Early Feeding and the Development of Immune System in Neonatal Chicks

Q1998 Applied Poultly Science, I n c

EARLY FEEDING AND OF THE IMMUNE

DEVELOPMENT SYSTEM IN

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

DESCRIPTION OF PROBLEM In modern poultry production, the sepa-

ration 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

hatchery practice. In some parts of the world, producers strive to place the neonates within 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 ini- tiate a vaccine response while the birds are under low immunological challenge from other antigens. Often the producer has no

1 Presented at the 1998 Poultry Science Association Informal Poultry Nutrition Symposium:

2 To whom correspondence should be addressed "Impact of Early Nutrition on Poultry."

426 EARLY FEEDING AND IMMUNITY

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 pro- cessing has an additional benefit. Just before the hatching process begins, the bird inter- nalizes 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 anti- bodies 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 place- ment 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 trans- port. Producers may feel that feeding is not essential during this period because conven- tional 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 ac- count 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 gastro- intestinal system may be necessary to trigger full differentiation of the primary immune cells, particularly the B lymphocytes. Com- plete differentiation of these cells is critical for the eventual development of secondary im- mune structures such as germinal centers or cecal tonsils, along with the associated ability

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 evalua- tion of cell proliferation and immunoglobulin isotype expression by lymphocytes. The nutri- ent 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 1 and 2). Control birds were fasted and given no water over the same period. Be- ginning on Day 3, all birds were given water and fed an identical corn-soy starter diet for- mulated to meet or exceed National Research Council recommendations for starter feed [6]. Effects on performance, organ weights, im- munoglobulin 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 intes- tine, 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 folli- cle area were determined. Hematoxylin and eosin staining and immunocytochemical methods for bromodeoxyuridine and IgA have previously been described and those for immunoglobulins M (IgM) and G (IgG) differ

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 corn- soy starter diet ad libitum. Buds were chal- lenged 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.

only slightly [A.

DIBNER et al. Symposium

427

RESULTS AND DISCUSSION IMMUNE SYSTEM ONTOGENY

The immune system of the bird is partly developed at hatch. The primary immune or- gans, 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 pro- pria 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 re- sponse, 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,

the present studies focused the effects of early nutrition on the bursa and on develop- ment of responses requiring cell-cell interac- tions 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 pre- cursor 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 capability to 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

428 EARLY FlEEDING A N D IMMUNITY

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

h cn - 125

cn E .- S ’ a

0.75 m

Y 8 I

a

- - Fasted *Fed HNS

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 DIBNER et al. 429

0.3

0.25 F E 0 0.2 s E .$ 0.15 s

m

- m $ 0.1

0.05

0 0 1 2 3 8 15 21

Day of Age

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 IPe .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 as- sociated with involution of primary lymphoid organs, even in young poultry [25].

Low substrate availability or low oral in- take of antigen could also cause the results shown in Figures 2 and 3. Although the im- mune response does not appear to be limited by substrate availability later in life, nutrient requirements for the development of the im- mune system have not been similarly exam- ined. During the frrst week of life, substrate availability may well be limiting. The growth of certain systems, particularly the gastro- intestinal, 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 sur- face area, and digestive enzyme levels have been found to be adequate but not in excess of early need for nutrients [30]. These obser- vations 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 con- trast to these primary immune organs, a criti- cal 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 differ- entiation and at the extreme may be limited to an IgM response with very low antigen- binding 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 microorganisms are 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].

430 EARLY FEEDING AND IMMUNITY

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 substituted for oral intake. First, note that the animals in this study were not deutectomized and all had residual yolk available a5 a source of nutrients. Appar- ently, there was not enough nutrient capacity in the yolk to maintain lymphocyte prolifera- tion 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.

DEVELOPMENT O F SECONDARY

TANCE The demonstration that early feeding in-

creases bursa weight and the amount of bursal

RESPONSES AND DISEASE RESIS-

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 depriva- tion. 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 ap- pearance 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 lympho- cytes in which T cells, B cells, and antigen- presenting 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

1 Bursa Stained for Proliferating Cells 72 Hours after Hatch I

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 DIBNER et al. 431

r- 0.35-

0.3 -

0 2 5 - 4 0

0.2 - zj w

0.15 -

0.05 O . l ~ , ---. '.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.

- -Fasted +Fed 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 lday 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 dis- ease challenge was tested. In the study shown in Figure 7, a challenge using a commercial coccidiosis vaccine [a] was used to evaluate

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

432 JAPR

EARLY FEEDING AND IMMUNITY

0.75

m 5 0.5 E m .- i z 3 0.25 m

0

b

12.0

Body Weight Cumulative Feed to Gain

H Fasted, No Challenge Fasted, Challenge a, b, c P< 05

0 Fed HNS, No Challenge El Fed HNS, Challenge

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 im- proved 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 formu- lation 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 enter- itis and mortality syndrome [41].

NUTRITION AND IMMUNITY The studies reported here are intended to

clarify the effect of early nutrition on the de- velopment of the immune system. It is import- ant 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 in- crease 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 im- mune cells [MI.

Development of the avian immune system has been widely studied, but little informa- tion has been published on the effects of early feeding on its development. There are three ways in which early feeding could affect im- mune development. First, nutrients provide substrates for cell proliferation and differ- entiation; second, nutrients can be im- munomodulators 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 gen- eration of immunoglobulin diversity in the bursa [45,46,47l.

Symposium DIENER et al. 433

USE OF RESIDUAL YOLK AS A NUTRIENT SOURCE

Implications of using residual yolk con- tents 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 pro- tein. 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 incu- bation [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 choles- terol esters represent about one-third of the residual yolk lipid and are not efficient sources of energy [52]. The synthesis of both choles- terol and phospholipids requires energy, and both are essential components of cell mem- branes. It would be extremely inefficient to catabolize these lipids and then resynthesize them unless survival was at stake. The remain- ing yolk lipids are triacylglycerols, but even if these were made totally available on the day of hatch and were metabolized at 100% effi- ciency, the total energy yield on Day 0 would be at most about 9 kcal- less than the 11 kcal

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 inter- preted 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 trans- port of lipid after hatch. It does not represent a significant energy depot for the hatchling. In addition, the portion of the hepatic lipid avail- able 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 mem- brane from other yolk fatty acids [a]. The relative levels of these two polyunsaturated fatty acids can modify eicosanoid metabolism and in this way affect the associated inflam- matory 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 hatch- ling 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 evolu- tion and particularly the long history of domes- tication 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 macro- molecules that it is unable to synthesize for itself. As a result, prompt oral intake of nutri- ents may be essential for the realization of the modern bird's genetic potential for growth and disease resistance.

JAPR 434 EARLY FEEDING AND IMMUNITY

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.

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 prolifera- tion. 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 capability to 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 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 post- hatched 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 bat- tery 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 gal- vanized 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 tempera- tures 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. Fluores- cent 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 1:20 in O.OSM carbonate bicarbonate buffer. (Optimal antibody concentrations were deter- mined 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 anti- goat IgG eroxidase conjugate diluted 1:lOOO (Sigma Chemical h.). Aliquots of the carbonate bicarbonate 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 Labware, Lincoln Park, d) and incubated for 2 hr at 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,

s AND NOTES

Symposium DIBNER et al. 435

For immunoglobulin staining of IgG (mouse anti- chicken IgG, Accurate Chemical & Scientific Corp. Westbury, NY) and IgM (goat anti-chicken I

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 anti- mouse 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%bn Buffer (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.

8. For the coccidiosis challenge study, birds were fasted (Treatments 1 and 2) or fed the hydrated nutri- tional 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. The coccidiosis challenge was ad- ministered 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 100 doses/100 g body weight.

9. Bucy, RP., C.H. Chen, J. Cihak, U. Losch, and M.D. Cooper, 1988. Avian T cells expressing$ receptors localize in the splenic sinusoids and the intestinal epithe- lium. J. Immun. 141:2200-2205.

10. Lilleho], H.S. and J.M. Trod, 1996. Avian gut- associated 1 phoid tissues and intestinal immune re- sponses to g e n a parasites. Clin. Micro. Rev. 9349-360.

11. Lilleho], H.S. and K.S. C h u g , 1992. Postnatal development of T-lymphocyte subpopulations in the in- testinal intraepithelium and lamina propria in chickens. Vet. Immunol. and Immunopathology 3137-360.

12. Peaulf B., F. Dieterlen-Lievre, and N.M. Le Dourarin, 1987. Cellular interactions occurring during primary lymphoid organ ontogeny in birds. Pa es 39-64 in: Avian Immunology: Basis and Practice. A. %oivanen and P. Toivanen, eds. CRC Press, Boca Raton, FL.

13. Leslie, G.A. and LN. Marlin, 1973. Suppression of chicken immunoglobulin ontogeny by F(ab’)2 fragments of anti+ chain and by anti-L chain. Intl. Arch. Allergy 45:42!U38.

14. Schat, K.A. and T.J. Myers, 1991. Avian intestinal immunity. Crit. Rev. Poultry Biol. 319-34.

15. Leslie, G.A., 1975. Ontogen of the chicken humoral immune system. Am. J. Vet. ges. W482485.

16. Perey, D.Y. and J. Bienenstock, 1973. Effects of bursectomy and thymectomy on ontogeny of fowl IgA, IgG, and IgM. J. Immun. 11k633-637.

rate Chem. & Sci. Corp.), slides were deparaf P inked Accu- and

17. McCorkle, F.M., G.H. Luginbuhl, D.G. Simmons, G.W. Morgan, and J.P. Thaxton, 1983. Ontogeny of de- layed h rsensitivity in young turkeys. Dev. and Comp. I m m u n T ~ 17-524.

18. Schaflner, T., M.W. Hess, and H. Cottier, 1974. A reappraisal of bursal functions. Ser. Hemat. 7568-592.

19. WeiU, J.C. and C.A. Reynaud, 1987. The chicken B cell compartment. Science 2381094-1098.

20. Grossi, C.E, P.M. Lydard, and M.D. Cooper, 1977. Ontogeny of B cells in the chicken. J. Immun. 119:749- 756.

21. Bderley, J. and W.A. Hemmings, 1956. The selec- tive transport of antibodies from the olk sac to the circulation of the chick. J. Embryol. Exp. Lorph. 4:34-41.

22. Marlin, LN. and G.A. Leslie, 1973. Ontogeny of IeA in normal and neonatallv bursectomized chickens. Z t h corroborative data on Ig? and IgM. Proc. SOC. Exp: Biol. Med. 143:241-243.

23. Shawky, S A , Y.M. Saif, and D.E Swayne, 1993. Role of circulating maternal anti-rotavirus IgG in protec- tion of intestinal mucosal surface in turkey poults. Avian Dis. 371041-1050.

24. Conslantin, N., J. Raszyk, A. Holub, and V. Kolrbacek, 1977. Effect of adrenocorticotrophic hor- mone and starvation on adrenocortical function in chick- ens. Acta. Vet. Bmo. 4687-93.

25. Nir, I., Z. Nilsan, EA. Dunnlngton, and P.B. Siegel, 1996. As ects of food intake restriction in young domestic fowl: k etabolic and genetic considerations. World’s Poultry Sci. J. 52:251-266.

26. Siegel, H.S., 1980. Physiological stress in birds. Bioscience 30529-534.

27.Parmenlier,H.K.,M.G.B.NieuwIand,E Rijke,G. De Vdcs Reilingh, and J.W. Schrama, 1996. Divergent antibody responses to vaccines and divergent body weights of chicken lines selected for high and low humoral responsiveness to sheep red blood cells. Avian Dis. a634-644.

28. Sell, J.L, C.R Angel, FJ. Piquer, EG. Mallmko, and H A Al-Batshan, 1991. Developmental patterns of selected characteristics of the gastrointestinal tract of young turkeys. Poultry Sci. 701200-1205.

29. Nitsan, Z., G. Ben-Avraham, Z. Zoref, and I. Nir, 1991. Growth and development of the di estive organs and some enzymes in broiler chicks after hatching. Br. Poultry Sci. 32515-523.

30. Noy, Y. and D. Sklan, 1995. Digestion and absorp- tion in the young chick. Poultly Sci. 74366-373.

31. Glick, B., 1997. The bursa of Fabricius and immunoglobulin synthesis. Intl. Rev. Cytol. 48345402.

32. Rao, D.S.V., F.C. McDuffie, and B. Glick, 1978. The regulation of IgM production in the chick Roles of the bursa of Fabricius, environmental antigens and plasma IgG. J. Immun. 12078S787.

33. Hedge, S.N., EA. R o b , A. Turvey, and M.E Coates, 1982. Influence of gut microflora on the 1 phoid tissue of the chicken (a domestlcus ) and g a n e s e

uail (w w). Comp. Biochem.

34. Hoqjo, K., T. Hagiwara, K. Itoh, E Takahashi, and Y. Hirota, 1993. Immunohistochemical analysis of tissue distribution of B and T cells in ermfree and con- ventional chickens. J. Vet. Med. Sci. 5f:1031-1034.

35. Thorbecke, G.J., H.A. Gordon, B. Wostman, M. Wagner, and J.A. Reynicrs, 1957. Lymphoid tissue and

3 . hyslol. 72A:205-209.

serum gamma globulin in young germfree chickens. J. Infect. Dis. 101:237-251.

436 EARLY FEEDING AND IMMUNITY

36. Kincade, P.W. and M.D. Cooper, 1971. Develop- ment and distribution of immuno obulin-containing cells in the chicken. J. Immun. 106:3#-382.'

37. Huang, H.V. and W.D. Dryer, 1978. Bursectomyh p41p blocks the generation of immunoglobulin diversity. J. Immun. 121:1738-1747.

38. Ekino, S.K. Matsuno, S. Harada, H. Fuji, Y. N m 4 and M. Ko(anl, 1979. Amplification of plaque-forming cells in the spleen after intra-cloacal antigen stimulation in neonatal chickens. Immun. 37811-815.

39. Lupelti, M., A. Dol@ and T. Michelucci, 1984. A contribution to the study of the replato w t e m of local immune res onse in m- .%ev. and Comp. Immun. 8693-672.

40. Coccivac - D, Mallinckrodt Veterinary, Inc., Millsboro, D E 19966.

41. Edens, F.E, RE Doeder, LD. Cain, and A.F. Giesen, 1998.. Prefeeding of a navel hydrated nutritional supplement (Oasis) im roves early performance in tur- key poults. Poultry Sci. h 4 1 (Abs).

42. Klasing, K.C., 1988. Influence of acute starvation or acute excess intake on immunocompetence of broiler chicks. Poultry Sci. 67626-634.

43. Fritsche, N.L, N.A. Cassity, and S. Huang, 1991. Effect of dietary fat source on antibody production and lymphocyte proliferation in chickens. Poultry Sci. 70611- 617.

44. Dieterl, RR, K.A. Golemboski, H. Kwak, and RE Austic, 1993. Nutrient-determined immunomodula- tion. Pages 106-113 in: Proc. 1993 Cornel1 Nutr. Conf., Rochester, NY.

45. Schaffner, T., J. Mueller, M.W. Hess, H. Collier, B. Sordat, andC. Ropke, 1974, The bursa of Fabricius: A central organprovidingfor contact between the lymphoid system and intestinal content. Cell. Immun. 13304-312.

46. Ekino, S., Y. Nnw4 K. Kanaka, K. Matsuno, H. Fuji, and M. Ko(ani, 1980. Suppression of immune response by isolation of the bursa of Fabricius from envi- ronmental stimuli. AJEBAK 58289-296.

47. Jeurissen, S.H.M., E.M. Janse, G. Koch, and G.F. De Boer, 1989. Postnatal development of mucosa-

associated lymphoid tissues in chickens. Cell Tis. Res. 258:119-124.

48. Kramer, T.T. and H.C. Cho, 1970. Transfer of immunoglobulins and antibodies in the hen's egg. Immun.

49. Losch, U., I. Schranner, R. Wanke, and L. Jurgens, 1986. The chicken egg, an antibody source. J. Vet. Med. 33609419.

50. Sailo, Z., W.G. Martin, and W.H. Cook, 1965. Changes in the major macromolecular fractions of e yolk during embryogenesis. Can. J. Biochem. 43175g 1770.

51. Knight, P.F. and A.M. Schechlman, 1954. The passage of heterologous serum roteins from the circula- tion into the ovum of the fowl. f F q . Zool. 127271-304.

52. Noble, RC. and M. Cocchi, 1990. Li id metabo lism and the neonatal chicken. Prog. Lipid k s . 291071 140.

53. Akib4 Y. and H. Murakemi 1995. Partitioning of energy and protein during early growth of broiler chicks and contribution of vitteline residue. Pages 45-52 in: Proc. 10th European Symposium on Poultry Nutr., Antalya, Turkey.

54. Ding, S.T. and M.S. Lib- 1996. Characteriza- tion of changes in yolk sac and h e r lipids during embry- onic and earl p t h a t c h development in turkey poults. Poultry Sci. 7$478-483.

55. Korver, D.R, P. Wakenell, and K.C. Klasing, 1997.Dietaryfishoil orlofrin,aS-li oxygenaseinhibitor, decrease the growth su pressing effects of coccidiosis in broiler chicks. Poultry lei. 76:1355-1363.

56. Anderson, GJ., W E Connor, and J.D. Corks, 1990. Docosahexanoic acid is the preferred dietary n-3 fatty acid for development of the brain and retina. Ped. Res. 278S97.

19:157-167.

ACKNOWLEDGEMENTS The authors gratefully acknowledge animal manage-

ment and experimental conduct by M.E. Wehmeyer and C.W. Wuelling.