LIBRARY Michigan State University PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE $311235 M9 6/01 c:/CIRCDateDuo.p65-p.15 UTILIZATION OF LOW PHYTIC ACID CORN WITH PHYTASE TO REDUCE PHOSPHORUS EXCRETION FROM GROWING TURKEYS AND PIGS By Michael Willard Klunzinger A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 2002 ABSTRACT UTILIZATION OF LOW PHYTIC ACID CORN WITH PHYTASE TO REDUCE PHOSPHORUS EXCRETION FROM GROWING TURKEYS AND PIGS By Michael Willard Klunzinger A series of experiments were conducted to determine if a low phytic acid, high protein corn (NutriDense Low Phytate'" (NDLP), Exseed Genetics L. L. C.) could replace yellow dent corn (YD) to reduce phosphorus excretion without affecting growth or bone parameters in growing turkeys and pigs. Phosphorus (P) bioavailability of NDLP was estimated to be 90%. Analyses of coms showed that 90% of total phosphorus (tP) in NDLP was in the form of non-phytate P (in) (0.32% and 0.29%, respectively). Analyzed YD contained 33% of tP as in (0.25% and 0.08%, respectively). Replacing YD with NDLP, with or without Natuphos" phytase, reduced P excretion by 30 to 45% (and 56% with phytase) in finishing toms. The NDLP can be safely formulated assuming a 90% P availability in growing-finishing pig diets. Also, NDLP can reduce P excretion by about 45% when fed instead of YD to grow-finish and weanling pigs. I would like to devote this work to my family and friends who helped me get through it. They were all there to support me every step of the way, whether they realized it or not, especially after very long days and times of fi'ustration. I especially thank my girlfriend, Raclene Charbeneau, whom I love clearly. I would also like to dedicate this work to Alpha Gamma Rho, who helped to shape me into the man I am today. . .”to make better men and through them a broader and better agriculture”. I am very proud to continue the family Spartan tradition as a fourth generation graduate of Michigan State University. ACKNOWLEDGMENTS I wish to thank Dr. Kevin Roberson and Dr. Maynard Hogberg for giving me the opportunity to work with Dr. Roberson to obtain my Masters Degree. I am very appreciative of the Corn Marketing Program of Michigan for helping to finance this research. I thank Exseed Genetics, specifically Jerry Weigel and Dr. Christopher Peter for the donation of low phytic acid, high amino acid corn and for their support along the way. I would also like to thank the good people at BASF for supplying phytase and technical assistance. I thank Dr. Thomas Crenshaw and staff at the University of Wisconsin-Madison for assisting in sample analyses of swine femurs. I thank those on my graduate committee for their guidance (Drs. Gretchen Hill, Michael Orth, Alan Rahn, and Robert von Bemuth). I thank all of the graduate students and staff who helped with data collection, advice, or any other number of essential tasks. Specifically, I want to thank Jane Link, David Main, Dr. Bradley Marks, Dr. Diana Rosenstein, Yin (Jackie) Yan, Raelene Charbeneau, Martin Ledwaba, Michelle Martinez, Jessica Green, Dana Dvoracek-Driksna, Daniel Shaw, Jason Rowntree, Michael Rincker, and Bob Bumette. I thank sheep farm manager George Good for his guidance and support both as an undergraduate and Masters student. I thank poultry farm manager Angelo Napolitano for his guidance and excellent husbandry skills with the turkeys. I thank Dr. Richard Balander for spurring my interest in the poultry field. I thank poultry farm staff for their help with projects and being there as a listening ear, especially Diane Karsten and Jeff Greenley. I thank the swine farm staff and manager Al Snedegar for their help in setting up the swine studies. I thank the meats lab management team of Tom Forton, Jennifer Dominguez, and staff for producing a tasty end product and helping to collect bone samples. I thank the secretaries and support staff, specifically Jackie Christie and Jeaniva Floyd for their assistance. Finally, I thank God and country for giving me the strength and perseverance to continue on, even in times of great tragedy and stress. TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ............................................................................................................ x KEY TO SYMBOLS AND ABBREVIATIONS .............................................................. xi INTRODUCTION .............................................................................................................. 1 LITERATURE REVIEW ................................................................................................... 4 Introduction ..................................................................................................................... 4 Nutrient attributes of conventional yellow dent corn ...................................................... 4 Nutrient composition of yellow dent corn ....................................................................... 8 Calcium and Phosphorus Nutrition in Swine and Poultry ............................................... 9 Phytase ........................................................................................................................... 22 History of Low-Phytic Acid Corn ................................................................................. 32 Nutrient Composition of NutriDenseTM Low Phytate Corn .......................................... 36 Bone Parameters in Poultry and Swine Nutrition .......................................................... 38 Conclusion ..................................................................................................................... 41 References ..................................................................................................................... 42 CHAPTER 1: IMPACT OF FEEDING LOW PHYTATE CORN TO GROWING- FINISHING LARGE WHITE MALE TURKEYS ........................................................... 57 Summary ........................................................................................................................ 57 Description of Problem .................................................................................................. 57 Materials and Methods .................................................................................................. 59 Results and Discussion .................................................................................................. 78 Conclusions and Applications ....................................................................................... 98 References and Notes .................................................................................................... 99 CHAPTER 2: IMPACT OF FEEDING LOW PHYTATE CORN TO WEANLING AND GROWING-FINISHING PIGS ...................................................................................... 104 Introduction ................................................................................................................. 105 Materials and Methods ................................................................................................ 107 Results ......................................................................................................................... 115 Discussion .................................................................................................................... 120 Conclusion ................................................................................................................... 124 References ................................................................................................................... 124 SUMMARY AND GENERAL CONCLUSION ............................................................ 128 VITA ............................................................................................................................... 129 APPENDICES ................................................................................................................ 131 vi LIST OF TABLES LITERATURE REVIEW ................................................................................................... 4 Table 1. Nutrient Composition of Yellow Dent Corn Grain, Zea mays indentata, for Poultry Diets (as-fed basis). ................................................................................................ 8 Table 2. Nutrient Composition of Yellow Dent Com Grain, Zea mays indentata, for Swine Diets (as—fed basis) ................................................................................................... 9 Table 3. Suggested calcium requirements for growing turkeys as percentage of diet (90 percent dry matter) ............................................................................................................ 19 Table 4. Suggested non-phytate phosphorus requirements for growing turkeys as percentage of diet (90 percent dry matter) ........................................................................ 20 Table 5. Calcium and non-phytate phosphorus requirements of growing turkeys fed ad libitum as percentage of diet (90% dry matter)a ............................................................... 20 Table 6. Calcium, total phosphorus, and available phosphorus requirements of growing pigs fed ad libitum as percentage of diet (90% dry matter) or daily requirement"l ........... 21 Table 7. Impact of microbial phytase on bodyweight (BW) gain, feed conversion ratio (F CR), phosphorus retention (P ret.), phosphorus excretion (P excr), tibia ash, tibia breaking strength (BS), and toe ash in broiler chickens fed com-soybean based diets 26 Table 8. Impact of microbial phytase on body weight (BW) gain, feed conversion ratio (FCR), phosphorus retention (Phos ret), and bone ash in growing turkeys fed com-soybean meal based diets .......................................................................................... 27 Table 9. Impact of microbial phytase supplementation on body weight (BW) gain, feed conversion ratio (FCR), phosphorus retention (P ret.), phosphorus excretion (P excr.), bone ash, and bone breaking strength (BS) in pigs fed com-soybean based diets ........... 28 Table 10. Impact of microbial phytase supplementation on body weight (B\V) gain, feed conversion ratio (FCR), phosphorus retention (P ret.), phosphorus excretion (P excr.), bone ash, and bone breaking strength (BS) in growing broilers, turkeys, and pigs fed low phytic acid com-soybean meal based diets .................................................. 30 Table 11. Research highlights of studies utilizing low phytic acid corn varieties as an alternative to conventional yellow dent corn in growing meat-type poultry diets ........... 33 Table 11 (cont’d). Research highlights of studies utilizing low phytic acid corn varieties as an alternative to conventional yellow dent corn in growing meat-type poultry diets .. 34 Table 12. Research highlights of studies utilizing low phytic acid corn varieties as an alternative to conventional yellow dent corn in growing swine diets ............................... 35 Table 13. Analyzed nutrient content of NutriDense" Low Phytate (NDLP) and Yellow Dent (YD) corn .................................................................................................... 38 CHAPTER 1: IMPACT OF FEEDING LOW PHYTATE CORN TO GROWING- FINISHING LARGE WHITE MALE TURKEYS ........................................................... 57 Table 1: Environmental temperature target guidelines. .................................................... 60 Table 2. Analyzed nutrient content of NutriDensem Low Phytate and Yellow Dent com 62 Table 3. Composition and selected nutrient content of Prestarter diets (Experiment 1).. 63 Table 4. Composition and selected nutrient content of Starter l diets (Experiment 1) 64 Table 5. Composition and selected nutrient content of Starter 2 diets (Experiment 1).... 65 Table 6. Composition and selected nutrient content of Grower 1 diets (Experiment 1) .. 66 Table 7. Composition and selected nutrient content of Grower 2 diets (Experiment 1) .. 67 vii Table 8. Composition and selected nutrient content of Finisher diets (Experiment 1) ..... 68 Table 9. Composition and selected nutrient content of Starter 2 diets (Experiment 2) 71 Table 10. Composition and selected nutrient content of Grower 1 diets (Experiment 2) 72 Table 11. Composition and selected nutrient content of Grower 2 diets (Experiment 2) 73 Table 12. Composition and selected nutrient content of Finisher diets (Experiment 2)... 74 Table 13. Growth performance of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (N DLP) corn for Starter 1 (STl), Starter 2(ST2), Grower 1 (GRl), Grower 2 (GR2), and Finisher (FIN) phases (Experiment 1) ............... 78 Table 14. Growth performance of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (NDLP) corn for Starter 2(ST2), Grower 1 (GRl), Grower 2 (GR2), and Finisher (FIN) phases (Experiment 2) .......................................... 79 Table 15. Bone fracture force and bone strength (stress) of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (N DLP) corn (Experiment 1) ................ 85 Table 16. Bone fracture force and bone strength (stress) of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (N DLP) corn (Experiment 2) ................ 87 Table 17. Bone cortical wall thickness and bone ash of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (N DLP) corn (Experiment 1) ................ 91 Table 18. Bone cortical wall thickness, bone ash, and bone mineral density of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (NDLP) com (Experiment 2) .......................................................................................................... 92 Table 19. Litter phosphorus and excreta phosphorus of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (N DLP) corn (Experiment 1) ................ 94 Table 20. Litter phosphorus and excreta phosphorus of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate (N DLP) corn (Experiment 2) ................ 95 CHAPTER 2: IMPACT OF FEEDING LOW PHYTATE CORN TO WEAN LING AND GROWING-FINISHING PIGS ...................................................................................... 104 Table l. Analyzed nutrient content of NutriDensem Low Phytate (NDLP) and Yellow Dent (YD) maize ........................................................................................ 106 Table 2. Composition and nutrient content of Grower 1 (GRl), Grower 2 (GR2), and Finisher (FIN) dietsA (Experiment 1) ....................................................................... 108 Table 3. Composition of Phase I dietsA (Experiment 2) ................................................. 111 Table 4. Composition of Phase II dietsA (Experiment 2) ................................................ 112 Table 5. Effects of dietary treatments on body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed efficiencyA (GzF) (Experiment 1) ............. 115 Table 6. Effects of dietary treatments on bone fracture force, bone ultimate stress (strength), average cortical wall thickness, serum osteocalcin, and bone ash (Experiment 1) ............................................................................................................... 116 Table 7. Effects of dietary treatments on faecal phosphorus (Experiment 1) ................. 117 Table 8. Effects of dietary treatments on body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed efficiencyA (GzF) (Experiment 2) .............. 118 Table 9. Effects of dietary treatments on plasma phosphorus, faecal phosphorus, and apparent phosphorus digestibility (Experiment 2) .......................................................... 119 APPENDICES ................................................................................................................ 131 Table 1. Ultimate stress equation variables from swine study (Experiment 1) .............. 132 Table 2. Ultimate stress equation variables from turkey study (Experiment 1) ............. 132 viii Table 3. Ultimate stress equation (measurements taken using computed tomography (CT) technology) with variables from turkey study (Experiment 2) ............................ 133 Table 4. Ultimate stress equation (measurements taken using a digital caliper) with variables from turkey study (Experiment 2) ................................................................... 134 Table 5. Feed cost savings by replacing yellow dent (YD) corn with NutriDense Low Phytatem (N DLP) corn in turkey Finisher diets (Experiment 1) ..................................... 135 Table 6. Feed cost savings by replacing yellow dent (YD) corn with NutriDense Low Phytatem (NDLP) corn in turkey Finisher diets (Experiment 1) ..................................... 136 LIST OF FIGURES Figure 1. Structure of phytic acid proposed by Anderson (1914) ..................................... 15 Figure 2. Structure of phytic acid chelatc at neutral pH (Erdman, 1979. ......................... 16 ADF ADFD ADG AMP ADPD ANOVA ASAE ATP BMD BUTA BW Ca cm CP CT Cu KEY TO SYMBOLS AND ABBREVIATIONS cross-sectional area acid detergent fiber average daily feed disappearance average daily gain adenosine diphosphate adenosine monophosphate average daily phosphorus disappearance Analysis of Variance American Society of Agricultural Engineers adenosine triphosphate major outside diameter of a bone cross section bone mineral density British United Turkeys of America body weight Celsius calcium cubic centimeter crude protein computed tomographic copper day xi dL DNA et a1. Fe FIN FTU GF GLM GR] GR2 h, hr i.e. kcal kg kN L LP lsmeans ME minor outside diameter of a bone cross section deciliter deoxyribonucleic acid “and others” maximum force required to break a bone iron Finisher BASF Natuphos® Aspergillus niger phytase units gram gainzfeed General Linear Model Grower 1 Grower 2 hour “that is” iodine kilocalorie kilogram kiloNewton distance between fulcra points Low Phytate least square means metabolizable energy xii Mg magnesium mg milligram min minute MJ MegaJoule mL milliliter mm millimeter Mn manganese MPa MegaPascal N Newton NCIUBMB Nomenclature Committee of the International Union of Biochemistry and Molecular Biology NDLP NutriDensem Low Phytate ng nanogram in non-phytate phosphorus NRC National Research Council P phosphorus Pa Pascal pP phytate phosphorus PTH parathyroid hormone PTU Alko Ltd. Biotechnology F inasem Aspergillus niger phytase activity units PU Alko Ltd. Biotechnology yeast phytase activity units qCT quantitative computed tomography RNA ribonucleic acid xiii 5 second Se selenium SEM standard error of the mean stderr standard error sP soluble phosphorus TME true metabolizable energy tonne metric ton tP total phosphorus Trt treatment US United States USA United States of America USDA United States Department of Agriculture USDA-ARS United States Department of Agriculture-Agricultural Research Service vs. versus w average cortical wall thickness wk week X distance from neutral axis to outer fiber of a bone YD yellow dent Zn zinc xiv INTRODUCTION As the human population has increased in the USA, so has the demand for food. The expansion in the size of farms has increased to partially meet that demand, with either crop or livestock production, and ofien both. The current trend is that the number of farms is decreasing while the average size of the farming operation is on the rise. In 1900 there were approximately 5.7 million farms in production with an average acreage of 147 acres. In 1997, there were about 1.9 million farms in production with an average size of about 487 acres. As examples of this trend, in 1978 the number of swine operations in existence was 635,000 and in 1992 it was 240,150. Similarly, the number of turkey operations in existence was 26,638 in 1978 and 13,766 in 1992. The increase in the number of animals raised for production from 1978 to 1992 was 12% for swine (from 88,512,000 to 99,142,000 pigs) and 109% for turkeys (fiom 138,939,000 to 289,880,000). From these numbers, clearly the number of animals raised and the number of animals raised per operation is increasing. The use of domestic livestock wastes as sources of potential nutrients for crop production has been a long-standing tradition in the USA. Many years of manure application to crop lands on or around animal feeding operations has, in some instances, - led to a saturation of soils with nutrients. If the nutrient saturation exceeds the soils’ ability to retain nutrients, a potential for water pollution may exist. This may occur in one or more of several ways. Surface runoff is one major pathway. In this scenario, the loss of soil nutrients may occur as sediment bound or dissolved nutrients associated with organic material or soil particles erodes away from the soil and is washed away with surface water runoff. Rainfall or irrigation can then take this runoff into other bodies of water including streams, rivers, ponds and lakes. Another pathway is by way of artificial drainage systems and subsurface water flows. Loss of nutrients by leaching down through the soil, especially in periods of drought followed by periods of rainfall, is another pathway. Nutrients in this situation could end up in groundwater and drinking water and become a concern for human health. Eventually the manure-derived nutrients may end up in large bodies of water such as ponds and lakes. Coupled with the influx of nutrients from other sources, a potential for a nutrient overload of the aquatic system could lead to an increase in biological oxygen demand and a condition known as eutrophication. As organic material (manure particles or sediment-bound nutrients, for instance) enter bodies of water, plant growth will increase often preventing adequate oxygenation. Also, bacteria may act to decompose the organic matter, using dissolved water oxygen in the process. Dissolved oxygen concentrations can drop below sustainable concentrations for fish and other organisms in the aquatic system, leading to kills. Eutrophication can occur when an overabundance of phosphorus and other nutrients lead to a surge in aquatic plant growth, especially algae. Too much decaying algae can lead to depletion in dissolved oxygen in the water and may eventually cause acute fish kills. As the size of the animal feeding operation, proximity to watersheds, and soil nutrient saturation from applied manure increase so does the potential for water pollution. When managed properly, through a variety of different mechanisms, livestock feeding operations can pose only minimal risk for water pollution. Phosphorus utilization from grain feedstuffs in poultry and swine diets is limited due to a difference in gastrointestinal metabolism. Most phosphorus in grains is part of the phytic acid (phytate) complex. Turkeys and swine have limited phytase, an enzyme which splits phosphorus fi'om the phytate complex, increasing its availability. Reducing manure nutrient concentrations allows for more manure application with a reduced risk of water pollution. Since corn is the leading cereal grain produced in the USA, and is the primary constituent of most USA turkey and swine diets, a reduced phytate phosphorus corn variety could be a potential feedstuff to reduce manure phosphorus. Addition of exogenous phytase enzyme could also reduce manure phosphorus by making more phytate-bound phosphorus available to turkeys and pigs. The focus in the following chapters will address how feeding a variety of low-phytic acid corn with phytase and reducing dietary sources of inorganic phosphorus (dicalcium phosphate) in turkey and swine diets could reduce manure concentrations of phosphorus. The impacts on growth performance and skeletal attributes will also be examined. Thesis Organization The following thesis is organized as a literature review followed by two papers as thesis chapters. The first chapter is written in the style and format of the Journal of Applied Poultry Research. The second Chapter is written in the style and format of Animal Feed Science and Technology. Michael W. Klunzinger completed the research - reported in the papers under the direction of Kevin D. Roberson, Gretchen M. Hill, Michael W. Orth, Allan P. Rahn, and Robert D. von Bemuth. Included in the appendices is an estimation of cost of dietary treatments used in the experiments. LITERATURE REVIEW Introduction More than 95% of the corn that is harvested in the USA is marketed as mature commodity yellow dent corn, with 55% being used in livestock feeding (Hallauer, 2001). Corn is the leading cereal grain feedstufl fed in livestock diets in the USA. The goal to reduce livestock manure phosphorus, more specifically poultry and swine manure phosphorus, has led to the advent of corn varieties which have a higher available phosphorus content than conventional yellow dent corn. The use of the enzyme phytase to increase phosphorus availability of poultry and swine feeds, which results in the reduction of inorganic phosphorus supplementation in poultry and swine feeds, has also been proven to reduce poultry and swine manure phosphorus. Nutrient attributes of conventional yellow dent corn Mature corn is used in livestock feeding because its nutrient content better matches the needs of livestock than immature com. A mature kernel of corn commonly contains 70-75% starch, 8-10% protein, 4-5% oil, and 10-18% water, fiber, vitamins, and minerals (Boyer and Hannah, 2001). A kernel of com includes four main parts: tip cap, germ, endosperm, and pericarp. Components of the kernel can be genetically modified to create a new corn nutrient profile. Energy In livestock, energy can be separated into two categories: (1) heat energy to maintain body temperature; and (2) molecular energy available for work and biochemical processes in the body (but energy is stored as fat). When estimating the amount of potential energy available in feed, the common unit is kilocalories (kcal), or the amount of heat energy required to raise the temperature of one gram of water by one degree centigrade, when the feed is completely oxidized in a bomb calorimeter. This value is then referred to as Gross Energy (GE). (Scott et al., 1982) When formulating diets for swine and poultry, the energy requirements are generally based on Metabolizable Energy (ME), which is defined as the gross amount of potentially oxidizable energy (GB) in a consumed feed minus the oxidized Fecal Energy (FE) minus Gaseous Products of Digestion (GDP) minus Urinary Energy (UE). In poultry feeding, some nutritionists use True Metabolizable Energy (TME) in dietary formulation rather than apparent Metabolizable Energy (ME) (NRC, 1994). The difference is that TME is unaffected by variations in feed intake and accounts only for the gross energy of the feces, urine or mates, and gaseous products that are of feed origin, rather than endogenous. Because corn is rather high in digestible carbohydrates and fats, it is a good source of energy (Scott et al., 1982). Carbohydrate A majority of the carbohydrate in the corn kernel occurs as starch. Starch is the storage form of nutritionally available carbohydrate for the germinating corn seed, most of which (80-90%) is found in the endosperm. About 80% of the total mature corn kernel dry weight is comprised of starch. Starch has a GE value of approximately 4 kcal/g and is readily digested by poultry (Moran, 1985a). After pigs are two to three weeks old, they can utilize starch (Becker and Terrill, 1954; Cunningham, 1959; Sewell and Maxwell, 1966). Prior to that time, pigs lack the amounts of pancreatic amylase and intestinal disaccharides to digest cornstarch (Cunningham, 1959; Sewell and Maxwell, 1966). Starch is made up of primarily or-l,4 linkages of glucose units arranged in a linear manner. A small amount of branching occurs as ot-l,6 linkages of glucose units. Starches are made up of two distinct types of polymers: (1) amylose and (2) amylopectin. (Scott et al., 1982) Standard yellow dent corn starch is composed of about 25% amylose and 75% amylopectin. Amylose is a molecule made up of 100 to 1000 glucose subunits arranged by linear ot-1,4 linkages and branched ot-1,6 linkages about every 200 glucose subunits. Amylopectin molecules contain up to 200,000 glucose units. Approximately 8,000-10,000 of the total number of glucose subunits in amylopectin are arranged by ot- 1,6 linkages, with 19,000-192,000 arranged by or-l,4 linkages. The linear regions of amylopectin are either 10-20 or 30-50 glucose units long. (White, 2001) Amylases in swine and poultry hydrolyze starches in the saliva, crop, and small intestine, through a series of reactions to eventually break them down to glucose units. Monosaccharides can then be absorbed across the small intestine and converted to glucose in the intestinal cell. Glucose is then transported through the blood and can serve as an energy source to any tissue or organ in the body through a variety of different mechanisms. Glucose can be stored, to a certain extent, in the liver and muscle as glycogen. Glycogen is similar in structure to amylose. Glucose can also be used in the formation of adipose tissue. (Pond . et al., 1995) Digestion and absorption of starch can be affected by many factors, including com particle size, starch. configuration (amylase and amylopectin proportions), interactions with protein and fa t, and the presence of antinutritional factors such as phytate (Pond et al., 1995). Protein Most of the protein in a corn kernel is found in the endosperm and germ. Standard yellow dent corn kernel protein is made of 7% albumins, 5% globulins, 52% prolamines (zeins), and 25% glutelins on a percent nitrogen basis. The endosperm may contain up to 80% of the total protein in a com kernel, and accounts for 34% of kernel glutelins and 60% of kernel prolamines. The germ contains 60% albumins and. 5-10% prolamines. (Boyer and Hannah, 2001) Albumins, globulins, and glutelins are more soluble than prolamines and provide a more desirable balance of essential amino acids for animals. The prolamine (zein) fraction of corn contains extremely low portions of lysine (an essential amino acid) and higher portions of glutamine, proline and alanine (non-essential amino acids), as compared to other protein fractions of corn (V asal, 2001). Because . standard yellow dent corn contains higher concentrations of prolamines and contributes more non-essential amino acids (e. g. glutamine, proline, and alanine) and less essential amino acids (e.g. lysine and tryptophan), it would be desirable to feed corn that would provide less zein protein and more albumin, globulin, and glutelin proteins. Antinutritional factors, such as phytate, can have a detrimental effect on protein digestion and absorption (de Rahm and Jost, 1979; Cosgrove, 1980; Caldwell, 1992). Lipids The majority (83-85%) of the lipid (oil) contained in a kernel of conventional yellow dent corn is found in the germ portion. The endosperm, tip cap, and pericarp contain 10-13% of the kernel oil. Corn oil is comprised of mainly triacylglycerides, which is a mixture of saturated and unsaturated fatty acids. Typical high quality yellow dent corn oil should contain approximately 50% linoleic acid, 40% oleic acid, 12% palrnitic acid, 2% stearic acid, and 1% linoleic acid (Boyer and Hannah, 2001) Because there is a large percentage (50%) of linoleic acid and a low percentage (1%) of linolenic acid (an essential fatty acid) in corn oil, it is considered a good source of dietary fatty acid and, therefore, energy (Boyer and Hannah, 2001; Scott et al., 1982). Nutrient composition of yellow dent corn Table 1 illustrates a nutrient profile of US #2 Grade yellow dent corn grain used for poultry diet formulation (NRC, 1994). Table 2 illustrates a nutrient profile of US #2 Grade yellow dent corn grain used for swine diet formulation (NRC, 1998). Nutrient values listed are those of particular interest for this thesis. Grade US #2 is the standard for most animal feeds (Leeson and Summers, 1997). Table 1. Nutrient Composition of Yellow Dent Corn Grain, Zea mays indentata, for Poultry Diets (as-fed basis).' Nutrient Units Values Nutrient Units Values Moisture % 12.00 Histidine % 0.23 TME kcal/kg 3,470 Isoleucine % 0.29 MB kcal/kg 3,350 Leucine % l .00 Crude Protein % 8.50 Lysine % 0.26 Ether Extract % 3.80 Methionine % 0.18 Crude Fiber % 2.20 Cystine % 0.18 Calcium % 0.02 Phenylalanine % 0.38 Total % 0.28 Tyrosine % 0.30 Phosphorus Non-phytate % 0.08 Threonine % 0.29 phosphorus Arginine % 0.38 Tryptophan % 0.06 Glycine % 0.33 Valine % 0.40 Serine % 0.37 ‘Based on NRC Nutrient Requirements of Poultry (1994) values. Table 2. Nutrient Composition of Yellow Dent Corn Grain, Zea mays indentata, for Swine Diets (as-fed basis)! Nutrient Units Values Nutrient Units Values Moisture % 1 1.00 Isoleucine % 0.28 MB kcal/kg 3,420 Leucine % 0.99 Crude Protein % 8.30 Lysine % 0.26 Ether Extract % 3 .90 Methionine % 0.17 ADF % 2.80 Cystine % 0.19 Calcium % 0.03 Phenylalanine % 0.39 Total % 0.28 Tyrosine % 0.25 Phosphorus Available % 0.04 Threonine % 0.29 phosphorusb Arginine % 0.37 Tryptophan % 0.06 Histidine % 0.23 Valine % 0.39 'Based on NRC Nutrient Requirements of Swine (1998) values. I’Based on a phosphorus bioavailability estimate of 14%. Calcium and Phosphorus Nutrition in Swine and Poultry Although the focus of this thesis centers around phosphorus, the discussion of calcium is also essential. Calcium and phosphorus must be discussed in conjunction with one another because they have close associations in animal metabolism, especially in bone formation. Calcium Approximately 99% of the calcium found in animal stores is in the skeleton as part of bones and teeth (Pond et al., 1995). Calcium is found in roughly a 2:1 ratio with phosphorus in bone, mainly as crystals of hydroxyapatite [Calo(PO4)5(OH)2]. Most of the blood calcium is in the extracellular plasma fraction as one of three forms (Lloyd et al., 1978): 60% as a free ion, 35% protein bound, and 5—7% as complexed with organic (e.g. citrate) or inorganic acids (e.g. phosphate). It is important that blood calcium is maintained at a relatively constant concentration so that calcium is available for fimctions such as neurotransmission, muscle contraction, and blood clotting. The parathyroid gland is the calcium control point for calcium regulation in the body. A decrease in plasma calcium results in the stimulation of the parathyroid gland to release parathyroid hormone (PTH), which induces biochemical activation of vitamin D3 eventually to 1,25- dihydroxycholecalciferol [l-25(OH2) D3] by the kidney, thereby causing increases in calcium absorption from the gastrointestinal tract (primarily in the duodenum of the small intestine) and resorption of calcium from bone (Lloyd et al., 1978; Groff and Gropper, 1998; Underwood and Suttle, 1999). Absorption of calcium across the small intestine is facilitated by the carrier protein calbindin. The kidney is also a very important organ in calcium reabsorption. An increase in plasma calcium stimulates the thyroid gland C-cells to release calcitonin, which is a hormone that represses calcium resorption from bone (Groff and Gropper, 2000). Bone growth occurs by bone forming cells called osteoblasts. In general, bone formation occurs as a process in which mineral elements are deposited onto an organic matrix. Dicalcium phosphate is of great importance in the process. There are several events that take place during the formation of bone, outlined as follows: 1) dicalcium phosphate accumulates; 2) three molecules of dicalcium phosphate congregate to form one molecule of tricalcium phosphate, leaving one molecule of phosphoric acid; 3) ions of carbonate, fluorine, or hydroxyl attach to the unstable tricalcium phosphate to complete a characteristic crystalline structure of apatite; 4) further mineral complex additions contribute to structure and insolubility (Lloyd et al., 1978). 10 v.4; ‘ fi Bone resorbing cells called osteoclasts are responsible for bone surface resorption Bone resorption also takes place deep within the bone by a process known as osteocytic resorption. Bone is in a constant state of flux, as more bone accretion occurs and changes in shape and density take place. Bone resorption also influences the structure and shape of bone. Calcium is also necessary for normal blood coagulation and has functional roles in muscle and nervous tissue, regulation of cell membrane permeability, enzyme activation, and vitamin B12 absorption (Lloyd et al., 1978). Dietary calcium is absorbed Primarily in the duodenum and jejunum of the small intestine of swine and poultry. The efficiency of absorption is difficult to ascertain due to endogenous sources of calcium re- excreted into the intestine. Apparent digestibility values (those which does include endogenous losses) are therefore used, when the measurement of net absorption values (that which does not include endogenous losses) are not possible. Net absorption values involve very intensive balance studies. Absorption of calcium occurs by energy ’ dependent active tranSport and by passive diffilSion transport across the intestinal epithelium. Acid conditions of the stomach or proventriculus and gizzard help to promote calcium absorption. (Pond et al., 1995) Active transport calcium absol'Ption is ' controlled by the vitamin D dependent calcium binding protein calbindin (Hurwitz, 1996)- About 50% of plasma calcium is filtered by the kidneys and 99% is reabsorbed- AS a general rule, an increase in dietary calcium will result in a decrease in percentage . calcium absorbed. Absorption of calcium can be adversely affected by the presence 0f phytic acid, Which can form insoluble complexes with calcium in poultry and swine. (Wise, 1983; Pond et al., 1995) When high dietary concentrations of radio-labeled ll calcium were fed to rats, insoluble calcium-phytate complexes were formed, which had a negative effect on phosphorus utilization (Nahapetian and Young, 1980). Excretion of dietary calcium is by way of feces from the intestine and urine (pigs) or uric acid (poultry) from the kidneys. Fecal excretion of calcium includes both endogenous calcium and that which was not absorbed. Urinary or uric acid excretion of calcium is low due to filtration of plasma calcium and reabsorption by the kidneys. (Pond et al., 1995) The calcium content of yellow dent corn grain is generally quite low not only in concentration, but also in bioavailability as calcium can be an integral part of the structure of phytin, a calcium-magnesium salt of phytic acid, which is mostly unavailable to swine and poultry. As a result, swine and poultry corn based diets must be supplemented with ingredients that will provide bioavailable calcium to prevent a deficiency. The bioavailability of calcium found in monocalcium phosphate, dicalcium phosphate, tricalcium phosphate, defluoronated phosphate, calcium gluconate, and calcium sulfate is very high, 90-100% (Baker, 1991; Scares, 1995), based on poultry research as compared to the calcium in calcium carbonate, which is assumed to be 100% bioavailable. Phosphorus Approximately 80% of the phosphorus in the body is present in the skeleton and teeth as phosphate phosphorus in the crystal hydroxyapatite and calcium phosphate essential to bone ossification. The remaining 20% of body phosphorus is found throughout the rest of the body tissues. In blood serum, phosphorus occurs in an organic form as part of blood lipids or as inorganic phosphorus ( 10% bound to protein, 50-60% 12 ionized, and about 35% complexed with Na, Ca, and/or Mg). Osloyd et al., 1978; Pond et al., 1995; Underwood and Suttle, 1999) The major role of dietary phosphorus in the animal is as a constituent of the skeleton and its involvement with bone metabolism, as outlined in the section on calcium. Phosphorus is also found in phospholipids, which have roles as cell membrane structure and lipid transport. Another very important function of phosphorus is its role in energy metabolism as a component of adenosine monophosphate (AMP), ADP, ATP, and creatine phosphate. Phosphorus has a functional role in. protein synthesis as part of the structure of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Phosphorus also has an integral role in many enzyme systems including carbohydrate, amino acid, and lipid metabolism. (Scott et al., 1982; Pond et al., 1995; Groff and Gropper, 1998; Underwood and Suttle, 1999) Phosphorus concentrations in the blood can fluctuate more than that of calcium because phosphorus is not as tightly regulated. The primary control point of phosphorus regulation in the body is the kidney. Low dietary phosphorus will increase phosphorus reabsorption in the proximal tubule of the kidney. Phosphorus reabsorption is upregulated by IGF-l, growth hormones, and thyroid hormones, particularly in growing - animals that are increasing muscular mass. Parathyroid hormone downregulates phosphorus reabsorption in the kidney. The kidney is the key organ in keeping calcium and phosphorus under the saturation point in blood. Parathyroid hormone influences resorption of phosphate from bone. In contrast to calcium, PTH stimulates excretion of phosphorus in urine or uric acid sufficient to transcend bone resorption of phosphorus to effect a net decrease in plasma phosphorus. Calcitriol and the active for of vitamin D3 13 (1,25-dihydroxycholecalciferol [l-25(OH2) D3]) enhances phosphate absorption across the gastrointestinal tract (primarily in the duodenum of the small intestine) and phosphate resorption from bone. (Groff and Gropper, 1998) Although there are some endogenous losses of phosphorus, fi'om secretion of phosphorus into the intestinal lumen, the losses are not as great as those seen with calcium. Apparent digestibility values for dietary phosphorus are, therefore, somewhat more reliable than apparent digestibility values for dietary calcium. Absorption of phosphorus occurs by energy-dependent active transport and by passive diffusion transport across the intestinal epithelium. G’ond et al., 1995) Acid conditions of the stomach or proventriculus and gizzard help to promote phosphorus absorption. In relation to calcium, an excess of dietary phosphorus can impair calcium absorption, presumably by the formation of insoluble calcium phosphate salts (Pond et al., 1995). If dietary phosphorus is present as phytic acid, absorbability will be very low (Pond et al., 1995). Calcium and phosphorus deficiency If calcium and phosphate concentrations in fluids surrounding bone fall below a critical level necessary for calcium phosphate precipitation into the crystalline lattice structure of apatite, proper ossification (bone mineralization) fails to transpire. In growing animals, this results in a condition known as rickets. Rickets generally results from inadequate dietary calcium, vitamin D3, phosphorus, or a very narrow or wide calcium: available phosphorus ratio. Symptoms often include growth failure, enlarged ends of long bones, “rubbery” or “springy” bones leading to bowed legs, beaded ribs, curved spine, curved sternum, with an increasing amount of loss of appetite, weakness, l4 and ataxia as the condition advances and could eventually result in death. (Lloyd et al., 1978; Scott et al., 1982; Pond et al., 1995) Phytic acid structure and function Phytic acid is the principal progenitor of inositol and inositol phosphates and the storage form of phosphorus in plant seeds (Cosgrove, 1980). Phytic acid is the common name for myo-inositol l,2,3,4,5,6-hexakis dihydrogen phosphate (C6I‘Ingz4P6). The term “phytic acid” refers to the free acid; “phytate” refers to the free salt of phytic acid; and “phytin” refers to the calcium/magnesium salt of phytic acid. Anderson (1914) first prOposed the most generally accepted chemical structure of phytic acid (Figure l). Figure 1. Structure of phytic acid proposed by Anderson (1914) Adapted from Sebastian et al. (1998). This structure best explains the biochemical properties, interactions, and nutritional effects of phytic acid. At a neutral pH, the phosphate groups on the phytic acid molecule generally have one or two negatively charged oxygen atoms. For this reason, positively charged cations are able to strongly bond between two phosphate groups or weakly with an individual phosphate group on the phytic acid molecule to form insoluble salts (Figure 2) (Erdman, 1979). 15 Figure 2. Structure of phytic acid chelate at neutral pH (Erdman, 1979) Adapted from Sebastian et a1. (1998). In the plant, phytic acid serves two major roles as the supply of inositol phosphates for signal transduction as well as storage of phosphorus in dormant seeds later used for ATP synthesis during germination. Concentrations of phytic acid (as a proportion of the total phosphorus) in various plants and plant seeds are somewhat variable, ranging between 60 and 80% (Simons et al., 1990). About two-thirds of the total phosphorus present in yellow dent corn (Zea mays indentata) grain is in the form of phytate phosphorus and about one-third is represented as non-phytate phosphorus (Nelson et al., 1968; NRC, 1994, 1998; Simons et al., 1990; Cromwell, 1992). Phytate as an antinutritional factor The phosphorus and other essential elements in phytate have lead to the recognition of phytate as a nutrient. Phytate has also been considered to be an antinutritional factor in poultry and swine diets because it binds other essential elements and reduces bioavailability of those elements (Erdman et al., 1979; Reddy et al., 1982). Nutritionally essential cations able to bond to the phytic acid molecule include calcium, magnesimn, manganese, iron, zinc, and potassium. Phytate can reduce the solubility of other essential dietary constituents of poultry and swine diets including proteins (Saio et al., 1967; Rojas and Scott, 1969; de Rahm and Jost, 1979; Cosgrove, 1980; Cheryan, 1980; Prattley and Stanley, 1982)..Phytate can also cause a reduction in pepsin activity (Deshpande and Cheryan, 1984), a reduction in trypsin activity (Singh and Krikorian, 1982; Caldwell, 1992), and the inhibition of ot-amylase activity by binding the calcium necessary for its stability and activation resulting in low starch digestibility and the reduction of available dietary energy (Deshpande and Cheryan, 1984; Knuckles and Betschart, 1987). Phytate digestibility and bioavailability For dietary phytate to be digested and utilized by poultry and swine, it must first be hydrolyzed to release inorganic phosphorus and other bound essential elements, such as calcium, magnesium, zinc, iron, manganese, and potassium. Although non-enzymatic hydrolysis of phytate has been suggested (Hegsted et al., 1954), the liberation of phytate- derived inorganic phosphorus and associated elements is strongly dependent on the presence of phosphatase enzymes, including phytase. Dietary phytate is poorly utilized by poultry and swine because they lack the quantities of phytase that would be sufficient ' to hydrolyze large amounts of dietary phytate and is, therefore, relatively poor in bioavailability of phosphorus fi'om- the phytate molecule; (Taylor, 1965; Nelson, 1967, 1976; Peeler, 1972; Cromwell, 1979). Phosphorus in grain feedstufifs Reported phytate phosphorus concentrations in yellow dent corn are somewhat variable ranging from 59-73%, when expressed as a percentage of total phosphorus l7 (Nelson et al., 1968; Cromwell, 1979; Reddy et al., 1989; Eeckhout and De Paepe, 1994; Ravindran et al., 1995a). The non-phytin, or non-phytate phosphorus content of dent corn is listed as 0.08%, with phytate phosphorus as 0.20% and total phosphorus as 0.28%, by the NRC (1994). Swine and poultry corn based diets must be supplemented with ingredients that will provide more bioavailable sources of phosphorus to avoid deficiency. The phosphorus from inorganic supplements, such as monocalcium phosphate, dicalcium phosphate, monosodimn phosphate, defluoronated rock phosphates and animal byproduct ingredients is considered to be highly bioavailable, often approaching 100% bioavailability (Komegay, 1972b; Hays, 1976; Clawson and Armstrong, 1982; Partridge, 1981; Tunmire et al., 1983; Cromwell et al., 1987; Cromwell, 1992). Steamed bone meal is often less bioavailable andmore variable in phosphorus bioavailability (Cromwell, 1992). High-fluorine rock phosphates, soft phosphate, Curacao Island phosphate, and colloidal clay are relatively low in phosphorus bioavailability compared to other inorganic sources (Chapman et al., 1955; Plumlee et al., 1958; Hays, 1976). Dietary calcium and phosphorus requirements for turkeys and pigs The ratio of calciumzphosphorus normally found in bone is approximately 2:1 (Lloyd et al., 197 8). Consequently, the recommendations for most starting, growing, and finishing diets for turkeys have a calciumznon-phytate phosphorus ratio of around 2:1 (NRC, 1994). For weanling pigs, the recommendations for nursery diets have a calciumzavailable phosphorus ratio of around 2:1 (NRC, 1998). For growing-finishing pigs, the recommended calciumzavailable phosphorus ratio increases from 2:1 to 3:1 as the pigs age and increase body weight (Jongbloed, 1987; Ketaren et al., 1989; Qian et al., 18 1996; NRC, 1998). The reason why higher calcium:availab1e phosphorus ratios are suggested (NRC, 1998) as pigs age and increase in body weight is not entirely clear, especially since some studies investigating the calciumzphosphorus ratio, growth, bone parameters, and serum measurements were not influenced by calciumzphosphorus ratio, even at a ratio as high as 3.1:1 (Koch et al., 1984). Other reports have stated that responses to higher (greater than 2: l) calcium:phosphorus ratios had adverse effects (Qian et al., 1996). Satisfactory calcium and phosphorus nutrition for swine and poultry is contingent upon an appropriate dietary calciumzavailable phosphorus ratio, adequate dietary vitamin D, and an adequate concentration of each macromineral in a bioavailable form in the diet. Other minerals, such as magnesium, zinc, and iron'can‘bind calcium and phosphorus and must be fed in the right amounts (NRC, 1994; NRC, 1998) so as not to have a negative impact on calcium and phosphorus nutrition. A limited amount of information is published on the dietary calcium and phosphorus recommendations for turkeys (Tables 3 and 4). Table 3. Suggested calcium requirements for growing turkeys as percentage of diet 90 percent dry matter) Source Recommendation (%) Age of Turkey Motzok and Slinger, 1948 1.70 Starting poults (0-5 wks) Wilcox et al., 1953 1.50 Starting poults (0-4 wks) t Slinger et al., 1961 1.00 Starting poults (0-8 wks) Formica et al., 1962 0.81 Starting poults (0-8 wks) Neagle et al., 1968 1.20‘ Starting poults (0-4 wks) Nelson et al., 1961 1.20‘ Growing turkeys Sullivan et al., 1962 Formica et al., 1962 Nelson et al., 1984 Can increase Growing turkeys requirements when diets contain high levels of phytate P. ' Requirement when dietary total P and vitamin D levels were 0.80% and 1,100 ICU/kg diet, respectively l9 Table 4. Suggested non-phytate phosphorus requirements for growing turkeys as . Ercentage of diet (90 percent dry matter) Source Recommendation (%) AEOf Turkey Almquist, 1954 0.60 Starting poults (0-6 wks) Bailey et al., 1986 0.60 Starting poults (0-3 wks) Stevens et al., 1986 0.60 Starting poults (0-3 wks) Day and Dilworth, 1962 non-phytate P requirement Growing turkeys Sullivan, 1962 decreases with age from (9-16,17-24, and 8-20 0.60 to 0.45 wks, respectively) The National Research Council (NRC, 1994) has suggested recommendations for the dietary calcium and phosphorus requirements of growing turkeys based on research by Day, Dilworth, and Sullivan (1962) (Table 5). The NRC (1994) recommendations are based on male turkeys phase fed in four-week intervals from previous research. However, the NRC (1994) states that genetic improvements in growth performance and body weight gain would suggest that the dietary recommendations be implemented at an earlier age as 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, and 18-21 weeks, rather than the previously suggested four-week intervals. This is because as turkeys reach market weight faster, feed intake increases and physiological age increases at an earlier chronological age, and so the nutritional requirements must also increase. libitum as Jgercenggg of diet (90% dry matter)‘ Table 5. Calcium and non-phytate phosphorus requirements of growing turkeys fed ad Growing turkeys, males hosphorus Nutrient 0-4 wksb 4-8 wksb 8-12 wksb 12-16 wks1r 16-2bO 20-24 wits" wks Calcium 1.20 1.00 0.85 0.75 0.65 0.55 Non-phytate 0.60 0.50 - 0.42 0.38 0.32 0.28 ‘Based on NRC recommendations (1994). 17 weeks, respectively. l’Age intervals are based on previous research of actual chronology. Genetic improvements in body weight gain necessitate an earlier implementation of these levels, at 0-3, 3-6, 6-9, 9-12, 12-15, and 15- "Organic phosphorus is considered to be of limited bioavailability as it is associated with phytin. Non- hytate phosphorus is, therefore, considered in the phogrhorus requirements of growing turlgys. 20 A considerable amount of research has been published concerning the calcium and phosphorus requirements of weanling pigs (Rutledge et al., 1961; Combs and Wallace, 1962; Combs et al., 1962, 1966; Miller et al., 1962, 1964a,b, 1965b,c,d; Menehan et al., 1963; Zimmerman et al., 1963; Blair and Benzie, 1964; Mudd et al., 1969; Coalson et al., 1972, 1974; Mahan et al., 1980; Mahan, 1982) and growing- finishing pigs (Chapman et al., 1962; Libal et al., 1969; Cromwell et al., 1970, 1972b; Stockland and Blaylock, 1973; Doige etal., 1975; Pond et al., 1975, 1978; Fammatre et al., 1977; Komegay and Thomas, 1981; Thomas and Komegay, 1981; Maxson and Mahan, 1983; Combs et al., 1991a,b). The National Research Council (NRC) has suggested recommendations for the dietary calcium and phosphorus requirements of weanling pigs and growing-finishing pigs (Table 6). The NRC (1998) recommendations are based on pigs phase fed within particular body weight ranges (3-5, 5-10, 10-20, 20- 50, 50-80, and 80-120 kg body weight). Table 6. Calcium, total phosphorus, and available phosphorus requirements of growing pigs fed ad libitum as percentage of diet (90% dry matter) or daily requirement‘ Growingpi gs body weight (kg) Nutrient 3-5 5-10 10-20 20-50 50-80 80-120 Calcium” 0.90 / 0.80 / 0.70 / 7.00 0.60 / 11.13 0.50 / 0.45 / 2.25 4.00 12.88 13.84 Total 0.70 / 0.65 / 0.60 / 6.00 0.50 / 9.28 0.45 / 0.40 / phosphorusb 1.75 3.25 11.59 12.30 Available 0.55 / 0.40/ 0.32 / 3.20 0.23 / 4.27 0.19 / 0.15 / hosphorus" 1.38 2.00 4.89 4.61 ‘Based on NRC recommendations (1998). 1’Percentages of calcium, total phosphorus, and available phosphorus should increase by 0.05 to 0.10 percentage units for developing boars and replacement gilts in the 50 to 120 kg body weigh_tgrowth period. 21 Phytase The enzymatic dephosphorylation and hydrolysis of phytic acid occurs primarily by the action of a family of enzymes known as phytases (myo-inositol hexaphosphate phosphorohydrolases). Phytases have the ability to catalyze the removal of inorganic orthophosphates from the phytic acid molecule in a stepwise fashion (N ayni and Markalds, 1986). Phytases consequently also have the ability to release other elements or compounds that may be bound to phytate, such as the cations that may be bound (calcium, magnesium, manganese, etc.), phytate-associated proteins, and phytate- associated starch. There are two phytases that have been classified by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC- IUBMB). These two phytases fall under the recommended names of 3-phytase (myo- inositol hexakis phosphate 3-phosphorohydrolase; EC 3.1.3.8) and 6-phytase (myo- inositol hexakis phosphate 6-phosphorohydrolase; EC 3.1.3.26), where the numbers refer to the carbon positions of phytase removal of the phosphate groups.- Eeckhaute and De Paepe (1994) reported that of the 50 feedstuffs they analyzed for phytase activity, only rye (5130 units/kg), triticale (1688 units/kg), wheat (1193 units/kg), and barley (582 units/kg) were phytase-rich. Maize, oats, sorghum. and oilseeds contained little or no phytase. Phytases present in the gastrointestinal tract of poultry and swine emanate from dietary sources of plant matter, gut microflora, and endogenous secretions fi'om the intestinal mucosa for the hydrolysis of the phytate molecule. Very low concentrations of phytase have been gleaned from the brush border region of the small intestine of mammals, such as pigs (Cooper and Gowing, 1983). The intestinal phytase activity in 22 poultry is rather controversial. Bitar and Reinhold (1972) and Maenz et al. (1995) showed that phytase activity exists in the small intestine of poultry. Davies and Motzok (1972) reported that a homogenate of chick intestinal mucosa was able to hydrolyze a sample of sodium phytate. In studies by Moore and Veum (1983), the presence of intestinal phytase was not confirmed. High concentrations of phytase are normally present in certain species of yeast, ftmgi including Aspergillus ficuum, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Aspergillusfitmigatus, Emericella nidulans, Myceliophthora thermophila, and T alaromyces thermophilus, as well as certain species of bacteria, including Escherichia coli, Pseudomonas, and Bacillus subtilis (Nelson et al., 1968; Sebastian et al., 1998). Aspergillus niger has been the most active fungal species isolated in terms of phytase activity (Sebastian et al., 1998). Aspergillusficuum, a variant of Aspergillus niger has been shown to produce the largest concentrations of phytase and was found to be very therrnostable (Nelson et al., 1967; Sebastian et al., 1998). Microbial phytases isolated from previously mentioned fungal species have a broader range in pH activity than plant phytases and have consequently been more effective in the lower pH gastrointestinal environments of swine and poultry (Simons et al., 1990; Sebastian et al., 1998). Nelson et al. (1968, 1971) conducted extensive studies investigating fungal phytase supplementation in com-soybean based poultry feeds. Cromwell and Stahly (1978) conducted an experiment in which a dried live yeast culture (Saccharomyces cerevisiae) was added to a com-soybean based diet for pigs. The anticipation was that the live yeast culture would contain phytase enzyme to liberate phytate phosphorus fi'om the feed. They concluded, however, that the yeast culture did not improve phytate 23 utilization because growth rate, feed conversion (as intake/ gain), and bone strength were not impacted. Similar conclusions were drawn by Chapple et al. (1979) from a similar swine experiment. Shurson et al. (1984), however, did conclude that growing pigs had an improved growth rate due to yeast phytase supplementation. The researchers were not able to improve phytate phosphorus utilization by supplementing a yeast phytase in com- soybean meal based diets in balance studies and feeding trials with piglets. In the past, production costs of fimgal-derived phytase would have been high and the cost of inorganic somces of phosphate (such as dicalcium phosphate) would have been relatively low. Because of these reasons, the commercialization of phytase supplementation was not realized (Swick and Ivey, 1992). Increased environmental awareness and advances in fermentation technology have renewed interest in feed phytase enzyme production and supplementation for poultry and swine diets. The use of phytase as a feed additive is enticing to poultry and swine producers in countries that have strict regulations. on land application of animal waste phosphorus, such as in the Netherlands (Sirnell et al., 1989; Campbell and Bedford, 1992). In the US, concerns about surface runoff water contamination from poultry production in areas such as the Delaware-Maryland-Virginia area have spurred interest in- phytase supplementation (Swick and Ivey, 1992). In Maryland, legislation has made it compulsory for poultry producers to feed phytase to reduce excreted phosphorus (Hansen, 2000). Recent developments in Michigan indicate that large animal feeding operations (greater than 1000 animal units [1,000 beef cattle = 3,000 pigs = 55,000 turkeys]) will need to develop a phosphorus-based comprehensive nutrient management plan (personal communication, Dr. Robert von Bemuth, Michigan State University). Over the past decade or so, research involving the use of phytase supplementation in com-soybean meal based diets for swine and poultry has intensified. Microbial phytase supplementation improves phytate availability in broiler chickens fed com-soybean meal based diets (Nelson et al., 1968; Denbow et al., 1995; others summarized in Table 7). On average, phytase supplementation of broiler diets improves phytate phosphorus availability by 20 to 40 percent. The amount of phytate phosphorus liberated from com-soybean meal based broiler diets depends on the concentration and source of added phytase and phytate (Simons et al., 1990; Komegay et al., 1996; Yi et al., 1996), calcium (Schoner et al., 1993; Sebastian et al., 1996), vitamin D3 (Edwards, 1993; Roberson and Edwards, 1994; Ravindran et al., 1995b; Yi et al., 1995; Qian et al., 1995, 1997), and the calciumzphosphorus ratio (Schoner er al., 1993; Qian et al., 1993). 25 awe he 3m 3 mm SEN—“$33 em a E 808mm,: 0.8 05 8 Eco wee: vow 203 who—Emu .055:— _ E Eon 03.93 88..— mEosamosamo .95.. _ «83.2 35 bréoa 05 m_ 38b... he ES r. .2383: 2: 5.8 03235 8: 33 83 889.2 85 88:2: mafia—co E 3:80. 11 l 2 mm 11 an 5 e2. 8e.oaoemaoz a. agree :5 88 3. 11 It 2 : we we _ .3 Sewaofieez mm .36 mass 82 l. e.» 2 ll 1. I 2 Re 26 .oaoeeaez a. .._e so :28 on: 11 l .I. 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Impact of microbial phytase on body weight (BW) gain, feed conversion ratio (FCR), phosphorus retention (Phos ret), and bone ash“ Ill growing turkeys fed corn-soybean meal based diets , % Improvement‘ Source Bird Phytase Diet BW gain FCR Phos ret. Bone age (d) source, in" ash units/kg” 4%) Yi et 29 NatuphoseT 0.45 11 _ _ _ al., 750 l 996 Qian et 21 Natuphos®, 0.27 83 32 24 al., 600 49 l 996 ' Atia et 49 Natuphos®, 52% of . . . — — 9.4 al., 500 NRC 1996 112 (1994) 12 aDashes in columns indicate that response data was not available from the literature. b1 unit of phytase is the activity that releases 1 umol of phosphorus from phytic acid in 1 minute. °in = nonjhytate mosphorus Additionally, Ravindran et al. (1995) reported that when turkey poults were fed 800 FTU/kg for 3 weeks, they had increased body weights, a 24% increase in bone ash, and a 35% increase in bone strength over poults fed no phytase with diets that contained 0.27% non-phytate phosphorus. Atia et al. 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Pigs fed [pal-1 corn diets had carcasses with less back fat and higher percentage lean. Diets containing Ipa] -1 corn with reduced phosphorus supplementation could be fed to growing-finishing swine (27-122 kg body weight) under experimental and commercial conditions with no deleterious effects on pig performance, bone strength, or carcass characteristics. In general, feeding low-phytic acid corn varieties, in place of conventional yellow dent corn, to poultry and swine on com-soybean meal based diets can reduce phosphorus excretion. When diets contain lower than required phosphorus levels, using low-phytic acid corn in place of dent com, can have an effect of increased phosphorus retention in the animal and could possibly improve bone strength relative to low phosphorus diets. Nutrient Composition of NutriDenseTM Low Phytate Corn NutriDenseTM Low Phytate (NDLP) Corn is a variety of corn that was developed by Exseed Genetics, L.L.C. (owned by BASF, Inc.) is lower in phytic acid and higher in total phosphorus and non-phytate phosphorus, amino acids, energy, crude protein, and oil content than conventional yellow dent corn. The marketing strategy behind NDLP corn, with respect to poutlry and swine nutrition, was to be able to provide an alternative variety of corn that would reduce excreted phosphorus and other minerals that might otherwise be chelated by the phytic acid complex, as well as providing a better amino acid balance than conventional dent corn. In doing so, the need for supplementing diets 36 with inorganic sources of phosphate, such as dicalcium phosphate, would decrease. The need for phytase supplementation might also decrease because of the lower levels of phytic acid in NDLP corn based diets. Other nutritional benefits from NDLP corn include the possibility of improvements in protein digestibility, weight gain, and feed efficiency. Studies involving feeding NDLP corn to meat type poultry were outlined in the previous section. The specific nutrient profile of NDLP com, as compared to YD com is given in Table 12. Corn analysis was obtained from the University of Missouri- Columbia nutrition laboratory. Estimated ME value of NDLP for pigs (3,498 kcal/kg) and poultry (3,490 kcal/kg) came from Exseed Genetics, L.L.C. Estimated ME value for YD (3,480 kcal/kg) came by personal communication from Vernon Felts, Goldsboro Milling, Raleigh, NC. Amino acid composition of the two corn types was analyzed at the University of Missouri-Columbia. Phytate phosphorus composition of the two corn types was analyzed at the University of Georgia. 37 Table 13. Analyzed nutrient content of NutriDense" Low Phytate (NDLP) and Yellow Dent (YD) corn NutrientA NDLP YD ME,” kcal/kg 3,480 3,410 Crude Protein 10.00 8.20 Crude Fat 2.60 2.20 Arginine 0.45 0.36 Cystine 0.22 0.17 Glycine 0.37 0.30 Histidine 0.29 0.23 Isoleucine 0.34 0.25 Leucine 1.31 0.97 Lysine 0.32 0.28 Methionine 0.21 0.16 Phenylalanine 0.51 0.39 Proline 0.87 0.68 Serine 0.43 0.33 Threonine 0.33 0.27 Tryptophan 0.07 0.05 Tyrosine 0.29 0.24 Valine 0.47 0.37 Ca 0.03 0.01 pP 0.03 0.1 7 in 0.29 0.08 tP 0.32 0.25 ANutrient values are expressed as % unless otherwise specified. BEstimated values. Bone Parameters in Poultry and Swine Nutrition Because phosphorus is a major constituent of bone tissue and has a prominent role in its structure, several techniques to surmise the nutritional contribution of phosphorus to bone have been devised. These include bone breaking strength, bone ash, and computed tomography. Bone breaking strength Animal nutritionists have used bone-breaking strength as a measure of response to nutritional regimens for many years, including dietary phosphorus concentrations. A very good review of bone strength as a measure of bone mineralization of swine was 38 reported by Crenshaw et al. (1981). The authors define “bone breaking strength” as the force per unit area required for breaking a bone. Most publications, which include “bone breaking strength” only involve a measure of force, or mass, with no consideration for the area of bone over which the force is applied. Many types of tests have been devised to determine the strength of materials. Modern devices used to measure bone strength have a better capability to reduce variations in bone strength than those in the past. An Instron Universal Testing Machine, for instance, has the capability to sustain a constant rate of deformation, electronically. A deformation rate of 5 mm/min is reported to be optimal for plotting a force deformation curve with an Instron Universal Testing Machine (Crenshaw et al., 1981). Variation also exists in the procedures used for bone preparation. Freezing bones before testing will not negatively impact the mechanical properties of the bones. Changes in temperature at the time of bone testing may slightly affect bone strength (Seldin, 1965). Wet bones bend more than dry fat extracted bones when comparing bone strain to the point of ultimate stress and consequent breakage (Crenshaw et al., 1981). As wet bones are exposed to air at room temperature, the bones begin to dry and begin to show an increase in strength (Seldin and Hirsch, 1966; Crenshaw et al., 1981). Wet bones are preferable during bone testing as they more closely resemble the state of the bones as they exist inside the living animal. Consideration must also be given to the inside hollow cross section of the bone as well as the bone shape (Crenshaw et al., 1981). Altered dietary calcium and phosphorus concentrations can result in changes in bone wall thickness without changing outside bone diameters (Cromwell et al., 1972; Tanksley et al., 1976; and Crenshaw et al., 1981). 39 In recent years, an attempt to standardize procedures for bone strength testing has been reported by the American National Standards Institute (March, 1998) for the shear and three-point bending test of animal bone. The outlined standards were developed by the American Society of Agricultural Engineers (ASAE) Physical Properties of Agricultural Products Committee approved by the ASAE Food and Process Engineering Institute Standards Committee and published in 1999. The standards outlined include ultimate shear stress (strength) and ultimate bending stress (strength). Standards for testing instrumentation are outlined. Bone ash Bone ash has been used as a response variable in countless studies investigating dietary phosphorus regimens for many years. Bone ash is the inorganic mineral portion of the bone and is most often reported as a percentage of fat-free dry bone. Fat-free bone is obtained by using solvents, such as ether, to extract fat and marrow out of bones and allowing to dry by removing the moisture in an oven set to greater than 100°C. Bone ash is obtained by burning bone in an oven at high temperature (600°C) for several hours (usually greater than 4, depending on the size of the bone) until every component of bone is combusted except “ash”, which is made up of bone minerals. Computed Tomography Computed tomography has been used as a radiological tool for the medical community since the 1970’s. Computed tomography (CT) scans employ the use of 360- degree x-ray beams accompanied by the computer production of images. Computed tomography scans allow for cross-sectional views of body tissues and organs, including bones. The images from a CT scan are much more pronounced, sharp, focused, than 40 standard x-ray equipment. This allows for a better view of body tissues (Hathcock and Stickle, 1993). The use of CT scans for studies involving the effects of dietary calcium and phosphorus on bone development in growing turkeys has been reported (Rosenstein et al., 2000). Computed tomography has also been utilized in studies involving vitamin and mineral supplement withdrawal and wheat middling inclusion in growing pig diets (Shaw, 2001). Bone mineral density (BMD) can be estimated using CT scanning equipment and have been found to correlate with turkey bones from turkeys fed different concentrations of dietary calcium and phosphorus (Rosenstein et al., 2000). Hydroxyapatite crystal standards can be scanned with bone samples simultaneously and analyzed using a bone mineral density software package to compare the x-ray linear attenuation coefficient of bone to the hydroxyapatite crystal standards. Conclusion From the review of literature, it is apparent that nutritional as well as environmental benefits exist for phytase supplementation and low phytic acid corn inclusion in swine and turkey diets. Although the amount of swine and turkey research in the area of phytase supplementation and low phytic acid corn usage is limited, the potential benefits they may offer are worthy of investigation. The objective of this thesis is to investigate the impact of low phytic acid com (NutriDensem Low Phytate) substitution for yellow dent (YD) com with and without microbial-derived phytase supplementation on turkey and swine grth performance, bone parameters, and reduce phosphorus excretion. 4l References Almquist, H. J ., 1954. 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Speer, V. W. Hays, and D. V. Catron, 1963. Effect of calcium and phosphorus levels on baby pig performance. J. Anim. Sci. 22: 658-661. 56 CHAPTER 1: IMPACT OF FEEDING LOW PHYT ATE CORN TO GROWING- FINISHING LARGE WHITE MALE TURKEYS Summary Two studies were conducted to verify the availability of P in a high amino acid, low-phytic acid corn (N DLP) variety as compared to conventional yellow dent corn (YD) fed to large white (British United Turkeys of America ‘Big 6’ breed) male turkeys. The NDLP contained 0.29% non-phytate P (in), which is assumed to be available, and 0.32% total P (tP). The YD contained 0.08% in and 0.25% tP. Results of these studies verify that P in NDLP is more available than P in YD. The NDLP can be safely formulated with an assumption of a 90% P availability without compromising growth or bone attributes, and feeding NDLP in place of YD can reduce excreted P by 30 to 44% without phytase and by up to 56% with 600 FTU/kg NatuphosO 600 phytase supplementation. Description‘of Problem Approximately two-thirds of the P in cereal grains and oilseed meals is bound to phytate, which is generally considered to have limited availability to poultry [1]. Because poultry diets in the US are based primarily on corn and soybean meal, they need- to be supplemented with inorganic P in order to meet the P requirements of the bird. The limited ability of poultry to utilize phytate from corn and soybean meal based diets can result in a large amount of P in the manure [2]. Manure is often disposed of by applying it to cropland as a nutrient source for the crops. In areas of highly concentrated poultry numbers, manure application can exceed the nutritional needs of the planted crop. In this case, manure derived nutrients such as P can pose an environmental risk. Runoff of P 57 into water bodies can contribute to the process of eutrophication, which can be perceived as environmental pollution. Scientists have recently developed corn mutants that contain less phytate P (pP) and more in, without reducing the concentration of tP [3]. Ifthese corn mutants are fed in place of YD, less supplemental dietary inorganic P (i.e., dicalcium phosphate) is necessary to achieve the same dietary concentrations of in. Ertl et al. [4] first reported that a variety of corn with the [pa] -1 mutation for low-phytic acid had a P availability of 75% when fed to broiler chicks and the chicks showed a reduction in excreted P ranging from 9 to 40%. Huff et al. [5] reported that total P can be reduced by 11% in the diet when using a high available P (HAP) [6] corn variety over YD in broiler diets. Kersey et al.[7] showed that excreted P could be reduced by 41.4% when a HAP corn diet was fed to broiler chicks with in reduced by 0.10% as compared to a YD corn diet. Li et al. [8] reported that broiler chicks fed HAP corn diets retained more calcium and phosphorus than chicks fed a YD corn diet. Yan et al. [9] showed that broilers fed HAP corn diets had reductions in excreted P ranging from 8 to 55% as compared to broilers fed YD corn diets. Komegay et al. [10] showed that young turkeys fed HAP corn in place of YD com in P reduced diets had a linear increase in body weight, toe ash, tibia ash, and P retention.- Waldroup et al. [11] reported that the greatest need for in was for maximum tibia ash (0.39% for YD and 0.37% for HAP) and excreted P was reduced by 28% when broilers were fed HAP corn in place of YD corn. Yan et al. [12] showed that when young turkeys and broilers were fed HAP corn in place of YD corn, there was a decrease in excreted P. Douglas et a1. [13] showed that a different variety of low-phytic acid (LP) com [14] had an average phosphorus availability of 86.2% with a range of 59 to 95%, based 58 on chick tibia response curves. A variety of low-phytic acid, high amino acid corn has been developed under the trade name NutriDense" LP corn (NDLP) [14]. This low- phytic acid corn has more in and tP and less pP concentration as compared to YD. Saylor et al. [15] reported that broilers fed NDLP corn based diets had a 24% decrease in excreted total P (tP) and a 26% decrease in soluble P (sP) as compared to broilers fed YD corn diets. Phytase enzyme addition is also a way to make dietary P more available to poultry [l6]. Atia et al. [17] reported an increase (P<0.05) in body weight (in 4, 8, 12, and 16 wk old male turkeys), bone ash (of 7 wk old turkeys), and bone density (of 7, 11, and 15 wk old male turkeys) when 500 FTng Natuphos” phytase was added to diets containing in at 52% of NRC [1]. Ravindran et al. [18] reported that when poults were fed 800 FTU/kg Natuphos” phytase added to diets containing 0.27% in for 3 wk, linear increases in body weight, bone ash, and bone strength were observed. Yan et al. [19] reported that when 1000 FTU/kg Natuphos“) was supplemented to young turkey diets (0-18 d of age) with in reduced by 0.15 percentage units from NRC [l], excreted P was reduced and bone ash was increased. The objectives of the two experiments were to evaluate what P availability of NDLP for toms grown to market age for consumer toms (ca. 16 kg) and whether phytase could be used in conjunction with NDLP to further reduce P excretion without reducing growth performance and bone strength. Materials and Methods The Michigan State University All University Committee for the Use and Care of Animals approved all animal experimentation procedures. Both experiments were 59 conducted from April to August using large white male turkeys [20]. All turkeys were procured from a commercial hatchery [21]. Experiment 1 BIRDS AND HUSBANDRY Four hundred twenty one 3-wk old poults were randomly assigned to four treatments. A completely randomized design was used with four dietary treatments and four replicate pens of 25 to 27 poults allotted randomly to each dietary treatment from 3 to 17 wk of age. Prior to allotment, 428 day-old poults were group brooded with the same four dietary treatments and two replicate pens of 53 to 55 poults placed randomly to each dietary treatment from day-old to 3 wk of age. Supplemental heat was provided for the first 8 d of the experiment with heat lamps, hung 45.72 cm above the pen floor in the center of the pen. For the first 6 d, turkeys were maintained on 24 h of light. On Days 7 and 8, turkeys were given 1 h of darkness. On Day 8, heat lamps were removed. Turkeys were given 1 more h of light and 1 less h of darkness each day, until 16 h of light and 8 h of darkness were achieved on Day 13. Turkeys were maintained on a 16-h light 8-h dark lighting schedule for the rest of the experiment. One 40-watt incandescent bulb at the center of each pen provided light for the entire study. Light intensity was approximately 60 lux. Environmental temperature was set to provide temperature targets (Table 1) using computerized propane gas powered heating and electronic ventilation. Table 1: Environmental temperature tagge_t_guidelines. Days of age Room Temperature (C) 21-28 26.67 i 2.22 29-35 23.88 i 2.22 36-42 fl 21.11 i 2.78 43-49 ' 18.33 i 2.78 49-119 15.56 i 2.78 60 Temperature settings were gradually decreased each day by approximately 056°C until the final temperature target setting was reached. Turkeys were housed in 2.46m X 3.08m floor pens with fresh pine shavings for bedding. Water was provided ad libitum using automatic red plastic bell type waterers. Feed was provided ad libitum using trough type feeders (day-old to 1 wk of age) and plastic tube round type feeders from day old-9 wk of age and galvanized steel tube round type self feeders from 9 wk until 17 wk of age. EXPERIMENTAL DIETS Diets were phase fed in mash form on a 3-wk interval from day old-3wk, 3-6, 6-9, 9-12, 12-15, and 15-17 wk of age. Dietary treatments included Treatment 1: YD-soybean meal based diet with a control level of tP Treatment 2: YD-soybean meal based diet with a negative control level of tP (-0.10% of treatment 1 tP) Treatment 3: NDLP-soybean meal based diet with the same tP as treatment 2, and a 75% availability assumption for in in NDLP Treatment 4: NDLP-soybean meal based diet with the same tP as treatment 2 and a 90% availability assumption for in in NDLP All treatments maintained a Cazin ratio at approximately 2: l . Nutrient composition of YD and NDLP are listed in Table 2 as analyzed [22, 23]. The ME values listed for YD and NDLP were estimated [24,25]. 61 Table 2. Analyzed nutrient content of NutriDense" Low Phytate and Yellow Dent corn Nutrient” NDLP YD M13,B kcal/kg 3,480 3,410 Crude Protein 10.00 8.20 Ether Extract 2.60 2.20 Arginine 0.45 0.36 Cysteine 0.22 0.17 Glycine 0.37 0.30 Histidine 0.29 0.23 Isoleucine 0.34 0.25 Leucine l .3 1 0.97 L sine 0.32 0.28 Methionine 0.21 0.16 Phenylalanine 0.5 1 0.39 Proline 0.87 0.68 Serine 0.43 0.33 Threonine 0.33 0.27 Tryptophan 0.07 0.05 Tyrosine 0.29 0.24 Valine - 0.47 0.37 Ca 0.03 0.01 W 0.03 0.17 grPt 0.29 0.08 tPt 0.32 0.25 ANutrient values are expressed as % unless otherwise specified. BEstimated values; YD estimate by personal communication with V. Felts, Goldsboro Milling, Goldsboro, NC; NDLP estimate from equation for ME from N. Dale (2000 Midwest Poultry Federation Conference, St. Paul, MN) CValues analyzed from the University of Georgia Poultry Nutrition Laboratory Athens, GA 30602) The ingredient concentration and selected nutrient composition of Experiment 1 diets are given in Table 3 for Prestarter (day-old to 3 wk) Table 4 for Starter 1 (3-6 wk) diets, Table 5 for Starter 2 (6-9 wk), Table 6 for Grower l diets (9-12 wk), Table 7 for Grower 2 diets (12-15 wk), and Table 8 for finisher diets (15-17 wk). Treatment 1 dietary calcium and phosphorus are considered to be the levels required for sufficient growth and 62 adequate bone strength, as determined in a previous experiment [26]. Washed fine sand was added to NDLP diets to maintain similar fat additions and ME values for the diets. Table 3. Composition and selected nutrient content of Prestarter diets xperiment 1) Prestarter diets (day old-3 wk) Ingredient, % 1 2 3 4 YD cornA 42.29 42.94 — — NDLP cornB - - 44.49 44.38 Soybean meal 48.44 48.33 46.26 46.28 (48% CP) Choice White 1.81 1.58 1.19 1.23 Grease Limestone 1.47 1.50 1.78 1 .71 Menhaden Meal 1.00 1.00 1.00 1.00 Blood Meal 1.00 1.00 1.00 1.00 Sodium 0.30 0.30 0.30 0.30 Bicarbonate Dicalcium 2.89 2.54 2.41 2.52 phosphate Salt 0.14 0.14 0.14 0.14 DL-Methionine 0.27 0.27 0.25 0.25 L-Lysine-HCl 0.09 0.09 0.14 0.14 Trace mineral 0.12 0.12 0.12 0.12 . mixC VitaminmixD 0.13 0.13 0.13 0.13 Choline 0.05 0.05 0.05 0.05 Washed sand - - 0.75 0.75 Nutrients ME, kcal/kg 2,850 2,850 2,850 2,850 CP, % 28.50 28.50 28.50 28.50 Ca, % 1.44 1.38 1.44 1.44 Calculated tP, % 0.98 0.91 0.91 0.94 Analyzed tP, % 1.04 0.94 0.92 0.92 Calculated in, % 0.72 0.66 0.72 0.72 AYellow Dent corn BNutriDenseI“ Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 I. DVitamin mix provided (/kg diet): vitamin A, 11,000 IU; vitamin D3, 5,000 IU; vitamin E, 35 IU; menadione, 2.75 mg; pantothenic acid, 20 mg; riboflavin, 10 mg; niacin, 80 mg; thiamine, 2.9 mg; pyridoxine, 4.3 mg; folic acid, 2.2 mg; biotin, 0.20 mg; vitamin B12, 0.025 mg; vitamin C, 0.10 mg; selenium, 0.275 mg; ethoxyquin, 125 mg. “ 63 Table 4. Composition and selected nutrient content of Starter l diets (Experiment 1) Starter 1 diets (3-6 wk) LEgredient, % 1 2 3 4 YD cornA 46.49 47.24 - - NDLP comB - - 48.66 48.54 Soybean meal 45.56 45.43 43.21 43.23 (48% CP) Choice White 2.59 2.32 1.99 2.03 Grease Limestone 1.44 l .47 1.75 1 .67 Dicalcium 2.85 2.46 2.29 2.42 phosphate Salt 0.38 0.38 0.38 0.38 DL-Methionine 0.29 0.29 0.27 0.27 L-Lysine-HCl 0.12 0.12 0.18 0.18 Trace mineral 0.12 0.12 0.12 0.12 mixc VitaminmixD 0.13 0.13 0.13 0.13 Choline 0.03 0.03 0.03 0.03 Washed sand - - 1.00 1.00 Nutrients ME, kcal/kg 2,933 2,933 2,933 2,933 CP, % 26.00 26.00 26.00 26.00 Ca, % 1.34 1.27 1.34 1.34 Calculated tP, % 0.92 0.85 0.85 0.88 Analyzed tP, % 0.91 1.05 0.98 1.00 Calculated in, % 0.67 0.60 0.67 0.67 AYellow Dent corn BNutriDenseTM Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 I. DVitamin mix provided (/kg diet): vitamin A, 11,000 IU; vitamin D3, 5,000 IU; vitamin E, 35 IU; menadione, 2.75 mg; pantothenic acid, 20 mg; riboflavin, 10 mg; niacin, 80 mg; thiamine, 2.9 mg; pyridoxine, 4.3 mg; folic acid, 2.2 mg; biotin, 0.20 mg; vitamin Bu, 0.025 mg; vitamin C, 0.10 mg; selenium, 0.275 mg; ethoxyquin, 125 £18- Table 5. Composition and selected nutrient content of Starter 2 diets (Experiment 1) Starter 2 Diets (6-9 wk) ingredient, % 1 2 3 4 YDcom” 52.92 54.12 - - NDLP comB - - 55.53 55.39 Soybean meal 38.23 38.04 35.53 35.56 (48% CP) Choice White 3.81 3.39 3.08 3.13 Grease Limestone 1.32 1.16 1.71 1.63 Dicalcium 2.62 2.20 2.01 2.16 phosphate Salt 0.38 0.38 0.38 0.38 DL-Methionine 0.30 0.30 0.30 0.30 L-Lysine-HCl 0.17 0.17 0.24 0.24 Trace mineral 0.12 0.12 0.12 0.12 mixC VitaminmixD 0.13 0.13 0.13 0.13 Choline - - - - Washed sand - - 1.00 1.00 Nutrients ME, kcal/kg 3,080 3,080 3,080 3,080 CP, % 23.00 23.00 23.00 23.00 Ca, % 1.24 1.09 1.24 1.24 Calculated tP, % 0.85 0.78 0.78 0.80 Analyzed tP, % 0.79 0.80 0.89 0.89 Calculated MP, % 0.62 0.54 0.62 0.62 AYellow Dent corn BNutriDenseT" Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 I. DVitarnin mix provided (/kg diet): vitamin A, 11,000 IU; vitamin D3, 5,000 IU; vitamin E, 35 IU; menadione, 2.75 mg; pantothenic acid, 20 mg; riboflavin, 10 mg; niacin, 80 mg; thiamine, 2.9 mg; pyridoxine, 4.3 mg; folic acid, 2.2 mg; biotin, 0.20 mg; vitamin B12, 0.025 mg; vitamin C, 0.10 mg; selenium, 0.275 mg; ethoxyquin, 125 mg. 65 Table 6. Composition and selected nutrient content of Grower l diets (Experiment 1) Grower l diets (9-12 wk) ingredient, % 1 2 3 4 YD cornA 57.50 58.79 - - NDLP cornB - - 60.59 60.44 Soybean meal 33.46 33.25 30.47 30.50 (48% CP) Choice White 5.36 4.91 4.48 4.53 Grease Limestone 0.95 0.77 1.37 1.28 Dicalcium 1.70 1.25 1.03 1.19 phosphate Salt 0.38 0.38 0.36 0.36 DL-Methionine 0.26 0.26 0.23 0.23 L-Lysine-HCl 0.15 0.15 0.23 0.22 Trace mineral 0.12 0.12 0.12 0.12 mixc VitaminmixD 0.13 0.13 0.13 0.13 Washed sand - - 1.00 1.00 Nutrients ME, kcal/kg 3,250 . 3,250 3,250 3,250 CP, % 21.00 21.00 21.00 21.00 Ca, % 0.88 0.71 0.88 0.88 Calculated tP, % 0.66 0.58 0.58 0.61 Analyzed tP, % 0.70 0.60 0.48 0.56 Calculated in, % 0.44 0.36 0.44 0.44 AYellow Dent corn BNutriDenseT” Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 1. [)Vitamin mix provided (/kg diet): vitamin A, 9,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; menadione, 1.5 mg; pantothenic acid, 15 mg; riboflavin, 6 mg; niacin, 70 mg; thiamine, 1.4 mg; pyridoxine, 3.0 mg; folic acid, 2.0 mg; biotin, 0.10 mg; vitamin B12, ' 0.014 mg; selenium, 0.25 mg; ethoxyquin, 125 mg. 66 Table 7. Composition and selected nutrient content of Grower 2 diets (Experiment 1) Grower 2 diets (12-15 wk) Ingredient, % 1 2 3 4 YD eom" 62.40 63.80 - - NDLP eorn'3 - - 65.94 65.76 Soybean meal 28.72 28.49 25.44 25.47 (48% CP) Poultry Fat 5.38 4.89 4.36 4.41 Limestone 0.92 0.73 1 .39 l .29 Dicalcium 1.63 1.14 0.90 1.07 phosphate Salt 0.38 0.38 0.36 0.36 DL-Methionine 0.26 0.26 0.23 0.23 L-Lysine-HCl 0.05 0.05 0.14 0.14 Trace mineral 0.12 0.12 0.12 0.12 mixC VitaminmixD 0.13 0.13 0.13 0.13 Washed sand - - 1.00 1.00 Nutrients ME, kcal/kg 3,300 3,300 3,300 3,300 CP, % 19.00 19.00 19.00 19.00 Ca, % 0.84 0.66 0.84 0.84 Calculated tP, % 0.63 0.54 0.54 0.57 Analyzed tP, % 0.60 0.62 0.51 0.52 Calculated in, % 0.42 0.33 0.42 0.42 AYellow Dent corn BNutriDenseT“ Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 I. DVitamin mix provided (/kg diet): vitamin A, 9,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; menadione, 1.5 mg; pantothenic acid, 15 mg; riboflavin, 6 mg; niacin, 70 mg; thiamine, 1.4 mg; pyridoxine, 3.0 mg; folic acid, 2.0 mg; biotin, 0.10 mg; vitamin B12, 0.014 mg; selenium, 0.25 mg; ethoxyquin, 125 mg. 67 Table 8. Composition and selected nutrient content of Finisher diets (Experiment 1) Finisher diets (15-17 wk) Medient, % 1 2 3 4 YD cornA 62.49 63.50 - - NDLP cornB — - 65.97 65.81 Soybean meal 26.82 26.65 23.54 23.57 (48% CP) Choice White 7.45 7.10 6.45 6.51 Grease Limestone 0.88 0.93 1.35 1.25 Dicalcium 1.54 1.00 0.82 0.99 phosphate Salt 0.36 0.36 0.36 0.36 DL-Methionine 0.21 0.21 0.18 0.18 L-Lysine-HCl - 0.002 0.09 0.09 Trace mineral 0.12 0.12 ‘ 0.12 0.12 mixC VitaminmixD 0.13 0.13 0.13 0.13 Washed sand - - 1.00 1.00 Nutrients ME, kcal/kg 3,430 3,430 3,430 3,430 CP, % 18.00 18.00 18.00 18.00 Ca, % 0.80 0.70 0.80 0.80 Calculated tP, % 0.60 0.50 0.51 0.55 Analyzed tP, % 0.63 0.46 0.45 0.42 Calculated nLP, % 0.40 0.30 0.40 0.40 AYellow Dent corn BNutriDenseT“ Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 1. DVitamin mix provided (/kg diet): vitamin A, 9,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; menadione, 1.5 mg; pantothenic acid, 15 mg; riboflavin, 6 mg; niacin, 70 mg; thiamine, 1.4 mg; pyridoxine, 3.0 mg; folic acid, 2.0 mg; biotin, 0.10 mg; vitamin B12, 0.014 mg; selenium, 0.25 mg; ethoxyquin, 125 mg. 68 Turkeys were weighed at the end of each dietary phase and feed disappearance was recorded. Feed conversion (feed: gain) was calculated after correcting for mortality. Experiment 2 BIRDS AND HUSBANDRY Four hundred 6-wk old turkeys were randomly assigned to four treatments. A completely randomized design was used with four dietary treatments and four replicate pens of 25 turkeys allotted randomly to each dietary treatment from 6 to 18 wk of age. Dietary treatmens started at 6 wk of age due to a low availability of NDLP for this experiment. Hence, the turkeys were fed a common control diet adequate in P during the brooding period (day old-6 wk of age). The same husbandry practices used for Experiment 1 were used for Experiment 2. EXPERIMENTAL DIETS Diets were phase fed on a 3-wk interval from 6-9, 9-12, 12-15, and 15-18 wk of age. Dietary treatments included: Treatment 1: YD-soybean meal based diet with a control level of tP, Treatment 2: YD-soybean meal based diet with a negative control level - of tP (-0.20% of treatment 1 tP) Treatment 3: NDLP-soybean meal based diet with the same tP as treatment 2 Treatment 4: NDLP based diet with the same tP as treatment 2 plus Natuphos®600 phytase (600 FTU/kg). 69 A 90% P availability assumption (90% of tP as in) for NDLP was included in the dietary ingredient formulation matrix. Treatment 1 and treatment 3 diets maintained a Cazin ratio of 2: l . The same nutrient compositions for NDLP and YD were used as in Experiment 1 (refer to Table 2), with the exception of ME values. The ME values used in Experiment 2 for NDLP and YD were 3,490 (TME by analysis [23]) and 3,390 kcal/kg (ME poultry industry average [25]), respectively. The ingredient composition and selected nutrient concentrations of Experiment 2 diets are given in Table 9 for Starter 2 diets (6-9 wk), Table 10 for Grower 1 diets (9-12 wk), Table 11 for Grower 2 diets (12- 15 wk), and Table 12 for finisher diets (15-16.5 wk). 70 Table 9. Composition and selected nutrient content of Starter 2 diets (Experiment 2) Starter 2 diets (6-9 wk) Jpgredient, % l 2 3 4 YD cornA 54.14 55.69 - - NDLP comB - - 60.96 60.96 Soybean meal 37.86 37.58 34.32 34.32 (48% CP) Choice White 3.61 3.06 1.13 1.13 Grease Limestone 1 .25 l .38 1 .47 1 .47 Dicalcium 2.02 1.15 0.97 0.97 phosphate Salt 0.35 0.35 0.33 0.33 DL-Methionine 0.29 0.29 0.28 0.28 L-Lysine-HCl 0.23 0.24 0.29 0.29 Trace mineral 0.10 0.10 0.10 0.10 mixC VitaminmixD 0.15 0.15 0.15 0.15 Natuphos® 600E - - - + Nutrients ME, kcal/kg 3,080 3,080 3,080 3,080 CP, % 23.00 23.00 23.00 23.00 Ca, % 1.00 0.86 0.86 0.86 Calculated tP, % 0.76 0.60 0.60 0.60 Analyzed tP, % 0.59 0.59 0.59 0.52 Calculated in, % 0.50 0.34 0.43 0.53 AYellow Dent corn BNutriDenseT" Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 I. DVitamin mix provided (/kg diet): vitamin A, 11,000 IU; vitamin D3, 5,000 IU; vitamin E, 35 IU; menadione, 2.75 mg; pantothenic acid, 20 mg; riboflavin, 10 mg; niacin, 80 mg; thiamine, 2.9 mg; pyridoxine, 4.3 mg; folic acid, 2.2 mg; biotin, 0.20 mg; vitamin B12, 0.025 mg; vitamin C, 0.10 mg; selenium, 0.275 mg; ethoxyquin, 125 mg. _ E2 lb/ton phytase was added = 600 FTU/kg to release 0.10% in, according to the manufacturer (BASF Corp., Mount Olive, NJ) 71 Table 10. Composition and selected nutrient content of Grower l diets mfiment 2) Grower 1 diets (9-12 wk) Ingredient, % l 2 3 4 YD cornA 57.26 58.83 - - NDLP tomB - - 64.40 64.30 Soybean meal 33.25 32.97 29.52 29.52 (48% CP) Choice White 5.75 5.19 3.16 3.16 Grease Limestone 1.08 1.22 1 .32 1 .32 Dicalcium 1.63 0.74 0.54 0.54 phosphate Salt 0.35 0.35 0.34 0.34 DL-Methionine 0.26 0.26 0.24 0.24 L-Lysine-HCl 0.21 0.21 0.27 0.27 Trace mineral 0.10 0.10 0.10 0.10 mixc Vitamin mixD 0.12 0.12 0.12 0.12 Natuphos® 600E - - - + Nutrients ME, kcal/kg 3,250 3,250 3,250 3,250 CP, % 21.00 21.00 21.00 21.00 Ca, % 0.84 0.70 0.70 0.70 Calculated tP, % 0.66 0.50 0.50 0.50 Analyzed tP, % 0.54 0.42 0.50 0.46 Calculated in, % 0.42 0.25 0.35 0.45 AYellow Dent corn BNutriDenseT“ Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 1. DVitamin mix provided (/kg diet): vitamin A, 9,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; menadione, 1.5 mg; pantothenic acid, 15 mg; riboflavin, 6 mg; niacin, 70 mg; thiamine, 1.4 mg; pyridoxine, 3.0 mg; folic acid, 2.0 mg; biotin, 0.10 mg; vitamin B12, 0.014 mg; selenium, 0.25 mg; ethoxyquin, 125 mg. E2 lb/ton phytase was added = 600 FTU/kg to release 0.10% in, according to the manufacturer (BASF Com., Mount Olive, NJ) 72 Table 11. Composition and selected nutrient content of Grower 2 diets (Experiment 2) Grower 2 diets (12-15 wk) Moment, % 1 2 3 4 YD corn"l 62.63 64.25 - - NDLP cornB - - 70.32 70.32 Soybean meal (48% 28.33 28.05 24.28 24.28 CP) Choice White 5.64 5.07 2.85 2.85 Grease Limestone 0.99 1.17 1.28 1.28 Dicalcium phosphate 1.45 0.51 0.29 0.29 Salt 0.35 0.35 0.34 0.34 DL-Methionine 0.26 0.26 0.24 0.24 L-Lysine-HCl 0.12 0.13 0.19 0.19 Tracemineral mixC 0.10 0.10 0.10 0.10 Vitamin mixD 0.12 0.12 0.12 0.12 Natuphos“) 600E - - - + Nutrients ME, kcal/kg 3,300 3,300 3,300 3,300 CP, % 19.00 19.00 19.00 19.00 Ca, % 0.76 0.62 0.62 0.62 Calculated tP, % 0.61 0.44 0.44 0.44 Analyzed tP, % 0.48 0.39 0.40 0.43 Calculated on, % 0.38 0.21 0.31 0.41 AYellow Dent corn BNutriDenseT" Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 I. DVitamin mix provided mtg diet): vitamin A, 9,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU ; menadione, 1.5 mg; pantothenic acid, 15 mg; riboflavin, 6 mg; niacin, 70 mg; thiamine, 1.4 mg; pyridoxine, 3.0 mg; folic acid, 2.0 mg; biotin, 0.10 mg; vitamin Bu, 0.014 mg; selenium, 0.25 mg; ethoxyquin, 125 mg. E2 lb/ton phytase was added = 600 F '1ng to release 0.10% in, according to the manufacturer (BASF Corp., Mount Olive, NJ) 73 Table 12. Composition and selected nutrient content of Finisher diets (Experiment 2) Finisher diets (15-18 wk) Ingledient, % 1 2 3 4 YD com“ 63.25 64.86 - - NDLP cornB - - 70.99 70.99 Soybean meal 26.31 26.03 22.23 22.23 (48% CP) Choice White 7.58 7.01 4.77 4.47 Grease Limestone 0.86 l .04 1 .14 1 .14 Dicalcium 1.15 0.21 - - phosphate Salt 0.35 0.36 0.34 0.34 DL-Methionine 0.20 0.20 0.18 0.18 L-Lysine-HCI 0.07 0.08 0.13 0.13 Trace mineral 0.10 0.10 0.10 0.10 mixC Vitamin mixD 0.12 0.12 0.12 0.12 Natuphos® 600E - - - + Nutrients ME, kcal/kg 3,430 3,430 3,430 3,430 CP, % 18.00 18.00 18.00 18.00 Ca, % 0.64 0.50 0.50 0.50 Calculated tP, % 0.54 0.37 0.37 0.37 Analyzed tP, % 0.53 0.42 0.44 0.36 Calculated in, % 0.32 0.15 0.25 0.35 AYellow Dent corn BNutriDenseTM Low Phytate corn CMineral mix provided (mg/kg diet): 100 Mn, 100 Zn, 50 Fe, 10 Cu, 1 I. DVitamin mix provided (/kg diet): vitamin A, 9,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; menadione, 1.5 mg; pantothenic acid, 15 mg; riboflavin, 6 mg; niacin, 70 mg; thiamine, 1.4 mg; pyridoxine, 3.0 mg; folic acid, 2.0 mg; biotin, 0.10 mg; vitamin B12, 0.014 mg; selenium, 0.25 mg; ethoxyquin, 125 mg. E2 lb/ton phytase was added = 600 FTU/kg to release 0.10% in, according to the manufacturer (BASF Corp., Mount Olive, NJ) 74 Turkeys were weighed every 3 wk and at 16.5 wk and again at 18 wk. Feed disappearance was recorded every 3 wk. All performance measurements were calculated in the same way for Experiment 2 as for Experiment 1. BONE ANALYSES At the end of both experiments (17 wk for Experiment 1 and 16.5 wk for Experiment 2, three and six birds, respectively, per pen were chosen for slaughter at the Michigan State University USDA inspected Meat Laboratory to obtain bone samples. Birds were chosen to match target body weight fi'om breeder guidelines [20]. Bone Breaking Strength Procedures for bone breaking strength were followed using shear and three-point bending tests of animal bone guidelines from the ASAE Standards guide [27]. The standards guide does not give equations for cross-sectional area. Ulna, tibia, and femur cross sectional shape most closely resembled a hollow quadrant of an ellipse. The cross sectional area of a hollow ellipse was determined by the following equation: A(m2)=:11.x7tx[(BD-bv)+[vx Bz-b)+[bx DZ-vj], where A = cross sectional area, m2; D = minor outside diameter of the bone cross section (m); B = major outside diameter of the bone cross section (m); w = average cortical bone wall thickness (m); v = D — 2w (m); and b = B — 2w (m). Bone wall thickness and cross-sectional diameters were measured at the point of breakage with a digital caliper [28] in Experiment 1 and with computed tomography [29] in Experiment 2. All breaking tests were performed using an Instron Universal Testing Machine [30] equipped with a 10 kN load cell that had 75 a crosshead speed of 5 mm/min. Bone fracture force values, measured in Newtons (N) of force, were recorded. Ultimate shear stress (strength) was calculated for ulna and femur bone samples according to the ASAE Standards guide [27] equation: Ultimate shear stress (Pa) = 1.‘ = 5%, where F = maximum force required to break a bone (N); and A = cross sectional area of the bone (m2). Ultimate bending stress (strength) was calculated for tibia bone samples according to the ASAE Standards guide [27] equation: . . FLC . . Ultrmate bendrng stress (MPa) = ou = 4—1 , where F = maxrrnum force required to break a bone (N); L = distance between fulcra supports (m), which was set to 0.10 m; C = distance from neutral axis to outer fiber = 0.57559D (m); and I = moment of inertia (m4), which was determined by the equation 0.0549 x (BD3 — bv3 ). Computed Tomography Computed tomographic (CT) scanning was performed on the ulna, humerus, tibia, and femur bones from Experiment 2 when machinery became available at MSU Veterinary Radiology to acquire cross-sectional images in the transverse plane. Individual bone samples, of the same type, were positioned five at a time on a bone density phantom pad with built-in hydroxyapatite standards for bone mineral density (BMD) analysis. One slice of 10 mm thickness was acquired at the mid-diaphysis using the bone algorithm at a 512 x 512 matrix. Table height was 142 cm to position the specimens in the center of the imaging field. Measurements of cortical bone thickness, total bone diameter (major and minor) and cross-sectional area (cortical and medullary) were performed on the CT computer. Image data was transferred to a remote CT console with BMD analysis software. The CT density values of four selected regions of cortical 76 bone were compared to the CT density of the hydroxyapatite standards within each image to estimate average cortical BMD. This technique is known as quantitative CT (qCT) [3 1]. Bone Ash Broken bones (all pieces) were soaked in ethanol for a minimum of 1 wk, dried, extracted with ether [32], oven dried at 106°C for 24 h, and ashed in a muffle furnace at 600°C for 24 h. TOTAL PHOSPHORUS ANALYSIS Feed samples were collected during the mixing of each dietary phase for tP analysis. During the last week of each study, raw excreta samples (four pens per treatment) were collected for 3 consecutive days for tP analysis. Litter samples (four pens per treatment) were collected at the end of each study according to the method described by Smith and Lacy [33]. Excreta and litter samples were dried in an oven at 50°C for 24 h or until no moisture accumulated when warm samples were placed in plastic storage bags. Feed, excreta, and litter samples were ground first to pass through a 1.0-mm sieve [34] then through a 0.5-mm sieve [3 5]. Duplicate samples of feed, excreta, and litter were digested by a nitric acid microwave wet digestion system [36]. Phosphorus concentrations in feed, excreta, and litter samples were analyzed colorimetrically [37] by a molybdo-sulfuric method [38]. STATISTICAL ANALYSIS All data were analyzed using the General Linear Models (GLM) procedure of SAS® software [39]. Pen was defined as the experimental unit. Significantly different dietary treatment pen means were separated using the least square means option of GLM. 77 Standard errors for treatment pen means were determined using the standard enor option of GLM. Treatment means were considered significantly different at P < 0.05. Results and Discussion Growth Performance Results of Experiment 1 diets on growth performance are reported in Table 13. Table 13. Growth performance of turkeys fed diets containing yellow dent (YD) or NutriDense" Low Phytate (NDLP) corn for Starter l (STl), Starter 2(ST2), Grower IAGRI), Grower 2 (GR2), and Finisher (FIN) phases (Experiment 1) TREATMENT PHASE Diet Corn inA P availability STl $12 OR] GR2 FIN Source of corn (%) (3-6 (6—9 (9-12 (12-15 (15-17 wk) wk) wk) wk) wk) BODY WEIGHT Lkgf 1 YD Control 32 2.20 5.30 8.93 12.29 14.47 2 YD Control- 32 2.21 5.27 8.82 12.34 14.35 0.10% 3 NDLP as Diet 2 75 2.29 5.46 9.13 12.56 14.71 4 NDLP as Diet 2 90 2.27 5.44 9.17 12.72 14.77 SEM 0.03 0.06 0.09 0.13 0.13 Probability 0.07 0.09 0.05 0.14 0.12 FEED INTAKE kr 1 YD Control 32 2.75 5.90 8.59 10.45' 6.85 2 YD Control- 32 2.70 5.68 8.50 1 1.14" 6.53 0.10% 3 NDLP as Diet 2 . 75 2.80 5.76 8.64 11.04“ 6.85 4 - NDLP as Diet 2 - 90 2.65 5.85 8.60 11.41” 6.41 SEM 0.05 0.09 0.1 1 0.15 0.22 Probability 0.30 0.38 0.85 0.01 0.39 . FEED CONVERSION (lrflrp)13c ‘ 1 YD Control 32 r 1.76 1.93 2.41 3.12 3.22 2 YD Control- 32 1.69 1.86 2.47 3.22 3.25 . 0.10% 3 NDLP as Diet 2 75 1.70 1.86 2.47 3.22 3.25 4 NDLP as Diet 2 90 1.62 1.85 2.30 3.26 3.24 SEM 0.04 0.04 0.05 0.06 0.12 Probability 0.15 0.42 0.15 0.49 0.95 Ain = non-phytate P B . Data are means of four repllcate pens F1 -. CFeed conversion (feedzgain) was calculated by ——G— , where F I = feed intake for the phase and G = mortality adjusted pen gain for the phase “For the same varible, mean values in the same column with unlike superscripts are significantly different (P<0.05) 78 Results of Experiment 2 diets on growth performance are presented in Table 14. Table 14. Growth performance of turkeys fed diets containing yellow dent (YD) or NutriDense" Low Phytate (N DLP) corn for Starter 2(ST2), Grower l (GRl), Grower 2 (GR2), and Finisher (FIN) phases (Experiment 2) TREATMENT PHASE Diet Corn inA Phytase ST2 GRl GR2 FIN Source (FTU/kg”) (6-9 (9-12 (12-15 (15-18 wk) wk) wk) wk) BODY WEIGHT (kg C 1 YD Control 0 5.72 9.37 12.74 16.78 2 YD Control- 0 5.60 9.21 12.59 16.46 0.20% 3 NDLP as Diet 2 0 5.54 9.15 12.45 16.28 4 NDLP as Diet 2 600 5.53 9.33 12.58 16.49 SEM 0.06 0.07 0.12 0.20 Probability 0.12 0.17 0.44 0.40 FEED INTAKE (k )C 1 YD Control 0 5.99 9.30 8.24 l 1.98 2 YD Control- 0 5.91 9.03 8.15 12.17 0.20% 3 NDLP as Diet 2 0 5.97 9.24 8.23 11.93 4 NDLP as Diet 2 600 5.92 9.09 8.47 11.72 SEM 0.17 0.17 0.12 0.61 Probability 0.98 0.66 0.28 0.96 FEED CONVERSION Lkgzkgf" 1 YD Control 0 1.88 2.55 2.49 3.50 2 YD Control- 0 l .94 2.51 2.54 3.28 0.20% 3 NDLP as Diet 2 0 1.98 2.55 2.54 3.69 4 NDLP as Diet 2 600 1.98 2.43 2.68 3.30 SEM 0.05 0.06 0.07 0.21 Probability 0. 45 0. 45 0. 33 0. 49 "in= non-phytate P BNatuphosO phytase (BASF Corp., Mount Olive, NJ), 1 FTU 1S defined as the amount necessary to liberate 1 mole of 1norganic P per minute from 0. 0015 mole of sodium phytate at 37°C and pH 5.5 (Data are means of four replicate pens DFeed conversion (feed: gain) was calculated by 21 , the phase and G = mortality adjusted pen gain for the phase where F I = feed intake for 79 Diet 1 in both experiments was designed to insure that control diets were meeting nutritional needs to support growth parameters (body weight, feed intake, and feed conversion). The standard expected growth responses used in our studies were brwder [ ] performance goals. Across dietary phases, in Experiment 1, turkeys fed control diets had body weights that were numerically lower than breeder [20] performance goals (2.20 vs. 2.65 kg for Starter l; 5.30 vs. 5.63 kg for Starter 2; 8.93 vs. 9.14 kg for Grower 1; 12.29 vs. 12.77 kg for Grower 2; 14.47 vs. 15.12kg for Finisher). 'In Experiment 2, turkeys fed control diets had body weights that were numerically higher for all phases except the Grower 2 phase as compared to breeder [20] recommendations. Although numerical differences in body weight exist between turkeys from our experiments and breeder [20] performance goals, the differences may not be statistically significant, - indicating that our control diets were adequate to support similar body weight as breeder [20] performance goals. Compared to growing-finishing turkey studies at other - » universities, such as those reported by Atia et al. [17] or Waldroup et al. [40], turkeys . grown in our facilities generally had higher body weight, probably due to differences in facilities. Turkeys in Experiment 1 did not grow as well as in other studies [26,41] at Michigan State University. However, turkeys fiorn Experiment 2 were comparable in body weight to what we have seen in previous studies [26,41]. The calculated in concentrations in our control diets were less than typical in concentrations fed in the Midwestern USA [42] or Southeastern USA. [43] commercial torn diets. Due to warmer temperatures in the Southeastern USA compared to the Midwestern USA, turkeys would be eating less feed and would therefore require a greater concentration of in in the diet to meet their in requirements. Even though our diets 80 contained less than typical concentrations of in, growth performance was still adequate, based on breeder performance goals [20] for body weight. In Experiment 1, turkeys fed control diets consumed more feed for Starter 2, Grower 1, and Grower 2 as compared to breeder [20] estimates for feed consumption and numerically less feed for Starter l and Finisher phases. In Experiment 2, turkeys fed control diets consumed more feed for Starter 2 and Grower 1 phases and numerically less feed for Grower 2 and Finisher phases, as compared to breeder [20] estimates for feed consumption. Numerical differences in feed consumption between turkeys in our studies as compared to breeder [20] estimates for feed consumption might fluctuate due to environmental temperature or the form of feed that was fed, since the turkeys were fed a mash form of diet in this study. In Experiment 1, turkeys fed Diets 2 and 4 consumed more (P<0.02) feed than turkeys fed Diet 1 (11.14 and 11.41 vs. 10.45 kg) for the Grower 2 phase. Such differences in feed intake were unexpected and cannot be explained. Turkeys fed in Experiment 2 appeared to have eaten slightly more feed than turkeys fed in Experiment 1 for the Starter 2 and Grower 1 periods. Turkeys fed in Experiment 1, however, ate more (P<0.05) feed than turkeys fed in Experiment 2 for the Grower 2 phase. This may have - been compensatory feed consumption if poults were of lower quality and matured later as compared to poults from Experiment 2. Also, in Experiment 2, feed consumption did not increase for the Grower 2 phase right after a period of higher growth (Grower 1) possibly because those turkeys matured earlier than those in Experiment 1. Turkeys fed the NDLP diets tended (P<0.10) to be heavier at the end of Starter 1, Starter 2, and Grower 1 phases (Table '13) in Experiment 1. The increase in body weight 81 dming these phases was most likely due to incorrect estimates of ME for YD and NDLP. The higher BW may have been due to higher energy consumption if ME was underestimated for NDLP and/or overestimated for YD. The estimated ME value for NDLP in Experiment 1 was 3,480 kcal/kg and 3,410 kcal/kg for YD. The estimate of ME for YD is one used in the commercial turkey industry [24]. The estimated ME value for NDLP was determined using the equation described by Dale et al. [25] for oil content and added energy content in consideration of the increased CP compared to YD. Starch was not accounted for by this equation in Experiment 1, which likely resulted in an underestimation of ME. Prior to the start of Experiment 2, a sample of NDLP was sent to the University of Georgia for a cecectomized rooster assay to obtain an analyzed TME value, which was 3,490 kcal/kg [23]. From these analyzed values, our original estimate for ME (Experiment 1) of NDLP may have been Slightly underestimated, which could have resulted in heavier body weights, keeping in mind that dietary treatments were designed to be isorritrogenous and isocaloric. Estimated ME for YD in Experiment 2 was decreased to the value reported by Dale et al. [25]. There was no differences in body weight due to corn type in Experiment 2, suggesting that ME values in Experiment 2 were correct and dietary treatments were isocaloric. In both experiments, Diet 2 was designed to be a negative control diet for P and turkeys fed this diet were expected to be lighter than turkeys fed other diets, if analyzed dietary P had met calculated dietary P. Atia et al.[17], Yan et al. [19], and Ledoux et al.[44] showed that when turkeys are fed lower concentrations of dietary P (30 and 52% < NRC [1]; a low P concentration; and NRC [l] — 0.15% for Atia et al. [17], Yan etal. [19], and Ledoux et al. [44], respectively), decreased body weight can result. Our experiments 82 did not yield lower turkey body weights for Diet 2 fed birds, although turkeys in Experiment 1 fed Diet 2 tended (P<0.10) to be lighter than turkeys fed all other diets at 17 wk of age. Estimates for tP intake were determined for each phase by multiplying estimated feed intakes by the analyzed concentrations of dietary tP. From these estimates, turkeys fed Diet 2 did not necessarily consume the least amount of dietary tP, as compared to other diets. In Experiment 1, turkeys fed Diet 2 consumed less (P<0.0001) dietary tP than turkeys fed Diet 1 (223.98 vs. 237.51 g), but more (P<0.0001) dietary tP than Diets 3 and 4 (223.98 vs. 207.25 and 212.94 g). In Experiment 2, turkeys fed Diet 2 consumed less (P<0.0001) dietary tP than turkeys fed Diet 1 (155.71 vs. 188.60 g), but consumed the same amount of dietary tP as Diets 3 and 4 (155-71 vs. 166.80, and 151.22 g). Because turkeys fed Diet 2 in both experiments did not necessarily consume the least amount of dietary tP, body weights were not necessarily lighter, as would have been expected. Some variation in the P concentration of YD used for diets may have contributed to deviation from calculated dietary P concentrations. Mixing error may have also contributed to variations in dietary P concentrations as compared to calculated values. Growth performance of turkeys fed Diets 3 and 4 were Similar (P>0.05) to turkeys fed Diet 1 in both experiments. These results matched expectations for growth performance, which was that replacing YD with NDLP with or without phytase should not negatively impact growth performance. 83 Bone Parameters Bone parameters (fiacture force, strength, ash, and mineral density) were investigated as indirect measures of P status. Because approximately 80% of the P found in the body exists as part of the skeleton, bone characteristics should be indicators of P status [45]. The femur was selected as a bone for testing because it has been observed as the bone that is often broken in the commercial turkey industry [46]. Lilbum [47] reported that the femur and tibia are sensitive bones in investigating leg development in growing- finishing turkeys (8 to 16 wk of age). Crenshaw et al.[48] reported that the femur was a sensitive bone for measuring responses to dietary P in pigs. Observations from previous experiments [48] at Michigan State University indicated that the femur, tibia, and ulna are sensitive bones for measuring responses to dietary P. Studies involving the investigation of dietary P on performance [17,44] of growing-finishing turkeys have reported the tibia as a bone useful as an indication of dietary P utilization. The effects of dietary treatrrients on bone fracture force and ultimate stress (strength) for Experiment 1 are presented in Table 15. No treatment differences in bone fracture force were observed in Experiment 1. When an equation to determine ultimate shear stress (strength) was applied, using bone fracture force values, treatment differences (P<0.02) in ulna ultimate shear stress were observed. Ulnas from turkeys that were fed Diet 2 withstood less pressure than ulnas from turkeys that were fed Diet 1 (19.10 vs. 16.82 MPa). Ulna ultimate Shear stress values from turkeys that were fed Diets 3 and 4 (17.30 and 17.99 MPa) did not differ fiom Diets 1 or 2. No treatrrrent differences in bone ultimate stress were observed for other bones in Experiment 1. 84 311". Table 15. Bone fracture force and bone strength (stress) of turkeys fed diets containing yellow dent (YD) or NutriDense" Low Phytate (NDLP) corn xperiment l) TREATMENT BONE TYPE Diet Corn inA P avail. Ulna F emur Tibia Source of corn (Shear) (shear) (bend) (%) BONE F RACTURE FORCE (N)BC Fs Fs Fb 1 YD Control 32 2578.97 1968.46 938.50 2 YD Control- 32 2415.71 2170.63 878.33 0.10% 3 NDLP as Diet 2 75 2290.07 1894.99 913.89 4 NDLP as Diet 2 90 2366.83 2012.69 837.61 SEM 76.78 130.85 48.32 Probability 0.1 1 0.52 0.50 BONE STRENGTH (MPa)"" 1 r on 1 YD Control 32 19.10‘ 7.97 105.52 2 YD Control- 32 16.82" 8.65 104.01 0.10% 3 NDLP as Diet 2 75 17.30" 8.15 106.53 4 NDLP as Diet 2 90 17.99‘” 8.40 95.39 SEM 0.43 0.27 8.08 Probability 0.02 0.36 0.76 Ain = non-phytate P BData are means of four replicate pens of three turkeys each CFracture force is the force required to break a bone, measured in Newtons (N); 1 N = 0.102 kgf (kilograms force) = 9.8 kg (kilograms mass) DBone strength (ultimate stress, a"), measured in Pascals (Pa) is determined by the . F LC . . equatron a, = ’2” for ultrmate bendrng stress and r = :2 , where F b= bone bending fracture force (N), F s = bone shear fracture force (N), A = cross-sectional area of the bone (m2), L = distance between fulcra supports (tibia = 0.10 m), C = distance from neutral axis to outer fiber (m), I = moment of inertia (m‘); l MegaPascal (MPa) = Pax(lx10'°); 1 Pa= l-IL2 m “P or the same variable, mean values in the same column with unlike superscripts are _S_igrrificantly different (P<0.05) 85 Results from Experiment 2 for the effects of dietary treatments on bone fiacture force and ultimate stress (strength) are presented in Table 16. Turkeys fed Diet 3, in Experiment 2 had femurs that withstood less breaking force than femurs from turkeys fed Diet 2 (1511.35 vs. 1685.40 N, respectively). When ultimate stress values were calculated from bone cross-sectional measurements using a digital micrometer, no treatment differences were observed. When qCT was used to measure bone cross- sectional area, however, treatment differences for femur samples were observed (P<0.02). This would indicate that perhaps using qCT technology is a more accurate way to determine cross-sectional area than by hand measurements with a digital caliper. The advantage of qCT is that bones do not need to be broken to take cross-sectional measurements. Femurs fi'om turkeys fed Diet 3 withstood less pressure than femurs from turkeys fed Diet 2 (18.36 vs. 20.96 MPa). Ultimate stress values for femurs from turkeys fed Diets 1 and 4 (18.21 and 20.12 MPa) did not differ from ultimate Stress values for femurs from turkeys fed Diet 3 or Diet 2. These results were not consistent with the reSultS from Experiment 1 ulna stress, which may be partially explained by inconsistencies in dietary tP concentrations, particularly in Starter 2 and Grower 1. Diet 1 contained 0.15% less tP than expected (0.76%), which may partially explain why femurs from turkeys fed Diet 1 were not stronger than femurs from turkeys fed the negative control diet. Similarly, in Grower 2, Diet 1 contained 0.12% less tP than expected. Why Diet 2 femurs were stronger than Diet 3 femurs is questionable Since they were formulated to contain the same concentration of tP, and Diet 3 was calculated to ' contain a greater concentration of in than Diet 2. This should have yielded stronger femurs in Diet 3. No other bone strength differences were observed in Experiment 2. 86 Table 16. Bone fracture force and bone strength (stress) of turkeys fed diets containing yellow dent (YD) or NutriDense" Low Phytate (N DLP) corn (Experiment 2) TREATMENT BONE TYPE Diet Corn up? Phytase Ulna F emur Tibia Source (FTU/kgB) Ashear) (shear) (bend) BONE FRACTURE FORCE (NfD F, F, F, 1 YD Control 0 2542.90 2919.54ab 702.65 2 YD Control-0.20% 0 2414.48 2929.51: 654.60 3 NDLP as Diet 2 0 2439.05 2863.38T 688.28 4 NDLP as Diet 2 600 2326.81 2810.20” 683.18 SEM 71.45 160.05 21.30 Probability 0.25 0.95 0.47 BONE STRENGTH (MPa)CE, USING qCT" t t 0'u 1 YD Control 0 21.26 18.21ab 71.46 2 YD Control- 0 20.72 20.96' 63.56 0.20% 3 NDLP as Diet 2 0 20.87 18.36” 69.30 4 NDLP as Diet 2 600 20.26 20.12ab 64.62 SEM 0.69 1.15 2.34 Probability 0.79 0.30 0.10 BONE STRENGTH (MPa)CE t r G., 1 YD Control 0 32.79 15.66 74.57 2 YD Control- 0 3 l .42 15.83 66.06 0.20% 3 NDLP as Diet 2 0 31.52 16.69 73.38 4 NDLP as Diet 2 600 " 28.79 15.72 67.85 SEM 1.40 0.57 2.97 Probability 0.28 0.56 0.18 Ain = non-phytate P ”Natuphos" phytase (BASF Corp., Mount Olive, NJ ); 1 FTU is defined as the amount necessary to liberate 1 mole of inorganic P per minute from 0.0015 mole of sodium phytate at 37°C and pH 5.5 cData are means of four replicate pens of six turkeys each DFracture force is the force required to break a bone, measured in Newtons (N); 1 N = 0.102 kgf (kilogram force) = 9.8 kg (kilograms mass) EBone strength (ultimate stress, 0,), measured in Pascals (Pa) is determined by the F LC F equation a" = 21 for ultimate bending stress and T = 2: , where F l,= bone bending fracture force (N), F, = bone shear fracture force (N), A = cross-sectional area of the bone (m2), L = distance between fulcra supports (tibia = 0.10 m), C = distance from neutral axis to outer fiber (m), I = moment of inertia (m‘); 1 MegaPascal (MP3) = Pa x (1 x 10.6 )i 1 Pa = 1 1&2 . m FqCT = quantitative computed tomography for cross-sectional x-ray scan of a bone to determine cross- sectional area and wall thickness prior to bone breakage u’For the same variable, mean values in the same column with unlike superscripts are significantly different (P<0.05) 87 The ulna was the most consistent bone for breaking tests. The ulna appeared to be more uniform in shape and did not shift noticeably in position during Instron testing. The femurs and tibias were more likely to slide on the fulcra points or shift in position while testing. However, attempts were made to minimize this fi'orn occurring in Experiment 2. The breaking pattern of the femurs were much different than the other bones. Femurs would shatter into many different pieces whereas ulnas would have a clean shear through the midsection of the bone and tibias would begin to break along the length of the bone. The breaking pattern of the femur may indicate that it might be a more sensitive bone for phosphorus utilization, but a lack of expected responses to dietary treatments in the current study does not lend support to this theory. Bones fi'om turkeys fed Diet 2 would have been expected to be weaker than bones from turkeys fed other diets. Failure to obtain expected bone responses in Experiment 2 may have resulted because analyzed dietary P did not match calculated dietary P in the Starter 2 through Grower 2 periods. Ulna responses in Experiment 1 may have been partly attributed to proper dietary implementation of treatments during the 3-week Prestarter group brooding period. Previous studies have Shown that differences in cortical thickness of turkey bones were only Si gnificant in younger turkeys [49]. Why there were no responses fi'om other bones, however, is puzzling. Other evidence indicates that dietary treatment differences should have been observed. Roberson et al. [50] observed linear increases in femur, tibia, and 1111121 bone strength when 300-600 F TU/kg Natuphos” phytase was added to low in diets (0.45, 0.40, or 0.35% dietary in in Grower l, Grower 2, and Finisher diets, respectively) and fed to male turkeys up to 17 wk of age. Since Roberson et al. [50] observed 88 treatment differences in bone strength when dietary treatments were started when turkeys were 9 wk of age, their findings do not lend support to the theory that dietary treatments should be implemented in the Starter periods. Ledoux et al. [44] reported that when turkey hens were fed either 0 or 1000 FTU of NatuphosO phytase to diets containing either NRC [1] or NRC [1] —0.15% concentrations of dietary in, no dietary treatment differences (P=0.082) were observed in tibia breaking strength taken fiom 15 wk old hens. Mean tibia-breaking strength ranged from 70.6 to 76.6 kg force across treatment groups, which would equate to a range of 692.3 to 751.2 N. Tibia fiacture force values appeared to be greater (in Experiment 1) than values reported by Ledoux et al. [44] with mean tibia bending strength values ranging fiom 837.6 to 938.5 N (or 85.4 to 95.7 kg force). In Experiment 2, our mean tibia strength values ranged from 654.6 to 701.65 (or 66.8 to 71.5 kg force), which appeared to be less than what Ledoux et al. [44] had reported. Our calculated diets should have contained more in than NRC [1] recommendations. Previous research has indicated that the in requirement for grth may not necessarily be the optimum for bone ash and bone strength [11]. If that is the case, then that may be why Ledoux et al. [44] did not see responses for bone strength. Atia et al. [1 7] observed that when 500 FTU/kg Natuphos“ phytase was added to diets containing 30% of the NRC [1] recommendation of dietary in, tibia strength of 7 wk old male turkeys was improved. Because our dietary in concentrations were formulated to contain more in than NRC [1] recommendations and NDLP with or without phytase should have improved P utilization, we Should have seen more responses in bone strength in both experiments. Other research investigating turkey bone responses to dietary P [12,18,19] lends support that the current studies should have shown more responses in 89 bone fracture force and strength. Analysis of dietary pP was not performed in the cunent study and we cannot determine exactly what in concentrations were fed, so it is not known precisely where the discrepancies exist in the analyzed vs. calculated dietary P concentrations. Most values of bone “strength” are reported as kg. This is not the preferred way to report bone “strength”, according to the ASAE standards guide [27]. The preferred units for reporting bone fiacture force is as Newtons (N). One Newton is the force required to accelerate a mass of one kilogram 1 m/s’; force equals mass times acceleration. The acceleration due to gravity on Earth is 9.80665 m/S2 , so 1 N is associated with 0.10141 kg mass. Another unit of force is kgf, or kilogram force. One kgf is equivalent to 1/9.80665 of a Newton, so 1 N = 0.10197 kgf. Also, what has often been reported as “strength” may actually be “fracture force”. The correct measure of bone “strength”, according to the ASAE Standards guide [27] is as “ultimate stress”, which is the fracture force applied per unit area, measured in Pascals (Pa), which is equivalent to 1 —N—,— . This value can be converted to MegaPascalS (MPa) for the ease of looking at m" numbers. One MPa is equivalent to Pa x (1x 104’). No treatment differences in bone average cortical wall thickness or percentage bone ash (Table 17 for Experiment 1 and Table 18 for Experiment 2) were observed. 90 Table 17. Bone cortical wall thickness and bone ash of turkeys fed diets containing yellow dent (YDJor NutriDense" Low Phytate (NDLP) corn (Experiment 1) TREATMENT BONE TYPE Diet Corn in‘ P Ulna Femur Tibia Source availability of corn (%) ‘ BONE WALL THICKNESS (mm? 1 YD Control 32 1.22 1.50 1.55 2 YD Control- 32 l .32 1.5 1 1 .45 0.10% 3 NDLP as Diet 2 75 1.18 1.39 1.50 4 NDLP as Diet 2 90 1.19 1.45 1.46 SEM 0.05 0.06 0.03 Probability 0.20 0.52 0.15 BONE ASH (%) 1 YD Control 32 45 .85 41.20 45.71 2 YD Control- 32 45.92 41.48 45.39 0.10% 3 NDLP as Diet 2 75 44.74 40.98 46.74 4 NDLP as Diet 2 90 45.01 43.07 47.23 SEM 1.05 1.10 1.36 Probability 0.81 0.55 0.76 2‘in = non-phytate P Data are means of four replicate pens of three turkeys each 91 Ft‘shr'l- filth!“ Also, no treatment differences in average bone mineral density for Experiment 2 (Table 18) were observed. Table 18. Bone cortical wall thickness, bone ash, and bone mineral density of turkeys fed diets containing yellow dent (YD) or NutriDensem Low Phytate LP) corn (Experiment 2) TREATMENT BONE TYPE Diet Corn inZr Phytase Ulna Femur Tibia Source (FTU/k B) BONE WALL THICKNESS (mm)" 1 YD Control 0 1 .24 2.06 2. 12 2 YD Control- 0 1.16 2.01 2.04 0.20% 3 NDLP as Diet 0 1.18 1.88 2.07 2 4 NDLP as Diet 600 1.17 1.98 2.13 2 SEM 0.03 0.08 0.04 Probability 0.29 0.53 0.30 BONE ASH (%) 1 YD Control 0 43.59 49.04 49.24 2 YD Control- 0 42.79 58.62 50.40 0.20% 3 NDLP as Diet 0 46.33 50.22 49.43 2 4 NDLP as Diet 600 44.11 47.57 49.22 2 SEM 1.51 1.79 0.99 Probability 0.42 0.77 0.81 BONE MINERAL DENSITY (mg/cmbw 1 YD Control 0 617.44 538.12 765.64 2 YD Control- 0 596.64 537.61 741.90 0.20% 3 NDLP as Diet 0 616.45 519.08 722.36 2 4 NDLP as Diet 600 588.88 527.66 731.17 2 SEM 37.54 35.19 26.41 Probability 0.93 0.98 0.69 Ain = non-phytate P l’Natuphos" phytase (BASF Corp., Mount Olive, NJ); 1 FTU is defined as the amount necessary to liberate 1 uncle of inorganic P per minute from 0.0015 mole of sodium phytate at 37°C and pH 5.5 CData are means of four replicate pens of six turkeys each I)Bone mineral density determined by computed tomography technology 1|‘t’For the same variable, mean values in the same column with unlike superscripts are Significany different (P<0.05) The lack of response to dietary treatments for cortical wall thickness, percentage bone ash, or bone mineral density was surprising, since bones from turkeys fed Diet 2 in both experiments should have had lower bone ash and bone mineral density values as wall as thinner wall thickness compared to turkeys fed control diets. Crenshaw et al. [50] reported that when pigs were fed higher concentrations of Ca and P (0.8 and 0.8 vs. 0.4 and 0.4 %, respectively), bone wall thickness and percentage bone ash increased (P<0.01). From this data, the current study may not have yielded differences in bone wall thickness because the differences in dietary P between treatments were not as wide as those reported by Crenshaw et al. [50]. Atia et al. [17], Ledoux et al. [44], Roberson et al. [48] and others [11,12,18,19] reported that feeding higher concentrations of dietary P to turkeys and broilers, as compared to diets containing lower concentrations of dietary P, would have higher (P<0.05) bone ash values. Atia et al. [17] also reported that feeding higher concentrations of dietary P could increase bone mineral density (P<0.05). Tibias ashed in the current study appear to contain a higher percentage of ash than values reported by Atia et al. [17]. Atia et al. [1 7] reported tibia mean ash values ranging fiom 31.3 to 49.0% from 7 wk old male turkeys. In the current study, tibia mean ash values ranged from 45.71 to 50.40% from 17 wk old male turkeys. Tibia ash values in the current study are higher than those observed by Atia et al. [1 7] because bones were taken 10 wk later than those collected in the study reported by Atia et al. [17], and that our diets contained higher concentrations of P. Why no treatment differences were observed in our study might be again due to analyzed dietary P concentrations not matching calculated dietary P concentrations. 93 Excreted Phosphorus The effects of dietary treatments on litter P and excreta P for Experiment 1 are shown in Table 19. Table 19. Litter phosphorus and excreta phosphorus of turkeys fed diets containing yellow dent (YD) or NutriDense" Low Phytate (NDLP) corn (Experiment 1) TREATMENT LITTER P (%)B EXCRETA P (%)B Diet Corn inA P 17 wk 17 wk Source availability of corn %) 1 YD Control 32 1.75' 1 .87' 2 YD Control- 32 1.42" 1.4315 0.10% 3 NDLP As Diet 75 1.42b 1.27“ 2 4 NDLP As Diet 90 1.42” 1.301) 2 SEM 0.05 0.07 Probability 0.001 0.0002 Ain = non-phytate P 18Data are means of four replicate pens, samples were collected as random grab samples during 15 and 17 wk of age "’F or the same variable, mean values in the same column with unlike superscripts are sigm'ficantly differentiP<0.05) Turkeys fed Diets 2, 3. and 4 in Experiment 1 had litter (collected at 116 d of age) that contained 19% less (P<0.002) tP than litter from turkeys that were fed Diet 1 (1.42 vs. 1.75%). Compared with turkeys fed Diet 1, turkeys fed Diets 2, 3, and 4 excreted 24, 32, and 30% less (P<0.0002) tP, respectively (1.87 vs. 1.43, 1.27, and 1.30%). By multiplying feed intake by the percentage of analyzed dietary tP, turkeys fed Diet 1 ate more (P<0.0001) tP than turkeys fed Diets 2, 3, and 4 in the Finisher phase (43.17 vs. 30.03, 30.81, and 26.91 g/bird) which matches results of excreta tP. 94 The effects of dietary treatments on litter P and excreta P for Experiment 2 are shown in Table 20. Table 20. Litter phosphorus and excreta phosphorus of turkeys fed diets containing yellow dent (YD) or NutriDense“ Low Phytate (NDLP) corn (Experiment 2) TREATMENT LITTERP (%)C EXCRETA P (%)C Diet Corn inA Phytase 18 wk 18 wk Source (FTU/kg”) 1 YD Control 0 1.48‘ 1.30- 2 YD Control- 0 1.14” 0.89” 0.20% 3 NDLP As Diet 2 0 1.11” 0.72“ 4 NDLP As Diet 2 600 0.89“ 0.57= SEM 0.40 0.07 Probability <0.0001 <0.0001 Ain = non-phytate P BNatuphosO phytase (BASF Corp., Mount Olive, NJ); 1 FTU is defined as the amount necessary to liberate ] mole of inorganic P per minute from 0.0015 mole of sodium phytate at 37°C and pH 5.5 CData are means of four replicate pens, samples were collected as random grab samples during the last wk of the experiment (18wk of age) "*‘For the same variable, mean values in the same column with unlike superscripts are iignificantly different (P<0.05) Turkeys fed Diet 4 had litter that contained 40% less (P<0.0001) than litter from tlu'keys fed Diet 1 (0.89 vs. 1.48%). Litter from turkeys fed Diets 2 and 3 had 23 and 25% less (P<0.0001) P than litter from turkeys fed Diet 1, respectively (1.14 and 1.11 vs. 1.48%). Excreta from 18 wk-old turkeys fed Diet 4 had 58% less (P<0.0001) tP than excreta from turkeys fed Diet ] (0.57 vs. 1.30%). Also, excreta from turkeys fed Diets 2 and 3 had 32 and 45% less (P<0.0001) tP than excreta from turkeys fed Diet 1, respectively (0.89 and 0.72 vs. 1.30%). Turkeys fed Diet 1 consumed more dietary tP than Diets 2 and 3 or 4 (188.60 vs. 155.71 and 166.80 or 151.22 g/bird). This relationship closely matches results for litter and excreta tP. A further decrease in excreted tP in Diet 4 can be 95 attributed to an increase in available (np) dietary P by the addition of 600 FTU/kg Natuphos“ phytase. Ledoux et al. [44] reported higher litter P values from birds fed diets that contained NRC [1] concentrations of in than we observed (mean reported values ranged from 1.62 to 2.25%). Why we saw lower tP concentrations in our turkey litter may be due to differences in the type of shavings used or procedures for analysis employed. We used pine shavings whereas Ledoux et al. [44] used cedar shavings. We used a nitric acid microwave wet digestion procedure whereas Ledoux et al. [44] used a nitric perchloric digestion procedure. Ledoux et al. [44] reported that the P content of the cedar shavings was subtracted from the litter P value. This may have resulted in a lower than actual value of litter P. The P content of the cedar shavings was not reported in the publication. Furthermore, percentage of litter as shavings vs. feathers vs. wasted feed and excreta is not reported. These values would be nearly impossible to ascertain. Because the percentage of litter as wasted feed, feathers, and excreta is difficult, if not impossible to determine, we collected raw excreta in addition to litter. The tP concentration of pine shavings was found to be 0.03% in our studies. An average book value for turkey litter phosphorus [51] is 1.67%. Our control level turkey litter values were 1.75 and 1.48% at the end of Experiments 1 and 2, respectively, which agrees with normal expected values. Similar results were also observed in studies where broilers were fed NDLP [15]. One of the objectives of these studies was to evaluate phosphorus availability of NDLP for toms grown to market age. Diets 3 and 4 of Experiment 1 were designed to investigate this objective, with Diet 3 assuming 75% P availability for NDLP and Diet 4 assuming 90% P availability for NDLP. Because dietary analyzed P values did not 96 always match calculated dietary P values, claims about P availability are inconclusive. However, results from ulna stress data in Experiment 1 did provide some support to the 90% P availability assumption from NDLP analysis. Deviation fi'om dietary treatment regimen may have been attributed to fluctuations in ingredient P concentrations or errors in adding the right amount .of each ingredient during mixing. Concentrations of P in YD are known to vary by as much as 0.10 percentage units [52]. Deviations from prescribed dietary treatment regimen have been reported in other studies [17,53]. Another objective of these studies was to investigate whether dietary phytase supplementation could further reduce excreted P when added to the NDLP diet. Diet 4 in Experiment 2 was designed to address this objective. According to the manufacturer’s [54] recommendation, 2 lb/ton Natuphos” was added to the Diet 4 treatments, in which dietary P was the same as Diet 3 (except for phytase addition), to give 600 FTU/kg phytase activity, which Should be adequate to replace 0.10% dietary in. This objective was apparently met with a 20% decrease in litter P as compared to litter from turkeys fed Diet 3. Other researchers [18,55,56] have found that feeding 600-800 FTU/kg Natuphos° . to poults fed diets containing low concentrations of dietary P was adequate (compared to - 0 FTU/kg) to improve body weight gain and toe ash, which demonstrates that phytase is an effective way to improve dietary P utilization. Atia et al. [17] demonstrated that 500 FTU/kg Natuphoso fed to male growing-finishing turkeys on low P diets was able to improve tibia ash and tibia strength as well as body weight. Roberson et al. [48] observed that 300 FTU/kg Natuphos” phytase was adequate to maintain growth and bone strength of commercial toms gaining 178 g/d fed from 9-17 wk of age. 97 In summary, results from these studies suggested that P from NDLP was more available than YD, when comparing ulna data from Experiment 1. Other experimental results indicate that good growth and bone strength can be obtained with low P diets. Results showed that turkeys fed NDLP and phytase excreted 56% less excreta P and had 40% less litter P than turkeys fed YD with an adequate level of dietary P for growth. Cost of the Finisher diet (Experiment 1, Diet 1 vs. 4) was reduced by about $6.00/ton, just as an example of how replacing YD with NDLP might result in feed cost savings. Costs of ingredients are based on current sources and our assumptions [57, 58]. Conclusions and Applications 1. Laboratory analyses Showed that in made up 90% of the tP in NDLP, whereas YD had 33% of the tP as in. Ulna ultimate stress results imply that an estimate of 90% P availability for NDLP in Experiment 1 may be appropriate. Further conclusions about P availability of NDLP are limited in this study because analyzed P values in feed at various phases did not always agree with calculated values. 2. Feed cost could be reduced by approximately 5% if NDLP were fed in place of YD corn, based on our assumptions for ingredient costs and that the market price of NDLP is assumed to be the same as YD. Reduction in feed cost would arise due to the higher energy, crude protein and available P added value of NDLP, which would replace some ingredient supplementation, such as fat, soybean meal, and dicalcium phosphate, respectively. 3. Replacing YD with NDLP with or without phytase can reduce excreted P by approximately 56 (with phytase) or 40% (without phytase) in finishing toms, based on results from Experiment 2. 98 References and Notes 10. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Sweeten, J.M., 1992. Livestock and Poultry Waste Management A National Overview. In: J. Blake, J. Donald, and W. Magette (Ed) National Livestock, Poultry, and Aquaculture Waste Management, 4-15. 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Anim. Sci. 52: 1319-1329. 49. Leblanc, B., M. Wyers, F. Cohn-Bendit, J.M. Legall, E. Thibault, and J.M. Florent, 1986. Histology and histomorphometry of tibia growth in two turkey strains. Poult. Sci. 65: 1 787-1 795. 50. Roberson, ‘K.D., M.W. Orth, M.W. Klunzinger, R.A. Cbarbeneau, and T.L. Peters, 2002. Evaluation of phytase level needed for growing-finishing commercial toms. lntemational Poultry Scientific Forum, Atlanta, GA. 51. Leeson, S. and J.D. Summers, 1997. Nutrition and world poultry production. Page 7 in: Commercial Poultry Nutrition. S. Leeson and J .D. Summers, 2nd ed. University Books, Guelph, Ontario, Canada. 102 52. Roberson, KD., 2001, Michigan State University, East Lansing, MI 48824. Personal Communication. 53. Ibrahim, S., J.P. Jacob, and R. Blair, 1999. Phytase Supplementation to reduce phosphorus excretion of broilers. J. Appl. Poult. Res. 8:414-425. 54. BASF Corp., Mount Olive, NJ 07828. 55. Yi, Z., E.T. Komegay, and D.M. Denbow, 1996. Effect of microbial phytase on nitrogen and amino acid digestibility and nitrogen retention of turkey poults fed com- soybean meal diets. Poult Sci. 75:979-990. 56. Qian, H., E.T. Komegay, and D.M. Denbow, 1996. Phosphorus equivalence of microbial phytase in turkey diets as influenced by calcium to phosphorus ratios and phosphorus levels. Poult. Sci. 75:69-81. 57. Ingredient Market, 2002. Feedstuffs 74(22):42. 58. We assumed $91.43/ton for corn, $181/ton for soybean meal (48% protein), $230/ton for choice white grease, $2200/ton for DL-Methionine, $1000/ton for L-Lysine HCl, $250/ton for dicalcium phosphate, and S30/ton for limestone. ACKNOWLEDGEMENTS We appreciate the donation of NutriDense" LP corn by Exseed Genetics, LLC and advice from Jerry Weigel and Dr. Christopher Peter. The Corn Marketing Program of Michigan supported this study. We thank BASF Corp. for the donation of NatuphosO phytase. We also thank the MSU Poultry Farm manager Angelo Napolitano and his staff for their cooperation during this study. We thank the MSU Meats Lab Manager Tom Forton and his staff for their cooperation during the study. We thank the Tom Otto Turkey Farm, Middleville, MI and Michigan Turkey Producers Cooperative, Wyoming, M1 for the purchase of finished toms. We also thank Dr. Rob Tempehnan for his advice in Statistical analysis. 103 CHAPTER 2: IMPACT OF FEEDING LOW PHYTATE CORN TO WEANLING AND GROWING-FINISHING PIGS Abstract Two studies were completed to determine if phosphorus in NutriDense" Low Phytate maize (NDLP) can be formulated assuming a 90% phosphorus availability without affecting growth or bone parameters (for growing-finishing pigs only) when fed to crossbred weanling and growing-finishing pigs as compared to yellow dent maize (YD). The NDLP contained 2.9 g kg’1 non-phytate phosphorus (in) and 3.2 g kg" total phosphorus (tP), by analysis. The YD contained 0.8 g kg" in and 2.5 g kg" tP, by analysis. In Experiment 1, Treatment 1 diets contained YD with 5.7 g kg" calculated tP for Grower 1 (0-33 d), 5.0 g kg" calculated tP for Grower 2 (33-58 (I), and 4.7 g kg"1 calculated tP for Finisher (58-91 d). Treatment 2 diets contained NDLP with 4.7 g kg" calculated tP for Grower l, 4.0 g kg" calculated tP for Grower 2, and 3.6 g kg" calculated tP for Finisher. NO treatment differences (p>0.05) in bone or growth parameters were observed. F aecal phosphorus decreased (p<0.01) by 41, 47, and 49% in the three phases, respectively, when NDLP was fed. In Experiment 2, Treatment 1 diets contained YD with 6.1 g kg" calculated tP for Phase 1 (1-14 (1) and 5.3 g kg" calculated IP for Phase II (14-28 (1). Treatment 2 diets contained YD with 4.2 g kg" calculated tP for Phase I and 3.5 g kg" calculated tP for Phase 11. Treatment 3 and 4 diets contained NDLP with 4.5 g kg" calculated tP for Phase I and 3.9 g kg" calculated tP for Phase 11. Treatment 4 diets also contained 500 units (FTU) kg" of phytase (NatuphosTM) for Phase I and 250 FTU kg" for Phase 1]. Growth parameters were not different (p>0.05) were observed. Serum P was decreased in Treatment 2 as compared to Treatments 1, 3, and 4 104 (39.18 vs. 53.25, 48.65, and 51.60 g kg"). Faecal phosphorus decreased (p<0.006) by up to 55% when NDLP (with phytase) was fed, as compared to YD control. F aecal phosphorus was reduced by 35% when NDLP was fed in place of YD. Introduction Phytate phosphorus is of limited availability to weanling and growing-finishing pigs. Diets fed to pigs containing maize and soyabean meal have two-thirds of the phosphorus in the form of phytate phosphorus. To assure adequate growth, bone mineralization, and the prevention of phosphorus deficiency inorganic phosphorus must be supplemented. F aecal phosphorus is a challenge in swine waste management when manure phosphorus content exceeds the plants’ phosphorus requirement. Water runoff can carry excess phosphorus into streams, so phosphorus could contribute to eutrophication. This phosphorus excess can be partially alleviated by increasing the bioavailability of dietary phosphorus to swine by reducing dietary phytate phosphorus or using exogenous phytase. Raboy first described maize with a non-lethal mutation on the [pal-1 allele, known as low-phytate maize (Raboy et al., 2000). This mutation contained reduced phytate phosphorus without affecting the total amount of phosphorus. Phosphorus availability of low phytate maize ranges from 57 to 75% (Cromwell et al., 1998; Pierce et al., 1998; Spencer et al., 2000a) and reduced phosphorus excretion and increase phosphorus retention (Pierce et al., 1998; Veum et al., 1998; and Spencer et al., 2000a). Exogenous phytase addition to maize-soyabean meal based pig diets has been shown to improve phosphorus availability (Cromwell et al., 1993; Lei et al., 1993a, 105 1993b; Young et al., 1993; Mroz et al., 1994; Adeola, 1995; Cromwell et al., 1995; Komegay et al., 1995; Roberson, 1999). In 1999, a variety of maize, known as NutriDense" LP (NDLP) that was 82% lower in phytate phosphorus, 72% higher in non-phytate phosphorus, 18% higher in crude protein, and 15% higher in crude fat than conventional maize became commercially available. Table 1 compares the analyzed nutrient compositions of NDLP and YD maize. Table 1. Analyzed nutrient content of NutriDense" Low Phytate (NDLP) and Yellow Dent (YD) maize Nutrient" NDLPC YDD ME,B (MJ kg") 14.74 14.31 Crude Protein 100.0 82.0 Ether Extract 26.0 22.0 Arginine 4.5 3.6 Cysteine 2.2 l .7 Glycine 3.7 3.0 Histidine 2.9 2.3 Isoleucine 3.4 2.5 Leucine 13.1 9.7 Lysine 3.2 2.8 Methionine 2. l 1 .6 Phenylalanine 5.1 3.9 Proline 8.7 6.8 Serine 4.3 3.3 Threonine 3.3 2.7 Tryptophan 0.7 0.5 Tyrosine 2.9 2.4 Valine 4.7 3.7 Ca 0.3 0.1 Phytate P 0.3 1.7 Available (non-phytate) P 2.9 0.8 total P 3.2 2.5 ANutrient values are expressed as g kgTunless otherwise Specified. BEstimated values. The objectives of these studies were to 1) determine if NDLP phosphorus was 90% available in growing-finishing and weanling pigs, 2) to determine if exogenous 106 phytase fed with NDLP would further improve dietary phosphorus utilization, and 3) to determine if substitution of NDLP for YD maize would alter growth parameters. Materials and Methods Two experiments were conducted. In Experiment 1, 64 castrates and female crossbred (Musclorm ;Multigene USA, L.L.C., Clearfield, Iowa, USA 50840 x [Yorkshire-Landrace]) pigs (mean of 59 d of age; mean body weight of 20.48 kg). Pigs were allotted based on weight and sex. to eight pigs per pen, four pens per treatment and fed for 106 (1. Treatment 1 diets with YD met phosphorus requirements of NRC (1998). Treatment 2 diets with NDLP were formulated to contain 1.0 g kg" less total phosphorus than in Treatment 1, and the same concentration of available phosphorus. Three dietary phases were fed with calculated total phosphorus at 5.7 and 4.7 for Grower l, 5.0 and 4.0 for Grower 2, and 4.7 and 3.6 g kg’I for Finisher for Treatments 1 and 2, respectively. Diet compositions are given in Table 2. 107 mEosamofi 3.3.9.9205 vie—ES u mam maesamoi .88 .1. ate .5 8. .om m... as. e. a... 8. m. 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Feed and faecal samples were ground through a 1.0-mm sieve (Thomas Wiley Mill Model AH2151X, Artlrrup H. Thomas Co., Philadelphia, Pennsylvania, USA 19019) and then through a 0.5-mm sieve (Cyclotec Model 1093 Sample Mill, Foss North America, Eden Prairie, Minnesota, USA 55344). At termination of the study, blood was collected into 8.5 mL collection tubes with no anticoagulant fiom the vena cava of one castrate and one gilt, chosen at random per pen. Samples were maintained on ice until centrifuged at 4°C at 3,000 revolutions per minute for 5 minutes, using a Beckman 6S-6KR centrifuge (Beckman Instruments, Inc., Fullerton, Califomia, USA 92634). Serum samples were harvested and frozen at -18°C until analyzed. At the termination of the study, one gilt per pen was selected. based on mean pen body weight (92.93 kg), for slaughter at the Michigan State University USDA-inspected Meat Laboratory. All four feet and both femurs from each pig were collected for bone analyses. In Experiment 2, 96 castrate and gilt crossbred Duroc x (Yorkshire-Landmce) pigs were weaned (mean of 22 d of age; 6.56 kg mean body weight) and assigned to one of four treatment based on sex and weight. There were Six pigs per pen and four pens per treatment. Treatment 1 diets with YD and calculated total phosphorus of 1.0 g kg" less than the NRC (1998) recommendations werved as a negative control. Treatment 2 diets with YD had 2.0 g kg" and 1.5 g kg" less total phosphorus (calculated) in Phase I and 109 Phase II, respectively than diets in Treatment 1. Treatment 3 diets contained NDLP with the same calculated total phosphorus concentration as Treatment 2 diets. Treatment 4 diets were the same as Treatment 3 diets with the addition of 500 FTU kg" NatuphosO 600 phytase (BASF, Mount Olive NJ 07828). Dietary compositions are given in Tables 3 and 4 for Phase 1 diets (1 -14 d) and phase II diets (14-28 d), respectively. 110 Table 3. Composition of Phase I dietsA (Experiment 2) Phase I diets (1-14 (1) Ingrediean kg") 1 2 3 4 Yellow Dent maize 474.8 482.4 - - NutriDense" Low Phytate - - 500.7 500.07 maize Soyabean meal 283.7 282.9 274.0 274.0 (440 g kg" CP) Dried whey 100.0 100.0 100.0 100.0 Spray dried plasma 30.0 30.0 30.0 30.0 Spray dried blood cells - - - - Lactose 50.0 50.0 50.0 50.0 Corn oil 18.5 15.6 6.3 6.3 L-Lysine-HCI 1 .5 1.5 1.5 1 .5 DL-Methionine 1.1 1.1 1.1 1.] Salt 3.5 3.5 3.5 3.5 Copper sulfate 0.5 0.5 0.5 0.5 Dicalcium phosphate 10.3 - - - Limestone 8.8 15.2 15.1 15.1 Trace mineral premixB 5.0 5.0 5.0 5.0 Vitamin premixC 6.0 6.0 6.0 6.0 Zinc oxide 2.8 2.8 2.8 2.8 AntibioticD 2.5 2.5 2.5 2.5 Natuphos" phytaseE - - - 0.63 Chromic oxide 1.0 1.0 1.0 1.0 Nutrient (g kg") Crude Protein 198.2 198.4 204.0 204.0 ME (MJ kg") 13.66 13.66 13.66 13.66 Lysine 13.5 13.5 13.5 13.5 Calcium 8.0 8.0 8.1 8.0 Calculated tPF 6.1 4.2 4.5 4.5 Analyzed tPF 5.9 3.9 4.5 4.6 Calculated aPG 4.0 2.1 3.1 4.1 ADietary phase was based on NRC (1998) body weight intervals and counted as days from the start of the experiment (Phase 1: 5-10 kg fi'om Day 1 to 14) BMineral premix provided (mg kg" diet): 670 Ca; 10 Cu; 0.2 I; 100 Fe; 10 Mn; 0.3 Se; 100 Zn. CVitamin premix provided (kg" diet): Vitamin A 5511 IU; Vitamin D3 551 IU; Vitamin E 66 IU; Vitamin K activity 13 mg; menadione 4.4 mg; Vitamin B12 0.03 mg; riboflavin 3.7 mg; d- pantothenic acid 18 mg; niacin 26 mg; thiamine 1.1 mg; pyridoxine 0.9. I)Mecadoxm (Carbadox) (Pfizer, Inc., Exton, Pennsylvania, USA 19341) is commonly used in the US to maintain healthy status. E1.0 kg tonne" will provide 500 FTU kg" phytase activity accordin (BASF Corp., Mount Olive, New Jersey 07828) to replace 1.0 g kg hosphorus. tP = total phosphorus GaP = available (non-phytate) phosphorus II] to the manufacturer available dietary Table 4. Composition of Phase II dietsA (Experiment 2) Phase II diets (14-28 d) Ingredient (g kg") 1 2 3 4 Yellow Dent maize 652.1 659.5 - - NutriDense“ Low Phytate - - 665.9 665.2 maize Soyabean meal 223.8 223.] 213.0 213.0 (440 g kg" CP) Dried whey 50.0 50.0 50.0 50.0 Spray dried plasma - - - - Spray dried blood cells 20.0 20.0 20.0 20.0 Lactose - - - - Corn oil 12.5 9.8 4.0 4.0 L-Lysine-HCl l .5 1 .5 1.5 1.5 DL-Methionine 1.1 1.1 1.1 5.0 Salt 3.5 3.5 3.5 5.0 Copper sulfate 0.5 0.5 0.5 0.5 Dicalcium phosphate 9.6 - - - Limestone 8.1 14.1 13 .9 13.9 Trace mineral mixIB 5.0 5.0 5.0 5.0 Vitamin mixC 6.0 6.0 6.0 6.0 Zinc oxide 2.8 2.8 2.8 2.8 Antibiotic” 2.5 2.5 2.5 2.5 Natuphos°°° phytaseE - - - 0.7 Chromic oxide 1.0 1.0 1.0 1.0 Nutrient Crude Protein 174.7 175.0 181.7 181.7 ME (MI kg") 13.66 13.66 13.66 13.66 Lysine 11.5 11.5 11.5 11.5 Calcium 7.0 7.0 7.0 7.0 Calculated tPF 5.3 3.5 3.9 3.9 Analyzed tPF 4.2 3.2 3.3 3.6 Calculated aP” 3.2 1.4 2.7 3.7 ADietary phase was based on NRC (1998) body weight intervals and counted as days from the start of the experiment (Phase 11: 10-20 kg from Day 14 to 28) 8 Mineral premix provided (mg kg" diet): 670 Ca; 10 Cu; 0.2 I; 100 Fe; 10 Mn; 0.3 Se; 100 Zn. CVitamin premix provided (kg" diet): Vitamin A 5511 IU; Vitamin D3 551 IU; Vitamin E 66 IU; Vitamin K activity 13 mg; menadione 4.4 mg; Vitamin Biz 0.03 mg; riboflavin 3.7 mg; d- pantothenic acid 18 mg; niacin 26 mg; thiamine 1.1 mg; pyridoxine 0.9. DMecadox" (Carbadox) (Pfizer, Inc., Exton, Pennsylvania, USA 19341) is commonly used in the US to maintain healthy status. E1.0 kg tonne" will provide 500 FTU kg" phytase activity according to the manufacturer (BASF Corp., Mount Olive, New Jersey 07828) to replace 1.0 g kg’ available dietary phosphorus. tP = total phosphorus GaP = available (non-phytate) phosphorus 112 Pigs were housed in an environmentally controlled facility with steel slatted flooring, nipple waterers, and stainless steel feeders. Feed and water were supplied ad libitum. Pigs and feed were weighed every 14 d. Chromic oxide was added (1.0 g kg") to estimate apparent phosphorus digestibility. At the end of each phase, faecal samples were collected randomly as in Experiment 1. Laboratory analyses were the same as in Experiment 1, plus chromium concentration was determined by atomic absorption spectrometry. Analyses Feed and faecal samples were wet-ashed in duplicate using a nitric acid microwave digestion procedure (Model 907055 Microwave Accelerated Reaction System 5 with HP-500 Plus Vessel Accessory sets (CEM Corp., Matthews, North Carolina, USA 28105)). Feed, faecal, and serum phosphorus was measured colorimetrically (Gomori, 1942), using a Beckman DU-7400 spectrophotometer (Beckman Instruments, Inc., Fullerton, Califonria, USA 92634). Feed and faecal samples were digested in duplicate for chromic oxide determination by the phosphoric acid method (Williams et al., 1962). Digested samples were read on a Unicam 989 atomic absorption Spectrometer (Unicam Atomic Absorption, Cambridge CBIYF, UK). Third and fourth metacarpal and metatarsal bone strength was measured using the Shear method (ASAE, 1999) with an Instron Universal Testing Machine (Model 4202, Instron, Canton, Massachussetts, USA ) fitted with a 10 kN load cell with a crosshead speed of 5 mm min". Femur bone strength was measured using the bend method (ASAE, 1999) with a Texture Expert (Model TA-Hdi, Texture Technologies Corp., Scarsdale, New York, USA 10583). Bone ash was determined with third and fourth metacarpal and 113 metatarsal bones and 20-mrn cross sections of both femurs were soaked in ethanol for a minimum of 7 d, dried, extracted with ether in a Soxhlet extraction apparatus (Pyrex® Model 3885, Coming Glass Works, Corning, New York, USA 14831), oven dried (Model DN-81 Constant Temperature Oven (American Scientific Products Co., Erie, Pennsylvania, USA 16506)) at 106°C for 24 hr, and ashed (Thennolyne Type 30400 muffle fumace (Barnsted/Thermolyne, Dubuque, Iowa, USA 52044)). Osteocalcin in serum was measured in duplicate using a commercially available ELISA testing kit (Novocalcin®; Metra Biosystems, Mountain View, California, USA 94043). Calculations and Statistics Apparent phosphorus digestibility was calculated by a method described by Kersey et al. (1995) using analyzed chronric oxide and total phosphorus values fi'om faecal and feed samples. Ultimate bone stress (strength) was calculated according to the ASAE Standards guidelines (ASAE, 1999). The cross-sectional area of a quadrant of a hollow ellipse, which most closely matched the shape of the cross-section of bones tested, was estimated by the following equation: A = ixrthBx D)—(bxv)]=[vx(8;b]]+[bx(D—2——v-]], where A =cross-sectiona1 area of the bone (m2), D = minor outside diameter of the bone cross section (In), B = major outside diameter of the bone cross section (m), V = D - (2 x w), b = B —- (2 x w), and w = average bone wall thickness (m). Experiment 1 data were analyzed by the T-TEST procedure of SAS” software (1999) for a randomized complete block design. Experiment 2 data were analyzed by the least squares (lsmeans) Analysis of Variance (ANOVA) with the General Linear Model 114 (GLM) procedure of SAS” software (1999) for a randomized complete block design. Pen was defined as the experimental unit. Significantly different dietary treatment pen means were adjusted with a Tukey multiple pairwise comparison method for Experiment 2 data. Standard errors for pen means were ascertained using the standard error (stderr) option of GLM. Differences in treatment means were considered Significant at P<0.05. Results The effects of dietary treatments on growth performance for Experiment 1 are presented in Table 5. No dietary treatment differences were observed for body weight, average daily gain, average daily feed intake, Or feed efficiency. For overall estimated average daily feed intake, pigs fed Treatment 2 consumed more (P=0.04) feed than pigs in Treatment 1. ° ° Table 5. Effects of dietary treatments on body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed efficiencijd") (Experiment 1) Treatment Probability 1 2 SEM Trt Source of maize YDA NDLP” ADG (kg day") Grower 1 0.478 0.490 0.01 0.52 Grower 2 0.933 1 .01 0.06 0.30 Finisher 0.797 0.837 0.07 0.60 Overall 0.736 0.798 0.04 0.32 ADF] (kg day") Grower 1 1.05 1.13 0.03 0.14 Grower 2 2.14 2.38 0.1 1 0.17 Finisher 2.83 2.93 0.08 0.30 Overall 1 .98 2.14 0.09 0.04 G:F Grower 1 0.454 0.434 0.01 0.06 Grower 2 0.438 0.423 0.01 0.43 Finisher 0.281 0.286 0.02 0.80 Overall 0.366 0.363 0.02 0.73 AFeed efficiency as gainzfeed (GzF) BYellow Dent maize CNutriDense°°° Low Phytate maize 115 The effects of dietary treatments on bone measurements for Experiment 1 are Shown in Table 6. No differences in third and fourth metacarpal and metatarsal or femur fiacture force or ultimate stress (strength), average cortical bone wall thickness, percentage bone ash, or serum osteocalcin were observed. Table 6. Effects of dietary treatments on bone fracture force, bone ultimate stress (strength), average cortical wall thickness, serum osteocalcin, and bone ash (Experiment 3 Treatment Probability 1 2 SEM Trt Source of maize YDA NDLPE; Bone fiacture force (N)C 3rd and 4th metacarpal and 4077.72 3810.20 138.74 0.13 metatarsals-shear (F,)C Femur-bend (F.,)C 4654.24 4793.61 308.70 0.67 Ultimate stress (1100)D 3" and 4" metacarpal and 15.04 13.42 0.61 0.60 metatarsals-shear (T)D Femur-bend (6..)D 5.26 5.28 0.93 0.98 Average wall thickness (mm) 3rd and 4th metacarpal and 1.38 1.43 0.04 0.44 metatarsals Femur 5.01 5.63 0.53 0.83 Bone ash (g kg") 3rd and 4th metacarpal and 573.08 574.7 9.85 0.22 metatarsals Femur cross section 707.13 704.48 3.18 0.24 Serum osteocalcin 296.99 275.90 28.46 0.49 (ng mL") AYellow Dent maize ”NutriDense" Low Phytate maize CFracture force is the force required to break a bone. measured in Newtons (N); I N = 0.102 kgf (kilograms force) = 9.8 kg (kilograms mass) DBone strength (ultimate stress, 0;), measured in Pascals (Pa) is determined by the F equation 0'" = for ultimate bending stress and 1’ = 2i , where F ,,= bone bending fracture force (N), F , = bone shear fracture force (N), A = cross-sectional area of the bone (m2), L = distance between fulcra supports (tibia = 0.10 m), C = distance from neutral axis to outer fiber (at), l = moment of inertia (m4); 1 MegaPascal (MPa) = Pa x (1 x 10.6 ): 1 P8 = 1 3%" m 116 The effects of dietary treatments on faecal phosphorus excretion for Experiment 1 are included in Table 7. Pigs excreted less (P<0.01) phosphorus when fed Treatment 2 for all phases, as compared to pigs fed Treatment 1 diets. Table 7. Effects of dietary treatments on faecal phosphorus (Experiment 1) Treatment Probabilin 1 2 SEM Trt Source of maize YDA NDLPB F aecal phosphorus (g kg") Grower 1 15.9' 9.4” 5.25 0.008 Grower 2 15.0‘ 7.9" 1.44 0.009 Finisher 18.9‘ 9.6" 3 .37 0.0001 AYellow Dent maize ”NutriDense" Low Phytate maize “For the same variable, mean values in the same row with unlike superscripts are significantly different (P < 0.05) The effects of dietary treatments on growth performance for Experiment 2 are summarized in Table 8. Pigs fed Treatment 1 diets during Phase I] gained more (P<0.01) weight per day than pigs fed Treatments 2 and 4. No differences in average daily gain were observed for Phase I. For the entire study, pigs fed Treatment 1 had greater average daily gain (P<0.03) than pigs fed Treatment 2. No treatment differences were observed for feed intake in either dietary phase. During Phase 1], pigs fed Treatment 1 were more efficient (P=0.01) than pigs fed Treatments 3 and 4. 117 Table 8. Effects of dietary treatments on body weight (BW), average daily gain (ADG), avenggdmy feed intake (ADFI), and feed efficiency‘(G:F) (Experiment 2) Treatment Probability 1 2 3 4 SEM Trt Source of maize Y1?F YD”— NDLP? NDLPC Dietary P Control control — control - control - 2.0 g kg" 2.0 g kg" 2.0 g kg" Phytase 0 0 0 500 (FTU kg")° ADG (kg day") Phase I 0.232 0.170 0.227 0.209 0.02 0.29 Phase 11 0.3968 0.259b 0.346ab 0.293b 0.02 0.004 Overall 0.314a 0.214b 0.287ab 0.251” 0.02 0.02 ADF] (kg day") Phase I 0.416 0.402 0.498 0.467 0.03 0.17 Phase [1 0.908 0.742 1.08 1.09 0.09 0.05 Overall 0.662 0.572 0.790 0.777 0.05 0.05 G:F Phase 1 0.561 0.429 0.455 0.449 0.05 0.35 Phase 11 0.439“ 0.350ab 0.324b 0.279b 0.03 0.01 Overall 0477' 0.376'b 0.364“b 0.332b 0.03 0.04 AFeed efiiciency as gainzfeed (GzF) BYellow Dent maize CNutriDense" Low Phytate maize I)Natuphos‘) phytase (BASF Corp., Mount Olive, New Jersey, USA 07828); 1 FTU is defined as the amount necessary to liberate l umole of inorganic P per minute from 0.0015 mole of sodium phytate at 37°C and pH 5.5 “For the same variable, mean values in the same row with unlike superscripts are Significantly different (P < 0.05) The effects of dietary treatments on serum phosphorus, faecal phosphorus, and apparent phosphorus digestibility for Experiment 2 are given in Table 9. Pigs fed Treatment 2 had lower (P=0.03) plasma concentrations of phosphorus compared to pigs fed Treatments 1, 3, and 4 while pigs fed Treatment 2 diets excreted less (P<0.0001) phosphorus than pigs fed Treatment 1 diets during Phase 1 period. However, pigs fed Treatments 3 and 4 excreted less (P<0.0001) phosphorus than pigs fed Treatments 1 or 2 during Phase I. Pigs fed Treatments 3 and 4 had greater (P<0.0001) apparent phosphorus digestibilities than pigs fed Treatments 1 and 2 for Phase I . 118 Table 9. Effects of dietary treatments on plasma phosphorus, faecal phosphorus, and apparent phosphorus digestibility (Experiment 2) Treatment Probability 1 2 3 4 SEM Trt Source of maize YDA YDA NDLPB NDLPB Dietary P Control control — control — control — 2.0 g kg" 2.0 g kg" 2.0 g kg" Phytase 0 0 0 500 (FTU k8")C Plasma P (mg dL") 5.33' 3.92” 4.87‘ 5.16‘ 0.22 0.003 Faecal P” (g kg’) Phase I 14.1' 11.8" 7.6c 6.3‘ 0.45 <0.0001 Apparent P digestibilityD (%) Phase I 33.90‘ 38.30‘ 60.29b 62.30” 2.89 <0.0001 "Yellow Dent maize ”NutriDense" Low Phytate maize CNatuphos" phytase (BASF Corp., Mount Olive, NJ); 1 FTU is defined as the amount necessary to liberate 1 mole of inorganic P per minute from 0.0015 mole of sodium phytate at 37°C and pH 5.5 I)Faecal P and apparent P digestibility values for Phase 11 were not obtained due to technical difficulties “"F or the same variable, mean values in the same row with unlike superscripts are significantly different (P < 0.05) 119 Discussion Growth performance Treatment 1 diets in Experiment 1 met all nutritional needs. Growing-finishing pigs in Experiment 1 had comparable growth performance to previous studies where growing-finishing pigs were fed low phytic acid maize (Spencer et al., 2000). Weanling pigs fed Treatment 1 control diets in Experiment 2 had comparable feed efficiencies to a similar study by Roberson et al. (1999). Findings by Spencer et al. (2000b) are in agreement with our study where high available phosphorus (HAP) maize can be fed in place of YD maize without having deleterious impacts on growth parameters. Although HAP maize is a different variety than NDLP and has a lower (62%) phosphorus availability than NDLP (90%), it was still useful in improving maize phosphorus availability. Pierce et al. (1998) reported that 0.9 g kg'l less dietary phosphorus was needed to maximize performance. Treatment 2 in Experiment 2 was designed as a negative control diet for phosphorus and resulted in decreased gain. Treatments 3 and 4 with NDLP improved growth Similar to our positive control diets. However, this positive improvement when phytase was fed was not observed. A decrease in Phase II gainzfeed for Treatments 3 and 4 as compared to Treatment 1 was unexpected. Data for the overall study indicates that phosphorus availability limited feed efficiency. This observation necessitates validation with further experimentation. Bone Parameters Bone breaking load (fracture force), stress-testing of bone, and percent bone ash are sensitive indicators of dietary P availability (Crenshaw et al., 1981; Cromwell, 1992). 120 The percent ash of 3rd and 4th metacarpal and metatarsal bones of finishing swine have been reported to be between 570.0 to 600.0 g kg" ash (Doige et al., 1975; Crenshaw et al., 198]; Spencer et al., 2000a, 2000b), which is similar to ours. Bone strength has been reported as kg. However, the 1999 ASAE Standards suggests that bone strength is ultimate stress, or the force per unit area required to break a bone. Therefore, Pascals (Pa), which is equivalent to l , are the appropriate unit. ? Bone fiacture force or breaking load is reported in Newtons (N). One Newton is the force required to accelerate a ] kilogram mass by l m/sz. Combs et al. (199]) reported a 4th metacarpal and metatarsal ultimate shear stress value of 13.42 MPa for pigs fed to 113 kg fed NRC (1979) recommendations for dietary calcium and phosphorus. This is in agreement with our findings. Values for bone strength reported by Crenshaw et al. (1981), Carter and Cromwell (1998), and Spencer et al. (2000a, 2000b) ranging from 490 to 1863 N (converted from kgf). These values were smaller than values reported in our study. Carter et al. (1996) reported that serum concentration of osteocalcin was a good indicator of bone turnover in pigs. Osteocalcin was inversely correlated with growth rate, bone strength, metacarpal ash, femur ash (Carter et al., 1996). AS osteocalcin concentrations in serum increase, bone strength, growth rate, and bone ash decrease (Carter et al., 1996). If osteocalcin concentrations are elevated, that would indicate that there is a greater degree of bone turnover and the strength and ash of bones are decreasing indicating a greater need for phosphorus than supplied by diet. Osteocalcin concentrations reported by Carter et al. (1996) are lower than the values from our study (4.74-6.07 ng mL" vs. 275.9—296.99 ng mL"). The Differences may be due to differences 121 in assay, genetics, diets, or age of animals. Because serum osteocalcin concentrations in Experiment 1 were found to be the same for both treatments, it would appear that there was not a change in bone turnover in pigs on the four diets. Likewise, no differences in bone strength or bone ash indicate that dietary available phosphorus was probably adequate in the NDLP diets. Plasma phosphorus Plasma concentration of phosphorus has been reported as an indicator of dietary phosphorus utilization (Lei et al., 1993). Our plasma inorganic phosphorus concentrations were Similar for controls. When dietary phosphorus was reduced by 2.0 g kg" (Diet 2), plasma phosphorus concentration was reduced. Also, because pigs fed Diets 3 and 4 had Similar plasma concentrations of phosphorus as pigs fed Diet 1, NDLP with or without phytase appear to be effective in increasing dietary phosphorus utilization. F aecal phosphorus and phosphorus digestibility Several researchers have shown that maize with low phytic acid concentration fed with or without phtase reduced faecal phosphorus and increased apparent phosphorus digestibility (Pierce et al., 1998; Veum et al., 1998; Spencer et al., 2000). In these studies, low phytic acid maize reduced faecal phosphorus excretion by 13 to 50%, which is in agreement with our studies having a 46% reduction in faecal phosphorus. Although several researchers have used genetically different pigs of different ages and body weight, low phytic acid maize varieties (HAP or NDLP) can reduce faecal phosphorus excretion significantly. 122 Phytase The NDLP maize in Treatment 3 replaced YD maize was designed to increase available phosphorus by 1.0 g kg" in Experiment 2. NatuphosQ phytase was added (500 FTU kg" in Phase I and 250 FTU kg" in Phase 11 so available phosphorus concentrations should be similar to control (Treatment 1) diets. The NDLP addition in Treatment 3 diets may have been adequate enough to increase available phosphorus to control levels (Treatment 1) but phytase addition may not have had an effect. Roberson (1999) reported that 500 FTU kg" was adequate to replace dietary available phosphorus by 1.0 g kg". Manufacturer analysis for phytase activity of Phase I and 11 Treatment 4 diets yielded 640 and 400 FTU kg". This confirmed that phytase addition to Treatment 4 diets yielded a minimum 500 FTU kg" for Phase I and 250 FTU kg" for Phase II. The lack of response by feeding phytase was unexpected and the interaction between NDLP and phytase warrants further investigation. Qian et al. (1996) suggested that a decrease in the calciumztotal phosphorus ratio from 2.0 to 1.2:] increases phytase efficiency. In our analyzed diets, calciumztotal phosphorus for Treatment 4 diets was 1.76 and 1.94:] for Phases I and II, respectively. Qian et al. (1996) recommended that a calciumzavailable phosphorus ratio of between 1.5:] and 2.5:] should be efficacious for the use of 500 FTU kg" Natuphos” phytase. Our calculated calcium:phosphorus ratios for Phases 1 and 11 Treatment 4 diets were 1.6:] and 2.6: 1, which falls into the range suggested by Qian et al. (1996). 123 Conclusion The results of the two experiments clearly Show that NDLP maize can safely replace YD maize using a 90% phosphorus availability assumption for NDLP, without affecting growth performance and serum phosphorus of weanling pigs or growth performance and bone traits of growing-finishing pigs. NDLP maize can reduce phosphorus excretion by about 45% when fed in place of YD corn to weanling and growing-finishing crossbred pigs. Cost of finisher diets was reduced by US$3.71 tonne" due to replacement of soyabean meal, choice white grease and dicalcium phosphate by the addition of NDLP maize in place of YD maize, assuming the cost of the two maize varieties to be the same. References American Society of Agricultural Engineers, 1999. Shear and three-point bending test of animal bone. Pages 584-586 in: ASAE Standards. Adeola, O., 1995. Digestive utilization of minerals by weanling pigs fed copper- and phytase-supplemented diets. Can. J. Anim. Sci. 75, 603-610. 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Influence of age, sex and calcium and phosphorus levels on the mechanical properties of various bones in swine. J. Anim. Sci. 52, 1319-1329. Cromwell, G.L., 1992. The biological availability of phosphorus in feedstuffs for pigs. Pigs News Info. 13(2), 75N-79N. Cromwell, G.L., T.S. Stahly, R.D. Coffey, H.J. Monegue, and J.H. Randolph, 1993. Efficacy of phytase in improving the bioavailability of phosphorus in soybean meal and com-soybean meal diets for pigs. J. Anim. Sci. 71, 1831-1840. Cromwell, G.L., R.D. Coffey, G.R. Parker, H.J. Monegue, and J .H. Randolph, 1995. Efficacy of a recombinant-derived phytase in improving the bioavailability of phosphorus in com-soybean meal diets for pigs. J. Anim. Sci. 73, 2000-2008. Cromwell, G.L., J.L. Pierce, T.E. Sauber, D.W. Rice, D.S. Ertl, and V. Raboy, 1998. Bioavailability of phosphorus in low-phytic acid corn for growing pigs. J. Anim. Sci. (Suppl. 2), 115. Doige, C.E., B.D. Owen, and J .H.L. Mills, 1975. Influence of calcium and phosphorus on growth and skeletal development of growing swine. Can. J. Anim. Sci. 55, 147 164. Douglas, M.W., C.M. Peter, S.D. Boling, C.M. Parsons, and DH. Baker, 2000. Nutritional evaluation of low phytate and high protein corns. Poult. Sci. 79,1586 1591. Gomori, G., 1942. A modification of the colorimetric phophorus determination for use with the photoelectric colorimeter. J. Clin. Lab. Med. 27, 955-960. Komegay, E.T., D. Rhein-Welker, M.D. Lindemann, and C.M. Wood, 1995. Performance and nutrient digestibility in weanling pigs as influenced by yeast culture additions to starter diets containing dried whey or one of two fiber sources. J. Anim. Sci. 73, 1381-1389. Lei, X.G., P.K. Ku, E.R. Miller, and M.T. Yokoyama, 1993a. Supplementing corn soybean meal diets with microbial phytase linearly improves phytate utilization by weanling pigs. J. Anim. Sci. 71, 3359-3367. Lei, X.G., P.K. Ku, E.R. Miller, M.T. Yokoyama, and DE. Ullrey, 1993b. Supplementingcom-soybean meal diets with microbial phytase maximizes phytate phosphorusutilization by weanling pigs. J. Anim. Sci. 71, 3368-3375. 125 Liu, J ., D.W. Bollinger, D.R. Ledoux, and T.L. Veum, 1998. Lowering the dietary to total phosphorus ratio increases phosphorus utilization in low-phosphorus corn soybean meal diets supplemented with microbial phytase for growing-finishing pigs. J. Anim. Sci. 76, 808-813. Mroz, Z., A.W. Jongbloed, and P.A. Kemme, 1994. Apparent digestibility and retention of nutrients bound to phytate complexes as influenced by microbial phytase and feeding regimen in pigs. J. Anim. Sci. 72, 126-132. National Research Council, 1998. Nutrient Requirements of Swine, 10th Ed., National Academy Press, Washington, DC. National Research Council, 1979. Nutrient Requirements of Swine, 8th Ed., National Academy Press, Washington, DC. Pierce, J .L., G.L. Cromwell, T.E. Sauber, D.W. Rice, D.S. Ertl, and V. Raboy, 1998. Phosphorus digestibility and nutritional value of low-phytic acid corn for pigs. J. Anim. Sci. 76 (Suppl. 2), 117. Qian, H., E.T. Komegay, and DE. Conner, Jr., 1996. Adverse effects of wide calcium:phosphorus ratios on supplemental phytase efficacy for weanling pigs fed two dietary phosphorus levels. J. Anim. Sci. 74, 1288-1297. Raboy, V., P.F. Gerbasi, KA. Young, S.D. Stoneberg, S.G. Pickett, A.T. Bauman, P.P.N. Murphy, W.F. Sheridan, and D.S. Ertl, 2000. Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1. Plant Physiol. 124, 355-368. Roberson, KD., 1999. Estimation of the phosphorus requirement of weanling pigs fed supplemental phytase. Anim. Feed Sci. Technol. 80, 91-100. Rozeboom, D.W., GA. May, B.R. Hines, S.L. Hoover, T.J. Johnson, J. F.'Kelpinski, J.P. LeCreux, ML. Nagelkirk, D.B. Rajzer, N.A. Rector, D.J. Rossman, D.T. Shaw, R.J. Tempelman, N.L. Trottier, and P.W. Wylie, 2001. High-oil corn: crop production and swine feeding demonstration. Department of Animal Science Report, Michigan State University, East Lansing, MI, File No. 19.47, ANS Mimeo No. 402. SAS Institute, 1999. SAS® User’s Guide: Statistics. Version 8.2 SAS Institute, Cary, NC. Spencer, J .D., G.L. Allee, and TE. Sauber, 2000a. Phosphorus bioavailability and digestibility of normal and genetically modified low-phytate corn for pigs. J. Anim. Sci. 78, 675-681. Spencer, J.D., G.L. Allee, and TE. Sauber, 2000b. Growing-finishing performance and carcass characteristics of pigs fed normal and genetically modified low-phytate corn. J. Anim. Sci. 78, 1529-1536. 126 Veum, T., V. Raboy, D. Ertl, and D. Ledoux, 1998. Low phytic acid corn improves calcium and phosphorus utilization for growing pigs. J. Anim. Sci.,76 (Suppl. 1), 684. Williams, David, and Iismaa, 1962. The determination of chronric oxide in faeces samples by atomic absorption spectrophotometry. J. Agric. Sci. 59:381-385. Young, L.G., M. Leunissen, and J .L. Atkinson, 1993. Addition of microbial phytase to diets of young pigs. J. Anim. Sci. 71, 2147-2150. 127 SUMMARY AND GENERAL CONCLUSION The objective of this thesis was to verify the phosphorus bioavailability of NutriDense Low Phytate" corn as 90% bioavailable when fed to growing-finishing commercial tom turkeys and growing-finishing and weanling crossbred pigs. Another goal was to investigate the degree to which phosphorus excretion could be reduced without negatively impacting growth performance or bone characteristics. Although there were inconsistencies in dietary treatments between analyzed and calculated phosphorus concentrations, the increased nutrient profile of NutriDense Low Phytate" corn was adequate to replace dietary phosphorus to reduce excreted phosphorus by 30 to 45% and up to 56% (with phytase addition) in commercial finishing toms when fed in place of conventional yellow dent corn. Feeding NutriDense Low Phytate°°° corn reduced fecal phosphorus excretion by approximately 45% in both weanling and growing-finishing crossbred pigs when substituted for conventional yellow dent corn. In conclusion, this work suggests that NutriDense Low Phytate” has the potential to reduce phosphorus excretion from turkeys and swine. Economics will be dictated by market prices of ingredients and dietary percentage composition of those nutrients. AS Michigan animal feeding operations move towards more comprehensive nutrient management plans, lowered dietary phosphorus, NutriDense Low Phytate°°° corn, and/or phytase enzyme may become useful as tools in the planning process. 128 VITA Michael W. Klunzinger is originally from Grosse Pointe, Michigan where he was raised part of his life in the country and part of his life in the city. His mother’s side hails from Alrnont, Michigan and has been actively involved in farming for centuries in fi'uit production, crop production, livestock, and poulhy production. His father’s Side hails from East Lansing, Michigan, home of the Spartans. His interest in animals probably began as a small child when he was exposed to sheep, cattle, and poultry. In third grade, he moved to the city. His interest in animals never waned as he became employed at a veterinary hospital throughout high school. He came to Michigan State University in the fall of 1995 as a fifth generation Michigan State University student. He was actively involved as a member of the Alpha Gamma Rho fiatemity and took interest in the Animal Science department as a sophomore and decided to pursue a Bachelor’s degree in Animal Science. He worked at the MSU Sheep farm, where he gained the Skill of sheep shearing and has run a successful business travelling throughout the state of Michigan. He worked at the MSU poultry and mink farm as well. Summer experiences included working at Bennett Farms Dairy in Prescott, Michigan; study abroad through the College of Agriculture and Natural Resources in Australia and New Zealand; and attending the Midwest Poultry Consortium program at the University of Wisconsin-Madison with an invaluable internship at Jennie-O Foods, Inc. in Willmar, Minnesota. Upon graduation with a BS. in Animal Science in 1999, he continued on for his Masters degree in turkey and swine nutrition at Michigan State University under the direction of Dr. Kevin D. Roberson. While in graduate school, he met the love of his life, Raelene A. Charbeneau. 129 APPENDICES Appendix A Calculations for ultimate bending stress of bone: Adapted from ASAE Standards, 1999 Ultimate bending stress (strength): a, = 41:—’35, C = 0.57559x D, and I = 0.0549x[(Bx D3)-(bxv3)J X Where: a, = ultimate bending stress, Pa F = bone fracture force, N L = distance between fulcra supports (0.1000 mm for turkey tibia, 0.0596 mm for swine femur) C = distance fiom neutral axis to outer fiber of bone, m I = moment of inertia, In4 B = outside major diameter of the bone, m D = outside minor diameter of the bone, m b = inside major diameter of the bone, m = B x (2 x w) v = outside major diameter of the bone, m = D x (2 x w) w = average cortical bone wall thickness, m Ultimate shear stress (strength): T F A =%xflx[((BxD)—(bxv))+[vx BZ—b]+(bx D-v)] =2xA° Where: r= ultimate shear stress, Pa A = cross-sectional area of a quadrant of a hollow ellipse (bone), m2 B = outside major diameter of the bone, m D = outside minor diameter of the bone, m b = inside major diameter of the bone, m = B x (2 x w) v = outside major diameter of the bone, m = D x (2 x w) w = average cortical bone wall thickness, m 131 Table 1. Ultimate stress equation variables from swine study (Experiment 1) Variable Femur 3“I and 4m Metacarpals and Metatarsals Diet 1 Diet 2 Diet 1 Diet 2 F 4654.24 4793.61 4077.72 3810.20 L 0.0596 0.0596 C 0.012362 0.012615 1 1.73818 x 10'7 1.74738 x 10' A 1.376x 10" 1.429x10" B 0.0183188 0.0185650 D 0.0149250 0.0152469 w 0.0019156 0.0019521 b 0.0144880 0.1474600 v 0.01 10940 0.01 13430 Table 2. Ultimate stress equation variables from turkey study (Experiment 1L Tibia Variable Diet 1 Diet 2 Diet 3 Diet 4 F 938.50 878.33 913.89 837.61 L 0.1 0.1 0.1 0.1 C 0.008302886 0.008312479 0.008192564 0.008322072 1 1.87137 x 10'9 1.79150 x 10'9 1.78699 x10"r 1.84714 x10"— Ulna F 2578.97 2415.713 2290.06 2366.83 B 0.014433 0.014533 0.014567 0.014625 D 0.010708 0.015000 0.010800 0.010483 w 0.001219 0.001317 0.001178 0.001186 b 0.01 1994 0.01 1900 0.122110 0.012253 v 0.008269 0.007867 0.008444 0.0081 1 1 Femur F 1968.46 2170.63 1894.97 2012.69 B 0.018967 0.018958 0.01900 0.018842 D 0.017458 0.017275 0.01755 0.017075 w 0.001503 0.00151 1 0.001389 0.001453 b 0.015961 0.015936 0.016222 0.015936 v 0.014453 0.014253 0.014772 0.014169 132 Table 3. Ultimate stress equation (measurements taken using computed tomography (Cffiechnolggy with variables from turkey study (Experiment 2) Variable Tibia Diet 1 Diet 2 Diet 3 Diet 4 F 702.66 654.60 688.28 683.17 L 0.1 0.1 0.1 0.1 c 0.007965 0.008389 0.008133 0.008329 I 1.97055 x 10‘9 2.2623 x 10"r 2.05658 x 10" 2.23946 x 10”— Ulna F 2548.78 2411.52 2408.82 2372.20 A, by CT scan 3.90952 x 10'5 3.90909 x 10" 4.00952 x 10" 4.06818 x 11? A, using CT 8.2187 x 10'5 8.4576 x 10'T 8.1046 x 10" 8.9272 x 11? measurements then equation B 0.014329 0.014450 0.014514 0.014555 D 0.010519 0.010614 0.010614 0.010682 w 0.001727 0.001771 0.001671 0.001877 b 0.010875 0.010908 0.011171 0.010800 v 0.007065 0.007071 0.007271 0.006927 Femur F 1628.54 1685.40 151 1.35 1549.12 A, by CT scan 7.3250 x 10'5 7.2750 x 10" 7.2375 x 10'5 7.0083 x 10'5 A, using CT 0.00016252 0.00016805 0.00016082 0.0001622] measurements then equation B 0.018796 0.019058 0.018721 0.018792 D 0.017363 0.017792 0.017375 0.017325 w 0.002307 0.002331 0.002290 0.002311 b 0.014182 0.014397 0.014140 0.014169 v 0.012749 0.013131 0.012794 0.012703 133 Table 4. Ultimate stress equation (measurements taken using a digital caliper) with variables from turkey study (Experiment 2) Variable Tibia Diet 1 Diet 2 Diet 3 Diet 4 F 702.66 654.60 688.28 683.17 L 0.1 0.1 0.1 0.1 C 0.007787 0.008128 0.007895 0.0081 16 1 1.85849 x 10"” 2.08919 x 10'9 1.88643 x 10" 2.091437 x 10”" Ulna F 2548.78 2411.52 2408.82 2372.20 A 7.8487 x 10‘5 8.2439 x 10'5 7.7273 x 10'5 8.4718 x 10" B 0.013919 0.014186 0.014019 0.013977 D 0.010157 0.010405 0.010243 0.010277 w 0.001727 0.001767 0.001671 0.001877 b 0.010465 0.010652 0.010676 0.010223 v 0.006703 0.006871 0.006900 0.006523 Femur F 1628.54 1685.40 1511.35 1549.12 A 0.00015914 0.00015958 0.00015952 0.00015051 B 0.0184 0.018375 0.0184375 0.0187667 D 0.016525 0.017033 0.0164875 0.0165208 w 0.002367 0.002297 0.0023780 0.0021970 b 0.013667 0.014143 0.0136820 0.0140720 v 0.01 1792 0.012439 0.0117320 0.0121260 134 Appendix B Example of feed cost savings from substiflng’ NutriDense Low Phym"I corn for nv '0 Yellow at m in turk and ° fini ' diets: Ingredient cost assumptions arriced on a per ton basis): $91.43 corn $181.00 soybean meal (48% protein) $230.00 choice white grease $2200.00 DL-Methionine $1000.00 L-Lysine HC] $250.00 dicalcium phosphate $30.00 limestone *other ingredients not considered because they remained the same between treatments“ Table 5. Feed cost savings by replacing yellow dent (YD) corn with NutriDense Low Phytate°°° (NDLP) corn in turkey Finisher diets (Experiment 1) $/ton $/ton Itema % in Diet Item price Cost in YD % in Diet Item price Cost in NDLP control diet ($lton) diet (90% P bioavailability) Corn 62.49 x $91.43 = $57.13 65.81 x $91.43 = $60.17 SBM 26.82 x $181.00 = $48.54 23.57 x $181.00 = $42.66 CWG 7.45 x $230.00 = $17.14 6.5] x $230.00 = $14.97 DL- 0.21 x $2200.00 = $4.62 0.18 x $2200.00 = $3.96 Met L-Lys 0.00 x $1000.00 = $0.00 0.09 x $1000.00 = $0.90 DP 1.54 x $250.00 = $3.85 0.99 x $250.00 = $2.48 Lime 0.88 x $30.00 = $0.26 1.25 x $30.00 = $0.38 Total =$l3l.55 =$125.51 Total cost savings = $131.55/ton - $125.51/ton = $6.03/ton 'Itern abbreviations: SBM= soybean meal (48% protein); CWG = choice white grease; DL-Met = DL- Methionine; L-Lys = L-Lysine; DP = dicalcium phosphate; lime = limestone 135 Table 6. Feed cost savings by replacing yellow dent (YD) corn with N utriDense Low Phytate" (NDLP) corn in turkey Finisher diets (Experiment 1) $/ton $/ton ItemIll % in Diet Item price Cost in YD % in Diet Item price Cost in NDLP control diet ($/ton) diet (90% P bioavailability) Corn 77.45 x $91 .43 = $70.81 84.97 x $91.43 = $77.69 SBM 17.41 x $181.00 =$3l.51 12.40 x $181.00 =$22.44 CWG 2.27 x $230.00 = $5.22 0.11 x $230.00 = $0.25 L-Lys 0.00 x $1000.00 = $0.00 0.09 x $1000.00 = $0.90 DP 0.85 x $250.00 = $2.13 0.00 x $250.00 = $0.00 Lime 0.72 x $30.00 = $0.22 1.13 x $30.00 = $0.34 Total = $109.89 = $104.68 Total cost savings = $109.89/ton - $101.63/ton = $8.26/ton aItem abbreviations: SBM= soybean meal (48% protein); CWG = choice white grease; L-Lys = L-Lysine; DP = dicalcium phosphate; lime = limestone 136 MMMMMM will11111111711111 31 111111 09 '