Q1998 Applied Poultly Science, I n c

E R Y FEEDING DEVELOPMENT AL AND 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 PROBLEM producers 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
1 Presented at 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

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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 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

to respond to a vaccine with the development of immune memory. Studies will be discussed with reference to these three postulates.

MATERIALS METHODS AND

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 and by evaluaimmunoglobulin A (I@) [4], tion 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 9, per treatment on Days 0, 1,2, 3,6, 7,8, 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 l for the day of hatch and the day after. Al birds were then allowed to consume the same cornsoy starter diet ad libitum. Buds were chalOx lenged orally with a 1 O dose of a commercial coccidiosis vaccine [8] on Day 14, i.e. after 12 days on ad libitum feed. Birds and feed and were weighed on Days 7,14, 20.

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the RESULTS DISCUSSION present studies focused theoneffects of AND early nutrition on the bursa and 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

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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.

E .h

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*Fed
HNS

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-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).

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DIBNER et al. 429

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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 *Means was sionificantlv heavier I P e .003)for the birds fed HNS from Day 3 through Day 21 (SEM = .03; 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 birds 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 criticl influence on the development of secondary a 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].

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EARLY FEEDING AND IMMUNITY 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

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). T i may be due to hs 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

Bursa Stained for Proliferating Cells 72 Hours after Hatch

IFasted Control, mag = 20x

HNS, mag = 20x

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.

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431

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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 coccidiosis vaccine [a] 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 iiu. buds were consuming the same diet ad l b t m In addition, there was a significant difference

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432 EARLY FEEDING AND IMMUNITY

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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 w s also observed in turkey poults a 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.

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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 [ I should not be assumed that the balance S It . 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 1 be at most about 9 kcal- less than the 1 kcal
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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 nlm and in this way affect the associated i f a 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 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.

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CONCLUSIONS AND APPLICATIONS

Development of the immune system is initiated during embryogenesis but is not complete hs until weeks or months after hatch. T i 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 NOTES s AND

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 d y and held constant thereafter. Com lete exchange a of room air with fresh air was provided lxh. !/r 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., g Birmingham, AL), goat anti-chicken Y A 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.).

' % 1

fat (from egg yolk).

d)

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.

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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 and Accuhydrated 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 0 % bovine serum albumin (Sigma Chemical Co.) .1 for 10 min. Prima antibody was ap lied diluted in the above blocking buxer at 1:1600 for & 4 and 1:1800 for h 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.

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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. 119:749756. 21.Bderley, J. and W.A. Hemmings, 1956.The selective 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 protection 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 hormone and starvation on adrenocortical function in chickens. 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

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 ( 2 % 1,@ 5 min) and the pellet was diluted with water to give a coccidial challenge of 100doses/100 g body weight.

domestic fowl: etabolic and genetic considerations. Worlds Poultry Sci. J. 52:251-266.

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ACKNOWLEDGEMENTS
The authors gratefully acknowledge animal management and experimental conduct by M.E. Wehmeyer and C.W. Wuelling.