Accepted Manuscript

Title: Effect of feed supplementation with a thymol plus

carvacrol mixture, in combination or not with an
NSP-degrading enzyme, on productive and physiological
parameters of broilers fed on wheat-based diets
Author: H. Hashemipour V. Khaksar L.A. Rubio T. Veldkamp
M.M. van Krimpen
PII:
DOI:
Reference:

S0377-8401(15)30033-X
http://dx.doi.org/doi:10.1016/j.anifeedsci.2015.09.023
ANIFEE 13386

To appear in:

Animal

Received date:
Revised date:
Accepted date:

18-3-2015
24-9-2015
25-9-2015

Feed

Science

and

Technology

Please cite this article as: Hashemipour, H., Khaksar, V., Rubio, L.A., Veldkamp, T.,
Krimpen, M.M.,Effect of feed supplementation with a thymol plus carvacrol mixture,
in combination or not with an NSP-degrading enzyme, on productive and physiological
parameters of broilers fed on wheat-based diets, Animal Feed Science and Technology
(2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.09.023
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Effect of feed supplementation with a thymol plus carvacrol mixture, in combination or not

with an NSP-degrading enzyme, on productive and physiological parameters of broilers fed

on wheat-based diets

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H. Hashemipoura,*, V. Khaksara, L. A. Rubiob, T. Veldkampc, and M. M. van Krimpenc

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Excellence Centre for Animal Sciences and Department of Animal Science, Faculty of

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Agriculture, Ferdowsi University of Mashhad, P.O. Box 91775-1163, Mashhad, Iran;

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Fisiologa y Bioqumica de la Nutricin Animal (EEZ, CSIC), Profesor Albareda, 1, 18008

Granada, Spain

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Wageningen UR Livestock Research, P.O. Box 338, 6700 AH Wageningen, The Netherlands

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Corresponding author: Tel. and Fax: +98 513 879 6845.

E-mail address: hamideh_hashemipour@yahoo.com (H. Hashemipour).

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ABSTRACT
The current study was conducted to evaluate the effect of feed supplementation with a

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phytogenic product (equal mixture of thymol plus carvacrol; T+C) on performance, nutrient

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retention, volatile fatty acid (VFA) profiles, cecum microbial ecosystem, serum parameters and

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characteristics of gastrointestinal tract of broilers fed on wheat-based diets with or without an

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NSP-degrading enzyme product (xylanase plus -glucanase; E) from d 0 to 42. Six dietary

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treatments were arranged according to a factorial design with three levels of T+C (0, 100 and

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200 mg/kg of diet) and two levels of E (0 and 0.5 g/kg of diet). Each treatment was replicated

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five times with 12 chicks per replicate. There was no interaction effect between E and T+C on

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any of the measured parameters. Compared with the control group, birds fed diets containing E

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or T+C had a higher (P < 0.01) average daily gain and feed efficiency at d 42. Digesta viscosity

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was reduced (P < 0.05) in treatments with E addition in all parts of the small intestine. In

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treatments with T+C inclusion digesta viscosity was reduced in jejunum and ileum at d 24. E or

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T+C treated birds showed an increased (P < 0.05) retention of DM, protein and gross energy.

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Dietary supplementation with E and T+C increased (P < 0.01) total VFA and acetate levels at d

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24 and 42, whereas the level of butyrate decreased (P < 0.01). E. coli and C. perfringens counts

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were lower (P < 0.01) than controls, and Lactobacilli counts were higher (P < 0.01), in birds fed

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on diets supplemented with enzyme or T+C at the rate of 200 mg/kg. E supplementation

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increased (P < 0.05) serum triglyceride, total cholesterol, total protein (TP), albumin and

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globulin concentrations, while T+C supplementation decreased (P < 0.05) total cholesterol, TP

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and albumin at d 40. E supplementation decreased (P < 0.01) the relative length of duodenum,

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jejunum and ileum of broilers. Moreover, carcass, liver and pancreas relative weights decreased

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(P < 0.05) with E supplementation at d 42. T+C dietary supplementation only affected carcass

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relative weight and jejunum and ileum relative lengths. The present study showed thatthymol +

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carvacol, in combination or not with an NSP-degrading enzyme, improved growth performance,

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enhanced nutrients retention, increased total VFA, reduced cholesterol and modulated intestinal

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microbial counts in broilers fed on a wheat-based diet.

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Keywords: wheat, enzyme, thymol, carvacrol, broiler

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Abbreviations: ADFI, average dairy feed intake; ADG, average dairy gain; AGP, antibiotic

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growth promoter; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatin

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kinase; CP, crude protein; DM, dry matter; E, enzyme; EO, essential oil; GGT, gamma

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glutamyltransferase; HDL, high density lipoprotein; LDL, low density lipoprotein; NSP, non-

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starch polysaccharide; PFA, phytogenic feed additive; T+C, thymol+carvacrol; TP, total protein;

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TTAR, total tract apparent retention; VFA, volatile fatty acid.

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1. Introduction
Wheat is an important ingredient in broiler diets as energy source, and is often the only cereal

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in grower and finisher diets. However, compared with corn, wheat contains higher amounts of

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anti-nutritional factors consisting mainly of water soluble and insoluble non-starch

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polysaccharides (NSP). Soluble NSPs have been shown to detrimentally increase digesta

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viscosity (Lazaro et al., 2003), stimulate the growth of some pathogenic bacteria species,

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including Escherichia coli and Clostridium perfringens (Collier et al., 2003), and adversely affect

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villus height, width, surface area, and shape (Mathlouthi et al., 2002). Compelling evidence

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indicates that broilers fed diets based on wheat, barley, or rye suffer from reduced crude protein

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(CP) and fat digestibility, and a reduced apparent metabolizable energy content (Mathlouthi et

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al., 2002), which resulted in depressed body weight gain and feed conversion ratio (Lazaro et al.,

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2003). The benefits of exogenous enzyme supplementation to NSP-rich diets are well

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documented. These enzymes can partially hydrolyze NSP, reduce the viscosity of gut contents,

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and result in improvements in nutrient digestion and absorption (Almirall et al., 1995; Yu et al.,

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1997). Several studies have also demonstrated improvements of nutritive value, feed utilization,

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body weight gain, composition and activity of intestinal microbiota, and reduction in excreta

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volume after supplementation of wheat-based diets with NSP-degrading enzymes such as

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cellulases, pectinases, hemicellulases, arabinoxylanases and -glucanases (Bedford and

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Apajalahti, 2001).

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Increasing insight into the potentially beneficial activities of the gastrointestinal microbiota,

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together with increasing public concern about antibiotic resistance and residues in animal

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products, have resulted in the search for alternatives to antibiotic growth promoters (AGP) such

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as prebiotics, probiotics, phytogenics and other feed additives. Phytogenic feed additives (PFA)

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may positively affect poultry health and productivity. Hashemipour et al. (2013a) indicated that

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the use of PFA-containing compounds, such as essential oils (EO) or spices, stimulate digestive

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enzyme production and activity, and induce a higher secretion of bile acids. The antimicrobial

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properties of EO have encouraged their use as a natural replacement for AGP in animal feeds.

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Positive effects of PFA were observed on daily weight gain and feed conversion ratio of

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chickens when fed a diet supplemented with a mixture containing capsaicin, cinnamaldehyde and

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carvacrol (Jamroz and Kamel, 2002). Jamroz et al. (2005) showed that the addition of a plant

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extract to broiler diets had no influence on apparent ileal digestibility of nutrients, but on the

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other hand, Hernandez et al. (2004) reported that essential oils and a Labiatae extract added to a

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starter diet increased ileal DM and starch digestibility, but not CP digestibility. Furthermore,

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active plant compounds are the potential effectors on microbial communities (Hashemipour et

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al., 2013b), and could therefore be considered as the alternatives in controlling the intestinal

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microbial population. Thymol and carvacrol, the main bioactive components in thyme essential

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oil, are appetite- and digestion-stimulating, and have considerable antimicrobial and antifungal

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activity, which have been reported to promote the growth of beneficial bacteria and inhibits the

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growth of potentially deleterious intestinal bacteria (Akyurek and Yel, 2011). Given their

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antimicrobial activity, it would be expected (Wenk, 2000) that thymol and carvacrol could have

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positive effects on growth and performance in broilers. The two compounds have the status of

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generally recognized as safe (GRAS), which is endorsed by the Flavor and Extract

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Manufacturers Association (FEMA) and the Food and Drug Administration (FDA) of the

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U.S.A. (Furia and Bellanca, 1975).

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Accordingly, the present study was conducted to determine the effect of feed

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supplementation with a thymol plus carvacrol mixture, in combination or not with an NSP-

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degrading enzyme, on productive and physiological parameters of broilers fed wheat-based diets.

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2. Materials and methods

2.1. Birds, Housing, and Diets

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All experimental procedures were approved by the Animal Welfare Committee of the

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Department of Animal Science, Ferdowsi University of Mashhad, Iran. A total of 360 day-old

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Ross-308 male broiler chicks were obtained from a local hatchery and distributed in 30 groups of

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12 birds each. Six treatments were arranged according to a factorial desgin with 3 levels (0, 100

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and 200 mg/kg) of thymol plus carvacrol (T+C) (Next enhance150, 1:1 thymol:carvacrol;

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Novus International, Inc., St. Louis, MO) and 2 levels (0 and 0.5 g/kg diet) of the enzyme

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product (E) (Endofeed W, GNC Bioferm Inc., Saskatoon, Saskatchewan, Canada). According to

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the manufacturer, Next enhance150 contains 50% of the active components thymol and

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carvacrol, and enzyme Endofeed W holds 2250 and 700 fungal arabinoxylanase and -glucanase

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units/g of enzyme activity, respectively.

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Each diet was randomly fed to five groups of chicks. The feeding regimen consisted of a

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starter (1-10 d), grower (11-24 d), and finisher (25-42 d) diet. The basal diet was formulated to

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meet the nutrient requirements according to Ross-308 rearing guideline (Aviagen, 2007). Mash

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feed and water were provided ad libitum throughout the experiment. The ingredients and

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chemical composition of the basal diets are shown in Table 1. Next enhance150 and Endofeed

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W were added to 100 g of wheat bran and were subsequently blended with premix. Finally, the

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premix was mixed with the basal diet. Feed was prepared weekly and stored in airtight

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containers. Birds received a continuous lighting regimen during the first week and 23 h light per

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day afterward. Broiler chickens were kept at 32C during the first day of age. Thereafter,

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temperature was gradually decreased by 0.5C per day until 21C was reached. After that, they

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were maintained at approximately 21C until the end of the experiment.

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2.2. Bioactive components analysis

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Samples (4 g) of grinded T+C supplemented diets were weighed into a centrifuge tube,

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mixed with distilled water (2.5 mL) and ethanol (1 mL), and allowed to stand for 15 min. Diethyl

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ether (12 mL) was then added, and the samples were shaken for 16 h and centrifuged at 15,000

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g for 5 min at 4C. The calibration samples were prepared with control feed which was

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supplemented with standard solutions of carvacrol and thymol at 5 different concentrations (5,

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10, 20, 40, and 100 mg/L in ethanol). Feed supplemented with ethanol free of T+C was used as a

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blank.

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Gas chromatographic analyses were performed using a GC PU 4500 system (Shimadzu

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Corp., Kyoto, Japan) equipped with a flame ionization detector and an E30 (30 m 0.32 mm ID,

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5% phenyl methyl silicone, phase thickness 0.5 mm) capillary column. The column temperature

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ranged from 80 to 202C with increments of 8C per minute. Helium was used as the carrier gas

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at a flow rate of 1.5 mL/min. Sample injection was carried out in splitless mode at 200C with

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splitless time of 1 min with a sample injection volume of 0.5 L. Temperature of the detector

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was 202C. Oven temperature was maintained initially at 80C for 2 min, then raised at a rate of

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8C/min to 125C, maintained for 10 min, then raised at a rate of 25C/min to 200C, and

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maintained for 10 min. The 5 concentration linear calibration curves were calculated by using

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internal standards (Carvacrol). Using the peak areas, the concentrations (mg/kg) of the analysts

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in the samples were calculated from the calibration curves.

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2.3. Performance
The bird experimental period lasted for 42 d. Feed consumption and body weight were

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measured on a pen basis on d 0, 10, 24, and 42. Average daily feed intake (ADFI), average daily

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gain (ADG), and feed efficiency (G:F) were calculated for each period (d 0-10, d 11-24, d 25-42,

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and d 0-42). The chickens were inspected daily and dead birds were removed following

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registration of date and BW. When calculating feed efficiency, the BW of dead birds was also

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taken into account.

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2.4. Sampling procedures for intestinal digesta viscosity and pH

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On d 24, two birds per replicate (i.e. 10 birds per treatment) were randomly selected and

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euthanized using sodium thiopental. The small intestine was removed, the digesta contents of the

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duodenum, jejunum, and ileum was immediately collected, and samples from the two birds were

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pooled and placed into clean tubes. The small intestine was divided into three segments:

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duodenum (from gizzard to pancreo-biliary ducts), jejunum (from pancreo-biliary ducts to

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Meckels diverticulum), and ileum (from Meckels diverticulum to ileo-caecal junction). The

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samples were mixed with deionized water (1:10 wt/vol), and used to measure the pH of each

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segment of the gastrointestinal tract in duplicate by using a digital pH meter (Model 827,

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Metrohm, Herisau, Switzerland).

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Other samples taken from duodenum, jejunum and ileum of the 2 birds/pen were pooled and

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mixed to achieve a homogenous mixture, which was then centrifuged at 9,000 g at room

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temperature (4C) for 10 min. The supernatant was withdrawn, and viscosity was determined at

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40C using a Brookfield digital viscometer model DV- III as described by Bedford and Classen

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(1993).

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2.5. Total tract apparent retention of nutrients

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For determination of total tract apparent retention (TTAR) of nutrients, 2 birds per replicate

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were moved to metabolism cages (2 birds in each) with a wire mesh bottom and excreta

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collection trays (60 30 30 cm, length width height) on d 20. Each cage was equipped

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with a metal feeder and drinker placed outside the cage. Experimental diets were the same as in

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the growth experiment, except that 0.3% of chromic oxide (Cr2O3) was added and mixed to

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facilitate determination of nutrient retention. The metabolizable trial included a 3-d preliminary

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adaptation period at 20 to 22 d of age followed by 2 d of total excreta collection. Contamination

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(e.g., feathers and down) was carefully removed and the collected excreta was dried at 60C until

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constant weight, homogenized and finely ground to pass through a 1-mm screen, and stored in

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airtight plastic containers until analysis. The following equation was used to calculate TTAR

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(Scott et al., 1976):

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TTAR (%) = 100 [(diet Cr2O3/excreta Cr2O3) (nutrient in excreta/nutrient in diet)] 100.

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2.6. Volatile fatty acids analysis

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On d 24 and 42, cecal contents from two birds per replicate (i.e. 10 birds per treatment) were

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gently squeezed into a tube and stored at -80C until analysis. Approximately 1.5 g of thawed

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digesta was diluted with distilled water (1:1 wt/vol) in a screw-capped tube. After

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homogenization and centrifugation, 1 mL of clear supernatant was transferred into an ampulla,

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and 0.2 mL metaphosphoric acid solution was added. The sample was again homogenized and

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placed in an ice bath for at least 30 min to allow the protein to settle completely. Finally, samples

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were centrifuged at 10,844 g for 10 min, and the supernatant was analyzed for VFA with gas

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chromatography (Zhang et al., 2003).

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2.7. Enumeration of bacteria

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On d 42, cecal digesta (10 g) from two birds per replicate (i.e. 10 birds per treatment) were

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aseptically transferred into 90 mL of sterile peptone containing 0.5% cysteine hydrochloride and

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serially diluted. For C. perfringens enumeration, dilutions were placed on Perfringens agar base

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(OPSP, Oxoid Inc., Nepean, Ontario, Canada) containing supplements SR 76 and SR 7 (Oxoid

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Inc.), and incubated at 38C for 48 h in jars containing gas generation kits (BBL GasPak Plus,

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Becton Dickinson, Sparks, MD). The population of Bifidobacteria in cecal samples was

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determined by using a standard laboratory method (Ibrahim and Salameh, 2001). Briefly, ileal

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samples (10 g) were diluted with 90 ml sterilized 0.1% peptone water and homogenized using

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Stomacher 400 Lab System 4 (Seward, Norfolk, UK) for 2 min, and 100 ml of appropriate

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dilution was surface plated onto modified BIM 24 agar. The level was determined at the serial

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dilution of 10-5. Plates were incubated at 37C for at least three days. Lactobacilli were

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enumerated on De Man-Rogosa-Sharpe (MRS) agar, and E. coli was counted on Mac Conkey

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(MC) agar after incubation at 37C in an anaerobic chamber for 48 h, and in an aerobic chamber

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for 24 h, respectively. All samples were plated in duplicate.

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2.8. Serum parameters

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After 4 h starvation, two birds per replicate were randomly selected and their blood samples

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were collected by syringe from the wing vein on d 40. Blood samples were collected in labelled

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sterile test tubes and centrifuged at 1,000 g for 15 min at 4C to isolate serum. After

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centrifugation, serum was collected and stored at -20C until analysis. The levels of serum

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triglyceride (mg/dl), total cholesterol (mg/dl), high density lipoprotein (HDL, mg/dl), low

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density lipoprotein (LDL, mg/dl), aspartate aminotransferase (AST, IU/L), alanine

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aminotransferase (ALT, IU/L), gamma glutamyltransferase (GGT, IU/L), creatin kinase (CK,

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U/L), total protein (TP, g/dl) and albumin (g/dl) were measured by an autoanalyzer (Selectra E

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vital scientific, Netherlands). Globulin value was obtained from the difference between serum TP

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and albumin concentrations. All tests were carried out in duplicate.

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2.9. Size of different organs

At the end of the experiment, 2 birds per replicate whose body weights were closest to the

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mean weight of the pen were selected and humanly killed by cervical dislocation, plucked, and

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eviscerated of gastrointestinal tract, giblets and other inner organs to determine the carcass

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characteristics.

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2.10. Chemical analysis

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Diets and excreta samples were analyzed for DM content (method 930.15; AOAC, 1995) and

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fat [method 920.32 (AOAC, 2000) by a 1043 Soxtec HT system, Foss Tecator AB, Hoganas,

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Sweden]. Crude protein was calculated as nitrogen 6.25. Nitrogen was determined by using a

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Kjeltec Auto 1030 Analyzer (Tecator AB, Hoganas, Sweden). Gross energy of diets and excreta

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samples was measured with a bomb calorimeter (IKA-Kalorimeter, Model C400, IKA, Staufen,

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Germany). Chromium oxide content in the experimental diets and excreta were measured

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according to Saha and Gilbreath (1991).

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2.11. Statistical analysis

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Data were analyzed as a 23 factorial arrangement (2 levels of enzyme and 3 levels of

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thymol+carvacrol) using PROC GLM of SAS (SAS Institute, 2001). Data were analysed

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considering the pen of birds as the experimental unit for performance parameters, and the

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individual chicken as the experimental unit for the rest of the parameters measured. Treatment

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means were separated using Tukeys multiple comparison tests. Statistical significance was

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declared at a probability of P < 0.05. Microbiological counts were subject to base-10 logarithm

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transformation before analysis.

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3. Results

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3.1. Chemical composition of phytogenic product and diets

Calculated and analysed carvacrol and thymol contents of Next enhance150 and diets

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(mg/kg) are shown in Table 2. The GC-MS results indicated that the 2 phenols, carvacrol and

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thymol, were the sole components of the phytogenic product (Table 2). Analysis of Next

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enhance150 by gas chromatography revealed the components to be 54.13% carvacrol and

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45.87% thymol.

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3.2. Performance

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All birds were healthy during the entire experimental period. Mortality was lower than 1.4%

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with no differences between the groups. The effect of dietary NSP-degrading enzyme and

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thymol+carvacrol supplements on growth performance traits of broilers fed wheat-based diet at

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different phases is shown in Table 3. Both E and T+C supplementation significantly (P < 0.05)

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affected ADG and G:F of broilers throughout the trial, but no significant effects were observed

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for ADFI. Addition of E and 200 mg T+C/kg of diets improved ADG by 9.9 and 11.3%,

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respectively, and G:F by 11.4 and 17.1%, respectively, at 10 d of age compared with birds fed

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the control diet. From d 11-24 and d 25-42, ADG and G:F was increased (P < 0.05) by the

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inclusion of E and T+C while ADFI was not affected. For the whole period, E and the two levels

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of T+C supplementation improved (P < 0.05) ADG by 5.5 and 5.3 and 6.2%, respectively, and

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G:F by 5.8 and 5.7 and 7.1%, respectively, of birds fed wheat-based diets.

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3.3. Intestinal digesta viscosity and pH

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The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on intestinal

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digesta viscosity and pH of broilers fed wheat-based diet at d 24 is shown in Table 4. Digesta

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viscosity was reduced (P < 0.05) after enzyme addition in all parts of small intestine and also

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after inclusion of phytogenic in jejunum and ileum. Neither E nor T+C supplementation had any

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effect on intestinal digesta pH of broilers at d 24.

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3.4. TTAR of nutrients

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The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on total

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tract apparent retention (TTAR, %) of nutrients of broilers fed wheat-based diet at d 24 is shown

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in Table 5. NSP-degrading enzyme or phytogenic treated birds showed an increased (P < 0.05)

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retention of DM, protein and gross energy. In the E supplemented group, the retention of DM,

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protein and gross energy was increased (P < 0.01) by 8.0, 9.5% and 10.7, respectively. Enzyme

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addition did not affect fat retention. Inclusion of 100 and 200 mg of T+C/kg increased (P < 0.05)

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retention of DM by 7.3 and 8.8%, protein by 6.8 and 8.6%, and gross energy by 6.9 and 8.7%,

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respectively, while fat retention was unaffected.

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3.5. VFA production

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The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on volatile

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fatty acid profile, and total VFA amounts in the cecal contents of broilers fed wheat-based diet at

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d 24 and 42 is shown in Table 6. Dietary supplementation of E and T+C increased (P < 0.01)

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total VFA and acetate levels at d 24 and 42, whereas level of butyrate decreased (P < 0.01).

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Enzyme decreased propionate at d 24 and 42 (P < 0.01), isobutyrate at d 42 (P < 0.05) and

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valerate at d 24 (P < 0.01). Phytogenic product decreased (P < 0.01) isovalerate at d 42.

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Proportion of isovalerate was not changed by E supplementation at d 24 and 42. A similar

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pattern was observed for propionate, isobutyrate and valerate by T+C.

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3.6. Intestinal bacterial numbers

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The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on cecal

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microbial population of broilers fed wheat-based diet at d 42 is shown in Table 7. E. coli and C.

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perfringens counts were lower (P < 0.01) than controls, and Lactobacilli counts higher, in birds

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fed E or T+C at the rate of 200 mg/kg. Bifidobacteria counts were not affected by E and dropped

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(P < 0.01) with increasing T+C dosages.

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3.7. Serum parameters

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The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on serum

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lipid metabolites of broilers fed wheat-based diet at d 40 is shown in Table 8. The inclusion of E

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elevated serum triglyceride (P < 0.05) and total cholesterol (P < 0.01), while there was no

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significant effect of enzyme on LDL and HDL cholesterol. Chickens fed diets supplemented with

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T+C decreased (P < 0.01) serum total cholesterol and T+C at the rate of 200 mg/kg decreased (P

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< 0.05) LDL, while no effect was found on triglyceride and HDL values. The effect of dietary

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NSP-degrading enzyme and thymol+carvacrol supplements on serum biochemical parameters of

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broilers fed wheat-based diet at d 40 is shown in Table 9. There was no significant difference

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among groups in serum levels of aspartate aminotransferase, alanine aminotransferase, gamma

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glutamyltransferase and creatin kinase. However, dietary E supplementation increased (P < 0.01)

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TP, albumin and globulin, while dietary T+C (P < 0.01) elevated TP and albumin.

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3.8. Organ weights and intestinal lengths

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The effect of dietary NSP-degrading enzyme or thymol+carvacrol supplements on relative

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weights of carcass, fat pad, liver and pancreas and relative lengths of duodenum, jejunum and

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ileum of broilers fed wheat-based diet at d 42 is shown in Table 10. Enzyme supplementation

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decreased (P < 0.01) the relative size of digestive organs of broilers. Moreover, carcass, liver and

299

pancreas relative weights decreased (P < 0.05) with E supplementation at d 42. No effects were

Page 14 of 42

15

observed with T+C dietary supplementation except for carcass relative weight and jejunum and

301

ileum relative lengths.

302

4. Discussion

303

4.1. Growth performance

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According to our results, dietary supplementation with either the phytogenic product or the

305

NSP-degrading enzyme modified the performance of broilers fed on wheat-based diets by

306

increasing ADG and G:F over the whole grower period. Wheat can be a more cost effective feed

307

ingredient compared to corn as the major cereal in broiler diets, especially during the harvest

308

season in various regions of the world. However, the use of wheat is limited due to a number of

309

nutritional disadvantages: varying nutrient contents, lower metabolisable energy than corn, and

310

the presence of soluble NSPs such as arabinoxylans and -glucans (Basmacioglu et al., 2010).

311

Pentosan solubilisation results in a viscous condition of the digesta that has been shown to

312

interfere with nutrient assimilation within the chicks' intestine (Friesen et al., 1992). It has been

313

reported that these effects result in wet droppings, increased intestinal microbiota load, depressed

314

growth and feed efficiency (Knarreborg et al., 2002).

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315

Various treatments including enzyme supplementation, antibiotic addition and the use of

316

bioactive substances have proved to be beneficial in improving the nutritive value of wheat

317

(Choc et al., 2004; Amad et al., 2011). The present study showed that the addition of an enzyme

318

complex (xylanase and -glucanase) to broiler wheat-based diets led to improved performance.

319

These positive results were probably associated with a reduction in digesta viscosity as

320

previously reported (Bedford and Apajalahti, 2001), and are in line with McCracken and Quintin

321

(2000), who reported that xylanase addition to broiler diets improved live weight gain and

322

gain:feed. The viscosity reduction in the digestive content observed in birds fed on enzyme

Page 15 of 42

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323

supplemented diets has been reported to allow a faster transit of the digesta, a greater feed intake,

324

and a facilitated contact between nutrients and digestive enzymes to improve nutrient

325

digestibility (Lzaro, 2003).

Diets supplemented with the phytogenic T+C improved broilers performance compared to

327

the control diet. Oregano and thyme oils are usually composed of the monoterpenes thymol and

328

carvacrol in varying proportions (Daferera et al., 2000). Next enhance150 is a commercial

329

supplement based on a 1:1 ratio of thymol:carvacrol, and has been shown to improve average

330

weight gain and G:F of broilers (Hashemipour et al., 2013a,b). Oregano essential oil, alone or in

331

combination with a multi-enzyme, significantly increased body weight gain during the first week

332

of life in broilers (Basmacioglu et al., 2010). These authors noted that dietary supplementation

333

with oregano essential oil (250 mg/kg diet) may possess an antioxidant effect which has been

334

associated with an effect on body weight gain at early life of broiler chicks fed wheat-based diets

335

as a nutritional stress factor. Given their antimicrobial activity (Wenk, 2000), it would be

336

expected that thymol + carvacrol could have positive effects on growth performance in broilers.

337

Cross et al. (2003) noted that thyme oil together with an enzyme mix (xylanase and glucanase) in

338

diets is likely to improve performance synergistically that this was not the case in the current

339

experiment.

340

4.2. Intestinal digesta viscosity and pH

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341

Arabinoxylans and -glucans have been shown to increase digesta viscosity (Lzaro et al.,

342

2003), which can be overcome by adding NSP-degrading enzymes (Bedford and Apajalahti,

343

2001). Enzyme supplementation significantly decreased viscosity of intestinal contents in

344

duodenum, jejunum and ileum, but did not result in significant alterations in pH values of either

345

duodenal, jejunal or ileal digesta. These results are in agreement with those reported by

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Mathlouthi et al. (2002), who showed that the pH of the intestinal contents in birds fed wheat and

347

barley-based diet was not significantly affected by the addition of xylanase and -glucanase.

348

Earlier research demonstrated that NSP-degrading enzymes were effective in both viscosity

349

reduction and degradation of the cell wall structure, which resulted in increased digestibility of

350

nitrogen, fat, starch, and NSP in the small intestine of young broiler chickens fed wheat-based

351

diets (Basmacioglu et al., 2010).

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Thymol+carvacrol supplementation significantly reduced viscosity of jejunum and ileum

353

contents, but there was no effect on the intestinal digesta pH in the current work. To some extent,

354

this was surprising given that the fermentation of carbohydrates usually leads to an increased

355

production of VFA, which tend to lower intestinal lumen pH values. However, fatty acids

356

production and absorption takes place mainly in the ceca (where it actually increased, Table 6)

357

by bacterial fermentation of undigested NSP, and very low bacterial fermentation takes place

358

within the small intestine of broilers (Svihus et al., 2012).

359

4.3. Nutrients retention

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360

Performance has a close relationship with energy metabolism in birds. Consequently, a

361

metabolism trial was conducted to determine the effects of enzyme and phytogenic inclusion on

362

total tract apparent retention of nutrients (TTAR). In our study, we found that enzyme

363

supplementation improved DM, protein, and gross energy retention by 8.0, 9.5 and 10.7%,

364

respectively. Exogenous xylanase can partially hydrolyse the arabinoxylans and release the

365

enclosed nutrients for the birds to use (Bedford and Apajalahti, 2001), where after birds can

366

digest and absorb the nutrients more easily and achieve better growth performance. The

367

improved CP retention with added xylanase in wheat based diets may be partly due to lowering

368

the endogenous amino acid losses, resulting from the elimination of adverse effects of wheat

Page 17 of 42

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pentosans (Angkanaporn et al., 1994). Xylanase did not affect fat retention determined at d24 in

370

our study, which is in agreement with the findings of Juanpere et al. (2005). This may be partly

371

due to the age of the birds and the type of fat (soybean oil) used in this study. In young chickens,

372

fat digestion increases with age and reaches optimal capacity after 2 wk of age (Nitsan et al.,

373

1991).

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The phytogenic product significantly increased the TTAR of nutrients at d 24 in the current

375

study. Kamel (2001) mentioned that there is evidence to suggest that herbs, spices, and various

376

plant extracts have appetite- and digestion-stimulating properties and antimicrobial effects.

377

Therefore, the improvement of the TTAR of nutrients in this study could be caused by the

378

stimulation effect of the phytogenic on endogenous digestive enzymes activity, and/or an

379

increased absorption surface area, which were previously reported (Hashemipour et al., 2013a,b).

380

4.4. VFA production

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It is well known that the amount and type of fermentable substrates, especially

382

carbohydrates, reaching the large intestine affects volatile fatty acids (VFA) concentration and

383

profile (Svihus et al., 2012). Volatile fatty acids are the major end products of microbial

384

fermentation. Their levels could be used indirectly to monitor gut microbe populations in broilers

385

(Taylor, 2002), and they are efficiently absorbed by the colonic mucosa. In our current study,

386

birds consuming the control diet generally had lower total VFA levels in the ceca than birds

387

consuming diets containing supplemental enzyme at d 24 and 42, which is consistent with the

388

results reported by Choct et al. (1999). The concentration of acetate in the ceca was clearly

389

higher than the concentration of the other acids. When supplemental enzyme was added to the

390

wheat based control diet, it might partially degrade these larger molecular polysaccharides into

391

smaller ones, even oligosaccharides, and at the same time the enzymes might alleviate the

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392

viscous property of the digesta (Mathlouthi et al., 2002), increase the digesta flow rate, thus

393

stimulating the production of VFA and the growth of specific beneficial bacteria within the ceca

394

(Choct et al., 1999).

At d 24 and 42, the concentration of butyric acid was lower in the cecal contents of the

396

phytogenic-treated chicks, which may be related with the observed effect (Table 7) in the counts

397

of C. perfringens in the chicken ceca (Elwinger et al., 1992). Sakata (1987) demonstrated in rats

398

that intraluminally infused VFA accelerated the crypt cell production rate and increased the gut-

399

wall mass. The stimulation was most efficient with butyrate. The positive effect of dietary

400

antibacterials appears to be related at least in part with the elimination of fermentative

401

microorganisms, mainly butyric acid producers (especially Clostridia), from the small intestine

402

(Choct et al., 1999). This effect has been shown to decrease the gut-wall mass and stimulate

403

nutrient absorption (Parker and Armstrong, 1987), which supports the improved nutrient

404

retention found in the present study.

405

4.5. Intestinal bacterial numbers

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406

High intestinal viscosity reduces nutrient absorption by the host animal, decreases the rate of

407

feed passage, and may enhance mucus production (Piel et al., 2005), which could lead to

408

increased numbers of anaerobic bacteria in the small intestine, particularly C. perfringens

409

(Collier et al., 2003). Bedford and Apajalahti (2001) demonstrated that in birds fed wheat-based

410

diets, the addition of a xylanase based enzyme preparation resulted in a 60% reduction in

411

bacterial numbers. In accordance with these findings, the present investigation resulted in lower

412

counts of E. coli and C. perfringens, and higher counts of Lactobacilli, in birds fed the NSP-

413

degrading enzyme. In the process of depolymerizing various polysaccharides in the diet,

414

exogenous enzymes may produce galacto-, gluco-, manno-, or xylo-oligomers, which are similar

Page 19 of 42

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to prebiotics and which may facilitate the proliferation of health-promoting bacteria such as

416

Bifidobacteria and Lactobacilli (Monsan and Paul, 1995). The inclusion of thymol+carvacrol in

417

the diets improved the microbial counts in birds compared to those fed the control diet. In line

418

with the present results, the dietary supplementation with an encapsulated product containing

419

capsaicin, carvacrol and cinnamaldehyde, reduced the numbers of E. coli and C. perfringens in

420

broiler rectal contents to the same extent as avilamycin (Jamroz et al., 2003). The mechanism of

421

action of thymol and carvacrol is probably linked to their effect of bacterial membrane integrity

422

disruption, which further affects pH homeostasis and equilibrium of inorganic ions (Lambert et

423

al., 2001).

424

4.6. Serum parameters

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The inclusion of E increased serum triglyceride, total cholesterol, total protein (TP), albumin

426

and globulin concentrations, while T+C decreased total cholesterol, TP and albumin at d 40.

427

Similar results were observed by Hajati et al. (2009) who reported that dietary multi-enzyme

428

inclusion increased the blood concentrations of total cholesterol, HDL-cholesterol and

429

triglyceride. The addition of the multi-enzyme may alleviate the limitations for the function of

430

bile salts and their emulsifying properties in intestinal chyme due to undigested NSP, which may

431

result in increased total fat in blood (Hajati et al., 2009).

Ac
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432

Case et al. (1995) indicated that thymol decreased the serum cholesterol concentration in

433

leghorn chickens fed a corn based diet, due to a hypocholesterolaemic effect of thymol, in

434

contrast with Lee et al. (2003) who reported no hypocholesterolaemic activity of dietary

435

carvacrol and thymol. The hypocholesterolemic effect of thymol and carvacrol has been ascribed

436

to the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Elson,

437

1995), the rate controlling enzyme of the cholesterol synthetic pathway. The absence or presence

Page 20 of 42

21

438

of cholesterolaemic effects of dietary components in an animal is also dependent on factors such

439

as breed, gender and age, and also on the composition of the feed (Lee et al., 2003).
The liver plays an important role in metabolic processes, and the metabolic activity of the

441

liver is important for the normal functioning of cellular events (Cornellus, 1980). Serum AST

442

and ALT are indicators of normal liver functioning. In the present study, there was no significant

443

alteration in the serum levels of AST, ALT and CK, and so no evidence of hepatocyte and

444

muscle injury was determined. Also, no significant difference in serum GGT concentration was

445

observed among treatment groups, suggesting that cholestasis and duct hyperplasia (Tennant,

446

1997) did not occur in this experiment. In contrast, Traesel et al. (2011) suggested a dose-

447

dependent effect of essential oil on serum concentration of AST in which the increase in serum

448

levels of AST is caused by hepatocyte injury.

449

4.7. Organ weights and intestinal lengths

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Enzyme supplementation decreased the relative size of the digestive organs. Moreover,

451

carcass, liver and pancreas relative weights decreased with E supplementation at d 42. No effects

452

on organ weights and intestinal lengths were observed with T+C dietary supplementation except

453

for carcass relative weight and jejunum and ileum relative lengths. The presence of viscous

454

grains such as wheat in diets can increase the viscosity of the digesta and inhibit the effective

455

contact between the digestive enzymes and their corresponding substrates, thereby leading to

456

significant modifications of the structure and function of intestine and organs (Dworkin et al.,

457

1976). Sarica et al. (2005) noted that thyme oil and xylanase-based enzyme complex

458

significantly decreased small intestine weight or ileum length when these feed supplements were

459

used together in wheat-based diets. When supplementing exogenous enzymes in the wheat

460

control diet, a greater proportion of NSP may be hydrolyzed, which might attenuate the secretory

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Page 21 of 42

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461

function of the responding organs and GIT segments, and then the organ sizes may decrease. The

462

reduction in digestive organs relative weight has direct economic implications, as the dressing

463

yield of broilers increased proportionally.

In conclusion, the present study showed that the phytogenic product thymol + carvacol, and

465

NSP-degrading enzyme independently improved growth performance, enhanced nutrients

466

retention, increased total VFA, reduced cholesterol and modulated intestinal microbial counts in

467

broilers fed on a wheat-based diet. These results have both productive and health implications for

468

the broiler industry and warrant further investigation.

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Acknowledgments

471

The authors are grateful to Dr. Khaksar for providing the experimental facilities for this work.

470

472

References

474

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475

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Almirall, M., Francesch, M., Perez-Vendrell, A.M., Brufau, J., Esteve-Garcia, E., 1995. The

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an

601

Sarica, S., Ciftci, A., Demir, E., Kilinc, K., Yildirim, Y., 2005. Use of an antibiotic growth

605

promoter and two herbal natural feed additives with and without exogenous enzymes in

606

wheat based broiler diets. South Afri. J. Anim. Sci. 35, 61-72.

604

SAS Institute. 2001. SAS user's guide. Release 8.2 Ed. SAS Institute Inc., Cary, Nc.

608

Scott, M.L., Nesheim, M.C., and Young, R.J., 1976. Nutrition of the Chicken. M. L. Scott and

Ac
ce
pt
e

607

Associates, Ithaca, NY.

609
610

Svihus, B., Choct, M., Classen, H.L., 2012. Function and nutritional roles of the avian caeca: a
review. Worlds Poult. Sci. J. 69, 249-263.

611
612

Taylor, M., 2002. Hindgut function in laying hens. Publication 02/043. Rural Industries Research
and Development Corporation, Newcastle, Australia.

613
614

Tennant, B.C., 1997. Hepatic function. In: Kaneko JJ, Harvey JW, Bruss ML (eds) Clinical
biochemistry of domestic animals, 5th edn. Academic Press, London, pp 327-352.

615

Page 28 of 42

29

Traesel, C.K., Schmidt, C., Silva, C.B., Paim, F.C., Wolkmer, P., Rosa, A.P., Alves, S.H.,

617

Santurio, J.M., Lopes, S.T.A., 2011. Serum biochemical profile and performance of broiler

618

chickens fed diets containing essential oils and pepper. Comp. Clin. Pathol. 20, 453-460.

619

Wenk, C., 2000. Recent advances in animal feed additives such as metabolic modifiers,

620

antimicrobial agents, probiotics, and enzymes and highly available minerals. Review. Asian-

621

Aus. J. Anim. Sci. 13, 86-95.

growth performance of broilers. Anim. Feed Sci. Technol. 70, 353-361.

623

Zhang, W.F., Li, D.F., Lu, W.Q., Yi, G.F., 2003. Effects of isomalto-oligosaccharides on broiler

an

624

cr

Yu, B., Hsu, J.C., Chiou, P.W.S., 1997. Effects of -glucanase supplementation of barley diets in

us

622

ip
t

616

performance and intestinal microflora. Poult. Sci. 82, 657-663.

625

Ac
ce
pt
e

626

Page 29 of 42

30

Finisher
(25-42 d)
614.7
290.5
1.0
61.4
12.0
10.0
3.4
0.8
1.1
0.1
2.5
2.5

ip
t

Table 1
Composition and calculated analysis (g/kg as fed) of the basal diet.
Starter
Grower
Ingredients (g/kg)
(1-10 d)
(11-24 d)
Wheat
574.7
600.0
Soybean meal, 440 g/kg CP
341.2
308.7
Wheat bran
1.0
1.0
Vegetable oil
40.0
56.0
Limestone
14.5
12.5
Dicalcium phosphate
13.5
11.0
Salt
3.7
3.6
HCL-Lys
3.3
0.2
DL-Met
1.9
1.5
Thr
1.2
0.5
2.5
2.5
Vitamin permix1
2.5
2.5
Mineral permix2

us

cr

626
627

628
629
630
631
632
633
634

Ac
ce
pt
e

an

Calculated chemical composition

ME (MJ/kg diet)
11.97
12.47
12.68
CP
221
208
200
Ca
10.0
8.5
8.0
Available P
4.7
4.2
3.9
Sodium
1.8
1.8
1.7
Lys
13.5
11.7
10.3
Met
4.8
4.2
3.9
Met + Cys
10.1
9.0
8.1
Thr
8.9
7.8
7.0
1
Vitamin premix provided the following per kilogram of diet: vitamin A (trans-retinyl acetate),
10,000 IU; vitamin D3 (cholecalciferol), 3500 IU; vitamin E (DL--tocopheryl acetate), 60 mg;
vitamin K (menadione), 3 mg; thiamine, 3 mg; riboflavin, 6 mg; pyridoxine, 5 mg; vitamin B12
(cyanocobalamin), 0.01 mg; niacin, 45 mg; pantothenic acid (D-calcium pantothenate), 11 mg;
folic acid, 1 mg; biotin, 0.15 mg; choline chloride, 500 mg; ethoxyquin (antioxidant), 150 mg.
2
Mineral permix provided the following per kilogram of diet: Fe, 60 mg; Mn, 100 mg; Zn, 60
mg;
Cu,
10
mg;
I,
1
mg;
Co,
0.2
mg;
Se,
0.15
mg.

Page 30 of 42

31

635
636 Table 2
637 Calculated and analyzed carvacrol and thymol contents of the experimental diets (mg/kg).

us

an
M
d
Ac
ce
pt
e

638
639
640
641

cr

ip
t

Calculated
Analyzed
Experimental diets1
Carvacrol
Thymol
Carvacrol
Thymol
Control
E
T+C100
54.13
45.87
51.55
43.23
T+C200
108.26
91.74
106.23
88.65
E plus T+C100
54.13
45.87
53.81
44.27
E plus T+C200
108.26
91.74
104.24
89.21
1
Control, wheat-based diet contained neither thymol+carvacrol (T+C) nor enzyme (E). E, 0.5
g/kg of enzyme Endofeed W; T+C100, 100 mg/kg of Next enhance150; T+C200, 200 mg/kg of
Next enhance150; E plus T+C100, 0.5 g/kg of enzyme and 100 mg/kg of Next enhance150; E
plus T+C200, 0.5 g/kg of enzyme and 200 mg/kg of Next enhance150.

Page 31 of 42

32

Ac
ce
pt
e

an

us

cr

ip
t

642

Page 32 of 42

cr
an

Table 3
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on growth performance traits of broilers fed
wheat-based diet at different phases.
0 to10 d
11 to 24 d
25 to 42 d
0 to 42 d
G:F1
ADG1 ADFI1 G:F1
ADG1 ADFI1 G:F1
ADG1 ADFI1
G:F1
ADG1 ADFI1
(g)
Treatment
(g)
(g)
(g/kg)
(g)
(g)
(g/kg)
(g)
(g)
(g/kg)
(g)
(g/kg)
E, g/kg
0
24.3b
30.0
810.8b
62.1b
92.6
662.9b
98.9b 191.2 517.2b
68.6b 120.3 572.2b
29.8
902.9a
63.9a
93.2
685.9a
104.4a 190.2 549.0a
72.4a 119.7 605.2a
0.5
26.7a
SEM
0.48
0.55
19.34
0.57
0.64
7.21
0.91
0.31
4.89
0.60
0.33
5.04
784.7b
866.5ab
919.3a
23.69

60.3b
64.0a
64.6a
0.70

94.0
93.1
93.1
0.78

641.4b
688.4a
693.4a
8.83

98.6b
102.9a
103.6a
1.11

191.1
190.9
190.2
0.38

P-value
E
**
NS
**
*
NS
*
**
NS
T+C
**
NS
**
**
NS
**
*
NS
E T+C
NS
NS
NS
NS
NS
NS
NS
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01.
a,b
Means within the same column with no common superscripts differ significantly (P < 0.05).
1
ADG: average daily gain; ADFI: average daily feed intake; G:F: gain to feed ratio.

Ac

646
647
648

30.5
30.1
29.2
0.68

pt

23.9b
26.0ab
26.6a
0.59

ce

T+C, mg/kg
0
100
200
SEM

ed

643
644
645

33

us

515.8b
539.0a
544.4a
5.98

68.0b
71.6a
72.2a
0.74

120.5
120.0
119.5
0.40

564.5b
597.0a
604.6a
6.18

**
**
NS

**
**
NS

NS
NS
NS

**
**
NS

Page 33 of 42

cr

34

us

Ac

ce

pt

ed

an

649

Page 34 of 42

35

2.42
2.13
1.76
0.22

2.97a
2.38b
2.06b
0.12

3.76a
3.08b
2.86b
0.12

**
**
NS

**
**
NS

cr

T+C, mg/kg
0
100
200
SEM

ip
t

Table 4
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on
intestinal digesta viscosity and pH of broilers fed wheat-based diet at d 24.
Viscosity (cPs)
pH
Treatment
duodenum jejunum ileum
duodenum jejunum
ileum
E, g/kg
0
2.43a
3.04a
4.24a
6.00
6.23
6.30
b
b
1.91
2.11b
5.96
6.31
6.35
0.5
1.77
SEM
0.18
0.10
0.10
0.12
0.06
0.10
6.01
5.96
5.98
0.14

6.31
6.23
6.28
0.07

6.21
6.36
6.39
0.12

NS
NS
NS

NS
NS
NS

NS
NS
NS

us

650
651
652

different superscripts differ significantly (P < 0.05).

Ac
ce
pt
e

653
654

an

P-value
E
*
T+C
NS
E T+C
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01.
a,b
Means within the same column with

Page 35 of 42

36

63.96b
69.13a
69.61a
0.65

56.05b
59.87a
60.88a
0.72

cr

T+C, mg/kg
0
100
200
SEM

ip
t

Table 5
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on total
tract apparent retention (%) of nutrients of broilers fed wheat-based diet at d 24.
Treatment
DM
Protein
Fat
Gross energy
E, g/kg
0
64.98b
56.26b
78.07
65.14b
a
a
61.61
77.37
72.09a
0.5
70.16
SEM
0.53
0.59
1.44
0.84
79.19
78.15
75.81
1.77

us

655
656
657
658

65.23b
69.74a
70.88a
1.03

P-value

NS
NS
NS

**
*
NS

an

**
**
NS

superscripts differ significantly (P < 0.05).

Ac
ce
pt
e

659
660

E
**
T+C
**
E T+C
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01.
a,b
Means within the same column with different

Page 36 of 42

37

Table 6
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on
volatile fatty acid profile (VFA, %), and total VFA amounts (mmol/L) in the cecal contents of
broilers fed wheat-based diet at d 24 and 42.
Individual VFA
Total
VFA
Treatment
Acetate Propionate Butyrate Isobutyrate Isovalerate Valerate
d 24
E, g/kg
0.91a
17.47a
3.05
0.59
4.95a
14.75b
0
73.02b
0.5
78.32a
0.39b
11.27b
4.83
0.49
4.69b
17.33a
SEM
0.75
0.11
0.50
0.60
0.12
0.06
0.18

T+C, mg/kg
0
100
200
SEM

666
667

**
**
NS

**
NS
NS

**
**
NS

4.61
3.24
3.99
0.74

0.82
0.36
0.43
0.15

4.74
4.90
4.81
0.08

14.76b
16.60a
16.78a
0.22

P-value
NS
NS
NS

NS
NS
NS

**
NS
NS

**
**
NS

an

16.78a
14.06b
12.27b
0.62

Ac
ce
pt
e

E, g/kg
0
0.5
SEM

0.55
0.75
0.66
0.14

E
T+C
E T+C

72.49b
76.69a
77.83a
0.92

T+C, mg/kg
0
100
200
SEM

us

cr

ip
t

661
662
663
664
665

d 42

74.07b
79.36a
0.73

8.62a
6.46b
0.44

11.94a
7.25b
0.38

0.91a
0.71b
0.05

0.64
0.75
0.08

3.81
5.46
0.66

48.36b
55.08a
0.46

73.54b
77.79a
78.89a
0.90

7.21
7.76
7.65
0.54

11.42a
9.35b
8.00b
0.47

0.77
0.85
0.81
0.07

1.11a
0.50b
0.48b
0.10

5.93
4.12
3.84
0.81

48.39b
53.16a
53.62a
0.56

**
**
NS

P-value
*
NS
NS

NS
**
NS

NS
NS
NS

**
**
NS

E
**
**
T+C
**
NS
E T+C
NS
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01.
a,b
Means within the same column with

different superscripts differ significantly (P < 0.05).

Page 37 of 42

38

Table 7
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on cecal
microbial population (log CFU/g of digesta) of broilers fed wheat-based diet at d 42.
Lactobacilli
Bifidobacteria
C. perfringens
E. coli
Treatment
E, g/kg
0
7.60b
6.47
2.55a
6.29a
a
b
6.44
2.29
5.90b
0.5
7.84
SEM
0.03
0.07
0.05
0.07
7.62b
7.73a
7.80a
0.03

6.92a
6.52b
5.93c
0.09

2.66a
2.51a
2.10b
0.06

us

T+C, mg/kg
0
100
200
SEM

cr

ip
t

668
669
670
671

6.46a
6.22a
5.60b
0.09

d
Ac
ce
pt
e

672
673

an

P-value
**
E
**
NS
**
T+C
**
**
**
**
E T+C
NS
NS
NS
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01.
a,b,c
Means within the same column with different superscripts differ significantly (P < 0.05).

Page 38 of 42

39

Table 8
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on serum
lipid metabolites of broilers fed wheat-based diet at d 40.
Triglyceride
Cholesterol
HDL1
LDL2
Treatment
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
E, g/kg
0
71.6b
118b
82.9
32.8
a
a
120
82.9
34.3
0.5
80.4
SEM
2.76
0.40
0.78
0.85
121a
119b
117b
0.49

77.1
75.3
75.5
3.39

82.2
83.2
83.3
0.95

us

T+C, mg/kg
0
100
200
SEM

cr

ip
t

674
675
676
677

36.1a
33.2ab
31.5b
1.04

Ac
ce
pt
e

678
679
680
681

an

P-value
E
*
**
NS
NS
T+C
NS
**
NS
*
E T+C
NS
NS
NS
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01.
a,b
Means within the same column with different superscripts differ significantly (P < 0.05).
1
HDL = High density lipoprotein.
2
LDL
=
Low
density
lipoprotein.

Page 39 of 42

40

Table 9
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on serum
biochemical parameters of broilers fed wheat-based diet at d 40.
AST1
ALT2
GGT3
CK4
TP5
Albumin Globulin
Treatment
(IU/L)
(IU/L)
(IU/L)
(U/L)
(g/dl)
(g/dl)
(g/dl)
E, g/kg
0
134
19.9
9.95
3223
3.89b
1.67b
2.22b
a
a
1.88
2.65a
0.5
132
20.0
9.81
3242
4.53
SEM
4.76
0.57
0.29
145.91
0.10
0.07
0.07
132.7
133.3
132.8
5.83

19.6
20.2
19.9
0.70

9.9
9.5
10.3
0.35

3037
3274
3386
178.7

3.83b
4.34a
4.45a
0.12

us

T+C, mg/kg
0
100
200
SEM

cr

ip
t

682
683
684
685

1.58b
1.87a
1.89a
0.09

2.25
2.46
2.56
0.09

Ac
ce
pt
e

686
687
688
689
690
691
692

an

P-value
E
NS
NS
NS
NS
**
*
**
T+C
NS
NS
NS
NS
**
*
NS
E T+C
NS
NS
NS
NS
NS
NS
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01.
a,b
Means within the same column with different superscripts differ significantly (P < 0.05).
1
AST = Aspartate aminotransferase.
2
ALT = Alanine aminotransferase.
3
GGT = Gamma glutamyltransferase.
4
CK = Creatin kinase.
5
TP
=
Total
protein.

Page 40 of 42

41

Table 10
Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on
relative weights of carcass, fat pad, liver and pancreas (g/100 g of BW) and relative lengths of
duodenum, jejunum and ileum (cm/100 g of BW) of broilers fed wheat-based diet at d 42.
Relative weight
Relative length1
Treatment
Carcass Fat pad
Liver Pancreas
Duodenum Jejunum Ileum
E, g/kg
0
61.8b
1.22
2.33a
0.30a
1.56a
2.70a
2.78a
0.5
64.7a
1.29
2.07b
0.26b
1.29b
2.54b
2.55b
SEM
0.82
0.04
0.08
0.01
0.06
0.03
0.04
1.24
1.23
1.30
0.05

2.12
2.22
2.27
0.10

0.28
0.28
0.28
0.01

1.38
1.45
1.44
0.07

us

60.6b
64.5a
64.7a
1.01

2.77a
2.51b
2.57b
0.04

2.83a
2.60b
2.56b
0.05

an

T+C, mg/kg
0
100
200
SEM

cr

ip
t

693
694
695
696
697

P-value
**
NS
NS

Ac
ce
pt
e

698
699
700
701
702
703
704
705
706

**
E
*
NS
*
**
**
T+C
**
NS
NS
NS
**
**
E T+C
NS
NS
NS
NS
NS
NS
NS: P > 0.05, *: P < 0.05, **: P < 0.01
a,b
Means within the same column with different superscripts differ significantly (P < 0.05).
1
The small intestine was divided into three segments: the duodenum (from gizzard to pancreobiliary ducts), the jejunum (from pancreo-biliary ducts to Meckels diverticulum) and the ileum
(from Meckels diverticulum to ileo-caecal junction).

We test the potential of thymol plus carvacrol in broiler diet.

Broilers were fed on wheat-based diets with or without NSP-degrading enzyme.

707

Cecal populations of E. coli and C. perfringens were modulated by two additives.

708

Dietary thymol + carvacol enhanced oxidative status of broilers.

709

Thymol + carvacol, and enzyme independently improved growth performance.

710
711

712

713

Page 41 of 42

42

715

Ac
ce
pt
e

an

us

cr

ip
t

714

Page 42 of 42