Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. SOHE ASPECTS OF COPPER TOXICITY IN SHEEP GRAZING NE\-l ZEALAND PASTURES A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Hassey University. Bruce Campbell Farquharson B .V .Sc . ( Queensland ) December 1 984 ii ABSTRACT Several authors have suggested a role for different predisposing factors in cases of copper toxicity , but any association between these factors and biochemical changes in the sheep have not been studied. Therefore the objective of this study was to look for the changes that occurred in sheep treated regularly with a parenteral form of copper . After identifying some of these parameters, further sheep were subjected to stressors that may be encountered in normal farming practice and the changes in response to copper treatment were compared to those of sheep not subjected to that stesscr but treated with the same dose of copper . Eleven Romney rams were regularly monitored to measure any changes that occurred in certain biochemical parameters when the sheep received a weekly subcutaneous injection of 50mg of copper calcium edetate . The biochemical parameters included serum levels of sorbitol dehydrogenase (SDH ) and serum glutamate transaminase (SGOT) , blo.od copper concentration , and content . At fortnightly intervals, liver biopsy collected for an estimation of the copper content histopathological examination . oxaloacetate wool copper samples were and for a It was found that the activity of SDH and SGOT in the serwn became elevated as the liver accumulation of copper increased . SDH levels were the first to change and became elevated up to six weeks earlier than SGOT . Although the pathological changes in the hepatocytes developed progressively , their severity and extent did not have a direct relationship to liver copper content . There was no significant change in the copper content of e ither blood or of wool during the period that the liver copper content increased . iii The determination of indicators of early changes in cases of toxicity allowed stressors to be superimposed on copper therapy to assess whether such stressors might influence copper toxicity . The sheep involved received 3mg Cu/kg bodyweight as copper calcium edetate on the premise that sheep of low bodyweight might receive a double injection of the currently recommended dose of 50 mg per sheep . The same biochemical parameters of SDH and SGOT activity , b lood copper concentration and liver copper content were useq to assess the potentiating effects on copper toxicity of sheep first treated with copper calcium edetate parenterally and then subjected to various stressors . For this purpose 56 sheep, with additional control animals where appropriate , were divided into their respective groups . The stressors included dehydration by removing 25% of blood volume , starvation by fasting for 48 hours , exposure to cold ( 5°C ) for 5 days , and exposure to heat ( 40QC ) for 5 days . Other sheep were e ither immet•sed in an organophosphate insecticide , o t' treated with thiabendazole anthelmintic at a dose rate of 1 00 mg/kg bodyweight . Pregnant sheep , and others heavily parasitised ( e . p . g . > 1 570) were s imilarly treated . The stressors of dehydration application of insecticide or the and cold , administration pregnancy , the of anthelmintic showed no evidence of enhancing the toxic effects of copper . However the stressors of starvation, heat , and parasitism did potentiate toxicity and resulted in approximately half of the sheep in each group dying from copper toxicity . A further series of experiments used 60 sheep, divided into 1 4 groups ; each group being given a different schedule of copper administration which consisted of one o f the stressors and/or one of a series of formulations ·of copper consisting of salts made up in various bases . Blood samples were collected hourly for 1 6 hours and the rate of change of blood copper concentration was measured . In the sheep that vrere starved , the rate of change of blood copper concentration increased to 0 . 1 41 mg Cu/1/hr for animals starved for 72 hours in comparison Hith .a rate of 0 . 056 mg Cu/1/hr for animals iv given access to food and water . Those sheep that received 50 mg of copper calcium edetate in either of two proprietary formulations ; one containing polyvinyl pyrrolidine ( PVP) and the other without PVP but the same dose contained in half the volume , showed a mean blood copper concentration rate of increase of 0 . 0 1 7mg Cu/1/hr . An increase in the dose to 1 00mg Cu increased the rate of uptake of copper to 0 . 022mg Cu/1/hr , whereas a 50mg dose diluted in an equivalent amount of water showed an increase in the rate of translocation of copper to 0 . 036mg Cu/1/hr . The four sheep subjected to heat stress or given copper by mouth as copper edetate at a dose rate of 0 . 33mg/kg showed a blood copper concentration increase to 0 . 025mg Cu/1/hr , whereas 7gm of oral copper oxide needles administered to three 50kg sheep did not produce any increase in blood copper concentration during the period of study . Starved sheep also showed changes in their blood concentrations of glucose and albumin . Blood glucose reduced from a mean of 4 . 7gm/ 1 00ml to a mean of 2 . 4gm/ 1 00ml in the nine sheep starved over 72 hours , plasma albumin increased from 1 . 30gm/1 00ml to 2.26gm/ 1 00ml , and total protein rose by 1 0 .2%. Deaths following the administration of copper therapeutically have been reported on many occasions . Therefore it was decided to measure the effects of copper therapy on the liver copper storage of sheep which initially had a range of liver copper concentrations . A "copper deficient" farm which regularly reports lambs with enzootic ataxia , and a "copper sufficient" farm with no reported signs of copper deficiency in sheep , 'ire r e selected . Two hundred sheep on the copper deficient farm and fifty sheep on the copper sufficient farm were treated once annually with 50mg of copper calcium edetate given subcutaneously . This dose was adequate to maintain the liver copper content of all treated sheep on the copper deficient farm above 70 ppm Cu D .M . However in the sheep grazing the copper sufficient farm , liver biopsy samples indicated that copper , apparently surplus to requirements , was stored in the liver resulting in copper concentrations in all sheep in excess of 5 1 0 ppm Cu D . M . V Another study measured the uptake of copper by the liver in groups of four sheep of four different breeds common in New Zealand . These breeds were the Border Leicester , N . Z . Romney , Suffolk and Merino . There was no significant difference between the former three breeds, but the Merinos retained less copper in their livers after grazing pasture for 3 months ( 88ppm vs 1 6 4ppm) , and also following administration of copper by subcutaneous injection ( 2 1 5ppm vs 330ppm ) . The results of this work indicate that certain common stressors met with in everyday sheep management , may enhance copper toxicity . Copper should never be administered to sheep unless the requirement has been confirmed , and at the time of administration particular attention should be paid to avoiding those circumstances that might lead to starvation of the sheep . vi ACKNOWLEDGEMENTS I would like to thank sincerely my two supervisors , Prof . A . N . Bruere and D r B . S . Cooper for their guidance , inspiration and ready willingness to discuss this project . I am grateful to Dr J . Lee o f Applied Biochemistry Division of D . S . I . R . for his assistance with analytical methods and in the operation of spectrometer . the inductively-coupled argon plasma emission The technical a2sistance received from Miss Karen Armitage and Miss Gaeleen Bell was appreciated . Mr P . H . Whitehead supplied most of the sheep used in this study and Mr C .K . Barnett spent many hours feeding them and caring for them . The farmers , Mr D . J . Duncan , Hunterville , Mr G . Craine , Feilding, and Mr H.J . Ellison , Tangimoana , were most cooperative and interested in this project . To these people I express my appreciation . Many people in the Faculty of Veterinary Science gave me willing assistance and I thank them for their cooperation . In particular I thank Mr J . V . Pauli and Mrs E . Davies for their assistance with the c linical pathology , Mrs P . Slack for the preparation of the histological sections , Dr R . D . Jolly for the assistance in the interpretation of the histological sections , and Mrs Janice Tan for the preparation and photography of the electronmicrographs . This work was financed by Glaxo ( N . Z . ) , Ltd and would not have been possible without their support and encouragement . vii Finally , to my wife , Rosemary , and my children Anne , Kirsty , and Stuart , I offer my greatest appreciation for their interest and reassurance throughout the entire period of the project . TABLE OF CONTENTS Chapter 1 : Literature Review 2 : Establishing biochemical parameters to monitor the effects of excessive copper used therapeutically 3: Assessment of the effect of various stressors on the potential for toxicity from copper toxicity 4 : Absorption rates of different copper formulations under various conditions 5 : Monitoring the effect of copper supplementation on animals grazing copper sufficient and copper deficient farms 6: The differences in the absorption of copper amongst four viii 36 67 82 97 d ifferent breeds of sheep in New Zealand 1 1 8 7 : Copper content of wool from sheep of known copper status 1 32 General discussion 1 39 Appendices 1 50 Bibliography 1 55 ix LIST OF TABLES Table 1 . 1 : Essential copper-containing enzymes and their functions 1 . 2 : Copper absorption from various feeds Page 1 2 1 4 1 . 3 : Reported cases of copper toxicity in sheep in New Zealand taken from "Surveillance" reports of Animal Health Laboratories 1 . 4 : List of workers and the serum enzyme analyses they used in the investigation of copper toxicity 2 . 1 : Results of copper analysis and histology of tissues 26 32 collected from sheep used in copper toxicity studies 47 2 . 2 : Changes in blood parameters of sheep no . 237 during a haemolytic crisis 49 3 . 1 : Details of housing , management , stressors , and copper administration , and schedules to all groups of sheep 69 3 . 2 : Results of haemolysis , liver enzyme increases , and number of deaths in animals from all groups 73 .3 . 3 : Summary of results of Table 3 . 2 combining stressor groups exhibiting deaths and stressor groups in \-lhich no deaths occurred 3 . 4 : Results of bodyHeight , serum pepsinogen levels , faecal egg counts , and total worm burdens of hoggets given copper calcium edetate at a dose rate of 3 mg Cu/kg bodyweight 3 . 5 : Results of bodyweight , faecal egg count , and total worm counts of hoggets given copper calcium edetate at a 74 75 dose rate of 2 mg/kg bodyweight 76 3 . 6 : Results from post-mortem material of sheep that died 77 4 . 1 : Schedule of food and water deprivation of sheep used when determining copper uptake rate 84 4 . 2 : Design of an experiment to compare translocation rates of different injectable c .opper formulations 86 4 . 3 : Mean blood parameter changes of sheep undergoing various periods of starvation immediately prior to copper supplementation 4 . 4 : Liver copper content of sheep after administration of copper oxide needles 4 . 5 : Mean uptake rates of copper in sheep administered different copper therapeutic agents 4 . 6: Mean uptake rates of copper in sheep administered copper calcium edetate after being subjected to different periods of starvation· 5 . 1 : Mineral content of pastures taken from area 1 on the copper deficient farm 5.2 : Mineral content of pastures taken from area 2 on the copper deficient farm 5 . 3 : Mineral content of pastures taken from the copper sufficient farm 5 . 4 : Bodyweights of sheep grazing the copper deficient farm 5 . 5 : Bodyweights of sheep grazing the copper sufficient farm 5 . 6: Liver copper content of sheep grazing the copper deficient farm 5 . 7 : Liver copper content of sheep grazing the copper sufficient farm 6. 1 : Composition of basal low copper diet 6. 2 : Copper content of liver samples from different breeds of sheep given four different diets , followed by parenterally administered copper 6. 3 : Bodyweights and bodyweight changes of different breeds 88 89 9 1 9 1 1 03 1 04 1 08 1 09 1 09 1 1 0 1 1 1 1 2 1 1 25 of sheep when eating different diets 1 26 6. 4 : Mineral content of respective diets used 1 27 7 . 1 : Copper content of fleeces determined by other workers 1 33 X xi LIST OF FIGURES Figure 1 . 1 : Intestinal absorption of copper � 8 1 . 2 : Copper homeostasis in the normal hepatocyte 1 0 2 . 1 : Path o f the liver biopsy probe shah� using a dissected specimen 38 2 . 2 : Average blood parameters of all sheep recorded from six daily .bleedings prior to haemolytic crisis or death 43 2 . 3 : Changes in sorbitol dehydrogenase activity in serum of eleven sheep subjected to weekly injections of copper calcium edetate 2 . 4 : Changes in glutamate oxaloacetate transaminase activity in serum eleven sheep subjected to weekly injections of of copper calcium edetate 2 . 5 : Serum from sheep no . 237 collected during the haemolytic crisis 2 . 6 : Electronmicrograph of normal erythrocytes 2 . 7 : Electronmicrograph of erythrocytes taken from sheep no . 237 during the haemolytic crisis 2 . 8: Jaundiced conjuncti�al membranes in a sheep that died 4 5 4 6 48 50 50 from copper toxicity 5 1 2 . 9 : Petechial haemorrhages in the pectoral muscle planes of a sheep that died from copper toxicity 5 1 2 . 1 0 : Typical ' gun-metal ' kidneys from a sheep that died from copper toxicity 2 . 1 1 : Histology of normal liver tissue 2 . 1 2 : Histology of liver from a sheep that died from c�pper toxicity 2 . 1 3 : Histology of liver from a sheep that died from copper toxicity , showing greenish/black granules of copper 2 . 1 4 : Histology of normal kidney tissue 2 . 1 5 : Histology of kidney tissue from a sheep that died from copper toxicity 52 54 54 55 56 56 xii 2 . 1 6: Histology of normal brain tissue 58 2 . 1 7 : Histology of brain tissue from a sheep that died from copper toxicity 58 5 . 1 : Location of the two experimental farms 99 5 . 2 : Relationship between soil copper and pasture copper on the copper sufficient farm 1 02 5 . 3 : Copper , molybdenum and sulphur content of pastures on the two areas of the copper deficient farm 1 05 5 . 4 : Copper, iron , zinc and manganese content of pastures on the two areas of the copper deficient farm 1 0 6 6 . 1 : Probable origins and relationships of British breeds of sheep 1 1 9 7 . 1 : Copper content of wool samples 1 36 7 . 2 : Copper content of liver tissues 7 . 3 : Copper content of blood samples 1 36 1 36 xiii APPENDICES Appendix I: Technique for analysis of blood samples 1 50 II: Technique for analysis of tissue samples 1 5 1 III: Technique for demonstrating copper in tissue sections 1 5 3 I V : Technique for analysis o f wool samples 1 54 1 CHAPTER 1 : LITERATURE REVIEW Historical Chronic copper poisoning in farm animals was first recorded in 1 883 by Ellenberger & Hofmeister who induced the condition experimentally . The first confirmed field case in sheep occurred in animals grazing in orchards that had recently been sprayed l-lith Bordeaux mixture ( Schaper & Luetje , 1 93 1 ;Beijers , 1 932) . Since that time clinical and experimental cases have been reported from natural and accidental causes and from most countries of the world. The first report of possible copper toxicity in New Zealand sheep was made by Hopkirk , ( 1 9 3 1 ) , who described cases of ' epizootic jaundice ' which occut�red in January and March . These sheep were grazing land that Has growing excessive clover . In the subsequent reports ( Hopkirk , 1 932 , 1 933 , 1 934 & 1 937 ) deaths due to epizootic icterus were reported and the sheep referred to in the 1 934 outbreak were suspected of having eaten ( 1 938 ) , analysed the livers ragwort of sheep ( Senecio jacobea ) . Fitch , which had died of enzootic icterus , and he found that these had copper concentrations as high as 3 , 280 ppm D .M . Subsequent work at Wallaceville b y Buddle i n 1 939 showed that sheep dosed with 1 00ml of 1 % CuS04 daily , developed signs and showed post-mortem lesions similar to those described in epizootic jaundice . The liver analysis from these sheep revealed 2 , 940 , 4 , 030 , 3 , 500 , & 1 , 950 ppm D .M . of copper , ( Hopkirk , 1 939 ) . 2 Copper as an essential element Soil as a source of copper The earth's crust contains between 45 ppm Cu and 70 ppm Cu ( Hodgson , 1 963; Norrish , 1 975 ) . Most soils of the \.-orld contain about 20 ppm but estimates ranging frqm 2 1 ,000 ppm have been recorded ( Allaway , 1 968; Aubert & Pinta , 1 977 ) . In New Zealand , soils commonly contain about J7 . 5 ppm of copper . Some of these soi ls have levels as low as 2 ppm but the upper limit rarely exceeds 25 ppm (Wells , 1 957 ) , apart from the yellow brown loams and brown granular loams and clays derived from andesitic ash which have a higher copper content ( Rolt , 1 962 ) . The copper content of the soil depends on the parent material from which it was derived ( Ermolenko , 1 9 72 ) , and on the amount of weathering , that has subsequently taken place . The greater the amount of weathering ; Ermolenko, 1 975) . the lower the copper content (Wells , 1 9 57; Copper may be bound· in the soil (a) in a water-soluble form, ( b ) as exchangeable ions, ( c ) in a non-exchangeable form as an organic complex , ( d ) in association with iron , aluminium and sometimes manganese oxides , or ( e ) as a constituent of crystal lattices of other sand- , silt- , and clay-sized minerals ( Le Riche et al . , 1 9 63; Mitchell , 1 972; Ermolenko , 1 975; Norrish , 1 975; Aubert & Pinta , 1 977; McBride , 1 98 1 ) . Up to 99% of coppe r in soil solution may be complexed with organic matter ( Hodgson et al . , 1 9 66; Mitchell , 1 972 ) . This formation of insoluble organic complexes may act as a reserve supply of copper for the plant ( Ermolenko , 1 972 ) , and such copper may be liberated in a form accessible to plants in conditions liable to cause the copper to precipitate ( e .g . in calcareous soils ) . The complexes may act as a transport and carrier agent of copper for plant use , or they may reduce the concentration of available ionic cu++ to a non-toxic level when excess copper is 3 present (McBride, 1 981 ; Stevenson & Fitch, 1981 ) . The mobility and availability of texture, drainage, redox potential, ( Thornton, 1 979 ) . copper is related to soil pH, and organic matter status Wet soils are usually anaerobic and they tend to convert copper to various insoluble sulphides . If the soil is low in organic matter, the soluble copper compounds are leached out ( Ermolenko, 1 972 ) . Wet-dry cycles tend to increase the amount of exchangeable copper in chelated form (Ng Siew Kee & Bloomfield, 1 962 ) but · this does not necessarily impair its availability to plants ( Tiller et al . , 1 972 ; McLaren et al., 1 974) . Drainage o f wet soils may limit the availability of copper by favouring the formation of more highly oxidized forms ( Allaway, 1 968) , but drainage may still increase the uptake of copper by clovers by as much as twenty-five percent (Mitchell , 1 972 ) . Increasing soil acidity by fertilizers ( Ermolenko, 1 975 ; the application Loneragan, 1 975 ) , of nitrogen increases the mobili ty of copper ( Ermolenko, 1 972 ; Loneragan, 1 975 ) , whereas increasing the soil pH with lime decreases the mobility of copper ( Ermolenko, 1 972 ) 1 975 ) by increasing the adsorption of cu++ ions on to iron and manganese oxides and clay lattices (Murray et al . , 1 9 68; Murray, 1 975 ; James & Barrow, 1 981 ) . It has been reported that overliming can render copper unavailable in those soils liable to induce copper toxicity ( Peech, 1 94 1 ; Aubert & Pinta, 1 977) . The application of copper fertilizers to soils may increase the concentration of copper in plants (Wells, 1 9 57 ; Branion, 1 960 ; Peive, 1 959) . The increase has been found to be as high as 30% in some instances (Mitchell et al . , 1 9 57 ) . As copper is not a mobile e lement in soil (Hodgson, 1 963 ) the effect of copper supplementation depends on the establishment of a large number of copper depots in the soil (Gilkes, 1 981 ) , and on the soil pH, fertilizer particle s ize and the root interception by · the plant . 4 In the diagnosis of copper deficiency in either plants or animals , the measurement of the total amount of copper in the soil is of little value , as it is seldom correlated with the amount of copper available to plants ( Le Riche et al . , 1 963 ; Mitchell , 1 97 4 ) . It is the extent to which it has been mobilized and is present in the soil in an available form that is significant ( Mitchell , 1 972 ) . Plants as a source of copper Copper is required by the more highly developed p+ants for incorporation into enzymes that assist in respiration , photosynthesis and the formation of proteins required for lignification . It is also involved in anabolic metabolism , cellular defense mechanisms and hormone metabo�ism ( Nicholas , 1 975 ; Walker & Webb , 1 98 1 ) . The amount of copper absorbed by the roots of the plant depends on the mass flow of copper in soil solution , the diffusion of copper down the osmotic gradient , and the amount of root interception ( Mi tchell , 1 972 ; Ermolenko , 1 975 ; Loneragan , 1 975 ; Graham , 1 98 1 ; Jarvis , 1 98 1 ) . The root interception is determined by the root length per plant , and by the dens ity of root hairs and the amount of root exudate secreted to mobilise cu++ from the soil organic complexes ( Ermolenko , 1 972 ; Loneragan , 1 975 ; Norrish , 1 975 ; Graham , 1 98 1 ) . The roots of plants accumulate copper in a bound form in the cell ·Halls (Graham , 1 98 1 ; Jarvis & Jones , i979 ) , and when soil copper content is high , they may contain 1 0- 1 00 times as much copper as in the plant solution ( Chandry & Loneragan , 1 970 ) . This excess copper is not translocated to the plant shoots , unless the roots are damaged , in which case.·copper may be released to enter the plant (Graham , 1 98 1 ) . Plant tissues normally contain 5-20 ppm D . M. of copper . Copper deficiency usually occurs when the concentration falls below 4 ppm while toxicity occurs Hhen levels rise above 20 ppm ( Jones , 1 972 ; Robson & Reute�, 1 98 1 ) . In New Zealand very few areas grow pastures which contain more than 1 3 ppm of copper ( Cunningham et al . , 1 956 ; 5 Rolt , 1 962 ) . The young shoots of plants contain the highest amounts of copper but levels decrease steadily as the plant matures (Adams & Elphick , 1 956 ; Cunningham et al . , 1 956 ; Allaway , 1 968 ; Mitchell , 1 972 ; Loneragan , 1 975 ; Loneragan , 1 981 ) . This movement of copper from old leaves during senescence parallels the loss of nitrogen until at full senescence, leaves may only contain 20% of the original copper content (Loneragan , 1 975 ; Loneragan et al . , 1 980 ; Loneragan , 1 981 ) . As the seed heads o f plants mature they gain copper at a rate similar to the decline in the concentration of copper in the leaves and stems (Loneragan , 1 981 ) . Legumes have a higher affinity for copper than do grasses. (Beeson & MacDonald , 1 95 1 ; Adams & Elphick , 1 956 ; Mitchell et al . , 1 9 57 ; Branion , 1 960; Gladstones & Loneragan , 1 970; Patil & Jones , 1 970 ) , especially when they are grO\iing in soils of higher copper content or in soils that have been supplemented with copper . The application of copper fertilisers to copper deficient soils will increase the copper content of plants growing in that soil (Loneragan , 1 975 ) , but copper application will have little effect on plants growing et al . , 1 957 ; in soils containing adequate copper levels (Mitchell Allaway , 1 9 68) . Fertilisers such as nitrogen that promote plant growth and alter pasture composition tend to reduce the pasture copper content (Lone ragan , 1 97 5 ; Reith et al . , 1 97 9 ) . High concentrations of copper are toxic to most plants . The symptoms of copper poisoning include reduced shoot vigour , poorly developed and discoloured root systems , and leaf chlorosis (Smith & Specht , 1 953 ; Delas , 1 963 ; Reuther & Labanauskas , 1 96 6 ; Daniels et al� , 1 972 ) . Toxicity commonly occurs in very acid soils (pH< 5 ) which have low cation exchange capacity (Reuther & Smith , 1 954 ; Leeper , 1 978) , or it may be induced by the application of copper to soils with a low organic matter content (Delas , 1 9 63 ; Page , 1 974 ; Purves , 1 977) • 6 Many plant species , especially herbaceous plants , can tolerate high copper levels by immobilis ing copper complexes in cell vacuoles or in non-diffusable metal-protein complexes , or by excluding the uptake of the metal (Woolhouse & Walker , 1 98 1 ) . The copper content of plants should be measured by the analysis of plant material , and whenever concentrations in whole shoots exceed 20 ppm D .M . , the plant must be treated as a potential source of copper toxicity to animals ( Robson & Reuter , 1 98 1 ) . Absorption of copper by the animal- There have been many studies on the absorption of copper by sheep and cattle . Most of these have dealt with circumstances of the normal physiological process and those involving toxicity . There are clearly species and age differences of importance . The newborn lamb absorbs copper very efficiently . Immediately after birth almost 1 00% of the lamb ' s copper intake is absorbed , but uptake decreases to 1 0% by weaning ( Suttle , 1 973; Suttle , 1 975; Suttle , 1 979 ) . This decrease in absorption is due to both the lowering of the concentration of copper in the ewe ' s milk as lactation advances ( from 1 . 2- 1 . 4 ppm in colostrum to 0 . 05-0 . 2 9 ppm at weaning ) ( Beck , 1 94 1 ; Polidori , 1 960; Ash ton & Williams , 1 977; Lonnerdal et al . , 1 98 1 ) , and the decreased absorption of copper via the ileal mucosa ( Suttle , 1 975 ) . The lamb foetus begins to accumulate copper in its liver at the twelfth week of gestation , while maternal liver stores start to decrease in the fourteenth week of pregnancy ( Russanov et al . , 1 98 1 ) . The newborn lamb has low concentrations of liver copper but rapidly accumulates copper in the abundant mitochondrial hepatocuprein ( Suttle , 1 975) . Adult sheep absorb less than 1 0% of dietary copper ( Suttle , 1 979 ) , and only about 5% of the dietary intake is stored in the liver (Dick , 1 954; Hemingway & ·MacPherson , 1 967; Suttle , 1 973; Lee , 7 1 97 4 ; Moodie , 1 975 ) . The absorption o f copper appears to be a physiologically controlled process (Neethling et al . , 1 968 ) , as during lactation ewes temporarily increase their efficiency of absorption ( Suttle , 1 979 ) . A similar phenomenon is found in sheep with hypocupraemia ( Neethling et al. , 1 9 68 ; Hill et al . , 1 969 ) , and it was proposed by Evans & Johnson ( 1 97 8 ) and by Bremner ( 1 9 8 1 ) that it is the protein metallothionein in the intestinal mucosa that is largely responsible for the control of copper . Metallothionein determines the rate at which copper is transported across the intestinal mucosa . Copper can only be absorbed in the ionic form by attachment to a binding site on metallothionein (Mills , 1 96 1 ) . Metallothionein provides binding sites for copper within the intestinal mucosa and acts as a temporary storage site pending subsequent absorption or excretion of this element (Evans , 1 973 ) , but it does have an upper threshhold level ( Saylor et al . , 1 980 ) . At the serosal surface of the intestine the copper dissociates from metallothionein and either diffuses directly into the plasma as ionic copper or becomes bound to either albumin or a copper-amino acid complex for transport to the liver via the portal circulation (Figure 1 . 1 ) ( Bush et al . , 1 9 56 ; Beaten & McHenry , 1 964, Evans , 1 973 ) . The efficiency vrith which copper is assimilated from the diet depends on the solubi lity of the copper (Mills , 1 9 6 1 ) , which in turn is largely dependent upon pH ( Bremner , 1 970 ; Kirchgessner & Grassman , 1 970 ) . In the rumen most o f the copper is present either in an insoluble form or is pound to receptors in the rumen microorganisms (Mills , 1 9 6 1 ; Bremner , 1 970 ) . As the copper complexes are more stable · at lowe r pH , abomasal absorption is minimal , but with the rising pH of ingesta distally , copper is freed and absorbed in the ileum ( Bremner , 1 970 ) and in the large intestine (Grace , 1 975 ) . The amount absorbed in the ileum is small , and is similar in amount to that secreted in the biliary and pancreatic secretions (Grace , 1 975 ) . 8 Lumen Mucosa Plasma [Faecyl · Cu-metallothionein Cu-albumin !t ii cu..:.MM Cu2+ + AA� Cu2+ Cu2+ �t !I 1! Cu2+ Cu-AA AA�_ t /\ Cu-AA ATP AA = amino acid MM = macromolecule Figure 1 . 1 : Intestinal absorption of copper ( Evans , 1 973 ) Approximately 72% of ingested copper is excreted in the faeces and about 2% in the urine ( Scheinberg & Sternlieb , 1 9 60 ; Neethling et al . , 1 968 ; Evans , 1 973 ; Grace , 1 975 ; Grace & Gooden , 1 980 ) . The remainder of endogenous copper is lost in the secretions of saliva ( Bertoni et al . , 1 976; Stevenson & Unsworth, 1 978 ; Grace et al . , 1 98 1 ) , bile ( van Ravesteyn , 1 944 ; Grace & Gooden , 1 980 ) , and pancreatic juices (Grace & Gooden , 1 980 ) ; none of which is reabsorbed from the gastrointestinal tract as the copper ions are bound in organic complexes (Grace , pers . comm . ) . The difference in the absorption rate and in the excretion rate between species . may affect the copper requirements and the copper reserves of these animals . Sheep are more susceptible than other domestic ruminants to copper accumulation and poss ible toxicity , for three reasons . First , they have less control of the intestinal copper homeostatic mechanism ( Bremner & Davies , 1 979 ) , secondly , they have l imited capacity to excrete copper from the liver ( Beck , 1 96 3 ; Neethling 1 968; Corbett et al . , 1 978 ; Bremner , 1 98 1 ) , and thirdly , their lysosomes are unable to sequester large amounts of copper 9 (Corbe tt et al . , 1 978 ) . Metabolism of copper by ruminan ts Copper is essential for the metabolism of most plant and animal cells ( Scheinberg & Sternlieb , 1 960 ) . In the animal body it is an essential part of many enzymes and structural and carrier proteins . The highest concentrations of copper are found in the live r , brain , heart and kidney . The lung , intestine and spleen carry inte rraediate levels , while the endocrine glands , muscle and bone have · the lowest levels ( Adelstein & Vallee , 1962 ; Evans , 1 973 ) . After intestinal absorption , much o f the copper is rapidly deposited in the liver . Deposition occurs within minutes of absorption and continues for several hours ( Scheinberg , 1 96 1 ) . In the liver , copper undergoes a chain of metabolic processes preparing copper ions for subsequent incorporation into proteins for storage , transport and excretion of copper . There are three distinct pathways involved in this process : ( i ) the preparation of copper for secretion into bile for excretion , ( ii ) the temporary storage of copper , and ( iii ) the incorporation of copper into caeruloplasmin for distribution throughout the body ( Evans , 1 973 ) . Biliary copper enters the bile canaliculi bound to amino-acid ( Evans , 1 973 ) , and its resorption is negligible . Copper is stored in the liver hepatocytes : the distribution being 20% in the nuclear fraction , 1 0% in microsomes , and 20% in the large granules of mitochondria · and lysosomes . The remainder is stored in the cytosol as either copper-dependent en zymes or metallothionein ( Evans , 1973 ; Bloomer & Sourkes , 1974; Davies & Wahle , 1978 ; Saylor & Leach , 1980 ; Saylor et al . , 1980 ; Russanov · et al . , 198 1 ) . The copper concentration o f the subcellular fractions of the liver is related to the total copper content of the liver rather than to the physiological state of the animal such as age , species or copper status (Gregoriadis & Sourkes , 1967 ) . Once 1 0 concentrations reach a certain threshhold , any copper surplus to requirements is seques tered into the lysosomes so that copper ions are not available to initiate toxic effects (Lindquist , 1 968 ; \-lorwood & Taylor , 1 9 69 ; Evans et al . , 1 973 ; Sternlieb & Goldfischer , 1 976 ) ( Figure 1 . 2 ) . As the liver becomes saturated with copper then the kidneys become the secondary s ite of copper deposition (Walshe , 1 968 ) . Cu-metallothionein i� Cu2+ + AA �1 Lysosomes ti Cu-albumin� Cu2+9= � Cu-AA AA j Cu-enzymes J AA = amino acid Figure 1 . 2 : Copper homeostasis in the normal hepatocyte ( Evans , 1 973 ) Caeruloplasmin , a globulin , is a mul ti functional metalloprotein respons ible for copper transport . It also facilitates the mobi lization of iron from iron storage sites to plasma for haem synthes is , and acts as a regulator of circulating biogenic amine levels through its oxidase activity ( Beaten & McHenry , 1 9 64 ; Evans , 1 973 ; Evans & Abraham , 1 973 ; Frieden & Hsieh , 1976; Mills et al., 1 976 ; Frieden , 1 978) . Caeruloplasmin is synthesized in the liver , and its rate of formation depends on the hepatic copper concentration ( Bush et al . , 1 956 ; Evans , 1 97 3 ; Moodie , 1 975 ) . Thus , it aids in the regulation of hepatic copper concentration . It has a half-life of about 50 hours in plasma ( Frieden & Hsieh , 1 97 6 ) . Blood contains copper in erythrocyte superoxide dismutase five which 1 1 separate fractions : in contains 60% of erythrocyte copper , in an erythrocyte copper complex , in plasma caeruloplasmin which comprises 60-95% of plasma copper ( Shields £!__al . , 1 96 1 ; Westerfield , 1 96 1 ; McCosker , 1 968a ; Weser , 1 974 ) , in albumin-copper , and bound to plasma amino acids ( Brown et al. , 1 968 ; Evans , 1 973 ; Moodie , 1 975 ; Frieden & Hsieh , 1 97 6 ) . The erythrocyte and plasma copper contents are approximately equal (Westerfield , 1 96 1 ) . The total content of copper in the erythrocyte does not fluctuate in spite of variations in the copper status of tDe animal ( Evans , 1 973) and erythrocytes are not involved in copper transport ( Evans , 1 97 3 ; Frieden & Hsieh , 1 976 ) . Copper i� incorporated into the molecular structures of five major enzymes , and several less important ones , as well as being part of several proteins and amino-acids ( Evans , 1 9 73 ) . Caeruloplasmin is a major copper enzym� because of its ferroxidase activity and involvement in iron transport ( Table 1 . 1 ) . Cytochrome oxidase is the te rminal enzyme in the oxidative phosphorylation process and is si ted in mitochondria ( Scheinberg & Sternlieb , 1 960 ; Gallagher & Reeve , 1 97 1 ; Evans , 1 97 3 ; Mills et al . , 1 97 5 ) . Dietary copper deficiency may resul t in a 40-50% reduction in the rate of cytochrome oxidation activity (Davies & Wahle , 1 978) affecting especially the liver and intestinal mucosa ( Boyne , 1 978) , and myelin formation in the central nervous system (Gallagher & Reeve , 1 97 1 ; Evans , 1 973 ) . Monamine oxidase ( lysyl oxidase ) is the enzyme required for the cross-linkage of the peptide chains of collagen and elastin ( Bornstein et al . , 1 966 ; Partridge , 1 9 66 ; Carnes , 1 97 1 ; Mills et al . , 1 976 ; Harris & Rayton , 1 978) which maintain the structural integrity of both vascular and skeletal tissue ( Evans , 1 973 ) . Copper deficiency is characterized by fragili ty of the skeletal system (Carnes , 1 97 1 ; Evans , 1 973 ) . 1 2 Enzyme Activity Source Function Caeruloplasmin ferroxidase plasma iron transport Cytochrome oxidase terminal mitochondria energy metabolism oxidase & phosphorylation Lysyl oxidase peptide cross- aorta & . collagen & elastin linkage cartilage formation Tyrosinase oxidase melanocytes tyrosine to melanin Supe roxide dismutase all aerobic 0�+0�+H20�H202+02 dismutase cells Dopamine-B- oxygenase adrenal gland dopamine to hydroxylase nor-epinephrine Table 1 . 1 : Essential copper-containing enzymes and their functions . Tyrosinase is an oxidase required for the conversion o f tyrosine to melanin needed for pigmentation ( Scheinberg & Sternlieb, 1 960; Walshe, 1 968; Evans, 1973 ) . Superoxidase dismutase (which includes all the cupreins ) is present in all aerobic cells ('Vleser, 1 974 ; Fridovich, 1 975 ). The enzyme catalyses the dismutation of supe roxide free radical anions to hydrogen peroxide and oxygen (McCord & Fridovich, 1 9 69b; Evans, 1 973 ; Fridovich, 1 975b ) . It is the primary defence mechanism against the toxic singlet oxygen radical ( Evans, 1973 ; Weser, 1 974; Fridovich, 1 975a ) • . Copper requirements by the sheep If copper associated with the liver is excluded, a fully fleeced sheep, contains about 60mg of copper; each kilogram of bodyweight containing 0.8mg of copper and each kilogram of wool containing 6-8mg of copper (Grace, 1 9 83 ) , to 9- 1 1 mg of copper (Cunningham & Hogan, I I I I 1 3 1 958 ) . A newborn lamb contains a total of 1 0mg of copper (Pryor , 1 964; Williams et al . , 1 97 8 ) . The nett requirements for maintenance rarely exceed 4ug of copper per kilogram of bodyweight per day and show no relationship to metabol ic rate ( Suttle , 1 974b; Smith, 1 98 1 ; Grace, 1 98 3 ) . Growth requires approx imately 1 . 1 mg copper per ki logram of bodyweight increase ( Smith , 1 98 1 ; Grace , 1 983 ) . The foetus requires 2 . 8mg of copper per kilogram of bodyweight (Grace, 1 983 ) , and a lactating ewe requires an extra 0 . 3mg copper for every litre of milk produced ( Grace , 1 983 ) . Factors influencing copper absorption and metabolism Newborn lambs have a higher total copper concentration than their dams ; up to 50% of their copper baing present in the liver ( Cunningham, 1 93 1 ; Adelstein & Vallee, 1 9 62 ; Pryor, 1 964 ; Hartmann et a�, 1 978 ) . Such copper is stored as neonatal hepatic mitochondrocuprein which has either a storage o r a detoxifying role, or both (Walshe, 1 968 ; Porter, 1 974 ) . Plasma copper levels in the neonate are lowe r than those of the dam, as the foetus is unable to synthesize caeruloplasmin (McCosker, 1 9 68 ; Walshe, 1 9 68 ; Moodie, 1 97 5 ) and caeruloplasmin does not cross the placental barrier ( Sternlieb & Scheinberg, 1 960 ). However caeruloplasmin levels of the lamb rise rapidly to adult levels wi thin seven days of birth ( Hov1ell et al . , 1 968 ; McCosker, 1 9 68 ), while foetal m i tochondrocuprein liver reser·ves decrease (Wiener et al . , 1 974 ) • Young lambs absorb copper very efficiently while they are on a liquid cllet . This high rate of absorption is due to the higher absorptive capacity of the large intestine, and the lack of interference by sulphide from rumen microflora . Thus the increased requirements of a rapidly growing lamb on a low copper-content milk diet are satisfied ( Suttle, 1 982b ) . The amount of copper absorbed from natural food varies considerably, depending on the type of food and the season of the year . The copper contained within stored fodder such as hay or silage is absorbed at a higher rate than that of pasture ( Table 1 . 2 ) (Mills, 1 961 ; Suttle, 1979; · Suttle, 1 98 1 a ; Suttle, 1 982b ) . Feed summer pasture autumn pasture hay silage leafy brassicas root brassicas % Absorption 2 . 3 1 . 2 1 . 2 4 . 9 1 2 . 8 6 . 7 Table 1 . 2 : Copper absorption from various feeds ( Suttle, 1 98 1 a ) 14 Although MacPherson and Hemingway ( 1 96 5 ) cohsidered that pro tein retards the absorption of copper, it is more probable that the high sulphur content of most protein diets is the factor limiting copper absorption . can adapt to the variations of copper and absorptive mechanisms· Metabolic processes availabi lity as can ( Kirchgessner et al., the excretory 1 98 1 ) . During periods of high copper requirements ther·e can be, for example, active bone resorpt.ion and bone demineralization to meet the copper demands of the animal ( Ljubashevsky, 1 978 ; Anon, 1 98 1) . Higher plasma levels of copper are seen in response to increased oestrogen levels, as in ovulation ( Hidiriglou et _al., 1 982 ) , pregnancy ( Scheinberg & Sternlieb, 1 9 60 ; Scheinberg, 1 96 1 ; Adelstein & Vallee, 1 9 62 ; Pryer, 1 9 64 ; Moss et al . , 1 97 4 ; Howell, 1 9 68 ; Russanov et al . , 1 98 1 ) . Plasma concentrations of copper may also increase release of corticosteroids ( Henkin, 1 97 4 ; following the use and Andrewartha & Caple, 1 978 ) There is considerable genetic variation between breeds and strains of sheep in their abi lity to absorb copper . This has been shown from the variable incidence of enzootic ataxia recorded in different breeds of sheep {Wiener, 1 96 6 ; Wiener & Field, 1 9 69 ; 15 Poole , 1970 ; Wiener , 197 1) , and in their susceptibility to copper toxicity ( Edgar et al . , 194 1 ; Mar3ton & Lee , 1948b ; Luke & Weirman , 197 0 ; �iener & Field , 197 1 ; Luke & Marquering , 1972 ; Wiener , 1973 ; Schmitten et al. 1978 ; van der Berg et al . , 1983 ) . The maternal effect may influence copper levels for the first twelve weeks of life (Wiener , Herbert & Field , 1976 ; Wiener et al . , 1977 ; Wiener et al . , 1978 ) , whereas after weaning the breed of the sire strongly influences the concentration of liver copper in the progeny (Wiener , Hayter & Field , 1976 ; Suttle , 1982b ; Woolliams �al . , 1982 ) . There is a greater seasonal variation in copper uptake and storage in those strains or breeds of sheep that have a lower ability to accumulate copper (Wiener et al . , 19 69 ; Wiener et al . , 1970 ; Hayter. et al . , 1973 ) . This di fference between breeds diminishes as the molybdenum content of the diet is increased ( Suttle , 198 1b ) . The genetic variation in copper levels between breeds of sheep is due to differences in the absorption of copper rather than its utilisation or excretion (Herbert et al . , 1978 ; Wiener et al . , 1978) . The rumen is probably the site at which the change in copper availability is ini tiated because dosing sheep with copper oxide needles into the abomasum eliminates any differences determined by the breed of sheep (Wiener , 1980) . In ruminants the presence of certain elements interferes with the absorption and utilisation of copper and may result in the development of copper deficiency . When such inhibiting elements are not present be induced . or compete the animal . or are in low concentrations , copper toxici ty may still These elements either inhibit the absorption of copper directly with copper at its copper binding sites within In the rumen molybdenum combines with sulphur to form insoluble thiomolybdates which may form copper thiomolybdates and render copper insoluble . It has been shown that levels of molybdenum in the diet seriously Dowdy & in excess of 5 ppm D . M . , i f present with sulphur ions , may affect the uptake of copper by ruminants (Dick , 1953 ; Matrone , 1968a & 1968b ; Smith et al . , 1968 ; Spais et al . , 1968 ; 1 6 Suttle & Field , 1 968a , 1 9 68b , 1 9 68c ; Suttle , 1 974b , 1 974c ; Smith & Wright , 1 975 ; Suttle , 1 97 5 ; Suttle & Field , 1 9 83) . Other ions which may affect the absorption of copper at this site include zinc ( Evans 1 973 ; Bremner , 1 979 ; Reynolds , 1 979 ) , iron (Dick , 1 954 ; Anthony & Nix , 1 965 ; Sourkes et al . , 1 9 68 ; Standish et al . , 1 971 ) , cadmium ( Evans , 1 973 ; Hennig et al . , 1 974 ; Doyle & Pfander , 1 975 ; Ghergariu , 1 978 ; Bremner , 1 979 ) , lead ( Hemingway et al . , 1 964 ; Alloway , 1 9 69 ; Ghergariu , 1 978 ) and calcium ( Dick , 1 9 54 ; Adelstein & Vallee , 1 9 62 ) . Other heavy metals may compete with copper for certain binding sites . These include cadmium ( Evans , 1 973 ) , silver ( Evans , 1 973 ) , nickel ( Evans , 1 973) , cobalt ( Evans , 1 97 3 ) and zinc ( Evans , 1 97 3 ; Bremner & Marshall , 197 4 ; Bremner et al . , 1 976 ; Bremner et al . , 1 977) . Manganese and molybdenum may also depress copper levels by increasing its urinary excretion ( Gubler e t al . , 1 954 ; Dowdy & Matrone , 1 9 68b ) . Ingested soil is a source of many mine rals to the grazing sheep ( Field & Purves , 1 964 ; Ghergariu , 1 978) and during the winter months soil may comprise up to 30% of the diet ( Healy , 1 969 ; Suttle et al . , 1 97 5 ) . The ingestion of such large quantities of soil may decrease by up to 50% the soluble copper concentration of the duodenal liquor (Healy , 1 970) . Copper deficiency of sh eep Many conditions have been diagnosed in ruminants as being the result of copper deficiency , or they have responded to copper therapy . Copper deficiency may affect growth , development of the skeletal and nervous systems , formation of erythrocytes and wool fibres , or the function of leucocytes , the gastrointestinal tract and the reproductive system . These s igns may occur singly or in combination and not necessarily in any particular sequence . 17 Lambs which are raised on milk having a low copper content , have a lower plasma copper concentration and show a reduced growth rate ( Lee , 1 951a; Whitelaw et al . , 1977; Whitelaw & Evans , 1979; Whitelaw et al . , 1979; �fuitelaw et al . , 198 1) . Weaned lambs provided with a low copper diet supplemented with copper , may achieve good weight gains in comparison to control animals ( Bennetts & Beck , 1942; HO\-iell , 1968; Hogan et al . , 197 1 ) . Under different circumstances copper deficient lambs may continue to have good growth rates but if for any other reason their growth is impaired , the effect is much more dramatic ( Lewis , 1970 ) . Prolonged copper deficiency results in light and brittle long-bones because of the thinning of their cortices ( Hogan , 1973; Hidiroglou , 1980; Hurley , 1 981 ) . Os teoporos is , especially in the central metaphyseal region ( Cunningham, 1944; Suttle et al . , 1972; Whitelaw et al . , 1979; Hidiroglou , 1980 ) and rib fractures (Whitelaw et al . , 1979; Hurley, 198 1 ) may also be seen . Sheep with mandibular osteopathy and resulting dental abnormalities , have been found to have low liver copper concentrations ( Bruere et al . , 1979 ) . The osteoblasts in lambs which have been depleted of coppe r bo th in utero and during the suckling period , are pa rticularly sens itive to copper deficiency ( Suttle et al . , 1972 ) , but they will respond rapidly to an increased copper intake (Doyle , 1979) . Enzootic ataxia is caused by copper deficiency and is the most characteristic manifestation of copper deficiency . It affects lambs up to four months of age and is characte rised by hindlimb paralysis , severe incoordination , and sometimes blindness ( Bennetts , 1932; Innes & Shearer , 1940; Bennetts & Beck , 1942; Hurley , 1981; Smith et al . , 198 1 ) . The neuronal changes o f enzootic ataxia are present in the brain stem and the grey matter of the spinal cord . The predilection si tes are the red and vestibular nuclei , the reticular formation of the brain , and the ventral horns of the spinal cord ( Barlow 19 60b; Howell et al . , 1964; Fell et al . , 19 65; Howell , 1970; Howell e t al . , 1 981; Smith et al . , 1981 ) . Cerebral cavitation occurs in 20-60% of cases with accompanying cerebral expansion due to ( Roberts et al . , the 1 966; 1 8 pressure o f entrapped cerebrospinal fluid Howell , 1 970; Ho-...1ell et al . , 1 98 1 ) . The main central nervous system les ions are the result of hypomyelination associated with a reduction of cy tochrome oxidase activity ( Barlow et al . , 1 9 60b; Barlow, 1 963b; Howell et al . , 1 964; Fell et al . , 1 965; Howell, 1 970; Prohaska, 1 98 1 ) . There are two peak periods for the laying down of myelin . The first is during the last fifty days of pregnancy when the major growth phase of the cer·ebrum occurs and the second pePiod is dur ing the first five weeks of neonatal life when the spinal cord doubles in size without an increase in cell numbers ( Barlow, 1 9 63a; Smith et al . , 1 977; Howell et al . , 1 98 1 ; Prohaska, 1 98 1 ) . If one twin is affected with enzootic ataxia (also referred _ to as swayback ) , then the other twin wil l develop the condition although not necessarily at the same time ( Lewis et al ._, 1 98 1 ) . The criteria used for the diagnosis of enzoo tic ataxia are ( i ) ataxia in the young o r newborn lamb, ( ii ) cavitation or gelatinous lesions of the cerebral white matter often accompanied by histological evidence of chromatolysis and myelin degeneration in the brain stem and spinal cord, and ( iii ) low copper status ( Barlow et al . , 1 9 60a ) . The copper content o f the spinal cord i s cons idered to be the best ind icator of the copper status of these animals ( Howell, 1 970 ) . Anaemia has been reported in association with copper deficient sheep ( Bennetts & Chapman, 1 9 37; Benne tts & Beck, 1 942; Suttle & Field, 1 967; Hogan, 1 973; White law et al . , 1 979 ); as copper is required in ferroxidase to mobi lize iron for haemoglobin synthesis (Gallagher et al . , 1 956 ; Blunt, 1 975; Holmes & Dargie, 1 975 ) . The first sign of depletion of copper reserves is often loss of crimp in the fleece ( Lee, 1 9 5 1 a; Fearn & Habel, 1 9 6 1 ; Suttle & Field, 1 967 ) . With further depletion wool production is seve rely diminished and there is a deterioration in the wool staple with the appearance of straight lustrous fibres typical of "steely -.. mol" (Marston & Lee, 1 94 8a; Lee, 1 9 5 1a, 1 9 5 1 b; Whitelaw et al . , 1 97 9 ) . 1 9 The copper deficiency causes an impediment in the keratinization process by reducing cross linkage of disulphide bonds between polypeptide chains (Danks et al . , 1 972 ) . Achromo trichia also occurs because there is insufficient copper dependent tyrosinase to produce melanin . This may result in either an overall pallor in black o r coloured fleeces , o r a banding effect o n the fibre ( Lee , 1 95 1 b ; Suttle & Field , 1 970a ; Hogan , 1 973 ) • Copper deficiency may influence the susceptibility of ruminants to infection as it has been shown to impair the phagocytic activity of leucocytes ( Boyne & Arthur , 1 98 1 ; Jones & Suttle , 1 98 1 ) and the immune response of other mammals ( Newberne et al . , 1 9 68 ; Gross & Newberne , 1 980; Nauss & Newberne , 1 98 1 ; Prohaska & Lukase�jcz , 1 98 1 ; Jones & Suttle , 1 983 ) . Bennetts and Beck ( 1 942 ) observed diarrhoea and loss of condition during late gestation and lactation in ewes of low copper status . This may have been due to partial villus atrophy in the duodenum and jejuneum as has been recorded in copper deficient cattle ( Fell et al . , 1 975 ) . Low plasma copper levels have been reported to reduce libido in rams (Wiener et al . , 1 976 ) . In ewes on very low copper semi-synthetic diets , barrenness , foetal resorption and abortion have been reported (Howell , 1 968 ; Howell & Hall , 1 970 ; Suttle & Field , 1 970a) , with the margin in copper status between that causing infertility and that causing production of swayback lambs being a very small one ( Suttle & Field , 1 970a ) . Therapy of copper deficiency Since the recognition of copper defi ciency , many copper compounds have been tested at a variety of dose rates and administered by different routes . The results have varied . In the treatment of whole flocks of animals , the margin between adequate pro tection and toxicity appears to be narrow . 20 Ha�vey and Sutherland ( 1 953) describe the qualities desirable for an inj ectable copper preparation as ( i ) producing minimal damage at the site of injection , ( ii ) causing satisfactory storage in the liver of about 90% of the administered copper ( iii ) having a safety margin between the therapeutic and toxic dose rate , and ( i v ) being easily prepared and economical . Therapeutic and safety differences amongst the available compounds depend on the rate of translocation from the injection si te to the liver (Mahmoud & Ford , 1 98 1 ; Suttle , 1 98 1 b ) . Injected copper exhibits a much longer period of therapeutic effect than orally adminis tered copper ( Comar , 1 950) . Only 1 . 6- 1 . 8% of orally adminis tered copper sulphate is stored in the live r (MacPherson & Hemingway , 1 968 ; ARC , 1 980 ) , and 8 . 3% for copper oxide needles ; this is reduced to 3 . 8% if sheep are being fed on a high molybdenum diet ( Suttle , 1 98 1 a ) . On the other hand , the subcutaneous injection o f copper edetate as an example , resulted in an 80-90% storage of copper in the liver ( Camargo et al . , 1 9 62 ) . The protection from hypocupraemia afforded by a parenteral dose of copper reflects the completeness of transfer from the injection site to the liver . The speed of translocation may determine the extent of any local tissue reaction . However it may be difficult to combine the properties of rapid translocation with an adequately slow arrival at the storage site ( Suttle , 1 98 1 b ) . Deaths may occur in compounds , due to rapid certain flocks ( Ishmael association with the use translocation , but deaths et al . , 1 969 ; Hendy & of parenteral only occur in Evans , 1 977 ; Gardiner , 1 978 ) . To reduce the risk of losses , it is recommended that "stress" at the time of inj ection should be reduced and that other animal remedies that are detoxified by the liver are not used at the same time as the copper inj ection ( Hendy & Evans , 1 977 ) . Lambs are more susceptible than adult sheep to copper toxicity yet a non-toxic dose of parenteral copper may be insufficient to prevent delayed swayback ( Lewis et al . , 1 98 1 ) . Copper oxide needles given to lambs •..Till elevate plasma copper concentrations (Hhitelaw et . 2 1 al . , 1 980 ) , but will not alleviate the clinical condition affecting lambs (Whitelaw et al . , 1 982 ) . To prevent swayback in lambs a relatively high dose of some co pper compound must be given to the ewe during mid-pregnancy to increase the transplacental transfer of copper to the lamb . This early and high dose of copper is essential as the placental t ransfer of copper from dam to foetus is relatively ineffic ient ( Hendy & Evans , 1977 ) . Many different copper compounds have been tested and most rejected , often because of their potential to cause lo9al tissue reaction ( Harvey & Sutherland , 1 953; Cunningham , 1 959; Uvarov , 1 970 ) . Or·al copper compounds vary in their absorption rates and in the faecal and urinary excretion rates ( Lassitter & Bell , 1 960 ) . In general the absorption rate of oral copper is very low and repeated dosing requires cons iderable labour , vlhich is expensive; which make its use unattractive ( Fearn & Habel , 1 9 6 1 ) . factors Copper glycinate ( aminoac etate ) at a dose rate of 45mg for ewes , effectively raises plasma and l iver copper levels ( Fearn & Habel , 1 96 1 ; Hemingway et al . , 1 970 ) ; 80% o f the available copper being stored in the liver ( Camargo et al . , 1 9 62 ) . The disadvantages of this material are that it causes va rious degrees of local necros is ( Harvey & Sutherland , 1 953; Al lcroft et al . , 1 9 59) and doses of 1 00mg have proven very toxic ( Harvey , 1 953 ) : eight out o f eleven sheep were killed by that dose in one experiment . Copper methionate given a s a 50rog dose to adult ewes was shown to rais e plasma and liver copper leve ls ( Hemingway et al . , 1 970; Whitelaw , 1 980; Mahmoud & Ford , 1 98 1 ; Suttle , 1 98 1 b; Mahmoud & Ford , 1 982 ) . The product has a slow uptake ( Suttle , 1 98 1 b ) , and causes very severe local reactions ( Camargo et al . , 1 962; Suttl e , 1 98 1 b ) . On the other hand animals can tole rate a higher dose rate of copper methionate as , only 1 0-50% is retained in the live r (Camargo , 1 9 62 ) . 22 Copper oxyquinoline sulphonate , as a solution containing 6 mg Cu/ml , is rapidly translocated from the i njection s ite ( Suttle , 1 98 1b ; Mahmoud & Ford , 1 9 82 ) , and nearly 1 00% is retained in the liver ( Cunningham , 1 959 ) . It causes minimal tissue reaction ( Harvey & Sutherland , 1 9 53 ) but causes toxicity at doses of · 1 -2mg per kilogram . ' bodyweight (Mahmoud & Ford , 1 98 1 ; Suttle , 1 98 1b) , and accordingly only relatively small total amounts of copper can be given in this form : the recommended dose is only 6mg for ewes ( Brue re , 1 980) . Copper oxyquinoline sulphonate appears to afford less protection to sheep feeding on a diet high in molybdenum ( Suttle , 1 98 1b) . Copper edetate ( versenate ) has been the most favoured parente ral . copper compound as it effec tively raises plasma and live r copper levels ( Harvey_ & Sutherland , 1 953 ; Camargo et al . , 1 9 62 ; Hemingway et al . , 1 970; Suttle , 1 98 1b; Mahmoud & Ford , 1 9 82 ) . This product has an intermediate trans location rate when compared to copper glycinate and copper oxyquinoline sulphonate ( Suttle , 1 982b) and causes minimal tissue damage at the site of injection ( Harvey & Sutherland , 1 953 ) . It has caused deaths experimentally at dose rates of 3mg per kg (Mahmoud & Ford , 1 982 ) and even at loHer dose rates on some farms when used therapeutically ( Harvey & Sutherland , 1 95 3 ; Ishmael e t al . , 1 969 ) . Howeve r these losses have not exceeded 1 in 2 , 000 , and only 3% of all flocks treated were affected ( Ishmael et al . , 1 9 69 ; Hendy & Evans , 1 977 ) . More recently copper oxide needles , which -v;hen given orally are retained in the abomasum , have been shown to increase plasma and liver copper concentrations both in lambs (Whitelaw , 1 980 ) and in adult sheep ( El lis , 1 981 ; Whitelaw , 1 980 ; Suttle , 1 98 1 a ; Judson et al . , 1 9 82 ) . They have a liver copper reten tion of 8 . 3% ( Suttle , 1 98 1b) . Due to the slow re lease of copper over 7-8 v1eeks (Dewey , 1 977 ; El lis , 1 9 81) there is minimal risk of toxicity using doses up to 1 0gm and even when pretreatment liver copper concentrations exceed 1 , 000 ppm D .M . ( Ellis , 1 9 81 ) . 23 Copper oxide suspended in olive oil and inj ected intramuscularly has been shown to raise plasma and liver copper concentrations ( Lamand , 1 978b) as has insoluble coppe r dust ( Lamand , 1 978a ) . Although both agents produce a moderate degree of inflammation , the fact that they are administered intramuscularly makes their use undesirable . In 1 980 , Mallinson et al . , and in 1 9 82 , Moore et al . , experimented with controlled release glasses as a medium for copper therapeutic agents . These glasses are implanted subcutaneously and dissolve in from 1 to 1 5 days , depending on their compos ition . Up to 80% of the incorporated copper was retained in the liver and in . addition plasma copper concentrations were raised . The glasses that dissolve over a longer period ( 5 days and grea te r ) , tend to produce a reaction at the s ite of implan tation for up to 28 days after implantation . This medium for supplementation has promise as doses of 1 0 0 mg of copper can be given to sheep without any apparent indication of toxici ty . Copper toxicity in sheep Sources of Copper There are numerous reports of copper toxicity in sheep . These have been caused in many different ways because of the wide variety of sources from which copper may be derived . Environmental contamination of soil and plant material occurs naturally in cupri ferous soil outcrops . Contamination has also been caused by mine tai lings and from corrosion of metallic copper . This contamination is the result of a high concentration of copper in the superficial soil layers as copper is not a mobile element ( Ti ller & Merry , 1 98 1 ) . Sheep have also been poisoned from ingesting pasture contaminated by copper which has been depos ited from industrial pollution such as mine tailings ( Lander , 1 9 1 2 ; Bisset , 1 934 ; Wiemann , 1 939 ; Bischoff & Haun , 1 939 ; Tiller & Merry , 1 98 1 ) . 24 Some plants , such as " skeleton weed" ( Chondrilla juncea ) ( Bull , 1 9 56 ) , become potentially toxic because of their ability to accumulate high levels of copper ( Skinner , 1 9 60 ; Woolhouse & Walker , 1 98 1 ) . Legumes , such as clovers and lucerne , frequently have high concentrations of copper ( up to 20 ppm D . M . ) , when growing in soils of high copper con tent ( Bull , 1 9 56; Robson & Reuter , 1 98 1 ) . Other plants , such as Senecio spp ( ragwort) ( Bull , 1 956 ; Mortimer & White , 1 975 ) , Heliotropeam europeum (heliotrope ) ( Bull , 1 9 56 ; Bull �t al . , 1 956 ) , lup in ( Bennetts , 1 9 57 ; Allen et al . , 1 970 ) and Echium spp ( Paterson ' s curse ) ( St George et al . , 1 9 62 ; Connors , 1 979 ) , may indirectly cause coppe r poisoning due to the he pato toxic action o f their alkaloids . Such alkaloids can produce hepatic necros is leading to the release of liver copper and the precipi ta tion of a haemolytic cris is . The application of copper to the soil has been responsible for copper toxicity . Possible sources are orchard sprays ( Bordeaux mixture ) ( B aum & Seelinger , 1 898 ; Schaper & Luetj e , 1 93 1 ; Beijers , 1 932 ; Lafenetre et al . , 1 9 35 ; Fincham , 1 945 ; Muth , 1 952 ; Ogilvie , 1 95 4 ) , copper topdressing ( Pryer , 1 9 59 ; Tiller & Merry , 1 98 1 ) , pasture spraying of pig slurry ( Loosmore , 1 9 69 ; Batey et al . , 1 9 '{2 ; Feenstra & van Ulsen , 1 973 ; Kneale & Howell , 1 97 4 ; Dalgarno & Mills , 1 97 5 ; Suttle & Price , 1 976 ; Tiller & Me rry , 1 98 1 ) , and of sewage sludge ( Tiller & Me rry , 1 98 1 ) , and copper sulphate which has been used as a molluscicide in liver fluke control (Gracey & Todd , 1 9 60 ) . Housed animals have been poisoned frequently by copper . This has occurred when feed concentrates containing a high copper con tent , but o ften a low molybdenum content , have been fed continuously (Clegg , 1 9 56 ; Pear son , 1 9 56 ; Bracewell , 1 9 58 ; Senior , 1 9 59 ; Ross , 1 964 ; Hil l & Williams , 1 9 65 ; Watt , 1 966 ; Hogan et al . , 1 9 68 ; Adamson et al . , 1 969 ; Todd , 1 9 69 ; Buck , 1 97 0 ; Tait �t al . , 1 97 1 ; Hartmanns , 1 975 ; Arora et al . , 1 977 ; Bundza et al . , 1 9 82 ) . 25 Ironically the use of copper therapeutically has also produced a number o f animal deaths . Reports state that all these losses have been caused by the careless use of a wide range of coppe r containing products . These include copper sulphate-nicotine sulphate mixture as an anthelmintic ( Bo�ghton & Hardy , 1 934 ) , copper sulphate as a copper supplement ( Boughton & Hardy , 1 934 ; Eden , 1 940; Naerland , 1 94 8 ; Sharman , 1 969 ) , and copper chelates a s copper edetate ( Allcroft et al . , 1 965 ; Ishmael et al . , 1 9 69 , Wiener & Field , 1 97 0 ; Wiener & MacLeod , 1 970 ) and copper oxyquinoline sulphonate ( Cooper , pers . comm . , Cunningham , 1 959) . Some cases of copper toxicity have occurred when sheep have been moved on to pasture containing "normal " amounts of copper : for example , as in the seaweed eating �orth Ronaldsay (Wiener et al . , 1 977 ; MacLachlan & Johnston , 1 982 ) , and in Texel sheep eating normal concentrate feed ( Arora et al . , 1 977 ) . The cases of copper toxicity in sheep confirmed in New Zealand by the Animal Health Laboratories s ince 1 973 , are set out in Table 1 . 3 . Clinical signs of copper toxicity The signs of copper poisoning are dramatic and in most cases death follows rapidly . Animals a ffected with copper toxicity show depression and loss of appetite , which may continue for seve ral days , but more often it lasts about 24 hours before the haemolytic cris is occurs . Once intravascular haemolysis begins , severe clinical signs develop and these include dyspnoea , increased heart rate , and haematuria ; the latter being no ticed in woolly sheep by the charac teristic staining of the crutch . The mucous membranes become muddy-brown and the sheep may pass watery brown faeces . In most cases these symptoms result in death in 1 -2 days . Occasionally sheep may recover from the haemolytic cris is , or they may die as a result of kidney damage or even from subsequent haemolytic crises . 26 Year No . dead Age Source or predisposing cause 1 973 seve ral 9 mths parenteral 1 974 2 cases ewes orchard spr·ay 1 0/ 1 00 ewes ragwort 2 lambs concentrate feed 1 976 3 cases e\ves copperised salt lick copperised superphosphate 1 977 1 5/ 3000 ewes ragHort & copperised superphosphate 1 980 case 9 mths penfed on concentrate case lambs copperised salt lick & pig mash 1 case 9 mths lucerne meal 1 98 1 3/week ove r 9 mths oral supplementation 10 weeks 1 982 6/ 1 200 lambs oral copper & ewes sup�lemented Table 1 . 3 : Reported cases of copper toxicity in sheep in N . Z . taken from "Surveillance" reports of Animal Health Laboratories . Post-mortem changes in copper toxicity Animals which have died of copper toxicity show pathological changes vThich are typical of a haemolytic cris is . There is a marked jaundice throughout all tissues , and the mucous membranes o ften have a muddy-brown appearance . The liver is swollen , with rounded edges and is pale yellow in appearance . The kidneys often have a black metallic sheen , the so called "gun-metal" kidney . Ecchymotic and petechial haemorrhages are often seen between the muscle planes and subcutaneously . These changes are more evident in a�imals which have been handled or accidentally injured prior to death . Such post � mortero changes appear cons istently and have been reported by the many 27 workers in this field . Although copper poisoning is often classified into acute and chronic forms , depending on the duration and cause of accumulation of copper by the bod y , there is only one main event which initiates poisoning . It is the accumulation o f excessive amounts of copper in the lysosomes which causes release of the lysosomal contents and these initiate the extensive hepatic necros is . I shmael et al . ( 1 97 1 a ) have reported sudden within 24 hours of copper administration . In deaths in sheep these cases the condition is more typical of a heavy me tal poisoning and it is charac terised by excess fluid in the serous cavities , subendocardial haemorrhages , a congested abomasal mucosa , fluid intestinal contents and sometimes rec tal haemorrhage . There is no generalised jaundice of the carcase . Histology of the liver reveals some centrilobular necros is , but on chemical analysis liver copper levels are only 300-500 ppm D .M . Changes in blood in coppe r toxicity In copper poisoning the changes in blood parameters only occur near to the haemolytic cris is . Blood copper concentrations rise ve ry quickly . Within 48 hours they may have risen to ten times the normal concentration (Marston , 1 952 ; Barden & Robertson , 1 9 62 ; Todd & Thompson , 1 9 63 ; Ishmael et al . , 1 972 ) . However in some cases elevated blood copper concentrations have been recorded up to 28 days before the crisis ( Sutter et al . , 1 9 58 ; McCosker , 1 9 68 ; MacPherson & Hemingway , 1 9 69 ) . In other cases a roil� "cris is " has probably occurred without vis ible haemolys is or haematuria although McCoske r ( 1 968 ) has reported a two-fold increase in blood copper concentration seven days prior to the cris is period . 28 Circulating copper ions appear to be the cause of haemolysis as the resolution of haemolysis parallels the fall in the blood copper concentrations ( Ishmael et al . , 1 972 ) . The increase in copper content of whole blood is much greater than the increase in copper levels in plasma . This is due to the pronounced increase in erythr·ocyte copper concentration ( Ishmael et al . , 1 97 1 a ) . The increase in plasma copper is due to an increase in copper ion concentration . Caeruloplasmin activity alters very l ittle (Maribei , 1 978 ) , although Ishmael et al . ( 1 972 ) have reported a two-fold increase in caeruloplasmin just prior to the haemolytic cri�is . An important prodromal feature o f copper poisoning is the marked e levation of the packed cell volume ( PCV ) , sometimes to as much as 55% , and occurring up to seven days in advance of the haemolytic cris is (McCosker , 1 9 68 ; MacPherson & Hemingway , 1 9 69 ; Ishmael et al . , 1 9 72 ) . The dramatic increase may be caused e ither by e rythrocytic enlargement or by dehydration . Once the haemolytic cris is becomes evident the PCV drops in direct relation to the severity of haemolysis . Should the animal survive , the PCV usually returns to normal concent rations over a ten day period . During the period of intravascular haemolysis , methaemoglobin is present in the blood as evidenced by its characteristic chocolate-brown colour . Todd & Thompson ( 1 9 6 1 ) found that this methaemoglobin is intracorpuscular and cons titutes up to 35% of the to tal haemoglobin . As erythrocytes become lysed , the haemoglobin and methaemoglobin are released into the plasma . Soli & Froslie ( 1 977 ) also demonstrated that the methaemoglobin formation during the terminal cris is is an intracorpuscular process and that it occurs before any osmotic fragility of red cells can be detected . The first change detected before the onset of the haemolytic cris is is a fall in erythrocyte glutathione concentration . Two to four hours prior to the appearance of haemoglobin in the plasma there is a build up of Heinz bodies in the erythrocytes ( from 1 % in normal blood to 50% in pre-haemolytic blood ) ( Soli & Froslie , 1 977 ) whereas 29 during t�e cris is proper , Heinz bodies appear in up to 90% of the erythrocytes and free Heinz bodies are seen in the plasma . Although the red blood cells maintain their osmo tic status before the haemolytic cris is , they soon begin to lose shape and become distorted ( Soli & Froslie , 1 977 ) . Some erythrocytes are cont racted or crenated and some appear as ghost cells devoid of haemoglobin but still contain Heinz bodies . Reticulocytes and normoblasts do not appear in blood until · 20-24 hours after the commencement the peripheral of the cris is . Leucocytes appear to be unaffected by copper toxicity although Soli & Froslie ( 1 976 ) have reported an increase in the number of monocytes within 24 hours of the onset of the crisis . There is an increase in neutrophils as cellular debris is released into the circulation during haemolysis . Changes in liver in copper toxici ty Because the liver is the main storage organ for copper , its tissues are the first to be affected in copper poisoning . The changes that occur in the live r during the process of coppe r accumulation have been observed by several means . Histological changes have been monitored , and the '' leakage " of hepatic enzymes into the circulation has been measured . It has been found that the activity of these enzymes a re correlated with the concentration of liver copper , and blood copper , as well as to the to tal copper intake . Centrilobular necrosis is the first observed histological change and may be seen within 48 hours following the administration of copper ( Ishmael & Gopinath , 1 972 ) . As the live r copper concentrations increase , the zones of conspicuously altered cells widen and become extended towards the central ve ins ( King & Bremner , 1 979 ) . Just prior to haemolysis most liver parenchyma! cells appear to be severely affected ( Ishmael et al . , 1 97 1 b ) . Neutrophils start to invade the necro tic a reas and the re is centrilobular 30 congestion and haemorrhage . Rubeanic acid-posi tive granules are seen both in the cytoplasm of hepatic parenchyma! cells and in swollen Kuppfer cells as the copper content increases ( Gooneratne et al . , 1 980 ) . As the number of necrotic cells increases , the reticular framework in these areas of the liver starts to collapse ( Ishmael et a l . , 1 97 1 b ) . At the time of haemolytic crisis there are further changes ; such as the appearance of large foci of necrotic live r cell s , often associated with foci of polymorphonuclear leucocytes . Eventually bile pigments start to accumulate in the canaliculi and numerous fat droplets are present in the viable cells ( Ishmael et al . , 1 97 1 b ) . The Kuppfer cells appear large and numerous , and they contain eosinophilic and brown granular or globular cytoplasm, especially in the areas adjacent to the central vein (Gooneratne et al . , 1 980 ) . In sheep that survive the haemolytic cris is , cords of new parenchymal cells start to appear . Necrosis , polymorphonuclear infi ltration and ballooning are no longer evident . These regenerative changes may be visible within nine days after copper administration ( Ishmael & Gopinath , 1 972 ) and sheep surviving up to three haemolytic crises may , after 40 days , show an increase in portal connective tissue . This may even extend into the periportal zone of the lobules (Gopinath & Howell , 1 97 5 ) . King and Bremner ( 1 979 ) saw evidence of apoptosis ( Kerr et�, 1 972 ) in the form of ovoid masses of condensed cytoplasm , nuclear remnants and other cellular debris . Apoptosis is a mechanism of controlled cell deletion involving the phagocytosis and degradation of these ovoid masses by other cells ( Kerr et al . , 1 972 ) . Changes in live r enzymes in copper toxicity Early tissue damage may often be detected by measuring the activity of cellular enzymes that have escaped from the damaged cell into the serum . Necrosis of hepatic cells or an alteration to the permeability of the cell wall will cause leakage of cellular 3 1 contents , i n particular cellular enzymes (Kramer , 1 9 80 ) . Several enzymes are found predominantly in hepatic cells . Although they may be present in other tissues their levels in those tissues are insignifican t . These enzymes can b e monitored i n serum . It has been found that the quantity of enzyme present in serum bears a relationship to the amount of liver damage . The increase in serum enzyme activity is dependent upon three factors . These are the enzyme measured , the hal f life of that enzyme in serum , and the molecular s ize of the enzyme . The amount of alteration in cell wall permeability determines the amount of enzyme entering the blood stream . The smaller the molecular s ize of the enzyme the more readily it tdl l permeate the cell wall . Table 1 . 4 gives a summary of speci fic liver enzyme analyses that have been used by workers in this field . Changes in kidney in copper toxicity The his topathological changes in the kidneys of sheep poisoned by copper are seen mainly as degenerative changes in the proximal convoluted tubules and indicated by epithelial necrosis , desquamation and vacuolation ( Ishmael et al . , 1 97 1 b ) . The tubular lumina contain a granular eosinophilic material ( Soli & Nafstad , 1 976 ) which is positive to rubeanic acid and Perl ' s iron stain (Gopinath & Howell , 1 975 ) . During the pre-haemolytic phase of copper toxicity when copper is accumulating in the tissues , the only d iscernible histological change is the presence of eos inophilic intracytoplasmic granules in the epithelium of the proximal convoluted tubules . The number and size of these granules is dependent upon the duration o f exposure to copper excess (Gopinath et al . , 1 974 ) . 32 SGOT SDH GD LDH arg Todd & Thompson ( 1 963 ) * * Ross ( 1 9 64 ) * van Adrichem ( 1965 ) * * Ross ( 1 966 ) * * MacPherson & Hemingway ( 1 9 69 ) * Ishmael ,Gopinath & Treeby ( 1 97 1 ) * * * * Ishmael ,Gopinath & Ho-v1ell ( 1 97 1 ) * * * * Ishmael & Gopinath ( 1 972 ) * * * Ishmael et al . ( 1 972 ) * * * Todd & Thompson ( 1 973 ) * * Thompson & Todd ( 1 974 ) lf Gopinath & Howell ( 1 975 ) * * * Bath ( 1 979 ) * Buckley & Tait ( 1 98 1 ) * SGOT = Serum glutamate oxaloacetate transaminase SDH = Sorbitol dehydrogenase GD = Glutamate dehydrogenase LDH = Lactate dehydrogenase arg .. Arginase Table 1 . 4 : List of workers and the serum enzyme analyses they have used in the investigation of copper toxicity . Once the haemolytic crisis occurs , the cells o f the proximal convoluted tubules show increased eosinophilic and intracytoplasmic granules , many of which stain pos itively for haemoglobin , copper and iron . The cortical tubules become dilated and contain many eosinophilic and granular casts (Gopinath et a l . , 1 974 ) . 33 In the post-haemolytic phase the same htstological changes are s till eviden t . During the accumulation of copper in the kidney it is thought that the increase in the s ize and number of intracytoplasmic granules is a lysosomal response to the increased · copper s torage . The sequestrat ion of copper into the lysosomes protects the remainder of the cell from the toxic effects of the metal (Goldfischer et al . , 1 970 ) . It is believed that the accumulation of numerous casts of haemoglobin in the kidney tubules cause an impairment o f function that produces the renal failure in copper toxicity . Changes in brain in copper toxicity The histopathological changes in the tissues of the central nervous system of sheep which have d ied from copper poisoning , have been examined and reported upon by Doherty et al . , ( 1 9 69 ) , Ishmael et a l . , ( 1 97 1 c ) , Hooper ( 1 9 72 ) , Morgan ( 1 9 73 ) , Howell et al . , ( 1 974 ) , Gopinath & Howell ( 1 975 ) , and Gooneratne & Howell ( 1 979 ) . Doherty et al . , ( 1 9 69 ) , described the lesions seen in the brains of six sheep poisoned by copper sulphate , as a marked spongy transformation predomi nantly in the white matter, especially the midbrain , pons and cerebellum . Ishmael et al . , ( 1 97 1 c ) , confirmed these findings . Hooper , ( 1 972 ) , described this spongy degeneration as the occurrence of large empty vacuoles in or along myelin sheaths in the white matter ; and i n s ingle axons trave rsing adjacent grey matter of the brain and spinal cord . The brain stem was commonly and severely affected . He called this condition status spongiosus and compared it to the changes seen in ruminants poisoned by . hepatotoxic pyrroli zidine alkaloids of plants and other hepatic diseases such as severe liver fluke infestation , ischaemic l iver necros is , facial eczema , and heart failure associated with liver necrosis . 34 Using electron microscopy Morgan , ( 1 973 ) , found that the clear spaces around the large myelinated axons vrere intramyelinic vacuoles produced by the separation of lamellae at the intraperiod line . He found that the oligodendroglia , neurones and their processes and blood vessels were �pparently normal as were the axons within the severely distended myelin sheaths . Howell et al . , ( 1 974 ) , produced vacuolation in the myelin sheaths of 12 out of 20 sheep that e ither died of copper toxicity , or were killed during the haemolytic cris is . Ultrastructur�l studies revealed that the vacuoles associated with the nerve fibres were in the outer tongue of oligodendrocyte cytoplasm and the swollen astrocytes contained more reticulum than . normal . glycogen , mitochondria They concluded that the and endoplasmic changes in the central nervous system due to copper poisoning were the result of the · effects of altered metabolic processes on glial transport mechanisms . The brains of s ix out of seven copper-poisoned sheep examined by Gopinath and Howell , ( 1 975 ) , showed lesions varying from swelling o f the myelin sheath i n the medulla , cerebellum , and dorso-lateral area of the thalamus , to pronounced vacuolation of white matter in all sections of the brain . The levels of copper , iron and zinc in brains of normal sheep were compared with those from sheep which had been poisoned with copper (Gooneratne and Howell , 1 979 ) . Surprisingly , there was no measureable increase in copper or the other two elements in sheep vlhich had d ied from copper poisoning . As c laimed by Morgan , ( 1 973 ) , and by Howell et al . , ( 1 97 4 ) , it is likely that the degenerative changes which occur in the brain of sheep poisoned with copper are terminal and due to other toxins released as a result o f liver damage . Hooper et al . , ( 1 974) , poisoned fourteen sheep with the hepato toxic alkaloid lasiocarpine . All sheep developed severe hepatic necrosis : seven of these sheep deve loped spongy degeneration 35 of the central nervous system . They found a positive correlation between the appearance of spongy degeneration and the development o f elevated blood levels of ammonia and of glutamine i n the cerebro-spinal fluid . This further showed that status spongiosus is a terminal effect associated with severe liver necrosis . Changes in muscle in copper toxicity Muscle changes have been studied by Thompson and Tod d , ( 1 97 4 ) , and by Gooneratne and Howell , ( 1 980 ) . There appear �o be no detectable h istological changes in the muscle tissue but during the period of haemolytic crisis there is a rapid rise in creatine phosphokinase blood levels which in the sheep that survive the haemolytic crisis return to normal in about three days . The increase in serum enzyme activity appears to be the result of cellular enzyme escaping through the cell membrane . This increase in cell membrane permeability also occurs in hepatocytes , erythrocytes , and cells in the kidney and brain . The suggested causes are those of hypoxia , hypercupraemia and possibly a decrease of selenium and or vitamin E content in the blood and muscle tissues . CHAPTER 2 . ESTABLISHING BIOCHEMICAL PARAMETERS TO MONITOR THE EFFECTS OF EXCESSIVE COPPER U SED THERAPEUTICALLY INTRODUCTION 36 Copper preparations that are intended for parenteral administration in sheep are often used to prevent or to treat copper deficiency . Certain of these preparations under special circumstances have caused deaths . In order to evaluate the effects of copper therapy on the animal , a number of different biochemical parameters were measured . The alteration in the biochemical parameters should indicate the earliest s igns o f impending toxicity . In this experiment repeated doses of a copper therapeutic agent were administered to sheep , while the appropriate biochemical parameters were under constant surveillanc e . Supplementation with copper was con tinued until each sheep underwent a haemolytic crisis . The copper compound used in this expel�iment was copper calcium edetate as it is a chelate with a medium translocation rate when compared to other copper salts ( Suttle , 1 98 1 b ) . The parameters used in this study included liver copper concentrations , s erum levels of live r enzyme activity , histopathology of liver tissue , and haematological changes . The liver is the main storage organ for copper , and regular determination of the liver copper concentration will indicate the changes in copper status of the animal . Because hepatotoxic damage occurs in copper toxicity and because the hepatocellular enzymes pass out of the damaged hepatocytes and into the plasma , changes in the levels of the two liver-specific enzymes , serum glutamate oxaloacetate transaminase ( SGOT ) ( EC 2 . 6 . 1 . 1 ) and sorbitol dehydrogenase ( SDH ) ( EC 1 . 1 . 1 . 1 4 ) should indicate early changes in liver cells . SGOT is the conventional enzyme used to assess liver damage , while SDH is live r speci fic and has a short half-life o f 36 - 4 8 hours . ( Traces of SDH may be found in the testis and retina but are inconsequential ) . 37 Histopathological examination of liver may help to demonstrate when liver damage has taken place . Al teration in the conventional blood pararneters such as packed cell volume , haemoglobin , plasma protein , and the icteric index , are indicative of any haemolytic changes , while changes in the blood cells may provide some indication of the body ' s response to any toxic e ffects caused by the e xcess copper . MATERIALS AND METHODS Two e ighteen month-old Perendale rams were used initially to · determine whether the taking of a l iver biopsy under general anaesthesia , using sodium pentobarbitone1 , might cause changes in the levels of SGOT and SDH activity in the serum . Enzyme activity levels in the sera were estimated prior to the liver biopsy and then twice daily for seven days . The results showed a slight rise above normal in enzyme activity at between 24 and 36 hours , but these levels had returned to normal within 48 hours . I t was concluded that the effects of liver biopsy would be insignificant at 72 hours . On completion of this initial investigation these same two rams , maintained on a hay d iet containing 1 . 67 ppm D .M . of coppe r , were injected once a week with 50mg of copper as copper calcium edetate contained in a 2ml dose 2 • The injections were given subcutaneously in the dorsal region of the neck . The s ites chosen in the neck were the anterior and posterior dorsal areas , and they alternated between left and right sides . Blood and serum samples were collected from the jugular vein immediately before the injection and at 1 2 , 24 , 36 , 48 , and 96 hours after the injection . Every second week , on the fifth day after the copper injection , each sheep was anaethetised with sodium pentobarbitone . Liver biopsy samples \V"e re taken by aspiration from the diaphragmatic surface of 1 Anathal , V . R . Laboratories (Aus t . ) P ty . Ltd . 2 Coprin Mult ido s e , Glaxo (N . Z . ) Ltd . 38 the d orsal lobe of the live r . The live r b iopsy technique used was based on the aspiration technique developed by Dick ( 1 944 ) , but modi fied so that the liver was entered from the diaphragmatic surface . The point of entry for the biopsy probe was at the right paralumbar fossa approximately three centimetres posterior to the angle of the last rib ( Figure 2 . 1 ) . Each liver sample was d ivided into two aliquo ts . One sample was immediately frozen at -4° C for later chemical analysis and the other sample was placed in 1 0% buffered formol saline for subsequent histopathological examination . Figure 2 . 1 : Path of the l iver b iopsy probe shmm us ing a dis sec ted specimen As the blood samples from each sheep were collected , the injection sites of the copper calcium edetate were palpated to assess any tissue reaction and this was recorded . At the time o f each liver biopsy both sheep were we ighed . 39 The copper calcium edetate injections continued to be given weekly un ti l s igns of chronic copper poisoning appeared , or death occurred . The signs of copper poisoning were l istlessness , hyperpnoea , haematuria , and yellow grey mucous membranes . The " preliminary outlined would allow investigation" showed that the procedures the appropriate measurement of any changes to the animal ' s body in response to repeated copper inj ections . A ful� experiment was set up , utilizing a further nine e ighteen-month old Romney rams . These rams were grazed on pasture containing on average 3 . 66 ppm D .M . of copper . Pasture samples were analysed for copper content monthly . Afte r an initial live r biopsy , each ram Has injected with 1 00mg of copper as copper calcium edetate contained in 4 ml , and then each subsequent week wi th a fur ther 50mg of copper contained in 2ml of the same product . Blood and serum samples were collected for the measurement of the appropriate enzyme activities and blood parameters immediate ly before and at 48 and 96 hours after injection . Also at each bleeding , the injection site was palpated and any reaction recorded . Every second week live r biopsy samples were obtained for chemical analys is and for histopathological examination . Wool samples Here also retained after shaving the biopsy site , and on each occas ion the rams were weighed . The Caprin injections were copper poisoning developed , or given weekly until the signs of death occurred . All rams that d ied were autopsied . Samples of liver , kidney , and brain we re collected into 1 0% buffered formal saline for histopathological examination and aliquots of the same tissues were deep frozen to await chemical analysis . The three rams that developed a haemolytic crisis but survived , were examined and sampled three times weekly until live r enzyme concentra tions in the serum had returned to the pre-injection concentrations . 40 Blood analys is All blood samples Here analysed for haemoglobin content by the cyanomethaemoglobin method ( Schalm et a l . , 1 97 5 ) and the packed cell volu..'lle of each was e stimated by the haematocrit method ( Schalm et al . , 1 97 5 ) . In each case the mean corpuscular haemoglobin content (MCHC ) was calculated from the former t'fiO readings . The plasma protein content was d e te rmined us ing a refractome ter ( Schalm �-� a l . , 1 975 ) , and the i cterus index was dete rmined by comparing the colour of the plasma with potassium d ichromate s tandards ( Schalm et .... '-' H � '-'� 70 Stressors The initial groups of sheep were not subjected to any stressor but received either a single dose of coppe r at 3 mg Cu/kg bodyweight ( triple dose ) , or three separate doses of 1 mg Cu/kg bodyweight given at hourly intervals ( 3 x 1 ) ( Table 3 . 1 ) . The cold-stressed animals were shorn , placed in crates in the cold room at a temperature varying between 5 . 0 and 6 . � c , and rectal temperatures were recorded daily . Those sheep subjected to the ho t environment were also placed in crates and had rectal temperatures and respiratory rates recorded twice daily . In this experiment "dehydration" was achieved by bleeding . Each sheep had 750 ml of blood removed (approximately 25% of total blood volume ) immediately before the treatment with copper . In these dehydrated sheep the plasma protein decreased by 0 . 52 gm/ 1 00ml ( 7% ) at 24 hours and then returned to normal . Liver biopsy samples were not collected from the group of pregnant ewes because of their advanced pregnancy . In the experiment involving parasitised animals , nine-month old Romney hoggets were used . The "worm-free" control animals were selected from animals with a zero faecal egg coun t , and then each sheep was treated once weekly for six weeks with 1 80 mg oxfendazo1i · . The " parasite-infected" animals had faecal egg counts in excess of 1 57 0 e . p .g . Each hogget that died , and the two parasitised hoggets that received 3 mg Cu/kg bodyweight and survived , had their gastrointestinal contents submitted for total worm counts . The surviving lambs used for total worm counts were killed after the second liver biopsy had been taken . It had previously been determined that benzimidazole anthelmintics had no effect on the biochemical parameters measured in this experiment . 2synanthic , Syntex (N . Z . ) Ltd . 7 1 Those sheep treated with anthelmintic received an oral dose of 30 ml of oxfendazole containing 22 . 65 gm of active ingredient per litre . The sheep treated with insecticide were immersed for 30 seconds in a bath containing the organophosphate , diazinon , at a concentration of 300 mg per litre . Collection of blood samples Blood and serum samples were taken from each animal immediately before treatment and at 48 hours and 96 hours after the 90pper was administered . The blood samples were analysed for haemoglobin , packed cell volume , plasma protein and icteric index , and the MCHC was calculated . Blood was also digested in hydrogen peroxide and nitric acid apd analysed for the copper content . Serum samples were analysed for SDH and SGOT activity as described in Chapter 2 . Liver biopsies , for chemical analysis of the copper content , were taken three days before the time of the copper injection and again four days after the copper injection . The bodyweight of each sheep was recorded at the same time . Any sheep that died during the period of experimentation was subjected to a post-mortem kidney and brain ·were placed examination . Tissue samples of liver , in 1 0% buffered formol saline for histological examination , and further samples of the same tissues were frozen awaiting analysis for their copper content ( Appendix · 1 ) . RESULTS In those ewes subjected to cold or dehydration , those which were pregnant , or those which had been treated concurrently with insecticide or anthelmintic , no deaths occurred and no significant findings were recorded . In the groups subjected to the stressors of heat , starvation or parasitism , the results were much more dramatic . Nine of these 23 sheep died . 72 The results showing the number of animals that exhibited intravascular haemolysis , the changes in liver enzyme activity and the number of deaths are given in Table 3 . 2 . These results are further summarised in Table 3 . 3 into groups of sheep in which deaths did occur and those in which no deaths occurred . There were no significant differences in bodyweight change for any sheep throughout its experiment . Assuming that liver is 1 . 1 3% of bodywe ight and that it is 29 . 4% dry mat ter ( van Ryssen , 1 980) , then the calculated mean liver retention of the parenteral copper was 65% . There was no significant variation in retention rate between any of the groups . Blood copper increased by a mean of 0 . 5 ppm at 48 hours for all experiments . Of the sheep in the initial group that received 3 mg Cu/kg bodyweight , one died of copper toxicity . There were no significant differences between those sheep receiving a triple dose of copper , and those sheep receiving the same dose but divided into three equal doses and given at hourly intervals . The rectal temperatures of the sheep in the cold environment were reduced by 0. 6.9C after 36 hours and they remained at this level throughout · the experiment . The food intake of these sheep was high at approximately 1 . 6 kg D .M . /day . The liver enzyme concentrations of these sheep did not peak until 96 hours after treatment ; a delay that also occurred with the rise in copper concentrations of the blood . In those sheep exposed to the hot environment , rectal temperatures were raised by 0 . 6 0c after four hours , and remained at this elevated level while the sheep remained in this ho t environment . The respiratory rates increased �rom a mean of 98/minute to 1 72/minute in the first four hours , and then returned to a steady rate of 1 30/minute . These sheep drank large volumes of water , but food intake was severely reduced to approximatly 0 . 2 kg D . M . /day . Two o f the four sheep in the . group receiving 3 mg Cu/kg bodyweight 73 Cu doseNo . of No . show S . D . H . s . G . 0 . T . No . Stress mg/kg AnimalsHaemol . norm mid high norm mid high died triple dose3 5 0 2 2 1 2 2 1 3 X 1 3 5 3 1 1 3 1 0 starve 3 4 1 1 2 0 0 4 2 3x 1 starve 3 4 2 1 2 0 3 2 dehydr 3 4 0 2 0 2 2 0 2 0 heat t.l oc 2 4 0 4 0 0 0 4 0 0 heat 4l°C 3 3 0 2 0 0 2 0 cold b0G 2 4 0 0 2 2 0 4 0 0 cold "oc 3 4 0 2 1 1 0 3 1 0 preg 3 3 0 2 0 3 0 0 0 insect 3 4 0 2 1 1 0 1 3 0 anthel 3 4 0 1 2 1 2 0 2 0 paras it 2 4 0 1 0 3 0 3 2 paras it 3 4 0 2 0 2 2 0 2 2 S . D . H . levels S .G . O . T . levels norm = < 20 i . u . /ml norm = < 75 i . u . /ml mid = 2 1 - 200 i . u . /ml mid = 76 - 400 i . u . /ml high = 20 1 + i . u . /ml high = 40 1 + i . u . /ml Table 3 . 2 : Results of haemolys is , live r enzyme increases , and number of deaths in �nimals from all groups . Effect of Deaths No .with S D H S G 0 T stressors per group haemal . norm mid high norm mid high No deaths 0/23 0 9 7 7 7 Some deaths 9/23 4 9 4 9 3 ( a ) (b) Note : one sheep died before serum was collected . ( a ) and ( b ) : see text . 8 7 Table 3 . 3 : Summary of results of Table 3 . 2 combining stressor groups exhibiting deaths and stessor groups in which no deaths occurred . 8 1 2 7 4 died before treatment started, . and the other 36 hours after copper administration . Neither sheep showed evidence of copper toxicity either in histological changes of tissues taken after death , or from chemical analysis . Hyperaemia of the intestinal tract and enlargement of the gall bladder were seen in both sheep . These findings were suggestive of salmonellos is but this was not confirmed bacteriologically . Fifty percent ( four out of eight ) of the sheep that were starved died from copper toxicity . These four sheep showed an increase in plasma protein of 1 . 1 25 gm/ 1 00 ml ( 1 4% ) , in comparison with a 0 . 27 5 gm/ 100 ml ( 4% ) increase in those sheep that survived . Plasma albumin levels did not increase in the surviving sheep , but could not be measured in those sheep that subsequently died , because of the extensive intravascular haemolysis . Blood copper concentrations at 48 hours had increased by 1 . 84 ppm Cu in those sheep that died , in comparison with 1 . 32 ppm Cu in those sheep that survived . 75 "vlorm-free" "Infected" Sheep no . 1 20 1 23 1 24 1 28 1 04 105 1 22 1 26 Bodyweight : kg . 20 . 5 20 . 9 25 . 0 20 . 9 25 . 5 23 . 2 1 9 . 1 20 . 0 Serum pepsinogen 0 . 2 0 2 . 0 0 0 . 4 0 0 . 2 0 . 8 Faecal egg count · 0 0 0 0 3 , 650 2 , 400 7 , 050 4 , 600 Fate sl . sl . died died died died sl . sl . Haemonchus 0 0 0 0 1 00 600 400 1 00 Ostertagia 0 0 0 0 1 0 0 700 700 1 , 1 00 Trichostrongylus 0 0 0 0 1 , 000 800 300 800 I Cooperia 0 0 0 0 0 400 1 , ooo 1 0 0 Trichostrongylus 0 0 0 0 600 400 1 , 300 200 Nematodirus 0 0 0 0 300 800 0 0 Chabertia 0 0 0 0 20 20 30 20 Oesophagostomum 0 0 0 0 60 20 2 1 0 20 Trichuris 0 0 0 0 0 0 30 0 Moniezia 0 0 0 0 pres pres present 0 s l . = slaughtered Table 3 . 4 : Results of bodyweight , serum pepsinogen levels , faecal egg counts and total worm burdens of hoggets given copper calcium edetate at a dose rate of 3 mg Cu/kg bodyweight . "Worm-free" " Infected" Sheep no . 1 03 1 0 6 1 07 1 08 1 09 1 1 0 Bodyweight : kg . 27 . 3 23 . 6 30 . 0 1 7 . 3 24 . 1 2 4 . 5 Faecal egg count 0 0 1 . 570 1 , 600 2 , 1 00 2 , 7 ?0 Fate survived died survived died Ostertagia 300 200 Trichostrongylus 2 , 000 5 , 900 Cooperia 1 , 1 00 300 Trichostrongylus 1 , 600 1 , 1 00 Nematodirus 1 00 1 , 700 Oesophagostomum 0 1 0 Chabertia 20 0 Trichuris 0 20 Moniezia present present Table 3 . 5 : Results of bodyweight , faecal egg count and to tal worm counts of hoggets given copper calcium edetate at a dose rate of 2 mg Cu/kg bodyweight . 76 Sheep no . Stressor Copper dose mg/kg Live r Cu ppm Liver score H&E Kidney Kidney Cu score ppm O.M. H&E 1 1 . 05 1 3 . 75 1 3 . 1 55 1 4 . 496 1 4 . 1 086 1 9 . 6 1 2 6 . 1 07 2 6 . 1 1 0 24 . 1 04 24 . 1 05 24 . 1 24 24 . 1 28 triple starve starve starve starve heat parasite parasite parasite parasite "worm-free" "worm-free" Histology key : Liver H & E 1 . No significant findings 2 . Occasional macrophages & regular nuclei 3. Necrotic cells present 4 . Pyknos is & necros is , & aggregations of macrophages 5 . 50% of nuclei enlarged , vacuolation present & aggregations of macrophages 3 3 3 66 1 833 1 1 2 9 735 667 2 35 1 2 3 752 587 80 1 5 4 4 4 4 2 4 4 3 3 5 4 1 34 4 1 66 54 3 4 4 4 5 4 4 3 3 3 4 . 3 3 3 2 2 3 3 3 3 I 7 47 Kidney H & E 143 21 123 1 68 52 92 I 82 3 * no tissue analysed 1 . Some protein in proximal tubules 2 . Some necrosis & vacuolation in cells of proximal tubules 3 . Pyknosis & necrosis in proximal tubules & macrophages present 4 . Pyknosis and necrosis of tubules & macrophages infiltrating 5 . Most tubules necrotic or dis tended , some pyknosis and invading macrophages Table 3 . 6 : Results from post-mortem material of sheep that died . 77 78 The hoggets in the parasite trial were the most adversely affected of any groups of sheep , following the copper supplementation . Two of the infected and two of the "worm-free" hoggets , which received 3 mg Cu/kg bodyweight , died 1 8-24 hours after treatment . Copper toxicity was confirmed as the cause of death . The two surviving infected sheep had an increased blood copper concentration of 0 . 9 ppm Cu at 48 hours , in comparison with the two surviving "worm-free" hoggets which had an increase of 0 . 4 ppm Cu . Table 3 . 4 gives the bodyweight , faecal egg count and serum pepsinogen levels at the time of injection , and the total worm burdens at the time o f death or at slaughter . Two o f the infected sheep that received 2 mg Cu/kg bodyweight died at 45 apd 72 hours respectively . The deaths were confirmed as being the result from copper toxicity . The two hoggets and the two "vrorm-free" hoggets concentration increases of 0 . 7 ppm Cu at 48 surviving infected had blood copper hours , whereas the infected hogget that died at 72 hours had an increase in blood copper concentration of 1 . 7 ppm Cu at 48 hours . The three infected hoggets which were still alive at 48 hours had an icteric index of 50-75 units as compared with the non-infected hoggets which had 5 and 1 0 units respectively . MCHC levels did not indicate intravascular haemolysis . Table 3 . 5 gives the bodyweights , faecal egg counts and total worm burdens of these six hoggets . Deaths : Post-mortem examination , histopathology and chemical analysis of tissues confirmed that the deaths were the result of copper toxicity . Table 3 . 6 summarizes these findings . 79 DISCUSSION It would appear from the results of these experiments , that the dose rate of copper used ( 3 mg Cu/kg bodyweJ.ght ) is close to a toxic dose . One sheep which received this dose rate , but which was deliberately not subjected to any additional stress , died as a result of copper poisoning . Mahmoud & Ford ( 1 9 82 ) have reported losses in the field in adult ewes that also had each received a dose of 3 mg Cu/kg bodyweight as copper calcium edetate . Therefore it is of be suggested that any stressor that will enhance the toxic potential copper calcium edetate administered at this dose rate may identified by measuring the changes in certain biochemical parameters ­ in sheep subjected to that stressor and treated with copper calcium edetate . The responses may be death , or high increases in the biochemical parameters used to assess hepatotoxic damage . In these experiments losses occurred in those groups subjected to heat , starvation , and to gastrointestinal parasites . In the other stressed groups there were no deaths as a result of the copper supplementation . In fact , the increases in the biochemical parameters that indicat� . hepatotoxicity were significantly lowe r in these sheep , than in the surviving sheep from the groups in which deaths occurred . There was no apparent difference in the biochemical parame ters measured , between those sheep receiving 3 mg Cu/kg bodyweight as a s ingle dose , and those receiving it in three doses of 1 mg Cu/kg bodyweight , each an hour apart . Forty-eight hours resulted in death in published accounts starvation prior to the copper injection four out . of the eight sheep treated . In of deaths following parenteral copper administration , it been subjected to transport ( Skinner , is apparent that i n some cases the animals have stressors , and especially to the stress of 1 960) or handling ( Ross , 1 964 ) . It would appear likely that sheep subjected to these stressors have been deprived o f food and water for 24 to 48 hours before treatment : therefore the precipitating cause of toxicity is most likely to be starvation . The 80 sheep subjected to heat also showed an adverse response to the parenteral copper . These sheep were in a state of partial starvation as their feed intake Has low. In the sheep which died there was a marked increase in plasma protein , and in particular in the plasma albumin fraction . This factor is of special interest as albumin is the carrier of copper in the blood , from the injection site to the liver . The enhancement of toxicity may be the result of increased amounts of copper being presented to the liver because of the increased plasma albumin . When the effects o f copper supplementation were measured in sheep affected with gastrointestinal parasitism , seve ral points arose . In the. group receiving 3 mg Cu/kg bodyweight , half o f the animals died of copper toxicity but the deaths occurred in both the infected and the worm-free animals . The predispos ing cause was probably one of age as these animals were only nine months old . Lewis et al . , ( 1 981 ) , reported that young sheep are more susceptible to copper toxicity . It seems likely that this is the result of younger animals absorbing drugs more rapidly from subcutaneous sites than older animals ( Ballard , 1 978 ) . Ballard suggested that the difference is due to an increase in the thickness of subcutaneous tissue with age , as well as to changes in the composition of subcutaneous fat tissues . However when the dosage of copper was only 2 mg Cu/kg bodyweight , losses from copper toxicity still occurred , but only in those animals affected with a heavy burden of gastrointestinal parasites . The blood copper content of these sheep that died increased by 1 . 7 ppm in comparison with 0 . 7 ppm in those that survived . The parasi te burden carried in the infected sheep ( Tables 3 . 3 & 3 . 4 ) would not be . uncommon under some New Zealand farming situations . In these experiments the faecal egg count was much higher than that expected from animals carrying this total worm burden . This is probably due to a large increase in faecal egg concentration as a result of the clinically affected sheep los ing their appetite prior to death or slaughter . the sheep treated with copper may have 8 1 I t is also poss ible that lost some of their worm population a fter the copper treatment ( Charleston , pers . comm. ) . High parasite burdens can result in a large loss of protein from the intestine ( Symons e t al . , 1 974 ) . This loss may produce modified protein metabolism leading to increased induced inappetance caused by the parasite plasma burden albumin , ( Sykes & or the Coop , 1 976 ; Sykes & Coo p , 1 977 ) , may influence protein metabolism, to produce the same effect . As suggested earlier , this increased plasma albumin may enhance copper toxicity by increasing the uptake of copper from the injection site and presenting copper to the liver at _ a higher concentration . In seven of the nine sheep that died from copper poisoning in these experiments , the serum levels of SOH activity and of SGOT activity did not rise significantly . In each of these cases intravascular haemolys is had occurred ( Table 3 . 3 ( a ) and ( b ) ) and the technique for measuring SOH activity specifies that the serum must be free of haemolys is , but the same condition is not specified for the SGOT technique . Presumably in both cases the free haemoglobin in the haemolysed serum uti lized the enzyme substrate provided for the test so that little enzymic reaction was able to occur . In those sheep in which the MCHC value did not rise above 40% , it is possible that any free haemoglobin in the serum was bound to the serum haptoglobin , and therefore was not available to interfere with the reaction . CONCLUSION When copper calcium edetate is injected into sheep its toxicity may ' be potentiated if the animal is subjected to certain management stressors . These include 48 hours deprivation of food and water , exposure in an environment in excess of 40�C , and the presence of gastrointestinal nematodes . Young sheep are more susceptible to the toxic effects of copper calcium edetate than are adult sheep . 82 it CHAPTER 4 : ABSORPTION RATES OF DIFFERENT COPPER FORMULATIONS ADMINISTERED UNDER VARIOU S CONDITIONS INTRODUCTION When all other variables are controlled , the differences in toxicity between the various parente ral copper preparations is dependent on the rate of translocation of copper from the injection site ( Suttle , 1 98 1 b ) . The translocation rate of a drug is affected by the volume injected , the solids to vehicle ratio , and the particle size of the drug in suspension . It is also affected by the presence or absence of pharmaceutical agents such as suspending agents and by the pH , the . tonicity of the formulation , the surface area of the depot formed at the injection site , the physicochemical properties of the drug , and the nature of the vehicle (MacDiarmid , 1 983 ) . In 1 977 a formulation of copper calcium edetate at a concentration of 25 mg Cu/ml , in a multidose container 1 , was released for the correction of copper deficiency in cattle , in New Zealand . Unfortunately some 35 deaths in the 600 , 000 cattle injected were recorded , not as a result of copper toxicity , but from an anaphylactoid reaction . The agent that appeared respons ible for this reaction was polyvinyl pyrrolidine ( PVP ) , ( Easingwood , 1 98 1 ) . Polyvinyl pyrrolidine was included as a suspending agent and also used to delay the absorption of copper from the injection site . Because of the ability of this product to translocate copper from the s ite of injection to ruminant liver without causing undue reaction at the injection site , the manufacturers reformulated the product by excluding the PVP and they doubled the concentration of the copper calcium edetate . The translocation rate of this new formulation� , (hereafter referred to as the high concentration formula ) , had to be assessed , and its safety for use in sheep evaluated . When evaluating the toxicity of copper calcium edetate in sheep which had been subjected to various stressors (Chapter 3 ) , it was apparent that heat and starvation enhanced toxicity . It was decided 1 Coprin Mul tidos e , Glaxo ( N . Z . ) Ltd . 2 Caprin , Glaxo (N . Z . ) Ltd . * In thi s chapter absorption refers to the movement of copper from the site of administration to the blood circulation . 83 to determine whether this enhanced toxicity was the result of an increased translocation rate , or due to some other factors . Differences in formulation may alter the translocation rate of copper (MacDiarmid , 1983 ) , but other factors have not been described . MATERIALS AND METHODS In this series of experiments several proprietary copper preparations were administered . The low concentration formulation of copper calcium edetate contained 25mg of elemental copper per millilitre , and the high concentration formulation of copper calcium edetate contained 50mg of elemental coppe r per milli litre . Stressors The two sheep used · in the pilot experiment described in Chapter 3 , were injected subcutaneously with 50 mg of copper as copper calcium edetate in the low concentration formulation , ( approximately 1 mg/kg bodyweight ) , 48 hours after entering the hot room which was maintained at a temperature of 40 . 0 - 4 1 . 5°C . Blood samples were collected immediately before treatment , and then at hourly intervals for 9 hours . The blood parameters of PCV , haemoglobin , plasma protein were measured for each sample , and the MCHC calculated . Blood samples were also digested and analysed for copper content . Blood uptake rates of copper were expressed as changes in the blood copper concentration over time , using the regression line from the start of sampling to the peak of blood copper concentration . As described in Chapter 3 , starvation and deprivation of water for 48 hours were the stressors that appeared to make sheep most susceptible to copper toxicity . A pilot experiment was carried out in which two sheep were deprived of food and water for 48 hours . These sheep were then injected subcutaneously with copper calcium edetate in the. low concentration formulation at a dose rate of 2 mg Cu/kg bodyweight . Blood samples were collected immediately prior to 84 the copper injection and then hourly for 1 2 hours . Each blood sample was analysed for copper content . Regression lines were fitted to the raw data to assess the increase in blood copper over time . The result achieved in the pilot experiment warranted an expanded trial . This was designed to confirm the early resul t , and to determine the duration of starvation required before blood copper uptake rates were significantly affected . Twenty-eight adult sheep were randomly placed into six groups and subjected to varying periods of starvation as set out in Table 4 . 1 . No . in group Duration of Access to starvation water 5 72 hours no 4 12 hours yes 5 48 hours no 5 24 hours no 5 1 2 hours no 4 Control yes Table 4 . 1 : Schedule of food and water deprivation of sheep used when determining copper uptake rate . A liver biopsy was taken from each sheep six days before the copper supplementation , and again at the end of the experiment seven days later . The sheep had access to adequate pasture from the time of the first liver biopsy until the commencement of the starvation period . Only the control group had access to feed during the period of blood collection . Each sheep was injected subcutaneously with copper calcium edetate in the low concentration formulation at a dose rate of 2 mg Cu/kg bodyweight . Blood samples were collected immediately prior to the copper injections and then at hourly . - 8 5 intervals for 1 2 hour s . Blood samples were also collected at the start of the respective period of starvation . Bodyweights of each sheep were recorded at the start of the period of starvation , and at the completion of the experiment . The blood samples collected a t the commencement of the starvation period and at the time of copper injection were used in an attempt to quantify the e ffects of starvation . Packed cell volume of ­ the blood was measured . Plasma was assessed for glucose content using glucose oxidase 3, and for the presence of ketones using the nitroprusside method 4 • Total plasma protein was determined using a refractrometer , and plasma albumin using the bromocresol reaction. green The bloods were also digested and analysed for copper content . Regression lines were fitted to the copper concentration data from the pre-injection time to the peak blood copper levels to assess the increase in blood copper concentration over time . Copper formulations The first experiment was designed to evaluate the increase ih blood copper concentration , and the total uptake of copper from two oral copper preparations , namely , a "Mineral Premix" containing copper edetate 5, ( a copper chelate ) , and copper oxide needles9 . Two groups of four , aged , Romney ewes were selected for this experiment . Liver biopsies were obtained on days 0 and 5 of the experiment . The sheep were penned and maintained on a diet of hay containing 3 . 6 ppm D . M . of coppe r . The mineral premix-treated group received an oral dose containing 0 . 33 mg Cu/kg bodyweight - the manufacturer ' s recommended dose ; and the copper oxide group were dosed with 2 . 86 gm of copper contained in 7 gm of coppe r oxide needles , in a gelatine capsule . 3 Mercktes t 339 3 , E . Merck , Germany 4 Ace test tab l e t s , Ames Divis ion , Mil es Laboratories , Aus tralia 5 Mineral Premix , May & Baker (N . Z . ) Ltd . 6 " Copper oxide , Merck Ltd . 86 Blood samples were collected immediately prior to copper supplementation and then hourly for 1 2 hours . The sheep in the copper oxide group had liver biopsies taken every fourteen days for 84 days . Blood parameters measured on the collected samples were PCV , haemoglobin content and total plasma protein . Each blood sample was also digested and analysed for copper content . Regression lines were fitted to the data from pre-injection time to peak blood copper levels to assess the increase in blood copper concentration over time . Liver biopsy samples were analysed for copper content . To assess the uptake rate of the high concentration formulation as described in the introduction , twenty aged Romney ewes which weighed approximately 50 kg were used . The numbers of sheep used and the dosage of copper used are set out in Table 4 . 2 . Formulation No . of sheep Copper dosage Dose volume Low cone . 4 50 mg 2 ml High cone . 5 50 mg 1 ml High cone . 6 1 00 mg 2 ml High + water 5 50 mg 2 ml Table 4 . 2 : Design of an experiment to compare translocation rates of different injectable copper formulations . Blood samples were collected at the start of copper supplementation , hourly for 1 6 hours , and then at 24 , 36 , and 48 hours . These blood samples were digested and analysed for copper concentration . Regression lines were fitted to measure the increase in blood copper concentration . 87 Five of the sheep receiving 1 00 mg of copper contained in the high concentration formulation were a lso used to assess the toxicity of this new formulation . These sheep had a liver biopsy taken before · the copper supplementation and again 7 days after treatment . The live r tissue was analysed for copper · content . Serum from blood samples taken from these sheep at 0 , 4 8 , and 96 hours , was assessed . for SOH and SGOT activity. These sheep were penned throughout the experiment and had access to water and meadmo� hay . The statistical analysis applied to the recorded data was an , analysis of variance one-way command . The F-ratio was used as the test statistic . RESULTS Heat The blood parameters of plasma protein , PCV and haemoglobin varied within 5% of the mean throughout the 9 hour bleeding period , while the MCHC remained almost constant . The regression line o f increase i n blood copper concentration ove r time had for the two sheep slopes of 0 . 0387 and 0 . 02 1 1 mg Cu/litre of blood per hour respectively , with a mean slope o f 0 . 0299 mg Cu/litre/hour . Starvation The regression line describing copper uptake for the two pilot sheep had a slope of 0 . 0897 and 0 . 07 86 mg Cu/litre of blood per hour respectively , with a mean of 0 . 0842 mg Cu/litre/hour . 00 Table 4 . 3 gives the results of the blood parameters measured , and the changes in bodyweight . The regression lines of copper uptake are given in Table 11 . 6 . The liver biopsies taken before and after treatment showed that the liver stored 56% of the administered copper . Premix Starvation period in hours Parameter 72 72+H20 48 24 1 2 Kg loss 6 . 3 5 . 1 5 . 0 5 . 6 3 . 7 PCV change +0 . 1 0 +0 . 1 0 +0 . 04 +0 . 06 +0 . 1 0 Plas prot change�my�+0. 7 +0 . 9 +0 . 5 -0 . 3 +0 . 3 Glucose change '!"."3/oo ,...\.,.2 . 3 -2 . 5 - 1 . 5 - 1 . 4 - 1 . 0 Albumin change rsJKX>,..,\ +0 . 9 6 +0 . 75 +0. 48 - 0 . 1 0 - 0 . 1 4 Alb . · % change + 1 0 . 2 +7 . 0 +4 . 5 -0 . 3 +2 . 2 Table 4 . 3 : Mean blood parameter changes of sheep undergoing various periods of starvation immediately prior to copper supplementation . 0 0 0 0 0 0 0 The blood parameters measured showed no significant variation throughout the 24 hours of uptake . .Liver biopsies showed a mean increase of 52 ppm of copper . Assuming a total l iver weight of 1 . 1 3% of bodyweight and a 29 . 4% dry matter content ( van Ryssen , 1 980 ) , the liver retention of the copper was 46% of the administered dose . The hay diet of these sheep contained 3 . 7 ppm copper , < 0 . 8 ppm molybdenum , 0 . 1 6% sulphur , and 60 ppm of iron . Blood copper concentration peaked at a mean of 7 hours <±1 hour) after copper administration , and the increase in blood copper concentration had a mean regression line of 0 . 02 1 0 mg Cu/litre/hour . ', 89 The serum enzyme levels of SDH and of SGOT remained within the normal l imits in every sheep . Copper oxide needles The blood parameters of PCV , haemoglobin and plasma protein did not vary significantly throughout the experiment . Blood copper levels remained almost constant during the 1 2 hours of sampling and showed a mean regression line of -0 . 0093 mg Cu/litre/hour . The liver biops ies taken throughout the experiment gave liver copper concentrations as seen in Table 4 . 4 . ·, Liver copper content : ppm DM Sheep no . 7 1 63 237 274 Day 0 443 562 1 203 353 1 4 1 0 1 6 507 979 1 78 28 1 1 06 1 284 1 275 3261 42 1 284 1 4 1 0 1 1 84 1 484 * 56 1 4 22 1 280 1 060 70 1 423 1 1 06 1 1 40 84 1 306 1 1 04 1 08 1 * = death Table 4 . 4 : Liver copper conten t of sheep after administration of copper oxide needles . Shee p 274 failed to recover from the anaesthetic given 6 weeks after the treatment with copper oxide needles . On examination of the liver , lesions typical of facial eczema were seen . The abomasal washings were recovered , and analysed for copper content . The 1 5 . 77 gm of abomasal washings contained 2854 ppm of copper , indicating that 90 6 . 73 gm of the 7 gm of the copper oxide needles had been absorbed or lost over the 6 week period . The estimated amount of copper retained in the live r was 4 . 9% of the administered dose , after 6 weeks . Only two of the four sheep were considered in these calculations as sheep no . 274 had liver lesions of facial eczema , and sheep no . 2 37 had erratic liver copper levels on analysis of biopsy samples , possibly also associated with facial eczema changes . Copper calcium edetate The uptake rate of copper measured in blood samples showed a variation when different formulations of copper calcium edetate were used . There was a non-significant difference between low and high dose rates , but a s ignificant difference occurred when the formulation was diluted with water , ( P > 0 . 0 1 ) , or when the sheep were starved for a period before treatment greater than 24 hours . The mean regression lines of the changes in blood copper concentrations , the numbers of sheep used , · and the copper dosage administered are shown in Table 4 . 5 and Table 4 . 6 . In the sheep that received 1 00 mg of the high concentration formulation , the liver enzyme activity concentrations in serum did not rise beyond the normal limits in any sheep . In these same sheep , using the liver biopsy results , 54% of the administered dose was present in the liver after 7 days . There was a statistical significance in the uptake rate of copper between those sheep that were starved for 48 hours and those starved for only 1 2 hours or not starved at all ( Table 4 . 6 ) . Treatment Heat + 50mg Oral chelate CuO needles Low cone form High cone form High cone form High cone + water Starve 48 hrs No . sheep 2 4 4 4 5 6 5 2 Dose rate Uptake mg Cu/1/hr 4 mg/kg . 0299 33 mg/kg . 02 1 0 2 . 86 gm - . 0093 50 mg . 0 1 5 4 5 0 mg . 0 1 97 1 00 mg . 02 1 9 50 mg . 0356 2 mg/kg . 0842 Table 4 . 5 ; Mean uptake rates o f copper in shee p administered different copper therapeutic agents . Treatment Starve Starve Starve Starve Starve Starve 72hrs 72 + H20 48 hrs 24 hrs 1 2 hrs 0 hrs No . sheep 5 4 5 5 5 4 Dose rate Uptake F mg Cu/1/hr 2 mg/kg • 1 27 6 B 2 mg/kg . 1 4 1 0 B 2 mg/kg . 0862 AB 2 mg/kg . 09 1 8 AB 2 mg/kg . 0688 A 2 mg/kg . 0556 A Table 4 . 6 : Mean uptake rates of copper in sheep administered copper calcium edetate after being subjected to different periods of starvation . 9 1 . 14 . 12 . 10 Jptake � Cu/ 1 /hr . 08 . 06 . 04 . 0 2 0 7 2 h r s + water 7 2 hrs Period o f 2 4 hrs 48 hrs 12 hrs 0 hrs Time Supp l ement to tab le 4 . 6 : Graph o f mean uptake s o f copper in sheep admini s terd copper calc ium edeta te a fter be ing sub j ected to d i f ferent periods o f s t arva t ion . St arvat ion 92 DISCUSSION For the purposes of this discussion , the sheep subjected to the various treatments can be classed conveniently into three groups of experiments • . The first group of experiments includes those sheep subjected to heat and those sheep treated orally with copper edetate and copper oxide needles . The copper oxide needles are slowly absorbed (Dewey , 1 977 ) and so did not affect blood copper concentrations . The liver uptake was s imilarly slow as shown by the low serum enzyme activity . This supports the work of Ellis ( 1 97 9 ) which showed that the uptake . of copper from copper oxide needles was slow and appeared non-toxic to sheep . The oral copper edetate dose is also non-toxic and yet it has an uptake rate s imilar to that of 50 mg of copper calcium edetate given as the low concentration formulation by subcutaneous injection . However only an estimated 7 . 6 mg of this oral dose was retained in the live r , and this is approximately 20% of the amount expected to be retained after a single recommended dose of parenterally administered copper calcium edetate . Nevertheless this high retention rate for o rally administered copper probably resulted from the low concentrations of the inhibiting e lements molybdenum and sulphur ( < 0 . 2 ppm and < 0 . 01 % respectively ) . These latter concentrations are not typical of most New Zealand pastures on which copper deficiency is diagnosed • . Sheep given this same dose rate of oral copper edetate , and on the same diet , but also given a daily supplement of 50 mg of molybdenum and 1 00 mg of sulphur did not demonstrate any change in liver copper concentration ( Farquharson , unpublished data ) . The uptake rate of the copper in the sheep in the hot environment was almost double that of a s imilar dose administered to sheep in a normal environment ( Table 4 . 5 ) . This increase was probably the result of two mechanisms . The skin temperature of these animals probably increased · the subcutaneous blood supply which in 93 turn enhanced the absorption rate ( Harvey , 1 970 ) . The other factor possibly responsible for the increased uptake was partial starvation , as these sheep had a greatly reduced feed intake over the two days previous to the copper supplementation . The mechanism of starvation is decribed later in the text . The second group involved those sheep that received the different formulations of copper calcium edetate given parenterally . The lowest uptake rate occurred in the group that received 50 mg of copper as copper calcium edetate in the low concentration formulation . Those sheep that received 50 mg of copper in the high concentration formulation of copper calcium edetate , and those that received 1 0 0 mg of copper in this same formulation had similar mean uptake rates of 0 . 0 1 97 and 0 . 02 1 9 mg Cu/litre/hour respectively . This was only slightly higher than that obtained following 50 mg of copper in the low concentration formulation and this contained PVP which delays drug absorption ( Ballard & Nelson , 1 970) . The high concentration formulation contained copper calcium edetate at double the concentration of the low concentration formulation . The retarded absorption of the concentrated suspension was most likely the result of a compact poorly soluble deposit formed subcutaneously ( Ober et al . , 1 958 ) . The effect of concentration on absorption in this experiment was comparable to the retarding effect of PVP . When the new formulation was diluted with its own volume of water , the uptake rate almost doubled . This increase was probably due to the less concentrated suspension presenting a greater surface area to the absorptive capillary bed (Yeoman , 1 977 ) , to its lower viscos ity (Dowrick , 1 980 ) , and to more copper being present in solution in a more dilute suspension (MacDiarmid , 1 983 ) . The higher uptake rate would greatly enhance the toxicity of copper . The third group o f experiments comprised those sheep which had been subjected to starvation , but had all received the same dose rate of the low concentration formulation at 2 mg Cu/kg bodyweight . The uptake rate . in �th groups of sheep that were starved for 48 hours was almost identical . Howeve r in the control sheep that were not 94 starved the uptake rate was 0 . 0556 mg Cu/litre/hour , which exceeds the 0 . 02 1 9 of the group receiving 1 00 mg of the new formulation . The control group was on pasture before the supplementation with coppe r , and then had access to hay during the hourly bleedings . These sheep did not eat their food on offer and therefore were fasting from the time of the copper injection . Unfortunately no parameters were measured at the completion of bleeding to confirm this effect . The bodyweight losses and the blood glucose depression were typical of fasting ruminants as reported by Kirton et al . , ( 1 965 ) , and by Bouchat et al . , ( 1 980 ) . Liver protein metabolism alters during fasting ( Aronson , 1 980) ; the liver losing up to 40% of its total weight over 72 hours ( Furner & Feller , 1 97 1 ) . Most of this loss is protein. In these experiments after 72 hours of fasting , plasma protein had increased by approximately 1 0 % , whereas plasma albumin had increased by approximately 70% . This can be explained by the fact that albumin is the smaller protein molecule and perhaps permeated through the hepatocyte cell wall more readily during times of increased demand for hepatic protein. The increase in blood copper concentration during translocation of copper from the depot site to the liver may have resulted from any or all of three phenomena . These are , an increased blood supply at the site of deposition , an increase in the plasma protein that is the carrier protein ( for copper it is albumin ) , or the liver receptor sites may not immediately accept the copper ions from the protein carrier complex . I t is difficult to imagine that subcutaneous vascularity would alter in fasted animals when there was no variation in environmental temperature , or in local irritation at the depot site . Total blood albumin had increased dramatically and it can be assumed that hepatic protein metabolism was altered . In copper toxicity there is increased hepatic damage resulting from the increased copper content of hepatocytes as copper in the blood is presented to the liver at a higher concentration (Chapter 3 ) . During ··- 95 the accumulation phase of potential copper toxicity , there is an increase in hepatocyte copper content . It is proposed that during periods of starvation copper is presented to the liver in the copper-albumin complex at higher concentrations than in animals that have not been subjected to starvation , and therefore there is a more rapid accumulation of copper in the hepatocyte . The uptake rate of copper in those animals not starved , and which were used as control sheep in the starvation experiment , was greater than expected . These sheep were penned for the �xperiment after grazing pasture and may not have eaten the hay provided . Therefore by the tenth hour of blood collection , some effects of starvation may have influenced the uptake rate of copper. Because of the difficulty_ in controlling all the variables in this experiment it would appear inadvisable to compare results from experiments carried out at different times . The greater than two-fold increase in uptake rate of copper in sheep starved for 72 hours , when compared to sheep that were not starved , suggests that translocation rate is important when assessing factors that may potentiate copper toxicity . Therefore in developing new copper therapeutic compounds and their route of administration it is essential to determine the translocation rate of the copper and also to identify those factors that may alter the translocation rate . From this data it is suggested that sheep should not be injected with copper calcium edetate at dose rates exceeding mg Cu/kg bodyweight if they have been deprived of feed and water for 24 hours or more . Better still , they should not be deprived of either about the time of copper administration . 96 CONCLUSION The toxicity of copper calcium edetate , after injection into sheep , may be enhanced when the uptake of copper from the depot site to the bloodstream is elevated . This increase may occur either when sheep are placed in a hot environment or when the concentration of the parenteral copper product is reduced , but the total volume injected increased . More importantly , the toxicity is greatly enhanced if sheep have been fasted . Sheep that have been deprived of food and water for 24 hours or longer should not be injected with copper calcium edetate • . CHAPTER 5 : MONITORING THE EFFECT OF COPPER SUPPLEMENTATION ON ANIMALS GRAZING COPPER SUFFICIENT AND COPPER DEFICIENT FARMS . INTRODUCTION 91 In the past , the administration of copper to domestic animals has often been based on inadequate information . There are now a number of ways in which the copper status of a farm and its animals can be assessed accurately , and from this data , the best advice on supplementation can be given . I t is also necessary to appreciate the effect of copper therapeutic agents on animals of sufficient and ·­ deficient copper status . Because of the potential effects of a number of management , climatic , this is best monitored in conditions . feeding and geographical factors , animals grazing under typical farm For the purposes of this study , two farms were selected . On farm A , copper deficiency had been regularly diagnosed i n both sheep and cattle . This was indicated by a low but regular occurrence o f swayback in lambs , and . �oor growth and reproductive performance in cattle . The cattle also showed the classical signs of coat colour change (achromotrichia ) . On farm B , copper deficiency had never been diagnosed in sheep , but young cattle showed signs of copper deficiency in some years . In other years , the copper levels in cattle , as determined by regular liver biopsy , were adequate , and no clinical signs of copper deficiency were apparent . Three years prior to the commencement of the present study , this farm had been topdressed with copperised superphosphate at a rate of 5 kg copper per hectare . The apparent need for periodic copper supplementation of cattle on this farm made it a valuable property to monitor as it is typical of many New Zealand North Island sheep and beef cattle properties on which copper deficiency occurs in cattle , but not in sheep . 98 To calculate the availabil ity of copper in pasture to grazing ruminants , Suttle ( 1 974a ) , has developed a formula to i ncorporate the influence of mol ybdenum and sulphur on copper availability . This formula i s : A = 0 . 07 5 - 0 . 0 30 Mo - 0 . 0 1 34 S where A i s the availability of copper Mo is in mg/kg S is in gm/kg and is used in this experiment to assess the amount of copper available to the sheep on these farms . Farm Description Farm A , the copper deficient farm , is situated in the Turakina River valley , 20 km west of Hunterville in the Rangitikei County ( Figure 5 . 1 ) . The farm is 6 34 ha in area and consists of two distinct areas . Area 1 ' known as Mill Block , i s of 320 ha and is divided into nine major paddocks . This is rolling hill country with a soil type classified as Mangatea clay loam . 'This soil is derived from a consolidated rubbly sandy mudstone (Campbell , 1 979 ) . Only 500 ha of this soil type are in the Rangitikei County . The pasture on this area is improved with a high content of perennial ryegrass ( Lolium perenne ) and white clover ( Trifolium repens ) . The area of land located at the back of the farm is area 2 and known as Wallies Block . It is 3 1 4 ha in area and is poorly subdivided into four paddocks . This is steep hill country classified as Turakina steepland soil derived from a consolidated rubbly sandy mudstone ( Campbell , 1 979 ) . This land which is typical o f the area (with 20 , 000 ha in the Rangitikei County) contains poorer quality pasture species such as brown top ( Agrostis tenuis ) , timothy ( Phleum pratense ) , and crested dogstail ( Cynosurus cristatus ) , and only small quantities of ryegrass and clover . 99 Figure 5 . 1 : Location of the two experimental farms The farm is situated approximately 500 metres above sea level and enjoys a regular annual rainfall of approximately 1 200 mm . There is pasture growth all year round , except on the dry faces exposed to the hot dry winds in summer . This farm is run at a stocking rate of 1 1 . 0 s tock units per hectare . . . 1 00 The "copper sufficient" farm , farm B , is located at Tangimoana , near the coast on the western boundary of the Manawatu County ( Figure 5 . 1 ) . The farm is 1 1 89 ha i n area subdivided into 39 paddocks , and has a stocking rate of 9 . 4 stock units per hectare . The farm is coastal sand country of the Hokio-Waitarere association ( Cowie , 1 9 58 ) , which makes up approximately 1 00 , 000 ha of this coastal strip of country . The yellow-brown sand is derived from wind-blown coastal sand of predominantly feldspar and quartz . This soil is very low in clay and organic matter . The area is mainly sand plains , with some sand dunes ( Cowie , 1 977 ) . The pasture sward contains _ryegrass , Yorkshire fog ( Holcus lanatus ) , crested dogstail , and white and strawberry clovers (Trifolium fragiferum ) . Although the a rea has a regular annual rainfall of approximately 1 000 mm , the main growth period for pa�ture is in the winter and spring as the pastures tend to dry out in summer . · MATERIALS AND METHODS On farm A , ( to be known as the copper deficient farm ) , the liver copper levels of the sheep and the mineral content of the pasture were monitored ove r an 1 8 month period . Four hundred Romney sheep , comprising 1 00 ewe hoggets , 1 00 two-year old ewes , and 200 mixed-age ewes , were individually identified using numbered ear tags . Eight vis its were made to this farm . On farm B , ( the copper sufficient farm ) , the liver copper levels of the sheep and the mineral content of the pasture were monitored four times over a 1 2 month period . One hundred Romney ewe hoggets were individually identified and used for this purpose . At the initial visit to each farm , soil samples from the paddocks to be grazed by the trial sheep . was extracted using 2N hydrochloric acid ( Fiskell , were collected The soil copper 1 965) , and the copper content estimated using atomic emission spectrometry . 1 0 1 On farm A the sheep grazed seven paddocks . Pasture samples from these paddocks were collected for analysis on twelve occasions over the 1 8 month period of the trial . On farm B pasture samples were collected from the paddocks in which the identified sheep grazed , and these samples were collected on seven occasions . In each case the pasture samples were digested in nitric acid and analysed for mineral content using atomic emission spectrometry ( Appendix II ) . At each farm visit the tagged sheep were drafted out and weighed . Four sheep in every group of 1 00 were selected at random and liver biopsy samples and blood samples were collected . These samples were analysed for copper content ( Appendix I & I I ) . In addition the blood samples were analysed for PCV , haemoglobin content and plasma protein content , and the MCHC was calculated . At the initial vis it to each farm , half of the sheep in each group were injected with 50 mg of copper calcium edetate 1 , contained in a 2 ml volume . On farm A these treated animals were given a further 2 ml of copper calcium edetate , 1 3 months after the first injection . Eleven months after the start of the trial , the fleece weights were recorded at shearing from some of the identified animals on farm B . The F-ratio was the test statistic applied to an analysis of variance command , to determine significance . RESULTS On both farms the concentration of extractable copper in the soil was similar . Amounts ranged from 2 . 00 ppm Cu to 1 6 . 98 ppm Cu , and the range is s imilar to levels recorded in many New Zealand soils (Well s , 1 957 ) . The re was a positive correlation of 0 . 9 94 between soil extractable copper and pasture copper on farm B ( the copper sufficient farm ) ( Figure 5 . 2 ) . The data in Tables 5 . 1 and 5 . 2 1 Coprin Mul t idos e , Gl axo (N : Z . ) Ltd . Pas ture Copper ppm 1 5 1 2 9 6 3 Soil copp e r : PP� , Figure 5 . 2 : Relat ionship b e tween soil copper and pas ture copper on the copper s uff icient farm 1 02 represent the average mineral content of the pasture from the two areas of the copper deficient farm . On both areas o f the farm the pasture copper levels as estimated from the samples analysed , were lowest over the winter period and highest in the summer , whereas the amounts of molybdenum and sulphur were highest in the early spring period , with molybdenum as high as 4 . 74 ppm D .M . , and sulphur as high as . 32% D .M . ( Figure 5 . 3 ) . The lowest molybdenum levels measured were 1 . 4 1 ppm D .M . , with a mean of 3 . 1 3 ppm D .M . . I 1 0 3 Dates Cu Mo s Fe Zn Mn S/McL pp m pp m % ppm ppm pp m ppm 1 2 . 6 . 8 1 4 . 36 1 . 4 1 . 1 4 5920 39 . 3 1 37 5 25 . 6 . 8 1 4 . 08 2 . 70 • 1 5 34 1 2 43 . 4 22 1 5 1 3 . 7 . 8 1 5 . 43 2 . 85 . 20 6485 52 . 4 296 6 25 . 9 . 8 1 6 . 52 3 . 2 9 . 2 3 6552 85 . 7 322 7 1 3 . 1 0 . 8 1 6 . 40 3 . 69 . 22 3449 54 . 3 309 7 20 . 1 1 . 8 1 5 . 34 2 . 35 . 22 1 428 4 1 . 1 246 6 3 . 3 . 82 8 . 9 1 3 . 60 . 26 1 5 1 2 77 . 8 1 32 8 2 5 . 5 . 82 3 . 68 2 . 02 • 1 7 1 529 86 . 1 273 6 1 5 . 6 . 82 6 . 79 3 . 50 . 23 . 1 6 1 2 58 . 1 250 7 1 6 . 7 . 82 5 . 78 3 . 8 1 . 22 220 1 6 1 . 6 1 9 1 7 Mean 5 . 73 2 . 92 . 20 340 1 60 . 0 238 6 . 4 S/McL is the est�mated copper content of pasture to meet dietary requirements ( Suttle & McLauchlan formula ) . Table 5 . 1 : Mineral content of pastures taken from area 1 on the copper deficient farm . 1 0 4 Dates Cu Mo s Fe Zn Mn S/McL pp m pp m % ppm ppm ppm ppm 1 2 . 6 . 8 1 7 . 42 2 . 00 . 24 250 1 66 . 0 1 77 7 25 . 6 . 8 1 6 . 1 0 2 . 4 3 • 1 9 2565 36 . 6 1 1 6 6 1 3 . 7 . 8 1 " 6 . 85 2 . 32 . 22 4309 60 . 5 1 4 1 6 1 3 . 1 0 . 8 1 6 . 84 4 . 74 . 36 1 668 49 . 0 1 27 1 0 20 . 1 1 . 8 1 4 . 87 4 . 54 . 2 1 367 28 . 7 57 7 1 0 . 1 2 . 8 1 7 . 69 2 . 98 . 32 344 59 . 7 46 8 3 . 3 . 82 8 . 93 2 . 83 . 29 422 56 . 4 98 8 25 . 5 . 82 7 . 1 0 2 . 06 . 23 545 56. 6 1 1 2 6 1 5 . 6 . 82 9 . 30 4 . 72 . 28 1 45 1 52 . 5 1 03 9 1 6 . 7 . 82 7 . 4 9 4 . 69 . 26 1 67 1 42 . 0 8 1 8 22 . 1 0 . 82 1 1 • 1 3 3 . 1 5 . 34 2877 54 . 5 1 4 1 9 Mean 7 . 6 1 3 . 3 1 . 27 1 704 5 1 . 1 1 09 7 . 6 S/McL is the estimated copper content o f pasture to meet dietary requirements ( Suttle & McLauchlan formula ) . Table 5 . 2 : Mineral content of pastures taken from area 2 on the copper deficient farm . s pp m · Mo pp m Cu pp m 4000 0 1 0 0 --- - Area 1 (Mill Block) Area 2 (Wal l ies Block) /--- - - - - -- """? - - -... -..... _J ..... --/ -- - .... 1 05 I' J J A S 0 N D J F M A M J J A S 0 N Period of Year Figure 5 . 3 : Copper , molybdenum and sulphur cont ent of · pas tures on the t\vo areas o f the copper deficient farm · · ..... Zn pp m Mn pp m Fe pp m Cu pp m 1 00 0 400 0 5000 0 1 0 0 Area 1 (Mil l Block) Area 2 (Wallies Block) ,- - - - -, ..... ... , \ \ - - ­ , � - \ \ \ \ :;.-, "" " ,"" ' .,., ' .,., ' , ... ' I '• ' I ',r J J A S O N D J F M A M J J A S O N Period of Year 106 Figure 5 . 4 : Copper , iron , zinc and manganes e content of pas tures dn · th e two areas o f the _copper . 'deficient farm 1 07 The major d ifference between the pasture mineral content of the two areas of farm A is the concentration of the soil-contaminating elements ( iron , zinc , and manganese ) ( Figure 5 . 4 ) ; elements which may inhibit the uptake of copper by the animal . On area 2 the concentrations of these elements were higher than those on area 1 , at all times of the year . Over the winter-early spring period , the concentrations of these elements in pasture rose to almost three times the content of the same elements in pasture from area 1 . By contrast , the copper sufficient farm provided adequate copper ( Suttle & Mclauchlan , 1 97 6 ) throughout most of the year , with a mean pasture content of 8 . 97 ppm D .M . ( Table 5 . 3 ) . Molybdenum concentrations . . recorded were below 1 . 3 1 ppm D .M . , and the sulphur levels averaged . 2 3% D .M . Iron , zinc , and manganese had mean concentrations o f 1 p62 , 52 . 3 , and 1 3 1 ppm D . M . respectively . Their influence on copper uptake was not knovm but probably at the concentrations recorded they would not influence the copper absorption significantly . There were no significant differences between bodyweights o f the copper treated and the control groups of animals ( Tables 5 . 4 and 5 . 5 ) . On both farms the copper-treated sheep had a higher mean live r copper concentration than the untreated sheep ( Tables 5 . 6 and 5 . 7 ) . There was some variability in the means of the liver copper concentrations due to individual animal variation , as on each occasion a random selection of sheep from each group was used . On the copper deficient farm the liver copper levels fell rapidly during the winter , but by early summer the difference between the treated and the untreated groups was not great ; 84 ppm and 67 ppm Cu D .M . respectively . However on the copper sufficient farm the liver copper levels showed little variation throughout the year . Also on this farm the copper treated sheep did have an overall higher liver copper concentration initially , and they maintained this higher concentration throughout the year . Dates Cu ppm 8 . 1 0 . 8 1 7 . 1 9 { 1 4 . 1 1 6 . 99 4 . 1 1 • 8 1 8 . 59 { 6 . 84 7 . 70 2 1 . 1 . 82 { 9 . 22 2 . 78 3 . 09 { 1 4 . 69 9 . 6 . 82 1 1 . 36 1 5 . 1 0 Mean 8 . 97 Mo ppm . 53 1 . 23 • 59 . 74 . 76 . 10 . 42 . 2 1 • 1 1 . 28 1 . 3 1 1 . 00 . 66 S/McL is the estimated dietary requirements s Fe Zn Mn S/McL % pp m ppm pp m ppm . 28 574 36 . 5 203 6 . 1 2 6407 38 . 5 223 5 . 24 7 1 2 38 . 0 1 85 6 • 1 8 1 93 1 34 . 9 1 22 5 . 22 808 40 . 9 1 3 1 6 . 2 1 1 053 82 . 7 1 60 6 . 2 1 5 1 7 7 1 . 3 73 5 . 28 552 28 . 2 76 6 . 24 204 37 . 3 59 5 . 3 1 3006 94 . 4 93 5 • 1 9 2534 66 . 1 1 59 6 . 27 1 645 59 . 4 85 7 . 23 1 662 52 . 3 1 3 1 5 . 7 copper content of pasture to meet ( Suttle & McLauchlan formula ) . Table 5 . 3 : Mineral content o� pastures taken from the copper sufficient farm . 1 08 Bodyweight : kg Hoggets Two Year Old Mi xe9 Age Dates No Cu Cu No Cu Cu No Cu Cu 22 . 4 . 8 1 2 1 . 5 . 8 1 1 0 . 8 . 8 1 20 . 1 1 . 8 1 1 0 . 1 2 . 8 1 29 . 0 29 . 8 ;tO . 65 ;tO . 7 1 32 . 6 33 . 6 ±0 . 63 ;t0 . 69 36 . 2 36. 0 ±0 . 62 ±0 . 67 39 . 9 39 . 5 ±0 . 57 ±0 . 6 1 26 . 5 . 82 49 . 4 49 . 8 ±0 . 67 ±0 . 9 1 8 . 1 2 . 82 50 . 0 49 . 4 ± 1 . 22 ± 1 . 07 Weight change 21 . 0 1 9 . 6 44 . 8 4 6 . 2 ;�:0 . 54 ±0 . 68 4 6 . 9 47 . 0 ±0 . 42 ±0 . 53 4 6 . 8 48 . 2 ±0 . 50 ±0 . 66 50 . 5 5 1 . 2 ±0 . 5 1 ±0 . 73 50 . 7 5 1 . 1 ±0 . 65 ±0 . 90 50 . 6 5 1 . 8 ±0 . 76 ±0 . 92 5 . 8 5 . 6 55 . 0 48 . 8 ±0 . 45 ±0 . 63 53 . 4 5 1 . 1 ;t0 . 46 ;�:0 . 48 53 . 6 5 2 . 4 ;t_0 . 49 ±0 . 52 57 . 8 5 6 . 1 ±0 . 57 ±0 . 63 55 . 7 54 . 5 ±0 . 66 ±D o 7 1 5 3 . 3 53 . 1 ±0 . 99 ±0 . 85 - 1 . 7 4 . 3 No Cu = untreated Table 5 . 4 : Bod yweights of sheep grazing the copper defic ient farm . Bodyweight : kg Two Year Old Dates No Cu Cu 1 7 . 1 2 . 8 1 54 . 0 52 . 3 1 0 . 2 . 82 59 . 4 58 . 2 5 . 5 . 82 56 . 5 56 . 0 2 9 . 1 1 . 82 55 . 1 52 . 2 Weight change 1 . 1 - 0 . 1 No Cu = untreated Table 5 . 5 : Bodyweights of sheep grazing the copper sufficient farm . 1 0 9 1 1 0 Liver copper content : ppm D .M . Hoggets Two Year Old Mi x ed Age Flock Date No Cu Cu No Cu Cu No Cu Cu No Cu Cu 22 . 4 . 8 1 58 1 76 1 76 37 37 77 77 ± 1 9 . 8 ±7 1 ±7 1 ±1 2 . 4 ± 1 2 . 4 2 1 . 5 . 8 1 99 1 65 264 87 1 34 1 1 4 1 60 ±75 ± 1 7 . 3 ±39 . 3 ±28 . 8 ±38 . 7 1 0 . 8 . 8 1 80 73 59 30 92 59 8 1 ±28 . 8 ±33 . 5 ± 1 9 . 0 ±8 . 2 ±40 . 7 1 4 . 1 0 . 8 1 65 2 1 0 2 4 83 67 84 ±30 . 1 ±33 . 2 .:t-9 . 8 ± 1 4 . 5 20 . 1 1 . 8 1 1 69 83 1 69 83 ±46 . 9 ± 1 4 . 7 1 0 . 1 2 . 8 1 25 3 7 22 3 1 22 ±2 . 7 ± 1 2 . 0 ±3 . 8 26 . 5 . 82 34 49 88 37 1 7 39 53 ± 1 7 . 1 ± 1 3 . 0 ±29 . 5 ±25 . 0 ±8 . 5 8 . 1 2 . 82 1 9 69 45 50 39 3 1 34 50 ±4 . 5 ±30 . 6 ± 1 4 . 0 ± 1 4 . 5 ± 1 1 . 0 ±6 . 5 Mean 55 69 1 28 90 42 70 62 77 No Cu = un treated = no sample Table 5 . 6 : Liver copper content o f sheep grazing the copper deficient farm . was no correlation between the blood copper content and the l iver copper content , on either farm . Those sheep supplemented with coppe r did not have higher blood concentrations , and there was n o difference in the blood copper concentra tion between those sheep grazing the copper sufficient farm and those sheep grazing the copper deficient farm . The blood parameters of packed cell volume , haemoglobin content , MCHC , and plasma pro tein stayed within normal l imits at all times and were not affec ted by the liver copper status of the animal , or the farm on which the sheep grazed . Liver Dates 4 . 1 1 . 8 1 1 7 . 1 2 . 8 1 1 0 . 2 . 8 1 5 . 5 . 82 29 . 1 1 . 82 Mean coppe r : ppm D .M . Two Year Old No Cu Cu 33 1 . 429 530 480 627 627 530 553 367 440 5 1 4 No Cu = untreated = no sample Table 5 . 7 : Liver copper content of sheep grazing the copper sufficient farm . 1 1 1 Of the forty fleeces weighed on the copper sufficient farm , the copper treated sheep had a mean fleece weight of 4 . 1 5 kg , compared to the control sheep which averaged 4 . 2 9 kg . This difference was not s ignificant . DISCUSSION Due to the high organic matter content and the high clay content of the soil , the amount of soil copper extracted using 2N hydrochloric acid bears little relationship to the total amount of copper present in pasture ( Robson & Reuter , 1 98 1 ) . The data recorded on these farms support this belief , particularly on farm B, the copper deficient farm . However on the coppel' sufficient farm the correlation between extractable soil copper and pasture copper content was surprisingly high (r = 0 . 9 94 ) . This high correlation is probably due to· the nature of the soil on this farm . The soil is yellow brown sand which is low in both organic matter and in clay 1 1 2 content . Such soils are known to have more freely available copper i . e . in an unbound state ( Thornton , 1 979 ; James & Burrow, 1 981 ) . However , in the light of the mineral analysis of pasture , the reason for the pr�sence of copper deficient animals on farm A ( the copper deficient farm) and not on farm B ( the copper sufficient farm) appears more obvious . The copper sufficient farm provided adequate concentrations of pasture copper throughout the year ( as judged by the Suttle & McLauchlan formula , 1 976 ) . There were also lower levels present of those elements known to . inhibit the absorption of copper . On farm B , the other elements which are believed to inhibit copper uptake , namely , iron , zinc , and manganese were all at low levels in .. the pasture throughout the year . However according to the work by Mills , ( 1 980 ) ; . Reynolds , ( 1 979 ) , and Grace , ( 1 973 ) , iron, zinc , and manganese probably had little effect on copper absorption . Applying this data to the graph of Cornforth ( 1 980 ) from the formula of Suttle & McLauchlan ( 1 976 ) , in only two paddocks was the pasture unable to provide adequate copper for sheep ( Table 5 . 3 ) . On the copper deficient farm there was considerable variation in the mineral content of pasture between paddocks . On area 1 (Mill Block ) , the pasture copper concentration was adequate throughout the year ( 3 . 68 - 8 . 9 1 ppm D .M . ) . Further , the inhibiting elements remained low throughout the year , except for molybdenum which rose in late spring in one year and in winter the following year ( Figure 5 . 3 ) . When this data is e xamined in the light of the Cornforth graph , 77% of the grazed area was able to supply adequate pasture copper for sheep . Area 2 of farm A , known as Wallies Block , had low copper concentrations in pasture throughout the year . These dropped to their lowest concentrations during winter , while conversely the molybdenum and sulphur concentrations rose to their highest during this period . The other elements which depress copper absorption by animals were also at their highest level during the winter . This is probably the result of soil contamination of this area ; 50% of which 1 1 3 faces south . This aspect , together with the high rainfall and heavy continual grazing generated the mud which caused pasture contamination . Again if the Cornforth modification of the Suttle & MacLachlan formula is applied to this data , then only 34% of these paddocks were able to supply adequate copper for sheep . Unfortunately this is the area of the farm on which the main ewe - flock was wintered . At this time of the year the demand for copper by the ewe is highest (Grace , 1 983 ) and the availability of copper the lowest . Further these paddocks species of low digestibility which availability o f absorbable copper . are large and contain grass is likely to further lower the The concentration o f copper relative to the concentration of the other inhibiting elements on the copper deficient farm suggests an explanation for the regular appearance of swayback lambs and the signs of copper deficiency in the cattle . It must also be noted that cattle have a 50% higher requirement than sheep for copper in pasture ( Suttle , 1 98 1 b ) . However from the data collected from the copper deficient farm ( Tables 5 . 1 , 5 . 2 , & 5 . 6 ) , it does appear that an annual supplementation of 50 mg of copper as copper calcium edetate was sufficient to prevent the appearance of copper deficiency in sheep . These data also confirm the importance of knowing the levels o f pasture copper and other minerals o n various parts of the farm where copper deficiency exists . With such information greater utilization o f copper sufficient areas may be made during the periods when the copper requirements of grazing animals are high . There was no significant difference in the bodyweights between the treated and Wltreated sheep on each farm , and between the sufficient and deficient farms . This reaffirms that \-Teight gain response trials are probably of little value in assessing copper status of animals . Only Hogan et al . ( 1 97 1 ) , Whitelaw et al . ( 1 979 ) , and Whitelaw et al . ( 1 980 ) have been able to measure a s ignificant weight response to copper · supplementation . However the latter two 1 1 4 trials were carried out using lambs , and the first trial was with sheep on a diet which was very high in molybdenum . No s ignificant reduction in bodyweights of sheep was found on the copper deficient farm even though some of the non-treated ewes produced lambs that subsequently developed enzootic ataxia . Whitelaw et al . ( 1 979) also noted significant differences in fleece structure and in wool characteristics in their trial . However in the trial on the copper sufficient farm there was no difference in either of these two features or in the weight of wool shorn .from each ewe . None of the sheep showed signs of copper deficiency or had liver or blood tissues which had low concentrations of copper at any .. stage of the trial . The liver copper content of the sheep on the respective farms was different . On the copper deficient farm the liver copper concentrations fell during the winter , rose slightly during the summer , only to fall again during the next winter . On this farm , liver copper concentrations declined rapidly after treatment , but maintained a slightly higher concentration throughout the rest of the year in comparison with the control animals . Obviously during the winter there is a very high demand for stored live r copper to maintain copper homeostasis . This is due in part to the higher demand for copper in late pregnancy ( Suttle , 1 98 1 b ) , and from the low availability of pasture copper , due to the inhibiting factors already discussed . The degree of absorption of copper from the diet can be calculated from the results of the pasture analysis , the liver analysis of the untreated animals on both farms , and from assuming that the copper requirements for a ewe for maintenance are 0 . 0 1 mg Cu/kg bodyweight/day (Grace, 1 983 ) . · Using the criteria above , on the copper sufficient farm , 4 . 7% of dietary copper was absorbed , whereas on the copper deficient farm 6 . 4% of dietary copper was absorbed . The copper deficient farm had pasture high in inhibiting elements which affected the absorption and retention of pasture copper . The 1 1 5 sheep on the copper sufficient farm however , had high liver copper l evels and maintained these at a relatively constant level . In addition , this latter farm had pastures of adequate copper levels and a lower content of inhibiting elements . This difference in intestinal absorption of copper would suggest that sheep are able to control their copper absorption , which supports the work of Neethling et al . , ( 1 968 ) , and of Hill et al . ( 1 969 ) . A mechanism may operate whereby the animal is able to control the absorption of intestinal copper , regulate the excretion of copper , or control the function o f both processes . The haematological results were similar to those reported for , normal sheep . In addition , the blood copper concentration was within the normal range ; the sheep which were treated with parenteral copper had liver copper concentrations similar to those found in the untreated sheep . In all cases the blood copper concentrations bore no relationship to the storage concentrations of copper in the liver . This lack of correlation suggests that unless an animal is hypocupraemic , then blood samples are of little value in assessing its copper reserves . In the sheep which regularly grazed farms A and B , there w�re marked differences in the liver storage concentrations of copper . On farm B , which was copper sufficient , the sheep maintained high liver concentrations of copper throughout the year ( 33 1 - 627 ppm D . M . ) . Furthermore , on this farm the elements which may have prevented the uptake and retention of copper by the animals were at low concentrations in the pasture . In contrast , the liver copper concentrations of the sheep grazing the copper deficient farm showed a substantial variation throughout the year . The lowest liver copper concentration occurred in the winter when pasture copper concentrations were low and the content of inhibiting elements high . This suggests that the measurement of copper requirements for animals may be evaluated most e ffectively in the winter , particularly as this is a time when advice on copper supplementation may be required . 1 1 6 It would appear that on a copper sufficient farm , similar to the one studied in this experiment , the copper status assessed from the animals and the plants , may be confirmed at any time of the year . Ho�ever on a potentially copper deficient farm , it is important to know the status of the animals prior to the period of greatest demand , that is , the winter . Assessment of liver copper stores , and of pasture mineral content , will ass ist in making a decision about any need for copper supplementation at this time of the year . The supplementation of the sheep on the copper deficient farm with 50 mg of copper , as copper calcium edetate , was effective in elevating liver copper concentrations immediately after injection , . . although these concentrations steadily decreased during the ensuing year . The timing of the injection was important as it ensured that the animals had high copper stores during the period o f greatest copper demand and lowest dietary copper availability . There were no lambs with enzootic ataxia born to the ewes supplemented with coppe r . However there were three known cases o f swayback in lambs born to the untreated ewes . Conversely , in the ewes grazing the copper sufficient farm and injected with 50 mg of copper as copper calcium edetate , . the liver copper stores rose immediately after the injection and remained at this elevated level throughout the duration of the experiment . CONCLUSION Regular monitoring of the mineral content of pastures copper concentrations of animals using liver biopsies , understanding the dynamic status of copper on that farm . information , supplementation procedures can be considered . and the helps in From this Liveweight changes in response to copper supplementation are not an effective method of diagnosing copper deficiency . Similarly blood copper concentrations do not indicate how adequate are the copper s tores of the animal . 1 1 7 Supplementation of sheep with 50 mg of copper as copper calcium edetate was effective in elevating live r copper storage leve ls . It is important that the liver copper stores are elevated prior to the periods of high copper demand . ·., 1 1 8 CHAPTER 6 : THE DIFFERENCES IN THE ABSORPTION OF COPPER AMONGST FOUR DIFFERENT BREEDS OF SHEEP IN NEW ZEALAND . INTRODUCTION There are considerable differences between breeds of sheep in their ability to absorb copper from the diet . This interesting phenomenon has been reported by several workers , experimenting with British breeds of sheep (Wiener & Field ( 1 969 ) , Wiener et a l . ( 1 969 ) , Wiener & Field ( 1 970 ) , Wiener , Herbert & Field ( 1 976 ) , Wiener , Wilmut & Field ( 1 978 ) , and Herbert , Wiener & Field ( 1 978 ) ) . Luke & Weirman ( 1 970) and van der Berg et al . ( 1 9 83 ) have also shown differences between the European breeds of sheep . Any variation between breeds diminishes as the molybdenum content of the diet increases ( Suttle , 1 98 1 b ) , and it would seem the rumen is the site at which interference in copper absorption occurs (Wiener , 1 980 ) . No such comparative work on the uptake and retention of copper has been done using the breeds of sheep commonly farmed in New Zealand . In order to establish whether any significant differences in copper absorption by sheep of different breeds occurred in New Zealand , four breeds o f sheep were selected . All of these have played a major role in our sheep industry . Each of these breeds was considered to be "genetically dive rse" , according to the thes is of Ryder ( 1 964 ) that proposed the probable lines of evolution o f British breeds of sheep ( Figure 6 . 1 ) . The four breeds selected were the Border Leicester , Merino , New Zealand Romney , and Suffolk . The aim of the experiment was to reduce liver copper reserves of the sheep to a stage when the majority had copper concentrations below 1 00 ppm Cu D .M . The sheep were then grazed on a farm where the availability of copper in the pasture was known to be high . In fact , the farm that was selected had a history of copper toxicity in sheep on several occasions . Following grazing on this farm to allow significant increases in liver copper content (approximately three months ) , each sheep was dosed with copper parenterally and the liver She t land Scot·s B lackface Cheviot ' . B�der We�s �eyd� / ' Dartmoor & Devon V ees� / 1 1 9 Leice�t� r T ter� � Leices ter 1 . 1 t �0 n Northe rn Black-faced Midland Lotgwoo l Breeds Nedieval_�R _, omney Welsh -----------"1>- Radnor Hountain J. /f � d Cot swold Cl . Longwlo� < /.. . Ryel"an Ox�rd Devon Ker Y. hropsh ).,re,. r ff�k . �-.� �- ... .. Norfolk Hereford • • • · .+ .•• South mm HaFE�l fe · · · . • "-. Dorse"'! Be rkshire W�i re South\ves t T Wh . t d ...._____ an or 1 e Horne -� Face d . Horn�� ' . · Down ·. . . . · . . . . . . . . ·.· . . . ·. . . . . . Exmoor Dorset • . J. Horn • . Hainly Horn- Closewool less Whi te- B lack-Faced Horned So�y Face Figure 6 . 1 : Probab l e origins and relat ionships of British b re eds (Ryder , 1964) copper content was measured before and after each dose . The increase in liver copper content was then a measure of the copper retained by that particular sheep , and enabled comparisons to be made between breeds . MATERIALS AND METHODS The sheep used in this experiment included four four-year-old Border Leicester ewes , five three-year-old Merino ewes , four three-year-old Romney ewes , and four four-year-old Suffolk ewes . At the commencement of the experiment a liver biopsy was collected from each sheep . content ( Appendix II ) . These samples were analysed for copper 1 20 During the stage o f liver copper depletion , all the sheep were housed and fed on a diet of hay containing 6 . 2 ppm D .M . o f copper , and allowed free access to water . All sheep were dosed daily with 2 0 ml of a solution of . ammonium molybdate and sodium sulphate , containing 50 mg molybdenum and 450 mg of sulphur , to help reduce the liver copper reserves ( Suttle & Field , 1 968b ) . This treatment continued for two months after which time the second liver sample was taken . From the results o f the analyses of the copper concentration o f the second liver biopsy , the sheep were divided into two groups . Those sheep with live r copper levels below 1 50 ppm D .M . were . maintained on the hay diet and the daily dosing with ammonium molybdate and sodium sulphate was continued . The remaining sheep with liver copper concentrations above 200 ppm D .M . were given a low copper diet similar to that described by Suttle & Field ( 1 968b ) ( Table 6 . 1 ) . On chemical analysis this diet contained 1 . 3 ppm Cu D .M . Each sheep received a daily allowance of 1 . 0 kg of this basal low copper diet and in addition received a daily dose of 20 ml of the solution containing ammonium molybdate and sodium sulphate . This treatment of the two groups of sheep continued for 8 weeks . When the third series of liver samples found that instead of the anticipated were analysed , decrease in it was copper concentrations , the latter had actually risen . At explanation could be given , but the possibility absorbed from bedding straw eaten by these sheep , or content in the drinking water , could not be excluded . this stage no of copper being a high copper In an attempt to overcome this lack of liver depletion , all sheep were moved , and grazed on a known copper deficient farm for s ix months . This grazing period was from June to December . At the end of this time liver biopsy samples from each sheep were analysed for copper content and were found to have fallen below 1 50 ppm D . M . in Whole oats 20 % Oat husks 1 7 % Starch 1 7 % Sucrose 1 7 % Dried skim milk powder 1 6 % Urea 2 % Peanut oil 2 . 5 % Potassium b icarbonate 2 % Sodium chloride 1 . 6 % Magnesium oxide 0 . 5 % Vitamin A 1 000 i . u . /kg Vitamin D 1 4 0 i . u . /kg Vitamin E 36 i . u . /kg Iron 1 00 mg/kg Manganese 50 mg/kg Zinc 20 mg/kg Iodine 8 mg/kg Cobalt 2 mg/kg Daily allowance 1 . 0 kg/sheep/day Table 6 . 1 : Composition of basal low copper diet ( Suttle � Field , 1 968b ) 1 2 1 all except three o f the sheep . At this stage of the study , depletion of liver copper was considered to have reached a sufficiently low level to act as a baseline for subsequent copper supplementation and · estimations of uptake . All the sheep were ·then grazed · on a farm of known high copper status , where deaths from copper poisoning had previously occurred . These sheep remained on this farm for four months before a fifth liver biopsy was taken and analysed . At this time the sheep were returned to Massey University . 1 2 2 Copper calcium edetatel : , at a rate of 1 mg/kg bodyweight was then injected into each sheep . The sheep were grazed on pasture containing 6 . 6 ppm D .M . of copper and after two weeks liver biopsy samples were again taken for copper estimations . At the time of each liver biopsy , wool samples were collected after shaving over the biopsy site . The samples were cleaned , and analysed for copper content . Sheep bodyweights were also recorded at the time of each liver biopsy . While the sheep were on the two farms , pasture samples were collected monthly and analysed for mineral content . For the calculation of the absorption o f copper from the diet it was assumed that the requirements for copper are 0 . 0 1 mg/kg bodyweight/day for maintenance , and 4 mg for each kilogram increase in bodyweight (Grace , 1 9 83) . It was also assumed that any loss o f copper from liver was used i n general metabolism and any increase in liver copper was from a dietary source . Liver mass was estimated to be 1 . 1 3% of to tal bodyweight and contained 2 9 . 4% as dry matter ( van Ryssen , 1 980 ) , and the estimated daily intake of pasture was 1 . 2 kg D .M . /day and of the synthetic diet 0 . 9 kg D .M . /day . The equation used to calculate the percentage absorption o f copper from the d iet was : absorption = mx ( b+f )/2x . 0 1 + ( f-b )x4 + { ( exf)- (axb ) } 1 . 1 3%x29 . 4% x 1 00 m X y X Z 1 Coprin Multidos e , Glaxo (N . Z . ) Ltd . where a = mean live r copper content at start ( ppm D .M . ) b = mean bodyweight at start ( kgs ) e = mean liver copper content at end ( ppm D .M . ) f = mean bodyweight at end (kgs ) m = no . of days on diet y = copper content of diet ( ppm D .M . ) z = feed intake (kg D . M . /day ) 1 23 An example of the calculation for the absorption of copper from the copper high pasture is set out below : - Mean liver copper at start = 86 ppm Mean bodyweight at start = 60 . 2 kg Total liver copper at start = 86 X 60 . 2 X 1 . 1 3 % X 2 9 . 4 % = 1 7 . 2 mg Mean liver copper at end = 289 ppm Mean bodyweight at end = 64 . 9 kg Total liver copper at end = 289 X 64 . 9 X 1 . 1 3 % X 29 . 4 % = 62 . 1 mg Copper increase stored in liver = 62 . 1 - 1 7 . 2 = 4 4 . 9 mg Copper requirements Dietary intake = 1 1 2 days x + 4 . 7 kg X + 44 . 9 mg = 1 33 . 6 mg 62 . 45 kg x . 0 1 metabolism 4 mg weight increase increased liver store = 1 . 2 kg x 1 1 2 days x 6 . 9 ppm = 927 . 4 mg % absorbed from diet = 1 33 . 6/927 . 4 = 1 4 . 3% 1 24 The F...:ratio applied to an analysis of variance one-way command was the test used to determine the s ignificance of differences . RESULTS AND DISCUSSION The changes in bodyweight that occurred were directly related to food intake ( Table 6 . 3 ) . On the two farms the sheep were give n access to surplus pasture . The one Merino that lost weight o n the high copper pasture farm became infected with foot-rot . All sheep lost weight when on the synthetic diet as it was no.t highly palatable . The analyses of the diets used throughout these experiments are shown in Table 6 . 4 . The straw and water were analysed as the sheep had access to these during the depletion phase , but the analysis showed their copper content was not sufficiently high to affect significantly the to tal . dietary copper content . The results of the wool analyses are discussed in Chapter 1 . On the initial hay diet , containing 6 . 4 ( Table 6 . 4 ) , the liver copper content of ppm D .M . of most sheep copper , actually increased . Only the Merinos showed a significant decrease . The supplementation with ammonium molybdate and sodium sulphate appeared to have little effect on copper uptake . However these sheep were on a hay diet , and according to Suttle ( 1 98 1 a) 7 . 2% of dietary copper is absorbed from hay in comparison with 2 . 3% from summer pasture . The analyses of l iver biopsy samples are given in Table 6 . 2 . The increase in live r copper content for sheep on the basal low copper diet was in direct contrast to the findings of Suttle & Field , ( 1 968b ) , yet analysis of the diet showed s imilar levels in both cases , namely , 1 . 3 ppm Cu D .M . From the analysis , straw and water contained insufficient copper to have any influence on the increased storage levels of copper . The only. apparent difference in the diets , was that the diet used by Suttle & Field was pelleted , while the diet in this experiment was fed as a powder . This difference in feed 1 25 Liver copper concentration ppm D . M . Change Change Breed Sheep Hay Basal De f . onhigh Pasture fromCu no . 1 2 3 4 copper 5 6 inject 2 1 1 9 1 233 370 36 +282 3 1 8 867 +54 9 Border 22 39 < 1 2 89 2 1 + 1 42 1 63 509 +34 6 Leicester23 355 347 5 1 1 55 +276 33 1 6 1 8 +287 24 1 38 428 653 1 38 Mean +233 +394 31 1 0 1 1 2 9 1 6 1 76 +284 360 671 +3 1 1 Suffolk 38 1 1 2 293 327 1 98 + 1 58 356 650 +294 39 1 54 289 40 256 363 557 Mean +22 1 +302 5 1 294 499 670 1 47 + 1 4 1 288 N . Z . 52 1 8 5 488 606 264 +272 536 1 1 49 +6 1 3 Romney 53 1 40 4 1 4 738 1 64 +3 1 2 476 55 1 +75 54 30 22 80 1 5 + 1 28 1 4 3 34 1 + 1 98 Mean +237 +29 5 63 1 50 < 1 2 200 1 27 64 2 9 < 1 2 < 1 2 1 5 +4 1 56 293 +237 Merino 65 1 4 9 1 4 5 1 11 1 4 + 1 34 1 4 8 340 + 1 92 66 24 1 246 3 1 4 67 2 9 < 1 2 60 Mean. +88 +2 1 5 Basal = Basal low copper diet De f . = ' Copper deficient ' farm Table 6 . 2 : Copper content of liver samples from different breeds of sheep given four different diets , followed by parenterally administered copper ; and the change in 1i ver copper concentration while on the low and high (!opper regimes . 1 26 Bodyweight and weight changes : kgs . Hay Basal Both Breed Sheep Weight Diet Weight Diet Weight Farms rle igh t no . 1 Change 2 Change 3 Change 4 2 1 5 1 . 4 +6 . 3 57 . 7 -5 . 7 5 1 . 8 . + 1 6 . 4 6 8 . 2 Border 22 58 . 6 +3 . 7 62 . 3 +1 . 3 63 . 6 +9 . 6 73 . 2 Leicester23 4 6 . 8 +4 . 6 5 1 . 4 -7 . 3 4 4 . 1 + 1 4 . 1 58 . 2 24 5 1 . 4 +3 . 1 54 . 5 - 8 . 1 46 . 4 Mean +4 . 4 - 5 . 0 + 1 3 . 4 . .._ 37 50 . 0 +3 . 2 5 3 . 2 -7 . 3 45 . 9 +28 . 6 74 . 5 Suffolk 38 47 . 3 +5 . 9 53 . 2 -9 . 6 43 . 6 +28 . 7 72 . 3 39 4 3 . 2 + 1 5 . 0 58 . 2 40 4 5 . 5 +8 . 1 53 . 6 -7 . 2 46 . 4 Mean +8 . 1 - 8 . 0 +28 . 7 - 5 1 59 . 5 -2 . 7 56 . 8 - 9 . 1 4 7 . 7 N . Z . 52 7 4 . 1. -8 . 6 65 . 5 -7 . 8 57 . 7 +20 . 5 78 . 2 Romney 53 6 0 . 0 -0 . 5 59 . 5 -8 . 1 5 1 . 4 + 1 5 . 9 67 . 3 54 62 . 7 - 9 . 1 5 3 . 6 -2 .2 5 1 . 4 +9 . 5 60 . 9 Mean - 5 . 2 -6 . 8 + 1 5 . 3 63 4 2 . 3 -5 . 9 36 . 4 36 . 4 64 5 1 . 5 + 1 . 2 52 . 7 - 7 . 2 45 . 5 - 1 . 4 4 4 . 1 Merino 65 4 5 . 7 - 0 . 2 45 . 5 - 1 . 9 43 . 6 +6 . 4 50 . 0 66 46 . 1 - 1 . 6 4 4 . 5 - 1 0 . 0 34 . 5 67 4 5 . 8 - 1 . 3 44 . 5 -2 . 2 42 . 3 Mean - 1 . 6 - 4 . 3 +2 . 5 Table 6 . 3 : Bodyweights and bodyweight changes of different breeds o f sheep when eating different diets . 1 2 7 Diet Cu Mo s Fe Zn Mn pp m ppm % pp m pp m pp m Hay 6 . 4 0 . 8 . 1 4 30 1 9 5 5 Basal low diet 1 . 3 <0 . 2 . 07 40 25 48 Bedding straw 2 . 1 < 0 . 2 ·• 1 8 24 33 1 1 Water <0 . 0 1 tr 0 . 6 0 . 1 0 . 03 Copper deficient pasture 9 . 3 4 . 2 . 29 2000 49 1 08 High copper· pasture 6 . 9 1 . 1 . 22 281 2 1 4 0 Massey pasture 6 . 6 0 . 9 • 36 1 075 4 5 8 3 Table 6 . 4 : Mineral content o f respective diets used . texture appears to be the factor that could have caused the variation in absorption of copper for the following reasons . Inh ibition of copper absorption occurs in the rumen ( Suttle , 1 98 1c ; Whitelaw et al . , 1 982) . The passage o f dietary contents through to .the reticule-omasum from the rumen is dependent on particle s ize . Those particles of diameter less than 0 . 6 mm apparently pass straight through to the reticula-omasum ( Ellis et al . , 1 979 ) and on to the abomasum. The majority of the particles in the synthetic diet we re less than 0 . 6 mm , thus the rapid passage of ingesta would leave little opportunity for copper ions to be rendered insoluble in the rumen by the thiomolybdate complexes . The absorption of copper from the three major diets , namely , the synthetic diet , the ' copper deficient ' pasture , and the ' high copper ' pasture , was calculated according to the formula described previously , and by using the data from Tables 6 . 2 , 6 . 3 , and 6 . 4 . These calculations gave the following absorption rates :- synthetic diet copper deficient pasture high coppe r pasture 76 . 6 % 4 . 8 % 1 4 . 3 '}. 1 28 The difference in absorption rates of copper from the pasture between the copper deficient farm and the high copper farm was substantial . The copper deficient farm had pasture of a higher copper content ( 9 . 3 vs 6 . 9 ) , but also it contained higher levels of the copper inhibiting elements , molybdenum , sulphur , iron , zinc and manganese . By applying the Cornforth modification of the Suttle & McLauchlan formula to these pastures , using copper , molybdenum and sulphur only ; both diets would appear to have provided adequate copper . However , the mean differences between the two farms for iron , zinc and manganese were great ; viz . , 2000 , 40 and 1 08 ppm respectively for the copper deficient farm , and 281 , 2 1 and 40 ppm respectively for the high copper pasture farm . These minerals then may still have had a contributory effect . There was also a considerable difference in _the pasture quality between the two farms . The copper deficient farm had pasture consisting of poor quality species of low digestibility , for example , brown top , danthonia , and crested dogstail , whereas the pasture on the high copper farm contained species of higher digestibility , especially perennial ryegrass and white clover . The pasture of low digestibility would have spent a greater amount of time in the rumen until it was broken down into particles sufficiently small to pass on to the reticule-omasum (Kay , 1 983 ) . This l onger period of time may have allowed a greater opportunity for the inhibiting elements to be effective in restricting the uptake of copper . The feeding o f the synthetic diet resulted in a calculated absorption of approximately 76 . 6% of the copper . This absorption rate is very high and approximates that of the pre-ruminant lamb ( Suttle , 1 98 1 b ) . This similarity - would suggest that the components of this diet either spent a very brief period of digestion in the rumen or that they bypassed the rumen almost completely . 1 29 The differences in the absorption and retention of copper measured in this experiment justify its repetition and extension , using a greater number o f animals , in an attempt to verify the preliminary results . There was no s ignificant difference between the three British breeds i . e . the Border Leicester , N . Z . Romney , and Suffolk ( Table 6 . 2 ) when considering the uptake of copper from the high copper pasture diet , and also the liver retention of copper after the parenteral supplementation with coppe r . The lack o f breed d ifferences may be supported from field work in New Zealand where investigations into clinical enzootic ataxia have never implicated a particular breed . The Merino , howeve r , differs from the three British breeds . The uptake of dietary copper was significantly lower ( P < 0 . 05 ) in the Merino , and also the retention in the liver of parente rally administered copper was lower than for the other three breeds used in this study . The lower absorption and hepatic storage of copper suggests that this may be associated with the higher incidence o f ' steely wool ' i n Merino fleeces (Marston , 1 9 55 ) , compared to the British breeds grazing the same pastures . Edgar et al . , ( 1 94 1 ) , also found that Merinos were less susceptible than British breeds of sheep to copper toxicoses as they accumulate less copper in the live r . The original work b y Wiener e t al . , ( 1 9 69 ) , which was concerned with the absorption o f copper from the diet , indicated a difference between breeds in plasma copper concentration , but on intravenous repletion of similar sheep they found no differences between breeds . Herbert et al . , ( 1 97 8 ) , studied the liver retention of copper absorbed from the diet and found breed differences and concluded that the difference was in the absorption of copper from the diet and not as a result of selective grazing . The breeds of sheep used in the experiments especially the North Ronaldsay , Welsh Mountain, and Britain , and the Texel and the White-headed Mutton sheep in Europe , Cheviot in in Europe , have been in almost complete isolation for many years , and in some 1 30 cases centuries , and have adapted to a particular habitat . The North Ronaldsay , for example, has become a very efficient absorber o f copper when its principal diet for nine months o f the year is seaweed . Herbert et al . , ( 1 978 ) , suggest that seaweeds may have a high sulphur content which will inhibit copper absorption , but analys is of twelve varieties of seaweed found on the New Zealand coast showed a copper content of 0 . 6-0 . 9 ppm D .M . ( Farquharson , unpublished data ) , which would suggest that the North Ronaldsay may have become very efficient at absorbing copper due to the low copper content of the diet . The three British breeds of sheep used in this experiment have all been farmed in New Zealand for over 1 00 years , and have shared the same environment and had access to a similar diet . Therefore it is not surprising that there is no significant difference in their absorption rates of copper from the d iet . The Merino breed however has been adapted to a drier environment and in New Zealand is mainly confined to the South Island high country . This is a distinctive environment which may provide sufficient copper to meet dietary requirements for Merinos , so that Merinos do not need to be as e fficient in the absorption of copper as other breeds of sheep , which generally graze a different type of pasture . Although the differences in the absorption of copper between the Merino and the British breeds of sheep may be explained by environmental adaptation, this cannot be used to explain the suspected difference in liver uptake of copper after parenteral copper supplementation . As the Merinos appeared to have accumulated less copper from both the diet and from parenteral copper , it is suggested that two mechanisms could be involved . Either , the Merino has a higher metabolic requirement for copper , or it has a higher e xcretory rate of copper from the liver , or both mechanisms may operate . Woolliams et a l . , ( 1 983 ) , suggested that breed variation in copper requirements was the result o f the difference in the rate of loss of endogenous coppe r . 1 3 1 From the results obtained in this experiment , it would appear that there is little merit in changing the breed of sheep , when farming British breeds , to help alleviate potential copper deficiency . In this experiment insufficient animals were present to establish whether differences in the absorption of coppe r , between breeds of shee p , did exis t , although a difference between the Merino and the British breeds of sheep was suggested . Therefore it would be appropriate to repeat this experiment using greater numbers of animals to confirm these findings . It would also be important to treat similar groups of animals with parenteral copper to determine ._ whether there is any breed difference in the retention of copper in the liver storage cells , in addition to a poss ible diference in the intestinal absorption of copper . CONCLUSION There appears to be some difference in copper pharmacokinetics between the Merino breed of sheep and the Bri tish breeds of Border Leicester , N . Z . Romney , and Suffolk . The difference is probably associated with the absorption rate of copper from the diet , as well as the retention of copper in the liver following the administration of copper parenterally . CHAPTER 7 : COPPER CONTENT OF WOOL FROM SHEEP OF KNOWN COPPER STATUS I NTRODUCTION 1 32 Copper is essential for the growth and formation of crimp in the wool fibre (Underwood , 1 97 1 ) . It is required in the keratinization process , when the cross-linking of amino-acid disulphide groups form to produce keratin ( Burley & de Kock , 1 957 ; Kapoor et al . , 1 972 ) . The enzyme required in this process is cytochrome oxidase (Gillespie , 1 964 ) . In copper deficiency there is an associated reduction in wool growth (Underwood , 1 97 1 ) . Such wool has reduced fibre strength , lacks crimp , loses its lustre , and is known as ' steely wool ' (Marston , 1 955) . Copper deficiency also causes a reduction of pigment in black-coloured sheep . This is the result of a reduction in the conversion of the amino-acid tyrosine to melanin ; the conversion being catalysed by the copper-containing enzyme , polyphenol oxidase ( Underwood , 1 97 1 ) . The two changes of wool seen during a severe d ietary deficiency of copper ; namely , steely wool and decrease in pigmentation , are reversed within two days of returning to a diet supplying adequate copper (Marston , 1 955 ) . Estimations of copper concentrations of wool from sheep in various parts of the world differ markedly ( Table 7 . 1 ) . Stevenson & Wickham ( 1 976) noted that during the winter the copper content fell as the feed intake was reduced , while Kapoor et al . , ( 1 972 ) , found no correlation between dietary copper content and wool copper content . Healy et al . , ( 1 964) suggested that wool may be a secondary excretory pathway for copper for those sheep that had adequate dietary copper , and Rish ( 1 970 ) also supported this theory i n relation to zinc and molybdenum . This latter theory suggested that when sheep have low concentrations of stored copper , zinc and molybdenum become stored at higher concentations in the wool . 1 33 No . of Range or Workers Sheep mean in ppm o.n . Cunningham & Hogan ( 1 958 ) 6 8 . 3- 1 3 . 3 Burns et al . ( 1 9 64) 50 25 Healy et al . ( 1 964 ) 30 42- 1 47 Healy & Zieleman ( 1 966) 32 22-81 Rish ( 1 970 ) n . s . 8- 1 0 Kapoor et al . ( 1 972 ) 36 3 . 95- 1 2 . 55 Stevenson & Wickham ( 1 976 ) 66 25 . 5-37 . 3 Langlands et al . ( 1 98 1 ) 1 2 5 . 2 Grace ( 1 9 83 ) 50 7 . 0�0 . 3 1 Woolliams et al . ( 1 983 ) 32 3 . 48�0 . 1 6 Suttle & McMurray ( 1 983 ) 5 3 . 6�0 - 35 n . s . = not stated Table 7 . 1 : Copper content of fleeces determined by other workers . As many of the experiments carried out in this study were to assess the amount of stored copper in the shee p , it seemed appropriate in the current set of experiments to determine the copper concentration of the wool . The investigation was attractive too because wool is a convenient t issue to collect and might possibly indicate the copper status of the keratinization of the wool fibre . animal MATERIALS AND METHODS at the time of Reference has been made previously to the collection of wool samples from certain sheep during the process of the other experiments in this study . In summary , samples were collected from the two pilot sheep and the nine other sheep used in setting up the 1 34 toxicity model ( Chapter 2 ) . The sheep in the experiment comparing copper uptake and retention between diffe rent breeds of sheep were used (Chapter 6 ) , and also those sheep involved in monitoring the copper deficient and the copper sufficient farms ( Chapter 5 ) . The samples of wool were collected by close-clipping over the site of entry for the liver biopsy ( the right paralumbar fossa ) . In the sheep which were subjected to the taking of repeated liver biopsy samples , only that woo l that had grown since the previous occasion was collected . Each wool sample was washed at least three times in a detergentl , to remove the wool grease and any extraneous matter . The sample was then washed a further three times in deionized water , dried in the oven at 1 00 °C for 72 hours , cooled to room temperature in a dessicator and weighed . Each weighed sample of approximately 300mg was digested in concentrated nitric acid and taken to dryness . The digest was then taken up in 5ml of 2N hydrochloric acid ready for copper estimation using the atomic emission spectrometer ( Appendix 4 ) . The estimates of the copper content of the wool for each sheep were compared against both the liver copper concentrations of that shee p , and blood copper concentrations of samples collected on that same day . The linear correlation coefficient was computed using the Pearson product moment correlation coefficient . 1 Pyroneg , D iversey Wallace Ltd . , New Zealand 1 35 RESULTS The values for copper concentrations from the analysis of the 1 68 wool samples ranged from 1 . 1 0 - 1 1 . 56 ppm Cu D .M . , with a mean of 4 . 75�0 . 1 4 ppm ( Fig�re 7 . 1 ) . Although the correlation between copper content of wool and the copper content of liver was low ( r = 0 . 4 1 2 ) ; o f the 39 liver copper values below 50 ppm Cu D .M . only two sheep had wool values in excess of the mean wool value of 4 . 7 5 ppm. The histograms of the liver copper concentration and the copper concentration are shown in Figures 7 . 2 and 7 . 3 . correlation between wool copper concentration and blood concentration from the same sheep was low ( r = · 0 . 0 6 1 ) . blood The copper __ Even in those sheep that received weekly injections o f 50mg of copper , there was only a very small increase in the copper content of . the wool . Of the 6 3 · sheep with a liver copper concentration in excess of 500ppm Cu D .M . , fifty of these animals had wool containing copper in excess of the mean of 4 . 7 5ppm . The low correlation between wool copper concentration and liver copper concentration was most evident in animals of lower liver copper concentration , but as liver concentrations increased so did the wool copper concentration ; the regression line for this relationship had a slope of y = 4 . 1 9 + 0 . 000874 x . DISCUSSION The copper content of wool samples collected during this series of experiments was generally lower than those amounts obtained by most of the workers listed in Table 7 . 1 . This is significant especially as many of the sheep in the current study had high liver copper concentrations . Rish ( 1 970 ) and Healy et al . ( 1 96 4 ) suggested that wool may be a secondary excretory pathway for copper . On the basis of the . results obtained in this series of experiments this premise must be doubted , as although 50 out of the 6 3 sheep with 1 2 �. 10 8 6 I I pp m 4 r D. M. I 2 I w 0 10 20 30 4 0 . 50 Percentage of samples Figure 7 . 1 : Copper concentration of wool samples 320 2 80 200 pp m D.M 160 pp m 80 0+---'----. 40LH-----L-----. or---.---.---�--�--�--�--�--�--�-L� 10 20 30 40 so Percentage of s amples F igure 7 . 2 : Copper concentrat ion of liver tissues 1 . 0 0 . 8 � I 0 . 6 I I 0 . 4 1 I o : 2 I 1---J 0 1 0 20 30 40 Percentage of samples Figure 7 . 3 : Copper concentration of blood s amples so 1 36 1 37 liver copper concentrations in excess of 500 ppm had copper concentrations in wool greater than 4 . 75 ppm , the highest copper concentration found in any wool sample was only 1 1 . 6 ppm . Similarly the results of Burns et al . ( 1 96 4 ) , Healy & Zieleman ( 1 966 ) , and Stevenson & Wickham ( 1 976) must be treated with reserve . For a sheep to deposit in excess of 20ppm of copper into a 3kg clean fleece annually would require at least 2 . 7mg Cu/day in the diet ( assuming an absorption of 6%) just to support wool growth . This is almost 50% of the estimated copper requirements for body ma.intenance (Grace , 1 983 ) . The results of analyses of wool samples performed s ince 1 980 gave the lowest values for copper content ( Table 7 . 1 ) . Some of the earlier workers used unwashed samples of wool and as the fleece may co�lect foreign material , variable results may have occurred . Normal , soft , animal tissues ( Grace , 1 983 ) , with the exception of live r , contain a mean copper content of 4 . 2 ppm D .M . (Underwood , 1 97 1 ) . It is suggested that wool utilises copper similarly to a normal soft tissue and has a normal copper requirement , and thus the mean copper content obtained in this study of 4 . 75 ppm D . M . appears to fall in the expected range (Grac e , 1 983) . Woolliams et al . , ( 1 983 ) , using unwashed wool samples found a correlation between wool and liver copper concentration ( r = 0 . 46 ) , which agrees closely with the correlation of 0 . 4 1 2 obtained in this study . They also found that copper concentrations in wool rarely exceeded 4 . 5 ppm , but that the concentration did continue to rise slightly in sheep having liver copper levels in excess of 20 ppm . A s imilar relationship occurred in this study . The analysis of wool for copper content appears to have little merit in indicating the copper status of animals which have adequate or high copper reserves . When the liver concentrations were below 20 ppm , Kellaway et al . ( 1 978) found that the copper content o f both bovine hair and bovine plasma were sensitive to changes . Suttle & -- 1 38 McMurray ( 1 983 ) , in their repletion experiments found that the copper status of wool changed following copper depletion and with subsequent repletion of the animal . They concluded that wool was a good indicator of the copper status of the animal as wool copper concentration changed less rapidly than either live r or plasma copper concentration . Langlands et al . , ( 1 98 1 ) , when investigating the relationship between wool copper content and hepatic copper storage found that wool copper content appeared to be a reliable indicator o f induced copper deficiency . Insufficient numbers o f sheep of low copper status were used in these Massey experiments to ascertain the value of wool analysis in detecting copper deficiency . CONCLUSION The analyses of 1 68 wool samples from sheep of known liver copper status are presented . The wool samples had a mean coppe r concentration o f 4 . 75 � 0 . 1 4 ppm . Sheep o f high copper status d o not accumulate copper in the wool fibres . An insufficient number of animals of low copper status were used in these experiments to indicate the value of wool in the diagnosis of copper deficiency . ·., 1 39 GENERAL DISCUSSION The work described in this thesis covers two aspects of copper metabolism. First the factors that may enhance the toxicity o f copper administered therapeutically are considered , and secondly observations have been made on the changes in copper status o f animals grazing on New Zealand pastures . As pasture is the stable diet of sheep in New Zealand , it was preferable for the sheep used in these experiments to be grazed on pasture whenever possible . This is in contrast to studies in other countries where many of the experiments involved animals which were housed and fed concentrate diets (Dick , 1 954 Wiener , 1 980 ; Suttle , 1 98 1 b ) . Similarly in the earlier work in New Zealand of Hogan et al . , ( 1 9 68) , pen-fed sheep were used . As trace element concentrations in pasture seasonally and . anticipated and also vary ultimately under shown other that circumstances , some of the change it was copper concentrations recorded in sheep proved to be different from those of previous workers . These differences were largely attributable to the effects of pasture feeding . In both the area of toxicity and seasonal copper status of animals , new information has been obtained , and where possibl e , explanations have been offered for the variations reported . Further , these variations open up challenging new areas for investigation which need to be undertaken before a more complete understanding of copper supplementation to livestock in New Zealand is available . In addition to confirming the results of previous studies on the development of copper toxicity , namely that serum concentrations of sorbitol dehydrogenase and serum glutamate oxaloacetate transaminase become e levated , and that liver copper concentrations increase ; the present study highlighted responses to the repeated parenteral administration of copper . It would appear that sorbitol dehydrogenase is the earlier indicator of hepatic cellular damage as 1 40 serum concentrations of this enzyme are elevated from one to four weeks before any s ignificant rise in serum concentrations of serum glutamate oxaloacetate transaminase activity can be detected . The latter is the enzyme that has been measured most often by previous workers ( Table 1 . 4 ) . There was a regular increase in both the number of hepatocytes damaged and in the accumulation of copper granules as the number of copper treatments increased , until a haemolytic crisis or death occurred . At this stage approximately 5 0% of the hepatocytes were destroyed . These histological changes did not appear to be related to the number of treatments administered , nor to the copper concentration of the liver at the time of that injection . The amount of copper present in the hepatocytes before any change in the serum enzyme concentrations was observed , and before there were alterations in the cellular structure , varied between sheep . This suggests that there is an individual threshhold of tolerance to copper for each sheep and the mechanism may be either an inherent ability of the hepatocytic lysosomes to retain the accumulated excess copper , or some undefined resistance factor operating elsewhere . The analysis of liver tissue for copper content at the time of death or during the haemolytic crisis showed that the copper concentration was approximately half of that recorded at the previous sampling . This suggests that either half of the hepatocytes are destroyed and release their copper contents , during a crisis , or there is a massive release of lysosomal copper from the intact hepatocytes . Alternatively both phenomena could have taken place . In those sheep that reduced their feed intake for two or three days and then died , from causes o ther than copper toxicity , there was also a reduction of almost 50% in their liver copper concentration at the time of death . This reduction may be important when field assessments of the copper status of grazing animals are being undertaken . Not infrequently , veterinarians have submitted samples of liver for copper analysis from animals which are already suffering from other diseases and are in poor body condition . The results from the current work show that in the determination of copper 1 4 1 concentrations in grazing animals , the selection o f those individuals which are free of concurrent disease and in good body condition is very important. Accordingly it should be emphasised that the liver samples collected for copper estimation should be taken either by liver biopsy , or from healthy animals destroyed for that purpose . The liver biopsy technique has been performed regularly in this series of experiments and has proved both reliable and safe . The individual variations in liver copper content may also pose difficulties in the diagnosis of copper toxicity . It would appear that some sheep can tolerate in excess of 4 , 000 ppm D .M . of copper in the liver , without showing any untoward e ffects , whi le in others , ·­ with concentrations as low as 1 400 ppm D .M . o f copper , clear s igns of copper toxicity are present . Therefore the diagnos is of copper poisoning is dependent on a cons ideration of the clinical signs confirmed by the presence o f elevated liver copper . It should be noted also that during the haemolytic crisis , the kidneys accumulate large amounts of copper , the measurement of which is a better confirmatory criterion for copper poisoning than the liver estimation . To confirm that death has - resulted from coppe r poisoning the history , clinical signs and autopsy findings should be considered in relation to the histopathological changes in the liver and the kidney , and the copper concentrations measured in particularly the kidney , as well as the liver . Work from this thesis suggests that the analysis o f wool samples is not a suitable method for estimating the copper status of sheep which are absorbing copper in excess of their requirements . In previous reports ( Skinne r , 1 9 60 ; Ross , 1 964 ) it has been stated that copper poisoning has been initiated by stress following the use of copper therapeutics . On many New Zealand farms sheep are placed under temporary stress as a result of routine management procedures , and it is often during these periods that sheep are dosed with coppe r ¥ A. series of expe riments was designed in a n attempt to identify those factors which may potentiate the toxicity of 1 42 parenterally administered copper . The effects of anthelmintic administration , insecticide application , pregnancy , dehydration and exposure to a cold environment appeared to have no potentiating e ffect . However some factors did enhance the toxic effects of copper preparations given parenterally . These included starvation , exposure to a hot environment , and the harbouring of a heavy burden of gastrointestinal parasites . These latter three conditions probably affected sheep in a s imilar manner ; that is , by a reduction in the intake of nutrients . Also in those sheep affected with gastrointestinal parasitism there was a greater susceptibility to copper toxicity as these animals were of a younger age . finding was recorded by Lewis et al . , ( 1 98 1 ) . A s imilar In this study it was shown that when sheep are fasted for 24 hours or more there is an alteration in the plasma protein concentration , and after the administration of copper , the blood copper concentration became signi ficantly elevated . A most likely explanation for this phenomenon may be as follows . During fasting the body maintains gluconeogenes is by utilising amino-acids . These immediately available amino-acids are held as a pool in the hepatic lysosomes ( Aronson , 1 980 ) • demand for amino-acids by lysosomes occurs , which It would appear that because of this the may fasting animal , also cause the autophagy of the release of their accumulated copper , or render hepatocytes unable to take up circulating albumin-complexed copper for storage , or both mechanisms may be . involved . At the same time there is an increase in plasma albumin , which is presumably released from the hepatocytes , which will transport the copper ions from the site o f injection . Therefore it would seem that to minimise the risk o f poisoning following parenteral administration of copper , animals should be in good body condition and not starved as a result of prolonged periods of yarding or droving without food . Young animals are more susceptible to copper poisoning than adults . Therefore to reduce the risk of death following dosing , care must be given to . the dose rate used . Alternatively the foetus can 1 43 gain a store of copper in utero by dosing the dam at an appropriate stage of pregnancy . This will ensure an adequate supply of copper to the foetus during pregnancy and subsequently during lactation . The measurement of the blood copper concentration at hourly intervals after the administration of parenteral copper appeared to be an effective technique for measuring the rate of uptake , and therefore the translocation rate , of copper formulations given under various controlled conditions . It also indicated the potential toxicity of copper compounds when their translocation rate was compared to the translocation rate uptake rate . The measurement of of safe the formulations activity of of known sorbitol dehydrogenase and glutamate oxaloacetate transaminase enzymes in the serum after 48 and 96 hours , indicated the amount of cellular damage to hepatocytes caused by parenteral copper formulations . By measuring the rate of uptake of a copper compound and by measuring the activity of the liver enzymes in serum , it should be possible to determine the potential toxicity of that copper compound in any particular formulation . It should also be possible to determine which stressors may enhance the toxicity of copper . In this study starvation was the stressor found most likely to enhance copper toxicity , and it would be appropriate now for further biochemical studies to be undertaken to investigate more precisely the changes that occur in liver metabolism in response to starvation . The understanding of this mechanism is important as many therapeutic agents are initially stored in the liver , and their ultimate fate is influenced by variations in the metabolism of the liver . The determination of the 'copper status of animals grazed on pasture , and make up that experimental the estimation of the copper content of the plants that pasture , have not been regularly recorded . Most investigations have used animals in a controlled environment and on a diet of known composition . In this study the regular monitoring of animals and pastures on New Zealand farms has highlighted the d ynamic nature of copper metabolism . It has also 1 4 4 identified factors that may influence the absorption of copper from pasture and its retention in the liver . This study has also shown the poor correlation which exists between the copper content of the soil and the copper content of the pasture growing on that soil . Like the uptake of copper from pasture by animals , the uptake of copper from soil by plants is influenced by a variety of factors ( Le Riche et al . , 1 963 ; Mitchell , 1 974 ) . Although there are variations in pasture copper concentrations between farms , these are not as great as the variation in liver copper concentrations found in the animals grazing those pastures . _ As outlined in this thes is , and as reported in previous work , pasture copper concentrations usually bear little relationship to liver storage concentrations in sheep . The factors that appear to influence the absorption and storage of copper are the inhibiting elements of molybdenum and sulphur incorporated in the plant , and the soil contaminating elements of zinc , iron and manganese . Other important factors which influence copper uptake are the species of plant ingested , the digestibility of that plant and its stage of maturity , and the copper status of the sheep grazing that pasture . The elements that inhibit copper absorption , namely molybdenum and sulphur , have been well investigated in sheep on hand-fed diets , and their effects quantified ( Suttle & Field , 1 968b ) . However the effect of these two elements does not completely explain the changing copper status of animals grazing pasture . It has been noted again in this study that the soil elements of zinc , iron and manganese may affect copper uptake and storage . These elements contaminate pasture during winter in particular , and therefore may have a marked influence on copper absorption and retention ( Field & Purves , 1 9 64 ; Ghergariu , 1 978 ) . Further , winter is the time of the year when pasture copper content is at its lowest . Reynolds ( 1 979 ) and Standish et al . , ( 1 97 1 ) have reported on the influence of zinc and iron respectively . other New Zealand In this study and from the analysis of pasture on farms , it has been noted that when pasture 1 45 manganese concentrations rise above 200 ppm D .M . that sheep grazing such pasture frequently have low liver copper concentrations . On the other hand sheep grazing pasture with a lower manganese content ( > 1 00 ppm D .M . ) usually show an adequate liver storage o f copper . These observations are worthy of further study particularly as manganese is stoichiometrically similar to copper in the ionic state and may be able to replace some copper ions in their protein complex without those proteins losing any of their activity . The role of manganese as a possible antagonist to copper requires further investigation . On investigation of the farms where the animals had adequate or high copper storage , it was established that the predominant species of plant in the pasture were perennial ryegrass . ( Lolium perenne ) and white clover ( Trifolium repens ) . This contrasted with the farms on which the concentrations of liver copper in the sheep were low . On these farms the predominant pasture species were the less digestible grasses such as crested dogstail ( Cynosurus cristatus ) , brown top ( Agrostis tenuis ) , and danthonia ( Sieglingia decumbens ) . Probably it was not so much a marked variation in the copper content of the species in these pastures per se , but their differing digestibility which affected copper uptake . The digestibility of plant material is influenced by the species and also by the stage of maturity ( Church , 1 977 ) . The liver copper concentrations of sheep showed not only a variation between farms , but also a seasonal variation . The copper content of the pasture was lowest in the winter and early spring on all farms studied . During this same period , pasture molybdenum concentrations were highest and the soil contaminating elements o f iron , zinc and manganese were at their highest in the pasture . This seasonal variation was most marked on farm A ( the copper deficient farm described in Chapter 5 ) where the sheep were set-stocked on the poorer pastures during the winter . As the winter progressed these sheep would only have had access to the less digestible pasture species , and then at a time when requirements for copper were highest . 1 4 6 In this work the measurement of the amount o f copper retained in the liver was used in calculating the absorption of coppe r from the diet . In sheep grazing pasture , the amount of copper retained in the liver varied . from 4 . 7% to 1 4 . 3% of dietary copper . Although the pasture content of inhibiting elements , and the species of plants that comprise that pasture influence the absorption rate , it would appear from this work that the digestibility of the plant species , and the copper status of the animals grazing that pasture have a dominant influence on the absorption of copper from the diet . The farm on which the sheep retained 1 4 . 3% of dietary copper had pastures containing mainly perennial ryegrass and white clover ; both in an active growing state . The contrast in copper retention between the ­ " copper deficient" and the "copper sufficient" farms is the most surprising. Those sheep grazing the "copper deficient" farm and being of low copper status retained 6 . 4% of the dietary copper , whereas those sheep grazing the "copper maintaining their copper status retained sufficient" farm and only 4 . 7% of the dietary copper . These results are similar to those of Kirchgessner et al . , ( 1 981 ) which showed that the excretory and absorptive mechanisms for copper tend to adapt to the variations in copper requirements and the copper status of the animal , thus altering the absorption rate of copper from the diet . The sheep that were fed on the synthetic diet of very small particle size , retained 76 . 6% o f the dietary copper in the live r . This high retention rate is comparable to that o f the pre-ruminant lamb ( Suttle , 1 979 ) and suggests that in the digestion o f this diet it almost completely by-passed the rumen , which is the major s ite of interference of copper absorption ( Suttle , 1 98 1 c ) . Suttle , ( 1 98 1 a ) , has estimated the absorption of copper from various diets ( Table 1 . 2 ) , and has also shown the effect o f molybdenum and sulphur content in the diet on the amount of dietary copper available to the animal . In New Zealand where sheep are grazed continuously on pasture of varying digestibility and copper content , the amount of copper available to the animal from the diet 1 47 is extremely variable . When investigating farms to establish their copper status , it is imperative that plant and animal tissue copper concentrations should be evaluated at regular intervals . From this data and from information on the availability of copper in various plant species , it should be possible to determine the copper requirements of the animals relative to the available copper of the pasture . If the pasture is unable to supply sufficient copper to meet metabolic requirements , then copper supplementation should be provided . Alternatively i t may be possible to supply adequate copper from pasture by changing the grazing management of that farm . From the data collected on the "copper deficient" farm and on the "copper sufficient" farm , it appears that 50 mg of parenteral copper given to ewes in early winter , is sufficient to maintain adequate blood and liver copper concentrations during pregnancy . In sheep with adequate copper reserves at the outset , it appears that liver copper concentrations usually increase after treatment and remain at this higher leve l . On any farm where copper supplementation is practised the copper status of the farm and of its animals should be assessed at least annually , prior to medication . If sheep of adequate copper status are maintained on a diet providing sufficient copper to meet metabolic requirements and are also given supplementary copper , toxicity may result . The supplementary copper will accumulate in the liver and may reach the threshhold level for copper storage and precipitate copper toxicity . This accumulation of copper sufficient to reach the threshhold leve l to precipitate toxicity may involve more than one treatment . The variation in the absorption of copper from the diet and the retention in the liver between the British breeds of sheep ( the Border Leicester , the Suffolk , and the New Zealand Romney ) , used in this study , was non-significant . Therefore manipulating the breed of 1 4 8 sheep to graze pastures of a lower copper content appears to be of little value . The Merinos used in this experiment appeared to have a higher turnover of copper and therefore a higher requirement for copper , as was found by Edgar et al . , ( 1 94 1 ) . However in New Zealand the British breeds of sheep are not adapted to the environment grazed by Merinos and so a change of breed to counter copper deficiency would be impractical . The copper requirements of sheep of different breeds have been shown to be variable in experiments carried out in Britain (Wiener , 1 980) and in Europe ( van der Berg , 1 983 ) . Howeve r , the breeds of sheep used in these European experiments have adapted to their respective environments over many years , whereas the British breeds of sheep used in this study have shared a common environment . This experiment should be repeated using a greater number of animals from a greater range of breeds , in an . endeavour to establish the variability in copper absorption that exists in the breeds of sheep farmed in New Zealand . Similar experiments using cattle of different breeds , and also of deer , would provide valuable information for farming in this country . The information gained from this thes is suggests that copper toxicity resulting from the treatment o f sheep with therapeutic agents can . be avoided . Before initiating copper copper therapy for shee p , the copper status o f the animals and the pasture o n which they are grazing must be established . It is suggested that in New Zealand , the pasture should provide sufficient copper to meet the daily requirements of the animals grazing that pasture , and that the liver copper concentration should be in excess of 50 ppm Cu D .M . If these criteria can not be met then copper supplementation must be considered . Further , the therapeutic copper formulation used must have an adequate margin of safety . This can be determined by using copper compounds of a known translocation rate that will not produce damage to hepatic cells . The formulation of the copper therapeutic agent should not be altered until it is increase the translocation rate established that of the copper . this My 1 49 will not suitable p reparation must be administered at the appropriate dose rate based on the age and on the bodyweight of the animals being treated . In addition , any stressors that may potentiate the toxicity of copper by increasing its translocation rate should be avoided . The stessors known to enhance toxicity are those that reduce feed intake , which can be the result of starvation , exposure to a hot environment , or when animals are affected with gastrointestinal parasites . While copper given parenterally is the most reliable and the most commonly used form of copper therapy , its use can result in toxicity . It is important that in prescribing and using these products that their potential toxicity is recognised but as this toxicity can be avoided , the pre-requis ites for safe copper medication should be widely promoted . Although study should continue to establish those factors which may enhance copper toxicity , it is equally important to develop copper therapeutics that are safer than existing products . . Such work may lead to different formulations , different routes of administration , or the development of slowly absorbed formulations in controlled release devices . 1 50 APPENDICES APPENDIX I Technique for the Analysis of Blood Samples The following steps were used : 1 . One ml of blood was placed in a test tube 2 . Half one ml of 2 5 volume hydrogen peroxide was added and left for 4 hours 3 . Two ml of concentrated nitric acid was added and the solution was taken to dryness 4 . The residue was dissolved i n 5 ml o f 2N hydrochloric acid , containing 5 ppm Ni to act as an internal standard , and warmed for 4 5 minutes 5. A blank , containing deionised water instead of blood , was run with each batch of samples 6. The solution was analysed for mineral content using the inductively-coupled argon plasma emission spectrometer All glassware was washed , soaked in Pyroneg detergent (Diversey Wallace Ltd ) for 1 2 hours , soaked in 2N hydrochloric acid for 8 hours and then triple rinsed in deionised water . They were air-dried . 1 5 1 APPENDIX II Technique for the Analysis of Tissues and Pastures . The following steps were used : 1 . A sample of tissue was placed in a weighed crucible and dried at 1 ooP.c for 1 5 hours 2 . The sample and crucible were weighed 3 . The sample was ashed at 48S°C for 1 5 hours 4 . The ash was washed into a test-tube with approximately 5 of 2N hydrochloric acid and taken to dryness ml 5 . Two ml o f concentrated nitric acid was added and taken to dryness 6 . The residue was dissolved in 5 ml of 2N hydrochloric acid and warmed for 45 minutes 1 . A blank sample was prepared using every step except the sample 8 . The solution was analysed for mineral content using the inductively-coupled argon plasma emission spectrometer 9 . 0 Pasture samples were dried at 1 00 C for 1 5 .hours and milled to 2 mm . Approximately 0 . 5 gm o f sample was weighed into a crucible and ashed at 485°C for 1 5 hours . The ash was then digested as the tissue samples . 1 0 . Pasture samples used to estimate sulphur content were wet-ashed in 2 �1 of concentrated nitric acid instead of dry-ashed at 485°C . ', 1 52 1 1 . In every batch of samples analysed a standard of known mineral content was used . This was the National Bureau o f Standards Bovine Liver Standard Reference Material No . 1 577 which contained 1 93 � 1 0 ppm of copper 1 2 . All glassware and crucibles were prepared as set out in Appendix I . APPENDIX III Technique for Demonstrating Copper in Tissue Sections Staining solution a . 50 mg rubeanic acid ( BDH Ltd ) in 50 ml ethanol 1 53 b . 1 0 gm sodium acetate in 1 00 mls distilled water staining solution Use 2 . 5 ml of solution a and 50 ml of solution b . Method : 1 . Sections of fixed tissues were placed into distilled water 2 . Sections were placed in covered Coplin jars containing stain , at 37 't , ove rnight 3 . The sections were rinsed in 70% ethanol for 2 to 3 minutes 4 . The sections were then rinsed in ethanol coloured with eosin , until the sections were light pink 5 . Each section was cleared i n xylol and mounted in DPX . Note : To get consistent results fresh solution should be used . This technique was supplied by Mr B . J . Young , Ruakura Animal Health Laboratory . 1 54 APPENDIX IV Technique for Analysis of Wool Samples The following steps were used : 1 . A sample of approximately 2 gm of wool was triple washed in hot detergent ( Pyroneg , Diversey Wallace Ltd ) and triple rinse� in deionised water 0 2 . Each sample was dried a t 1 0o�c for 3 days , and cooled in a dessicator 3 . Approximately 0 . 5 gm of wool was weighed into a dried weighed test-tube 4 . Two ml of concentrated nitric acid was added and the mixture was taken to dryness . This step was repeated up to four times until the digestion was completed 5 . The residue was dissolved i n 5 ml of 2N hydrochloric acid and warmed for 45 minutes 6 . A blank solution was prepared using every step except the sample 1 . The solution was analysed for mineral content using the inductively-coupled argon plasma emission spectrometer 8 . All glassware was prepared as set out in Appendix I . I I 1 55 BIBLIOGRAPHY Adams , A . F . R . , & Elphick , B . L . ( 1 956 ) The copper content of some soils and pasture species in Canterbury • . N . z . J . Sci . Tech . Sect . 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