|
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 --
U) O
r-4
CO
-
-ri
0
-s
C'
ito
-C)
0
C
'
E-1
Cl Z
:N
iO N ]
C1 CO
CO r-
m m
1.0U
01 01
**~L
L)
Co
fr M
0 0
O
'
v3
$
0
-
~ o
o
t7~~
r*7
1- ul
COO
. .
mon
m vI
coH
NHr
0 C
LA LD
LA U
v to
LnN
d 0
-H 0
-0
c, o
-9 U 0
(n '
O 5 4
O O -c -W
r0 O in u)
;r. TC .I- r
* *3 *
) U
In I o
r- rl t
x it
x x x
[n CA
r-INZZZ
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 *
21 HC Lt) i' 0 O
I O cn N
~J-I- 1 4l3 t C? 0* *X
IJ oco w
(rd LO 01 Ci (
'~s k mi o0
't- O rT1 r 0
C1O mU U) \) C3 o l
!I I
ti -4 I O- O- 0D
rt o
iir I I Lol
LO- * * c
oc 01 01
Wc ul oo ,
m~c at Cc(3O 0
Lo ii Ir D t0t l
E- o
9 0)
uN a
ct I II o iv c Hc 01 to U
8 v > c 0 p .
ON C 01 ,
" 3 t=1 S O
GT, O> O N 0 < O C
Jm M U \ -r~
fi3 3 0 r0 u cu a
O O
N 0
O~ GoX
mN m 9
-- 0 -
a
O
CO
c
* '
r'- O
G1 CO
o 0
N co
(r3 r0
co r-
3~ r
( 0
[r-l C
io
O~
r0
*H
---
- v
CO
P-''
r3 s
E-1
rr
n3
O-
ro
co
'U3
0
O ,
r;-Q
XU
s
W '+
co ~
... cI
m ao>
4 ,S
RO
a
0
o
rT
O
-H
a
rj
I
In In
rN u7
ON
r03
003
H co
LO
rJ p
**
m D k
Od r
O i=
0- k
r -1 h r
m 'd
ev m
O
c
to
IP I
C 0 0
to c to
(3 CH W
to
O
k '
cr.
x
rr
X X
5 2
- 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 --
(''- ci T Ln r 0~ 1.07 (V (? C3 (0i CC CO3
N H N r ~~~~ r-IN VO .^OPl~~t 7 t ) O WN!
\U j J J N i CI N *NNGHH\ ';!` -Ir
i O -I co 0 3CC m *G)MON *MO
OPM OO adam LOO!C:r~'-~r JN ~?
N--INNH H *HO3 N *040
Co
Ot
00s
-
PI
CM R
TQQ
'0 F
XO
OR
o n
I~ n rl 01 c3 C\ *
YOB~OUDO1ND
~NN~NCMCNNNN
11 C0 OD LT; I0 I
OOO Nr CN
m rm cOTmM
I`D cJ 0I (3\ 010 Ln CO m rx.-, w NCH
NNNmmm~mmNNNNNN
WLTo r) ecno m3emb e a me r L
omm~obeavowomome
ONNN NN N N ~C ~Cl N N C JNN
~mOHHOMMOHO~Cmwm
MON~mubmwtomemNO
cim (-;N van.NN C:ClCl N N irm
`s m n 3 -1oc \caca e an N
MHNHO Hl rl r- >>I0 SI~I ~ N O
(? 17m mm M MN ~N N NN Nm o
<0 e. y
%<~~~C~ ~~ eA
r N
m (
x
-J,
m
uT)
Z
..- .4 4 ..
I)
rJ
n4
O rl N' (9 C3
CN N CJ CV
rV 01 (1 N OU
N~ ( m m N
(0 LO OOcU
ra m m m N L`
CID W Hn LOi
m
0~: .c < t
L~ZZm
O
V3
LO
Q\ v
's
to
i)
E
-C
16
*
aO
I -
ri 3
9 O c
O .0
E -H
E nr
CO CO
(6
Tr.
r
ra
O
k t
- 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 --
N HM( r ~ O N O uLn o Cjc
~oo HH mHH Loem emm
to
Gl
13
to
0
00
r ~
~PI~O
~ N m ,i N m ~ c~l m
r
n
LOm
CO1 -
\nr-i c O NUO 0000 CTOO~ \
NO r-- O CI NH r
r-Ir-I -
r-I r-- H
--sam .-aim --asm
T I
U U
x x
CO O')
0F n
* *
Lo Io r-I cyt
crl cy
.- am .-, iam
~-J U
O
k
mo
O
0 2'
I:
a,
- 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 .
r-4 m co ah rm
U1~0 CO NnI
.)j CIN LO NCI
) C m NI to
L, II to 'D 1.
Da 1n '9 LO N o
w O) Lo m m 01
-0 Lo u
I I
rd I IQ
O o c
-i~a II 1 uo *0
~'0
0 M
c~~ r l o co i0 mc
co r-- c o -H 4 m
r34 Ii O
ii1 C) '0 &
o I 'J- I rd i
03 <3 rg f? CO
to u1 c 01- O 0 -
C- (? U-- O -4 U
a !I II I a
om x xrcJ m rlr
O XX r
mM Co r
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 -
03 CO
O C
a a,
C)
r--1
TO
i*c*
PJ
Sco
c
LO
O
A
-0
1
4-,
FI
O,
0
ot
e 9
r-4
N- 0
I
yr; 0
E-1 0
I-H
a,
c-7 tl
r-( CT
*~ *-
cS C
O N
I*
CJ 01
co as
I
U
(D)
O) -H
to~
ri
O
4E
un to co c
r-1 r-1 H
X X
03 C
0 0
0
H
to
u,
SO
-P
a
V)
I
Oo)
* W
O r
0 3
CO ; L1
...)
:.0 CO Ns
r! 01 01
ZI co
LO [U
LO 0 1
n *
0\ 'o
*CO
CO
0 a0
C01 CO- ri
H 0
coo
b00
0300
an! on Cr
ri H H
2;
x x
cmZ
I-(
- n
c
- N
C
I-H
C
a
r
9
O H
m
O
)
r.
to
SC~
O O
m 00
IN B
r(O
- 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
a co a~ asmr- ves
N~N NNM NM Nc7
H NO i H r--
1 ~ - 0 A O 3 O
LIO HH i?(3O C\J
to
Cc,
re
54
On CO tD LO
cO rr rH LO
LT I--O
r-1
UH 03 r)
rlQ~H r
r10
rl
r--
Cr
SQ)
.L
ag
m iP
to <
co~ > 0
H 01 01
NCO a t
C NJO
N O O N H
N-- r1 H 0 N -
r- r H r--I
NH! NO H H
1 -( I 0
-, t
a
-0
+
UIN
,.4
O
Ur
*
E r
w k
rd
J 3
9O
m GT
NO
r
NrH Nr HH
ri r- r
~1
i 0
- 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 -
Nr-40 WO 'InN 0
Nama~C'
N~r Lo N\ < 17 r-4 NV2 r-i `= 10 r-i
NNNNd
'j to ~ m rA ls o LO P-- O f cr 0
CV N r--I NH
h)
Ot
ega
O
Lo
;1)
3
O
t
s
r-1
o)
EN
o)
co
O
Ni nd al1 FImH ~
WHH~ UtrO
O
r(
r( ri m ct cJ ~', r~ C1 crl ~~ CI .")
4 J
9
O
* -J
k -Hr.c
'3 *
Od u 01
',- 1 -
00
1Q 3 g
Z 0'
< CLO r- V
N ~r-
d
C
mNc
04
H r-4
9H~rO LnCO
r-
e m a \
C; CA cU CN
O
- 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
,, ***'
ny
~eas *am
Pln.~**Mana,
'"'""~ .. S
"""
S.~
to Z
-r-I
dP
rd!
O
a r 1
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.
|
