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

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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  degree at the University of  of  the  requirements  for  an advanced  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 department  or  by  his  or  her  representatives.  It  is  understood  that  copying  my 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  ABSTRACT Two studies were conducted to evaluate the effectiveness of moist heat and lignosulfonate ( L S 0 ) in increasing the rumen undegradable fraction of canola meal 3  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  (wt wt* ), for 0, 30, 60, 90 or 120 min. 1  3  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 C P compared to untreated canola meal.  The corresponding intestinal C P  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. samples were taken during the third and fifth weeks.  Milk, blood and rumen fluid  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" ) than U-CM 1  diet (34.8 kg d' ), but did not differ from H T - C M diet (35.4 kg d" ). Cows supplemented 1  1  with LSO3-CM showed reduced (P<0.05) apparent C P 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 H T - C M 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 H T - C M diets. The results of these studies showed that treatment of canola meal with 5% L S O 3 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 L S O 3 - C M was used more efficiently and was more effective as a rumen undegraded protein source than was the protein in H T - C M or UCM.  iv TABLE OF CONTENTS ABSTRACT  II  TABLE OF CONTENTS  IV  LIST O F T A B L E S  VI  LIST O F F I G U R E S  VIII  ACKNOWLEDGMENTS  IX  LIST O F S Y M B O L S , N O M E N C L A T U R E A N D A B B R E V I A T I O N S  X  I. G E N E R A L I N T R O D U C T I O N  1  1.1 DIETARY PROTEIN INTAKE AND DIGESTION 1.2 C A N O L A M E A L 1.3 PROTEIN P R O T E C T I O N 1.3.1 Formaldehyde 1.3.2 Heat Treatment 1.4 EVALUATION O F R U M E N PROTEIN DEGRADABILITY 1.4.1 Disadvantages of the Nylon Bag Technique 1.4.2 Advantages of the Nylon Bag Technique 1.5 EVALUATION O F INTESTINAL AND T O T A L T R A C T DEGRADABILITY 1.5.1 Disadvantages of the Mobile Nylon Bag Technique 1.5.2 Advantages of the Mobile Nylon Bag Technique 1.6 S U M M A R Y 1.7 O B J E C T I V E S 1.8 R E F E R E N C E S II. T H E E F F E C T S O F H Y D R O T H E R M A L C O O K I N G O F C A N O L A M E A L O N IN SITU R U M E N A N D INTESTINAL D I S A P P E A R A N C E O F D R Y M A T T E R A N D C R U D E PROTEIN 2.1 INTRODUCTION 2.2 MATERIALS AND M E T H O D S 2.2.1 Canola Meal Treatments 2.2.2 Animals and Basal Diet 2.2.3 Rumen Disappearance 2.2.4 Intestinal Disappearance 2.2.5 Chemical Analyses 2.2.6 Calculations and Statistical Analyses 2.3 R E S U L T S 2.3.1 Rumen Disappearance of DM and C P . . 2.3.2 Total Tract and Intestinal Disappearance of DM and C P 2.4 DISCUSSION 2.5 R E F E R E N C E S  2 3 4 5 6 9 10 11 12 12 13 13 14 15  21 21 23 23 24 24 25 26 26 27 27 28 29 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 3.2 MATERIALS AND M E T H O D S 3.2.1 Canola Meal Treatments 3.2.2 Experimental Design 3.2.3 Diets 3.2.4 Sampling 3.2.4.1 Feed 3.2.4.2 Milk 3.2.4.3 Rumen Fluid 3.2.4.4 Blood 3.2.5 Total Collections 3.2.6 Efficiency Calculations 3.2.7 Statistical Analysis 3.3 R E S U L T S 3.3.1 Feed Composition 3.3.2 Dietary Intakes and Milk Production and Composition 3.3.3 Blood Composition 3.3.4 Rumen Fluid 3.3.5 Water Intake and Waste Excretion 3.3.6 Apparent Digestibility of Nutrients 3.3.7 Nitrogen Balance 3.3.8 Nitrogen Efficiency 3.3.9 Energy Efficiency 3.4 DISCUSSION 3.5 R E F E R E N C E S  46 48 48 49 50 50 50 51 51 52 52 53 53 54 54 56 56 57 57 57 58 58 59 59 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 ) 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 ) and moist heat at 100°C  38  Table 2.3. DM disappearance (% of initial) of heat and lignosulfonate ( L S 0 ) treated canola meal and Soy Pass® in the rumen (n=4)  39  Table 2.4. C P disappearance (% of initial) of heat and lignosulfonate ( L S 0 ) treated canola meal and Soy Pass® in the rumen (n=4)  40  Table 2.5. The intestinal DM and C P disappearance (% of initial) of heat and lignosulfonate (LS0 ) treated canola meal and Soy Pass® (n=8)  41  Table 2.6. The total tract DM and C P disappearance (% of initial) of heat and lignosulfonate (LS0 ) 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  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 C P 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 C P 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  3  3  3  3  3  3  3  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 ( N H N) and V F A concentrations  86  +  4  Table 3.12.  The effects of diets supplemented with different canola meal treatments on the water intake and urine and fecal outputs by cows  Table 3.13. The apparent digestibilities of diets supplemented with different canola meal sources Table 3.14.  87  88  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 (LSO -120) or without 5% L S 0 (heat-120) in the rumen of non-lactating cows  43  The C P disappearance of untreated canola meal and canola meal heated with (LSO -120) or without 5% L S 0 (heat-120) in the rumen of non-lactating cows  44  Partitioning of total tract C P disappearance of untreated, heat treated and lignosulfonate (LS0 ) plus heat treated canola meal between rumen and intestines following rumen incubation for 8 h  45  3  Figure 2.2.  3  Figure 2.3.  3  3  3  ix ACKNOWLEDGMENTS I would like to express my sincere thanks to my supervisors Dr. J . A. Shelford of the Animal Science Department at U B C and Dr. L. J . Fisher of Agriculture and AgriFood 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 U B C has been appreciated. I am grateful to the dairy staff and summer students at P A R C (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 P A R C (Summerland, B.C.) for statistical advice and Gay Wilson of P A R C (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 N S E R C (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 LS0 NH N 3  +  4  calcium-sodium lignosulfonate ammonium nitrogen  Abbreviations ADF ADIN AA BUN CM CP d dl DM DMI FCM h HT-CM LSO3-CM min mM MUN NEL N NDF NPN RDP RUP SBM SCC SE TMR U-CM VFA wt  acid detergent fibre acid detergent insoluble nitrogen amino acid blood urea nitrogen canola meal crude protein day decalitre dry matter dry matter intake fat corrected milk hour heat and water treated canola meal heat and lignosulfonate treated canola meal minute millimolar milk urea nitrogen net energy for lactation nitrogen neutral detergent fibre non-protein nitrogen rumen degradable protein rumen undegradable protein soybean meal somatic cell counts standard error total mixed ration untreated canola meal volatile fatty acid 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 A A 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 A A 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 A A 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 N R C (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 A N D 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 ( N H  + 4  N) promotes microbial growth up to the limit of the  microbial N requirement, which is set by the available fermentable carbohydrate, the A T P yield, and the efficiency of conversion to microbial cells (Van Soest 1994). Excess N H NH  + 4  + 4  diffuses from the rumen to the blood where the liver rapidly converts  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* of excess N 1  (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% C P , 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, C M  has an excellent balance of A A and is a rich source of methionine, cysteine, histidine and threonine (Christensen and McKinnon 1993). However, C M 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 C M range from 44.3 (Kendall et al. 1991) to 74.9% (McAllister et al. 1993).  The use of C M use as a RUP source is limited because it is a highly  degradable protein, leading to surplus N H intestinal A A availability.  + 4  production in the rumen and reduced  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 A A 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" to 0.2 mg kg" . 1  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, costeffectiveness, 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). moderate  Mild or  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 (Amadori)  rearrangement  of the  compound (Labuza  Schiffs et  al.  base to  1977).  the  These  1-amino-1-deoxy-2-ketose 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) 1977).  digestion and making it available for intestinal absorption (Erbersdobler  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 C M is insufficient to maximize the escape of protein and A A in C M 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 C M 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 S B M with xylose was more effective in reducing in vitro degradation of S B M by rumen micro-organisms than was glucose, lactose or fructose. Calcium-sodium lignosulfonate (LS0 ), a non-toxic by-product of the wood pulp 3  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 S B M and C M 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 C M 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 S B M by Windschitl and Stern (1988). Heating S B M at 95°C for 45 min with 15-20% moisture had no effect on protein degradability compared to untreated  SBM, but 5%  LS0  3  and heat resulted in  9 significantly lower degradation (Windschitl and Stem 1988).  Similarly, McAllister et  al. (1993) found treatment of C M with 5% L S 0 followed by heat at 1 0 0 ° C for 60 min 3  with 25% moisture caused a larger reduction in effective rumen degradability of protein than heat treatment without L S 0 . 3  combination of L S 0  3  Stanford et al. (1995) concluded that a  and heat is necessary to achieve large increases in rumen  undegradable protein when C M is heated at temperatures between 9 5 ° 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 N R C (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).  A s 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). reported  that the  discrepancies between  Kirkpatrick and Kennelly (1984)  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 C P without consideration of R U P 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 C M 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 C M on 1) in situ ruminal and intestinal disappearance of DM and C P ; 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 International Symp. on Ruminant Physiology. Academic Press, Inc. th  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 nutritionreproduction 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. 3333-3340.  J . Dairy Sci. 80:  Hurrell, R. F. and Finot, P. A. 1985. Effects of food processing on protein digestibility and A A 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 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 Int. Symp. on Protein Metabolism and Nutrition. EAAP Publication No. 59, Vol. 2. Nat. Inst. of Anim. Sci., Research Center Foulum. 15  th  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 A A s 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 6 2 Annual Feeders' Day Report. Agric. For. Bull. Univ. of Alberta, Edmonton, Canada. nd  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 degradability of canola meal protein. Can. J . Anim. Sci. 73: 211-215.  rumen  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  th  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  nd  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., 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.  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: 12641271.  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 microorganisms. Br. J . Nutr. 50: 463-470.  Storm, E., Brown, D. S., and Orskov, E. R. 1983. The nutritive value of rumen microorganisms 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: 479485.  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. protein degradability of roasted soybeans spectroscopy. J . Dairy Sci. 79: 276-282. Tyrrell, H. F. and Moe, P. W. 1975. Sci. 58: 1151-1163.  using  near  Estimating ruminal infrared reflectance  Effect of intake on digestive efficiency. J . Dairy  Van Soest, P. J. 1994. Nutritional ecology of the ruminant. 2  nd  edition. Cornell  University Press, Ithaca, NY.  Varvikko, T. and Lindberg, J. E. 1985. Estimation of microbial nitrogen in nylon-bag residues by feed N dilution. Br. J . Nutr. 54: 473-481. 15  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. 153:16031614.  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: 493507.  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 R U P 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 A A 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 C M 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 R U P content  include  formaldehyde and acids. Formaldehyde treatment (5% wt wt" ) has been successful in 1  reducing CM's in situ rumen C P degradability by 75% (Bailey and Hironaka 1984). Similarly, treatment of C M with acetic acid (3% vol wt" ) followed by oven drying has 1  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 A A 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 C M protein by 76% following 10 h of rumen incubation. total tract digestibility was also reduced by 15%.  However,  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 ). The L S 0 , containing 24% xylose, 3  3  is derived from the spent sulphite liquor produced during sodium sulphite digestion of hardwoods. McAllister et al. (1993) decreased the C P degradability of C M treated in the laboratory by 20% with L S 0  (5% wt wt" ) 1  3  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 C M by 52%. A s a means to study the ability of treatments to alter protein degradation, the in situ nylon bag technique has received the most extensive evaluation Doreau and Ould-Bah 1992).  (Michalet-  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 C M 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 L S 0 WI).  3  (LignoTech USA, Inc., Rothschild,  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 C M 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.  C M 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  C M , C M before heating and after heating and the final C M 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 L S 0 on C M compared to S B M , 3  the commercial product Soy Pass® (LignoTech USA, Inc.,  Rothschild, WI)  was  included in the in situ trial as a positive control. Soy Pass® is S B M treated with 5% L S 0 and heated at 100°C for an undisclosed period of time. 3  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% C P , 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; A N K O M 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 6 0 ° 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 C P disappearance during passage through the intestine. Duodenal nylon bags (3.5 x 5 cm; pore size: 50 + 15 pm; A N K O M , 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 3 9 ° 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 6 0 ° 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. within cows was not measured.  Therefore variation  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 C P 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  measured in this experiment.  into the nylon bag contents was  not  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 C P 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 C P 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 C P 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  while  and  total  tract  disappearances  were  direct  measurements  disappearances in the intestines were calculated values. Effective degradabilities of DM and C P 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 S A S (1990). Least square means between treatments were used to test for significant differences.  2.3 RESULTS The addition of 5% L S 0 reduced the DM content from 87.49% in untreated C M 3  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 L S 0  3  plus heat  treated C M , respectively. Thus, the L S 0 plus heat treated products were cooked at 3  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 L S 0 plus heat treatments, respectively. 3  2.3.1 Rumen Disappearance of DM and CP Results of the in situ rumen DM and C P evaluation of treated C M are shown in Tables 2.3 and 2.4, respectively.  For heat treatment, the extents of ruminal DM and  C P 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 C M and fell between disappearance values of untreated C M and heat-120.  Zero, 4, 12 and 24 h DM disappearances of C M were unaffected by  heat treatment, except for significant reductions with heat-30 relative to untreated C M at 4 and 24 h. Application of 5% L S 0 in the absence of heat reduced the extent of DM and C P 3  disappearance at 0, 4 and 8 h compared to untreated, but was not different at 12, 16  28 and 24 h. DM and C P disappearance of LS0 -120 was lower than L S 0 treatment at 3  3  0, 4 and 8 h of rumen incubation with disappearances of LS0 -30, LS0 -60 and L S 0 3  90 in between.  3  3  Relative to the control, few consistent reductions in 12, 16 and 24 h  rumen disappearances of DM and C P were noted for L S 0 and increasing levels of 3  heat.  Disappearance of Soy Pass® DM and C P was significantly lower at all lengths  of rumen incubation. The DM and C P degradation characteristics of untreated, heat120 and LS0 -120 C M treatments are illustrated in Figures 2.1 and 2.2. 3  illustrate  that  treatment  differences,  in  particular  with  LS0 -120, 3  They clearly 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 L S 0 plus heat treatment of C M had appreciable effects on total 3  tract disappearance of DM or C P (Table 2.6).  The total tract DM disappearance of  Soy Pass® was greater than all C M treatments at 12 and 16 h and all but untreated, heat-120 and LS0 -120 at 8 h rumen incubation. 3  Total tract disappearance of Soy  Pass® C P was lower than all C M 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" with a 1  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 C P 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 L S 0 plus 60, 90 and 120 min of heat increased intestinal disappearance of C M C P 3  29 at 8 and 16 h relative to untreated C M . There were no effects of treatments observed at 12 h and significant reductions in degradability of the L S 0 and LS0 -30 treated C M 3  3  were only observed at 8 h of rumen incubation. The greatest increases in intestinal protein  disappearance  occurred  with  LS0 -120 3  treatment,  which  increased  disappearance from 15.9 to 34.2% at 8 h rumen incubation for untreated and L S 0 3  120, respectively. Soy Pass® intestinal DM and C P disappearances were greatest at all levels of incubation, with 8 h intestinal C P disappearance of 73.7% compared to 34.2% for LS0 -120. 3  2.4 DISCUSSION The rumen C P 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). trends.  Disappearance values of DM followed similar  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 (MichaletDoreau and Ould-Bah 1992). Different commercial processing methods may account for some of the discrepancy as Kendall et al. (1991) reported effective degradabilities of C M protein ranged from 44 to 59%, depending on the source.  Basal diets with  higher roughage or lower C P contents have been associated with increased protein degradation (Orskov 1992).  McAllister et al. (1993) reported that the  effective  degradability of C M 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) (1984).  and lower observed values relative to Bailey and Hironaka  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 C M was more degradable in the rumen than most treatments.  Heat  treatment of C M for 2 h at 100°C compared with lower levels of heat decreased rumen C P disappearance.  Heat treatment of C M at 125°C or 145°C reduced in situ C P  disappearance of C M , but potentially compromised the post ruminal supply of C P (McKinnon et al. 1991).  These higher processing temperatures are less desirable  because of the formation of indigestible protein (McKinnon et al. 1991).  The L S 0  3  treatment of C M followed by 2 h of heat at 100°C, compared with lower levels of heat,  31 reduced rumen C P disappearance.  Using similar heat conditions McAllister et al.  (1993) treated C M with 10% L S 0 and 2% xylose, equivalent to the xylose content of 3  10%  LS0  3  and found C P degradability was reduced a further  respectively, over the reduction observed with 5% L S 0 treatment. 3  28  and  20%,  However, because  of two and three fold increases in ADIN, which is negatively associated with digestibility, they concluded 10% L S 0 and 2% xylose levels were too high. 3  McAllister et al. (1993) reported that heating C M with L S 0 for 2 h at 100°C 3  also reduced C P degradability more than heating without LS0 . Windschitl and Stern 3  (1988) found no difference in C P degradability of untreated S B M and heat treated S B M , but lower degradation in L S 0 plus heat treated S B M . Thus, a combination of 3  L S 0 and heat is necessary to produce large increases in R U P when C M or S B M are 3  heated at temperatures of 100°C for 1-2 h. The less pronounced response of C M to L S 0 treatment relative to SBM, supports the results of others (Windschitl and Stern 3  1988) and our results for Soy Pass®. This may reflect the lower lysine content of C M relative to that of S B M as discussed by McAllister et al. (1993). A s lysine is the main reactive site for reducing sugars during the Maillard reaction, lowered C M responses in degradability to L S 0 may be expected. 3  Total tract disappearances of untreated C M DM and C P 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 C M relative to untreated.  The higher total tract DM  disappearances of Soy Pass® compared to C M , reflect S B M fibre level, which is considerably lower than in C M (NRC 1989). Soy Pass® protein, relative to untreated  32 S B M protein, may be overprotected as reflected in the consistently lower total tract C P disappearance. With exposure of C M 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  C M fractions  disappearances of heat and L S 0  3  and  the  marginal  separation  between  treated C M may be attributed to shorter than  anticipated exposures to 100°C heat.  In the present study, timing of heat exposure  began when C M first entered the hydrothermal cooker. However, the period of time for C M 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 C M 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 C M with or without L S 0 followed by heating at 100°C for 2 h increases the resistance of C M protein to 3  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 A A 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 canola meal. Can. J. Anim. Sci. 64: 183-185.  and  untreated  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 International Symp. on Ruminant Physiology. Academic Press, Inc.  th  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, O N .  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 A D F 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 degradability of canola meal protein. Can. J. Anim. Sci. 73: 211-215.  rumen  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  th  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  nd  ed. Academic Press, London.  SAS Institute, Inc. 1990. SAS/STAT® user's guide: statistics. Version 6. 4 Edition. th  Vol. 1 and 2. S A S 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 University Press, Ithaca, NY.  nd  edition. Cornell  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 ( L S 0 ) and moist heat at 100°C Treatment L S 0 added (%, wtwt" ) Duration of heat (min) 3  1  3  Untreated  0  0  heat-30  0  30  heat-60  0  60  heat-90  0  90  heat-120  0  120  LS0  5  0  LSO -30  5  30  LSO -60  5  60  LSO -90 3  5  90  LSO -120  5  120  3  3  3  3  38 Table 2.2. Changes in DM content that occurred during processing of canola meal with graded levels of lignosulfonate (LSQ ) and moist heat at 1 0 0 ° C DM (%) 3  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  LSO3-6O  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 ( L S 0 ) treated canola meal and Soy Pass® in the rumen (n=4) Incubation time (h) 3  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  LS0  33 7c  50 6cd  61 6cd  77 4a  79 4a  83 4ab  LSO -30  31 3de  46 6de  57 5de  73 1abc  77 3ab  82 3ab  LSO -60  31 6d  45 2e  58 1de  72 7abcd  76 1abc  81 9ab  LSO -90  30 9de  44 4e  57 6de  71 7bcde  76 8ab  82 7ab  LSO -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  0 3  14  17  1. 8  1. 4  0 8  3  3  3  3  3  SE  a-f Means within columns with different letters differ significantly (P<0.05)  40 Table 2.4. C P disappearance (% of initial) of heat and lignosulfonate ( L S 0 ) treated canola meal and Soy Pass® in the rumen (n=4) Incubation time (h) 3  Treatment  0  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  LS0  33.4bc  56.5bc  68.1c  87.2a  88.9a  92.7ab  LSO -30  29.1f  52.6cd  63.6de  81.8abc  86.4abc  92.5ab  LSO -60  30.8de  49.6de  65.4cd  81.3abc  84.8abc  90.8abc  LSO -90 3  27.8g  46.7e  63.4def  79.5bcd  84.4abc  91.9ab  LSO -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  SE  0.5  1.4  1.6  2.1  1.7  1.2  3  3  3  3  4  8  12  16  a-i Means within columns with different letters differ significantly (P<0.05)  24  41 Table 2.5.  The intestinal DM and C P disappearance (% of initial) of heat and  lignosulfonate (LSQ ) treated canola meal and Soy Pass® (n=8) Parameters Dry Matter (%) Crude Protein (%) 3  Incubation time (h)  8  12  16  8  12 9.9de  16 4.6f  3.5cd  15.9hi  11.3bc  4.8bcd  15.3i  14.8bc  8.2bcd  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  LS0  17.3cd  3.8f  3.5cd  24.2def  5.9e  LSO3-3O  21.6c  7.3de  4.8bcd  28.8cd  10.7cd  6.6def  LSO -60  19.4cd  6.7ef  4.8bcd  26.2cde  10.8cd  7.6cde  LSO -90 3  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  1.6  1.2  0.9  1.5  1.6  untreated  12.2ef  heat-30  11.6f  heat-60  3  3  SE  6.1ef  a-i Means within columns with different letters differ significantly (P<0.05)  5.0ef  54.5a 1.0  42  Table 2.6. The total tract DM and CP disappearance (% of initial) of heat and lignosulfonate (LSQ ) treated canola meal and Soy Pass® (n=8) Parameters Dry Matter (%) Crude Protein (%) 3  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  LS0  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  LSO3-6O  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  LSO3-I2O  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  0.7  0.5  0.9  0 8  0.7  SE  3  1.0  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 (LSO -120) or without 5% L S 0 (heat-120) in the rumen of non-lactating cows 3  3  100  n  80  60  40  20  0 0  4  8  12  16  20  24  Incubation time (h)  Figure 2.2. The C P disappearance of untreated canola meal and canola meal heated with (LSO -120) or without 5% L S 0 (heat-120) in the rumen of non-lactating cows 3  3  45  to  • c  CD  E  Z3  a:  (o/ ) aouBJBaddBSjQ u j s j o j d e p n j Q 0  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. quality  protein,  however  they  Ruminal micro-organisms are a good source of  cannot  always  supply  adequate  amounts  metabolizable protein to support production and maintenance (NRC 1989).  of  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' , some studies  (Kung and Huber 1983; Broderick et al. 1990)  1  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 A A profiles of RUP presented for absorption lack sufficient amounts of a limiting A A (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. most successful treatments  The  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  S B M protein  in  (Windschitl and Stern 1988b) and in vivo (Windschitl and Stern 1988a). supplemented with L S 0  3  vitro Diets  treated S B M have supported similar milk production when  untreated S B M was replaced with one-half as much protein from L S 0  3  treated S B M  (Nakamura et al. 1992). Canola meal (CM), the predominant ruminant protein source in Canada, has a good A A profile relative to milk protein, but its small quantities of R U P limit its usefulness (Christensen and McKinnon 1993). Combinations of heat and L S 0  3  have  successfully reduced the rumen degradability of C M without affecting its digestibility (McAllister et al. 1993; Stanford et al. 1995).  While L S 0  3  treated C M 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 C M was left untreated or was processed with either water or calcium-sodium lignosulfonate (LS0 ) (LignoTech USA, Inc., Rothschild, WI). 3  Either  5% L S 0 or 2% water was added (wt wt" ) to C M and thoroughly mixed for 10 min prior 1  3  to heating.  The water was added to increase the moisture content of C M 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, B C . Forced air drying at ambient temperature was used to remove the moisture added prior to heating through water or L S 0 and the moisture added during heating through steam. 3  The C M 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 L S 0 addition, before heating, following heating and post cooling. 3  Four batches of each treatment,  each about 3 tonnes in quantity,  prepared over the course of the experiment.  were  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 6 0 ° C until constant weight were then ground (1 mm screen) and stored until subsequent chemical analysis. The C M 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 C M sources: A) untreated (LICM); B) heat and water treated (HT-CM); or C) heat and lignosulfonate treated ( L S 0 3  CM). To provide an isonitrogenous contribution to total C P intake from each protein source, the amounts of C M in each diet were 20.0, 20.6 and 21.5% for cows fed UC M , H T - C M and L S 0 - C M , respectively (DM basis). Concentrates were prepared in a 3  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 T M R 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 T M R 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 6 0 ° C until constant weight was achieved. Samples were then ground (1 mm screen) and stored until subsequent chemical analysis of NDF, A D F 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. calculated using the equation:  Fat corrected milk (4% FCM) production was  4% F C M (kg d" )= (0.4 + (0.15 * % fat)) * kg milk d" 1  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 approximately immediately. (NH  + 4  2  and 35 of each period between  h after  morning feeding.  Rumen  fluid  1030 pH  h and 1130 was  h,  determined  A proportion of each sample was frozen at - 1 0 ° C until ammonium N  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" , with an initial time of 1  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 - 1 0 ° 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 - 1 0 ° C .  Fecal samples were later dried in a forced air oven at 6 0 ° C until constant  weight was achieved for the determination of DM. ground (1  Dried fecal samples were then  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' ) = 2.2[41.84(% milk fat) + 22.29(% milk solids not fat) 1  - 25.28] (Tyrrell and Reid 1965). kcal N E L k g  1  BW  0 7 5  Maintenance requirement was assumed to be 73  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" for B W loss and 5.12 Meal NEL kg' of B W gain (NRC 1989). 1  1  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 G L M procedure of S A S (1990). Effects were considered to be significant at P<0.05 unless otherwise specified. The model used for this experiment was: YjjKi = U, + 0t + Xj + p| + Yi ) + 6ij| ) k  u, = overall mean,  (k  (k  54 a = effect of square (k = 1 ,...,6), k  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 HTC M processing reduced the DM content on average by 2.98 percentage points, whereas the addition of L S 0  3  in L S 0 - C M processing reduced the DM content on 3  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 H T - C M and L S 0 - C M , respectively. 3  The cooling process increased the DM content on average by 2.17 and 2.68 percentage points to yield the HT-CM and L S 0 - C M final products, respectively. 3  However, the final DM content for both HT-CM and L S 0 - C M was less than that of the 3  original untreated C M .  The DM content of batch 3 of the H T - C M and to a lesser  degree L S 0 - C M were too low, which resulted in the growth of mold. 3  As a  consequence none of the H T - C M batch 3 and only half of the L S 0 - C M batch 3 were 3  fed during the lactation trial. Heat in combination with L S 0  3  (LS0 -CM) increased NDF content to 33.4%, 3  whereas heat alone (HT-CM) marginally increased the NDF to 25% from 24% in U-  55 C M (Table 3.3).  The ADF levels were increased in L S 0 - C M and to a lesser degree 3  in H T - C M over that in U-CM.  ADIN levels were increased from 1.8% in U-CM to  2.0% in H T - C M and markedly increased to 4.1% in L S 0 - C M . 3  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 H T - C M DM was not different than U-CM except at 8 h incubation where there was greater disappearance of H T - C M (50 and 59%, for U-CM and H T - C M , respectively) (Table 3.4). was  Other than no difference at 0 h, the disappearance of DM from L S 0 - C M  consistently  3  lower  than  that of  U-CM  and  HT-CM.  While  the  rumen  disappearance of C P followed the same trend as DM, the differences between L S 0 3  C M and that of U-CM and H T - C M were markedly pronounced (Table 3.5).  At all  incubation times the rumen disappearance of L S 0 - C M C P was at least 45% below 3  that of U-CM and H T - C M . The total tract disappearances of both DM and C P following 8 h rumen incubation were not different across treatments (Table 3.6).  L S 0 - C M had the 3  greatest 8 h intestinal DM disappearance at 45% compared to 26% for U-CM and 18% for H T - C M (Table 3.6).  As C P accounts for 35% of the DM content of C M , the 8  h disappearance value for L S 0 - C M was also greatest 3  at 72%  relative to the  disappearance values of 36 and 26% for U-CM and H T - C M , respectively.  The C P 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 C P level in L S 0 - C M diet was 17.5%, which was greater 3  56 than H T - C M diet (17.2%), but not different than U-CM diet at 17.3%.  The L S 0 - C M 3  diet contained higher NDF at 34.4% compared to 32.5 and 33.2% for U-CM and HTC M diets. A D F was greater in L S 0 - C M diet than in U-CM diet but not different from 3  HT-CM  diet  (19.8,  20.3  and  20.8%  for  U-CM,  HT-CM  and  L S 0 - C M diets, 3  respectively). The higher fibre levels in L S 0 - C M diet reflect the higher fibre levels in 3  the L S 0 - C M supplement. 3  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 L S 0 - C M treated C M in the diet increased DMI 3  over cows fed diets supplemented with U-CM or H T - C M (Table 3.9).  Milk production  in cows fed L S 0 - C M diet was greater than those fed the U-CM diet, but not different 3  from those fed the H T - C M diet (Table 3.9).  The F C M production was higher in cows  fed L S 0 - C M diet compared to HT-CM diet, but not different from the U-CM diet. 3  The  MUN level was lowered in cows fed the L S 0 - C M diet relative to those fed the U-CM 3  and H T - C M 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" in cows fed L S 0 - C M diet 1  3  compared to 18.6 and 18.2 mg dl" for cows fed U-CM and H T - C M supplemented 1  diets.  57  3.3.4 R u m e n Fluid The  pH of the rumen fluid was not affected by dietary treatment (Table 3.11).  The rumen N H CM  + 4  N levels were lower for cows fed L S 0 - C M diet than those fed the U3  diet, but were not different for those fed HT-CM diet.  The molar proportion of  acetate was higher in cows fed L S 0 - C M diet than those fed U-CM and H T - C M diet. 3  Cows fed L S 0 - C M diet produced a lower proportion of propionate than cows fed HT3  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 LS0 -CM 3  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 LS0 -CM diet due to the higher acetate and 3  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 L S 0 - C M diet had a 6% greater output of fecal DM than those fed U3  C M diet, but were not different than HT-CM diet. This reflects the increased DMI of 7% in cows fed L S 0 - C M diet over that of U-CM diet. 3  3.3.6 A p p a r e n t Digestibility of Nutrients The The  effect of diet on apparent digestibility of nutrients is given in Table 3.13.  apparent digestibility of C P was decreased in cows fed L S 0 - C M diet compared 3  to cows fed U-CM or HT-CM diets (74, 73 and 71% for U-CM, H T - C M and L S 0 - C M , 3  respectively). However, the apparent digestibility of NDF and A D F was increased in  58 cows fed L S O 3 - C M diet over that of the U-CM and H T - C M 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 H T - C M diet (Table 3.14). This reflects the higher C P content of the L S 0 - C M diet relative to H T - C M (Table 3.8) and the 3  increased DMI in cows fed L S 0 - C M over that of cows fed U-CM and H T - C M diets 3  (Table 3.9). Fecal N excretion was greater in cows fed L S 0 - C M diet than U-CM and 3  H T - C M diet.  Conversely, urinary N excretion was lower for cows fed L S 0 - C M diet 3  (0.227 kg d" ) when compared to cows fed U-CM and H T - C M diets (0.258 and 0.258 1  kg d" , respectively). The excretion of N in milk was unaffected by dietary treatment. 1  Cows fed LSO3-CM diet retained more N than cows fed the U-CM diet, but was not different than those fed H T - C M diet.  3.3.8 Nitrogen Efficiency The percentage of N intake excreted in feces (Table 3.15) was significantly greater for cows fed LS0 -CM diet than those fed U-CM and H T - C M (26, 27 and 29%, 3  for U-CM, H T - C M and LS0 -CM diets, respectively). 3  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 H T - C M diets, respectively. Cows fed LSO3-CM diet tended (P=0.06) to retain greater amounts of N, as a percentage  of N intake  respectively). treatment.  (10,  10 and 14% for U-CM, H T - C M and LS0 -CM, 3  Milk N excretion, as a percentage of dietary N, was not affected by  59  3.3.9 Energy Efficiency Cows on L S O 3 - C M diet tended (P=0.10) to gain weight faster at 0.337 kg d"  1  than those fed U-CM and H T - C M at gains of 0.158 and 0.125 kg d" , respectively 1  (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 L S 0 - C M diet than those 3  fed U-CM and H T - C M 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 C M on DMI, milk production and digestibility. Heat and L S 0 treatments were designed to shift the digestion of the C M 3  protein from the rumen to the small intestine through the enhancement of the Maillard browning reaction.  During the processing of C M , the formation of primary Maillard  reaction products resistant to rumen proteolysis was reflected in the elevated NDF  levels and reduced rumen C P degradability in L S 0 - C M . 3  (39%)  Heat treatment alone  produced few differences in levels of NDF, A D F and ADIN or in in situ degradability between H T - C M 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 C M 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 C M 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 A D F (22%)  and associated ADIN in L S 0 - C M suggest that 3  indigestible terminal Maillard reaction products were formed. This was not apparent in the in situ analysis of L S 0 - C M , as total tract disappearances of DM and C P were 3  not significantly different in L S 0 - C M compared to that of U-CM.  Similarly in the  3  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 C M in vivo apparent C P 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, H T - C M or L S 0 - C M . Analysis of 3  the total mixed rations showed significant differences in C P content (range  17.2-  17.5%); however the biological significance of these relatively small differences might be questionable.  Supplementation with L S 0 - C M increased milk yield by 1.8 kg d'  1  3  which may have resulted from the increased amount of protein passing to the intestine or from a direct effect of the L S 0 - C M protein on microbial growth and rumen 3  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 R U P sources have shown increased flows of essential A A 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 S B M 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 L S 0 - C M diet were not 3  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 C P 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 L S 0 - C M diet produced more milk, they also had increased 3  DMI of 1.8 kg d" over that of the control. 1  increased R U P and DMI  There may be confounding effects of  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 A A  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 R U P diets relative to low R U P diets. In the present study, higher DMI with L S 0 - C M supplementation may 3  have improved the supply of metabolizable energy and accounted for the increase in milk yield. Increased R U P in the diet results in less A A deamination and lowered ruminal NH  + 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 C M protein was shifted to the intestine as the intestinal disappearance of L S 0 - C M C P was 72%, a 3  100%  increase over that of U-CM (36%). The reduction in BUN levels by 10%, to 16.7 mg dl' and MUN levels by 12%, to 13.9 mg dl" in cows fed L S 0 - C M diets relative to 1  1  3  cows fed control diets is in agreement with results of Roseler et al. (1993) who fed increasing levels of RUP. numerically  lower,  were  The BUN and MUN levels for cows fed H T - C M while 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" or 1  corresponding BUN concentrations of greater than 8 to 10 mg dl" are required. 1  Correspondingly, Wohlt et al. (1978) observed higher DM digestibility when N H  + 4  N  levels were greater than 5 mg dl" compared with concentrations of less than 5 mg dl" 1  \  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 L S 0 - C M . 3  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" h" (Gustafsson and Palmquist 1993). 1  1  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 and reflected the efficiency of N utilization.  + 4  loss from the rumen  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 C P 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  concentrations  low  Veen  degradability.  (1986)  To  suggested that  explain under  the the  lowered  influence  propionate 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 L S 0 - C M diet, a corresponding increase in butyrate was not observed. Other 3  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 L S 0 treated SBM. 3  A depression in branched chain V F A concentrations were observed in cows supplemented with L S O 3 - C M compared to U-CM and HT-CM.  In contrast, others  (Windschitl and Stern 1987; Windschitl and Stem 1988a) found branched chain V F A concentrations were not affected when xylose or L S 0 treated S B M were fed. Similar 3  65 to the present study, Veen (1986) and Windschitl and Stern (1988b) observed decreases in branch chained V F A when low degradable proteins were fed.  The  branched chain V F A depression may be attributed to the lower protein degradation found with L S 0 - C M , 3  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 H T - C M and L S 0 - C M diets, the total 3  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 V F A concentrations when low degradable proteins were fed. Stern (1984) and Windschitl and Stern (1988b) reported decreased cellulose and A D F digestion and lower total V F A flows from continuous culture fermenters when L S 0 treated S B M was used in 3  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 S B M 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 digestibility.  of cows supplemented with L S 0 - C M would tend to decrease 3  However, cows fed L S 0 - C M had increased apparent digestibilities of 3  NDF and A D F over that of the control and H T - C M diets. This suggests that the rapid rate and high degradability of protein in U-CM and H T - C M 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 L S 0 - C M diet 3  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 V F A 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 A D F with the L S 0 - C M diet 3  might have improved ruminal conditions for fibre fermentation, as reflected in the increased rumen concentrations of acetate. Based on studies with fish meal, S B M 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 H T - C M diets suggests uncoupled ruminal fermentation of carbohydrate and protein due to high RDP (McAllan and Griffith 1987). Treatment with L S 0 may have ensured that 3  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 C P digestibility from 74 and 73% for U-CM and H T - C M to 71% with LSO3-CM diet supports the fibre and ADIN levels observed for the respective diets and suggests that L S 0 - C M protein was overprotected to a small 3  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 S B M compared to untreated S B M .  Cunningham et al.  68 (1996) reported no differences in apparent total tract N digestion between S B M based diets  of  low and  overprotected.  high  RUP and concluded that the  treated  S B M was  not  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 R U P 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 A A into milk and body proteins (Tamminga 1992).  Rumen loss can be decreased by reducing  dietary C P , 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 A A deamination yielding the highest N excesses, thus the observed higher urinary N losses with the control and H T - C M . 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 L S 0 - C M diet 3  (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 S B M 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 N H volatilization, nitrate leaching or nitrous oxide emissions (Hof et al. 1997).  3  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 C P has been shown to result in decreased N H emission from the manure and decreased 3  N volatilization as a fraction of the excreted N (Paul et al. 1998). The primary source of N H emissions has been shown to be the urine, with the feces contributing very low 3  emissions (Paul et al. 1998).  In the present study lower N H  3  emissions from the  waste of cows fed L S 0 - C M diet would be expected, as the urinary N excretion was 3  reduced 17%, even though fecal N excretion was increased 10%. The increased energy expenditure of cows supplemented with  LS0 -CM 3  reflects their increased milk production and their numerically higher B W gain and retention of N relative to cows fed control and H T - C M 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 C M supplements. Results of this study indicate that the degradation of C M , treated with 5% L S 0  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 L S 0 - C M . The 3  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. 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A., Bailey, D. R. C , Pickard, M. D., and Gilbert, R. P. 1993. Use of lignosulfonate to decrease the degradability of canola meal protein. Can. J. Anim. Sci. 73: 211-215.  rumen  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  th  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 midlactation. 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. 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Ingredient composition of the total mixed rations Diet Ingredient  U-CM  1  HT-CM  LS0 -CM 3  (%, 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  0  20.6  0  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  0.10  0.10  0.10  HT canola meal  2  L S 0 canola meal  3  3  mineral-vitamin premix  4  U - C M = untreated canola meal, H T - C M = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal Canola meal with 2% added water hydrothermally cooked at 100°C for 120 min. Canola meal treated with 5% lignosulfonate and hydrothermally cooked at 100°C for 120 min Premix contained 40,000 mg kg" of Mn, 40,000 mg kg" of Zn, 16,000 mg kg" of Fe, 12,000 mg kg' of Cu, 640 mg kg" of I, 240 mg kg" of Se, 160 mg kg" of Co, 4,000 KIU kg" of vitamin A, 800 KIU kg" of vitamin D and 10 KIU kg" of vitamin E on a DM basis 1  2  3  4  1  1  1  1  1  1  1  1  1  1  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  HT-CM  Final  1  1  88.66  87.53  85.35  88.75  2  88.99  —  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  3  LS0 -CM  2  3  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  heat (100°C, 120 min) and water (2%) treated canola meal heat (100°C, 120 min) and lignosulfonate (5%) treated canola meal Wing data 1  2  HT-CM = LS0 -CM = 3  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 Batch DM (%) NDF (%) C P (%) A D F (%) ADIN (%) 3  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  2  3  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 H T - C M = heat (100°C, 120 min) and water (2%) treated canola meal L S 0 - C M = heat (100°C, 120 min) and lignosulfonate (5%) treated canola meal 3  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  U-CM 1  21.5  40.9  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  HT-CM 1  19.8  45.5  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  LSO3- -CM  1  15.2  24.8  30.6  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  1.1  1.8  1.3  1.7  1.3  SE  1.4  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 C P 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  U-CM 1  15.7  45.5  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  HT-CM 1  12.6  47.7  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  L S 0 - -CM 3  1  0  8.8  13.3  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  SE  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 C P 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 (%) Treatment Batch  Intestinal  Total tract  C P disappearance (%) Intestinal  Total Tract  U-CM 1  26.6  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  HT-CM 1  16.5  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  LSCVCM 1  47.1  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  SE  1.2  1.3  2.2  a-c Means within columns with different letters differ significantly (P<0.05)  0.4  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 Diet Nutrient  n  U-CM  1  HT-CM  LSO3-CM  SE  (%, DM basis) CP  19  17.3  NDF  19  32.5  ADF  19  19.8  17.2  a  17.5  b  0.1  a  33.2  a  34.4  b  0.4  a  20.3  b  0.2  ab  ab  20.8  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, H T - C M = heat (100°C, 120 min) and water (2%) treated canola meal, and L S O 3 - C M = heat (100°C, 120 min) and lignosulfonate (5%) treated canola meal a b  1  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  LSO3-CM  SE  25.1  26.4  0.2  DMI kg d"  53  24.6  % of B W  52  3.8  3.8  3.9  <0.1  52  665.2  666.4  672.9  3.4  1  b  b  a  Body wt kg gain, kg d"  51  1  Milk, kg d"  1  4% F C M , kg d"  1  0.158  52  34.8  52  33.8  0.125 35.3  b  33.4  ab  0.337  0.093  ab  36.6  a  0.6  a  35.2  b  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  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  Component Yield, kg d"  1  Milk S C C , x 1 0 m r 3  MUN, mg dr  1  1  53 53  223 15.68  229 3  15.34  372 a  13.86  105 b  0.27  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, H T - C M = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal  a b  1  85 Table 3.10. The effects of diets supplemented with different canola meal treatments on blood composition Diet 1  n  U-CM  53  31.1  Blood glucose, mg dl"  53  68.2  BUN, mg dl"  53  18.6  Hematocrit, % 1  1  LSO3-CM  SE  30.6  31.1  0.3  70.2  69.1  0.9  HT-CM  a  18.2  a  16.7  b  0.3  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, H T - C M = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal  a , b  1  86 Table 3.11. The effects of diets supplemented with different canola meal treatments on ruminal pH, ammonium nitrogen ( N H / N) and V F A concentrations DieT n  U-CM  53  6.79  1  53  11.24  Total V F A , mM  53  PH NH  + 4  N, mg dl"  LSO3-CM  HT-CM 6.83 3  100.6  6.84  9.45  ab  0.03  8.00  b  95.1  95.7  SE  0.73 2.6  VFA, mol 100mol"  1  Acetate (A)  53  59.5  Propionate (P)  53  22.1  Isobutyrate  53  Butyrate (B)  53  Isovalerate  53  2.32  Valerate  53  1.85  Caproate  53  0.50  b  0.53  b  0.64  a  0.03  A:P  53  2.73  b  2.70  b  2.88  a  0.04  A+B:P  53  3.32  b  3.28  b  3.49  a  0.05  b  a  1.07  59.4  b  60.4  22.2  a  21.3  1.06  a  12.7  2.30  0.3  b  0.95  a  12.6 a  0.2  a  b  12.8 a  1.87  2.10  0.01 0.1  b  1.79  0.04 0.03  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, H T - C M = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal  a b  1  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 Diet 1  Measurement  n  Water intake, I d"  1  Urine output, kg d'  1  Fecal output, kg of DM d"  1  U-CM  LSO3-CM  HT-CM  SE  15  65.62  68.87  66.55  5.26  27  18.84  19.63  18.92  0.47  27  7.38  a  7.78  ab  7.89  b  0.14  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, H T - C M = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal  a b  1  88 Table 3.13. The apparent digestibilities of diets supplemented with different canola meal sources  Die? Apparent digestibility  n  U-CM  HT-CM  LSO3-CM  SE  (%, DM basis) DM  27  69.6  CP  27  73.6  NDF  27  50.1  ADF  27  45.2  69.1 a  b  b  69.3  0.4  73.0  a  70.9  b  0.4  50.9  b  54.0  a  0.6  45.0  b  48.3  a  0.9  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, H T - C M = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal  a b  1  89 Table 3.14. Nitrogen balance measurements for cows fed three different canola meal treatments  Diet  1  Measurement  n  U-CM  LSO3-CM  HT-CM  SE  (kg d" ) 1  N intake  27  0.68  Fecal N  27  0.179"  0.189"  0.209  Urinary N  27  0.258  0.258  0.227"  0.008  MilkN  27  0.174  0.182  0.004  27  0.065  0.102"  0.011  Retained N  2  0.70  a  3  0.72"  ab  a  0.181 3  0.071  ab  0.01 3  0.004  " Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, H T - C M = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal Retained N = Intake N - (fecal N + urinary N + milk N)  a  1  2  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  LS0 -CM 3  SE  (% of N intake, DM basis) Fecal N  27  26.4  Urinary N  27  38.3  MilkN  27  25.8  25.9  25.4  0.9  27  9.6  10.1  13.8  1.5  Retained N  2  b  27.0  b  29.1  a  0.4  a  36.9  a  31.6  b  1.1  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal d e t a i n e d N = 100((lntake N - (fecal N + urinary N + milk N)) / Intake N) a , b  1  91  Table 3.16. The effect of canola meal treatment on energy expenditure and efficiency Diet 1  n  U-CM  Energy output , Meal of NEL  50  Efficiency, Meal NEL k g DMI  50  2  1  HT-CM  LSO3-CM  37.1b  36.9b  39.5a  1.51  1.47  1.49  SE 0.7 0.03  Means within rows with different letters differ significantly (P<0.05) U - C M = untreated canola meal, HT-CM = heat and water treated canola meal, and L S O 3 - C M = heat and lignosulfonate treated canola meal Energy output = NEL for maintenance, milk yield, and B W change  a b  1  2  92 IV. G E N E R A L CONCLUSIONS In the first experiment the rumen protein degradability of untreated C M was moderately decreased by heating and substantially decreased by L S 0  3  plus heat  treatment. Treatment of C M with 5% L S 0 followed by heating at 100°C for 2 h was 3  the most effective at decreasing rumen microbial degradation and increasing the amount of C P and DM delivered to the intestines. supplemented with 5% L S 0  3  In the second experiment cows  plus heat (100°C, 120 min) treated C M 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 treated C M has the potential to produce a 3  commercially viable RUP source for lactating cows. In addition to the direct benefits measured, the formulation of diets with L S 0 - C M also has positive implications for 3  health, fertility and reduced environmental impact.  

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