Effects of dietary gallic acid supplementation on performance, antioxidant status, and jejunum intestinal morphology in broiler chicks

Effects of dietary gallic acid supplementation on performance, antioxidant status, and jejunum intestinal morphology in broiler chicks K. G. Samuel,∗ J. Wang,∗ H. Y. Yue,∗ S. G. Wu,∗ H. J. Zhang,∗,1 Z. Y. Duan,† and G. H. Qi∗,1 ∗

Key Laboratory of Feed Biotechnology of Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; and † Kemin Industries (Zhuhai) Co., Ltd., Zhuhai, Guangdong 519040, China hibit higher breast muscle ratio at 42 d (P = 0.043). Interestingly, dietary GA inclusion level from 50 to 100 mg/kg reduced the crypt depth (P = 0.009) and increased the villus height:crypt depth ratio (VCR) of the birds (P = 0.006). Dietary supplementation of GA at 100 mg/kg decreased plasma malondialdehyde (MDA) content at 42 d of age (P = 0.030). Moreover, dietary addition of GA linearly increased plasma total antioxidant capacity (P = 0.039) and plasma total superoxide dismutase activities (P = 0.049) at 21 d of age. However, analysis of plasma biochemical markers revealed that dietary supplementation of GA did not exhibit beneficial health effects. Overall, we conclude that 75 to 100 mg/kg of GA are suitable for enhanced growth performance and health benefits in a broiler diet.

Key words: gallic acid, broiler chick, growth performance, intestinal morphology, antioxidant status 2017 Poultry Science 96:2768–2775 http://dx.doi.org/10.3382/ps/pex091

INTRODUCTION Antibiotics have been used for a long time to advance the profitability of the poultry industry by improving weight gain, feed conversion ratio, and flock uniformity (Wenger et al., 1998). However, the negative effect of feed antibiotics has become increasingly prominent with the feed industry’s development. Because of the consumer concerns over safety and the ban imposed by the European Union and other countries on antibiotic growth promoters used in feed, recent investigations have targeted the naturally occurring plant extracts as an alternative. Phenolic compounds, generally regarded as major bioactive phytochemicals, are one of the best options given that they are widely distributed in plants, exhibit various antioxidant properties (Salah et al., 1995; Bravo, 1998; Brown et al., 1998), and have health benefits (Nour et al., 2012). Although plant ex C 2017 Poultry Science Association Inc. Received December 14, 2016. Accepted March 17, 2017. 1 Corresponding authors: [email protected] (H. J. Zhang); [email protected] (G. H. Qi)

tracts as a dietary additive are showing promising results in a new “post-antibiotic” era, for many of them the optimal dosages and mode of action are still not well known. The naturally occurring phenolic compound identified as 3,4,5-trihydroxybenzoic acid, also known as gallic acid (GA), is found in several fruits, vegetables, and derived products, where it is present either in free form or, more commonly, as an ingredient of hydrolysable tannins (Niemetz and Gross, 2005; Jung et al., 2010; Lee et al., 2012). GA is a yellowish white crystal (molecular mass 170.12 g/mol) with a melting point at 250◦ C and water solubility 1.1% at 20◦ C (Polewski et al., 2002). According to numerous studies, the pharmacological and health benefits of GA include its being antioxidant (Golumbic and Mattill, 1942; Kim et al., 2002), anti-inflammatory (Kroes et al., 1992), ¨ celik et al., 2011), antiantibacterial and antiviral (Oz¸ allergic (Jung et al., 2010), anti-mutagenic (Gichner, 1987; Hour et al., 1999), and anti-carcinogenic (Mirvish et al., 1975; Inoue et al., 1995), and it is also hepato protective against carbon tetrachloride toxicity (Kanai and Okano, 1998). Despite the benefits, studies in human and animal models suggested that the usage of GA is

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ABSTRACT Gallic acid (GA), widely distributed in plants and feeds, is known to have a diverse range of activities such as anti-oxidant, anti-inflammatory, antibacterial, anti-allergic, anti-mutagenic, and anticarcinogenic. The purpose of this study was to investigate the efficacy of inclusion of dietary GA at levels 0, 25, 50, 75, 100, or 150 mg/kg on growth performance, antioxidant status, and jejunum intestinal morphology of broiler chicks. In total, 630 one-day-old Arbor Acres (AA) male broiler chicks were randomly allotted to 6 treatment groups for a period of 6 weeks. The results indicate that dietary addition of GA at 75 to 100 mg/kg improved feed conversion efficiency in both the grower (d 21 to 42, P = 0.045) and overall (d 1 to 42, P = 0.026) periods. Dietary addition of GA at a concentration ≥100 mg/kg was able to ex-

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MATERIALS AND METHODS Dietary Treatments and Animal Management A total of 630 one-day-old male broiler chicks Arbor Acres strain was supplied by Beijing Huadu Broiler Company (Beijing, China). Chicks were weighed and randomly assigned to one of the 6 treatment groups that were fed typical corn-soybean meal basal diets supplemented with 0, 25, 50, 75, 100, or 150 mg/kg of GA. Each treatment contained 7 cages of 15 chicks each. Chicks were provided with water and feed ad libitum. The basal diets were formulated to meet nutrient requirements of broiler chicks (National Research Council, 1994), for the starter period from 1 to 21 d and grower period from 21 to 42 d of age. The composition and nutrient levels of the basal diets are shown in Table 1. Samples of GA (purity ≥ 90%) were supplied by Kemin Industries (Zhuhai) Co., Ltd (Zhuhai, Guangdong, China). Chicks were raised in wire floor cages (cage size 110 x 100 x 55 cm3 ) in a 4-level battery in an environmentally controlled room with continuous light using incandescent light bulbs. Room temperature was maintained at 33◦ C for the first wk and then gradually decreased by 3◦ C each consecutive wk until it reached 24◦ C. All management of birds was in accordance with the guidelines of raising Arbor Acres broilers. All the experimental procedures have been approved by the Animal Care and Use Committee of the Feed Research Institute of the Chinese Academy of Agricultural Sciences.

Growth Performance Measurements Growth performance was evaluated by calculating the average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (FCR) at d 21 and d 42. Body weight and feed intake were recorded at the beginning of the trial, at d 21 and d 42, on a per replicate basis. FCR was calculated as the ratio of ADFI to ADG. Mortality was recorded as it occurred.

Table 1. Dietary composition and nutrient level of the basal diets. Age (days) Items

Starter diet (1 to 21)

Grower diet (21 to 42)

56.68 31.86 2.5 2 2.78 1.81 1.28 0.35 0.21 0.12 0.02 0.2 0.1 100

60.37 27.32 2.5 2.5 3.57 1.51 1.22 0.35 0.12 0.13 0.02 0.2 0.1 100

2950 20.50 (20.81) 1.00 (0.94) 0.71 0.45 (0.45) 1.10 0.49 0.81

3050 19.00 (18.92) 0.90 (0.85) 0.64 0.40 (0.36) 1.0 0.39 0.69

Ingredients (%) Corn Soybean meal Rapeseed meal Cottonseed meal Soybean oil Dicalcium phosphate Limestone (CaCO3 ) Salt DL-methionine (99%) L-lysine HCL (99%) Vitamin premixa Mineral premixa Choline chloride (50%) Total (kg) Nutrient compositionb AME (Kcal/kg) CP, % Ca, % Total P, % Available P, % Lys, % Met, % Met + Cys, %

a Premix supplied per kg of diet: vitamin A 12500 IU, vitamin D3 2500IU, vitamin E 18.75 IU, vitamin K3 2.65 mg, vitamin B1 2 mg, vitamin B2 6 mg, vitamin B12 0.025 mg, biotin 0.0325 mg, folic acid 1.25 mg, Ca-pantothenate 12 mg, niacin 50 mg, Cu 8 mg, Zn 75 mg, Fe 80 mg, Mn 100 mg, Se 0.15 mg, I 0.35 mg. b Nutrient levels listed are calculated values. Numbers in parenthesis are analyzed values.

The ADG, ADFI, and FCR were corrected for dead birds.

Blood Sampling On d 21 and 42, birds were fasted overnight, and one bird per replicate, close to the average body weight, was selected for sampling and data collection, and was slaughtered by cutting the jugular vein. Blood samples were collected into heparinized test tubes, quickly centrifuged at 1,800 × g for 10 min at 4◦ C to separate plasma, and stored at −20◦ C until analysis.

Carcass Characteristics Measurements After blood sampling, the birds were exsanguinated, defeathered, and then the carcasses were eviscerated manually by cutting around the vent. The eviscerated weight was measured after the heads, paws, giblets, and abdominal fat were removed. Pairs of breast muscles (including pectoralis major and minor), leg muscles (including thigh and drumstick), and the abdominal fat pad (fat trimmed from proventriculus up to cloaca) were removed and weighed. The relative eviscerated yield was calculated as a proportion of pre-slaughter weight multiplied by 100, whereas the breast muscle, leg muscle, and the abdominal fat pad were calculated as the proportion of the BW.

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restricted due to its poor absorption, low bioavailability, and rapid elimination from the body (Shahrzad et al., 2001; Ferruzzi et al., 2009). Given that different phenolic compounds have many useful features, their effects have been extensively investigated and thus used in medical applications and intestinal health modulation. However, there is limited published information concerning the effect of GA on growth performance and intestinal morphology of broiler chicks in the field of alternative poultry growth promoters. Thus, the present study was designed to investigate the efficacy of dietary supplementation of GA on production performance, antioxidant status, some biochemical responses, and jejunum intestinal morphology of broiler chicks.

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Plasma Antioxidant Indices Measurement

Plasma Biochemical Indices Measurement Plasma samples were analyzed in duplicate for total protein (TP), albumin (ALB), urea nitrogen (UN), uric acid (UA), and Creatinine (CRE) using an automatic biochemical analyzer (Model 7020, Hitachi, Tokyo, Japan).

Jejunum Intestinal Morphology On d 42, one bird from each replicate (n = 7/diet) was killed after overnight fasting to limit intestinal throughput. The whole length of the small intestine was removed and a length of approximately 5 cm of the jejunum was cut from the midpoint between the point of bile duct entry and Meckel’s diverticulum. The samples were gently flushed several times with physiological saline (1% NaCl) to remove intestinal contents and placed in 10% formalin in 0.1 M phosphate buffer (pH = 7.0) for fixation. The samples were then processed for 24 h in a tissue processor with anhydrous ethanol as the dehydrant, and samples were embedded in paraffin. Three slices of 4 μm were made from the tissue and were stained with hematozylin-eosin. Histological sections were examined with a positive optical microscope (NIKON DS-U3, Tokyo, Japan) coupled with a digital camera (NIKON ECLIPE CI, Tokyo, Japan). The variables measured were villus height, crypt depth, and villus height:crypt depth ratio (VCR). Villus height (μm) was measured from the tip of the villus to the villus crypt junction, and crypt depth was defined as the depth of the invagination between adjacent villi. For the purpose of statistical analysis, the average of these values was used.

Statistical Analysis All analyses were performed using the SPSS version 16.0 for windows (Statistical Packages for the Social Sci-

RESULTS Growth Performance and Carcass Characteristics Dietary GA supplementation did not affect ADG, ADFI, or FCR during the starter period (d 1 to 21, P > 0.05, Table 2). However, dietary supplementation at 75 and 100 mg/kg improved feed conversion ratio compared with the control group in both the grower (d 21 to 42, P = 0.045) and overall (d 1 to 42, P = 0.026) periods. With increasing dietary GA levels, a linear effect in FCR also was observed in both the grower (P = 0.017) and overall (P = 0.007) periods. Table 3 shows the effect of dietary GA supplemental levels on slaughter characteristics of broiler chicks. Inclusion of graded concentration of dietary GA at 100 mg/kg or 150 mg/kg increased the relative breast muscle percentage compared with those fed the control diet (P = 0.043) at 42 d of age. In addition, a linear effect (P = 0.002) on relative breast muscle percentage was noted with increasing dietary GA concentration. Dietary addition of GA in the chick’s diets, however, did not change the relative percentage of eviscerated yield, leg muscle yield, and abdominal fat (P > 0.05).

Jejunum Intestinal Morphology The results for the effect of dietary GA on jejunum intestinal morphology of broiler chicks at 42 d of age are displayed in Table 4. The ratio of villus height to crypt depth was increased (P = 0.006) in chicks fed dietary GA at 50 to 100 mg/kg compared with those fed the control diet. Likewise, crypt depth was reduced (P = 0.009) in chicks fed dietary GA at levels 50 to 150 mg/kg compared with those fed the control diet. Both linear (P = 0.035) and quadratic (P = 0.018) effects were observed on crypt depth in response to dietary GA addition. However, villus height was not affected by addition of GA to the diet of broiler chicks (P > 0.05).

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Colorimetric kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) were used to measure total anti-oxidative capacity (T-AOC), enzymatic activity of total superoxide dismutase (T-SOD), and levels of malondialdehyde (MDA) in plasma samples. Each plasma sample was analyzed in duplicate. T-SOD activities in the plasma were assayed using the xanthine/xanthine oxidase system for superoxide anion generation as described by Sun et al. (1988). A unit of T-SOD activity was defined by the amount of the enzyme required to inhibit the rate of formazen dye formation by 50% under a defined condition. The extent of lipid oxidation was assayed by measuring the thiobarbituric acid reactive substances with a spectrophotometer at 535 nm (Wills, 1966). Thiobarbituric acid material was described as nM of MDA per mL plasma.

ences, Chicago, IL). The replicate (each replicate represented one cage) was the experimental unit for the analysis of growth performance data, and individual bird was the experimental unit for other parameters. The normality of data was initially tested. The dependent variables were analyzed using one-way ANOVA. When the differences among treatments were significant (P < 0.05), the means of treatments were separated by post hoc Duncan’s multiple range tests. The dose related effect of supplemental GA was computed by GLM, using contrast command for the linear and quadratic effects. Differences were considered statistically significant at P ≤ 0.05. Data are presented as mean and pooled SEM.

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GALLIC ACID IN BROILERS Table 2. Effect of dietary gallic acid supplementation on growth performance of broiler chicks. Gallic acid level (mg/kg feed) Items1

50

75

100

150

SEM

ANOVA

Linear2

Quadratic2

741.6 31.5 46.7 1.485

756.7 32.8 47.9 1.461

780.0 34.7 49.3 1.431

759.7 33.3 48.7 1.468

769.4 33.1 48.6 1.474

9.958 0.492 0.584 0.009

0.775 0.357 0.192 0.772

0.808 0.728 0.407 0.680

0.475 0.536 0.149 0.348

2,405.0 78.9 149.7 1.897a,b

2,394.8 76.9 147.0 1.914a,b

2,451.2 77.5 145.2 1.872b

2,436.9 77.3 143.1 1.851b

2,403.4 76.6 145.2 1.899a,b

14.255 0.557 1.146 0.014

0.839 0.297 0.603 0.045

0.514 0.621 0.137 0.017

0.479 0.130 0.850 0.055

53.8 93.5 1.763a,b

53.4 93.0 1.764a,b

54.8 93.5 1.719b

53.9 91.3 1.717b

53.5 92.2 1.749a,b

0.390 0.799 0.009

0.765 0.461 0.026

0.751 0.077 0.007

0.759 0.458 0.052

Data are the mean of 7 replicates with 15 birds each. 1 FCR: feed conversion ratio. 2 Orthogonal polynomial contrasts were used to determine the effect of dietary gallic acid levels. a,b Means with no common superscripts within a row differ significantly (P < 0.05).

Table 3. Effect of dietary gallic acid supplementation on carcass characteristics of broiler chicks at 42 d of age. Gallic acid level (mg/kg feed) Items Eviscerated yield (%)2 Breast muscle yield (%)2 Leg muscle yield (%)2 Abdominal Fat (%)2

P-value

0

25

50

75

100

150

SEM

ANOVA

Linear1

Quadratic1

71.40 25.97b 20.01 2.20

71.57 26.52a,b 20.40 2.01

70.81 26.49a,b 21.07 1.79

72.41 26.83a,b 20.42 1.56

72.50 27.96a 20.97 1.94

71.08 27.91a 19.84 1.84

0.243 0.230 0.235 0.079

0.213 0.043 0.593 0.346

0.564 0.002 0.969 0.158

0.388 0.728 0.133 0.117

Data are the mean of 7 replicates (one bird from each replicate). 2 Orthogonal polynomial contrasts were used to determine the effect of dietary gallic acid levels. 3 Percentage of body weight. a,b Means with no common superscripts within a row differ significantly (P < 0.05).

Table 4. Effect of dietary gallic acid supplementation on jejunum intestinal morphology of broiler chicks at 42 d of age. Gallic acid level (mg/kg feed) Items1 Villus Height Crypt Depth VCR

P-value

0

25

50

75

100

150

SEM

ANOVA

Linear2

Quadratic2

642.90 173.26a 3.73c

655.82 157.57a,b 4.16b,c

669.11 141.48b,c 4.81a,b

610.56 138.17b,c 4.55a,b

587.53 119.58c 4.96a

574.10 139.81b,c 4.14b,c

16.877 4.488 0.111

0.521 0.009 0.006

0.342 0.035 0.188

0.175 0.018 0.087

Data are the mean of 7 replicates (one bird from each replicate). 1 VCR: villus height:crypt depth ratio. 2 Orthogonal polynomial contrasts were used to determine the effect of dietary gallic acid levels. a–c Means with no common superscripts within a row differ significantly (P < 0.05).

Plasma Antioxidant Indices Table 5 shows the effect of dietary GA supplementation on blood antioxidant indices of broiler chicks at 21 and 42 d of age. As shown in the table, the plasma T-AOC, T-SOD activity, and MDA content were not affected by dietary addition of GA in broiler chicks at d 21 (P > 0.05). At 42 d of age, dietary addition of GA at 100 mg/kg reduced plasma MDA content (P = 0.030), but did not affect the plasma T-AOC or T-SOD activity compared with the control group (P > 0.05). The trend analysis indicates that a linear effect on plasma T-SOD activity (P = 0.049) at d 21,

MDA content at d 42 (P = 0.003), and T-AOC at both d 21 and 42 (P = 0.039 and 0.042, respectively) was observed with an increase of inclusion level of dietary GA, in which, 100 mg/kg dose group recorded the highest value.

Plasma Biochemical Indices Table 6 presents the results of the effect of dietary GA on plasma biochemical indices of broiler chicks. Dietary GA supplementation did not affect the plasma TP, ALB, UN, UA, or CRE contents of the broiler

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25

0

Day (1 to 21) BW d 21 (g) 796.0 ADG (g) 35.3 ADFI (g) 52.1 FCR (g:g) 1.477 Day (22 to 42) BW d 42 (g) 2,392.0 ADG (g) 74.7 ADFI (g) 147.8 FCR (g:g) 1.980a Day (1 to 42) ADG (g) 53.6 ADFI (g) 96.5 FCR (g:g) 1.801a

P-value

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Table 5. Effect of dietary gallic acid supplementation on plasma antioxidant indices of broiler chicks. Gallic acid level (mg/kg feed) Items1

0

25

50

Day 21 T-AOC (U/mL) T-SOD (U/mL) MDA (nmol/mL)

11.30 105.75 4.19

11.11 138.26 3.63

13.78 145.50 3.36

14.71 138.20 3.87

Day 42 T-AOC (U/mL) T-SOD (U/mL) MDA (nmol/mL)

8.34 144.87 4.37a

8.73 127.47 4.02a

10.14 138.76 3.73a,b

10.25 169.73 3.81a,b

P-value 150

SEM

ANOVA

Linear2

Quadratic2

15.64 163.93 3.29

13.63 137.77 2.75

0.616 5.810 0.195

0.197 0.110 0.365

0.039 0.049 0.059

0.238 0.084 0.789

11.82 184.08 2.97b

10.54 133.22 3.47a,b

0.477 9.107 0.129

0.326 0.543 0.030

0.042 0.445 0.003

0.470 0.360 0.434

75

100

Table 6. Effect of dietary gallic acid supplementation on plasma biochemical indices of broiler chicks. Gallic acid level (mg/kg feed) Items1

P-value ANOVA

Linear2

Quadratic2

0

25

50

75

100

150

SEM

Day 21 TP (g/L) ALB (g/L) UN (mmol/L) UA (μ mol/L) CRE (μ mol/L)

32.33 15.71 0.66 605.71 35.44

33.77 15.94 0.81 777.29 42.77

31.14 15.17 0.60 736.57 38.91

34.60 16.51 0.81 897.50 43.60

30.39 15.20 0.63 777.86 42.41

30.43 15.78 0.67 754.60 37.73

0.628 0.302 0.042 39.773 0.996

0.248 0.831 0.181 0.479 0.099

0.205 0.935 0.372 0.535 0.439

0.341 0.953 0.108 0.728 0.025

Day 42 TP (g/L) ALB (g/L) UN (mmol/L) UA (μ mol/L) CRE (μ mol/L)

36.47 16.35 0.56 571.86 55.49

40.00 17.33 0.59 402.57 53.90

35.64 16.06 0.54 455.71 44.76

33.07 15.26 0.66 490.14 50.54

36.12 16.44 0.69 621.43 49.71

36.47 15.26 0.60 557.17 55.67

1.193 0.236 0.018 26.334 1.208

0.210 0.091 0.155 0.144 0.060

0.430 0.062 0.097 0.247 0.802

0.081 0.842 0.482 0.152 0.008

Data are the mean of 7 replicates (one bird from each replicate). 1 TP: total protein; ALB: albumin; UN: urea nitrogen; UA: uric acid; CRE: creatinine. 2 Orthogonal polynomial contrasts were used to determine the effect of dietary gallic acid levels.

chicks at either 21 or 42 d of age (P > 0.05). However, the trend analysis shows a quadratic effect of dietary GA supplementation on plasma CRE content at 21 (P = 0.025) and 42 (P = 0.008) d of age.

DISCUSSION To date, there are few publications about using GA as a supplement in broiler chicks’ diets. Thus, any effort towards improving their performance without compromising health would be of fundamental importance to the productive poultry chain. In the present study, dietary addition of GA did not affect growth performance during the starter phase. However, broiler chicks fed diets supplemented with 75 to 100 mg/kg of GA significantly improved FCR in the grower phase and indeed in the overall period, indicating efficient utilization of nutrients. In agreement with our study, Starˇcevi´c et al. (2015) reported that GA improved feed efficiency in broiler chicks. The improved FCR in the grower phase, but not in the starter phase, observed in the present study is in line with our previous work that grape proanthocyanidins (GPC), which most of the time are oligomers of catechin, epicatechin, and their GA esters,

supplemented at 7.5 and 15 mg/kg (Yang et al., 2016) enhanced feed efficiency in a time-dependent manner. On the contrary, it has been reported that dietary inclusion of polyphenol rich grape pomace concentrate (GPC) improved FCR in broiler chicks in the starter phase (Viveros et al., 2011) and did not change the FCR in the grower phase (Brenes et al., 2008). In addition, supplementation of broiler chicken diets with low concentration (up to 3.6 g/kg) of grape seed extract (GSE), known to contain GA as its major active agent, did not change FCR in either the starter or grower phase (Brenes et al., 2010). A substantial increase in relative weight of breast muscle was also apparent in the current study, suggesting that dietary GA may improve carcass yield in a favorable manner. Boka et al. (2014) observed that heavier chicks tended to have longer villus, shallower crypts, and higher VCR as compared to their lighter counterparts. Therefore, it can be suggested that the positively modulated jejunal morphology and substantially inhibited plasma MDA (lipid peroxidation by-product) content observed in the present study may have contributed either independently or synergistically to the efficacy of dietary GA towards efficient feed utilization and high carcass

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Data are the mean of 7 replicates (one bird from each replicate). 1 T-SOD: total superoxide dismutase; MDA: malondialdehyde; T-AOC: total antioxidant capacity. 1 Orthogonal polynomial contrasts were used to determine the effect of dietary gallic acid levels. a,b Means with no common superscripts within a row differ significantly (P < 0.05).

GALLIC ACID IN BROILERS

ural antioxidant capable of eliminating ROS such as superoxide anions, hydrogen peroxide, and hydroxyl radicals (Polewski et al., 2002; Yen et al., 2002). In the present study, antioxidant markers were investigated to evaluate the antioxidant activity dietary GA, and the reduced plasma MDA content suggests that supplemental GA at 100 mg/kg can improve redox status of broiler chicks. The free radical scavenging action of GA is already well established (Polewski et al., 2002; Kim, 2007). Our results confirmed that dietary GA has potential to enhance antioxidant status of broiler chicks and is in keeping with a report that GA can improve broilers’ muscle antioxidant capacity (Lee et al., 2012; Starˇcevi´c et al., 2015). Moreover, both Wang et al. (2008) and Yang et al. (2016) have reported inhibition of lipid peroxidation in broiler chicks in response to GPC supplementation. Thus, the inhibitory activity of dietary GA on plasma MDA content may be due to the enhancement of the body’s antioxidant status or directly neutralizing ROS and free radicals or both. Ignea et al. (2013) showed that the role of physiologically relevant levels (5 and 50 μM GA equivalents) of GSE in yeast cell and yeast cell recovery assay varied from antioxidant to pro-oxidant, depending on the cellular antioxidant deficiency; in cells deficient in catalase activity or glutathione level, GSE augmented cell growth, whereas GSE operated as pro-oxidant agents in cells lacking SOD activity. In the current study, plasma MDA content was not significantly influenced by dietary GA supplementation in the starter phase but was significantly reduced in the grower phase, suggesting that maybe the short dietary treatment period was not sufficient enough to significantly modify the intensity of lipid peroxides or was due to the sensitive balance between the antioxidant and pro-oxidant roles of GA. Similarly Yang et al. (2016) demonstrated that addition of 15 mg/kg of GPC showed pro-oxidant leniency and a higher level evoked the rise of MDA at d 42. Dietary GA supplementation had no significant effect on plasma biochemical indicators, suggesting no influence on blood biochemical metabolism of broiler chicks. Overall, the present study shows that dietary GA supplementation at levels 75 to 100 mg/kg generally improved the performance of broiler chicks. It improved feed utilization, breast muscle yield, and oxidative stability, and positively modulated jejunum intestinal morphology. It is evident that the positively modulated intestinal morphology and enhanced anti-oxidant activities have partly contributed to the beneficial effect of dietary GA on growth performance. These findings justify that under normal experimental conditions the efficacious dietary supplementation levels of GA in broiler chicks’ diets are around 75 to 100 mg/kg. Differences in the effect of the same phenolic compound (GA) but different doses were observed in the present study. Therefore, further research on diverse dosages and situations is essential in order to attain more comprehensive results and fully understand the mechanism of action.

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yield of the broiler chicks. However, further studies are needed to test this hypothesis and the specific mechanism underlying. Jejunum intestinal morphology can be used as an indirect measurement for feed efficiency (Varel et al., 1987), as it is the main region for nutrient absorption in the broiler intestine (Leeson and Summers, 2001). Although it has been suggested that thinner intestinal epitheliums promote nutrient absorption and decline the metabolic demands of the gastrointestinal system (Visek, 1987), it is assumed that an increased villus height is paralleled by an increased digestive and absorptive function of the intestine, expression of brush border enzymes, nutrient transport system (Caspary, 1992), and increased body weight gain (Zijlstra et al., 1996). On the other hand, the crypt (another measure of gut integrity) can be regarded as the villus cells producer, and a small crypt shows slower tissue turnover and so a lower need for new tissue regeneration (Yason et al., 1987). However, reports on the effect of dietary GA on intestinal structure and function in broiler chicks and their contribution to changes in performance are limited. In the present study there was no significant effect of dietary GA on villus height. However, a significant reduction in crypt depth accompanied by a substantial increase in VCR was evident in broiler chicks fed dietary GA at 50 to 100 mg/kg as compared with those fed the control diet. The VCR is a histological index for digestive capacity of the small intestine and enhancement in this ratio improves the digestion and absorption process (Montagne et al., 2003; Mahdavi et al., 2010). Although inclusion of polyphenol rich grape products had an inhibitory effect on the growth of jejunum villi of broiler chicks, Viveros et al. (2011) confirmed that broiler chicks fed GPC and GSE slightly reduced crypt depth whereas, GPC increased VCR of the jejunum and was in line with our findings. In addition, the current findings are in accordance with our previous studies that GPC significantly reduced jejunum crypt depth and increased VCR in broiler chicks (Yang et al., 2016). It is well documented that a lengthening of the villus and a short crypt can lead to better nutrient absorption, decreased secretion in the gastrointestinal tract, increased disease resistance, and greater overall performance (Viveros et al., 2011). Accordingly, the increased VCR observed in the current study may be partly the reason for improved production performance in broiler chicks supplemented with dietary GA. In physiological conditions, animals produce reactive oxygen species (ROS), which are essential for life and growth. To regulate cellular concentration of ROS, the organism has to dispose of both endogenous and nutritional antioxidants (Favier, 1997). Previous studies have noted that MDA measured as the thiobarbituric acid reactive substances index is produced as a result of unsaturated fatty acid oxidation (Jensen et al., 1997), and use of dietary antioxidants is recommended to limit lipid peroxidation and ensure animal health (Wood and Enser, 1997). GA has been described as a powerful nat-

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ACKNOWLEDGMENTS This study was supported by an earmarked fund for China Agriculture Research System – Beijing Team for Poultry Industry and Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.

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