figfifikm a . . I . H u . é! ... Amiga . a. «“35“.»me 1 in. r. a? :r: .. .3. :Emfin . “E ,r {m— r V m w..— 'x. 1, V {a ,.%..M..: ‘. c .7 3mm» lit. .. . i. i 1 . ‘ .7... .,..:.u Whvfi 454 w tsm- --t 'k ,v.r yt’l: gratin... u LIBRARY Michlgar State University This is to certify that the thesis entitled REDUCING DIETARY CRUDE PROTEIN CONCENTRATION: EFFECTS ON NITROGEN METABOLISM AND ODOROUS COMPOUND PRODUCTION IN SWINE MANURE presented by Emily Rae—Dianne Otto has been accepted towards fulfillment of the requirements for M.S. degreein Animal science Major professor DateFebruarx 19, 2001 . - . . .- . n . . . MSU ,. an ‘1‘“ ..... n- 1 v“, I hunt-um- 0—7639 PMCE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 cJClRC/DateDuepssoi 5 REDUCING DIETARY CRUDE PROTEIN CONCENTRATION: EFFECTS ON NITROGEN METABOLISM AND ODOROUS COMPOUND PRODUCTION IN SWINE MANURE Emily Rae-Dianne Otto A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE 2001 H i'-‘ ~ptu a; c . _ ‘ l ‘ It ‘ '1 ("u 3.1! i,g'v.t.x,'(.} ut‘illnuttifxflideu (wheat? 26 " 1‘ -. 543-: a": a; i3 ri’fi‘iNW‘lWiIMNi' ‘ l l .t‘ :3 _ _»_ wit» u 6'.‘ ‘1." lgl‘ [h1:l'i‘ ‘1': l :.‘:a ...4..' ‘ ”our it."'-.I‘|I I'Il.\lo'." a". 1:13. I“ t',.". l :‘A “e. ABSTRACT REDUCING DIETARY CRUDE PROTEIN CONCENTRATION: EFFECTS ON . NITROGEN METABOLISM AND ODOROUS COMPOUND PRODUCTION IN SWINE MANURE i"- . i' i < ' “ ‘ int j 'el‘ v I. ‘ 'I'VWH "Ind 01“". t.r.l| ...,,, ”U"... i' (’"ii' w o . By Emily Rae-Dianne Otto Reducing nitrogen (N) excretion by reducing dietary crude protein (CP) in swine diets will reduce total N output and ammonia (NH3) emissions from manure slurries. It has not been determined whether reducing CP and supplementing crystalline amino acids (CAA) at digestible requirements will maintain N retention of pigs. Additionally, it is hypothesized that reducing dietary CP will reduce odorous compounds generated in the hindgut by microbial fermentation of undigested proteins and unabsorbed amino acids (AA). Two experiments were conducted to determine the effects of reduced dietary CP and CAA supplementation on N balance and AA digestibility. Each experiment used six barrows arranged in 6 x 6 Latin squares to determine N retention and AA ileal digestibility of the reduced dietary CP. Results of the N balance indicated that CP could be reduced from 15 to 9% while maintaining N retention. Samples of feces and urine from the N balance were mixed together to form slurries for in vitro experiments to determine NH3 emissions and conduct an odor panel. Ammonia emissions were reduced by 65% but did not decrease odor offensiveness when dietary CP was decreased from 15 to 6%. Results of the digestibility study found that apparent and standardized AA digestibility increased in the 6 and 9% when CP was reduced from 15 to 6% CP and supplemented with CAA. ACKNOWLEDGMENTS First, I would like to acknowledge National Pork Producers Council and Michigan Agriculture Experiment Station for providing financial support of this research. I would like to express great appreciation to my advisor, Dr. Nathalie Trottier, for giving me the opportunity to participate in her laboratory. I would also like to thank her for her guidance, trust, and encouragement in further developing my skills as a researcher, student, and animal science professional during my Master’s program. Additionally, I would like to thank my co-advisor, Dr. Melvin Yokoyama, for his guidance throughout my program, including the use of the microbiology laboratory and the opportunity to actively participate with his lab group. And, a thank you to my additional committee members, Dr. Dale Rozeboom, Dr. Robert von Bemuth, and Dr. Kevin Roberson, with . their inputs I gained greater appreciation of the scope and impact of my research. I would also like to thank Al Snedegar and the MSU Swine Farm Crew for providing the animals I used to complete this research and for their time in assisting with the care of the animals and providing equipment and facilities. In addition, I would like to extend a thank you to Dr. Pao Ku, Dr. Sue Hengemuehle, Jane Link, and Bob Burnett for their guidance and training in the laboratories. Sincerest thanks and appreciation to the many friends, students, and faculty members that made my experience as a graduate student at MSU fulfilling, exciting, and memorable. Finally, thank you to my parents, family, and friends for supporting and encouraging me to pursue my degree and giving me the strength to see it through. iii A l in .4 '4. .. P c-’.' ‘Ai 3“ J .- ht” l N I 'F'D'I'd‘dfltwir‘. .L P ri Or. I. _ i _ 'u u 1mm," ' I ‘ Uh. . 'I‘I".J-“y .‘s ’IN‘, 'JH‘ In .I i i-JIA.‘ I . lie. ..4 {mi-Hi... ‘in 1H. u TABLE OF CONTENTS LIST OF TABLES .................................................. vi LIST OF FIGURES ................................................. vii LIST OF ABBREVIATIONS ......................................... viii CHAPTER 1 Introduction ................................................. 2 1.0 Odorous compounds in swine manure .......................... 3 1.1 Origin and classification of odor ........................ 3 1.2 Ammonia .......................................... 4 1.3 Phenolics .......................................... 5 1.4 Volatile fatty acids ................................... 5 1.5 Quantifying odor .................................... 5 1.6 Odor reduction ...................................... 6 2.0 Diet and nitrogen excretion .................................. 7 2.1 Protein digestion .................................... 8 2.2 Amino acid and peptide absorption ...................... 8 2.3 Amino acid digestibility ............................... 10 3.0 Post-gut nitrogen metabolism ................................ 1 l 3.1 Urea Cycle ......................................... 12 3.2 Nitrogen utilization .................................. 13 3.2.1 Dietary amino acid intake ...................... 13 3.2.2 Dietary amino acid balance~ ..................... 14 3.2.3 Dispensable amino acids ....................... 14 Literature Cited .............................................. 17 CHAPTER 2 Abstract: Effect of reduced intact crude protein and supplementation of crystalline amino acids on nitrogen balance in growing pigs .............. 25 Introduction ................................................. 26 Materials and Methods ......................................... 27 Results and Discussion ........................................ 31 Implications ............................. -. ................... 37 Literature Cited .............................................. 47 CHAPTER 3 Abstract: Amino acid digestibility of reduced concentrations of intact dietary crude protein fed to growing pigs .......................... 52 Introduction ................................................. 53 Materials and Methods ......................................... 54 Results and Discussion ........................................ 57 Implications ................................................. 62 Literature Cited .............................................. 70 CHAPTER 4 Abstract Effect of dietary crude protein reduction on ammonia, volatile fatty acids, phenolics and swine manure odor offensiveness ................ 73 Introduction ................................................. 74 Materials and Methods ......................................... 75 Results ..................................................... 79 Discussion .................................................. 81 Implications ................................................. 87 Literature Cited .............................................. 97 SUMMARY AND CONCLUSIONS Summary and Conclusions ..................................... 100 Implications ................................................. 102 T / LIST OF TABLES Table l. Ingredient composition of experimental diets (as fed) ............... 38 Table 2. Nutrient composition of experimental diets (as fed) ................. 39 Table 3. Ingredient composition of casein and protein-free diets (as fed) ....... 40 Table 4. Nutrient composition of casein and protein-free diets (as fed) ......... 41 Table 5. Nitrogen utilization and digestibility of experimental diets ........... 42 Table 6. Comparison of nitrogen excretion of experimental diets ............. 43 Table 7. Apparent ileal amino acid digestibility of experimental diets with crystalline amino acids, % ..................................................... 63 Table 8. Endogenous amino acid losses determined by two methods, mg/kg . . . . 64 Table 9. Standardized ileal amino acid digestibility of complete diets with crystalline amino acids using protein-free diet to estimate endogenous amino acid losses, % . 65 Table 10. Standardized ileal amino acid digestibility of complete diets with crystalline amino acids using casein diet to estimate endogenous losses, % .............. 66 Table 11. Apparent ileal amino acid digestibility of intact protein without crystalline amino acids, % ..................................................... 67 Table 12. Standardized ileal amino acid digestibility of intact protein without crystalline amino acids using protein-free diet to estimate endogenous losses, % .......... 68 Table 13. Volatile compounds in feces and urine from feeding experimental diets .................................................................. 90 Table 14. Proportion of individual volatile fatty acids to total volatile fatty acids 91 vi LIST OF FIGURES Figure 1. Proportion of urinary and fecal nitrogen excreted. ................. 45 Figure 2. Ammonia emission of fermented manures ....................... 93 Figure 3. Index of odor offensiveness of fermented manures ................. 95 vii l LIST OF ABBREVIATIONS AA amino acid AAd amino acid concentration in digesta AAf amino acid concentration in feed ACE acetate AID apparent ileal digestibility AIDI apparent ileal digestibility of intact protein ALA alanine ARG arginine ASP aspartate BUTY butyrate CAA cystalline amino acids CP crude protein Crd chromium concentration in digesta Crf chromium concentration in feed CYS cysteine EAL endogenous amino acid losses FN fecal nitrogen GLU glutamate GLY glycine HIS histidine ISOB isobutyrate ILE isoleucine viii .Mw- _. .n. .t I . ”In ‘- 1‘! P . ..3I{)‘,‘.ii.:‘.t:.; .' .5 a -3. ’1‘ "V .- ’u L. '; a s 1* o ISOV L‘EU LYS MET ND NH3 NH4 + NPA NPI NR PHE PRO PROP SER SID SIDI TNO THR TRP TYR UN VAL isovalerate leucine lysine methionine nitrogen nitrogen digestibility ammonia ammonium nitrogen retained as a percent of absorbed nitrogen retained as a percent of intake nitrogen retention phenylalanine proline propionate serine standardized ileal digestibility standardized ileal digestibility of intact protein total nitrogen output threonine tryptophan tyrosine urinary nitrogen valerate, valine ix VFA volatile fatty acid VOC volatile organic compound The chapters in this thesis are written according to 2000 Journal of Animal Science format guidelines. xi -I'!n$'-‘i'. 'a‘ I nn_~rrpy.unol>r¢ .L'idlv'.) ..".~J.n‘.c.'oc. CHAPTER 1 Introduction Research involving amino acid nutrition in pigs extends beyond meeting requirements for maximum growth and(or) performance. Growing concerns about environmental pollution stemming from expanding intensified livestock production facilities, especially in the swine industry, are driving the industry’s research efforts to alleviate potential problems. Research has shown that dietary ingredients directly influence the excretion of potential environmental pollutants found in pig manure (Gatel and Grosjean, 1992; Hobbs et al., 1996; Sutton etal., 1999). Environmental pollutants in swine manure include nitrogen, phosphorus, and trace mineral concentrations that can potentially exceed. the loading capacity of the soils when applied (Miner, 1999). Land applied manure can lead to the contamination of water supplies by elements leaching out of soils or from surface run-off. Ammonia and noxious odors emanating from manure storage pits, land application, and swine housing facilities also contribute to air pollution and are considered a nuisance to surrounding residences (Schiffman et al., 1998; Hankins et al., 2000). The overall goal of this thesis is to understand the relationship of nitrogen metabolism in growing pigs to ammonia and odor production from swine manure. In the following chapters, three specific questions are addressed. First, does reduction of dietary crude protein concentration adversely affect nitrogen retention of growing pigs when crystalline amino acids are supplemented to meet requirements on a digestible basis? Second, does reduction of dietary crude protein concentrations of a complete diet reduce apparent and standardized ileal amino acid digestibility? Third, can reduced crude protein diets fed to growing pigs decrease ammonia emission, volatile organic Compound concentrations, and odor offensiveness to levels similar to those contributed from endogenous secretions. The objective of this introduction is to provide information to the reader regarding nitrogen metabolism and excretory products, thus allowing a better understanding of the research questions and results discussed in chapters 2, 3, and 4. l. Odorous compounds in swine manure 1.1 Origins and classification of odorous compounds Odorous compounds originate from the degradation and fermentation of proteins and carbohydrates (Irnoto and N amioka, 1978; Spoelstra, 1980; Hartung and Phillips, 1994; Hobbs et al., 1996). Odorous compounds resulting from protein degradation are phenolics, i.e., indole and skatole (Yokoyama and Carlson, 1979; Yokoyama et al., 1982; Yasuhara et al., 1984) and ammonia (Debuyckere and Vansteelant, 1992). Volatile fatty acid production results from protein and carbohydrate fermentation (Argenzio and Southworth, 1974; Imoto and Namioka, 1978). Over 200 odorous compounds have been identified in pig manure slurries and classified into six categories: ammonia, sulfides, volatile fatty acids, phenols, alcohols and aldehydes (Miner, 1977, Yasuhara and Fura, 1980, Zahn et al., 1997). Odorous compounds used as manure odor indicators must meet specific criteria (Spoelstra, 1980). Criteria are (1) be products of protein or carbohydrate degradation, (2) remain stable under normal waste storage conditions, (3) compound formation must reflect the kinetics of degradation, (4) components must respond to environmental changes, and (5) concentrations must be easily measured (Spoelstra, 1980). Odorous compounds that do not adhere to all the criteria, such as ammonia, sulfides, and phenolics, are not considered g00d odor indicators (Spoelstra, 1980). Additional studies agree that ammonia is not a good indicator of manure odor (Lunn and Van de Vyver, 1977; Williams, 1984; Yasuhara and Fuwa, 1983). However, other studies find that sulfides and(or) phenolics are good indicators of manure odor offensiveness (Schaefer, 1977; Williams, 1984). Swine farm odors are being classified as environmental pollutants to air quality and are potential health risks (Zahn et al., 1997; Schiffman, 1998; Miner, 1999) when animals and humans are exposed for extended periods of time. In the United States, industrial entities must follow government regulations regarding exposure threshold limits and toxic release limits of hazardous compounds such as ammonia, sulfides, and volatile organic compounds (US. EPA, 1992; EPA, 2000a,b). However, minimal to no regulations exists regarding toxic release limits on the very same compounds produced and emitted into the environment from agriculture (Zahn et a1, 1997). 1.2 Ammonia Ammonium originates from oxidative deamination of amino acids through removal of a-amino groups (Jackson et al. 1986). Deamination occurs when amino acids are in excess of supply for protein synthesis or from microbial fermentation in the gastrointestinal tract, especially the hindgut (Debuyckere and Vansteelant, 1992). Ammonia metabolism will be further described later in this review. I .3 Phenolics Tyrosine and tryptophan are substrates for microbial organisms in the hindgut, which produce phenolics, i.e., phenol, p-cresol, and p-ethylphenol, aromatic indole and 3- methyl indole. These phenolics are absorbed, converted to glucuronide conjugates, and excreted in urine (Yokoyama and Carlson, 1979). 1.4 Volatile fatty acids Volatile fatty acids are a result of microbial fermentation of incompletely digested endogenous secretions and dietary proteins. These proteins provide amino acids as the substrates for microbial organisms. The main amino acids undergoing significant degradation by microbial organisms in the hindgut are valine, leucine, tyrosine, and tryptophan. Of these, microbial degradation of valine and leucine produce two malodorous volatile fatty acids, isobutyric and isovaleric, respectively (Yasuhara et al., 1983). 1.5 Quantifying odors Odors emanating from livestock facilities, particularly swine production units, have been measured with highly variable results (Warner et al., 1990; Debruyckere and Vansteelant, 1992; O’Neill and Phillips, 1992; Zahn et al., 1997). Quantifying odorous compound concentrations has been done effectively (Yokoyama et al., 1982; Yasuhara et al., 1984), however, the qualitative evaluation of the compounds in questions is much more difficult. Qualitative evaluation has two primary components, i.e., intensity and quality (Lunn and Van de Vyver, 1977; Miner, 1977; Williams, 1984; Yasuhara et al.,l984; O’Neill and Phillips, 1992). Odor intensity is associated with the odor strength, i.e., weak or strong, and can be altered by the concentration of the compound in question Yap C0nj mon qudi. al..1\ 1 .3 Phenolics Tyrosine and tryptophan are substrates for microbial organisms in the hindgut, which produce phenolics, i.e., phenol, p-cresol, and p-ethylphenol, aromatic indole and 3- methyl indole. These phenolics are absorbed, converted to glucuronide conjugates, and excreted in urine (Yokoyama and Carlson, 1979). 1.4 Volatile fatty acids Volatile fatty acids are a result of microbial fermentation of incompletely digested endogenous secretions and dietary proteins. These proteins provide amino acids as the substrates for microbial organisms. The main amino acids undergoing significant degradation by microbial organisms in the hindgut are valine, leucine, tyrosine, and tryptophan. Of these, microbial degradation of valine and leucine produce two malodorous volatile fatty acids, isobutyric and isovaleric, respectively (Yasuhara et al., 1983). 1.5 Quantifying odors Odors emanating from livestock facilities, particularly swine production units, have been measured with highly variable results (Warner et al., 1990; Debruyckere and Vansteelant, 1992; O’Neill and Phillips, 1992; Zahn et al., 1997). Quantifying odorous compound concentrations has been done effectively (Yokoyama et al., 1982; Yasuhara et al., 1984), however, the qualitative evaluation of the compounds in questions is much more difficult. Qualitative evaluation has two primary components, i.e., intensity and quality (Lunn and Van de Vyver, 1977; Miner, 1977; Williams, 1984; Yasuhara et al., 1984; O’Neill and Phillips, 1992). Odor intensity is associated with the odor strength, i.e., weak or strong, and can be altered by the concentration of the compound in question (Williams, 1984). Odor quality is defined as how the odor is associated to a positive or negative smell regardless of concentration (Williams, 1984). 1.6 Odor reduction Odor reduction of swine manure has been attempted several waysthrough dietary manipulation and formulation. The inclusion of feed additives such as botanical plant extracts, anti—microbial agents, and alteration of dietary fiber content and protein have yielded variable results. Most recent work on odor control has focused on reducing volatile fatty acids, sulfides, ammonia and phenolic concentrations (Hobbs et al., 1996; Hegel, 1997; Hankins et al., 2000; Kendall et al., 2000). However, the correlation between compound concentration and odor offensiveness is not consistent (Hobbs et al., 1996; Hegel, 1997; Hankins et al., 2000; Kendall et al., 2000). The major difficulty in assessing odors and controlling odor emission from livestock facilities is that certain VOC concentrations immeasurable by gas chromatography can still be detected by the human nose (O’Neill and Phillips, 1992). One of the greatest problems is that many of the most offensive smelling compounds have very low odor thresholds and the smallest concentrations still emit potent odor (Spoelstra, 1980; Yasuhara and Fuwa, 1983). Endogenous protein secretions may contribute to the odorous compound pool found in swine manure and thus counteract to a certain degree the potential benefits of dietary manipulation. nm (ICC pro and Ofie absc Crya in di acidS Critic 2- Diet and nitrogen excretion Nitrogen found in pig manure originates from the degradation of protein and amino acid catabolism by digestive and metabolic processes, respectively. Any nitrogen not utilized by the body is excreted in either feces or urine. Fecal nitrogen consists of unabsorbed, undigested dietary and endogenous proteins, bacterial proteins and ammonia. Urinary nitrogen is found primarily as urea, but also as ammonia, amines and amino acids. Nitrogen excretion can be altered and controlled to some extent by diet. Limiting nitrogen intake reduces nitrogen excretion. Reducing nitrogen intake can be accomplished by feeding complete diets that have a reduced concentration of dietary protein. While this results in reducing the excess dispensable amino acids, indispensable amino acids may become limiting, depending on the extent of dietary crude protein reduction. Correcting for the reduction of indispensable amino acids can be accomplished by supplementing synthetic amino acids. Synthetic amino acids are manufactured by chemical and fermentation processes, and are available in crystalline form mainly as an L-isomer. The crystalline amino acids offer nutritional advantages over dietary feed ingredients in that they are nearly 100% absorbed in the small intestine (Chung and Baker, 1992; Butts et al., 1993a, b). Thus, crystalline amino acids can be supplemented to alleviate amino acids deficiencies found in diets with reduced crude protein concentrations. For these reasons, crystalline amino acids can play a key role in reducing nitrogen excreted in pig manure. Further research is critical to provide reliable information on the rate of crystalline amino acid inclusion. sul l9: enz 511 We Vein l99. ' Capd lfan\ 2.1 Protein digestion Dietary protein degradation occurs during digestion throughout the gastrointestinal tract. Enzymes secreted from the salivary glands, stomach, lumen mucosa cells, and pancreas hydrolyze both proteins and peptides from dietary and endogenous origin and release amino acids and small peptides (Zebrowska et al., 1983; Corring et al., 1982;1mbeah et al., 1988). Enzymatic secretion and activity is important since not all amino acids or peptides are hydrolyzed from protein at the same time or same location along the intestinal tract (Casirola et al., 1994). Enzyme digestion is dependant on the presence of substrate and residence time in the digestive tract (Ravindran et al., 1984; Imbeah et al., 1988). The greater amount of substrate present in the gastrointestinal tract, the higher the enzyme secretion (Casirola et al.,1994). Dietary protein concentration (high vs. low) also stimulates enzyme activity along the small intestine of mice (Casirola et al., 1994). In pigs, the volume and activities of enzyme secretions are not different when comparing protein—rich grains versus low-protein grains (Imbeah et al., 1988; Pohland et al., 1993). 2.2 Amino acid and peptide absorption Amino acids and peptides are absorbed by the enterocytes throughout the small intestine by competitive active-transport systems, and delivered to the liver via the portal vein (Baumrucker and Davis, 1980; Casirola et al., 1994; Soriano Garcia, et al., 1998, 1999; Torras- Llort, et al., 1996, 1998; Bertolo et al., 2000). Each transport system is capable of transporting different amino acids, however, some amino acids can be transported by more than one system (Soriano Garcia et al., 1998, 1999; Torras-Llort et al., 1996, 1998). and 1 SN limp Amino acids are classified into four categories according to their structure type and characteristics. The dibasic includes lysine and arginine, the dicarboxylic includes aspartate and glutamate, the neutral includes leucine, threonine, and alanine, and the imino includes proline. Transport of amino acids is ion(cation) gradient dependant and correlates to the categories of amino acids (Silk et al., 1985; Gardner, 1988). However, transport systems do not strictly adhere to transporting amino acids within a single category. This creates competition for transporters amongst free amino acids. A pr0per balance of amino acids included in crystalline form in reduced crude protein diets may be critical to minimize competition for absorption across the gut epithelium. Free amino acid transport systems are different from peptide transport systems (Gardner, 1988). Two transport systems, a transcellular and a paracellular have been suggested for peptides (Gardner, 1988). Transcellular transport may be more prevalent compared to paracellular transport in humans (Gardner, 1988). Transcellular transport of peptides occurs when they become bound to the membrane surface receptors. The peptide is enveloped by the membrane to form a vesicle. The vesicle and protein contents are degraded by proteolysis resulting in small peptide fragments. The fragments are either fused, removed via additional pathways, or are expelled by exocytosis at the basolateral membrane (Gardner 1988). It is believed that transcellular transport minimizes entrance of proteins and large peptides into circulation (Gardner 1988). Paracellular transport occurs by peptide movement through tight junctions between cells and is involved with fluid and ion transport (Gardner, 1988). This peptide transport system may be more significant in situations of enteral disease or damaged intestinal lining (Gardner, 1988). quc end. llca.‘ Transporter location and density, and age of the animal affect the rate of amino acid and peptide absorption (Baumrucker and Davis, 1980; Casirola et al., 1994; Soriano and Planas, 1998). Casirola et al. (1994) found that amino acid transport is greater in the proximal and middle section of small intestine in mice, while the capacity to uptake amino acids in the distal segment of the small intestine is greater compared to the proximal and middle sections in chickens (Soriano and Planas, 1998). Amino acid uptake increases with age and development of the epithelium of the small intestine in both mice and chickens (Casirola et al., 1994; Soriano and Planas, 1998). 2.3 Amino acid digestibility Amino acid digestibility is a measure of amino acid disappearance from the gastrointestinal tract. Apparent digestibility of a protein depends on its dietary concentration (Donkoh and Moughan, 1994; Fan and Sauer, 1997). A positive curvilinear response has been found with increasing concentrations of dietary protein and apparent amino acid digestibility in rats and pigs (Donkoh and Moughan, 1994; Fan and Sauer, 1997). In contrast, when the apparent amino acid digestibility of a protein is corrected for endogenous losses, it is independent of dietary protein concentration in question. The terminology for when apparent ileal digestibility is corrected for endogenous losses is true ileal digestibility, or more recently referred to as standardized ileal digestibility. Endogenous losses are any of the proteins and amino acids resulting from digestive secretions or sloughing of cells lining the gastrointestinal tract that are not reabsorbed and recycled by the animal (Danfoer et al.,1996). It is estimated that nearly 75% or more of endogenous secretions are absorbed and recycled (Souffrant et al., 1993). 10 i3. ' 12% rd 3 ’U’A ; I 9-" II 1.".‘tl? file‘vl. .. Jig: , H ‘0 I “bin.“ 1 I v . 9 i133“ .'. «55:; ‘W 4I 90 y" tiff-mi . I.“ .1“ ,l'i‘i . . . . . .l' .l ' Ml MM Ali's-415'! a I_H.'¢ I "’le u Endogenous losses can be classified into two categories: non-specific basal losses and specific losses . Non-specific basal losses are those that will occur regardless of diet. composition or ingredient characteristics and are only dependent on dry matter intake, therefore assumed to be constant (Boisen and Moughan, 1996). Specific losses are all those dependant on the characteristics of the dietary ingredients (Boisen and Moughan, 1996). Fiber and anti-nutritional factors are two main components of ingredients known to increase specific endogenous losses (Ravindran et a1, 1984; Grala et al., 1998). Indigestible fiber increases cell sloughing through abrasive action against the intestinal lumen (Ravindran et al., 1984). Fiber can also inhibit enzyme activity on substrates (Ravindran et al., 1984), and increase the passage rate of digesta, thereby reducing absorption of amino acids and peptides (Ravindran et al., 1984; Sauer etal., 1991). Some ingredients contain anti-nutritional factors that inhibit enzyme activity (Grala et al., 1998). Trypsin inhibitors and tannins are two examples of anti-nutritional factors that will bind to the pancreatic enzymes and prevent protein digestion (Grala et al., 1998). Because enzymes cannot hydrolyze proteins and release peptides and amino acids, increased enzyme secretions will also occur (Grala et al., 1998). Dietary crude protein reduction and crystalline amino acid inclusion may allow for an optimum balance of amino acids, and thus minimize competition of uptake across the gastrointestinal tract, minimize endogenous losses, and maximize digestibility. 3. Post-gut nitrogen metabolism Nitrogen metabolism is the cycle of protein accretion and degradation, more specifically, protein synthesis and amino acid catabolism (Christensen et al., 1986; Groff et al., 1995). Protein accretion occurs as long as a sufficient supply of balanced amino 11 't . I .. w ,1- V i‘. ~ ‘ '1 . mum”. M- him i . i" " v" " “ e ‘I 'éf.r-.‘_'!Hu‘ir»{‘tl_:'. {ILA hulnfi‘fil‘v g5 "I‘ll; 1 1Q -‘, r. .j'.1>» a“ :Is':::"."|._ I:. ' flfibuzfig'h - .. . . .. ta- ‘ WWW . .. I -I ~ I .'- w ‘F‘H’vh, 5y lit. ska 3./ {Hill fa“ hi acids are present (Mnilk et al., 1996). When amino acids become limiting for accretion, body proteins are degraded and the amino acids are released into plasma. These amino acids will be re-utilized for protein synthesis (Mnilk et al., 1996). The cycle of protein accretion and degradation is called protein turnover. Protein turnover rate is more rapid in organ tissues and the gastrointestinal tract as compared to skeletal muscle (Benevenga et al., 1993; Seve and Ponter, 1997; Nyachoti, et al 2000).. The high rate of protein turnover in organs such as the liver and small intestine is due to the constant synthesis of enzymes and replacement of epithelial cells, respectively (Seve and Ponter, 1997; Nyachoti et al., 2000). The primary organ responsible for most amino acid catabolism and protein synthesis is the liver (Christensen et al., 1986; Groff et al., 1995). The liver is responsible for synthesizing dispensable amino acids, proteins, enzymes, and urea. The liver removes excess amino acids by oxidizing them into free ammonia and a carbon skeleton (Groff et al., 1995). 3.1 Urea Cycle The process of catabolizing excess amino acids and removing the resultant ammonia is known as the urea cycle. Arginine, glutamate, and glutamine are fundamentally important amino acids involved in removing excess ammonia from the body (Jackson et al., 1986). Any excess amino acids not needed for synthesis of proteins and dispensable amino acids are deaminated. The carbon skeleton is used for glucose and lipid synthesis (Broquist, 1984). The remaining amino group is transferred by a series of enzymatic reactions to arginine, which is cleaved to produce urea and omithine. 12 0x1 Son lilo gasu pro» SOUl' Cndo nnro. bdar‘ iZJ efficl. exCeg. The urea cycle consists of five enzymatic reactions that occur in the periportal hepatocytes (Morris, 1992) and it has been demonstrated in rats, pigs, and humans that urea cycle activity is increased with increasing dietary protein intake (Edmonds et al., 1987; Matthews and Campbell, 1992; Bertolo et al., 2000). The concentrations of amino acids in the portal circulation are higher than those circulating in the plasma (Bertolo et al., 2000). In order to maintain balance of the amino acids in the plasma pool, the liver oxidizes the excess amino acids. Urea is transported out of the liver to the kidneys to be excreted in the urine. Some of the urea has been shown to be secreted into the small intestine and stomach (Mosenthin et al., 1992a; Pohland et al., 1993). However, recycling of urea in the gastrointestinal tract is not a significant source of nitrogen for protein synthesis to pigs provided adequate dietary protein (Thacker et al., 1982), but does supply a nitrogen source for bacterial protein synthesis in the hindgut (Mosenthin et al., 1992b). 3.2 Nitrogen utilization Nitrogen utilization is a measurement an animal’s ability to retain dietary and endogenous nitrogen for protein synthesis and accretion. Several studies have shown that nitrogen utilization is directly affected by dietary protein intake and dietary amino acid balance (Wang and Fuller, 1989; Gate] and Grosjean, 1992; Lopez et al., 1994; Lenis et al., 1999; Seve and Ponter, 1997). 3.2.1 Dietary amino acid intake Amino acids limiting in relation to other amino acids are conserved, and their efficiency of utilization are increased as compared to when all amino acids are present in excess (Mnilk et al., 1996). Utilization of amino acids provided in excess of 13 reclu'lrements for protein synthesis decreases because they are oxidized instead of being incorporated into body proteins (Benevenga et al 1993). 3.2 .2 Dietary amino acid balance The optimal dietary amino acid balance corresponds to the whole body protein composition of the pig (Wang and Fuller, 1989; Chung and Baker, 1992; NRC, 1998). This balance is also referred to as the ideal protein or ideal amino acid pattern, whereby no deficiency or excess exists and all amino acids are equally limiting. Because lysine is the first limiting amino acid in growing pigs, lysine is used as the reference amino acid to express other amino acids as a ratio to lysine (NRC, 1998). Because dispensable amino acids can be derived from indispensable amino acids, the ideal pattern represents only the indispensable amino acids. An ideal dietary amino acid pattern is one that results in the highest nitrogen retention for a given nitrogen intake (Wang and Fuller, 1989). 3.2.3 Dispensable amino acids Even though certain indispensable amino acids can be efficiently converted todispensable amino acids, dispensable amino acid demand cannot always be met, even in the presence of excess indispensable amino acids (Lenis et al., 1999). Some organs have specific requirement for dispensable amino acids. The gut has a high requirement for glutamine, as demonstrated from first-pass metabolism studies, where glutamate and glutamine concentrations were much lower compared to all other amino acid concentrations in portal blood following a meal (Matthews and Campbell, 1992; Bertolo et al., 1999). Certain dispensable amino acids also play an important role as they provide a sparing effect for the indispensable amino acids (Heger et al., 1998; Lenis et al., 1999). 14 and Iota (\ 5 I . l ‘. I ”ran. _ . I - v. "v"“‘ " I L» "#97791. . ‘J'i‘d" hi . "I 59. ‘txiriitt‘iifiéi‘ti’r *- . <'. a“: a. £6. .‘ . 4' a... . ,. - ‘ i.'.'- "urns-ne'- 0. Mn. Reduced crude protein diets may not provide sufficient dispensable amino acids, thus sinii'PI resulting in transamination of indispensable amino acids to dispensable amino acids and compromising protein accretion (Lenis, 1999). Heger et a1. (1998) and Lenis et a1. (1999) both showed that a'minimum concentration of dietary nitrogen is necessary for optimal nitrogen utilization and nitrogen retention in pigs. Both studies showed that as the ratios of indispensable to total amino acid nitrogen increased, nitrogen retention decreased in pigs fed the higher dietary crude protein concentrations (Heger et al.,1998; Lenis et al., 1999). Heger et a1. (1998) and Lenis et al. (1999) suggested that reduced nitrogen retention occurred because either total nitrogen or dispensable amino acids became limiting. 15 LITERATURE CITED l6 Ben Ben Bols Broq Butt. 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'r;- .. fi 0’ '4' - ~11 \w'A-c‘lh” :‘."n'1v. ‘-‘|v.\.1."&e"»c‘a Zx-‘v4‘5’u' t‘nU-fl -vQ--d- '_ '-.~. 5.0 1.. f CHAPTER 2 24 ABSTRACT EFFECT OF REDUCED INTACT CRUDE PROTEIN AND SUPPLEMENTATION OF CRYSTALLINE AMINO ACIDS ON NITROGEN BALANCE IN GROWING PIGS By Emily Rae-Dianne Otto It is not known whether nitrogen retention (NR) required for maximum growth in pigs can be achieved with crude protein (CP) reductions of intact protein greater than 3% and supplementing crystalline amino acids (CAA) to meet the AA requirements using an ideal amino acid pattern. Six barrows were allotted to one of six dietary treatments in a Latin square design. Pigs were housed individually in metabolism pens to allow total collection of feces and urine, separately, in an environmentally controlled room. Dietary treatments were offered three times daily and consisted of 15, 12, 9, and 6% intact CP from com-soybean meal (CSBM) with CAA included in the 12, 9 and 6% CP diets, a 15% CP casein based, and a protein-free diet. The 15% CP casein and protein-free diets served as positive and negative controls, respectively. Crude protein concentrations were reduced by adding cornstarch to maintain the CSBM ratio. The indispensable to dispensable amino acid ratio was maintained at 45:55 with addition of L—glutamate to the 9 and 6% CP diets. Nitrogen retention with 6% CP was less (P < 0.01) compared to retentions with the 12 and 15% CP diets. Nitrogen retention was similar when the 9 and 12% CP diets were compared individually to the 15% CP diet. Moreover, the NR in pigs was less (P < 0.01) when the 9% CP diet was fed compared to the 12% CP diet. Nitrogen digestibility was greater (P < 0.01) when feeding a 6% CP diet compared to feeding a 9, 12 or 15% CP diet. Nitrogen utilization efficiency was increased (P < 0.01) as dietary CP was reduced from 15 to 6%. These results indicate that CP concentrations of dietary 25 intact protein can be reduced from 15 to 9% while supplementing CAA to an ideal pattern without adversely affecting nitrogen retention in growing pigs. Introduction Nitrogen is a major component of pig manure that when in excess in any one locality, has negative effects on the environment (Zahn et al., 1997). Environmental pollution results from nitrogen contamination of surface and ground water (Miner, 1999). Decreasing dietary CP by 3 to 4 percentage units and ensuring adequate amino acid balance through crystalline amino acid supplementation does not reduce growth performance of growing and finishing pigs (Lopez et al., 1994; Tuitoek et al., 1997). I hypothesize that feeding reduced intact CP diets balanced to meet both the ideal digestible amino acid profile (NRC, 1998) and the nitrogen requirement of the growing pig (Lenis et al., 1999) decreases nitrogen excreta amount while maintaining nitrogen balance equal to that of pigs fed a non—reduced CP diet. This study focused on the following three objectives. First, test whether nitrogen retention can be maintained in pigs fed reduced CP diets containing 80, 60 and 40% of the intact protein concentration contained in the control diet. Second, quantify the endogenous protein contribution to the total nitrogen excretion. And third, determine the minimum dietary CP needed to attain nitrogen excretion rate equivalent to that contributed by endogenous proteins alone. 26 Materials & Methods Animals, Experimental Design and Diets The Michigan State University All University Committee on Animal Use and Care approved the experiment. Six barrows ((Yorkshire x Landrace) x Duroc), with an initial BW of 44.7 kg :1: 1.8 kg were allocated to six dietary treatments in a 6 x 6 Latin square design. Pigs were penned individually in stainless steel metabolism pens (1.2 m x 0.75 m) equipped with low-pressure water nipples providing free access to water. Pens had wire flooring that allowed feces to be caught on a fine-mesh wire screening below the flooring. Metal catch pans were placed below the screens to funnel urine into plastic collection vessels. Pigs were housed in an environmentally controlled room maintained at 21°C. The six diets consisted of 15, 12, 9 and 6% CP com-soybean meal-based (CSBM) diets, a 15% CP casein-based diet and a protein-free diet. Obligatory endogenous protein output was detemiined using the protein—free and the casein-based diet. The protein-free diet was chosen to determine the non-specific endogenous protein losses, and the casein-based diet to determine non-specific endogenous protein losses in the presence of a highly digestible dietary protein. Diet ingredient and nutrient composition are provided in Tables 1 and 3, and 2 and 4, respectively. The 6, 9 and 12% CP concentrations of CSBM in diets were obtained by diluting the 15% CP CSBM diet with corn starch in order to maintain equal AA profile arising from intact protein sources, i.e., corn and soybean meal. In order to meet the amino acid requirements of the growing pig, diet formulation was based on the NRC (1998) ideal apparent amino acid digestibility pattern. Crystalline amino acids were added to the 6, 9 and 12% CP diets. Among them, glutamic acid was added to balance 27 the indispensable amino acid (LAA) to dispensable amino acid (DAA) nitrogen ratio to 45:55 (IAAzDAA). Analyzed total CP and values for total amino acid content closely matched the calculated values, except for the lysine and threonine content of the 12 and 15% CP diets, which were lower than expected. Lenis et al. (1999) determined that optimal nitrogen utilization and nitrogen retention for growing pigs is attained in low CP diets when 1AA nitrogen and DAA nitrogen are balanced to 50:50 or slightly less. Diets were also formulated to contain similar energy levels. Feed was provided at 3.5% BW and divided into three equivalent meals per day (800, 1200 and 1600 h). To reduce feed wastage, water was added to the meal (approximately 100 mL/ 300 g) and mixed to form a gruel. Body weights were measured on d 1 of each period. Sample Collection The experiment consisted of six collection periods. Each period lasted 10 d. Feces and urine were collected for a duration of 5 d from each pig following a 5-d adaptation period to the diets. Ferric oxide was used as an indigestible marker to indicate the initiation and termination of collection for fecal matter. Each pig received 5 g of ferric oxide that was added to 100 g of feed, and mixed thoroughly with 125 mL of water at the first meal on d 1 and d 6. The remaining meal allotment was fed after the ferric oxide-feed mixture was consumed completely. Total daily fecal samples were collected once daily, weighed and stored at 4 °C for 5 d. Fecal samples were homogenized for 15 min using a Hobart mixer (Model A-200, Hobart Manufacturing, Troy, Ohio). Sub- samples were collected (500 g) and stored in 490-mL plastic containers and frozen at -20 °C. For urine collection, 10 mL of 6 N HCl was added daily to each of the lO-L 28 collection vessels to reduce the pH of the urine and prevent volatilization of NH," (Russell et al., 1983). Urine was collected into a 10-L plastic bucket. The funnel attached to the collection bucket had glass wool to prevent fecal and other particulate matter from contaminating the urine. The urine was filtered again through four layers of cheesecloth into a clean bucket. Approximately 20% of daily urine volumes were stored in 4-L plastic bottles throughout the collection period at 4 °C. At the end of each collection period, the urine was homogenized, and a sub-sample of pooled urine was frozen at -20 °C. Sample Analysis For chemical analysis, fecal samples were freeze-dried (VirTis Model 25-SRC, VirTis Co., Gardiner, NY). Fecal and feed samples were finely ground using a cyclone mill (Cyclotec Sample Mill 1093, Sweden) with a 1-mm mesh screen. Nitrogen content in feces and feed was determined using an automated nitrogen analyzer (LECO FP-2000, LECO Co., St. Joseph, MI, AOAC #99003). Dry matter of feces and feed was determined following a 12-h drying period at 60 °C using a vacuum oven (model 583/Full View, National Appliance Co., Portland, Oregon). Urine was filtered (Whatman filter paper No.4, Whatman Int’l. Ltd. Maidstone, England) and nitrogen concentration measured using the same procedure as described for feed and feces. Amino acid analysis was performed on feed samples using the Pico-Tag method (Waters Co., Milford, MA) following a 24-h acid hydrolysis in 6 N HCl at 105 °C and 121 mm Hg. Samples were brought up to volume (40 mL) and filtered (Whatman filter paper No.2). The amino acid filtrate was sub-sampled and dried using vacuum centrifugation (ATR CS3 drying vacuum system, Appropriate Technical Resources, Laurel, MD). The amino acid 29 hydrosylate was reconstituted, dried again by vacuum centrifuge, derivatized with phenylisothiocyanate and separated using a Waters high-pressure liquid chromatograph (Waters Co., Milford, MA) fitted with a 15-cm hydrosylate column. Statistical Analysis Nitrogen balance data were analyzed by analysis of variance using PROC MIXED of SAS (1999) (SAS Inst. Inc., Cary, SC). The dependent variables nitrogen intake (NI), fecal nitrogen output (FN), urinary nitrogen output (UN), and total nitrogen output (TNO) were analyzed for a 6 x 6 Latin square. The fixed effects of pig, period, and diet were included in the statistical model. Differences between least squares means (LSM) values for diet were evaluated using Adjusted Tukey-Kramer Honestly Significantly Difference (Younger, 1998) for the dependant variables NI, FN, UN, TN 0 . Residual plots of the response variables for each dietary treatment revealed heterogeneity of variances for the protein-free and casein-based diets compared to the 6, 9, 12, and 15% CP diets. Due to variance heterogeneity, the protein-free and casein- based diets were removed prior to analysis of the following dependant variables: nitrogen retained (NR), nitrogen absorbed (NA), nitrogen digestibility (ND), nitrogen retained as percent of intake (N PI), and nitrogen retained as percent of absorbed (NPA). Additionally, response variables NR, NA, ND, NPI, and NPA were most relevant for comparing the four diets containing the CSBM. Therefore, a 4 x 6 Latin rectangle including fixed effects of pig, period, and diet was used to analyze the nitrogen balance data. Differences between LSM, for the dependant variables NR, NA, ND, NPI, and NPA were evaluated using the Bonferroni (Dunn) Test (Younger, 1998). The Bonferroni test was used because non—orthogonal comparisons were made between the 6, 9, 12 and 30 15% CP diets. Hence, level of significance was determined to be 0.0083, where P = 0.05 and number of contrasts made were 6. Nitrogen intake, TNO, NR, ND, NPI and NPA were regressed against dietary protein concentration (6, 9, 12, and 15% CF) to determine linear and(or) quadratic relationship using PROC MIXED. The fixed effects included in the regression model were pig, period, and diet. Results and Discussion Nitrogen Balance The primary aim of this study was to determine the effect of CP reduction on nitrogen balance and nitrogen excretion. I hypothesized that decreasing dietary CP from an intact protein source and including crystalline amino acids would maintain nitrogen retention, yet reduce total nitrogen excretion to similar levels contributed by endogenous proteins. Nitrogen Retention Nitrogen retention of the pigs fed the 6% CP diet was similar to feeding the 9% CP diet (Table 5). Lower (P<0.01) NR in pigs resulted from feeding the 6% CP compared to the 12 and 15% CP diets. Pigs fed the 9 and 12% CP diets had similar NR when compared individually to when the 15% CP diet was fed. Interestingly, the 12% CP diet resulted in greater NR compared to the 9% CP diet. Comparable results were reported by Shriver et al. (2000). In that study there was no difference in NR of 40-kg pigs fed com-soybean meal based diets when dietary CP concentrations were lowered from 15% to 11%, and supplemented with crystalline lysine, methionine, threonine, tryptophan, isoleucine, and valine on an ideal basis (Carter, 2000; Shriver et al., 2000). 31 Similar results were also found in 21-kg pigs where NR was equivalent for a 16% CP diet and a 12% CP including crystalline lysine, tryptophan, threonine, glutamic acid and glycine (Kerr and Easter, 1995). In contrast, NR in 32-kg growing pigs decreased when diets containing 16.5 and 13.6% CP were offered compared to a control diet containing 19.5% CF (Zervas et al., 2000). The reduction in NR occurred despite the fact that crystalline lysine, methionine, tryptophan, threonine, isoleucine, or valine were included in the 13.6 and 16.5% CF. The inconsistent results from the literature suggest that balancing total digestible amino acids to an ideal pattern is only one part of the solution to maximizing nitrogen retention in pigs with reduced crude protein diets. Another important component to explain the differences in results is how much of the total amino acids are contributed from crystalline amino acids. In the present study, NR was decreased significantly in the 6% CP diet in comparison to the 12 and 15% diets and nearly 30% of the total dietary amino acids were from synthetic origin in that diet. This percentage of free amino acids may have caused an imbalance in plasma amino acid concentrations. Crystalline amino acids are available for absorption as soon as they enter the small intestine. Whereas amino acids from intact protein must be released by digestive enzymes prior to absorption. The crystalline amino acids may have been oxidized and not used for protein accretion because not enough of the other amino acids and peptides from intact protein sources were absorbed and available at the same time. In addition to nitrogen retention measurements, growth parameters of ADG, ADFI, G/F, in growing pigs were measured by Shriver et al. (2000). The CP of diets was reduced from 18, 16 and 14% to 14, 12 and 10% for the three stages of growth, 32 respectively. Shriver el al. (2000) found no differences in growth performance. Similar results have been reported by Liu et al. (2000) in 59.8-kg pigs fed diets containing 10.4% CF and supplemented with crystalline lysine, threonine, tryptophan, methionine, isoleucine and valine compared to a 15.4% CF. In contrast, earlier studies report feed efficiency reductions and reduced daily gains in 18 to 35-kg pigs when dietary CP was lowered from 16 to 12% (Russell et al., 1983) or from 17 to 11% (Kephart and Sherritt, 1990). Nitrogen Digestibility Nitrogen digestibility increased linearly (P<0.01) as dietary intact protein CP was reduced from 15 to 6% (Table 5). Nitrogen digestibility was greater (P<0.01) in the 6% compared to the 9 and 15% CP diets. Similar ND was found between 9, 12, and 15% CP diets. Nitrogen digestibility was equal between a 17.4% CP com-soybean meal based and a 14.8% CP corn-peas-soybean meal based diet with lysine, methionine, threonine and tryptophan supplementation (Gatel and Grosjean, 1992). Kephart and Sherritt (1990) found that ND was not different between growing pigs fed an 11, 12 and 13% CP diets compared to a 17% CP diet when crystalline lysine, methionine, threonine, tryptophan, isoleucine, valine and(or) glutamic acid were included and diets were formulated to exceed amino acid requirements by 115%. In contrast, ND was higher when indispensable and dispensable CAA were added to a 12% CP basal diet to match the total amino acid profile and total nitrogen of a 16% CP com-soybean meal diet (Kerr and Easter, 1995). One explanation why the 12% CP basal diet had greater ND compared to the 16% CP diet, is because both indispensable and dispensable CAA were included in 33 the 12% basal diet to match the total amino acid content of the 16%. This large amount of CAA would significantly increase the ND of the diet. In our study, ND improvement in the 6% CP compared to the 15% CP diet may be explained by three possible reasons. First, the improved ND occurred because of reduced endogenous nitrogen secretions when feeding the 6% CP diet as compared to feeding the 9, 12 and 15 % CP diets. Recovery of endogenous nitrogen influences apparent digestibility of feed ingredients and it has been documented that the proportion of endogenous secretions increases when dietary CP concentrations are decreased (Donkoh and Moughan, 1994). Second, passage rate was observed to be 18 to 24 h for the 9, 12 and 15% CP diets while the 6% CP passed after 36 to 48 h (results not shown). Reduced passage rate increases residence time in the intestinal tract and may increase disappearance of N, thereby increasing ND. Thirdly, approximately 30% of the total amino acids in the 6% CP diet were of a crystalline source compared to 11 and 1.5% in the 9 and 12% CP diets, respectively. Therefore, the proportion of free amino acids available for immediate absorption was greater in the 6% CP diet compared to the 9 and 12% CP diets. Nitrogen Utilization Nitrogen utilization increased linearly (P<0.001) as intact CP was reduced from 15 to 6%. The highest nitrogen utilization was achieved when the 6% CP diet was fed. lrnproved NPI and NPA resulted from the inclusion of CAA to balance the ideal amino acid pattern of the growing pig. Our results agree with Lenis et al. (1999) that utilization of total nitrogen increased in pigs fed 11.8 and 14.3% CP diets compared to feeding and 18.8% CF. 34 Nitrogen Excretion Total Nitrogen Output Reducing dietary CP from 15% to 6% resulted in a linear decrease (P<0.001) of TNO (Table 5). Total nitrogen output was decreased 63% when feeding the 6% CP diet in comparison to the 15% CP diet. Results comparing TNO of all experimental diets are reported in Table 6. The 6% CP and the protein-free diets had similar TNO and the lowest (P<0.05) compared to the 15% CP casein, 9, 12 and 15 % CP diets. This result ‘ indicates that TNO was mainly of endogenous origin when pigs were fed the 6% CP diet. The casein—based, 9, and 12% CP diets had similar TNO, but were all lower in comparison to the 15% CP diet. Similar findings have been reported by Hobbs et al. ( 1996), and Kephart and Sherritt (1990). In those experiments, total nitrogen excretion in growing pigs was reduced 40% and 80% when dietary CP was decreased from 16 to 14% CP (Hobbs et al., 1996) and from 17 to 11% (Kephart and Sherritt, 1990), respectively. Fecal Nitrogen Output Fecal nitrogen was similar between the protein-free and casein-based diet, 1.63 and 2.02 g/d, respectively, indicating that endogenous nitrogen losses were similar at the gut level and that casein was 100% digested and absorbed. The protein-free diet had lower (P<0.05) FN compared to the 6, 9, 12 and 15% CP diets. Feeding pigs the 6% CP and casein-based diets resulted in similar FN output, but lower (P<0.05) in comparison to the 9, 12 and 15% CP diets. Fecal nitrogen was not different between the 9, 12 and 15% CP diets. Because FN remained unchanged when CP was reduced from 15% to 9% in this study, the contribution of FN to TNO reduction was not significant. Similar findings by Kerr and Easter (1995) and Kephart and Sherritt (1990) have been reported. Fecal 35 nitrogen excretion was not different when dietary CP concentrations were reduced from 17 to 11%, (Kephart and Sherritt, 1990) and from 16 to 12% (Kerr and Easter, 1995). The undigested corn and soybean meal proteins were determined to be the primary source of FN (Kephart and Sherritt, 1990; Kerr and Easter, 1995). While FN did not contribute significantly to the decrease in TNO, decreasing UN output was the major factor in reducing total nitrogen excretion in those studies. Urinary Nitrogen Output Urinary nitrogen was dramatically reduced (P<0.05) by approximately 50% when dietary CP was decreased from 15 to 9%, but no additional significant decrease occurred with further CP reduction (Table 6). Similarly, UN was decreased 40 to 50% in growing pigs fed diets containing 12 and 13 % CP, as compared to diets containing 16 and 17% CP, respectively (Russell et al., 1983; Kephart and Sherritt, 1990; Kerr and Easter, 1995). In this study, reducing dietary CP from 15 to 9% was sufficient to minimized UN output similar to levels that occur with basal endogenous nitrogen loss. Partitioned Excreta Nitrogen . Partitioned fecal and urinary nitrogen as a percent of TNO (FN% and UN%) of the 6, 9, 12, 15% CP, casein-based and protein—free diets is presented in Figure 1. As dietary crude protein was decreased from 15 to 6%, the percent of N excreted in feces was increased (P<0.05) and percent of urinary N decreased (P<0.05)of TNO. These results indicate that nitrogen utilization was improved in pigs fed reduced CP diets with CAA supplementation. And, the decline in TNO resulted primarily from decreases in UN output. The high percentage of urinary N compared to fecal N resulting from feeding the casein diet shows that even though casein is highly digestible, the efficiency of 36 utilization of amino acids was not as great. Excess amino acids from casein were catabolized and N excreted into the urine. In conclusion, reducing intact dietary CP and supplementing CAA to meet digestible amino acid requirements can maintain similar nitrogen retention in growing pigs compared to feeding a 15% CP com-soybean meal diet, formulated to meet the digestible lysine requirement. Furthermore, reducing intact dietary CP from 15 to 6% significantly decreases total nitrogen excretion in swine manure. But there is a limit to reducing intact dietary CF to minimize total nitrogen excretion and maintain nitrogen retention. For practical diet formulation, the 12% CP diet can be recommended because it can maintain nitrogen retention and reduce nitrogen excretion in pigs. To maximize the reduction of nitrogen excretion and still maintain adequate nitrogen retention, the 9% CP diet can be recommended. However, the 9% CP diet may not be practical or economically feasible because of the commercial availability and costs of some of the crystalline amino acids. Implications With growing environmental concerns regarding water, soil, and air quality, environmental pollution by nitrogen must be lessened. This study demonstrated that feeding reduced CP diets supplemented with CAA according to an ideal digestible amino acid pattern can reduce total nitrogen output. 37 Table 1. Ingredient composition of experimental diets (as fed) Ingredient, % Corn Soybean meal, 44% CP Corn starch“ Sucrose Corn oil Solka flocb Dicalcium Phosphate Limestone Vitamin premixc Mineral premixd Salt Sowpac vitamin premixc Amino Acids L-Lysine-HCI, 78.8% L—Threonine L-Valine L-Isoleucine L-Leucine L-Phenylalanine DL-Methionine L-Tryptophan L—Histidine L—Glutamate 15% 74.56 20.00 2.44 0.70 0.70 0.60 0.50 0.50 12% 59.65 16.00 12.57 5.00 3.00 0.60 0.70 0.70 0.60 0.50 0.50 0.149 0.035 9% 44.74 12.00 29.87 5.00 3.00 1.20 0.70 0.70 0.60 0.50 0.50 0.03 0.296 0.119 0.072 0.041 0.042 0.024 0.572 6% 29.82 8.00 46.33 5.00 3.00 1.75 0.70 0.70 0.60 0.50 0.50 0.05 0.444 0.202 0.185 0.140 0.150 0.125 0.084 0.049 0.063 1.609 38 a Argo Foods, CPC International, Inc., Englewood Cliffs, NJ 07632-9976. b Harland Teklad, Madison, WI 53744-4220. ° Provided per kg of premix: vit. A 918583 IU, vit. D3 91858 IU, vit. E 11023 IU, vit. K 735 mg, riboflavin 735 mg, pantothenic acid 2939 mg, niacin 4409 mg, vitamin Bl2 5512 mcg, thiamin 184 mg, pyridoxine 165 mg. dProvided per kg premix: Cu 2000 mg, Fe 20000 mg, Zn 20000. Mg 2000 mg, I 30 mg. Se 60 mg. ° Provided per kg premix: vit. A 918583 IU, biotin 73487 mcg, choline 128602 mg, folic acid 551 mg. Lid-l. 5. mkm .. .. T TH'I I' .‘ im€ p‘.’r:. .‘ Table 2. Nutrient composition of experimental diets (as fed) Items 15% 12% 9% 6% Composition, calculated Crude protein, % 14.95 12.10 10.23 9.10 ME, kcal/kg 3365 3414 3415 3385 IAA:DAA 45:55 45:55 45:55 45:55 Amino Acids“, % Arginine 0.92 0.74 0.55 0.37 Histidine 0.41 0.32 0.24 0.22 Isoleucine 0.61 0.49 0.41 0.38 Leucine 1.42 1.14 0.85 0.72 Lysine 0.76 0.73 0.69 0.65 Methionine + cystine 0.55 0.44 0.37 0.30 Phenylalanine + tyrosine 1.04 0.83 0.63 0.54 Threonine 0.56 0.48 0.46 0.42 Tryptophan 0.17 0.13 0.12 0.12 Valine 0.70 0.56 0.49 0.46 Composition, analyzedb Dry Matter, % 90.40 91.74 92.02 92.09 Crude protein, % 14.64 12.25 11.03 9.85 Amino Acids Indispensable Arginine 0.89 0.81 0.65 0.52 Histidine 0.37 0.33 0.27 0.26 Isoleucine 0.56 0.51 0.46 0.48 Leucine 1.39 1.26 1.07 0.95 Lysine 0.58 0.59 0.63 0.66 Methionine 0.22 0.20 0.20 0.21 Phenylalanine 0.68 0.61 0.51 0.56 Threonine 0.49 0.46 0.44 0.44 Valine 0.64 0.58 0.56 0.61 DISpensable Alanine 0.87 0.78 0.66 0.53 Aspartate 1.28 1.18 0.95 0.72 Cystine 0.1 1 0.10 0.09 0.08 Glutamate 2.68 2.42 2.39 2.83 Glycine 0.57 0.51 0.41 0.33 Proline 0.97 0.87 0.73 0.58 Serine 0.70 0.63 0.51 0.41 Me 0.52 0.47 0.38 0.30 “Calculated based on NRC (1998) ingredient composition. 39 . ,r l- . ,7 “-?~ ‘ trusts- Table 3. Ingredient composition of casein based and protein-free diets (as fed) Ingredient, % Casein Protein-free Corn starch“ 64.52 81.52 Caseinb 17.00 - Sucrose 5.40 5.40 Corn oil 4.00 4.00 Solka flocb 4.00 4.00 Dicalcium phosphate 2.70 2.70 Vitamin premixc 0.60 0.60 Mineral premixd 0.50 0.50 Potassium chloridec 0.40 0.40 Limestone 0.30 0.30 Salt 0.25 0.25 SOWpac vitamin premixc 0.25 0.25 Magnesium oxide 0.08 0.08 ‘ Argo Foods, CPC International, Inc., Englewood Cliffs, NJ 07632-9976 b Harland Teklad, Madison, WI 53744-4220 ° Provided per kg of premix: vit. A 918583 IU, vit. D3 91858 IU, vit. E 11023 IU, vit. K 735 mg, riboflavin 7 35 mg, pantothenic acid 2939 mg, niacin 4409 mg, vitamin B,2 5512 mcg, thiamin 184 mg, pyridoxine 165 mg. d Provided per kg premix: Cu 2000 mg, Fe 20000 mg, Zn 20000, Mg 2000 mg, I 30 mg, Se 60 mg. ° Provided per kg premix: vit. A 918583 IU, biotin 73487 mcg, choline 128602 mg, folic acid 551 mg. 40 Items Composition, calculated Crude protein, % 15.08 0 ME, kcal/kg 3470 3496 Amino Acids, % Arginine 0.55 - Histidine 0.48 — Isoleucine 0.79 - Leucine 1.49 - Lysine 1.25 - Methionine + Cystine 0.53 - Phenylalanine + Tyrosine 1.63 - Threonine 0.68 - Tryptophan 0.19 - Valine 1.04 - Composition, analyzeda Dry Matter, % 92.85 91.78 Crude protein, % 14.67 0 Amino Acids Indispensable Arginine 0.62 - Histidine 0.44 - Isoleucine 0.82 - Leucine 0.06 - Lysine 1.03 - Methionine - - Phenylalanine 0.80 - Threonine 0.66 - Valine 1.05 - Dispensable Alanine 0.59 - Aspartate 1.20 - Cystine - - Glutamate 3 .91 - Glycine 0.31 - Proline 1.85 - Serine 0.94 - Tyrosine 0.78 - ' Mean of three batches of experimental diets. 41 _OO.OVn— .18 ~90 VA— 9.. modvm ... 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Proportion of urinary and fecal nitrogen excreted. Comparison of feeding 15, 12, 9, 6% CP com-soybean meal based, protein-free, or 15% CP casein-based diets to growing pigs, on fecal nitrogen (FN) or urinary nitrogen (UN) as a percent of total nitrogen output. Gray bars indicate UN and black bars indicate FN. Comparisons are made within UN or FN. Bars with different letters differ at P < 0.05. rig 35 5:0 l Excreta Nitrogen,% 100 - - Fecal N % 80 _ E Urine N % 15% 12% 9% 6% Prot-free 15% Cas Dietary Treatments 45 LITERATURE CITED 46 Literature Cited Argenzio, R. A., and Southworth, M. 1974. Sites of organic acid production and absorption in gastrointestinal tract of the pig. American Journal of Physiology 228:454-460. Canh, T. T., A. L. Sutton, A. J. A. Aamink, M. W. A. Verstegen, J. W. Schrama, and G. C. M. Bakker. 1998. Dietary carbohydrates alter the fecal composition and pH and the ammonia emission from slurry of growing pigs. J. Anim. Sci. 76:1887- 1895. Chung, T. K. and D. H. Baker. 1992. Apparent and True Amino Acid Digestibility of a Crystalline Amino Acid Mixture and of Casein: Comparison of Values Obtained with Deal—Cannulated Pigs and Cecectomized Cockerels. J. Anim. Sci. 70:3781- 3790. Cromwell, G. L., Turner, L. W., Gates, R. S.,Taraba, J. L., Lindemann, M. D., Traylor, S. L., Dozier III, W. A. 1999. Manipulation of swine diets to reduce gaseous emission from manure that contribute to odor. J. Anim. Sci. Supplement 1 :#187. Debruyckere, M. and B. Vansteelant. 1992. Livestock Housing and Environment - Ammonia Emission and Odour Nuisance. Zemedelska Technika 38:279-290. Gatel, F. and F. Grosjean. 1992. Effect of protein content of the diet on nitrogen excretion by pigs. Livestock Production 31: 109-120. Purdue University. (2000). Reduction of odorous sulfide and phenolic compounds in pig manure through diet modification. Hankins, 8., Sutton, A., Patterson, J ., Adeola, L., Richert, B., Heber, A., Kelly, D., Kephart, K., Mumma, R., and Bogus, E. pp. 142-151. August 31, 2000. Hartung, J. and V. R. Philips. 1994. Control of Gaseous Emissions from Livestock Buildings and Manure Stores. J. Agric. Engng Res. 57:173-189. Hobbs, P. J. and B. F. Pain. 1996. Reduction of Odorous Compounds in Fresh Pig Slurry by Dietary Control of Crude Protein. J. Sci. Food Agric. 71:508—514. Irnoto, S., and Namioka, S. 1978. VFA production in the pig large intestine. Journal of Animal Science 47:467-478. Jackson, M. J ., A. L. Beaudet, and W. E. O'Brien. 1986. Mammalian urea cycle enzymes. Ann. Rev. Genet. 20:431—464. 47 Kerr, B. J. and R. A. Easter. 1995. Effect of Feeding Reduced Protein, Amino Acid- Supplemented Diets on Nitrogen and Energy Balance in Grower Pigs. J. Anim. Sci. 73:3000-3008. Lenis, N. P., H. T. M. v. Diepen, P. Bikker, A. W. Jongbloed, and J. v. d. Meulen. 1999. Effect of the Ratio Between Essential and Nonessential Amino Acids in the Diet on Utilization of Nitrogen and Amino Acids by Growing Pigs. J. Anim. Sci. 77: 1777-1787. ' Liu, H., Allee, G. L., Touchett, K.J., Frank, J. W., and Spencer, J. D. 2000. Effect of reducing protein and adding amino acids on performance, carcass characteristics, and nitrogen excretion, and the valine requirement of early-finishing barrows. J. Anim. Sci. Supplement 2 78:#184, p. 166. Lopez, J ., R. D. Goodband, G. L. Allee, G. W. Jesse, J. L. Nelssen, M. D. Tokach, D. Spiers, and B. A. Becker. 1994. The Effects of Diets Formulated on an Ideal Protein Basis on Growth Performance, Carcass Characteristics, and Thermal Balance of Finishing Gilts Housed in a Hot, Diurnal Environment. J. Anim. Sci. 72:367-379. Miner, J. R. 1999. Alternative to Minimize the Environmental Impact of Large Swine Production Units. J. Anim. Sci. 77:440-444. Officer, D. I., E. S. Batterham, and D. J. Farrell. 1997. Comparison of growth performance and nutrient retention of weaner pigs given diets based on casein, free amino acids or conventional proteins. British Journal of Nutrition 77:731- 744. O'Neill, D. H. and V. R. Phillips. 1992. A Review of the Control of Odour Nuisance from Livestock Buildings: Part 3, Properties of the Odorous Substances which have been Identified in Livestock Wastes or in the Air around them. J. Agric. Engng Res. 53:23-50. Radecki, S. V. and M. T. Yokoyama. Intestinal Bacteria and Their Influence on Swine Nutrition. In Factors Influencing Swine Nutrition, pp. 439-447. Richert, B. 2000. Nutritional strategies for reducing manure DM, N, and P concentrations [Online] Available http://pasture.ecn.purdue.edu/~epados/swinelpubs/nutriman.htm, September 14, 2000. Sutton, A. (ed."eds.), pp. 1-7. 48 Russell, L. E., G. L. Cromwell, and T. S. Stahly. 1983. Tryptophan, Threonine, Isoleucine and Methionine Supplementation of a 12% Protein, Lysine- Supplemented, Com-Soybean Meal Diet for Growing Pigs. J. Anim. Sci. 56:1115-1123. Schiffman, S. S. 1998. Livestock Odors: Implications for Human Health and Well- Being. J. Anim. Sci. 76:1343-1355. Shriver, J. A., Carter, S.D., Pettey, LA, and Senne, B.W. 2000. Effects of adding fiber sources to low protein, amino acid-supplemented diets on nitrogen excretion and performance of finishing pigs. J. Anim. Sci. Supplement 2 78:#191, p. 168. Spoelstra, S. F. 1980. Origin of Objectionable Odorous Components in Piggery Wastes and the Possibility of Applying Indicator Components for Studying Odour Development. Agriculture and Environment 5:241-260. Sutton, A. L., K. B. Kephart, M. W. A. Verstegen, T. T. Canh, and P. J. Hobbs. 1999. Potential for Reduction of Odorous Compounds in Swine Manure Through Diet Modification. J. Anim. Sci 77:430—439. Tuitoek, K., L. G. Young, C. F. M. d. Lange, and B. J. Baker. 1997. The Effect of Reducing Excess Dietary Amino Acids on Growing-Finishing Pig Performance: An Evaluation of the Ideal Protein Concept. J. Anim. Sci. 75:1575-1583. Yasuhara, A., K. Fuwa, and M. Jimbu. 1984. Identification of Odorous Compounds in Fresh and Rotten Swine Manure. Agric. Biol. Chem. 48:3001-3010. Yokoyama, M. T., Carlson, J .R. 1979. Microbial metabolites of tryptophan in the intestinal tract with special reference to skatole. The American Journal of Clinical Nutrition 32: 173-178. Yokoyama, M. T., C. Tabori, E. R. Miller, and M. G. Hogberg. 1982. The effects of antibiotics in the weanling pig diet on growth and the excretion of volatile phenolic and aromatic bacterial metabolites. The American Journal of Clinical Nutrition 35:1417-1424. Zahn, J. A., J. L. Hatfield, Y. S. Do, A. A. DiSpirito, D. A. Laird, and Pfeiffer. 1997. Characterization of Volatile Organic Emissions and Wastes From a Swine Production Facility. J. Environ. Qual. 26:1687-1696. Zervas, S. a. Z., R. T. 2000. Effect of dietary protein and carbohydrates on internal nitrogen flow and excretion patterns in growing pigs. J. Anim. Sci. Supplement 2 78:#185, p. 166. 49 Zhang, Y., Tanaka, A., Dosman, J. A., Senthilselvan, A., Barber, E. M., Kirychuk, S. P., Holfeld, L. E., and Hurst, T.S. 1998. Acute respiratory responses of human subjects to air quality in a swine building. J. Agric. Engng Res. 70:367-373. 50 CHAPTER 3 51 ABSTRACT AMINO ACID DIGESTIBILIT Y OF REDUCED CONCENTRATIONS OF INTACT DIETARY PROTEIN FED TO GROWING PIGS By Emily Rae-Dianne Otto Reducing intact dietary crude protein (CP) concentrations of swine diets can reduce N excretion, but the digestibility of intact proteins may be adversely affected. The objective of this study was to test whether reducing dietary CP of com-soybean meal based diets (CSBM) decreases apparent (AID) and standardized (SID) ileal amino acid (AA) digestibilities when crystalline amino acids (CAA) are provided to meet digestible AA requirements. Six barrows were surgically fitted with a T-cannula at the terminal ileum and allocated to six diets in a Latin square design. Diets consisted of 15, 12, 9, and 6% CP CSBM, a 15% CP casein based, and a protein-free diet. The casein based and protein-free diets were used to determine basal endogenous AA losses (EAL). Results show that amino acid AID and SID in the 6 and 9% diets were higher (P < 0.01) for all indispensable amino acids (IAA) compared to the 12 and 15% diets. Apparent and standardized amino acid digestibility of intact CP (AIDI and SIDI, respectively), i.e., CSBM alone, was estimated by mathematically removing the CAA contribution in the 12, 9, and 6% CP diets. Calculated AIDI and SIDI were similar when feeding the 6,12, or 15% CP diets, with all three being less (P < 0.01) than the AIDI and SIDI when the 9% CP diet was fed. These results indicate that reducing intact dietary CP concentration does 52 not reduce apparent and standardized ileal amino acid digestibilities when CAA are included in the diets. Introduction Our research has shown that reducing dietary crude protein (CP) concentration can reduce total nitrogen excretion and ammonia emission from fermenting swine manure (Chapters 1 and 3). But, reductions of dietary crude protein may adversely affect protein digestibility. Amino acid apparent ileal digestibility (AID) of meat and bone meal and soybean meal (used as sole dietary protein sources) were decreased when dietary CP concentrations were below 9.5 and 8%, respectively (Donkoh and Moughan, 1994; Fan et al., 1994). At these low CP concentrations, amino acid AID is decreased because endogenous amino acid loss (EAL) is proportionately larger than dietary amino acid loss (Fan et al., 1994; Donkoh and Moughan, 1994). Thus, when amino acid AID was corrected for EAL, the amino acid digestibility was not affected by the dietary CP levels of a single protein ingredient diet (Fan et al., 1994; Donkoh and Moughan, 1994). When reducing CP concentrations of complete diets containing more than one feed ingredient, apparent and standardized ileal amino acid digestibility were decreased (Stein, 1998). The objective of this study was to test whether reducing dietary CP concentration of a com—soybean meal based diets decreases amino acid apparent and standardized ileal digestibility when crystalline amino acids (AA) are provided to meet digestible amino acid requirements. 53 Materials and Methods Animals, Diets and Experimental Design The Michigan State University All University Committee on Animal Use and Care approved the experiment. Six barrows ((Landrace x Yorkshire) x Duroc), with an initial BW of 35.5 :t 0.1 kg were surgically fitted with stainless steel T-cannula at the terminal ileum according to the procedures of Stein et al. (1998). Penicillin G procaine (3 x 105 IU / mL) and flunizin meglumine (50 mg/ 1 mL, Shering-Plough Animal Health, Kenilworth, NJ) were administer I.M. at recommended dosage (6615 IU / kg and 1 mg/ kg, respectively) three days following surgery. Pigs were allowed to recover for three weeks in individual pens (1.5 m x .75m) with smooth sided panels made of polyvinyl chloride. Each pen was equipped with a suspended water line fitted with a low-pressure nipple and wire flooring. The environmental temperature of the room was maintained at 21°C. Pigs were randomly allocated to six dietary treatments in a 6 x 6 Latin square design. The average BW at initiation of the experiment was 53.1 :t 1.8 kg. The six dietary treatments consisted of 15, 12, 9 and 6% CP com-soybean meal based diets, a 15% CP casein based and a protein-free diet (see Chapter 1, Tables 1 to 4). Feed was provided in three equivalent meals per'day (800, 1200 and 1600) at five times the energy requirement for maintenance (106 kcal ME / kg BW'75). The casein-based and protein- free diets were mixed with water (500 mL/ meal) to increase palatability and reduce feed wastage. Adjustments to feed allotment were made prior to each collection period. Amount of feed provided was based on average daily gain and weekly body weight of 54 Materials and Methods Animals, Diets and Experimental Design The Michigan State University All University Committee on Animal Use and Care approved the experiment. Six barrows ((Landrace x Yorkshire) x Duroc), with an initial BW of 35.5 :t 0.1 kg were surgically fitted with stainless steel T-cannula at the terminal ileum according to the procedures of Stein et al. (1998). Penicillin G procaine (3 x 105 IU / mL) and flunizin meglumine (50 mg / 1 mL, Shering—Plough Animal Health, Kenilworth, NJ) were administer I.M. at recommended dosage (6615 IU / kg and 1 mg/ kg, respectively) three days following surgery. Pigs were allowed to recover for three weeks in individual pens (1.5 m x .75m) with smooth sided panels made of polyvinyl chloride. Each pen was equipped with a suspended water line fitted with a low-pressure nipple and wire flooring. The environmental temperature of the room was maintained at 21°C. Pigs were randomly allocated to six dietary treatments in a 6 x 6 Latin square design. The average BW at initiation of the experiment was 53.1 i 1.8 kg. The six dietary treatments consisted of 15, 12, 9 and 6% CP com-soybean meal based diets, a 15% CP casein based and a protein-free diet (see Chapter 1, Tables 1 to 4). Feed was provided in three equivalent meals pervday (800, 1200 and 1600) at five times the energy requirement for maintenance (106 kcal ME / kg BW'75). The casein-based and protein- free diets were mixed with water (500 mL / meal) to increase palatability and reduce feed wastage. Adjustments to feed allotment were made prior to each collection period. Amount of feed provided was based on average daily gain and weekly body weight of 54 pigs studied in a previous experiment (Chapter 1). Those pigs were of similar age, size and breed to those in this experiment. Sample Collection The experiment consisted of six collection periods. Chromic oxide (0.25% of diet) was used as an indigestible marker for determination of amino acid digestibility. Digesta was collected from each pig over two consecutive days for 12 h each day following a 5-d adaptation period to the test diets. Digesta samples were collected as described by Stein (1998). Digesta was kept on ice throughout the sampling period, pooled into 4-L plastic bottles, and stored at 47C until the 2-d sampling period was completed. Pooled digesta was homogenized for each pig, stored in plastic bottles and frozen. Sample Analysis For chemical analysis, digesta samples were freeze dried (Tri-Philizer MP, FTS Systems, Stone Ridge, New York). Feed samples and freeze-dried digesta were finely ground using a cyclone mill (Cyclotec Sample Mill 1093, Sweden) through a 1-mm mesh screen. Dry matter of digesta and feed was determined following a 12-h drying period at 60 °C using a vacuum oven (Model 583/Full View, National Appliance Co., Portland, Oregon). Amino acid analysis was performed on feed and freeze-dried digesta samples by reverse-phase high-performance liquid chromatography (Waters Co., Milford,MA) following a 24-h acid hydrolysis in 6 N HCl at 110 °C and 121 mm Hg. In short, samples were brought up to volume (40 mL) and filtered (Whatman filter paper, No. 4). The amino acid filtrate was sub-sampled and dried using vacuum centrifugation (ATR C83 55 drying vacuum system, Appropriate Technical Resources, Laurel, MD). The amino acid hydrosylate was reconstituted, dried again, derivatized with phenylisothiocyanate (Pico-Tag method, Waters Co., Milford MA) and separated using a Waters high—pressure liquid chromatograph (Waters Co., Milford, MA) fitted with a lS-cm hydrosylate column. Chromium concentration in the diets and digesta was determined using a modified procedure for perchloric digestion described by Arthur ( 1970). Briefly, feed and digesta (0.3 g) were digested using 20 mL concentrated nitric acid and heated. After digestion, 10 mL of perchloric acid was added to oxidize Cr3+ to Cr“. Completion of oxidation was determined when the solution changed from a bright orange to a clear, pale yellow. Solutions were then weighed, diluted and analyzed by atomic absorption spectrophotometry (Unicam 93, TJ A Unicam Co., Cambridge, UK). Digestibility Calculations Apparent ileal amino acid digestibility (AID), standardized ileal amino acid digestibility (SID), and endogenous amino acid losses (EAL) estimated using both the protein-free and the casein based diets were calculated in the following manner: AID = (AAf - AAd) / AAf EAL = AAd ' (Cr,/ Crd) . SID = AID + (EAL/AA,) Estimation of apparent ileal amino acid digestibility of intact protein (AIDI) and standardized ileal amino acid digestibility of intact protein (SIDI), i.e. com-soybean meal portion only, were calculated in the following manner: AIDI = [(AAf- CAAf) - AAd] / (AA,- CAAf) SIDI = AIDI + (EAL / (AAf- CAA,» 56 Where AAf is the amino acid content in diet, AAd is the amino acid content in digesta, Crf is the chromium concentration in diet, Crd is the chromium concentration in digesta, and CAAf is the concentration of crystalline amino acid supplemented in the diet. Statistical Analysis The effect of dietary treatment on amino acid digestibility was analyzed by analysis of variance using PROC MIXED of SAS (2000) (SAS Inst., Inc., Cary, SC). The fixed effects of pig, period and diet were included in the model. Differences between least square means were evaluated using Bonferroni’s (Dunn) test (Younger, 1998), because non-orthogonal comparisons were made between the 6, 9, 12 and 15% CP diets. Hence, level of significance was determined to be 0.0083, where P = 0.05 and number of contrasts made were 6. Results and Discussion The primary aim of this experiment was to determine whether amino acid digestibility is decreased when dietary CP concentration of a complete diet is reduced. Apparent ileal amino acid digestibility of complete diets Apparent ileal digestibility for all indispensable amino acids was similar when the 12% CP was compared to the 15% CP diet, and when the 6% CP was compared to the 9% CP diet (Table 7). The AID for most of amino acids were greater (P < 0.01) in the 6 and 9% CP diet compared to the 12 and 15% CP diets, with AID for Arg, Gly and Pro not being different across all dietary treatments. The lack of difference of the AID of Arg is not surprising because Arg presence in all com-soybean meal diets was found to be in excess of the digestible Arg requirement. Therefore, no crystalline Arg was supplemented. The 12% diet had crystalline Lys and 57 Thr added, but not to the extent that would increase AID of amino acids greater than that of the 15% diet. The 9% diet had the highest AID for all but two dispensable amino acids (DAA), Gly and Pro, as compared to the 6, 12 and 15% diets. Currently, no information is available on AID of amino acids in reduced CP, CAA supplemented diets. In the present study, the amino acid digestibility improved in the 6 and 9% CP diets because of the large contribution of free amino acids from CAA supplementation. The 6 and 9% CP diets had approximately 30% and 11% of total amino acids supplied in crystalline form, respectively. Chung and Baker (1992) and Butts et al. (1993) demonstrated that CAA are nearly 100% absorbed before reaching the hindgut of the pig. Therefore, amino acids available for absorption from intact protein and CAA combined will be greater than amino acids from intact protein alone. Endogenous amino acid losses Endogenous amino acid losses found in digesta of pigs consuming the protein-free diet (Table 8) were in agreement with values reported by Leterme et al (1992) and Stein et al.(l999). However, the results of EAL from feeding pigs a casein-based diet (Table 10) were lower than published values of Liebholz (1982), Chung and Baker (1992), and Butts et al. (1993). Most amino acids in the endogenous losses estimated by the protein- free and casein-based diets were similar. Endogenous losses of Arg, Ala, and Tyr were all higher (P < 0.05) when the protein-free diet was fed compared to the casein-based diet. Similarly, no differences in EAL between feeding a protein—free or a casein-based diets were found in pigs (Wang and Fuller, 1989). In contrast, Butts et al. (1993) and Donkoh et al. (1995) reported higher EAL when using enzymatically hydrolyzed casein (EHC) 58 diets compared to protein-free diets to determine EAL in growing pigs and rats, respectively. Standardized ileal amino acid digestibility of complete diets Standardized digestibility of indispensable amino acids, using either the protein- free or the casein diet to estimate EAL, were increased when CP of complete diets was reduced from 15 to 6% (Tables 9 and 10). Indispensable amino acid SID was similar between the 12% and the 15% CP, and between the 6% and 9% CP diet, except for Arg and Thr. Again, the improvement in SID of indispensable amino acids for the 6 and 9% CP diets compared to the 12 and 15% CP diets was due to of the inclusion of crystalline AA. Arginine was not supplemented in crystalline form and therefore did not increase the Arg SID of the 6% CP diet greater than that of the 12 and 15% CP diets. Threonine SID improved as dietary CP concentration was reduced from 15 to 6%, and most likely resulted from crystalline AA supplementation. Standardized ileal digestibility for most dispensable amino acids, estimated using the protein-free and casein diets, were similar when the 6% CP was compared to the 9% CP diet and when the 12% was compared to the 15% CP diet. Dispensable amino acid SID was greater (P < 0.01) in the 6 and 9% CP diets compared to the 12 and 15% CP diets. Glycine and Pro SID were not different across all dietary treatments. Differences between the dispensable amino acids SID reflect the differences found between the EAL estimates determined from the protein-free or casein-based diet. 59 / vii i I \- I . / 4 . . '1' f/ S l, l I // ' I “"‘-L:.v. ., _, I" ./ if f - ,x i7 , I . l ’ J. J Apparent ileal amino acid digestibility of intact protein Apparent ileal amino acid digestibility of intact protein is dependent on dietary CP concentrations (Donkoh and Moughan, 1994; Fan et al., 1994). In growing pigs fed soybean meal based diets, with varied levels of dietary CP ranging from 4 to 24%, amino acid AID decreased at CP concentrations below 8%, but remained unchanged at CP concentrations above 12% (Fan et al., 1994). Similarly, in rats fed diets consisting of meat and bone meal, and ranging from 2.5 to 20% CP, amino acid AID decreased when dietary CP concentrations below 9.5% were fed (Donkoh and Moughan, 1994). In contrast, in this study, amino acid AIDI was not reduced when CP concentrations were reduced from 15 to 6% (Table 11). Moreover, amino acid AIDI was greater (P < 0.01) in the 9% CP compared to the 12 and 15%. The majority of dispensable amino acid AIDI were also greater (P < 0.01) in the 9% compared to the 6, 12, and 15% CP diets, and similar between the 6, 12 and 15% CP diets. Fan et al (1994) and Donkoh and Moughan (1994) reported that true amino acid digestibility was not decreased when dietary CP was decreased. Those results agree with the results of the present study. Stein (1998) found that all amino acid AID and most amino acid SID were decreased in a 10% CP complete diet compared to a 16% CP complete diet. The difference between the results of our study and Stein (1998) may be due to the presence of CAA in our reduced CP diets. Crystalline amino acids may have had a digestive and absorptive stimulatory effect on intact protein digestibility. Digestibility of some amino acids are improved when the observed true digestibility amino acid values are compared to calculated values in diets containing multiple feed ingredients (Imbeah et al., 1988). This additivity effect may also occur with 60 CAA. Currently, there is no information available on the additivity effect of CAA and feed ingredients on amino acid digestibility. Standardized ileal amino acid digestibility of intact protein All amino acids standardized ileal digestibility within intact protein (SIDI) were similar in the 12% compared to the 15% CP diet. Standardized ileal digestibility of all amino acids was similar in the 6% compared to the 12 and 15% CP diet. The SIDI of most amino acids in the 9% CP were greater (P < 0.01) compared to the 6, 12 and 15% CP diets (Table 12). Fan et al. (1994) and Donkoh and Moughan (1994) found that dietary CP concentrations had no effect on true ileal amino acid digestibility of single ingredient diets. In contrast, most amino acid SIDI in the present study were improved when feeding the 9% CP compared to the 6, 12 and 15% CP diets. The amino acid AIDI and SIDI improved in the 9% CP diet for two possible reasons. First, the 9% CP diet may have contained the best balance of amino acids and intact proteins that allowed for optimum efficiency of amino acid uptake. The balance of amino acids and proteins may have allowed minimum competition for amino acid transport across enterocytes. Amino acid transport systems can interact with multiple amino acids (Torras Llort, 1996; Soriano Garcia et al., 1998,1999). For example, it has been demonstrated that L-Methionine and L—Lysine share transport systems with cationic and neutral amino acids in the intestine of chickens. Inhibition or competition for uptake in the presence of other amino acids occurs amongst L-methionine and L-lysine (Torras Llort, 1996; Soriano Garcia et al., 1998,1999). Second, CAA, when present in optimum balance, may be involved in stimulating gut secretions that increase digestibility of intact 61 protein. In conclusion, reducing intact dietary CP from 15 to 6% and supplementing CAA to meet digestible amino acid requirements improves AID and SID. Both AIDI and SIDI, com-soybean meal protein, are not adversely affected by reducing dietary CP from 15 to 6%. Implications This study indicates that reducing dietary crude protein concentrations from 15 to 6% and supplementing CAA will not decrease amino acid digestibility of intact protein within a complete diet. The benefit of not adversely affecting amino acid digestibility of reduced CP diets provides further support to the reduction of dietary CP in swine diets in order to decrease nitrogen excretion from growing pigs. 62 / V //4’- [A ' / I g.- A ,r .t f ' I. I a / a" f ' I "|‘-LI.. - . ,‘~ I. Table 7. Apparent ileal amino acid digestibility of experimental diets with crystalline amino acids, % (dry matter basis)a 15% 12% 9% 6% SEM" Amino Acid Indispensable Arginine 88.54” 87.56’ 91.20" 86.77’ 0.73 Histidine 85.88’ 85 .39’ 90.12" 89.38" 0.57 Isoleucine 83.10’ 83.1 1’ 90.18" 90.09" 0.71 Leucine 82.48’ 82.22’ 89.49" 88.82" 0.73 Lysine 81.26’ 84.98’ 92.22" 91.63" 0.86 Methionine 81.39’ 82. 10’ 89.76" 90.05" 0.82 Phenylalanine 84.25’ 83.92’ 9044* 90.40" 0.68 Threonine 75.322 79.49’ 86.01" 86.19" 0.88 Valine 79.94’ 80.31’ 88.81" 89.14" 0.86 Dispensable Alanine 75.78’ 77 .92’ 86.22" 80.70’ 1.17 Aspartate 78.72’ 79.53’ 88.56" 83.09’ 1.09 Cystine 78.51’ 79.14’ 85.89" 83.76" 0.71 ' Glutamate 84.00’ 83.50’ 91.81" 92.84" 0.63 Glycine 66.19 62.20 72.01 62.25 3.05 Proline 71.44 61.14 70.24 33.37 10.29 Serine 80.26’ 80.26’ 86.31" 81 .60’ 0.82 Tyrosine 80.81’ 80.09’ 87.13" 81 .47’ 0.95 aData are reported as least squares means bStandard error of least squares means. ""‘zLeast square means within row with different superscripts are different at P < 0.01. 63 / VA \' / ./‘ . , I // 4’ ‘ '.. I A .' I r l ‘1 a If , ‘—._.,““>."‘ t. Table 8. Endogenous losses estimated using two methods“, mg/kg DMI Amino Acid Casein Protein Free SEM Indispensable Arginine 325.8" 163.5’ 18.9 Histidine 93 .8 71.5 17.4 Isoleucine 230.7 214.8 9-2 Leucine 393.2 283.5 230.0 Lysine 234.1 161.4 21.0 ' Methionine 76.3 067.3 13.0 Phenylalanine 206.5 132.9 13.5 Threonine 377.2 364.5 21.9 Valine 315.8 265.2 17.7 Dispensable Alanine 464.5" 315.7’ 14.1 Aspartate 351.7 246.9 52.1 Cystine 25.6 0.00 9.6 Glutamate 660.5 781.4 1 14.5 Glycine 799.7 362.5 97.4 Proline 2,082.4 463.4 455-7 Serine 346.4 387.5 19.8 Tyrosine 229.2" 144.5’ 7. 5 " Analysis of ileal digesta from feeding of either a protein-free or casein-based diet b Standard error of the mean "" Least square means within rows with different superscripts are different (P<0.05). 64 Table 9. Standardized amino acid ileal digestibility with crystalline amino acids using rotein-fre diet to estimate endo enous ami 0 acid losses % d matter basis “ 15% 12% 9% 6% SEM" Amino Acid Indispensable Arginine 91 .70’ 91.44’ 95.93" 92.92"’ 0.73 Histidine 88.14’ 88.23’ 93.52" 93.02" 0.57 Isoleucine 86.73’ 87.59’ 94.98" 94.69" 0.71 Leucine 84.98’ 85.35’ 93.09" 92.79" 0.73 Lysine 84.77’ 88.52’ 95.86" 94.97" 0.86 Methionine 84.06’ 85.36’ 92.93" 93.07" 0.82 Phenylalanine 86.91’ 87.24’ 94.07" 94.18" 0.68 Threonine 81.852 86.89’ 93.78" 93.98" 0.88 Valine 84.32’ 85.68’ 94.34" 94.32" 0.86 Dispensable Alanine 80.45’ 83.21’ 92.36" 88.86" 1.15 Aspartate 81 .27’ 82.47’ 92.04" 88.24" 1.09 Cystine 80.80’ 81.78’ 89.13" 87.79" 0.71 Glutamate 86.21’ 86.22’ 94.36" 95.07" 0.63 Glycine 78.88 78.02 90.97 87.24 3.05 Proline 90.82 87.84 100.55 73.75 10.34 Serine 84.59’ 85.70’ 92.79" 90.36" 0.82 Tyrosine 84.67’ 84.97’ 93.05" 89.73" 0.95 " Data are least squares means. bStandard error of mean """ Least square means within row with different superscripts are different P < 0.01. 65 Table 10. Standardized ileal amino acid digestibility of experimental diets with crystalline amino acids using casein to estimate endogenous losses, % (d matter asis)" 15% 12% 9% 6% SEMb Amino Acid Indispensable Arginine 90.15’ 89.51’ 93.58" 89.82’ 0.74 Histidine 87.63’ 87.56’ 92.72" 92.12" 0.59 Isoleucine 86.51’ 87.28’ 94.65" 94.34" 0.74 Leucine 84.32’ 84.47’ 92.09" 91.65" 0.76 Lysine 83.72’ 87.42’ 94.73" 93.91" 0.89 Methionine 83.78’ 84.97’ 92.55" 92.68" 0.85 Phenylalanine 85.99’ 86.05’ 92.67" 92.80" 0.71 Threonine 81 .682 86.65’ 93.52" 93.67" 0.92 Valine 83.66’ 84.82’ 93.46" 93.45" 0.90 Dispensable Alanine 81.40’ 81.52’ 90.40" 86.18"’ 1.03 Aspartate 80.55z 81.60’Z 91.01" 86.64"’ 1.1 1 Cystine 78.55’ 79. 15’ 85.89" 83.70" 0.74 Glutamate 86.64’ 86.72’ 94.83" 95.46" 0.65 Glycine 72.02 69.42 80.61 73.45" 3.06 Proline 72.70 67.10 77.03 42.20" 10.36 Serine 85.15’ 86.34’ 93.57" 91.34" 0.84 Tyrosine 83.28’ 83. 17’ 90.86" 86.62"’ 0.98 " Data are least squares means. bStandard error of least squares means. ""'" Least 'square means within rowwith different superscripts are different P < 0.01. 66 Table 11. Apparent ileal amino acid digestibility of experimental diets without crystalline amigo agidsI % (dgy matte; basis)" 15% 12% 9% 6% SEMb Amino Acid Indispensable Arginine 88.54" 87. 16" 90.03" 82.68’ 0.93 Histidine 85.88’ 85.28’ 88.85" 83.21’ 0.64 Isoleucine 83. 10’ 82.66’ 87.41" 80.1 1’ 0.80 Leucine 82.48’ 81 .97’ 87.63" 82. 12’ 0.81 Lysine 81.26’ 81.23’ 87.27" 77.63’ 0.93 Methionine 81.39"’ 81.40"’ 85.36" 77.64’ 1.05 Phenylalanine 84.25’ 83.63’ 88.66" 82.74’ 0.75 Threonine 75.32’ 77.03"’ 80.05" 70.52" 1.00 Valine 79.94’ 79.65’ 84.97" 76.61’ 0.97 Dispensable Alanine 75.78’ 74.76’ 81.88" 71 .29’ 1.24 Aspartate 78.72’ 77.45’ 85.76" 78.65’ 1.23 Cystine 78.51’ 77.01’ 83.08" 76.47’ 0.84 Glutamate 84.00’ 82.99’ 87.96" 81 .94’ 0.76 Glycine 66.19 61.55 68.24 51.22 3.71 Proline 71.44 64.94 68.37 20.23 12.52 Serine 80.26’ 80.06’ 84.49" 76.86’ 0.88 Tyrosine 80.81’ 79.98’ 85.73" 77.91y . 0.99 "Data are least squares means. bStandard error of least squares means. ""'" Least squares means within row with different superscripts are different P < 0.01. 67 Table 12. Standardized amino acid ileal digestibility without crystalline amino acids usin rotei -fr ediet to estimate endo enous amin ac’d losses % d matt r i S a 15% 12% 9% 6% SEMb Amino Acid Indispensable Arginine 91 .70"y 91 . 17"’ 95.39" 90.73’ 0.93 Histidine 88. 14’ 88. 14’ 92.68" 88.96’ 0.64 Isoleucine 86.73’ 87.26’ 93.57" 89.34’ 0.80 Leucine 84.98’ 85. 14’ 91.86" 88.48"’ 0.81 Lysine 84.77’ 85.58’ 93.22" 86.57’ 0.93 Methionine 84.06’ 84.79’ 89.89" 84.44’ 1.05 Phenylalanine 86.91’ 87.01’ 93.18" 89.53y 0.75 Threonine 81.85" 85.31’" 91.13" 87.16“ 1.00 Valine 84.32’ 85 .21’ 92.40" 87.76’ 0.97 Dispensable Alanine 80.15’ 80.80’ 89.96" 83.42’ 1.24 Aspartate 81.27’ 80.69’ 90.09" 85.15"’ 1.23 Cystine 80.80’ 79.91’ 86.97" 82.31’ 0.84 Glutamate 86.21’ 85.80’ 91.71" 87.57’ 0.76 Glycine 78.88 77.64 89.76 83.52 3.71 Proline 90.84 89.02 100.58 68.58 12.52 Serine 84.59’ 85.56’ 91.84" 87.88"’ 0.88 Tyrosine 84.67’ 84.88’ 92.29" 87.76"’ 0.99 "Data are least squares means. bStandard error of least squares means. "' "" Least square means within row with different superscripts are different P < 0.01. 68 ~"- I I . -. - —- -~. ‘--— .- ..._ . 1 ,‘L‘.‘??'t LITERATURE CITED 69 Literature Cited Arthur, D. 1970. The Determination of chromium in animal feed and excreta by atomic absorption spectrophotometry. Canadian Spectroscopy 15: 134-140. Chung, T. K. and D. H. Baker. 1992. Apparent and True Amino Acid Digestibility of a Crystalline Amino Acid Mixture and of Casein: Comparison of Values Obtained with Ileal-Cannulated Pigs and Cecectomized Cockerels. J. Anim. Sci. 70:3781- 3790. Donkoh, A. and P. J. Moughan. 1994. The effect of dietary crude protein content on apparent and true ileal nitrogen and amino acid digestibilities. British Journal of Nutrition 72:59-68. Fan, M. Z. and W. C. Sauer. 1997. Determination of true ileal amino acid digestibility in feedstuffs for pigs with the linear relationships between distal ileal outputs and dietary inputs of amino acids. J. Sci. Food Agric. 73:189-199. Fan, M. Z., W. C. Sauer, R. T. Hardin, and K. A. Lien. 1994. Determination of Apparent Ileal Amino Acid Digestibility in Pigs: Effect of Dietary Amino Acid Level. J. Anim. Sci. 72:2851-2859. Fan, M. Z., W. C. Sauer, and M. L. McBumey. 1995. Estimation by Regression Analysis of Endogenous Amino Acid Levels in Digesta Collected from the Distal lleum of Pigs. J. anim. Sci. 73:2319—2328. Furuya, S. and Y. Kaji. 1989. Estimation of the true ileal digestibility of amino acids and nitrogen from their apparent values for growing pigs. Animal Feed Science and Technology 26:271-285. Imbeah, M., W. C. Sauer, and R. Mosenthin. 1988. The Prediction of the Digestible Amino Acid Supply in Barley-Soybean Meal or Canola Meal Diets and Pancreatic Enzyme Secretion in Pigs. J. Anim. Sci. 66:1409-1417. Leterme, P., L. Pirard, and A. T hewis. 1992. A note on the effect of wood cellulose level in protein-free diets on the recovery and amino acid composition of endogenous protein collected from the ileum in pigs. Anim. Prod. 54:163-165. Nyachoti, C. M., C. F. M. d. Lange, and H. Schulze. 1997. Estimating Endogenous Amino Acids Flows at the Terminal lleum and True Heal Amino Acid Digestibility in Feedstuffs for Growing Pigs Using the Homoarginine Method. J. Anim. Sci. 75:3206-3213. Soriano Garcia, J. F., M. Torras Llort, R. Ferret, and M. Moreto. 1998. Multiple pathways for L-methionine transport in brush-border membrane vesicles from chicken jejunum. J Physiol Lond 509:527-539. 70 Soriano Garcia, J. F., M. Torras Llort, M. Moreto, and R. Ferrer. 1999. "Regulation of L- methionine and L-lysine uptake in chicken jejunal brush-border membrane by dietary methionine. Am J Physiol 277:R1654-1661. Stein, H. H. 1998. Comparative amino acid digestibilities in growing pigs and sows. Ph. D, University of Illinois. Stein, H. H., N. L. Trottier, C. Bellaver, and R. A. Easter. 1999. The Effect of Feeding Level and Physiological Status on Total Flow and Amino Acid Composition of Endogenous Protein at the Distal lleum in Swine. J. Anim. Sci. 77:1180—1187. Torras Llort, M., J. F. Soriano Garcia, R. Ferret, and M. Moreto. 1998. : Effect of a lysine-enriched diet on L-lysine transport by the brush-border membrane of the chicken jejunum. : Am J Physiol 274:R69-75. 71 I“ .. \ 5‘ ' v. a; t v I- ' s-rhk' .\J.F'& taro L\.:\;.¢-kt~g1"u £221.45“- . 7 §‘(vot-sp~nt.;u-‘ "D“ -'|-..» r _ _ - CHAPTER 4 72 ABSTRACT EFFECT OF DIETARY CRUDE PROTEIN REDUCTION ON AMMONIA, VOLATILE FATTY ACIDS, PHENOLICS AND SWINE MANURE ODOR OFFENSIVENESS By Emily Rae-Dianne Otto The objective of this study was to investigate the effect of crude protein (CP) reductions with amino acid (AA) supplementation on volatile fatty acids (VFA), phenolics, ammonia emission (NH3), and manure odor. Six barrows were used to test six diets consisting of 15, 12, 9, and 6% CP com-soybean meal based, a 15% CP casein and a protein-free diet in a 6 x 6 Latin square. Total feces and urine were collected and pooled within pig and period. Feces and urine were analyzed for VFA and phenolic concentrations, respectively. Feces and urine were mixed, stored and fermented at room temperature for 31d. Headspace air was sampled for NH3 at 24 h, 48 h and 72 h from slurries. Slurry samples were placed into vials, capped and double randomized prior to odor panel evaluation. Odor offensiveness was classified on severity: (1) non-offensive, (2) mild, (3) moderate, (4) strong, and (5) extreme. Results of fecal VFA concentrations showed that reducing dietary CP increased (P<0.01) VFA concentrations. No differences were found in phenolic concentrations in urine. Slurry NH3 emission was reduced (P<0.05) as dietary CP levels decreased from 15 to 0%. Odor results showed that the odds of the15% CP CSBM and 12% CP being in lower offensiveness category as 6% CP were 2.41 and 1.79, respectively. Comparing treatment 6% CP to protein-free showed that 6% was .51 times more likely to be in a lower odor category than protein-free. These 73 results indicate that NH3 emission can be reduced by reducing dietary CP, but odor offensiveness and VFA concentrations are increased with reduced dietary CP. Introduction Nitrogen (N), ammonium (NH,*) and volatile organic compounds (VOC) are major components of pig manure that have negative effects on the environment (Zahn et al., 1997). Environmental pollution results from N contamination of surface and ground water, and emission of noxious odors, ammonia (NH3), and VOC into the air (Miner, 1999). People exposed to noxious odors and NH3 experienced eye and respiratory irritations, headache and drowsiness (Schiffman et al., 1995; Schiffman, 1998). Elevated aerial NH3 concentrations in confinement swine facilities delayed the onset of puberty in gilts (Malayer et al., 1987). Odorous compounds in feces and urine originate from fermentation and metabolism of undigested and(or) unabsorbed proteins and amino acids not utilized by the animal (Hobbs et al., 1996). Ammonium originates from oxidative deamination of amino acids through removal of Ot-amino groups. Toxic NH; is converted to urea in the liver and excreted in the urine (Jackson et al., 1986). Both NH3 and VOC generated from manure slurry can be decreased by reducing dietary crude protein (CP) concentrations (Sutton et al., 1999; Hobbs et al., 1996). Our previous reseach showed that intact dietary protein concentrations reduced from 15 to 9% did not compromised nitrogen retention in growing pigs. Thus, this study addresses whether reductions of intact dietary protein concentration would contribute significantly to the reduction in NH3 and VOC. 74 Thus, we hypothesized that the minimum NH3 emission and VOC concentrations found in pig manure, solely contributed by endogenous proteins, and can also be attained by reductions of intact dietary protein concentrations. The objective was to determine the minimum dietary CP needed to attain NH3 emission, odor offensiveness score and VOC concentrations equivalent to that contributed by endogenous proteins alone. Materials & Methods Sample Collection Experimental design, animals, diets and excreta collection procedures are as described in Chapter 1. Experimental design, animals, diets and excreta collection procedures are as described in Chapter 1. At the conclusion of each collection period, stock slurries were prepared in the following manner. A sub-sample (200 g) of fresh homogenized feces were added to 1000 mL of fresh homogenized urine for each respective pig. The stock slurries were stored in 3.8-L plastic containers at 21 °C and fermented for a period of 30 d. Ammonia Emission Testing After the fermentation period, slurries were shaken vigorously and 25 mL transferred into lOO-mL glass beakers in duplicate. Beakers were covered tightly with aluminum foil and allowed to ferment for an additional 24 h. Using Gastec ammonia detector tubes (Gastec Corp., Gastec Detector Tube No.3M, Japan) the foil seal was punctured and head space air was sampled approximately 2.5 cm above the slurry surface at a rate of 100 mL/ min. Ammonia was recorded as ppm. Following the air sampling, each slurry beaker was gently agitated manually with a wooden stick applicator to disrupt 75 any crusting, covered with aluminum foil, and the procedure was repeated at 48 and 72 h. Odor Panel Approval for use of human subjects was granted by the University Committee on Research Involving Human Subjects. For the odor panel, six sub-samples of slurry per diet (lO-mL each) were prepared from the stock slurries. Each sub-sample was poured into a plastic vial containing a cotton ball and capped. Every sample set contained all diets per collection period. The six sets of vials were allowed to rest for 1 h and double randomized prior to panelist evaluation. The laboratory where the odor panel was conducted had mechanically controlled temperature (20 - 22 °C) and ventilation. The laboratory air had no detectable odors that would interfere with the panel evaluation. Four evaluation stations were located on lab benches throughout the laboratory. A total of 34 volunteers (22 males and 12 females) participated throughout the duration of the experiment. Volunteers were not trained. Volunteer selection criteria was that the panelist be familiar with livestock manure odors. The number of participants per panel average 13, with the number ranging from 7 to 17 participants. Thirteen of the volunteers participated three times or more. The panel participants were asked to sniff each sample individually and pause for a minimum of three minutes before sniffing the next sample. Individuals were asked to classify the severity of odor offensiveness. Offensiveness was classified on a 1 to 5 scale 0f Severity: (1) none, (2) mild, (3) moderate, (4) strong, or (5) extreme offensiveness. Responses were recorded following the evaluation of each sample. 76 Volatile Organic Compounds Volatile Fatty Acids Volatile fatty acids (VFA) were chemically analyzed from feces of each pig, period and diet. Previously frozen fecal samples were thawed and a 2-g sample was taken. Each sample was diluted with 8 mL of distilled water and 2 drops of concentrated HCl in a centrifuge tube, mixed using a vortex and centrifuged at 4° C and 17,400 g for 10 min (Centrifuge model and Source). The supernatant was filtered using .22 1.1m filter (Millipore Co., Bedford, MA) and pipetted into sterile 2.0 mL gas chromatography vials (SUPELCO, Cat. No. 27265, Supelco Park, Bellefonte, PA). The VFA concentrations were determined using a gas chromatograph (Varian Model 3700 FID, Varian, Inc., Walnut Grove, CA 94598) equipped with a 1.85 m x 32 mm column (15% SP1220 ?l% phosphoric acid on 80-100 Chromosorb, SUPELCO, Supelco Park, Bellefonte, PA 16823). Nitrogen was used as a carrier gas with a flow rate of 25 mL / min. Air and hydrogen gas were used for combustion with a flow rate at 30 mL / min. Samples were manually injected (2 11L) at a temperature of 110 °C. Following each sample injection, the temperature was held at 110 °C for 5 min and increased to 127 °C at a rate of 3 °C/ min. The final temperature was held at 127 °C for 3 min to insure complete VFA volatilization. A standard solution containing 10 mmol / mL of each of the following VFA: acetate, propionate, isobutyrate, butyrate, isovalerate and valerate ,was injected (2 ML) and a standard curve determined. Phenolic Compounds Urine samples were thawed and centrifuged at 27,200 g in a refrigerated centrifuge for 25 min. Supematants were filtered (Whatman filter paper, No. 4) and 1 mL 77 transferred in duplicate into 2-mL gas chromatograph crimp top vials (SUPELCO, 2-mL, Cat. No. 27058, Supelco Park, Bellefonte, PA 16823-0048) containing 0.5 mL of a 3-ppm sodium chloride solution. The samples were analyzed using a gas chromatograph-mass spectrophotometer (Varian Gas Chromatograph CP-3800, Varian Mass Spectrophotometer Saturn 2000, Varian, Inc.,Walnut Creek, California 94598). Two different polar solvent absorption fibers (polyacylate and polydimethylsioxane) of similar polarity were used. The polyacylate fiber broke away from the sampling needle apparatus at the conclusion of sampling urines from the protein-free, 6 and 9% CP dietary treatments. The replacement fiber was a polydimethylsiloxane solvent absorption fiber and used to sample urines from the casein, 12 and 15% CP dietary treatments. Statistical Analysis Analysis of variance within the dependant variables NH3, total VFA, acetate (ACE), propionate (PROP), isobutyrate (ISOB), butyrate (BUTY), isovalerate (ISOV), and valerate (VAL), p-cresol and p-ethylphenol were performed using the PROC MIXED procedure of SAS (1999) (SAS Inst., Inc., Cary, S. C.) in a 6 x 6 Latin square. The model included the fixed effects of pig, period, and diet. Least square mean (LSM) differences for NH, VFA, total VFA, individual VFA as a percent of total VFA, and phenolics were separated using Tukey's multiple comparison test (Younger, 1998) and the difference established at P < 0.05. Isobutyrate was not detected in feces of pigs fed the 15% CP diet and a value of zero was used to determine LSM estimates for total VFA and individual VFA as percent of total VFA. 78 Dietary CP concentration (0, 6, 9, 12 and 15%) was regressed against NH3 emission to determine linear and(or) quadratic relationships. The fixed effects of pig, period, and diet were included in the model and performed using PROC MIXED. The odor panel results were analyzed using the GENMOD modeling procedures of SAS (1999) to estimate differences within a qualitative response variable. Estimates with significant differences (P < 0.05) between dietary treatments were reported as log odds ratios. The statistical model included the fixed effects of panelist, gender of panelist, order in which sample was sniffed, pig, period, and diet to test the dependent variable of odor offensiveness. The significant difference between dietary treatments was determined at P < 0.05. The log odds ratio comparisons of LSM were transformed to an index to elucidate the differences in odor. Odor offensiveness indices resulting from feeding the protein- free, casein-based, 6, 9, 12, and 15% CP diets were non-orthogonally compared. Manure odor offensiveness from feeding the 15% CP diet served as the control and had an index value of 100. Multiplying each respective log odds ratio derived the indices for the casein-based, protein-free, 6, 9, and 12% CP diets by the 15% CP index value. Results Ammonia Emissions Ammonia emission declined quadratically (P<0.001) as dietary CP was reduced from 15 to 0% (Figure 3). Ammonia emission produced from feeding the protein-free, 6 and 9% CP diets were similar, and all lower (P < 0.05) when compared to the casein: based, 12, and 15% CP diets. The NH3 emission from feeding the 12% CP diet was 79 "j?! lower (P<0.05) compared to the casein-based and 15% CP diets. The 15% CP and casein—based diets produced similar NH3 emission. Volatile Organic Compounds Results of volatile fatty acid concentrations in feces are reported in Table 13. Total VFA increased (P < 0.05) when feeding 12, 9 and 6% CP diets as compared to feeding the 15% CP, casein-based and protein-free diets. Acetate concentration was not different between diets. Propionate concentration was higher (P < 0.05) when feeding the 9 and 6% CP diets than the 15% CP diet. Similar propionate concentration was found between the12 and 15% CP diets. Propionate concentration was decreased (P < 0.05) to nearly non-detectable levels from feeding the casein-based and protein-free diets compared to the concentration levels in manure resulting from the 15, 12, 9 and 6% CP diets. Isobutyrate concentration in feces was higher when the 6, 9 and 12% CP diets were fed as compared to feeding the casein-based and protein-free diets. Isobutyrate was not detected in feces when the 15% CP was fed. Butyrate, ISOV, and VAL concentrations were all higher in feces when the 6, 9 and 12% CP CSBM diets were fed compared to feeding the protein-free, casein-based, and 15% CP diets. p-Cresol and p-ethylphenol were the only phenolics detected in urine samples and differences between diets were not found (Table 13). The results of the percentage of individual VFA to total VFA concentration in feces are presented in Table 14. The proportion of ACE of total VFA fecal concentration Was lower (P < 0.05) from feeding the 6, 9, 12 and 15% CP diets as compared to the CaSein-based and protein-free diets. Proportion of propionate to total VFA decreased in feces when the casein-based and protein-free diets were fed as compared to feeding the 6, 80 9. 12 and 15% CP diets. Isobutyrate as a percentage of total VFA was lower in feces when pigs were fed the casein-based and protein-free diets compared to 6, 9, 12 and 15% CP diets. There was no effect of diet on proportion of BUTY to total VFA concentration in feces. The 12% CP diet resulted in higher (P<0.05) percentage of VAL to total VFA fecal concentration compared to 9 and 6% CP diets. The 12, 9 and 6% CP diets resulted in higher VAL fecal concentration as a percent of total VFA as compared to the 15% CP, casein—based, and protein-free diets. Odor Panel The results of the odor panel showed an increase in odor offensiveness as intact CP decreased (Figure 4). The 15% CP diet was the least offensive manure slurry with LSM equal to 2.58. This LSM value is equivalent to the qualitative ranking of “mild- moderately” offensive. Comparing reduced intact CP diets to the 15% CP diet, only the 9 and 6% CP diets were found to be more offensive (P < 0.05) with qualitative rankings of “moderately” offensive. The order of offensiveness (least to most) for the remaining treatments were as follows: protein—free, 12% CP, and the casein-based diet, 2.70, 2.77, and 2.81, respectively, with qualitative rankings equivalent to “mild-moderately” offensive. Discussion The primary aim of this study was to determine the extent of intact dietary protein reduction on NH3 emission and VOC concentrations in pig manure and fresh feces and urine, respectively. Furthermore, reducing N H3 and VOC would also improve odor Offensiveness of fermenting swine manure. We hypothesized that decreasing dietary CP 81 from an intact protein source and including crystalline amino acids would reduce NH, emission and VOC concentrations tosimilar levels contributed by endogenous protein. Decreased concentrations of urinary nitrogen results from the reduced CP concentrations in the experimental diets (Chapter 1) can significantly lessen the total N contamination to the environment and therefore reduce NH, emission. It is known that NH, is a major pollutant (Debruyckere and Vansteelant, 1992) and decreases air quality (Zhang, et al., 1998). Ammonia is produced by the bacterial and enzymatic degradation of urea and other nitrogenous components found in urine (Jackson, et al., 1986; Hartung and Phillips, 1994). Attempts have been made to separate manure solids from the liquid. because bacterial urease present in feces hydrolyzes the urea in the urine into NH]. Both ionized (NH,") and unionized (NH,) forms of ammonia are present in the slurry, but the unionized form is also volatile, hence found in the air (Spoelstra, 1980). Ammonia emission decreased nearly 80% by reducing CP from 15 to 9%. Crude protein reductions beyond 9% did not yield any further reduction in NH, emission. Ammonia emission reductions were estimated between 28 to 79% when dietary CP was reduced by 4 to 7 percentage units in growing and finishing pig diets (Sutton et al., 1999). Reductions of 29 and 33% in NH, emission were found when dietary CP was reduced from 16.5 to 10.5% (Turner et al., 1999) and from 14 to 10% CP (Richert and Sutton, 2000), r65pectively. Canh et al. (1998) estimated that for each percentage point decrease of dietary CP, 3 10 to 12% reduction in NH, emission occurred. In this study, NH, emission decreased approximately 16% for each unit of CP reduction from 15 to 9% CP. 82 The second goal was to determine whether reducing CP would reduce VFA and phenolic concentrations to levels similar to those produced when only endogenous protein contributes to the formation of these products. It was observed that total VFA concentrations in feces dramatically increased when pigs were fed the 6, 9 and 12% CP diets compared to being fed the 15% CP, casein- based and protein-free diets. The increasing pattern of total VFA concentrations reported in the present study were consistent with findings in the literature (Irnoto and N amioka, 1978; Hankins et al., 2000). A recent study found that total VFA concentration was higher in fresh feces when a 10% CP diet with crystalline lysine, threonine and tryptophan, and 5% cellulose was fed as compared to feeding a 13% CP diet (Hankins et al., 2000). However, in that same study no differences in total VFA concentration in fresh feces were found when feeding pigs the 10% CP diet with crystalline amino acids supplemented but without cellulose as compared to feeding the 13% CP diet (Hankins et al., 2000) . In this study, ACE concentrations in feces were similar across all dietary treatments. And, PROP, ISOB, BUTY, ISOV and VAL were all found in higher concentrations in feces when the 6, 9, 12% CP diets were fed compared to feeding pigs the 15% CP, casein-based and protein-free diets. Similarly, only ACE, PROP and BUTY concentrations were reported to be greater in fresh feces when pigs were fed a 10% CP diet with crystalline lysine, threonine and tryptophan supplementation with 5% cellulose (Hankins, et al., 2000). However, the actual concentrations of VFA determined in Hankins et al. (2000) study were lower than the VFA concentrations found in our study. Imoto and Namioka (1978) measured VFA concentrations in fresh fecal matter of 23-kg 83 ' A growing pigs fed either a low or a high carbohydrate diet containing 20.9 or 14.7% CP, respectively. Similar to the results of this study, ACE, PROP, and BUTY concentrations were all higher in feces from pigs fed the high carbohydrate, 14.7% CF diet (Irnoto and Namioka, 1978). Concentrations of PROP and BUTY reported by Irnoto and Namioka (1978) were equivalent to concentrations measured in the feces from feeding pigs the 15% CP diet in this study. Production of certain volatile fatty acids is the result of anaerobic microbial fermentation of soluble carbohydrates (Argenzio and Southworth, 1974). However, it was found that additional corn starch and glucose added to their diets were nearly 100% digested in the small intestine of pigs and therefore were not appreciably available for hindgut fermentation (Irnoto and Namioka, 1978; Lin et al., 1987; Shi and Noblet, 1994). Irnoto and Namioka (1978) found that digesta entering the large intestine was similar in composition between the low and high carbohydrate dietary treatment groups. Therefore, they concluded that changes in the microflora number or microbial activity were most likely responsible for the increases in VFA concentrations in feces from feeding the high carbohydrate diet (Irnoto and Namioka, 1978). A similar explanation for increased VFA concentrations in the reduced CP diets can be made. Even though significant amounts of corn starch were added to the diets in this study to dilute the intact CP concentration, it was assumed that the corn starch was almost completely digested and absorbed before 1‘6 aChing the hindgut. Approximately 94% of total starch in a com-based diet was di gested by pigs at the point of the ileum (Keys and DeBarthe, 1974). Furthermore apparent ileal digestibility of starch in corn grain is 90 to 99.2% when fed to growing pigs [Li n et al., 1987; Andersen et al., 2000). 1A 84 Iii .I ll The distribution of the VOC in relation to total VOC mixture, and more importantly the proportion of individual VFA to total VFA concentration in relation to odor offensiveness has been deemed extremely significant (O’Neill and Phillips, 1992). In the present study, the proportion of ACE and PROP were reduced in feces from pigs fed the 12, 9 and 6% CP as compared to pigs fed 15% CP, casein-based, and protein-free diets. The proportions of ISOB, BUTY, ISOV, and VAL increased in the feces from feeding the 12, 9 and 6% CP diets. Previous research found the proportion of VFA to be about 50:40:10 for acetate, propionate and butyrate, respectively, for pigs fed either a low or high carbohydrate diet (Irnoto and Namioka, 1978). The ratio of 50:40:10 for acetate, propionate, and butyrate, respectively is fairly consistent when pigs are fed any cereal grain based diet, but can fluctuate with inclusion of dietary fibers (Radecki and Yokoyama, 1991). In this study, pigs fed the 15% CP diet had similar proportions of VFA in fecal matter as reported by Irnoto and Namioka, for pigs fed a normal diet (1978). In contrast, all six VFA were more evenly distributed in feces from pigs fed the 6, 9 and 12% CP diets. Acetate and PROP decreased as percentage of total and ISOB, BUTY, IS 0V, VAL increased as a percentage of the total VFA. This change in proportion of VFA can be explained by possible changes in the microflora populations and(or) their activity and an increase in degradation of proteins and(or) amino acids that escaped absorption in the small intestine. The other volatile components found primarily in urine, p-cresol and p- 6 thy 1 phenol, were detected in very small concentrations, however, no differences in C: O nCentrations were found across diets. Concentrations of these phenolic compounds W ere significantly lower than values reported in the literature (Yokoyama et al., 1932; 85 1A Yasuhara, et al., 1984 ). Analysis of fresh and aged urine from grow-finish pigs was conducted and concentrations of phenolics increased as the urine aged (Yasuhara et al., 1984). The increase in phenolic concentrations was explained by trace amounts of fecal matter contaminating urine at the time of collection and phenolics being released by bacterial enzymatic act on glucuronides (Yasuhara et al., 1984). Current research comparing urinary phenolic compound concentrations by feeding growing pigs reduced dietary CP diets has not been reported. Only one study to date has analyzed manure slurries from pigs fed reduced CP diets. In that study, only skatole and 4-ethylphenol concentrations were found to be reduced as dietary CP was reduced (Hobbs et al., 1996). One possible reason for the low concentration and non-detection of phenolic compounds in this study is that the phenolics were not produced to any extent due to lack of substrate. Some phenolic compounds are by—products of amino acid catabolism, especially the amino acids tryptophan and tyrosine (Yokoyama and Carlson, 1979; Yokoyama et al., 1982). The diets in this study were formulated to match the digestible amino acid requirement of pigs. Therefore, tryptophan and tyrosine were not fed in excess, and thus reduced the potential for phenolic production from amino acid catabolism to occur. Long chain VFA and phenolic compounds have been determined to contribute to manure odor quality (Spoelstra, 1980). Acetic and propionic acid concentrations have been deemed unimportant when investigating odor quality (Spoelstra, 1978; Spoelstra, 1 9 80). Therefore any decrease in the proportion of long-chain and branched-chained VFA relative to short chain VFA has potential to reduce the odors emanating from pig m anUre. 86 After analysis of the odor panel responses, it was found that manures resulting from feeding the 6 and 9% CP diets were actually more offensive compared to the 15% CP. Relating the VFA results to the odor panel, it was determined that the increased concentrations of ISOB, BUTY, ISOV, and VAL in the reduced CP diets may have contributed to the increased odor offensiveness. The role of phenolics contributing to the odor offensiveness were less significant and inconclusive because only p-cresol and p-ethylphenol were identified in urine. Because the concentrations were found equally across all dietary treatments, they may have contributed equally to the odor quality. Additionally, the slurries were made with fixed proportion of urine volume and fecal matter from each pig and dietary treatment, possibly resulting in similar amounts of phenolic compounds in each slurry. In conclusion, reducing intact dietary CP and supplementing CAA to meet digestible amino acid requirements significantly decreases ammonia emission from swine manure. However, reducing dietary CP from intact protein sources, i.e., corn and soybean meal, did not improve odor quality of manure or decrease fecal VFA concentration. This suggests as well that endogenous proteins may contribute to a great extent to odor and VFA production. In fact, feeding the reduced CP diets compared to the 15% CP com- SOybean meal diet increased odor offensiveness and fecal VFA. In the case of ammonia, endOgenous proteins contributed significantly, as the minimum NH, emission was attained by feeding a 9% CP diet. Implications With growing environmental concerns regarding water and air quality, and fi 1 1 vironmental pollution to soil, water and air must be lessened. This study demonstrated 87 'btllll that feeding reduced CP diets minimized ammonia emission, but not VOC concentrations in fresh feces or urine. Furthermore, reduction to 9% was sufficient to minimize NH, emission. Manure odor quality originating from feeding pigs reduced CP diets did not improve. Additional research to determine microflora populations and their activity of VFA production in the hindgut needs to be conducted. 88 r * * H . ‘ . ' ‘ . ' ' ‘ ‘ " 4' '7'nf-Ou.-§"~4~ r“; .‘--M{“.8éto" a Table 13. Volatile organic compounds in feces and urine from feeding experimental diets. 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The regression analysis was performed without inclusion of the casein-based diet. A quadratic effect was found at P < 0.01. Least square means with different letters differ at P < 0.05. -l Ammonia Emissions, ppm per 100mL min 500 1 450 '4 400 . 350 . 300 . 250 . 200 . 150 .. 100 . 15% 112% 9% 6% PF 15% Cas Dietary treatments 93 5‘” a— ‘ w ‘ Ava-fl, Figure 3. Index of odor offensiveness of fermented manures. Log odds index indicates likelihood of increased odor offensiveness. The index values were determined by standardizing the mean response of manure odor offensiveness from feeding the 15% CP com-soybean meal based diet to growing pigs. Higher index value represents increased odds that odors are more offensive. Indices with different letters differ P < 0.05. 94 Log Odds Index 300 250 200 150 100 50 ab 15% 12% 9% 6% Dietary Treatments 95 PF ab "L 15% Cas LITERATURE CITED 96 Eli! i i Literature Cited Canh, T. T ., A. L. Sutton, A. J. A. Aarnink, M. W. A. Verstegen, J. W. Schrama, and G. C. M. Bakker. 1998. Dietary carbohydrates alter the fecal composition and pH and the ammonia emission from slurry of growing pigs. J. Anim. Sci. 76:1887-1895. Hankins, 8., Sutton, A., Patterson, J ., Adeola, L., Richert, B., Heber, A., Kelly, D., Kephart, K., Mumma, R., and Bogus, E. 2000. Reduction of odorous sulfide and phenolic compounds in pig manure through diet modification. Research report pp. 142-151. Purdue University, West Lafayette, Indiana. Hartung, J., and V. R. Philips. 1994. Control of Gaseous Emissions from Livestock Buildings and Manure Stores. J. Agric. Engng Res. 57:173-189. Hobbs, P. J ., and B. F. Pain. 1996. Reduction of Odorous Compounds in Fresh Pig Slurry by Dietary Control of Crude Protein. J. Sci. Food Agric. 71:508-514. Irnoto, S., and Namioka, S. 1978. VFA production in the pig large intestine. Journal of Animal Science. 47:467-478. ' Jackson, M. J., A. L. Beaudet, and W. E. O'Brien. 1986. Mammalian urea cycle enzymes. Ann. Rev. Genet. 20:431-464. Keys, J. E. a. D., J. V. 1974. Site and extent of carbohydrate, dry matter, energy and protein digestion and the rate of passage of grain diets in swine. J. Anim. Sci. 39:57-62. Malayer, J. R., Kelly, D. T., Diekman, M. A., Brandt, K. E., Sutton, A. L., Long, G. G., and Jones, D. D. 1987. Influence of Manure Gases on Puberty in Gilts. J. Anim. Sci. 64:1476-1483. O'Neill, D. H., and V. R. Phillips. 1992. A Review of the Control of Odour Nuisance from Livestock Buildings: Part 3, Properties of the Odorous Substances which have been Identified in Livestock Wastes or in the Air around them. J. Agric. Engng Res. 53123-50. Radecki, S. V., and M. T. Yokoyama. Intestinal Bacteria and Their Influence on Swine Nutrition. (ed.) Factors Influencing Swine Nutrition. p 439-447. Richert, B. 2000. Nutritional strategies for reducing manure DM, N, and P concentrations [Online] Available http://pasture.ecn.purdue.edu/~epados/swine/pubs/nutriman.htm, September 14, 2000. A. Sutton. 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T., C. Tabori, E. R. Miller, and M. G. Hogberg. 1982. The effects of antibiotics in the weanling pig diet on growth and the excretion of volatile phenolic and aromatic bacterial metabolites. The American Journal of Clinical Nutrition. 35: 1417-1424. Younger, M. S. 1998. SAS Companion for RV. Rao's Statistical Research Methods in the Life Sciences. Zahn, J. A., J. L. Hatfield, Y. S. Do, A. A. DiSpirito, D. A. Laird, and Pfeiffer. 1997. Characterization of Volatile Organic Emissions and Wastes From a Swine Production Facility. J. Environ. Qual. 26:1687-1696. Zhang, Y., Tanaka, A., Dosman, J. A., Senthilselvan, A., Barber, E. M., Kirychuk, S. P., Holfeld, L. E., and Hurst, T.S. 1998. Acute respiratory responses of human subjects to air quality in a swine building. J. Agric. Engng Res. 70:367-373. 98 SUMMARY AND CONCLUSIONS 99 Summary and Conclusion Growing concerns about environmental pollution are stemming partially from expanding intensified swine production. Research designed to alleviate environmental pollution is critical. Some factors causing environmental pollution include elemental nitrogen pollution of soil and water, as well as air pollution caused by ammonia and noxious odors emanating from manure storage areas and pig housing facilities. Several dietary and managerial techniques are currently being investigated. In this thesis, the effect of dietary crude protein concentration reduction on nitrogen excretion, ammonia and odor emissions was addressed. In addition, two challenges associated with reducing dietary crude protein on the nitrogen economy of the pig were evaluated. One, can growing pigs maintain a nitrogen balance equivalent to pigs fed a non-reduced crude protein diet? There were three major findings from this experiment. First, nitrogen balance was not adversely affected when dietary crude protein was reduced from 15 to 9%, however, further reduction beyond 9% resulted in lower nitrogen retention in the growing pig. Second, nitrogen utilization was dramatically improved as Crude protein concentrations were decreased and crystalline amino acids were supplemented. And third, total nitrogen excretion significantly declined as crude protein concentrations were reduced. In relation to total nitrogen excretion reduction, manure ammonia emission was also decreased. Ammonia emission was drastically reduced by 80% when dietary crude protein was reduced from 15 to 9%. Even though the 6% CP diet achieved equivalent reductions of ammonia emission to those produced by the 9% CP diet, the nitrogen retention in pigs fed the 6% diet was decreased in comparison to the 15% CP diet. These 100 findings indicate that reducing crude protein beyond 9% is not practical to maintain growth performance as measured by nitrogen balance. The second question, does the amino acid digestibility of a reduced crude protein diet with crystalline amino acid supplementation decrease? The results showed that on both an apparent and a standardized basis, amino acid digestibility of the diets increased as crude protein decreased. In fact, further investigation of the results warranted the question of whether the presence of crystalline amino acids in a reduced crude protein diet improved the amino acid digestibility of the intact protein. Amino acid digestibility of the intact protein was therefore mathematically derived. Results showed that amino acid digestibility was not decreased, and furthermore, improved in the 9% CP diet on both apparent and standardized basis. These results suggest that the presence of amino acids in an appropriate balance along with peptides from the intact protein source may provide an additive effect on amino acid digestibility. The final question answered in this thesis was whether dietary crude protein reductions decreased the manure concentrations of volatile organic compounds associated with malodor emission. The results from volatile organic compound analysis indicated that as dietary crude protein concentrations were reduced, malodorous volatile fatty acid concentrations were increased. Concurrently, odor offensiveness of manure was increased as dietary crude protein concentration was reduced. These findings indicate that the endogenous protein fraction may be a greater contributor to odor production in the hindgut of the pig than dietary crude protein. 101 Implications Findings in this thesis could benefit the swine industry and the environment in two ways. First, by reducing manure nitrogen content, the amount of land required to apply manure for disposal, and the risks of nitrogen contamination of water are reduced. And second, by reducing ammonia emission, lower incidences of respiratory illness in both humans and animals may result. The dramatic reductions of total nitrogen excretion and ammonia emission as seen when the 9% CP diet was fed to pigs may not be fully realized until more crystalline amino acids become commercially available. Until then, recommendations of reducing dietary crude protein of corn-soybean meal diets to 12% with L-lysine and L-threonine supplementation is feasible to attain nearly 35% reduction in nitrogen excretion and nearly 65 % reduction of ammonia emission compared to a 15% CP com-soybean meal diet. 102 . an» '1 I A? “1" .4 _‘ .'« .I'I--v .h.‘-