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1 SOURCE AND DURATION OF TRACE MINERAL SUPPLEMENTATION AND THEIR EFFECTS ON EARLY POSTPARTUM REPRODUCTIVE PERFORMANCE IN MARES AND INNATE AND ACQUIRED IMMUNITY IN FOALS By JEROME GEORGE VICKERS IV A THESIS PRESENTED TO THE GRAD UATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008
2 2008 Jerome George Vickers IV
3 To my mother and father, Terry and Mike Vickers, because they always told me I could, and to the two truest friends I have and will ever know, brothers Brandon and Chase.
4 ACKNOWLEDGMENTS First and foremost, my utmost gratitude and thanks goes to my major professor, Dr. Lori K. Warren. Her guidance, wisdom, patience, and most of all the example she sets, have been invaluable during my tenure as a graduate student. Her humanness coupled with her uncanny ability to communicate well the ideas of scientific merit makes her an in dividual I am proud to call my mentor. She truly raises the bar, and ultimately redefines what it means to be a professor and mentor I am forever grateful. I thank Jan Kivipelto for being my source of reference and often refuge. Her abilities and assis tance in and out of the laboratory have been crucial for the completion of this project. Her generosity and selflessness will not be forgotten. In addition, a special thanks goes to Nancy Wilkinson for her aid and support in the laboratory she will be missed by many. I also thank fellow graduate students Kelly Vineyard, Sarah Dilling, and Drew Cotton for their advice, comradeship, and help along the way. To my committee members, Dr. McDowell and Dr. Crawford, I thank you for your time and effort in re viewing my thesis and guidance during my graduate work. My sincere appreciation and thanks goes to the farm team at the Horse Research Center at the University of Florida, namely Chris and Joss Cooper, Richard, Adel, and Larry. Without their dedicated ef fort during foal wrangling, blood draws, and daily reproductive exams, this research would not have been possible. I also thank Dr. Sharp and his graduate students Luciano Silva and Michelle Eroh for their enthusiasm and daily participation necessary for t he completion of the reproductive exams, and ultimate success of this project. Words cannot express the sincere sentiment I have towards my family and friends who have supported me not only through graduate school, but also along the path that has led me
5 h ere. We are often told as young children that we can become whatever we want to be, and I thank my parents for this promise of greatness. Their promise has given me strength for the journey I have already completed and for that which is still unknown. T o my siblings, thank you for your support and most of all good humor. To my grandparents, whose encouragement and words of wisdom have never ceased, thank you. You have all lived Christ led lives, and it is this example I look up to and hope to embody so meday.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ....................... 10 ABSTRACT ................................ ................................ ................................ ................................ ... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 13 2 REVIEW OF LITERATURE ................................ ................................ ................................ 15 Trace Mineral Source, Absorption, and Metabolism ................................ .............................. 15 Source ................................ ................................ ................................ .............................. 15 Absorption ................................ ................................ ................................ ....................... 17 Bioavailability ................................ ................................ ................................ ................. 20 Trace Mineral Supply to the Nursing Foal ................................ ................................ ............. 21 Placental Transfer of Trace Minerals ................................ ................................ .............. 21 Trace Mineral Content of Mare Colostrum and Milk ................................ ..................... 21 Overview of the Immune System ................................ ................................ ........................... 23 Innate Immunity ................................ ................................ ................................ .............. 23 Acquired Immunity ................................ ................................ ................................ ......... 24 Passive Immunity in the Foa l ................................ ................................ ................................ 27 Importance of Mare Colostrum ................................ ................................ ................ 27 Failure of Passive Immunity in the Foal ................................ ................................ .. 30 Effect of Trace Mineral Supplementation on Immune Function ................................ ............ 31 Foal Heat Ovulation ................................ ................................ ................................ ............... 34 Uterine Involution ................................ ................................ ................................ ........... 35 Uterine Fluid ................................ ................................ ................................ .................... 36 Uterine Edema ................................ ................................ ................................ ................. 36 Effect of Dietary Trace Mineral Sup plementation on Reproductive Performance ................ 37 Conclusions ................................ ................................ ................................ ............................. 39 3 MATERIALS AND METHODS ................................ ................................ ........................... 41 Animals ................................ ................................ ................................ ................................ ... 41 Diets and Treatments ................................ ................................ ................................ .............. 41 Blood Sample Collection and Handling ................................ ................................ ................. 44 Passive Transfer of Immunity ................................ ................................ ................................ 44 Tetanus Antibody Titers ................................ ................................ ................................ ......... 45 Neutrophil Function ................................ ................................ ................................ ................ 46 Vitamin B12 Status ................................ ................................ ................................ ................. 47
7 Serum Trace Minerals ................................ ................................ ................................ ............. 48 Reproductive Performance ................................ ................................ ................................ ..... 48 Bodyweights ................................ ................................ ................................ ........................... 50 Statistical Analyses ................................ ................................ ................................ ................. 50 4 RESULTS ................................ ................................ ................................ ............................... 59 General Observations ................................ ................................ ................................ .............. 59 Passive Transfer of Immunity ................................ ................................ ................................ 59 Tetanus Antibody Titers ................................ ................................ ................................ ......... 61 Neutrophil Function ................................ ................................ ................................ ................ 61 Trace Mineral Concentrations in Umbilical Cord Serum ................................ ....................... 62 Trace Mineral Concentrations in Mare and Foal Serum ................................ ........................ 62 Vitamin B12 Status ................................ ................................ ................................ ................. 64 Postpartum Reproductive Performance ................................ ................................ .................. 65 5 DISCUSSION ................................ ................................ ................................ ......................... 86 Passive Transfer of Immunity ................................ ................................ ................................ 86 Tetanus Antibod y Titers ................................ ................................ ................................ ......... 88 Neutrophil Function ................................ ................................ ................................ ................ 91 Serum Trace Minerals ................................ ................................ ................................ ............. 92 Serum Vitamin B12 ................................ ................................ ................................ ................ 93 Early Postpartum Reproductive Performance ................................ ................................ ........ 95 6 IMPLICATIONS ................................ ................................ ................................ .................... 99 APPENDIX A PROCEDURE FOR A SSESMENT OF EQUINE NEUTROPHIL FUNCTION ................ 101 B PROCEDURE FOR SERUM MANGANESE ANALYSIS ................................ ................ 104 C PROCEDURE F OR SERUM COPPER AND ZINC ANALYSIS ................................ ...... 105 D PROCEDURE FOR IMMUNOGLOBULIN ANALYSIS ................................ .................. 106 E MARE AND FOAL SERUM FOLATE ................................ ................................ ............... 108 LIST OF REFERENCES ................................ ................................ ................................ ............. 110 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 120
8 LIST OF TABLES Table page 2 1. AAFCO feed ingredient definitions for organic trace mineral products ................................ 16 3 1. The exp ected and actual concentrations of zinc, manganese, copper and cobalt in the SULF and 4PLEX supplement pellets ................................ ................................ ............... 53 3 2. Nutrient composition of each of the four batch mixes of SULF and 4PLEX su pplement pellets (100% DM basis) ................................ ................................ ................................ .... 54 3 3. Nutrient composition of whole oats, Coastal bermudagrass hay and bahiagrass pasture in the basal diet (100% DM basis) ................................ ................................ ..................... 55 3 4. Mineral concentrations in the total diet based on actual intake of supplement pellets and oats and estimated intake of pasture and/or hay for mares in late gestation and lactation. ................................ ................................ ................................ ............................. 56 4 1. Bodyweights of mares and foals ................................ ................................ ............................. 69 4 2. Effects of dietary trace mineral (TM) source and duration of supplementation on immunoglobulin concentrations in mare c olostrum ................................ .......................... 70 4 3. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on immunoglobulin concentrations in foal serum at 24 and 36 h of age .................. 71 4 4. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on tetanus specific IgG titers (IU/mL) in foal serum ................................ ................ 72 4 5. Effects of dietary trace mineral (TM) source and duration of supplementation on neutrophil phagocytosis and oxidative burst in the mare 56 d before and 56 and 112 d after foaling ................................ ................................ ................................ ........................ 73 4 6. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on neutrophil phagocytosis and oxidative burst in the foal at 1 2, 56, and 112 d of age ................................ ................................ ................................ ................................ .. 74 4 7. Effe cts of dietary trace mineral (TM) source and duration of supplementation on copper, zinc, and manganese concentrations in umbilical cord serum .............................. 75 4 8. Effects of dietary trace mineral (TM) sour ce and duration of supplementation on copper, zinc, and manganese concentration in the mare 56 d before and 56 d after foaling ................................ ................................ ................................ ................................ 76 4 9. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on copper, zinc, and manganese concentrations in foal serum at 1 2 and 112 d of age ................................ ................................ ................................ ................................ .. 77
9 4 10. Effects of dietary trace mineral (TM) source and duration of suppleme ntation on serum cobalamin concentrations in the mare 56 d before and 56 and 112 d after foaling ................................ ................................ ................................ ................................ 78 4 11. Effects of dietary trace mineral (TM) source and duration of supplementation in the mar e on serum cobalamin concentrations in the foal at 1 2, 56, and 112 d of age ............ 79 4 12. Effects of dietary trace mineral (TM) source and duration of supplementation on follicle diameter in the fi rst postpartum cohort ................................ ................................ .. 80 4 13. Effects of dietary trace mineral (TM) source and duration of supplementation on follicle growth rate in the first postpartum cohort ................................ ............................. 81 4 14. Effects of dietary trace mineral (TM) source and duration of supplementation on number of follicles developing in the first postpartum cohort and day of follicle deviation ................................ ................................ ................................ ............................. 82 4 15. Effects of dietary trace mineral (TM) source and duration of supplementation on day of foal heat ovulation ................................ ................................ ................................ ......... 83 4 16. Effects of dietary trace mineral (TM) source an d duration of supplementation on postpartum uterine morphology ................................ ................................ ......................... 84 4 17. Effects of dietary trace mineral (TM) source and duration of supplementation on peak serum concentrations of LH and FSH a nd day of peak concentration relative to ovulation ................................ ................................ ................................ ............................ 85 E 1. Effects of dietary trace mineral (TM) source and duration of supplementation on serum folate concentrations in the mare 56 d before an d 56 and 112 d after foaling ................. 1 08 E 2. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on serum folate concentrations in the foal at 1 2, 56, and 112 d o f age ................. 109
10 LIST OF FIGURES Figure page 3 1. Timeline of sample collection ................................ ................................ ................................ 57 3 2. Representative scatter plot generated from one mare illustrating neutrophil phagocytosi s and oxidative burst determined by flow cytometry. ................................ .......................... 58
11 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 SOURCE AND DURATION OF TRACE MINERAL SU PPLEMENTATION AND THEIR EFFECTS ON EARLY POSTPARTUM REPRODUCTIVE PERFORMANCE IN MARES AND INNATE AND ACQUIRED IMMUNITY IN FOALS By Jerome George Vickers IV December 2008 Chair: Lori K. Warren Major: Animal Science A study was conducted to investigat e the effects of source and duration of trace mineral supplementation on foal heat ovulation and innate and acquired immunity in nursing foals. Mares were blocked by age, breed, and expected foaling date and randomly assigned to dietary treatments in a 2 x 2 factorial arrangement. Mares were supplemented with isoelemental amounts of Zn, Mn, Cu and Co in one of two forms: 1) inorganic (SULF; n=18); or 2) amino acid complexes (4PLEX; n=18), supplied in amounts to provide 1X Zn and Mn, 1.5X Cu and 44X Co requi rements for mares in late gestation and lactation. Within each form of trace mineral supplementation, mares originated from one of two populations: 1) mares that had been maintained on a similar program of inorganic or amino acid complex trace mineral supp lementation for 6 mo as part of a previous experiment (LONG; n=18); or 2) mares that had no prior exposure to amino acid complex trace minerals (SHORT; n=18). This arrangement resulted in four treatments: 1) SULF LONG (n=9); 2) SULF SHORT (n=9); 3) 4PLEX L ONG (n=9); and 4) 4PLEX SHORT (n=9). Supplementation began 84 d before expected foaling and continued through 112 d post foaling. Neutrophil function was not affected by trace mineral source or duration of supplementation in mares or their foals. Colostr um IgG and IgM were not
12 affected by trace mineral treatment. Colostrum IgA was higher (P<0.05) in mares receiving 4PLEX and serum IgA at 24 h was higher (P<0.05) in foals nursing 4PLEX mares. Foals received a series of three tetanus toxoid vaccinations beg inning at 112 d of age. Tetanus specific IgG titers did not differ in foals nursing 4PLEX or SULF mares. Improved transfer of trace minerals in utero was not evident based on similar trace mineral composition of umbilical cord blood among treatments. Trace mineral treatment had no effect on mare serum Zn, Mn, or Cu. Foals from SULF mares had higher Zn levels at 1 2 d of age (P<0.05) and higher Cu levels at 112 d of age (P<0.05). Serum vitamin B12 was higher in 4PLEX mares at 112 d post foaling (P<0.05) and in foals from 4PLEX mares at 1 2 d of age (P<0.05). Dietary trace mineral source and duration of supplementation had no effect on growth rate of the six largest follicles, days to follicle deviation, days to ovulation, or the number of mares that ovulated within or after 10 d post foaling. In addition, temporal changes in intrauterine luminal fluid score, endometrial edema score, uterine body diameter, and non gravid horn diameter were not affected by trace mineral treatment. Growth rate of the largest foll icle (P<0.05) and rate of gravid horn involution (P<0.05) were greater in 4PLEX SHORT mares than 4PLEX LONG mares, but did not differ among other treatments. Based on the results of this study, it appears supplementing mares with trace mineral amino acid c omplexes in late gestation may positively affect transfer of passive immunity in the foal. In addition, this study illustrates the potential for amino acid complexes supplemented in late gestation to more rapidly repare the uterus for rebreeding at foal h eat.
13 CHAPTER 1 INTRODUCTION Trace minerals are important nutrients in the diets of all animal species, as they are essential components of certain enzymes and transport proteins, and they play fundamental roles in optimal immune and reproductive funct ions (Power and Horgan, 2000) While the effic acy of organic trace mineral supplementation has been studied in most farm animal species (Spears, 1996; Acda and Chae, 2002; Hostetler et al., 2003) research in horses is lacking. The small number of studies usin g horses have principally examined organic trace mineral supplementation in weanlings and yearlings, with little focus on supplementation of the broodmare and its effect on the foal. The physiologic state of the broodmare is unique from other classes of h orses, due to an increased systemic demand for nutrients during pregnancy, parturition, and lactation. Research has shown that deposition of the trace minerals copper, zinc, and manganese in foal tissues is greater at birth than during the first year of th (Meyer and Ahlswede 1978) indicating accretion of trace minerals during gestation. Increasing the trace mineral content of the diet above NRC requirements during lactation has little effect on the trace mineral content of the (Lewis, 1995) thus making in utero transfer of trace minerals important. Therefore, supplementing the broodmare with organic trace minerals during gestation may serve as an appropriate means to improve immune function and trace mineral status of the foal. The reproductive success of the mare during foal month foaling interval. To the authors knowledge, only two studies (Ley et al., 1990; Ott and Asquith, 1994) have investigated the effects of organic trace mineral supplementat ion on reproduction in horses. By comparison, several studies in ruminants have shown enhanced reproductive function compared to inorganic
14 trace mineral supplementation. Organic trace mineral supplementation of broodmares in late gestation through the fir st postpartum estrous cycle may provide a method for enhancing reproductive performance during foal heat. The objectives of this research were to determine the effects of dietary trace mineral source and duration of supplementation in the mare on: 1) pass ive transfer of immunity to the foal; 2) innate and humoral immunity in nursing foals; 3) circulating trace mineral concentrations in mares and foals; 4) vitamin B12 status of mares and foals; and 5 ) reproductive performance of the mare during the first po stpartum estrous.
15 CHAPTER 2 REVIEW OF LITERATURE Trace Mineral Source, Absorption, and Metabolism Source Trace minerals are generally available in two molecular forms: inorganic or organic. Traditionally, trace mineral supplementation has been achieved th rough the use of inorganic trace minerals. The trace metal ion in the inorganic form is combined with an inorganic salt, largely oxide, sulfate, chloride, or carbonate. More recently, there has been interest in using organic trace minerals or a combination of inorganic and organic trace minerals in equine diets. The term organic is used to describe the molecule formed when a metal ion reacts with some organic ligand. The ligand in the metal ligand complex can potentially be any one or more amino acids, pro teins of varying size, polysaccharides, or propionate. As such, organic trace mineral sources encompass a wide variety of metal ligand arrangements, which have been defined by the Association of American Feed Control Officials (AAFCO, 2005) (Table 2 1).
16 Table 2 1. AAFCO feed ingredient definitions for organic trace mineral products 1 Product Fe ed Ingredient Number Description Metal amino acid complex 57.150 Product resulting from complexing of a soluble metal salt with amino acid(s). Declared as a specific metal amino acid Metal (specific amino acid) compl ex 57.151 Product resulting from complexing a soluble metal salt with a specific amino acid. Declared as a specific metal, specific amino Metal amino acid chelate 57.142 Product resulting from the reaction of a metal i on from a stable metal salt with amino acids with a mole ratio of one metal to one to three moles of amino acids to form coordinate covalent bonds and heterocyclic ring(s). Declared as a specific metal amino acid M etal polysaccharide complex 57.29 Product resulting from complexing of a soluble salt with a polysaccharide solution. Metal proteinate 57.23 Product resulting from chelation of a soluble salt (mineral) with amino acids and/or partially hydrolyzed protein. Declared as 1 Adapted from AAFCO, 2005 The terms c helates and complexes are often used interchangeably when referring to organic trace min erals, but in fact the terms identify different complexes. In general, a complex describes the product formed when a metal ion reacts with a ligand. The ligand is a molecule or ion that contains an atom which has a lone pair of electrons. Donor atoms suc h as oxygen, nitrogen, or sulfur serve to bond the metal ion to the ligand, forming a complex. Hynes and
17 Kelly (1995) describe ligands based on the number of donor atoms present. Ligands that contain a single donor atom are referred to as monodentate, while those containing two or more donor atoms cap able of bonding to a metal ion are referred to as bi tri or tetradentate, collectively known as polydentate ligands. Amino acids are examples of bidentate ligands, whereby amino acid(s) are bound to metal ions via an oxygen of the carboxylic acid grou p and the nitrogen of the amino group (Hynes and Kelly, 1995) The complex resulting from the use of polydentate ligands and metal ions creates one or more heterocyclic rings containing the metal atom, and is referred to as a chelate. Therefore, all chelates are complexes, but not all complexes are chelates. Mult iple chelates may form the organic trace mineral referred to as a proteinate. Proteinates are formed by partially hydrolyzing a protein source through enzymatic or acid procedures. The hydrolyzing of the protein results in the formation of an amino acid /peptide hydrolysate consisting of varying chain lengths. The reaction of this hydrolysate with a metal salt under appropriate conditions yields a complex containing chelated metal ions, called a proteinate (Hynes and Kelly, 1995). Inorganic and organic trace mineral supplementation strategies are both widely implemented, and important differences exist in their molecular forms. As such, important differences also exist in uptake mechanisms and the ultimate bioavailability of different sources. Absorpt ion The absorption of ingested minerals is often the primary factor determining their ultimate utilization. Ingested minerals can be separated into two categories which aid in distinguishing their absorptive capacities. First are those minerals soluble throughout the varying pH of the gastrointestinal tract. Minerals such as magnesium, sodium, and calcium fall into this first category, in which hydroxy polymerization is not a concern. Minerals that undergo hydroxy
18 polymerization in the gastrointestinal tract fall into the second category, and include most trace minerals. Acid soluble cations like copper, manganese, and zinc are least soluble when exposed to the neutral pH of the small intestine (Whitehead et al., 1996) Because of their hydrolytic nature, water molecules tend to coordinate around the metal io n, subsequently losing protons in the neutral pH of the small intestine. This results in a hydroxy metal species, ultimately forming insoluble precipitates in the absence of soluble binding ligands. For normal metal ion uptake to occur, it has been propo sed that both endogenous soluble ligands and mucosally associated ligands are required (Ashmead, 1993; Whitehead et al., 1996) Gastroferrin, albumin, lactoferrin, citrate and soluble mucins have all been identified as possible secreted soluble ligands, preventing the hydroxy polym erization of susceptible metal ions. The predominant mucosally associated ligand is thought to be mucin. Mucus is chiefly produced by goblet cells and released throughout the digestive tract, and constitutes two separate phases (Hunter et. al., 1989), th e aforementioned soluble mucin and the insoluble mucous gel layer adherent to the mucosal surface. Luminal nutrients must pass the mucosally adherent mucous layer before actual presentation to the enterocyte and subsequent absorption occurs. The solubl e mucins present throughout the gastrointestinal tract have a strong affinity for metal binding, in which highly charged metals are bound more readily than more neutrally charged metals. The affinity of metal binding to mucus in the mucosally adherent muc us layer follows the same pattern, and the pattern for mineral absorption is its inverse (i.e., the greater the binding of minerals to mucus, the less absorption) (Whitehead et al., 1996). In short, the mucous layer acts as a filter in regulating metal up take and the strength of binding to and rate of passage across the mucosally adherent mucus layer could be important in determining the overall absorption of a metal (Powell et al.,
19 1999a ) Highly charged metals would be bound tightly by mucus, having kinetically slow rates of ligand exchange, rendering the metal unable to pass quickly through this layer. Metals that remain bound are shed with the mucus back into the lumen, and excreted (Powell et al., 1999b) This is illustrated by toxic Al 3+ in which the metal is so tightly bound by the mucus layer that it rarely manages to traverse it, and is sloughed off into the lumen during mucosal turnover. The tight binding of triv alent cations also serves to explain the decreased absorption of ferric iron (Fe 3+ ) compared to ferrous iron (Fe 2+ ) (Whitehead et al., 1996). Thus, metal ions must avoid hydroxy polymerization and also penetrate two functional barriers before presentation to the enterocyte for absorption. While hydroxy polymerization accounts for much of the metal ion loss in the gastrointestinal tract, antagonistic relationships among minerals also occur that inhibit their absorption and utilization. Antagonism can occur between two different minerals, as well as among minerals and other nutrients present in the gastrointestinal tract. For example, in ruminants the formation of insoluble thiomolybdates can occur in the presence of sulfur and high intakes of molybdenum, re sulting in a copper deficiency (McDowell, 2003). In the horse, an antagonistic relationship exists between zinc and copper, such that when high levels of zinc are supplemented, competition among minerals for binding sites can result in a copper deficiency (Bridges et al., 1984) In addition, phytic acid and dietary fibers have been shown to bind trace minerals, thus negatively influencing their absorption (Forbes and Erdman, 1983) While inorganic trace minerals are subject to a host of physiochemical factors affecting absorption, organic trace minerals are proposed to avoid such factors. Although not fully elucidated, organic trace minerals are believed to utilize alternate uptake mechanisms in the gastrointestinal tract. Amino acid complexes and proteinates have been proposed to utilize
20 amino acid uptake mechanisms rather than normal metal ion uptake pathways. The theory, as described by Power and Horgan (2000), is that the metal in question is protected within the complex in a chemically inert form by covalent and ionic bonding by the amino acid ligand. Therefore, the metal is not susceptible to dissociation because of the metal ligand high stability constant, preventing exposure to the physiochemical factors mentioned earlier. It is believed that the organic trace mineral is absorbed intact through the intestinal mucosa, effectively pulling the metal along with it. However, if organic trace minerals have strong stability constants such that dissociation fails to occur once absorbed, the metabolism of the mineral may be compromised (Miles and Henry, 2000) Regardless of uptake mechanism, ligand binding can reduce the charge on mineral ions. By reducing charge, metal ions may arrive at the mucosally adherent mucus layer in a non precipitated form, avoid negative interactions with dietary phytates and phenols, speed the passage of the metal through the mucus layer, and avoid competition with unprotected metal ions for binding sites on mucins (Power, 2006) Bioavailability Bioav ailability, defined differently by many, can be described as the involvement of both the absorption and ultimate metabolic utilization of nutrients within the cell (Power and Horgan, 2000). The bioavailability of various inorganic trace mineral sources ha s been reviewed elsewhere (Ammerman et al., 1995; McDowell, 2003) and will not be described here. The alternate uptake mechanisms as well as protective mechanisms exhibited by organic trace minerals may result in increased absorption, ultimately enhancing their bioavailability. By increasing the availability of the trace mineral, it may be possible to elicit different and often optimal physiol ogic responses in the animal, which may subsequently redefine what constitutes normal performance. Evidence for greater bioavailability of organic trace minerals has been
21 sought from a variety of different measures, which will be discussed in relevant sec tions throughout this chapter. Trace Mineral Supply to the Nursing Foal Placental Transfer of Trace Minerals The equine placenta is characterized as having an epitheliochorial design. The epitheliochorial design is such that the uterine epithelium, connec tive tissue and blood vessel endothelium separate the maternal blood from the placental absorbing surface, similar to the ruminant placenta. This is unlike the hemochorial placenta of most higher primates, in which there is direct contact between the chor ionic villi and a circulating pool of maternal blood. As a result, the equine fetus is completely dependent on the dam for its supply of nutrients, including trace minerals The trace minerals copper, manganese, and zinc have all been proposed as limiti ng trace elements required by the fetus for normal growth (Abdelrahaman and Kincaid, 1993) Little research exists describing the placental transfer of trace minerals in the horse. In one of the few studies completed, Meyer and Ahlswede (1978) reported that almost half of the fetal copper, manganese, and zinc deposited in the developing equine fetus occurs during the last 2 months of gestation. In contrast, Abdelrahman and Kincaid (1993) evaluated copper concentrations in fetal tissue at different stages of pregnancy in cattle, and found that stage of gestation did not affect copper concentrations in the fetal liver or kidney. However, others have noted an accumulation of copper in the ovine fetal liver with the progression of gestation (Hostetler et al., 2003) Trace Mineral Con tent of Mare Colostrum and Milk In addition to placental transfer and subsequent storage of trace minerals occurring in utero mare colostrum and milk serve as sources of trace minerals available to the nursing foal. st milk, and is more viscous than milk. During the last
22 trimester of gestation, the mare produces colostrum and it is secreted for a very short duration after parturition (Lavoie et al., 1989) Within 24 h after foaling, mammary secretions have transitioned from viscous colostrum to milk (Ul l rey et al., 1966) Trace mineral concentrations are generally highest in colostrum and decrease as colostrum transitions to milk. Schryver et al. (1986) examined the trace mineral composition of mare milk from 1 to 17 wk postpartum. They found that the concentrations of total solids, ash and minerals were highest during the first week of lactation. Zinc concentration progressively declined by 41% from wk 1 to wk 5, and remained relatively unchanged through wk 17 of lactation. Copper displ ayed the greatest decrease in concentration, declining 35% from wk 1 to 4, further declining by 47% from wk 5 to 8, and remaining relatively constant from wk 9 to 17. Ullrey et al. (1974) reported similar declines in the trace mineral concentrations of mare milk wit h the progression of lactation. The authors obtained frequent samples from mares beginning at parturition and continuing through 16 wk of lactation. Zinc concentration was highest in colostrum obtained at parturition, but declined 44 56% between 12 and 24 h postpartum. Thereafter, zinc concentration progressively declined an additional 39% by wk 5 and remained relatively unchanged through 16 wk of lactation. Similar to zinc, copper concentration was highest in colostrum and declined 16 26% between 12 and 24 h postpartum. Copper concentration declined a further 60% in the first week of lactation and an additional 48% between wk 1 and 5 of lactation. Based on comparisons between zinc and copper concentrations in milk and estimates of foal requirements, the aut hors concluded that milk copper concentrations were low, but zinc from milk may be sufficient to meet the needs of the nursing foal. A study by Grace et al. (1999) in New Zealand confirmed that mare milk is low in trace minerals relative to that required by t he foal. Milk was sampled from pasture fed Thoroughbred mares at various times during early, mid, and late lactation. Results showed that
23 milk provided adequate amounts of magnesium, sodium, potassium, sulfur, and zinc to the foal, but appeared to be ina requirements. Other than a study demonstrating manganese concentration in mare milk to be similar to that of cow milk (Anderson, 1992) little information exists regarding the manganese concentration in mare milk. It is unknown whether the stage of lactation affects the manganese concen tration of mare milk, or whether the manganese supplied in mare milk is capable of meeting the requirements of the nursing foal. has been reported to have little effect (Baucus et al., 1987; Breedveld et al., 1988; Kavazis et al., 2002) In contrast, there is some evidence that supplementation of the pregnant mare in late gestation may effective ly increase fetal trace mineral stores, which may be of use after birth (Pearce et al., 1998). Therefore, natal reserves resulting from placental transfer of trace minerals in utero and consumption of solid feeds likely contribute significantly towards mee ting the trace mineral requirements of the foal in addition to milk. Overview of the Immune System Innate Immunity The innate immune system, unlike that of adaptive and acquired immunity, consists of all the immune defenses that lack immunologic memory. T hus, innate immunity is characterized by responses that remain unchanged however often the antigen is encountered (Mackay and Rosen, 2000) The nonspecific nature of the innate immune system provides a defense mechanism for antigens, as described by Goldsby et al. (2003) in anatomical, physiologic, phagocytic, and inflammatory ways. The skin and the surface of mucous membranes serve as anatomic defense mechanisms, preventing entry of most microorganisms. T he physiologic barriers contributing to
24 innate immune function are, among others, temperature, pH, and various cell associated molecules that act as chemical mediators of innate immunity. Once pathogens gain access to the body, many are recognized, ingest ed, and killed by phagocytes. Macrophages are the first cells to encounter pathogens, but are quickly reinforced as neutrophils are recruited to the site of infection (Janeway et al., 2005) Functioning as phagocytic cells, macrophages and neutrophils act by phagocytosis, in which the bound pathogen is surrounded by the phagocyte membrane and t hen internalized. Upon phagocytosis, the neutrophils and macrophages of the innate immune system also produce toxic products like nitric oxide, superoxide anion, and hydrogen peroxide to aid in killing bacteria. Superoxide is generated by a multicomponen t, membrane associated NADPH oxidase process termed respiratory burst (Janeway et al., 2005). Unlike macrophages, neutrophils are short lived cells, dying after the completion of oxidative burst, and comprise the majority of pus that forms with an infecti on. Lastly, inflammation represents a complex sequence of events that stimulates immune responses, known as the inflammatory response. The end result of inflammation is often the marshalling of specific immune responses to the invasion or clearance of th e invader by components of the innate immune system (Goldsby et al., 2003). Acquired Immunity The acquired immune system involves a number of different cells and proteins that respond to a specific antigen. Acquired immunity, unlike innate immunity, is hi ghly specific. The term acquired is used because the immune cells involved must be exposed to an antigen to develop memory of that antigen, known as a primary response. In a subsequent exposure to the antigen, immune cells will react with greater compete nce because specificity for the antigen has already been established (Goldsby et al., 2003). The responses elicited by the acquired immune system involve the proliferation of antigen specific B and T lymphocytes (also referred to as B and T cells). For pr oliferation to occur, surface receptors of the B and T cells must bind to the
25 antigen (Mackay and Rosen, 2000) Specialized cells termed antigen presenting cells display the antigen to lymphocytes, and work in concert with them during response to the antigen. Developing from pluripotent stem cells in the fetal liver and bone marrow, B and T cells will mature and eventually circulate throughout the extracellular fluid (Mackay and Rosen, 2000). B cells mature within bone marrow, while T cells migrate to the thymus where their development is completed. The B cells within t he immune system function to secrete immunoglobulins, the antigen specific antibodies responsible for eliminating extracellular microorganisms. T cells aid in the antibody production by B cells, and also function to eliminate intracellular pathogens by ac tivating macrophages and by killing virally infected cells (Mackay and Rosen, 2000). The acquired immune system is comprised of, among others, the humoral immune system. The humoral immune system is mediated by B cells, and describes the type of immuni ty associated with extracellular fluids, including plasma and lymph (Goldsby et al., 2003). The antigen recognition molecules of B cells are the immunoglobulins, produced in a vast range of antigen specificities (Janeway et al., 2005). Antigen specific i mmunoglobulins are secreted as antibodies by the differentiated B cells known as plasma cells. Antibodies are primarily found in blood but are also located in mucosal secretions and in fluids such as milk. The immunoglobulin repertoire of the horse consi sts of IgG, IgM, IgA and IgE (Nezlin, 1998) Immunoglobulin G is the smallest of the immunoglobulin isotypes (Nezlin, 1998), moving easily fro m the blood to other tissues. Therefore, IgG is the major immunoglobulin constituent present in blood and milk (Tizard, 1996) In the horse, four IgG subclasses have been described, consistin g of IgGa, IgGb, IgGc, an IgG[T] (Sheoran et al., 2000) The subclasses chain sequence (amino acid), with each IgG subclass having different antigenic properties (Goldsby et al., 2003). More recently, the nomenclature of equine
26 immunoglobulin subclasses has been changed to reflect their corresponding immunoglobulin heavy chain constant genes. Seven genes have been identified for encoding gamma heavy chains; thus, more IgG isotypes occur than the four described above (Wagner, 2006) The newer nomenclature designates the IgG isotypes as IgG1 through IgG7 (Wagner, 2006). Regardless of the subclass, IgG functions predominantly by bindi ng to specific antigens like those found on the surfaces of bacteria (Tizard, 1996). While mainly present in the blood, IgG may also provide mucosal protection by paracellular passive transfer (Snoeck et al., 2006) Immunoglobulin M is second to IgG in its concentration in the blood, and is the largest of the immunoglobulin isotypes (Nezlin, 1998). Immunoglobulin M is secreted by plasma cells as a pentamer in which five monomer units are held together by disulfide bonds. Thus, the valency of IgM is greater than any other immunoglobulin, which is capabl e of binding up to five molecules of large antigens simultaneously (Goldsby et al., 2003). During the primary immune response to antigen, IgM is the first immunoglobulin class produced, and is also the first immunoglobulin synthesized by the neonate. IgM also plays an important role in the first line of defense, having an accessory role to IgA as a secretory immunoglobulin (Goldsby et al., 2003). Unlike the mode of transport utilized by IgG, IgM and IgA are bound to polymeric immunoglobulin receptor (pIg R), enabling the transport of the IgM and IgA to the mucosa (Snoeck et al., 2006). Immunoglobulin A is chiefly known for its role in mucosal immunity. The quantity of IgA produced exceeds that of all other immunoglobulin isotypes combined (Nezlin, 1998 ). Although present in serum, IgA is the predominant antibody present in mucosal secretions, as well as in saliva, milk, and tears. The mucosal predominance of the antibody depends on cooperation between local plasma cells that produce IgA and mucosal ep ithelial cells that
27 express the pIgR receptor (Snoeck et al., 2006). Once released from the plasma cell, the IgA molecule diffuses through the stroma, and becomes bound by the pIgR receptor. This facilitates the transport of IgA across mucosal epithelial cells for extrusion into external secretions (Snoeck et al., 2006). Secretory Ig A secreted on epithelial surfaces throughout the body serves in the first line of defense against pathogens, in large part by preventing adherence of bacteria and viruses to epithelial surfaces (Widmann and Itatani, 1998) Immunoglobulin E is present in the blood in very minute concentrations, and is the immunoglobuli n most associated with allergic reactions. Multivalent antigens bound by IgE antibodies triggers the release of histamine, leukotrienes, prostaglandins, and chemotatic factors from cells (Nezlin, 1998). Therefore, allergic and inflammatory reactions are initiated, such that serum IgE levels become elevated. This is exemplified when conditions like hay fever and chronic parasitic infections are present, which result in increased serum IgE concentrations (Nezlin, 1998). Passive Immunity in the Foal Impo rtance of Mare Colostrum The epitheliochorial placenta of the mare restricts transplacental passage of large molecules, including the large glycoproteins that are immunoglobulins (Jeffcott, 1974). Due to the absence of transfer of maternal antibodies in u tero the maternal supply of passive immunity occurs postnatally from immunoglobulins in colostrum and milk of the mare. Therefore, newborn foals, while immunocompetent, are immunologically nave due to the lack of placental transfer of immunoglobulins. For successful passive transfer of immunity, lactational secretions must contain adequate quantities of immunoglobulins, and the immunoglobulins must be delivered intact to the site of absorption and subsequently delivered to circulation (Rooke and Bland, 2002) Mares begin
28 synthesizing co lostrum during the last trimester of gestation, and it is secreted for a very short duration after parturition (Lavoie et al., 1989) Average immunoglobulin concentrations found in mare colostrum, milk, and serum are presented in Table 2 2. The immunoglobulin co ntent of mare colostrum is characterized by high concentrations of IgG, and relatively lower concentrations of IgA and IgM (Lavoie et al., 1989) Sheoran et al. (2000) reported that IgGb was the dominant isotype in pre suckle colostrum of mares, followed by IgGa, IgG(T), IgA, and IgGc in order of descending concentration; colostral IgM was not determined in their study. There is a rapid decline in all colostral i mmunoglobulins during the first 24 h after parturition (Pearson et al., 1984) Sheoran et al. (2000) observed a more than 40 fold decline in colostrum immunoglobulin concentration from birth to 1 d postpartum. Rouse and Ingram (1970) reported 20 fold decreases in IgG concentrations in mammary secretions of ponies between 0 to 3 hr and 9 to 24 hr after parturition. Such a rapid decline differs from other livestock, and suggests that transfer of immunoglobulins from serum to mammary secretions ceases at or possibly before parturition in the mare. Although IgG concentration continues to decline throughout lactation, IgA concentration remains relatively constant after about d 7 postpartum (Sheoran et al. 2000). As a result, IgA becomes the predominant immunoglobulin in mare milk (Norcross, 1982; Sheoran et al., 2000) Table 2 2. Immunoglobulin concentrations in colostrum, milk, and serum of adult horses 1 Immunoglobulin, mg/dL Sample IgG IgM IgA Serum 1000 1500 100 200 60 350 Colostrum 1500 5000 100 350 500 1500 Milk 20 50 5 10 50 100 1 Adapted from Tizar d, 1996.
29 As described above, foals are born essentially agammaglobulinemic and, thus, depend on passive transfer of maternal immunoglobulins from colostrum. Foals can, however, synthesize their own immunoglobulins, although de novo immunoglobulin producti on fails to yield serum concentrations similar to those of adult horses until 3 to 6 months of age (Jeffcott, 1974, 1975). The onset of endogenous immunoglobulin production by the foal has been shown to vary by isotype. Foals were observed to produce mucos al IgA between 4 and 8 wk of age (Sheoran et al. 2000). Production of IgGa and IgG(T) did not begin t o occur until 4 to 6 wk of age (Sheoran et al., 2000; Holznagel et al., 2003) whereas production of IgGb was delayed beyond 16 wk of age (Ho l znagel et al. 2003). Endogenous synthesis of immunoglobulins by the foal may be inhibited by the presence of maternal antibodies, which, prevent the foal from producing antigen specific antibodies tha t maternal antibodies are already specific for (Wilson et al., 2001) Therefore, active immunization may be hindered in the presence of maternal ant ibodies. A series of studies have demonstrated that IgGb is a critically important immunoglobulin for resistance to viral and bacterial pathogens (Sheoran et al., 1997; Nelson et al., 1998) T herefore, although the uptake of all immunoglobulins in colostrum is crucial for successful passive transfer in the foal, the supply and uptake of large amounts of IgGb in colostrum are particularly critical, due to the late onset of endogenous IgGb production in the foal. Because colostrum secretion is short lived and immunoglobulin concentrations decline rapidly, there is a narrow window of opportunity to ensure passive transfer of immunity to the foal. Immunoglobulins ingested shortly after birth are absorbed by the epithelial cells of the small intestine (Jeffcott, 1975). The epithelial cells of the newborn intestine are highly proliferating, but are replaced by mature cells withi n 38 h of life (Jeffcott, 1975). Thus, the
30 sensitive process. Known as gut closure, permeability of colostral immunoglobulins is terminated by approximately 24 h of age in t he foal (Jeffcott, 1971) Failure of Passive Immunity in the Foal Under normal circumstances, colostrum derived immunoglobulins provide protection for the foal until that time when the foal is capable of synthesizing its own. However, ingestion of insufficient amounts of immunoglobulins or a delayed ingestion may result in the fail ure of passive transfer of immunity in the foal. Defined by Kohn et al. (1989) as the failure of absorption of maternal immunoglobulins, failure of passive transfer of imm unity often predisposes the foal to infection and death (Pearson et al., 1984). The causative factors of failure of passive transfer in foals are described as: 1) failure by the foal to ingest a sufficient volume of colostrum, 2) delayed access to colostr um resulting in an inability of the intestinal epithelial cells to absorb immunoglobulins, and 3) low colostral immunoglobulin concentrations (Pearson et al., 1984). The concentrations of immunoglobulins that confer failure of passive transfer have been equivocal. Pearson et al. (1984) suggested that foals over 24 h of age having serum IgG concentrations of less than 200 mg/dL were classified as having failure of passive transfer, while others have suggested failure of passive transfer when serum IgG is less than 400 mg/dL (LeBlanc et al., 1986) Partial failure of passive transfer has been defined when foal serum IgG is between 200 and 400 mg/dL (McGuire et al., 1977) An increased risk of bacterial infection indicative of failure of passive transfer was associated with foals having serum IgG levels of less than 400 mg/dL, while 800 mg/dL was considered satisfactory (Tyler M cGowan et al., 1997) Regardless of values indicative of failure of passive transfer, the value of ensuring passive
31 transfer from mare to foal is realized as it is the most commonly recognized immune deficiency in neonatal foals, often resulting in septi cemia and death (Raidal et al., 2005) Effect of Trace Mineral Supplementation on Immune Function The role of trace minerals in immune function has been well documented. Zinc affects the immune response by mediating T and B cell res ponses to antigens, and is also known to influence neutrophil and natural killer cell function (Calder et al., 2002; McDowell, 2003) In zinc deficiency, the aforementioned immune functions have all been suppressed (Calder et al., 2002). In addition, the immune system as a highly proliferating system depends on the availability of zinc, in which reduced circulating lymphocyte counts have been observed in zinc deficient livestock ( McDowell, 2003). Furthermore, elevated zinc intake has been shown to potentiate immune function above basal levels. Salvin et al. (1987) found that mice fed a high zinc diet had increased numbers of splenic plaque forming cells in response to T lymphocyt e dependent antigens. In a study by Singh et al. (1992), mice that received supplemental doses of zinc showed increased T lymphocyte and macrophage function. High zinc diets have also been shown to decrease the amount of lipid peroxidation in the livers of mice infected with Plasmodium berghei (Arif et al. 1976). During the acute phase in infection, decreasing plasma zinc levels are often observed in humans (Ibs and Rink, 2003). Although zinc is required by human beings for the proliferation of immune c ells, the same is true for the necessity of zinc for the proliferation of pathogens. Thus, decreasing plasma zinc levels may serve as a defense mechanism to inhibit the proliferation of pathogens (Ibs and Rink, 2003). Even so, zinc supplementation has re sulted in a reduced duration and severity of cold symptoms (Prasad et al., 2000), while other studies have shown that zinc has limited effectiveness for treatment of the common cold (Turner and Cetnarowski, 2000).
32 Similar to zinc, copper metabolism affec ts T and B cells, neutrophils, and macrophages, and in a copper deficient state, have been shown to be depressed (McDowell, 2003). In humans, copper deficiency results in neutropenia (Percival, 1995) and in animals has been shown to result in decreased phagocytic and killing function of neutrophils (Boyne and Arthur, 1981) However, Arthington et al. (1995) found no depression of neutrophil bactericidal function during copper depletion or repletion of heifers. In addition to copper, a deficiency in zinc has been shown to impair chemotaxis, phagocytosis, and generation of the oxidative burst by neutrophils in humans and primates (Allen et al., 1983; Keen and Gershwin, 1990) Although less is known about manganese and its role in immune f unction, the interaction of manganese with cells such as neutrophils and macrophages has been demonstrated (McDowell, 2003). In addition, abnormalities of cell function and ultrastructure occur in manganese deficiency (McDowell, 2003), having the potentia l to compromise cellular responses and activity in the immune system. Although most of the known impact of trace minerals occurs when animals are deficient, the an tagonistic relationships associated with inorganic trace mineral supplementation, organic trace minerals may meet animal requirements more efficiently, enabling further support of the immune function and reproductive performance of animals in high producti on and high stress situations. However, evidence for the benefits of organic trace minerals over inorganic sources for their effects on immune function has been somewhat equivocal. Dorton et al (2003) observed an increased antibody response to vaccination with ovalbumin in feedlot steers supplemented with copper lysine compared to copper sulfate. Ferket and Qureshi (1992) reported greater antibody titers in young turkey s supplemented with zinc and manganese amino acid complexe s compared with inorganic trace minerals. In recently transferred feedlot c attle supplementation
33 with zinc methionine tended to result in a greater primary humoral response to bovine herpesvirus 1 vaccination compared to cattle fed zinc oxide or no zinc supplementation (Spears et al., 1991) Weanling horses supplemented with zinc, manganese, and copper amino acid complexe s exhibited greater serum IgM concentrations in response to a single injection of pig red blood cells compared to weanlings fed isoelemental amounts of inorganic trace minerals (Siciliano et al., 2003) In contrast to the positive responses described above, an equal number of studies have found either no benefit to organic trace m inerals over inorganic sources or greater responses to inorganic trace mineral supplementation. Dorton et al. (2003) reported that feedlot steers supplemented with copper sulfate had greater total IgM and IgG concentrations after vaccination with pig red b lood cells compared to steers supplemented with copper lysine Ward et al. (1993) observed no difference in humoral immune response between steers suppleme nted with copper sulfate or copper lysine in response to an injection of ovalbumin. Spears and Kegley (2002) vaccinated cattle with infectious bovine rhinotracheitis and found no difference in serum antibody titers between steers sup plemented with zinc oxide or zinc proteinate. The lack of consistency between studies likely results from several factors, including the specific antigen durat ion of trace mineral supplementation, how closely the diet met animal requirements, and the different organic and inorganic trace mineral sources fed. Several studies have also reported on other aspects of immune function. Working with calves, Saker et al. (1994a) observed an increase d expression of histocompatability complex II for copper lysine fed cattle compared to those supplemented with copper sulfate. Copper lysine has also been found to increase monocyte phagocytic activity in calves compared to those supplemented with copper sulfate, although copper sulfate was supplemented at one half the rate
34 of copper lysine (Saker et al., 1994b) George et al. (1997) noted an increase in both cell mediated and humoral immune response when heifers were supplemented with trace mineral amino acid chelates compared to heifers that underwent isoelemental supplementation with the same inorganic trace minerals. In converse, Stanton et al (2000) found no difference in cell mediated immune resp onse as measured by injection with phytohaemaglutinin (PHA) between calves fed trace minerals from organic or inorganic sources. In a study by Heugten et al. (2003) weanling pigs supplemented with zinc lysine had greater lymphocyte proliferation in response to PHA and pokeweed mitogen compared to pigs supplemented with zinc sulfate. In lactating dai ry cattle, decreased somatic cell counts in milk have been observed when supplemented with zinc methionine. A p ooled statistical analysis of eight studies using each experiment as a replication indicated a significant improvement in somatic cell count as well as milk production in dairy cattle supplemented with zinc met hionine (Spears, 1996) Foal Heat Ovulation The first postpartum estrous in mares is co mmonly referred to as foal heat. The majority of mares show signs of estrus within 5 to 12 d postpartum (Ginther, 1992) The reproductive state of the prepartum mare is governed by progesterone; how ever, once parturition has occurred, progesterone is undetectable in the early postpartum mare (Hillman and Loy, 1969) Concentrations of luteinizing hormone (LH) are low immediately after foaling due to the suppressive effects of fetoplacental derived progestins ( Ginther, 1979). A surge of follicle stimulating hormone (FSH) be gins a few days before parturition and peaks just on or shortly after the day of parturition, accounting for the development of follicles during the first postpartum estrous cycle (Ginther, 1992). Serum LH concentrations increase during the periparturient period, usually occurring 72 h after foaling. Serum LH generally peaks near the day of ovulation; if LH does not peak on the day of ovulation, it will peak the day after.
35 Ovulation during foal heat estrous generally occurs between 9 and 12 d postpartum. Due to the mo gestation, conception would need to occur within 1 mo after parturition in order for the mare to foal on a yearly basis. Thus, the long gestation of the mare necessitates breeding during foal heat, particularly for mares foaling late in the season, in order to achieve maximum economic returns. However, pregnancy rates achieved during this time are reported to be 10 to 20% lower than rates achieved during subsequent estrous periods (Ginther, 1992). The decreased conceptio n rates associated with breeding on foal heat are thought to be due to incomplete involution of the uterus (Gyax et al., 1979; Griffin and Ginther, 1991) Uterine Involution Uterine involution is the restoration of the endometrium to a condition where conception can take place again, ensuring an optimal climate for embryonic development. During involution, degenerating or detached epithelial cells are replaced and the uterine epithelium redifferentiates into ciliated and secretory cell types (Steven et al., 1979) as the endometrium returns to prepartum histological appearance by 14 d postpartum. Accompanying these morphological changes is a change in uterine size, in which the uterus returns to its pre gravid size by 21 d postpartum (McKinnon et al., 1988) The return of the endometrium to its pre gravid morphological state is critical for the support of the next pre gnancy. This can be illustrated by the observation that pregnancy rates when bred at foal heat are higher in mares that ovulate 10 or more days after foaling than in mares that ovulate on or before 10 d after foaling (Loy, 1980) On average, the equine embryo enters the uterus 5 d after ovulation. Therefore, ovulation 10 or more days after foaling ensures that the endometrium has returned to normal prior to arrival of the embryo (Arrott et al., 1994)
36 Uterine Fluid The process of uterine involution is crucial for the expulsion of uterine fluids associated with foaling. Postpartum luminal fluids incl ude a mucus like material present 3 d after foaling, which is reported to be gone by 6 d postpartum if uterine involution progresses normally (Ginther, 1992). It has been suggested that some of the fluid represents an influx of fluid from the uterine wall into the lumen (Ginther, 1992). Ultrasonic monitoring of mares during the first postpartum ovulatory period has indicated that mares with detectable uterine fluid accumulations was associated with decreased pregnancy rates (McKinnon et al., 1988) Mares often exhibit a profound increase in the amount of uterine fluid immediatel y after foaling, representing both the fluid associated with foaling, as well as that resulting from the process of uterine involution. Intrauterine fluids decrease in temporal association with decreased uterine tone and diameter (i.e., uterine involution ). Without successful uterine involution and subsequent elimination of the luminal fluid, the ability of the mare to achieve and sustain pregnancy is compromised. Uterine Edema The appearance and disappearance of endometrial edema is related to the onset of estrus and ovulation in mares. During estrous, the mare is under the influence of estrogen, and will have an increase of edema in the reproductive tract associated with the development and progression of follicles. Substantial edema occurs during estru s, and is identified by trans rectal ultrasonography as dark (non echogenic) centrifugal rays between the lumen and peritoneal surface. When ovulation is imminent, a sudden decline of uterine edema occurs with the already declining estrogen decreasing sod ium retention in the lamina propria. The presence of uterine edema has been shown to be a reliable indicator of the estrogenic competence of the dominant follicle (Samper, 1997) Thus, the rise and subsequent decline in uterine edema is often used as a tool to aid in timing of breeding. Once the mare has ovulated the reproductive tract is under the
37 influence of progester one and uterine edema is no longer present under normal conditions (Samper, 1997; Watson et al., 2003) Effect of Dietary Trace Mineral Supplementation on Reproductive Performance The exact biological roles of trace minerals in reproduction are largely unknown. However, reports of compromised reproduction during dietary trace mineral deficiencies suggest their necessity for optimal reprod uctive performance. In copper deficient animals, estrous cycles and conception rates appear to remain unaffected, however, reproductive failure due to fetal death and resorption has been known to occur (U nderwood, 1977) Copper is also necessary for the formation of connective tissue (McDowell, 2003), and as such would be necessary for proper fetal development. Zinc is known to be centrally involved in cell division, suggesting its importance during fetal growth and during the physiologic events such as uterine involution that occur in the postpartum female. In addition, zinc is intimately associated with several hormones; for example, the steroid hormones androgen and estrogen function by binding to trans cription factors that contain zinc fingers (McDowell, 2003). Zinc deficiency in the female has been shown to impair the synthesis and or secretion of FSH and LH, as well as cause abnormal ovarian development and disruption of the estrous cycle (Bedwal and Bahuguna, 1994) Doisey (1974) pr oposed that insufficient manganese interrupts the synthesis of cholesterol and its precursors, which would ultimately inhibit the synthesis of sex hormones, adversely affecting reproduction. Hostetler et al. (2003) hypothesized that manganese may play a ro le in the secretion of progesterone based on the findings by Hidirolgou and Shearer (1976 ) that the manganese concentration in the corpus luteum of ewes increased during early pregnancy, and that inadequate progesterone concentrations are known to cause ea rly embryonic loss. While much is known regarding the effects of trace mineral deficiency on reproductive performance, little is known about what optimal levels of trace mineral supplementation achieve
38 optimum reproductive performance. Because organic tra ce minerals are generally considered to be more bioavailable to the animal, their supplementation may redefine what optimal reproductive performance is as it relates to optimal supplementation. The majority of studies investigating the effects of organic trace mineral supplementation on reproductive performance have been conducted in species other than the horse. Boland et al. (1996) reported that dairy cattle receiving proteinated trace minerals had a non significant reduction in days to follicle deviation, and 5 fewer days to first ovulation. Manspeaker et al. (1987) found that dairy cattle suppl emented with chelated trace minerals had increased ovarian activity, greater postpartum uterine involution of the pregnant horn, and less embryonic mortality, although results were not significantly different from cattle receiving no trace mineral suppleme ntation. Toni et al. (2007) replaced inorganic minerals with amino acid complexed trace minerals in dairy cows 60 d prior to calving and through 200 d of lactation, and observed that cows receiving the amino acid complexed trace minerals tended to have an increase in first service to conception compared to those cows receiving inorganic trace minerals. Campbell et al. (1999) found that dairy cattle supplemented with complexed trace minerals from parturition to 154 days in milk showed fewer days to first est rus than cattle receiving a control diet consisting of no organic trace minerals during the same time, although days to first service, days open, days from first service to conception, and services per conception were similar between control and supplement ed cows. Siciliano Jones et al. (2008) supplemented dairy cattle 3 wk prepartum through 35 wk postpartum with isoelemental amounts of trace minerals as sulfates or amino acid complexes and foun d no differences in days to first service, services per conception, or number of days open.
39 Reports addressing the effects of organic trace mineral supplementation on reproductive performance of mares are limited. Ott and Asquith (1994) observed a reduction in the number of cycles bred an d services per mare when mares were provided proteinated vs. inorganic trace minerals. Similarly, Ley et al. (1990) found that barren mares supplemented with inorganic trace minerals experienced no first cycle pregnancie s, two early embryonic losses, and higher number of services per 17 d conception compared to mares receiving chelated trace mineral supplementation. Neither the observations of Ott and Asquith (1994) nor Ley et al. (1990) were found to be significant after statistical analyses. Nonetheless, these studies, along with the positive responses seen in other species, provide impetus for further investigation of the effects of organic trace mineral supplementation on reproductive performance in the mare. Conclusio ns Evidence for improved bioavailability of organic trace minerals exists for many animal species (Spears, 1996; Acda and Chae, 2002; Hostetler et al., 2003). Although responses have varied, animals supplemented with organic trace minerals have often show n enhanced immune function when compared to animals supplemented with inorganic trace minerals. This improved response is particularly evident in animals stressed by transport or a high level of production, and in those placed in an environment with a high level of pathogen exposure. Similarly, reports on the effect of trace mineral source on reproductive performance have been conflicting, but several studies have described improvements in ovulation and conception when animals are supplemented with organic trace minerals. Most of these studies have considered conception rates or pregnancy rates as the ultimate end points, but have not attempted to identify the factors that work to enhance conception rates in animals fed organic trace minerals. Few studies have investigated the use of organic trace minerals in equine diets. In particular, it is unknown whether supplementation of the pregnant or lactating mare with organic
40 trace minerals will also benefit the health of her foal. Based on studies in other spe cies, supplementing the mare with organic trace minerals may have potential to affect foal immunity. on milk mineral composition; therefore, supplementing the pregnant mare with a more bioavailable form of trace mineral may promote greater placental transfer of trace minerals in utero and greater natal reserves Because of the role of trace minerals in immune function, these reserves may act to enhance inna te and humoral immunity in the foal. In addition, supplementing the pregnant mare with organic trace minerals may improve colostrum immunoglobulin composition, thereby augmenting passive transfer of immunity and disease resistance in the foal. Finally, or ganic trace mineral supplementation could improve early postpartum reproductive performance, permitting the mare to be rebred earlier to align with industry standards. The study presented in this thesis was conducted to better elucidate the potential benef its of substituting inorganic zinc, manganese, copper and cobalt with amino acid complexes of these trace minerals in the diets of broodmares. The objectives of this research were to determine the effects of dietary trace mineral source and duration of sup plementation in the mare on: 1) passive transfer of immunity to the foal; 2) innate and humoral immunity in nursing foals; 3) circulating trace mineral concentrations in mares and foals; 4) vitamin B12 status of mares and foals; and 5 ) reproductive perform ance of the mare during the first postpartum estrous.
41 CHAPTER 3 MATERIALS AND METHOD S Animals T hirty six pregnant Thoroughbred (n=20), Quarter Horse (n=14), and Standardbred (n=2) mares and their resulting foals were used to determine the effects of org anic or inorganic trace mineral sources on reproductive performance at foal heat and innate and acquired immunity in nursing foals. The age of m are s ranged from 5 to 22 yr with mean SE of 12.0 0.7 yr M ares and foals were housed at the Institute of F ood and Agricultural Sciences, Horse Research Center located in Ocala, FL Other than 1 to 2 wk surrounding parturition, m ares were housed on pasture. Within 1 wk of expected foaling, each mare was moved to a small, dry lot paddock Milk testin g for calcium concentration was performed during this time to better gauge time of foaling. As signs of impending parturition were evident, the mare was moved into a large 4.3 x 4.9 m foaling stall. After foaling, the mare and foal were kept in the stall for 24 h and then turned out into a small paddock for approximately 1 wk before returning to pasture. Throughout the experiment, horses received routine anthelmintic treatment and vaccinations according to the protocols established for pregnan t and lactating mares at the Horse Research Center. In addition, all foals were evaluated at 12 h postpartum for failure of passive transfer by using a semi quantitative enzyme immunoassay to detect immunoglobulin G in whole blood (SNAP Foal IgG Test kit; IDEXX Laboratories, Inc., Westbrook, M E). The experiment was performed in accordance with the regulations and approval of the Institutional Animal Care and Use Committee at the University of Florida. Diets and Treatments Mares were blocked by age, breed, and expected date of foaling and randomly assigned to dietary treatments in a 2 x 2 factorial arrangement. Mares were supplemented with
42 isoelemental amounts of trace minerals in one of two forms: 1) inorganic zinc, manganese, copper and cobalt (SULF; n=18 ); or 2) amino acid complexes of zinc, manganese, and copper and cobalt glucoheptonate (4PLEX; n=18). Within each form of trace mineral supplementation, mares originated from one of two populations: 1) mares that had been maintained on a similar program of inorganic or amino acid complex trace mineral supplementation for 6 mo as part of a previous experiment (LONG; n=18); or 2) mares that had no prior exposure to amino acid complex trace minerals (SHORT; n=18). Collectively, this design resulted in the foll owing four treatments: 1) SULF LONG (n=9); 2) SULF SHORT (n=9); 3) 4PLEX LONG (n=9); and 4) 4PLEX SHORT (n=9). Mares began receiving their respective treatments 84 d prior to estimated foaling date and continued through 112 d postpartum Foaling season las ted from January through May 2007 ; thus, the experiment spanned the months of October 2006 to December 2007. Supplemental trace mineral sources were incorporated into separate protein/vitamin/mineral supplement pellets (Table 3 1). Sources of trace minera ls in SULF treatments were zinc sulfate, manganese sulfate, copper sulfate, and cobalt sulfate. Trace mineral sources in 4PLEX treatments included zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate provided via 4Plex (Zinpro C orporation, Eden Prairie, MN), along with additional manganese in the form of manganese sulfate to provide isoelemental amounts of manganese compared to SULF treatments. The nutrient composition of the pellets was similar; the only difference was the sourc e of added zinc, manganese, copper and cobalt (Table 3 2). The amounts of sulfate and amino acid complex trace minerals added to each supplement pellet were f ormulated to provide 1X the daily zinc and manganese requirements, 1.5X the daily copper requireme nt, and 44X the daily cobalt requirement for mares in late gesta tion and lactation
43 (NRC, 2007). The estimated daily intake of trace minerals in mares in late gestation and lactation are presented in table 3 4. Throughout the experiment, mares were housed in one of two 16.2 ha (40 acre) pastures of similar forage composition, with mares on all treatments equally distributed among pastures. The basal diet for all treatment groups consisted of whole oats pasture and hay. From November to March (late gestatio n, early lactation), mares were offered Coastal bermudagrass hay ad libitum. From April to October (lactation) mares only h ad access to bahiagrass pasture. Twice daily at 0700 and 1500 h mares were individually fed whole oats and the protein/vitamin/miner al pellets containing either 4PLEX or SULF trace minerals. The amount of oats was adjusted to maintain body condition and ranged from 4.5 kg/d during late gestation and 8.5 kg/d during lactation (as fed basis). The supplement pellets were fed at a rate of 0.24% BW/d during late gestation and 0.32% BW/d during lactation (as fed basis). The diet was designed to meet or slightly exceed the nutrient requirements of mares in late gestation and lactation (NRC, 2007). All feeds were periodically sampled for subs equent analysis of nutrient composition. Pasture samples were collected from areas on or near where there was evidence of grazing and analyzed at 1 mo intervals from April to October. Four cores from each round hay bale fed from November to March, as well as random samples of oats collected from each weekly delivery, were composited and each analyzed as a single sample. Random samples of the SULF and 4PLEX supplement pellets were collected from each of the four batch mixes and composited and analyzed by b atch. Composite feed samples were dried (60C for 48 h) and stored at 20C until further analysis. Feed analysis consisted of DM at 100C (AOAC 1990), crude protein by an automated nitrogen analyzer (Elementar vario Max; Elementar Americas, Inc., Mt. Lau rel, NJ ), and NDF and ADF using an Ankom 200 Fiber Analyzer (Ankom Technologies, Fairfield, NY).
44 Copper, calcium, zinc, manganese and cobalt in feeds were determined by atomic absorption spectrophotometry (AAnalyst800; Perkin Elmer, Norwalk, CT). Phosphor us was determined by colorimetry (AOAC, 1990). The nutrient composition of the supplement pellets is presented in Table 3 2. The nutrient composition of the oats, hay, and pasture are presented in Table 3 3. Blood Sample Collection and Handling As summariz ed in Figure 3 1 and described in further detail in the sections below, blood samples were collected from mares and foals at specific intervals for the analysis of immunoglobulin trace mineral, cobalamin, and hormone concentrations. In all cases, b lood sa mples were collected by jugular venipuncture into evacuated blood collection tubes (BD Vacutainer Becton Dickinson Franklin Lakes, NJ) containing either sodium heparin or potassium EDTA for the harvesting of whole blood or plasma, or no anticoagulant fo r harvesting of serum. W ith the exception of samples obtained from foals at 24 and 36 h after parturition, all b lood samples were co llected between 0700 and 0900 h After collection, blood samples were immediately placed on ice and transported approximatel y 30 min to the Animal Nutrition Laboratory for further processing. Blood samples containing no anticoagulant were allowed to clot a minimum of 1 h before centrifugation to facilitate separation of serum. Blood samples were centrifuged at 2058 x g for 15 m in for separation of plasma or serum. Serum and plasma were harvested with plastic disposable pipettes and aliquoted into polypropylene cryogenic vials (3 4 vials, 1.0 2.0 mL each). Samples were frozen at whole blood used in the neutrophil function assay which w as analyzed on the day of collection Passive Transfer of Immunity At 30 d prior to expected foaling, all mare s received a booster vaccination containing tetanus, Eastern E quine E ncephalitis (EEE), Western E quine E ncephalitis (WEE), West Nile
45 Virus (WNV) influenza, E quine H erpes V irus 1 (EHV 1) and E quine H erpes V irus 4 (EHV 4) in order to facilitate maternal transfer of an tibodies in colostrum. Passive transfer of immunity was assessed by measuring immunoglobulin (Ig) concentrations in postpartum mare colostrum and foal serum. A pproximately 100 mL of pre suckle colostrum was obtained by hand milking shortly after foaling an d further processing. Colostrum was processed by gentl e mixing and strain ing through four layers of cheese cloth to remove any d irt and debris Samples were remixed and subsamples of colos trum were aliquoted into polypropolene cryogenic vials and stored at of Ig concentrations Approximately 10 mL of blood was obtained from foals by jugular venipuncture at 24 and 36 h after parturition into evacuated tubes containing no anticoagulant. Blood samples were processed for serum and stored until analyses of Ig concentrations were performed, as described above. Concentrations of IgG, IgA, and IgM were determined in mare colostrum and foal serum by sin gle radial immunodiffusion using commercially available kit s (VMRD, Inc., Pullman, WA). Serum and colostrum s amples were diluted to an appropriate level to ensure values were within measurable limits of the assay The detection ranges were 200 160 0, 31 250, and 25 200 mg/dL for IgG, IgA, and IgM, respectively. Four standards supplied by the manufacturer of known concentration for each Ig were used to create a standard curve, and unknown Ig concentrations in samples were determined using the Metra Fit computer program (Metra Biosyste ms, Inc., Mountain View, CA). Tetanus Antibody Titers Humoral immune function in foals was evaluated in response to tetanus vaccination. The same inactivated, multivalent vaccine containing tetanus, EEE, WEE, WNV, influenza A 2, EHV 1, and EHV 4 antigens (Fort Dodge Laborat ories, Fort Dodge, IA) used for the mares was administered to foals. A primary vaccination was administered i.m. at 112 d of age, followed by
46 a second and third vaccination at 140 and 168 d of age, respectively. Approximately 10 mL of bl ood was obtained by jugular venipuncture prior to the administration of vaccinations at 112, 140 and 168 d of age and 4 wk following the vaccination at 196 d of age. Blood was collected into evacuated tubes containing no anticoagulant, processed to obtain serum, and stored until later analysis, as described above. Tetanus specific IgG titers were determined in serum using an ELISA (Scintilla Development Co., Bath, PA). In addition to determining antibody titers in response to vaccination, maternal influence over tetanus specific IgG was also evaluated in foal serum obtained 1 2 d postpartum and at 56 and 112 d of age. Neutrophil Function P olymorphonuclear (PMN) neutrophil function was assessed in mares and foals as a measure of innate immunity. Blood was col lected by jugular venipuncture at 56 d prior to and 56 and 112 d after foaling in mares, and at 1 2, 56, and 112 d of age in foals. On each day of sampling, approximately 10 mL of blood were collected into evacuated tubes containing sodium heparin (for neu trophil function) and approximately 10 mL were collected into tubes containing potassium EDTA (for white blood cell differential analysis) as the anticoagulants. Neutrophil function in whole blood was assessed using a simultaneous phagocytosis and oxidativ e burst d ual color flow cytometric assay as described by Vineyard et al. (2007) Briefly, w hole blood in 100 L aliquots from each horse was loaded with 4 M dihydrorhodamine (DHR) for 10 min at Heat killed St aphylococcus aureus b acteria labeled with propidium iodide (PI) were added to achieve a bacteria:PMN ratio of 30:1. White blood cell differential analysis was used to determine the PMN concentration in whole b lood in order to achieve an accurate bacteria:PMN ratio. DHR loaded whole blood without bacteria served as the negative control, while DHR loadedwhole blood stimulated with phorbol miristate acetate (PMA) (5g/mL) served as the positive control. After incu
47 constant mixing, samples were placed on ice to immediately halt the p hagocytosis and oxidative burst processes. Samples were prepared for flow cytometry utilizing a Q prep automated lysing system (Coulter Corp., Miami, FL). F or completion of hemolysis, 500 L of de ionized water was added, and 10 L of 0.4% trypan blue added to quench extracellular fluorescence Preparation of the blood for flow cytometry was completed within 4 h of collection. A FACSort flow cytometer (Becton Dickinson, San Jose, CA) was utilized to measure the fluorescent intensi ty in the prepared sample after processing was complete. Data were collected from 10,000 cells/sample and analyzed using CellQuest software (Becton Dickinson San Jose, CA ) to quanti fy the per centage of neutrophils that phagocytosed bacteria, as well as the percentage of neutrophils with a phago cytosis induced oxidative burst, as shown in Figure 3 2. Due to an apparent decrease in the fluorescence of the bacteria over time, no attempt was made to examine the effect of time on neutrophil function. Vitamin B12 Status Cobalamin concentrations were determined in mare and foal serum a s a measure of the gastrointestinal microbial synthesis of vitamin B12 from supplemental cobalt. Approxima tely 10 mL of blood was obtained by jugular venipuncture from mares at 56 d prior to and at 56 and 112 d after foaling and from foals at 1 2, 56, and 112 d of age. Blood was collected into evacuated tubes containing no anticoagulant, processed to obtain se rum, and stored until later analysis, using the procedures described above. Serum was analyzed for cobalamin using an IMMULITE 2000 solid phase, competitive chemiluminescent enzyme immunoassay involving an automated alkaline denaturation procedure ( Gastroi ntestinal Laboratory, Department of Small Animal Clinical Sciences, Texas A & M University, College Station, TX).
48 Serum Trace Mineral s Maternal transfer of trace minerals to the foal in utero was estimated by analyzing the zinc, copper, and manganese conc entrations of umbilical cord blood collected at foaling In addition, serum t race mineral concentrations were determined in mare s 56 d prior to and 56 d after foaling and in foals at 1 2 and 112 d of age. Umbilical cord blood was collected during parturiti on before the umbilical cord had separated from the foal using a sterile syringe and needle. Subsequent blood samples obtained from mares and foals were obtained by jugular venipuncture. Umbilical cord and venous blood were processed to obtain serum and st ored until later analysis, as described above. The concentrations of Zn, Cu, and Mn were determined using atomic absorption spectrometry (AOAC 1990) To determine Zn and Cu concentrations, ser um was combined with de ionized water in a 1:1 ratio and read us ing a flame atomic abs orption spectrometer (AAnalyst 800 ; PerkinElm er, Inc., Shelton, CT) at wavelength s of 213.9 nm (Zn) and 324.8 nm (Cu). To determine Mn concentration, serum was combined with diluent (8 mL Triton X 100, 5 g of sodium EDTA per liter of de ionized water ) in a 1:1 ratio and analyzed using a graphite furnace atomic absorption spectrometer (AAnalyst 800 with THGA Graphite Furnace and AS 800 Autosampler ; PerkinElmer, Inc., Shelton, CT) at a wavelength of 279.5 nm Reproductive Performance To determine the effect of source and duration of trace mineral supplementation on reproductive performance mares were examined daily by the same investigator with trans rectal ul trasonography beginning 1 d post foaling and continuing through 1 d post ovulation. Reproductive exams were discontinued on mares failing to ovulate within 21 d post foaling in order to analyze the reproductive data more effectively.
49 The size and number of follicles developing in the first postpartum cohort were recorded daily without maintaining the identity of individual follicles, as described by (Ginther and Bergfelt, 1992) Follicle development data were used to de termine the following: 1) total number of follicles that developed from both ovaries; 2) average size of the 6 largest follicles that developed (3 from each ovary); 3) size of the largest follicle that developed; 4) days to foal heat ovulation; 5) occurren ce of ovulation less than or equal to 10 d or greater than 10 d post foaling; 6) days to follicle deviation, defined as the day in which the growth rate of the dominant follicle deviated significantly from the growth rate of the subordinate follicles (Ginther et al., 2002) ; 7) growth rate (mm/d) of the largest follicle that developed; and 8) average growth rate (mm/d) of the 6 largest follicles that developed (3 from each ovary). Assessment of intrauterine luminal fluid was recorded daily using a 4 point scale, where 1 = no fluid and 4 = extensive fluid occupying the uterine lumen through the body and both horns. Similarly, intrauterine endometrial edema was evaluated daily using a 4 point scale, where 1 = homogenous app earance or no edema and 4 = extreme heterogeneous appearance or excess edema. These data were used to determine the daily change in intrauterine luminal fluid and endometrial edema from foaling to foal heat ovulation. The diameters of the uterine body (m idway between horn bifurcation and cervix) and the gravid and non gravid uterine horns (at mid point between uterine bifurcation and the ovarian tip of the horn) were recorded and used to determine the rate of postpartum uterine involution. Daily blood sa mples (10 mL) were collected by jugular venipuncture from 1 d postpartum through 1 d post ovulation for the determination of serum luteinizing hormone (LH) and follicle stimulating hormone (FSH). Hormone concentrations were measured using a radio
50 immuno as say (RIA) assay (Colorado State University Endocrine Laboratory, Fort Collins, CO). Data were used to determine peak hormone levels relative to day of ovulation. Bodyweights Bodyweights were obtained from mares 84 d prior to foaling, within 1 2 d of part urition, and at 28 d intervals through 140 d post foaling. Bodyweights were obtained from foals within 1 2 d of birth and at 28 d intervals through 168 d of age. All bodyweights were obtained using a calibrated digital livestock scale with an accuracy of 0.5 kg. Statistical An alyse s One foal (mare on 4PLEX LONG) had a serum IgG level indicative of failure of passive transfer at 12 h of age (as determined by SNAP testing) and was given a plasma transfusion. The same foal was euthanized due to angular l imb deformities at 7 wk of age. As a result, the only data included in the analysis from this foal were umbilical trace mineral concentration, serum cobalamin and trace mineral concentrations at birth, and neutrophil function at birth. Another foal (mare on SULF LONG) was euthanized within 4 wk post foaling due to severe angular limb deformities. Only serum Ig to assess passive transfer, umbilical trace mineral concentrations, serum cobalamin and trace mineral concentrations at birth, and neutrophil functi on at birth were included in the analysis for this foal. A third foal (mare on SULF LONG) died due to complications from Rhodococcus pneumonia at approximately 4 wk of age. The data included in the analysis from this foal included serum Ig for assessment o f passive transfer of immunity, serum cobalamin and trace mineral concentrations at birth, and neutrophil function at birth were included in the analysis for this foal. It is unlikely the angular limb deformities or pneumonia were resultant of dietary trea tment. In all cases of foal loss, mares remained on treatment and all data collected from the mare remained in the analysis.
51 Three mares required a uterine lavage in the early postpartum period due to a retained placenta (mare on 4PLEX SHORT), placentitis (mare on 4PLEX LONG), or extreme intrauterine fluid (mare on SULF SHORT). As a result, no reproductive data from these mares were included in the statistical analyses. Two mares receiving 4PLEX LONG, two mares receiving SULF SHORT, and one mare receiving S ULF LONG failed to ovulate within 21 d postpartum. With the exception of ovulation occurring 10 d post foaling, data from these mares was omitted from all other reproductive analyses. Three mares receiving 4PLEX SHORT double ovulated and were removed fro m day of deviation analysis. The gravid and non gravid horn of two mares on SULF SHORT and one mare on 4PLEX SHORT could not be distinguished and thus, were not included in the analysis of these measures. Collectively, the number of mares included in the a nalysis of various reproductive performance measures was as follows: ovulation occurring 10 d post foaling (n=33; 4PLEX LONG (n=9), 4PLEX SHORT (n=6), SULF LONG (n=9), and SULF SHORT (n=9)); day of deviation (n=25; 4PLEX LONG (n=6), 4PLEX NEW (n=4), SULF LONG (n=9), and SULF SHORT (n=6)); all other reproductive measures (n=28; 4PLEX LONG (n=6), 4PLEX SHORT (n=7), SULF LONG (n=9), and SULF SHORT (n=6)). Statistical analyses of colostrum and foal Ig concentrations, neutrophil phagocytosis and oxidative bu rst, trace mineral concentrations in umbilical cord serum, and early postpartum reproductive performance were performed using the mixed procedure of SAS (Version 9.1; SAS Institute, Inc., Cary, NC). The model for foal serum Ig concentrations included sourc e, duration, time, and the source x duration, source x time, duration x time, and source x duration x time interactions, whereas the model for the other variables included source, duration, and the source x duration interaction. In addition, the freq proce dure of SAS was used to analyze whether the
52 day of ovulation occurred less than or equal to 10 d or greater than 10 d post foaling. Tetanus antibody titers, mare and foal serum trace mineral concentrations, and mare and foal serum cobalamin were analyzed u sing the mixed procedure of SAS (Version 9.1; SAS Institute, Cary, NC) with a covariance test suitable for repeated measures (Littell et al. 1998). The covariance structure was first order autoregressive, with horse within treatment used as the subject eff ect. The model for each variable included source, duration, time, and the source x time, duration x time, source x duration, and source x duration x time interactions. All data are expressed as the tr discussed as a trend.
53 Table 3 1. The expected and actual concentrations of zinc, manganese, copper and cobalt in the SULF and 4PLEX supplement pellets Expected Anal ysis 1 Actual Analysis 2 SULF 4PLEX SULF 4PLEX Nutrient As fed DM As fed DM As fed DM As fed DM DM, % 89.9 100 90.5 100 89.7 100 90.5 100 Zn, ppm 325 361 325 359 449 501 466 515 Mn, ppm 325 361 325 359 422 470 472 521 Cu, ppm 117 130 117 129 13 3 149 139 153 Co, ppm 23 26 23 25 25 28 26 29 1 Expected Analysis does not include the Zn Mn Cu and Co that naturally occur in the feeds included with in the supplement pellet. These endogenous levels were estimated at 44 ppm Zn, 38 ppm Mn, 27 p pm Cu, and 0.11 ppm Co (100% DM basis). 2 Mean of four batches of 4PLEX and SULFATE pellets (see Table 2 for individual batch analyses).
54 Table 3 2. Nutrient composition of each of the four batch mixes of SULF and 4PLEX supplement pelle ts (100% DM basis) SULF 1 4PLEX 2 Nutrient 1 2 3 4 Mean 1 2 3 4 Mean Moisture, % 10.3 10.1 10.4 10.3 10.3 9.0 9.6 9.7 9.7 9.5 DE, Mcal/kg 3.8 3.8 3.8 3.7 3.8 3.5 3.5 3.5 3.5 3.5 Crude Protein, % 36.0 36.4 37.0 36.0 36.4 35.5 36.4 36.4 35.8 36.0 ND F, % 9.2 9.8 9.4 9.3 9.4 15.8 16.1 15.5 17.6 16.3 ADF, % 5.1 5.6 4.6 6.4 5.4 11.2 9.9 10.0 10.9 10.5 Ca, % 3.3 3.1 3.4 3.6 3.4 3.5 3.5 3.6 3.7 3.6 P, % 1.9 1.8 2.0 2.0 1.9 1.9 1.9 2.0 1.9 1.9 Zn, ppm 500 545 455 502 501 521 493 540 505 515 Mn, pp m 475 529 418 456 470 583 454 574 474 521 Cu, ppm 142 165 147 141 149 184 136 147 146 153 Co, ppm 30 29 27 27 28 28 31 27 30 29 1 Represents Zn, Mn, Cu, and Co as sulfate forms. 2 Represents amino acid complexes of Zn, Mn, Cu, and Co.
55 Table 3 3. Nutri ent composition of whole oats, Coastal bermudagrass hay and bahiagrass pasture in the basal diet (100% DM basis) Pasture Nutrient Oats Hay April May June July Aug Sept Mean DE, Mcal/kg 3.3 2.0 2.6 2.4 2.6 2.4 2.0 2.1 2.4 Crude Protein, % 11.9 12.1 19 .7 16.8 21.4 17.7 13.3 14.5 17.2 NDF, % 28.0 73.1 58.6 62.3 62.0 59.0 69.8 69.8 63.6 ADF, % 13.2 38.6 29.6 32.0 30.2 31.3 39.9 39.7 33.8 Ca, % 0.07 0.33 0.45 0.56 0.47 0.34 0.37 0.40 0.43 P, % 0.34 0.22 0.31 0.26 0.32 0.37 0.30 0.31 0.31 Zn, ppm 29 41 35 25 32 31 29 22 29 Mn, ppm 48 113 123 120 148 89 70 66 103 Cu, ppm 6 8 10 9 10 11 10 8 10 Co, ppm 0.19 0.46 0.49 0.33 0.26 0.29 0.26 0.29 0.32
56 Table 3 4. Mineral concentrations in the total diet based on actual intake of supplement pellets and oats and estimated intake of pasture and/or hay for mares in late gestation and lactation. Nutrient 1 Zn, ppm Mn, ppm Cu, ppm Co, ppm Late Gestation 2 SULF 3 84 126 22 3.2 4PLEX 4 85 131 22 3.3 NRC requirement 5 40 40 12.5 0.05 Percent of requirement m et by supplement pellet 130 % 128 % 125 % 5470 % Lactation 6 SULF 3 80 115 23 3.3 4PLEX 4 82 120 23 3.4 NRC requirement 5 40 40 10 0.05 Percent of requirement met by supplement pellet 138 % 134 % 164 % 6185 % 1 All nutrient concentrations are pr esented on a DM basis. 2 Total daily ration includes oats (0.7% BW) and Coastal bermudagrass hay (estimated at 1.1% BW) and either the SULF or 4PLEX supplement pellet (0.20% BW). 3 Zn, Mn, Cu, and Co added to the supplement pellet are provided in sulfate for ms. 4 Zn, Mn, and Cu added to the supplement pellet are provided as amino acid complexes and Co provided as glucoheptonate complex. 5 Nutrient requirements based on total daily intake of 2.0% BW in late gestation and 2.5% BW in lactation (NRC, 2007). 2 Total daily ration includes oats (0.7% BW) and fresh bahiagrass pasture (estimated at 1.1% BW) and either the SULF or 4PLEX supplement pellet (0.28% BW).
57 Figure 3 1. Timel ine of sample collection
58 Figure 3 2 Representative scatter plot generated from one mare illustrating neutrophil phagocytosis and oxidative burst determined by flow cytometry. ( A ) Neutrophils were gated for analysis on the basis of cell size (forward scatter) and complexity (side scatter) Panels B, C, and D show the propidium iodide (PI) labeled y axis which represents the intensity of red fluorescence from bacteria labeled with PI. The dihydrorhodamine ( DHR ) labeled x axis represents the int ensity of green fluorescence generated by conversion of nonfluorescent DHR to fluorescent rhodamine by the oxidative burst response of neutrophils ( B ) N egative control t he lower left quadrant contains DHR loaded neutrophils in the absence of PI labeled b acteria. ( C ) P ositive control t he lower right and left quadrants contain DHR loaded PMN in the absence of bacteria stimulated with phorbol miristate acetate ( PMA ). T he lower right quadrant contain s cells that have undergone an artificial oxidative burst. ( D ) The lower left quadrant contains PI labeled bacteria that have not yet undergone phagocytosis or subsequent oxidative burst. The upper left quadrant contains DHR loaded neutrophils that have phagocytosed PI labeled bacteria T he upper right quadrant co ntains DHR loaded neutrophils that have phagocytosed PI labeled bacteria and have undergone a subsequent oxidative burst. A B C D
59 CHAPTER 4 RESULTS General Observations The body weight s of mares and foals are presented in Table 4 1 Dietary trace mineral source and duration of supplementation had no effect on mare or foal body weights. Mares lost an average of 67 kg at foaling, but subsequently maintained or gained body weight during lactation Mean bodyweight of foals SE at birth was 56.91.3 kg. From birth to 24 wk of age, average daily gain of foals was 1.2 kg/d. Routine observations at feeding time, as well as the lack of feed refusals, indicated the mares readily consumed the protein/vitamin/mineral supplement pellets containing either sulfate or amino acid complex trace mineral sources. In addition, foals were observed to share their Passive Transfer of Immunity Immunoglobulin concentrations in mare colostrum are presented i n Table 4 2. Dietary trace mineral source had no effect on colostrum IgG or IgM concentrations, but did affect IgA. Colostrum from mares receiving 4PLEX had higher (P=0.03) IgA levels than colostrum from mares receiving SULF. Long term versus short term d u ration of trace mineral supplementation had no effect on colostrum IgG, IgM or IgA concentrations. Similarly, trace mineral source x duration interactions were not detected for any of the colostrum immunoglobulins. Nonetheless, evaluation of the response among the four treatment combinations revealed that colostrum IgA was greater in mares receiving 4PLEX LONG (P=0.05) and tended to be higher in mares receiving 4PLEX SHORT (P=0.09) compared to that observed in mares fed SULF SHORT (Table 4 2). Similarly, c olostrum IgA concentrations were numerically higher in 4PLEX LONG
60 (P=0.14) and 4PLEX SHORT (P=0.22) compared to SULF LONG, but these differences were not significant. Colostrum IgG and IgM were not different among the four treatments. The Ig concentrations in foal serum mimicked those observed in colostrum (Table 4 3). Trace mineral source fed to the mare had no effect on foal serum IgG or IgM, but trace mineral source did affect foal serum IgA (P=0.02). Foals nursing 4PLEX mares had higher serum IgA concen trations at 24 h of age (P=0.05) and numerically higher IgA concentrations at 36 h of age (P=0.13) compared to foals nursing SULF mares. Duration of trace mineral supplementation in the mare showed a trend to affect foal serum IgG (P=0.10) and IgA (P=0.09 ), whereby foals from long term supplemented mares had higher serum IgG and IgA than foals from short term supplemented mares (Table 4 3). Foal serum IgM concentrations were not affected by the duration mares had been supplemented with trace minerals. Trac e mineral source x duration and source x duration x time interactions were not significant for foal serum immunoglobulins. However, differences in foal serum IgG and IgA were found among the four treatment combinations (Table 4 3). Foals nursing 4PLEX LONG mares had higher serum IgG across all time points (P=0.03) and specifically at 36 h (P=0.04) compared to foals from SULF SHORT mares, with foals from 4PLEX SHORT and SULF LONG exhibiting intermediate concentrations of serum IgG. Foal serum IgG did not dif fer among treatments at 24 h. Across time, serum IgA was higher in foals suckling 4PLEX LONG mares compared to 4PLEX SHORT (P=0.05), SULF SHORT (P=0.005), and SULF LONG (P=0.01). Most of this difference among treatments occurred at 24 h, where serum IgA wa s higher in foals nursing 4PLEX LONG mares compared to foals nursing mares fed SULF SHORT (P=0.02) or SULF LONG (P=0.04). In addition, serum IgA tended to be higher at 24 h in foals from 4PLEX LONG mares compared to 4PLEX SHORT (P=0.09). At 36 h, serum IgA continued to show a trend to
61 be higher in foals nursing 4PLEX LONG mares compared to foals nursing SULF SHORT mares (P=0.10). Foal serum IgM was not affected by trace mineral treatment of the mare at either 24 or 36 h of age. Tetanus Antibody Titers Teta nus specific IgG titers measured in the serum o f foals are presented in Table 4 4 Time had the greatest effect on antibody titers (P=0.0001). Titers were highest at 1 2 d of age, reflecting the uptake of maternal antibodies in colostrum. Titer levels had decreased by 56 d of age (P=0.001) and subsequently remained unchanged through 112 d of age. Although foals received their first tetanus vaccination at 112 d of age, antibody titers were not different from 112 to 140 d of age and, in fac t, continued to dec line (Table 4 4 ). Thus, a serological response to the first vaccination was not observed. Trace mineral source fed to the mare and duration of supplementation had no overall effect on foal serum tetanus antibody titers. However, a marginal response to the second vaccination administered at 140 d was observed in foals from SULF mares, in which antibody titers tended to increase (P=0.10) from 140 to 168 d of age. A similar response to the second vaccination was not observed in foals from 4PLEX mares (Table 4 4) When these responses were analyzed with respect to the four treatment combinations, only the foals from SULF SHORT mares showed an increase in antibody titers from d 140 to 168 (P=0.03), resulting in higher titers at d 168 compared to foals from 4PLEX SHORT mares (P=0.05). All foals, regardless of the mare treatment, failed to show a serologic response to the third vaccination administered at 168 d, as antibody titers were similar between d 168 and d 196. Neutrophil Function The phagocytic and oxida tive burst activities of neutrophils in mares and foals are presented in Tables 4 5 and 4 6, respectively. To avoid potential differences arising from the use of multiple Staphylococcus aureus cultures, the same batch of labeled bacteria were utilized in
62 t he neutrophil function assay throughout this 11 mo study. As a result, some loss of bacteria fluorescence was noted over time. Therefore, no attempt was made to analyze the effect of time on mare or foal neutrophil function. When data were analyzed within each day, source and duration of dietary trace mineral supplementation of the mare had no effect on the percentage of neutrophils that underwent phagocytosis or a subsequent oxidative burst in either mares or foals. Similarly, a trace mineral source x dura tion interaction was not observed for neutrophil function in mares or foals. However, at 56 d of age, foals nursing SULF SHORT mares tended to have a greater percent phagocytosis (P=0.06) and oxidative burst (P=0.06) compared to foals nursing SULF LONG mar es. Trace Mineral Concentrations in Umbilical Cord Serum Trace mineral concentrations in umbilical cord serum are presented in Table 4 7. Dietary trace mineral source had no effect on Cu or Mn concentrations in umbilical cord serum, but mares receiving SULF tended to have higher cord serum Zn than mares supplemented with 4PLEX (P=0.07). Duration of trace mineral supplementation and the trace mineral source x duration interaction did not affect umbilical cord serum Cu, Zn or Mn. Trace Mineral Concentrat ions in Mare and Foal Serum Serum trace mineral concentrations measured during late gestation and early lactation in th e mare are presented in Table 4 8 Dietary trace mineral source and duration of supplementation had no effect on mare serum Cu, Zn, or M n concentrations. Similarly, an overall interaction between trace mineral source x duration of supplementation was not observed for any of these minerals in mare serum. However, SULF LONG mares had greater serum zinc levels 56 d prior to foaling than 4PLE X LONG mares (P=0.04), while values remained similar between treatments at 56 d after foaling. An overall effect of time was detected for mare serum Mn (P=0.0002). Serum Mn declined from 56 d before to 56 d after foaling in mares
63 supplemented with SULF (P= 0.0003) and tended to decline in mares supplemented with 4PLEX (P=0.06). Copper and Zn concentrations in mare serum did not differ between pre and post foaling samples. Serum trace mineral concentrations measured in foals at 1 2 and 112 d of age are pre sented in Table 4 9. An overall effect of time was noted for all trace minerals, describing an increase in serum Cu (P=0.0001) and Zn (0.0001) and a decrease in serum Mn (P=0.002 ) in foals from 1 2 d to 112 d of age. Source of trace mineral supplemented to the mare and duration of supplementation had no effect on foal serum Cu, Zn, or Mn concentrations. No interactions between trace mineral source, duration of supplementation, and/or time were significant for foal serum Zn or Mn. In contrast, trends for sou rce x duration (P=0.06), source x time (P=0.10), and source x duration x time (P=0.11) interactions were detected for foal serum Cu. Although serum Cu concentrations were not different among treatments at birth, foals belonging to SULF mares had higher ser um Cu levels at 112 d of age than foals from 4PLEX mares (P=0.03) When all four treatment combinations were examined, foals from SULF SHORT mares had higher serum Cu at 112 d of age compared to SULF LONG (P=0.01), 4PLEX SHORT (P=0.002), and 4PLEX LONG (P= 0.02) foals. Foal serum Zn increased with age in foals from 4PLEX SHORT (P=0.03), 4PLEX LONG (P=0.0001), and SULF LONG (P=0.02) mares, but not SULF SHORT mares (P=0.14). Numeric decreases in serum Mn were observed in foals from SULF SHORT (P=0.27), 4PLEX L ONG (P=0.15), and SULF LONG (P=0.13) supplemented mares, but the decrease was only significant in foals from 4PLEX SHORT mares (P=0.01). The high variability in serum Mn measured at 1 2 d of age, coupled with the numerically higher initial serum Mn concent rations in foals from 4PLEX SHORT mares likely accounted for these responses.
64 Vitamin B12 Status Serum cobalamin concentrations measured during late gestation and early and late lactation in the mare are presented in Table 4 10. An effect of trace mineral source (P=0.03), time (P=0.0001), and source x time (P=0.05) were found for mare serum cobalamin. Serum cobalamin was similar among treatments 56 d before and 56 d after foaling. Mares supplemented with 4PLEX exhibited a 2 fold increase in serum cobalamin (P=0.001) from 56 to 112 d post foaling, whereas serum cobalamin remained unchanged over time in SULF supplemented mares. As a result, serum cobalamin was greater in mares fed 4PLEX at 112 d post foaling (P=0.001) compared to mares receiving SULF. Durati on of trace mineral supplementation had no effect on mare serum cobalamin; however, there was a trend for an effect of source x duration x time (P=0.10). Serum cobalamin concentrations did not change from 56 d pre to 56 d post foaling in any of the four t reatment combinations and were similar among treatments. From 56 to 112 d post foaling, 4PLEX SHORT (P=0.001), 4PLEX LONG (P=0.07), and SULF LONG (P=0.07) supplemented mares had an increase in serum cobalamin, while serum cobalamin remained unchanged in SU LF SHORT mares. At 112 d post foaling, mares fed 4PLEX SHORT had higher serum cobalamin concentrations than mares fed SULF SHORT (P=0.0001) and SULF LONG (P=0.02), and tended to have higher serum cobalamin than mares fed 4PLEX LONG (P=0.10). In addition, s erum cobalamin concentrations were higher in 4PLEX LONG mares (P=0.01) and tended to be higher in SULF LONG mares (P=0.07) at 112 d compared to mares receiving SULF SHORT. Table 4 11 lists serum cobalamin concentrations measured in foals. Although foals n ursing 4PLEX mares had numerically higher serum cobalamin concentrations than SULF foals at birth, the source of trace mineral supplied to the mare had no overall effect on foal serum cobalamin. An overall effect of time was noted (P=0.06). Serum cobalamin decreased from 1 2 to 56 d of
65 age in foals nursing 4PLEX mares (P=0.02) and remained unchanged from 56 to 112 d of age. A progressive decline in serum cobalamin from birth through 112 d of age was also observed for foals belonging to SULF mares, but these changes were not significant. Duration of trace mineral supplementation in the mare had no overall effect on foal serum cobalamin; however, foals nursing mares that underwent long term trace mineral supplementation had higher serum cobalamin concentration s at 1 2 d of age than foals from mares that underwent short term supplementation (P=0.04). The combination of trace mineral source and duration of supplementation in the mare had no overall effect foal serum cobalamin. Nonetheless, at 1 2 d of age, serum cobalamin concentration was greater in foals from 4PLEX LONG mares compared to foals from 4PLEX SHORT (P=0.04) and SULF SHORT (P=0.01) mares. Serum cobalamin in foals from SULF LONG mares did not differ from 4PLEX LONG foals at 1 2 d of age, and while nume rically higher, also did not differ from SULF SHORT or 4PLEX SHORT foals. No differences in foal serum cobalamin were observed among the four treatment combinations at 56 or 112 d of age. Postpartum Reproductive Performance Data obtained to ascertain repr oductive performance in the early postpartum period are presented in Tables 4 12 4 17. Source of dietary trace mineral had no effect on the average diameter of all follicles, the diameter of the largest follicle, or the average diameter of the 6 largest follicles that developed in the early postpartum period (Table 4 12). Similarly, duration of trace mineral supplementation had no effect on the average diameter of all follicles or the diameter of the largest follicle, but tended have an effect on the aver age diameter of the 6 largest follicles that developed (P=0.10), whereby short term supplemented mares tended to have a greater average size of the 6 largest follicles than mares receiving long term supplementation
66 (Table 4 12). There was no overall sourc e x duration effect, but 4PLEX SHORT mares tended to have greater average size of the 6 largest follicles than 4PLEX LONG mares (Table 4 12). There was no effect of source or duration of trace mineral supplementation on the growth rate of the largest foll icle or the average growth rate of the six largest follicles that developed in the early postpartum period (Table 4 13). Although there was also no overall effect of trace mineral source x duration of supplementation on either of these measures, the growt h rate of the largest follicle that developed did differ between the four treatment combinations. The growth rate of the largest follicle was greater in 4PLEX SHORT mares than 4PLEX LONG mares (P=0.04), and tended to be greater than SULF LONG (P=0.08) and SULF SHORT (P=0.08) mares (Table 4 13). Across treatments, the number of follicles that developed in the first postpartum cohort averaged 20.5 1.3. The number of follicles that developed were not affected by dietary trace mineral source, but showed a t rend to be affected by the duration of supplementation (P=0.10) and the source x duration interaction (P=0.09) (Table 4 14). Short term supplemented mares had greater number of follicles develop in the first postpartum cohort than did long term supplemente d mares (P=0.10). Comparison of the four treatment combinations revealed that mares fed 4PLEX LONG had fewer follicles develop than mares supplemented with 4PLEX SHORT (P=0.02), and tended to have fewer follicles compared to SULF LONG (P=0.09). The day of follicle deviation, where there is a departure in the growth rate between the dominant and subordinate follicles, averaged 6.1 0.7 d post foaling. Dietary trace mineral source, duration of supplementation, and the source x duration interaction had no eff ect on the day of follicle deviation (Table 4 14).
67 The number of days from foaling to foal heat ovulation is presented in Table 4 15. Across treatments, ovulation occurred 12.1 0.5 d post foaling. Dietary trace mineral source and the duration of supplem entation had no effect on the day of foal heat ovulation. Across treatments, ovulation occurred within 10 d postpartum for 24% of the mares, whereas 76% of mares ovulated greater than 10 d postpartum. There was no difference in the number of mares ovulatin g within or after 10 d post foaling between 4PLEX or SULF trace mineral sources, or among the source and duration combinations. Changes in uterine dynamics in the early postpartum period are presented in Table 4 16. In all treatments, the net negative cha nge in fluid score, uterine body diameter, and gravid and non gravid horn diameters were indicative of the process of uterine involution. The net positive change in edema score in all treatments reflected the increase in edema associated with follicle deve lopment and impending ovulation. Dietary trace mineral source and duration of supplementation had no effect on the change in intrauterine luminal fluid score or endometrial edema score from foaling to foal heat ovulation. Similarly, the source and duration of trace mineral supplementation had no effect on the change of the uterine body or non gravid horn from foaling to foal heat ovulation (Table 4 16). However, the change in diameter of the grav i d horn was affected by duration of supplementation, where mar es that had received short term supplementation had a greater rate of involution compared to mares that had been supplemented long term (P=0.05). Although the rate of change in diameter of the gravid horn was not affected by trace mineral source or the sou rce x duration interaction, differences were observed among the four treatment combinations. The gravid horn in mares receiving 4PLEX LONG had a slower rate of involution compared to 4PLEX SHORT (P=0.02), with SULF SHORT and SULF LONG mares exhibiting inte rmediate rates of involution (Table 4 16).
68 Dietary trace mineral source and duration of supplementation had no effect on peak LH or FSH concentrations in mare serum, or the day on which the peak in these hormones occurred relative to foaling (Table 4 17). Across treatments, peak LH concentration averaged 74.2 7.2 ng/mL and peaked on or near the time of ovulation (0 0.33 d). Serum FSH concentrations averaged 172.7 24.8 ng/mL and peaked 7.7 1.0 d prior to ovulation. A trace mineral source x duration of supplementation interaction was detected for the day of FSH peak (P=0.06). Mares receiving 4PLEX LONG tended to have FSH peak closer to foaling (and further from ovulation) than 4PLEX SHORT (P=0.06) and SULF LONG mares (P=0.07).
69 Table 4 1 Bodyweigh ts of mares and foals Day post foaling Mare bodyweight, kg Foal bodyweight, kg SULF 4PLEX SULF 4PLEX ( )56 644.0 (11. 6 ) 62 5.0 ( 11. 8) ----1 2 575.7 ( 10. 1) 558.3 ( 10.3 ) 56.7 ( 1.3 ) 57. 1 ( 1.3 ) 28 581.3 ( 10.3 ) 568.4 ( 11.8 ) 9 6.5 ( 2. 8) 95. 6 ( 2. 8) 56 588.8 (12. 9 ) 566.6 (12. 9 ) 133.4 ( 2.9 ) 143. 8 ( 13. 9) 84 590. 8 ( 10.0 ) 564. 2 ( 10. 1) 169.6 ( 4. 4) 163.4 ( 4. 1) 112 593.9 ( 11.8 ) 570. 2 (1 2.5 ) 196.0 ( 5. 7) 198. 4 ( 5.3 ) 140 596.0 ( 11.0 ) 570. 7 ( 12. 7) 224. 3 ( 5. 4) 224.2 ( 6.1 ) 168 ----252. 4 ( 5.6 ) 252. 3 ( 6.5 ) Values presented as mean (SE).
70 Table 4 2. Effects of dietary trace mineral (TM) source and duration of supplementation on immunoglobulin concentrations in mare colostrum Variable Colostrum immunoglobulins, mg/dL IgG IgA IgM TM Source SULF 10 268 ( 741 ) 464 ( 56 ) z 161 ( 17 ) 4PLEX 10,660 (741 ) 647 ( 56 ) y 153 ( 17 ) Duration SHORT 11,025 (741) 533 (56) 1 47 (17) LONG 9,903 (741) 577 (56) 166 (17) Source x Duration SULF SHORT 11,011 (1048) 436 (80 ) 151 (23) SULF LONG 9,526 (1048) 491 (80 ) y,z 155 (23) 4PLEX SHORT 11,039 (1048) 632 (80 ) 144 (23) 4PLEX LONG 10,281 (1048) 662 (80 ) y 178 (23) P values TM Source (S) 0.71 0.03 0.74 Duration (D) 0.29 0.60 0.42 S x D 0.73 0.87 0.54 Values presented as lsmeans (SE). y,z Within a column, means with different superscripts differ (P<0.05).
71 Table 4 3 Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on immunoglobulin concentrations in foal serum at 24 and 36 h of age Foal serum immunoglobulins, mg/dL IgG IgA IgM Variable 24 h 36 h 24 h 36 h 24 h 36 h TM Source SULF 2,404 (177 ) 2,510 (177 ) 46 z (11) 43 (11) 45 (4) 45 (4) 4PLEX 2,600 (177 ) 28 28 (177 ) 76 y (11) 66 (11) 51 (4) 47 (4) Duration SHORT 2,413 (177) 2,460 (177) 49 (11) 47 (11) 48 (4) 44 (4) LONG 2,592 (177) 2,879 (177) 72 (11) 61 (11) 49 (4) 49 (4) So urce x Duration SULF SHORT 2,236 (251) 2, 350 z (251) 41 z (16) 40 (16) 47 (6) 44 (6) SULF LONG 2,571 (251) 2 ,671 y, z (251) 50 z (15) 46 (15) 44 (6) 46 (6) 4PLEX SHORT 2,589 (251) 2,570 y, z (251) 56 y, z (16) 54 (16) 48 (6) 44 (6) 4PLEX LONG 2,612 (251) 3,087 y (251) 96 (16) 78 (16) 54 (6) 51 (6) P values TM Source (S) 0.15 0.02 0.33 Duration (D) 0.10 0.09 0.49 S x D 0.87 0.27 0.42 Time 0.35 0.57 0.62 S x Time 0.73 0.73 0.65 D x Time 0.50 0.68 0.69 S x D x Time 0.48 0.79 0.75 Values are presented as lsmeans (SE). y z Within a column, means with different superscripts differ (P<0.05). ns differ (P<0.10).
72 Table 4 4. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on tetanus specific IgG titers (IU/mL) in foal serum Foal age, d 1 Variable 1 2 56 112 140 168 196 TM Source SULF 2.83 a (0.27) 1.98 b (0.27) 1.94 b (0.27) 1.34 (0.27) 1.76 (0.27) 1.47 b (0.27) 4PLEX 3.10 a (0.26) 2.18 b (0.26) 1.86 b,c (0.27) 1.41 c (0.26) 1.29 c (0.27) 1.28 c (0.26) Duration SHORT 2.99 a (0.25) 1.97 b (0.25) 1. 83 b,c (0.25) 1.25 c (0.25) 1.54 b,c (0.25) 1.30 c (0.25) LONG 2.94 a (0.28) 2.19 b (0.28) 1.97 b,c (0.29) 1.51 b,c (0.28) 1.50 c (0.29) 1.46 c (0. 28) Source x Duration SULF SHORT 2.87 a (0.36) 1.91 b,c (0.36) 1. 72 b,c (0.36) 1.19 c (0.36) 2.03 a,b,y (0.36) 1.72 b,c (0.36) SULF LONG 2.80 a (0.41) 2.06 a,b (0.41) 2.16 a,b (0.41) 1.50 b (0.41) 1.49 b,y,z (0.41) 1.23 b (0.41) 4PLEX SHORT 3.12 a (0.36) 2.03 b (0.36) 1.93 b,c (0.36) 1.31 b,c (0.36) 1.06 c,z (0.36) 0.88 c (0.36) 4PLEX LONG 3.09 a (0.38) 2.33 a,b (0.38) 1.78 b (0.4 0) 1.51 b (0.38) 1.52 b,y,z (0.40) 1.69 b (0.38) P values TM Source (S) 0.87 Duration (D) 0.61 S x D 0.51 Time 0.0001 S x Time 0.61 D x Time 0.94 S x D x Time 0.43 Values are presented as lsmeans (SE). 1 Foals rece ived a primary tetanus vaccination at 112 d of age, followed by booster vaccinations at 140 and 168 d of age. a,b,c Within a row, means with different superscripts differ (P<0.05). y,z Within a column, means with different superscripts differ (P<0.05). in a row, means differ (P<0.10).
73 Table 4 5. Effects of dietary trace mineral (TM) source and duration of supplementation on neutrophil phagocytosis and oxidative burst in the mare 56 d before and 56 and 112 d after foaling Phagocytosis, % Oxidative burst, % Variable ( ) 56 d 56 d 112 d ( ) 56 d 56 d 112 d TM Source SULF 91.3 (2.5) 80.7 (3.3) 65.8 (2.8) 65.0 (3.1) 62.9 (3.8) 45.5 (2.6) 4PLEX 89.6 (2.7) 80.7 (3.4) 64.7 (2.8) 68.4 (3.3) 62.5 (3.9) 46.3 (2.6) Duration SHORT 9 1.1 (2.6) 80.4 (3.4) 65.6 (2.9) 65.0 (3.1) 59.8 (3.9) 45.1 (2.6) LONG 89.7 (2.7) 81.1 (3.3) 64.9 (2.7) 68.5 (3.3) 65.5 (3.8) 46.7 (2.5) Source x Duration SULF SHORT 92.0 (3.6) 79.5 (5.0) 68.1 (4.0) 63.2 (4.4) 59.7 (5.7) 45.5 (3.7) SULF LON G 90.5 (3.6) 82.0 (4.4) 63.6 (3.8) 66.9 (4.4) 66.0 (5.1) 45.5 (3.5) 4PLEX SHORT 90.2 (3.6) 81.3 (4.7) 63.2 (4.0) 66.7 (4.4) 59.9 (5.4) 44.6 (3.7) 4PLEX LONG 89.0 (4.1) 80.1 (5.0) 66.3 (3.8) 70.2 (4.9) 65.0 (5.7) 47.9 (3.5) P values TM Sou rce (S) 0.20 0.99 0.78 0.46 0.94 0.84 Duration (D) 0.72 0.88 0.86 0.44 0.31 0.65 S x D 0.97 0.70 0.34 0.99 0.92 0.66 Values presented as lsmeans (SE).
74 Table 4 6. Effects of dietary trace mineral (TM) source and duration of supplementation in t he mare on neutrophil phagocytosis and oxidative burst in the foal at 1 2, 56, and 112 d of age Phagocytosis, % Oxidative burst, % Variable 1 2 d 56 d 112 d 1 2 d 56 d 112 d TM Source SULF 83.3 (3.5) 70.7 (3.8) 62.9 (3.8) 63.6 (4.2) 56. 6 (4.2) 38.9 (5.2) 4PLEX 79.8 (3.6) 68.2 (3.9) 65.0 (4.1) 57.1 (4.3) 55.0 (4.3) 41.8 (5.7) Duration SHORT 83.4 (3.7) 73.3 (3.9) 64.5 (3.9) 62.5 (4.4) 60.2 (4.3) 38.2 (5.4) LONG 79.6 (3.4) 65.6 (3.8) 63.3 (3.9) 58.2 (4.0) 51.4 (4.2) 42.4 ( 5.4) Source x Duration SULF SHORT 82.6 (5.1) 78.1 (5.8) 66.5 (5.3) 67.1 (6.1) 64.7 (6.4) 37.6 (7.3) SULF LONG 83.9 (4.8) 63.3 (4.9) 59.2 (5.3) 60.1 (5.7) 48.4 (5.4) 40.2 (7.3) 4PLEX SHORT 84.2 (5 .4) 68.5 (5.3) 62.6 (5.8) 57.9 (6.5) 55.6 (5.8) 38.9 (8.0) 4PLEX LONG 75.3 (4.8) 67.9 (5.8) 67.4 (5.8) 56.3 (5.7) 54.4 (6.4) 44.6 (8.0) P values TM Source (S) 0.49 0.64 0.71 0.29 0.80 0.71 Duration (D) 0.45 0.17 0.82 0.48 0.16 0.59 S x D 0.31 0.21 0.29 0.65 0.22 0.84 Values presented as lsmeans (SE)
75 Table 4 7. Effects of dietary trace mineral (TM) source and duration of supplementation on copper, zinc, an d manganese concentrations in umbilical cord serum Umbilical cord serum trace minerals Variable Copper, mg/L Zinc, mg/L Manganese, g/L TM Source SULF 0.17 (0.01) 1.30 (0.09) 62.2 (37.0) 4PLEX 0.18 (0.01) 1.05 (0.09) 90.3 (37.0) Duration SHORT 0.17 (0.01) 1.16 (0.09) 45.4 (37.0) LONG 0.18 (0.01) 1.20 (0.09) 107.1 (37.0) Source x Duration SULF SHORT 0.17 (0.01) 1.33 (0.13) 70.7 (52.4) SULF LONG 0.17 (0.01) 1.27 (0 .13) 53.7 (52.4) 4PLEX SHORT 0.18 (0.01) 0.98 (0.13 ) 20.1 (52.4) 4PLEX LONG 0.18 (0.01) 1.13 (0.13 ) 160.4 (52.4) P values TM Source (S) 0.32 0.07 0.59 Duration (D) 0.62 0.76 0.24 S x D 0.87 0.43 0.14 Val ues presented as lsmeans (SE).
76 Table 4 8. Effects of dietary trace mineral (TM) source and duration of supplementation on copper, zinc, and manganese concentration in the mare 56 d before and 56 d after foaling Mare serum trace minerals Copper, mg/L Zinc, mg/L Manganese, g/L Variable ( )56 d 56 d ( )56 d 56 d ( )56 d 56 d TM Source SULF 1.38 (0.07) 1.34 (0.07) 0.74 (0.02) 0.69 (0.02) 190.9 a (31.8) 23.6 b (31.8) 4PLEX 1.45 (0.07) 1.43 ( 0.07) 0.69 (0.02) 0.70 (0.02) 140.3 (31.8) 56.8 (31.8) Duration SHORT 1.42 (0.07) 1.41 (0.07) 0.72 (0.02) 0.69 (0.02) 156.5 a (31.8) 48.2 b (31.8) LONG 1.41 (0.07) 1.35 (0.07) 0.71 (0.02) 0.69 (0.02) 174.6 a (31.8) 32.1 b (31.8) So urce x Duration SULF SHORT 1.43 (0.10) 1.31 (0.10) 0.72 y,z (0.03) 0.71 (0.03) 167.3 a (45.0) 38.1 b (45.0) SULF LONG 1.32 (0.10) 1.37 (0.10) 0.75 y (0.03) 0.67 (0.03) 214.4 a (45.0) 9.0 b (45.0) 4PLEX SHORT 1.41 (0.10) 1.51 (0.10) 0.71 y,z (0.03) 0.68 (0.03) 145.8 (45.0) 58.3 (45.0) 4PLEX LONG 1.49 (0.10) 1.34 (0.10) 0.66 z (0.03) 0.71 (0.03) 134.8 (45.0) 55.2 (45.0) P values TM Source (S) 0.24 0. 34 0.79 Duration (D) 0.63 0.78 0.97 S x D 0.89 0.86 0.80 Time 0.66 0.43 0.0002 S x Time 0.95 0.21 0.19 D x Time 0.73 0.90 0.59 S x D x Time 0.13 0.08 0.51 Values presented as lsmeans (SE). a,b Within a mineral, means in the same r ow with different superscripts differ (P<0.05). y,z Within a mineral, means in the same column with different superscripts differ (P<0.05). Within a mineral, means in the same row differ (P<0.10).
77 Table 4 9. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on copper, zinc, and manganese concentrations in foal serum at 1 2 and 112 d of age Foal serum trace minerals Copper, mg/L Zinc, mg/L Manganese, g/L Variable 1 2 d 112 d 1 2 d 112 d 1 2 d 112 d TM Sour ce SULF 0.28 a (0.06) 1.75 b,y (0.06) 0.51 a (0.04) 0.67 b (0.04) 61.4 a (22.3) 1.7 b (22.3) 4PLEX 0.30 a (0.06) 1.57 b,z (0.06) 0.47 a (0.04) 0.73 b (0.04) 84.7 a (20.8) 2.0 b (21.5) Duration SHORT 0.27 a (0.06) 1.72 b (0.06) 0.53 a (0.04) 0.67 b (0.04) 76.7 a (20.8) 1.7 b (20.8) LONG 0.31 a (0.06) 1.61 b (0.06) 0.46 a (0.04) 0.73 b (0.04) 69.4 a (22.2) 1.9 b (22.9) Source x Duration SULF SHORT 0.27 a (0.08) 1.91 b,y (0.08) 0.53 (0.06) 0.65 (0.06) 47.9 (29.5) 1.5 (29.5) SULF LONG 0.29 a (0.08) 1.59 b,z (0.09) 0.49 a (0.06) 0.69 b (0.06) 75.0 (33.4) 1.8 (33.4) 4PLEX SHORT 0.27 a (0.08) 1.53 b,z (0.08) 0.52 a (0.06) 0.69 b (0.06) 105.5 a (29.5) 2.0 b (29.5) 4PLEX LONG 0.32 a (0.08) 1.62 b,z (0.09) 0.43 a (0.06) 0.77 b (0.06) 63.9 (29.5) 1.9 (31.2) P values TM Source (S) 0.20 0.88 0.59 Duration (D) 0.51 0.95 0.87 S x D 0.06 0.99 0.43 Time 0.0001 0.0001 0.002 S x Time 0.10 0.26 0.60 D x Time 0.22 0.13 0.87 S x D x Time 0.11 0.61 0.44 Values presented as lsmeans (SE). a,b Within a mineral, means in the same row with differen t superscripts differ (P<0.05) y,z Within a mineral, means in the same column with different superscripts differ (P<0.05).
78 Table 4 10. Effects of dietary trace mineral (TM) source and duration of supplementation on serum cobalamin concentrations in the m are 56 d before and 56 and 112 d after foaling Mare serum cobalamin, ng/L Variable ( )56 d 56 d 112 d TM Source SULF 2,507 (428) 2,471 (428) 3,289 (428) z 4PLEX 2,644 (428) a 2,772 (428) a 5,286 (428) b,y Duration SHORT 2,602 (4 28) a 2,406 (428) a 4,248 (428) b LONG 2,549 (428) a 2,837 (428) a 4,328 (428) b Source x Duration SULF SHORT 2,465 (606) 2,349 (606) 2,501 (606) z SULF LONG 2,548 (606) 2,593 (606) 4,078 (606) 4PLEX SHORT 2,738 (606) a 2,46 3 (606) a 5,996 (606) b.x 4PLEX LONG 2,550 (606) a 3,082 (606) a,b 4,577 (606) b,x,y P values TM Source (S) 0.03 Duration (D) 0.68 S x D 0.20 Time 0.0001 S x Time 0.05 D x Time 0.82 S x D x Time 0.10 Values presented as lsmeans (SE). a,b Within a row, means with different superscripts differ (P<0.05). x,y,z Within a column, means with different superscripts differ (P<0.05).
79 Table 4 11. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on ser um cobalamin concentrations in the foal at 1 2, 56, and 112 d of age Foal serum cobalamin, ng/L Variable 1 2 d 56 d 112 d TM Source SULF 3,272 (429) 2,879 (429) 2,790 (429) 4PLEX 4,127 (413) a 2,7569 (413) b 2,865 (413) b Duration SHORT 3,073 (401) z 2,834 (401) 2,876 (401) LONG 4,325 (440) a .y 2,804 (440) b 2,779 (440) b Source x Duration SULF SHORT 2,848 (567) z 2,961 (567) 2,805 (567) SULF LONG 3,965 (643) y,z 2,796 (643) 2,774 (643) 4PLEX SHORT 3,298 ( 567) z 2,707 (567) 2,946 (567) 4PLEX LONG 4,955 (601) a ,y 2,811 (601) b 2,784 (601) b P values TM Source (S) 0.44 Duration (D) 0.28 S x D 0.65 Time 0.06 S x Time 0.48 D x Time 0.21 S x D x Time 0.85 Values presented as lsmeans ( SE). a,b Within a row, means with different superscripts differ (P<0.05). y,z Within a column, means with different superscripts differ (P<0.05).
80 Table 4 12. Effects of dietary trace mineral (TM) source and duration of supplementation on follicle diameter in the first postpartum cohort Follicle diameter, mm Variable All follicles 2 Largest six follicles 3 Largest follicle 4 TM Source SULF (n=14) 12.4 (0.3) 20.4 (0.7) 49.6 (1.1) 4PLEX (n=14) 12.7 (0.3) 20.3 (0.7) 49.1 (1.1) Duration SH ORT (n=14) 12.7 (0.3) 21.2 (0.7) 50.0 (1.1) LONG (n=14) 12.4 (0.3) 19.5 (0.7) 48.9 (0.1) Source x Duration SULF SHORT (n=6) 12.7 (0.5) 20.8 (1.1) 51.2 (1.6) SULF LONG (n=8) 12.2 (0.4) 19.9 (0.9) 48.1 (1.4) 4PLEX SHORT (n=8) 12 .8 (0.4) 21.5 (0.9 ) 48.6 (1.4) 4PLEX LONG (n=6) 12.5 (0.5) 19.1 (1.1) 49.7 (1.6) P values TM Source (S) 0.65 0.94 0.76 Duration (D) 0.45 0.10 0.51 S x D 0.87 0.48 0.18 Values presented as lsmeans (SE). 2 Average of all follicles fro m the left and right ovaries. 3 Average of the 3 largest follicles from the left and 3 largest from the right ovary. 4 Largest follicle from either ovary. Within a column, means with different superscripts differ (P<0.10).
81 Table 4 13. Effects of dietary t race mineral (TM) source and duration of supplementation on follicle growth rate in the first postpartum cohort Follicle growth rate, mm/d Variable Largest six follicles 2 Largest follicle 3 TM Source SULF (n=14) 0.33 (0.09) 2.62 (0.16) 4PLEX ( n=14) 0.46 (0.09) 2.83 (0.16) Duration SHORT (n=14) 0.39 (0.09) 2.89 (0.16) LONG (n=14) 0.40 (0.09) 2.56 (0.16) Source x Duration SULF SHORT (n=6) 0.34 (0.13) 2.60 (0.25) SULF LONG (n=8) 0.33 (0.11) 2.64 (0.22) 4PLE X SHORT (n=8) 0.45 (0.11) 3.19 (0.22) 4PLEX LONG (n=6) 0.47 (0.13) 2.47 (0.25) z P values TM Source (S) 0.31 0.37 Duration (D) 0.96 0.16 S x D 0.92 0.11 Values presented as lsmeans (SE). 2 Growth rate of the 3 largest developing fo llicles from the left and 3 largest from the right ovary. 3 Growth rate of the largest developing follicle regardless of ovary. y,z Within a column, means with different superscripts differ (P<0.05). Within a column, means with different superscripts diffe r (P<0.10). Within a column, means with different superscripts differ (P<0.10).
82 Table 4 14. Effects of dietary trace mineral (TM) source and duration of supplementation on number of follicles developing in the first postpartum cohort and day of follicle deviation Variable Number of follicles 2 Day of deviation 3 TM Source SULF (n=14) 20.9 (1.3) 6.0 (0.7) 4PLEX (n=11) 19.6 (1.3) 6.3 (0.8) Duration SHORT (n=12) 21.8 (1.3) 6.4 (0.8) LONG (n=13) 18.8 (1.3) 5.9 (0.7) Source x Dura tion SULF SHORT (n=6) 20.8 (1.9) y,z 6.8 (1.1) SULF LONG (n=8) 21.0 (1.7) y,z, 5.3 (0.9) 4PLEX SHORT (n=8) 22.8 (1.7) y 6.0 (1.1) 4PLEX LONG (n=6) 16.5 (1.9) z, 6.6 (1.2) P values TM Source (S) 0.48 0.81 Duration (D) 0.10 0.65 S x D 0.09 0.32 Values presented as lsmeans (SE). 2 Number of follicles developing in the left and right ovaries 3 The day post foaling in which there was a departure in growth rate between the dominant and subordinate follicles y,z Within a column, va lues with different superscripts differ (P<0.05). Within a column, values differ (P<0.10).
83 Table 4 15. Effects of dietary trace mineral (TM) source and duration of supplementation on day of foal heat ovulation Percentage of mares ovulating Variable Day 2 (n=28) 3 (n=34) >10 days 4 (n=34) TM Source SULF 12.5 (0.7) 22.2 77.8 4PLEX 11.9 (0.7) 31.3 68.8 Duration SHORT 11.9 (0.7) 35.3 64.7 LONG 12.5 (0.7) 17.7 82.4 Source x Duration SULF SHORT 13.0 (1.0) 22.2 77.8 SULF LONG 12.0 (0.9) 22.2 77.8 4PLEX SHORT 10.9 (0.9) 50.0 50.0 4PLEX LONG 13.0 (1.0) 12.5 87.5 P values TM Source (S) 0.56 0.55 Duration (D) 0.56 0.24 S x D 0.11 0.35 Values presented as lsmeans (SE). 2 Day of ovulation. 3 P ercentage of mares that ovulated within 10 d of foaling. 4 Percentage of mares that ovulated more than 10 d after foaling.
84 Table 4 16. Effects of dietary trace mineral (TM) source and duration of supplementation on postpartum uterine morphology Change 2 in score /d Change 3 in diameter, mm/d Variable Fluid (n=28) Edema (n=28) Uterine body (n=28) Gravid horn (n=25) Non gravid horn (n=25) TM Source SULF 0.07 (0.02) 0.02 (0.02) 0.9 (0.2) 2.8 (0.4) 2.7 (0.4) 4PLEX 0.08 (0.02) 0.03 (0.02) 1.3 (0 .2) 2.7 (0.3) 2.9 (0.4) Duration SHORT 0.08 (0.02) 0.03 (0.02) 1.1 (0.2) 3.3 y (0.4) 3.1 (0.5) LONG 0.07 (0.02) 0.03 (0.02) 1.1 (0.2) 2.3 z (0.3) 2.5 (0.4) Source x Duration SULF SHORT 0.07 (0.03) 0.01 (0.03) 0.8 (0.3) 3.0 y,z (0.5) 2.8 (0.7) SULF LONG 0.06 (0.02) 0.02 (0.03) 1.0 (0.3) 2.6 y,z (0.4) 2.5 (0.5) 4PLEX SHORT 0.09 (0.02) 0.04 (0.03) 1.4 (0.3) 3.5 y (0.4) 3.4 (0.5) 4PLEX LONG 0.07 (0.03) 0.03 (0.03) 1.1 (0.3) 2.0 z (0.5) 2.5 (0.6) P values TM Source (S) 0.63 0.61 0.30 0.90 0.64 Duration (D) 0.60 0.99 0.98 0.05 0.32 S x D 0.79 0.86 0.49 0.25 0.58 Values presented as lsmeans (SE). 2 Daily change in in trauterine luminal fluid score and endometrial edema s core from 1 d post foaling to day of ovulation. 3 Daily change in d iameter (mm/d) of the uterine body previously pregnant uterine horn, and previously non pregnant uterine horn from 1 d post foaling to day of ovulation. y,z Within a column, means with di fferent superscripts differ (P<0.05).
85 Table 4 17 Effects of dietary trace mineral (TM) source and duration of supplementation on peak serum concentrations of LH and FSH and day of peak concentration relative to ovulation LH FSH Variable Peak, ng/ mL Day Peak, ng/mL Day TM Source SULF (n=15) 74.4 (10.6) 0.2 (0.5) 157.2 (34.8) 7.3 (1.3) 4PLEX (n=13) 75.5 (11.0) 0.2 (0.5) 195.1 (36.3) 8.7 (1.4) Duration SHORT (n=13) 74.6 (10.6) 0.6 (0.5) 140.4 (34.8) 7.1 (1.3) LONG (n=15) 75.2 (11.0) 0.2 (0.5) 211.9 (36.3) 8.8 (1.4) Source x Duration SULF SHORT (n=6) 73.9 (15.4) 0.9 (0.7) 136.0 (50.9) 8.3 (1.9) y,z SULF LONG (n=9) 74.9 (14.4) 0.4 (1.7) 178.4 (47.6) 6.3 (1.8) z 4PLEX SHORT (n=7) 75.4 (14.4) 0.4 (0.7) 144.8 (47.6) 6.0 (1.8) z 4PLEX LONG (n=6) 75.5 (16.6) 0.0 (0.8) 245.3 (55.0) 11.3 (2.1) y P values TM Source (S) 0.94 0.94 0.45 0.47 Duration (D) 0.97 0.25 0.16 0.39 S x D 0.98 0.54 0.56 0.06 Values presented as lsmeans (SE). y,z Within a column, means with different superscripts differ (P<0.10).
86 CHAPTER 5 DISCUSSION Passive Transfer of Immunity The mammary gland of the mare is capable of selectively concentrati ng a wide range of antibodies into colostrum prior to foaling (Jeffcott, 1974, 1975). Immunoglobulin concentrations found in mare colostrum in the current study were similar to those observed by others (Pearson et a l., 1984; Kohn et al., 1989; Spearman, 2004; Stelzleni, 2006) Using an inhibition ELISA with specific monoclonal antibodies against equine IgG and IgA subisotypes, Sheoran et al. (2000) reported that IgGb was the dominant isotype in pre suckle colostrum of mares, followed by IgGa, IgG(T), IgA, and IgGc in order of descending concentration. Although the SRID assays used to determine Ig concentrations in the current study do not differentiate subclasses, the total Ig of colostrum consisted of 93.5% IgG, 5% IgA, and 1.5% IgM. While there were no treatment differences observed for colostral IgG or IgM, mares supplemented with trace mineral amino acid complex es, regardless of duration of supplementation, had higher colostral IgA than mares supplemented with sulfate trace minerals. The IgA found in mammary secretions can originate from either humoral sources (i.e., serum) or can be produced locally by plasma ce lls located adjacent to the secretory epithelium (Larson et al., 1980) Tracer studies in sows have found that approximately 60% of IgA in colostrum is produced within the mammary gland, with the remaining colostrum IgA originating from serum (Bourne and Curtis, 1973) Although the proportion of colostral IgA that originates locally has not been studied in the mare, McGuire and Crawford (1972) found a greater mixture of IgA with differing molecular weights in the colostrum of a pony mare compared to that in milk, indicating a significant portion of IgA in colostrum was serum derived, whereas IgA in milk was almost solely prod uced within the mammary gland. Serum derived IgA appears predominantly as a
87 dimeric, non secretory form in the horse (Porter, 1973; McGuire and Crawford, 1972). In contrast, l ocally produced IgA contains secretory component which is acquired during the t ransport of IgA across glandular and mucosal epithelia. Secretory component is derived from proteolytic cleavage of five Ig like extracellular domains of the epithelial cell surface polymeric Ig receptor, which is widely distributed in the epithelia lining of the digestive, respiratory and genital tracts, as well as in most exocrine glands, including the mammary gland (Kraehenbuhl and Neutra, 1992). Although not directly demonstrated in the horse, several cytokines and steroid hormones have been shown to re gulate expression of this receptor in other animals and, thus, indirectly control the amount of IgA that is delivered into external secretions (Kraehenbuhl and Neutra, 1992). In the current study, supplementation with trace mineral amino acid complexes may have enabled greater quantities of IgA to be produced or, more likely, allowed greater quantities of IgA to be released into colostrum. Zinc is essential for the highly proliferating cells of the epithelium (Maggini et al., 2007) ; thus, adequate dietary zinc is needed to help support and maintain the integrity of the secretory epithelium of the mammary gland. Zinc is also required for protein synthesis, and manganese plays a role in cholesterol biogenes is and potentially steroid synthesis (Underwood, 1977). If the trace mineral amino acid complexes found in diet of 4PLEX mares were more bioavailable than sulfate trace minerals, they may have been better able to support the health of mammary tissue, as we ll as influenced polymeric Ig receptor functioning, resulting in greater transepithelial transport of local and humoral IgA into colostrum. The greater IgA levels in colostrum from mares supplemented with trace mineral amino acid complexes was directly r eflected in their foals, whose serum IgA concentrations were greater than those from SULF mares. In the pig, there is selectivity against the absorption of secretory IgA in colostrum with preferential absorption of monomeric, non secretory IgA
88 (Porter, 197 3). In contrast, there appears to be no selectivity between secretory and non secretory IgA in the calf, as the IgA profile of serum rapidly becomes similar to the colostrum ingested (Porter, 1973). Based on the low ratio between colostrum IgA and serum Ig A in 1 d old foals, Sheoran et al. (2000) suggested uptake of secretory IgA in foals is minimal, similar to that observed in pigs. In the current study, the higher serum IgA concentrations observed in foals nursing 4PLEX mares, coupled with the higher IgA concentrations in colostrum suggests that the colostrum of 4PLEX mares may have contained greater quantities of non secretory IgA that was capable of being absorbed by the foal. Secretory Ig A secreted on epithelial surfaces throughout the body serves a rol e in the first line of defense against pathogens, in large part by preventing adherence of bacteria and viruses to epithelial surfaces (Widmann an d Itatani, 1998) In contrast, the function of serum IgA, regardless of form, has been largely un elucidated. Sheldrake et al. (1984) suggested that selective transepithelial transport of serum IgA occurs at a number of mucosal sites, but is dependent on secretory component availability. While the presence of secretory component in neonatal foals has been well documented, Sheoran et al. (2000) found no evidence of IgA in the nasal secretions of foals before 28 d of age. Alternatively, it has been sugge sted in many species that the hepatobiliary transport of serum IgA serves to reinforce the secretory IgA produced locally in the gastrointestinal tract (Sheldrake et al., 1984) In the current study, the greater supply of IgA in colostrum of mares fed trace mineral a mino acid circulating IgA in the foal may function as a second line of defense or serve to further strengthen intestinal mucosal immunity. Tetanus Antib ody Titers Several trace minerals are thought to play a role in antibody production; however, evidence for the benefits of organic trace minerals over inorganic sources has been somewhat
89 equivocal. Ferket and Qureshi (1992) reported greater antibody titers in young turkeys sup plemented with amino acid complexes of zinc and manganese compared to supplementation with similar amounts of inorganic trace minerals. Dorton et al. (2003) supplemented feedlot steers with copper lysine or copper sulfate and observed an increased antibody response to vaccination with ovalbumin, but not in response to pig red blood cells. Spears et al. (1991) reported a trend for greater primary humo ral immune response to vaccination with bovine herpes virus in newly received feedlot cattle fed zinc methionine compared to zinc oxide or no zinc supplementation; however, no differences in antibody titers were observed among the three zinc treatments in response to parainfluenza vaccination. Other studies in cattle have found no difference in humoral immune response between copper sulfate and copper lysine (Ward et al., 1993) or zinc oxide and zinc proteinate (Spears and Kegley, 2002). Only one study has investigated humoral immune response in horses supplemented with organic trace minerals. The investigators reported that weanlings fed zinc, manganese, and copper amino acid complexes had greater primary humoral immune response to a single injection of pig red blood cells compared to weanlings fed isoelemental amounts of these minerals in inorganic form (Siciliano et al. 2003). The lack of consistency between studies likely results from several factors, including the specific antigen used to elicit the humo antigen, the rate and duration of trace mineral supplementation, and the different organic and inorganic trace mineral sources fed. In the current study, tetanus specific IgG titers were highest at 1 2 d of age in all foals, indicating the uptake of maternal antibodies from colostrum, and progressively declined through dietary treatment, failed to respond serol ogically to the primary vaccination given at 112 d of
90 age. Furthermore, this priming of the immune system had no effect on the response to the second vaccination given at 140 d of age, with the exception of foals from mares supplemented short term with SUL F. The ability of these foals, but not the others, to show a marginal seroconversion is not clear, although these foals did have the lowest titers at the time of the second vaccination. Subsequently, foals from all treatment groups failed to respond to the third vaccination. Similar results were found by Wilson et al. (2001), in which IgGa, IgGb and IgG(T) titers remained low in 3 mo old foals given a series of three vaccinations of tetanus toxoid. By comparison, a similar tetanus vaccination strategy initi ated in foals at 6 mo of age resulted in the desired antibody response. The authors concluded that maternal antibodies, still present in 3 mo old foals, exerted an inhibitory effect on the response of foals to tetanus vaccination (Wilson et al., 2001). In the current study, foals still had significant tetanus titers at 4 mo of age when the first vaccination was administered; thus, the presence of maternal antibodies is likely the cause of the inability of the foals to seroconvert in response to vaccination. Traditionally, it has been recommended that vaccination of foals begin at 3 or 4 mo of age in order to complete the primary series before weaning (Ardans, 1982) But, as demonstrated by Wilson et al. (2001), as well as the current study, beginning vaccinations at this time may be ineffective in the face of antigen specific maternal immunity. Unfortunately, the presenc e of maternal antibodies at the time of vaccination also prevented accurate assessment of the influence of trace mineral source fed to the mare on the humoral immune response in the foal. eries of vaccinations would be advantageous for farm herd health management. However, the powerful and relatively long lasting influence of maternal antibodies, coupled with the fact that most foals are consuming significant quantities of solid food by 6 m o of age, suggests it may be more
91 appropriate to supplement foals directly with trace mineral amino acid complexes when evaluating their response to routine vaccinations. To assess primary and/or secondary humoral immune response in foals younger than 6 mo of age, a novel antigen, in which there are no preexisting maternal antibodies, would have to be used. Neutrophil Function In the current study, foal neutrophil function was similar to that observed in adult mares, demonstrating comparable phagocytic and oxidative burst activity throughout the study. These findings agree with Flaminio et al. (2000) who found no ag e dependent maturation of phagocyte function in neutrophils isolated from foals. Supplementation of trace minerals as amino acid complexes appeared to offer no advantage in support of neutrophil function over trace minerals supplied in sulfate form. In h umans, copper deficiency results in neutropenia (Percival, 1995) and in animals has been shown to re sult in decreased phagocytic and killing function of neutrophils (B oyne and Arthur, 1981) However, Arthington et al. (1995) found no depression of neutrophil bactericidal function during copper depletion or repletion of heifers. In addition to copper, a deficiency in zinc has be en shown to impair chemotaxis, phagocytosis, and generation of the oxidative burst by neutrophils in humans and primates (Allen et al., 1983; Keen and Gershwin, 1990) In the current study, supplemental trace mineral sources were supplied to mares in amounts to meet 100% of their zinc and manganese requirements and 150% of their copper requirements in late gestation and lactation. These minerals, coupled with those naturally occurring in forage and oats, presumably met the requirements for normal neutrophil function in the mare. Further, because neutrophil function in foals was not different from mares or among treat ments, it is likely that they too received adequate trace minerals in utero from milk, and from the consumption of
92 Serum Trace Minerals In the current study, umbilical cord serum zinc concentrat ions were greater than zinc concentrations observed in both mare and foal serum. However, mare and foal serum manganese values were similar to those manganese concentrations observed in umbilical cord serum. Umbilical cord serum copper was less than serum copper concentrations observed in the mare, and similar to serum concentrations observed in the foal at birth. Mare serum zinc and copper concentrations were relatively similar during late gestation and early lactation, whereas serum manganese decreased. In the foal, serum zinc and copper increased and serum manganese decreased from birth to 112 d of age. These findings differ from that of Ott and Asquith (1994) in which a decrease in serum copper and zinc was observed in foals from birth to 112 d of age. Okumura et al. (1998) reported an increase in foal serum copper until 1 mo of age, with little variation in serum zinc and manganese. Tawatsin et al. (2002) also observed an increase in serum copper concentrations in foals from birth to 25 wk of age. Bell et al. (1987) reported that during the first year of extrauterine life, concentrations of zinc in foal serum are variable and apparently unrelated to the age of the animal. However, copper and its major transport protein, ceruloplasmin, increased in parallel from birth to 28 d of age. The comparative differences found in postnatal development of serum trace minerals in foals is likely due to several factors, including placent al transfer in utero and the resulting body stores of trace minerals at birth, as well as trace mineral intake from solid feeds made available to the foal in addition to milk. The source of trace mineral supplied to the mare appeared to have no effect o n serum zinc and manganese concentrations in either mares or their foals. However, while not different at birth, serum copper in foals belonging to mares supplemented with sulfate trace minerals was higher at 112 d age compared to foals nursing mares suppl emented with trace mineral amino acid complexes. It is difficult to determine whether this was a result of supplementation of the mare
93 or the result of external factors that were not controlled for, such as greater forage or concentrate consumption by the (Baucus et al., 1987; Kavazis et al., 2002) and Ull rey et al. (1974) concluded that when trace mineral concentration in mare milk is compared to foal requirements, milk values are low. In contrast, there is some evidence that supplementation of the pregnant mare in late gestation may effectively increase fetal trace mineral stores, which may be of use after birth (Pearce et al., 1998) Therefore, placental transfer of trace minerals in utero concentrate likely contributed significantly as sources of tra ce minerals available to the foal in the current study. Although assessment of trace mineral status is routinely completed using blood measurements, especially in horses, the predictive relationship between tissue and blood levels and the presence of a deficient or non deficient state is variable. Trace minerals are distributed into a number of different pools, including storage, transport, and biochemical function pools (Suttle, 1986) Thus, the serum levels in the current study most likely represent the transport pool of trace minerals. Serum Vitamin B12 The only known function of dietary cobalt is as a component of vitamin B12 (cobalamin), which can be synthesized by microorganisms in the hindgut of the horse. Cobalt dependent vitamin B12, along with iron and copper, are involved in hematopoiesis (Ammerman, 1970) Vitamin B12 has a long biological half life and is stored primarily in the liver of most a nimals (McDowell, 2000). Mean serum cobalamin concentrations measured in mares and foals in the current study were similar to those observed by Roberts (1983) Roberts (1983) observed a decrease in serum
94 cobalamin in mares during gestation with a subsequent rise during lactation, which suggests possible transfer of vitamin B12 from mare to fetus i n utero Because only one sample was taken during gestation in the current study, it is not known if gestation affected vitamin B12 status similarly. Nonetheless, serum cobalamin concentration in the foal was highest at 1 2 d of age compared to 56 and 112 d of age, particularly in foals born to mares that had been supplemented with trace mineral for a longer duration. Therefore, it is reasonable to presume some placental transfer of vitamin B12 occurred in the current study. Although cobalamin concentration s were not measured in colostrum or milk, higher serum cobalamin in 1 2 d old foals could also represent uptake from early postpartum nursing. Kincaid et al. (2004) found that serum cobalamin reserves are depleted in high producing dairy cows in early to mid lactation, despite the provision of cobalt well above the requirement, reflecting losses of vitamin B12 in milk. A decline in mare serum cobalamin from late gestation to early lactation was not observed in the current study, but cobalamin concentrations were significantly lower at these time points compared to late lactation. Milk production in the mare progressively declines after approximately 2 mo (NRC, 2007), which may help to conserve v itamin B12 in the mare. In addition to changes that occurred over time, serum cobalamin in mares and foals was influenced by dietary trace mineral treatment. Foals born to mares that underwent long term cobalt supplementation had higher serum cobalamin co ncentrations than foals from mares that underwent short term supplementation, suggesting that long term supplementation of the broodmare with cobalt may enhance the vitamin B12 status of the foal at birth. In addition, mares fed trace mineral amino acid co mplexes had greater serum cobalamin in late lactation than mares fed sulfate trace minerals. The combination of trace mineral source and duration of supplementation also appeared to affect serum cobalamin in the mare in late lactation. Serum
95 cobalamin was greater in mares receiving short term 4PLEX supplementation compared to either short or long term SULF supplementation. Mares receiving long term 4PLEX had greater serum cobalamin concentrations that short term SULF, but intermediate between short term 4P LEX and long term SULF. Finally, mares receiving long term SULF had considerably higher serum cobalamin than short term SULF, although this difference was not found to be significant. Collectively, t hese findings suggest greater availability of cobalt gluc oheptonate during repletion of serum vitamin B12 after the losses that occurred during gestation and lactation. Furthermore, these findings suggest that mares may benefit from long term cobalt supplementation to better support placental transfer during pre gnancy, as well as the supply of vitamin B12 in colostrum and milk. Although others have shown that horses respond to cobalt supplementation (above the NRC requirement) with an increase in serum cobalamin ( Alexander and Davies 1969) this is the first stu dy describing a greater response with cobalt glucoheptonate compared to cobalt sulfate. Griffiths et al. (2007) reported higher serum cobalamin in lactating dairy cows supplemented with trace mineral amino acid complexes including cobalt glucoheptonate, from 35 d prior to calving through 270 d of lactation. Ho wever, the control diet in their study contained no supplemental trace minerals ; therefore, it is unclear whether the increase resulted from greater availability of cobalt glucoheptonate or a greater supply of cobalt in the diet Early Postpartum Reprodu ctive Performance The exact biological roles of trace minerals in reproduction are largely unknown. However, reports of compromised reproduction during dietary trace mineral deficiencies suggest their necessity for optimal reproductive performance. In co pper deficient animals, estrous cycles and conception rates appear to remain unaffected, however, reproductive failure due to fetal death and resorption has been known to occur (Underwood, 1977) Copper i s also necessary for the formation of connective tissue (McDowell, 2003), and as such would be necessary for proper
96 fetal development. Zinc is known to be centrally involved in cell division, suggesting its importance during fetal growth and during the phy siologic events such as uterine involution that occur in the postpartum female. In addition, zinc is intimately associated with several hormones; for example, the steroid hormones androgen and estrogen function by binding to transcription factors that cont ain zinc fingers (McDowell, 2003). Zinc deficiency in the female has been shown to impair the synthesis and or secretion of FSH and LH, as well as cause abnormal ovarian development and disruption of the estrous cycle (Bedwal and Bahuguna, 1994) Doisey (1974) proposed that insufficient m anganese interrupts the synthesis of cholesterol and its precursors, which would ultimately inhibit the synthesis of sex hormones, adversely affecting reproduction. Hostetler et al. (2003) hypothesized that manganese may play a role in the secretion of pro gesterone based on the findings by Hidirolgou and Shearer (1976) that the manganese concentration in the corpus luteum of ewes increased during early pregnancy, and that inadequate progesterone concentrations are known to cause early embryonic loss. Repo rts addressing the effects of organic trace mineral supplementation on reproductive performance of mares are limited. Ott and Asquith (1994) observed a reduction in the number of cycles bred and services per mare when mares were provided proteinated vs. inorganic trace minerals, although t he differences were not statistically significant. Similarly, Ley et al. (1990) found that barren mares supplemented with inorganic trace minerals experienced no first cycle pregnancies, two early embryonic losses, and h igher number of services per 17 d conception compared to mares receiving chelated trace mineral supplementation; but again, these differences were not statistically different. Studies evaluating reproduction in response to organic trace mineral supplementa tion in other species of livestock are more numerous but not necessarily consistent. Boland et al. (1 996) reported that dairy cattle receiving proteinated trace minerals had
97 a non significant reduction in days to follicle deviation, and 5 fewer days to first ovulation. Manspeaker et al. (1987) found that dairy cattle supplemented with chelated trace minerals had increased ovarian activity, greater postpartum uterine involution of the pregnant horn, and less embryonic mortality, although results were not significantly different from cattle receiving no trace mineral supplementation. More recently, Sic iliano Jones et al. (2008) supplemented dairy cattle 3 wk prepartum through 35 wk postpartum with isoelemental amounts of trace minerals as sulfates or amino acid complexes and found no differen ces in days to first service, services per conception, or number of days open. In the current study, mares that underwent short term supplementation with trace mineral amino acid complexes exhibited a faster rate of uterine involution in the gravid horn, an earlier peak in FSH, developed a greater number of follicles, had a faster growth rate of the largest follicle, and fewer days to foal heat ovulation compared to mares supplemented long term with amino acid complexes and mares supplemented short or lo ng term with trace mineral sulfates. When bred on foal heat, a higher pregnancy rate has been observed in mares that ovulate 10 or more days after foaling compared to mares that ovulate on or before 10 days after foaling (Loy, 1980) Thus, enhancing follicular development and subsequent ovulation may not always be beneficial. The return of the endometrium to its pre gravid morphological state is critical for support of the next pregnancy. On average, the equine embryo enters the uterus 5 days after ovulation. Therefore, ovulation 10 or more days after foaling ensures that the endom etrium has returned to normal prior to arrival of the embryo (Arrott et al., 1994) However, if a more rapid rate of uterine involution occurs concomitantly with faster follicular development and ovulation, as occurred in short term supplemented 4PLEX mares, the potential exi sts for the pregnancy to be successful.
98 Mares in the present study were managed as a commercial herd, which included breeding mares by artificial insemination (Quarter Horses and Standardbreds ) or live cover (Thoroughbreds) to several different stallions, as well as strategic timing of breedings to yield a subsequent foal crop from mid January through March in order to be economically competitive in an equine market. Because of these extraneous variables, conception rates, number of cycles per conception, a nd services per conception were not evaluated. Although follicle dynamics and uterine involution were marginally altered in mares supplemented with trace mineral amino acid complexes, conception should be considered the ultimate end point. A synergy of man y physiologic factors and events must take place in order f or conception to occur Many of the physiologic events that occur in preparation for early postpartum conception were enhanced by substituting trace mineral amino acid complexes in place of sulfate trace minerals in the diet. Therefore, further investigation is warranted to evaluate the impact trace mineral amino acid complexes have on mare conception rates, as well as early embryonic loss.
99 CHAPTER 6 IMPLICATIONS The present study demonstrated that supplementing mares in late gestation with trace mineral amino acid complexes can influence immunoglobulin concentrations in mare colostrum and subsequently influence passive transfer of immunity in the nursing foal. Immunoglobulin A concentrations were greater in mares supplemented with trace mineral amino acid complexes compared to mares supplemented with trace mineral sulfates. This was further reflected in the foal, in which foals nursing mares supplemented with trace mineral amino acid complexe s had greater serum IgA concentrations than foals nursing mares supplemented with trace mineral sulfates. Increased IgA levels may render the foal more immunologically competent by making greater amounts of the immunoglobulin available to act as both a se cond, and more importantly, a first line of defense in the neonatal foal. The presence of maternal antibodies precluded the evaluation of the effect of trace mineral supplementation of the mare on humoral immune response to routine tetanus vaccination in the foal. Further research is warranted to determine if supplementing the foal directly with organic trace minerals will elicit a greater response to routine vaccinations administered after 6 mo of age. Neutrophil function in mares and foals was not affect ed by dietary trace mineral treatment of the mare, suggesting that innate immunity was not limited by trace mineral supply The current study suggests greater availability of cobalt glucoheptonate during repletion of serum vitamin B12 after the losses that occurred during gestation and lactation. Furthermore, these findings suggest that mares may benefit from long term cobalt supplementation to better support placental transfer during pregnancy, as well as the supply of vitamin B12 in colostrum and milk. Fi nally, variable effects of trace mineral source and duration of supplementation were observed with respect to early postpartum reproductive performance. The ability to detect such effects may have been limited by the small sample size
100 resulting from the el imination of mares from statistical analysis for various reasons. Nonetheless, follicular development and uterine involution appeared to be marginally enhanced in mares receiving short term supplementation with trace mineral amino acid complexes. Such phys iological events may better prepare the mare for conception when breeding at foal heat. Additional research is needed to reevaluate the role that duration of supplementation plays in reproductive performance, as well as to determine if supplementation with trace mineral amino acid complexes influences conception rates in the mare.
101 APPENDIX A PROCEDURE FOR ASSESS MENT OF EQUINE NEUTR OPHIL FUNCTION Neutrophil function was assessed in mare and foal whole blood as described b y Vineyard et al. (2007). The neutrophil function assay procedure was as follows: 1. Label 3 tubes for each horse: negative control (DHR only), positive control (DHR + PMA), and SA (DHR + Staphylcoccous aureus) or 6 tubes for duplicates. 2. Prepare 50 M working solution of DHR from 500 M stock solution (100 L DHR stock + 900 L PBS = 1000 L of 50 M DHR). 3. Add 100 L of heparanized whole blood to each tube. 4. Add 10 L of 50 M DHR into all the tubes (final concentration/tube = 4 M). 5. I neutrophils. 6. Prepare 5 g/L working solution of PMA from 1 mg/mL stock solution (5 L PMA stock + 995 L PBS = 1000 L 5 g/mL PMA solution). Store working solution on ic e pending use. 7. Add 10 L of the PMA working solution to the positive control. The final concentration of PMA per tube = 50 ng. 8. Add the appropriate amount of bacterial suspension (10 6 cells/L) to the SA tubes for a bacterial:neutrophil ration of 3 0:1. 9. 10. Immediately place tubes on ice to stop phagocytosis and oxidative burst activity. 11. Process the tubes for flow cytometry using the automated Q Prep Epics immunology workstation set on the 35 second cycle (600 L reagent A, 265 L reagent B, 100 L reagent C). 12. Add 500 L of cold distilled water to each tube for completion of hemolysis. 13. Add 10 L of 0.4% trypan blue to each tube to quench extracellular fluorescence.
102 DHR wo rking stock : To make 500 M stock solution, add 11.5 mL DMSO to 2 mg DHR. To make 500 M working solution, add 100 L DHR stock to 900 L PBS Q Pre p Reagents: Reagent A Formic Acid: 1.2 mL/1000 mL de ionized water Reagent B Sodium carbonate: 6.0 g/1000 mL de ionized water Sodium chloride: 14.5 g/1000 mL de ionized water Sodium sulfate: 31.3 g/1000 mL de ionized water Reagent C 1% Paraformaldehyde : 10.0 g/1000 mL PBS Preparation of Bacterial Targets: 1. Obtain a prepara tion of Staphylcoccus aureus bacteria grown in tryptic soy broth at final volume of 10 mL and final concentration of 5x10 9 cells/mL. 2. 60 min. Harvest the heat killed bacteria by centrifugation at 2000 rpm for 15 min. 3. Decant the supernatant and re suspend the bacterial pellet in 10 mL sterile PBS. Vortex. Centrifuge a t 2000 rpm for 15 min. 4. Decant the supernatant and re suspend the bacterial pellet in 10 mL of 300 g/mL PI solution (7mL PBS + 3 mL PI stock solution (1 mg/mL)). 5. Cover tube in aluminum foil to protect against light and mix by continuous rotation at 6. Harvest the PI labeled bacteria by centrifugation at 2000 rpm for 15 min. Decant the supernatant and re suspend the bacteria in 10 mL sterile PBS.
103 Flow Cytome try Settings: Cytometer Type: FACSort Detectors/Amps: Param Detector Voltage AmpGain Mode P1 FSC E00 1.20 Lin P2 SSC 370 1.00 Lin P3 FL1 400 1.00 Log P4 FL2 485 1.00 Log P5 FL3 412 1.00 Log P6 FL2 A 1.00 Lin P7 FL2 W 1.00 Lin Threshold: Parameter: FSC Value: 120 Compensatio n: FL1 3.60 % FL2 FL2 99.9 % FL1 FL2 0.00 % FL3 FL3 0.00 % FL2
104 APPENDIX B PROCEDURE FOR SERUM MANGANESE ANALYSIS Equipment: AAnalyst 800 Atomic Absorption Spectrometer with THGA Graphite Furnace and AS 800 Autosampler (PerkinElmer, Inc., Shelto n, CT). The THGA is a transversely heated graphite furnace for electrothermal atomization in atomic absorption spectrometry. The furnace incorporates the electromagnet required for the application of the Zeeman effect background correction. Standard P rep aration: Stock manganese solution of 1000 ppm (1g/L) and deionized water was used to prepare two intermediate solutions of 10 g/mL and 100 g/L. The two intermediate solutions were used to prepare the 20 g/L working standard in a diluent of 8 mL Triton X 100 and 5 g of sodium EDTA per liter of deionized water. From this working standard another standard of 5 g/L was also prepared in diluent. Sample Preparation : 0.5 mL serum diluted with 0.5 mL of diluent. A 20 L injection volume was used for AA analys is. Automatic recovery of spiked samples was programmed for every 10 samples. The furnace program was modified to include a special gas type ( air) for steps preceding atomization step. Method : Spec t rometer Element: Mn Wavelength (nm): 279.5 Slit wid th (nm): 0.2L Signal Type: AA BG (Zeeman) Furnace Program Step Ramp Time Hold Time Internal Flow Gas Type 1 110 2 30 250 Air 2 150 5 20 250 Air 3 300 15 15 250 Air 4 950 10 30 250 Argon 5 1900 0 3 0 Argon 6 2600 2 2 250 Argon
105 APPENDIX C PROCEDURE FOR SERUM COPPER AND ZINC ANAL YSIS Equipment: AAnalyst 800 Flame Atomic Absorption Spectrometer and AS 800 Autosampler (PerkinElmer, Inc., Shelton, CT). Standard P reparation: Copper: Automatic Zero (AZ) Standard contained 50 mL serum matrix, 2 0 mL 50% glycerol, and 0 mL 100 ppm Cu stock. Standard ( S1 ) Standard contained 50 mL serum matrix, 20 mL 50% glycerol, and 5 mL 100 ppm Cu stock. Zinc: AZ Standard contained 50 mL serum matrix, 20 mL 50% glycerol, and 0 mL 100 ppm Zn stock. S1 Standa rd contained 50 mL serum matrix, 20 mL 50% glycerol, and 1 mL 100 ppm Zn stock. All standards brought to final 100 mL total volume with deionized water. Standards consisted of 50 % glycerol so that their viscosity matched that of the serum. Sample Prepar ation : Serum samples were prepared using a 1:1dilution with deionized water for the determination of Cu and Zn. Method : Spec t rometer Element: Cu Zn Wavelength (nm): 324.8 213.9
106 APPENDIX D PROCEDURE FOR IMMUNO GLOBULIN ANALYSIS Immunoglobulin ( Ig) G, A, and M were analyzed using a single radial immunodiffusion (SRID) kit (VMRD, Inc., Pullman, WA). Detection ranges were 200 160 0, 31 250, and 25 200 mg/dL for IgG, IgA, and IgM, respectively. The procedure used for IgG, A, and M was as follows: 1. Samples were allowed to come to room temperature and vortexed thoroughly. 2. Samples with high expected Ig values were diluted with deionized water to ensure that readings were within measurable levels. Dilution ratios (sample:deionized water) were as f ollows IgG Colostrum: 1:25 Foal serum (24 h): 1:5 Foal serum (36 h): 1:5 IgA Colostrum: 1:8 Foal serum (24 h): No dilution Foal serum (36 h): No dilution IgM Colostrum: 1:4 Foal serum (24 h): No dilution Foal serum (36 h): No dilution 3. Three L of S tandards A through D for each Ig were pipetted into wells 1 through 4 of plate 1. The pipette was lifted off the bottom of the well simultaneously as the plunger of the pipette was depressed to ensure excess sample did not overflow the well. 4. Three L of each sample to be tested was pipetted into the remaining wells of all plates using the method discussed above. Sample identification specifying well number and dilution rates of each sample were recorded. 5. Plate covers were reattached and plates wer e left undisturbed, and incubated rightside up at room temperature for 18 24 h outside of their mylar pouches. 6. After the incubation period, ring diameter was recorded, with measurements taken in mm using a monocular comparator (VMRD, Inc., Pullman, W A). Used plates were inverted and placed back into their mylar pouches and stored at 4 reference.
107 7. Immunoglobulin concentrations were determined by entering standard and sample diameters along with the appropriate sample dilutio ns into the computer program MetraFIT (Metra Biosystems, Inc., Mountain View, CA). Concentrations were determined using a standard model equation provided by the program. Standards: IgG IgA IgM Standard A: 200 mg/dL Standard A: 31 mg/dL Standa rd A: 25 mg/dL Standard B: 400 mg/dL Standard B: 62 mg/dL Standard B: 50 mg/dL Standard C: 800 mg/dL Standard C: 125 mg/dL Standard C: 100 mg/dL Standard D: 1600 mg/dL Standard D: 250 mg/dL Standard D: 200 mg/dL
108 APPENDIX E MARE AND FOAL SERUM FOLATE T able E 1. Effects of dietary trace mineral (TM) source and duration of supplementation on serum folate concentrations in the mare 56 d before and 56 and 112 d after foaling Mare serum folate, ng/L Variable ( ) 56 d 56 d 112 d TM Source SULF 8.0 5 (0.40) 7.80 x (0.40) 7.42 (0.40) 4PLEX 7.97 a (0.40) 9.24 b,y (0.40) 7.03 a,b (0.40) Duration SHORT 8.09 a (0.40) 8.93 b (0.40) 7.11 c (0.40) LONG 7.93 a,b (0.40) 8.11 a (0.40) 7.35 b (0.40) Source x Duration SULF SHORT 7.91 (0.57) 7.74 z (0.57) 7 .17 (0.57) SULF LONG 8.18 (0.57) 7.86 z (0.57) 7.67 (0.57) 4PLEX SHORT 8.29 (0.57) 10.12 b,y (0.57) 7.04 (0.57) 4PLEX LONG 7.67 a (0.57) 8.37 b,x,z (0.57) 7.02 a,c (0.57) P values TM Source (S) 0.47 Duration (D) 0.58 S x D 0.23 Time 0 .0001 S x Time 0.001 D x Time 0.13 S x D x Time 0.41 Values presented as lsmeans (SE). a,b,c Within a row, means with different superscripts differ (P<0.05). Within a row, means differ (P<0.10). x,y,z Within a column, means with different superscrip ts differ (P<0.05).
109 Table E 2. Effects of dietary trace mineral (TM) source and duration of supplementation in the mare on serum folate concentrations in the foal at 1 2, 56, and 112 d of age Foal serum folate, ng/L Variable 1 2 d 56 d 112 d TM So urce SULF 6.75 a (0.50) 5.02 x,b (0.52) 5.63 a,b (0.52) 4PLEX 5.79 (0.50) 6.74 y (0.50) 6.32 (0.55) Duration SHORT 6.70 (0.49) 5.80 (0.49) 6.08 (0.49) LONG 5.84 (0.54) 5.97 (0.54) 5.87 (0.58) Source x Duration SULF SHORT 6.05 (0. 69) 4.46 (0.69) 5.23 (0.69) SULF LONG 7.45 (0.78) 5.59 (0.78) 6.03 (0.78) 4PLEX SHORT 7.34 x,z (0.69) 7.13 x (0.69) 6.93 (0.69) 4PLEX LONG 4.24 (0.73) 6.35 (0.73) 5.74 (0.73) P values TM Source (S) 0.28 Duration (D) 0 .51 S x D 0.003 Time 0.72 S x Time 0.03 D x Time 0.58 S x D x Time 0.34 Values presented as lsmeans (SE). a,b Within a row, means with different superscripts differ (P<0.05). Within a row, means differ (P<0.10). x,y,z Within a column, means with different superscripts differ (P<0.05). Within a column, means differ (P<0.10).
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120 BIOGRAPHICAL SKETCH ton, Texas along with his twin brother. In a childhood most aptly described as n omadic, horses seemed to be the one constant, and his passion for the equine industry ensued. J.G. graduated third in his class from Liberty County High School in 2001, and attended Chipola College in Marianna, FL shortly thereafter. He then moved to Wi lliston, FL to work at Gainesville, FL. He enrolled in the University of Fl orida, declaring animal science s as a major. While in school, he discovered in himsel f a great passion for science in general, and for equine nutrition in particular. The chance to complete a Master of Science in equine nutrition with Dr. Lori K. Warren presented itself just prior to the completion of his B.S. During his tenure as a grad uate student, J.G. worked as a teaching assistant for multiple equine science courses. He also served as Vice President to the Animal Sciences Graduate Student Asso ciation during the 2007 and 2008 terms During this time, he was also a representative for the C ollege of Agricultural and Life Sciences Ag Council, giving insight and instruction on issues facing the agricultural industry. Upon graduation, J.G. will continue with the pursuit of his passion, and enter into the equine feed industry, with the possibil ity of furthering his education one day in the future.