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Grow th and Skeletal D evelopm ent

Growth and Skeletal Development in Great Dane PupsFed Different Levels of Protein Intake1RICHARD C. NAP,*2 HERM AN A. W . HAZEW INKEL* G EORG E VO ORHO uTjW ALTER E. VAN DEN BRO M * SINUS A. GO EDEG EB REJAND ARIE TH. VAN T KLO OSTERS"D ep artm en t o f C lin ic al S cien ce s o f C ompa nio n A nimals, ^D ep artm ent o f V ete rin ary R ad io log y,^D ep artm en t o f V ete rin ary P ath olo gy, a nd ^D ep artm en t o f H usb an dry a nd N utritio n,F aculty o f V ete rin ary Medic in e, Un iv ersity o f U tre ch t, T he N eth erla nd s

A BSTR AC T Feeding a dog of a large breed w ith a dietexce eding th e N ational R esearch C ouncil (1974) recommendat ions fo renergy , p rotein, ca lc ium, phosphorusand vitam in D m ay result in disturbances of skeletald ev elo pment. T he e ffe cts o f e xc es s e ne rg y and v ario usc alc ium rp ho sp ho ro us ra tio s p er s e h av e b een re po rte dby o thers. T he ro le o f dietary protein, especially w ithregard to calcium m etabolism and skeletal developm ent, in large breed-dogs is reported in this article.S even teen G reat D ane pups, 7 w k of age, w ere divid edinto three groups. D uring 18 w k each group receivedisoene rgetic dry food (~ 15 kJ m etabo lizable energy/g ) c on ta in in g 3 1.6 ,2 3.1 o r 1 4.6% p ro te in o n d ry m atte rbasis. N o differences w ere found am ong the high (H -P r), n orma l (N -P r) a nd low p ro te in (L -P r) g ro up s fo r th eheight at the shoulder. S ign ificant differen ces w erefo un d b etw een th e H -P r and L -P r g ro up s fo r b od y weig htand plasm a album in and am ong all three groups forplasm a urea. T he differe nces in protein intake per seh ad n o d emons tra ble c on sequ en ce s fo r c alc ium me tabolism and skeletal d evelopm en t. A causative role ford ie ta ry p ro te in in th e d ev elo pment o f o ste ocho nd ro sisin dogs is unlikely. J. N utr. 121: S 107-S113, 1991.INDEXINGKEY WORDS:symposium dog p ro te in cal ci ummetabo li sm growthIn young dogs of large breeds, disturbances in en-ch on dra l o ssifica tio n may le ad to sev ere alteratio ns inboth a rtic ula r a nd phy se al c artila ge , c lin ic ally k nownas o steo ch on drosis (OC) an d resu ltin g in sev ere lam en ess a nd sk ele ta l d efo rm itie s (1 ,2 ). D ie t c ompositio np lay s a n impo rta nt ro le in e nch on dra l o ssifica tio n. O fthe m any possible variables in the diet, attention hasthus far been focused on the influence of the total inta ke , e ne rg y and c alc ium -to -p ho spho rous ra tio .Hedhammer et al. (3) induced OC in Great Danepup s by fe ed in g exc ess e ne rg y, p ro te in , c alc ium , pho s-

0022- 3166 /91 $3. 00 1991 Amer ic an In stitu te o f Nu tri tio n.

phorous and vitam in D . Excess energy per se in a balanced diet did not cause an increased incidence ofsk eletal ab no rm alities (4 ). B y in crea sin g o nly th e c alcium content of the diet, H azew inkel et al. (5) foundinc re ase d o ccu rre nce an d sev erity o f OC in G rea t D an epups. R esults of follow -up studies w ith various calc ium an d p ho sp ho ro us in tak es d emon stra ted th at h ig hcalcium intake (independent of the ratio to phosphorous) is an im portant determ inant of disturbances inenchondral ossification (6). A nother im portant dietc omponen t, i.e ., p ro te in , h as not y et b een in ve stig ate das a single variable w ith regard to the skeletal developm ent in large breeds of dogs.T here are at least tw o reasons to pursue this m atter.F irst, from the m ulti-variable study of H edham mer etal. (3) it w as suggested that a high protein content inthe diet contributed to the developm ent of OC. Second, there is evidence from studies in hum ans and ratsthat protein excess influences calcium absorption,sk ele ta l m in era liz atio n and c alc ium exc re tio n (7 -1 4).There have been no studies on the protein requirem ents in grow ing dogs of large breeds (15).The present study w as prim arily designed to testthe hypothesis that high protein intake plays a causativ e ro le in th e p ath og en esis o f d istu rbe d en ch on dralo ssifica tio n. T he seco nd o bjec tiv e w as to inc re ase u nderstanding of protein requirem ents for grow th inla rg e b re ed s o f d og s. In th is re po rt, th e c lin ic al, ro utin elabo ra to ry , b iochemical, r ad iogr aphie and h is to logical1P re se nte d a s p an o f th e Wa lth am In te rn atio na l S ympo sium onNutr it ion of Sma ll Compan ion Anima ls , a t Un ive rs it y o f Ca li fo rn ia ,D avis, C A 95616, on Septem ber 4-8, 1990. G uest editors for thesy mp osiu m w ere Jam es G . M orris, D 'A nn C . F in ley an d Q uintonR . Roger s.2 T o whom c orre sp on de nc e s ho uld b e a dd re ss ed : D ep artm en t o fC lin ic al S cie nc es o f C ompa nio n Anim als , F ac ulty o f V ete rin aryMedic ine, Un ive rs it y o f U t recht , P .O . Box80 .154, 3508 TD Ut recht ,The Netherlands .

S107

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S108 NAP ET AL.results, as w ell as results of calcium kinetic studies,in G reat D ane pups fed different levels of protein w illbe presen ted .

MATERIALS AND M ETHODSAnimal s. Seven teen Great Dane dogs (11 males,6 females), 7 wk of age and originating from threelitte rs, were ra ndom ly d iv id ed in to th re e g ro up s: a h ig hprotein group (H-Pr; n = 6), a low protein group (L-Pr; n = 6) and a control group (N-Pr; n = 5). In allgroups the sexes w ere represented as equally as possible. At the end of the study, i.e., 27 wk of age, alld og s were k ille d fo r p ath olo gic al in ve stig atio n, b y u seo f an in trav eno us o verd ose o f so dium th io pen ta l.Housi ng. T he dogs wer e hou sed i n i ndi vi dual metabolism cages for 2 w k during each calcium kineticstudy (W 1+2, W 7+8, W 13+14, and W 19+20). Betw een these 2-w k periods the anim als w ere housed inin div id ual c ag es, h ad acc ess to a n ou tsid e ru n an d wereallowed free exercise once a week for 4 h.Diet and water . The dry diet was formulated tom eet the recom mendations of the U .S. N ational Research Council's Nutrient Requirements of Dogs(1974) (16). D uring the first 2 w k all dogs received theN -P r fo od w ith 2 3.1% p ro tein e xp re ssed o n d ry matte rbasis (% DM). From W 3 onward, dogs of the H-Prgroup received diet w ith 31.6% protein, and those ofthe L -P r g rou p 1 4.6% p ro te in (T ab le 1 ). T he th re e d ie tsw ere isoenergetic, w ith ~ 15 kj m etabolizable energy(M E) p er g D M. T his w as achieved by exchanging carbohydrate for protein in the H -Pr diet and the reversein the L-Pr diet, as com pared w ith the N -Pr group (Table I). The protein sources of the diets are given inT ab le 3. T he p r otein of th e d iet s u sed had a ly sin econtent of 6 g/100 g of protein, calculated from standard reference values. The relatively low content ofsu lp hu r-c on tain in g amin o ac id s in th e p ro tein -rich ingredients w as com pensated by the addition of m ethi-onine to the diet. L ysine w as added to the ingredientsof the L-Pr diet to maintain a lysine content of 6 g/1 00 g p ro te in .F or re stricte d fe ed in g, th e d aily amou nt offered , e xpressed as kj (M E) p er unit of m etabolic body w eight(kJ/BW75),w as decreased stepw ise from 1500 at W1-4 to 1200 at W 16-20. The nonconsumed food was

T A BL E 1Pr otein content of hi gh (H -Pr i, nor mal (N -Pr )an d l ow (L -Pr ) pr otei n di ets

Pro te in c on te nt ' H-Pr N-Pr L-Pr%DMg/1000kJMEPr:E r at io

31.619.50.2923.114.30.21

14.69.00.131 % DM ; percentage of dry matter; M E, metabolizable energy;

P r:E ratio, protein energy to total energy ratio.

T A BL E 2Proximate (Weende) ana lysi s o f h igh (H-Pr ),normal ( f f-Pr )and l ow L -Pr !r otei n f ood

H-Pr N-Pr L-PrCruderotein1Crudefat1Crudefiber1N-freeextract1Ash1Moisture1Ca1P2Vitamins3309.72.841.46.110109219.93.049.56.

'g/lOOg.3 E ach kg of diet contained 5000 IU of retinyl acetate, 1000 IUo f ch olec alcifero l a nd 50 IU o f d l-a-to co phe ry l ac etate a s g uara nte edb y t he p ro du ce r.

w eighed, and food intake w as calculated. The proxim ate com position (W eende analysis) of the diet w asdeterm ined in triplicate at the beginning and at theend of the experim ent (Table 2). D ogs had free accessto d rink ing water.Physi cal ex am inati on . T he dogs wer e obser vedtw ice daily at feeding tim e. T he height at the shoulderwas m easured in all dogs once w eekly. Body w eightw as reco rd ed th re e tim es w eek ly a t reg ula r in terva ls,and a physical examination was performed onceweekly.Chemi str y. B lood sampl es wer e col lected on ce aw eek by jugular venapuncture w ith the dogs in sittingposition after an overnight 9-h fast. This was donew ithout prolonged occlusion of the vein. T he following m easurem ents (by the m ethods in parentheses)w ere carried out in blood, serum or plasm a, as appropriate: packed cell volum e (PCV ); w hite blood cellcount (W BC) (Sysmex system F800, Sysmex-ToAMed ic al E lectro nic s C o. L td ., K ob e, Ja pan ) an d d iffe re ntia tio n; to ta l p ro te in (b iu re t); a lb um in (b romcre so l-green); protein electrophoresis (cellulose acetate,s ta in ing by Ponceau S); to ta l ca lc ium (o-cresolph ta le in );in org an ic p ho sp hate (moly bd ate w ith ou t d ep ro te in i-z atio n); u re a (u re ase g lu tam ic d ehyd rogena se ); c re at-in in e (Jaffmeth od , in itia l rate at 3 0 C );a lk alin ephospha ta se [(AP)EC 3 .1 .3 .1 ] and a lani ne aminotrans -ferase [(A LT) E C 2.6.1.2] (both kinetic according toInternational Federation of Clinical C hem istry recommendatio ns a t 3 0C ); -g lu tamyltra nsfe ra se , 7 -GT(EC 2 .3 .2 .2 ) ( kinet ic , L -7 -g lu tamate -3 -5 -d ib romo-4-hyd roxyan il id e, 30C) .S erum c alcium c onc en tratio ns w ere ad ju ste d u sin gthe form ula: calcium adjustcd= total plasm a calcium(m mol/L) + 0.875 - 0.025 X (album in concentration,Stati sti cs. D i ff er ences between two groups wereinvestigated w ith the Student's t test. O ne-w ay anal-

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DIETA RY PROTEIN INFLUENCES ON SKELETA L GROW TH S 1 0 9TABL E 3

I ngre die nts o f th e h igh ( H- Pr) ,no rm al ( N- Pr)and low ( L- Pr)pro te in f oodIngredient I.F.N.1 H-Pr N -Pr L-Pr

g/100 g DMBloodm ealCasein(dehydrated]SoyabeanflourCornglutenmealHominyfeedPotatostarchW heatmiddlingsSugarTallowSoyabeanoilCellulosedJ-MethionineJ-Ly sineVitamin/m ineralsupplement2B utylated

hydroxytoluene5-00-3815-01-1625-04-5935-02-9004-02-8874-07-8504-05-2054-08-1274-07-9838.17.813.68.08.521.514.72.55.92.01.00.46.00.15.24.88.75.012.521 I .F.N ., In te rnatio nal Fe ed N um bers.z V itam in, m ineral and trace-elem ent m ix (6 g; T rouw , Putten,

T he N etherlands] contained the follow ing: V it A , 6250 IU (retiny lace tate ); v it D 3, 1 00 IU |ch ole calc if ero l); v it E , 6 .9 IU (dJ-a-to co ph -ery l acetate); v it C , 3.7 m g (ascorbic acid); thiam in, 0.7 m g; biotin,2 1 f ig ; V i t B -1 2, 5 f ig (c yan o co balam in ); -p an tho ten ic acid , 1.9 m g ;nicotinic acid, 5 m g; f olie acid, 0.12 m g; py ridox ine-H Cl, 0.7 m g;m e nad io ne -so diu m b isu lf ide , 0 .2 5 m g ; ch olin e ch lorid e, 8 3 m g ; iron ,7.3 m g; copper, 0.5 m g; z inc, 10 m g; m anganese, 4.5 m g; cobalt,0.025 m g; iodine, 0.11 m g; selenium , 0.01 m g; sodium chloride,0.5 g; lim estone, 0.6 g; dicalcium phosphate, 2.75 g; potassium chloride, 0.6 g.y sis of variance (A N OV A ) w as applied w hen threeg ro up s w e re in vo lv ed . W h en sig nif ican t, th is an aly sisw as follow ed by S tudent-N ew m an-K euls m ultiplecom parisons m ethod. P = 0.05 w as chosen as a lev elof s ignif icance.R adiology. M ediolateral radiographs of the rightradius and ulna w ere m ade w ith the dogs in right lateral recum b ency , using a conv entional diagnostic X -ray sy stem (Max imu s M - 15 0, Ph ilip s NV , E in dh ov en ,T he N etherlands), on regular black -and-w hite f ilm s(Cronex 4 DO S, DuPont de N em ours Gm bH , Frankf urt, Federal R epublic of G erm any ), in com b inationw ith h ig h-d etail in ten sif y in g sc re en s (C ro nex D etailS creens). E xposure settings ranged f rom 48 to 54 k V pand 10 to 16 m A , depending on siz e of the object, w itha focus-f ilm distance of 100 cm and table-top technique. R adiographs w ere m ade f our tim es, at 6-w k interv als, i.e., W 3 , 9 , 1 5 and 2 1. T h ese rad io graph s w e reused to assess the dev elopm ent of the ulnar sty loidpro ce ss and th e an co ne al p ro cess, th e arch itectu re o fthe distal ulnar m etaphy sis and the grow th in lengthof the radius and ulna. T he radial diaphy seal lengthw as m easured betw een the prox im al and distal m e-

taphy seal border through the ax is of the bone (18).T he length of the ulna w as m easured betw een theprox im al border of the olecranon, adjacent to theapo ph yse al g row th p late , an d th e e pip hy seal b ord er o fthe distal ulnar grow th plate, through the ax is of thebone. M easurem ents w ere m ade w ith a curv ed ruler,c orre cte d f or g eometric magn if ic atio n and e xpre sse din c en time te rs. T h e av e rag e le ng th ( SD)f th e rad iu sand ulna w as calculated for each group of dogs at diff e rent ages .Calcium kinetics. Calcium kinetic studies w ith45Ca were perform ed four tim es (W 1+2, W 7+8, W13+14 and W 19+20) in all dogs, as described elsew h ere (6 ).Pathology. C ostochondral junctions of the ninthribs w ere obtained by surgical resection in W 3 (lef trib, n = 7), W 9 (lef t rib, n = 8) and W 15 (right rib, n= 17). A t necropsy the right prox im al hum rusandtibia, the right distal radius and ulna and the costo-ch on dral ju nctio n o f th e 1O th rib w e re c olle cte d. A f te rremo v al o f so ft tissu es, 2-mm - th ick m id sag ittal slicesw e re c ut f rom th ese bon es. T issu e p re paratio n f or lig htm icro sco pic stu dies w as as d esc rib ed p rev io usly (19 ).H istom o rphom etric studies in bone tissue w ere donein u nd ec alc if ied rib se ctio ns. T h e f ollow in g d eterm inations w ere m ade: total v olum e of bone (V% b), relat ive osteob las t- covered (Obs ) and osteoc las t- covered(O cls) trabecular bone surf ace and the num b er of os-teoclasts per m icroscopic f ield (Ocl; the m ean of 15f ields, 25X objectiv e) (20). A v e rage w idth of the phy -seal grow th plate of the rib w as determ ined from them e an o f f iv e m e asu rem e nts at f ix ed sp ace d site s, u sin gan ocular m icrom e ter. S ections of heart, lung, liv er,k idney , spleen, gut, thy roid, parathy roid, adrenalg lan ds an d ce rv ic al sp in al co rd w e re p ro cesse d (1 9).

RESULTST he m e an daily f ood intak e w as not dif ferent am o nggroups and decreased gradually f rom ~ 1400 k j M E/k g075 in W l to 1100 k j M E/k g075 in W 20 in allg ro up s. Ph y sic al e x am i natio n re v eale d no abno rmalities. N o signif icant dif ferences w ere found for the

height at the shoulder throughout the study . T he increase in body w eight w as low er in the L -Pr dogs thanin the H -Pr dogs throughout the study , although onlystatistic ally sig nif ican t in th e p erio d o f W 6 -8 (Fig . 1 ).A f ter W 3, signif icant dif f erences occurred in theH -Pr and L -Pr groups f or plasm a album in (Fig. 2) andin all th re e g ro up s f or u re a (Fig . 3 ). L in ear re gre ssio ndem o nstrated a signif icant increase in plasm a totalprotein and creatinine concentration during the experim ent f or all groups. D if ferences betw een groupsf or p lasma creatin in e (Fig . 4) an d total p ro te in (Fig . 5 )w ere not signif icant at any tim e . H ow ev er, as alreadysuggested by Figures 4 and 5, the m ean dif f erences

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suo NA P ET A L .BODY WE IGHT UREA

-- low normal *ow normal50

40

3020

1 0

10 15 20 25e xp t im e (w e e k s )

F IGURE 1 Mean (SD)ody weights (BW) of Great Danepups fed high (H-Pr, n = 6, 31.6%), norm al (N -Pr, n = 5;23.1% ) or low (L -Pr, n = 6; 14.6% protein on dry m atterb asis, % DM ) du rin g th e ex perim e nt. (* )sig nif ican t d if ferenc e(P : 0.05) betw een H -Pr and L -Pr group. For clarity , S D o fthe N -Pr group is not printed.

were sig nif ic an tly d if f ere nt f rom z ero . D if f ere nc es b etw een groups w ere not signif icant for PCV , W B C , 7-g lo bu lin , to tal p lasma calc ium , c alc iumad j, p ho sphorous, A P, A L T and 7-GT . In all dogs, the shape of theulnar sty loid process dev eloped f rom rectangular orsquare in W 3 to cone-shaped in W 9. Partial or complete ossif ication of the apex of the sty loid processw as present in W 9, w hile at that tim e com plete fusionof the ossif ied apex w ith the sty loid process had occurred in one H -Pr dog. T he latter stage w as reached

ALBUMIN*ow *ormal

35

3O

25

2O

1510 15 2O 25

e x p t im e (w e e k s )F IGURE 2 Mean (SD)pla sm a a lbumin c once ntra ti on sof Great Dane pups fed three dif f erent protein levels. S eelegend to Figure 1 for further ex planation.

1086

E

10 15 20 25e xp t im e (w e e k s )

F IGURE 3 Mean (SD)plasma urea concentration ofGreat Dane pups fed three dif f erent protein lev els. (+) signif icant dif ference am ong all three groups. S ee legend to Figu re 1 f or f urther ex plan atio n.

in all d og s in W 15. T h e an co neal p ro ce ss w as p artiallyor com pletely ossif ied in all dogs in W 9 and fusionof the anconeal process w ith the olecranon had occurred in one N -Pr and one H-Pr dog. In W 15, theanconeal process w as com p letely ossif ied in all dogsand fused w ith the olecranon in all f iv e N -Pr, f iv e H -Pr dogs and three L -Pr dogs. In all dogs ex cept f or oneL -Pr dog, radiologically detectable f usion of the anconeal process w ith the olecranon had occurred in W21. Flattening or indentation of the phy seal border ofCREATININE- *- low normal

8O7060504O30

10 15 20 25exp time (weeks)

F IGU RE 4 M ea n ( SD )la sma crea tin in e co ncen tra tionof Great Dane pups fed three dif ferent protein lev els. N osignif icant dif ferences w ere f ound am ong groups at any tim e.S ee legend to Figure 1 for further ex planation.

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DIETA RY PROTEIN IN FLUENCES ON SKELETA L GROW TH Sill low normal

7O

6O

50

4O

3O 5 10 15 xp tim e (w e eks )

20 25

F I GURE 5 M ean (SD )lasm a total p rotein con cen trations of G reat D ane p ups f ed three dif ferent p rotein lev els.N o sig nif ican t d if fere nces w e re f ou nd amo ng g ro up s at an ytim e . S ee leg en d to Fig ure 1 f or f urth er ex plan atio n.

of the grow th plate cartilage of the costochondralju nc tio n o f th e rib s d ec re ase d w ith in cre asing age (T able 5; phy sis). A l low ing f or age, no signif icant dif feren ces w e re c alc ulate d b etw e en gro up s o f d og s. T h erew ere also no dif f erences in the am ount or thick nessof the m etaphy seal bone trabeculae of both prim aryand secondary spongiosa betw een the groups. T hesam e applied f or the am o unt and thick ness of osteoidseam s on the trabeculae and the am o unt of rem ainingcart ilage matri x in the t rabecu lae . Histomorphometri cdata on specif ic areas of trabecular bone in the rib aregiv en in T able 5. N either the V % b nor the Obs, Oclsan d Ocl w e re d if feren t amo ng th e g ro up s. T h e amo un tof paraf ollicular cells in the thy roid and the activ ityo f th e parath y ro id g lands d id not d if f er h isto lo gic allyam o ng the three groups. In cerv ical spinal cord therew e re m in imal d eg en erativ e ch an ge s, e spe cially in th ese gments b etw e en th e th ird and f if th c erv ic al v e rte braein som e dogs, but these lesions occurred equally in allthree groups. T here w ere no histological lesions ino ther s of t t is sues .

th e d is tal u lnar me taphys is , o r even a re tai ned cartilagecone in the distal ulnar m etaphy sis, w as present in alldogs at som e point in W 3, 9 or 15. Im prov em ent ofarch ite ctu re o f th e d istal uln ar m e tap hy sis w as n otedin W 21, resulting in a norm al shape of the distal ulnarm etaphy sis in four N -Pr dogs, three H-Pr dogs andfour L -Pr dogs. T he abnorm alities in the rem ainingdogs w ere conf ined to a sm all rem nant of a cartilagecone in one H -Pr dog and m inor f lattening of the distalulnar m etaphy sis in the other dogs. A distinct bonyspur at the palm ar aspect of the distal ulnar m etaphy sis, w hich w as present in all dogs at som e point inW 3, 9 or 15, had disappeared in W 21. T here w as nodif f erence in the m ean length of the radius and theulna betw een the groups in W 3, 9, 15 and 20.In all groups food intak e w as such that there w ereno signif icant dif ferences in calcium intak e (V (). Inad ditio n, no sig nif ican t d if feren ces w e re f ou nd in calcium k inetics am ong all groups, including the percentage calcium absorption f rom the intestinal tract(a), calcium - accretio n (V o ), an d calcium r s orp tionf rom the sk eleton (V o ) (T able 4).A t pathological ex am inations no m acroscopic lesions w ere present in the v arious organs of any dog.In one dog in the L -Pr group an ununited anconealprocess (U A P) w as found. A long the m idsagittal cutsurf ace of the long bones, irregularities of retainedcartilag e o f v ary in g d eg ree s of b oth articu lar and p hy -se al g row th p late cartilag e w e re p re sen t. S uc h osteo -c hond ral le sio ns w e re e sp ec ially se en in th e c audo cen-tral p art o f th e p ro xim al h um e ral articu lar c artilag e,in the distal ulnar phy seal grow th plate cartilage andin the grow th plate of the ribs. T he sev erity of thesele sio ns d if f ere d b etw e en dog s and was equally p re se ntin e ac h g rou p. H istomorp ho lo gic ally th e m e an w id th

DISCUSSIONProtein requirem ents in dogs hav e been discussedfor ov er 50 y and num erous ex perim ents hav e beencarried out to determ ine the optim al protein content

TABLE 4C alciu m k in etic m easu res in th ree grou ps of G reat D an e pu psf ed dif feren t levels o f p rotein in ta ke1MeasureV,,mmol/kg075per

daya,%VJ,

mmoIAg075perdayVi,

mmol/kg075perdayW171319171319171319171319H-Pr

|n =)20.212.49.47.87766747236.232.223.116.221.924.916.7+10.90.51.71.21.58107124.02.33.51.95.32.53

(n =)19.312.510.18.37970606733.833.624.716.419.925.818.811.32.01.50.71.1875191.7

=)18.513.710.78.5+7750+55+6734.329.

322.216.020.624.017.410.9+1.71.50.80

1 V alues are m eans S D C a intak e (V ,), percentage intestinalC a absorption (a), C a accretion (V 5 ) and ar sorption f rom thesk eleton (V at W 1, 7, 13 and 19 in Great Dane pups fed high (H-Pr), norm al (N -Pr) or low protein |L -Pr) f ood. N o statistically signif icant dif f erences w ere found. V ,, V Q and V are expressed asm m ol/k g m etabolic body w eight (k g0 7 S).

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SllZ NAP ET AL.T A BL E S

H i st omorphometr i c measu res of th r ee groups of G reat Danepups f ed di f fer en t l evel s of pr ot ei n1MeasurePhysis,i/mObsOclsOclV%bW391521391521391521391521391521H-Pr109 83)96

143)92 146)69 2611)49.0.3(3)47.8.0(3)59.0 8 .16)53.0.1(11)16.5 3 .63)16.4.8(3)14.8 4 .66)11.84.8(11)14.3 2 .13)12.8.8(3)10.3.9(6)7.0.1(11)20.9.0(3)20.8.9(3)20.9 3 .06)23.5 2.5(11)N-Pr116

111)100 93)94 155)78 257|45.4

(1)49.7.4(3)56.5.4(5)60.6.7(7)10.5(1)15.2.6(3)13.8 4 .85)10.2.7(7)10.3(1)11.9.3(3)9.4.5(5)6.2.5(7)17.2(1)19.5.6(3)18.8.9(5)22.7 5 .7 (7)L-Pr117

293|96 113|87 186)72 2410)44.0 2 .13)46.9 3 .13)56.6.0(6)56.0.5(10)14.4 4 .83)13.0.7(3)12.5 2 .36)11.8.3(10)1.4.9(3)10.1.8(3)9.2.5(6)6.9.6(10)18.8.6(3)17.6.7(3)17.9.9(6)19.5 5 .3 (1 0)

' V alu es a re m ea ns S Dj n o. o f b io ps ie s e va lu ate d in p are nth ese s. B io psie swere taken of the 9th or 10th rib at the following times: W 3 |9th left), W 9( 9th le ft) , W 1 5 ( 9th r ig ht) a nd W 2 1 ( 1O th ). P hy se al h eig ht ( ph ysis), p erc en ta geof trabecular bone covered by osteoblasts (O bs) and by osteoclasts (O cls),n um ber of osteoclasts (obje ctive 25X ) pe r m icroscopic field |O cl) a nd the volu me percenta ge of trabecular bone (V %b) did not differ signific antly am onggroups.

of the food and the optim al protein sources (21-25).The use of different bases in the literature to expressthe protein content of the diet, i.e., as percentage inthe product or on a dry m atter basis, as gram s protein/1000 kj, or as protein to energy ratio is confusing.The m inim al protein requirem ents reported in theliterature differ betw een studies from 11.5% (26) to22% DM . (27). The protein requirem ent depends onfa cto rs su ch a s d ig estib ility , am ino a cid compositio n,p ro pe r ra tio s among th e e sse ntia l amino a cid s a nd th eirav ailab ility from th e p ro te in so urc e, en erg y d en sity o fthe food and physiological state of the dog (26).The growth in length of the dogs receiving foodonly differing in protein content did not differ, as revealed by m easurem ents of height at the shoulder andlen gth o f th e ra diu s an d u lna measu red o n ra dio gra phs.The significant differences in body w eight in W 6-8betw een the H-Pr and the L-Pr groups m ay have beenthe result of the high protein requirem ents at that veryyoung age (26, 28, 29), the protein supply in the L-Prgroup being suboptim al. The body w eights finallyreached were about the same as those observed byH edham mer et al. (3) in G reat D anes fed ad libitum .In this study the differences in protein intake definitely had consequences on som e biochem ical m easures. The serum album in concentration of the L-Pr

g ro up was lowe r th an th at o f th e H -P r g ro up. A lth ou ghthe values in the L-Pr group were still w ithin the reference range, this finding indicates that the proteincontent in the food of 14.6% (% DM), i.e., 13% ofenergy as protein, w ith the protein quality as used ino ur ex perim en t, w as ju st b elow o ptim al req uirem en tsfor growing dogs of giant breeds under 27 w k of age.This is in agreem ent w ith recom mendations of a m inimum requirement of 16% of energy as protein forg row ing dog s (2 9-3 1).Ther e were a ls o d if fe rences i n me tabo lite s o f p ro te inmetab olism , i.e., u re a a nd c re atin in e. In th e H -P r d og s,plasm a urea concentration w as higher and the creatinine concentration was low er than in the L-Pr dogs.This is in accordance w ith the finding that excessivedietary protein is m etabolized and increases the glo-m erular filtration rate (32, 33). There w as no histo-logical evidence for kidney damage in any of thegroups.The alterations in protein m etabolism had no demon stra ble c on se qu en ce s on ca lcium metab olism , o s-teoblastic activity and calcium accretion. Thus, theinfluence of changes in dietary protein on calcium kinetics observed in several other species (7, 9-13) w asnot seen in this study. It seem s unlikely that the protein content of the food is an im portant determ inantof disturbances in enchondral ossification in largeb ree ds o f d og s.T he rad io grap hie a nd h isto lo gie ex am in ation s n eve rth ele ss re ve ale d chang es c ompatib le w ith d istu rb edenchondral ossification. The changes w ere equallydistributed am ong the groups, indicating that theyw ere not related to protein intake but rather to geneticfactors or another food constituent, m ost likely calcium , as w as dem onstrated in previous experim entsw ith G reat Danes (5, 6, 19).It is concluded that in this study the differences inprotein intake per se did not affect the occurrence ofd istu rb ed sk ele ta l d ev elo pmen t in young Gre at Dan es,and that an etiologic role for dietary protein in thedevelopm ent of osteochondrosis in dogs is unlikely.F rom th e d iffe re nce s in b od y weig ht a nd th e re la tiv elylow p la sm a a lb um in c on ce ntra tio ns in th e L -P r g ro up ,it is concluded that 14.6% protein on a dry matterb asis (1 3% o f en erg y a s p ro tein ) in th e foo d is m arg in alfor giant breeds of dogs during grow th.

ACKNOWLEDGMENTSThe technical assistance of H . S. W outerse (D ept.Husb. and Nutr.) in the m orphom etric analysis andA. van W ees (Dept. Clin. Sci. Comp. Anim .) in thecalcium kinetic studies and the preparation of theman usc rip t w as g reatly ap prec iate d by th e a uth ors.

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1 . OL S SON ,. E . (1 98 2) Mo rp ho lo gy an d p hy sio lo gy o f th e g row thc artilag e u nd er n ormal an d p ath olo gic c on ditio ns. In : B o ne inCl in ica l Or thopaed ics (Sumner -Smi th G., d.) ,p . 159-196,W . B . Saund ers , Phi lad elp hia, PA .2. H A Z EW I N KE L,. A . W . (19 89 ) N u tritio n in relatio n to sk eletalg row th d ef orm i tie s. /. Small A n im . Piac t. 3 0: 6 25 -6 30 .3. H EDHAMMER ,., W u , F., K .R OOK , ., S CH R Y V E R ,. F., D E L A -HUNTA ,A . , WHALEN , . P., K A L L FE L Z,., NUNEZ ,E . A . , H INTZ ,H . F., S HEFFY , . E . & . R Y A N ,A . D . (1 97 4) O v ern utritio n an ds k ele tal d is eas e: an e x pe rimen tal s tu dy in g row ing Great Daned og s. C orn ell V e t. 6 4, S u pp l. 5 : 1 1-1 60 .4. L A V E L LE ,. B . (1989) T he ef fects of the ov erf eeding of a balan ced c om p lete co mm ercial diet to a g rou p o f g ro w in g G reatDan es. In : N u tritio n o f th e Dog an d Cat. (B u rg er, I . H . & R iv ers,J. P .W ., e ds .) , p p. 3 03 -315 . Camb rid ge Un iv e rs ity Pre ss , Cambridge , England.5. H A Z EW I N KE L,. A . W . , G OEDEGE BUUR E,. A ., POULOS ,P. W .&W OLV EKA MP ,. TH . C . ( 1985 ) I nf lu en ce s o f c hronic c alc iume xc ess o n th e sk ele tal d ev elo pme nt o f g row in g G re at Dan es. /.Am. Anim. Hosp. Assoc. 21: 377-391.6. H A Z EW I N K EL ,. A . W . , V A NDENBROM ,W . E ., V A N 'T K LOOS TER ,A . T H ., V o oR HOU T,G . & V A N WEES ,A . (1991) C alcium m etab olis m in G re at Dan e d og s f ed d ie ts w ith v ario us c alc ium an dpho spho ru s le v el s. / . Nu tr. 1 21 : S99-S106 .7. EN GS TR OM ,. W . & D EL UC A ,H . F. (1 96 3) E ff ect o f e gg w h iteo n c alc ium me tab olis m in th e rat. /. N u tr. 8 1: 2 18 -2 22 .8. S HE NO L IK A R ,. S . (1974) Protein nutrition and calcium abs orp tio n. Nu tr. Me tab . 1 6: 1 0- 14 .9. BELL,R. R., ENGELMANN,D . T., SI E, T -L . & DRAPER,H . H .(1 975 ) E ff ect o f a high p ro tein in tak e o n calciu m m e tab olismin th e rat. /. N u tr. 1 05 : 4 7 5-4 83 .10. A L L EN ,L . H . & HA L L ,T . E . (1978) C alcium m etabolism , intestinal calciu m b in ding pro tein an d b on e g ro w th o f rats f edh ig h p ro te in d ie ts. /. N u tr. 1 08 : 9 6 7-9 72 .11 . WH I TING ,S . J. & DRAPER ,H . H . (1980) T he role of sulphatein th e calciu ria of h ig h p ro tein diets in ad ult rats. /. N u tr. 11 0:212-222.12. CA L V O ,M . S ., B EL L,R . R . & FO RB ES , . M . (1982) Effect ofprotein-induced calciuria on calcium metabol ism and bone s ta tusin ad ult rats . /. N u tr. 1 12 : 1 40 1-1 41 3.1 3. S PENCER , . , KRA MER , ., DEBARTOLO , . , NORR I S,C . & .Osis,D . (1 98 3) Fu rth er stu dies o f th e ef fect of a h ig h p ro tein d iet asm e at o n c alc ium me tab olis m . Am . /. C Jin . N u tr. 3 7: 9 24 -9 29 .1 4. K ER S TE T TER ,. E. & A L L EN ,L . H . (1989) D ietary protein inc re as es u ri nary c alc ium . /. Nu tr. 1 20 : 134 -136 .1 5. SCHAEFFER ,. C ., ROGER S, .R . & MORR IS ,.G . (1 989 ) Pro teinin the nutrition of dogs and cats. In: N utrition of the D og andCat, (Burger , I . H . & River s, J. P .W., eds.) , pp. 159-205, Cambri dge Un ive rs it y Pre ss , Cambri dge , Eng land .16 . NA TIONA LRESEA RCHOUNCIL(1974) Nu t ri en t Requ iremen tsof Dogs, Nat ional Academy Press, Washington DC.

17. M EUT EN ,D . J., C HEW ,D . J., C A PE N,C . C . & . K OCIB A ,G . J.( 1982 ) Re lati on sh ip i n s erum to tal c alc ium to alb um i n and t ot alpro tein in d ogs. /. Am . V e t. M e d. A s soc. 1 80 : 6 3-67 .18 . VOORHOUT ,. & HAZEW INKEL ,. A . W . (1 98 7) A rad io grap hiestu dy on th e d ev elo pm e nt o f th e an teb rach iu m in G reat D an ep up s o n d if fe re nt c alc ium in tak es. V e t. R ad io l. 2 8: 1 52 -1 57 .19. GOEDEGEBUURE,. A. & HAZEWINKEL,H. A. W. (1986)Mo rp ho lo gical f in ding s in y oun g do gs ch ro nically f ed a dietc on tain in g e x ce ss c alc ium . Ve t . Path ol . 2 3: 5 94 -605 .20 . A NDER SON , . (1 98 2) H istom o rp hom etry . In : M a nu al f or theExaminat ion of Bone. p. 93, CRC-Press, Boca Raton, FL.21 . W EECH ,A . A . , GOETTSCH,. & REEVES ,. B . ( 1935 ) Nu triti on aledem a in the dog. I. D ev elopm ent of hy poproteinaem ia on ad ie t d ef ic ie nt in p ro te in . /. E x p. M e d. 6 1: 2 99 -3 17 .2 2. HEGSTED ,D. M ., K EN T,V ., T SON GA S ,A . G. & STA RE,F. J.(1947) A com parison of the nutritiv e v alue of the proteins inm i xe d d ie ts f or d og s, rats, an d h uman b ein gs. /. L ab . C lin . M e d.32: 403-407.2 3. GESSERT ,. F. & PH ILL IPS ,. H . (1 95 6) Pro te in in th e n utritio no f th e g row in g d og . /. N u tr. 5 8: 4 15 -4 21 .24. W A NN EM ACHE R,. W . S i M c C oy , J. R . (1 966 ) D eterm in ationo f o ptimal d ie tary p ro te in re qu irem en ts o f y ou ng an d o ld d og s./. N u tr. 8 8: 6 6-7 4.25. B U RN S ,R . A ., L EFA IV R E , . H . S i M I LN ER ,J. A . (1982) E ff ectsof dietary protein quantity and quality on the grow th of dogsan d rats . /. N u tr. 1 12 : 1 84 3-1 85 3.26 . NA TIONA LRESEA RCHOUNCIL(1985) Nu t ri en t Requ iremen tsof Dogs, Nat ional Academy Press, Washington, DC.2 7. ONTKO,J. A . , WURTH IE R ,R . E. & PH IL LIPS ,. H . (1957) T hee ff ec t o f in cre as ed d ie tary f at u po n th e p ro te in re qu irem en t o fth e g row in g d og . /. N u tr. 6 2: 1 63 -1 69 .28. M E Y E R,H . ( 1983 ) En erg ie und N hrs to f fe S t of f wec hs el ndB e darf . In : E rn hru ngd es Hun de s, p p. 9 2-1 74 , E ug en U lme rV erl ag , S t u tgart , Germany .2.9. CASE,L . P. & CZARNECKI-MAULDEN,. L . (1990) Protein req uirem e nts o f g row in g p up s f ed p ractical d ry -ty pe d ie ts contai ni ng m i x ed -p ro te in s ou rc es . A m . / . V e t . R e s. 5 1: 8 08 -812 .3 0. SHEFFY ,B . E . (1979) M eeting energy -protein needs of dogs.C omp . Con t. E d. 1 : 3 45 -3 54 .3 1. SHEFFY ,. E . ( 1989 ) Th e 1985 re v is io n o f th e Na tio nal Re se arc hC ouncil nutrient requirem ents of dogs and its im pact on thepetf ood industry . In: N utrition of the D og and C at. (B urger,I. H . & R iv ers , J. P. W ., e ds.), p p. 1 1-2 6, C amb rid ge Univ ers ityPress, Cambridge , England.3 2. ROM SOS ,D . R ., B EL O,P. S ., B ENN INK ,M . R ., B ERG EN ,W . G . &LEVE ILLE ,. A . (1 97 6) E ff ec t o f d ie tary c arb oh y drate , f at an dpro te in on growth, body compos it ion and b lood me tabo li te l eve lsin th e d og . /. N u tr. 1 06 : 1 45 2-1 46 4.33. HOSTETTER , T . H., M EY ER , T . W ., R ENNKE, H . G. & BRENNER ,B .M . (19 86 ) C hron ic ef fects o f d ietary p rotein in th e rat w ithin tac t and re du ce d re nal ma ss . K i dn ey I nt. 3 0: 5 09 -517 .