Evaluating the Efficacy of Dietary Organic and Inorganic Trace Minerals in Reproducing Female Pigs on Reproductive Performance and Body Mineral Composition

Home , Mineral absorption, Organic mineral

EVALUATING THE EFFICACY OF DIETARY ORGANIC AND INORGANIC TRACE MINERALS IN REPRODUCING FEMALE PIGS ON REPRODUCTIVE PERFORMANCE AND BODY MINERAL COMPOSITION

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

James C. Peters, M.S.

*****

The Ohio State University 2006

Dissertation Committee: Dr. Donald C. Mahan Approved by Dr. William F. Pope Dr. Steven K. St. Martin Dr. Sandra G. Velleman Adviser Dr. Henry N. Zerby Graduate Program in Animal Sciences

ABSTRACT

Sow nutrient demands have increased during past decades, yet mineral recommendations for reproducing females have remained essentially unchanged. To compensate for the greater needs of high producing sows, diets are commonly formulated to levels that are in excess of NRC mineral recommendations. However, the efficacy of these higher levels has not been evaluated. Additionally, the use of organic trace minerals is increasing, however their utility in sow diets has not been widely investigated. Organic trace minerals are expected to be more available and may affect mineral retention in tissues, even at lower supplementation levels. In our first experiment 375 farrowings over 6 parities were used to evaluated diets which contained either inorganic or organic sources of trace minerals provided at 1998 NRC recommended levels (NRC) or higher levels (Ind.). Two additional treatments evaluated the Ind. level of both sources, but with additional Ca and P (Ind+Ca/P). Sows fed the organic trace minerals farrowed more total and live pigs, with heavier litters at birth. Sows fed the Ind+Ca/P level tended to have fewer (P < 0.10) total pigs born and lower (P < 0.05) litter birth weights. These results suggest that feeding organic trace mineral resulted in more pigs born. However, there was no improvement in reproductive performance when feeding higher dietary levels. In our second experiment trace mineral sources and levels were evaluated on sows in a commercial setting. The organic and inorganic trace mineral sources were provided to sows at NRC and Ind. levels over 6 parities. Numbers of live pigs born were similar for all treatment. Feeding the Ind. level of both sources resulted in more pigs weaned and heavier litter weaning weights, suggesting a response to the higher dietary trace minerals level. Sow retention was similar for all treatments. The effects of sow dietary trace mineral source on the Fe status of neonatal pigs and the postnatal carry-over of Fe to the progeny were evaluated in our third experiment. The results suggested that there was minimal carryover of Fe from sows fed either trace mineral source and that pigs required a Fe injection during the initial days of birth to prevent anemia. ii An experiment evaluated the effects of dietary trace mineral source and level on the mineral status of sows. After weaning at parities 1, 2, 4, and 6, sows from our first experiment were selected for determination of body mineral content. In addition, the effects of the sow trace mineral treatments on mineral transfer to the progeny were evaluated. Total sow body and liver Se content were greater (P < 0.05) when the organic source and the Ind. and Ind.+Ca/P levels were fed. Overall there were no other effects of trace mineral source on sow mineral content, however total body Cu content was greater (P < 0.05) at the higher trace mineral levels. Sow liver Cu and Zn contents were lower (P < 0.05) at the NRC level. There were no effects of trace mineral source or level on neonatal pig mineral contents. Colostrum and milk Se increased at the higher dietary levels when the organic source was fed, but not when the inorganic source was fed. Milk Cu and Zn concentrations were lowest at the NRC level. Total body mineral concentrations of Cu, Fe, and Mn were higher (P < 0.05) for pigs from the sows fed the organic source. Concentrations of Mn and Zn were greater when the higher trace mineral levels were fed. However, liver concentrations were not affected by trace mineral source or level. These results suggest that trace mineral source and level had little effect on sow mineral content. However, the organic mineral source and higher Cu and Zn levels seemed to improve transfer of trace minerals to the nursing pig. Our final experiment evaluated the effects of dietary trace mineral source and level on the mineral status of gilts maintained in a non-gravid state from 8 to 35 months of age which is equivalent to a six parity period. The content of all minerals increased between 8 and 35 months of age. When organic trace minerals were fed to non-gravid gilts, total body and liver Cu content were lower, but total body Fe content was greater. Trace mineral source did not affect Mn or Zn contents. Total body Cu and liver Cu, Mn, and Zn contents were greater at the higher trace mineral levels. Total sow body Se content were greater (P < 0.05) when the organic source and the Ind. and Ind.+Ca/P levels were fed. Total empty body and liver macro-mineral quantities were not affected by trace mineral source and level. The overall results of this research suggest that feeding reproducing sows organic trace minerals increased sow reproductive performance. However, sow mineral status was not consistently affected by trace mineral source, suggesting that other factors may be involved in the increased sow productivity. Feeding higher dietary levels of either trace mineral source demonstrated that except for a few trace minerals, particularly Se and Zn, being higher in the sow liver, milk, and progeny, there were few beneficial effects.

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Dedicated to my wife Susan and daughter Madison Gayle

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to my advisor Dr. Donald C. Mahan for his guidance and belief in my abilities throughout my graduate studies. He has provided great support and direction my research project. He also exhibited great patience as I made strides to become a swine nutritionist. I would like to thank Ken Mays and Larry Warnock, the managers at the OSU Swine Center; for helping conduct these experiments. I would also like to thank the student farm employees, especially Jason Beagly, who helped with these experiments. Thanks go to Frank Cihla, Mauria Watts, Starr Fischer, and Yadira Malavez for assistance in the laboratory and with sow processing. I would also like to thank Jack Bardall for aid in feed manufacturing, Kevin Jewell for mineral analysis, and Burt Bishop for statistical analysis. Thanks to Dr. Steve Moeller for providing the ultrasound equipment used in this project and for interpreting the images. I thank Dr. Henry Zerby and Gary Dunlap of the OSU Meat Lab for their assistance with arranging and conducting the harvesting of the sows. Thanks to Allen Bridges, Rachel Howdyshell, and other graduate and undergraduate students for assisting in the rather unpleasant chore of separating and cleaning visceral organs. I would like to thank Kim Turnley (Alltech Australia) and Dr. David Henman (QAF Meat Industries, Corowa, NSW) for conducting the commercial sow study. My sincere appreciation is expressed to Dr. Karl Dawson (Alltech, Nicholasville, KY) for providing generous financial support I am greatly indebted to Nathan Fastinger and Ted Wiseman for assisting with my graduate work and expanding my thinking about swine nutrition. They were of tremendous help in my work and provided never-ending support and friendship. Most of all, I want like to thank my wife, Susan, for her unwavering support and encouragement. Without her, none of this would have been possible. She has unselfishly made numerous sacrifices so that I could continue my education. v Susan has exhibited extreme patience while I continued my education. Most importantly, she believes in me. Additionally, I want to thank my daughter Madison for providing me with further motivation to succeed and another smiling face to come home to at night.

VITA

February 4, 1976 ...... Born, Steubenville, Ohio

1998 ...... B.S. Agriculture, The Ohio State University

1998-2000 ...... Graduate Research Associate, Department of Animal Sciences The Ohio State University

2000 ...... M.S. Agriculture, The Ohio State University

2000 to present ...... Graduate Research Associate, Department of Animal Sciences The Ohio State University

PUBLICATIONS

Mahan, D.C. and J.C. Peters. 2004. Long-term effects of dietary organic and inorganic selenium sources and levels on reproducing sows and their progeny. J. Anim. Sci. 82:1343-1358.

Mahan, D.C., N.D. Fastinger, and J.C. Peters. 2004. Effects of diet complexity and dietary lactose levels during three starter phases on postweaning pig performance. J. Anim. Sci. 82:2790-2797.

FIELDS OF STUDY

Major Field: Animal Sciences

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TABLE OF CONTENTS Page Abstract...... ii Dedication...... iv Acknowledgments ...... v Vita ...... vii List of Tables...... xii List of Figures...... xvi

Chapters:

1. Review of literature ...... 1 1.1. Introduction ...... 1 1.2. Plant distribution...... 2 1.2.1. Grain distribution ...... 2 1.2.2. Geographical location ...... 4 1.2.3 Effects of soil conditions...... 5 1.2.3.1. Soil pH ...... 5 1.2.3.2. Plant microenvironment...... 5 1.2.3.3. Soil fertility ...... 6 1.2.4. Phytic acid...... 9 1.3. Tissue distribution ...... 11 1.3.1 Visceral organs...... 11 1.3.2 Bone ...... 13 1.3.3 Muscle...... 14 1.3.4 Other tissues...... 15 1.3.5 Milk...... 16 1.4. Trace mineral metabolism ...... 18

viii 1.4.1 Absorption...... 18 1.4.2 Transport and storage...... 19 1.4.3 Excretion...... 21 1.4.4 Deficiency...... 21 1.4.5 Toxicity ...... 22 1.5. Trace mineral biological functions ...... 24 1.5.1 Metalloproteins...... 24 1.5.2 Growth and development ...... 26 1.5.3 Immunity...... 26 1.5.4 Reproduction...... 28 1.6. Mineral-nutrient interactions ...... 32 1.6.1 Interactions involving trace minerals...... 32 1.6.2 Interactions in the intestinal lumen ...... 34 1.7. Mineral nutrition requirements ...... 35 1.8. Supplemental dietary trace mineral forms ...... 37 1.8.1 Inorganic salts ...... 37 1.8.2 Organic sources...... 38 1.8.2.1 Bioavailability...... 38 1.8.2.2 Organic trace minerals and reproduction...... 42 1.9. Research objectives and rationale...... 44

2. Effects of dietary organic and inorganic trace minerals at various levels in reproducing sows over six parities...... 45 2.1 Abstract...... 45 2.2 Introduction ...... 46

2.3 Materials and methods...... 46

2.3.1 Experimental design and treatments ...... 46 2.3.2 Grower phase ...... 47 2.3.3 Breeding and postbreeding period ...... 47 2.3.4 Farrowing and lactation period ...... 48 2.3.5 Diet composition...... 49 2.3.6 Mineral intake ...... 49 ix 2.3.6 Analytical methods ...... 49 2.4 Results ...... 51 2.4.1 Reproductive performance...... 51 2.4.2 Gilt and sow weight, feed intake, and backfat thickness ...... 52 2.4.3 Parity responses...... 53 2.4.4 Sow daily mineral intake...... 54 2.5 Discussion ...... 56 2.6 Implications ...... 57

3. Commercial evaluation of dietary trace mineral sources and levels in reproducing sows over six parities...... 71 3.1 Objective...... 71 3.2 Rationale...... 71 3.3 Materials and methods...... 71 3.3.1 Experimental design and treatments ...... 71 3.3.2 Grower-development phase ...... 72 3.3.3 Reproducing phase...... 72 3.3.4 Diet composition...... 73 3.3.5 Statistical evaluation ...... 74 3.4 Results...... 74 3.5 Discussion...... 76 3.6 Implications ...... 78

4. Effect of sow dietary organic and inorganic trace minerals on the iron status of neonatal pigs and the effect of iron injections to their progeny at birth and weaning...... 85 4.1 Abstract...... 85 4.2 Introduction ...... 86 4.3 Materials and Methods...... 87 4.3.1 Experimental design and treatments ...... 87 4.3.2 Lactation period...... 87 4.3.3 Nursery period...... 88 4.3.4 Diet compositions ...... 88

x 4.3.5 Analytical methods ...... 89 4.4 Results...... 89 4.5 Discussion...... 92 5. Effects of dietary trace mineral sources and levels on changes in sow body mineral content and their progeny over six parities...... 98 5.1 Abstract...... 98 5.2 Introduction ...... 99 5.3 Materials and Methods...... 100 5.3.1 Experimental design and treatments ...... 100

5.3.2 Mineral transfer to progeny...... 101 5.3.3 Slaughter and sampling procedures ...... 101 5.3.4 Analytical methods ...... 102 5.4 Results...... 103 5.4.1 Sow and liver mineral content...... 103 5.4.2 Neonatal pig and liver mineral content ...... 105 5.4.3 Milk mineral content...... 105 5.4.4 Weaned pig (17 d) mineral content...... 107 5.5 Discussion...... 108 5.6 Implications ...... 110

6. Long-term effects of dietary organic or inorganic trace minerals on the body mineral composition of non-reproducing gilts...... 125 6.1 Abstract...... 125 6.2 Introduction ...... 126 6.3 Materials and Methods...... 126 6.3.1 Experimental design and treatments ...... 126 6.3.2 Statistical analysis...... 128 6.4 Results...... 128 6.5 Discussion...... 130 6.6 Implications ...... 132

List of references ...... 137

LIST OF TABLES

Table Page

1.1. Average analysis of grains and by-products ...... 3

1.2. Trace mineral concentration in various organic and inorganic fertilizers...... 8

1.3. Total mineral content of growing pigs from birth to 145 kg body weight and parity 3 sows ...... 11

1.4. Comparison of trace mineral concentrations of visceral organs and brains of pigs, cattle, and sheep...... 13

1.5. Comparison of trace mineral concentrations of lean muscle of various species (wet basis)...... 15

1.6 Comparison of typical mineral concentrations in milk of various species ...... 17

1.7. Historical NRC mineral recommendations for reproducing sows...... 36

1.8. AAFCO definitions for organically bound mineral compounds ...... 39

2.1 Calculated mineral composition of treatment diets ...... 58

2.2. Composition of basal experimental diets fed during gilt development, (%, as-fed basis)...... 59

2.3. Composition of experimental basal diets fed during reproduction (%, as-fed basis) ...... 60

2.4. Treatment responses of dietary trace mineral source and level on pig during the growing-finishing period...... 61

2.5. Treatment responses of dietary trace mineral source and level on litter and pig measurements...... 62 xii

2.6. Treatment responses of dietary trace mineral source and level on sow reproductive measurements over six parities...... 63

2.7. Parity effects on sow reproductive measurements ...... 64

2.8. Parity responses of litter and pig measurements...... 65

2.9. Effect of trace mineral level and parity on calculated daily macro- and trace mineral intake during gestation and lactation ...... 66

2.10. Effect of trace mineral level and parity on calculated daily macro- and trace mineral intake per kg body weight during gestation and lactation ...... 69

3.1. Composition experimental basal diets (%, as-fed basis)...... 79

3.2. Effects of parity and trace mineral source and level on litter numbers, sow retention and the number and percentage of litters containing 5 or fewer pigs ...... 80

3.3. Effect of trace mineral sources and levels on sow reproductive performance...... 81

3.4. Main effects of trace mineral source and level on sow reproductive performance ...... 82

3.5. Parity effects on sow reproductive performance...... 83

3.6. Effects of parity, trace mineral source, and level on pig and litter measurements ...... 84

4.1. Composition of nursery diets (%, as fed basis) ...... 93

4.2. Effect of dietary trace mineral source fed to second parity sows on sow reproductive performance and composition of trace minerals in colostrum and milk ...... 94

4.3. Effect of sow dietary trace mineral source and neonatal iron injections on piglet performance and blood parameters during the nursing period ...... 95

4.4. Effect of sow dietary trace mineral source and neonatal iron injection on postweaning pig performance and blood parameters...... 96

4.5. Effect of iron injections in neonatal pigs and weanling pigs on postweaning performance and blood parameter responses...... 97 xiii

5.1. Effects of trace mineral source and level on total liver and body mineral content of reproducing sow...... 111

5.2. Effect of parity on the macro-mineral content of the individual components and total body ...... 112

5.3. Effect of parity on the trace mineral content of the individual components and total body...... 113

5.4. Effects of trace mineral level and parity on body mineral content of reproducing sow...... 114

5.5. Effect of trace mineral level and parity on total liver mineral content of reproducing sow...... 115

5.6. Effects of trace mineral source and level on total body and liver mineral contents of neonatal pigs...... 116

5.7. Effects of trace mineral source and level on relative mineral content of neonatal pigs and livers ...... 117

5.8. Effects of parity on mineral content of neonatal pigs and livers ...... 118

5.9. Effect of parity on relative mineral content of neonatal pigs and livers ...... 119

5.10. Effects of trace mineral source and level on colostrum and milk mineral concentrations ...... 120

5.11. Effects of trace mineral source and parity on colostrum mineral concentrations...... 121

5.12. Effects of trace mineral source and parity on milk mineral concentrations...... 122

5.13. Effects of trace mineral source and level on empty body and liver mineral content of 17 day old pigs...... 123

5.14 Effects of trace mineral source and level on the relative amount of minerals in the liver and empty body of 17 day old pigs...... 124

6.1. Effect of trace mineral source and level on total liver and body mineral content of gilts at 8 months of age...... 133

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6.2. Effect of trace mineral source and level on total liver and body mineral content of non-gravid females from 12 to 35 months of age...... 134

6.3. Effect of dietary trace mineral source and parity in non-gravid gilts on body mineral composition from 8 to 35 months of age ...... 135

6.4. Effect of dietary trace mineral source and parity in non-gravid gilts on total liver mineral composition from 8 to 35 months of age...... 136

LIST OF FIGURES

Figure Page

1.1. Selenium distribution in the U.S. soils ...... 4 1.2. Effect of production phase on lagoon trace minerals concentrations ...... 8 1.3. Structure of phytic acid and phytic acid chelate ...... 9 1.4 Interactions involving trace minerals...... 32

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CHAPTER 1

REVIEW OF LITERATURE

1.1. Introduction The classification of the trace mineral elements is within the 73 elements of the periodic table that are found immediately after the 11 major elements (C, Ca, Cl, H, K, Mg, N, Na, O, P, and S). Only a few trace minerals have been determined as essential for animal life; they currently include Co, Cr, Cu, Fe, I, Mn, Se, and Zn. As research is extended, more may be added. Cobalt is an essential component of vitamin B12, but mammals lack the enzyme necessary to synthesize this vitamin in vivo, therefore vitamin B12 is supplemented to swine diets. Iodine is supplemented by iodized salt to meet the animal’s requirement. Therefore only Cu, Fe, Mn, Se, and Zn are currently important in non-ruminant nutrition and are in most trace mineral premixes, though Cr may shortly be added (NRC, 1998). Essential trace minerals are constituents of proteins and enzymes that are involved in a variety of metabolic functions. They have functional implications in growth and development, reproduction, and health. Sows mineral reserves have been shown to decline over several reproductive cycles and depletion is exacerbated when sows support larger litter growth rates (Mahan and Newton, 1995). Possibly exacerbating this reduction in reserves are suspected interactions between a trace mineral with other minerals or nutrients, which may decrease its availability or utilization by the animal. A decline may lead to marginal mineral deficiencies which may affect growth, reproduction, and health, but otherwise have no outward signs. Under modern intensive management practices, animals receive a supplemental supply of dietary trace minerals in addition to those contributed by dietary plant and/or animal sources. However, trace mineral premix sources vary greatly in their bioavailability. Consequently, under certain conditions it may be possible to achieve maximum reproductive performance at lower dietary levels when sources with higher bioavailabilities are fed.

1.2. Plant distribution Trace minerals are found incorporated in a variety of plant enzymes and involved in enzymatic reactions which primarily occur in the leaves. Iron is important to the plant’s oxidation-reduction reactions. Up to 75 to 80% of plant Fe is associated with the chloroplasts, where Fe is a structural component of the cytochromes, heme, and electron-transfer systems (Miller and Donahue, 1990). Cytochromes are the site of various photosynthetic reduction processes and ferredoxin, the first stable electron acceptor in the photosynthetic electron transport chain (Tisdale et al., 1993). Deficiencies of Fe therefore lead to a decrease in the photosynthetic pigments, resulting in the characteristic condition chlorosis or yellowing of plant leaves. Zinc has an essential function in numerous plant enzyme systems, including those involved in plant protein and carbohydrate synthesis. Enzymatically bound Cu is involved in redox reactions, like cytochrome oxidase (Marschner, 1995). Copper-Zn superoxide dismutase (SOD) is found in the cytoplasm of the mitochondria and chloroplasts. Manganese is also involved in redox processes, primarily as Mn-SOD, and is a cofactor in numerous other enzymatic reactions (Marschner, 1995). Manganese may substitute for Mg in many phosphorylating reactions (Tisdale et al., 1993).

1.2.1 Grain distribution The grain is the reproductive end-product of the plant, therefore nutrients are deposited in the seed to be used for germination and initial growth of seedlings. Grain mineral concentrations reflect the plant’s nutritional status, thus plants grown under deficient conditions will have lower grain concentrations and potentially lower production. Trace mineral concentrations varies widely between grain types (Table 1.1) and varieties. The content and/or availability of trace minerals in grain are most often too low to meet the animal’s metabolic requirements; therefore dietary supplementation of most trace minerals is often necessary. In corn, trace minerals are primarily found in the germ (78%) and secondarily in the endosperm (18%) portions of the kernel (O’Dell et al., 1972; Watson, 1987). Other cereal grains (i.e. wheat and barley), have minerals concentrated in the bran portion which is the outer covering of the kernel (Lorenz and Kulp, 1991). Wheat germ and barley hulls contain trace minerals, but at lower concentrations than the bran (O’Dell et al., 1972). The concentration of minerals in most cereal grains is affected by the ratio of bran to endosperm. As the endosperm

(carbohydrate) portion increases there is a decreased mineral concentration. Bran is higher in fiber content and therefore the availability of minerals for utilization by the animal may be low, especially in non-ruminants. Seeds from leguminous plants generally have higher mineral concentrations than the cereal grains, especially the macro-minerals (Underwood and Suttle, 1999). For example soybeans have higher trace mineral concentrations, especially compared to corn. A large portion of these minerals are in the soybean hull or seed coat. For instance, 32% of the Fe in soybeans is in the hull (Levine et al., 1982). However, the soybean meal fed to non-ruminants often has the hulls removed, thus reducing the trace minerals consumed by non-ruminants. Extracting specific portions of grains (i.e. oils and carbohydrates) alters the composition of the remaining product (Table 1.1). For example, soybean meal, the primary protein source used in non-ruminant diets, is the by-product of oil extraction and therefore has higher mineral concentrations. Distiller’s dried grains with solubles (DDGS) and Brewer’s grains have higher mineral concentrations then the original grain because the starch portion of the grain has largely been utilized for alcohol production, thus the bran constitutes a much larger proportion of the by-product used in animal feed.

Mineral, mg/kga Feedstuff Cu Fe Mn Se Zn Corn 3 29 7 0.07 18 Wheatb 6 39 34 0.33 40 Barleyc 8 88 16 0.10 15 Soybeansd 16 80 30 0.11 39 Soybean meale 20 176 36 0.27 55 DDGSf 57 257 24 0.39 80 aAs-fed basis bHard red winter wheat cSix row variety dSeeds, heat processed eSolvent extracted, without hulls fDistiller’s dried grains with solubles

Table 1.1. Average analysis of grains and by-products (NRC, 1998)

1.2.2. Geographical location When soil minerals are low, plants either reduce the mineral concentrations in the seed, reduce plant growth, or a combination of both situations. Soil generally contains, on average 4% Fe, 800 ppm Mn, 50 ppm Zn, 20 ppm Cu, and 0.01 ppm Se (Bohn et al., 1985). However, soil mineral concentrations vary widely between regions of the world due to various factors. A primary factor affecting soil mineral concentrations is the type of parent rock (i.e. sedimentary, igneous, and metamorphic) from which the soil originated. The degree of weathering and the leaching of minerals from the topsoil also affects the soil mineral concentrations. The best example of geographic variations in soil content is Se (Figure 1.1). Soil Se concentration depends on the rocks from which the soil was derived. Soils found in South Dakota, Nebraska, Kansas, and Colorado, originated from cretaceous shale and tend to have higher (2 to 10 ppm) soil Se concentrations. However, many states in the Great Lakes, Northwest, and Southeast have low (<0.05 ppm) soil Se concentrations because soils in these regions were derived from volcanic deposits or well-washed coastal deposits. The Se content of corn and soybean meal (Cromwell et al., 1999) originating from these regions and the tissues of pigs fed these feedstuffs (Mahan et al., 2005) reflect the soil Se content.

Low: Approximately 80% of all forage and grain contain <0.05 ppm Se Variable: Approximately 50% of all forage and grain contain > 1.0 ppm Approximately 80% of all forage and grain contain >1.0 ppm Se • Local areas where selenium accumulator plants contain >50 ppm

Figure 1.1. Selenium Distribution in the U.S. Soils. (From Kubota et al., 1967.) 4

1.2.3. Effects of soil conditions Nutrients must reach the root before they can be absorbed, which is accomplished by nutrients moving in or with the soil solution. Roots grow to the nutrient source, reducing the length of nutrient transport. The concentration of nutrients in the soil solution and the buffer capacity of the soil, or ability of the soil to resist change in pH, are important in order for the plant to utilize the minerals (Marschner, 1995). Mineral concentrations in the soil solution vary widely and are affected by many factors including soil moisture, pH, soil depth, cation- exchange capacity, and organic matter (Marschner, 1995).

1.2.3.1. Soil pH Iron and Zn deficiencies are the two most common trace mineral deficiencies in plants and are typically associated with reduced solubility of minerals at neutral or alkaline pH, rather than simply having a low soil mineral content (Crowley and Rengel, 1999). A decrease in soil pH or in redox potential increases both the concentrations and availability of cations, like Cu, Mn, Fe, and Zn, in the soil solution (Sanders, 1983; Sims and Patrick, 1978), whereas anions like Cr and Mo are less available in acidic soils (Bohn et al., 1985). Reduced ferrous iron (Fe2+), formed under anaerobic conditions, is more mobile in the soil and more available for the plant’s incorporation into tissues than the ferric iron (Fe3+; Tisdale et al., 1993). But Fe2+ is readily oxidized to Fe3+ in aerated soils. For example, at pH 3 Fe is soluble enough to meet plant needs, but at a pH of 5.5 to 6.5 Fe has a lower solubility and may be inadequate to meet the plant’s requirement. Soybeans grown in the high pH soils of the Upper Midwest are susceptible to Fe deficiency (Tisdale et al., 1993). Similarly Mn deficiency is common in wheat and soybeans grown in high pH soils, especially if organic matter is high (Marschner, 1995). Conversely, Mn toxicity has been observed in soybeans growing in extremely acidic soils, but increasing the pH with limestone corrects the problem (Tisdale et al., 1993).

1.2.3.2. Plant microenvironment Plant root exudates are secreted into the root zone and alter the surrounding soil environment in response to nutrient deficiencies (Mori, 1994). The exudate is composed of soluble organic acids, sugars, phenolics, and amino acids (Curl and Truelove, 1986). The organic acids, typically citric, malic, acetic, fumic, succinic, and oxalic, have a major role in the

5 solubilization and plant uptake of minerals (Crowley and Rengel, 1999). Microbial populations and their activities increase in response to the exudate, which influences soil pH and redox. Furthermore, soil microorganisms increase mineralization of the organic matter by forming chelates between soil minerals and degraded nutrients from organic matter. Adequate uptake of trace minerals, to meet the relatively small micronutrient requirement for plant growth, can be achieved if a small portion of the root zone is sufficiently acidic (Bohn et al., 1985). Plants alter the pH of the root zone by differential uptake of cations and by using inducible proton pumps (Crowley and Rengel, 1999).

1.2.3.3. Soil fertility Soil organic matter is an accumulation of decaying plant and animal residues, worms, and microorganisms. Soils vary greatly in organic matter content, but the majority of soils have a low organic matter content, ranging from 0.5 to 5% (Bohn et al., 1985). Prairie grasslands may contain thick topsoil that is 5-6% organic matter, while the contents of poorly drained soils are often greater than 10%. Organic matter content can be increased by incorporating crop residues into the top soil. Soil amendments like livestock manure and sewage sludge also provide organic matter content. Organic matter influences plant growth by altering soil chemical, biological, and physical properties. Soluble organic substances, like fulvic acid, form natural complexes or chelates with cations (i.e. Fe, Cu, Mn, and Zn). These natural chelates are products of the organic matter and microbial activity. Chelates shield the cations from hydrolysis and precipitation reactions (Bohn et al., 1985), which increases their solubility and allows the metals to move to the roots (Tisdale et al., 1993). This is particularly beneficial in alkaline pH soils where cation solubility is reduced. Typical commercial fertilizers are low in most essential trace minerals (Table 1.2). However, fertilizers may have trace minerals added when necessary. For instance, fertilizers containing added Zn doubled wheat Zn concentrations compared to plants grown on the same soil without Zn fertilizer (Hambidge et al., 1986). Synthetic chelates may be used in foliage sprays or soil additives to provide Fe, Zn, Mn, and Cu. Limestone applied to adjust soil pH also contributes trace minerals (McBride and Spiers, 2001). Livestock manure and sewage sludge contribute organic matter, but equally important are the trace minerals they provide. Livestock manures are relatively high in trace mineral

6 content, but vary between species (Table 1.2). Feed wastage and the animal’s water source contribute to manure levels. Zinc and Cu are often used in high concentrations in swine diets as growth promoters (Hill et al., 2000) resulting in high manure levels. Manure is usually applied to fields on the basis of phosphorus and nitrogen content, without regard to trace mineral contributions. Consequently, without monitoring, it is possible that high levels of trace minerals could accumulate over time with heavy manure application. Swine manure is often stored and applied to farm land in liquid form and if expressed on a dry-matter basis would have much higher concentrations than those listed in Table 1.2. Variation in manure mineral concentrations between different phases of production (Figure 1.2) is mainly due to the dietary levels fed to the animals, but dilution by water could alter wet-basis concentrations. The percentage of solids in sow lagoons was 0.5%, whereas nursery and finishing lagoons contained approximately 1.3% solids (DeRouchey et al., 2002). This suggests that the manure from sow lagoons was more diluted. Sewage sludge is also high in trace minerals, especially Zn (Table 1.2). Land disposal of sewage compost and sludge resulted in increased levels of available Zn for up to six years after application (Leriche, 1968). However, sewage sludge also contains much higher levels of toxic heavy metals like Pb and Hg (McBride and Spiers, 2001). Because sludge is rarely limited to the P requirement of the crop, the accumulation of toxic metals is a concern (McBride and Spiers, 2001).

Mineral, ppm Soil amendment Cu Fe Mn Se Zn Mo Commercial fertilizerab 7.0 - - <1.0 94.0 2.6 Commercial fertilizerac 1.0 - - 1.0 25 4.1 Dairy manuread 64 - - 3 191 2.5 Swine manuree 3.1 32 2.5 - 16.8 - Broiler litterf 450 2073 388 5.5 399 0.9 Sewage sludgeg 109 - 420 - 845 12.3 aMcBride and Spiers, 2001. b10% N, 20% P, and 20% K. c12% N, 16% P, and 16% K. dDry basis. eWet basis, swine lagoons, DeRouchey et al., 2002. fAir dried 48h, Kpomblekou et al., 2002. gBalkcom et al., 2001.

Table 1.2. Trace mineral concentration in various organic and inorganic fertilizers

Cu Mn Fe Zn 60 50 40 30 20

mg/kg wet basis mg/kg wet 10

Mineral concentration, 0 Sow Nursery Finisher Phase of Production

Figure 1.2. Effect of Production Phase on Lagoon Trace minerals Concentrations. (Wet basis; DeRouchey et al., 2002)

1.2.4 Phytic acid Phytic acid (inositol hexaphosphoric acid) and phytic acid salts occurring together in seeds are generally referred together as phytate (Sathe and Reddy, 2002). Phytic acid is composed of an inositol ring with six phosphate groups (Figure 1.3). Phytate, which is an important source of P for plant establishment, accumulates during seed development and is highest at maturity.

Figure 1.3. Structure of Phytic acid (a) and Phytic acid Chelate (B) (Adapted from Schinckel, 2005).

In cereal grains, phytate accumulates in the aleurone layer located between the endosperm and bran. In legumes and many other seeds, phytate is located in globoid crystals which are associated with membrane-bound intracellular protein bodies. The endosperm of wheat and many other cereal grains is nearly devoid of phytate, while approximately 86% is in the aleurone layer associated with the bran. However, corn is unique because 90% of the phytate is found in the germ portion of the kernel (O’Dell et al., 1972). In soybeans, phytate is distributed throughout the protein bodies in the cotyledons, while the hull is low in phytate. The phytate content of corn is 8.3 to 22.2 g/kg, for wheat it is 3.9 to 13.5 g/kg, and soybeans contain 10.0 to 22.2 g/kg (Lolas et a., 1976; Reddy et al., 1989). Phytates complex with charged carboxyl groups on amino acids and proteins (O’Dell and de Boland, 1976; Sathe and Reddy, 2002). Furthermore, phytate frequently forms insoluble 9 salts with di- and trivalent minerals like Ca, Cu, Mg, Zn, and Fe (Figure 1.3). Under normal physiological conditions these chelates are poorly soluble and not readily absorbed from the intestine. A major portion of the P in grain is associated with phytate. Approximately 80% of the P in corn is associated with phytate (Spencer et al., 2000; Viveros et al., 2000; Veum et al., 2001) and for soybeans it is approximately 55 to 60% (Lolas et al., 1976; Ravindran et al., 1994). The percentage of phytate P in wheat (64 -79) and barley (61 -63%) is somewhat lower than corn (Viveros et al., 2000; Shen et al., 2005). Ruminants possess anaerobic bacteria in the rumen which produce the phytase enzyme that allows them to degrade phytate (Yanke et al., 1998) and liberate the P and trace minerals. However, non-ruminants lack phytase and therefore phytate is considered essentially unavailable. Cromwell and coworkers (1992) estimated that only 15% of the P in a corn-soybean meal diet is available for use by pigs. Consequently, phytate’s impact on phosphorus availability has received much attention in controlling phosphorus excretion (Knowlton et al., 2004). The primary method of reducing phytate’s antinutritional properties in non-ruminants is by supplementing diets with exogenous phytase. Phytase addition to swine (Cromwell et al., 1993; Yi et al., 1996) and poultry (Leske and Coon, 1999; Rutherford et al., 2004) diets improves phosphorus bioavailability and thus reduces phosphorus excretion. Phytase supplementation has also been demonstrated to increase trace mineral utilization by pigs fed diets with no supplemental trace minerals (Shelton et al., 2005). Corn (Spencer et al., 2000; Veum et al., 2001) and barley (Veum et al., 2002) varieties have been developed that have reduced phytic acid contents. Researchers have developed transgenic pigs that produce saliva containing phytase (Golovan et al., 2001), but these transgenic pigs are not yet approved for human consumption.

1.3. Tissue distribution The total body content of minerals in swine and other animals increases from birth to maturity. This is not only because the animal’s mass is larger, but because tissue concentrations increase, especially in bone. Table 1.3 demonstrates the increase in body mineral content in pigs to maturity. The increase in Ca and P content is expected as bones become more ossified. Iron and Zn are the trace minerals with the highest content in body. The large difference between Fe contents (Table 1.3) of the 145 kg pig (1.9 g) and third parity sow (77 g) may be related to different methods used to calculate the contribution of blood minerals to whole animal content. Of the remaining essential trace minerals, Cu has the highest concentrations and Cr, Mn, and Se the lowest. These patterns of prevalence are typically reflected in the individual tissues.

Mineral Birtha Weaninga 75 kga 145 kga Sowbc Ca, g 15.2 88.3 483.2 1078.4 1327.0 P, g 8.8 54.0 318.6 637.3 768.3 Fe, g 0.03 0.19 1.41 1.90 77.33 Zn, g 0.02 0.11 0.97 1.75 3.75 Cr, mg 0.62 6.86 27.08 71.90 124.33 Cu, mg 2.45 9.64 56.29 93.96 466.33 Mn, mg 0.65 3.43 25.49 31.05 141.00 Se, mg 0.11 0.66 10.09 33.50 20.33 aCalculated from data of Mahan and Shields, 1998. bMahan and Newton, 1995. cParity 3, 185 kg BW.

Table 1.3. Total mineral content of growing pigs from birth to 145 kg body weight and parity 3 sows

1.3.1 Visceral Organs Trace mineral concentrations vary between body tissues, however the liver and other internal organs have higher concentrations than the remaining body tissues. The variation between organs is mostly related to individual organ functions and the need for the trace minerals in that tissue (Table 1.4). The liver acts as the major storage tissue for trace minerals in the body, whereby it typically has the highest concentrations and its reserves are more labile. For example Fe is

11 stored in hepatocytes as ferritin, until the Fe is processed into transferrin for transportation in the blood. Furthermore, the liver is a site of Fe recycling during erythrocyte degradation. The liver is also involved in mineral homeostasis because of its role in enterohepatic recycling, whereby minerals are diverted into the bile, emptied into the duodenum, and then are available for reabsorption. The kidney is both a storage organ and excretory route for minerals. The kidney typically increases its trace mineral concentration with higher dietary mineral intakes. The high mineral concentrations of the spleen, particularly Fe, relates to its function in filtering out and degrading aged or abnormal erythrocytes, while storing Fe obtained from destroyed cells. Consequently, the spleen has the second highest concentration of Fe in the body. In the brain and heart, Cu is an essential constituent of enzymes like peptidylglycine-α-aminating monooxygenase (Prohaska et al., 2005), which regulates posttranslational modification of several neural peptides and cytochrome C oxidase, which is involved in cellular respiration (Strausak et al., 2001). Homeostasis of Cu, Fe, and Zn is controlled in the epithelial cells of the small intestine, whereupon the absorbed minerals are stored, rather then entering the blood stream (McDowell, 2003). Eventually the cells are sloughed and digested, while some minerals are reabsorbed. Organ trace mineral concentrations are generally similar between pigs, cattle, and sheep, with the exception of liver Cu (Table 1.4). Ruminants have distinctly higher liver Cu levels than non-ruminants. Ruminants have the ability to store large quantities of Cu in response to their susceptibility to Cu deficiency (Underwood and Suttle, 1999). This susceptibility is partially related to the propensity for Cu to precipitate with sulphide in the rumen, preventing its absorption (Bird, 1970). Molybdenum exacerbates this precipitation with the formation of thiomolybdates in the rumen (Dick et al., 1975).

Organa Mineralb Liver Kidneys Heart Pancreas Spleen Lungs Brain Pig Cu, ppm 6.77 6.22 4.08 0.90 1.31 0.83 2.40 Fe, ppm 233.0 48.9 46.8 21.3 223.2 189.00 16.00 Mn, ppm 3.40 1.23 0.63 1.57 0.72 0.17 2.40 Se, ppm 0.53 1.90 0.10 0.41 0.33 0.18 0.16 Zn, ppm 57.6 27.5 28.0 26.2 25.4 20.3 12.7

Cattle Cu, ppm 97.50 4.26 3.96 0.60 1.68 2.60 2.78 Fe, ppm 49.0 46.0 43.1 22.2 445.5 79.5 25.5 Mn, ppm 3.10 1.42 0.35 1.50 0.73 0.19 0.26 Se, ppm 0.40 1.41 0.22 0.25 0.62 0.44 0.21 Zn, ppm 40.0 19.2 17.0 25.8 21.1 16.1 10.2

Sheep Cu, ppm 69.79 4.46 3.97 0.61 1.21 2.50 2.40 Fe, ppm 73.7 63.8 46.0 23.0 418.9 64.0 17.5 Mn, ppm 1.84 1.18 0.46 4.00 0.50 0.19 0.44 Se, ppm 0.82 1.27 0.32 0.34 0.32 0.18 0.09 Zn, ppm 46.6 22.4 18.7 19.3 28.4 18.0 11.7 aSource: USDA National Nutrient Database for Standard References bExpressed as mg per kg (ppm) of wet tissue.

Table 1.4. Comparison of trace mineral concentrations of visceral organs and brains of pigs, cattle, and sheepa.

1.3.2 Bone Within the body, 96 to 99% of the Ca and 60 to 80% of the P are present in skeletal tissue (Crenshaw, 2001). The variability in the proportion of whole body P located in bone relates to the quantity of P located in soft tissues. Calcium and P are deposited into hydroxyapatite crystals (Ca10 (PO4)6 • (OH)2) in the bone, with a typical Ca:P ratio of 2.5:1. Mineralization increases with age, as more hydroxyapatite is formed on a cartilage framework. Trace minerals are deposited into hydroxyapatite at a ratio to one another that seems to follow the same pattern as the soft tissues, but can be influenced by dietary intake. For example, bone Zn concentration increases as Zn intake increases (Wedekind et al., 1994).

Trace mineral concentrations in the bone ash of growing pigs are approximately 9 ppm Cu, 55 ppm Fe, 4 ppm Mn, and 300 ppm Zn (Pond et al., 1975, 1978; Ward et al., 1991). Slightly lower concentrations were reported for the bones of cattle and sheep (Doyle, 1979). Bone does not appear to be a reliable labile source of trace minerals. In addition to the adsorption of trace minerals during the formation of hydroxyapatite, trace minerals are also present in cartilage and bone marrow. Copper, Zn, and Mn are constituents of various enzymes critical for forming and maintaining the collagen framework of bone (McDowell, 2003). In the marrow, hemosiderin stores Fe for erythropoiesis and hemoglobin synthesis.

1.3.3 Muscle Muscle is not considered a storage tissue for minerals, rather the minerals are primarily constituents of enzymes or muscle proteins. Consequently, muscle contains lower concentrations of minerals than visceral organs and bone. However, because muscle mass contributes a large proportion of the body mass, an appreciable portion of total body minerals reside in muscle. For example, 65% of the Zn in the fleece-free empty body of sheep is in the muscle (Grace, 1983). Comparisons of muscle trace mineral concentrations for various domestic and wild animals are presented in Table 1.5. Of the common domestic meats, chicken has the lowest concentration of trace minerals on a wet basis, followed by pork, with beef and lamb having the highest concentrations. The largest differences between species appear to be in Zn and Fe contents. Manganese and Se muscle concentrations are low, but similar between species. In contrast to the domesticates, wild animals seem to have higher muscle trace mineral concentrations. Because most wild animals obtain their minerals exclusively from forages, they likely have a lower daily mineral intake than domestic food animals. Additionally, the differences in wet muscle concentrations may also be related to intramuscular fat contents. Differences between species in muscle mineral concentrations may be partially related to the color and myglobin content of the muscle. Myoglobin, which contains Fe, is in a greater concentration in darker red meats. The content of myoglobin in muscle differs between species, with cattle having the highest concentration (8 mg/g), followed by lamb (6 mg/g), pigs (2 mg/g) and poultry (2 mg/g). Meat color of these species reflects this myoglobin content with cattle being the darkest red and pork and chicken being the palest in color. Beef muscles also have

14 higher Zn and Fe contents than pork, especially when longissimus dorsi muscles (LDM) are compared (Leonhardt and Wenk, 1997). Trace mineral and myoglobin concentrations differ between the various muscles of the body. The pale colored LDM of pigs contains approximately one-third of the Zn, Fe, and heme iron as the darker muscles from the shoulder region (Leonhardt and Wenk, 1997). Soluble myglobin content of LDM also differs between breeds of pigs (Newcom et al., 2004), suggesting that the muscle mineral content may also differ between breeds and within species. Muscle mineral content usually is not affected by dietary mineral levels as in other body tissues, with the exception of Se. Muscle concentrations reflect the dietary Se source and level fed because Se is incorporated into muscle proteins as selenomethionine. Mahan in a regional study (2005) reported that loin Se concentrations are highly correlated with dietary Se levels. Feeding organic Se sources (grain or selenized yeast), which contains selenomethionine, increases the loin Se content compared to feeding sodium selenite (Mahan and Parrett, 1996; Mahan et al., 1999).

Domestic Food Animals Wild Species Mineral Pork Chicken Beef Lamb Bison Deer Elk Antelope Cu, ppm 0.61 0.53 0.71 1.08 1.30 2.50 1.20 1.80 Fe, ppm 11.0 8.9 18.3 16.2 26.0 34.0 27.6 31.9 Mn, ppm 0.18 0.19 0.10 0.20 0.09 0.40 0.12 0.20 Se, ppm 0.45 0.16 0.18 0.20 0.19 0.10 0.10 0.10 Zn, ppm 29.7 5.4 35.7 35.2 42.9 20.9 24.0 12.8 aSource: USDA National Nutrient Database for Standard References.

Table 1.5. Comparison of trace mineral concentrations of lean muscle of various species (wet basis)a

1.3.4 Other Tissues Blood provides the circulatory route for minerals to reach the various body tissues. A large proportion of total body Fe content is located in the hemoglobin in red blood cells (Miller et al., 1961). Appreciable quantities of other trace minerals are also present in blood and typically reflect dietary mineral intakes.

Appreciable concentrations of minerals are found in the skin, hair, wool and hoof, particularly Zn. In sheep, the highest tissue Zn concentrations (275 mg/kg) are in the wool and approximately the same quantity of Mn and Zn are in the fleece as in the empty-fleece free body (Grace, 1983). Trace minerals in the skin appear critical for cartilage synthesis and maintenance. Zinc has a role in skin nucleic acid and collagen synthesis, while a Zn deficiency is characterized by parakeratosis, or the thickening and hyperkeratinization of skin epithelial cells. The Cu-dependent enzyme lysyl oxidase is a rate-limiting enzyme in the cross-linking of collagen and elastin fibers. Manganese-dependent glycosyl transferase adds carbohydrates to core proteins of proteoglycans, which play an important role in cartilage integrity.

1.3.5 Milk Comparison of milk mineral concentrations from various species is presented in Table 1.6. Of the species represented, sow milk has the highest macro- and trace mineral concentrations. Cows have higher macro-mineral concentrations than humans, but the trace mineral levels of dairy cow and human milk are similar, with the exception of Cu. Zinc is the trace mineral with the highest milk concentration in all species. Copper and Fe contents seem to be the most variable of the trace minerals between species. Within each of these species there are however, large variations in milk trace mineral concentrations reported in the literature, which is likely attributed to dietary levels of trace minerals consumed and breed differences. The mineral concentration of colostrum differs markedly from mature milk (Hill et al., 1983a; Csapo et al., 1996). In sows, the concentration of Ca and Mg are lower in colostrum compared to milk, whereas Cu and Zn are higher in the colostrum and Fe and Mn are similar. However, in dairy cattle colostrum concentrations of all minerals decline rapidly within 24 h of parturition as the mature milk begins to be secreted (Kume and Tanabe, 1993). The majority of Ca and P in milk is associated with casein micelles. Over 50% of the Cu, Mn, and Zn in cow’s milk are bound to casein (Renner et al., 1989). The remainder is associated with whey proteins, low molecular weight fractions of the milk, and the hydrophilic outer fat globule membrane (Fransson and Lonnerdal, 1984; Jenness, 1988; Renner et al., 1989). Iron and Mn are bound to lactoferrin, which is associated with the whey fraction of milk (Renner et al., 1989), however it has been estimated that only 30% of milk Fe is associated with lactoferrin (Fransson and Lonnerdal, 1980). Trace minerals are also found in certain enzymes present in milk, for example Zn is present in lactate dehydrogenase in fat globules (Jenness,

1988). Selenium may be incorporated into all milk protein fractions as selenomethionine. Feeding organic Se (selenomethionine) has increased milk Se in sows (Mahan, 2000) and cattle (Givens et al., 2004). Little is known about the form and distribution of Cr in milk, but concentrations up to 0.177 µg/g have been reported (Garcia et al., 1999). Several researchers have observed that milk Ca and P concentrations are not affected by the dietary levels fed (Harmon et al., 1975; Mahan and Fetter, 1982), suggesting genetic regulation. The concentration of Fe in milk is also believed to be genetically controlled by lactoferrin or transferrin in the milk. Increased dietary Fe levels (Pond et al., 1965) and Fe injections to sows (Pond et al., 1961) failed to elevate milk Fe concentrations. However increasing the dietary levels of Zn (Hill et al., 1983a), Mn (Plumlee et al., 1956; Christianson et al., 1990), and Se (Mahan, 2000; Mahan and Peters, 2004) does increase their concentrations in both colostrum and milk.

Mineral Humanab Pigcde Cowabfgh Horseij Ca, mg/l 330 1900 1210 835 P, mg/l 170 1300 960 550 Mg, mg/l 40 110 120 63 Cu, ppm 0.31 1.20 0.12 0.26 Fe, ppm 0.26 2.0 0.30 1.21 Mn, ppm 0.01 0.07 0.04 0.05 Se, ppm ― 0.03 0.07 ― Zn, ppm 2.15 6.12 3.90 2.00 aAnderson, 1992. bMacy et al., 1953. cHill et al., 1983a. dCsapo et al., 1996. eMahan and Peters, 2004. fRenner, 1983. gJenness, 1988. hSahih et al., 1987. iCsapo-Kiss et al., 1995. jSchryver et al., 1986.

Table 1.6 Comparison of typical mineral concentrations in milk of various species.

1.4. Trace mineral metabolism 1.4.1 Absorption Trace mineral absorption occurs in the small intestine, primarily in the duodenum, although Zn and Cu are also absorbed in the rumen and the proventriculus of chickens (McDowell, 2003). Selenium absorption occurs in the duodenum, cecum, and colon compared to other trace minerals (NRC, 1983). Hydrochloric acid in the stomach solubilizes Fe by reducing it from the ferric (Fe3+) to the ferrous (Fe2+) state for absorption. However, Fe can complex with gastric secretions (Conrad and Umbreit, 2002) allowing it to remain soluble at more alkaline pH environments of the intestine. Various dietary chelates such as ascorbic acid, histidine, and lysine have been shown to increase Fe absorption (Beard and Dawson, 1997). Heme Fe from animal sources is soluble in the small intestine and do not react with dietary constituents (Conrad and Umbreit, 2002). Heme seems to be absorbed by a receptor-mediated transport mechanism, allowing it to be absorbed differently than the inorganic source (Grasbeck et al., 1979). Zinc and Cu are not free ions at the neutral pH of the intestine, but rather are often associated with low molecular weight binding ligands. They include citrate, picolinate, histidine, and glutamic acid and they enhance mucosal uptake of these trace minerals (Hambridge, 1986; Harris, 1997). Mucin secreted by intestinal goblet cells seems to facilitate the absorption of Fe (Conrad and Umbreit, 2002). At least two active transport pathways have been identified for Fe uptake by the mucosal cell (Conrad and Umbreit, 2002). Zinc and Cu are transported across the brush border by both carrier mediated systems and paracellular movement (Cousins, 1989). The active transport pathway predominates at low dietary concentrations, whereas paracellular movement increases with greater intakes. The Cu transporters (hCtr1 and Nramp2) have been identified (Pena et al., 1999). Manganese absorption is not well understood but is believed to share many absorption mechanisms with Fe (Leach and Harris, 1997). Copper and Zn homeostasis seems to be partially controlled at the intestinal epithelial cell level, whereas Fe homeostasis is strictly regulated at the intestinal level. Absorption of Zn and Cu are influenced by mucosal cell metallothionein saturation (Chesters, 1997; Harris, 1997; Davis and Mertz, 1986). Metallothionein contributes to the regulation of the Cu and Zn flux between the intestinal lumen and portal circulation in response to plasma Zn and Cu concentrations (Hambridge, 1986. Because Zn and Cu are regulated by the same metalloprotein, one mineral can reduce the absorption and/or transfer of the other mineral (Davis and Mertz, 18

1986; Harris, 1997). Iron absorption is controlled by intestinal cell Fe content whereby Fe is stored by ferritin in the mucosal cell until it is transferred across the basolateral membrane into the circulation. When diets are low in Cu, Fe, or Zn, their relative efficiency of absorption is greater, conversely at high intakes the percentage absorbed is reduced (Krebs, 2000; McDowell, 2003). In non-ruminants, Se is absorbed with a high efficiency, suggesting there is no regulation of Se absorption. Patterson and coworkers (1989) reported that approximately 84% of selenite was absorbed at relatively high levels (200 µg) administered to humans. However, selenite absorption is much lower in ruminants, due in part to the formation of insoluble compounds in the rumen (Spears, 2003). Organic Se forms, selenocysteine and selenomethionine, are believed to be absorbed later in the small intestine as intact amino acids utilizing the same transport pathways as cysteine and methionine. The mechanism of trivalent Cr is poorly understood, but is believed to be absorbed primarily by passive diffusion (McDowell, 2003), although active transport may be involved (Offenbacher et al., 1997). Physiological state (i.e. rapid growth and reproduction) has been shown to increase absorption (Chesters, 1997; Krebs, 2000). Various dietary components such as high Ca and P levels and phytate may also react with trace minerals which can reduce their absorption. Furthermore, trace minerals also compete with one another for absorption sites on the enterocyte. These trace mineral nutrient interactions will be discussed more in depth in section 1.6.

1.4.2 Transport and storage Trace minerals typically have specific carriers in the circulating blood to ensure that elemental ions are not in a free-state. Body Fe levels are strictly regulated so that a deficiency is prevented, while also preventing free Fe from forming insoluble complexes with hydroxide ions that may participate in the generation of free radicals (Baynes and Stipanuk, 2000). Transferrin is the primary carrier of Fe and facilitates body Fe distribution. Transferrin accepts Fe entering the blood from the intestines, released from storage sites, and from hemoglobin degradation. Iron may then be redistributed for storage, incorporation into Fe-containing enzymes, or for hemoglobin synthesis in bone marrow. Iron uptake by cells is accomplished by transferrin receptor mediated endocytosis (Baynes and Stipanuk, 2000).

The liver synthesizes a metalloprotein, ceruloplasmin, which binds Cu in the blood and contains six Cu atoms per unit. Albumin and histidine also participate in transporting Cu, but to a lesser extent than ceruloplasmin (Pena et al., 1999). Zinc is primarily carried in the circulation by albumin and α2-macroglobulin, however some is bound to high molecular weight ligands (Chesters, 1997; Fleet, 2000). Absorbed Mn is transported to the liver in the portal blood via albumin (Leach and Harris, 1997). After reaching the liver, Mn is primarily transported by transferrin and α2-macroglobulin. Similarly, Cr is transported by transferrin, but when transferrin is saturated, serum albumin will transport Cr (Offenbacher et al., 1997). Selenium is believed to be transported by glutathione peroxidase in erythrocytes and by selenoprotein P (Burk and Hill, 2005). The primary storage organ for trace minerals is the liver, although other organs also possess some storage functions. Trace minerals are adsorbed onto bone, but they are not considered readily available because they can not be specifically mobilized (Fleet, 2000). Trace minerals are present in low concentrations in skeletal muscle, but due to its mass a large portion of total body trace mineral content is in muscle. Ferritin and hemosiderin are the storage compounds of Fe and are located largely in the parenchymal liver cells, bone marrow, and spleen. At high tissue Fe levels hemosiderin is the predominant form, whereas at low levels ferritin predominates. Transfer of Fe from transferrin to ferritin is reversible and allows for redistribution between tissues (McDowell, 2003). The mobilization of Fe from ferritin requires ceruloplasmin (ferroxidase I). Metallothionein, the major Zn storage protein is primarily concentrated in the liver, but is also found in the kidney, pancreas, and intestine (Chesters, 1997) and metallothionein increases with Zn intake (Carlson et al., 1999). Of the trace minerals, Zn has the highest bone concentration and bone is responsive to Zn intake (Wedekind et al., 1994). Copper is stored by metallothionein in the liver and the major extrahepatic storage form is mitochondrial cuprein, though Cu is also in the cytosol bound to metallothionein-like proteins (McDowell, 2003). Tissue Se concentration are highest in the kidney and liver, but Se is also stored in the spleen and pancreas (Jenkins and Winter, 1973; Kim and Mahan, 2001). Selenium deposition increases with intake, but to a greater extent when organic sources are fed (Mahan and Parrett, 1996; Mahan et al., 1999). Chromium is not stored in high concentrations in the body, but the kidney and liver have the highest concentrations (Anderson et al., 1997).

1.4.3 Excretion Trace minerals are primarily excreted in the feces. Fecal minerals are composed of those not absorbed from the diet and endogenous losses. In most species, trace minerals are poorly absorbed and the extent of absorption is influenced by chemical form, dietary level, and nutrient demand. Under normal physiological conditions and at adequate intakes, only 15% of dietary Cu, Fe, and Zn are retained by the body (McDowell, 2003). Absorption and retention of Mn (Finley et al., 1997) and Cr (Anderson, 1987; Offenbacher et al., 1997) is even lower. Endogenous mineral losses by bilary and pancreatic secretions and sloughing of intestinal mucosal cell contribute to maintaining body mineral balance. These losses increase as Cu, Mn, and Zn intakes increase and the minerals have the potential for enterohepatic recycling (Fleet, 2000). However due to the body’s strict control of Fe metabolism, less than 3% of fecal Fe is from endogenous origins (McDowell, 2003). As previously indicated, non-ruminants effectively absorb Se, however very little fecal Se originates from endogenous sources (McDowell, 2003). Although trace minerals are excreted in the urine, their urinary concentrations, with the exception of Se, are generally low (Underwood and Suttle, 1999). In non-ruminants, some Se is excreted through urine, feces, and exhalation. Because the absorption of Se is not regulated, the primary method for maintaining Se homeostasis is through urinary excretion. But at toxic intakes, Se is also removed from the lungs as dimethylselenide by exhalation (NRC, 1983).

1.4.4 Deficiency Because minerals are associated with many metabolic pathways, their deficiency often results in impaired growth, reproduction, and immunity. Marked deficiencies are unlikely to occur in modern commercial production systems, however marginal deficiencies could occur under certain conditions (i.e. poor feed formulation, low feed intake). The occurrence and severity of mineral deficiencies are influenced by length of time that deficient diets are fed, prior mineral status, and physiological state (Hill and Spears, 2001). Pigs fed plant diets that are not supplemented with trace minerals could lead to mineral deficiencies because phytate binds the minerals making them unavailable to the animal (Reddy et al., 1989). During deficiency, the efficiency of mineral absorption is increased because homeostatic controls are down regulated. Iron deficiency is the most common mineral deficiency in swine (Hill and Spears, 2001), but is far more common in humans. Inadequate Fe leads to anemia, or decreased 21

hemoglobin synthesis, and is most prevalent in neonatal pigs. Neonates are born with low hepatic Fe stores. The combination of rapid postnatal growth and low milk Fe content results in anemia in young pigs. Attempts to increase placental and mammary Fe transfer via the diet or Fe injections have been unsuccessful (Pond et al., 1961; 1965). Consequently, injecting pigs with 200 mg of iron dextran at birth is a standard practice (Hill and Spears, 2001). Oral Fe administration is most effective within the first 24 hr postpartum before gut closure occurs. In older pigs, anemia may result from inadequate dietary Fe intake or heavy parasitic load. Copper-deficiency results in connective and vascular tissue damage related to a reduction in lysyl oxidase activity. Skeletal defects in growing pigs result from Cu deficiency because development of the connective tissue framework necessary for bone development is impaired, though bone Ca and P concentrations are normal (Hill et al., 1983d). Moreover, incidences of osteochondrosis were increased in deficient sows (Hill et al., 1983b). Aortic rupture resulting from reduced lysyl oxidase activity and weakened blood vessel walls have been observed in Cu-deficient pigs (Hill et al., 1983d). The classic Zn deficiency sign is skin hyperkeritinization, or parakeratosis. Decreased feed intake and poor growth are also hallmarks of Zn deficiency. In addition to reduced nutrient intake, impaired growth also results from compromised RNA, DNA, and protein synthesis. In humans, “adolescent nutritional dwarfism”, a syndrome previously common in Middle Eastern countries, is related to Zn deficiency (Prasad et al, 1961). Selenium deficiency results in excessive oxidative damage to the cellular membranes and in the cytoplasm and the release of the cellular contents as a result of inadequate glutathione peroxidase activity. In swine, Se deficiency is characterized by cardiac myopathy, coronary capillary degeneration, liver and skeletal muscle degeneration (White muscle disease), hemorrhaging, and gastric ulcers (Sunde, 1997; Mahan, 2001) and can often result in death. Manganese and Cr deficiencies are rare in swine, suggesting that naturally occurring quantities of Cr in feedstuffs are sufficient (Hill and Spears, 2001).

1.4.5 Toxicity Mineral toxicities are also rare in modern swine production, but when they do occur they are typically associated with feed mixing errors or animals consuming plants and grains grown on soils high in a particular mineral. As with deficiencies, severity is influenced by

22 mineral status, physiological state, age, and duration. When toxicities do occur they often result in reduced feed intake, growth, and efficiency of growth and severe toxicity could result in death. It is possible that a high Cu or Fe status could result in tissue damage. Free ionic Fe and Cu have been determined in vitro to cause free radical damage to cells. The redox potential of Fe and Cu allows these minerals to participate in reactions that result in the production of highly reactive oxygen species (ROS) including hydroxyl radical (Halliwell and Gutteridge, 1984). Membrane lipid peroxidation, protein oxidation, and cleavage of DNA and RNA molecules are believed to be the result of hydroxyl radical production. Adding free Cu to an in vitro system generates the formation of free radicals, but adding histidine and albumin at physiological concentrations appears to prevent the formation (Rowley and Halliwell, 1983). Cellular chaperones, ATX1 and CCS, are involved in partitioning Cu within the cell and preventing the ion from being free in the cytosol (Pena et al., 1999). However, it appears that Fe and Cu associate with both high and low-molecular-weight cellular components allowing them to remain in the cytosol (Powell, 2000). In this state, the association site with which Fe and Cu are bound serves as site for free radical production. Selenium is commonly considered the most toxic of the essential trace minerals. Unlike the others, Se has a high efficiency of absorption because there appears to be no regulating mechanism in the gut. Tissue Se concentrations increase linearly as dietary Se intake increases. Consequently, high levels of Se result in toxicity. In addition to reduced feed intake and gain, signs of Se toxicity include anorexia, alopecia, separation of the hoof and skin at the coronary band, fatty infiltration of the liver. However, feeding organic Se reduced the severity of selenosis signs compared to when pigs were fed the same toxic level of sodium selenate (Kim and Mahan, 2001).

1.5. Trace mineral biological functions

1.5.1 Metalloproteins

Many trace minerals functions in the body are mediated through their roles as constituents in metalloproteins. Metal-containing enzymes (metalloenzymes) are the largest class of metalloproteins. Trace minerals function in metalloenzymes to provide structural integrity to the protein and/or directly participate in the reaction as catalysts. Zinc is recognized as a constituent of over 200 proteins, where it has both structural and catalytic roles in numerous metalloenzymes (Hambridge et al., 1986). Typically Zn’s structural role is to stabilize the quaternary structure of the enzyme. Zinc finger motifs have a structural function, in which Zn facilitates the binding of transcription factors to DNA sequences at promoter regions initiating transcription and gene expression (Hambidge, 2000; Maret, 2000). Other notable structural roles include alcohol dismutase and ferrodoxin (McCall et al., 2000). As a catalyst, Zn is redox stable, unlike Fe, Cu, and Mn which are transition metals involved in redox reactions (McCall et al., 2000). Zinc acts as a Lewis acid to accept a pair of electrons, which enables it to function as a cofactor in reactions that require stable ions without risk of oxidant damage. Consequently, there are many enzymes in which Zn has a catalytic role, including hydrolases (alkaline phosphatase) and lyases (carbonic anhydrase). Copper containing enzymes include cytochrome c oxidase, lysyl oxidase, dopamine hydroxylase, and tyrosinase. In the mitochondria, the Cu and Fe containing enzyme, cytochrome c oxidase, is the last enzyme in the electron transport chain that reduces oxygen to water. Lysyl oxidase initiates the cross-linking of collagen by adding hydroxyl group to lysine residues, an essential process necessary for forming and maintaining bone, skin, and blood vessels. Iron is present in the body as a constituent of heme enzymes and hemoproteins which have critical roles in oxygen transportation and storage, electron transport, and substrate oxidation (Beard and Dawson, 1997). Heme globulin complexes (hemoglobin and myoglobin) are responsible for transporting O2 and CO2 to support respiration. Mitochondrial cytochromes have a heme active site and Fe-porphyrin ring which accepts electrons during the reduction of the ferrous ion to ferric ion (McDowell, 2003). The electron transport chain links substrate

24 oxidation with oxygen metabolism responsible for adenosine triphosphate (ATP). Furthermore, the tricarboxylic acid (TCA; Kreb’s) cycle has many enzymes in which Fe is an active center or cofactor. Arginase, pyruvate carboxylase, carbonic anhydrase, alcohol dehydrogenase, alkaline phosphatase are examples of Mn containing enzymes (Leach and Harris, 1997). Numerous other enzymes are activated by Mn, including hydrolases, kinases, decarboxylase, and transferase (Leach and Harris, 1997). However, Mg is capable of replacing Mn as a cofactor in many enzymatic reactions, with the exception of glycosyl transferase. This is the only enzyme in which Mn is specifically required, whereby the important cartilage molecule chondroitin sulfate is formed (McDowell, 2003). Chromium is involved in carbohydrate, lipid, and protein metabolism (Anderson, 1987). The exact mechanism requires further elucidation, but it appears that the mechanism is a glucose tolerance factor containing Cr. Chromium supplemented as Cr picolinate increases insulin sensitivity on the cell membrane and glucose clearance rate in growing pigs after they receive intravenous glucose (Aimoken et al., 1995). Several metalloenzymes are involved in reducing free radical metabolism. Free radicals, or reactive oxygen species, are any species that contain one or more unpaired electrons and may interact with fatty acids, proteins, and DNA causing oxidative damage (Jones and Delong. 2000). Examples of free radicals include superoxide, hydrogen peroxide, hydroxyl radicals, and ozone. Superoxides are generated by ionic Fe and Cu, leukocytes phagocytizing pathogens, or normal cellular metabolism. The superoxide dismutase (SOD) enzyme present in the blood and cytosol contains both Zn and Cu, while Mn-SOD is present in the mitochondria. Superoxide dismutase converts superoxide radicals to hydrogen peroxide and dioxygen (O2). Catalase (an iron-containing enzyme) and glutathione peroxidase then convert the peroxide to water. The primary functional role for Se in the body is as selenocysteine in glutathione peroxidase production (Sunde, 1997). Glutathione peroxidase, in concert with vitamin E, protects cells from oxidative damage. Glutathione peroxidase acts in the cytosol to destroy peroxides before they react with the membrane. Vitamin E also acts within the membrane as a fat soluble antioxidant (Levander, 1987). Glutathione peroxidase activity has been determined in a variety of tissues, fluids, cells and subcellular fractions.

1.5.2 Growth and Development Because many trace minerals have roles in protein, carbohydrate, and lipid metabolism their essentiality is demonstrated by growth retardation during deficiency states. Many early discoveries of trace mineral functions involved experiments in which growth was restored when specific minerals were provided to deficient animals. Growth is more severely retarded during Zn deficiency, because of Zn’s involvement in numerous metalloenzymes. Zinc’s role in protein synthesis includes functions in synthesis of nucleotides, RNA catabolism, and assisting in transcriptional regulation. Reverse transcriptase, DNA and RNA polymerase, and nucleoside phosphorylase are also dependent on Zn. Furthermore, Zn may be involved in the function of insulin-like growth factor-I (IGF-1) which mediates the anabolic effects of growth hormone actions (Zapf and Froesch, 1986). Circulating levels of IGF-1 decline during Zn deficiency (MacDonald, 2000). Copper, Mn, and Zn play important roles in bone development. Manganese-dependent glycosyl transferase adds carbohydrates to core proteins of proteoglycans to form chondroitin sulfate which plays an important role in cartilage integrity. Lysyl oxidase (Cu) is involved in cross-linking of collagen and elastin fibers. Consequently, the cartilage framework on which hydroxyapatite is deposited is malformed when Mn or Cu is deficient. Alkaline phosphatase is a Zn-containing enzyme found in bone and blood. In bone, osteoblasts produce alkaline phosphatase which is involved in forming hydroxyapatite. Pharmacological concentrations of Cu and Zn are used as growth promoters in nursery pig diets (Hill et al., 2000), however their mode of action has not been determined. Zinc oxide was initially used to prevent Escherichia coli scours (Poulsen, 1995), however E.coli numbers are not greatly effected, whereas growth is improved dramatically (Jensen-Waern et al., 1997). The increased villus height and intestinal metallothionein when zinc oxide was fed (Carlson et al., 1999) may contribute improvements in metabolism and gut health.

1.5.3 Immunity Extensive research has established the importance of adequate trace minerals for proper immune function (Failla, 2003). Both innate (“nonadaptive”) and acquired (“adaptive”) immunity are compromised during Zn, Cu, and Se deficiencies. Marginal mineral deficiencies in which growth or productivity are not impacted may however result in an impaired immune response. Although nutrient requirements set by NRC (1998) can be defined as levels which permit maintenance of normal health and productivity, it is unclear if higher levels are required 26 to maximize immunity, especially when animals are challenged (Cromwell, 2001). Consequently, exact nutrient requirements to maximize immunocompetence have yet to be determined. This is due to a lack of specific response criterion to evaluate immunity and the complexities of factors effecting health (Cromwell, 2001). However, in vivo studies examining mineral deficiencies and supplementation and in vitro studies examining the role of minerals in cellular mechanisms have furthered our knowledge to make better estimates (Shanakar and Prasad, 1998). Through its involvement in a myriad of metabolic processes, Zn plays a critical role in immune function, disease resistance, and general health (Shanakar and Prasad, 1998). The skin acts as a physical barrier to insults. Epidermal damage resulting from Zn deficiency leads to skin lesions and parakeratosis. During Zn deficiency, precursor T and B cells in the thymus and bone marrow decrease, which leads to thymic atrophy and lymphopenia (Fraker et al., 2000). This may be related to a chronic production of glucocorticoids resulting in apoptosis of precursor cells. Additionally, the hormone thymulin, produced by thymic epithelial cells, requires Zn for its role in aiding in T-lymphocyte maturation (Mocchegiani and Muzzioli, 2000). Consequently there is an overall decrease in T and B- lymphocytes, whereas neutrophils increase when Zn is deficient, suggesting a shift from acquired immunity to a greater reliance on innate immunity (Failla, 2003). Zinc may mediate the relationship between cell-mediated and humoral immunity through regulating cytokine production. Zinc deficiency has been shown to decrease the secretion of interferon-γ, tumor necrosis factor-α, and interleukin (IL)-2 in blood mononuclear cells, but not the secretion of Il-4, IL-6, or IL-10 (Beck et al., 1997). The effects Cu has on immunity are similar to those of Zn. During Cu deficiency, decreased respiratory burst and antimicrobial activity of neutrophils and macrophage cells occurs (Failla and Hopkins, 1998). Unlike Zn, Cu seems to influence T lymphocytes more than B- lymphocytes. T cell numbers decline and their antigen processing and presentation capabilities are impaired (Bala et al., 1990; 1991). This is likely mediated through the reduced production of IL-2 by T cells (O’Dell, 1993). Selenium deficiency results in decreased proliferation of T and B cells, lymphocytes, and immunoglobulins (Arthur et al., 2003). Of particular importance is the role of Se in neutrophils function. Neutrophils produce superoxide radicals as a means of destroying pathogens. Glutathione peroxidase, in conjunction with Cu/Zn-SOD and catalase, supports the neutrophils ability to produce free radicals, thus optimizing its activity and preventing the cell

27 from being damaged. Consequently, during Se deficiency neutrophil numbers are not changed but their effectiveness is decreased (Arthur et al., 2003). Selenium deficiency may also affect the virulence of certain viruses. A benign strain of the Coxsackie virus became virulent in Se deficient mice, causing myocarditis (Beck et al., 1995). Reduced glutathione peroxidase activity likely resulted in oxidative damage to the RNA-viral genome, leading to virulence (Beck et al., 1998). 1.5.4 Reproduction The biological roles for trace minerals mentioned in previous sections likely also play important functions in reproduction. The complexities of mineral metabolism, combined with those of reproduction, have lead to few clear conclusions being drawn for specific roles of trace minerals in reproductive function. The few experiments which have investigated the mechanistic relationship between trace minerals and reproduction have involved feeding diets resulting in deficiencies or excesses. Mineral requirements for specific reproductive tissues to support reproduction are poorly defined (Hostetler et al., 2000) Litter size at parturition is largely determined within the first 30 d of pregnancy and is influenced by ovulation number and embryo survival (Wilmut et al., 1986), whereas the number of stillbirths is determined by fetal death after 30 d postcoitum or during the birth process. Previous experiments have demonstrated that litter size was increased when the basal (deficient) diet was supplemented with Cu (Cromwell et al., 1993), Mn (Plumlee et al., 1956), or Zn (Hill et al., 1983b). Some researchers (Mahan et al., 1974; Chavez and Patton, 1986) have reported increased litter size with Se supplementation, whereas others have not (Chavez, 1985; Mahan and Kim, 1996; Mahan and Peters, 2000). Hostetler et al. (2000) reported higher concentrations of Cu, Mn, and Zn in the conceptus products then in the surrounding endometrium and ovaries between days 12 and 30 postcoitum. This suggests an increased uptake and/or utilization of trace minerals by the embryo and fetus early in pregnancy. The occurrence of stillbirths seems to increase during trace mineral deficiency. Fetal death and resorption have been reported in Cu-deficient rats (Davis and Mertz, 1987). A similar response was observed in swine, where gestating sows fed 2 ppm of Cu farrowed more stillborn pigs than sows fed 9.5 ppm (Kirchgessner et al., 1980). Early work by Plumlee and coworkers (1956) demonstrated that prolonged feeding of 0.5 ppm of Mn resulted in more resorbed fetuses and small, weak pigs at birth. Although sows fed Zn-deficient diets farrowed fewer pigs, there was no difference in the number of stillbirths (Hill et al., 1983b). The mechanism(s) by which

28 trace minerals affect reproduction have yet to be identified. However, the roles of trace minerals in hormone production, oxidant metabolism, and general metabolism are likely involved. Several trace minerals are essential components or activators of enzymes involved in steroidogenesis (Hurley and Doane, 1989). Manganese is believed to have a role in steroid synthesis, whereby cholesterol and cholesterol precursor synthesis is inhibited during Mn deficiency (Hurley and Doane, 1989). Nuclear receptors for steroid hormones are a type of zinc finger transcription factor. However, it has not been determined what, if any, effect Zn deficiency has on their activity. Activity of the pituitary gonadotropins, follicle-stimulating hormone and luteinizing hormone, may also be influenced by Zn status (Root et al., 1979). Zinc enzymes are involved in the arachidonic acid cascade of prostaglandin formation (Chanmugam et al., 1984; Wauben et al., 1999). Secretion of PGF2α into the uterine lumen and away from the corpus luteum is necessary to maintain pregnancy in swine (Kraeling et al., 1985). It is unclear, however, if Zn concentration in the uterus effects prostaglandin synthesis. Uterine concentrations of IGF-1 are high in pregnant sows (Simmen et al., 1992) and circulating levels of IGF-1 decline during Zn deficiency (MacDonald, 2000) suggesting a potential role in pregnancy. Tissue differentiation and cell proliferation are stimulated by IGF-1 (Zapf and Froesch, 1986) and IGF-1 may be involved in uterine remodeling at the time of implantation. Furthermore, in vitro experiments indicate that Zn increases the binding affinity of IGF-1 to IGF receptors (McCusker et al., 1998). The importance of antioxidant protection for embryonic and fetal survival has not been widely investigated. Excessive free radical production is known to cause damage to cellular membranes and the DNA, possibly resulting in cell death. Elevated expression of Cu/Zn-SOD and glutathione peroxidase has been demonstrated in the mouse fetus (deHaan et al., 1994), while transcription and expression of Mn-SOD has been demonstrated in bovine ovaries (Lequarre et al., 2001). In vitro work with Cu-deficient rat embryos demonstrated decreased Cu/Zn SOD activity resulting in elevated levels of superoxide and a higher incidence of malformations (Hawk et al., 2003). However, there have been few reports of the in utero effects of oxidative status on reproductive performance. Hostetler and Kincaid (2004a) reported that the fetuses of sows fed Se-deficient diets had lower liver Se contents, which decreased throughout gestation. Although fetal liver glutathione peroxidase activity was not affected by maternal Se intake, liver hydrogen peroxide and malondialdehyde concentrations in the fetus were elevated, suggesting increased oxidative stress was occurring in the developing fetus.

Neonatal pig mineral reserves reflect the dietary levels of Cu, Zn (Hill et al., 1983c), Mn (Gamble et al., 1971), and Se (Mahan and Peters, 2004; Hostetler and Kincaid, 2004b) fed to the sow during gestation, demonstrating their effective placental transfer. An exception is Fe, whose transfer across the placenta is regulated by uteroferrin and not affected by dietary Fe levels (Pond, 1961). Restricting the supply of minerals to the developing conceptus may compromise fetal metabolism and can detrimentally effect prenatal growth and development. Sow Cu deficiency (Hill et al., 1983b; Cromwell et al., 1993) results in reduced birth weights. Similarly, feeding low Mn gestation diets reduced birth weight, but had no effect on number of pigs born (Plumlee et al., 1956; Christianson et al., 1989; 1990). In addition to their composition in milk, trace minerals are involved in protein, carbohydrate, and fatty acid metabolism for milk synthesis through their role in metalloenzymes and as cofactors. Mineral absorption increases during lactation in response to the high nutrient demand for milk production. However when dietary mineral intake is insufficient, the sow will mobilize her body mineral reserves, particularly from the liver to meet the demand. Sow mineral status decreases with each reproductive cycle and this is exacerbated at higher levels of production (Mahan and Newton, 1995). Milk mineral concentration of Zn (Hill et al., 1983a), Mn (Plumlee et al., 1956), and Se (Mahan, 2000; Mahan and Peters, 2004), but not Fe (Pond et al., 1965), reflect the dietary intake and/or mineral status of the sow. Consequently, mineral status of the offspring at weaning may be affected by the sow’s mineral status. One would suspect that sow mineral status at weaning may also affect her subsequent reproductive performance (Mahan, 1990). Because trace minerals are involved in immunity, their status may also affect the occurrence of mastitis and other health conditions. Although the impact of mineral nutrition on mastitis has not been extensively investigated in sows, it has been in dairy cattle. Selenium and/or vitamin E deficiencies increase the incidence and severity of intramammary infections, number of mastitis cases, and somatic cell counts (Smith et al., 1997). A low Cu status has been found to increase somatic cell count and the severity of intramammary infections (Scaletti et al., 2003). For both Se and Cu, the effects on mastitis seem to be mediated by impaired neutrophil activity. Zinc’s importance for maintaining the keratin layer which lines the streak canal of the teat may contribute to preventing infection (Capuco et al., 1992). Although, the effects of Zn

30 status on preventing mastitis have not been widely investigated, there has been some investigation of the effects of Zn source. Feeding Zn-methionine (Kellogg, 1990) and Zn- proteinate (Harris, 1995) decreased somatic cell counts. There is minimal information regarding the effects of mineral nutrition in boars. However, Zn and Se seem to have important roles in male fertility. Zinc deficiency impairs testicular development (Liptrap et al., 1970), causing the cessation of spermatogenesis. Zinc deficiency has been associated with reduced production of pituitary gonadotropins and androgens in the rat (Kellokumpu and Rajaniemi, 1981). Zinc is associated with sulfhydryl groups and disulfide linkages in the tail region of the sperm (Calvin et al., 1973; 1975) and has involvement in ATP contraction and phospholipid energy reserves may affect motility (Hidiroglou and Knipfel, 1984). Marin-Guzman and associates (1997) demonstrated the impact of Se deficiency on boar semen quality and subsequent fertilization rate. Boars fed Se-deficient diets since weaning had lower testicular and semen Se contents and decreased glutathione peroxidase activity compared to boars fed 0.5 ppm Se. Additionally, Se-deficient boars had more abnormal sperm and lower sperm motility, which contributed to a lower fertilization rate when gilts were inseminated with their semen. These effects are related to the spermatozoa having abnormal tail morphology, decreased ATP concentration, and mitochondrial alterations (Marin-Guzman et al., 2000).

1.6. Mineral-nutrient interactions

1.6.1 Interactions involving trace minerals There are numerous interactions that occur between trace minerals and other nutrients. Some of these interactions are synergistic and positive, whereas others are antagonistic. Figure 1.4 illustrates the most common interactions involving trace minerals.

Phytic acid - P Fe Mn

Ca Vitamin E

Zn Cu Se

Mo S

Figure 1.4 Interactions involving trace minerals. (Adapted from O’Dell, 1997).

The most common and complex interaction is the 3-way interaction involving Cu, Fe, and Zn. Metallothionein binds both Zn and Cu, so saturation of the metalloprotein by one of these minerals can reduce absorption and retention of the other mineral in the mucosal and hepatic cells (Harris, 1997). Zinc induces metallothionein synthesis more so than Cu (Oestreicher and Cousins, 1985), but metallothionein has a higher affinity for the Cu ion. Therefore with a high Zn intake metallothionein content would increase, but would preferentially bind Cu and prevent its release from the erythrocyte. The reduction in Cu status by high dietary levels of Zn has been confirmed in young pigs (Hill et al., 1983d; Adeola et al., 1995) and sows (Hill et al., 1983b). Pharmacological Zn levels (3000 ppm) were shown to increase Zn-metallothionein concentrations in the hepatocyte and enterocyte (Carlson et al., 1999). Kidney Cu concentrations are also higher when pharmacological levels of Zn are fed suggesting that urinary conservation of Cu was occurring (Carlson et al., 1999). Veum and coworkers (2004) demonstrated that high Cu intake decreased Zn retention in pigs. Iron

32 metabolism is affected because ceruloplasmin’s ferroxidase activity is required to catalyze the conversion of Fe in liver ferritin from the ferrous state to the ferric state (Baynes and Stipanuk, 2000). This diminishes the ability of Fe to bind to tranferrin and be transported for utilization in hemoglobin synthesis. Therefore during a Cu deficiency, Fe is sequestered in the liver, even though liver Fe content is increased (Hill et al., 1983c; 1983d). Conversely, high Cu may decrease liver Fe concentrations (Bradley et al., 1983) and cause anemia in pigs (Gipp et al., 1973). High Fe appears to also affect Cu status in ruminants (Spears, 2003). High dietary levels of Mn can reduce Fe absorption while an Fe deficiency enhances Mn absorption (Leach and Harris, 1997). Excess Fe does not seem to affect Mn absorption (Baker, 2001). This interaction between Fe and Mn is believed to be related to shared absorption and transport pathways (Leach and Harris, 1997). Dietary fiber has been known to affect mineral absorption from the gastrointestinal tract. However, it may be more appropriate to identify the phytate associated with fiber as the more important antinutritional factor. Phytate is a strong acid and produces insoluble salts with metals at physiological pH. Zinc and Cu seem to have a high affinity to form insoluble complexes (Reddy et al., 1989), though Fe (O’Dell, 1997) and Mn (Halpin et al., 1986) also interact with phytate. Some researchers have suggested that phytate may increase Cu absorption by removing the antagonist effects of Zn (Lee et al., 1988). Non-ruminants lack phytase, whereas in the rumen the bacteria produce phytase (Yanke et al., 1998). Adding phytase to pig diets has been shown to increase the absorption and tissue retention of Ca, P, Zn, and Cu (Adeola et al., 1995), hemoglobin concentration, and packed cell volume (Stahl et al., 1999). Dietary Ca and P are believed to reduce the absorption of Cu, Zn, and Fe in non- ruminants by facilitating the formation of insoluble complexes with phytate. Several studies have reported reduced absorption of Zn (Morris and Ellis, 1980), Fe (O’Dell, 1997), and Mn (Wedekind and Baker, 1990a) independent of phytate, whereas others have not (Lonnerdal, 2000). It is possible that the negatively charged P will react with cations (Cu, Fe, and Zn) to form insoluble phosphate complexes making them less available. Additionally Ca, a cation, may compete with other cations for negatively charged ligands. Selenium and vitamin E have a mutual sparing effect because they partially share antioxidant roles. Selenium is a constituent of glutathione peroxidase which acts in the cytosol to destroy peroxides before they react with the membrane, whereas vitamin E acts as a fat

33 soluble antioxidant within the membrane to prevent fatty acid hydroperoxide formation (Levander, 1987). Due to these common biological functions and shared intimacy in the cell, deficiency of each nutrient can result in similar clinical deficiency signs. Selenium and S also have chemical similarities which allow them to replace one another in animal and plant proteins. Selenium replaces S in methionine and cysteine to form the Se-containing analogs, selenomethionine and selenocysteine. The multiple interactions of Cu-Mo-S are important in ruminants, but appear to be of little significance in non-ruminants. Copper precipitates with sulphide in the rumen, preventing its absorption (Bird, 1970). Molybdenum seems to exacerbate this precipitation with the formation of thiomolybdates in the rumen which also bind Cu (Dick et al., 1975). Proteins and amino acids aid in absorption by acting as ligands and preventing chelation with phytate. Ascorbic acid has been shown to decrease Cu absorption and its utilization in young chicks (Aoyagi and Baker, 1994), but it increases Fe absorption by maintaining Fe in a soluble state (Monsen, 1988). Additionally, minerals may also interact with fatty acids to form insoluble soaps, thus reducing the absorption of the mineral.

1.6.2 Interactions in the intestinal lumen The solubility of most minerals in an aqueous solution is pH dependent. Hydrochloric acid in the stomach increases the solubility of most trace minerals, however at the higher pH of the intestine, solubility is decreased. Soluble metal ions are free to bind to ligands, which will then allow them to remain soluble in the intestine. If metal ions fail to interact with these ligands, water molecules may coordinate around it at a neutral pH to form an insoluble hydroxyl-metal species (Whitehead et al., 1996) or form insoluble salts with phytate. The primary ligands with which metal ions interact include dietary components, such as amino acids and carbohydrates, or endogenous ligands, such as pancreatic and bilary secretions and mucin (Whitehead et al., 1996). Mucin is a large glycoprotein secreted throughout the digestive tract that aids in nutrient absorption (Powell et al., 1999). The metal ions then can bind to and cross the pH stable mucosally-adherent mucus layer of the intestine (Powell et al., 1999). The valence of the ion seems to be important, whereby the pattern of efficiency is M+> M2+ > M3+, where M represents the ionic state of the metal (Whitehead et al., 1996). For example Zn2+ crosses more effectively then Zn3+. The acidic environment of the luminal basal membrane allows the metal to disassociate from its ligand.

When metal ions reach the epithelial cell, uptake is likely mediated by a mucosal transport system. A divalent cation transporter utilizing a proton-coupled process has been identified that may be involved in Cu2+, Mn2+, and Zn2+ absorption (Gunshin et al., 1997).

1.7. Mineral nutrition requirements Mineral recommendations for reproducing females have remained essentially unchanged since 1973 (Table 1.7) and for some minerals, like iodine, requirements were extrapolated from experiments with growing pigs (NRC, 1998). Few experiments have recently been conducted to evaluate the reliability of these mineral requirements for sows. For example, Mn was the only trace mineral requirement to change from 1988 to 1998 (Table 1.7). This change was based on two abstracts (Christianson et al., 1989; 1990) that were presented in the interim, which added to essentially one previous experiment in sows (Plumlee et al., 1956). Sow nutrient demands have increased during the past 30 years with genetic selection resulting in larger litters and increased milk production. Furthermore, sows are currently managed under more intensive systems (i.e. indoor production and < 21d lactation) and are retained in the herd for longer periods. It has been determined that reductions in sow body mineral reserves intensify with higher productivity (Mahan and Newton, 1995). Lactation feed intakes are often low, resulting in the requirement for more nutrient dense diets. Faced with these challenges swine nutritionists frequently formulate sow diets with macro- and trace mineral levels which exceed NRC (1998) recommendations. Dietary requirements are estimates of the minimum nutrients required to support normal metabolic functions or at the very least prevent deficiency signs. Interactions involving trace minerals complicate their determination (Sect. 1.6). Lillie and Frobish (1978) reported no effect on total or live pigs born over 4 parities when sows were fed diets containing 100 or 200 ppm Fe and either 0, 15, 30, or 60 ppm Cu. However, few other experiments have evaluated reproductive responses to multiple trace minerals fed at different dietary levels. Further complicating the matter, trace mineral sources vary in their bioavailability. However, it may be possible to achieve maximum reproductive performance at lower dietary levels when sources with higher bioavailabilities are fed. Because less trace minerals could be fed, the potential for the formation of insoluble complexes between trace minerals and other

35 minerals within the lumen of the gastrointestinal tract may be reduced. Consequently, the availability of all minerals may be improved, and therefore the ratio of all dietary minerals needs to be examined. The last NRC publication (1998) did not establish a Cr requirement for swine. However, recent work by Lindemann’s Lab at the University of Kentucky suggests that feeding 0.2 ppm of Cr picolinate (1995) or Cr tripicolinate (2004) increases litter size.

NRC Mineral 1973 1979 1988 1998 Calcium, % 0.75 0.75 0.75 0.75 Phosphorus, total % 0.50 0.50 0.60 0.60 Phosphorus, avail % . . 0.35 0.35 Cu, mg/kg 6 5 5 5 Fe, mg/kg 80 80 80 80 Mn, mg/kg 20 10 10 20 Se, mg/kg 0.10 0.15 0.15 0.15 Zn, mg/kg 50 50 50 50

Table 1.7. Historical NRC mineral recommendations for reproducing sows

1.8. Supplemental dietary trace mineral forms Oftentimes the indigenous trace minerals present in most cereal grains or their by- products are either inadequate or they are rendered unavailable by other dietary factors such as phytate. Diets are frequently supplemented with trace minerals in order to meet the animal’s dietary requirement to avoid a deficiency.

1.8.1 Inorganic salts Bioavailability estimates are expressed as a percentage of a recognized standard and are not the actual quantity absorbed or retained. In addition to source, mineral bioavailability is affected by other factors including dietary phytate content, mineral status, and physiological state of the animal. Few experiments have evaluated the bioavailability of trace minerals in sows and most of the studies conducted used younger swine. Sow physiology is different in regards to transit time within the gut and length of the digestive tract. Sulfate salts are generally the most commonly used sources for most trace minerals for non-ruminants because they are considered to be the most bioavailable forms. They are also typically the standard by which other forms are compared. Conversely, oxide mineral sources have the poorest utilization (except for Mg). Cupric sulfate and tribasic Cu chloride are well utilized in pigs (Cromwell et al., 1998). The availability of Cu oxide salts is lower (Cromwell et al., 1989), while Cu sulfide is almost completely unavailable to the pig (Cromwell et al., 1978). Copper carbonate is intermediate (Baker and Ammerman, 1995). Due to the prevalence of Fe deficiency in humans, a large body of data on the bioavailability of different Fe sources using chicks and rats as models has been compiled. Blood hemoglobin content, especially after Fe depletion, is the typical bioavailability response criteria

(Henry and Miller, 1995). Ferrous sulfate heptahydrate (FeSO4 • 7H2O) and ferrous sulfate

(FeSO4 • H2O) are considered the standards upon which comparisons are made. In pigs, ferric citrate and Fe choline citrate are essentially equal in their bioavailability to the Fe sulfate sources (Baker, 2001) and ferric oxide is almost completely unavailable (Henry and Miller, 1995). The availability of Zn from oxide salts are low compared to sulfates (Sandovol et al., 1997; Edwards and Baker, 1999). In chicks, Zn oxide is only 61% available compared to Zn

37 sulfate when weight gain was used and 44% when using tibia Zn content (Wedekind and Baker, 1990b). The low availability of Zn oxide may allow higher levels (2000 to 3000 mg Zn/kg) to be included in nursery diets for growth promotion, without inducing toxic effects. The majority of experiments regarding Mn bioavailability have utilized chicks. Manganese chloride is nearly equal to Mn sulfate (Southern and Baker, 1983b). Manganese monoxide is 75% as available as Mn sulfate (Ammerman et al., 1998), while Mn carbonate and Mn dioxide are utilized less effectively (Henry, 1995). Only sodium selenite and sodium selenate are approved as the inorganic sources of Se for livestock in the United States (FDA, 1987) and are considered to have high availability. Sodium selenite is more commonly used due to its lower cost (Mahan, 2001). Although organic Se is efficiently absorbed, it is retained more in protein tissue and is more readily incorporated into milk protein. Both inorganic and organic Se efficiently produce glutathione peroxidase. Thus the bioavailability of Se clearly depends upon biological function ascribed and the physiological state of the animal. Chromium has not typically been supplemented in the inorganic form. Chromium absorption from the oxide source is considered to be 0%. Chromium oxide is commonly used as an indigestible marker in digestibility experiments. Limestone and dicalcium phosphate, added as Ca and P sources, also contain appreciable amounts of trace minerals (Baker, 2001). These trace minerals are often in forms which are not highly available for absorption. Dietary contaminants from soil, rust, and galvanized steel contribute Fe and Zn, usually in an oxide form, and are relatively unavailable.

1.8.2 Organic sources

1.8.2.1 Bioavailability Organically bound minerals (“organic minerals”) have been developed in recent years as dietary alternatives to traditional inorganic sources and are termed metal- chelates, complexes, or proteinates. In 1997 the Association of American Feed Control Officials (AAFCO) developed definitions (Table. 1.8) for organically bound mineral compounds (Ammerman et al., 1998). These classes of organic trace minerals differ by type and specificity of the nonmetal ligands and the method of binding.

1. Metal Amino Acid Chelates: Product resulting from the reaction of a soluble metal salt with amino acids with a molar ratio of one mole of metal to one to three moles (preferably two) of amino acids to form coordinate covalent bonds. The average molecular weight of the hydrolyzed amino acids must be approximately 150 and the resulting molecular weight of the chelate must not exceed 800. 2. Metal Amino Acid Complex: Product resulting from complexing a soluble metal salt with an amino acid(s) 3. Metal (Specific amino acid) Complex: Product resulting from complexing a soluble metal salt with a specific amino acid

4. Metal Proteinate: Product resulting from the chelation of soluble salt with amino acids and/or partially hydrolyzed protein. 5. Metal Polysaccharide Complex: Product resulting from complexing a soluble salt with a polysaccharide solution declared as an ingredient as the specific metal complex

Table 1.8. AAFCO definitions for organically bound mineral compounds (Ammerman et al., 1998).

The premise for using organic trace minerals in animal diets and replacing traditional inorganic sources, is that their bioavailability is greater because they remain stable in the digestive tract and do not form insoluble chelates with other dietary components, like phytate. It has been proposed that absorption from the intestinal lumen could be accomplished using other mechanisms in addition to those for metal ions (Ashmead, 1993; Du et al., 1996). Although one proposed mechanism is that mineral-ligand chelates are absorbed intact by utilizing the uptake mechanism of the ligand, like an amino acid or peptide transporter (Ashmead, 1993), this theory has yet to be proven. Similar to inorganic sources, bioavailability is different between the various organic sources of a given trace mineral. An important aspect for physiological function is the degree to which the metal binds with the organic ligand under physiological conditions. The bond between the metal ion and ligand must be strong enough to withstand the low pH of the stomach, but not too strong to prevent detachment before absorption. This is assuming that an alternative absorption pathway is not utilized, in which case disassociation would be detrimental. However, definitive tests for measuring the strength of metal-ligand bond have yet 39 to be developed. Cao and coworkers (2000) reported a high negative correlation between Zn bioavailability and organic Zn solubility in a buffer solution at pH 5. In another study by these same researchers (Guo et al., 2001), the solubility of organic Cu in a pH 2 buffer was shown to be the best predictor of bioavailability. Additionally, all organic Cu sources were highly soluble at pH 2 and 5 and the relationship between Cu solubility and bioavailability was positive. While these researchers found differences in chelation and solubility characteristics of the organic trace mineral sources using several other indicators, they proved to be poor predictors of mineral bioavailability. This demonstrates the difficulty of using chemical methods to predict bioavailability and that the use of these methods is inconsistent between minerals Organic Zn sources have received more attention than other trace minerals, however with variable results. Wedekind and coworkers (1992) demonstrated that Zn-methionine bioavailability in chicks was increased as compared to Zn sulfate when phytate content of the diet increased. Studies comparing organic Zn sources to Zn sulfate in pigs have indicated higher (Matsui et al., 1996) or equal (Hill et al., 1986; Swinkels et al., 1996; Cheng et al., 1998) bioavailabilities when growth performance and/or tissue concentrations were the response criterion. Lower organic Zn bioavailability has also been reported in pigs; however pharmacological levels were fed, complicating the interpretation (Schell and Kornegay, 1996). Furthermore, in a study in which four Zn complexes and four Zn chelates were evaluated in chicks and lambs, only one of the Zn proteinates had a bioavailability greater than Zn sulfate, whereas the remaining seven were considered to be equal to Zn sulfate (Cao et al., 2000). The results of the few experiments evaluating organic Cu are also inconsistent. In some studies with pigs, Cu-Lysine improved growth performance as compared to Cu sulfate, however liver Cu contents were not effected (Coffey et al., 1994; Zhou et al., 1994). In another experiment with pigs, Cu-lysine was not different from Cu sulfate when Cu absorption and retention were measured (Apgar and Kornegay, 1996). Copper absorption and retention was greater in weanling pigs when 100 ppm of Cu from a Cu-proteinate was fed, compared to when 250 ppm of Cu sulfate (Veum et al., 2004). In a study using rats, liver Cu levels were higher when Cu-Lysine and Cu-proteinate were fed (Du et al., 1996). Liver Fe and Zn were also higher in this study when the organic sources were fed, suggesting that Cu interference with Fe and Zn was reduced. In cattle, Cu and Zn proteinates failed to prevent the adverse effects of high Fe diets (Mullis et al., 2003). Copper status was reduced in all animals and liver concentrations of Cu, Zn, and Fe were similar between organic and inorganic treatments.

Several experiments have evaluated organic Mn in chicks, due to the greater importance of Mn in poultry nutrition for preventing leg abnormalities and thin egg shells, whereas there have been few studies in swine. Using cardiac Mn-SOD as an assessment of bioavailability in broilers, Li and coworkers (2004) reported that the chelation strength of Mn-methionine affects bioavailability. Sources with moderate and high chelation strength are more bioavailable than Mn sulfate. Furthermore, Mn methionine was more effective when higher dietary Ca levels were fed. Manganese proteinate appears to be more available than Mn sulfate when bile and bone concentrations of chicks were used as response criterion (Baker and Halpin, 1987). If organic minerals do actually take advantage of alternative absorption pathways, organic Fe would be a logical candidate for utilization, because Fe absorption is strictly regulated at the intestinal mucosa. Feeding 300 mg/day of Fe-proteinate or 650 mg/day of glutamic acid-chelated iron for three weeks prior to farrowing failed to increase hemoglobin content or packed cell volume in neonatal pigs. Yu and coworkers (2000) reported that nursery pigs fed a Fe-amino acid complex had higher packed cell volume (+9%) and hemoglobin concentrations (+14%) after 5 weeks than those fed Fe sulfate, suggesting a higher bioavailability of the chelate. Organic Se differs significantly from other trace minerals because Se will not artificially complex with ligands due to its electron chemistry. Selenium-enriched yeast is produced by cultivating yeast (Sarccharomyces cerevisiae) in a sulfur-deficient cane molasses media, so that Se will be incorporated into cellular proteins as selenomethionine (FDA, 2003). Residual Se salts are removed from the yeast with repeated washing. This is the only Se yeast (Selplex, Alltech, Inc) approved for use in swine, poultry, and cattle diets in the U.S. (FDA, 2003). Selenium yeast contains a minimum of 40% selenomethionine (Kelly and Power, 1995). Selenium-enriched yeast has been demonstrated to increase tissue and milk Se concentrations in pigs and cattle (Mahan and Parrett, 1996; Mahan, 2000; Givens et al., 2004. Yet, inorganic Se (sodium selenite) seems to be more effective at enhancing glutathione peroxidase production and activity at lower dietary levels than Se yeast (Mahan and Parrett, 1996; Mahan et al., 1999; Mahan, 2000). Although it is likely that selenomethionine is incorporated directly into tissue proteins before having the opportunity to have metabolic activity, it is possible that the conversion from selenomethionine to selenocysteine for incorporation into glutathione peroxidase may be less efficient than Se derived from inorganic sources.

Several organic Cr sources are available, including Cr picolinate, Cr tripicolinate, Cr nicotinate, Cr propionate, and Cr yeast. Few experiments exist where two or more Cr sources are fed at similar concentrations in order to evaluate availability.

1.8.2.2 Organic trace minerals and reproduction Researchers at Washington State University conducted a series of experiments evaluating the partial replacement of inorganic sources of Cu, Mn, and Zn with proteinate sources. Multiparous sows with a history of poor reproductive performance (< 10 pigs born) were fed a control diet with Zn oxide, Mn sulfate, and Cu sulfate, or a treatment in which 25% of the inorganic minerals were replaced by mineral proteinates during one lactation through 30 days postcoitum of the subsequent reproductive cycle (Mirando et al., 1993). There was no effect of the organic trace minerals on lactation performance, however more sows conceived when fed the organic trace minerals. Furthermore, there were more live fetuses and fewer dead embryos when the organic source was fed. The number of corpora lutea were similar between treatments, suggesting that the organic minerals improved embryo and or fetal survival. In another experiment, these treatments were fed to gilts beginning at 105 days of age through day 15 of pregnancy (Hostetler and Mirando, 1998). Gilts fed the organic treatment reached puberty 13 days sooner than the inorganic treatment, although pregnancy and ovulation rates were not effected. Finally, gilts fed Cu, Mn, and Zn proteinates had higher concentrations of these minerals in the conceptus products at day 12 postcoitum and higher Cu at day 30 (Hostetler et al., 2000). This suggests that the organic trace minerals have higher bioavailabilities and appear more able to meet mineral requirements for reproduction during early pregnancy. Flowers and coworker (2001) fed Cu, Fe, Mn, and Zn proteinates to reproducing females over three parities. The control diet contained inorganic trace minerals at higher industry levels. Two other treatments included the trace mineral at 25% of the control diet (below NRC), with one consisting entirely of inorganic source and the other provided as metal- proteinates. The number of pigs born live and weaned were increased when sows were fed the reduced level of the inorganic trace minerals compared to the control, however the organic treatment was similar to the control. Despite weaning fewer pigs than the reduced inorganic treatment, sows fed the reduced organic treatment had heavier litter weaning weights. Selenium yeast has demonstrated more consistent effects in reproducing females than the other “organic” trace minerals. There was no effect of feeding organic Se on total or live

42 pigs born, however there were more stillborn pigs when inorganic Se was fed (Mahan and Peters, 2000). The major benefit of feeding organic Se seems to be the greater transfer of Se to the progeny. Neonatal pigs from sows fed organic Se are born with greater liver Se content then when sows are fed inorganic sources (Mahan and Kim, 1996; Mahan and Peters, 2000). Several experiments have reported higher Se contents in colostrum and milk with organic Se supplementation (Mahan and Kim, 1996; Mahan, 2000; Mahan and Peters, 2004). This is likely related to selenomethionine being directly incorporated into the milk proteins. The effectiveness of Se incorporation into milk secretions is further demonstrated by the feeding Se yeast for only six days prior to parturition increasing colostrum Se content (Mahan, 2000). The greater transfer of Se in the milk results in pigs having greater liver Se concentrations at weaning (Mahan and Kim, 1996).

1.9 Research objectives and rationale Although energy and amino acid recommendations for reproducing sows have increased over past decades, NRC (1998) mineral recommendations have remained essentially unchanged. Low conception rates and high culling rates (> 40%) remain persistent problems for swine producers. Although this poor performance might be linked to several factors, trace minerals may be involved in this malady. Sow body mineral reserves decline over several reproductive cycles and larger litter growth rates exacerbate this depletion (Mahan and Newton, 1995). In order to compensate for the greater needs of high producing sows, gestation and lactation trace mineral levels are commonly formulated to exceed NRC (1998) mineral recommendations. However, the efficacy of these higher levels has not been evaluated. The research to determine mineral recommendations was largely conducted using inorganic mineral sources. However, several organic trace minerals are available, but their utility in sow diets has not been widely investigated. Organic trace minerals are expected to remain bound to a ligand in the digestive tract, preventing the formation of insoluble chelates with other minerals and nutrients. Consequently, the increased mineral bioavailability would improve mineral retention in tissues, even at lower supplementation levels. The objectives of the experiments in this dissertation are to evaluate sow reproductive performance and estimate the changes in sow mineral reserves over a six parity period when diets contain either inorganic or organic sources of trace minerals. In addition, both sources are fed at or above NRC (1998) recommended levels. The effects of feeding the higher trace mineral levels provided in conjunction with higher Ca and P during the reproductive cycle are also evaluated. The hypothesis is that organic trace minerals may reduce the depletion of sow mineral reserves and improve reproductive performance by reducing the interactions between trace minerals and other dietary nutrients which reduce the availability of trace minerals. These interactions may be intensified at higher dietary trace mineral levels and especially when high Ca and P are fed.

CHAPTER 2

EFFECTS OF DIETARY ORGANIC AND INORGANIC TRACE MINERALS AT VARIOUS LEVELS IN REPRODUCING SOWS OVER SIX PARITIES

2.1 Abstract

The effects of dietary trace mineral source and levels on sow reproductive performance were evaluated over six parities using a total of 375 farrowings. The experiment was a 2 x 3 factorial, conducted in a randomized complete block design. The first factor evaluated organic and inorganic trace mineral sources (Cu, Fe, Mn, Se, and Zn) fed to developing gilts and reproducing sows. The second factor evaluated dietary mineral levels, with one at NRC recommendations (NRC) and the second reflected normal industry recommendations (Ind.). During the development phase, gilts were fed one of the four dietary treatments. However, at the initial breeding through parity six, two treatments were included that evaluated the effects of adding higher levels of Ca and P to the Ind. trace mineral level (Ind+Ca/P). Litters were equalized within 3 d postpartum. There were no effects (P > 0.10) of trace mineral source or level on gains, daily feed intakes, or gain:feed ratio from 30 to 110 kg BW. During the reproductive phase, sows fed the organic trace mineral source farrowed more (P < 0.05) total (12.2 vs. 11.3) and live pigs (11.3 vs. 10.6). Although litter weights were heavier (P < 0.05) at birth when sows were fed the organic source, individual pig weights were similar for the various treatment groups. Sows fed the Ind+Ca/P level tended to have fewer (P < 0.10) total pigs born and lower (P < 0.05) litter birth weights. There were more stillbirths (P < 0.10) and mummies (P < 0.05) when sows were fed the NRC level. Nursing pig daily gains tended to be greater (P < 0.10) when sows were fed the organic trace mineral source. Sow lactation feed and mineral intakes, litter size at birth, and litter growth rates increased in a curvilinear manner (P < 0.01) as parity advanced. These results suggest that feeding sows the organic trace mineral source

45 increased sow reproductive performance, but not sow weight gains or feed intakes. There were no improvements in reproductive performance over six parities when elevated dietary levels (Ind. and Ind+Ca/P) were fed. 2.2 Introduction Despite increasing energy and amino acid recommendations for reproducing sows over the past decade, low conception rates and high culling rates continue to persist in most swine herds. Although inadequate energy and amino acid nutrition has been associated with these maladies, poor reproductive performance might also be linked to other factors. For example, sow mineral reserves, particularly Ca and P, decline after several reproductive cycles and their depletion can be exacerbated when sows produce larger litter growth rates (Mahan and Newton, 1995). The NRC (1979; 1988; 1998) mineral recommendations have remained essentially unchanged even with increased sow productivities and greater mineral needs. As a result, dietary levels of both macro and trace minerals for sows are generally elevated beyond current NRC (1998) recommendations. These higher levels are thought to compensate for the greater needs of high producing sows, but scientific evidence is lacking. Mineral recommendations have largely been determined using inorganic mineral sources. Interactions between inorganic trace minerals and Ca and P may occur in the lumen of the digestive tract, reducing their absorption (Morris and Ellis, 1980; O’Dell, 1997). Several trace minerals chelated with peptides, amino acids, or carbohydrates are available to the feed industry. By providing one of these organic trace minerals forms, their interactions in the intestine may be minimized because the trace minerals would not be expected to be in the free ionic form until at the site of absorption (Ammerman et al., 1998). Their effectiveness in sow nutrition has however, not been widely investigated. This experiment evaluated gilt and sow reproductive performance over six parities when diets contained either inorganic or organic sources of trace minerals provided at or above 1998 NRC recommended levels. The effects of adding elevated dietary trace mineral levels in conjunction with higher Ca and P levels were also evaluated. 2.3 Materials and Methods

2.3.1 Experimental Design and Treatments This experiment evaluated the effects of dietary trace minerals (Cu, Fe, Mn, Se, and Zn) on sow reproductive performance. The experiment was conducted as a 2 x 3 factorial arrangement of treatments in a randomized complete block (RCB) design in seven replicates.

The first factor evaluated trace mineral form (organic or inorganic) and the second factor evaluated trace mineral levels. The organic minerals (Bioplex) were provided by Alltech Inc. (Nicholasville, KY) and were metal proteinates, whereby trace minerals were chelated with partially hydrolyzed soybean protein, while the organic Se source was Se yeast (Sel-plex). Most inorganic trace minerals were provided in the sulfate form, but the Se source was sodium selenite. The first level evaluated the NRC (1998) recommendations, while the second evaluated a higher trace mineral level that was considered to be typical of university and commercial feed industry recommendations (Ind.). The dietary trace mineral levels are presented in Table 2.1. These four treatment diets contained 0.75% Ca and 0.60% P (total). A third level (Ind. + Ca/P) evaluated the effects of additional Ca and P, but only with the higher trace mineral level of each source, during the reproductive phase of the experiment. Gestation Ca and P levels in these latter treatments were 1.00% and 0.75% (total), respectively, while 1.20% Ca and 0.90% P (total) during lactation. These latter diets started at the initial breeding and continued through the six parity period. 2.3.2 Grower Phase Two hundred five gilts (Yorkshire x Landrace) were obtained (Temple Genetics, Gentryville, IN) in 7 groups at approximately 21 d of age and fed common starter diets for approximately 5 wk. At approximately 30 kg BW, the gilts were moved to grower pens and allotted to dietary treatments based on weight and ancestry. During the development phases, gilts were fed either the NRC or the Ind. level of the trace mineral sources (4 treatment diets). Because the two additional treatments were not incorporated until the initial breeding and were fed only with the higher trace mineral levels, more gilts were developed on the Ind. treatment levels. Gilts fed the NRC and Ind. level of both trace mineral source were housed in 4 total pens during the development phase. However, because greater numbers of gilts on the higher trace mineral level treatments were needed, a total of 6 pens were used in each replicate. Prior to the initial breeding, gilts fed the higher trace mineral level treatments during the development phases were randomly allotted and received either the Ind. or Ind.+Ca/P treatment diets. 2.3.3 Breeding and Postbreeding Period At 8 mo of age, an equal number of gilts from each of the 6 treatment group within each of the seven groups were bred for a total of 24 gilts. During gestation, the females were housed in individual stalls, each with a stainless-steel feeder and a nipple waterer. Gilts and sows were

47 artificially inseminated at the onset of estrus and 12 h later with semen from PIC (280 genetic line). Semen was obtained twice weekly, stored at 18º C, rotated twice daily, but used within 4 d of collection. Body weights were collected at breeding and at 110 d postcoitum. Sonoray backfat measurements using a single transducer (Lean Meater, Renco Corp., Minneapolis, MN) were determined, approximately 40 mm off the midline at the last rib at each weighing. Backfat thickness and longissimus muscle area (LMA) at the 10th rib were also determined at 30 and 90 d postcoitum using real-time ultrasound equipment (Aloka 500-V, Corometrics Medical Systems, Wallingford, CT). Sows were bred on their first estrus postweaning, with the wean-to-estrus interval and breeding weights recorded. Sows not exhibiting estrus within 10 d of weaning were placed in the next breeding group and if they failed to cycle in this subsequent breeding group (45 d from initial weaning) they were removed from the study. Sows that were determined not pregnant by ultrasound examination after 30 d postcoitum were removed from the experiment. Pre-selected sows from each treatment group were terminated after weaning at parities 1, 2, 4, and 6, where body composition was evaluated. Consequently, the number of litters in the subsequent parities does not reflect sow longevity. Upon weaning, the sows were moved to individual gestation crates and fed their gestation treatment diet at 1.5 times their gestation feeding level until bred. Upon breeding, their gestation treatment diet was provided at 1.8 kg/d (5890 kcal/d) for 30 d and increased for the remainder of pregnancy. Gestation feed intake from d 30 to farrowing was 2 kg/d (6540 kcal/d) for parity 1 gilts and increased by 135 g/d (440 kcal/d) for each subsequent parity to parity 4. Because of declining body condition after parity 4, the gestation ration was increased by 270 g/d (880 kcal/d) during parities 5 and 6 in all treatment groups. 2.3.4 Farrowing and Lactation Period Sows were moved to individual farrowing crates at d 110 of gestation and lactation diets were fed at the same quantity provided during gestation. Upon parturition sows were fed their treatment diet at 2.0 kg on d 1, then cumulatively increased by 2.0 kg/d, such that by d 3 sows were provided feed on an ad libitum basis. Within 12 h postpartum sows were weighed, sonoray backfat thickness (last rib) measured, and their litters processed (i.e., pigs weighed, ear- notched, teeth clipped, and injected with 200 mg of Fe (iron dextran). Within 3 d postpartum litter size was equalized across treatments. It was assumed that sow milk production was not influenced greatly by the trace mineral level fed and that

48 utilization of labile tissue mineral reserves would be more uniform across treatment groups if litter size was standardized. Sow weights, feed intakes, and litters weights were measured at d 7 postpartum and at weaning (17 d). Sow sonoray backfat thickness (last rib) and 10th rib LMA and backfat thickness were determined at weaning using real-time ultrasound. Milk was collected from all functional glands (30 to 50 ml) at weaning after an i.m. injection (40 U.S.P. units) of oxytocin. Milks were frozen and stored at -4º C for later analysis. 2.3.5 Diet Composition Corn-soybean meal (C-SBM) mixtures were fed during the 30 to 55, 55 to 85, and 85 to 110 kg BW growth phases and provided dietary lysine (total) levels of 1.30, 1.10, and 0.90%, respectively (Table 2.2). The latter diet was also used to limit-feed gilts at 1.8 kg/d from 110 kg BW to initial breeding. To allow for continued muscle development of younger reproducing females, gestation diets during parity 1 and 2 were formulated to 0.75% lysine (total), but during parity 3 to 6 the C-SBM mixture gestation diet was formulated to 0.53% lysine (total). Lactation diets were C- SBM mixtures containing 5% added fat and were formulated to 1.00 % lysine (total). The NRC and Ind. trace mineral level treatments of both sources were formulated to contain 0.75% Ca and 0.60% P (total) during both gestation and lactation, but was increased to 1.00% Ca and 0.75% P (total) during gestation and 1.20% Ca and 0.90% P (total) during lactation for the Ind.+Ca/P treatment groups. All diets were fortified with vitamins that met or exceeded NRC (1998) nutrient recommendations. The percentage compositions of the gestation and lactation basal diets are presented in Table 2.3. 2.3.6 Mineral Intake Daily intakes of supplemental trace minerals and macro-minerals were calculated for each gestation and lactation period. The calculated dietary concentration of each mineral was multiplied by the sow’s average daily feed intake for each reproductive phase. Mineral intakes were expressed on a total daily intake and on a mg per kg sow BW basis. Breeding and farrowing weights were used for calculating average gestation weight, while lactation weight was the average of the farrowing and weaning weights. 2.3.7 Analytical Methods Premixes and composites of the diets were analyzed for the major elements (Cu, Fe, Mn, Zn, Ca, P, Mg, K, S, and Na) using the Inductively Coupled Plasma method (PS 3000, Leeman Labs, Inc. Hudson, NH). Chlorides were analyzed by using the chloride ion

49 electrode method of LaCroix et al. (1970). Selenium samples were wet ashed in nitric and perchloric acid and analyzed using the fluorometric method outlined by AOAC (2000). Milk from all weaned sows was analyzed for fat content using the Babcock method (AOAC, 2000). Data for the development phase were analyzed as a RCB design in seven replicates using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). Within each replicate, the two treatment pens fed the high trace mineral level were averaged. Because only the NRC and Ind. levels of each trace mineral source were fed during gilt development, the data were analyzed as a 2 x 2 factorial arrangement of treatments. The model included the effects of trace mineral source and level, blocks (replicates), and treatment x block (error). Block was considered the random effect. Pen was the experimental unit for analysis of the performance data. The reproductive and mineral intake data were analyzed as a 2 x 3 factorial in a RCB design in seven replicates using the MIXED procedure of SAS. The data were analyzed according to the following model:

Y ijklm = µ + Si + Lj + SLij+ Pk + SPik + LPjk + sl + gm + gSLijm + eijklm where Y ijklm is the dependent, continuous variable, µ is the overall mean, Si is the fixed effect of the ith trace mineral source (i = 1,2), Lj is the fixed effect of the jth trace mineral level (j =

1,2,3), SLij is the fixed effect of the ith trace mineral source by the jth trace mineral level interaction, Pk is the fixed effect of the kth parity (k = 1,…,6), SPik is the fixed effect of the ith trace mineral source by kth parity interaction, LPjk is the fixed effect of the jth trace mineral level by kth parity interaction, sl is the random effect of the lth sow, gm is the random effect the mth block (m = 1,…..,7), g(SL)ijm is the random effect of the mth block by the ith trace mineral source by the jth trace mineral level interaction, and eijklm is the residual error. The block x treatment interaction was included because sows from specified replicates were killed after a predetermined number of litters (i.e. 1, 2, 4, 6), therefore number of observations were not equal between replicates. Repeated measures were also included in the analysis. The subject of the repeated measure was individual sow nested within treatment and the first-order autoregressive covariance structure was used. This structure consistently gave the lowest Bayesian information criteria for the covariance structures tested. Individual sow and litter measurements were considered the experimental units. Parity effects were partitioned into linear and curvilinear components using orthogonal polynomial contrasts. A P value of ≤ 0.05 was considered significant and a P < 0.10 was considered a trend. Least square treatment means are presented in tabular form.

2.4 Results Analyzed trace mineral concentrations of the premixes were similar to the calculated values (Table 2.1). Mineral contents of the complete diets were slightly higher than the targeted values (data not shown), likely due to the mineral contributions from other dietary constituents or contaminants. Iron content was particularly higher and likely reflected the Fe contributed by the supplemental macro mineral sources (limestone and dicalcium phosphate) and environmental contaminates (feed and mixing equipment, stalls, floor, water, etc.). 2.4.1 Reproductive Performance There were no effects of trace mineral source or level on gilt weights, gains, daily feed intake, or gain:feed ratios during the development period of 30 to 110 kg BW (Table 2.4). The effect of trace mineral source and level on reproductive performance is presented in Table 2.5. The number of pigs born (total and live) at each trace mineral level was greater (P < 0.05) when sows were fed the organic trace mineral source. The highest numbers of total and live pigs born were from those sows fed the organic trace mineral source at the NRC and Ind. level. There were fewer total and live pigs born when sows were fed the Ind.+Ca/P trace mineral level for both trace mineral sources, but the interaction was not significant. The number of stillbirths (P < 0.10) and mummies (P < 0.05) were lower as mineral levels of both sources increased, but the interaction was not significant. In general, a decline in total and live pigs born occurred when the inorganic trace mineral source was fed and was exacerbated as the mineral level increased, but the response was more stable between levels when the organic mineral source was fed. Because more pigs were born when sows were fed organic trace minerals, litter birth weights were heavier (P < 0.05) but not when expressed on an individual pig weight basis. Sows fed the Ind.+Ca/P level of both sources had the lightest (P < 0.05) litter birth weights, but not individual pig weights, again the measurements probably reflecting smaller litters sizes at birth. Pig birth weights were heavier (P < 0.05) when sows were fed the Ind. level treatments, particularly for those fed the inorganic trace mineral source, but the source x level interaction response was not significant (P > 0.10). Because litter size was standardized within 3 d postpartum, the numbers of pigs at d 7 and at weaning (17 d) were similar. Both litter and pig weights at weaning (17 d) did appear to be somewhat greater when sows were fed organic trace minerals, but the response was not significant. However, daily gains of nursing pigs tended to be greater (P < 0.10) when sows

51 were fed the organic trace mineral source. Although the reason for these higher pig gains may reflect greater sow milk productions, it may also reflect other benefits of the organic minerals. There were no trace mineral source x level interactions for pig and litter weaning weights or daily gain criteria.

2.4.2 Gilt and Sow Weight, Feed Intake, and Backfat Thickness Sow weights, gains, weaning-to-estrus intervals, milk composition, and backfat thicknesses are presented in Table 2.6. There was no effects of mineral source on breeding or 110 d weights, or gestation gains (P > 0.10), but there was an effect of dietary trace mineral level (P < 0.05) on these responses. Most of this was due to the lighter weights at breeding, 110 d, and lower gestation gains of sows fed inorganic minerals at the Ind.+Ca/P levels. These sows had lighter breeding weights, gestation gains, but there was no effect on lactation feed intake (total or ADFI), estrus intervals, or milk fat content. This resulted in a breeding weight interaction response (P < 0.05), whereas there was no other significant interaction response. These results suggest that trace mineral source did not have an apparent effect on lactation feed intakes (total or ADFI), milk fat composition, or return to estrus interval, but when high levels of Ca and P were fed with the inorganic trace mineral source, lower sow performances tended to result. Sonoray and ultrasound backfat thicknesses at the various measurement periods were greater when sows were fed the inorganic trace mineral source at the NRC and Ind. trace mineral levels, but were similar for both trace mineral sources at the Ind.+Ca/P treatment level. This resulted in a trace mineral source x level interaction response (P < 0.05). Backfat measurements at the 10th rib and last rib demonstrated that both measurements increased from breeding to the end of gestation, but declined at weaning, suggesting that sows mobilized fat reserves during lactation. As expected, Sonoray backfat thickness measurements (last rib) were approximately 65% lower than the real-time ultrasound measurements (10th rib). Because of the fat deposition pattern in swine, backfat thicknesses are generally thinner at the last rib than at the more anterior region of the animal’s back (Tess et al., 1986). In all treatment groups, the loin muscle area (LMA) measured by real-time ultrasound at the 10th rib increased from 30 to 90 d of gestation and then declined by weaning. This suggests that muscle protein and intramuscular fat accretion were occurring during pregnancy,

52 but then one or both of these loin components were catabolized after 90 d postcoitum to weaning in order to support late fetal or milk production. Sows fed the inorganic source at the Ind. and Ind.+Ca/P levels had somewhat larger LMA at 30 d postcoitum and at weaning than sows fed the organic source suggesting that they mobilized more body protein at those levels. The results also indicated that sows fed the organic NRC treatment had larger LMA than those fed inorganic NRC treatment, resulting in a source x level interaction (P < 0.05).

2.4.3 Parity Responses The reduction in sow numbers after parity 1, 2, and 4 is also attributed to sows being removed from each treatment group to determine sow body compositions. The parity results presented in Table 2.7 demonstrated as expected that sow weights at breeding, 110 d postcoitum, gestation gain, farrowing and weaning weights increased (P < 0.01) as parity increased. Gestation weight gains were lowest (P < 0.01) during parity 3 and 4, but increased during parity 5 and 6, as were the subsequent farrowing weights. This response is largely due to the additional feed (energy intake) fed during gestation. Sows lost more body weight during lactation 1 and 2 and a minimal amount during parity 3 to 5. There was a greater weight loss during parity 6 possibly reflecting their higher body fat accretion during gestation and subsequent lower lactation feed intake. The negative relationship between gestation feed level with lactation feed intake is well documented (Mullan and Williams, 1989; Dourmad, 1991; Weldon et al., 1994). Return-to-estrus intervals were similar at all parities. Sonoray and real-time ultrasound backfat thickness, at the various measurements periods demonstrated a decreased thickness from parity 1 to 4 and an increase thereafter resulting in a quadratic response (P < 0.01), again the latter increase is attributed largely to the increased gestation feed intake. There was a quadratic decline by parity in milk fat contents at weaning from parity 1 to 4 and an increased thereafter (P < 0.10). The corresponding decline in both milk fat and the declining backfat thickness to parity 4 suggests that body fat probably made a large contribution to milk fat content in earlier parities. Longissimus muscle area increased curvilinearly from 30 d to 90 d postcoitum (P < 0.05), but there was a large increase at weaning (P < 0.01) as parity advanced, with the greatest increase after parity 4. The large increase during the latter parities suggests that muscle catabolism was less and that possibly compensatory responses to muscle development were

53 occurring as sow productivity declined with advancing parities. The higher backfat thickness and LMA during parity 5 and 6 also reflected the higher gestation feeding levels and greater sow BW during these parities. Total pigs born increased (P < 0.05) in a curvilinear manner to parity 5, whereupon it declined at parity 6 (Table 2.8). Live pigs born also increased quadratically (P < 0.01) by parity, plateauing at parity 4, and then declined by parity 6. There was no apparent difference in the number of stillbirths or mummies by parity. Litter birth weights quadratically increased (P < 0.01) to parity 5, followed by a decrease at parity 6, the latter reflecting the smaller litter sizes. Pig birth weights increased in a curvilinear manner by parity (P < 0.05). Litter and pig weights at weaning (17 d) and litter daily gains increased in a curvilinear manner (P < 0.05), with parity 1 having the lowest pig weights and gains, parities 2 to 4 being only slightly heavier and gains being lower during parity 5 and 6. Pig daily gains increased quadratically (P < 0.01) to parity four and were lower thereafter. The lower litter and pig weaning weights and gains at parity 5 and 6 is probably attributed to reduced sow milk production resulting from the lower lactation feed intakes (King and Dunkin, 1985; 1986).

2.4.4 Sow Daily Mineral Intake Calculated daily trace mineral (mg/d) and macro-mineral (g/d) intakes during gestation and lactation are presented in Table 2.9 and on a mg/kg BW in Table 2.10. Because gestation feeding levels were equal for all treatments within each parity, there was little variation within treatment group, but the amount consumed during lactation varied by feed intakes and thus were statistically analyzed. The main effects of trace mineral intakes were similar for both sources during gestation and lactation (data not shown). The NRC model recommendations for gestation mineral intakes presented in Table 2.9 were somewhat lower than the mineral intakes for the NRC trace mineral level treatments. These differences between calculated mineral intakes for the NRC level treatments and mineral recommendation are largely attributed to gestation feed intakes being generally higher than those calculated by the NRC model, especially at later parities. However, during lactation, trace mineral intakes for the NRC treatments were lower than NRC recommendations, largely because lactation feed intakes were lower than those predicted by the NRC model. As expected, sows fed the Ind. and Ind.+Ca/P treatments

54 consumed considerably more trace minerals than the NRC gestation and lactation recommendations. Calcium and P intakes for the Ind.+Ca/P treatments were also much higher than the NRC recommendations. Sows fed the Ind. and Ind.+Ca/P treatment levels (P < 0.01) consumed more trace minerals during both gestation and lactation than sows fed the NRC level (Table 2.9). Daily intake of Ca, P, Mg, and S (g/d) were also greater (P < 0.01) during both gestation and lactation when the Ind.+Ca/P treatments were fed, whereas K, Na, and Cl intakes were similar for all levels. Gestation daily macro-mineral intakes increased with parity, except for K. Daily K intakes increased at parity 2, decreased at parity 3, and increased thereafter. Because gestation diets were formulated to a lower lysine level during parities 3 to 6, the K contributed by soybean meal was lower during these parities. Daily mineral intakes for each trace mineral level were approximately 2-fold greater during lactation than gestation, reflecting the higher lactation feed intakes (Table 2.9). During gestation, daily mineral intakes increased each parity as feeding level increased, with larger increases occurring at parity 5 and 6 due to incrementally higher feeding levels. For all macro and trace minerals levels, sow lactation mineral intakes generally increased in a quadratic manner (P < 0.01) to parity 4 and declined thereafter. The decrease in lactation trace mineral intakes at parity 6 were greater for the NRC and Ind.+Ca/P trace mineral levels then the Ind. level, which resulted in a trace mineral level x parity interaction response (P < 0.01). The trace mineral level x parity interaction response was not significant for lactation daily feed intake (P > 0.10). When expressed relative to average gestation weight, daily trace mineral intakes during both gestation and lactation were greater (P < 0.01) for the Ind. and Ind.+Ca/P treatment levels (Table 2.10). With the exception of K, gestation trace and macro-mineral intakes per kg sow BW decreased quadratically (P < 0.01) with parity, whereby these intakes were similar but lower after parity 1. This suggests that while mineral intake during gestation increased each parity, this increase was not at the same rate as sow BW increased. During lactation, mineral intakes per kg BW increased quadratically (P < 0.01) to a maximum at parity 3 for each trace mineral level. Due to the decreasing feed intakes and heavier BW at parity 5 and 6, intakes relative to sow BW were lower than at parity 1. There were no trace mineral level x parity interaction responses for trace mineral intakes expressed per kg BW during either gestation of lactation (P > 0.10).

2.5 Discussion None of the dietary treatments demonstrated any difference in performance during the grower-finisher period. Creech and coworkers (2004) reported no difference in performance during the grower-finisher period when gilts were fed diets which contained supplemental inorganic trace minerals in excess of NRC requirements and two diets which were formulated to approximately NRC recommendation levels. One lower level treatment contained inorganic trace minerals whereas the other treatment contained an equal combination of organic and inorganic trace minerals. Our results and those of Creech et al.(2004) suggest that trace mineral sources and levels fed to developing gilts do not greatly affect their growth performance. However, it would be expected that tissue mineral reserves at the beginning of reproduction would reflect the mineral level fed during gilt development (Mahan, 1990). Feeding sows the organic trace mineral source resulted in more total (12.2 vs. 11.3) and live (11.3 vs. 10.6) pigs born. Although litter sizes were larger at each dietary level when the organic source was provided, the difference seemed to be greatest at the Ind. level. For both trace mineral sources, there tended to be fewer (P < 0.10) total pigs born when the higher trace mineral level was fed in conjunction with higher Ca and P. This suggests that an interaction between trace minerals and high levels of Ca and P may have negatively affected sow reproductive performance for both trace mineral sources, though these effects were not as great when trace minerals are provided in an organic form. There were more stillbirths and mummies at the NRC level, which resulted in similar numbers of live pigs born for all levels. Pigs which nursed sows fed the organic treatments tended to have greater daily gains, suggesting that milk production may have been greater for sows fed these treatments or that there were other benefits from feeding the organic trace minerals. Limited research has previously investigated the effectiveness of organic trace minerals in reproducing sow and the mechanisms by which trace mineral sources and levels affect reproduction have yet to be identified. Selenium yeast does not appear to greatly affect the number of total pigs born, although it may reduce the number of stillbirths (Mahan and Peters, 2000). Partial replacement of inorganic sources of Cu, Mn, and Zn with their proteinate from resulted in more live fetuses and fewer dead embryos at 30 d postcoitum (Mirando et al., 1993). The number of corpora lutea were similar between treatments, suggesting that the organic minerals may have improved embryo and/or fetal survival. Higher concentrations of Cu, Mn, and Zn have been reported in the conceptus products then in the surrounding endometrial tissues

56 and ovaries between days 12 and 30 postcoitum (Hostetler et al., 2000). This suggests an increased uptake and/or utilization of trace minerals by the embryo and fetus early in pregnancy. However, it is unclear if the increased litter size in the current study resulted from differences in conceptus product trace mineral concentrations or other factors. The relationship between trace minerals and sow antioxidant status is another mechanism by which reproductive performance may be affected. Excessive free radical production is suspected to cause damage to cellular membranes and possibly result in cell death. Elevated levels of free radicals have been reported in embryos during Cu (Hawk et al., 2003) and Se (Hostetler and Kincaid, 2004) deficiencies. Additionally, the redox potential of some trace minerals (i.e. Fe and Cu) allows them to participate in reactions which may produce free radicals (Halliwell and Gutteridge, 1984). However it is unclear if sows and their litters in the present study differed in their oxidative status.

2.6 Implications Feeding reproducing sows organic trace minerals resulted in more total and live pigs at all levels of supplementation. However, there was no apparent advantage to feeding the higher trace mineral levels to reproducing sows, especially when additional Ca and P was fed. Gilt performance between 30 and 110 kg BW was not affected by trace mineral source and level. The mechanisms by which trace mineral sources and levels effect reproduction deserves further investigation.

Trace mineral level, mg/kg diet Trace mineral NRCabcd Ind.abef Cu 5 15 Fe 80 120 Mn 20 40 Se 0.15 0.30 Zn 50 120 a Inorganic treatments: copper sulfate (25.2% Cu), ferrous sulfate (30% Fe), manganese sulfate (32% Mn), selenium selenite (0.1% Se), and zinc sulfate (35.5% Zn). In a rice hull carrier. b Organic treatments: Bioplex Cu (10% Cu), Bioplex Fe (15% Fe), Bioplex Mn (15% Mn), Sel- plex (10% Se), and Bioplex Zn (15% Zn). In a finely ground corn carrier. c Analyzed values for Inorganic trace mineral source at NRC level supplied per kilogram of diet: 4.8 mg Cu, 79.5 mg Fe, 21 mg Mn, 0.15 mg Se, and 49 mg Zn. d Analyzed values for Organic trace mineral source at NRC level supplied per kilogram of diet: 4.7 mg Cu, 76 mg Fe, 26 mg Mn, 0.15 mg Se, and 56.5 mg Zn. e Analyzed values for Inorganic trace mineral source at Industry treatment level supplied per kilogram of diet: 16 mg Cu, 126 mg Fe, 43 mg Mn, 0.30 mg Se, and 122 mg Zn. f Analyzed values for Organic trace mineral source at Industry treatment level supplied per kilogram of diet: 14.6 mg Cu, 119 mg Fe, 40.5 mg Mn, 0.30 mg Se, and 131 mg Zn.

Table 2.1 Calculated mineral composition of treatment diets

Weight range, kg BW Ingredient 30 to 55a 55 to 85b 85 to breedingc Corn 53.15 61.30 67.60 Soybean meal, 48% CP 38.40 30.50 24.50 Fatd 5.00 5.00 5.00 Dicalcium phosphate 1.30 1.15 1.05 Limestone 1.05 1.05 0.90 Salte 0.35 0.35 0.35 Vitamin premixf 0.25 0.25 0.25 Trace mineral premixg,h,I,j ± ± ± Mannanoligosaccharidesk 0.10 0.10 0.10 Mycotoxin adherentl 0.10 0.10 0.10 Antibioticm 0.20 0.10 0.05 a Formulated to 1.30% Lys (total), 0.81% Ca, and 0.65% P (total). b Formulated to 1.10% Lys (total), 0.75% Ca, and 0.60% P (total). c Formulated to 0.90% Lys (total), 0.66% Ca, and 0.50% P (total). dChoice white grease. eIodized salt. fSupplied per kilogram diet: 12 IU vitamin E (dl α-tocopheryl acetate); 1450 IU vitamin A (acetate); 167 IU vitamin D3; 0.6 mg vitamin K (menadione); 8.9 mg of d-pantothenic acid; 2.8 mg of riboflavin; 11 mg of niacin; 0.3 mg of folacin; 0.06 mg of d-biotin; and 11 µg of vitamin B12. gOrganic trace mineral source (Bioplex) at NRC level supplied per kilogram of diet: 5 mg Cu; 80 mg Fe; 20 mg Mn; and 0.15 mg Se (Se yeast); 50 mg Zn; in a finely ground corn carrier. Added at the expense of corn. hInorganic trace mineral source at NRC level supplied per kilogram of diet: 5 mg Cu (sulfate); 80 mg Fe (ferrous sulfate); 20 mg Mn (sulfate); 0.15 mg Se (selenite); 50 mg Zn (sulfate); in a rice hull carrier. Added at the expense of corn. iOrganic trace mineral source (Bioplex) at Industry treatment level supplied per kilogram of diet: 15 mg Cu; 120 mg Fe; 40 mg Mn; 0.30 mg Se; 120 mg Zn; in a finely ground corn carrier. Added at the expense of corn. jInorganic trace mineral source at Industry treatment level supplied per kilogram of diet: 15 mg Cu (sulfate); 120 mg Fe (ferrous sulfate); 40 mg Mn (sulfate); 0.30 mg Se (selenite); 120 mg Zn (sulfate); in a rice hull carrier. Added at the expense of corn. kMannanoligosaccharides (Biomoss). lMycotoxin adherent (MTB-100). mTylosin supplied at the rate of 20, 10, and 5 mg/kg, respectively, for each phase.

Table 2.2. Composition of basal experimental diets fed during gilt development, (%, as-fed basis)

Phase and Parity: Gestation (1 and 2) Gestation (3 to 6) Lactation (1 to 6) Ingredient Ca and P level: NRCa Highb NRCc Highd NRCe Highf Corn 78.30 77.15 85.90 84.65 58.75 56.70 Soybean meal, 48% CP 18.00 18.10 10.25 10.50 32.45 32.60 Fatg - - - - 5.00 5.00 Dicalcium phosphate 1.40 2.25 1.60 2.40 1.15 2.80 Limestone 1.00 1.20 0.95 1.15 1.05 1.30 Salth 0.40 0.40 0.40 0.40 0.50 0.50 Vitamin premixi 0.50 0.50 0.50 0.50 0.50 0.50 Trace mineral premixj,k,l,m ± ± ± ± ± ± Mannanoligosaccharidesn 0.10 0.10 0.10 0.10 0.10 0.10 Mycotoxin adherento 0.10 0.10 0.10 0.10 0.10 0.10 Antibioticp,q 0.05 0.05 0.05 0.05 0.25 0.25 aFormulated to 0.75% Lys (total), 0.75% Ca, and 0.60% P (total). bFormulated to 0.75% Lys (total), 1.00% Ca, and 0.75% P (total). cFormulated to 0.53% Lys (total), 0.75% Ca, and 0.60% P (total). dFormulated to 0.53% Lys (total), 1.00% Ca, and 0.75% P (total). eFormulated to 1.00% Lys (total), 0.75% Ca, and 0.60% P (total). fFormulated to 1.00% Lys (total), 1.20% Ca, and 0.90% P (total). gChoice white grease. hIodized salt. iSupplied per kilogram gestation and lactation diet: 49 IU vitamin E (dl α-tocopheryl acetate); 4455 IU vitamin A (acetate); 223 IU vitamin D3; 0.6 mg vitamin K (menadione); 13 mg of d- pantothenic acid; 4 mg of riboflavin; 11 mg of niacin; 1.5 mg of folacin; 1.1 mg of thiamine; 0.22 mg of d-biotin; 17 µg of vitamin B12; 1.1 g of choline; and 1.1 mg of pyridoxine. jOrganic trace mineral source (Bioplex) at NRC level supplied per kilogram of diet: 5 mg Cu; 80 mg Fe; 20 mg Mn; 0.15 mg Se (Se yeast); 50 mg Zn. Added at the expense of corn. kInorganic trace mineral source at NRC level supplied per kilogram of diet: 5 mg Cu (sulfate); 80 mg Fe (ferrous sulfate); 20 mg Mn (sulfate); 0.15 mg Se (selenite); 50 mg Zn (sulfate). Added at the expense of corn. lOrganic trace mineral source (Bioplex) at Industry treatment level supplied per kilogram of diet: 15 mg Cu; 120 mg Fe; 40 mg Mn; 0.30 mg Se; 120 mg Zn. Added at the expense of corn. mInorganic trace mineral source at Industry treatment level supplied per kilogram of diet: 15 mg Cu (sulfate); 120 mg Fe (ferrous sulfate); 40 mg Mn (sulfate); 0.30 mg Se (selenite); 120 mg Zn (sulfate). Added at the expense of corn. nMannanoligosaccharides (Biomoss). oMycotoxin adherent (MTB-100). pAureomycin was added at 55 mg/kg of gestation diet. qBacitracin methylene disalicylate was added at 330 mg/kg of lactation diet.

Table 2.3. Composition of experimental basal diets fed during reproduction (%, as-fed basis)

TM Source: Organic Inorganic Item TM Level: NRCa Ind.bc NRCa Ind.b SEM No. giltsc 36 66 36 67 ― Final weight, kgd 111.5 110.8 111.8 110.9 2.19 Daily gain, g 894 886 891 887 12 Daily feed intake, kg 2.38 2.40 2.38 2.41 0.06 Gain:feed, g/kg 378 371 377 370 7 aNRC trace mineral treatments formulated to NRC (1998) recommendations. (See Table 2.1). bIndustry (Ind.) trace mineral treatments formulated to higher levels commonly fed in the swine industry. (See Table 2.1). cSows fed Ind.+Ca /P treatments during reproduction were fed the Ind. level of the appropriate source during development. dAverage initial BW, 30.2 kg.

Table 2.4. Treatment responses of dietary trace mineral source and level on pig performance during the growing-finishing period

TM Source: Organic Inorganic Item TM Level: NRCa Indb Ind+Ca/Pc NRCa Indb Ind+Ca/Pc SEM Litter, no. 62 65 60 76 54 58 ― No. of pigs/litters Totald,e 12.40 12.66 11.52 11.97 11.14 10.82 0.50 Stillborne 1.02 0.55 0.40 0.45 0.60 0.42 0.18 Mummiesf 0.23 0.25 0.06 0.33 0.06 0.10 0.09 Lived 11.08 11.79 10.99 11.14 10.44 10.27 0.46 7 d 10.29 10.18 10.29 10.13 10.51 10.07 0.35 Weaning, 17 d 10.28 10.10 10.29 10.06 10.38 9.91 0.35 Litter weight, kg 0 dd,f 20.3 20.5 18.7 18.6 19.5 17.5 0.8 7 d 34.2 35.0 34.0 33.2 35.9 33.8 1.0 Weaning, 17 d 64.0 63.7 64.0 62.0 65.5 62.6 2.2 Gain (0 to 17 d), kg 47.9 46.4 47.5 45.5 46.6 45.8 1.6 ADG, kg 2.80 2.73 2.78 2.66 2.73 2.69 0.09 Pig weight, kg 0 df 1.66 1.68 1.66 1.65 1.82 1.68 0.05 7 d 3.34 3.40 3.30 3.23 3.38 3.33 0.07 Weaning, 17 d 6.33 6.35 6.29 6.17 6.33 6.36 0.13 Gain (0 to17 d), kg 4.71 4.67 4.67 4.53 4.53 4.69 0.10 ADG, gg 277 275 275 267 267 275 5 aNRC trace mineral treatments formulated to NRC (1998) recommendations. bIndustry (Ind.) trace mineral treatments formulated to higher levels commonly fed in the swine industry. cInd.+Ca/P treatments contained the same trace mineral level as Ind., however with higher Ca and P levels. dTrace mineral source response (P < 0.05). eTrace mineral level response (P < 0.10). fTrace mineral level response (P < 0.05). gTrace mineral source response (P < 0.10).

Table 2.5. Treatment effects of sow dietary trace mineral source and level on litter and pig measurements

TM Source: Organic Inorganic Item TM Level: NRCa Indb Ind+Ca/Pc NRCa Indb Ind+Ca/Pc SEM No. litters 62 65 60 76 54 58 ― Sow weight, kg Breedingd,e 182 182 183 184 189 178 2.8 110 d 229 226 228 231 230 225 4.3 Gaind 50 47 49 52 44 46 2.5 Farrowe 217 213 221 221 223 214 3.4 Weaning, 17 de 211 205 212 214 215 209 3.6 Change -6.1 -6.2 -8.2 -6.3 -6.6 -4.0 2.1 Sonoray BF, last rib mm Breedingd,f,g 12.3 11.8 13.4 14.4 12.5 13.6 0.6 110 dd,e,g 14.8 14.3 16.4 17.7 15.8 16.2 0.7 Farrowd,e,g 14.3 13.4 15.9 22.7 14.3 14.8 1.2 Weaning, 17 df,h 13.0 12.5 14.5 17.7 17.7 14.1 1.5 Ultrasound BF, 10th rib, mm 30 d postcoitumd,e,h 19.81 15.75 22.10 24.64 18.03 20.07 1.78 90 d postcoitumd,e 24.13 22.86 27.69 29.97 23.11 25.91 1.52 Weaning, 17 dd,e,h 21.84 18.03 23.62 26.42 20.57 22.10 1.27 Ultrasound LMA, 10th rib, cm2i 30 d postcoitume 49.42 46.32 47.55 46.06 51.74 48.97 1.29 90 d postcoitum 50.39 48.84 49.29 48.84 51.81 49.48 1.35 Weaning, 17 deg 47.42 44.84 47.35 46.32 50.71 49.42 1.29 Return-to-estrus, d 5.64 5.77 4.93 5.51 5.26 5.64 0.43 Sow feed intake, kgj 0 to 7 d 28.4 31.2 31.9 30.2 32.6 33.6 2.00 7 to 17 d 61.5 60.4 59.6 59.7 59.7 61.4 2.93 Total 89.7 91.4 91.3 89.6 92.0 94.8 2.57 ADFI 5.28 5.37 5.37 5.27 5.41 5.57 0.25 Milk fat, % 8.12 7.70 7.65 7.85 7.78 8.33 0.41 aNRC trace mineral treatments formulated to NRC (1998) recommendations. bIndustry (Ind.) treatments formulated to higher levels commonly fed in the swine industry. cInd.+Ca /P treatments contained the Ind. trace mineral level., but with higher Ca and P levels. dTrace mineral level response (P < 0.05). eTrace mineral source x level interaction response (P < 0.05). fTrace mineral source x level interaction response (P < 0.10). gTrace mineral source response (P < 0.05). hTrace mineral source response (P < 0.10). iLongissimus muscle area. jAs-fed basis.

Table 2.6. Treatment responses of dietary trace mineral source and level on sow reproductive measurements over six parities 63

Paritya Item 1 2 3 4 5 6 SEM No. litters 134 92 60 48 23 18 ― Sow weight, kg Breedingb 145 162 178 188 203 221 2.7 110 db 191 212 217 228 255 265 4.0 Gainc 49 52 41 41 54 50 2.2 Farrowc 176 195 203 215 249 271 3.3 Weaning, 17 db 165 186 201 214 245 256 3.4 Changec -9.4 -8.0 -0.5 -0.9 -3.6 -15.1 2.0 Sonoray BF, last rib, mmd Breedingc 14.6 12.0 11.8 11.7 12.5 15.5 0.5 110 dc 15.7 14.2 14.3 14.2 17.6 19.2 0.7 Farrowc 14.7 13.5 12.9 13.5 16.3 24.5 0.9 Weaning, 17 dc 13.1 12.2 12.1 13.7 16.3 17.7 1.4 Ultrasound, BF, 10th rib, mme 30 d postcoitumc 24.64 18.80 17.53 16.51 18.03 24.64 1.78 90 d postcoitumc 27.18 24.13 22.10 18.80 24.64 34.04 1.27 Weaning, 17 dc 21.08 19.56 19.05 19.05 24.89 28.96 1.27 Ultrasound, LEA, 10th rib, cm2ef 30 d postcoitumg 45.48 45.35 45.61 46.97 54.26 52.45 1.16 90 d postcoitumg 46.90 45.42 47.55 48.39 56.00 54.45 1.23 Weaning, 17 db 42.39 43.81 45.35 47.48 52.90 54.19 1.16 Return-to-estrus, d - 5.53 5.88 5.33 5.73 4.83 0.39 Sow feed intake, kgh 0 to 7 dc 24.0 32.9 35.0 25.1 35.6 25.2 2.0 7 to 17 dc 50.6 61.1 64.2 66.2 62.1 58.0 2.9 Totalc 74.4 93.7 99.1 101.1 97.6 83.1 4.2 ADFIc 4.37 5.515.81 5.94 5.74 4.89 0.25 Milk fat, %i 7.92 8.017.60 7.06 8.83 8.01 0.39 aEighteen sows were killed after parity 1, 2, 4, and 6 to determine body composition. bLinear response (P < 0.01). cQuadratic response (P < 0.01). dDetermined at the last rib. eDetermined at the 10th rib. fLongissimus muscle area. gCubic response (P < 0.05). hAs-fed basis. iQuadratic response (P < 0.10).

Table 2.7. Parity effects on sow reproductive measurements

Parity Item 1 2 3 4 5 6 SEM Litter, no. 134 92 60 48 23 18 ― No. pigs/litters Totala 11.62 11.10 12.17 12.45 12.44 10.72 0.52 Stillborna 0.68 0.34 0.52 0.49 0.86 0.55 0.17 Mummies 0.25 0.11 0.17 0.13 0.20 0.18 0.09 Liveb 10.69 10.60 11.45 11.80 11.30 9.89 0.44 7 db 9.74 10.32 10.52 10.37 10.42 10.09 0.34 Weaning, 17 db 9.72 10.30 10.43 10.33 10.24 10.02 0.34 Litter weight, kg 0 db 17.4 19.1 19.0 20.4 20.8 18.4 0.7 7 da 30.8 36.1 34.7 35.1 34.3 35.1 1.2 Weaning, 17 da 56.9 66.0 65.2 66.4 63.2 64.2 2.1 Gain (0 to 17 d), kga 41.5 47.9 48.6 49.1 46.4 46.2 1.6 ADG, kg a 2.44 2.81 2.85 2.88 2.72 2.71 0.10 Pig weight, kg 0 da 1.56 1.76 1.63 1.70 1.71 1.81 0.04 7 da 3.12 3.46 3.26 3.37 3.29 3.48 0.06 Weaning, 17 da 5.90 6.45 6.28 6.48 6.22 6.48 0.12 Gain (0 to 17 d), kgb 4.35 4.69 4.70 4.80 4.58 4.68 0.10 ADG, gb 256 276 276 283 270 276 5.9 aCubic response (P < 0.05). bQuadratic response (P < 0.01).

Table 2.8. Parity responses of litter and pig measurements

Gestation Lactation Parity Parity Item TM Levelb,c,d 1 2 3 4 5 6 1 2 3 4 5 6 SEMa ADFI, kge NRC 2.04 2.18 2.31 2.45 2.72 2.99 4.86 6.01 6.36 6.36 6.13 4.72 0.25 Ind. 2.04 2.18 2.31 2.45 2.72 2.99 4.70 5.89 6.43 6.66 5.85 5.79 Ind+Ca/P 2.04 2.18 2.31 2.45 2.72 2.99 5.18 6.23 6.48 6.55 6.31 5.05 NRC predicted ADFI, kg 1.94 2.19 1.96 2.02 2.41 2.45 5.48 6.71 7.36 7.54 7.21 5.93 NRC recommended daily mineral intake Trace mineral intake, mg/d Cu 9.7 10.9 9.8 10.1 12.0 12.3 27.4 33.6 36.8 37.7 36.0 29.7 Fe 154.6 174.8 156.9 161.5 192.7 196.3 438.7 537.0 589.1 603.5 576.6 474.5 Mn 38.71 43.7 39.2 40.4 48.2 49.1 109.7 134.3 147.3 150.9 144.2 118.6

66 Se 0.29 0.33 0.29 0.30 0.36 0.37 0.82 1.01 1.10 1.13 1.08 0.89 Zn 96.8 109.3 98.1 100.9 120.4 122.7 274.2 335.6 368.2 377.2 360.4 296.5

Continued

aGestation feed levels within each parity were identical for all treatments, therefore they were not be statistically analyzed. bNRC trace mineral treatments formulated to NRC (1998) recommendations. cIndustry (Ind.) trace mineral treatments formulated to higher levels commonly fed in the swine industry. dInd.+Ca/P treatments contained the same trace mineral level as Ind., however with higher Ca and P levels. eQuadratic parity response, lactation (P < 0.01). fTrace mineral level response, lactation (P < 0.01). gTrace mineral level x parity interaction response, lactation (P < 0.01).

Table 2.9. Effect of trace mineral level and parity on calculated daily macro- and trace mineral intake during gestation and lactation

Table 2.9 continued

Macro-mineral intake, g/d Ca 14.5 16.4 14.7 15.1 18.1 18.4 41.1 50.4 55.2 56.6 54.1 44.5 P 11.6 13.1 11.8 12.1 14.5 14.7 32.9 40.3 44.2 45.3 43.2 35.6 Mg 0.77 0.87 0.78 0.81 0.96 0.98 2.2 3.7 3.0 3.0 2.9 2.4 S K 3.9 4.4 3.9 4.0 4.8 4.9 11.0 13.4 14.7 15.0 14.4 11.86 Na 2.9 3.3 2.9 3.9 3.0 3.7 11.0 13.4 14.7 15.0 14.4 11.86 Cl 2.3 2.6 2.4 2.4 2.4 2.9 8.8 10.7 11.8 12.1 11.5 9.49

Trace mineral intake, mg/d Cue,f,g NRC 10.2 10.9 11.6 12.3 13.6 15.0 24.3 30.0 31.8 31.9 30.8 23.6 3.2 Ind. 30.6 32.7 34.7 36.8 40.8 44.9 70.4 88.5 96.7 100.1 87.8 86.8

67 Ind+Ca/P 30.6 32.7 34.7 36.8 40.8 44.9 77.8 93.5 97.0 98.0 94.6 75.4 Fee,f,g NRC 163.2 174.4 184.8 196.0 217.6 239.2 388.8 480.9 508.7 509.5 490.9 378.2 27.3 Ind. 244.8 261.6 277.2 294.0 326.4 358.8 563.4 707.5 773.1 800.0 702.4 694.7 Ind+Ca/P 244.8 261.6 277.2 294.0 326.4 358.8 621.8 748.2 776.9 784.8 756.8 604.6 Mne,f,g NRC 40.8 43.6 46.2 49.0 54.4 59.8 97.2 120.2 127.2 127.5 122.8 94.5 7.3 Ind. 81.6 87.2 92.4 98.0 108.8 119.6 187.8 235.9 257.8 266.7 234.1 231.6 Ind+Ca/P 81.6 87.2 92.4 98.0 108.8 119.6 207.4 249.4 258.8 261.5 252.3 201.3 See,f,g NRC 0.31 0.33 0.35 0.37 0.41 0.45 0.73 0.90 0.95 0.96 0.92 0.71 0.06 Ind. 0.61 0.65 0.69 0.74 0.82 0.90 1.41 1.77 1.93 2.00 1.76 1.74 Ind+Ca/P 0.61 0.65 0.69 0.74 0.82 0.90 1.56 1.87 1.94 1.96 1.89 1.51 Zne,f,g NRC 102.0 109.0 115.5 122.5 136.0 149.5 242.9 300.4 318.0 318.9 307.2 236.2 25.6 Ind. 244.8 261.6 277.2 294.0 326.4 358.8 563.5 707.8 773.4 800.4 702.3 694.6 Ind+Ca/P 244.8 261.6 277.2 294.0 326.4 358.8 622.2 748.3 776.3 784.2 756.8 603.4

Continued

Table 2.9 continued

Macro-mineral intake, g/d Cae,f NRC 15.3 16.3 17.4 18.4 20.4 22.5 36.5 45.1 47.1 47.8 46.1 35.5 2.3 Ind. 15.3 16.3 17.4 18.4 20.4 22.5 35.2 44.2 48.3 49.9 44.0 43.5 Ind+Ca/P 20.4 21.8 23.1 24.5 27.2 29.9 62.2 74.8 77.6 78.8 75.7 60.4 Pe,f NRC 12.3 13.1 13.9 14.7 16.3 18.0 29.2 36.1 38.2 38.2 36.9 28.4 1.8 Ind. 12.3 13.1 13.9 14.7 16.3 18.0 27.2 35.3 38.6 39.9 35.2 35.2 Ind+Ca/P 15.3 16.3 17.4 18.4 20.4 22.5 46.7 56.1 58.2 58.8 56.8 45.3 Mge,f NRC 3.7 3.9 3.8 4.1 4.5 5.0 9.7 12.0 12.7 12.7 12.3 9.5 0.5 Ind. 3.7 3.9 3.8 4.1 4.5 5.0 9.4 11.8 12.9 13.3 11.7 11.6 Ind+Ca/P 3.9 4.1 4.1 4.3 4.8 5.3 11.1 13.4 13.9 14.1 13.6 10.9 Se,f NRC 3.9 4.2 3.9 4.1 4.6 5.1 11.0 13.6 14.4 14.4 13.9 10.7 0.6 Ind. 3.9 4.2 3.9 4.1 4.6 5.1 10.6 13.3 14.6 15.1 13.2 13.1

68 Ind+Ca/P 4.0 4.3 4.1 4.3 4.8 5.2 12.3 14.8 15.4 15.5 15.0 12.0

Ke NRC 13.2 14.1 11.7 12.4 13.8 15.2 43.3 53.5 56.6 56.6 54.6 42.1 2.3 Ind. 13.2 14.1 11.7 12.4 13.8 15.2 41.8 52.5 57.3 59.3 52.1 51.5 Ind+Ca/P 13.2 14.1 11.7 12.4 13.8 15.2 46.1 55.5 57.7 58.3 56.1 45.0

Nae NRC 3.2 3.4 3.6 3.8 4.2 4.7 9.2 11.4 12.1 12.1 11.7 9.0 0.5 Ind. 3.2 3.4 3.6 3.8 4.2 4.7 8.9 11.2 12.2 12.7 11.1 11.0 Ind+Ca/P 3.2 3.4 3.6 3.8 4.2 4.7 9.8 11.8 12.3 12.4 12.0 9.6

Cle NRC 5.3 5.7 6.0 6.4 7.1 7.8 14.8 18.3 19.4 19.4 18.7 14.4 0.8 Ind. 5.3 5.7 6.0 6.4 7.1 7.8 14.3 18.0 19.6 20.3 17.9 17.7 Ind+Ca/P 5.3 5.7 6.0 6.4 7.1 7.8 15.8 19.0 19.8 20.0 19.2 15.4

Gestation Lactation Parity Parity Item TM Levelabc 1 2 3 4 5 6 SEM 1 2 3 4 5 6 SEM Average BW, kgde NRC 168 187 200 210 229 248 3 182 202 215 226 252 268 5 Ind. 167 186 198 209 232 251 179 200 212 224 252 268 Ind+Ca/P 164 182 193 205 231 250 179 198 210 222 255 268 Trace mineral intake, mg/kg BW Cud,e,f,g NRC 0.061 0.058 0.058 0.059 0.060 0.060 0.003 0.134 0.149 0.148 0.143 0.123 0.087 0.017 Ind. 0.185 0.176 0.176 0.177 0.177 0.179 0.395 0.442 0.462 0.449 0.350 0.327 Ind+Ca/P 0.187 0.181 0.181 0.180 0.177 0.180 0.434 0.475 0.466 0.442 0.372 0.281 Fed,e,f,g NRC 0.972 0.933 0.927 0.937 0.954 0.969 0.024 2.142 2.386 2.368 2.268 1.949 1.409 0.151 Ind. 1.476 1.412 1.407 1.413 1.414 1.434 3.156 3.540 3.701 3.587 2.803 2.614 Ind+Ca/P 1.498 1.444 1.446 1.442 1.418 1.445 3.468 3.797 3.732 3.559 2.980 2.254 d,e,f,g 69 Mn NRC 0.243 0.233 0.232 0.234 0.239 0.241 0.008 0.536 0.596 0.593 0.569 0.488 0.352 0.049 Ind. 0.492 0.471 0.469 0.471 0.471 0.478 1.052 1.180 1.234 1.196 0.934 0.871 Ind+Ca/P 0.499 0.481 0.482 0.481 0.473 0.482 1.157 1.266 1.244 1.186 0.993 0.750

Continued

aNRC trace mineral treatments formulated to NRC (1998) recommendations. bIndustry (Ind.) trace mineral treatments formulated to higher levels commonly fed in the swine industry. cInd.+Ca/P treatments contained the same trace mineral level as Ind., however with higher Ca and P levels. d Quadratic parity response, gestation (P < 0.01). eQuadratic parity response, lactation (P < 0.01). fTrace mineral level response, gestation (P < 0.01). gTrace mineral level response, lactation (P < 0.01).

Table 2.10. Effect of trace mineral level and parity on calculated daily macro- and trace mineral intake per kg body weight during gestation and lactation

Table 2.10 continued

See,f,h,i NRC 0.002 0.002 0.002 0.002 0.002 0.002 0.000 0.004 0.004 0.004 0.004 0.004 0.003 0.000 Ind. 0.004 0.004 0.004 0.004 0.004 0.004 0.008 0.009 0.009 0.009 0.009 0.009 Ind+Ca/P 0.004 0.004 0.004 0.004 0.004 0.004 0.009 0.009 0.009 0.009 0.007 0.006 Znd,e,f,g NRC 0.607 0.582 0.579 0.579 0.579 0.604 0.024 1.339 1.491 1.482 1.425 1.224 0.880 0.231 Ind. 1.476 1.415 1.407 1.413 1.414 1.434 3.157 3.541 3.703 3.590 2.802 2.613 Ind+Ca/P 1.498 1.444 1.446 1.442 1.418 1.445 3.470 3.798 3.730 3.556 2.980 2.248

Macro-mineral intake, mg/kg BW Cae,f,g,h NRC 91 87 87 88 89 91 1.8 201 224 222 213 183 132 11 Ind. 92 88 88 88 88 90 197 221 231 224 176 164 Ind+Ca/P 125 120 120 120 118 120 347 380 373 356 298 225 Pd,e,f,g NRC 73 70 70 70 72 73 1.4 161 179 178 170 146 106 9 Ind. 74 71 70 71 71 72 158 177 185 179 141 131 70 Ind+Ca/P 94 90 90 90 89 90 260 285 280 267 223 169 Mgd,e,f,g NRC 21.9 21.0 19.2 19.4 19.8 20.1 0.4 54 60 59 57 49 35 3 Ind. 22.1 21.2 19.5 19.6 19.6 19.8 53 59 62 60 47 44 Ind+Ca/P 23.6 22.8 21.2 21.2 20.8 21.2 62 68 67 64 53 40 Sd,e,f,g NRC 23.2 22.3 19.6 19.8 20.2 20.5 0.4 61 67 67 64 55 40 3 Ind. 23.5 22.5 19.8 19.9 19.9 20.2 59 67 70 67 53 49 Ind+Ca/P 24.7 23.7 21.1 21.0 20.7 21.1 68 75 74 70 59 45 Kd,e NRC 78.6 75.4 58.8 59.3 60.5 61.4 1.2 238 266 263 252 216 157 11 Ind. 79.5 76.0 59.5 59.7 59.7 60.6 234 262 274 266 208 194 Ind+Ca/P 80.7 77.8 60.1 60.9 59.9 61.0 257 282 277 264 221 168 Nad,e NRC 19.0 18.2 18.1 18.3 18.6 18.9 0.3 51 57 56 54 46 33 2 Ind. 19.2 18.4 18.3 18.4 18.4 18.6 50 56 59 57 44 41 Ind+Ca/P 19.5 18.8 18.8 18.7 18.4 18.8 55 60 59 56 47 36 Cld,e NRC 31.6 30.3 30.1 30.4 31.0 31.5 0.6 82 91 90 86 74 54 4 Ind. 32.0 30.6 30.5 30.6 30.6 31.1 80 90 94 91 71 67 Ind+Ca/P 32.5 31.3 31.3 31.2 30.7 31.3 88 96 95 91 76 58

CHAPTER 3

COMMERCIAL EVALUATION OF DIETARY TRACE MINERAL SOURCES AND LEVELS IN REPRODUCING SOWS OVER SIX PARITIES

3.1 Objective This experiment evaluated the effects of organic and inorganic sources of trace minerals fed at or above NRC (1998) recommendations on sow reproductive performance over a six parity period in a commercial swine herd in Australia.

3.2 Rationale Organic trace minerals have been previously considered to have higher bioavailabilities than inorganic sources (Ammerman et al., 1998). If true, lower dietary organic trace mineral levels may be necessary to meet the reproductive needs of the sow. This study was conducted in collaboration with a commercial swine producer in Australia to evaluate such effects. The study evaluated two trace mineral sources each provided at two dietary levels, over a 6 parity time period similar to the study conducted at The Ohio State University (Chapter 2). The study allowed a greater number of animals per treatment to be evaluated.

3.3 Materials and methods 3.3.1 Experimental Design and Treatments The effects of trace mineral source and levels on sow reproductive performance were evaluated over six parities using a total of 219 sows involving 633 farrowings. The experiment was conducted in collaboration with a large swine integrator (QAF Meat Industries, formerly Bunge, Corowa, New South Wales, Australia). This experiment was conducted as a 2 x 2 factorial, in a randomized complete block (RCB) design in 4 groups or replicates. The first factor evaluated organic or inorganic trace mineral sources, while the second factor evaluated the dietary inclusion level. The trace minerals evaluated were Cu, Fe, Mn, Se, and Zn. The

71 organic sources (Bioplex) provided by Alltech Inc. (Nicholasville, KY) were metal proteinates, whereby trace minerals were chelated with partially hydrolyzed soybean protein, while the organic Se source was Se yeast (Sel-plex). Most inorganic trace minerals were provided in the sulfate form, but the Se source was sodium selenite. The second factor evaluated the level of trace mineral level added to the diet. One level evaluated the suggested NRC (1998) recommendation level of each trace mineral (NRC), while the second level incorporated a higher dietary trace mineral level. The levels in these two treatment groups added trace minerals commonly recommended by university and the commercial feed industry professionals (Ind.). The NRC treatment group was supplemented with 5 mg Cu, 60 mg Fe, 50 mg Zn, 0.15 mg Se, and 20 mg Mn/kg diet, whereas the Industry treatment groups contained diets adding 15 mg Cu, 100 mg Fe, 120 mg Zn, 0.30 mg Se, and 40 mg Mn/kg diet. 3.3.2 Grower-Development Phase A total of 400 gilts, at approximately 25 kg body weight and 9 weeks of age, were initially moved to grower-finisher pens and allotted to dietary trace mineral treatments based on weight and ancestry in two replicates. Within each group, 50 gilts were allotted per dietary treatment for 100 total gilts per treatment. The two groups differed in age by approximately 2 wk. Gilts were fed their initial grower treatment diets containing the treatment mineral source and levels for 9 weeks and then the developer diet until mating (Table 3.1). Gilts were allowed ad libitum access to feed during the grower and developer periods. Growth performance during this period was not determined. Gilts were sexually stimulated with boar contact from five weeks prior to breeding until mating. 3.3.3 Reproducing Period At approximately 7 mo of age, the gilts were bred in the grower facility. The breeding period was 30 d and gilts were allotted into four farrowing groups based upon the week bred. Gilts and weaned sows were artificially inseminated at the onset of estrus and 12 h later. Semen was obtained from the farm’s boar stud and refrigerated until used. The number of gilts bred per treatment was not regulated, thus variable numbers of gilts bred per treatment were evaluated. Bred gilts were moved to gestation pens at the end of the breeding period. During gestation, females were housed in large pens, in covered (hoop) outdoor structures with rice hull bedding. Females were fed their dietary treatments daily in individual crates. Color-coded ear tags on each animal identified the dietary treatment that each animal

72 received. Gestation feeding level was set at 2.2 kg/d for parity 1 and increased to 2.4 kg/d for subsequent parities. The feeding level was constant throughout a gestation period. Sows were moved to individual farrowing crates at approximately 110 d of gestation, whereupon lactation diets were fed at the quantity provided during gestation. Postpartum sows were allowed ad libitum access to their treatment diet upon farrowing and sow feed intakes recorded daily. By 24 h postpartum, litters were processed (i.e. weighed and injected with 200 mg iron dextran). Within 3 d postpartum litter sizes were equalized within dietary treatments. Litters were allowed access to the sow’s dietary treatment. Sow and litter weights and sow backfat thickness were measured at weaning. All sows within a farrowing group were weaned on the same day, with lactation lengths differing between treatment groups. The average lactation length was 24.8 ± 0.3 d. Consequently, litter and pig weaning weights, litter and pig weight gains, and total sow feed intake were adjusted to a standard 28 d period for each sow and litter assuming that feed intake was the average of that previously consumed. Pig gains were also adjusted to a 28 d lactation length. Pig gain adjustments (for the 28 d period) were calculated using the equation: pig gain, adjusted 28 d = [(PWW - PBW) / LL] x 28. In this equation PWW is the average pig weaning weight measured, PBW is the average pig birth weight, and LL is the lactation length in days. Adjusted pig weaning weights (28d) were the pig birth weights added to the adjusted pig gain. Litter weaning weight and litter gains (28 d) were calculated by multiplying the adjusted pig weaning weights and adjusted pig gains by the number of pigs weaned. Adjusted total sow lactation feed intakes were the average daily feed intakes multiplied by 28 d. These estimates assume that a longer lactation period would not greatly affect sow and litter performance. Weaned sows were moved to individual crates and provided ad libitum access to their lactation diet until mated. Sows were bred in their first estrus postweaning, with the weaning-to- estrus intervals recorded, moved by group to the gestation pens within a week of breeding. Sows not exhibiting estrus within 7 d of weaning and those were determined not pregnant after 30 d postcoitum were removed from the experiment. 3.3.4 Diet Composition Wheat-lupin based mixtures were used to formulate diets fed during the grower (9 to 18 wk of age) and developer (18 wk to breeding) phases. They provided dietary lysine (total) levels of 1.14 and 0.82%, respectively. Calcium levels were 0.89% and 0.82% for the two phases, respectively, while diets in both phases contained 0.60% P (total). Gestation diets contained a

73 mixture of wheat, barley, and canola meal and formulated to contain 0.62% lysine (total), 0.86% Ca, and 0.71% P (total). Lactation diets were wheat-barley based mixtures formulated to 1.00 % lysine (Total), 0.80% Ca, and 0.60% P (total). All diets also contained meat and bone meal and tallow, but at differing proportions during each phase. Diets were fortified with vitamins that met or exceeded NRC (1998) nutrient recommendations. Exogenous phytase (Natuphos-5000) was added to all diets at 500 FTU/kg diet. The percentage compositions of the basal diets are presented in Table 3.1.

3.3.5 Statistical Evaluation

The reproductive data were analyzed as a 2 x 2 factorial in a RCB design in four replicates using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC) with sow treatment nested within parity. The statistical model included trace mineral source, trace mineral level, trace mineral source x level interactions, group, parity, trace mineral source x parity interactions, trace mineral level x parity interactions, and trace mineral source x level x parity interactions. Individual sow and litter measurements were considered the experimental unit. Parity effects were evaluated using regression analysis. Least square treatment means are presented in tabular form.

3.4 Results Analyzed trace mineral concentrations in the various dietary treatments were within targeted values, except for Fe contents which were higher (data not shown). This is likely due to the Fe contributed by supplemental limestone and dicalcium phosphate and environmental contamination from mixing and feeding equipment. The number of litters farrowed for each treatment by parity and the percentage farrowing at the subsequent parity are presented in Table 3.2. Because fewer gilts were bred and farrowed during parity 1 when the Organic Ind. and Inorganic NRC treatments were provided, there were fewer litters farrowed for these treatment groups during the 6 parity study. The percentage of sows which farrowed at the subsequent parity after parity 1 and 2 seemed to be greater when sows were fed the Organic trace mineral treatments (Table 3.2). However, the percentage of sows completing the 6 parity study (~11 %) was similar for all treatments, but appeared to favor the Ind. trace mineral level treatments.

74 The effects of trace mineral source and level on sow reproductive and litter performance are presented in Table 3.3 and Table 3.4. When the Organic trace mineral source was fed to sows, total pigs born increased when the Ind. level was provided, however litter size decreased at the Ind. level of the Inorganic source (Table 3.3). This resulted in a trace mineral source x level interaction response (P < 0.05). Live pigs born showed a similar pattern, but the interaction was not significant (P > 0.15). Total and live pigs born increased linearly (P < 0.05) as parity increased (Table 3.5) and the treatment differences in litter size were more apparent at the later parities (Table 3.6). The trace mineral source x level x parity three-way interaction, however, was not significant (P > 0.15). The number of stillbirths and mummies born were not affected by trace mineral source or level (P > 0.15) and the source by level interaction was not significant. Overall, there was no difference in number of total, live, or stillborn pigs between the trace mineral sources or levels (Table 3.4). Pig and litter birth weights were also not affected by trace mineral source or level, likely because litter sizes at birth did not differ between dietary treatments. Litter and individual pig birth weights increased in a curvilinear manner (P < 0.05) to parity 5, with the largest increase occurring at parity 2. Lactation lengths were shorter (P < 0.05) at parities 2 and 3 and were somewhat longer (P < 0.05) when sows were fed the Ind. level of trace minerals (25.9 vs. 24.2 d). Because of these differences in lactation lengths, we standardized lactation data to a 28 d length to more appropriately compare the treatment effects and parity responses. Number of weaned pigs increased to parity 6 when sows were fed the Organic NRC and Inorganic Ind. treatments (Table 3.6), whereas when the Organic Ind. and Inorganic NRC treatments were fed, pig numbers weaned increased quadratically (P < 0.05) to parity 5, but decreased at parity 6. This resulted in a trace mineral source x level x parity three-way interaction (P < 0.01) response. Overall, more (P < 0.05) pigs were weaned when the Ind. level was fed (Table 3.4). Because litters were standardized within dietary treatments by 3 d postpartum, the difference in number of pigs weaned may suggest a slight improvement in pig survivability. Pig and litter weaning weights and average daily gains adjusted to a standard 28 d length appeared to be greater when the Organic trace mineral treatments were fed (Table 3.4), but the effect was not significant (P < 0.15). Litter weaning weights (P < 0.05) and litter daily gains (P < 0.10), but not pig weights and gains, were greater when the Ind. level had been fed.

75 This likely reflects their larger litter size at weaning. There was also a trace mineral source x level x parity interaction (P < 0.05) response for litter weaning weights (28 d, adjusted). However, this interaction is likely explained by the difference in number of pigs weaned, because individual pig weaning weights (28 d) were not affected by trace mineral source or level. Litter and pig weaning weights and daily gains increased in a curvilinear manner (P < 0.05) to parity four and were lower thereafter (Table 3.5). Sow body weights and backfat thickness at weaning, lactation feed intakes (total and ADFI), and return-to-estrus intervals are presented in Table 3.3 and Table 3.4. Total (28 d, adjusted) and daily lactation feed intakes were similar for both Organic trace mineral treatments, however sows fed the Inorganic NRC treatment consumed less feed than those fed the Inorganic Ind. treatment, resulting in a trace mineral source x level interaction response (P < 0.05). Although total feed intake was greater (P < 0.05) when sows were fed the Ind. level (Table 3.4), these sows had lighter weaning weights (P < 0.05) and longer return-to-estrus intervals (P < 0.10). These responses were more evident for sows fed the Inorganic Ind. treatment (Table 3.3), which resulted in a trend (P < 0.10) for a trace mineral source x level interaction response for weaning weight, but not return-to-estrus interval (P > 0.15). Furthermore, backfat thickness at weaning appeared to be lower when the Inorganic Ind. treatment was fed (Table 3.3), but the interaction response was not significant (P > 0.15). Because sows fed the Ind. trace mineral level weaned larger and heavier litters, they may not have increased their lactation feed intake enough to support milk production and consequently they mobilized more body reserves. The parity results presented in Table 3.5 demonstrated that sow weights at weaning increased (P < 0.05) as parity increased, with the largest weight increase occurring at parity 3. Lactation feed intake (total, 28 d or ADFI) increased quadratically (P < 0.05) to parity 4 and was lower thereafter. Sow backfat thickness at weaning decreased to parity 3, was higher at parity 4, and decreased to parity 6. Return-to-estrus interval did not differ by parity.

3.5 Discussion Trace mineral source and level may have affected gilt reproductive performance, as demonstrated by the treatment differences in parity 1 farrowings, but the results were inconclusive. It should be noted that gilts were initially bred during mid-Summer and that this

76 was a particularly hot and dry Australian summer with temperatures frequently exceeding 35º C (> 100º F) during and around the breeding period. Upon reviewing the data (Table 3.2), it was obvious that several sows had litters with 5 or fewer pigs born during parity 1 (n = 24 sows) and 2 (n = 10 sows), whereas there were only 11 litters with fewer than 6 pigs after parity 2. These smaller litters represent 11% and 7% of the litters which farrowed during parity 1 and 2, respectively. These small litters obviously make litter size interpretations regarding the efficacy of trace minerals difficult. Consequently, trace mineral source and level did not seem to greatly affect the percentage of litters with 5 or fewer pigs born during these early parities. This suggests that other reproductive problems may have influenced the response to the dietary treatments during parity 1 and 2 and the experiment did not effectively test the trace mineral sources and levels. The demonstrated increase in the number of total pigs born when the Industry level of the Organic source was fed implies that the Organic trace minerals may have been required to maximize pigs born in this difficult reproduction study, whereas the decreased litter size for the Inorganic Ind. treatment suggests the possibility of negative effects on reproduction by feeding higher Inorganic levels. The treatment effects seemed to be more evident at the later parities (Table 3.6), though the trace mineral source x level x parity interaction was not significant (P > 0.15). However, the overall response suggests that litter size at birth was not affected by trace mineral source or level. The greater litter weaning weights and gains for the Ind. level treatments can partially be explained by larger litter sizes at weaning, because pig weaning weights were similar for both trace mineral levels. However, the interpretation of the effects of sow dietary treatments on litter and pig weaning weights and gains is complicated because litters were provided access to creep feed. In our previous study (Chapter 2), we observed a decrease in the number of pigs born (total and live) when the higher levels of the Inorganic sources, but not when the Organic trace mineral sources were fed. In that study litter sizes were also greater for both levels of the Organic treatments than the Inorganic treatments. The reasons for the discrepancy between experiments are not clear, but may have been related to differences in sow productivity and or herd environmental conditions. Diets at each of the two sites contained different feedstuffs (i.e. wheat vs. corn-soybean meal based diets), potential interactions between the trace minerals and

77 other dietary nutrients may have affected trace mineral availability differently, however this effect is not known. The trend for greater litter and pig daily gains when the Organic treatments were fed is in agreement with our previous experiment (Chapter 2).

3.6 Implications Sow retention during the early parities seemed to favor the Organic trace mineral treatments but overall the percentage of sows completing the 6 parity study was similar for all treatments. The number of total pigs born was similar for the Organic Ind. and Inorganic NRC treatments, however total pigs born decreased when the Inorganic Ind. level was fed. However, numbers of live pigs born, were in general similar for all treatment. Feeding the Ind. level of both sources resulted in more pigs weaned and heavier litter weaning weights, suggesting a benefit by providing higher sow dietary trace minerals on subsequent pig survivability.

Ingredient Phase: Growera Developmentb Gestationc Lactationd Wheat 62.19 67.87 45.12 46.36 Barley - - 11.27 20.50 Lupin kernels 8.43 8.47 - 12.50 Mill mix 7.20 16.00 25.90 7.73 Canola meal 10.00 - 5.00 2.03 Meat and bone meal 6.00 2.00 3.00 3.23 Blood meal 1.00 - - 1.00 Hominy - - 5.00 - Molasses - - - 0.33 Fate 2.00 1.60 1.40 2.20 Fish oil - - - 0.30 Dicalcium phosphate - 0.67 0.50 0.70 Limestone 0.63 0.97 0.97 0.57 Salt 0.20 0.20 0.30 0.37 Vitamin premix 0.30 0.30 0.30 0.30 Lysine-HCl 0.40 0.40 0.03 0.38 DL-Methionine 0.10 0.07 - 0.11 Water 1.00 1.00 1.00 1.00 Phytasef 0.01 0.01 0.01 0.01 Threonine 0.13 0.15 - 0.14 Trace mineral premixghij ± ± ± ± aFormulated to 1.14% Lys (total), 0.89% Ca, and 0.60% P (total). bFormulated to 0.82% Lys (total), 0.82% Ca, and 0.60% P (total). cFormulated to 0.62% Lys (total), 0.86% Ca, and 0.71% P (total). dFormulated to 1.00% Lys (total), 0.80% Ca, and 0.60% P (total). eTallow. fNatuphos-5000 (500 FTU/kg diet). gOrganic trace mineral source (Bioplex) at NRC level supplied per kilogram of diet: 4 mg Cu; 60 mg Fe; 50 mg Zn; 20 mg Mn. hInorganic trace mineral source at NRC level supplied per kilogram of diet: 4 mg Cu; 60 mg Fe; 50 mg Zn; 20 mg Mn. iOrganic trace mineral source (Bioplex) at Industry treatment level supplied per kilogram of diet: 15 mg Cu; 100 mg Fe; 120 mg Zn; 40 mg Mn. jInorganic trace mineral source at Industry treatment level supplied per kilogram of diet: 15 mg Cu; 120 mg Fe; 120 mg Zn; 40 mg Mn.

Table 3.1. Composition of experimental basal diets, (% as-fed basis)

Parity Item 1 2 3 4 5 6 Overall Sows farrowing, no. Organic NRC 63 45 34 18 11 6 177 Inorganic NRC 54 35 25 22 15 5 156 Organic Ind. 39 28 21 19 9 5 121 Inorganic Ind. 63 43 29 22 14 8 179 Sow retention, %ab Organic NRC - 7176536155 9.5 Inorganic NRC - 6571886833 9.3 Organic Ind. - 72 75 90 47 56 12.8 Inorganic Ind. - 68 67 76 64 57 12.7 Litters, ≤ 5 pigs born (total), no Organic NRC 7 5 2 0 0 0 14 Organic Ind. 3 1 1 1 0 0 6 Inorganic NRC 5 2 0 2 1 0 10 Inorganic Ind. 9 2 1 1 1 1 15 Litters, ≤ 5 pigs born, %c Organic NRC 11.1 11.1 5.9 0.0 0.0 0.0 7.9 Organic Ind. 7.7 3.6 4.8 5.3 0.0 0.0 5.0 Inorganic NRC 9.3 5.7 0.0 9.1 6.7 0.0 6.4 Inorganic Ind. 14.3 4.7 3.4 4.5 7.1 12.5 8.4 a Percentage of sows that farrowed in the subsequent parity. b The overall percentage is the percentage of sows farrowing at parity 1 completing the experiment. c Percentage of total litters which contained 5 or fewer pigs within each treatment group.

Table 3.2. Effects of parity and trace mineral source and level on litter numbers, sow retention and the number and percentage of litters containing 5 or fewer pigs

TM Source: Organic Inorganic Item TM Level: NRCa Indb NRCa Indb SEM No. of sows 63 39 54 63 ― No. of litters 177 121 156 179 ― Pigs per litter, no. Totalc 10.81 11.43 11.61 10.89 0.32 Stillborn 0.72 0.83 1.05 0.83 0.12 Mummy 0.27 0.27 0.32 0.20 0.05 Live 9.94 10.33 10.22 9.88 0.30 Weanedd 8.59 8.87 8.37 8.93 0.17 Litter weights, kg Birth 15.3 15.8 15.7 15.7 0.40 Weaning d 63.9 69.1 61.1 68.2 2.2 Weaning (28 d, adj) d 73.7 76.2 69.1 75.2 2.1 Gain (0 to 28 d) e f 60.0 62.1 56.0 60.7 1.8 ADG, kgef 2.14 2.22 2.00 2.17 0.06 Pig weights, kg Birth 1.60 1.58 1.56 1.61 0.03 Weaning f 7.49 7.68 7.40 7.66 0.14 Weaning (28 d, adj) 8.51 8.56 8.27 8.40 0.14 Gain (0 to 28 d) e 6.92 6.99 6.72 6.79 0.15 ADG, g e 247 250 240 242 4 Lactation length, dd 24.6 24.4 24.8 25.5 0.3 Return-to estrus, df 5.0 5.8 4.7 7.7 1.0 Sow feed intake, kgg Total d h 154.0 157.6 152.1 166.4 2.8 Total (28 d, adj) c d 181.2 180.4 176.2 187.0 2.5 ADFIc 6.47 6.44 6.29 6.80 0.09 Sow weaning wt., kg d h 237.0 234.8 238.7 227.1 3.0 Sow weaning BF, mm e 15.0 15.6 14.8 14.1 0.5 aNRC trace mineral treatments formulated to NRC (1998) recommendations. bIndustry (Ind.) trace mineral treatments formulated to higher levels commonly fed in the swine industry. cTrace mineral source x level interaction response (P < 0.05). dTrace mineral level response (P < 0.05). eTrace mineral source response (P < 0.15). fTrace mineral level response (P < 0.10). gAs-fed basis. hTrace mineral source x level interaction response (P < 0.10).

Table 3.3. Effect of trace mineral sources and levels on sow reproductive performance 81

Source Level Item Organic InorganicSEM NRCa Indb SEM No. of sows 102 117 - 117 102 - No. litters 298 335 - 356 300 - Pigs per litter, no. Total 11.12 11.25 0.22 11.21 11.17 0.23 Stillborn 0.76 0.95 0.08 0.89 0.83 0.08 Mummy 0.27 0.26 0.04 0.29 0.24 0.04 Live 10.13 10.05 0.21 10.08 10.11 0.21 Weaned c 8.73 8.65 0.13 8.48 8.90 0.13 Litter weights, kg Birth 15.6 15.7 0.3 15.5 15.7 0.3 Weaningc 66.5 64.7 1.6 62.5 68.7 1.5 Weaning (28 d, adj) c 74.9 72.2 1.4 71.4 75.7 1.4 Gain (0 to 28 d)de 61.0 58.4 1.2 58.0 61.4 1.3 ADG, kgde 2.18 2.08 0.04 2.07 2.19 0.04 Pig weights, kg Birth 1.59 1.59 0.02 1.58 1.60 0.02 Weaninge 7.58 7.52 0.10 7.44 7.67 0.10 Weaning (28 d, adj) 8.53 8.33 0.10 8.39 8.48 0.10 Gain (0 to 28 d) d 6.96 6.75 0.10 6.82 6.88 0.10 ADG, g d 241 224 5 244 246 3 Lactation length, dc 24.3 24.6 0.2 25.9 24.2 0.3 Return-to estrus, de 5.4 6.2 0.7 4.9 6.7 0.7 Sow feed intake, kgf Totalc 155.8 159.2 2.0 153.0 162.0 2.0 Total (28 d, adj)c 180.8 181.6 1.8 178.7 183.7 1.8 ADFI 6.38 6.56 0.06 6.38 6.56 0.06 Sow weaning wt., kgc 235.9 232.9 2.0 237.8 230.0 2.0 Sow weaning BF, mm d 15.3 14.5 0.4 14.9 14.9 0.4 a NRC trace mineral treatments formulated to NRC (1998) recommendations. b Industry (Ind.) trace mineral treatments formulated to higher levels commonly fed in the swine industry. c Trace mineral level response (P < 0.05). d Trace mineral source response (P < 0.15). e Trace mineral level response (P < 0.10). f As-fed basis.

Table 3.4. Main effects of trace mineral source and level on sow reproductive performance

Parity Item 1 2 3 4 5 6 SEM Pigs per litter, no. Totala 9.55 10.49 11.18 11.53 12.15 12.23 0.32 Stillborna 0.49 0.58 1.09 1.050.77 1.19 0.13 Mummya 0.10 0.24 0.24 0.260.31 0.41 0.06 Livea 8.95 9.66 9.85 10.36 11.13 10.61 0.26 Weanedb 7.88 8.59 8.54 9.059.25 8.80 0.16 Litter weights, kg Birthb 12.6 15.3 15.9 16.1 17.6 16.2 0.5 Weaningc 56.7 63.3 56.7 75.3 72.4 69.2 1.6 Weaning (28 d, adj)b 60.2 74.7 72.2 80.5 79.2 74.6 1.7 Gain (0 to 28 d)b 48.7 60.7 58.0 66.2 63.9 60.6 1.7 ADG, kgb 1.74 2.17 2.07 2.362.28 2.16 2.0 Pig weight, kg. Birthc 1.45 1.62 1.66 1.591.63 1.59 0.03 Weaningc 7.24 7.41 6.68 5.347.66 8.02 0.15 Weaning (28 d, adj)c 7.66 8.70 8.49 8.92 8.33 8.50 0.13 Gain (0 to 28 d)c 6.21 7.08 6.83 7.34 6.74 6.94 0.14 ADG, gc 222 253 244 263 241 248 6 Lactation length, dc 26.0 22.9 20.4 25.7 25.6 25.9 0.24 Return-to estrus, d 6.5 5.6 5.2 7.3 4.3 - 1.1 Sow feed intake, kgd Totalc 149.4 151.2 128.7 185.2 166.7 164.0 2.6 Total (28 d, adj)b 160.4 184.5 175.6 201.8 185.2 179.5 2.6 ADFIb 5.75 6.59 6.27 7.216.62 6.40 0.10 Sow weaning wt., kgb 179.9 201.2 236.4 257.1 258.0 273.7 1.8 Sow weaning BF, mmc 15.3 13.6 13.9 17.0 14.0 15.0 0.4 aLinear response (P < 0.05). bQuadratic response (P < 0.05). cCubic response (P < 0.05). dAs-fed basis.

Table 3.5. Parity effects on sow reproductive performance

Parity Item Treatment 1 2 3 4 5 6 SEM Total pigs born, no. Organic NRC 9.32 9.87 10.93 11.73 12.21 10.79 0.63 Inorganic NRC 9.65 10.75 11.83 11.19 12.11 14.15 Organic Ind. 9.51 10.62 11.04 11.46 13.10 12.89 Inorganic Ind. 9.71 10.71 10.91 11.75 11.18 11.11 Live pigs born, no. Organic NRC 8.68 9.33 9.61 10.48 11.57 9.97 0.63 Inorganic NRC 9.06 9.83 10.70 9.75 11.09 10.92 Organic Ind. 8.79 9.71 9.51 10.51 11.79 11.64 Inorganic Ind. 9.29 9.75 9.60 10.70 10.06 9.91 Pigs weaned, no.a Organic NRC 7.66 8.13 8.60 9.00 8.96 9.64 0.29 Inorganic NRC 7.71 8.53 8.66 8.79 8.87 7.67 Organic Ind. 8.06 9.12 9.06 8.82 9.94 8.21 Inorganic Ind. 8.10 8.57 8.32 9.61 9.25 9.70 Pig birth wt, kg Organic NRC 1.43 1.63 1.64 1.58 1.62 1.68 0.06 Inorganic NRC 1.42 1.61 1.66 1.55 1.59 1.54 Organic Ind. 1.42 1.65 1.71 1.62 1.61 1.47 Inorganic Ind. 1.52 1.59 1.63 1.59 1.70 1.66 Litter birth wt, kg Organic NRC 12.06 14.63 15.13 16.38 17.95 15.95 0.86 Inorganic NRC 12.68 15.60 17.62 14.56 17.43 16.55 Organic Ind. 12.15 15.74 15.68 16.56 18.16 16.26 Inorganic Ind. 13.70 15.34 15.34 16.72 16.67 16.14 Pig weaning wt. (28 d adj.), kg Organic NRC 7.72 8.69 8.44 8.86 8.67 8.65 0.27 Inorganic NRC 7.51 8.78 8.12 8.88 8.13 8.20 Organic Ind. 7.69 8.79 8.68 8.94 8.31 8.97 Inorganic Ind. 7.74 8.54 8.71 9.00 8.22 8.17 Litter weaning wt. (28 d adj.), kgb Organic NRC 58.76 70.97 69.09 79.52 81.01 83.00 3.3 Inorganic NRC 58.25 74.61 70.59 78.21 72.52 60.60 Organic Ind. 61.78 79.83 77.04 78.95 83.81 75.59 Inorganic Ind. 61.85 73.38 72.04 85.26 79.55 79.04 aTrace mineral source x level x parity interaction response (P < 0.01). bTrace mineral source x level x parity interaction response (P < 0.05).

Table 3.6. Effects of parity, trace mineral source, and level on pig and litter measurements 84

CHAPTER 4

EFFECT OF SOW DIETARY ORGANIC AND INORGANIC TRACE MINERALS ON THE IRON STATUS OF NEONATAL PIGS AND THE EFFECT OF IRON INJECTIONS TO THEIR PROGENY AT BIRTH AND WEANING

4.1 Abstract A total of 68 pigs from 7 second parity sows were used to evaluate the effect of sow dietary trace mineral source on the Fe status of neonatal pigs and the postnatal carry-over effect of Fe to the progeny. The experiment was a 2 x 2 x 2 factorial in a split plot design. Sow dietary trace mineral source (organic or inorganic) served as the main plot. Pig Fe injection (0 or 200 mg of Fe dextran) at birth and weaning was the subplot. All pigs were bled and weighed on d 2, 7, 9, 13, and 17 postpartum. Each group was subdivided at weaning and half received a Fe injection. All pigs were fed the same postweaning diets, which contained 90 ppm of supplemental Fe. Pigs were bled and weighed on d 7, 10, 14, 17, 21 and 28 postweaning. Hemoglobin (Hb), hematocrit (Hct), and ceruloplasmin oxidase activity were determined at all measurement periods. Neonatal pigs from both sow dietary groups not injected with Fe had lower growth rates and lower weaning body weight (P < 0.05). Blood Hb and Hct in the non-Fe injections groups declined from d 2 to weaning in both sow treatment groups, but increased when neonatal pigs received Fe injections. Blood Hb and Hct values were somewhat lower when sows were fed the organic trace minerals. Ceruloplasmin activity was initially low in all neonates, but increased to weaning, whether or not Fe was injected. During the postweaning period, weights and daily gains were lower (P < 0.01) when pigs did not receiving a Fe injection at birth. Blood Hb and Hct were initially lower in the non-Fe injected group but increased to 28 d postweaning. Weight gains, blood Hb, and Hct values were greater at each measurement period for pigs not injected with Fe at birth but that were injected at weaning, but their blood

85 values were still lower at d 28 than pigs receiving Fe at birth. For pigs which received 200 mg Fe at birth, weights, daily gains, blood Hb, and Hct were not affected by an additional Fe injection at weaning, resulting in an interaction response (P < 0.01). There was no effect of sow trace mineral source during the postweaning period on blood values, regardless of neonatal Fe injection. Ceruloplasmin activity in weaned pigs increased from weaning to 28 d postweaning, but was not affected by sow trace mineral source or Fe injections at birth or weaning. These results suggest that there was minimal carryover of Fe from sows fed either trace mineral source and that pigs required a Fe injection during the initial days of birth to prevent anemia.

4.2 Introduction Nursing pigs require approximately 7 mg of Fe per d to support erythropoiesis and other metabolic activities in body tissue. To meet the pig’s need during the nursing phase, the young animal must use the 40 to 50 mg of Fe body reserves accumulated during gestation and the approximate 1 mg supplied daily from sow milk. However, unless an exogenous source of Fe is administered within a few days of birth anemia will generally occur within 7 to 10 d of age. Larger and faster growing pigs would be expected to exhibit signs of anemia sooner due to their greater Fe requirement because of increasing blood volume when greater amounts of tissue are being formed. Attempts to increase neonatal Fe reserves (Pond et a., 1961) and milk Fe content (Pond et al., 1965) by increasing dietary inorganic Fe levels in sow diets have not been successful. Therefore, to prevent anemia in the young pig, neonatal pigs routinely receive exogenous Fe generally by an i.m. injection at birth (Ullrey et al., 1959; Kernkamp et al., 1962). Nursing pigs may, however, practice coprophagy and absorb some of the Fe ingested from sow fecal residue. Reports have suggested that providing an organic Fe source to the sow may increase fetal Fe stores (Ashmead and Graff, 1982), increase birth weights, reduce postnatal mortality and result in heavier pig weaning weights (Ashmead, 1996). Other research has shown fewer stillbirths, higher blood hemoglobin concentrations in nursing piglets, increased sow feed intakes, and sow productivities when sows are provided an organic Fe source (Close, 1999). The bioavailability of organic Fe is reported to be 125 to 185% compared to inorganic Fe (Henry and Miller, 1995). These collective results imply that organic Fe may be a potentially excellent source of Fe for reproducing sows than inorganic Fe sources.

This experiment evaluated the Fe status of young pigs when sows were fed either organic or inorganic trace minerals including Fe, during their reproductive cycle and the postnatal Fe carry-over response in the progeny. The effects of Fe injections administered at birth and weaning were evaluated with pig blood and growth performances evaluated.

4.3 Materials and Methods

4.3.1 Experimental Design and Treatments A total of 7 second parity sows (Yorkshire x Landrace) were used to evaluate the effect of dietary mineral source on the subsequent mineral status of the progeny at birth and the carry- over postnatal effect of Fe reserves in the progeny. Sows were fed one of two diets incorporating an organic or inorganic trace mineral source. The organic trace mineral source (Bioplex, Alltech Inc., Nicholasville, KY) and the inorganic trace minerals had been initiated when the females weighed 30 kg BW, with the treatment diets continued after they successfully completed their first reproductive cycle and rebred within 7 d postweaning. The organic trace minerals provided in the sow’s diet were metal proteinates chelated with partially hydrolyzed soybean protein and comprised a combination of organic trace minerals, while organic Se was Se yeast (Sel-plex). Inorganic trace minerals were provided in the sulfate form except Se which was provided as Na selenite. Trace mineral levels added to both gestation and lactation diets were: 15 ppm Cu, 120 ppm Fe, 40 ppm Mn, 0.30 ppm Se, and 120 ppm Zn. The composition of sow diets was largely a corn-soybean meal mixture and had been presented earlier (Chapter 2). Pigs within each litter were randomly selected and either non-injected or injected with 200 mg Fe dextran. Within each pig group, they were subdivided at weaning and half injected with 200 mg Fe dextran or non-injected. The experiment was conducted as 2 x 2 x 2 factorial in a split plot design using sow treatment as the main plot and pig injections as subplots.

4.3.2 Lactation Period Gestating sows were fed once daily 2.1 kg of their treatment diet in individual stalls. At 110 d of gestation they were moved to individual farrowing crates and fed their lactation treatment diet at 2.18 kg/d. Upon parturition sows were offered their treatment diet at 2.0 kg on d 1, then the ration was cumulatively increased by 2.0 kg/d, such that by d 3 feed was provided on an ad libitum basis. Within 12 h postpartum sows were weighed and litters processed (i.e.

87 ear-notched, teeth clipped). Sow weights and feed intakes were also collected at weaning (17 d). Sow colostrum (0 d) and milk at weaning (17 d) was collected from all functional glands (30 to 50 ml) after an i.m. injection (40 U.S.P. units) of oxytocin. Milks were frozen and stored at -4º C for later analysis. A total of 68 ((Yorkshire x Landrace) x PIC (line 280)) pigs were used in this experiment. Within each litter some pigs were randomly bled prior to the consumption of colostrum to determine initial hematological values. Within 12 h of birth the pigs in each litter were randomly selected to receive either no Fe or an i.m. injection of 200 mg of Fe dextran. All pigs were subsequently bled and weighed on d 2, 7, 9, 13, and 17 postpartum. Blood samples (< 2 mL) were by cardiac puncture, collected into 3-mL heparinized (45 UCP units of sodium heparin) vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) with 20-gauge, 3.81 cm needles. Blood was placed on ice until transported to the laboratory for analysis for the determination of hemoglobin (Hb), percent hematocrit (Hct), and ceruloplasmin oxidase activity.

4.3.3 Nursery Period At weaning (17± 1 d), half the pigs from each of the two neonatal Fe injection groups within each litter were randomly selected and injected with 200 mg of Fe dextran. Pigs were allotted in three replicates to nursery pens based on their treatment group and body weight. Each pen contained 4 to 5 pigs per nursery pen which provided approximately 0.30 m2 of woven-wire floor space per pig, one nipple water, and a four-hole stainless steel feeder. Environmental temperature was established at 28º C, but adjusted as needed to meet the comfort zone of the pigs. Pigs were bled and weighed initially, and on d 7, 10, 14, 17, 21 and 28 postweaning. Blood samples (< 2 mL) were collected into 3-mL heparinized vacutainer tubes from cardiac puncture, and placed on ice until transported to laboratory for analysis.

4.3.4 Diet Compositions Sow gestation and lactation diets were previously reported (Chapter 2). All pigs were fed the same diets postweaning, which contained a supplemental level of 90 ppm of Fe (Fe sulfate). From 0 to 14 d postweaning the nursery diet was comprised of corn-soybean meal containing dried whey, lactose, fat, and blood plasma. During the 14 to 28 d postweaning period the diet contained the same ingredients, except that blood plasma was removed. The calculated lysine (total) of the diets was 1.55 and 1.45% during the 0 to 14 d and 14 to 28 d periods,

88 respectively. All diets met or exceeded NRC (1998) nutrient recommendations for vitamins and minerals. In both diets the trace mineral mixture was provided from inorganic salts. The composition of the nursery diets are presented in Table 4.1.

4.3.5 Analytical Methods Total Hb concentration (g/dL) in whole blood was determined by conversion of Hb and its derivatives to cyanmethemoglobin and read on a spectrophotometer set at 540 nm. Hematocrit was determined on fresh blood using the microhematocrit method. Whole blood samples were inverted several times before Hb and Hct were determined. The remaining blood was centrifuged at 2200 x g at 5º C for 15 min, with plasma collected and stored at 4º C. Plasma ceruloplasmin oxidase activity analysis was performed within 24 hr of blood sample collection. Plasma ceruloplasmin oxidase activity was determined by spectrophotometry at room temperature using the method of Lehmann et al. (1974). Ceruloplasmin oxidase activity was calculated using the change in absorbance between tubes incubated for 5 and 15 min and the molar absorptivity of the substrate (o-dianisidine dihydrochloride) consumed. Ceruloplasmin oxidase activity is expressed in International Units (U) per mL plasma. Milk and colostrum samples were analyzed on freeze dried milk samples for the trace elements (Cu, Fe, Mn, Se, and Zn) using the Inductively Coupled Plasma (ICP) method (PS 3000, Leeman Labs, Inc. Hudson, NH). Selenium was analyzed from fresh milk after being wet ashed in nitric and perchloric acid using the fluorometric method outlined by AOAC (2000). Nursery diets were analyzed for their trace mineral content by ICP. Performance and hematological data were analyzed as a split-plot design (Steel and Torrie, 1980) using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). Sow trace mineral source was the main plot and Fe injection treatments at birth and weaning were the subplots. The litter was considered the experimental unit while nursing the sow, but individual pigs were used as the experimental unit in the nursery. Least square treatment means are presented in tabular form.

4.4 Results Reproductive performance of sows presented in Table 4.2 tended to show the same results as previously reported (Chapter 2). Generally, the data of this experiment showed that the number of pigs born (total and live) was greater when sows were fed the organic trace mineral mixture with similar pig birth weights for both sow groups. The responses were not 89 significant attributable to the low number of observations. Because litter size was standardized between sows postpartum, the weaning pig and litter data were similar for both sow groups. Sow colostrum trace mineral composition was similar for Cu, Fe, and Zn for both sow treatment groups, except that Se was greater (P < 0.01) when the organic mineral mixture was provided. Consistent with our other report (Chapter 5), the trace mineral content of milk at 17 d (weaning) tended to be higher in Cu, Fe, and Zn when the inorganic trace mineral mixture was fed to lactating sows. As with colostrum, Se content in milk (17 d) was greater (P < 0.01) when the organic trace mineral mixture was fed. Our other results (Chapter 5) also indicated that the trace mineral status, particularly Fe, in the liver and total body content in the neonate was similar regardless of the trace mineral source fed to the sow. The effect of the postnatal responses when neonatal pigs received either no injection or were injected with 200 mg Fe dextran is presented in Table 4.3. When Fe was not injected in neonatal pigs, their growth rate was lower by 7 d of age in the pigs of both sow dietary groups (P < 0.05). This lowered growth rate continued until weaning (P < 0.01) and resulted in lower body weight at weaning (P < 0.05). Within 2 d of age there was also a decline in blood Hb in all pigs, but the decline was somewhat greater when pigs were from sow fed the organic mineral source. A decline in blood Hb in the non-Fe injections groups continued from d 2 to weaning in both sow groups. When Fe was injected into pigs the Hb concentration increased from d 2 to weaning. The interaction between sow mineral source and Fe injection was only significant at 13 d of age (P < 0.05), where non-Fe injected pigs from sows fed the organic trace minerals had lower Hb concentrations than those pigs from sows fed the inorganic minerals. This suggests no additional Fe carry-over in pigs from sows the organic mineral source. Although no attempt was made to prevent coprophagy within the farrowing crate, there was no indication that it prevented the decline in blood Hb concentration or Hct. These results suggest that there was a similar carry-over response of Fe from sows fed either mineral source, that the organic mineral source of Fe was similar in response to the inorganic supplement, and that pigs required a Fe injection during the initial days of birth to prevent anemia. Hematocrit values showed the same general results as did Hb concentrations. A declined in Hct occurred within 2 d of age regardless of sow mineral level, largely attributable to neonatal pig dehydration. The Hct continued to decline in non-Fe injected pigs and were lower when sows had been fed the organic mineral source. When neonatal pigs received 200 mg

90 Fe injections, Hct initially declined upon birth but steadily increased during the nursing period in the pigs from both sow mineral groups. There was no interaction between sow mineral level and Fe injections at any stage during the nursing period (P > 0.15). Ceruloplasmin activity was extremely low in the neonate in all sow groups, being non detectable in many samples. Within 2 d of age, the activity of this oxidase enzyme increased in the plasma of pigs from both sow groups whether or not Fe was injected. Ceruloplasmin oxidase activity continued to increase to weaning in all pigs. Although there was a somewhat higher activity in pigs not injected with Fe at 13d of age (P < 0.05) and ceruloplasmin activity was higher (P < 0.01) at weaning when sows had been fed the organic mineral source, there were no consistent responses by sow dietary treatment level or Fe injections in the pigs. These results imply, however, that during Fe deficiency that this enzyme’s activity may be increased, at least somewhat, to compensate for the lowered antioxidant activity contributed from Fe. The effect of sow mineral source and the two neonatal Fe injection treatments on their subsequent responses during the postweaning period when fed nursery diets supplemented with an exogenous Fe source is presented in Table 4.4. Neonatal pigs that did not receive an Fe injection had lower (P < 0.01) weights and daily gains than those who had been injected with 200 mg Fe dextran. Blood Hb and Hct were initially lower in the non-Fe injected group but during the 28 d postweaning period they both increased, attributed largely from the dietary Fe provided in the nursery diet. At 28 d postweaning, blood Hb and Hct values for the non-Fe injected group were similar to the group injected with 200 mg Fe, although they still had a lighter (P < 0.01) body weight. The postweaning responses of pigs not injected with Fe at birth were similar regardless of sow mineral source. For those neonatal pigs injected with 200 mg Fe dextran, both blood Hb concentrations and Hct values were similar between the two sow treatment groups at each measurement period postweaning. There was no consistent interaction between these variables at any measurement period. Ceruloplasmin activity increased from weaning to 28 d postweaning, but there was no effect of sow dietary trace mineral source, neonatal Fe injection or an interaction of these variables postweaning. The combined treatment effects of Fe injection at birth and at weaning presented in Table 4.5 demonstrates that pigs administered 200 mg of Fe at birth did not show any further advantage when an additional 200 mg Fe was injected at weaning compared with those not

91 injected with Fe at weaning. Weight, daily gains, blood Hb, Hct, and ceruloplasmin activity was similar for both groups at each measurement period. For pigs not injected with 200 mg Fe at birth, but administered an injection at weaning, there were greater weight gains and blood Hb and Hct values at each measurement period, perhaps in an increasing rate or compensatory response. Whether this was due to the Fe injection, the dietary Fe, or both in combination cannot be ascertained from this experiment. Although both groups increased in their blood Hb and Hct as the length of the nursery period continued they did not attain the weights or hematological values of pigs injected with Fe at birth. This response resulted in an interaction between these two variables (P < 0.01) at each measurement period. Ceruloplasmin activity in weaned pigs increased from weaning to 28 d postweaning, with no effect of sow trace mineral source fed, Fe injection at birth or weaning, nor was there an interaction between the variables on these measurement criteria.

4.5 Discussion The results of our experiment suggests that the Fe status of the neonatal pig, sow milk composition, and subsequent carryover effect from gestation was not additionally enhanced whether sows had been fed organic or inorganic trace mineral sources. This is in contrast to other reports which indicated higher bioavailabilities, tissue stores, increased sow and progeny performances and higher blood hematology when organic Fe was fed to reproducing sows. Injection of Fe at 200 mg appears to be beneficial to pigs, whether sows are fed organic and inorganic trace minerals. Although we did not test if greater quantities of Fe should be administered, the research of Hill et al. (1999) and our data imply that hematological values were optimized. There was no advantage of an additional injection at weaning. Hemoglobin and Hct values have been previously shown to decline when pigs are not administered Fe during the nursing period. Our results are consistent with those responses. The results of Rincker et al. (2004) suggested that postweaning diets that contained 150 mg Fe (Fe sulfate) per kg diet were necessary to maintain blood parameters supplied by Fe. Our data and those of Rincker et al. (2004) suggest that supplementing the nursery diet with higher dietary levels of Fe sulfate are required to maintain or increase blood Hb and Hct values.

Nursery diets Item 0 to 14 da 14 to 28 db Corn 28.40 22.15 Soybean meal, 48% CP 28.25 34.60 Dried whey 25.00 25.00 Blood plasma 4.00 - Lactose 8.00 10.00 Fatc 3.00 5.00 Dicalcium phosphate 1.30 1.10 Limestone 0.75 0.85 Se premixd 0.15 0.15 Trace mineral premixe 0.10 0.10 Vitamin premixf 0.25 0.25 Salt 0.25 0.25 Lysine HCl 0.15 0.15 DL Methionine 0.10 0.10 Zinc oxide, 72% Zn 0.25 0.25 Antibioticg 0.05 0.05 aFormulated to 1.54% Lys (total), 0.85% Ca, and 0.71% P (total). bFormulated to 1.45% Lys (total), 0.86% Ca, and 0.68% P (total). cChoice white grease. dSodium selenite in a limestone carrier provided 0.30 mg Se/kg of diet. eSupplied per kilogram nursery diet: 90 mg of Fe (ferrous sulfate); 8 mg of Cu (sulfate); 20 mg of Mn (oxide); 100 mg of Zn (sulfate). fSupplied per kilogram nursery diet: 18 IU of vitamin E (dl α-tocopheryl acetate); 2450 IU of vitamin A (acetate); 245 IU of vitamin D3; 0.6 mg of vitamin K (menadione); 1.7 mg of thiamine; 2.8 mg pyridoxine; 13.5 mg of d-pantothenic acid; 4.5 mg of riboflavin; 22.3 mg of niacin; 0.3 mg of folacin; 0.1 mg of d- biotin; 22.3 µg of vitamin B12; and 0.70 g choline. gTylosin was added at 50 mg/kg of nursery diet.

Table 4.1. Composition of nursery diets (%, as fed basis)

Sow trace mineral source Item Organic Inorganic SEM No. of sows 3 4 - Weight, kg Breeding 153 163 5.3 Farrow 195 200 6.0 Weaning 185 193 6.2 Parturition Pigs born/litter, no. Total 12.3 9.8 1.8 Live 11.7 9.5 1.4 Pig weight, kg 1.79 1.79 0.08 Weaning, 17 d No. of pigs 10.3 10.0 0.40 Pig weight, kg 6.14 5.88 0.25 Composition, mg/L Colostrum Cu 2.51 2.43 0.45 Fe 1.81 1.72 0.16 Sea 0.16 0.10 0.01 Zn 13.99 13.33 1.48 Milk (17 d) Cu 0.62 0.80 0.09 Fe 1.88 2.11 0.45 Mn Se1 0.08 0.05 0.01 Zn 7.10 7.80 0.47 aTrace mineral source response, P < 0.01.

Table 4.2. Effect of dietary trace mineral source fed to second parity sows on sow reproductive performance and composition of trace minerals in colostrum and milk

Sow mineral Source: Organic minerals Inorganic minerals Item Neonatal Fe, mg: 0 200 0 200 SEM No. of pigs 16 15 19 18 — Weight, kg 0 d (Birth) 1.83 1.78 1.78 1.76 0.07 7 d 3.25 3.45 3.27 3.42 0.13 17 d (Wean)a 5.50 6.51 5.56 6.22 0.34 Daily gain, g 0 to 7 da 203 238 212 238 13 7 to 17 db 216 329 228 279 14 0 to 17 db 201 261 222 262 12 Hemoglobin, g/dl 0 dc 8.72 9.73 10.07 9.25 0.46 2 dd 6.45 7.36 7.67 7.74 0.28 7 db,e 4.77 8.81 5.58 9.15 0.23 9 db,d 4.04 9.12 4.82 9.61 0.21 13 db,c,e 3.35 9.66 4.20 9.69 0.20 17 db 3.89 12.03 4.29 10.93 0.38 Hematocrit, % 0 d 33.4 37.2 38.0 35.2 1.8 2 da,e 24.0 27.5 27.6 28.5 1.0 7 db,e 19.7 35.7 22.6 35.7 0.7 9 db,e 18.1 36.3 20.5 36.8 0.6 13 dd 16.0 37.3 18.7 38.3 0.6 17 db 15.9 39.5 17.7 37.7 1.1 Ceruloplasmin activity, U/mLf 0 d 0.001 0.007 0.004 0.005 0.002 2 d 0.026 0.026 0.027 0.026 0.003 7 d 0.087 0.132 0.169 0.101 0.038 9 d 0.101 0.114 0.109 0.099 0.006 13 da 0.121 0.112 0.117 0.094 0.007 17 dd 0.135 0.134 0.112 0.095 0.009 aEffect of Fe injection, P < 0.05. bEffect of Fe injection, P < 0.01. cTrace mineral source x Fe injection interaction response, P < 0.05. dTrace mineral source response, P < 0.01. eTrace mineral source response, P < 0.05. fCeruloplasmin activity units = units/mL plasma.

Table 4.3. Effect of sow dietary trace mineral source and neonatal iron injections on piglet performance and blood parameters during the nursing period

Sow mineral source: Organic Inorganic Item Neonatal Fe, mg: 0 200 0 200 SEM No. of pigs 16 15 19 18 — Weight, kg 0 d (wean)a 5.32 6.21 5.55 6.27 0.24 14 da 7.76 9.56 8.46 9.87 0.35 28 da 14.44 17.07 15.46 17.66 0.55 Daily gain, g 0 to 7 d 25 61 74 78 18 7 to 14 da 324 418 342 437 16 14 to 21 da 436 478 447 529 18 21 to 28 db 518 594 552 583 26 14 to 28 da 477 536 500 556 18 0 to 28 da 326 388 354 407 15 Hemoglobin, g/dl 0 da 3.57 10.32 4.30 10.94 0.28 7 da 6.65 12.51 6.99 11.87 0.29 14 da 7.64 11.00 8.01 10.05 0.19 17 da 9.14 11.22 9.16 10.08 0.21 21 da 9.78 11.76 9.93 11.36 0.22 28 da 11.08 11.83 11.00 11.38 0.17 Hematocrit, % 0 da 15.5 36.8 17.6 37.6 0.84 7 da 25.3 41.6 26.4 38.8 0.77 14 da 30.5 38.4 31.1 35.9 0.42 17 da 33.9 39.3 34.2 37.7 0.60 21 da 35.9 40.9 36.5 38.8 0.60 28 da 37.2 39.9 38.2 39.1 0.43 Ceruloplasmin activity, U/mL c 0 db 0.127 0.111 0.114 0.095 0.008 7 d 0.132 0.126 0.134 0.128 0.010 14 d 0.155 0.152 0.141 0.147 0.008 17 d 0.157 0.174 0.156 0.149 0.010 21 d 0.170 0.161 0.163 0.162 0.009 28 d 0.171 0.160 0.160 0.153 0.008 aEffect of neonatal Fe injection, P < 0.01. bEffect of neonatal Fe injection, P < 0.05. cCeruloplasmin activity units = units/mL plasma

Table 4.4. Effect of sow dietary trace mineral source and neonatal iron injection on postweaning pig performance and blood parameters

96 Neonatal Fe, mg: 0 200 Item Weaning Fe, mg: 0 200 0 200 SEM No. of pigs 13 18 19 18 — Weight, kg 0 d (wean)a 5.58 5.28 6.186.29 0.24 7 da 5.85 5.71 6.726.73 0.27 14 da 7.92 8.31 9.719.72 0.35 28 da 14.52 15.38 17.43 17.29 0.55 Daily gain, g 0 to 7 d 39 61 76 63 18 7 to 14 da,b 295 371 428 427 17 14 to 21 da,b 413 470 519 489 18 21 to 28 dc 530 540 584 593 26 14 to 28 da 471 505 552 541 18 0 to 28 da 319 361 402 393 15 Hemoglobin, g/dl 0 da 3.85 4.02 10.24 11.02 0.28 7 da,d,e 5.16 8.48 11.90 12.48 0.29 14 da,d,e 6.11 9.55 10.40 10.64 0.18 17 da,d,e 8.06 10.23 10.96 11.06 0.21 21 da,d,e 8.90 10.76 11.81 11.31 0.23 28 da,d,f 10.67 11.41 11.61 11.60 0.17 Hematocrit, % 0 da 16.15 16.99 36.50 37.98 0.84 7 da,d,e 20.73 30.97 39.43 40.95 0.76 14 da,d,e 26.09 35.51 36.56 37.69 0.52 17 da,d,e 31.48 36.62 38.73 38.35 0.59 21 da,d,e 34.19 38.14 40.12 39.57 0.60 28 da,d,e 36.92 38.45 39.91 39.09 0.43 Ceruloplasmin activity, U/mL g 0 dc 0.116 0.125 0.105 0.102 0.008 7 d 0.134 0.131 0.116 0.138 0.010 14 d 0.146 0.150 0.145 0.155 0.008 17 d 0.152 0.161 0.163 0.16 0.010 21 d 0.166 0.167 0.148 0.175 0.009 28 d 0.165 0.166 0.154 0.159 0.008 aEffect of neonatal Fe injection, P < 0.01. bNeonatal Fe and weaning Fe injection interaction, P < 0.05. cEffect of neonatal Fe injection, P < 0.05. dNeonatal Fe and weaning Fe injection interaction, P < 0.01. eEffect of weaning Fe injection, P < 0.01. fEffect of weaning Fe injection, P < 0.05. gCeruloplasmin activity units = units/mL plasma.

Table 4.5. Effect of iron injections in neonatal pigs and weanling pigs on postweaning performance and blood parameter responses 97

CHAPTER 5

EFFECTS OF DIETARY TRACE MINERAL SOURCES AND LEVELS ON CHANGES IN SOW BODY MINERAL CONTENT AND THEIR PROGENY OVER SIX PARITIES

5.1 Abstract The effects of trace mineral source and level on sow mineral status were evaluated over a six parity period using 68 sows and 20 gilts. This experiment was a 2 x 3 x 4 factorial arrangement of treatments in a randomized complete block design. Organic and inorganic sources of trace minerals were evaluated at three dietary levels. The first level met NRC recommendations (NRC), while the second level was set at a level that was considered commonly recommended by universities and the commercial feed industry (Ind.). Two additional treatments evaluated the Ind. level of both sources, but with additional Ca and P (Ind+Ca/P). Three sows per treatment were selected for determination of liver and total body mineral content after weaning at parities 1, 2, 4, and 6. Body composition at the initiation of reproduction was also determined. Mineral content in neonatal pigs, colostrum and milk, and pigs at parity 6 weaning were evaluated. Total sow body and liver Se content were greater (P < 0.05) when the organic source and the Ind. and Ind.+Ca/P levels were fed. The only other effect of trace mineral source on sow mineral status was that liver Zn content tended (P < 0.10) to be greater organic trace minerals were fed. The total sow body Cu content was greater when the Ind. and Ind.+Ca/P levels were fed. Sow liver contents of Cu and Zn were lower (P < 0.05) at the NRC level. There were no effects (P > 0.10) of trace mineral source or level on neonatal total pig or liver trace mineral contents. Colostrum and milk Se increased at the higher dietary levels when the organic source was fed, but not when the inorganic source was fed. Milk Ca, P, Cu and Zn contents were higher (P < 0.05) when the inorganic source was fed. Milk Cu, Se, and Zn concentrations were lowest when the NRC trace mineral level was fed (P < 0.05). Total pig

98 mineral concentrations of weaned pigs were not affected by trace mineral source or level. However, empty body Cu, Fe, and Mn on a mg/kg basis were higher (P < 0.05) for pigs from the sows fed the organic source. Empty body concentrations of Mn and Zn were greater (P < 0.05) when the higher trace mineral levels were fed, particularly at the Ind.+Ca/P level. These results suggest that trace mineral source and level had minimal effect on the mineral content of reproducing sows. However, the organic trace mineral source seemed to improve transfer of trace minerals to the nursing pig through the milk.

5.2 Introduction A previous study by Mahan and Newton (1995) demonstrated that sow body mineral reserves had decreased after the females had completed three parities, when compared to contemporary non-gravid gilts, and this depletion was exacerbated when sows had supported larger litter growth rates. Sow nutrient demands have increased during the past 30 years as genetic selection has resulted in larger litters and higher milk production. Yet, mineral recommendations for reproducing females have remained essentially unchanged during this period (NRC, 1979; 1988; 1998). In order to compensate for the greater needs of high producing sows, gestation and lactation diets are commonly formulated to macro and trace mineral levels beyond current NRC (1998) recommendations. However, the efficacy of these higher levels has not been evaluated. Although the use of organic trace minerals is increasing, their utility in sow diets has not been widely investigated. Organic minerals are expected to remain bound to a ligand in the digestive tract until at the site of absorption (Ammerman et al., 1998), preventing the formation of insoluble chelates with other minerals and nutrients. Consequently, the increased mineral availability could potentially improve mineral retention in tissues, even at lower supplementation levels. This experiment evaluated the effects of dietary trace mineral source and level on the mineral content of sows and their progeny over a six parity period when diets contained either inorganic or organic sources of trace minerals fed at or above 1998 NRC recommended levels. The effects of feeding the higher trace mineral levels in conjunction with higher Ca and P were also evaluated. Furthermore, the effects of sow dietary trace mineral sources and levels on the transfer of minerals to the progeny were also evaluated.

99 5.3 Materials and Methods

5.3.1 Experimental Design and Treatments This experiment evaluated the effects of trace minerals (Cu, Fe, Mn, Se, and Zn) added to the diet on total body mineral content of reproducing sows and their progeny. The experiment was conducted as a 2 x 3 x 4 factorial arrangement of treatments in a randomized complete block (RCB) design. The first factor evaluated trace mineral forms (organic or inorganic). The second factor evaluated trace mineral levels. The first level evaluated the established NRC (1998) recommendations (NRC), while the second evaluated a higher trace mineral level that was considered commonly recommended by universities and the commercial feed industry (Ind.). The organic minerals (Bioplex) were provided by Alltech Inc. (Nicholasville, KY) and were metal proteinates, whereby trace minerals were chelated with partially hydrolyzed soybean protein, while the organic Se source was Se yeast (Sel-plex). Most inorganic trace minerals were provided in the sulfate form, but the Se source was sodium selenite. These four treatment diets contained 0.75% Ca and 0.60% P (total) and represented the NRC (1998) recommendations. A third level (Ind.+Ca/P) evaluated providing additional Ca and P with the higher trace mineral level of each source, but only during the reproductive phase of the experiment. Gestation Ca and P levels in these latter treatments were 1.00% and 0.75% (total), respectively, while at 1.20% Ca and 0.90% (total) P during lactation. These diets started at the initial breeding and were continued through six parities. Diet composition, feeding programs, and management conditions were described previously (Chapter 2). After weaning at parities 1, 2, 4, and 6, three weaned sows per treatment (n = 68) were selected to be killed for determination of body mineral content in three replicates. These weaned sows were selected from a previous experiment (Chapter 2). The sows had nursed a typical number of pigs for their treatment group during each of the previous lactation periods. Sows were killed within 24 h of weaning. Additionally, 20 gilts (5/treatment) were killed at 8 mo of age for determination of mineral composition at the initiation of reproduction. Only the NRC and Ind. levels of each trace mineral source were fed during development because the additional 2 treatments containing higher Ca and P levels were not incorporated until the initial breeding. Gilts which were allotted to the Ind.+Ca/P treatments had been fed the Ind. trace mineral level of their respective trace mineral source during development (Chapter 2).

100 5.3.2 Mineral Transfer to Progeny During parity 1, 3, and 5, stillborn pigs were weighed and frozen (-4º C) for later mineral analysis. Whole livers from these pigs were collected, weighed, and frozen separated from the body. During parity 6, two pigs per litter from each of three sows from each treatment group were killed at weaning (17 d) to determine body mineral composition. The pigs were randomly selected from those that were born and nursed by the sow. Pigs were electrically stunned and killed by exsanguination, the digestive contents removed, and the entire carcass was frozen (-4º C) for later mineral analysis. Livers were collected from each pig, weighed, and frozen separately. The effects of trace mineral source and level on the mineral composition of the colostrum (within 12 h parturition) and milk at weaning were evaluated at parity 1, 2, 4, and 6. The samples analyzed at each of these parities were collected from the reproducing sows which were killed at parity 6. Colostrum and milk samples (30 to 50 ml) were collected after an i.m. injection (40 U.S.P. units) of oxytocin. Samples were frozen and stored at -4º C for later analysis.

5.3.3 Slaughter and Sampling Procedures Upon transfer to the OSU Meat Laboratory, animals were fasted overnight, but provided with access to water. Animals were weighed prior to slaughter, electrically stunned, and killed by exsanguination, but blood was not collected. Digesta in the intestinal tract was manually removed and residue was rinsed from the tract with water. Fresh livers were weighed and ground, with a sample retained and frozen. The remaining visceral organs were composited into a 20-l plastic container, weighed, and frozen at -20º C for 96 h. Animals were skinned and hides were split longitudinally at the midline. Perirenal fat, both feet, and the skin, including hair, from the right half of the animal were combined, weighed, and frozen in plastic containers. The head and right half of the mammary tissue from the sows were weighed and frozen separately. Mammary tissue was not separated from the body of the gilts used for determining initial body composition. Hot carcasses were split longitudinally at the midline, weighed, and chilled for 24 h at 2º C. The carcass was weighed after chilling to determine evaporative water loss. The right side of the carcass was then reduced in size, placed in plastic containers, weighed, and frozen for 72 h at -20º C.

101 Frozen components were weighed prior to grinding to determine the quantity of water loss during storage. The frozen head was split longitudinally using a band saw (Model 5212, Hobart, Troy, OH) and the right half was weighed and combined with the frozen carcass, skin, feet, and perirenal fat. This composite of the animal will be referred hereafter as the carcass. Frozen components were reduced in size using a band saw. Carcass and viscera components were ground independently through an industrial meat grinder (Autio 801, Autio Co., Astoria, OR) using a 9 mm die, mixed, and then reground through a 4 mm die. Mammary tissues and neonatal and weaned pigs were ground through a 12 mm die and again through a 3 mm die using a Stimpson grinder (Model 5412, Louisville, KY). All tissues were subsampled and a representative sample of each was placed in plastic petri dishes, sealed with tape, and frozen at - 20º C for later analysis. The saw and grinders were dismantled, washed, and dried after grinding each tissue component.

5.3.4 Analytical Methods All frozen tissue samples were freeze-dried and fat was removed by the ether extraction method (AOAC, 2000). Fat-free dry samples were ground through a 1mm screen before mineral analysis. Tissues, colostrum, and milk samples were analyzed for the major elements using the Inductively Coupled Plasma (ICP) spectroscopic method (PS 3000, Leeman Labs, Inc. Hudson, NH). Total mineral content of the individual body components were calculated by multiplying their analyzed compositional values by the components total fat-free dry weight. Total mineral content of the sow was the sum of the carcass, liver, viscera and mammary components. Blood mineral contents were not estimated, therefore no adjustments were made for its contribution to total body mineral content. Empty body weight (EBW) was the weight of the animal with the digesta and blood removed. For the neonatal and weaned pigs, liver mineral contents were determined separately from body tissue and this quantity was also added to the body content to obtain the total pig mineral content. Mineral concentrations expressed on an amount/kg EBW were calculated by dividing the total empty body mineral content by the EBW. Data were analyzed as a RCB design in three replicates using the GLM procedure (SAS Inst., Inc., Cary, NC). Individual sow and pig measurements were considered the experimental

102 unit. Parity effects were evaluated using regression analysis. Initial animals were analyzed separately as a 2 x 2 factorial. A P value of ≤ 0.05 was considered significant and a P < 0.10 was considered a trend. Least square treatment means are presented in tabular form.

5.4 Results 5.4.1 Sow and Liver Mineral Content The effects of trace mineral source and level on the weights and mineral contents of reproducing sows and their livers are presented in Table 5.1. Sow body weight at slaughter and EBW were not affected by trace mineral source or level (P > 0.10). Sow liver weights were not affected by trace mineral source, while livers seemed to be lighter when sows were fed the Ind.+Ca/P level, however the effect was not significant. Trace mineral source and level had no effect on the total macro-mineral content of the empty bodies of sows. Feeding the higher Ca and P level did not affect body Ca and P content. However the liver content of Ca, P, Na, and Cl were greater (P < 0.05) when sows were fed the organic trace minerals. Liver content of K also tended (P < 0.10) to be greater when the organic trace minerals were fed, whereas the content of Mg and S were similar for both sources. In general, liver macro-mineral content appeared to be lower when the Ind.+Ca/P level was fed, however the effect was only significant (P < 0.05) for liver P content. This can partially be explained by the slightly lower liver weight for the Ind.+Ca/P level. The trace mineral content of the total body and liver were similar for both trace mineral sources, except that the Se content (P < 0.05) in the total body and liver and the Zn content of the livers (P < 0.10) were greater for sows fed the organic trace minerals. The mineral contents presented are for the empty body and do not include the minerals present in the blood and therefore are somewhat lower than if the blood contributions were included, particularly for Fe content. The total body content of Cu, Mn, and Zn appeared to be greater when the Ind. and Ind.+Ca/P levels were fed, however the response only approached significance (P < 0.10) for Cu (Table 5.1). Body Fe content tended (P < 0.10) to be the lowest when the Ind. level was fed. Total body and liver Se content were greater (P < 0.05) when the Ind. and Ind.+Ca/P levels were fed, but the effect on total body Se content was greater when the organic source was fed, resulting in a trace mineral source x level interaction response (P < 0.01). There were no other trace mineral source x level interaction responses. The liver contents of Cu and Zn were lower at the NRC level (P < 0.05). The Fe content of the liver appeared to be somewhat lower at the 103 Ind.+Ca/P level, but the effect was not significant (P > 0.10). These results suggest that feeding higher levels of Se, Cu and Zn resulted in greater sow liver and body contents, but the effects were more variable for Fe. The change in macro-mineral content of the body components from initial breeding to parity 6 are presented in Table 5.2. The largest portions of macro-minerals were present in the carcass, followed by the viscera, mammary tissue, and liver. Total macro-mineral contents of the carcass, liver, viscera, and total empty body increased quadratically (P < 0.01) with advancing parity. Macro-mineral content of each component and the total empty body were higher at parity 1, decreased or were similar at parity 2, and generally were higher at parity 4 and 6. Mammary macro-minerals increased in a curvilinear manner (P < 0.01), whereby contents were lower at parity 2, increased at parity 4 and were slightly lower at parity 6. Mammary tissue was not collected separate from the carcass for the gilts killed for determining initial mineral composition. As with the macro-minerals, the largest proportion of trace minerals were present in the carcass (Table 5.3). However the liver had the second highest trace mineral content and the highest concentration (mg/kg) of trace minerals (data not presented). In general, the trace mineral content of each tissue component increased in a curvilinear manner (P < 0.01) with advancing parity, except for the Zn content in the carcass and empty body, which increased linearly (P < 0.01) with parity. Carcass and total empty body Cu and Fe content were lower at parity 1 than in the initial animals, but increased at parity 2 and were similar thereafter. Total empty body Se content increased to parity 2, appeared to plateau at parity 4, and then was higher again at parity 6. The Mn content of the carcass and total empty body were higher in the reproducing sows. Total empty body Se content increased from parity 1 to parity 6 when the organic trace minerals were fed (Table 5.4), with the largest increases occurring at parity 2 and 6, however when the inorganic trace minerals were fed these increases were of a lesser magnitude. This resulted in a trace mineral source x parity interaction response (P < 0.05). The interaction response was similar for liver Se content (Table 5.5), however was not significant. There were no other trace mineral source x parity interactions (P > 0.10) for empty body or liver mineral contents. This suggests that the responses for Cu, Fe, Mn, and Fe were generally consistent for both trace mineral sources over 6 parities. Additionally, there were no trace mineral level x parity interaction responses (data not presented).

104 5.4.2 Neonatal Pig and Liver Mineral Content The effects of trace mineral source and level on total mineral content in neonatal pigs and their liver are presented in Tables 5.6 and when expressed on a mg/kg tissue basis presented in Table 5.7. There were no effects (P > 0.10) of trace mineral source or level on the trace or macro-minerals contents in the liver or total body when expressed on either a total or mg/kg tissue basis. Additionally, there were no trace mineral source x level interaction responses. This suggests that the transfer of minerals to the progeny in utero was not greatly affected by the form or level of minerals fed to sows during gestation. Total neonatal body Ca and P content decreased in a curvilinear manner (P < 0.01) as parity advanced, whereby contents were lower after parity 1, particularly at parity 3 (Table 5.8). The total content of the other macro minerals were generally similar at each parity. The total content of Cu, Mn, and Zn decreased as parity advanced (P < 0.01). Iron content also appeared to be lower after parity 1, but the response was not significant (P > 0.10). When expressed on a mg/kg tissue basis (Table 5.9), all macro-mineral concentrations appeared to be somewhat lower at parity 3 and 5. Total content of the macro-mineral in the liver, except for Ca, increased in a curvilinear manner (P < 0.01) with parity (Table 5.8). These increases, however, were likely attributed to slightly heavier (P < 0.10) liver weights at parity 3 and 5, because when expressed on a mg/kg liver weight basis (Table 5.9), only K, S and Cl increased (P < 0.05) with parity. Liver Ca decreased with parity on both a total (P < 0.01) and mg/kg BW basis (P < 0.05). Neonatal liver trace mineral contents were generally similar at each parity, on both a total and wet basis. There were no parity x trace mineral source or parity x trace mineral level interaction responses for any neonatal mineral criteria.

5.4.3 Milk Mineral Content The effects of trace mineral source and level on colostrum and milk mineral content are presented in Tables 5.10. Colostrum macro-mineral content was generally higher for sows fed the organic trace minerals, except that Na and Cl were lower. However, the effect was only significant (P < 0.05) for Cl. Colostrum Ca, S, and Na contents tended to be higher (P < 0.10) when the NRC trace mineral level was fed. Iron contents of the colostrum tended (P < 0.10) to be higher when sows were fed the organic trace mineral source. Copper and Zn appeared to be higher when the inorganic source

105 was fed, however the response was not significant (P > 0.10). Trace mineral level did not effect the concentrations of Cu, Fe, Mn, or Zn in the colostrum. Sows fed the organic source had greater (P < 0.05) colostrum Se concentrations. Colostrum Se increased at the higher dietary levels when the organic source was fed, but Se concentrations were similar at all levels when the inorganic source was fed, resulting in a trace mineral source by level interaction response (P < 0.01). Colostrum macro-mineral concentrations increased in a curvilinear manner as parity advanced, however the response was only significant for P, Mg, and K (Table 5.11). The increases were more variable between trace mineral sources for colostrum Ca, K, and Na, which resulted in a trend (P < 0.10) for a trace mineral source x parity interaction response. Colostrum Zn increased quadratically (P < 0.05) to parity 4, then was lower at parity 6 (Table 5.11). Colostrum Fe appeared to decline as parity advanced for both sources, whereas Cu seemed to increase, however the effects were not significant. Concentrations of Se in the colostrum seemed to increase with advancing parity when the organic source was fed, but for the inorganic source colostrum Se decreased to parity 4 and then were higher at parity 6, resulting in a trend (P < 0.07) for a trace mineral source x parity interaction response. There were no other trace mineral source x parity interaction responses for colostrum trace mineral concentrations. Milk concentrations of Ca and P were higher in the mature milk than in the colostrum, whereas as all other minerals were generally lower in the milk (Tables 5.10), which is in agreement with previous reports (Hill et al., 1983a; Csapo et al., 1996). Milk macro-mineral contents appeared to be higher when the inorganic source was fed, however the response was only significant (P < 0.05) for Ca, P and Cl. Trace mineral level had no effect on milk macro- mineral concentrations. As in the colostrum, milk Se concentrations were greater and increased when the higher dietary levels of the organic source were fed, whereas milk Se was similar at all levels when the inorganic source was fed, resulting in a trace mineral source by level interaction (P < 0.01). Milk Cu and Zn were higher (P < 0.05) when the inorganic source was fed and were also greater (P < 0.05) at the higher trace mineral levels. Iron concentrations appeared to increase at the higher dietary levels, however the effect was not significant.

106 Milk macro-mineral concentrations, except for Na, increased quadratically as parity advanced (Table 5.12). Milk trace mineral content generally were lower at later parities, however the effect was not significant (P > 0.10).

5.4.4 Weaned Pig (17 d) Mineral Content The effects of trace mineral source and level on total body and liver mineral contents of weaned pigs (17 day old) at parity 6 are presented in Tables 5.13 and 5.14. Parity 6 pigs were used to evaluate the effects of trace mineral source and level after 6 parities on the transfer of minerals to the nursing pig. Total macro-mineral contents of pigs from sows fed the inorganic source were greater. This is likely attributed to their heavier (P < 0.05) empty BW, because when expressed on a mg/kg BW basis, there was no effect of trace mineral source on the macro- mineral concentration of the body (Table 5.14). There were no effects of trace mineral level on the macro-minerals when expressed on a total or mg/kg basis. Although the total Zn content of the pig at weaning tended (P < 0.10) to be higher when the inorganic source was fed, there was no effect (P > 0.10) of source on the total body content of Cu, Fe, or Mn (Table 5.13). However, total body content Cu, Fe, and Mn on a mg/kg basis were higher (P < 0.05) for the pigs from the sows fed the organic source, whereas Zn was not affected. This likely related to the heavier empty BW of the pigs from sows fed the inorganic trace minerals. All pigs had been administered an injection of 200 mg of Fe (iron dextran) within 12 hr of birth, which likely contributes to the similar Fe contents at weaning. Total liver macro- and trace minerals were generally higher when sows were fed the inorganic source (Table 5.13), however this is likely attributed to their slightly (P < 0.10) heavier liver weights. Therefore, liver macro-mineral concentrations were not effected by trace mineral source (Table 5.14). There also were no differences in liver body trace mineral concentrations, except that Cu seemed to be somewhat higher (P < 0.15) when the organic trace minerals were fed (Table 5.14). Total liver macro- and trace minerals were generally higher for pigs from sows fed the Ind. level, however these pigs seemed (P < 0.10) to have heavier liver weights (Table 5.13). Liver concentrations of Mn and Zn were greater (P < 0.05) when the higher trace mineral levels were fed, particularly at the Ind.+Ca/P level (Table 5.14), but Cu and Fe concentrations were similar.

107 5.5 Discussion Sow total macro- and trace mineral contents increased to parity 6. These increases seemed to primarily occur in the carcass component (Tables 5.4 and 5.5), presumably as bone and muscle mass and bone mineralization increased. The mineral contents of the liver, viscera, and mammary tissue also increased with parity, but these components made smaller contributions to total empty body mineral contents. The liver serves as the major labile storage tissue for trace minerals in the body, whereby it typically has the highest concentrations and is typically considered to be good indicator of mineral status. When mineral contents were expressed on a mg/kg tissue basis, the effects of parity on mineral concentrations were similar to the effects on total mineral values (data not presented). The total macro- and trace mineral contents of 9 month old gilts and parity 3 sows reported by Mahan and Newton (1995) are similar to our initial gilts (8 mo old) and parity 2 sows. These researchers included blood minerals in their total body content, whereas we did not, which likely explains their slightly higher values, particularly for Fe. There were no apparent effects of complete replacement of inorganic trace mineral with an organic source (mineral proteinate) on the content of macro- and trace minerals in the empty body or livers of sows. However feeding organic Se (Se yeast) did increase empty body and liver Se content compared to feeding the inorganic source (sodium selenite) particularly when the higher dietary levels were fed, which is in agreement with previous research with sows (Mahan and Kim, 1996; Mahan and Peters, 2004).The response of total empty body Se content when the organic trace minerals were fed was greater at the later parities, which may reflect the long-term incorporation of selenomethionine into muscle proteins. However, neither source demonstrated an effect on Cu, Fe, Mn, or Zn content at later parities, suggesting that for these minerals each source’s response was consistent over 6 parities. Previous experiments with grower-finisher pigs demonstrated that organic trace minerals may have higher availability (Stalder et al., 2005) when trace minerals were provided in the diet below NRC recommendations. This indicates that the proposed function of organic trace minerals to reduce mineral interactions are more evident when dietary levels are below the animals requirement, whereas when dietary levels exceed the animals physiological needs the benefits are less obvious. Feeding the Ind. and Ind.+Ca/P trace mineral levels of both sources resulted in higher sow total empty body and liver Cu and liver Zn contents. Although total empty body Se content

108 increased to greater extent when the higher dietary levels of the organic source were fed, there were no other trace mineral source by level interactions. This suggests that both trace mineral sources provided sufficient trace minerals to support sow growth and reproduction when provided at or higher than NRC recommended dietary levels. Additional Ca and P did not affect total mineral content of the sows over 6 parity period. This suggests that feeding dietary Ca and P levels in excess of NRC recommendations does not increase deposition of these minerals and/or prevent depletion of body reserves. These results in combination with our previous results (Chapter 2), which indicated that feeding the higher Ca and P levels resulted in decreased reproductive performance, suggest that higher Ca and P provide little or no advantage and possibly have detrimental effects on sow performance and their use deserves further evaluation. Neonatal pig and liver mineral concentrations were not affected by trace mineral source and level. Neonatal pig mineral reserves will reflect large differences in sow gestation dietary levels of Cu, Zn (Hill et al., 1983c), and Mn (Gamble et al., 1971), however they may be less sensitive to smaller changes in levels. Iron is not affected by dietary Fe levels, because transfer across the placenta is regulated by uteroferrin (Pond, 1961). The lack of difference in neonatal pig and colostrum mineral concentrations suggest that the trace mineral source and level provided to gestating sows did not greatly affect the transfer of minerals to the progeny in utero or in the colostrum. Colostrum and milk mineral concentrations were similar to previous reports (Csapo et al., 1996; Mahan and Newton, 1995). The greater colostrum and milk Se contents when sows were fed the organic trace mineral source is in agreement with our earlier reports (Mahan, 2000; Mahan and Peters, 2004). Milk concentrations of Ca, P, Cu and Zn were higher when sows were fed the inorganic trace mineral source. However, despite having lower milk concentrations of Cu and Zn when sows were fed organic trace minerals, the 17 day old pigs of these sows had greater empty body concentrations of Cu, Fe and Mn, and the concentration of all other minerals did not differ between sources. Furthermore, milk Ca and P contents are generally believed to be under genetic regulation and therefore not expected to be affected by the diet. This suggests that the milk from the organic treatments may have had a lower percentage of solids, which would act to dilute mineral concentrations. However, milk percentage of solids was not measured in this experiment.

109 Our results agree with previous research which demonstrated that colostrum and milk concentrations of Ca and P (Harmon et al., 1975; Mahan and Fetter, 1982) and Fe (Pond et al., 1965) do not increase when higher dietary levels of these minerals are fed to sows. Milk Cu and Zn increased when higher levels were fed to sows, which is also in agreement with previous reports (Hill et al., 1983a). These higher milk concentrations resulted in greater weaned pig and liver concentrations of Zn, as well as Mn, when the Ind. and Ind.+Ca/P treatments were fed, however liver Cu concentrations were similar at all levels.

5.6 Implications

These results suggest that except for the increased Se content when the organic trace minerals were fed, trace mineral source did not have an apparent effect on the mineral content of reproducing sows over 6 parities. However, the organic trace mineral source seemed to improve transfer of trace minerals to the nursing pig through the milk. The progeny of sows fed the higher trace mineral levels seemed to have greater trace mineral contents, however only sow body and liver Cu, Se and Zn contents increased. Feeding higher Ca and P levels had no effect on sow or pig mineral concentrations.

110

Source Level Item Organic Inorganic SEM NRC Ind Ind+Ca/P SEM No. of sows 34 34 ― 23 22 23 ― Body weight, kg 205 204 2 208 204 202 2 Empty BW, kg 199 199 2 203 199 197 3 Macro-minerals , ga Ca 1,638 1,600 51 1,580 1,618 1,659 63 P 963 946 24 935 955 973 30 K 379 370 5 381 370 372 7 Mg 47 45 1 46 46 46 1 S 252 246 3 252 248 246 4 Na 167 166 3 168 165 167 3 Cl 157 157 3 157 156 158 3 Trace minerals, mga Cub 268 271 18 235 266 309 22 Feb 3,922 4,105 165 4,019 3,697 4,326 201 Mn 53.5 57.4 5.3 50.7 60.5 55.1 6.5 Sec,d 30.9 19.5 0.4 22.4 26.9 26.3 0.5 Zn 4,693 4,581 65 4,546 4,654 4,710 80 Liver wt., kg 3.44 3.33 0.07 3.41 3.43 3.27 0.08 Macro-minerals, (liver) g Cac 0.15 0.14 0.01 0.14 0.14 0.15 0.01 Pc,d 10.87 10.22 0.21 10.96 10.74 9.94 0.26 Ke 10.29 9.84 0.19 10.31 10.17 9.72 0.23 Mg 0.60 0.58 0.01 0.60 0.59 0.57 0.01 S 7.22 6.90 0.16 7.13 7.12 6.91 0.19 Nac 2.56 2.38 0.06 2.46 2.56 2.39 0.07 Clc 3.53 3.30 0.08 3.37 3.56 3.31 0.10 Trace minerals, (liver) mg Cud 64 71 7 38 80 84 8 Fe 467 467 27 507 474 420 33 Mn 9.3 8.8 0.3 9.0 9.4 8.7 0.4 Sec,d 1.94 1.71 0.04 1.61 1.96 1.90 0.05 Znd,e 264 239 10 221 272 262 12 aQuantity of minerals in the empty body. bTrace mineral level response (P < 0.10). cTrace mineral source response (P < 0.05). dTrace mineral level response (P < 0.05). eTrace mineral source response (P < 0.10).

Table 5.1. Effects of trace mineral source and level on total liver and body mineral content of reproducing sow 111 Parity Item Tissue 0ab 1246 SEM No. of sows 20 18 18 17 15 ― Macro-minerals, g/component Ca Carcassc 1,098 1,325 1,273 1,720 2,084 79 Liverc 0.09 0.14 0.13 0.15 0.17 0.01 Viscerac 1.4 3.1 4.6 7.7 7.5 0.4 Mammaryd ― 13.1 10.5 15.3 12.4 0.6 Totalc 1,099 1,341 1,288 1,744 2,104 82 P Carcassc 636 772 756 962 1,149 37 Liverc 5.1 9.7 9.2 11.6 11.6 0.2 Viscerac 9.0 11.9 14.0 16.2 16.6 0.4 Mammaryd ― 19.5 16.0 22.4 20.2 0.6 Totalc 650 813 795 1,012 1,197 37 K Carcassc 251 283 304 339 381 8 Liverc 4.5 8.9 8.9 11.1 11.4 0.2 Viscerac 14.4 16.7 19.2 21.4 20.7 0.6 Mammaryd ― 18.4 15.4 20.7 19.3 0.6 Totalc 268 327 346 392 433 8 Mg Carcassc 32.7 36.5 36.8 45.2 51.3 1.2 Liverc 0.3 0.5 0.5 0.7 0.6 0.1 Viscerac 0.8 1.2 1.2 1.6 1.7 0.1 Mammaryd ― 1.5 1.2 1.8 1.6 0.1 Totalc 33.8 39.7 39.8 40.2 55.2 1.3 S Carcassc 164 179 196 227 262 5 Liverc 3.2 6.5 6.2 7.7 7.8 0.2 Viscerac 9.4 12.2 13.3 16.2 15.8 0.4 Mammaryd ― 11.0 9.3 12.8 12.2 0.4 Totalc 177 209 225 264 298 6 Na Carcassc 102 115 120 147 172 4 Liverc 1.31 2.32 2.22 2.59 2.75 0.07 Viscerac 8.3 10.9 11.0 15.8 14.9 0.4 Mammaryd ― 11.8 10.1 13.5 14.1 0.5 Totalc 112 140 144 179 204 4 Cl Carcassc 88 98 101 126 143 3 Liverc 1.72 3.11 2.84 3.68 4.01 0.10 Viscerac 12.2 15.8 16.7 20.9 21.8 0.5 Mammaryd ― 17.3 15.5 20.2 18.4 0.7 Totalc 102 134 136 171 187 3 aInitial gilts were killed at the initial breeding (8 months of age). bMammary tissue was not collected separate from the carcass for the initial gilts. cQuadratic parity response (P < 0.01). dCubic parity response (P < 0.01).

Table 5.2. Effect of parity on the macro-mineral content of the individual components and total body 112

Parity Item Tissue 0ab 1246 SEM No. of sows 20 18 18 17 15 ― Trace minerals, mg/component Cu Carcassc 276 115 200 165 175 20 Liverd 34 63 90 99 98 29 Viscerad 8.7 1.2 3.6 1.4 1.5 0.3 Mammaryc ― 17.5 14.1 18.4 16.8 0.7 Totale 319 197 308 284 291 31 Fe Carcassc 2,358 1,835 2,875 2,614 3,370 193 Livere 456 453 375 356 684 50 Viscerac 380 378 679 512 592 36 Mammary ― 322 319 351 340 12 Totale 3,227 2,989 4,248 3,833 4,985 198 Mn Carcass 29.9 34.5 34.1 30.6 36.8 6.8 Livere 3.5 8.0 8.6 10.4 9.2 0.4 Viscerac 4.2 6.0 7.4 7.9 8.8 0.7 Mammaryf ― 4.4 4.7 5.6 4.9 0.2 Total 37.6 52.9 54.9 54.4 59.6 6.8 Se Carcassc 16.12 14.33 19.54 18.78 22.90 0.66 Livere 0.91 1.53 1.75 1.99 2.07 0.59 Viscerae 1.59 2.12 2.72 3.30 3.93 0.15 Mammaryc ― 1.57 1.59 1.80 1.71 0.59 Totale 18.61 19.56 25.47 25.85 30.69 0.64 Zn Carcassg 2,650 3,263 3,620 4,355 5,164 90 Livere 133 210 250 237 310 16 Viscerac 107 140 181 186 213 8 Mammaryc ― 103 93 114 111 4 Totalg 2,890 3,715 4,144 4,891 5,797 91 aInitial gilts were killed at the initial breeding (8 months of age). bMammary tissue was not collected separate from the carcass for the initial gilts. cCubic parity response (P < 0.01). dQuadratic parity response (P < 0.05). eQuadratic parity response (P < 0.01). fCubic parity response (P < 0.05). gLinear parity response (P < 0.01).

Table 5.3. Effect of parity on the trace mineral content of the individual components and total body

113

Trace mineral source: Organic Inorganic Item Parity: 1 2 4 6 1 2 4 6 SEM No. of sows 9 9 8 8 9 9 9 7 ― Live weight, kga,b 177 180 217 246 173 181 206 255 4 Empty BW, kgb,c 174 174 212 238 170 177 201 249 4 Macro-minerals, g Cab 1,457 1,294 1,663 2,139 1,226 1,282 1,825 2,069 103 Pb 871 796 970 1,214 755 794 1,055 1,180 48 Kc 335 346 395 439 319 346 389 427 11 Mgb 42.3 39.8 48.4 55.9 37.2 39.8 50.0 54.4 1.6 Sb 214 223 268 301 203 227 260 295 6 Nab 142 145 176 206 139 143 183 201 5 Clb 134 138 169 188 136 134 173 186 5

114 Trace minerals, mg Cub 203 291 283 295 190 325 284 286 36 Feb 2,863 4,414 3,851 4,561 3,115 4,082 3,815 5,409 328 Mn 41.9 59.7 55.3 57.2 63.9 50.0 53.6 62.0 10.5 Seb,c,d 24.1 30.3 31.9 37.3 14.4 20.6 19.5 23.7 0.8 Zne 3,845 4,126 4,883 5,917 3,584 4,161 4,900 5,678 131 aTrace mineral source x parity interaction (P < 0.10). bQuadratic parity response (P < 0.01). cTrace mineral source x parity interaction (P < 0.05). dTrace mineral source response (P < 0.05). eLinear parity response (P < 0.01).

Table 5.4. Effects of trace mineral level and parity on body mineral content of reproducing sow

Trace mineral source: Organic Inorganic Item Parity: 1 2 4 6 1 2 4 6 SEM No. of sows 9 9 8 8 9 9 9 7 ―

Liver wt., kga 3.00 3.08 3.66 4.01 2.92 2.93 3.71 3.63 0.13 Macro-minerals, g Caa,b 0.14 0.14 0.16 0.18 0.13 0.12 0.14 0.14 0.01 Pa,b 10.04 9.57 11.91 11.95 9.39 8.85 11.33 11.34 0.42 Ka,d 8.99 9.06 11.11 12.02 8.73 8.66 11.09 10.87 0.38 Mga 0.55 0.53 0.65 0.67 0.53 0.50 0.65 0.62 0.02 Sa 6.67 6.44 7.65 8.12 6.36 6.00 7.67 7.56 0.32 Naa,b 2.37 2.34 2.63 2.91 2.26 2.11 2.55 2.60 0.12 Cla,b 3.16 2.93 3.73 4.29 3.05 2.76 3.64 3.75 0.16 115 Trace minerals, mg Cua 52 71 65 70 43 73 84 83 13 Fea 428 214 368 757 479 435 345 611 54 Mna 8.0 8.9 10.4 10.0 8.0 8.2 10.3 8.5 0.7 Sea,b 1.65 1.88 1.99 2.23 1.34 1.61 2.00 1.88 0.08 Znc,d 219 264 250 325 200 236 224 295 20 aQuadratic parity response (P < 0.01). bTrace mineral source response (P < 0.05). cCubic parity response (P < 0.01). dTrace mineral source response (P < 0.10).

Table 5.5. Effect of trace mineral level and parity on total liver mineral content of reproducing sow

Source Level Item Organic Inorganic SEM NRC Ind Ind.+Ca/P SEM No. of pigs 37 32 ― 23 24 22 ― Pig wt., kg 1.52 1.55 0.04 1.53 1.59 1.48 0.05 Macro-mineral, g/pig Ca 11.87 11.52 0.50 11.73 11.78 11.58 0.62 P 6.99 6.91 0.27 6.92 7.04 6.88 0.33 Mg 0.31 0.31 0.01 0.32 0.31 0.31 0.01 K 2.56 2.57 0.08 2.53 2.66 2.51 0.09 S 1.67 1.69 0.05 1.66 1.73 1.65 0.06 Na 2.69 2.73 0.09 2.65 2.80 2.68 0.10 Cl 3.01 3.04 0.09 2.94 3.15 2.99 0.12 Trace mineral, mg/pig Cu 3.64 4.03 0.20 3.84 4.05 3.62 0.24 Fe 54.60 55.92 2.95 52.42 59.83 53.53 3.62 Mn 0.86 0.87 0.05 0.87 0.92 0.81 0.06 Zn 27.73 26.54 1.08 26.59 27.85 26.98 1.34 Liver wt., g 49.41 52.65 2.43 50.73 52.10 50.27 3.00 Macro-mineral, mg/liver Ca 6.2 5.7 0.8 6.2 6.7 5.0 1.0 P 82.0 86.4 4.2 86.0 81.6 85.0 5.2 Mg 5.3 5.8 0.3 5.7 5.4 5.6 0.4 K 92.6 103.6 5.7 96.8 98.4 99.1 7.1 S 47.1 50.7 2.4 50.5 47.8 48.3 2.9 Na 62.9 64.8 3.6 66.0 64.5 61.0 4.5 Cl 158.1 166.5 10.5 167.7 161.3 157.8 13.1 Trace mineral, mg/liver Cua 1.76 2.04 0.14 2.05 1.88 1.77 0.17 Fe 10.13 10.75 0.74 10.73 10.05 10.54 0.91 Mna 0.04 0.05 0.003 0.04 0.04 0.04 0.003 Zn 4.51 4.27 0.41 4.56 4.08 4.52 0.52 aTrace mineral source response (P < 0.15).

Table 5.6. Effects of trace mineral source and level on total body and liver mineral contents of neonatal pigs

116

Source Level Item Organic Inorganic SEM NRC Ind Ind.+Ca/P SEM No. of pigs 37 32 ― 23 24 22 ― Pig wt., kg 1.52 1.55 0.04 1.53 1.59 1.48 0.05 Macro-mineral, g/kg BW Ca 7.79 7.40 0.22 7.62 7.43 7.73 0.28 P 4.59 4.44 0.11 4.51 4.43 4.60 0.13 Mg 0.20 0.20 0.01 0.21 0.20 0.21 0.01 K 1.69 1.66 0.02 1.66 1.67 1.69 0.03 S 1.10 1.09 0.01 1.09 1.09 1.11 0.02 Na 1.76 1.77 0.02 1.73 1.76 1.80 0.03 Cl 1.98 1.97 0.03 1.93 1.97 2.01 0.04 Trace mineral, mg/kg BW Cu 2.4 2.6 0.1 2.5 2.6 2.4 0.1 Fe 35.9 36.1 1.5 35.0 37.3 35.7 1.9 Mn 0.58 0.56 0.03 0.58 0.59 0.54 0.04 Zn 18.3 17.1 0.5 17.3 17.6 18.1 0.7 Liver wt., g 49.4 52.7 2.4 50.7 52.1 50.3 3.0 Macro-mineral, g/kg liver Ca 0.13 0.12 0.02 0.13 0.14 0.10 0.02 P 1.67 1.66 0.05 1.71 1.58 1.71 0.07 Mg 0.11 0.11 0.01 0.11 0.10 0.11 0.01 K 1.86 1.94 0.05 0.10 0.10 0.10 0.01 S 0.95 0.96 0.02 0.98 0.93 0.96 0.03 Na 1.26 1.24 0.05 1.27 1.27 1.21 0.06 Cl 3.11 3.15 0.13 3.16 3.13 3.09 0.16 Trace mineral, mg/kg liver Cu 36.2 37.8 2.0 38.8 35.8 36.3 2.5 Fe 196 198 11 199 186 206 14 Mn 0.83 0.87 0.04 0.84 0.80 0.90 0.05 Zn 86 80 7 80 81 89 8

Table 5.7. Effects of trace mineral source and level on relative mineral content of neonatal pigs and livers

117

Parity 1 3 5 SEM No. of pigs 23 24 22 ― Pig wt., kg 1.51 1.54 1.56 0.05 Macro-mineral, g/pig Caa 13.12 10.87 11.11 0.60 Pa 7.61 6.54 6.69 0.32 Mg 0.32 0.31 0.31 0.01 K 2.58 2.53 2.58 0.09 S 1.69 1.66 1.69 0.06 Na 2.80 2.66 2.67 0.10 Cl 3.02 2.94 3.12 0.11 Trace mineral, mg/pig Cua 4.34 3.38 3.80 0.24 Fe 59.22 55.45 51.07 3.53 Mnb 1.00 0.82 0.77 0.05 Zna 31.17 25.18 25.06 1.30 Liver wt., gc 46.1 54.9 52.1 2.9 Macro-mineral, mg/ liver Cac 7.27 6.12 4.54 0.92 Pb 74.59 86.01 91.96 5.05 Mgb 4.94 5.69 6.03 0.34 Ka 82.38 111.64 100.26 6.89 Sa 40.85 52.51 53.26 2.85 Naa 56.17 72.30 62.96 4.37 Cla 120.16 198.64 168.01 12.68 Trace mineral, mg/ liver Cu 1.83 1.87 2.01 0.16 Fe 9.77 11.95 9.60 0.91 Mn 0.04 0.04 0.05 0.01 Zn 4.63 4.57 3.95 0.51 aQuadratic parity response (P < 0.01). bLinear parity response (P < 0.01). cQuadratic parity response (P < 0.10).

Table 5.8. Effects of parity on mineral content of neonatal pigs and livers

118

Parity 1 3 5 SEM No. of pigs 23 24 22 ― Pig wt., kg 1.51 1.54 1.56 0.05 Macro-mineral, g/kg BW Caa 8.69 6.95 7.14 0.27 Pa 5.04 4.20 4.30 0.13 Mga 0.21 0.20 0.20 0.01 K 1.71 1.65 1.66 0.02 S 1.12 1.08 1.09 0.02 Naa 1.86 1.72 1.72 0.03 Clb 2.01 1.90 2.01 0.04 Trace mineral, mg/kg BW Cua 2.85 2.20 2.45 0.13 Fea 34.95 37.26 35.72 1.86 Mna 0.66 0.55 0.49 0.04 Zna 20.56 16.35 16.17 0.64 Liver wt., gb 45.1 54.9 52.1 2.9 Macro-mineral, g/kg liver Cac 0.16 0.12 0.09 0.02 P 1.63 1.61 1.77 0.06 Mg 0.11 0.11 0.12 0.01 Ka 1.77 2.00 1.93 0.06 Sc 0.89 0.96 1.02 0.03 Na 1.23 1.31 1.21 0.05 Cla 2.59 3.55 3.24 0.15 Trace mineral, mg/kg liver Cu 38.21 34.05 38.64 2.37 Fe 206.9 200.5 183.6 13.7 Mn 0.89 0.79 0.86 0.05 Znb 95.9 77.6 75.5 7.9 aQuadratic parity response (P < 0.05). bQuadratic parity response (P < 0.10). cLinear parity response (P < 0.05).

Table 5.9. Effect of parity on relative mineral content of neonatal pigs and livers

119

Source Level Item Organic Inorganic SEM NRC Ind Ind+Ca/P SEM No. of samples 36 35 ― 24 24 23 ― Colostrum Macro-minerals Ca, mg/kga 902 873 31 934 918 810 40 P, mg/kg 1,365 1,313 24 1,340 1,379 1,298 31 Mg, mg/kg 108 103 3 110 102 104 3 K, mg/kga 1,336 1,270 34 1,227 1,317 1,365 44 S, mg/kga 950 890 46 1,034 876 849 59 Na, mg/kga 654 738 46 801 675 612 59 Cl, mg/mlb 937 1031 24 1,026 952 973 30 Trace minerals Cu, mg/kg 2.46 2.71 0.21 2.58 2.66 2.50 0.27 Fe, mg/kgc 1.88 1.63 0.10 1.98 1.61 1.68 0.13 Mn, mg/kg 0.014 0.004 0.001 0.013 0.002 0.013 0.01 Se, mg/kgb 0.15 0.10 0.01 0.12 0.14 0.13 0.01 Zn, mg/kg 14.49 14.90 0.78 15.80 15.11 13.16 1.00

Milk, 17d Macro-minerals Ca, mg/kgb 1,830 2,058 42 1,920 2,005 1,907 55 P, mg/kgb 1,429 1,515 17 1,490 1,476 1,449 22 Mg, mg/kg 113 112 3 116 110 112 4 K, mg/kg 808 820 18 821 815 806 24 S, mg/kgc 498 527 11 515 516 508 14 Na, mg/kg 326 351 14 323 364 328 18 Cl, mg/mla,b 577 647 24 571 671 593 31 Trace minerals Cu, mg/kgb,d 0.40 0.58 0.04 0.37 0.51 0.58 0.05 Fe, mg/kgc 1.38 1.70 0.12 1.33 1.68 1.62 0.16 Mn, mg/kg 0.04 0.03 0.01 0.04 0.03 0.04 0.01 Se, mg/kg,b,d 0.07 0.04 0.01 0.05 0.06 0.06 0.01 Zn, mg/kgb,d 5.73 8.20 0.19 6.30 7.90 6.70 0.25 aTrace mineral level response (P < 0.10). bTrace mineral source response (P < 0.05). cTrace mineral source response (P < 0.10). dTrace mineral level response (P < 0.05).

Table 5.10. Effects of trace mineral source and level on colostrum and milk mineral concentrations 120 Parity Item Trace mineral source 1 2 4 6 SEM No. of samples Organic 9 9 9 9 ― Inorganic 9 9 9 8 ― Macro-minerals Ca, mg/kga Organic 881 956 782 988 60 Inorganic 832 845 949 864 P, mg/kgb Organic 1,267 1,374 1,422 1,396 47 Inorganic 1,239 1,280 1,493 1,242 Mg, mg/kgb Organic 116 115 93 106 5 Inorganic 125 119 98 107 K, mg/kga, b Organic 1,431 1,353 1,215 1,343 66 Inorganic 1,390 1,265 1,321 1,105 S, mg/kg Organic 781 902 1,143 973 88 Inorganic 838 929 826 966 Na, mg/kga,c Organic 619 630 660 708 88 Inorganic 618 799 591 1,007 Cl, mg/mld Organic 987 972 919 870 45 Inorganic 1,018 1,034 967 1,104

Trace minerals Cu, mg/kg Organic 2.28 2.18 2.89 2.48 0.39 Inorganic 3.062.80 2.26 2.71 Fe, mg/kge Organic 2.10 1.88 1.90 1.63 0.19 Inorganic 1.761.73 1.69 1.34 Mn, mg/kg Organic 0.02 0.02 0.01 0.02 0.01 Inorganic 0.020.01 0.01 0.01 Se, mg/kga,f Organic 0.15 0.16 0.17 0.16 0.01 Inorganic 0.120.11 0.09 0.12 Zn, mg/kga,b Organic 10.41 13.64 18.33 15.57 1.46 Inorganic 13.15 15.40 14.15 16.88 aTrace mineral source x parity response (P < 0.10). bQuadratic parity response (P < 0.05). cCubic parity response (P < 0.05). dTrace mineral source response (P < 0.05). eTrace mineral source response (P < 0.10). fTrace mineral source x parity response (P < 0.05).

Table 5.11. Effects of trace mineral source and parity on colostrum mineral concentrations 121

Parity Item Trace mineral source 1 2 4 6 SEM No. of sows Organic 9 9 9 9 ― Inorganic 9 9 9 8 ― Macro-minerals Ca, mg/kga,b,c Organic 1,775 1,956 1,643 1,946 81 Inorganic 2,274 2,142 1,767 2,049 P, mg/kg a,b,c Organic 1,315 1,404 1,560 1,438 32 Inorganic 1,483 1,519 1,566 1,490 Mg, mg/kga Organic 116 120 103 114 6 Inorganic 125 119 97 107 K, mg/kga Organic 759 742 808 924 35 Inorganic 754 787 845 894 S, mg/kgc,d Organic 484 506 512 490 20 Inorganic 568 542 503 498 Na, mg/kg Organic 316 352 276 362 27 Inorganic 380 354 325 343 Cl, mg/mlb,e Organic 576 611 539 580 46 Inorganic 754 661 539 589 Trace minerals Cu, mg/kg,b Organic 0.45 0.60 0.28 0.28 0.09 Inorganic 0.750.74 0.29 0.51 Fe, mg/kgd Organic 1.42 1.49 1.21 1.41 0.24 Inorganic 2.061.80 1.53 1.42 Mn, mg/kg Organic 0.03 0.05 0.04 0.03 0.01 Inorganic 0.040.02 0.04 0.03 Se, mg/kgb,e Organic 0.08 0.07 0.07 0.07 0.01 Inorganic 0.050.04 0.04 0.04 Zn, mg/kgb,e Organic 5.62 5.19 6.41 5.70 0.35 Inorganic 8.338.30 8.53 7.63 aQuadratic parity response (P < 0.05). bTrace mineral source response (P < 0.05). cTrace mineral source x parity response (P < 0.10). dTrace mineral source response (P < 0.10). eQuadratic parity response (P < 0.10).

Table 5.12. Effects of trace mineral source and parity on milk mineral concentrations 122

Source Level Item Organic Inorganic SEM NRC Ind. Ind+Ca/P SEM No. of pigs 17 15 ― 12 10 10 ― Live wt., kga 6.56 7.41 0.28 6.59 7.27 7.05 0.35 Empty BW, kga 6.46 7.29 0.28 6.49 7.18 6.96 0.35 Macro-mineral, g/pig Caa 40.24 47.34 2.07 40.29 45.27 45.82 2.55 Pa 28.80 33.37 1.38 29.02 32.36 31.87 1.70 Mga 1.59 1.82 0.07 1.58 1.77 1.77 0.09 Kb 14.41 16.15 0.62 14.71 15.91 15.22 0.76 Sb 9.05 10.17 0.39 9.04 9.99 9.80 0.49 Naa 7.26 8.22 0.32 7.42 8.03 7.78 0.40 Cla 8.81 10.03 0.41 9.30 10.05 8.93 0.51 Trace mineral, mg/pig Cu 23.86 23.65 1.37 21.12 26.05 24.10 1.68 Fec 198.6 191.4 7.3 182.2 196.7 206.2 8.9 Mnd 5.04 4.26 0.49 2.50 4.38 7.07 0.60 Znb,c 139.6 157.9 7.1 134.8 153.0 158.4 8.7 Liver wt., gb 170.7 188.9 7.6 174.2 191.6 173.7 8.7 Macro-mineral, mg/liver Cac 9.7 9.7 0.6 9.1 11.1 8.9 0.8 P 488 522 23 482 547 486 27 Mgc 30.4 32.7 1.6 29.6 35.2 29.9 1.9 K 467 504 22 469 515 472 27 S 271 287 15 264 311 262 18 Na 158 165 12 152 180 152 15 Cl 448 477 29 432 521 435 36 Trace mineral, mg/liver Cu 14.42 13.71 1.22 12.17 16.30 13.73 1.50 Fe 21.48 26.84 2.79 19.64 29.79 23.05 3.53 Mn 0.35 0.40 0.04 0.31 0.44 0.38 0.05 Zn 22.89 23.69 2.10 20.33 25.16 24.39 2.57 aTrace mineral source response (P < 0.05). bTrace mineral source response (P < 0.10). cTrace mineral level response (P < 0.15). dTrace mineral level response (P < 0.01).

Table 5.13. Effects of trace mineral source and level on empty body and liver mineral content of 17 day old pigs

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Source Level Item Organic Inorganic SEM NRC Ind. Ind.+Ca/P SEM No. of pigs 17 15 - 12 10 10 - Macro-mineral, g/kg EBW Ca 6.21 6.45 0.15 6.17 6.27 6.56 0.19 P 4.45 4.55 0.08 4.46 4.48 4.57 0.10 Mga 0.25 0.25 0.01 0.24 0.25 0.25 0.01 Ka 2.23 2.22 0.02 2.27 2.22 2.19 0.03 S 1.40 1.39 0.01 1.39 1.39 1.41 0.02 Na 1.13 1.13 0.01 1.14 1.12 1.12 0.02 Clb 1.36 1.38 0.02 1.43 1.40 1.28 0.03 Trace mineral, mg/kg EBW Cuc 3.71 3.24 0.14 3.32 3.62 3.49 0.18 Fec 30.85 26.32 0.83 28.46 27.58 29.71 1.02 Mnb,c 0.78 0.58 0.07 0.41 0.61 1.02 0.09 Znb 21.62 21.45 0.42 20.81 21.16 22.64 0.51

Macro-mineral, mg/kg liver Ca 0.06 0.05 0.01 0.05 0.06 0.05 0.01 P 2.86 2.77 0.07 2.79 2.84 2.81 0.08 Mg 0.18 0.17 0.01 0.17 0.18 0.17 0.01 K 2.74 2.66 0.06 2.71 2.67 2.73 0.08 S 1.58 1.52 0.04 1.53 1.61 1.51 0.05 Na 0.92 0.87 0.05 0.88 0.94 0.87 0.06 Cl 2.61 2.53 0.12 2.49 2.73 2.49 0.14 Trace mineral, mg/kg liver Cud 83.9 72.1 5.4 71.7 84.0 78.4 6.7 Fe 121.1 141.2 13.4 108.9 156.3 128.2 17.0 Mn 2.03 2.14 0.19 1.80 2.30 2.16 0.24 Zn 133.1 120.8 10.3 119.4 124.4 137.0 12.7 aTrace mineral level response (P < 0.15). bTrace mineral level response (P < 0.05). cTrace mineral source response (P < 0.05). dTrace mineral source response (P < 0.15).

Table 5.14 Effects of trace mineral source and level on the relative amount of minerals in the liver and empty body of 17 day old pigs

124

CHAPTER 6

LONG-TERM EFFECTS OF DIETARY ORGANIC OR INORGANIC TRACE MINERALS ON THE BODY MINERAL COMPOSITION OF NON-REPRODUCING GILTS

6.1 Abstract A total of 90 gilts were used in this study to evaluate the effects of dietary trace mineral source and level on the total body mineral content of females maintained in a non-gravid state until 35 months of age. The experiment was conducted as a 2 x 3 x 5 factorial arrangement of treatments in a randomized complete block design in 3 replicates. Organic and inorganic sources of trace minerals were evaluated at three dietary levels. The first level met NRC recommendations (NRC), while the second level was set at a level that was considered commonly recommended by universities and the commercial feed industry (Ind.). Two additional treatments evaluated the Ind. level of both sources, but with additional Ca and P (Ind+Ca/P). Five gilts from the NRC and Ind. levels of each trace mineral source were killed at 8 mo of age. Three gilts per treatment were killed for determination of liver and total body mineral content after weaning at approximately 12, 17, 26, and 35 months of age. Total empty body and liver macro-mineral quantities were not affected by trace mineral source and level. Total body content of Cu was lower (P < 0.05) when organic trace minerals were fed and body Fe was greater (P < 0.05) at each level of the organic source. Total body and liver contents of Cu increased at the Ind. and Ind.+Ca/P levels of both sources. The quantities of total body Mn and Zn were not affected by trace mineral source or level, but liver Mn and Zn contents increased (P < 0.05) at the Ind. level, but not the Ind.+Ca/P level of both source. Total sow body Se content were greater (P < 0.05) when the organic source and the Ind. and Ind.+Ca/P levels were fed. Total Ca and P content was not affected by feeding the Ind.+Ca/P level of either source. Body and liver weights and macro-mineral contents increased linearly (P < 0.01) with

125 advancing age for gilts fed both trace mineral sources. Body and liver Cu increased in a curvilinear manner (P < 0.05), with increases to 26 months. Body Fe content increased in a cubic manner (P < 0.05), with a large increase at 17 months, a decrease at 26 months, and large increase again at 35 months, whereas liver Fe increased linearly (P < 0.01). Body and liver Zn content increased linearly (P < 0.01), whereas Mn content was relatively constant (P > 0.10) from 8 to 35 mo of age.

6.2 Introduction Mahan and Newton (1995) demonstrated that sow mineral content increased when gilts were maintained in a non-gravid state from 9 to 24 months of age. Reproducing females which had completed 3 parities had lower total mineral quantities then the contemporary females at 24 months of age suggesting that sow mineral reserves declined over several reproductive cycles Our previous research has demonstrated that trace mineral source or level had little effect on sow empty body mineral content over a 6 parity period (Chapter 5), although liver Cu and Zn content did increased when higher trace mineral levels were fed. By maintaining gilts in a non-gravid state, the effects of trace mineral source and level on body mineral content can be evaluated in adult females without including the effects reproduction. This experiment evaluated liver and empty body mineral content of females maintained in a non-gravid state up to 35 months of age when diets contained either inorganic or organic sources of trace minerals provided at or above 1998 NRC recommended levels. The effects of feeding higher trace mineral levels in conjunction with higher Ca and P levels were also evaluated.

6.3 Materials and Methods 6.3.1 Experimental Design and Treatments A total of 90 gilts selected were used in this study to evaluate the effects of dietary trace mineral source and level on the total body mineral contents of females maintained in a non- gravid state over 35 months (six parity period). The experiment was conducted as a 2 x 3 x 5 factorial arrangement of treatments in a randomized complete block (RCB) design in 3 replicates. The first factor evaluated trace mineral forms (organic or inorganic). The second factor evaluated trace mineral levels. The first level evaluated the established NRC (1998) recommendations (NRC), while the second evaluated a higher trace mineral level that was considered commonly recommended by universities and the commercial feed industry (Ind.). 126 The organic minerals (Bioplex) were provided by Alltech Inc. (Nicholasville, KY) and were metal proteinates, whereby trace minerals were chelated with partially hydrolyzed soybean protein, while the organic Se source was Se yeast (Sel-plex). Most inorganic trace minerals were provided in the sulfate form, but the Se source was sodium selenite. A third level (Ind.+Ca/P) evaluated providing additional Ca and P with the higher trace mineral level of each source, but only after 8 months age not incorporated until 8 months of age, when contemporary reproducing sows were initially breed. The Ca and P levels in these latter treatments were 1.00% and 0.75% (total). Diet composition, feeding programs, and management conditions were described previously (Chapter 2). Twenty gilts (5/treatment) were killed at 8 mo of age for determination of initial mineral composition. Only the NRC and Ind. levels of each trace mineral source were fed during development because the additional 2 treatments containing higher Ca and P levels were not incorporated until 8 months of age. Gilts which were allotted to the Ind.+Ca/P treatments had been fed the Ind. trace mineral level of their respective trace mineral source during development (Chapter 2) The gilts used in this experiment were contemporaries of the reproducing females used in Chapter 2. At approximately 8 mo of age, 12 gilts from each of the 6 dietary treatments were selected to remain in a non-gravid state. During gestation, non-gravid females were fed the same treatment diets and at the same quantity/d as the reproducing females during each parity (Chapter 2). When the reproducing females were lactating, the non-gravid females continued to be fed the same diet at the gestation level until the reproducing female group was rebred. At approximately 12, 17, 26, and 35 months of age, three gilts per dietary treatment were killed for determination of body mineral content. These ages correspond to parities 1, 2, 4, and 6, respectively. Slaughter and laboratory procedures were described previously (Chapter 5). Total mineral contents of the individual body components were calculated by multiplying their analyzed compositional values by the components total fat-free dry weight. Total empty body mineral content of the non-gravid gilt was the sum of the carcass, liver, viscera and mammary components. Only the total mineral contents of the animal and livers are presented. Blood mineral contents were not estimated, therefore no adjustments were made for its contribution to total body mineral content.

127

6.3.2 Statistical Analysis Data were analyzed as a RCB design in three replicates using the GLM procedure (SAS Inst., Inc., Cary, NC). The model included the effects of trace mineral source and level, age, source x age interactions, level x age interactions, and blocks (replicates). Individual gilt was considered the experimental unit. Age effects were evaluated using regression analysis. Initial animals were analyzed separately as a 2 x 2 factorial. A P value of ≤ 0.05 was considered significant and a P < 0.10 was considered a trend. Least square treatment means are presented in tabular form.

6.4 Results The effect of trace mineral source and level on total mineral contents of the body and liver in the gilts at 8 mo. of age are presented in Table 6.1 and the non-gravid gilts from 12 to 35 mo. of age are presented in Table 6.2. Because the Ind.+Ca/P level treatments were not incorporated until the contemporary sows’ initial breeding (8 mo.), they were initially fed the Ind. level of their respective trace mineral source during development (Chapter 2). Consequently, only the NRC and Ind. levels of organic and inorganic trace mineral were fed until 8 months of age. Gilt live and empty body weights were not affected by trace mineral source or level in the 8 month old gilts (Table 6.1) or gilts from 12 to 35 months (Table 6.2). Liver weights of the 8 month old gilts seemed to be higher for gilts fed the organic trace minerals, however the effects were not significant (P > 0.10). For the gilts aged 12 to 35 months (Table 6.2), liver weights of the organic Ind. and inorganic Ind.+Ca/P treatments were similar, but they were higher than the other treatments, resulting in a source x level interaction response (P < 0.05). Total empty body macro-mineral quantities in the 8 month old gilts (Table 6.1) and gilts from 12 to 35 months (Table 6.2) were not affected by trace mineral source and level. Feeding additional Ca and P did not affect the content of these minerals in the bodies of the older gilts (Table 6.2). In the older gilts, liver macro-mineral contents seemed to reflect the trace mineral source x level interaction response for liver weight, however the source x level interaction was only significant for Cl (P < 0.05). There were no other trace mineral source x level interactions. For both sets of gilts, total body Cu contents were greater (P < 0.05) when the inorganic trace mineral source was fed. Liver Cu contents appeared to be somewhat higher (P > 0.10) for the inorganic source in the 8 month old gilts (Table 6.1), however the effect was significant (P < 128 0.05) for the gilts from 12 to 35 months of age (Table 6.2). Total and liver Cu contents of the older gilts increased (P < 0.05) at the Ind. level and increased further when additional Ca and P were fed, particularly for the inorganic source, however the interaction was not significant (Table 6.2). There were no trace mineral source x level interaction responses for total or liver Cu contents. In the gilts from aged 12 to 35 months (Table 6.2), empty body Fe was greater (P < 0.05) at each level of the organic trace mineral source, however there was no effect of trace mineral source or level in the gilts at 8 month (Table 6.1). These results suggest that the effect of trace mineral source on total body Fe content were more apparent as the gilts aged. The effects of trace mineral source and level on liver Fe contents were not consistent between the two groups of gilts. The gilts at 8 months of age (Table 6.1), liver Fe content was greater when the NRC level of inorganic trace minerals was fed compared to the Organic NRC treatment, but at the Ind. level of the inorganic was lower, resulting in a trend (P < 0.10) for a trace mineral source x level interaction response. However in the older gilts, liver Fe content was greater at the NRC and Ind. levels of inorganic minerals, whereas content was lower for the Inorganic Ind.+Ca/P, resulting in a trend (P < 0.10) for a source x level interaction response (Table 6.2). Total body Mn and Zn contents were not affected by trace mineral source or level in either set of gilts. The total body quantity of Mn seemed to be somewhat greater (P < 0.10) at the Ind. level in the 8 month old gilts (Table 6.1), but not for the older gilts (Table 6.2). The quantity of liver Mn and Zn was not affected by trace mineral source or level in the 8 month old gilts (Table 6.1). However in the older gilts, liver Mn and Zn contents were highest (P < 0.05) when gilts were fed the Ind. trace mineral level (Table 6.2). There were no trace mineral source x level interaction responses for total or liver contents of Mn and Zn. For both sets of gilts, total body Se content was greater (P < 0.05) at each level when the organic trace minerals were fed, however liver Se content was not effected by source. Total body and liver Se contents were greater when the higher levels were fed. However for the gilts aged 12 to 35 months (Table 6.2) total body Se increased to a greater extent when the Ind. and Ind.+Ca/P levels of the organic source were fed, resulting in a trace mineral source x level interaction response (P < 0.05). The effects of trace mineral source x parity on total body mineral content are presented in Table 6.3 and Table 6.4, respectively. Live and empty body weights and liver weights

129 increased linearly (P < 0.01) with advancing age. Body and liver macro-mineral increased linearly (P < 0.01) with advancing age for gilts fed both trace mineral sources. Liver macro- mineral content was greater at 35 months for gilts fed the inorganic minerals, resulting in a trace mineral source x parity interaction effect for Ca (P < 0.05) and Mg (P < 0.10). However, this is largely because of the heavier liver weights for the inorganic source at 35 months of age. These results suggest that when maintained in a non-gravid state, bone growth and mineralization likely continued to 35 months of age. Total empty body and liver Cu increased in a curvilinear manner (P < 0.05), with increases to 26 months (Table 6.4). Empty body Fe content increased in a cubic manner (P < 0.05), with a large increase at 17 months, a decrease at 26 months, and large increase again at 35 months, whereas liver Fe increased linearly (P < 0.01). The contents of Se and Zn in the total empty body and liver increased linearly (P < 0.01), whereas Mn content only increased somewhat (P > 0.10) from 8 to 35 mo of age. There were no trace mineral level x parity interaction responses for trace or macro-mineral content in the total body or liver.

6.4 Discussion The content of all minerals increased from 8 to 35 months of age, which is consistent with the research of Mahan and Newton (1995). Gilt weights and quantities of many minerals (i.e. Ca, P, and Zn) at 35 months of age were nearly two times that of the 8 month old gilts. These increases indicate continued bone mineralization to 35 months of age, in addition to increases in both bone and lean tissue mass. Feeding organic Se (Se yeast) increased total empty body Se content compared to feeding the inorganic source (sodium selenite) particularly when the higher dietary levels were fed, which is in agreement with previous research with reproducing sows (Mahan and Kim, 1996; Mahan and Peters, 2004) and grower-finisher pigs (Mahan and Parrett, 1996; Mahan et al., 1999). This increased content is likely the result of long-term selenomethionine incorporation into muscle proteins. In contrast, however, liver Se content was similar at each level for both trace mineral sources. Because these were adult non-reproducing females, it unclear if their liver protein metabolism was decreased compared to growing or reproducing females, thus resulting in decreased selenomethionine incorporation into liver proteins. Total body Cu content was lower (P < 0.05) when organic trace minerals were fed, but body Fe was greater (P < 0.05) at each level of the organic source. Daily feed intake levels were

130 equal for all treatments within each parity (Chapter 2), therefore mineral intakes were similar for both sources at each dietary level. This suggests that the differences in Cu and Fe contents between the trace mineral sources may have been related to mineral availability. To our knowledge the effects of organic trace minerals on mineral retention in adult females has not been previously evaluated. However in weanling pigs, Cu absorption and retention was greater when 100 ppm of Cu from a Cu-proteinate was fed, compared to when 250 ppm of Cu sulfate (Veum et al., 2004). It is unclear if the discrepancy between experiments is because the organic Cu source used in this trial had a lower bioavailability or if mineral interactions reduced the availability of the organic Cu source we used. Total body and liver Cu contents were greater (P < 0.05) for both trace mineral sources when the Ind.+Ca/P level was fed compared to the Ind. level, whereas liver Zn contents increased (P < 0.05) at the Ind. level, but not the Ind.+Ca/P level. These dietary treatments only differed in their Ca and P content, which suggests that interactions involving trace and macro- minerals were possibly occurring, which may have affected the availability of these trace minerals. Some researchers have suggested that phytate P may increase Cu absorption by removing the antagonist effects of Zn (Lee et al., 1988). Previous research (Morris and Ellis, 1980) has demonstrated that interactions between Zn and dietary P, independent of phytate P, may also reduce Zn absorption. In mucosal and hepatic cells, metallothionein binds both Zn and Cu. Saturation by one of these minerals can reduce the absorption and retention of the other mineral (Harris, 1997). Zinc induces metallothionein synthesis more so than Cu (Oestreicher and Cousins, 1985), but metallothionein has a higher affinity for the Cu ion. At high Zn intakes, metallothionein content would therefore increase, but would preferentially bind Cu and prevent its release from the erythrocyte. Therefore a reduction in Zn absorption as the result of interactions with dietary P, could potentially explain the increase in Cu content at the Ind.+Ca/P level, whereas Zn decreased. Total body and liver Ca and P contents were not affected by feeding the Ind.+Ca/P level of either source. It is unlikely that interactions between the trace and macro-minerals had a large effect on the availability of the macro-minerals, largely because of the magnitude of their dietary concentrations. These results suggest that because the gilts did not require nutrients to support reproduction, their Ca and P demands were likely lower than if they were reproducing

131 and that NRC (1998) Ca and P recommendations were sufficient. Dietary vitamin D levels (223 IU) were in excess of NRC (1998) recommendations in all treatment diets, however it is unclear if higher vitamin D levels would have had any effect.

6.5 Implications

When organic trace minerals were fed to non-gravid gilts, total body and liver Cu content were lower, but total body Fe content was greater. Trace mineral source did not affect Mn or Zn contents. Total body and liver Cu contents were higher at the dietary levels, whereas liver contents of Mn and Zn were higher at the Industry trace mineral level. Total empty body and liver macro-mineral quantities were not affected by trace mineral source and level. The content of all minerals increased between 8 and 35 months of age.

132

Trace mineral source: Organic Inorganic Item Trace mineral level: NRC Ind NRC Ind SEM No. of gilts 10 10 10 10 ― Body weight, kg 150 139 145 141 6 Empty BW, kg 144 136 141 140 1 Macro-minerals, ga Ca 950 1,126 1,074 1,248 112 P 591 649 648 712 51 K 271 254 282 264 12 Mg 32 34 34 35 2 S b 182 169 185 170 8 Na 111 109 115 111 6 Cl 100 103 105 103 5 Trace minerals, mga Cuc,d 116 305 320 491 85 Fe 2,762 3,333 3,444 3,273 235 Mnc 33 39 36 42 3 Sec,d 17 24 15 18 2 Zn 2,785 2,817 3,009 2,948 107

Liver wt., kg 1.69 1.55 1.54 1.49 0.09 Macro-minerals, g Ca 0.09 0.09 0.09 0.08 0.01 P b 5.82 4.97 5.10 4.57 0.37 Kc 5.25 4.31 4.61 4.00 0.32 Mg 0.31 0.27 0.28 0.25 0.02 S 3.56 3.19 3.18 2.91 0.21 Na 1.34 1.30 1.29 1.30 0.11 Cl 1.82 1.66 1.73 1.66 0.14 Trace minerals, mg Cu 24.4 29.3 27.2 38.4 7.1 Fee 434 472 567 350 67 Mn 4.0 3.4 3.3 3.2 0.3 Seb 0.90 1.00 0.820.92 0.06 Zn 150 145 119 120 18 aQuantity of minerals in the empty body. bTrace mineral level response (P < 0.10). cTrace mineral level response (P < 0.05). dTrace mineral source response (P < 0.05). eTrace mineral source x level interaction response (P < 0.10).

Table 6.1. Effect of trace mineral source and level on total liver and body mineral content of gilts at 8 months of age

133 TM Source: Organic Inorganic Item TM Level: NRC Ind. Ind+Ca/P NRC Ind. Ind+Ca/P SEM No. of gilts 12 12 11 11 12 12 ― Body weight, kg 222 227 228 230 232 235 6 Empty BW, kg 217 223 223 226 227 230 6 Macro-minerals, ga Ca 1,763 1,679 1,840 1,667 1,749 1,771 108 P 1,029 987 1,058 976 1,026 1,044 49 K 393 372 375 374 375 397 10 Mg 50 47 48 47 47 50 2 S 269 257 257 259 261 273 7 Na 156 151 160 153 156 163 5 Cl 140 138 140 136 144 146 4 Trace minerals, mga Cub,c,d,e 231 294 316 289 323 498 45 Feb 4,316 4,537 4,278 3,875 3,937 3,718 190 Mn 42 55 40 37 38 38 8 Seb,c,f 26 34 32 19 19 20 1 Zn 4,999 4,967 4,821 4,726 5,026 5,065 111 Liver wt., kgf 2.08 2.32 2.07 2.12 2.19 2.32 0.07 Macro-minerals, g Ca 0.10 0.10 0.10 0.11 0.10 0.11 0.01 P 6.68 7.09 6.85 7.10 7.11 7.30 0.24 K 6.37 7.02 6.31 6.51 6.66 6.92 0.23 Mgg 0.38 0.42 0.38 0.40 0.40 0.42 0.12 Sh 4.28 4.91 4.56 4.67 4.75 4.90 0.05 Nag 1.41 1.68 1.50 1.47 1.48 1.65 0.83 Clf,h 2.08 2.49 2.14 2.13 2.20 2.45 0.11 Trace minerals, mg Cub,c,d,e 31 85 130 105 128 218 21 Fef 588 770 898 761 787 701 74 Mnc,d,e 3.7 4.4 3.7 3.5 4.1 3.7 0.20 Sec 1.07 1.45 1.48 1.16 1.32 1.57 0.06 Znc,d,e 195 292 221 207 234 193 21 aQuantity of minerals in the empty body. bTrace mineral source response (P < 0.05). cTrace mineral level response (P < 0.05). dNRC vs. Ind. and Ind.+Ca/P (P < 0.05). eInd. vs. Ind.+Ca/P (P < 0.05). fTrace mineral source x level interaction response (P < 0.05). gTrace mineral source x level interaction response (P < 0.10). hTrace mineral level response (P < 0.10).

Table 6.2. Effect of trace mineral source and level on total liver and body mineral content of non-gravid females from 12 to 35 months of age 134

Organic trace minerals Inorganic trace minerals Item Age, mo.: 8 12 17 26 35 8 12 17 26 35 SEM No. of gilts 10 9 9 9 8 10 9 9 9 8 ― Body weight, kga,b 144 183 198 246 276 144 181 207 250 291 7 Empty BW, kga,b 140 172 194 240 270 140 179 201 245 284 7 Macro-minerals, g Caa,b 1,038 1,493 1,391 1,975 2,184 1,161 1,359 1,364 2,067 2,127 123 Pa,c 620 903 862 1,105 1,227 680 845 842 1,159 1,217 57 Ka,b 262 334 364 390 431 273 330 358 399 441 11 Mga,c 33 42 43 52 57 35 40 42 53 57 2 Sa,b 175 214 241 278 310 178 211 245 284 318 8 Naa,c 110 135 135 168 184 113 131 139 176 184 6 Cla,c 101 124 120 150 164 104 125 127 151 166 5 Trace minerals, mg 135 Cua,c,d 172 250 261 307 301 259 238 407 454 380 52 Fea,c,d 3,048 3,319 4,615 4,317 5,257 3,359 2,694 3,839 4,104 4,736 220 Mna 36 30 70 41 41 39 28 40 40 43 9 Sea,b,d 20 25 30 31 36 17 14 21 20 23 1 Zna,b 2,801 3,889 4,392 5,388 6,048 2,979 3,885 4,300 5,321 6,250 128 aGilts, 8 month of age vs. gilts, 12 to 35 month of age. bLinear age response (P < 0.01). cCubic age response (P < 0.05). dTrace mineral source response (P < 0.05).

Table 6.3. Effect of dietary trace mineral source and parity in non-gravid gilts on body mineral composition from 8 to 35 months of age

Organic trace minerals Inorganic trace minerals Item Age, mo.: 8 12 17 26 35 8 12 17 26 35 SEM No. of gilts 10 9 9 9 8 10 9 9 9 8 ― Liver wt., kga,b 1.62 1.97 2.12 2.21 2.32 1.54 1.90 2.05 2.29 2.60 0.09 Macro-minerals, g Caa,b,c 0.09 0.10 0.10 0.10 0.10 0.08 0.09 0.09 0.11 0.14 0.01 Pa,b 5.40 6.59 6.48 7.16 7.25 4.84 6.33 6.66 7.44 8.24 0.27 Ka,b 4.78 5.95 6.39 6.75 7.17 4.30 5.61 6.17 6.98 8.04 0.26 Mga,b,d 0.29 0.37 0.38 0.41 0.42 0.27 0.36 0.36 0.42 0.49 0.01 Sa,b 3.37 4.36 4.30 4.79 4.88 3.05 4.23 4.45 4.88 5.54 0.18 Naa,b 1.32 1.51 1.43 1.58 1.59 1.30 1.47 1.38 1.51 1.79 0.09 Cla,b 1.74 2.11 2.06 2.33 2.45 1.70 2.04 1.96 2.34 2.69 0.12 Trace minerals, mg Cua,e,f 27 86 61 116 66 33 100 169 198 135 24 136 Fea,b 453 553 651 858 947 458 532 626 834 1,008 86 Mna 3.7 4.4 3.8 3.9 3.6 3.3 3.7 3.6 3.5 4.3 0.24 Sea,b 1.0 1.2 1.3 1.4 1.4 0.9 1.1 1.4 1.4 1.5 0.1 Znb 147 205 217 274 248 120 186 188 206 266 24 aGilts, 8 month of age vs. gilts, 12 to 35 month of age. bLinear age response (P < 0.01). cTrace mineral source x age interaction response (P < 0.01). dTrace mineral source x age interaction response (P < 0.10). eTrace mineral source response (P < 0.05). fQuadratic age response (P < 0.05).

Table 6.4. Effect of dietary trace mineral source and parity in non-gravid gilts on total liver mineral composition from 8 to 35 months of age

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