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POST-RUMINAL STARCH INFUSION IN DAIRY CATTLE:  IMPLICATIONS FOR INFLAMMATORY RESPONSE AND ANIMAL HEALTH

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POST-RUMINAL STARCH INFUSION IN DAIRY CATTLE: IMPLICATIONS FOR INFLAMMATORY RESPONSE AND ANIMAL HEALTH
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Acidosis ( jstor )
Blood ( jstor )
Cattle ( jstor )
Digestion ( jstor )
Endotoxins ( jstor )
Histamines ( jstor )
P values ( jstor )
pH ( jstor )
Rumen ( jstor )
Starches ( jstor )

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POST-RUMINAL STARCH INFUSION IN DAIRY CATTLE: IMPLICATIONS FOR INFLAMMATORY RESPONSE AND ANIMAL HEALTH By HEIDI ANN BISSELL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2002

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Copyright 2002 by Heidi Ann Bissell

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This thesis is dedicated to my mother, Patricia Bissell, for always encouraging and believing in me and for her sense of humor. It is for my grandfather, Harry B. Bissell, Jr., whose support and bear hugs helped me immensely along the way, and to my aunt and uncle, Gael Bissell and Richard Mace, who showed me the beauty of nature and encouraged my interest in science.

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ACKNOWLEDGMENTS Many people deserve special recognition for their help throughout my studies. First, I would like to thank Dr. Mary Beth Hall for serving as my major advisor and mentor, for helping me through all stages of my Master’s work, and for introducing me to the wonderful world of cows. Her guidance and instruction in numerous areas, ranging from public speaking to statistics and beyond, have been invaluable to me. I thank Dr. John Arthington for serving as cochair of my committee, for advising on the complex subject of immunology, and for allowing me to spend a lovely month in the serene beauty of Ona, Florida. I thank Dr. Charles Wilcox, for his expert advice on statistical design and analyses. I would also like to thank Dr. Douglas Levey for sparking my interest in wildlife, birds, and research. I wish to thank all the faculty, graduate students, and staff who helped day and night during my experiment. I would especially like to thank those students working in our laboratory who have been friends as well as coworkers: Adenike Akinyode, Alexandra Amorocho, Coleen Casey, and Lucia Holtshausen. Jocelyn Jennings deserves special recognition for her guidance and assistance with running analyses in the laboratory and discussions on topics near and far. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES...........................................................................................................ix ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION..........................................................................................................1 2 LITERATURE REVIEW...............................................................................................5 Starch Digestion............................................................................................................5 Structure of Starch...................................................................................................5 Ruminal Fermentation of Starch..............................................................................7 Post-Ruminal Digestion of Starch............................................................................8 Ruminal Acidosis........................................................................................................12 Buffering................................................................................................................13 Motility...................................................................................................................13 Acid Production.....................................................................................................14 Absorption of VFA................................................................................................14 Microbial Changes.................................................................................................15 Changes in Starch Fermentation............................................................................16 Gastrointestinal Permeability......................................................................................16 Luminal Factors Affecting Permeability................................................................16 Extra-luminal Factors Affecting Permeability.......................................................19 Physiology of Gut-Induced Laminitis.........................................................................23 Hematological Causes of Laminitis.......................................................................24 Role of Absorbed Endotoxin and Histamine in the Onset of Laminitis................26 Inflammatory Response and the Onset of Laminitis...................................................29 Summary.....................................................................................................................30 v

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3 POST-RUMINAL STARCH INFUSION IN DAIRY CATTLE: IMPLICATIONS FOR INFLAMMATORY RESPONSE AND ANIMAL HEALTH...........................32 Materials and Methods................................................................................................32 Cows, Diet, and Facilities......................................................................................32 Treatments..............................................................................................................33 Sampling................................................................................................................34 Laboratory Analyses..............................................................................................34 Statistical Analyses................................................................................................36 Results and Discussion...............................................................................................39 Indices of Animal Health.......................................................................................39 Fecal pH and fecal [H + ]...................................................................................39 Fecal consistency and indications of gut damage............................................40 Respiration rate................................................................................................42 Rectal temperature...........................................................................................42 Hematocrit........................................................................................................43 Acute Phase Proteins..............................................................................................44 Ceruloplasmin..................................................................................................44 Haptoglobin......................................................................................................44 Fibrinogen........................................................................................................45 Alpha acid glycoprotein...................................................................................46 Other Measurements..............................................................................................46 Liver panel enzymes........................................................................................46 Complete blood count......................................................................................47 Ruminal pH......................................................................................................47 Conclusions............................................................................................................47 APPENDIX A FIGURES......................................................................................................................74 B NECROPSY REPORT..................................................................................................85 C RAW DATA.................................................................................................................90 SOURCES CITED.............................................................................................................95 BIOGRAPHICAL SKETCH...........................................................................................105 vi

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LIST OF TABLES Table page 1-1. Incidence of laminitis in dairy herds...........................................................................4 2-1. Reported critical limits of acute and chronic forms of acidosis................................31 3-1. Composition of TMR and intake...............................................................................51 3-2. Error terms used for certain terms the model............................................................51 3-3. Fecal characteristics by hour from start of infusion for four treated cows. ..............52 3-4. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II, containing all data, 2 treatment descriptors, and including cow 2245.....................................................................53 3-5. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II containing all data, 2 treatment descriptors, and excluding cow 2245.....................................................................54 3-6. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II, containing all data, 3 treatment descriptors, and including cow 2245.....................................................55 3-7. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II, containing all data, 3 treatment descriptors, and excluding cow 2245.....................................................57 3-8. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 2 treatment descriptors, and including cow 2245.....................................................59 3-9. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 2 treatment descriptors, and excluding cow 2245.....................................................60 3-10. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 3 treatment descriptors, and including cow 2245.....................................................61 vii

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3-11. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 3 treatment descriptors, and excluding cow 2245.....................................................63 3-12. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 2 treatment descriptors, and including cow 2245.....................................................65 3-13. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 2 treatment descriptors, and excluding cow 2245.....................................................66 3-14. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 3 treatment descriptors, and including cow 2245.....................................................66 3-15. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 3 treatment descriptors, and excluding cow 2245.....................................................69 3-16. Alkaline phosphatase arithmetic means by treatment and day................................71 3-17. Alanine aminotransferase arithmetic means by treatment and day.........................71 3-18. Aspartate aminotransferase arithmetic means by treatment and day......................72 3-19. Biliruben arithmetic means by treatment and day...................................................72 3-20. Albumin arithmetic means by treatment and day....................................................73 3-21. Gamma-glutamyltransferase arithmetic means by treatment and day.....................73 3-22. Red and white blood cell counts at hour 66 from three cows..................................73 C-1. Raw Data....................................................................................................................90 viii

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LIST OF FIGURES Figure page A-1. Diagram of catheter..................................................................................................74 A-2. Fecal pH....................................................................................................................75 A-3. Hydrogen ion concentration.....................................................................................76 A-4. Normal fecal samples collected from control cows 5781 (left) and 2521 (right) at hour 81...................................................................................................................77 A-5. Foamy fecal sample collected from cow 2811 at hour 81........................................78 A-6. Diarrhea collected from cow 2245 at hour 81..........................................................79 A-7. Pasty fecal sample collected from cow 2686 at hour 81..........................................80 A-8. Haptoglobin raw data showing elevated (detectable) levels in all cows, especially cow 2245................................................................................................................81 A-9. Fibrinogen raw data showing elevated levels in cow 2245......................................82 A-10. AGP raw data showing elevated levels in cow 2245..............................................83 A-11. Comparison of fecal consistency at hour 81 among control cows (dark brown), ST-1 cows (medium brown), and ST-2 cows (pale yellow)..................................84 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science POST-RUMINAL STARCH INFUSION IN DAIRY CATTLE: IMPLICATIONS FOR INFLAMMATORY RESPONSE AND ANIMAL HEALTH By Heidi Ann Bissell August 2002 Chair: Dr. Mary Beth Hall Cochair: Dr. John Arthington Department: Animal Sciences Post-ruminal starch fermentation may be related to health disorders associated with ruminal acidosis in cattle. The effects of post-ruminal starch digestion on animal health and inflammatory response were measured in six ruminally cannulated, multiparous, nonpregnant, nonlactating Holstein cows. Starch-treated (ST) animals were abomasally infused with 4 kg of starch suspended in 8 L of 0.9% saline solution. Control animals were infused with 8 L of the saline solution. Infusates were administered on days 2 through 4. Every 4 h (offset daily by one hour) for six days, blood samples, fecal pH (analyzed as fecal hydrogen ion concentration ([H + ]), rectal temperature, and respiration rate were collected for each cow. Blood samples were analyzed for the acute phase proteins haptoglobin (Hp), ceruloplasmin (Cp), fibrinogen, and -acid glycoprotein (AGP), as well as the liver proteins alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, biliruben, albumin, and -glutamyltransferase. x

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Starch infusion caused a decrease in fecal pH (increase in fecal [H + ]), declining as low as 4.32 in ST animals. Indications of gut damage included the presence of mucous, blood, and tissue in the feces of ST animals. Indices of animal health, including respiration rate and rectal temperature were influenced by treatment or fecal [H + ]. Of the acute phase proteins, only fibrinogen was affected by treatment. However, with the exception of one value from the cow that died, fibrinogen levels did not exceed the normal range for cattle. There was little evidence for gut damage as measured by enzymes in the liver panel, although gut damage was evident in the fecal material. There were differences among the ST cows in their response to treatment, with the fecal pH of two cows returning to normal levels even while infusions continued to be administered. Another cow recovered more slowly and not until after treatments were discontinued. One ST cow was euthanized because of severe enteritis and toxicosis. Post-ruminal starch infusions lowered fecal pH and altered fecal characteristics, likely due to increased post-ruminal bacterial fermentation and damage to the gut. The limited number of cows and the large amount of variation among them made determination of a systemic acute phase response difficult. The results of this study begin to explain how some symptoms of ruminal acidosis (i.e., diarrhea, foamy feces, and mucin casts). These results also show distinct variation in how animals respond to this dietary challenge. This study offers the basis to further explore the effects of diet on animal health. xi

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CHAPTER 1 INTRODUCTION Carbohydrates comprise an integral part of ruminant diets, yielding energy and microbial mass essential for maintenance, growth, and production. The amount and type of carbohydrate are critical in determining whether the cow thrives on the diet, or succumbs to carbohydrate-associated disorders such as ruminal acidosis or laminitis. Hopefully, by understanding the digestion of carbohydrate throughout the digestive tract and the potential effects of carbohydrate malabsorption, producers can develop and feed diets that provide the cow with the substrate needed for efficient milk production, yet maintain the cow’s health and lower the incidence of diet-related disorders. Ruminal acidosis, a condition of lowered ruminal pH, is a widespread disorder affecting numerous beef and dairy cattle. It is found more commonly in animals that are fed or that select diets high in concentrate or grain. The economic implications of ruminal acidosis include the cost of treating the acidotic cow, the loss associated with decreased milk production, and the increased incidence of other disorders, including reduced reproductive efficiency and laminitis. Laminitis, an inflammation of the membranes surrounding the foot, is a common and economically significant problem in cattle, especially dairy cows. Although the incidence of laminitis within dairy herds varies widely depending on many factors, estimates range from 0-55%, with a mean of 17% of dairy cattle in the United States and Great Britain having laminitic symptoms at any given time (Nocek, 1997; Table 1-1). This presents a major economic issue as well as an animal welfare issue. Laminitis is 1

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2 painful, reduces the animal’s mobility, and causes declines in feed intake, milk production, and reproduction (Bergsten, 2001; Cattell, 2001; Oldruitenborgh-Oosterbaan, 1999). Cows suffering from laminitis are culled more often than cows with healthy hooves (44% vs. 25%; Cattell, 2001). These costs, in addition to treatment and extra labor costs, may total $128-$627 per case (Shearer et al., 1996). Acidosis and laminitis are correlated with one another and with the inclusion of increased levels of rapidly-fermentable carbohydrates (RFC) in the diet (Brent, 1976; Nocek, 1997; Nocek, 2001). Although acidosis itself may not cause laminitis, the conditions that favor the development of acidosis, such as high-grain diets and a lack of effective neutral detergent fiber (NDF), may trigger other metabolic processes that may lead to laminitis. Other species, such as humans, horses, and rodents, also share gut-joint problems. People suffering from bacterial infections of their small intestine, genetic predisposition to gut inflammation, or bowel stasis due to surgery or trauma often experience reactive arthritis in their joints (Li, 1999; Stenson, 1999; Toivanen and Toivanen, 2000). In horses, the feeding or infusion of a large dose of grain can clinically induce laminitis. There is a mouse model of Crohn’s disease (HLA-B27 knockout mice) in which a high RFC diet induces arthritis (Mielants and Veys, 1990; Stenson, 1999). Interestingly, the species for which gut-induced arthritis or laminitis has been well documented (horses, rodents, humans) are not ruminants. Yet the same dietary factors – namely levels of RFCs that apparently exceed the animal’s capacity to digest it, cause the resulting hoof or joint pain.

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3 Several theories exist for how disturbances in the gut can influence seemingly unrelated tissues such as those found in the joints. Although joints are not equivalent to hoof tissue, the underlying cause and mechanisms may be similar. In general, disturbances in the gut are thought to lead to an increased intestinal permeability or a disruption of the immune barrier in the gut. The contents of the gut are technically located outside the body. The animal must be protected from the luminal contents, which are a potential source of toxins and antigens. This is accomplished by the presence of the mucosa and its secretions, and the large amount of lymph and immune cells associated with the gut (Cohen and Giannella, 1991; Cordain et al., 2000; Li, 1999; Moore et al., 1981). If these barriers are compromised, bacteria, bacterial antigens, or toxic compounds from the lumen may be able to cross the intestinal wall and enter the bloodstream, causing various hematological changes linked to laminitis. The central element to all hypotheses is a derangement of normal gut functioning, and this in turn is influenced to a large degree by diet. The interaction between the gut and the hoof, or the gut and the joint, depends on two related events. The first is an increase in gastrointestinal permeability, likely due to acidification of the gut as a result of bacterial fermentation of undigested carbohydrate. The second is the onset of the inflammatory response in response to substances that are absorbed due to this increased permeability. The inflammatory response may lead to the changes in the hemodynamics of the hoof that are presumed to cause laminitis.

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4 Table 1-1. Incidence of laminitis in dairy herds Source Herd location % of cows affected (Russell et al., 1982) United Kingdom 5.5 (Eddy and Scott, 1980) United Kingdom 7.33 (Cattell, 2001) USA 13.9 (Whitaker et al., 1983) United Kingdom 25.0 (Prentice and Neal, 1972) United Kingdom 30.0 (Shearer et al., 1996) USA 35 (Smilie et al., 1996) USA > 62

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CHAPTER 2 LITERATURE REVIEW Starch Digestion The digestive tract of cattle is adapted for the digestion of roughages. Modern production systems feed highly digestible carbohydrate sources such as concentrates to allow greater energy consumption and use by their cattle. However, there are limits to the ability of cows to digest and absorb these compounds. High-producing animals are often fed high concentrate diets in an effort to meet their expanded energy requirement. These animals are particularly vulnerable to acidosis and laminitis, especially if certain aspects of the diet are not managed properly. Under normal conditions and a balanced diet, the rumen fermentative processes are capable of digesting nearly all of the RFCs that enter the digestive tract. Readily-fermentable carbohydrates include sugars, oligosaccharides, starches, glycogens and pectins. In general, digestion of RFCs is faster, is more complete, and yields more net energy than fiber digestion (Armstrong, 1965; Huntington, 1988). Because of this, RFCs may play a key role in the onset of carbohydrate-related disorders. In dairy cattle diets commonly fed in the United States, the most common RFC is starch, mostly found in grains. It is the main nutrient implicated in the onset of acidosis. Structure of Starch Starch is a storage polysaccharide synthesized by plants. Although it is found in highest concentrations in the seed, as a source of nutrients for the developing plant embryo, it is also found in other parts of the plant including the leaves, roots, and stems 5

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6 (Rooney and Pflugfelder, 1986). Starch is composed of a mixture of two types of glucose polymers: amylose, in which the glucose molecules are linked -(1,4) in long linear chains, with a small amount of -(1,6) branching, and amylopectin, which is also composed of -(1,4) glucosidic linkages, but contains a greater number of -(1,6) branches (Huntington, 1997; Rooney and Pflugfelder, 1986; Van Soest, 1994). The physical structure of the grain as well as that of the starch molecule has a great deal to do with digestibility of starch in the gastrointestinal tract. The outer layers of grains, such the cuticle, pericarp, and peripheral endosperm of corn, are often impermeable to water and bacterial degradation (Kotarski et al., 1992; McAllister et al., 1994; Rooney and Pflugfelder, 1986). In order to be digested, care must be taken that either processing or mastication cracks the outer layers. The starch granules are crystalline and insoluble in cold water. Rooney and Pflugfelder (1986) noted that the digestibility of starch decreases as the amount of amylose increases. The proportion of amylose to amylopectin in the granules is variable and depends on the genetics and environment of the plant. Waxy varieties of grains contain little or no amylose, and are considered highly digestible. Another factor that increases digestibility of the starch is processing of grain via steam-flaking or other methods where heat, mechanical action and/or water are applied. With the application of heat and moisture, the crystalline structure of the granule is altered and the starch becomes gelatinized, increasing its digestibility (Huntington, 1997; Rooney and Pflugfelder, 1986; Van Soest, 1994). The proportion of starch digested ruminally versus post-ruminally has been of considerable interest to animal scientists, as the end products and implications for animal

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7 performance differ. Ruminal digestion of starch is important for bacterial growth, rumen health and the production of volatile fatty acids. Intestinal starch digestion provides free glucose, which can be used directly by the mammary tissue for milk production (Huntington, 1997; Knowlton et al., 1998), and is estimated to yield 42% more energy than ruminally digested starch (Owens et al., 1986). Feeding large amounts of grain leads to increased amounts of starch escaping ruminal fermentation, and, theoretically, greater milk production (Armstrong and Smithard, 1979; Huntington, 1997; Hurtaud et al., 1998; Knowlton et al., 1998). However high levels of grain, especially finely ground or highly processed, can also lead to excessive ruminal fermentation of carbohydrate and ruminal acidosis (Allison et al., 1975; Crichlow and Chaplin, 1985; Dirksen, 1970; Dougherty et al., 1975b; Dunlop, 1972; Hungate et al., 1952; Meyer et al., 1965; Owens, 1998; Slyter, 1976). If the amount of grain is sufficiently large, it may lead to further digestive upset in the lower portions of the digestive tract. Producers must take care to feed not only the proper amounts of grain, but in a form that is neither so rapidly digestible that there is an increased risk of acidosis, nor so indigestible that little benefit is gained from the feed. Ruminal Fermentation of Starch Ruminal fermentation of starch is accomplished by diverse species of bacteria, protozoa and fungi working in concert. The most numerous and significant for starch digestion are the bacteria, although protozoa also digest large amounts of starch, and fungi aid in bacterial attachment (Huntington, 1997; McAllister et al., 1994; Mendoza et al., 1993; Van Soest, 1994). The cow and her diet largely influence the total and relative numbers of microbes and microbial species. Microbial populations adjust over time to the diet being fed and conditions within the rumen. Those most adapted to amylolytic

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8 digestion predominate in numbers in cows on high starch diets. Conversely, cows on diets with high forage contents have increased numbers of cellulolytic bacteria (Russell and Wallace, 1997; Warner, 1965). Ruminal starch digestion begins with bacterial attachment to the surface of the starch molecule. The hyphae of the fungi are often instrumental in breaking through the outer layers of unprocessed grains to allow bacterial access to the inner starch granules (McAllister et al., 1994; Mendoza et al., 1993; Van Soest, 1994). Once attached, diverse species of bacteria begin to secrete amylases and other digestive enzymes. Different species contain different enzymes, and it is thought that a monoculture of rumen bacteria would not be as efficient at carbohydrate digestion as the mixture naturally found in the rumen because of the symbiotic nature of the bacteria (Church, 1988; Cotta, 1992; Van Soest, 1994). As hydrolysis of the starch molecule occurs, the glucose molecules released are absorbed by the bacterial cells and fermented. As the amount of starch in the diet increases, the amount of starch digested in the rumen increases as well, however the proportion of starch digested in the rumen, compared with total tract digestion, decreases (Huntington, 1997; Russell et al., 1981b). Most starch digestion in the rumen occurs within five hours of ingestion (Owens, 1988). Passage of starch from the rumen depends on overall motility patterns, the overall rate of passage, and particle size. If particle size is sufficiently small, undigested starch may pass from the rumen into the omasum and abomasum before complete digestion has occurred. Post-Ruminal Digestion of Starch Due to the active ruminal fermentation, only a fraction of the starch ingested by the animal reaches the small intestine. Owens et al. (1986) and Huntington (1997) concluded

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9 from their reviews of the literature that the approximate theoretical maximum ability of the cow to digest starch in the rumen was between 50 and 94%. Thus, between 6 and 50% of dietary starch passes to the small intestine. In addition to this unfermented dietary starch, the small intestine also receives microbial glycogen, a storage compound similar to starch, from the ruminal microbes. Active starch digestion by the cow herself begins in the proximal portion of the small intestine, where pancreatic -amylase secreted into the lumen breaks down the large starch molecules at the -(1,4) linkages. The major products of luminal starch digestion are -limit dextrins, maltotrioses, and maltose. No free glucose is formed at this stage. These smaller polysaccharides are then digested by enzymes found in the brush border, such as maltase and isomaltase. The released glucose is then absorbed by the epithelial cells lining the small intestine for use by the body (Church, 1988; Owens et al., 1986; Van Soest, 1994). While cows have a large ability to break down starch in the small intestine, this ability is not unlimited. Unlike many monogastric animals, in which the digestion of starch is considered nearly complete, in cattle only 45 to 85% of the starch that enters from the rumen is digested in the small intestine (Huntington, 1997; Owens et al., 1986). As the amount of starch in the diet increases, the amount of starch digested in the small intestine increases as well. However, the percentage of the total starch digested decreases (Kreikemeier, 1995; Owens et al., 1986). Because of the loss in feed efficiency involved with incompletely digested feed, as well as the potential health problems associated with undigested starch in the lower gut, several research efforts have been focused on determining what factors limit starch digestion in the small intestine.

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10 One of the more common factors hypothesized to limit starch digestion is limited activity of pancreatic -amylase. Amylase activity is decreased when the pH in the lumen differs from the optimum of 6.7 to 7.2 (Johnson et al., 1982; Russell et al., 1981c). The pH in the small intestine of cattle on high-grain diets is between 5.6 and 6.8 (Wheeler and Noller, 1977), thus a decrease in starch digestion might be expected. However, infusing amylase with and without bicarbonate buffer into the duodenum of three steers consuming high-grain diets did not increase starch digestion significantly in their small intestine (Remillard and Johnson, 1984). Owens et al.(1986), in his review of limits to starch digestion in the ruminant small intestine, stated that there was no evidence that incomplete starch digestion was due to insufficient amylase activity. A factor that has not been extensively studied is that brush border enzymes such as maltase and isomaltase may also limit starch digestion by failing to break down the diand tri-saccharides released by amylase. Russell et al. (1981c) found that maltase activity is influenced by pH, but not by diet. Because they found little free sugars in the feces of their animals, they concluded that inadequate intestinal maltase was not the limiting factor. However, they neglected large intestinal bacterial fermentation, which may have removed much of the free sugars. Another factor that may limit starch digestion is the ability of the epithelial cells to absorb free glucose. Huntington (1984) found a negative net absorption of glucose in both lactating and non-lactating dairy cows. However, Hurtaud et al. (1998) infused up to 1500g/d of glucose into lactating Holstein cows, and stated that the absorption of glucose appeared to be complete. However, the effects of the 1500 g/d infusion on milk composition were less than that of the lower infusion doses (500 and 750 g/d), suggesting

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11 that 1500 g/d may have begun to exceed the limits for glucose digestion or metabolism. Kreikemeier et al. (1991) infused 20, 40, or 60 g/h (480, 960, 1440 g/d) of glucose into eight Holstein steers and found that glucose absorption into the portal blood increased linearly with glucose infusion. However, they also found that there was a negative flux of glucose when no glucose was infused, likely due to the glucose demands of the epithelial tissue. Their work suggests that glucose absorption is not the rate-limiting step in starch digestion, at least not at the low levels of glucose normally found in the small intestine. One problem with infusion studies is that the glucose is infused into the lumen, whereas during digestion, the glucose is only released at the brush border. This may lead to differences in absorption that may not be biologically accurate (Owens et al., 1986). Perhaps the most important factor in limiting starch digestion in the small intestine is the structure of the starch itself. The proportion of amylose to amylopectin, presence of the pericarp on corn grain, and particle size all influence the accessibility of amylase to the starch molecule (Oliveira et al., 1995; Owens et al., 1986; Zinn, 1990). On forage-based diets, little, if any, dietary starch reaches the cecum or large intestine. On high-concentrate diets, however, starch can escape the rumen and small intestine. If this starch is bound inside the pericarp of uncracked corn or lignified, it may escape digestion in the large intestine as well, and escape degradation completely (Galyean et al., 1981). If readily digestible forms of starch reach the large intestine, they can be fermented by the bacterial population. This is the least efficient site of carbohydrate digestion for the cow, as much of the energy is lost to the cow via either fecal outflow or for microbial protein synthesis which is not absorbed by the cow (Hoover, 1978).

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12 Ruminal Acidosis Ruminal fermentation is a dynamic process, affected by chemical factors such as pH and osmolarity, the complex population dynamics of the ruminal microbes, the cow, her diet, and the passage rate of digesta. Alterations in these factors cause changes in fermentation. Ruminal acidosis can be a result or a cause of alterations in any number of these factors that results in a reduction in ruminal pH below desirable levels. The measured pH of the rumen varies widely depending on the diet, the animal, location in the rumen, the amount eaten per meal by the animal, and how the sample is taken (via stomach tube, ruminocentesis, or through a cannula) (Garrett et al., 1999; Ortolani, 1995). In a healthy animal, the ruminal pH is normally between 5.5 and 7.2 (Church, 1988). Healthy grain-fed cattle can have ruminal pHs sporadically below 5.5. Ruminal acidosis occurs when the pH drops below certain critical levels, which are different for the acute and chronic (subacute) forms (Table 2-1). Acute acidosis can occur when the percentage of concentrate in the diet is rapidly increased. The cows become suddenly and seriously ill as the pH in the rumen drops below 5.0 in a short period of time (Brown et al., 2000; Church, 1988; Ortolani, 1995; Owens, 1998). In chronic acidosis, animals may appear healthy, yet experience bouts of reduced feed intake, reduced production, diarrhea, mucin casts in the feces, liver abcesses, keratinization of the rumen, and laminitis. In these cases, the pH of the rumen is below 5.5 for significant amounts of time (Garrett et al., 1999; Huntington, 1988; Nocek, 1997; Ortolani, 1995; Owens, 1998; Slyter, 1976; Van Soest, 1994). The most common cause of this economically costly condition is a high-starch diet, especially when coupled with poor management practices such as allowing cows to consume large quantities of grain in a short period of time or sort their feed, feeding

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13 inadequate levels of NDF, or grinding feed too finely (Meyer et al., 1965; Nocek, 1997). Cattle with larger body weights and metabolic weights are also more susceptible to acidosis (Ortolani, 1995). These factors can influence the physiology of the rumen by causing changes in buffering capacity, motility, acid production, acid absorption, changes in the microbial population, and starch digestion. Buffering During feeding and rumination, the cow produces copious amounts of saliva, roughly 180 L/day (Van Soest, 1994). The saliva contains phosphate and bicarbonate buffers which buffer between pH 5.5 and 7.5 (Church, 1988), which is in the range of normal ruminal pH. Rumination is stimulated by the large particle sizes normally found in forage (Ruckebusch, 1988). On diets with large amounts of small particulate matter, such as high grain diets, time spent eating and ruminating is decreased, leading to a decrease in salivation. This reduces the buffering efficiency of the rumen, as both the amount of salivary buffers and the total volume of liquid (which dilutes the acids) in the rumen is decreased (Owens, 1998; Slyter, 1976). In addition, during acidosis, ruminal pHs of less than 5.5 are lower than the buffering capacity of saliva and are in the buffering range of the volatile fatty acids (VFAs) (Van Soest, 1994). Due to its cation exchange capacity, forage itself also acts as a buffer (McBurney and Van Soest, 1991). On high grain diets, the amount of physically effective fiber is often inadequate for performing this role. Motility Rumino-reticular motility is decreased during acidosis, although this may be more of an issue in acutely acidotic animals than chronically acidotic animals, as ruminal contractions stabilize over time. On high grain diets, rumino-reticular contractions have a lower amplitude (i.e., are weaker), but this does not affect the frequency or rate of

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14 contractions. The reduction may be caused by particle size affecting mechanoreceptors, inhibitory action of VFAs, or both (Ruckebusch, 1988). Lactate in the duodenum decreases rumino-reticular motility as well. This may lead to ruminal stasis in acutely acidotic animals (Goad et al., 1998). Acid Production Increased levels of RFCs initially cause a surge in microbial fermentative activity, both of amylolytic bacteria as well as cellulolytic, as some cellulose digesters are capable of fermenting RFC as well. There is a rapid increase in the amount of VFAs produced 0.5 to 4 hours after a high-RFC meal (Van Soest, 1994). Generally, as the ratio of carbohydrate to forage increases, the proportion of propionate to acetate increases (Owens, 1988). As the amount of VFAs produced exceed the ability of the cow to either buffer or absorb and metabolize them, the pH of the rumen decreases. Below pH 6.0, some bacterial species begin to produce lactate. Below pH 5.0 to 5.5, lactate production is greatly increased, and production of VFAs is reduced (Owens, 1998; Slyter, 1976). Because lactate is a much stronger acid than the VFAs (pKa of 3.8 vs 4.8-4.9), it causes a greater drop in pH than the VFAs alone (Van Soest, 1994). Absorption of VFA Volatile fatty acids are readily absorbed across the rumen wall via passive transport. The undisassociated form of VFAs, which predominates at low pHs, is believed to be preferentially absorbed by the rumen epithelium (Owens, 1998; Slyter, 1976). However, Krehbiel et al. (1995), found that VFA absorption decreased during acidosis in lambs, possibly due to gut stasis. They replaced rumen contents with an isolated VFA solution, which may not be representative of true ruminal absorption. They also did not report the

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15 pH of this solution. Keratinization of the rumen epithelium may also decrease VFA absorption under chronic conditions (Church, 1988). Even at normal ruminal pH, the absorption of VFAs is still efficient, most likely because of the constant removal of the free acid and its re-formation based on laws of equilibrium (mass action) (Church, 1988; Van Soest, 1994). In addition, VFA pass with digesta into the omasum and abomasum, where they are almost entirely absorbed. Once in the blood, the acids can be buffered with the bicarbonate found circulating in the blood, but if excessive levels of acid are transferred to the blood, metabolic acidosis can occur (Owens, 1998; Van Soest, 1994). Very little VFA reaches the small intestine (Van Soest, 1994) Microbial Changes As the starch content of the diet increases and/or the pH decreases, the composition of the microbial population changes. Cellulolytic organisms which thrive at high pHs are either inhibited, killed, or adjust to lower pHs by shifting their fermentation mechanisms. Many cellulolytic bacteria are capable of digesting both cellulose and starch. Streptococcus bovis, a common rumen bacterium, produces VFA from cellulose and starch at high pHs, but as the pH is lowered, or the amount of free glucose in the rumen fluid increases, S. bovis preferentially produces lactate (Van Soest, 1994). There appears to be a limit to how quickly microbes can utilize the freed glucose. Free glucose increases in the rumen during acidosis (Owens, 1998). This may allow the growth of normally uncompetitive bacteria, such as S. bovis, in addition to inhibiting lactate utilizers, such as Megasphera elsdenii. Thus, there is a shift from cellulolytic bacteria to amylolytic, and a concurrent shift from VFA production to lactate. Below pH 5.0 to 5.5, growth of many ruminal microbes is inhibited, including amylolytic organisms. Ruminal

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16 protozoa are inhibited at low pH as well. This may be due to a lack of substrate of sufficient size to bind to, such as forage particles (Cotta, 1992; Kistner, 1965; Russell et al., 1981a ; Warner, 1965). Changes in Starch Fermentation The optimum pH for bacterial amylases is not known, but the optimal pH for mammalian amylases is 6.8 (Russell et al., 1981c). Ruminal amylase activity, and hence, starch degradation, may be inhibited at the low pHs seen during acidosis as well. Strobel and Russell (1986) reported a 15% decrease in in vitro starch digestion when the medium had a pH of 6.0 as compared to a medium with a pH of 6.7. Below pH 6.0, cellulose digestion is inhibited as well. The low pH also shifts digestion from predominately VFA production to lactate production, which provides less energy for microbial growth. Gastrointestinal Permeability Gastrointestinal permeability is affected by factors within the lumen, such as pH and inflammation, as well as extraluminal factors including blood endotoxin levels and a genetic predisposition towards increased gut permeability(Sehesteed et al., 1999; Batt et al., 1992; Deitch et al., 1989; Stenson, 1999). Luminal Factors Affecting Permeability pH. A drop in luminal pH has been implicated in increasing gut permeability in a variety of species. This drop in pH may be caused by several factors. Ruminal acidosis is known to damage the rumen epithelium and may increase its permeability (Church, 1988; Huntington, 1997; Sehested et al., 1999; Slyter, 1976). Aschenbach and Gbel (2000) found that ruminal tissue electrical conductance, an estimate of ruminal permeability, was inversely proportional to the pH of the medium. In

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17 this study, a decrease in ruminal pH resulted in greater amounts of histamine being absorbed across isolated mucosa. Using isolated bovine rumen epithelial tissue, Sehested et al. (1999) showed that I decrease in pH increased the flux rate of volatile fatty acids across the mucosal-serosal as well as the serosal mucosal border. Sehested et al. (2000) found that increased grain feeding increased the absorption of Na + and Cl ions across bovine ruminal epithelium in vitro. They speculated that the increased permeability was due to changes in cell structure wrought by the short chain fatty acid burst feeding strategy used in the studies, not by a sudden change in pH. They did not measure pH directly, although the authors mention that the feeding strategy employed was known to lower ruminal pH significantly (Sehested et al., 2000). Ahrens (1967) found that the continuous presence of an isotonic solution of pH 3.90 in the rumen caused epithelial damage and an infiltration of neutrophils in the microvesicles of the epithelium. He did not measure permeability, but speculated that such damage would make the mucosa more susceptible to bacterial and fungal invasion leading to ruminitis. Intestinal bacteria can multiply rapidly, causing a small intestinal bacterial overgrowth (SIBO) and producing the same products as ruminal bacteria: organic acids, gasses (CO 2 , CH 2 , H 2 ), and microbial mass. Animals are also susceptible to SIBO after a course of antibiotics (Li, 1999). In the small intestine, Batt et al. (1992) showed that gastrointestinal permeability is increased during bacterial overgrowth in dogs, although pH was not measured. The pH of the gastrointestinal tract is known to decrease during SIBO in other species, however (Li, 1999). Saltzman et al. (1994) induced ileal mucosal

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18 acidosis by making mice either hypoxic (low O 2 ) or hypercapnic (high CO 2 ) and found that ileal permeability increased . Blood flow to the gastrointestinal tract is reduced during acidosis (Huber et al., 1961). In the large intestine, under conditions of carbohydrate malabsorption in humans (lactose intolerance), or when experimental subjects were given unabsorbable carbohydrates such as lactulose, the pH of the colon has been observed to fall as low as 3.5 to 5.5, causing mucosal injury, increased permeability, and eventually degradation and sloughing of the mucosal epithelial layer (Bown et al., 1974). Interestingly, evidence suggesting previous mucosal damage is often found in the feces of acidotic animals. Argenzio and Meuten (1991) conducted several studies to elucidate whether it was the low pH or the VFA that produced the damage. They concluded that acetate and lactate were more effective in causing damage than did an acidified sodium chloride solution. It is unknown whether this may be true for the small intestine as well. Gut inflammation. Chronic genetic conditions, such as inflammatory bowel disease in humans, can cause inflammation in the intestinal tract (Stenson, 1999) as can bacterial overgrowth due to stasis of the intestines (Batt et al., 1992; Li, 1999) or malabsorption of nutrients (Bown et al., 1974; Rutgers et al., 1996), and some toxins (Buchanan et al., 1991). This inflammation is thought to cause increased permeability in the small intestine (Batt et al., 1992; Fink et al., 1991; Krueger et al., 1986; Nusrat et al., 2000; Rutgers et al., 1996; Salzman et al., 1994; Shao et al., 2001). It is unclear whether the inflammation causes the increase in permeability or vice versa. According to Shao et al. (2001), stimuli such as bacterial infection or the presence of enterotoxins could lead to disturbance of the tight junctions between the cells (i.e., increased permeability), and

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19 therefore lead to uncontrolled inflammation in the gut. Inflammation in the small intestine has been detected during ruminal acidosis in cattle (Wheeler and Noller, 1977). Extra-luminal Factors Affecting Permeability Endotoxin. Endotoxins, also referred to as enterotoxins, are toxic lipopolysaccharide compounds found within the outer cell wall of Gram-negative bacteria, including Clostridium spp, Escherichia coli, Salmonella spp, Shigella spp, Staphylococcus spp, Klebsiella spp, Proteus, spp, and Pseudomonas spp (Banwell, 1975; Moore et al., 1981). They occur wherever there are colonies of Gram-negative bacteria. The normal level of endotoxin in the blood is approximately zero. If endotoxin does enter the bloodstream, it is rapidly removed by the liver (Sprouse et al., 1987). Despite this, small, transient levels of endotoxin are occasionally found in the blood of animals (Aschenbach and Gbel, 2000; Ketchum et al., 1997) and these levels are correlated with various health disorders including fever, laminitis, gastrointestinal upset, shock, and death (Lohuis et al., 1998; Moore et al., 1981; Sprouse et al., 1987) Endotoxin is routinely found in the digestive tract of animals, especially in those areas with high numbers of bacteria. In healthy ruminants, colonies of endotoxin-containing Gram-negative bacteria are located primarily in the rumen, cecum and colon (Church, 1988). In one study, hayand concentratefed animals had ruminal endotoxin concentrations of 148 and 1,019 EU/mL, respectively (Andersen et al., 1994). Small amounts of bacteria can be found in the small intestine of many species (Batt et al., 1992; Li, 1999; Rutgers et al., 1996), and endotoxin levels of 80 g/mL have been found in the cecum of clinically normal horses (Moore et al., 1979). Endotoxin may also be present at sites of infection including mastitis and uterine infections. Endotoxin is released from the cell wall of the bacteria during death as well as growth (Nagaraja et al., 1978a).

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20 Endotoxin, both within the gut and systemically, is strongly correlated with the incidence of laminitis (Moore et al., 1979; Sprouse et al., 1987; Vermunt and Leach, 1992). Nagaraja et al. (1978b) found that the number of Gram-negative bacteria in the rumens of individual cows receiving the same diet were similar, and that cows fed grain had higher numbers of Gram-negative bacteria than those fed hay. However, they found that the amount of endotoxin in the rumen fluid (as determined by an LD50 analysis) varied among individual animals receiving the same diet, although the amount in grain-fed animals was always greater than hay-fed animals. From this they concluded that something in addition to diet influenced the amount of free endotoxin in the rumen. In an in vitro study, Nagaraja et al. (1978a) found that rumen fluid incubated with corn had an 18-fold increase in endotoxin concentration, however that incubated with alfalfa increased only 3-fold. The researchers also attempted to quantify endotoxin in the rumens of acidotic and non-acidotic cattle, but were unsuccessful using the LD50 technique, as both control and experimental rumen fluid was lethal to mice. Andersen et al.(1994) adjusted the diets of cows that had been fed hay to include concentrate. One group had the concentrate in their ration increased in stages to 6 kg of concentrate, and the other were dosed with an acidosis-inducing bolus of roughly 24-28 kg of a high-starch diet (the percentage of starch in the diet was not specified). The first group showed a dramatic increase in ruminal endotoxin, while the second showed little increase in the amount of endotoxin (measured using the Limulus Amebocyte Lysate assay). Both groups of researchers concluded that concentrate-fed animals tend to have higher levels of ruminal endotoxin than hay-fed animals, although it is unclear how the level of

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21 ruminal endotoxin is related to diet and the number of bacteria. It is possible that gradual increase in concentrate allows time for rapid growth of Gram-negative bacteria, releasing free endotoxin into the rumen. The more rapid changes associated with the acute acidosis may have killed the Gram-negative bacteria before they produced much endotoxin. Endotoxin is extremely stable and is not affected by acidic pHs (Nagaraja et al., 1978b). Although luminal endotoxin has not been shown to cause an increase in intestinal permeability (Beeken et al., 1974), intraperitoneal, intramuscular, and intravenous injections of endotoxin have been shown to increase intestinal permeability (Deitch et al., 1991; Deitch et al., 1989; Fink et al., 1991; Salzman et al., 1994; Xu et al., 1993). Two interrelated mechanisms for this effect have been proposed. The first implicates xanthine oxidase, a free-oxygen radical generator, in damaging the intestinal mucosa. This allows for increased translocation of bacteria from the lumen of the gut into the mesenteric lymph tissue. Several studies with mice have shown that endotoxin is a potent activator of xanthine oxidase activity (Deitch et al., 1991; Deitch et al., 1989; Xu et al., 1993). Although it is not clear whether the association between endotoxin, xanthine oxidase, and mucosal injury is a causal or a casual relationship, Deitch et al. (Deitch et al., 1991) and Xu et al. (1993) found that xanthine oxidase-induced mucosal injury and the resulting intestinal permeability were inhibited by allopurinol, a competitive inhibitor of xanthine oxidase, implying that xanthine oxidase is involved in the mucosal damage. The exact mechanism by which endotoxin increases xanthine oxidase activity is unclear, but Deitch et al. hypothesized in 1989 that it may occur when endotoxin stimulates macrophages to release interferons, which promote xanthine oxidase activity. However,

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22 when Deitch et al. (1991) later tested the effects of complement and macrophage activity on bacterial translocation using complement and macrophage-deficient strains of mice, they found that these two factors had no effect on the level of bacterial translocation. They did not propose a different mechanism. The second mechanism proposed for how endotoxin can increase intestinal permeability is by causing intestinal ischemia (lack of blood flow). Ischemia of the small intestine, whether caused by endotoxin or mechanical means, has been shown to acidify the mucosa and increase intestinal permeability. Xu et al. (1993) found that after intraperitoneal injection of endotoxin, the blood flow to the ileum and cecum was decreased 35-50%, while that to the duodenum, jejunum, and large intestine remained unchanged. Permeability was increased only at sites of decreased blood flow. Salzman et al. (1994) showed that mucosal permeability was directly correlated with [H + ] in ischemic and endotoxin-injected pigs and that an increased [H + ] independent of ischemia also caused increased permeability. Fink et al. (1991) showed that ileal permeability in pigs increased during administration of endotoxin as well as during mechanically-induced 100% ischemia. Gut permeability did not increase in cases of only 50% ischemia or in control animals, implying that the partial ischemia caused by endotoxin alone does not cause the increased permeability seen in endotoxic animals. It is possible that both mechanisms occur together to cause the increased permeability seen in endotoxic animals. Another element to note is the potentially cyclic nature of these reactions (Xu et al., 1993). If extraluminal endotoxin causes increased intestinal permeability, then it is possible for luminal endotoxin to enter the blood, beginning a cycle of increased endotoxin levels and increased permeability. Whether this happens or

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23 not may depend on the liver's ability to remove endotoxin from circulation (Moore et al., 1981) as well as the amount entering from the lumen. Genetic predisposition. In humans, several gastrointestinal and arthritic disorders have been linked to a genetic predisposition to greater gut permeability. These include patients with Crohn’s disease, a disorder marked by inflammation of the entire small intestinal wall (mucosa to serosa), patients with ulcerative colitis, a disorder marked by inflammation of the colonic mucosa (Stenson, 1999), celiac disease (sprue), a disease in which the mucosa is damaged in response to ingestion of gluten and similar proteins (Schulzke et al., 1995; van Elburg et al., 1993), and ankylosing spondylarthropathy, a degenerative condition of the spine (Chou et al., 1998). Defects in the gene HLA-B27 are thought to play a role in these disorders, as many of these patients and their relatives have a variation of HLA-B27. The rodent models of these disorders are HLA-B27 knock-out animals (Mielants and Veys, 1990; Stenson, 1999). Relatives of patients with Crohn’s disease and celiac disease often have higher-than-average indices of gut permeability (Stenson, 1999; van Elburg et al., 1993). Although rodent models are being studied, nothing has been done to determine the genetic influence on gastrointestinal permeability in ruminants. Physiology of Gut-Induced Laminitis Once the mucosa is breached, substances from inside the lumen, including bacteria, toxins, and other molecules can be absorbed from the lumen into systemic circulation. These foreign antigens in the bloodstream may trigger a host of physiological and immunological changes in the animal. The two molecules most often linked with the physiological onset of laminitis are endotoxin and histamine, bacterial products normally found in the lumen of the gut and

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24 increased luminally and systemically during acidosis. (Ahrens, 1967; Andersen et al., 1994; Dain et al., 1955; Dougherty et al., 1975a; Maclean, 1970; Moore et al., 1979; Nagaraja et al., 1978a; Nagaraja et al., 1978b; Sprouse et al., 1987; Suber et al., 1979). However, it is not yet clear how endotoxin and histamine are involved and whether they trigger laminitis or are just metabolites released during laminitis. Besides endotoxin and histamine, antigens may be absorbed during periods of increased gastrointestinal permeability. Even if they have no direct physiological effect themselves, the antigens can initiate an inflammatory response from the body. The inflammatory response is a coordinated series of reactions which stimulate the animal’s immune system to respond to foreign material. During normal reactions, this foreign material is removed, thereby diminishing the inflammatory response and helping to heal the area (Baumann and Gauldie, 1994). However, the gut has a unique immunophysiology, due to the large amount of antigenic material continuously present in the lumen. It is thought that the inflammatory response is somewhat diminished in the vicinity of the gut (Matsuura and Fiocci, 1993). The study of gastrointestinally absorbed antigens and their role in arthritis is a current topic of investigation in humans and rodents. Hematological Causes of Laminitis The pathophysiological changes that occur during the onset of laminitis have been extensively studied in horses. During the onset of laminitis in horses, changes in the blood circulation to the hoof occur. The pathophysiology of these changes has been the subject of much recent research. These changes appear to be aggravated by high circulating levels of endotoxin and histamine in the blood. However, much of the research has focused on the onset of acute laminitis. Chronic laminitis, a common, yet

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25 less obvious type of laminitis may occur via a different mechanism. In addition, there may be differences between horses (non-ruminants) and cattle (ruminants) and how their bodies react to these substances. Three main theories have been put forth as to how changes in blood flow may produce laminitis: microthrombosis, vasoconstriction, and arteriovenous anastomoses. Microthrombosis. Microthrombi are tiny blood clots in the capillaries of the hooves. Weiss et al. (1994) found that horses suffering from carbohydrate-induced laminitis had large numbers of blood clots in their hoof capillaries. Andersson and Bergman (1980) found microthrombi in the hooves of laminitic cattle. In a later study, Weiss et al. (1998) determined that laminitis in carbohydrate-overloaded horses could be prevented by using a competitive inhibitor of platelet aggregation, RPR 110885. This implies that the onset of laminitis involved platelet aggregation or blood clotting. However, a retrospective study done with horses at the University of Georgia showed no effect of the administration of another anticoagulant, heparin, on the development of laminitis (Belknap and Moore, 1989). In addition, Prasse et al. (1990) found that blood indices of clotting ability did not change during the beginning stages of carbohydrate-induced laminitis in horses. It is unclear at this point whether microthromboses are involved in the pathogenesis of laminitis. Vasoconstriction. The second theory proposed is that constriction of the blood vessels of the hoof causes a local increase in blood pressure. This forces serum out of the capillaries and into the surrounding space, causing edema and swelling of the lamina. This may cause blood clots as well. If this continues, blood supply to the tissue is cut off,

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26 damaging the tissues and causing the release of still more vasoactive hormones (Moore and Allen, 1996; Nocek, 1997). Arteriovenous anastomoses. Arteriovenous anastomoses (AVAs) occur where blood is shunted away from the capillaries by direct connections between arterioles and venules (Moore and Allen, 1996; Nocek, 1997). Using corrosion casts, Pollitt and Molyneux (1990) found over 500 anastomoses/cm 2 in the laminar region of the hoof of healthy horses. These AVAs may exist to shunt blood away from the capillary bed at times of either high activity or cold stress (Pollitt and Molyneux, 1990). If blood is shunted away from the capillary bed during the onset of laminitis, as was suggested by Hood et al. (1978) the lack of blood flow to the capillaries may cause ischemia and tissue damage, similar to the other two hypotheses (Pollitt and Molyneux, 1990). Role of Absorbed Endotoxin and Histamine in the Onset of Laminitis Endotoxin. Endotoxin may play a role in the formation of microthrombi. Endotoxin is a known stimulant of coagulation, and is responsible for precipitating the release of vasoactive mediators such as thromboxane and prostaglandins, which are also implicated in increasing blood clotting. Sprouse et al. (1987) hypothesized that acute laminitis is the result of a Schwartzman reaction. In this experimental reaction, two injections of a reactive substance, such as endotoxin, are given. The first injection sensitizes the cells and encourages granulocytes to infiltrate the afflicted tissue. The second provokes a strong reaction that causes the immune cells to degranulate, releasing histamine and other vasoactive substances, and producing dermal hemorrhagic necrosis, including the formation of thrombi (Sprouse et al., 1987). In a study done by Sprouse et al., (1987) horses that were previously sensitized to endotoxin developed laminitis after carbohydrate overload in a manner similar to the Schwartzman reaction. Thus, an initial

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27 exposure to endotoxin could come from a systemic infection, such as some form of enteritis or mastitis. This increases the permeability of the gut, while allowing time for the Schwartzman reaction to develop. It may then follow that, when endotoxin later enters from the acidotic gut, a localized Schwartzman reaction may be generated and laminitis could develop. Histamine. Histamine is produced via decarboxylation of the amino acid, histidine, and is found at low levels in rumen fluid of healthy animals (Chavance, 1946; Sanford, 1963; Sjaastad, 1967). The formation of histamine from histidine is favored at low pHs (Dain et al., 1955). Normal rumen fluid concentrations of histamine are low and range from 0.0 g/mL (Dain et al., 1955) to 0.3 g/mL (Suber et al., 1979). During acidosis, ruminal levels of histamine can rise as high as 3.0 70.0 g/mL (Suber et al., 1979). It should be noted that the fluorometric assay used by Suber et al.(1979) and Ahrens (1967) does not include metabolites of histamine, while the paper chromatographic technique used by Dain et al. (1955) includes these. These metabolites are not thought to be vasoactive, therefore including them inflates the true amount of vasoactive material in the circulation. Histamine is also produced systemically at sites of inflammation or infection when mast cells degranulate (Chavance, 1946; Maclean, 1970; Nocek, 1997; Suber et al., 1979). Histamine can be absorbed, in at least small quantities, across the rumen wall (Sjaastad, 1967). There is disagreement over whether this is of sufficient amount to significantly influence blood histamine levels. Brent (1976) noted that histamine is unlikely to be absorbed from the gut at an acidic pH, because it would be disassociated at low pHs and therefore less likely to be absorbed. Recent research by Aschenbach and

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28 Gbel (2000), however, suggests that acidic pHs and their damage to the epithelial layer actually increase the absorption of histamine. Most circulating histamine probably comes from the degranulation of mast cells at an area of injury within the animal (Chavance, 1946; Maclean, 1970; Nocek, 1997; Suber et al., 1979). Histamine has been implicated as a major effector of laminitis by causing changes in blood flow. Although it is generally thought to be a vasodilator, it may also cause arterial constriction (Chavance, 1946). Nocek (1997) noted that it is irrelevant whether it causes arterial constriction or arterial dilation because both would have the same effect at the capillary level – edema. Few studies have been conducted to experimentally address the effect of histamine on digital blood flow in either horses or cattle. In Japan, Takahashi and Young (1981) experimentally overfed grain to a group of bulls, which induced a mild hoof pain. However, when histamine was injected into an artery in the hooves of the overfed animals, the cattle experienced severe laminitis. Injection of histamine alone, without grain overfeeding, did not result in laminitis. Likewise, feeding of histamine in large doses to cattle had no effect, presumably because the histamine was metabolized by either the microbes or the gut wall (Bergsten, 2001; Takahashi and Young, 1981). Like endotoxin, histamine is rapidly cleared from circulation by the liver (Nocek, 1997), and therefore, a chronic condition implies a continuing influx of histamine from either an inflamed area, such as the hoof, or from the gut. Histamine may be more active in chronic than in acute laminitis. Maclean (1970) found that the circulating histamine level in chronically laminitic cattle was nearly four times higher than acutely laminitic cows (0.2027 vs. 0.0589 g/mL). The amount of histamine circulating during acute laminitis was not significantly different from that of

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29 healthy adult cows. Little research has been done on chronically laminitic cows. Methods have been developed to induce experimental acute laminitis in both horses and cattle, but similar methods do not exist for the induction of chronic laminitis. Inflammatory Response and the Onset of Laminitis The gut and the immune system are very closely related. The endothelial tissue of the body, both internal and external, is the major barrier to invasive pathogens. In contrast to the external skin, however, the gut is responsible for the absorption of nutrients vital to the animal, yet must still exclude potential pathogens. As such, the gut has been termed the "largest immune organ in the body" (Matsuura and Fiocci, 1993; Shao et al., 2001). Because of the high antigenic load on the luminal surface of the gut, the gut exists in a persistent state of inflammation and immune system activity (Shao et al., 2001; Watkins, 1999), although it is at the same time thought to be hyporesponsive (Shao et al., 2001). However, under conditions of gut damage, a heightened or acute immune response may be seen. The response may be due to the damage itself, or in reaction to the absorbed antigenic compounds and microorganisms. Most likely, the inflammatory response seen occurs in reaction to both of these events. The inflammatory response begins with the migration of neutrophils and macrophages (monocytes) to the area of tissue damage. These immune cells then produce pro-inflammatory cytokines – molecules important in initiating as well as ending the inflammatory response (Baumann and Gauldie, 1994). The three pro-inflammatory cytokines most commonly associated with the inflammatory response are Interleukin 1 (IL-1), Interleukin 6 (IL-6), and Tumor Necrosis Factor Alpha (TNF-) (Baumann and Gauldie, 1994; Breazile, 1988). Although the

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30 three molecules exhibit a diverse array of effects on the body, functionally the three compounds are very similar. Some of their more general functions include the induction of fever and anorexia, alteration of protein, lipid, mineral, and carbohydrate metabolism, inhibition of growth, alteration of blood flow and the enhancement of the local inflammatory response (Baumann and Gauldie, 1994; Johnson, 1997). In response to increased levels of IL-1 and IL-6, the liver produces acute phase proteins, such as 1 -glycoprotein , antiproteases, ceruloplasmin, fibrinogen, haptoglobin, seromucoid, and serum amyloid A. These levels are known to be elevated during times of immunological stress in cattle (Baumann and Gauldie, 1994; Conner et al., 1988). Interestingly, the pro-inflammatory cytokines are also involved in extinguishing the acute inflammatory response, by acting on neurotransmitters in the brain to release glucocorticoids, which quell inflammation (Breazile, 1988). However, if the triggering antigen is not removed, the inflammatory response continues in the chronic form. In this form, macrophages continue to enter the area, stimulate collagen production and the production of IL-1. Thus, the systemic effects of IL-1 may continue for extended periods of time (Baumann and Gauldie, 1994). IL-1 also causes vasoconstriction(Breazile, 1988). Vasoconstriction is one theory for the mechanism of laminitis, and thus is a possible connection between gut damage and hoof problems. Summary Acidosis causes disruptions in the gut that may interfere with normal gut processes and bacterial populations. These perturbations in gut functioning can lead to acidosis and/or intestinal bacterial overgrowth, both of which are implicated in increasing the

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31 permeability of the gut. Because of the toxic nature of gut contents, luminal substances such as histamine and endotoxin may enter systemic circulation, triggering vascular changes. Laminitis is thought to be caused by minute hemodynamic changes in the hoof and small changes in blood flow caused by small amounts of endotoxin or histamine. It seems likely that both endotoxin and histamine are involved in the onset of laminitis, although their roles may change in chronic versus acute forms. Endotoxin may initiate laminitis when it is formed by the cow’s immune cells during a systemic infection or injury. This would act to not only initiate the Schwartzman reaction, and stimulate histamine release, but also an increase in the permeability of the gut. The possible chronic nature of endotoxin, whereby endotoxin increases permeability of the gut, allowing luminal endotoxin to enter circulation, may lead to a chronic mild endotoxemia. Histamine release from injured tissues is also stimulated during chronic laminitis. The two together may form the basis for chronic laminitis. Further research is needed to determine if blocking one of the two, for example, histamine via antihistamines, would be of use and how chronic differs from acute laminitis. Table 2-1. Reported critical limits of acute and chronic forms of acidosis Acute Chronic (subacute) 5.2 (Owens, 1998) 5.5 (Garrett et al., 1999) 3.9 – 4.2 (Ortolani, 1995) 5.6 (Owens, 1998)

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CHAPTER 3 POST-RUMINAL STARCH INFUSION IN DAIRY CATTLE: IMPLICATIONS FOR INFLAMMATORY RESPONSE AND ANIMAL HEALTH Materials and Methods Cows, Diet, and Facilities Six ruminally cannulated (10 cm i.d., Bar Diamond, Inc. Parma, ID), multiparous, nonpregnant, nonlactating Holstein cows (BW 792 96 kg) were used in a single period experiment with two treatments. The animals had been previously culled from the dairy herd at the University of Florida for reasons which included udder, feet, and reproductive problems and low production. Animals were cared for under protocols approved by the University of Florida Institutional Animal Care and Use Committee. The experiment consisted of 14 days of acclimatization to a standard diet and six days of data collection. Treatments were administered on days 2, 3, and 4 of the six-day sampling period. Cows were initially housed in outdoor pens during the acclimatization period and group fed a TMR (Table 3-1) once a day. Three days before the onset of data collection, the cows were moved into tie-stalls at the University of Florida Dairy Research Unit in Hague, FL, where they were individually fed the same TMR as in the acclimatization period until the end of the sampling period. The cows were fitted with jugular catheters on day 1 of the sampling period. On day 0, each cow was fitted with an abomasal infusion catheter consisting of a length of the Tygon tubing (0.3175 cm i.d. 0.635 cm o.d; Saint-Gobain Performance Plastics Corp., Akron, OH) ending in a 13 cm diameter rubber flange and a perforated 60 32

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33 mL Nalgene bottle (used to prevent digesta from blocking the end of the tube; Figure A-1). The end of the tube external to the cow was passed through a hole in the rumen cannula and was connected to a length of Tygon tube descending from a peristaltic pump. Treatments The two treatments consisted of abomasal infusions of either 0.9% saline solution (control; CTRL) or a starch and 0.9% saline suspension (ST). A saline solution was used in both infusions to minimize effects of osmolality and hypotonicity of a water solution. The starch suspension was prepared by blending a mixture of 1 kg of cornstarch, 4 g of xanthan gum (an emulsifier and suspending agent; CP Kelco ApS, Chicago, IL), and 18 g of sodium chloride with 1.25 L of tap water in a KitchenAid mixer (model K45SS, Hobart Corp. Troy, Ohio). On each of sampling days 2, 3, and 4, approximately 8 L of infusate was abomasally infused into each animal over a 12-hour period using a peristaltic pump (Haake Buchler Multistaltic Pump model 426-2100, Saddle Brook, NJ), followed by a 12-hour period with no infusions. The amount of starch infused daily in this study (5.0 g/kg BW per day) was higher than that infused in other studies with cattle, in which no indications of laminitis or digestive disturbances were reported (4.8 g/kg BW per day, Kreikemeier et al., 1991 1 ; 3.7 g/kg BW per day, Kreikemeier and Harmon, 1995). However, it was less than the amount of starch commonly given to horses to induce laminitis (15.0 g/kg BW per day, Garner, 1975). For the infusions, the appropriate infusate (ST or CTRL) was placed in a 19-L container on a platform suspended over the feed bunks. Tygon tubing, weighted to stay 1 Kreikemeier et al. (1991) observed a decline in ileal pH in the starch infused cows in their study.

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34 near the bottom of the container, ran from each container, through the peristaltic pump, over a line suspended over the cows’ stalls, and culminated in a reducing connector. This connector was then attached to the external portion of a cow’s abomasal infusion catheter. Sampling Every four hours (offset daily by one hour), beginning at hour 0 on day 1, blood and fecal samples were collected and rectal temperature and respiration rate (breaths per 30 seconds, multiplied by 2) were recorded on each cow. Approximately 20 mL of blood was withdrawn from the jugular catheter into a sterile syringe. Patency of the jugular catheters was maintained by flushing with sodium citrate after each collection. When several of the jugular catheters became non-patent, samples were obtained by caudal venipuncture. The blood was then injected into four vacutainers (two heparinized and two containing EDTA, Becton Dickinson, Franklin Lakes, NJ), slowly inverted several times, and kept chilled on ice. The blood was centrifuged at 1586 x g for 30 min at 5 C and frozen at -20 C until analysis. Fecal material was collected in 100 mL Fisher sampling cups with a minimum of headspace for pH analysis. Rectal temperature was measured with an electronic thermometer (model MT1681-BMWF, Florida Medical Industries, Inc., Leesburg, FL). Diet and orts samples were collected daily. Laboratory Analyses One-gram feed and orts samples were dried in a forced-air oven at 105C overnight to determine dry matter content. Feed and orts samples for nutrient analysis were dried at 55C in a forced-air oven to constant weight and ground in a Wiley mill (Arthur A. Thomas, Philadelphia, PA) to pass a 1 mm screen. Diet samples were composited on a

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35 DM basis by cow. Composited diet and un-composited orts samples were analyzed for DM (105C for 8 h), OM (512C for 8 h), and NDF using heat-stable, -amylase (Goering and Van Soest, 1970). Crude protein was determined by a modification of the AOAC procedure (AOAC, 1995) in which a digestion mixture of 96% Na 2 SO 4 and 4% Cu 2 SO 4 was used in the digestion, and distilled ammonia was recovered in a 4% boric acid solution. The pH of the fecal material was measured as soon as possible after collection, using 80 g of fecal material mixed with 40 mL of distilled water. Blood samples were analyzed for the acute phase proteins haptoglobin (Hp), ceruloplasmin (Cp), fibrinogen, and -acid glycoprotein (AGP). Haptoglobin was analyzed using the method of Makimura and Suzuki (1982) using a Jenway 6300 spectrophotometer (Jenway Ltd., Dunmow, England). Ceruloplasmin was analyzed according to the method of Demetriou et al.(1974), using a PowerWave 340 microplate spectrophotometer (Biotek Instruments, Winooski, VT; 1974). Fibrinogen was analyzed with a Fibrinogen kit (Sigma Diagnostics Inc., Procedure No. 886, St. Louis, MO), per manufacturer’s instructions, and AGP was analyzed using a commercial bovine radioimmunodiffusion kit (Bovine AGP kit, Cardiotech Services, Louisville, KY) per manufacturer’s instructions. A liver panel was run on a selected set of blood samples from three treated cows and two control cows collected on days 1 and 2. These enzymes included alkaline phosphatase (AP), measured in IU/L; alanine aminotransferase (ALT), measured in IU/L; aspartate aminotransferase (AST), measured in IU/L; Biliruben, measured in mg/dL; Albumin, measured in g/dL; and gamma glutamyltransferase (GGT), measured in IU/L. The liver panel proteins were analyzed by the University of Florida Veterinary Medical

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36 Teaching Hospital, Department of Clinical Pathology using a Hitatchi 911 system (Boehringer Mannheim Corp. Indianapolis, IN). Statistical Analyses Data were analyzed as a split plot design in time. Type III sums of squares were used to develop tests of hypotheses (F-tests). For statistical analyses, the untransformed hydrogen ion concentration [H+] was used instead of pH (Murphy, 1982). Error terms for elements in the model are listed in Table 3-2. All model terms containing “cow” were included in the RANDOM statement. The following two models were used to analyze all dependent variables in the complete data set (ALL): Model I Yijkl = + Ti + Cj(Ti) + Dk + TiDj + Cj(Ti)Dk + Kl + TiKl + Cj(Ti)Kl + DkKl + DkKlTiijkl where: Yijkl = the dependent variable = overall mean Ti = infusion treatment (i = CTRL or ST in analyses using 2 treatments) or (i = CTRL, ST-1, or ST-2 when using 3 treatments) Cj(Ti) = cow within treatment (j = 1) Dk = day (k = 1) TiDk = interaction term for treatment and day Cj(Ti)Dk = interaction term for cow within treatment by day Kl = collection (within each day; l = 1) TiKl = interaction term for treatment and collection Cj(Ti)Kl = interaction term for cow within treatment by collection DkKl = interaction term for day and collection DkKlTi = interaction term for day, collection, and treatment ijkl = residual error term

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37 Because fecal [H+] was a sensitive descriptor for the effect of starch infusion treatment on the cow, it was used in Model II as a descriptor of the impact of treatment. Model II Yijkl = + Hi + Cj + Dk + Kl + DkKl ijkl where: Yijkl = the dependent variable = overall mean Hi = fecal hydrogen ion concentration Cj = cow (j = 1) Dk = day (k = 1) Kl = collection (within each day; l = 1) DkKl = interaction term for day and collection ijkl = residual error term Two other data sets – the daily minima (MIN) and maxima (MAX) for each dependent variable were analyzed using a third model: Model III Yijkl = + Ti + Cj(Ti) + Dk + TiDj ijk where: Yijkl = the dependent variable = overall mean Ti = infusion treatment (i = CTRL or ST in analyses using 2 treatments) or (i = CTRL, ST-1, or ST-2 when using 3 treatments) Cj(Ti) = cow within treatment (j = 1) Dk = day (k = 1) TiDk = interaction term for treatment and day ijk = residual error term Many of the blood chemistry values of cow 2245 were elevated on day 1, before the initiation of treatments, indicating that she may have already had undetected compromised health. Therefore, data were analyzed both with and without cow 2245. In

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38 addition, although there were only two treatments, there were three apparent responses. Cows 2811 and 5329 returned to a normal fecal pH more rapidly than did cows 2245 and 2686 (Figure 3-2). Because of this, for some analyses, a third descriptor for treatment was added. The treatments for these analyses consisted of control, starch-infused “faster” recoverers (ST-1), and starch-infused “slower” recoverers (ST-2). Thus, four analyses were conducted within each model. These analyses are named in the text with a numeral indicating the number of treatment descriptors used (2 or 3) and a letter indicating the presence (Y) or absence (N) of cow 2245 in the analyses. Thus, the four analyses are termed 2Y, 2N, 3Y, and 3N. Data were analyzed by the MIXED procedure of SAS (SAS, 2001). Orthogonal contrasts for treatment, day, and treatment x day were performed (Tables 3-8 through 3-15). To assess the relative direction of change in dependent variables with fecal [H + ], the coefficients in model I were estimated using the SOLUTION option. Because all other independent variables in the model statement were classification variables, their coefficients became part of the intercept, leaving the coefficient for fecal [H + ] as the only descriptor of slope. The sign of the slope then, is indicative of the direction of change between the dependent variable and fecal [H + ]. Significance was set at a P-level of < 0.10 and a tendency was declared to exist at a P-level of < 0.15. All means are reported as least squares means. Means reported without a standard error were calculated manually from the least squares means of higher-order interactions. Results of all analyses are summarized in Tables 3-4 to 3-23.

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39 Results and Discussion Indices of Animal Health During this study, dramatic changes in animal health and fecal appearance were noted in the cows receiving starch treatments. Two starch treated (ST) cows, 2245 and 2686, became ill showing decreased feed intake, diarrhea, and obvious discomfort. Cow 2245 was euthanized after sampling day 4. A necropsy showed locally extensive severe hemorrhagic enteritis and bacterial overgrowth in the small intestine, along with preexisting endoparasitism of her lungs, esophagus and myocardium (Appendix B). Because of the preexisting medical conditions revealed at the necropsy of cow 2245, and because some of her acute phase protein concentrations were increased even on day 1 before starch infusions began, it appeared that this cow was experiencing inflammatory response before the onset of the trial. However, she responded to starch infusion treatment and was not euthanized until after day 4. Cow 2686 also became ill on day 4, but recovered after the cessation of starch infusion and the transfer of rumen contents from a healthy cow and intramuscular injections of 4 cc of MuSe (selenium and vitamin E; Schering-Plough, Union, NJ), 12 cc of vitamin B complex and 12 cc of Banamine (Schering-Plough, Union, NJ) and an oral dose of Probios (Chr. Hansen BioSystems, Milwaukee, WI), a probiotic. Fecal pH and fecal [H + ] There was a dramatic decrease in fecal pH or increase in fecal [H + ] for ST cows, whereas CTRL cows remained near initial levels throughout the experiment (Figures A-2 and A-3). The fecal pH of ST cows ranged from 4.32 to 7.66 during the experiment. Following the conclusion of treatments or slightly before, the fecal pH of the ST cows began to return to initial levels.

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40 Fecal [H + ] was increased (2Y P = 0.05; 2N P = 0.10; 3Y P = 0.04; and 3N P = 0.03) in ST animals in all analyses. The fecal [H + ] of two ST cows, 2811 and 5329, differed (P = 0.07) from the other two ST cows, returning to initial levels much more rapidly. The cows that evidenced a more rapid decrease in fecal [H + ] were termed ST-1 and the slower recovering cows were termed ST-2. Fecal [H + ] differed by treatment x day in the 2Y, 3Y, and 3N analyses, by treatment x collection in all four analyses, and by treatment x day x collection in the 3Y and 3N analyses (Tables 3-4 to 3-7). The change in fecal [H + ] is highly suggestive of increased hindgut fermentation related to an increase in undigested, fermentable starch passing to the large intestine. Fecal [H + ] minima were affected by treatment in all four analyses and by the interaction of treatment x day in analyses 2Y, 3Y, and 3N. The fecal pH of ST cows was elevated over CTRL cows on days 2, 3, and 4 (P = 0.15), and on day 4 over day 3 (P = 0.1388). Fecal [H + ] maxima were affected by treatment in the 3Y and 3N analyses, and tended to be affected by treatment in the 2Y analysis. All orthogonal contrasts for treatment and the treatment x day interaction were significant in the 3Y and 3N analyses, indicating that cows in the different treatment descriptor groups differed from one another and that the different groups responded differently on different days. The decreased fecal pH is an indication of bacterial fermentation in the intestinal tract. Based on the pKas of the VFAs (4.76 – 4.87) and lactate (3.86; Biochemical calculations, 1976), it is likely that both VFA and lactate were present in the feces, in order to bring about the fecal pHs below 4.8 seen in this experiment. Fecal consistency and indications of gut damage Fecal material of ST animals collected throughout the trial changed in consistency and content. Initially all cows showed what appeared to be normal fecal material for dairy

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41 cattle receiving a high forage diet, which was dark brown in color, relatively solid, and with only minor amounts of undigested feed present (Figure A-4). Starch treated cows, however, exhibited marked changes in fecal consistency (Table 3-3) over the course of the experiment. During collections in which the fecal pH was below 6.5, feces were often foamy or frothy, and contained minute trapped gas bubbles (Figure A-5). This is likely due to fermentation gasses produced by hindgut bacteria. The feces also became lighter in color. Segments of pink tissue 1 to 1.5 cm in length, mucus and strands of mucin or fibrin 1 to 2 mm wide and 3 to 5 mm long were seen in the feces of the ST cows. Cow 2245 had watery diarrhea (Figure A-6), and other cows’ feces became pasty or sticky (Figure A-7). No determination was made to confirm the composition (mucin or fibrin) of strands and casts passed in the feces. Symptoms of possible gut damage were seen in the form of sloughed mucin casts, mucus, and fleshy tissue found in the feces. When the fecal pH was below 6.0, segments of tissue, mucus, and mucin casts or watery diarrhea often were seen in the feces, indicating possible onset of damage to the gastrointestinal tract. This agrees with Saunders and Sillery (1982), who found that sloughing of the epithelium occurred at a pH of 5.0 in the ileum and colon of the rat. Argenzio and Meuten (1991) found in vitro epithelial damage to colonic tissue of swine at pH of 4.0 and below. In that study, at pH 4.0, the colonic epithelium had separated from the lamina propria, was filled with fluid, and blistering of the epithelium was seen. Prolonged exposure resulted in large segments of sloughed epithelium and a thickening of the mucus layer. These signs were also observed during the necropsy of cow 2245. The damage observed in the current study appears to have occurred at a higher pH than in Argenzio and Meuten’s study and is more

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42 similar to that of Saunders and Sillery (1982), if fecal pH is an accurate indicator of colonic pH. In a study by Wheeler and Noller (1977) fecal pH was not significantly different from colonic pH. . Respiration rate Respiration rate typically is increased during certain stresses in animals, including heat stress, vigorous exercise, illness and pain. The normal range in cattle is 25 to 50 respirations per minute (The Merck Veterinary Manual, 1997). In the current study, respiration rate ranged from 12 to 90 respirations per minute. Respiration rate was increased numerically with starch infusion, but was not affected by treatment or the interaction of treatment x day in any analysis. When cow 2245 was included in the model, respiration rate increased (P = 0.02) with increasing fecal [H + ] in the 2Y analysis. Daily minima tended (P = 0.15) to be elevated in ST cows over CTRL cows on days 2, 3, and 4. Rectal temperature As with respiration rate, rectal temperature is often elevated during illness, especially bacterial infection and toxemia. The normal range in dairy cattle is 38.0 to 39.0 C (The Merck Veterinary Manual, 1997). In this experiment, rectal temperatures ranged from 37.9 to 40.2 C. Rectal temperature tended to differ among treatments in the 3Y and 3N analyses, with ST-2 cows having the highest rectal temperature, ST-1 intermediate, and CTRL the lowest. ST-2 cows had numerically elevated rectal temperatures on day 1, however. Thus, the elevated rectal temperatures in this group are likely not due to the starch infusion treatment. That initial differences in rectal temperature occurred between ST-1

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43 and St-2 animals suggests that the ST-2 animals were suffering from preexisting conditions that may have compromised their ability to handle the starch challenge. In no analyses for mean rectal temperature, were treatment x day interactions detected. As fecal [H + ] increased, rectal temperature tended (P = 0.12) to decrease in the analyses which included cow 2245. Rectal temperature minima were elevated (P = 0.05) in the 3Y analysis and tended (P = 0.13) to be elevated in the 3N analyses, with ST-2 cows having higher minima than ST-1 cows, and both ST groups being higher than CTRL cows. The daily maximum temperatures were increased (P = 0.10) in CTRL cows over both ST treatment descriptor groups on day 4 in the 3N analysis. Hematocrit Hematocrit, or percent packed cell volume, is an indicator of the state of hydration or of anemia in the animal. The normal range for cattle is between 24 and 37% (The Merck Veterinary Manual, 1997). In this study, hematocrit ranged from 22 to 40%. Starch-treated animals had a lower hematocrit than control animals (2Y P = 0.02; 2N P = 0.03). Treatment also affected (3Y P = 0.13; 3N P = 0.13) hematocrit in the 3Y and 3N analyses (Tables 3-6 and 3-7). The lower hematocrit in the ST animals is likely not explained by hemodilution, as these animals received a smaller quantity of fluids (5L of saline in the starch suspension versus 8L in the saline-infused animals), and diarrhea or loose stools were observed in all ST animals, which would tend to increase the hematocrit values due to loss of fluids.

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44 Acute Phase Proteins Ceruloplasmin Ceruloplasmin is an indicator of bovine inflammatory response, although the concentration of Cp in plasma may not be indicative of the level of inflammatory response in the animal (Conner et al., 1988). The normal range for cattle is between 16.8 and 34.2 mg/dL (The Merck Veterinary Manual, 1997). In this trial, Cp concentrations ranged from 25.5 to 62.5 mg/dL. The Cp concentrations of cow 2245 were elevated above the other cows throughout the entire trial, including the time before treatments were administered (Figure A-10). However, all cows exceeded these normal limits during the course of the experiment. No effects of fecal [H + ], treatment, or interaction terms containing treatment on Cp concentrations were detected. Minimum Cp concentrations were increased (P = 0.05) in ST-2 cows on days 3 and 4 over day 2. Haptoglobin Haptoglobin is normally undetectable in bovine serum, except in animals suffering from acute inflammatory conditions. Detectable concentrations, therefore, are indicative of an inflammatory response. Haptoglobin was elevated in two cows, 2245 and 2521, throughout the trial (Figure A-8). Cow 2245 had a history of parasitic infection and chronic enteritis. It is unknown whether cow 2521, a CTRL cow, had a preexisting inflammatory condition. The Hp concentrations in this trial ranged from 2 to 58 mg HbB/dL. Excluding cows 2245 and 2521, the range lay between 2 and 42 mg HbB/dL. Regardless, these are considered elevated concentrations of Hp for cattle. No effects due to fecal [H + ] or treatment were detected in any analysis.

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45 Fibrinogen Elevated fibrinogen concentrations in the blood are another indicator of inflammatory response. In cattle, fibrinogen is a more sensitive indicator of inflammation than the white blood cell count used in other species (The Merck Veterinary Manual, 1997; Morris and Johnston, 2002). Increased plasma fibrinogen concentrations are found in the blood during massive internal hemorrhage or tissue damage (Morris and Johnston, 2002). Normal values in cattle range from 100 to 600 mg/dL (The Merck Veterinary Manual, 1997; Morris and Johnston, 2002). In this study fibrinogen values ranged from 25 to 629 mg/dL, which is only marginally outside the normal range. Treatment tended (P = 0.12) to affect fibrinogen concentrations in the 3N analysis (Table 3-7), with means of 156.50 2 , 162.10 14.26, and 66.23 for CTRL, ST-1, and ST-2 cows, respectively. With increasing fecal [H + ], fibrinogen concentrations tended (P < 0.01) to decrease in analyses that excluded cow 2245 (Table 3-5). Minimum fibrinogen concentrations were affected by the interaction of treatment x day in analyses 2N, 3Y, and 3N (Tables 3-9 to 3-11) . Like Cp, fibrinogen minimum concentrations decreased on days 3 and 4 in ST cows, while increasing in CTRL cows in the 2Y and 2N analyses (Tables 3-4 and 3-5). Cows in the ST-2 group had elevated concentrations of fibrinogen over ST-1 cows on days 2, 3, and 4 in the 3Y analysis (Table 3-11). Only the fibrinogen concentrations of cow 2245 exceeded the normal range of fibrinogen in cattle, and only at one collection (Figure A-9). This occurred at hour 62, 30 h before she was euthanized. After hour 62, fibrinogen concentrations in this cow 2 Values reported without standard error terms were calculated manually from the least squares means of higher order interactions.

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46 declined. Because fibrinogen functions in blood clotting, it may have been depleted in animals that had gut damage (as evidenced by tissue and blood clots in the feces), and thus be lower than expected. Alpha acid glycoprotein Alpha-acid glycoprotein (AGP) is an acute phase protein elevated in cows experiencing inflammatory response. The normal range is from 200 to 460 g/mL. In this study, only cow 2245 exceeded the normal range (Figure A-10). Starch-treated animals had lower (P = 0.13) daily maximum AGP concentrations in the 2N analysis than CTRL animals. Differences in AGP concentrations due to treatment, treatment x time interactions, and fecal [H + ] were not detected in any other analysis. Other Measurements Liver panel enzymes Among the enzymes measured in a standard liver panel, AST is most likely to show an increase in animals experiencing gastrointestinal distress, as it is a indicator of tissue damage. AP, although normally considered indicative of liver damage, is also released from the gastrointestinal mucosa (The Merck Veterinary Manual, 1997). Albumin is expected to decrease during gut damage due to a protein losing enteropathy. The remaining liver panel enzymes are not normally considered indicators of gut damage (Carlson, 2002). Of these enzymes, only AP tended to increase in ST animals on day 2 (Tables 3-16 through 3-21).

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47 Complete blood count Selected blood samples from hour 66 (day 3) were analyzed for red and white blood cells (Table 3-22). The white blood cell count was abnormally low (2.22 k/L) in cow 5329, a ST cow. Ruminal pH Ruminal pH remained near neutrality ( x = 6.75 0.07) throughout the trial. This was expected as treatments were infused post-ruminally. Conclusions Post-ruminal starch infusion had a significant impact on fecal pH, fecal characteristics, respiration rate, rectal temperature and on fibrinogen levels. Fecal pH declined dramatically in ST animals, likely as a result of bacterial fermentation of the infused starch in the hindgut. Coincident with declines in fecal pH were changes in fecal consistency, with feces becoming softer, lighter in color, and containing evidence of gut damage (sloughed tissue, mucus, and blood). The evidence for an inflammatory response was less clear. Ceruloplasmin showed an increase in minimum (baseline) concentrations on days 3 and 4 in ST animals. Haptoglobin concentrations were not affected by treatment. Although treatment affected fibrinogen concentrations in the 3N analysis, the results were difficult to interpret as the mean of the control group was intermediate between that of the fast recoverers (ST-1) and the slow recoverers (ST-2). This may have been due to the use of fibrinogen by the damaged gut in the ST-2 group. However, with one exception, the concentrations of fibrinogen did not leave the normal range for fibrinogen in cattle. The AGP daily maxima were decreased in ST animals in one analysis, but differences were not detected

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48 in other analyses. Acute phase protein concentrations, except for those of cow 2245, rarely left the normal range (with the possible exception of haptoglobin which was detectable (i.e., not normal) in all cows). This may be due to the characteristic immunological hyporesponsiveness of the gastrointestinal tract (Matsuura and Fiocci, 1993; Shao et al., 2001). Fagliari et al. (1998) observed an increase in Hp, Cp, fibrinogen, and AGP in ponies between 4 and 28 hours after administration of 16 g/kg BW of starch. The difference in response to starch infusion between these horses and the cattle in the current study may be due to differences in the immune response within the gastrointestinal tract between horses and cattle. Also, the horses in the study of Fagliari et al. were dosed with a higher amount of starch per unit body weight (15g/kg BW in the ponies (administered in a single dose) versus 5 g/kg BW per day infusion in the cattle in the current study). Indices of time, which included day, collection, and the day x collection interaction, were significant for all dependent variables in at least one analysis. There were changes throughout the trial which may have been related to stress in the new environment as well as due to treatment. The cows were moved into the tie-stall barn three days before the initiation of collections, which may have stressed these animals that were not accustomed to indoor confinement housing. Respiration rate was increased in all animals at the beginning of the trial, and the acute phase proteins ceruloplasmin and haptoglobin were highest on day 2, before the significant declines in fecal pH and evidence of gut damage occurred. The liver panel enzymes failed to show conclusive evidence of gut damage. However, only samples from days 1 and 2 (through hour 43) were analyzed, and evidence of

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49 damage to the gut began to be detected in fecal samples at hours 31-39. Thus, the liver panel may not have included the time of maximum gut damage. One notable observation is the difference in response among the four ST cows. Two cows became severely ill. One of these cows needed to be euthanized, and another required medical attention. The surviving cow returned to a normal fecal pH more slowly than either of the other ST cows. The decline in fecal pH in the severely affected cows (ST-2) occurred later than in the less affected cows (ST-1). This may have been due to a higher rate of passage in the ST-1 cows, allowing less time for bacterial fermentation, and hence, lowered pH. This may explain why these cows did not become as ill as the ST-2 cows. The difference between the two groups was also evident in the fecal samples. The manure of CTRL cows was a solid dark brown color throughout the trial. The manure of ST-1 cows changed from the dark brown color to a light brown color. The manure of ST2 cows changed to a golden yellow color (Figure A-11). These differences in response speak to individual animal variation and the observations in other studies that animals receiving similar treatments responded differently. In 1977 Garner et al. infused a cornstarch and woodflour gruel into 31 horses via stomach tube. In this study, five horses died, 21 developed lameness, and five did not develop lameness. The factors that allow animals treated similarly to show disparate responses are unknown, but may be related to the rate of passage of material through the gut, the presence of healthy symbiotic bacterial colonies in the gut, the ability to digest and absorb carbohydrates rapidly, and the immune response of the animal. Although there were a limited number of animals on this study and a large amount of variation among animals, there is evidence to support the fact that post-ruminal starch

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50 infusions influence fecal pH and animal health, and may alter indices of the inflammatory response. The results of this study begin to explain how some symptoms of ruminal acidosis (i.e., diarrhea, foamy feces, and mucin casts). These results also show distinct variation in how animals respond to this dietary challenge. This study offers the basis to further explore the effects of diet on animal health.

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51 Table 3-1. Composition of TMR and intake Composition % of Diet DM Sorghum silage 70.8 Corn silage 6.1 Alfalfa Hay 3.3 Cottonseed hulls 1.4 Citrus pulp 2.6 Prolak 0.6 Corn meal 6.1 Soybean meal 2.7 Whole cottonseed 3.8 Mineral mix 1.1 Chemical Composition % of Diet DM Intake (kg/d) DM 29.6 (% of fed diet) 6.24 CP 11.7 0.74 NDF 54.5 3.42 Starch 15.0 0.93 Ash 6.0 0.38 Table 3-2. Error terms used for certain terms the model 1 Model Term Associated Error Term Treatment Cow (treatment) Day Cow (treatment) x day Treatment x day Cow (treatment) x day Collection Cow (treatment) x collection Collection x day Cow (treatment) x collection 1 Model terms not listed utilized the residual error term

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52 Table 3-3. Fecal characteristics by hour from start of infusion for four treated cows 1 Hour 2245 2686 2811 5329 12 Mucus Loose feces 16 Foamy diarrhea; tissue 20 Foamy diarrhea Foamy diarrhea Foamy diarrhea 24 Mucus; foamy Tissue; foamy Tissue 39 Watery diarrhea; tissue 43 Diarrhea 47 Tissue , mucus; pasty Mucin casts Mucin casts Pasty 58 Watery diarrhea 62 Watery diarrhea Pasty 66 Transfaunation from cow 2521, banamine injection 77 (Euthanized) 1 Control cows did not exhibit these symptoms.

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53 Table 3-4. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II, containing all data, 2 treatment descriptors, and including cow 2245 All 2Y Hematocrit Fecal [H + ] RR RT Cp Hp FB AGP Model I P-values trt 0.0202 0.0472 0.2805 0.2922 0.5625 0.8846 0.7896 0.9948 day 0.2996 0.1015 0.0007 0.0016 0.0244 0.2272 0.0247 1.0000 day*trt 0.7846 0.0988 0.3889 0.8956 0.7343 0.7760 0.2933 1.0000 coll 0.6266 0.1119 0.0321 0.0014 0.0272 0.8119 0.4430 0.5657 trt*coll 0.9550 0.1028 0.7898 0.4384 0.8015 0.2246 0.9714 0.6773 day*coll 0.0002 0.5991 0.0070 0.0103 0.0001 0.1232 0.0011 0.9147 day*trt*coll 0.3902 0.6228 0.6952 0.9938 0.9035 0.4277 0.7212 0.1619 Treatment % [H + ] respirations/ min C mg/dL mg HbB/dL mg/dL 0 33.4537 1.09E-07 1.95E-06 38.29 3.08 38.50 0.16 34.6 14.57 158.67 324.42 1 29.4782 6.87E-06 1.38E-06 43.00 2.18 38.74 0.12 38.13 3.42 16.34 6.01 193.25 56.50 416.45 7703.47 Day * Treatment 1 * 0 34.60 1.64E-07 3.49E-06 47.50 3.89 38.45 0.18 31.55 4.96 12.82 8.72 151.11 81.70 270.33 11268.00 1 * 1 33.41 1.18E-07 2.46E-06 50.75 2.75 38.68 0.13 36.63 3.51 15.42 6.17 181.03 57.77 360.75 7967.73 2 * 0 32.11 8.87E-08 3.47E-06 36.75 3.89 38.46 0.18 36.08 4.97 17.91 8.75 112.73 82.05 335.88 11268.00 2 * 1 33.70 2.87E-06 2.45E-06 38.29 2.75 38.64 0.13 39.75 3.51 18.29 6.17 183.33 57.85 418.24 7967.73 3 * 0 30.24 7.86E-08 3.47E-06 34.50 3.89 38.34 0.18 35.94 4.97 15.71 8.75 179.41 82.05 359.58 11268.00 3 * 1 28.14 1.00E-05 2.45E-06 38.83 2.75 38.64 0.13 38.49 3.52 15.50 6.18 181.92 57.95 424.35 7967.73 4 * 0 28.25 1.06E-07 3.45E-06 34.42 3.89 38.75 0.18 34.81 11.82 191.42 331.89 4 * 1 30.98 1.40E-05 2.45E-06 44.13 2.79 39.01 0.13 37.66 3.52 16.16 6.18 226.70 57.95 462.45 7967.73 Model II P-values [H + ] ——— ——— 0.0245 0.1235 0.4537 0.4021 0.9924 0.4248 day ——— 0.0335 0.0001 0.0008 0.0288 0.116 0.1625 0.2673 coll ——— 0.0273 0.0073 0.0015 0.0960 0.8984 0.6983 0.3721 day*coll ——— 0.0830 0.0047 0.0085 0.0001 0.0921 0.0006 0.6584 Direction of change with [H + ] ——— ——— + + + + 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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Table 3-5. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II containing all data, 2 treatment descriptors, and excluding cow 2245 All 2 N Hematocrit Fecal [ H + ] RR RT Cp Hp FB AGP Model I P val u es trt 0.0321 0.1022 0.3865 0.3281 0.8518 0.2773 0.4698 0.1594 day 0.1779 0.2677 0.0028 0.0043 0.0793 0.1307 0.0420 0.3857 day*trt 0.9246 0.2612 0.6048 0.7277 0.4864 0.4902 0.1221 0.1816 coll 0.4523 0.1218 0.0856 0.0097 0.0210 0.8116 0.1295 0.2415 trt*coll 0.8303 0.1125 0.9361 0.7039 0.7406 0.1848 0.9809 0.1748 day*coll 0.0002 0.6793 0.0072 0.0248 0.0001 0.2884 0.0016 0.4272 day*trt*coll 0.2854 0.6977 0.6370 0.9797 0.3275 0.1754 0.6489 0.0528 Treatment % [H + ] respirations/ min C mg/dL mg HbB/dL mg/dL g/dL 0 33.4695 1.09E-07 2.01E-06 38.29 3.55 38.50 0.08 34.6 14.5 158.3 4 .29 1 29.8092 6.14E-06 1.64E-06 42.93 2.90 38.62 0.07 34.13 0.88 9.69 2.29 133.07 25.75 5 .03 16.97 Day * Treatment 1 * 0 34.60 1.64E-07 3.52E-06 47.50 4.32 38.45 0.11 31.55 1.51 12.82 3.15 151.11 34.74 0 .33 29.47 1 * 1 33.46 1.35E-07 2.84E-06 51.11 3.52 38.54 0.09 33.39 1.23 10.47 2.57 129.04 28.37 1 .33 24.06 2 * 0 32.10 8.85E-08 3.49E-06 36.75 4.32 38.46 0.11 36.06 1.54 17.81 3.21 112.02 35.34 5 .59 29.76 2 * 1 33.71 3.29E-06 2.85E-06 38.83 3.52 38.49 0.09 35.41 1.24 10.81 2.60 126.01 28.61 8 .90 24.18 3 * 0 31.18 7.84E-08 3.49E-06 34.50 4.32 38.34 0.11 35.94 1.54 15.61 3.21 179.21 35.34 9 .57 29.97 3 * 1 29.26 9.51E-06 2.87E-06 38.50 3.52 38.55 0.09 34.23 1.26 9.58 2.63 127.50 28.89 4 .03 24.71 4 * 0 29.14 1.06E-07 3.48E-06 34.42 4.32 38.75 0.11 34.84 11.76 190.88 1 .65 4 * 1 29.68 1.20E-05 2.86E-06 43.28 3.52 38.90 0.09 33.49 1.26 7.90 2.63 149.74 28.89 285.86 24.35 Model I I P val u es [H + ] — — — --0.2691 0.2074 0.2825 0.8185 0.0051 0.8731 day — — — 0.1797 0.0008 0.0021 0.1275 0.1339 0.0314 0.6873 coll — — — 0.0962 0.0406 0.0125 0.0477 0.9941 0.2534 0.4657 day*coll — — — 0.2487 0.0084 0.0221 0.0001 0.4643 0.0007 0.7002 Direction of change with [H + ] ——— --+ + + 54 1 Coll = collection; RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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55 Table 3-6. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II, containing all data, 3 treatment descriptors, and including cow 2245 All 3Y Hematocrit Fecal [H + ] RR RT Cp Hp FB AGP Model I P-values trt 0.1272 0.0037 0.5959 0.1049 0.3625 0.6506 0.8709 0.4729 day 0.0590 0.0001 0.0015 0.0027 0.0237 0.2162 0.0393 0.3008 day*trt 0.1518 0.0001 0.6141 0.4506 0.2812 0.4443 0.5010 0.5578 coll 0.4358 0.0050 0.0171 0.0015 0.0277 0.7539 0.1946 0.2524 trt*coll 0.2582 0.0438 0.6264 0.7375 0.7244 0.1546 0.3493 0.2458 day*coll 0.0001 0.0012 0.0045 0.0076 0.0001 0.0351 0.0118 0.3892 day*trt*coll 0.1514 0.0008 0.7380 0.9402 0.8132 0.4094 0.9501 0.0710 Treatment % [H + ] respirations/min C mg/dL mgHbB/dL mg/dL g/dL 0 33.45 1.10E-07 0.00E+00 38.29 3.55 38.50 0.10 34.59 14.55 158.21 324.34 1 29.45 4.07E-06 0.00E+00 42.69 3.55 38.55 0.10 33.87 4.15 10.43 8.51 162.10 89.79 266.81 161.85 2 29.71 9.61E-06 0.00E+00 43.48 3.56 38.93 0.10 42.79 4.16 22.31 8.52 222.37 89.92 566.53 161.90 Day * Treatment 1 * 0 34.60 1.66E-07 1.12E-06 47.50 4.31 38.45 0.13 31.55 4.28 12.82 8.70 151.11 91.58 270.33 168.60 1 * 1 33.42 1.27E-07 1.06E-06 52.00 4.31 38.45 0.13 33.91 4.28 11.12 8.70 157.00 91.58 277.92 168.60 1 * 2 32.09 -6.73E-08 1.16E-06 49.50 4.31 38.91 0.13 39.36 4.28 19.73 8.70 205.06 91.58 443.58 168.60 2 * 0 33.69 8.93E-08 1.09E-06 36.75 4.31 38.46 0.13 6 .10 4.29 17.87 8.73 111.73 91.92 335.71 168.75 2 * 1 31.38 3.65E-06 1.09E-06 37.58 4.31 38.37 0.13 35.85 4.28 12.64 8.70 142.65 91.58 271.17 168.60 2 * 2 29.45 2.33E-06 1.09E-06 39.00 4.31 38.91 0.13 43.73 4.29 24.32 8.73 221.78 91.92 573.21 168.75 3 * 0 27.70 7.89E-08 1.09E-06 34.50 4.31 38.34 0.13 35.93 4.29 15.66 8.73 179.29 91.92 359.57 168.86 3 * 1 29.31 8.14E-06 1.09E-06 38.75 4.31 38.43 0.13 33.30 4.28 10.12 8.70 161.19 91.58 254.62 168.75 3 * 2 29.10 1.20E-05 1.11E-06 38.92 4.31 38.85 0.13 44.50 4.31 9 .79 8.77 191.75 92.31 597.42 168.97 4 * 0 26.96 1.06E-07 1.06E-06 34.42 4.31 38.75 0.13 34.78 11.85 190.71 331.74 4 * 1 28.57 4.38E-06 1.12E-06 42.42 4.31 38.95 0.13 32.43 4.28 7.83 8.70 187.55 91.58 263.55 168.60 4 * 2 33.45 2.40E-05 1.09E-0 46.49 4.46 39.07 0.14 43.56 4.31 25.38 8.77 270.88 92.31 651.89 168.97

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Table 3-6. Continued 56 All 3Y Hematocrit Fecal [H + ] RR RT Cp Hp FB AGP Model II P-values [H+] — —— ——— 0.0245 0.1235 0.4537 0.4021 0.9924 0.4248 day — —— 0.0335 0.0001 0.0008 0.0288 0.1160 0.1625 0.2673 coll — —— 0.0273 0.0073 0.0015 0.0960 0.8984 0.6983 0.3721 day*coll — —— 0.0830 0.0047 0.0085 0.0001 0.0921 0.0006 0.6584 Direction of change with [H + ] — —— ——— + + + + 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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57 Table 3-7. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for models I and II, containing all data, 3 treatment descriptors, and excluding cow 2245 All 3N Hematocrit Fecal [H + ] RR RT Cp Hp FB AGP Model I P-values trt 0.1258 0.0283 0.7424 0.1421 0.9214 0.5848 0.1219 0.3901 day 0.2539 0.0007 0.0180 0.0242 0.0829 0.4835 0.3078 0.6589 day*trt 0.4679 0.0019 0.8336 0.3405 0.2919 0.6516 0.3219 0.4783 coll 0.6307 0.0144 0.1110 0.0292 0.0799 0.9873 0.3064 0.2068 trt*coll 0.6079 0.1092 0.6371 0.8917 0.7571 0.5487 0.9960 0.5159 day*coll 0.0002 0.0067 0.0003 0.0876 0.0009 0.5242 0.0356 0.9073 day*trt*coll 0.0760 0.0016 0.0725 0.7891 0.6624 0.7607 0.9232 0.3100 Treatment % [H + ] respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 33.4644 1.10E-07 0.00E+00 38.29 4.34 38.50 0.05 34.58 14.6 156.5 324 1 29.4524 4.05E-06 0.00E+00 42.69 4.34 38.55 0.05 33.87 1.26 10.43 3.28 162.10 14.26 266.75 23.16 2 30.6912 9.86E-06 43.42 6.14 38.77 0.06 35.03 8.27 66.23 295.2 Day * Treatment 1 * 0 34.60 1.66E-07 1.30E-06 47.50 5.08 38.45 0.09 31.55 1.54 12.82 3.61 151.11 20.46 270.33 32.63 1 * 1 33.46 1.27E-07 1.25E-06 52.00 5.08 38.45 0.09 33.91 1.54 11.12 3.61 157.00 20.46 277.92 32.63 1 * 2 32.09 1.49E-07 1.77E-06 49.33 7.19 38.71 0.13 32.36 2.17 9.18 5.10 73.13 28.94 288.17 46.15 2 * 0 33.70 8.95E-08 1.27E-06 36.75 5.08 38.46 0.09 36.10 1.57 17.77 3.67 111.48 21.64 335.60 32.94 2 * 1 31.38 3.66E-06 1.27E-06 37.58 5.08 38.37 0.09 35.85 1.54 12.64 3.61 142.65 20.46 271.17 32.63 2 * 2 29.45 2.37E-06 41.33 7.19 38.73 0.13 35.17 6.8 84.88 271 3 * 0 27.70 7.92E-08 1.27E-06 34.50 5.08 38.34 0.09 35.93 1.57 15.60 3.67 179.10 21.64 359.58 33.16 3 * 1 29.31 8.07E-06 1.27E-06 38.75 5.08 38.43 0.09 33.30 1.54 10.12 3.61 161.19 20.46 254.39 32.94 3 * 2 30.80 1.14E-05 38.00 7.19 38.81 0.13 36.35 7.98 53.55 298.6 4 * 0 29.31 1.06E-07 1.25E-06 34.42 5.08 38.75 0.09 34.77 11.72 189.8 331.7 4 * 1 31.75 4.34E-06 1.30E-06 42.42 5.08 38.95 0.09 32.43 1.54 7.83 3.61 187.55 20.46 263.55 32.63 4 * 2 30.88 2.50E-05 1.77E-06 45.00 7.19 38.82 0.13 37.54 9.05 45.25 332.6

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Table 3-7. Continued 58 All 3N Hematocrit Fecal [H + ] RR RT Cp Hp FB AGP Model II P-values [H + ] ——— ——— 0.2691 0.2074 0.2825 0.8185 0.0051 0.8731 day ——— 0.1797 0.0008 0.0021 0.1275 0.1339 0.0314 0.6873 coll ——— 0.0962 0.0406 0.0125 0.0477 0.9941 0.2534 0.4657 day*coll ——— 0.2487 0.0084 0.0221 0.0001 0.4643 0.0007 0.7002 Direction of change with [H+] ——— ——— + + + 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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59 Table 3-8. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 2 treatment descriptors, and including cow 2245 MIN 2Y Fecal [H + ] RR RT Cp Hp FB AGP Model P-values trt 0.0178 0.2751 0.2071 0.5769 0.7016 0.8676 0.6855 day 0.1415 0.0001 0.0992 0.0365 0.8654 0.2910 0.2154 day*trt 0.1337 0.2305 0.3900 0.3598 0.6706 0.1720 0.9897 Treatment [H + ] respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 1.75E-07 2.26E-06 27.13 2.06 38.10 0.11 31.15 4.33 7.84 6.04 106.70 62.02 276.97 141.40 1 1.10E-05 1.60E-06 30.31 1.46 38.31 0.08 34.36 3.06 10.88 4.27 120.20 43.86 352.44 99.98 Day * Treatment 1 * 0 2.87E-07 4.51E-06 37.00 2.50 38.31 0.16 27.13 4.54 9.20 6.66 103.71 64.26 219.50 149.46 1 * 1 1.74E-07 3.19E-06 37.50 1.77 38.42 0.11 32.59 3.21 10.10 4.71 124.01 45.44 279.00 105.69 2 * 0 1.38E-07 4.51E-06 23.50 2.50 38.06 0.16 31.36 4.54 8.30 6.66 66.66 64.26 282.50 149.46 2 * 1 9.26E-06 3.19E-06 27.25 1.77 38.21 0.11 35.58 3.21 12.48 4.71 121.45 45.44 360.00 105.69 3 * 0 1.13E-07 4.51E-06 25.50 2.50 38.03 0.16 32.50 4.54 7.80 6.66 116.52 64.26 307.20 149.46 3 * 1 1.50E-05 3.19E-06 27.50 1.77 38.14 0.11 35.11 3.21 8.30 4.71 120.77 45.44 380.29 105.69 4 * 0 1.62E-07 4.51E-06 22.50 2.50 38.03 0.16 33.60 4.54 6.05 6.66 139.90 64.26 298.67 149.46 4 * 1 1.90E-05 3.19E-06 29.00 1.77 38.47 0.11 34.16 3.21 12.65 4.71 114.55 45.44 390.45 105.69 Contrasts P-values t0 vs. t1 x d1 vs. d2,3,4 0.0428 0.1480 0.4955 0.2090 0.5444 0.7471 0.7923 t0 vs. t1 x d2 vs. d3,4 0.2700 0.8422 0.5200 0.2923 0.8998 0.0445 0.9540 t0 vs. t1 x d3 vs. d4 0.5864 0.1388 0.1518 0.4721 0.2983 0.3962 0.8503 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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60 Table 3-9. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 2 treatment descriptors, and excluding cow 2245 MIN 2N Fecal [H + ] RR RT Cp Hp FB AGP Model P-values trt 0.0435 0.4379 0.3310 0.9175 0.4234 0.2893 0.3144 day 0.3041 0.0002 0.2061 0.0777 0.4448 0.2888 0.1181 day*trt 0.2897 0.3250 0.4637 0.4234 0.9206 0.0105 0.3730 Treatment [H + ] respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 1.75E-07 2.30E-06 27.13 1.84 38.10 0.10 31.15 1.33 7.84 1.48 106.70 21.06 276.96 24.83 1 1.00E-05 1.88E-06 29.25 1.51 38.25 0.08 30.95 1.08 6.07 1.21 71.79 17.19 238.33 20.27 Day * Treatment 1 * 0 2.87E-07 4.57E-06 37.00 2.44 38.31 0.15 27.13 1.97 9.20 1.99 103.71 23.97 219.50 31.88 1 * 1 2.04E-07 3.73E-06 36.00 1.99 38.33 0.12 29.25 1.61 7.03 1.62 84.35 19.57 228.33 26.03 2 * 0 1.38E-07 4.57E-06 23.50 2.44 38.06 0.15 31.36 1.97 8.30 1.99 66.66 23.97 282.50 31.88 2 * 1 1.00E-05 3.73E-06 26.33 1.99 38.11 0.12 31.74 1.61 5.93 1.62 80.94 19.57 232.00 26.03 3 * 0 1.13E-07 4.57E-06 25.50 2.44 38.03 0.15 32.50 1.97 7.80 1.99 116.52 23.97 307.19 31.88 3 * 1 1.40E-05 3.73E-06 26.67 1.99 38.15 0.12 32.42 1.61 5.87 1.62 74.40 19.57 243.72 26.03 4 * 0 1.62E-07 4.57E-06 22.50 2.44 38.03 0.15 33.60 1.97 6.05 1.99 139.90 23.97 298.66 31.88 4 * 1 1.60E-05 3.73E-06 28.00 1.99 38.41 0.12 30.40 1.61 5.43 1.62 47.48 19.57 249.25 26.03 Contrasts P-values t0 vs. t1 x d1 vs. d2,3,4 0.0793 0.1639 0.4334 0.2509 0.8221 0.3207 0.0991 t0 vs. t1 x d2 vs. d3,4 0.5445 0.8676 0.3645 0.4688 0.6624 0.0036 0.8743 t0 vs. t1 x d3 vs. d4 0.8015 0.2302 0.2988 0.3386 0.6485 0.0669 0.7465 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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61 Table 3-10. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 3 treatment descriptors, and including cow 2245 MIN 3Y Fecal [H] + RR RT Cp Hp FB AGP Model P-values trt 0.0055 0.5129 0.0464 0.3157 0.5587 0.8200 0.4217 day 0.0002 0.0001 0.0646 0.0394 0.7366 0.5049 0.0883 day*trt 0.0004 0.4250 0.6693 0.2038 0.7145 0.0878 0.3204 Treatment [H] + respirations/min C mg/dL mg HbB/dL mg/dL 0 1.75E-07 0.00E+00 27.13 2.25 38.10 0.06 31.15 3.56 7.84 5.86 106.70 67.30 276.97 125.31 1 7.88E-06 0.00E+00 29.38 2.25 38.15 0.06 30.06 3.56 6.26 5.86 90.19 67.30 2 1.40E-05 0.00E+00 31.25 2.25 38.47 0.06 38.66 3.56 15.50 5.86 150.20 67.30 480.62 125.31 Day * Treatment 1 * 0 2.87E-07 2.01E-06 37.00 2.69 38.31 0.12 27.13 3.76 9.20 6.54 103.71 68.77 219.50 131.72 1 * 1 1.83E-07 2.01E-06 36.00 2.69 38.22 0.12 29.37 3.76 6.50 6.54 112.57 68.77 1 * 2 1.64E-07 2.01E-06 39.00 2.69 38.61 0.12 35.82 3.76 13.70 6.54 135.44 68.77 329.00 131.72 2 * 0 1.38E-07 2.01E-06 23.50 2.69 38.06 0.12 31.36 3.76 8.30 6.54 66.66 68.77 2 * 1 1.10E-05 2.01E-06 25.50 2.69 38.11 0.12 31.91 3.76 6.65 6.54 96.95 68.77 227.00 131.72 2 * 2 7.37E-06 2.01E-06 29.00 2.69 38.31 0.12 39.25 3.76 18.30 6.54 145.95 68.77 3 * 0 1.13E-07 2.01E-06 25.50 2.69 g/dL 224.25 125.31 229.00 131.72 282.50 131.72 493.00 131.72 38.03 0.12 32.50 3.76 7.80 6.54 116.52 68.77 307.20 131.72 3 * 1 1.20E-05 2.01E-06 28.00 2.69 37.97 0.12 31.25 3.76 6.00 6.54 92.69 68.77 220.59 131.72 3 * 2 1.80E-05 2.01E-06 27.00 2.69 38.31 0.12 38.98 3.76 10.60 6.54 148.85 68.77 540.00 131.72 4 * 0 1.62E-07 2.01E-06 22.50 2.69 38.03 0.12 33.60 3.76 6.05 6.54 139.90 68.77 298.67 131.72 4 * 1 8.00E-06 2.01E-06 28.00 2.69 38.31 0.12 27.72 3.76 5.90 6.54 58.55 68.77 220.40 131.72 4 * 2 3.10E-05 2.01E-06 30.00 2.69 38.64 0.12 40.60 3.76 19.40 6.54 170.56 68.77 560.50 131.72

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Table 3-10. Continued 62 MIN 3Y Fecal [H + ] RR RT Cp Hp FB AGP Contrasts P-values A. t0 vs. t1,2 0.0032 0.3316 0.0728 0.5139 0.7001 0.8803 0.6566 B. t1 vs. t2 0.0238 0.5975 0.0371 0.1857 0.3463 0.5731 0.2438 C. d1 vs. d2,3,4 0.0001 0.0001 0.0506 0.0061 0.9691 0.8544 0.0174 D. d2 vs. d3,4 0.0052 0.5066 0.5395 0.9496 0.4690 0.1547 0.4923 E. d3 vs. d4 0.1050 1.0000 0.0545 0.8202 0.4192 0.7910 0.9205 Contrast A x C 0.0007 0.1711 0.4913 0.1676 0.5614 0.7047 0.7554 Contrast A x D 0.0291 0.8492 0.5157 0.2445 0.9038 0.0260 0.9455 Contrast AxE 0.2417 0.1616 0.1527 0.4230 0.3208 0.3241 0.8231 Contrast B x C 0.0360 0.6029 0.6248 0.2451 0.6315 0.0971 0.0235 Contrast B x D 0.0006 0.3359 0.5313 0.2559 0.6648 0.2475 0.4525 Contrast B x E 0.0021 0.4015 1.0000 0.1018 0.2170 0.1224 0.8303 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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63 Table 3-11. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily minima, using 3 treatment descriptors, and excluding cow 2245 MIN 3N Fecal [H + ] RR RT Cp Hp FB AGP Model P-values trt 0.0337 0.7865 0.1349 0.5430 0.7647 0.1529 0.4570 day 0.0045 0.0030 0.1340 0.0371 0.5625 0.7771 0.1618 day*trt 0.0139 0.5551 0.4071 0.1255 0.9627 0.0686 0.4056 Treatment [H + ] respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 1.75E-07 1.15E-06 27.13 2.25 38.10 0.06 31.15 1.20 7.84 1.80 106.70 12.55 276.97 25.05 1 7.88E-06 1.15E-06 29.38 2.25 38.15 0.06 30.06 1.20 6.26 1.80 90.19 12.55 224.25 25.05 2 1.50E-05 1.63E-06 29.00 3.19 38.44 0.08 32.74 1.70 5.68 2.55 35.00 17.75 266.48 35.42 Day * Treatment 1 * 0 2.87E-07 2.30E-06 37.00 2.83 38.31 0.11 27.13 1.61 9.20 2.35 103.71 17.20 219.50 31.66 1 * 1 1.83E-07 2.30E-06 36.00 2.83 38.22 0.11 29.37 1.61 6.50 2.35 112.57 17.20 229.00 31.66 1 * 2 2.46E-07 3.26E-06 36.00 4.00 38.56 0.16 29.02 2.28 8.10 3.33 27.90 24.33 227.00 44.77 2 * 0 1.38E-07 2.30E-06 23.50 2.83 38.06 0.11 31.36 1.61 8.30 2.35 66.66 17.20 282.50 31.66 2 * 1 1.10E-05 2.30E-06 25.50 2.83 38.11 0.11 31.91 1.61 6.65 2.35 96.95 17.20 227.00 31.66 2 * 2 9.12E-06 3.26E-06 28.00 4.00 38.11 0.16 31.39 2.28 4.50 3.33 48.93 24.33 242.00 44.77 3 * 0 1.13E-07 2.30E-06 25.50 2.83 38.03 0.11 32.50 1.61 7.80 2.35 116.53 17.20 307.20 31.66 3 * 1 1.20E-05 2.30E-06 28.00 2.83 37.97 0.11 31.25 1.61 6.00 2.35 92.69 17.20 220.59 31.66 3 * 2 1.70E-05 3.26E-06 24.00 4.00 38.50 0.16 34.77 2.28 5.60 3.33 37.84 24.33 290.00 44.77 4 * 0 1.62E-07 2.30E-06 22.50 2.83 38.03 0.11 33.60 1.61 6.05 2.35 139.90 17.20 298.67 31.66 4 * 1 8.00E-06 2.30E-06 28.00 2.83 38.31 0.11 27.72 1.61 5.90 2.35 58.55 17.20 220.40 31.66 4 * 2 3.20E-05 3.26E-06 28.00 4.00 38.61 0.16 35.78 2.28 4.50 3.33 25.34 24.33 306.94 44.77

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Table 3-11. Continued 64 MIN 3N Fecal [H + ] RR RT Cp Hp FB AGP Contrasts P-values A. t0 vs. t1,2 0.0181 0.5605 0.1219 0.8873 0.5150 0.1173 0.4409 B. t1 vs. t2 0.0744 0.9322 0.0969 0.3257 0.8680 0.1264 0.4329 C. d1 vs. d2,3,4 0.0015 0.0005 0.0995 0.0079 0.2329 0.6198 0.0547 D. d2 vs. d3,4 0.0405 0.8430 0.1603 0.3418 0.7345 0.5171 0.2461 E. d3 vs. d4 0.1468 0.8637 0.2147 0.7010 0.5734 0.5670 0.9008 Contrast A x C 0.0053 0.2244 0.4247 0.2511 0.9881 0.5200 0.1685 Contrast A x D 0.0966 0.9402 0.2005 0.6487 0.6631 0.0104 0.9041 Contrast A x E 0.2606 0.2245 0.3347 0.3504 0.7377 0.1157 0.7001 Contrast B x C 0.0951 0.9033 0.8144 0.1565 0.4370 0.1978 0.2352 Contrast B x D 0.0138 0.3238 0.1336 0.0541 0.7480 0.8942 0.2325 Contrast B x E 0.0145 0.4397 0.4537 0.1874 0.8235 0.5394 0.7651 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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65 Table 3-12. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 2 treatment descriptors, and including cow 2245 MAX 2Y Fecal [H + ] RR RT Cp Hp FB AGP Model P-values trt 0.1437 0.2621 0.4488 0.5654 0.9212 0.7479 0.6761 day 0.1953 0.2314 0.0010 0.0393 0.2419 0.2426 0.5673 day*trt 0.1968 0.7371 0.6519 0.9819 0.8374 0.7108 0.9108 Treatment respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 5.83E-08 1.45E-06 49.75 6.65 38.97 0.21 38.52 5.46 21.63 11.22 228.60 114.45 372.66 208.79 1 3.28E-06 0.00E+00 60.38 4.70 39.18 0.15 42.71 3.86 23.07 7.93 276.88 80.93 487.70 147.64 Day * Treatment 1 * 0 7.09E-08 2.61E-06 56.00 8.82 38.72 0.29 36.30 5.79 17.50 12.17 233.45 126.60 326.00 220.20 1 * 1 7.17E-08 1.85E-06 68.50 6.24 38.97 0.21 40.22 4.10 19.48 8.61 229.10 89.52 414.25 155.71 2 * 0 4.92E-08 2.61E-06 48.00 8.82 38.86 0.29 42.18 5.79 23.40 12.17 139.49 126.60 385.00 220.20 2 * 1 5.85E-08 1.85E-06 52.50 6.24 38.94 0.21 46.75 4.10 29.45 8.61 260.08 89.52 489.75 155.71 3 * 0 3.84E-08 2.61E-06 48.00 8.82 38.53 0.29 38.31 5.79 26.70 12.17 249.89 126.60 403.50 220.20 3 * 1 4.23E-06 1.85E-06 56.50 6.24 39.03 0.21 41.72 4.10 24.36 8.61 286.14 89.52 494.37 155.71 4 * 0 7.46E-08 2.61E-06 47.00 8.82 39.78 0.29 37.31 5.79 18.90 12.17 291.57 126.60 376.14 220.20 4 * 1 8.77E-06 1.85E-06 64.00 6.24 39.79 0.21 42.14 4.10 19.00 8.61 332.21 89.52 552.43 155.71 Contrasts P-values t0 vs. t1 x d1 vs. d2,3,4 0.2490 0.7962 0.8813 0.9141 0.9286 0.4425 0.7599 t0 vs. t1 x d2 vs. d3,4 0.1129 0.4274 0.6327 0.8958 0.3971 0.3980 0.8160 t0 vs. t1 x d3 vs. d4 0.3207 0.4776 0.2573 0.7180 0.8004 0.9683 0.5529 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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66 Table 3-13. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 2 treatment descriptors, and excluding cow 2245 MAX 2N Fecal [H + ] RR RT Cp Hp FB AGP Model P-values trt 0.2589 0.2354 0.7449 0.9185 0.3219 0.6966 0.1338 day 0.3907 0.3617 0.0019 0.0899 0.4111 0.3285 0.7147 day*trt 0.3916 0.8185 0.5540 0.8535 0.6990 0.7303 0.4832 Treatment [H + ] respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 5.83E-08 1.52E-06 49.75 6.85 38.97 0.11 38.52 1.27 21.63 4.65 228.60 61.88 372.66 20.79 1 2.79E-06 1.24E-06 62.83 5.59 39.02 0.09 38.34 1.04 14.52 3.80 194.31 50.52 317.86 16.98 Day * Treatment 1 * 0 7.09E-08 2.56E-06 56.00 9.47 38.72 0.22 36.30 2.20 17.50 6.74 233.46 83.86 326.00 39.04 1 * 1 7.64E-08 2.09E-06 71.33 7.74 38.69 0.18 37.11 1.80 13.00 5.50 160.88 68.47 319.67 31.88 2 * 0 4.92E-08 2.56E-06 48.00 9.47 38.86 0.22 42.18 2.20 23.40 6.74 139.50 83.86 385.00 39.04 2 * 1 6.13E-08 2.09E-06 56.00 7.74 38.80 0.18 41.47 1.80 21.50 5.50 182.50 68.47 340.67 31.88 3 * 0 3.84E-08 2.56E-06 48.00 9.47 38.53 0.22 38.31 2.20 26.70 6.74 249.90 83.86 403.50 39.04 3 * 1 4.49E-06 2.09E-06 57.33 7.74 38.94 0.18 36.44 1.80 13.15 5.50 171.74 68.47 285.49 31.88 4 * 0 7.46E-08 2.56E-06 47.00 9.47 39.78 0.22 37.31 2.20 18.90 6.74 291.57 83.86 376.14 39.04 4 * 1 6.53E-06 2.09E-06 66.67 7.74 39.67 0.18 38.33 1.80 10.43 5.50 262.11 68.47 325.59 31.88 Contrasts P-values t0 vs. t1 x d1 vs. d2,3,4 0.3322 0.7961 0.7302 0.6776 0.6886 0.6131 0.2853 t0 vs. t1 x d2 vs. d3,4 0.1819 0.5998 0.5492 0.9325 0.3327 0.3733 0.5246 t0 vs. t1 x d3 vs. d4 0.6550 0.4732 0.2236 0.4646 0.6326 0.6928 0.3582 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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67 Table 3-14. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 3 treatment descriptors, and including cow 2245 MAX 3Y Fecal [H + ] RR RT Cp Hp FB AGP Model P-values trt 0.0111 0.5874 0.3932 0.4765 0.7352 0.9061 0.4940 day 0.0002 0.1942 0.0010 0.0370 0.2048 0.2693 0.3300 day*trt 0.0002 0.8780 0.3196 0.7965 0.7454 0.6560 0.3467 Treatment [H + ] respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 5.83E-08 0.00E+00 49.75 7.68 38.97 0.19 38.52 5.16 21.63 11.71 228.60 129.77 372.66 195.36 1 1.32E-06 0.00E+00 60.25 7.68 39.01 0.19 38.27 5.16 16.28 11.71 246.22 129.77 314.66 195.36 2 5.25E-06 0.00E+00 60.50 7.68 39.36 0.19 47.14 5.16 29.86 11.71 307.54 129.77 660.74 195.36 Day * Treatment 1 * 0 7.09E-08 0.00E+00 56.00 9.93 38.72 0.26 36.30 5.52 17.50 12.65 233.45 140.74 326.00 204.48 1 * 1 6.23E-08 0.00E+00 71.00 9.93 38.61 0.26 37.47 5.52 14.15 12.65 187.78 140.74 326.00 204.48 1 * 2 8.11E-08 0.00E+00 66.00 9.93 39.33 0.26 42.97 5.52 24.80 12.65 270.42 140.74 502.50 204.48 2 * 0 4.92E-08 0.00E+00 48.00 9.93 38.86 0.26 42.18 5.52 23.40 12.65 139.49 140.74 385.00 204.48 2 * 1 7.69E-08 0.00E+00 48.00 9.93 38.67 0.26 42.71 5.52 27.10 12.65 208.74 140.74 357.50 204.48 2 * 2 4.02E-08 0.00E+00 57.00 9.93 39.22 0.26 50.78 5.52 31.80 12.65 311.43 140.74 622.00 204.48 3 * 0 3.84E-08 0.00E+00 48.00 9.93 38.53 0.26 38.31 5.52 26.70 12.65 249.89 140.74 403.50 204.48 3 * 1 3.28E-06 0.00E+00 56.00 9.93 38.83 0.26 35.24 5.52 14.08 12.65 223.33 140.74 274.74 204.48 3 * 2 5.19E-06 0.00E+00 57.00 9.93 39.22 0.26 48.19 5.52 34.65 12.65 348.95 140.74 714.00 204.48 4 * 0 7.46E-08 0.00E+00 47.00 9.93 39.78 0.26 37.31 5.52 18.90 12.65 291.57 140.74 376.14 204.48 4 * 1 1.87E-06 0.00E+00 66.00 9.93 39.92 0.26 37.66 5.52 9.80 12.65 365.04 140.74 300.39 204.48 4 * 2 1.60E-05 0.00E+00 62.00 9.93 39.67 0.26 46.63 5.52 28.20 12.65 299.38 140.74 804.47 204.48

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Table 3-14. Continued 68 MAX 3Y Fecal [H + ] RR RT Cp Hp FB AGP Contrasts P-values A. t0 vs. t1,2 0.0138 0.3407 0.4279 0.5552 0.9260 0.7812 0.6635 B. t1 vs. t2 0.0119 0.9831 0.2773 0.3109 0.4722 0.7602 0.2991 C. d1 vs. d2,3,4 0.0022 0.0693 0.0613 0.0620 0.2059 0.3616 0.0973 D. d2 vs. d3,4 0.0002 0.3565 0.0227 0.0168 0.2025 0.1198 0.6376 E. d3 vs. d4 0.0051 0.4523 0.0004 0.9789 0.2049 0.4078 0.6159 Contrast A x C 0.0148 0.8135 0.8677 0.9154 0.9303 0.4506 0.7256 Contrast A x D 0.0022 0.4690 0.5951 0.8973 0.4103 0.4067 0.7891 Contrast A x E 0.0302 0.5170 0.2144 0.7222 0.8050 0.9687 0.4974 Contrast B x C 0.0117 0.5703 0.1864 0.2535 0.6756 0.7883 0.0784 Contrast B x D 0.0015 0.4263 0.2144 0.4784 0.1573 0.5214 0.1205 Contrast B x E 0.0002 0.7391 0.1627 0.3997 0.8486 0.1628 0.6532 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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69 Table 3-15. P-values for model effects, least squares means ( standard error) for treatment and the treatment x day interaction for the model containing daily maxima, using 3 treatment descriptors, and excluding cow 2245 MAX 3N Fecal [H + ] RR RT Cp Hp FB AGP Model P-values trt 0.0502 0.4957 0.8874 0.9928 0.5840 0.2790 0.4086 day 0.0030 0.4766 0.0089 0.2258 0.6352 0.6473 0.8055 day*trt 0.0085 0.8330 0.2482 0.7925 0.7876 0.6956 0.6633 Treatment [H + ] respirations/min C mg/dL mg HbB/dL mg/dL g/dL 0 5.83E-08 0.00E+00 49.75 7.77 38.97 0.10 38.52 1.55 21.63 5.27 228.60 41.24 372.66 25.16 1 1.32E-06 0.00E+00 60.25 7.77 39.01 0.10 38.27 1.55 16.28 5.27 246.22 41.24 314.66 25.16 2 5.73E-06 0.00E+00 68.00 10.98 39.06 0.14 38.48 2.19 11.00 7.45 90.48 58.33 324.25 35.58 Day * Treatment 1 * 0 7.09E-08 1.08E-06 56.00 10.48 38.72 0.19 36.30 2.43 17.50 7.44 233.46 71.05 326.00 43.40 1 * 1 6.23E-08 1.08E-06 71.00 10.48 38.61 0.19 37.47 2.43 14.15 7.44 187.78 71.05 326.00 43.40 1 * 2 1.05E-07 1.52E-06 72.00 14.82 38.83 0.27 36.40 3.44 10.70 10.52 107.10 100.49 307.00 61.38 2 * 0 4.92E-08 1.08E-06 48.00 10.48 38.86 0.19 42.18 2.43 23.40 7.44 139.50 71.05 385.00 43.40 2 * 1 7.69E-08 1.08E-06 48.00 10.48 38.67 0.19 42.71 2.43 27.10 7.44 208.74 71.05 357.50 43.40 2 * 2 3.02E-08 1.52E-06 72.00 14.82 39.06 0.27 38.99 3.44 10.30 10.52 130.02 100.49 307.00 61.38 3 * 0 3.84E-08 1.08E-06 48.00 10.48 38.53 0.19 38.31 2.43 26.70 7.44 249.90 71.05 403.50 43.40 3 * 1 3.28E-06 1.08E-06 56.00 10.48 38.83 0.19 35.24 2.43 14.08 7.44 223.33 71.05 274.74 43.40 3 * 2 6.92E-06 1.52E-06 60.00 14.82 39.17 0.27 38.85 3.44 11.30 10.52 68.57 100.49 307.00 61.38 4 * 0 7.46E-08 1.08E-06 47.00 10.48 39.78 0.19 37.31 2.43 18.90 7.44 291.57 71.05 376.14 43.40 4 * 1 1.87E-06 1.08E-06 66.00 10.48 39.92 0.19 37.66 2.43 9.80 7.44 365.04 71.05 300.39 43.40 4 * 2 1.60E-05 1.52E-06 68.00 14.82 39.17 0.27 39.68 3.44 11.70 10.52 56.24 100.49 376.00 61.38

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Table 3-15. Continued. 70 MAX 3N Fecal [H + ] RR RT Cp Hp FB AGP Contrasts P-values A. t0 vs. t1,2 0.0397 0.2967 0.6881 0.9495 0.3707 0.3845 0.2510 B. t1 vs. t2 0.0420 0.6227 0.7978 0.9445 0.6214 0.1611 0.8462 C. d1 vs. d2,3,4 0.0103 0.1860 0.0403 0.2222 0.5552 0.7602 0.4854 D. d2 vs. d3,4 0.0019 0.8285 0.0563 0.0989 0.3644 0.3975 0.7699 E. d3 vs. d4 0.0474 0.4871 0.0051 0.7267 0.5212 0.4002 0.5814 Contrast A x C 0.0311 0.9077 0.7976 0.7627 0.6897 0.7479 0.3863 Contrast A x D 0.0074 0.8272 0.7177 0.7088 0.5398 0.3016 0.7450 Contrast A x E 0.1133 0.5347 0.0964 0.5404 0.6236 0.8600 0.3660 Contrast B x C 0.0355 0.5994 0.5691 0.7081 0.8470 0.4822 0.6571 Contrast B x D 0.0082 0.2685 0.1933 0.2049 0.2501 0.3216 0.2734 Contrast B x E 0.0078 0.9232 0.0610 0.7746 0.7635 0.3829 0.6799 1 RR = Respiration rate; RT = rectal temperature; Cp = Ceruloplasmin; Hp = Haptoglobin; FB = Fibrinogen; AGP = Alpha-glycoprotein. 2 LS Means without error terms denote terms manually calculated from higher order interactions.

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Table 3-16. Alkaline phosphatase arithmetic means by treatment and day. days treatments 1 2 0 25.75 1.37 a 28 2.08 1 35.5 2.84 38.5 2.36 2 21 1 23.33 1.6 a IU/L SE Table 3-17. Alanine aminotransferase arithmetic means by treatment and day. 71 days treatments 1 2 0 12.25 1.88 a 12.77 0.92 1 16 1.08 14.08 0.45 2 4.16 1.62 a IU/L SE

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Table 3-18. Aspartate aminotransferase arithmetic means by treatment and day. days treatments 1 2 0 43.5 4.62 a 42.22 2.39 1 45.5 2.25 45.33 1.75 2 63.5 0.5 52.83 2.27 a IU/L SE Table 3-19. Biliruben arithmetic means by treatment and day. 72 days treatments 1 2 0 0.12 0.07 a 0.12 0.02 1 0.12 0.04 0.04 0.02 2 0.3 0 0.21 0.03 a mg/dL SE

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Table 3-20. Albumin arithmetic means by treatment and day. days treatments 1 2 0 2.85 0.02 a 2.83 0.06 1 2.77 0.17 2.75 0.05 2 2.3 0 2.2 0.23 a g/dL SE Table 3-21. Gamma-glutamyltransferase arithmetic means by treatment and day. 73 days treatments 1 2 0 23.25 0.25 a 21.88 1.07 1 20.25 2.46 21.25 0.97 2 21 1 18 1.39 a IU/L SE Table 3-22. Red and white blood cell counts at hour 66 from three cows. Cow RBC (million/L) WBC (thousand/L) 5329 5.92 2.22 2811 5.71 12.4 5781 6.14 8.26 Normal range 5 – 10 4 – 12

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APPENDIX A FIGURES Figure A-1. Diagram of catheter 74

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44.555.566.577.58020406080100120140160HourpH t2245 c2521 t2686 t2811 t5329 c5781 75 Figure A-2. Fecal pH

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76 0.0E+001.0E-052.0E-053.0E-054.0E-055.0E-056.0E-05020406080100120140160Hour[H+] h2245 h2521 H2686 H2811 h5329 h5781 Figure A-3. Hydrogen ion concentration

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77 Figure A-4. Normal fecal samples collected from control cows 5781 (left) and 2521 (right) at hour 81.

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78 Figure A-5. Foamy fecal sample collected from cow 2811 at hour 81.

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79 Figure A-6. Diarrhea collected from cow 2245 at hour 81

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80 Figure A-7. Pasty fecal sample collected from cow 2686 at hour 81

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010203040506070020406080100120140160HourHp (mg HbB/dl) t2245 c2521 t2686 t2811 t5329 c5781 81 Figure A-8. Haptoglobin raw data showing elevated (detectable) levels in all cows, especially cow 2245.

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0.00100.00200.00300.00400.00500.00600.00700.00020406080100120140160HourFibrinogen (mg/dl) t2245 c2521 t2686 t2811 t5329 c5781 82 Figure A-9. Fibrinogen raw data showing elevated levels in cow 2245

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0200400600800100012001400020406080100120140160HourAGP (ug/ml) t2245 c2521 t2686 t2811 t5329 c5781 83 Figure A-10. AGP raw data showing elevated levels in cow 2245

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84 Figure A-11. Comparison of fecal consistency at hour 81 among control cows (dark brown), ST-1 cows (medium brown), and ST-2 cows (pale yellow)..

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APPENDIX B NECROPSY REPORT 85

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89

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APPENDIX C RAW DATA Table C-1. Raw Data cow trt totHr Day fecpH RR RT Cp Hp AGP FB AP ALT AST Bili Alb GGT 2245 1 0 1 7.13 42.00 101.60 42.61 33.25 431.00 396.86 . . . . . . 2245 1 4 1 . 60.00 101.70 44.43 23.90 632.00 433.74 22.00 . 63.00 0.30 2.30 20.00 2245 1 8 1 7.24 48.00 103.70 49.22 19.30 698.00 416.54 . . . . . . 2245 1 12 1 7.08 42.00 102.70 44.96 34.70 632.00 245.83 20.00 3.00 64.00 0.30 2.30 22.00 2245 1 16 1 . 48.00 102.60 49.54 31.60 611.00 286.03 . . . . . . 2245 1 19 1 . 58.00 102.00 47.36 38.90 590.00 242.98 . . . . . . 2245 1 23 2 7.24 38.00 102.30 51.06 44.90 862.00 242.98 19.00 3.00 61.00 0.20 1.50 16.00 2245 1 27 2 7.16 38.00 101.30 53.35 32.10 838.00 331.15 21.00 12.00 51.00 0.30 2.90 15.00 2245 1 31 2 7.30 36.00 102.10 62.57 33.80 744.00 393.15 24.00 2.00 52.00 0.20 2.30 17.00 2245 1 35 2 7.16 36.00 102.90 53.00 34.40 838.00 287.99 30.00 4.00 47.00 0.30 2.80 24.00 2245 1 39 2 5.47 30.00 102.80 48.94 44.20 937.00 393.13 21.00 3.00 48.00 0.10 1.70 16.00 2245 1 43 2 5.25 42.00 102.70 47.11 53.30 937.00 492.84 25.00 1.00 58.00 0.20 2.00 20.00 2245 1 46 3 5.46 30.00 101.40 50.55 58.00 1067.00 309.72 . . . . . . 2245 1 50 3 . 47.00 100.60 49.68 36.40 1121.00 379.11 . . . . . . 2245 1 54 3 4.95 54.00 102.70 53.66 16.25 790.00 286.51 . . . . . . 2245 1 58 3 4.75 44.00 102.50 57.53 39.50 937.05 277.06 . . . . . . 2245 1 62 3 4.88 32.00 102.20 43.19 40.05 814.05 259.86 . . . . . . 2245 1 66 3 4.79 32.00 102.70 48.19 15.60 790.32 629.33 . . . . . . 2245 1 69 4 4.81 32.00 102.40 49.87 34.30 1067.23 542.52 . . . . . . 2245 1 73 4 4.69 56.00 102.20 45.42 44.70 1014.29 437.49 . . . . . . 2245 1 77 4 4.59 56.00 103.10 47.25 40.45 814.05 516.51 . . . . . . 2245 1 81 4 4.54 42.00 101.60 51.02 40.50 838.07 461.06 . . . . . . 2245 1 85 4 4.64 54.00 104.30 49.96 36.40 1232.94 315.79 . . . . . . 2245 1 89 4 . . . 53.57 43.90 1040.62 456.46 . . . . . . 2521 0 0 1 . 46.00 101.10 25.72 11.20 227.00 296.12 . . . . . . 2521 0 4 1 6.60 38.00 101.50 28.07 10.80 258.00 124.98 24.00 9.00 52.00 0.30 2.80 23.00 2521 0 8 1 6.98 42.00 101.70 29.69 13.00 197.00 193.65 . . . . . . 2521 0 12 1 6.78 42.00 101.20 30.21 13.00 212.00 123.15 23.00 9.00 51.00 0.20 2.80 23.00 2521 0 16 1 . 32.00 101.70 38.04 20.60 227.00 128.43 . . . . . . 2521 0 19 1 6.98 44.00 101.10 35.48 15.60 242.00 169.41 . . . . . . 2521 0 23 2 7.12 23.00 101.30 32.65 24.60 307.00 115.67 19.00 11.00 51.00 0.20 2.80 20.00 2521 0 27 2 6.93 32.00 100.40 35.46 25.30 394.00 86.36 22.00 10.00 50.00 0.20 2.80 20.00 2521 0 31 2 6.96 42.00 101.40 39.26 30.30 376.00 70.88 23.00 9.00 50.00 0.10 2.80 21.00 2521 0 35 2 7.49 30.00 102.10 40.51 32.00 358.00 156.75 40.00 15.00 46.00 0.10 2.80 16.00 2521 0 39 2 7.04 30.00 102.20 . . . . . . . . . . 90

PAGE 102

91 cow totHr Day fecpH RR RT Cp Hp AGP FB AP ALT AST Bili Alb GGT 2521 0 43 2 7.19 36.00 100.20 30.35 8.40 258.00 154.18 . . . . . . 2521 0 46 3 7.19 28.00 100.90 35.40 32.80 324.00 142.86 . . . . . . 2521 0 50 3 7.16 25.00 101.40 37.09 18.90 394.00 150.77 . . . . . . 2521 0 54 3 7.28 36.00 101.50 37.64 16.10 431.00 145.41 . . . . . . 2521 0 58 3 7.66 20.00 100.50 . . . . . . . . . . 2521 0 62 3 6.99 20.00 101.30 35.84 13.50 412.25 179.86 . . . . . . 2521 0 66 3 7.10 32.00 101.40 33.30 11.30 393.98 213.28 . . . . . . 2521 0 69 4 7.11 32.00 101.60 38.04 7.80 393.98 296.63 . . . . . . 2521 0 73 4 7.04 28.00 102.80 36.60 16.20 358.30 160.85 . . . . . . 2521 0 77 4 7.05 32.00 102.10 . . . . . . . . . . 2521 0 81 4 6.98 21.00 100.60 35.80 22.00 306.94 189.80 . . . . . . 2521 0 85 4 7.18 42.00 102.00 39.40 30.40 290.39 262.72 . . . . . . 2521 0 89 4 7.01 24.00 101.60 . . . . . . . . . . 2521 0 92 5 7.09 20.00 101.40 38.32 34.70 412.25 63.07 . . . . . . 2521 0 96 5 7.17 24.00 100.70 . . . . . . . . . . 2521 0 100 5 7.24 28.00 101.40 . . . . . . . . . . 2521 0 104 5 7.40 24.00 101.30 . . . . . . . . . . 2521 0 108 5 7.02 20.00 101.40 . . . . . . . . . . 2521 0 112 5 7.12 30.00 101.40 41.83 19.25 393.98 106.68 . . . . . . 2521 0 115 5 7.10 36.00 101.60 . . . . . . . . . . 2521 0 119 6 7.13 24.00 101.20 . . . . . . . . . . 2521 0 123 6 7.01 12.00 100.40 . . . . . . . . . . 2521 0 127 6 7.04 24.00 100.90 . . . . . . . . . . 2521 0 131 6 7.09 32.00 101.70 . . . . . . . . . . 2521 0 135 6 7.07 26.00 101.40 . . . . . . . . . . 2521 0 139 6 7.07 20.00 101.40 . . . . . . . . . . 2686 1 0 1 6.98 44.00 101.50 30.71 9.90 307.00 98.54 . . . . . . 2686 1 4 1 6.95 42.00 101.80 29.02 10.70 274.00 70.28 . . . . . . 2686 1 8 1 6.79 36.00 101.80 33.06 9.50 307.00 107.10 . . . . . . 2686 1 12 1 6.80 72.00 101.90 32.37 8.10 227.00 30.77 . . . . . . 2686 1 16 1 6.61 42.00 101.70 36.40 8.50 307.00 27.90 . . . . . . 2686 1 19 1 6.95 60.00 101.40 32.61 8.40 307.00 104.18 . . . . . . 2686 1 23 2 7.52 28.00 101.90 32.34 6.40 307.00 57.22 . . . . . . 2686 1 27 2 6.83 34.00 101.30 37.19 7.00 242.00 71.97 . . . . . . 2686 1 31 2 6.76 42.00 101.90 31.39 4.50 274.00 130.02 . . . . . . 2686 1 35 2 5.62 36.00 102.30 38.99 5.80 242.00 48.93 . . . . . . 2686 1 39 2 5.04 36.00 102.30 . . . . . . . . . . 2686 1 43 2 . 72.00 100.60 35.93 10.30 290.00 116.27 . . . . . . 2686 1 46 3 5.16 60.00 101.70 34.77 11.30 307.00 65.07 . . . . . . 2686 1 50 3 5.09 42.00 101.30 35.04 9.30 290.00 68.57 . . . . . . 2686 1 54 3 4.93 30.00 102.20 . . . . . . . . . . 2686 1 58 3 . 24.00 101.50 . . . . . . . . . . 2686 1 62 3 4.88 44.00 102.50 36.74 5.70 290.39 37.84 . . . . . . 2686 1 66 3 4.76 28.00 101.90 38.85 5.60 306.94 42.73 . . . . . . 2686 1 69 4 4.80 28.00 101.50 37.28 4.50 323.77 25.34 . . . . . . trt

PAGE 103

92cow trt totHr Day fecpH RR RT Cp Hp AGP FB AP ALT AST Bili AlbGGT 2686 1 73 4 4.72 52.00 101.50 37.43 8.80 306.94 47.66 . . . . . . 2686 1 77 4 4.63 68.00 101.70 . . . . . . . . . . 2686 1 81 4 4.56 44.00 102.40 35.78 11.20 323.77 51.77 . . . . . . 2686 1 85 4 4.49 30.00 101.60 39.68 11.70 376.00 56.24 . . . . . . 2686 1 89 4 4.54 48.00 102.50 . . . . . . . . . . 2686 1 5 4.53 20.00 92 100.20 . . . . . . . . . . 2686 1 96 5 4.43 76.00 101.30 . . . . . . . . . . 2686 1 100 5 4.32 . . . . . . . . . . . . 2686 1 104 5 4.73 . . . . . . . . . . . . 2686 1 108 5 . 32.00 101.70 . . . . . . . . . . 2686 1 112 5 4.98 42.00 101.50 . . . . . . . . . . 2686 1 115 5 7.04 48.00 101.20 . . . . . . . . . . 2686 1 119 6 . . . . . . . . . . . . . 2686 1 123 6 6.72 . . . . . . . . . . . . 2686 1 127 6 . . . . . . . . . . . . . 2686 1 131 6 6.41 . . . . . . . . . . . . 2686 1 135 6 . . . . . . . . . . . . . 2686 1 139 6 6.83 . 101.50 . . . . . . . . . . 2811 1 0 1 6.63 48.00 101.40 28.44 12.40 227.00 163.47 . . . . . . 2811 1 4 1 6.73 42.00 100.70 34.63 11.40 168.00 170.10 44.00 19.00 52.00 0.20 3.30 16.00 2811 1 8 1 6.74 48.00 101.40 32.98 11.20 258.00 181.90 . . . . . . 2811 1 12 1 7.27 50.00 100.60 31.23 7.80 212.00 142.38 32.00 14.00 42.00 0.20 2.50 16.00 2811 1 16 1 6.67 36.00 101.20 33.65 8.60 227.00 151.10 . . . . . . 2811 1 19 1 6.99 52.00 101.00 33.42 4.60 228.00 132.49 . . . . . . 2811 1 23 2 7.05 23.00 100.90 32.63 8.50 258.00 94.76 44.00 15.00 49.00 0.20 3.00 17.00 2811 1 27 2 6.65 42.00 101.00 34.32 8.40 227.00 160.96 48.00 14.00 49.00 0.00 3.00 20.00 2811 1 31 2 6.56 42.00 101.20 38.39 5.90 374.00 185.11 47.00 13.00 47.00 0.00 2.90 18.00 2811 1 35 2 5.44 48.00 101.50 31.88 2.00 227.00 84.28 46.00 14.00 47.00 0.10 2.80 17.00 2811 1 39 2 4.80 42.00 100.90 30.18 9.60 227.00 182.25 49.00 16.00 48.00 0.00 3.00 17.00 2811 1 43 2 5.70 42.00 100.70 34.98 40.10 307.00 90.19 23.00 10.00 60.00 0.20 2.80 22.00 2811 1 46 3 5.52 36.00 100.20 31.57 7.00 227.00 150.77 . . . . . . 2811 1 50 3 5.73 28.00 100.50 29.41 6.20 242.00 159.59 . . . . . . 2811 1 54 3 6.62 60.00 101.60 30.70 9.10 183.00 145.41 . . . . . . 2811 1 58 3 . 44.00 101.80 32.38 11.95 227.09 208.98 . . . . . . 2811 1 62 3 5.11 44.00 101.00 30.06 8.80 242.48 145.93 . . . . . . 2811 1 66 3 5.31 28.00 101.80 31.17 11.10 242.48 98.14 . . . . . . 2811 1 69 4 5.15 40.00 101.90 30.41 4.80 242.48 142.86 . . . . . . 2811 1 73 4 5.16 32.00 102.00 33.57 6.20 227.09 143.36 . . . . . . 2811 1 77 4 5.68 52.00 101.30 25.45 4.90 227.09 173.78 . . . . . . 2811 1 81 4 5.30 28.00 101.70 30.63 6.20 182.63 166.73 . . . . . . 2811 1 85 4 5.49 42.00 103.40 39.03 6.20 211.98 180.65 . . . . . . 2811 1 89 4 5.65 36.00 102.30 29.44 5.70 227.09 42.66 . . . . . . 2811 1 92 5 5.64 28.00 101.80 31.64 5.10 211.98 139.36 . . . . . . 2811 1 96 5 5.90 32.00 100.30 30.88 4.27 227.09 95.33 . . . . . . 2811 1 100 5 6.74 52.00 100.60 32.20 6.44 211.98 134.27 . . . . . .

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93 cow totHr Day fecpH RR RT Cp Hp AGP FB AP ALT AST Bili Alb GGT 2811 1 104 5 6.90 28.00 101.50 34.67 5.89 258.17 198.71 . . . . . . 2811 1 108 5 6.62 32.00 101.50 34.53 6.64 227.09 115.19 . . . . . . 2811 1 112 5 6.76 24.00 101.60 35.29 7.25 168.38 125.09 . . . . . . 2811 1 115 . 5 6.95 32.00 101.60 37.56 9.62 227.09 177.24 . . . . . 2811 1 119 6 6.97 60.00 101.60 37.39 16.20 211.98 118.58 . . . . . . 2811 1 123 6 6.65 44.00 101.40 35.61 11.93 227.09 161.90 . . . . . . 2811 1 127 6 6.64 36.00 101.40 37.49 12.67 227.09 172.36 . . . . . . 2811 1 131 6 6.91 28.00 101.90 36.59 16.77 242.48 129.53 . . . . . . 2811 1 135 6 6.76 34.00 101.90 34.52 15.32 258.17 161.20 . . . . . . 2811 1 139 6 6.72 28.00 101.20 26.51 19.46 306.94 175.59 . . . . . . 5329 1 0 1 7.15 56.00 101.50 30.29 11.30 324.00 174.34 . . . . . . 5329 1 4 1 6.93 90.00 101.40 34.84 12.70 290.00 163.47 33.00 16.00 43.00 0.10 2.60 24.00 5329 1 8 1 6.88 60.00 101.10 35.48 15.90 394.00 193.65 . . . . . . 5329 1 12 1 7.05 42.00 101.60 36.39 8.40 324.00 134.18 33.00 15.00 45.00 0.00 2.70 25.00 5329 1 16 1 7.15 36.00 101.60 40.30 13.80 376.00 184.29 . . . . . . 5329 1 19 1 7.12 64.00 101.00 35.27 15.30 307.00 92.66 . . . . . . 5329 1 23 2 7.19 28.00 100.80 33.64 11.30 341.00 117.29 34.00 14.00 38.00 0.00 2.60 22.00 5329 1 27 2 7.05 40.00 100.90 38.08 11.50 307.00 114.41 35.00 14.00 41.00 0.00 2.40 24.00 5329 1 31 2 5.24 48.00 101.70 39.80 12.70 227.00 146.36 31.00 14.00 39.00 0.00 2.70 23.00 5329 1 35 2 . 30.00 101.40 47.03 13.60 227.00 109.62 37.00 16.00 44.00 0.00 2.60 26.00 5329 1 39 2 5.25 30.00 101.30 34.21 14.00 242.00 232.36 32.00 15.00 42.00 0.00 2.70 26.00 5329 1 43 2 5.19 36.00 100.50 35.06 14.10 290.00 194.26 36.00 14.00 40.00 0.00 2.60 23.00 5329 1 46 3 5.06 52.00 100.50 34.40 16.10 274.00 218.91 . . . . . . 5329 1 50 3 5.20 35.00 100.90 36.58 13.30 307.00 129.25 . . . . . . 5329 1 54 3 4.97 42.00 102.00 37.60 16.20 . 87.23 . . . . . . 5329 1 58 3 4.79 36.00 101.10 33.08 8.70 258.17 188.93 . . . . . . 5329 1 62 3 4.98 32.00 101.00 34.53 5.80 306.94 163.41 . . . . . . 5329 1 66 3 4.78 28.00 101.60 38.10 7.20 306.94 237.67 . . . . . . 5329 1 69 4 5.05 28.00 100.60 36.29 8.00 306.94 348.00 . . . . . . 5329 1 73 4 5.34 52.00 101.40 35.20 13.40 306.94 160.22 . . . . . . 5329 1 77 4 . 80.00 101.90 32.52 11.90 306.94 549.43 . . . . . . 5329 1 81 4 5.20 29.00 102.40 33.69 8.70 358.30 166.06 . . . . . . 5329 1 85 4 . 54.00 104.30 32.90 11.00 258.17 74.43 . . . . . . 5329 1 89 4 5.78 36.00 102.10 29.98 7.00 306.94 102.43 . . . . . . 5329 1 92 5 5.25 28.00 101.40 35.29 9.56 376.00 147.73 . . . . . . 5329 1 96 5 . 48.00 100.30 35.23 12.06 323.77 91.89 . . . . . . 5329 1 100 5 6.48 44.00 100.60 36.67 15.25 393.98 143.15 . . . . . . 5329 1 104 5 7.21 48.00 101.60 . . . . . . . . . . 5329 1 108 5 6.80 28.00 100.70 . . . . . . . . . . 5329 1 112 5 7.02 42.00 101.60 37.68 19.52 430.81 145.41 . . . . . . 5329 1 115 5 6.92 50.00 102.30 40.14 21.15 290.39 188.80 . . . . . . 5329 1 119 6 7.10 63.00 101.70 . . . . . . . . . . 5329 1 123 6 6.85 48.00 102.10 32.40 11.93 306.94 242.92 . . . . . . 5329 1 127 6 7.11 64.00 101.70 . . . . . . . . . . 5329 1 131 6 6.85 40.00 101.70 . . . . . . . . . . trt

PAGE 105

94 cow totHr Day fecpH RR RT Cp Hp AGP FB AP ALT AST Bili Alb GGT 5329 1 135 6 7.00 28.00 101.40 . . . . . . . . . . 5329 1 139 6 6.92 26.00 100.90 . . . . . . . . . . 5781 0 0 1 6.65 50.00 101.70 28.53 11.60 242.00 155.01 . . . . . . 5781 0 4 1 6.49 66.00 100.90 31.14 11.50 307.00 170.79 27.00 15.00 35.00 0.00 2.90 24.00 5781 0 8 1 6.79 48.00 101.20 31.98 14.40 307.00 157.34 . . . . . . 5781 0 12 1 6.77 60.00 100.90 31.25 7.60 324.00 108.49 29.00 16.00 36.00 0.00 2.90 23.00 5781 0 16 1 6.96 42.00 100.80 34.55 11.60 394.00 84.28 . . . . . . 5781 0 19 1 7.43 60.00 100.80 33.93 12.90 307.00 101.65 . . . . . . 5781 0 23 2 7.10 26.00 101.10 33.02 11.20 307.00 62.43 30.00 16.00 41.00 0.20 2.90 23.00 5781 0 27 2 6.80 44.00 101.00 41.26 14.80 307.00 77.43 30.00 17.00 38.00 0.10 2.90 24.00 5781 0 31 2 6.90 54.00 100.80 39.95 11.60 376.00 118.96 29.00 11.00 32.00 0.00 2.40 21.00 5781 0 35 2 . 48.00 101.50 43.84 8.20 307.00 114.41 27.00 13.00 36.00 0.10 3.10 26.00 5781 0 39 2 7.04 24.00 101.70 32.36 13.10 358.00 122.24 32.00 13.00 36.00 0.10 3.00 26.00 5781 0 43 2 7.18 52.00 101.00 33.36 11.90 307.00 116.60 . . . . . . 5781 0 46 3 7.18 44.00 101.20 36.18 7.40 324.00 90.19 . . . . . . 5781 0 50 3 . 31.00 100.40 37.70 12.80 307.00 286.51 . . . . . . 5781 0 54 3 7.06 42.00 100.90 34.00 8.10 . 181.45 . . . . . . 5781 0 58 3 7.26 60.00 101.20 38.98 20.60 290.39 207.93 . . . . . . 5781 0 62 3 6.91 36.00 101.00 31.70 12.45 340.89 145.41 . . . . . . 5781 0 66 3 6.92 40.00 100.50 33.54 4.30 376.00 182.25 . . . . . . 5781 0 69 4 7.08 40.00 100.80 32.53 4.30 323.77 286.51 . . . . . . 5781 0 73 4 6.94 28.00 100.30 32.74 6.00 358.30 173.05 . . . . . . 5781 0 77 4 6.93 52.00 101.10 . . . . . . . . . . 5781 0 81 4 6.66 24.00 102.20 32.56 5.80 323.77 133.93 . . . . . . 5781 0 85 4 7.08 42.00 104.40 35.21 7.40 306.94 139.36 . . . . . . 5781 0 89 4 6.91 48.00 101.40 31.40 4.30 323.77 118.96 . . . . . . 5781 0 92 5 6.97 36.00 100.70 31.56 6.50 340.89 105.12 . . . . . . 5781 0 96 5 . 36.00 101.30 32.63 4.61 393.98 99.58 . . . . . . 5781 0 100 5 6.84 60.00 100.50 31.74 4.67 258.17 113.74 . . . . . . 5781 0 104 5 7.08 20.00 101.40 37.02 5.35 323.77 139.36 . . . . . . 5781 0 108 5 6.78 40.00 101.40 32.34 5.69 274.14 68.70 . . . . . . 5781 0 112 5 6.94 54.00 101.30 33.25 6.30 242.48 115.19 . . . . . . 5781 0 115 5 6.81 26.00 101.40 36.47 5.96 258.17 58.16 . . . . . . 5781 0 119 6 . 56.00 101.00 39.82 4.94 323.77 106.68 . . . . . . 5781 0 123 6 6.85 44.00 101.10 32.67 4.81 393.98 108.61 . . . . . . 5781 0 127 6 7.01 48.00 101.00 31.34 5.01 340.89 91.89 . . . . . . 5781 0 131 6 7.02 48.00 101.70 31.97 7.32 323.77 61.16 . . . . . . 5781 0 135 6 6.95 32.00 101.90 33.58 7.93 227.09 69.95 . . . . . . 5781 0 139 6 7.11 32.00 101.90 31.87 7.39 323.77 102.14 . . . . . . trt

PAGE 106

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BIOGRAPHICAL SKETCH Heidi Ann Bissell was born on October 23, 1976 in Munich, Germany. After moving to the United States with her mother, Patricia Bissell, she completed elementary, middle, and high schools in Bradenton, Florida. She enrolled at the University of Florida in 1994, where she became interested in veterinary medicine. Heidi began volunteering at veterinary clinics, rehabilitating local wildlife, and assisting graduate students with their research. While volunteering at the veterinary school’s wildlife ward, she noticed the large number of animal clients whose problems stemmed from poor nutrition and became interested in exotic animal nutrition, especially as so little was known about it. Heidi received her B.S. in zoology in 1997. Her veterinary plans were completely sidetracked one day while walking across campus, when she saw a Peace Corps information table. That same day, she withdrew her application from the vet school and signed up for the Peace Corps in Africa. Fate sent her to Poland instead, where she spent two years teaching English to a talented and memorable group of high school students as well as assisting graduate students from the University of Poznan with their research projects at a local wildlife reservation. After returning from the Peace Corps, she was accepted by the Graduate School at the University of Florida and began studying dairy cattle nutrition. After graduation she hopes to work at a zoo conducting nutritional research. 105