The Use of Isotopes to Detect Moderate Mineral Imbalances Farn I M Animals

IAEA-TECDOC- 267

THE USE OF ISOTOPES TO DETECT MODERATE MINERAL IMBALANCES FARN I M ANIMALS

RESULT CO-ORDINATEA F SO D RESEARCH PROGRAMME ON THE USE OF ISOTOPE TECHNIQUES FOR DETECTION OF MODERATE MINERAL IMBALANCES IN FARM ANIMALS ORGANIZEE TH Y DB JOINT FAO/IAEA DIVISIO ISOTOPF NO RADIATIOD EAN N APPLICATIONS OF ATOMIC ENERGY FOR FOOD AND AGRICULTURAL DEVELOPMENT AND PRESENTED AT A RESEARCH CO-ORDINATION MEETING HELD IN NICOSIA, CYPRUS, 6-10 OCTOBER 1980

A TECHNICAL DOCUMENT ISSUED BY THE INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1982 THE USE OF ISOTOPES TO DETECT MODERATE MINERAL IMBALANCES IN FARM ANIMALS IAEA, VIENNA, 1982 IAEA-TECDOC-267

Printed by the IAEA in Austria September 1982 PLEAS AWARE EB E THAT ALL OF THE MISSING PAGES IN THIS DOCUMENT WERE ORIGINALLY BLANK The IAEA does not maintain stocks of reports in this series. However, microfiche copies of these reports can be obtained from INIS Microfiche Clearinghouse International Atomic Energy Agency Wagramerstrasse 5 P.O. Box 100 A-1400 Vienna, Austria prepaymenn o f Austriao t n Schillings 40.0 r agains0o IAEe on t A microfiche service coupon. FOREWORD

It has long been recognized that gross deficiencies and excesses of mineral elements can have marked effects upo abilite nth f faryo m animal surviveo t s , gro produced wan . Detectioa f no gross imbalance is relatively easy because symptoms appear rapidly or the imbalance is readily detectable by means of chemical analysis. However, in situations where an element or elements ma e onlb y y moderately deficien r moderatelo t y excessiv e diet imbalance th th ,n ei e expresses itself nonspecificall reduces ya d growth rate, productivity, reproductive level viabilityr ,o . Chemical analyses of the diet may not provide the clue to a potential problem since the imbalance may be e unavailabilitth du o t e elemene th interaction a f yo r o t n with other element y diee th ma t f o s reduc availabilite eth y and/or utilizatio primare th f no y element. Broad supplementation of the diet to bring all elements up to a specified level may not succeed in situations where interactions between elements reduce availability and utilization. Furthermore, broad supplementation is of no avail, and in fact may result in further complications, if the basic problem is one of moderate excess of a particular dietary element. Methods of detecting moderate mineral imbalances are not generally available, and so the intent of the Joint FAO/IAEA Division was to establish a programme that would bring together scientists from developed and developing countries who might through collaboration elaborate methods useful in detecting moderate trace element imbalances in farm animals. This co-ordinated research programm e Jointh f t o eFAO/IAE A Division bega n 196i n 7 wit intene hth studo t t y selective trace elements, to explore various procedures for trace element assay, to develop technique possiblf i procedured e th san e verifus d solvo st yan e field problems. The first meeting of the contract and agreement holders in this co-ordinated research programm hels Ankaran dewa i , Turkey 1976n i , secone th , d meetin hels Washingtonn gdwa i , DC , USA, in 1978, while the third and final meeting took place in Nicosia, Cyprus, in October 1980. programmee Oveth e liff o th er , some eleven contract holders from nine different countriesd an , five agreement holders from four countries have taken part in the programme. The papers containe thesn di e proceedings represen e findingth t s from those investigators associated wite hth projec terminations it t a t . While no single method achieved the goal of being readily available for field use, significant progress was made on several procedures, which would suggest the degree of usefulness which migh e achievedb t n additionI . , significant scientific observations have been made that have contribute fiele f th traco d o dt e element metabolis animalsmn i . Accompanyin papere gth a s si lis f recommendationo t s resulting from discussion participante th f so programmee th n si . Thi f isotopiso publicatioe us ce techniqueth n no deteco st t moderate mineral imbalancen si animals should be of value to scientists working on mineral metabolism, as well as veterinarians and field nutritionists involved in improving animal nutrition and increasing animal productivity. CONTENTS

A study of the calcium metabolism of dairy sheep using radioisotope and balance techniques..... 7 S. Economides phosphorus-3f o The eus diagnosie th n 2i phosphoruf so s deficienc sheep...... n yi 3 3 . A.O. Lobao, D.M.S.S. Vitti Marcondes, J.W. Lemos, M.I.B. Peixoto Escubedo, A.A.D. Oliveira,de BinnertsT. W. Heat-induced mineral imbalances in animals ...... 49 T.H. Kamal Possible role of vitamin D compounds in the calcinogenic action of Trisetum flavescens...... 63 M. Peterlik, D.-S. Regal, H. Kohler Problem diagnosie th n si tracf so e element deficiency ...... 9 6 . M. Kirchgessner, F.J. Schwarz Effec f seleniuo t m deficienc selenium-7n yo calcium-4d 5an 5 metabolis chicks...... mn i 1 9 . J.S. Edwardly Importance of mineral imbalances in animal production and health and some diagnostic and corrective trials ...... 101 K. Goksoy, Cetinkaya,N. Gul,Orkiz,F. M. A.I. Gucus Copper and cobalt status of sheep and cattle grazing the central highlands of Peru, elucidated by enzyme analysis and nuclear techniques ...... 113 /. Kalinowski Ferritin in cattle serum ...... 123 Lengemann,F.W. J.A. Lopera, F.A. Kallfelz, Lengemann,W. F. Jr. Absorption, distribution and excretion of arsenic-76 in hens and ruminants ...... 135 M. Anke, G. Hoffmann, M. Griin, B. Groppel, E. Riedel Assessmen chromiuf o t animals...... d man n statuma f so 7 14 . R.A. Anderson, MertzW. Marginal mineral deficiencie theid an s r importanc productivite th r efo reproductivitd yan y of domestic animals ...... 3 16 . /. Wegger

Conclusions and Recommendations ...... 189 List of Participants ...... 193 CALCIU E STUDA TH F YO M METABOLIS DAIRF MO Y SHEEP USING RADIOISOTOPE AND BALANCE TECHNIQUES*

S. ECONOMIDES Agricultural Research Institute, Nicosia, Cyprus Abstract

The Ca metabolism of dairy sheep was studied using radioisotope and balance techniques.

The level of Ca intake increased the rate of Ca absorption and decreased the efficiency of Ca absorption in dry sheep. The net Ca requirements of dry sheep were estimated to 870 mg/day.

The endogenous faeca urinard an l lossea yC s decreased and the efficiency of Ca absorption increased in pregnant sheep on a Ca-deficient diet compared! to pregnant sheep on a normal Ca diet. Ca balance was positive in ewes on both diets.

The pre-partum level of Ca intake had similar effect on e efficienc th e rat d th a absorptioan eC f balanca o yC d an ne was positiv n earli e y Icatation endogenoue Th . s faecaa C l linearls loswa s y relatemattey dr o t dr intake.

INTRODUCTION

The calcium metabolis n dairmi y cows beeha sn extensively studied particularly in relation to parturient paresis (l,2,3,U, 5,6). The demand for calcium is large in late pregnancy and early lactation. During the early stages of lactation high yielding dairy cows are in negative calcium balance and despite the increased absorption of calcium, extra calcium has to be provided through mobilizatio f calciuo n m froe bonemth .

Thmetabolisa C e f muttomo n breed f sheeo s s beeha p n studied in pregnant (7, 8,9,10), lactating (11,12) and dry sheep (13,1U), whereas work with dairy sheep is very limited.

IAEA Research Contract No. 2085/RB At variance with mutto na requirement C shee e th p f dairo s e sh y nahighee b y earln i r y lactation tha latn i n e pregnancyn I . additio e lonnfh g lactating period e higanfth Lh leve f milo l k production may delay the replenishment of skeletal Ca reserves

e limite th n vieI f wo d amoun f dato t a availabl n dai:o e y . e existinth ahee d an p g discrepancie e recommendeth n i s d allowance differeny b a C f o st agencie e presens th (15 ) 16 ,t studies were undertaken to determine the endogenous faecal calciue efficiencth d man f calciuo y m absorptio dietn i n f o s dry, pregnan lactatind an t ge effec sheeth f d prepartuo tpan m dietary calcium on the rate of Ca absorption in early lactation

MATERIALS AND METHODS

Animals^._housing and^ diejts non-prepcnany Dr Tria . 1 l t ewes.

Sixteen dry non pregnant Chios ewes having lambed at least twice were divided into four groups on the basis of liveweigh werd an et randomly f allocatefouo e r on diet o dt s providing similar intakes of energy, protein, phosphorus and Vitamin D-, per kg liveweight and four levels of Ca intake ewee Th (Tabls wer . l) ee housed individuall r fouyfo r weeks before they were transferred to metabolic crates designed for e separatth e collectio f faeceo n urined an s . Animals remain^ n metabolii weeksc o firs e cratetw Th r t. fo wees k serven a s a d adaptation period and during the second week radioisotope and calcium balance techniques were used.

The diets consisted of chopped barley straw (300g per head/daya pellete f o d dan ) concentrate mixture with barley

grain and soyabean meal being the basic ingredients. Limestone and disodium hydrogen orthophosphatP e d v/eran ea useC s a d supplements , mixturvitamiD A d . s usean o wa eprovidt ndA e about l|50 I.U. of vitamin A per animal/day. A known amount of -.vater was given to each animal. . Trial 2. L act at ing ewe_s

Twenty one Chios ewes lambing within one week were used The lambs were separated from their dams immediately after birth and were reared artificially amoune Th f colostru.o t m produced recordes v/ithiwa e h firsd sampleth n8 an dU tl f m abouo s 0 20 t froe firsmth t milking were collecte analyzed an d r differenfo d t constituents ewee Th s . were divide day0 t-partus 1 d po s m into three groups on the basis of milk yield and liveweight and were randomly allocated to one of three treatment diets (Table 2). The three diets provided the same Ca, energy, protein and vitami^ f finisheo g k r dDpe ~ feed differenan d t phosphorus levels (Table 3). The ewes remained in individual pens until day 28 post-partum 9 post-partu2 y da n O m. twelve ewes (four from each treatment) v/ere balance^ on the basis of milk yield and liveweight and were transferred in metabolic crates for a further two-week e periodseconth n I d . week (35-^4-2 days post- partum) radioisotop balancd an e e techniques were used. Each ewe v/as offered 0.6 kg of barley hay and 1.5 kg of the appropriate pelleted diet (Table 2). All ewes were hand milkec twice dail theid yan r individual milk yields v/ere recorded.

Trial 5

The trial v/as divided into three phases.

HU^s i g_i Sixt y ewes which lambe n Septembei d r 1978 v/ere mate; again after weaning (1+2-3 days) in 17 ov ember 1978. Prom matin., an averag0 day f pregnanc10 o s f o e e eweth y s v/ere kep n t'li t e S3.r,n undepe e r similar feedin managemend an g t conditions. During this period! milk yield, calcium conten f milko t , feed intake and calcium intake v/ere recorded.

s,e_I^I_. Prom day 100 of pregnancy until lambing a feeding an a metabolid c trial v/ere carried out. Feeding trial. Forty eight ewes were divided intgroupo otw s basie oth n f expecteo s d dat f lambino e d liveweighan g werd an te randomly allocated to either a control (A) or a Ga-deficient (3) diet (Table U). Each ewe was offered 200g of barley hay and 200g of barley straw daily until lambing. Ewes on the control diet were offered 1.03 kg of the pelleted diet (A) daily until lambing. Ewes on the Ga- deficient diet were offered daily 1.015 kg of diet B until day 137 of pregnancy. From day 138 until lambing they were offered 1.015kg per head daily of diet C (Table k) .

The ewes were kept in two adjacent pens and concentrates c.n-1 roughage were group fed. Individual liveweight l eweal f so s were recorde e commencementh t a d e triath f lo tf (100to y hda pregnancy) and immediately after lambing. Individual lamb birth weights were also recorded. Feed residues were collected f pregnanco 7 13 d immediately an ydailyda n O . y after lambing blood samples were taken from each ev/ r blooefo d calcium determination. Sample f wate o f feed o sd ran swer e also taken for Ca determination.

The remaining 12 ev;es were divided in two groups on the basis of expected date of lambing ancl liveweight and were randomly allocated to either a control (D) or Ca- deficient (_. diet (Table U). The ev/es "/ere transferred to metabolic crat^e and remaine 5 weeks e firs r Th weekfo dU t . s serves a d adaptation period, while the fifth week (130-137 day of pregnancy s use r radioisotopwa fo d) d balancan e e techniques. Lach ewe was offered U50c of barley hay daily and a measured amoun f concentrateo t o providt s e energth e y requirementf o s pregnant ev/es kept indoor carryind an s g twin lambs e intakTh . e of different nutrients in either diet D or E is shown in Table 5.

10 On completion of the blood sampling and the collection period adjacen o e ewetw th , sn i wer tt werpend pu e an se group fed. Feeding and management of ewes in treatments D and E were similar to those of ewes in treatments A and B, feedine oth f g trial respectively.

Phase III. From lambing to six weeks post-partum a feeding and a metabolic trial were carried out. Ewes lambing from treatments A or B of the feeding trial and from treatments D or E of the metabolic trial in late pregnancy were kept separately during lactation. 'The lambs were separated from their dams immediately after lambing. The production of colostrus recorde l ewe 9 frowa e L\ firs(1 h sth mr U8 fo n dmti the control and 22 from the Ca- deficient diet). Samples of colostrum froe firsmth t milkin g) wer (abouml e 0 take20 tr fo n the determinatio f differeno n t constituents.

Ten days post partum 10 ev/es (5 from the control diet an fro5 de Ca-deficien mth t diet were transferre o metabolit d c crates. Three from the control and 4 fron the Ca-deficient diet were used earlier for the metabolic studies during late pregnancy. They remained for k weeks In the crates and the h v/eeuses r kt radioisotopwa kdfo balancd an e e techniques. The ev/es were offsre e collectioth d f o fro d men n lambine th o t g f o r g heaf barlek o pe dy g 5 perio k 1. dailha y 6 d 0. dyan concentrate mixture F (Table U) . The intake of different nutrient n eithei s e controCa-deficiene th rth r o l t diete ar s shown in Table 6. While in the cratas they were hand milked twice daily and individual milk yields were recorded. Milk samples from the morning and a fternoon milkings were taken twice for Ca determination.

Feeding trial e remaininTh . 3 ewe fro3 gU e contro (l mth s d an l - deficienCa 9 fro e 1 mth t diet) were offered die (TablC t e 14.), unti day7 3 l s post partum. They were machine milked twice dail individuad yan l milk yields were recorded once weekly.

11 I-iilk samples for Ca determination were taken three times during the experimental period. The ev/es were offered 0.5 kg of barley hay and about 2.0 kg of concentrates (Diet C, Table 4),

Ewe liveweights were recorded on the last day of the experimental period and blood samples were taken on the same day for Ca determination. Feed! intake and Ca output in milk were also measured.

JRnciioisotope and calcium balance _technioue_s

In all trials feed and water intake, faeces and urine output and in lactating ev/es milk yield were recorded daily for oovcn consecutive days. Urine v/as collecte a vesse n i d l containing 25 ml of 50£"o HgSO, . The collection of faeces started 5h after the administration of radioactive calcium. 45 45 A know s a n a CaClactivitC n aqueoui o f o y s solutior. (c:bou 300yCd an r tpregnan y fo 250uCi dr lactatind r fo an it g animals) v/as injected inte jugulath o r vei f eaco n h sheed an p blood samples were withdrawn at 0, 30, 60, 90, 120, 130, 300, 420, 600 and 780 minutes and at 2Uh, 2,3,4,5,6,7 days after the injection.

Prepar at ion of samples for analysis

Blood samples were allowed to clot at room temperature and were subsequently contrifuge t 2,500Xa d r serufo g m separation. Feeds, faece mild an sk v/ere processe d analyzean d d accordin o MAPt g F (17). Urine samples v/ere analyzed without any treatment. Total Ca in each sample was measured by atomic absorption spectrophotometr n foodi y , urine, faece mild an sk n serui d (18man )(19) .

Measurement of radioactivity

Radioactivity in serum and faeces was measured in a Tri- Carb Liquid Scintillation Spectrometer (Packard, Model 3255). Serum (iml acidified with 3 drops of 2N-HC1) and of the solution of ashed faeces in 2N-HC1 (iml), v/ere counted in lOnl

12 oa scintillatof r solution containing toluen Tritond 0 an e0 l .X- (9:U d 0.2v/van gO n )whici ^OPO PP g r litrU hpe P e were added.

_ jlcrtions _,nc\ogenous faecal calcium was calculates from the eou?.J._cr, •pp Vf= T T Tj5fractio3 , th -."hers i f aosf o n R f etrace o e r excreted e faeceth n i s after injectio e meaSAd th nan nRs i specific i- -f.ioactivity of calcium in serum. The rate of Ga absorption (Va) was calculated from the Cifferencc between calcium intake (Vi) an-." total faecal calcium (VF) correcte r enfogcn'fo d - 1 _^3cal calcium; Va=Vi-(VF-Vf ) . The efficiency of Ca abs o:?-r ••as taken as the ratio of the rate of Ga absorption to Ca irt 1'- (in percentage units).

non-pregnany TriaDr . 1 l t .e\ves n treatmeni Informatioe ew e 3 witton n ho n very lo.v feed. wite ew h e completintakon d an ee anorexi n treatmeni a werC t e discarded. The endogenous faecal Ga loss was similar in the four diets. Total faecal Ga loss proportionally increased with increasing calcium intake (Table 7). The efficiency of Ga absorptio s highese dieth nwa tn o v/it t e lowesth h intaka C t e and decreased with increase a intakedC a e G rat th f o e; absorption tended to increase from low to high Ca intaKe, but higheo tw similas e r intakth levela wa G n t i i rf eo s (Tabl. 7) e Calcium balance was positive for all four levels of Ca intake •anc" followed the pattern of the rate of Ca absorption. The me cm Ca loss (mg/kg L'.TT/day n urini )s 2.92^2.32 wa e a intakC s . wa e not related to urine Ca loss. No relationship was found_/bet'.vecn OT. intak d endogenouan e s faeca a losC l s either. However, tne endogenous faecal Ca loss (mg/kgLrT?T/day) was linearly relatec. to dry matter intake. The linear regression describing the above relationship was Y= 3.20+0.76X(r=0.73), where Y is endogenous faecal Ca loss in mg/kgL VT/day and X is dry matter intake in : g/kg L'VT/day. Total faecal Ca loss was also positively related to Ca intake. The relationship was Y=3.55+0.78X( r=0.99) wnere Y= total faecal Ca loss (ng/kgL'VT/ciay) and X= calcium intake (mg/kgL^T/day) .

The rate of Ca absorption was positively related to the Ca retention. Tne relationship was Y = 13.12+1.072 X (r-O.S5/, w.iere Y= the rate of Ca absorption (mg/kg LV/T/d) and X= Caiciu.: retention (mg/kgL.VT/d) .

13 It is 'vorth mentioning that one ewe in treatment C -vent "off feed" during the collection period and the mean daily r'ry matter intak s onl a balancwa eC y e UOg f Th thio e . s s ev/wa e negative and the endogenous faecal Ca loss was 3.8 mg/kgL'VT/d . 'She suffered from rume ne balanc th aton d an ye results showed tha froa t G tabsorbem no fees wa t d alla bloode levea Th .C d l was 10.03 mg/10. 0ml

^Eiai_?js. L act at ing ewes

dien o tA e showe ew e d On inappetanc dats s it awa d ean discarded.

colostruMeah US n m e levelyieldth d f differeno san s t constituent e show ar intakea s C n Tabli a n rate C . 8 th f e, o e absorption and Ca balance tended to be lower in treatment A, than thos f eitheo e t differencer bu treatmen , C r o sD t wert no e significant (Tabl. 9) e

Ca int alee was not related to either endogenous faecal Ca or urine Ca loss. The cn^o^enous faocal Ca loss was positively related to C.-ry natter intake. The rate of Ca absorption -.7 as also positively relate o calciut d m retention. Total faocaa C l s positivelwa los) (Y s y relate a intak Y=21|.9+0.71( C ) o dt (X e X (r=0.9l).

The ewer were mate t a 5k-IOd days post lambing. They v/srs r:l&ed for 115^17 days (plus 2 days of colostrum) followed by - c'ry perio f 35-1o d 1 days after whic e eweth h s were divided _n t /o groups for the trials during Phase II (Late Pregnancy).

The total mil a contenkC yiele s 195-5r f th ev/o t pe dwa , e 6kg s 0.217-0wa -ilk 5?1 .J0 (N=2UO) e totaan

14 Bacconsumee ew h d durin f concentrateo g gk Phas2 25 I e s (61%) and 120 kg of roughage (33/0 providing 2.8 kg of Ca per ewe. The protein content of the total die't was 13.5%. £we liveweigh t lambina t g v/as 65.U-7.Li increased an g k - o 67.6t d - f Phaso d . I e en e th t a g k 1 3.

Phase II. Late_ pregnancy .

tri.al

Three ev/fcs frcrn the Ca-deficient diet and six from the control diet remained barren. In audition two ewes from the control diet aborc-ed resulte Th . s thereforee refeth o t r remaining 21 and 16 ewes from the Ga-deficient and control diets, respectively.

Tha intakC e e (g/day e contro f th eweo )n o s l dies wa t 9.U ( including Ca in water) and that of ewes on the Ca-def icier.t 2.14dies .wa t unti f pregnanco e 137t th y l da hd increase an y o t d 16.3 from the 138th until lambing.

Liveweigh f tpregnanc o los 0 s10 froy y mda til l immediately after lambing was similar for bot h- treatments. Litter size and1. la-nb birth weight per cw was also similar. (Table 10). However males were heavier than female singled an s s heavier t'.ian t\f/ins. Protein, phosphoru vitamid an s intaknD r ev/pe e e per clay were similar for both treatments.

.- Ixi ^b olic tr_ial

Two ewes e froon m, each treatment went "off feed" during e collectio th e controth n o le n die) aborteperiodew (D e t On d. and another on the Ca- deficient (E) was barren during the sane period. The data of all ewes v/ere therefore' excluded from all analyses. Endogenous faecal Ca loss, total faecal and urinary Ca losses a absorptio e C rat th ,f balanca eo C d mg/kgLYi/T/daynan ( e ) were highe e contro th n ewe i rdie) n o s(D lt compare o thost d e on the Ca-def icient(E) diet (Table ll). The efficiency of Ca absorptio highes e Ca-deficienwa n th n ewei r n o s t diet.

IS There was a positive relationship between Ca intake and either endogenous faecal Ca loss or urinary Ca loss. The Ca intak alss owa e positively relate o totat d e l Th faeca . Ga l rate of Ca absorption v/as linearly and positively related to Ca retention. But there was no relationship betv/een endogenous mattey faecadr losa d C rlai s intake .

Phase III Metabolic tr_ial

Two ewes, one from each treatment went "off feed" and their data were discarded.

There v/er o significann e t differences bet\7een evvcs -.vhic'.i had receive n lati d e -ore^nancy eithe- r Ca contror o ) (D l cioficient (^) diets in endogenous faecal Ca loss, the rate of Ca absorption ano e othe.th r measurements show n Tabli n. 12 e There was no relationship between Ca intake, and endogenous faeca lossa C l , urinar losa yC r mil o s loss a t kC ther bu , e v;as a positive relationship betwee a intaknC totad an e l faecan O l loss. The rate of Ca absorption was also positively related to Ca retention.

Feeding trial

diee Onew ed from pneumoni o mortw e d ev/ean a s suffered from mastitis in ewes which had received the control (A) Ciet e feedinth n i g trial during late pregnancy. Another tv/o ewes died (one frcm peritonitis and one from gangrenous mastitis) and four more ev/es suffered from mastiti n ewei s s whicd hha receive Ca-deficiene th d e feedin th ) die n i (B gt trial during late pregnancy. The data presented are therefore from the remainin ewe U l 2 g(l fros e contro3 mth fro1 e ) diemth d (A an lt Ca-deficient (B) diet.

Ev/es v/hic h had received either the control (A) or Ca- deficient (B) diet had similar yields of colostrum and of similar chemical composition. The results were therefore poolec. e presenteanar d Tabln i d. 13 e

16 There was no significant effect of the prepartum dietary Ga level on the milk yield, liveweight loss and Ca output in mil f eweo kn earli s y lactation (Table 1U).

The Ga level in scrum at day 137 of pregnancy was higher n ewei s whic received ha h e contros th dwa ) die t (A lbu t similar wit e ewehh t thas f whico t received ha h Ca-deficiene th d t diet immediately post-partum7 afte3 y r da lambin t a d . an g(Tabl ) 15 e

e pre-partu Thlevea th C e n i l m e lameffeco th n diet bd n i tha s birth weight or weaning weight.

DISCUSSION

Calcium metabolism is influenced by other nutritional factors suc s phosphorusa h ,e level vitamith d f energo san nD y ".nd protein intake o avoi T y interactio.an d n betweea nG .nctabolism and particularly Ca absorption from the intestine, the experimental diets v/ere balanced for these nutrients. n additionI e concentratth , e mixtures were pellete o achievt d e uniform intake of nutrients.

Some ewes v/ere unable to adapt to the conditions of the metabolic crates and the first symptom they exhibited was very iow feed intake Trian I . (dr1 l y non-pregnant ewes) rumen atony absorptioa C r he nils d nwa an . e s Atondiagnoseew e yon n i d of the digestive tract v/as reported to impair Ca metabolism (20). The endogenous faecal Ca loss in this ewe was 3.8 nig/k^ L'7T/da d thian yrepresenalus y v ema e actuath t l endogenoua C s excretion.

Trial 1. Dry non-prej^nant ewes.

Th ea absorptio C rat f o e n increase e efficiencth d an d f o y Ca absorption decreased with increasing level of Ca intake. These results are in agreement v/ith those reported for wether or youn maturd an g e sheep (ill, 21). Hov/evea e ratC th f r o e balanca absorptioC e th e d increasean n d unti intaka C l e reached level 133mg/kgL1.ifT/d, despite the fact that Ca balance was positive froe lowesmth t intaka leveC f o el (6l/mg/kgLWT/d).

17 This may indicate that ev/es, contrary to wethers or mature sheep continu o replenist e lossesa hC , which incurred from e previouth s lactation absorptioa C rate f Th eo . n (mg/kgL\VT/d) v/as highly relate a retentioC o t d n (mg/kgL".YT/d) equatioe Th . n describing this relationship v/as Y=13.10+1.07X; setting X=0, e resultinth g valu f 13.1o e 0 mg/kgL'.VT/ represeny ma d e th t endogenous faecal and urinary Ca losses of dry non-pregnant c--/es. This experiment was carried out at daily temperatures of ove lossea rC 32°d san C froe respiratormth y tracd an t s.vcating might have been another source of Ca loss. Therefore, endogenous losses might have been increased slightl o 1U.yt / 5ng o account e t r ew requirement fo ne tg k ivgir/T/e 0 th 6 d a an df o s suc hmg/day0 losse87 e ar .s

.Tria . 2 Lactatinl g ewes.

The rate a nd the efficiency of Ca absorption for the two diets with a Ca:P ratio of 2.3:1 and 1.2:1 were similar and slightly higher thae correspondinth n g e valueclieth tf o swit h a Ca:P ratio of 3.2:1. Though ruminants can tolerate a wide range of Ca:P ratio in their diets, if vitamin D and Ca and P intakes arc adequate (l)the present results suggest that the lo\v phosphorus leve f dieo l (Ca^A t P ratio 3.2:1) might enhanced e excretio n th faecei a C sf o n(22 resulted )an o lowet d r efficienc absorptiona C f o y .

The yield and composition of colostrum varied considerably among ev/es. The fat, protein and phosphorus were higher and lactos d calciuan e m content were lov/er than normal milk from sheep of the same breed (Economides, unpublished).

Tria . 3 Phase_I^J?hasl . PhasII e e III.

Ev/es wer d adequatfe e e quantitie f energyo s , proteid nan calcium from lambin 0 dayn matin f ra o matin10 t o gsf o d t g an g pregnancy (whe e dietarth n levela yC s were imposed o overcomt ) e the increased stress from lambing twice in one year and to replenis l possiblal h e losse f nutriento s s frohigd mth h demands of lactation.

18 The Da-deficient die n lati t e pregnancy causee r a d e endogenouth " c. s faecal and. urinar a lossesyC , v/hile th e efficiency of Ca absorption increased to 58%. The lav enc faeca urinard an l lossea yC s coul e explainedb adaptation a s a d n - deficienCa e th t o t die e increaset th (13d an ) d efficiency of Ca absorption may be partly due to lov; Ca intake or to a stimulation fron the feeding of the Ca-deficient fiet and the balance (mother+foetuses; increased demand for Ca in late pregnancy (23). The positive C: / found in this trial is in agreement with reports for pregnant e-wes fed 8 gCa/head daily (ll). It is possible that in the ewes fed the Ca- deficient die6 the rate of bone resorption increased considerably, resulting in a negative Ca balance for the mother alone.

The rate and the efficiency of Ca absorption were similar and the Ca balance was positive in early lactation in ewes which had received either the control or the Ca-deficient diet until f pregnancyo 7 day13 t I seem. s tha increasen a t d consumption of Ca in the diet in early lactation resulted in an increase of the net Ca absorbed (l68 mg/kg LV7T/d). Sansom (214.) had also suggested that high feed intake is essential for dairy cows after parturitio e maintenancth r nfo adequatn a f o esuppla eC y for peak lactation. The results on Ca balance are in agreement with thos f Sykeo e Dingwal& s l (12) whea e C rat th nf o e absorption was high (115 mg/kg L'7T/d) but at variance with thos f Braithwaito e absorptioa ratC w f elo o e (23a t n)a (U3 mg/kgL\7T/d) .

e feedinth n I g trial e pre-partusth m intak a leveC f o el had no effect on ewe liveweight changes until lambing and from lambing to day 37 post partum and on the lamb birth v/eight and weaning weight. The yield and chemical composition of colostrun •and the milk yield of ewes were also not affected. Blood Ca levels immediately after lanbing and on day 37 post partum were not affected by the prepartum level of Ca intake.

19 f pregnanco 7 13 Howeve y yda bloon o ra leve e dC f th eweo ln o s control diet was higher tnan that of ewes on the Ca deficient diet, but both blood values v/ere normal.

The level of Ca intake was not related to the faecal or urinary Ca losses or Ca output in milk, \7hen the data from dry, pregnant, and lactating sheep v/ere pooled, the regression equation describin e relationshith g p between total faecaa intakC ) botd ln (X emg/kgLWT/daan i h los) ($ s y was: 9.1LH-0.7UX(R= $ . ) Tota 29 =.9 l faeca losa alss C l swa o related absorptiona e C rat th f o e o equatioe t Th . n resulting from e pooleth d dat f dryo a , pregnan lactatind an t g shees pwa Y=-2.i46+0.l4X(R2=.G9), where Y= the rate of Ca absorption and e totaXth = l faeca losa C l s (bot n mg/kgLWT/day)i h .

The Ca retention and the rate of Ca absorption were also related equatioe Th . n describin e relationshigth p froe mth pooled data was: Y=l4.1.8+1.6x(R2=.{4-5) where, $= the rate of Ca a retentioC absorptio e th X n d (botan n n mg/ki h g LV/T/day).

The mean endogenous faeca losa C ln dry i s , pregnand an t lactating animal 11.67-3.10s wa s , 13.32^3. 29.3d 0an 0 rag/kgL'VT/d, respectively. In ail trials dry matter intake was related to the endogenous faecal Ca loss. The pooled regression equation resulting frcm the dry, pregnant (excluding the U animals on the Ca-deficient diet) and the lactating sheep was: 2,U+O.8X(Y= * R =.8U) wher = endogenoue$ s faeca a losC l s (y matterng/kgl/VT/dr = X r d intakan d) e g/kg LWT/d.

The author wishes to thanl: Dr. D.G. Braithwaite of the ITational Institut r Researcfo e n Dairyingi h , Shinfield, U.K., for his assistance in the processing of the data, Dr.^L. nacljisenonos , of the Veterinary Department, Cyprus, and the staff of the Chemistry Laboratory and the Animal Prediction 3:ction of the Agricultural Research Institute for their technical assistance.

20 . 1 Todd, J.R. (1976). Calcium, phosphoru magnesiud an s m nctai bu v/ith particular referenc o milt e k fever (parturient hypocalcaemia) and grass tetany (hypomagnesaemic tetany) in ruminant animals. In "Nuclear Techniques in Animal Productio d Health"an n . IAEA Vienna.

. 2 Ramberg, C.P. (Jr.), Kronfeld, D.S Wilsond .an , G.D.A. (1-7T) Regulatio f calciuo n m metabolis n cattlmI e during grov/th, gestation, lactation and changes in diet. In "Digestion ar.c1. metabolis e ruminant"th n mi . I.'/VEd . . McDonal A.G.Id an d . Warner. '-The University of Hew England Publishing Unit.

. 5 Wiggers, K.D., Nelson, D.K Jacobd .an , N.Lson . (1975). Preventio f parturieno n tcalciu w paresilo a m y b sdie t prepartum: A field study. J. Dairy Sci., 58: U30-U31.

U. Goings, R.L., Jacobson, N.L., Beitz, B.C., Litlldike, E.T. and Wiggers, K.D. (197U). Preventio f parturieno n t paresis by a prepartum calcium deficient diet. J. Dairy Sci., 57: 118U-1188.

. 5 Pickard, D.V7. (1975) apparenn A . t reductio e .incidencth n i n e of milk fever achieved by regulation of the dietary intake of calcium and phosphorus. Br. Vet. J. 131 : 7L\l

. 6 Symonds, H.W., Manstcn , PayneR. , , J.M d Sansoman . , B.F. (1966). Changes in the calcium and phosphorus requirements of the dairy cow at parturition v/ith particular reference e amountth o t s supplie foetue th uteron i o s. dt Vet Br . . , 122J. : 196-200.

. 7 Symonds, K.\V ., Sans on,' ii.PTv/ardockd an . , A.R. (1972). T.ie measuremen e transfeth f o tf calciuo r d phosphoruman s frora e sheeth n pi usinm foetuda y/hola ^o t s e body counter, lies. Vet. Sci., 13:272-275.

21 6. Braithwaite, G.D., Glascock, R.F. and Riazuddin, Sh. Calcium metabolis pregnann mi . t J Nutr ewes. k Br 2 . . 661 - 669.

9. Field, A.G. and Suttle, N.P. (196?). Retention of calcium, phosphorus, magnesium, sodium and potassium by the developing sheep foetus . AgricJ . . Sci., Camb. : 1|17-U2369 , .

10. Twardock, A.R., Symonds, H.W. , Sansom, B.F. and Rowlands, G.J. (1973) effece f Th litteo .t r size upon foetal growth e placentath ratd an e l transfe f calo r cphosphoru d iuran a s in superovulated Scottish half-bred ewes. Br. J. Nutr.,

11. Braithwaite, G.D., Glascock, R.F. and Riazuddin, Sh. Calcium metabolism in lactating ewes. Br. J. Nutr., 23:827-83U.

12. Sykes, A.R. and Dingwall, B .A. (1975). Calcium absorption during lactation in sheep with de mineralized skeletons. . AgricJ . Sci., Camb. 2U: 5- 8U 2i|8 , .

13. Braithwaite, G.D., (197U). The effects of changes of clietr. calcium concentration on calcium metabolism in sheep. Br. J. Nutr. 31: 319-331.

14. Braithwaite, G.D. and Riazuddin, Sh. (1971). The effect leved an f dietar o e l oag f y calcium intak calciun o e m metabolism in sheep. Br. J. Nutr., 26:215-225.

, " NationaI l Research Council (1975) Nutriont requirementf o s . 5 sheepnationa . No . l Academ f Sciencesc y . Washington, B.C.

16. Agricultural Research Council (1965). The nutrient requirements of farm livestock. No. 2. Ruminants, LonScn: . 0 . HM3 . .

22 I/-. I'!A7F (1973) Analysie Th . f Agriculturao s l Materials. Technical Bulleti . Ministrn27 f Agricultureo y , Fisheries and Pood. London. K.I-i.S.O.

18. '.Villis, J.B. (I96l). Determination of calcium and magnesium n urini y atomib e c absorption spectroscopy. Anal. Chem. 22 556-559.

. Willis19 , J.B. (I960) determinatioe Th . f metalo n "bloodn i s , serum by atomic absorption spectroscopy - I Calcium Spectrochimica Acta. 16: 259-272.

20. Payne, J.M. De;?,, S.H., Mansion, B. ana Vagg, K.J. (1970). Jietabolic disorder e ruminantth f o s : Hypocalcaemid an a hypomagnesaenia n "PhysiologI . f Digestioyo Metabolicd an n n in the Ruminant". Oriel Press.

21. Braithwaite, G.P. (1975). Studies on the absorption and retentio f calciuo n d phosphoruman y youn b smaturd an g e Ca-deficient . sheepJ Nutr. . 311-321;: Br .3U , .

22. Chicco, C.P., Ammernan, C.B., Peaster, J.E. and Dunavant, E.G. (1973) Nutritional Intcrrelitionship f dietaro s y calcium, phosphorus, magnesium in sheep. J. Anim. Sci. ^6 986-993.

. Braithwaite23 , G.D. (1978) effece Th f dietaro . t y calciuir. intake of cv/es in pregnancy on their calcium an-1 phosphcr^z: metabolis lactation mi . Nutr.J . : 213-218nBr 39 , .

2ii. Sansora, B.P. (1969) Calcium metabolism of cov/s at parturition and. during milk production. J. An;r. Sci. Canb. 2 U55-U582 .

23 C—lA ThG intake of different nutrients of dry non pregnant ev/e Trian i s . 1 l

Treatments ——— i A B C D

". Jt'.'DJr of animals u 3 3 U :ry natter intake (kg/day) 0.71 0.70 0.71 0.69 ". -cccin intake (g/day) 71 69 7U 67 "7itamin intake (l.U./day) i|80 U20 U90 U70 55

Phosphoriis intake (g/day) 2.70 2.70 2.90 2.60 Calcium intake (g/day) 3.50 5.50 8.20 9.80 1.1C

Ja'ol . 2 ePercen t compositiof o n cliets in Trial 2.

T5iet

' irley grain 79.9 79.0 76.25 "oyabean meal 16.0 16.0 16,0 Hicalciura phosphate - 0.9 3.0 sodiui Tr m phosphate - 0.6 3.0 Limestone 3.6 3.0 1.25 Salt 0.5 0.5 0.5 Y it. -I'.; in. nix. (kg/ton finished 2 2 2 feed) Zn30.(g/ton finished feed) 50 50 50

24 able The Intake of different nutrients of lactating ewe Trian i s . 2 l

Treatment

A B C dumber of animals 3 k U Initial weight (kc) 53.3 51.9 5C.L. Dry natter intake (kg/day) 1.79 1.68 1.77 Protein intake (g/day) 27U 260 267 Ca intake (g/day) 22.1 23.5 22.2 Phosphorus intake (g/day) 6.9 10.2 17.9 Vitami intaknD e (l.U./day) 1500 1U56 1U70 Ca:P 3.2:1 2.3:1 1.25:1

25 "abl-j L[. Percent composition of iU<. ts in Tri'U.

Diets I - ——————— -1 A B c D E p

''"'arlc^ y 87.7 90.0 79.5 70.35 72.6 78.0 loyibean moil 9.8 9.5 16.0 26.0 26.0 16.0 2.0 - 3.0 2.25 - U.5 Trisodium phosphate - - 1.0 1.0 1.0 1.0 Salt 0.5 0.5 0.5 O.k o.u 0.5 Trace elements (kg/ton finished feed) 1 1 1 1 1 1 ZnSOr (fxton finished feed) - - 50 - - 50 Vitani (l.U./knD c finished feed) too uoo 800 UOO £4.00 800 e intakeTh f differeno -. 5 t nutrient pre^nn.ny b s t ev/es in Trial 3, Phase II.

Diet

Control Ca-cief icicm

of animals u U -

Initial weight (kg) 6U.9 70.2 ^ - • . - Dry matter intake (kg/c!.ay) 1.01 1.01 o.ou Crude protein intake (a/day) 16U 157 7 Ca intake (g/day) 8.2 3.0 C.I Phosphorus intake (a/day) U.9 5.2 0.2 Vitami intaknD e (l.U./ciay) U26 W-U o

The intake f cliffercno - t nutrient y lactatinb s g c-.v_ in Trial 3. Phase III.

Diet 3.3. i Control C a-deficient

I'urcbcr of animals u U - Initial v;eight (kg) 5U.3 U9.3 U.I Dry matter intake (kg/day) 1.65 1.65 0.07 Crude protein intake (g/day) 235 238 10 Ca intake (g/day) 27.2 27. U o.U Phosphorus intake (g/day) 10.3 10.3 0.2 Vitamin D intake (l.U./day) 1182 1193 20

27 Tabl . 7 eEndogenou s faeca a lossC l ,absorptioa C rat f o e n anc.

Ca "balanc f o ^.r e n pregnanyno t ev/cs (mg/kg I.'iTJ/c'.ay) (Tria) l l

Treatments c- —* D C 3 A

- U 3 3 U rur.b;, f animalo r s Ca intake 6l.lt 96.7 132.7 165.3 23.1 Endogenous faecal Ca loss 13.7 H.5 11.6 11.9 2.6 Total faecal loss 51.1 78.0 103.6 135.1 19.5 a absorptioRatC f o e n 2*4..0 32.1 U0.6 U2.7 08. Efficiency of Ca absorption 39.3 32.6 30.9 25.6 6.5 (%) 9 7. 17.0 Ca .25.balanc7e 27. 2 87.

. 6 Yiel f colostruo d m ancs chenicait L l composition

(c/10 ; 0; milk) . (Tria) 2 l

Mean

Total yield (kg/i4.8h) 3.53 1.50 0.0V 7. 0 - rat 10.8 u.i 5.6 - L:.: Protein 1U.U 5 2. 10.6 - 19.5 Lactose 3-3 0.8 1.5 - -4-. 3 3 1. - 7 0. 2 0. 0 1. Ash Total solids 29. 8 65. 18. - 7^0. 6 Calcium 0.15 O.OU 0.0 7- 0.22 Phosphorus 0.1? 0.03 0.08 - 0.23

28 Endogenous f';ccal Ci loss, rate of Ca absorption and Ga balance of lactating ewes (mg/kg L'.VT/clay) .

(Trial 2)

Diet

A B C

rimbe f animalo r s 3 k k - L'VT of animals (kg) 53.3 51.9 50.U 3.U- tlilk yield (kg/day) 1.88 1.78 1.81 0.52 Ca intake U19.2 U53.1 UU1.1 3U.3 Urine output 7 0. 3 1.1. 2 1 1. Ga output in milk 79.3 68. k 79.8 23.3 ZncLogenous faecal Ga loss 25.7 2U.3 28.3 3. 3 Total faecal loss 337.8 3UU.O 336.9 32.1 absorptioa C Sat f o e n 107.0 133.U 133.0 26.2 Lfficiency of Ga absorption(?o) 25.3 29.U 30.1 5.1 Ca balance 0.9 39.6 23.1 32.6

-iVole 10. Performance data of CY/*-* s a u uwo levels of ' Ca intake in late pregnancy. (Tria , 3 Phasl ) II e . Diet i i A B Control G a -deficient

ITur.ibcr of animals 16 21 Day n experimeno s t 52.1 51.5 2.7 Gestation length (days) 151.7 9 1. 150.7 Dry matter intake (kg) 1.2U - 1.22 Protein intak) (g e 1U3 1U7 Phosphorus intak) (g e U.7 U.5 1'iveweight changes (kg) - 3.3U 7. 0. k - Wof lambo . s born/ewe 2.12 1.76 0.£2 No. of lambs born alive/ ewe 1.87 1.57 O.cC Birth weigh f lambo t s (kg) 3.6(3U) 3.9(37) C,?i Birth weigh f lambo t s a s a lambine percenew f o gt weight 11.9 11.1 i:,'c 29 Tabl j ll eEndogenou s faeca lossa C l ,absorptioa C rat f o e n anbalanca G d f pregnano e t cv/es (m^kg LV/T/day) (Trial 3,Phase II).

Diet 3,1;.. D E Control Ca-:7ef icient

1'T umber of anir.als k k - Ca intake 12k.7 W-.8 3.8 r.ndogenous faecal loss 13.3 8.8 1.6 Total faecal loss 101.7 26.k 2.1 Urine Ca loss 1.2 0.5 O.U absorptioa RatC f o e n 36.3 2U.1 33. Efficiency of Ca absorption($) 29.1 57.6 2.9 Ca balance 21.8 1U.9 2.U

.'' cl^, 12. The effect of propartun dietary Ca level on the absorptioa C rat balancf a o eC d f an lactatinino e 1

cv/cs (me/kg L'TT/day). (Trial 3, Phase III)

Diet

Control Ca-def icient c?,~

h U /•OTibei f o animal* s Ca intake 500 551 39 Drilocenous faecal loss 30.9 33.2 3.3 Total faecal loss 369 W>9 2S 3 1 5 17 2 16 a absorptioj^atC f o e n Efficiency of Ca absorption (%) 32.k 31.8 C.7 Urine Ca loss 2.0 2.0 l.C Ca outpu n mili t k 75.2 79.7 1C.6 2 2. 1 6 k 5 Ca balance

30 '-'-ble li|« ^ effect of prepartum Ca level in the diet nc e performancoth n f cv/eo e n earli s y lactation.

(Trial 3, Phase III).

Diet ———————————— r —^ • 1 I.D. Ca-def icicnt Control

of animals 13 11 Ililk yield (kg) 70.6 73.3 30.0 Ca output in milk (s/day) 3.67 3.66 1.C6 Initial weight (kg) 68.3 67.6 5.6 Vifeight changes (kg) -4.0 -2.7 3.6 Concentrate intake (kg) 58.7 61.6

Barle intaky ha y e (kg) 18.4 18.6 Ca intake (g/day) 26.5 27.8

Tabl . Yiel13 e f colostruo d itechcnicad man l composition (g/lOOg milk). (Trial 3. Phase III).

Mean S.D.

Total yield (k,yU8h) 3.62 1.26 0.1 - 5.^5 "•at 12. UO 4.98 6.U - 2ii.O Protein 16.10 3.65 8.5b- 23.45 Lactose 2.87 0.69 1.3 - a.2 -ish 1.17 0.23 0. 8- 1.7 9 Total solids 32.40 7.00 21.96- 1+5.6k Calcium 0.17 0.06 0.04- 0.33 Phosphorus 0.19 0.03 O.Ik- 0.27

31 THE USE OF PHOSPHORUS-32 IN THE DIAGNOSIS OF PHOSPHORUS DEFICIENCY IN SHEEP

A.O. LOBÃO, D.M.S.S. VITTI MARCONDES, J.W. LEMOS, M.I.B. PEIXOTO ESCUBEDO Centr Energie od a Nuclea Agricultura rn a (CENA), Piracicaba. P . S , A.O. LOBÃO, A.A.D. DE OLIVEIRA Instituto de Zootecnia (IZ), Nova Odessa, S.P., Brazil W . BINNERT.T S Landbouwhogeschool, Wageningen, The Netherlands

ABSTRACT

Phosphorus deficienc s beeha y n recorde cattln i d mosn i e t states of Brazil and while clinical deficiencies are easily recognised and corrected, subclinical deficiencies often go undetected. The development of a method for the diagnosis of borderline deficiencies would have considerable value since such deficiencie e economicallar s y very important. In the experiments reported here, a variety of methods were employed for diagnosing phosphorus deficienc n ruminantsi y , includin e uptakth g f o e y rumeb p n32 microorganism y erythrocytesb d san e resultTh . s show that P deficiency depresses the synthetic capacity of rumen microflora, indicating that P uptake might be of some value in the detection of such deficiency. By contrast, the uptake of P by erythrocytes was unre- lateanimale P statu th o t df o s.

n With an area of 8,511,965 km Brasil ranks as the third largest cattle breeder in the world. Although considered as 'the fourth neat producer, the herds' productivity is very low due to incidenc infecto-contagiouf o e s diseases, parasite qualitativd san e d quantitativan e nutritional deficiencies. Literature published so far in Brasil reveals that extensively bred cattl n severai e l regions deficiene ;ar n phosphorusi t , copper, cobalt and iodine (TOKARNIA & DÖBEREINER 1976). Phosphorus deficiency in cattle has been detected in almost e Statesth l somn al ,i e regions seriously affectin animalse gth , which show los appetitef so , poor development, rickets, osteo- nalformation, ingestio bonef no s foun pasturn o d whic- e h pro- vokes botulis animalse th n i fertilitw m ,lo y etc.

The authors wish to thank Inger Wegger, Dep of Physiology Endocrinolog d Bloodgroupingyan Royae ,Th l Veterinard yan Agricultural College, Copenhagen, Denmar assistancr kfo n i e preparing the manuscript.

33 When clinical symptoms of deficiency or toxicity are clear, corrective measures can be taken immediately, the animals soon becoming satisfactorily productive again. However, borderline deficiencies are economically more important. Due to the lack of clinical symptoms in such deficiencies no special care is given to the animals to improve their potential productivity. The development of a method for the detection and diagnosis of borderline deficiencies is very important because correction of these disturbances in their initial phase can be made with more adequate diets. This will improv e productivitth e herde th f so y (UNDERWOOD 1969). Research workers have indicate e leveth df inorgani o l c phosphoru n plasm i sparametea e diagnosis a th r fo rpho'nphoruf o s o deficienc n animalsi y . Experiments carrie t witou d h lamb- re a vealed that there is a positive correlation between phosphorus intake per unit body weight and blood phosphorus concentration. However, the level of this element in blood does not always reflect phosphorus intake n caseI . f starvationso , inadequat- in e gestio f proteio n d energnan y resultin losn i gweightf o s e th , conten f inorganio t c phosphoru e blooth n di s increases. These levels can also vary with age, behaviour of the animals (feeding, ruminatin resting)r o g , stress etc. (MOODIE 1975). Considerin w availabilitlo e gth f phosphoruo y Brasilian i s n soil resulting in a low content of this element in forages and a high incidenc f phosphoruo e s deficienc n animalsi y t seemi , - im s portant to develop more efficient methods for diagnosing borderline deficiencies regarding this mineral. The main objectiv thif o e s research projeco compart s wa te different method f detectino s g phosphorus deficienc ruminantn i y d an s to study the possibility of using radioisptopes for this purpose.

EXPERIMENTAL ANIMALS AND METHODS Twelve castrated sheep were divided in 3 groups (A, B, C) and kept metabolin i c 3 weeks1 cage e dier Th .fo st comprised grass hay, molasses and casein. Phosphorus was supplemented as sodium phosphate (NapHPOx) at the levels of 2 and 4 g per day to animals in respectively, C group d an sB , animal t receivno groun d si di e pA phosphorus supplementation e sodiuTh . m contene dietth f so t was.

34 balanced with NaCl e treatmenTh .three th ef o tanima l groups si e schemshowth n nei below.

Animal Na?HP04 Phosphorus NaCl Sodium group g/day S/day g/day A - 0 15.26 6.0 B 11.50 2 7.60 6.0 C 23.00 4 - 6.0

The animals were weighed weekly, feed and water ingestion was recorded daily. Urine and faeces were collected each day mid weekly po.ol samples prepared. Sample rumef so n fluid (20) 0ml were taken onc weea e k usin plastiga c tube fluie s filte,th wu d - d througre h cheese-cloth befor measuremenH p e d analysan t ewera e performed. Blood stabilize) sampleml 0 (5 s d with heparin were also collected each week. The concentratio inorganif o n c phosphoru plaamn i s d rumeaan n fluid - diluted 20 times with distilled water - was measured colo- rimetrically (PISKE & SUBBAROW 1925) and the alkaline phosphatase activit plasmn i y determines awa methoe th KIN f y o d b d KIND& G (1954). Sample f faeceso s were grae groundon m d aliquotean , s were pla- cecruciblen i d determinatior fo s mattey dr f ro n (100°Ch as d )an (500°C) content. The ash was dissolved in 5 ml HC1 and diluted to 100 ml with distilled water whereafter the phosphorus content s determinewa e above.mentionemeany b th d f o s d e methodcalth -d 'an cium concentratio y atominb c absorption spectroscopy. Thvitrn i e rumey b o P uptakn microorganism f o e measures swa •50 d in the following way. One ml of the filtered rumen liquid was transferre tesa o tdt tube containin glucoseg m ureg 0 m 30 ga0 3 , and 19 ml artificial saliva free from phosphorus (DURAND et al 1976). Triplicate determinations were made on each sample. The tubes were

place watea bubbles n wa i dr 9 batdC0 t 39°througd a h Can mixture th h e minutes5 r fo n aliquo.A P (0.32 f 1o t pCi addes )wa eaco dt h tube e incubatioth d an s continue nwa houre on .r fo dAfte r coolino gt 8°C 1 ml HpSO. ( 5 N) was added to each tube and the liquid cen- e supernatanth f trifuge o minutes0 2 l m r te t 2000fo a On d . m 0rp o o s take wa determinatior nfo activitP f o n y usin Cerenkoe gth - ef v r measuremenfo fecd an t stablf o t e phosphorus e precipitatTh . e was suspended in 10 ml of saline solution (0.85 %} and centrifuged for 5 minutes at 2000 rpm, the supernatant was then centrifuged •50 at 20000 rpm for 20 minutes and the J P activity in the precipitate was determined after addition of distilled water.

35 The calculation of phosphorus uptake was based on the woik of VAN NEVE DEMEYE& L R (1973):

P inc. • - phosphorus incorporated dpi.i inc. - disintegrations per minute in precipitate Sa poo speciii= l c activit e phosphatth f o y e pool n i r.'ipc'tt in " nit mg P

n vrtrI y erythrocyteob P uptak f o s eals wa os determined. Ten ml of hcparinized blood (3 replicates per treatment) were p] u- ced in Erlenmeyer flasks and ^0 ml of a solution containing 0.2'j yd -> p were added. The samples were incubated at 39°C in a sha- o hourstw r .fo r Durinke e fir^th g 5 minutet a mixturs 5 '/<>9 f o e

02 and 5 % COp was bubbled through the flasks and this procedure wae incubatioth s f repeateo d en n e dperio th afte t e houA don rr e sampleth s were centnfuged (3000 rpm) under refrigeratione th , d bloore d cells were washed three times with salin d ashean e d s treatewa h (500°C)das wite e Th h-solutio th .t-^SO d an , n filtered before determinatio e radioactivityth f o n . The uptake of P by the erythrocyt^s was calculated and correcte r differencefo d n celi s l e volumoriginath n i e l blood samplee equatiomeany th b s f o s n establishe l a (1967)y BUR b dt e K :

40 uptake, correcte activit= d x n cellO i y 1Q x s total activity cell volume % Twenty four hours prior to termination of the experiments most of the animals were dosed intravenously with approximately 130 •j o of carrier- free P as Na^HPO , . Blood samples were collected 5, 10, ?0, 30, 40, 50, 60, 120 and 240 minutes after the injection and the radioactivity in plasma and erythrocytes was determined. Thn vivi ey erythrocyte ob P uptak f o e s calculateswa e th n i d 32 same way as the in vitro uptake. After twenty four hour e animalth s s were sacrifice y bleedinb d g d samplee followinan th f o s g tissues were obtained: liver, kidney, heart, tongue, left rib, metacarpus and wool. The intestinal content was also collected. All samples were ground and ashed as described earlier ( LOBAO 1973) before determination of•)O P activity, the activity was expressed as percentage of dose retained per gram of dry matter.

36 RESULT DISCUSSIOD SAN N The average pH values in rumen fluid from the three groups of sheep e showar n Figuri n . 1 eStatistica l analysis showed thavarie th t - e rumes treatmentth ou f t ninfluenco no fluid H d p di e s. th e Thij n agreemeni s i t wite resultth h f PONNESBECo s . (1970al o t e K)wh found that different levels of phosphorus did not change the pll of rumen content a linea t bu ,r decreas concentratioe th n i e f volao n - tile fatt e additioth y o t acid f phosphoruo e n sdu a noteswa 3 d hours after feeding. e rumeth usualls n i i 0 ThH p 7. e y o t withi 0 range 5- th nf o e e variationth d an s diete e naturdepenth th ,f n o o edtim e after feedin d watean g r ingcstion e bufferinTh . g capacit e rumeth f no y fluid is considerable'primarily due to the bicarbonate and phosphate content (SWENSON 1970; CHURCH et al. 1971). EMMANUEL et al. (1970) also observed that the buffering capacity of phosphate maintained a high pH value in the rumen, especially 2 to 3 hours after feeding. The phosphorus concentration in the rumen fluid varied with the dietary t emergelevei s a l s e Pishefroth md rTablan tes, 1 et showed thae differenceth t s betwee e threth n e treatment -groups were significanlevel% 1 e . th EVANt a t DAVIS& S (1961 PRESTOd )an N& PPANDER (1964) have also reported that the phosphorus content in rumen liquid change n proportioi d e dietarth o nt y level, while CLARK (1953) observed that a high concentration of water-soluble phosphorus was maintained in the rumen irrespective of the dietary conten. HOFLUWD & HEDSTROM (1948) postulated that the presence of water soluble phosphate in the ruminal content was essential for the maintenance of a normal flora. They stated that, on a phosphorus deficient diet, it was necessary to supplement with water-soluble phosphate in order to maintain ruminal digestion. ANDER30N et al. (1956) also demonstrated by means of in vitro experiments that ru- microorganismn me s hav phosphorua e s requirement. •>2 The in vitro incorporation of P by rumen microorganisms is show n Tablni . 2 eStatistica l analysi resulte th f so s showed that the overall average incorporation in groups D and C was significantly higher (P<0.05) tha groun ni .wherea, pA differenco sn s wa e found between treatments B and C. Although the same trend was seen n individuao l sampling daye variatioth s n within group sheef o s p s considerablwa e hencd (Tabl differencee an th e) 2 e s between groups on a single occasion were not statistically significant. The ability to secure a high level of biosyr.thesis of microbial proteir. ir, the rumen is a good quality criterion for ruminant feedstuffs (NIKOL1C cited by HARMEYER et al. 1975). In most cases it

37 is difficult to determine the net rate of microbiul protein synthesis directly due to the fact that both microtial ami Toed proteins are present in rumen contents and these typos of proLoiu usually cannoe b t distinguishe y chemicab d l methods (HARMEYEt e K . 1975)al . Instead indirect methods suc s incorporatioa h f isoo n - topic markers into rumen microorganism e use b s indicator a dy ma s s for microbial protein synthesis capacity. Several procedures for this purpose usin incorporatioe th g havP e f beeo n n described->2 (DURAN. 1975al . 1975t NEVEN e D;al VA t ,Le 1976; HARMEYE. al t e R 1975). e resultTh s show n Tabli n 2 suppore e abovth t e mentioned view that phosphorus deficiency depresses the synthetic capacity of the ruminal microflora and, on the other hand, also indicate that the n vitri o incorporatiomicroorganisme th y b P f o ns f migho e b t 32 some value in the detection of phosphorus deficiency in ruminants although hardly applicable under field conditions. in plasma The concentration f inorganio s c phosphorus/are show n Tabli n . 3 e d significantlha Shee C groupn i d p an sB y higher plasma phosphorus levels than thos n grouP i e( 0.001) A p t increasin,bu daile th g y phosphoru ) cause(C o furtheg n d s 4 intak- o t in r ) e(B frog 2 m creas n plasmi e a phosphate content. The various treatments had no influence on the activity of alkaline phosphatase in plasma. Thus the inverse relationship between inorganic phosphorus concentratio d alkalinephosphatasan n e activity reported for cattle' (LOPES et al. 1973) was not seen in the present investigations, isotope In vitro/methods for the diagnosis of mineral deficiencies have been develope r zind seleniufo dan c m . (BERR1966al t ;e Y BURt e K al. 1967). The procedures are based on the uptake of Zn or ' Se by erythrocytes which was shown to Le inversely related to dietary intak d plasman e a concentratio f theso n e trace elementss ha t I . •>o been known for many years that P is also taken up by erythrocytes n vitri o (HEVES l 1944)a t t apparentlYe , bu r fa o attempo n ys s ha t been made to utilize this uptake as a measure for phosphorus status in animals. The results of the present study are shown in Table 4 from which s obviouii t s thae differenth t t dietary phosphorus t levelno d di s ->2 affect the J P uptake by erythrocytes in vitro. Thus in contrast e tracth o et elements mentioned above thio valun ss f a eo tes s i t a diagnostic aid for detecting phosphorus status. One might speculate whether this difference between phosphorus e sidd zincon an en o and'seleniu e othe th o dift n ro e m - couldu e b d ference metabolin i s c fate after accumulatio erythrocytee th n ni .

38 Zinc and selenium in erythrocytes are mainly present as components enzymee th f o s carbonic anhydras glutathiond an e e peroxidase- re , spectively e tracTh . e element e presumablar s y incorporate then i d - se enzyme n clossi e connectio e synthesith e proteio t nth f o s n moi- ety n othei , r e formatiowordth e erythrocyto t sth f no e itself. As far as phosphorus is concerned it is on the contrary well known that phosphorylation processes take place also in mature erythrocyte s weln a vitrosi n vivi ls a o. vitrn i Thu e o sth accumulation f zind o seleniuan c erythrocyten i m s migh e regardeb t mainls a d y due to properties of the plasma (concentration of the element in exchangeable pools), while that of phosphorus could be due primarily to properties of the red blood cells such as their content of phosphorylating enzymes. n accuratA e metho measurinr fo d g phosphorus intak graziny b e g animals has not yet been developed (MeDONALD 1968). BROMFIELD & JONES (1970) however, foun relationshia d p between phosphoru- in s takd faecaan e l phosphorus excretion e existencTh . - f sucre o e a h lation is confirmed by the results summarized in Tables 5 and 6. A statistic analysis (Tu/key test) of the results showed that the faecal content of phosphorus increased significantly from group A over B to group C sheep. Furthermore a significant positive correlation (r = 0.83) was found between intake and excretion of phosphorus. e differenTh t dietary level f phosphoruo s sa sligh alsd - ha oin t fluence on faecal calcium excretion but in a non-systematic way since grou B showep highese th d t o valuedifferencn d an s s seewa e n between groups A and C (Table 7). No good correlation was found between ingestion of calcium and excretion in faeces (r = 0.40). The results of the short term study of radiophosphorus motabo- •30 lism after intravenou se showinjectio ar Figurn d i nP an 2 ef o n . 9 Tabled an s8 Blood is considered the central metabolic pool in which phosphorus is transported in the processes of absorption, exchange, mobilization and excretion. The mixing of an intravenously injec- d radio'te opt iso e with plasm s i usualla y complete withi minute5 n s (COMA WASSERMA& R N 1957). -)r) Figur show2 e disappearance th s P fro J mf o eplasm a whicj a; h also observe y . KOIILE(1971b dal t e R) followed exponential curves. The disappearance rate was funtcst in the r.hcop rcM-civin^ rx diet whilP n o differenci n e w lo s observewa e d between shee groun i p p B and C. In Table 8 is shown the average in vivo uptake of ^ P by erythro- e threcyteth n evitrn si i groupe oth uptak f animalsr o s fo es A . o influencn f phosphoruo e s intak s seen might wa eI .e noteworth b t y

39 that the percentage of P accumulated in erythrocytes is approximatel e sam n th vivoyi n vitr i ed .an o An analysis of variance showed that the treatment affected the amount of the P dose retained by the animals. Group A (0 g P/ g P/day 4 ( C ) d showedayan e highes) th d t values e averageth , r fo s werC treatmend e an 0.040 B , A ,t 0.02 d 0.04an ? , respectively% 3 . Table 9 shows the distribution of radioactive phosphorus among tissues. The lowest values were in most cases found in the sheep f grouo , pB however n agreemeni , t wit e resultth h f ABOU-HUSSEIo s N ->2 (1968 e ordeth ) f tissueo r s regardin gs simila retentiowa P r f o n l fothreal r e groups, irrespectiv f dietaro e y intake, namely: liver - kidney heart = tongue - rib = intestinal content metacarpus - wool. This orde f tissueo r s comparabli s e resultth o f t eo s SMITH et al. (1951), while AHRINGTON et al. (1963) found the high- t retentioes n boni n e tissu n theii e r studie cattlen o s . However, LOBAO (1973) found thae amounth t f phosphoruo t s retaine tisy b d - sues was affected by the age of the animals, the level of phospho- e duratio th n serue i experiments d th s man f ru o n .

CONCLUSIONS The data obtained suggest thae followinth t g methode useb dn ca s in the diagnosis of phosphorus deficiency in sheep: Measuremen f phosphoruo t s contene rumeth n ni t fluid. In vitro uptake of ^2P by the rumen microorganisms. Phosphorus conten n faecesi t . Further experiments wil e carrieb l t usinou d g another sourcf o e phosphorus supplement (CaHPO.).

40 Tabl : Leve1 e f phosphoruo l e rumesth (nig/10 nn i flui) ml 0d from sheep subjecte o differendt t dietary phosphorus levels. Averag S.D± e . is shown.

oup w^\f A B C of expt. ^\.

0 49 ± 11 3 ± 1 6 71 ± 8 1 9 ± 1 4 3 62± ' ? 8± 2 2 37+6 70 + 3 6 1 + 2 10 3 46 ± 10 58 + 3 80 ± 19 4 38 ± 6 60 + 2 6 1 ± 1 8 5 35 ± 2 64 ± 5 3 1 ± 7 8 6 3 ± . 44 4 + 7 6 2 1 ± 4 9 7 3 ± 9 3 68 ± 9 5 1 7± 9 8 2 ± 8 3 59 ± 6 83 ± 16 9 34+2 57 + 5 76 ± 8 10 37 ± 3 59 ±11 89 ± 21 11 3 ± 0 3 4 ± 0 5 79 + 11 12 30 ± 3 55 ± 6 69+2

TOTAL 2 ± 8 3 61+2 82 ± 4

Table 2: Incorporation of 32P (mg) by rumen microorganisms from sheep fed different level f phosphoruso s , averag S.D± e .

^\. Group Wee^ ^\ k A B C of expt. ^^\^

0 0.083 ± 0.022 0.091 ± 0.017 0.09 0.00+ 3 8 1 0.10 0.03± 1 9 ± 0.020.1202 ± 0.010.134 5 2 0.077 ± 0.024 0.10 0.012± 3 0.01+ 0.1503 3 0.09 20.01± 8 0.104 + 0.010 0.01± 0.1370 4 0.109 ± 0.026 0.128 ± 0.008 ± 0.010.152 9 5 0.107 + 0.026 0.158 + 0.030 0.20 ± 0.020 2 6 0.089 + 0.038 0.112 ± 0.045 0.12 0.04+ 5 1 7 0.08 0.03+ 6 6 0.115 ± 0.040 0.138 ± 0.043 8 0.083 ± 0.020 0.116 + 0.033 0.14 0.04± 0 2 9 0.103 ± 0.031 0.04+ 0.1488 0.164 ± 0.041 10 0.10 0.04+ 3 2 0.122 + 0.052 0.143 ± 0.050 11 0.079 ± 0.029 0.08 0.02± 6 8 0.09 0.02+ 3 5 12 0.07 0.03± 2 3 0.08 0.03+ 5 3 0.07 ± 0.026 3

TOTAL 0.091 ± 0.007 0.11 0.00± 4 8 0.134 ± 0.009

41 Tabl : Concentratio3 e f phosphoruo n plasmn i s a (mg/10 ) fro0ml m sheep subjected to different dietary levels of phosphorus, average ± S.D.

^^"-v. Group ^ Wee^\ k A B C of^ expt^^ .

0 5.2 ± 0.3 7.9 ± 0.6 0 . 1 ± 9 7. 1 5 0. ± 5 5. 7.7 ± 0.3 8.3 ± 0.3 2 7 0. ± 2 6. 7.2 ± 0.5 7.9 ± 0.8 3 5 0. ± 3 6. 7.8 ± 0.6 8.3 ± 0.9 4 5.6 ± 0.6 6.6 ± 0.3 7.5 ± 0.5 5 5.3 ± 0.3 6.3 ± 0.4 7.2 ± 0.5 6 5.0 ± 0.4 3 0. ± 3 6. 3 0. ± 1 7. 7 6.2 ± 0.5 1 1. ± 9 7. 7.0 ± 1.1 8 4.8 ± 0.4 6.7 ± 0.5 7.3 ± 0.6 9 4 0. ± 7 4. 6.9 ± 0.7 6.6 ± 0.3 10 4.9 ± 0.3 7.6 ± 1.1 3 0. ± 8 6. 11 6 0. ± 3 5. 7 0. ± 6 7. 6.9 ± 0.3 12 5.0 ± 0.5 7.9 ± 1.1 4 0. ± 7 6.

TOTAL 5.4 ± 0.1 7.3 ± 0.2 7.3 ± 0.2

vitrn TablI } byerythrocytesfro : o(% 4 e uptakP 32 f o e m sheep subjecteo t d different levels of phosphorus in the diet, average ± S.D.

^ Grou^\ p Week ^~\^^ A B C of expt. ^\^^ 0 7.2 ± 1.8 7.8 ± 2.6 0 1. ± 2 6. 1 9 0. 5. 5± 5.2 ± 0.7 4 0. 5. ± 1 2 7.2 ± 0.8 3 0. ± 0 6. 7 0. ± 2 6. 3 6.4 ± 0.7 5.8 ± 0.5 6.3 ± 0.8 4 5 0. ± 5 5. 5.3 ± 0.3 5.3 ± 0.4 5 5.6 ± 0.3 4 0. ± 3 5. 6.3 ± 1.3 6 5 0. + 3 5. 5.3 ± 0.5 5.4 ± 0.4 7 6.8 ± 0.7 7.1 ± 0.8 6.4 ± 0.6 8 5.2 ± 0.7 4.6 ± 0.4 5.0 ± 0.4 9 5 0. ± 8 5. 4.9 ± 0.3 5.5 ± 0.3 10 5.1 ± 0.6 5.0 ± 0.2 7 0. ± 3 5. 11 6.3 ± 1.6 4.9 ± 0.3 9 5.0. 9± 12 6.2 ± 1.0 4.8 ± 0.3 5.9 ± 0.9

TOTAL 6.0 ± 0.2 5.6 ± 0.3 5.8 ± 0.2

42 Table 5: Phosphorus intake (g/day) by sheep diets with different levels of phosphorus, average ± S.D. for the entire experimental period.

Group A B C

P intake, g/day 0.688 ± 0.024 2.720 ± 0.020 4.650 ± 0.020

Table 6: Concentration of phosphorus in faeces (%) from sheep subjected to different levels of dietary phosphorus, average ± S.D.

^"\. Group Week ^^\^ A B C of expt. ^^\^^ Q 0.17 ± 0.03 0.2 0.0± 5 4 0.30 ± 0.04 1 0.13 ± 0.02 0.20.0± 6 4 0.39 ± 0.03 2 0.1 0.0± 3 4 0.29 ± 0.05 0.36 ± 0.05 3 0.1 0.0± 5 5 0.32 ± 0.04 0.36 ± 0.07 4 0.14 ± 0.04 0.3 0.0± 1 4 0.39 ± 0.03 5 0.13 ± 0.02 0.3 0.0± 5 6 0.43 ± 0.07 6 0.14 ± 0..01 0.38 ± 0.07 0.50 ± 0.05 7 0.14 ± 0.01 0.3 0.0± 2 5 0.41 ± 0.07 8 0.12 ± 0.02 0.3 0.0± 4 4 0.40.0± 6 6 9 0.12 ± 0.01 0.3 0.0± 3 5 0.45 ± 0.04 10 0.11 ± 0.01 0.35 ± 0.04 0.49 ± 0.04 11 0.1 0.0± 1 3 0.28 ± 0.06 0.42 ± 0.08 12 0.10 ± 0.01 0.3 0.00± 7 0.4 20.0± 9

TOTAL 0.13 ± 0.01 0.3 0.0± 1 1 0.41 ± 0.02

Tabl : Concentratio7 e f calciuno mn faecei fro) m(% s sheep subjecteo t d different levels of dietary phosphorus, average ± S.D. for the entire experimental period.

Group A B C

Cn i faecesa % , 0.4 0.07± 2 0.53 ± 0.01 0.46 ± 0.01

43 n TablvivI y erythrocyte: ob 8 e uptakP f o e) fro (% ms shee d dietfe p s 32 with different levels of phosphorus, average ± S.D.

T. ^\ Group Time \^ A B C after i . v-^\^ injectio f 3ZP\no ^

5 min. 6.59 ± 1.29 6.49 ± 1.65 8.1 ± 3.24 6 10 min. 6.00 ± 1.46 6.56 ± 1.24 8.42 ± 2.40 20 min. 5.0 ± 0.87 9 5.9 ± 1.15 4 7.80 ± 1.99 30 min. 5.05 ± 1.15 5.43 ± 1.25 6.88 ± 1.75 40 min. 5.32 ± 1.25 5.83 ± 0.93 7.39 ± 2.14 50 min. 4.86 ± 1.00 5.3 ± 1.15 3 7.4 ± 2.30 2 60 min. 4.8 ± 1.26 0 5.63 ± 1.10 5.5 ± 82.3 0 120 min. 5.6 ± 61.7 9 5.31 ± 1.44 ± 5.72.64 4 240 min. 5.7 ± 21.5 2 6.7 ± 2.17 7 5.47 ± 2.07 24 hrs. 5.87 ± 1.45 5.28 ± 0.15 4.97

TOTAL 5.4 ± 0.39 6 5.8 ± 0.36 8 7.06 ± 0.66

Tabl : Percentag9 e f doso e e retaine y matte r dr gra f dpe n mtissue o i r f o s sheep fed different levels of phosphorus 24 hours after intravenous injection of 32P, average ± S.D.

^^•^^ Group A B C Tissue ^^^^

Liver 0.111 ± 0.031 0.075 ± 0.014 0.111 ± 0.026 Kidneys 0.081 ± 0.023 0.05 ± 0.006 9 0.114 ± 0.052 Heart 0.05 ± 0.012 3 0.03 ± 0.007 7 0.04 ± 0.002 7 Tongue 0.03 ± 0.002 6 0.019 ± 0.005 0.03 ± 0.000 7 Ribs 0.035 ± 0.018 0.014 ± 0.004 0.01 ± 0.009 8 Intestinal content 0.01 ± 0.004 7 0.01 ± 0.007 6 0.035 ± 0.014 Metacarpus 0.003 ± 0.001 0.00 ± 0.003 1 0.002 ± 0.001 Wool 0.0002 + 0.0001 0.0002 ±0.0001 0.0003 ±0.00003

44 8,1 • . 0 8, •O 5 7,9 - 7,8 c 7-7 Q> 7,6 E 7,S- 2 7.4

M- 7' O 7,2 I 7''' Q. 7,O

1 0 2345 2 6781 I I 0 1 9 Week f experimeno s t

Fig 1— pH of rumen fluid from sheep subjected to different dietary level f phosphoruo s s

IOO-

to 'O

1 05102030405060 120 240 Tim minutes( e )

DisappearancFig— 2 . e p frocurvm f o eplasm f sheeo a p subjecte"V? o dt different dietary levels of phosphorus.

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48 HEAT-INDUCED MINERAL IMBALANCES IN ANIMALS*

T.H. KAMAL Radiobiology Department, Nuclear Research Center, Atomic Energy Establishment, Cairo, Egypt

ABSTRACT

iuineral balance was determined on 14- Friesian heifers maintaine metabolin o d cclimatie cagech n i s c laboratort a y 19°C for 5$ days followed by 11 days at 40°C-5 hr daily-and 19°C-the rest of the day . The data of the last 9 days of mild climate were compared wit ht climatethosho e f o e Th . data revealed that in early days of heat exposure the ash, Ca, balancP d Man g e decreased significantly tha n mili n d climate by 17.6JS, 24.1%, 65.7% and 11.5#, respectively as a result of the increase in their urinary and fecal excretions. At the enf heao d t exposure they decreased also significantly than ni mild climat t greatebu e r tha t earla n y heat exposure. These result e attributear s e destructioth o t d f tissueo n s under heat stress with concomitbant washinf mineralo t ou gy b s increased water turnover rate, itfa and K balances, on the other hand, wer t affecteno e heay b d t stres o maintait s e osmotinth c pressur f bodo e y fluids, which increas n heai e t stress.

I-wuole body turnover rate constant (K) and half life value (TK) were determined in 9 albino rats maintained at 2B°C and in another 9 albino rats maintained at ^5°G for 8 days using a whole body counter with l\fal detector. The data revealed that n mili d climat larges s shortee Kth e1wa wa r y T (P<0.05r e th d an ) 0.05) t climate valueho thaK e n ni Th s . were 6.2 910~X 5

* This work was supported by the FAO/IAEA (Research Contract RC/2209-EGY) and by the USA-NSF (Research Project INT-75-22294).

49 an drespectivel, 6.8" 10 value; 41 X T d syan were 84. 103.d 6an 1 hrs, respectively. Tnese results suggest that iodine allowances for animals should be increased under mild climate than under hot climate.

INTRODUCTION

e maintenancTh mineraf o e l balanc farn i e m animalf o s i s a profound importance for their growth, production, reproduction and resistance to disease and parasites. This can be realized from the role they play in bone and teeth formation, blood clotting, proper functioning of nerve tissue, regulation of osmotic pressur bodn ei y fluius, maintenanc homeostasie th f o e s of acid-base balanc moderatind an e enzymatie th g c reactiony sb acting as cofactors of enzymes or as catalysts in enzymatic reactions, thus functioning as stabilizers of the biochemical structure bodye th .n i s These element e interrelatesar d dan balanced against each other in body function and cannot be considered as single elements with circumscribed functions. i?or example iron, copper, cobalt sulfate and molybdenum' are interrelate anaemin i d c diseases. Cobal texcretioe th aid n si n of seleniu d higman h dietary manganese decreases calciud man phosphorus balances. Calcium, potassiu d sodiuman m show d.efinite relationship maintenance th n si acid-basf o e e balance. e significancTh mineralf o e theid san r interrelationshin pi body functions have been extensively studied by many workers . 4) , 3 (1 , ,2 When the exotic European-evolved breeds of cattle are exporte tropicao t d l countries, thefacee yar d with many problems relateclimatt ho o dt e namely heat-stress, poor- quality pastures, tropical diseases,and parasites. These factors are thought to be the cause of the impairment noted in performance of the highly productive cattle in the tropics. Of these, factors the effect of hot climate on many biological functions in ruminants has been investigated (5» 6, 7t 8).

SO However, insufficient informatio available nar e concerning suc effecn ha mineran o t l balance thin I .s stud intakee yth , excretio d bodnan y retentio mineralf o n s were determinen i d Friesian heifers under controlled mild and hot climates in attempt to detect moderate mineral imbalances. The rate of whole body ^ I disappearance was also determined for this purpose in rats under controlled mild and hot climates.

MATERIALS AKD JtETHODS

Balanc- 1 e trial;

Fourteen Friesian heifers of about 12 months old bodg k y0 an weigh20 d t were kep metabolin o t c cagen i s the climatic chambers for 3:5 days under mild climate

9 H.H.0 +/ _5 (1 4°c + ,,5 followe6 1 day1 sy b dunde t rho y durins da hr e H.H% 5 5 th g climat r + .1°C+ fo p 0 ti ,(4 e and 19 ± 4°C, 65 + 5% R.H. during the rest of the day). The cages were constructed in a way to collect feces and urine partially seperated without restrainin e animalgth s with harnesse alloo t d w san convenien t sidy movementwa e s e animalTh e abl staso ar t e e animalsytth o * on these cages for more than 2 months without apparant physiological disturbances. o possiblThern s wa e e contact of water, feed and excreta with any metal or concrete in orde avoio t r d their contamination with external minerals. The balance trials were carried out during the last 9 days f eaco h climatic conditio date th a d wernan e collected durin firse lasd gth an tday$ t thesf so days9 e .

The animals were fed individually in the metabolic cages with 2 kg pelleted concentrates, followed by 2 kg clover hay in the morning and 2 kg pelleted concentrates followericg k e2 afternoone stray th b d n wi e pelleteTh . d concentrates contained 46% undecorticated cotton seed cake, 25% rice milling, 20% wheat bran, 2% calcium carbonate, 1% sodium chloride, 1% trace mineralized salt and

51 vitamins A and D mixture and 5% molasses. The daily intake level per animal was about 12 Meal metabolizable energy and 40 g digestible protein per Heal metabolizable energy e amoun.Th f concentrates,nao t d straan y w left over from each animal was weighed daily and their intakes were deduce d averagelase an d th day9 t r f eaco sfo d h climatic condition. Water was provided freely through as automatic water bowl, which was fitted in the metabolic cage and connected to a plastic water reservoir with a plastic hose e reservoirTh . s were filled with water every mornine amounth watef d o t an g r intak s rea glasn ewa o d s manometer reservoire fittetu n o d s next morning. However, the amoun mineralf o t s daily intake from wate s founwa r d neglegibl t use mineran no i d thu s an ewa s l balance calculations.

Daily urine and feces excreted during the first and last $ uays of the last 9 days of mild and hot climates were collecte d weighedan d A sampl. e representinf o % g2 this daily urine or ieces excretion was collected and the samples of each 2-oays period of each animal were mixed togethe e compositon s a r e sample.

The feed and mixed urine and feces samples were analysed for dry matter and ash determinations as described in A.O.A.C. (9 ). 3odium and potassium were analysed by flame photometer. Calciu d magnesiuan m m were determined using a complexometric titralion method developed by Kainal (10). Phosphorus was determined by spectrophotometric method develope y Fisk b dSubbarod an e e wTh (11). y matteconcentratiodr s multiplien i wa r e h th as y f b o dn dry matter intake arid by urine and feces dry matter excretion to h intakobtaias d urinare an eth n d fecayan h las excretion, whic deductes hwa d froh intakmas o obtait e n the amount of ash retention in the body including the

52 h amounexcreteas f o tn swea i d t and. saliva outside th e body e concentratioTh . f eaco n h element determinen i d ash was multiplied by the ash intake and by the urinary d feca an h excretiolas o obtait n n each element intake d excretionan e latteTh .deductes wa r d froe elementh m t intak o obtait e amoune th n f elemeno t t retained froe th m gastrointestinal tract and kidneys in the body including that excrete n sweatini d d salivationan g e intakeTh . , urinary d elementexcretioan e balanch th as s d f o nan e studied unuer mild and hot climates for the first 3-aays (Periolasa an uayt3 t ) I d; showe (Perioar n i n) dII Taole 1 and 2, respectively. The differences between mild and hot climates daca are tested statistically using the "t" test (12).

Hadioisolop- 2 e turnover rate;

Eighteen Albino rat g oodf aoou o s0 y 15 tweigh t were injected intraperitoneally with 1.0 nil of 50 uCi fta ^ I. The rat^ were divided into 2 groups of ^ animals each, one served as a control mai> lai'eo. under mild a.nbient temperature (2o°'J) wherea e othetn s r grou s suojeciewa p a hig o t ha a.abicnt tempera tare (>>°'0« rt whole body counter constructe r thifo d s purport s use o wa -count d t daily for 8 days each animal in a confined box to keep the geometry constant throughout the experiment. The empty box background was deducted from the total counts wholI ' et o bodt obtaine y e activityth n e dailTh . y 15] net activities of all animals of each group through the 8 days were averagee th d an ds weru plotted against time on a semilogarithmic grid (-Fig. 1). Th.e rate constants and T,. of each group were calculated and e differenceth s between groups were tested statistically usin studene gth test(12" "t t shows )a Tabln i n e (3).

53 RESULTS AND DISCUSSION

1 - Balance trial;

It is noted from Tables 1 and 2 that the 9-days average of ash intake in Friesian heifers was 10.54$ lower unde t climatrho e than that under mild climate. This decrease occured as a result of che heat-induced decreas n feei e d intake mostl d ricn clovei yan e y rha straw s expecteminerale A .th l al ds studied showed similar lower averages under heat stress than under mild climate.

I'uble 1 shows tnab at early heat exposure ( j5r - 5 day) the total urinary and i'ecal excretion of ash and sodium decreased while the excretion of potassium calcium, magnesium and phosphorus increased irrespective of their depressed intakes. This resulted howeven ri significant decrease totan i sbalanch as l f 17.5%o e , calcium balance of 24-. 1%, magnesium balance of 65.7% and phosphorus balance of 11.5%» These decreases were significant at P< 0.10,<0.05,<0.01 a:id<0.10, respectively. These results indicate that these Friesian heifers which are the fourth generation in JSgypt were still intolerant to hot climate n accordancI . e with these results Kamad an l Abdelaal (15) observed thabalancee th t f intravenouslo s y injected Ca and P in Friesian cows were lower in hot summer than under mild winter climate in Egypt. Hoistein- Friesian calves, which were raised in cold climate in U.S.A. (!<+) showed significant decreases not only in Ca, . balancesK P balanced d an an a N t .alsg sM bu n i o

Tabl 2 showe s that with longer heat exposuree th , animals became more heat stressedd an . Durin9 e th g 11 day of hot climate (Period II), there were greater decreases tha n Perio i nn totai I dl P ashd , I»lan g,Ca balances reaching 22%, o5%, 35% and 6%, respectively.

54 These decreases were significan 0.05,<0.05^ P- t a t , <0.01 and<0.05, respectively.

The above mentioned mineral imbalance observed in heat-stressed heii'ers may be attributed to the fact that these temperate-climate evolved cattle undergo tissue destructio t climatho n i ns indicatea e y negativb d e nitrogen balance (15), lean body mass loss (16), bodt fa y loss • (17-Accordingly) , mineral thesn si e destructed tissues and bones are excreted thus causing the observed increas n urinari e d fecayan l excretio f calciumo n , magnesium and phosphorus and probably other elements not determined in this investigation reasoe Th K balance. d n an tha a sN t did not decrease significantly in the heat-stressed heii'ers may be attributed to their increased reabsorption from guts and kidneys to maintain the osmotic pressure of extracellular d intracellulaan r fluid s theia s r volume e knowar so t n increase under heat stress (18). Under heat stress sweating d salivatioan e alsar no increased thus ucilizdn a considerablg e amoun f theso t e reabsorbed electrolytes. Base n theso d e balance trial 'e suggeste b resultn ca t i ds that minerals allowanc r Friejiafo e n cattle shoul e increaseb d d undet rho climate.

2 - Radioisotope turnover rate:

Figure 1 shows tuat I-wnole body activity decreased with a faster rate in the control group of rats under mild climate than those exposed to hot climate. Table ;> shows that the rate constant of ^ I-wnole body activity was larger significantly (P) iu chu concrol group than in the

heat-streSDud group. This indicates that 13^ 1I was utilized and excreted from the rats at a faster rate under mild climate than under hot climate. This is attributed to the fact t-rit thyroid f-;land activity uptakes

55 more I and secretes it as •* I-thyroxine at a faster rat miln i e d climat b eclimate no tna n - i n I turI . n thyroxin s utilizei e y tissueb d d degredate, an sI ^ o t d part of which, is recycled tnrough the thyroid gland and the res s excretei t d outsid a bode faste th t ea y r rate under mild climate bnan under hot climate.

It is noted that the rate constants and Ty values in Table 3 are slightly different from those on Fig. 1. This d 'I1,/ an becaus meanK e n Tabli sth e wer3 e e calculatey b d averaging the K and T,< values of all animals in the groups, whereas in Fig. 1. The K and T,, values were calculated by averaging the activity of all animals in the group for each time after dosing and the tl average values of the grou d s T,/user .deducinan pwa fo dK e th g

These results suggest thaiodine th t e allowance th n i e feed should be increased lor animals raised under mild climate than undet ho r

1- Comar, C.L. and Lronner, F. ...in oral metabolism. An advanced treatise. Academic Press. Jew iork and London. (1962). - FAO/IAEA2 . i.iineral studies with isotope n domestii s c animals, Proceeding a panel f o s , Vienna, 1970, IAEA, Vienna 1971, STI/PUB/293. 3>- Ammerman ,« G.bSymposiumal t e . : Trace elements . DairJ . y Sci. £2(1970) 1097. - FAO/IAEA4 . -Lsotope studiee physiolth domestif n o o s y o c animals, Proc S.ymp.f o . , Atneas, 1972, IAEA, Vienna, (19?2) STI/PJb/309. 5- Kamal, T.il. , Johnson, H.D. , lOgsdalu, A.G. Environmental physiolog d sheltean y r engineering with special referenco t e domestic animals. Missouri Ag-r. Exp. Sta. Res. Bull.(1962)785, - Kamal6 , T.il. Uadioisotop n animai os l nutritio d physiologyan n . Proc. Series, IAEA, Vienna (Isu^). p. 767. 7- Kamal, T.H. Progress in animal bio ..eteorology. The effect of weather and-climate on animals. Swecs Si Zeitlinger, Ainestei-dam Vol. I (1975). y. ^65-

56 Hafez- 8 , E.S.E. Adaptatio domestif no c animals Febiger& e Le . , Philadelphia (1968). 9- A.O.A.C. Official methods of analysis. A.O.A.C., Washington, B.C., U.S.A. (I960). - Kamal10 , T.HAgr . FooJ d ..an d Chemistry.8(1960) 156. - Fisk11 , SubbarowC.Hd an .Biol. J .. ,Y Chem . 66(1925) 374. 12- Steel, H.G.D., Torrie, J.M. Principles and procedures of statistics. wcCiraw-Hill BooInc. * Yorw kGo Ne ,k (I960). 13- Kamal, T.H., Abdelaal, A.S. Proc. Series, IAEA, Vienna (1972)95 STI/PUii/30y. 14- Kaunal, T.h., Johnson, H.D. Trace elements in drinking water, agriculture and human life. Proc. Symp. Cairo, 1977• Middle Eastern Reg. Had. Center. Dokki, Cairo (1977. )54

Kamal- 15 , T.H., Clark, J.L., Johnson, H.D. Int Biometeor. J . . 11(1970) 301. 16- Kamal, T.H., Johnson, H.D. J. Dairy Sci. ^5_(1970) 1734. 17- Kamal, T.H., Johnson, H.D. J. Animal Sci- ,3_2(1971) 306. 18.- Kamal, T.H., Shehata, 0., Elbanna, I.M. Proc. Series, IAEA, Vienna (1972. )95

57 TABLE (1): Mineral balanc n heiferi e s maintained under controlled t climateho mild an d s (Perio. I) d

Intake » & Urinary and Fecal Balance, Excretion, 8 & Items Mild Hot Mild Hot Mild Hot »t"

Ash 873 781 390 365 489 403 (13) 279 24~.3 2T.6 22.1 2T.8 29.7 2.066* Sodium 44.4 41.3 27.3 22.9 18.5 17.3 (11) 073 079 275 275 179 176 0.549 Potassium 83.1 7b.8 46.3 48.5 36.6 26.8 (ID 072 179 373 573 373 379 1.583 Calcium 67.2 63.2 16.8 22.0 50.4 38.3 (11) 071 270 371 277 370 373 3.090** magnesium 27.4 26.3 17.2 22 lu.2 3.5 (11) O7l 077 o76 170 076 l7o 3.950*** Phosphorus 47.2 44.0 5-3 6.0 41.8 37.0 (12) 071 o7i 076 o75 075 173 1.945*

z Significant at P<^0.10 aat Significant at P<0.05 30EX Significant at P<0.01 ) (Number f animalo s s t useclimati ho n mili d r o d c conditions. TABLE (2): Mineral balanc heifern i e s maintained under controlled mild and hot climates (Period II).

Intake, g Urinard yan Fecal Balance Excretion, g , g Items Mild Hot Mild Hot Mild Hot ntn

Ash 872 781 370 384 509 397 (14) 279 2T.3 2^.6 3T.1 2$. 6 30.8 2.789** Sodium 44.4 41.3 34.8 30.2 9.6 12.4 (13) 0703 079 270 277 272 272 0.769 Potassium 83.1 76.8 45.5 43.0 35.9 35.9 (13) 072 179 278 377 271 379 0.002 Calcium 67.3 62.9 43.7 56.2 23.6 8.3 (13) 071 177 2Tu 47^ 270 3^6 2.883** Magnesium 27.4 26.2 11.7 24.2 15.3 2.6 (13) 071 076 076 -072 076 174 4.917*** Phosphorus 47.2 43.9 4.0 4-4 43.2 40.5 •f (14) O7l 071 075 075 075 078 2.827**

3E Significan 0.1^ P- 0t a t Significant at P<0.05 Significant at P< 0.01 ( ) Numbers of animals used in mild or hot climatic conditions. TABLE (3)

Effect of controlled hot climate on ^ I-whole body activit ratsn yi .

Control (mild) Heat stress

Mea x ICT- K n 5* S.fi. X8-2 0.2± 9 6 #6.84 +3 _0.

Mean TV + S.E.(hr) 84.6 + 2.69 103.11 + 4.9

9 9 N

"t" (K x 10~3) 2.733*

"" (Twt ) 3.227 *

Significan 0.0< P 5. t a t

60 Control= • * - Heat stress

2 U d I , X/ O I/ 6 9 72 21C 240 264 2&Q 3:2 Hours after dosing

-tig,f : hi 1 effect .f o .tuii r>t temperatar I whol ^ . e or ebod y cliba^ \ eara.nct; rate . i.a - in

61 POSSIBLE ROLE OF VITAMIN D COMPOUNDS IN THE CALCINOGENIC ACTION OF Trisetum flavescens*

M. PETERLIK Departmen f Generao t Experimentad an l l Pathology, University of Vienna Medical School, Vienna D.-S. REGAL, H. KOHLER Department of Pathology and Forensic Veterinary Medicine, Veterinary University, Vienna, Austria

ABSTRACT

The occurrence of endemic calcinosis in grazing animals has been linked to the ingestion of certain plants (notably Solanum malacoxyj^n, Cestrum diurnum and grasses, e.g., Trisetum flavescens. Whereas 'the calrinogenic action of plants has been linked to the presence of the vitamin D metabolite 1,25-dihydroxyvitamin D, an explanation has still to be found for the mode of actio f calcinogenio n c grasses. This paper describe e contenth s f o t viatmin D-related substance n Trisetui s m flavescens (T.f.) through measurements base n bioassao d y techniques e resultTh . s clearly demonstrate th e ability of T.f. to induce remineralisation of rachitic bone in Japanese quail, and to produce an increase in bone ash, plasma total Ca and plasma inorganic phosphate in chicks fed a high strontium diet. These findings indicate thae planth t t contains some 1,25-dihydroxyviatmin D-like activity n additioi t o vitamifinat nbu , l 03 proon f thio f s requires further isolation and characterisation studies.

Introduction

Fndeinic calcinosi n grazini s g animal a diseas s i s e occurring n varioui se worlpartth f do s (cf. Tab . Characteristi.1) c symptoms displaye e afflicteth y b d d animals include chronic wasting, stiffness of joints, lameness and severe pain. Underlying these signs of illness is an excessive ectopic calcification of heart, lungs, main blood vessels, kidneys, tendon d ligamentsan s . Although e diseasth s beeha en recognize r quitfo d a elon g time s (1)wa t ,i not until recently that its etiology has become known. In three instances, endemic calcinosis coul e e traceingestiob dth o t d f o n certain plants: Three different species e shruth , b Solanum mala- Qo_XYl_P_Q.* the ornamental plant Cestrum diurnum, and the grass Tri- setum flavescens were e responsiblshowb o t n r calcinosico e n i s grazing animals in so distant and different areas as the grasslands of South America (Argentina, Brazil) e Uniteth , d States (Florida) e anAlpinth d e pasture f Centrao s l Europe (Austria, Germany) (2,3,4) n additionI e betteth , r understandin f vitamio g metabolisD n d man action evolving over the past years, brought aoout a deeper insight into the pathogenesis of calcinosis.

Research Agreement No. 2169/CF: "Investigation on the calcinogenic factors of Trisetum flavescens"

63 Table 1: Calcinosis in grazing animals Location Afflicted Species Term apllied to Responsible the disease plant

Jamaica Cattle; sheep Red earth disease Hawaii Cattle Naalehu disease Argentina Cattle; sheep; horses Enteque seco . malacoxyloS r Brazil Cattle Espichamento . malacoxyloS r Florida Horses . diurnuC m Austria, Cattle Weidekrankheit T. flavescens Cermany Enzootische Cal- cinose

w welno ls Ii testablishe d thae mosth t t active metabolite of vitami s 1,25-dihydroxyvitamii D n s synthesiIt . D n s froe th m precursor 25-hydroxyvitamin D taking place exclusively in the kidne strictls i y y controlle y serub d m level f calciumo s , phosphate and parathyroid hormone according to the need of the organism e respectivth r fo e ions. 1,25 Dihydroxyvitami D nitsel f playa s n e regulatioformatioow th lol s n i eit y controllinb f no n e th g synthesis of the converting enzyme 25-hydroxyvitamin D-1-hydroxylase. To meet additional demands of calcium and phosphorus during particular periods such as growth, pregnancy, lactation or in the egg-laying circle, a stimulatory action on the synthesis of the active vitamin D metabolite by sex steroids and proteohormones (estrogen progesterone prolactin growth hormone) is superimposed on the normal control mechanism preventing excess formatio f 1,25-dihydroxyvitamio n D n (for review cf. 5). By s activvirtuit f eo e metabolite, vitami D nstimulate s the absorption of calcium and phosphate from the intestine. Excessive absorptio f theso n e ionn onlca s y occua consequenc s a r f bypassino e g the intricate control mechanism which regulates the endogenous synthesis of 1,25-dihydroxyvitamin D. This would take place e. g. in the case of administration of the active metabolite from exo- genous sources n fact I .e calcinogeni th , c actio f Solanuo n m mala- coxylo d Cestruan n m diurnu s beeha mn e occurenclinketh o t d f o e 1,25-dihydroxyvitami n thesi _ D en plants (6,7). Ingestioe th f o n active vitami D compounn d certainly reduce s endogenouit s s renal synthesis but due to the excessive amount absorbed induces hyper- absorptio f calciuo n d phosphatan m e eventually leadin o ectopit g c depositio e excessivelth f o n y absorbed minerals.

This paper describes the efforts to provide a reasonable explanation for the calcinogenic action of Trisetum flavescens (T.f.) s contenbaseit n f vitamio do t n D-related substances.

Endemic calcinosis and Trisetum flavescens In their report on the appearance of calcinosis in Austria, Kohle d Libisellean r ) describe(8 r e extensivth d e searcr fo h possible causes of the disease. Since mineral analysis of soil, fertilizer f suspecteso used an d d plant material provideo n d e causativclueth o t s e agent n unknowa , n organic compoun f plano d t origi s suspectewa n f inducino d g calcificatio e afflicteth n i n d animals. Independently, s coworkerDirksehi d ) describean n (4 s e th d disease in Germany and subsequently identified the grass Trisetum flavescens as the plant species containing the unknown calcinogenic substance. That T.f. actually contains some vitamin D-like activity was shown by Wasserman et al. (9), who fed dried plant material to vitamin D-deficient chicks. In these animals, T.f. induced the synthesis of intestinal calcium-binding protein (CaBP), a response strictly related to the action of vitamin D. This finding stimulated attempt t isolatioa s d characterizatioan n e vitamith f o nD n related compound(s) of T.f. in several laboratories.

64 Bioassav of vitamin D-like activity of T.f. e effecTh f T.fo t .n bono e mineralization (antirachitic activity s exploitewa ) r quantitatiofo d s vitamiit f o nn D-like activity. Female Japanese quail were raised on a starter diet until their g productioeg e theoreticalln th average f o % 8O d y possible rate. They were then divided into two groups. One group was fed a normal diet containing 26OO e otheI.Uth e on , r. kg vitamir pe - D n was fed a basal vitamin D-free diet. After 16 days, the now vitamin D- deficient animals were further divided into t.iree groups: Some were continued on the rachitogenic (basal) diet, others were fed the basal diet supplemented with 1OOO I.U. vitamin D.,/kg, whil thira e d group receive e basath d l diet fortifie df lyophilize o wit% IO h d T_. .f e quaiTh l were sacrificed afte additiona3 r le experimen weekth n o s - tal diets. Bone ash determinations were taken from defatted tibiae. e pertinenTh t date give ar aTabn i n .2

Tabl : Effec2 e f T.fo t .n bono e mineralization Diet Addition Bone ash (% weight of defatted bone)

Basal None 6L.9±O.4

Basal 100O I.Ug /k .D 61.9+O.5

Basal 10% T.f. 63.3+O.3 +)

Normal 26OO I.U. D /kg 66.3±1.O +'

Statistically significant difference from control group receiving basal diet only (P

The results of this bioassay clearly demonstrate the ability of T.f. to induce remineralization of rachitic bone in Japanese quails. A rough estimate puts the biopotency of the T.f. -supplemented diet to a value distinctly higher than 1OOO I.U. vitamin D~ Hence. pe kg re tota th , l vitami D contenn f T.fo t . amounto t s approximately r.ore than 1OOOO I. U./kg lyophilj.zed material. A comparison with estimates obtained in other laboratories is given in Tab. .3

Table : Estimate3 d value f totao s l vitami D contenn t of'T.f. Method Vitami D n(I.U./k g Reference y plandr t material) Direc O t O determinatio O 4 Rambec. n(13 al )t e k (GC and HPLC)

Inductio f CaBo nn i P vitamin D-deficient chicks 8 0 0 O Peterlik et al. (1O) Induction of CaBP in vitamin D-deficient chicks 8 O O O Wasserman et al.(ll) Bonh determinatioas e n in Japanese quails > 1 0 O O O Peterlik et al., (unpublished)

65 Though estimate f totao s l vitami D contenn f T.fo t . show considerable variations due to the method applied, there is agreement among researchers in the field that the obtained values o providt w lo n explanatioa e o e massivto th r r fa fo ene calcinosiar s observed after ingestion of T.f. by grazing animals. This consideration is valid, however, when applied strictly to unmetabolized a vitami o t t n no D-relate d an . D vitami dr o metabolit ^ D n e capable of overriding tne stringent control of 1,25-dihydroxyvitamin D formation.

In fact, some 1,25-dihydroxyvitamin D-like activit s beeha yn detected in T.f. grown on a pilot plot in Lower Austria (1O). Feeding T.f. to chicks, whose endogenous production of the active metabolite had been blocke y higb d h dietary strontium, induced intestinal CaBP and produced an increase in calcium and phosphate absorption froe smalth m l intestine.

Bioactivity of Trisetum flavescens in strontium-fed chicks

Bioactivity of T.f. in Sr-fed chicks was confirmed by the following experiments d chickol y sda e werOn .e a starteplace n o d r diet for 2-3 weeks and were then divided into two groups. A number of chicks were put on an experimental diet adequate in vitamin D^ (12OO I.U./kg t supplementebu ) d with 2.5% strontium ("high strontium" diet o prevent ) t endogenous synthesi f 1,25-dihydroxyvitamio s . D, n Other chicks were fed the "high strontium" diet containing 22% lyophilized T.f. Aftee experimentao weekth tw rn o s l diets plasma calcium, phosphate, and bone ash were determined (Tab. 4).

Tabl : Influenc4 e n Sr-fei d bon f h T.fan o edas e n plasmP o . , Ca a chicks

Diet Plasn a (mMC a ) Plasm P a (mM) % lonh as e

"Hig" Sr h 2.45 + 0.03 2.78 + 0.09 38.8 + 1.2

"High Sr" + T.f. (22%) 2.61 + 0.08 3.8 0.1+ 8 3 4 43.1. + 8

Date expressear a s mean+.S.Ea d . fro t leasa m t five determinations (•+)Significantly different from corresponding valu f "higo e" Sr h grou

Inclusion of high amounts of T_._f . in a "high strontium" diet produce n increasa d n boni e e ash, plasm ad mos totaan t a C notablyl , plasma inorganic phosphate. This again suggests thae planth t t might contain some 1,25-dihydroxyvitamin D-like activity. e otheth n rO hand ) wer,03 e Rambec . ablal o isolatt et a k e authentic vitami . andfro, f . D. nn additionT m i , , even showed that biosynthesis of cholecalciferol takes place in this plant species unde e influencth r V lighU f to e (12). These authors, howeverd di , not detect any other vitamin D~-related compounds by the isolation procedure used. This finding, of course, explains the bioactivity of T.f n vitamii . n D-deficient chick t cannobu s t e accounth r fo t biological response elicite n strontiui de th r d chickfo fe m r o s massive calcinosis encountered in grazing animals. Thus, the search for the "true" calcinogenic compound has to be continued. Froe relativelth m y high amoun f T.fo t . o t whic d ha h e mixeb d inte "higth o h strontium" die o demonstratt t e some bioactivity (cf. also lo), the actually calcinogenic vitamin D compound must comprise only a small fraction of the total vitamin D content of T.f. To facilitate the bioassay procedure we tried o obtait n fractions enriche n calcinogenii d c activit y extractiob y n of lyophilized plant material with organic solvents.

66 Bioactivit f T.fo v . extracts We found that an active fraction could be prepared by exhaustive extractio f drieo n d T.f. wit methanoa h l :CHC1 mixture- ex e .Th traction procedure was as follows: T.f. was stored at -2O C before s thewa nd lyophilizean e us r 36-4fo d 8f lyophilizeo hoursg o 5o . d material was finely chopped in a household blender and stirred with 5 vol. of methanol: chloroform 2:1 (v/v) at 4 C for 4 h in a Nn atmosphere. After filtration the residue was washed twice 1 vol. solvent. The combined organic extract was evaporated in a rotator e averagef yo th evaporatog n O 3 1 ,. C ° vacun 37 i r t a o an oily residue were obtained from BOO g lyophilized T.f . The ability of this extract to counteract the strontium effect on bone mineralization and on plasma calcium and phosphate was tested as follows: Chicks raised on a commercial starter diet for 18 days were divided into three groups which were fed the diets detailed in Tab. 5: The "high strontium" diet was fortified with additional vitami 3 accordinD n e steroth concentratios o lt g' n i n methariol/chloroe Th . f . T solublm for es extracwa f . T tg froO 5O m e "higmixeth f ho d strontiumg witk 1 h " diet e normaTh . l diet containe e samth d e amoune aforementioneth f vitamis o ta . D- n d diets.

Table 5; Composition of experimental diets

Diet Strontium Vitamin D3 (I.U./kg diet) From T.f. Synthetic Total

High Sr 2.5% 36OO 36OO

High Sr. + T.f. I £.O • . 1200 2400 36OO

Normal 360O 3600 Calculation of vitamin D, content of lyophilized T.f. was on ref.. &

Chick e experimentasth wer d fe e ldaysu l diet i .fu sResult f o s bone ash P ,determination plasmd an a C a e show ar sTabn i n . .6 e combineTh d data suggest1that Trisetum flavescens does contaia n caDcinogenic substance in addition to vitamin D. which to sonie degree prevents strontium-induced demineralizatlo f bonno s wela e l as hypophosphatemia and hypocalcemia. This finding raises some

Tabl : Effec 6 eT.fa f .o t extrac n bono t e mineralization, plasma Ca and P in strontium-fed chicks Diet % Bone ash Plasma Ca (mM) Plasm P a (mM)

High strontium 43.4 O. 3+ 1.91 ± 0.10 2.1 0.0+ O 8 High strontium + T.f. extract 3 44.O. 8+ 2.04 + 0.01 2.51 + 0.08

Normal 47.9 + O.8 2.3 O.O+ O 4 2.2 O.O+ 4 9 Data are expressed as mean + S.E. from five determinations. ) Significantl+ y different from corresponding valu n "higi e" Sr h group

67 question e unknowe naturth th f n o o esn calcinogenic factor, whether it might be 1,25-dihydroxyvitamin D or even some other biologically active vitamin D derivative. Until now no vitamin D metabolite other than 1,25-dihydroxyvitami s beeha nD n isolate r synthesizeo d d that is active in strontium-fed animals, i.e. in a condition resembling the nephrectomized state as far as the synthesis of the active metabolite is concerned. Until evidence to the contrary is provided, s reasonablii t o assumt e e thae calcinogenith t c s factoit n i r molecular structure might closely resemble 1,25-dihydroxyvitamin D. Final proof of this assumption, however, will ultimately depend on the isolation and characterization of the vitamin D-related compound^) oresen Trisetun i t m flavescen n additioi s o vitamit n . D_ n

Acknowledgements Par f thio t s wors supportewa k y Granb e d. 3O3th No t f 1o Fond r Forderunzu s r wissenschaftlichede g n Forschun n Osterreichi g . e capablTh e technical assistanc Pete. Mr r- f o eAn Wyskovsk . Mr d an y dreas Kock is thankfully acknowledged.

References . CollierA (1. W ) , Zeitschr . Infektionskrankhf . . 31:81, 1927 . WorkeA d . B.J.CarilloN an r) (2 , Nature 215:72, 1967 (3 . Krook. L Wasserman)H . R , . McEnteeK , . BrokkeD . T d , an n . TeiglandMB . , Cornell Vet. 65:557, 1975 (4) G. Dirksen, P. Plank, U. Simon, T. Hanichen, P. Daniel and A. Spiess, Dtsch. tierarztl. Wschr. 81:1, 1974 (5) J. L. Omdahl and H. F. DeLuca, Physiol. Rev. 53:327, 1973 (6) M. R. Haussler, R. H. Wasserman, T. A. McCain, M. Peterlik, K. M. Bursac and M. R. Hughes, Life Sci. 18:1O49, 1976 (7) M. R. Hughes, T. A. McCain, S. Y. Chang, M. R. Haussler, M. Villareal. WassermanH . R d an e, Nature 268:373, 1977 (R) H. Kohler and R. Libiseller, Zbl. Vet. Med. A 17:289, 197O (9) R. H. Wasserman, M. Peterlik, M. Haussler, M. Hughes, K. Bursac an. KrookL d , Isr . MedJ . . Sci. 12:1492, 1976 . Peterlik(10M ) , D.-S. . KohlerH Rega d an l, Biochem. Biophys. Res. Comm. 77:775, 1977 (11) R. H. Wasserman, L. Krook, and G. Dirksen, Cornell Vet. 67:333, 1970 (12) U. Zucker, II. Stark and W. A. Rafibeck, Nature 283:68, 198O . Rambeck(13W ) . OesterheltW , . Zucker. VecchH M , d an i, Biochem. Biophys. Res. Comm. 87:743, 1979

68 PROBLEMS IN THE DIAGNOSIS OF TRACE ELEMENT DEFICIENCY

M. KIRCHGESSNER, F.J. SCHWARZ Institut fur Ernahrungsphysiologie der Technischen Universitat Miinchen, Freising-Weihenstephan, Federal Republic of Germany

ABSTRACT

This paper reviews current knowledge on the metabolism of trace elements (e.g., zinc, copper and manganese) and describes the problems of diagnosing deficiencies of such elements. At the moment, the most promising and sensitive Lests for these elements are the response techniques or functional tests wher e s animaseruit th e r s mi o treatel d wite elementh h t under study and the response to this treatment measured. Radioisotopes have particular advantage f stablo n e suci e sfuture us th eh e n testsi th ,t bu , isotope techniques will become increasingly important.

. 1 Introduction

Analytical studiee tracth n eo s element conten n feedstuffi t s give only limited informatio e actuath n o ln suppl f faro y m animals. No conclusion can be drawn on the amount truly available for biological function e bodyth f o . s Therefore, criteri r minerafo a l imbalance mainly have to be examined in animal studies and suitable methods mus e developeb t o diagnost d a esuboptima l supply status. This intentio t wit me n onlh e ca nb succesy s when simultaneously more knowledg n traco e e element metaboliss mi available. On this basis the coordinated program was formed between the Atomic Energy Agency and our research institution. e followinTh g paper will giv a more e general statemenn o t problems in the diagnosis of trace element deficiency. The work is focussed on the elements zinc, copper and manganese.

2. Trace element conten n bodi t y fluid d organan s s

Blood e measuremenTh e tracth f eo t element concentratio n bloodi n , plasm r seruo a s mveri y often use o chect d e supplth k y status. In this case, it is very simple to take the samples. Under definite experimental conditions of feeding an extremely Zn- deficient diet, the serum Zn content will be reduced significantly within a few days (Pallauf and Kirchgeflner, 1972). Similarly, milking cows will show a lower Zn concentration in serum after two weeks of feeding a semi-synthetic diet with 6 mg Zn/kg diet (Schwar d KirchgeBneran z e otheth , n r 197O hand . e a) 5th ,

69 serum Zn concentration reflects quite well the different nutritional Zn supply afte a fourteen-dar y perion repletioZ - f o Zn d f o n depleted rats (tabl , 1 Roted Kirchgefiner an h , 197 . Also9a) ,

Table 1 Response of the Zn concentration in the serum to different Zn supplements during a fourteen-day repletion

Zn supplements 0 2 2 1 0 1 8 6 4 2 (ppm)

Zn concentra- e th tio n i n 0.38 0.34 0.34 0.48 0.80 0.78 1.11 serum (/ug/ml) ±0.13 ±0.03 ±0.04 ±0.04 ±0 13 ±0.13 ±0.14

the Cu concentration in the serum diminishes rapidly after a lowered Cu intake (Spoerl and Kirchgeflner, 1975 a). Under practical conditions, however, serum zinc and copper will not change in a short period of suboptimal supply. Due to higher Cu or Zn amount e dieth t n i aftes a depletior n period, seru n concentratiomZ n will reach a plateau earlier than storage organs such as liver or bone These results depend upon the homeostatic regulation mechanism e organisth n i s m attemptin o maintait g a nstead y balanc n serui e m concentration (Kirchgefine , 1980). al t e .r Therefore, the diagnosis of a suboptimal trace element deficiency only on the basis of the serum content as the sole parameter will not be sensitive enough. Also, there are some factors which will change the trace element concentration independent e nutritivoth f e amounte . th Thesinfluencem g ma ee. e ar s time of taking blood samples, age of the animal, gravidity and lactation, stress, infection r otheo s r disease n additioI s o t n these problems, the question comes up in what range serum e differenlevelth f o s t trace element e "normal"ar s .

In orde o overcomt r e these difficultie sensitivw ne a s e parameter for diagnosing the early stage of marginal Zn supply could be the in vitro analysis of the percent Zn-binding capacity of serum. This procedur s beeha e n workey Rotd b Kirchgefine t an h ou d r s i show t i n tabli ns A 2 (198(Rote . d Kirchgefinera) an 0h , 198. 0a)

70 Table 2: Zn-binding capacity and Zn content of the serum in respons o dietart e n supplZ y y

Dietary zinc bindin- Zn g Zn concentration capacity

(ppm) < % ) (pg Zn/ml serum)

1.3 87. 1 ± 2. 7 0. 40 ± 0. 09 6 84. 3 ± 2. 1 0. 42 ± 0. 14 10 74. 2 ± 4. 8 0. 61 ± 0. 11 20 69. 2 ± 3. 7 0. 84 ± 0. 08 100 58. 9 ± 5 . 3 1. 26 ± 0. 10

e bindinth g capacit e rang th f seru f o yn o i es nearl m% wa 0 9 y severely e dietdeficienth t I n .i n supplm Z t pp 4 y o frot 1 m e dietarth s n contena Z fely o about % s raisel 0 wa 6 t tp u d n vitri e o0 ppmth incubatio10 r o .t e Fo seruth f mo n zinc-65 could also be used.

In different studies (Chesters and Will, 1978; Roth and Kirchgef3ner, 1979 b) it has been shown that the in vitro uptake of zmc-65 of the erythrocytes is negatively correlated to the n supplZ y status (tabl , 3 Roted Kirchgefiner an h , 197. b) 9

65 Table 3- Zn uptake of erythrocyte in vitro in response tn odos Z dietar) e e th y f n supplo Z % ( y

Dietar2 y zinc 6 10 20 100 (ppm)

6bZ7 6 1 2 n uptak ) (% 8 e 1 2 2 3 3 ±9. 5 . 3±2.2 t ±4 68 . ±1.7

However e opinioth n i f Chestero ,n d Wilan s l (1978) this method o advantagn f o s i n comparisoi e e seruth n concentrationo t mZ n . The zmc-65 uptake of the erythrocytes corresponds to similar factors as the serum zinc.

71 Uj-me

The renal excretion of the trace elements zinc, copper or manganese is very low. The findings on the amount of Zn excretion in relation e nutritivth o t e supply status show only small differences (Weigand and Kirchgeflner, 1977). Therefore e urinarth , n excretioZ y n seems usefuo n e b l parameteo t e diagnosisth r fo r n differenI . t diseases the Zn excretion of the urine is raised significantly (Sandstead et al., 1976).

Milk

n contenZ e f milo Th t k reflect e supplth s y status quite well , (Fig1 Schwar. d Kirchgefineran z , 197 . Thereforeb) 5 , this measurement coula usefu e b d l additional parametee th r fo r diagnosis. The Cu content of milk is diminished only after a long perio f severo d u deficiencC e y (Spoer d Kirchgefineran l , 197; b 5 KirchgeRner et al., 1981). High Cu supplementation of the diet does not u contenchangC e f milth o et k (Schwar d Kirchgefinan z , er 1978),

Liver, bone, hair

Organs use o measurt d e deficiencie f traco s e elements should reflect the supply status of the whole organism and should change the concentration rapidly wite dietarth h y amount e maiTh .n orga o stort n e coppee liverth Cu-deficiens A i .r t diet reduces u thconcentratioC e e liveth n ri n significantly (Fig , .2 Grafiman n and Kirehgefiner, 1973). However, especiall d alsan o e ag y gravidity will influence the liver Cu concentration. Similarly n contene coppero livet M th e f ro th t varie, s with different Mn dietary content (Kirchgefiner and Heiseke, 1977 —see also table 9). By contrast, the Zn amount in the liver changes little with the supply status (see also table 5 and 10).

One of the best parameters for diagnosing Zn deficiency is the bone Zn concentration (see also table 5 and 10). Different findings confirm a nearly linear relationship between bone Zn content and dietary zinc over a wide range of Zn supplements (Momcilovic and Shah, 1976; Forbes and Parker, 197J1; Kirchgefiner et al. , 1980). Also, Mn concentration of the bone gives a good answer n supplM e yto th status (Kirchgeflne d Heisekean r , 197 e alsse 7 o- table 9). Nevertheless, some caution is necessary due to interaction

72 problems between different trace elements r exampleFo . n i , Cu deficiency there is an iron accumulation in the liver and spleen despit a hypochromice , microcyti e anemiaF cn Z n I . deficiency, Cu retention is raised, whereas Mn retention is lowered. Further details are reported in a newer review (KirchgeCne t al.e r , 197. 9a)

Much work has been spent to use the trace element content in the hair as an indicator for the supply status (Anke and Risch, 1979). One advantage is that it is very easy to take samples. Fig- ure 3 (Pallauf and Kirchgeftner, 1973) illustrates the relationship between dietary zinc and hair Zn content. In the suboptimal supply status hair zinc closely corresponds to Zn supply, where- o significann s a t relationship exists with sever n deficiencZ e y r wito h highe n amountsZ r . Also e dynamicth , f storeo s d hair zin s veri c y low. Hence, this methot alwayno f s greai do s t benefit to characterize the supply status.

3. Biochemical parameters

e activit e Cu-contaimnTh th f o y g coerulo plas mn changei s very rapidly with different dietary Cu content (Fig. 2, Grafimann and Kirchgeftner, 1973). Nearly 60 to 99 % of the copper in plasma, depending upon animal species s i boun, o coeruloplasmit d n (Evan d Wiederandersan s , 1967) e half-lifTh 5 .day r f thio fo se s enzyme is short. Therefore, the activity of coeruloplasmin is a very sensitive paramete f marginao r u supplC l y- als e shoroth t term differences - and responds very early to Cu supplements after deficiency (Grafimann and KirchgeGner, 1973).

Amonn metalloenzymesZ l al g e alkalinth , e phosphatasf o e serum shows the strongest and fastest loss in its activity in respons o inadequatt e n supplyZ e . Therefore, this enzyme could be used as sensitive parameter to indicate Zn supply status. Again, there might be some problems with this indicator as is e casth e wite seruth h n concentratiomZ n becaus e questioth e n arises as to what are "normal values" during optimal Zn supply in different species. Also, the activity of alkaline phosphatase may change due to influences other than dietary zinc. To overcome these problems, Rotd Kirchgefinean h r (197 , 9a 1980) b proposed an in vivo response technique. As is shown in table 4

73 Table 4 Alkaline phosphatase activity before and after Zn injection responding to dietary Zn supply

Dietary Zinc Alkaline phosphatase in the serum Increase in (ppm) (mil/ml) the activity before Zn injection after Zn injection ( % )

2 8 2± 3 152 ± 36 555 6 1 4 ± 8 8 195 ± 61 122 10 139 ± 29 3 3 ± 3 19 39 20 9 3 ± 5 17 6 6 ± 9 24 42 10U 7 2 23 ± 9 246 ± 50 3

(Roth and KirchgeBner, 1979 a) the activity of this enzyme was measured befor d threan e e days afte n injectionZ r e responsTh . e in the activity of the enzyme correlates quite well with the supply status. However, a diagnosis oi Zn deficiency due to a rise in the activity of alkaline phosphatase in vitro in response to adding a Zn-contaming reaction buffer is not possible (Roth and Kirchgefiner, 1979 c). r manganeseFo , specifi n metalloenzymeM c t unknownye e ar s . How- ever, ther e somar e e enzymes whic e activatear h y manganeseb d . A good indicator seems to be the liver argmase. According to results from newer findings e liveth , r arginas s reducei e y b d more thao thirdtw n n Mn-deficieni s t rat n comparisoi s o t n adequately fed animals (KirchgeBner and Heiseke, 1978).

For a more detailed description of biochemical parameters, referenc s madi e o somt erevieww ne e s (KirchgeBne d Rothan r , 1976; KirchgeBner et al. , 1979 b).

. 4 Diagnosi l traco s e element deficienc y isotopeb y s

The organism attempts to maintain a steady balance of trace elements and, therefore, counteract y changean s n dietari s y intake by making adjustments. According to several results it may be postulated thae tracth t e elements zinc, copper, manganesed an , within limitations iron are underlying strict homeostatic regulatory mechanisms. For further explanations figure 4

74 (Kirchgefiner et al., 1974; Kirchgefiner and Schwarz, 1976) should be used as a model of the trace element metabolism. After an oral uptak e tracth e e element wil e absorbeb l e intestinth n i d e and ente e distributioth r n pool e (stebloo , th thar 1) po s d i t e storeb y serumdma (retainedt i , e liverth n i ,) bon r otheo e r e use b r differen fo dy depot ma t i ts r syntheses(steo ) 2 p , e.g. for metalloenzymes, - or it may be excreted via the intestinal wall as endogenous fecal excretion (step 3). These three steps play together in order to maintain a steady balance and a certain trace element level in the distribution pool. This balance is calle e homeostatith d c regulatio f traco n e element metabolism. Expressed in a simplified way, in the case of a lack of nutritional intake trace element t firsa e t ar sreplenishe d from depots. These depots are gradually depleted. The absorption increases and the endogenous excretio s limitedi n . When n abundanthera s i e t dietary e tracintakth f eo e elements e resultinth , g adjustmente th e ar s opposit f theso e e mad n deficiencyi e . Afte e e depotth th r f o s organ e replenishedar s e rat f th intestinao e, l absorption declines, and at the same time, the endogenous excretion increases. Consequently, there is a negative feedback from the supply status e absorptio oe th endogenoun th a positivn o d e an n on es secretion. These mechanisrn-S for e a modebasi mth r fo o diagnosst l e deficiencie f traco s e element e organisth n i s m using isotopes. The extent of absorption, retention and excretion are dependent upon the state of supply and can thus be applied to measure the suppl ye tracstatth f eo e element.

e suppositioTh r thifo n s mode e repors giveth wa lf n 197i no t 6 (Kirchgefine d Schwarzan r , 1976) where results from several experiments were submitted. In figure 5 the absorption and excretion of zinc (Weigand and Kirchgefiner, 1978) - measured wit n isotopia h c dilution technique (Weigan d Kirchgeflneran d , 1976) - is shown. Again, it clearly indicates the adaptation to a different dietary supply status. As it was pointed out in the earlier report (Kirchgeflner and Schwarz, 1976) the measurement e endogenouoth f s fecal excretion afte a singlr e radioisotop. v . i e dose has the advantage of a very simple sampling. Therefore, n repeatei d experiments, rats wit a hvaryin n supplZ g y status in the range of a marginal, suboptimal, adequate and high dietar n amounZ y t receive a dsingl e i.v. dos f o e Zn-ZnSO P - (abou g zinc)2 /jCm t 0 i. 30 Afte zin d 4 hours2 an cr e th ,

75 radioactivit f o y s zinmeasure wa ce iaeceth d expressen i an ds d as percentage of the i. v. dose. There is a highly significant relationship (B = 0.98, PC 0.001) between the endogenous fecal excretion and the dietary Zn supply as it is shown by the regression in figure 6 (KirchgeGner et al. 1979 b).

The measurements of the absorption and especially of the endogenous fecal excretion of trace elements may be used as functional tests for the supply status. These methods are very sensitive because the pathways of absorption and excretion adapt much earliee dietarth o t ry supply thae retentioth n f storeo n d trace elements or the concentration in body fluids would change. The above model studies were done under definite conditions (e.g. semi-synthetic diets r wito ) h animal e samth f eo s breed and age. From these the questions arise, firstly whether interactions between different trace elements would change the metabolic pathway e tracth f eo s elements and, secondly whether the physiological status (gravidity or lactation) influences the absorption, excretio d retentioan n a singl f o n e element. Both factors are of special interest for using the parameters oi absorption or excretion to detect a mineral imbalance.

4. 1 Interactions of zinc and copper

Imbalances between zind coppe an cy occu ma r r becaus f eitheo e r a deficienc f zino yr coppeo c re counteractineveth f i n g element remains adequate. Consequently, whe e supplth n s lowi y , copper will be present at a relatively high level compared to zinc. In th eu deficienc C cas t o e e oppositth y e situation occurs.

The effects of Zn depletion on Cu metabolism are shown in the nex o tablesn deficiencZ tw t u concentrationn C I . e th y n livei s r and bone increase along with reductions in Zn concentrations (table 5, Roth and Kirchgefiner, 1977). These two trace elements also showed opposing responses regardin e leveth gf theio l r excretio e mil th f highl o kn i n y Zn-depleted dairy cows (Kirchgefiner et al. , 1978). The supply status of zinc also influence u absorptioC s ne absorptio Th (tabl . G) e f boto n h zinc n contentZ Tabl d : an f live5 eo sd u bon C Changean r ee th n i s during Zn deficiency

Zinc deficiency Control (pair-fed) (1.2 mg Zn/kg (96 mg Zn/kg + 5 mg Cu/kg + 5 mg Cu/kg e dietth )n i in the diet)

Liver (/ug/g DMa) Zinc 4 ± 8 9 100 ± 4 Copper 26 ± 4 22 ± 2

Bone (ug/g DM) Zinc 52 ± 6 232 ± 14 Copper 4 ± 8 1 10 ± 3

DM = dry matter

Tabl : 6 e Interaction e intestinath n i s l transfe f coppeo r d an r zinc durin u deficiencC g r eitheo n Z yr (ng/intestinal sac • 60 mm)

Intestinal Intestinal Cu transfer Zn transfer

Trial 1 Cu deficiencg m 5 . (0 y

Cu/kg diet) 29 ± 7 22 ± 5

Control (4 mg Cu/kg diet) ± 2 19 ± 4

Trial 2 n deficiencZ g Zn/km 2 ( yg 3 2 ± 7 7 diet) 1 1 6± 8

Control (70 mg Zn/kg diet) 17 ± 4 2 ± 7 1

and copper, administered separately, is improved by Zn deficiency (Schwar d Kirchgefineran z , 197. b) , 4a

During Cu deficiency the zinc status is affected little. Usually, a slight increase of Zn retention is indicated in Cu-deficient animals as compared to controls given a normal Cu supply. Table 7 shows

77 Table 7 Changes in the Zn and Cu contents of serum, live d bonan r e durin u deficiencC g y

Cu deficiency Control g Cu/km 7 g7 ( g Cu/k(0.2m 4 g 30.g Zn/kgm 0 ) 30. g Zn/kgm 0 )

Copper Serum (pg/ml) 0.10 ± 0.03 1.03 ± 0.08 Liver (/Jg/g DM) 2.53 ± 0.77 9.00 ± 1.55 Bone (ng/g DM) 6.16 ± 1.12 7.51 ± 1.36

Zinc Serum (jug/ml) 1.83± 0.20 1.70± 0.20 Liver (/ug/ ) DM g 42. 7 4. 1 ± 39.0 4. 2 ± Bone (/ug/) DM g 246.0 ±12.8 236.0 ±11.3

n contentZ thae th n tbonei s , liver d seruan ,f Cu-deficienmo t rats were e increasefoun, b wherea % u o t C d0 1 e y abouo b th dt s 5 t contents were reduced dramatically (Schwarz and Kirchgefiner, . Unde197a) 9e samth r e feeding conditio u deficiencC n y shows no interaction with the absorption and excretion of zinc, but for copper these pathways adapted quite well to the supply status (Schwar d Kirchgeflneran z , 197 , 9b tabl . 8) e

Tabl 8 e Absorptio d excretioan n f coppeo n d zinan rc after Cu depletion

Cu depletion Control u C m (0.2pp u 4 C (7. m 7pp 30 ppm Zn) 30 ppm Zn)

Absorption (% of the intestinal dose)

Copper 17.8 ± 5.7 12. 6 ± 7.1 Zinc 43.7 ± 6.7 44.7 ± 4.1

Excretion (% of the i.v. dose)

Copper 22.7 ± 4.6 42.7 ± 5.7 Zinc 8 1. ± 1 . 14 4 12.2. 2±

78 4. 2 Interactions of zinc and manganese

Concerning manganese onlinteractionw fe y s with other essential trace element e knowar o n Accordin o resultt g f Heiseko s d an e Kirchgefiner (1978) slight reductions of zinc concentrations could be noted in liver, kidneys, spleen and bone in Mn deficiency when the Mn contents were markedly diminished Furthermore, there are several indications that Zn retention may increase in response to diets highly supplemented with manganese as compare o unsupplementet d d rations n despitsupplZ - e re yth e maining the same (Grace, 1973, Ivan and Grieve, 197t>, Jarvmen d Ahlstroman , a 197t>recen n I ) t experiment (Schward an z KirchgefJner, 198U n contentZ ) f live o d sbon an r e responded differently to deficient Mn supply (table 9), while the Mn

Table 9 Mn and Zn concentration in liver and femur in relation to Mn nutrition

Pretreatment Mn deficiency Pair-fed control

) Zn m pp 8 8 ; Mn m pp 7 (6 ) Zn m pp 6 8 ; Mn m pp (1

Manganebe (pg/) gDM 2 0 t 1 3. 67±04Liver Femur 0 9 ± 0 1 13. 0 t 0 6

Zinc (pg/) gDM Liver 63.3 ± 5.o 68 0 ± 8 5 Femur 482 9 t 12.1 446 6 ± 12.7

concentration was lowered significantly.

Durin n deficiencyZ g t juse concentrationo th ,t f zint o alsn bu co of manganese decreased in the liver, small-intestinal wall and bone (tabl , Schwar10 e d Kirchgefineran z , 1980 n contentM ) s increased again with appropriat n supplementZ e s althoug n supplM h y remained the same (Roth and Kirchgeflner, 1977).

79 Table 10 Zn and Mn concentrations in liver and femur in relatio n nutritioZ o t n n

Pre treatment Zn deficiency Pair-fed control (3 ppm Zn, ) 6Mn 4 ppm mpp 7 Mn6 ) , Zn m pp

Zinc (/ig/) DM g

Liver 0 4. 72. ± 4 bo. 0 ± 3.0

Femur 70. 4 ± 6.1 0 8. 206± 0 .

Manganese (;ug/g DM1!

Liver 7 0. ± 7 . 10 13. 2 ± 0. 8 Femur o O. ± o . 6 20.4 ±2.6

On the basis of these concentration measurements in various organs e overalth , l findin s i thag t zind manganesan c e respond equidirectionall o differencet y n suppli s f thes o yo metals tw e . Thi s i alss o supporte y studieb d s showing tha a deficienct f o y either manganese or /me augments the retention of i.v. administered zind manganesan c d loweran e e extenth s l theio t r endogenous excretion (see table 11, Schwarz and Kirchgefiner, 1980) Effects of this interaction on the extent of absorption are not evident Thus n absorptioM , n doe t changno s e wit n deficiencZ h y Like- wise, the intestinal uptake of zinc and transport of zinc is not affecten depletioM y b d n (tabl , Schwar12 e d KirchgeGneran z , 1980).

Taol1 1 e Enteral excretion of an i.v dose of Zn-ZnCl 54 or Mn-MnCl following different Zn and Mn iLt nutrition (values in % of dose)

Zn deficiency Control Mn deficiency Control

Zn excretion o. 2 25.0 16.9 24.4 ±0.8 ±1.6 ± 1 9 ± i 7

Mn excretion 7j.8 77.0 75.7 79.6 ±1.0 ±46 ± 2.8 ±3.2

80 Table 12: Absorption of zinc and manganese after Zn or Mn depletion

Zn absorption Mn absorption in vitro in vitro transfer uptake transfer uptake ng/ml yug/lOU mg DM ng/ml ng/lOUmgDM

6 2. Zn 3 depletio18 n 4.4 131 Control (Pair-fed) 6.4 0.5 4.5 111

Mn depletion 6.3 0.4 5.5 244 Control 6 0. 4 8. (Pair-fed) 2 16 3 5.

4. 3 Influence of gravidity and lactation on Zn metabolism

Gravidity and lactation significantly influence metabolism. There- fore, change e retentionth n i s , absorptio d excretioan n f traco n e elements could be possible. As it is shown in table 13, Zn

Table 13: Zn content in liver and serum of rats during gravidity and lactation

Gravid Control After Lactation Control S (da21 y

Liver (fj g Zn total) Dietary zinc m 2pp 0 270 183 188 182 100 ppm 256 185 190 193

Live g Zn/) (n r gDM Dietary zinc m 2pp 0 84 83 87 81

100 ppm 89 87 85 87

Serum (/ug Zn/ml) Dietary zinc 20 ppm 0. 74 1.47 1.24 1.35 m 10pp 0 1.24 1. 53 1.26 1.46

81 retention in the liver clearly increases during gravidity (Kirchgefiner and Schneider, 1978). This is mainly caused by an additional growte liveth f ro h becausn concentratioZ e th e s i n nearly unchanged. Under the same condition the serum Zn concentratio s diminishedi n , whereau concentratioC e th s n increases (Kirchgefiner and Spoerl, 1975). This reduction of the Zn content in serum during gravidity is clearly depending upon the dietary Zn content. A high Zn amount has a beneficial effect on the Zn concentration in serum.

Gravidit d lactatioan y n change alse absorptioth o n rat f zinco e . The experimental studies were done with the in vitro method using Zn-ZnCl (Schwarz et al. , 1981). As it is shown in n transfe Z e serosatabl , th 14 o et e rn uptak lZ th fluiy d b ean d intestinal wall is somewhat higher in the second third of the gravidity period in comparison to the control group, whereas in the last third there ifa a tremendous increase of the absorption rate. During the lactation period, the absorption declines again and therefore s i highe, r tha n nonlactatini n g controle s th onl n i y first days.

Table 14 Zn transfer to the serosal solution and uptake by the intestinal wall of gravid and lactating rats

Zn transfer Zn uptake (pg/ml) ) (pg/DM g

Control 11 4 ± 1. 3 4. 71 ± 0. 21 Gravidity (days) 6 8. 6 ± 1. 5 3. 73 ± 0. 28 12 19. 6 ± 3. 5 5. 68 ± 0. 51 18 168. 8 ± 15. 0 11. 56 ± 0. 47 20 112. 8 ± 13. 8 9. 83 ± 0. 89

Lactation (days) 1 31. 7 ± 5 . 7 6. 28 ± 0. 74 3 22. 0 t 6. 6 6. 14 ± 0. 68 8 8. 6 ± 1. 6 3. 93 ± 0. 46 21 7. 0 ± 1. 8 3. 72 ± 0. 47

82 9. Conclusions

In orde o fine t besr th d t method r detectinfo s a gminera l imbalance, t firsa t model studies under definite condition e necessarar s y with a supply e staturangth f deficientn o ei s , suboptimal, adequatd an e high supplements. As it is evident from the response the criteria of the trace element content in body fluids and organs, of biochemical parameters or of functional tests with isotopes there is no "method of choice". Therefore, to give an unequivocal diagnosis more than one parameter must be tested. Also, there is a difference in the importance of the criteria to different trace elements.

Problems could arise especially in using "normal" or "standard" value f traco s e element content n bodi s y fluidse , th organf o r o s enzyme activities.. As it was shown in the coordinated research work wite agencyth h , factors like interactions between different trace elements or the physiological status can change these "normal" valuese momentth t e mosA .th , t promisin d mosan g t sensitive methods for zinc, copper and manganese are the response techniques or functional tests. In these diagnoses the animal t i lika e parseru f r th s etreateo i o t m- - d wite elementh h t under investigation and the response to this treatment is used as parameter. Radioisotopes can be of special advantage. But in the future, much more interest should be given to stable isotopes. Also, more experienc e differenth r fo e t methods under field conditions i s necessary.

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Depletion Repletion Repletion 6 mg Zn/kg Jjp22-130mgZn/kg j^" D >lOOmg Zn/kg 10

E CT 6

3 1 0 1 8 16 19 Experimental period (weeks)

Fig. 1: Zn content in the milk of dairy cows depending upon the dietary Zn supply

87 Copper content of the liver

0 3 0 2 10 50 Experimental period (days)

Fig. 2: Relative decrease of Cu content in the liver and coeruloplasmm activity of rats during Cu depletion

220 y=2U8-110x+1.12x2-0031x3 210 R=0.2A P

160

150 _i——i——1_ 0 5 0 2 5 1 2 1 0 1 8 6 i 2 100 500 Zinc concentration of the diet(pgZn/gDM)

n contenZ f haio t : rFig3 dependin. g upo e dietarth n n Z y supply Pool for distribution ~^Z. + (Synthesis) (blood) J

Urine (Milk)

Fig. 4: Scheme for homeostatic regulation of trace element metabolism

100

160 c "o

20 Endogenous fecal excretion

0 20 40 60 80 100 120 140 Zinc concentration of the diet (pg/gDM)

Fig.: 5 Homeostatic respons n absorptioZ f o e d an n endogenous fecal excretio o different n n supplZ t n i y the diet

89 18

01 2 y= 5.0 20.15-0.0004x+ 2 (SI B= 0.98 10

0 13 0 11 0 9 0 7 0 5 0 3 0 1 Zinc concentration of the diet (pg Zn/g DM)

Fig, 6: Zn excretion of an i.v. Zn dose in response n supplZ e yth o t

90 EFFECT OF SELENIUM DEFICIENCY ON SELENIUM-75 AND CALCIUM-45 METABOLISM CHICKN I S

J.S. EDWARDLY Centre for the Application of Isotopes and Radiation, National Atomic Energy Agency, Jakarta, Indonesia

ABSTRACT Two hundred, 1 day old broiler chicks were divided into five groups. One group was fed a purified basal diet low in selenium ( ^O.O3 ppm). Three other groups were fed the basal diet supplemented with O.O5, O.I or 0.15 ppm basafifte e Th th lh d . diegroufe Se s tp wa supplemente r VitdU I pe wit .O . E h1O dietkf go .

After thre four eo r experimenta e weekth n so l diets, chicks were given an oral dose of 75S e or 45Ca. Blood, and tissue levels of radioisotopes, as well as total body excretion of radioactivity were determined.

While body retention of Se was significantly higher (p^ O.O5) in Se deficient chicks than in the supplemented groups. Liver Se level was also consistently highe unsupplementen ri d chicks.

Retention of 45Ca was significantly higher in selenium deficient chicks than in supplemented animals. Total Ca concentration in muscle was also higher in this group.

Studie celd re l demonstratee f uptaksS o f eo cleada r difference between Se repleted and selenium deficient groups. Selenium deficient chicks demonstrated poor growt feathersf ho , reduced weight gain and a high mortality as compared to the supplemented groups.

INTRODUCTION

Selenium is an essential nutrient for animals, including poultry. In the chicken adequate selenium is required for prevention of exudative diathesis, pancreatic fibrosi musculad san r dystrophy (Crie Scottd san , 1972)n i s A . other species, selenium deficiency disease in poultry occurs primarily in the young growing animal. Selenium levels in various tissues have been measured in selenium deficien repletd tan e animals (Scot Cantord tan , 1971, Arnol al.t de , 1974), However, adequate diagnostic criteri seleniur afo m deficienc chickn yi e sar still needed.

91 In bee s lambha n t sshowi n tha disturbancta musclf eo e metabolism characterize increasey db d level calciu f musclese o th n mi , occur earln a s ysa sig nutritionaf no l muscular dystrophy. (Godwi al.,1974)t ne . This increase in muscle calcium occurs clinica y prioan o rt l sign diseasef so . The objective thesf so e studies wer determino et e tissue concentrations of 75S e and 45Ca in selenium deficient chicks and also in chicks supplemented wi various levels of selenium after oral administration of the radioisotopes. Red celalss wa l o e uptakevaluatedS develof o eo t goa e s Th .lwa p practical diagnostic test evaluatinr sfo g selenium statu chickensf so .

Materials and Methods Two hundred broiler chicks (1 day old) were used for these experiments. The chicks were randomly allotte fivo dt e dietary groups basae Th .l d groufe s pwa purifiea d diet containing ^0.0 selenium 3pp m (Tabl . Three1) e other groups were fed the basal diet supplemented with 0.05, 0.1O or O.15 ppm selenium. The fifth group was fed the basal diet supplemented with vitamin E (100 IU alpha tocopherol per kg of feed).

Chicks were weighed and examined for clinical signs of selenium deficiency diseas weekl9 n eo y basis. week4 Afte r feedinf o s o r3 experimentae gth l diets chickn ,te s from each group were give orasodiun s na a le AiCS dos4 m if selenite eo . Heparinized blood samples were taken by cardiac puncture at 2, 4, 6, 24 and 48 hours after dosin determinatior gfo plasme th f ano disappearanc. e Se curv f eo Excreta including urine and faeces, was dried, ground and ashed at 550 C for three hours. hourA8 t4 s after dosing animale ,th s were kille varioud dan s tissues including liver, heart and muscle were collected for determination of 75Se retention. determinatior Fo heparinizef o uptakecel e d S l m re l 5 f no ,4- d blood was incubated in air at room temperature with ^20/uCi of Se as H^SeO.,. At 3O, 60, 120 and 18O minutes, 0.5 ml was removed from the incubation mixture. cellThd ere s were washed volume n twicte n eisotonii f s o c saline solution before counting. The uptake was normalized to a PCV of 4O in all cases.

bloodn i e LevelS , tissues f so excretd ,an a were determinen a n di automatic well-type gamma counter with a Nal crystal.

For determination of 45Ca retention, ten chicks from each group were give oracalciun s na a ,uCl4 a C dos3- im f echlorideo . Excret collectes awa 45 d and 7$Seprocessee th n epxerimentsi s da ashee Th .d excret dilutes awa a o dt constant volume in 6 NHC1. The level of 45Ca was obtained in an automatic liquid scintillation counte previousls ra y described (Godwi al.t ne , 1974).

92 Stable selenium in blood and excreta was determined by neutron activation analysis.

Itesult Discussiod san n

The effect of selenium deficiency on the plasma disappearance curve of Se after an oral dose is shown (Fig. 1).

It can be seen that Se deficient animals maintained a consistently higher plasme th (+2O% n ai )whee S leven comparef lo three th eo dt supplemente d groups. This suggests a greater intestinal absorption of Se in the deficient as compared supplementee th o t d animals.

Tissue levels of Se in the various groups are given (Table 2). The data show that the selenium deficient chicks retained a higher fraction of Se in all three tissues than did any of the supplemented groups. It can be seen tha livee tth r retaine higheda r fractio radioactivitf no y thaheard ndi r to muscle tissue in all groups, the liver level approximately 2-4 times that in the heart and 8-10 times that found in skeletal muscle. The increased level of S etissue e founth n seleniudf i s o m deficient animals sugges greateta r demand for Se in the deficient animals. There were no apparent differences in Se retention among the three supplemented groups.

Selenium supplementation significantly increase hour8 4 e s dth excretio n of Se (Table 3). Animals supplemented with O.O5 ppm Se excreted 43% more of the dose than did those in the deficient group (29.3% vs 19.8%). Increasing level supplementaf so l selenium resulte non-significann di t increasen si excretion when compared to the O.05 ppm group. This data strongly implies an increased absorptio deficienn i e S f no t animal compatibls i d san e wite hth plasm date S a (Fig. .1)

Red cell uptake of Se revealed a clear difference between deficient and supplemented chicks (Fig . Animalbasa.e 2) th ld sdie fe thos d tan e supplemented with either O.O5 ppm Se or vit. E had peak red cell Se uptake values above 23%, while animals supplemented withe S 0.1eithe r o m 5pp I rO. demonstrated peak uptak uptakless e r eshapo e th value Th % .f e 15 o curvef so s differed somewhat between deficient and replete groups. The deficient groups but not the vit. E supplemented group demonstrated a peak uptake at 12O minutes while the supplemented groups peaked at thirty minutes and decreased thereafter. This data suggests that perhaps O.05 ppm Se supplementation of the basal diet is not sufficient to ensure adequate selenium status. Further studies are needed in this area.

Excretion of 45Ca in the five experimental groups is shown in Table 4. Selenium deficient animals retained significantly more 45Ca than did those with selenium supplementatio evidences na significantla y db y reduced excretion ni

93 unsupplemented animals and vit. E. supplemented animals cotpared to animals supplemented with 0.1 or 0.15 ppm of selenium. Animals supplemented with O.O5 ppm Se demonstrated a retention less than that of deficient animals (76% vs 84%) but greater than that of animals supplemented with O.I or O.15 ppm Se (76% vs 52%). The data is conpatible with the results of the Se red cell uptake studies which also suggest that O.O5 Se supplementation may be inadequate.

45 Interestingly excretioa C e ,th n data indicate possibilite sth y that intestinal calcium absorption may be increased in selenium deficiency. However, since urine and faeces were collected and analysed together, another possibility is that while intestinal calcium absorptio unchanges ni seleniun di m deficiency, urinary excretio greatls ni y reduced. Additional experiment needee sar o dt clarify this question. 45 Muscle calcium levels reflect the excretion values for Ca; deficient animals show a higher level of total muscle calcium than the supplemented animals thin I (Tabls . casee5) , however, significan o thern e ear t differences among the supplemented groups.

45 The Ca retention data in the study agrees with the work done in lambs 45 (Godwin et al., 1974) where Ca retention was found to be significantly higher in dystrophic lambs date Th a. reported here thus demonstrate tha disturbancta e in calcium distribution is also founa in selenium deficient chicks. Further study is needed to detertrone Lhe temporal sequence between the increase in muscular calcium concentratio appearance th d nan clinicaf eo histologicad lan l change selenion si m deficient chicks.

The data in this study indicate several possible diagnostic indicators for selenium deficiency. Radioselenium retention in liver, total body Se retention, calcium level in muscle, 45Ca retention, and red cell uptake of Se all appear to hold promise as diagnostic metncds. Perhaps the red cell bein, Se uptak non-destructivga f eo vitrn i n oa techniqued ean , deserves additional consideration.

In addition, selenium deficient chicks demonstrated poor growtf ho feathers, reduced waight gain and a high mortality as compared to the supplemented groups mortality)% (251 d % comparan % 5 .o et

These studies also raise several basic questions regardin pathoe gth - physiolog seleniuf yo m deficiency answere ,th whico st h will require further investigations.

94 ACKbOiGLEDGBMENT

We wis egreso ht s appreciatio Internatione th o nt l Atonic Energy Agency for partial suppor thif to s work through Research Contrac 1930/K. tNo B from the Animal Production and Health Section of the Joint FAD/IAEA Division. A special thanks to Dr. Haryoto from Reactor Atom Bandung for his help in Se analysis of basal diet and to Dr. Yuyu Wahyu for suggestions regarding composition of the basal diet. We also would lik acknowledgo et technicae eth l assistanc. J . Mr f eo Gobel and Eddy Irawan, and to thank Michardi S. for typing the manuscript.

Tabl Compositio. I e Basaf no l Diet

dief o t% Ingredient

Corn 50

Rice bran 8

Coconut cake 10

Soya bean cake 30

Premi * (VitamixA Mineran+ l Mix) 0.25

Grit + Limestone 1.75

100

- Pfizer Product. Per kg Pfizer Premix A contain Vit. A. 2.0OO.OOO I.U. Vit D .2OO.OO O ; I.Umg .O Vit; Vit20 mg . 1.40. O E .B Vit ; 2O Omg K , . J J •J

_ (riboflin) 1.20 Vit; 0 mg r (pyridosin.B Niaci; mg 0 n )10 2.00 g 0m £. 6 Panthothenic acid 1.1O Chotin; Omg e chlorid; mg e O 2.00 8O Vit; 0mg .B DL-methemin 45.40 Santoquir; 0mg a 10.OOg 200e 2.00M F ; ; 0; mg 0Omg mg Cu 400 mg; Mn 3.0OO mg; Zn 2.0OO mg; I 20 mg.

- Se analysis of basal diet is less than O.03U q/q - Crude Protein level of basal diet is - Metabolisable Energy, Kcal/kg 2012.

95 Table 2. Effect of dietary Se level en 48 hr tissue retention of an oral dose of Se

Tissue Se concentration Treatment Liver , Heart Muscle (% dose/g) dose/g)*% -( * - (% dose/g) - Basal diet 0.69 0.27 0.09

Basal diee S O,Ot+ m 5pp 0.29 0.10 0,03

Basal diet + O.1O ppm Se 0.27 0.07 0,03

Basal diet + 0.15 ppm Se 0.28 0.09 0.03

7able 3. Effect of dietary selenium level on Se excretion during 48 hours after dosing.

Time faeces Basal diet- Basal diet Basal diet Basal diet collecting + 0.05 ppm Se + 0.1 ppm Se +0.15 ppm Se

Hours % dose % dose % dose % dose 24 18.6 27.6 29 31.4

48 1.2 1.7 1.6 1.72

Total 19.8 29.3 30.6 33.12

Tabl . Effec4 e dietarf to houleve8 e y4 S rn l o excretio f no 45.Ca afte oran ra l dose.

Group 24 hours 48 hours Total

Basal diet 14.88 - 1.29 1.43 - 0.30 16.30 - 0.84

BD + 0.05 ppm Se 21.59 - 4,69 2.O7 i O.44 23.80 - 3.45

, + . BD + 0.10 ppm Se 44.97 - 5,80 2.53 - 0.63 47.50 - 5,67

^ + , BD + 0.15 ppm Se 44.53 - 4.94 3.61 - 0,46 48.1 4.7- 4 8 , , , BD 1OO I.U 14,21 - 1.59 1,37 - 0.5O 15.5 1.8- 9 1 tocopherol

96 Tabl . 5 eEffec dietarf to y selenium concentratio e leveth n lo f no * musclen i a C .

Group Treatment Pectoralis Semi-tendenosis

(ppm) (HN

I Basal diet 7.50 6.75 II Basal diet + O.05 ppm Se 4.75 4.8 III Basal diee S 0.1t + m 0pp 4.0 5.2 IV Basal diee 0.1tS + m 5pp 4.75 5.0

Significantly differen XO.05tp basat ,a l diet group.

97 -O-

Figur Plasm: aftee 1 eS oran aa r l dose 75

98 Figur« 2 : Red cell af

99 Reference

GRIES. 1 , C.L M.Ld .an . SCOTT., Patholog Seleniuf yo m deficienc chicksn yi . J. Nutr (19722 .10 ) 2187-2196.

SCOTT. 2 , M.L A.Hd .an . COUTOR., Tissue selenium level chickn si s receiving graded amount of dietary selenium. Fed. Proc. 30 (1971) 237.

3. ARNOLD, R.L., O.E. OLSON and C.W. CARLSON., Tissue selenium content and serum Tocapherols as Influenced by Dietary Type, selenium and vitami Poultr. nE y Scienc (197U3 5 e ) 2185-2192.

h, GODWIN, K.O., JENN EDWARDL. YS CHRISTINEd Yan FUSS.. ,N , Retentiof no U5 Ratn CLambd ai an s s associated wit onsee hth Nutritionaf to l Muscular dystrophy. Aust. J. Biol. Sci. 28 (1975) ^59.

5. THOMPSON, J,N SCOTTd an , , M.L,, Rol seleniuf eo Nutritioe th n i m f o n the chick Nutr. .J 7 (1969.9 ) 335-3^2.

WRIGHT. 6 , P.L M.Cd .an . BELL., Seleniu vitamid man nInfluencE e upoe nth invitr y y Ovinb b o e e eS uptakS e Bloo f f eo o d Cells75 . Proc. Soc. Exp, Biol Medd .an . (1963) 379-382.

100 IMPORTANCE OF MINERAL IMBALANCES IN ANIMAL PRODUCTION AND HEALTH AND SOME DIAGNOSTIC AND CORRECTIVE TRIALS

K. GOKSOY, N. gETINKAYA, F. GUL Turkish Atomic Energy Commission, Lalahan Nuclear Research Institute in Veterinary Medicine and Animal Science, Ankara M. ORKIZ, A.I. GUCU§ Ministry of Agriculture, Lalahan Zootechnique Research Institute, Ankara, Turkey

ABSTRACT

Importanc Mineraf o e l Imbalance Animan si l Productio Healtd nan n hi Turkey, and Some Diagnostic and Corrective Trials. Mineral imbalances of farm animals in Turkey are a limiting factor in animal production and health. Apart from a few severe deficiency syndromes it is assumed that many moderate mineral imbalances (deficiencies and toxicities) stile exisar ld twaitinan investigatede b o gt . From this poin vief to w some diagnostic and corrective trials were carried out to develop a system for future application. For this purpose (i) an in vitro red cell radiomineral uptake tes developeds t wa founs wa dt I .tha modifieta vitrn di celd ore l radiomineral uptake can be used to determine the mineral status of farm animals under field conditions. The percentage of red cell radiomineral uptake is inversely proportiona seruo l t dietar d man y mineral levels, (ii) copper deficienc sheen yi p in the middle part of the Black Sea region of Turkey was investigated using several approaches. Results show that it is a conditioned (i.e. secondary) copper deficiency. Comparison of different parameters such as ceruloplasmin levels, seru levelu concentrationmS C d ,an forago M , eCu wel s s a environmentas la l conditions are useful indicators to evaluate the copper status of ewes.

pgTRODUCTION

Present knowledg tracf eo e element imbalance farf so m animal Turken si s yi derived fro stude mth sever f yo e deficiency syndromes. Example sheen si e par deficiencies of copper in the middle part of the Black Sea region and of selenium Interioe inth r Anatolia. Phosphorous deficienc cattln yi e occure th n si European part (1,2,3). Phosphorous deficienc firss ywa t diagnose botuliss da m in cattle but now it is known that the etiologic factor of the disease is phosphorous deficiency deficiene Th . t animals were scavenging bones contaminated with botulism happenin s organiswa t i othes n mga i r countries.

101 In 195 ove l Interio4e al rth r Anatolia white muscle diseas sees en wa n i lairbs (2). Thi followes swa outbreaky db enzootif so c ataxi e lambn ai th n si middle part of the Black Sea region in 1961 (4). Thus mineral deficiencies in farm animals became serious after a change in the agricultural system. Before 195 countre Oprimitiva th d yha e agricultural enoug d systeha d hman grassland to prevent mineral deficiencies by transferring herds from one pasture to another. Since 1950 the situation has changed with the use of motor vehicles and fertilizers and very many grasslands being ploughed throughout the country. Perhap severar sfo l years mineral deficiencie somn si e areas were occurring on a marginal level and then became serious with the change in the system.

In Turkey the production of good quality food for animals is not usual. Animal suppliee sar d with little concentrated food during winter. The main food during the long period of winter in the east, south east and Interior Anatolia is straw with a little hay or bran. Certainly with this feeding system the problem is not only mineral deficiency but the production of good quality food; the supplementation of deficient minerals makes the present food more profitable to animals to obtain more produce. For this purpose some diagnostic and corrective trials were carried out to use in our future studies to overcome mineral imbalances in farm animals.

MATERIALS AND METHODS

For diagnostic studies of mineral imbalances in farm animals relation between the percentage of in vitro red cell radicmineral uptake,serum mineral concentratio enzymd nan e activity; percentag vitrn i cel d f oeo re l radicmineral uptakes of the mineral supplemented and unsupplemented groups of animals; serum mineral concentratio enzymd nan e activity were determine comparisor dfo n whether in vitro test is suitable for the diagnostic studies or not.

Measuremen vitrn i cel d f otre o l radicMmjieral uptakes

The methods use Wrighy db Beld tan somd l an e(5)) (6 , . Perlal t ae other investigators (7,8,9,10,11) were modified by adding citrate-phosphate- dextrose (CPD) blood preserving solution (12) before incubation of red cells.

First Experiment: According to the local district veterinarian at the village of SUNNI, about 4O km from Ankara a few selenium deficiencies in lambs (white muscle disease) were reported during the last few years. It was learned that sheep were not supplemented with any selenium salt. In one farm ewes were marked with ear tags and separated into two groups. Three weeks before the in vitro seleniug tesm 0 t1 mg/ml0 (1 m sodius )a m selenite solutio SeO-.St^Oa n(N ) were injected intramuscularly int firse oth t group.

102 Three weeks later about 12 ml blood was taken from each animal into heparinized test tubeboxviv e n vitr celn i d kepic d I . ore d sl an n to i an radio-selenium uptake, whole blood Se concentration by thermal neutron activation analysi plasmd san a clearanc radio-seleniuf eo m were determined e botS n hi injected and non-injected groups (13).

Second Experiment: In the second year the first group of sheep was supplemented with zinc

sulphate (ZnS04.7H20) and sodium selenite (Na_SeO4 .5H2O). The supplement dailye S estimates g m ,wa thre4 a mixe O. r ed dfo d montan n Z h g periom 8 s da with concentrated diet and fed six tines every fifteen days starting on the 1st of November until end of February. In vitro red cell uptakes of radio- selenium and radio-zinc were carried out starting at the middle of March to the middl Mayf eo . Alkaline phosphatase activit seruf yo spectrophotometriy mb c (14) and Zn concentration by AAS were also measrued to compare with the in vitro red cell radio-zinc uptake.

Third Experiment: Red cel vitrn li o radio-zin radio-seleniud can m uptakes were carried out in Angora goats before and after supplementation with zinc and selenium. Lalahan state farm Angora goats were used as experimental animals. After the firs vitrn ti o testn (ZnSCL.Vl^OZ f ,o dailg m O y3 ) estimate animan a r ldfo perioy da 8 d n evera r yfo morninand dfe g admixed with theig m r 0 diet1 d ,an

Se/animal (Na2SeO,.SH-O) was injected intramuscularly 1O days before the secon vitrn di o test. Befor afted ean r supplementation blood seru corpusd man - cula concentrationn rZ serud san m alkaline phosphatase activity were measured comparisofoe rth vitrnn i wit e oh th tes t results.

In vitr celd ore l radio-mineral uptake test procedure To measure red cell radio-mineral uptake 0.7 ml CPD solution were pipetted into an 25 ml Erlenmayer flask and 5 ml blood was added, and gently radioactivl mixedm 3 O. . e solution containing abou ,uCt1 i radioactivits ywa added using a micrc-pipetter. All the flasks were covered with plastic stoppers an syringda e needl pushes ewa d throug stoppere n i hth r emptai o s t e yth

the flasks while approximatel O% 2 +5y2 95 wer %O0 e injecte tubes d ga fro se mth connected with syringe needles flaske Th . s were incubate houro tw n si r dfo a shaking water bath at 38°C. After two hours incubation period all flasks were transferre refrigeratoe th o dt theid ran r microhematocrits were measured. blool m 2 d were drawn from each scintillatio e flasth o kt n counting tubes. They were centrifuge minutes 5 250 m r rp plasm0e fo t dTh a discarde.s awa d dan the packed countinbotto e e cellth th f n mso i g tubes were washed twice with cold isotonic saline. Gamma counting was carried out using well-type scintillation counter, and calculated as the percentage of initial activity (5).

103 Measurements of in vitro red cell radio-mineral uptakes were carried out in different times after transferrin containinD CP g e blooth o dgt Erlenmayer flasks as below:-

(i) Blood samples in heparinized test tubes were taken to the laboratory and transferred to the CPD containing Erlenmayer flasks and incubated after injectioisotops hour4 ga r d o s ean 3 pas( n drao st w blood dan to start incubation).

(ii) Blood samples were transferre containinD CP e th o dgt flask soos sa n as bloo draws dwa n froanimalse mth .

(iii) Blood samples were transferre containinD CP e th o e dgt th flask n i s sa second procedure but kept 24 hours at 4° - C and then incubated.

STUDIES CN COPPER DEFICIENCY

Copper deficienc middl e sheen yth i n Blacepe o parregioa th kSe f t o f no Turke s investigatedywa . Blood serum copper concentratio ceruloplasmid nan n enzyme activity related with hemoglobin type ewesf so , prophylactic effecu C f to supplementation on the incidence of Enzootic ataxia (swayback) in lambs, and Cu, Mo and S concentration of pastures and diet, ratio of Cu to Mo were measured (15).

Percentag vitrn i celd f oere o l radio-selenium uptake result showe sar n eweie n th sTabl n supplementei e1 intra-musculay b e S f o d g witm O rh1 injection 3 weeks before the test and unsupplemented groups. Percentage of in vitro red cell uptake of radio-selenium in supplemented group was found lower than the unsupplemente dceld vitrgroupn re l i e oTh . radio-seleniu m uptak controf eo l grou founs pwa d lower than bot supplementee hth unsupplemented dan d group. This difference cones from the breeding condition of Lalahan state farm ewes which were thought to be in better condition since they were supplemented with concentrated diet during winter.

Exp. % of in vitro whole blood Groups______red ceTl uptake______Se Gone. ppm/DM Unsupplemented 7.8 2.964- a 0.7 00.04- a (36)x Supplemented 6.23 - 1.88b 1.13 - 0.096 (36) Control 5.39 - 1.27b ______(18)______

Table l.aiean percentag f in_vitro e o radio-selenium uptake injecS f o e- ted, non-injected and Control group of ewes (uiean-S.D) (x) figuree bracketth n i s s shov; sample numbers a,b. means with different lettere samth e t a scolum n statistically significant.

104 160

140

120 100

U " 40

fathh 48thh 48thh 72adh

Erythrocyte radio-selenium Plasma Clearenc f radio-seleo e - uptake nium (13)

Diagram shows erythrocyte uptake and plasma clearence of radio-selenium.

Selenium supplemented group,

Unsupplemented group,

Differenc e mea concentratioth e S nn i e f wholno e blood betwee e supplemententh d and unsupplemented group was found statistically significant as well and reflec vitr n i same e th toe th tesresuln i ts a XTablt . 1) e

Result of plasma clearance and in vitr^o cell radio-selenium of

supplemente unsupplemented dan d group e show(Tablar Fign ) ni . 1 e 1 Plasm. a rec clearanc f radio-seleniueo e unsupplemente fastee founs b th m wa o n t d i r d group than in the group supplemented with 1O mg of Se by the intramuscular injection three weeks befor trialse th e . However vivn i , o radio-selenium uptak f eryo e - throcyte was found to be higher in the unsupplemented group than in the supplemented. Results obtained from plasma clearance of radio-selenium in vivo an vitrn di celd ore l radio-selenium uptake supplementeth f eo unsupplemented dan d groups of ewes clearly reflect the expected results (Table 1, Figure 1).

105 The results of the second experiment are shown in Table 2. There was a difference between Se and Zn supplemented and unsupplemsnted groups of the ewes in in vitro red cell radio-selenium and radio-zinc uptakes as in the first experiment onle Th .y difference found betwee firse seconnth d tan d experiment lowee th rs percentagwa vitrn i cel d f eoo re l uptake radio-seleniuf so e th f mo second experiment than the first. The test procedure was the same except the blood samples were transferred to the Erlenmayer flasks containing 0.7 ml CPD solutio blooe bees sooth s n a dha s n na drawn fiel e frc eweth e dmth n s i instea d laboratorye th on fi difference Th . uptaken ei s betwee firse nseconth d tan d experiments is quite clear and it emerges whether the blood samples were transferred to the CPD containing flasks as soon as they were drawn or not. thir Fo s purpose secone ,th d experiment test procedur repeates ee wa th n di third experiment. f Zn-6o % 5f Se-7o % 5Alkalin Exe p Serui mZf Groups red-cell uptake red cell uptake phosphatase cone, activitf yo ______serum(SU/ml)______Supplemented 2.30-0.73a 1.69-0.493 5.35^1.91a 1.28±0.47a ______(50)*______Unsupplemented 2.99±1.21b 1.88io.57b 4.11-1.74b 1.02±0.33b ______C50)______Table 2.Percentage of in vitra red cell radio-selenium,radio-zinc uptakes,and serum Alkaline phosphate activit d Zinyan c cone.(Mean-S.D). (x)Figure e bracketth n i s s ahow sample numbers a,b.mean values with different letters at the same column statistically significant.

However, mea vitrn ni celd valuee ore th l f uptakeso f radio-seleniumo s d an , radio-zinc between supplemented and unsupplemented groups were found significant. s knowi t I n thergooa s dei correlation between serum zinc concentratiod nan alkaline phosphatas r experimenou en i activit d e samth an t e) resuly(7 s wa t obtaine s showda Tabln ni . e2 Serum alkaline phosphatase activit zind yan c concentration were found to be lower in the unsupplemented group of ewes than the supplemented v.v. with the in vitro red cell radio-zinc uptakes. The third experiment was carried out to check our previous studies for the in vitro red cell uptakes of radio-zinc and radio-selenium. This time amount of mineral salts supplemented to Angora goats was raised to 3O mg of Zn daily and fed for 8 days supplementing every day early in the morning instead of periodi- injectes wa e S md O f gcally1 o intramuscularl d an , e firsth n ti experiments ya . In vitro radio-selenium and radio-zinc red cell uptakes were measured before and after supplementation. Results are shown in Table 3. Serum Zn concentration and alkaline phosphatase activity of serum was also measured to compare the Zn statue animalth f so s (Tabl. 3) e

106 f Zn-6o % 5 f Se-7o % 5Seru m Exp. phosphata8e Groups uptake uptakConc.Hg/ran Z e l activity ______(SU/ml) Before 3.53 - 0.14b 1.53 - 0.07b 1.10 ± 0.05a 3.33-0.24 Supplement 8t8 8O± .l6(e) 9.3 O?± .ll(s) (45)*______

After 2.64 - 0.09a 1.61 i 0.10a . . . Supplement 2.2 0.232± ° 4.11-0.21° (45) 9*85 " °*12 8'77± 0-15(0)

Tabl . Percentag3 e f in-vitro e d celore l radio-seleniu radio-zind man c uptakes before and after suplementatior with in on £e of Angora goats (Mean i SE) (x) Figures in the bracket show sample numbers to, .a mean values with different lettere samth e t sa colum n statistically significant. (s) Serum activity

In vitro red cell radio-selenium and radio zinc uptakes mean values were found to be significantly different befor afted ean r supplementation with related minerals. Serum zinc concentration and alkaline phosphatase activities before and after supplementation were also found to be different as in the in vitro red cell uptakes. These results show that the red cell in vitro uptakes of radio-minerals reflect the minerals status of Angora goats. Measurements of serum activity after in vitro red cell uptakes were found to be high after supplementation. This suggests tha radio-minerae tth bindins celld li re so g t durin g incubation period proportionally with the contact of diet supplied to animals.

It seems that "th vitrn e i celd ore l radicMmneral uptake usee b dy "ma adiagnostisa c tes determino tt minerae eth l statu farf so m animals since the result secone th third f dan so d experiments were simila therd ran s ewa little variation among the percentage of uptakes even if the test applied in different species and condition. The use of CPD solution to keep blood samples test from hemolysis and to extend the time to to be applicable under field condition is quite convenient according to their hematocrit values. Nfean hema- tocrits obtained before and after supplementation are 28.00 - O.38 and 28.22 - O.39 respectively. It means that Ht. values (Corpusculaer part) before and after supplementation trials in 5 ml blood 1.4O and 1.41 ml respective- dilute e ly value. caluclate s th percentag,x a Ht f l o m ds . 5 bloo Ht y df b eo d samples are:

un .• • * amoun. c± TT.Lf .corpusculat o O l bloo1O rm X par5 dn ti Ht after dilution = blood samples + 0.7 CPD + 0.3 isotope sol.

A resul sa founs i t dti that only 4.7 2.O9d 8an % hemolysis occurred durine gth incubation period.

107 modificatioe th vitr n f i cel o d e om re l th ai radio-^minera f no e Th l uptake test is to develop a method to determine mineral status of farm animals under field condition. The in vitro red cell radio-mineral uptake test results obtaine somy db e investigators reflec dietare tth y intak tracf eo e mineralf so farm animals. However, their studies are related to the laboratory condition. It was thought that if the method used by Wright and Bell (5) and by some other investigators (6,7,8,9 modifies )wa might di applicable tb fiele th do e t condition . Under field condition the difficulty is to keep the blood fresh until testing, i.e. from one to several days. For this purpose citrate-^phosphate-dextrose solutio useds ni . This solutio humar fo ns n i bloo d preservation until transfusion. Gibso. (12)al t ncomparee d clinical significanc D (acide-citrate-dextroseAC f yo ) f anradioactivsolutionD o dCP e us e vitrn sth i eshowey d o b chromiuan ) d Cr ( m that the mean survival time of red cells at the 28th day is 70% for ACD and 76% for CPD when they were kept at 4 -1C.

The studies on the distribution of radio-selenium in heme and non-hems fractions of mice bone-marrow and spleen show that selenium binds to red cell in the circulation instead of during erythropoiesis, and the excretion of selenium takes place both by the urine and faeces (17). The same trial must be radio-zincarrier fo t dou determino ct amoune eth zinf to c bindind re o gt cell during erythropoiesis and in the circulation.

STUDIE COPPEM SC R METABOLISM (15)

It was found that Karakaya ewes in the copper deficient area mostly hemoglobicarryinB A d an gA n types. Mean seru concentratiou mC ewee th s f no related with their hemoglobin types were found about at the same concentration ceruloplasmie th an si n O.D. (Tabl. e4)

Hb typeb Lumbe ewef ro e Ceruloplaemin Seru u C mlumbe f ro P.P. • •• • — r — A 161 0.086-0.005 0.75*0.03 15

AB 125 0.087-0.009 0.73-0.03 11

B 11 0.059-0.003 0.66*0.04 2

Table 4. dumber of the ewes carrying different hemoglobin types and their ceruloplasmi d nseru an O.Dconcentratiou mC . d numbean n f o r ataxic lamp born (mea - nL.L) .

Thergooa s di e correlation betwee e numbenth f ataxie o r th d can lambm bo s hemoglobin types of ewes. It was found that there is no prophylactic effect of 1O mg Cu supplementation (as a total for 45 days) fed to ewes three times every fifteen days durin e lasgth t trimeste f pregnancyo r incidence Th . f eo

108 ataxic lamb bom was 8.6%. When the amount of Cu supplementation was increased timex si twsd oever fe fold ydan fifteen days starting 15th Octobe folloe th n r-i wing sam e yeath e o experimentart l animal incidence sth ataxif eo c lambm sbo decreased to 2%. Ceruloplasmin O.D. and percentage of serum total protein were found ewe e loweth s n witri h ataxic lambs than ewes with healthy lambs (Tabl supplemente) e5 d withO S coppe u C s ra

Serum Ewes with ataxic Ewes with normal parameters lambs lambs

ceruloplasmin 0. 078±0. 011(14) 0.156±0.96(43) O.D.

seruf o m% total 6.27-0.30(19) 7.30±0.13(4~) protein

Table 5. Ceruloplasmin O.D. and percentage of serum total protein in ewes with ataxi healtd an c y lambs (means-S.E)

The amoun supplementeu C f to mg/daO 1 eacr s yfo dwa h ewesupplemene Th . t d everfe s y wa fiftee ndaystota5 a 4 day r f ,lso fo admixing wit diee hth t and applied 6 times starting on the 15th October. Forage samples collecte Aprin di l fro differenm7 t placee th n si diseased area contain about 18.9 ppm Cu (D.M.) 9.2 ppm Mo forage and diet samples collecte Novemben di o rM contaim pp 2 n4. aboud an tu C 11.m Opp (Table 6).

Forage samples______Cu ppm/DM______Mo ppm/DM______hovamber 1973 11.0 4.2

April 1979 18.9 9.2

Table 6. Forage and diet Cu and Mo concentration collected from the diseased area.

Mean sulphur conten forag6 7 f o et samples collected from different parte e diseaseoth f d O.31 e arefouns b - O.O17%8ao wa t d . Seru concentratiou mC ceruloplasmid nan n O.D f sampleo . s collected from non-diseased area different breed f eweso s (Interior Anatolia) were shown i n Table 7. It is clear that the Cu concentration and ceruloplasmin O.D. of the ewes from diseased are e loweaar r tha e non-diseasenth d area different bree^ o d ewes. Forage samples collected from 7 different places around Lalahan State farm contained about 7 ppm Cu, 1.6 ppm Mo and O^2O3% S, concentrated diet fed animals during winter contained about 7.41 ppm Cu, 2.05 ppm Mo and 0.211% S.

109 Specie f Breedo s s Seru (ffg/mlu mC ) Ceruloplaamin O.D.

Diseased area 0.72 ± 0.03/,19i 0.077 - 0-003/ ^ (samsun) (312) (280) KARAKAYA SHEEP

gubuk-Ankara 1.24 - 0.07 0.110i 0.005 j AKKARAMAK SHEEP (150) (4Q

Lalahan State farm 1 "7*7 i f\ ~]r\ 0.122 - 0.004 Ankara Merino sheep (36)

Table 7. Diseased and non-diseased area different breed of ewes serum Cu cone d ceruloplasmian , n O.D. (mean-S.E)

Concentration of Cu, Mo and S in the diet are lower in the non-diseased area than diseasee th d are t diseasee serusheen i th a bu u mn pS i d lowe e arefouns b ao wa rt d than state farm sheep serum Cu. Ratio of Mo to Cu is about between 3.6 : l.O and 4.4 : l.O and they are in the normal level according to the classification of Lesperance (18).

Copper deficiency conditions classifed by Lesperance are(i) sinple copper deficiency ppO m5. Mo- , .1 die0. Ihi d t s an ppcontainin conditiou O mC 5. - I gnO. is not suitable for sheep in the middle part of the Black Sea region in Turkey since diee th t contains enoug meeo t u thC copper requirement : M s u changinoi C . g from 1 : \ii) 8 2. norma o t . 1 l condition: 1 2. , diet containin - 15.O Og5. 1.O-5.pp, mCu O : Mresult pp d u s ochanginmi an C Md 1 so3: an obtainego t r studie fro1 ou m5: y db s do not resemble this condition, (iii) molybdenum toxicity, diet containing 5.O - 8.0 , 5.O-50.ppmCu t possibl therOno y molybdenua sa s ppi s ei o m et t i Mod m,an toxicity for middle sheeth n pe'i Blace regioa parth f kSe o t n accordin resultse th o gt . The results obtained here resemble closely the studies classified by Bingley and Carillo (19). These worker diesd statetan 1 : M d: u o 8 thaC equa 2. f i t o t l containing enough sulphate hypocuprosis is clear. In our studies in the diseased area the dietary Cu and Mo ratio was found to be between 2.1 and 2.8 and the diet contained enouah S (0.318 - O.O17 %); and ecological conditions is quite available consumo t e fresh foor sheefo d evern pi y seasoe yeath rf importan n no whica s hha t role in the development of copper deficiency as stated by Ward, Hartmans and Barshad (20,21,22,23). V/ard (20) states that the animal can't be safely grasse n Colaraddo o grasslands containin higheo gM r tha0 2 n ppm and if it is fed ae hay there is no problem for animals. In Ho- land causo M em hypocuprosipp 5 eve f i n n animali s s (low n Colarad i founs B wa I. d y t rathei oha s a r d fe s i t i f i t bu live ) Cu r safe (23). As a result we can say "Copper deficiency in sheep on the middle part of the Black sea region of Turkey is a conditioned copper deficienc r proved ou an pointeUrma s y yy b ) b d wa statust (1 n t ou i d s a " studies.

110 References arkadae v Urman . 1 . K þ. lar,H ý (1971) Vet.Fak.Dergisi 18,276 Baran. 2 (1966. ,S ) A.U.Vet.Fak.Dergis. 25 ) (1 3 i1 3. Pamukçu, A. M. (1954) Türk Vet.Hek.Dern.Derg. 96-97. 4. Karagözoðlu, A. G.(1962),Türk Vet.Hek.Der.Dergisi,190,191,324. 5. Wright.^.L.and Bell,M (1963. C ) Stroc.Soc.Exp.Biol.Med,114-379 6 .l (1968 a Perlat e Kut.-.. )J . ,L , 219 94 , . 7. Me Gunnel, K.P.and Cooper, B.J.(1950) J.Biol.Chera.Vol. 183, 459. Schwarz. 8 (I960. ,K ) Nutr. Rew. 193, ,18 . 9. Burk, R. F et al (1967) Am. j. Cl. Nutr. 20 723. 10. Berry, R. X. et al (1966) J. fcutr. 83, 284. 11. Chester, J. K. and Will, M. (1978) 38, 297. 12. Gibson, G. J. et al (1961) Transfusion, l, 280. 13. Göksoy, K. et al (1980) (Ý.Ü.Vet.rak.Derg., 1-2 282. 14. Sigma Tech bulletin No.104 (10-74) If. Göksoy, K^Çalýþlar, T. (1980) Summary of the Report Submitted to the Turkish Scientific and Technical Council. 16. Kirchgessner, M. et al (1976) Nuclear Techniques in Animal Production and Health, IAEA-SM-205/102 . Gzsar17 , GöksoyS. , . BoundinK , f Seleniuo g o Erythrocytmt n i e Mice, (in press). . Lesperance18 , A.C.(1967) Winnin e Battlth g e against Molybdenosis, Levada Ranc d homan h e review. 14 » 3 , 19. Bingley, J. B. and Carillo, B.j. (1966) Nature, 201 834-335 20. Kard, G.1Â. (1977) J. Animal t>ci. (cited from kc Dowell and Conrad J.H., World Animal Review, SAG pub. (1977) 24,24. . Hartmans21 . J (1976, ) Nuclear Technique n Animai s l Productiod an n Health, IAEA, SM-205/24, 123. 22. Barshad . (1948)L , , boil Sci. , 187,06 . . . (1976WardM 23 . G ), Nuclear Technique n Animai s l Productiod an n Health, IAEA, SwI-205/24, 123.

Ill COPPE COBALD RAN T STATU SHEEF SCATTLO D PAN E GRAZIN CENTRAE GTH L HIGHLAND PERF SO U ELUCIDATE ENZYMY DB E ANALYSI NUCLEAD SAN R TECHNIQUES

J. KALINOWSKI Universidad Nacional Agraria La Molina, Lima, Peru

ABSTRACT

Studiee coppeth d cobalf o an sr t statu f grazino s g ruminants were made through measurements of blood concentrations, plasma caerulo- plasmin levels, and by the determination of vitamin 8^2 levels by radioassay. The results demonstrate not only the importance of sample handling techniques on the values obtained, but also the limitations of currently available technique s indicatora s f minerao s l status.

Introduction

The Peruvian Central Highland is inainly a sheep raising area,

which also support n importana s t populatiow f cattlo nNe d an e

World Cameloids. Feedine stocth f ko g depends entireln o y

the use of natural grasslands composed of grasses of relative^ nutritivw lo y l e value (Echevarri a_l^ et .a, 1970) .

Mineral analysi f nativo s e grasses froe Centramth l Highland

grasslands (Echevarri , 1970 . al , t 1970; e . Soikea al )t e r

~~vealed relativel concentrationw lo y f coppeo s cobald an r n i t~~

~~some specien certaii d an sn localities e pastth n ,I neona. -~~

~~l ataxita widespreas wa a n somi d e areas; having been control^~~

~~led in well organized farms by copper supplementation and~~

~~paddock selection, involving the elimination of affected pad_~~

~~docks frora use for grazing, or the use of such paddocks for~~

~~grazing of store lambs and steers for short periods of "time.~~

~~Copper blood levels were initially used to determine normal~~

~~and deficient states. When it vras shown that blood copper~~

~~levels were maintained -at the expense of liver reserves, in_ 113 ter-est changed towards the detertiination of copper in snro~~

~~pies of liver tissue taken by biopsy. Cobalt levels in~~

~~sair.ples of liver have also been used to determine the co-~~

~~balt statu f ruminanto s s (House t aj.e r . 1978), . However,~~

~~livestock keepers are reluctant to allow liver sampling by biopsy whereas there is no resistance to blood sampling.~~

~~Plasma ceruloplasmin levels determined by oxidase activity~~

~~measurements arc considered to he of greater value than copper blood levels in assessing copper status (Bingley and Anderson, 1972). Since vitanin B^2 is the functional form~~

f cobalto t seeni t s e determinatioreasonablth e us o t y f o n vitamin indicaton serua i s j a m B^ n f cobalo r t statuf o s ruminants n recenI . t years radioassays have largel- re y placed microbiologica lmeasuremene assayth r fo s f seruo t m

~~vitami• T BI n~~

~~Material d Methodpn s s~~

~~Sampling Several surveys were carrieo collect t ou dt blood samples~~

~~from livestock grazin e Peruviath g n Central Highlands.~~

~~e firsTh t too ke Jaujplacth n ai e provinc e begininth t a e g~~

~~e rainoth f y season (December 1979). Blood samples were collected froo calleds me sheeth f Huaccho p a type, raised~~

~~mainl y peasantsb y .~~

~~In February 1980, a second survey was carried out in the~~

~~same area but samples vere taken this time from Corricdale~~

~~shee pg cooperativ rearebi a y b d e farm (SAIS Ramon Casti- raine th yf o seaso d en ne (Marcth lla) t A .h 1980 thira ) d surve s carriewa y t oveou da larger r are f Junio a n Depart-~~

~~ment, where animals reared in organized farms and animals kept by peasants were sampled.~~

~~114 Serum samples taken during December 197d Februaran 9 y 1980 were insufficien o carr t t bot ou yh ceruloplasmi d vitan n - ami2 assaysB^ n , therefore only ceruloplasmin analysis were performed.~~

Ceruloplasmin oxidase activity According to the procedure reported by Ravin (I960), samples of 0.1 ml of serum were incubated for 1 hour at 37°C in 8 ml of 0.4 H acetate buffer at pH 5.5 with 1 ml of 0.5% p-phenylemediamine dihydrochloride substrate (FPD). The reactio s stoppe a 0.5wa n f %o y addin b dsodiul m 1 g m azide solution, and the absorbance of the coloured solutio . n Ceruloplasmimu measure 0 53 t a d n concentration wfis calculated multiplying the optical density attained in the PPD-oxidas ee constanassath y b y t factor 87.5, whics i h e Holmberg-Laurelbaseth n o d l facto r conversiofo r _ op f o n

~~tical density at 605 mra to ceruloplasmin concentration~~

~~{Ravin, 19,60)~~

~~Serum vitamin IK ^ concentration~~

~~The radioactive methods for the determination of vitamin B._ in serum have been described by several authors (Lau et al.,~~

~~1965; Henry and Haibach, 1974).~~

Principle of the procedure e radioassaTh y procedure r vitamifo s determination- B. n e ar s based on the principle of saturation analysis. A standard amoun f vitamio t B n binding agent (Intrinsic Factor) IF , is added to a mixture of a standard amount of labelled vita-

~~min Bj_ and the serum extract containing an unknown amount~~

~~f unlabelleo d endogenous vitamin B.?. When bindins ha g taken place the B not bound is removed by adsorption to al i0 JL —~~

buminized charcoal e bounTh .d radioactive vitami 2 varieB- n s inversely wite amounth h f unlabelleo t d vitamin B._e conTh ._ centratio serue th f vitamimn o n i extrace deterb B nn ca t- mine y comparinb d percentage th g f bouno e d radioactivity with a standard curve that is obtained by replacing the serum ex-

~~tract by various vitamin B12 solutions of known concentration.~~

~~Preparation of serum extract f redisto l m -5 2. d an % mg O U N KC f o f serum* o l m l m 55 ,0. illed water were mixed in a 25 ml beaker and titrated~~

with IN HCL to pH U. 8-5.0. Then it was transfered quantitatively into a 25 ml volume flask and after letting it stan t rooa d m temperatur r aboufo e 1 hou ts steamerwa n i d an autoclave for 30 minutes at 100°C with no pressure in

~~the chamber. After heat treatment samples were let to~~

~~cool at room temperature for about 1 hour. Volume was brought to 25 ml with redistilled water. Coagulated prp_~~

~~teins were e supernatanbroketh d an n t flui s separatewa d d~~

~~from the protein deposit by centrifugation at 3000 rpm 0 minutes2 r e supernatfo Th . s freezwa e e stored.~~

~~Assay protocol Tabl 1 showe procedure th s e followee estimatioth r fo d n~~

~~of vitamin 6^2 ^n serum extracts.~~

Graphic analysis Graphic analysis requires the following data: Percent binding by IF.- Is calculated by dividing "Total counts" into the average count for the "aero saline" counts correcte e counte "zerth th of r o s extract"fo d . B/A ratio. s calculateI - y dividinb d e correcteth g d "cero

~~saline" by the average count for each 8^2 standard and for each sample.~~

* When les l wersm serue 5 availables use1. wa md d an 5 ,2. l reagentanal d s were adjusted proportionally.

~~116 Data for the standards are plotted in linear graph paper~~

~~having the (B/A-1) ratio on the Y axis and the amount of axisX e r e .th stabltubP n 2 o e 8^ e~~

~~Mathematical calculation The mathematical calculation requires a knowledge of the~~

~~amount of B12 in the radioisotopic solution. The basic formula is:~~

~~X = Y (B/A-1) where X = weight of B^j in a sample Y = weight of Bj2 in the amount of radioactive~~

~~e assa th 2 use n yBj i d B = CPM of 57Co Bi2 in the "zero saline" r samplfo 2 eBj supernatano C f o M tCP = A c «7~~

~~RESULT D DISCUSSIOAN S N~~

Ceruloplasmin concentration No differences were found in ceruloplasmin concentration among Huacch d Corriedalan a e sheep surveye e Jaujth t aa d province. Ceruloplasmin concentrations wert affecteno e d by sexa tendenc t ,bu y towards lower level f ceruloplaso s _

~~min was observed in adult sheep as compared to young~~

~~stock (Tabl . Variabilit2) e y tendee higheb o t dr among~~

~~Huaccha sheep.~~

~~117 TABLE 1, ASSAY PROTOCOL FOR THE DETERMINATION OF SERUM B12 LEVEL~~

~~Plasma Col2 Bl d 0.9% Saline Buffer3 Tubes Extract Sol A .Sol B 4 . P0 SolutioCoBnIF 12 1 2 575 4~~

1 + 2 0 00/ 6 0 l m 0 1. 0 0 0 4 3+ l / m 7 0 1. 0 l al o t l m 0 2. 0 l 1m 5 5 + 6 0 00 1 0 ml to all tubes 0.5 ml 8/ 0 l m 3 . 0 7 + 8 0 0 7 tubes to all 0 1 + 9 0 0.2 0 0 8 tubes 2 11 1+ 0 0.15 0 0 85 4 11 3+ 0 0.1 0 0. 9 . 6 11 00 5+ 0 75 ml 0 25 17 + 18 0 0 0.5 0 5 0 2 + 9 1 0 0 0 .2 0 8 2 22 1+ 0 0.1 0 9 4 22 3+ 1.0 ml etc to all samples tubes

I\ \ .' / \I\ >' After all additions samples were let stand for 1 hour at room temperature. 1 ml of albuminized charcoal was then added to all tubes except 1+2, mixing well and centri^ fuging for 15 minutes at 3000 rpm. After centrifugation the radioactivity of the supernatant flui s countedwa a .

1 1 nvug B12/ml 5 15/agIF/ml 1 nvu0. g B2 12/ml 6 Total counts 3 0.26 M KH2POn Buffer at pH 7.0 7 "zero extract" <+ 0.05 mug/ml (JO.0091 uci) 8 "zero saline" TABLE 2. CERULOPLASMIN CONCENTRATIOHS (tng/100 ml) IN BLOOD SERUM OF CORRIEDALE AND HUACCHA SHEEP SAMPLEE JAUJTH N AI D PROVINCE

~~Huaccha sheep Corriedale sheep Yearlings (Y) Adults (A) Yearlings (Y) Adults (A )~~

~~Sex M F M F M F M F f sampleo . No s 18 20 U 8 9 12 • 2 10 Ceruloplasmin~~

~~Jc 18.5 18.6 13.8 14.2 17.0 19.9 26.9 15.9~~

~~SD 6.2 6.5 4.4 9.4 2.6 3.5 2.2~~

~~Similar results were obtaine n Junini d ,e high excepth _r fo t er coruloplasnin values found for adult sheep (Table 3). Brown Swiss cattle sample n Laivi d e shovred higher average~~

ceruloplasmin concentration than crosse f Browo s n Swisx s Criolio sample Runatulln i d w ceruloplalo e o Th (Tabl £. 4) e min values obtained from cattle in Runatullo could be a cha_ racteristic of the riollo cattle, since similar values (7.8 mg/100 ml) were found in serum samples taken from 12 rio_ llo steers fed on a different diet when the ceruloplasmin procedure was in the setting up phase in our laboratory.

Ceruloplasmin concentrations found suggest that copper supply for ceruloplasrain synthesis nay not be adequate. Since both, mineral supplemented (Corriedale) and unsupplemented sheep (Huaccha), show similar ceruloplasmin values, and copper content in forages available for both type f sheeo s s similari p , varyin s ppm8 gi o frot t i ; 6 m likely that the copper supply by the mineral supplements e adequateb t no y ,ma unde e and/ous r r that impairmenf o t copper utilization by other elements may overrun the sup- plementary copper. The possibility that handling of samples before the determination of cerulcplasmin activity itself might affect results should not be ruled out.

~~119 TABLE 3. CERULOPLASMIN COKCENTRATIOHS (mg/100 ml) IN BLOOD SERUM OF CORRIEDALE AND HUACCHA SHEEP SAMPLE N THREI D E LOCALITIE F JUNIO S N DEPARTMENT~~

~~Localities Acopalca Laive Runatullo Huaccha Corriedale Huaccha Corriedale Corriedale~~

~~A Age y Y . * A F MS F M6 x Se F M M F 2 1 f sampleo . 2 No 1 s 12 8 17 18 Ceruloplasroin j? 18.2 18.2 15.9 15..9 19.3 19..U~~

~~6 5. SD 8 5. 5.4 7 5.4. 8 3. .5~~

~~TABLE 4. CERULOPLASMIN CONCENTRATION (mg/100 ml) IN BLOOD SERUM OF CATTLE SAMPLED FROM TWO LOCALITIES OF JUNIH DEPARTMENT~~

~~Localities Laive Runatullo~~

~~Breed Brown Swiss B.S. x Criollo~~

~~Age, years 2-3 4-6~~

~~Sex F F~~

~~f sampleo . Ho s 8 17~~

~~Ceruloplasmin~~

~~D S 1 X 14.10.t 7 0 » 7 1. 5. i 5~~

~~TABLE 5. VITAMIN B12 CONCENTRATION (mug/ml) IN BLOOD SERU F O M CORRIEDAL D HUACCHAN E A SHEEP SAMPLED FROM THREE LOCALITIES OF JUNIN DEPARTMENT~~

~~Localities Acopalca Laive Runatullo~~

~~Breed Huaccha Corriedale Huaccha Corriedale Corriedale~~

~~5 1 5 1 8 1 1 f sampleo 0 2 . Ho s~~

~~Vitamin 12 x 1 SD 1.02+0.5 0.9810.62 1.11±0.73 1.07±0.59 1.05+0.65~~

~~120 Serum Vitami? BI n Table 5 shows the concentrations of vitanin 8^2 determined in samples collected from Huacch d Corriedalan a e sheep.~~

~~Values foun e extremelar d w comparelo y o reporte t d th f o s~~

~~literature (Sutto d Elliotan n , 1972; Hedric al_-t_ et h 1973). Levels of vitamin B._ found are even lower than serum vit-~~

~~amin D-2 concentrations obtained when cobalt content in the diet of sheep was as low as 0.06 ppm (Hedrich et a. 1., 1973).~~

~~These results might not reflect a cobalt defficiency, but probabl n inadequata y e procedur o obtait e e seruth n ex m tract in addition to some deficiencies in sampling. It~~

~~is likely that liberatio bounf o n d vitami 2 1 mighB n t no t be complete, and/or some binding proteins might not be completely remove r inactivateo d d durin e extractioth g n~~

process alss i ot I .likel y that vitami 2 1 contene B n th n i t samples migh e loweretb d since reasonable volume f seruo s m are only obtained long periods after sampling under the environmental condition e highlandsth f o s d transpor,an f o t samples to the laboratory takes from 6 to 8 hours after survey have been completed.

~~The questions that have arisen from results obtained point t onle importancno th y t ou f studyino e e effecth g f hando t -~~

~~ling of samples upon results when 'deficiencies for blood sampling and ready processing are faced, but also the limit_~~

ations for the use of procedures assesing biological acri_ vity or concentration of biological compounds related to trace elements as indicators of mineral status of animals under the circumstances described.

~~121 LITERATURE CITED~~

BINGLEY, J.B. and H. Anderson. (1972). Clinically silent hypocuprosis and the effect of molybdenum loading on beef calve Gippslandn si , Victoria. Australian Jour- nal of Agricultural Research, 23: 885. ECHEVARRÍA, H., R. SOIKES, K.C. BEESOU y J. KALIHOHSKI. (1970). Interrelaciónos Suelo-Planta-Nutrició. nX Composición químic s forrajelo e d a JunSne sd . Anales Científicos, 8: 179. HEDRICH, M.F., J.H. FLLIOT and J.E. LOKE. (1973). Response in vitamin B-12 production and absorption to increasing cobalt intake in the sheep. Journal of Nutrition, 103: 16*6. HENRY, R.E. and H. HA.IEACH. (197H). Radioassay for vitamin 8^2 a Radioassal - Clinican i y l Medicine. C.C. Thomas, Springfield, 111. pp 83-10»*. HOUSER, R.H., K.R. TICK and L.R. HcDOWELL. (1978). Cobalt in ruminant nutrition. Proceedings Latin American Symposiu n ninerao m l nutrition research with grazing ruminants. Edited by J.H. Conrad and L.R. McDowell. University of Florida, Gainesville. LAU, K.S., C. GOTTLIEB, L.R. WASSERMAH and V. HERBERT. (1965). Measurement of serum 812 level using radioisotope dilutio d coateen n d charcoal. Bloo - 20226 d . RAVIN . ,H (1960) n inproveA . d colorimetric enzinatic assay of ceruloplasroin. 1'etional Institute of Neuro- logical Disease Blindnessd an s . U.S.A. Public Service. SOIKES. KALINOV7SKIJ , R. »ECHEVARRÍA. H y . (1970). Interre_ laciones Suelo-Planta-Nutrici6n XII. Composición quírn^ ca de especies forrajeras nativas de In Sierra Central. Reunión de Especialistas e Investigadores Forrajeros del Perú.. 35 Lina . p .

~~SUTTOH, A.L d J.!'.?n . FLLIOT. (1Í172). Kffcc f ratio t f o roughage to concentrate «nd levftl of feeft intake on ovine rurainal vitotfii 2 productionFj n . Journa- Hu f lo trition, 102; 1341.~~

~~122 FERRITIN IN CATTLE SERUM~~

F.W. LENGEMANN*, J.A. LOPERA1", F.A. KALLFELZ§ New York State College of Veterinary Medicine, Cornell University, Ithaca, New York F.W. LENGEMANN, Jr. Rome Veterinary Clinic, Rome, Pennsylvania, United States of America

~~ABSTRACT~~

A commercially available radioimmunoassay kit for human serum ferritin was used to determine the ferritin concentration in serum or plasma of 41 calves from 0 to 106 days of age, 192 cows and 35 bulls from 2 to 11 years of age. The geometric average concentration of ferritin was 2.1,

12.6 and 4.5 ng/ml for the calves, cows and bulls, respectively. The cows were statistically different from the calves and bulls; there were no differences betwee calvee th nd bulls an s . Withi cowe e hers th nson dwa found to have lower serum ferritin levels than all the other herds (P<0.05) but no differences in packed cell volumes were present. The data suggest that a radioimmunoassay procedure with a ferritin antibody specific for bovine ferritin coul e studusefue b d th f iro o yn i ln metabolis n cattlei m .

~~INTRODUCTION~~

As expressed by Underwood, "There is no convincing evidence that iron deficiency ever occurs in sheep or cattle grazing under natural conditions... excep consequenca s a t parasitif eo c infestatio diseaser no • Sinc O e C " th e demand f growthso , lactatio d pregnancan n e concurrenb y n animalma ya n i t , this implies that sheep and cattle, unlike the human, must be very well adapte extractino t d e needeth g d iron from their diets during these repeated stress periods. Nevertheless, littl knows i e n abou e availabilitth t f o y dietary iron to ruminants or even the nutritional requirements of ruminants for iron.

* Department of Physiology ' Present address: Director, Technical Section, Dept. of Agriculture, Medellin, Colombia * Departmen Largf to e Animal Medicine Obstetric Surgerd san y

~~123 Anemias do occur in young and mature sheep and cattle as a result of~~

~~parasitism (2,3), as a result of a deficiency of another nutrient important utilizatioe th n i ironf no , i.e. copper deficiency situationn i (4) d ,an s~~

~~suc wintes a h r anemi ) wher causae (5 a th e l agen obscures i t . Procedures~~

~~that have been availabl studyinr fo e g iron metabolis ruminann i m t animals~~

~~include determinatio packee th blood f re dno d cell volume (PCV) hemoe ,th -~~

~~globin content of blood, the total serum iron, the serum unbound iron, the~~

~~transferrin-bound iron and the transferrin saturation ratio and radioiron~~

~~disappearance studies. Unfortunately none of these procedures measures the~~

~~e storagamoun th s chang f it o tbod e d eth iroan y oven ni e rth time t Ye .~~

~~reductio n iroi n n stores woul e firs e expecteth b d te n b a sig o f t do n~~

~~incipient iron deficiency.~~

~~In human medicine an important advance in the diagnosis of nutritional~~

~~iron deficienc s beedevelopmene yha th n radioimmunoassaf o t y (RIAr fo )~~

~~ferritin. Ferritin, along with hemosiderin storage th s ,i e for f iromo n i n~~

~~the body. Ferritin, although water soluble, resides mostly within the cells~~

~~bodye oth f , particularly liver, splee bond nan e marrow (6). Minute quantities~~

~~of ferritin circulate in the blood in an amount that has been judged to be~~

~~proportionat e iroth n o t storee f ferritin/mso huma e i.eg th n 1 n .i f o l~~

~~serum correspond froo t s m f storag8-1o g 0m e iron (7) .o datT e there appears~~

~~o application e tb o e ferritith f o nA procedur nRI o ruminant e t animals.~~

~~Early in the study of ferritin radioimmunoassay it was found that~~

~~antibodies to ferritin of one species could interact with ferritin of other~~

~~species (8) e reaction.Th , however s sensitiv a crose ,e th b migh sr t fo eno t~~

~~reacting species. Sinc commerciaa e l ferritin radioimmunoassa s offerewa t dyki r testinfo s tu e odecidegw investigato t d e a possiblferriti s a A nRI e toon i l~~

~~studying iron metabolism in ruminants. We wished to determine the relative~~

~~level f ferritio s ne ferriti presenth w ho n serun i t d levelan m s change with~~

~~age, sex, stage of lactation and herd differences. The information developed~~

~~could point out areas that would be worthy of study.~~

~~METHODS~~

~~e assa Th f ferritio y n level n plasmi s r seruao f cattlmo s carrieewa t ou d~~

~~using a commercially available radioimmunoassay kit . The kit had been~~

~~standardize manufacturee th y b d r using crystalline human liver ferritir fo n~~

~~RIANEN ™ FERRITIN [ I] RIA KIT, New England Nuclear, Boston, Massachusetts. 1 125 124 serum standards, iodinated trace d productioan r primare th f no y antibodyA .~~

~~solid phase second antibody was used to facilitate separation of the antigen •~~

~~antibody complex. Determination of 125I was by gamma counting with a Nal~~

~~crystal well detector.~~

~~The plasma and serum samples used in the assay were obtained primarily~~

~~from commercial herds bule Th .l serum samples were from single commercial~~

~~artificial breeders cooperative located in Ithaca, New York. The veal calf~~

~~serum samples all came from a single calf raising operation near Ithaca, New~~

~~York and represent calves approximately 106 days of age that had been fed~~

~~commercial high energy milk replacer diet for this entire period. The~~

~~serum and plasma samples of new-born and the 10 week old calves on solid feed~~

~~were divided between calves born into the Cornell University herd and random~~

~~vicinite calveth n i sRomef yo , Pennsylvania f theso l e Al .animal s were~~

~~thought to be healthy at the time of sampling.~~

~~The lactating cattle included in this study comprised some 27 cows~~

~~used for nutritional studies at a farmers' cooperative research farm and 165~~

~~serum samples from 8 different commercial herds where some ill defined health~~

~~problems were thought to exist. The sampling in these 8 herds was to include~~

~~7 cows with the highest daily milk production, 7 cows of medium daily pro-~~

~~ductio non-lactatin7 d nan g cows e pertinenTh . t characteristic f thesso e~~

~~herd showe e standardize sar Th Tabln ni . 1 e d metabolic profile tests also blooe th rudn o nsample f theso s e cows included packed cell volume (PCV),~~

~~total protein, albumin, globuling M , P urea , Ca , , creatineCl , K , ,Na~~

~~and glucose. The feeding and maintenance of these herds varied but would~~

~~represent husbandry practices prevelan Northeastere th n i t n sectioe th f no~~

~~United States.~~

~~The data for plasma ferritin were highly skewed, therefore, a log~~

~~transform [log statisticae (X+l)datth e s useth r awa fo f d]o l analysis;~~

~~all means are thus geometric means. After transformation the data were~~

~~subjected to "t" tests, analysis of variance and/or correlation analyses~~

~~using standard procedures (9).~~

~~RESULTS~~

~~ferritie th f o r firs nOu e assaus t t showeki y d tha e coultw d detect~~

~~ferritin in the plasma or serum of cattle. This is not unexpected since~~

~~it was early demonstrated that cross reactions do occur (8). Since there is~~

~~125 a possibility that other substances might be cross reacting with the ferritin~~

~~antibody serum samples from 5 cows were diluted 3:1, 1:1 and 1:3 with bovine~~

~~seru t showm no thand tha detectabl e amount ferritinf so e tesTh .t sera~~

~~s determinef ferritia o l m g range n r 0 npe 9 d o t frostandare 5 th m4 d~~

~~curve. The results of this test are shown in Table 2. The geometric average~~

~~concentratio ferritif no undilutee th n i n d ser s 71.awa 9 ng/ml% wit66 ha~~

~~confidence interval (C.I.)> equivalent to plus or minus 1 standard deviation,~~

~~from 55-95 ng/ml. The dilution of the sera in the proportions indicated~~

~~should have produced averages of 54, 36 an(j 13 ng ferritin/ml, respectively.~~

~~As shown in Table 2 the diluted sera agreed well with these expectations.~~

~~Using the "t" test no significant differences were found to exist between~~

~~the theoretical mean and the observed means suggesting that the assay was~~

~~measuring ferritin.~~

~~In another study 8 cows were each sampled twice 1 week apart. The~~

~~mean difference (d) between the consecutive samples was 1.15 ng/ml~~

~~(t = 0.63) and was not significant statistically. Fifteen cows sampled~~

~~twice one month apart showed d equal to 1.05 ng ferritin/ml (t = 0.42) and~~

~~again was not significant. These data indicated the repeatability of the~~

stabilite th assa d an yf ferriti o y n level cattln i s e over timee Th . Coefficient of Variation (S2- x 100) averaged 23% between duplicates in one ^y_ series of samples and 3.2% in another possibly reflecting developing operator

~~skill.~~

~~Figure 1 is a bar graph of the ferritin levels in sera or plasma of~~

~~calves, cowbullsd san e calves Th . , rangin106dayso e frot ag ma 1 n d i g ,ha~~

~~geometri cferritin/mg n averag1 2. f eo l wit% C.I60 h .10.3o t fro9 .0. m Adult~~

~~cows, fro + year year2 m11 agef o so t s , showe geometria d c mea 12.f no 6 with~~

~~42.o t 32 % ng/mlC.I3. 66 f .o a . Bulls year1 ,1 rangino froe st ag 1 m n i g~~

~~with an average of 5.6 years, had a geometric mean of 4.5 with a 66% C.I. of~~

~~24.o t 02 ng/ml0. . These graphs shovere th wy wide rang ferritin i e n con-~~

~~centrations between animals and justifies the log transform of the data.~~

~~bulle d calveth Mos an sf o t s showed ferritin level ng/m5 f r slesso lo ;~~

~~cowe th moss f o showet d mor f serumo f ferritieo l g m tha n . r 5 npe n~~

~~transformeg lo Usin e th g waye on d ,cel3 a dat n li a analysi variancf so e~~

~~s determineiwa t d thae cowth ts were statistically signficantly different~~

~~(P<0.05) frobulle calvee d calveth mt differth an sd bullno t an sd s bu di s.~~

~~126 regressioA n bull e analysith date sr th showefo a f so o correlation d n of ferritin serum concentrations with age (r = 0.009).~~

~~The blood samples of the calves had been taken with the idea that~~

calves fed an artificial milk based diet might show levels of ferritin lower than calves of similar age but with access to a normal iron diet. ferritie th e dat r Th d packefo anan d cell volume cale th f f sgroupo e sar

~~shown in Table 3. The PCV's for the newborn and solid diet calves are~~

~~withi normae nth l rang cattlr . S fo e . Y e experience . baseN th e n o dth f eo~~

~~Colleg Veterinarf eo y Medicine showed ,an differenco dn e betwee newbore nth n~~

~~oldee anth d r calves r ferritinFo . "veae ,th l calves" tende havo t d e~~

~~higher levels but due to the wide dispersion of values there were no significant differences between groups.~~

~~The majority of samples from lactating cows were from herds where a metabolic profile tess desiredtwa . Becaussampline th f eo g desige th n~~

~~results coul subjectee db statisticaa o t d l analysis involving herd, production level, and their interaction. The ferritin data for these cows~~

~~are shown in Table 4 along with the PCV data. For the PCV the overall~~

~~averag s 31.7ewa % wit standarha d deviatio 1.45%f no analysie ;th f o s~~

~~variance showed no statistically significant differences between herds,~~

~~lactation groups or their interaction. For ferritin, however, Herd 1 was~~

~~found to be significantly lower than all other herds (P<0.05) and Herd 2~~

~~s significantlwa y different tha differenco nN Her . 7 d e existed between~~

~~lactation groups or the interaction of lactation and herd. The geometric~~

~~average for ferritin was 11.9 ng/ml with a 66% C.I. of 8.0 to 17.8 ng/ml. Other statistical tests revealed thacowr fo ts o significann ther s ewa t~~

~~correlatio V levelPC ferritio f nt o s n concentration 0.05f = o r ( d s )an~~

~~ferritin levels to age (r = 0.08).~~

~~DISCUSSION~~

~~A first objective for this study was to determine the feasibility of~~

~~measuring ferritin in the blood of cattle using an RIA kit developed for~~

~~the detection of ferritin in human serum. The demonstration here that there~~

~~is a wide range of ferritin in the serum of cattle, that the results are~~

~~repeatable over time and that the dilution of samples produces predictable~~

~~decrease serun i s m ferritin values suggest appreciabln sa e cross reaction~~

~~betwee humae nth n ferritin d bovinantibod an assae t th e ki y f yo ferritin .~~

~~127 The particularl w ferritilo y n concentrations see n calveni bulld an s- to s~~

~~gether with the finding of values below the detection limits may indicate~~

~~that the interaction of the ferritin antibody of this assay kit is not as~~

~~intense with bovine wits ferritii ht i huma s na n ferritin. Addiso. al t ne~~

~~(8) have observed that while the antibody to horse ferritin crossreacts~~

~~well with human ferritin it could not be used to develop an assay to detect~~

~~levels of ferritin normally present in human serum.~~

~~The values for ferritin as presented in this paper are expressed in~~

~~ng/ml only for convenience and probably are not quantitatively accurate but~~

~~do reflect qualitative differences between animal d groupssan .~~

~~n reviewinI e resultth g f thiso s preliminary study with cattls i t i e~~

~~evident that mature females have higher serum ferritin levels than mature~~

~~males similaA . x relatese r d differenc s see i eratsn i n , mice, chickens~~

~~and eels (10,11)humane th n I ,. rabbi d guinean t a pig d percsan h (11)~~

~~the male show highee th s r serum ferritin humae values th e lowe th n I r.~~

~~concentration of ferritin of the female has been ascribed to the repeated~~

~~losse irof so n during menstrual flows (12).~~

~~The calves in this study had much lower ferritin levels than the cows~~

~~in general agreement with other species wher differencee ag e s occur (13),~~

~~but the calves were not different from the bulls. The extremely low ferritin~~

~~values for the calves and for the bulls, in particular, are difficult to~~

~~explain A firs. e t th though o t e s thaassae i tdu th tw s readin i ylo o to g~~

~~differing immunological propertie humaf o sd bovin an n e ferritin. Another~~

~~possibility for the bulls is that they represent only a single husbandry~~

~~unit whose practices somehow resulted in a marginal metabolic availability~~

for iron.*

~~A third explanation involve findinge sth s thaimmunologicae th t l~~

~~properties of ferritin are affected by species and by the tissue of origin.~~

~~The ferritins of heart, liver, spleen and tumors show different responses~~

~~to the same antibody (14); moreover the proportions of these ferritins~~

~~found in serum are dependent upon tissue destruction. There also appear to~~

~~be "fetal d "adult"an " typ f ferritio e n tissui n e suc s cardiaa h d can~~

Addendu* m These bulls have been intentionall w qualitlo a d y fe roughagy r severafo e l years. The PCV's have been found to be at the low end of the normal range whic s unusuai h r non-lactatinfo l g cattle e totaTh . l dies beeha t n determined to contain 50 ppm Fe, a concentration below the recommended 90 ppm.

~~128 skeletal muscle cardiae (15)th n .I c muscl adulf eo t rat"slowe th s "~~

~~ferritin predominate female "faste th th male n d e"i th an e (16) n i s . With~~

~~thi minn e couli s on d d speculat w valueelo r ferritithae fo sth t serun i n m~~

~~of bulls and calves, in comparison to the cows, could indicate the presence~~

~~of differing ferritin serue th r plasma o mn i s . Thi f courseo s , needo t s~~

~~be substantiated.~~

~~herd8 e sth e datassayer Th fo a thin i d s paper show that even whero en~~

~~statistically significant differences are seen in PCV, between herd differences serue n existh ca m n productio r i to ferritin e ag o N .n level differencen i s~~

~~ferriti werV PC e r o n presenanimals e th n e herti Th .d differencer fo s~~

~~ferriti vere ar ny interestin mort bu ge informatio needes i n d n beforca e on e~~

~~ascribe these difference w irolo no t sstores .~~

~~The chief use of a ferritin radiolmmunoassay at present is to detect~~

~~depletio storagf no e iron befor frana e k iron deficienc manifests i y .~~

~~Another possible use would be to study the changes in iron stores as a~~

~~physiological stress suc ages a h , pregnancy, and/or lactatio s appliedni .~~

~~Kineti pharmacokinetid can c studies would benefits wa greatl t i f i y~~

~~possibl determino t e e iro th e sizn eth f eo poo l over time. Before these~~

~~studiee carrieb n ca sd out, however woult i , e necessarb d develoo t y a p~~

~~more specific antibod d the an relatyo t n e serum amoune levelth f o o t s~~

~~iron in storage.~~

~~e determinatioTh ferritif no n cattlni s raiseeha d some very interesting~~

~~questions, however, the only conclusion that can be drawn from this study~~

~~is thacommerciae th t l ferritit useki dA cannoRI n t provide definitive~~

~~answerexistence th n so f imbalanceeo f iro so n cattle i n . Thi t appearski s~~

~~to lacneedee th k d sensitivity, moreover variatioe ,th n between animaln i s~~

~~the same husbandry groupin o larg to permio t es i g t settin limia g t below~~

~~whic iroe th hn consideree storeb n ca s d deficient.~~

~~129 REFERENCES~~

~~. Underwood1 . TracJ . ,E e Element Human si Animad nan l Nutrition, Fourth Edition, Chapter 2, Academic Press, New York. 1977.~~

~~2. CampbellGardinerd an . . A VetC . . ,,J A . Rec : 100.77 6 (1960).~~

~~3. Rice, R. W., Nelms, G. E., and Schoonover, C. 0. J. Animal Sci. 26; 613 (1967).~~

4. Marcilese, M. A., Valsecchi, R. M., Rossi, F. M., Rudelli, M.D. and Figueiras, H. D. Review of Research on Mineral Metabolism and Diseases in Farm Animal Argentinn si Mineran ai l Studies with Isotopen si Domestic Animals. Proceeding Panela f so , 28th Oct.,1970Septd 2n .- , STI/PUB/293. IAEA, Vienna. 1971.

~~5. Whitlock, R. H., Little, W. and Rowlands, G. J. Res. Vet. Sci. 16_: 122 (1974).~~

~~. Granick6 Biol. J .. ,S Chem . 149 7 (1943);15 .~~

~~7. Norwoodd Walters an Clin. , Miller J , 0. M. . . ,M ,G . Path ,F . J260 :77 (1973).~~

~~8. Addison, G. M., Beamish, M. R. , Hales, C. N., Hodgkins, M., Jacobs, A. and Llewellin, P. J. Clin. Path. 25_: 326 (1972).~~

~~. 9 Snedecor . StatisticaW . ,G l Methods, Fifth Editio Iowe nTh a State University Press, Ames, Iowa. 1961.~~

~~. Kaldor 10 Powelld an . ,I . , AustralM Exptl. .J . Biol. Med 3 .(1957) Sci12 : ..35~~

~~. Widdowson McCance11 d (1948) 7 an . Blochem57 A . M ; . ,R . ., 42 E . .J~~

~~12. Sturgeon, P. and Schoden, A. Am. J. Clin. Nutr. 24; 469 (1971).~~

~~Munro d . 1 (1972). LinderEnzyman C 13 11 . . ,: C H .e15 . , M~~

~~14. Munro, H. N. and Linder, M. C. Physiol. Rev. _58_: 317 (1978).~~

~~Munro d . BiochemN an . Linder. Scott, , ,15 MoorH , R. E. C. . . . .J ,. J L , M 129; 455 (1972).~~

~~16. Vulimiri, L., Linder, M. C. , and Munro, H. N. Biochim. Biophys. Acta 497: 280 (1977).~~

~~130 TABLE 1. CHARACTERISTICS OF HERDS SAMPLED FOR THE METABOLIC PROFILE TEST~~

For Cows Sampled Herd Date Herd 2 305 Day Daily Number Location Sampled Size Breed Age Milk Prod. Milk Prod. Health Problem Number Yrs. Kg Kg/Day Cows off feed every 1 Bergen, June '79 107 H 4.1 5779 Hi 31+5.4 three weeks; displaced N.Y. +2.7 +1095 Med 19+4.5 abomasums; retained placentas.

~~Springville0 7 , 5.7 5075+ Hi 22+1.4 Dysentary every 6 N.Y. Sept.'79 17+1.d Me 4 1 82 wks.+2.2, milk feven i r 10-15% of cows.~~

~~3 Groton, Oct., '79 57 H 1 6. 6250sho% 32+5.i H 10 w+ 4 mil k fever, N.Y. +2.6 1183 Med 13.6+3.2 liver problems.~~

~~4 Charlton Dec., '78 70 H 8 4. 802235+5.i H +9 Fai produco t l s a e City, N.Y. +1.6 119 22+6.d 4Me 8 expected retaine% 20 ; d placentas; 20% ketosis.~~

~~8 4. 5 Sheffield H 2 ,9 Dec. 8 ,'7 941337+8.i H +6 100% ketosis, 5% Mass. +3.18903 Med24+2.7 displaced abomasums.~~

~~6 Batavia, Jan., '79 29 4.4 8688+ Hi 38+2.3 Breeding problem N.Y". +2.9 1993 Med 22+1.4~~

~~7 Stamford, - Sept. 9 ,'7 N.Y.~~

~~Canajoharie, Sept., '79 - 7.2 Low milk production N.Y. +2.1 with 4.8% fat test.~~

~~Abbreviations H = Holstein Friesen; J = Jersey; - = Breed not stated.~~

~~AgYearn i e sStandar+ d Deviation~~

~~131 TABL . DILUTIOE2 BOVIN% F NO E PLASMA SAMPLE EFFECD SAN FERRITIN TO N (FERR)~~

~~CONCENTRATION AS DETERMINED BY RADIOIMMUNOASSAY1~~

~~Dilution Actual 0 3:1 1:1 1:3 ng/Ferr/ral 71.9 58.7 41.5 29.3~~

~~66% Ci/ng'/ml 55.2 to 47.o t 0 31.o t 5 22.4 to 94.6 73.0 55.2 61.0~~

~~Theoretical~~

~~ng/Ferr/ml 71.9 53.9 36.0 18.0~~

For statistical testing the data were transformed using Loge(X+l). Using the " tes "T o significann t t differences wer hypothesie th foun r fo d s thae th t actual value did not differ from the calculated value for a particular dilution.

~~TABLE 3. FERRITIN LEVELS (FERR) AND THE PACKED CELL VOLUME (PCV) of BLOOD~~

~~OF NEWBORN, 3-MONT VEAD HOL L CALVE D 3-MONTSAN D CALVEA HOL D FE S~~

~~SOLID DIET.~~

~~New Born Calves Veal Calves Solid Diet Calves~~

~~FERR PCV FERR PCV FERR PCV ng/ml % ng/ml % ng/ml % n 16 16 14 11 11~~

~~X 1.3 34.4 4.2 2.2 36.6~~

~~66% Ci -.34 to 29.o t 2 0.3 to -0.05 to 32.o t 1 7.1 39.6 12.2 9.4 42.1~~

~~The statistical analysis of Ferritin concentration used a Loge(X+l) transform originae oth f lV dat PC data ae analyseth ; d withou g transformlo t .~~

~~132 TABLE A. PACKED CELL VOLUME (PCV) AND PLASMA FERRITIN (FERR) OF HIGH PRODUCING, MEDIUM~~

~~LEVEL PRODUCING, AND DRY COWS AS A FUNCTION OF HERD3,~~

~~HERD Average By Level of Production~~

~~LACTATION PCV FERR PCV FERR PCV FERR PCV FERR PCV FERR PCV FERR PCV FERR PCV FERR PCV FERR LEVEL % ng/ml % ng/ml % ng/ml % ng/ml % ng/ml % ng/ml % ng/ml % ng/ml | % ng/ml~~

~~HIGH 30. 6 330.5. A 131.A. A 15.0 29.3 16.7 33.0 10.1 29.2 2A.7 33.3 25. - 722. 31., 8 0 11.8~~

~~AVERAGE 27.6 1.7 31.0 9.A 30.A 25.6 31.3 17.9 31.9 7.0 31.5 15.3 35.3 41.3.19 | 31.3 11.3~~

~~DRY 2 32.1. 3A.9 6 10.3 33.7 21.2 31.A 33.1 33.0 3A.3 19.7 3A.1 16. - A15. 7 32.' 8 12.6~~

~~HER. DAV 30. 9 2.Ab 31.2 7.6° 30.2 20.1 30.7 13.9 32,7 13,5 32,0. 15,1 3A,2 26,0 12,A 31,7d 11.9e~~

A 2-way ANOV uses statisticar wa Afo d l analysi datae e originath Th .f so l dats awa whilV Ferritie PC th useer fo d n dats transformeawa analysi e th r fo ds using Loge(X+l). b HERD itl ferritin levels were different frol othe % al levem5 re f lo herdth t a s significance.

~~° HERD #1 differed from herd #7 at the 5% level of significance.~~

~~The standard deviatio 1.45%+ s nwa .~~

C.I.% 66 e , Th equivalen 6 standar1 o t d deviation 17.o t fros 80 ,i 8. mng/ml . 60 1 =1 CALVE N S 50 x - 2,1 10 T9 O66K 0. 10. - I3 30 20 I 10 >»* 30 N =19COW2S x = 12.6 20 ^> 66ZC1 = 3.2 TO 13.3 SSS^tf^jSSSl 10 §SS«58§SS§§§J^SSi5' 95 sssssssssssssssssSJSSS^^S^v^

~~FIGURE 1. Distribution of ferritin levels in populations of calves,~~

~~cowbullsd san .~~

~~134 ABSORPTION, DISTRIBUTIO EXCRETIOD NAN ARSENIC-7F NO HENN 6I D SAN RUMINANTS~~

M. ANKE . HOFFMANNG , . GRUNM , . GROPPELB , . RIEDEE , L Karl Marx Universitat Leipzig, Sektion Tierproduktion und Veterinarmedizin, Wissenschaftsbereich Tierernahrungschemic, Jena, German Democratic Republic

~~ABSTRACT~~

The absorption, distributio excretiod nan f arsenino c were investigate resulte goatn Th di layind . san sAs indicateg f heno d s ai witd e hth 7fi that hens incorporated 7'6 As considerably faster than the goats. The preferential incorporation of arsenic into skin, hair and feathers may be important for the identification of As deficiency as well as As exposure. Moreover, the high incorporation demonstrated into epididymis, ovary and eggs could also be important in connection with disorders of reproduction.

~~INTRODUCTION~~

Afte e demonstratioth r f As-essentialito n n mini-pigsi y , ratd an s goats (Nielson 1975, Nielsen l a 1977_ et , , . 1976 al Ank ^ et e, 1977, 1980, Uthus and Nielsen 1980), the biological interest in this trace element has considerably increased. These were first indications of the biological importance of this element only known as toxic up to that time. Four hundred and fifty years ago, the father of modern pharmacology, Teophrastus Bombastu n Hohenheimvo s , called Paracelsus s alread,ha y indicated depen- denc n arsenico e .

In the meantime, several authors (Lanz e_t al. 1950, Tsutsumi 1971, Levande . 1977al ^ , ej rDutkiewic z 1977) also repeatedly e facpointeth t t ou d that ther e speciear e s specific differences with regar o absorptiont d , distribution, incorporation and excretion. This dependence of the As effect on the species is considerably more marked in sea organisms than in mammals and birds (Benson e_t al. 1980, Andreae 1980, Abdelghani et_ al. 1980). In order to be in a position to better evaluate differences resulting from the species specific characte s effecto A identift f o d r an sn environ a y - menta s exposurA l e more clearly s absorptionA e th , , distributio d excrean n - tion were investigated with the help of As in ruminants (goats) and poultry (laying hens). Furthermore, there was an opportunity to test the above mentioned parameters in goats with a normal As supply and in animals with an extremely poor As ration (Anke ej^ al. 1980).

~~135 MATERIA METHODD LAN S~~

Forty nine month old laying hens and 6 control and 6 As deficient goats (Anke et_ al. 1977) were used as test animals (3 adult goats and 3 group)r pe e 0 ag .day 10 f kido Fivsf o se hens were slaughtere t 0.75a d , 6 hour9 d san afte8 4 r, 24 placin , e 12 isotopcrope th g, th 6 1.5n ., i e3 , The As was placed in the rumen of the goats which were then sacrificed 48 and 96 hours later. The hens were given 0.2 mCi (6.1/*g As) As, the goats 7.36 mCi (244 /<-g As) As per animal. As was in the form of AsnOj, s dissolvewa d an n dilutei d d HC1.

~~RESULTS~~

~~s incorporatioA e Th d excretioan n n dependen n timeo t , species statuA d an s~~

Forty-five minutes aftee hend e absorbeorath ha sth r , l % As intak34 d f o e e dosd probabloth fan e d alreadha y y passe e pea th f absorptiondo k % 63 . of the As was in the digestive tract and its contents. Only 3% could be detected in the urine - faeces mixture.

~~Table 1: The As incorporation and the As content of the digestive e dos th r cen etracpe f o tn i t~~

~~measuring point body digestive tract~~

~~s X X s~~

~~45 minutes 21 34 63 20 90 minutes 7.5 27 66 23 3 hours 6.5 28 35 15 6 hours 4.5 22 18 11 12 hours 3.0 11 22 19 4 hour2 s 0.7 3.0 4.3 3.9 48 hours 0.4 1.4 0.9 0.3 6 hour9 s 0.6 1.2 0.3 0.08~~

~~e x hourth Si e isotopn si th aftes f o s e doseorawa eA th % rl 60 ,~~

faeces - urine mixture, 2270 in the body and only 18% in the digestive tract. Thuss excretio A half-time e th , th r henn i nfo es amounte 6 hour , d ^ an so t d correspond o tha t sf dog o t s (Hollin . 1979)al _ et s. Withi 4 hours2 n % 93 , s excretewa s A d e wite ofaeceth fth h — surin e e mixtureth n i s , wa onl % 3 y body at that time, and 4% was in the digestive tract. At the end of the tes d tlefe body ha perioth ts .A dThi e 98.5s greatei th s f %o r thas wa n found with dogs by Hollins e_t al. (1979).

s incorporatioA n e FigurI th , 1 e n rat n heni es graphicall i s y represented. The extraordinary speed of the incorporation and excretion of this element becomes apparent in spite of the fact that the first measurement at 45 minutes after the oral application was apparently too late. 136 incorporatios A . •/ n (body) As excretion '/.

~~0.75 3 96 hours~~

~~Figure 1: The incorporation and excretion of 76,As in hens, as a function of time after the oral intake, in per cent of the applied As amount (log jga).~~

Goats probably absorbed As considerably slower than hens. The average of the control and As deficient goats was 4.1 and 7.2% of the As dose, respectively 8 hour4 , s afte e dosinth r g inte rumeth o n (Tabl. 2) e At that time, control goats deficien A d 587 d sha e diges ,an th tn -i goat % 67 s s excretee wa % th respectivels 26 A a vi d tiv e an eth 7 3 tracf o d y an t s e fourtingestionA urineth y afte t da e A h .th re goat th , s retained n i s A e th f o % 38 o t 0 bode 3 d stil th 76e an d y A n th ha li s f abouo % 4 t the intestines. In this, they differ considerably from hens at that time; comparable values from other specie f ruminanto st availableno e ar s . Afte n oraa r l ^As dose, monkeys excret a faecee vi onl % 2 sy (Charbonneau 7 1978). al ^ ,e dog % (Hollin4 s 1979) . al t ,e s rat % (Dutkiewic8 s z 1977).

As deficiency influenced As incorporation, but the difference was not significant (Table 2). Goats with an As poor ration incorporated 76 and 30% respectively at 48 and 96 hours. This was greater than for the control animals. The difference between the groups became significantly smaller with timee n ATablth I s . , incorporatio3 e f hen o ncons i s - 76 trasted with that of goats 96 hours after the dosing. At that measuring point, goats stored three times as much As in the body as hens.

~~137 Table 2: As incorporation by control and As deficient goats 6 hour9 d san afte8 4 r dosing.~~

~~hours after control goats As deficient p % dosing goats~~

~~S X X S~~

~~48 2.4 4.1 7.2 4.0 >0.05 176 96 2.0 3.7 4.7 2.1 >0.05 130~~

~~P >0.05 >0.05 7. 90 67~~

~~s incorporatioA e Th Tabl : f hen 3 eo nd goat san 6 hour9 s s after ora e dosel th r cenintak pe f .o tn i e~~

~~hen _ _ goat S X X S~~

~~0 2. 8 30 <0.00 7 3. 1 2 1. 6 0.~~

~~The As distribution in the body dependent on time, species and As status~~

Skeleton, liver, lungs and heart had the highest As activity at 45' after the As dose. Blood and kidneys had the highest As level 90' afte e applicationth r , whereas muscles, feather d ovaran s y reachee th d highest 76As incorporation considerably later (Table 4). It can be derived therefro e firsm th tha t a tmeasurin g poin 5 minutes)(4 t highese th , t rate s oabsorptioA f d probablha n y just been reache r exceededo d n conformitI . y with s store. (1942)Huntewa al t s n skeletoA i de_ r , e n th muscles f o % 60 ; retaine 4 day s sA afted e intak th rs locate wa e d heree skeletoTh . d ha n abou e samth te proportio e firsth e 2 daystr f retaineth o n fo n i ;s A d followin 8 hours4 g , this proportion decrease abouy b d t 50%.

~~138 s concentratioA e Th Tabl : f 4 differenteo n bode partf th o y f o s hens in per cent of the As retained at the particular measuring points~~

~~organ 45' 90' 3h 6h 12h 24h 48h 96h~~

skeleton muscle 31 37 46 50 35 53 36 60 skeleton 25 19 20 19 17 20 20 9 liver 11 10 7 5 4 4 2 2 blood 9 12 8 6 4 2 2 2 lungs 8 4 3 5 2 1 0 0 kidneys 7 8 6 4 2 2 2 1 feathers 4 7 5 9 32 14 28 18 ovary 3 2 3 3 3 5 8 6 heart 1 1 1 1 1 0 0 0

~~sum 100 100 99 101 100 101 98 98~~

Liver, blood, lungs, kidneys d hear,an t reduced their relativs A e concentration continuously with timd almosan e t reache e limith df detec o t - tion after 48 hours. Feathers and ovary showed the highest proportion 12 and 48 hours respectively after the dosing with labelled As. The rich As incorporation into the ovary and the developing eggs must be taken into consideration. Ther s apparentli e s incorporatioA o barrien e y th o t r n into eggs as there is for Cd and no involution of ovaries as seen with Cd (Anke £t al . 1976). Arsenic is preferentially stored in all proteins as Lowry . (1942 l e_a ts stated)ha n thesI . e investigations, only s A trace f o s coul e foun b dn clearl i n lipidsi ds A y .measurabl e amounts coule b d detected onl eggn i y s lai 4 hour2 d s after dosin 3 egg gn sI (Tabl . 5) e lai 2 hour1 d s after dosing, onl ys coulA trace e foundb d e f eggo sTh .s laid 1 to 3 days after the As intake contained only 0.2% of the ingested amount of isotope, this is equivalent to 0.1 juu% of As. The low standard deviation shows the homogeneity of the incorporated amount of As.

The long term As transfer to the eggs is noteworthy when it is considered that after a As dose, only insignificant amounts are found in milk (Peoples 1964, 1967, Marshal . 1963al _ et l, e . 1939)th Fitcal t t A e .h end of the experiments, ovary and eggs had the highest radioactivity of all tissues examined.

~~139 s incorporatio*A . V n (skeleton) 30 r~~

~~6 0,7 3 5 12 96 hours~~

s incorporatioA e FigurTh : 2 ne inte skeletoth o f henso n , dependenn o t the time after the oral intake, in per cent of the amount of 7 6s storee bodyA th n .di s incorporatio A a functio s a e r Th g Tablpe f tim o eg n n : i r 5 ee npe cent of the dose.

~~parameter 45' 90' 3h 6h 12h 24h 48h 96h~~

~~X 0.00 0.00 0.00 0.00 0.01 0.19 0.23 0.18 s 0.00 0.00 0.00 0.00 0.01 0.03 0.02 0.04~~

e proportionatTh s distributioA e e differenth n i n t organ f goato s s is similar to that in hens (Table 6). Four days after the application in the rumen muscles and fatty tissue, a lot of which is found in adult goats, stored about 40% of the retained As. This percentage corresponds with that found in hens. The same is true for skeleton, kidneys, and heart. On the other hand, liver, blood and lungs proved to be considerably richer in As than the same organs of hens at the same measuring point. Considerable As is apparently excreted via the gall bladder into the intestines, a fact that Cikr d Benckan t o (1974 d Klaasse)an n (1974) pointe s wella t .ou d e tim6 hours) 9 Ath f sacrifict o e d e galan th , l8 (4 bladdere f goato s s contained remarkabl e skiTh ne . pluamountAs s haif so f goat o r s contained considerable As — up to 14% of the retained As.

~~140 Table 6: The As concentration of different parts of the body of,control and As deficient goats in per cent of the incorporated As amount 48 hours after the dose.~~

~~organ control goats As deficient P % goats s X X s~~

skeleton muscle 11 38 37 7 >0.05 97 skeleton 6 16 17 7 >0.05 106 liver 4 15 14 3 ^•0.05 93 blood 1 7 7 0.4 >0.05 100 lungs 0.2 4 7 5 >0.05 175 kidneys 1 3 3 0.6 >0.05 100 skin 3 14 11 3 >0.05 79 heart 0.2 0.8 1. 1 0.3 >0.05 138 spleen 0.1 0.5 0. 9 0.5 >0.05 180 cerebrum 0.2 0.5 0. 5 0.2 >0.05 100

~~sum 99 101 -~~

e cerebruth e n proportiod i spleeTh an m s s A insignificantwa n f o n . The same is true for testicles, the data for which are not presented in Table 6. The As concentration in lungs is noteworthy (p < 0.1). There is o biologican l explanatios accumulatioA e th e cerebrumr th fo nn i n , spleen and lungs. According to the results, species can influence time dependent As distribution, regardles e fac s th incorporatei tf o s thaA t d intl al o tissues. As deficiency did not significantly influence the As distribution (see Table 6). s incorporatioA e Th n into feather d haian sr

e possibilitieTh f identifyino s s exposurA n a g e wite helf th ho p the analysis of hair and feathers has been summarized by Levander et al. (1977) among others. In our experiments, hens stored 3.57= of the applied As amoung in feathers (Figure 3) 12 hours after the oral As dose. This percentag e experimenth f o ed decreaseten e th t o 0.4a 4 hourt d2 d %y an b s wa0.2%t a s .

s incorporatioA e Th n inte hai f th goatoo r s retardewa s d compared to the feathers of hens (Figure 4), with the maximum As incorporation being reached only alter 48 hours. The difference between feathers and hair is statistically significant at all measuring points. The removal of As from hair between the 48th and the 96th hour after the dose was less for hair than for feathers. Hence, it follows that hair and feathers take

~~141 % 'Vl'mcofporolion IfWthtr]~~

~~4.0~~

~~1.0~~

~~0.5~~

~~3 6 12 6 hour9 s £8 s incorporatioA e Figure Th featherth : n 3 ei n f henso s .~~

~~•/«« 76As incorporation in I0g ha 0.25~~

~~0,20~~

~~0,10~~

~~2 1 1.6 3 5 96 hours~~

~~s incorporatioA e Figure Th hai th f goats o r: n 4 ei n .~~

142 part in As metabolism, just as seen for other trace elements (Anke and Risch 1979). This metabolism must be taken into consideration when inter- preting the As concentration in different parts of the hair as was done in the case of Napoleon's hair by Leslie and Smith (1978).

~~Discussio e resultth f o n s~~

There are differences between the speed of the As absorption and excretio n hen i d ngoat an s s apparently e differencecauseth y e b d th n i s anatome digestivth f o y e tract. Hens incorporate d excretean d s A d considerably faster than goats.

~~Table 7: The As concentration in different parts of the body of hens and goats in per cent of the incorporated As 48 hours after the oral application~~

~~organ hens goats P s X X s~~

skeleton muscle 9 36 38 11 > 0.05 skeleton 5 20 16 6 >0.05 liver 1 2 15 4 < 0.001 blood 2 2 7 1 <0.01 lungs 0.1 0.3 4 0.2 < 0.001 kidneys 4 3 3 1 >0.05 feathers, skin 15 28 14 3 >0.05 heart 0.1 0.3 0.8 0.2 < 0.001

A remarkable e florpercentagth f o s e ay seemtakeA b b o p t u snf o e the rume d absorbean n d incorporatean d e abomasue smalth th n i ld an mintes - tine only after digestion. This aspect must be taken into consideration in further investigations. In both species, the incorporation of the absorbed 76As follows approximately the same order (Table 7). Two days after the oral intake, both species had stored about the same percentage of the absorbed n skeletai s A l muscles, skeleton, lungs, kidney d cardiaan s c muscle. There were species specific differences only for liver, blood and feathers and skin respectively, which might have the following reasons. In hens, at 2 days, the As excretion via the gall bladder into intestines is already finished, wherea s continuini t i s n ruminantsi g r thaFo .t s concentraA reason e th , - tion in the liver and the blood, which is serving as the transport route, is still high. The difference in the As concentration of blood between r goatles fa d hen san s i thas n that between ratd rabbitan s s founa s y b d Marafante ejt al. (1980). Rats are distinguished by a particularly intensive As storage in erythrocytes and particularly in haemoglobin (Hunter et^ al. 1942). Other species distribut bode th moso y t s f A tissueso ts A e e Th . accumulatio n skii n n plus haid featheran r s alswa s o note micn i dy Lind b e - gren and Dencker (1980). In mice and golden hamsters they found As only

~~143 e epididymisth n i , thyroid gland, crystalline lend skian s n four days after ths injectionA e .~~

The results presented in this paper showing the preferential As incorporation into skin, haid featheran r e importan e sar identi th r fo -t ficatio s deficiencA f s o exposurenA d an e yhig Th .h incorporation into epididymis, ovary and eggs can also be of importance in connection with disorder f reproductiono s . Meyhofe d Knotan r h (1966) have reportee th d decreased libid d los an f opotenco s n humani y s afte a rtherap y with Fowler's solution. . Benck(1968al t e o) found thae germinath t l epitheliue th f o m testis was considerably damaged in mice after chronic exposure to As. An increased frequenc f spontaneouo y s abortions, malformatio d decreasean n d birth weight in children of female workers at a smelter in Sweden was recently reported e smelteTh . e abortiorTh emit . sAs n frequency among exposed females was even higher when the father was also employed at the smelter (Nordstro . . 197b) al , t 9a e m

45 minutes after the As was placed in the crop, hens retained 34% dosee oth f . This percentag 6 hourse9 decreasey b .% 1 Withio t d hour6 n s 50% of the As had again left the body of the hen and 48 hours after the dose e digestivth , e tract contained , onl7% . yo Witt As trace 4 h f o s s doseA ,e ogoatth f s retained considerabld an s dosA 8 4 e ye th mor f o e 6 hour9 s after dosing thae hensth n .

n skeletai e mai s Th wa n ls storags A retentioA muscles f e o e Th n. in skeletal musclretaine the cen hensof per edosAs of t in de, increased over time. Ovary, eggs, feathers d ski,an n store t earla d s mucA y htim e intervals t latebu , r theis proportionatA r e uptake decreased n mosI .t other s tissuesconcentratioA e th , n decreased continuously fro 5 minute4 m s to 96 hours after the intake.

Apart froe s retardatioincorporatioA th m e th n i n n into organf o s goats, there wer o principan e l differences between hend goatsan se Th . preferential As incorporation into skin, hair and feathers is important for the identification of the As status. The high As incorportation into ovary and epididymis might also influence reproductio s exposureA n casi nf o e .

~~144 Short title Absorption, distributio henn i d d excretioruminant an nan s A f o sn~~

~~References~~

Abdelghani, A.A., Anderson, A.C., Hughesd Englandean , J. , , A.J: .In Arsen Symposium 3 (Anke, M., Schneider, H.-J. and Bruckner, C., eds.), 147 (1980). Andreae, M.O. In: Arsen Symposium 3 (Anke, M., Schneider, H.-J. and Bruckner, C., eds.), 131 (1980). Anke, M., Groppel, B., Griin, M., Hennig, A., and Meissner, D. In: Arsen Symposium 3 (Anke, M., Schneider, H.-J. and Bruckner, C., eds.) (1980)5 2 , . Anke, M., Grun, M. and Partschefeld, M. In: Trace Substances in Environ- mental Health (Hemphill, D.D., ed. , 403)10 , Univ. Missouri, Colombia (1976). Anke Grun, M. , Partschefeld ,M. , GroppelHennigd ,M. an , . ,A G. Trac e Element Metabolism in Man and Animals (Kirchgessner, M., ed) 3_, 248 (1977). Anke, M., Hennig, A., Schneider, H.-J., Groppel, B., Grun, M., Partsche- Liidked an feld . , H Wiss, M. , . KarZ . l Marx Univ. Leipzig, Math- . Naturviss . 2_5 R .1 (1976),24 . d RischAnkean . . ,M M Haaranalys d Spurenelementstatusun e B GustaVE , v Fischer Verlag, Jena (1979). Bencko, V., Nejedly, K., and Somora, J. Cs. Hyg. j_3, 344 (1968), Benson, A.A., Cooney, R.V., and Summons, R.E. In: Arsen Symposium 2 (Anke , Schneider,M. , H.-J. Brucknerd ,an eds.). C , 9 (1980)13 , . Charbonneau, S.M., Spencer d Sandi, Brycean K. , . , E ,BullF. , . Environm. Contam. Toxicol0 (1978)47 , 20 .. d BenckoCikrt. Hygan J . M . ,V , Epid., Microbiol d Immunologan . , 18 y 129 (1974). Dutkiewics, T. Environ. Health Perspect. 19, 173 (1977). Fitch. L.W., Grimmett, R.E.R., and Wall, E.M. N.Z. J. Sci. Techn. Sect. A. 2J., 146A (1939). Hollins, J.G., Charbonneau, S.M., Bryce, F., Ridgeway, J.M., Tarn, G.K.H., and Willes, R.F. Toxicology Letter 7 (1979) , 4 s . Hunter, F.T., Kip, A.F., and Irvine, J.W. Jr. J. Pharmacol. Exp. Ther. 7 1±(1942)20 , . Klaassen, C.D. Tox. and Appl. Pharm. 2_9, 447 (1974). Lanz , WallaceH. , , P.C d Hamiltonan . , J.G. Univ. Calif. Pub. Pharmacol. 3 (1950)226 , . Leslie, A.C.D. and Smith, H. Arch. Toxicol. 41, 163 (1978). Levander ,. ArsenicO.Aal . .et , National Academ f Scienceso y , Washington, D.C. (1977)7~ Lindgren, A., and Dencker, L. In: Arsen Symposium 3 (Anke, M., Schneider, H.-J. Brucknerd an , , eds.),C. 7 5 ,(1980) .

Lowry, O.H., Hunter, F.T., Kip, A.F. and Irvine, J.W. Jr. J. Pharmacol. Exp. Ther. 76, 221 (1942). Marafante , Rade E. , Pietra J. , , SabbioniR. ,d Bertoleroan : . In E , . F , Arsen Symposium 3 (Anke, M., Schneider, H.-J. and Bruckner, C., eds.), 49 (1980). Marshall, S.P., Hayward, F.W. and Meagher, W.R. J. Dairy Sci. 46, 580 ~ ~ (1963). Meyhofer r Hautarz 9 Knothd De (1966)30 an , . . ,W W 17 t .

145 Nielsen, F.H., Givand, S.H. and Myron, D.R. Fed. Proc. .34, 923 (1975). Nielsen, F.H., Myron, D.R.Uthusd an , , E.G : Trac.In e element metabolism in man and animals (Kirchgessner, M.), .3, 244 (1977). Nordstrom, S., Beckman, L., and Nordenson, I. Hereditas 90, 297 (1979a). Nordstrom Beckman, Nordensond ,S. an , ,L. . ,I Heredita 1 (1979b)29 , 90 s . Peoples, S.A. Ann. N.Y. Acad. Sci. Ill 4 (1964),64 . Peoples, S.A. Fed. Proc. 2£, 359 (1969). Tsutsumi . S Act, a Paed. Jap. _75 7 ,(1971)89 . Uthus, E.O. and Nielsen, F.H. In: Arsen Symposium 3 (Anke, M., Schneider, H.-JBrucknerd an . eds.), ,C. (1980)3 ,3 .

~~146 ASSESSMEN CHROMIUF TO ANIMALD MAN STATUN SMA F SO~~

~~R.A. ANDERSON . MERTW , Z USDA, SEA, Human Nutrition, Beltsville Human Nutrition Research Center, Beltsville, Maryland, United States of America~~

~~ABSTRACT~~

Direct methods involving determination of Cr content of blood, urin d tissuean e f n animalappeao sma d o offet ran s r marginal promise to assess Cr status. Hair analysis may have the best potential of the direct methods but has not been verified. Measurements involving glucose tolerance are quite reliable since most Cr deficient animals and humans do have impaired glucose tolerance but impaired glucose tolerance can be caused by a number of substances not related to Cr metabolism. e levelTh f seruo s m cholesterol, triglycerides, insulin, glucose, fertility and sperm count appear to be improved by chromium deficiency, but none of these parameters are individually specific for chromium. Improvemen n thesi t e parameters afte r supplementatioC r n appearo t s b^ the best present indicator of Cr status of man and animals.

Introduction: Suboptimal chromiu d animalan m n y statuleama o ma st df o s detrimental health and economic implications. In man, marginal chromium intak s beeha e n linke o diabetest d , cardiovascular problems, weight loss and nerve disorders (Anderson, 1980). In animals, chromium deficiency lead o impairet s d glucose metabolisd an m decrease n longevityi s , reproduction, litter size g qualiteg , d an y growth (Schwarz and Mertz, 1959; Davidson and Blackwell, 1968; Britto t al.e n , 1968; Steel t al.e e , 1977, Jense t al.e n , 1978; Steel d Rosebroughan e , 1979). Margina r deficiencC l n appearma n i y s e quittb o e widesprea y affecma d e s mana tan th s d hala yf o f individuals in certain population groups (Glinsmann and Mertz, 1966; Hopkin d Pricean s , 1968; Levin t al.e e , 1968); chromium deficiency in farm animals has not been actively investigated but several reports have appeared indicating that several specie o respond s o t d supplemental chromium with, increased performance.

147 Direct and indirect methods can often be used to assess trace element statrs. Direct methods would include determination f traco s e element concentratio f biologicao n l samples suc s blooda h , urine, hair and body tissues. Indirect methods would include determinations of physiological and biochemical parameters e.g. determination of glutathione peroxidas r hemoglobio e n concentratio r seleniufo n d an m iron status, respectively. However there are no real good indirect indicators presently available to assess Cr status. Insulin level, glucose tolerance, and blood lipid profiles have been correlated with Cr status but these parameters are influenced by a number of othe t specifi. no AdvantagerCr e factorr ar fo c d f an sspecifio s c methods to assess Cr status of man and animals will be discussed. Chromium analysis: To assess Cr status by direct measurement of Cr in specific tissue d bodan s y fluids a ,suitabl e metho r analysifo d f totao s r C l is essential. The analytical methodology for the determination of Cr in biological materials is only recently attaining a level of acceptance such that inter laboratory values for Cr in biological tissues can be evaluated. In the past, absolute values for Cr in biological materials varied considerably from laboratoro t y laborator d evean y n withi e samth ne laboratory r examplFo . e reported values for serum Cr range from several hundred parts per billion (ppb) to the presently accepted value of less than 1 ppb (see sectio n seruo n m Cr) r analysi.C s complicatei s y extremb d e matrix effects, low concentration in biological samples, possible volatilit f somo yr complexeC e e inherenth d an s t propertr C f o y complexes to bind nonspecifically to reaction vessels, graphite tubes, etc. Contaminatio s als i nn extrem a o e problem especially where tissue samples are collected by use of scalpels, trays, needles, and other stainless steel utensils made of approximately . 18Cr % Improved methods of digestion and the use of standard reference materials with Cr concentration certified by the U.S. National Bureau of Standard d internationaan s l agencies have helpeo t d

148 promote the analytical determination of Cr in biological materials to a level of acceptance. At present there is still no single method tha s suitabll biologicai t al r fo e l materials e method; th man f o y s r analysiC user fo d s have been summarized (Towil al.t e l , 1977). Standard reference materials certified for Cr content should be employel analyticaal r fo d l studies involvin . MorCr g e thae on n reference material should be utilized since Cr from different sources behaves differently during digestion and analysis and the levels of Cr in different reference materials vary considerably (Anderson, 1980). Values for urine and serum of 1 ppb or less obtained in the late seventie r normafo s l individuals (Guthri1978, . al ;t Kayne e t al.e e , 1978; Versiec t al.e k , 1978, Veillo t al.e n , 1979;) appea y presenb r t standards to be correct (vide infra). Earlier values should be interpreted with great caution. Chromium conten t f tissueo tno s ha s been verified and may also decrease when newer quality control standards and procedures are applied. Values for Cr in hair appear e correcb o t t neebu t d fur-ther verification. n indicatoa - Hair contentC r C rf o r : Hai r contenC r s beeha t n suggeste a possibl s a d e indicatof o r bod r contenC y t (Hambidge, 1974). Included amon e potentiath g l advantage f haio s r analysi e relativelth s i s y large concentratiof o n chromium in this tissue. This permits analyses of total chromium by less sensitive techniques thae lesar ts affecte y backgrounb d d contamination and matrix effects (Hambidge et al., 1968). Hair Cr concentratio s beeha n n correlated with disease states e.g. head hair f diabetico s lowei s r than tha f normao t l subjects (Schroeder 1968; Hambidg t al.e e , 1968). Direct assessmen r statuC f o ts using hair chromium analysis to monitor the Cr status of pregnant and parous women has been used. Nulliparous women have significantly higher levels of hair chromium than parous women but additional children t furthedino d r decreas r statuC e s judgea s y haib d r chromium concentration (Mahalk d Bennionan o , 1976). Hai r contenC r t alonn a g individual hair shaft may also be indicative of Cr status. For example e hai r th ,concentratioC r n dista8 l1 fron e scala th mf o p

149 month child whose hair had never been cut was 940 ppb, reflecting the high levels at birth, but the Cr concentration in the 2 cm region closese scal s th onl4 ppbwa po 14 t yta valu, e similao t r that of older children and adults (Hambidge, 1971). Hair Cr content of premature infants is indicative of gestational age; presumably Cr status is considerably lower in premature infants than full-term babies. Hambidge (1971) reporte a haid r concentratioC r f babieo n s aa gestationat 2 weeke mor3 b o f et so thae ag 6 ntimel s lower than that of babies at a gestational age of 36 weeks. Hai r contenC r a woman f o t , later demonstrate e overtlb o t d y Cr-deficient, was also a useful indicator of Cr status; her hair chromium level was more than 2.5 times lower than the lowest level observe r tha fo f normado t l control subjects (Jeejeebho t al.e y , 1977). However, hair chromium conten f malnourisheo t d adults wa s normal but these individuals appeared to be chromium deficient since their glucose tolerance improved after Cr supplementation (Gurson et al., 1975). As malnutrition is known to retard or stop hair growth, hair is not accepted as an indicator of trace element status of malnourished subjects. Studies are in progress in our laboratory to determine the usefulness of hair Cr as a predictor of Cr status and to ascertain the effect of Cr supplementation on hair Cr content. Analysi f hai o sr conten C r f faro t m animals a appear e b o t s productive area of research that has not been actively pursued. The y studiet authorawaran no f o e e sar s correlating hai r contenC r f o t farm animals with any growth or production parameters. n indicatoa s r a statusC Bloo- f o r C d : e usefulnesTh r contene wholC th f o sef o tbloo r serua o d s i m controversial subject. Since blood Cr is not in equilibrium with tissue stores e actuath ,a goo t lde bloo no leve th indicatos i n di l r of overall chromium status. However, in the 2 reported cases of over r deficiencC t n humani yr C e value w th s lo s2 obtainee th r fo d subjects were considerably lower than those obtaine r controfo d l subjects (Jeejeebhoy et al., 1977; Freund et al., 1979). Neuman et al., (1978) compared the serum chromium content of subjects with coronary artery diseas d thosan e e apparently fre f symptomo e d an s

150 found that subjects with lower serum chromium conten muca d hha t higher incidence of coronary artery disease than subjects with higher serum chromium levels. Coronary artery disease was not statistically correlated with cholesterol, blood pressure or weight index but there was some correlation (P < 0.05) with triacylglycerols and a highly significant correlation (P < 0.01) with serum Cr. Therefore, serum Cr in that study was the best predictor of coronary artery disease. While the actual Cr content in the blood, except in extreme cases, may not be an indicator of Cr status, the rise in blood Cr followin e administratioth g f glucoso n r insulio ea e y provb ma n o t e useful indicator of chromium status. When young subjects were given a test dose of glucose or insulin there was an acute rise of plasma n olde i C0 minr 9 t rwithi o Bu midle .t subjectsb 0 y 3 ny ma o ,wh chromium deficient ,o simila n ther s wa er increas bloon i e. Cr d Supplementation of older subjects with chromium chloride for several weeks caused the reappearance of the acute increase in circulating Cr indicating that lack of a rise in Cr may be due to a depletion of bod r storeC y s (Hambidge, 1971; Glinsman t al.e n , 1966; Behnd an e Diehl, 1972). However, some workers have reporte a decreasd n i e plasma Cr following administration of glucose or insulin (Davidson and Burt, 1973) while others have found increase n circulatini s r C g after oral administratio f glucoso na decreas t bu e n thii er afte C s r intravenous administration of glucose (Pekarek et al., 1973). Liu and Morris (1978) reported that the ratio of the serum Cr concentration 1 hour after a glucose load divided by the fasting Cr concentra- meaningfua tio s i n l indicato r status;C f o r they reported than i t respons a glucos o t e e load, serum chromium levels decreasen i d sujects with presumably inadequate chromium storagw d thalo an ea t Cr ratio indicates suboptimal Cr status. The apparent discrepancies in the values for circulating chromium reported by various investigators may be due to the chromium status of the individual and (or) time of sampling since the acute increase e precedeb n initiay a ma y b dr i C nl declin a leve o t el below that observed e administratioprioth o t r e testh t f o nloa d (Behnd an e Diehl, 1972). Studies are presently in progress to ascertain whether

151 the rise or fall in serum Cr following a test load can be used as an indicato r statuC f t eveo rbu sf verifie i n e rapith d d transient increas r concentratioC n i e d variabilitan ne th e tim th f o en i y increase make these change n blooa veri s r yC d difficult clinical test for Cr nutriture. Bloo r contenC d f faro t m t beeanimalno ns reportedha s s A . mentioned, serum Cr does not appear to be in equilibrium with body stores of Cr and, except in quite severe cases of Cr deficiency, would not appear to be useful indicator of status. n Urinarindicatoa - r statusC r C yf o r : Unlike blood chromium, urinar r contenC y t appear e relateb o t s d to carbohydrate metabolism and may be a meaningful indicator of Cr status of higher animals and man. Part of the Cr increment appearing in the circulation in response to a glucose or insulin challenge is subsequentl e yurine th los n i t;e majo urinth s ri e excretory route e functiona th e sin th f o ks i r absorbe fo ld an plasm r C d a dialyzable Cr (Saner, 1979). The transient rise in blood Cr, which may go undetected unless several blood samples are taken, would appear in the urine in a cummulative fashion. Therefore, increased Cr content followin a gglucos r insulio e n challengmeaningfua e b y ma e l indicator of Cr nutriture. Individuals with deplete r storeC d r thoso s e otherwise unablo t e utilize Cr do not appear to display increased levels of blood Cr in response to insulin challenge (see section on Blood Cr - an indicator r ostatusC f d therefor)an e shoul t shono d w increased urinary output . oCr f

To ascertain whether the change in urinary Cr could be used as a meaningful indicator of Cr status, the urinary excretion of 76 subjects administere f glucoso g 1 ds e wa g orallbodk . wt r y pe y determined. Subjects ranged in age from 21 to 66 years and were administered glucose after an overnight fast. Blood and urine sample s0 min 9 wer d .e an Urinartake0 t a nr conten C y 0 tim t a et ranged from .05-.58 ppb with a mean of 0.195 + .12 ppb and 90 rain afte a glucosr e loa excretioCr d n ranged fro-1.1.05 m wit6ppb a h b (Tablpp . 1 Supplementatio1) e.2 _ f + o 4 g y .3 mea 0 f o n20 f o n

152 chromium chloride daily for 2 or 3 months led to more than a 4-fold increas r excretioC n i e e ratit (Tablth bu nf chromiu o o) 2 e m excreted afte a glucosr e load versu e pretesth s tt increasperiono d d di d an e actually decreased. For example, in the pretest period 80 percent (61/76) of the subjects showed an increase in chromium excretion following a glucose load; after 2 and 3 months of Cr supplementation only 56 percent of the subjects displayed increased Cr excretion. These data suggest that an increase in Cr excretion following a glucose load may not be a meaningful indicator of Cr status or that r excretioC e e rati th t th onl f no s o ni y afte a glucosr e load versus before important but also the fasting Cr excretion. Since subjects supplemented with Cr excrete approximately 4 times more Cr than control subjects, little additionae needeb y o combat ma d r C lt a glucose challenge. However, diabetics also have higher urinary excretion than control subjects (Hambidge, 1971; Vanderlind, . al t e e 1979) but do not appear to be able to utilize the absorbed Cr. This e cas th y alse ma e b wito h individuals supplemented with inorganic chromium, sufficient Cr appears to be present but may not be in a useable form. However most normal individuals appeae ablb o o t et r convert inorganic Cr to a useable form e.g. in the 2 reported cases r deficiencC f o humann i y s both subjects responde o inorganit d r C c and special forms of Cr were not required. There is ample evidence regardin e essentialitth gt littlbu r C e f evidenco y e regardine th g essentiality of certain forms of Cr (Anderson, 1980). The suggested r chromiufo t certaiA no bases RD i md n totaan o dn r organiC l c forms. To our knowledge, urinary Cr excretion has not been used as an indicatoe difficultth r statuC o t f o f e animalro f sdu o y d an s analysis, problems in obtaining uncontaminated samples, and difficulty in interpretation of the data, does not appear to be a suitable metho o assest d r statuC s f animalso s . Tissue Cr - and indicator of Cr status: There are not enough data available to ascertain the validity of Cr tissue conten meaningfua s a t l indicato r statusC f o r . Most values in the literature date from a period when concentrations reported for urine and serum ranged from 100 to more than 1000 times

153 higher than wha presentls i t y accepted, making statementr C e th n o s conten f tissueo t s very dubious. Geographical variatiod an n decreasing chromium content f bodo y tissues wit e alsag h oe b nee o t d verified. Cr supplementation - A means to assess Cr status: r statusC , excep n extremi t e cases s veri , y difficulo t t evaluate. Direct analytical determination e subjecar s o samplt t e preparation, contamination and matrix effects but even if all these problems are resolved there are still serious limitations with regard to the tissues or body fluids that should be analyzed and the interpretatio e resultsth f o n . However r supplementatio,C n ma f o n and animals can be used as a means to assess Cr status. In three separate studies involving humans approximately 50% of the maturity- onset diabetics (Glinsman Mertzd an n , 1966) middle aged (Hopkind san Price, 1968 d elderl)an y subjects (Levin al.t e e , 1968) showed improvement in glucose tolerance after supplementation of 150 vg of Cr daily to their diets. Liu and Morris (1978) reported that an even higher percentage of normal and hyperglycemic women showed significant improvement in blood glucose and circulating insulin after supplementation with yeast thas hig wa n biologicallti h y activ . SupplementatioCr e o childret 0 pg/dar C 25 n Jordaf f i o no y n suffering from kwashiorkor (Hopkin d Pricesan , 1968) d childre,an n i n Turkey suffering from protein-calorie malnutrition (Gursen and Saner, 1971; 1973) lead to restoration of normal glucose tolerance. Malnourished children in Egypt, that were consuming sufficient levels of Cr, did not respond to additional Cr (Carter et al., 1968). Other investigators reported little difference between subjects treated with placebo or chromium (Sherman et al., 1968; Wise, 1978). However numbea , f factorso r s onlwhicf i o , yr C hone , affect glucose removal rates, therefore Cr should not be construed as the sole criterio r glucosfo n e tolerance. Limited researc s beeha h n reporte e directh n o td assessmenf o t experimental and farm animals; however, a number of reports have appeared demonstrating improved performanc f animalo e s after C r

154 supplementation. Animals responsiv r supplementatioC o t e e ratar n s (Schwarz and Mertz, 1959) diabetic mice (Tuman et al., 1978) sheep (Britto t al.e n , 1968), squirrel monkeys (Davidso d Blackwellnan , 1968), swine (Steele et al., 1977) laying hens (Jensen et al., 1978) and turkey poults (Steel d Rosebroughan e , 1979). Presto. al t ne (1976) reported that dietar r doet C affecy no s t guinea pig r theio s r offspring; this may indicate a difference among rodents or that the conditions employed wert conducivno ee demonstratioth o t e r C f o n deficiency. Physiological functions of man and animals affected by Cr are listed in table 3. In assessing the Cr status of rats, we observed that while the absorption of radioactive Cr was not significantly affected by Cr status (Table 4), which is consistent with previous workers (Hopkins, 1965, Mertz et al., 1965), the fertility and sperm count were adversely effected by low levels of dietary Cr (Table 5). If the reproductive capacit f fary'o m animal s alsi s o decreasey b d marginal Cr intakes, this may have serious economic implications. e especiallb Thi y ma s y applicable sinc e studieth e s involving improved growth and improvement in other physiological parameters by additional dietar r pertaineC y o fart d m animals that were raisen o d standard diets with no measures to control Cr contamination. Supplemental chromium significantly improved the growth rate of turkey poulta corn-soybea d fe s n diet (Steel d Rosebroughan e , 1979), g qualiteg d f eggo yan s from leghor na corn-soybean-alfalf hend fe s a diet (Jense t al.e n , 1978) A comple. r potentiateC f o x e th d hypoglycemic response observed following an intravenous insulin challeng n swini ed stocfe e k diet (Steel t al.e , 1977); nitrogen utilization by sheep was also increased by addition of Cr to a basal diet which containe n apparentla d y complete mineral mixture (Britton et al., 1968). This may indicate that large numbers of farm animals, like numbers of humans (Glinsmann and Mertz, 1966; Hopkins and Price, 1968; Levin t al.e e , 1968 e sufferin)ar g from sign f marginao s r C l deficiency.

~~155 Tabl : Urinar1 e r ExcretioC y 0 min9 nd .prioan o t r following a glucose load~~

0 time n mi 0 9 Subject Cr, (ppb) Range [Cr]b pp , Range M, F (80) 2 .19.1 + 5 0.58- 5 .0 ) ( .34 + 0.21 ( .05 - 1.16) M (51) .20 + .12 ( .05 - .58) .28 + .16 ( .05 - 0.80) F (29) 1 .1 + 8 .1 ( .05 - .5) .38 + 0.27 ( .05 - 1.16)

~~After the initial urine sample, subjects were given 1 g of glucose orally per kg body wt; second urine sample was 90 min. after ingesting glucose test solution.~~

~~Table 2: Effect of Cr supplementation on Cr Excretion~~

~~Pretest 2 months 3 months Cone, (ppb) .195 + 0.12 .84 + 0.60 (41) 1.1 + .74 (41) Range (.0 5- .47 ) (.0 52.7- ) (.0 53.1- )~~

~~Subjects ingeste g CrCly 0 e time3 20 dth dail r s fo yindicated . Urinary Cr concentrations were determine n urino d e samples from individuals after an overnight fast.~~

~~156 Tabl : Physiologica3 e l Functions Improve y Chromiub d m~~

~~Function Animal~~

Glucose tolerance Human, rat, mouse, squirrel monkey Circulating insulin Humant ra , Insulin response Human, rat, swine Nerve disorders Human Brain disorders Human Glycogen formation Rat, turkey Glucose oxidation Rat Glucose uptake Rat Serum cholesterol Human, rat, mouse Serum triglycerides Human, rat, mouse Aortic lipid d plaquean s s Rat Fertility and sperm count Rat Corneal opacity Rat Protein synthesis Rat Nitrogen utilization Sheep Egg Quality Chicken Growth Human, rat, turkey Life span Rat, mouse, hamster

~~157 Tabl : Effec4 e r absorptio C f die o tn o t n~~

~~Diet Absorptio) (% n~~

~~Lor C w 0.3 50.1+ 6 (24) Lor pluC w sn fee i 2pp dr C m 0.3 10.1+ 3 (24) Stock 0.52 (SO0. 0)+~~

Rats weighing 200-250 grams were 8 hoursfaste1 r fo d, stomach-tubed i chromiuyC wit 0 6 h m chlorid t zera e o time d sacrificean , d afte4 2 r hours. Absorption refers to the counts retained in the carcass minus entire gastrointestinal tract. Rats were raised in plastic cages with purified diean tr componentai d s (Polansk d Andersonan y , 1979).

~~Table 5: Effect of Cr on Sperm Count and Fertility~~

~~Fertility (I)1 Sperm Count2 r +C r -C r +C r -C f rato e s Ag 0 (8/8 0 10 (8/810 )4 )month s44. 411.+ 3 46. 8^ 11. 2 5 (2/82 0 (8/8 10 )+ 12. )8 3 month 5 3 s 28.8 9. 1 +~~

Rats were raised as described in table 4 1-Eacmal4 f eo h rats matewa s d wit 2 femalh e5 days ratr .fo s Female rats were 3 months old and raised on a stock diet for 2 1/2 months; 2 weeks prior to mating all female rats were switched to a r die C o minimiz t w t lo e contamination. After mating l femalal , e rats were switche a stoc o t dk diet containing adequat. Cr e Numbe n parenthesii r s denotes numbe f rato r s impregnated. ^Number of sperm cells was determined after adding 5 ml of physiologica t caudacu l e salinlth portion o e t epididymidee th f o s s of each rat. Sample was shaken, diluted 50-fold and 10 microliters were place a hemacytomete n o d d countean r n duplicatei d . Counts given are for a 0.04 mm2 area of the diluted samples. Sperm count e standare meath i th s_ + n d deviatio f spero n m samples fro 8 ratm s (Anderson and Polansky, 1980). ^Significantly different from each other (p<^

~~158 References:~~

Anderson, R.A. Nutritional rol f chromiumo e . Science Totath lf o e Environmen , 198016 t . Anderson, R.A. and Polansky, M.M. Role of chromium in sperm cell production and fertility. Fed. Proc. 39, 787, 1980. d BehneDiehlan . . D ,F Relation s between carbohydrat d tracan e e element metabolism investigated by neutron activation analyses. : NucleaIn r Activation Technique e Lifth en i Sciencess . IAEA-SM-157/11, Vienna 1972, 407-413. Britton, R.A., McLaren, G.A d Jettan . , D.A. Influenc f cano e e molasses on NPN utilization and fiber degestibility. J. Animal Sci. 27, 1510, 1968. Carter, J.P., Kattob, A., Abd-El-Hadi, K., Davis, J.T., El Cholmy, A., and Pathwardhan, V.N. Chromium (III) in hypoglycemia and in impaired glucose utilisation in kwashiorkor. Am. J. Clin. Nutr. 21, 195-202, 1968. Davidson, I.W.F d Blackwellan . , W.F. Change n carbohydrati s e metabolis f squirreo m l monkeys with chromium dietary supplementation. Proc. Soc. Exp. Biol. Med. 127, 66-70, 1968. Davidson, I.W.F d Burtan . , R.L. Physiological change plasmn i s a chromiu f normao m d pregnanan l t women: Effec f glucoso t e load. Am. J. Obstet. Gynecol. 116, 601-608, 1973. Freund, H., Atamian, S., Fischer, J.E. Chromium deficiency during total parenteral nutrition . AmerJ . . Med. Assoc. 241, 496-498, 1979.

Glinsmann, W.H., Feldman, J.F. and Mertz, W. Plasma chromium after glucose administration. Science 152, 1243-1245, 1966. Glinsmann, W.H. and Mertz, W. Effect of trivalent chromium on glucose tolerance. Metabolis , 510-51715 m , 1966. Gurson, C.T. and Saner, G. Effect of chromium on glucose utilization in marasmic protein - calorie malnutrition. Amer. . ClinJ . Nutr , 1313-131924 . , 1971.

159 Gurson, C.T d Saner.an . G ,Effec f chromiuo t m supplementation o n growth in marasmic protein - calorie malnutrition. Amer. J. Clin. Nutr. 26, 988-991, 1973. Guthrie, B.E., Wolf, W.R., Veillon, C. and Mertz, W. Chromium in urine. Hemphill ,: Trac D.D.In e. ed Substance, n i s Environmental Health XII, Univ. of Missouri, Columbia, Missouri, 1978, 490-492. Gurson, C.T., Saner , MertzG. , , Wolf,W. , W.R d Sokucii.an . S , Nutritional significanc f chromiuo e n differeni m t chronological age groups and in populations differing in nutritional backgrounds. Nutr. Rep. Int , 9-1712 . , 1975. Hambidge, K.M. Use of static argon atmosphere in emission spectrochemical determinatio f chromiuo n n biologicai m l materials. Anal. Chem , 103-10743 . , 1971. Hambidge, K.M. Chromium nutritio . Clinn J mani n . .. Am Nutr, 27 . 505-514, 1974. Hambidge, K.M., Rodgerson, D.O d O'Brien.e concentratioan Th . D , n of chromium in the hair of normal children and children with juvenile diabetes mellitus. Diabetes 17, 517-519, 1968. Hopkins, L.L. . f physiologicao DistributioJr , t ra e th n i nl amounts of injected Cr51 (III) wit . hPhysiolJ time. Am . . 209, 731-735, 1965. Hopkins, d PriceL.L.an . ,Jr , M.G . Effectivenes f chromiuo s m (III) in improving the impaired glucose tolerance of middle-aged Americans. In: Proceedings of Western Hemisphere Nutrition Congr., Puerto Rico, 1968, Vol. II, 40-41, 1968. Jeejeebhoy, K.N., Chu, R.C., Marliss, E.B., Greenberg, G.R.d an , Bruce-Robertson, A.. Chromium deficiency, glucose intolerance, and neuropathy reversed by chromium supplementation in a patient receiving long-term total parenteral nutrition . ClinJ . .Am . Nutr. 30, 531-538, 1977. Jensen, L.S., Maurice, D.V d Murray.an , M.W..w Evidencne a r fo e biological functio f chromiumo n . Fed. Proc , 40437 . , 1978.

160 Kayne, F.J., Komar, G., Laboda, H., Vanderlinde, R.E.. Atomic absorption spectrometr f chromiuo y n seru i md urinan m e wita h modified Perkin-Elme 3 atomi60 r c absorption spectrometer. Clin. Chem : 2151-215424 . , 1978. Levine, R.A., Streeten, D.H.P. and Doisy, R.J.. Effects of oral chromium supplementation on the glucose tolerance of elderly human subjects. Metabolis , 114-12517 m , 1968. Liu, V.J.K. and Morris, J.S.. Relative chromium response as an indicator of chromium status. Am. J. Clin. Nutr. 31, 972-976, 1978. Mahalko, J.R d e Bennioneffecan .Th f parit. o t ,M. d tim an y e between pregnancie n maternao s l hair chromium concentration. Amer. J . Clin. Nutr , 1069-107229 . , 1976. Mertz Roginski, . ,W , E.E d Rebaan . , R.C.. Biological activitd an y fat f traco e e quantitie f intravenouo s s chromium (IIIe th n i ) rat. Amer. J. Physiol. 209, 489-493, 1965. Newman, H.A.I., Leighton, R.F., Lanese, R.R. and Freedland, N.A.. Serum chromiu d angiographicallan m y determined coronary artery disease. Clin. Chem. 24, 541-544, 1978. Pekarek, R.L., Hauer, E.G., Wannamacher, R.W. Jr. d Beiselan , , W.R.. e direcTh t determinatio f seruo n m chromiu n atomia y b mc absorption spectrometer wit a heateh d graphite atomizer. Anal. Biochem , 283-29259 . , 1973. Polansky, M.M d Andersonan . , R.A.. Metal-free housing unitr fo s trace element studie n ratsi s . Lab. Animal Sci , 357-3593 . , 1979. Preston, A.M., Dowdy, R.P., Preston, M. and Freeman, J.N.. Effect of dietary chromium on glucose tolerance and serum cholesterol in guinea pigs. J. Nutr. 106, 1391-1397, 1976. Saner . ,G Chromiu d glucosan m e metabolis n childreni m : ChromiuIn . m in Nutritio d Metabolisan n m Shapcott . d HuberEd an , . ,D J. , Elsevier/North Holland Biomedical Press, 1979, 129-144. Schroeder, H.A. The role of chromium in mammalian nutrition. Amer. J. Clin. Nutr. 21, 230-244, 1968.

161 Schwarz d Mertzan . . ,K ,W Chromiu me glucos (IIIth d )ean tolerance factor Arch. Biochem. Biohphys , 292-29585 . , 1959. Steele, N.C., Alien, T.G. and Frobish, L.T.. Biological activity of glucose tolerance factor in swine. J. Animal Sci. 45, 1341-1345, 1977. Steele, N.C. and Rosebrough, R.W.. Trivalent chromium and nicotinic acid supplementatio e turketh r y fo npoult . Poultry Sci, .58 983-984, 1979. Towill, L.E., Shriner, C.R. and Drury, J.S.. Reviews of the environmental effects of pollutants: III. Chromium. U.S. Environmental Protection Agency, Cincinnati, Ohio, 1977. Tuman, R.W. Bilbo, J.T. and Doisy, R.J.. Comparison and effects of natural and synthetic glucose tolerance factor in normal and genetically diabetic mice. Diabetes 27, 49-56, 1978. Vanderlinde, R.E., Kayne , KomarF. ,, Simmons G. , , M.J., Tsore, J.Y. and Lavine. Serum and urine levels of chromium. In: Chromium in Nutrition and Metabolism. D. Shapcott and J. Hubert, ed., Elsevier/North Holland Biomedical Press, 1979, 49-57. Veillon, C., Wolf, W.R., Guthrie, B.E.. Determination of chromium in biological materials by stable isotope dilution. Anal Chem. 51, 1022-1024, 1979. Versieck . HosteJ , , BarbierJ. , , SteyaertF. , , DeRudderH. ,d an , J. , Michels . Determinatio. H , f chromiuo n d cobalan m n humai t n serum by neutron activation analysis. Clin. Chem. 24, 303-308, 1978. Wise, A.. Chromium supplementation and diabetes. J. Am. Med. Assoc. 240, 2045-2046, 1978.

162 MARGINAL MINERAL DEFICIENCIES AND THEIR IMPORTANCE FOR THE PRODUCTIVITY AND REPRODUCTIVITY OF DOMESTIC ANIMALS*

~~I. WEGGER Departmen f Physiologyo t , Endocrinolog Bloodgroupingd yan , Royal Veterinary and Agricultural University, Copenhagen, Denmark~~

~~ABSTRACT~~

Using selenium as an example some aspects of trace element metabolism especiall swinn i y e have been discusse precee th n -i d ding paper. Several investigations indicate that the chemical n elemen e feea fory influencth f ma do m n i t availabilits it e y to and metabolism by the animals. Results lending further support to this are presented and discussed. A normal physiological condition which greatly enhances the requirement for nutrients including trace elements is pregnancy. The placental transfer of selenium is discussed in some details and a survey e theoreticaoth f l minimum requirement r severafo s l elements at various stages of pregnancy is given. The possible relationship between marginal subclinical deficiencies and animal health especially the resistance towards infectious diseases is also touched upon. Durin pase decadew gth fe t s marginal mineral deficiencies or imbalances have become prominent problem mann i s y partf o s the worl unded an d r widely varying animal production systems. The possible cause f thio s s unfortunate developmen e conar t - sidered and the advantages and disadvantages of various methods to detect such conditions discussed. Special emphasis is laid upon screening methods suitable for depicting geogra- phically limited areas where marginal deficiencie suse b y - sma pecte o occurt d . n recenI t year e existencth s geneticallf o e y controlle- va d riation in the trace element content of blood and tissues i.e. in the biochemical profile of animals belonging to the same race has become evident. These findings makes it difficult to speak about "normal" values in blood and tissues. They also hamper the possibility for establishing normal requirements for

* The investigations were supported by grants from the Danish Agricultural and Veterinary Research Council and from the Danish International Development Agency. The Danish group comprises: I. Wegger, P. Fogd J^rgensen . Hyldgaard-JensenJ , . PalludaB , . MoustgaardJ d nan resulte Th . s presente thin di s report were partly obtaine co-operation di n wit followine hth g guest scientists: S.D. Tunce. ErgiinA d an r, Turkey; D. Vitti, Brazil; T. Kosla, Poland and N. Gazia, Egypt.

163 the various trace elements possibls o doubi N . t i t breeo t e d animals which posses requiremenw lo a higsa give a r o hr nfo t trace element such as for example copper or selenium. This underlines the importance of a close cooperation between genetically orientated biochemist d nutritionistssan .

~~INTRODUCTION~~

Prank deficiencies of macro or micro minerals have been known for lon resulo gt n characteristii t c disease farn i s m animals leading to severe production losses. Research during the past few deca- des has, however, shown that marginal deficiencies or imbalances in mineral intake are of equal or even greater importance for the productivity and economy of animal husbandry in many parts of the world. Such borderline condition r morfa e e difficular s - de o t tect and hence to correct than a manifest deficiency state since they t provokofteno o y significand nan e t clinical symptoms. None the less marginal deficiencies especially of trace elements can seriously impair meat, milk, wool and egg production and also the fertility in both male and female animals. The occurrence of such deficiencies may be caused by several factors alone or in combination e soid henc Th an crope l. th ey contai sma n insufficient amounts of one or more elements or the availability of a mineral may be low either due to its chemical form or to the pre- senc f interferino e g substance e feedth .n si These factore ar s especially important in areas with extensive management systems where livestoc n locall o oftes i kd fe ny grown crops withou- mi t neral supplementation. However, several examples of marginal trace element deficiencies are also known in countries with intensive animal production systems e increasinth o t . e Thidu g s si deman r animafo d l productivity e intensifieth , confinef o e us d d management systems and industrially produced feedstuffs often of non-conventional character.

In order to detect and combat trace element deficiencies in a rational way ,thorouga h knowledg metabolise th f o e biod an m - chemical function of the elements is essential. Also the normal physiological variatio mineran ni l statu farf o s m animalo t s sha b efollowin e knownth n I . g studies along this line wil dise lb - cussed with special emphasis on selenium. Items to be dealt with ) seleniuA e ar m metabolis n swinei m ) geneti;B c aspect tracf o s e element metabolism, and C) methods for evaluating trace element status of domestic animals under practical conditions.

~~164 Seleniu. A m metabolis swinn i m e~~

Absorptio d distributionan selenitf no d seleno-methioninean e . Selenium in plants is incorporated in the protein fraction mainly as seleno-methionine (Olson et al. 1970) and several studies have shown that animals utilize native selenium better than selenite (cf. Moustgaard 1977)• This lea investigationo t d whethen o s r the chemical for seleniuf o m m influence intestinas sit l absorption and distribution in the animal body (Wegger et al. 1978). Six fasting pigs weighing 20.5-22.0 kg were dosed per orally with radioactive selenium eithe s ra Se-selenit75 r o eSe-methio - 75 nine pige Th .s were place metabolin i d animace on cage n d o l san each treatmen killes days6 wa t d d. an afte3 Faece , r1 urind san e was collected quantitatively as was intestinal content after kil- ling retentioe Th . seleniuf o n s calculatemwa e S s dose a df o 75 minus activity recovered in faeces, urine and intestinal content. Tabl show1 e retentioe sth d excretionan seleniuf o ncenr pe t n i m of dose. As seen the retention of 75Se was higher from seleno- methionine than from selenite primarile , du whic s greaa hwa o t y- ter urinary excretion of selenite during the first three days of the experiment. The daily excretion of 75Se thereafter was low d practicallan y constant irrespectiv e th chemica f o e l form the isotop s givenwa e . The retention of 75Se-selenite found in our experiment is of the same order of magnitude as reported by Buescher et al. (1961). pign I s give foddena r containin nativf o m epp g sele 5 0.00. -o 3t niu lineama r correlatio founs nwa d between selenium concentration in feed and muscle tissue (Ku et al. 1972). But if selenite was used as selenium source the increase in muscle selenium content ceased whe concentratioe nth fodden ni r m reachepp 1 0. d (Groce et al. 1973). This agrees well with the higher retention of seleno-methionine than of selenite, as reported here. Simi- lar results have been obtained with rats (Thomso nStewar& t 1973) and lambs 1967). (Ehlial t ge . Th varioun ei activite S s tissuef o y shows i s7 5tabln ni . 2 e The kidney d especiallsan cortee th y xhige possesseth -r fa y db hest activity in all the pigs. The second highest 75Se activity s foun wa liven i d r followe kidnee th y ydb medulla, pancread san adrenals, spleen, thymu d lympsan h node, heart centrae ,th l nervous system and skeletal muscle tissue. The differences between the various parts of the brain were small but the spinal cord accumulated significantly less selenium than the rest of the central nervous tissues. Maximum activity in liver was reached within the first 24 hours and in most other tissues 3 days after the administration of radioactive selenium.

~~165 Table 1. Excretion and retention of 75-Se in per cent of dose after per oral administration of 75-Se-metlonine or 75-Se-selenite to 6 Danish Landrace pigs.~~

~~75-Se-methionine 75-Se-selenite Days 1 3 6 1 3 6~~

~~Urine 5.8 6.6 7.6 10.8 18.0 16.6 Faeces 0.3 3.1 10.9 0.1 6.2 18.5 Intestinal 2.2 content 4.6 3.4 1.4 20.6 1.2~~

~~Retention 89-3 86.9 80.1 68.5 73.6 63.7~~

Table 2. 75-Se activity (ncpm x 10 /g tissue) corrected for differences in dose and glutathione peroxidase activity (mean + s.e.) in various tissues from 6 pigs given 75-Se-selenite or 75-Se-methionine per orally.

75-Se -me onini th e 75-Se-selenite aSH-Px Days 1 3 6 1 3 6 u/g Liver 92.,0 72.6 71.1 81.1 73.1 59.2 31.1 + 1.5 Spleen 4?.,1 46 .7 36 9 • 27 .9 37.8 28.8 18.5 + 1.0 Kidney cortex 272.,1 319 .5 253 .7 309 .7 463.8 278.1 29. i 0.6 6 Kidney medulla 73,.3 80 .3 52 .6 68 .5 87.9 46.8 10. + 7 7. Pancreas 58,.0 68 .4 50 .8 28 .0 34.7 27.2 00. + 6 2. Thymus 26,.7 30 .3 28 .8 15 .4 21.6 21.3 20. + 0 8.

Lymph node 33,.1 32 .0 25 .6 17 .6 27.6 20.7 3 0. 9. 8± Heart 22.,5 25 .0 25 .2 13 .8 14.3 12.3 3.62 + 0.12 M. long, dorsi 13,.3 16 .1 13 .3 2 .9 3.3 3-0 0.7 0.0+ 8 5 M. flex. dig. 11,.4 9 .1 9 .4 4 .2 4.9 4.0 1.26 + 0.04 Cerebrum 15,.8 17 .1 15 .2 4 .0 6.0 6.0 1.19 + 0.02 Cerebellum 16,.1 17 .9 15 .2 5 .1 7.6 7.6 2.05 + 0.04 Med. oblong. 13,.9 16 .3 13 .2 5 .2 7.7 6.4 1.70 + 0.08 Med. spinal. 6..9 7 .7 7 .2 3 .1 4.0 3.8 0.48 + 0.06 Adrenals 63..6 65 .8 39 .7 45 .8 55.3 27.9 0.60 + 0.12

The cortex of kidneys accumulated considerably more 75Se from selenite than from seleno-methionine. Contrary to this all other lowese tissueth d t sha activit pige th s n dosei y d with selenite. Thus, the kidneys seem to have a central position in the metabolism and excretion of selenium in monogastric animals (cf. Klevay 1976) differencee Th . activitn i s y levels amon tissuee gth s studied are in agreement with earlier results from experiments on as well pig othes sa r species (Orstadiu Abers& g 1961, Buescher et al. 1961, Thomson & Stewart 1973)• The high activity in adrenals and spleen is interesting when seen in connection with the finding that these organs contai mucna h higher activity thay nan other tissue afte administratioe rth radioactivf no e vitaminE (Gallo-Torres 1973)• In this context it should be mentioned that

166 ther certaia e e b see o t nm parallelit abilite th n variouf i yo y s organs except the kidney to accumulate selenium and vitamin E. The last column of table 2 shows the glutathione.peroxidase (GSH-Px) activit varioun i y s tissues highese Th . t activit s founwa yn i d liver and kidney cortex followed by spleen, lymph node, thymus, medull kidneyf ao , heart, pancreas, brain, muscle, adrenald san spinal cord. The order of the tissues regarding GSH-Px activity and 75Se activity is, as it appears, roughly the same. Exceptions from thikidneye sar , pancrea d adrenalsan GSHw lo s -a whic d hha x activitP s compare a yhige th h o t seleniud s ma uptaker fa s A . the kidneys are concerned a possible explanation could be that onl yaccumulatee th som f o e d seleniu f functionao s i m l importance e laswhil th rese e excretiot s takes th eth i a stet f p o pnu n process. The relative distribution of retained 75Se is shown in figure 1. It appears that the selenium metabolism in some tissues depends on the chemical form in which the element was ingested. Se- lenite-S s concentratei e kidneyn i d greatea o st r extent thas ni seleno-methionine-Se, while pancreas, muscl braid an e n tissue take up less selenium from selenite. The differences in liver, spleen and thymus are less pronounced and disappear after 6 days. Regardin distributioe gth retainef o n d seleniu e orgath -n i m nise resultth m s froe literaturth m e contradictoryar e . Gart e y . (1973al ) foun highea d r selenium concentratio musclen i n t sbu not in other tissues from rats fed seleno-methionine than from those fed selenite for 50 days. In contrast to this Thomson & Stewart t finy (1973chemicae effecno an dth d f )di o t l forn i m which radioactive seleniu distributioe s giveth wa m n o ne S f no 75 in various tissues. Experiments with sheep (Jenkins & Hidiroglou 1971) and chickens indicate, however, that the differences in uptake and metabolism of selenit d seleno-methioninan e have ew e foun n severai d l organs n swini e also occu n othei r r animal species. Canto. (1975al t e r) r examplfo e found that selenit s bes preveno wa et t e developth t - ment of exudative diathesis in chicken while seleno-methionine protected more efficiently against pancreas fibrosis. One of the most frequent symptoms of vitamin E-selenium deficienc pign i ymuscls i s e degenerations which affec wels a te th l skeletal muscles as the heart. As shown here the selenium uptake in these tissues is considerably lower from selenite than from seleno-methionine. This means, other things being equal, that one should use higher doses of selenite than of seleno-methionine in orde obtaio t r e samnth e effec f seleniuo t muscln o m e dystrophy. practicae Th l significanc thif o e s canno deducee tb d fropree th m - sent results. Suc n evaluatioha n requires knowledg whethef o e r o formthtw ef seleniu o s e e incorporateth ar m n i e samy th wa e n i d

167 Tabl - Mea3 e n selenium concentration (,ug/g n foetuses)i , placentd an a foetal tissue t variousa s times during pregnancy. Numbe f foetusero s is shown in brackets; other values are means of determinations on three foetuses. The last column shows mean selenium concentrations in tissues from six pigs, age four months.

~~Foetal age, Pigs days 42 55 76 95 102 4 months~~

Foetus 0.116 0.084 0.076 0.072 0.072 - (7) (6) (6) (6) (5) Placenta 0.077 0.041 0.061 0.070 0.050 Liver - 0.280 0.266 0.200 0.241 0.32? Kidneys - 0.262 0.249 0.193 0.243 1.111 Heart - 0.143 0.124 0.100 0.086 - M. Longiss. dorsi - - - 0.040 0.042 0.131

~~Tabl . 4 Daile y depositio nitrogenf o n , calcium, phosphorus, iron,zin d seleniuan c n porcini m e foetuses calculatea r fo d litter size of 10 foetuses.~~

Day of gestation 40 60 80 100 110 115 N, g/day 0.16 0.7 2.7 11.0 22.1 31.3 Ca, g/day 0.03 0.2 1.2 7.3 17.9 28.1 P, g/day 0.03 0.2 0.8 4.2 9.3 13.9 Fe, rag/day 0.63 1.9 5.7 17.1 29.7 39.1 Zn, mg/day 0.31 1.0 3.1 9.9 17.7 23.7 Se, ^g/day 1.55 3.5 8.0 18.1 27.3 33.5

Table 5• Variation in copper, iron and selenium status among Danish Landrace pigs reare progena t a d y testing station. Selenium status was evaluated by means of G-SH-Px activity in erythrocytes. Variation between litter s significantlwa s y greater than within l litteral r fo s parameter e P-valuess showa th s y nb .

~~Copper cone, Iron cone, Total iron GSH-Px act iv. in plasma plasmn i a binding capac. n erythrocytei s No. of litters 25 40 38 52 Nof o . pigs 100 146 138 207 P < 0.001 0.05 0.001 0.0005~~

~~168 compounds through which selenium exerts its biological function. In other words whether the two forms per molecule have the same biological activity after being taken up by the tissues.~~

Placental transfer of selenium. During pregnancy the require- menr essentiafo t l nutrients including trace element greats i s - ly enhanced e suppl mineraa th f f o yI .insufficiens i l coveo t r the requirement wels a lr maternasfo l maintenanc s foetaa e l production maternal reserves of that element will at least to a certain degree be mobilised and transferred to the foetuses, they posses firssa t priority. Consequentl latena y t deficiencf o y the mother animal will be the result. A relatively simple method for estimating the amount of the normal placental transfer of a nutrient is to determine the foetal content of that substance at various times during pregnancy. Such investigations have been carried through in swine comprising several nutrients including the trace elements iron (Moustgaard 1959) and zinc (Palludan & Wegger 1972). Recentl e studiew y placentae th d l transfe f seleniuro e th n i m same manner . 1980)(Koslal t foetuse2 A totaae .4 f o l s from five litters obtained at a slaughterhouse were analysed. The age of the foetuses was estimated on basis of their length. In the four oldest litters liver, kidneys and heart and in the two oldest also muscle tissu . longissimu(m e s dorsi) were analysed separatel thren i y e foetuses e remaininTh . g foetuses were homo- genized and used for determination of the total selenium content. Furthermore three placentae from each litter were analysed. Se- lenium determinations were made according to Olson (1969)• Table 3 shows the selenium concentrations in whole foetuses, placenta d foetaan e l organs A decreas. n seleniui e m concentration with increasin s see i wholn i ne gag e foetu d heartan s , whereas the concentration in other foetal tissues seems nearly constant durin e secongth dgestatioe th hal f fo n period n tabls I i . 3 e also shown the average selenium concentration in tissues from six fouf o re monthsag e e seleniuth pigTh .t sa m concentration ni foetal liver is comparable to that in liver from growing swine while muscl d especiallan e y kidneys contain considerably less selenium than the corresponding tissues of growing pigs (Nielsen & Rasmussen 1979, Wegger et al. 1979, Wegger & Chnstensen 1979). These results indicate that pigs have a relatively low selenium status in several tissues at birth, and that a significant deposition of selenium in excess of that determined by growth itself takes place during the growth period. This is further supported by the fact that plasma selenium concentratio birtt a w hlo s ni (0.05 ug/ml) and gradually increases during the first 3-4 months of life to the normal level of 0.1-0.2 ug/ml in adult pigs.

169 Table 9. Zinc, copper and iron concentration, and glutathione peroxidase activity in swine liver from four slaughterhouses, mea s.e._ + n Values within each column followe e samth ey b d letter are significantly different (P < 0.05 - O.Ol).

~~Zinc Copper Iron GSH-Px Region ug/g ,ug/g U/g~~

Northwest 58+2 a .9+0 183+5. 6a 40. a Zealand .3 7 .5 2+15 . Lolland- b 173+7.8b 6+1.6a'b Palster 56+2 .3 7 .6+0 .3 45. North 61+2 .2 .6+0 a b?C> 3+2.4b'c Jutland 7 .4 206+7. 9 ' 37. West a b 175+6.2C 1+2 . Oc Jutland 66+2 .6 ' 7 .4+0 .5 44.

~~Kidney, cortex D "Se-mKhionint 0 ™Se-selenite~~

~~Kidney, medulla is 30~~

~~1 6 13 6 13~~

~~Days after administration~~

~~Spleen Thymus~~

~~M lortfli* dorsi C*rtbrum~~

~~6 13 6 1313 6~~

~~Days nisi m aftt i d ratioa r n~~

~~Figure 1. Relative distribution of retained radioselenium in various organ d tissuesan s from pig different sa t times after oral dosing with labelled selenite or seleno-methionine.~~

~~170 TOi St/fo«tu»«0.734-e«>g >j — «* - >igS*/day«0030-«oo«it 60-~~

~~40-~~

~~20- t 31 10-~~

~~40 50 60 70 80 90 100 110 Days after conception (t)~~

~~Figure 2. Selenium accumulation in pig foetuses. The fully drawn curve show e totasth l depositio r foetuspe n calcue th , - lated daily deposition is shown as a stippled curve.~~

~~l diseaseAl a a s b • Diarrhoea c a Lung diseases f n d s Other diseases n I * p.~~

~~I 30 i r- «* | 20 1~~

\ 1 O5 10'" - i j. | 1 3 m-A i 1 fl none none none GSH-Px i 2 3 4 5 6 7 8 9 10 It 12 Tola I no. 12 33 10 155 19 04 72 it 25 K 2 1 of animals Figur . 3 eDiseas e frequency durin growte th g h perio pigf o d s n relatioi erythrocyto nt e GSH-Px activity e enzymTh . e activity increases gradually from GrouGrouo t withi2 p1 p1 e th n range 7-288 U/g of haemoglobin, the class width being 24 U/g of haemoglobin.

171 A numerical value for the selenium deposition in foetal life can be obtained by calculating the deposition curves shown in e solifigurTh d lin. 2 ee curve depicts selenium accumulation wholn i case eother th efo foetus s ri micrs A d .macr an o o elements the selenium deposition increases exponentially with time after conception (Moustgaard 1959, Palluda Weggen& r 1972, 1976). It appears froe equatiocurve th mth er nfo thae relativ th t e rate by whic foetae hth l selenium poo lcenr r increasepe pe t 1 4. s si day. For comparison it can be mentioned that the relative rate of deposition of the trace elements iron and zinc is 5.5 and 5.8 pey respectivelrda cenr tpe y considerabls whili t i e y higher rfo the macro elements calcium (9.0 per cent), phosphorus (8.1 per cent) and nitrogen (6.9 per cent)(Palludan & Wegger 1972). The absolute amount of selenium transferred to the foetus per e calculateb n daca y y differentiatiodb e equatioe th th f o nr nfo deposition curve e resulTh .f suc o t calculatioa h represens ni - ted by the broken line in figure 2. During the last trimester the daily deposition of selenium increases from 0.8 ûg on day 80 to 3-3 p-S on day 115 corresponding to an average of 2 ûg per day in this period of gestation. Supposing a litter size of ten foetuses - thire se mean ar seleniuf o y sg da thaû r 0 mpe 2 t quired exclusivel r foetafo y l production pregnanA . w so t given 2.5-3 kg of feed containing 0.1 ppm selenium receive 250-300 )ig of Se per day. Thus, during the last trimester approximatel ingestee th r cen f pe o t 8 d 6- yseleniu m must be transferred to the foetuses. To this shall be added the amoun f seleniuo t m deposite placentn i d d uterinaan e fluids. Furthermore it must be kept in mind that not all of the ingested selenium is absorbed from the intestine. Therefore, the selenium requirement for foetal production is higher than the above mentioned theoretica dayr pe .g l^. 0 valu2 f o e Table 4 gives a survey of the daily deposition of various elements in foetuses at different stages of gestation. The figure nitrogenr sfo , calcium, phosphoru d iro san calculates ni d froresulte th m s publishe Moustgaary db d zinr (1959fo c d )an from those of Palludan & Wegger (1972). Although the daily de- minerale positioth l al s f nstudieo d increases steeply during the last trimester a characteristic difference exists between the macro elements calcium and phosphorus on one side and the trace elements on the other. From day 80 to 115 of pregnancy the deposition of calcium and phosphorus is increased approximately twentyfold while that of the trace elements is only enhanced by a factor 4-8. This difference is due to the fact that most of the calcium and phosphorus in the organism is found in the skeleton the ossification of which does not become significant unti e laslth t trimester e tracTh . e element e otheth n ro s

~~172 hand are incorporated in compounds of direct physiological and biochemical importance such as enzymes in all tissues. Many of these substances are presumably needed early in foetal life.~~

Glutathione peroxidase and health. As already touched on in the preceding section trace elements exert their biological function primarily as members of enzyme systems. Selenium is an in- tegral part of the enzyme glutathione peroxidase (GSH-Px) which participates in the mechanisms protecting cells against damage due to peroxides. During the phagocytic action of macrophages d leucocytean s considerable amount hydrogef so n peroxid generatee ear d in these cells, which normally have high GSH-Px activity (Serfass & Ganther 1976). Since GSH-Px activit mann i y y tissues closely reflect seleniue th s m statun animala f so mighe ,on t expec- se t lenium deficienc caus. to y e decreased resistanc infectionso t e . Such an effect was found in rats where macrophages isolated from selenium deficient animal d onlsha y GSH-Pe 5-1 th cenr f 0pe xo t activity found in cells from control rats, and their microbici- dal activity was lower than that of normal macrophages (Serfass . 1974)eal t . Similar results were reporte leucocyter fo d s from selenium deficient steers (Boyne & Arthur 1978). Selenium supplementation of selenium-vitamin E deficient pigs has also been found to prolong the incubation period in experimental swine dysentery 1978). (Teigal t e . A high incidenc infectiouf o e s diseases suc pneumonis ha d aan majoe th rf diarrhoeo problem e productiog on pi s an i i s n today. Besides seleniue th , m statu Danisf so h Landrace pigs varies considerably (Jargense . 1977al t ne , Wegge. 1979)al t e r. These facts together with the results cited above lead us to investigate the incidence of disease in swine in relation to their selenium status (J0rgensen & Wegger 1979)• The study comprise pig1 Danis69 f dso h Landrace animale Th . s were reare progena t a d y testing statio- n bo fro f o m g abouk 2 2 t dy weight until slaughter at a body weight of 90 kg, thus feeding and environmental conditions were identical for all pigs. Glu- tathione peroxidase activit erythrocyten i y measures - swa de s a d scribe y J0rgenseb d . (1977al t ne ) whe e pige d beeth nth s ha n no station for 3 weeks. Morbidity and diagnosis were recorded for each statione individuath e resultt a Th .g lpi s were groupen i d classes according to GSH-Px activity, the class width being 24 haemoglobinf o g u/ . Within each clas percentage sth pigf o e s affecte y diseasdy b tim an et a edurin growte gth h perios wa d calculated. The result e showsar n figurni « Diarrhoe3 e d pneumoniaan a were the predominant diagnosis in all groups, but as seen from the figure, pigs wit higha h leve GSH-Pf o l x activity, i.e.a high selenium status, were less susceptibl diseaso t e e than those

173 witGSH-Pxw hlo tere Th .m "Other diseases" comprise widsa - eva riety of disorders each occurring with a low and inconsistent frequency not related to GSH-Px classes. The correlation between GSH-Px activity and total percentage of pigs with disease remarks was highly significant (P < 0.001). It should be underlined that the animals were fed a diet considered selenium-vitami nadequatE clinicao n d an e l symptomf so selenium deficiency were seen. Thus the results indicate that individual differences in selenium requirement exist in pigs. Taking into accoun standardizee th t d managemen animale th f o ts the most likely explanatio existence th s ni genetif o e c variation between pig thein si r ability eithe absoro rt b selenium- in o ,t corporate this elemen GSH-P n i synthetizto t r xo apoenzymee th e .

~~These differences may result in marginal selenium deficiency in some pigs, a condition which lowers the animal's resistance to infections.~~

. E Genetic aspect tracf o s e element metabolism During recent years it has become evident that mineral meta- d animalbolisan certaia d n statuo an msma t n i sn - extenge s i t netically controlled which presumably implies inherited differences in requirement among animals. A very convincing example of the existence of such variations is Scottish investigations e susceptibilitth n o f differeno y t sheep breed o coppest - rde ficiency and intoxication (Wiener et al. 1978). The cause of the variations was shown to be differences in the animals' abilit absoro t y b copper froe intestinemth . Inherited difference tracn si e element status may, however, also occur within the-same breed. For example genetic variation in plasma copper concentration was found between groups of halfsib calves of the Danish cattle breeds EDM and SDM (Wegger & Larsen 1978) reare progena t a d y testing station variatioe Th . n in plasma copper concentration between halfsib group s sigswa - nificantly higher than between related animals in both breeds (P < 0.001). Plasma copper concentration was correlated not only with ceruloplasmin activity (Todd 1970 alst )bu o witn a h animal's phenotype regarding this polymorph protein (Wegger& Larsen 1978) Danisn I . h cattle three electrophoretically detectable ceruloplasmin phenotypes are known AA, AC and CC. As can be seen from figures 4 and 5, the AA phenotype was associated not only with the highest ceruloplasmin oxidase activity but also with the highest plasma copper concentration. The CC phenotype possesse lowese th d t enzyme activit d coppean y r concentration C typwhilA s intermediary e wa e th e . Whetherf o thi s i s y biochemicaan practicar o l l importanc r coppefo e r metabolism remains unknown.

174 Pigs of Danish Landrace all possess the same ceruloplasmin phenotype. However spitn ,i thif o e s their plasma copper concentration is partly genetically determined since the variation between litters is significantly greater than within litters even when the animals are reared in the same environment and fed the same die e tsam Th (table applie. 5) e plasmo st a iron concentration and total iron binding capacity. Also the selenium statu pigf so determines sa GSH-Py b d x activit erythrocyten i y s (see below) is subject to genetic control. The practical im- plication thesf t fullo sno eyt ye findingunderstood s a e sar .

C. Methods for evaluation of trace element status of domestic animals under practical conditions. Estimatio nutritionae th f no l statu individuaf so l animalr o s local herds regarding essential nutrients such as trace elements may serve as well theoretical experimental as practical econo- mi clatte e purposeth n rI cas methode s. th e s applied should possibls a r afa s e fulfil followine lth g claims ) sampl(a :- ema terial should be easy to obtain, (b) the analyses should be as simple as possible and not require too specialized equipment, ) sample(c s shoul storable db r sendinfo e regionao gt l laboratories, (d) last but not least the results should be independent of factors others than the mineral in question. For many years analyses of mineral content in feedstuffs were use ensuro t d e thae requirementh t animalf o t r tracsfo e element mets swa . Such determinations are, however limif o , - ted value for the diagnosis of marginal mineral deficiencies wher o clinicaen l symptoms occur mucA . h more relevant approach to the problem is determination of mineral concentration in samples taken from the feed consumers. When deciding which tissue analyser fo s importani e tus ot s i realizo t s a r e fa tha s a t trace elements are concerned some tissues serve as depots (i.e. liver) and some as transport media (blood, milk) while others have specific trace element dependent functions (gl. thyreoidea, bone marrow). Hair is a readily available sample material from most farm animals even under field conditions d severa,an l experiments have been concerne tracf o e ed us elemenwit e hth t concentration n indicato haia n i s ra minerar rfo l statu animaln f so ma d san (Lewis et al. 1957, Miller et al. 1965, Klevay 1970). However, since an element once incorporated in hair cannot be reutilized organisme bth y , hair mineral concentration doet reflecsno t the actual nutritional and metabolic status of an animal, but rather the status 1-2 months ago when the sampled hair was grown (Kristianse . 1971al t ,ne Mille. 1965)al t e r. Never- theless, analyses of hair samples should not be completely a-

175 bandoned as a tool for investigating trace element status, especially in geographic areas where long lasting exposure to "mar- ginally deficient" crops is suspected. Under such, conditions the analyses of hair might be of diagnostic value. The analysi bloof so plasmd - diagnostidan a de s a n i d ai c tecting mineral deficiencies has been and st^ll is widely used. It should, however, be kept in mind that the trace element content in plasma is the result of a continuous exchange between labile tissue pool d plasmasan mobilizatioe Th . tracf no e elements from storage organ initiae sth suc n i lives lha y starma - ge mas marginaka l deficiency sinc wilt i e l keeplasme th p a concentratio normaa n no l certaia leve r fo l n perio timef o d . Besi- des, normal physiological conditio y causnma e fluctuatione th n i s trace element conten plasmf o t primarilt ano y relate minerao t d l intake. Well known examplechangee th e plasmn i sar s a zind an c copper concentrations during pregnancy (Palluda Weggen& r 1976). Finally bees ha nt ,i show humann ni s that infectious diseases are accompanie y significantldb y decreased zind increasean c d copper concentration plasmn i s a (Sandstea . 1976)al t e .d Whe- ther similar changes occur in farm animals is not known. In experimental studies on trace element status several isotope techniques have provee verb o yt dvaluabl e tools. These include measuremen intestinaf o t l absorption, endogenous excretion, whole body retention, plasma clearence rate etc. However, all these methods imply administration of isotopes to the animals case whicf radioactivth o e n i h e isotopes render e anisth - mal and its products such as egg and milk unfit for human consumption. Furthermore ,considerabla e amoun radioactivf o t e costlmethodo e to th e disposed b e d an yo sar an t wast f s o d eha laboriou usee b larg n o o dt s e number animalsf o s e isotopOn . e technique vitrn nameli e o th yuptak tracf o e e element y erysb - throcytes usefue b y l,ma also under practical conditions. It has been known for several years that erythrocytes can accumulate zinc in vitro as well as in vivo and Berry et al. 1966 have suggested to use the in vitro uptake of zinc-65 as measura zinf o e c status. This possibilit s testewa y conn i d - nection with experimental zinc deficiency in pigs. The results from such an experiment are shown in table 6. Erythrocytes from one normal and four zinc deficient pigs were incubated with zinc-6 zins 5a c chlorid firs e shows th A t 37° a etn . i n three columntable th ef so cells frodeficiene th m t animals takp eu significantly more zinc-65 than those from the control when the incubatio performes ni pig'e plasman th ow sn however, i d If . , cells from both deficient and normal pigs are incubated in plas- wita m normaha l zinc conten differenco n t isotopn i e e uptake is observed, as shown in the last column of table 6. In accor- dance with this Cheste Wilr& l (1977) found thazinc-6e th t 5

~~176 Table 6. In vitro uptake of Zn in erythrocytes from four zinc deficient and one normal pig in relation to plasma zinc concentration and incubation time.~~

Plasma Per cent Zn in erythrocytes Pig no. zinc pg % 15 min. 30 min. min0 6 . 60 min.* 535 112 3.5 7.4 12.1 11.7 531 40 7.9 15.6 28.3 - 532 44 8.2 15.2 25.5 9-5 533 48 8.7 16.3 29.1 10.6 534 30 7.5 14.7 27.3 9.6 x: Erythrocytes from all pigs incubated in a standard plasm. a Zn containin% g ^ 2 g11

Table 7. In vitro uptake of 75Se in erythrocy- s incubatete d with labelled seleno-methionine after storage for various periods of time at 4, 20 or 37°C. The uptake is expressed in per cent of uptake on day zero.

~~Storage Lengt f storageho , days temperature 0 1 2 5 4°C 100 75 77 88 20°C 100 72 74 88 37°C 100 68 73 168~~

~~Tabl . 8 eStabilit f erythrocytyo e G-SH-Px activity (U/g haemoglobin) under various conditions of storage. Zero value: average of 9 determinations + S.D.~~

~~„ Length of storage, days Pre treatment Sure ————————————————————————————— Q rature123 4~~

~~Erythrocytes washed - 20°C 161 174 164 169. twice in saline + 4°C 148 162 156 153 6 1 + 8 16 Untreated + 4°C 160 159 162 150 blood sample + 20°C 161 157 159 162~~

~~177 BK. ssa.~~

~~AC AA AC CC AC AA AC~~

Ctnjloplasmin typ« Ctruloplocmin typ« Figure 4.._ Average ceruloplasmi- nac -Figur . Averag5 e e total copper tivit n relatioi y ceruloplasmio nt n concentration in plasma in rela- phenotype o cattltw n si e breeds, ver- tio o ceruloplasmit n n phenotypes. tical lines show s.e e figure.Th s r detailFo legene se s figuro t d e show number of calves. 4.

~~25- 30 • '^ Se - seleno - methionine o 75 Se - selenite 25 20-~~

~~f, '5~~

~~I u »~~

1 12345 Hours Hours re 6. In vitro uptake of radio- vitrn FigurI o. 7 euptak f o e selenium by porcine erythrocytes in- radio seleniu porciny b m e erythro- cubated with either labelled selenite cytes incubated with labelled or seleno-methionin 37°Ct a e . eeleno-methionine at various temperatures.

178 uptak s inverslewa y proportiona plasme th o alt zinc concentration. One could therefore speculate if the degree of Zn up- other o y r wa depend e n "isotopa on tak n n so i e e dilution" occur- labiln i g ein zinc plasmae poolth shoult f sI o . d als mene ob - tioned that considerable species differences exist, the zinc-65 uptake by bovine erythrocytes for example being only half as high as that by porcine cells. These differences seem to be related to -properties of the erythrocytes rather than plasma. The same technique has been used to elucidate selenium status of ewes and pregnant sows (Wright & Bell 1963, Wilkinson et al. 1977). In both cases the isotope was added to the blood as selenium-75-selenite. Some experiments with blood from normal pigs were carried out to stud e influencth y variouf o e uptake S s factore th n so 75 (cf. Moustgaard 1977) mentiones A . d before seleno-methionine is utilized more efficiently than selenite when given orally to animals. To elucidate whether the same applies to the in vitro uptak y erythrocyteeb millilitee son r aliquot blooa f o sd sample were incubated with either 75-seleno-methionine or 75-sele- appeart nitei s A .s from figur significantle6 e y th mor f o e isotope was accumulated by the cells from the amino acid than froinorganie th m c compound. These results indicate thae th t sensitivit e increaseb e tesy th ma t f y replacino yb d g selenite with seleno-methionine. It can be mentioned that the results with selenite shown here agree with the earlier experiments on sows and also with the results obtained with ewe Wrighy sb tBel& l (1963) after one hour of incubation. In contrast to the present results, however, these authors found increasing uptake of 75Se for as long as 4 hours. The reason for this difference could be the differenc incubatioe th n ei n methods used r incubationOu . s were performe normaa n i d l atmosphere which- containni ° i 9 s7 trogen while Wright and Bell used a mixture of 95 % oxygen and carbo° i 5 n dioxid d alsan e o demonstrated that nitrogen inhibi- ted the uptake. The accumulation of 75Se from seleno-methionine seems less dependen oxygen o t n since s show,a figurn ni e 7, the red cells continue to take up the isotope at least for 5 hours. It also emerge from figur , thauptake 7 th t highls i e y temperature dependent which indicate that it is an active process and not merely a result of passive diffusion. For both zinc and selenium the majority of the isotope accumulated by the erythrocyte transferres i s cele th l o t lumed d onlnan y very little adsorbed to or bound on the cell membrane. This is il- lustrated by figure 8.

~~179 30 • Erythrocytes o -Ghosts"~~

~~Hours~~

Figure 8. In vitro uptake of radioselenium by erythrocytes and "ghosts" from labelled seleno-methionin t 37°Ca e . After incubatio e cellnth s were washed twice wit huptake salinth d ean e measured. The cells were the haemolysed, the membranes isola- tey centrifugatiob d e bounth dd nactivitan y estimated.

~~005-~~

~~004-~~

~~003-~~

~~E 002~~

~~001- r=0 559 P~~

~~50 too 150 GSH-Px activity in trythrocytts. U/g Hb Figur,._. 9 e. The relationship between erythrocyte GSH-Px activity and plasma selenium concentratio Egyptian i n n water buffaloes (33)= n .~~

180 Finall influence yth uptake S s stu storagf ewa e o e -th n o e 75 died, as shown in table 7. Blood samples were stored at 4, 20 and 37 for up to 5 days before the selenium uptake test was performed. Although the erythrocytes lost some of their abilit accumulato t y e selenium usable results could - stilob e lb tained after two days storage at any of the temperatures applied. When kept for longer time especially at the high temperature the uptake rate increased considerably, probably due to diffusio e isotopth f no e into mor lesr o e s damaged cells. Results obtained by means of the methods mentioned so far n easilca e impaireyb y contaminatiodb - samplee ex th f d no san tensive precautions must be taken to avoid this. It might, therefore, ofteadvantagn a e determinatioe nb us o et tracf no e element dependent enzyme asseso st e statusth animalf o s - sre garding these minerals. Moreover, this might give a more correct and sensitive measure since it is mainly through the action of such enzymes the trace elements exert their essential biological functions. In the choice of suitable enzymes it must be taken into consideration that not all enzymes are equally affected by deficiency of the element on which their activity depends. This can either be due to localization of the enzymes in tissues not equally affected by the deficiency or to differences in affinity towards the trace element (cf. Kirchgessne . 1976)al t re . Useful enzymes in this context are for example alkaline phosphatase and ceruloplasmin as indicators for zinc and copper status respectively (Agergaar Palludad& n 1974, Todd 1971, Wegger Larse& n 1978 glutathiod )an n peroxidas sensitiva s a e e marker for selenium statu varioun si s animal species (Hafema. al t ne 1974, Omaye & Tappel 1974, Alien et al. 1975, Sivertsen et al. 1977) . In the lat'ter case it has been discussed whether whole blood, erythrocytes or plasma should be used for analysis (cf. J0rgense 1977. erythrocyteal f o t ). ne Againse us e r th t (o s whole blood) speaks the fact that the selenium content and hence the GSH-Px activity of these cells does not reflect the immediate selenium status of the animal but rather the situation some months ago. Plasma selenium content on the other hand is known to respond very rapidly to changes in selenium intake (Thomp- . 1980)al sot ne . certaia Eve , nso n relationship exist- sbe tween GSH-Px activity in erythrocytes and selenium concentration in plasma as seen in figure 9 (Gazia & Wegger 1980). How- ever correlatioe th , n coefficien considerabls i t y lower than those reporte wholr fo d e blood seleniu d GSH-Pan m x (Arthut re al. 1979, Schol Hutchinsoz& n 1979). For screening purposes the GSH-Px activity determination can be simplified by using the socalled "spot test" (Board & Peter 1976). In figure 10 is shown the results of a comparison be-

~~181 03~~

~~I 0.2~~

~~0 1-~~

~~12345~~

~~Tim < d*fluoresc»ncO * ) m (m « Figure 10. Relationship between glutathione peroxidase activity in porcine erythrocytes as measured by a spectrophotometric method e spoth t d testan , respectively.~~

~~to f S. 30 S 20~~

~~01 02 03 05 Selenium, pg/g relationshie Th Figur . e11 p between selenium concentratiod nan GSH-Px activit porcinn i y e live =50n ( r 0.8?= , r < 0.001) ,P .~~

182 tween the quantitative spectrophotometric method and the spot test (J0rgense . 1977) al usefulnese t Th ne . spoe th t f so tes t under field condition s recentlsha y been studied with cattle and sheep (Backall & Scholz 1979, Berrett & Hebert 1979). A prerequisite for using enzymes as indicators of trace element statu n screeninsi g studie thas enzyme i s th t question i e n is stable enough to allow transport of the samples over a certain distance preferably without cooling or other precautions. The stability of porcine erythrocyte GSH-Px was investigated under various conditions A heparinise. d blood- sampldi s wa e vided into subsamples the half of which was left untreated at othee th r, C hal 0 s eentrifuged2 eithef wa r o 4 r erythroe ,th - cytes washed twice with saline and stored at 4 or -20°C. As show tabln significano ni n 8 e t los GSH-Pn i s x activits wa y seen even after storag r foufo er day t 20°Csa . Marginal mineral deficiencies often affect whole herdd san mann i y cases occu geographicalln i r y limited areas. Methods suitable to point out such localities are, therefore, of prac- 'tical interest. One possibility could be to analyse samples of animal tissues collected at local slaughterhouses. The liver contain mose sth t labile poo manf o l y trace elements, hence it seems relevant to choose this organ for the purpose. Such a screening experimen s conductewa t evaluato t d zince eth , copper, iron and selenium status of Danish pigs. A further purpose was to examine the possibility for using liver GSH-Px activity as indicator for the selenium content of the organ (Wegger & Ergiin 1979, Wegger et al. 1979). Liver samples from 50 pigs randomly selected on each of four slaughterhouses representing differen countre t th par f o ty were analysed samplee th n I s. froe slaughterhouson m e both selenium concentration and GSH-Px activity were determined. As shown in figure 11 a highly significant linear correlation (r = 0.87, 0.001< P founs )wa d parameterbetweeo tw GSH-Pe e th nth d x san activity was therefore used as a measure for selenium content remainine inth 0 liveg15 r samples. Tabl esummarize9 resulte sth s obtained. Onlyn i smal t lbu some cases significant difference zincn si , iro d seleniunan m status were found betwee varioue nth s regions, wherea cope sth - per statu s simila l fouswa al r n country e ri part th f e so Th . rather uniform picturt surprisingno s i e , whe widespreae nth d use in Denmark of standardized mineral mixtures and industrially processed feedstuff s consideredi s e existencTh . geof o e - graphic differences although small in mineral status of farm animals even under Danish conditions, however, indicates the potential usefulnes sucf so h screening method countrien i s s with more varied geographic and climatic conditions. It can

~~183 50 100 ISO Zinc jig/g~~

~~) OX 1030507 0 30 0 20 100 0 x unitP H g */ OS Iron jig/g~~

~~Figur . Frequenc12 e y distributio 0 swin20 ef o n liver samples regarding zinc, coppe d iroan r n concentrations and GSH-Px activity.~~

be mentioned that screening method variouf o s s kind usee sar d in some South American countries where mineral imbalances are prominent problems in animal husbandry (Kiatoko et al. 1978, Sousa 1978). Furthermore, similar studies have been carried out in England, Scotland and the Netherlands in order to lo- calize areas where marginal selenium deficiencies occurred in cattle and sheep either by determining GSH-Px in blood (An- derson & Patterson 1977, Arthur et al. 1979) or selenium in milk (Binnerts 1977). The overall frequency distributio zincf no , coppe d iroran n concentratio d GSH-Pnan 0 xlive 20 activit e r th sample n i y s si shown in figure 12. As it appears the range of values is very wide for all four parameters. Whether this is due to difference tracn si e element intake alone canno deducee tb d from the present results seemt i t s ,bu unlikely . Several investigations have shown individual differences among pigs in their abilit accumulato t y e livee coppeth rn i r (Caste. 1975al t ,e l Meyer et al. 1976, Fr0slie & Norheim 1977). Similar variations have been foun n sheei d p (Suttle 1974) whicn ,i h species

184 also breed differences in copper retention are known to exist (Wiener et al. 1978, Herbert et al. 1978). Furthermore, earlier investigations indicated great individual variation among pigs in their susceptibility to zinc deficiency (Wegger & Pal- ludan 1977). Taken together and viewed in the light of the genetic variations in trace element content of blood discussed above these results strongly indicat hereditarea y influence on trace element metabolism and status.

~~References~~

AGERGAARD, N. & B. PALLUDAN, Ann. Rep. Steril. Res. Inst., Co- penhagen, 17 (1974) 47. ALLEN, W.M., W.H. PARR, P.H. ANDERSON, S. BERRETT, R. BRADLEY & D.S. PATTERSON, Vet. Res. 96. (1975) 360. ANDERSON, P.H. & D.S.P. PATTERSON, Proc. 3rd Int. Conf. Prod. Dis. Farm Animals, Wageningen 1977, p. 129. ARTHUR, J.R. PRIC. J , C.FE& . MILLS, Vet. Rec 4 (197910 . ) 340. BACKALL, K.A. & R.W. SCHOLZ, Amer. J. vet. Res. .40 (1979) 733- BERRETT C.N& . ,S . HEBERT, Vet. Rec 5 (197910 . ) 145. BERRY, R.K., M.C. BEL P.LL& . WRIGHT Nutr. ,J (19668 .8 ) 284. BINNERTS, W.T., Proc. 3rd Int. Conf. Prod. Dis. Farm Animals, Wageningen 1977, p. 131. BOARD, P.G. & D.W. PETER, Vet. Rec. 99 (1976) 144. BOYNE, R. & J.R. ARTHUR, J. Comp. Pathol. 89 (1979) 151. BUESCHER, R.G., M.C. BEL R.KL& . BERRY Anim. ,J . Sci 0 (19612 . ) 368. CANTOR, A.H., M.L. LANGEVIN NOGUCH. ,T M.LI& . SCOTT Nutr. ,J . 105 (1975) 106. GARY, E.E., W.H. ALLAWA MILLER. M Y& Anim. ,J . Sci (19736 .jl ) 285. CASTELL, A.G., R.D. ALLEN, R.M. BEAMES, J.M. BELL, R. BELZILE, J.P. BOWLAND, J.I. ELLIOT, M. IHNAT, E. LARMOND, T.M. MALLARD, D.T. SPURR, S.C. STOTHERS, S.B. WILTON & L.G. YOUNG, Can. J. Anim. Sci . (197555 . ) 113. CHESTER, J.K. & M. WILL, In: Trace Element Metabolism in Man and Animals 3, Ed. M. Kirchgessner, Munchen 1977, p. 211. EHLIG, C.F., D.E. HOGUE, W.H. ALLAWAY & D.J. HAMM, J. Nutr. 9_2 (196?) 121. FR0SLIE NORHEIM. G & . ,A , Acta vet. scand 8 (1977.1 ) 471. GALLO-TORRES, H.E., ActaAgric. Scand. suppl. 19 (1973) 97- GAZIA, N. & I. WEGGER, Acta vet. scand. 21 (1980) 137. GROCE, A.W., E.R. MILLER, D.E. ULLREY, P.K K.K, .KU . KEAHEY& D.J. ELLIS Anim. ,J . Sci 1 (1973.2 ) 948.

~~185 HAFEMAN, D.G., R.A. SUNDE& W.G. HOEKSTRA, J. Nutr. 104 (1974) 580.~~

HERBERT, J.G. WIENE. ,G A.CR& . FIELD, Anim. Prod (19786 .2 ) 193. JENKINS, K.J. & M. HIDIROGLOU, Can. J. Anim. Sci. 51 (1971) 389. J0RGENSEN, P.F. HYLDGAARD-JENSE. ,J MOUSTGAARD. J N& , Acta vet. scand. 18 (1977) 323. J0RGENSEN, P.P WEGGER. I .& , Acta vet. scand (19790 .2 ) 610. KIATOKO L.R, ,M. . McDOWELL, K.R. PICK FONSECA. ,H CAMACHO. ,J , J.K. LOOSLI & J.H. CONRAD, J. Dairy Sci. 61 (1978) 324. KIRCHGESSNER H.P, ,M. . WEIGANDROT. E H& Trac: ,In e Elementn i s Human Health and Disease. Voll. Ed. A.S. Prasad, Acad. Press, New York 1976, p. 189. KLEVAY, L.M., Amer. J. Clin. Nutr. _23_ (1970) 284. KLEVAY, L.M., Pharmac. Ther I (1976 .A ) 211. KOSLA, T., B. PALLUDAN & I. WEGGER, Ann. Rep. Steril. Res. Inst., Copenhagen (19803 ,2 . )26 KRISTIANSEN, P.H., I.H. PEDERSE WEGGER. I N& , Ann. Rep. Steril. Res. Inst., Copenhagen 4 (1971,1 ) 111. KU, P.K., W.T. ELY, A.W. GROCE & D.E. ULLREY, J. Anim. Sci. 3_4 (1972) 208. LEWIS, P.K., W.G. HOEKSTR R.HA& . GRUMMER Anim. ,J . Sci.6 1 (1957) 578. MEYER, H., H. KROGER & K. von BENTEN, Dtsch. Tierarztl. Wschr. 83. (1976) 401. MILLER, W.J., G.W. POWEL, W.J. Pitt H.Fs& . PERKINS Dair. ,J y Sci. 48 (1965) 1091. MOUSTGAARD, J., In: Reproduction in Domestic Animals. Eds. H.H. Cole & P.T. Cupps. Acad. Press, New York 1959, p. 169. MOUSTGAARD Ann, ,J. . Rep. Steril. Res. Inst., Copenhagen0 ,_2 (1977) 157. NIELSEN, H.E. O.K& . RASMUSSEN, Acta Agric. Scand. suppl1 2 . (1979) 246. OLSON, O.E., J. Ass. Off. Anal. Chem. 5£ (1969) 617. OLSON, O.E., E.J. NOVACEK, E.J. WHITEHEAD & J.J. PALMER, Phyto- chem. 9 (1970) 1181. OMAYE, S.T. & A.L. TAPPEL, J. Nutr. 104 (1974) 747. ORSTADIUS ABERG. B & ,. ,K Act a vet. scand 2 (1961. . )60 PALLUDAN, B. & I. WEGGER, Ann. Rep. Steril. Res. Inst., Copen- hagen, 15 (1972) 27. PALLUDAN, B. & I. WEGGER, In: Nuclear Techniques in Animal Pro- ductio Healthd nan . Proc. Series, IAEA, Vienna 1976 191. ,p . SANDSTEAD, H.H., K.P. VO-KHACTU & N. SOLOMONS, In: Trace Ele- ments in Human Health and Disease. Vol. I. Ed. A.S. Prasad, Acad. Press, New York 1976, p. 33.

186 SCHOLZ,R.W. & L.J. HUTCHINSON, Amer.J.vet. Res. 40 (1979) 245. SERFASS, R.E. & H.Eo GANTHER, Life Sci. 19 (1976) 1139. SERFASS, R.E., R.D. HINSDIL LH.E& . GANTHER, Fed. Proc3 3 . (1974) 694. SIVERTSEN J.T, ,T. . KARLSE FR0SLIE. A N& , Acta vet. scand3 Ij . (1977) 494. SOUSA, J.C. De, Interrelationships Among Mineral Levels in Soil, Forag d Animaan e l Tissue Ranchen o s Northern i s n Mato Grosso, Brazil. Thesis, Universit Floridaf o y , 1978. SUTTLE, N.F., Br. J. Nutr. _3_2 (1974) 395. TEIGESAXEGAAR. P , FR0SLIE,. J. A D& , Acta vet. scand 9 (1978.1 ) 133. THOMPSON, K.G., A.J. FRASER, B.M. HARROP & J.A. KIRK, Res. Vet. Sci 8 (19802 . ) 321. THOMSON, C.D R.D.H.& . STEWART Nutr. J 0 (1973. .^ ,Br ) 139. TODD, J.R. Trac: In , e Element Metabolis Animalsn i m C.F. .Ed . Livingstone. MillsS & . ,E , Edinburgh 1970 448. ,p . TODD, J., In: Mineral Studies with Isotopes in Domestic Animals. Panel Proc. Series, IAEA Vienna 1971 159. ,p . WEGGER, I. & I.D. CHRISTENSEN, unpublished results 1979. WEGGER, I. & A. ERGUN, Ann. Rep. Steril. Res. Inst., Copenhagen _22 (1979) 135. WEGGER, I. & B. LARSEN, Ann. Rsp. Steril. Res. Inst., Copenhagen, 21 (1978. )1 PALLUDANWEGGER. B & . Trac: I , ,In e Element Metabolisn Ma n i m and Animals 3. Ed. M. Kirchgessner, Munchen 1977, p. 428. WEGGER, I., K. RASMUSSEN & P.P. J0RGENSEN, Ann. Rep. Steril. Res. Inst., Copenhagen, 22 (1979) 127. WEGGER S.D, ,I. . TUNCER, P.P. J0RGENSE RASMUSSEN. K N& , Ann. Rep. Steril. Res. Inst., Copenhagen, 21 (1978) 57.

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~~187 CONCLUSION RECOMMENDATIOND SAN S~~

~~Conclusions~~

(1) The co-ordinated research programme on the use of isotopes to detect moderate mineral imbalances in farm animals advanced the research of contract holders throug e provisioth h f fundo n r suppliefo s d equipmenan s t and provided contact with scientist f similao s r interest coulo wh sd hele th p contract holders attain their research objectives.

(2) The programme resulted in greater government interest in the mineral status of farm animals in many of the contract and agreement holders' countries and was responsible for stimulating government and other support for research. The programme and the subsequent stimulation of research will result in increased food production in these and other countries. e beginninth e t programmA th f o g e ) objectivth (3 e f developino e g diagnostic procedure o detect s t moderate mineral imbalances seemee b o t d attainabl e programme onseth th f o t tt a e upo bu e n investigation turnet ou d a ver e b y compleo t x undertaking. Furthermore, this objectiv taky a ema e great dea f efforo l o attait t r particulafo n r trace elements n hindsightI . , e programmth overls wa e y optimisti o broato n scopei d an c .

(4) The concept of the co-ordinated research programme itself was endorsed by the group. Some shortcomings were noted, however, particularly in relation to understanding the needs of contract holders, some insufficienc f communicatioo y n between contract holders, agreement holders and the Agency, and some loss of the programme objectives with time.

(5) Problems existing in developing countries which need to be faced and overcome by contract holders ara not always fully recognized and may resul n excessivi t e optimism regarding potentia r progresfo l n researchi s . Perhaps review of contract proposals by an expert who has previously visited a facility could provide better insight int e goaloth whethet s no r o r propose! are realistic.

(6) The duration of contracts was felt to be not long enough as it frequently o thret take o etw s year o develot s a programmp a poin o t e t where meaniningful results can be obtained. Contracts should be in effect for at least five years.

~~(7) Whil o diagnostin e c procedure achieve e statuch i f beino s g immediately available for field studies, some procedures were felt to be~~

189 nearly read r testinfo y g their validit d applicatioan y n preliminari n y field trials. These procedure celd o statussre C l includee r S fo , dB uptake for Se status, ferritin.RIA for Fe status and hair content for As status. programme Th ) e (8 also resulte e recognitioth n i d n that non-isotopic techniques, e.g. serum mineral analyse enzymr so e assays occasion o y ,ma e b n more appropriate than isotopic techniques for diagnosis of particular mineral imbalances. e programm Th t progres no poine d th f developin ) o di eto st (9 a g protocol for diagnosing moderate mineral imbalances.

~~A seriou(10) s problem existe n somi d e labs with maintaining equipmen d obtaininan t e serviceth g f traineo s d personne maintaio t l n sophisticated counting equipment.~~

~~Recommendations~~

(1) The interaction of agreement holders and contract holders within a co-ordinated research programme should be promoted as much as possible to ensure dissemination of new iueas, suggestions, protocols and new techniques s quickla s possibla y e scientistth o t e hol o dwh s contracts. This should include agreement holders assisting in the review of progress reports before submissio e Agencyth o t n .

f substantiaO (2) l importanc e continuitth s i e f leadershio y e th f o p co-ordinated research programme. The Head of the Animal Production and Health Sectio a Senio r o n r Research Agreement Holder shoul e designateb d s a d e "Programmth e Co-ordinator e responsiblb o t " r maintaininfo e a grevie f wo each project including visits, where possible, to laboratories of contract holders. This position should be held by the appointee for the life of the co-ordinated research programme.

(3) In developing a programme there should be a mix of both agreement holder contracd an s t holders wherei e researcth n h intereste th f o s agreement holders must match those of the contract holders.

e researcTh (4) h co-ordination programme should hav a etime-spa n long enough to allow investigators to develop methods and obtain supplies before expecting significant results to be obtained from their efforts.

(5) Circulatio f progreso n s reports, research summaries, newsletters e distributio th s wela ,s a l f pertineno n t publications among members of the co-ordinated research programme would assist communication among members. Furthermore, papers to be presented at co-ordinated research meetings shoul distributee b d l participantal o t d se meeting prioth o t r .

190 e regulath t A r(6 meeting) e co-ordinateth f o s d research programme agreement holders should be asked to present review papers of direct relevanc e programme goalth th f o o st e e rather than original research data.

(7) Two years before the termination of a programme efforts should be mad o determint e e futurth e e direction f e i anylin, th f researcho ef ,o . This two-year lead time should allow for the planning and implementing of new programme(s e continuatioth d an ) f supporo n r worthfo t y projects.

(8) While the present programme is now ending, a mechanism should be sought to provide continued support for those contractors whose work within this programm s showha e n potentia r meaningfufo l l contribution withie th n yearnexw fe o increasintt s g food production.

(9) It is recommended that a future co-ordinated programme encompassin e broath g d aspect e metabolisth f o n elemensa f mo r element o t s be developed. Such a programme might centre on phosphorus or on the micro-elements selenium, cobalt and zinc. Alternatively, a programme should

~~be considered to investigate the use of stable isotopes or short-liveJ isotope o replact s e long-lived radioisotope n large-animai s l research.~~

~~(10) Consideration should be given to consolidation of information obtaineJ from this programme and from other sources to provide descriptive protocol f choiceo s r detectinfo s g mineral imbalances.~~

~~191 LIST OF PARTICIPANTS~~

~~. GoksoK . Dr y 1869/RB Ankara Nuclear Researcd han Training Center "In-vitro use of radio-minerals to Besevler determine mineral imbalance n fari s m Ankara, animals". TURKEY~~

Mrs. J.S. Edwardly 1930/RB Pasar Jumat Research Centre P.O.Box 2 "Study of Se deficiency and its dia- Kebayoran Lama gnosi poultrn si y farm Indonesian si ; Jakarta, Selatan e effecTh e f vitamiS o t d an nE INDONESIA deficienc n '^Syo e metabolisd an m pathological changes in chicks".

Dr. A. Lobao 1894/RB Centr e Energid o a Nucleaa n r Agricultura "Research within the framework of a Caixa Posta6 9 l f isotopeo coor e us d o e st prog th n o r 13.400 Piracicaba, S.P. diagnose moderate mineral imbalances BRAZIL farn i m animals".

~~. J.EDr . Kalinowski 1968/RB Universidad Nacional Agraria Department of Nutrition "Detection of mineral imbalances in Apartad6 45 o grazing livestock". La Molina Lima, PERU~~

~~Prof. T.H. Kamal 1487/RB Chairman, Radiobiology Department Atomic Energy Establishment "Heat-induced mineral imbalance in Cairo, ruminant s diagnosea s radioisoy b d - EGYPT tope turnover rates".~~

. GiacintoP . Dr v (Dr . BusA . ) 1948/RB Central State Veterinary Institute Me reov4 5 a "Utilization of radioisotopes in 612 38 Brno, veterinary medicine to diagnose CZECHOSLOVAKIA mineral imbalances".

. AnkM e. Dr 2079/CF Karl-Marx-Universitaet Leipzig Sektion Tierproduktiod nan "Research within the framework of a Veterinaermedizin f isotopeo coore us d o e st progth n o r Wissenschaftsberelch Tierer- diagnose moderate mineral imbalances naehrungschemie in farm animals". Dornburger Str4 .2 69 Jena, GERMAN DEMOCRATIC REPUBLIC

Dr. M. Peterlik 2169/CF Institut fuer allgemeine und ex- perlmentelle Pathologir de c "Investigation on the calcinogenic Universitaet Wien factors of Trisetum flavescens". Waehringerstrasse 13 A-1090 Vienna AUSTRIA 193 Dr. Walter Mertz 1856/CF Nutrition Institute Human Nutrition Center "Role of chromium in human and animal Beltsville, MD 20705 health". USA

Moustgaar. J . Dr d 1850/CF Dept. of Physiology, Endocrino- logBloodgroupind an y g "Research within the coord prog on Royal Veterinar Agriculturad an y l f isotopeo e thus e o diagnosst e University moderate imbalances in farm animals" Bulowsve, 13 j 1870 Copenhagen V, DENMARK

Dr. S. Economides 2085/RB Agricultural Research Institute Ministr Agriculturf o y & e "The use of isotopic techniques in Natural Resources studie mineraf so l imbalance dairn si y Nicosia, breed f sheep"so . CYPRUS

~~Prof. F.W. Lengemann 1849/CF Cornell University P.O.BoH xD "Use of radioisotopes to diagnose Ithaca, New York 14853 moderate trace element excess or USA deficiency".~~

Dr. M. Kirchgessner 1848/CF Institut fuer Ernaehrungsphysio- logie "Research withi e frameworth n a f ko Technische Universitaet Muenchen f isotopeo e coorus o e dst th pro n o g D-8050 Freiseing-Weihenstephan, diagnose moderate mineral imbalances FEDERAL REPUBLI F GERMANO C Y in farm animals".

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