*Department of Animal Science, University of Missouri, Columbia, Missouri 65211; and Novus International, Inc., St. Louis, Missouri 63304

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1 Impact of Glutamine and Oasis Hatchling Supplement on Growth Performance, Small Intestinal Morphology, and Immune Response of Broilers Vaccinated and Challenged with Eimeria maxima 1,2 G. F. Yi,*,3 G. L. Allee,* C. D. Knight, and J. J. Dibner *Department of Animal Science, University of Missouri, Columbia, Missouri 65211; and Novus International, Inc., St. Louis, Missouri ABSTRACT Seven hundred and twenty hatchling broilers in the Feed groups had the highest gain during d 0 to 21 were allotted to 12 treatment groups. Groups 1 and 2 were fasted for 48 h posthatch; groups 3 and 4 were fasted for 48 h followed by ad libitum access to a 1% glutamine (Gln) diet; groups 5 and 6 had ad libitum access (P < 0.01). During d 22 to 28, birds in the Fast groups had the lowest BW and livability (P < 0.01), and the nonvaccinated birds had lower gain and feed efficiency relative to vaccinated birds (P < 0.01). Birds in the Feed and Oasis to a common diet; groups 7 and 8 had access to a 1% Gln groups had higher villus height (VH) of mid small intestine than Fast groups at d 2 and 7 (P < 0.05), and nonvacci- diet posthatch; groups 9 and 10 were fed regular Oasis hatchling supplement; and groups 11 and 12 were fed nated birds had higher VH than vaccinated birds (P < 0.01) at d 7 after hatch. On d 14, there were differences Oasis sprayed with 1% Gln for the first 48 h posthatch. in serum interferon-γ (P < 0.05) levels among treatments. The birds in treatment groups 2, 4, 6, 8, 10, 11, and 12 During d 22 to 28, vaccinated birds had lower lesion were vaccinated with Eimeria maxima posthatch, and all scores in the mid small intestine than nonvaccinated birds birds were orally challenged with high dose E. maxima (P < 0.01), and birds in the Feed or Oasis groups had on d 22. During the first 2 wk, birds in group 7 had the lower lesion scores compared with the Fast groups (P < highest gain and feed efficiency among treatments (P < 0.02). These results indicated the importance of immediate access to feed posthatch, the beneficial effects of feed- 0.01). Compared with birds in the nongln groups, birds in the Gln group had higher gain, feed efficiency, and livability (P < 0.05). Among the Fast (groups 1 to 4), Feed (groups 5 to 8), and Oasis (groups 9 to 12) groups, birds ing Oasis hatching supplement and Gln after hatch, as well as the necessity of the vaccination program against coccidiosis challenge. (Key words: glutamine, hatchling supplement, intestinal morphology, immune response, Eimeria maxima) 2005 Poultry Science 84: INTRODUCTION Under commercial situations, newly hatched broiler chickens often do not have access to water and feed for 36 to 48 h before they are transported from hatchery to production facilities, which greatly increases their susceptibility to stress and mortality (Dibner and Knight, 1999). Access to feed and water immediately after hatch is very important for the overall growth performance of poultry. The consequences of delayed intake of feed and water include slow gastrointestinal tract and immune system 2005 Poultry Science Association, Inc. Received for publication April 20, Accepted for publication October 13, Paper was partially presented at 2003 Poultry Science annual meeting, Madison, Wisconsin (abstract no. 120). 2 Oasis Hatchling Supplement is a trademark of Novus International, Inc., and is registered in the United States and other countries. 3 To whom correspondence should be addressed: iangyi@ novusint.com. development as well as poor growth performance (Dibner and Knight, 1999). Klasing (1998) reported that cell-mediated immunity and humoral antibody responses are enhanced in chicks that have been deprived of feed for short periods of time (12 to 24 h); however, longer periods without feed result in elevated corticosterone levels and impaired humoral and cellular immune response. Oasis is a hydrated nutritional supplement for newly hatched poultry, which contains a minimum of 25% moisture, 20% protein, 0.5% fat, and a maximum of 3% crude fiber. Dibner et al. (1998) found that feeding Oasis for 48 h after hatch, followed by a corn-soy starter diet, enhanced the maturation of the bursa of Fabricius and its associated humoral immune system, improved resistance to an oral Abbreviation Key: CD = crypt depth; CFG = cumulative feed:gain ratio; CGAIN = cumulative weight gain; CLIV = cumulative livability; IFN-γ = interferon-γ; IL-2 = interleukin 2; MLT = muscular layer thickness; PERLIV = period livability; VCR = villus height:crypt depth ratio; VH = villus height. 283

2 284 YI ET AL. challenge with coccidial Eimeria maxima, and improved growth performance of 20-d-old broilers. Recently, the beneficial effect of feeding Oasis for the first 24 to 48 h posthatch on improving the growth performance, energy use, and meat yield of young chickens and turkey poults has been reported (Batal and Parsons, 2002; Mozdziak et al., 2002). Glutamine is an important precursor for the synthesis of amino acids, nucleotides, nucleic acids, amino sugars, proteins, and many other biologically important molecules (Souba, 1993). Glutamine is the main energetic substrate for rapidly proliferating cells such as intestinal enterocytes and activated lymphocytes (Calder and Yaqoob, 1999), and is considered a conditionally essential amino acid in some species under inflammatory conditions such as infection and injury (Newsholme, 2001). Glutamine is used at a high rate by isolated cells of the immune system in culture and is required to support optimal lymphocyte proliferation and cytokine production by lymphocytes and macrophages (Calder and Yaqoob, 1999). The immune response of broiler chicks infected with E. maxima includes generation of superoxide and nitric oxide free radicals at infection sites (Allen, 1997). However, Gln may play a role in eliminating those free radicals because it is also a precursor for the antioxidant glutathione synthesis (Wu, 1998). Poor weight gain and feed efficiency are characteristic of coccidiosis in broiler chicks, particularly through the 4- to 9-d period after infection when the parasite is rapidly reproducing in the upper small intestine (Allen, 2000; Matthews and Southern, 2000; Zhu et al., 2000). Eimeria maxima are considered the most immunogenic coccidia that infect chickens (Rose and Long, 1962) and they mainly parasitize the jejunal mucosa of the mid small intestine causing nutrient malabsorption and decreased weight gain (Ruff and Wilkins, 1980). Eimeria maxima infection can cause significant economic losses to producers by impaired digestive and absorptive function of the jejunum of broiler chicks, and decreased growth performance and poor feed efficiency (Allen, 1997; Zhu et al., 2000). Immune stress exposure of poultry could stimulate the cytokine production of macrophages, which act on the central nervous system and lead to anorexia, fever, hypersomnia, lethargy, and neuroendocrine changes, and on the peripheral target tissues that lead to metabolic changes and acute phase protein synthesis (Klasing, 1988). Byrnes et al. (1993) reported that macrophages from chickens infected with either E. maxima or Eimeria tenella coccidiosis secreted significantly higher amounts of proinflammatory cytokines interleukin-1 and tumor necrosis factor-α than macrophages from uninfected controls. The objectives of this study were to investigate the impact of a 48-h fast and E. maxima vaccination after hatch on growth performance and gastrointestinal tract development of broiler chicks; to explore the effects of 4 Novus International, St. Louis, MO. feeding glutamine (48 h or 28 d) and Oasis hatchling supplement (48 h) on growth performance, small intestinal morphology, and immune responses of posthatch broilers after vaccinated and challenged with E. maxima. MATERIALS AND METHODS Chickens, Housing, and Management Seven hundred and twenty Cobb Cobb hatchling broilers (half male and half female) were used in the current study. Chicks were hatched in our own hatchery and put on test immediately after the 21 d of incubation. All birds were raised in electrically heated battery brooders at ambient temperature with continuous light. Each pen was provided with water and an individual feeder. Room temperature was maintained at 32 C from 0 to 5 d and then gradually reduced according to standard brooding practice. The study lasted 28 d. The animal protocol for this research was in accordance with the Standard Operating Procedure of Novus International, Inc., and agreed by the University of Missouri Animal Care and Use Committee. On d 0 posthatch, experimental birds were allotted to 12 treatment groups in a complete randomized design, with 6 replicate pens per treatment (3 of each sex) and 10 birds per pen (Table 1). Groups 1 and 2 were fasted for 48 h after hatch followed by ad libitum access to a common starter diet and water. Groups 3 and 4 were fasted for 48 h after hatch followed by ad libitum access to a 1% Gln supplemented starter diet and water. Together, groups 1 to 4 comprised the Fast group. Groups 5 and 6 had ad libitum access to a common starter diet and water after hatch. Groups 7 and 8 had ad libitum access to a 1% Gln supplemented starter diet and water after hatch. Together, groups 5 to 8 comprised the Feed group. Groups 9 and 10 were fed regular Oasis Hatching Supplement 4 Oasis for 48 h posthatch followed by ad libitum access to a common starter diet and water. Groups 11 and 12 were fed Oasis sprayed with 1% Gln for 48 h followed by ad libitum access to a common starter diet and water. Together, groups 9 to 12 comprised the Oasis group. All diets were fed in mash form, and were formulated to meet or exceed NRC (1994) estimated nutrient requirements. The common starter diet was a typical cornsoybean meal diet (chemical analysis of nutrients is presented in Table 2). The diet with 1% Gln was similar to the common starter diet but was supplemented with 1% glutamine. The birds in groups 2, 4, 6, 8, 10, 11, and 12 were vaccinated with 50 viable sporulated oocysts of E. maxima after hatch by gavage, whereas the birds in groups 1, 3, 5, 7, and 9 were not vaccinated. Additionally, birds in group 11 were orally injected with Propionibacterium acnes after hatch. Propionibacterium acnes is an immunostimulant (Yoshihiro et al., 2004) used to boost immune resistance to E. maxima. Birds in all treatments were orally challenged with viable sporulated oocysts per bird on d 22 posthatch.

3 EIMERIA MAXIMA VACCINATED AND CHALLENGED BROILERS 285 TABLE 1. Experimental treatments 1,2 E. maxima P. acnes 3 E. maxima Treatment Treatment in First 48 h Days 2 to 28 vaccination challenge challenge group first 48 h 1% Gln 1% Gln (d 0) (d 0) (d 22) 1 Fast; water ad libitum No No No No Yes 2 Fast; water ad libitum No No Yes No Yes 3 Fast; water ad libitum No Yes No No Yes 4 Fast; water ad libitum No Yes Yes No Yes 5 Feed; water ad libitum No No No No Yes 6 Feed; water ad libitum No No Yes No Yes 7 Feed; water ad libitum Yes Yes No No Yes 8 Feed; water ad libitum Yes Yes Yes No Yes 9 Oasis; water ad libitum No No No No Yes 10 Oasis; water ad libitum No No Yes No Yes 11 Oasis; water ad libitum Yes No Yes Yes Yes 12 Oasis; water ad libitum Yes No Yes No Yes 1 Glutamine was supplemented in common starter diets or sprayed onto Oasis at 1% for first 48 h after hatch or at 28 d for studying period posthatch. 2 Birds were vaccinated with 50 viable sporulated oocysts of Eimeria maxima after hatch (d 0) and challenged with 40,000 viable sporulated oocysts of E. maxima on d 22 after hatch. 3 Propionibacterium acnes is an immunostimulant, which is used in coccidial vaccination studies to boost immune resistance to E. maxima. All birds were observed at least twice daily and any mortality was recorded to calculate period livability (PER- LIV) or cumulative livability (CLIV). Body weight and feed intake of each pen were measured on d 0, 7, 14, 21, and 28 and numbers of birds were recorded each weigh day to calculate weight gain, cumulative weight gain (CGAIN), feed:gain ratio (FG), and cumulative feed:gain ratio (CFG). On d 2, 7, and 14, one bird from each pen (6 birds per treatment) was randomly selected and killed to collect mid small intestine tissues for intestinal morphology measurement. On d 14 and 21, one bird from each pen (6 birds per treatment) was randomly selected and killed to collect blood sample for serum cytokine interferon-γ (IFN-γ) and interleukin-2 (IL-2) concentration analysis. The serum IFN-γ and IL-2 measurements were conducted at the Parasite Biology, Epidemiology and Systematics Laboratory of USDA-ARS, Beltsville, Maryland, using methodology described by Dalloul et al. (2003). Briefly, flat-bottom microtiter plates 5 were coated with 100 µl of0.2m sodium carbonate buffer for 18 h at 4 C and washed 3 times with PBS containing 0.05% Tween-20 (PBS-T). This step was followed by blocking with PBS-2% BSA 6 for 1 h at room temperature, and then plates were washed 3 times with PBS-T. To each well, 100 µl of mouse antichicken IFN-γ monoclonal antibody or mouse antichicken IL-2 antibody were added to measure IFN-γ and IL-2, respectively, and allowed to incubate for 1 h at room temperature on a plate shaker. The plates were washed with PBS-T, and 100 µl of horseradish peroxide-conjugated goat antimouse immunoglobulin G antibody (dilutions were 1:1000 for IFN-γ and 1:2000 for IL- 2) was added to each well and allowed to incubate for 30 min at room temperature with shaking. The plates 5 Dynex Technologies, Chantilly, VA. 6 Sigma Chemical Co., St. Louis, MO. 7 Bio-Rad Clinical Diagnostics, Hercules, CA. were washed again, 100 µl of the substrate tetramethylbenzidine 5 was added, and the optical density was read at 450 nm by an automated microtiter plate reader. 7 On d 28, 4 birds from each pen (24 birds per treatment) were randomly removed and killed for lesion evaluation in the mid small intestine (near the yolk stalk). Intestinal Morphology and Lesion Scoring Method On d 0, 2, 7, and 10 after hatch, birds were killed to collect 4-cm mid small intestinal (2 cm proximal to 2 cm distal to Meckel s diverticulum) segments. The intestinal segments were flushed clean with and fixed in 10% No- TABLE 2. Chemical analysis of broiler starter diet (as-fed basis) 1 Amino acid composition Percentage Lysine 1.14 Methionine 0.60 Cysteine 0.39 Methionine + cysteine 0.99 Threonine 0.96 Tryptophan 0.25 Isoleucine 0.86 Valine 1.05 Arginine 1.38 Glycine + serine 1.95 Proline 1.53 Glutamic acid 4.23 Proximate analysis Crude protein Moisture Fat 5.35 Fiber 2.01 Ash 5.84 Total calcium 0.95 Total phosphorous 0.82 Sodium 0.24 Metabolizable energy, kcal/kg 3,040 1 Proximate and amino acid analyses were conducted by the chemical laboratory of Novus International, Inc., St. Louis, MO, according to the methodology of AOAC (1999).

4 286 YI ET AL. ToX (a nonformalin tissue fixative). 5 Histological slides were prepared from 3 cross-sections (5 µm thick) of each intestinal sample, which were processed in low-melt paraffin and stained with hematoxylin-eosin. Villus height (VH) and crypt depth (CD) were measured using the Image-Pro Plus as described in details by Touchette et al. (2002), and VH:CD ratio (VCR) were calculated. The ten longest and straightest villi and associated crypts were measured from each segment. In addition, the 3 thickest muscular layers (MLT) were measured from the submucosal layer to the serosal layer of the intestinal walls. Mean VH, CD, VCR, and MLT were calculated and used for statistical analysis. The lesion scoring method used in this study was modified from that described by Johnson and Reid (1970). It is a 5-point system scored in half points, based on the small reddish lesions formed in the mid small intestine (near the yolk stalk) on d 6 after challenge with E. maxima. The higher the lesion score, the more severe the infection of the mid small intestine. Statistical Analysis All the data were subjected to ANOVA procedures for completely randomized designs using the GLM models procedure of the SAS program (SAS Institute, 1996). Statistical significances of differences were assessed among dietary treatments by using the least significant difference (LSD) test (Steel and Torrie, 1980). Single degree of freedom orthogonal contrasts were performed to compare the treatment effects of nonvaccinated vs. vaccinated groups (groups 1, 3, 5, 7, 9 vs. 2, 4, 6, 8, 10), Fast vs. Feed groups (groups 1, 2, 3, 4 vs. 5, 6, 7, 8), Fast vs. Oasis groups (groups 1, 2, 3, 4, vs. 9, 10, 11, 12), Feed vs. Oasis groups (groups 5, 6, 7, 8 vs. 9, 10, 11, and 12), Oasis vs. Oasis/ Gln (group 10 vs. 12), Oasis vs. Oasis/Propionibacterium challenge (group 10 vs. 11), and nongln vs. Gln groups (groups 1, 2, 5, 6, 10 vs. 3, 4, 7, 8, and 12). A level of P < 0.05 was used as the criterion for statistical significance. Growth Performance RESULTS The growth performance of broiler chicks in different treatments from d 0 to 28 posthatch are presented in Table 3, and the P value of growth parameters for different contrast groups are presented in Table 4. During d 0 to 7 after hatch, birds in group 7, which had ad libitum access to 1% Gln supplemented starter diet and water after hatch without E. maxima vaccination had the highest BW, gain, and feed efficiency among treatments (P < 0.01). Birds given feed or oasis with 1% Gln (groups 7, 8, 9, and 12) had the highest, whereas birds in the fasted, vaccinated, and no Gln group (group 2) had the lowest PERLIV among treatments (P < 0.02). Compared with the vaccinated groups, the birds in the nonvaccinated groups had heavier BW (P < 0.02), gain (P < 0.01), and PERLIV (P < 0.05). Among the Fast, Feed, and Oasis groups, birds in the Feed groups had the highest, and birds in the Fast groups had the lowest BW, gain, and feed efficiency (P < 0.01). Additionally, birds in the Feed groups had higher PERLIV relative to the Fast groups (P < 0.03). Compared with birds in the nongln groups, birds in the Gln-fed groups had higher BW (P < 0.01), gain (P < 0.01), feed efficiency (P < 0.02), and PERLIV (P < 0.05). During d 7 to 14 posthatch, similar to d 0 to 7 posthatch, birds given feed with Gln (group 7) had the highest BW, gain, CGAIN, and cumulative feed efficiency (P < 0.01) among treatments (P < 0.01). Birds in treatments fed with Gln-supplemented starter feed or Oasis (groups 7, 8, 9, and 12) had the highest CLIV among treatments (P < 0.02). Among all groups, birds in the Feed groups had the highest, whereas birds in the Fast groups had the lowest BW, gain, and CGAIN (P < 0.01). Additionally, birds in the Feed groups had higher cumulative feed efficiency relative to the Fast or Oasis groups. Compared with the E. maxima vaccinated groups, birds in the nonvaccinated groups had higher CGAIN (P < 0.05), feed efficiency (P < 0.05), and cumulative feed efficiency (P < 0.01). Compared with birds in the nongln groups, birds in the Gln groups had higher BW, gain, CGAIN, cumulative feed efficiency, and CLIV (P < 0.05). During d 14 to 21 after hatch, birds in the fasted treatments (groups 1 to 4) had lower BW, and CGAIN relative to all other treatments fed ad libitum (groups 5 to 8) or fed Oasis (groups 9 to 12) for 48 h posthatch (P < 0.01). Similar to d 0 to 7 and d 7 to 14, birds in groups given feed or Oasis with supplemental Gln (groups 7, 8, 9, and 12) had the highest CLIV among treatments (P < 0.01). Among the Fast, Feed, and Oasis groups, birds in the Feed groups had the highest BW, CGAIN, and CLIV (P < 0.01). Compared with the vaccinated groups, the birds in the nonvaccinated groups had higher CLIV (P < 0.01). In addition, compared with birds in the nongln fed groups, birds in the Gln fed groups had higher CLIV (P < 0.01). During the postchallenge period with E. maxima (d 22 to 28 posthatch), birds in the fasted treatments (groups 1 to 4) had lower BW and CGAIN relative to all other treatments fed ad libitum (groups 5 to 8) or fed Oasis (groups 9 to 12) for 48 h posthatch, regardless of vaccination (P < 0.01). Birds in groups 7, 8, and 12, which all had access to Gln-supplemented feed or Oasis posthatch, had the highest PERLIV among treatments (P < 0.01). In contrast to d 0 to 7 and d 7 to 14,during the postchallenge period with E. maxima (d 22 to 28 posthatch), the birds in the nonvaccinated groups had lower BW, gain, CGAIN, feed efficiency, and cumulative feed efficiency relative to the vaccinated groups (P < 0.01). In addition, compared with birds in the nongln groups, birds in the Gln groups had higher PERLIV (P < 0.01). Intestinal Morphology The intestinal morphology of the mid small intestine of broilers in different treatments on d 2, 7, and 14 post-

5 EIMERIA MAXIMA VACCINATED AND CHALLENGED BROILERS 287 TABLE 3. Growth performance of posthatch broilers in different treatment groups (d 0 to 28) 1 Treatment group Variable SEM P-value Days0to7 BW, g 104 a 101 a 103 a 106 a 151 c 148 c 160 d 151 c 135 b 130 b 130 b 132 b 2.48 <0.01 Gain, g 62 a 59 a 62 a 63 a 110 c 105 c 120 d 109 c 93 b 89 b 89 b 91 b 2.28 <0.01 FG, g/g 1.11 a 1.19 a 1.13 a 1.10 b 1.07 b 1.08 b 0.97 c 1.00 c 1.00 c 1.07 b 1.01 c 1.01 c 0.03 <0.01 PERLIV, % 99 a 91 b 96 a 98 a 98 a 98 a 100 a 100 a 100 a 96 a 98 a 100 a Days7to14 BW, g 306 a 297 a 311 a 307 a 386 c 388 c 416 d 390 c 358 b 351 b 352 b 358 b 7.28 <0.01 CGAIN, g 264 a 256 a 270 a 265 a 345 c 346 c 375 d 348 c 316 b 310 b 311 b 317 b 7.03 <0.01 Gain, g 202 a 197 a 208 ab 202 a 235 c 241 cd 256 d 239 c 223 bc 221 bc 222 bc 226 c 5.48 <0.01 CFG, g/g 1.22 bc 1.25 ab 1.21 bc 1.25 ab 1.23 bc 1.22 bc 1.18 c 1.20 c 1.21 bc 1.28 a 1.23 bc 1.23 bc FG, g/g 1.26 ab 1.28 abc 1.24 a 1.31 bc 1.32 c 1.29 bc 1.31 bc 1.32 bc 1.32 bc 1.39 d 1.33 c 1.33 c 0.02 <0.01 CLIV*, % 98 ab 92 a 96 ab 98 ab 98 ab 96 ab 100 b 100 b 100 b 94 ab 94 ab 100 b Days 14 to 21 BW, g 666 a 647 a 680 a 675 a 750 bc 772 c 764 bc 765 bc 725 b 749 bc 737 bc 743 bc 15.6 <0.01 CGAIN, g 625 a 606 a 639 a 633 a 709 bc 731 c 723 bc 723 bc 683 b 708 bc 697 bc 702 bc 15.4 <0.01 Gain, g 361 a 350 a 369 ab 368 ab 364 a 385 b 348 a 374 ab 367 ab 399 b 386 b 385 b CFG, g/g 1.24 ab 1.26 bc 1.20 a 1.25 bc 1.29 c 1.26 bc 1.29 c 1.27 bc 1.26 bc 1.27 bc 1.24 a 1.27 bc FG, g/g 1.26 a 1.27 a 1.20 a 1.25 a 1.36 bc 1.30 b 1.42 c 1.34 bc 1.31 b 1.27 a 1.26 a 1.32 b 0.03 <0.01 CLIV, % 98 b 83 a 95 b 95 b 98 b 95 b 100 b 100 b 100 b 93 b 93 b 100 b 2.76 <0.01 Days 22 to 28 BW, g 1,041 a 1,109 ab 1,074 ab 1,137 b 1,166 c 1,261 d 1,156 bc 1,249 d 1,156 bc 1,247 d 1,231 cd 1,252 d 32.2 <0.01 CGAIN, g/g 999 a 1,067 ab 1,033 ab 1,095 b 1,125 bc 1,219 d 1,116 bc 1,207 d 1,145 bc 1,206 d 1,189 cd 1,211 d 32.0 <0.01 Gain, g 374 a 462 bcd 394 a 462 bcd 416 ab 488 cd 392 a 484 cd 431 abc 498 d 493 cd 509 d 23.2 <0.01 CFG, g/g 1.58 bc 1.55 a 1.55 a 1.53 a 1.61 c 1.54 a 1.61 c 1.55 a 1.57 abc 1.56 ab 1.53 a 1.54 a 0.01 <0.01 FG, g/g 2.95 ab 2.44 a 3.81 b 2.31 a 2.98 ab 2.45 a 2.99 ab 2.38 a 2.69 a 2.39 a 2.40 a 2.25 a PERLIV, % 94 b 81 a 92 b 94 b 97 b 94 b 100 b 100 b 97 b 92 b 92 b 100 b a d Means in a row without a common superscript are different (P < 0.05). 1 Birds of treatment groups 2, 4, 6, 8, 10, 11, and 12 were vaccinated with 50 viable sporulated oocysts of E. maxima after hatch (d 0), and all birds were challenged with 40,000 viable sporulated oocysts of E. maxima on d 22 after hatch. Groups 1 and 2 were fasted for 48 h after hatch followed by ad libitum access to a common starter diet and water. Groups 3 and 4 were fasted for 48 h after hatch followed by ad libitum access to a 1% Gln supplemented starter diet and water. Together, groups 1 to 4 comprised the Fast group. Groups 5 and 6 had ad libitum access to a common starter diet and water after hatch. Groups 7 and 8 had ad libitum access to a 1%Gln supplemented starter diet and water after hatch. Together, groups 5 to 8 comprised the Feed group. Groups 9 and 10 were fed regular Oasis for 48 h posthatch followed by ad libitum access to a common starter diet and water. Groups 11 and 12 were fed Oasis sprayed with 1% Gln for 48 h followed by ad libitum access to a common starter diet and water. Together, groups 9 to 12 comprised the Oasis group. 2 CGAIN = cumulative weight gain; Gain = period weight gain; CFG = cumulative feed:gain ratio; FG = period feed:gain ratio; CLIV = cumulative livability; PERLIV = period livability.

6 288 YI ET AL. TABLE 4. Probability values of growth parameters for different contrast groups 1 Contrast (groups) Nonvac. vs. vac. Fast vs. feed Fast vs. Oasis Feed vs. Oasis Oasis vs. Oasis vs. NonGln vs. Gln (1, 3, 5, 7, 9 vs. (1, 2, 3, 4 vs. (1, 2, 3, 4 vs. (5, 6, 7, 8 vs. Oasis/Gln Oasis/P 3 (1, 2, 5, 6, 10 vs. Variable 2 2, 4, 6, 8, 10) 5, 6, 7, 8) 9, 10, 11, 12) 9, 10, 11, 1) (10 vs. 12) (10 vs. 11) 3, 4, 7, 8, 12) Days0to7 BW 0.02 <0.01 <0.01 < GAIN <0.01 <0.01 <0.01 < <0.01 FG, g/g 0.11 <0.01 < <0.01 PERLIV, % Days7to14 BW, g 0.07 <0.01 <0.01 < CGAIN, g 0.05 <0.01 <0.01 < GAIN, g 0.17 <0.01 <0.01 < CFG, g/g < FG, g/g < CLIV, % Days 14 to 21 BW, g 0.62 <0.01 < CGAIN, g 0.65 <0.01 < GAIN, g CFG, g/g 0.57 < FG, g/g 0.23 < < CLIV, % <0.01 < <0.01 Days 22 to 28 BW, g <0.01 <0.01 < CGAIN, g <0.01 <0.01 < GAIN, g < < CFG, g/g < FG, g/g < PERLIV, % 0.08 < Birds of treatment groups 2, 4, 6, 8, 10, 11, and 12 were vaccinated with 50 viable sporulated oocysts of E. maxima after hatch (d 0), and all birds were challenged with 40,000 viable sporulated oocysts of E. maxima on d 22 after hatch. Groups 1 and 2 were fasted for 48 h after hatch followed by ad libitum access to a common starter diet and water. Groups 3 and 4 were fasted for 48 h after hatch followed by ad libitum access to a 1% Gln supplemented starter diet and water. Together, groups 1 to 4 comprised the Fast group. Groups 5 and 6 had ad libitum access to a common starter diet and water after hatch. Groups 7 and 8 had ad libitum access to a 1%Gln supplemented starter diet and water after hatch. Together, groups 5 to 8 comprised the Feed group. Groups 9 and 10 were fed regular Oasis for 48 h posthatch followed by ad libitum access to a common starter diet and water. Groups 11 and 12 were fed Oasis sprayed with 1% Gln for 48 h followed by ad libitum access to a common starter diet and water. Together, groups 9 to 12 comprised the Oasis group. 2 CGAIN = cumulative weight gain; Gain = period weight gain; CFG = cumulative feed:gain ratio; FG = period feed:gain ratio; CLIV = cumulative livability; PERLIV = period livability. 3 Oasis/P = oral challenge with Propionibacterium acnes after hatch; P. acnes is an immunostimulant used to boost immune resistance to E. maxima. hatch are shown in Table 5 (the P value of intestinal morphology parameters for different contrast groups are not shown). On d 2 posthatch, in the mid small intestine, there were no differences in VH, VCR, and MLT among treatments (P > 0.10); however, there was a difference in CD among treatments (P < 0.01). Compared with the Fast groups, birds in the Feed and Oasis groups had higher VH of mid small intestine (P < 0.05). Among all groups, birds in the Feed groups had the highest, and birds in the Fast groups had the lowest CD (P < 0.01) and MLT (P < 0.05). On d 7 posthatch, there was difference in VH of the mid small intestine among treatments (P < 0.01), whereas there were no differences in CD, VCR, and MLT among treatments (P > 0.10). Compared with the vaccinated groups, birds in the nonvaccinated groups had higher VH (P < 0.01). Birds in the Feed groups had higher VH (P < 0.01) and VCR (P < 0.02) compared with the Fast groups, and higher VH (P < 0.01) compared with the Oasis groups. Additionally, birds in the Oasis groups had higher VH compared with the Fast groups (P < 0.01). However, on d 14 posthatch, there were no differences in VH, CD, VCR, and MLT of the mid small intestine among treatments or contrast groups (P > 0.07). The cross-sections and intestinal morphology of mid small intestine of birds in groups 1, 4, 8, and 12 at d 7 posthatch are shown in micrographs A, B, C, and D of Figures 1 and 2, respectively. Figure 1A clearly demonstrated that fasting for 48 h posthatch (group 1) delayed the growth of mid small intestine and led to the shortest villus height and smallest absorptive surface area. Figure 1B indicates that feeding 1% Gln following a 48-h fast (group 4) was able to help recovery of small intestine development, but was not able to match the development of birds fed starter feed with 1% Gln ad libitum posthatch (group 8) or fed Oasis sprayed with 1% Gln for 48 h posthatch followed by ad libitum access to a common starter diet (group 12). In Figure 2A, fasted birds in groups 1 and 4 had shorter villus height and disrupted intestinal integrity compared with birds fed starter feed with 1% Gln ad libitum posthatch (group 8) or fed Oasis sprayed with 1% Gln for 48 h posthatch followed by ad libitum access to a common starter diet (group 12). The E. maxima infection in the intestinal villi was observed in the birds

7 EIMERIA MAXIMA VACCINATED AND CHALLENGED BROILERS 289 TABLE 5. Intestinal morphology of the mid small intestine of broilers in different treatments (d 0 to 14) 1 Treatment group Item SEM P-value Day 2 VH, µm CD, µm 93 ab 90 a 92 ab 84 a 111 c 110 c 106 bc 101 abc 100 abc 94 ab 106 bc 96 abc VCR MLT, µm Day 7 VH, µm 570 b 530 ab 560 b 481 a 656 c 612 c 632 c 607 c 614 c 570 b 557 b 619 c <0.01 CD, µm VCR MLT, µm Day 14 VH, µm CD, µm VCR MLT, µm a c Means in a row without a common superscript are different (P < 0.05). 1 Birds of treatment groups 2, 4, 6, 8, 10, 11, and 12 were vaccinated with 50 viable sporulated oocysts of E. maxima after hatch (d 0), and all birds were challenged with 40,000 viable sporulated oocysts of E. maxima on d 22 after hatch. Groups 1 and 2 were fasted for 48 h after hatch followed by ad libitum access to a common starter diet and water. Groups 3 and 4 were fasted for 48 h after hatch followed by ad libitum access to a 1% Gln supplemented starter diet and water. Together, groups 1 to 4 comprised the Fast group. Groups 5 and 6 had ad libitum access to a common starter diet and water after hatch. Groups 7 and 8 had ad libitum access to a 1%Gln supplemented starter diet and water after hatch. Together, groups 5 to 8 comprised the Feed group. Groups 9 and 10 were fed regular Oasis for 48 h posthatch followed by ad libitum access to a common starter diet and water. Groups 11 and 12 were fed Oasis sprayed with 1% Gln for 48 h followed by ad libitum access to a common starter diet and water. Together, groups 9 to 12 comprised the Oasis group. 1 VH = villus height; CD = crypt depth; VCR = VH:CD ratio; MLT = muscular layer thickness. vaccinated with low dosage of viable E. maxima in groups 4, 8, and 12, but not observed in group 1 (not vaccinated), indicating the success of the vaccination program as applied in the current study. FIGURE 1. Light microscopy (20 by 10 ) of the cross-sections of mid small intestine of birds on different treatments (scale bar = 2 mm) at d 7 posthatch. A. Birds fasted for 48 h posthatch followed by ad libitum access to a common starter diet and water (group 1). B. Birds fasted for 48 h followed by ad libitum access to 1% glutamine starter diet and water (group 4). C. Birds with access to 1% glutamine starter diet and water posthatch (group 8). D. Birds fed Oasis sprayed with 1% glutamine for 48 h followed by ad libitum access to a common starter diet and water. Serum Cytokine Concentration and Lesion Scores of the Mid Small Intestine Serum cytokine IFN-γ and IL-2 concentrations and lesion scores of the mid small intestine of broilers in different treatments are shown in Table 6 (the P value of serum IFN-γ and IL-2 and lesion scores for different contrast groups are not shown). On d 14 posthatch, there was a difference in serum IFN-γ among treatments, in which birds in groups 4 and 11 had the highest serum IFN-γ (P < 0.01). Birds in the Feed groups had lower serum IFN-γ compared with the Oasis groups (P < 0.03). Birds fed Oasis and vaccinated with E. maxima (group 10) had lower serum IFN-γ compared with birds fed Oasis and inoculated with P. acnes along with E. maxima vaccination (group 11) (P < 0.04) at d 14 posthatch. On d 21 posthatch, there was no difference in serum IFN-γ among treatments (P < 0.31). Birds fed Oasis and vaccinated with E. maxima (group 10) had lower serum IFN-γ compared with birds fed Oasis and injected with P. acnes along with E. maxima vaccination (group 11) at d 21 after hatch (P < 0.02). There were no differences in serum IL-2 among treatments or contrast groups (P < 0.16) on d 14 and d 21 posthatch. After the E. maxima challenge on d 22 posthatch, i.e., at d 28 posthatch, there were differences in lesion scores of the mid small intestine among treatments (P < 0.01), in which birds in group 1 had the highest, and those in groups 6, 8, and 12 (fed starter feed or Oasis and vaccinated with E. maxima) had the lowest lesion scores. Compared with the vaccinated groups, birds in the

8 290 YI ET AL. TABLE 6. Serum interferon-γ (IFN-γ) and interleukin-2 (IL-2) concentrations and lesions scores of the mid small intestine of birds 1 Treatment group SEM P-value IFN-γ (ng/ml) d c 156 c 160 bc 189 a 181 ab 152 c 154 c 154 c 168 abc 164 bc 187 a 171 abc 7.21 <0.01 d IL-2 (ng/ml) d d Lesion score 3 d a 0.19 c 1.06 b 0.40 c 0.85 b 0.00 d 1.03 b 0.00 d 0.99 b 0.25 c 0.21 c 0.04 d 0.13 <0.01 a d Means in a row without a common superscript are different (P < 0.05). 1 Birds of treatment groups 2, 4, 6, 8, 10, 11, and 12 were vaccinated with 50 viable sporulated oocysts of E. maxima after hatch (d 0), and all birds were challenged with 40,000 viable sporulated oocysts of E. maxima on d 22 after hatch. Groups 1 and 2 were fasted for 48 h after hatch followed by ad libitum access to a common starter diet and water. Groups 3 and 4 were fasted for 48 h after hatch followed by ad libitum access to a 1% Gln supplemented starter diet and water. Together, groups 1 to 4 comprised the Fast group. Groups 5 and 6 had ad libitum access to a common starter diet and water after hatch. Groups 7 and 8 had ad libitum access to a 1%Gln supplemented starter diet and water after hatch. Together, groups 5 to 8 comprised the Feed group. Groups 9 and 10 were fed regular Oasis for 48 h posthatch followed by ad libitum access to a common starter diet and water. Groups 11 and 12 were fed Oasis sprayed with 1% Gln for 48 h followed by ad libitum access to a common starter diet and water. Together, groups 9 to 12 comprised the Oasis group. 2 On d 14 and 21 after hatch, 1 bird per pen (6 birds per treatments) was randomly selected and pooled blood samples by cardiac puncture to measure serum IFN-γ αand L-2. 3 On d 28 after hatch, 4 birds per pen (24 birds per treatment) were randomly selected and killed to evaluate mid small intestine (close to yolk stalk) lesion score. nonvaccinated groups had higher lesion scores (P < 0.01). Among the Fast, Feed, and Oasis groups, birds in the Feed or Oasis groups had lower lesion scores compared with the Fast groups, regardless of vaccination (P < 0.02). Birds fed Oasis and vaccinated (group 10) had higher lesion scores compared with birds fed Gln-sprayed Oasis and vaccinated (group 12). For group 12, Gln was present only during the Oasis feeding period (48 h posthatch) FIGURE 2. Light microscopy (100 by 10 ) of the cross-sections of mid small intestine of birds on different treatments (scale bar = 200 µm, and the arrows indicate E. maxima infection) at d 7 posthatch. A. Birds fasted for 48 h posthatch followed by ad libitum access to a common starter diet and water (group 1, no vaccination). B. Birds fasted for 48 h followed by ad libitum access to 1% glutamine starter diet and water (group 4, with E. maxima vaccination). C. Birds with access to 1% glutamine starter diet and water posthatch (group 8, with E. maxima vaccination). D. Birds fed Oasis sprayed with 1% glutamine for 48 h followed by ad libitum access to a common starter diet and water (group 12, with E. maxima vaccination). and absent in subsequent feedings. However, the benefits of Gln on lowering the lesion score was observed at 6 d postchallenge, which may indicate some beneficial effect of Gln on immune modulation against E. maxima infection. DISCUSSION Under commercial situations, it may take 36 to 48 h for newly hatched broiler chickens to be transported and given access to feed and water in production facilities. Dibner and Knight (1999) reported that delayed intake of feed and water can impair gastrointestinal tract and immune system development as well as growth performance. In the current study, fasting for 48 h after hatch dramatically decreased the weight gain and feed efficiency during the first 2 wk. However, having ad libitum access to feed or feeding Oasis hatchling supplement for 48 h followed by feeding ad libitum are beneficial for the early growth performance of broiler chicks. During the prechallenge period (d 0 to 21), among the Fast, Feed, and Oasis groups, birds in the Fast groups had the lowest cumulative weight gain and the highest mortality. During the postchallenge period, birds in the Fast groups had greatly reduced BW, gain, CGAIN, and PERLIV relative to the Feed or Oasis groups. These results indicated that fasting for a short time (48 h in this study) after hatch can dramatically impair growth performance and increase the mortality of young birds. Consistent with our findings, Noy and Sklan (1999) reported that compared with fasted chicks (34 h) or fasted turkey poults (48 h), birds having ad libitum access to feed or Oasis immediately after placement had significantly higher BW through 21 d of age. Knight and Dibner (1998) reported that, compared with turkey poults fasted for 72 h posthatch, feeding Oasis immediately after placement resulted in higher growth

9 EIMERIA MAXIMA VACCINATED AND CHALLENGED BROILERS 291 rates of the small intestine, liver, and pancreas, and better growth rate. The results of the current study emphasize the importance of having immediate access to nutrientsufficient starter diets for broiler chicks after hatch. Noy and Sklan (2001) reported that feed deprivation for 48 to 96 h posthatch resulted in reduced plasma T3 levels and dramatically decreased glucose and amino acid uptake, whereas immediate feed intake after hatch stimulated yolk secretion to the small intestine and triggered uptake mechanisms for hydrophilic compounds. Therefore, immediate access to feed and water posthatch may shuttle more nutrients and immunoglobulin from the yolk sac to promote the early development of the gastrointestinal tract, and enhance the digestive and absorptive functions of the small intestine. That is probably why the birds fed ad libitum or fed Oasis followed by ad libitum feeding resulted in better growth than fasted birds in the current study. Factors related to the genetics of poultry, the frequency of their exposure to pathogens, the virulence of the pathogens, and the efficiency of vaccination programs are primary detriments of the incidence of infectious disease in poultry (Klasing, 1998). Poor weight gain and feed conversion efficiency are characteristic of coccidiosis-infected broiler chicks, particularly through the 4- to 9-d period after infection, when the parasite is rapidly reproducing in the upper small intestine (Allen, 2000; Matthews and Southern, 2000; Zhu et al., 2000). In the present study, it was clear that on d 7 posthatch, E. maxima infection was observed in the intestinal villi of mid small intestine of birds that were vaccinated with E. maxima (Figure 2). Vaccination with E. maxima sporulated oocysts immediately after placement decreased the growth performance and increased the mortality of broiler chicks during the first 2 wk. However, during the postchallenge period with E. maxima (d 22 to 28), the growth performance was reversed, in which the vaccinated birds had higher growth and feed efficiency compared with the nonvaccinated birds, indicating the success and efficiency of the vaccination program used in this study. The high dosage E. maxima challenge caused detriments to birds as reflected by the decreased feed efficiency and livability. However, birds with access to Gln-supplemented feed or Gln-sprayed Oasis immediately after placement maintained livability as observed during the pre- and postchallenge period, indicating the beneficial effects of Gln supplementation on improving the livability of broilers vaccinated and challenged with E. maxima. Glutamine has been reported to increase survival rate and reduce bacterial translocation in mice following immunological challenge (Adjei et al., 1994). During the first 2 wk posthatch, birds in the treatment given immediate access to feed supplemented with Gln (group 7) had the highest BW, gain, CGAIN, and cumulative feed efficiency among all treatments. Additionally, birds in the Gln groups had higher BW, gain, cumulative feed efficiency, and CLIV compared with birds in the nongln groups. In a previous study with 1-d-old broiler chicks, we did not see the beneficial effects of feeding a 1% Gln diet on growth performance compared with a corn-soy starter diet (Yi et al., 2001), which may indicate the beneficial effect of Gln under more stressed or immune-challenged situations (Newsholme, 2001). The small intestine of newly hatched chicks is immature and undergoes dramatic morphological, biochemical, and molecular changes during the first week posthatch (Uni et al., 1998). Eimeria maxima are considered the most immunogenic coccidia that infect chickens (Rose and Long, 1962), and mainly parasitize the jejunal mucosa of the mid small intestine, causing nutrient malabsorption and decreased weight gain (Ruff and Wilkins, 1980). In the current study, the mid small intestine, which is close to the yolk stalk of the distal jejunum, is selected for intestinal morphology measurement. On d 2 posthatch, birds in the Feed groups had the highest, and birds in the Fast groups had the lowest VH of the mid small intestine. On d 7 posthatch, it was shown that fasting for 48 h posthatch resulted in less developed intestinal villi and disrupted intestinal integrity compared with birds fed ad libitum or fed Oasis for 48 h immediately after placement (Figures 1 and 2). Consistent with our findings, Uni et al. (1998) found that delayed access to feed after hatch depressed mucosal development for several days. In the current study, the improved VH of the mid small intestine of birds in the Feed and Oasis groups was in agreement with the enhanced growth performance compared with the Fast groups. On d 14 posthatch, there were no differences in VH, CD, VCR, and MLT among treatments or contrast groups, which may indicate the relative maturity of the small intestine at 14 d after hatch. During the first 2 wk, birds in the group given Gln-supplemented feed (group 7) after placement had the best growth performance. These birds also had higher CD on d 2 posthatch and higher VH on d 7 posthatch compared with birds fasted for 48 h after hatch. However, there was no difference between fed birds with or without Gln providing the birds had access to the starter feed immediately after placement. Therefore, there may be other factors contributing to the increased growth performance when feeding Gln. Those factors may include the supply of enteral Gln as an energy substrate for rapidly proliferating enterocytes and a nitrogen source for nucleotide synthesis (Calder and Yaqoob, 1999), and the importance of Gln in maintaining mucosal structure, especially in the maintenance of the tight junction and permeability of intestinal mucosa (Panigrahi et al., 1997). Glutamine may also act as a signal or regulator of metabolic demands, increasing protein synthesis and decreasing protein degradation in skeletal muscle (Haussinger et al., 1994) for young broilers. The principal defense mechanism against protozoa (E. maxima in this case) that survive within macrophages is cell-mediated immunity, particularly activation of T H 1 CD + 4 T cells. Interleukin-2 is a growth factor for antigenstimulated lymphocytes and is responsible for T cell clonal expansion; IFN-γ is the macrophage-activating cytokine that provides the means by which T H 1CD + 4 T cells activate macrophages to kill intracellular parasites (Abbas

10 292 YI ET AL. et al., 2000). In the current study, on d 14 posthatch, birds in Oasis groups had the highest serum IFN-γ, which might be attributed to more vaccinated birds in the Oasis groups and activated IFN-γ production of T H 1CD + 4 T cells. On both d 14 and 21, birds given Oasis with no P. acnes (group 10) had lower serum IFN-γ compared with birds in the Oasis treatment inoculated with the P. acnes (group 11). Birds in group 11 were orally injected with P. acnes immediately after placement (P. acnes is an immunostimulant used in coccidial vaccination studies for a boost in immune resistance to E. maxima). This study indicated that P. acnes injection might be an effective immunostimulant to increase IFN-γ production and activate macrophages to kill intracellular E. maxima. On d 14 and 21, there were no differences in serum IL-2 among treatments or contrast groups. Byrnes et al. (1993) reported that macrophages from chickens infected with either E. maxima or E. tenella coccidiosis secrete significantly higher amounts of the proinflammatory cytokines, interleukin- 1 and tumor necrosis factor-α, than macrophages from uninfected controls. Unfortunately, we did not measure serum IFN-γ, IL-2, or other cytokines after the E. maxima challenge. During the prechallenge period, there was no difference in serum IFN-γ or IL-2 between vaccinated or nonvaccinated groups. Eimeria maxima is a parasite that develops in the mid small intestine, and can cause pathological symptoms such as bloated mid small intestine, watery diarrhea, mucosal reddish lesions, and mucosal sloughing (Allen and Fetterer, 2002). Body weight gain, feed conversion, lesion scores, and oocyst shedding are commonly used to evaluate the resistance or susceptibility of poultry to coccidiosis challenge (Zhu et al., 2000). In the current study, during the challenge period (d 22 to 28 posthatch), birds in the nonvaccinated groups had higher lesion scores compared with the vaccinated groups, indicating more severe intestinal infection of E. maxima in the nonvaccinated groups and the success of the vaccination program in alleviating the deleterious effect of E. maxima infection. Among all groups, birds in the Fast groups had higher lesion scores of the mid small intestine relative to the Feed and Oasis groups, which indicates that fasting for a short period of time immediately after placement (48 h in this study) impairs gastrointestinal tract development and immune system maturation. Among the Feed and Oasis groups vaccinated with E. maxima immediately after placement, birds with access to the Gln-supplemented diet or Gln-sprayed Oasis had the lowest lesion scores, indicating the beneficial effect of glutamine in promoting the maturity of gutassociated immune system and maintaining the intestinal integrity. In conclusion, having access to feed and water after hatch is very important for broiler chicks to promote gastrointestinal tract development, maintain small intestinal integrity, and enhance growth performance during pre- and postchallenge with E. maxima. Early access to a 1% glutamine supplemented diet can greatly improve the growth performance and decrease the mortality of broilers having access to feed immediately after placement. Compared with birds being fasted for 48 h after hatch, ad libitum feeding or feeding of Oasis for 48 h followed by ad libitum access to feed are both effective in advancing small intestine development, enhancing growth performance, and decreasing the severity of E. maxima infection. Compared with nonvaccinated birds, birds vaccinated with a low dose of viable E. maxima sporulated oocysts immediately after placement had slightly decreased growth performance during the prechallenge period. Growth depression associated with high dosage E. maxima challenge was alleviated, indicating the success and importance of the vaccination program used in the current study. ACKNOWLEDGMENTS Appreciation is extended to Novus International, Inc., for financial and product support. The authors express their gratitude to S. Hampton, C. W. Wuelling, M. E. Wehmeyer, S. W. Richter, and M. L. Kitchell for their technical support and animal care. REFERENCES Abbas, A. K., A. H. Lichtman, and J. S. 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