Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The evaluation of heat and lignosulfonate treated canola meal as sources of rumen undegradable protein… Wright, Chad Frederick 1999

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1999-0652.pdf [ 3.83MB ]
Metadata
JSON: 831-1.0088993.json
JSON-LD: 831-1.0088993-ld.json
RDF/XML (Pretty): 831-1.0088993-rdf.xml
RDF/JSON: 831-1.0088993-rdf.json
Turtle: 831-1.0088993-turtle.txt
N-Triples: 831-1.0088993-rdf-ntriples.txt
Original Record: 831-1.0088993-source.json
Full Text
831-1.0088993-fulltext.txt
Citation
831-1.0088993.ris

Full Text

THE EVALUATION OF HEAT AND LIGNOSULFONATE TREATED CANOLA MEAL AS SOURCES OF RUMEN UNDEGRADABLE PROTEIN FOR LACTATING COWS by CHAD FREDERICK WRIGHT B.Sc, The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1998 © Chad Frederick Wright, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) Department The University of British Columbia Vancouver, Canada DE-6 (2/88) II A B S T R A C T Two studies were conducted to evaluate the effectiveness of moist heat and lignosulfonate (LS0 3) in increasing the rumen undegradable fraction of canola meal for use as a protein supplement for dairy cattle. In the first study, the in situ nylon bag and mobile nylon bag techniques were utilized to determine the dry matter (DM) and crude protein (CP) degradability of canola meal treatments in the rumen, intestines and total tract. Treatments consisted of untreated canola meal and canola meal heated at 100°C, with or without 5% L S 0 3 (wt wt*1), for 0, 30, 60, 90 or 120 min. Results indicated that treatment of canola meal with or without 5% L S 0 3 and heated at 100°C for 120 min was effective in reducing (P<0.05) the rumen degradation of DM and CP compared to untreated canola meal. The corresponding intestinal CP disappearances for untreated, heat treated, and L S 0 3 plus heat treated canola meal following 8 h rumen pre-incubation were 15.9, 23.5 and 34.2%, respectively. The shift in degradability from the rumen to the intestines was accomplished without reducing total tract disappearance. The second study was undertaken to evaluate the effects of feeding diets containing three canola meal protein supplements, varying in rumen degradability, on nutrient digestibility and animal performance. Eighteen lactating multiparous Holstein cows were randomly assigned to treatment sequences in a 3 x 3 Latin Square design, replicated six times. Total mixed rations were formulated to be isonitrogenous and to contain approximately 30% corn silage, 20% grass silage, 24% barley, 4% soybean meal, 1% mineral and vitamin mix and 21% of one of the following canola meal sources: 1) untreated (U-CM); 2) heat treated (HT-CM); or 3) L S 0 3 plus heat treated Ill (LSO3-CM) on a DM basis. Milk production and dry matter intake (DMI) were measured daily during each 42 d experimental period. Milk, blood and rumen fluid samples were taken during the third and fifth weeks. Total collections of urine and feces from nine cows occurred during the last 5 d of each experimental period. Milk production was greater (P<0.05) for cows fed LSO3-CM diet (36.6 kg d"1) than U-CM diet (34.8 kg d'1), but did not differ from HT-CM diet (35.4 kg d"1). Cows supplemented with LSO3-CM showed reduced (P<0.05) apparent CP digestibility and concentrations of rumen ammonium N, blood urea nitrogen and milk urea nitrogen compared to cows supplemented with U-CM and HT-CM. DMI and apparent digestibilities of neutral and acid detergent fibre were increased (P<0.05) in cows fed LSO3-CM diet when compared to cows fed U-CM and HT-CM diets. The urinary excretion of N, as a percentage of N intake, was reduced (P<0.05) in cows fed LSO3-CM diet relative to cows fed U-CM and HT-CM diets. The results of these studies showed that treatment of canola meal with 5% LSO3 followed by heating at 100°C for 120 min was an effective means of increasing the proportion of protein digested in the intestines. When supplemented in the diets of lactating cows, the protein in LSO3 -CM was used more efficiently and was more effective as a rumen undegraded protein source than was the protein in HT-CM or U-CM. iv T A B L E OF CONTENTS A B S T R A C T II T A B L E OF C O N T E N T S IV LIST OF T A B L E S VI LIST OF FIGURES VIII A C K N O W L E D G M E N T S IX LIST OF S Y M B O L S , N O M E N C L A T U R E AND ABBREVIATIONS X I. G E N E R A L INTRODUCTION 1 1.1 DIETARY PROTEIN INTAKE AND DIGESTION 2 1.2 C A N O L A M E A L 3 1.3 PROTEIN PROTECTION 4 1.3.1 Formaldehyde 5 1.3.2 Heat Treatment 6 1.4 EVALUATION OF RUMEN PROTEIN DEGRADABILITY 9 1.4.1 Disadvantages of the Nylon Bag Technique 10 1.4.2 Advantages of the Nylon Bag Technique 11 1.5 EVALUATION OF INTESTINAL AND TOTAL TRACT DEGRADABILITY 12 1.5.1 Disadvantages of the Mobile Nylon Bag Technique 12 1.5.2 Advantages of the Mobile Nylon Bag Technique 13 1.6 SUMMARY 13 1.7 OBJECTIVES 14 1.8 R E F E R E N C E S 15 II. THE E F F E C T S OF HYDROTHERMAL COOKING OF C A N O L A M E A L ON IN SITU RUMEN AND INTESTINAL D ISAPPEARANCE OF DRY M A T T E R AND C R U D E PROTEIN 21 2.1 INTRODUCTION 21 2.2 MATERIALS AND METHODS 23 2.2.1 Canola Meal Treatments 23 2.2.2 Animals and Basal Diet 24 2.2.3 Rumen Disappearance 24 2.2.4 Intestinal Disappearance 25 2.2.5 Chemical Analyses 26 2.2.6 Calculations and Statistical Analyses 26 2.3 RESULTS 27 2.3.1 Rumen Disappearance of DM and CP. . 27 2.3.2 Total Tract and Intestinal Disappearance of DM and CP 28 2.4 DISCUSSION 29 2.5 R E F E R E N C E S 34 V III. PRODUCTION AND DIGESTION RESPONSES OF LACTATING DAIRY COWS TO THE FEEDING OF HEAT AND LIGNOSULFONATE TREATED CANOLA MEAL 46 3.1 INTRODUCTION 46 3.2 MATERIALS AND METHODS 48 3.2.1 Canola Meal Treatments 48 3.2.2 Experimental Design 49 3.2.3 Diets 50 3.2.4 Sampling 50 3.2.4.1 Feed 50 3.2.4.2 Milk 51 3.2.4.3 Rumen Fluid 51 3.2.4.4 Blood : 52 3.2.5 Total Collections 52 3.2.6 Efficiency Calculations 53 3.2.7 Statistical Analysis 53 3.3 RESULTS 54 3.3.1 Feed Composition 54 3.3.2 Dietary Intakes and Milk Production and Composition 56 3.3.3 Blood Composition 56 3.3.4 Rumen Fluid 57 3.3.5 Water Intake and Waste Excretion 57 3.3.6 Apparent Digestibility of Nutrients 57 3.3.7 Nitrogen Balance 58 3.3.8 Nitrogen Efficiency 58 3.3.9 Energy Efficiency 59 3.4 DISCUSSION 59 3.5 R E F E R E N C E S 70 IV. GENERAL CONCLUSIONS 92 vi LIST O F T A B L E S Table 2.1. Canola meal processed with graded levels of lignosulfonate (LS0 3) and moist heat at 100°C 37 Table 2.2. Changes in DM content that occurred during processing of canola meal with graded levels of lignosulfonate (LS0 3) and moist heat at 100°C 38 Table 2.3. DM disappearance (% of initial) of heat and lignosulfonate (LS0 3 ) treated canola meal and Soy Pass® in the rumen (n=4) 39 Table 2.4. CP disappearance (% of initial) of heat and lignosulfonate (LS0 3 ) treated canola meal and Soy Pass® in the rumen (n=4) 40 Table 2.5. The intestinal DM and CP disappearance (% of initial) of heat and lignosulfonate (LS0 3) treated canola meal and Soy Pass® (n=8) 41 Table 2.6. The total tract DM and CP disappearance (% of initial) of heat and lignosulfonate (LS0 3) treated canola meal and Soy Pass® (n=8) 42 Table 3.1. Ingredient composition of the total mixed rations 76 Table 3.2. Changes in moisture content that occurred during processing of individual batches of treated canola meal cooked in a hydrothermal cooker for 120 min at 100°C 77 Table 3.3. Chemical composition of individual batches of untreated canola meal and canola meal cooked in a hydrothermal cooker for 120 min at 100°C, with or without 5% L S 0 3 78 Table 3.4. The influence of processing batch on the rumen DM disappearance (% of initial) of untreated and treated canola meal fed during the lactation trial (n=4) 79 Table 3.5. The influence of processing batch on the rumen CP disappearance (% of initial) of untreated and treated canola meal fed during the lactation trial (n=4) 80 Table 3.6. The intestinal and total tract DM and CP disappearance (% of initial) following 8 h rumen incubation of different processing batches of untreated and treated canola meal fed during the lactation trial (n=8) 81 Table 3.7. Chemical composition of major diet components 82 vii Table 3.8. Chemical composition of the total mixed rations 83 Table 3.9. The effects of diets supplemented with different canola meal treatments on DMI, body weight, milk yield and milk composition 84 Table 3.10. The effects of diets supplemented with different canola meal treatments on blood composition 85 Table 3.11. The effects of diets supplemented with different canola meal treatments on ruminal pH, ammonium nitrogen (NH 4 + N) and V F A concentrations 86 Table 3.12. The effects of diets supplemented with different canola meal treatments on the water intake and urine and fecal outputs by cows 87 Table 3.13. The apparent digestibilities of diets supplemented with different canola meal sources 88 Table 3.14. Nitrogen balance measurements for cows fed three different canola meal treatments 89 Table 3.15. The utilization of N, as a percentage of N intake, in cows fed different canola meal sources 90 Table 3.16. The effect of canola meal treatment on energy expenditure and efficiency 91 viii LIST O F F I G U R E S Figure 2.1. The DM disappearance (% of initial) of untreated canola meal and canola meal heated with (LSO3-120) or without 5% L S 0 3 (heat-120) in the rumen of non-lactating cows 43 Figure 2.2. The CP disappearance of untreated canola meal and canola meal heated with (LSO3-120) or without 5% L S 0 3 (heat-120) in the rumen of non-lactating cows 44 Figure 2.3. Partitioning of total tract CP disappearance of untreated, heat treated and lignosulfonate (LS0 3) plus heat treated canola meal between rumen and intestines following rumen incubation for 8 h 45 ix A C K N O W L E D G M E N T S I would like to express my sincere thanks to my supervisors Dr. J . A. Shelford of the Animal Science Department at UBC and Dr. L. J. Fisher of Agriculture and Agri-Food Canada's Pacific Agri-Food Research Centre (PARC) in Agassiz, B. C. for their advice, encouragement, and assistance in carrying out this research and for their valued comments on the thesis. Gratitude is extended to Dr. R. M. Tait for his advice throughout the project. I would also like to thank Drs. J. R. Thompson and Z. Xu for the time they dedicated to reading this thesis and for their constructive comments. Appreciation is extended to Mary Lou Swift of Agro Pacific Industries for her initiation of the project and her support throughout it. I am grateful to Dr. Marina von Keyserlingk for her organization of and perseverance throughout this project. The friendship and valued input from my colleagues at UBC has been appreciated. I am grateful to the dairy staff and summer students at PARC (Agassiz, B.C.) for the care of the animals used in the studies and their assistance with the experiments. The expertise of Dr. John Hall of PARC (Summerland, B.C.) for statistical advice and Gay Wilson of PARC (Agassiz, B.C.) for her assistance with laboratory analysis has been gratefully appreciated. Financial support for this research was provided by Agro Pacific Industries in partnership with NSERC (Industrial Postgraduate Scholarship). Above all I am grateful to Nicola Ashurst and my family, for the completion of this project was made possible through their love, patience and everlasting support. LIST OF SYMBOLS, NOMENCLATURE AND ABBREVIATIONS Nomenclature L S 0 3 calcium-sodium lignosulfonate N H 4 + N ammonium nitrogen Abbreviations ADF acid detergent fibre ADIN acid detergent insoluble nitrogen AA amino acid BUN blood urea nitrogen CM canola meal CP crude protein d day dl decalitre DM dry matter DMI dry matter intake FCM fat corrected milk h hour HT-CM heat and water treated canola meal L S O 3 - C M heat and lignosulfonate treated canola meal min minute mM millimolar MUN milk urea nitrogen NEL net energy for lactation N nitrogen NDF neutral detergent fibre NPN non-protein nitrogen RDP rumen degradable protein RUP rumen undegradable protein SBM soybean meal S C C somatic cell counts S E standard error TMR total mixed ration U-CM untreated canola meal V F A volatile fatty acid wt weight 1 I. GENERAL INTRODUCTION As a consequence of their foregut fermentation, a ruminant's amino acid (AA) requirements are provided by two major sources. One is microbially synthesized protein of good quality in terms of its AA content (Storm and Orskov 1983) and digestibility (Storm et al. 1983), while the other comes from dietary protein that escapes rumen fermentation. Digestion of this mixture of microbial protein and undegraded dietary protein in the abomasum and small intestine yields the AA supply to the ruminant. Early research demonstrated that cattle fed purified diets with only non-protein nitrogen as a nitrogen (N) source gained 65% of the weight of the cattle fed conventional energy ingredients and protein supplements (Oltjen 1969). When lactating dairy cattle were fed protein free diets, they produced 4000 kg milk per lactation from microbial protein (Virtanen 1966; Virtanen et al. 1972). However, by supplying protein for 20% and 40% of the N needed, milk production increased 1000 kg and 1500 kg, respectively. The importance of dietary protein in beef and dairy cattle production was soon recognized. Postruminal administration of AA that were thought to limit milk protein synthesis has shown positive effects on milk protein production (Clark 1975; Schwab et al. 1976; Guinard and Rulquin 1994). The undegradable protein concept was introduced by the NRC (1989) where requirements for rumen degradable (RDP) and rumen undegradable protein (RUP) were separately defined. Use of RUP sources in ruminant diets has become a common practice in diet formulation. 2 1.1 DIETARY PROTEIN INTAKE AND DIGESTION The younger the animal or the higher its milk production, the greater its requirement for undegradable protein (Orskov et al. 1980). Quality protein supplements are fed to meet the protein requirements of high producing dairy cows. However, the rumen microflora are highly proteolytic and thus ensure that most protein entering the rumen is degraded to peptides and AA, most of which are subsequently deaminated (Orskov 1992). Consequently, protein supply may still be inadequate to meet the requirements of production. The difficulty arises in part because microbial growth and protein degradation are not coupled directly (NRC 1989). The rate at which energy is generated for microbial growth is not synchronized with the more rapid degradation of protein. Thus, much of the value of protein supplements is lost because dietary protein N is converted to ammonium N. Ammonium N (NH 4 + N) promotes microbial growth up to the limit of the microbial N requirement, which is set by the available fermentable carbohydrate, the ATP yield, and the efficiency of conversion to microbial cells (Van Soest 1994). Excess N H 4 + diffuses from the rumen to the blood where the liver rapidly converts N H 4 + to urea, which is eventually recycled or excreted as urea in urine (NRC 1989). Increasing excesses of N in the rumen leads to reduced use of the recycled endogenous urea and more excretion in urine and milk (Hof et al. 1997). This results in the inefficient utilization of dietary protein. Large excesses of dietary protein may decrease the energy supply to the cow as the consumption of excess crude protein (CP) increases energy requirements by 13.3 kcal of digestible energy g*1 of excess N (NRC 1989). 3 Urea equilibrates in body water, and kinetic analysis suggests the passive transfer of urea along with water from blood into other body fluids, including saliva, uterine fluid and milk (Baker et al. 1992). High concentrations of urea in body fluids have been associated with negative impacts on health (Carroll et al. 1997), impaired fertility (Ferguson et al. 1993; Butler et al. 1996), reduced metabolic efficiency of milk yield (Tyrrell and Moe 1975), environmental pollution concerns as greater than 95% of endogenous urea is excreted in urine (Baker et al. 1992; Tamminga 1992), and economic loss. In cows fed isonitrogenous diets, blood urea nitrogen (BUN) was elevated with imbalances in RDP and RUP (Baker et al. 1995). BUN concentrations have also been shown to decrease when more optimal levels of ruminally fermentable carbohydrate were supplied to enhance the incorporation of RDP into microbial protein (Roseler et al. 1993). BUN may serve as an indicator of ruminal protein degradability and postruminal protein supply; however, sampling requires invasive techniques, and routine analysis is expensive. Milk urea N has a high correlation with BUN (Roseler et al. 1993; Broderick and Clayton 1997) and has the potential to be utilized as an economical noninvasive measurement to assist in monitoring the protein status of dairy cows fed a particular diet. 1.2 CANOLA MEAL The economic value of supplemental protein used in rations is largely determined by the amount of RUP that is available for digestion and absorption in the small intestine (Tremblay et al. 1996). Canola meal (CM), a readily available plant protein supplement (36-40% CP, DM basis) rich in calcium, phosphorous and B vitamins, is the principal protein source used in ruminant rations in Canada (Christensen and McKinnon 1989). Relative to the composition of milk protein, CM has an excellent balance of AA and is a rich source of methionine, cysteine, histidine and threonine (Christensen and McKinnon 1993). However, CM has a relatively low protein efficiency because of extensive rumen degradation relative to by-product feed ingredients such as meat, bone and blood meal. Reported effective rumen degradabilities of CM range from 44.3 (Kendall et al. 1991) to 74.9% (McAllister et al. 1993). The use of CM use as a RUP source is limited because it is a highly degradable protein, leading to surplus N H 4 + production in the rumen and reduced intestinal AA availability. Several procedures, including chemical and physical treatments, have been studied to alter the rate and extent of rumen microbial protein degradation. 1.3 PROTEIN PROTECTION The production of insoluble protein through heat (physical) or by complexing (chemical) decreases the rate of proteolytic hydrolysis. This is not only through reduced accessibility of the substrate, but also through the formation of linkages resistant to enzyme attack. Chemical treatments can be divided into methods in which the chemicals actually combine with the proteins, e.g. formaldehyde treatment, and those in which the chemicals denature the proteins, e.g., alcohol, sodium hydroxide and propionic acid. Physical treatments include heat, applied with a wide variation of application methods, or physical encapsulation, in which a protein supplement is coated with a product such as blood that is resistant to rumen degradation (Broderick et al. 1991). Producing resistant linkages with heat or formaldehyde treatment risks permanently reducing the availability of protein, which 5 then becomes a part of the ultimately indigestible residue. The most successful chemical treatment has been formaldehyde and the most successful and commonly used physical treatment has been heat. 1.3.1 Formaldehyde Aldehydes, of which formaldehyde is the cheapest and most reactive, form complexes with AA residues rendering proteins less soluble at ruminal pH and more resistant to microbial degradation (Weakley et al. 1983). Formaldehyde treatments are designed upon the presumption that linkages resistant to rumen degradation will be broken by acidic conditions (pH 2-3) in the abosmasum and by enzymatic digestion, making the protein available for digestion and absorption in the lower tract (Van Soest 1994). Protection of this type results in biomanipulation in which digestive processes of supplements fed to ruminants can be modified to resemble those of a monogastric animal in terms of protein composition of the tissue or milk. Overprotection of protein is a major problem in the application of formaldehyde treatment as increasing amounts have resulted in progressively reduced digestibilities (Broderick et al. 1991). As formaldehyde has been recognized as a carcinogen, there is a concern that formaldehyde used in protein protection may be transferred to milk and therefore present a health hazard. However, Atwal and Mahadevan (1994) reported that feeding formaldehyde treated soybean meal (SBM) did not increase formaldehyde levels above those naturally occurring in milk ranging from 0.02 mg kg"1 to 0.2 mg kg"1. Despite other studies that have not indicated any adverse health problems due to 6 feeding formaldehyde protected proteins, industry and consumer concerns about the potentially adverse health risks limit the use of formaldehyde (Broderick et al. 1991). 1.3.2 Heat Treatment Heat is the most commonly used treatment because of its efficacy, cost-effectiveness, safety and ease of application (Hussein et al. 1995). The application of heat causes coagulation or denaturation of the protein reducing the solubility and accessibility of the substrate. However, heat treatment primarily decreases protein solubility due to cross-linking between peptide chains and carbohydrates in the Maillard or non-enzymatic browning reaction. Amino acids such as lysine with a free amino group in the peptide form are very reactive (Van Soest 1994). Mild or moderate heating of proteins in the presence of carbonyl compounds, usually reducing sugars, begins with a simple addition to form a Schiff's base, followed by the Amadori rearrangement of the Schiffs base to the 1-amino-1-deoxy-2-ketose (Amadori) compound (Labuza et al. 1977). These intermediate amino-sugar complexes represent the cross-linkage of a sugar aldehyde group to a free epsilon amino group of lysine, while the alpha amino group is bound in the protein structure (Erbersdobler 1977). This complex is more resistant than normal peptides to enzymatic hydrolysis, thus this is the major process by which heating protects protein from rumen degradation. Of critical importance to nutrition is that this reaction is reversible, yielding utilizable lysine from the Amadori compounds following abomasal (pH 2-3) digestion and making it available for intestinal absorption (Erbersdobler 1977). 7 The advanced stages of the Maillard reaction, precipitated by excessive heat, result in permanent cross-linking of the peptide and carbohydrate chains yielding nutritionally unavailable brown melanoidin polymers (Labuza et al. 1977). These compounds prevent enzyme penetration or mask the sites of enzyme attack (Hurrell and Finot 1985; Ames 1992) which leads to reduced post-rumen availability of sugars and AA. These end products, containing about 11 % nitrogen, possess many of the physical properties of lignjn and are recoverable in lignin and acid detergent fibre (Van Soest 1994). The rate and the extent of the Maillard reaction are affected by a number of determinants, the most important being: temperature and length of heat exposure; availability of free amino groups and reducing sugars; moisture content; and pH (Ames 1992). The degree of heating practiced by commercial canola seed processing plants during oil extraction and preparation of CM is insufficient to maximize the escape of protein and AA in CM from the rumen (Moshtaghi Nia and Ingalls 1995). Moshtaghi Nia and Ingalls (1995) found that 15 min of moist heat treatment (autoclaving, 127°C) on CM had no significant effect on in situ total tract disappearance of AA, but 45 min of heat treatment resulted in the classic decrease in digestibility that occurs with extended heating. Moshtaghi Nia and Ingalls (1992) concluded that the beneficial effects of the decreased rate of protein degradation in the rumen appeared to be greater than the decreased protein digestibility in the total Gl tract caused by limited heat damage. The rate of the Maillard reaction is also highly dependent on moisture content, with less heat energy required at higher moisture levels (Lingnert 1990). Maximum reaction rates have been reported at moisture levels of 30%, but the effect of water is 8 variable (Van Soest 1994). However, high moisture levels are not practical as the energy required to dry a product for extended storage surpasses the energy conserved by heating at high moisture levels. The most reactive carbohydrates in plants are the hemicelluloses and soluble sugars with cellulose and starch being comparatively more stable (Marounek and Brezina 1993). The order of reactivity of reducing sugars in the Maillard reaction is xylose > arabinose > glucose > lactose > maltose > fructose, with fructose being only one-tenth as reactive as glucose (Ames 1992). Cleale et al. (1987) found that treatment of SBM with xylose was more effective in reducing in vitro degradation of SBM by rumen micro-organisms than was glucose, lactose or fructose. Calcium-sodium lignosulfonate (LS0 3), a non-toxic by-product of the wood pulp industry, is derived from the spent sulphite liquor that is generated during the sodium sulphite digestion of wood. It contains hemicellulose and reducing sugars of which approximately 24% are xylose. Traditionally lignosulfonate products have been used to precipitate protein from packing house waste water, bind to protein in the leather tanning process, and bind feed pellets in the manufacture of animal feeds (Windschitl and Stern 1988). Treatment of SBM and CM with 5% L S 0 3 in combination with heat has been shown to successfully reduce protein degradation by ruminal microbes (Windschitl and Stern 1988; McAllister et al. 1993). The reductions in CM protein degradation in situ occurred without substantially increasing indigestible protein as assessed by ADIN (McAllister et al. 1993). However, the decreased degradation was less than that observed for SBM by Windschitl and Stern (1988). Heating SBM at 95°C for 45 min with 15-20% moisture had no effect on protein degradability compared to untreated SBM, but 5% L S 0 3 and heat resulted in 9 significantly lower degradation (Windschitl and Stem 1988). Similarly, McAllister et al. (1993) found treatment of CM with 5% L S 0 3 followed by heat at 100°C for 60 min with 25% moisture caused a larger reduction in effective rumen degradability of protein than heat treatment without L S 0 3 . Stanford et al. (1995) concluded that a combination of L S 0 3 and heat is necessary to achieve large increases in rumen undegradable protein when CM is heated at temperatures between 95°C and 110°C for 1 to 2 h. Thus, if the Maillard reaction can be controlled in such a manner that the formation of reversible primary products is maximized without the formation of the unavailable terminal products, the value of a supplemental protein could be enhanced. 1.4 EVALUATION OF RUMEN PROTEIN DEGRADABILITY Accurate determination of the RDP and RUP fractions is of critical importance in the evaluation of the supply of nutrients for absorption and utilization by high yielding cows. The efficacy of treatments designed to alter ruminal protein digestion has been evaluated using in vivo, in vitro and in situ techniques. Conventional in vivo digestibility measurements are believed to accurately reflect the feeding value of total diets (Orskov 1992), however, in addition to being laborious and time consuming, they are limited to a single feedstuff or combination of feedstuffs. A quick, effective and widely accepted method to determine the RDP and RUP fractions of a feedstuff is the dacron polyester or nylon bag technique. This popular technique has been accepted by A R C (1984) and NRC (1989) as the method of choice to determine the protein degradability of a feedstuff (Orskov 1992). The nylon bag technique involves the incubation of a feedstuff in a synthetic nylon bag, which is suspended in the rumen for 10 varying lengths of time. The pore size (50 um) of the bag allows bacteria to enter and to digest the feed. The nylon bag technique permits a large number of samples to be evaluated at one time and is suitable for the initial evaluation of a feedstuff. The value for the degradation is used to determine both the degradable portion available for micro-organisms and the undegradable protein which may be available for enzymatic digestion in the intestine. 1.4.1 Disadvantages of the Nylon Bag Technique Care must be exercised in the interpretation of results as the nylon bag technique is used to measure disappearance, and we assume that disappearance is synonymous with degradation (Kennelly and Ha 1983). The strongest criticism of this technique is its low repeatability as suggested by the diversity of values obtained by different researchers for a similar feed sample (Michalet-Doreau and Ould-Bah 1992). Potential explanations for this discrepancy could be: 1) microbial growth within the nylon bag may underestimate N loss (Nocek and Grant 1987); 2) the actual number of microbes within the bag may be an under representation of the level present in the rumen leading to lower loss (Meyer and Mackie 1986); and 3) the rapid removal of soluble materials and very fine particles may overestimate N loss (England et al. 1997). The greatest error is believed to arise from the net migration of microbes from the rumen into the bag as a result of their attachment to the feedstuff. The challenge is trying to accurately quantify the bacterial component of nylon bag residues following rumen incubation. Bacterial contamination can be a serious problem leading to underestimation of protein degradation for forages (Varvikko and Lindberg 1985; 11 Nocek and Grant 1987). Correspondingly, others (Mathers and Aitchison 1981; Hof et al. 1990) stated that the problem appears not to be associated with concentrates and that the extent of microbial contamination was nutritionally insignificant. Thus, corrections for bacterial contamination are most appropriate for high fibre, low protein feeds (Waters and Givens 1992). Several factors identified by Michalet-Doreau and Ould-Bah (1992) and Stern et al. (1994) that need to be controlled if this technique is to be standardized include: 1) the porosity of the nylon bag (must be large enough for the degradation process to take place normally within the bag and sufficiently small to limit the efflux of undegraded feed particles); 2) fineness of the ground sample (must mimic the effect of mastication); 3) the ratio of sample weight to bag surface area; 4) method of bag placement in the rumen; 5) basal diet of the animal; and 6) degree of bacterial attachment to feed residues remaining in the bag. While requiring relatively little equipment, this technique does require surgical preparation of an animal with a ruminal cannula and facilities for its maintenance which may be expensive. 1.4.2 Advantages of the Nylon Bag Technique Although the in situ nylon bag technique is imperfect in ways that cannot be fully corrected for, it is rapid, fairly reproducible, and requires minimal apparatus (Broderick et al. 1988). The technique has gained popularity due to its versatility and ability to give reliable estimates of in vivo degradation across a wide range of feedstuffs. Most importantly, unlike in vitro procedures, it involves digestive processes that occur in the rumen of a living animal (Nocek 1988). As a relatively 12 quick and simple procedure, it allows many feedstuffs to be evaluated simultaneously (Stern et al. 1983). 1.5 EVALUATION OF INTESTINAL AND TOTAL TRACT DEGRADABILITY Low rumen degradability of a feedstuff assessed using the in situ technique does not necessarily mean it is available for digestion and absorption in the lower Gl tract. Determination of the proportion of a feed degraded in the intestine can be evaluated using the in situ mobile nylon bag technique. The mobile bag technique developed by Hvelplund (1985) and modified by de Boer et al. (1987) involves a sequence of pre-incubating a feedstuff in a small nylon bag in the rumen for 8 h; incubating the bag in vitro in an acid-pepsin solution; inserting the bag into the duodenum via a cannula; and collecting the bag in the feces. The proportion of protein degraded in the intestines is equal to total tract digestion minus the protein degraded in the rumen. 1.5.1 Disadvantages of the Mobile Nylon Bag Technique During incubation, microbes attach to the feedstuff and break it down. The degree of contamination of the residue by microbial protein due to sustained attachment has been a subject of controversy (Hvelplund et al. 1992). While Varvikko and Lindberg (1985) reported this to be substantial, others (Hvelplund 1985; Jarosz et al. 1991; Kohn and Allen 1992) have reported limited microbial contamination of feed residues. This technique has also been criticized because disappearance of the feedstuff in the intestine does not infer that the animal absorbed the material. Estimates of protein degradability using the mobile nylon bag technique are considerably greater 13 than those observed using conventional in vivo techniques (Broderick 1994). The substantially higher in situ estimates relative to apparent digestibility values reported for conventional in vivo studies, suggests that the endogenous cost of feeding a protein source is significant (Robinson et al. 1992). Kirkpatrick and Kennelly (1984) reported that the discrepancies between conventional and mobile nylon bag digestibility results can be eliminated if endogenous and other contaminating N sources are not removed through the washing of the bags collected in the feces. Similar to the rumen nylon bag technique, this technique requires animals to be surgically prepared with cannulas both in the rumen and duodenum, which can be time-consuming and expensive. 1.5.2 Advantages of the Mobile Nylon Bag Technique The mobile nylon bag technique is not as time-consuming or expensive as alternative infusion techniques (Hvelplund et al. 1992). In comparison to infusion studies using sheep, Hvelplund et al. (1992) have shown the mobile bag technique produced comparable results. This technique has also been identified by others (de Boer et al. 1987; Varvikko and Vanhatalo 1991) as a promising technique for evaluating the intestinal degradability of feedstuffs. 1.6 Summary Diets formulated according to requirements for CP without consideration of RUP and RDP lead to higher urea N concentrations and lowered efficiency of N utilization for milk production and growth. Controlling the rate and extent of degradation of dietary protein to balance the protein supply from microbial synthesis is of great interest to ruminant nutritionists, because inefficient utilization necessitates 14 the over feeding of protein, the most costly ingredient of a diet. Enhancement of the early stages of the Maillard browning reaction through the use of controlled levels of heat and reducing sugar shows potential to increase the feeding value of CM as a RUP source. Cows supplemented with increased levels of RUP have the potential to increase production and show increased efficiency of N utilization. 1.7 Objectives The objectives of this study were to ascertain the effects of heat and L S 0 3 treatment of CM on 1) in situ ruminal and intestinal disappearance of DM and CP; and 2) in vivo apparent digestibility, milk production and milk composition. 15 1.8 References Ames, J. A. 1992. The Maillard reaction. Pages 99-153 in B. J. F. Hudson, ed. Biochemistry of food proteins. Elsevier Applied Science, London, U.K. ARC (Agricultural Research Council) 1984. The nutrient requirements of ruminant livestock. C. C. Balch, D. G. Armstrong, J. F. D. Greenhalgh, E. L. Miller, E. R. Orskov, J . H. B. Roy, R. H. Smith, and J. C. Taylor, eds. Commonwealth Agricultural Bureaux, Slough, England. Atwal, A.S. and Mahadevan, S. 1994. Formaldehyde in milk not affected by feeding soybean meal coated with chemically treated zein. Can. J. Anim. Sci. 74: 715-716. Baker, L. D., Ferguson, J. D., and Chalupa, W. 1995. Responses in urea and true protein of milk to different protein feeding schemes for dairy cows. J. Dairy Sci. 78: 2424-2434. Baker, L. D., Ferguson, J. D., and Ramberg, C. F. 1992. Kinetic analysis of urea transport from plasma to milk in dairy cows. J. Dairy Sci. 75 (Suppl. 1): 181. Broderick, G. A. 1994. Quantifying forage protein quality. Pages 200-208 in G. C. Fahey Jr., ed. Forage Quality, Evaluation, and Utilization. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc. Madison, WI. Broderick, G. A. and Clayton, M. K. 1997. A statistical evaluation of animal and nutritional factors influencing concentrations of milk urea nitrogen. J. Dairy Sci. 80: 2964-2971. Broderick, G. A., Wallace, R. J., and Orskov, E. R. 1991. Control of rate and extent of protein degradation. Pages 541-592 in T. Tsuda, Y. Sasaki, and R. Kawashima, eds. Physiological Aspects of Digestion and Metabolism in Ruminants. Proc. of the 7 t h International Symp. on Ruminant Physiology. Academic Press, Inc. Broderick, G. A., Wallace, R. J., Orskov, E. R., and Hansen, L. 1988. Comparison of estimates of ruminal degradation by in vitro and in situ methods. J. Anim. Sci. 66: 1739-1745. Butler, W. R., Calaman, J. J., and Beam, S. W. 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating cattle. J. Anim. Sci. 74: 858-865. Carroll, D. J., Barton, B. A., and Thomas, D. R. 1997. Review of protein nutrition-reproduction studies. J. Dairy Sci. 80 (Suppl. 1): 139. 16 Christensen, D. A. and McKinnon, P. J. 1989. Canola meal for beef and dairy cattle. Pages 19-22 in D. R. Clandinin, ed. Canola meal for livestock and poultry. Canola Council of Canada, Winnipeg, MB. Christensen, D. A. and McKinnon, J. J. 1993. Canola meal for beef and dairy cattle. Pages 21-26 in D. Hickling ed. Canola Meal: Feed Industry Guide. Canola Council of Canada, Winnipeg, MB. Clark, J.H. 1975. Lactational response to postruminal administration of proteins and amino acids. J. Dairy Sci. 58: 1178-1197. Cleale, R. M., Klopfenstein, T. J., Britton, R. A., Satterlee, L. D., and Lowry, S. R. 1987. Induced non-enzymatic browning of soybean meal I. Effects of factors controlling non-enzymatic browning on in vitro ammonia release. J. Anim. Sci. 65: 1312-1318. de Boer, G., Murphy, J. J., and Kennelly, J. J. 1987. Mobile nylon bag for estimating intestinal availability of rumen undegradable protein. J. Dairy Sci. 70: 977-982. England, M. L., Broderick, G. A., Shaver, R. D., and Combs, D. K. 1997. Comparison of in situ and in vitro techniques for measuring ruminal degradation of animal by-product proteins. J. Dairy Sci. 80: 2925-2931. Erbersdobler, H. F. 1977. The biological significance of carbohydrate-lysine crosslinking during heat-treatment of food proteins. Pages 367-378 in M. Friedman, ed. Protein crosslinking: nutritional and medical consequences. Plenum Press, NY. Ferguson, J. D., Gallighan, D. T., Blanchard, T., and Reeves, M. 1993. Serum urea nitrogen and conception rate: the usefulness of test information. J. Dairy Sci. 76: 3742-3746. Guinard, J. and Rulquin, H. 1994. Effect of graded levels of duodenal infusions of casein on mammary uptake in lactating cows. 2. Individual amino acids. J. Dairy Sci. 60: 1706-1724. Hof, G., Kouwenberg, W. J. A., and Tamminga, S. 1990. The effect of washing procedure on the estimation of the in situ disappearance of amino acids from feed protein. Neth. J. Agric. Sci. 38: 719-724. Hof, G., Vervoorn, M. D., Lenaers, P. J., and Tamminga, S. 1997. Milk urea nitrogen as a tool to monitor the protein nutrition of dairy cows. J. Dairy Sci. 80: 3333-3340. Hurrell, R. F. and Finot, P. A. 1985. Effects of food processing on protein digestibility and AA availability. Pages 233 - 246 in J. W. Finley and D. T. Hopkins, eds. Digestibility and amino acid availability in cereals and oilseeds. American Assn. Of Cereal Chemists, Inc., MN. 17 Hussein, H. S., Demjanec, B., Merchen, N. R., and Aldrich, C. G. 1995. Effect of roasting on site and extent of digestion of soybean meal by sheep: II. Digestion of Artifacts of Heating. J. Anim. Sci. 73: 835-842. Hvelplund, T. 1985. Digestibility of rumen microbial protein and undegraded dietary protein estimated in the small intestine of sheep and by in sacco procedure. Acta Agric. Scand. 25 (Suppl.): 132-144. Hvelplund, T., Weisberg, M. R., and Anderson, L. S. 1992. Estimation of the true digestibility of rumen undegraded dietary protein in the small intestine of ruminants by the mobile nylon bag technique. Acta. Agric. Scand. Sect. A. Anim. Sci. 42: 34-39. Jarosz, L., Weisbjerg, M. R., Hvelplund, T., and Borg Jensen, B. 1991. Digestibility of nitrogen and 1 5 N from different roughages in the lower gut of cows estimated with the mobile nylon bag procedure. Pages 113-115 in B. O. Eggum, S. Boisen, C. Borsting, A. Daufer, and T. Hvelplund, eds. Protein metabolism and nutrition. Proceedings of the 6 t h Int. Symp. on Protein Metabolism and Nutrition. EAAP Publication No. 59, Vol. 2. Nat. Inst. of Anim. Sci., Research Center Foulum. Kendall, E. M., Ingalls, J. R., and Boila, R. J. 1991. Variability in the rumen degradability and postruminal digestion of the dry matter, nitrogen and AAs of canola meal. Can. J. Anim. Sci. 71: 739-754. Kennelly, J. J. and Ha, J. K. 1983. Protein quality for ruminant animals. Use of nylon bag technique as a rapid, reliable indicator of protein quality. Pages 113-114 in 62 n d Annual Feeders' Day Report. Agric. For. Bull. Univ. of Alberta, Edmonton, Canada. Kirkpatrick, B. K. and Kennelly, J. J. 1984. Prediction of digestibility in cattle using a modified nylon bag technique. Can. J. Anim. Sci. 64: 1104. Kohn, R. A. and Allen, M. S. 1992. Storage of fresh and ensiled forages by freezing affects fibre and crude protein fractions. J. Sci. Food Agric. 58: 215-220. Labuza, T. P., Warren, R. M., and Warmbier, H. C. 1977. The physical aspects with respect to water and non-enzymatic browning. Pages 379-418 in M. Friedman, ed. Protein crosslinking: nutritional and medical consequences. Plenum Press, NY. Lingnert, H. 1990. Development of the Maillard reaction during food processing. Pages 171-185 in Finot et al., eds. The Maillard reaction in food processing, human nutrition and physiology. Birkhauser Verlag, Berlin, Germany. Marounek, M. and Brezina, P. 1993. Heat-induced formation of soluble maillard reaction products and its influence on utilization of glucose by rumen bacteria. Arch. Anim. Nutr. 43: 45-51. 18 Mathers, J. C. and Aitchison, E. M. 1981. Direct estimation of the extent of contamination of food residues by microbial matter after incubation with synthetic fibre bags in the rumen. J. Agric. Sci. (Camb) 96: 691-693. McAllister, T. A., Cheng, K. -J., Beauchemin, K. A., Bailey, D. R. C , Pickard, M. D., and Gilbert, R. P. 1993. Use of lignosulfonate to decrease the rumen degradability of canola meal protein. Can. J. Anim. Sci. 73: 211-215. Meyer, J. A. F. and Mackie, R. I. 1986. Microbiology of feed samples incubated in nylon bags in the rumen of sheep. S. Afr. Tydskr. Veek. 13: 220-222. Michalet-Doreau, B. and Ould-Bah, M. Y. 1992. In vitro and in sacco methods for the estimation of dietary nitrogen degradability in the rumen: a review. Anim. Feed Sci. Technol. 40: 57-86. Moshtaghi Nia, S. A. and Ingalls, J. R. 1992. Effect of heating on canola meal protein degradation in the rumen and digestion in the lower gastrointestinal tract of steers. Can. J. Anim. Sci. 72: 83-88. Moshtaghi Nia, S. A. and Ingalls, J. R. 1995. Evaluation of moist heat treatment of canola meal on digestion in the rumen, small intestine and total digestive tract of steers. Can. J. Anim. Sci. 75: 279-283. Nocek, J. E. 1988. In situ and other methods to estimate ruminal protein and energy digestibility: A review. J. Dairy Sci. 71: 2051-2069. Nocek, J. E. and Grant, A. L. 1987. Characterization of in situ nitrogen contamination of hay crop forages preserved at different dry matter percentages. J. Anim. Sci. 64: 552-564. NRC (National Research Council) 1989. Nutrient requirements of dairy cattle. 6 t h rev. ed. National Academy Press, Washington, DC. Oltjen, R. R. 1969. Effects of feeding ruminants non-protein nitrogen as the only nitrogen source. J. Anim. Sci. 28: 673-682. Orskov, E. R. 1992. Protein nutrition in ruminants. 2 n d ed. Academic Press, London. Orskov, E. R., Hughes-Jones, M., and McDonald, I. 1980. Degradability of protein supplements and utilization of undegraded protein by high-producing dairy cows. Page 85-98 in W. Haresign, ed. Recent Advances in Animal Nutrition. Butterworth & Co. London, U.K. Robinson, P. H., Okine, E. K., and Kennelly, J. J. 1992. Measurement of protein digestion in ruminants. Pages 121-144 in S. Nissen, ed. Modern Methods in Protein Nutrition and Metabolism. Academic Press, Inc. 19 Roseler, D. K., Ferguson, J. D., Sniff en, C. J., and Herrema, J. 1993. Dietary protein degradability effects on plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J . Dairy Sci. 76: 525-534. Schwab, C. G., Satter, L. D., and Clay, A. B. 1976. Response of lactating dairy cows to abomasal infusion of AAs. J. Dairy Sci. 59: 1254-1270. Stanford, K., McAllister, T. A., Xu, Z., Pickard, M., and Cheng, K. J. 1995. Comparison of lignosulfonate-treated canola meal and soybean meal as rumen undegradable protein supplements for lambs. Can. J. Anim. Sci. 75: 371-377. Stern, M. D., Ortega, M. E., and Satter, L. D. 1983. Retention time in the rumen and degradation of protein supplements fed to lactating dairy cattle. J. Dairy Sci. 66: 1264-1271. Stern, M. D., Varga, G. A., Clark, J. H., Firkins, J. L, Huber, J. T., and Palmquist, D. L. 1994. Evaluation of chemical and physical properties of feeds that affect protein metabolism in the rumen. J. Dairy Sci. 77: 2762-2786. Storm, E. and Orskov, E. R. 1983. The nutritive value of rumen micro-organisms in ruminants 1. Large-scale isolation and chemical composition of rumen micro-organisms. Br. J . Nutr. 50: 463-470. Storm, E., Brown, D. S., and Orskov, E. R. 1983. The nutritive value of rumen micro-organisms in ruminants 3. The digestion of microbial amino and nucleic acids in, and losses of endogenous nitrogen from, the small intestine of sheep. Br. J. Nutr. 50: 479-485. Tamminga, S. 1992. Nutrition management of dairy cows as a contribution to pollution control. J. Dairy Sci. 75: 345-357. Tremblay, G. F., Broderick, G. A., and Abrams, S. M. 1996. Estimating ruminal protein degradability of roasted soybeans using near infrared reflectance spectroscopy. J. Dairy Sci. 79: 276-282. Tyrrell, H. F. and Moe, P. W. 1975. Effect of intake on digestive efficiency. J. Dairy Sci. 58: 1151-1163. Van Soest, P. J. 1994. Nutritional ecology of the ruminant. 2 n d edition. Cornell University Press, Ithaca, NY. Varvikko, T. and Lindberg, J. E. 1985. Estimation of microbial nitrogen in nylon-bag residues by feed 1 5 N dilution. Br. J. Nutr. 54: 473-481. 20 Varvikko, T. and Vanhatalo, A. 1991. Intestinal nitrogen degradation of hay and grass silage estimated by the mobile nylon bag technique. World Rev. Anim. Prod. 26: 73-76. Virtanen, A. I. 1966. Milk productin of cows on protein-free food. Science. 153:1603-1614. Virtanen, A. J., Ettala, T., Makinen, S., and Virtanen, A. 1.1972. Milk production by cows fed on a purified diet free of protein. Zootechnia. 21: 171-199. Waters, C. J. and Givens, D. I. 1992. Nitrogen degradability of fresh herbage: Effect of maturity and growth type, and prediction from chemical composition and by near infrared reflectance spectroscopy. Anim. Feed Sci. Tech. 38: 335-349. Weakley, D. C , Stern, M. D., and Satter, L. D. 1983. Factors affecting disappearance of feedstuffs from bags suspended in the rumen. J. Anim. Sci. 56: 493-507. Windschitl, P. M. and Stern, M. D. 1988. Evaluation of calcium lignosulfonate-treated soybean meal as a source of rumen protected protein for dairy cattle. J. Dairy Sci. 71: 3310-3322. 21 II. THE EFFECTS OF HYDROTHERMAL COOKING OF CANOLA MEAL ON IN SITU RUMEN AND INTESTINAL DISAPPEARANCE OF DRY MATTER AND CRUDE PROTEIN 2.1 INTRODUCTION Combinations of both rumen microbial protein and RUP are necessary to provide sufficient protein to support the high milk yields achieved in dairy cattle. Recommendations for RUP levels within diets have been developed to increase the amount of AA presented to the small intestine for utilization (NRC 1989). The extent to which protein is degraded depends primarily upon microbial proteolytic activity in the rumen, microbial access to the protein, and ruminal retention time of the protein (Stern et al. 1994). Protein structure influences accessibility by proteolytic enzymes, thereby affecting the degradability of protein in the rumen. Some dietary feed ingredients are naturally resistant to ruminal microbial degradation while other ingredients may have greater or lower resistance to microbial degradation because of prior chemical and physical processing (Van Soest 1994). Canola meal (CM) is used as a supplemental protein in dairy cattle diets because of its high protein quality and its competitive pricing compared to other protein supplements (Christensen and McKinnon 1993). Although, the use of CM is limited by its high rumen degradability, its excellent balance of amino acids relative to milk protein make it a promising candidate for research aimed at increasing its RUP content. Chemical treatments administered to increase RUP content include formaldehyde and acids. Formaldehyde treatment (5% wt wt"1) has been successful in reducing CM's in situ rumen CP degradability by 75% (Bailey and Hironaka 1984). Similarly, treatment of CM with acetic acid (3% vol wt"1) followed by oven drying has 22 been shown to reduce its in situ rumen protein degradability by 29%, while reducing its total tract digestibility by only 3% (Khorasani et al. 1993). The most successful physical treatment has been heat (Broderick et al. 1991). Heat promotes the Maillard reaction, which decreases the solubility of proteins by creating cross-linkages between sugar aldehyde and free amino groups. These bonds, while resistant to rumen microbial degradation, are cleaved during acid hydrolysis of protein in the abomasum, yielding AA available for absorption in the duodenum. Moshtaghi Nia and Ingalls (1992) used 90 min of moist heat treatment (autoclaving) at 127°C to reduce disappearance of CM protein by 76% following 10 h of rumen incubation. However, total tract digestibility was also reduced by 15%. In subsequent work, Moshtaghi Nia and Ingalls (1995) reported that 45 min of moist heat (127°C) did not affect total tract disappearance of N, with rumen N disappearance still significantly reduced by 68%. Significant reductions in rumen degradability have resulted from combinations of heat and calcium-sodium lignosulfonate (LS0 3). The L S 0 3 , containing 24% xylose, is derived from the spent sulphite liquor produced during sodium sulphite digestion of hardwoods. McAllister et al. (1993) decreased the CP degradability of CM treated in the laboratory by 20% with L S 0 3 (5% wt wt"1) and heat at 100°C for 60 min. Beauchemin et al. (1995), using similar treatment levels but at a commercial feed manufacturing facility, reduced rumen protein degradability of CM by 52%. As a means to study the ability of treatments to alter protein degradation, the in situ nylon bag technique has received the most extensive evaluation (Michalet-Doreau and Ould-Bah 1992). It is the most widely used technique for assessing nutrient degradability because it is rapid, fairly reproducible, and generally provides reasonable estimates of protein degradation (Stern et al. 1994). 23 The objective of this study was to evaluate the effects of the treatment of CM with L S 0 3 and graded levels of heat delivered in a hydrothermal cooker on in situ ruminal, intestinal and total tract nutrient degradability. 2.2 MATERIALS AND METHODS 2.2.1 Canola Meal Treatments Commercially available solvent extracted canola meal (CM) was processed with combinations (Table 2.1) of heat and LS0 3 (LignoTech USA, Inc., Rothschild, WI). Moist heat was applied at 100°C using a hydrothermal cooker (Amandus Kahl Nachf, Hamburg, Germany) installed at Agro Pacific Industries Ltd. (Chilliwack, BC). The hydrothermal cooker is an insulated cylinder, with capacity to cook 2000 kg of CM at once, that uses steam to control and maintain a pre-set temperature. Four probes located at different positions within the cooker measure temperatures, which are monitored by a computer, which in turn controls steam input. CM samples were removed from the cooker after heating for 30, 60, 90 or 120 min (denoted as heat-30, heat-60, heat-90 and heat-120, respectively). Samples were dried with forced air at ambient temperature (20°C) and then stored in plastic bags. Samples of untreated CM, C M before heating and after heating and the final CM product were collected for DM determination. Dry matter content was determined by drying samples in a forced air oven at 105°C for 24 h. To evaluate the effects of LS0 3 on CM compared to SBM, the commercial product Soy Pass® (LignoTech USA, Inc., Rothschild, WI) was included in the in situ trial as a positive control. Soy Pass® is SBM treated with 5% LS0 3 and heated at 100°C for an undisclosed period of time. 24 2.2.2 Animals and Basal Diet Two non-lactating Holstein cows, fitted with rumen fistulas and T-shaped duodenal cannulas, were fed a diet consisting of 5.5 kg of grass hay and 3.0 kg of 14% CP, commercially prepared dairy concentrate per day (as fed basis). The cows were fed equal portions of the diet at approximately 0730 h and 1630 h. The animals were cared for according to the standards set by the Canadian Council on Animal Care (1993) and the experimental protocol was approved by The University of British Columbia Animal Care Committee. 2.2.3 Rumen Disappearance Nylon bags ( 5 x 1 0 cm; pore size: 50 + 15 um; ANKOM Tech Corp., Fairport, NY) were filled with approximately 3 g of material and sealed using elastic bands. Each sample was incubated in duplicate in the rumen for 24, 16, 12, 8, 4 and 0 h in each of two cows. Nylon bags were suspended in the rumen in a polyester mesh bag (25 x 40 cm; pore size 3 mm) attached to the end of a 50 cm line weighted with a sand filled bottle. Bags were removed at a common time in order to minimize the variation in time that the bags were exposed to air after incubation and to enable simultaneous washing of all bags (Nocek 1985). Following ruminal incubation, the polyester mesh bags containing all the nylon bags (including the zero bags) were rinsed with cold water to remove particulate material. The nylon bags were removed from the mesh bags and placed in a conventional clothes washing machine (von Keyserlingk et al. 1996). The machine was allowed to fill with water and to agitate for 5 min prior to draining, without allowing the bags to be spun. This procedure was repeated until the rinse water remained clear, normally four to five washes. Nylon bags were dried in a 25 forced air oven at 60°C until constant weight was achieved for the determination of DM disappearance. Nylon bag replicates within cows were pooled and residues were ground through a 0.5 mm screen prior to N analysis. 2.2.4 Intestinal Disappearance The mobile nylon bag technique, as described by de Boer et al. (1987), was used to measure the DM and CP disappearance during passage through the intestine. Duodenal nylon bags (3.5 x 5 cm; pore size: 50 + 15 pm; ANKOM, Fairport, NY) were filled with 0.5 g of dry ground (2 mm screen) sample and heat-sealed. Samples were pre-incubated in quadruplicate in the rumen of each cow for 16, 12 and 8 h. Following rumen incubation, the bags were left unwashed and stored at - 2 0 ° C until the day of insertion into the duodenum. On the day of incubation the bags were thawed at room temperature (20°C) and incubated in a pepsin-HCI solution (1 g of pepsin per L of 0.01 N HCI) for 3 h at 39°C to simulate abomasal digestion (Kirkpatrick and Kennelly 1984). Mobile nylon bags were kept at 4°C until they were randomly inserted into the duodenum every 20-30 min. Duodenal bags were collected from the feces (16 h mean passage time) then hand washed until the rinse water remained clear. Dry matter disappearance was determined for all replicates by drying them in a forced air oven at 60°C until constant weight was achieved. All replicates were ground through a 0.5 mm screen. Due to limited amounts of sample remaining after in situ incubation, replicates within cows were pooled prior to nitrogen analysis. Therefore variation within cows was not measured. 26 2.2.5 Chemical Analyses Feeds were analyzed for acid detergent fibre (ADF) and neutral detergent fibre (NDF) using the modified method of Van Soest et al. (1991) called the filter bag technique (ANKOM Co., Fairport, NY; Komarek et al. 1994). Nitrogen determinations, for CP disappearance calculations, were performed using a Leco FP-428 N analyzer (Leco Corp., St. Joseph, Ml). 2.2.6 Calculations and Statistical Analyses Incorporation of microbial protein into the nylon bag contents was not measured in this experiment. This decision was based on the work of Mathers and Aitchison (1981) who stated that in the case of concentrates, the extent of microbial contamination is so minimal that it is nutritionally unimportant. The rumen disappearance of DM and CP at each individual time was calculated as the difference between the feed and the portion remaining after incubation in the rumen. Total tract DM and CP disappearance from each of the nylon bags was calculated as the difference between the feed and the amount remaining (DM basis) after collection of the bag in the feces. The amount of DM and CP that disappeared in the small intestine were calculated for each individual bag by subtracting the amount that disappeared in the rumen from the amount that disappeared in the total tract. Thus, rumen and total tract disappearances were direct measurements while disappearances in the intestines were calculated values. Effective degradabilities of DM and CP have not been included, because the maximum degradation was not reached at 24 h of ruminal incubation (Figures 2.1 and 2.2). Without the establishment of a plateau in degradability values, it was not possible to adequately fit 27 disappearance data to the effective degradability equations (McDonald 1981). Statistically, the data were analyzed using the General Linear Model procedure of SAS (1990). Least square means between treatments were used to test for significant differences. 2.3 RESULTS The addition of 5% LS0 3 reduced the DM content from 87.49% in untreated CM to 86.00% (Table 2.2). Steam heating in the hydrothermal cooker reduced the DM on average by 2.76 and 3.67 percentage points for heat treated and LS0 3 plus heat treated CM, respectively. Thus, the LS0 3 plus heat treated products were cooked at higher moisture levels than were the heat treated products. Forced air drying at ambient temperature increased the DM of the feeds on average by 1.97% and 2.20% for the heat and LS0 3 plus heat treatments, respectively. 2.3.1 Rumen Disappearance of DM and CP Results of the in situ rumen DM and CP evaluation of treated C M are shown in Tables 2.3 and 2.4, respectively. For heat treatment, the extents of ruminal DM and CP disappearance at 8 and 16 h of ruminal incubation decreased with heat-120. Values for 30, 60 and 90 min of heat were generally not significantly different from values for the untreated CM and fell between disappearance values of untreated CM and heat-120. Zero, 4, 12 and 24 h DM disappearances of CM were unaffected by heat treatment, except for significant reductions with heat-30 relative to untreated CM at 4 and 24 h. Application of 5% LS0 3 in the absence of heat reduced the extent of DM and CP disappearance at 0, 4 and 8 h compared to untreated, but was not different at 12, 16 28 and 24 h. DM and CP disappearance of LS0 3-120 was lower than LS0 3 treatment at 0, 4 and 8 h of rumen incubation with disappearances of LS0 3-30, LS0 3-60 and LS0 3 -90 in between. Relative to the control, few consistent reductions in 12, 16 and 24 h rumen disappearances of DM and CP were noted for LS0 3 and increasing levels of heat. Disappearance of Soy Pass® DM and CP was significantly lower at all lengths of rumen incubation. The DM and CP degradation characteristics of untreated, heat-120 and LS0 3-120 CM treatments are illustrated in Figures 2.1 and 2.2. They clearly illustrate that treatment differences, in particular with LS0 3-120, were most pronounced following 4 and 8 h of rumen incubation. 2.3.2 Total Tract and Intestinal Disappearance of DM and CP Neither heat nor LS0 3 plus heat treatment of CM had appreciable effects on total tract disappearance of DM or CP (Table 2.6). The total tract DM disappearance of Soy Pass® was greater than all CM treatments at 12 and 16 h and all but untreated, heat-120 and LS0 3-120 at 8 h rumen incubation. Total tract disappearance of Soy Pass® CP was lower than all CM treatments at all times of rumen incubation. Few consistent differences in intestinal DM disappearances were found (Table 2.5). The outflow rate of protein supplements from the rumen is about 10% h"1 with a high level of feeding of mixed diets to dairy cows (Orskov 1992). Thus, intestinal disappearance results following 8 h rumen incubation were chosen to represent the degradation that would occur in a high producing cows (Figure 2.3). The highest level of heat treatment increased intestinal CP disappearance the most from 15.9 to 23.5% at 8 h and 4.6 to 10.8% at 16 h for untreated and heat-120, respectively. The addition of LS0 3 plus 60, 90 and 120 min of heat increased intestinal disappearance of CM CP 29 at 8 and 16 h relative to untreated CM. There were no effects of treatments observed at 12 h and significant reductions in degradability of the LS0 3 and LS0 3-30 treated CM were only observed at 8 h of rumen incubation. The greatest increases in intestinal protein disappearance occurred with LS0 3-120 treatment, which increased disappearance from 15.9 to 34.2% at 8 h rumen incubation for untreated and LS0 3 -120, respectively. Soy Pass® intestinal DM and CP disappearances were greatest at all levels of incubation, with 8 h intestinal CP disappearance of 73.7% compared to 34.2% for LS0 3-120. 2.4 DISCUSSION The rumen CP disappearance values at 12 h of incubation for untreated C M were similar to those reported by de Boer et al. (1987), but were 6% lower than those of Bailey and Hironaka (1984). Conversely, the protein disappearance results of untreated C M following 16 h of rumen incubation are 19% greater than those reported by Moshtaghi Nia and Ingalls (1992) and 28% greater than those reported by Moshtaghi Nia and Ingalls (1995). Disappearance values of DM followed similar trends. Discrepancies in reported in situ disappearance values can be attributed to varietal or processing differences in the samples of meal incubated (Kendall et al. 1991) or the in situ technique employed. Differences in the basal diet fed to cows in which the nylon bags are placed (Orskov 1992) or variation in the extent of microbial contamination of the incubated samples (Nocek 1988) might also have contributed to differences in this study compared to published observations. The washing procedure utilized in the present study was similar to that used by de Boer et al. (1987), but longer than that used by others (Bailey and Hironaka 1984; Moshtaghi Nia and Ingalls 30 1992, 1995), which may have explained the observed differences. Increased agitation may wash away more small particles or be strong enough to stretch pores in the bags allowing for increased escape of undigested substrate through bag pores (Michalet-Doreau and Ould-Bah 1992). Different commercial processing methods may account for some of the discrepancy as Kendall et al. (1991) reported effective degradabilities of CM protein ranged from 44 to 59%, depending on the source. Basal diets with higher roughage or lower CP contents have been associated with increased protein degradation (Orskov 1992). McAllister et al. (1993) reported that the effective degradability of CM ranged from 62% with an 80% concentrate basal diet to 75% with an alfalfa hay basal diet. The concentrate portion of the diet fed to cows was 30% in the present study, 0% in the study of Bailey and Hironaka (1984), and 50% in the studies of Moshtaghi Nia and Ingalls (1992, 1995). Thus, this may have also contributed to higher observed values in this study relative to Moshtaghi Nia and Ingalls (1992, 1995) and lower observed values relative to Bailey and Hironaka (1984). When comparing in situ results between studies from different labs, it is important to consider the in situ method used, in particular the washing technique and basal diet. Untreated CM was more degradable in the rumen than most treatments. Heat treatment of CM for 2 h at 100°C compared with lower levels of heat decreased rumen CP disappearance. Heat treatment of CM at 125°C or 145°C reduced in situ CP disappearance of CM, but potentially compromised the post ruminal supply of CP (McKinnon et al. 1991). These higher processing temperatures are less desirable because of the formation of indigestible protein (McKinnon et al. 1991). The LS0 3 treatment of CM followed by 2 h of heat at 100°C, compared with lower levels of heat, 31 reduced rumen CP disappearance. Using similar heat conditions McAllister et al. (1993) treated CM with 10% LS0 3 and 2% xylose, equivalent to the xylose content of 10% LS0 3 and found CP degradability was reduced a further 28 and 20%, respectively, over the reduction observed with 5% LS0 3 treatment. However, because of two and three fold increases in ADIN, which is negatively associated with digestibility, they concluded 10% LS0 3 and 2% xylose levels were too high. McAllister et al. (1993) reported that heating CM with LS0 3 for 2 h at 100°C also reduced CP degradability more than heating without LS0 3 . Windschitl and Stern (1988) found no difference in CP degradability of untreated SBM and heat treated SBM, but lower degradation in LS0 3 plus heat treated SBM. Thus, a combination of LS0 3 and heat is necessary to produce large increases in RUP when CM or SBM are heated at temperatures of 100°C for 1-2 h. The less pronounced response of CM to LS0 3 treatment relative to SBM, supports the results of others (Windschitl and Stern 1988) and our results for Soy Pass®. This may reflect the lower lysine content of CM relative to that of SBM as discussed by McAllister et al. (1993). As lysine is the main reactive site for reducing sugars during the Maillard reaction, lowered CM responses in degradability to LS0 3 may be expected. Total tract disappearances of untreated CM DM and CP are in agreement with those of others (Moshtaghi Nia and Ingalls 1992, 1995). It was apparent that the levels of moist heat treatments were not excessive as reflected in the unaltered total tract disappearances of treated CM relative to untreated. The higher total tract DM disappearances of Soy Pass® compared to CM, reflect SBM fibre level, which is considerably lower than in CM (NRC 1989). Soy Pass® protein, relative to untreated 32 SBM protein, may be overprotected as reflected in the consistently lower total tract CP disappearance. With exposure of CM to moist heat for 2 h, a small depression in total tract digestibility might be expected (McAllister al. 1993). However, this was not observed in the present study and suggests the heat delivered was insufficient. Relatively high rumen degradabilities of CM fractions and the marginal separation between disappearances of heat and LS0 3 treated CM may be attributed to shorter than anticipated exposures to 100°C heat. In the present study, timing of heat exposure began when CM first entered the hydrothermal cooker. However, the period of time for CM within the cooker to reach 100°C varied depending upon the initial temperature of the cooker and the length of time necessary for all of the CM to enter. In future work, to ensure that feeds are heated for specific periods of time, the feed should be added to a preheated cooker in as short a time as possible and timing initiated when 100°C is reached. In conclusion, the present study indicates that treatment of CM with or without LS0 3 followed by heating at 100°C for 2 h increases the resistance of CM protein to microbial digestion, likely through the formation of primary Maillard products. This shift in degradability from the rumen to the intestines increases the amount of RUP available for digestion and absorption post ruminally. Of greater importance, this resistance to microbial degradation is apparently accomplished without the formation of intestinally indigestible terminal Maillard products. The increased rumen undegradable protein and the availability of this protein for digestion and absorption in the small intestine could benefit rapidly growing ruminants or high producing cows. Further research is recommended to investigate the repeatability of the treatments, 33 due to the discrepancy in results compared to those of others, and to study the profile and availability of essential AA in the small intestine. 34 2.5 REFERENCES Bailey, C. B. and Hironaka, R. 1984. Estimation of the rumen degradability of nitrogen and of nonprotein organic matter in formaldehyde-treated and untreated canola meal. Can. J. Anim. Sci. 64: 183-185. Beauchemin, K. A., Bailey, D. R. C , McAllister, T. A., and Cheng, K. -J. 1995. Lignosulfonate treated canola meal for nursing beef calves. Can. J. Anim. Sci. 75: 559-565. Broderick, G. A., Wallace, R. J., and Orskov, E. R. 1991. Control of rate and extent of protein degradation. Pages 541-592 in T. Tsuda, Y. Sasaki, and R. Kawashima, eds. Physiological Aspects of Digestion and Metabolism in Ruminants. Proc. of the 7 t h International Symp. on Ruminant Physiology. Academic Press, Inc. Canadian Council on Animal Care. 1993. Guide to the care and use of experimental animals. Vol. 1. E. D. Olfert, B. M. Cross, and A. A. McWilliam, eds. 2nd Edition, C C A C , Ottawa, ON. Christensen, D. A. and McKinnon, J. J. 1993. Canola meal for beef and dairy cattle. Pages 21-26 in D. Hickling ed. Canola Meal: Feed Industry Guide. Canola Council of Canada, Winnipeg, MB. de Boer, G., Murphy, J. J., and Kennelly, J. J. 1987. Mobile nylon bag for estimating intestinal availability of rumen undegradable protein. J. Dairy Sci. 70: 977-982. Kendall, E. M., Ingalls, J. R., and Boila, R. J. 1991. Variability in the rumen degradability and postruminal digestion of dry matter, nitrogen and amino acids of canola meal. Can J. Anim. Sci. 71: 739-754. Khorasani, G. R., Robinson, P. H., and Kennelly, J. J. 1993. Effects of canola meal treated with acetic acid on rumen degradation and intestinal digestibility in lactating dairy cows. J. Dairy Sci. 76: 607-616. Kirkpatrick, B. K. and Kennelly, J. J. 1984. Prediction of digestibility in cattle using a modified nylon bag technique. Can. J. Anim. Sci. 64: 1104. Komarek, A. R., Robertson, J. B., and Van Soest, P. J. 1994. A comparison of methods for determining ADF using the Filter Bag Technique versus conventional filtration. J. Dairy Sci. 77 (Suppl. 1): 114. Mathers, J. C. and Aitchison, E. M. 1981. Direct estimation of the extent of contamination of food residues by microbial matter after incubation with synthetic fibre bags in the rumen. J. Agric. Sci. (Camb) 96: 691-693. 35 McAllister, T. A., Cheng, K. -J., Beauchemin, K. A., Bailey, D. R. C , Pickard, M. D., and Gilbert, R. P. 1993. Use of lignosulfonate to decrease the rumen degradability of canola meal protein. Can. J. Anim. Sci. 73: 211-215. McDonald, I. M. 1981. A revised model for the estimation of protein degradability in the rumen from incubation measurements weighted according to the rate of passage. J. Agric. Sci. (Camb.) 96: 251-252. McKinnon, J. J., Olubobokun, J. A., Christensen, D. A., and Cohen, R. D. H. 1991. The influence of heat and chemical treatment on ruminal disappearance of canola meal. Can. J. Anim. Sci. 71: 773-780. Michalet-Doreau, B. and Ould-Bah, M. Y. 1992. In vitro and in sacco methods for the estimation of dietary nitrogen degradability in the rumen: a review. Anim. Feed Sci. Technol. 40: 57-86. Moshtaghi Nia, S. A. and Ingalls, J. R. 1995. Evaluation of moist heat treatment of canola meal on digestion in the rumen, small intestine, large intestine and total digestive tract of steers. Can. J. Anim. Sci. 75: 279-283. Moshtaghi Nia, S. A. and Ingalls, J. R. 1992. Effect of heating on canola meal protein degradation in the rumen and digestion in the lower gastrointestinal tract of steers. Can. J. Anim. Sci. 72: 83-88. NRC (National Research Council) 1989. Nutrient requirements of dairy cattle. 6 t h rev. ed. National Academy Press, Washington, DC. Nocek, J. E. 1985. Evaluation of specific variables affecting in situ estimates of ruminal dry matter and protein digestion. J. Anim. Sci. 60: 1347-1358. Nocek, J. E. 1988. In situ and other methods to estimate ruminal protein and energy digestibility: A review. J. Dairy Sci. 71: 2051-2069. Orskov, E. R. 1992. Protein nutrition in ruminants. 2 n d ed. Academic Press, London. SAS Institute, Inc. 1990. SAS/STAT® user's guide: statistics. Version 6. 4 t h Edition. Vol. 1 and 2. SAS Institute, Inc., Cary, NC. Stern, M. D., Varga, G. A., Clark, J. H., Firkins, J. L., Huber, J. T., and Palmquist, D. L. 1994. Evaluation of chemical and physical properties of feeds that affect protein metabolism in the rumen. J. Dairy Sci. 77: 2762-2786. Van Soest, P. J. 1994. Nutritional ecology of the ruminant. 2 n d edition. Cornell University Press, Ithaca, NY. 36 Van Soest, P. J., Robertson, J. B., and Lewis, B. A. 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3583-3597. von Keyserlingk, M. A. G., Swift, M. L., Puchala, R., and Shelford, J. A. 1996. Degradability characteristics of dry matter and crude protein of forages in ruminants. Anim. Feed Sci. Technol. 57: 291-311. Windschitl, P. M. and Stern, M. D. 1988. Evaluation of calcium lignosulfonate-treated soybean meal as a source of rumen protected protein for dairy cattle. J. Dairy Sci. 71: 3310-3322. 37 Table 2.1. Canola meal processed with graded levels of lignosulfonate (LS0 3 ) and moist heat at 100°C Treatment L S 0 3 added (%, wtwt"1) Duration of heat (min) Untreated 0 0 heat-30 0 30 heat-60 0 60 heat-90 0 90 heat-120 0 120 L S 0 3 5 0 LSO 3 -30 5 30 LSO 3 -60 5 60 LSO 3 -90 5 90 LSO 3-120 5 120 38 Table 2.2. Changes in DM content that occurred during processing of canola meal with graded levels of lignosulfonate (LSQ 3) and moist heat at 100°C DM (%) Treatment Untreated Before cooking After cooking Final Heat Treatments heat-30 87.49 87.49 85.24 88.24 heat-60 87.49 87.49 85.25 86.50 heat-90 87.49 87.49 84.07 86.06 heat-120 87.49 87.49 84.34 86.01 Mean 87.49 87.49 84.73 86.70 LSO3 + Heat Treatments LSO3-30 87.49 86.00 81.86 84.41 L S O 3 - 6 O 87.49 86.00 84.17 85.21 LSO3-90 87.49 86.00 81.88 84.94 LSO3-120 87.49 86.00 81.42 83.56 Mean 87.49 86.00 82.33 84.53 39 Table 2.3. DM disappearance (% of initial) of heat and lignosulfonate (LS0 3 ) treated canola meal and Soy Pass® in the rumen (n=4) Incubation time (h) Treatment 0 4 8 12 16 24 untreated 36 2a 58 2a 69 7a 74 5ab 79 5a 84 2a heat-30 35 3ab 51 5bc 65 Oabc 67 1e 72 6c 78 2c heat-60 34 7b 55 4ab 64 9abc 69 2cde 78 1ab 82 6ab heat-90 35 5ab 56 8a 68 1ab 68 Ode 75 1bc 82 5ab heat-120 35 Ob 55 3ab 64 3bc 70 7bcde 74 1bc 82 7ab L S 0 3 33 7c 50 6cd 61 6cd 77 4a 79 4a 83 4ab LSO 3 -30 31 3de 46 6de 57 5de 73 1abc 77 3ab 82 3ab LSO 3 -60 31 6d 45 2e 58 1de 72 7abcd 76 1abc 81 9ab LSO 3 -90 30 9de 44 4e 57 6de 71 7bcde 76 8ab 82 7ab LSO 3-120 30 5e 45 6e 54 3e 74 Oabc 76 6ab 81 8b Soy Pass® 27 7f 31 2f 34 2f 46 Of 54 8d 67 5e S E 0 3 1 4 1 7 1. 8 1. 4 0 8 a-f Means within columns with different letters differ significantly (P<0.05) 40 Table 2.4. CP disappearance (% of initial) of heat and lignosulfonate (LS0 3) treated canola meal and Soy Pass® in the rumen (n=4) Incubation time (h) Treatment 0 4 8 12 16 24 untreated 31.3de 61.9a 76.8a 83.4ab 88.5a 92.9a heat-30 34.8a 57.6b 75.0a 77.1cd 82.9c 88.5c heat-60 33.5ab 60.6b 73.1ab 78.3bcd 88.0ab 92.2ab heat-90 33.2bc 60.3b 75.2a 74.9d 83.3bc 91.6abc heat-120 31.6d 58.5b 68.9bc 78.0bcd 81.8c 91.2abc L S 0 3 33.4bc 56.5bc 68.1c 87.2a 88.9a 92.7ab LSO 3 -30 29.1f 52.6cd 63.6de 81.8abc 86.4abc 92.5ab LSO 3 -60 30.8de 49.6de 65.4cd 81.3abc 84.8abc 90.8abc LSO 3 -90 27.8g 46.7e 63.4def 79.5bcd 84.4abc 91.9ab LSO 3-120 26.5h 49.4de 58.8f 82.8abc 84.6abc 89.5bc Soy Pass® 6.1 i 7.8f 10.3g 22.8e 32.1d 47.9d S E 0.5 1.4 1.6 2.1 1.7 1.2 a-i Means within columns with different letters differ significantly (P<0.05) 41 Table 2.5. The intestinal DM and CP disappearance (% of initial) of heat and lignosulfonate (LSQ 3) treated canola meal and Soy Pass® (n=8) Parameters Dry Matter (%) Crude Protein (%) Incubation time (h) 8 12 16 8 12 16 untreated 12.2ef 6.1ef 3.5cd 15.9hi 9.9de 4.6f heat-30 11.6f 11.3bc 4.8bcd 15.3i 14.8bc 8.2bcd heat-60 15.8def 10.7bcd 2.5d 19.9gh 14.1 bed 4.6f heat-90 11.6f 12.3b 6.3b 17.2hi 17.2b 9.7bc heat-120 16.0de 9.4bcde 7.2b 23.5efg 14.1 bed 10.8b L S 0 3 17.3cd 3.8f 3.5cd 24.2def 5.9e 5.0ef LSO3 -3O 21.6c 7.3de 4.8bcd 28.8cd 10.7cd 6.6def LSO 3 -60 19.4cd 6.7ef 4.8bcd 26.2cde 10.8cd 7.6cde LSO 3 -90 21.9c 8.5cde 4.7bcd 28.9c 12.8bcd 8.3cde LSO3-120 27.4b 6.6ef 5.2bc 34.2b 9.5de 8.3cde Soy Pass® 48.5a 43.2a 33.6a 73.7a 66.7a 54.5a S E 1.6 1.2 0.9 1.5 1.6 1.0 a-i Means within columns with different letters differ significantly (P<0.05) 42 Table 2.6. The total tract DM and CP disappearance (% of initial) of heat and lignosulfonate (LSQ3) treated canola meal and Soy Pass® (n=8) Parameters Dry Matter (%) Crude Protein (%) Incubation time (h) 8 12 16 8 12 16 untreated 81.9abc 80.7b 82.9b 92.8a 93 3a 93.0ab heat-30 76.6f 77.8c 77.4e 90.3b 91 Oab 91.1b heat-60 79.9cde 79.9b 80.6d 92.3ab 92 3a 92.6ab heat-90 79.6cde 80.6b 81.3cd 92.4ab 92 6a 93.0ab heat-120 80.4bcd 80.3b 81.2cd 92.4ab 92 4a 92.8ab L S 0 3 79.1def 81.1b 82.9b 92.6ab 93 1a 94.0a LSO3-30 79.1def 80.4b 82.2bc 92.2ab 92 6a 93.3a L S O 3 - 6 O 77.5ef 79.4bc 80.4d 91.6ab 92 1a 92.4ab LSO3-90 79.5cde 80.2b 81.5bcd 92.1ab 92 3a 92.7ab L S O 3 - I 2 O 81.7abc 80.0b 81.8bcd 93.0a 92 3a 92.9ab Soy Pass® 82.6ab 89.0a 89.2a 84.0c 89 2b 87.8c S E 1.0 0.7 0.5 0.9 0 8 0.7 a-e Means within columns with different letters differ significantly (P<0.05) 43 100 n 0 0 4 8 12 16 20 24 Incubation time (h) Figure 2.1. The DM disappearance (% of initial) of untreated canola meal and canola meal heated with (LSO3-120) or without 5% L S 0 3 (heat-120) in the rumen of non-lactating cows 100 n 80 60 40 20 0 0 4 8 12 16 20 24 Incubation time (h) Figure 2.2. The CP disappearance of untreated canola meal and canola meal heated with (LSO3-120) or without 5% L S 0 3 (heat-120) in the rumen of non-lactating cows 45 to • c CD E Z3 a: (o/0) aouBJBaddBSjQ u js jo jd epn jQ 46 III. PRODUCTION AND DIGESTION RESPONSES OF LACTATING DAIRY COWS TO THE FEEDING OF HEAT AND LIGNOSULFONATE TREATED CANOLA MEAL 3.1 INTRODUCTION High producing dairy cows, in addition to their need for sufficient N to optimize microbial growth and function in the rumen, require an appropriate amount of good quality protein that contains the desired amounts of essential amino acids (AA) to be presented to the small intestine to support lactational and metabolic functions. Protein requirements of ruminants are met by microbial protein produced from dietary protein degraded in the rumen (RDP) and undegraded protein (RUP) which escapes microbial degradation in the rumen. Ruminal micro-organisms are a good source of quality protein, however they cannot always supply adequate amounts of metabolizable protein to support production and maintenance (NRC 1989). Dietary RUP can substantially increase the amount of protein for digestion by increasing the outflow and balance of AA to the duodenum for absorption. The potential benefits from feeding increased RUP include: increased level of milk production (Broderick et al. 1990); more efficient use of protein sources (Nakamura et al. 1992); enhanced reproductive performance (Ferguson and Chalupa 1989); and reduced environmental impact (Tamminga 1992). For cows producing 31-37 kg of milk d' 1, some studies (Kung and Huber 1983; Broderick et al. 1990) have shown that increasing the amounts of RUP in the diet increased milk production, while other studies (Robinson et al. 1991; Henson 1997) showed no improvement. The lack of response to high RUP may be caused by less than optimum protein protection; decreased microbial protein synthesis from increased RUP leaving insufficient RDP; low postruminal 47 digestibility of RUP sources; and essential AA profiles of RUP presented for absorption lack sufficient amounts of a limiting AA (Henson et al. 1997). Treatment of dietary proteins with various agents, including heat, formaldehyde, sodium hydroxide, acids and reducing sugars, has successfully decreased the susceptibility of protein to rumen microbial proteolytic activity. The most successful treatments have incorporated heat and reducing sugars which facilitate the Maillard browning reaction. If the Maillard reaction between the sugar aldehyde group and the free amino groups could be controlled in such a manner that degradation is shifted from the rumen to the small intestine, then animal performance evaluated by either N retention or milk production would be increased (Chalupa 1975). Treatment of soybean meal (SBM) with calcium-sodium lignosulfonate (LS0 3) has successfully decreased the rumen degradability of SBM protein in vitro (Windschitl and Stern 1988b) and in vivo (Windschitl and Stern 1988a). Diets supplemented with L S 0 3 treated SBM have supported similar milk production when untreated SBM was replaced with one-half as much protein from L S 0 3 treated SBM (Nakamura et al. 1992). Canola meal (CM), the predominant ruminant protein source in Canada, has a good AA profile relative to milk protein, but its small quantities of RUP limit its usefulness (Christensen and McKinnon 1993). Combinations of heat and L S 0 3 have successfully reduced the rumen degradability of CM without affecting its digestibility (McAllister et al. 1993; Stanford et al. 1995). While L S 0 3 treated CM has not been fed to lactating cows, its use in feeding trials with lambs and calves has not increased performance (Stanford et al. 1995; Beauchemin 1995). 48 The objective of this trial was to evaluate the effects of CM protein supplements, varying in ruminal degradability, fed to high producing dairy cows on: 1) dry matter intake, milk yield and composition; 2) in situ and in vivo digestibility; and 3) efficiency of nitrogen utilization. 3.2 MATERIALS AND METHODS 3.2.1 Canola Meal Treatments Solvent extracted CM was left untreated or was processed with either water or calcium-sodium lignosulfonate (LS03) (LignoTech USA, Inc., Rothschild, WI). Either 5% LS0 3 or 2% water was added (wt wt"1) to CM and thoroughly mixed for 10 min prior to heating. The water was added to increase the moisture content of CM to an amount equivalent to the moisture added from LS0 3 . The mixture was heated at 100°C for 120 min using moist heat in a hydrothermal cooker (Amandus Kahl Nachf, Hamburg, Germany) located at Agro Pacific Industries Ltd. (Chilliwack, BC. Forced air drying at ambient temperature was used to remove the moisture added prior to heating through water or LS0 3 and the moisture added during heating through steam. The CM was stored in unsealed one tonne bags until it was incorporated in diet concentrate mixtures. Samples for moisture determination were collected prior to water or LS0 3 addition, before heating, following heating and post cooling. Four batches of each treatment, each about 3 tonnes in quantity, were prepared over the course of the experiment. Each batch was sub-sampled and DM content was determined by drying in a forced air oven at 105°C for 24h. Sub-samples dried at 60°C until constant weight were then ground (1 mm screen) and stored until subsequent chemical analysis. The CM samples were analyzed for acid detergent 49 fibre (ADF) and neutral detergent fibre (NDF) using the modified method of Van Soest et al. (1991) called the filter bag technique (ANKOM Co., Fairport, NY; Komarek et al. 1994) and for acid detergent insoluble nitrogen (ADIN) and N, using a Leco FP-428 N analyzer (Leco Corp., St. Joseph, Ml). Each batch was also assessed for rumen and intestinal disappearance as described in Chapter II, sections 2.2.2, 2.2.3 and 2.2.4, except intestinal disappearance was only assessed with samples pre-incubated in the rumen for 8 h. The basal diets fed the host cows were different. The non-lactating cows were fed 7.1 kg grass hay (17.1% CP) and 6.9 kg rolled barley (13.0% CP) per day (DM basis). 3.2.2 Experimental Design Eighteen mulitiparous, lactating Holstein cows, averaging 60 ± 7 days in milk were used in the production trial. Cows were milked twice daily and housed in a free stall barn located at the Pacific Agri-Food Research Centre (Agassiz, BC). Cows were blocked according to milk production, calving date, body weight and age, and randomly assigned to three treatment groups in a 3 x 3 Latin square design, replicated six times. Cows were randomly assigned to subsequent treatments in second and third periods. Experimental periods were 42 d in length. Individual feed intakes were monitored using Calan feeding doors (American Calan Inc., Northwood, NH). The animals were cared for according to standards set by the Canadian Council on Animal Care (1993) and the experimental protocol was approved by the Research Centre and The University of British Columbia Animal Care Committees. 50 3.2.3 Diets The animals were fed isonitrogenous total mixed rations (TMR) consisting of 30% corn silage, 20% grass silage and 50% grain concentrate (DM basis) (Table 3.1). Diets were formulated to contain one of the following CM sources: A) untreated (LI-CM); B) heat and water treated (HT-CM); or C) heat and lignosulfonate treated (LS0 3 -CM). To provide an isonitrogenous contribution to total CP intake from each protein source, the amounts of CM in each diet were 20.0, 20.6 and 21.5% for cows fed U-CM, HT-CM and L S 0 3 - C M , respectively (DM basis). Concentrates were prepared in a mash form and were mixed with the silages to minimize sorting. The diets for each treatment group were prepared once daily and cows were fed equal portions at 0830 h and 1530 h. Orts were weighed and removed daily, prior to the 0830 h feeding and the amount of TMR offered was adjusted to maintain about 10% orts (as fed basis). Feed intakes were measured daily for each cow, averaged by week, and corrected for DM content of the TMR in order to calculate dry matter intake (DMI). 3.2.4 Sampling 3.2.4.1 Feed Total mixed rations were sampled three times per week, composited weekly, and frozen. Weighback samples were taken twice weekly from each cow and composited by week for each diet. Forage and concentrate samples were taken every two weeks. The DM content was determined by drying samples in a forced air oven at 60°C until constant weight was achieved. Samples were then ground (1 mm screen) and stored until subsequent chemical analysis of NDF, ADF and N. 51 3.2.4.2 Milk Milk yield was electronically recorded twice daily for all cows. Milk samples were collected from each cow from four consecutive milkings on days 13-14, 20-21 and 34-35 of each treatment period. Milk samples were assayed for fat, protein and lactose content by infrared spectroscopy and analyzed for somatic cell counts (SCC) using a Fossimatic cell counter (BC DHIS Lab, Chilliwack, BC). Milk urea nitrogen (MUN) was also determined after defatting by centrifugation at 1500 x g for 5 min at 4°C before analysis using colorimetric procedures employing a Kodak Ektachem DT 60 Analyzer with Disc Two Module (Clinical Products Division, Eastman Kodak Co., Rochester, NY). Total daily yields of milk components were calculated using the A.M. and P.M. milk yield for the test date. Fat corrected milk (4% FCM) production was calculated using the equation: 4% FCM (kg d"1)= (0.4 + (0.15 * % fat)) * kg milk d"1 (NRC 1989). 3.2.4.3 Rumen Fluid Rumen fluid samples (300 ml) were collected by vacuum rumen tube from each cow on days 21 and 35 of each period between 1030 h and 1130 h, approximately 2 h after morning feeding. Rumen fluid pH was determined immediately. A proportion of each sample was frozen at - 1 0 ° C until ammonium N (NH 4 + N) analysis (Fawcett and Scott 1960). The remainder of each sample was acidified to pH 2 with 50% sulphuric acid, centrifuged and the supernatant frozen until volatile fatty acids (VFA) were analyzed by gas chromatography. The V F A were determined using a Shimadzu gas chromatograph equipped with a capillary column (30 m x 0.25 mm I.D. Stabilwax-DA). The injection port temperature was set at 170°C. 52 The column temperature was set at 120 to 180°C at 10°C min"1, with an initial time of 1 min and a final time of 2 min. The internal standard used was isocaproic acid (0.70 g in 200 ml water). 3.2.4.4 Blood Samples of blood (20 ml) were taken in heparinized Vacutainers by jugular venipuncture on day 21 and 35 of each period between 1030 h and 1130 h. Hematocrit was determined and then blood was centrifuged and plasma was frozen for later analysis for urea nitrogen (BUN) and glucose by colorimetric procedures using a Kodak Ektachem DT 60 Analyzer with Disc Two Module (Clinical Products Division, Eastman Kodak Co., Rochester, NY). 3.2.5 Total Collections For the last 5 d of each period, half of the cows on each treatment were confined to metabolism stalls for the total collection of urine and feces, and the measurement of individual water intakes. Foley bladder catheters (French 75, Rusch of Canada, Scarborough, ON) were used to collect urine. Flexible rubber tubing connected the catheter to a stainless steel container holding sufficient sulphuric acid (50%) to acidify the urine. Urine was sampled daily from each cow (250 ml), composited for each 5 d collection period and frozen at -10°C until further analysis. Kjeldahl N contents of the urine samples were determined after digestion on a block digester (Parkinson and Allen 1975; Fukumoto and Chang 1982). Feces were collected in large pans placed in the gutter behind the cows. Fecal material for each cow was removed from the pans every 4 h and placed in a covered garbage can. Daily fecal composites were mixed using a Hobart mixer, sampled (700 g) and frozen 53 at -10°C. Fecal samples were later dried in a forced air oven at 60°C until constant weight was achieved for the determination of DM. Dried fecal samples were then ground (1 mm screen), composited for each cow-treatment period, and stored. Samples were later analyzed for ADF, NDF and N. Body weights for each cow were recorded on three consecutive days at the beginning of each period and at the end of the experiment, and means for individual cows were used to provide an estimate of change in body weight. 3.2.6 Efficiency Calculations Energy expenditure, expressed in Meal of net energy for lactation (NEL), was calculated as the sum of the energy output for milk, maintenance requirements, and body weight change. The energy value of milk was calculated using the following equation: energy (kcal NEL kg'1) = 2.2[41.84(% milk fat) + 22.29(% milk solids not fat) - 25.28] (Tyrrell and Reid 1965). Maintenance requirement was assumed to be 73 kcal NEL k g 1 B W 0 7 5 with a 10% allowance for activity added (NRC 1989). The amount of energy spared or required respectively, was assumed to be 4.92 Meal NEL kg"1 for BW loss and 5.12 Meal NEL kg'1 of BW gain (NRC 1989). Efficiency of DM utilization was expressed in terms of the daily energy expenditure divided by DMI. 3.2.7 Statistical Analysis Statistical analysis was via least squares ANOVA, following the GLM procedure of SAS (1990). Effects were considered to be significant at P<0.05 unless otherwise specified. The model used for this experiment was: YjjKi = U, + 0tk + Xj + p| + Yi(k) + 6ij| (k) u, = overall mean, 54 a k = effect of square (k = 1 ,...,6), Tj = effect of treatment (j = 1 ,...,3), pi = effect of period, (I = 1 ,...,3) Yi(k) = effect of cow within square, (i = 1 ,...;3) £iji(k) = experimental error 3.3 RESULTS 3.3.1 Feed Composition The DM content of U-CM from different batches, derived from different rail car shipments, was consistent at about 89% (Table 3.2). The addition of 2% water in HT-CM processing reduced the DM content on average by 2.98 percentage points, whereas the addition of LS0 3 in LS0 3 -CM processing reduced the DM content on average by 1.88 percentage points. The use of steam to heat and maintain 100°C cooking temperature in the hydrothermal cooker further reduced the DM content on average by 1.73 and 3.24 percentage points for HT-CM and LS0 3 -CM, respectively. The cooling process increased the DM content on average by 2.17 and 2.68 percentage points to yield the HT-CM and LS0 3 -CM final products, respectively. However, the final DM content for both HT-CM and LS0 3 -CM was less than that of the original untreated CM. The DM content of batch 3 of the HT-CM and to a lesser degree LS0 3 -CM were too low, which resulted in the growth of mold. As a consequence none of the HT-CM batch 3 and only half of the LS0 3 -CM batch 3 were fed during the lactation trial. Heat in combination with LS0 3 (LS0 3-CM) increased NDF content to 33.4%, whereas heat alone (HT-CM) marginally increased the NDF to 25% from 24% in U-55 CM (Table 3.3). The ADF levels were increased in LS0 3 -CM and to a lesser degree in HT-CM over that in U-CM. ADIN levels were increased from 1.8% in U-CM to 2.0% in HT-CM and markedly increased to 4.1% in LS0 3 -CM. The 8 h rumen, intestinal and total tract disappearances of U-CM were similar to values reported by Moshtaghi Nia and Ingalls (1995). The rumen disappearance of HT-CM DM was not different than U-CM except at 8 h incubation where there was greater disappearance of HT-CM (50 and 59%, for U-CM and HT-CM, respectively) (Table 3.4). Other than no difference at 0 h, the disappearance of DM from LS0 3 -CM was consistently lower than that of U-CM and HT-CM. While the rumen disappearance of CP followed the same trend as DM, the differences between LS0 3 -CM and that of U-CM and HT-CM were markedly pronounced (Table 3.5). At all incubation times the rumen disappearance of LS0 3 -CM CP was at least 45% below that of U-CM and HT-CM. The total tract disappearances of both DM and CP following 8 h rumen incubation were not different across treatments (Table 3.6). LS0 3 -CM had the greatest 8 h intestinal DM disappearance at 45% compared to 26% for U-CM and 18% for HT-CM (Table 3.6). As CP accounts for 35% of the DM content of CM, the 8 h disappearance value for LS0 3 -CM was also greatest at 72% relative to the disappearance values of 36 and 26% for U-CM and HT-CM, respectively. The CP levels of corn silage, grass silage and barley were 8.5, 11.7 and 10.5%, respectively (Table 3.7). Throughout the experiment the composition of each diet remained fairly constant, as reflected in the low standard errors for nutrient analysis (Table 3.8). The CP level in LS0 3 -CM diet was 17.5%, which was greater 56 than HT-CM diet (17.2%), but not different than U-CM diet at 17.3%. The LS0 3 -CM diet contained higher NDF at 34.4% compared to 32.5 and 33.2% for U-CM and HT-CM diets. ADF was greater in LS0 3 -CM diet than in U-CM diet but not different from HT-CM diet (19.8, 20.3 and 20.8% for U-CM, HT-CM and LS0 3 -CM diets, respectively). The higher fibre levels in LS0 3 -CM diet reflect the higher fibre levels in the LS0 3 -CM supplement. 3.3.2 Dietary Intakes and Milk Production and Composition Reported DMI, milk production and milk composition results represent data collected during day 15-35 of each period. No significant differences were apparent within the first 14 d. The inclusion of LS0 3 -CM treated CM in the diet increased DMI over cows fed diets supplemented with U-CM or HT-CM (Table 3.9). Milk production in cows fed LS0 3 -CM diet was greater than those fed the U-CM diet, but not different from those fed the HT-CM diet (Table 3.9). The FCM production was higher in cows fed LS0 3 -CM diet compared to HT-CM diet, but not different from the U-CM diet. The MUN level was lowered in cows fed the LS0 3 -CM diet relative to those fed the U-CM and HT-CM diets. Dietary treatment had no impact on body weight, body weight change, milk composition and component yield or S C C . 3.3.3 Blood Composition Hematocrit and blood glucose values were not influenced by treatment (Table 3.10). The values for BUN were lower at 16.7 mg dl"1 in cows fed LS0 3 -CM diet compared to 18.6 and 18.2 mg dl"1 for cows fed U-CM and HT-CM supplemented diets. 57 3.3.4 Rumen Fluid The pH of the rumen fluid was not affected by dietary treatment (Table 3.11). The rumen N H 4 + N levels were lower for cows fed LS0 3 -CM diet than those fed the U-CM diet, but were not different for those fed HT-CM diet. The molar proportion of acetate was higher in cows fed LS0 3 -CM diet than those fed U-CM and HT-CM diet. Cows fed LS0 3 -CM diet produced a lower proportion of propionate than cows fed HT-CM diet, but were not different from the U-CM diet. Molar proportions of branched chain fatty acids, isobutyrate and isovalerate, were lower in cows fed the LS03-CM diet. There was no apparent influence of dietary treatment on rumen butyrate or valerate concentrations. The ratios of acetate to propionate and acetate plus butyrate to propionate were higher in cows fed LS03-CM diet due to the higher acetate and lower propionate proportions. 3.3.5 Water Intake and Waste Excretion Water intake and urine output were not affected by dietary treatment (Table 3.12). Cows fed LS0 3 -CM diet had a 6% greater output of fecal DM than those fed U-CM diet, but were not different than HT-CM diet. This reflects the increased DMI of 7% in cows fed LS0 3 -CM diet over that of U-CM diet. 3.3.6 Apparent Digestibility of Nutrients The effect of diet on apparent digestibility of nutrients is given in Table 3.13. The apparent digestibility of CP was decreased in cows fed LS0 3 -CM diet compared to cows fed U-CM or HT-CM diets (74, 73 and 71% for U-CM, HT-CM and LS0 3 -CM, respectively). However, the apparent digestibility of NDF and ADF was increased in 58 cows fed L S O 3 - C M diet over that of the U-CM and HT-CM diets. No differences were observed among treatment groups in the apparent digestibility of DM. 3.3.7 Nitrogen Balance Nitrogen intakes were higher for cows fed the LSO3-CM diet than for cows fed U-CM diet, but not different than those fed HT-CM diet (Table 3.14). This reflects the higher CP content of the LS0 3 -CM diet relative to HT-CM (Table 3.8) and the increased DMI in cows fed LS0 3 -CM over that of cows fed U-CM and HT-CM diets (Table 3.9). Fecal N excretion was greater in cows fed LS0 3 -CM diet than U-CM and HT-CM diet. Conversely, urinary N excretion was lower for cows fed LS0 3 -CM diet (0.227 kg d"1) when compared to cows fed U-CM and HT-CM diets (0.258 and 0.258 kg d"1, respectively). The excretion of N in milk was unaffected by dietary treatment. Cows fed LSO3-CM diet retained more N than cows fed the U-CM diet, but was not different than those fed HT-CM diet. 3.3.8 Nitrogen Efficiency The percentage of N intake excreted in feces (Table 3.15) was significantly greater for cows fed LS0 3-CM diet than those fed U-CM and HT-CM (26, 27 and 29%, for U-CM, HT-CM and LS0 3-CM diets, respectively). Conversely, the urinary excretion of N, as a percentage of N intake, was significantly reduced at 32% in the LSO3-CM diet compared to 38 and 37% for U-CM and HT-CM diets, respectively. Cows fed LSO3-CM diet tended (P=0.06) to retain greater amounts of N, as a percentage of N intake (10, 10 and 14% for U-CM, HT-CM and LS0 3-CM, respectively). Milk N excretion, as a percentage of dietary N, was not affected by treatment. 59 3.3.9 Energy Efficiency Cows on L S O 3 -CM diet tended (P=0.10) to gain weight faster at 0.337 kg d"1 than those fed U-CM and HT-CM at gains of 0.158 and 0.125 kg d"1, respectively (Table 3.9). The output of energy for maintenance, milk production and body weight gain or loss was greater at 39.5 Meal of NEL in cows fed the LS0 3 -CM diet than those fed U-CM and HT-CM diets (37.1 and 36.9 Meal of NEL, respectively) (Table 3.16). The energy efficiency of DM utilization by cows was unaffected by treatment. 3 . 4 DISCUSSION This study was designed to evaluate the influence of the amount of supplementary RUP from untreated and treated CM on DMI, milk production and digestibility. Heat and LS0 3 treatments were designed to shift the digestion of the CM protein from the rumen to the small intestine through the enhancement of the Maillard browning reaction. During the processing of CM, the formation of primary Maillard reaction products resistant to rumen proteolysis was reflected in the elevated (39%) NDF levels and reduced rumen CP degradability in LS0 3 -CM. Heat treatment alone produced few differences in levels of NDF, ADF and ADIN or in in situ degradability between HT-CM and U-CM. This suggests that the extent and rate of the Maillard reaction was not sufficient to alter digestion and thus marginal lactation responses to heat treatment would be anticipated. The significantly greater responses of CM to treatment in the present study relative to the initial in situ study (Chapter II) suggests the heat applied in the present study was longer than that in the first. In the present study, 120 min of heat treatment represented the length of time the CM was exposed 60 to 100°C. It did not include the time required for the temperature to reach 100°C which was included in the 120 min of heat treatment in the initial study. The elevated ADF (22%) and associated ADIN in LS0 3 -CM suggest that indigestible terminal Maillard reaction products were formed. This was not apparent in the in situ analysis of LS0 3 -CM, as total tract disappearances of DM and CP were not significantly different in LS0 3 -CM compared to that of U-CM. Similarly in the evaluation of heat treated soybean proteins Faldet et al. (1991) failed to observe any reductions in total tract N disappearance despite small increases in ADIN with extended heating. They concluded that the mobile nylon bag technique may be insensitive to small differences in intestinal digestibility. A small reduction in treated CM in vivo apparent CP digestibility and a corresponding increase in fecal N output as a percentage of N intake also suggest the formation of indigestible Maillard reaction products. Commercial application of this technology would require routine quality control analysis to ensure a consistent product of high quality. Replacement of the cooler used in the current study with a more efficient one would dry the final product more effectively and prevent the spoilage of the feed that was observed with the third processing batch. The diets were formulated to be isonitrogenous such that the only differences between diets reflected the inclusion of either U-CM, HT-CM or LS0 3 -CM. Analysis of the total mixed rations showed significant differences in CP content (range 17.2-17.5%); however the biological significance of these relatively small differences might be questionable. Supplementation with LS0 3 -CM increased milk yield by 1.8 kg d' 1 which may have resulted from the increased amount of protein passing to the intestine or from a direct effect of the LS0 3 -CM protein on microbial growth and rumen 61 digestion. Stabilizing ruminal fermentation, through improvement in the synchrony of protein and organic matter degradation in the rumen, can stimulate microbial growth and protein synthesis (Khorasani et al. 1994). High producing cows supplemented with RUP sources have shown increased flows of essential AA critical for milk protein synthesis to the small intestine resulting in increased milk and milk component production (Baker et al. 1996; Wright et al. 1998). However, other researchers (Lundquist et al. 1986; Keery and Amos 1993) reported no improvement in milk yield from cows fed diets containing SBM with low ruminal degradability compared with those fed untreated SBM. Numerical increases in milk composition and component yields of fat, protein, lactose and total solids for cows fed LS0 3 -CM diet were not significant in the present study. This is in agreement with results of Oldham et al. (1985) and Khorasani et al. (1996) but differ from those of Erfle et al. (1983) and Sloan et al. (1988), who reported that increased dietary RUP reduced milk fat percentage and milk fat yield. An absence of RUP influence on milk protein and lactose concentrations was also observed by others (Erfle et al. 1983; Khorasani et al. 1996). In a study by Baker et al. (1995) in which diets were fed containing different levels of RDP and RUP no differences in CP content were observed. However, the milk from cows fed the higher levels of RUP had lower proportions of non-protein nitrogen and higher proportions of true protein. While cows fed the LS0 3 -CM diet produced more milk, they also had increased DMI of 1.8 kg d"1 over that of the control. There may be confounding effects of increased RUP and DMI on energy status that contributed to increased milk production. Cunningham et al. (1996) observed tendencies (P = 0.08) for increased milk and milk component yields for high RUP diets, however, the flow of essential AA 62 to the duodenum was unchanged relative to low RUP diets. The researchers attributed the increased yields to improved energy status of cows due to an 8.5% (not significant) increase in organic matter intake for cows on high RUP diets relative to low RUP diets. In the present study, higher DMI with LS0 3 -CM supplementation may have improved the supply of metabolizable energy and accounted for the increase in milk yield. Increased RUP in the diet results in less AA deamination and lowered ruminal N H 4 + N concentrations. The 29% reduction in rumen N H 4 + N concentrations in cows fed LSO3-CM diet compared to U-CM diet is in agreement with the in situ results showing reduced ruminal degradation. The degradation of CM protein was shifted to the intestine as the intestinal disappearance of L S 0 3 -CM CP was 72%, a 100% increase over that of U-CM (36%). The reduction in BUN levels by 10%, to 16.7 mg dl' 1 and MUN levels by 12%, to 13.9 mg dl"1 in cows fed L S 0 3 -CM diets relative to cows fed control diets is in agreement with results of Roseler et al. (1993) who fed increasing levels of RUP. The BUN and MUN levels for cows fed HT-CM while numerically lower, were not significantly different, confirming minimal rumen protection of the protein. To maximize organic matter digestion in the rumen Journet et al. (1983) stated rumen N H 4 + N concentrations of greater than 8 to 15 mg dl"1 or corresponding BUN concentrations of greater than 8 to 10 mg dl"1 are required. Correspondingly, Wohlt et al. (1978) observed higher DM digestibility when N H 4 + N levels were greater than 5 mg dl"1 compared with concentrations of less than 5 mg dl" \ As urea concentration is a metabolic indicator of N wastage (Baker et al. 1992), the lowered BUN and MUN levels for cows fed the low RUP treatment supports a theory for higher efficiency of N utilization in cows fed LS0 3 -CM. 63 Due to the solubility of urea, it appears that MUN behaves very similarly to BUN (Baker et al. 1992). However, urea levels go through peaks and valleys, depending on time of the last feeding and number of feedings, and whether or not forages and concentrates were fed separately or together as a TMR. Rumen N H 4 + N levels have been reported to peak at 2 h (Rodriguez et al. 1997) or 1 h after feeding followed by a decline to a baseline value by 6 h after feeding (Gustafsson and Palmquist 1993). The BUN concentrations of high producing cows have been reported to peak 1.5-2.0 h after the rumen N H 4 + N peak, at levels 70-85% higher than the lowest concentrations of BUN (Gustafsson and Palmquist 1993). Levels of MUN equilibrated with BUN after a lag of 1-2 h when the rate of BUN change was 3-6 mg dl"1 h"1 (Gustafsson and Palmquist 1993). In the present study, higher BUN values than MUN values were expected as BUN values reflect blood samples taken 2 h following feeding whereas MUN values reflect a composite of milk samples taken 2 h prior to the morning feeding and 0.5 h prior to the afternoon feeding. Despite differences in magnitude, blood and milk urea nitrogen levels showed the same trends across treatments and were both useful indicators of N H 4 + loss from the rumen and reflected the efficiency of N utilization. Others (Roseler et al. 1993; Hof et al. 1997) have also suggested that MUN is closely related to the ratio of dietary protein to energy intake and a correct balance between RDP and energy intake is essential for the efficient synthesis of milk protein. Thus, high urea levels can either be absolute (just too much protein) or indirect (not enough available rumen energy to match the available RDP). As percentages of CP in milk are not adequate to assess the efficiency of diet formulation and BUN analysis is invasive and expensive, routine 64 analysis of MUN content could be useful to assess efficiency of protein feeding on dairy farms. Molar proportions of acetate were higher and propionate were lower for cows fed L S O 3 - C M compared to other treatments. In contrast, Cunningham et al. (1996) found the molar percentages of acetate, propionate, isobutyrate, isovalerate, and valerate were unaffected by increasing dietary RUP. In agreement with the present study, Veen (1986), Windschitl and Stern (1988a), and Arieli et al. (1996) reported increases in acetate and decreases in propionate concentrations when cows were fed diets of relatively low degradability. To explain the lowered propionate concentrations Veen (1986) suggested that under the influence of bacterial fermentation, proteins give rise to the formation of relatively more propionate, and therefore, when proteins are less degradable or fermentable, propionate concentrations are decreased. With enhanced fibre digestion and increased proportions of acetate in cows fed the LS0 3 -CM diet, a corresponding increase in butyrate was not observed. Other researchers (Veen 1986; Cunningham et al. 1996) have reported increased molar percentages of butyrate as dietary RUP increased, while Folman et al. (1981) reported a decrease. Valerate concentration was unaffected by dietary treatment in the present study, however in contrast, Windschitl and Stern (1988a) found valerate was reduced with the feeding of xylose or LS0 3 treated SBM. A depression in branched chain VFA concentrations were observed in cows supplemented with L S O 3 -CM compared to U-CM and HT-CM. In contrast, others (Windschitl and Stern 1987; Windschitl and Stem 1988a) found branched chain VFA concentrations were not affected when xylose or LS0 3 treated SBM were fed. Similar 65 to the present study, Veen (1986) and Windschitl and Stern (1988b) observed decreases in branch chained VFA when low degradable proteins were fed. The branched chain V F A depression may be attributed to the lower protein degradation found with LS0 3 -CM, since isobutyrate and isovalerate are derived from the branched-chain AA, valine and leucine, respectively (Harwood and Canale-Parola 1981). While numerically lower in animals fed the HT-CM and LS0 3 -CM diets, the total V F A levels were not significantly different from cows fed the U-CM diet. Others (Folman et al. 1981; Veen 1986) have observed in vivo decreases in total VFA concentrations when low degradable proteins were fed. Stern (1984) and Windschitl and Stern (1988b) reported decreased cellulose and ADF digestion and lower total V F A flows from continuous culture fermenters when LS0 3 treated SBM was used in the diet. With high amounts of RUP, a shortage of RDP may limit the degradative activity of the rumen microbes, resulting in a combination of reduced intake, energy supply and protein supply (Tamminga 1992). Thus, it is important to include a source of RDP when high RUP supplements are fed, to ensure sufficient ruminally available N to meet the requirement for optimum growth of cellulolytic bacteria (Waltz et al. 1989; Wohlt et al. 1991). By infusing urea into culture fermenters, Windschitl and Stern (1988b) demonstrated that in vitro organic matter digestion could be enhanced for corn based diets containing protein of relatively low rumen degradability. In the lactation study by Cunningham et al. (1996), diets containing protected SBM in combination with urea supplementation appeared to provide sufficient RDP to meet the N needs of ruminal micro-organisms for protein synthesis. Other researchers (McCarthy et al. 1989; King et al. 1990; Keery and Amos 1993) have reported that the 66 source and amount of RUP in diets fed to lactating dairy cows appears to have only small effects on the digestion of fibre in the rumen and total tract. The increased flow of digesta through the gastrointestinal tract resulting from the higher DMI of cows supplemented with LS0 3 -CM would tend to decrease digestibility. However, cows fed LS0 3 -CM had increased apparent digestibilities of NDF and ADF over that of the control and HT-CM diets. This suggests that the rapid rate and high degradability of protein in U-CM and HT-CM diets was not synchronized with the rate at which energy was generated for microbial growth. Hence, this resulted in uncoupled fermentation and inefficient protein utilization (Broderick et al. 1991). Additionally, the increased apparent fibre digestibilities of the LS0 3 -CM diet suggests that N H 4 + N levels were not limiting with regard to supporting optimal fibre digestion as they were in other studies (Stern 1984; Windschitl and Stern 1988b). This apparent inconsistency, between our results and those of others, on the influence of high RUP on VFA concentrations and fibre digestion may have been due to our use of barley versus corn. Barley is more rapidly and efficiently colonized by rumen microbes than is corn (McAllister et al. 1990), resulting in a more rapid digestion of barley protein and higher production of N H 4 + with barley based than corn based diets. Additionally, the greater intake of NDF and ADF with the LS0 3 -CM diet might have improved ruminal conditions for fibre fermentation, as reflected in the increased rumen concentrations of acetate. Based on studies with fish meal, SBM and urea-casein, McAllan and Griffith (1987) suggested that ruminal fibre digestibility might be enhanced with proteins that are more slowly degraded in the rumen. Slowly fermentable proteins may result in a more gradual release of N H 4 + N, peptides, and branched chain fatty acids; therefore, 67 these essential growth factors are available to the cellulolytic bacteria for an extended period of time after feeding (Veen 1986). While cows in this study had access to feed the majority of the time, lower fibre digestion with U-CM and HT-CM diets suggests uncoupled ruminal fermentation of carbohydrate and protein due to high RDP (McAllan and Griffith 1987). Treatment with LS0 3 may have ensured that the necessary N growth factors were available to the cellulolytic bacteria on a relatively constant basis. As N H 4 + N, BUN and MUN levels of all three dietary groups were sufficiently high to indicate that all diets contained ample amounts of RDP and as milk protein concentrations were normal (> 3.2%), cows in the present study probably consumed an adequate supply of ruminally fermentable energy. Had milk protein concentrations been lower (< 3.2%), this would have indicated reduced bacterial crude protein supply caused by an inadequate supply of fermentable energy (Kaufmann 1982). Fecal N losses result from the excretion of undigested feed N, undigested microbial N and endogenous N. The 17% increase in fecal N excretion for cows fed LSO3-CM is unlikely to be due to differences in microbial N digestibility or endogenous N, but more likely due to feed N factors (Tamminga 1992). An increase in N intake of 6% and a reduced apparent N digestibility were probably the contributing factors. The reduction in apparent CP digestibility from 74 and 73% for U-CM and HT-CM to 71% with LSO3-CM diet supports the fibre and ADIN levels observed for the respective diets and suggests that L S 0 3 - C M protein was overprotected to a small degree. The study of Windschitl and Stern (1988a) showed increased fecal N output due to a large reduction (77 to 71%) in total tract apparent N digestibility with the inclusion of L S 0 3 treated SBM compared to untreated SBM. Cunningham et al. 68 (1996) reported no differences in apparent total tract N digestion between SBM based diets of low and high RUP and concluded that the treated SBM was not overprotected. It has been suggested by Owens and Bergen (1983) that a slight depression in total tract N digestion may be necessary in order to maximize the supply of digestible N to the small intestine. In addition, they suggested that N retention, rather than fecal N loss, must be used as an index for the value of treated proteins. In agreement with Wright et al. (1998), efficiency for retained N, expressed as a percentage of N intake, tended (P=0.06) to increase with increased RUP levels. Losses of N in urine originate from various sources including: rumen loss; the replacement of metabolic losses in the gut; the incorporation of dietary protein into microbial nucleic acids which are not available for metabolism by the cow; loss in maintenance; and loss caused by the inefficient conversion of absorbed AA into milk and body proteins (Tamminga 1992). Rumen loss can be decreased by reducing dietary CP, by reducing protein degradability, and by enhancing microbial protein synthesis by increasing the capture of rumen degraded protein. The greater the rumen degradability, the greater will be the rumen AA deamination yielding the highest N excesses, thus the observed higher urinary N losses with the control and HT-CM. The higher urinary N excretion may also be indicative of insufficient energy substrates available in the rumen for productive use of the N (Tamminga 1996; Wright et al. 1998). An improved balance between the quantity of carbohydrate and protein and improved synchrony between the rate at which they were degraded may have contributed to lower N losses from the rumen in cows fed the LS0 3 -CM diet (Stern et al. 1994; Tamminga 1996). Similar to the present study, Wohlt et al. (1991) reported a decrease in urinary N, as a percentage of absorbed nitrogen, from 42% for 69 cows fed SBM to 38% for cows fed fish meal which is a less rumen degradable protein. The improvement in N efficiency has important implications for dairy producers and the environment. Not only is excess dietary N expensive, there is the energy cost associated with conversion of ammonium to urea. A considerable part of the excreted N in feces and urine, is lost by NH 3 volatilization, nitrate leaching or nitrous oxide emissions (Hof et al. 1997). These losses contribute to environmental deterioration through eutrophication of water, acidification of soil and depletion of the ozone layer (Tamminga 1996). Decreased N concentrations in the manure (feces and urine) from dairy cows fed diets lower in CP has been shown to result in decreased NH 3 emission from the manure and decreased N volatilization as a fraction of the excreted N (Paul et al. 1998). The primary source of NH 3 emissions has been shown to be the urine, with the feces contributing very low emissions (Paul et al. 1998). In the present study lower NH 3 emissions from the waste of cows fed LS0 3 -CM diet would be expected, as the urinary N excretion was reduced 17%, even though fecal N excretion was increased 10%. The increased energy expenditure of cows supplemented with LS0 3 -CM reflects their increased milk production and their numerically higher BW gain and retention of N relative to cows fed control and HT-CM diets. In agreement with the study of Keery and Amos (1993) the energy efficiency was not affected by the level of UIP supplied by the different CM supplements. Results of this study indicate that the degradation of CM, treated with 5% LS0 3 and heated at 100°C for 2 h in a hydrothermal cooker, was shifted from the rumen to the small intestine. This increased supply of RUP resulted in increased DMI, milk 70 production and efficiency of N utilization in cows supplemented with LS0 3 -CM. The lower rumen degradation of protein may have resulted in a better balance of protein and carbohydrate breakdown that led to greater synchrony in the release of ruminally degraded N and microbial N capture. The level of undegradable protein makes LS0 3 -CM an effective source of bypass protein for high producing cows. 3.5 R E F E R E N C E S A r i e l i , A . , S h a b i , Z . , B r u c k e n t a l , I., Tagar i , H., A h a r o n i , Y., Z a m w e l l , S . , a n d Voet , H. 1996. Effect of the degradation of organic matter and crude protein on ruminal fermentation in dairy cows. J. Dairy Sci. 79: 1774-1780. B a k e r , M. J . , A m o s , H. E., N e l s o n , A . , W i l l i a m s , C . C , a n d F r o e t s c h e l , M. A . 1996. Undegraded intake protein: Effects on milk production and amino acid utilization by cows fed wheat silage. Can. J. Anim. Sci. 76: 367-376. B a k e r , L. D., F e r g u s o n , J . D., a n d C h a l u p a , W. 1995. Responses in urea and true protein of milk to different protein feeding schemes for dairy cows. J. Dairy Sci. 78 : 2424-2434. B a k e r , L. D., F e r g u s o n , J . D., a n d R a m b e r g , C . F. 1992. Kinetic analysis of urea transport from plasma to milk in dairy cows. J. Dairy Sci. 75 (Suppl. 1): 181. B e a u c h e m i n , K. A . , B a i l e y , D. R. C , M c A l l i s t e r , T. A . , a n d C h e n g , K. - J . 1995. Lignosulfonate treated canola meal for nursing beef calves. Can. J. Anim. Sci. 75 : 559-565. B r o d e r i c k , G . A . , R i c k e r , D. B., a n d Driver, L. S . 1990. Expeller soybean meal and corn by-products versus solvent soybean meal for lactating dairy cows fed alfalfa silage as sole forage. J. Dairy Sci. 73 : 453-462. B r o d e r i c k , G . A . , W a l l a c e , R. J . , a n d O r s k o v , E. R. 1991. Control of rate and extent of protein degradation. Pages 541-592 in T. Tsuda, Y. Sasaki, and R. Kawashima, eds. Physiological Aspects of Digestion and Metabolism in Ruminants. Proc. of the 7 t h International Symp. on Ruminant Physiology. Academic Press, Inc. But le r , W . R., C a l a m a n , J . J . , a n d B e a n , S . W. 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74 : 858-865. C a n a d i a n C o u n c i l o n A n i m a l C a r e . 1993. Guide to the care and use of experimental animals. Vol. 1. E. D. Olfert, B. M. Cross, and A. A. McWilliam, eds. 2nd Edition, C C A C , Ottawa, ON. 71 Chalupa, W. 1975. Rumen bypass and protection of proteins and amino acids. J. Dairy Sci. 58: 1198-1218. Christensen, D. A. and McKinnon, J. J. 1993. Canola meal for beef and dairy cattle. Pages 21-26 in D. Hickling ed. Canola Meal: Feed Industry Guide. Canola Council of Canada, Winnipeg, MB. Cunningham, K. D., Cecava, M. J., Johnson, T. R., and Ludden, P. A. 1996. Influence of source and amount of dietary protein on milk yield by cows in early lactation. J. Dairy Sci. 79: 620-630 Erfle, J. D., Mahadevan, S., Teather, R. M., and Sauer, F. D. 1983. The performance of lactating cows fed urea-treated corn silage in combination with soybean or fish meal containing concentrates. Can. J. Anim. Sci. 63: 191-199. Faldet, M. A., Voss, V. L, Broderick, G. A., and Satter, L. D. 1991. Chemical, in vitro, and in situ evaluation of heat-treated soybean proteins. J. Dairy Sci. 74: 2548-2554. Fawcett, J. K. and Scott, J. E. 1960. A rapid and precise method for the determination of urea. J. Clin. Path. 13: 156-159. Ferguson, J. D. and Chalupa, W. 1989. Impact of protein nutrition on reproduction in dairy cows. J. Dairy Sci. 72: 746-766. Folman, Y., Neumark, H., Kaim, M., and Kaufmann, W. 1981. Performance, rumen and blood metabolites in high-yielding cows fed varying protein percents and protected soybean. J. Dairy Sci. 64: 759-766. Fukumoto, H. E. and Chang, G. W. 1982. Manual salicylate-hypochlorite procedure for determination of ammonia in kjeldahl digests. J. Assoc. Off. Anal. Chem. 65: 1076-1079. Gustafsson, A. H. and Palmquist, D. L. 1993. Diurnal variation of rumen ammonia, serum urea, and milk urea in dairy cows at high and low yields. J. Dairy Sci. 76: 475-484. Harwood, C. S. and Canale-Parola, E. 1981. Adenosine 5'-triphosphate-yielding pathways of branched-chain amino acid fermentation by a marine spirochete. J. Bacteriol. 148: 117-123. Henson, J. E., Schingoethe, D. J., and Maiga, H. A. 1997. Lactational evaluation of protein supplements of varying ruminal degradabilities. J. Dairy Sci. 80: 385-392. 72 Hof, G., Vervoorn, M. D., Lenaers, P. J., and Tamminga, S. 1997. Milk urea nitrogen as a tool to monitor the protein nutrition of dairy cows. J. Dairy Sci. 80: 3333-3340. Journet, M., Champredon, C , Pion, R., and Verite, R. 1983. Physiological basis of the protein nutrition producing dairy cows. Critical analysis of the allowances. 4 t h Int. Symp. Protein Metab. Nutr. Colloques de I'lNRA. No. 16. Vol. 1. Versailles, France INRA Publications p.433. Kaufmann, V. W. 1982. The significance of using special protein in early lactation (also with regard to the fertility of the cow). Page 117 in Protein and energy supply for high production of milk and meat. Pergamon Press. New York. Keery, C. M. and Amos, H. E. 1993. Effects of source and level of undegraded intake protein on nutrient use and performance of early lactation cows. J. Dairy Sci. 76: 499-513. Keery, C. M., Amos, H. E., and Froetschel, M. A. 1993. Effects of supplemental protein source on intraruminal fermentation, protein degradation, and amino acid absorption. J. Dairy Sci. 76: 514-524. Khorasani, G. R., de Boer, G., and Kennedy, J. J. 1996. Response of early lactation cows to ruminally undegradable protein in the diet. J. Dairy Sci. 79: 446-453. Khorasani, G. R., de Boer, G., Robinson, B., and Kennedy, J. J. 1994. Influence of dietary protein and starch on production and metabolic responses of dairy cows. J. Dairy Sci. 77: 813-824. King, K. J., Huber, J. T., Sadik, M., Bergen, W. G., Grant, A. L, and King, V. L. 1990. Influence of dietary protein sources on the amino acid profiles available for digestion and metabolism in lactating dairy cows. J. Dairy Sci. 73: 3208-3216. Komarek, A. R., Robertson, J. B., and Van Soest, P. J. 1994. A comparison of methods for determining ADF using the Filter Bag Technique versus conventional filtration. J . Dairy Sci. 77 (Suppl. 1): 114. Kung, L, Jr. and Huber, J. T. 1983. Performance of high producing cows in early lactation fed protein of varying amounts, sources, and degradability. J. Dairy Sci. 66: 227-234. Lundquist, R. C , Otterby, D. E., and Linn, J. G. 1986. Influence of formaldehyde treated soybean meal on milk production. J. Dairy Sci. 69: 1337-1345. McAllan, A. B. and Griffith, E. S. 1987. The effects of different sources of nitrogen supplementation on the digestion of fibre components in the rumen of steers. Anim. Feed Sci. Technol. 17: 65-73. 73 McAllister, T. A., Cheng, K. -J., Beauchemin, K. A., Bailey, D. R. C , Pickard, M. D., and Gilbert, R. P. 1993. Use of lignosulfonate to decrease the rumen degradability of canola meal protein. Can. J. Anim. Sci. 73: 211-215. McAllister, T. A., Rode, L. M., Major, D. J., Cheng, K. - J . , and Buchanan-Smith, J. G. 1990. Effect of ruminal microbial colonization on cereal grain digestion. Can. J. Anim. Sci. 70: 571-579. McCarthy, R. D., Jr., Klusmeyer, T. H., Vicini, J. L., and Clark, J. H. 1989. Effects of source of protein and carbohydrate on ruminal fermentation and passage of nutrients to the small intestine of lactating cows. J. Dairy Sci. 72: 2002-2016. Moshtaghi Nia, S. A. and Ingalls, J. R. 1995. Evaluation of moist heat treatment of canola meal on digestion in the rumen, small intestine, large intestine and total digestive tract of steers. Can. J. Anim. Sci. 75: 279-283. Nakamura, T., Klopfenstein, T. J., Owen, F. G., Britton, R. A., and Grant, R. J. 1992. Nonenzymatically browned soybean meal for lactating dairy cows. J. Dairy Sci. 75: 3519-3523. NRC (National Research Council) 1989. Nutrient requirements of dairy cattle. 6 t h rev. ed. National Academy Press, Washington, DC. Oldham, J. D., Napper, D. J., Smith, T., and Fulford, R. J. 1985. Performance of dairy cows offered isonitrogenous diets containing urea or fish meal in early and mid-lactation. Br. J. Nutr. 53: 337-345. Owens, F. N. and Bergen, W. G. 1983. Nitrogen metabolism of ruminant animals: historical perspective, current understanding and future implications. J . Anim. Sci. 57 (Suppl. 2): 498. Parkinson, J. A. and Allen, S. E. 1975. A wet oxidation procedure suitable for the determination of nitrogen and mineral nutrients in biological material. Commun. Soil Sci. Plant Anal. 6: 1-11. Paul, J. W., Dinn, N. E., Kannangara, T, and Fisher, L. J. 1998. Protein content in dairy cattle diets affects ammonia losses and fertilizer nitrogen value. J. Environ. Qual. 27: 528-534 Robinson, P. H., McQueen, R. E., and Burgess, P. L. 1991. Influence of rumen undegradable protein levels on feed intake and milk production of dairy cows. J. Dairy Sci. 74: 1623-1631. 74 Rodriguez, L. A., Stallings, C. C , Herbein, J. H., and McGilliard, M. L. 1997. Diurnal variation in milk and plasma urea nitrogen in Holstein and Jersey cows in response to degradable dietary protein and added fat. J. Dairy Sci. 80: 3368-3376. Roseler, D. K., Ferguson, J. D., Sniffen, C. J., and Herrema, J. 1993. Dietary protein degradability effects on plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J. Dairy Sci. 76: 525-534. SAS Institute, Inc. 1990. SAS/STAT® user's guide: statistics. Version 6. 4 t h Edition. Vol. 1 and 2. SAS Institute, Inc., Cary, NC. Sloan, B. K., Rowlinson, P., and Armstrong, D. G. 1988. The influence of a formulated excess of rumen degradable protein or undegradable protein on milk production in dairy cows in early lactation. Anim. Prod. 46: 13-22. Stanford, K., McAllister, T. A., Xu, Z., Pickard, M., and Cheng, K. -J. 1995. Comparison of lignosulfonate-treated canola meal and soybean meal as rumen undegradable protein supplements for lambs. Can. J. Anim. Sci. 75: 371-377. Stern, M. D., Varga, G. A., Clark, J. H., Firkins, J. L, Huber, J. T., and Palmquist, D. L. 1994. Evaluation of chemical and physical properties of feeds that affect protein metabolism in the rumen. J. Dairy Sci. 77: 2762-2786. Stern, M. D. 1984. Effect of lignosulfonate on rumen microbial degradation of soybean meal protein in continuous culture. Can. J. Anim. Sci. 64 (Suppl.): 27-28. Tamminga, S. 1992. Nutrition management of dairy cows as a contribution to pollution control. J. Dairy Sci. 75: 345-357. Tamminga, S. 1996. A review on environmental impacts of nutritional strategies in ruminants. J . Anim. Sci. 74: 3112-3124. Tyrrell, H. F. and Reid, J. T. 1965. Prediction of energy value of cow's milk. J . Dairy Sci. 48: 1215-1223. Van Soest, P. J., Robertson, J. B., and Lewis, B. A. 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3583-3597. Veen, W. A. G. 1986. The influence of slowly and rapidly degradable concentrate protein on a number of rumen parameters in dairy cattle. Neth. J. Agric. Sci. 34: 199-216. Waltz, D. M.j Stern, M. D., and lllg, D. J. 1989. Effect of ruminal protein degradation of blood meal and feather meal on the intestinal amino acid supply to lactating cows. J. Dairy Sci. 72: 1509-1518. 75 Windschitl, P. M. and Stern, M. D. 1987. In vivo evaluation of lignosulfonate-treated soybean meal as a source of rumen protected protein. J. Anim. Sci. 65 (Suppl 1): 165. Windschitl, P. M. and Stern, M. D. 1988a. Evaluation of calcium lignosulfonate-treated soybean meal as a source of rumen protected protein for dairy cattle. J. Dairy Sci. 71: 3310-3322. Windschitl, P. M. and Stern, M. D. 1988b. Effects of urea supplementation of diets containing lignosulfonate-treated soybean meal on bacterial fermentation in continuous culture of ruminal contents. J. Anim. Sci. 66: 2948-2958. Wohlt, J. E., Chmiel, S. L, Zajac, P. K., Backer, L, Blethen, D. B., and Evans, J. L. 1991. Dry matter intake, milk yield and composition, and nitrogen use in Holstein cows fed soybean, fish, or corn gluten meals. J. Dairy Sci. 74: 1609-1622. Wohlt, J. E., Clark, J. H., and Blaisdell, F. S. 1978. Nutritional value of urea versus preformed protein for ruminants. II Nitrogen utilization by dairy cows fed corn based diets containing supplemental nitrogen from urea and/or soybean meal. J. Dairy Sci. 61: 916-931. Wright, T. C , Moscardini, S., Luimes, P. H., Susmel, P., and McBride, B. W. 1998. Effects of rumen-undegradable protein and feed intake on nitrogen balance and milk protein production in dairy cows. J. Dairy Sci. 81: 784-793. 76 Table 3.1. Ingredient composition of the total mixed rations Diet1 Ingredient U-CM HT-CM L S 0 3 - C M (%, DM basis) corn silage 30.0 30.0 30.0 grass silage 20.0 20.0 20.0 barley 25.0 24.4 23.5 untreated canola meal 20.0 0 0 HT canola meal 2 0 20.6 0 LS0 3 canola meal 3 0 0 21.5 soybean meal 3.67 3.68 3.68 limestone 0.96 0.95 0.94 salt 0.25 0.25 0.25 mineral-vitamin premix4 0.10 0.10 0.10 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 2 Canola meal with 2% added water hydrothermally cooked at 100°C for 120 min. 3 Canola meal treated with 5% lignosulfonate and hydrothermally cooked at 100°C for 120 min 4Premix contained 40,000 mg kg"1 of Mn, 40,000 mg kg"1 of Zn, 16,000 mg kg"1 of Fe, 12,000 mg kg'1 of Cu, 640 mg kg"1 of I, 240 mg kg"1 of Se, 160 mg kg"1 of Co, 4,000 KIU kg"1 of vitamin A, 800 KIU kg"1 of vitamin D and 10 KIU kg"1 of vitamin E on a DM basis 77 Table 3.2. Changes in moisture content that occurred during processing of individual batches of treated canola meal cooked in a hydrothermal cooker for 120 min at 100°C DM (%) Batch Untreated Before cooking After cooking Final HT-CM 1 1 88.66 87.53 85.35 88.75 2 88.99 — 3 85.31 87.13 3 89.02 84.08 81.69 82.84 4 88.48 85.83 83.95 86.27 Mean 88.79 85.81 84.08 86.25 L S 0 3 - C M 2 1 88.66 87.01 84.46 86.72 2 88.99 87.09 83.41 85.99 3 89.02 86.69 82.56 85.70 4 88.48 86.85 84.26 87.00 Mean 88.79 86.91 83.67 86.35 1 HT-CM = heat (100°C, 120 min) and water (2%) treated canola meal 2 L S 0 3 - C M = heat (100°C, 120 min) and lignosulfonate (5%) treated canola meal Wing data 78 Table 3.3. Chemical composition of individual batches of untreated canola meal and canola meal cooked in a hydrothermal cooker for 120 min at 100°C, with or without 5% L S Q 3 Batch DM (%) CP (%) NDF (%) ADF (%) ADIN (%) U-CM 1 1 88.66 34.8 24.7 19.0 1.8 2 88.99 37.2 22.8 17.0 1.8 3 89.02 36.2 22.9 17.4 1.9 4 88.48 35.0 25.6 19.0 1.8 Mean 88.79 35.8 24.0 18.1 1.8 HT-CM 2 1 88.75 35.5 24.6 18.0 1.9 2 87.13 35.7 24.2 17.4 2.2 3 82.84 34.6 26.9 21.5 1.9 4 86.27 35.1 24.2 18.1 1.9 Mean 86.25 35.2 25.0 18.8 2.0 LSO3 -CM 3 1 86.72 34.7 33.6 21.3 4.2 2 85.99 36.3 34.2 21.9 4.6 3 85.70 35.3 32.4 20.7 2.8 4 87.00 34.0 33.5 24.6 4.9 Mean 86.35 35.1 33.4 22.1 4.1 U-CM = untreated canola meal 2 H T - C M = heat (100°C, 120 min) and water (2%) treated canola meal 3 L S 0 3 - C M = heat (100°C, 120 min) and lignosulfonate (5%) treated canola meal 79 Table 3.4. The influence of processing batch on the rumen DM disappearance (% of initial) of untreated and treated canola meal fed during the lactation trial (n=4) Incubation time (h) Treatment Batch 0 4 8 12 16 24 1 21.5 40.9 U-CM 49.9 64.6 68.3 76.8 2 20.4 40.3 51.2 63.6 71.9 80.2 3 19.9 45.2 48.7 60.7 69.5 79.1 4 17.7 39.9 49.4 65.1 66.9 74.6 Mean 19.9 41.6a 49.8b 63.5a 69.2a 77.7a 1 19.8 45.5 HT-CM 60.3 64.1 70.6 76.7 2 16.9 50.6 59.8 67.2 71.5 78.9 3 11.6 37.4 53.6 60.2 64.3 72.2 4 16.9 42.9 61.0 65.3 70.8 77.8 Mean 16.3 44.1a 58.6a 64.2a 69.3a 76.4a 1 15.2 24.8 LSO3-30.6 -CM 39.8 49.5 55.5 2 14.3 23.2 30.2 42.3 44.1 54.9 3 16.8 28.1 34.6 47.6 53.8 59.4 4 15.7 24.7 27.9 40.7 45.8 55.5 Mean 15.5 25.2b 30.8c 42.6b 48.3b 56.3b S E 1.1 1.8 1.3 1.4 1.7 1.3 a-c Means within columns with different letters differ significantly (P<0.05) 80 Table 3.5. The influence of processing batch on the rumen CP disappearance (% of initial) of untreated and treated canola meal fed during the lactation trial (n=4) Incubation time (h) Treatment Batch 0 4 8 12 16 24 1 15.7 45.5 U-CM 55.8 73.7 77.4 88.2 2 20.6 44.3 56.1 69.8 80.1 90.2 3 15.8 50.5 53.7 66.5 77.0 88.9 4 10.0 42.6 54.6 75.4 78.2 87.0 Mean 15.5a 45.7a 55.0b 71.3a 78.2a 88.6a 1 12.6 47.7 HT-CM 66.6 70.9 79.5 86.5 2 10.3 53.4 65.3 74.2 80.1 88.9 3 7.7 43.4 62.9 71.0 75.4 83.0 4 7.6 45.4 66.5 70.6 77.6 84.7 Mean 9.6b 47.5a 65.3a 71.7a 78.3a 85.8a 1 0 8.8 LS0 3 -13.3 -CM 22.5 33.1 41.5 2 1.1 10.2 18.1 27.1 30.2 42.3 3 2.7 23.7 29.0 44.7 50.4 54.6 4 1.2 7.0 11.5 25.1 31.5 42.1 Mean 1.3c 12.4b 18.0c 29.9b 36.3b 45.1b S E 1.5 2.7 2.3 3.2 2.8 2.0 a-c Means within columns with different letters differ significantly (P<0.05) 81 Table 3.6. The intestinal and total tract DM and CP disappearance (% of initial) following 8 h rumen incubation of different processing batches of untreated and treated canola meal fed during the lactation trial (n=8) DM disappearance (%) CP disappearance (%) Treatment Batch Intestinal Total tract Intestinal Total Tract 1 26.6 U-CM 76.5 35.4 91.2 2 27.3 79.2 35.8 92.6 3 27.9 77.1 37.6 91.8 4 23.4 72.7 35.5 90.1 Mean 26.3b 76.4 36.1b 91.4 1 16.5 HT-CM 76.2 25.7 90.9 2 19.5 79.5 28.2 92.4 3 17.4 73.4 25.8 90.6 4 17.5 79.0 25.5 92.0 Mean 17.7c 77.0 26.1c 91.5 1 47.1 L S C V C M 77.7 77.4 90.7 2 46.0 76.2 71.4 89.5 3 39.4 74.0 61.4 90.3 4 45.7 73.6 77.7 89.1 Mean 44.6a 75.4 72.0a 89.9 S E 1.2 1.3 2.2 0.4 a-c Means within columns with different letters differ significantly (P<0.05) 82 Table 3.7. Chemical composition of major diet components Nutrient corn silage grass silage barley (%, DM basis) DM 36.88 30.30 90.25 CP 8.49 11.73 10.46 NDF 39.19 61.73 16.01 ADF 22.61 37.54 5.76 83 Table 3.8. Chemical composition of the total mixed rations Nutrient n Diet1 S E U-CM HT-CM L S O 3 -CM (%, DM basis) CP 19 17.3a b 17.2a 17.5b 0.1 NDF 19 32.5a 33.2a 34.4b 0.4 ADF 19 19.8a 20.3 a b 20.8b 0.2 a b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat (100°C, 120 min) and water (2%) treated canola meal, and LSO3 -CM = heat (100°C, 120 min) and lignosulfonate (5%) treated canola meal 84 Table 3.9. The effects of diets supplemented with different canola meal treatments on DMI, body weight, milk yield and milk composition n U-CM HT-CM L S O 3 -CM S E DMI kg d"1 53 24.6b 25.1 b 26.4a 0.2 % of BW 52 3.8 3.8 3.9 <0.1 Body wt kg 52 665.2 666.4 672.9 3.4 gain, kg d"1 51 0.158 0.125 0.337 0.093 Milk, kg d"1 52 34.8b 35.3 a b 36.6a 0.6 4% FCM, kg d"1 52 33.8 a b 33.4a 35.2b 0.6 Milk compositon, % Fat 53 3.80 3.68 3.82 0.06 Protein 53 3.25 3.28 3.31 0.04 Lactose 53 4.41 4.50 4.54 0.05 Total solids 53 12.70 12.69 12.92 0.12 Component Yield, kg d"1 Fat 53 1.33 1.27 1.36 0.03 Protein 53 1.14 1.14 1.18 0.02 Lactose 53 1.56 1.57 1.63 0.03 Total solids 53 4.47 4.42 4.62 0.09 Milk S C C , x10 3 m r 1 53 223 229 372 105 MUN, mg dr1 53 15.683 15.34a 13.86b 0.27 a b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 85 Table 3.10. The effects of diets supplemented with different canola meal treatments on blood composition Diet1 n U-CM HT-CM L S O 3 -CM S E Hematocrit, % 53 31.1 30.6 31.1 0.3 Blood glucose, mg dl"1 53 68.2 70.2 69.1 0.9 BUN, mg dl"1 53 18.6a 18.2a 16.7b 0.3 a , b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 86 Table 3.11. The effects of diets supplemented with different canola meal treatments on ruminal pH, ammonium nitrogen ( N H / N) and VFA concentrations DieT n U-CM HT-CM L S O 3 -CM S E PH 53 6.79 6.83 6.84 0.03 N H 4 + N, mg dl"1 53 11.243 9.45 a b 8.00b 0.73 Total VFA, mM 53 100.6 95.7 95.1 2.6 VFA, mol 100mol"1 Acetate (A) 53 59.5b 59.4b 60.4a 0.2 Propionate (P) 53 22.1a 22.2a 21.3 b 0.3 Isobutyrate 53 1.07a 1.06a 0.95b 0.01 Butyrate (B) 53 12.7 12.6 12.8 0.1 Isovalerate 53 2.32a 2.30a 2.10b 0.04 Valerate 53 1.85 1.87 1.79 0.03 Caproate 53 0.50b 0.53b 0.64a 0.03 A:P 53 2.73b 2.70b 2.88a 0.04 A+B:P 53 3.32b 3.28b 3.49a 0.05 a b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 87 Table 3.12. The effects of diets supplemented with different canola meal treatments on the water intake and urine and fecal outputs by cows Diet1 Measurement n U-CM HT-CM L S O 3 -CM S E Water intake, I d"1 15 65.62 68.87 66.55 5.26 Urine output, kg d' 1 27 18.84 19.63 18.92 0.47 Fecal output, kg of DM d"1 27 7.38a 7.78a b 7.89b 0.14 a b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 88 Table 3.13. The apparent digestibilities of diets supplemented with different canola meal sources Die? Apparent digestibility n U-CM HT-CM L S O 3 -CM SE (%, DM basis) DM 27 69.6 69.1 69.3 0.4 CP 27 73.6a 73.0a 70.9b 0.4 NDF 27 50.1b 50.9b 54.0a 0.6 ADF 27 45.2b 45.0b 48.3 a 0.9 a b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 89 Table 3.14. Nitrogen balance measurements for cows fed three different canola meal treatments Diet1 Measurement n U-CM HT-CM L S O 3 -CM S E (kg d"1) N intake 27 0.68a 0.70a b 0.72" 0.01 Fecal N 27 0.179" 0.189" 0.2093 0.004 Urinary N 27 0.2583 0.258a 0.227" 0.008 MilkN 27 0.174 0.181 0.182 0.004 Retained N 2 27 0.0653 0.071 a b 0.102" 0.011 a " Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 2Retained N = Intake N - (fecal N + urinary N + milk N) 90 Table 3.15. The utilization of N, as a percentage of N intake, in cows fed different canola meal sources DieT Measurement n U-CM HT-CM L S 0 3 - C M S E (% of N intake, DM basis) Fecal N 27 26.4b 27.0b 29.1 a 0.4 Urinary N 27 38.3a 36.9a 31.6b 1.1 MilkN 27 25.8 25.9 25.4 0.9 Retained N 2 27 9.6 10.1 13.8 1.5 a , b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal detained N = 100((lntake N - (fecal N + urinary N + milk N)) / Intake N) 91 Table 3.16. The effect of canola meal treatment on energy expenditure and efficiency Diet1 n U-CM HT-CM L S O 3 -CM SE Energy output2, Meal of NEL 50 37.1b 36.9b 39.5a 0.7 Efficiency, Meal NEL k g 1 DMI 50 1.51 1.47 1.49 0.03 a b Means within rows with different letters differ significantly (P<0.05) 1 U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 -CM = heat and lignosulfonate treated canola meal 2Energy output = NEL for maintenance, milk yield, and BW change 92 IV. GENERAL CONCLUSIONS In the first experiment the rumen protein degradability of untreated CM was moderately decreased by heating and substantially decreased by L S 0 3 plus heat treatment. Treatment of CM with 5% L S 0 3 followed by heating at 100°C for 2 h was the most effective at decreasing rumen microbial degradation and increasing the amount of CP and DM delivered to the intestines. In the second experiment cows supplemented with 5% L S 0 3 plus heat (100°C, 120 min) treated CM had increased milk production and showed increased efficiencies of N utilization which were reflected in lowered rumen N H 4 + N levels, circulating urea N levels and urinary N excretion, as a percentage of intake. Dietary N supplied to the rumen in excess of microbial needs is wasted as urea and reduces the efficiency of N utilization for product formation. Thus, the formulation of rations with slowly degraded proteins will improve utilization of dietary N by balancing carbohydrate breakdown with the rate of crude protein breakdown (Tamminga 1992). With modest changes in processing methods the hydrothermal cooking of L S 0 3 treated CM has the potential to produce a commercially viable RUP source for lactating cows. In addition to the direct benefits measured, the formulation of diets with LS0 3 -CM also has positive implications for health, fertility and reduced environmental impact. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0088993/manifest

Comment

Related Items