Q1998 Applied Poultly Science, I n c
EARLY FEEDING
AND DEVELOPMENT
SYSTEM
IN
OF THE IMMUNE
NEONATAL
POULTRY'
J. J. DIBNER~,c.D. KNIGHT, M. L. KITCHELL,c.A. ATWELL,
A. C. DOWNS, and E J. IVEY
Novus International, Inc., 20 Research Park Drive, Missouri Research Park
St. Charles, MO 63304
Phone: (314) 9267410
F M : (314) 926-7405
Primary Audience: Nutritionists, Immunologists, Hatchery Managers,
Primary Breeders
hatchery practice. In some parts of the world,
DESCRIPTION
OF PROBLEMproducers strive to place the neonates within
In modern poultry production, the separation of the hatchery from the production
facility means that the hatchling will spend a
period of time without provision of feed or
water. The time period between processing
and placement is highly variable. It depends on
the availability of transport equipment, the
distance to the placement facility, and the
a period of hours. This reduces stress and gets
the birds to feed and water, and into the
brooding environment. In other parts of the
world, the practice is to hold the birds for
12-24 hr, to allow them to mature and to initiate a vaccine response while the birds are
under low immunological challenge from
other antigens. Often the producer has no
1998 Poultry Science Association Informal Poultry Nutrition Symposium:
"Impact of Early Nutrition on Poultry."
2 To whom correspondence should be addressed
1 Presented at the
426
options, for example during shipment of
birds over long distances.
Producers are well aware that post-hatch
holding and bird processing are hard on the
hatchlings and take steps to minimize their
effects. Many hatcheries attempt either to
place the birds within hours of removal from
the hatcher or to hold them for a sufficient
period of time to mature and recover from
processing. This rest and recovery from processing has an additional benefit. Just before
the hatching process begins, the bird internalizes what is left of the yolk sac, which has
nourished it during incubation. The residual
yolk protein is the source of antibodies from
the hen [l]. To be effective, maternal antibodies must not only move from the residual
yolk into the bloodstream but must also diffuse
to sites of vulnerability - in particular to the
mucosal surfaces where organisms are most
likely to enter the body. Thus a delay in placement can leave the hatchlings better able to
respond to the environment once they are
placed.
The attendant problem is that birds are
generally not fed in the hatchery - even when
held overnight - nor are they fed during transport. Producers may feel that feeding is not
essential during this period because conventional wisdom says that the bird can survive on
its residual yolk [2]. This is a valid statement
but does not completely represent the modern
chick or poult. While the survivalof a hatchling
may indeed depend, in the absence of other
feed, upon its use of residual yolk as a nutrient
source, the research described in this report
indicates that this is not the optimum use for
residual yolk. In addition, data indicate that
the development of the immune system in
particular appears to respond to early feeding.
Three mechanisms are proposed to account for the dramatic effect of oral nutrition
on the hatchling immune system. First, early
nutrition may provide limiting substrates;
second, feeding may affect endogenous levels
of hormones or other immunomodulators;and
third, the presence of antigen in the gastrointestinal system may be necessary to trigger
full differentiation of the primary immune
cells, particularly the B lymphocytes. Complete differentiationof these cells is critical for
the eventual development of secondary immune structures such as germinal centers or
cecal tonsils, along with the associated ability
EARLY FEEDING AND IMMUNITY
to respond to a vaccine with the development
of immune memory. Studies will be discussed
with reference to these three postulates.
MATERIALS
AND METHODS
In these studies, the effect of early feeding
on the immune system of broiler chicks [3]
was evaluated by measuring the weights of
immune organs and levels of serum and biliary
immunoglobulin A (I@) [4],and by evaluation of cell proliferation and immunoglobulin
isotype expression by lymphocytes. The nutrient source fed to these neonatal chicks was
a hydrated nutritional supplement (Oasis
hatchling supplement) consisting of 70%
water, 10% protein, 20% carbohydrate, and
less than 1% fat [q.This hydrated nutritional
supplement (HNS) was replenished daily and
was fed to the treated birds ad libitum on the
day of hatch (Day 0) and the two subsequent
days (Days 1and 2). Control birds were fasted
and given no water over the same period. Beginning on Day 3, all birds were given water
and fed an identical corn-soy starter diet formulated to meet or exceed National Research
Council recommendations for starter feed [6].
Effects on performance, organ weights, immunoglobulin expression, and levels of serum
and biliary IgA were measured in four birds
per treatment on Days 0, 1,2, 3,6, 7,8,9, 10,
13, 14, and 21. Tissue sections of small intestine, ileocecal junction, bursa, and thymus
were prepared and stained using hematoxylin
and eosin for purposes of morphometry. To
evaluate microscopic structure, villus length,
mid-villus width, crypt depth, and bursa follicle area were determined. Hematoxylin and
eosin staining and immunocytochemical
methods for bromodeoxyuridine and IgA
have previously been described and those for
immunoglobulinsM (IgM) and G (IgG) differ
only slightly [A.
In a separate study, birds were fasted or
were fed the hydrated nutritional supplement
for the day of hatch and the day after. All birds
were then allowed to consume the same cornsoy starter diet ad libitum. Buds were challenged orally with a 1OOx dose of a commercial
coccidiosis vaccine [8] on Day 14, i.e. after
12 days on ad libitum feed. Birds and feed
were weighed on Days 7,14,and 20.
Symposium
DIBNER et al.
427
the present studies focused the effects of
RESULTS
AND DISCUSSION
early nutrition on the bursa and on developIMMUNE SYSTEM ONTOGENY
The immune system of the bird is partly
developed at hatch. The primary immune organs, the thymus and bursa, are both present,
and are populated by lymphoid tissue. The
migration of lymphocytes to the thymus occurs
in several waves, beginning at Day 6 of
embryogenesis. These cells pass through the
thymus and populate peripheral tissues [9].
The thymocytes are CD3+ (avian homologue)
and develop CD4 or CD8 antigens during
embryogenesis [lo]. In peripheral organs,
however, development of T cell receptor
specificities (as or yS) and of CD4 and
CD8 markers occurs after hatching [ll]. The
seeding of the bursa by lymphocytes occurs
between embryonic Days 10 and 15[12]. These
cells are committed B cells but are capable
of only IgM expression at hatch [13]. The
secondary immune organs, such as the
spleen, cecal tonsils, Meckel's diverticulum,
Harderian gland, and the diffuse lymphoid
tissue of the gut and respiratory systems are
incomplete at hatch [14]. There are B cells in
the cecal tonsils, but these only express IgM.
Similarly, there are T cells in the lamina propria and epithelium of the gut and in other
secondary immune organs, but these do not
develop helper or cytotoxic capability until
some period after hatch. The ability to mount
a secondary response, as indicated by the
presence of germinal centers or circulating
IgG and IgA, begins to appear between 1
and 4 wk of post-hatch life in the broiler chick
[151*
The effect of thymectomy or bursectomy
on the development of the immune response is
one indicator of its functional status at hatch.
Neonatal thymectomy does not result in severe
impairment of cell-mediated responses or the
development of T-cell diversity, indicating a
fairly high degree of development during
embryogenesis [16, 11. Bursectomy of the
neonate results in an impaired humoral response, particularly in the areas of isotype
differentiation and development of antibody
diversity [la]. Bursectomy as late as Day 18
of incubation can result in a total loss of
circulating IgG and IgA, leaving a primary
IgM response of very limited diversity as the
only humoral immune capability [19]. Because
humoral immunity is less developed at hatch,
ment of responses requiring cell-cell interactions between the humoral and cell mediated
systems.
Figure 1 shows sections of bursa on the
day of hatching. Sections were stained using
antibodies to chicken IgM, IgG, or IgA.
Clearly, the dominant immunoglobulin in the
bursa of the hatchling is IgM (Figure M).
These IgM-bearing lymphocytes are the precursor cells for those expressing IgG or IgA
[u)].When the bursa was stained for IgG
(Figure lB), the only positive areas were
found in the interfollicular connective tissue,
specifically in the blood vessels. The origin of
this immunoglobulin is the residual yolk sac
[21]. The IgG detected in the bursa of this
hatchling represents maternal IgG deposited
in the yolk by the hen. The hatchling does not
have the capabilityto produce IgG at this age,
and is entirely dependent on the maternal
antibody for humoral immune protection
(22, 231. Finally, the section of bursa stained
for IgA (Figure 1C) clearly indicates that the
bird is not yet able to synthesize IgA. Similar
sections of cecal tonsil and other secondary
immune organs confirmed that the humoral
immune system of the neonate consists of IgM
and maternal IgG only (data not shown).
EARLY FEEDING AND IMMUNE
ORGAN DEVELOPMENT
Data presented in this section are from a
study in which the HNS was fed on Days 0, 1,
and 2 of life. Control birds were fasted and
given no water. The treatment resulted in a
significant improvement in body weight over
fasted controls during the first 3 wk of life
(data not shown). Figure 2 shows the effect of
these treatments on bursa weight. There was a
significant effect of treatment that persisted
through 21 days. Figure 3 shows the effect on
bursa weight as a percentage of body weight.
The bursa lost weight as a percentage of body
weight in the fasted birds. The provision of
feed on Day 3 did not result in the return of
bursa weight to match that seen in the birds
fed the HNS. This significant difference
persisted until Day 21.
The mechanism by which the deprivation
of feed affects bursa weight more than the
rest of the body is not known. One possible
explanation for these negative effects on
EARLY FlEEDING A N D IMMUNITY
428
IBursa Stained on the Day of Hatch1
I
FIGURE 1. Bursa of Fabriciuson the day of hatch stained for: A) immunoglobulin M (IgM); B) immunoglobulin
G (IgG); and C) immunoglobulin A (IgA). The B lymphocytes of the neonatal chick can synthesize only IgM. All
of the IgG is found in blood vessels and represents maternal antibody. There is no indication of IgA, of either
maternal or chick origin.
I
E
.h
cn
125
- - Fasted
cn
Sa ’
0.75
*Fed
Y
HNS
8
I
m
a
0.5
8
a
-1GURE 2. Bursa weight as a function of age in fasted birds or birds fed a hydrated nutritional supplement
:HNS)for post-hatch Days 0, 1, and 2. Bursa weights were significantly heavier (P c .OOOl) for the 21-day period
or the birds fed HNS (SEM= .05; *Means significantly different, P < .05).
Symposium
429
DIBNER et al.
0.3
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0.25
0.2
.$
0.15
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0.1
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0.05
0
0
1
2
3
8
Day of Age
15
21
FIGURE 3. Bursa weight as a percentage of body weight in fasted birds or birds fed a hydrated nutritional
supplement (HNS) for post-hatch Days 0, 1, and 2. Bursa weight as a percentage of body weight
was sionificantlv heavier I P e .003)for the birds fed HNS from Day 3 through Day 21 (SEM = .03;*Means
significantly diffkrent, P e .05).
’
bursa weight may simply be the rise in
glucocorticoids associated with fasting [24].
Glucocorticoids have been reported to be associated with involution of primary lymphoid
organs, even in young poultry [25].
Low substrate availability or low oral intake of antigen could also cause the results
shown in Figures 2 and 3. Although the immune response does not appear to be limited
by substrate availability later in life, nutrient
requirements for the development of the immune system have not been similarly examined. During the frrst week of life, substrate
availabilitymay well be limiting. The growth of
certain systems, particularly the gastrointestinal, cardiovascular, and respiratory, is
critical to the achievement of the genetic
potential of the bird for growth. Modern
production systems and genetic selection for
performance in poultry may have diminished
the bird’s immune responsiveness and may
also have influenced partitioning of nutrients
in the neonate [26, 271. The growth rate of
the gastrointestinal system over the first week
of life has been estimated at three to five
times that of the rest of the body [28, 291. In
addition, nutrient transport systems, gut surface area, and digestive enzyme levels have
been found to be adequate but not in excess
of early need for nutrients [30]. These observations suggest that there may not be a nutrient
surplus even in birds that are fed immediately
after hatching.
The development of secondary immune
tissues such as the spleen, cecal tonsils, and
Harderian gland is clearly dependent first on
the bursa and thymus [31]. However, in contrast to these primary immune organs, a critical influence on the development of secondary
tissues appears to be antigen exposure [32,33].
The cecal tonsils are a very good example of
this, with germ-free animals demonstrating
small cecal tonsils devoid of germinal centers
[34]. This effect lasts through about 4 wk, after
which a few small germinal centers can be seen
[35]. Eventually, some of the animals may
exhibit low levels of serum IgG, but there are
no reports of the presence of IgA. Germ-free
animals can also be deficient in isotype differentiation and at the extreme may be limited
to an IgM response with very low antigenbinding diversity [%, 371. It is interesting that
this difference between conventional and
germ-free animals exists, because the feed
itself, even if sterile, should provide some
antigen. It may be that antigens from living
microorganismsare required for this function.
In any case, feeding on the day of hatch may
provide an early antigen stimulus and thus
facilitate rapid differentiation of the humoral
response [38,39].
EARLY FEEDING AND IMMUNITY
430
Figure 4 shows sections of bursa from
birds fasted or given the HNS for 3 days. The
sections are stained to detect proliferating
cells, whose nuclei appear dark. Note that in
the bursa from the fed bird, virtually every
lymphocyte is stained. The bursal lymphocytes
undergo an explosive proliferation in the
neonate, as long as substrate and antigen are
present. Figures 3 and 4 illustrate, and it is
important to emphasize, that the contents of
the residual yolk cannot be substitutedfor oral
intake. First, note that the animals in this study
were not deutectomized and all had residual
yolk available a5 a source of nutrients. Apparently, there was not enough nutrient capacity
in the yolk to maintain lymphocyte proliferation at the same level as seen in birds receiving
oral nutrition (Figure 4). This may be due to
the priority use of nutrients by the gut and
cardiovascular and respiratory systems but
may also be related to a lack of antigen from
the gastrointestinal system.
D E V E L O P M E N T O F SECONDARY
RESPONSES AND DISEASE RESISTANCE
The demonstration that early feeding increases bursa weight and the amount of bursal
1
lymphocyte proliferation does not prove that
it results in an improvement in development of
immunocompetence. Other observations,
however, suggest that there may be long-term
consequences of early feed and water deprivation. Figure 5 shows the effect of early feeding
on the appearance and levels of biliary IgA.
This immunoglobulin is a part of the mucosal
immune system and is the last of the major
isotypes to appear. Thus, presence of IgA is a
sign that the humoral immune system is fully
developed. As is clear from Figure 5, early
feeding was associated with a more rapid appearance of biliary IgA and a generally higher
level of the immunoglobulin over the entire
21-day study.
Another indicator of immune maturation
is the appearance of germinal centers [14].
These are local concentrations of lymphocytes in which T cells, B cells, and antigenpresenting cells form an organized structure
associated with the development of immune
memory to antigen, such as is required for a
vaccine response. In the study reported here,
germinal centers in the cecal tonsils were used
to indicate full immune maturation and the
capability to mount an anamnestic response.
Figure 6 shows the effects of early feeding on
I
Bursa Stained for Proliferating Cells 72 Hours after Hatch
IFasted Control, mag = 20x
I
I
HNS, mag = 20x
I
FIGURE 4. Bursa tissue sections stained for proliferating cells at the end of the treatment period. Birds given
the hydrated nutritional supplement (HNS) showed many more lymphocytes in DNA synthesis than the fasted
birds.
Symposium
431
DIBNER et al.
r0.35-
4
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0.3
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025
-
0.2
-
0.15
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0.05
O
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l
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,
a
.
'
0
0
2
4
6
8
10
12
14
16
18
20
22
Age (Days)
FIGURE 5. Biliary immunoglobulin A (IgA) levels as a function of age in fasted birds or birds fed a hydrated
nutritional supplement (HNS) for post-hatch Days 0, 1, and 2. Data points represent a pooled sample from four
birdsfireatment/day, and no estimate of variability was obtained.
+Fed
-Fasted
HNS
FIGURE 6. Number of germinal centers in sections of cecal tonsil as a function of age in fasted birds or birds
fed a hydrated nutritional supplement (HNS) for post-hatch Days 0, 1, and 2. Number of germinal centers was
significantly greater (P<.OoOl) for the 2 l d a y period for the birds fed HNS (SEM=.9; *Means significantly
different, P < .05).
the appearance of germinal centers. It is clear
that there is an effect of early feeding and that
once these structures appear they undergo a
linear increase with age.
Finally, the effect of early feeding on disease challenge was tested. In the study shown
in Figure 7, a challenge using a commercial
was used to evaluate
coccidiosis vaccine [a]
disease resistance. These birds had not been
immunized for coccidiosis and all effects
shown were due to the presence or absence of
nutrients on Days 0 and 1.As can be seen in
Figure 7, the fasted birds were not as heavy as
the early fed birds, even 18 days after all the
buds were consuming the same diet ad libitum.
In addition, there was a significant difference
JAPR
EARLY FEEDING AND IMMUNITY
432
12.0
0.75
b
m
5
0.5
Em
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3
m
0.25
0
Body Weight
Cumulative Feed to Gain
Fasted, Challenge
0 Fed HNS, No Challenge El Fed HNS, Challenge
H Fasted, No Challenge
a, b, c P< 05
ZIGURE 7. Performance of birds fed or fasted over post-hatch Days 0 and 1 and then placed on a corn-soy diet
&libitum. (Treatmenteffects: bodyweightP<.0003, SEM=.Ol; cumulativefeedefficiencyP=.l4,SEM=.W;
=Means significantly different, P < .05).
in performance between fasted and fed birds
following the coccidiosis challenge. Birds fed
the HNS on Days 0 and 1 retained the improved performance associated with feeding
even during a disease challenge. It should be
emphasized that this effect was not exclusive
with respect to coccidiosis. The oral challenge
was simply used as a model for a non-specific
stress or disease challenge. The data suggest
that birds given the optimum nutrient formulation immediately after hatch are better able
to respond to the variety of physiological and
environmental challenges of the production
facility.This was also observed in turkey poults
exposed to a challenge model for poult enteritis and mortality syndrome [41].
NUTRITION AND IMMUNITY
The studies reported here are intended to
clarify the effect of early nutrition on the development of the immune system. It is important to distinguish immune development from
the immune response. Numerous publications
have covered the subject of nutrition as it
affects the ability of the animal to respond to
an immune challenge. Nutrition can affect the
magnitude of the response and the nature of
the response. For example, a period of feed
restriction in poultry has been reported to increase the cellular and humoral response to
sheep red blood cells [42]. Specific nutrients
affecting the immune response have also been
identified. As an example, dietary fatty acids
have been reported to affect the levels and
types of responses to an immune challenge
[43]. Dietary immunomodulators can amplify
or diminish the magnitude of the reaction to
a challenge through their effects on other immune cells [MI.
Development of the avian immune system
has been widely studied, but little information has been published on the effects of early
feeding on its development. There are three
ways in which early feeding could affect immune development. First, nutrients provide
substrates for cell proliferation and differentiation; second, nutrients can be immunomodulators themselves or can affect
their endogenous synthesis; and third, oral
intake provides many of the antigens that drive
both the development of isotypes and the generation of immunoglobulin diversity in the
bursa [45,46,47l.
Symposium
DIENER et al.
USE OF RESIDUAL YOLK AS A
NUTRIENT SOURCE
Implications of using residual yolk contents to provide amino acids or energy for
growth should be examined in light of our
current understanding of the nature of the
residual yolk. First, maternal immunoglobulin
represents up to 20% of the residual yolk protein. It should be noted that this fraction of
yolk protein is not used during embryogenesis
and as a result, the antibody titer of the yolk
actually increases over the course of incubation [ a ] . The yolk antibody is a pool of
macromolecules from highly differentiated
cells that the hatchling cannot provide for
itself [49]. It is clear that using this material
for amino acids would deprive the neonate of
the maternal immunoglobulin that is its sole
source of high specifcity antibodies over the
first week or more of life. Their digestion for
amino acids can be interpreted as a survival
mechanism only - not as a routine metabolic
pathway.
The rest of the residual yolk protein is
composed of serum proteins present in the hen
during the time the yolk was formed [50].
These can include soluble protein antigens
from the hen to which the chick would be
exposed shortly after hatch. These may play a
role in development of the secondary immune
organs, particularly the Meckel's diverticulum
[SI
It.should not be assumed that the balance
of the residual yolk protein is best used as an
amino acid source until more is known of the
nature and function of these proteins.
A similar argument can be made for the
residual yolk lipids. Phospholipids and cholesterol esters represent about one-third of the
residual yolk lipid and are not efficient sources
of energy [52]. The synthesis of both cholesterol and phospholipids requires energy, and
both are essential components of cell membranes. It would be extremely inefficient to
catabolize these lipids and then resynthesize
them unless survivalwas at stake. The remaining yolk lipids are triacylglycerols, but even if
these were made totally available on the day of
hatch and were metabolized at 100% efficiency, the total energy yield on Day 0 would
be at most about 9 kcal- less than the 11kcal
433
maintenance requirement estimated for the
first day of life [53].
The possibility of using hepatic lipids for
energy in the neonate should also be viewed in
this context. Over 80% of the hepatic lipids at
the time of hatch are cholesterol esters. The
esterification of the cholesterol can be interpreted as a relatively nontoxic way of storing
the large amounts of cholesterol required for
lipid transport during embryogenesis [a].
This material can be used either structurally
in cell membranes or functionally in the transport of lipid after hatch. It does not represent
a significant energy depot for the hatchling. In
addition, the portion of the hepatic lipid available for energy, i.e. the triacylglycerol (4%)
and phospholipid (14%) fractions, contains a
high concentration of arachidonic and
docosahexanoic acids. Ding and Lilburn have
reported similar findings in turkey poults [54].
Arachidonic and docosahexanoic acids are
synthesized by the cells of the yolk sac memThe
brane from other yolk fatty acids [a].
relative levels of these two polyunsaturated
fatty acids can modify eicosanoid metabolism
and in this way affect the associated inflammatory and immune responses of the neonate
[SS]. Finally, the observation that
docosahexanoic acid is the preferred n-3 fatty
acid for the development of the chick hatchling central nervous system and retina may
explain the selective incorporation of this fatty
acid in neonatal hepatic triacylglycerols and
phospholipids [56]. As with the maternal
antibody fraction of the yolk protein, these
residual yolk components are much more
valuable intact than catabolized.
It is hypothesized that during the evolution and particularly the long history of domestication of poultry, hatchlings have received
feed promptly so that postnatal survival has
not depended on the use of yolk for energy and
amino acids. This has allowed the residual
components of yolk to become an important
means of providing the neonate with macromolecules that it is unable to synthesize for
itself. As a result, prompt oral intake of nutrients may be essential for the realization of the
modern bird's geneticpotential for growth and
disease resistance.
JAPR
EARLY FEEDING A N D IMMUNITY
434
CONCLUSIONS
AND APPLICATIONS
Development of the immune system is initiated during embryogenesis but is not complete
until weeks or months after hatch. This development may be limited by nutrient availability
in fasted hatchlings.
Early feeding was associated with larger bursa welghts and greater lymphocyte proliferation. Residual yolk did not provide the required level of nutrition to fully support immune
system maturation during the first two days after hatch.
Appearance of biliary IgA and germinal centers occurred earlier and in larger amounts in
birds given early nutrition, indicating a more rapid development of the capabilityto respond
to vaccine administration. Early feeding was associated with improved bird performance
following a disease challenge.
The chick or poult should be provided with an optimum nutrient formulation and a source
of water immediately after hatching. This initiates immune development and spares yolk
macromolecules such as yolk antibodies for passive immunity. Biochemically, residual yolk
lipids are ideal for Lipid transport, for cell membrane and immunomodulator synthesis, and
for development of the central nervous system and retina.
REFERENCE
s AND NOTES
1. Larsson, A, RM. Bdow, T.L LindahI, and P.O.
Forsberg, 1993. Chicken antibodies: Taking advantage of
evolution - A review. Poultry Sci. 72:1807-1812.
2. Esteban, SJ.M. Rayo, M. Moreno, M. Sastre, RV.
Mal, and J.A. Tor, 1991. A role played by the vitelline
diverticulum in the yolk sac resorption in p u n posthatched chickens. J. Comp. Phyiol. B 160:645-64k
3. Ross HyY broilers were incubated and hatched at
Nows International. Cockerels used in this study were
feather sexed. Chicks were housed eight per cage in battery rooms. Cages were 51 cm wide X 69 cm long X 36 cm
high and made of olyvinylchloride-coated wire
mesh. Mesh size was
X 2.5 cm for sides and top and
1.25 X 1.25 cm for the floor. Feed was supplied in a galvanized trough feeder and water was supplied in sanitary
type plastic and stainless water nip les. Temperatures
were maintained starting at 3 3 ~J . e r 3 days temperatures were decreased w t h a linear function to 22 C at
21 day and held constant thereafter. Com lete exchange
of room air with fresh air was provided l!x/hr.
Fluorescent light with an intensity of 45 lux was provided for
23 hr/day. Chicks were euthanized using carbon dioxide
inhalation.
4. Chicken Serum and bile were collected and frozen
for determination of IgA levels at a later date. Serum was
diluted 1:lO and bile 1:20in O.OSM carbonate bicarbonate
buffer. (Optimal antibody concentrations were determined using a chessboard titration as described in
Crowther, J.R, 1995. Methods in Molecular Biology:
ELISA Theory and Practice, Humana Press, Totowa,
NJ). The antibodies used were mouse anti-chicken IgA
diluted 1:100 (Southern Biotechnolo Associates, Inc.,
Birmingham, AL), goat anti-chicken YgA diluted 1:10oO
(Sigma Chemical Co., St. Louis, MO), and rabbit antigoat IgG eroxidase conjugate diluted 1:lOOO (Sigma
Aliquots of the carbonate bicarbonate
Chemical
buffer solution (50pL, Sigma Chemical Co.)and mouse
anti-chicken IgA (50pL) were placed in Falcon Pro-Bind
96 flat-bottom well assa plates (Becton Dickinson
and incubated for 2 hr at
Labware, Lincoln Park,
room temperature. After the 2 hr incubation, plates were
washed four cycles with phosphate-buffered saline
(PBS, Sigma Chemical Co.) using a Bio-Tek EL404
Automated Microplate Washer (Bio-Tek Instruments,
h.).
d)
'%1
fat (from egg yolk).
6. National Research Council, 1994. Nutrient
Requirements of Poultry. Natl. Acad. Press, Washington,
DC.
7. Dibner, JJ., M.L Kitchell, C.A. Atwell, and F.J.
hey, 1996. The effect of dietary ingredients and age on
the microscopic structure of the astrointestinal tract in
poultry. J. Appl. Poultry Res. 5:&77.
Symposium
435
DIBNER et al.
For immunoglobulin staining of IgG (mouse antichicken IgG, Accurate Chemical & Scientific Corp.
Westbury, NY) and IgM (goat anti-chicken I
rate Chem. & Sci. Corp.), slides were deparaf inked
Accuand
hydrated and placed on the Shandon Cadenza (Shandon,
Inc., Pittsburgh, PA) slide rack where they were allowed
to drain for 5 min. Slides were washed with Shandon
Cadenza Buffer (Shandon, Inc.) for 10 rnin and then
blocked with antibody dilution media containing PBS
with 0.01% bovine serum albumin (Sigma Chemical Co.)
for 10 min. Prima antibody was ap lied diluted in the
above blocking buxer at 1:1600 for &h4 and 1:1800 for
IgG. After a 2 hr incubation with primary antibody, slides
were washed with Shandon Cadenza Buffer for 10 rnin
followed by a 30 rnin incubation with biotinylated antimouse IgG (Vector Elite ABC Kit, Vector Laboratories,
Burlingame, CA). Slides were washed with Shandon
Buffer for 10 min, and the avidin-biotin complex (Vector
Elite ABC Kit) was a lied for 30 min. Slides were
washed again with Shan%bnBuffer (Shandon, IC.) for
10 min and developed with the Vector Laboratories VIP
Peroxidase Kit (Vector Laboratories) for 5 min, followed
bya wash in running tapwater. Slideswere counterstained
with methyl green counterstain and coverslipped.
P
8. For the coccidiosis challenge study, birds were
fasted (Treatments 1 and 2) or fed the hydrated nutritional supplement (Treatments 3 and 4) on the day of
hatch (Day 0) and the subsequent day (Day 1). Beginning
on Day 2,all birds were given water and fed
a corn-soy diet formulated to meet or exceed NRC [6]
recommendations. T h e coccidiosis challenge was administered to Treatments 2 and 4 by gava e on Day 14.
An oral coccidiosis vaccine was used &occiVac D,
Mallinckrodt Veterin
Millsboro, DE). The vaccine
was centrifuged (12,%@
5 min) and the pellet was
diluted with water to give a coccidial challenge of
100doses/100 g body weight.
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ACKNOWLEDGEMENTS
The authors gratefully acknowledge animal management and experimental conduct by M.E. Wehmeyer and
C.W. Wuelling.