19U. aubaittod la partial fulfiliawst of the OF SCIE5CB. Department of Poultry Husbandry OF ACGRICULTUBE AMD APPLIED 8CIEHC1

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1 EFFECT Of VAKTHG LBFBLB OF CALCUM Of THE CALCUM BALANCE, SHELL TEJUBS, AMD BLOOD CALCUM LEVEL OF W8XTB U8B0K FULLETS B. «., Stat* Collec* of WuhlAgfeoa* 1959 aubattod la partal fulflawst of the requr*a«tb for tha degree of OF SCE5CB Department of Poultry Husbandry OF ACGRCULTUBE AMD APPLED 8CEHC1 19U

2 * fc Document LD H * Tt &l m c^l TABLE OF CONTENTS Page HTRODUCTON «... 5*1 REVEW OF LTERATURE 2 EXPERMENTAL PROCEDURE. 5 EXPERMENTAL RESULTS 8 Calcum Balance Studes. 6 Egg Producton. «16 *... Egg Shell Thckness x Calcum n Egg Contents. Blood Calcum. *. DSCUS3QB SUMMARY.. ACKNOWLEDGlffNT LTERATURE CTES APPENDX a «

3 NTRODUCTON The detary requrement of varous farm anmals for oalcum, n terms of total raton, s about 0.29 per cent for the laotatng oow, 0.2^5 per oent for the sow, and 0*23 per oent for the ewe, accordng to Mtchell and LcClure (1937)* The level of detary caloum that wll sustan a hgh level of egg producton, promote good hatchablty and satsfactory shell texture has been set at 1.65 per cent by Norrs et al (193U), 2.25 per oent or less by Mller and Bearse (93k)» ejxd U»5 per oent by Brd (1937). Although t s qute apparent that the oalcum requrements of layng hens are consderably greater than those of other farm anmals, the fndngs of the varous nvestgators show wde dsagreement. Undoubtedly these dfferences are due to varatons n the fowls and n the expermental procedures used, varatons exstng n number of factors studed, sources of caloum, duraton of test, and possbly others. The value of these experments n establshng the mnmal level of caloum requred by the layng hen must be questoned, however, because no consderaton has been gven to the changes whch occur n the caloum stores durng the perod of egg producton. That the changes n caloum stores must be consdered n an attempt to arrve at the caloum requrements of layng hens was frst suggested by Hanan (1925), as quoted by Russel and McDonald (1929), when he reported that brds n heavy producton may be n a negatve caloum balance, or n other words drawng on ther oalcum stores to obtan oalcum for shell formaton. Smlar results have been reported by Russel and McDonald (1929) and by Common (1935, 1936, 1938)- The extent to whoh a layng brd may deplete her oalcum stores has been studed by a few nvestgators. Deobald et al (1936), Edn and Andersson

4 2 (1937)* and Common (1938) h&v employed dfferent expermental procedures n arrvng at values of 10, 20, and 25 per cent respectvely as the amount of body calcum nhoh a brd may wthdraw for shell formaton* The present experment was conducted to determne by r.eans of calcum balance studes the amount of calcum whch a brd xaay wthdraw for shell formaton, and to study the effect of varyng levels of calcum ntake on the calcum balance, shell thckness, and blood calcum level of layng pullets. t was hoped that the data obtaned would present more evdence concernng the calcum requrements of layng hens. REVEW OF LTERATURE Evdence has been presented by Common (1933* 1936, 1938)* Morgan and Mtchell (1938), Deobald, Lease, and Hart (1936), and Edn and Andersscn (1937) whoh suggests that chckens n producton may use the calcum stored n ther skeletons for egg shell formaton Common (1933< 1936) showed that calcum was retaned or stored n the bone as troaloum phosphate and that t was lost from the body also as a calcum phosphate salt. Hs data confrm the vew that when the calcum carbonate ntake s suffcently hgh, lme s transferred drectly from the gut to the ovduct va the blood stream and that no extra phosphorus excreton occurs under these condtons* VJhen the calcum carbonate ntake s low, an nsuffcent amount of detary calcum s avalable durng the perod of shell formaton to provde calcum for an egg shell. Under these oondtona phosphorus excreton was accelerated. Ths would ndoate that n leu of detary calcum, prevously stored oaloum n the form of troaloum phosphate was utlsed for shell formaton and ths accentuated the excreton of

5 3 the lberated phosphorus. These results were confrmed by Morgan and Mtchell (1938). They concluded also that the ablty of a hen to utlse calcum seems to be an ndvdual matter, the hgher producng hens as a rule utlsng the greater percentage of the ngested calcum. Hanan (1925) as quoted by Russel and McDonald (1929) concluded that oaloum was not stored to any great extent n advance of sexual maturty and that the brd's need for calcum after the onset of layng was almost completely met by the ncreased utlzaton of calcum from the avalable food supply. Negatve oaloum balances were recorded durng perods of heavy egg producton* Common (1938) noted, however, that as much as 9»3k8 grams of calcum was stored durng the 20-day perod just precedng the layng of the frst egg when a raton contanng approxmately 3*0 per cent oaloum oxde was fed. Russel and McDonald (1929) obtaned a few slghtly negatve but ohefly slghtly postve balances wth layng pullets an a 1.2, per cent oaloum raton for a perod of fve weeks. The brds, however, were n comparatvely low producton. Morgan and Mtohell (1938) $ however, demonstrated by balance studes that layng brds consumng lberal quanttes of oaloum n the form of oyster shell could have cumulatve negatve oaloum balances as great as grams at the concluson of 10 ffteen-day perods. The dfferences n the oaloum ntake and calcum outgo through egg shells of brds recevng a low calcum det have been studed by Edn and Andersson (1937)* They concluded that brds may wthdraw 20 per cent of ther body calcum for shell formaton before producton decreases to the pont where the ntake and outgo are the same. Deobald, Lease, end lart (1936) found that 10 per oent of the oaloum of the bone could be used for egg shell formaton snce the percentage ash contort

6 of the breast bones and tbae of Brown Leghorn hens on a low calcum det was 10 per cent less than that of the control groups recevng adequate mounts of calcum. t must be ponted out, however, that whle a decrease n the percentage skeletal ash can serve as a measure of calcum loss from the skeleton, an accurate measurement of the loss can be made only by determnng the total amount of calcum present n the body before and after depleton* The concluson of Deobald end coworkers s vald provded that (l) the ntal percentage of ash was the some for both brds and (2) the control brd nether stored nor lost oaloum. Common (1933) conducted oaloum balance studes wth Whte Wyandotte pullets usng hgh and low levels of detary calcum. Data were obtaned for calcum retenton (total oaloum ntake durng the experment less total calcum voded n eggs and the excreta). Over a perod of 73 days one of hs brds on the low calcum det (.365 per cent CaO) had a negatve oaloum oxde balanoo of 9^3kk grams. A slght postve or negatve balance was mantaned by the brds on the hgh oaloum det. At the concluson of the balance trals the carcasses of the brds were analysed for calcum content, and from the values obtaned, together wth the balance data, he was able to calculate the ntal amount of calcum n the bodes of the brds at the begnnng of the experment. A knowledge of the ntal body oaloum and the gan or loss of oaloum made t possble to determne the percentage loss or gan of body calcum durng the expermental perod. On the bass of the data obtaned for hs brds 2Q. and LUL). he concluded that layng brds may utlse 25 per cent of ther body calcum for shell formaton, and ths wthn a surprsngly short perod of tme. As Common ponts out, however, the data for one of hs brds may not be of great value n determnng the maxmum possble loss of body oaloum. Ths brd had not been layng for some tme before she

7 klled and -terefbre had stored calcum to replace that whoh she had lost. EXPERMENTAL PROCEDURE Balance studes -were conducted on a plan smlar to that used by (1958). However, all brds were kept on the experment for 30 days, and the calcum balance was determned by three-day perods. As a result, t was possble to calculate the maxmum loss or gan rather than the loss over the entre expermental perod. Expermental data were obtaned for 18 Sngle Comb fthte Leghorn pullets of the Kansas State College flock* n order that the brds mght become accustomed to the envronmental condtons pecular to the experment, and n order to determne the date on whch the brds lad ther frst egg, those pullets used were taken from the range and placed n the expermental cages a short tme prevous to sexual maturty. The cages used n the experment conssted of a seres of 16 hen batteres separated each from the other by sold parttons. ndvdual feces contaners were placed under each brd. Feed and water contaners were constructed n such a manner as to mnmse the loss of feed. The brds were led an all mash basal raton consstng of ground yellow corn 35 parts, ground wheat 25, ground oats 10*3, wheat shorts 10. 3, wheat bran 8.23, dehydrated alfalfa 2.6, meat and bone meal 2,6, fsh meal 2.6, soybean ol meal 2.6, salt 0.5, and cod lver ol * To three lots of the basal raton, whch contaned 0*562 per oent calcum, was added suffcent calcum carbonate 2 to ncrease the calcum content of the three dets *The ood lver ol used contaned 4OO A.O.A.C. unts of vtamn D and 3,000 U.S. P. unts of vtamn A per gram. 2 Lmeolth, a preoptated calcum carbonate analysng 99»»3 P«r oent caloum carbonate, was used.

8 to.42, 2.111, and J.17U por oent respectvely. The ratons contaned 0.70 per oent phosphorus and 370 p. p.m. of manganese. Although the calcum to phosphorus rato was dfferent n each det t waa beleved that there was suffcent vtamn D n the ratons to nsure the proper utlzaton of the oaloum and phosphorus regardless of the rato of the two. Sxteen pullets, whch were dvded nto four lots of four brds each, were started an the experment. Lot 1 was fed the basal raton only. Lot 2 receved the det contanng 1 per cent calcum, Lot 3 the det contanng 2 per cent oaloum, and Lot k receved the 3 per cent calcum raton. The ratons, n pellet form, and dstlled water wero before the brds at all tmes. One brd n each of Lots 1 and h ceased egg producton entrely and were klled before the duraton of the study. Two other brds were subsequently placed on experment to replace these two brds. A feces marker consstng of approxmately 1 gram of ron oxde, gven by means of a capsule, was used to determne the begnnng of the balanoe study. Data for computng the oaloum balance of each brd were obtaned perodcally for ten 3-day perods. Collecton of data began wth the frst egg and durng the perod mmedately followng. Data on calcum ntake and the calcum outgo, through egg shells, egg contents and feces, were obtaned. Caloum oxde n the shells was determned by ashng the shells n oovered poroelan dshes. A whte ash of caloum oxde wth constant weght was obtaned by heatng to 900 C. for three hours. The weght of contents of the egg was determned by subtractng the weght of the damp shell and membranes, whch had been wped as free as possble from all adherng albumn, from the total weght of the egg. A 2-gram sample of a mxture of the contents of all eggs lad by each brd durng each three-day perod was analyzed for oaloum. n analyzng, the sample

9 7 ashed, the ash dssolved n 2 oo. of concentrated HC1 and dluted to 50 oc. wth dstlled water. Calcum was deternned on a 5 oo. alquot by the Wang (1935) method for determnng the calcum of preoptated whole blood. The collected feces were placed n 12-nch porcelan evaporatng dshes and dred n a forced-draft electrc oven at 100 C. for 2U hours. The dred feces were then ground n a Wley mll and a two-gram sample taken for analyss. The calcum n the feces and also n samples of the feed was determned by the Assocaton of Offcal Agrcultural Chemsts (lsfyo) method for calcum oxde n mneral feeds. At the concluson of the balance study the brds were klled and the flesh oarefully removed from the skeleton- n an endeavor to determne f the loss of calcum from the skeleton of the brds on the low caloum det was from any partcular porton of the skeleton or was lost equally from all portons, the skeleton was dvded nto ts appendcular and axal parts. The bones were ground wth a food chopper, ashed, the ash dssolved n 200 cc. of 1-3 EC1 and dluted to 10 lters wth dstlled water. Calcum was determned on a 25 cc. alquot by the same method used for analyss of the feeds. n vew of the fact that Mtchell, Card and Hamlton (1931) have shown that the total calcum of both the flesh and feathers of four-pound Whte Leghorn pullets amounts to only 2 per cent of the total body caloum, and that Common (1933) has shown that the calcum n the flesh of brds on both hgh and low calcum ntake s vrtually the same, the oalcum n the flesh and feathers of the brds was not used n calculatng the percentage loss of body caloum. Blood caloum was determned by a slght modfcaton of the Wang (1935) method, the modfcaton beng made n order to take nto consderaton the dfference n blood calcum between layng brds and man.

10 8 Shell thckness was determned for eaoh shell wth membranes ntact by means of a mcrometer calper calbrated to thousandths of an nch. Four measurements were taken near the equatoral plane of each shell. Body weghts were recorded at the end of every sx days. Warren and Schnepel (19^) end Conrad (1939) reported that temperatures greater than 90 F. wll adversely affect egg shell thokness and the blood calcum level of fowls. For ths reason the temperature of the room n whch the brds were kept was mantaned as nearly as possble between 70 and 75 degrees Fahrenhet. Temperatures were recorded on a Tyoos thermograph. EXPERMENTAL RESULTS Calcum Balance Studes The balance data whch are presented n Tables 7 to 2k of the appendx show that the brds whoh receved 0.5 or 1.0 per cent oalcum n the det were n negatve cumulatve oalcum balance, whereas all brds recevng the 2 and 3 V er C6rt calcum dets, wth the excepton of Nos. 56(1) and 59 > were n postve cumulatve oalcum balance. Table 1 presents a summary of the balance data for each lot. The brds n Lot 1 had an average negatve balance of 7*556 grams and wthdrew an average of per oentx of ther skeletal calcum for shell formaton. ndvdual wthdrawals vared from J4 to 51 P r cent. The brds la Lot 2 utlzed an average of 3«U«38 per oent of ther skeletal calcum, and had average negatve calcum balances of grams. ndvdual varatons from 27 to JUU per cent. n Lots 3 and k postve balances of grama ^Percentage loss or gan of skeletal calcum was determned from loss or gan from perod of greatest negatve or postve cumulatve balance and not from total balance for 10 perods.

11 9 and grams respectvely were mantaned. Ths represents gans n skeletal calcum of and per cent respectvely. n Lot 3 the varatons were from -O.j. to +28 per cent, and n Lot * from 21+ to +50 per cent. The brd on negatve balance n Lot h was Ho. 58(1) and she ceased layng after four perods and was klled. Fgures 1 and 2 show that the average rate of wthdrawal of skeletal calcum n Lots 1 and 2 was rapd. The maxmum wthdrawal n Lot 1 had been nearly reached by the end of the ffth perod. Fgures 3 and k, however, show that the rate of deposton of skeletal oaloum n Lots 3 and k was slow and was dstrbuted over the entre expermental perod. Table 1. The balance data for groups of brds recevng varyng amounts of calcum. Lot 1 Lot 2 Lot 5 Lot k Av. Total Ca ntake, grans Av. Ca n Shells, grams * l 29.01*0 Av. Ca n Egg Contents, grama.2*4.3»^90.1*92 «381 Av. Ca n Feoes 8.70U CAjB Av. Ca Outgo U J4 Av. Ca Balance -7* *2.838 Av. Percentage Loss or Gan of Skeletal Ca* -1*6.71*. -5l* Percentage loss or gan of skeletal calcum was determned from loss or gan from perod of greatest negatve or postve cumulatve balance and not from total balance for 10 perods. That 1 6»7 per cent s not the maxmum skeletal calcum whch fthte Leghorn pullets may wthdraw s ndoatod by the fact that n most oases when the brds n Lot 1 started layng after havng been out of producton for a few days they wthdrew more calcum for the formaton of egg shells than that whch they had stored whle out of producton. The faot that some of the brds on the basal det were stll layng at the close of the experment suggests that further depleton mght be possble.

12 k ". 16 ^V 15 1U - _ [l V 13 % X1 'v ^ ''"- ' 1 A '" 10 9 Grams of calcum n skelet< Mg. of oalcum per 100 cc. of vhc>le "blood Shell thckness n thousan< ths o an noh m U Perod No. Fg. 1. Mean expermental data for Lot No

13 : * ^ -v l* " N V * -., s.. - X - c= Gr ams of calcum n skeleton Mg. of calcum per 100 oc. of -whole "blood ell thckness n thousandths of an nch 6 2 k 7 Perod No. 10 'g. 2. Mean expermental data for Lot No. 2. j

14 . ' ' 12 2> 2h 2'J ^ ^ - " 1U " Q y G rams of calcum n skeleton ME Of Calcum ne* 3 CO CO. nf TurVnla hlnnl S dell thckness n thousandths of an nch c o 2 3 f Perod No. 10 Fg. 3. Mean expermental data for Lot No. 3.

15 V k r y 1 _ V V ^ _ S wm * mmm ^ ll 10 9 Grams of calcum n skeleton Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch o l 2 3 h Perod No. 10 Fg. 4.. Mean expermental data for Lot No. 4..

16 Bl The average percentage lows of calcum for the brds n Lot 1 s much greater than that reported by Bdn and Andersscn (1937), Beobald ot al (1936), or Conaon (1930)* Th» results of the present oxporlment are n varance wth thnt of Deobald ot al end Edln and ^ndersaon probably due to dfferences n expermental methods and methods of statatoal analyss enployed. These dfferences xavc been prerously enumerated. The faot that the percentage lose n the present study was calculated fran a pont not more then three days frob whch the brd was n ts greatest negatve balance, rather than from the lose resultng durng the entre expermental perod s probably responsble for a wthdrawal value nearly twoe that reported by Canaan. The procedure followed n the present study elmnated the factor of calcum storage whch results when brds are not layng. n the rent that layng was not resumed so as to afford an opportunty to convert the stored calcum nto egg shell the extent of wthdrawal would be greatly obscured. That the stook need by Conaon was nferor wth respect to egc layng ablty s suggested by the fact that two brds on the low oaloun det produced only 13 and 11 eggs n 73 end 1*9 days respectvely end hence had lttle opportunty to deplete ther body reserves of calcum. n the present experment the brds produced an average of 12 eggs n 30 days even though losng more skeletal caloum than dd the brds of Common. The level of oaloun n the det may also have been a factor snce the det used by Cocoon contaned only per cent calcum* t s possble tlmt ths level of calcum n the det was so low as to provde nsuffcent calcum for the normal physologcal proceases of the brds. Ths oould result n reducng the rate of lay, ahoh n turn would make t mpossble to determne the amount of caloum actually susceptble of beng wthdraw.

17 15 That the skeletal oaloum of the brds n negatve calcum balanoe wthdrawn equally from all portons of the skeleton s shown by the data presented n Table 2. Table 2. Percentage oaloum n appendcular and axal skeleton of brds reoevng varyng amounts of oaloum. Percentage Calcum Percentage Calcum Lot Ho. Brd No. Appendcular Axal (1) 5U.1* (2) Average Average y»! <4.o Average *4.2 h 58(1) (2) 55.4 su Average The rato of calcum n the axal or appendcular skeleton to the total calcum present n the skeleton was approxmately the same for all brds, the average rato of all skeletons beng 43*6 per cent axal and 56.4 per cent appendcular. Ths ndcates that calcum s wthdrawn from and stored n both dvsons of the skeletal structure at the same rate.

18 16 Egg Producton The effect of a low oaloum det on subsequent layng performance was studed n growng pullets by Massengale and Patt (1930)* A det unsupplemented by calcum was fed durng the frst ten weeks. Two per oent of lmestone was added to tho raton for the next eght weeks followng whch the lmestone was agan removed from the raton. The average producton of ths group at 33 weeks of age was 0.5 egg per brd, whereas brds vhoh lad receved 4 per oent or more of oyster shell n the raton for a correspondng perod lad an average of 20.8 eggs per brd, Buokner and Martn (1920) observed that the feedng of lmestone or oyster shell ad lb ncreased egg producton per oent over a control group of pullets recevng an unsupplemented det. The brds fed no mneral supplements contnued to lay eggs untl there was a general depleton of magnesum, phosphorus, and calcum n ther bones, the breakdown occurrng n about sx months These results were confrmed by Buokner, Martn and Peter (1923, 1925). n 1925 they observed that the producton of yearlng hens whch were fed a low calcum det for two months and then gven oaloum oarbonate ad lb for 31 days ncreased the rate of producton from 11 to 58 per cent. Deobald, Lease, and Hart (1936) have reported that brds vrtually ceased layng wthn 12 days after the abrupt removal of calcum supplements from ther det. Smlar results had prevously been reported by Buokner and assocates (1930). They fed lvhte Leghorn hens for 4. days on a det consstng of yellow corn, skm mlk, end oaloum carbonate fed ad lb and then removed the oaloum carbonate from the det of half of the brds, the other half beng contnued on the calcum supplemented det. n a perod of

19 17 sx weeks the producton of the brds deprved of oaloum carbonate decreased 78 per cent whle brds kept on the calcum supplemented det ncreased 30 per cent n producton. Lkewse, Buokner, Martn, and Peter (1925) observed that when oyster shell was removed from the det of confned hens havng receved t for sx months, egg producton decreased 50 per cent wthn 3 months. Brds whch had access to range and were suddenly deprved of the calcum oontnued to lay at a satsfactory rate. Apparently suffcent mneral matter was obtaned from the range to mantan good egg producton. The addton of oyster shell to the dets of hens not havng receved any calcum supplements for sx months trebled egg producton wthn 3 months. nablty to consume and utlse suffcent oalcum to mantan a postve balance of calcum was shown by Morgan and Mtchell (1938) to result n a cessaton of layng. Edn and Andersson (1937) reported that when the body oalcum of a brd on a low calcum ntake has decreased to 60 per cent of normal, due to loss of calcum through egg shells, egg producton decreases. Deorease3 n producton of from 65 to 8 per cent were noted wthn 25 days. Common (1938) observed that the normal egg layng cycle s markedly nterfered wth from the outset n brds on low oaloum dets where detary oaloum must be supplemented wth body oalcum for egg formaton. He suggests as a reason for ths the more rapd moblsaton of ngested oalcum as oompared wth the moblzaton of skeletal oaloum for egg shell formaton. The results of the present study show that the average producton per brd n Lots 1, 2, 3, and k for the 30-day perod was 12, 21, 20, and 15 eggs respectvely.

20 18 All brds n Lot 1, whoh receved the 0.5 per cent calcum det, ceased producton for perods of at least 5 or more days one or more tmes durng the experment. The average number of days that the brds were on experment before producton ceased was 13 wth "the greatest number of days for any ndvdual brd before producton ceased beng 16. The results obtaned wth brds 57, 71, and 73 as shown n fgures 5* 6, and 7 of the appendx ndcate that after the brds ceased layng, they stored calcum and when suffcent was stored, egg producton contnued untl there was a depleton of the accumulated reserve. n some nstanoes a return to the layng condton resulted n a wthdrawal of calcum whoh exceeded the ecaount stored durng sexual nactvty. Normal egg producton was mantaned by all the brds n Lots 2 and 3«he average producton of Lot 2 was ncreased due to the hgh producton of brd 61. The lower average producton of the brds n Lot k s dffcult to understand. The fact that brd 75 lad 20 eggs, and that other non-expermental brds kept under dentcal condtons, except for expermental handlng, and fed the 3*0 per cent raton lad at a satsfactory rate ndcates that the low producton was due to some factor such as an unfortunate choce of brds. These results suggest that ratons contanng as lttle as 1*0 per oent caloum wll mantan satsfactory egg producton for a perod of at least 30 days followng date of onset of sexual maturty. Egg Shell Thckness Buokner and lartn (1920) reported a decrease n shell weght from 5*1 grams to 2*9 grams when hens were lmted for a perod of sx months to the calcum naturally occurrng n the food* However, the percentage of calcum and phosphorus n the egg shells remaned constant thus ndcatng that there

21 19 s a stable equlbrum between the two elements. These results were confrmed by Buokner and coworkers (1930) when they observed that feedng a noncalcum supplemented det decreased shell weght 21 per cent n a perod of sx weeks. Deobald et al (1936) reported a gradual dmnuton of the COg free ash of the eggshells untl the ash content of the shell was less than 25 per cent of normal when brds were abruptly deprved of the calcum n the raton. However, they were unable to obtan any shell-less eggs before producton ceased. The results obtaned by Common (1936) also show that brds on a low calcum det (O.365 per cent calcum oxde) lay eggs whose shells become progressvely thnner. Hs data show that a brd on a low calcum raton wll lay two or three eggs whch oontan about two grams, or a normal amount of calcum oxde, and then may decrease as much as from 2*039 grams to grams. Edn and Anc ersson (1937) observed that when the ealcum content of the det of layng brds was abruptly reduced, a decrease n the relatve calcum content of the eggs s notceable even the day after reducton of calcum n the feed. They report a deorease of 4B per cent n the calcum oxde content of eggs wthn a perod of 25 days. Rddle and Hanke (1921) observed n ther studes on egg-layng rng doves that thn-shell eggs cannot be attrbuted to an nadequate supply of calcum n the feed. n the present experment the qualty of the eggshell s expressed n terms of (l) measured shell thoknese, and (2) the mllgrams of calcum present per square oentmeter of surface area of eggshell. These methods should provde a more accurate measure of shell qualty than total shell weght or weght of calcum n the shell because varablty due to dffer-

22 20 enoes n egg sce s elmnated. A summary of the measured shell thckness (n thousandths of an noh) and also calculatons of the amount of calcum per square centmeter of surface area of the shells s presented n Table 3* Surfaoe area of the shells, expressed as square centmeters, was computed by the formula U.*6fSr^^, where V la the weght of the egg n grams. Ths method for determnng surface area has been shorn by Mueller and Scott (19^0) to yeld a smaller standard error than methods prevously presented by Marshall and Cruokshank (1938) and Edn and Andersson (1937)* Table 3* Shell thckness of eggs from brds recevng varyng amounts of calcum- Lot 1* Lot 2* Lot 3* Lot k Av. Shell Thckness, thousandths of an nch, Perod Av. Shell Thckness, thousandths of an nch, Perod Av. mg. Ca per sq. cm*, Perod Av* mg. Ca per sq. cm., Perod ; *Data for Lots 1, 2, and 3 begn wth the perod durng whch brds were frst placed an ther respectve dets. t does not nclude Perod 1 when on 3 P r cent calcum. ncreases n average shell thckness of 5*3 and 9*1 per oent were obtaned n Lots 3 and k respectvely. The ncrease n Lot 4. was made even though Brd 79 of the lot had a consderable decrease n shell thckness. A decrease n shell thckness of 17*3 per oent resulted n Lot 2. n Lot 1 the average decrease n thckness was from 12 to 9.2 thousandths of an nch. Ths represents a decrease of 23*3 per oent. ndvdual shells only 7*4 thousandths of an nch n thckness were obtaned but n no oase were shell-less egg6 obtaned before producton ceased. Ths confrms the

23 21 results of Deobald et al (1936) The actual average thckness and amount of calcum per square oentmeter of surface area also show that the level of detary calcum affects the composton of the eggshells. The amount of oaloum per unt of surface area ncreased slghtly on dets contanng 2.0 and 3.0 per cent calcum. Sgnfcant decreases n calcum were secured on the 0.5 and 1*0 per cent levels of ntake. The mean mllgrams of calcum per square centmeter was 20.3, 22.8, 27.9, and 3O.J4. for Lots 1 to k respectvely. That shell thckness tends to ncrease and decrease as skeletal calcum and blood calcum ncrease and decrease s shown n Fgs* 1 to 22. Calcum n Egg Contents That the level of calcum n the raton does not apprecably affect the percentage of proten nor the percentage of calcum n the whte and yolk of the egg was shown by Buckner, Martn and Peter (1925)* Common (1936) and Buckner et al (1923) were unable to show any notoeable effect of detary calcum an the amount of calcum oxde n the egg contents. Data for the mllgrams of calcum per gram of egg contents as obtaned n the present study are shown n Table U«The amount of calcum per gram of egg contents vares n many cases from perod to perod, and there s consderable varaton between the averages of brds wthn the same lots. Lot U, however, had an average of 4. per cent more calcum n egg contents than dd Lot 1. Lot 2 averaged 5*2 per oent more oaloum n egg contents than Lot 1, Lot 3 averaged 2.3 per oent more than Lot 2, and Lot j., k»k P*r oent more than Lot 3- An analyss of the varanoe of the oalcum n the egg contents of the eggs produced by the brds n the dfferent lots s presented

24 22 3 <.H W rr» -tf 3 «3 1 1 O o o 1 3 G» o O n N~ 0J sj { «m mm $m t» «. o cu j ONfQO^Cr 2>k,-jo t->-k> -4Z3-UN-4 rr»-4-4 } fmfmf ft 14»l * * t S TJs j fr^s^q -3"tAvO q OaW k> _=f_3-v3_g- fttr0s-d- * 3ssrr *! S2M { * $SS SMS! d? 3 ta -3"» UN'S 3LfMT>3- tr 5m3J rv j (»! Fv-3-tr7 _d-ltusm so u>-3- r t? t*.» HA fs 1-4 ssb hh nn m] H$t ss hh hm $;3& $33 sj3 1?^^ SrtO-tfCM 0{tWrt W K>vO CO K>cu UNXN-d- ^'ltu ^ N o^r KWrs NMT.5jrs«ka3 -=r-3"-=r 0ruS_3- c^-r0-4 c- r-35 UMA ]6-1 2 SHKSf-N LPQrHCO Q O QJ v0 CO CO <M UN ON

25 25 n Table 5. An F value, Snedecor (1938, P* 18U), of 8.8 s hghly sgnfcant wth the degrees of freedom represented n the analyss. Table 5. Analyss of varance of calcum n egg contents of eggs of 18 brds classfed by ndvduals and lots. Degrees of Source of varaton freedom Sum of squares Mean square Total Between Ca levels Between brds wthn Ca levels Wthn brds P Blood Calcum Buokner and assocates (1930) studed the effect of varyng the level of calcum ntake on the blood calcum of layng hens. They found that brds whch were kept on a det of yellow oorn, skm mlk, and calcum carbonate ad lb for sx weeks mantaned a constant blood calcum level, whereas the blood calcum of brds whch were deprved of the calcum oarbonate decreased from 20.9 mg. to 16.1 mg. per 100 co. of blood. Deobald et al (1938) demonstrated that as long as the calcum content of the raton was suboptmal and nhbted egg producton, the blood calcum shows regular, almost oyolo, varatons whch are qute characterstc for each ndvdual hen. When the calcum was removed from the raton of heavly layng pullets, layng contnued for about ten days wth the blood calcum content decreasng from 16 mg. to a level of about 10 mg. per 100 co. of whole blood. The effect of fastng upon the calcum content of blood plasma of layng

26 2U hens was nvestgated by Laskowsk (193U) Short fasts of 2 to h days dd not nterrupt egg layng or plasma oalcum whle fasts of k to 6 days nterrupted egg layng for several days and reduced the plasma caloua level to that of non-layng brds. n the present experment all data for blood calcum are presented as mllgrams of oalcum per 100 oc. of whole blood. Data are not avalable for sozne perods because of dffcultes arsng n expermental procedure. Results obtaned for ndvdual brds are presented n Tables 7 to 21* of the appendx. A summary of the results s presented n Table 6. Table 6. nfluence of dfferent levels of calcum ntake on blood oalcum. Lot No Mean mg. blood Ca, Perod la 16.6H* Moan mg. blood Ca, Perod * 15.1* Blood oaloum values decreased from 7.8 mg. to 9.1* mg., from 19.7 mg. to 13.8 mg., and from 19.1 mg. to 15«5 mg. on dets contanng 0.5, 1.0 and 2.0 per cent oalcum respectvely. Three per oent oaloum n the det enabled the hens of Lot J* to mantan a hgh level of blood calcum, a slght ncrease beng noted. The blood calcum for all brds n Lot 1 decreased from the frst to the last perod. n Lot 2 the blood caloum of all brds except No. 6l decreased. The records show that "ths brd consumed consderably more feed than the other brds n the lot. Ths would ncrease the oalcum ntake, makng t possble to mantan a hgh level of blood caloum. The blood oalcum of all ndvdual brds n Lot 3 deoreased. n two nstances slght decreases n blood oaloum were noted n Lot 1* brds. The remanng three brds of ths lot showed slght noreases n blood oalcum.

27 25 Of partcular nterest are the ncreases n blood caloum assocated wth perods of non-laya and postve oaloum balanoes. That varatons n blood calcum nay be related to perods of wthdrawal and storage of calcum s suggested by the present study. Ths s clearly shown n Fgs. 1 to 22 where t wll be noted that the blood oaloum curve tends to parallel the curve representng changes n skeletal calcum. DSCUSSOH The data obtaned n ths experment show that the level of calcum n the det has a defnte effect on the calcum balance, egg producton, shell thckness, calcum n egg oontents, and blood oaloum level of layng pullets. Dets of 0.5 and 1.0 per oent oaloum dd not furnsh suffcent calcum to meet the needs of the brds whereas 2.0 and 3*0 per cent calcum n the det were suffcent to E»et the requrements for the physologcal processes studed except that 2.0 per oent oaloum was nsuffcent to mantan the blood calcum level. The present study has shown that layng brds are capable of storng detary calcum n ther skeletons and of drawng upon ther skeletal calcum for shell formaton. t was also shown that the amount of detary oaloum avalable to the brd determnes whether oaloum s stored or wthdrawn and the rate at whch those changes take place. Brds recevng a det contanng only 0.5 par oent oaloum wthdrew an average of 4.7 per cent of ther skeletal calcum for shell formaton and dd so wthn about 15 days. Brds recevng 1.0 per oent calcum utlzed an average of 3k per cent of thor skeletal calcum and requred an average of about 25 days to wthdraw the greater porton of ths. The brds fed 2.0 and 3»0 per oent oaloum dets ncreased n skeletal calcum by 12 and 21 per cent, respectvely.

28 26 Lmtng the caloum n the det fed to pullets just enterng producton to that occurrng n feed materals wth no oalcum supplements has been shorn n the present study to cause at least a temporary cessaton of layng wthn 13 days. That brds can contnue producton for some tme whle n a negatve oalcum balance has been demonstrated, however. Brds wth a cumulatve negatve caloum balance of 9 grams obtaned over a perod of 30 days oontnued satsfactory egg producton durng the entre perod. Ko doubt, however, the brds whch were n negatve balance for the entre experment, but contnued to lay at a satsfactory rate, would have ceased layng, temporarly at least, when they had wthdrawn all of ther skeletal calcum whch was avalable for shell formaton. A det contanng 0.5 per cent calcum decreased shell thckness from 012 nch to.0092 nch. A 1.0 per cent oalcum det lkewse caused a decrease n shell thckness. ncreases n shell thckness, however, resulted from feedng 2.0 and 3*0 per cent oalcum dets. The average thckness of the shells obtaned on the 3*0 per oent oalcum det was greater than the average thckness of those obtaned on the 2.0 per cent caloum det. The rate of ncrease n shell thckness durng the expermental perod, however, was practcally the same n the shells obtaned by feedng the two hgher levels of calcum. Although prevous workers were unable to show an effect of detary caloum on the amount of calcum n the egg contents, the results of the present study show sgnfcant dfferences as a result of varyng the Ca level of the det. The fact that a dfference of H4. per oent between brds recevng 0.5 and 3*0 per oent caloum was noted and that graded ncreases n detary calcum caused correspondng ncreases n the amount of calcum n the egg contents suggests that the level of detary calcum nfluences the calcum found

29 n the egg oontents. Although 2.0 per oent oaloum n the det was suffcent to mantan a postve skeletal oaloum balance and shell thckness t was not suffcent to mantan the blood calcum level of the brds of ths experment. Feedng dets contanng 0.5 and 1.0 per oent oaloum lkewse caused decreases n blood oaloum. The greatest deorease occurred n the brds fed 0.3 per cent oaloum, n whch case the deorease n blood calcum tended to follow the wthdrawal of skeletal oaloum untl the blood calcum content reached the level for non-layng brds, whch resulted when they had wthdrawn as much skeletal oaloum for shell formaton as possble. Three per oent calcura n the det was suffcent to mantan normal blood oaloum values. On the bass of the results obtaned t nay be concluded that the calcum requrement of layng hens as measured by (l) balance studes, (2) egg producton, (3) shell thckness, and (4.) blood oaloum, and under the condtons descrbed, rests between the 2 and 3 P9 ** «n* level. 1. The effeot of varyng levels of detary oaloum on the oaloum bal- *> «g producton, shell thckness, calcum n egg contents, and blood calcum of Whte Leghorn pullets has been studed. 2. Layng pullets reoevng O.56 and l.llj. per oent detary caloua were n negatve calcum balanoe whereas brds recevng 2.11 and per cent oaloum were n postve oaloum balance. 3. Data are presented whch show that layng brds may utlze at least 4.7 per oent of ther skeletal oaloum for shell formaton. J4.. Satsfactory egg producton was mantaned for a perod of 30 days by brds recevng 1, 2, and 3 P ** 0 * oaloum. Brds recevng only 0.5

30 per cent calcum n the raton ceased producton temporarly at least after a perod of 13 days. 5» Shell thckness decreased 23 and 17 ]?or cent respectvely n the lots where 0.5 and 1*0 per cent oaloum were fed and ncreased slghtly n lots where 2 and 3 per cent calcum were ncluded n the raton. 6. A dfference of 4. per oent n the amount of calcum n the egg contents of eggs produced on the 0.5 and 3*0 per cent oaloum ratons was observed. 7. Blood calcum levels of brds recevng 0.5, 1.0, and 2.0 per cent oaloum were decreased. She decrement was n drect relaton to the amount of calcum n the det. Three per oent calcum n the det was suffcent to mantan the blood oaloum level. 8. The oaloum requrement of layng pullets confned to layng oages can be met by the ncluson of slghtly more than 2.1 per oent calcum (5.2 per oent calcum carbonate) n the raton. acejowlewmht ndebtedness s gratefully acknowledged to Dr. B. M. Scott, Assocate Professor of Poultry Husbandry, for hs valuable counsel and assstance durng ths study, and to Dr. R. H. Conrad, Assstant Professor of Poultry Chemstry, for hs assstance n the techncal analyses nvolved n securng data.

31 29 LTERATURE CTED Assocaton of Offcal Agrcultural Chemsts, Washngton, D. C. Methods of analyss, 5th ed. 19^ p. Brd, 8. Fecundty and reproductve ablty n closely confned fowl. So. Agr. 17* Feb Buckner, G. D., and Martn, J. E. She effeot of oalcum on the composton of the eggs and oaroass of layng hens. Jour, of Bol. Chen. ljll Feb Buckner, G. D., Martn, J. H., and nsko, W. M. Jr. The blood caloum of layng hens vared by the calcum ntake. Amer. Jour. Physol. 9k»692, 695- Sept Buckner, G. D., Martn, J. E., and Peter, A. X. Calcum metabolsm n the layng hen. By. Agr* Expt. Sta. Bu. 250* Effect of a caloum carbonate supplement n the det of hens, on the weght, proten content, and caloum oontent of the whte end yolk of ther eggs. Amer. Jour. Physol. 72tk5B-l&!>. May, Common, E. E. Observatons on the mneral metabolsm of pullets. Jour. Agr. So. 23* Oct , Observatons on the mneral metabolsm of pullets. Jour. Agr. So. 26t Jan Observatons on the mneral metabolsm of pullets. Jour. Agr. So. 28t5U July, Conrad, Ealph X. The effeot of hgh temperatures on the blood oalcum of the layng hen. Poultry So July, 1939* Deobald, E. J., Chrstansen, J. B., Hart, E. B., and Halpn, J. (. The relatonshp between blood oalcum end blood phosphorus and the effeot of varatons n the caloum oontent of the raton on ovulaton and blood caloum changes n the layng pullet. Poultry Sol. 17llt>»119* March, Deobald, H. J., lease, E. J., and Hart, E. B. Studes on the oalcum metabolsm of layng hens. Poultry So. 15* March, 1936.

32 30 Edxt, H., and Andersson, A. Kalkstensgrus, ostronskal ooh musselshal som kompletterngsfoder tll kalkfattga foderstater vd hog aggprodukton. Kungl. Lantbruks. Akademens Handlngar ooh Tdskrft, 76 l^-^- 1957* Laekowsk, H. Caloum and phosphorus content of blood plasma of layng hens durng fastlog. nter. Rev. Poul. Sol. 82l. 193k«Marshall, W,, and Cruckahank, D. B. The functon of the cutcle n relaton to the porosty of eggs. Agr. Sol. 28»2M42. Jan Jour. Massengale, 0. N., and Patt, C. S. Effect of caloum from dfferent sources on the growth and egg producton of poultry. Poultry So. 9t2M)-2l<6. May, Mller, Wayne M., and Bearse, G. S. Phosphorus requrements of layng hens. Wash. Agr. Eacpt. Sta. Bu U- Mtchell,. H., Card, L. B., and Hamlton, T. 8. A techncal study of the growth of -whte leghorn chckens Agr. Espt. Sta. Bu. 367« Mtchell, H. B., and MoClure, 7. J. Mneral nutrton of farm anmals. Hatl. Res. Councl Bu. 99* 135 P* Aprl, Morgan, C. L., and Mtchell, J. H. The calcum and phosphorus balance of layng hens. Poultry So ^. March, Mueller, Clyde D., and Scott, H. M. The porosty of the egg shell n relaton to hetohabllty , 19L 0. Poultry Sol. Morrs, L. C, Beuser, 0. F., Bngrose, A. J., end Wlgus, H. 6. Jr. Studes of the ealoum requrements of layng hens. Poultry So Mot. 193U- Rddle, 0., and Hanks, M. C. B. Effect of feedng soluble calcum salts upon reproductve secretons and upon the total norganc consttuents of the egg shell. Amor. Jour. Physol. 57»26U-27*. Sept Rusael, Walter C, and McDonald, F. G. The utlsaton of the oaloum of calcum carbonate end ctrate by layng and non-layng pullets. Jour. Bol. Cham ^3-473 Cot. 1929* Snedeoor, George W. Statstcal methods. Ames, owa. Collegate Press. 378 p. 1938*

33 31, c c» mprovements la the methods for oaloum determnatons n hologcal natter. Jour. Bol. Chen. ukk5-h * Warren, D. C, and Sohnepel, R. L. The effect of ar temperature on egg shell thckness n the fowl. Poultry Sol *0.

34

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53 1 ^ ' k A _ ' 15 ^. ^^ H* l S X >., s^ ^ "* s^^ ^N^ **-* ^ J -JV' Grams of calcum n skeleton V Mg. of calcum per 100 cc. of whole blood ' Shell thckness n thousandths of an nch 6 123U Perod No. Fg. 5* Expermental data for Brd No. 57 > Lot 1.

54 ! x ' k r. v v j _ l_ J -~ (> Mg. of calcum per 100 cc. of whole blood S aell thckness n thousandths of an nch fc Perod No. Fg. 6. Expermental data for Brd No. 71. Lot

55 *« lb J; -1. l h- - -V 7 l 13 ^ ^ n 9 to " ^^*^^"^~-~^ "' vy l Grams of calc um n skelet< Mg. of oalcum per 100 oo. of whole blood Shell thckness n thousancths of an nch 123 k Perod No. Fg. 7. Expermental data for Brd No. 73* Lo*! m 10

56 1 1 5k 25 ol. 2U A ^ - rv x.. 1 N 10 f- 9 Grams of calc um n skeleton Mg. of calcum per 100 oc. of whole blood Shell thckness n thousandths of an nch Perod No. Fg. f3. Expermental data for Brd No. 77(l)» Lot 1.

57 * 25 2k ~ ^ ( V 1 V h 1 r s Grams of calcum n skeleton Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch 6 2 k Perod No. ]? g.. 9 Expermental data for Brd No. 77(2), Lot 1.

58 b 23 9? P ^ ^ _ 19 lb s lu L> * 1 l N^ 10 9 Grams of calcum n skeleton Mg. of caloum per 100 cc. of whole blood Shell thckness n thousandths of an nch 123U Perod No. 10 Fg. 10. Expermental data for Brd No. 55» Lot 2.

59 - 25 2k " ' -V 1 V 11+ s _ 13 **>.. "> > 1 p^ Grams of calcum n skeleton Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an noh 23U Perod No. 6 Fg. 11. Expermental data for Brd No. 60, Lot 2.

60 N V 15 s. 1U Grams of calcum n skeleton Hg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch 123^56789 Perod No. Fg. 12. Expermental data for Brd No. 6l, Lot 2. 10

61 1 2k v. N 1J V N * ^^ p x'" V., ^5678 Grams of calcum n skeleton Mg. of calcum per 100 cc. of whole blood RVp Hr?«Tm«Bs n +.> m RATdths of an nch 9 10 Perod No. Fg. 13. Expermental data for Brd No. 78, Lot 2.

62 1 60 2? 2k A V. A x V A V u, _ [_ S s 10 9 G. rams of calcum n skeleton M 5. of oalcum per 100 cc. of whole blood 4H xell thckness n thousandths of an nch b 123^ Perod No. 6 Fg. ]1.. Expermental data for Brd No. 56, Lot 3-

63 r MB 61 1$ 2k 23 dd lb 1 _ J 7 U+ d ^.**"" -.. * "' -- l" 7 -.X"' y Grams of calcum n skeleton Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch rod M 0. 8 sr* " 0 Fg. 15. Expermental data for Brd No. 59 j Lot 3.

64 1 J k J '. N 13 ' X Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch 6 2 k o Perod No. 'g. 1< S. Expermental data for Brd No. 62, Lot J.

65 ! A- 20 A ] L k v '**-.. s'_ y'',- x ^--... ^, Grams of calcum n skeleton Mg. of calcum per 100 cc. of -whole blood Shell thckness n thousandths of an nch Perod No. Fg. 17. Expermental data for Brd No. 76, Lot 3-

66 1 6U 25 2k V ^ _ -A , _ V " Grams of calcum n. skeleton Mg. of calcum per 100 cc. of whole blood Shell thckne ss n thousandths of an nch 6 ' Perod No. D Fg. 18. Expermental data for Brd No. 58(l), Lot k-

67 k f l s x- V. -' -f " 12 _ Grams of calcum n skeleton Mg. of oalcum per 100 cc. of whole blood Shell thckness n thousandths of an nch Perod No. Fg. :L9. Expermental data for Brd No. 58(2), Lot.

68 c J _,-'"" "* Grams of calcum n skeleton Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch 6 L 2 3 J Perod No. 10 Pg. 2D. Expermental data for Brd No. 72, Lot k'

69 h do y y. NT1 ^.--" 15 v f'"t *>. * [ Grams of calcum n. skeleton Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch U Perod No. 10 Fg. 21. Expermental data for Brd No. 75> Lot l..

70 h A ' _,,. v^.'v H 13 N ^ v - > V- l Mg. of calcum per 100 cc. of whole blood Shell thckness n thousandths of an nch 123U Perod No. 6 :Fg. 2>2. Expermental data for Brd Mo. 79, Lot k-