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Effect of Mannan Oligosaccharide (MOS) Supplementation on the Immune Status of Mares and their Foals


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EFFECT OF MANNAN OLIGOSACCHAR IDE (MOS) SUPPLEMENTATION ON THE IMMUNE STATUS OF MARES AND THEIR FOALS By KELLY ROBERTSON SPEARMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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This thesis is dedicated to my parents, Steve and Charlotte Robertson, who have continuously provided me with unconditi onal love and support for the past 26 years.

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iii ACKNOWLEDGMENTS I am very fortunate to be able to work under Dr. Edgar A. Ott, who served as both chairperson of my committee and a constant source of wisdom. I also extend gratitude to Dr. Saundra H. T enBroeck and Dr. Steeve Giguere, who served on my committee and dedicated their time and effort to reviewing my thesis. I also would like to thank others who c ontributed to the completion of this project, including the staff at the University of Florida Horse Research Center and Jan Kivipelto, Kylee Johnson, and Tony a Stevens; and of course, the mares and their foals. I also thank Joel McQuagge and Dr. Tim Ma rshall for their encouragement and friendship. Finally, I thank my parents, brother, grandparents, and ext ended family for all of their love and support during the cour se of my study at the University of Florida. I am extremely fortunate to have such a wonderful family, whose faith in me sometimes exceeds the faith I have in myself. They have taught me about the unconditional love of God, who gives me the peace and strength necessary to accomplish anything.

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iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................. iii LIST OF TABLES ............................................................................................................ vi LIST OF FIGURES ........................................................................................................ viii ABSTRACT ...................................................................................................................... ix CHAPTER 1 INTRODUCTION ........................................................................................................1 2 REVIEW OF LITERATURE ......................................................................................4 The Immune System .. ...............................................................................................4 Passive Immunity .......................................................................................................8 Colostrum ....................................................................................................................9 Prelactation ........................................................................................................10 Breed ..................................................................................................................10 Age ......................................................................................................................11 Foal Diarrhea ............................................................................................................12 Rotavirus ............................................................................................................12 Salmonella .........................................................................................................13 Clostridium .........................................................................................................13 Other Bacteria ...................................................................................................14 Mannan Oligosaccharides ......................................................................................14 In vitro Agglutination Studies ..........................................................................15 Intestinal Environment Studies .......................................................................16 Performance ......................................................................................................17 Immune Function ..............................................................................................21 3 MATERIALS AND METHODS ...............................................................................26 Animals ......................................................................................................................26 Housing and Management .....................................................................................26 Diets ...........................................................................................................................27 Body Measurements ................................................................................................27 Colostrum and Blood Samples ..............................................................................28 Feed Sample Analysis .............................................................................................28

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v Statistical Analysis ...................................................................................................29 4 RESULTS AND DISCUSSION ..............................................................................31 Feed Analysis ...........................................................................................................31 Growth Analysis .......................................................................................................31 Mare Serum Immunoglobulins ...............................................................................35 IgG .......................................................................................................................35 IgA .......................................................................................................................35 IgM ......................................................................................................................36 Discussion ..........................................................................................................36 Mare Colostrum Immunoglobulins ........................................................................37 IgG .......................................................................................................................37 IgA .......................................................................................................................38 IgM ......................................................................................................................39 Discussion ..........................................................................................................40 Foal Serum Immunoglobulins ................................................................................43 IgG .......................................................................................................................43 IgA .......................................................................................................................46 IgM ......................................................................................................................49 Discussion ..........................................................................................................52 5 SUMMARY AND CONCLUSIONS ........................................................................54 LITERATURE CITED ......................................................................................................55 BIOGRAPHICAL SKETCH ............................................................................................62

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vi LIST OF TABLES Table page 2-1. Immunoglobulin concentrati on in serum of mature horses .................................5 2-2. Immunoglobulin content of mare’s colostrum and milk .....................................10 3-1. Composition of Concentrate (HR-136) ................................................................30 3-2. Concentrate feeding rates for mares ...................................................................30 4-1. Concentrate (HR-136) and Coastal bermudagrass hay nutrient composition analysis ...................................................................................................................31 4-2. Influence of treatment on mare weight and body condition scores .................33 4-3. Influence of treatment on foal growth ...................................................................33 4-4. Influence of sex on foal growth .............................................................................34 4-5. Influence of breed on foal growth .........................................................................34 4-6. Influence of treatment on mare serum IgG concentration ................................35 4-7: Influence of treatment on mare serum IgA concentration .................................36 4-8. Influence of treatment on mare serum IgM concentration ................................36 4-9. Influence of treatment, prelactati on occurrence, age, and breed on colostrum IgG ...........................................................................................................................38 4-10. ANOVA generated P values for colostru m IgG from a statistical model which included treatment, prelactati on, age, breed, with treatment*age and treatment*breed interactions ................................................................................38 4-11. Influence of treatment, prel actation occurrence, age, and breed on colostrum IgA .........................................................................................................39 4-12. ANOVA generated P values for colostru m IgA from a statistical model which included treatment, prelactation, age, breed, with treatment*age and treatment breed*interactions ................................................................................39

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vii 4-13. Influence of treatment, prel actation occurrence, age, and breed on colostrum IgM .........................................................................................................40 4-14. ANOVA generated P values for colostru m IgM from a statistical model which included treatment, prelactati on, age, breed, with treatment*age and treatment*breed interactions ................................................................................40 4-15. Influence of treatment on foal serum IgG concentration .................................44 4-16. Influence of treatment on fo al serum IgA concentration .................................47 4-17. Influence of treatment on foal serum IgM concentration ................................50

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viii LIST OF FIGURES Figure page 4-1: Mean foal serum IgG concentration .....................................................................45 4-2: Mean foal serum IgA concentration .....................................................................48 4-3: Mean foal serum IgM concentration .....................................................................51

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ix Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science EFFECT OF MANNAN OLIGOSACCHAR IDE (MOS) SUPPLEMENTATION ON THE IMMUNE STATUS OF MARES AND THEIR FOALS By Kelly Robertson Spearman May 2004 Chair: Edgar A. Ott Major Department: Animal Sciences Newborn foals are susceptible to m any pathogens that can cause health problems such as diarrhea, sepsis, and even death. The foal obtains the antibodies necessary to combat the onslaught of these pathogens from the mares colostrum when it is ingest ed within the first 24 hours of life. Previous research in other specie s suggests that mannan oligosaccharide (MOS) supplementation to the diet has positive effects on immune function, including increased serum and colostrum im munoglobulin levels. An experiment was designed to identify the effects of MOS supplementation to the diet on colostrum and serum immunoglobulin conc entrations in the pregnant mare and serum immunoglobulin concentration in her foal. Twenty-six pregnant Thoroughbred (n=21) and Quarter Horse (n =5) mares were paired by expected foaling dates and assigned at random to the treatment or control group. Treatment mares received 10 g of MOS mixed in 45 g of ground corn in the

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x morning ration. Control mares received 55 g of ground corn in the morning ration. All mares were fed a concentrate des igned to provide NRC recommended or higher nutrient intake when fed with Coastal bermudagrass hay or bahiagrass pasture ad libitum in season. Both treatments began 56 days before the expected foaling date (Day -56) for each mare and continued through 28 days post-parturition (Day +28). The IgG, Ig M, and IgA values were determined on mare serum at Days -56, 0, and +28. The IgG, Ig M, and IgA values were determined on colostrum collected before the foal had nursed. IgG, IgM and IgA values were determined on foal serum co llected at 0 hour (before foals had nursed), 6 to 10 hours post-parturition, and at Day +7, +14, +28, +56, and +112 of age. The mares receiving MOS supplement ation had higher colostrum IgA (p=0.008) and IgG (p=0.033); and t ended to have higher IgM (p=0.076) concentrations when controlled for prelac tation colostrum loss, age, and breed. Prelactation adversely affected colo strum IgG (p=0.006) and IgA (p=0.008) immunoglobulin concentration, but had no effect on IgM concentration. There were no significant differences between treatments for mare IgG, IgM, and IgA serum levels at any collection period. F oals from control mares tended to have higher serum IgA concentration at 6 to 10 hours post-parturition than did foals from mares fed MOS (p=0.09) There were no other significant differences in foal serum immunoglobulin concentrations at any collection period. This trial suggests that MOS supplementation to pr egnant mares increases colostrum immunoglobulin content.

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1 CHAPTER 1 INTRODUCTION Suckling foals are susceptible to many pathogens that cause various health problems such as diarrhea, enterit is, septicemia, and even death. These problems can result in major veterinar y expenses and financial loss for horse breeding operations. The diarrhea that is as sociated with foal heat occurs in foals 7 to 12 days after birth and is considered the most common cause of diarrhea in young foals (Cohen 1997). This generally caus es minimal stress for the foal and can resolve itself with little to no medical treatment. The diarrhea that occurs just after birth or later during lactation is o ften pathological in nature, and is a major health concern, because it can result in severe dehydration, reduced growth, and even death. Many organisms have been in dicated in the development of diarrhea, including Clostridium perfringens (East et al. 2000), Clostridium difficile (Jones et al. 1988) Salmonella typhimurium and other Salmonella spp. (Spier 1993) and rotavirus (Dwyer 1993). The foal obtains the antibodies necessary to combat the onslaught of these pathogens from the mare’s colostrum when it is ingested within the first 24 hours of life (Jeffcott 1974). The immunoglobul in found in the greatest quantity in mare colostrum is IgG, fo llowed by IgA and IgM (McGuire et al. 1973). Studies have shown that colostral IgG concentrati on is highly correlated with foal serum IgG concentration 18 hours after birth (LeBlanc et al. 1992). Failure of the mare to provide the foal with adequate antibodie s via the colostrum may necessitate

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2 the administration of supplemental colostru m or plasma to the foal shortly after birth. When included as a supplement to the diet, mannan oligosaccharides (MOS) have been shown to have a positive effect on immune response in several species. Mannan oligosacchar ides are indigestible complex polysaccharide molecules derived from yeast cell walls. Mannan oligosaccharides are commercially av ailable as BioMos, a nutritional supplement manufactured by Alltech, Inc. (Nicholasville, KY). Supplemental MOS in poultry diets increased both plasma IgG and bile IgA (Savage et al. 1996). In dogs supplemented with MOS, total lym phocyte count was increased, and serum IgA concentrations tended to be greater (Swanson et al. 2002). Mannan oligosaccharide supplement ation increased serum IgM and tended to increase colostral IgG levels in sows (Newm an 2001). In addition to the positive immune response elicited from MOS, they also serve as alte rnate attachment sites in the gut for gram-negative pathogenic organi sms with mannose-specific type-1 fimbriae that adhere to intestinal epithel ial cells to initiate disease (Ferket et al. 2002). These pathogens will bind to MOS pr esent in the intestinal tract and pass through the gut, instead of attaching to host epithelial cells. Previous studies have demonstrated that MOS reduces in vitro attachment of Salmonella typhimurium to cultured intestinal cells (Oyofo et al. 1989) and decreases fecal concentrations of Clostridium perfringens in poultry (Finuance et al. 1999). Other in vitro studies have demonstr ated agglutination of Escherichia coli, Salmonella typhimurium, and S. enteritidis in the presence of MOS (Spring et al. 2000).

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3 There has recently been an incr eased emphasis on the reduction of antibiotic use in production diets becaus e of the associated potential negative environmental and health issues. The swi ne, poultry, and cattle industries are interested in supplemental MOS because t hey may serve as a viable alternative to antibiotic use in ration formulation. Mo st of the previous research involving MOS has investigated the positive per formance benefits seen with MOS addition to production diets. Studies have demonstr ated that the addition of MOS to the diet results in increased average daily ga in (Hooge 2003), increased gain-to-feed ratio (Davis et al. 2002), and heavier litter birth and weaning weights (O’Quinn et al. 2001). The immune response elicited by MOS supplementation in swine, poultry, and cattle has only recently begun to be investigated. To the author’s knowledge, there have been no previous equine studies involving MOS supplementation. Results obtained in previous resear ch with other species suggest that MOS supplementation to the diet of the pregnant mare may increase the immunoglobulin content in her colostrum and protect her from colonization of pathogenic organisms in the gut. Greater immunoglobulin content in the colostrum will result in more protection for the foal from disease initiated by pathogenic organisms. The reduced occurr ence of diarrhea and other problems caused by these organisms in suckling f oals would result in healthier foals and decreased financial loss due to veteri nary expenses for horse breeding operations.

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4 CHAPTER 2 REVIEW OF LITERATURE The Immune System The immune system of the horse is a versatile defense mechanism that provides protection from a daily ons laught of pathogenic organisms. The body must be prepared to combat this invasion with an arsenal of cells capable of recognizing and eliminating these fore ign microbes. The immunoglobulins are a group of molecules exhibiting this proper ty, through their ability to effectively recognize and bind foreign antigen. Thes e large glycoprotein molecules are present on B-cell membranes, and are also secreted by plasma cells. They are found throughout the body in the blood, muco sal tissues, and external secretions. Immunoglobulins synthesized by the pregnant mare will affect the survivability of her foal, because the foal relies on passive transfer to provide the major source of antibodies for a period of at least 1 month after birth (McGuire and Crawford 1973). After that, the foa l’s own immune system is able to begin producing immunoglobulins in a quantity that can mo unt an immunologic response that will provide protection from pathogenic organisms. Immunoglobulins The immunoglobulins are a large group of glycoprotein molecules found in the seru m of the blood and other body fluids. They are part of the fraction of serum pr oteins termed the “globulins” and play an integral role in the immune response (Peakman and Vergani 1997). An antibody is an immunoglobulin (Ig) that exhibits antigen-bind ing ability. Therefore all

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5 antibodies are Igs but not all Igs are antibodies. Howe ver, the two terms are commonly used interchangeably. The functi ons of antibodies include targeting foreign molecules, recruitment of effect or responses, neutralization of toxins, and binding and removal of foreign antigens Antibodies also serve as useful diagnostic tools. For exampl e, to determine whether su ccessful passive transfer of maternal antibodies in newborn foals has occurred, the IgG concentration in the foal’s serum can be measured. The four major equine Ig isotypes are IgG, IgM, IgA, and IgE (Nezlin 1998). The average Ig concentrations found in the serum of mature horses are presented in Table 2-1. Table 2-1. Immunoglobulin concentr ation in serum of mature horses IgG IgA IgM Concentration (mg/dL) 1000 to 1500 60 to 350 100 to 200 Adapted from Tizard 1996: Veterinary Immunology: An Introduction p. 155 Table 13-2. W.B. Saunders Co., Philadelphia. IgG. IgG is the most abundant Ig found in the serum and in the colostrum. It is made and secreted by plasma cells found in the spleen, lymph nodes, and bone marrow (Tizard 1996). Plasma cells are the antibody-secreting cells that are differentiated from B ly mphocytes (B-cells). IgG is the smallest of the Ig classes, therefore it is easily able to migrate from the blood into other tissues. IgG readily binds foreign antigen it come s into contact with. This leads to agglutination and opsonizati on, the process that makes foreign particles susceptible to phagocytosis by neutrophils. IgG antibodies also play a role in activating the complement system, a comple x enzymatic pathway resulting in the ultimate destruction of invading microor ganisms. There are five subclasses of equine IgG, which are IgG2a, IgG2b, Ig G2c, IgG(B), and IgG(T), which are also

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6 divided into two subclasses, IgG(T) a and IgG(T)b (Tizard 1996). These IgG subclasses are distinguished by their different -chain sequences and slight differences in biological function (Goldsby et al. 2003). IgM. IgM is the second most abundant Ig found in the serum and the third most abundant in colostrum. IgM is the fi rst class of Ig detected in a primary immune response and the first Ig produced by the neonate (Goldsby et al. 2003). The secreted form of IgM is the largest of the Igs and also has more antigen binding sites than the other isotypes. Because of its high affinity for antigen, IgM is more efficient than IgG at causing agglutination, neutralizing virus particles, and activating complement. The larger size of IgM restricts its ability to diffuse from the blood to other tissues. Through spec ialized binding sites, secretory cells in the respiratory and gastrointestinal tr act are able to transport IgM molecules across mucosal linings. Once released into the intestinal lumen, they play an important accessory role to IgA, the most prevalent antibody found in mucosal secretions. IgA. IgA is the third most abundant serum Ig and second most abundant colostrum Ig. However, as production shifts from colostrum to milk production, IgA becomes the predominant antibody f ound milk. IgA present in colostrum, milk, and other external secretions, incl uding gastrointestinal tract secretions, primarily exists in the form of secretory IgA. Secretory IgA is different from the circulating monomeric form of IgA in serum. It is a complex molecule made up of the dimeric form of IgA attached to a glycoprotein chai n called secretory component. Secretory component mediates the transport of IgA across mucosal

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7 epithelium surfaces and provides protecti on from degradation by proteases that are abundant in the mucosal environment. T he primary function of IgA is to prevent attachment of antigens to body surfaces. IgA can also serve as an opsonin and activate the complement syst em, although not as efficiently as IgG. Mucosal immunity. The majority of IgA is produced by plasma cells in mucosal lymphoid tissues, which are located underneath the respiratory and gastrointestinal epithelium. The daily produc tion of secretory Ig A is greater than that of any other Ig isotype, mainly bec ause of the sheer size of the intestine (Abbas et al. 2000). Most invasions by pathogenic organisms occur through ingestion or inhalation. In the inte stine, secretory IgA binds to pathogenic organisms and provides protection by prev enting their attachment to mucosal cells. Secretory IgA has been shown to successfully prevent attachment of bacteria such as Salmonella Vibrio cholerae and Neisseria gonorrhoeae in the gastrointestinal tract (Goldsby et al. 2003). IgE. IgE is a minor class of Ig found in very low concentrations in the serum of a healthy horse. IgE, like IgA, is primarily synthesized by plasma cells beneath epithelial surfaces (Tizard 1996). The primary function of IgE is to activate mast cells, which are responsible for the reactions characteristic of a hypersensitivity reaction, such as hive s or anaphylactic shock. IgE is also responsible for immunity to parasitic worms. Each class of Ig plays a unique role in the protection of both mare and foal from disease. An immune system functioning at optimum capacity is essential for the mare to produce a healthy and viable foal. Because the mare has a diffuse

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8 epitheliochorial placenta, no significant transfer of Ig molecules across the placental barrier can occur during gestati on (Jeffcott 1974). The main vehicle for transfer of immunologic protection from the mare to the foal is the colostrum. Colostrum rich with maternal antibodies wi ll increase the chances for the foal to successfully deal with antigenic stimul ation it faces soon after birth. Passive Immunity Foals are born with essentially no circulating Igs, although measurable quantities of IgG and IgM may be detected in the serum at birth (LeBlanc 1990, Vivrette 2001). The acquisition of maternal antibodies by the newborn foal within the first 24 hours of life is essential for t he foal’s survival. Prior to parturition, selective concentration of Igs from t he blood occurs in the mare’s mammary gland to form the antibody component of t he colostrum (Jeffcott 1972). When the foal ingests colostrum, specialized epithel ial cells of the small intestine absorb the large Ig molecules present in the colostrum through pinocytosis (Jeffcott 1974). Passive immunity obtained by the foal from the ma re is dependant upon the colostrum Ig content, the quantity of colostrum ingested, and the successful absorption of Igs by the new born foal’s digestive tract (Tizard 1996). Failure of any of these processes is known as “failu re of passive transfe r” (FPT). Important factors associated with the colostrum that influence successful passive transfer of maternal antibodies include colostra l Ig concentration, time of colostrum ingestion, and occurrence of prelac tation colostrum loss (McGuire et al. 1977, LeBlanc et al. 1992). If the foal is not able to nurse or the colostrum is of poor quality, administration of colostrum from a colostrum bank or a colostrum substitute is

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9 important to insure that t he foal receives the essential antibodies that provide protection against pathogens. After 24 hours, the mechanism for absorption of large immunoglobulin molecules in the sm all intestine is no longer functional. This cease in function is referred to as “gut closure.” There are therapies available for a foal that has not su ccessfully ingested an adequate quantity of colostrum before gut closure occurs, incl uding IV administration of equine plasma or commercially available Ig supplem ents. However, colostrum contains beneficial factors including leukocyt es, hormones, growth factors, and constituents that inhibit bacterial coloni zation in the intestine, which makes it preferable to IV immunoglobul in therapy for the treatment of FPT (Vivrette 2001). Colostrum The mare secretes colostrum for only a re latively short period of time. It is manufactured in the mammary gland duri ng the last two weeks of pregnancy and is secreted the first time the foal su ckles (McCue 1993). The colostrum contains high concentrations of three classes of Igs. IgG concentration is high at birth, but rapidly declines within the first 24 hours post-parturition (Pearson et al. 1984). Colostral IgA and IgM are lower than Ig G at birth (McCue 1993). As lactation shifts from colostrum to milk production during the first day of lactation, IgA becomes the predominant class of Ig found in mare’s m ilk (Norcross 1982). The average content of the thr ee classes of Igs found in mare’s colostrum and milk are shown in Table 2-2.

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10 Table 2-2. Immunoglobulin content of mare’s colostrum and milk Fluid IgG IgA IgM Colostrum (mg/dL) 1500 to 5000 500 to 1500 100 to 350 Milk (mg/dL) 20 to 50 50 to 100 5 to 10 Adapted from Tizard 1996: Veterinary Immunology: An Introduction p. 242 Table 19-1. W.B. Saunders Co., Philadelphia. Prelactation Many factors can affect the Ig concent ration of the colo strum. One of the main determinants of colostral Ig content at parturition is whether or not the mare experienced prelactation colostrum loss prio r to parturition. Premature lactation, or “prelactation” is one of the most important causes of FPT due to colostrum loss (McCue 1993). It is relatively co mmon for mares to lactate prior to parturition, with the caus e presumably associated with hormonal changes or certain conditions such as impending aborti on, twin pregnancy, placentitis, and premature placental separation (Je ffcott 1974, McCue 1993). Mares that prematurely lactate for longer than 24 hours before foaling tend have lower colostral IgG concentrations than in ma res that do not pr ematurely lactate (Leblanc 1990). Morris et al. (1985) found a significant upw ard linear trend in the percentage of mares that prelacta ted as colostral IgG decreased. Breed There have been reports demonstrating t hat breed of the mare can affect colostrum Ig content. In a study including Thoroughbred, Arabian, and Standardbred mares, breed of mare signi ficantly affected colostral IgG concentration (LeBlanc et al. 1992). In another st udy, the mean IgG

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11 concentration in the colostrum was 9, 6911,639 mg/dL in 14 Arabian mares and 4,6082,138 mg/dL in 22 T horoughbred mares (Pearson et al. 1984). Kohn et al. (1989) reported a mean colostral IgG conc entration of 8,3296,206.8 mg/dL in 36 Standardbred mares. This value is within the range reported in a study of 136 Standardbred mares by Morris et al. (1985). In another study, the mean IgG colostral concentration in 21 QH mares was found to be 5,843722 mg/dL (LeBlanc et al. 1986). More investigation is needed to conclusively determine the exact degree of influence that breed has on colostral Ig content. Age The age of the mare may also correla te to colostrum quality. Pearson et al. (1984) suggests that age of t he dam is a possible factor that influences colostral Ig concentration. In a study that in cluded 293 mares, mean colostral IgG concentration was highest in mares bet ween 3 and 10 years old, and FPT was most prevalent in foals whose da ms were >15 years old (LeBlanc et al. 1992). Clabough et al. (1991) reported a possible asso ciation of an age >12 years old with FPT. However, other studies s uggest that mare age does not have a significant effect on colo strum Ig content. Morris et al. (1985) reported that mare age did not significantly affect colo strum IgG content in a study of 136 Standardbred mares aged 3 to 24. The di screpancies between these reports may be due to variations in the time of colo strum sample collection. Future studies with greater sample sizes and less variati on may further elucidate the effect of age on colostrum Ig content.

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12 Foal Diarrhea Diarrhea is one of the most common health problems experienced by foals. It is characterized by an increase in the water content of t he feces and/or an increase in the frequency of defecation. Enteritis is a similar condition characterized by diarrhea along with inflamma tion of the intestinal tract. If left untreated for more than a few days, other problems may arise such as dehydration, electrolyte im balance, and even death. Identifying the cause can be a challenge because there is a myriad of pathogenic organisms that can cause diarrhea in sucking foals. The most commo n noninfectious cause of foal diarrhea is associated with foal heat of the mare which occurs between 7 and 12 days of age (Cohen 1997). This is usually self-limit ing and can resolve itself with minimal medical treatment. Other noninfectious caus es of diarrhea in young foals include nutritional causes, gastric ulceration, and antibiotic administration (Cohen 1997). Rotavirus Diarrhea that is pathogenic in nature pr esents a major concern for horse operations, primarily because of the infe ctious nature of t he organisms that cause diarrhea. One extremely contagious viral cause of diarrhea in young foals is rotavirus Rotavirus is the most common cause of foal enteritis in central Kentucky, Ireland, and Great Britain (Dwyer 1993). Alt hough the mortality rate of rotavirus infection is low, there is a signifi cant cost involved for treatment with fluid and drug therapy, increased labor for the care of sick foals, and disinfection of facilities to contain the outbreak (Dwyer 1993).

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13 Salmonella Diarrhea can also be caused by many different species of bacteria. The most common cause of bacterial diarrhea a nd enteritis in foals is considered to be Salmonella (Cohen 1997, Spier 1993). The genus Salmonella is a diverse population of Gram-n egative bacteria. Salmonella typhimurium is the most common strain that causes diseas e, although many other strains of Salmonella have been implicated in cases of salmonellosis. In the host, Salmonella is capable of colonizing in the intestinal tract where it can invade the mucosal epithelium and spread to other locations (Spier 1993). When bacterial invasion occurs in other parts of the body, this condition is termed septicemia. This is a serious condition with a survival rate of only 26% reported in a study of 38 cases of septicemic foals admitted to a ve terinary hospital for treatment (Koterba et al. 1984). Septicemia can also occur from in vasion by many other bacterial species besides Salmonella Clostridium Clostridium perfringens and Clostridium difficile are two species of Grampositive bacteria t hat have been associated with enter itis and diarrhea in foals (Traub-Dargatz and Jones 1993, Jones et al. 1988). Infection with C. perfringens in foals was associated with a high case-mo rtality risk of 68% in a retrospective case study investigating 125 foals admi tted to a veterinary teaching hospital (East et al. 2000). Another study r eported a mortality risk of 54% with this infection (East et al. 1998). The majority of foals reported to have C. perfringens associated enteritis hav e been under 3 days of age (Traub-Dargatz and Jones 1993).

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14 Other Bacteria The Gram-negative bacteria Escherichia coli is rarely associated with diarrhea in foals (Cohen 1997). However, E. coli accounted for 56% of all bacteria cultured from the blood in a study that examined 38 septicemic foals (Koterba et al. 1984). Another study found that E. coli was one of the most frequent causes of death in septicemic foals less than one week old (Platt 1973). Other less common bacterial causes of f oal diarrhea that hav e been reported are Rhodococcus equi Bacteroides fragilis and Compylobacter jejuni but the clinical significance of these organi sms is not notable (Cohen 1997). Mannan Oligosaccharides Carbohydrates play a unique role wit hin living systems. The function of a carbohydrate will vary depending on its stru cture and location within a biological system. Carbohydrates are important stru ctural components of the majority of cell-surface and secreted proteins of animal cells (Osborn and Khan 2000). Carbohydrates are also a major source of metabolizable energy in the diet. Oligosaccharides are formed when 2-10 monosaccharide molecules are joined together to form a larger molecule. More than 10 monosaccharide molecules joined together would constitute a polysa ccharide. Mannose is a monosaccharide that forms the building block of MOS. The small intestine does not contain the digestive enzymes required to br eak down mannan oligosaccharide bonds, therefore they arrive at the large inte stine intact after ingestion and passage through the small intestine (Strickling et al. 2000). Mannose-based oligosaccharides occur naturally in cell walls of the yeast Saccharomyces cerevisiae and are relatively easy to obtain by centrifugation from a lysed yeast

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15 culture (Spring et al. 2000). The commercially available product Bio-Mos (Alltech, Inc., Nicholasville, KY) is a source of MOS from Saccharomyces cerevisiae cell walls. This product was introduced in 1993 as a feed additive for broiler chickens (Hooge 2003). Lectins are carbohydrate-binding proteins that mediate interactions of cells with their environment through their initia l interactions with other cell surface carbohydrates (Osborn and Khan 2000). Ma nnose residues on the surface of intestinal epithelial cells serve as re ceptor binding sites for certain pathogens with type-1 fimbriae that contain mannos e-specific lectins (Ofek and Beachey 1978, Oyofo et al. 1989b, Spring et al. 2000, Rckendorf et al. 2002). Adherence to the intestinal cell wall is a prerequisite for the initiation of colonization by pathogenic organisms in the gastr ointestinal tract (Ferket et al. 2002). Once binding by the pathogenic organism occurs translocation across the intestinal wall and subsequent enteric infection can occur (Iji et al. 2001, Ferket et al. 2002). In vitro Agglutination Studies Mannan oligosaccharide preparations have been shown to agglutinate pathogens with mannose-spec ific type-1 fimbriae in vitro Spring et al. (2000), in an attempt to investigate the ability of diffe rent enteric pathogens and coliforms to trigger MOS agglutination, showed t hat MOS agglutinated 7 of 10 strains S. typhimurium and S. enteritidis and 5 of 7 strains of E. coli in vitro Strains of S. cholerasuis S. pullorum and Campylobacter did not result in MOS agglutination. Another study using several human isolates of E. coli showed high mannosebinding activity of the bacterial cell s with the addition of D-mannose (Ofek and

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16 Beachey 1978). This same study al so demonstrated that D-mannose could displace over 90% of E. coli that had already adhered to in testinal epithelial cells in vitro In another study, E. coli with type-1 mannose-specific lectins did not attach to mammalian cells in the pres ence of supplemental mannose (Salit and Gotschileh 1977). Intestinal Environment Studies Efforts to demonstrate that MOS has the same effect on bacterial populations in the intestinal envir onment have proven successful. Oyofo et al. (1989b) investigated the adherence of S. typhimurium to the small intestine of one-day-old chicks and found that adherence wa s significantly inhibited in the presence of D-mannose. Droleskey et al (1994) found that incubation of S. typhimurium with cultured chick intestinal segments resulted in the loss of mucosal epithelial integrity evidenc ed by the complete shedding of the epithelium. It was found in this study that the addition of 2. 5% D-mannose to the incubation medium inhibit ed the loss of epithelial ce lls. When provided in the drinking water of chicks, mannose significant ly reduced intestinal colonization of S. typhimurium (Oyofo et al. 1989a). When supplemented to the diet of hens, MOS affected the birds’ intestinal microflora by increasing the Bifidobacterium spp. and Lactobacillus spp., while decreasing colonization of S. enteritidis (Fernandez et al. 2002). The addition of 4,000 ppm of MOS to the diet of threeday-old chicks that were orally challenged with S. typhimurium significantly reduced cecal S. typhimurium concentrations on day 10 when compared with controls (Spring et al. 2000). In a separate trial using S. dublin as the challenge organism, the number of chicks that tested positive for Salmonella in the cecum

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17 at day 10 was less in chicks that were consuming the MOS supplemented diet (Spring et al. 2000). In growing turkeys younger than six weeks of age, MOS supplemented birds had a higher total anaerobe count and a lower level of C. perfringens in cecal cultures (Finuance et al. 1999). These studies demonstrate that pathogens with the mannose-specific type-1 fimbriae adsorb to MOS instead of attaching to intestinal epithelial cell walls and, therefore, move through the intestine with less probabilit y of initiating disease. There have also been investigations into the intestinal environment effects of MOS supplementation to the diet of a companion animal species. Strickling et al. (2000) found that in dogs, fecal C. perfringens tended to be lower when supplemented with 5g MOS/kg diet DM. Th e same study found no diet effects on fecal bifidobacteria numbers or ileal bacteria colony forming units. Dogs supplemented with 2 g MOS/ day had significantly lower fecal total aerobe and tended to have greater Lactobacillus populations (Swanson et al. 2002). 1 g/kg BW/day of MOS supplem entation to the diets of 4 fema le beagle dogs resulted in a lower fecal pH (Zentek et al. 2002). Performance The use of antibiotics in food animal diets is a common practice in the industry. Antibiotics have been shown to improve growth, feed efficiency, and overall herd health when used in poultry, s wine, and cattle production diets. Due to consumer concerns and increasing r egulatory restrictions, producers have begun searching for alternatives to the us e of antibiotic growth promotants in production diets. Mannan oligosaccharide suppl ementation has been

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18 investigated as an alternative to antibiotic supplementation to enhance performance characteristics. Poultry. Numerous studies have been cond ucted in poultry, because MOS was first introduced in 1993 as a feed addi tive for broiler chicken diets (Hooge 2003). Over 150 broiler chicken pen trials were analyzed to collectively determine the effects of MOS-supplem ented diets versus negative and/or positive control (antibiotic) diets. The conclusion wa s that MOS supplementation results in bodyweight and feed conversion ratios co mparable to antibiotic supplementation while significantly lowering mortalit y rate (Hooge 2003). Fritts and Waldroup (2000) reported that turkey poults fed 0.10% MOS had the same feed conversion as poults fed 55 ppm of the antibiotic bacitracin methylene disalicyclate (BMD) and significantly better feed conversion than negative controls. In a study conducted to determine growth effects in turkey hens with diets supplemented with MOS or antibiotics (BMD and virginia mycin), investigators found that birds fed 0.5g/ kg MOS supplemented birds had improved feed efficiency over birds fed the control or antibioti c-supplemented diet (Hulet et al. 2000). Mannan oligosaccharide was shown to be a suitable alternative to terramycin as a growth enhancer in turkey diets when no difference in bodyweight was seen between control and treatment animals after 105 days of supplementation (Stanley et al. 2000). Both MOS and antibiotic growth pr omoters enhance the efficiency of nutrient utilization by reduc ing the competition between the host and intestinal pathogens. Without microbial competition fo r energy and other nutrients, there

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19 are more nutrients available for absor ption and metabolism by the host (Ferket et al. 2002). It is well documented that antibio tic supplementation to poultry diets increases the utilization of dietary energy (Buresh et al. 1985, Harms et al. 1986, Ferket et al. 2002). Although MOS supplementation has proved to be as effective as antibiotics in improving utilization of dietary energy the mechanism is unclear and likely different than that used by antib iotic growth promotant s. Possibly it is related to the improvement of characteri stics of the intesti nal lining (Ferket et al. 2002) or changes in digestive enzyme activiti es that are stimul ated by MOS (Iji et al. 2001). Swine. Pregnant sows fed 0.20% MOS thr ee weeks prior to farrowing and 0.10% MOS throughout the 21-day lactatio n period produced piglets with heavier litter birth and weaning weights (O’Quinn et al. 2001). In a factorial experiment conducted to determine the effects of tw o levels of MOS (0 and 0.10%) and three levels of protein (20, 23, and 26%) in piglet diets, MOS supplementation improved weight gain and f eed consumption regardless of protein level (Kim et al. 2000). The addition of minerals such as Zn and Cu in excess of NRC recommendations to swine diets is a co mmon practice to improve performance (NRC 1998). However, this may result in an undesirable effect on the bacteria responsible for waste degradation in lagoons (Gilley et al. 2000). The addition of 0.20% MOS to the diets of nursery pigs increased average daily gain and average daily feed intake in the absence of excess zinc but had no effect or a negative effect in the presenc e of excess zinc (LeMieux et al. 2003). In a separate trial of the same study, t he interactive effects of antibiotics

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20 (oxytetracycline and neomycin) and MOS and of Zn and MOS were evaluated. Mannan oligosaccharide improved pig perfo rmance only when fed in combination with an antibiotic and no excess Zn. There was no effect or a negative effect in the presence of excess Zn or in the absence of an antibiotic (LeMieux et al. 2003). Mannan oligosaccharides have also been considered as an alternative to excess Cu supplementation in swine di ets for performance enhancement. The effects of MOS fed at either basal or excess levels of Cu in the diets of weanling and growing-finishing pigs were de termined in an experiment by Davis et al. (2002). From day 0 to day 10, average da ily gain, average daily feed intake, and gain : feed increased when MOS was added to diets containing basal levels of Cu. From day 10 to day 38, pigs fed diets containing excess Cu had greater ADG and ADFI regardless of MOS addition (Davis et al. 2002). The researchers concluded that MOS addition to swine diet s results in a moderate improvement in gain and feed efficiency, but the magnitude of response is not as great as that seen with the addition of excess levels of Cu (Davis et al. 2002). Should trace mineral supplementation restrictions on swine diets come into effect, MOS supplementation may provide a viable performance-enhancing alternative. Cattle. The production-enhancement effects of MOS supplementation in cattle diets have received relatively less attention than supplementation of poultry or swine diets. Heinrichs et al. (2003) investigated the effects of MOS or antibiotics in dairy calf milk replacer diets, and found the addition of 4 g MOS/ day was as effective as antibiotic use to maintain normal fecal fluidity and

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21 consistency and to decrease scours severi ty. Feed consumption increased when MOS was included in the diet, but this di d not result in a difference in growth measures (Heinrichs et al. 2003). Immune Function After MOS supplementation to producti on diets proved to increase weight gain and feed efficiency, identifying the mech anism of the physiological response associated with the positive growth respons es was the next logical step. To do this, studies focused on measuring the par ameters that are r epresentative of a functional immune system. These parameter s include Ig content of the serum, lymphocyte proliferation, and response to antigenic stimulation. The main antigenic components of yeast cells are mannans present in the isolated cell wall (Ballou 1970). Mannans found in the cell walls of S. cerevisiae have been shown to induce an antigenic response in humans (Young et at. 1998) Therefore some MOS-immune system interacti on would be expected (Ferket et al. 2002). Poultry. Savage et al. (1996) fed 0.11% MOS to male turkeys for 53 days and obtained blood and bile samples at t he end of the period. The samples were analyzed using both radial imm unodiffusion (RID) and rocket immunoelectophoresis (RI). Using RID, no signifi cant differences were found, but RI analysis showed that concentrations of both blood and bile IgG and IgA were significantly increased in turkeys fed MOS (Savage et al. 1996). In a trial investigating the effects on humoral immunity in commercial laying hens, investigators injected the hens with sheep red blood cells (SRBC) suspended in a solution of bovine serum albumin ( BSA) and obtained serum samples one, two, and four weeks post-sensitization. Hens supplemented with 0.05% MOS had

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22 higher SRBC titers than controls at one week post-sensitization (Malzone et al. 2000). The BSA titers of the MOS-fed hens were numerically greater at week one and week two, but the differences were not statistically significant (Malzone et al. 2000). In broiler breeder diets, the additi on of MOS significantly increased the antibody response to infectious bursal di sease virus and also increased maternal antibody titers in the breeders’ pr ogeny (Shashidhara and Devegowda 2003). Swine. Positive immune response effect s have also been observed with MOS supplementation to swine diets. Newman and Newman (2001) supplemented sow diets with 5g MOS/ day for appr oximately 14 days prefarrowing and continued supplementation throughout lactation. At farrowing, MOS treated sows had significantly higher serum IgM and colostrum IgM levels and numerically higher colostrum IgG leve ls (Newman and Newman 2001). The piglets from the MOS treat ed sows also weighed more on day 7, 14, and 21 postfarrowing than those from unsupplement ed sows (Newman and Newman 2001). In another study evaluating sow and litte r performance, concentrations of IgA, IgG, and IgM in pre-suckle colostrum samples were increased by MOS addition to the diet. IgG showed the greatest response, followed by IgM and IgA respectively (O’Quinn et al. 2001). As found in the previous trial, the piglets from the MOS treated sows also had heavier litte r birth and weaning weights (O’Quinn et al. 2001). To determine whether MOS modulated the cell-mediated immune response of the weaned pig, Davis et al. (2002) obtained blood samples from MOS supplemented growing-fini shing pigs and measured ly mphocyte proliferation in

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23 vitro Lymphocyte proliferation did not diffe r significantly between the control and MOS supplemented pigs (Davis et al. 2002). Although not dem onstrated in this study, other researchers have demonstrat ed that MOS may have an inhibitory effect on certain lymphocyte functions (Muchmore et al. 1990, Podzorski et al. 1990). It is conceivable t hat immune function suppression could also be a means by which MOS improves gain and efficien cy, because of the shift in metabolic activity to support the body’s defense agai nst foreign antigens that occurs during immune response activation (Spurlock 1997). Another mechanism of growth enhancement in swine may be through the altera tion of intestinal microflora, as is what happens with supplementat ion with pharmacological levels of Cu (Davis et al. 2002). Cattle. The addition of 10 g MOS/ day to the diet of 40 dairy cows resulted in numerically greater serum Ig levels in calves 24-hours post-calving than in the calves of unsupplemented cows (Franklin et al. 2002). In the same study, antibody titers to rotavirus vaccination following calving were numerically greater in claves from MOS supplemented cows (Franklin et al. 2002). Dogs. Adult female dogs were supplemented with 1 g MOS per day for a 14 day period, and serum IgA concentra tions tended to be greater and the percent of white blood ce lls that were lymphocyt es was greater in dogs supplemented with MOS. Total white blood cell count and neutrophil concentration were unaffected by treatment (Swanson et al. 2002). The authors hypothesized that because serum IgG and IgM were not affected, a systemic immune response may not have occurr ed and was not the cause of the

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24 increased lymphocytes and serum IgA. T he trends for increased serum IgA and lymphocyte concentration may be due to the increased pro liferation of Blymphocytes and secretory IgA in the intestinal tract (Swanson et al. 2002). A study performed in rats reported increas ed cecal IgA contents and an increase in the proportion of IgA-presenti ng lymphocytes present in the cecal mucosal of rats fed glucomannans at 5% for three weeks (Kudoh et al. 1999). These studies have demonstrated the positive effects of MOS on Ig concentration in serum and colostrum and on immune response to antigen challenge. However, a mechanism for th is action has yet to be demonstrated. Some studies suggest that MOS supplementat ion stimulates intestinal lymphoid tissue resulting in increased dev elopment or activation (Guigoz et al. 2002, Ferket et at. 2002). The stimulatory effect may occur through a healthy population of gut microflora or “drag effects” of the indigestible oligosaccharide molecules as they move along the lengt h of the intestine (Cunningham-Rundles and Lin 1998). The activation of lymphoid ti ssue may result in greater plasma cell production by B-cells found in underlying lymphoid follicles. These plasma cells then would be able to secrete Igs that can either be secreted into the intestinal lumen when associated with secretory co mponent or end up in the circulation via transport through the lymphatic system. To determine if MOS supplementation to the diet of pregnant mares would result in a change in the total Ig concent ration of the mare’s colostrum and the serum of the mare or foal the current experiment was proposed. Previous results from work in other species suggest that MOS supplementation will increase

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25 colostrum Ig content, and therefore translate into increased foal serum Ig content after absorption of maternal antibodies is complete. Growth measurements of both mares and foals will indicate any negat ive effects of MOS supplementation on physical development. Determination of Ig content in serum and presuckle colostrum samples will indicate any c hange of immune status in the mare. Serum Ig concentration in the foals will reflect any effect on absorption of colostral Igs and initial serum Ig concentration and any long-term effect on immune status of the foal due to MOS suppl ementation of the dam.

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26 CHAPTER 3 MATERIALS AND METHODS Animals Twenty-six pregnant Thoroughbred (n=21) and Quarter Horse (n=5) mares and their subsequent foals were used in this trial. The mares ranged from 3 to 24 years of age with a mean age of 9 (STD=6 .1). The pregnant mares were paired by expected foaling dates and assigned at random to one of two treatment groups 56 days prior (d-56) to expected date of parturition. They continued on the treatment diet until 56 days post-parturi tion (d+56). The foals remained on the trial until 112 days of age (d+112). One mare leaked mi lk for 3 weeks prior to foaling, and her foal acqui red septicemia and was hospi talized for one week after birth. No data from this ma re or foal were used. Housing and Management During the course of the trial, the ma res and their foals were housed at the University of Florida’s Horse Research Center in Ocala, Florida. Pregnant mares were kept at pasture until pre-foaling signs were evident. They were then moved to a dry lot where they remained until they foaled. After foaling, the mare and her foal were moved to a small paddock for approximately one week and then were returned to pasture. A routine vaccinat ion and anthelmintic schedule for all animals on trial was followed by farm management. The University of Florida Institutional Animal Care and Use Committe e approved the protocol for this trial.

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27 Diets Treatment group 1 (n=13) served as controls and received supplement A, which consisted of 55g of ground corn as a placebo. Treatm ent group 2 (n=12) received supplement B, which consist ed of 10g of MOS (Bio-Mos, Alltech, Nicholasville, KY) mixed in 45g of gr ound corn. Supplements A and B were top dressed on the morning ration and fed to the mares from day -56 until day +56. Feeding time was at 0700 hours (AM feeding) and 1500 hours (PM feeding). Mares and foals were brought into stalls for individual feeding for both AM and PM feedings. Foals remained in the sta lls with their dam and potentially could have consumed some of her feed, depending upon her temperament and willingness to allow the foal access to her feed bucket. Both treatment groups were fed the same concentrate, HR-136, which was formulated to meet or exceed requirements for late gestating and lactating mares based on NRC recommendations (NRC 1989) when fed with bahiagrass pasture ( Paspalum notatum ) or Coastal bermudagrass hay ( Cynodon dactylon ) (see table 3-1). The amount of concentrate fed wa s adjusted according to each mare’s body condition score (BCS) to maintain a minimum BCS of 5 (see table 3-2). The mares were also fed ad-libitum Coastal bermudagrass hay and/or bahiagrass pasture in season. Trace mineralized salt blocks and fres h water were available at all times. A creep-feeder was introduced when the olde st foal was 2 months of age, and HR-136 was provided as the creep feed. Body Measurements The mares were weighed and assessed for body condition scores every 28 days. Foals were weighed at birth, d+ 7, d+14, d+28, d+56, and d+112. Foal

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28 body measurements taken at the same ti me were withers height, hip height, body length, and heart girth. The scale used was a digital walk-on scale. Body measurements were made with a sliding st ick made specifically for the purpose of taking accurate body length and height measurements. Colostrum and Blood Samples Colostrum samples were obtained from the mare after the foal was born but before it was allowed to nurse. Th ree 1 ml aliquots from each colostrum sample were placed in cryogenic tubes and frozen at -80C until further analysis. Jugular blood samples were collect ed from the mares between 0700 and 0900 hours on d-56, d-28, and d+28. Jugular bl ood samples were collected from the foals at birth before the foal was allo wed to nurse, 6 -10 hours post-parturition (referred to as 8 hour sample), and bet ween 0700 and 0900 hours on d+7, d+14, d+28, d+56, and d+112. Precision Gli de Vaccutainer brand blood collection needles (20G, 1 in.) were used to collect blood into Beckton Dickinson Vaccutainers. Samples were allowed to clot for one to two hours and then centrifuged at 3000 x G for 10 minutes to allow for separation and collection of serum. Three 1 ml aliquots from ea ch serum sample were placed in polypropylene cryogenic vials and fr ozen at -80C until further analysis. Colostrum samples and serum samples from both the mares and foals were analyzed for IgG, IgA, and Ig M content using a commercially available single radial immunodiffusion kit (SRID Kit, VMRD, Inc., Pullman, WA). Feed Sample Analysis Monthly samples were taken of HR -136 and the Coastal bermudagrass hay for the duration of the experiment. To determine dry matte r content of the

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29 samples, the concentrate and hay were fi rst put through a Wile y mill fitted with a 1mm screen to assure uniform particle size. 1 to 2 gr ams of the sample were then weighed to 4 decimal places on a Mettler balance and placed into ceramic crucibles. They were dried in a 105 C drying oven overnight and equilibrated for 1 hr. in a dessicator before weig hed again to 4 decimal places. The samples were analyzed for calcium, copper, manganese, zinc, and iron content by atomic absorpt ion spectrophotometry (Miles et al. 2001) using the Perkin-Elmer Model 5000 Atomic Absorp tion Spectrophotomet er (Perkin-Elmer Corp., Norwalk, CO). Crude protein cont ent was analyzed by first digesting the sample according to the procedure put forth by Gallaher et al. (1975) and then determining the nitrogen content of the sample using the Alpkem auto analyzer (Alpkem Corp., Clackemas, OR). Phosphor us content was determined by using a calorimetric procedure (Technicon Indus trial Systems, Tarrytown, NY) on the automated Alpkem analyzer (Alpke m Corp., Clackemas, OR). Neutral and acid detergent fiber content was determined using the Ankom fiber analyzer (Ankom Technology, Fairport New York). Prior to fat content analysis, carbohydr ates were first extracted from the sample (AOAC 1995). Fat content was t hen determined by ether extraction using a soxhlet apparatus. Statistical Analysis The treatment effect on Ig concentra tion in the serum of the mares was analyzed using PROC GLM procedures with repeated measures in SAS (SAS 1989). Ig content of the mare’s colo strum was analyzed using PROC GLM in SAS controlling for age, breed, and prelac tation. The treatment effect on Ig

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30 concentration in the serum of the foals was analyzed using PROC GLM procedures with repeated measures in SAS (SAS 1989). Treatment, sex, and breed effects on foal growth measur ements were analyzed using PROC GLM procedures with repeated measures in SAS. Significance was considered to be p<0.05, and p<0.10 was considered a trend. Table 3-1. Composition of Concentrate (HR-136) Ingredient Amount (%) Corn, cracked Oats, crimped Soybean meal (48% CP) Wheat bran Molasses, blackstrap Alfalfa meal pellets (17% CP) Limestone, ground Monocalcium phosphate Salt Vitamin premixa Vitamin Eb Lysine 98% Luprosil (mold inhibitor) Trace mineral premixc 34.25 26.50 10.00 10.00 8.00 7.50 1.50 0.80 0.75 0.30 0.15 0.05 0.10 0.10 aProvides 4,400,000IU Vit A, 440,000IU Vit D, and 35,200IU Vit E/ kg premix bProvides 44,200IU Vit E/ kg premix cProvides 7,200mg Cu, 28,000mg Zn, 28,000mg Fe, 28,000mg Mn, 80mg Co, 80mg I, and 80mg Se/ kg premix Table 3-2. Concentrate feeding rates for mares Stage Rate Late Gestation -56 d to -28 d -28 d to parturition Early Lactation Parturition to +84 d Late Lactation +84 d to +112 d 0.75% BWa 1.0% BWa 1.5% BWa 1.0% BWa aAdjust concentrate feeding for body condition score (-20% above BCS 5/ +20% below BCS 5)

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31 CHAPTER 4 RESULTS AND DISCUSSION Feed Analysis The average nutrient compositi on of HR-136 (n=7) and the Coastal bermudagrass hay (n=3) from monthly samp les taken throughout the trial period are presented in Table 4-1. Table 4-1. Concentrate (HR-136) and Coastal bermudagrass hay nutrient composition analysis Nutrient HR-136 Hay Dry Matter (%) Crude protein (%) Fat (%) ADF (%) NDF (%) Calcium (%) Phosphorus (%) Cu (ppm) Mn (ppm) Zn (ppm) Fe (ppm) 94.78 1.1 15.07 1.0 2.62 0.8 9.46 1.0 25.01 1.5 1.16 0.4 0.62 0.2 48.17 2.3 124.83 4.7 137.67 4.3 267.50 4.8 88.44 3.6 5.15 1.1 1.64 0.4 39.21 1.5 80.35 1.1 0.46 0.3 0.15 0.1 2.29 0.9 51.67 4.3 23.00 1.7 101.67 7.4 All values Mean SE Dry matter basis (except dry matter) Growth Analysis For the duration of the experiment, mares maintain ed good body condition and remained at a healthy body weight during both gestation and lactation (See Table 4-2). Mares from treatment 1 (cont rol) foaled 6 fillies and 7 colts, and mares from treatment 2 (MO S) foaled 6 fillies and 6 colts. There were no statistically significant differences (p > 0.05) between contro l and foals from MOS supplemented mares for any of the growth paramet ers measured (see Table 4-

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32 3). Control foals weighed 50. 31.7 kg at birth and gained a total of 140.04.1 kg during the 112-day trial. Foals from MOS supplemented mares weighed 48.90.8 kg at birth and gained 142.64.4 kg over the trial period. Control foals grew 26.41.0 cm in height, 29.61.0 cm in hip height, 44.21.3 cm in length, and 47.11.1 cm in heart girth. Foals from MOS supplemented grew 25.41.0 cm in height, 28.51.1 cm in hip height, 46.01. 4 cm in length, and 48.21.2 cm in heart girth. Average daily gain measurements for both treatments were consistent with previously published data (Kavazis and Ott 2003, Lawrence et al. 1991). The influence of sex on foal growth was minimal during the 112-day trial period. Because there was no significant treatment effect on growth, the data from the two treatment groups were pooled (see Table 4-4) to determine any influence of sex on growth. Average height was the only growth parameter that showed any trend towards significant diffe rence between males and females. At d+112, colts tended to be taller than fillies (p=0.08). To determine the influence of breed on foal growth, data from the two treatment groups were pooled (see Table 45). There was a trend for TB foals to be taller than QH foals at d+112 (p=0.08). TB foals had greater birth body length than QH foals (p=0.04). The total gain in body length tended to be greater for QH foals than TB foals (p=0.06).

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33 Table 4-2. Influence of treatment on mare weight and body condition scores Weight (kg) Body Condition Score Day Treatment 1 (Control) Treatment 2 (MOS) Treatment 1 (Control) Treatment 2 (MOS) d-56 567.611.7 577.19. 3 4.80.1 4.80.1 d-28 580.410.3 586.99. 7 4.60.2 5.00.1 d0 514.110.4 526.110. 2 4.60.1 4.70.1 d+28 528.012.5 538.2 10.6 4.60.1 4.80.1 d+56 529.412.0 543.1 11.0 4.60.2 4.80.1 d+84 535.211.6 547.3 11.1 4.60.2 4.90.2 d+112 531.811.0 544.99. 7 4.60.2 4.60.1 All values are Mean SE Table 4-3. Influence of treatment on foal growth Growth parameter Treatment 1 (Control) Treatment 2 (MOS) Birth weight (kg) 50.3 1.7 48.91.8 d+112 weight (kg) 190.25.0 191.55.4 Total weight gain (kg) 140.04.1 142.64.4 Birth withers height (cm) 98.21.0 99.91.1 d+112 withers height (cm) 124.60.7 125.30.8 Total withers height gain (cm) 26.41.0 25.41.0 Birth hip height (cm) 100.41.1 102.41.2 d+112 hip height (cm) 130.00.9 131.00.9 Total hip height gain (cm) 29.61.0 28.51.1 Birth length (cm) 73.31.0 72.81.1 d+112 length (cm) 117.51.1 118.81.2 Total length gain (cm) 44.21.3 46.01.4 Birth heart girth (cm) 80.11.2 80.01.3 d+112 heart girth (cm) 127.21.5 128.21.6 Total heart girth gain (cm) 47.11.1 48.21.2 All values are LSMean SE

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34 Table 4-4. Influence of sex on foal growth Growth parameter Colts Fillies Birth weight (kg) 49.9 1.7 49.2.8 d+112 weight (kg) 188.3.6 190.7.9 Total weight gain (kg) 140.8.1 191.0.4 Birth withers height (cm) 100.0.0 98.2.1 d+112 withers height (cm) 125.8.7 124.1.8 Total withers height gain (cm) 26.0.0 25.9.0 Birth hip height (cm) 102.0.1 100.8.2 d+112 hip height (cm) 130.7.9 130.3.9 Total hip height gain (cm) 28.6.0 29.5.1 Birth length (cm) 72.4 1.0 73.7.1 d+112 length (cm) 117.1.1 119.1 Total length gain (cm) 44.7.3 45.5.4 Birth heart girth (cm) 79. 5.2 80.6.3 d+112 heart girth (cm) 127.4.5 128.0.6 Total heart girth gain (cm) 47.9.1 47.4.2 All values are LSMean SE Table 4-5. Influence of breed on foal growth Growth parameter QH TB Birth weight (kg) 50.0.4 49.2.2 d112 weight (kg) 195.2.5 186.5.0 Total weight gain (kg) 145.2.3 137.3.0 Birth withers height (cm) 97.8.5 100.3.8 d112 withers height (cm) 124.0.0 126.0.5 Total withers height gain (cm) 26.0.0 25.8.0 Birth hip height (cm) 100.3.6 102.6.8 d112 hip height (cm) 129.9.2 131.1.6 Total hip height gain (cm) 30.0.5 28.6.7 Birth length (cm) 71.2.5* 74.9.7* d112 length (cm) 118.4.6 117.8 Total length gain (cm) 47.2.9 42.9.0 Birth heart girth (cm) 80.1.7 80.0.9 d112 heart girth (cm) 129.1.2 126.4.1 Total heart girth gain (cm) 49.0.6 46.3.8 All values are LSMean SE *p=0.04

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35 Mare Serum Immunoglobulins Mare serum Ig content was analyzed wit h treatment as the only source of variation. IgG For IgG serum concentration, cont rol mares averaged 1807.6.8 mg/dL on d, 1525.1.5 mg/dL on d 0, and 1 929.1.6 mg/dL on d+28. Mares fed MOS had an average serum IgG conc entration of 1789.8.8 mg/dL on d 56, 1405.5.2 mg/dL on d 0, and 1874. 7.1 mg/dL on d+28 (see Table 46). Although control mares had numerically higher serum IgG concentration at d56, d 0, and d+28, the differences were not significant. The control mares had a numerically higher IgG concentration at t he start of the exper iment, and this is the likely reason control mare IgG concentration remained slightly above IgG concentration in mares fed MOS fo r the duration of the trial. Table 4-6. Influence of treatment on mare serum IgG co ncentration Day Treatment 1 (Control) Treatment 2 (MOS) d-56 (mg/dL) 1807.5 130.8 1789.8 125.8 d0 (mg/dL) 1525.0 191.5 1405.5 108.2 d+28 (mg/dL) 1929.1 163.6 1874.7 96.1 All values are Mean SE IgA Average serum IgA concentration for c ontrol mares was 349.2.7 mg/dL at d, 424.9.1 mg/dL at d 0, and 378.6.9 mg/dL at d+28. Mares fed MOS had an average serum IgA concentra tion of 360.1.4 mg/dL at d, 419.0.0 mg/dL at d 0, and 412.0.2 mg /dL at d+28 (see Table 4-7). Mares fed MOS had numerically higher serum IgA concentration than control mare

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36 serum IgA concentration throughout the durat ion of the experiment. Because this difference was present at the start of t he experiment, this effect was not likely due to MOS supplementation. Table 4-7: Influence of treatment on mare serum IgA concentration Day Treatment 1 (Control) Treatment 2 (MOS) d-56 (mg/dL) 349.2 38.7 360.1 40.4 d0 (mg/dL) 424.9 31.1 419.0 44.0 d+28 (mg/dL) 378.6 31.9 412.0 68.2 All values are Mean SE IgM Serum IgM concentration for control mares averaged 109.213.8 mg/dL on d–56, 115.612.7 mg/dL on d 0, and 101.921.6 mg/dL on d+28. Mares fed MOS averaged 98.88.8 mg/dL on d–56, 113.1 10.3 mg/dL on d 0, and 89.115.8 mg/dL on d+28 (see Table 4-8). Control ma re serum IgM concentration remained numerically above mares fed MOS for the duration of the experiment, and this was not likely due to the treatment. Table 4-8. Influence of treatment on mare serum IgM co ncentration Day Treatment 1 (Control) Treatment 2 (MOS) d-56 (mg/dL) 109.2 13.8 98.8 8.8 d0 (mg/dL) 115.6 12.7 113.1 10.3 d+28 (mg/dL) 101.9 21.6 89.1 15.8 All values are Mean SE Discussion There were no significant differences fo r IgG, IgA, or IgM concentration in samples obtained from the ma res at d-56, d0, or d+28. This result agrees with results obtained in a previous study performed in 40 pregnant dairy cows to evaluate the effect of MO S supplementation on the immune status of dairy cows

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37 and their calves. No overt differences in serum Ig levels were observed between cows that were supplement ed with 10 g/MOS/day and t he control group (Franklin et al. 2002). Savage et al. (1996) reported an increase in plasma IgG and bile IgA in male turkeys after 53 days of MOS supplem entation at 0.11% of the total diet. These turkeys were started on the suppl ementation protocol immediately after birth at one day of age. The data was analyzed using two different assays, and only one assay, rocket immuno-electr ophoresis, revealed any significant difference in plasma IgG and bile IgA le vels between the two treatment groups. The assay that did not reveal any di fference was radial immunodiffusion, the same assay that is used in the current experiment. Mare Colostrum Immunoglobulins Mare colostrum data were anal yzed to determine the treatment, prelactation occurrence, age, breed, treatment*age interaction, and treatment*breed interaction effects. Previ ous research suggests that prelactation, age, and breed can affect the Ig concentra tion in mare colostrum (McCue 1993, Leblanc 1990, Morris et al. 1985, LeBlanc et al. 1992, Pearson et al. 1984, LeBlanc et al. 1986, and Clabough et al. 1991), therefore it is important to consider these factors when evaluating colostrum content. IgG Colostrum IgG concentration for mare s fed MOS was significantly higher than in control mares (p=0.05) when all sources of variation were taken into consideration in the overall ANOVA model. Colostrum IgG concentration for mares fed MOS was significantly higher than control mares due to treatment

PAGE 48

38 (p=0.03), prelactation (p=0.006), and treatm ent*age (p=0.02). All other sources of variation were not significantly different between treatments (see Tables 4-9 and 4-10). Table 4-9. Influence of tr eatment, prelactation occu rrence, age, and breed on colostrum IgG Source of Variation Mean s.e. Treatment Control (n=13) (mg/dL) 10242.2 1181.1 MOS (n=12) (mg/dL) 12824.0 2245.6 Prelactation Y (n=4) (mg/dL) 6934.2 1174.0 N (n=21) (mg/dL) 12555.5 1429.0 Age <12 years (n=18) (mg/dL) 11663.8 985.7 >12 years (n=7) (mg/dL) 11253.0 3585.5 Breed TB (n=20) (mg/dL) 12388.2 1542.8 QH (n=5) (mg/dL) 8627.1 1461.0 Table 4-10. ANOVA generated P values for colo strum IgG from a statistical model which included treatment prelactation, age, breed, with treatment*age and treatm ent*breed interactions Model p=0.05 Treatment p=0.0334 Prelactation p=0.0063 Age p=0.1377 Breed p=0.4803 Treatment*Age p=0.0163 Treatment*Breed p=0.7593 IgA Colostrum IgA concentration for mare s fed MOS was significantly higher than in control mares (p=0.05) when all sources of variation were taken into consideration in the overall ANOVA model. Colostrum IgA concentration for mares fed MOS was significantly higher than control mares due to treatment

PAGE 49

39 (p=0.008), prelactation (p=0.008), age (p =0.02), and treatment *age (p=0.04). All other sources of variation were not signi ficantly different bet ween treatments (see Tables 4-11 and 4-12). Table 4-11. Influence of treatment, pr elactation occurrence, age, and breed on colostrum IgA Source of Variation Mean s.e. Treatment Control (n=13) (mg/dL) 47.7 9.5 MOS (n=12) (mg/dL) 112.1 38.9 Prelactation Y (n=4) (mg/dL) 43.6 12.6 N (n=21) (mg/dL) 88.0 24.9 Age <12 years (n=18) (mg/dL) 67.5 15.9 >12 years (n=7) (mg/dL) 106.6 57.5 Breed TB (n=20) (mg/dL) 85.0 26.4 QH (n=5) (mg/dL) 62.7 19.8 Table 4-12. ANOVA generated P values for colo strum IgA from a statistical model which included treatment prelactation, age, breed, with treatment*age and treat ment breed*interactions Model p=0.05 Treatment p=0.0080 Prelactation p=0.0079 Age p=0.0177 Breed p=0.1796 Treatment*Age p=0.0356 Treatment*Breed p=0.7746 IgM Colostrum IgM concentration for mare s fed MOS tended to be higher than in control mares (p=0.06) when all s ources of variation were taken into consideration in the overall ANOVA model. Colostrum IgM concentration for mares fed MOS tended to be higher t han control mares due to treatment

PAGE 50

40 (p=0.08). The treatment*age interaction was significantly higher for mares fed MOS (p=0.04). All other sources of vari ation were not significantly different between treatments (see Tables 4-13 and 4-14). Table 4-13. Influence of treatment, pr elactation occurrence, age, and breed on colostrum IgM Source of Variation Mean s.e. Treatment Control (n=13) (mg/dL) 133.2 12.2 MOS (n=12) (mg/dL) 154.1 8.1 Prelactation Y (n=4) (mg/dL) 126.3 23.0 N (n=21) (mg/dL) 147.5 7.7 Age <12 years (n=18) (mg/dL) 154.7 6.9 >12 years (n=7) (mg/dL) 120.0 15.6 Breed TB (n=20) (mg/dL) 149.1 7.4 QH (n=5) (mg/dL) 125.0 20.6 Table 4-14. ANOVA generated P values for colo strum IgM from a statistical model which included treatment, prelactation, age, breed, with treatment*age and treatment *breed interactions Model p=0.06 Treatment p=0.0764 Prelactation p=0.2994 Age p=0.2195 Breed p=0.9598 Treatment*Age p=0.0350 Treatment*Breed p=0.9545 Discussion The colostrum Ig concentration for a ll isotypes was highly variable. This could be due to many factors, some of which could not be accounted for in the statistical model. Ig content was dete rmined by single radial immunodiffusion (SRID) using raw colostrum samples. This method has been used in previously

PAGE 51

41 published reports (Zou et al. 1998, Turner et al. 2003). However, there have been other published reports that describe extracting the colostral whey (located between the superficial fat layer and the pr ecipitate) to remove cellular debris and fat for use in the SRID assay (Waelchli et al. 1990, Pearson et al. 1984, LeBlanc et al. 1986, LeBlanc et al. 1992). It is possible that using colostral whey for the determination of Ig content coul d minimize the extreme variation in colostrum Ig values. One of the QH mares from the control treatm ent was dropped from the statistical analysis because the Ig concentration in her colostrum was a significant outlier to the average distributi on of expected Ig concentration in mare colostrum. The Ig content of her co lostrum was 45,409.5 mg/dL for IgG, 278.5 mg/dL for IgA, and 220 mg/dL for IgM. These values were much higher than those from the other mare s in the study and average values reported in the literature (Tizard 1996, LeBlanc et al. 1992, Morris et al 1985, Pearson et al. 1984, LeBlanc et al. 1986). In order to maintain a representative sample of the mare population, her data was not used for colostrum analysis. When controlled for variation due to pr elactation colostrum loss, age, and breed, IgG and IgA content of colostru m was significantly enhanced by MOS supplementation, and IgM content tended to be enhanced. This result agrees with previous findings of two other st udies evaluating the effect of MOS supplementation on colostrum immunoglo bulin content. Newman and Newman (2001) reported significantly increased presuckle colostrum IgM levels (p=0.04) in MOS supplemented sows and numerically gr eater IgM levels in colostrum 24-

PAGE 52

42 hour post-farrowing. They also reported numerically increased presuckle and 24hour post-farrowing colostrum IgG levels in MOS supplemented sows when compared to controls, but there was no effect on colostrum IgA concentration (Newman and Newman 2001). In another st udy involving sows, the addition of MOS resulted in significantly incr eased IgG (p=0.007) and IgM (p=0.03) concentration in presuckle colostrum (O’Quinn et al. 2001). Presuckle IgA levels tended to be greater in MOS supple mented sows (p=0.06) (O’Quinn et al. 2001). There was a significant effect due to treatment (p=0.03), prelactation (p=0.006), and treatment*age interaction (p =0.02) for IgG colostrum content. The highly significant prelactation effect is expected, because lost colostrum cannot be replaced due to its limited production. The negative effect of prelactation on colostrum Ig content has previous ly been well documented (McCue 1993, Jeffcott 1974, Leblanc 1990, and Morris et al. 1985). There was a significant effect due to treatment (p=0. 008), prelactation (p=0.008), age (p=0.02), and treatment* age interaction (p=0.04) for IgA colostrum content. The highl y significant prelactation effect is expected, for reasons stated previously. Age effect on co lostrum content is not well defined, however some reports show that mean colostrum Ig concentration was highest in mares between 3 and 10 years old and lower in mares over 12 years old (LeBlanc et al. 1992, Clabough et al. 1991). Other reports s how no effect of age on colostral Ig content (Morris et al. 1985, Kohn et al. 1989). In this experiment, mares that were >12 years old had hi gher mean colostrum IgA concentration. This may be due to the fact that many of the mares used in this study were

PAGE 53

43 maiden mares. There was one maiden mare in the control treatment group and seven maiden mares in the MOS treatm ent group. It has been reported that primiparous (maiden) mares have lowe r colostrum Ig concentrations than multiparous mares, and this may explain the significant age effect on IgA content (Jeffcott 1972, Erhard et al. 2001). Although no significant age effect was seen for colostrum IgG or IgM, a significant treatment*age in teraction was seen for all three isotype concentrations, and the unbalanc ed distribution of maiden mares in the treatment groups may have contributed to this effect. The treatment effect approached signifi cance (p=0.08) and there was a significant effect due to treatment*age in teraction (p=0.02) for IgM colostrum content. The occurrence of prelactati on did not significantly affect IgM concentration, possibly because the over all concentration of IgM in equine colostrum is relatively low (McCue 1993). Foal Serum Immunoglobulins Foal serum immunoglobulin concentra tion was analyzed with treatment as the only source of variation. IgG A detectable amount of IgG was present in foal serum at birth prior to colostrum ingestion. There were no signifi cant differences in IgG concentration for any of the foal serum samples collected. The mean foal serum IgG concentration for each sample collection is presented in Table 4-15. Figure 4-1 presents this data in graphic format to illustrate the change in foal serum IgG concentration over time.

PAGE 54

44 Table 4-15. Influence of treatment on foal serum Ig G concentration Day/hour Treatment 1 (Control) Treatment 2 (MOS) 0 hour (mg/dL) 82.6 11.4 88.2 11.4 8 hour (mg/dL) 1478.8 238.0 1420.0 227.6 d+7 (mg/dL) 1431.7 172.8 1322.5 159.4 d+14 (mg/dL) 1275.6 146.8 1229.7 121.6 d+28 (mg/dL) 1234.1 121.7 1322.7 129.7 d+56 (mg/dL) 930.1 65.0 907.3 58.1 d+112 (mg/dL) 653.9 27.6 648.7 15.2 All values are Mean SE

PAGE 55

45 02004006008001000120014001600 0h 8h d+7 d+14 d+28 d+56 d+112IgG, mg/dl Control MOS Figure 4-1: Mean foal serum IgG concentration

PAGE 56

46 IgA There was no detectable amount of IgA in foal serum at birth prior to colostrum ingestion. There were no statisti cally significant differences in foal serum IgA concentration, however, foal s from control mares tended to have higher serum IgA concentration than foal s from mares fed MOS at 6 -10 hours post-parturition (p=0.09). The mean foal serum IgA concentration for each sample collection is presented in Table 4-16. Figure 4-2 presents this data in graphic format to illustrate the change in fo al serum IgA concentration over time.

PAGE 57

47 Table 4-16. Influence of treatment on foal serum IgA concentration Day/hour Treatment 1 (Cont rol)Treatment 2 (MOS) 0 hour (mg/dL) 0 0 8 hour (mg/dL) 214.7 30.8 122.8 27.9 d+7 (mg/ dL) 81.3 7.6 84.7 21.7 d+14 (mg/ dL) 59.6 4.1 62.3 10.0 d+28 (mg/ dL) 67.5 7.5 61.4 3.5 d+56 (mg/dL) 98.9 8.5 93.9 9.4 d+112 (mg/dL) 140.4 11.2 119.1 7.3 All values are Mean SE

PAGE 58

48 050100150200250 0h 8h d+7 d+14 d+28 d+56 d+112IgA, mg/dl Control MOS Figure 4-2: Mean foal serum IgA concentration

PAGE 59

49 IgM A detectable amount of IgM was present in foal serum at birth prior to colostrum ingestion. There were no signifi cant differences in IgM concentration for any of the foal serum samples collected. The mean foal serum IgM concentration for each sample collection is presented in Table 4-17. Figure 4-3 presents this data in graphic format to illustrate the change in foal serum IgM concentration over time.

PAGE 60

50 Table 4-17. Influence of treatment on foal serum IgM co ncentration Day/hour Treatment 1 (Control) Treatment 2 (MOS) 0 hour (mg/dL) 17.0 2.0 17.5 1.5 8 hour (mg/dL) 40.2 6.8 41.0 5.0 d+7 (mg/dL) 33.9 3.9 35.5 4.1 d+14 (mg/dL) 37.1 4.0 40.9 3.3 d+28 (mg/dL) 46.7 6.9 41.3 3.3 d+56 (mg/dL) 77.8 8.3 67.3 8.6 d+112 (mg/dL) 109.2 9.0 119.5 7.6 All values are Mean SE

PAGE 61

51 020406080100120140 0h 8h d+7 d+14 d+28 d+56 d+112IgM, mg/dl Control MOS Figure 4-3: Mean foal serum IgM concentration

PAGE 62

52 Discussion There were no statistically significant differences for any serum concentration at any hour or day sampling period. Because foals were not fed the MOS directly, any immune response woul d be expected to come from the ingestion of colostrum with a higher concentration of immunoglobulins and predominantly be apparent in the fi rst weeks of life. Franklin et al. (2002) reported numerically greater serum IgG and IgM concentration 24 hours postcalving in calves from cows supplement ed with MOS, but the differences were not significant. LeBlanc et al. (1986) reported that mean foal serum IgG concentration increases concurrently with increasing colostral IgG concentration. Morris et al. (1985) reported similar results and showed a highly significant correlation between colostral IgG and foal serum IgG concent ration (r=0.584, p<0.001). The positive asso ciation between colostru m Ig and foal serum Ig concentration after colostrum ingestion is well documented. Ho wever, there was no noticeable difference between the two tr eatment groups in this experiment, even with significantly higher Ig concentra tion in colostrum of mares fed MOS. This is most likely due to the fact that peak values of passively obtained maternal antibodies are reached ar ound 18 hours after birth (Jeffcott 1972). The foal serum samples taken in this experiment to determine successful passive transfer were obtained between 6 and 10 hours postparturition. At this time, full absorption of maternal antibodi es is not yet complete (Kohn et al. 1989). Evaluation of foal serum from 6 – 12 hours post-partu rition is appropriate to determine if proper absorption of mater nal antibodies is occurring so that a treatment protocol for suspected FPT c an be implemented if necessary (Erhard

PAGE 63

53 et al. 2001, Vivrette 2001). However, obtaining a 24 to 36 hour post-parturition serum sample would have more accurately reflected the complete absorption of Igs from the mare’s colostrum (Morris et al. 1985). Serum IgA concentration in foals fr om control mares tended to be higher than in foals from mares fed MOS 6 10 hours post-parturition (p=0.09). The reason for this trend for foal serum IgA c oncentration to be higher in control foals is unclear. Intestinal permeability is selective in the horse, with IgG and IgM preferentially absorbed while IgA remains in the intestine (Tizard 1996). The IgA content of colostrum was most signific antly increased by MO S supplementation, but this was not reflected in the 6 –10 hour foal serum samples. The principal form of IgA in human colostrum is secret ory IgA, which is resistant to the proteolytic effects of enzymes present in the neonatal gut (Chapel et al. 1999). Perhaps the increased quantity of IgA in the colostrum of mares fed MOS was primarily in the form of secretory IgA, and significant amounts could not be immediately absorbed across the intestinal epithelium. There is evidently some absorption of colostrum IgA as shown by the initial increase in serum IgA concentration and subsequent decrease for bot h treatment groups over the first 7 days of life. A foal serum sample obt ained 24 to 36 hours post-parturition may have reflected higher peak absorption of IgA in the foals from MOS supplemented mares due to t he higher content of IgA in the colostrum of mares fed MOS.

PAGE 64

54 CHAPTER 5 SUMMARY AND CONCLUSIONS Supplementing pregnant mares with 10 g/MOS/day 56 days prior to expected date of parturition through the fi rst 56 days of lactation significantly increased IgG and IgA content and tended to increase IgM content in the colostrum. Supplementation had no effect on serum Ig c ontent of mares or foals, except at 8 hours after birth when control foals had significantly higher serum IgA concentration than foals from mares fed MO S. Because the timing of foal serum sampling at 8 hours after birth was not ideal, this may have prevented an accurate portrayal of full absorption of maternal antibodies. However, no ill effects were seen as a result of MOS s upplementation, and greater Ig content in the colostrum increases the chance for successful passive transfer to occur. Supplementation of pregnant mare diet s with MOS may be a beneficial practice to help protect the mare from pathogenic organisms and to boost the Ig content of her colostrum. Although not investigated in this experiment, MOS supplementation of sucking and weanling diets ma y be beneficial as well. Foal s that are provided a source of MOS may be better protect ed from pathogenic organisms present in the environment and theref ore may have a reduced incidence of illness caused by these organisms. This is a prom ising area for further research.

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55 LITERATURE CITED Abbas, Abul K., Lichtman, A. H., and Pober, Jordan S. (2000) Cellular and Molecular Immunology W.B. Saunders Co., Philadelphia. Association of Offici al Analytical Chemists (AOAC) (1995) Official Methods of Analysis of AOAC. AOAC Internationa l, Arlington, VA. Ballou, C.E. (1970) A study of the immunochemistr y of three yeast mannans. J Biol Chem 245(5) 1197-1203. Buresh, R.E., Miles, R.D., and Harms, R. H. (1985) Influence of virginiamycin on energy utilization when turkey poults were fed ad libitum or restricted. Poult Sci 64 1041-1042. Chapel, H., Haeney, M., Misbah, S., and Snowden, N. (1999) Essentials of Clinical Immunology. Blackwell Science, Oxford. Clabough, D.L., Levine, J.F., Grant, G.G ., and Conboy, H. S. (1991) Factors associated with failure of passive tr ansfer of colostral antibodies in Standardbred foals. J Vet Intern Med 5(6) 335-340. Cohen, N.D. (1997) Diarrheal Diseases of Foals. In: Current Therapy in Equine Medicine Ed: N.E. Robinson, W. B. Saunders Co., Philadelphia. Cunningham-Rundles, S. and Lin, D. H. (1998) Nutrition and the immune system of the gut. Nutrition 14 573-579. Davis, M.E., Maxwell, C.V., Brown, D. C., de Rodas, B.Z., Johnson, Z.B., Kegley, E.B., Hellwig, D.H. and Dvorak, R.A. (2002) Effect of dietary mannan oligosaccharides and(or) pharmacologica l additions of copper sulfate on growth performance and imm unocompetence of weanling and growing/finishing pigs. J Anim Sci 80 2887-2894. Drolesky, R.E., Oyofo, B.A., Hargis, B.M., Corrier, D.E., and DeLoach, J.R. (1994) Effect of mannose on Salmonella typhimurium -mediated loss of mucosal epithelial integrity in cu ltured chick intestinal segments. Avian Diseases 38 275-281. Dwyer, R.M. (1993) Ro taviral diarrhea. Vet Clin North Am Equine Pract 9 311319.

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56 East, L.M., Dargatz, D.A., Traub-Dargatz, J.L. and Sav age, C.J. (2000) Foalingmanagement practices associated with the occurrence of enterocolitis attributed to Clostridium perfringens infection in the equine neonate. Preventive Veterinary Medicine 46 61-74. East, L.M., Savage, C.J., Tr aub-Dargatz, J.L., Dickinson C.E., and Ellis, R.P. (1998) Enterocolitis associated with Clostridium perfringens infection in neonatal foals: 54 cases (1988-1997). J Am Vet Med Assoc 212 17511756. Erhard, M. H., Luft, C., Remler, H. P., and Stangassinger, M. (2001) Assessment of colostral transfer and systemic av ailability of immunoglobulin G in newborn foals using a newly developed enzyme-linked immunosorbant assay (ELISA) system. J Anim Phys Anim Nutr 85 164-173. Ferket, P.R., Parks, C.W. and Grimes, J.L. (2002) Benefits of Dietary Antibiotic and Mannanoligosaccharide Supp lementation for Poultry. Proc Multi-State Poultry Meeting Indianapolis, Indiana, May 14-16, 2002. Fernandez, F., Hinton, M., and Van Gils, B. (2002) Dietary mannanoligosaccharides and their effect on chi cken caecal microflora in relation to Salmonella enteritidis colonization. Avian Pathology 31(1) 49-58. Finuance, M.C., Dawson, K.A., Spring, P ., and Newman, K.E. (1999) The effect of mannan oligosaccharide on the composition of the microflora in turkey poults. Poult Sci 78(Suppl 1) ,77. Franklin, S.T. Newman K.E., and Newman, M.C. (2002) Evaluation of mannan oligosaccharide on the i mmune status of dairy cows and their calves. J Anim Sci 80 (Suppl) 192. Fritts, C.A. and Waldroup, P.W. (2 000) Utilization of Bio-Mos mannan oligosaccharide in turkey diets. Poult Sci 79(Suppl 1 ) 126. Gallaher, R. N., Weldon, C. O., and Frut al, J. G. (1975) An aluminum block digester for plant and soil analysis. Soil Sci Soc Amer Proc 39 803-806. Gilley, J.E., Spare, D.P., Koelsch, R.K., Schulte, D.D ., Miller, P.S., and Parkhurst, A.M. (2000) Phototrophic anaerobic lagoons as affected by copper and zinc in swine diets. Trans Am Soc Agric Eng 43 1853-1859. Goldsby, R.A., Kindt, T.J., Osbor ne, B.A., and Kuby, J. (2003) Immunology W.H. Freeman and Co., New York.

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57 Guigoz, Y., Rochat, F. Perruisseau-Carri er, I. Rochat, I., and Schiffrin, E.J. (2002) Effects of oligosaccharide on the faecal flora and non-specific immune system in elderly people. Nutr Res 22 13-25. Harms, R.H., Ruiz, N., and Miles, R.D. (1986) Influence of virginiamycin on broilers fed four levels of energy. Poult Sci 65 1984-1986. Heinrichs, A. J., Jones, C. M., and He inrichs, B.S. (2003) Effects of mannan oligosaccharide or antibiotics in neonatal diets on health and growth of dairy calves. J Dairy Sci 86(12) 4064-4069. Hooge, D.M. (2003) Bro iler chicken performance may improve with MOS. Feedstuffs 75(1) Hulet, R.M., Lorenz, E.S., and Saleh, T.M. (2000) Turkey hen growth response to diets supplemented with either antibiotic or mannan oligosaccharide. Poult Sci 79 (Suppl 1) S186. Iji, P. A., Saki, A. A., and Tivey, D. R. (2001) Intestinal structure and function of broiler chickens on diets supplem ented with a mannan o ligosaccharide. J Sci Food Agric 81 1186-1192. Jeffcott, L.B. (1972) Passive immunity and its transfer with s pecial reference to the horse. Biol Rev 47 439-464. Jeffcott, L.B. (1974) Some practical aspects of the transfer of passive immunity to newborn foals. Equine Vet J 6 109-115. Jones, R.L., Shideler, R.K. and Cocke rell, G.L. (1988) Association of Clostridium difficile with foal diarrhea. Equine Infectious Diseases V: Proc. of the 5th International Conference 236. Lexington, KY, October 1998. Kavazis, A. N. and Ott, E.A. (2003) Grow th rate in Thoroughbred horses raised in Florida. J Eq Vet Sci 23(8) 353-357. Kim, J.D., Hyun, Y., Sohn, K.S., Kim, T.J., Woo, H.J. (2000) Effects of mannan oligosaccharide and protein levels on growth performance and immune status in pigs weaned at 21 days of age. J Anim Sci Tech 42(4) 489-498. Kohn, C. W., Knight, D., Hueston, W. Jacobs, R., and R eed, S. M. (1989) Colostral and serum IgG, IgA, and IgM concentrations in Standardbred mares and their foals at parturition. JAVMA 195(1) 64-68. Koterba, A.M., Brewer, B.D., and Tarplee, F.A. (1984) Clinical and clinicopathological characteristics of the septicaemic neonatal foal: Review of 38 cases. Equine Vet J 16(4) 376-383.

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58 Kudoh, K., Shimizu, J., Ishiyama, A., Wada, M., Takita, T., Kanke, Y., and Innami, S. (1999) Secretion and excret ion of immunoglobulin A to cecum and feces differ with type of indigestible saccharides. J Nutr Sci Vitaminol (Tokyo) 45(2) 173-181. Lawrence, L. A., Murphy, M., Bump, K. Weston, D., Key, J. (1991) Growth responses in hand-reared and naturally raised Quarter Horse foals. Eq Pract 13 19-26. LeBlanc, M. M. (1990) Imm unologic Considerations. In: Equine Clinical Neonatology Ed: A.M. Koterba, W.H. Drummond, and P.C. Kosch, Lea & Febiger, Philadelphia. LeBlanc, M. M., McLaurin, B.I., and Bo swell, R. (1986) Relationships among serum immunoglobulin in foals, colost ral specific gravity, and colostral immunoglobulin concentration. JAVMA 189(1) 57-60. LeBlanc, M.M., Tran, T., Bald win, J.L. and Pritchard, E. L. (1992) Factors that influence passive transfer of immunoglobulins in foals. JAVMA 200 179183. LeMieux, F.M., Southern, L.L. and Bidner T.D. (2003) Effect of mannan oligosaccharides on growth performance of weanling pigs. J Anim Sci 81 2482-2487. Malzone, A., Paluch, B., Lilburn, M.S. and Sefton, A.E. ( 2000) Modulation of humoral immunity in commercial la ying hens by a dietary probiotic. Poult Sci 79(Suppl 1) 165. McCue, P.M. (1993) Lactation. In: Equine Reproduction Ed. McKinnon, A.O. and Voss, J.L., Lea & Febiger, Philadelphia. McGuire, T.C. and Crawford, T.B. ( 1973) Passive immunity in the foal: measurement of imm unoglobulin classes and specific antibody. Am J Vet Res 34 1299-1303. McGuire, T.C., Crawford, T.B., Hallowell, A.L., and Macomber, L.E. (1977) Failure of colostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals. JAVMA 170 1302-1304. Miles, P. H., Wilkinson, N. H., and McDowell, L. R. (2001) Analysis of Minerals for Animal Nutrition Research. Departm ent of Animal Sciences, University of Florida, Gainesville, FL. Morris, D.D., Meirs, D.A ., and Merryman, G.S. (1985) Pa ssive transfer failure in horses: Incidence and causativ e factors on a breeding farm. Am J Vet Res 46(11) 2294-2299.

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59 Muchmore, A.V., Sathyam oorthy, N., Decker, J., and Sherblom, A.P. (1990) Evidence that specific high-mannose o ligosaccharides can directly inhibit antigen-driven T-cell responses. J Leuko Biol 48 457-464. National Research Council (NRC). 1989. Nutrient Requirements of Horses 5th ed. National Academy Press, Washington, DC. National Research Council (NRC). 1998. Nutrient Require ments of Swine 10th ed. National Academy Press, Washington, DC. Newman, K.E. and Newman, M.C. (2001) Evaluation of Mannan Oligosaccharide on the microflora and immunoglobulin status of sows and piglet performance. J Anim Sci 79 189. Nezlin, R. (1998) The Immunoglobulins: st ructure and function. Academic Press, San Diego. Norcross, N.L. (1982) Secretion and co mposition of colostrum and milk. JAVMA 181(10) 1057-1060. Ofek, I. and Beachey, E.H. (1978) Mannos e binding and epit helial cell adherence of Escherichia coli Infection and Immunity 22(1) 247-253. O'Quinn, P.R., Funderburke, D.W. and Tibbe tts, G.W. (2001) Effects of dietary supplementation with mannan oligosaccharides on sow and litter performance in a commercial production system. J Anim Sci 79 212. Osborn, H.M.I. and Khan, T.H. (2000) Oligosaccharides: Their synthesis and biological roles. Oxford University Press Inc., New York. Oyofo, B.A., DeLoach, Corrier, D.E ., Norman, J.O., Ziprin, R.L., and Mollenhauer, H.H. (1989a) E ffect of carbohydrates on Salmonella typhimurium colonization on broiler chicks. Avian Diseases 33 531-534. Oyofo, B.A., Droleskey, R.E., Norman, J.O., Mollenh auer, H.H., Ziprin, R.L., Corrier, D.E. and DeLoach, J.R. (1989b) Inhibition by mannose of in vitro colonization of chicken small in testine by Salmonella typhimurium. Poult Sci 68 1351-1356. Peakman, M. and Vergani, D. (1997) Basic and Clinical Immunology Churchill Livingstone, New York. Pearson, R.C., Hallowell, A.L., Bayley, W.M., Torbeck, R.L ., and Perryman, L.E. (1984) Times of appearance and disappear ance of colostral IgG in the mare. Am J Vet Res 45(1) 186-190. Platt, H. (1973) Septicaemia in t he foal. A review of 61 cases. Br Vet J 129 221229.

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60 Podzorski, R.P., Gray, G.R. and Nelson, R. D. (1990) Different effects of native Candida albicans mannan and mannan-derived o ligosaccharides on antigen-stimulated ly mphoproliferation in vitro J Immunol 144 707-716. Rckendorf, N., Sperliing, O., and Lindhors t, T.K. (2002) Trivalent cluster mannosides with aromatic partial struct ure as ligands for the type-1 fimbrial lectin of Escherichia coli Aust J Chem 55 87-93. Salit, I.E. and Gotschlich, E.C. ( 1977) Type 1 Escherichia coli pili: characterization of binding to monkey kidney cells. J Exp Med 146 11821194. SAS (1989) Statistical Analysis System: A Users Guide SAS Inst., Version 6, 4th ed. Cary, NC. Savage, T.F., Cotter, P.F. and Zakrzewska E.I. (1996) The effect of feeding a mannanoligosaccharide on i mmunoglobulins, plasma IgA, and bile IgA of Wrolstad MW male turkeys. Poult Sci 75 (Suppl 1) Shashidhara, R.G., and Devegowda, G. (2003) Effect of dietary mannan oligosaccharide on breeder pr oduction traits and immunity. Poult Sci 82(8) 1319-1325. Spier, S.J. (1993) Salmonellosis. In: Veterinary Clinics of North America: Equine Practice Ed: W.B. Saunders Co., Philadelphia. pp 385-397. Spring, P., Wenk, C., Dawson, K.A. and Newman, K.E. (2000) The effects of dietary mannaoligosaccharides on ceca l parameters and the concentrations of enteric bacteria in the ceca of salmonella-chall enged broiler chicks. Poult Sci 79 205-211. Spurlock, M.E. (1997) R egulation of metabolism and growth during immune challenge: an overview of cytokine function. J Anim Sci 75 1773-1783. Stanley, V.G., Brown, C., Sefton, A.E. (2000) Compar ative evaluation of yeast culture, mannanoligosacc haride and antibiotic on per formance of turkeys. Poult Sci 79 (Suppl 1) S186. Strickling, J.A., Harmon, D.L., Dawson, K.A., Gross, K.L ( 2000) Evaluation of oligosaccharide addition to dog diets: influences on nutrient digestion and microbial populations. An Feed Sci & Tech 86 205-219. Swanson, K.S., Grieshop, C.M., Flicki nger, E.A., Bauer, L.L., Healy, H.P., Dawson, K.A., Merchen, N.R. and F ahey, G.C., Jr. (2002) Supplemental fructooligosaccharides and mannanol igosaccharides influence immune function, ileal and total tract nutrient digestibilities, microbial populations and concentrations of protein catabol ites in the large bowel of dogs. J Nutr 132 980-989.

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61 Tizard, I.R. (1996) Veterinary Immunology: An Introduction W.B. Saunders Co., Philadelphia. Traub-Dargatz, J.T. and Jones R.L. (1993) Clostridia-associated enterocolitis in adult horses and foals. In: Veterinary Clinics of North America: Equine Practice Ed: W.B. Saunders Co., Ph iladelphia. pp. 411-421. Turner, J. L., Arns, M.J., and Minton, J. E. (2003) Ca se study: Effects of nonspecific immunostimulation of prepartum mares on colostral quality and foal immune function. Prof Anim Sci 19 62-67. Vivrette, S. (2001) Colostrum and oral i mmunoglobulin therapy in newborn foals. Compendium 23(3) 286-291. Waelchli, R.O., Hssig, M., Eggenberger Nussbaumer, M. (1990) Relationships of total protein, specific gravity, viscosity, refractive index and latex agglutination to immunoglobulin G c oncentration in mare colostrum. Eq Vet J 22(1) 39-42. Young, M., Davies, M.J., Bailey, D., Gradw ell, M.J., Smestad-Paulsen, B., Wold, J.K., Barnes, R.M.R., and Hounsell, E.F. (1998) Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae Glycoc J 15 815-822. Zentek, J., Marquart, B., and Pietrzak T. (2002) Intestinal effects of mannanoligosaccharides, transgalact ooligosaccharides, lactose, and lactulose in dogs. J Nutr 132 1682S-1684S. Zou, S., Brady, H. A., Hurley, W. L. ( 1998) Protective factors in mammary glan secretions during the peripar turient period in the mare. J Eq Vet Sci 18(3) 184-188.

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62 BIOGRAPHICAL SKETCH Kelly Robertson Spearman was born on August 28, 1977, in Tuscaloosa, Alabama. She lived there fo r 3 years until her family moved to Montevallo, AL, in 1980. In 1986, they moved to Anniston, AL which is where Kelly’s interest in horses began. Her 5th grade English teacher also taught riding lessons, and although her family knew nothing about horse s, they agreed to bi-monthly riding lessons that would fit into an alr eady busy schedule of piano lessons, choir practice, gymnastics, and church activi ties. Kelly and her family moved back to Tuscaloosa in 1989, and her interest in hor ses continued to grow, as she started taking dressage lessons at a local barn. This sparked an enduring fascination with the art of dressage and its training philosophies. The family moved to Montgomery, AL, in 1994, just before her senior year, and she graduated with a 3.9 GPA from Jeff erson Davis High School. While in high school, she also worked as a pharma cist assistant, attended a performing arts school for piano, and was the accom panist for the school’s jazz choir. She received a freshman academic scholarship to Auburn University, and graduated in 1999 with a B.S. degree in animal and dairy sciences, with cum laude honors. While at Auburn, she was a member of t he university honors program, a charter member of the Auburn Equestrian Team, and vice president of the horseman’s club. For three summers during her coll ege career, she was the wrangler for a working cattle ranch in northwest Colo rado. She is a member of Alpha Zeta

PAGE 73

63 honorary fraternity, and Gamma Sigma Delta honor society. After graduating from Auburn University, Kelly moved to Missouri for 1 year and worked as an assistant trainer, working with young horses. She moved back to Alabama, and became a North America Handicapped Riding Association certified instructor, and she was an instructor for Special Equestrians in Birmingham, AL. In 2001, Kelly received a presidential fellowship to study Equine Nutrition at the University of Florida under Dr. Edgar A. Ott. While at the University of Florida, Kelly taught numerous undergraduate equine classes, and participated in many equine nutrition research projects. S he will continue her education at the University of Florida, as she works toward a Ph.D. in equine nutrition.


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Title: Effect of Mannan Oligosaccharide (MOS) Supplementation on the Immune Status of Mares and their Foals
Physical Description: Mixed Material
Copyright Date: 2008

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EFFECT OF MANNAN OLIGOSACCHARIDE (MOS) SUPPLEMENTATION ON
THE IMMUNE STATUS OF MARES AND THEIR FOALS













By

KELLY ROBERTSON SPEARMAN


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

U NIVE RS ITY OF FLORI DA


2004


































This thesis is dedicated to my parents, Steve and Charlotte Robertson, who have
continuously provided me with unconditional love and support for the past 26
years.















ACKNOWLEDGMENTS

I am very fortunate to be able to work under Dr. Edgar A. Ott, who served

as both chairperson of my committee and a constant source of wisdom. I also

extend gratitude to Dr. Saundra H. TenBroeck and Dr. Steeve Giguere, who

served on my committee and dedicated their time and effort to reviewing my

thesis.

I also would like to thank others who contributed to the completion of this

project, including the staff at the University of Florida Horse Research Center and

Jan Kivipelto, Kylee Johnson, and Tonya Stevens; and of course, the mares and

their foals. I also thank Joel McQuagge and Dr. Tim Marshall for their

encouragement and friendship.

Finally, I thank my parents, brother, grandparents, and extended family for

all of their love and support during the course of my study at the University of

Florida. I am extremely fortunate to have such a wonderful family, whose faith in

me sometimes exceeds the faith I have in myself. They have taught me about the

unconditional love of God, who gives me the peace and strength necessary to

accomplish anything.





















TABLE OF CONTENTS

Page


AC K NOWLE DG EM ENTS ................. ................. iii........ ....


LI ST OF TABLE S ................. ................. vi......... ...


LI ST OF FIG URE S................. .............. viii


ABSTRACT ................. ................. ix.............


CHAPTER


1 INTRO DU CTION ............... .............


2 REVIEW O F L ITE RATU RE ........._... ......___ ...............4.


The Immune System .............. ...............4.....
Passive Im unity ........._.._ ..... .___ ...............8.....
Col ostru m .............. ...............9.....
Pre lactation .........._.._ ....._.. ...............10.....
B reed .............. ...............10....

A g e ................. ................. 11..............
Foal Diarrhea .........._.._ ....._.. ...............12.....
Rotavirus .............. ...............12....
Salmonella .............. ...............13....
Clostridium .............. ...............13....
Other Bacteria .............. ...............14....
M annan Ol igosaccharid es ................. ......... ...............14......
In vitro Agglutination Studies .............. ...............15....
Intestinal Environment Studies .............. ...............16....
Performance .............. ...............17....
Immune Function .............. ...............21....


3 MATERIALS AND METHODS .............. ...............26....


A n im als ................ ......... ..... ............2
Housing and Management .............. ...............26....
D iets ........................... ..............2

Body Measurements................. .............2
Colostrum and Blood Samples .............. ...............28....
Feed Sample Analysis............... ...............28












Statistical Analysis .............. ...............29....


4 RE SU LTS AN DDISCUSSION .............. ...............3 1....


Feed Analysis .............. ...............3 1....
Growth Analysis ................. .. .. ...............31
Mare Serum Immunoglobulins .............. ...............35....

I gG............... ...............3 5..
I gA ................ ...............3.. 5..............
IgM .............. ...............36....
Discussion ................... ................ ...............36......

Mare Colostrum Immunoglobulins .............. ...............37....

I gG............... ...............37..
I gA ............. ....._.. ...............3 8...
IgM .............. ...............39....
D discussion ........._................_.. ...............40....

Foal Serum Immunoglobulins .............. ...............43....

I gG............... ...............43..
I gA ............. ....._.. ...............46....
IgM .............. ...............49....
Discussion ............._. ...._... ...............52....


5 SU MMARY AN D CON CLU SIONS ............... .............5


LITE RATU RE CITED............... ...............55.


BIOGRAPHICAL SKETCH ............. ..............62.....

















LIST OF TABLES


Table page

2-1. Immunoglobulin concentration in serum of mature horses............... .................5

2-2. Immunoglobulin content of mare's colostrum and milk............... ..................1

3-1. Composition of Concentrate (H R-1 36) ........._.. ........ ......__ ..........30

3-2. Concentrate feeding rates for mares .............. ...............30....

4-1. Concentrate (HR-136) and Coastal bermudagrass hay nutrient composition
analysis .............. ...............3 1....

4-2. Influence of treatment on mare weight and body condition scores .................33

4-3. Influence of treatment on foal growth............... ...............33.

4-4. Influence of sex on foal growth .............. ...............34....

4-5. Influence of breed on foal growth .............. ...............34....

4-6. Influence of treatment on mare serum IgG concentration ............... ...............35

4-7: Influence of treatment on mare serum IgA concentration............... ..............3

4-8. Influence of treatment on mare serum IgM concentration ............... ...............36

4-9. Influence of treatment, prelactation occurrence, age, and breed on colostrum
IgG .............. ...............38....

4-10. ANOVA generated P values for colostrum IgG from a statistical model
which included treatment, prelactation, age, breed, with treatment*age and
treatment*breed interactions............... ..............3

4-11. Influence of treatment, prelactation occurrence, age, and breed on
colostrum I gA .............. ...............39....

4-12. ANOVA generated P values for colostrum IgA from a statistical model
which included treatment, prelactation, age, breed, with treatment*age and
treatment breed*interactions...........................3










4-13. Influence of treatment, prelactation occurrence, age, and breed on
colostrum IgM ................. ...............40.......... .....

4-14. ANOVA generated P values for colostrum IgM from a statistical model
which included treatment, prelactation, age, breed, with treatment*age and
treatment*breed interactions............... ..............4

4-15. Influence of treatment on foal serum IgG concentration............... ..............4

4-16. Influence of treatment on foal serum IgA concentration ................ ...............47

4-17. Influence of treatment on foal serum IgM concentration .............. ................50

















LIST OF FIGURES

Fiogrure page

4-1: Mean foal serum IgG concentration .............. ...............45....

4-2: Mean foal serum IgA concentration .............. ...............48....

4-3: Mean foal serum IgM concentration ................. ...............51...............















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

EFFECT OF MANNAN OLIGOSACCHARIDE (MOS) SUPPLEMENTATION ON
THE IMMUNE STATUS OF MARES AND THEIR FOALS

By

Kelly Robertson Spearman

May 2004

Chair: Edgar A. Ott
Major Department: Animal Sciences

Newborn foals are susceptible to many pathogens that can cause health

problems such as diarrhea, sepsis, and even death. The foal obtains the

antibodies necessary to combat the onslaught of these pathogens from the

mare's colostrum when it is ingested within the first 24 hours of life.

Previous research in other species suggests that mannan oligosaccharide

(MOS) supplementation to the diet has positive effects on immune function,

including increased serum and colostrum immunoglobulin levels. An experiment

was designed to identify the effects of MOS supplementation to the diet on

colostrum and serum immunoglobulin concentrations in the pregnant mare and

serum immunoglobulin concentration in her foal. Twenty-six pregnant

Thoroughbred (n=21) and Quarter Horse (n=5) mares were paired by expected

foaling dates and assigned at random to the treatment or control group.

Treatment mares received 10 g of MOS mixed in 45 g of ground corn in the









morning ration. Control mares received 55 g of ground corn in the morning ration.

All mares were fed a concentrate designed to provide NRC recommended or

higher nutrient intake when fed with Coastal bermudagrass hay or bahiagrass

pasture ad libitum in season. Both treatments began 56 days before the

expected foaling date (Day -56) for each mare and continued through 28 days

post-parturition (Day +28). The IgG, IgM, and IgA values were determined on

mare serum at Days -56, 0, and +28. The IgG, IgM, and IgA values were

determined on colostrum collected before the foal had nursed. IgG, IgM and IgA

values were determined on foal serum collected at 0 hour (before foals had

nursed), 6 to 10 hours post-parturition, and at Day +7, +14, +28, +56, and +112

of age.

The mares receiving MOS supplementation had higher colostrum IgA

(p=0.008) and IgG (p=0.033); and tended to have higher IgM (p=0.076)

concentrations when controlled for prelactation colostrum loss, age, and breed.

Prelactation adversely affected colostrum IgG (p=0.006) and IgA (p=0.008)

immunoglobulin concentration, but had no effect on IgM concentration. There

were no significant differences between treatments for mare IgG, IgM, and IgA

serum levels at any collection period. Foals from control mares tended to have

higher serum IgA concentration at 6 to 10 hours post-parturition than did foals

from mares fed MOS (p=0.09). There were no other significant differences in foal

serum immunoglobulin concentrations at any collection period. This trial suggests

that MOS supplementation to pregnant mares increases colostrum

immunoglobulin content.














CHAPTER 1
INTRODUCTION

Suckling foals are susceptible to many pathogens that cause various health

problems such as diarrhea, enteritis, septicemia, and even death. These

problems can result in major veterinary expenses and financial loss for horse

breeding operations. The diarrhea that is associated with foal heat occurs in foals

7 to 12 days after birth and is considered the most common cause of diarrhea in

young foals (Cohen 1997). This generally causes minimal stress for the foal and

can resolve itself with little to no medical treatment. The diarrhea that occurs just

after birth or later during lactation is often pathological in nature, and is a major

health concern, because it can result in severe dehydration, reduced growth, and

even death. Many organisms have been indicated in the development of

diarrhea, including Clostridium perfringens (East et al. 2000), Clostridium difficile

(Jones et al. 1988), Salmonella typhimurium and other Salmonella spp. (Spier

1993), and rotavirus (Dwyer 1993).

The foal obtains the antibodies necessary to combat the onslaught of

these pathogens from the mare's colostrum when it is ingested within the first 24

hours of life (Jeffcott 1974). The immunoglobulin found in the greatest quantity in

mare colostrum is IgG, followed by IgA and IgM (McGuire et al. 1973). Studies

have shown that colostral IgG concentration is highly correlated with foal serum

IgG concentration 18 hours after birth (LeBlanc et al. 1992). Failure of the mare

to provide the foal with adequate antibodies via the colostrum may necessitate









the administration of supplemental colostrum or plasma to the foal shortly after

birth.

When included as a supplement to the diet, mannan oligosaccharides

(MOS) have been shown to have a positive effect on immune response in

several species. Mannan oligosaccharides are indigestible complex

polysaccharide molecules derived from yeast cell walls. Mannan

oligosaccharides are commercially available as BioMos@, a nutritional

supplement manufactured by Alltech, Inc. (Nicholasville, KY). Supplemental MOS

in poultry diets increased both plasma IgG and bile IgA (Savage et al. 1996). In

dogs supplemented with MOS, total lymphocyte count was increased, and serum

IgA concentrations tended to be greater (Swanson et al. 2002). Mannan

oligosaccharide supplementation increased serum IgM and tended to increase

colostral IgG levels in sows (Newman 2001). In addition to the positive immune

response elicited from MOS, they also serve as alternate attachment sites in the

gut for gram-negative pathogenic organisms with mannose-specific type-1

fimbriae that adhere to intestinal epithelial cells to initiate disease (Ferket et al.

2002). These pathogens will bind to MOS present in the intestinal tract and pass

through the gut, instead of attaching to host epithelial cells. Previous studies

have demonstrated that MOS reduces in vitro attachment of Salmonella

typhimurium to cultured intestinal cells (Oyofo et al. 1989) and decreases fecal

concentrations of Clostridium perfringens in poultry (Finuance et al. 1999). Other

in vitro studies have demonstrated agglutination of Escherichia coli, Salmonella

typhimurium, and S. enteritidis in the presence of MOS (Spring et al. 2000).









There has recently been an increased emphasis on the reduction of

antibiotic use in production diets because of the associated potential negative

environmental and health issues. The swine, poultry, and cattle industries are

interested in supplemental MOS because they may serve as a viable alternative

to antibiotic use in ration formulation. Most of the previous research involving

MOS has investigated the positive performance benefits seen with MOS addition

to production diets. Studies have demonstrated that the addition of MOS to the

diet results in increased average daily gain (Hooge 2003), increased gain-to-feed

ratio (Davis et al. 2002), and heavier litter birth and weaning weights (O'Quinn et

al. 2001). The immune response elicited by MOS supplementation in swine,

poultry, and cattle has only recently begun to be investigated. To the author's

knowledge, there have been no previous equine studies involving MOS

supplementation.

Results obtained in previous research with other species suggest that

MOS supplementation to the diet of the pregnant mare may increase the

immunoglobulin content in her colostrum and protect her from colonization of

pathogenic organisms in the gut. Greater immunoglobulin content in the

colostrum will result in more protection for the foal from disease initiated by

pathogenic organisms. The reduced occurrence of diarrhea and other problems

caused by these organisms in suckling foals would result in healthier foals and

decreased financial loss due to veterinary expenses for horse breeding

operations.














CHAPTER 2
REVIEW OF LITERATURE

The Immune System

The immune system of the horse is a versatile defense mechanism that

provides protection from a daily onslaught of pathogenic organisms. The body

must be prepared to combat this invasion with an arsenal of cells capable of

recognizing and eliminating these foreign microbes. The immunoglobulins are a

group of molecules exhibiting this property, through their ability to effectively

recognize and bind foreign antigen. These large glycoprotein molecules are

present on B-cell membranes, and are also secreted by plasma cells. They are

found throughout the body in the blood, mucosal tissues, and external secretions.

Immunoglobulins synthesized by the pregnant mare will affect the survivability of

her foal, because the foal relies on passive transfer to provide the major source

of antibodies for a period of at least 1 month after birth (McGuire and Crawford

1973). After that, the foal's own immune system is able to begin producing

immunoglobulins in a quantity that can mount an immunologic response that will

provide protection from pathogenic organisms.

Immunoglobulins. The immunoglobulins are a large group of

glycoprotein molecules found in the serum of the blood and other body fluids.

They are part of the fraction of serum proteins termed the "globulins" and play an

integral role in the immune response (Peakman and Vergani 1997). An antibody

is an immunoglobulin (lg) that exhibits antigen-binding ability. Therefore all









antibodies are Igs but not all Igs are antibodies. However, the two terms are

commonly used interchangeably. The functions of antibodies include targeting

foreign molecules, recruitment of effector responses, neutralization of toxins, and

binding and removal of foreign antigens. Antibodies also serve as useful

diagnostic tools. For example, to determine whether successful passive transfer

of maternal antibodies in newborn foals has occurred, the IgG concentration in

the foal's serum can be measured. The four major equine Ig isotypes are IgG,

IgM, IgA, and IgE (Nezlin 1998). The average Ig concentrations found in the

serum of mature horses are presented in Table 2-1.

Table 2-1. Immunoglobulin concentration in serum of mature horses
IgG IgA IgM
Concentration
1000 to 1500 60 to 350 100 to 200
(m g/d L)
Adapted from Tizard 1996: Veterinary Immunology: An Introduction, p. 155 Table
13-2. W.B. Saunders Co., Philadelphia.

IgG. IgG is the most abundant Ig found in the serum and in the colostrum.

It is made and secreted by plasma cells found in the spleen, lymph nodes, and

bone marrow (Tizard 1996). Plasma cells are the antibody-secreting cells that

are differentiated from B lymphocytes (B-cells). IgG is the smallest of the Ig

classes, therefore it is easily able to migrate from the blood into other tissues.

IgG readily binds foreign antigen it comes into contact with. This leads to

agglutination and opsonization, the process that makes foreign particles

susceptible to phagocytosis by neutrophils. IgG antibodies also play a role in

activating the complement system, a complex enzymatic pathway resulting in the

ultimate destruction of invading microorganisms. There are five subclasses of

equine IgG, which are IgG2a, IgG2b, IgG2c, IgG(B), and IgG(T), which are also









divided into two subclasses, IgG(T)a and IgG(T)b (Tizard 1996). These IgG

subclasses are distinguished by their different y-chain sequences and slight

differences in biological function (Goldsby et al. 2003).

IgMI. IgM is the second most abundant Ig found in the serum and the third

most abundant in colostrum. IgM is the first class of Ig detected in a primary

immune response and the first Ig produced by the neonate (Goldsby et al. 2003).

The secreted form of IgM is the largest of the Igs and also has more antigen

binding sites than the other isotypes. Because of its high affinity for antigen, IgM

is more efficient than IgG at causing agglutination, neutralizing virus particles,

and activating complement. The larger size of IgM restricts its ability to diffuse

from the blood to other tissues. Through specialized binding sites, secretary cells

in the respiratory and gastrointestinal tract are able to transport IgM molecules

across mucosal linings. Once released into the intestinal lumen, they play an

important accessory role to IgA, the most prevalent antibody found in mucosal

secretions.

IgA. IgA is the third most abundant serum Ig and second most abundant

colostrum Ig. However, as production shifts from colostrum to milk production,

IgA becomes the predominant antibody found milk. IgA present in colostrum,

milk, and other external secretions, including gastrointestinal tract secretions,

primarily exists in the form of secretary IgA. Secretory IgA is different from the

circulating monomeric form of IgA in serum. It is a complex molecule made up of

the dimeric form of IgA attached to a glycoprotein chain called secretary

component. Secretory component mediates the transport of IgA across mucosal









epithelium surfaces and provides protection from degradation by proteases that

are abundant in the mucosal environment. The primary function of IgA is to

prevent attachment of antigens to body surfaces. IgA can also serve as an

opsonin and activate the complement system, although not as efficiently as IgG.

Mlucosal immunity. The majority of IgA is produced by plasma cells in

mucosal lymphoid tissues, which are located underneath the respiratory and

gastrointestinal epithelium. The daily production of secretary IgA is greater than

that of any other Ig isotype, mainly because of the sheer size of the intestine

(Abbas et al. 2000). Most invasions by pathogenic organisms occur through

ingestion or inhalation. In the intestine, secretary IgA binds to pathogenic

organisms and provides protection by preventing their attachment to mucosal

cells. Secretory IgA has been shown to successfully prevent attachment of

bacteria such as Salmonella, Vibrio cholerae, and N~eisseria gonorrhoeae in the

gastrointestinal tract (Goldsby et al. 2003).

IgE. IgE is a minor class of Ig found in very low concentrations in the

serum of a healthy horse. IgE, like IgA, is primarily synthesized by plasma cells

beneath epithelial surfaces (Tizard 1996). The primary function of IgE is to

activate mast cells, which are responsible for the reactions characteristic of a

hypersensitivity reaction, such as hives or anaphylactic shock. IgE is also

responsible for immunity to parasitic worms.

Each class of Ig plays a unique role in the protection of both mare and foal

from disease. An immune system functioning at optimum capacity is essential for

the mare to produce a healthy and viable foal. Because the mare has a diffuse









epitheliochorial placenta, no significant transfer of Ig molecules across the

placental barrier can occur during gestation (Jeffcott 1974). The main vehicle for

transfer of immunologic protection from the mare to the foal is the colostrum.

Colostrum rich with maternal antibodies will increase the chances for the foal to

successfully deal with antigenic stimulation it faces soon after birth.

Passive Immunity

Foals are born with essentially no circulating Igs, although measurable

quantities of IgG and IgM may be detected in the serum at birth (LeBlanc 1990,

Vivrette 2001). The acquisition of maternal antibodies by the newborn foal within

the first 24 hours of life is essential for the foal's survival. Prior to parturition,

selective concentration of Igs from the blood occurs in the mare's mammary

gland to form the antibody component of the colostrum (Jeffcott 1972). When the

foal ingests colostrum, specialized epithelial cells of the small intestine absorb

the large Ig molecules present in the colostrum through pinocytosis (Jeffcott

1974). Passive immunity obtained by the foal from the mare is dependant upon

the colostrum Ig content, the quantity of colostrum ingested, and the successful

absorption of Igs by the newborn foal's digestive tract (Tizard 1996). Failure of

any of these processes is known as "failure of passive transfer" (FPT). Important

factors associated with the colostrum that influence successful passive transfer

of maternal antibodies include colostral Ig concentration, time of colostrum

ingestion, and occurrence of prelactation colostrum loss (McGuire et al. 1977,

LeBlanc et al. 1992).

If the foal is not able to nurse or the colostrum is of poor quality,

administration of colostrum from a colostrum bank or a colostrum substitute is









important to insure that the foal receives the essential antibodies that provide

protection against pathogens. After 24 hours, the mechanism for absorption of

large immunoglobulin molecules in the small intestine is no longer functional.

This cease in function is referred to as "gut closure." There are therapies

available for a foal that has not successfully ingested an adequate quantity of

colostrum before gut closure occurs, including IV administration of equine plasma

or commercially available Ig supplements. However, colostrum contains

beneficial factors including leukocytes, hormones, growth factors, and

constituents that inhibit bacterial colonization in the intestine, which makes it

preferable to IV immunoglobulin therapy for the treatment of FPT (Vivrette 2001).

Colostrum

The mare secretes colostrum for only a relatively short period of time. It is

manufactured in the mammary gland during the last two weeks of pregnancy and

is secreted the first time the foal suckles (McCue 1993). The colostrum contains

high concentrations of three classes of Igs. IgG concentration is high at birth, but

rapidly declines within the first 24 hours post-parturition (Pearson et al. 1984).

Colostral IgA and IgM are lower than IgG at birth (McCue 1993). As lactation

shifts from colostrum to milk production during the first day of lactation, IgA

becomes the predominant class of Ig found in mare's milk (Norcross 1982). The

average content of the three classes of Igs found in mare's colostrum and milk

are shown in Table 2-2.












Table 2-2. Immunoglobulin content of mare's colostrum and milk
Fluid IgG IgA IgM
Colostrum
1500 to 5000 500 to 1500 100 to 350
(m g/dL)
Milk (mg/dL) 20 to 50 50 to 100 5 to 10
Adapted from Tizard 1996: Veterinary Immunology: An Introduction, p. 242 Table
19-1. W.B. Saunders Co., Philadelphia.

Prelactation

Many factors can affect the Ig concentration of the colostrum. One of the

main determinants of colostral Ig content at parturition is whether or not the mare

experienced prelactation colostrum loss prior to parturition. Premature lactation,

or "prelactation" is one of the most important causes of FPT due to colostrum

loss (McCue 1993). It is relatively common for mares to lactate prior to

parturition, with the cause presumably associated with hormonal changes or

certain conditions such as impending abortion, twin pregnancy, placentitis, and

premature placental separation (Jeffcott 1974, McCue 1993). Mares that

prematurely lactate for longer than 24 hours before foaling tend have lower

colostral IgG concentrations than in mares that do not prematurely lactate

(Leblanc 1990). Morris et al. (1985) found a significant upward linear trend in the

percentage of mares that prelactated as colostral IgG decreased.

Breed

There have been reports demonstrating that breed of the mare can affect

colostrum Ig content. In a study including Thoroughbred, Arabian, and

Standardbred mares, breed of mare significantly affected colostral IgG

concentration (LeBlanc et al. 1992). In another study, the mean IgG









concentration in the colostrum was 9,69111,639 mg/dL in 14 Arabian mares and

4,60812, 138 mg/dL in 22 Thoroughbred mares (Pearson et al. 1984). Kohn et al.

(1989) reported a mean colostral IgG concentration of 8,32916,206.8 mg/dL in 36

Standardbred mares. This value is within the range reported in a study of 136

Standardbred mares by Morris et al. (1985). In another study, the mean IgG

colostral concentration in 21 QH mares was found to be 5,8431722 mg/dL

(LeBlanc et al. 1986). More investigation is needed to conclusively determine the

exact degree of influence that breed has on colostral Ig content.

Age

The age of the mare may also correlate to colostrum quality. Pearson et al.

(1984) suggests that age of the dam is a possible factor that influences colostral

Ig concentration. In a study that included 293 mares, mean colostral IgG

concentration was highest in mares between 3 and 10 years old, and FPT was

most prevalent in foals whose dams were >15 years old (LeBlanc et al. 1992).

Clabough et al. (1991) reported a possible association of an age >12 years old

with FPT. However, other studies suggest that mare age does not have a

significant effect on colostrum Ig content. Morris et al. (1985) reported that mare

age did not significantly affect colostrum IgG content in a study of 136

Standardbred mares aged 3 to 24. The discrepancies between these reports may

be due to variations in the time of colostrum sample collection. Future studies

with greater sample sizes and less variation may further elucidate the effect of

age on colostrum Ig content.









Foal Diarrhea

Diarrhea is one of the most common health problems experienced by foals.

It is characterized by an increase in the water content of the feces andlor an

increase in the frequency of defecation. Enteritis is a similar condition

characterized by diarrhea along with inflammation of the intestinal tract. If left

untreated for more than a few days, other problems may arise such as

dehydration, electrolyte imbalance, and even death. Identifying the cause can be

a challenge because there is a myriad of pathogenic organisms that can cause

diarrhea in sucking foals. The most common noninfectious cause of foal diarrhea

is associated with foal heat of the mare, which occurs between 7 and 12 days of

age (Cohen 1997). This is usually self-limiting and can resolve itself with minimal

medical treatment. Other noninfectious causes of diarrhea in young foals include

nutritional causes, gastric ulceration, and antibiotic administration (Cohen 1997).

Rotavirus

Diarrhea that is pathogenic in nature presents a major concern for horse

operations, primarily because of the infectious nature of the organisms that

cause diarrhea. One extremely contagious viral cause of diarrhea in young foals

is rotavirus. Rotavirus is the most common cause of foal enteritis in central

Kentucky, Ireland, and Great Britain (Dwyer 1993). Although the mortality rate of

rotavirus infection is low, there is a significant cost involved for treatment with

fluid and drug therapy, increased labor for the care of sick foals, and disinfection

of facilities to contain the outbreak (Dwyer 1993).









Salmonella

Diarrhea can also be caused by many different species of bacteria. The

most common cause of bacterial diarrhea and enteritis in foals is considered to

be Salmonella (Cohen 1997, Spier 1993). The genus Salmonella is a diverse

population of Gram-negative bacteria. Salmonella typhimurium is the most

common strain that causes disease, although many other strains of Salmonella

have been implicated in cases of salmonellosis. In the host, Salmonella is

capable of colonizing in the intestinal tract where it can invade the mucosal

epithelium and spread to other locations (Spier 1993). When bacterial invasion

occurs in other parts of the body, this condition is termed septicemia. This is a

serious condition with a survival rate of only 26% reported in a study of 38 cases

of septicemic foals admitted to a veterinary hospital for treatment (Koterba et al.

1984). Septicemia can also occur from invasion by many other bacterial species

besides Salmonella.

Clostridium

Clostridium perfringens and Clostridium difficile are two species of Gram-

positive bacteria that have been associated with enteritis and diarrhea in foals

(Traub-Dargatz and Jones 1993, Jones et al. 1988). Infection with C. perfringens

in foals was associated with a high case-mortality risk of 68% in a retrospective

case study investigating 125 foals admitted to a veterinary teaching hospital

(East et al. 2000). Another study reported a mortality risk of 54% with this

infection (East et al. 1998). The majority of foals reported to have C. perfringens-

associated enteritis have been under 3 days of age (Traub-Dargatz and Jones

1993).









Other Bacteria

The Gram-negative bacteria Escherichia coli is rarely associated with

diarrhea in foals (Cohen 1997). However, E. coli accounted for 56% of all

bacteria cultured from the blood in a study that examined 38 septicemic foals

(Koterba et al. 1984). Another study found that E. coli was one of the most

frequent causes of death in septicemic foals less than one week old (Platt 1973).

Other less common bacterial causes of foal diarrhea that have been reported are

Rhodococcus equi, Bacteroides fragilis, and Compylobacter jejuni, but the clinical

significance of these organisms is not notable (Cohen 1997).

Mlannan Oligosaccharides

Carbohydrates play a unique role within living systems. The function of a

carbohydrate will vary depending on its structure and location within a biological

system. Carbohydrates are important structural components of the majority of

cell-surface and secreted proteins of animal cells (Osborn and Khan 2000).

Carbohydrates are also a major source of metabolizable energy in the diet.

Oligosaccharides are formed when 2-10 monosaccharide molecules are joined

together to form a larger molecule. More than 10 monosaccharide molecules

joined together would constitute a polysaccharide. Mannose is a monosaccharide

that forms the building block of MOS. The small intestine does not contain the

digestive enzymes required to break down mannan oligosaccharide bonds,

therefore they arrive at the large intestine intact after ingestion and passage

through the small intestine (Strickling et al. 2000). Mannose-based

oligosaccharides occur naturally in cell walls of the yeast Saccharomyces

cerevisiae and are relatively easy to obtain by centrifugation from a lysed yeast









culture (Spring et al. 2000). The commercially available product Bio-Mos@

(Alltech, Inc., Nicholasville, KY) is a source of MOS from Saccharomyces

cerevisiae cell walls. This product was introduced in 1993 as a feed additive for

broiler chickens (Hooge 2003).

Lectins are carbohydrate-binding proteins that mediate interactions of cells

with their environment through their initial interactions with other cell surface

carbohydrates (Osborn and Khan 2000). Mannose residues on the surface of

intestinal epithelial cells serve as receptor binding sites for certain pathogens

with type-1 fimbriae that contain mannose-specific lectins (Ofek and Beachey

1978, Oyofo et al. 1989b3, Spring et al. 2000, Rocckendorf et al. 2002). Adherence

to the intestinal cell wall is a prerequisite for the initiation of colonization by

pathogenic organisms in the gastrointestinal tract (Ferket et al. 2002). Once

binding by the pathogenic organism occurs, translocation across the intestinal

wall and subsequent enteric infection can occur (lji et al. 2001, Ferket et al.

2002).

In vitro Agglutination Studies

Mannan oligosaccharide preparations have been shown to agglutinate

pathogens with mannose-specific type-1 fimbriae in vitro. Spring et al. (2000), in

an attempt to investigate the ability of different enteric pathogens and coliforms to

trigger MOS agglutination, showed that MOS agglutinated 7 of 10 strains S.

typhimurium and S. enteritidis and 5 of 7 strains of E. coli in vitro. Strains of S.

cholerasuis, S. pullorum, and Campylobacter did not result in MOS agglutination.

Another study using several human isolates of E. coli showed high mannose-

binding activity of the bacterial cells with the addition of D-mannose (Ofek and









Beachey 1978). This same study also demonstrated that D-mannose could

displace over 90% of E. coli that had already adhered to intestinal epithelial cells

in vitro. In another study, E. coli with type-1 mannose-specific lectins did not

attach to mammalian cells in the presence of supplemental mannose (Salit and

Gotschileh 1977).

Intestinal Environment Studies

Efforts to demonstrate that MOS has the same effect on bacterial

populations in the intestinal environment have proven successful. Oyofo et al.

(1989b3) investigated the adherence of S. typhimurium to the small intestine of

one-day-old chicks and found that adherence was significantly inhibited in the

presence of D-mannose. Droleskey et al. (1994) found that incubation of S.

typhimurium with cultured chick intestinal segments resulted in the loss of

mucosal epithelial integrity evidenced by the complete shedding of the

epithelium. It was found in this study that the addition of 2.5% D-mannose to the

incubation medium inhibited the loss of epithelial cells. When provided in the

drinking water of chicks, mannose significantly reduced intestinal colonization of

S. typhimurium (Oyofo et al. 1989a). When supplemented to the diet of hens,

MOS affected the birds' intestinal microflora by increasing the Bifidobacterium

spp. and Lactobacillus spp., while decreasing colonization of S. enteritidis

(Fernandez et al. 2002). The addition of 4,000 ppm of MOS to the diet of three-

day-old chicks that were orally challenged with S. typhimurium significantly

reduced cecal S. typhimurium concentrations on day 10 when compared with

controls (Spring et al. 2000). In a separate trial using S. dublin as the challenge

organism, the number of chicks that tested positive for Salmonella in the cecum









at day 10 was less in chicks that were consuming the MOS supplemented diet

(Spring et al. 2000). In growing turkeys younger than six weeks of age, MOS

supplemented birds had a higher total anaerobe count and a lower level of C.

perfningens in cecal cultures (Finuance et al. 1999). These studies demonstrate

that pathogens with the mannose-specific type-1 fimbriae adsorb to MOS instead

of attaching to intestinal epithelial cell walls and, therefore, move through the

intestine with less probability of initiating disease.

There have also been investigations into the intestinal environment effects

of MOS supplementation to the diet of a companion animal species. Strickling et

al. (2000) found that in dogs, fecal C. perfringens tended to be lower when

supplemented with 5g MOS/kg diet DM. The same study found no diet effects on

fecal bifidobacteria numbers or ileal bacteria colony forming units. Dogs

supplemented with 2 g MOS/day had significantly lower fecal total aerobe and

tended to have greater Lactobacillus populations (Swanson et al. 2002). 1 g/kg

BW/day of MOS supplementation to the diets of 4 female beagle dogs resulted in

a lower fecal pH (Zentek et al. 2002).

Performance

The use of antibiotics in food animal diets is a common practice in the

industry. Antibiotics have been shown to improve growth, feed efficiency, and

overall herd health when used in poultry, swine, and cattle production diets. Due

to consumer concerns and increasing regulatory restrictions, producers have

begun searching for alternatives to the use of antibiotic growth promotants in

production diets. Mannan oligosaccharide supplementation has been









investigated as an alternative to antibiotic supplementation to enhance

performance characteristics.

Poultry. Numerous studies have been conducted in poultry, because MOS

was first introduced in 1993 as a feed additive for broiler chicken diets (Hooge

2003). Over 150 broiler chicken pen trials were analyzed to collectively determine

the effects of MOS-supplemented diets versus negative andlor positive control

(antibiotic) diets. The conclusion was that MOS supplementation results in

bodyweight and feed conversion ratios comparable to antibiotic supplementation

while significantly lowering mortality rate (Hooge 2003). Fritts and Waldroup

(2000) reported that turkey poults fed 0. 10% MOS had the same feed conversion

as poults fed 55 ppm of the antibiotic bacitracin methylene disalicyclate (BMD)

and significantly better feed conversion than negative controls. In a study

conducted to determine growth effects in turkey hens with diets supplemented

with MOS or antibiotics (BMD and virginiamycin), investigators found that birds

fed 0.5g/ kg MOS supplemented birds had improved feed efficiency over birds

fed the control or antibiotic-supplemented diet (Hulet et al. 2000). Mannan

oligosaccharide was shown to be a suitable alternative to terramycin as a growth

enhancer in turkey diets when no difference in bodyweight was seen between

control and treatment animals after 105 days of supplementation (Stanley et al.

2000).

Both MOS and antibiotic growth promoters enhance the efficiency of

nutrient utilization by reducing the competition between the host and intestinal

pathogens. Without microbial competition for energy and other nutrients, there









are more nutrients available for absorption and metabolism by the host (Ferket et

al. 2002). It is well documented that antibiotic supplementation to poultry diets

increases the utilization of dietary energy (Buresh et al. 1985, Harms et al. 1986,

Ferket et al. 2002). Although MOS supplementation has proved to be as effective

as antibiotics in improving utilization of dietary energy, the mechanism is unclear

and likely different than that used by antibiotic growth promotants. Possibly it is

related to the improvement of characteristics of the intestinal lining (Ferket et al.

2002) or changes in digestive enzyme activities that are stimulated by MOS (lji et

al. 2001).

Swine. Pregnant sows fed 0.20% MOS three weeks prior to farrowing and

0. 10% MOS throughout the 21-day lactation period produced piglets with heavier

litter birth and weaning weights (O'Quinn et al. 2001). In a factorial experiment

conducted to determine the effects of two levels of MOS (0 and 0. 10%) and three

levels of protein (20, 23, and 26%) in piglet diets, MOS supplementation

improved weight gain and feed consumption regardless of protein level (Kim et

al. 2000). The addition of minerals such as Zn and Cu in excess of NRC

recommendations to swine diets is a common practice to improve performance

(NRC 1998). However, this may result in an undesirable effect on the bacteria

responsible for waste degradation in lagoons (Gilley et al. 2000). The addition of

0.20% MOS to the diets of nursery pigs increased average daily gain and

average daily feed intake in the absence of excess zinc but had no effect or a

negative effect in the presence of excess zinc (LeMieux et al. 2003). In a

separate trial of the same study, the interactive effects of antibiotics









oxytetracyclinee and neomycin) and MOS and of Zn and MOS were evaluated.

Mannan oligosaccharide improved pig performance only when fed in combination

with an antibiotic and no excess Zn. There was no effect or a negative effect in

the presence of excess Zn or in the absence of an antibiotic (LeMieux et al.

2003).

Mannan oligosaccharides have also been considered as an alternative to

excess Cu supplementation in swine diets for performance enhancement. The

effects of MOS fed at either basal or excess levels of Cu in the diets of weanling

and growing-finishing pigs were determined in an experiment by Davis et al.

(2002). From day 0 to day 10, average daily gain, average daily feed intake, and

gain : feed increased when MOS was added to diets containing basal levels of

Cu. From day 10 to day 38, pigs fed diets containing excess Cu had greater ADG

and ADFI regardless of MOS addition (Davis et al. 2002). The researchers

concluded that MOS addition to swine diets results in a moderate improvement in

gain and feed efficiency, but the magnitude of response is not as great as that

seen with the addition of excess levels of Cu (Davis et al. 2002). Should trace

mineral supplementation restrictions on swine diets come into effect, MOS

supplementation may provide a viable performance-enhancing alternative.

Cattle. The production-enhancement effects of MOS supplementation in

cattle diets have received relatively less attention than supplementation of poultry

or swine diets. Heinrichs et al. (2003) investigated the effects of MOS or

antibiotics in dairy calf milk replacer diets, and found the addition of 4 g MOSI

day was as effective as antibiotic use to maintain normal fecal fluidity and









consistency and to decrease scours severity. Feed consumption increased when

MOS was included in the diet, but this did not result in a difference in growth

measures (Heinrichs et al. 2003).

Immune Function

After MOS supplementation to production diets proved to increase weight

gain and feed efficiency, identifying the mechanism of the physiological response

associated with the positive growth responses was the next logical step. To do

this, studies focused on measuring the parameters that are representative of a

functional immune system. These parameters include Ig content of the serum,

lymphocyte proliferation, and response to antigenic stimulation. The main

antigenic components of yeast cells are mannans present in the isolated cell wall

(Ballou 1970). Mannans found in the cell walls of S. cerevisiae have been shown

to induce an antigenic response in humans (Young et at. 1998) Therefore some

MOS-immune system interaction would be expected (Ferket et al. 2002).

Poultry. Savage et al. (1996) fed 0. 11% MOS to male turkeys for 53 days

and obtained blood and bile samples at the end of the period. The samples were

analyzed using both radial immunodiffusion (RID) and rocket immuno-

electophoresis (RI). Using RID, no significant differences were found, but RI

analysis showed that concentrations of both blood and bile IgG and IgA were

significantly increased in turkeys fed MOS (Savage et al. 1996). In a trial

investigating the effects on humoral immunity in commercial laying hens,

investigators injected the hens with sheep red blood cells (SRBC) suspended in

a solution of bovine serum albumin (BSA) and obtained serum samples one, two,

and four weeks post-sensitization. Hens supplemented with 0.05% MOS had









higher SRBC titers than controls at one week post-sensitization (Malzone et al.

2000). The BSA titers of the MOS-fed hens were numerically greater at week one

and week two, but the differences were not statistically significant (Malzone et al.

2000). In broiler breeder diets, the addition of MOS significantly increased the

antibody response to infectious bursal disease virus and also increased maternal

antibody titers in the breeders' progeny (Shashidhara and Devegowda 2003).

Swine. Positive immune response effects have also been observed with

MOS supplementation to swine diets. Newman and Newman (2001)

supplemented sow diets with 5g MOSI day for approximately 14 days pre-

farrowing and continued supplementation throughout lactation. At farrowing,

MOS treated sows had significantly higher serum IgM and colostrum IgM levels

and numerically higher colostrum IgG levels (Newman and Newman 2001). The

piglets from the MOS treated sows also weighed more on day 7, 14, and 21 post-

farrowing than those from unsupplemented sows (Newman and Newman 2001).

In another study evaluating sow and litter performance, concentrations of IgA,

IgG, and IgM in pre-suckle colostrum samples were increased by MOS addition

to the diet. IgG showed the greatest response, followed by IgM and IgA

respectively (O'Quinn et al. 2001). As found in the previous trial, the piglets from

the MOS treated sows also had heavier litter birth and weaning weights (O'Quinn

et al. 2001).

To determine whether MOS modulated the cell-mediated immune response

of the weaned pig, Davis et al. (2002) obtained blood samples from MOS

supplemented growing-finishing pigs and measured lymphocyte proliferation in









vitro. Lymphocyte proliferation did not differ significantly between the control and

MOS supplemented pigs (Davis et al. 2002). Although not demonstrated in this

study, other researchers have demonstrated that MOS may have an inhibitory

effect on certain lymphocyte functions (Muchmore et al. 1990, Podzorski et al.

1990). It is conceivable that immune function suppression could also be a means

by which MOS improves gain and efficiency, because of the shift in metabolic

activity to support the body's defense against foreign antigens that occurs during

immune response activation (Spurlock 1997). Another mechanism of growth

enhancement in swine may be through the alteration of intestinal microflora, as is

what happens with supplementation with pharmacological levels of Cu (Davis et

al. 2002).

Cattle. The addition of 10 g MOSI day to the diet of 40 dairy cows resulted

in numerically greater serum Ig levels in calves 24-hours post-calving than in the

calves of unsupplemented cows (Franklin et al. 2002). In the same study,

antibody titers to rotavirus vaccination following calving were numerically greater

in claves from MOS supplemented cows (Franklin et al. 2002).

Dogs. Adult female dogs were supplemented with 1 g MOS per day for a

14 day period, and serum IgA concentrations tended to be greater and the

percent of white blood cells that were lymphocytes was greater in dogs

supplemented with MOS. Total white blood cell count and neutrophil

concentration were unaffected by treatment (Swanson et al. 2002). The authors

hypothesized that because serum IgG and IgM were not affected, a systemic

immune response may not have occurred and was not the cause of the









increased lymphocytes and serum IgA. The trends for increased serum IgA and

lymphocyte concentration may be due to the increased proliferation of B-

lymphocytes and secretary IgA in the intestinal tract (Swanson et al. 2002). A

study performed in rats reported increased cecal IgA contents and an increase in

the proportion of IgA-presenting lymphocytes present in the cecal mucosal of rats

fed glucomannans at 5% for three weeks (Kudoh et al. 1999).

These studies have demonstrated the positive effects of MOS on Ig

concentration in serum and colostrum and on immune response to antigen

challenge. However, a mechanism for this action has yet to be demonstrated.

Some studies suggest that MOS supplementation stimulates intestinal lymphoid

tissue resulting in increased development or activation (Guigoz et al. 2002,

Ferket et at. 2002). The stimulatory effect may occur through a healthy

population of gut microflora or "drag effects" of the indigestible oligosaccharide

molecules as they move along the length of the intestine (Cunningham-Rundles

and Lin 1998). The activation of lymphoid tissue may result in greater plasma cell

production by B-cells found in underlying lymphoid follicles. These plasma cells

then would be able to secrete Igs that can either be secreted into the intestinal

lumen when associated with secretary component or end up in the circulation via

transport through the lymphatic system.

To determine if MOS supplementation to the diet of pregnant mares would

result in a change in the total Ig concentration of the mare's colostrum and the

serum of the mare or foal, the current experiment was proposed. Previous results

from work in other species suggest that MOS supplementation will increase









colostrum Ig content, and therefore translate into increased foal serum Ig content

after absorption of maternal antibodies is complete. Growth measurements of

both mares and foals will indicate any negative effects of MOS supplementation

on physical development. Determination of Ig content in serum and presuckle

colostrum samples will indicate any change of immune status in the mare. Serum

Ig concentration in the foals will reflect any effect on absorption of colostral Igs

and initial serum Ig concentration and any long-term effect on immune status of

the foal due to MOS supplementation of the dam.














CHAPTER 3
MATERIALS AND METHODS

Animals

Twenty-six pregnant Thoroughbred (n=21) and Quarter Horse (n=5) mares

and their subsequent foals were used in this trial. The mares ranged from 3 to 24

years of age with a mean age of 9 (STD=6.1i). The pregnant mares were paired

by expected foaling dates and assigned at random to one of two treatment

groups 56 days prior (d-56) to expected date of parturition. They continued on

the treatment diet until 56 days post-parturition (d+56). The foals remained on the

trial until 112 days of age (d+112). One mare leaked milk for 3 weeks prior to

foaling, and her foal acquired septicemia and was hospitalized for one week after

birth. No data from this mare or foal were used.

Housing and Management

During the course of the trial, the mares and their foals were housed at the

University of Florida's Horse Research Center in Ocala, Florida. Pregnant mares

were kept at pasture until pre-foaling signs were evident. They were then moved

to a dry lot where they remained until they foaled. After foaling, the mare and her

foal were moved to a small paddock for approximately one week and then were

returned to pasture. A routine vaccination and anthelmintic schedule for all

animals on trial was followed by farm management. The University of Florida

Institutional Animal Care and Use Committee approved the protocol for this trial.









Diets

Treatment group 1 (n=13) served as controls and received supplement A,

which consisted of 55g of ground corn as a placebo. Treatment group 2 (n=12)

received supplement B, which consisted of 10g of MOS (Bio-Mos, Alltech,

Nicholasville, KY) mixed in 45g of ground corn. Supplements A and B were top

dressed on the morning ration and fed to the mares from day -56 until day +56.

Feeding time was at 0700 hours (AM feeding) and 1500 hours (PM feeding).

Mares and foals were brought into stalls for individual feeding for both AM and

PM feedings. Foals remained in the stalls with their dam and potentially could

have consumed some of her feed, depending upon her temperament and

willingness to allow the foal access to her feed bucket. Both treatment groups

were fed the same concentrate, HR-136, which was formulated to meet or

exceed requirements for late gestating and lactating mares based on NRC

recommendations (NRC 1989) when fed with bahiagrass pasture (Paspalum

notatum) or Coastal bermudagrass hay (Cynodon dactylon) (see table 3-1). The

amount of concentrate fed was adjusted according to each mare's body condition

score (BCS) to maintain a minimum BCS of 5 (see table 3-2). The mares were

also fed ad-libitum Coastal bermudagrass hay andlor bahiagrass pasture in

season. Trace mineralized salt blocks and fresh water were available at all times.

A creep-feeder was introduced when the oldest foal was 2 months of age, and

HR-136 was provided as the creep feed.

Body Measurements

The mares were weighed and assessed for body condition scores every

28 days. Foals were weighed at birth, d+7, d+14, d+28, d+56, and d+112. Foal









body measurements taken at the same time were withers height, hip height, body

length, and heart girth. The scale used was a digital walk-on scale. Body

measurements were made with a sliding stick made specifically for the purpose

of taking accurate body length and height measurements.

Colostrum and Blood Samples

Colostrum samples were obtained from the mare after the foal was born

but before it was allowed to nurse. Three 1 ml aliquots from each colostrum

sample were placed in cryogenic tubes and frozen at -80oC until further analysis.

Jugular blood samples were collected from the mares between 0700 and 0900

hours on d-56, d-28, and d+28. Jugular blood samples were collected from the

foals at birth before the foal was allowed to nurse, 6 -10 hours post-parturition

(referred to as 8 hour sample), and between 0700 and 0900 hours on d+7, d+14,

d+28, d+56, and d+112. Precision Glide Vaccutainer brand blood collection

needles (20G, 1%/ in.) were used to collect blood into Beckton Dickinson

Vaccutainers. Samples were allowed to clot for one to two hours and then

centrifuged at 3000 x G for 10 minutes to allow for separation and collection of

serum. Three 1 ml aliquots from each serum sample were placed in

polypropylene cryogenic vials and frozen at -80oC until further analysis.

Colostrum samples and serum samples from both the mares and foals were

analyzed for IgG, IgA, and IgM content using a commercially available single

radial immunodiffusion kit (SRID Kit, VMRD, Inc., Pullman, WA).

Feed Sample Analysis

Monthly samples were taken of HR-136 and the Coastal bermudagrass

hay for the duration of the experiment. To determine dry matter content of the









samples, the concentrate and hay were first put through a Wiley mill fitted with a

1mm screen to assure uniform particle size. 1 to 2 grams of the sample were

then weighed to 4 decimal places on a Mettler balance and placed into ceramic

crucibles. They were dried in a 105oC drying oven overnight and equilibrated for

1 hr. in a dessicator before weighed again to 4 decimal places.

The samples were analyzed for calcium, copper, manganese, zinc, and iron

content by atomic absorption spectrophotometry (Miles et al. 2001) using the

Perkin-Elmer Model 5000 Atomic Absorption Spectrophotometer (Perkin-Elmer

Corp., Norwalk, CO). Crude protein content was analyzed by first digesting the

sample according to the procedure put forth by Gallaher et al. (1975) and then

determining the nitrogen content of the sample using the Alpkem auto analyzer

(Alpkem Corp., Clackemas, OR). Phosphorus content was determined by using a

calorimetric procedure (Technicon Industrial Systems, Tarrytown, NY) on the

automated Alpkem analyzer (Alpkem Corp., Clackemas, OR).

Neutral and acid detergent fiber content was determined using the

Ankom fiber analyzer (Ankom Technology, Fairport New York).

Prior to fat content analysis, carbohydrates were first extracted from the

sample (AOAC 1995). Fat content was then determined by ether extraction using

a soxhlet apparatus.

Statistical Analysis

The treatment effect on Ig concentration in the serum of the mares was

analyzed using PROC GLM procedures with repeated measures in SAS (SAS

1989). Ig content of the mare's colostrum was analyzed using PROC GLM in

SAS controlling for age, breed, and prelactation. The treatment effect on Ig












































Table 3-2. Concentrate feeding rates for mares
Stage Rate
Late Gestation
-56 d to -28 d 0.75% BWe
-28 d to parturition 1.0% BWe
Early Lactation
Parturition to +84 d 1.5% BWe
Late Lactation
+84 d to +112 d 1.0% BWe
aAdjust concentrate feeding for body condition score
(-20% above BCS 51 +20% below BCS 5)


concentration in the serum of the foals was analyzed using PROC GLM

procedures with repeated measures in SAS (SAS 1989). Treatment, sex, and

breed effects on foal growth measurements were analyzed using PROC GLM

procedures with repeated measures in SAS. Significance was considered to be

p<0.05, and p<0.10 was considered a trend.

Table 3-1. Composition of Concentrate (HR-136)
Ingredient Amount (%)
Corn, cracked 34.25
Oats, crimped 26.50
Soybean meal (48% CP) 10.00
Wheat bran 10.00
Molasses, blackstrap 8.00
Alfalfa meal pellets (17% CP) 7.50
Limestone, ground 1.50
Monocalcium phosphate 0.80
Salt 0.75
Vitamin premixa 0.30
Vitamin Eb 0. 15
Lysine 98% 0.05
Luprosil (mold inhibitor) 0. 10
Trace mineral premixo 0. 10
aProvides 4,400,0001U Vit A, 440,0001U Vit D, and 35,2001U Vit El kg
remix
bProvides 44,2001U Vit El kg premix
CProvides 7,200mg Cu, 28,000mg Zn, 28,000mg Fe, 28,000mg Mn,
80mg Co, 80mg 1, and 80mg Se/ kg premix














CHAPTER 4
RESULTS AND DISCUSSION

Feed Analysis

The average nutrient composition of HR-136 (n=7) and the Coastal

bermudagrass hay (n=3) from monthly samples taken throughout the trial period

are presented in Table 4-1.

Table 4-1. Concentrate (HR-136) and Coastal bermudagrass hay nutrient
composition analysis
Nutrient HR-1 36 Hay
Dry Matter (%) 94.7811.1 88.4413.6
Crude protein (%) 15.0711.0 5. 1511.1
Fat (%) 2.6210.8 1.6410.4
ADF (%) 9.4611.0 39.2111.5
NDF (%) 25.0111.5 80.3511.1
Calcium (%) 1.1610.4 0.4610.3
Phosphorus (%) 0.6210.2 0. 1510. 1
Cu (ppm) 48. 1712.3 2.2910.9
Mn (ppm) 124.8314.7 51.6714.3
Zn (ppm) 137.6714.3 23.0011.7
Fe (ppm) 267.5014.8 101.6717.4
All values MeantSE
Dry matter basis (except dry matter)

Growth Analysis

For the duration of the experiment, mares maintained good body condition

and remained at a healthy body weight during both gestation and lactation (See

Table 4-2). Mares from treatment 1 (control) foaled 6 fillies and 7 colts, and

mares from treatment 2 (MOS) foaled 6 fillies and 6 colts. There were no

statistically significant differences (p > 0.05) between control and foals from MOS

supplemented mares for any of the growth parameters measured (see Table 4-









3). Control foals weighed 50.31.7 kg at birth and gained a total of 140.014. 1 kg

during the 112-day trial. Foals from MOS supplemented mares weighed 48.910.8

kg at birth and gained 142.614.4 kg over the trial period. Control foals grew

26.411.0 cm in height, 29.611.0 cm in hip height, 44.21.3 cm in length, and

47. 111.1 cm in heart girth. Foals from MOS supplemented grew 25.411.0 cm in

height, 28.511.1 cm in hip height, 46.011.4 cm in length, and 48.21.2 cm in

heart girth. Average daily gain measurements for both treatments were

consistent with previously published data (Kavazis and Ott 2003, Lawrence et al.

1991).

The influence of sex on foal growth was minimal during the 112-day trial

period. Because there was no significant treatment effect on growth, the data

from the two treatment groups were pooled (see Table 4-4) to determine any

influence of sex on growth. Average height was the only growth parameter that

showed any trend towards significant difference between males and females. At

d+112, colts tended to be taller than fillies (p=0.08).

To determine the influence of breed on foal growth, data from the two

treatment groups were pooled (see Table 4-5). There was a trend for TB foals to

be taller than QH foals at d+112 (p=0.08). TB foals had greater birth body length

than QH foals (p=0.04). The total gain in body length tended to be greater for QH

foals than TB foals (p=0.06).









Table 4-2. Influence of treatment on mare weight and body condition scores
Weight (kg) Body Condition Score
Treatment 1 Treatment 2 Treatment 1 Treatment 2
Day (Control) (MOS) (Control) (MOS)
d-56 567.6111.7 577. 1 9.3 4. 810. 1 4. 810. 1
d-28 580.4110.3 586.919.7 4.610.2 5. 0 0. 1
dO 514. 1110.4 526. 1110.2 4.610.1 4.710. 1
d+28 528.0112.5 538.210.6 4.610. 1 4.810. 1
d+56 529.412.0 543. 1111.0 4.610.2 4.810. 1
d+84 535.211.6 547.311.1 4.610.2 4.910.2
d+112 531.8111.0 544.919.7 4.610.2 4.610. 1
All values are Mean a SE

Table 4-3. Influence of treatment on foal growth
Treatment 1 Treatment 2
Growth parameter
(Control) (MOS)
Birth weight (kg) 50.31.7 48.911.8
d+112 weight (kg) 190.25.0 191.515.4
Total weight gain (kg) 140. 014. 1 142.614.4


Birth withers height (cm)
d+112 withers height (cm)
Total withers height gain (cm)

Birth hip height (cm)
d+112 hip height (cm)
Total hip height gain (cm)

Birth length (cm)
d+112 length (cm)
Total length gain (cm)

Birth heart girth (cm)
d+112 heart girth (cm)
Total heart girth gain (cm)
All values are LSMean + SE


98.21.0
124.610.7
26.411.0

100.41.1
130. 00.9
29.611.0

73.31.0
117.51.1
44.21.3

80. 11.2
127.21.5
47.111.1


99.911.1
125. 30. 8
25.411.0

102.41.2
131.00.9
28.511.1

72.81.1
118.81.2
46.011.4

80.011.3
128.21.6
48.21.2






































TB
49.21.2
186.514. 0
137.33. 0

100.30.8
126.00.5
25.811.0

102.610.8
131.110.6
28.610.7

74.910.7*
117.818
42.911.0

80.00.9
126.41.1
46.30.8


Birth withers height (cm)
d112 withers height (cm)
Total withers height gain (cm)

Birth hip height (cm)
d112 hip height (cm)
Total hip height gain (cm)

Birth length (cm)
d112 length (cm)
Total length gain (cm)

Birth heart girth (cm)
d112 heart girth (cm)
Total heart girth gain (cm)
All values are LSMean + SE *p=0.04


97.811.5
124.011.0
26.011.0

100.31.6
129.91.2
30.011.5

71.21.5*
118.41.6
47.21.9

80.111.7
129. 112.2
49.011.6


Table 4-4. Influence of sex on foal growth
Growth parameter
Birth weight (kg)
d+112 weight (kg) 1
Total weight gain (kg) 1

Birth withers height (cm) 1
d+112 withers height (cm) 1
Total withers height gain (cm)

Birth hip height (cm) 1
d+112 hip height (cm) 1
Total hip height gain (cm)

Birth length (cm)
d+112 length (cm) 1
Total length gain (cm)

Birth heart girth (cm)
d+112 heart girth (cm) 1
Total heart girth gain (cm)
All values are LSMean + SE


Colts
49.911.7
88.34.6
40.814. 1

00.011.0
25.810.7
26.011.0

02.011.1
30.710.9
28.611.0

72.411.0
17. 111.1
44.711.3

79.511.2
27.41.5
47.911.1


Fillies
49.21.8
190.714.9
191.015.4

98.21.1
124. 110.8
25.911.0

100.81.2
130.30.9
29.511.1

73.71.1
119. 112
45.511.4

80.611.3
128.01.6
47.41.2


Table 4-5. Influence of breed on foal growth
Growth parameter
Birth weight (kg) 5(
d112 weight (kg) 19~
Total weight gain (kg) 14~


QH
0.012.4
5.216.5
5.25.3










Mare Serum Immunoglobulins

Mare serum Ig content was analyzed with treatment as the only source of

variation.

IgG

For IgG serum concentration, control mares averaged 1807.6130.8 mg/dL

on d-56, 1525.11191.5 mg/dL on d 0, and 1929.11163.6 mg/dL on d+28. Mares

fed MOS had an average serum IgG concentration of 1789.81125.8 mg/dL on d-

56, 1405.51108.2 mg/dL on d 0, and 1874.7196.1 mg/dL on d+28 (see Table 4-

6). Although control mares had numerically higher serum IgG concentration at d-

56, d 0, and d+28, the differences were not significant. The control mares had a

numerically higher IgG concentration at the start of the experiment, and this is

the likely reason control mare IgG concentration remained slightly above IgG

concentration in mares fed MOS for the duration of the trial.

Table 4-6. Influence of treatment on mare serum IgG concentration
Treatment 1 Treatment 2
Day (Control) (MOS)
d-56 (mg/dL) 1807.51130.8 1789.81125.8
dO (mg/dL) 1525.01191.5 1405.5~1108.2
d+28 (mg/dL) 1929. 1163.6 1874.7196. 1
All values are Mean + SE

IgA

Average serum IgA concentration for control mares was 349.238.7 mg/dL

at d-56, 424.9131.1 mg/dL at d 0, and 378.6131.9 mg/dL at d+28. Mares fed

MOS had an average serum IgA concentration of 360. 1140.4 mg/dL at d-56,

419.0144.0 mg/dL at d 0, and 412.0168.2 mg/dL at d+28 (see Table 4-7). Mares

fed MOS had numerically higher serum IgA concentration than control mare









serum IgA concentration throughout the duration of the experiment. Because this

difference was present at the start of the experiment, this effect was not likely

due to MOS supplementation.

Table 4-7: Influence of treatment on mare serum IgA concentration
Treatment 1 Treatment 2
Day (Control) (MOS)
d-56 (mg/dL) 349.2138.7 360. 1140.4
dO (mg/dL) 424.9131.1 419.0144. 0
d+28 (mg/dL) 378.6131.9 412.0168.2
All values are Mean + SE

IgMI

Serum IgM concentration for control mares averaged 109.213.8 mg/dL on

d-56, 115.612.7 mg/dL on d 0, and 101.9121.6 mg/dL on d+28. Mares fed MOS

averaged 98.818.8 mg/dL on d-56, 113.1110.3 mg/dL on d 0, and 89.1115.8

mg/dL on d+28 (see Table 4-8). Control mare serum IgM concentration remained

numerically above mares fed MOS for the duration of the experiment, and this

was not likely due to the treatment.

Table 4-8. Influence of treatment on mare serum IgM concentration
Treatment 1 Treatment 2
Day (Control) (MOS)
d-56 (mg/dL) 109.2113.8 98.818.8
dO (mg/dL) 115.6112.7 113. 1110.3
d+28 (mg/dL) 101.9121.6 89. 1115.8
All values are Mean + SE

Discussion

There were no significant differences for IgG, IgA, or IgM concentration in

samples obtained from the mares at d-56, dO, or d+28. This result agrees with

results obtained in a previous study performed in 40 pregnant dairy cows to

evaluate the effect of MOS supplementation on the immune status of dairy cows









and their calves. No overt differences in serum Ig levels were observed between

cows that were supplemented with 10 g/MOS/day and the control group (Franklin

et al. 2002).

Savage et al. (1996) reported an increase in plasma IgG and bile IgA in

male turkeys after 53 days of MOS supplementation at 0.11% of the total diet.

These turkeys were started on the supplementation protocol immediately after

birth at one day of age. The data was analyzed using two different assays, and

only one assay, rocket immuno-electrophoresis, revealed any significant

difference in plasma IgG and bile IgA levels between the two treatment groups.

The assay that did not reveal any difference was radial immunodiffusion, the

same assay that is used in the current experiment.

Mlare Colostrum Immunoglobulins

Mare colostrum data were analyzed to determine the treatment,

prelactation occurrence, age, breed, treatment*age interaction, and

treatment*breed interaction effects. Previous research suggests that prelactation,

age, and breed can affect the Ig concentration in mare colostrum (McCue 1993,

Leblanc 1990, Morris et al. 1985, LeBlanc et al. 1992, Pearson et al. 1984,

LeBlanc et al. 1986, and Clabough et al. 1991), therefore it is important to

consider these factors when evaluating colostrum content.

IgG

Colostrum IgG concentration for mares fed MOS was significantly higher

than in control mares (p=0.05) when all sources of variation were taken into

consideration in the overall ANOVA model. Colostrum IgG concentration for

mares fed MOS was significantly higher than control mares due to treatment





































Table 4-10. ANOVA generated P values for colostrum IgG from a statistical
model which included treatment, prelactation, age, breed,
with treatment*age and treatment*breed interactions
Model p=0.05
Treatment p=0.0334
Prelactation p=0.0063
Age p=0. 1377
Breed p=0.4803
Treatment*Age p=0.0163
Treatm ent*B reed p=0.7593


(p=0.03), prelactation (p=0.006), and treatment*age (p=0.02). All other sources of

variation were not significantly different between treatments (see Tables 4-9 and

4-10).

Table 4-9. Influence of treatment, prelactation occurrence, age, and breed on
colostrum IgG
Source of Variation Mean a s.e.


Treatment
Control (n=13) (mg/dL)
MOS (n=12) (mg/dL)


10242.211181.1
12824. 012245.6


6934.2+1174.0
12555.5~11429. 0


11663.8+985.7
11253.0+3585.5


12388.2~11542.8
8627.1+1461.0


Prelactation
Y (n=4) (mg/dL)
N (n=21) (mg/dL)


Age
<12 years (n=18) (mg/dL)
>12 years (n=7) (mg/dL)

Breed
TB (n=20) (mg/dL)
QH (n=5) (mg/dL)


IgA


Colostrum IgA concentration for mares fed MOS was significantly higher

than in control mares (p=0.05) when all sources of variation were taken into

consideration in the overall ANOVA model. Colostrum IgA concentration for

mares fed MOS was significantly higher than control mares due to treatment





































Table 4-12. ANOVA generated P values for colostrum IgA from a statistical
model which included treatment, prelactation, age, breed,
with treatment*age and treatment breed*interactions
Model p=0.05
Treatment p=0.0080
Prelactation p=0.0079
Age p=0.0177
Breed p=0. 1796
Treatment*Age p=0.0356
Treatm ent*B reed p=0. 7746


(p=0.008), prelactation (p=0.008), age (p=0.02), and treatment*age (p=0.04). All

other sources of variation were not significantly different between treatments (see

Tables 4-11 and 4-12).

Table 4-11. Influence of treatment, prelactation occurrence, age, and breed on
colostrum IgA
Source of Variation Mean a s.e.


Treatment
Control (n=13) (mg/dL)
MOS (n=12) (mg/dL)

Prelactation
Y (n=4) (mg/dL)
N (n=21) (mg/dL)

Age
<12 years (n=18) (mg/dL)
>12 years (n=7) (mg/dL)

Breed
TB (n=20) (mg/dL)
QH (n=5) (mg/dL)


47.719. 5
112. 1138.9


43.6112.6
88.0124. 9


67.5115.9
106.6157.5


85.0126.4
62.7119.8


IgMI


Colostrum IgM concentration for mares fed MOS tended to be higher than

in control mares (p=0.06) when all sources of variation were taken into

consideration in the overall ANOVA model. Colostrum IgM concentration for

mares fed MOS tended to be higher than control mares due to treatment









(p=0.08). The treatment*age interaction was significantly higher for mares fed

MOS (p=0.04). All other sources of variation were not significantly different

between treatments (see Tables 4-13 and 4-14).

Table 4-13. Influence of treatment, prelactation occurrence, age, and breed on
colostrum IgM
Source of Variation Mean a s.e.
Treatment
Control (n=13) (mg/dL) 133.2112.2
MOS (n=12) (mg/dL) 154. 118. 1

Prelactation
Y (n=4) (mg/dL) 126.3123.0
N (n=21) (mg/dL) 147.517.7

Age
<12 years (n=18) (mg/dL) 154.716.9
>12 years (n=7) (mg/dL) 120.015.6

Breed
TB (n=20) (mg/dL) 149.117.4
QH (n=5) (mg/dL) 125.0120.6

Table 4-14. ANOVA generated P values for colostrum IgM from a statistical
model which included treatment, prelactation, age, breed, with
treatment*age and treatment*breed interactions
Model p=0.06
Treatment p=0.0764
Prelactation p=0.2994
Age p=0.2195
Breed p=0.9598
Treatment*Age p=0.0350
Treatm ent*B reed p=0.9545

Discussion

The colostrum Ig concentration for all isotypes was highly variable. This

could be due to many factors, some of which could not be accounted for in the

statistical model. Ig content was determined by single radial immunodiffusion

(SRID) using raw colostrum samples. This method has been used in previously









published reports (Zou et al. 1998, Turner et al. 2003). However, there have

been other published reports that describe extracting the colostral whey (located

between the superficial fat layer and the precipitate) to remove cellular debris

and fat for use in the SRID assay (Waelchli et al. 1990, Pearson et al. 1984,

LeBlanc et al. 1986, LeBlanc et al. 1992). It is possible that using colostral whey

for the determination of Ig content could minimize the extreme variation in

colostrum Ig values.

One of the QH mares from the control treatment was dropped from the

statistical analysis because the Ig concentration in her colostrum was a

significant outlier to the average distribution of expected Ig concentration in mare

colostrum. The Ig content of her colostrum was 45,409.5 mg/dL for IgG, 278.5

mg/dL for IgA, and 220 mg/dL for IgM. These values were much higher than

those from the other mares in the study and average values reported in the

literature (Tizard 1996, LeBlanc et al. 1992, Morris et al 1985, Pearson et al.

1984, LeBlanc et al. 1986). In order to maintain a representative sample of the

mare population, her data was not used for colostrum analysis.

When controlled for variation due to prelactation colostrum loss, age, and

breed, IgG and IgA content of colostrum was significantly enhanced by MOS

supplementation, and IgM content tended to be enhanced. This result agrees

with previous findings of two other studies evaluating the effect of MOS

supplementation on colostrum immunoglobulin content. Newman and Newman

(2001) reported significantly increased presuckle colostrum IgM levels (p=0.04) in

MOS supplemented sows and numerically greater IgM levels in colostrum 24-









hour post-farrowing. They also reported numerically increased presuckle and 24-

hour post-farrowing colostrum IgG levels in MOS supplemented sows when

compared to controls, but there was no effect on colostrum IgA concentration

(Newman and Newman 2001). In another study involving sows, the addition of

MOS resulted in significantly increased IgG (p=0.007) and IgM (p=0.03)

concentration in presuckle colostrum (O'Quinn et al. 2001). Presuckle IgA levels

tended to be greater in MOS supplemented sows (p=0.06) (O'Quinn et al. 2001).

There was a significant effect due to treatment (p=0.03), prelactation

(p=0.006), and treatment*age interaction (p=0.02) for IgG colostrum content. The

highly significant prelactation effect is expected, because lost colostrum cannot

be replaced due to its limited production. The negative effect of prelactation on

colostrum Ig content has previously been well documented (McCue 1993,

Jeffcott 1974, Leblanc 1990, and Morris et al. 1985).

There was a significant effect due to treatment (p=0.008), prelactation

(p=0.008), age (p=0.02), and treatment*age interaction (p=0.04) for IgA

colostrum content. The highly significant prelactation effect is expected, for

reasons stated previously. Age effect on colostrum content is not well defined,

however some reports show that mean colostrum Ig concentration was highest in

mares between 3 and 10 years old and lower in mares over 12 years old

(LeBlanc et al. 1992, Clabough et al. 1991). Other reports show no effect of age

on colostral Ig content (Morris et al. 1985, Kohn et al. 1989). In this experiment,

mares that were >12 years old had higher mean colostrum IgA concentration.

This may be due to the fact that many of the mares used in this study were









maiden mares. There was one maiden mare in the control treatment group and

seven maiden mares in the MOS treatment group. It has been reported that

primiparous (maiden) mares have lower colostrum Ig concentrations than

multiparous mares, and this may explain the significant age effect on IgA content

(Jeffcott 1972, Erhard et al. 2001). Although no significant age effect was seen

for colostrum IgG or IgM, a significant treatment*age interaction was seen for all

three isotype concentrations, and the unbalanced distribution of maiden mares in

the treatment groups may have contributed to this effect.

The treatment effect approached significance (p=0.08) and there was a

significant effect due to treatment*age interaction (p=0.02) for IgM colostrum

content. The occurrence of prelactation did not significantly affect IgM

concentration, possibly because the overall concentration of IgM in equine

colostrum is relatively low (McCue 1993).

Foal Serum Immunoglobulins

Foal serum immunoglobulin concentration was analyzed with treatment as

the only source of variation.

IgG

A detectable amount of IgG was present in foal serum at birth prior to

colostrum ingestion. There were no significant differences in IgG concentration

for any of the foal serum samples collected. The mean foal serum IgG

concentration for each sample collection is presented in Table 4-15. Figure 4-1

presents this data in graphic format to illustrate the change in foal serum IgG

concentration over time.









Table 4-15. Influence of treatment on foal serum IgG concentration
Treatment 1 Treatment 2
Day/hour
(Control) (MOS)
0 hour (mg/dL) 82.6111.4 88.2111.4
8 hour (mg/dL) 1478.81238. 0 1420.01227.6
d+7 (mg/dL) 1431.71172.8 1322.5~1159.4
d+14 (mg/dL) 1275.6~1146.8 1229.71121.6
d+28 (mg/dL) 1234. 11121.7 1322.71129. 7
d+56 (mg/dL) 930. 1165.0 907.3158. 1
d+112 (mg/dL) 653.9127.6 648.7115.2
All values are Mean + SE





I

I


I


Oh

8h

d+7

d+14

d+28

d+56

d+112

0 200 400 600 800 1000

IgG, mg/di

H Control 0MOS

Figure 4-1: Mean foal serum IgG concentration


1200 1400


1600






46


IgA

There was no detectable amount of IgA in foal serum at birth prior to

colostrum ingestion. There were no statistically significant differences in foal

serum IgA concentration, however, foals from control mares tended to have

higher serum IgA concentration than foals from mares fed MOS at 6 -10 hours

post-parturition (p=0.09). The mean foal serum IgA concentration for each

sample collection is presented in Table 4-16. Figure 4-2 presents this data in

graphic format to illustrate the change in foal serum IgA concentration over time.











Table 4-16. Influence of treatment on foal serum IgA concentration


Day/hour


Treatment 1 (Control) Treatment 2 (MOS)


0 hour (mg/dL) 0
8 hour (mg/dL) 214.7130.8
d+7 (mg/ dL) 81.317.6
d+14 (mg/ dL) 59.614.1
d+28 (mg/ dL) 67.517.5
d+56 (mg/dL) 98.918.5
d+112 (mg/dL) 140.411.2
All values are Mean + SE


0
122.8127.9
84.7121.7
62.3110.0
61.413. 5
93. 919.4
119. 117.3











Oh


8h


d+7


d+14


d+28


d+56


d+112


0 50 100 150 200 250

IgA, mg/dl

SControl O MOS


Figure 4-2: Mean foal serum IgA concentration






49


IgMI

A detectable amount of IgM was present in foal serum at birth prior to

colostrum ingestion. There were no significant differences in IgM concentration

for any of the foal serum samples collected. The mean foal serum IgM

concentration for each sample collection is presented in Table 4-17. Figure 4-3

presents this data in graphic format to illustrate the change in foal serum IgM

concentration over time.











Table 4-17. Influence of treatment on foal serum IgM concentration
Treatment 1 Treatment 2
Day/hour
(Control) (MOS)
0 hour (mg/dL) 17.012.09 17.51.5
8 hour (mg/dL) 40.216.8 41.015.0
d+7 (mg/dL) 33.913.9 35.514. 1
d+14 (mg/dL) 37. 114.0 40.913.3
d+28 (mg/dL) 46.716.9 41.313.3
d+56 (mg/dL) 77.818.3 67.318.6
d+112 (mg/dL) 109.219.0 119.517.6
All values are Mean + SE










Oh


8h


d+7


d+14


d+28


d+56


d+112

0 20 40 60 80 100 120 140

Ig M, mg/dl

SControl O MOS


Figure 4-3: Mean foal serum IgM concentration









Discussion

There were no statistically significant differences for any serum

concentration at any hour or day sampling period. Because foals were not fed the

MOS directly, any immune response would be expected to come from the

ingestion of colostrum with a higher concentration of immunoglobulins and

predominantly be apparent in the first weeks of life. Franklin et al. (2002)

reported numerically greater serum IgG and IgM concentration 24 hours post-

calving in calves from cows supplemented with MOS, but the differences were

not significant. LeBlanc et al. (1986) reported that mean foal serum IgG

concentration increases concurrently with increasing colostral IgG concentration.

Morris et al. (1985) reported similar results and showed a highly significant

correlation between colostral IgG and foal serum IgG concentration (r=0.584,

p<0.001). The positive association between colostrum Ig and foal serum Ig

concentration after colostrum ingestion is well documented. However, there was

no noticeable difference between the two treatment groups in this experiment,

even with significantly higher Ig concentration in colostrum of mares fed MOS.

This is most likely due to the fact that peak values of passively obtained maternal

antibodies are reached around 18 hours after birth (Jeffcott 1972). The foal

serum samples taken in this experiment to determine successful passive transfer

were obtained between 6 and 10 hours post-parturition. At this time, full

absorption of maternal antibodies is not yet complete (Kohn et al. 1989).

Evaluation of foal serum from 6 12 hours post-parturition is appropriate to

determine if proper absorption of maternal antibodies is occurring so that a

treatment protocol for suspected FPT can be implemented if necessary (Erhard









et al. 2001, Vivrette 2001). However, obtaining a 24 to 36 hour post-parturition

serum sample would have more accurately reflected the complete absorption of

Igs from the mare's colostrum (Morris et al. 1985).

Serum IgA concentration in foals from control mares tended to be higher

than in foals from mares fed MOS 6 10 hours post-parturition (p=0.09). The

reason for this trend for foal serum IgA concentration to be higher in control foals

is unclear. Intestinal permeability is selective in the horse, with IgG and IgM

preferentially absorbed while IgA remains in the intestine (Tizard 1996). The IgA

content of colostrum was most significantly increased by MOS supplementation,

but this was not reflected in the 6 -10 hour foal serum samples. The principal

form of IgA in human colostrum is secretary IgA, which is resistant to the

proteolytic effects of enzymes present in the neonatal gut (Chapel et al. 1999).

Perhaps the increased quantity of IgA in the colostrum of mares fed MOS was

primarily in the form of secretary IgA, and significant amounts could not be

immediately absorbed across the intestinal epithelium. There is evidently some

absorption of colostrum IgA as shown by the initial increase in serum IgA

concentration and subsequent decrease for both treatment groups over the first 7

days of life. A foal serum sample obtained 24 to 36 hours post-parturition may

have reflected higher peak absorption of IgA in the foals from MOS

supplemented mares due to the higher content of IgA in the colostrum of mares

fed MOS.














CHAPTER 5
SUMMARY AND CONCLUSIONS

Supplementing pregnant mares with 10 g/MOS/day 56 days prior to

expected date of parturition through the first 56 days of lactation significantly

increased IgG and IgA content and tended to increase IgM content in the

colostrum. Supplementation had no effect on serum Ig content of mares or foals,

except at 8 hours after birth when control foals had significantly higher serum IgA

concentration than foals from mares fed MOS. Because the timing of foal serum

sampling at 8 hours after birth was not ideal, this may have prevented an

accurate portrayal of full absorption of maternal antibodies. However, no ill

effects were seen as a result of MOS supplementation, and greater Ig content in

the colostrum increases the chance for successful passive transfer to occur.

Supplementation of pregnant mare diets with MOS may be a beneficial practice

to help protect the mare from pathogenic organisms and to boost the Ig content

of her colostrum.

Although not investigated in this experiment, MOS supplementation of

sucking and weanling diets may be beneficial as well. Foals that are provided a

source of MOS may be better protected from pathogenic organisms present in

the environment and therefore may have a reduced incidence of illness caused

by these organisms. This is a promising area for further research.















LITERATURE CITED


Abbas, Abul K., Lichtman, A. H., and Pober, Jordan S. (2000) Cellular and
Molecular Immunology. W.B. Saunders Co., Philadelphia.

Association of Official Analytical Chemists (AOAC) (1995) Official Mllethods of
Analysis of AOAC. AOAC International, Arlington, VA.

Ballou, C.E. (1970) A study of the immunochemistry of three yeast mannans. J
Bio/ Chem 245(5), 1197-1203.

Buresh, R.E., Miles, R.D., and Harms, R.H. (1985) Influence of virginiamycin on
energy utilization when turkey poults were fed ad libitum or restricted. Poult
Sci 64, 1041-1042.

Chapel, H., Haeney, M., Misbah, S., and Snowden, N. (1999) Essentials of
Clinical Immunology. Blackwell Science, Oxford.

Clabough, D.L., Levine, J.F., Grant, G.G., and Conboy, H. S. (1991) Factors
associated with failure of passive transfer of colostral antibodies in
Standardbred foals. J Vet Intern Miled 5(6), 335-340.

Cohen, N.D. (1997) Diarrheal Diseases of Foals. In: Current Therapy in Equine
Medicine, Ed: N.E. Robinson, W. B. Saunders Co., Philadelphia.

Cunningham-Rundles, S. and Lin, D. H. (1998) Nutrition and the immune system
of the gut. Nutrition 14, 573-579.

Davis, M.E., Maxwell, C.V., Brown, D.C., de Rodas, B.Z., Johnson, Z.B., Kegley,
E.B., Hellwig, D.H. and Dvorak, R.A. (2002) Effect of dietary mannan
oligosaccharides and(or) pharmacological additions of copper sulfate on
growth performance and immunocompetence of weanling and
growing/finishing pigs. J Anim Sci 80, 2887-2894.

Drolesky, R.E., Oyofo, B.A., Hargis, B.M., Corrier, D.E., and DeLoach, J.R.
(1994) Effect of mannose on Salmonella typhimurium-mediated loss of
mucosal epithelial integrity in cultured chick intestinal segments. Avian
Diseases 38, 275-281.

Dwyer, R.M. (1993) Rotaviral diarrhea. Vet Clin N~orth Am Equine Pract 9, 311-
319.











East, L.M., Dargatz, D.A., Traub-Dargatz, J.L. and Savage, C.J. (2000) Foaling-
management practices associated with the occurrence of enterocolitis
attributed to Clostridium perfringens infection in the equine neonate.
Preventive Veterinary Mlledicine 46, 61-74.

East, L.M., Savage, C.J., Traub-Dargatz, J.L., Dickinson, C.E., and Ellis, R.P.
(1998) Enterocolitis associated with Clostridium perfringens infection in
neonatal foals: 54 cases (1988-1997). J Am Vet Miled Assoc 212, 1751-
1756.

Erhard, M. H., Luft, C., Remler, H. P., and Stangassinger, M. (2001) Assessment
of colostral transfer and systemic availability of immunoglobulin G in new-
born foals using a newly developed enzyme-linked immunosorbant assay
(ELISA) system. J Anim Phys Anim N~utr 85, 164-173.

Ferket, P.R., Parks, C.W. and Grimes, J.L. (2002) Benefits of Dietary Antibiotic
and Mannanoligosaccharide Supplementation for Poultry. Proc Multi-State
Poultry Mlleeting. Indianapolis, Indiana, May 14-16, 2002.

Fernandez, F., Hinton, M., and Van Gils, B. (2002) Dietary mannan-
oligosaccharides and their effect on chicken caecal microflora in relation to
Salmonella enteritidis colonization. Avian Pathology 31(1), 49-58.

Finuance, M.C., Dawson, K.A., Spring, P., and Newman, K.E. (1999) The effect
of mannan oligosaccharide on the composition of the microflora in turkey
poults. Poult Sci 78(Suppl 1),77.

Franklin, S.T. Newman K.E., and Newman, M.C. (2002) Evaluation of mannan
oligosaccharide on the immune status of dairy cows and their calves. J
Anim Sci 80 (Suppl), 192.

Fritts, C.A. and Waldroup, P.W. (2000) Utilization of Bio-Mos mannan
oligosaccharide in turkey diets. Poult Sci 79(Suppl 1), 126.

Gallaher, R. N., Weldon, C. O., and Frutal, J. G. (1975) An aluminum block
digester for plant and soil analysis. Soil Sci Soc Amer Proc 39, 803-806.

Gilley, J.E., Spare, D.P., Koelsch, R.K., Schulte, D.D., Miller, P.S., and
Parkhurst, A.M. (2000) Phototrophic anaerobic lagoons as affected by
copper and zinc in swine diets. Trans Am Soc Agric Eng 43, 1853-1859.

Goldsby, R.A., Kindt, T.J., Osborne, B.A., and Kuby, J. (2003) Immunology. W.H.
Freeman and Co., New York.









Guigoz, Y., Rochat, F. Perru isseau-Carrier, 1. Rochat, I., and Schiffrin, E.J.
(2002) Effects of oligosaccharide on the faecal flora and non-specific
immune system in elderly people. N~utr Res 22, 13-25.

Harms, R.H., Ruiz, N., and Miles, R.D. (1986) Influence of virginiamycin on
broilers fed four levels of energy. Poult Sci 65, 1984-1986.

Heinrichs, A. J., Jones, C. M., and Heinrichs, B.S. (2003) Effects of mannan
oligosaccharide or antibiotics in neonatal diets on health and growth of dairy
calves. J Dairy Sci 86(12), 4064-4069.

Hooge, D.M. (2003) Broiler chicken performance may improve with MOS.
Feedstuffs 75(1).

Hulet, R.M., Lorenz, E.S., and Saleh, T.M. (2000) Turkey hen growth response to
diets supplemented with either antibiotic or mannan oligosaccharide. Poult
Sci 79 (Suppl 1), S186.

lji, P. A., Saki, A. A., and Tivey, D. R. (2001) Intestinal structure and function of
broiler chickens on diets supplemented with a mannan oligosaccharide. J
Sci Food Agric 81, 1 186-1 192.

Jeffcott, L.B. (1972) Passive immunity and its transfer with special reference to
the horse. Bio/ Rev 47, 439-464.

Jeffcott, L.B. (1974) Some practical aspects of the transfer of passive immunity to
newborn foals. Equine Vet J 6, 109-115.

Jones, R.L., Shideler, R.K. and Cockerell, G.L. (1988) Association of Clostridium
difficile with foal d iarrhea. Equine Infectious Diseases V: Proc. of the 5th
International Conference, 236. Lexington, KY, October 1998.

Kavazis, A. N. and Ott, E.A. (2003) Growth rate in Thoroughbred horses raised in
Florida. J Eq Vet Sci 23(8), 353-357.

Kim, J.D., Hyun, Y., Sohn, K.S., Kim, T.J., Woo, H.J. (2000) Effects of mannan
oligosaccharide and protein levels on growth performance and immune
status in pigs weaned at 21 days of age. J Anim Sci Tech 42(4), 489-498.

Kohn, C. W., Knight, D., Hueston, W., Jacobs, R., and Reed, S. M. (1989)
Colostral and serum IgG, IgA, and IgM concentrations in Standardbred
mares and their foals at parturition. JA VM~IA 195(1), 64-68.

Koterba, A.M., Brewer, B.D., and Tarplee, F.A. (1984) Clinical and
clinicopathological characteristics of the septicaemic neonatal foal: Review
of 38 cases. Equine Vet J 16(4), 376-383.









Kudoh, K., Shimizu, J., Ishiyama, A., Wada, M., Takita, T., Kanke, Y., and
Innami, S. (1999) Secretion and excretion of immunoglobulin A to cecum
and feces differ with type of indigestible saccharides. J N~utr Sci Vitaminol
(Tokyo) 45(2), 173-181.

Lawrence, L. A., Murphy, M., Bump, K., Weston, D., Key, J. (1991) Growth
responses in hand-reared and naturally raised Quarter Horse foals. Eq
Pract 13, 19-26.

LeBlanc, M. M. (1990) Immunologic Considerations. In: Equine Clinical
N~eonatology, Ed: A.M. Koterba, W.H. Drummond, and P.C. Kosch, Lea &
Febiger, Philadelphia.

LeBlanc, M. M., McLaurin, B.I., and Boswell, R. (1986) Relationships among
serum immunoglobulin in foals, colostral specific gravity, and colostral
immunoglobulin concentration. JA VMlrA 189(1), 57-60.

LeBlanc, M.M., Tran, T., Baldwin, J.L. and Pritchard, E.L. (1992) Factors that
influence passive transfer of immunoglobulins in foals. JA VM~A 200, 179-
183.

LeMieux, F.M., Southern, L.L. and Bidner, T.D. (2003) Effect of mannan
oligosaccharides on growth performance of weanling pigs. J Anim Sci 81,
2482-2487.

Malzone, A., Paluch, B., Lilburn, M.S., and Sefton, A.E. (2000) Modulation of
humoral immunity in commercial laying hens by a dietary probiotic. Poult
Sci 79(Suppl 1), 1 65.

McCue, P.M. (1993) Lactation. In: Equine Reproduction, Ed. McKinnon, A.O. and
Voss, J.L., Lea & Febiger, Philadelphia.

McGuire, T.C. and Crawford, T.B. (1973) Passive immunity in the foal:
measurement of immunoglobulin classes and specific antibody. Am J Vet
Res 34, 1299-1303.

McGuire, T.C., Crawford, T.B., Hallowell, A.L., and Macomber, L.E. (1977)
Failure of colostral immunoglobulin transfer as an explanation for most
infections and deaths of neonatal foals. JA VM~A 170, 1302-1304.

Miles, P. H., Wilkinson, N. H., and McDowell, L. R. (2001) Analysis of Minerals
for Animal Nutrition Research. Department of Animal Sciences, University
of Florida, Gainesville, FL.

Morris, D.D., Meirs, D.A., and Merryman, G.S. (1985) Passive transfer failure in
horses: Incidence and causative factors on a breeding farm. Am J Vet Res
46(11), 2294-2299.









Muchmore, A.V., Sathyamoorthy, N., Decker, J., and Sherblom, A.P. (1990)
Evidence that specific high-mannose oligosaccharides can directly inhibit
antigen-driven T-cell responses. J Leuko Bio/ 48, 457-464.

National Research Council (NRC). 1989. N~utrient Requirements of Horses. 5th
ed. National Academy Press, Washington, DC.

National Research Council (NRC). 1998. N~utrient Requirements of Swine. 10Oth
ed. National Academy Press, Washington, DC.

Newman, K.E. and Newman, M.C. (2001) Evaluation of Mannan Oligosaccharide
on the microflora and immunoglobulin status of sows and piglet
performance. J Anim Sci 79, 189.

Nezlin, R. (1998) The Immunoglobulins: structure and function. Academic Press,
San Diego.

Norcross, N.L. (1982) Secretion and composition of colostrum and milk. JA VMlrA
181(10), 1057-1060.

Ofek, 1. and Beachey, E.H. (1978) Mannose binding and epithelial cell adherence
of Escherichia coli. Infection and Immunity 22(1), 247-253.

O'Quinn, P.R., Funderburke, D.W. and Tibbetts, G.W. (2001) Effects of dietary
supplementation with mannan oligosaccharides on sow and litter
performance in a commercial production system. J Anim Sci 79, 212.

Osborn, H.M.I. and Khan, T.H. (2000) Oligosaccharides: Their synthesis and
biological roles. Oxford University Press Inc., New York.

Oyofo, B.A., DeLoach, Corrier, D.E., Norman, J.O., Ziprin, R.L., and
Mollenhauer, H.H. (1989a) Effect of carbohydrates on Salmonella
typhimurium colonization on broiler chicks. Avian Diseases 33, 531-534.

Oyofo, B.A., Droleskey, R.E., Norman, J.O., Mollenhauer, H.H., Ziprin, R.L.,
Corrier, D.E. and DeLoach, J.R. (1989b3) Inhibition by mannose of in vitro
colonization of chicken small intestine by Salmonella typhimurium. Poult Sci
68, 1351-1356.

Peakman, M. and Vergani, D. (1997) Basic and Clinical Immunology. Churchill
Livingstone, New York.

Pearson, R.C., Hallowell, A.L., Bayley, W.M., Torbeck, R.L., and Perryman, L.E.
(1984) Times of appearance and disappearance of colostral IgG in the
mare. Am J Vet Res 45(1), 186-190.

Platt, H. (1973) Septicaemia in the foal. A review of 61 cases. Br Vet J 129, 221-
229.









Podzorski, R.P., Gray, G.R. and Nelson, R.D. (1990) Different effects of native
Candida albicans mannan and mannan-derived oligosaccharides on
antigen-stimulated lymphoproliferation in vitro. J Immunol 144, 707-716.

Rocckendorf, N., Sperliing, O., and Lindhorst, T.K. (2002) Trivalent cluster
mannosides with aromatic partial structure as ligands for the type-1 fimbrial
lectin of Escherichia coli. Aust J Chem 55, 87-93.

Salit, I.E. and Gotschlich, E.C. (1977) Type 1 Escherichia coli pili:
characterization of binding to monkey kidney cells. J Exp Miled 146, 1182-
1194.

SAS (1989) Statistical Analysis System: A User's Guide. SAS Inst., Version 6, 4th
ed. Cary, NC.

Savage, T.F., Cotter, P.F. and Zakrzewska, E.I. (1996) The effect of feeding a
mannanoligosaccharide on immunoglobulins, plasma IgA, and bile IgA of
Wrolstad MW male turkeys. Poult Sci 75 (Suppl 1).

Shashidhara, R.G., and Devegowda, G. (2003) Effect of dietary mannan
oligosaccharide on breeder production traits and immunity. Poult Sci 82(8),
1319-1325.

Spier, S.J. (1993) Salmonellosis. In: Veterinary Clinics of N~orth America: Equine
Practice, Ed: W.B. Saunders Co., Philadelphia. pp 385-397.

Spring, P., Wenk, C., Dawson, K.A. and Newman, K.E. (2000) The effects of
dietary mannaoligosaccharides on cecal parameters and the concentrations
of enteric bacteria in the ceca of salmonella-challenged broiler chicks. Poult
Sci 79, 205-211.

Spurlock, M.E. (1997) Regulation of metabolism and growth during immune
challenge: an overview of cytokine function. J Anim Sci 75, 1773-1783.

Stanley, V.G., Brown, C., Sefton, A.E. (2000) Comparative evaluation of yeast
culture, mannanoligosaccharide and antibiotic on performance of turkeys.
Poult Sci 79 (Suppl 1), S186.

Strickling, J.A., Harmon, D.L., Dawson, K.A., Gross, K.L (2000) Evaluation of
oligosaccharide addition to dog diets: influences on nutrient digestion and
microbial populations. An Feed Sci & Tech 86, 205-219.

Swanson, K.S., Grieshop, C.M., Flickinger, E.A., Bauer, L.L., Healy, H.P.,
Dawson, K.A., Merchen, N.R. and Fahey, G.C., Jr. (2002) Supplemental
fructooligosaccharides and mannanoligosaccharides influence immune
function, ileal and total tract nutrient digestibilities, microbial populations
and concentrations of protein catabolites in the large bowel of dogs. J N~utr
132, 980-989.









Tizard, I.R. (1996) Veterinary Immunology: An Introduction W.B. Saunders Co.,
Philadelphia.

Traub-Dargatz, J.T. and Jones, R.L. (1993) Clostridia-associated enterocolitis in
adult horses and foals. In: Veterinary Clinics of N~orth America: Equine
Practice, Ed: W.B. Saunders Co., Philadelphia. pp. 411-421.

Turner, J. L., Arns, M.J., and Minton, J. E. (2003) Case study: Effects of non-
specific immunostimulation of prepartum mares on colostral quality and foal
immune function. ProfAnim Sci 19, 62-67.

Vivrette, S. (2001) Colostrum and oral immunoglobulin therapy in newborn foals.
Compendium 23(3), 286-291.

Waelchli, R.O., Haissig, M., Eggenberger, Nussbaumer, M. (1990) Relationships
of total protein, specific gravity, viscosity, refractive index and latex
agglutination to immunoglobulin G concentration in mare colostrum. Eq Vet
J 22(1), 39-42.

Young, M., Davies, M.J., Bailey, D., Gradwell, M.J., Smestad-Paulsen, B., Wold,
J.K., Barnes, R.M.R., and Hounsell, E.F. (1998) Characterization of
oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae.
Glycoc J 15, 815-822.

Zentek, J., Marquart, B., and Pietrzak, T. (2002) Intestinal effects of
mannanoligosaccharides, transgalactooligosaccharides, lactose, and
lactulose in dogs. J N~utr 132, 1682S-1684S.

Zou, S., Brady, H. A., Hurley, W. L. (1998) Protective factors in mammary glan
secretions during the periparturient period in the mare. J Eq Vet Sci 18(3),
184-188.















BIOGRAPHICAL SKETCH

Kelly Robertson Spearman was born on August 28, 1977, in Tuscaloosa,

Alabama. She lived there for 3 years until her family moved to Montevallo, AL, in

1980. In 1986, they moved to Anniston, AL, which is where Kelly's interest in

horses began. Her 5th grade English teacher also taught riding lessons, and

although her family knew nothing about horses, they agreed to bi-monthly riding

lessons that would fit into an already busy schedule of piano lessons, choir

practice, gymnastics, and church activities. Kelly and her family moved back to

Tuscaloosa in 1989, and her interest in horses continued to grow, as she started

taking dressage lessons at a local barn. This sparked an enduring fascination

with the art of dressage and its training philosophies.

The family moved to Montgomery, AL, in 1994, just before her senior year,

and she graduated with a 3.9 GPA from Jefferson Davis High School. While in

high school, she also worked as a pharmacist assistant, attended a performing

arts school for piano, and was the accompanist for the school's jazz choir. She

received a freshman academic scholarship to Auburn University, and graduated

in 1999 with a B.S. degree in animal and dairy sciences, with cum laude honors.

While at Auburn, she was a member of the university honors program, a charter

member of the Auburn Equestrian Team, and vice president of the horseman's

club. For three summers during her college career, she was the wrangler for a

working cattle ranch in northwest Colorado. She is a member of Alpha Zeta









honorary fraternity, and Gamma Sigma Delta honor society. After graduating

from Auburn University, Kelly moved to Missouri for 1 year and worked as an

assistant trainer, working with young horses. She moved back to Alabama, and

became a North America Handicapped Riding Association certified instructor,

and she was an instructor for Special Equestrians in Birmingham, AL.

In 2001, Kelly received a presidential fellowship to study Equine Nutrition at

the University of Florida under Dr. Edgar A. Ott. While at the University of Florida,

Kelly taught numerous undergraduate equine classes, and participated in many

equine nutrition research projects. She will continue her education at the

University of Florida, as she works toward a Ph.D. in equine nutrition.