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Title: Effect of feed supplementation with a thymol pluscarvacrol mixture, in combination or not with anNSP-degrading enzyme, on productive and physiologicalparameters of broilers fed on wheat-based diets
Author: H. Hashemipour V. Khaksar L.A. Rubio T. VeldkampM.M. van Krimpen
PII: S0377-8401(15)30033-XDOI: http://dx.doi.org/doi:10.1016/j.anifeedsci.2015.09.023Reference: ANIFEE 13386
To appear in: Animal Feed Science and Technology
Received date: 18-3-2015Revised date: 24-9-2015Accepted date: 25-9-2015
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 physiologicalparameters of broilers fed on wheat-based diets, Animal Feed Science and Technology(2015), http://dx.doi.org/10.1016/j.anifeedsci.2015.09.023
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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Effect of feed supplementation with a thymol plus carvacrol mixture, in combination or not 3
with an NSP-degrading enzyme, on productive and physiological parameters of broilers fed 4
on wheat-based diets 5
6
H. Hashemipoura,*, V. Khaksara, L. A. Rubiob, T. Veldkampc, and M. M. van Krimpenc 7
8
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aExcellence Centre for Animal Sciences and Department of Animal Science, Faculty of 10
Agriculture, Ferdowsi University of Mashhad, P.O. Box 91775-1163, Mashhad, Iran; 11
bFisiología y Bioquímica de la Nutrición Animal (EEZ, CSIC), Profesor Albareda, 1, 18008 12
Granada, Spain 13
cWageningen UR Livestock Research, P.O. Box 338, 6700 AH Wageningen, The Netherlands 14
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*Corresponding author: Tel. and Fax: +98 513 879 6845.
E-mail address: [email protected] (H. Hashemipour).
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ABSTRACT 17
The current study was conducted to evaluate the effect of feed supplementation with a 18
phytogenic product (equal mixture of thymol plus carvacrol; T+C) on performance, nutrient 19
retention, volatile fatty acid (VFA) profiles, cecum microbial ecosystem, serum parameters and 20
characteristics of gastrointestinal tract of broilers fed on wheat-based diets with or without an 21
NSP-degrading enzyme product (xylanase plus β-glucanase; E) from d 0 to 42. Six dietary 22
treatments were arranged according to a factorial design with three levels of T+C (0, 100 and 23
200 mg/kg of diet) and two levels of E (0 and 0.5 g/kg of diet). Each treatment was replicated 24
five times with 12 chicks per replicate. There was no interaction effect between E and T+C on 25
any of the measured parameters. Compared with the control group, birds fed diets containing E 26
or T+C had a higher (P < 0.01) average daily gain and feed efficiency at d 42. Digesta viscosity 27
was reduced (P < 0.05) in treatments with E addition in all parts of the small intestine. In 28
treatments with T+C inclusion digesta viscosity was reduced in jejunum and ileum at d 24. E or 29
T+C treated birds showed an increased (P < 0.05) retention of DM, protein and gross energy. 30
Dietary supplementation with E and T+C increased (P < 0.01) total VFA and acetate levels at d 31
24 and 42, whereas the level of butyrate decreased (P < 0.01). E. coli and C. perfringens counts 32
were lower (P < 0.01) than controls, and Lactobacilli counts were higher (P < 0.01), in birds fed 33
on diets supplemented with enzyme or T+C at the rate of 200 mg/kg. E supplementation 34
increased (P < 0.05) serum triglyceride, total cholesterol, total protein (TP), albumin and 35
globulin concentrations, while T+C supplementation decreased (P < 0.05) total cholesterol, TP 36
and albumin at d 40. E supplementation decreased (P < 0.01) the relative length of duodenum, 37
jejunum and ileum of broilers. Moreover, carcass, liver and pancreas relative weights decreased 38
(P < 0.05) with E supplementation at d 42. T+C dietary supplementation only affected carcass 39
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relative weight and jejunum and ileum relative lengths. The present study showed that thymol + 40
carvacol, in combination or not with an NSP-degrading enzyme, improved growth performance, 41
enhanced nutrients retention, increased total VFA, reduced cholesterol and modulated intestinal 42
microbial counts in broilers fed on a wheat-based diet. 43
Keywords: wheat, enzyme, thymol, carvacrol, broiler 44
Abbreviations: ADFI, average dairy feed intake; ADG, average dairy gain; AGP, antibiotic 45
growth promoter; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatin 46
kinase; CP, crude protein; DM, dry matter; E, enzyme; EO, essential oil; GGT, gamma 47
glutamyltransferase; HDL, high density lipoprotein; LDL, low density lipoprotein; NSP, non-48
starch polysaccharide; PFA, phytogenic feed additive; T+C, thymol+carvacrol; TP, total protein; 49
TTAR, total tract apparent retention; VFA, volatile fatty acid. 50
51
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1. Introduction 51
Wheat is an important ingredient in broiler diets as energy source, and is often the only cereal 52
in grower and finisher diets. However, compared with corn, wheat contains higher amounts of 53
anti-nutritional factors consisting mainly of water soluble and insoluble non-starch 54
polysaccharides (NSP). Soluble NSPs have been shown to detrimentally increase digesta 55
viscosity (Lazaro et al., 2003), stimulate the growth of some pathogenic bacteria species, 56
including Escherichia coli and Clostridium perfringens (Collier et al., 2003), and adversely affect 57
villus height, width, surface area, and shape (Mathlouthi et al., 2002). Compelling evidence 58
indicates that broilers fed diets based on wheat, barley, or rye suffer from reduced crude protein 59
(CP) and fat digestibility, and a reduced apparent metabolizable energy content (Mathlouthi et 60
al., 2002), which resulted in depressed body weight gain and feed conversion ratio (Lazaro et al., 61
2003). The benefits of exogenous enzyme supplementation to NSP-rich diets are well 62
documented. These enzymes can partially hydrolyze NSP, reduce the viscosity of gut contents, 63
and result in improvements in nutrient digestion and absorption (Almirall et al., 1995; Yu et al., 64
1997). Several studies have also demonstrated improvements of nutritive value, feed utilization, 65
body weight gain, composition and activity of intestinal microbiota, and reduction in excreta 66
volume after supplementation of wheat-based diets with NSP-degrading enzymes such as 67
cellulases, pectinases, hemicellulases, arabinoxylanases and β-glucanases (Bedford and 68
Apajalahti, 2001). 69
Increasing insight into the potentially beneficial activities of the gastrointestinal microbiota, 70
together with increasing public concern about antibiotic resistance and residues in animal 71
products, have resulted in the search for alternatives to antibiotic growth promoters (AGP) such 72
as prebiotics, probiotics, phytogenics and other feed additives. Phytogenic feed additives (PFA) 73
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may positively affect poultry health and productivity. Hashemipour et al. (2013a) indicated that 74
the use of PFA-containing compounds, such as essential oils (EO) or spices, stimulate digestive 75
enzyme production and activity, and induce a higher secretion of bile acids. The antimicrobial 76
properties of EO have encouraged their use as a natural replacement for AGP in animal feeds. 77
Positive effects of PFA were observed on daily weight gain and feed conversion ratio of 78
chickens when fed a diet supplemented with a mixture containing capsaicin, cinnamaldehyde and 79
carvacrol (Jamroz and Kamel, 2002). Jamroz et al. (2005) showed that the addition of a plant 80
extract to broiler diets had no influence on apparent ileal digestibility of nutrients, but on the 81
other hand, Hernandez et al. (2004) reported that essential oils and a Labiatae extract added to a 82
starter diet increased ileal DM and starch digestibility, but not CP digestibility. Furthermore, 83
active plant compounds are the potential effectors on microbial communities (Hashemipour et 84
al., 2013b), and could therefore be considered as the alternatives in controlling the intestinal 85
microbial population. Thymol and carvacrol, the main bioactive components in thyme essential 86
oil, are appetite- and digestion-stimulating, and have considerable antimicrobial and antifungal 87
activity, which have been reported to promote the growth of beneficial bacteria and inhibits the 88
growth of potentially deleterious intestinal bacteria (Akyurek and Yel, 2011). Given their 89
antimicrobial activity, it would be expected (Wenk, 2000) that thymol and carvacrol could have 90
positive effects on growth and performance in broilers. The two compounds have the status of 91
generally recognized as safe (GRAS), which is endorsed by the Flavor and Extract 92
Manufacturers’ Association (FEMA) and the Food and Drug Administration (FDA) of the 93
U.S.A. (Furia and Bellanca, 1975). 94
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Accordingly, the present study was conducted to determine the effect of feed 95
supplementation with a thymol plus carvacrol mixture, in combination or not with an NSP-96
degrading enzyme, on productive and physiological parameters of broilers fed wheat-based diets. 97
98
2. Materials and methods 99
2.1. Birds, Housing, and Diets 100
All experimental procedures were approved by the Animal Welfare Committee of the 101
Department of Animal Science, Ferdowsi University of Mashhad, Iran. A total of 360 day-old 102
Ross-308 male broiler chicks were obtained from a local hatchery and distributed in 30 groups of 103
12 birds each. Six treatments were arranged according to a factorial desgin with 3 levels (0, 100 104
and 200 mg/kg) of thymol plus carvacrol (T+C) (Next enhance150®, 1:1 thymol:carvacrol; 105
Novus International, Inc., St. Louis, MO) and 2 levels (0 and 0.5 g/kg diet) of the enzyme 106
product (E) (Endofeed W, GNC Bioferm Inc., Saskatoon, Saskatchewan, Canada). According to 107
the manufacturer, Next enhance150® contains 50% of the active components thymol and 108
carvacrol, and enzyme Endofeed W holds 2250 and 700 fungal arabinoxylanase and β-glucanase 109
units/g of enzyme activity, respectively. 110
Each diet was randomly fed to five groups of chicks. The feeding regimen consisted of a 111
starter (1-10 d), grower (11-24 d), and finisher (25-42 d) diet. The basal diet was formulated to 112
meet the nutrient requirements according to Ross-308 rearing guideline (Aviagen, 2007). Mash 113
feed and water were provided ad libitum throughout the experiment. The ingredients and 114
chemical composition of the basal diets are shown in Table 1. Next enhance150® and Endofeed 115
W were added to 100 g of wheat bran and were subsequently blended with premix. Finally, the 116
premix was mixed with the basal diet. Feed was prepared weekly and stored in airtight 117
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containers. Birds received a continuous lighting regimen during the first week and 23 h light per 118
day afterward. Broiler chickens were kept at 32°C during the first day of age. Thereafter, 119
temperature was gradually decreased by 0.5°C per day until 21°C was reached. After that, they 120
were maintained at approximately 21°C until the end of the experiment. 121
2.2. Bioactive components analysis 122
Samples (4 g) of grinded T+C supplemented diets were weighed into a centrifuge tube, 123
mixed with distilled water (2.5 mL) and ethanol (1 mL), and allowed to stand for 15 min. Diethyl 124
ether (12 mL) was then added, and the samples were shaken for 16 h and centrifuged at 15,000 × 125
g for 5 min at 4°C. The calibration samples were prepared with control feed which was 126
supplemented with standard solutions of carvacrol and thymol at 5 different concentrations (5, 127
10, 20, 40, and 100 mg/L in ethanol). Feed supplemented with ethanol free of T+C was used as a 128
blank. 129
Gas chromatographic analyses were performed using a GC PU 4500 system (Shimadzu 130
Corp., Kyoto, Japan) equipped with a flame ionization detector and an E30 (30 m × 0.32 mm ID, 131
5% phenyl methyl silicone, phase thickness 0.5 mm) capillary column. The column temperature 132
ranged from 80 to 202°C with increments of 8°C per minute. Helium was used as the carrier gas 133
at a flow rate of 1.5 mL/min. Sample injection was carried out in splitless mode at 200°C with 134
splitless time of 1 min with a sample injection volume of 0.5 μL. Temperature of the detector 135
was 202°C. Oven temperature was maintained initially at 80°C for 2 min, then raised at a rate of 136
8°C/min to 125°C, maintained for 10 min, then raised at a rate of 25°C/min to 200°C, and 137
maintained for 10 min. The 5 concentration linear calibration curves were calculated by using 138
internal standards (Carvacrol). Using the peak areas, the concentrations (mg/kg) of the analysts 139
in the samples were calculated from the calibration curves. 140
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2.3. Performance 141
The bird experimental period lasted for 42 d. Feed consumption and body weight were 142
measured on a pen basis on d 0, 10, 24, and 42. Average daily feed intake (ADFI), average daily 143
gain (ADG), and feed efficiency (G:F) were calculated for each period (d 0-10, d 11-24, d 25-42, 144
and d 0-42). The chickens were inspected daily and dead birds were removed following 145
registration of date and BW. When calculating feed efficiency, the BW of dead birds was also 146
taken into account. 147
2.4. Sampling procedures for intestinal digesta viscosity and pH 148
On d 24, two birds per replicate (i.e. 10 birds per treatment) were randomly selected and 149
euthanized using sodium thiopental. The small intestine was removed, the digesta contents of the 150
duodenum, jejunum, and ileum was immediately collected, and samples from the two birds were 151
pooled and placed into clean tubes. The small intestine was divided into three segments: 152
duodenum (from gizzard to pancreo-biliary ducts), jejunum (from pancreo-biliary ducts to 153
Meckel’s diverticulum), and ileum (from Meckel’s diverticulum to ileo-caecal junction). The 154
samples were mixed with deionized water (1:10 wt/vol), and used to measure the pH of each 155
segment of the gastrointestinal tract in duplicate by using a digital pH meter (Model 827, 156
Metrohm, Herisau, Switzerland). 157
Other samples taken from duodenum, jejunum and ileum of the 2 birds/pen were pooled and 158
mixed to achieve a homogenous mixture, which was then centrifuged at 9,000 × g at room 159
temperature (4°C) for 10 min. The supernatant was withdrawn, and viscosity was determined at 160
40°C using a Brookfield digital viscometer model DV- III as described by Bedford and Classen 161
(1993). 162
2.5. Total tract apparent retention of nutrients 163
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For determination of total tract apparent retention (TTAR) of nutrients, 2 birds per replicate 164
were moved to metabolism cages (2 birds in each) with a wire mesh bottom and excreta 165
collection trays (60 × 30 × 30 cm, length × width × height) on d 20. Each cage was equipped 166
with a metal feeder and drinker placed outside the cage. Experimental diets were the same as in 167
the growth experiment, except that 0.3% of chromic oxide (Cr2O3) was added and mixed to 168
facilitate determination of nutrient retention. The metabolizable trial included a 3-d preliminary 169
adaptation period at 20 to 22 d of age followed by 2 d of total excreta collection. Contamination 170
(e.g., feathers and down) was carefully removed and the collected excreta was dried at 60°C until 171
constant weight, homogenized and finely ground to pass through a 1-mm screen, and stored in 172
airtight plastic containers until analysis. The following equation was used to calculate TTAR 173
(Scott et al., 1976): 174
TTAR (%) = 100 – [(diet Cr2O3/excreta Cr2O3) × (nutrient in excreta/nutrient in diet)] × 100. 175
2.6. Volatile fatty acids analysis 176
On d 24 and 42, cecal contents from two birds per replicate (i.e. 10 birds per treatment) were 177
gently squeezed into a tube and stored at -80°C until analysis. Approximately 1.5 g of thawed 178
digesta was diluted with distilled water (1:1 wt/vol) in a screw-capped tube. After 179
homogenization and centrifugation, 1 mL of clear supernatant was transferred into an ampulla, 180
and 0.2 mL metaphosphoric acid solution was added. The sample was again homogenized and 181
placed in an ice bath for at least 30 min to allow the protein to settle completely. Finally, samples 182
were centrifuged at 10,844 × g for 10 min, and the supernatant was analyzed for VFA with gas 183
chromatography (Zhang et al., 2003). 184
2.7. Enumeration of bacteria 185
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On d 42, cecal digesta (10 g) from two birds per replicate (i.e. 10 birds per treatment) were 186
aseptically transferred into 90 mL of sterile peptone containing 0.5% cysteine hydrochloride and 187
serially diluted. For C. perfringens enumeration, dilutions were placed on Perfringens agar base 188
(OPSP, Oxoid Inc., Nepean, Ontario, Canada) containing supplements SR 76 and SR 7 (Oxoid 189
Inc.), and incubated at 38°C for 48 h in jars containing gas generation kits (BBL GasPak Plus, 190
Becton Dickinson, Sparks, MD). The population of Bifidobacteria in cecal samples was 191
determined by using a standard laboratory method (Ibrahim and Salameh, 2001). Briefly, ileal 192
samples (10 g) were diluted with 90 ml sterilized 0.1% peptone water and homogenized using 193
Stomacher 400 Lab System 4 (Seward, Norfolk, UK) for 2 min, and 100 ml of appropriate 194
dilution was surface plated onto modified BIM 24 agar. The level was determined at the serial 195
dilution of 10-5. Plates were incubated at 37°C for at least three days. Lactobacilli were 196
enumerated on De Man-Rogosa-Sharpe (MRS) agar, and E. coli was counted on Mac Conkey 197
(MC) agar after incubation at 37°C in an anaerobic chamber for 48 h, and in an aerobic chamber 198
for 24 h, respectively. All samples were plated in duplicate. 199
2.8. Serum parameters 200
After 4 h starvation, two birds per replicate were randomly selected and their blood samples 201
were collected by syringe from the wing vein on d 40. Blood samples were collected in labelled 202
sterile test tubes and centrifuged at 1,000 × g for 15 min at 4°C to isolate serum. After 203
centrifugation, serum was collected and stored at -20°C until analysis. The levels of serum 204
triglyceride (mg/dl), total cholesterol (mg/dl), high density lipoprotein (HDL, mg/dl), low 205
density lipoprotein (LDL, mg/dl), aspartate aminotransferase (AST, IU/L), alanine 206
aminotransferase (ALT, IU/L), gamma glutamyltransferase (GGT, IU/L), creatin kinase (CK, 207
U/L), total protein (TP, g/dl) and albumin (g/dl) were measured by an autoanalyzer (Selectra E 208
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vital scientific, Netherlands). Globulin value was obtained from the difference between serum TP 209
and albumin concentrations. All tests were carried out in duplicate. 210
2.9. Size of different organs 211
At the end of the experiment, 2 birds per replicate whose body weights were closest to the 212
mean weight of the pen were selected and humanly killed by cervical dislocation, plucked, and 213
eviscerated of gastrointestinal tract, giblets and other inner organs to determine the carcass 214
characteristics. 215
2.10. Chemical analysis 216
Diets and excreta samples were analyzed for DM content (method 930.15; AOAC, 1995) and 217
fat [method 920.32 (AOAC, 2000) by a 1043 Soxtec HT system, Foss Tecator AB, Hoganas, 218
Sweden]. Crude protein was calculated as nitrogen × 6.25. Nitrogen was determined by using a 219
Kjeltec Auto 1030 Analyzer (Tecator AB, Hoganas, Sweden). Gross energy of diets and excreta 220
samples was measured with a bomb calorimeter (IKA-Kalorimeter, Model C400, IKA, Staufen, 221
Germany). Chromium oxide content in the experimental diets and excreta were measured 222
according to Saha and Gilbreath (1991). 223
2.11. Statistical analysis 224
Data were analyzed as a 2×3 factorial arrangement (2 levels of enzyme and 3 levels of 225
thymol+carvacrol) using PROC GLM of SAS (SAS Institute, 2001). Data were analysed 226
considering the pen of birds as the experimental unit for performance parameters, and the 227
individual chicken as the experimental unit for the rest of the parameters measured. Treatment 228
means were separated using Tukey’s multiple comparison tests. Statistical significance was 229
declared at a probability of P < 0.05. Microbiological counts were subject to base-10 logarithm 230
transformation before analysis. 231
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232
3. Results 233
3.1. Chemical composition of phytogenic product and diets 234
Calculated and analysed carvacrol and thymol contents of Next enhance150® and diets 235
(mg/kg) are shown in Table 2. The GC-MS results indicated that the 2 phenols, carvacrol and 236
thymol, were the sole components of the phytogenic product (Table 2). Analysis of Next 237
enhance150® by gas chromatography revealed the components to be 54.13% carvacrol and 238
45.87% thymol. 239
3.2. Performance 240
All birds were healthy during the entire experimental period. Mortality was lower than 1.4% 241
with no differences between the groups. The effect of dietary NSP-degrading enzyme and 242
thymol+carvacrol supplements on growth performance traits of broilers fed wheat-based diet at 243
different phases is shown in Table 3. Both E and T+C supplementation significantly (P < 0.05) 244
affected ADG and G:F of broilers throughout the trial, but no significant effects were observed 245
for ADFI. Addition of E and 200 mg T+C/kg of diets improved ADG by 9.9 and 11.3%, 246
respectively, and G:F by 11.4 and 17.1%, respectively, at 10 d of age compared with birds fed 247
the control diet. From d 11-24 and d 25-42, ADG and G:F was increased (P < 0.05) by the 248
inclusion of E and T+C while ADFI was not affected. For the whole period, E and the two levels 249
of T+C supplementation improved (P < 0.05) ADG by 5.5 and 5.3 and 6.2%, respectively, and 250
G:F by 5.8 and 5.7 and 7.1%, respectively, of birds fed wheat-based diets. 251
3.3. Intestinal digesta viscosity and pH 252
The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on intestinal 253
digesta viscosity and pH of broilers fed wheat-based diet at d 24 is shown in Table 4. Digesta 254
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viscosity was reduced (P < 0.05) after enzyme addition in all parts of small intestine and also 255
after inclusion of phytogenic in jejunum and ileum. Neither E nor T+C supplementation had any 256
effect on intestinal digesta pH of broilers at d 24. 257
3.4. TTAR of nutrients 258
The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on total 259
tract apparent retention (TTAR, %) of nutrients of broilers fed wheat-based diet at d 24 is shown 260
in Table 5. NSP-degrading enzyme or phytogenic treated birds showed an increased (P < 0.05) 261
retention of DM, protein and gross energy. In the E supplemented group, the retention of DM, 262
protein and gross energy was increased (P < 0.01) by 8.0, 9.5% and 10.7, respectively. Enzyme 263
addition did not affect fat retention. Inclusion of 100 and 200 mg of T+C/kg increased (P < 0.05) 264
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%, 265
respectively, while fat retention was unaffected. 266
3.5. VFA production 267
The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on volatile 268
fatty acid profile, and total VFA amounts in the cecal contents of broilers fed wheat-based diet at 269
d 24 and 42 is shown in Table 6. Dietary supplementation of E and T+C increased (P < 0.01) 270
total VFA and acetate levels at d 24 and 42, whereas level of butyrate decreased (P < 0.01). 271
Enzyme decreased propionate at d 24 and 42 (P < 0.01), isobutyrate at d 42 (P < 0.05) and 272
valerate at d 24 (P < 0.01). Phytogenic product decreased (P < 0.01) isovalerate at d 42. 273
Proportion of isovalerate was not changed by E supplementation at d 24 and 42. A similar 274
pattern was observed for propionate, isobutyrate and valerate by T+C. 275
3.6. Intestinal bacterial numbers 276
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The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on cecal 277
microbial population of broilers fed wheat-based diet at d 42 is shown in Table 7. E. coli and C. 278
perfringens counts were lower (P < 0.01) than controls, and Lactobacilli counts higher, in birds 279
fed E or T+C at the rate of 200 mg/kg. Bifidobacteria counts were not affected by E and dropped 280
(P < 0.01) with increasing T+C dosages. 281
3.7. Serum parameters 282
The effect of dietary NSP-degrading enzyme and thymol+carvacrol supplements on serum 283
lipid metabolites of broilers fed wheat-based diet at d 40 is shown in Table 8. The inclusion of E 284
elevated serum triglyceride (P < 0.05) and total cholesterol (P < 0.01), while there was no 285
significant effect of enzyme on LDL and HDL cholesterol. Chickens fed diets supplemented with 286
T+C decreased (P < 0.01) serum total cholesterol and T+C at the rate of 200 mg/kg decreased (P 287
< 0.05) LDL, while no effect was found on triglyceride and HDL values. The effect of dietary 288
NSP-degrading enzyme and thymol+carvacrol supplements on serum biochemical parameters of 289
broilers fed wheat-based diet at d 40 is shown in Table 9. There was no significant difference 290
among groups in serum levels of aspartate aminotransferase, alanine aminotransferase, gamma 291
glutamyltransferase and creatin kinase. However, dietary E supplementation increased (P < 0.01) 292
TP, albumin and globulin, while dietary T+C (P < 0.01) elevated TP and albumin. 293
3.8. Organ weights and intestinal lengths 294
The effect of dietary NSP-degrading enzyme or thymol+carvacrol supplements on relative 295
weights of carcass, fat pad, liver and pancreas and relative lengths of duodenum, jejunum and 296
ileum of broilers fed wheat-based diet at d 42 is shown in Table 10. Enzyme supplementation 297
decreased (P < 0.01) the relative size of digestive organs of broilers. Moreover, carcass, liver and 298
pancreas relative weights decreased (P < 0.05) with E supplementation at d 42. No effects were 299
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observed with T+C dietary supplementation except for carcass relative weight and jejunum and 300
ileum relative lengths. 301
4. Discussion 302
4.1. Growth performance 303
According to our results, dietary supplementation with either the phytogenic product or the 304
NSP-degrading enzyme modified the performance of broilers fed on wheat-based diets by 305
increasing ADG and G:F over the whole grower period. Wheat can be a more cost effective feed 306
ingredient compared to corn as the major cereal in broiler diets, especially during the harvest 307
season in various regions of the world. However, the use of wheat is limited due to a number of 308
nutritional disadvantages: varying nutrient contents, lower metabolisable energy than corn, and 309
the presence of soluble NSPs such as arabinoxylans and β-glucans (Basmacioglu et al., 2010). 310
Pentosan solubilisation results in a viscous condition of the digesta that has been shown to 311
interfere with nutrient assimilation within the chicks' intestine (Friesen et al., 1992). It has been 312
reported that these effects result in wet droppings, increased intestinal microbiota load, depressed 313
growth and feed efficiency (Knarreborg et al., 2002). 314
Various treatments including enzyme supplementation, antibiotic addition and the use of 315
bioactive substances have proved to be beneficial in improving the nutritive value of wheat 316
(Choc et al., 2004; Amad et al., 2011). The present study showed that the addition of an enzyme 317
complex (xylanase and β-glucanase) to broiler wheat-based diets led to improved performance. 318
These positive results were probably associated with a reduction in digesta viscosity as 319
previously reported (Bedford and Apajalahti, 2001), and are in line with McCracken and Quintin 320
(2000), who reported that xylanase addition to broiler diets improved live weight gain and 321
gain:feed. The viscosity reduction in the digestive content observed in birds fed on enzyme 322
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supplemented diets has been reported to allow a faster transit of the digesta, a greater feed intake, 323
and a facilitated contact between nutrients and digestive enzymes to improve nutrient 324
digestibility (Lázaro, 2003). 325
Diets supplemented with the phytogenic T+C improved broilers performance compared to 326
the control diet. Oregano and thyme oils are usually composed of the monoterpenes thymol and 327
carvacrol in varying proportions (Daferera et al., 2000). Next enhance150® is a commercial 328
supplement based on a 1:1 ratio of thymol:carvacrol, and has been shown to improve average 329
weight gain and G:F of broilers (Hashemipour et al., 2013a,b). Oregano essential oil, alone or in 330
combination with a multi-enzyme, significantly increased body weight gain during the first week 331
of life in broilers (Basmacioglu et al., 2010). These authors noted that dietary supplementation 332
with oregano essential oil (250 mg/kg diet) may possess an antioxidant effect which has been 333
associated with an effect on body weight gain at early life of broiler chicks fed wheat-based diets 334
as a nutritional stress factor. Given their antimicrobial activity (Wenk, 2000), it would be 335
expected that thymol + carvacrol could have positive effects on growth performance in broilers. 336
Cross et al. (2003) noted that thyme oil together with an enzyme mix (xylanase and glucanase) in 337
diets is likely to improve performance synergistically that this was not the case in the current 338
experiment. 339
4.2. Intestinal digesta viscosity and pH 340
Arabinoxylans and β-glucans have been shown to increase digesta viscosity (Lázaro et al., 341
2003), which can be overcome by adding NSP-degrading enzymes (Bedford and Apajalahti, 342
2001). Enzyme supplementation significantly decreased viscosity of intestinal contents in 343
duodenum, jejunum and ileum, but did not result in significant alterations in pH values of either 344
duodenal, jejunal or ileal digesta. These results are in agreement with those reported by 345
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Mathlouthi et al. (2002), who showed that the pH of the intestinal contents in birds fed wheat and 346
barley-based diet was not significantly affected by the addition of xylanase and β-glucanase. 347
Earlier research demonstrated that NSP-degrading enzymes were effective in both viscosity 348
reduction and degradation of the cell wall structure, which resulted in increased digestibility of 349
nitrogen, fat, starch, and NSP in the small intestine of young broiler chickens fed wheat-based 350
diets (Basmacioglu et al., 2010). 351
Thymol+carvacrol supplementation significantly reduced viscosity of jejunum and ileum 352
contents, but there was no effect on the intestinal digesta pH in the current work. To some extent, 353
this was surprising given that the fermentation of carbohydrates usually leads to an increased 354
production of VFA, which tend to lower intestinal lumen pH values. However, fatty acids 355
production and absorption takes place mainly in the ceca (where it actually increased, Table 6) 356
by bacterial fermentation of undigested NSP, and very low bacterial fermentation takes place 357
within the small intestine of broilers (Svihus et al., 2012). 358
4.3. Nutrients retention 359
Performance has a close relationship with energy metabolism in birds. Consequently, a 360
metabolism trial was conducted to determine the effects of enzyme and phytogenic inclusion on 361
total tract apparent retention of nutrients (TTAR). In our study, we found that enzyme 362
supplementation improved DM, protein, and gross energy retention by 8.0, 9.5 and 10.7%, 363
respectively. Exogenous xylanase can partially hydrolyse the arabinoxylans and release the 364
enclosed nutrients for the birds to use (Bedford and Apajalahti, 2001), where after birds can 365
digest and absorb the nutrients more easily and achieve better growth performance. The 366
improved CP retention with added xylanase in wheat based diets may be partly due to lowering 367
the endogenous amino acid losses, resulting from the elimination of adverse effects of wheat 368
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pentosans (Angkanaporn et al., 1994). Xylanase did not affect fat retention determined at d24 in 369
our study, which is in agreement with the findings of Juanpere et al. (2005). This may be partly 370
due to the age of the birds and the type of fat (soybean oil) used in this study. In young chickens, 371
fat digestion increases with age and reaches optimal capacity after 2 wk of age (Nitsan et al., 372
1991). 373
The phytogenic product significantly increased the TTAR of nutrients at d 24 in the current 374
study. Kamel (2001) mentioned that there is evidence to suggest that herbs, spices, and various 375
plant extracts have appetite- and digestion-stimulating properties and antimicrobial effects. 376
Therefore, the improvement of the TTAR of nutrients in this study could be caused by the 377
stimulation effect of the phytogenic on endogenous digestive enzymes activity, and/or an 378
increased absorption surface area, which were previously reported (Hashemipour et al., 2013a,b). 379
4.4. VFA production 380
It is well known that the amount and type of fermentable substrates, especially 381
carbohydrates, reaching the large intestine affects volatile fatty acids (VFA) concentration and 382
profile (Svihus et al., 2012). Volatile fatty acids are the major end products of microbial 383
fermentation. Their levels could be used indirectly to monitor gut microbe populations in broilers 384
(Taylor, 2002), and they are efficiently absorbed by the colonic mucosa. In our current study, 385
birds consuming the control diet generally had lower total VFA levels in the ceca than birds 386
consuming diets containing supplemental enzyme at d 24 and 42, which is consistent with the 387
results reported by Choct et al. (1999). The concentration of acetate in the ceca was clearly 388
higher than the concentration of the other acids. When supplemental enzyme was added to the 389
wheat based control diet, it might partially degrade these larger molecular polysaccharides into 390
smaller ones, even oligosaccharides, and at the same time the enzymes might alleviate the 391
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viscous property of the digesta (Mathlouthi et al., 2002), increase the digesta flow rate, thus 392
stimulating the production of VFA and the growth of specific beneficial bacteria within the ceca 393
(Choct et al., 1999). 394
At d 24 and 42, the concentration of butyric acid was lower in the cecal contents of the 395
phytogenic-treated chicks, which may be related with the observed effect (Table 7) in the counts 396
of C. perfringens in the chicken ceca (Elwinger et al., 1992). Sakata (1987) demonstrated in rats 397
that intraluminally infused VFA accelerated the crypt cell production rate and increased the gut-398
wall mass. The stimulation was most efficient with butyrate. The positive effect of dietary 399
antibacterials appears to be related at least in part with the elimination of fermentative 400
microorganisms, mainly butyric acid producers (especially Clostridia), from the small intestine 401
(Choct et al., 1999). This effect has been shown to decrease the gut-wall mass and stimulate 402
nutrient absorption (Parker and Armstrong, 1987), which supports the improved nutrient 403
retention found in the present study. 404
4.5. Intestinal bacterial numbers 405
High intestinal viscosity reduces nutrient absorption by the host animal, decreases the rate of 406
feed passage, and may enhance mucus production (Piel et al., 2005), which could lead to 407
increased numbers of anaerobic bacteria in the small intestine, particularly C. perfringens 408
(Collier et al., 2003). Bedford and Apajalahti (2001) demonstrated that in birds fed wheat-based 409
diets, the addition of a xylanase based enzyme preparation resulted in a 60% reduction in 410
bacterial numbers. In accordance with these findings, the present investigation resulted in lower 411
counts of E. coli and C. perfringens, and higher counts of Lactobacilli, in birds fed the NSP-412
degrading enzyme. In the process of depolymerizing various polysaccharides in the diet, 413
exogenous enzymes may produce galacto-, gluco-, manno-, or xylo-oligomers, which are similar 414
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to prebiotics and which may facilitate the proliferation of health-promoting bacteria such as 415
Bifidobacteria and Lactobacilli (Monsan and Paul, 1995). The inclusion of thymol+carvacrol in 416
the diets improved the microbial counts in birds compared to those fed the control diet. In line 417
with the present results, the dietary supplementation with an encapsulated product containing 418
capsaicin, carvacrol and cinnamaldehyde, reduced the numbers of E. coli and C. perfringens in 419
broiler rectal contents to the same extent as avilamycin (Jamroz et al., 2003). The mechanism of 420
action of thymol and carvacrol is probably linked to their effect of bacterial membrane integrity 421
disruption, which further affects pH homeostasis and equilibrium of inorganic ions (Lambert et 422
al., 2001). 423
4.6. Serum parameters 424
The inclusion of E increased serum triglyceride, total cholesterol, total protein (TP), albumin 425
and globulin concentrations, while T+C decreased total cholesterol, TP and albumin at d 40. 426
Similar results were observed by Hajati et al. (2009) who reported that dietary multi-enzyme 427
inclusion increased the blood concentrations of total cholesterol, HDL-cholesterol and 428
triglyceride. The addition of the multi-enzyme may alleviate the limitations for the function of 429
bile salts and their emulsifying properties in intestinal chyme due to undigested NSP, which may 430
result in increased total fat in blood (Hajati et al., 2009). 431
Case et al. (1995) indicated that thymol decreased the serum cholesterol concentration in 432
leghorn chickens fed a corn based diet, due to a hypocholesterolaemic effect of thymol, in 433
contrast with Lee et al. (2003) who reported no hypocholesterolaemic activity of dietary 434
carvacrol and thymol. The hypocholesterolemic effect of thymol and carvacrol has been ascribed 435
to the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Elson, 436
1995), the rate controlling enzyme of the cholesterol synthetic pathway. The absence or presence 437
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of cholesterolaemic effects of dietary components in an animal is also dependent on factors such 438
as breed, gender and age, and also on the composition of the feed (Lee et al., 2003). 439
The liver plays an important role in metabolic processes, and the metabolic activity of the 440
liver is important for the normal functioning of cellular events (Cornellus, 1980). Serum AST 441
and ALT are indicators of normal liver functioning. In the present study, there was no significant 442
alteration in the serum levels of AST, ALT and CK, and so no evidence of hepatocyte and 443
muscle injury was determined. Also, no significant difference in serum GGT concentration was 444
observed among treatment groups, suggesting that cholestasis and duct hyperplasia (Tennant, 445
1997) did not occur in this experiment. In contrast, Traesel et al. (2011) suggested a dose-446
dependent effect of essential oil on serum concentration of AST in which the increase in serum 447
levels of AST is caused by hepatocyte injury. 448
4.7. Organ weights and intestinal lengths 449
Enzyme supplementation decreased the relative size of the digestive organs. Moreover, 450
carcass, liver and pancreas relative weights decreased with E supplementation at d 42. No effects 451
on organ weights and intestinal lengths were observed with T+C dietary supplementation except 452
for carcass relative weight and jejunum and ileum relative lengths. The presence of viscous 453
grains such as wheat in diets can increase the viscosity of the digesta and inhibit the effective 454
contact between the digestive enzymes and their corresponding substrates, thereby leading to 455
significant modifications of the structure and function of intestine and organs (Dworkin et al., 456
1976). Sarica et al. (2005) noted that thyme oil and xylanase-based enzyme complex 457
significantly decreased small intestine weight or ileum length when these feed supplements were 458
used together in wheat-based diets. When supplementing exogenous enzymes in the wheat 459
control diet, a greater proportion of NSP may be hydrolyzed, which might attenuate the secretory 460
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function of the responding organs and GIT segments, and then the organ sizes may decrease. The 461
reduction in digestive organs relative weight has direct economic implications, as the dressing 462
yield of broilers increased proportionally. 463
In conclusion, the present study showed that the phytogenic product thymol + carvacol, and 464
NSP-degrading enzyme independently improved growth performance, enhanced nutrients 465
retention, increased total VFA, reduced cholesterol and modulated intestinal microbial counts in 466
broilers fed on a wheat-based diet. These results have both productive and health implications for 467
the broiler industry and warrant further investigation. 468
469
Acknowledgments 470
The authors are grateful to Dr. Khaksar for providing the experimental facilities for this work. 471
472
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626
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Table 1 626 Composition and calculated analysis (g/kg as fed) of the basal diet. 627
Ingredients (g/kg)
Starter (1-10 d)
Grower (11-24 d)
Finisher (25-42 d)
Wheat 574.7 600.0 614.7 Soybean meal, 440 g/kg CP 341.2 308.7 290.5 Wheat bran 1.0 1.0 1.0 Vegetable oil 40.0 56.0 61.4 Limestone 14.5 12.5 12.0 Dicalcium phosphate 13.5 11.0 10.0 Salt 3.7 3.6 3.4 HCL-Lys 3.3 0.2 0.8 DL-Met 1.9 1.5 1.1 Thr 1.2 0.5 0.1 Vitamin permix1 2.5 2.5 2.5 Mineral permix2 2.5 2.5 2.5 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
1Vitamin premix provided the following per kilogram of diet: vitamin A (trans-retinyl acetate), 628 10,000 IU; vitamin D3 (cholecalciferol), 3500 IU; vitamin E (DL-α-tocopheryl acetate), 60 mg; 629 vitamin K (menadione), 3 mg; thiamine, 3 mg; riboflavin, 6 mg; pyridoxine, 5 mg; vitamin B12 630 (cyanocobalamin), 0.01 mg; niacin, 45 mg; pantothenic acid (D-calcium pantothenate), 11 mg; 631 folic acid, 1 mg; biotin, 0.15 mg; choline chloride, 500 mg; ethoxyquin (antioxidant), 150 mg. 632 2Mineral permix provided the following per kilogram of diet: Fe, 60 mg; Mn, 100 mg; Zn, 60 633 mg; Cu, 10 mg; I, 1 mg; Co, 0.2 mg; Se, 0.15 mg.634
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635 Table 2 636 Calculated and analyzed carvacrol and thymol contents of the experimental diets (mg/kg). 637
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 1Control, wheat-based diet contained neither thymol+carvacrol (T+C) nor enzyme (E). E, 0.5 638 g/kg of enzyme Endofeed W; T+C100, 100 mg/kg of Next enhance150®; T+C200, 200 mg/kg of 639 Next enhance150®; E plus T+C100, 0.5 g/kg of enzyme and 100 mg/kg of Next enhance150®; E 640 plus T+C200, 0.5 g/kg of enzyme and 200 mg/kg of Next enhance150®.641
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Table 3 643 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on growth performance traits of broilers fed 644 wheat-based diet at different phases. 645 0 to10 d 11 to 24 d 25 to 42 d 0 to 42 d Treatment
ADG1 (g)
ADFI1 (g)
G:F1 (g/kg)
ADG1 (g)
ADFI1 (g)
G:F1 (g/kg)
ADG1 (g)
ADFI1 (g)
G:F1 (g/kg)
ADG1 (g)
ADFI1
(g) G:F1
(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 0.5 26.7a 29.8 902.9a 63.9a 93.2 685.9a 104.4a 190.2 549.0a 72.4a 119.7 605.2a ±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 T+C, mg/kg
0 23.9b 30.5 784.7b 60.3b 94.0 641.4b 98.6b 191.1 515.8b 68.0b 120.5 564.5b 100 26.0ab 30.1 866.5ab 64.0a 93.1 688.4a 102.9a 190.9 539.0a 71.6a 120.0 597.0a 200 26.6a 29.2 919.3a 64.6a 93.1 693.4a 103.6a 190.2 544.4a 72.2a 119.5 604.6a ±SEM 0.59 0.68 23.69 0.70 0.78 8.83 1.11 0.38 5.98 0.74 0.40 6.18
P-value
E ** NS ** * NS * ** NS ** ** NS ** T+C ** NS ** ** NS ** * NS ** ** NS ** E × T+C NS NS NS NS NS NS NS NS NS NS NS NS NS: P > 0.05, *: P < 0.05, **: P < 0.01. 646 a,b Means within the same column with no common superscripts differ significantly (P < 0.05). 647 1ADG: average daily gain; ADFI: average daily feed intake; G:F: gain to feed ratio.648
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649
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Table 4 650 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on 651 intestinal digesta viscosity and pH of broilers fed wheat-based diet at d 24. 652 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 0.5 1.77b 1.91b 2.11b 5.96 6.31 6.35 ±SEM 0.18 0.10 0.10 0.12 0.06 0.10 T+C, mg/kg
0 2.42 2.97a 3.76a 6.01 6.31 6.21 100 2.13 2.38b 3.08b 5.96 6.23 6.36 200 1.76 2.06b 2.86b 5.98 6.28 6.39 ±SEM 0.22 0.12 0.12 0.14 0.07 0.12
P-value
E * ** ** NS NS NS T+C NS ** ** NS NS NS E × T+C NS NS NS NS NS NS NS: P > 0.05, *: P < 0.05, **: P < 0.01. 653 a,b Means within the same column with different superscripts differ significantly (P < 0.05). 654
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655 Table 5 656 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on total 657 tract apparent retention (%) of nutrients of broilers fed wheat-based diet at d 24. 658 Treatment DM Protein Fat Gross energy E, g/kg 0 64.98b 56.26b 78.07 65.14b 0.5 70.16a 61.61a 77.37 72.09a ±SEM 0.53 0.59 1.44 0.84 T+C, mg/kg
0 63.96b 56.05b 79.19 65.23b 100 69.13a 59.87a 78.15 69.74a 200 69.61a 60.88a 75.81 70.88a ±SEM 0.65 0.72 1.77 1.03
P-value
E ** ** NS ** T+C ** ** NS * E × T+C NS NS NS NS NS: P > 0.05, *: P < 0.05, **: P < 0.01. 659 a,b Means within the same column with different superscripts differ significantly (P < 0.05). 660
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661 Table 6 662 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on 663 volatile fatty acid profile (VFA, %), and total VFA amounts (mmol/L) in the cecal contents of 664 broilers fed wheat-based diet at d 24 and 42. 665 Individual VFA
Total VFA
Treatment Acetate Propionate Butyrate Isobutyrate Isovalerate Valerate d 24
E, g/kg 0 73.02b 0.91a 17.47a 3.05 0.59 4.95a 14.75b
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 72.49b 0.55 16.78a 4.61 0.82 4.74 14.76b
100 76.69a 0.75 14.06b 3.24 0.36 4.90 16.60a
200 77.83a 0.66 12.27b 3.99 0.43 4.81 16.78a
±SEM 0.92 0.14 0.62 0.74 0.15 0.08 0.22
P-value
E ** ** ** NS NS ** ** T+C ** NS ** NS NS NS ** E × T+C NS NS NS NS NS NS NS
d 42
E, g/kg 0 74.07b 8.62a 11.94a 0.91a 0.64 3.81 48.36b
0.5 79.36a 6.46b 7.25b 0.71b 0.75 5.46 55.08a
±SEM 0.73 0.44 0.38 0.05 0.08 0.66 0.46
T+C, mg/kg
0 73.54b 7.21 11.42a 0.77 1.11a 5.93 48.39b
100 77.79a 7.76 9.35b 0.85 0.50b 4.12 53.16a
200 78.89a 7.65 8.00b 0.81 0.48b 3.84 53.62a
±SEM 0.90 0.54 0.47 0.07 0.10 0.81 0.56
P-value
E ** ** ** * NS NS ** T+C ** NS ** NS ** NS ** E × T+C NS NS NS NS NS NS NS NS: P > 0.05, *: P < 0.05, **: P < 0.01. 666 a,b Means within the same column with different superscripts differ significantly (P < 0.05).667
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668 Table 7 669 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on cecal 670 microbial population (log CFU/g of digesta) of broilers fed wheat-based diet at d 42. 671 Treatment
Lactobacilli Bifidobacteria C. perfringens E. coli
E, g/kg 0 7.60b 6.47 2.55a 6.29a 0.5 7.84a 6.44 2.29b 5.90b ±SEM 0.03 0.07 0.05 0.07 T+C, mg/kg
0 7.62b 6.92a 2.66a 6.46a 100 7.73a 6.52b 2.51a 6.22a 200 7.80a 5.93c 2.10b 5.60b ±SEM 0.03 0.09 0.06 0.09
P-value
E ** NS ** ** T+C ** ** ** ** E × T+C NS NS NS NS NS: P > 0.05, *: P < 0.05, **: P < 0.01. 672 a,b,c Means within the same column with different superscripts differ significantly (P < 0.05). 673
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674 Table 8 675 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on serum 676 lipid metabolites of broilers fed wheat-based diet at d 40. 677 Treatment
Triglyceride (mg/dl)
Cholesterol (mg/dl)
HDL1 (mg/dl)
LDL2 (mg/dl)
E, g/kg 0 71.6b 118b 82.9 32.8 0.5 80.4a 120a 82.9 34.3 ±SEM 2.76 0.40 0.78 0.85 T+C, mg/kg
0 77.1 121a 82.2 36.1a 100 75.3 119b 83.2 33.2ab 200 75.5 117b 83.3 31.5b ±SEM 3.39 0.49 0.95 1.04
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. 678 a,b Means within the same column with different superscripts differ significantly (P < 0.05). 679 1HDL = High density lipoprotein. 680 2LDL = Low density lipoprotein.681
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682 Table 9 683 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on serum 684 biochemical parameters of broilers fed wheat-based diet at d 40. 685 Treatment
AST1 (IU/L)
ALT2 (IU/L)
GGT3 (IU/L)
CK4 (U/L)
TP5 (g/dl)
Albumin (g/dl)
Globulin (g/dl)
E, g/kg 0 134 19.9 9.95 3223 3.89b 1.67b 2.22b 0.5 132 20.0 9.81 3242 4.53a 1.88a 2.65a ±SEM 4.76 0.57 0.29 145.91 0.10 0.07 0.07 T+C, mg/kg
0 132.7 19.6 9.9 3037 3.83b 1.58b 2.25 100 133.3 20.2 9.5 3274 4.34a 1.87a 2.46 200 132.8 19.9 10.3 3386 4.45a 1.89a 2.56 ±SEM 5.83 0.70 0.35 178.7 0.12 0.09 0.09
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. 686 a,b Means within the same column with different superscripts differ significantly (P < 0.05). 687 1AST = Aspartate aminotransferase. 688 2ALT = Alanine aminotransferase. 689 3GGT = Gamma glutamyltransferase. 690 4CK = Creatin kinase. 691 5TP = Total protein.692
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693 Table 10 694 Effect of dietary NSP-degrading enzyme (E) and thymol+carvacrol (T+C) supplements on 695 relative weights of carcass, fat pad, liver and pancreas (g/100 g of BW) and relative lengths of 696 duodenum, jejunum and ileum (cm/100 g of BW) of broilers fed wheat-based diet at d 42. 697 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 T+C, mg/kg
0 60.6b 1.24 2.12 0.28 1.38 2.77a 2.83a 100 64.5a 1.23 2.22 0.28 1.45 2.51b 2.60b 200 64.7a 1.30 2.27 0.28 1.44 2.57b 2.56b ±SEM 1.01 0.05 0.10 0.01 0.07 0.04 0.05
P-value
E * NS * ** ** ** ** T+C ** NS NS NS NS ** ** E × T+C NS NS NS NS NS NS NS NS: P > 0.05, *: P < 0.05, **: P < 0.01 698 a,b Means within the same column with different superscripts differ significantly (P < 0.05). 699 1The small intestine was divided into three segments: the duodenum (from gizzard to pancreo-700 biliary ducts), the jejunum (from pancreo-biliary ducts to Meckel’s diverticulum) and the ileum 701 (from Meckel’s diverticulum to ileo-caecal junction). 702 703 704 705
We test the potential of thymol plus carvacrol in broiler diet. 706
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
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714
715
