¢ .mumo :o_m:m:_ “039 newswummxm mm manna 1E33 ~_.uaa _ oo.nmo. »¢= mug on.nu 50.na ~o.n nm.a_ oo.a hm." a..mno_ o..mno— n~.-. a~.moo_ so.. as.” a..n_a mu¢¢u>¢ uuuuuuuuuuuuuuuu uuuuuuuuuuuununuuuuuuuuuunuuuuuuauugunocuunnuoouguuuuuu-soonnuoou---onouuuuuuuuuunuunuuuuuuuuuuuuuuuuuuuuuuuuuuuuununuu«nun ofi.nu .n.~m on.q m~.o_ odqa Ne.” . ~m.~w oa.o~m om.-~ ow.o~u m~.. on.m on.m~m caoouo_ u~.«~ on.nm ca.n a...“ ~5.~ an.” ma.om o~.a«u . ov...« oo.«.o_ .... no." o...oo ¢;ooum u.nu o—.wm .m.n va.~_ Nu.c no." ~a.- o..h¢~ o~.amn om.-~ °~.m -.m o~.-m caconm uu.n~ . nn.n¢ om.n a .«g mm.“ ,no.~ an.m— co.oon o~.nnw oo.ooo_ on.. ~N.N on.-~ c.0ou oh.ou oo..m no.n mo.n~ o~.n co." au.o~ o...mo on.cnv oo.omo_ «o.. no.m om.-o taco." mo.nu aa.«a -.¢ aw.o~ _ nn.‘ as.“ _~.N" on.ano oo._~n oo.¢~o .~.v mm.“ om. nu cgooum. nn.¢u .a.na mo.n on.o_ o«.n n~.n on.m~ co.om- oq.~o. oc.m-— .m.v mm.m. om.~no :aoouo~ u...” u ..o n«.« ¢~.a_ no.“ n~.n -.un °°.¢o~. oc.mon oo.mmo a..n cm.“ o~.uoo gaooum na.nu .m.nm «a.n ~n.a— oo.m No.n ~_.o. oo.~.~_ on..o. on..mo. o~.. mm." om.mmn :uooua n.nu « .mm .4.” oe.n_ Kn.. am.m v_.«~ . on.~nv— oc.oo« on.mn- u~.. co." oo.omn cmocu. on.nu oo.na oo.v aa.n. on.” oq.n ao.nu oo.om- o«.-n on.-n~ o_.m oa.~ om.unm caooun on.nu co.n um.v v~.o_ «a.» mg.» 09.9" on.nm«— o<.nnv oo.o——— an.n on.“ oo.~no swoon“. :...... .323 33:3 33:3::..:....38:332.... 3.3 3.3 3...: g ::..:...zggis as: a: 3.. 9.... .6 2a .5. Us a... 2-2.. as 5. ea ..a as 3 a... 5 ea 2. as .22: as a. “.:......" 3.. :..3 n a: 5.33 a...“ uo_o _moz cmon>0m .omm .ma_c< .N uo_gom .muma :o_m:uc_- "039 ucmsfiummxm mm manna BIBLIOGRAPHY BIBLIOGRAPHY Aderibigbe, A.0. and D.C. Church. 1983. Feather and hair meals for ruminants. I. Effect of degree of processing on utilization of feather meal. J. Anim. Sci. 56:1198. Allen, M., P.J. Van Soest, M.I. McBurney and P.J. Horvath. 1982. Rare earth elements: Affinity for and binding by cell wall fractions. Proc. XVI Rumen Function 'Conference. 1 Allison, M.J. and M.P. Bryant. 1963. Biosynthesis of branched-chain amino acids from branched-chain fatty acids by rumen bacteria. Arch. Biochem. Biophys. 101:269. Allison, M.J., J.A. Bucklin and I.M. Robinson. 1966. The importance of isovalerate carboxylation pathway of leu- cine of biosynthesis in the rumen. Appl. Microbiol. 14:807. Alvarez v.0. 1950. An Introduction to Gastroenterology. Hoeber, New York, USA. Chapter 27. Bailey, L.H., R.G. Caper and J.A. LeClerc. 1935. The composition and characteristics of soybeans, soybean flour and soybean bread. Cereal Chem. 12:441. Balch, C.C., A. Kelley and G. Heim. 1951. Factors affecting the utilization of food by dairy cows. 4. The action of the reticulo-rumenal orifice. Brit. J. Nutr. 5:206. Balch, C.C., D.A. Balch, V.W. Johnson and J. Turner. 1953. Factors affecting the utilization of food by dairy cows. VII. The effect of limited water intake on the digest- ibility and rate of passage of hay. Brit. J. Nutr. 7:212. Balch, C.C. 1957. Use of the lignin ratio technique for determining the extent of digestion in the reticulo-rumen of the cow. Brit. J. Nutr. 11:213. Beever, D.E., R.C. Kellaway, D.J. Thomson, J.C. MacRae, C.C. Evans and A.S. Wallace. 1978. A comparison of two non- radioactive digesta marker systems for the measurement nutrient flow at the proximal duodenum of calves. J. Agric. Sci. 90:157. 18h 185 Bergeim, 0. 1926. Intestinal Chemistry. V. Carbohydrates and calcium and phosphorus absorption. J. Biol. Chem. 70:29. Bergen, H.G., E.H. Cash and H.E. Henderson. 1974. Changes in nitrogenous compounds of the whole corn plant during ensiling and subsequent effects on dry matter intake by sheep. J. Anim. Sci. 39:629. Black, J.L. 1971. A theoretical consideration of the effect ,of preventing rumen fermentation on efficiency of util- ization of dietary energy and protein in lambs. Brit. J. Nutr. 25:31. Blaxter, K.L., N. HeGraham and F.w. Wainman. 1956. Some observations on the digestibility of food by sheep and on related problems. Brit. J. Nutr. 10:69. Briggs, P.K. 1961. The influence of lipids on the utiliza- tion of dietary nitrogen by the ruminant. Thesis. University of Aberdeen. Broderick, G.A. 1978. In-vitro procedures for estimating rates of ruminal protein degradation and proportions of protein escaping the rumen undegraded. J. Nutr. 108:181. Brohult, S. and E. Sandegren. 1954. Seed proteins. In: H. Neurath and K. Baily (Ed.) The Proteins. Vol. 2, part A, pp. 493-500. Academic Press, New York. Bryant, M.P. and R.M. Doetsch. 1955. Factors necessary for ' growth of bacteroides succinogenes in the volatile acid fraction of rumen fluid. J. Dairy Sci. 38:340. Chalupa, W. 1975. Rumen bypass and protection of proteins and amino acids. J. Dairy Sci. 58:1198. Church, D.C., D.A. Daugherty and W.H. Kennick. 1982. Nutritional evaluation of feather and hair meals as protein sources for ruminants. J. Anim. Sci. 54:337. Clark, J.H. 1975. Nitrogen metabolism in ruminants. Pro- tein solubility and rumen bypass of protein and amino acids. Nutr. Clin. Nutr. 1:261. Clemens, E.T. 1982. Comparison of polyethylene glycol and dye markers in nutrition research. Nutr. Res. 2:323-324. Cline, T.R., U.S. Garrigus and E.E. Hatfield. 1966. Addi- tion of branched- and straight-chain volatile fatty acids to purified lamb diets and effects on utilization of certain dietray components. J. Anim. Sci. 25:734. 186 Corbett, J.L. 1981. Determination of the utilization of energy and nutrients by grazing animals. In: J.L. Wheeler ‘ and R.D. Mochrie (Ed.). Forage Evaluation: Concepts and Techniques.. pp. 383-398. CSIRO, Melbourne, Australia. Corbett, J.L. and F.S. Pickering. 1983. Estimation of daily flows of digesta in grazing sheep. Aust. J. Agric. Res. 34:193. Cottrill, B.R., D.E. Beever, A.E. Austin and D.F. Osbourn. 1982. The effect of protein and nonprotein nitrogen supplements to maize silage on total amino acid supply in young cattle. Brit. J. Nutr. 48:527. Crickenberger, R.G., V.G. Bergen, D.C. Fox and L.A. Gideon. 1979. Effect of protein level in corn-corn silage diets on abomasal nitrogen passage by steers. J. Anim. Sci. 49:177. Crooker, B.A., J.H. Clark and R.D. Shanks. 1982. Rare earth elements as markers for rate of passage measurements of individual feedstuffs through the digestive tract. J. Nutr. 1353:1361. De Huelenaere, H.J.H., M.L. Chen and A.E. Harper. 1967. Assessment of factors influencing estimation of lysine in cereal products. J. Sci. Food Chem. 15:310. Dixon, R.H. and J.V. Nolan. 1982. Studies of the large in- testine of sheep. 1. Fermentation and absorption in sections of the large intestine. Brit. J. Nutr. 47:289. Downes, A.E. and I.W. McDonald. 1964. The chromium 51 ' complex of EDTA as a soluble rumen marker. Brit. J. Nutr. 18:153. Drennan, H.JL, J.H.G. Holmes and W.N. Garrett. 1970. A Com- parison of markers for estimating the magnitude of rumen digestion. Brit. J. Nutr. 24:961. Driedger, A. and E.E. Hatfield. 1972. Influence of tannins on the nutritive value of soybean meal for ruminants. J. Anim. Sci. 31:1038 (Abstr.). Ellis, N.C. and J.E. Huston. 1967. Caution concerning the stained particle technique for determining gastrointes- tinal retention time of dietary residues. J. Dairy Sci. 50:1996-1999. Ellis, V.C- 1968- Dysprosium as an indigestible marker and its determination by radioactivation analysis. J. Agr. Food Chem. 16:220. Ellis, W.C. and J.E. Huston. 1968. Ce-141 Pr-144 as a parti- culate digesta flow marker in ruminants. J. Nutr. 95:67-78. 187 Ellis, W.C., J.H. Matis and C. Lascano. 1979. Quantitating ruminal turnover. Fed. Proc. 38:2702. Ellis, H.G., C. Lascano, R. Teeter and F.N. Owens. 1980. Solute and particulate flow markers. Protein Requirements for Cattle Symposium. Oklahoma State Univ- ersity MP-109:37. Ellis, W.C., G.T. Schelling and L.W. Green. 1983. The acid resistant binding of ytterbium with feedstuffs. J. Anim. Sci. 57 (suppl. 1):429 (Abstr.). Engelhardt, W. 1974. In: Tracer Techniques in Trapical Ani- mal Production. pp. 111-124. International Atomic Energy Agency. Vienna, Austria. Evans, C.C., J.C. MacRae, S. Wilson. 1977. Determination of ruthenium and chromium by X-ray flourescence spectrometry and use of inert ruthenium (II) phenantholine as a solid phase marker in sheep digestion studies. J. Agri. Sci. 89:17. Evans, E. 1981a. An evaluation of the relationships between dietary parameters and rumen liquid turnover rate. Can. J. Anim. Sci. 61:91. Evans, E. 1981b. An evaluation of the relationships between dietary parameters and rumen solid turnover rate. Can. J. Anim. Sci. 61:97. Fahey, G.C. and H.G. Jung. 1983. Lignin as a marker in digestion studies: A review. J. Anim. Sci. 57:220. Faichney, G.J. 1971. The effect of formaldehyde treated casein on the growth of ruminant lambs. Aust. J. Agric. Res. 22:453. Faichney, G.J. 1974. The use of_markers to partition digestion within the gastrointestinal tract of ruminants. In: I.W. McDonald and A.C.I. Warner (Ed.) Digestive Phy— siology and Metabolism in Ruminants. pp. 277—291. Univ. New England Publ., New York. Faichney, G.J. 1975. The effect of formaldehyde treatment of a concentrate diet in the passage of solute and par- ticle markers through the gastrointestinal tract of sheep. Aust. J. Agric. Sci. 26:319. Faichney, G.J. 1980a. The use of markers to measure digesta flow from the stomach of sheep fed once daily. J. Agric. Sci. (Camb.) 94:313-318. 188 Faichney, G.J. 1980b. Measurement in sheep of the quantity and composition of rumen digesta and of the fractional outflow rates of digesta constituents. Aust. J. Agric. Sci. Res. 31:1129é1137. ' Ferguson, K.A. 1974. In: I.W. McDonald and A.C.I. Warner (Ed.) Digestion and Metabolism in the Ruminant. p. 448. Univ. New Engl. Publ. Unit. Frape, D.L., H.G. Tuck, N.H. Sutcliffe and D.B. Jones. 1982. The use of inert markers in the measurement of the di- gestibility of cubed concentrates and of hay given in several proportions to the pony, horse and white rhino- ceras (Diceros simas). Comp. Biochem. Physiol. 72:77. _Furuichi, Y. and T. Takahashi. 1981. Evaluation of acid insoluble ash as a marker in digestion studies. Agric. Biol. Chem. 45:2219-2284. Galyean, M.L., D.C. Wagner and F.N. Owens. 1979. Corn par- ticle size and site and extent of digestion in steers. J. Anim. Sci. 49:204. Ganev, G., E.R. Orskov and R.I. Smart. 1979. Effect of type of substrate and retention time on rate and extent of degradation of protein supplements in the rumen. J. Agric. Sci.(Camb.) 93:651. ' Gill, J.L. 1973. Current status of multiple comparisons of means in designed experiments. J. Dairy Sci. 56:973. Gill, J.L. 1978. Design and analysis of experiments in the animal and medical sciences. Vol. 1. The Iowa State University Press. Ames, Iowa. Goering, H.K. and P.J. Van Soest. 1970. Forage Fiber Analy- sis. Agriculture Handbook No. 379.. Agricultural Research Service, USDA. Goering, H.K., J. Menear and I.L. Lindahl. 1974. Growth and nitrogen metabolism of sheep fed alfalfa dehydrated at different temperatures. J. Dairy Sci. 57:621. (Abstr.). Goetsch. A.L. and M.L. Galyean. 1983. Influence of feeding frequency on passage of fluid and particulate markers in steers fed a concentrate diet. Can. J. Anim. Sci. 63:727. Goodall, E.D. and R.N.B. Kay. 1973. The use of the chromium complex of ethylenediaminetetracetic acid for studies of digestion in sheep. Proc. Nutr. Soc. 32:22A-23A (Abstr.). Gordon, J.C. and 1.x. McAllister. 1970. The circadium rhythum of rumination. J. Agric. Sci. (Camb.) 74:291. 189 Grovum, W.L. and G.D. Phillips. 1973. Rate of passage of digesta in sheep. 5. Theoretical condsiderations based on a physical model and computer simulation. Brit. J. Nutr. 30:377. Grovum, W.L. and V.J. Williams. 1973a. Rate of passage of digesta in sheep. 3. Differential rates of passage of water and dry matter from the reticulo-rumen, abomasum and caecum and proximal colon. Brit. J. Nutr. 30:231. Grovum, W.L. and V.J. Williams. 1973b. Rate of passage of digesta in sheep. 4. Passage of marker through the alimentary tract and the biological relevance of rate- constants derived from changes in concentration of marker in faeces. Brit. J. Nutr. 30:313. Grovum, W.L. and V.J. Williams. -1977. Rate of passage of digesta in sheep. 6. The effect of level of food intake on mathematical predictions of the kinetics of digesta in the reticulo-rumen and intestines. J. Nutr. 38:425. Harris, L.E. and A.T. Phillipson. 1962. The measurement of the flow of food to the duodenum of sheep. Anim. Prod. 4:97. Hartnell, ‘G.F. and L.D. Satter. 1979. Determination of rumen fill, retention time and ruminal turnover rates of ingesta at different stages of lactation in dairy cows. J. Anim. Sci. 48:381. Hayes, B.W., C.O. Little and G. E. Mitchell, Jr. 1964. In- fluence of ruminal, abomasal and intestinal fistulation on digestion in steers. J. Anim. Sci. 23:764. Hogan, J.P. and R.H. Weston. 1967. The digestion of chopped and ground roughages by sheep. II. The digestion of nitrogen and some carbohydrate fractions in the stomach and intestines. Aust. J. Agric. Res. 18:803-819. Hembry, F.G., W.H. Pfander and R.L. Preston. 1975. Utilization of nitrogen from soybean, casein, zein and urea by mature sheep. J. Nutr. 105:267. Hume, I.D., R.J. Moir and M. Somers. 1970. Synthesis of microbial protein in the rumen. I. Influence of the level of nitrogen intake. Aust. J. Agric. Res. 21:283. Huston, J.E. and W.C. Ellis. 1968. Evaluation of certain properties of radiocerium as an indigestible marker. J. Agric. Food Chem. 16:225. Hyden, S. 1955. A Turbimetric method for determination of higher polyethylene glycols in biological material. Kungl. Lantbr. Hogsk. Ann. 22:139-145. 190 Isaacs, J. and F.N. Owens. 1972. Protein soluble in rumen fluid. 35:267 (Abstr.). Ivan, M. and D.W. Johnston. 1981. Reentrant cannulation of small intestine in sheep: cannula and surgical method. J. Anim. Sci. 55:849. James, 8., J.B. Rowe and A.W.J. Brooms. 1981. A method for measuring short-term changes in duodenal outflow rate. Nutr. Soc. Proc. 40:104A. Johnson, G.T. and G.C. Kyker. 1966. The mechanism of cerium uptake by Saccharomyces cerevisiae. Mycologia 58:91. Kay, R.N.B. 1969. Effects of tannic acid on the solubility of PEG. Proc. Nutr. Soc. 28:22A. Kleiber, M. 1961. The Fire of Life. pp. 254-255. John Wiley and Sons. New York, London. Klopfenstein, T., W. Rounds and J. Waller. 1976. Distillers feeds as protein sources for beef cattle. Proc. Distill. Feed Conf. 31:52. Klopfenstein, T., J. Waller, N. Merchen and L. Petersen. 1978. Distillers grains as a naturally protected pro- tein for ruminants. Proc. Distill. Feed Conf. 33:38. Komarek, R.J. 1981a. Rumen and abomasal cannulation of sheep with specially designed cannulas and a cannula in- sertion instrument. J. Anim. Sci. 53:790. Komarek, R.J. 1981b. Intestinal cannulation of cattle and sheep with a T-shaped cannula designed for total diges- tion collection without externalizing digesta flow. J. Anim. Sci. 53:796. Kotb, A.E. and T.D. Luckey. 1972. Markers in nutrition. Nutr. Abstr. and Reviews. 42:813-845. Kropp: J.R., R.R. Johnson and F.N. Owens. 1976. Microbial protein synthesis from urea and soybean meal. J. Anim. Sci. 43:327 (Abstr.). Kunihara, M. and M. Teruhiko. 1981. Measurement of gastro- intestinal transit of solid food using a colestipol- phenal red complex as a marker. J. Pharm. Dyn. 4:916- 921. Kyker, G.C. 1961. Rare Earths in Mineral Metabolism. C.L. Comar and F. Bronner (Ed.) Chap. 36, Vol. 2, part B. Academic Press, New York. 191 Laughren, L.C. and A.W. Young. 1979. Duodenal nitrogen flow in response to increasing dietary crude protein in sheep. J. Anim. Sci. 49:211. Lemanager, H.P., F.N. Ovens, B.J. Shockey, K.S. Lusby and R. Totusek. 1978. Monensin effects on rumen turnover rate, twenty-four hour VFA pattern, nitrogen components - and cellulose disappearance of various protein sources. J. Anim. Sci. 56:206. “36386: J.C. 1974. The use of intestinal markers to measure digestive functions in ruminants. Proc. Nutr. Soc. Mahadevan. S., J.D. Erfle and F.D. Saven. 1980. Degradation of soluble and insoluble proteins by bacteroides amylo- philus protease and by rumen microorganisms. J. Anim. Sci. 50:723. - ' Malawar, S.J. and D.W. Powell. 1967. An improved tubimetric analysis of polyethylene glycol utilizing an emulsifier. Gaestroent. 53:250. Mehrez, A.Z. and E.R. Orskov. 1976. A study of the artifi- cial fiber bag technique for determining the digestibility of feeds in the rumen. J. Agric. Sci. (Camb.) 88:645. Mehrez, A.Z., E.R. Orskov and I.W. McDonald. 1977. Rates of rumen fermentation in relation to ammonia concentra- tion. Brit. J. Nutr. 38:437. Merchen, N., T. Hanson and T. Klopfenstein. 1979. Ruminal bypass of brewers dried grains protein. J. Anim. Sci. 49:177. Misra, H.P. and L.M. Potter. 1970. Available lysine, digestible protein and metabolizable energy of corn gluten meal as measured in diets of young turkeys. Poultry Sci. 49:1416. Mueller, W.J. 1956. Feasibility of the chromium oxide and lignin indicator methods for metabolism experiments with chickens. J. Nutr. 58:89. Murphy, M.R., R.L. Baldwin, M.J. Ulyatt and L.J. Koong. 1983. A quantitative analysis of rumination patterns. J. Anim. Sci. 56:1236. Miller, O.L. and H.P Schedle. 1970. Total recovery studies of nonabsorbable indicators in rat small intestine. Gastroent. 58:40-46. Miller, E.L. 1983. Symposium on nitrogen utilization by the ruminant. Proc. Nutr. Soc. 32:79. WM”, \//\__\ . A , 192 Mudgal, U.D., R.M. Dixon, P.M. Kennedy and L.P Milligan. 1982. Effect of two intake levels on retention time of liquid, particle and microbial markers in the rumen of sheep. J. Anim. Sci. 54:1051. Muntifering, R.B., R.M. DeGregorio and L.E. Deetz. 1981. Ruminal and post-ruminal lignin digestion in lambs. Nutr. Reports International 24:543-549. Muntifering, R.B., B. Theurer and T.H. Noon. 1981. Effects of monensin on site and extent of whole corn digestion and bacterial protein synthesis in beef steers. J. Anim. Sci. 53:1565. Neudoerffer, T.S., D.R. McLaughlin, S.J. Slinger and I.D. Horney. 1973. Difficulties associated with the use of triated polyethylene glycol as a water soluble di- ,gestion marker in ruminants. J. Anim. Sci. 36:749-743. Neumark, H., A. Halevi, S. Amir and S. Yerushalmi. 1975. Assay and use of magnesium ferrite as a reference in ab- sorption trials with cattle. J. Dairy Sci. 58:1476. Neumark, H., R. Bielorai and B. Iosif. 1982. Magnesium ferrite as a marker in absorption trials with chicks. J. Nutr. 112:387. Ngo, T.T., A.P.H. Phan, C.F. Yam and R.M. Lenhoff. 1982. Interference in determination of ammonia with the hypochlorite-alkaline phenol method of Berthelot. Anal. Chem. 54:46. Nishimutu, J.P., D.C. Ely and J.A. Boling. 1973. Nitrogen metabolism in lambs fed soybean meal treated with heat, formalin and tannic acid. J. Nutr. 103:49. Orskov, E.R. and I.W. McDonald. 1979. The estimation of protein degradability in the rumen from incubation meas- urement weighted according to rate of passage. J. Agric. Sci. (Camb.) 92:499. Orskov, E.R., F.D. DeB Hovell and F. Mould. 1980. The use of the nylon bag technique for the evaluation of feed- stuffs. Trop. Anim. Prod. 5:195. Q Orskov, E.R. 1982. Protein Nutrition in Ruminants. Academ- ic press, London. Osborne, T.D. 1924. The Vegetable Proteins. Longmous, Green and Co., London. ‘ Oyaert, W. and J.H. Bouckaert. 1961. A study of the passage of fluid through the sheeps omasum. Res. Vet. Sci. 2:41. 193 Pearce, G.R. and R.J. Hair. 1964. Rumination in sheep. I. The influence of rumination and grinding upon the passage and digestion of food. Australian J. Agr. Res. 15:635. ' Penning. F.D. and R.H. Johnson. 1983. The use of internal markers to estimate herbage digestibility. 2. Indigest- ible acid detergent fiber. J. Agric. Sci. (Camb.) 100:133. Peter. A.P., E.E. Hatfield, F.N. Owens and U.S. Garrigus. 1971. Effects of aldehyde treatment of soybean meal on in vitro ammonia release, solubility and lamb perform- ance. J. Nutr. 101:605. Phillips, G.D. and G. W. Dyck. 1964. The flow of digesta into the duodenum of sheep.. Can. J. Anim. Sci. 44:220. Phillips, G.C. 1961. Physiological comparisons of European and Zebu steers. II. Effects of restricted water in- take. Res. Vet. Sci. 2:209. Pilgrim, A.F., F.V. Gray, R.A. Weller and 0.3. Belling. 1970. Synthesis of microbial protein in sheep's rumen and proportion of dietary nitrogen converted into micro- bial nitrogen. Brit. J. Nutr. 24:589. Poncet, C., M.Ivan and m. Leveille. 1982. Electromagnetic measurements of duodenal digesta flow in cannulated sheep. Reprod. Nutr. Develop. 22:651. Prigge, E.C., G.A. Varga, J.L. Vicini and R.L. Reid. 1981. Comparison of ytterbium chloride an dichromium sesquioxide as fecal indicators. J. Anim. Sci. 53:1629- 1633. Purser, D.B. and R.J. Mori. 1966. Rumen volume as a factor involved in individual sheep difference. J. Anim. Sci. 25:509. Putnam, P.A. and R.E. Davis. 1965. Post-ruminal fiber digestibility. J. Anim. Sci. 24:826. Reiners, R.A. and S.A. Watson. 1975. Latest Analytical Data on corn gluten feed, corn gluten meal and other feed ingred- ients from corn wet milling. Feedstuffs. 47:19. Rock, D.W., T.J. KlOpfenstein, J.K. Ward, R.A. Britton and M.L. McDonald. 1983. Evaluation of slowly degraded proteins: Dehydrated alfalfa and corn gluten meal. J. Anim. Sci. 56:476. 191+ Sachtleben, 8.3. 1980. Studies of the effect of diet on nitrogen passage to the lower gastrointestinal tract in steers. Ph.D. Dissertation. Mich. State Univ., East Lansing. . Santos, K.A.S. and L.D. Satter. 1980. Flow of amino acids to the small intestine of dairy heifers consuming rations having variable amounts of distillers dried grains with solubles. Dairy Sci. Res. Rep., Univ. Wisc., Madison. p. 55. ‘ Satter, L.D. and L.L. Slyter. 1974. Effects of ammonia concentrations on rumen microbial protein production in vitro. Brit. J. Nutr. 32:199. Schedl,H.R., D. Miller and D. White. 1966. Use of polyethylene glycol and phenol red as unabsorbed indicators for in- testinal absorption studies in man. Gut 7:159-163. Sharma, H.R., J.H. Ingalls and J.A. McKirdy. 1972. Nutri- tive value of formaldehyde treated rapeseed meal for dairy calves. Can. J. Anim. Sci. 52:363. Shipley, A.E. and R.E. Clark. 1972. Tracer Methods for In Vivo Kinetics. Academic Press, New York and London. SiddonS’ E.C., D.E. Beaver and JoVo NOlano 19820 A compar- ison of methods for the estimation of microbial nitrogen in duodenal digesta of sheep. Brit. J. Nutr. 48:377. Singleton, A.G. 1961. The electromagnetic measurement of the flow of digesta through the duodenum of the goat and sheep. J. Physiol. 155:184. Sissons, J.W. and R.H. Smith. 1982. Effect of duodenal cannulation on abomasal emptying and secretion in the preruminant calf. J. Physiol. 322:409. Sklan, 0., D.Dubrov, U. Eisner and S. Hurwitz. 1975. Cr-51 EDTA, Yb-91 and Ce-141 as nonabsorbable reference substances in the gaStrointestinal tract of the chicken. J. Nutr. 105:1549-1552. smith, Jro, CORO, FOR. Earle and IOAO Wolff. 1959. compar- ison of solubility characteristics of selected seed proteins. J. Agric. Food. Chem. 7:133. Sniffen, C.J. and W.H. Hoover. 1978. Amino acid profile of ‘ Dietary bypass protein and its importance to ruminmants. Proc. Distill. Feed Conf. 33:61. Spears, J.W., E.E. Hatfield and J.H- Clark. 1980. Influence of formaldehyde treatment of soybean meal on performance of growing steers and protein availability in the chick. J. Anim. Sci. 50:750. 195 Stanley, M.M. and S.H. Cheng. 1957. Excretion from the gut and gastrointestinal exchange: Studied by means of inert marker method. Amer. J. Dig. Diseases. 2:628. Stern, M.D. and L.D. Satter. 1982. In vivo estimation of rumen degradability in the rumen. In: Protein Require- ments for Cattle: Symposium. Division of Agr., Oklahoma State University. MP-107:57. Stern, M.D., L.M. Rode, R.W. Prange, R.H. Stauffacher and L.D. Satter. 1983. Ruminal protein degradation of corn gluten meal in lactating dairy cattle fitted with duo- denal T-type cannulae. J. Anim. Sci. 56:194. Sulzman, P.M. 1982. Microcomputer monitering of circadian rhythms. Comput. Bio. Med. 12:253. Sutton, A.L. and R.L. Vetter. 1971. Nitrogen studies with lambs fed alfalfa (Medicago satava) as hay, low moisture and high moisture silage. J. Anim. Sci. 32:1256. Tamminga, S. 1979. Protein degradation in the forestomach of ruminants. J. Anim. Sci. 49:1615. Tamminga, S., C.J. Van der Koelen and A.M. Van Vuuren. 1979. The effect of level of feed intake on nitrogen entering ~ the small intestine of dairy cows. Livestock Prod. Sci. 6:225. Tan, T.N., R.H. Weston and J.P. Hogan. 1971. Use of Ru-103 labelled tris (1,10-phenanthroline) ruthenium II chloride as a marker in digestion studies with sheep. Int. J. Appl. Radiation and Isotopes. 22:301-308. Teeter, H.G., F.N. Owens and G.W. Horn. 1979. Ytterbium as a ruminal marker. J. Anim. Sci. 49:412 (Abstr.). Teeter, H.G., F.N. Owens and T.L. Mader. 1981. Ytterbium chloride as a particulate phase marker for ruminants. J. Anim. Sci. 52:436 (Abstr.). Teeter, H.G., F.N. Owens and M.W. Sharp. 1981b. Ruminal and fecal passage rates of particulate matter varying in size, density and dietary concentration. Proc. 73rd. Annual ASAS Meetings. Abstr. 756 pp 437. Thompson, F. and G.E. Lamming. 1972. The flow of digesta, dry matter and starch to the duodenum in sheep given rations containing straw of varying particle sizes. Brit. J. Nutr. 28:391. Thompson, F. 1973. The effect of frequency of feeding on flow and composition of duodenal digesta in sheep given straw-based diets. Brit. J. Nutr. 30:87. 196 Thonney, M.L., D.J. Duhaime, P.W. Moe and J.T. Reid. 1977. Acid insoluble ash and permanganate lignin as indicators to determine digestibility of cattle rations. J. Anim. Sci. 49: 1112. ~ i. Topps, J.H., R.N.B. Kay and E.D. Goodall. 1968. Digestion of concentrates and hay diets in the stomach and intes- tines of ruminants. 1. Sheep. Brit. J. Nutr. 22:261. Uden, P. 1978. Comparative Studies on Rate of Passage, Particle Size and Rate of Digestion in Ruminants, Equines, Rabbits and Man. Ph.D. Thesis, Cornell Univer- sity, Ithaca, New York. . Uden, P., P.E. Colucci and P.J. Van Soest. 1980. Investiga- tion of chromium, cerium and cobalt as markers in digesta rate of passage. J. Sci. Food Agric. 31:625- 632. Ulyatt, M.J. 1964. The use of polyethylene glycol as a marker for measuring rumen water volume and rate of flow of water from the rumen of grazing sheep. N.Z. J. Agric. Res. 7:713. Utley, P.R., N.W. Bradley and J.A. Boling. 1970. Effect of water restriction on nitrogen metabolism in bovine fed two levels of nitrogen. J. Nutr. 100:551. Van Keulen, J. and B.A. Young. 1977. Evaluation of acid- insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 44:282. Van Soest, P.J. 1963. Use of detergent in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. J. Assoc. Official Agr. Chem. 46: 829. Van Soest, P.J. 1965. Use of detergents in analysis of fibrous feeds. III. Study of the effects of heating and drying on yield of fiber and lignin in forages. J. Assoc. Official Agr. Chem. 48:785. Van Soest, P.J. and R.H. Wine. 1967. Use of detergents in the analysis of fibrous feeds..IV. Determination of plant cell wall constituents. J. Assoc. Official Anal. Chem. 50:50. van't Klooster, A.Th., P.A.M. Rogers and H.R. Sharma. 1969. Observations on the rate of flow of digesta through the duodenum of sheep and on the recovery of polyethylene glycol and chromium sesquioxide from duodenal contents. Netherlands J. Agr. Sci. 17:60. 197 Waller, J.C. 1978. Low solubility protein sources for cattle. Ph.D. dissertation. Univ. of Nebraska, Lincoln. Waller, J., N. Merchan, T. Hansen, T. Klopfenstein. 1980. Effect of sampling intervals and digesta markers on abomasal flow determinations. J. Anim. Sci. 50:1122. Waller,J.C., T. Klopfenstein and M. Poos. 1980. Distillers feeds as protein sources for ruminants. J. Anim. Sci. 51:1154. ' Warner, A:C.I. 1981. The mean retention times of digesta markers in the gut of the tammar, Macropus eugenni. Aust. J. Zool. 29:759-771. Welch, J.G. and A.M. Smith. 1978. Particle sizes passed from the rumen. J. Anim. Sci. 46:309. Wenham, G. and R.S. Wyburn. 1980. A radiological investi- gation of the effects of cannulation on intestinal motility and digesta flow in sheep. J. Agric. Sci. (Camb.). 95:539- Wilkinson, J.H. and J.H.D. Prescott. 1970. The use of Chromic oxide in the measurement of individual feed intake in cattle fed silage and barley. Anim. Prod. 12:71. 14 Winne, D. and H. Gorig. 1982. Appearance of C- Polyethylene glycol 4000 in intestinal venous blood: Influence of osmolarity and laxatives, effect on net water flux determination. Arch. Pharmacol. 321:149- 156. Wohlt, J.E., C.J. Sniffen and W.H. Hoover. 1973. [Measure- ment of protein solubility in common feedstuffs. J. Dairy Sci. 56:1052. Wohlt, J.E., C.J. Sniffen, W.H. Hoover, L.L. Johnson and C.K. Walker. 1976. Nitrogen metabolism in wethers as affected by dietary protein solubility and amino acid profile.) J. Anim. Sci. 42:1280. Young, M.C., B. Theurer, P.R. Ogden, G.W. Nelson and W.H. Hale. 1976. Dysprosium as an indicator in cattle digestion trials. J. Anim. Sci. 43:1270. Yu, Y. 1976. Relationship between measurement of heating and acid detergent insoluble nitrogen in heat damaged fresh alfalfa, haylage and hay. J. Dairy Sci. 59:1845. Zinn, R.Q. 1978.‘ Studies on supplemental protein degrada- tion in the rumen. Ph.D. thesis. University of Kentucky, Lexington. 198 Zinn, R.A., L.S. Bull and R.W. Hemken. 1981. Degradation of supplemental proteins in the rumen. J. Anim. Sci. 52: 857- Zinn. R.A. and F.N. Owens. 1983. Influence of feed intake level on site of digestion in steers fed a high concen- trate diet. J. Anim. Sci. 56:471.