Feed supplementation with inulin on broiler performance and meat quality challenged with Clostridium perfringens: Infection and prebiotic impacts

Journal Pre-proof Feed supplementation with inulin on broiler performance and meat quality challenged with Clostridium perfringens: Infection and prebiotic impacts Andréia Guaragni, Marcel Manente Boiago, Nathieli B. Bottari, Vera Maria Morsch, Thalison F. Lopes, Aleksandro Schafer da Silva PII:

S0882-4010(19)31740-1

DOI:

https://doi.org/10.1016/j.micpath.2019.103889

Reference:

YMPAT 103889

To appear in:

Microbial Pathogenesis

Received Date: 1 October 2019 Accepted Date: 21 November 2019

Please cite this article as: Guaragni André, Boiago MM, Bottari NB, Morsch VM, Lopes TF, Schafer da Silva A, Feed supplementation with inulin on broiler performance and meat quality challenged with Clostridium perfringens: Infection and prebiotic impacts, Microbial Pathogenesis (2019), doi: https:// doi.org/10.1016/j.micpath.2019.103889. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Feed supplementation with inulin on broiler performance and meat quality

2

challenged with Clostridium perfringens: infection and prebiotic impacts

3 4

Andréia Guaragni1, Marcel Manente Boiago2,*, Nathieli B. Bottari3, Vera Maria

5

Morsch3, Thalison F. Lopes3, Aleksandro Schafer da Silva2,3,*

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1

8

Pinhalzinho, SC, Brazil

9

2

Department of Science and Food Technology, University of Santa Catarina State,

Department of Animal Science, University of Santa Catarina State, Chapecó, SC,

10

Brazil

11

3

12

Maria, Santa Maria, Brazil.

Graduate Program in Toxicological Biochemistry, Universidade Federal de Santa

13 14 15 16 17 18 19 20 21 22 23 24 25

*

Author for correspondence: [email protected] ; [email protected]

26 27

ABSTRACT

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Following the ban on the use of antibiotics as growth enhancers in 2006 by the

29

European Union, alternative products have been sought. Inulin is a prebiotic that is

30

found naturally in many plants. It reaches large intestine of animals unaltered, where it

31

is fermented by beneficial bacteria that comprise the intestinal microbiota. Inulin also

32

inhibits the growth of pathogenic bacteria. Consumption of inulin in chicken diets

33

improves performance at slaughter; nevertheless, little is known about its effects on

34

poultry meat. Therefore, the objective of this study was to evaluate the effects of inulin

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on feeding of broilers challenged with Clostridium perfringens (4.0 x 108 CFU) and its

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consequences on the quality of breast meat. Four hundred Cobb male broiler chickens

37

were distributed in a completely randomized design with four treatments and five

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replications each, as follows: T1: control treatment, basal diet (DB); T2: DB + 21-day

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challenged with C. perfringens orally; T3: DB + 21-day challenge with C. perfringens

40

orally + 25 mg/kg inulin; T4: DB + 21-day challenge by C. perfringens orally + 4.4

41

mg/kg lincomycin. There were no significant differences between treatments in terms of

42

pH, color parameters (L, a*, b*), water retention capacity, or shear force cooking weight

43

loss. However, we found that the meat of poultry challenged by C. perfringens showed

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lower lipid peroxidation and increased activity of the antioxidant enzymes SOD and

45

CAT, suggesting improvement in antioxidant profile. Nitrate/nitrite levels were lower

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with T3 and higher with T4 than with T1. We therefore conclude that inulin can replace

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antibiotics as growth promoters without causing changes in the physicochemical

48

characteristics of meat. C. perfringens challenge caused lower lipid peroxidation and

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stimulated antioxidant responses in breast meat.

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Keywords: Birds; Prebiotic; Lipid peroxidation; Antioxidant; Pathogenesis.

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1. INTRODUCTION

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Chicken meat is one of the most popular sources of animal protein for human

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consumption worldwide. The increased demand for chicken has required the

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development of various strategies to optimize production [1]. Advances in genetic

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selection, production practices and nutrition have made production more efficient;

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however, these more intensive conditions have also created stressful conditions for

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animals, making them more susceptible to disease [2].

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Clostridium perfringens infection causes necrotic enteritis in birds [3]. This

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condition develops in conjunction with other predisposing factors, and is controlled by

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antimicrobial supplementation in broiler feed. In recent decades, antibiotics have been

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widely used to prevent enteric disease in broilers, to maintain health and promote

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growth. Nevertheless, the continued indiscriminate use of antibiotics has given rise to

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antibiotic resistant microorganisms, with transfer of resistance to humans, whether due

66

to ingestion of resistant microorganisms or their residues [4]. This led the European

67

Union in 2006, followed by other countries, to ban antibiotics as growth enhancers in

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animal feed; this move to some extent undermined the efficiency of intensive livestock

69

systems [5].

70

Since then, there has been study of alternatives to antibiotics, including

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prebiotics that are nothing more than sets of carbohydrates, derived from plants or

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synthesized by microorganisms, capable of promoting beneficial intestinal microflora

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growth and providing health benefits [6]. In particular, attention has been focused on

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inulin, a natural component of many plants. Inulin is a soluble fermentable fiber that is

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not digested by host digestive enzymes and serves as a substrate for beneficial bacteria

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in the gut of birds, where it acts selectively on pathogenic bacteria. Its benefits include

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improved feed conversion and conversion parameters, mainly through changes in the

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structure of the intestinal mucosa, as well as improved weight gain and strengthened

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skeletal systems, improving carcass yield [7]. The same author also suggested that

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inulin improved absorption of some minerals and improved hormonal regulation in

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chickens.

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Few studies have evaluated the characteristics and quality of inulin-fed chicken

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meat, especially in birds challenged with pathogens. Therefore, the aim of this study

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was to evaluate the effects of inulin use on broiler chickens challenged with C.

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perfringens and its effects on meat quality.

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2.

MATERIALS AND METHODS 2.1. Setting and animals

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A total of 400 one-day-old male Cobb chicks were reared over a period of 42

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days, divided into three phases: initial (1–21 days), growth (22–35 days) and finishing

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(36–42 days). The birds were housed in an experimental house divided into 1.80 m2

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boxes, with 20 birds allocated per box based on the average weight of the flock, aiming

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at homogeneity among the birds in each plot. We followed management guidelines

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indicated by the pedigree manual. Water and feed were provided ad libitum throughout

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the experiment. The basal diet was formulated based on the requirements presented in

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the Brazilian Tables for Poultry and Swine [8].

97 98

2.2. Experimental design

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A completely randomized design with four treatments and five replications of 20

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birds each was used. All groups received the same basal diet (BD), the treatments were

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divided into: T1 - control treatment, BD; T2: DB + oral challenge with C. perfringens;

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T3: DB + oral C. perfringens challenge + 25 mg/kg inulin; T4: DB + oral C. perfringens

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challenge + 4.4 mg/kg lincomycin. The challenge was performed at 21 days, when each

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bird individually received 1.0 mL of inoculum (4.0 X 108 CFU/mL).

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2.3. Bird Performance Parameters

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To evaluate the performance parameters of the birds, the study was divided into

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two experimental periods, 1 to 35 days and 1 to 42 days of growth, where the animals

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were weighed at the beginning and end of each period, resulting in obtaining the

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average weight gain (g), average daily bird weight gain (g) and the average bird weight

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at the end of each period (g). The diets were also weighed at the beginning and end of

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each experiment period, and the average feed intake per bird (g) was calculated,

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obtained from the poultry feed intake of each batch, in each period, divided by the

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number of feeds birds from each batch. Feed consumption in each period also served as

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the basis for calculating feed conversion of birds, calculated by dividing feed

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consumption in the period by the average weight of birds in the same period. The

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number of birds that died in each period was also calculated and determined as mortality

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(%).

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2.4. Slaughter

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At 42 days, 48 birds were separated and identified based on the average weight,

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three birds per experimental plot, including 12 birds per treatment. After an 8-hour fast,

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they transported and slaughtered in a commercial slaughterhouse. After slaughter and

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evisceration, the carcasses were boned and the breasts were separated, identified,

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packaged in plastic bags, packaged in ice-boxes and sent to the Products Technology

126

Laboratory. After evisceration, the carcasses were not subjected to pre-cooling and

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cooling processes using chillers, so as not to generate changes in parameters that may be

128

caused by water absorption.

129 130

2.4.1. Physicochemical characteristics of meat

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2.4.1.1. pH

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The pH analysis of the chicken breasts was performed within five hours of

133

slaughter using a Testo 205® digital pH meter: we inserted the probe into the cranial

134

part of the chest muscle (Pectoralis major).

135 136

2.4.1.2. Color

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The evaluation of meat color was determined in breast muscle (Pectoralis

138

major) after boning, using a Minolta Chroma Meter model CR-400, to obtain the

139

following parameters: luminosity (L*), red intensity (a*) and yellow intensity (b*).

140 141

2.4.1.3. Water retention capacity

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To determine water retention capacity (%), a sample of 2 g (± 0.15) was taken

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from each boneless breast. These samples were placed between two filter papers and

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acrylic plates, where they were subjected to pressure exerted by a 10.0 kg weight for

145

five minutes. They were then re-weighed to calculate water retention capacity (WRC) as

146

described by Hamm [9].

147 148

2.4.1.4. Cooking losses

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Cooking losses were determined according to methodology proposed by Honikel

150

[10], with modifications, taking samples from each boneless breast. Samples were

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weighed, then packed in plastic bags, identified and placed in a water bath at 85 ºC for

152

30 minutes. They were then removed from the bags to eliminate water and cool.

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Samples were re-weighed and these weights were compared with initial weights,

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thereby determining percentage of losses during cooking.

155 156

2.4.1.5 Shear force

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The same samples used to measure cooking losses were reduced to known

158

measurement sizes and were placed with the fibers oriented perpendicular to a Warner-

159

Bratzler blade coupled to a Texture Analyzer TA-XT2i apparatus to measure shear

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force, expressed as g/f, according to Lyon, Lyon and Dickens [11].

161 162

2.4.2. Oxidative profile of the meat

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2.4.2.1. Sample preparation

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Samples were placed in 10 mM Tris-HCl pH 7.4 solution to analyze for

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superoxide dismutase (SOD), catalase (CAT), nitrite/nitrate (NOx) and gluthatione S-

166

transferase (GST) analysis. Tissue samples were gently homogenized in a glass Potter

167

homogenizer in specific buffer. The homogenates were centrifuged at 10,000 g at 4 °C

168

for 10 min to produce an S1 that was used for analysis. Aliquots of homogenates were

169

stored at -80 ° C until use. Prior to analyses, protein concentrations were determined

170

using the Coomassie blue method according to Bradford [12] with bovine albumin as

171

the standard.

172 173

2.4.2.2. Lipid peroxidation - TBARS

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The analysis was performed using the method described by Pikul et al. [13],

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with samples packed and stored refrigerated for five days. We measured muscle

176

oxidation by quantifying thiobarbituric acid reactive substances (TBARS) formed

177

during the decomposition of lipid peroxides, using a spectrophotometer at 532 nm. The

178

compound 1,1,3,3 tetramethoxypropane (TMP) was used as standard TBARS, and

179

results were expressed as nmol TMP/g sample.

180 181

2.4.2.3. Antioxidant profile

182

Superoxide dismutase (SOD) activity was determined by inhibiting the O2

183

reaction with adrenaline as described by McCord and Fridovich [14]. One unit of SOD

184

enzyme is defined as the amount of enzyme that inhibits the adrenaline oxidation rate by

185

50%. This leads to the formation of a red-colored product, adrenochrome, that is

186

detected using a spectrophotometer. SOD activity was determined by measuring the rate

187

of adrenochrome formation observed at 480 nm in a reaction medium containing 50

188

mM glycine-NaOH, pH 10 and 1 mM adrenaline. Results were expressed as IU

189

SOD/mg protein.

190

Catalase (CAT) activity was measured according to the modified method of Nelson

191

and Kiesow [15]. This assay involves the change in absorbance at 240 nm for 2 min due

192

to the decomposition of hydrogen peroxide-dependent catalase (H2O2). Enzyme activity

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was calculated using the molar extinction coefficient (0.0436 cm2/µmol) and the results

194

were expressed as nmoles/mg protein.

195

Glutathione S-transferase (GST) activity was measured using spectrophotometry at

196

340 nm according to Habig et al. [16]. The mixture contained muscle homogenate

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supernatant as a test, 0.1 M potassium phosphate buffer (pH 7.4), 100 mM GSH and

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100 mM CDNB, used as substrate. Enzyme activity was expressed as µmol/CDNB/mg

199

protein.

200

201

2.4.3. Nitrate/nitrite (NOx) levels in meat

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To determine NOx, an aliquot (200 µL of samples) was homogenized in 200 mM

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Zn2SO4 and acetonitrile (96%, HPLC grade). Thereafter, the homogenate was

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centrifuged at 16,000 g for 20 min at 4 °C and the supernatants were separated for

205

analysis of NOx content as described previously [17]. The resulting pellets were

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suspended in NaOH (6 M) for protein determination and expressed as µmol/mg protein.

207 208

2.5. Ethics committee

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This study was approved by the Animal Use Ethics Committee (CEUA) of the

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State University of Santa Catarina (UDESC), protocol number 3369060819, under the

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rules of the National Council for Animal Experimentation Control (CONCEA).

212 213

2.6. Statistical analysis

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Data were subjected to normality testing followed by analysis of variance. In

215

cases of significant differences, the means were subjected to the Tukey test accepting

216

5% as significant.

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3. RESULTS

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3.1 Zootechnical Performance

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Performance results of broilers are shown in Table 1. There were no significant

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differences among treatments for zootechnical performance.

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3.2 Physicochemical characteristics of meat

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Physicochemical characteristics of breast meat are displayed in Table 2. There

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were no significant differences among treatments in terms of final pH, luminosity (L*),

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red intensity (a*), yellow intensity (b*), shear force, water retention capacity or cooking

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losses.

228 229

3.3 Oxidant and antioxidant status of the meat

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There were significant differences in TBARs levels among groups, with lower

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TBARS in broiler meat of chickens that had undergone treatments (T2, T3 and T4), that

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is, chickens infected by C. perfringens. Levels reactive oxygen species (ROS) in meat

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were higher when birds were fed with inulin (T3) than in other groups. Chicken meat

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showed higher SOD activity in treatments challenged with C. perfringens (T2, T3 and

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T4) than in T1; similar results were observed for CAT activity in treatments T3 and T4

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when compared to that of control (T1). GST activity did not differ significantly among

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treatments. NOx levels in the meat of chickens that consumed inulin were lower (T3),

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while T4 NOx levels were higher than that of control (Table 3).

239 240

4. DISCUSSION

241

Differences between treatments for bird performance variables were not

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observed in our study, unlike that reported by Buclaw [7]. Since the birds were reared in

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new wonderland, the challenge may have been small; reason for not checking difference

244

between treatment; even the group of birds that ingested feed without conventional or

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alternative growth promoter.

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Inulin supplementation in broiler diets did not affect meat quality. The absence

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difference in pH between treatments agrees with the findings of Tavaniello et al. [18]

248

who fed chickens with prebiotics. pH is a basic parameter used for meat quality

249

evaluation, that indicates the level of glycolytic transformations [19]. It is also related to

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other attributes of meat quality such as tenderness, coloration, shelf-life and water

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retention capacity [4].

252

In the present study, color was not altered by C. perfringens infection, or by with

253

the presence of inulin in poultry feed. Similar results were described by

254

Dankowiakowska et al. [19] using different symbiotics and prebiotics in chicken feed,

255

including inulin at 1.760 mg/kg. However, Park and Park [20] found higher levels

256

luminosity (L*) and yellow intensity (b*) in poultry meat supplemented with 0.25 g/kg

257

of microencapsulated inulin and vitamin E. Cho et al. [21] found that birds were

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challenged with 5.0 mL of inocula of C. perfringens (107 CFU/mL) that were

259

supplemented with phytogenic additives showed no change in meat color. This is

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important because meat coloration is an important commercial feature. Color most often

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affects consumer decisions to purchase the product, because they associate color with

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freshness and quality [22].

263

The shear force (texture) we found in our study was similar to that described by

264

Silva et al. [23] for broiler chickens. However, they differ from those proposed by

265

Poorbaghi et al. [24] who reported a reduction in shear force when birds consumed

266

0.1% inulin/kg feed per day. Several factors modify the texture of meat, including pre-

267

slaughter factors such as poultry age, breed, feeding, and post-slaughter factors such as

268

postmortem glycolysis and development of rigor mortis, scalding temperature, cooling

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and boning time [22]. Based on this information, researchers tested whether injection of

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1.5% prebiotic into chicken breasts stored at nine days of refrigeration temperature

271

could maintain meat texture; they found that the shear force was not changed by the

272

treatment [25].

273

Water retention capacity was not affected by bacterial infection and the use of

274

inulin. This is important because it suggests that the meat was able to retain some or all

275

of its own water [22]. Cho et al. [26] and Tavaniello et al. [27], using different

276

prebiotic-containing symbiotics, also found no change in water retention capacity.

277

Dankowiakowska et al. [19] observed high water losses when birds were fed inulin. By

278

contrast, Park and Park [20] reported increased retention capacity when birds were

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supplemented with microencapsulated inulin and vitamin E. With respect to water

280

retention, Cho et al. [26] and Cheng et al. [4] reported a reduction in cooking water

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losses when birds were fed prebiotics, differing from the findings of the present study.

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These differences in meat-related studies may be attributed to a number of factors,

283

including pH, sarcomere length, ionic strength, osmotic pressure, and rigor mortis

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development, all of which alter cellular and extracellular components. Water retention

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capacity has a direct influence on meat color and tenderness. It is one of the most

286

important functional properties of raw meat. Increasing muscle water content,

287

increasing sensitivity, juiciness, firmness and appearance, improves meat quality and

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economic value [22]. Therefore, we believe that inulin is a potential additive to replace

289

conventional growth promoters.

290

Poultry meat is particularly susceptible to lipid peroxidation, mainly due to

291

higher levels of unsaturated fatty acids that facilitates its deterioration. As a

292

consequence, its shelf life decreases. Lipid peroxidation begins shortly after slaughter,

293

mediated post mortem biochemical changes related to cessation of blood flow;

294

consequently, natural antioxidant system failures occur [28]. In our study, we found

295

lower lipid peroxidation in all chickens challenged by bacterial infection; this was an

296

unexpected result, because the literature reports contrary results in cases of infection. In

297

the present study, we did not associate the reduction of TBARS with conventional

298

inulin or antimicrobial consumption, because levels in the positive control (infected)

299

birds were also lower. Park and Park [20] and Cheng et al. [4] reported that birds

300

supplemented with 0.25 g/kg microencapsulated inulin and vitamin E and 1.5 g/kg-1

301

prebiotic-containing symbiotic had lower TBARS levels. One of the possible

302

explanations is the antioxidant capacity of inulin as well as the activity of inulin

303

fermenter microflora in the gastrointestinal tract [29]. By resisting the breakdown of

304

digestive enzymes, inulin arrives intact in the digestive system because it is mainly

305

fermented by lactic acid bacteria and bifid bacteria, that in turn have been attributed to

306

the host [30]. We believe that the effects inulin on reducing infection-mediated lipid

307

peroxidation are beneficial to meat quality; nevertheless, the explanations for this result

308

are not known.

309

In our study, we found lower NOx levels in inulin-fed chickens; however, there

310

were high levels of NOx in the meat of broilers that consumed the antibiotic diet.

311

Nitrites and nitrates are the main stable metabolites of endogenous nitric oxide (NO)

312

[31], a molecule that reacts with oxygen species and biological molecules such as

313

dioxygen, superoxide anion and oxyhemoglobin to form a variety of products, including

314

nitrites and nitrates that are toxic at high doses [32]. According to the literature,

315

cytotoxicity of NO may be due to its ability to generate peroxynitrite, initiating a variety

316

of oxidative reactions, including modifications of nucleic acids, lipids and proteins that

317

lead to tissue injury [33]. NO levels can be an important tool for assessing immune

318

responses because NO is a proinflammatory molecule [34] as well as a marker of

319

oxidative status [35]. Knowing the properties of nitric oxide in the animal organism, we

320

believe that inulin had an antioxidant effect, neutralizing the production of the free

321

radical peroxynitrite. We cannot rule out an anti-inflammatory effect of this prebiotic.

322

The increase in NOx in birds that consume lincomycin has attracted attention, and

323

deserves further investigation, because this antibiotic is commonly used as a growth

324

enhancer in broiler diets.

325

SOD and CAT activity increased significantly in birds supplemented with inulin

326

and challenged with C. perfringens; this was a similar result to what occurred with the

327

use of lincomycin. This increase in antioxidant enzymes may be associated with lower

328

levels of lipid peroxidation in meat, a characteristic effect of antioxidant protection

329

described by Shang et al. [29]. GST activity did not differ in this study. GST performs a

330

wide range of functions in cells, including the removal of reactive oxygen species,

331

protecting cells from free radical-induced cell death [36], primarily in the liver.

332 333

5. CONCLUSION

334

Inulin can replace antibiotics as growth promoters without causing changes in

335

the physicochemical characteristics of meat, even in the context of C. Perfringens

336

infection. Challenge with C. perfringens caused lower lipid peroxidation and increased

337

activity of antioxidant enzymes in the breast meat, a desirable biochemical characteristic

338

of meat that gives rise to longer shelf life. Inulin had a positive effect on meat nitric

339

oxide levels by stimulating the inflammatory process that is desirable in farm animals,

340

as well as by increasing free radical levels in meat.

341 342

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[35] Beckman, J.S.; Koppenol, W.H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. American Journal of Physiology-Cell Physiology 271 (5) (1996) 1424-1437. 10.1152/ajpcell.1996.271.5.C1424.

486 487 488 489

[36] Sheehan, D. et al. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochemical Journal 360 (1) (2001) 1-16. 10.1042/0264-6021:3600001. Table 1 - Mean values obtained for feed intake (FI, g/bird), average weight (AW,

490

g/bird), average weight gain (AWG, g/bird), daily weight gain (DWG, g/bird/day), feed

491

conversion (FC) and mortality (MORT, %) of birds submitted to different treatments in

492

the periods from 1 to 35 days and from 1 to 42 days.

493

Period: 1 to 35 days AW AWG GPD FC MORT 2433 2388 68.23 1.59 4.00 2451 2406 68.74 1.58 3.75 2502 2456 70.18 1.57 3.00 2440 2395 68.44 1.59 1.25 0.064 0.061 0.061 0.865 0.578 2.64 2.68 2.68 3.78 11.48 Period: 1 to 42 days T1 5271 3163 3117 74.22 1.69 4.5 T2 5194 3142 3097 73.74 1.67 4.0 T3 5088 3187 3143 74.83 1.62 3.0 T4 5028 3065 3020 71.92 1.72 3.75 P-value 0.144 0.364 0.349 0,349 0.148 0.541 CV (%) 4.15 2.81 2.84 2,84 3.42 10.78 Note: CV = coefficient of variation. T1 = control; T2 = positive control for Clostridium

494

perfringens; T3 = alternative growth enhancer; T4 = commercial growth enhancer.

Treatment T1 T2 T3 T4 P-value CV (%)

495 496 497 498

FI 3800 3818 3850 3808 0.101 3.21

499 500 501 502 503 504 505 506 507

Table 2 – Mean values of final pH (pH), luminosity (L*), red intensity (a*), yellow

508

intensity (b*), shear force (SF, g/f), water retention capacity (WRC, %) and cooking

509

losses (CL, %) in samples of breast muscle (Pectoralis major) of birds subjected to

510

various treatments. Treatments

pH

L*

a*

b*

SF

WRC

CL

T1

5.80

51.20

-0.13

7.80

1994

71.75

15.54

T2

5.75

52.16

-1.22

8.65

2261

72.91

13.54

T3

5.78

52.60

-0.518

8.13

2590

75.36

14.21

T4

5.89

52.43

0.373

8.39

2198

76.20

16.04

P-value

0.181

0.525

0.523

0.417

0.245

0.369

0.146

CV (%)

1.95

3.34

34.56

10.77

33.93

6.46

13.25

511

CV = coefficient of variation. T1 = control; T2 = positive control for Clostridium

512

perfringens; T3 = alternative growth enhancer; T4 = commercial growth enhancer.

513 514 515 516

517 518 519 520 521 522 523 524 525

Table 3 – Mean values obtained for thiobarbituric acid reactive substances (TBARS,

526

mmol

527

µmol/CDNB/min), SOD (U SOD/mg protein), CAT (nmol CAT/mg of protein) in

528

breast muscle (Pectoralis major) of birds subjected to various treatments.

TMP/g),

Treatments

NOx

(µmol/mg

protein),

glutathione

S-transferase

(GST,

TBARS

NOx

GST

SOD

CAT

T1

3.92 A

3.59 B

440.67

0.22 B

4.80 B

T2

2.19 B

3.47 B

428.53

0.36 A

5.60 A B

T3

2.24 B

1.76 C

450.08

0.43 A

6.17 A

T4

1.84 B

7.18 A

358.33

0.39 A

6.64 A

Valor de P

0.001

0.0001

0.063

0.0013

0.0022

CV (%)

12.81

16.76

14.56

21.45

11.98

529

A, B –

530

significant difference for P < 0.05. CV = coefficient of variation. T1 = control; T2 =

531

positive control for Clostridium perfringens; T3 = alternative growth enhancer; T4 =

532

commercial growth enhancer.

Mean values followed by different letters overwritten in the same column indicate