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Invited Review: Assessing trace mineral status in ruminants, and factors that affect measurements of trace mineral status

      ABSTRACT

      Purpose

      The purpose of this article is to review criteria for assessing copper, zinc, manganese, and selenium status in ruminants. Factors that affect measurements of trace mineral status also will be discussed.

      Sources

      Published scientific literature was the primary source of information reviewed.

      Synthesis

      When assessing mineral status, it is always good to analyze the diet or forages being consumed for the mineral of interest as well as other minerals that may affect its requirement. Liver is the best indicator of both low and excess Cu status. Plasma Cu concentrations do not decrease below normal values until liver Cu stores are mostly depleted, but a plasma Cu concentration less than 0.4 mg/L suggests Cu deficiency. Severe Zn deficiency can be diagnosed based on extremely low plasma or serum Zn concentrations (less than 0.5 mg/L) or on clinical signs of Zn deficiency that respond to Zn supplementation. It is important to note that infections or acute stress may cause plasma Zn concentrations to temporarily decrease to levels consistent with Zn deficiency. There is currently no reliable indicator of marginal Zn deficiency. Several criteria have been measured in an attempt to assess Mn status. However, no criteria have been demonstrated to accurately predict Mn deprivation. Whole blood or liver Se concentrations are useful in assessing Se status. When interpreting whole blood or liver Se concentrations, it is important to consider whether dietary Se is being derived from organic or inorganic sources.

      Conclusions and Applications

      The most appropriate measurement criteria to assess trace mineral status in ruminants depend on the trace mineral being considered. Liver copper is generally a good measure of low as well as excess Cu status. Liver or whole blood Se concentrations are reliable measures of Se status if one takes into account the source of dietary Se. Unless clinical deficiency signs are apparent, it is more difficult to assess Zn and Mn status. In the absence of disease or stress, plasma or serum Zn concentrations below 0.5 mg/L suggest possible severe deficiency. No reliable predictor of marginal Zn deficiency has been determined. Currently, no reliable indicator of Mn status has been identified.

      Key words

      INTRODUCTION

      Analyzing the diet or forages being grazed for the trace mineral in question, as well as other minerals that can affect the requirement of the mineral, is always a good place to start when assessing mineral status. The trace mineral content of forages (

      Mortimer, R. G., D. A. Dargatz, and L. R. Corah. 1999. Forage analysis from cow-calf herds in 23 states. #N303.499. USDA, Animal and Plant Health Inspection Service, Veterinary Services, Centers for Epidemiology and Animal Health. Accessed Mar. 15, 2021. https://handle.nal.usda.gov/10113/33403.

      ) and by-product feeds (
      • DePeters E.J.
      • Fadel J.G.
      • Arana M.J.
      • Ohanesian N.
      • Etchebarne M.A.
      • Hamilton C.A.
      • Hinders R.G.
      • Maloney M.D.
      • Old C.A.
      • Riordan T.J.
      • Perez-Monti H.
      • Pareas J.W.
      Variability in the chemical composition of seventeen selected by-product feedstuffs used by the California Dairy Industry..
      ) varies considerably. In some areas mineral analysis of water can also be useful in assessing trace mineral status. The most appropriate measurement criteria in the animal vary depending on the trace mineral. Liver and plasma or serum are the most common criteria used to predict trace mineral status. Red blood cell concentrations of iron (Fe), zinc (Zn), manganese (Mn), and selenium (Se) are considerably greater than concentrations in plasma (

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). Therefore, hemolysis of red blood cells will result in falsely elevated levels of these trace minerals in plasma or serum. In the literature liver trace mineral concentrations are expressed either on a DM basis or wet weight basis. In our opinion liver trace mineral concentrations should be expressed on a DM basis because of dehydration and other factors that may affect liver moisture content. For example, liver biopsy samples obtained from cull dairy cows before being transported to a commercial slaughter plant analyzed 22.6% DM, whereas liver samples collected from these cows following slaughter averaged 31.6% DM (T. E. Engle, Colorado State University, Fort Collins, personal communication). For the purpose of this article, liver concentrations in ruminants (except fetal liver) expressed in publications on a wet tissue basis have been converted to a DM basis assuming a liver DM content of 28% (
      • Herdt T.H.
      • Hoff B.
      The use of blood analysis to evaluate trace mineral status in ruminant livestock..
      ). The DM content of fetal liver will vary depending on the stage of fetal development. Therefore, fetal liver trace mineral concentrations are reported as they were expressed in the original publication.
      Concentrations of certain trace minerals reported in older literature are likely less accurate than those measured using newer and more sensitive analytical methods. This is especially true for reported serum or plasma Mn concentrations. Low concentrations of Mn can be accurately measured today using flameless (graphite furnace) atomic absorption spectrophotometry or inductively coupled plasma mass spectrometry.
      Reference values indicative of deficiency or marginal trace mineral status vary in textbooks and scientific literature (
      • Kincaid R.L.
      Assessment of trace mineral status of ruminants: A review..
      ;

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ) and among diagnostic laboratories. Diagnostic ranges reported in Mineral Levels in Animal Health by R.

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      are frequently used in diagnostic laboratories. In this book Puls defined deficient as “levels at which subclinical or pathological signs of deficiency should be apparent,” marginal as “levels at which subclinical effects may prevail, such as reduced immune response or growth rate,” and adequate as “levels sufficient for optimum functioning of all body mechanisms with a small margin of reserve to counteract commonly encountered antagonistic condition.” The objective of this article is to review criteria for assessing copper (Cu), Zn, Mn, and Se status in ruminants. Factors that affect measurements of trace mineral status will also be discussed.

      COPPER

      Copper deficiency is considered one of the most widespread trace mineral deficiencies, especially in grazing cattle. In a study involving 2,007 beef cows and heifers from 256 herds in 18 states, 38.9% were classified as marginally deficient in Cu, based on a serum Cu concentration equal to or less than 0.65 mg/L (
      • Dargatz D.A.
      • Garry F.B.
      • Clark G.B.
      • Ross P.F.
      Serum copper concentrations in beef cows and heifers..
      ). Bioavailability of Cu is greatly affected by sulfur (S), molybdenum (Mo), and Fe. Therefore, when deciding whether and how much supplemental Cu is needed, it is important to not only consider Cu but also dietary S, Mo, and Fe concentrations in forages and other feed ingredients.

      Mortimer, R. G., D. A. Dargatz, and L. R. Corah. 1999. Forage analysis from cow-calf herds in 23 states. #N303.499. USDA, Animal and Plant Health Inspection Service, Veterinary Services, Centers for Epidemiology and Animal Health. Accessed Mar. 15, 2021. https://handle.nal.usda.gov/10113/33403.

      reported that 66.7% of forage samples (n = 709) from 23 states analyzed below the beef cattle recommendation of 10 mg of Cu/kg of DM (

      NASEM (National Academies of Sciences, Engineering, and Medicine). 2016. Nutrient Requirements of Beef Cattle. 8th ed. The National Academies Press. https://doi.org/10.17226/19014.

      ). Over 40% of forage samples in this study had a Cu:Mo ratio that suggested at least marginal Cu deficiency. Liver Cu concentrations are generally considered the best indicator of Cu status in ruminants. Plasma, serum, and hair Cu concentrations, and activities of superoxide dismutase (SOD) in erythrocytes and diamine oxidase in plasma, have also been used to evaluate Cu status. Markers that have been used to assess Cu status will be discussed in more detail below.

      Liver Cu (Adults and Growing Ruminants with Developed Rumens)

      Liver is the major storage organ for Cu, and Cu can readily be mobilized from the liver into the blood to supply Cu for biochemical functions. Depending on liver Cu concentrations and level of dietary Cu antagonist, liver Cu can maintain normal plasma Cu concentrations for months even in ruminants fed low-Cu diets. In nonlactating Holstein cows with low initial liver Cu status, liver Cu decreased from approximately 61 to 22 mg/kg of DM after 161 d without Cu supplementation (
      • Grace N.D.
      • Knowles S.O.
      • West D.M.
      • Smith S.L.
      The role of liver Cu kinetics in the depletion of reserves of Cu in dairy cows fed a Cu-deficient diet..
      ). Liver Cu concentrations in cows with moderate initial liver Cu decreased from approximately 123 to 92 mg/kg during the 161-d study. Cows in this study were fed silage, pasture, and hay that analyzed 6.5 to 9 mg of Cu/kg of DM, 0.5 to 0.6 mg of Mo/kg, and 0.17 to 0.28% S (
      • Grace N.D.
      • Knowles S.O.
      • West D.M.
      • Smith S.L.
      The role of liver Cu kinetics in the depletion of reserves of Cu in dairy cows fed a Cu-deficient diet..
      ). In beef cows, plasma Cu concentrations did not decrease below normal concentrations until liver Cu concentrations were less than 40 mg/kg of DM (
      • Claypool D.W.
      • Adams F.W.
      • Pendell H.W.
      • Hartmann Jr., N.A.
      • Bone J.F.
      Relationship between the level of copper in the blood plasma and liver of cattle..
      ). Studies in growing cattle indicate that plasma Cu concentrations do not decrease substantially until liver Cu is less than 20 mg/kg of DM (
      • Phillippo M.
      • Humphries W.R.
      • Garthwaite P.H.
      The effect of dietary molybdenum and iron on copper status and growth in cattle..
      ;
      • Hansen S.L.
      • Schlegel P.
      • Legleiter L.R.
      • Lloyd K.E.
      • Spears J.W.
      Bioavailability of copper from copper glycinate in steers fed high dietary sulfur and molybdenum..
      ).
      Liver Cu is affected by dietary Cu, age, species, and breed and possibly by sex and gestation. Increasing dietary Cu increases liver Cu even in ruminants already receiving adequate Cu. Newborn bovines generally have much greater liver Cu concentrations than older cattle (

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). This is not true for sheep as liver Cu generally increases with age (

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). Most sheep breeds retain more Cu in their livers than cattle when fed diets adequate in Cu. Large differences exist among sheep breeds in their ability to accumulate Cu in their livers (
      • Littledike E.T.
      • Young L.D.
      Effect of sire and dam breed on copper status of fat lambs..
      ;
      • Suttle N.F.
      • Lewis R.M.
      • Small J.N.W.
      Effects of breed and family on rate of copper accretion in the liver of purebred Charollais, Suffolk and Texel lambs..
      ). Liver Cu concentrations also differ among cattle breeds, especially when diets are deficient or marginally deficient in Cu (
      • Mullis L.A.
      • Spears J.W.
      • McCraw R.L.
      Estimated copper requirements of Angus and Simmental heifers..
      ;
      • Dermauw V.
      • De Cuyper A.
      • Duchateau L.
      • Waseyehon A.
      • Dierenfeld E.
      • Clauss M.
      • Peters I.R.
      • Du Laing G.
      • Janssens G.P.J.
      A disparate trace element metabolism in zebu (Bos indicus) and crossbred (Bos indicus × Bos taurus) cattle in response to a copper-deficient diet1..
      ). Sex also appears to affect liver Cu in cattle. Male fetuses (
      • Fry R.S.
      • Spears J.W.
      • Lloyd K.E.
      • O’Nan A.T.
      • Ashwell M.S.
      Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and use in Angus and Simmental cows and fetuses..
      ) and fattening males (
      • Miranda M.
      • Cruz J.M.
      • López-Alonso M.
      • Benedito J.L.
      Variations in liver and blood copper concentrations in young beef cattle raised in north-west Spain: Associations with breed, sex, age and season..
      ) had greater liver Cu concentrations than females. Liver Cu decreases in late pregnancy in beef (
      • Fry R.S.
      • Spears J.W.
      • Lloyd K.E.
      • O’Nan A.T.
      • Ashwell M.S.
      Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and use in Angus and Simmental cows and fetuses..
      ) and dairy cows (
      • Xin Z.
      • Waterman D.F.
      • Hemken R.W.
      • Harmon R.J.
      Copper status and requirement during the dry period and early lactation in multiparous Holstein cows..
      ) fed diets marginal (5.5 to 6.6 mg of Cu/kg) in Cu but not in those supplemented with 10 mg of Cu/kg of DM.

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      has defined minimal liver Cu concentrations in ruminants, with developed rumens, based on the likelihood of a Cu-responsive disorder. Using this definition liver Cu less than 7 mg/kg of DM indicates a high risk, and concentrations of 7 to 20 mg/kg of DM suggest a possible risk for disorders that respond to Cu supplementation. In growing cattle liver Cu concentrations of approximately 10 mg/kg of DM or lower have been associated with reduced growth rate, compared with Cu-supplemented animals (
      • Phillippo M.
      • Humphries W.R.
      • Garthwaite P.H.
      The effect of dietary molybdenum and iron on copper status and growth in cattle..
      ;
      • Spears J.W.
      • Kegley E.B.
      • Mullis L.A.
      Bioavailability of copper from tribasic copper chloride and copper sulfate in growing cattle..
      ;
      • Hansen S.L.
      • Schlegel P.
      • Legleiter L.R.
      • Lloyd K.E.
      • Spears J.W.
      Bioavailability of copper from copper glycinate in steers fed high dietary sulfur and molybdenum..
      ). The growth response to Cu supplementation is most evident in cattle fed diets high in Mo and S (
      • Phillippo M.
      • Humphries W.R.
      • Garthwaite P.H.
      The effect of dietary molybdenum and iron on copper status and growth in cattle..
      ).
      Diagnostic laboratories generally use considerably greater liver Cu concentrations as indicators of deficiency or marginal Cu status.

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      suggests liver Cu concentrations of 2 to 36 mg/kg as deficient, 18 to 90 mg/kg as marginal, and 90 to 360 mg/kg as adequate.

      Liver Cu (Newborn Ruminants)

      Liver Cu is generally greater in newborn calves than in older cattle. Copper stored in the liver serves to provide the young animal with Cu when receiving primarily milk, which is low in Cu. Elevated liver Cu at birth also is due to the inability of the fetal liver to synthesize ceruloplasmin (Cp;

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). A high percentage (70–90%) of Cu in plasma leaving the liver is present in Cp. Plasma Cu concentrations are extremely low at birth but increase within 1 to 2 d of age as the liver starts synthesizing Cp.
      The critical concentration of Cu in the newborn calf liver needed to prevent deficiency, during the nursing period, has been suggested at 300 mg/kg of DM (
      • Gooneratne S.R.
      • Buckley W.T.
      • Christensen D.A.
      Review of copper deficiency and metabolism in ruminants..
      ). Fetal liver Cu concentrations are not affected by stage of gestation (
      • Abdelrahman M.M.
      • Kincaid R.L.
      Deposition of copper, manganese, zinc, and selenium in bovine fetal tissue at different stages of gestation..
      ). However, the total amount of Cu deposited in fetal liver increases throughout fetal development. Transfer of Cu to fetal liver is especially high in the last trimester when most of the fetal growth occurs. Liver Cu is lower than normal in fetuses from cows with low Cu status (
      • Gooneratne S.R.
      • Buckley W.T.
      • Christensen D.A.
      Review of copper deficiency and metabolism in ruminants..
      ;
      • Fry R.S.
      • Spears J.W.
      • Lloyd K.E.
      • O’Nan A.T.
      • Ashwell M.S.
      Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and use in Angus and Simmental cows and fetuses..
      ). As indicated earlier, fetal liver Cu is greater in male than female fetuses (
      • Fry R.S.
      • Spears J.W.
      • Lloyd K.E.
      • O’Nan A.T.
      • Ashwell M.S.
      Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and use in Angus and Simmental cows and fetuses..
      ).
      Young ruminants may be more susceptible to Cu toxicosis than older animals. Calves are born with high liver Cu concentrations, if their dams were adequate in Cu. Absorption of Cu in lambs is considerably greater before rumen development compared with after rumen development (
      • Suttle N.F.
      Effects of age and weaning on the apparent availability of dietary copper to young lambs..
      ). Based on a study of calves (from birth to 1 yr of age) necropsied at the California Animal Health and Food Safety Laboratory, dairy calves had liver Cu concentrations 175 to 215 mg/kg of DM greater than beef calves (
      • Puschner B.
      • Thurmond M.C.
      • Choi Y.-K.
      Influence of age and production type on liver copper concentrations in calves..
      ). In this study the estimated liver Cu concentration at birth was approximately 130 mg/kg of DM greater for dairy than beef calves. The greater liver Cu in dairy calves at birth likely relates to greater Cu supplementation to maternal diets and perhaps greater bioavailability of Cu from dairy versus beef cow diets. The high liver Cu concentrations generally observed in dairy calves raises questions regarding Cu supplementation to milk replacers used for heifer development and veal calf production.
      A study with Holstein and Ayrshire-Holstein crossbred male calves indicated that they could tolerate up to 500 mg of Cu/kg of DM (from CuSO4) in milk replacers for up to 6 wk (
      • Jenkins K.J.
      • Hidiroglou M.
      Tolerance of the calf for excess copper in milk replacer..
      ). However, gain was reduced and liver Cu concentrations were greatly increased in calves supplemented with 200 or 500 mg of Cu/kg of DM. Liver histology was not evaluated in this study. Copper toxicosis was diagnosed in 7 veal calves, 10 to 16 wk of age, based on liver damage and high liver and kidney Cu concentrations (
      • Sullivan J.M.
      • Janovitz E.B.
      • Robinson F.R.
      Copper toxicosis in veal calves..
      ). Liver Cu concentrations in the calves ranged from approximately 950 to 2,400 mg/kg of DM, and the severity and extent of liver lesions appeared to correlate with liver Cu concentrations. The Cu content of the diet was not indicated in the case report, but it did indicate that the calves had been supplemented with various Cu-containing hematinics.
      • Robinson F.R.
      • Sullivan J.M.
      • Brelage D.R.
      • Sommers R.L.
      • Everson R.J.
      Comparison of hepatic lesions in veal calves with concentrations of copper, iron and zinc in liver and kidney..
      reported that 3 to 5% of livers from veal calves in Indiana were condemned due to abnormalities in color and texture. To investigate this problem, 258 Holstein calves were purchased from Wisconsin dairy farms at 1 to 3 d of age and transported to a veal facility in Indiana. Calves were given milk replacer that analyzed 12.8 mg of Cu/kg of DM. Some of the calves in this study underwent a clinical hemolytic crisis before 8 wk of age that resulted in generalized icterus and decreased red blood cell counts. These calves either died or recovered following treatment (treatment not described). Livers from slaughtered calves and calves that died were divided into 4 categories based on severity of histopathologic alterations. Concentrations of hepatic Cu tended to decrease and kidney Cu concentrations increased as the severity of liver lesions increased, especially in calves with severe liver damage. They postulated that elevated liver Cu resulted in hepatic damage that reduced the hepatocellular mass capable of storing Cu (
      • Robinson F.R.
      • Sullivan J.M.
      • Brelage D.R.
      • Sommers R.L.
      • Everson R.J.
      Comparison of hepatic lesions in veal calves with concentrations of copper, iron and zinc in liver and kidney..
      ).
      High winter mortality in female Jersey calves characterized by mild hepatopathy and enteropathy was reported in Scotland (
      • Hunter A.G.
      • Suttle N.
      • Martineau H.M.
      • Spence M.A.
      • Thomson J.R.
      • Macrae A.I.
      • Brown S.
      Mortality, hepatopathy and liver copper concentrations in artificially reared Jersey calves before and after reductions in copper supplementation..
      ). Calves that died had liver Cu concentrations of approximately 1,200 mg/kg of DM and also high kidney Cu concentrations. Newborn male calves on this farm were found to have liver Cu concentrations of approximately 800 mg/kg of DM. The high liver Cu concentrations at birth were due to their dams being supplemented at high (41–60 mg of Cu/kg of DM) Cu levels. In addition to already high liver Cu concentrations at birth, female calves were fed milk replacer supplemented with 10 mg of Cu/kg of DM and creep feed supplemented with 35 mg of Cu/kg of DM.

      Plasma and Serum Cu

      Absorbed Cu is transported to the liver bound to albumin. Copper not excreted in bile, stored, or used for Cu-dependent enzymes in the liver leaves the liver largely bound to Cp. Ceruloplasmin is a multifunctional protein (
      • Healy J.
      • Tipton K.
      Ceruloplasmin and what it might do..
      ). It has ferroxidase activity and is important in the oxidation of ferrous Fe (Fe+2), stored in the liver in ferritin, to ferric Fe (Fe+3). This conversion is necessary for the release of stored Fe+2 because transferrin, the Fe transport protein in blood, only binds Fe+3. Ceruloplasmin is also an acute phase protein that increases during an infection or during acute stress, if liver Cu is adequate. In Cu-deficient calves, Cp increased only slightly following a viral and bacterial respiratory disease challenge (
      • Stabel J.R.
      • Spears J.W.
      • Brown Jr., T.T.
      Effect of copper deficiency on tissue, blood characteristics, and immune function of calves challenged with infectious bovine rhinotracheitis virus and Pasteurella hemolytica..
      ). Ceruloplasmin may also act as an antioxidant by scavenging oxygen radicals, and it has been proposed to transport Cu to certain cellular sites.
      Activity of Cp is highly correlated with plasma Cu concentrations (
      • Legleiter L.R.
      • Spears J.W.
      Plasma diamine oxidase: A biomarker of copper deficiency in the bovine..
      ). Serum Cu concentrations are widely reported in the literature. However, plasma Cu represents a more accurate measure of circulating Cu concentration. Copper should be measured in plasma rather than serum because in the clotting process, some of the Cu present in Cp is sequestrated into the clot (
      • Laven R.A.
      • Lawrence K.E.
      • Livesey C.T.
      An evaluation of the copper sequestrated during clotting in cattle: Is it just caeruloplasmin?.
      ). Ceruloplasmin activity is 18 to 35% lower in serum than in plasma, and Cu concentrations are 14% lower in serum compared with plasma (
      • Kincaid R.L.
      • Gay C.C.
      • Krieger R.I.
      Relationship of serum and plasma copper and ceruloplasmin concentrations of cattle and the effects of whole blood sample storage..
      ).
      The normal range for plasma Cu concentration is 0.6 to 1.5 mg/L, with values usually between 0.8 and 1.2 mg/L (

      Underwood, E. J. 1981. The Mineral Nutrition of Livestock. 2nd ed. Commonwealth Agricultural Bureaux.

      ). Plasma Cu concentrations less than 0.6 mg/L may indicate marginal Cu status, and values less than 0.4 mg/L suggest Cu deficiency. As indicated earlier, plasma Cu concentrations do not decrease until liver Cu concentrations decrease below 40 mg/kg of DM (
      • Claypool D.W.
      • Adams F.W.
      • Pendell H.W.
      • Hartmann Jr., N.A.
      • Bone J.F.
      Relationship between the level of copper in the blood plasma and liver of cattle..
      ).
      Plasma Cu concentrations can be affected by dietary Cu, gestation, disease, and possibly the occurrence of estrus. Copper supplementation to Cu-deficient animals greatly increases plasma Cu, whereas Cu supplementation to diets adequate in Cu results in little or no increase in concentration. Plasma Cu decreases slightly in late pregnancy and then increases during lactation in dairy cows (
      • Xin Z.
      • Waterman D.F.
      • Hemken R.W.
      • Harmon R.J.
      Copper status and requirement during the dry period and early lactation in multiparous Holstein cows..
      ). The increase in plasma Cu during lactation, relative to plasma concentrations in gestation, is even observed in beef heifers fed Cu-deficient diets (
      • Gengelbach G.P.
      • Ward J.D.
      • Spears J.W.
      Effect of dietary copper, iron, and molybdenum on growth and copper status of beef cows and calves..
      ). Induction of Cp synthesis in the liver by disease or acute stress results in increased plasma Cu concentrations in cattle adequate in Cu (
      • Stabel J.R.
      • Spears J.W.
      • Brown Jr., T.T.
      Effect of copper deficiency on tissue, blood characteristics, and immune function of calves challenged with infectious bovine rhinotracheitis virus and Pasteurella hemolytica..
      ). Serum Cu concentrations were greater when beef heifers and cows were bred at estrus than at 21 d after breeding (
      • Small J.A.
      • Charmley E.
      • Rodd A.V.
      • Fredeen A.H.
      Serum mineral concentrations in relation to estrus and conception in beef heifers and cows fed conserved forage..
      ).

      Interaction of Molybdenum and Sulfur with Plasma Cu

      It is well documented that high concentrations of Mo and S reduce Cu bioavailability. Sulfide can react with molybdate in the rumen to form various thiomolybdates. Thiomolybdates (especially tri- and tetrathiomolybdates) reduce Cu absorption by forming insoluble complexes with Cu that do not release Cu even under acidic conditions (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ). This can lead to reduced liver Cu and eventually reduced plasma Cu concentrations. Certain thiomolybdates also can be absorbed and affect systemic metabolism of Cu (
      • Gooneratne S.R.
      • Buckley W.T.
      • Christensen D.A.
      Review of copper deficiency and metabolism in ruminants..
      ). Thiomolybdate absorption is most likely when the production of thiomolybdates exceeds the amount that can react with Cu to form insoluble complexes in the rumen. One of the systemic effects is thiomolybdates binding strongly to plasma Cu bound to albumin, which results in reduced transport of available Cu for biochemical processes (
      • Gooneratne S.R.
      • Buckley W.T.
      • Christensen D.A.
      Review of copper deficiency and metabolism in ruminants..
      ). This can result in high plasma Cu concentrations even in ruminants deficient in Cu. Although plasma Cu concentration is above normal, Cp activity remains low. Increased plasma Cu due to absorption of thiomolybdates is rare. Reduced plasma Cu in ruminants receiving diets high in Mo and S is much more common.

      Copper Metalloenzymes

      Erythrocyte (
      • Gengelbach G.P.
      • Spears J.W.
      Effects of dietary copper and molybdenum on copper status, cytokine production, and humoral immune response of calves..
      ;
      • Ward J.D.
      • Spears J.W.
      Long-term effects of consumption of low-copper diets with or without supplemental molybdenum on copper status, performance, and carcass characteristics of cattle..
      ) SOD activity has been measured as an indicator of Cu status. Plasma Cu and Cp activity were reduced much earlier in Cu deficiency than erythrocyte SOD activity (
      • Ward J.D.
      • Spears J.W.
      Long-term effects of consumption of low-copper diets with or without supplemental molybdenum on copper status, performance, and carcass characteristics of cattle..
      ). Reduced SOD activity appears to indicate a prolonged Cu deficiency.
      Plasma diamine oxidase is another Cu-containing enzyme that has been used to assess Cu deficiency. Activity of diamine oxidase was reduced in Cu-deficient calves and was highly correlated with liver and plasma Cu concentration, and plasma Cp activity (
      • Legleiter L.R.
      • Spears J.W.
      Plasma diamine oxidase: A biomarker of copper deficiency in the bovine..
      ).
      Copper-dependent enzymes can be assayed in a research setting. However, they are much more involved to measure than measuring plasma or liver Cu concentrations, and most diagnostic laboratories do not offer these services. It is also difficult to standardize enzyme assays. Measuring SOD involves repeated washing and centrifugation of red blood cells followed by lysing cells and further processing. The diamine oxidase assay needs to be run with fresh plasma that has not been frozen (
      • Legleiter L.R.
      • Spears J.W.
      Plasma diamine oxidase: A biomarker of copper deficiency in the bovine..
      ).

      Hair and Milk Cu Concentrations

      Hair and milk Cu concentrations are not reliable indicators of Cu status. Copper concentrations in hair decrease slowly in Cu deficiency, and low values indicate prolonged deficiency (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ). Variation among individual animals is also high. Copper deficiency has been reported to decrease milk Cu concentrations (

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). However, milk Cu concentrations are affected by stage of lactation and are generally quite low.

      Assessing Excess Liver Cu Concentrations

      Copper toxicosis is a major problem in sheep and is of increasing concern in dairy cattle. Problems involved in providing adequate dietary Cu but not supplying concentrations that may lead to Cu toxicosis were recently reviewed (
      • López-Alonso M.
      • Miranda M.
      Copper supplementation, a challenge in cattle..
      ). Chronic Cu toxicosis is characterized by gradual accumulation of Cu in the liver over a period of time. As Cu accumulates in the liver, no clinical signs are generally evident until shortly before the hemolytic phase occurs. During the hemolytic phase in sheep, Cu is rapidly released from the liver into the blood, resulting in hemolysis, hemoglobinemia, hemoglobinuria, and elevated kidney Cu concentrations. Elevated kidney Cu concentrations are consistently observed in cows that die from Cu toxicosis (
      • Bradley C.H.
      Copper poisoning in a dairy herd fed a mineral supplement..
      ;
      • Bidewell C.A.
      • Drew J.R.
      • Payne J.H.
      • Sayers A.R.
      • Higgins R.J.
      • Livesey C.T.
      Case study of copper poisoning in a British dairy herd..
      ). Clinical signs that may be observed following the hemolytic phase include anorexia, dullness, jaundice of mucous membranes, excessive thirst, dark-colored urine, and frequently death (

      Howell, J. M., and S. R. Gooneratne. 1987. The pathology of copper toxicity in animals. Pages 53–78 in Copper in Animals and Man. Vol. II. J. M. Howell and J. M. Gawthorne, ed. CRC Press.

      ). Liver Cu concentrations may decrease slightly following the hemolytic crisis (

      Howell, J. M., and S. R. Gooneratne. 1987. The pathology of copper toxicity in animals. Pages 53–78 in Copper in Animals and Man. Vol. II. J. M. Howell and J. M. Gawthorne, ed. CRC Press.

      ). Before the hemolytic phase or crisis, liver damage occurs and enzymes concentrations indicative of liver damage may become high, but plasma or serum Cu is normal. In sheep histological and biochemical evidence of liver damage may be seen with liver Cu as low as 350 mg/kg of DM (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ). Clinical signs of Cu toxicosis usually do not occur until liver Cu concentrations reach 1,000 mg/kg of DM or greater. Samples of liver obtained by biopsy or from cull cows at slaughter in the United States (
      • Strickland J.M.
      • Herdt T.H.
      • Sledge D.G.
      • Buchweitz J.P.
      Short communication: Survey of hepatic copper concentrations in Midwest dairy cows..
      ) and the United Kingdom (
      • Kendall N.R.
      • Holmes-Pavord H.R.
      • Bone P.A.
      • Ander E.L.
      • Young S.D.
      Liver copper concentrations in cull cattle in the UK: Are cattle being copper loaded?.
      ) have indicated that approximately 40% of dairy cows have liver Cu concentrations above 500 mg/kg of DM. Studies have indicated that high liver Cu concentrations may be associated with increased hepatic oxidative stress, even in dairy cows not showing clinical signs of toxicosis (
      • Lyman D.
      • Clark L.J.
      • Campbell K.
      Copper accumulation in Wisconsin Holsteins with indications of oxidative liver damage..
      ;
      • Strickland J.M.
      • Lyman D.
      • Sordillo L.M.
      • Herdt T.H.
      • Buchweitz J.P.
      Effects of super nutritional hepatic copper accumulation on hepatocyte health and oxidative stress in dairy cows..
      ). In animals not showing clinical signs of toxicosis, liver Cu concentrations considered high vary from greater than 450 mg/kg of DM in cattle (
      • Grace N.D.
      • Knowles S.O.
      • Hittmann A.R.
      High and variable copper status identified among dairy herds in the Waikato region by concentrations of Cu in liver sourced from biopsies and cull cows..
      ) to 700 to 2,000 mg of Cu/kg of DM in cattle and sheep (

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      ).

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      suggested a marginal toxic concentration for liver Cu of 350 to 1,500 mg/kg of DM in both cattle and sheep. The concentration of Cu in the liver likely to result in toxicosis varies with breed (especially in sheep), feed toxins that cause hepatic damage, and stressors (

      NASEM (National Academies of Sciences, Engineering, and Medicine). 2005. Mineral Tolerance of Animals. 2nd ed. The National Academies Press.

      ). Liver Cu concentrations in Holstein cows that die from Cu toxicosis generally exceed 1,000 mg/kg of DM (
      • Perrin D.J.
      • Schiefer H.B.
      • Blakley B.R.
      Chronic copper toxicity in a dairy herd..
      ;
      • Bradley C.H.
      Copper poisoning in a dairy herd fed a mineral supplement..
      ). Jersey cows are more susceptible to Cu toxicosis than Holsteins. Recent case reports from New Zealand have indicated Cu toxicosis in Jersey cows with liver Cu concentrations of 525 to 885 mg of Cu/kg of DM (
      • Johnston H.
      • Beasley L.
      • MacPherson N.
      Copper toxicity in a New Zealand dairy herd..
      ;
      • Morgan P.
      • Grace N.
      • Lilley D.
      Using sodium molybdate to treat chronic copper toxicity in dairy cows: A practical approach..
      ). In a herd of 139 Jersey and 249 Holstein cows, 8 died from Cu toxicosis over a 17-mo period (
      • Bidewell C.A.
      • Drew J.R.
      • Payne J.H.
      • Sayers A.R.
      • Higgins R.J.
      • Livesey C.T.
      Case study of copper poisoning in a British dairy herd..
      ). Seven of the 8 that died were Jersey cows.
      Liver Cu concentrations can be assessed by obtaining liver biopsies or obtaining liver samples from cull cows at slaughter to determine whether cows are at risk for chronic Cu toxicosis. Liver damage that occurs before clinical signs of Cu toxicosis often results in increased serum activities of liver enzymes. Glutamate dehydrogenase has been shown to be high in serum of dairy calves (
      • Hunter A.G.
      • Suttle N.
      • Martineau H.M.
      • Spence M.A.
      • Thomson J.R.
      • Macrae A.I.
      • Brown S.
      Mortality, hepatopathy and liver copper concentrations in artificially reared Jersey calves before and after reductions in copper supplementation..
      ) and dairy cows (
      • Laven R.A.
      • Livesey C.T.
      • Offer N.W.
      • Fountain D.
      Apparent subclinical hepatopathy due to excess copper intake in lactating Holstein cattle..
      ;
      • Johnston H.
      • Beasley L.
      • MacPherson N.
      Copper toxicity in a New Zealand dairy herd..
      ) with high liver Cu concentrations.
      • Laven R.A.
      • Livesey C.T.
      • Offer N.W.
      • Fountain D.
      Apparent subclinical hepatopathy due to excess copper intake in lactating Holstein cattle..
      reported that high glutamate dehydrogenase activities observed in cows fed high dietary Cu decreased by 6 wk following removal of supplemental Cu. Serum aspartate aminotransferase activity has also been positivity correlated with high liver Cu concentrations in lambs (
      • Woolliams J.A.
      • Suttle N.F.
      • Wiener G.
      • Field A.C.
      • Woolliams C.
      The effect of breed of sire on the accumulation of copper in lambs, with particular reference to copper toxicity..
      ) and calves (
      • López-Alonso M.
      • Crespo A.
      • Miranda M.
      • Castillo C.
      • Hernández J.
      • Benedito J.L.
      Assessment of some blood parameters as potential markers of hepatic copper accumulation in cattle..
      ). Serum or plasma Cu is not high until clinical signs of Cu toxicosis are apparent. However, whole blood Cu concentrations were positively correlated with liver Cu concentrations in cattle (
      • López-Alonso M.
      • Crespo A.
      • Miranda M.
      • Castillo C.
      • Hernández J.
      • Benedito J.L.
      Assessment of some blood parameters as potential markers of hepatic copper accumulation in cattle..
      ). Erythrocyte Cu concentrations were increased in calves supplemented with high Cu in milk replacer (
      • Jenkins K.J.
      • Hidiroglou M.
      Tolerance of the calf for excess copper in milk replacer..
      ).
      Removing supplemental Cu and supplementing with Mo (
      • Bradley C.H.
      Copper poisoning in a dairy herd fed a mineral supplement..
      ;
      • Morgan P.
      • Grace N.
      • Lilley D.
      Using sodium molybdate to treat chronic copper toxicity in dairy cows: A practical approach..
      ) or Mo and S (
      • Johnston H.
      • Beasley L.
      • MacPherson N.
      Copper toxicity in a New Zealand dairy herd..
      ) has been very effective in alleviating Cu toxicosis and reducing liver Cu concentrations in dairy cows. Supplementing 200 mg of Mo/cow per day for 26 d reduced liver Cu by 61% in Jersey cows in a herd experiencing chronic Cu toxicosis (
      • Morgan P.
      • Grace N.
      • Lilley D.
      Using sodium molybdate to treat chronic copper toxicity in dairy cows: A practical approach..
      ). When a Cu antagonist is not supplemented, the rate of decrease in liver Cu, following the removal of supplemental Cu, is slow in dairy cows with high liver Cu concentrations (
      • Grace N.D.
      • Knowles S.O.
      • West D.M.
      • Smith S.L.
      The role of liver Cu kinetics in the depletion of reserves of Cu in dairy cows fed a Cu-deficient diet..
      ;
      • Hittmann A.R.
      • Grace N.D.
      • Knowles S.O.
      Loss of reserves of Cu in liver when Cu supplements are withdrawn from dairy herds in the Waikato region..
      ). Nonlactating Holstein cows lost liver Cu at a rate of 0.57%/d over a 161-d period when fed a non-Cu-supplemented diet containing 6 to 7 mg of Cu/kg of DM (
      • Grace N.D.
      • Knowles S.O.
      • West D.M.
      • Smith S.L.
      The role of liver Cu kinetics in the depletion of reserves of Cu in dairy cows fed a Cu-deficient diet..
      ). In Holstein calves fed milk replacer low in Cu followed by a semi-purified diet containing 1 to 2 mg of Cu/kg of DM, approximately 150 d has been required to reduce plasma Cu concentrations to levels consistent with low or marginal Cu status (
      • Stabel J.R.
      • Spears J.W.
      • Brown Jr., T.T.
      Effect of copper deficiency on tissue, blood characteristics, and immune function of calves challenged with infectious bovine rhinotracheitis virus and Pasteurella hemolytica..
      ;
      • Gengelbach G.P.
      • Spears J.W.
      Effects of dietary copper and molybdenum on copper status, cytokine production, and humoral immune response of calves..
      ).

      ZINC

      Severe Zn deficiency has been observed in ruminants (

      Underwood, E. J. 1981. The Mineral Nutrition of Livestock. 2nd ed. Commonwealth Agricultural Bureaux.

      ) and can be diagnosed based on extremely low plasma or serum Zn concentrations (less than 0.5 mg/L) or presence of clinical Zn deficiency signs that respond to Zn supplementation. However, severe Zn deficiency in ruminants is rare. A marginal Zn deficiency is more likely under practical conditions. Unfortunately, there is no reliable indicator of marginal Zn deficiency, other than a positive production response (growth, reproduction, milk production, or health) of the animal to increased dietary Zn. Analyzing diets and forages for Zn is important in predicting the adequacy of dietary Zn. Of 709 forage samples collected from 23 states, 70% were below the beef cattle recommendation of 30 mg of Zn/kg of DM (

      NASEM (National Academies of Sciences, Engineering, and Medicine). 2016. Nutrient Requirements of Beef Cattle. 8th ed. The National Academies Press. https://doi.org/10.17226/19014.

      ). However, factors that affect bioavailability of Zn in ruminants are poorly defined (
      • Spears J.W.
      Trace mineral bioavailability in ruminants..
      ).

      Plasma and Serum Zn

      Normal Zn concentrations in serum or plasma generally range between 0.6 and 1.2 mg/L (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ). Low plasma or serum Zn generally reflects either Zn deficiency or acute stress or infection. Plasma Zn concentrations below 0.60 mg/L may indicate Zn deficiency in the absence of acute stress or infection (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ). Under conditions of infection or acute stress, plasma Zn may decrease to concentrations (less than 0.40 mg/L) consistent with Zn deficiency (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ). Plasma Zn decreases around parturition in beef (
      • Dufty J.H.
      • Bingley J.B.
      • Cove L.Y.
      The plasma zinc concentration of nonpregnant, pregnant and parturient Hereford cattle..
      ) and dairy cows (
      • Goff J.P.
      • Stabel J.R.
      Decreased plasma retinol, α-tocopherol, and zinc concentration during the periparturient period: Effect of milk fever..
      ) and returns to baseline levels by d 3 of lactation (
      • Goff J.P.
      • Stabel J.R.
      Decreased plasma retinol, α-tocopherol, and zinc concentration during the periparturient period: Effect of milk fever..
      ). Decreases in plasma Zn at parturition are greatest in cows exhibiting dystocia (
      • Dufty J.H.
      • Bingley J.B.
      • Cove L.Y.
      The plasma zinc concentration of nonpregnant, pregnant and parturient Hereford cattle..
      ) or milk fever (
      • Goff J.P.
      • Stabel J.R.
      Decreased plasma retinol, α-tocopherol, and zinc concentration during the periparturient period: Effect of milk fever..
      ). The decrease in plasma Zn during stress or disease is a normal physiological response, and increasing plasma Zn under these conditions may actually be detrimental to animal health (
      • Chesters J.K.
      • Will M.
      Measurement of zinc flux through plasma in normal and endotoxin-stressed pigs and the effects of Zn supplementation during stress..
      ). The decrease in circulating Zn concentrations is due to stress hormones or cytokines inducing the Zn binding protein metallothionein, which temporarily binds Zn in the liver (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ).
      In addition to acute stress or disease, plasma Zn concentrations are affected by dietary Zn and age. In ruminants fed diets severely deficient in Zn, plasma or serum Zn concentrations decrease below 0.5 mg/L before the development of clinical signs of deficiency. Serum Zn was decreased below 0.4 mg/L in growing lambs fed a diet containing 3.7 mg of Zn/kg of DM by 14 d (
      • Droke E.A.
      • Spears J.W.
      In vitro and in vivo immunological measurements in growing lambs fed diets deficient, marginal or adequate in zinc..
      ). However, deficiency signs such as reduced feed intake and gain, and parakeratotic lesions, were not observed until after 28 d (
      • Droke E.A.
      • Spears J.W.
      In vitro and in vivo immunological measurements in growing lambs fed diets deficient, marginal or adequate in zinc..
      ). In young calves fed a diet containing 3 mg of Zn/kg of DM, serum Zn concentrations decreased to 0.18 mg/L by 2 wk (
      • Ott E.A.
      • Smith W.H.
      • Stob M.
      • Parker H.E.
      • Beeson W.M.
      Zinc deficiency syndrome in the young calf..
      ). Decreased weight gain was observed by 3 wk, and parakeratotic lesions started to develop by 4 wk (
      • Ott E.A.
      • Smith W.H.
      • Stob M.
      • Parker H.E.
      • Beeson W.M.
      Zinc deficiency syndrome in the young calf..
      ). The decrease in plasma Zn is less rapid in older sheep fed a Zn-deficient diet, probably because their greater BW provides more mobilization of Zn from tissue turnover (
      • Masters D.G.
      • Moir R.J.
      Effect of zinc deficiency on the pregnant ewe and developing foetus..
      ). Ewes fed a diet containing 4 mg of Zn/kg of DM throughout gestation had reduced feed intake and produced lambs with lighter birth weight (
      • Masters D.G.
      • Moir R.J.
      Effect of zinc deficiency on the pregnant ewe and developing foetus..
      ). However, parakeratosis and other clinical signs of Zn deficiency were not observed, although plasma Zn concentrations decreased below 0.5 mg/L by 6 wk of pregnancy (
      • Masters D.G.
      • Moir R.J.
      Effect of zinc deficiency on the pregnant ewe and developing foetus..
      ). Collectively, these studies indicate that plasma or serum Zn concentrations below 0.5 mg/L may indicate severe Zn deficiency, and that observed clinical signs of Zn deficiency such as parakeratosis are less likely to be apparent in older animals.
      Moderate Zn supplementation (20–150 mg/kg) to diets marginal or adequate in Zn has little or no effect on plasma or serum Zn concentrations in calves (
      • Kincaid R.L.
      • Chew B.P.
      • Cronrath J.D.
      Zinc oxide and amino acids as sources of dietary zinc for calves: Effects on uptake and immunity..
      ;
      • Wright C.L.
      • Spears J.W.
      Effect of zinc source and dietary level on zinc metabolism in Holstein calves..
      ) and pregnant ewes (
      • Masters D.G.
      • Fels H.E.
      Zinc supplements and reproduction in grazing ewes..
      ). However, supplementing Zn-adequate diets with high (300–500 mg/kg) Zn concentrations will increase plasma or serum Zn levels in calves (
      • Kincaid R.L.
      • Chew B.P.
      • Cronrath J.D.
      Zinc oxide and amino acids as sources of dietary zinc for calves: Effects on uptake and immunity..
      ;
      • Wright C.L.
      • Spears J.W.
      Effect of zinc source and dietary level on zinc metabolism in Holstein calves..
      ). In sheep Zn concentrations are greater in neonatal than in maternal plasma regardless of Zn status (
      • Masters D.G.
      • Moir R.J.
      Effect of zinc deficiency on the pregnant ewe and developing foetus..
      ;
      • Apgar J.
      • Fitzgerald J.A.
      Measures of zinc status in ewes given a low zinc diet throughout pregnancy..
      ). Plasma Zn concentrations in calves shortly after birth are also greater than in their dams (
      • Dufty J.H.
      • Bingley J.B.
      • Cove L.Y.
      The plasma zinc concentration of nonpregnant, pregnant and parturient Hereford cattle..
      ). In Holstein calves, plasma Zn concentrations were high at birth and decreased with age up to 9 wk (
      • Kincaid R.L.
      • Hodgson A.S.
      Relationship of selenium concentrations in blood of calves to blood selenium of the dam and supplemental selenium..
      ).
      There are several possible sources of Zn contamination during the collection of blood. Contamination can occur from evacuated tubes, lubricants and anticoagulants added to tubes, and rubber stoppers (
      • King J.C.
      • Brown K.H.
      • Gibson R.S.
      • Krebs N.F.
      • Lowe N.M.
      • Siekmann J.H.
      • Raiten D.J.
      Biomarkers of Nutrition for Development (BOND)—Zinc review..
      ). Evacuated tubes designated for trace mineral analysis with siliconized rather than rubber stoppers should be used. Some rubber stoppers contain Zn and can result in falsely high plasma or serum Zn concentrations. Red blood cells are much greater in Zn than plasma (
      • King J.C.
      • Brown K.H.
      • Gibson R.S.
      • Krebs N.F.
      • Lowe N.M.
      • Siekmann J.H.
      • Raiten D.J.
      Biomarkers of Nutrition for Development (BOND)—Zinc review..
      ). Therefore, hemolysis of red blood cells can greatly increase Zn concentrations in serum or plasma. Collection of plasma rather than serum is preferred for Zn determination because the extent of hemolysis is usually less when collecting plasma.

      Tissue Zn Concentrations

      Zinc concentrations in pancreas and bone appear to decrease the most in severe Zn deficiency in calves (
      • Miller J.K.
      • Miller W.J.
      Experimental zinc deficiency and recovery of calves..
      ;
      • Ott E.A.
      • Smith W.H.
      • Stob M.
      • Parker H.E.
      • Beeson W.M.
      Zinc deficiency syndrome in the young calf..
      ). Liver Zn concentrations in weaned calves and cows generally range from 80 to 130 mg/kg of DM. Fetal liver Zn concentrations are considerably greater than dam liver when expressed a DM basis (
      • Gooneratne S.R.
      • Christensen D.A.
      A survey of material and fetal tissue zinc, iron, manganese, and selenium concentrations in bovine..
      ) or wet tissue basis (
      • Graham T.W.
      • Thurmond M.C.
      • Mohr F.C.
      • Holmberg C.A.
      • Anderson M.L.
      • Keen C.L.
      Relationships between maternal and fetal liver copper, iron, manganese, and zinc concentrations and fetal development in California Holstein dairy cows..
      ). Liver Zn is not a reliable indicator of Zn status in cattle. Liver Zn concentrations were only slightly decreased (107 vs. 130 mg of Zn/kg of DM;
      • Miller J.K.
      • Miller W.J.
      Experimental zinc deficiency and recovery of calves..
      ) or not decreased at all (23.9 vs. 24.4 mg of Zn/kg of wet tissue;
      • Ott E.A.
      • Smith W.H.
      • Stob M.
      • Parker H.E.
      • Beeson W.M.
      Zinc deficiency syndrome in the young calf..
      ) in Zn-deficient calves that received a semi-purified diet containing 3 mg of Zn/kg of diet, compared with those receiving adequate Zn. Lactating dairy cows fed diets containing 39.5 or 16.6 mg of Zn/kg of DM for 6 wk had similar liver Zn concentrations (
      • Neathery M.W.
      • Miller W.J.
      • Blackmon D.M.
      • Gentry R.P.
      Performance and milk zinc from low-zinc intake in Holstein cows..
      ). Supplementing high Zn levels (300–600 mg of Zn/kg of diet) will increase liver Zn concentrations in calves above normal values (
      • Kincaid R.L.
      • Miller W.J.
      • Fowler P.R.
      • Gentry R.P.
      • Hampton D.L.
      • Neathery M.W.
      Effect of high dietary zinc upon zinc metabolism and intracellular distribution in cows and calves..
      :
      • Kincaid R.L.
      • Chew B.P.
      • Cronrath J.D.
      Zinc oxide and amino acids as sources of dietary zinc for calves: Effects on uptake and immunity..
      ;
      • Wright C.L.
      • Spears J.W.
      Effect of zinc source and dietary level on zinc metabolism in Holstein calves..
      ). In nonlactating cows, supplementation of a control diet, containing 41 mg of Zn/kg of DM, with 600 mg of Zn/kg for 23 d did not affect liver Zn concentrations, suggesting that homeostatic control mechanisms for regulating liver Zn levels are more effective in mature animals (
      • Kincaid R.L.
      • Miller W.J.
      • Fowler P.R.
      • Gentry R.P.
      • Hampton D.L.
      • Neathery M.W.
      Effect of high dietary zinc upon zinc metabolism and intracellular distribution in cows and calves..
      ). Liver Zn concentrations were reduced in lambs fed a diet severely deficient in Zn (
      • Ott E.A.
      • Smith W.H.
      • Stob M.
      • Beeson W.M.
      Zinc deficiency syndrome in the young lamb..
      ) and in neonatal lambs born to ewes fed a diet containing 4 mg of Zn/kg during pregnancy (
      • Masters D.G.
      • Moir R.J.
      Effect of zinc deficiency on the pregnant ewe and developing foetus..
      ).

      Hair Zn Concentrations

      Severe Zn deficiency reduces hair (
      • Miller W.J.
      • Powell G.W.
      • Pitts W.J.
      • Perkins H.F.
      Factors affecting zinc content of bovine hair..
      ) and wool (
      • Apgar J.
      • Travis H.F.
      Effect of a low zinc diet on the ewe during pregnancy and lactation..
      ) Zn concentrations under controlled conditions. Hair Zn can be affected by color of hair, age, and the procedure used to wash hair (
      • Miller W.J.
      • Powell G.W.
      • Pitts W.J.
      • Perkins H.F.
      Factors affecting zinc content of bovine hair..
      ). Hair samples must be washed with detergents to remove Zn contamination from soil and manure. Hair Zn is also quite variable from one animal to another. Although some studies have observed a relationship between Zn intake and hair Zn, the variation in hair Zn concentrations among studies has been greater than differences due to dietary Zn (
      • Perry T.W.
      • Beeson W.M.
      • Smith W.H.
      • Mohler M.T.
      Value of zinc supplementation of natural rations for fattening beef cattle..
      ;
      • Beeson W.M.
      • Perry T.W.
      • Zurcher T.D.
      Effect of supplemental zinc on growth and on hair and blood serum levels of beef cattle..
      ;
      • Mayland H.F.
      • Rosenau R.C.
      • Florence A.R.
      Grazing cow and calf responses to zinc supplementation..
      ).

      Milk Zn Concentrations

      Zinc concentrations in milk are affected by dietary Zn and stage of lactation, with Zn concentrations decreasing with advanced stage of lactation (

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). Milk Zn concentrations were 23% lower in Holstein cows fed 16.6 mg of Zn/kg compared with cows receiving 39.5 mg of Zn/kg of diet (
      • Neathery M.W.
      • Miller W.P.
      • Blackmon D.M.
      • Gentry R.P.
      • Jones J.B.
      Absorption and tissue zinc content in lactating dairy cows as affected by low dietary zinc..
      ). In a more recent study, increasing dietary Zn from 41 to 63 mg/kg of diet did not alter milk Zn concentrations in dairy cows (
      • Cope C.M.
      • MacKenzie A.M.
      • Wilde D.
      • Sinclair L.A.
      Effects of level and form of dietary zinc on dairy cow performance and health..
      ). The addition of 1,000 or 2,000 mg of Zn/kg to a control diet greatly increased milk Zn concentrations (
      • Miller W.J.
      • Amos H.E.
      • Gentry R.P.
      • Blackmon D.M.
      • Durrance R.M.
      • Crowe C.T.
      • Fielding A.S.
      • Neathery M.W.
      Long-term feeding of high zinc sulfate diets to lactating and gestating dairy cows..
      ). Zinc concentrations in colostrum are 3 to 4 times greater than normal milk (

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). Colostrum Zn concentrations were markedly lower in ewes fed a diet extremely (<1.0 mg/kg) deficient in Zn throughout pregnancy (
      • Apgar J.
      • Fitzgerald J.A.
      Measures of zinc status in ewes given a low zinc diet throughout pregnancy..
      ) but not in ewes fed a diet containing 4 mg of Zn/kg (
      • Masters D.G.
      • Moir R.J.
      Effect of zinc deficiency on the pregnant ewe and developing foetus..
      ) compared with ewes receiving adequate Zn.

      Marginal Zn Deficiency

      There is no reliable indicator of marginal Zn deficiency. In some studies Zn supplementation has increased gains in growing cattle (
      • Price J.
      • Humphries W.R.
      Investigation of the effect of supplementary zinc on growth rate of beef cattle on farms in N. Scotland..
      ;
      • Spears J.W.
      • Kegley E.B.
      Effect of zinc source (zinc oxide vs zinc proteinate) and level on performance, carcass characteristics, and immune response of growing and finishing steers..
      ) or nursing calves (
      • Mayland H.F.
      • Rosenau R.C.
      • Florence A.R.
      Grazing cow and calf responses to zinc supplementation..
      ) with normal plasma or serum Zn concentrations (0.7–1.2 mg/L).
      • Price J.
      • Humphries W.R.
      Investigation of the effect of supplementary zinc on growth rate of beef cattle on farms in N. Scotland..
      conducted field trials on 21 farms in Scotland to determine whether supplemental Zn would affect growth of growing and fattening cattle fed typical winter rations. Cattle on each farm were divided into 2 groups, based on weight, breed, and sex, with one group receiving the standard (control) farm diet and the other group receiving 60 mg of supplemental Zn/kg of diet. Mean initial plasma Zn concentrations on the different farms ranged from 0.73 to 1.10 mg/L, and Zn concentrations of diets ranged from 13 to 32 mg/kg of DM. Body weight gain responses to Zn supplementation on the 21 farms ranged from −0.14 to +0.22 kg/d during the 100- to 140-d feeding period. Gain responses to Zn supplementation were not related to plasma Zn concentration or the Zn content of the basal diet (
      • Price J.
      • Humphries W.R.
      Investigation of the effect of supplementary zinc on growth rate of beef cattle on farms in N. Scotland..
      ).

      MANGANESE

      Several criteria including plasma or serum Mn; whole blood, liver, and hair Mn concentrations; and Mn-dependent SOD activity have been measured to assess Mn status in ruminants. However, measures of Mn status have been extremely variable among studies, and no criteria have been demonstrated to accurately predict Mn deprivation. Even in animals exhibiting classical signs of Mn deficiency, such as impaired reproduction or skeletal disorders, measures of Mn status have not always differed from controls. Furthermore, age, sex, genetics, and other factors may affect measures of Mn status. Analyzing feedstuffs for Mn also has not always been reliable in determining whether diets are adequate in Mn.
      It is difficult to formulate a diet using practical feed ingredients that will guarantee that animals will become Mn deficient. In young lambs fed a semi-purified diet, containing 0.8 mg of Mn/kg of DM, clinical signs of deficiency including joint pain, with poor locomotion and balance, appeared by 12 wk (
      • Lassiter J.W.
      • Morton J.D.
      Effects of a low manganese diet on certain ovine characteristics..
      ). At the end of the 22-wk study, liver, heart, and wool Mn concentrations were reduced in lambs fed the low Mn diet compared with those receiving 29.9 mg of Mn/kg of DM. Ewes fed a semi-purified diet containing 8 mg of Mn/kg of DM from 5 mo before breeding and throughout gestation had reduced whole blood Mn concentrations and required more services per conception (2.5 vs. 1.5) than ewes supplemented with 60 mg of Mn/kg of DM (
      • Hidiroglou M.
      • Ho S.K.
      • Ivan M.
      • Shearer D.A.
      Manganese status of pasturing ewes, of pregnant ewes and doe rabbits on low manganese diets and of dairy cows with cystic ovaries..
      ).
      • Hidiroglou M.
      • Ho S.K.
      • Standish J.F.
      Effect of dietary manganese levels on reproductive performance of ewes and on tissue mineral composition of ewes and day-old lambs..
      fed ewes a semi-purified diet containing either 5 or 60 mg of Mn/kg of DM after breeding until lambing. Whole blood Mn (22.1 vs. 12.0 µg/L) and liver Mn concentrations (9.5 vs. 6.8 mg of Mn/kg of DM) were greater in Mn-supplemented ewes at lambing. Newborn lambs from Mn-supplemented ewes also had greater liver Mn concentrations (15.1 vs. 12.2 mg/kg of DM). Considerable variation in liver Mn among ewes and lambs within a treatment was observed in this study (
      • Hidiroglou M.
      • Ho S.K.
      • Standish J.F.
      Effect of dietary manganese levels on reproductive performance of ewes and on tissue mineral composition of ewes and day-old lambs..
      ).
      In a long-term study, Holstein heifers were assigned to a low-Mn diet (7–10 mg of Mn/kg of DM) or Mn-supplemented diet (30 mg of Mn/kg of DM;
      • Bentley O.G.
      • Phillips P.H.
      The effect of low manganese rations upon dairy cattle..
      ). This study lasted almost 4 yr and covered 2 calf crops. Heifers fed the low-Mn diet exhibited delayed first estrus and increased number of services per conception. Four of 14 calves born to heifers fed low Mn had weak legs and pasterns. However, liver and whole blood Mn concentrations in cows and calves were not significantly affected by dietary Mn (
      • Bentley O.G.
      • Phillips P.H.
      The effect of low manganese rations upon dairy cattle..
      ).

      Diet Mn

      Analyzing feedstuffs for Mn is generally considered the best indicator of dietary Mn adequacy (

      Underwood, E. J. 1981. The Mineral Nutrition of Livestock. 2nd ed. Commonwealth Agricultural Bureaux.

      ;

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      ). It is also important to consider other minerals that may affect Mn bioavailability, especially Fe, which appears to share a common intestinal transporter with Mn (
      • Hansen S.L.
      • Ashwell M.S.
      • Moeser A.J.
      • Fry R.S.
      • Knutson M.D.
      • Spears J.W.
      High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves..
      ). High dietary Ca and P are well documented to affect Mn requirements in poultry and may affect requirements in ruminants.
      Other factors also may affect Mn bioavailability in ruminants. A condition referred to as congenital joint laxity and dwarfism has been described in young calves in several countries (
      • White P.J.
      • Windsor P.A.
      Congenital chondrodystrophy of unknown origin in beef herds..
      ). This condition has been linked to low serum or liver Mn concentrations, and clinical signs have included superior brachygnathism, disproportionate dwarfism, and joint problems that affect the ability of calves to stand and walk. Birth of calves with congenital joint laxity and dwarfism has been associated with prolonged drought conditions (
      • White P.J.
      • Windsor P.A.
      Congenital chondrodystrophy of unknown origin in beef herds..
      ) or feeding silage during gestation (
      • Hidiroglou M.
      • Ivan M.K.
      • Bryan C.S.
      • Ribble E.D.
      • Janzen J.G.
      • Proulx J.E.
      Assessment of the role of manganese in congenital joint laxity and dwarfism in calves..
      ). Beef cows wintered on red clover or grass silage in Canada had lower serum Mn concentrations than cows fed grass hay (
      • Hidiroglou M.
      • Ivan M.K.
      • Bryan C.S.
      • Ribble E.D.
      • Janzen J.G.
      • Proulx J.E.
      Assessment of the role of manganese in congenital joint laxity and dwarfism in calves..
      ). Incidence of congenital joint laxity and dwarfism in calves born to cows fed red clover or grass silage was 38 and 28%, respectively, whereas all calves born to cows fed hay were normal. Manganese concentration in the hay (51 mg/kg of DM) was actually lower than in the silages (64 or 63 mg of Mn/kg of DM).

      Blood Mn

      Manganese concentrations are considerably greater in red blood cells than in plasma (

      Underwood, E. J. 1977. Trace Minerals in Human and Animal Nutrition. 4th ed. Academic Press.

      ). Concentrations of Mn in blood are much lower than Cu and Zn. Whereas Cu and Zn concentrations in plasma or serum are expressed as milligrams per liter, Mn is only present in microgram-per-liter concentrations. The low concentrations of Mn in blood results in greater likelihood of errors in analysis, and this is evident in the wide variation of values reported in the literature. Accurately measuring Mn in whole blood, plasma, or serum requires the use of flameless atomic absorption spectrophotometry, neutron activation, or inductively coupled plasma mass spectrometry.
      There is little evidence that plasma or serum Mn is a reliable indicator of Mn status. The concentration of Mn in plasma leaving the liver is tightly regulated via biliary excretion. Controlling the amount of Mn in plasma leaving the liver is critical in preventing excess Mn from entering the brain, the major organ responsible for Mn toxicosis (
      • Roth J.A.
      Homeostatic and toxic mechanisms regulating manganese uptake, retention, and elimination..
      ). Plasma or serum Mn was measured by flameless atomic absorption spectrophotometry in all of the studies cited in the following. Reported plasma or serum Mn concentrations in lambs have varied from approximately 2.0 (
      • Masters D.G.
      • Paynter D.I.
      • Briegel J.
      • Baker S.K.
      • Purser D.B.
      Influence of manganese intake on body, wool and testicular growth of young rams and on the concentration of manganese and the activity of manganese enzymes in tissues..
      ) to 44 µg/L (
      • Black J.R.
      • Ammerman C.B.
      • Henry P.R.
      Effects of high dietary manganese as manganese oxide or manganese carbonate in sheep..
      ). In growing heifers and steers plasma Mn concentrations have varied from approximately 10 to 20 µg/L (
      • Legleiter L.R.
      • Spears J.W.
      • Lloyd K.E.
      Influence of dietary manganese on performance, lipid metabolism, and carcass composition of growing and finishing steers..
      ;
      • Hansen S.L.
      • Spears J.W.
      • Lloyd K.E.
      • Whisnant C.S.
      Growth, reproductive performance, and manganese status of heifers fed varying concentrations of manganese..
      ). Increasing dietary Mn from approximately 16 to 68 mg/kg of DM did not affect plasma Mn in growing heifers (
      • Hansen S.L.
      • Spears J.W.
      • Lloyd K.E.
      • Whisnant C.S.
      Growth, reproductive performance, and manganese status of heifers fed varying concentrations of manganese..
      ). However, plasma Mn concentrations were affected by sampling day in this 196-d study. The addition of graded levels of Mn up to 240 mg/kg to a growing diet (analyzed 29 mg of Mn/kg of DM) or finishing diet (analyzed 8 mg of Mn/kg of DM) did not affect plasma Mn concentrations in steers (
      • Legleiter L.R.
      • Spears J.W.
      • Lloyd K.E.
      Influence of dietary manganese on performance, lipid metabolism, and carcass composition of growing and finishing steers..
      ). Increasing dietary Mn from 13 to 45 mg/kg of DM increased plasma Mn on d 52 but not on d 28 or 84 in a study with ram lambs (
      • Masters D.G.
      • Paynter D.I.
      • Briegel J.
      • Baker S.K.
      • Purser D.B.
      Influence of manganese intake on body, wool and testicular growth of young rams and on the concentration of manganese and the activity of manganese enzymes in tissues..
      ). Plasma Mn is affected by age in lambs, with young lambs having much lower concentrations than 16-wk-old lambs (
      • Paynter D.I.
      • Caple I.W.
      Age-related changes in activities of the superoxide dismutase enzymes in tissues of the sheep and the effect of dietary copper and manganese in these changes..
      ).
      Whole blood Mn may better reflect Mn status than plasma or serum. As mentioned previously, ewes fed a semi-purified diet containing 5 (
      • Hidiroglou M.
      • Ho S.K.
      • Standish J.F.
      Effect of dietary manganese levels on reproductive performance of ewes and on tissue mineral composition of ewes and day-old lambs..
      ) or 8 mg of Mn/kg of DM (
      • Hidiroglou M.
      • Ho S.K.
      • Ivan M.
      • Shearer D.A.
      Manganese status of pasturing ewes, of pregnant ewes and doe rabbits on low manganese diets and of dairy cows with cystic ovaries..
      ) had lower whole blood Mn concentrations than ewes supplemented with 60 mg of Mn/kg of DM. Calves born to heifers fed a diet containing 15.8 mg of Mn/kg of DM throughout gestation, and exhibiting signs of Mn deficiency at birth, had lower whole blood Mn concentrations than calves from heifers supplemented with 50 mg of Mn/kg of DM (
      • Hansen S.L.
      • Spears J.W.
      • Lloyd K.E.
      • Whisnant C.S.
      Feeding a low manganese diet to heifers during gestation impairs fetal growth and development..
      ). However, at the end of the study, when calves averaged 67 d of age, whole blood Mn was slightly greater in calves from heifers fed the low-Mn diet. Increasing dietary Mn from approximately 43 to 60 mg/kg of DM in late gestation did not affect whole blood Mn concentrations in Holstein cows or their calves at birth (
      • Weiss W.P.
      • Socha M.T.
      Dietary manganese for dry and lactating Holstein cows..
      ). Whole blood Mn concentrations were greater in newborn calves than in cows in this study.

      Liver Mn

      As mentioned earlier, lambs (
      • Lassiter J.W.
      • Morton J.D.
      Effects of a low manganese diet on certain ovine characteristics..
      ) and ewes (
      • Hidiroglou M.
      • Ho S.K.
      • Standish J.F.
      Effect of dietary manganese levels on reproductive performance of ewes and on tissue mineral composition of ewes and day-old lambs..
      ) fed semi-purified diets low in Mn had reduced liver Mn concentrations compared with Mn-supplemented animals. Beef calves born to dams receiving 13 mg of Mn/kg of DM and showing skeletal abnormalities also had lower liver Mn concentrations at birth than those born to cows fed 21 mg of Mn/kg of DM (
      • Howes A.D.
      • Dyer I.A.
      Diet and supplemental mineral effects on manganese metabolism in newborn calves..
      ). However, liver Mn was not reduced in calves born to dairy cows receiving a diet containing 7 to 10 mg of Mn/kg of DM relative to those born to cows supplemented with Mn (
      • Bentley O.G.
      • Phillips P.H.
      The effect of low manganese rations upon dairy cattle..
      ).
      Supplementation of graded levels of Mn to increase dietary Mn from 13 to 45 mg/kg of DM did not affect liver Mn concentrations in ram lambs (
      • Masters D.G.
      • Paynter D.I.
      • Briegel J.
      • Baker S.K.
      • Purser D.B.
      Influence of manganese intake on body, wool and testicular growth of young rams and on the concentration of manganese and the activity of manganese enzymes in tissues..
      ). In growing heifers, supplementation of a control diet (15.8 mg of Mn/kg of DM) with 30 or 50 mg of Mn/kg of DM slightly increased liver Mn concentrations (
      • Hansen S.L.
      • Spears J.W.
      • Lloyd K.E.
      • Whisnant C.S.
      Growth, reproductive performance, and manganese status of heifers fed varying concentrations of manganese..
      ). However, differences among sampling days (d 98 and 196 of the study) were greater than treatment differences. Age has been shown to greatly affect liver Mn concentrations in sheep (
      • Paynter D.I.
      • Caple I.W.
      Age-related changes in activities of the superoxide dismutase enzymes in tissues of the sheep and the effect of dietary copper and manganese in these changes..
      ). The addition of graded concentrations of Mn (ranging from 0 to 240 mg/kg of DM) to growing and finishing steer diets resulted in a linear increase in liver Mn at slaughter (
      • Legleiter L.R.
      • Spears J.W.
      • Lloyd K.E.
      Influence of dietary manganese on performance, lipid metabolism, and carcass composition of growing and finishing steers..
      ). Although a linear response was observed, the increase in liver Mn was small ranging from 12.1 mg/kg of DM in controls up to only 15.1 mg/kg of DM in steers supplemented with 240 mg of Mn/kg of DM. Liver Mn is not affected by stage of gestation in cows (
      • Gooneratne S.R.
      • Christensen D.A.
      A survey of material and fetal tissue zinc, iron, manganese, and selenium concentrations in bovine..
      ;
      • Graham T.W.
      • Thurmond M.C.
      • Mohr F.C.
      • Holmberg C.A.
      • Anderson M.L.
      • Keen C.L.
      Relationships between maternal and fetal liver copper, iron, manganese, and zinc concentrations and fetal development in California Holstein dairy cows..
      ).
      Fetal liver Mn concentrations in the third trimester were reported to be 77% (DM basis;
      • Gooneratne S.R.
      • Christensen D.A.
      A survey of material and fetal tissue zinc, iron, manganese, and selenium concentrations in bovine..
      ) and 67% (wet weight basis;
      • Graham T.W.
      • Thurmond M.C.
      • Mohr F.C.
      • Holmberg C.A.
      • Anderson M.L.
      • Keen C.L.
      Relationships between maternal and fetal liver copper, iron, manganese, and zinc concentrations and fetal development in California Holstein dairy cows..
      ) of those found in maternal liver (
      • Gooneratne S.R.
      • Christensen D.A.
      A survey of material and fetal tissue zinc, iron, manganese, and selenium concentrations in bovine..
      ;
      • Graham T.W.
      • Thurmond M.C.
      • Mohr F.C.
      • Holmberg C.A.
      • Anderson M.L.
      • Keen C.L.
      Relationships between maternal and fetal liver copper, iron, manganese, and zinc concentrations and fetal development in California Holstein dairy cows..
      ). Maternal and fetal liver Mn concentrations are positively correlated (
      • Gooneratne S.R.
      • Christensen D.A.
      A survey of material and fetal tissue zinc, iron, manganese, and selenium concentrations in bovine..
      ;
      • Graham T.W.
      • Thurmond M.C.
      • Mohr F.C.
      • Holmberg C.A.
      • Anderson M.L.
      • Keen C.L.
      Relationships between maternal and fetal liver copper, iron, manganese, and zinc concentrations and fetal development in California Holstein dairy cows..
      ). Based on fetuses and stillborn calves submitted to the Minnesota Veterinary Diagnostic Laboratory in 2010, fetuses with skeletal abnormalities had lower liver Mn concentrations than fetuses with normal skeletal features (

      Schefers, J. 2011. Fetal and perinatal mortalities associated with manganese deficiency. Pages 70–74 in Proc. 2011 Minnesota Dairy Health Conf. University of Minnesota.

      ).
      Sheep appear to differ from cattle with regard to liver Mn concentrations. Newborn lambs had greater liver Mn concentrations than their dams, regardless of dietary Mn (
      • Hidiroglou M.
      • Ho S.K.
      • Standish J.F.
      Effect of dietary manganese levels on reproductive performance of ewes and on tissue mineral composition of ewes and day-old lambs..
      ). This is consistent with a study indicating that day-old and week-old lambs had greater liver Mn concentrations than adults (
      • Paynter D.I.
      • Caple I.W.
      Age-related changes in activities of the superoxide dismutase enzymes in tissues of the sheep and the effect of dietary copper and manganese in these changes..
      ).

      Hair Mn and Mn-Dependent SOD

      Hair Mn concentrations are not a useful measure of Mn status (

      Underwood, E. J. 1981. The Mineral Nutrition of Livestock. 2nd ed. Commonwealth Agricultural Bureaux.

      ;

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      ). Manganese-dependent SOD activity was increased in heart when dietary Mn was increased from 13 to 45 mg/kg of DM (
      • Masters D.G.
      • Paynter D.I.
      • Briegel J.
      • Baker S.K.
      • Purser D.B.
      Influence of manganese intake on body, wool and testicular growth of young rams and on the concentration of manganese and the activity of manganese enzymes in tissues..
      ). However, Mn-dependent SOD was not affected in liver, kidney, testes, or muscle in this study. Furthermore, liver, heart, and lung Mn-dependent SOD activity is affected by age in lambs (
      • Paynter D.I.
      • Caple I.W.
      Age-related changes in activities of the superoxide dismutase enzymes in tissues of the sheep and the effect of dietary copper and manganese in these changes..
      ).

      SELENIUM

      Forages and other feedstuffs produced in many areas contain Se concentrations that are deficient or marginal in terms of meeting requirements of ruminants. In the United States, the US FDA restricts the amount of Se that can be supplemented to diets to 0.3 mg of Se/kg of DM. Ruminants can ingest Se in organic and inorganic forms. Selenium occurring naturally in feedstuffs is present primarily as selenomethionine (SeMet). Most supplemental forms of organic Se, including selenized yeast, also contain largely SeMet. Inorganic Se is generally supplemented as sodium selenite (SeO3). Organic and inorganic Se are metabolized differently in the body, and measures of Se status are affected by Se source. Before discussing measures of Se status, it is important to understand the metabolism of inorganic and organic Se.

      Rumen Metabolism

      Selenium absorption is much lower in ruminants than in nonruminants. Low absorption of Se in ruminants is believed to result from reduction of dietary Se to insoluble forms such as elemental Se or selenide by ruminal microorganisms (
      • Spears J.W.
      Trace mineral bioavailability in ruminants..
      ). Organic Se is taken up by ruminal microorganisms to a greater extent than inorganic Se (
      • Van Ryssen J.B.J.
      • Deagen J.T.
      • Beilstein M.A.
      • Whanger P.D.
      Comparative metabolism of organic and inorganic selenium by sheep..
      ;
      • Mainville A.M.
      • Odongo N.E.
      • Bettger W.J.
      • McBride B.W.
      • Osborne V.R.
      Selenium uptake by ruminal microorganisms from organic and inorganic sources in dairy cows..
      ;
      • Galbraith M.L.
      • Vorachek W.R.
      • Estill C.T.
      • Whanger P.D.
      • Bobe G.
      • Davis T.Z.
      • Hall J.A.
      Rumen microorganisms decrease bioavailability of inorganic selenium supplements..
      ). Following incubation with SeO3, 42% of the Se in ruminal microorganisms was present as selenocysteine (SeCys) and only 20% as SeMet (
      • Van Ryssen J.B.J.
      • Deagen J.T.
      • Beilstein M.A.
      • Whanger P.D.
      Comparative metabolism of organic and inorganic selenium by sheep..
      ). In contrast, 79% of the Se present in ruminal microorganisms incubated with SeMet was present as SeMet. The greater uptake of organic Se by ruminal microorganisms may increase bioavailability of Se by reducing the amount of dietary Se reduced to insoluble forms in the rumen (
      • Galbraith M.L.
      • Vorachek W.R.
      • Estill C.T.
      • Whanger P.D.
      • Bobe G.
      • Davis T.Z.
      • Hall J.A.
      Rumen microorganisms decrease bioavailability of inorganic selenium supplements..
      ). Selenomethionine and Se yeast were approximately twice as bioavailable, based on erythrocyte glutathione peroxidase activity (GSHpx), as SeO3 when supplemented to Se-deficient heifers (
      • Pehrson B.
      • Knutsson M.
      • Gyllensward M.
      Glutathione peroxidase activity in heifers fed diets supplemented with organic and inorganic selenium compounds..
      ). Glutathione peroxidase activity is generally not affected by source when organic and inorganic Se are supplemented to diets adequate in Se (
      • Ortman K.
      • Pehrson B.
      Effect of selenate as a feed supplement to dairy cows in comparison to selenite and selenium yeast..
      ;
      • Juniper D.T.
      • Phipps R.H.
      • Jones A.K.
      • Bertin G.
      Selenium supplementation of lactating dairy cows: Effect on selenium concentration in blood, milk, urine, and feces..
      ;
      • Guyot H.
      • Spring P.
      • Andrieu S.
      • Rollin F.
      Comparative responses to sodium selenite and organic selenium supplements in Belgian Blue cows and calves..
      ). However, supplementation of organic Se results in greater concentrations of Se in blood, milk, liver, and muscle than inorganic Se, especially when supplemented to diets in quantities greater than requirements (
      • Juniper D.T.
      • Phipps R.H.
      • Jones A.K.
      • Bertin G.
      Selenium supplementation of lactating dairy cows: Effect on selenium concentration in blood, milk, urine, and feces..
      ,
      • Juniper D.T.
      • Phipps R.H.
      • Ramos-Morales E.
      • Bertin G.
      Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle1..
      ).

      Mammalian Metabolism

      In the body SeO3 is reduced to selenide, which then undergoes a series of reactions to form SeCys. Selenocysteine is the form of Se present in the active site of selenoenzymes, such as GSHpx (

      Sunde, R. A. 1997. Selenium. Pages 493–556 in Handbook of Nutritionally Essential Mineral Elements. B. L. O’Dell and R. A. Sunde, ed. Marcel Dekker Inc.

      ). Inorganic Se not used for synthesis of SeCys is largely excreted in the urine. There are 2 possible pathways for SeMet. Following intestinal absorption, SeMet enters the methionine pool, where it can be incorporated into nonspecific proteins in place of methionine or be further metabolized to selenide for synthesis of SeCys (

      Sunde, R. A. 1997. Selenium. Pages 493–556 in Handbook of Nutritionally Essential Mineral Elements. B. L. O’Dell and R. A. Sunde, ed. Marcel Dekker Inc.

      ). The amount of SeMet incorporated into nonspecific proteins is affected by dietary methionine (
      • Butler J.A.
      • Beilstein M.A.
      • Whanger P.D.
      Influence of dietary methionine on the metabolism of selenomethionine in rats..
      ). The greater Se concentrations in blood, milk, liver, and skeletal muscle in ruminants receiving organic Se versus inorganic Se is largely due to SeMet being incorporated into general proteins, when Se is supplemented to diets adequate or marginal in Se (
      • Juniper D.T.
      • Phipps R.H.
      • Jones A.K.
      • Bertin G.
      Selenium supplementation of lactating dairy cows: Effect on selenium concentration in blood, milk, urine, and feces..
      ,
      • Juniper D.T.
      • Phipps R.H.
      • Ramos-Morales E.
      • Bertin G.
      Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle1..
      ).

      Blood Se

      Whole blood Se is considered a more desirable measure of Se status than serum or plasma Se concentrations (
      • Maas J.
      • Galey F.D.
      • Peauroi J.R.
      • Case J.T.
      • Littlefield E.S.
      • Gay C.C.
      • Koller L.D.
      • Crisman R.O.
      • Weber D.W.
      • Warner D.W.
      • Tracy M.L.
      The correlation between serum selenium and blood selenium in cattle..
      ;
      • Herdt T.H.
      • Hoff B.
      The use of blood analysis to evaluate trace mineral status in ruminant livestock..
      ). Plasma or serum Se concentrations are sensitive to short-term changes in Se intake. In cattle fed free-choice minerals, intake can vary greatly from day to day, and this can cause plasma or serum Se concentrations to not accurately reflect Se status. Hemolysis of red blood cells during processing will also falsely increase serum Se concentrations because 60 to 70% of the total Se in blood is present in erythrocytes (
      • Maas J.
      • Galey F.D.
      • Peauroi J.R.
      • Case J.T.
      • Littlefield E.S.
      • Gay C.C.
      • Koller L.D.
      • Crisman R.O.
      • Weber D.W.
      • Warner D.W.
      • Tracy M.L.
      The correlation between serum selenium and blood selenium in cattle..
      ;
      • Herdt T.H.
      • Hoff B.
      The use of blood analysis to evaluate trace mineral status in ruminant livestock..
      ). Because of the slow turnover of erythrocytes, whole blood Se gives a better overall measure of long-term Se status. The majority of Se in erythrocytes is present in GSHpx. Whole blood Se is positively correlated (r = 0.85) with whole blood GSHpx activity (
      • Thompson K.G.
      • Fraser A.J.
      • Harrop B.M.
      • Kirk J.A.
      • Bullians J.
      • Cordes D.O.
      Glutathione peroxidase activity and selenium concentration in bovine blood and liver as indicators of dietary selenium intake..
      ;
      • Koller L.D.
      • South P.J.
      • Exon J.H.
      • Whitbeck G.A.
      • Maas J.
      Comparison of selenium levels and glutathione peroxidase activity in bovine whole blood..
      ).
      Fetal whole blood Se concentrations are similar to maternal values, whereas serum Se concentrations are considerably lower in fetal compared with maternal serum (
      • Van Saun R.J.
      • Herdt T.H.
      • Stowe H.D.
      Maternal and fetal selenium concentrations and their interrelationships in dairy cattle..
      ). Young beef calves have lower whole blood and plasma Se concentrations than their dams (
      • Pehrson B.
      • Ortman K.
      • Madjid N.
      • Trafikowska U.
      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..
      ;
      • Guyot H.
      • Spring P.
      • Andrieu S.
      • Rollin F.
      Comparative responses to sodium selenite and organic selenium supplements in Belgian Blue cows and calves..
      ).
      There is no agreement in the literature regarding Se concentrations in whole blood and plasma or serum that should be considered deficient, marginal, or adequate. Whole blood Se concentrations below 50 µg/L are generally considered deficient (
      • Dargatz D.A.
      • Ross P.F.
      Blood selenium concentrations in cows and heifers on 253 cow-calf operations in 18 states..
      ;
      • Pehrson B.
      • Ortman K.
      • Madjid N.
      • Trafikowska U.
      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..
      ;
      • Kincaid R.L.
      Assessment of trace mineral status of ruminants: A review..
      ). Clinical signs of nutritional muscular dystrophy (NMD) have been observed in cattle with whole blood Se levels up to 30 µg/L (
      • Pehrson B.
      • Ortman K.
      • Madjid N.
      • Trafikowska U.
      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..
      ). Unthriftiness with high mortality rates (25–45%) were observed in lambs with blood Se concentrations less than 5 µg/L, and milder cases of unthriftiness were seen in lambs with whole blood Se concentrations of 5 to 10 µg/L (
      • Sheppard A.D.
      • Blom L.
      • Grant A.B.
      Levels of selenium in blood and tissues associated with some selenium deficiency diseases in New Zealand sheep..
      ). Whole blood Se concentrations less than 10 µg/L were associated with increased fetal death losses in ewes (
      • Sheppard A.D.
      • Blom L.
      • Grant A.B.
      Levels of selenium in blood and tissues associated with some selenium deficiency diseases in New Zealand sheep..
      ). Selenium concentrations in whole blood considered marginal vary from 50 to over 100 µg/L (

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      ;
      • Dargatz D.A.
      • Ross P.F.
      Blood selenium concentrations in cows and heifers on 253 cow-calf operations in 18 states..
      ). Concentrations of Se in whole blood considered to be adequate vary from 81 to 200 µg/L (

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      ;
      • Dargatz D.A.
      • Ross P.F.
      Blood selenium concentrations in cows and heifers on 253 cow-calf operations in 18 states..
      ).
      Greater whole blood Se concentrations may be needed to maximize immunity rather than to prevent clinical signs of Se deficiency. A blood Se concentration of at least 100 µg/L appeared to be necessary in calves for optimal antibody production following injection of a foreign protein (egg lysozyme;
      • Swecker W.S.
      • Eversole D.E.
      • Thatcher C.D.
      • Blodgett D.J.
      • Schurig G.G.
      • Meldrum J.B.
      Influence of supplemental selenium on humoral immune responses in weaned beef calves..
      ). Dairy cows with whole blood Se levels of 33 µg/L were more susceptible to experimentally induced Escherichia coli mastitis than those with average blood Se concentrations of 132 µg/L (
      • Erskine R.J.
      • Eberhart R.J.
      • Grasso P.J.
      • Scholz R.W.
      Induction of Escherichia coli mastitis in cows fed selenium-deficient or selenium-supplemented diets..
      ).
      • Jukola E.
      • Hakkarainen J.
      • Saloniemi H.
      • Sankari S.
      Blood selenium, vitamin E, vitamin A, and β-carotene concentrations and udder health, fertility treatments, and fertility..
      reported that a whole blood Se concentration of 180 µg/L appeared to be critical for prevention of coagulase-negative staphylococci and Staphylococcus aureus mastitis in dairy cows.
      Plasma or serum Se concentrations less than 25 µg/L are considered deficient, and concentrations between 30 and 60 µg/L are considered marginal by

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      . Calves born to cows with plasma Se concentrations of approximately 15 µg/L exhibited an 8 to 11% incidence of NMD (
      • Hidiroglou M.
      • Proulx J.
      • Jolette J.
      Intraruminal selenium pellet for control of nutritional muscular dystrophy in cattle..
      ). Increased death loss due to diarrhea and unthriftiness was observed in calves born to cows with plasma Se concentrations of 40 µg/L before calving (
      • Spears J.W.
      • Harvey R.W.
      • Segerson E.C.
      Effects of marginal selenium deficiency and winter protein supplementation on growth, reproduction and selenium status of beef cattle..
      ).
      Source of Se should be considered when evaluating whole blood, and plasma or serum Se concentrations. When supplemented to diets marginal in Se (0.10–0.16 mg/kg) at supplemental levels of 0.10 to 0.20 mg of Se/kg of DM, whole blood Se concentrations have been 11 to 33% greater and plasma Se concentrations 22% greater in cattle supplemented with Se yeast compared with SeO3 (
      • Ortman K.
      • Pehrson B.
      Effect of selenate as a feed supplement to dairy cows in comparison to selenite and selenium yeast..
      ;
      • Juniper D.T.
      • Phipps R.H.
      • Jones A.K.
      • Bertin G.
      Selenium supplementation of lactating dairy cows: Effect on selenium concentration in blood, milk, urine, and feces..
      ,
      • Juniper D.T.
      • Phipps R.H.
      • Ramos-Morales E.
      • Bertin G.
      Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle1..
      ). The magnitude of differences in whole blood and plasma or serum concentrations between organic and inorganic sources becomes greater with greater supplemental levels. Ewes receiving a low-Se diet and supplemented with 0.7 mg of Se/d for 12 mo from Se yeast had a 35% greater whole blood and 24% greater serum Se concentration than ewes given the same amount of Se from SeO3 (
      • Hall J.A.
      • Van Saun R.J.
      • Bobe G.
      • Stewart W.C.
      • Vorachek W.R.
      • Mosher W.D.
      • Nichols T.
      • Forsberg N.E.
      • Pirelli G.J.
      Organic and inorganic selenium: I. Oral bioavailability in ewes..
      ). In ewes supplemented with 2.1 mg of Se/d for 12 mo, whole blood and serum Se concentrations were 61 and 40% greater, respectively, in ewes receiving Se yeast compared with those supplemented with SeO3 (
      • Hall J.A.
      • Van Saun R.J.
      • Bobe G.
      • Stewart W.C.
      • Vorachek W.R.
      • Mosher W.D.
      • Nichols T.
      • Forsberg N.E.
      • Pirelli G.J.
      Organic and inorganic selenium: I. Oral bioavailability in ewes..
      ).

      Liver Se

      Liver Se concentrations also reflect Se status. Selenium liver concentrations of fetuses and young calves are greater than in adults (
      • Herdt T.H.
      • Hoff B.
      The use of blood analysis to evaluate trace mineral status in ruminant livestock..
      ). Fetal liver Se concentrations are at least twice as high as maternal levels and are positively correlated with maternal liver Se concentrations (
      • Gooneratne S.R.
      • Christensen D.A.
      A survey of material and fetal tissue zinc, iron, manganese, and selenium concentrations in bovine..
      ;
      • Van Saun R.J.
      • Herdt T.H.
      • Stowe H.D.
      Maternal and fetal selenium concentrations and their interrelationships in dairy cattle..
      ). Concentrations of liver Se suggestive of deficiency in adult cattle and sheep vary greatly in the literature.

      Puls, R. 1994. Mineral Levels in Animal Health. 2nd ed. Sherpa International.

      reported that liver Se concentrations between 0.07 and 0.61 mg/kg of DM are deficient, and values between 0.43 and 0.89 mg/kg of DM are marginal. In New Zealand,
      • Thompson J.C.
      • Thornton R.N.
      • Bruere S.N.
      • Ellison R.S.
      Selenium reference ranges in New Zealand cattle..
      reported a reference range for liver Se in cattle of 0.17 mg/kg of DM as deficient (defined as responsive to Se supplementation) and 0.17 to 0.24 mg/kg of DM as marginal. A liver Se concentration of 2.2 mg/kg of DM has been suggested as adequate in the bovine fetus (
      • Van Saun R.J.
      • Herdt T.H.
      • Stowe H.D.
      Maternal and fetal selenium concentrations and their interrelationships in dairy cattle..
      ). Calves born to cows with a liver Se concentration of 0.47 mg/kg of DM had an 8 to 11% incidence of NMD (
      • Hidiroglou M.
      • Proulx J.
      • Jolette J.
      Intraruminal selenium pellet for control of nutritional muscular dystrophy in cattle..
      ). In New Zealand liver Se concentrations in lambs with NMD averaged 0.19 mg/kg of DM compared with 0.47 mg/kg in normal lambs (
      • Gabbedy B.J.
      • Masters H.
      • Boddington E.B.
      White muscle disease of sheep and associated tissue selenium levels in Western Australia..
      ).
      Increasing dietary Se from inorganic or organic sources from deficient to adequate concentrations increases liver Se in lambs (
      • Oh S.H.
      • Pope A.L.
      • Hoekstra W.G.
      Dietary selenium requirement of sheep fed a practical-type diet as assessed by tissue glutathione peroxidase and other criteria..
      ;
      • Ullrey D.E.
      • Brady P.S.
      • Whetter P.A.
      • Ku P.K.
      • Magee W.T.
      Selenium supplementation of diets for sheep and beef cattle..
      ) and cattle (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ;
      • Juniper D.T.
      • Phipps R.H.
      • Ramos-Morales E.
      • Bertin G.
      Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle1..
      ). Supplementing dairy cows grazing forage containing 0.035 mg of Se/kg of DM with 2 or 4 mg of Se/d, from SeO3, increased liver Se from 0.26 to 0.54, and 0.76 mg/kg of DM, respectively (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ). Increasing dietary Se from 0.02 to 0.12 mg/kg of DM, from SeO3, increased liver Se concentrations in lambs from 0.18 to 0.65 mg/kg of DM (
      • Oh S.H.
      • Pope A.L.
      • Hoekstra W.G.
      Dietary selenium requirement of sheep fed a practical-type diet as assessed by tissue glutathione peroxidase and other criteria..
      ). Lambs and steers receiving Se from natural feedstuffs have greater liver Se concentrations than those supplemented with a similar amount of Se from SeO3 (
      • Ullrey D.E.
      • Brady P.S.
      • Whetter P.A.
      • Ku P.K.
      • Magee W.T.
      Selenium supplementation of diets for sheep and beef cattle..
      ). Supplementation of Se yeast also results in greater liver Se concentrations than a similar quantity of Se from SeO3 (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ;
      • Juniper D.T.
      • Phipps R.H.
      • Ramos-Morales E.
      • Bertin G.
      Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle1..
      ).

      Milk Se

      Selenium concentrations in colostrum are approximately 4 times greater than in milk (
      • Weiss W.P.
      • Hogan J.S.
      Effect of selenium source on selenium status, neutrophil function, and response to intramammary endotoxin challenge of dairy cows..
      ;
      • Guyot H.
      • Spring P.
      • Andrieu S.
      • Rollin F.
      Comparative responses to sodium selenite and organic selenium supplements in Belgian Blue cows and calves..
      ). Colostrum Se concentrations are reduced by Se deficiency in cows (
      • Juniper D.T.
      • Rymer C.
      • Briens M.
      Bioefficacy of hydroxy-selenomethionine as a selenium supplement in pregnant dairy heifers and on the selenium status of their calves..
      ) and ewes (
      • Hidiroglou M.
      • Hoffman I.
      • Jenkins K.J.
      • MacKay R.R.
      Control of nutritional muscular dystrophy in lambs by selenium implantation..
      ). Cows supplemented with Se yeast (
      • Weiss W.P.
      • Hogan J.S.
      Effect of selenium source on selenium status, neutrophil function, and response to intramammary endotoxin challenge of dairy cows..
      ;
      • Guyot H.
      • Spring P.
      • Andrieu S.
      • Rollin F.
      Comparative responses to sodium selenite and organic selenium supplements in Belgian Blue cows and calves..
      ) and hydroxy-SeMet (
      • Juniper D.T.
      • Rymer C.
      • Briens M.
      Bioefficacy of hydroxy-selenomethionine as a selenium supplement in pregnant dairy heifers and on the selenium status of their calves..
      ) have greater Se concentrations in colostrum than those receiving SeO3.
      Milk Se concentrations are also affected by dietary Se level and source. Beef (
      • Ammerman C.B.
      • Chapman H.L.
      • Bouwman G.W.
      • Fontenot J.P.
      • Bagley C.P.
      • Moxon A.L.
      Effect of supplemental selenium for beef cows on the performance and tissue selenium concentrations of cows and suckling calves..
      ;
      • Hidiroglou M.
      • Proulx J.
      • Jolette J.
      Intraruminal selenium pellet for control of nutritional muscular dystrophy in cattle..
      ) and dairy cows (
      • Malbe M.
      • Klaassen M.
      • Fang W.
      • Myllys V.
      • Vikerpuur M.
      • Nyholm K.
      • Sankari S.
      • Suoranta K.
      • Sandholm M.
      Comparisons of selenite and selenium yeast feed supplements on Se-incorporation, mastitis and leukocyte function in Se-deficient dairy cows..
      :
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ) receiving diets containing less than 0.05 mg of Se/kg of DM have milk Se concentrations less than 10 µg/L. Milk Se concentrations are generally greater than 20 µg/L in cows receiving adequate Se. Increasing dietary Se, from inorganic Se, in cows fed adequate Se has little or no effect on milk Se concentrations. In Holstein cows increasing dietary Se (SeO3) from 0.2 to 0.3 mg/kg of DM for 13 wk slightly increased milk Se (
      • Maus R.W.
      • Martz F.A.
      • Belyea R.L.
      • Weiss M.F.
      Relationship of dietary selenium to selenium in plasma and milk from dairy cows..
      ). However, increasing dietary Se from 0.3 mg/kg of DM up to levels as high as 0.7 mg/kg using SeO3 did not further increase milk Se levels after 13 wk of supplementation (
      • Maus R.W.
      • Martz F.A.
      • Belyea R.L.
      • Weiss M.F.
      Relationship of dietary selenium to selenium in plasma and milk from dairy cows..
      ). Supplementing graded levels of SeO3 up to 53 mg of Se/d did not greatly affect milk Se concentrations in cows fed adequate Se (
      • Fisher L.J.
      • Montemurro J.
      • Hoogendoorn C.
      The effect of added dietary selenium on the selenium content of milk, urine and feces..
      ). Increasing dietary Se from 1 to 2 or 3 mg/d, from SeO3, also did not affect milk Se in Hereford cows (
      • Perry T.W.
      • Peterson R.C.
      • Beeson W.M.
      Selenium in milk from feeding small supplements..
      ). Supplementing 2 mg of Se/d, from SeO3, to dairy cows grazing forage containing 0.035 mg of Se/kg of DM increased milk Se concentrations (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ). Increasing Se from 2 to 4 mg of Se/d from SeO3 did not result in further increases in milk Se (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ).
      Cows supplemented with Se yeast have greater milk Se concentrations than cows given a similar concentration of Se from SeO3 (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ;
      • Juniper D.T.
      • Phipps R.H.
      • Jones A.K.
      • Bertin G.
      Selenium supplementation of lactating dairy cows: Effect on selenium concentration in blood, milk, urine, and feces..
      ;
      • Guyot H.
      • Spring P.
      • Andrieu S.
      • Rollin F.
      Comparative responses to sodium selenite and organic selenium supplements in Belgian Blue cows and calves..
      ). Increasing dietary Se above requirements with Se yeast further increased milk Se concentrations above those observed in cows supplemented with SeO3 (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ;
      • Givens D.I.
      • Allison R.
      • Cottrill B.
      • Blake J.S.
      Enhancing the selenium content of bovine milk through alteration of the form and concentration of selenium in the diet of the dairy cow..
      ). Greater milk Se concentrations in cows supplemented with Se yeast is due to greater Se concentrations in the casein fraction (
      • Knowles S.O.
      • Grace N.D.
      • Wurms K.
      • Lee J.
      Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows..
      ).
      • Juniper D.T.
      • Phipps R.H.
      • Jones A.K.
      • Bertin G.
      Selenium supplementation of lactating dairy cows: Effect on selenium concentration in blood, milk, urine, and feces..
      showed that the amount and percentage of total Se in milk present as SeMet was greater in cows supplemented with Se yeast than in those receiving SeO3. This is consistent with SeMet being incorporated into milk protein in place of methionine.

      Glutathione Peroxidase

      Glutathione peroxidase is a Se metalloenzyme that has been measured in erythrocytes (
      • Thompson K.G.
      • Fraser A.J.
      • Harrop B.M.
      • Kirk J.A.
      • Bullians J.
      • Cordes D.O.
      Glutathione peroxidase activity and selenium concentration in bovine blood and liver as indicators of dietary selenium intake..
      ;
      • Pehrson B.
      • Ortman K.
      • Madjid N.
      • Trafikowska U.
      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..
      ), whole blood (
      • Koller L.D.
      • South P.J.
      • Exon J.H.
      • Whitbeck G.A.
      • Maas J.
      Comparison of selenium levels and glutathione peroxidase activity in bovine whole blood..
      ;
      • Spears J.W.
      • Harvey R.W.
      • Segerson E.C.
      Effects of marginal selenium deficiency and winter protein supplementation on growth, reproduction and selenium status of beef cattle..
      ), and plasma (
      • Thompson K.G.
      • Fraser A.J.
      • Harrop B.M.
      • Kirk J.A.
      • Bullians J.
      • Cordes D.O.
      Glutathione peroxidase activity and selenium concentration in bovine blood and liver as indicators of dietary selenium intake..
      ) in research studies to assess Se status. Sex, age, and factors other than Se status that may affect GSHpx are not well defined. Temperatures and pH values used in GSHpx assays can vary and greatly affect reported enzyme activities (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI.

      ). Because of the difficulty involved in the GSHpx assay, most diagnostic laboratories do not offer this service.

      APPLICATIONS

      Providing ruminants with adequate amounts of trace minerals, without supplying excessive concentrations that may negatively affect productivity, can be challenging. Supplementation practices in the United States include (1) no supplemental trace minerals, (2) free-choice mineral supplements (containing trace minerals), (3) trace mineral additions to the TMR, and (4) injectable trace minerals. Trace mineral requirements are not static but can be affected by such factors as bioavailability of trace minerals from feedstuffs or supplemental sources, genetics, physiological status, and antagonists. Factors that may affect trace mineral requirements are discussed in detail in nutrient requirement publications.
      Severe trace mineral deficiencies are often associated with clinical signs of deficiency. However, marginal trace mineral deficiencies can affect animal performance and health in the absence of obvious clinical signs. Trace mineral status is often assessed to address problems or at least perceived problems in health or production. Based on this review, recommended criteria for assessing Cu, Zn, Mn, and Se status in ruminants is presented in Table 1.
      Table 1Recommended criteria for assessing Cu, Zn, Mn, and Se status in ruminants
      The question marks indicate some uncertainty with regard to whether these minerals affect bioavailability.
      Trace mineralAnimal criteriaDietary considerations
      CopperLiver Cu, plasma CuCu, Mo, S, Fe
      ZincPlasma ZnZn, Ca?
      Manganese?Mn, Fe, Ca?, P?
      SeleniumWhole blood Se, liver SeSe, source of Se
      1 The question marks indicate some uncertainty with regard to whether these minerals affect bioavailability.
      It is recommended that blood samples for trace minerals be collected in evacuated tubes designated for trace mineral analysis. Collecting plasma instead of serum will minimize hemolysis and should provide more accurate measures of circulating Cu concentrations. Liver trace mineral concentrations should be expressed on a DM basis because of factors that affect liver moisture content. Plasma Cu concentrations less than 0.6 mg/L or liver concentration less than 50 mg/kg of DM suggest marginal Cu status. Liver Cu concentrations less than 20 mg/kg of DM or plasma concentrations less than 0.4 mg/L are consistent with Cu deficiency. In the absence of disease or acute stress, plasma Zn concentrations less than 0.5 mg/L suggest possible severe Zn deficiency. No reliable indicator of marginal Zn status is currently known. Selenium concentrations in whole blood less than 50 µg/L or liver concentrations less than 0.50 mg/kg of DM are indictive of Se deficiency. Currently, there is no reliable indicator of Mn status. Research is needed to better define markers of Mn status, using improved analytical instrumentations for more accurately measuring Mn concentrations.
      When evaluating dietary Cu concentrations, it is important to also consider the potent Cu antagonists Mo and S, and perhaps dietary Fe. High dietary Fe, when present in an available form, reduces bioavailability of Cu (
      • Spears J.W.
      Trace mineral bioavailability in ruminants..
      ) and Mn (
      • Hansen S.L.
      • Ashwell M.S.
      • Moeser A.J.
      • Fry R.S.
      • Knutson M.D.
      • Spears J.W.
      High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves..
      ). However, analyzed dietary Fe concentrations are difficult to interpret because of unknown bioavailability from feed ingredients and possible soil contamination that can greatly increase analyzed values. Iron in most soil types is considered to be of low bioavailability to ruminants, but soil Fe appears to become more available when exposed to the acid environment during silage fermentation (
      • Hansen S.L.
      • Spears J.W.
      Bioaccessibility of iron from soil is increased by silage fermentation..
      ). Calcium and P are known to be Mn antagonists in poultry. Research is needed to determine whether dietary Ca and P affect Mn bioavailability in ruminants. Factors that affect Zn bioavailability in ruminants are also not clearly defined. Earlier research examining the interaction of Ca and Zn in ruminants has been inconsistent (
      • Spears J.W.
      Trace mineral bioavailability in ruminants..
      ). Studies are needed to determine whether high soluble Ca concentrations in the rumen affect Zn bioavailability and also degradation of phytate by microbial phytase. The interaction between Ca and phytate with Zn may be more important than previously thought if high soluble Ca concentrations reduce phytate degradation in the rumen.
      Studies have indicated that approximately 40% of dairy cows in the United States have liver Cu concentrations in excess of 500 mg/kg of DM. Research is needed to determine whether high liver Cu concentrations in dairy cows, not showing clinical signs of toxicosis, affect longevity in the herd, milk production, or reproduction. If longevity or production is affected, liver Cu concentrations necessary to cause impairment need to be better defined. Studies are also needed to determine whether high liver Cu concentrations in dairy cows are due to Cu over supplementation of cows, excess Cu supplementation in milk replacers and heifer development diets, or a combination of the 2.

      ACKNOWLEDGMENTS

      J. W. Spears acknowledges his former graduate students, who were responsible for much of the research cited in this review. He also acknowledges his long-term research specialist, Karen Lloyd, for her many contributions. This research was supported by state and federal funds appropriated to North Carolina State University.

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