Response to dietary supplementation of l -glutamine and l -glutamate in broiler chickens reared at different stocking densities under hot, humid tropical conditions

ENVIRONMENT, WELL-BEING, AND BEHAVIOR Response to dietary supplementation of L-glutamine and L-glutamate in broiler chickens reared at different stocking densities under hot, humid tropical conditions M. Shakeri,* I. Zulkifli,*1 A. F. Soleimani,* E. L. O’Reilly,† P. D. Eckersall,† A. A. Anna,* S. kumari,* and F. F. J. Abdullah‡ *Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; †Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, United Kingdom; and ‡Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia = 0.0007), and elevated heterophil to lymphocyte ratio (0.0005). Neither AminoGut supplementation nor stocking density affected cecal microflora counts. In conclusion, under the conditions of this study, dietary supplementation of AminoGut, irrespective of stocking density, had no beneficial effect on growth performance, intestinal morphology, and physiological adaptive responses of broiler chickens raised under hot and humid tropical conditions. However, AminoGut supplementation from d 1 to 42 was beneficial in reducing mortality rate. Also, the increased serum concentrations of a wide range of acute phase proteins together with elevated corticosterone and heterophil to lymphocyte ratio suggested that high stocking density induced an acute phase response either indirectly as a result of increased incidence of inflammatory diseases such as foot pad dermatitis or possibly as a direct physiological response to the stress of high stocking density.

Key words: l-glutamine, l-glutamic acid, stocking density, acute phase protein, well-being 2014 Poultry Science 93:1–9 http://dx.doi.org/10.3382/ps.2014-03910

INTRODUCTION

cause diseases that include leg weakness, ascites, sudden death syndrome, deep pectoral myopathy, and right ventricular hypertrophy. Incidence of these problems could also partly be associated with stocking density (Dozier et al., 2005; Estevez, 2007). There is considerable information available on the proper stocking density for broiler production (Estevez, 1999, 2007; Berg and Yngvesson, 2012); however, there is a paucity of information on the density of birds raised under the hot, humid tropical environment. It is well documented that heat stress-related problems in broiler chickens may be exacerbated by high stocking density (Imaeda, 2000). Although high stocking density has been closely associated with poor welfare of broiler chickens, many studies have failed to observe significant changes in physiological stress indicators such as plasma levels of

Commercial broiler chickens are among the fastest growing farm animal species. This unrivalled growth rate and large scale production is mainly due to the genetic selection for traits such as rapid growth, feed conversion ratio (FCR) and carcass yield. As reviewed by Bessei (2006), genetic selection together with environmental and management practices are reputed to

©2014 Poultry Science Association Inc. Received January 16, 2014. Accepted July 5, 2014. 1 Corresponding author: [email protected]

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ABSTRACT A study was conducted to determine whether supplementing AminoGut (a commercial dietary supplement containing a mixture of l-glutamine and l-glutamic acid) to broiler chickens stocked at 2 different densities affected performance, physiological stress responses, foot pad dermatitis incidence, and intestinal morphology and microflora. A randomized design in a factorial arrangement with 4 diets [basal diet, basal diet + 0.5% AminoGut from d 1 to 21, basal diet + 0.5% AminoGut from d 1 to 42, and basal diet + virginiamycin (0.02%) for d 1 to 42] and 2 stocking densities [0.100 m2/bird (23 birds/pen; LD) or 0.067 m2/bird (35 birds/pen; HD)]. Results showed that villi length and crypt depth were not changed by different dietary treatments. However, birds in the HD group had smaller villi (P = 0.03) compared with those of the LD group. Regardless of diet, HD consistently increased the serum concentrations of ceruloplasmin, α-1 acid glycoprotein, ovotransferin, and corticosterone (P

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MATERIALS AND METHODS Birds, Husbandry, and Housing The study was undertaken following the guidelines of the Research Policy on animal ethics of the Universiti Putra Malaysia. A total of 928 one-day-old Cobb 500 female broiler chicks were obtained from a local hatchery. On d 1, the chicks were weighed and randomly allocated to 32 floor pens (1.7 m × 1.4 m) with wood shavings deep litter and cyclic temperature (minimum, 24°C; maximum, 35°C). The RH was between 75 and 90%. Each pen had 2 tube feeders and 1 automatic bell drinker. The chicks received a live Newcastle disease vaccine (Sunvac ND Clone, Sunzen Biotech, Shah Alam, Selangor, Malaysia) intraocularly on d 7 and 21. Feed and water were provided ad libitum, and lighting was continuous.

Experimental Treatment The experimental treatments consisted of a 4 × 2 factorial arrangement with 4 levels of diets and 2 levels of stocking densities. Each diet-stocking density subgroup had 4 replicate pens. The number of chicks in each pen varied according to stocking density. Birds were raised at stocking densities of 0.100 m2/bird (23 birds/pen; LD) or 0.067 m2/bird (35 birds/pen; HD) from d 1 onward. Chicks from each stocking density were equally assigned to 1 of 4 feeding regimens: (i) basal diet (control), (ii) basal diet + 0.5% AminoGut from d 1 to 21 (AG3), (iii) basal diet + 0.5% AminoGut from d 1 to 42 (AG6), and (iv) basal diet + virginiamycin (0.02%) for d 1 to 42 (VM). Composition of experimental diets are shown in Table 1. The starter and finisher diets in mash form were fed from d 1 to 21 and d 22 to 42, respectively.

Growth Performance For each pen, BW, and feed intake were recorded on d 1, 21, and 42. Feed conversion ratios (feed/gain) (FCR) were calculated, and mortality was recorded daily.

Blood Parameters On d 42, 3 birds per pen were randomly selected and removed with minimum disturbance to flock mates. Immediately following capture, birds were killed, blood samples were collected and serum separated and stored at −20°C awaiting analysis for CORT, ceruloplasmin (CER), ovotransferin (OVT), α-1 acid glycoprotein (AGP), and leukocyte count. Blood smears were prepared using Wright’s stain; heterophil and lymphocyte were counted to a total of 60 cells (Gross and Siegel, 1983).

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corticosterone (CORT; Thaxton et al., 2006), heterophil/lymphocyte ratios (Cravener et al., 1992), and humoral immune response to sheep erythrocytes (Heckert et al., 2002). Thus, there is a need to assess other physiological stress indices to determine the effect of high stocking density on the well-being of broiler chickens. A group of blood proteins called acute phase proteins (APP) are involved in the restoration of homeostasis in animals subjected to external and internal challenges such as infection, inflammation, or stress (Murata, 2007). The APP are considered to be nonspecific innate immune components and shown by several studies as a possible indicator of stress in farm animals (Eckersall, 2000; Murata et al., 2004). For example, shipment of pigs by road increased the levels of major APP (haptoglobin, serum amyloid A, and C-reactive protein; Pineiro et al., 2007). Although there is substantial work on APP response to inflammation and infection in poultry (O’Reilly and Eckersall, 2014), no information is available on the effect of stressors such as stocking density on these proteins. Glutamine is a neutral free amino acid that is found in large quantity in muscle tissue and plasma. It has a role in the synthesis of amino acids, nucleotides, nucleic acids, amino sugars, and proteins. Glutamine can be synthesized from ammonia and glutamate (Glu) primarily in the skeletal muscle. Glutamine is the principal metabolic fuel for enterocytes, lymphocytes, macrophages, and fibroblast (Andrews and Griffiths, 2002). Although Gln is classified as a nonessential amino acid, its requirement may not be meet the optimum level for specific conditions such as stress, infection, or injury (Newsholme, 2001). Dietary supplementation of Gln has been shown to improve growth performance, development of the gastrointestinal tract, meat quality, and humoral immune response in poultry (Yi et al., 2001; Bartell and Batal, 2007; Dai et al., 2009). Dai et al. (2009) reported that Gln supplementation may reduce the detrimental effects of heat stress on growth performance, carcass characteristics, and meat quality in broiler chickens. On the contrary, Maiorka et al. (2000) and Sakamoto et al. (2006) failed to note any beneficial effects of supplementing Gln on the growth performance of broiler chickens. AminoGut is a commercial dietary supplement (Ajinomoto, Chuo-ku, Tokyo, Japan) containing a mixture of l-glutamine and l-glutamic acid. The benefits of AminoGut supplementation in improving growth, immune response, health, and survivability rate of piglets have been reported (Cabrera et al., 2013). Little information, however, is available on the effect of AminoGut supplementation in poultry. The present study was carried out to determine the effect of dietary AminoGut supplementation on growth performance, intestinal morphology and microflora, serum levels of APP, and other blood parameters in broiler chickens raised at 2 different stocking densities in the hot and humid tropical environment.

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GLUTAMINE AND ACUTE PHASE PROTEIN Table 1. Ingredient and nutrient composition of diets Starter (1 to 21 d) Item, % unless noted

AminoGut % 0.5

Virginiamycin % 0.02

Basal

AminoGut % 0.5

49.00 40.00 6.15 1.95 1.21 0.44 0.30 0.30 0.15 0.00 0.00 0.50 100

49.00 40.00 6.15 1.95 1.21 0.44 0.03 0.30 0.15 0.50 0.00 0.00 100

49.00 40.00 6.15 1.95 1.21 0.44 0.30 0.30 0.15 0.00 0.02 0.48 100

54.54 34.44 6.65 1.82 1.05 0.30 0.30 0.30 0.10 0.00 0.00 0.50 100

54.54 34.44 6.65 1.82 1.05 0.30 0.30 0.30 0.10 0.50 0.00 0.00 100

3,050.00 22.00 1.00 0.45

3,050.00 22.00 1.00 0.45

3,050.00 22.00 1.00 0.45

3,150.00 20.00 0.90 0.42

3,150.00 20.00 0.90 0.42

Virginiamycin % 0.02 54.54 34.44 6.65 1.82 1.05 0.30 0.30 0.30 0.10 0.00 0.02 0.48 100   3,150.00 20.00 0.90 0.42

1Supplied per kilogram of diet: vitamin A: 1,500 IU; cholecalciferol: 200 IU; vitamin E: 10 IU; riboflavin: 3.5 mg; pantothenic acid: 10 mg; niacin: 30 mg; cobalamin: 10 µg; choline chloride: 1,000 mg; biotin: 0.15 mg; folic acid: 0.5 mg; thiamine: 1.5 mg; pyridoxine: 3.0 mg; iron: 80 mg; zinc: 40 mg; manganese, 60 mg; iodine: 0.18 mg; copper: 8 mg; selenium: 0.15 mg; BHT: 100 mg.

The CORT was measured by a commercial ELISA kit (IDS, Boldon, UK). The intra- and interassay variability for this kit were less than 6.7% and less than 7.8%, respectively; and the detection limit was 27 ng/mL. The AGP concentration was determined by radial immunodiffusion using a commercial ELISA kit (Life Diagnostics Institute, West Chester, UK). Radial immunodiffusion method, modified from Mancini et al. (1965), was used to measure OVT. Briefly, 1% agarose gel (Sigma A9539) was prepared (0.13 g of agarose in 13 mL of TBS in a water bath at 56°C) and 260 µL of rabbit anti-chicken transferring antibody (RabMAbs Abcam, Cambridge, MA) was added to the mixture and poured onto a gel membrane (Flow-Mesh, Sigma Aldrich, St. Louis, MO) under room temperature. Nine wells were punched in each gel and 10 µL of standard or serum samples were loaded in each well. The OVT standards were prepared at 0, 0.3125, 1.250, and 5 mg/mL. Gels were incubated in a dark and humid environment for 48 h. Following incubation, size of the ring around each well was measured and calculated against standards. The concentration of CER was determined by the rate of formation of a colored product from CER and the substrate, 1,4-phenylenediamine dihydrochloride according to Sunderman and Nomoto (1970). Briefly, 20.375 g of sodium acetate trihydrate was dissolved in 250 mL of distilled water and adjusted to pH 6 using glacial acetic acid. Then 0.615 g of 1,4-phenylenediamine dihydrochloride (Sigma P1519) was added to the prepared buffer and kept in the dark for a minimum of 45 min. One hundred microliters of the above buffer and 100 µL of samples or standards were added to each microplate wells, shaken gently, and kept in the dark for 20 min. The absorbance was recorded spectro-

photometrically using a microplate reader at 450 nm. Standards were prepared with serial dilution of pig serum and saline buffer combination to achieve various concentration of 12.75 (20 µL of pig serum + 60 µL of saline buffer), 6.375, 3.1875, 1.59375, 0.79608, 0.39804, 0.199, and 0.099 µg/mL of CER.

Intestinal Microbial Populations and Morphology Immediately following blood sampling, approximately 1 g of the cecal content was collected and diluted with 9 mL of prereduced sterile 0.9% saline solution and homogenized by stomacher homogenizer in CO2 flushed plastic bags for approximately 2 min. Serial dilutions of each sample prepared using prereduced saline, and lactobacilli were quantified by spread plating on MRS agar (Merck, Darmstadt, Germany) incubated in anaerobic conditions at 37°C for 2 d. The total number of Escherichia coli were quantified by spread plating on MacConkey agar (Merck) incubated in aerobic conditions at 37°C for 1 d. The microbial counts were determined as cfu per gram of samples. The midpoint of the duodenum were excised and fixed in 10% neutral-buffered formalin solution and embedded in paraffin wax (Baddeley et al., 1986). Slides were prepared using 5-µm sections, stained by hematoxylin and eosin, and the villi length and crypt depth were measured using a light microscope (Bancroft and Gamble, 2002). The distance from the tip of the villus to the villus crypt junction represents the villus height, and crypt depth was defined as the depth of the invagination between adjacent villi. A total of 10 villi and 10 crypts per sample were measured.

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Corn Soybean meal Palm oil DCP Limestone Sodium chloride Vitamin premix1 Mineral premix dl-Methionine AminoGut Virginiamycin Sand Total Calculated analysis   ME (kcal/kg)  CP  Ca   P available

Basal

Grower (22 to 42 d)

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0.0002 0.0003 0.15 0.0001 0.0001 0.12

Statistical Analysis

0.31 0.15 0.43

Data were subjected to ANOVA using the GLM procedure of SAS (2003, SAS Institute Inc., Cary, NC). All data were analyzed using diet, stocking density, and their interactions as main effects. When interactions between main effects were significant, comparisons were made within each experimental variable. When significant effects were found, comparisons among multiple means were modeled by Duncan’s multiple range test. Mortality data were analyzed by chi-squared test.

0.0001 0.002 0.12

RESULTS

0.0001 0.0001 0.09

Performance

1AG3:

a–cMeans

within a column-subgroup with no common superscripts are different at P < 0.05. AminoGut d 1 to 21; AG6: AminoGut d 1 to 42; VM: virginiamycin. 2LD: low stocking density; HD: high stocking density.

0.15 0.17 0.54 0.0001 0.01 0.25 0.0001 0.01 0.55

0.05b 0.04a 0.01 0.01 37a 25b 35a 22b 11 9 22a 15b 21a 11b 6 7

0.67 0.16 0.85

0.02b 0.02a

0.02a 0.03a 0.03a 0.02b

  ± ± ± ±   1.86 ± 1.96 ± 1.91 1.96 1.95 1.80

0.04a 0.06a 0.09a 0.07b

  ± ± ± ±   2.12 ± 2.41 ± 2.33 2.35 2.36 2.03 0.01 0.02 0.01 0.01

  ± ± ± ±   1.49 ± 1.47 ± 1.47 1.49 1.46 1.46

68a 77a 47a 42b

  ± ± ± ±   3,649 ± 3,394 ± 3,619 3,547 3,512 3,408

64a 62b 62b 32c

  ± ± ± ±   2,438 ± 2,205 ± 2,423 2,332 2,339 2,193 15 19 3 16

  1,196 ± 1,215 ± 1,172 ± 1,214 ±   1,210 ± 1,188 ± 56a 43b 58b 39a

  ± ± ± ±   1,964 ± 1,731 ± 1,890 1,809 1,803 1,888

46ab 43b 56b 49a

  ± ± ± ±   1,152 ± 915 ± 1,045 996 1,002 1,090 8 7 4 13

  ± ± ± ±   812 ± 816 ± 811 812 801 832

 Basal  AG3  AG6  VM Stocking density2  LD  HD ANOVA (P-value)  Diet  Density   Diet × density

3–6 wk 1–3 wk 1–6 wk 3–6 wk Item

1–3 wk

3–6 wk

1–6 wk

1–3 wk

Feed intake (g) Weight gain (g)

Table 2. Mean (±SEM) weight gains (g), feed intake (g), and feed conversion ratios (FCR) by diet and stocking density

On d 42, 15 birds per pen were randomly selected and the incidence and severity of foot pad dermatitis (FPD) was scored by a visual ranking system (Nagaraj et al., 2007).

There were no diet × density interactions for weight gain, feed intake, and FCR during the starter period (1–3 wk; weight gain, P = 0.85; feed intake, P = 0.54; FCR, P = 0.43), finisher period (3–6 wk; weight gain, P = 0.55; feed intake, P = 0.09; FCR, P = 0.12), and over the whole trial period (1–6 wk; weight gain, P = 0.25; feed intake, P = 0.12; FCR, P = 0.15; Table 2). Diet and density affected weight gain (diet effect from 3 to 6 wk, P = 0.0001; diet effect from 1 to 6 wk, P = 0.0002; density effect from 3 to 6 wk, P = 0.01; density effect from 1 to 6 wk, P = 0.01), feed intake (diet effect from 3 to 6 wk, P = 0.0001; diet effect from 1 to 6 wk, P = 0.0001; density effect from 3 to 6 wk, P = 0.0001; density effect from 1–6 wk, P = 0.002), and FCR (diet effect from 3 to 6 wk, P = 0.0001; diet effect from 1 to 6 wk, P = 0.0002; density effect from 3 to 6 wk, P = 0.0001; density effect from 1 to 6 wk, P = 0.0003), of birds during the finisher and whole trial periods. The weight gains of AG3 and AG6 birds were similar to control during the finisher period but lower than control over the whole trial period. The VM birds showed greater weight gains than their AG3 and AG6 counterparts during both finisher (P = 0.0001) and whole trial periods (P = 0.0001). The total feed intake (1–6 wk) of control, AG3, and AG6 chickens was similar but lower (P = 0.0001) than the VM group. The VM birds showed the best FCR compared with other groups during both finisher (P = 0.0001) and whole experimental (P = 0.0002) periods. The FCR of control, AG3, and AG6 groups were similar throughout the experimental period. The mortality rate of AG3 (3.88%) birds was lower (P < 0.05) than those of control (9.90%) group. The mortality rates of AG6 (5.60%) and VM (4.74%) groups were similar (P > 0.05) with the other groups. No effect of stocking density was observed during the starter period for weight gain (P = 0.16), feed intake (P

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Diet1

FCR

1–6 wk

Foot Pad Dermatitis

GLUTAMINE AND ACUTE PHASE PROTEIN Table 3. Mean (±SEM) villi lengths (µm) and crypt depths (µm) by diet and stocking density Item Diet1  Basal  AG3  AG6  VM Stocking density2  LD  HD ANOVA (P-value)  Diet  Density   Diet × density

Villi length (µm)   ± ± ± ±   1,089 ± 1,008 ± 1,027 1,033 1,065 1,068

33 45 31 41 23a 28b

0.78 0.03 0.33

Crypt depth (µm)   ± ± ± ±   134 ± 144 ±

123 148 139 147

7 14 7 7 6 7

0.20 0.29 0.23

= 0.17), and FCR (P = 0.15). The HD resulted lower weight gain (3–6 wk, P = 0.01; 1–6 wk, P = 0.01) and feed intake (3–6 wk, P = 0.0001; 1–6 wk, P = 0.002), and poorer FCR (3–6 wk, P = 0.0001; 1–6 wk, P = 0.0003) when compared with the LD group during both finisher and whole experimental periods. Stocking density (HD = 6.78%; LD = 5.54%) had a negligible effect on mortality rate throughout the period of study.

Morphometric Variables There was no diet × density interaction for villi length (P = 0.33) and crypt depth (P = 0.23) on d 42 (Table 3). Villi length (P = 0.78) and crypt depth (P = 0.20) were not changed by different dietary treatments. However, birds in HD group had smaller villi (P = 0.03) compared with those of the LD group. Crypt depth was not affected by stocking density (P = 0.29).

Blood Parameters The levels of serum APP, plasma CORT, and heterophil to lymphocyte ratio (HLR) are presented in Table 4. No interactions for CER (P = 0.07), AGP (P = 0.14), OVT (P = 0.17), CORT (P = 0.29), and HLR (P = 0.79) was observed. Except for OVT, there was no effect of diet on levels of CER (P = 0.23), AGP (P = 0.47), CORT (P = 0.62), and HLR (P = 0.65). The birds of AG3 group had higher level of OVT compared with the control group (P = 0.023). Regardless of diet, stocking density consistently increased the serum concentrations of CER (P = 0.006), AGP (P = 0.0001), OVT (P = 0.002), and CORT (P = 0.0007), and increased HLR (P = 0.0005).

Enumeration of Bacteria The results showed no interaction between diet and density for intestinal population of lactobacilli (P =

0.74) and E. coli (P = 0.30; Table 5). Among the dietary treatment groups, VM treatment reduced (P = 0.0001) the E. coli population compared with other groups, but no effects (P = 0.31) were observed regarding lactobacilli. Stocking density had no effect on cecal populations of lactobacilli (P = 0.23) and E. coli (P = 0.11).

FPD There was no (P = 0.35) diet × density interaction for incidence of FPD on d 42 (Table 5). Only birds of AG3 group had lower (P = 0.01) incidence of FPD compared with other groups. Birds in the HD group were more susceptible (P = 0.0001) to FPD than their LD counterparts.

DISCUSSION Literature regarding the effect of Gln supplementation on growth performance of broiler chickens is inconsistent. Bartell and Batal (2007), Avellaneda et al. (2008), and Dai et al. (2009) reported a significant improvement in weight gain and feed efficiency in broiler chickens supplemented with Gln. Yi et al. (2001) found greater weight gain in turkey poults fed diets with Gln during the first week of life only. Conversely, Maiorka et al. (2000), Sakamoto et al. (2006), and Soltan (2009) found that Gln did not increase weight gain in poultry. The findings of this study indicated that, irrespective of duration of supplementation and stocking density, AminoGut supplementation at 0.5% did not enhance growth performance of broilers. There is no clear explanation for our failure to note the beneficial effect of Gln supplementation on broiler chickens. The dosage of Gln dietary supplementation used in the present study may have accounted for the discrepancies. However, Maiorka et al. (2000) and Sakamoto et al. (2006) reported that dietary Gln supplementation at 1% also had negligible effect on growth performance of broiler chickens. Avellaneda et al. (2008) supplemented birds with Gln at 0.5, 1.0, and 1.5% and concluded that the latter 2 dosages improved growth performance but not at 0.5%. On a cautionary note, however, the authors supplemented Gln for 1 or 3 wk and birds were raised only till 25 d of age. The beneficial effect of Gln supplementation on growth performance of chickens has been associated with better development of the intestinal mucosa (Yi et al., 2005b; Bartell and Batal, 2007). Glutamine is responsible for maintenance of the mucosal structure (Khan et al., 1999) and reconstitution after damage (Newsholme, 2001). Bartell and Batal (2007) reported that birds fed diets with Gln had significantly longer intestinal villi than those fed a control diet. Increase in intestinal villi height early in the chicks’ life may allow a more efficient utilization of nutrients and consequently improved growth performance.

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a,bMeans ± SEM within a column-subgroup with no common superscripts are different at P < 0.05. 1AG3: AminoGut d 1 to 21; AG6: AminoGut d 1 to 42; VM: virginiamycin. 2LD: low stocking density; HD: high stocking density.

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Table 4. Mean (±SEM) serum ceruloplasmin (CER), α-1 acid glycoprotein (AGP), ovotransferin (OVT), and corticosterone (CORT) concentrations, and heterophil to lymphocyte ratio (HLR) by diet and stocking density CER (mg/mL)

Item Diet1

  ± ± ± ±   7.52 ± 11.34 ±

 Basal  AG3  AG6  VM Density2  LD  HD ANOVA (P-value)  Diet  Density   Diet × density

11.11 7.41 9.86 9.75

1.72 0.71 1.61 1.31 0.64b 1.13a

AGP (mg/mL)   ± ± ± ±   0.71 ± 1.08 ±

0.87 0.95 1.03 0.79

0.23 0.006 0.07

0.08 0.07 0.13 0.09 0.05b 0.06a

0.47 0.0001 0.14

OVT (mg/mL)   ± 0.15b ± 0.33a ± 0.32ab ± 0.34ab   1.4 ± 0.16a 2.5 ± 0.25b

1.44 2.51 2.17 1.63

0.023 0.002 0.17

CORT (ng/mL)   ± ± ± ±   1.15 ± 1.77 ±

1.29 1.58 1.51 1.46

0.14 0.24 0.21 0.16 0.06b 0.14a

HLR   ± ± ± ±   0.52 ± 0.76 ± 0.67 0.67 0.64 0.57

0.62 0.0007 0.29

0.06 0.05 0.07 0.06 0.03b 0.02a

0.65 0.0005 0.79

a,bMeans

The preceding discussion suggested that dietary Gln and Glu supplementations have no beneficial effects on growth performance and intestinal morphology in broilers. There is no clear explanation for the phenomenon; however, previous studies in pigs (Yi et al., 2005b) and poultry (Yi et al., 2005a) suggested that the beneficial effects of Gln were more obvious during early age and under challenged conditions. In the present study, although chickens were under a challenged environment because of high stocking density, the adverse effects of such practice were only apparent when the birds reached the finisher period. AminoGut supplementation from d 1 to 21 but not from d 1 to 42 can reduce mortality rate compared with the control group. Although we did not measure any immunological parameters in the present study, DeMarco et al. (2003) indicated that Gln supplementation may improve the health of intestinal epithelial cell and thus reduce susceptibility to infections. Work in pigs suggested that Gln may alter immune cells in the mesenteric lymph nodes by supporting a Th1-type cytokine

response after T-cell stimulation (Johnson et al., 2006). Taken together, it appears that Gln supplementation may enhance immunity in both animals and human beings (see review by Calder and Yaqoob, 1999). Previous work has consistently demonstrated the adverse effect of high stocking density on growth performance of broiler chickens (Bessei, 2006). Work conducted in temperate climates suggest that the most severe effects of high density (reduction of BW and walking ability) occur when space available per bird drops below 0.07 or 0.0625 m2/bird and between 34 to 40 kg/ m2 (Estevez, 2007). The present study clearly showed that, under the hot and humid tropical climate, HD was detrimental to weight gain, FCR, and incidence of FPD when compared with the LD group. Houshmand et al. (2012) raised broilers at 0.0625 m2/bird and 0.1 m2/bird in battery cages under a similar environment as the present study. The authors indicated that the higher stocking density retarded overall feed efficiency but not BW at 42 d of age. The discrepancies could be attributed to the difference in housing system. Chickens

Table 5. Mean (±SEM) intestinal Escherichia coli and lactobacilli counts, and foot pad dermatitis (FPD) scores by diet and stocking density Item Diet1

 Basal  AG3  AG6  VM Density2  LD  HD ANOVA (P-value)  Diet  Density   Diet × density a,bMeans

E. coli (log cfu/g of digesta)   ± ± ± ±   9.73 ± 10.37 ± 11.36 10.82 11.48 6.48

0.11a 0.37a 0.28a 0.82b 0.73 0.47

0.0001 0.11 0.30

Lactobacilli (log cfu/g of digesta)   ± ± ± ±   7.44 ± 8.05 ±

8.24 6.86 8.02 7.78

0.54 0.36 0.58 0.57 0.36 0.38

0.31 0.23 0.74

FPD   ± 0.12a ± 0.11b ± 0.11ab ± 0.11ab   1.15 ± 0.06b 2.09 ± 0.06a

1.81 1.42 1.67 1.59

0.01 0.0001 0.35

± SEM within a column-subgroup with no common superscripts are different at P < 0.05. AminoGut d 1 to 21; AG6: AminoGut d 1 to 42; VM: virginiamycin. 2LD: low stocking density; HD: high stocking density. 1AG3:

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± SEM within a column-subgroup with no common superscripts are different at P < 0.05. AminoGut d 1 to 21; AG6: AminoGut d 1 to 42; VM: virginiamycin. 2LD: low stocking density; HD: high stocking density. 1AG3:

GLUTAMINE AND ACUTE PHASE PROTEIN

density elevated serum levels of OVT, AGP, and CER. To the best of our knowledge, this is the first documented work to show the effect of stocking density on APP in poultry. Murata (2007) suggested that the activation of APP synthesis from the liver was modulated by the hypothalamic-pituitary-adrenal axis. It can be concluded that other than inflammation, injury, and infection, stress attributed to stocking density may also alter serum levels of APP in chickens. However, because a higher incidence of FPD observed in this study, further studies would be needed to establish the absolute response of the APP to psychological stress in the complete absence of inflammatory processes. The present finding together with Meddings and Swain (2000), Mitchell and Carlisle (1992), and Burkholder et al. (2008) showed that environmental stress may profoundly affect intestinal morphology. Our data indicated that chickens subjected to 0.067 m2/bird had shorter intestinal villi length than those provided 0.100 m2/bird. The adverse effect of higher stocking density on intestinal morphology may have accounted for the poor growth performance of chickens raised in crowded environment. Although work by Burkholder et al. (2008) showed that feed restriction and high temperature may alter intestinal microbiota, the cecal population of E. coli and lactobacilli in the present study was not affected by stocking density. In conclusion, under the conditions of this study, dietary supplementation of AminoGut, irrespective of stocking density, had no beneficial effect on growth performance, intestinal morphology, and physiological adaptive responses of broiler chickens raised under hot and humid tropical conditions. However, AminoGut supplementation from d 1 to 21 was beneficial in reducing mortality rate. The results of this experiment clearly showed that high stocking density was detrimental to growth performance and intestinal morphology. The increased serum concentrations of a wide range of APP together with an elevated HLR indicate that high stocking density induced an acute phase response either indirectly as a result of increased incidence of inflammatory diseases such as FPD or possibly as a direct physiological response to the stress of high density stocking. The findings of this study, undertaken in a hot and humid tropical climate, reveal that high stocking density reduced overall performance of broilers, but did not increase mortality. The increased incidence of FPD, however, suggests that as well as overall performance, welfare at the high stocking density was also compromised. Furthermore, the high serum concentrations of OVT, AGP, and CER among HD birds indicate that APP are thus physiological indicators of health and welfare in broiler production. Further investigation within the context of other welfare concerns would fully determine and validate whether APP are biomarkers of not only disease and inflammation, but also overall health and wellbeing of broilers across production systems.

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in the present study were raised on deep litter whereas those of Houshmand et al. (2012) were in battery cages. It has been documented that the effect of stocking density on performance and welfare of broiler chickens acts through its influence on litter quality. High moisture content of the litter enhances microbial activity, which in turn leads to an increase in temperature and ammonia in broiler houses, and thus, growth performance and well-being will be affected (Miles et al., 2004). The higher incidence of FPD among the HD birds compared with their LD counterparts reflected the poor quality of the litter for HD birds. Higher incidence of FPD has previously reported to be associated with litter moisture content (Dozier et al., 2005; Estevez, 2007). It is well documented that high stocking density is a major welfare issue in intensive broiler production (Bessei, 2006; Estevez, 2007). However, findings on stocking density and physiological stress are inconsistent. Stocking density had no effect on HLR (Cravener et al., 1992; Heckert et al., 2002; Dozier et al., 2006; Houshmand et al., 2012) or plasma level of CORT (Thaxton et al., 2006; Dozier et al., 2006: Houshmand et al., 2012). On the contrary, based on heat shock protein 70 expression (Beloor et al., 2010), telomere length (a biomarker for oxidative stress; Beloor et al., 2010), and bursa weights (Heckert et al., 2002), higher stocking densities may elicit physiological stress response in chickens. The noted higher CORT and HLR in birds subjected to high stocking density suggested elicitation of physiological stress response. Pettit-Riley and Estevez (2001) reported that high stocking density may exacerbate mortality related to heat stress. Although the physiological indices measured in the present study clearly showed that the HD birds were stressed, stocking density had no significant effect on mortality rate. The inconsistent effect of stocking density on growth performance, mortality, and welfare status could be attributed to variations in housing conditions and management practices. Based on studies in commercial chicken farms in the United Kingdom, Dawkins et al. (2004) concluded that environmental conditions and stocksmanship were more important than stocking density per se in determining the welfare of broiler chickens. Work in cattle (Murata and Miyamoto, 1993; Arthington et al., 2003), dromedary camels (Baghshani et al., 2010), and pigs (Saco et al., 2003; Pineiro et al., 2007) suggested that transportation stress may induce APP response. Studies in chickens showed that inflammatory response to various type of infections may trigger and change APP response and concentration (Holt and Gast, 2002; Xie et al., 2002; Buyse et al., 2007; Rath et al. 2009; Georgieva et al., 2010; O’Reilly and Eckersall, 2014). Elevation of APP also has been reported in noninflammatory stress such as molting and different housing systems in hens (Holt and Gast, 2002; Salamano et al., 2010); however, to date, there is no report on the effects of psychophysical stress on APP in chickens. Results obtained in our study showed that higher stocking

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ACKNOWLEDGMENTS This research was funded by the Malaysian Ministry of Education, Putrajaya (Long Term Research Grant Scheme), and the Malaysian Ministry of Science, Technology and Innovation, Putrajaya (Science Fund).

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