Title: Dietary factors affecting blood spot incidence and changes in the vascular system of the hen
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Title: Dietary factors affecting blood spot incidence and changes in the vascular system of the hen
Physical Description: 111p.: ill. ;
Language: English
Creator: Merkley, John Wilburn, 1938-
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 1970
Copyright Date: 1970
 Subjects
Subject: Deficiency diseases in poultry   ( lcsh )
Eggs   ( lcsh )
Poultry -- Feeding and feeds   ( lcsh )
Genre: bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis (Ph. D.)--University of Florida, 1970.
Bibliography: Includes bibliographical references (leaves 103-110).
General Note: Description based on print version record.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by John Wilburn Merkley.
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Bibliographic ID: UF00097729
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 004817102
oclc - 503116210

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DIETARY FACTORS AFFECTING BLOOD SPOT INCIDENCE

AND CHANGES IN THE VASCULAR SYSTEMb OF THE HEN















JOHN WILBURN MERKLEY


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA


1970











ACKNOW~LEDGE~EMEN


Sincere appreciation is expressed to Dr. J'. L. Fry

for his understanding, guidance and assistance throughout

the duration of this research. Gratitude is extended to

Dr. R. H. Harms for his knowledgeable contributions in both

the planning and conducting of this work. Acknowledgement

is expressed to Dr. H-. R. Wiilson and Dr. R,. A. Voitle for

their many helpful suggestions and unselfish assistance.

The author thanks the members of his committee, Dr. R. C.

Robbins, Dr. R. H. Biggs and Dr. A. Z. Palmer, for their

counsel and interest.

The author is grateful for the assistance of Dr.

C, B. Amm~rermnan and Mrs. Sarah M"iller_ without who~cse assciEt-

ance the analyses of copper and iron would have been im-

possible.

Special thanks are due Dr. C. F. Simpson for the

hi~t~ologi~c~l. data reported in this work.

Fi~naly, the understanding, assistance and sacrifie~

orfi the au~thor's ri~fe , Mrgaret:, during these years of

graduate study aIre sinlcerely appreciated.








ii













TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS ... .., .... .... ..

LIST OF TABXLES .. .. .. .. . .. . .

LIST OF FIGURES .. .. .. .. . . . .. .

ABSTRACT .. . . . .. .. .. . .

INTRODUCTION .. .. .. .. . .. .


Chaperr

I. LITERATUR- E RiEVIEW ............

II. RELATION OF COPPER DEFICIENCY TO
BAPN TOXICITY .. .. . .. .. .


vILIiii


General Procedures
Experimenl;t I r....


...........
... .......
...........


III. R4~.TiE OFC; OVA't GROW~'TH AN~D OVAi SIZE~ IN
B3IRDS~af OF THE NO~RPAL AN~D BLOOD SPOT


In~outioduto .....
Pr~o c tdure .. .. .
Results and Discussion
Summannru ..... .


I ..... j


IV. THE RELATIONSHIP CT ELOOD PRESSURE
TO' THEii~ INCI(.DENCIE~E' OF OOD SPORTS
P~TR3OD CED . . . .. . .. . .


iii









TABLE OF CONTENTS---Continued


CharPter
Pag-e
IV. Results .,................................ as
SummarY .....,................,,...,..... 92

V. CHARACTERIZATION OF FOLLICLES FROM
HENS OF THE NORMAFL AND BLOOD SPOT
STRA~IN ....,....,.....,.................... 93

Introduction ............................~, 93
Procedure ................,,,............. 93
Results .....,......,..........,.....,..... 94
Summary .......,........................... 97

VI. GENERAL DISCUSSION3 AND SUMMARYRk .......,....... 49


RE:3:lF~ERENCES ................... ....,.............. ... 10~3

BTOGRAPHICAkL SKETCH ..,......................... .... 111












LISTI OFr TABLES


Table Page

1. Standard farm formulas .,....................9

2. Composition of low-copper layer
diet ...............................,........ 31

3. BAPNT~ starter diet .....,............. ........ 32

4.Percenti egg produc-tion of normal
and blood-spot hens fed a
copper-deficient diet ...................... 35

5. Average number of eggs produced
por hen during 30-day
experimental period ...................-..... 38Y

h. Percent fertility in normnal and
blood~-spot hens fed a copper-
deficient diet ............................. 39

'., Percent hiatchlability of fertile eggs
from normal and blood-spot hens
fed a copper-deficient diet ..............~.. 40

8. Five-week weight of chickens fed a starter
diet containing 0.06% EAPN from hens
fEed a lowJ-copper diet ................~. ..... 413

9. Severity of leg and toe deformities
in chicks at 5 weeks of age ....~............ 44

10. Mortalityv of chicks fed a starteer diet
containing 0.06% BAPN at 5 weeks
of age .................,.................... 46

11. Prcent egg production of normal
anid bliood-spo-t hensc fed a
coppe~r-deficient dtiet ................... ... 493

1.2. Percent fertility of normal and
blood-spot hens fed a copper-
defic~ient diet ...............~........~...~. 51









LIST OF TABLES--CSontinued


Tacble Pag~e

13. Percen-t hatchabhility of fertile
eggs from niormal and blood-
spot hens fed a copper-
deficient diet ............,...... ...,....... 52

14. Three-week weight of chickens fed
a starter diet con~taining 0.1_%
BA~PN from hens. fed a low-copper
diet ..........,............................. 54

15. Mfort~ality of chickIs fed a starter
diet containing 0.1% BAPN at 3
weeks of age ............................... 56

16. Percent egg production of normal
and blood-spo~t hens fed a
copper-deficient diet .............,......,.. 62

17, Percenzt- fertility of normal and
blood-spot hens fed a copper-
deficient diet ...,.....................~.... 64

18. Percent: hatchability of fertile eggs
from~; normalc7 and~ bl~ood--spot hens fed
a copper-deficient diet .................... 65

19. Copper levels in the liver of layi~ng
bens fed a copper-defcicint diet for
18 days ..................................... 66

20. Iron, levels in the liver of :l~aying
hens fed a copper-deficient diet
for 18 dayvs ................... ............. 68

21. Observations of electron micrographis of
m~or~tas from~ d~ay oldc clicks treat~ed
with a low-copper parental diet ...........~. 70

22. Composition ofE semisynthetic lw--copper
layeFr diets ..,................ ......~.....*. 73

22". Average size of yolk produced by 42 hens
of both--~ the- aicrmal and bl.ood-spot
stra~in dur-ing a 10-dayr periodC .............. 85








L~ST DF TA'FBLES--Cont Linuzed


Tabl~e Page

24. Th'!e effect of decreasing the!
systolic pressure upon
incidence and severity of
blood--spo-ts by feeding 0.06%
tapazole to 16 birds of the
blood-spot strain .......,,...........,.... 90

25. Comparison of ova characteristics
in h-ens of tl-he normal and b~lood-
spot strain .......~,...................,. 95


V11













LIST OF FIGURES


Figuree Page

1. Aortic rupture and mortality ofl- chicks
from? both strains fed a diet
containing 0.1% BAPN ........,.......... 58

2, Rat~e of' yolk row~t'h in. N and BS
strain ...........,.................... 83








Abstr~act-, of Dissertation Presented to the Graduate Council
of th~e Universit~y of Florida in Partial Fulfi~llment of the
Re-qu~iremnents- for the Degree of Doclor of Phil~osophy


DIETARY FACTORS AFFECTING BLOOD SPOT INCIDENCE A4ND
CHANGES IN THE VASCULAR SYSTEM OF THE HEN


By

John W~iilburn Merk~ley

December, 1970


Chiairmnan: J'ack L. Fryr
MajorT Departm~ent: Anrimal Science (Poultry Science)


One of the oldest problems of concern to thie egg-

producing industry is the occurrence of blood spots in eggs.

While mcany dietary factors have~ been reported to sEfect the

incidnce of eggs containing blood spots, these relationships

have not established the direct cause of this abnormality.

Studies were conducted to determine the relationship

of th~e vasculari integrity of birds to the percent; of blood-

spot 2ggs they produce. Both a normal commercial strain

and an experimental strain producing a high incidence of

blood-spot eggs were examined.

Four feeding trials w~ere conducted in which the

progenyr fr-om hen;s of- both strains treated with a coppear-

deficient d~iet were subjected to the stress of B-amin~opro-

picnitrilee (FBAP) -toxjicity. Production, fertility, hatch-~

abhilit~y and liver copper levels we~re used to determine the








effect of the iwaternal copper-deficient diet. Striking

differences w~ere~ detect'ed b~etw~een thle response of the~ two

str-ains to this dietary treatment. Egg production of tihe

normal strain was markedly affected, decreasing from 80%

to7 less, than. 6% in 5 days. Durijng the same period, pro-

duction in the blood-spot strain showed only a slight de-

c~rease.. Tihe level of copper in~ the livers of thle nor-mal

hens was reduced to a significantly lower level than in

the blood-spot strain by the dietary treatment. No dif-

ferences were detected between the response of chicks from

the two strains of henis to the BAPN toxicity. The i~ncidenzce

of death, a~ortic rupture, severity of legj disord~ers and

growth depression in the 2 strains was not s~tatcistically

different. Electron micrographs of thle aortas fro! day-old

chick:s treated with the parental lowJ copper diet indicatedl

similar disruptions in the vascular wall of both strains.

Lack of integrity in the follicular vascular system

in the blood-spot strain would not be the only abnormality

wJhichh could account for the high incidence of blood spots

produced. The critical period in the formation of blood

spots appears to be during the rapid phase of ova growth.

The rate of ova growth during the rapid phase and the ulti-

mate size of the miatur-e ova we~re compi~ared in the 2 strains.

NLo differences between the strain7s w~ere diectectd in the

rate of rapid -rvow.th. Hoeeit wars found that the size








of the mat,-ure Cva wJas signifi~cantly smal~le-r in the bloo~d-

spot strain.

Blood pressure is often m~entione~ldc as a possibleF

factor inlflue2ncing the occurr-ence of bloodJ spzotls.Te

systolic blood pressure in hens of the blood-spot strain

w:as sign~ificantly altered by feeding tapazole, a goitrogenic

agent. ~h~ile tih~e syistolic pressure wras significantly. de--

creased and ultimnately increased from thne normal. pretireatmenter l

pressure, no significant change in the incidence of blood

spots was observed,

A final study was conducted in which the physical

state of follicles remove-d from both~~ strains w~as comp~ared.

Of the characteristics compared, the high incidence of

blood clot~s in, the follicle wrass and wJide stigmas inn t~he

blood-spot strain are most noteworthy. The relationship

of these ch;arac~ter:isti~cs to the high incidence of blood

spots has not been established at this time.











INTRODUCTION


One of the oldest problems of concern to the

egg-producing industry is the occurrence of blood spots in

egigs. Today, witth thle de~velopme~nt of ~each n~ew strain of:

high-producing hens, this problem becomes more costly in

a highly competitive industry.

While the incidence- of blood soot~s can be mininmized

by proper manag~ement and the feeding of c~orrec~tly-balan~ce

diets, there has been no means discovered! to elimina-te or

entirelyr prevent their formation.

Since the first studli~es in the eaLrl:I 1940's, ve~ry

few? n~ew approaches in at.t~empting~ to solve this problems ha~ve

been report-ed, Many factors which apparently intenrify or

alleviate the abnormal. conditions associated with the

formation of blood s~pats have beeni publishedd but n~o single

factor or conditions di~rectly responsible for- th-eir formation

has been r~epo~rt-ea. at: is tle! c~omm~only held. theoiry_ today

thait blood spots result from~ a ruptured blocod vressel~: in th~e

foill~ice w~al. This rLp~ture and the accomp~anyi;ngg loss of

blood may occur either: at th~e time ofr ovulation or prior to

ovulati~on during the rapid phlase of ovz gr-owth. No hypothesic

canr be found-i .inr ther? lite~ratu"re, how~iever-, exp:la~ining the basic

cause for theil )ccurreCe~;' iJ? these~ rup;tuce; in :ith first p-lace!,


J








This lack of knowledge conceerning the reasons for the

occurrence of blood spots is evident when one realizes that

no means has been found of reducing the incidence below

the normally accepted range of from 2 to 6%.

It was the purpose of this research to not only

study new approaches in an effort to discover the cause of

preovu~latory intrafollicular hemorrhage, but: to reinvestigate

some of the cJommonl.y accepted ideas already developed.












CHAPTERS I

'LITER1ATURE~ REVIEW'


Since a large percentage of blood-spot eggs are

classed as inedible and reqluire special han~ding,, they

con~stitu~te a substantial financial loss to the poultry

industry e~ach year. W.ith the innovation of automatic blood

spo~t detectors, producers eliminate man;y perfectly sound

eggs rather than risk the occurrence of a blood-spot agg

among those sent to fresh market. The importance of blood

spo-rs as a factocr in consumer demand was pointed out by

Stadlman(1950). Results of a survey sh~owe!d that 7% of

all consumer complaints about egg quality con;cerncd bjloodt

and meat spots which found their wcay in~to market chainnels.

The p~resecie of detectable amounts of blood i~n eggs may

cau~e a consumer to stop using eggs until they recover from

this adverse psychological reaction.

':he oldest reference, often cited, referring to

the presence of blood spots in eggs dates back to around

3003 B.C. w~hen Arist,-otle explained the condition~ as being

due co a rem~atur~e expousion of -the yclkr. So~me of the

frSrt and mos-t. xtensive studies on the_ blood-spot problem

were conducted at tne University of Illinois 'oy Nalbandov

and Card (19 44L:) These authors r-eachecd the conclusioi n thrat


- 3




1
'x


blocod spots were the resu'Lt of a rupturing of blood

vessels in the follicle wall sometime prior to ovulation

and during the act'rive growing- stage of the follicle. They

state that if blee~ding is significant in amount, the;

r-esulting cl~otC will adhere to thae vitell~ine membrane a~nd

will remain atta~ched to the yolk while th;e latter passes

through the vid-rcuct. If this bleeding is extensive, t-he

blood wJil~l accumulate in the hollow stalk of trhe foll~icl~e

and the clo~t appearing in thie laid egg will not be attached

to the yolk but floating free in the albumen.

Jeffrey (1945) made an extensive study of the origin

of blood and meat spots using a different approach from

those physiological methods usually employed. By recordingy

the color and size of blood spots in each clutch position..

the author determined the time of blood spot formation.

Hi~s results In regard to the time of ovarian heimorrh!ages,

during formations of the yolk, support the conclusions reached

by other rIesearchers.

Stiles et al-. (1_958), using 32P-labeled red blood

cells, developed a procedure~t for- labeling blood spots in

eggs from hens. if labeled cells were injected into a bird

at knownuv~ times du~i~ng, the ovuilat~r~y cycle, thle =ime of

subsequent- blood spotI formnation, could be correla~t~ed to this

cyrcle. Blood spots for~ed after ?the la~beledi c~ells h~adr been

injected showed d~etectable radio~activity if th~e spots




- 5


contained mor~e than 0.024 mnl of blood. Although the

au~thors claim t:hat th~e time of blood sp~ot formatio n can be

determined! by this method, they report very- few results

aind state only thatl- blood spot for~mati~on occurs within a

9-hourli perioj-d prior to ovulation.

After the theory of bloodc spot -olrmatiojn in~ eggs

was developed various attempt~-s were made to control the

intrafollicular bleeding which is essential to their

formration. Possbble causes of the bleeding include abnormally

slowY blood--clotting timte, abnormally w;eak. capillary walls,

a vitamin deficiency which wJould result in either of th~e

first t~oJ and, finallyr,abn\orma~ll high blood pressure which

would lead to frequent rupture of blood vessels.

Halman and Day (1935) found no correlation between

productivity and the occur~rence of egg faults and i~nfer-red

that the condition had a hereditary basis. Quin~n and God-frey

(1940) report that thiey found no significant correlation

between percentage of blood spots and egg production, egg

weight or body weight. They state, however, that a statisti-

cal analysis indiicated significant breed and family differ-

cnces in both yolk and albureon spots in eggs of Rhode Island

Red, W4yand~otte, Whlite Leghorn and Fl crosses of these breeds.

Jeffrey and Pino (19413) concurredl in the opinion that. the

condition w4as heritable. Additionally, they- foun-d that

hens kept in layingj cages producca, 6.2% fewezr- blo.od spots




-- 6


th~an thi;-lr si~sters mainitained~c on thre flo~or. They! also tested

the effect of systematic scaring of the flock~ and found. that

this did not increase the frequency of blood spots produced.

NTo exiplan~ation w~as offered as to whly reduced exercise fromi

confinement_ reduced the inl~cidence of blood spots wJhile

violent exercise did not increase the incidence. The

importance~ of heicedi~ty in the production of blood spots wIas

illustrated by Lerner et al. (1951) wh~o estIimated the

hleritability of the characteristic to be 0.5 or equal to

all other factors combined. Sauter et al. (1952) reported

that on the basis of strain differences withinl the

NEW~ Hampshire breed. heredity appeared to be important in

blood spot incidence. Breed difference tended to confirm

this. The intensity and persistency of lay were reported

to have little effect on the incidence of blood spots.

Seasonal variation i~n blood spot inciden-e has

been reported. Lierner and Smith (1942) found significant

seasonal differences with a definite increase after the

first of April. Jeffrey (1945) reported the tendency to

be highest at the beginning of the laying year and to

decrease through the following; August. Lerner and Tiaylor

(1947) found an increase in incidence of blood spots until

June followed by a subsequent decrease through Septemb~er,

A similar, thoughl not identical, trend, w~as reported by

Sharma (1949). Deaton (1947), using 3 separate periods




S 7


during the year, could find no seasonal influence on the

p~resence of blood spots. Nalbando~v and Card (1944) found

a defi~nit-e increase in blood spots from Decembejr through

July.Sau~ter et a~l. (1952)! reported there is a definite

and statistically significant seasonal variation in incidence.

Percentages ranged from 3..3 to 10.3 for Wh~ite L~eghorns and

fromn 1.3..7 to 39.2: for Ndew Hampshires. Percentages of total

eggs having blood spots were lowest at the start of obser-

vations in December.

Stiles and Dawlson (19359) found a higher incidence

of blood spots in the first egg of a clutch than in

suceeding~ clutch posits~ios. Thie average wceight of eggs

containing blood spots was found tLo be- signifi;cantl~y greater

than the average w~eight of normal eggs when: comparisons were

made be~twe~en eggs of th;e first clutch position and wihen all

the eggs w~ere compared disregarding clutch position. This

difference w~as app=~rox;imatelyl 1.5 g. These authors further

reported there was no correlation between the barometric

pressure or net pressure changes for the area and the

incidence of bl~ood spots in eggs laid 24 to 418 hours later.

They also compared normal eggs teceggs containing blood

spots to determine if experierncesex:isted regarding th~e time

of oviposit~ion, position in clutch and egg weight. In

comparing normal and blood-spot eggs of a similar clutch




8


position, it. was determined that the average ovipositi~on

time of. bloo~d-spot eggs occurred approximately 30; to _50

minutes sooner thans normal eggs.

The effect of nutrition upon thiis defect hYas been

investigated by m~anly. Nalbandov and Ca~rd (1.944, 1.947)

reported that the feeding of dehydrated, unjointed, young

cereal grasses and per-mitt~'c~ng birds access to range

subst-antially Yeducedl the number of blood spots. Both the

size and numrrber of blood spots diminiished rapidly and

steadily when bens were turned on range. The authors stated

that vitamins A, C,, D, E, K and P supplements, administered

singly, had no effect. Dehydrated alfalfa and grass meals

are accepted components of poultry feeds. In addition to

supplying nutrients, they serve as an economical means of

providing dietary pigmnents for desired yolk color. The

effects of dehydrated alfalfa on blood spot incidence are

controversial. Sauter et al.. (1952) reported that birds

fed a ration containing 10% alfalfa consistently produced

a lower percentage of both total blood spots and of medium

and large blood spots. The average was 0.6% lower with

Leghorns and 5.9%~ lower wi~th- New H~ampshires. Carver and

Henderson (1948) reported that hens fed high alfalfa levels

produced few~er~ blood-spot eggs than those fed little or

none~. Bearse (1955) stated chat suboptimal levels of

vitamin Ai caused5 mo~re and above optimal levels caused fewser









blood spots than did the level recomm-rended by the National

Research Council (2000 U.S.P. units/1b). This does not

agree w~ith the ear-lier conclusions of N~albandov and Card

(1.9 4 1.9475) That vitamin A itself might: be involved w~as

indicated by Bearse et al. (1953). Subsequently,, ScottL etr al.1

(1957) reported more blood-spot eggs from hens fed 800 I.U.

of vitamin A per pound of diet than with hens fed higher

levels. Bearse et al. (1960) reported the results of

feeding experimnents conducted with WQhite Leghofrn chickens.

They found that: (1) blcod-spot incidence. was in~creased

at vit~am~in A levels below 1,100) U.S.P. units per poulnd of

feed;- (2) vitamnin A levels in the range of 1.,200 t"o 1,600O

un~t~s per pound result-ed in minimum blood-spot incidence

and that no further reduction occurred with levels up to

1,5;00 units; and (3) birds depleted of vitamin A reserv-es

decreased in incidence of blood spots more rapidly wh~en

fed diets containing 3,60O0 and 10,000 units per pound than

when fed diets contaa~inig 2,300 units per pound. Pope et al.

(19G1) conducted experiments in floor pens and in laying

bLatte~ries w\i~th W.hite Loghorns to- dete~nrmie th~e effects: of

certain minerals, vitamins and u~nidentified factor sources

on blood-spot inlcidence. Sodiumi chloride, potassiumn chloride;

sodium ca~rbonate., their comb~inatIion, vi~tamins a, E and K

and corn fermntnation products had n~o sjignlificant effect

upon1 bl~ood-sptc i~ncidenlce.. Birds fed diets~c containing no




-10


added vitamin A or very low levels produced a greater

incidence of blood spots than did birds fed much higher

level~s. This defect was not associated? with the individual.

hen's ability to mobilize vitamin A as determined by liver

analysiss. The authors further concluded that the extent

to which blood spots occur is influenced by genetic differ-

ences, nutrition and bird age, as wecll as season, management

and stress. March and Biely (1964) fed diets containing

excessive amounts of vitamin A and Vitamnin K to laying hens.

In both experiments, egg production was significantly

reduced and there wuas no evidence that the blood-spot

production was affected. This is in accord with the

observations of Bearse et al. (1960) and Hill et al. (1961)

wrho found that increasing the vitamin A, level in the diet

beyond the requirement for maximum egg production did not

lower the blood spot incidence.

Since it has been shown that in7trafollicular

hemorrhages are responsible for blood spots, the theory

has been postulated that extended bl~ood-clotti~ng time,, as

caused by dicumarol-like products, might cause an increase

in suchI hem~orrhages, thus r:esult-ing in a higher incidence

of bjlood spot; eggs. WCialdroup an~d Harms (19C62) addied

d~icumarol., a vi~tantin K antagonist, to a layer diet and

fail.edZ to sign~ificantly rinc-,ease blood spot-tingq. These




-11


w~orkers also concluded that blood prothrombin time was not

related to the incidence of blood spots. Siddiqui and F'ry

(1963) studied thle effect of warfarin in the, diet of laying

hens and reported that, while the blood prothrombhin time

was increased, the incidence of blood spots did not increase,

TIhe general conclusion, thereforet-, was that the incidence

ofl- blood spots and blood prothrombin times were independent.

Lowered blood prothrom~ibin time of layers, as a result of

adding menadione sodium bisulfite complex (MSBC) to the

diet, has been: associated with an increased incidence of

blood spots in eggs (Day and Wocody, 1964). In contrYast,

thec addition of anticoagulants to layer diets has reportedly

prolonged blood clotting times without affecting the incidence

of blood spots (WJaldroup and Har-ms, 1962; Sidd~iqui and Fry,

1963) These. author-s theorized that eggs from hens treated

with anticoagulants probably have blood in them but since

clotting is slow, the blood is diffused throughout the

albumen and not visible as a clot. D3ay et al. (1964) found

th-at thle incidence of blood spots in eggs w:as increased by

sul.faquinoxaline (SQ) and decreased by dicuu~:arol supp~lemnen-

tain loodJ prothrombn~jr time wa~s sign~ifica~ntl~y incr-eased

by dicumaroli supplementation andr tended to be3 increa~sed by

SQ. It is apparent from these data that thee corr~elati;on,

if any, betw~eenl blood prolthromb~in times ca, ~the in~cide~nce

of~ blood spots is de2pendentI~ upon)r .the~ cau~sative agent.-









Berruti and Didrtick~ (1961) and Day and Wuoody (1964) reported

tchat supplemnental vitamin K decreased the incidence o~f blood

spots in eggs. Sauter et al. (1963) indicated that supple-

mnental alfalfa mneal, a rich source of vitamin K, increases

the blood. spotting of eggs. Day- and W.oodyJ (1964) reported

an increase in the blood-spot incidence as a result of

su~pplemientingn cage layer diets wc~ith MiSBC or alfalfa meal.

Blood prothromrbin tim~e wJas decreased wiith supplemental. MSBCI

or alfalfa meal, suggesting a possible inverse relationship

between prothrombcin time of layers and blood-spot inci~en~ce.

Fry et al. (1968) studied the relationship of vitamiin Kc andc

vitamin A to blood-spot incidence and prothromb~in times.

They reported no definite relationship between prothr~ombin

times and blood~-spot incidence. Bears et al. (:1966) studied

the effect of adding various levels of dehydrated and sun-

cured alfalfa and dehydrated grass on blood-spot incidence.

Different: basal diets were for-mulated and fed, including

one without any of the usual sources of vitamin K and

containing sulfaquinoxaline. Regardless of amount or kind

added, they failed to significantly (P< 0.05) increase the

incidence or size of blood spots in the aggs produced.

These results from bi-ds m~ain!tained on litter-floored pens

are in contrast to finini~rgs orf Sauter et al. (1964, 1965)

and of Day and W~oody (1964/) w:ho observed the opposite effect





-13



with birds maintained in wire-floored cages. Both these

groups suggested that the rations fed resulted in marginal

or deficient levels of vitamin K;. Similarity in prothrombin

times between basal and alfalfa supplemented diets indicates

that the birds on the basal ration were receiving sufficient

uietary vitamin K. Cage-maintained birds did not have

supplemen"- arry vitamin K available from the litter. Sauter et ali.

(1964) conducted an experiment comparing 3 levels of dehydrated

alfalfa meal and 3 levels of vitamin K supplied as MSBC in a

corn-soybean meal basal diet. Blood-spot incidence increased

for all dietary treatments. Approximately half of the hens

inn each dietary treatment produced no blood spots during the

10-week~ 'pasal feeding p~eriod. In contrast, about 90% of all.

hens fed e~itierr alfalfa or vitamin K~ produced eggs with

blood spots. P~etersen et al. (1966) found that hens fed a

vitamin K deficient diet produced significantly fewer blood?

spots th-an similar hens maintained on a cozrn:-soy--alfalfa

mceal diet. The deficient diet, supplemnented with either 3%

alfalfa mie~al: or vit-amnin K; (7485 1g per k=il~ogram as MVSBC)

resulted in a 10% increase .in blood spots. Vitam~in K; add~it~ion:

tos th~-e brasal diet- also resulted inr a further increase of

bliood spots exrceeding! 15% of; all? eggs plroduJced.,

Recenti wJork by Bjearse (1962) has shlown blCOod-spot

inci.dence to increase as pr~ote~in leveli of Ll.e feed is




-14


increased from 12 to 18%. Pepper et al, (1967), while

conducting experiments for the purpose of determining

protein-calci~um interaction in hens, observed that. there

was a significant increase in large blood sports as the

protein level of h-igh calcium diets was increased. The

level of proteiin did! not significantly affect the increase

in large blood spots on the low calcium diets.

Since all the literature reviewed implicates the

ovarian capillaries as the origin of blood found in blood
sposfactors affecting the capll~aries adcnetv

tissue of birds have become of great interest. Shirley

(1965) gives a detailed description of the formation of

a blood spot during ovulation. After a laparotomy was

performed on an anaesthesized hen, the rupturing o-F a

capillary at the stigma was observed. The hemiorrh!-age wans

judged to be beneath the surface layer of follicular tissue

and occurred just prior to ovulation. In the study of any

hemnor.rhagic disorder, the application of capil~lary re~sist-ance

mieasurements is necessary in determining thie nature of the

bleeding. ILt would appear lo~gical that the relative strength

of capillaries would influence the occurrence of hemorrhages

during orvulat!.:ion~. The cause of preovulatory. intr af.011licular

he~morrhage, resu.t~in~g in bloosd spots, has not been detzermiined

however, it~ is; t:LhougIht toC be related ti-o capilayfai1t

by' many autho'"crs. Some repsort~s have shown -that_ hesperidini




- 5


and closely related compounds have a therapeutic effect in

cases of capi.llary fragility of m~anmmls (Scarbolrough and

Edin, 1938; Sokoloff et al., 1957). Hud~cspeth- et al. (1966),

after feeding varying amounts of ascorbic acid and hesperidin

to m~ale chickens, found that, in most cases, both compounds

tended to significantly decrease thie streng-th of capillaries

in the w-jng web as measured by a vacuum tech~niqcue. Carver

and H~enderson (1948), contrary to suggestions by Nalbandov

anid C~ard (194f7), found that the! addition of rutin anld vitamin

to the~ ration did not reduce the incidence of blood andl meat

spots. VJitamin P (bioflavonoids) in the form of lemon juice

was ineffective (Nalbanidov and Card, 1944) and rutin and

a~scorlbic acid fed to chickens, likewise, failed to low4er

incidence of blood spots (Carver and Hendersonn, 1943).

One of the most dramatic changes in bloo~d-spot

incidence wcas reported by Bigjland et al. (1964, 1965) wor~

found that daily subcutaneous injections of water-soluble

derivatives of flavone glucosides, chalcones, significantly

reduced the incidence of blood spots. Tlhe derivatives of

pyrrole-2-aldehyde chalcon~e reduced by twlc--thirds the j.incience

of a hi~gh- blood-spot producing line. TIhe same diose givten orally

had no significant effect. The chalcomes used in these

studies are believed. to be the~ first comp;oundse knownr t-hat

consistently reduce blood-spot inlcidence7. T:he aC1T th~at

chlalconess have no ef~fect on wholce blood cloattincg time,





- 16S -


prothrombcin time, hematocrit values, and erythrocyte count

confirms the observations of Nalbandov and Card (1947) and

Siddiqui and Fry (196.3) that blood spots are not caused by

a hemostatic: defect,

Highn blood pressure could be a contributory stress

in birds genetically susceptable to the hrigh incidence of

blood spots or in birds subjected to conditions whichl may

reduce capillary resistance. Weiss (1958) examined the

eggs from White Leghorn pullets whose systolic blood pressure

difFered widely and significantly in order to compare the

incidence of blood spot formation. No statistically signifi-

cant differences wecre found, .suggcsting; that th~e normal range

of pressure in the White Leghorn does not materially influ-

ence thie occurrence of blood spots in eggs. While arterial

and capillary pressure are generally correlated, they are

not necessarily so, and h~emodynamic controls loca~lized i~n

the ovary and oviduct could operate to maintain a uniforms

capillary pressure. Perhaps the complex venous drainage

and spiral arteries of the follicle play such a role. Ward

and Schaible (1963) report that the feeding of reserpine,

an antihypertensive agent, had no influence on blood-spot

incidence. Fry ati al. (1968) reported the correlation

coefficients of blood pressure with the percentage of

blood spot eggs prodc~uced (+ .156), blood spot scores (+ .059)!,

andl aver~age sc-ore of eggs containing blood (+5 .096j) from




- 17


223 birds. The authors concluded, from tie positive

correlaticn coefficients reported, that blood pressure Is

a factor in blood-spot incidence. HoweJver, since these

correl.ation coefficients are quite small, the effect would

app~-ear to be mn;inimax.

C~hin and Brant (1953) reported that the addition

of aureamyci~n to a laying diet showed s;omre tendency to

decrease the incidence of blood spots with added levels of

antibiotic. These findings are essentially in agreement

with those reported by Berg et. al. (1952) using terramycin.

Andrews et al.. (1966) found that 14 and 16% protein diiet~s

supplemented with 0.02% arasanilic acid increased signifi-

cantly the incidence of blood spots. To a lesser degree,

this was also true of birds in floor pens at all protein

levels u~sed. This may be attributed to the increased

requirement for vitamin K of caged layers fed arsanilic acid.

The authors also reported that protein level wJas no-t associ-

ated with blood-spot incidence.

Anly agent causing a hemorrhagic condition would be

of interest as to their effect on the incidence of blood

spots. W~aibel and PomaroyL (1958) reported that B-amurino-

proprionitrilec (BAPN) produced "dissecting aneurysm of the

posterior aorts and, consequently, death due =o internal

hemorrhagee! in grodinj .turke~ys. Earnett: et al. (1958)

reported thatr vitamin-K suipplemnentationi did no", consistently








redu~ce BAPN-induced hemorrhaging in chickens, thus

:indicating tha?-t. the BAPN effect is not: mediated by' causing

a vitamin K deficiency. Ward and Schaible (1963) found

that BAPN did not affect the number or size of blood spots

produced. They concluded that the effect of BAPN on the

small veins and capillaries of the ovary differs from its

effect on the larger vessels, or it is not of sufficient

magnitude to cause hemorrhaging.












!'HAP~TERF IT

RELAT`ION Or C~OPPER DEFrIC~IECYi TO' BAPN~ TiOXICITY`'





Avian follicles are probably thle fastest grow~ing

structures found in higher vertebrates. It is not surprising

then, tha~t,to accomplish thie task- of transportation anld

deposition of yclk material into the rapidly growing ova,

a veryi' compFlex circulatory system has been developed in

this follicullar area~, Nalbandr~cov and ames (1949) cla.~2s s ified

thle vJenous system: of th:e follicles into 3 lawyers: (1) a

capillary network in the theca of the f:ollicle that drains
by venules into (2) a complex newr peripheral to the

first layer thiat drains into (3) a third venious layer

consistingg mainly of a fewc large veins that drain intro the

follicular stalk. In contrast, the arterial supply is

poorly developed, theil systemir ap-parently depend~inrg on' getting~

the blood into th-e vencous network. When the largest follicle

ovulates, spiral arteries providing the main blood supply

in t-he follicle wall constrict wh~en the ruptured follicle

col~lapses; th:us, thei blood flow~ to the rnow~ emp~ty follicular

sac is greatly reduced anid little, if any, biceding occurs

at ovulation. Th'iis is trsue evren in thei aro tecr


-- 13 -




- ~r3 -


stigma through which the ovumm is ovulated. The stigmaa

i-tself is less vascular t~han the adjacent follicles wall

but is byl no means devoid of: bl~ood vessels (Nalband~ov,

1964),

Eradley and Grahame (1950) described the ci~rculatory

system~ of th~e foll.icle as being a thick player of dense

connective tissue containing an abundance of capillaries

lying beneath the germinal epithelium.

The functional importance of the connective tissue

associated with the blood vessels of the follicle wall in

facilitating the constriction of the spiral arteries at

ovulation and the prevention of ruptures during growth is

not known. The connective tissue of birds, especially that

associated with the circulatory system, has been the subject

of great interest recently. Both the presence of BAPN in

the diet and the deficiency of copper in the diet have been

studied in relation to their effect on the connective tissue

of birds.

In 1961., Hill and Mlatrone, investigat-ing the effect

of a copper deficiency in chicks, reported that a great

number of the copper-deficient chicks died even though the

anemnis waJRS not severe. O'Del~l et al.. (1961) presented

histological evidence which indicated there w~as a derange--

ment in elastic tissue in thle aortas of chicks fed a copper-

deficientt diet. T'he mortality found in these studies was




- 2.1 -


caused by a rupture of the major blood vessel~s. Subsequently,

Carlton and Henderson (1963) and Simpson and Hiarm~s (196;4)

reported that: the el~astice memabrane degeneratedc and aortic

rupjture occurred when thle copper-deficient diet was fed.

All of these findings clearly indicate that copper deficiency

affected metabolism in a manner which was reflected in

elastic tissue integrity.

Starechr et al. (1964~) stated tihat the elastin

content of the aorta of newly hatched chicks is approximately

5% of: the w~et w~eighit of the acrta, W~hen chicks were fed a

diet containing 25 ppm copper, the elastin content increased

to 12%5 by the seventeenth day. Whiren the diet contained less

than 2 ppm copper, the elastin content of the aorta increased

more slowly and never equalled that of the control chicks.

Amino acid analysis of el_3astin from copper-deficient and

control chicks revealed that: the lysine concentration of the

copper-deficient elastin was 3 times that of control elastin.

Hill et al. (19673 found simlar results w~hen following the

content of aortic elastin from the day of hatch to four

weeks of age in chicks fed a copper--defi~cient and a ~ont~rol

diet. These workers reported that the results of copper

deficiency could be reversed. After 16 days of copper

supplementation to the diet: of chicks that h~aj been fed the

deficient feed for 27 days, the e~lastin content of the.

aor-tas of these copper-deficient chick-s had retu.r~nea to normal.




3?
rL


Hill et al. (1967) reported that the lysinie content of the

elastinn from copp~er--deficientc chicks was miuch higher than

that from the controls.

In 1963,. an Engl~ish group under the direction of

Dr. S, MY. Part-ridge isolated and proposed a. struc~lture of

the cross-linkage groups in elastin (Thomas et al., 1963).

Thilis substance is called de~smosine anid its isomer .isodesmosine;

they are tetracariboxyic tetraamnino acids. Partridge et al.1

(1964) obtained isotope data suggesting that desmosine could

arise from the condensation of 4 lysine residues preexisting

in straight-chain elastic precursors. In order fo-~r this

reaction to take place, the auth-ors proposed that the epsilon-

ami~no group of the Lysine residues would have to be removed

and carbon oxidized, possibly to an aldehyde.

The role of copper in the conversion of lysine to

desmosine is considered to affect the o-idative deamination

of thve epsilon a~mino acid group of the lysine resi.due. Ti

type of reaction i~s ca-talylzed by a group of en7zymes, am~ine

oxidases, which contain copper.

Like all elastomers, natural or synthetic, elastin

is essentially a cross-linked gel, and analysis of the

stress-strain curves and other physical properties of moist

elastin shows it to be composed of random coils which are

kinetically free throughout the greater part of their length

bu~t are cross-ilnked~ by firm chemicals_ bonds.









Hill et al, (1967) reported that chicks hatch

without det-ectable amnine oxidase activity in the sorta, or.

liver. When t-he chicks were fed a dict continuing copper,

the activity appeared in both tissues by the third day at

levels which were essentially maintained throughout the

26 days of the experimen-tal period, W~hen th-e diet was

deficient in copper, the activity in the aorta remained

undetectable throughout the period while, in the liver,

activity remained substantially below that of the controls.

These data are in accord with the hypothesis that thie

biochemical lesion responsible for the decreased content

of aortic elastin in copper deficien:cy is a reduction in

amine oxidase activity. This reduction in enzymatic

activity ultimately results in fewer cross-linkages in the

elastin which, in turn, results inl less elasticity of the

aorta and a generaL decrease in the strength of the el.astin

fiber bundles.

Rucker et al. (1968) found in studies with 10- and

20-day old chicks t:hat low dietary copper (4 ~pp) increased

soluble bone collayen when compared with chicks fed adequate

copper (25 ppm). The collagen content was determined by

hydroxyproline concentrations in the soluble and insoluble

protein fractions from femurs. Using benzylamine as substrate,

these authors determined the3 level of amine exi:dace activity

of femurs from 10-day old chicks wais 36%" glreater in~ controls






-- 24y -


than deficient -hicks. No differences w~ere observed for

the calcium, phosphorus, magnesium and copper content of

bone ash fromn birds fed low copper di.:ets.

It has been known for many years that the consumnption

of seeds from certain leguminous plants of the genus Lathyrus

is associated w~~ith a disease that produces physical disa-

bility in humans. Following the isolation of the toxic

substance from Lathyrus odoratus and its identification as

6 -am inopr op i oni tr ile ( BAPN ) (Schilling and Strong,

1955), this compound has been widely used to produce experi-

mental lathyrism in various animals.

Barnett et al. (1957) first described the toxic

effect upon turkey poults by feedinJ BAPN-HC1. The compound

caused paralysis: degeneration of anterior motor neurons and

growJth depression when fed at levels of 0.12-0.25%, and

pericardial and pulmonary hemorrhage, ruptured acrtas, leg

and too deformities and growth depression at lower levels.

Bar-nett and Molrgan (1958) ob-tained similar results whe1n

chicks and chick embryos were used. They described in

de-tail the anatomical lesions of the skeleton and vascular

system induced by the BAPN. Roy and Bird (1959) found

similar resuilt-s when feeding 0.036% BAPN-fumarat~e to ch~icks.

They reported a decrease in growth, a high incidence of leg

deform!itiess and~- some deaths due to ruptured aortas,








TLher-e are m~any reports in thre .literature that, in

lathyrism, the amount of elastin in the large blood vessels

is reduced just as it is in copper deficiency, and that

dietary BAPN produces symptoms which are very similar to

copper deficiency. Partridge (1966) reported that;, in

lathiVrism, the am~ounti of elastin in the large blood vessels

is redued and that feeding with BAPN produces symptoms

which are very similar. Naber et al. (1967) studied the

nature of BAPN-induced lathyrism and showed that the

lathyrogen induced alterations in the molecular aggregation

of collagen which increased the fragility and solubi~lity of

connective tirssue. Page and Bendith (1967) agree ~thiat th~e

lathyrogen appears to exert its effect on connective tissue

by inhibiting the production of new covalent cross-li~nks in

collagen. They assayed, in the presence of BAPN, pig plasm~a

amine oxidase, an enzyme thought to closely resemble th!e

oxi~dase functional in collagen and elastin cross-linking.
The BAPN~ was shown to inhibit en y e a ti i y c mp t t v l

and reversibly forming a complex: wi'th the active site of thie

enzyme. It presently seems clear that BAPN prevents the

formation of normal cross-links betw;een the polypeptide

chains that become a part of the collagen fibril. Consequently,

connective tissue formed during BAP2N intox7;c~:iction in younrg

animlals is fragile and its collagen fibrils do not possess

the normal tensile strength.




Cr
L;O


Tihe hypothersis w~as formed, after a literature

-revi~ew concerned wJith bl~ood-spot i~ncid~lence in eggs, t;~hat

this could be a manifestation of abnormal or deficient

cornnective tissue in the follicle of the hen. An abnor-

mnality in the fiollicular connective tissue due to a de-

ficiency of amine oxidase, for example, could account for

the occurrence of ruptures in the vascular system of the

follicle during rapid growth. A second possibility could

be deficient connective tissue.associated with the spiral

arteries which would not sufficiently decrease the flow

of blood at the time of ovulation. In both cases, the

result could be the inherited characteristics of a high~

inicidence~ of blood spots in an experimental strain of bird..

As a dietary means of testing this hypothesis, an

expe~riment was devised using the effects of copper deficiency

and BAPN toxicity to produce stress conditions upon the

vascular connective tissue of an experimental high blood-

spot incident strain. In theory, if the connective tissue

of the blood-spot strain of bird was deficient in amount

or integrity of connective tissue, these birds would be

more susceptible to these stress conditions than would a

normal strain of bird.




z 2


Genesral Procedures


The experimental birds uszd in the following studies

were~ a strain of Singlez Comib W~hrite Leghorn h~ens which

produced a high percentage of blood-spot eggs. This strain

wias originally selected by the Wveste-rni Washington Experime~nt

Statioin (Pjuyall1.up)i and has be~en mrainanda the Florida

St~a tion since 1965. These birds will be referred to in the

~-tex ais -the blood3-spot strain. Control birds were selected

from a commercial strain. of Single Comb W~hite Leghorn.

In examining eggs for the presence of blood spots,

each egg was broken out on a glass-topped. stand. The2 yolk

was s3eparated f-rom the a~lbu-aen and its entire surac~e

carefully1 exami~ned under a m~agnifying3 lamp. The albumnen
was then cheked for the prsneo blood. Based o~n the

mnaximum dimensions of the largest single inclusion of blood,

the eggs w~ere scored on the following scale: (0) no blood;

(1) 1/16 inch or less; (2): 1/16 to 31/16 inch; (3) -- 3/16

to 1/2 inch; (4) greater th~an 1/2 inch;; and (5) -- presence

of bicod in the albumen. Some~ subjective adjustment wIas

made by increasing the score when large numbSers of blood

spots wierce present. The adjusted~ score wars, therefore,

more indicative of the t~ct-al amount of blood present in the

egg. All m~eat spots Jwere eliminated fromt thne s~o?-ring system.

All eggs to be examjrnd w~er~e stored at 55;'P for no longer

than 4 days anid no less than 1 day.





- 28 -


All birds used, both the blood -spot and the control

strains, were raised in the same manner. Whn17z not being fed

an expeirimental diet, thzey were maintained on the standard

farm feeds (Table 17) which were form~ulatied usingc a corn-

soybean meal base, Upon reaching maturity, all birds wiere

placed in individual cages conist~ruccted to facilitate egg

coll.ection and. identificationn and allowing for the mainte--

nance of production records for each individual hen. Both

the experimental and the standard farm feeds and water

were fed ad libitum.

The birds used in t-he studies were individually

selected on the basis of high production and a normal healthy

appearance. The birds from the blood-spot strain were

further selected on the basis of a high incidence of large

blood spots. After selection, birds were randomly placed

into the various groups used in the experiments.


Exper i imen7 t I


Thel first experiment was conducted to ascertain

the feasibility of using a copper-deficient diet and BAPN

toxicity in comparing vascu:lar integrity. These dietary

methods were used to make comparisons between birds of the

normal and bl~ood-spot strain. Additionally, the -elation-

ship of feeding a low-copper parental diet to chick

suscepFt~ibility- to BA4PN t-oxicity was observed.













TIABLE 1

Standar~d farm? formulas


Ingredienta % of diet

Starter Grower Layer

Yellow Corn Mleal 62.85 81.60 69.85

Soybean Meal (50%)- 31.00 12.25 19.00

Alfalfa Mleal (203) 2.50 2.50 2.50

Ground Limestone 1.00 1.00 6.00

Defluorinaited Phiosphrate 1.90 1..901 1.90

lodized Salt 0.25 0.25 0.25

Microingredient Mlix, 0.50 0.50) 0.50


% Protein 21.6 14.0 16.3

Productive Energy 977 1043 962

% Calcium 1.10 1.07 2.86

% Total Phosphorus .73 .66 .67


!I~upplied~ per~ 'Ii?:?gram~ of diet: vitamin A, 6600 I:.U.;
vitamnin Da,20 ...;vrai 2.2 :g.; rib~OflaVinr 4.4
m~g.; pantath-,enic aci2, 12.2 r:.:4.; r.- cin, 39. 6 rng. choline, 499.4
mg.; vitami Byz 22~ v. g; Sa-~n t-c7uin7, 0.01.23%; i~ar:angne se, 71.4
mg.; iron, 19.8 mg.* copper, 1..9S ag.; cobalt, 0.198 mg.; iodine,
1.1 mg.; and zinc, 99 mcg,





- 30


Th epeimntl roedure w~~as divided into tw~o

phases in preferenlce to superimposing the stress of copper

efficiency and BAPN (P-aminoproprionitrile) toxicity upon

the vascular system of birds simultaneously. In the first

phase, the effect of feeding the low-copper diet (Table 2)

upon t-he production, fertility and hiatchability of the

boo0d.-spot and normal strain of hen in the first year of

production was dete~rmined~. In the second phase, day-old

chicks hatched during the first phase of the experiments

were pl~aced on a standard starter diet modified by increasing

the added fat to 9% and containing 0.06% BRAPN-fumarate3

(Table 3), Barneett and MlorLgan (1959) reported that mortality

due to massive internal hemorrhages increased when the fat

level in the diet was increased even though the chicks fed

the hi h-fat diet consumed less feed. The parameters used

in determining the effect of BAPN toxicity in this phase of

the study wer-e toe and l~eg deformities, mortality, incidence

of aortic ruptures and body weight. Following this twoJ-

phase expersi~mentali procedure, the predisposition of the

chicks from hens fed the low-opper diet to the toxicity

of BAPN could be determined, Three consecutive studies

were conducted following this general experimental procedure.

Inl the first stud~y, 5 groups of laying hens of 20

birds each: were used'. TheiiLse groups conIsiC-ted? o~f~ blo-o





- 31. -


TABLE 2


Compositio of -o-cpe layer di

Ingredients % of diet

Dried sk~im milk 50.0

Cerelose 31.0

Corn oil 5.0

Glycine 0.5

DL-methionine 0.3

L;-arginine 1.0

Choline Chlorcide (50%) 0.1

Vitamrin A (250,000 10/gm) 1.0g

Vitamin Dg (3',000 ICU/gmn) 3.5gr

Vitamin E (20),000 10/1b) 20.0g

Santoquin (67%) 8.0g

NaH12PO (22.45%)P 4.0

CaCC3 (40%; Ca) 6.0

Microingredients2 0.5

Salt premix3 1.5


'By analysis, diet contained 2ppm copper.

F~urnishes per Kg diet (mg/ky): 0.03 vitamin Bi2, 0.30
biotin, 1.0 menadione, 8.0 pyridoxine HCL, 4.0 folic acid, 16.0
ri'coflavin, 100 nicotinic acid, 20 calciumn pan-otathenate, 24
th~iamrine.

3Furnishes per pound of premix< (gramns): 9. n~,90.7
MgSOu, 90.7 iodized salt, 18 FeSO, 7Ha0, 0.33 ZnCl2, 0.013 CoC12,
137KC1.





-- 32 -


TABLE 3

BAPN sbarter diet


Ingredients % of diet

Yellow corn 50.4

Soybean meal (50% protein) 25,0

Alfalfa (20% prote~in)30

Corn gluten 4.5

Animal fat 9.0

Fish meal 5.0

DL-methionine 22.7 q

Defluorinated phosphnat-e 2.0

Ground limestone O,2

Todized salt 0.4

Vitamin premnixl 0.5

S-aminopropionitrile 0.06


!Supplied per kilogram of diet: vitamin A, 6600 I.U.;
vitamin D3, 2200 I.C.U.; vitamin K, 2.2 mg.; riboflavin, 4.4 mg.;
pantothenic acid, 13.2 mg.; niacin, 39.6 mg..; cholin~e, 499.4 mg.;
vitamin B1e, 22 mcg.; Santoquin, 0.0125%; manganese, 71.4 mg.;
iron, 19.8 mg.; copper, 1.98 mg.; cobalt, 0.198 mg.; iodiine,
1..1 mg.; and zinc, 99 imcq.





-33


and _normal h.ens--ach insemrinated using semen from normal

males. A third group w~as composed of blood-spot hens

inseminated from blood-spot males, These 3 groups were

supplied the sem~isyntheticc low;-copper diet. The remaining

2 groups con~sisted of~ blood--spot h;ens and normal hens---eachl

insemilnat~ed from, their respective males, These last 2

groups were fed the regular farm laying feed and served as

a standard i~n comp~aringr the production, fertility and

hatchability during the experimental period.

All inseminatioins were made using 0.05 ml of pooled

semen, The first 2 inseminations were made at a 2-day

interval and thereafter the birds were inseminated every

7 days during the experimental period. Thle first

3 groups w~ere fed the low-copper diet for an 18-day period

imnmedilately following the second insemination. After this

feeding period, they wcere placed back on the regular farm

laying diet for a period of 12 dayrs. E:t~ensive precautions

w~ere taken: during the miiixing and feeding of the sem~isynlthetic

lowi-copper diet to prevent its adulteration w:ith copper ions.

All mixing apparatus and feed containers were carefully

washed, rinsed with a chelating agent and given a final

rinse with deionized, distilled water prior to use. Both

"the feed and water trroughs were lin;ed wih~th polyet~hylene

which had bteen wa~shed and rinsed as described ;Ibove. During

the 30-day experimental period, eggs were eolle~cted daily,





marked for, identification with thle date and hen nlumuerr and

inlcubated every 3 days. ECggs were candled on the fifth and

eighteenth day of incubatl-ion.

As the chicks hatched every 3 days, they were wing-

banded and placed in wire-floored bat-teries. All chicks

from the 5 groups were fed the modified starter diet con-

taining BAPNl fror a 5-jelek period. Each day th~e groups were

checked and thle dead birds posted to determine if death

could be attributed to aortic rupture. At the end of the

5-wieek growing period, the surviving birds werec visu-ally

scored for severity of leg and toe disorder and weighed.

The seve~rity of disorders for each bird w~as graded using

a scale from 1 to 3 for each leg. A score of 1 indicated

the absence of any disorder and a score of 3 indicated the

most severe cases. Scores for the legs were added; t-herefor-e,

each bird received a score of from 2 to 6. The birds were

weighed by groups and sex, the latter determined by secondary

sex chiaracteristics.


Results and Discussion

Production for the various groups are shown in

Table 4 and are presented as percentages based on the number

of hens during each 3-day period. The final number of birds

used as a source of data for each group was dependent upon

-the health; and performances of the bird during the entire




- 35 --


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30-day _period. All data collected from a bird which went

into a molt, died or failed to produce a single fertile egg

after the first 2 insemrinations were eliminated from the

statistical analysis. The final number of birds used in

each group axs a source of data are listed in the table.

The production of th~e normal. a normal and blood--

spot x2 blood-spot crosses wLhich remained on the farm laying

feed was comparablee and remained constant, indicating no

definite trendcs. Any large differences in the productiion

of these groups fromr one 3-day period to the following may

be attributed to the stress of handling during insemination

and normal variation expected when using relatively small

numbers of birds ini'each group. The actual production

percentages themselves are not as important as the trends

which they indicate.

The production in, all groups fed the lowJ-copper

diet decreased. This decrease in production is most evident

in the normal z norm~al cross. ThLe normal xe blood-spot

cross group displayed the lowest production rate during the

second 3-day period after removal from the low-copper diet.

In all glroups fed the low~-cop~peri diet, production retu.!rnred

to the pr-et-reatm~en~t level. after feeding the commercial type

d~iet* for a 12-day., periodi.

Using the2 number of eggs laid during thec 3-day~j

periods for Individuall hens in the groups as an observation,





- 37


a7n analysis of variance was conducted (Snedecor, 1961)~,

The production rates for the groups, periods anid the group x

period interaction were all significantly different at the

0.1%~ level. Thee interaction of group x period indicated

that as the copper reserves in the hen's body were depleted

on the low-c~oprper diet, production decreased si~gnificantly.

In Table 5, tihe means and the standard errors for

each group ar~e given. It canl be seen thiat -the production.

rates for both the= normal x normal and blood-spot z blood-

spot crosses were significantly lowcered when fed the low~-

copper diet as compared to the sa~me cross fed the farm~ feed.

No di~fference was found in the e~ffct of the low--copper diet

upon the normal x blood-spot and blood-spot blood-spot

crosses.

The fertility and the hatchability of the fertile

eggs inl each of the 5 groups during the ten 3-day periods

are lishced i~n Tables 6 and 7, respsectively. W~hen the

incidence of infertile eggs w~as analyzed using chi-square,

it was found that while the occurrence in the blood--spot

group was significantly greater (P < 0.001) th:ran in the

normal x normal group on the farm feed, only the normal

birds wiere affected by the dietary treatment. Thie increase

in incidence of infertile eggs in the normal xh normal group

on the low~-copper diet compared to the normal. x normal

control group was significant; at the 0,.5% level. In the




- 36


TABLE 5

Aver~age nuvmber of eggs produced pe-r he~n, duzring
30-day experimental period


6-day groups

Group 1 2 3 4

Cross B3S x BS' BS x BS N x N2 N x N Nx B:lS

Diet Cul L4 Cu- L C3u
F~ea5 :.:?.IQ e b 18,2C i.87
Mean 1111a17.170 13.63 1.3

SEM' 0.92 1.33 1.56 1.22 1 02


1B:lood-spot st~rain.

NMolrmal commerlrcial strain.,

3Semisynthetic low-copper diet.

"Standard farm layer diet.

sThe average number of eggs produced per hen in each group;
means witlh d~iffer-ent super-script are significantly differ~ent-.

6Standard error of the mean.










ul t ,- cc *
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- 41 -


normal ,- normal group during the first 3-diay period af;:er

being fed the low-copper diet, the fertility decreased to

42.9% wJhich represents approximcately~ 50% of the initial

fertility. T~The dietary treatment did not affect fertility

of the blood-spot strain, The occurrence of infelrtile eggs

in the normal xc blood-spo~t group decreased to a level

between that of the 2 parent strains and near that of the

normal x normal group,

The trend for hatchability of fertile eggs was

similar to that of fertility with the normal x normal cross

treated with thle low-copp~er diet showing a large decrease

during thle first 3-day period after being placed back on the

control diet. The normal xt blood-spot cross showoed a steady

decrease in hatch~ability wJith length of time fed the experi-

mentall diet, dropping from 95 to 45% in 18 days. Analysis

of the data by chi-square showed significant differences

between the hatchability of fertile eggs in t~he 5 groups

at the 0.5% level. The normal birds had a significantly

gjrea~ter- (P < 0.05) hatch than did the blood-spot birds.

However, in each cross, the feeding of the low-copper diet

did not affect hatchability. Hatchability of the normal x

blood-spot cross was almost identical to that of the blood-

spot z blood-spot cross fed the low-copper dict.

Thle results of the first phase of -ihiis stuldL are

n~ot in agreemnen~lt. withn those of Slim-rpson et al. (19657) wcho





- 42 -


reported a decrease in hatchability to 0 in 10 days whiren

hens were fed a similar low-copper diet. These authors

further reported that feeding a low-copper diet had no

significant. effect on production when using a normal. strain

of bird.

The 5-weekc weight of the chicks placed on the starter

diet containing 0.06% BAPN are presented as the average

weight for each-group during a 3-day per-iod (Table 8).Th

analysis of variance showed that the period effect was

signiificant at the 1%t level and that the period x: cross

interactions was signi-ficant: at the 5%k level. These factors;

were the only two found to be significant. There wars noG

detectable difference in body weight due to sex. It does

not appear that the lowJ-copper parental diet affected the

chick weights to any great extent. The chicks from the

blood-spot hens, while not significantly different in

weight from those of normal hens, were heavier regardless

of the parental diet. The lowest chick weights recorded

were fromr the normal a: normal cross and occurred when hens

were placed back on the parental control diet. These chicks

w~ere from -the sam~e group and period which had the lowest

fertility and b~atl-chcbril~ity in the first phase of th~is study.

The scores indicating the severity of leg and toe

deformities of the chicks are presented in Table 9. No

differences were detected in these scores as a result of




- 4 3 --


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- 45 -


parental t-reatmecnt- or strain of chick. OvJer 80% of the

birds had deformities which were att-r~ibu~ted to the BAPN

toxicity.

Thne incidence of' death and deaths specifically

caused by the acrtic rupture during the 5-week period on

the BAPN diet were relatively low and are shown in Table 10.

The post mnor-tem examinationi of the dead birds showed that

only a moderate percent of the deaths could be attributed to

a rupture of the aorta.

The incidence of death in the 5 groups as analyzed

by chi-square were significantly different at the 5% level,

indicating that the normal chicks were more susceptible to

the effects of BAPN than were the blood-spot chicks re-

gardless of dietary treatment. The incidence of death due

to aortic rupture was significant at the 0.5% level and the

normal x normal cross again appeared more susceptible than

the blood-spot ;:train to the toxic effects of BAPN. No

treatment differences between the 2 groups of each cross

were detected indicating that the parental diet had little

effect upon the susceptibility of the chicks.


Excp~2ierimet 11


The second study was conducted to clarify those

results previously obtained. The oinexpected loss in production

of hens fed a copper-deficient diet h~ad not previously been





- 46 --


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- 47 -


report-ed in the liter~aturte; thus, it'was necessary to

repeat thf-e first- study in an attempt to sjubstan~t~iate those

results.


Pr o c dure2;

The same general procedure outlined in the first

study was follow\ed wiith only a few~ m~inor Cchang~es. The

number of groups used was reduced to 4 with 20 birds each.,

Groups consisted of 2 of the normal and 2 of the blood-spot

strain crossed with their respective males. One group of

each cross was fed the low-copper di~et during thle first 15

days of the 30-day experimental period. The experimental

diet wras found by analysis to contain the same level of

copper present in the diet of the first study, 2 ppm. The

practical type diet was not analyzed. The remaining group

of each strain served as the control and remained on the

regular farm laying feed. To eliminated a possible source

of copper ions, all birds were provided with deionized,

distilled water ad libiiumn. During the second -phase of the

experiment, the chicks were raised in floor pens on peanut

hull litter. Th"is procedurall change was made to eliminate

the influence of the wire floor in the batteries upon! the

occurrence andi severity of leg and toe disord1Ers. The

amrount of EAPN added to thle modified, starter di.et was

increased fr~om the previous level of 0.06 to 0.13i in an

effort to increase the incidence of aortic rupturzes, h






10
CC


chicks were maintained on this diet for 21 days at which

time the study was tLerminated. Body? weights were obtained

by groups according to treatment (cross andf diet) for each

3-day period.


Results and Discussion

The production of the 4 groups during each of the

ten 3-day periods-are presented in Table 11. Tihe analysis

of variance for the number of eggs laid by individual hen~is

during each 3-day period revealed that thle effects of

treatment, periods and the treatment x period interaction

were al.l significant at the 1% level. Applying the error

of the mean squared showed that the average production of the

4 groups during the 30 days were all significantly different

(P < 0.01). The normal group of hens maintained on the

control diet had the highest rate of production. Feeding

the copper-deficient diet resulted in the greatest change

in production to the normal group, decreasing from 81 to 9%

after only 6 days. Production remained at this low level

until the birds had been returned to the control diet for

a period of 4r days. The control group of blood-spot birds

produced at a significantly lower rate than t~he normal

strain of conrt~rols. eWhile feeding the low-copper diet

significantly d~ecreased produc-cion in bocth strains, th're

decrease observed in the blood-spot strain was not as great





4 .


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C- (? U-- O -4 U


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Un in Q L





- 50y -


as that observed in the normal strain. Production of the

blood-spot group dropped to only 35.5% from the initial

rate of -73.3%. Prod~uc~tion of both treated groups returned

to the initial rate after being placed back on the control

diet for 9 days.

The fertiliity and hatchability of these 4 groups

during each 3-day period are given in Tables 12 and 13,

respectively. The chi-square analysis of the fertility data

showed that only the dietary treatment effects were signifi-

cant (<0.1.While it is not evident, from the per~centagres

given in Table 12, the only significant difference (P < 0.01)

was th~e decrease in fertility of the blood-spot group when

fed the low-cap-per diet. The decrease in fertility of the

normal strain of birds during the last 3 days on th~e experi-

mental diet was not shown to be significant. The production

of thi~s group during the seventh to the flif'teenth days on

the l~ow-copper diet was extremely low; this greatly influ-

enced the weight given to thie fertility and hatchability

results. The percent fertility and hatchab~iity during the

last 3 days on the low-copper diet were based on a total

of 3 aggs laid by the 18 birds in the normal group. No

significant differences were found in the hatchability of

the ~ groCUp=s.

The anal.ysis of varilance of the chick weights at

21 days of age showed them to be significantly different




..- 51r -


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- 53 -


at the 1% level. These weights are given in Table 14. The

birds in both normal groups were significantly (P < 0.0OL)

heavier thanl thle blood-spott birds in the remaining groups.

In both groups fed thle low-copper di~et, growth was s~ignrifi-

can~tly depress-edj (P < 0.01.) when each strain wqas compared to

it~s respective con-trol group. No differences we;re detected

in the effects of the parental diet on the susceptabiity of

the chick to the toxicity of the BAPN in the first study,

This difference in the results of the 2 studies could

possibly b~e affected by the increased level of BAIPN used

in the second study. The higher concentration of BAPN

surpassed the threshold level of both strains and, thus,

failed to indicate the differences previously observed,

The possibility that the differences in body weight can not

be detected until after the third week of age may also be

another con~sidera~ticn. The chicks from all 4 groups hatched

the last 3 days were placed on regular farm starter diets

without the addition of BAPN and, thus, served as positive

controls. Chick~s of the normal strain w~eighed mo~re than

those of -the blood-spot st-rain. All. 4 groups of the positive-

conitrol3 chicks wecre 20 g or approximately 20% heavier at

21 days than the comparable groups fed the BAPN-treated

diet.

No differences wer~Le found in the occurrence or

sev?~erit~y of leg3 and toe disorde~rs between bir-ds of th~e




.. 5


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- 55 -


4 groups. The- amocunti of varia-tion finhierent in the

subjective scoring mnetihod wnas probably so large that the~

sensitivity may have not been great enough to detect

differiences between groups if they did exist. The 2-w!eek

difference in age of the chicks at the time they were

weighed and scored complicated comparisons between those

in the first study raised in batteries and those in -the

second study raised on the floor. However, there appeared

to be no increase in these disorders as a result of

increasing the level of BAPN in the diet.

Since the chicks of the 4 groups in each 3-day

period were intermingled, nio data were obtained on feed

consumption. The incidence of leg and toe disorders

reduced the ability of the birds to reach food and water;

however: the actual reduction in feed intake could not be

determined. This effect probably accounted for the greater

variation in the weight of the birds in the first study at

5 weeks of age as compared with the variation in weight of

the second study at 3 weeks of age.

Mortality and the incidence of aortic rupture in

the 4, groups during t~he 21--day growth period were analyzed

by chi-square. No significant differences between the

groups were detected for either mortality or aortic rupture.

These percentages are given in Table 15 and compare favorably

with those reported by Barnet=t and M!organ (9).The






-56 -


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- 57 -


increased level of BAPN in the diet did not noticeably

increase either the mortality or the incidence of aortic

ruipture wrhen compared w~ith the first study, The. incidence

of death and death resulting fro~m aortic rupture are

pl~ottedJ against the age of the bird in Figure 1. Aortic:

ruptures were not detected until the sixteenth day and the

incid-'ence increased until the experiment texrminated. The

direct relationship, shown in the graph, between the

incidence of death and death attributed to aortic rupture

indicates that a much? larger number of deaths were probably

the result of aortic or other vascular disorders than

could be detected by gross post-mortem examination.

It can be concluded from these results, as it was

in the first study, that no vascular we~akness or abnolrmali--

ties were detected in the blood--soot strain as a result~ of

the stress placed uipon them by feeding a copper-deficient

diet or from the BAIPN toxicity. T'he results of this second

study confirm those of the previous one--that the blood-

spot strain of birds is not as susceptible as the normal

strain to the stress of a copper-deficient diet. The

response of the 2 strains of chicks to the stress of th~e

BAPN was not significantly different.

The strain of bird involved in nutritional studies

using semisynthetic or purified diets has a great effect

upon thie results obtained (Ne~sheLm 19G68) Variance in the





- 58 -


E.Y.
a ps a


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S59 -


nutritional requirements between strains of birds is well

documented (.Kondr~a and Hodgson, 1961, and Balloun and

Speers, 1969).

W~ith~out further evidence, the differences in

nutritional requirements between the normal and blood-spot

strain cannot, be directly or indirectly related to the

factors whrIich result in the high incidence of blood-s~pot

eggs produced.


Experiment Ill


A third study was conducted using a copper-sup21e-

mented, semisynthetic diet as the control in ascertaining

the effect of a low-copper diet upon laying hens. In

previous studies, the commercial type diet served as the

control. To obtain a better indication of the effects of

this low~-copper diet, analyses weJre conducted to determine

liver-copper levels. Furthermore, the aortas of the chicks

hatched during the first phase were examined at 1 day of

age for the presence of abnormlalitie~s in the morphology

of the connective tissue.


Procedure

Th7e procedure followed that used in th-e pr-evious

study. A total of 28 birds were used, 14 from both the

high blood-spot incident and normal strains. Half the birds





-; 60-


of each strain were fed the low-copper diet (basal_) for a

period of 18 days. The remaining 7 hens from each strain

were fed the basal diet supplemented with 16 ppm of copper

as copper sulfate. Dietary analyses showed that the basal

contained 3.0 ppm copper and the supplemented 18.0 ppm

copper. Production, fertility and hatchability were

determined during the 18-day period. At the end of the

experimental time, the hens were exanguinated and the intact

livers excised. Each liver was placed in a polyethylene

bag and frozen in a blast freezer at -180C where they were

subsequently stored. The livers were prepared for analysis

by partially thawing before dicing with surgical scissors.

Each diced liver was then equally divided into 2 crucibles.

Samples were dried overnight at 1100C, cooled in a dessicator

and the dry weights obtained. Using concentrated nitric

acid, the solid material was digested by boiling on a hot

plate. The crucibles were heated until contents were

almost dry and then placed in a muffle furnace for ashing.

After reaching 2000C, the temperature was raised 1000 hourly

until the desired 6000C was reached. This temperature was

maintained for 12 hours to complete ashing. The ash was

then dissolved in boiling 10% hydrochloric acid, filtered

and diluted to a known concentration. The iron and copper

levels wiere determined using an atomic absorption spectro-

photometer according to the method recomr-mended by the

manufacturer (Anonymous, 1964).





- 61 -


It has been shown that hiens fed a lowj-copper diet

produce chicks within abnormalities of the vascular system

(Sim-ipson e~t al,1r967), Howerver, it is not known if these

abnormalities are corrected by feeding a normal starter

diet during the first several weeks of growth. In Experiment

%III half the chicks hatched from the 4 groups of hens

during each 3-day period were killed at 1 day of age. The

hearts and aortas were excised and placed in 10% neutralized

formalin until prepared for examination using a Philiprs

EH' 200 electron microscope followiing the procedure of

Simpson et al. (1967). The remaining chicks were maintained

in batteries for 21 days and fed the regular practical type

diet. Whenn theyr reached 21 days of age, they were killed

and the hearts and aortas removed for comparison with those

remioved~ from birds of the sam~e group and period at 1. day

of ago.


Results and Discussion


Therates o~f production for -the 41 groups d~urin~g

each 3-day period are given as percentages in Table 15.

The analysis of' variance showed that the periods were

significantly different (P < 0.01) and that the period x:

treat~ment interact-iSon wJas significant at the 5%0 level.

While these r-esults showr a definite dietary-I; eff~ct, thet

ninuber of birds used ini each groupy wJas too low~V to showi




























Averages for 3-day periods

Nuo.
M F Diet Birds 1 2 3 4 5 6

BS x BS' CuT 5 40.0 46.7 40.0 33.3 33.3 45.0

N x N2 Cu+ 6 60.1 66,7 55.5 44.4 55.6 66.6

BS x BS Cu' 5 46.7 40.0 46.7 53.3 53.3 50.0

N x N Cu 7 71.4 71.4 66.7 42.9 28.6 21.4


- 62 -


TABLE 16i

Percent egg production of normaZ and blood-spot h2ens
fed a copper-deficient dies


'Blood-spot strain.

2Normal commercial strain.

3Basal diet supplemented with 18 ppm copper.

4Basal low-copper diet.




- 63 -


any treatment differences. Thez feritil~itiy and hatchabi~lity

are listed in Tab~le 17 and 18, respect:-ively. Again, as a

result of the small number of birds used and their com-

paratively low rate of producticn, no significant differ-

ences were detected between treatments or were any trends

indicated in the results obtained.

Results of the copper analyses on the livers are

presented in Table 19. Treatment was significant at the

1%~ level. The copper levels in th~e livers of the 2 strains

of birds fed the supplemented diet were almost identical.

The mean level in the normal group w~yas 21.16 ppm and in

the blood-spot group was 21.22 ppm. The copper levels of

the 2 groups fed the deficient diet were not only signifi-

cantly lowerr (P < 0.01) than their respective controls

but were significantly different (P < 0.01) from each other.

The livers from thle normal strain fed the basal diet con-

tained 13 ppm of copper as compared to the livers from

blood-spot birds which contained 16 ppm. These results

clearly indicate that the normal strain was more sensitive

to a dietary copper deficiency. The supplemental diet

containing 18 ppm of copper was well above the supplemental

level used by other workers (Hill and Mnatron~e 1958, 1961).

However, the 21 ppm of copper in the liver of the control

birds is below the 32 ppm found inl 3 livers from laying

hens of the n~ormnal str~ainn fed the standard type diet in




























Averages for 3-dayr periods

No.
M F Diet Birds 1 2 3 4 5 6

BS BS Cu 5 100 85.7 100 80.0 80.0 100

N x N2 Cu+ 6 100 100 100 100 100 100

BS x BS Cu 5 80.0 100 100 87.5 71.4 87.5

N x N Cu 7 100 100 100 100 100 100


IBlood-spot strain.

2Normal. commercial strain.

3Basal diet supplemented with 18ppm copper.

"Basal low-copper diet.


-- 6,4


TABLE 17

Percent fertiity of normal and blood-spot hens
fed a copper-deficient dief





- 65 -


000





00
. *O



Ll) 0 0' 0X









0 *















S to


+ 1 0
* * 0
ma00




m ta













m x






xx

03%mZ


v








O




m

rt


*O
SO




e
Ok


*

a,
O
O


co







-lJ

me





O-r(






Em


Sa




O r
z






- 66 -


O

V




















CO,

HI


a -r
a

C 0

U E C
Q)OO
0 4 *H
-0- M

'0 *H



O O -H



a, 3




Sa, m s


to-momb m <-I C~~
LO mC CO L N n CO \


+I








,Q


Inhecutomm eo H

+1








r
coman t:mL e m d r
er o ct a 6e O f 1 U

Hl~ototocom H H
NHNNNHMV~r (
+1













mmmoocome r( r
NNHHHNHl~r N
+1










1-I Um ytom ~o


Cto
I o

a ae
R








X i






~c





O ta


*


l 3
m m

















Qd +


t


E U3




- 6? -


this study. This decrease in the levels of liver copper

followsr the same pattern found in the rates of production.

The normal strain in both cases was more sensitive to the

low-copper diet.

Results of the iron analysis of these livers are

presented in Table 20. The analysis of variance indicated

that thie 4 group means were significantly different at the

1% level. The iron level in both strains was significantly

decreased by feeding the low-copper diet. The differences

in the level of liver iron between the 2 groups fed the

same diet were not significant. The normal bens showed

the greater decrease in liver iron due to the dietary

treatment, decreasing from the control level of 561 ppm

when fed the supplemental diet to 308 ppm in the group fed

the basal diet. This compares with the blood-spot strain

which decreased from a level of 496 ppm iron in the

controls to 377 ppm in the group fed the low-copper diet.

This decrease in the level of liver iron as a result of

feeding a low-copper diet is not unexpected. Maynard and

Loos~i (1969) state in their text that when copper is

deficient in the diet of swine, there is a decreased

absorption of iron, a lowering of its total body content

and a decrease in its mobilization from the tissues.

These results clearly indicate that the hens fed

the experimental diet were in a stress condition caused by






- 8


.--
O
.







14


C,

O


*H
W









a M



a, a



rd Cl




O O -4



a tP
OH < 0
00 0
O >O
a -r -I E
aaums


.Q
In ~ ~ ~ ~ L r c 0
000mNNC C

+1








.0

~~~~~ rfm c D
b~~~ n~Ca3 *
ci vincm e him m
< >






+1









n


C1O > llAr; N L 0
m un Io L m m to
+1















(U+ t










C Eer


U












0




*












0
-1




Q


O





co
2 0
Pr
vtI
Ob
n o

ScA
o 0
o -,

04
4'


CT
(




- G9


a copper deficiency. However, the observed effect of this

dietary deficiency of a single nutritional element may

ultimately result from its effect, in turn, upon the level

and availability of' other elements in the bird..

Except for 2 groups, all the samples were lost

prior to sectioning. These aortas were removed from the

chicks of henis which had been fed the supplemented diet.

A total of 19 aortas were sectioned and examined for

abnormalities using the electron microscope (Table 21).

The examination of the aorta from a blood-spot chick from

the fifth 3-day period, representing the thirteenth,

fouirteenth and fifteenth days the hen was fed the supple-

mental diet, was not conclusive. From this section, it

could not be determined if there was a definite change in

structure of the elastic fibers in the tunica of the

artery. When the 6 aortas from the normal chicks of the

same period were examined, 2 were normal and 2 were

definitely altered. Alterations in the vascular connective

tissue are generally characterized by fragmentation and

disruption of the elastic fibers. Of the 4 aortas examined

from the blood-spot strain taken from the sixth 3-day period,

representing the sixteenth, seventeenth and eighteenth day

on the supplemental control diet, all but one of the aortas

appeared to be altered. Of the 8 aortas rem~oved from normal

chicks during the same period, 1 was normnal and unchanged,






- 70 -


TABELE 21

Observations of electron micrographs of aortas from day old
chicks treated with a low-copper parent~al diet


aorta examined

Par-en-tal'
Strain period 1 2 3 4 5 6 7 8

Blood-spot 5 +-

Normal 5 + + .+ + -

Blood-spot 6 + + +-

Normal 6 + + + + + +- +- +-


Note: (+)Abnormal, change in structure of aorta wall.

(+-)Possibly abnormal.

(-)No change in structure detected.

'Period of parental dietary treatment, diet contained
18 ppm copper.




- 71 -


2 were border-line and their condition not definitely

determined, arnd the remaining 6 were definitely abnormal.

~It ir, scignificantt that such a large numbr~er of

aortas fromn chicks whose parental diet contained 18 ppm

copper possessed abnormalities characteristic of a copper

deficienicy. The National Research Council lists a re-

quirement of only G ppm copper for the chick, and, for

older birds, the requirement has not b~een determined.


Experiment IV


The 3 previous studies demo~nstLrated .that the

observed reaction of the laying hens fed the experimental

diet was a result of the stress condition induced by a

copper deficiency. The effects of the copper deficiency

reported in these studies were similar to those of Bird et al.

(1963) and Simpson et al. (1967) but they we:cre not identical

in all respects. These authors reported only slight de-

creases in the production of treated birds. While the

di-etary; effects of these studies are attributed entirely

to a dietary copper deficiency, other factors must be

involved which, if recognized, w~oul~d account for the differ-

ences in the observed results. The genetic backgyround~ of

the bir-ds used in conduicting nutritional studies is recog-

nized as a source of variation in the results obtained.

Washburn (19 69) found significant (P < 0.01) inter act ions









between genotype and the diet used when studying the

hematological response of different stocks of chickens to

iron-copper deficiencies. A comparison of the diet used by

Simpson et al. (1i967) and that used in the previous studies

revealed that these 2 diets not only differed in the form

in which some of the essential inorganic elements were

added but also In the presence of magnesium and cobalt.

This study was conducted to determine the effect

of differences in the mineral content of diets used in

studying copper deficiency upon a normal strain of White

L~eghorn~s.


Procedure

The effect of 3 semisynthetic low-copper diets

upon production, hatchability and fertility were compared

(Table 22). The first diet was formulated according to

Simpson et al. (1967). This was a low sulfate diet to

which neither magnesium or cobalt salts were added. The

second diet was identical to the first except for the

addition of sodium sulfate. The addition of this salt

increased the sulfate in the first diet to a level equal

to that present in the third diet. The third diet was

identical to that used in the previous studies which

included salts of magnesium and cobalt. A practical type

farm laying diet was used as a fourth treatment. It served





- 73 -


TABLE; 22

Composition of semi~synthet~ic Zou-copper Zayer diets'


Ingredients % of Diet

1 2 3


Dried skim milk 50.0 50.0 50.0
Cerelose 35.0 35.0 32.0
Corn oil 5.0 5.0 5.0
Glycine 0.5 0.5 0.5
DL-methionine 0.3 0.3 0.3
Choline C1 (25%) 0.2 0.2 0.2
CaCO, 5.0 5.0 6.0
NaH2PO4 . . 4.0
CaHPO, 2.0 2.0..
Mlicroingredients2 1.0 1.0 1.0


Salts (gramns/100 pounds)

NaCl 90.80 90.80 136.05
ZnCO, 20.43 20.43..
ZnC12 ... 0.50
Fe citrate 27.24 27.24..
FeS04 7H20 . . 27.00
MnCO3 14.07 14.07 .
KlnS04.. 15.25
KIO3 0.18 01
KC1 . . 205.50
CoC12 ... 0.02
MgSO4 ....136.05
NaS04 . 200.00..


IBy analysis contained 2.0ppm copper.

2Vitamins added per 100~g of diet: vitamin A, 2000 USP
units; vitamin D3, 433 ICU; and (in milligrams) m~enadione, 2.5;
a-tocophe~ry1 acetate, 2.5; Santoquin, 0.0125; thiamnine-HC1, 1.0;
riboflavin, 1.0; pyridoxine-HC1, 1.0; Ca pantothenate, 3.0;
niacin, 5.0; inositol, 50; biotin, 0.04; folic acid, 0.2; and
vitamin Bi2, 0.003.





- 74 -


as a positive control in det~ermini~ng the adaptability of

the hens to the copper-deficient diets. Each of the 3

low-copper and th;e positive control diets were fed to 2

replicate groups of 6 birds each for a period of 15 days

following the procedure outlined in thle previous studies.

The birds used in this study were a commercial strain

of White Leghorns.


Results and Discussion

The- analysis of variance showed that the 4 dietary

treatments differed significantly in their effect upon the

rate of production at the 0.5% level. The production of

those birds receiving the unsupplemented and sulfate-

supplemented diet of Simpson et al. (1967) during the

experimental period was significantly lower than the

production of the other groups. WhSen the results of the

2 replicates wiere combined, the birds in the first group

produced an average of 4.8 eggs during the 15-day experi-

mental period. This production rate is only slightly higher

than the average of 4.2 eggs produced by birds fed the same

diet supplemented with sulfate. The difference between

these groups was not significant. Those birds fed the

experimental diet: used in the previous studies produced an

average of 7.J7 eggs during the same period. All birds fe'd

the low-copper diets produced at a significantly lower





--75 -


(P < 0.01) rate tchan the positive cont-irols which produced

an average of 12,4 eggs during the 15-day period.

The effects of 'che 4 dietary treatments upon both

fertility and hat~chabilit~y w~ere significant at the 1% level

when analyzed by chi-square. The unsupplemented and sulfate-

su~pplemented diet of Simpson et al. (1967) resulted~ inl the

greatest incidence of infertile eggs produced as w~\ell as the

lowest hatchab~ility. While the effect of these 2 diets upon

fertility and hatchability were the same, they were signi~F-

can~tly different (P < 0.01) from the fertility and hatchability

of the remaining 2 dietary treatments. The positive control

hecns exhibited the lowest incidence of infertil~i~t and the

greatest hatchability. In both cases (fertility and

hatchability), the results were significant at the 1%1 level

when compared to the results obtained from those groups

receiving the semisynthetic diets.

Eird et al. (1963) do not give the complete

dietary formula used, reporting only that it w~as a nonfat

milk-solids diet and that only a slight depression in egg

production was observed after feeding the low-copper diet

for 20 wJeeks. Simpson et al. (1967) reported that egg

production, as a result of feeding a lowi-copper diet, was

not significantly affected. These authors do report: that a

group receiving the semisynthetic diet supplemented within

46 ppm of copper produced at a slightly low~er yate c~ormpared





S 76


to hens receiving a practical type diet. Both workers

report a reduction in the hat;chabilit~y of fertile eggs.

Bir-d et al_. (1963)\ observed a reduction in hatachability of

fertile eggs from 83 to 11%. Simpson et al.. (1967) stated

that ~thatcaility was reduced to zero after thie birds had

been fed the low3-coppe~r diet for 10 da~ys.

One of the major differences between the experimental

diet used in the previous studies and that used by Simpson

et al. (1967) was the form in which some of the essential

elements were added. In the diet of Simpson et al. (1967),

only the manganese was added as the sulfate salt. In the

low-copper diet of the previous studies, the manganese, as

welli as the iron, w~as added in the sulfate form. The

nutritional role of sulfate has been the subject of many

studies reported in the literature; good examples are those

of Mdachlin (1955) and But~ton et al. (1965). Brown et al.

(1965) reported that decreasing the level of dietary sulfate

decreased the production of' collagen by.preventing the

formation of cross-linkages. In 1967, Brown found that

the exten-dability of aortas was decreased when rats were

fed a diet low in sulfate.

In the present study, the sulfate level does not

a~ppear to be the dietary factor responsible for the difference

in results. Mo~rrison (1959) reports the need for magnesiumJT




- 77 -


supplementation when fee~dinig a diet of whcle milk as well

as for iron and copper. Magnesium was not added in the

diet of Simpson et al. (1967) but was added to the diet

used in the previous studies as the sulfate salt.


Summaryj


A series of experiments were conducted to determine

if differences could be detected between the integrity of

the.v~ascular connective tissue of a normal and blood-spot

strain of bird. These 2 strains were compared in stress

and nonstress environments. The definable stress environ-

ment was created by feeding both a copper-deficient diet

and a diet containing toxic levels of BAPNJ. Both these

diets are known to exert their effect upon the vascular

connective tissue, preventing the formation of normal collagen

and elastin. With deficiencies present in the connective

tissue of the blood-spot strain, it could be expected that

they would be more susceptable to stress placed on the

connective tissue.

The statistical analyses of the results showed

significant differences between the response of the 2 strains

to these dietary treatments. However, abnormalities in the

vascular system of the blood-spot strain w~ere not detected.

The normal strain proved to be the more susceptable of the






IC


two under the experimental conditions. The envioronmental

stress resulting from the experimental dietary procedure

did not pr-oduce any observabl.e response in the blood-spot

strain; such a response would have indicated an absence of

vascular integrity (abnormal or deficient connective

tissue).

Interprettion of the results must be made with

2 factors in mind. First, the difference between the 2

strains in their response to the dietary treatment may

result. entirely from a variation in their nutritionally

requirements and not be related in any way by factors

contributing to the incidence i~n blood-spot production.

Secondly, vascular abnormalities present in the follicular

wall mnay not be indicative of the state of th~e vascular

system throughout the body. If weakneisses- in the conn~ective

tissue of the vascular system in the blood-spot strain

exist, they are probably isolated in the follicle, Whaatever

deficiencies are present i~n the follicles of the bl~ood-spot

hens, these conditions do not appear to be characteristic

of' the ent-ire vascular system.

A more direct approach is necessary if the primary

factors resp-onsible for the occurrence of sl~ood spots i~s

to be~ resolved. I-t is obvious that,wvhen comparing the

results of feeding semisyn~thetic diets, the interaction of

elemient~s present, espe~-cially when the die-t is deficient in

an essential element, must be taken into consideration.











CHAPTER III

RATE OF OVA GROWTHCI AND OVA SIZE IN BIRDS OF THE
NORMAL AND BLOOD SPOT STRlAIN


Introduction


In studying the occurrence of blood spots, the

critical period is from the initiation of the rapid phase

of ova growth until ovulation. During this period, hemor-

rhage may occur, ultimately resulting in the presence of

a blood spot in the egg. Differences between the integrity

of the follicular vascular system of the two strains of

birds would not be the only abnormality which could account

for a high incidence of blood spots. A more accelerated

increase in the yolk size during the rapid phase of ova

growth or a larger yolk at maturity could produce a stress

upon the follicle wall resulting in ruptures of the blood

vessels.

Stiles and Dawson (1959), when comparing normal

and blood-spot eggs from a similar clutch position, found

that the average oviposition time of eggs containing blood

spots occurred 30 to 50 minutes sooner than normal eggs.

They also reported that the average weight of eggs con-

taining blood spots was significantly greater than the

average weight of normal eggs when comparisons were made


- 79 -





- 80 --


between eggs of the first clutch position and when all the

eggs were compared disregarding clutch position. The authors

concluded that eggs containing blood spots were ovulated

sooner, weighed more and that these were abnormal eggs at

or prior to ovulation. Total egg weight is of little value

in attempting to determine the condition which exists during

the critical period, since the albumren which accounts for

the majority of variation in egg size and the shell are

formed after the formation of blood spots.

The period of rapid development of the ovum has

been traced by using a fat-soluble dye incorporation tech-

nique in studies reported by -Warren and Conrad (1939) and

by Bacon and Skula (1968). After a thorough survey of the

literature, no reference was found where either the rate

of ova growth during the rapid phase or yolk size at

maturity were studied in relation to the occurrence of

blood spots.

Phy~siologicaally, interest in the problems of blood

spots is centered around the extremely rapid rate of fol-

licular growth and the attendant development of the necessary

blood supply. The avian follicle is the fastest growing

structure known, developing as it does, from a size of

about 2 mm in diameter to full ovulatory dimensions of

32 m~m in diameter during a period of 9 days (Sturkie, 1965).

It was desirable, therefore, to determine if the growth





- 81


rate and yclk size at maturity were abnormal in hens

producing a high incidence of blood-spot eggs.


Procedure

Based upon production records and a high incidence

of large blood spots, 45 birds in the first year of pro-

duction were selected. The same number of control hens of

the Babcock strain were selected on the basis of high

production.

A gelatin capsule containing approximately 30 mg

Oil Red O dye was orally administered to each of the 90

birds. Beginning on the date of dye administration, eggs

were collected daily for a period of two weeks, and indi-

vidually marked to identify each hen. All eggs were hard

cooked the day following collection and bisected by cutting

along the long axis through the latebra. Two perpendicular

measurements (to the nearest 0.5 mnm) were averaged in dete-

mining the diameter of both the whole yolk and the dye ring.

The percent of yolk development at the time of dye admin-

istration was determined by calculating the ratio of the

dye to the yolk diameter. The average of these ratios for

both groups were plotted each day to determine the rate of

yolk growth and the length of the rapid phase of growth.

Egg production and the mature yolk size were also determined

during the experimental period for the hens in each group.





- 832 -


Results and Discu~ssion

Th~e rates of ova growith for the normal and blood-

spot birds used in this study are plotted in F'igure 2

with the variance for each day during the test period.

The dye did not appear in the egg yolk until the third day

after administration of thle dye capsule. Thec data obtained

failed to indicate any differences betw-~ieen eggs of the

two strains in relation to ovarian function as measured

in terms of rapid development period. In both strains,

the o7va required a period of about 8.5 days to reach matulrity

after initiation of the rapid phase of ova development.

This period of rapid growth is indicated on the graph from

the third to the ninth day of the experimental period.

For the remainder of the experimental period, the size of

the ova at time of dye administration remained constant,

and results after the twelfth day are not show~n. These

two7 phases coincide with thle periods of slowJ and rapid

growth of the developing ova first reported by Warren and

Conrad (1939).

Bacon and Skula (1968) noted that fol.licular

ma-turation (cessation of rapid development) and ovulation

were not necessarily simultaneous events. They report that

differences exist in? the length of the res" period, the

tim~e from~ the. cessation of rapid growth until ovulatio~-n,

and; of eva in di~fferent clutch positions. From the data





-- 83


-DO-
E _I I


o 1---7 ir-i rj


'G 00 'O c t
O E
I! r- O C
ar 0 -.-? cn O
r: rr r----
ol -a -r( I

k r- co rl I

r; r to -H 1

r, '0C
FO k -
L1 rj re
*4 ri C--- -
O;1 U /-- O













e- r.- -.-re-- -- .-.--- ---- r -. - -- .-r -~-' "` 1

en CO 3 to m ca


vi71:~-~3 6





- 84 -


obtained in this study, no difference in the rest period

was detected between ova from the 2 strains, regardless

of clutch position.

The average yolk size during the experimental

period for each hen was treated as a unit and these units

were statistically analyzed (Table 23). Yolks from the

normal strain of hens were significantly larger (P < 0.01)

than the yolks from the blood-spot strain of hen. It is

possible that the relation of egg size and oviposition

time to the incidence of blood spots reported by Stiles

and Daw~son (1959) is not a cause-and-effect relationship.

Those factors responsible for blood-spot production may

also result in a shorter oviposition time and larger egg

size. Strain differences probably accounted for the

larger ova size in the normal strain rather than in effects

of factors which contribute to blood-spot incidence. These

data, together with the length and ra-te of rapid growth,

give no indication of differences between strains which

could account for the difference in incidence of blood

spots.

Suimmary

The rate of ova growth during the rapid phase of

maturation and the ultimate size of the ova were compared

in the normal and blood-spot strain. It is commonly

accepted that the hemorrhagee which results in the formation












TABLE 23

Average size of yolk produced by 42 hens of both the
normaZ and blood-spot strain during a 10-day period


- 85 -


Blood Spot


Normal


31.4
31.9
31.3
30.9
29.1
31.3
31.9
30.6
31.8
32.2
31.8
30.8
31.6
30.4
31.4
31.4
29.9
31.3
30.8
32.9
30.6


32.4
32.1
31.0
31.4
31.2
31.7
31.5
30.0
30.8
30.7
32.6
31.0
31.1
31.5
32.2
31.8
31.6
31.7
32.0
33.4
31.9


31.5
30.8
30.0
31.0
32.0
33.4
31.8
31.3
33.2
32.6
31.2
32.0
32.4
31.9
32.1
32.9
30.7
32.9
31.1
31.1
32.7


31.6
32.9
30.8
32.1
30.8
32.6
31.7
33.3
31.8
31.7
32.2
32.8
31.2
32.2
31.3
33.3
31.1
31.4
31.3
31.9
34.0


31.92 + 0.14b


mean + SEM


31.40 + 0.12a


'Yolk
M~ean
difference (P


diameter in mm.

yolk diameter, sulpersc~ripts denote significant
< j.01).




- 86 -


of a blood spot mnay occur during the last few hours of

rapid growJth o~r just prior to ovu~lat-ion. Stress sufficient~r.

to result inl a hemorrhage could conceivably result from an

excessive r1ate of ova growth or an abnormally large ovaumr

in the blood-spot strain. Using a dye technique, no detecta-

ble differences were observed between the rates of o~va growth

in -th-e 2. stra~inss of hens. In bo~th strains, the l~ength~ of

the rapid growth pha~se of ova maturation was 8.5; days,

which is within the expected range. While the mature ova

of the normal strain were significantly larger (P < 0.01)

than those of the blood-spot strain, this difference was

quite small and does not appear to be related to the inci-

dence of blood spots.










CHAPTER IV

THE RELATIONSHIP OF BLOO>D PRESSURE TO THE INCIDENCE
OF BLOOD SPOTS PRODUCED


Introduction


Blood pressure is often mentioned as a possible

factor influencing the occurrence of blood spots. Nalbandov

and Card (1944) reported a higher incidence of blood spots

in Sirds having a higher blood pressure. This difference

was not significant and the authors concluded that high

arterial pressure is not the primary cause of rupture in

follicular vessels. After comparing systolic blood pressure

and blood-spot occurrence, W2eiss (1958) concluded that the

normal range of pressure in the Wh~ite Leghorn does not

materially influence the incidence of blood spots. In 1.968,

Fry et al., using a strain of White Leghorns that produced

a large percent of blood-spot eggs, reported that blood

pressure was significantly and positively correlated with

both size and number of blood spots produced.

In this study, the effects of lowering the ~systolic

pressure of laying hens upon the formation of blood spots

was determined.


- 87 -





- 88 -


Procedure


Birds of the blood-spot strain were selected on

the basis of a high incidence of large blood spots. Sys-

tolic pressures wjere indirectly measured by a physiograph-

six; (manufactured by E-M~ Instruments Co., Inc.) using a

sphy gmomanome ter -The cuff was placed around the distal

end of the tibia and the pneumatic pulse pick-up on the

artery passing over the hock joint. Three determinations

were recorded for each bird, and the average was used in

subsequent calculations.

The systolic blood pressure was reduced using

methimazole, a goi~trogenic agent. The birds- were treated

by giving them free access to the standard farm laying

diet containing 0.06% methimazole, commonly referred to as

tapazole (registered trade-mark of Eli Lilly and Company

for 1-methyl-2-mercaptoimidazole). The level of 0.06%

was found, through a series of preliminary trials, to result

in the greatest decrease in blood pressure with the least

loss of production. W~vhen birds of the blood-spot strain

were fed a level of 0.03% tapazole for an 18-day period,

the systolic pressure was not significantly reduced. W~hen

the tapazole level was increased to 0.1% in the feed, the

rate of production immediately decreased. After 11 days,

all birds were out of production.




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