BIOAVAILABILITY OF MAJOR AND

TRACE MINERALS Dr. A.W. Jongbloed & Dr. P.A. Kemme

ID TNO Animal Nutrition, Lelystad, The Netherlands

Dr. G. De Groote & Dr. M. Lippens Department Animal Nutrition and Husbandry; Section Small Stock Husbandry; CLO-

Ghent, Belgium

Dr. F. Meschy UMR INRA INA-PG, Physiology of Nutrition and Animal Feeding, France

EMFEMA International Association of the European (EU) Manufacturers of Major, Trace and Specific Feed Mineral Materials

The study “Bioavailability of Major and Trace Minerals” was initiated by the members of EMFEMA, the EU association of producers of feed minerals. We thank former secretary-general Herman Liekens and present secretary-general Theo Dubois for the coordination and the completion of this study. EMFEMA also expresses its sincere gratitude to the authors for the excellent work performed as well as to Professor Marcel Vanbelle for his valuable scientific comments.

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CONTENT

PREFACE............................................................................................................................................................ 4

TERMS AND METHODS TO ASSESS AND EVALUATE THE BIOAVAILABILITY OF MINERALS FOR LIVESTOCK: A GENERAL INTRODUCTION................................................................................... 5

1. INTRODUCTION.............................................................................................................................................. 5 2. ABSORPTION OF MINERALS............................................................................................................................ 5 3. METHODS FOR EVALUATING THE BIOAVAILABILITY OF MINERALS ................................................................ 7 4. TERMS USED TO EXPRESS THE BIOAVAILABILITY OF MINERALS ................................................................... 10 5. RANKING OF VARIOUS CRITERIA TO ASSESS BIOAVAILABILITY .................................................................... 11 6. SELECTION OF LITERATURE ......................................................................................................................... 13 7. CONCLUSION ............................................................................................................................................... 13 8. REFERENCES................................................................................................................................................ 14

MAJOR MINERALS ...................................................................................................................................... 17

I. CALCIUM BIOAVAILABILITY ............................................................................................................... 18 CALCIUM BIOAVAILABILITY FOR PIGS ............................................................................................... 18 CALCIUM BIOAVAILABILITY FOR POULTRY ..................................................................................... 20 CALCIUM BIOAVAILABILITY FOR RUMINANTS ................................................................................ 23

II. MAGNESIUM BIOAVAILABILITY........................................................................................................ 26 MAGNESIUM BIOAVAILABILITY FOR PIGS......................................................................................... 27 MAGNESIUM BIOAVAILABILITY FOR POULTRY ............................................................................... 27 MAGNESIUM BIOAVAILABILITY FOR RUMINANTS.......................................................................... 27

III. SODIUM BIOAVAILABILITY ............................................................................................................... 33 SODIUM BIOAVAILABILITY FOR PIGS.................................................................................................. 33 SODIUM BIOAVAILABILITY FOR POULTRY ........................................................................................ 34 SODIUM BIOAVAILABILITY FOR RUMINANTS................................................................................... 36

IV. PHOSPHORUS BIOAVAILABILITY..................................................................................................... 37 PHOSPHORUS BIOAVAILABILITY FOR PIGS........................................................................................ 39 PHOSPHORUS BIOAVAILABILITY FOR POULTRY.............................................................................. 43 PHOSPHORUS BIOAVAILABILITY IN RUMINANTS ............................................................................ 48

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TRACE MINERALS....................................................................................................................................... 54

I. COBALT BIOAVAILABILITY .................................................................................................................. 55 COBALT BIOAVAILABILITY FOR PIGS.................................................................................................. 55 COBALT BIOAVAILABILITY FOR POULTRY........................................................................................ 55 COBALT BIOAVAILABILITY FOR RUMINANTS................................................................................... 56

II. COPPER BIOAVALABILITY................................................................................................................... 58 COPPER BIOAVAILABILITY FOR PIGS .................................................................................................. 59 COPPER BIOAVAILABILITY FOR POULTRY......................................................................................... 61 COPPER BIOAVAILABILITY FOR RUMINANTS ................................................................................... 63

III. IRON BIOAVAILABILITY ..................................................................................................................... 70 IRON BIOAVAILABILITY FOR PIGS........................................................................................................ 71 IRON BIOAVAILABILITY FOR POULTRY .............................................................................................. 73 IRON BIOAVAILABILITY FOR RUMINANTS......................................................................................... 77

IV. IODINE BIOAVAILABILITY ................................................................................................................. 79 IODINE BIOAVAILABILITY FOR PIGS.................................................................................................... 79 IODINE BIOAVAILABILITY FOR POULTRY .......................................................................................... 81 IODINE BIOAVAILABILITY FOR RUMINANTS..................................................................................... 81

V. MANGANESE BIOAVAILABILITY........................................................................................................ 84 MANGANESE BIOAVAILABILITY FOR PIGS ........................................................................................ 84 MANGANESE BIOAVAILABILITY FOR POULTRY............................................................................... 85 MANGANESE BIOAVAILABILITY FOR RUMINANTS ......................................................................... 87

VI. MOLYBDENUM BIOAVAILABILITY.................................................................................................. 90 MOLYBDENUM BIOAVAILABILITY FOR PIGS .................................................................................... 90 MOLYBDENUM BIOAVAILABILITY FOR POULTRY........................................................................... 90 MOLYBDENUM BIOAVAILABILITY FOR RUMINANTS ..................................................................... 90

VII. SELENIUM BIOAVAILABILITY ......................................................................................................... 92 SELENIUM BIOAVAILABILITY FOR PIGS ............................................................................................. 92 SELENIUM BIOAVAILABILITY FOR POULTRY.................................................................................... 94 SELENIUM BIOAVAILABILITY FOR RUMINANTS .............................................................................. 97

VIII. ZINC BIOAVAILABILITY ................................................................................................................. 101 ZINC BIOAVAILABILITY FOR PIGS ...................................................................................................... 102 ZINC BIOAVAILABILITY FOR POULTRY ............................................................................................ 103 ZINC BIOAVAILABILITY FOR RUMINANTS ....................................................................................... 106

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PREFACE

Major and trace minerals are essential nutritional elements of feed for livestock. They play a key role in the maintenance and development of the skeleton and in the maintenance of acid base balances. They are also essential components of a number of enzymes, vitamins and hormones, etc. EMFEMA, the EU association of producers of feed minerals, has published a guideline that presents an overview of essential major minerals, trace elements and specific minerals which are used in animal feed. This guideline offers useful information about the chemical, physical and technological characteristics of different mineral sources and is a practical manual to assist in the selection of mineral sources for feed formulations. However, this guideline does not provide information about the bioavailability of those major and trace minerals. This information is valuable to formulate feeds to ensure that the minerals are provided in sufficient quantities to achieve not only optimal health, welfare and growth of animals but also to minimize the excretion of minerals into the environment. Therefore, EMFEMA contacted different specialists at renowned institutes in the field of minerals for livestock to produce a literature overview of the relative bioavailability of major and trace minerals for poultry, pigs and ruminants. Dr. A.W. Jongbloed and Dr. P.A. Kemme were responsible for the general introduction, Dr. G. De Groote and Dr. M. Lippens were responsible for the poultry part, Dr. A.W. Jongbloed was responsible for the pig part and Dr. F. Meschy for the ruminant part. The authors used only those literature sources in which acceptable response criteria were used in order to make a meaningful comparison between the different mineral sources. In addition, only these studies, in which the mineral sources were accurately identified, were used in this study. The response criteria used and the ranking of importance for assessing the biological value is given for each mineral and for each type of animal. It is not the aim of this study to determine recommendations for the use of major and trace minerals but to provide scientific information about the relative bioavailability of minerals used in animal feeds. Professor M. Vanbelle 01/08/2002

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TERMS AND METHODS TO ASSESS AND EVALUATE THE BIOAVAILABILITY OF MINERALS FOR LIVESTOCK: A GENERAL

INTRODUCTION

1. INTRODUCTION

In the body of animals there are approximately 20 minerals that are essential for maintenance and normal functioning of the body. Lack or insufficient amounts of these minerals result in deficiency symptoms leading to reduced performance. Excess amounts, on the other hand, may also lead to a reduction in performance and toxicity may occur. Minerals are present in all organs and tissues in the body. The functions of the inorganic minerals are extremely diverse. They range from structural functions in some tissues to a wide variety of regulatory functions in other tissues (NRC, 1980; Underwood, 1981; McDowell, 1992; Underwood and Suttle, 1999). The major minerals calcium, magnesium, sodium, potassium, phosphorus, sulphur, and chlorine are present in the body in relatively larger amounts than the trace minerals, such as iron, copper, zinc, cobalt, molybdenum, manganese, iodine, and selenium. Absorption and utilization of major minerals may be, apart from passive transport through the gut wall, mediated by hormonal control, that is primarily based on their concentration in the extracellular fluid. Animals are able to maintain a homeostasis in the extracellular and intracellular fluids by means of several regulatory mechanisms. In consequence, large differences in absorption and utilization of minerals can be found, which can depend on a number of factors, including the nutritional status of the animal. As requirements for major minerals should be based on absorbable or utilisable/available minerals rather than on total amount, more attention should be directed to factors that affect mineral absorption. Actually, this holds true for trace minerals, although assessing their absorbability is much more difficult. It is obvious that a sufficient amount of (available) minerals should be supplied, because an insufficient supply impairs efficiency of animal production. In this introduction, an outline is given on factors that affect the bioavailability of minerals. Furthermore, evaluation methods and terms are discussed that are used to assess and express the bioavailability of minerals in mineral sources and feeds for livestock.

2. ABSORPTION OF MINERALS

Before absorption by the absorbing enterocytes from the gastrointestinal tract can take place, the minerals must become available in ionic form (as cations and anions), which is suitable for uptake and transport. In principal, the transepithelial transport consists of both, an active transcellular component which can be regulated and/or a passive paracellular component which depends on chemical and electrical gradients existing across the intestinal wall (Nys and Mongin, 1980; Schröder et al., 1996; Jongbloed and Mroz, 1997). The highly-soluble monovalent minerals, such as sodium, potassium, and chlorine can be transported easily. However, the solubility of various other minerals is often low at neutral pH. Their solubility is dependent on the presence of other compounds as they can relatively easy precipitate or form non-absorbable complexes. Well-known complexing food components are phytic acid and oxalic acid (Harland, 1989). In addition, there are several interactions among various minerals (e.g. calcium and phosphorus; calcium and zinc; copper and zinc; copper, molybdenum and sulphur) which complicate an easy understanding of absorption (Pointillart et al., 1987; Ashmead and Zunino, 1994; Van der Klis, 1994; Jongbloed et al., 1995). Also, viscosity of the chyme in the intestinal tract may negatively affect absorption of minerals (Van der Klis, 1993).

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A schematic representation of calcium metabolism is presented in Figure 1. This Figure shows that calcium metabolism is a dynamic process in which it may re-circulate in the body pool. To a large extent, this is also true for other minerals.

Figure 1: Main routes of calcium metabolism in pigs (I = ingested; FT = total faecal excretion; FI = faecal excretion from ingested origin; FE = endogenous faecal excretion; a = absorbed from ingested origin; aE = endogenous absorbed; U = urinary; R = retention; RI = retention of ingested mineral; RE = retention of endogenous mineral; adapted from Besançon and Guéguen, 1969). The numbers indicate grams of calcium.

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3. METHODS FOR EVALUATING THE BIOAVAILABILITY OF MINERALS

There are several factors that affect the bioavailability of minerals. In an excellent review, Guéguen (1961) discussed these factors for phosphorus, but they also apply to a large extent to various other minerals (see also Huyghebaert et al., 1980; Meschy and Guéguen, 1995). These factors, that will only be briefly mentioned here (see also Appendix 1), are as follows:

1. Factors related to the animal (species, sex, age, physiological state, health, differences between individual animals).

2. Factors related to the composition of the diet (amount of mineral intake, ratio between various individual minerals, vitamin levels, protein, fat, fibre and phytate levels).

3. Factors related to the mineral source (fineness, concentration of other minerals, crystallinity, production process, chemical-physical techniques applied, the source of the raw material, the presence as anion or cation, or in some cases in an organic complex).

4. Factors related to the technological treatment that has been applied to the final diet, e.g. mash feed or pelleted diet (DeGroote and Huyghebaert, 1997).

In addition, Jongbloed (1987) showed that even more dietary factors affect mineral digestibility. To give an idea of the complexity of these factors, some results of the research done by Huyghebaert et al. (1980; 1981) on phosphorus can be mentioned. They found that for broilers a sodium/chlorine ratio of 1:3 decreased the availability of phosphorus when compared with a ratio of 1:1. However, at different concentrations of fluorine, the effect of the sodium/chlorine ratio on phosphorus availability altered completely. There are two approaches to estimate the bioavailability of minerals for the animal.

In vivo techniques, which are expensive; In vitro techniques, which are relatively cheap.

Another approach is one in between the in vivo and in vitro techniques, the so-called semi in vivo techniques. These are techniques with cell cultures and tissues. One well-known technique is the Ussing chamber, which has been adopted from rumen absorption studies (Schröder et al., 1995). Estimation of the bioavailability of minerals from various sources for animals should be closely linked with those biological parameters, that are accurate, distinguishable, easy to execute and relatively inexpensive. They should also lead to tabulated values that can easily be applied in practice, or fit in an existing evaluation system. In order to judge the bioavailability of minerals it is important to know which method and criterion have been used. There are a large variety of methods and criteria used, sometimes leading to large differences. Methods and criteria will be discussed together, because they are often linked to each other.

3.1. In vivo experiments The following criteria in in vivo experiments can be used:

• animal performance (average daily gain, feed intake, feed conversion ratio, reproduction characteristics);

• digestion/absorption coefficients; • concentrations in several tissues (bone and organs, such as liver, kidneys, muscle, spleen); • total mineral content in the animal's body; • morphological characteristics in several tissues; • blood parameters (concentrations of the minerals, enzyme activities, hormones); • concentrations in secretory fluids (bile, pancreatic fluid); • concentrations in urine.

The several criteria will be further outlined in short.

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3.1.1. Animal performance Performance as a criterion for assessing the bioavailability of minerals is for several minerals probably the least sensitive response parameter. An effect on performance can only be expected if the animal is fed below its physiological requirement unless a pharmacological effect can be expected (e.g. for copper). Differences in performance can only be noted at large differences in bioavailability or large differences in mineral supply. Broilers are often more sensitive in their performance to differences in mineral supply than other animals, because they have low body reserves of minerals, in contrast to pigs and calves when born.

3.1.2. Digestion/absorption coefficients Assessing the bioavailability of mineral sources by means of digestion and absorption studies seems to be one of the best direct methods particularly for major minerals. This method is less effective for trace minerals due to the low inclusion levels, the low absorption coefficients and the relatively high endogenous secretion of the specific mineral. Under specific defined conditions, the potential value of products can be measured accurately. However, the problem is that the methods, although they look quite similar, may differ among each other. One large difference may be the basal diet: a more or less practical diet or a synthetic diet (Eeckhout and DePaepe, 1996; BASF, 1996; Pfeffer, 1996). Hence, altered digestibility coefficients may be measured e.g., by a lower faecal endogenous excretion. However, in experimental diets for poultry mostly (semi)-synthetic diets have to be used for assessing phosphorus digestion/absorption due to the fact that broilers are capable of hydrolysing phytate phosphorus with their endogenous enzymes. Otherwise, it may lead to underestimation in digestibility/availability of the phosphorus source under investigation. A prerequisite for a better harmonization is that the basal diets become more similar among research centres. Methods that are used are the balance technique and the slope ratio technique. Radio-labelled or stable isotopes of major minerals are seldom used, due to their high price. For trace minerals studies, they are used more commonly/frequently. By means of isotopes, excretion can be corrected for endogenous secretion, so that instead of apparent a true digestibility can be obtained.

3.1.3. Concentrations in several tissues In general, bone parameters are the most commonly used parameters for assessing the bioavailability of calcium, phosphorus, manganese and zinc in mineral sources. A large variety of bone parameters can be used such as fresh bone weight, dry bone weight, fat-free bone weight, bone ash weight, calcium, phosphorus, manganese or zinc content in bone ash, bone density, specific gravity, bone breaking strength, surface of epiphysial plate, and radiological or photometric parameters. Bone ash content and bone breaking strength are most commonly used for calcium and phosphorus, especially for poultry. Several bones can be chosen such as femur, tibia, metatarsals (3rd and 4th), metacarpals (3rd and 4th), tail vertebrae, and toes. The more distal the bones, the more sensitive they are found to be for differences in calcium and phosphorus supply. Because there is a rather large between-animal-variation, quite a large number of observations is necessary. Furthermore, it is an indirect parameter (reference source is generally included), that has to be converted to a direct (tabulated) value. This is one of the disadvantages of using bone parameters, as well as the price of sacrificing the animals. Moreover, the preparation of the bones is very laborious. For calcium, also egg shell strength can be used as a criterion as well as magnesium content in the egg shell or magnesium content in bone. Some trace minerals may accumulate in specific organs e.g. copper, cobalt, iron, manganese, molybdenum and zinc in the liver, kidney, spleen, or muscle. Furthermore, iodine concentration in thyroids or even thyroid weight can be used as a criterion for the iodine supply. Therefore, for evaluation of particular trace minerals some of these target organs are chosen. For large animals, biopsies can be taken from some organs and tissues. With regard to the liver it has been shown the site of sampling is also of importance (Götze et al., 1978). In human studies, hair or nails are used as indicators for the supply of certain minerals, e.g. selenium and copper, but this is not common for livestock. Concentration of ferritin in the liver is an indication for the iron supply.

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3.1.4. Total amount of minerals in the animal body It seems obvious that the total amount of a mineral that is retained in the animal body is one of the best response parameters for those minerals with a low mineral turnover such as calcium and phosphorus. However, this holds true only if the animals are fed below their mineral requirement. When fed above their mineral requirement, the surplus of the mineral is excreted in the faeces and urine and is, therefore, not retained in the body. This leads to underestimation of the nutritional value of the mineral source, although there are some exceptions such as copper (accretion in the liver) and zinc (accretion in the bones). There are several disadvantages of this method. Sample preparation from a whole body is not an easy task. Furthermore, the amount of the mineral present in the animal body should be doubled at least to obtain a reasonable accurate estimation of value of minerals when using cattle or pigs. Therefore, one should take care that there is a sufficient increase of the amount of mineral in the body. This is much easier when broilers are involved. Accordingly, both accuracy of sampling and amount of increase may result in a substantial variation of the estimated amount of the mineral in the body. Another disadvantage, in the case of large animals, is the price of the animals that have to be sacrificed.

3.1.5. Morphological measurements in various tissues Some minerals may have an effect on the morphology of tissues because of their interference with tissue formation. For instance manganese has an effect on connective tissue formation. Therefore the perosis severity index has often been taken as a measure for supply of manganese for birds. Furthermore, pancreatic fibrosis or exudative diathesis are used as a criterion for the supply of selenium.

3.1.6. Blood parameters Blood parameters such as concentration of minerals and activity of specific enzymes may also be used as a criterion for evaluation of the bioavailability of mineral sources. One should realise, however, that there is hormonal regulation to control homeostasis in the extracellular fluid. Therefore, differences in blood levels only occur when the regulatory mechanism is no longer able to maintain the extracellular content of the mineral within the narrow physiological range (Van der Velde et al., 1986). This means that serum mineral levels can only be used at large differences in bioavailability of the mineral sources under investigation. Also, diurnal variation in serum mineral content or enzyme activity may influence the results. For phosphorus, inorganic phosphorus content of the serum and alkaline phosphatase activity are used as a criterion. Boyd et al. (1981; 1983) showed that for phosphorus, serum alkaline phosphatase activity was highly correlated with bone breaking strength (r=-0.98), although the coefficients of variation for both parameters were high (23%). For copper, criteria may be blood or serum copper content, superoxide dismutase, cytochrome c oxidase or caeruloplasmin content in the liver. With regard to iron. haemoglobin content in blood or haemoglobin regeneration are well known criteria. The enzymes 5-iodothyronine deiodinase and glutathione peroxidase activity are important parameters for the selenium supply. In ruminants vitamin B12 synthesis is an indicator for the supply of cobalt.

3.1.7. Concentrations in secretory fluids In some cases concentrations of minerals in secretory fluids can be used as a criterion for the supply. This is the case for the content of copper in bile or the zinc content of zinc in pancreatic fluid.

3.2. In vitro techniques There have been several attempts to derive the bioavailability of minerals from in vitro techniques. So far, this approach has not resulted in satisfactory results, because it is very difficult to simulate the gastric and intestinal conditions properly. Guéguen (1976, 1977) developed the citric acid solubility of

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phosphorus sources, but came to the conclusion that this method was not accurate enough. Also, several studies at the ID-Lelystad showed that solubility in water and solubility in 2% citric acid were not discriminative enough to rank the phosphorus sources tested (Dellaert et al., 1990; Kemme et al., 1993). With these tests, only inferior sources can be distinguished from good quality sources (Guéguen, 1961; 1976). Recently, Havenaar et al. (1995) developed a method known as the TNO Intestinal Model (TIM) that simulates gastro-intestinal conditions and claims to be able to accurately simulate absorption conditions including minerals.

3.3. Conclusion methods for evaluating the bioavailability of minerals It appears from literature that there is no single satisfactory method for evaluating the bioavailability of minerals in mineral sources or in raw materials. The method(s) depend on the response parameter(s) as well as the specific mineral in study. For minerals such as calcium and phosphorus, digestibility/absorbability and bone parameters (bone ash content, bone breaking strength) are the best parameters. The digestibility/absorbability is preferred, because this is a direct method. For several trace minerals other parameters have to be chosen. The next step is to harmonise the methods, formulate the conditions for the experimental set up, and ensure the same response parameters are being measured.

4. TERMS USED TO EXPRESS THE BIOAVAILABILITY OF MINERALS

In literature, different terms are used to assess and express the nutritive value of minerals for animals e.g., digestibility, absorbability, (bio)availability or even bioefficacy (Partridge, 1980). The term digestibility and absorbability refer to the gastrointestinal tract (feed - faeces). The term (bio)availability, however, is used with several different meanings, and can therefore, be misleading. ARC (1981) defines (bio)availability as the fraction that is retained in the body [feed - (faeces + urine)]. The term (bio)availability is also used in studies assessing the nutritive value of mineral sources, whereby the result is compared with a reference that is assumed to be 100% available (NRC, 1998). In order to define availability in the latter situation, various response parameters that are not directly associated with absorption are used. In addition to absorbability or availability, another distinction can be made between apparent or true, and between ileal and faecal values. However, this will not be discussed further in this paper. In many cases, however, absolute values, which can differ from one experiment to the other have to be converted to relative values. This is often called the relative bioavailability (RBV).

4.1. Absorbability For minerals that are absorbed from the intestinal tract, the terms absorption and digestion can be used. From the scientific point of view, the term absorbability should only be used as the fraction that is absorbed from the gastrointestinal tract. In this case, digestibility is not the right term when related to minerals, although it is widely used in practice. The term availability should not be used in this case.

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4.2. Availability Availability can be used for all other measures not related to disappearance of minerals from the gastrointestinal tract. As in almost all studies the nutritive value of a mineral is related to a reference mineral, the term relative (bio)availability should be used. In addition, it should also be indicated which mineral source is the reference and which response parameter was used. The importance of the response parameter is illustrated in Table 1.

Table 1: Relative biological availability of Zn from different sources, in “practical” doses1) added to diets based on maize and soybean meal2) for weaned piglets (Wedekind et al., 1994) Dependent variable Zinc source Biological availability (%) Metacarpal Zinc sulphate monohydrate 100.0 Zinc methionine 60.4 Zinc lysine 37.5 Zinc oxide 66.7

Coccygeal bones Zinc sulphate monohydrate 100.0 Zinc methionine 84.4 Zinc lysine 24.3 Zinc oxide 69.5

Plasma Zn Zinc sulphate monohydrate 100.0 Zinc methionine 95.4 Zinc lysine 78.7 Zinc oxide 87.0 1)Added doses of zinc were 0, 5, 10, 20, 40 and 80 mg/kg diet. 2) Zinc content in basal diet was 27 to 32 mg/kg diet. Table 1 shows a large difference in bioavailability depending on the response criteria. Therefore, for many minerals it is of much more value if more than one response parameter is used. However, in practice for evaluating different phosphorus sources often only phosphorus digestibility is used, being a very discriminating criterion.

5. RANKING OF VARIOUS CRITERIA TO ASSESS BIOAVAILABILITY

As described above, several criteria are used to judge the effect on the animal’s mineral status of supplying a certain amount of a mineral. These response criteria should react sufficiently rapidly to variations in dietary supply of the mineral. A response criterion can reflect the actual supply (over a period of days or weeks) or the historical supply (over a period of months or years). Suitable response criteria should be sufficiently sensitive to variations in dietary mineral supply (preferably to both excess and insufficient supply), be sufficiently specific (reacting only on variations in one mineral; and be readily accessible (Sandoval et al., 1997; Jongbloed et al., 2001a). However, not all criteria are equally important. Therefore, criteria have to be ranked in order of their importance. This order may be different between the specific animal species or even animal categories. Beside this, it is important to note that the order of importance of the criteria may depend on the level of supply (below or above recommended requirements). Criteria and weighing factors of copper for pigs are presented in Table 2. The higher the ranking the more important is the criterion for judging the mineral status of the pig. This ranking of importance is mainly based on published literature and on the experience of the responsible expert group.

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Ranking of criteria of importance for phosphate sources in pigs were described by Dellaert et al. (1990). In their experiments they used several criteria, like blood phosphorus, alkaline phosphatase activity, bone mineral content, breaking strength, digestibility and total phosphorus retention. The highest R2 and lowest coefficients of variation were obtained with phosphorus digestibility, followed by ash content in fat-free dry matter of some reference bones like metatarsals and breaking strength. Ranking of criteria of importance for zinc sources were described by Sandoval et al. (1997). In their studies on broilers receiving different levels of supplementary zinc, several multiple linear equations were developed for estimation of concentrations of zinc in different tissues, like bone, liver, kidney and pancreas. They showed that concentration of zinc in bones gave the highest R2 , followed by zinc content in liver and pancreas. Swinkels et al. (1994) described for pigs the assessment of zinc bioavailability from several sources, and the complexity of using several response criteria. For humans, Delves (1985) concluded that a valid assessment of zinc status (in order of merit) can be made from plasma zinc, zinc tolerance test, plasma-bound zinc, plasma alkaline phosphatase and leukocyte zinc. However, they did not consider bone zinc. Furthermore Delves (1985) concluded that erythrocyte zinc, urinary zinc and hair zinc were of little value. For copper, liver copper concentration is the most important criterion, while many are less important or not suitable. A review of these response parameters has been given by Delves (1985).

Table 2: Ranking of criteria to judge the effects of a certain supply of copper on pig mineral status Cu Ranking of importance (weighing factors) Criterion Supply below requirements Supply above requirements Copper absorption 3 1 Hepatic copper content 4 3 Superoxide dismutase activity 1 1 Hepatic ceruloplasmin content 1 1 Animal performance 1 no Cytochrome oxidase activity no no Serum/plasma copper concentration no no Bile copper concentration no no When comparing different sources of a mineral, bioavailability has to be related to a reference mineral source. This source is defined to have a relative bioavailability of 100%. For copper, the reference source is usually copper sulphate pentahydrate (CuSO4⋅5H2O; reagent grade). An example of calculation of the relative bioavailability of three copper sources at a suboptimal copper supply is as follows. First the original data of the different relevant response criteria are compared with the reference source by assuming that the reference source has a bioavailability of 100. Subsequently these results are multiplied by the ranking factor as is presented in Table 2. The final results are presented in Table 3.

Table 3: Final evaluation of three different copper sources as compared with CuSO4⋅5H2O Cu sources Cu liver Cu absorption Performance Relative bioavailability CuSO4⋅5H2O 100 100 100 100 Source A 70 80 90 (4*70+3*80+1*90)/(4+3+1)=76.2 Source B 80 70 90 (4*80+3*70+1*90)/(4+3+1)=77.5 Source C 50 40 80 (4*50+3*40+1*80)/(4+3+1)=50 The final evaluation of this experiment is that the copper sources A, B and C have a relative bioavailability of 76, 77 and 50, respectively, as compared with the reference source copper sulphate.

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Important note: If in one experiment the reference source, e.g. in the above example copper sulphate, is not present, another copper source must be used as a reference to calculate the relative bioavailability of the other copper sources in the experiment. This will be clarified with an example in Table 4.

Table 4: Evaluation of three different copper sources as compared with CuSO4⋅5H2O or another reference Cu sources Cu 1 Cu 2 Cu 3 Cu 4 n Relative bioavailability CuSO4⋅5H2O 100 100 100 3 100 Source A 70 80 75 (75) 3 75 Source B 60 66 2 63 Source C 65 65 2 65 In experiments 1 to 3, copper sulphate is the reference source. In experiment 4, copper sulphate is not present but only copper sources A and B. One of those sources must be used as a reference source for the other copper source. Copper source A is taken as the reference source. Based on experiments 1 to 3, source A has a relative bioavailability of 75. The reference source is placed between brackets to show that this element was used as a reference and the relative bioavailability of copper source B is calculated based on the relative bioavailability of reference copper source A.

6. SELECTION OF LITERATURE

For the purpose of this publication a literature study was carried out. Literature used in this study had to comply to a number of selection criteria. First of all, only studies which were judged by the specialist as scientifically sound were used. Also, only original studies were considered and not results cited in other studies. Secondly, the method of evaluating bioavailability should be well defined and also acceptable response criteria had to be used. A third selection criterion was the fact that the mineral sources used in the studies should be (chemically) well defined. As a result, these selection criteria limited the amount of literature that could be used for this study. Information on the bioavailability of the group of minerals that are sometimes referred to as “complexed minerals”, such as mineral proteinates or chelates, is, therefore, limited because of the lack of product definitions.

7. CONCLUSION

In total there are 20 minerals that are essential for maintenance and normal functioning of the body. Insufficient supply of these minerals results in deficiency symptoms leading to reduced performance. Therefore, sufficient amounts should be supplied. Conversely, an oversupply of minerals may harm production efficiency together with a possible negative effect on the environment. Therefore, apart from the requirement for various minerals their availability should also be known to prevent oversupply. Different terms used to express the bioavailability of the minerals are described together with various factors that may affect their bioavailability. A description is given on how the bioavailability of minerals can be assessed, both in vivo and in vitro. Much attention has been paid to the different response criteria and how they can be evaluated to make a proper comparison between different mineral sources.

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8. REFERENCES

ARC, 1981 Agricultural Research Council. The nutrient requirements of pigs, C.A.B., Slough, 307pp. Ashmead, H.D., Zunino, H., 1994. Factors which affect the intestinal absorption of minerals. In:

Ashmead HD (ed.), The roles of amino acid chelates in animal nutrition. Park Ridge: Noyes Publications, p. 21-46.

BASF, 1996. Phosphor-Verdaulichkeit verschiedener Futterphosphate beim jungen Ferkel. Info-Service Tierernährung 43/96, 4 pp.

Besançon, P., Guéguen, L., 1969. [The main routes of calcium metabolism in growing pigs]. Ann. Biol. Anim. Bioch. Biophys. 9, 537-553.

Boyd, R.D., Hall, D., Wu, J.F., 1981. Plasma alkaline phosphatase as a criterion for determining biological availability of phosphorus for swine. Proc. Cornell Nutr. Conf., 58-63.

Boyd, R.D., Hall, D., Wu, J.F., 1983. Plasma alkaline phosphatase as a criterion for determining biological availability of phosphorus for swine. J. Anim. Sci. 57, 396-401.

Dellaert, B.M., Van der Peet, G.F.V., Jongbloed, A.W. and Beers, S., 1990. A comparison of different techniques to assess the biological availability of feed phosphates in pig feeding. Neth. J. Agric. Sc. 38, 555-566.

Delves, H.T., 1985. Assessment of trace element status. Clinics in Endocrinology and Metabolism 14, 725-760.

Eeckhout, W., DePaepe, M., 1996. The bio-availability of three calcium phosphates for pigs: digestibility as measured by difference and by slope-ratio assay. Paper 47th Annual Meeting EAAP, Lillehammer, Norway. Paper N3.4.

Götze, M., Steger, H., Brüssow, K.P., 1978. Über den Kupfereinsatz zur Unterstützung fortpflannungsphysiologischer Prozesse. Arch. Exper. Vet. Med. 32, 787-793.

Groote, G. de, Huyghebaert, G., 1997. The bio-availability of phosphorus from feed phosphates for broilers as influenced by bio-assay method, dietary Ca-level and feed form. Anim. Feed Sci. Technol. 69, 329-340.

Guéguen, L., 1961. Valeur comparée des phosphates minéraux comme sources de phosphore des animaux. Ann. Zootechn., 10, 177-196.

Guéguen, L., 1976. A propos du contrôle de la qualité du phosphore des composés minéraux; ses possibilités et ses limités. L'Élevage Bovin, 64, 49-51.

Guéguen, L., 1977. à propos du controle de la qualité du phosphore des composés minéraux. Elevage Porcin 65, 33-35.

Harland, B., 1989. Dietary fibre and mineral bioavailability. Nutr. Res. Rev. 2, 133-147. Havenaar, R.J., Minekus, M., Speckmann, A., 1995. Efficacy of phytase in a dynamic, computer-

controlled model of the gastro-intestinal tract. In: Proc. of second European Symposium on Feed Enzymes, W. van Hartingsveld, M. Hessing, J.P. van der Lugt and W.A.C. Sommers (Eds.), TNO Nutrition and Food Research Institute, Zeist, The Netherlands, p. 211-212.

Huyghebaert, G., Groote, G.D. de, Keppens, L., 1980. The relative biological availability of phosphorus in feed phosphates for broilers. Ann. Zootechn. 29, 245-263.

Huyghebaert, G., Groote, G.D. de, Keppens, L., 1981. Influence of varying calcium/and fluorine levels and sodium to chloride ratios on phosphorus utilization and bone strength of broiler chicks. Rev. Agric. 34, 311-330.

Jongbloed, A.W. ,1987. Phosphorus in the feeding of pigs: Effect of diet on the absorption and retention of phosphorus by growing pigs. PhD thesis, Landbouwuniversiteit Wageningen, Rapport IVVO-DLO no. 179, Lelystad, the Netherlands, 343pp.

Jongbloed, A.W., Kemme, P.A., Mroz, Z., Bruggencate, R. ten, 1995. Apparent total tract digestibility of organic matter, N, Ca, Mg and P in growing pigs as affected by levels of Ca, microbial phytase and phytate. In: W. van Hartingsveldt, M. Hessing, J.P. van der Lugt and W.A.C. Somers (Editors). Proceedings of Second Symposium on Feed Enzymes (ESFE2), Noordwijkerhout, Netherlands, TNO Nutrition and Food Research Institute, Zeist, p. 198-204.

Jongbloed, A.W. and Henkens, C.H., 1996. Environmental concerns of using animal manure - the Dutch case. In: E.T. Kornegay (ed.) Proceedings Symposium on Nutrient Management of Food

14

Animals to Enhance the Environment. Lewis Publishers/CRC Press, Boca Raton, FL, USA, pp. 317-333.

Jongbloed, A.W., Mroz, Z., 1997. Exchanges of macro- and microelements along the gastrointestinal tract of pigs. In: J.P. Laplace, C. Fevrier and A. Barbeau (Eds.), Proc. 7th Int. Symp. on Digestive Physiology in Pigs, INRA St Malo, France, EAAP Publication No. 88, p. 348-352.

Jongbloed, A.W., Top, A.M. van den, Beynen, A.C., Klis, J.D. van der, Kemme, P.A., Valk, H., 2001. Consequences of newly proposed maximum contents of copper and zinc in diets for cattle, pigs and poultry on animal performance and health. Report ID-Lelystad no. 2097, 73 pp.

Jongbloed, A.W., Kemme, P.A., Top, A.M. van de, 2001b. The role of nutrition in reducing the accumulation of minerals by pigs. Main paper N5.1, Book of Abstracts No. 7, 52nd Annual Meeting EAAP, Budapest, Hungary, 26-29 August 2001, p. 120.

McDowell, L.R., 1992. Minerals in Animal and Human Nutrition. Academic Press, San Diego, 524 pp.

Meschy, F., Gueguen, L., 1998. Les recommandations d’apport alimentaire en éléments minéreaux: analyse et perspectives. Rencontres Rech. Ruminants 5, 235-240.

NRC , 1980. Mineral Tolerance of Domestic Animals. National Academic Press, Washington (DC), USA, 577 pp.

NRC , 1994. Nutrient Requirements of Poultry. National Research Council, National Academy Press, Washington (DC), USA, 155pp.

NRC , 1998. Nutrient requirements of swine. National Academy of Sciences, National Academy Press, Washington (DC), USA, 93 pp.

Nys, Y., and Mongin, P., 1980. Jejunal calcium permeability in laying hens during egg formation. Reprod. Nutr. Dévelop. 20, 155-161.

Partridge, I.G., 1980. Mineral nutrition of the pig. Proc. Nutr. Soc. 39, 185-192. Pfeffer, E., 1996. Paper presented at seminar at Fulda, Germany. Pointillart, A., Fourdin, N. and Delmas, A., 1987. Consequences de l'exces de calcium chez des porcs

non supplementés en phosphore mineral. Journées Recherches Porcine en France 19, 281-287. Sandoval, M., Henry, P.R., Ammerman, C.B., Miles, R.D., Littell, R.C., 1997. Relative bioavailability

of supplemental inorganic zinc sources for chicks. J. Anim. Sci. 75, 3195-3205. Schröder, B., Kappner, H., Failing, K., Pfeffer, E., Breves, G., 1995. Mechanisms of intestinal

phosphate transport in small ruminants. Brit. J. Nutr. 74, 635-648. Schröder, B., Breves, G. and Rodehutscord, M., 1996. Mechanisms of intestinal phosphorus

absorption and availability of dietary phosphorus in pigs. Deutsch Tierärztliche Wochenschrift 103, 209-214.

Swinkels, J.W.G.M., Kornegay, E.T., Verstegen, M.W.A., 1994. Biology of zinc and biological value of dietary organic zinc complexes and chelates. Nutr. Res. Rev. 7, 129-149.

Underwood, E.J., 1981. The mineral nutrition of livestock, 2nd ed. Commonwealth Agricultural Bureaux, Slough, England, 237 pp.

Underwood, E.J., Suttle, N.F., 1999. The Mineral Nutrition of Livestock, 3rd edition. CABI Publishing, Wallingford, United Kingdom.

Van der Klis, J.D., 1993. Physico-chemical chyme conditions and mineral absorption in broilers. PhD thesis, Agricultural University Wageningen, The Netherlands.

Van der Klis, J.D., 1994. Mineral nutrition and broiler bone characteristics. In: Proc. of the 9th European Poultry Conference, Glasgow, United Kingdom, pp 211-214.

Van der Velde, J.P., van Ginkel, F.C., and Vermeiden, J.P.W., 1986. Patterns and relationships of plasma calcium, protein and phosphorus during the egg laying cycle of the fowl and the effect of dietary calcium. Br. Poultry Sci. 27, 421-433.

Wedekind, K.J., A.J. Lewis, M.A. Giesemann, P.S. Miller, 1994. Bioavailability of zinc from inorganic and organic sources for pigs fed corn-soybean meal diets. J. Anim. Sci. 72, 2681-2689.

15

Appendix 1: Factors affecting the bioavailability of mineral sources Aspects related to the diet are:

o feedstuff composition of the diet o chemical composition of the diet (proximate analysis and mineral contents) o vitamin content o presence of antimicrobial growth promoters or (organic) acids o chemical composition and purity of the mineral sources tested o level of supplementation of the minerals tested o particle size of the mineral source

Aspects related to the animal are:

o animal species, breed and physiological status of the animal o sex and age o production (performance) level and type of production

Environmental and management aspects are:

o health status of the animals o level of feeding expressed as energy level times maintenance requirement for energy o level of water supply o feeding method (dry or wet feeding; soaking) o housing and equipment

Evaluation method

o direct or indirect o criterion o reference mineral o levels of supplementation o number of replicates o model used for evaluation (doses-response; linear or non-linear). o period of feeding the test diet, duration of preliminary and test periods

16

MAJOR MINERALS

I. Calcium bioavailability

II. Magnesium bioavailability

III. Sodium bioavailability

IV. Phosphorus bioavailability

17

I. CALCIUM BIOAVAILABILITY

General function in the body Calcium is one of the most abundant elements in the body with 99 % being found in the skeleton. It is primarily present in bone tissue as the hydroxyapatite form of calcium phosphate. Indeed, the basic function of calcium is to provide a strong framework supporting and protecting delicate organs. The remaining one percent of the calcium is widely distributed in various soft tissues of the body. It occurs as the free ion (50-60 %), bound to serum proteins or complexed to organic and inorganic acids. It is the ionised form that is extremely important in cellular metabolism, blood clotting, enzyme activation and neuromuscular action (muscle contraction and nerve responses). For example in poultry, calcium has the unique function of protecting the egg through the deposition of an eggshell which has a high concentration of CaCO3. As about 1.5 g calcium/d is needed for egg production (Soares, 1987), eggshell deposition dominates the calcium metabolism in the laying hen. In comparison, a dairy cow in peak lactation mobilises 50 mg/kg body weight (Soares, 1987). The priority of all mammals is to maintain calcium concentrations in plasma and extracellular fluids. Homeostasis is achieved partly by the hormonal regulation of absorption, with the small intestine as the major absorptive site. When the supply of calcium is excessive, the homeostatic mechanisms are reversed. Equally important to the regulation of ionic calcium concentrations is the net flow of calcium from the enormous reserve in the skeleton. The modulation of excretion by the urinary route generally plays a minor role in calcium homeostasis. There are a lot of factors influencing the bioavailability of calcium sources. Among them are the vitamin D concentration, Ca/P-ratio, phytate or oxalate complexes, anion/cation-ratio, dietary magnesium and aluminium, particle size, etc. Disordered calcium metabolism can arise either as a result of an acute increase in demand (‘metabolic deprivation’) or as a result of chronic dietary deprivation. This can result in a range of symptoms including reduced growth rate, increased mortality, depression of milk yield and egg yield or quality, bone abnormalities and milk fever (parturient paresis or hypocalcaemia).

Use of calcium for livestock In general, practical diets need calcium supplementations to meet the requirements. Sources of supplemental calcium include calcium carbonate, limestone, oyster shells, calcium phosphates, calcium sulphate and bone and meat meals (if still allowed). Particle size of calcium sources has a distinct influence on the rate of solubility of the calcium. Coarse particles of calcite or oyster shells are preferred for egg production, because these are retained in the gizzard and release their calcium slowly during the period of shell formation.

CALCIUM BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs As described earlier, the sequence of importance of response criteria depends on whether the animals are fed suboptimal levels of calcium or if they are fed above their calcium requirement. The ranking of importance for assessing the relative biological value of calcium sources of the various criteria is listed in Table I.1.

18

Table I.1: Ranking of importance of various response criteria for assessing the relative biological value of calcium sources in pigs Criterion Suboptimal supply Above requirement Calcium absorption/digestibility 5 1 Calcium absorption (true) 5 2 Calcium retention 3 2 Bone ash content (g/kg) 3 2 Animal performance 1 0 Table I.1 shows that there are only a few criteria that can be used for evaluation of the bioavailability of calcium sources for pigs of which calcium absorption ranks highest. For our study the reference source is limestone (CaCO3) fine reagent grade (RG).

Comparison of calcium sources for pigs There were only four publications that were suitable for our study, which are listed in Table I.2. It was not always clear how many molecules of crystalline water were present in the source used. Therefore, these were regarded as one source. The mean was calculated of the four sources of calcium chloride used in the experiments by Kuznetsov et al. (1987). We did not calculate calcium availability of various feed phosphates due to the fact that calcium and phosphorus availability of these sources are strongly interrelated. Moreover, these experiments were not designed for this purpose, and also the calcium digestibility of the basal diet was not determined.

Table I.2: Overview of the experiments used for evaluation of the relative biological value of calcium sources in pigs Sources of calcium Animal

type Response criteria

Added Ca (g/kg)

Ref. no

Number of expts

Reference

Limestone Oyster shell Calcium sulphate

4–13 kg Performance 4.0, 8.0 Ca1 2 Combs and Wallace, 1962

Calcium carbonate Limestone Calcium chloride (4 types) Calcium citrate Calcium hydroxide Calcium lactate Calcium oxalate Calcium oxide Calcium propionate Calcium succinate Calcium sulphate

44-56 kg Digestibility 4.0 Ca2 1 Kuznetsov et al., 1987

Limestone Calcium benzoate Calcium chloride Calcium sulphate

40–65 kg Digestibility 3.0, 6.0 Ca3 2 Mroz et al., 1996

Limestone Calcite limestone Calcium sulphate Aragonite Dolomite Marble dust Oyster shell

15–28 kg PerformanceBone ash Bone breaking strength

3.0, 5.0 Ca4 2 Ross et al., 1984

19

Table I.3 shows that two or more observations were available for limestone, oyster shell and calcium sulphate. There was only one observation available of the other calcium sources. Most of the calcium sources had the same relative biological value as CaCO3 or limestone, except for calcium oxalate that had a lower bioavailability of 74. Furthermore, the results by Kuznetsov et al. (1987) suggest higher bioavailabilities for salts of calcium with organic acids (between 114 to 128) compared to limestone. Calcium hydroxide was also in this range. An experiment by Eggert et al. (1959) on weanling pigs also showed no differences between limestone and CaSO4·2H2O as supplemental calcium source (no details given). Furthermore, early studies by Thomas et al. (1933), and by McCampbell and Aubel (1934) showed no differences between limestone or gypsum; (cited by Ross et al., 1984). From the experiments by Ross et al. (1984) it can be concluded that no differences could be demonstrated between the particle sizes of three types of calcium sources (calcite limestone, marble dust and aragonite). Also, Pond et al. (1981) showed in pigs that particle size of limestone did not affect performance or bone traits.

Table I.3: Summarised results on the relative biological value of calcium sources for pigs Reference Ca1 Ca2 Ca3 Ca4 n Mean SD Number of experiments 2 1 2 2 7 CaCO3 precipitate 100 100 2 100 - Limestone 100 99 100 3 100 0.5 Calcite limestone A+B 104 1 104 - Calcium benzoate·2H2O 102 102 - Calcium chloride 117a 88 2 105 20.7 Calcium citrate 118 118 - Calcium hydroxide 116 116 - Calcium lactate 96 96 - Calcium oxalate 74 74 - Calcium oxide 96 96 - Calcium propionate 114 114 - Calcium succinate 128 128 - Calcium sulphate 108 106 101 100 4 104 4.1 Aragonite 100 1 100 - Dolomite A+B 94 1 94 - Marble dust 100 1 100 - Oyster shell 101 99 2 100 1.5 a different types were used

CALCIUM BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry Response criteria are ranked along their importance in Table I.4. Differences are made between suboptimal supply and feeding above requirement.

20

Table I.4: Ranking of importance of various response criteria for assessing the relative biological value of calcium sources in poultry Criterion Suboptimal supply Above requirement Ca absorption/digestibility (ileal/faecal) 5 1 Ca absorption (true) 5 2 Ca retention (balance or slaughter) 3 2 Tibia/toe/metatarsal ash or Ca 3 2 Bone breaking strength 2 2 Eggshell (only layers) (1) Performance 1 no There were 9 publications suitable for the study. They are listed in the Table I.5.

Table I.5: Overview of the experiments used for evaluation of the relative biological value of calcium sources in poultry Sources of calcium

Animal type

Response criteria

Added Ca (g/kg)

Number of expts

Ref. no

Reference

Calcium carbonate Ground limestone Tricalcium phosphate Dicalcium phosphate

Broiler Tibia ash Tibia Ca

unknown 1 Ca1 Blair et al., 1965

Pulverised limestone Aragonite Oyster shell

Laying hen

Mean shell weight Shell % Shell thickness

unknown 1 Ca2 Brister et al., 1981

Calcium carbonate Defluorinated phosphate

Broiler Tibia ash Tibia strength

(1.2), 2.4 1 Ca3 Burnell et al., 1990

Ground limestone Calcium sulphate

Broiler Tibia Ca unknown 1 Ca4 Hurwitz and Rand, 1965

Granular limestone Aragonite Oyster shell Egg shell

Laying hen

Shell thickness

unknown 1 Ca5 Muir et al., 1976

Calcium carbonate Ground limestone (5 origins) Oyster shell flour

Broiler

Performance Tibia ash

2.4, 4.4, 6.7, 8.7 (ref.) (diff: 4.2-9.7 for other sources)

1 Ca6a Reid and Weber, 1976

Ground limestone (5 origins) Oyster shell flour

Laying hen

Performance Ca retention Egg shell thickness

13.5, 17.6, 23.4 (ref.) (diff: 11.0-23.3 for other sources)

1 Ca6b Reid and Weber, 1976

21

Table I.5 (continued) Sources of calcium

Animal type

Response criteria

Added Ca (g/kg)

Number of expts

Ref. no

Reference

Calcium carbonate Dolomite limestone

Broiler Performance Tibia ash

unknown 1 Ca7 Stillmak and Sunde, 1971

Calcium carbonate Calcium sulphate Calcium gluconate Ground oyster shell Ground limestone (2 origins)

Broiler Performance Tibia ash

1.3, 2.3, 3.3, 4.3, 5.3

1 Ca8

Waldroup et al., 1964

Limestone Oyster shell

Laying hen

Performance Egg weight Breaking strength

7.5, 15.0 1 Ca9 Watkins et al., 1977

Comparison of calcium sources for poultry Table I.6 shows six studies with broilers using performance and tibia parameters as response criteria and four experiments with laying hens using eggshell parameters as response criteria. Since calcium plays an important and specific role in the metabolism for shell formation in the hen separate tables of comparison are given for chicks and laying hens. Table I.6 for chicks shows that calcium in the commonly used calcium sources, ground limestone and ground oyster shell, have a similar availability as of calcium carbonate, while it appears slightly lower in calcium sulphate and defluorinated phosphate. The calcium in dolomitic-high Mg-limestone is considerably less available. The calcium availability in di- and tricalcium phosphate is 8 % higher compared with the reference. It appears that within the normal range of particle size for ground calcium sources and calcium phosphates particle size is not an important factor influencing calcium availability for chicks and broilers. For laying hens (Table I.7) it appears that ground oyster shell has approximately 20% higher bioavailability compared to ground limestone based on one publication, while ungrounded oyster shells appear to have the same bioavailability as ground limestone. In general ungrounded oyster shell and calcite granules are considered to improve shell quality compared to limestone.

Table I.6: Summarised results on the relative biological value of calcium sources for broilers Reference Ca1 Ca3 Ca4 Ca6a Ca7 Ca8 n Mean SD Number of experiments 1 1 1 1 1 1 6 Calcium carbonate 100 100 100 100 100 5 100 - Calcium sulphate 861 99 2 93 8.9 Calcium gluconate 102 1 102 - Ground oyster shell 103 101 2 102 1.4 Ground limestone 99 (96)1 89 100 3 96 6.0 Dolomite limestone 65 1 65 - Defluorinated phosphate 95 1 95 - Tricalcium phosphate 108 1 108 - Dicalcium phosphate 108 1 108 - 1 recalculated using ground limestone as a reference

22

Table I.7: Summarised results on the relative biological value of calcium sources for laying hens Reference Ca2 Ca5 Ca6b Ca9 n Mean SD Number of experiments 1 1 1 1 4 Ground limestone 100 100 2 100 - Ground oyster shell 119 1 119 - Oyster shell 101 101 100 3 101 0.6 Granular limestone 100 1 100 - Aragonite 101 100 2 101 0.7 Egg shell 99 1 99 - Pulverised limestone 100 1 100 -

CALCIUM BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants As described earlier, the sequence of response criteria depends on whether the animals are fed suboptimal or if they are fed above their requirement. The ranking of importance for calcium of the various criteria to assess its bioavailability is listed in Table I.8.

Table I.8: Ranking of importance of various response criteria for assessing the relative biological value of calcium sources in ruminants Criterion Suboptimal supply Above requirement True absorption 5 2 Apparent absorption 3 Calcium balance 4 2 Bone parameters 3 2 Table I.8 shows that there are only few criteria that can be used for evaluation of calcium sources in ruminants. Five observations were based on apparent absorption and only one on calcium true absorption. For our study the reference source is calcium carbonate RG.

Comparison of calcium sources for ruminants There were in total six publications comprising nine experiments that were suitable for our study. This is listed in Table I.9.

Table I.9: Overview of the experiments used for evaluation of the relative biological value of calcium sources for ruminants Sources of calcium Animal

type Response criteria

Added Ca (g/kg)

Number of expts

Ref. no

Reference

Calcium carbonate RG Dolomite

Steers Apparent absorption

1.6 1 Ca1 Gerken and Fontenot, 1967

Calcium carbonate Dicalcium phosphate FG

Sheep Apparent absorption

3.5 1 Ca2 Guéguen and Bouchet, 1980

23

Table I.9 (continued) Sources of calcium Animal

type Response criteria

Added Ca (g/kg)

Number of expts

Ref. no

Reference

Calcium carbonate RG Bone meal Calcium chloride Dicalcium phosphate FG Dicalcium phosphate RG Limestone Monocalcium phosphate

Steers True absorption

2 Ca3 Hansard et al., 1957

Calcium carbonate Dolomite

Steers Apparent absorption

1.4 1 Ca4 Moore et al., 1971

Calcium carbonate Dolomite

Sheep Apparent absorption

2.2 1 Ca5 Rahnema and Fontenot, 1983

Calcium carbonate Aragonite (crystalline carbonate) Calcite (crystalline carbonate)

Cows Apparent absorption

6 and 9 g per day

3 Ca6 Wohlt et al., 1986

Comparisons of the different calcium sources have been summarised in Table I.10. This table shows that with the exception of dicalcium phosphate (n = 2) and dolomitic limestone (n = 3) only one observation was available for sources under investigation. The calcium phosphates show a higher calcium availability than the reference source. This table underlines the importance of purity of salts under investigation (e.g. RG vs FG), which probably also explains the quite low relative biological value (RBV) of limestone and the high SD for dolomite. We give the RBV of bone meal in spite of current EU regulations.

Table I.10: Summarised results of the relative biological value of calcium sources for ruminants References Ca1 Ca2 Ca3 Ca4 Ca5 Ca6 n Mean SD Number of experiments 1 1 2 1 1 3 9 Calcium carbonate RG 100 100 100 100 100 100 6 100 - Aragonite (CaCO3 crystalline) 128 1 128 - Bone meal 135 1 135 - Calcite (CaCO3 crystalline) 110 1 110 - Calcium chloride RG 125 1 125 - Dicalcium phosphate RG 125 1 125 - Dicalcium phosphate FG 110 120 2 116 7.1 Dolomite 66 94 83 3 81 14.1 Limestone 91 1 91 - Monocalcium phosphate RG 130 1 130 -

24

References Blair, R., English, P.R., Michie, W., 1965. Effect of calcium source on calcium retention in the young

chick. Brit. Poultry Sci. 6, 355-356. Brister, R.D., Linton, S.S., jr., Creger, C.R., 1981. Effects of dietary calcium sources and particle size

on laying hen performance. Poultry Sci. 60, 2648-2654. Burnell, T.W., Cromwell, G.L., Stahly, T.S., 1990. Effects of particle size on the biological

availability of calcium and phosphorus in defluorinated phosphate for chicks. Poultry Sci. 69, 1110-1117.

Combs, G.E., Wallace, H.D., 1962. Growth and digestibility studies with young pigs fed various levels and sources of calcium. J. Anim. Sci. 21, 734-737.

Eggert, R.G., Akers, W.T., Huhtanen, C.N., 1959. Chlortetracycline absorption and calcium utilization in growing swine as affected by terephthalic acid and calcium source. J. Anim. Sci. 18, 1505 (abstract).

Gerken, H.J., Fontenot, J.P., 1967. Availability and utilization of magnesium from dolomitic limestone and magnesium oxide in steers. J. Anim. Sci. 26, 1404-1408.

Guéguen, L., Bouchet, J.P., 1980. Compte rendu d'expérience sur l'utilisation digestive réelle de trois phosphates commerciaux. INRA Station de Recherches de Nutrition Jouy-en-Josas. 6pp.

Hansard, S.L., Crowder, H.M., Lyke, W.A., 1957. The biological availibility of calcium in feeds for cattle. J. Anim. Sci. 16, 437-451.

Hurwitz, S., Rand, N.T., 1965. Utilization of calcium sulfate by chicks and laying hens. Poultry Sci. 44, 177-183.

Kuznetsov, S.G., Kal’nitskii, B.D., Bataeva, A.P., 1987. Biological availability of calcium from chemical compounds. Soviet Agric. Sci. 3, 48-51.

Moore, W.F., Fontenot, J.P., Tucker, R.E., 1971. Relative effects of different supplemental magnesium sources on apparent digestibility in steers. J. Anim. Sci. 33, 502-508.

Mroz, Z., Jongbloed, A.W., Vreman, K., Canh, T.T., Diepen, J.Th.M. van, Kemme, P.A., Kogut, J., Aarnink, A.J.A., 1996. The effect of different cation-anion supplies on excreta composition and nutrient balance in growing pigs. Report ID-DLO no. 96.028.

Muir, F.V., Harris, P.C., Gerry, R.W., 1976. The comparative value of five calcium sources for laying hens. Poultry Sci. 55, 1046-1051.

Pond, W.G., Yen, J., Hill, D., Wheeler, W., 1981. Dietary source and level: Effects on weanling pigs. J. Anim. Sci. 53 (Suppl.1), 91.

Rahnema, S.H., Fontenot, J.P., 1983. Effect of supplemented magnesium from magnesium oxide or dolomitic limestone upon digestion and absorption of mineral in sheep. J. Anim. Sci. 57, 1545-1552.

Reid, B.L., Weber, C.W., 1976. Calcium availability and trace mineral composition of feed grade calcium components. Poultry Sci. 55, 600-605.

Ross, R.D., Cromwell, G.L., Stahly, T.S., 1984. Effects of source and particle size on the biological availability of calcium in calcium supplements for growing pigs. J. Anim. Sci. 59, 125-134.

Soares, J.H. jr. , 1987. Metabolic aspects of calcification in avians. J. Nutrition 117, 783. Stillmak, S.J., Sunde, M.L., 1971. The use of high magnesium limestone in the diet of the laying hen.

Poultry Sci. 50, 564-572. Waldroup, P.W., Ammerman, C.B., Harms, R.H., 1964. The utilization by the chick of calcium from

different sources. Poultry Sci. 43, 212-216. Watkins, R.M., Dilworth, B.C., Day, E.J., 1977. Effect of calcium supplement particle size and source

on the performance of laying chickens. Poultry Sci. 56, 1641-1647. Wohlt, J.E., Ritter, D.E., Evans, J.L., 1986. Calcium sources for milk production in Holstein cows. J.

Dairy Sci. 69, 2815-2824.

25

II. MAGNESIUM BIOAVAILABILITY

General function in the body Magnesium has been recognised as essential for mammals since 1926 (Leroy). Magnesium deficiency is quite uncommon in monogastric animals due to adequate magnesium levels in most practical diets. Consequently magnesium availability will be discussed here only for ruminants, nevertheless magnesium is important in poultry nutrition, especially for egg shell quality and sometimes it is used as anti-stress for pigs. In ruminants the importance of magnesium is predominantly related to grass tetany (hypomagnesaemia). This metabolic disorder occurs especially when the magnesium absorption decreases under nutritional conditions (high dietary potassium or soluble nitrogen). Moreover, all the stress factors (e.g. cold) increase the tetany risk by a large fall in blood magnesium content due to the effect of adrenaline secretion. Magnesium plays a structural role in the skeleton associated with hydroxyapatite crystals (60 to 70% of total magnesium of the body). Magnesium is also involved in functional roles such as nerve function and muscular contraction. Magnesium is a component of several enzymes implicated in the metabolism of carbohydrates, lipids and proteins. Hence, it is not surprising that magnesium deficiency may lead to a range of serious biochemical and functional problems. In ruminants, magnesium absorption occurs predominantly in the reticulo-rumen section of the gut (Thomas and Potter, 1976) and the range of its efficiency (5 to 30%, McDowell, 1992) indicates that rumen conditions play an important role. Magnesium absorption decreases with high ruminal potassium concentration (Greene et al., 1983) and increases with the level of available energy (Giduck and Fontenot, 1987).

Use of magnesium for livestock Magnesium dietary supply can be marginally deficient (e.g. in the early growth stage of forages such as grass and maize silage). Magnesium supplementation must is essential during periods when the risk of grass tetany is high (early spring or late autumn in Europe). The most usual recommendation is to provide dairy cows with an additional 25 g magnesium a day. Some magnesium salts (chloride, sulphate) are strongly bitter which limits their use. In association with sodium bicarbonate, magnesium oxide can also be used as rumen buffer in order to regulate rumen pH within a range which is favourable for cellulolytic activity of bacteria which maintains a good level of milk butterfat. Magnesium is sometimes used as anti-stress factor in pig nutrition. The magnesium requirements (expressed in terms of total- and unavailable Mg) for poultry ranges from 400-600 mg/kg dry matter. Since commonly used feedstuffs for poultry diets contribute Mg levels that are 2-4 times higher than the required level, it is unlikely that the commercial poultry diets would ever need supplementary magnesium for prolonged periods. The true absorption of Mg in selected commonly used feedstuffs and inorganic salts ranges for poultry from 42 to 83% (Guenter and Sell, 1974). In some studies with layers, supplemental MgSO4 increased shell weight and thickness of eggs produced by hens on a practical ration (Bastien et al., 1978), but this benefit was not observed by others (Sell, 1979). The most common magnesium source used is MgO (calcined magnesite) in granular form to avoid dust problems. Variations in bioavailability are influenced by processing methods (e.g. the temperature of calcinations) and particle size (Adam et al., 1996).

26

MAGNESIUM BIOAVAILABILITY FOR PIGS

There were only publications available from East-Europe of which details could not be obtained. Pustovoi (1989a, 1989b) carried out two experiments on pigs receiving different magnesium sources in which growth performance was the criterion. In the first experiment, magnesium chloride and magnesium chlorate were shown to have a slightly higher bioavailability than magnesium oxide (115 and 113 vs 100, respectively). The conclusion from the second experiment was that magnesium lactate, magnesium acetate and magnesium citrate were superior to magnesium oxide (on average 158 vs 100). It was, however, not possible to adequately evaluate the results.

MAGNESIUM BIOAVAILABILITY FOR POULTRY

There were no publications suitable for the study of magnesium bioavailability of poultry.

MAGNESIUM BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants The ranking of importance for magnesium of the various criteria is listed in Table II.1.

Table II.1: Ranking of importance of various response criteria for assessing the relative biological value of magnesium sources in ruminants Criterion Suboptimal supply Above the requirements Magnesium true absorption 5 5 Magnesium apparent absorption 3 3 Magnesium in urine 2 2 Magnesium balance 2 1 It can be seen in Table II.1 that there are only few criteria that can be used for evaluation of magnesium sources in ruminants of which magnesium absorption is the most important one. True absorption studies are very scarce due to technical reasons (half life of only 21.3 hr for 28Mg). We did not take into account studies based on magnesium blood analysis because of the very strong renal regulation of this parameter. In our data set, fourteen observations were based on apparent absorption and seven on magnesium urinary excretion. For our study the reference source is magnesium oxide reagent grade (high level of purity, Oxide RG in the Tables). In order to limit the wide variability of magnesium oxides availability due to their granulometry (Zervas and Papadopoulos, 1993; Adam et al., 1996), we have grouped magnesium oxides according to their particle size, i.e. granular (Oxide FGG) when 25% of particles were larger than 500µ or powder (Oxide FGP) if less than 500µ. This allows a significant reduction of the standard deviation (21, 8.3 and 5.2 % for all oxides, granular and powder oxides, respectively).

27

Comparison of magnesium sources for ruminants There were in total 18 publications that were suitable for our study. These are listed in Table II.2.

Table II.2: Overview of the experiments used for evaluation of the relative biological value of magnesium sources for ruminants Sources of magnesium

Animal type

Response criteria Added Mg (g/kg)

Number of expts

Ref. no.

Reference

Sulphate Carbonate RG Magnesite (ore) Oxide RG

Sheep Apparent absorption 0.65 1 Mg1 Ammerman et al., 1972

Hydroxide Oxide RG

Steers Apparent absorption 2 1 Mg2 Davenport et al.,1990

Oxide RG Mg phosphate Mg, Ca phosphate

Sheep Apparent absorption 1.7 1 Mg3 Fishwick and Hemingway, 1973

Dolomite Oxide RG

Steers Apparent absorption 1.8 1 Mg4 Gerken and Fontenot, 1967

Sulphate Oxide RG

Cattle Urinary excretion 1.8 1 Mg5 Grings and Males,1988

Oxide FG P Mg phosphate Mg,Ca, Na phosphate

Sheep Apparent absorption 1.5 1 Mg6 Guéguen and Bouchet, 1980

Oxide RG Mg phosphate

Sheep Apparent absorption 2 1 Mg7 Hemingway and Mc Laughlin, 1979

Citrate Hydroxide Mg mica Oxide FG G

Sheep Apparent absorption 2.6 1 Mg8 Hurley et al., 1990

Sulphate Mg mica Oxide RG

Sheep Apparent absorption 1.6 1.9 1.9

1 Mg9 Jackson et al., 1989

Oxide FG P Oxide FG G Mg phosphate Mg,Ca, Na phosphate

Sheep Apparent absorption 2 1 Mg10

Meschy, 1998

Oxide FG P Mg phosphate

Goat Apparent absorption 2 1 Mg11

Meschy et al., 2000

Carbonate Dolomite Oxide RG

Steers Apparent absorption 2 1 Mg12

Moore et al., 1971

28

Table II.2 (continued) Sources of magnesium

Animal type

Response criteria Added Mg (g/kg)

Number of expts

Ref. no

Reference

Hydroxide Oxide FG P Oxide FG G

Sheep Apparent absorption 2 1 Mg13

Parker et al., 1989

Dolomite Oxide RG

Sheep Apparent absorption 1.1 1 Mg14

Rahnema and Fontenot, 1983

Oxide RG Acetate Chloride Citrate Lactate Nitrate Trisilicate

Cattle Urinary excretion 2.6 4.0 1 Mg15

Storry and Rook, 1963

Sulphate Oxide FG P Oxide FG G

Sheep Urinary excretion 0.6 1.4 2 Mg16

Van Ravenswaay et al., 1989

Sulphate Oxide FG P Magnesite

Sheep Urinary excretion 1.4 3 Mg17

Van Ravenswaay et al., 1992

Oxide FG P Oxide FG G

Sheep Apparent absorption 1 1 Mg18

Zervas and Papadopoulos, 1993

Oxide RG: magnesium oxide reagent grade Oxide FG P: magnesium oxide feed grade powder (see the text) Oxide FG G: magnesium oxide feed grade granular (see the text) Comparisons have been summarised in Table II.3. This table shows that there was only one observation for magnesium acetate, magnesium chloride, magnesium hydroxide, magnesium lactate and magnesium nitrate. These salts are not commonly used for supplementation and their relative availability ranges between 90 and 100 compared to the reference source. Raw materials such as magnesites or dolomites show a low availability and the variations observed are probably related to the origin of ores. As expected previously, oxides present variations in terms of relative biological value according to their particle size. Granular and powdered oxides, magnesium carbonate RG and magnesium phosphates can be efficiently used for magnesium supplementation in ruminants. As mentioned before the most frequently used magnesium supplementation source is granular magnesium oxide. In order to justify this choice the commonly used magnesium sources (EMFEMA Guideline) with their “available magnesium”, taking into account both Mg content and the relative bioavailability, are summarised in Table II.4. This table shows that, except for magnesium hydroxide and magnesium oxide (FGP), which have limited use due to their dusty nature, magnesium oxide FGG has the highest “available Mg” per kg product. Also Mg phosphates have a high content of “available Mg”.

29

Table II.3: Summarised results on the relative biological value of magnesium sources for ruminants Magnesium source Mg 1 Mg

2 Mg 3

Mg 4

Mg 5

Mg 6

Mg 7

Mg 8 Mg 9

Mg 10

Mg 11

Mg 12

Mg 13

Mg 14

Mg 15

Mg 16

Mg 17

Mg 18

n Mean SD

No of experiments 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 1 21 Oxide Reagent Grade 100 10

0 100

100

100

100

100

100 100 100 10 100 -

Acetate 98 1 98 - Carbonate Reagent Grade

103 98 2 101 3.5

Chloride 89 1 89 - Citrate 92b 97 2 95 3.5 Dolomite 25 67 37 3 43 21.6 Hydroxide 99 (99)b (99)b 1 99 - Lactate 91 1 91 - Magnesite, magnesium ore

23 47a 2 38 17.0

Mg phosphate 73 92d 79 100d 88d 5 86 10.6 Mg, Ca phosphate 91 1 91 - Mg, Ca, Na phosphate 90d 2 103d 97 9.2 Mica, magnesium silicates

2 9 b 56 67 3 72 18.4

Nitrate 92 1 92 - Oxide Feed Grade Granular

9 5 b 64d 62b 49a 72d 5 61 8.3

Oxide Feed Grade Powder

(84)d (77)d,e (84)d 89b 87a 87a (84)d 5 84 5.2

Sulphate Reagent Grade

98 94 96 (96)a (96)a 3 96 2.0

a Recalculated with magnesium sulphate RG b Recalculated with magnesium hydroxide c Recalculated with magnesium phosphate d Recalculated with magnesium oxide FG P e One source of oxide feed grade powder was used as a reference

30

Table II.4: The relative biological value of different magnesium sources Mg content % Relative biological value Available Mg g/kg Chloride 12 89 106 Hydroxide 36 99 356 Mg phosphate 26 85 221 Mg, Ca phosphate 9 91 82 Mg, Ca, Na phosphate 5 99 49.5 Oxide FGG 51 61 311 Oxide FGP 51 84 428 Sulphate, 7H2O 10 96 96

References Adam, C.L., Hemingway, R.G., Ritchie, N.S., 1996. Influence of manufacturing conditions on the

bioavailability of magnesium in calcined magnesites measured in vivo and in vitro. J. Agric. Sci. 127, 377-385.

Ammerman, C.B., Chicco, C.F., Loggins, P.E., Arrington, L.R., 1972. Availability of different inorganic salts of magnesium to sheep. J. Anim. Sci. 34, 122-126.

Bastien, R.W. , Bradley, J.W., Pennington, B.Z., Ferguson, T.M., 1978. Bone strength and egg characteristics as affected by dietary minerals. Poultry Sci., 57, 1117 (Abstract).

Davenport, G. M., Boling, J. A., Gay, N., 1990. Bioavailability of magnesium in beef cattle fed magnesium oxide or magnesium hydroxide. J. Anim. Sci. 68, 3765-3772.

EMFEMA Guideline : Major, trace and specific minerals in animal feed and animal nutrition. EMFEMA, Brussels. 53 pp.

Fishwick, G., Hemingway, R.G., 1973. Magnesium phosphates as dietary supplements for growing sheep. J. Agric. Sci. 81, 441-444.

Gerken, H.J., Fontenot, J.P., 1967. Availability and utilization of magnesium from dolomitic limestone and magnesium oxide in steers. J. Anim. Sci. 26, 1404-1408.

Giduck, S.A., Fontenot, J.P., 1987. Utilization of magnesium and other macrominerals in sheep supplemented with different readily-fermentable carbohydrates. J. Anim. Sci. 65, 1667-1673.

Greene, L.W., Fontenot, J.P., Webb, K.E., 1983. Site of magnesium and other macromineral absorption in steers fed high levels of potassium. J. Anim. Sci. 57, 503-510.

Grings, E.E., Males, J.R., 1988. Performances, blood and ruminal characteristics of cows receiving monensin and a magnesium supplement. J. Anim. Sci. 66, 566-573.

Guéguen, L., Bouchet, J.P., 1980. Compte rendu d’essai sur l’utilisation digestive de deux phosphates magnésiens chez le mouton en croissance. Station de Recherches de Nutrition INRA France. 6 pp.

Guenter, N., Sell, J.L., 1974. A method for determining “true” availability of magnesium for feedstuffs using chickens. J. Nutr. 104, 1446-1457.

Hemingway, R.G., McLaughlin, A.M., 1979. Retention by sheep of magnesium, phosphorus and fluorine from magnesium and calcium phosphates. Br. Vet. J. 135, 411-415.

Hurley, L.A., Greene, L.W., Byers, F.M., Carstens, G.E., 1990. Site and extent of apparent magnesium absorption by lambs fed different sources of magnesium. J. Anim. Sci. 68, 2181-2187.

Jackson, K.E., Tucker, R.E., Mitchell,G.E., 1989. Bioavailability of magnesium and potassium in lambs fed different magnesium sources. Nutr. Rep. Int. 39, 493-501.

Leroy, J., 1926. Nécessité du magnésium pour la croissance de la souris. Compte Rendu des Séances de la Société de Biologie 94, 341.

McDowell, L.R., 1992. Minerals in animal and human nutrition. Academic Press, San Diego. Meschy, F., 1998. Mesure de la valeur nutritionnelle d'un phosphate triple sur agneau en croissance.

Renc. Rech. Ruminants 5, 250. Meschy, F., Beguin, J.M., Dagorne, R.P., 2000. Valeur nutritionnelle de quelques sources de

magnésium mesurée chez la chèvre laitière. Renc. Rech. Ruminants 7, 210.

31

Moore, W.F., Fontenot, J.P., Tucker, R.E., 1971. Relative effects of different supplemental magnesium sources on apparent digestibility in steers. J. Anim. Sci. 33, 502-506.

Parker, E.R., Ritchie, N.S., Hemingway, R.G., 1989. Dietary availability and rumen solubility of calcined magnesite. Proc. Nutr. Soc. 48, 7A.

Pustovoi, V.V., 1989a. [Biological availability of magnesium in early weaned piglets]. Byulleten’ Vsesoyuznogo Nauchno Issledovatel’skogo Instituta Fiziologii, Biokhimii i Pitaniya Sel’skokhozyaistvennykh Zhivotnykh 96, 31-35.

Pustovoi, V.V., 1989b. [Biological availability of magnesium from various compounds for pigs]. Byulleten’ Vsesoyuznogo Nauchno Issledovatel’skogo Instituta Fiziologii, Biokhimii i Pitaniya Sel’skokhozyaistvennykh Zhivotnykh 97, 32-35.

Rahnema, S.H., Fontenot, J.P., 1983. Effect of supplemented magnesium from magnesium oxide or dolomitic limestone upon digestion and absorption of mineral in sheep. J. Anim. Sci. 57, 1545-1552.

Sell, J.L., 1979. Magnesium nutrition of poultry and swine. In: Proc. of the 2nd Annual International Minerals Conference, St. Peterburg Beach, Florida, IMC, Illinois, 33-55.

Storry, J.E., Rook, J.A., 1963. Magnesium metabolism in the dairy cow. V. Experimental observations with a purified diet low in magnesium. J. Agric. Sci. 61, 167-171.

Thomas, F.M., Potter, B.J., 1976. The site of magnesium absorption from the ruminant stomach. Brit. J. Nutr. 36, 37-45.

Underwood, E.J., 1977. Trace elements in human and animal nutrition. Academic Press, New York. Underwood, E.J., Suttle N.F., 1999. The mineral nutrition of livestock. CABI Publishing, Wallingford

UK. Van Ravenswaay, R.O., Henry, P.R., Ammerman, C.B., Littell, R.C., 1989. Comparison of methods to

determine bioavailability of magnesium in magnesium oxides for ruminants. J. Dairy Sci. 72, 2968-2980.

Van Ravenswaay, R.O., Henry, P.R., Ammerman, C.B., Littell, R.C., 1992. Relative bioavailability of magnesium sources for ruminants as measured by urinary magnesium excretion. Anim. Feed Sci. Technol. 39, 13-26.

Zervas, G., Papadopoulos, G., 1993. The bioavailability of magnesium from different types of calcined magnesites of greek origin. Anim. Prod. 56, 351-358.

32

III. SODIUM BIOAVAILABILITY

General function in the body The importance of dietary salt (NaCl) for animals was known for hundreds of years before the metabolic effects of the compound were studied scientifically. Sodium (and chlorine) maintain osmotic pressure, regulate acid-base equilibrium and control water metabolism in the body. The presence of salt in a feed can contribute to the palatability of that feed, whereas the addition of salt to a feed replete with sodium can lower feed intake (De Waal et al. 1989 cited by Underwood and Suttle, 1999). Sodium uptake from the gut lumen is achieved by coupling to glucose and amino acid uptake and by exchange with hydrogen ions (H+). Sodium and chlorine are highly labile in the body. At the cellular level, the continuous exchange of sodium and potassium via ATP-dependent Na+-K+ pumps provides the basis for glucose and amino acid uptake by co-transport. Much of the sodium that enters the gastrointestinal tract comes from saliva, particularly in ruminants. Sodium, chlorine and potassium are also lost via skin secretions with major differences between species. In non-ruminants sodium is the major cation in sweat and salt concentrations in sweat can reach 4.5 %. Also with milk there is secretion of sodium. Regulation of sodium status in the face of fluctuations in sodium intake is achieved principally by the control of reabsorption in the proximal tubule of the kidney. Signs of sodium deficiency include pica, weight loss, inappetence, increased water consumption, reduced milk yield not accompanied by a decrease in milk sodium concentration, decreased egg production, and decreased sodium concentration in faeces, urine and saliva in ruminants. Due to the widespread practice of ignoring the contribution of sodium and chlorine from primary dietary ingredients and drinking water and adding supplemental salt to diets, there is generally little concern that a sodium or chlorine deficiency will occur. Sodium deficiency is, however, a frequent problem in unsupplemented grazing ruminants. Dietary excesses of sodium and chlorine can disturb body functions (e.g. induce oedema). Unnecessary oversupplementation with sodium and chlorine can cause elevated concentrations of these elements in animal wastes and may cause toxicity to plants and a build-up of these elements in the environment. Little research has been conducted to measure bioavailability of sources of either element due to the widespread commercial availability, palatability, and low cost of sodium chloride.

Use of sodium for livestock According to the type of diet, practical diets require less or more supplementary sodium. Common salt (NaCl) is mostly used for sodium supplementation, although for regulating acid-base balance and to optimise chlorine levels sodium bicarbonate is used. Other sources of sodium are sodium-containing phosphates and sodium sulphate.

SODIUM BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs Response criteria for sodium are ranked along their importance in Table III.1 with differences being made between suboptimal supply and feeding above requirement. Serum sodium content is not included in the list of response criteria.

33

Table III.1: Ranking of importance of various response criteria for assessing the relative biological value of sodium sources in pigs Criterion Suboptimal supply Above requirement Apparent sodium absorption 3 1

3 2 1 - 1 1

True sodium absorption Performance Urine sodium content Table III.1 shows that there are only a few criteria that can be used for evaluation of the bioavailability of sodium sources for pigs of which sodium absorption ranks highest. For our study the reference source is salt (NaCl).

Comparison of sodium sources for pigs There was only one publication that was suitable for our study, which is listed in Table III.2.

Table III.2: Overview of the experiment used for evaluation of the relative biological value of sodium sources in pigs Sources of sodium Animal

type Response criteria

Added Na (g/kg)

No. of expts

Reference

Sodium chloride, Sodium sulphate Sodium bicarbonate

18–63 kg Performance 0.5, 1.0 4 Cromwell et al., 1981

Table III.2 shows that sodium chloride, sodium sulphate and sodium bicarbonate have been compared in the study. Comparison of sodium sources for pigs is summarised in Table III.3. The results with pigs show that compared to sodium in sodium chloride, sodium bicarbonate and sodium sulphate were slightly less available ranging from 96 to 90 %, respectively.

Table III.3: Summarised results on the relative biological value of sodium sources for pigs Reference Cromwell et al., 1981 Number of experiments 4 Sodium chloride 100 Sodium bicarbonate 96 Sodium sulphate 90

SODIUM BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry Response criteria are ranked along their importance in Table III.4 with differences being made between suboptimal supply and feeding above requirement. Serum and urine sodium content are not included in the list of response criteria.

34

Table III.4: Ranking of importance of various response criteria for assessing the relative biological value of sodium sources in poultry Criterion Suboptimal supply Above requirement Apparent sodium absorption 3 1 True sodium absorption 3 2 Performance 1 - Egg production (layers) (1) no

Table III.5: Overview of the experiments used for evaluation of the relative biological value of sodium sources in poultry Sources of sodium Animal

type Response criteria

Added Na (g/kg) Number of expts

Ref. no

Reference

Sodium chloride Sodium bicarbonate

Broiler Performance 0.5, 1.0, 1.5, 2.0 (ref.) 0.72, 1.44, 2.16 (test)

2 Na1 Damron et al., 1986

Sodium chloride Defluorinated phosphates (3 origins) Monosodium phosphate Disodium phosphate

Broiler Performance 0.5, 1.0, 1.5, 2.0, 2.5 (ref.) unknown (test)

2 Na2 Damron et al., 1985

Sodium chloride Defluorinated phosphates

Broiler Performance 0.12, 0.20, 0.35, 0.61 (ref.) 0.20, 0.35, 0.61, 1.05 (test)

1 Na3 Nott and Combs, 1969

Sodium chloride Sodium sulphate

Broiler Performance 1 1 Na4 Ross, 1977

Comparison of sodium sources for poultry Comparison of sodium sources for poultry is summarised in Table III.6. Mainly based on the response criterion growth, the results with chicks show that sodium in sodium chloride, sodium bicarbonate and sodium sulphate is equally available; while in sodium phosphates and defluorinated phosphates it has a 5-10% lower bioavailability.

Table III.6: Summarised results on the relative biological value of sodium sources for poultry Reference Na1 Na2 Na3 Na4 n Mean SD Number of experiments 2 2 1 1 6 Sodium chloride 100 100 100 100 4 100 - Sodium bicarbonate 103 1 103 - Defluorinated phosphates 89 83 2 87 4.2 Monosodium phosphate 93 1 93 - Disodium phosphate 95 1 95 - Sodium sulphate 103 1 103 -

35

SODIUM BIOAVAILABILITY FOR RUMINANTS

There were no publications suitable for the study of sodium bioavailability of ruminants.

References Cromwell, G.L., Stahly, T.S., Moneque, H.J., 1981. Effects of source of sodium and chloride on

performance of pigs. J. Anim. Sci. 1981 (Suppl. 1), 237-238. Damron, B.L., Harms, R.H., Stepp, L.F., 1985. Sodium bioavailability from phosphate sources.

Poultry Sci. 64, 1772-1776. Damron, B.L., Johnson, W.L., Kelly, L.S., 1986. Utilization of sodium from sodium bicarbonate by

broiler chicks. Poultry Sci. 65, 782-785. Nott, H., Combs, G.F., 1969. Availability of sodium in defluorinated rock phosphate. Poultry Sci. 48,

482-485. Ross, E., 1977. Apparent inadequacy of sodium requirement in broiler chickens. Poultry Sci. 56, 1153-

1157. Underwood, E.J., Suttle, N.F., 1999. The mineral nutrition of livestock. 3rd edition. Moredun

Research Institute, Pentland Science Park, Midlothian, UK, p. 397-420.

36

IV. PHOSPHORUS BIOAVAILABILITY

General function in the body Phosphorus is essential for maintenance and normal functioning of the body. It is present in all kinds of organs and tissues. The functions of phosphorus are extremely diverse. They range from structural functions in some tissues (bones, membranes) to a wide variety of regulatory functions in other tissues (NRC, 1980; Jongbloed, 1987; McDowell, 1992; Underwood and Suttle, 1999). About 80% of the body's phosphorus is present in the skeleton. The remaining 20% is contained in nucleotides, such as ATP, nucleic acids, phospholipids, and many other phosphorylated compounds needed for metabolism. Inorganic phosphate is also found in the cell and is important in acid-base balance. Phosphorus is a constituent of some lipid pools in the body and in this capacity plays several important roles, among which is its function in formation of the cell membrane bilayer. Although phosphate is a minor component of the buffering system in the rumen, salivary phosphate contributes to the neutralisation of the short fatty acids that are produced after feeding. In rumen bacteria, phosphorus plays a structural role (rigidity of the cell wall of Gram-positive bacteria through teichoic acid) and a biochemical role (biosynthesis and enzymatic activities). The importance of phosphorus in carbohydrate degradation has been studied for many years. Some studies have underlined that phosphorus is involved specifically in the degradation of cell wall constituents, particularly cellulose (Durand et al., 1989). Phosphorus is also required for bacterial proteosynthesis but to a lesser extent than for cellulolysis. It must be emphasised that the bacterial requirements of phosphorus are much higher than those of the host animal (2 to 2.5 fold) and are mainly satisfied by phosphorus that is recycled through the saliva, which is unique to ruminants. Phosphorus deficiency will cause a reduction in bone mineralisation and thereby impair bone strength. In addition, deficiency lowers feed intake which reduces average daily gain and negatively affects feed conversion ratio. Furthermore, dificiency impairs reproduction efficiency of animals. So, sufficient amounts of phosphorus should be supplied. Animals can generally tolerate excessive intakes of phosphorus by excretion of excess phosphate via the urine. Problems arise through adverse interactions or cumulative effects with other minerals. High phosphorus intakes predispose animals to urinary calculi in sheep (Underwood and Suttle, 1999). Long-term consumption of dietary levels 2 to 3 times the requirement level will cause severe problems due to induced changes in calcium metabolism (NRC, 1980). Absorption and utilisation of phosphorus is mediated by hormonal control (parathyroid hormone, 1,25-dihydroxycholecalciferol), which is primarily based on the concentration in the extracellular fluid. Absorption takes place predominantly in the small intestine, while the contribution of the large intestine does not seem to be significant (Pointillart et al., 1984). The animal tries to maintain a homeostasis in the extracellular fluids, by means of several regulatory mechanisms (via the intestinal wall or kidneys, and for ruminants via saliva). Therefore, large differences in absorption and utilisation of phosphorus can be found, depending on the nutritional status of the animal. Several factors, such as intake level, age of the animal, levels of dietary mineral compounds, e.g., calcium, phytic acid and phytase, magnesium and intestinal pH, all greatly influence phosphorus availability (Jongbloed, 1987).

Use of phosphorus for livestock Practical diets require phosphorus supplementation in order to support optimal growth rate and bone quality of animals. Frequently used phosphate sources include dicalcium phosphate (anhydrous or hydrated), monocalcium phosphate, or mono-dicalcium phosphate. In diets for ruminants it is not always common to supplement with feed phosphates. Levels of supplementation are higher in diets for young animals (broilers and piglets) than for older animals. Lactating animals have higher levels of addition of phosphorus in their diets compared with pregnant animals. The supplementation rate of phosphorus for poultry is mostly higher than for pigs. It is important that sufficient phosphorus should be provided in available form. With regards to poultry, there is disagreement concerning their ability to utilize phytate phosphorus, which represents

37

60-70 % of the phosphorus in products of plant origin. Most studies indicate that the utilization of phytate phosphorus, by young or adult poultry, is negligible at dietary calcium concentrations near the birds’ requirement. Added phytases are able to liberate a significant amount of phytate phosphorus in available form, but supplementation of high quality feed phosphates with good phosphorus bioavailability is still required in poultry diets to meet the phosphorus requirements, which is generally expressed in terms of absorbable phosphorus. For laying hens, a high production rate increases phosphorus requirements more than would be expected from the small amount of phosphorus in the egg (about 120 mg), because the increased phosphorus catabolism from the medultary bone reserve increases endogenous phosphorus losses. Because a lot of different types of feed phosphates can be found in literature, a description of these types, the abbreviation used and, if possible, also the chemical formula is given in Table IV.1. Some of these phosphates are no longer used in practice.

Table IV.1: Types of feed phosphates used in this study, their chemical formula and abbreviations used Description Chemical formula Abbreviation Ammonium polyphosphate (NH4)n+2PnO3n+1 APP Calcium aluminum iron phosphate Undefined CaAlFeP Calcium magnesium sodium phosphate Undefined CaMgNaP Calcium sodium phosphate Undefined DFP Defluorinated phosphate Undefined DFP Dicalcium phosphate CaHPO4 DCP Dicalcium phosphate anhydrate CaHPO4·0H2O DCP·0H2O Dicalcium phosphate hydrate CaHPO4·xH2O DCP·xH2O Dicalcium phosphate dihydrate CaHPO4·2H2O DCP·2H2O Dicalcium phosphate bone CaHPO4·xH2O DCP·bone Dimonocalcium phosphate Undefined DMCP Disodium phosphate Na2HPO4·xH2O DSP Monoammonium phosphate NH4H2PO4 MAP Monocalcium phosphate Ca(H2PO4)2 MCP Monodicalcium phosphate Undefined MDCP Monopotassium phosphate KH2PO4 MPP Monosodium phosphate NaH2PO4 MSP Monosodium phosphate hydrate NaH2PO4·xH2O MSP·xH2O Monosodium phosphate (ref.) NaH2PO4.1H2O

(reagent grade; RG) MSP (ref.)

Phosphoric acid H3PO4 PPA Sodium calcium phosphate (high sodium content) Undefined SCP Tricalcium phosphate Ca3(PO4)2 TCP * if xH2O is used then the number of crystalline molecules of water is not known

38

PHOSPHORUS BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs The ranking of importance for phosphorus of the various criteria is listed in Table IV.2. Although alkaline phosphate or serum phosphate are sometimes used as a criterion, we regard them less suitable for this study.

Table IV.2: Ranking of importance of various response criteria for assessing the relative biological value of phosphorus sources in pigs Criterion Suboptimal supply Above requirement Phosphorus absorption/digestibility (faecal) 5 1 Phosphorus absorption (true) 5 3 Tibia/toe/metatarsal ash or P 3 2 Bone breaking strength 2 2 Phosphorus absorption/digestibility (ileal) - 1 Phosphorus retention (balance or slaughter) 3 1 Performance 2 no Urine phosphorus content no 1 Table IV.2 shows that there are several criteria that can be used for evaluation of the relative biological value of phosphorus sources for pigs of which phosphorus absorption ranks highest. Although alkaline phosphate or serum phosphate are sometimes used as a criterion, we regard them less suitable for this study. For our study the reference source is monosodium phosphate monohydrate (NaH2PO4·1H2O) RG. Because we know now that the chemical composition of feed phosphates is important it has been decided that only experiments that have been published after 1979 will be used. Prior to this only a limited description of the feed phosphates used has been presented. In contrast to other mineral sources, not only relative values are presented but also apparent digestibility values. This is done because in practice it has become more common to use apparent digestibility figures to express the nutritive value of feed phosphates.

Comparison of phosphorus sources for pigs There were in total 12 publications that were suitable for our study. Part of the reports by Jongbloed and Mulder (1985) and Peet et al. (1988) have been published by Dellaert et al. (1990). The publications used are listed in Table IV.3, which shows that in all experiments listed, digestibility of phosphorus was the minimum criterion used. Furthermore, in addition to phosphorus digestibility, performance and bone parameters had been used in some experiments. Therefore, we have presented a separate table showing the apparent digestibility of phosphorus of all feed phosphates (Table IV.5). Furthermore, in addition to phosphorus digestibility, performance and bone parameters had been used in some experiments. A summary of the comparisons with regard to relative bioavailability of phosphorus is presented in Table IV.4. In this table the same feed phosphates that have a different origin or brand names are indicated with A, B, C, etc. However, it is not clear for all phosphate sources what is the origin: in that case no further letter is added. Table IV.4 shows that several observations were available for several types and origins of DCP·0H2O, DCP·2H2O, MCP, and MSP, but few from DFP and MDCP. A difficulty is to convert the results of experiments in which no MSP (ref) was used. In order to harmonise this we also added in Table IV.4 the conversion factor to do this. This figure was in most cases derived from the relative value of monosodium phosphate (ref) to monocalcium phosphate⋅1H2O (A). Table IV.4 shows that in some cases, even for the same brand of feed phosphate, large differences

39

exist between experiments with regard to their relative value compared to the reference phosphate. Those differences may largely be attributed to the experimental design. It can also be seen in this table that the relative bioavailability of monocalcium phosphates is higher than that of dicalcium phosphates, while the relative bioavailability of monodicalcium phosphates is between these. This also applies to the apparent digestibility of phosphorus.

Table IV.3: Overview of the experiments used for evaluation of the relative biological value of phosphorus sources for pigs Sources of phosphorus

Animal type

Response criteria

Added P (g/kg)

Number of expts

Ref. no

Reference

MCP (2 origins) DFP (2 batches)

12-28 kg Performance Digestibility

2.0 1 P1 Beers et al., 1990

MCP DCP·2H2O DCP·0H2O MDCP

10-25 kg Digestibility 2.0 1 P2 Dellaert et al., 1990Experiment 3

MCP 30-40 kg Digestibility 1.4 1 P3 Düngelhoef et al., 1994

MCP DCP·0H2O DCP·2H2O

30-40 kg Digestibility 1.0 and 2.0

1 P4 Eeckhout and De Paepe, 1996

MCP DCP·0H2O (2 origins) DCP·2H2O (2 origins)

30-50 kg Digestibility 0.8 and 1.6

2 P5 Grimbergen et al., 1985

MSP (ref) MCP (3 origins) DCP·0H2O

10-26 kg Digestibility Bone ash % Breaking strength

0.6,1.2 and 1.8

1 P6 Dellaert et al., 1990Experiment 1

DSP·7H2O 26-110 kg Performance Digestibility Bone ash %

1.3 1 P7 Jongbloed, 1987

MSP (ref) MSP MCP (3 origins) DCP·0H2O (2 origins) DCP·2H2O DFP CaMgNaP

12-32 kg Performance Digestibility

1.8, 1.9 and 2.0

1 P8 Kemme et al., 1994a,b,c

MSP (ref.) MCP (2 origins) DCP·2H2O DCP·0H2O

10-26 kg Performance Digestibility Bone ash % Breaking strength

1.2 and 2.2

1 P9 Peet and Olijslagers, 1988

MSP MCP MDCP (2 origins) DCP·2H2O (2 origins) DFP

40-50 kg Digestibility 0.2, 1.1, 1.9, 2.7 and 3.4

1 P10 Poulsen, 1995

MSP (ref.) MCP DCP·0H2O

30-40 kg Digestibility 1.5 1 P11 Rodehutscord et al., 1994

40

Table IV.4: Overview of the results of experiments used for evaluation of the relative biological value of phosphorus sources in pigs Reference P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 n Mean SDNumber of experiments 1 1 1 1 2 1 1 1 1 1 1 12 Monosodium phosphate reference 100 100 100 100 100 - Calcium magnesium sodium phosphate 93 1 93 - Dicalcium phosphate·0H2O A 71a 3 65a 70a 69 3.5 Dicalcium phosphate·0H2O B 68a 77 81 86 91 5 80 8.7 Dicalcium phosphate·0H2O C 77 1 77 - Dicalcium phosphate·2H2O A 78a 75a 78a 86 73c 5 78 5.1 Dicalcium phosphate·2H2O C 86 64c 2 75 15.1 Defluorinated phosphate A 62c 1 62 - Defluorinated phosphate B 92a; 93a 2 90 1.4 Disodium phosphate (100)b 1 100 - Monocalcium phosphate (94)a 95 1 95 - Monocalcium phosphate A (94)a (94)a (94)a (94)a 82 106 2 94 16.6 Monocalcium phosphate B1 97 1 97 4.2 Monocalcium phosphate B2 82a 87 103 79c 4 88 10.6 Monocalcium phosphate C 97 1 97 - Monocalcium phosphate D 91 96 2 93 3.2 Mono-dicalcium phosphate A 84a 89c 2 86 3.7 Mono-dicalcium phosphate B 80c 1 80 - Monosodium phosphate 98 (98)c 2 98 - a recalculated with monocalcium phosphate A as a reference b the relative biological value of disodium phosphate is equal to the monosodium phosphate reference c recalculated with monosodium phosphate as a reference

41

Table IV.5: Overview of the results of experiments used for evaluation of the apparent digestibility of phosphorus sources in pigs Reference P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 n Mean SDNumber of experiments 1 1 1 1 2 1 1 1 1 1 1 Monosodium phosphate reference 93 89 88 96 91 4.0 Calcium magnesium sodium phosphate 81 1 81 - Dicalcium phosphate·0H2O A 63 63 64 3 63 0.6 Dicalcium phosphate·0H2O B 62 64 66 66 87 5 68 10.2 Dicalcium phosphate·0H2O C 61 1 61 - Dicalcium phosphate·2H2O A 69 73 71 69 59 5 68 5.4 Dicalcium phosphate·2H2O C 71 52 2 61 13.2 Defluorinated phosphate A 50 1 50 - Defluorinated phosphate B 86; 84 2 85 1.4 Disodium phosphate 91 1 91 - Monocalcium phosphate 88 91 2 89 2.5 Monocalcium phosphate A 84 83 92 86 81 82 6 85 3.8 Monocalcium phosphate B1 84 1 84 - Monocalcium phosphate B2 72 77 76 64 4 72 5.9 Monocalcium phosphate C 84 1 84 - Monocalcium phosphate D 83 83 2 83 0.3 Mono-dicalcium phosphate A 74 72 2 73 1.4 Mono-dicalcium phosphate B 65 1 65 - Monosodium phosphate 87 79 2 83 5.9

42

PHOSPHORUS BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry The sequence of the response criteria is listed in Table IV.6. Different weights are used if animals are fed phosphorus at a suboptimal level or above requirement. Alkaline phosphatase, serum/plasma phosphorus and urine phosphorus are criteria which are not suitable for determining phosphorus bioavailability. They are not mentioned in Table IV.6. From Table IV.7 it may be observed that most studies on phosphorus availability are based on sensitive response criteria like bone ash and bone breaking strength, followed by growth in trials with chicks and turkeys. More recently, apparent phosphorus absorption and phosphorus retention have also been used. The first category of response criteria provided only relative values of bioavailability related to a reference source, while the second category generates absolute values of phosphorus retention or phosphorus absorption, which can be recalculated to relative values.

Table IV.6: Ranking of importance of various response criteria for assessing the relative biological value of phosphorus sources in poultry Criterion Suboptimal supply Above requirement P absorption/digestibility (faecal) 4 1 P absorption (true) 5 3 Tibia/toe/metatarsal ash or P 3 2 Bone breaking strength 2 2 P absorption/digestibility (ileal) 5 1 P retention (balance or slaughter) 3 1 Performance 2 no

Table IV.7: Overview of the experiments used for evaluation of the relative biological value of phosphorus sources in poultry Sources of phosphorus

Animal type

Response criteria

Added P (g/kg)

Number of expts

Ref. no

Reference

MCP⋅H2O DCP (4 origins) MDCP (2 origins) MCP (2 origins) DFP (5 origins)

Turkey Tibia ash

(1.1), 2.1, (5.6)

1 P1 Akpe et al., 1987

MSP⋅H2O DFP (5 origins)

Broiler Tibia ash Bone strength

0.7, 1.4 (ref)1.4 (test)

1 P2 Burnell et al., 1990

MPP DCP

Broiler Tibia ash Bone strength

(0.5), 1.0 1 P3 Chung and Baker, 1990

MSP⋅H2O DFP (5 origins)

Broiler Tibia ash Bone strength

0.5, 1.0 1 P4 Coffey et al., 1994

MCP⋅H2O DCP⋅2H2O (3 origins) DCP⋅0H2O DCP bone

Broiler P retention Toe ash

0.6, 1.2, 1.8, 2.4, 3.0

3 P5 De Groote and Huyghebaert, 1997

43

Table IV.7 (continued) Sources of phosphorus

Animal type

Response criteria

Added P (g/kg)

Number of expts

Ref. no

Reference

DCP⋅2H2O DCP bone

Broiler Tibia ash Toe ash

1.0, 1.5, 2.0, 2.5, 3.5, 4.5

1 P6 De Groote et al., 1991

DCP⋅2H2O DCP (3 origins) DFP MAP

Broiler Performance Tibia ash Bone strength

0.8, 1.6 1 P7 Fernandes et al., 1999

DCP⋅2H2O DCP⋅0H2O

Turkey Tibia ash 1.0, 1.5, 2.0, 2.5

1 P8 Grimbergen et al., 1985

DCP⋅2H2O SCP DCP bone

Broiler Tibia ash Tibia P Bone strength

0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1

1 P9 Huyghebaert and De Groote, 1982

DSP MCP (2 origins) DCP⋅2H2O (2 origins) DCP⋅0H2O (2 origins) DFP (2 origins) CaMgNaP MSP CaAlFeP

Broiler Tibia ash Tibia P Bone strength

0.4, 0.7, 1.1, 1.4, 1.8, 2.1, 2.5, 2.8 0.4, 1.0, 1.5, 2.1, 2.6

2 P10 Huyghebaert et al., 1980

TCP DCP⋅2H2O DFP APP

Broiler Tibia ash 1, 2, 3, 4, 5 (reference) 1, 2, 3 (test)

2 P11 Jensen and Edwards, 1980

DCP⋅2H2O DCP⋅0H2O MCP⋅H2O MDCP

Broiler Apparent ileal absorption Multiple*

1.0, 1.5, 2.0, 2.5, 3.5, 4.5

1 P12 Ketels and De Groote, 1988

DCP (7 origins) DCP⋅2H2O

Broiler Performance Tibia ash Bone strength

1, 2, 3 1 P13 Lima et al., 1997

DCP⋅0H2O MCP DCP DFP (2 origins)

Broiler Tibia ash 0.5, 0.9, 1.3, 1.7, 2.1 (reference) 0.9, 1.7 (test)

2 P14 Nelson et al., 1990

DCP⋅2H2O MCP (3 origins) DCP (3 origins) DFP (7 origins)

Turkey Performance Toe ash

0.9, 1.9, 2.8, 4.6

2 P15 Potchanakorn and Potter, 1987

MCP DCP (2 origins) DFP (4 origins) Curacao P

Turkey Performance Toe ash

0.9, 1.8, 2.7 1 P16 Potter, 1988

44

Table IV.7 (continued) Sources of phosphorus

Animal type

Response criteria

Added P (g/kg)

Number of expts

Ref. no

Reference

DCP⋅2H2O MCP⋅H2O DCP⋅0H2O DCP⋅xH2O DFP MCP PPA MDCP

Broiler Performance Toe ash

0.5, 0.8, 1.2, 1.7, 2.3, 3.2, (4.4, 6.0)

1 P17 Potter et al., 1995

DCP⋅2H2O MCP⋅H2O DCP⋅0H2O DCP⋅xH2O DFP MCP PPA MDCP

Broiler Tibia ash Bone strength

0.5, 0.8, 1.2, 1.7, 2.3, 3.2, (4.4, 6.6)

1 P18 Ravindran et al., 1995

DCP⋅xH2O APP

Broiler Performance Tibia ash Toe ash Bone strength

3, 4 2 P19 Singh and Nagra, 1994

DCP⋅2H2O MDCP (9 origins) DMCP (12 origins) DFP (14 origins)

Turkey Multiple* 1.8, 2.4, 3.6 10 P20 Sullivan et al., 1992

DCP⋅2H2O MDCP DCP bone DMCP (3 origins) DFP (3 origins)

Turkey Multiple* 1.8, 2.4, 3.6 2 P21 Sullivan et al., 1994

MCP⋅0H2O (2 origins) DCP⋅2H2O (2 origins) CSP (2 origins) MCP⋅H2O MDCP (2 origins) DCP⋅0H2O (2 origins) MSP⋅H2O (3 origins)

Broiler P retention unknown 1 P22 Van der Klis and Versteegh, 1993

MDCP (8 origins) DCP (20 origins) DFP (20 origins) MCP⋅H2O

Turkey Tibia ash

1.1, 2.1, 3.1, 5.6 (ref.) 2.1 (test)

3 P23 Waibel et al., 1984

*tibia ash, toe ash and bone strength *BV=body weight gain/10+tibia ash %+10(gain/feed ratio)

45

Comparison of phosphorus sources for poultry The comparative ranking of mineral phosphorus sources is summarised in Table IV.8 for broilers and in Table IV.9 for turkeys. Compared to the reference source hydrated monosodium phosphate (=100), the following conclusions for broilers can be derived: The water-soluble sodium-, potassium-, ammonium- and monocalcium phosphates, together with the Ca-Mg-Na-phosphate and dicalcium phosphate⋅2H2O have the highest relative bioavailability values for P between 84 and 95%. Mono- and dicalcium phosphates, dicalcium phosphates⋅xH2O (unknown moles of water), dicalcium phosphates (bone) and phosphoric acid are intermediary, with relative values between 80 and 83 %. Finally, the P in defluorinated phosphates, dicalcium phosphates anhydrates and tricalcium phosphates are less available with values between 69 and 79 %. For turkeys no studies with monosodium phosphate⋅H2O are available. Using hydrated dicalcium phosphate as the reference it appears that monocalcium phosphate, bone dicalcium phosphate and mono/di-Ca-phosphate have approximately the same bioavailability. Defluorinated phosphates, dicalcium phosphate anhydrate and Curacao phosphate show noticeably lower availability values. Table IV.10 summarises the results of studies with broilers, where absolute values of P-retention or apparent ileal digestibility have been determined.

Table IV.10: Overview of the results of experiments with broilers, evaluated for P-retention and apparent ileal absorption (absolute values) Reference P5 P12 P22 Retention % Apparent ileal

absorption (%) Retention (%)

Crumbled diet Pelleted diet Monosodium phosphate⋅H2O

92

Dicalcium phosphate⋅2H2O

82 74 73 77

Dicalcium phosphate⋅0H2O

64 67 55

Monocalcium phosphate⋅H2O

86 78 71 84

Monocalcium phosphate⋅0H2O

81

Mono-dicalcium phosphate⋅xH2O

67 79

Calcium sodium phosphate

59

Dicalcium phosphate bone

80

46

Table IV.8: Summarised results on the relative biological value of phosphorus sources for broilers Reference P2 P3 P4 P5 P6 P7 P9 P10 P11 P12 P13 P14 P17 P18 P19 P22 n Mean SDNumber of experiments

1 1 1 3 1 1 1 2 2 1 1 2 1 1 2 1 22

MSP⋅H2O 100 100 100 3 100 - MSP 84d 1 84 - DSP 188d 88 - DCP⋅2H2O 87a (86)d (86)d (86)d (86)d (86)d (86)d (86)d (86)d (86)d 85 2 86 1.3 DCP⋅0H2O 79a 76d 74d (76)b 76d 91d 60 6 76 9.8 DCP bone 85a 379d 80d 82 2.9 DCP⋅xH2O 5(80)c 77d 83d 77b 82d 84d (80)c 80 3.4 MCP⋅H2O 84 (91)a d 95d 94d 91 4 91 5.0 MCP⋅0H2O 8 18 88 - MCP 483d 74b 89d 101d 85 11.3TCP 169d 69 - MDCP 77 d 78d 79d 86 4 80 4.1 CSP 6 14 64 - DFP 95 83 77d 79d 85d 77d 968b 71d 84d 79 8.6 APP 6 282d 7 c 79 4.0 MAP 1 94d 94 - PPA 79 2d 87d 83 5.5 MPP 192c 92 - CaMgNaP 190d 90 - CaAlFeP 112d 12 - a recalculated with MCP⋅H2O as a reference b recalculated with DCP⋅0H2O as a reference c recalculated with DCP⋅xH2O as a reference d recalculated using the relative value of DCP⋅2H2O against MSP⋅H2O of 85 (Van der Klis and Versteegh, 1993)

47

Table IV.9: Summarised results on the relative biological value of phosphorus sources for turkeys Reference P1 P8 P15 P16 P20 P21 P23 n Mean SD Number of experiments 1 2 1 10 2 2 19 DCP⋅2H O 2 100 100 100 100 4 100 - DCP bone 99 1 99 -

2 71 1 71 - MCP⋅H O 2 97 b 109b 2 104 8.2 MCP 92 b 92 (92) 2 92 0.2 DFP 71 b 73 77

1

DCP⋅0H O

c

c 91 87 76 b 6 82 8.0 MDCP b 98 96 95 a,b 3 97 DCP 85 b 81 90 c 88 b 86 4.1 Curacao phosphate 48 c 1 48 DMCP 94 95 2 94 0.6 a One source of MDCP (FG) was used as reference b recalculated using the relative value of MDCP against DCP⋅2H2O of 97 (cf. weighted mean of MDCP) c recalculated using the relative value of MCP against DCP⋅2H2O of 92 (cf. weighted mean of MCP)

PHOSPHORUS BIOAVAILABILITY IN RUMINANTS

(97) 1.1 4

-

Sequence of important response criteria and evaluation methods used for ruminants As described earlier, the sequence of response criteria depends on whether the animals are fed suboptimal or if they are fed above their requirement. The ranking of importance for phosphorus of the various criteria is listed in Table IV.11.

Table IV.11: Ranking of importance of various response criteria for assessing the relative biological value of phosphorus sources in ruminants Criterion Suboptimal supply Above requirement Measured true absorption 5 4 Calculated true absorption 4 3 Apparent absorption 3 2

3 2 Bone parameters 2 1 Phosphorus balance

Table IV.11 shows that there are only few criteria that can be used for evaluation of phosphorus sources in ruminants. In our data set, seven observations were based on true absorption (measured or calculated on the basis of theoretical value of phosphorus faecal endogenous loss), two on phosphorus balance and two on bone parameters. For this ruminant section of our study, the reference source is dicalcium phosphate due to the scarcity of publication where monosodium phosphate was a source under investigation: Tillman and Brethour, 1958 and Guéguen et al., 1976, moreover only the latter was a comparison study.

Comparison of phosphorus sources for ruminants There were in total 10 publications involving 10 experiments that were suitable for our study. These are listed in Table IV.12.

48

Table IV.12: Overview of the experiments used for evaluation of the relative biological value of phosphorus sources in ruminants Sources of phosphorus Animal

type Response criteria

Added P (g/kg)

Number of expts

Ref. no

Reference

Dicalcium phosphate anhydrous Diammonium phosphate

Sheep Phosphorus balance

3 1 P1 Godoy et al., 1995

Dicalcium phosphate anhydrous Calcium, magnesium, sodium phosphate Magnesium phosphate

Sheep True absorption 1.8 1 P2 Guéguen and Bouchet, 1980

Dicalcium phosphate dihydrate Monocalcium phosphate Diammonium phosphate

Sheep True absorption 1.6 1 P3 Guéguen and Durand, 1976

Monosodium phosphate Monoammonium phosphate

Sheep True absorption 2 1 P4 Guéguen et al., 1976

Dicalcium phosphate anhydrous Magnesium phosphate Monocalcium phosphate

Sheep Apparent absorption

1.75 1 P5 Hemingway and McLaughlin, 1979

Dicalcium phosphate anhydrous Monoammonium phosphate

Calves Bone parameters 1.4 1 P6 Jackson et al., 1988

Calcium, magnesium, sodium phosphate Magnesium phosphate Monodicalcium phosphate

Sheep Calculated true absorption

3 1 P7 Meschy, 1998

Dicalcium phosphate anhydrous Dicalcium phosphate dihydrate Magnesium phosphate Monocalcium phosphate Monodicalcium phosphate

Goats Calculated true absorption

4.4 1 P8 Meschy et al., 2000

Dicalcium phosphate anhydrous Diammonium phosphate

Calves Bone parameters 0.8 – 1.5 2 P9 Teh et al., 1982

Dicalcium phosphate anhydrous Monoammonium phosphate

Sheep True absorption 3 1 P10 Vitti et al., 1992

49

In contrast to phosphorus availability assessment for pigs and poultry, for ruminants we used some papers that have been published before 1980, because of the scarcity of recent publications. Some phosphates are not used in the European Union either because of their low availability or their high content of fluorine (higher than that allowed by EU regulations). Studies on these phosphates have not been included in this review. We combined anhydrous and hydrated dicalcium phosphates because no significant differences were found between these sources in ruminants in the literature. Comparisons have been summarised in Table IV.13. This table shows that differences between phosphates under investigation are minor. This can be explained by the fact we discarded very low availability phosphates (see above) as some untreated rock phosphates. Table IV.14 indicates absolute values of true absorption of phosphorus from sources under investigation. These values come from 32P measurements or calculations with a theoretical value of faecal endogenous loss of P (references P7 and P8).

Table IV.13: Summarised results on the relative biological value of phosphorus sources for ruminants

Reference P1 P2 P3 P4 P5 P6 P7 P8 P9 P 10

n Mean SD

Number of experiments 1 1 1 1 1 1 1 1 2 1 Dicalcium phosphate 10

0 100 10

0 10

0 100

100 100

100

8 100 -

Calcium magnesium sodium phosphate

103 102b 2 103 0.7

Diammonium phosphate 90 85 111

3 97 13.8

Magnesium phosphate 105 97 (101)b 100 3 101 4.0 Monoammonium phosphate (99)a 10

2 95 2 99 4.9

Monocalcium phosphate 96 87 102 3 95 7.5 Monodicalcium phosphate 93b 101 2 97 5.7 Monosodium phosphate 99a 1 99 -

a Recalculated with monoammonium phosphate b Recalculated with magnesium phosphate

Table IV.14: Absolute values of true digestibility % (measured or calculated) of phosphorus sources in ruminants P source n Mean SD Reference Calcium magnesium sodium phosphate

2 71.5 1.1 Guéguen and Bouchet, 1980; Meschy, 1998

Diammonium phosphate 1 63.0 - Guéguen and Durand, 1976 Dicalcium phosphate 4 67.6 5.3 Guéguen and Bouchet, 1980;

Guéguen and Durand, 1976, Meschy et al., 2000; Vitti et al., 1992

Magnesium phosphate 3 69.4 3.5 Guéguen and Bouchet, 1980; Meschy, 1998; Meschy et al., 2000

Monoammonium phosphate 2 65.6 4.8 Guéguen et al., 1976; Vitti et al., 1992

Monocalcium phosphate 2 69.5 3.3 Guéguen and Durand, 1976; Meschy et al., 2000

Monodicalcium phosphate 2 66.0 0 Meschy, 1998; Meschy et al., 2000 Monosodium phosphate 2 75.0 18.8 Guéguen et al., 1976; Tillman. and

Brethour, 1958

50

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Sullivan, T.W., Douglas, J.H., Gonzales, N.J., Bond, P.L. jr, 1992. Correlation of biological value of feed phosphates with their solubility in water, dilute hydrogen chloride, dilute citric acid, and neutral ammonium citrate. Poultry Sci. 71, 2065–2074.

Teh, T.H., Hemken, R.W., Bull, L.S., 1982. Evaluation of urea ammonium polyphosphate as a phosphorus source for dairy calves. J. Anim. Sci. 55, 174-179.

Tillman, A.D., Brethour, J.R., 1958. Ruminant utilization of sodium meta-, ortho- and polyphosphates.

52

J. Anim. Sci. 17, 792-796. Underwood, E.J., Suttle, N.F., 1999. The Mineral Nutrition of Livestock, 3rd edition. CABI

Publishing, Wallingford, United Kingdom. Van der Klis, J.D., Versteegh, H.A.J., 1993. De opneembaarheid van fosfor in grondstoffen bij

slachtkuikens. N & P in de voeding van éénmagige landbouwhuisdieren in relatie tot de milieu-problematiek - Reeks nr. 25 - CVB , 117-124.

Vitti, D.M.S.S., Abdalla, A.L., Meirelles, C.F., Dasilva, J.C., Louvandini, H., 1992. True phosphorus absorption from different sources for sheep, using radiophosphorus (P-32). Pesqu. Agropec. Bras. 27, 1405-1408.

Waibel, P.E., Nahorniak, N.A., Dziuk, H.E., Walser, M.M., Olson, W.G., 1984. Bioavailability of phosphorus in commercial phosphate supplements for turkeys. Poultry Sci. 63, 730–737.

53

TRACE MINERALS

I. Cobalt bioavailability

II. Copper bioavailability

III. Iron bioavailability

IV. Iodine bioavailability

V. Manganese bioavailability

VI. Molybdenum bioavailability

VII. Selenium bioavailability

VIII. Zinc bioavailability

54

I. COBALT BIOAVAILABILITY

General function in the body

Cobalt has been shown to be essential for ruminants in the 1930's in Australia and New Zealand as a component of vitamin B12 (Underwood and Filmer, 1935). Vitamin B12, also known as cobalamin contains about 4.5% cobalt. In ruminants cobalt deficiency leads to "wasting diseases" as a result of inadequate synthesis of vitamin B12 from dietary cobalt. It must be underlined that the efficiency of vitamin B12 conversion from dietary cobalt is very low (3-4%) when cobalt intake is adequate and higher (13 ± 5%) in cobalt deficient animals (Smith and Marston, 1970). This low conversion rate could be explained by rapid cobalt uptake by rumen micro-organisms (McDowell, 1992). Synthesis and metabolism of vitamin B12 are described in detail in the review by Smith (1987). Experimental results concerning a specific role of cobalt in rumen fibre digestion are inconsistent. There was an improvement in rumen bacterial fibre degrading activity in some studies as reviewed by Paragon (1993) and no effect in others (Hussein et al, 1994). Cobalt is distributed throughout the body with high concentrations in liver, bone and kidney (Underwood and Suttle, 1999).

Use of cobalt for livestock With regard to the animal requirements, cobalt deficiency is recognised throughout the world particularly in grazing animals, meaning that almost all natural ruminant diets must be supplemented with cobalt. In monogastric animals, there is no clear evidence that cobalt deficiency exists separately from vitamin B12 deficiency. For this reason, monogastrics must satisfy their vitamin B12 requirements by dietary supply. In ruminants, sources of cobalt must be partially soluble in the rumen. Cobalt is most commonly supplemented as cobalt sulphate, but some recent studies demonstrated its severe toxicity to the animals, which may justify the use of other sources for cobalt supplementation. For the study of cobalt bioavailability for pigs, there were only publications available from East-Europe of which details could not be obtained. No differences were observed in performance of growing pigs from 20 to 100 kg when fed cobalt dichloride or cobalt sulphate (Kunev and Pazardzhiev, 1975). There were no appropriate publications for the study of cobalt bioavailability for poultry.

COBALT BIOAVAILABILITY FOR PIGS

There were no publications suitable for the study of cobalt bioavailability of pigs.

COBALT BIOAVAILABILITY FOR POULTRY

There were no publications suitable for the study of cobalt bioavailability of poultry.

55

COBALT BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants As described earlier, the sequence of response criteria depends on whether the animals are fed suboptimal levels of cobalt or if they are fed above their cobalt requirement. The ranking of importance for cobalt of the various criteria is listed in Table I.1.

Table I.1: Ranking of importance of various response criteria for assessing the relative biological value of cobalt sources in ruminants Criterion Suboptimal supply Above requirement Cobalt absorption 2 1 Liver accumulation of cobalt 3 2 Kidney accumulation of cobalt 2 1 Vit B12 synthesis 4 2

Sources of cobalt

Table I.1 shows that there are only few criteria that can be used for evaluation of cobalt sources in ruminants of which vitamin B12 synthesis is the most important one. For our study the reference source is cobalt sulphate heptahydrate (CoSO4.7H2O) RG. Tissues cobalt concentrations have been transformed to 10log in order to maintain the RBV variation within physiological limits.

Comparison of cobalt sources for ruminants There were in total three publications that were suitable for our study. These are listed in Table I.2.

Table I.2: Overview of the experiments used for evaluation of the relative biological value of cobalt sources in ruminants

Animal type

Response criteria Added Co(mg/kg)

Number of expts

Ref. no

Reference

Cobalt sulphate RG Cobalt carbonate RG Cobalt carbonate FG Cobaltic oxide RG (Co3O4) Cobaltic oxide FG Cobalt oxide by-product (CoO)

Sheep Liver accumulation 40 1 Co1 Ammerman et al, 1982

Cobalt sulphate RG Co glucoheptonate

Sheep Liver accumulation Kidney accumulation

20, 40, 60 3 Co2 Kawashima et al, 1989

Cobalt sulphate RG Cobaltic oxide RG Cobaltic oxide FG Cobalt carbonate RG Cobalt carbonate FG Cobalt glucoheptonate

Sheep Liver accumulation Kidney accumulationVitamin B12 synthesis

20, 40, 60 3 Co3 Kawashima et al, 1997

56

Comparisons have been summarised in Table I.3, which shows that two observations were available for each salt except for cobalt oxide by-product. Moreover, this product was not well defined in the paper. Cobalt oxides had a markedly lower availability than the reference source. The relatively high availability of cobalt carbonate or cobalt glucoheptonate allows an efficient cobalt supplementation in ruminants

Table I.3: Summarised results on the relative biological value of cobalt sources for ruminants Reference Co1 Co2 Co3 n Mean SD Number of experiments 1 3 3 Cobalt sulphate heptahydrate 100 100 100 3 100 - Cobaltic oxide (Co3O4) RG 39 31 2 34 5.7 Cobaltic oxide (Co3O4) FG 65 68 2 67 2.1 Cobalt oxide by-product (CoO) 81 1 81 - Cobalt carbonate RG 99 102 2 101 2.1 Cobalt carbonate FG 106 106 2 106 0 Cobalt glucoheptonate 86 98 2 92 8.5

References Ammerman, C.B., Henry, P.R., Loggins, P.R., 1982. Cobalt bioavailability in sheep. J. Anim. Sci. 55,

403 [Abstract]. Henry, P.R., Little, R.C., Ammerman, C.B., 1997. Bioavailability of cobalt sources for ruminant. 1.

Effects of time and dietary cobalt concentration on tissue cobalt concentration. Nutr. Res. 17, 947-955.

Hussein, H.S., Fahey, G.C., Wolf, B.W., Berger, L.L., 1994. Effects of cobalt on in vitro fiber digestion of forages and by-products containing fiber. J. Dairy Sci. 77, 3432-3440.

Kawashima, T., Henry, P.R., Ammerman, C.B., Little, R.C., Price, J., 1997. Bioavailability of cobalt sources for ruminant. 2. Estimation of the relative value of reagent grade and feed grade cobalt sources from tissue cobalt accumulation and vitamin B12 concentrations. Nutr. Res. 17, 957-974.

Paragon, B.M., 1993. Readjustment of the supply of cobalt in ruminants. A critical approach. Rec. Méd. Vét. 169, 759-761.

Smith, R.M., Marston, H.R., 1970. Production, absorption, distribution and excretion of vitamin B12 in sheep. Brit. J. Nutr. 24, 857-877.

Kawashima, T., Ammerman, C.B., Henry, P.R., 1989. Tissue uptake of cobalt from glucoheptonate in sheep. J. Anim. Sci. 67, 503 [abstract].

Kunev, M., Pazardzhiev, A., 1975. [Effect of a supplement of cobalt in the feed for fattening pigs]. Zhivotnovudni Nauki 12, 61-65.

McDowell, L.R., 1992. Minerals in animal and human nutrition. Academic Press, San Diego.

Smith, R.M., 1987. Cobalt In: Trace element in human and animal nutrition. Academic Press, New York.

Underwood, E. J., Filmer, J. F., 1935. The determination of the biologically potent element (cobalt) in limonite. Aust. Vet. J. 11, 84-92.

Underwood, E. J., Suttle N.F., 1999. The mineral nutrition of livestock. CABI Publishing, Wallingford UK.

57

II. COPPER BIOAVALABILITY

General function in the body Copper is an essential mineral for all living organisms, but in contrast to zinc, it exhibits redox chemistry. Copper deficiency has detrimental effects on numerous organs and tissues, including the haematopoietic system, cardiovascular system, central nervous system, and the integumentem. Copper is an essential component of several metalloenzymes whose functional deficits give rise to the pathology associated with copper deficiency. Some of these enzymes can be directly associated with specific pathology, e.g., tyrosinase and lack of pigmentation, lysyl oxidase, and cardiovascular defects. Other copper metalloenzymes, such as cytochrome c oxidase, copper/zinc-superoxide dismutase and dopamine (-monoxygenase), play key roles in metabolism (O’Dell, 1976; McDowell, 1992; Ammerman et al, 1995). The anaemia of copper deficiency results from impairment of iron metabolism and is particularly manifested when dietary iron is marginal, but the specific copper-dependent enzyme or protein involved in iron metabolism is unclear. The metabolic functions of copper, which have been described, serve as indices of copper status and of copper bioavailability (Underwood and Suttle, 1999). Copper deficiency is a serious problem for grazing ruminants in many countries of the world. This is due to both low concentrations of the element in forage as well as to elevated amounts of molybdenum and sulphur, which interfere with copper utilisation. Despite the fact that most practical diets contain adequate copper for pigs and poultry, the element is still generally supplemented to complete diets for these species. Adult cattle copper tolerance level is 100 mg/kg and horses appear to be more resistant to copper intoxicosis than cattle (NRC, 1980). Copper toxicity has been described for sheep in particular. Hartmans (1975) indicated that diets containing 15 mg copper/kg of air-dry matter can cause copper poisoning, but NRC (1980) indicates a level of 25 mg/kg diet. Pigs, however are highly tolerant to copper overload (500 mg/kg of diet; NRC, 1980). For chickens and layers a toxic level of 300 to 500 mg/kg has been described (NRC, 1980). Copper is absorbed in the upper gastrointestinal tract, including the gastric mucosa (Rucker et al, 1994). Its absorption may be enhanced when intakes are low. Transport of copper into and across enterocytes involves multiple steps, and the redox state is important to entry into the enterocytes. At physiological concentrations (~ 1µmol) active transport occurs, which is energy-dependent and requires oxygen. Once inside the mucosal cell, copper binds to metallothionein and perhaps to other cellular transport protein and ligands. It seems that the relative concentration of apoceruloplasmin could serve as a signal in the movement of copper from the luminal content, for example albumin- or amino acid-copper complexes. Copper is lost from the intestine by desquamation of intestinal cells, and there is also evidence for basolateral transport (Rucker et al, 1994). A primary route of intraluminal copper secretion is bile, although salivary glands, gastric and pancreatic secretions occur.

Use of copper for livestock Copper is supplemented in diets for all species of livestock in the range of 10 to 30 mg/kg to cover their requirements (Jongbloed et al, 2001). The supplementation rate of copper for poultry is generally at the lower end of this range but at the higher end for pigs. In ruminating cattle, molybdenum is a well-known antagonist of copper together with sulphur, which leads to higher supplementation rates of copper for cattle (Underwood and Suttle, 1999). Apart from its essential metabolic function, copper is commonly included at a much higher rate in pig diets, up to 175 mg/kg, due to its growth promoting effect. This aspect, however, will not be discussed in this paper. For more information on this aspect the reader is referred to reviews by Meyer and Kröger (1973), Jongbloed et al (1998) and Lenis and Kogut (2000). Copper is usually supplemented as cupric sulphate pentahydrate (CuSO4·5Hanimal diets.

2O) in

58

COPPER BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs As described earlier, the sequence of importance of response criteria depends whether the animals are fed suboptimal levels of copper or if they are fed above their copper requirement. The ranking of importance for assessing the relative biological value of copper sources of the various criteria is listed in Table II.1.

Criterion

Table II.1: Ranking of importance of various response criteria for assessing the relative biological value of copper sources in pigs

Suboptimal supply Above requirement Copper absorption

Copper superoxide dismutase 1 1 Ceruloplasmin content in liver

1 no

3 1 Liver copper content (mg/kg DM) 4 3

1 1 Animal performance

Animal type

Response criteria

Added Cu (mg/kg)

Number of expts

Table II.1 shows that there are only a few criteria that are suitable for evaluation of the bioavailability of copper sources for pigs. Liver copper content ranks highest. The response criteria cytochrome c oxidase, serum/plasma copper, bile copper, haemoglobin content and hair copper content were considered not to be useful for assessing the relative biological value of the copper sources. For our study the reference source is cupric sulphate pentahydrate (CuSO4·5H2O) RG (reagent grade).

Comparison of copper sources for pigs In total, there were 13 publications that were suitable for our study. These are listed in Table II.2.

Table II.2: Overview of the experiments used for evaluation of the relative biological value of copper sources in pigs Sources of copper Ref.

no Reference

Cupric sulphate Cupric carbonate

22–96 kg Liver Cu 250 1 Cu1 Allen et al, 1961.

Cupric sulphate Copper lysine

8–20 kg Liver Cu 100, 150, 200

1 Cu2 Apgar et al, 1995.

Cupric sulphate Copper lysine

8–20 kg Cu3 Cu absorbed 200 1 Apgar et al, 1996.

Cupric sulphate Cupric sulphide

2 20-95 kg Liver Cu 250 Cu4 Barber et al, 1961

Cupric oxide 22-100 kg Liver Cu 250 1 Cu5 Bekaert et al, 1967

Cupric sulphate Cupric carbonate Cupric oxide

16-25 kg Cu absorbed 10 1 Cu6 Buescher et al, 1961

Cupric sulphate

59

Table II.2 (continued) Sources of copper Animal

type Response criteria

Added Cu (mg/kg)

Number of expts

Ref. no

Reference

Cupric sulphate Cupric oxide

6-22 kg Liver Cu 250 1 Cu7 Bunch et al, 1961

Cupric sulphate Cupric carbonate Copper methionine

5-25 kg Ceruloplasmin Liver Cu

250 1 Cu8 Bunch et al, 1965

Cupric sulphate Copper lysine

8-18 kg Liver Cu 50, 100, 200, 250

8 Cu9 Coffey et al, 1994

Cupric sulphate Cupric sulphide

15-95 kg Liver Cu 250 1 Cu10 Cromwell et al, 1978

Cupric sulphate Cupric oxide

7-14 kg Liver Cu 125, 250, 500

2 Cu11 Cromwell et al, 1989

Cupric sulphate Copper chloride, tribasic

9-24 kg Liver Cu 100, 150, 200

3 Cu12 Cromwell et al, 1998

Cupric sulphate Copper-molybdenum complex Copper citrate + sodium molybdate

1-21 d Liver Cu 20 1 Cu13 Dowdy and Matrone, 1968

Table II.3 shows that two or more observations were available for cupric carbonate, copper lysine, cupric sulphide and cupric oxide. There was only one observation available for the other copper sources. The sources cupric carbonate and copper lysine had the same relative biological value as cupric sulphate. Cupric sulphide had a lower availability than cupric sulphate (63%), whereas cupric oxide, which was expected to be lowest of all, was even higher than cupric sulphide but it had a very large standard deviation (74 ± 21). This unexpected high value is due to the results by Buescher et al (1961) who showed that cupric oxide had the same bioavailability as cupric sulphate, using labelled copper. When this observation is omitted then the relative bioavailability of copper in cupric oxide is 63 ± 11. The other copper sources were similar to cupric sulphate (reference). The chelated Cu sources, such as copper lysine and copper methionine, also showed equal availabilities as the reference source.

The liver copper content was converted using the 10log of this value in order to prevent a too large relative value. A summary of the comparisons is presented in Table II.3. To calculate the mean, the value obtained within an experiment was weighted according to the square root of the number of observations.

60

Table II.3: Summarised results on the relative biological value of copper sources for pigs Reference Cu1 u3 Cu8

11 13 Mean SDCu2 C Cu4 Cu5 Cu6 Cu7 Cu9 Cu

10 Cu Cu

12Cu n

Number of experiments

1 1 1 2 1 1 1 1 3 1 2 19 3 1

Cupric sulphate·5H2O

100 100 100 100 13 0 100 100 100 100 100 100 100 100 100 100

92 108 99 100 7.6 97 97 -

Copper citrate + Na2MoO4

99 1 99 -

Copper lysine 101 73 101 3 94 16.2Copper methionine

100 1 100 -

Copper-molybdenum complex

- 95 1 95

Cupric oxide 55 104 64 75 4 74 21.2Cupric sulphide 69 53 2 63 11.4

Cupric carbonate 3 Copper chloride, tribasic

1

COPPER BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry The sequence of the response criteria for copper is listed in Table II.4. Different weighing factors are used if animals are fed copper at a suboptimal level or above requirement. Cytochrome c oxidase, serum/plasma copper, bile copper, haemoglobin content and feather copper seemed to be unsuitable criteria and are not mentioned in the table.

Table II.4: Ranking of importance of various response criteria for assessing the relative biological value of copper sources in poultry Criterion Suboptimal supply Above requirement Copper absorption 3 1 Liver copper content (mg/kg DM) 4 3 Copper superoxide dismutase (SOD) 1 1 Ceruloplasmin content in liver 1 1

no Performance 1

61

Table II.5: Overview of the experiments used for evaluation of the relative biological value of copper sources in poultry Sources of copper

Animal type

Response criteria

Added Cu (mg/kg)

Number of expts

Ref. no

Reference

CuSO4⋅5H2O Cu-methionine Cu-lysine CuCl

Broiler Liver Cu 1 100, 200

2 Cu1 Aoyagi and Baker, 1993a

CuSO4⋅5H2O Cu-lysine

Broiler Performance 0.5, 1 1 Cu2 Aoyagi and Baker, 1993b

CuSO4⋅5H2O Cu-lysine CuO Cu2O

Broiler Liver Cu 75, 150 1 Cu3 Baker et al, 1991

CuSO4⋅5H2O CuO

Laying hen

Liver Cu 150, 300, 450, 600, 750

1 Cu4 Jackson and Stevenson, 1980

Cu acetate CuO Cu carbonate Cu sulphate

Broiler Liver Cu 150, 300, 450 1 Cu5 Ledoux et al, 1987

Cu acetate Cu oxide Cu sulphate Cu carbonate

Liver Cu Broiler 150, 300, 450 1 Cu6 Ledoux et al, 1991

Cu sulphate Cu oxide Cu iodide

Broiler Liver Cu 2 1 Cu7 McNaughton et al, 1974

CuSO4⋅5H2O Cu2(OH)3Cl

Broiler Liver Cu 150, 300, 450 200, 400, 600

2 Cu8 Miles et al, 1998

CuSO4⋅5H2O Cu-lysine

Broiler Liver Cu 150, 300, 450 1 Cu9 Pott et al, 1994

Cu acetate Cu carbonate

Broiler Liver Cu 5, 10, 20 1 Cu10 Zanetti et al, 1991

Comparison of copper sources for poultry The liver copper content was converted, if the original liver contents were available, using the 10log of this value in order to prevent too large differences in relative values. Results on copper bioavailability as summarised in Table II.6 show that for chicks (broilers) the relative bioavailability (RBV) of copper is about equal in CuSO4⋅5H2O, Cu2O and copper-lysine. Also CuSO4 FG and Cu-acetate have comparable bioavailability values. There were however no experiments comparing CuSO4⋅5H2O with CuSO4 FG. Cu shows a higher RBV than CuCl based on only one observation for each source. Copper carbonate and copper-oxide clearly have a much lower RBV compared to the reference hydrated CuSO

2(OH)3Cl

4.

62

Table II.6: Summarised results on the relative biological value of copper sources for broilers Reference Cu1 Cu2 Cu3 Cu5 Cu8 Cu9 Cu6 Cu7 Cu10 n Mean SD Number of experiments

1 1 1 1 1 1 2 1 1

4⋅ 100 100 100 100 5 100 Cu sulphate FG 100 100 100 3 100 -

112 (103)a 2 103 9.5 -5 1 76 4

Cu carbonate 64 61 68a 3 64 3.6 Cu iodide 82 1 82 - Cu2O 98 1 98 - Cu2(OH)3Cl 103 1 103 - CuCl 81 1 81 - Cu lysine 99 102 107 92 4 100 6.3 Cu methionine 91 1 91 -

CuSO 5H2O 100 -

Cu acetate 93 Cu oxide 0 24 38.7

a recalculated using copper acetate as a reference

Jackson and Stevenson (1980) compared copper oxide and CuSO4⋅5H2O in a laying hen experiment and found a RBV of 69 for copper oxide.

COPPER BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants As described earlier, the sequence of response criteria depends on whether the animals are fed suboptimal levels of copper or if they are fed above their copper requirement. The ranking of importance for copper of the various criteria for ruminants is listed in Table II.7.

Table II.7: Ranking of importance of various response criteria for assessing the relative biological value of copper sources in ruminants Criterion Suboptimal supply Above requirement Cu absorption 2 1 Liver accumulation 4 2 Kidney accumulation 3 1 Enzyme activity 2 2 Table II.7 shows that there are only few criteria that can be used for evaluation of copper sources in ruminants. Copper tissue accumulation was used as a response criterion in 12 publications, enzyme activity in six and only one publication used apparent absorption. Tissue copper accumulation has been transformed to 10log in order to maintain the RBV variation within physiological limits. For our study the reference source is copper sulphate pentahydrate (CuSO4⋅5H2O) reagent grade (RG).

Comparison of copper sources for ruminants There were in total 16 publications comprising 35 experiments that were suitable for our study. This is listed in Table II.8.

63

Comparisons of the several copper sources have been summarised in Table II.9. This table shows that there was only one observation for copper citrate and copper amino-acid-complex. For all other sources at least two observations were available. All copper sources under investigation had biological values that were quite close to each other; only copper oxide had a markedly lower availability than the reference source. The results obtained with organic copper sources can vary with the presence of interfering factors (S and Mo). The large SD observed for copper lysine is mainly due to the important difference in the apparent absorption efficiency compared to the reference source (Nockels et al, 1993). Without this observation the relative biological value of copper lysine becomes 98 ± 5.3.

64

Table II.8: Overview of the experiments used for evaluation of the relative biological value of copper sources in ruminants Sources of copper Animal

type Response criteria Added

Cu (mg/kg)

Number of expts

Ref no

Reference

Copper sulphate, 5 H20Copper acetate Copper chloride

Sheep Liver accumulation Kidney accumulation Enzyme activity (ceruloplasmin)

29 1 Cu1 Charmley and Ivan, 1989

Copper sulphate, 5 H20Copper lysine

Cows Liver accumulation 15 - 30 4 Cu2 Chase et al, 2000

Copper sulphate, 5 H20Copper proteinate

Cows Liver accumulation Enzyme activity

5 – 80 4 Cu3 Du et al,1996

Copper sulphate, 5 H20Copper proteinate

Ewes Liver accumulation Kidney accumulation Enzyme activity (ceruloplasmin)

10 – 20 – 30

3 Cu4 Eckert et al, 1999

Copper sulphate, 5 H20Copper chloride Copper citrate Copper proteinate

Steers Liver accumulation 20 2 Cu 5 Engle and Spears, 2000

Copper sulphate, 5 H20Copper AA complex

Ewes Liver accumulation 10 1 Cu 6 Hatfield et al, 2001

Copper sulphate, 5 H20Copper chloride

Cows Sheep

Liver accumulation 4 Cu 7 Ivan, 1990

Copper sulphate, 5 H20Copper oxide Copper lysine

Steers Enzyme activity (ceruloplasmin)

30 3 Cu 8 Kegley and Spears, 1994

Copper sulphate, 5 H20Copper proteinate

Calves Liver accumulation 18 1 Cu 9 Kincaid et al, 1986

Copper sulphate, 5 H20Copper carbonate Copper oxide Copper acetate Copper chloride

Sheep Liver accumulation 60 - 120 3 Cu 10

Ledoux et al, 1995

Copper sulphate, 5 H20Copper lysine

Sheep Liver accumulation 180 1 Cu 11

Luo et al, 1996

Copper sulphate, 5 H20Copper lysine

Calves Apparent absorption 1 Cu 12

Nockels et al, 1993

Copper sulphate, 5 H20Copper lysine

Sheep Liver accumulation 60, 120 180

3 Cu 13

Pott et al, 1994

Copper sulphate, 5 H20Copper lysine

Steers Enzyme activity (ceruloplasmin)

5 2 Cu 14

Ward et al, 1993

Copper sulphate, 5 H20Copper carbonate Copper proteinate

Heifers Liver accumulation 10 1 Cu 15

Ward et al, 1996

Copper sulphate, 5 H20Copper oxide

Calves Enzyme activity (SOD)

18 1 Cu 16

Xin et al, 1991

65

Table II.9: Summarised results on the relative biological value of copper sources for ruminants Reference Cu6 Cu16 Cu1 Cu2 Cu3 Cu4 Cu5 Cu7 Cu8 Cu9 Cu10 Cu11 Cu12 Cu13 Cu14 Cu15 n Mean SDNumber of experiments

1 4 4 3 2 1 4 3 1 3 1 1 3 2 1 1 35

Copper sulphate, 5 H20

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 16

100 -

Copper acetate 110 100 2 104 7.1 Copper carbonate 97 86 2 93 7.8 Copper citrate 101 1 101 - Copper chloride 96 118 98 102 4 102 10.0Copper lysine 104 102 97 153 97 89 6 104 23.2Copper oxide 64 81 86 3 76 11.5Copper amino acid complex

100 1 100 -

Copper proteinate 103 103 102 107 100 5 103 2.5

66

References

Barber, R.S., Bowland, J.P., Braude, R., Mitchell, K.C., 1961. Copper sulphate and copper sulfide (CuS) as supplements for growing pigs. Brit. J. Nutr. 15, 189-197.

Du, Z, Hemken, R.W., Harmon, R.J., 1996. Copper metabolism of Holstein and Jersey cows and heifers fed diets high in cupric sulphate or copper proteinate. J. Dairy Sci. 79, 1873-1880.

Allen, M.M., Barber, R.S., Braude, R., Mitchell, K.C., 1961. Further studies on various aspects of the use of high-copper supplements for growing pigs. Brit. J. Nutr. 15, 507-522.

Ammerman, C.B., Baker, D.H., Lewis, A.J., 1995. Bioavailability of nutrients for animals. Amino acids, minerals and vitamins. Academic Press, New York.

Aoyagi, S., Baker, D.H., 1993a. Nutritional evaluation of a copper-methionine complex for chicks. Poultry Sci. 72, 2309-2315.

Aoyagi, S., Baker, D.H., 1993b. Nutritional evaluation of copper-lysine and zinc-lysine complexes for chicks. Poultry Sci. 72, 165-171.

Apgar, G.A., Kornegay, E.T., 1996. Mineral balance of finishing pigs fed copper sulphate or a copper-lysine complex at growth-stimulating levels. J. Anim. Sci. 74, 1594-1600.

Apgar, G.A., Kornegay, E.T., Lindemann, M.D., Notter, D.R., 1995. Evaluation of copper sulphate and a copper-lysine complex as growth promoters for weanling swine. J. Anim. Sci. 73, 2640-2646.

Baker, D.H., Odle, J., Funk, M.A., Wieland, T.M., 1991. Research note: bioavailability of copper in cupric oxide, cuprous oxide, and in a copper-lysine complex. Poultry Sci. 70, 177-179.

Bekaert, H., Eeckhout, W., Buysse, F., 1967. [The effect of CuSO4 and CuO, the granulometric characteristics of CuSO4 and a zinc supplementation on performance, carcass quality and liver copper status of pigs]. Landbouwtijdschrift 20, 1571-1585.

Buescher, R.G., Griffin, S.A., Bell, M.C., 1961. Copper availability to swine from Cu64 labelled inorganic compounds. J. Anim. Sci 20, 529-531.

Bunch, R.J., McCall, J.T., Speer, V.C., Hays, V.W., 1965. Copper supplementation for weanling pigs. J. Anim. Sci. 24, 995-1000.

Bunch, R.J., Speer, V.C., Hays, V.W., Hawbaker, J.H., Catron, D.V., 1961. Effects of copper sulphate, copper oxide and tetracycline on baby pig performance. J. Anim. Sci. 20, 723-726.

Charmley, E.I., Ivan, M., 1989. The relative accumulation of copper in the liver and kidneys of sheep fed corn silage supplemented with copper chloride, copper acetate or copper sulphate. Can. J. Anim. Sci. 69, 205-214.

Chase, C.R., Beede, D.K., Van Horn, H.H., Shearer, J.K.,Wilcox, C.J., Donovan, G.A., 2000. Responses of lactating dairy cows to copper sources, supplementation rate, and dietary antagonist (iron). J. Dairy Sci. 83, 1845-1852.

Coffey, R.D, Cromwell, G.L., Monegue, H.J., 1994. Efficacy of a copper-lysine complex as a growth promotant for weanling pigs. J. Anim. Sci. 72, 2880-2886.

Cromwell, G.L., Hays, V.W., Clark, T.L., 1978. Effects of copper sulphate, copper sulfide and sodium sulfide on performance and copper stores of pigs. J. Anim. Sci. 46, 692-698.

Cromwell, G.L., Lindemann, M.D., Monegue, H.J., Hall, D.D., Orr, D.E., 1998. Tribasic copper chloride and copper sulphate as copper sources for weanling pigs. J. Anim. Sci. 76, 118-123.

Cromwell, G.L., Stahly, T.S., Monegue, H.J., 1989. Effects of source and level of copper on performance and liver copper stores in weanling pigs. J. Anim. Sci. 76, 2996-3002.

Dowdy, R.P., Matrone, G., 1968. A Copper-Molybdenum Complex: Its effects and movement in the piglet and sheep. J. Nutr. 95, 197-201.

Eckert, G.E., Greene, L.W., Carstens, G.E., Ramsey, W.S., 1999. Copper status of ewes fed increasing amounts of copper from copper sulphate or copper proteinate. J. Anim. Sci. 77, 244-249.

Engle, T.E., Spears, J.W., 2000. Effects of dietary copper concentration and source on performance and copper status of growing and finishing steers. J. Anim. Sci. 78, 2446-2451.

Hartmans, J., 1975. The frequency of occurrence of copper poisoning and the role of sheep concentrates in its merits enquiry. Tijdschr. Diergeneesk. 100, 379-382.

Hatfield, P.G., Swenson, C.K., Kott, R.W., Ansotegui, R.P., Roth, N.J., Robinson, B.L., 2001. Zinc and copper status in ewes supplemented with sulphate and amino-acid-complexed forms of zinc and copper. J. Anim. Sci. 79, 261-266.

67

Ivan, M., Proulx, J.G., Morales, R., Codagone, H.C.V., Dayrel, M.S., 1990. Copper accumulation in the liver of sheep and cattle fed diets supplemented with copper sulphate or copper chloride. Can. J. Anim. Sci. 70, 727-730.

Jackson, N., Stevenson, M.H., 1980. A study of the effects of dietary added cupric oxide on the laying, domestic fowl and a comparison with the effects of hydrated copper sulphate. Brit. J. Nutr. 45, 99-110.

Jongbloed, A.W., Klis, J.D. van der, Mroz, Z., Kemme, P.A., Prins, H., Zaalmink, 1998. Vermindering van koper, zink en cadmium in varkens- en pluimveevoeders. Een literatuuroverzicht. ID-DLO rapport 98.006. 57 pp.

Jongbloed, A.W., Top, A.M. van den, Beynen, A.C., Klis, J.D. van der, Kemme, P.A., Valk, H., 2001. Consequences of newly proposed maximum contents of copper and zinc in diets for cattle, pigs and poultry on animal performance and health. Report ID-Lelystad no. 2097, 73 pp.

Meyer, H., Kröger, H., 1973. Vergleichende Untersuchungen über Wachstumswirkungen von Kupfer- und Antibioticazulagen bei Ferkeln. Züchtungskunde 45, 439-446.

Kegley, E.B., Spears J.W., 1994. Bioavailability of feed-grade copper sources (oxide, sulphate or lysine) in growing cattle. J. Anim. Sci. 72, 2728-2734.

Kincaid, R.L., Blauwiekel, R.M., Cronrath, J.D., 1986. Supplementation of copper as copper sulphate or copper proteinate for growing calves fed forages containing molybdenum. J. Dairy Sci. 69, 160-163.

Ledoux, D.R., Ammerman, C.B., Miles, R.D., 1987. Biological availability of copper sources for broiler chicks. Poultry Sci. 66, 24 (abstract).

Ledoux, D.R., Henry, P.R., Ammerman, C.B., Rao, P.V., Miles, R.D., 1991. Estimation of the relative bioavailability of inorganic copper sources for chicks using tissue uptake of copper. J. Anim. Sci. 69, 215-222.

Ledoux, D.R., Pott, E.B., Ammerman, C.B., Merrit, A.M., Madison, J.B., 1995. Estimation of the relative bioavailability of inorganic copper sources for sheep. Nutr. Res. 15, 1803-1813.

Lenis, N.P., Kogut, J., 2000. Efficacy of copper sulphate as a growth promoter in pig diets. Confidential report ID-Lelystad no. 2010, 12 pp.

Luo, X.G., Henry, P.R., Ammerman, C.B., Madison, J.B., 1996. Relative bioavailability of copper in a copper-lysine complex or copper sulphate for ruminants as affected by feeding regimen. Anim. Feed Sci. Technol. 57, 281-289.

McDowell, L.R., 1992. Minerals in Animal and Human Nutrition. Academic Press, San Diego, California, 524 pp.

McNaughton, J.L., Day, E.J., Dilworth, B.C., 1974. Iron and copper availability from various sources. Poultry Sci. 53, 1325-1330.

Miles, R.D., O'Keefe, S.F., Henry, P.R., Ammerman, C.B., Luo, X.G., 1998. The effect of dietary supplementation with copper sulphate or tribasic copper chloride on broiler performance, relative copper bioavailability, and dietary prooxidant activity. Poultry Sci. 77, 416-425.

Nockels, C.F., Debonis, J., Torrent, J., 1993. Stress induction affects copper and zinc balance in calves fed organic and inorganic copper and zinc sources. J. Anim. Sci. 71, 2539-2545.

NRC, 1980. Mineral tolerance of domestic animals. National Research Council, National Academy of Sciences, Washington, D.C.

O’Dell, B.L., 1976. Biochemistry and physiology of copper in vertebrates. In: Trace elements in Human Health and Disease, A.S. Prasad and D. Oberleas (Eds), Vol 1, p. 15. Academic Press, New York.

Pott, E.B., Henry, P.R., Ammerman, C.B., Merritt, A.M., Madison, J.B., Miles, R.D., 1994. Relative bioavailability of copper in a copper-lysine complex for chicks and lambs. Anim. Feed Sci, Technol. 45, 193-203.

Rucker, R.B., Lönnerdal, B., Keen, J.L., 1994. Intestinal absorption of nutritionally important trace minerals. In: L.R. Johnson (Ed.). Physiology of the intestinal tract. 3rd edition, Raven Press, New York, 2183-2202.

Underwood, E.J., Suttle, N.F., 1999. The Mineral Nutrition of Livestock, 3rd edition. CABI Publishing, Wallingford, United Kingdom.

68

Ward, J.D., Spears, J.W., Kegley, E. B., 1993. Effect of copper level and source (copper lysine vs copper sulphate) on copper status, performance, and immune response in growing steers fed diets with or without supplemental molybdenum and sulfur. J. Anim. Sci. 71, 2748-2755.

Ward, J.D., Spears, J.W., Kegley, E. B., 1996. Bioavailability of copper proteinate and copper carbonate relative to Copper sulphate in cattle. J. Dairy Sci. 79, 127-132.

Xin, Z., Waterman, D.F., Hemken, R.W., Harmon, R.J., Jackson, J.A., 1991. Effects of copper sources and dietary cation-anion balance on copper availability and acid-base status in dairy calves. J. Dairy Sci. 74, 3167-3173.

Zanetti, M.A., Henry, P.R., Ammerman, C.B., Miles, R.D., 1991. Estimation of the relative bioavailability of copper sources in chicks fed on conventional dietary amounts. Brit. Poultry Sci. 32, 583-588.

69

III. IRON BIOAVAILABILITY

General function in the body Iron has been known as an essential element since ancient times and its beneficial effect on blood formation was recognised in the 17th century (Underwood, 1977). It plays a key role in many biochemical reactions. Practically all of the iron in the animal's body is organic in nature and only a very small percentage is found as free inorganic ions (Georgievskii, 1982). Haemoglobin (blood) iron represents approximately 60% of total body iron, whereas myoglobin represents only about 3-7% of total iron (McDowell, 1992). There are two kinds of organic iron, haemal and non-haemal. Haemal iron, which forms part of a porphyrin group, represents 70-75% of total iron and includes haemoglobin, myoglobin, cytochromes, cytochrome oxidase, catalase and peroxidase. Non-haemal iron includes iron transport and storage forms such as transferrin, ferritin, haemosiderin, and other iron proteinates. Iron content of the body varies with species, age, sex, nutrition and state of health and is controlled by adjustment in absorption rate (Finch and Cook, 1984). The body has a limited capacity to excrete iron and considerable recycling of the element occurs, especially as a result of the breakdown of senescent red blood cells. Iron is absorbed mainly from the duodenum after ferric iron is reduced to the ferrous form (Georgievskii, 1982). Iron absorption is affected primarily by the iron status of the body and age, and secondly by the chemical form of the iron ingested and the amounts and proportions in the diet of other minerals and compounds with which iron interacts (Underwood and Suttle, 1999). Iron of plant origin is more readily absorbed than nonhemal iron of animal origin. Protein-bound iron such as in non-haeme compounds must be released before absorption, whereas iron in heme compounds is absorbed as the haeme moiety without release from the bound form (Morris, 1987). Lactate and citrate seem to enhance iron absorption (Lonnerdal, 1988). Although iron is the most abundant of the trace elements in the animal’s body, practical deficiency problems in domestic animals are generally limited to nursing pigs and, to a lesser extent, other milk-fed animals. Iron deficiency in grazing animals is generally the result of blood loss from heavy parasite infestation rather than nutritional inadequacy. Toxicity of iron is seldom a problem in domestic animals and they have a high tolerance for iron. Pigs are more tolerant of excess iron than are cattle and poultry. The maximum tolerable levels of dietary iron are 3000 mg/kg for pigs and 1000 mg/kg for cattle and poultry (NRC, 1980). However, less than 1000 mg/kg iron can interact with copper and zinc metabolism and lead to reproduction problems in ruminants. Apparently, cattle can safely consume substantially more iron from natural feed sources than from soluble sources such as ferrous sulphate (Standish et al, cited by McDowell, 1992).

Use of iron for livestock Iron is supplemented in diets for all species of livestock in the range of 10 to 150 mg/kg to cover their requirements. The supplementation rate of iron for ruminants and poultry is generally at the lower end of this range but at the higher end for pigs. Ruminant diets are usually not supplemented with iron, because roughage contains already a high amount of iron due to contamination with soil particles. Therefore, no data on iron availability for cattle are reported in the literature available. No iron is added to diets of veal calves in order to obtain white coloured meat. Iron is most commonly supplemented as ferrous sulphate heptahydrate (FeSO4·7H2O) in animal diets.

70

IRON BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs As described earlier, the sequence of importance of response criteria to assess the relative biological value of iron sources for pigs, depends on the supply of suboptimal levels of iron or above the iron requirement. The ranking of importance for iron of the various criteria is listed in Table III.1.

Table III.1: Ranking of importance of various response criteria for assessing the relative biological value of iron sources in pigs Criterion Suboptimal supply Above requirement Iron absorption (apparent) 4 1 Iron absorption (true) 4 3 Haemoglobin regeneration (blood) 3 1 Haemoglobin content (blood) 2 1 Liver/spleen iron content 1 2 Performance 1 no Radio iron uptake no no Table III.1 shows that there are only a few criteria that can be used for evaluation of iron sources for pigs. True and apparent iron absorption are the most important ones. For our study, ferrous sulphate heptahydrate (FeSO sen as the reference source.

Table III.2: Overview of the experiments used for evaluation of the relative biological value of iron sources in pigs

Animal type

Added Fe (mg/kg)

4·7H2O) reagent grade (RG) was cho

Comparison of iron sources for pigs There were a total of eight publications that were suitable for our study. These are listed in Table III.2.

Sources of iron Response criteria

No. of expts

Ref. no

Reference

Ferrous sulphate 7H2O Ferrous carbonate

2 17 – 73 d Performance Hb content

40 or 80 Fe1 Ammermann et al, 1974

Electrolytic iron powder Sodium iron pyrophosphate Ferric polyphosphate powder Disodium iron EDTA

Mini pigs 5 – 33 d

Performance Hb Iron in organs

64 to 69 Fe2 Anderson et al, 1973

Ferrous sulphate 7H2O Ferric ammonium citrate

1 – 28 d Performance Hb content

Fe3 Harmon et al, 1967

125 3

Ferrous carbonate 3 – 39 d, 20 – 69 d Hb content

60 to 80 3 Fe4

Ferrous sulphate 7H2O Catalytically reduced iron

1

Ferrous sulphate 7H2O Performance Harmon et al, 1969

71

Table III.2 (continued) Sources of iron Animal

type Response criteria

Added Fe (mg/kg)

Fe5

Ferrous sulphate 7H2O Ferric choline citrate Ferric copper cobalt choline citrate

6 – 9 kg Hb content Iron in faeces

50 and 100 2 Fe6 Miller et al, 1981Performance

Ferrous sulphate 7H2O Ferrous sulphate H2O Ferrous carbonate

7 – 21 kg Hb content 320 to 360 1 Fe7 Poitevint, 1979

Ferrous sulphate Ferric citrate

2 – 21 d Performance Hb content Iron liver

100 2 Ullrey et al, 1973

Fe8

No. of expts

Ref. no

Reference

Ferrous sulphate 7H2O Ferrous sulphate H2O

28 – 61 d Performance Hb content

50 and 100 1 Miller, 1978

A summary of the comparisons is presented in Table III.3. This table shows that there were two or more observations only for ferrous sulphate monohydrate and ferrous carbonate. There was only one observation available for the other ferrous sources. Ferrous sulphate monohydrate was of the same availability as ferrous sulphate heptahydrate. Ferrous carbonate had a lower availability than ferrous sulphate (82%), whereas combinations with citrate showed a higher availability than ferrous sulphate. The sources containing catalytically reduced iron, such as electrolytic iron powder, sodium iron pyrophosphate and ferric polyphosphate powder, showed lower availabilities than ferrous sulphate. Furthermore, one experiment of Standish and one of Holt are cited by Ammerman and Miller (1972). Three feed grade ferrous carbonates studied were compared with reagent grade ferrous sulphate. The most available ferrous carbonate was similar to that of reagent grade ferrous sulphate, but the two other ferrous carbonates had a lower availability. In an experiment by Pickett et al (1961), in which several iron sources were tested ferrous sulphate resulted in higher haemoglobin values compared with ferrous carbonate. Young suckling piglets need supplementary iron when they are housed in concrete pens in order to prevent anaemia. Therefore, in practice these piglets are either injected with iron or iron is supplied orally. In the study by Pollmann et al (1983) it was shown that iron dextran and gleptoferron were similar in their effect to prevent anaemia.

72

Table III.3: Summarised results on the relative biological value of iron sources for pigs Reference Fe1 Fe2 Fe3 Fe4 Fe5 n SD Fe6 Fe7 Fe8 Mean

1 2 1 2 Ferrous sulphate 7H2O 100 8 100 100 100 100 100 100 100 100

2 100 2.0 Ferrous carbonate 81 74 98 3 82 14.1 Ferric choline citrate 118 118 1 Ferric citrate 114 1 114 Ferric copper cobalt choline citrate

114 1 114

Ferric ammonium citrate 102 1 102 1 78 1 86

81 1

Disodium iron EDTA 91 1 91

Number of experiments 2 1 3 3 15

Ferrous sulphate H2O 99 101

Catalytically reduced iron 78 Electrolytic iron powder 86 Sodium iron pyrophosphate 81 Ferric polyphosphate powder 91 1 91

IRON BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry As described earlier, the sequence of response criteria depends on whether the animals are fed suboptimal levels of iron or if they are fed above their iron requirement. The ranking of importance for iron of the various criteria is listed in Table III.4. Radio iron uptake is not included in the list of suitable criteria.

Above requirement

Table III.4: Ranking of importance of various response criteria for assessing the relative biological value of iron sources in poultry Criterion Suboptimal supply

Iron absorption (true) 4 3 Haemoglobin regeneration (blood)

Liver/spleen iron content 1 2 Performance 1 no

Iron absorption (apparent) 4 1

3 1 Haemoglobin content (blood) 2 1

73

Table III.5: Overview of the experiments used for evaluation of the relative biological value of iron sources in poultry Sources of iron Animal

type Response criteria

Added Fe (mg/kg)

Number of expts

Ref. no Reference

Ferrous sulphate⋅7H2O Ferric orthophosphate Sodium iron pyrophosphate Reduced iron Ferrous carbonate

Broiler Haemoglobin regeneration Haemoglobin content

10, 20, 40 diff. (5-60) diff. (5-60)

3 Fe1 Amine et al, 1972

Ferrous sulphate⋅7H2O Ferric sulphate FeSO4⋅H2O Zn-FeSO4⋅H2O Fe-ZnSO4⋅H2O

Broiler Performance Haemoglobin regeneration Haematocrit

10, 20 9.4, 18.8 10, 20

3 Fe2 Boling et al, 1998

Ferrous sulphate⋅7H2O FeSO4⋅H2O Fe methionine

Cao et al, 1996 Broiler Liver Fe 400, 600, 800

1 Fe3

Ferrous sulphate⋅7H2O Ferric ammonium citrate Ferric orthophosphate (3 origins) Ferric sulphate Ferric oxide Ferrous carbonate (4 origins) Reduced iron (4 origins) Sodium iron pyrophosphate (2 origins)

Broiler Haemoglobin regeneration

5, 10, 15, 20 (ref.) 20 (test)

1 Fe4 Fritz et al, 1970

Ferrous sulphate⋅7H2O Ferrous sulphate⋅H2O Mono/dicalcium phosphate Defluorinated phosphate

Broiler Haemoglobin regen.

7.5, 15.0, 22.5

1 Fe5 Henry et al, 1992

Ferric choline chloride Sequestered iron (3 origins) Ferric oxide

Broiler Haemoglobin content Performance Haematocrit

10, 20 5, 10

Fe6 Mc Naughton et al, 1974

Ferrous sulphate 2

74

Table III.5 (continued) Sources of iron Animal

type Response criteria

Added Fe (mg/kg)

Number of expts

Ref. no

Reference

Ferrous sulphate Sodium iron pyrophosphate Ferric orthophosphate

Broiler Haemoglobin regeneration Haemoglobin content

Unknown 6 Fe7 Pennell et al, 1976

Ferrous sulphate Ferrous chloride Ferric chloride Ferrous carbonate (2 origins)

Broiler Haemoglobin regeneration

5, 10, 15, 20 (ref.) 20 (test)

1 Fe8 Pla and Fritz, 1970

Ferrous sulphate⋅7H2O Ferric orthophosphates Sodium iron pyrophosphate Reduced iron Ferrous carbonate ore

Broiler HaemoglobinRepletion

5, 10, 15, 20 (ref.) 20 (test)

7 Fe9 Pla and Fritz, 1971

Encapsulated ferrous sulphate Solubilised ferric pyrophosphate Reduced iron (17 origins)

Broiler Haemoglobin regeneration

5, 10, 15, 20 (ref.) 20 (test)

1 Fe10 Pla et al, 1973

Ferrous sulphate⋅7H2O Ferrous sulphate monohydrate. Ferrous carbonate Ferric oxide

Broiler Haemoglobin content Performance Haematocrit

10, 20 1 Fe11 Poitevint, 1979

Ferrous sulphate

Comparison of iron sources for poultry A summary of comparisons on RBV of iron in different sources for chicks is presented in Table III.6. Ferrous sulphate⋅7H O and ferrous sulphate⋅1H O are used as references and have a RBV of 100, while the other sources such as ferrous chloride, Fe-ZnSO ⋅H O, ferric ammonium citrate and sequestered iron have a somewhat better (6-15 %) bioavailability.

2 2

2

By contrast, ferrous sulphate FG shows a slightly lower RBV than the hydrated iron-sulphates. The same conclusion is valid for Zn-FeZO ⋅H O and ferric choline chloride. 4The availability of iron in ferrous carbonate, sodium ferric pyrophosphate and ferric orthophosphate is very low. Ferric sources such as chloride, pyrophosphate and sulphate show intermediary availability.

4

2

75

Table III.6: Summarised results on the relative biological value of iron sources for poultry Reference Fe4 Fe5 Fe1 Fe2 Fe3 Fe6 Fe7 Fe8 Fe9 Fe10 Fe11 n Mean SDNumber of experiments 3 3 1 1 1 2 6 1 7 1 1 27Ferrous sulphate⋅7H2O 100 100 100 100 100 100 100 7 100 - Ferrous sulphate 100 100 100 100 4 100 - Ferrous sulphate FG 91 1 91 - Ferrous sulphate monohydrate

(103)b 102 103 2 103 0.7

Ferrous sulphate encapsulated

97 1 97 -

Ferric chloride 78 2 78 - Ferric pyrophosphate 86a 1 86 - Ferric orthophosphate 13 13 4 12 4 10 4.4 Ferric oxide 4 77 67 3 52 39.4 Ferric sulphate 37 65 2 47 19.8 Ferrous carbonate 55 3 5 3 88 5 27 39.0 Ferrous chloride 106 1 106 - Reduced iron 36 52 46 52 4 45 7.8 Sodium ferric pyrophosphate 22 8 5 13 4 12 7.5 Zn-FeSO4⋅H2O 96b 1 96 - Fe-ZnSO4⋅H2O 112b 1 112 - Mono-dicalcium phosphate 67 1 67 - Defluorinated phosphate 48 1 48 - Fe methionine 86 1 86 - Ferric ammonium citrate 115 1 115 - Ferric choline chloride 93 1 93 - Sequestered iron 106 - 1 106 a solubilised ferric pyrophophate=ferric pyrophosphate solubilised with sodium citrate recalculated using ferrous sulphate monohydrate as a reference

b

76

IRON BIOAVAILABILITY FOR RUMINANTS

There were no publications suitable for the study of iron bioavailability of ruminants.

Ammerman, C.B., Standish, J.F., Holt, C.E., Houser, R.H., Miller, S.M., Combs, G.E., 1974. Ferrous carbonate as sources of iron for weanling pigs and rats. J. Anim. Sci. 38, 52-58.

Finch, C.A., Cook, J.D., 1984. Iron deficiency. Amer. J. Clin. Nutr. 39, 471-477.

Harmon, B.G., Hoge, D.E., Jensen, A.H., Baker, D.H., 1969. Efficacy of ferrous carbonate as a hemanitic for young swine. J. Anim. Sci. 29, 706-710.

Henry, P.R., Ammerman, C.B., Miles, R.D., Littell, R.C., 1992. Relative bioavailability of iron in feed grade phosphates for chicks. J. Anim. Sci. 70 1, 228 (abstract).

Lonnerdal, B., 1988. Trace elements in infancy: a supply/demand perspective. In: L.S. Hurley, C.L. Keen, B. Lonnerdal and R.B. Rucker (Eds.), Proc. Sixth Intern. Symp. on Trace Elements in Man and Animals. Plenum Press, New York, pp. 189-195.

Morris, E.R., 1987. Iron In: W. Mertz (Ed), Trace Element in Man and Animal Nutrition, Vol 1, 5th ed., Academic Press, New York.

References Amine, E.K., Neff, R., Hegsted, D.M., 1972. Biological estimation of available iron using chicks or

rats. J. Agr. Food Chem. 20, 246-251. Ammerman, C.B., Miller, S.M., 1972. Biological availability of minor ions: a review. J. Anim. Sci.

35, 681-694.

Anderson, T.A., Filer, L.J., Fomon, S.J., Andersen, D.W., Nixt, T.L., Rogers, R.R., Jensen, R.L., Nelson, S.E., 1973. Bioavailability of different sources of dietary iron fed to Pitman-Moore miniature pigs. J. Nutr. 104, 619-627.

Boling, S.D., Edwards, H.M., Emmert, J.L., Biehl, R.R., Baker, D.H., 1998. Bioavailability of iron in cottonseed meal, ferric sulphate, and two ferrous sulphate by-products of the galvanising industry. Poultry Sci. 77, 1388-1392.

Cao, J., Luo, X.G., Henry, P.R., Ammerman, C.B., Littell, R.C., Miles, R.D., 1996. Effect of dietary iron concentration, age, and length of iron feeding on feed intake and tissue iron concentration of broiler chicks for use as a bioassay of supplemental iron sources. Poultry Sci. 75, 495-504.

Fritz, J.C., Pla, G.W., Roberts, T., Boehne, J.W., Hove, E.L., 1970. Biological availability in animals of iron from common dietary sources. J. Agr. Food Chem.18, 647-651.

Georgievskii, V.I., 1982. The physiological role of microelements. In: Mineral Nutrition of Animals. V.I. Georgievskii, B.N. Annenkov and V.T. Samokhin, (Eds)., p 171, Butterworths, London, UK.

Harmon, B.G., Becker, D.E., Jensen, A.H., 1967. Efficacy of ferric ammonium citrate in preventing anemia in young swine. J. Anim. Sci. 26, 1051-1053.

McDowell, L.R., 1992. Iron. In: Minerals in Animal and Human Nutrition. Academic Press, San Diego, CA, USA, p. 152-175.

McNaughton, J.L., Day, E.J., Dilworth B.C., 1974. Iron and copper availability from various sources. Poultry Sci. 53, 1325-1330.

Miller, E. R., 1978. Biological availability of iron in iron supplements. Feedstuffs 50, 20-21. Miller, E.R., Parsons, M.J., Ullrey, D.E., Ku, P.K., 1981. Bioavailability of iron from ferric choline

citrate and a ferric copper cobalt choline citrate complex for young pigs. J. Anim. Sci. 52, 783-787.

NRC, 1980. Mineral tolerance of domestic animals. National Research Council, National Academy of Sciences, Washington, D.C.

Pennell, M.D., Davies, M.I., Rasper, J., Motzok, I., 1976. Biological availability of iron supplements for rats, chicks and humans. J. Nutr. 106, 265-274.

Pickett, R.A., Plumlee, M.P., Beeson, W.M., 1961. Availability of dietary iron in different compounds for young pigs. J. Anim. Sci. 20, 946 (abstract).

Pla, G.W., Fritz, J.C., 1970. Availability of iron. J. Ass. Off. Anal. Chem. 53, 791-800.

77

Pla, G.W., Fritz, J.C., 1971. Collaborative study of the haemoglobin repletion test in chicks and rats for measuring availability of iron. J. Ass. Off. Anal. Chem. 54, 13-17.

Pla, G.W., Harrrison, B.N., Fritz, J.C., 1973. Comparison of chicks and rats as test animals for studying bioavailability of iron, with special reference to use of reduced iron in enriched bread. J. Ass. Off. Anal. Chem. 56, 1369-1373.

Poitevint, A.L., 1979. Determination of the true biological availability of ferrous carbonate. Feedstuffs 51, 31-33.

Pollmann, D.S., Smith, J.E., Stevenson, J.S., Schoneweis, D.A., Hines, E.H., 1983. Comparison of Gleptoferron with iron dextran for anemia prevention in young pigs. J. Anim. Sci. 56, 640-644.

Ullrey, D.E., Miller, E. R., Hitchcock, J. P., 1973. Oral ferric citrate vs ferrous sulphate for prevention of baby pig anemia. Michigan Agri. Exp. Station. Rep. 232, 34-38.

Underwood, E.J., 1977. Trace elements in Human and Animal Nutrition, 4th edition, Academic Press, New York.

Underwood, E.J., Suttle, N.F., 1999. The Mineral Nutrition of Livestock, 3rd edition. CABI Publishing, Wallingford, United Kingdom.

78

IV. IODINE BIOAVAILABILITY

General function in the body Iodine is an essential element for all animals. In the 19th century the link between iodine deficiency and goitre (enlargement of the thyroid gland in the neck) on the one hand and the correlation between the occurrence of endemic goitre and concentrations of iodine in soils, foods and waters on the other hand were first made. Indeed, plants, water or other animal feedstuffs have highly variable concentrations of iodine. This is due to differences in species and strains, climatic and seasonal conditions, the type of soil, the fertiliser treatment or possible interactions.

Iodine is present in tissues at concentrations of 0.1 µg/g body weight but is mainly concentrated in the thyroid gland at 400 µg/g body weight. It is an integral part of the thyroid hormones thyroxin (tetraiodothyronine) (T4) and triiodothyronine (T3). These hormones, particularly T3, control oxidation rate and protein synthesis in the cells. They set the basal metabolic rate. Also, they play a key role in the development of the foetus, digestion, muscle function, immune defence, circulation and seasonality of reproduction. Iodine is absorbed (and recycled) very efficiently in the gastrointestinal tract. The absorbed iodine is then transported in the bloodstream, bound to plasma proteins. As it passes through the thyroid gland, up to 90 % of the iodine is captured by a Na/K-dependent ATP-ase. The efficiency with which the iodine is taken up by the gland varies according to the need and is regulated by hormones. Excess dietary iodine is excreted, in the form of iodide, predominantly via urine, but also in milk of lactating animals. In addition to the environmental distribution of iodine, as an important factor influencing supply, some other factors influence availability. One of these factors is the presence of dietary components which interfere with thyroid hormone synthesis, so-called goitrogens. Goitrogenic feeds include brassicas and cruciferae, white and subterranean clover, some grass species (including ryegrass), maize silage, soybean and linseed. Also trace elements as selenium and iron influence metabolism. As cold stress increases basal metabolic rate, it is another factor which should be taken into account.

Use of iodine for livestock Iodine is usually supplemented in diets for cattle in the concentrate feed or mineral pellets. The predominant sources used by the animal feed industry are calcium iodate and ethyldiaminehydroiodide (EDDI) or organic forms of iodine. Potassium or sodium iodide are less stable sources.

IODINE BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs Response criteria for iodine are ranked along their importance in Table IV.1 with differences being made between suboptimal supply and feeding above requirement.

79

Table IV.1: Ranking of importance of various response criteria for assessing the relative biological value of iodine sources in pigs Criterion Suboptimal supply Above requirement Iodine absorption 3 1 Thyroid weight 2 2 Thyroid iodine content 2 2 Performance 1 no Table IV.1 shows that there are only a few criteria that can be used for evaluation of the bioavailability of iodine sources for pigs of which iodine absorption ranks highest. For our study the reference source is potassium iodide (KI).

Comparison of iodine sources for pigs There was only one publication that was suitable for our study, which is listed in Table IV.2.

Table IV.2: Overview of the experiments used for evaluation of the relative biological value of iodine in pigs Sources of iodine Animal

type Response criteria

Added I (mg/kg)

Number of expts

Reference

Potassium iodide Ethylenediamine dihydroiodide Iodine humate

32–41 kg Absorption 1.0 1 Herzig et al, 2000

Table IV.2 shows that potassium iodide, ethylenediamine dihydroiodide and iodine have been compared in the study. An experiment from Russia by Kuznetsov et al (1992) on iodine availability was carried out with 14 iodine compounds. Their response criteria were iodine content in the thyroid gland, thyroid forming hormones and excretion of cyclic nucleotides in the urine. Their conclusion was that there were no differences in iodine bioavailability between iodates and iodides, as well as from potassium iodate and calcium iodate. Comparison of iodine sources for pigs is summarised in Table IV.3. The results with pigs show that ethylenediamine dihydroiodide was similar to potassium iodide, while iodine humate was less available.

Table IV.3: Summarised results on the relative biological value of iodine sources for pigs Reference Herzig et al, 2000 Number of experiments 1 Potassium iodide 100 Ethylenediamine dihydroiodide 99 Iodine humate 71

80

IODINE BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry The ranking of the response criteria is listed in Table IV.4. The weights of the criteria used differ according to the fact if the supply of the mineral is suboptimal or above requirement.

Table IV.4: Ranking of importance of various response criteria for assessing the relative biological value of iodine sources in poultry Criterion Suboptimal supply Above requirement Iodine absorption 3 1 Thyroid weight 2 2 Thyroid I content 2 2 Performance 1 no

Table IV.5: Overview of the experiments used for evaluation of the relative biological value of iodine in poultry Sources of iodine Animal

type Response criteria

Added I (mg/kg)

Number of expts

Ref. number

Reference

Potassium iodide Calcium iodate

Broiler Thyroid weight 0.59 1 I1 Hixson and Rosner, 1957

Comparison of iodine sources for poultry Only one reference is available on iodine (Table IV.5), showing that iodine in calcium iodate is slightly less (5%) available than in potassium iodide for chicks.

IODINE BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants As described earlier, the sequence of response criteria depends on the fact whether the animals are fed suboptimal or if they are fed above their iodine requirement. The ranking of importance for iodine of the various criteria is listed in Table IV.6.

81

Table IV.6: Ranking of importance of various response criteria for assessing the relative biological value of iodine sources in ruminants Criterion Suboptimal supply Above requirement Iodine absorption or radio-iodine retained

3 1

Thyroid weight 2 2 Thyroid iodine content 2 2 Milk iodine content 2 1 It can be seen in Table IV.6 that there are only few criteria that can be used for evaluation of iodine sources in ruminant. In our study, five observations used radio-iodine metabolism and two used iodine content of milk. For our study the references sources are either sodium iodide or potassium iodide.

Comparison of iodine sources for ruminants There were in total 7 publications involving 13 experiments that were suitable for our study. This is listed in Table IV.7.

Sources of iodine Animal type

Response criteria Added I (mg/kg)

Number of expts

Reference

Table IV.7: Overview of the experiments used for evaluation of the relative biological value of iodine sources in ruminants

Ref. no

Sodium iodide Diiodosalicylic acid

Sheep Radio-iodine 12 µg/kg BW

Aschbacher et al, 1963 retained

1 I1

1.7 – 1.9 2 I3

Sodium iodide Calcium iodate Penta calcium orthoperiodate

Cows Radio-iodine iodine retained Labelled 1 I4 Miller et al,

1968

I5 Miller and Swanson, 1973

Calves Radio-iodine retained

Moss and Miller, 1970

Potassium iodide Ethyldiaminedihydroiodide

Cows 3 Milk iodine content

1 – 2 – 4 I7 Swanson et al, 1990

Sodium iodide Diiodosalicylic acid

Cows Radio-iodine retained

12 µg/kg BW

1 I2 Aschbacher et al, 1966

Potassium iodide Calcium iodide Ethyldiaminedihydroiodide

Cows Milk iodine content

Brzoska et al, 1999

Sodium iodide Ethyldiaminedihydroiodide

Cows Radio-iodine retained

Labelled iodine

3

Sodium iodide Calcium iodate Penta calcium orthoperiodate

Labelled iodine

2 I6

Comparisons have been summarised in Table IV.8. Most of the iodine sources under investigation show a good relative biological value which is slightly higher than the reference source ranking from 106 to 111. Only diiodosalicylic acid shows a very poor bioavailability (36) and cannot be recommended for iodine supplementation of ruminants. Some recent studies showed that the availability of ethyldiaminedihydroiodide, has a good relative biological value and a better stability than iodides.

82

Table IV.8: Summarised results on the relative biological value of iodine sources for ruminants Reference I1 I2 I3 I4 I5 I6 I7 n Mean SD Number of experiments 1 1 2 1 3 2 3 13 Potassium iodide - 100 100 2 100 Sodium iodide 100 100 100 100 100 5 100 - Calcium iodate 105 106 2 106 0.7 Calcium iodide 110 1 110 - Diiodosalicylic acid 43 29 2 36 9.9 Ethyldiaminedihydroiodide 100 118 114 3 111 9.5 Penta calcium orthoperiodate 99 120 2 111 14.8

References Aschbacher, P.W., Cragle, R.G., Swanson, E.W., Miller, J. K., 1966. Metabolism of oral iodide and

3,5 diiodosalicylic acid in the pregnant cow. J. Dairy Sci. 49, 1042-1045. Aschbacher, P.W., Miller, J.K., Cragle, R. G., 1963. Metabolism of diiodosalicylic acid in dairy

calves. J. Dairy Sci. 46, 1114-1117. Brzoska, F., Pyska, H., Pietras, M., Wiewiora, W., 1999. Effect of iodine source on iodine content in

milk and iodine status of dairy cows. Ann. Anim. Sci. 26, 93-103. Herzig, I., Pisarikova, B., Kursa, J., Suchy, P., 2000. Utilisation of iodine from different sources in

pigs. Archives Anim. Nutr. 53, 179-189. Hixson O.F., Rosner, L., 1957. Calcium iodate as a source of iodine in poultry nutrition. Poultry Sci.

36, 712-714. Kuznetsov, S.G., Bataeva, A.P., Ovcharenko, A.G., Aukhatova, S.N., 1992. [Biologic availability of

iodine for piglets and stability of its compounds in premixes]. Sel’skokhozyajstvennaya Biologya, No. 2, 31-39.

Miller, J.K., Moss, B.R., Swanson, E.W., Aschbacher, P.W., Cragle, R.G., 1968. Calcium iodate and pentacalcium orthoperiodate as sources of supplemental iodine for cattle. J. Dairy Sci. 51, 1831-1835.

Miller, J.K.and Swanson, E.W., 1973. Metabolism of ethylenediaminedihydroiodide and sodium or potassium iodide by dairy cows. J. Dairy Sci. 56, 378-384.

Moss, B.R., Miller, J.K., 1970. Metabolism of sodium iodine, calcium iodate and pentacalcium orthoperiodate initially placed in the bovine rumen or abomasum. J. Dairy Sci. 53, 772-775.

Swanson, E.W., Miller, J.K., Mueller, C.C., Bacon, J.A., Ramsey, N., 1990. Iodine in milk and meat of dairy cows fed different amounts of potassium iodide or ethylenediamine dihydroiodide. J. Dairy Sci. 73, 398-405.

83

V. MANGANESE BIOAVAILABILITY

General function in the body Manganese is one of the least abundant trace elements in all livestock tissues. In the early 1930s manganese was first recognised as an essential dietary nutrient for growth and reproduction in rats and mice (Kemmerer et al, 1931 ; Orent and McCollum, 1931 cited by Underwood and Suttle, 1999). About five years later, two diseases of poultry, known as perosis (slipped tendon) and nutritional chondrodystrophy were found to be prevented by sufficient manganese supplements (Lyons and Insko, 1937 ; Wilgus et al, 1936, 1937 cited by Underwood and Suttle, 1999 and Henry, 1995). Now, manganese is recognised to be essential for all animal species. The mineral forms a link in the chain of calcium metabolism. The major functions of manganese can be linked to the metalloenzymes. Insufficient manganese impairs the cartilage development, through the reduced synthesis of mucopolysaccharides, and causes skeletal disorders (Leach and Muenster, 1962). Impaired mucopolysaccharide synthesis may also result in subnormal egg production and poor shell formation. Manganese has an important function in blood clotting and lipid and carbohydrate metabolism. As manganese deficiency lowers the activity of the manganese superoxide dismutases, the element is very important for the protection of cells against damage by the free oxygen radical O2

-. Abnormal male and female reproductive functions are also related with a lack of sufficient manganese. Absorption is affected by manganese source, dietary antagonists (fibre, phytate, high levels of calcium and phosphorus, iron, magnesium) and depends on the concentration in the diet. Indeed, the efficiency of manganese absorption is raised when the concentration of the element in the diet is lowered. Absorbed manganese is transported to the liver from where any surplus can be excreted partially via the bile. As the amounts of manganese increase, progressively larger proportions are excreted via the faeces.

Use of manganese for livestock

Most diets for ruminants are likely to be deficient in manganese and there is a critical supplemental need for all categories of poultry. Manganese can be supplemented as manganese sulphate, manganese oxide or various organic forms. Although manganese sulphate has a somewhat higher bioavailability than manganese oxide, the latter form is mostly used in premixes.

MANGANESE BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs The ranking of importance for assessing the relative biological value for pigs of manganese sources of the various criteria is listed in Table V.1.

Above requirement

Table V.1: Ranking of importance of various response criteria for assessing the relative biological value of manganese sources in pigs Criterion Suboptimal supply

4 3 Bone manganese 2 2 Animal performance 2 no Liver/kidney manganese 1

Manganese absorption

no

84

Table V.1 shows that there are only a few criteria that are well suitable for evaluation of the bioavailability of manganese sources for pigs. Manganese absorption ranks highest. Bile manganese content and retention of manganese were not regarded as suitable response parameters for assessing the bioavailability of manganese. For our study the reference source is manganese sulphate monohydrate (MnSO4·1H2O) RG (reagent grade).

Comparison of manganese sources for pigs There was only one publication suitable for our study, which is listed in Table V.2.

Table V.2: Overview of the experiments used for evaluation of the relative biological value of manganese sources in pigs Sources of manganese Response criteria Added Mn

(mg/kg) Number of

experiments Reference

Manganese sulphate monohydrate, Manganese carbonate,

Absorption of Mn

Manganese oxide

10 2 Kayongo-Male et al, 1980

As there was only one experiment available for evaluation it is difficult to make firm conclusions. The relative bioavailability of the sources manganese sulphate monohydrate, manganese carbonate, and manganese oxide was 100, 95 and 96, respectively. The authors concluded that no significant differences among the sources tested could be demonstrated for the growing pig.

Table V.3: Summarised results on the relative biological value of manganese sources for pigs Reference Kayongo-Male et al, 1980

Manganese sulphate monohydrate 95

Manganese oxide 96

Number of experiments 2 100

Manganese carbonate

MANGANESE BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry The sequence of the response criteria is listed in Table V.4. Different weights are used if animals are fed manganese at a suboptimal level or above requirement. Bile manganese and manganese retention are not included in the list.

Table V.4: Ranking of importance of various response criteria for assessing the relative biological value of manganese sources in poultry

Above requirement Criterion Suboptimal supply True Mn absorption 4 3 Perosis severity index 3 - Bone Mn 2 2

no 1 -

Performance 2 Liver/kidney Mn

85

Table V.5: Overview of the experiments used for evaluation of the relative biological value of manganese sources in poultry Sources of manganese

Animal type

Response criteria

Added Mn (mg/kg)

Number of expts

Reference Ref. no

Mn-proteinate Broiler Tibia ash Mn 1000 1 Mn1 Baker and

Halpin, 1987 MnSO4⋅H2O MnCO3 MnO

Broiler Tibia ash Mn 1000, 2000, 4000

1 Mn2 Black et al, 1984

MnSO4⋅H2O MnO

Broiler Kidney Mn Tibia ash Mn Liver Mn

40, 80, 120 1 Mn3 Henry et al, 1986

MnSO4⋅H2O MnO Mn-methionine

Broiler Tibia ash Mn 700, 1400, 2100

1 Mn4 Henry et al, 1989

MnO MnO2 (2 origins)

Broiler Tibia ash Mn 1000, 2000, 3000

1 Mn5 Henry et al, 1987

MnSO4⋅H2O MnO⋅Mn2O3 (2 origins) MnO

Broiler Tibia ash Mn 50 1 Mn6 Korol et al, 1996

MnSO4⋅H2O MnO Mn proteinate

Broiler Tibia ash Mn 1000, 2000, 3000

1 Mn7 Smith et al, 1995

MnSO4⋅H2O MnCl2⋅4H2O

Tibia ash Mn Broiler 3000 1 Mn8 Southern and Baker, 1983

MnSO4⋅H2O MnO (4 origins)

Broiler Tibia ash Mn 1000, 2000, 3000

1 Mn9

Wong-Valle et al, 1989

MnSO4⋅H2O

Comparison of manganese sources for poultry The main response criterion used to measure manganese bioavailability is tissue or organ deposition of the element (bone, liver, kidney) as shown in Table V.5. The tissue and bone manganese content was converted, if the original contents were available in the article, using the 10log of this value in order to prevent too large relative values. Hydrated manganese sulphate was generally the reference standard. From the comparison of manganese bioavailability in different sources (Table V.6), it appears that MnCl2⋅4H2O has equal bioavailability as the reference source, while the manganese proteinate has about 10% higher bioavailability. Manganese oxide, manganese carbonate and especially manganese dioxide have lower RBV values than manganese sulphate. Manganese methionine has the same RBV as manganese sulphate.

86

Table V.6: Summarised results on the relative biological value of manganese sources for poultry Reference Mn1 Mn2 Mn3 Mn4 Mn5 Mn6 Mn7 Mn8 Mn9 n Mean SD Number of experiments

1 1 1 1 1 1 1 1 1 9

MnSO4⋅H2O 100 100 100 100 100 100 100 100 8 100 - 81 70 94 (85)a 74 99 91 6 85 11.7

MnO2 31a 31 1 - Mn-methionine 101 1 101 - MnO⋅Mn2O3 68 1 68 - Mn-proteinate 101 117 2 109 11.9

3 66 1 - MnCl2⋅4H2O 1 97 97 -

MnO

MnCO 66

a recalculated using MnO as a reference

MANGANESE BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants

Table V.7: Ranking of importance of various response criteria for assessing the relative biological value of manganese sources in ruminants Criterion

As described earlier, the sequence of response criteria depends on the fact whether the animals are fed suboptimal or if they are fed above their requirement. The ranking of importance for manganese of the various criteria is listed in Table V.7.

Suboptimal supply Above requirement True absorption 4 2

2 1 Kidney accumulation 2 1

3

Liver accumulation

Bone accumulation 1 It can be seen in Table V.7 that there are only few criteria that can be used for evaluation of manganese sources in ruminant. All the observations we used in this study were based on manganese tissue accumulation. Liver manganese accumulation has been transformed to aintain the RBV variation within physiological limits.

10log in order to m

For our study the reference source is manganese sulphate monohydrate RG.

Comparison of manganese sources for ruminants There were in total only 3 publications grouping 7 experiments that were suitable for our study. This is listed in Table V.8.

87

Table V.8: Overview of the experiments used for evaluation of the relative biological value of manganese sources in ruminants Sources of manganese Number

of expts Animal type

Response criteria Added Mn (mg/kg)

Ref. no

Reference

Manganese oxide FG Manganese carbonate RG

Sheep Liver accumulation Kidney accumulation Bone accumulation

2000 4000

Mn1

2 Black et al, 1985

Manganese sulphate RGManganese oxide FG Manganese methionine

Sheep 900, 1800, 2700

Mn2

Liver accumulation Kidney accumulation Bone accumulation

4 Henry et al, 1992

Manganese sulphate RGManganese carbonate RG Manganese oxide RG Manganese dioxide RG

Sheep Liver accumulation Kidney accumulation Bone accumulation

3000 1 Mn3

Wong-Valle et al 1989

Comparisons have been summarised in Table V.9. Dioxide and carbonate had markedly lower bioavailabilities than the reference source while manganese monoxide showed a substantial variation relating to the source tested (reagent or feed grade). Manganese methionine had a higher bioavailability when compared to manganese sulphate.

Mn2 SD

Table V.9: Summarised results on the relative biological value of manganese sources for ruminants Manganese source Mn 1 Mn3 n Mean Number of experiments 2 4 1 7 Manganese sulphate 100 100 2 100 - Manganese carbonate RG 74a 61 2 69 9.2 Manganese monoxide FG (91)a 91 1 91 - Manganese monoxide RG - 80 1 80 Manganese dioxide RG 67 1 67 - Manganese methionine FG 113 1 113 - a Recalculated with manganese monoxide FG as reference source

References Baker, D.H., Halpin, K.M., 1987. Research Note: Efficacy of a manganese-protein chelate compared

with that of manganese sulphate for chicks. Poultry Sci. 66, 1561-1563. Black, J.R., Ammerman, C.B., Henry, P.R., 1985. Effects of high dietary manganese as manganese

oxide or manganese carbonate in sheep. J. Anim. Sci. 60, 861-867. Black, J.R., Ammerman, C.B., Henry, P.R., Miles; R.D., 1984. Biological availability of manganese

sources and effects of high dietary manganese on tissue mineral composition of broiler-type chicks. Poultry Sci. 63, 1999-2006.

Henry, P.R., 1995. Manganese bioavailability. In : Bioavailability of nutrients for animals. Amino acids, minerals and vitamins. Eds. : Ammerman, C.B., Baker, D.H., Lewis, A.J.. Academic Press, San Diego, New York, Boston, London, Sydney, Tokyo and Toronto, USA p. , 239-256.

Henry, P.R., Ammerman, C.B., Littell, R.C., 1992. Relative bioavailability of manganese from a manganese-methionine complex and inorganic sources for ruminants. J. Dairy Sci. 75, 3473-3478.

Henry, P.R., Ammerman, C.B., Miles, R.D., 1986. Bioavailability of manganese sulphate and manganese monoxide in chicks as measured by tissue uptake of manganese from conventional dietary levels. Poultry Sci. 65, 983-986.

88

Henry, P.R., Ammerman, C.B., Miles, R.D., 1987. Bioavailability of manganese monoxide and manganese dioxide for broiler chicks. Nutrition Reports International 36 , 425-433.

Henry, P.R., Ammerman, C.B., Miles, R.D., 1989. Relative bioavailability of manganese-methionine complex for broiler chicks. Poultry Sci. 68, 107-112.

Kayongo-Male, H., Ullrey, D.E., Miller, E.R., 1980. Manganese (Mn) nutrition of the pig. 2. The availability of Mn from different sources to the growing pig. Bull. Anim. Hlth Prod. Afr. 28, 145-153.

Korol, W., Wojcik, S., Matyka, S., Hansen, T.S., 1996. Availability of manganese from different manganese oxides and their effect on performance of broiler chickens. J. of Anim. Feed Sci. 5, 273-279.

Leach, R.M. jr., Muenster, A.M. 1962. Studies on the role of manganese in bone formation. I. Effect upon the mucopolysaccharide content of chick bone. J. Nutri. 78, 51-56.

Smith, M.O., Sherman, I.L., Miller, L.C., Robbins, K.R., 1995. Relative biological availability of manganese form manganese proteinate, manganese sulphate and manganese monoxide in broilers reared at elevated temperatures. Poultry Sci. 74, 702-707.

Southern, L. L., Baker, D.H., 1983. Excess manganese ingestion in the chick. Poultry Sci. 62, 642-646.

Underwood, E.J., Suttle, N.F., 1999. The mineral nutrition of livestock. 3rd edition. Moredun Research Institute, Pentland Science Park, Midlothian, UK, p. 397-420.

Wong-Valle J., Ammerman, C.B., Henry P.R., Rao, P.V., Miles, R.D., 1989. Bioavailability of manganese from feed grade manganese oxides for broiler chickens. Poultry Sci. 68, 1368-1373.

Wong-Valle, J., Henry, P.R., Ammerman, C.B., Rao, P.V. 1989., Estimation of the relative bioavailability of manganese sources for sheep. J. Anim. Sci. 67, 2409-2414.

89

VI. MOLYBDENUM BIOAVAILABILITY

General function in the body Molybdenum, in the first place, has been considered as a toxic element because of its interference with the copper metabolism in ruminants (Underwood, 1977). Molybdenum has been recognised as being an essential element for animals (De Renzo et al, 1953; Richert and Westerfield, 1953) as a component of the enzymes xanthine oxidase, aldehyde oxidase and sulphite oxidase (Bray, 1974, cited by McDowell, 1992). Xanthine oxidase and aldehyde oxidase are involved in the electron transport chain in the cells, aldehyde oxidase in niacin metabolism and sulphite oxidase in sulphite to sulphate conversion for excretion in urine (McDowell, 1992). No data were available in the literature on the availability of molybdenum sources for pigs and poultry.

Use of molybdenum for livestock There is no evidence of characteristic symptoms of molybdenum deficiency in ruminants and animal performance was not affected by very low levels of molybdenum in the diet (McDowell, 1992). Nevertheless, Ellis et al (1958) showed improvement in cellulose digestibility and performances in lambs when dietary molybdenum increased from 0.36 to 2.37 mg/kg in the diet. The supplementation of ruminant diets is not usual for molybdenum but in certain cases it is added to ruminant diets to counteract toxicity risk due to high levels of copper supply (Underwood and Suttle, 1999). There were no publications suitable for the study of molybdenum bioavailability for pigs and poultry.

MOLYBDENUM BIOAVAILABILITY FOR PIGS

There were no publications suitable for the study of molybdenum bioavailability of pigs.

MOLYBDENUM BIOAVAILABILITY FOR POULTRY

There were no publications suitable for the study of molybdenum bioavailability of poultry.

MOLYBDENUM BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants As described earlier, the sequence of response criteria depends on the fact whether the animals are fed suboptimal levels of molybdenum or if they are fed above their molybdenum requirement. The ranking of importance for molybdenum of the various criteria is listed in Table VI.1.

90

Table VI.1: Ranking of importance of various response criteria for assessing the relative biological value of molybdenum sources in ruminants Criterion Suboptimal supply Above requirement Molybdenum apparent absorption 4 4

5 5 Molybdenum tissue accumulation 3 3 Performance 2 2 Molybdenum retention (balance) 2 2

Molybdenum true absorption

It can be seen in Table VI.1 that there are only few criteria that can be used for evaluation of molybdenum sources in ruminants of which absorption is the most important one. Molybdenum tissue accumulation is markedly influenced by the level of sulphate and wolfram (Underwood, 1977). For our study the reference source is mono sodium molybdate dihydrate (NaMoO4.2H2O) RG.

Comparison of molybdenum sources for ruminants There was only one publication that was suitable for our study (Pott et al, 1999). This work was based on liver and kidney molybdenum accumulation with 30 mg/kg molybdenum added to the diet. These tissue accumulations have been transformed to 10log in order to maintain the RBV variation within physiological limits. Results are reported in Table VI.2.

Table VI.2: Summarised results on the relative biological value of molybdenum sources for ruminants Reference Pott et al, 1999 Number of experiments 1 Mono sodium molybdate dihydrate 100 Ammonium molybdate 114 Molybdenum trioxide 121 Molybdenum metal 60 It is of course difficult to conclude with only one experiment but this result indicates that sodium molybdate, ammonium molybdate and molybdenum trioxide are good sources of molybdenum, but not molybdenum metal.

References De Renzo, E.C., Kaleita, E., Heytler, P., Oleson, J.J., Hutchings, B. L., Williams, J.H., 1953. The

nature of the xanthine oxidase factor. J. Am. Chem. Soc. 75, 753. Ellis, W.C., Pfander, W.H., Muhrer, M.E., Pickett, E.E., 1958. Molybdenum as a dietary essential for

lambs. J. Anim. Sci. 17, 180-188. McDowell, L.R. 1992., Minerals in animal and human nutrition. Academic Press, San Diego. Pott, E.B., Henry, P.R., Rao, P.V., Hindberger, E.J., Ammerman, C.B., 1999. Estimated relative

bioavailability of supplemental inorganic molybdenum sources and their effect on tissue molybdenum and copper concentrations in lambs. Anim. Feed Sci. Technol. 79, 107-117.

Richert, D.A., Westerfield W.W.,1953. Isolation and identification of the xanthine oxidase factor as molybdenum. J. Biol. Chem. 203, 915-923.

Underwood, E.J., 1977. Trace elements in human and animal nutrition. Academic Press, New York. Underwood, E.J., Suttle N.F., 1999. The mineral nutrition of livestock. CABI Publishing, Wallingford

UK.

91

VII. SELENIUM BIOAVAILABILITY

General function in the body Selenium was first, and for a long time, considered as a toxic element causing the lost of hair, nails and hooves. Most cases of chronic selenium toxicity have been related to consuming selenium-accumulating plants; nevertheless chronic toxicity can occur with a quite low selenium level (from 5 mg/kg), but acute toxicity needs a much higher selenium level in the diet (NRC, 2001). The finding that selenium may prevent liver necrosis in rats (Schwartz & Foltz, 1957) gave the first indication of the beneficial effects of selenium in nutrition and health. In the late 50’s selenium was recognised for preventing liver and muscular diseases. Selenium is a main constituent of glutathione peroxidase which, associated with vitamin E plays a role of “cellular scavenger” protecting cellular membranes from oxidative effects (peroxides and free radicals). Other roles of selenium have been reported predominantly for the optimisation of immune response in several species (McDowell, 1992). Some recent publications show that supplementation of selenium (and vitamin E), markedly above nutritional requirements, during late pregnancy allows a better control of mastitis and somatic cell count (NRC, 2001). Selenium may also protect the animal organism from detrimental effects of heavy metals including cadmium, mercury and silver (McDowell, 1992).

Use of selenium for livestock The selenium content of food shows wide variations, especially in forages, which depends on the soil concentration of selenium. Very large areas are selenium-deficient where a selenium supplementation is essential. Seeds and by-products have generally a higher selenium content than forages. Animal by-products including fish meals, but with the exception of milk products have high concentration of selenium. Selenium is mostly supplemented as sodium selenite. Due to the high toxicity of sodium selenite and the potential risks involved in handling it, other less toxic selenium sources with a good bioavailability must be investigated.

SELENIUM BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs As described earlier, the sequence of importance of response criteria depends on whether the animals are fed suboptimal levels of selenium or if they are fed above their selenium requirement. The ranking of importance for assessing the relative biological value of selenium sources of the various criteria is listed in Table VII.1.

Table VII.1: Ranking of importance of various response criteria for assessing the relative biological value of selenium sources in pigs Criterion Suboptimal supply Above requirement Selenium absorption/digestibility 2 1 Selenium absorption (true) 2 2 5-iodothyronine deiodinase 1 1 Glutathione peroxidase activity 4 2 Serum selenium 3 1 Animal performance 2 no Exudative diathesis 1 no Pancreatic fibrosis 1 no Milk selenium 2 2 Tissue selenium (liver, kidney) 2 2

92

Table VII.1 shows that there are several criteria that can be used for evaluation of the bioavailability of selenium sources for pigs of which glutathione peroxidase activity ranks highest. For our study the reference source is sodium selenite (Na2SeO3).

Comparison of selenium sources for pigs There were seven publications suitable for our study. These are listed in Table VII.2. Apart from the reference source sodium selenite, yeast selenium was used in most experiments. Only seleno methionine and calcium selenite were used in one experiment. The concentrations in organs were converted by using 10Log, with the restriction that the concentrations were first multiplied by 10 or 100 to obtain values above 1.0. Furthermore, in some experiments the same response parameters were measured both with lactating sows and their litters. Therefore, to obtain one figure for that criterion, the results from both sows and piglets were multiplied with one half of the weighing factor.

Table VII.2: Overview of the experiments used for evaluation of the relative biological value of selenium sources in pigs (GSH = glutathione peroxidase activity) Sources of selenium

Animal type

Response criteria used

Added Se (mg/kg)

No. of expts

Ref. no

Reference

Sodium selenite, Calcium selenite

12 – 33 kg Performance GSH Serum Se Tissue Se

0.3, 5.0 1 Se1 Mahan and Magee, 1991

Sodium selenite, Yeast selenium

Gilts lactating

Performance GSH Milk Se Tissue Se

0.1, 0.3 1 Se2 Mahan and Kim, 1996

20 – 105 kg GSH Tissue Se Digestibility

0.1, 0.3, 0.5

3 Se3 Mahan and Parrett, 1996

Yeast selenium 20 – 105 kg Performance

GSH Tissue Se

0.05, 0.1, 0.2, 0.3

1 Se4 Mahan et al, 1999

Sodium selenite, Yeast selenium

Sows lactating

Performance GSH Milk Se Tissue Se

0.15, 0.30 1 Se5 Mahan, 2000

Sodium selenite, Seleno methionine

6 – 105 kg Absorption GSH Tissue Se

0.1 2 Se6 Parsons et al, 1985

Sodium selenite, Seleno methionine (yeast)

22 – 110 kg Performance 0.3 1 Se7 Wolter et al, 1999

Sodium selenite, Yeast selenium

Performance

Sodium selenite,

Table VII.3 shows that two or more observations were available for yeast selenite. There was only one observation available of the other selenium sources. The yeast selenium had a slightly higher relative biological value than sodium selenite, which predominantly is caused by the sow experiment by Mahan (2000). In this experiment colostrum and milk Se content were substantially enhanced. Seleno methionine and calcium selenite had the same bioavailability as sodium selenite.

93

Table VII.3: Summarised results on the relative biological value of selenium sources for pigs Reference Se1 Se2 Se3 Se4 Se5 Se6 Se7 n Mean SD Number of experiments 1 1 3 1 1 2 1 10 Sodium selenite 100 100 100 100 100 100 100 7 100 - Calcium selenite 102 1 102 - Seleno methionine 104 100 2 102 2.3 Yeast selenium 106 97 103 137 4 108 17.8

SELENIUM BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry Response criteria are ranked along their importance in Table VII.4. Differences are made between suboptimal supply and feeding above requirement.

Table VII.4: Ranking of importance of various response criteria for assessing the relative biological value of selenium sources in poultry Criterion Suboptimal supply Above requirement

2 1 Se absorption (true) 2 2 5-Iodothyronine deiodinase 1 1 Glutathione peroxidase activity 4 2 Performance 2 no Exudative diathesis 1 no Pancreatic fibrosis 1 no Tissue Se (liver, kidney) 2 2 Egg yolk Se (layers) (2) 1

Se absorption/digestibility (ileal/faecal)

94

Table VII.5: Overview of the experiments used for evaluation of the relative biological value of selenium sources in poultry Sources of selenium Animal

Type Response criteria Added

Se (µg/kg)Number of expts

Ref. no

Reference

Na2SeO3 Se-methionine

Laying hen

Egg Se 100 1 Se1 Cantor and Scott, 1974

Na2SeO3 Sodium selenate Seleno-DL-cystine Seleno-DL-methionine Seleno-DL-ethionine

Turkey Glutathione peroxidase

200 1 Se2 Cantor and Tarino, 1982

Na2SeO3 Se-DL-methionine Seleno-DL-cystine 6-Selenopurine Seleno-DL-ethionine Na2SeO4 Sodium selenide Elemental Se

Broiler Exudative diasthesis 20, 40, 60 1 Se3 Cantor et al, 1975a

Na2SeO3 Se-DL-methionine Seleno-DL-cystine

Broiler Glutathione peroxidase

(10), 20, 40

1 Se4 Cantor et al, 1975b

Na2SeO3 Se-DL-methionine

Turkey

Glutathione peroxidase

40, 80, 120 1 Se5 Cantor et al, 1982

Na2SeO3 Selenodicysteine

Chick Liver Se Glutathione peroxidase

50, 100, 150

1 Se6 Cantor et al, 1983

Na2SeO3

3CaSeO3⋅4H2O Na2SeO3+carrier Na2SeO4 Se metal

Broiler Liver Se Kidney Se

3000, 6000, 9000

1 Se7 Echevarria et al, 1988

2SeO3 Se-methionine

Chick Glutathione peroxidase

20, 40, 60, 80, 100, 120

1 Se8 Gabrielsen and Opstvedt, 1980

Na2SeO3 L-Se-methionine

Chick Se absorption 40, 400 1 Se9 Humaloja and Mykkanen, 1986

2SeO3 Se-cystine Se-methionine

Laying hen

Se absorption Yolk Se

100 1 Latshaw and Osman, 1975

Na2SeO3 Se-methionine

Broiler Whole body Se 20, 50, 75, 100, 125, 500

1 Se11

Miller et al, 1972

Na2SeO3 Se-DL-methionine

Broiler Glutathione peroxidase

20, 40, 60 1 Se12

Noguchi et al, 1973

Na2SeO3 Se-DL-methionine Se-cystine

Broiler Liver Se Kidney Se

10, 20, 40, 60

1 Se13

Osman and Latshaw, 1976

Na2SeO3 Se-DL-methionine

Chick Glutathione peroxidase

30, 60, 90 1 Se14

Zhou and Combs, 1984

Na

Na Se10

95

Comparison of selenium sources for poultry The tissue selenium content was converted, if the original contents were available in the article, using the 10log of this value in order to prevent too large values. Summarised results on selenium bioavailability for broilers and layers are presented in Tables VII.6-VII.7. Sodiumselenite (ref.), 3CaSeO3⋅4H2O and selenodicysteine show the highest bioavailabilities, while Na2SeO4 has a slightly lower RBV. DL-seleno methionine and DL-seleno cystine have about 20% lower RBV compared to the reference sodium selenite. Metallic selenium, seleno-DL-methionine, seleno-purine and sodium selenide have less than half of the bioavailability of selenium in the reference source. For turkeys (Table VII.8) and according to the same authors (Cantor et al) DL-seleno cystine and sodium selenate show a much higher RBV than the reference source compared to chicks. Explanations for this are not available. In experiments with laying hens, no differences in RBV were found between the reference sodium selenite and seleno-DL-methionine and DL-selenocystine.

Table VII.6: Summarised results on the relative biological value of selenium sources for broilers Reference Se3 Se4 Se6 Se7 Se8 Se14 MeanSe9 Se11 Se12 Se13 n SDNumber of experiments

1 1 1 1 1 1 10

1 1 1 1

100 100 100 100 100 100 100 100 10

100

DL-seleno-methionine

37 68 78 105 122 85 8 78 27.153 76

DL-seleno-cystine

74 53 114 3 80 31.0

3CaSeO3⋅4H2O 104 104 - 1 Na2SeO3+carrier 102 102 - 1 Na2SeO4 74 109 2 92 24.7

55.9Selenodicysteine 99 1 99 -

44 1 44

20 - Sodium selenide 42 1 42 -

Sodium selenite 100 100 -

Se metal 7 86 2 47

Seleno-DL-ethionine

-

6-Selenopurine 20 1

Table VII.7: Summarised results on the relative biological value of selenium sources for layers Reference Se1 Se10 n Mean SD Number of experiments 1 1 2 Sodium selenite 100 100 2 100 - DL-selenomethionine 102 98 2 100 2.8 DL-selenocystine 101 1 101 -

96

Table VII.8: Summarised results on the relative biological value of selenium sources for turkeys Reference Se2 SD Se5 n Mean

100 100 2 100 -

DL-selenocystine 141 1 141 - Na2SeO4 205 1 205 - Seleno-DL-ethionine 74 74 - 1

Number of experiments 1 1 2 Sodium selenite DL-selenomethionine 81 115 2 98 24.0

SELENIUM BIOAVAILABILITY FOR RUMINANTS

Table VII.9: Ranking of importance of various response criteria for assessing the relative biological value of selenium sources in ruminants Criterion

Sequence of important response criteria and evaluation methods used for ruminants As described earlier, the sequence of response criteria depends on the fact whether the animals are fed suboptimal or if they are fed above their requirement. The ranking of importance for selenium of the various criteria is listed in Table VII.9.

Suboptimal supply Above requirement True absorption 5 3 Liver accumulation 4 2 Kidney accumulation 3 1 Enzyme activity 2 2 Table VII.9 shows that there are only few criteria that can be used for evaluation of selenium sources in ruminants. Three observations were based on selenium tissue accumulation, seven on enzymatic activity (glutathione peroxidase) and only one on true absorption. Tissue selenium accumulation has been transformed to in order to maintain the relative biological value variation in physiological limits. For our study the reference source is sodium selenite (Se

There were in total 10 publications involving 17 experiments that were suitable for our study. This is listed in Table VII.10.

10log 2O3) RG.

Comparison of selenium sources for ruminants

97

Table VII.10: Overview of the experiments used for evaluation of the relative biological value of selenium sources in ruminants

Added Se (mg/kg)

Reference Sources of selenium Animal type

Response criteria Number of expts

Ref. no.

Kidney accumulation

6/d* 1 Se1 Henry et al, 1988

Sodium selenite Yeast

Sheep True absorption Labelled selenium

2 Se2 Koenig et al, 1997

Sodium selenite Yeast

Calves Enzyme activity 1 4 Se3 Nicholson et al, 1991

Sodium selenite Yeast

Cows Liver accumulation Kidney accumulation Enzyme activity

3/d* 1 Se4 Ortman and Pehrson, 1997

Sodium selenite Sodium selenate Yeast

Cows Enzyme activity 0.3 1 Se5 Ortman and Pehrson, 1999

Sodium selenite Sodium selenate Yeast

Heifers Enzyme activity 0,25 1 Se6 Ortman et al, 1999

Sodium selenite Cobalt selenite

Enzyme activity to 1.21/d*

1 Se7 Pehrson et al, 1989

Selenomethionine Yeast

Heifers 0.45

Sodium selenite Yeast

Cows Enzyme activity 3.3/d* 2 Se9 Pehrson et al, 1999

Sodium selenite Sodium selenate

Cows Sheep

Enzyme activity 0.3 2 Se9 Podoll et al, 1992

Sodium selenite Calcium selenite

Sheep Liver accumulation Kidney accumulation

3/d* 2 Se10

Sodium selenite Calcium selenite Sodium selenate

Sheep Liver accumulation

Tarla et al, 1989

* Total selenium added per day, no information available on dry matter intake Comparisons have been summarised in Table VII.11. This table shows that except for sodium selenate (n = 4) and yeast cultivated on selenium-rich medium (n = 7) there are only few comparisons available. The relative biological values of salts under investigation ranks between 101 (cobalt selenite, only one study) and 124 (selenomethionine, only one study) meaning that all sources present in this study can be recommended for selenium supplementation in ruminants.

Table VII.11: Summarised results on the relative biological value of selenium sources for ruminants Reference Se1 Se2 Se3 Se4 Se5 Se6 Se7 Se8 Se9 Se10 n Mean SD Number of experiments 1 2 4 1 1 1 1 2 2 2 17 Sodium selenite 100 100 100 100 100 100 100 100 100 100 10 100 - Calcium selenite 120 105 2 111 10.6 Cobalt selenite 101 1 101 - Sodium selenate 124 102 106 98 4 107 11.5 Selenomethionine 124 1 124 - Yeast 90 106 103 113 118 128 114 7 109 12.1

98

References Cantor, A.H., Langevin, M.L., Noguchi, T., Scott, M.L., 1975b. Efficacy of selenium in selenium

compounds and feedstuffs for prevention of pancreatic fibrosis in chicks. J. Nutr. 105, 106-111. Cantor, A.H., Moorhead, P.D., Musser, M.A., 1982. Comparative effects of sodium selenite and

selenomethionine upon nutritional muscular dystrophy, selenium-dependent glutathione peroxidase, and tissue selenium concentrations of turkey poults. Poultry Sci. 61, 478-484.

Cantor, A.H., Scott, M.L., 1974. The effect of selenium in the hen’s diet on egg production, hatchability, performance of progeny and selenium concentration in eggs. Poultry Sci. 53, 1870-1880.

Cantor, A.H., Scott, M.L., Noguchi, T., 1975a. Biological availability of selenium in feedstuffs and selenium compounds for prevention of exudative diathesis in chicks. J. Nutr. 105, 96-105.

Cantor, A.H., Sutton, C.D., Johnson, T.H., 1983. Biological availability of selenodicysteine in chicks. Poultry Sci. 62, 2429-2432.

Cantor, A.H., Tarino, J.Z., 1982. Comparative effects of inorganic and organic dietary sources of selenium on selenium levels and selenium-dependent glutathione peroxidase activity in blood of young turkeys. J. Nutr. 112, 2187-2196.

Echevarria, M.G., Henry, P.R., Ammerman, C.B., Rao, P.V., Miles, R.D., 1988. Estimation of the relative bioavailability of inorganic selenium sources for poultry. 2. Tissue uptake of selenium from high dietary selenium concentrations. Poultry Sci. 67, 1585-1592.

Gabrielsen, B.O., Opstvedt, J., 1980. Availability of selenium in fish meal in comparison with soybean meal, corn gluten meal and selenomethionine relative to selenium in sodiumselenite for restoring glutathione peroxidase activity in selenium-depleted chicks. J. Nutr. 110, 1096-1100.

Henry, P.R., Echevaria, M.G., Ammerman, C.B., Rao, P.V., 1988. Estimation of the relative biological availability of inorganic selenium sources for ruminants using tissue uptake of selenium. J. Anim. Sci. 66, 2306-2312.

Humaloja, T., Mykkanen, H.M., 1986. Intestinal absorption of Se-labelled sodium selenite and selenomethionine in chicks: Effects of time, segment, selenium concentration and method of measurement. J. Nutr. 116, 142-148.

Koenig, K.M., Rode, L.M., Cohen, R.D.H., Buckley, W.T., 1997. Effects of diet and chemical form of selenium on selenium metabolism in sheep. J. Anim. Sci. 75, 817-827.

Latshaw, J.D., Osman, M., 1975. Distribution of selenium in egg white and yolk after feeding natural and synthetic selenium compounds. Poultry Sci. 54, 1244-1252.

Mahan, D.C., 2000. Effect of organic and inorganic selenium sources and levels on sow colostrum and milk selenium content. J. Anim. Sci. 78, 100-105.

Mahan, D.C., Cline, T.R., Richert, B., 1999. Effects of dietary levels of selenium-enriched yeast and sodium selenite as selenium sources fed to growing-finishing pigs on performance, tissue selenium, serum glutathione peroxidase activity, carcass characteristics, and loin quality. J. Anim. Sci. 77, 2172-2179.

Mahan, D.C., Kim, Y.Y., 1996. Effect of inorganic or organic selenium at two dietary levels on reproductive performance and tissue selenium concentrations in first-parity gilts and their progeny. J. Anim. Sci. 74, 2711-2718.

Mahan, D.C., Magee, P.L., 1991. Efficacy of dietary sodium selenite and calcium selenite provided in the diet at approved, marginally toxic, and toxic levels to growing swine. J. Anim. Sci. 69, 4722-4725.

Mahan, D.C., Parrett, N.A., 1996. Evaluating the efficacy of selenium-enriched yeast and sodium selenite on tissue selenium retention and serum glutathione peroxidase activity in grower and finisher swine. J. Anim. Sci. 74, 2967-2974.

McDowell, L.R., 1992., Minerals in animal and human nutrition. Academic Press, San Diego. Miller, D., Soares, J.H., Bauersfled, P. jr., Cuppett, S.L., 1972. Comparative selenium retention by

chicks fed sodium selenite, selenomethionine, fish meal and fish solubles. Poultry Sci. 51, 1669-1673.

National Research Council (NRC). 2001. Nutrient requirements of dairy cattle. National Academic Press, Washington.

99

Nicholson, J.W.G., St-Laurent, A.M., McQueen, R.E.,Charmley, E., 1991. The effect of feeding organically bound selenium and alpha-tocopherol to dairy cows on susceptibility of milk to oxidation. Can. J. Anim. Sci. 71, 135-143.

Noguchi, T., Cantor, A.H., Scott, M.L., 1973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103, 1502-1511.

Ortman, K., Andersson, R., Holst, H., 1999. The influence of supplements of selenite, selenate and selenium yeast on the selenium status of dairy heifers. Acta Vet. Scand. 40, 23-34.

Ortman, K., Pehrson, B., 1997. Selenite and selenium yeast as feed supplements for dairy cows. J. Vet. Med. 44, 373-380.

Ortman, K., Pehrson, B., 1999. Effect of selenate as a feed supplement to dairy cows in comparison to selenite and selenium yeast. J. Anim. Sci. 77, 3365-3370.

Osman, M., Latshaw, J.D., 1976. Biological potency of selenium from sodium selenite, selenomethionine, and selenocystine in chick. Poultry Sci. 55, 987-994.

Parsons, M.J., Ku, P.K., Ullrey, D.E., Stowe, H.D., Whetter, P.A., Miller, E.R., 1985. Effects of riboflavin supplementation and selenium source on selenium metabolism in the young pig. J. Anim. Sci. 60, 451-461.

Pehrson, B., Knutsson, M., Gyllensward, M., 1989. Glutathione peroxidase activity in heifers fed diets supplemented with organic and inorganic selenium compounds. Swed. J. Agric. Res. 19, 53-59.

Pehrson, B., Ortman, K., Madjid, N., Trafikowska, U., 1999. The influence of dietary selenium as selenium yeast or sodium selenite on the concentration of selenium in the milk of suckler cows and on the selenium status of their calves. J. Anim. Sci. 77, 3371-3376.

Podoll, K.L., Bernard, J.B., Ullrey, D.E., DeBar, S.R., Ku, P.K., Magee, W.T., 1992. Dietary selenate versus selenite for cattle, sheep, and horses. J. Anim. Sci. 70, 1965-1970.

Schwarz, K., Foltz, C.M., 1957. Selenium as an integral part of factor 3 against necrotic liver degeneration. J. Am. Chem. Society 79, 3292-3293.

Tarla, F.N., Henry, P.R., Ammerman, C.B., Rao, P. V., 1989. Effect of time on tissue deposition of selenium in sheep fed calcium selenite or sodium selenite. Nutr. Rep. Int. 39, 943-949.

Wolter, B., Ellis, M., McKeith, F.K., Miller, K.D., Mahan, D.C., 1999. Influence of dietary selenium source on growth performance, and carcass and meat quality characteristics in pigs. Can. J. Anim. Sci. 79, 119-121.

Zhou, Y., Combs, G.F. jr., 1984. Effects of dietary protein level and level of feed intake on the apparent bioavailability of selenium for the chick. Poultry Sci. 63, 294-303.

100

VIII. ZINC BIOAVAILABILITY

General function in the body The element zinc is considered to be essential for plants, animals, and humans (Hambidge et al, 1986). It activates several enzymes and is a component of many important metalloenzymes. Zinc is the most abundant intracellular trace mineral in animals and total body zinc content is similar to that of iron. However, within cells the concentration of free zinc is extremely low. The element is critically involved in cell replication and in the development of cartilage and bone. Signs of zinc deficiency in animals and humans include retarded growth, abnormal skeletal formation, delayed sexual development, alopecia, dermatitis, abnormal feathering, and impaired reproduction in both males and females. Foetal abnormalities occur and hatchability of eggs is reduced. Many animal diets require supplementation with zinc because of either low dietary levels or the presence of dietary factors that decrease bioavailability of the mineral (e.g. phytic acid). The critical importance of added dietary zinc for domestic animals was shown in 1955 when it was demonstrated that parakeratosis, a condition being observed in pigs, was caused by inadequate dietary zinc (Tucker and Salmon, 1955). It was the practice to feed high calcium levels along with plant proteins containing phytate and this apparently reduced the bioavailability of dietary zinc to the point that a severe deficiency of the element occurred. It was soon demonstrated (O'Dell and Savage, 1957; O'Dell et al, 1958) that zinc was required for normal growth and development in poultry. Results obtained with pigs and poultry probably led to the observations that zinc deficiency can occur in ruminants under grazing conditions in some areas of the world (Hambidge et al, 1986). Livestock exhibits considerable tolerance to high intakes of zinc, the extent depending partly on the species but mainly on the nature of the diet. Zinc toxicosis first appears when levels around 1000 mg/kg are incorporated into diets. For pigs and poultry levels above 2000 mg/kg zinc in the diet exerted a negative effect on body weight gain (NRC, 1980; Underwood and Suttle, 1999). Zinc is primarily absorbed from the small intestine. Current data from rat models indicate that zinc absorption occurs by pericellular and carrier-mediated processes, with uptake at the brush border membrane being rate limiting (Rucker et al, 1994). At concentrations of 0.2 mmol, zinc uptake by isolated brush border membranes is saturated and occurs via an energy-independent carrier-mediated process. At 1 mmol, zinc uptake is non-saturated, suggesting a passive diffusion. Intestinal perfusion studies showed that zinc absorption increased in a linearly between 0.1 and 1.5 mmol, after which it reaches a plateau. Balance studies of Poulsen and Larsen (1994) with gilts demonstrated that the apparent absorption of zinc is in a positive linear interrelationship with its daily consumption in the range from 44 up to 317 mg/day. Low-molecular-weight complexes of zinc facilitate its delivery to the luminal surface of mucosal cells, and newly absorbed zinc is then associated with proteins (e.g. metallothionein and cystein-rich intestinal protein). The transport across the basolateral surface is known to be energy dependent. Absorbed zinc retained by the mucosal cells can be lost by desquamation.

Use of zinc for livestock Zinc is supplemented in diets for all species of livestock in the range of 30 to 250 mg/kg to cover their requirements (Jongbloed et al, 2001). The supplementation rate of zinc is mostly at the lower end of the range for poultry and at the higher end for pigs. Apart from its essential metabolic function, zinc is sometimes used at much higher doses in pig diets up to 3000 mg/kg due to its claimed growth promoting effect. This aspect, however, will not be discussed in this paper. For more information on this aspect the reader is referred to reviews of Tokach et al (1992), Poulsen (1995) and Hill et al (1996). Zinc is mostly supplemented as zinc sulphate (ZnSO4·xH2O) or as zinc oxide in animal diets.

101

ZINC BIOAVAILABILITY FOR PIGS

Sequence of important response criteria and evaluation methods used for pigs As described earlier, the sequence of response criteria depends on the fact whether the animals are fed suboptimal levels of zinc or if they are fed above their zinc requirement. The ranking of importance for zinc of the various criteria is listed in Table VIII.1. Zinc salts, such as sulphate or carbonate, have commonly been used as standards in bioavailability studies.

Table VIII.1: Ranking of importance of various response criteria for assessing the relative biological value of zinc sources in pigs Criterion Suboptimal supply Above requirement Tibia/toe/metatarsal zinc 5 5 Serum/plasma zinc 4 3 Absorption of zinc (apparent) 3 3 Absorption of zinc (true) 3 3 Animal performance 3 no Pancreatic zinc 3 3 Table VIII.1 shows that bone zinc content ranks the highest followed by serum zinc content. Other response criteria, like zinc balance, urinary zinc content, erythrocyte zinc content, liver metalloproteins, hair zinc content and hair/feather condition were not considered to be useful parameters for the evaluation of zinc bioavailability of different zinc sources. The reference source is zinc sulphate monohydrate (ZnSO4·1H2O) RG (reagent grade) or zinc sulphate heptahydrate ZnSO4·7H2O RG.

Comparison of zinc sources for pigs There were a total of seven publications that were suitable for our study, which are listed in Table VIII.2.

Table VIII.2: Overview of the experiments used for evaluation of the relative biological value of zinc sources in pigs Sources of zinc Response criteria Added Zn

(mg/kg) Number of expts

Ref. no

Reference

Zinc sulphate·1H2O Zinc lysine

Absorption Serum zinc Bone zinc

100 4 Zn1 Cheng et al, 1998

Zinc sulphate·7H2O Zinc carbonate Zinc oxide

Absorption 127 1 Zn2 Hap and Zeman, 1994

Zinc sulphate·1H2O Zinc methionine

Performance Bone zinc Serum zinc

9, 12, 15 2 Zn3 Hill et al, 1986

Zinc oxide Metallic zinc dust

Performance Serum zinc

25, 50 1 Zn4 Miller et al, 1981

Zinc sulphate·xH2O Zinc methionine Zinc lysine Zinc oxide

Bone zinc Serum zinc

1000, 2000, 3000

3 Zn5 Schell and Kornegay, 1996

102

Table VIII.2 (continued) Sources of zinc Response criteria Added Zn

(mg/kg) Number of expts

Ref. no

Reference

Zinc sulphate·xH2O Zinc amino acid chelate

Pancreatic zinc 45 2 Zn6 Swinkels et al, 1996

Zinc sulphate·1H2O Zinc methionine Zinc lysine Zinc oxide

Performance, Bone zinc, Plasma zinc

5, 10, 20, 40, 80

2 Zn7 Wedekind et al, 1994

A summary of the comparisons has been presented in Table VIII.3. This table shows that two or more observations were available for zinc-lysine, zinc-methionine and zinc oxide. There was only one observation available of the other zinc sources. Almost all sources of zinc had the same availability as zinc sulphate except for zinc lysine. Surprisingly zinc oxide had only a slightly lower availability than zinc sulphate (94 %), which is mainly due to the value found by Hap and Zeman (1994; Zn2).

Table VIII.3: Summarized results on the relative biological value of zinc sources for pigs Reference Zn1 Zn2 Zn3 Zn4 Zn5 Zn6 Zn7 n Mean SD Number of experiments 4 1 2 1 3 2 1 14 ZnSO4·xH2O 100 100 100 100 100 100 6 100 - Zinc carbonate 98 1 98 - Zinc amino acid chelate 102 1 102 - Zinc lysine 98 92 64 3 89 18 Zinc methionine 101 95 86 3 95 8 Zinc metal dust 105a 1 105 - Zinc oxide 110 3 (92)a 87 82 92 15 a recalculated using zinc oxide as a reference

ZINC BIOAVAILABILITY FOR POULTRY

Sequence of important response criteria and evaluation methods used for poultry The sequence of response criteria depends on the fact whether the animals are fed suboptimal levels of zinc or if they are fed above their requirement. The ranking of importance for zinc of the various criteria is listed in Table VIII.4.

Table VIII.4: Ranking of importance of various response criteria for assessing the relative biological value of zinc sources in poultry Criterion Suboptimal supply Above requirement Absorption of zinc (apparent) 3 1 Absorption of zinc (true) 3 3 Tibia/toe/metatarsal zinc 5 5 Pancreatic zinc 3 3 Performance 3 No Serum/plasma zinc 4 No

103

Table VIII.5: Overview of the experiments used for evaluation of the relative biological value of zinc sources in poultry

Ref. no Sources of zinc Animal type

Response criteria

Added Zn (mg/kg)

Number of expts

Reference

ZnSO4⋅7H2O Zinc sulphate basic Zinc chloride basic Zinc oxide

Broiler Tibia ash Zn (200), 400 Cao et al, 2000 1 Zn1

ZnSO4⋅7H2O Zinc oxide (5 origins)

Zinc metal dust

Tibia ash ZnEdwards and Baker, 1999

ZnSO4⋅H2O (3 origins)

Zinc metal fume

Broiler Performance different according to sources*: 4.73-10.52

3 Zn2

ZnSO4⋅H2O Fe-ZnSO4⋅H2O Zn-FeSO4⋅H2O

4.88 (5.02), 9.75 (10.04) (reference)

Edwards et al, 1998

Broiler PerformanceTibia ash Zn

different according to sources : 5.01-10.22

2 Zn3

ZnSO4⋅7H2O ZnCO3 ZnO (2 origins) Zn metal ZnSO4 (2 origins)

Broiler

40, 80, 120

Tibia ash ZnPancreatic Zn

400, 800, 1200 300, 600, 900

3 Zn4 Sandoval et al, 1997

ZnSO4⋅H2O ZnO

Broiler PerformanceTibia ash Zn

7.5, 15 1 Zn5 Wedekind and Baker, 1990

4⋅ 2O ZnO Zn-methionine

Broiler PerformanceTibia ash Zn

7.5, 15 1 Wedekind and Baker, 1989

ZnSO4⋅H2O ZnO Zn-methionine

Broiler PerformanceTibia ash Zn

7.5, 15 3, 6, 9 5, 10 5, 10, (20)

4 Zn7 Wedekind et al, 1992

ZnSO4⋅7H2O ZnS

Performance

ZnCO3 (2 origins) ZnO (3 origins) (Fe,Mn,Zn,FeO2)2 Zn2SiO4 Zn metal-dust 2ZnO.SiO2

Broiler

10, 20, 40 (ref.) 10, 20

3 Zn8 Edwards, 1959

ZnSO4⋅7H2O ZnO ZnCO3

Broiler Performance 10, 20 1 Zn9 Roberson and Schaible, 1960

ZnSO H Zn6

* different according to the source

104

Comparison of zinc sources for poultry Bone (tibia) zinc accumulation and serum/plasma zinc appear to be reliable response parameters of relative zinc bioavailability and are mainly used in studies with chicks (Table VIII.5). The tissue zinc content was converted, if the original contents were available, using the 10log of this value in order to prevent unrealistic values. Compared to the reference source zinc sulphate⋅7H2O, hydrated zinc sulphate, basic zinc sulphate and basic zinc chloride are well utilised with RBV values around 100 %. The mean RBV of zinc oxide and metal zinc are lower, being respectively 67 and 46 percent. On the other side zinc-methionine has a significant higher RBV value of 130 % compared to the reference zinc sulphate⋅7H2O.

Table VIII.6: Summarised results on the relative biological value of zinc sources for broilers Reference Zn1 Zn2 Zn3 Zn4 Zn5 Zn6 Zn7 Zn8 Zn9 n Mean SD Number of experiments 1 3 2 3 1 1 4 3 1 19 ZnSO4⋅7H2O 100 100 100 5 100 100 100 - ZnSO4⋅H2O FG 87 (87)a (87)a (87)a (87)a 1 87 -

67 72 44a 36a 53a 99 105 8 67b 25.43 78 98 109 3 93 15.7

1 46 4 89

4⋅ 2O 99a 1 99 107a 107 -

67 2 36 1 36 -

Zn methionine 10.9121a 136a 2 131 Zn sulphate basic 101 1 101 - ZnCl basic 107 1 107 - ZnS 60 1 60 - (Fe, Mn, Zn, FeO2)2 70 1 70 -

2SiO4 103 1 103 - 2ZnO.SiO2.H2O 98 1 98 -

ZnO 49 ZnCOZn metal 46 - ZnSO 89 1 - Zn-FeSO H - Fe-ZnSO4⋅H2O 1 Zn metal dust 102 85 24.7Zn metal fume

Zn

a recalculated using ZnSO4⋅H2O FG as a reference b The RBV of ZnO-sources can vary between 22-97 % of the reference ZnSO4⋅7H2O (=100), according to origin and manufacturing process. It is important to consider that feed-grade sources (FG) of ZnO, having a weighted mean RBV of 67±15.4, are quite variable in colour, texture, Zn content, origin and processing method and also in relative bio-availability (Edwards and Baker, 1999). Compared to ZnSO4⋅7H2O (AG) a range in RBV’s of 22-97 was found. The Waelz-processed ZnO was particularly low in bioavailability of Zn. The reasons for this remain to be determined. In a recent study (Verdonck et al, 2002) with broilers, two sources of ZnO (Afox, Pharma-Umicore) were compared to ZnSO4, supplemented at 20 mg/kg to a corn-soybean diet (30 mg/kg). Apparent ileal absorption on the 3 diets ZnSO4, Afox and Pharma are respectively 9.9%, 14.9% and 12.6%. Another important consideration concerning the mean RBV of 131±10.9 for Zn-methionine is that among other factors, the type of the diet has a considerable effect on the relative bioavailability estimate (Wedekind et al, 1992). Relative to ZnSO4⋅H2O FG, the RBV for Zn-methionine assayed on an amino acid diet, a soy-isolate diet and a practical corn-soybean diet was found to be 117 over 177

105

to 208 respectively. It is assumed that this is due to the amount of phytate and soluble fibre, which forms complexes with the Zn of inorganic origin.

ZINC BIOAVAILABILITY FOR RUMINANTS

Sequence of important response criteria and evaluation methods used for ruminants

As described earlier, the sequence of response criteria depends on the fact whether the animals are fed suboptimal or if they are fed above their requirement. The ranking of importance for zinc of the various criteria is listed in Table VIII.7.

Table VIII.7: Ranking of importance of various response criteria for assessing the relative biological value of zinc sources in ruminants Criterion Suboptimal supply Above requirement Apparent absorption 2 1

4 2 3

Zinc plasma 2 1 Enzyme activity 2 2

Liver accumulation Kidney accumulation 1

Comparison of zinc sources for ruminants

Sources of zinc

Table VIII.7 shows that there are only a few criteria that can be used for evaluation of zinc sources in ruminant. Eight observations were based on zinc tissue accumulation, three on apparent absorption and two on zinc plasma content Tissue zinc accumulation has been transformed to 10log in order to maintain the relative biological value variation within physiological limits. For our study the reference source is zinc sulphate monohydrate or heptahydrate (ZnSO4⋅H2O or ZnSO4⋅7 H2O) RG.

There were in total 11 publications involving 13 experiments that were suitable for our study. This is listed in Table VIII.8.

Table VIII.8: Overview of the experiments used for evaluation of the relative biological value of zinc sources in ruminants

Animal type

Response criteria Added Zn (mg/kg)

Number of expts

Ref. no

Reference

Liver accumulation Kidney accumulation

1400 Zn1

Zinc lysine Zinc methionine

Calves Liver accumulation 23 1 Zn2 Engle et al, 1997

Liver accumulation 90 Zn3 Hatfield et al, 2001

Sheep Liver accumulationZinc plasma

25 1 Zn4 Ho and Hidiroglou, 1977

Zinc sulphate Zinc chelate Zinc proteinate Zinc methionine

Sheep 1 Cao et al, 2000

Zinc sulphate

Zinc sulphate Zinc-AA complex

Ewes 1

Zinc sulphate Zinc chelate Zinc sequestered (polysaccharide)

106

Table VIII.8 (continued) Sources of zinc Animal

type Response criteria Ref.

no Added Zn (mg/kg)

Number of expts

Reference

Zinc sulphate Zinc carbonate FG Zinc chloride Zinc oxide

Calves Zinc plasma 300 1 Kincaid., 1979 Zn5

Liver accumulation 300 1 Zn6 Kincaid et al, 1997

Zinc sulphate Zinc oxide

Calves Liver accumulationKidney accumulation Apparent absorption

600 1 Zn7 Miller et al, 1970

Zinc sulphate Zinc methionine

Calves Apparent absorption

30 1 Zn8 Nockels et al, 1993

Zinc sulphate Zinc oxide Zinc lysine Zinc methionine

Sheep Liver accumulation Kidney accumulation

360 1 Zn9 Rojas et al, 1995

Zinc sulphate Zinc carbonate RG Zinc oxide Zinc sulphate FG Zinc metal

Sheep Liver accumulation Kidney accumulation

1400 2 Zn10 Sandoval et al, 1997

Zinc sulphate Zinc oxide Zinc methionine

Sheep Heifers

Apparent absorption

20 2 Zn11 Spears et al, 1989

Zinc oxide Zinc methionine + lysine

Calves

Comparisons have been summarised in Table VIII.9. Except for the very low availability for feed grade zinc carbonate (but not for reagent grade) and for zinc chloride (these data have been obtained from only one experiment by Kincaid (1979) based on plasma zinc variation following the supply to animals of tested sources), no marked differences can be observed: zinc carbonate RG, zinc oxide, zinc sulphate FG, zinc metal and zinc organic compounds show relative biological values close to the reference source ranking from 95 to 107. The mean relative biological value of all organic zinc sources under investigation in this study (n = 13) was 101 ± 4.8. We must underline the difficulty in the assessment of relative biological values for organic zinc compounds because they are not always well defined in terms of chemical composition (e.g. zinc amino-acid complex, zinc proteinate, zinc sequestered or zinc chelate).

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Table VIII.9: Summarised results on the relative biological value of zinc sources for ruminants Zn6 SDReference Zn1 Zn2 Zn3 Zn4 Zn5 Zn7 Zn8 Zn9 Zn10 Zn11 n Mean

Number of experiments

1 1 1 1 1 1 1 1 1 2 2 13

Zinc sulphate RG

100 100 100 100 100 100 100 100 100 100 10

100 -

Zinc carbonate RG

105 1 105 -

Zinc carbonate FG

58 1 58 -

Zinc chloride 42 1 42 - Zinc metal RG - 95 1 95 Zinc oxide 98 (98)a 101 93 100 98 5 98 3.1Zinc sulphate FG

99 1 99 -

Zinc amino-acid complex

102 1 102 -

Zinc proteinate 102 1 102 - Zinc chelate 98 96 2 97 1.4Zinc lysine 100 107 114 2 9.9Zinc methionine 98 98 100 105 100 99 5 2.9

105a 1 105 -

1 97

Zinc lysine + methionine

Zinc sequestered 97 - a Recalculated using Zn oxide

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Whilst every care has been taken to ensure that the information given in the guideline is correct at the time of publication, EMFEMA cannot give any guarantee as to its accuracy or accept liability resulting from its use. Edition 1 / September 2002

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