The boiling process ensured that the inherent egg flavor was preserved. The group consisted of 6 probands in this research. The probands were selected and trained in accordance with LST ISO 8586-1. The estimation was performed according to the requirements of LST ISO 8589 in the institute cabins of the Laboratory of Sensory Analysis. For preparation of the sample for texture analysis, the modified WOODWARD and COTTERILL (1987) method. The samples texture characteristics were determined with the universal texture analyzer Universal Testing Machine Instron 3343 (Instron Engineering Group, High Wycombe, UK) equipped with a 1kN load cell and Bluehill software.

Extraction of lipids for fatty acid analysis was performed with chloroform/methanol (2:1 v/v) as described by FOLCH et al. (1957). Fatty acid methyl esters were prepared using the procedure of CHRISTOPHERSON and GLASS (1969). The FAMEs were analysed using a gas liquid chromatograph (GC–2010 SHIMADZU) fitted with flame ionization detector.

*Statistical analysis.* The results of the experiment were analysed using the 1-way ANOVA test, and significant differences between groups were determined by Duncan's multiple range test. Statistica 8.0. for Windows™ software was used. Differences were considered significant at P<0.05.

## Results

Productivity parameters of laying hens of 30–38 weeks of age in the Experimental I group the *Lumance®* increased egg laying intensity by 1.46–4.91% compared with the Control group. The results of increasing egg production percentage due to adding organic acids mixture is in agreement with those of SOLTAN (2008) who found that organic acid supplementation increased egg production by 5.77% compared to control group.

No significant difference in saturated, unsaturated and polyunsaturated fatty acids in egg yolk between the treatments and different days of storage was observed.

The results of albumen and yolk texture analysis are presented in Table 1. Due to the effect of *Lumance®*, the egg albumen after 28 days of storage was more harder in experimental groups by 2.99–4.49 N and resilient 15.53–26.23 N compared to the control group (P<0.05). Statistically significant differences were found in Experimental I group (0.5 kg/t *Lumance®*) after 28 days of egg storage, egg yolk springiness was by 0.95 mm less compared to the Control group.

Summing up the results of albumen and yolk texture analysis, it may be stated that the use of different *Lumance®* levels supplement in laying hen diet had a minor effect on the texture characteristics of yolk and albumen.

<table><caption>**Table 1.** Effect of dietary inclusion of medium chain fatty acids and butyrate on egg texture characteristics storage 1 and 28 days</caption><thead><tr><th rowspan="2">Characteristic</th><th rowspan="2">Storage time in days</th><th colspan="3">Groups</th></tr><tr><th>Control</th><th>Experimental I +0.5 kg/t Lumance®</th><th>Experimental II +1.5 kg/t Lumance®</th></tr></thead><tbody><tr><td colspan="5"><strong>Albumen</strong></td></tr><tr><td rowspan="2">Hardness, N</td><td>1</td><td>10.7±1.29<sup>a</sup></td><td>11.4±2.78<sup>a</sup></td><td>11.37±2.48<sup>a</sup></td></tr><tr><td>28</td><td>8.84±1.96<sup>a</sup></td><td>13.33±0.52<sup>c</sup></td><td>11.83±1.55<sup>bc</sup></td></tr><tr><td rowspan="2">Cohesiveness</td><td>1</td><td>0.47±0.02<sup>a</sup></td><td>0.46±0.10<sup>a</sup></td><td>0.44±0.09<sup>a</sup></td></tr><tr><td>28</td><td>0.52±0.04<sup>a</sup></td><td>0.57±0.02<sup>a</sup></td><td>0.56±0.04<sup>a</sup></td></tr><tr><td rowspan="2">Springiness, mm</td><td>1</td><td>8.64±0.44<sup>a</sup></td><td>8.03±0.57<sup>a</sup></td><td>7.64±0.60<sup>a</sup></td></tr><tr><td>28</td><td>8.11±0.39<sup>bc</sup></td><td>8.35±0.17<sup>c</sup></td><td>8.01±0.29<sup>abc</sup></td></tr><tr><td rowspan="2">Recilience, N</td><td>1</td><td>43.39±6.83<sup>a</sup></td><td>44.99±9.13<sup>a</sup></td><td>40.01±9.74<sup>a</sup></td></tr><tr><td>28</td><td>37.39±9.57<sup>ab</sup></td><td>63.62±1.76<sup>d</sup></td><td>52.92±5.76<sup>cd</sup></td></tr><tr><td colspan="5"><strong>Yolk</strong></td></tr><tr><td rowspan="2">Hardness, N</td><td>1</td><td>14.91±4.76<sup>ab</sup></td><td>22.78±6.42<sup>b</sup></td><td>24.88±10.30<sup>b</sup></td></tr><tr><td>28</td><td>19.40±3.26<sup>a</sup></td><td>22.17±2.48<sup>ab</sup></td><td>17.27±5.71<sup>a</sup></td></tr><tr><td rowspan="2">Cohesiveness</td><td>1</td><td>0.36±0.03<sup>bc</sup></td><td>0.36±0.05<sup>bc</sup></td><td>0.42±0.01<sup>c</sup></td></tr><tr><td>28</td><td>0.54±0.03<sup>ab</sup></td><td>0.59±0.01<sup>b</sup></td><td>0.58±0.02<sup>ab</sup></td></tr><tr><td rowspan="2">Springiness, mm</td><td>1</td><td>6.47±0.11<sup>d</sup></td><td>5.52±0.20<sup>c</sup></td><td>6.15±0.11<sup>d</sup></td></tr><tr><td>28</td><td>7.01±0.46<sup>a</sup></td><td>6.82±0.16<sup>a</sup></td><td>6.84±0.31<sup>a</sup></td></tr><tr><td rowspan="2">Recilience, N</td><td>1</td><td>34.33±7.76<sup>ab</sup></td><td>46.49±9.52<sup>b</sup></td><td>44.17±7.93<sup>b</sup></td></tr><tr><td>28</td><td>74.80±9.77<sup>a</sup></td><td>89.81±9.77<sup>a</sup></td><td>68.73±9.58<sup>a</sup></td></tr></tbody></table>

<footnote>a, b, c, d – values within each column with the same superscript are not different at P<0.05</footnote>
Determination of egg sensory attributes shows that the insertion of the different levels of *Lumance*® in laying hens compound feeds statistically influenced the hardness of the egg protein after 28 days of storage (from 0.75 to 1.17 N) compared to the Control group. Egg yolk hardness in the Experimental I group after 28 days of storage increased by 1 N compared to the Control group.

## Conclusions

Supplementation of different levels *Lumance*® and different time of storage had no significant impact on the laying hens performance, fatty acids profile and sensory properties except textural properties of eggs.

## Literature

GRASHORN, M., R. GRUZAUSKAS, A. DAUKSIENE, A. RACEVICIUTE-STUPELIENE, V. JARULE, A. MIEZELIENE, G. ALENCIKIENE, V. SLAUSGALVIS, 2013: Influence of dietary organic acids on quality and sensory attributes of chicken eggs. Arch. Geflügelk. **77**(1), 29–34.

HUOPALAHTI, R., R. LOPEZ-FANDINO, M. ANTON, R. SCHADE, 2007: Bioactive Egg Compounds. Springer-Verlag, Heidelberg, pp. 298.

FOLCH, J., M. LESS, G. H. SLOANE-STANLEY, 1957: A simple method for isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry. **226**, 497–509.

CHRISTOPHERSON, S. W., R. L. GLASS, 1969: Preparation of milk fat methylesters by alcoholysis in an essentially nonalcoholic solution. J. Dairy Sci. **52**, 1289–1290.

WOODWARD, S. A., O. J. COTTERILL, 1987: Texture and Microstructure of cooked whole egg yolks and heat - formed gels of stirred egg yolk. Journal of food Science **52**, 63–67.

NATIONAL RESEARCH COUNCIL, 1994: Nutrient Requirements of Poultry. 9th ed.; National Academy Press: Washington. DC.

SOLTAN, M. A., 2008: Effect of dietary organic acid supplementation on egg production, egg quality and some blood serum parameters in laying hens. International Journal of Poultry Science **7**(6), 613–621.
P-028 (ID 214)

CANTHAXANTHIN FROM CAROPHYLL® RED CAN IMPROVE THE VITELLINE MEMBRANE STRENGTH IN STORED EGGS FROM BREEDER HENS

C. Hamelin<fnref n="1" />, and F. Cisneros<fnref n="2" />

**Corresponding author:** catherine.hamelin@dsm.com

**Abstract**

Hatchability is certainly a key performance and profitability parameter for chick producers. Storing hatching eggs is a current practice and this can damage hatchability results. Canthaxanthin, as an antioxidant, is particularly effective for supporting hatchability. The vitelline membrane strength (VMS) is very important to the early embryo survival that canthaxanthin can promote it. The experiment involved a flock of 3,800 breeders (males and females) separated into two adjacent blocks. Feeding experiment started at 18 weeks of age with a control diet as 3000 IU/kg Vitamin D₃ and no Carophyll® supplementation and experimental diet as 1,600 IU/kg vitamin D₃, 37.5 µg/kg 25-hydroxy-vitamin D₃ (HyD®) & 6 mg/kg canthaxanthin (Carophyll® Red 10%). Vitamin D₃ or 25-hydroxy-vitamin D₃ were not expected to have any influence on the VMS. Eggs were collected at 40/45 weeks and stored 7 days. The VMS was evaluated using a compression machine. Stiffness and breaking strength were significantly increased with the supplementation of canthaxanthin. Thanks to the unique capabilities of high deposition rate in the yolk and singlet oxygen quenching capabilities, canthaxanthin from Carophyll® can improve the VMS in complement to Vitamin E and we hypothesized that it contributes to explain its influence on early embryo viability and hatchability in breeder performance.

**Keywords:** canthaxanthin, vitelline membrane, stored eggs, broiler breeders, strength

**Introduction**

Hatchability is certainly a key performance and profitability parameter for chick producers. At commercial level, there is often a bigger gap between fertility and hatchability than the genetics standard settings. It is well known that hatchability declines with age, partly explained by a lower egg quality. Storing hatching eggs for extended periods is a current practice in the broiler breeder industry due to uncoordinated demand for hatchlings in the market. The egg storage can also damage the hatchability and these increases with old flocks. Canthaxanthin is particularly effective for supporting hatchability. DSM was the first company to get the approval for canthaxanthin use in breeder diet (EU regulation No 684/2014). Canthaxanthin can improve early embryo survival as shown in a breeder study where the embryonic mortality was significantly reduced from 12.66% to 10.81% (Souza et al, 2008).

Vitelline membrane separates the yolk content from the albumen. The vitelline membrane strength (VMS) is reduced in aged eggs (Kirunda and McKee, 2000). The protein elasticity is crucial for VMS. Oxidation reduces the protein elasticity and the VMS. The vitelline membrane protects the embryo (pH 6) from the high pH of the albumen (pH 9.4). If it is too weak then the embryo will die. The embryo progressively gets surrounded by the yolk sac and is totally protected in the yolk sac between day 3 and 4 of incubation (Sadler, 1995).

**Material and Methods**

The objective of this experiment was to study the influence of Canthaxanthin from Carophyll® Red on the VMS in eggs from breeder hens. The experiment involved a flock of 3,800 breeders (males and females). They were placed in a laying house equipped with a floor system (1/3 slat, 2/3 litter) and separated into two adjacent blocks. Control diet contained 3,000 IU Vitamin D₃. It was based on corn, wheat, soya and sunflower meals; Vit E 60 IU/kg, Vit A 14,000 IU/kg, Se 0.3 ppm (including 0.05 organic Se). Supplementation with MaxiChick® started 3 weeks before the laying period (at 18 weeks), males and females both supplemented with 1,600 IU/kg vitamin D₃, 37.5 µg/kg 25-hydroxy-vitamin D₃ (HyD®) & 6 ppm canthaxanthin. Vitamin D₃ or 25-hydroxy-vitamin D₃ were not expected to have any influence on the VMS. Average hatchability rates (% fertile eggs) were 72.2 % in the control and 77.6 % in the MaxiChick® group from 21 to 51 weeks of age (the flock was killed at 51 weeks because of sanitary concerns). Eggs were collected at 40/45 weeks and stored 7 days. The VMS was evaluated using a compression machine MTS Synergy 200 a cylindrical probe with a diameter of 2 mm. Compression speed was 42 mm/mn. Each egg was cracked and the egg content was spread

<footnote n="1">¹DSM, 19 avenue Dubonnet, 92400 Courbevoie, France</footnote>

<footnote n="2">²DSM, Wurmisweg 576, Kaiseraugst 4303, Switzerland</footnote>
on a plastic plate. The probe moved towards the yolk to penetrate through the yolk vitelline membrane in the equatorial region and the VMS was measured. Stiffness (or rigidity) is the property of a solid body to resist deformation. Breaking strength (or fracture strength) is the maximum force applied on the yolk before the breaking of the membrane occurs. Statistics were run with R 3.0.2 software. Data were analyzed for normality test (Shapiro) and were significantly different when p<0.05.

## Results/discussion

The VMS was significantly increased by the MaxiChick® supplementation.

<table><caption>Table 1. Vitelline membrane strength according to the treatment</caption><thead><tr><th></th><th>Control</th><th>MaxiChick®</th><th>Statistics (T test)</th></tr></thead><tbody><tr><td>Number eggs</td><td>209</td><td>172</td><td></td></tr><tr><td>VM Stiffness (mN/mm)</td><td>7.0 +/- 1.0</td><td>7.2 +/- 0.9</td><td>P&lt;0.05</td></tr><tr><td>VM breaking strength (mN/mm)</td><td>42.4 +/- 8.2</td><td>44.4 +/- 7.7</td><td>P&lt;0.05</td></tr></tbody></table>

Oxidation occurs in any bodies living in an atmosphere with oxygen $O_2$. Radical oxygen species (ROS) are derived from the use of $O_2$. They are unstable and react with lipids, protein and cause damages to the tissues. Singlet oxygen $^1O_2$ is a very reactive ROS and canthaxanthin quenches the energy of $^1O_2$ and release it as heat. No chemical reaction is involved and the carotenoid is not consumed. Canthaxanthin has a high singlet oxygen quenching capacity among commercially available carotenoids (Cantrell et al, 2002) because of its long symmetric chain of double bonds carbon (Figure 1). Canthaxanthin is also used to regenerate oxidized Vitamin E to be able to capture a new ROS (Surai, 2012). During the first 3-4 days of egg incubation, the main generation of ROS occurs inside the yolk coming from the oxidation of unsaturated fatty acids and the cell division of the embryo (Surai, 2012). Increasing dosages of Vitamin E from 50 to 150 IU/kg could strengthen the vitelline membrane (Scheideler et al, 2010). Selenium had no significant effect on VMS (Scheideler et al, 2010; Monsalve, 2004). Selenium is not an antioxidant per se. Selenium forms part of the active site of the Glutathione peroxidase which main function is the protection of the albumen and not the yolk or the vitelline membrane (Surai, 2002). Vitamin C is a well-known antioxidant but it is not deposited in the egg and not produced by the embryo before 3 days of incubation. Beta-carotene is not efficiently transferred to the yolk of chicken eggs. All-trans-canthaxanthin from Carophyll® is well absorbed and deposited in the yolk. According to Grashorn and Steinberg, 2002, the deposition rate was 40% in the yolk. The diets for breeders do contain some carotenoids from corn but in a low amount. Therefore vitamin E and canthaxanthin are the only antioxidants present in high enough quantities to be of any protective use in the yolk during the first 3-4 days of incubation.

<figure><img src="image_28.png" /><figcaption>Figure 1. Canthaxanthin</figcaption></figure>

## Conclusions

Thanks to the unique capabilities of high deposition rate and singlet oxygen quenching capabilities, canthaxanthin from Carophyll® can improve the vitelline membrane strength in complement to Vitamin E and we hypothesized that it contributes to explain its influence on early embryo viability and hatchability in breeder performance.
## Literature

KLECKER, D., L. ZEMAN, S. SISKE, 2009: Effects of trace elements on eggshell quality. Br. Poult. Sci. **89**, 100–108.

ALJAMAL, A., 2011: The Effect of Vitamin E, Selenomethionine and Sodium Selenite Supplementation in Laying Hens, Master thesis. University of Nebraska-Lincoln, April 2011

CANTRELL, A., DJ. MCGARVEY, TG. TRUSCOTT, F. RANCAN, F. BOHM, 2003: Singlet oxygen quenching by dietary carotenoids in a model membrane environment. Arc. Of Biochemistry and Biophysics 412, 47-54

GRASHORN, M.A., W. STEINBERG, 2002: Deposition rates of canthaxanthin in egg yolks. Archiv für Geflügelkunde 66(6), 258-262

KIRUNDA, D.F.K., SR. MC KEE, 2000: Relating Quality Characteristics of Aged Eggs and Fresh Eggs to Vitelline Membrane Strength as Determined by a Texture Analyzer. Poultry Sci. 79:1189–1193

MONSALVE, D., 2004: Nutritional factors affecting yolk vitelline membrane strength, oxidation and potential effects on whipping performance of egg white during summer time conditions. Master thesis. University of Nebraska-Lincoln.

SADLER, WW., 1955: Chronological Relationship of the Disappearance of the Vitelline Membrane and the Closure of the Amnio-Chorion in Avian Embyros and Its Implications. Poultry Sc., 34 (4): 754-760

SCHEIDELER, SE., P. WEBER, D. MONSALVE, 2010: Supplemental vitamin E and selenium effects on egg production, egg quality, and egg deposition of α-tocopherol and selenium, J. Appl. Poult. Res. 19: 354–360

SOUZA, RA., PA. SOUZA, RC. SOUZA, 2008: Efeito da Utilização de Carophyll® Red nos índices reprodutivos de matrizes de frangos de corte. Proc. APINCO UNESP Brazil

SURAI PF., 2002: Natural antioxidants in avian nutrition and reproduction. Nottingham University Press

SURAI, PF., 2012: The antioxidant properties of canthaxanthin and its potential effects in the poultry eggs and on embryonic development of the chick. Part 1. World's Poultry Science Journal, 68:465-476. Part 2. 68:717-726
P-029 (ID 244)

SUPPLEMENTATION OF BUTYRIC ACID PRODUCING PROBIOTIC VERSUS DIETARY BUTYRATE

V. Hautekiet<fnref n="1" />, A. Kanora<fnref n="1" />, and M. Takahashi<fnref n="2" />

Corresponding author: veerle.hautekiet@huvepharma.com

**Abstract**

In this study the efficacy of Miya-Gold®, a probiotic containing viable spores of Clostridium butyricum, on foot pad lesions versus dietary butyrate supplementation was tested.

Foot pad lesions were compared between a group supplemented with coated calcium butyrate and a group which received the probiotic Miya-Gold® in the feed at a concentration of 2.5*10⁸ CFU/kg.

Broilers supplemented with butyrate showed a significant higher water to feed ratio, which resulted in a higher number of foot pad lesions compared to the group supplemented with Miya-Gold®. If any lesions were present, Miya-Gold® birds showed mainly signs of small superficial lesions while 35% of the butyrate group had deep large lesions.

**Keywords:** Clostridium butyricum, probiotic, foot pad lesions

**Introduction**

Contact dermatitis of the foot pads is an important commercial condition in the rearing of broilers. The condition impairs the welfare of the animals while economic costs are incurred as a result of carcass downgrading.

Foot pad dermatitis is mainly linked to wet litter. Enhanced gut health, by inhibiting pathogens to infiltrate the lower part of the gut, can reduce the incidence and severity of pododermatitis by affecting the litter moisture content.

Butyric acid is known to stimulate gut health (Guilloteau et al, 2010). However, targeted release at the hind part of the gut by dietary supplementation of butyrate is uncertain. The probiotic Miya-Gold®, which contains viable spores of the anaerobic bacteria Clostridium butyricum, delivers the butyric acid produced by the Clostridium at the lower part of the GIT (Okamoto et al., 2000).

**Material and Methods**

One thousand seven hundred, commercial, one-day-old broilers (male, Ross 308) were divided at random in two groups with 5 replicates per group. The broilers were housed in a standard environment at LVFZ Kitzingen (Germany) under commercial rearing conditions. A three-phase feed programme was used and the birds were kept to 35 days of age.

One group received feed supplemented with coated calcium butyrate (0.75 kg/mton of feed in starter; 0.5 kg/mton of feed in grower and finisher). The other group received feed supplemented with Miya-Gold® in starter, grower and finisher at a concentration of 2.5*10⁸ CFU/kg.

The animals were individually weighed at day 0 and at 35 days of age. Feed intake and water intake were registered daily on pen level. At the end of the trial foot pad lesions were scored on a scale of 0 to 2 (0=no lesions, 1=mild, superficial lesions, 2= severe lesions, ulcers or swollen foot pads).

**Results**

There were no significant differences in weight gain, feed conversion and survival rate between both treatments. On average, daily gain for both treatments was 59 g/day with a feed conversion of 1.55. Mortality was 2.8% for the butyrate and the Miya-Gold® group.

A significant lower water to feed ratio was seen for the birds supplemented with Miya-Gold® versus the butyrate group (table 1). As a result, Miya-Gold® birds had significant less foot pad lesions (56% showed no lesions) than the birds supplemented with butyrate (14% showed no lesions). If any lesions were present, Miya-Gold® birds showed mainly signs of small superficial lesions while 35% of the butyrate group had deep large lesions (table 2).

<footnote n="1">¹ Huvepharma nv, Uitbreidingstraat 80, 2600 Antwerpen, Belgium</footnote>

<footnote n="2">² Miyarisan Pharmaceutical Co. Ltd., 114-0016 Tokyo, Japan</footnote>
<table><caption>**Table 1.** Water/feed ratio*</caption><thead><tr><td></td><td>butyrate</td><td>Miya-Gold®</td></tr></thead><tbody><tr><td>Total feed intake (kg)</td><td>3.238<sup>a</sup></td><td>3.157<sup>b</sup></td></tr><tr><td>Total water intake (l)</td><td>6.702<sup>a</sup></td><td>6.142<sup>b</sup></td></tr><tr><td>Water/feed ratio</td><td>2.070<sup>a</sup></td><td>1.946<sup>b</sup></td></tr></tbody></table>

Different letters mean statistically different at p < 0.05.
* recalculated per bird

<table><caption>**Table 2.** Foot pad lesions</caption><thead><tr><td></td><td>butyrate</td><td>Miya-Gold®</td></tr></thead><tbody><tr><td>0: no foot pad lesions (% of birds)</td><td>14<sup>a</sup></td><td>56<sup>b</sup></td></tr><tr><td>1: small superficial lesions (% of birds)</td><td>51<sup>a</sup></td><td>35<sup>b</sup></td></tr><tr><td>2: deep and large lesions (% of birds)</td><td>35<sup>a</sup></td><td>9<sup>b</sup></td></tr></tbody></table>

Different letters mean statistically different at p < 0.05.

**Conclusions**

Although the dietary butyrate supplement was coated, probably the butyric acid was not released in the second part of the gut resulting in minimal influence. The obligate anaerobic *Clostridium butyricum* in Miya-Gold® produces butyric acid only in the anaerobic regions of the gut and thus at the targeted hind part.

A significant lower water to feed ratio was observed by supplementing Miya-Gold®, resulting in less and less severe foot pad lesions compared to the group receiving the coated butyrate.

In commercial management systems, Miya-Gold® exerts extra advantages versus dietary butyrate supplementation by improving the health of the birds.

**Literature**

GUILLOTEAU, P., L. MARTIN, V. EECKHAUT, R. DUCATELLE, R. ZABLIENSKI, F. VAN IMMERSEEL, 2010: From the gut to the peripheral tissues: the multiple effects of butyrate. Nut. Res. Rev. 23, 366-384.

OKAMOTO, T., M. SASAKI, T. TSUJIKAWA, Y. FUJIYAMA, T. BAMBA, M. KUSUNOKI, 2000: Preventive efficacy of butyrate enemas and oral administration of *Clostridium butyricum* M588 in dextran sodium sulfate-induced colitis in rats. J. Gastroenterol. 35, 341-346.
P-031 (ID 196)

EFFECT OF MEDIUM CHAIN FATTY ACIDS ON BROILER PERFORMANCE IN ENERGY REDUCED DIETS

Hermans David, De Laet Manu, Van Meenen Ellen

Nuscience, Booiebos 5, 9031 Drongen, Belgium

Corresponding author: manu.de.laet@nusciencegroup.com

Abstract

It is known that medium chain fatty acids (MCFA) have a positive effect on broiler performance when added on top of a control diet. In this experiment a total of 480 Ross 308 one-day-old male broilers were used to determine whether a carefully balanced mixture of medium chain fatty acids [Aromabiotic® Poultry containing 60% MCFA] can replace energy in the diet without impairing performance. A completely randomized design was applied with three experimental treatments. A standard corn/wheat/soy based diet was used as a positive control (PC) and Aromabiotic® Poultry was supplemented to a -150 kcal reduced control diet (NC) at a dosage of 0.16%, 0.12% and 0.08% to the starter, grower and finisher diet respectively. The experimental design was applied to 24 pens (20 broilers/pen) with 160 broilers per treatment until 38 d. The supplementation of the negative control diet (NC) with Aromabiotic® Poultry improves body weight significantly (2196 g vs 2108 g; P < 0.05) and brings back growth performance to the original level of the positive control (2196 g vs 2187 g). WAFCR is numerically improved with Aromabiotic® Poultry (1.65 vs 1.73; trend) and again no significant differences were observed with the positive control (1.65 vs 1.63). Although the latter difference is not significant the numerical difference is still there. It is concluded that Aromabiotic® Poultry is an interesting functional feed ingredient for replacing energy but further research is needed to calculate the exact energy equivalent.

**Keywords:** MCFA, Aromabiotic, reducing energy, antibacterial, intestinal morphology

Introduction

For years the focus of broiler breeding companies has been the improvement of growth rate and feed conversion. This 'less for more' strategy has put tremendous stress on the health of the animals. To be able to reach the increased potential of genetic selection birds not only need a well-balanced diet but also benefit to a large extent from functional feed ingredients. With oil and fat prices around 800 euro / MT, energy is the major cost factor in diets for high performing animals. Broiler chickens have traditionally been fed relatively high-energy diets, because, in addition to promoting efficient feed utilization, it is also assumed that this type of diet maximizes growth rate (Leeson and Summers, 1991). If medium chain fatty acids are added to a broiler feed, it is known that performances of broilers will be better. With an inclusion rate of 0.8 g/kg Aromabiotic® Poultry (containing 60 % medium chain fatty acids (MCFA)), an increase in weight of 90 g and a decrease of 5 points WAFCR have been shown (Deschepper et al., 2013). Since it is well known that FCR depends strongly on the energy density of the diet (Holsheimer and Veerkamp, 1992), it was the aim of this experiment to determine to what extent Aromabiotic® Poultry can replace energy in broiler diets.

**Material and Methods**

In the described trial, a total of 480 male broiler Ross 308 chicks were used (Origin was Belgabroed Hatchery, Merksplas, Belgium). At the beginning of the trial, all transport boxes are weighed and the average weight of the chickens is determined. After weighing the day-old chickens individually, 24 groups are made. Only the day-old chicks with a weight of the average weight + or - 3 g could participate in this trial. The birds were housed in an experimental farm in commercial conditions in Meigem, Belgium. There were 3 treatments: a positive control with 2800 kcal/kg, 2900 kcal/kg and 2950 kcal/kg in the starter, grower and finisher feed, a negative control (positive control - 150 kcal / kg) and a negative control + Aromabiotic® Poultry. Aromabiotic® Poultry is delivered by Nuscience and contains 60 % MCFA (C6, C8, C10, C12) on a support of silicium dioxide. The dosage of Aromabiotic® Poultry was respectively 1.6; 1.2 and 0.8 g/kg for the starter, the grower and the finisher. There were 8 replicates of 20 birds / replicate per treatment (480 birds in total). A randomized block design was used. The broilers were vaccinated the 1st day of age against Newcastle Disease (NDW, spray) and Infectious Bronchitis (Poulvac IB Primer, spray). At 16 days of age the vaccination against Newcastle Disease was repeated with La Sota (Clone 30, drinking water). No other treatments were administered to the animals during the entire study period. The diet was wheat/corn/soy based and was formulated as shown in table 1. All treatments had a 3-phase feeding scheme (starter-grower-finisher).
<table><caption>**Table 1:** Wheat/corn/soy based diets, positive control and negative control (-150 kcal / kg)</caption><thead><tr><th>Nutrient</th><th>Starter</th><th>Starter<br>-150 kcal/kg</th><th>Grower</th><th>Grower<br>-150 kcal/kg</th><th>Finisher</th><th>Finisher<br>-150 kcal/kg</th></tr></thead><tbody><tr><td>ME broiler (kcal/kg)</td><td>2800</td><td>2650</td><td>2900</td><td>2750</td><td>2950</td><td>2800</td></tr><tr><td>Crude protein (g/kg)</td><td>215</td><td>215</td><td>203</td><td>203</td><td>190</td><td>190</td></tr><tr><td>Crude fat (g/kg)</td><td>59.7</td><td>31.9</td><td>58.4</td><td>31.4</td><td>58.7</td><td>31.7</td></tr><tr><td>Ca (g/kg)</td><td>9.0</td><td>9.0</td><td>7.2</td><td>7.2</td><td>6.4</td><td>6.4</td></tr><tr><td>Dig P (g/kg)</td><td>4.1</td><td>4.1</td><td>3.2</td><td>3.2</td><td>2.8</td><td>2.8</td></tr><tr><td>Dig Lys (g/kg)</td><td>10.4</td><td>10.4</td><td>9.6</td><td>9.6</td><td>8.0</td><td>8.0</td></tr><tr><td>Dig M+C (g/kg)</td><td>7.6</td><td>7.6</td><td>7.3</td><td>7.3</td><td>6.1</td><td>6.1</td></tr><tr><td>Dig Thr (g/kg)</td><td>6.8</td><td>6.8</td><td>6.2</td><td>6.2</td><td>5.5</td><td>5.5</td></tr></tbody></table>

Daily the animals and housing facilities were inspected for the general health status, constant feed and water supply as well as temperature and ventilation, dead birds, and unexpected events. Daily mortality and cullings were recorded for each pen. Corrections for mortality calculating zootechnical performances were done using the number of ‘broiler days’ (number of broilers x days alive). For each trial, average pen weight was recorded at day 38. Feed intake was recorded. Feed conversion, daily growth rate and daily feed intake per bird were calculated. All parameters were subjected to a 2-factorial (treatment, block) analysis of variance (ANOVA, JMP).

## Results and discussion

At arrival, one-day old Ross 308 birds showed a good quality with an average body weight of 43.8 g. The results of the trial are summarized in Table 2.

<table><caption>**Table 2:** Zootechnical performances</caption><thead><tr><th></th><th>Positive control</th><th>Negative control<br>(-150 kcal / kg)</th><th>Negative control<br>+ Aromabiotic® Poultry</th></tr></thead><tbody><tr><td>Average daily gain<br>(g / bird / day)</td><td>54.9<sup>a</sup></td><td>52.9<sup>b</sup></td><td>55.2<sup>a</sup></td></tr><tr><td>Feed intake<br>(g / bird / day)</td><td>91.4</td><td>91.5</td><td>93.2</td></tr><tr><td>Body weight<br>(g / bird)</td><td>2187<sup>a</sup></td><td>2108<sup>b</sup></td><td>2196<sup>a</sup></td></tr><tr><td>WAFCR<br>(2500 g)</td><td>1.63<sup>a</sup></td><td>1.73<sup>b</sup></td><td>1.65<sup>ab</sup></td></tr></tbody></table>

As expected, reducing energy has a negative effect on the end weight (2108 g vs 2187 g, P < 0.05) and on the WAFCR (1.73 vs 1.63, P < 0.05). These results are similar to the observations of Holsheimer and Veerkamp (1992) who found a lower end weight and a higher FCR when low energy diets are used. If Aromabiotic® Poultry is supplemented to this low energy diet, technical performances are almost back at the same level of the positive control group. The weight of the Aromabiotic® Poultry group is significantly different from that of the Negative control group (2196 g vs 2108 g, P < 0.05) and not significantly different from the Positive control group (2196 g vs 2187 g). The WAFCR of the Aromabiotic® Poultry group is not significantly different from that of the Negative (1.65 vs 1.73) and Positive control group (1.65 vs 1.63). Numerically the WAFCR of the Aromabiotic® Poultry group is clearly improved, but the Aromabiotic® Poultry group was not able to reach the original level of the Positive control group.

<footnote><sup>a,b</sup> numbers on one row with different superscripts are significantly different (P < 0.05)</footnote>
It is clear that Aromabiotic® Poultry has a positive effect on the technical performances in an energy-reduced diet, possibly due to the antimicrobial activity of MCFA (Kabara et al, 1972; Skrivanova et al; 2006; Batovska et al, 2009). This may lead to a lower intestinal infection pressure and an improved intestinal morphology, resulting in better digestive and absorptive capacities (Deschepper et al, 2003). On the one side, the energy that the birds usually use for this antibacterial activity will be taken over by Aromabiotic® Poultry, and this spared energy can be used for extra growth and a better WAFCR. On the other side, the energy from the feed will be absorbed more efficiently due to the improved intestinal morphology. In this case, if we look at the WAFCR, the energy that can be replaced will be around 120 kcal / kg.

## Conclusions

In conclusion, Aromabiotic® Poultry proved to be very effective in replacing energy in broiler diets. Further research, however, is needed to determine the exact energy value Aromabiotic® Poultry is able to replace (without hampering zootechnical performance), in a given situation.

## Literature

BATOVSKA D.I., TODOROVA I.T., TSVETKOVA I.V., NAJDENSKI H.M., 2009: Antibacterial study of the medium chain fatty acids and their1-monoglycerides: Individual effects and synergistic relationships – Polish Journal of Microbiology 58, 43-47

DESCHEPPER K., LIPPENS M., HUYGHEBAERT G., MOLLY K., 2003: The effect of aromabiotic and gali d'or on technical performances and intestinal morphology of broilers – Proceedings of the 14th European Symposium of Poultry Nutrition, 189-190

DESCHEPPER K., GANTOIS I., MAERTENS L., VAN MEENEN E., 2013: A balanced mixture of medium chain fatty acids improves zootechnical performances and slaughter results of broilers in – Proceedings of the 19th European Symposium of Nutrition, PPV-10

HOLSHEIMER J.P., VEERKAMP C.H., 1992: Effect of dietary energy, protein, and lysin content on performance and yields of two strains of male broiler chicks – Poultry Science 71, 872-879

KABARA J.J., SWIECZKOWSKI D.M., CONLEY A.J., TRUANT J.P., 1972: Fatty Acids and Derivatives as Antimicrobial Agents – Antimicrobial Agents and Chemotherapy 2, 23-28

SKRIVANOVA E., MAROUNEK M., Benda V., BREZINA P., 2006: Susceptibility of Escherichia coli, Salmonella sp. and Clostridium perfringens to organic acids and monolaurin – Veterinary Medecine 51, 81-88
P-032 (ID 325)

GROWTH PROMOTING EFFECT OF ASPERGILLUS LUCHUENSIS IN BROILER

K. Hioki, C. Kawasaki, M. Yamamoto and K. Hayashi

Biogenkoji Research Institute

Corresponding author: hioki@genkoji.com

Abstract

Four experiments were conducted to assure the growth promoting effect of a fungus *Aspergillus luchuensis* in broiler chickens (Ross 308). After optimum feeding levels of the fungus culture (Koji) was determined in experiment 1, sex difference of the growth promoting effect was tested in experiment 2. In experiment 3, the effects of Koji with different xylanase activities on the growth of the chicken were examined, and the concerted effect of *A. luchuensis* and *Clostridium butyricum* was evaluated in experiment 4. The Koji of *A. luchuensis* and *C. butyricum* culture were mixed with the basal experimental diet. The basal diet was formulated mainly by corn and soybean meal. The birds were raised using steel cages in a temperature controlled room with 20h light and given experimental diets ad libitum during 16 days experimental period. Significant growth promoting effect was observed when the level of the Koji was 0.04% while there was little effect when the levels were 0.002, 0.02 and 0.2%. The growth promoting effect was higher but not significantly in male birds than female birds. The Koji of *A. luchuensis* contained significant xylanase activity and the growth rate of the birds given Koji with higher xylanase activity tended to be higher than those given Koji with lower activity. Finally, we showed that simultaneous dose of *A. luchuensis* and *C. butyricum* accelerated the growth more than the birds given *A. luchuensis* alone.

**Keywords:** broiler, growth performance, *Aspergillus luchuensis*

Introduction

The present Koji (fungus culture) is a solid state fermentation product of harmless *Aspergillus* using cereals such as rice, barley and soybean as substrates. In Japan, Koji has been widely used for processing foods such as Miso, Shoyu and Sake. Koji contains many kinds of enzymes such as amylase, protease and lipase and these enzymes play essen-
tial roles in processing foods. Black Koji (culture of black *Aspergillus*) is mainly used in Okinawa and southern Kyusyu for the production of shochu a kind of liquor. Black *Aspergillus* produces citric acid and thus lowers pH of the fermented products. We have already reported that dietary supplementation of a black Koji (*Aspergillus luchuensis*) improves per-
formance and meat quality in broiler (Saleh et al, 2011, 2012). We have also shown that digestibility is improved and intestinal profile of short chain fatty acids such as acetate, propionate and butyrate is changed by the Koji (Hioki et al in
press). The present study was conducted, 1)to determine the optimal feeding level of the Koji, 2)to examine the sex
difference of the effect, 3)to show the relation between xylanase activity and growth promoting effect and also 4)to show
the concerted effect of *A. luchuensis* and butyric acid bacteria (*C. butyricum*) on the performance in broiler.

Material and Methods

1. Peperation of black Koji

Rice was soaked and drained and then steamed for 1 hour. After cooling down to 36 °C the rice was inoculated with *A. luchuensis* (Kawachi genichiro shouten, Japan) and fermented at 34-38°C for 5 days.

2. Animal experiment

In all the experiments, Ross 308 broiler chickens and commercial basal diet (Japan Scientific Feeds Association) were
used. Water and feed were given ad libitum. Pairs of male and female birds (n=6) were raised in steel cages in the ex-
periments except experiment 2. In experiment 2, male and female birds (n=6) were raised individually. The experimen-
tal periods were from 7 to 23 days of age for experiments 1 and 2, from 15 to 31 days of age in experiment 3 and from
12 to 28 days of age in experiment 4. Body weight and feed intake were measured every 4 days and feed conversion
ratio was calculated. Experimental diets were prepared as shown below.
* Experiment 1 Basal diets supplemented with 0, 0.002%, 0.02%, 0.04% and 0.2% Koji (A. luchuensis) were prepared.

* Experiment 2 Basal diet supplemented with 0.04% Koji (A. luchuensis) was used.

* Experiment 3 Basal diets supplemented with 0.04% Koji (A. luchuensis) with high and low Xylanase (LX) activities were prepared

* Experiment 4 Basal diets supplemented with 0.04% Koji (A. luchuensis), 0.2% C. butyricum culture and both 0.04% Koji (A. luchuensis) and 0.2% C. butyricum culture were prepared

## Results

All the results were shown together in Figure 1. Figure 1-A showed that the growth performance was the best when 0.04% Koji diet was given. The difference between control and the 0.04% group was statistically significant (Experiment 1). Figure 1-B shows that the effects of Koji was stronger in male than female (Experiment 2). Figure 1-C (Experiment 3) shows that Koji with high xylanase activity improved the performance more than that with the low xylanase activity. Figure 1-D (experiment 4) shows the concerted effect of A. luchuensis and C. butyricum on the growth performance.

<figure><img src="image_29.png" /><figcaption>Figure 1. The differential effects of Koji on body weight gain due to feeding level(A), sex of chickens(B), xylanase activity(C) and combined supplementation with C. butyricum(D)</figcaption></figure>

## Conclusions

Growth promoting effect of Koji is highest at 0.04% feeding level especially in male chicken. The effect is higher when the xylanase activity is higher, and simultaneous feeding of C. butyricum stimulates the growth more.

## Literature

SALEH AA, YZ EID, TA EBEID, T KAMIZONO, A OHTSUKA, K HAYASHI. 2011. Effects of feeding *Aspergillus awamori* and *Aspergillus niger* on growth performance and meat quality in broiler chickens. Journal of Poultry Science, 48:201-206

SALEH AA, YZ EID, TA EBEID, A OHTSUKA, K HIOKI, M YAMAMOTO, K HAYASHI. 2012. The modification of the muscle fatty acid profile by dietary supplementation with *Aspergillus awamori* in broiler chickens. British Journal of Nutrition, 108:1546-1602
P-034 (ID 263)

EFFECT OF DIFFERENT LEVELS OF ARTICHOKE (CYNARASCOLYMUS L.) EXTRACT SUPPLEMENTATION IN DRINKING WATER ON THE IMMUNOLOGICAL RESPONSE AND YOLK RETENTION OF BROILER CHICKENS

Patipan Jaipeng, Jamlong Mitchaothai, Kornkamon Paditporn, Rachakris Lertpatarakomol and Sunisa Sirimongkolvorakul*

Mahanakorn University of Technology, Thailand

*Corresponding author: sirikul Sunii@hotmail.com

Abstract

This study was conducted to determine the effects of different levels of artichoke (Cynarascolymus L.) extract supplemented in drinking water on the immune response and yolk retention of broiler chickens. A total of 272 one-day-old broiler chicks (Ross 308) were randomly assigned to 16 battery cages and four treatment groups with 17 chickens per cage and 4 replicates per treatment. Treatments included the regular drinking water as control or the drinking water supplemented with 75 ppm. (T₁), 150 ppm. (T₂) and 300 ppm. (T₃) of artichoke. The drinking water was offered ad libitum throughout the study. Body weighted (BW) and yolk mass per body weight (YB) ratio were measured during experimental period. Blood samples were taken for measuring immune response. After 5 week, T₂ and T₃ showed higher hemagglutination inhibition titer against Newcastle disease virus (NDV) compared with control group (P<0.05). Other measured parameters, including BW, Y:B ratio and IgG against SRBC were not affected by the treatments (P>0.05). In conclusion, supplemented with 150 ppm. and 300 ppm. of artichoke in drinking water improved antibody-mediated immunity. Overall, high-level of artichoke appears to increase the immune response of broiler receiving the extract as supplement.

**Keywords:** Artichoke extract, broilers, body weight, yolk retention, immune response

Introduction

Rapid development of poultry production has increased stress and increased the risk of disease outbreaks in poultry industry. Under such conditions, antibiotics are used for therapy and growth-promotion in poultry drinking water, which have contributed to the increasing number of antibiotic resistant infection. Artichoke (Cynarascolymus L.) is a medicinal plant which rich in cynarin, flavonoids, and phenolic acids. Previously document nutritive effects of artichoke have been studied. Accordingly, the aim of this study was to evaluate different levels of artichoke in drinking water on the immune response and yolk retention of broilers.

Material and methods

A total of 272 one-day-old broiler chicks (Ross 308) were randomly assigned to 16 battery cages and four treatment groups with 17 chickens per cage and 4 replicates per treatment. Treatments included the regular drinking water as control or the drinking water supplemented with 75 ppm. (T₁), 150 ppm. (T₂) and 300 ppm. (T₃) of artichoke. The drinking water was offered ad libitum in two phases: starter (1-7 days) and grower (15-24 days). Chickens were vaccinated following the standard broiler vaccination schedule during the whole experimental period. The chicken were weighted and collected for determine yolk mass:body weight ratio (Y:B ratio) at 1 and 7 days. In order to assay antibody responses to sheep red blood cell (SRBC), two chickens in each replicate were injected intramuscularly with 1mL, suspension of 7%SRBC in phosphate buffer saline on days 28. The hemagglutination inhibition (HI) test against newcastle's disease virus (NDV) was also set up to determine the antibody titer.

Results

As Table 1 indicates, Y:B ratio of broilers feeding diets containing artichoke at 0, 75, 150, and 300 ppm had no significant different. However, decreased Y:B ratio were observed in the chickens fed diet T₂. The results obtained in this study suggest that the presence of the yolk sac in a chick beyond day 7 should be considered as retained. The suitable rate retained is necessary for initiation of growth and the utilization of diet.
<table><caption>**Table 1.** The effects of different dietary additives on yolk mass:body weight ratio (Y:B ratio) in broiler chickens at 1 and 7 day.</caption><thead><tr><th rowspan="3">Day</th><th colspan="4">Dietary treatments</th></tr><tr><th rowspan="2">Control</th><th colspan="3">Artichoke extract</th></tr><tr><th>75 ppm</th><th>150 ppm</th><th>300 ppm</th></tr></thead><tbody><tr><td>1</td><td>9.34±2.18</td><td>8.29±2.81</td><td>8.54±3.12</td><td>7.11±1.38</td></tr><tr><td>7</td><td>0.18±0.09</td><td>0.14±0.11</td><td>0.11±0.07</td><td>0.14±0.09</td></tr></tbody></table>

The effects of experimental treatments on HI-titer against NDV is summarized in Table 2. Administration of artichoke had no effects on HI-titer against NDV as similar to other studies that found that artichoke had no effect on immune response (Fallen *et al*, 2013 and Tajodini *et al*, 2014).

<table><caption>**Table 2.** The effects of different dietary additives on Hemagglutination inhibition titer (HI-titer) against newcastle's disease virus (NDV).</caption><thead><tr><th rowspan="3">Week</th><th colspan="4">Dietary treatments</th></tr><tr><th rowspan="2">Control</th><th colspan="3">Artichoke extract</th></tr><tr><th>75 ppm</th><th>150 ppm</th><th>300 ppm</th></tr></thead><tbody><tr><td>0</td><td>5.50±1.22</td><td>6.75±0.65</td><td>6.13±1.10</td><td>6.63±0.85</td></tr><tr><td>1</td><td>5.25±0.87</td><td>5.50±0.41</td><td>5.00±0.41</td><td>5.25±0.29</td></tr><tr><td>2</td><td>3.63±0.75</td><td>4.25±0.29</td><td>3.25±0.87</td><td>3.38±0.63</td></tr><tr><td>3</td><td>4.13±1.25</td><td>5.00±1.47</td><td>4.50±1.00</td><td>4.75±0.87</td></tr><tr><td>4</td><td>4.88±1.25</td><td>4.75±0.87</td><td>3.38±1.31</td><td>4.00±1.22</td></tr><tr><td>5</td><td>5.00±0.41</td><td>5.25±0.96</td><td>6.33±0.63</td><td>6.78±0.71</td></tr><tr><td>6</td><td>4.75±0.29</td><td>4.63±0.75</td><td>5.38±0.48</td><td>4.75±1.32</td></tr></tbody></table>

Antibody titer against sheep red blood cell (SRBC) is summarized in Table 3. There were no significant different (*p*<0.05) in antibody titer among groups.

<table><caption>**Table 3.** The effects of different dietary additives on antibody titer against sheep red blood cell (SRBC).</caption><thead><tr><th rowspan="2">Group</th><th colspan="2">IgG level (u/mL)</th></tr><tr><th>7 days</th><th>14 days</th></tr></thead><tbody><tr><td>Control</td><td>32.41±24.76</td><td>23.40±17.08</td></tr><tr><td>T<sub>1</sub> (75 ppm)</td><td>19.53±4.90</td><td>12.76±10.02</td></tr><tr><td>T<sub>2</sub> (150 ppm)</td><td>41.74±27.82</td><td>8.69±6.48</td></tr><tr><td>T<sub>3</sub> (300 ppm)</td><td>35.67±15.52</td><td>8.79±2.91</td></tr></tbody></table>

## Conclusions

Regarding the present study, the effects of artichoke on the immune function are rare. However, high-level of artichoke appears to increase the immune response of broiler receiving the extract as supplement.

## Literature

FALLAH, R., KIANI, A., AZARFAR, A., 2013: Effect of Artichoke Leaves Meal and Mentha Extract (*Mentha piperita*) on Immune Cells And Blood Biochemical Parameters of Broilers. GV. **10**, 99-102.

TAJODINI, M., SAMDI, F., HASHEMI, S.R., SHAMS-SHARGH, M., 2014: Effect of different level of Artichoke (*Cynarascolymus* L.) poder on the performance and immune response of broiler chickens. Int. J. Agr Sci. **4**: 66-73.
P-035 (ID 330)

EFFECT OF A PHYTOGENIC FEED ADDITIVE ON PROTEIN AND AMINO ACID DIGESTIBILITY IN BROILERS

L. Jungbauer, A. Müller, J.D. van der Klis

Delacon Biotechnik Gmbh, Steyregg, Austria

Corresponding author: leopold.jungbauer@delacon.com

Abstract

The objective of this study was to investigate the effects of a phytogenic feed additive (PFA; Biostrong 510, Delacon) consisting of essential oils, herbs and spices on ileal protein and amino acid digestibility in 21-day-old broilers. 72 day-old Cobb 500 broilers were randomly allocated to two dietary treatments. Each treatment was replicated 9 times with 4 birds per replicate. Birds were fed a corn/wheat/soy-based mash diet with 0 mg/kg PFA (control) or 150 mg/kg PFA (treatment). Feed and water were available for ad libitum intake. Titanium oxide was used as inert dietary marker (dose 5 g/kg) to enable measurement of the apparent ileal digestibility values on day 21. Ileal protein digestibility was significantly (P<0.05) improved by 5.1% in birds fed the PFA. Digestibility values for amino acids were increased by on average by 1 to 4 percentage units by the PFA, but effects were only statistically significantly demonstrated for four amino acids at P<0.05 and for three amino acids at P<0.10. Body weight gain and feed intake were not significantly different between the two treatments, whereas feed conversion ratio tended (P<0.10) to be better by dietary PFA supplementation. This study showed the tested PFA improved protein digestibility in broiler, being supported by amino acid digestibility values.

**Keywords:** phytogenic, broiler, digestibility, amino acids

Introduction

Phytogenic feed additives are plant derived and numerous growth and health promoting effects have been shown in poultry. These benefits mainly comprise effects on secretion of digestive enzymes and bile (JAMROZ et al., 2003; PLATEL and SRINIVASAN, 2004), and on intestinal morphology (AMAD et al., 2013). Positive effects on nutrient digestibility values were shown not to be age-depended in broilers (AMAD et al., 2011). Moreover, Utterback at al. (2013) demonstrated positive effects on amino acid digestibility in laying hens. However, data on the effects of phytogenic feed additives on ileal amino acid digestibility in broilers is limited. Therefore an experiment was done to investigate the effects of a phytogenic feed additive on amino acid digestibility in broiler. (UTTERBACK et al., 2013)

Material and Methods

A total of 72 day-old male Cobb 500 chickens were randomly allocated to two dietary treatments. Each treatment was replicated 9 times with each 4 broilers. Broilers were housed in stainless steel cages with a base area of 0.19 m².

Birds of the control group (CON) were fed a diet based on soybean meal, corn and wheat from day 1 till day 21. Treated birds (PFA) were fed the basal diet supplemented with a phytogenic feed additive (Biostrong 510, Delacon Biotechnik GmbH) at 150 mg/kg. Diets were formulated to meet the nutrient recommendations given by GFE (1999). Feed and water were available for ad libitum intake.

Production performance (body weight, feed intake and feed conversion ratio) was measured per cage from day 1 to 21 of age. For measuring apparent ileal amino acid digestibility birds were killed after stunning on day 21. Digesta were collected from the entire ileum (segment from Meckel's diverticulum to about 3 cm cranial to the ileocaecal junction). Ileal digesta were flushed with distilled water of the intestinal segment and were pooled per cage. Diets and ileal digesta were analyzed for crude protein, AA and Ti contents. Titanium Oxide (TiO₂) was supplemented to the diets as inert marker at 5 g/kg.

Performance and digestibility data were statistically analyzed using SAS. After checking the homogeneity of the variances, analysis of variance was performed (proc glm) and means were compared (Tukey test). The significance level was set at p ≤ 0.05.
## Results

The effects of the addition of the PFA on protein and amino acid digestibility are summarized in Table 1. PFA supplementation significantly (P<0.05) improved crude protein digestibility by 5.1% in 21 day broiler. Digestibility of total amino acids (TAA) was also significantly improved in birds fed the PFA. Digestibility of Lys, Ala, Gly and Glu were significantly improved (P<0.05) and digestibility of Pro, His, Leu and Asp tended (P<0.10) to be improved by the PFA.

<table><caption>**Table 1.** Effects of a phytogenic feed additive (PFA) on protein and amino acid digestibility in 21-day old broilers, when compared to an unsupplemeted control (CON) diet.</caption><thead><tr><th></th><th>CON</th><th>PFA</th><th>P-value</th></tr></thead><tbody><tr><td>Crude Protein</td><td>80.8 ± 2.3</td><td>84.9 ± 1.7</td><td>&lt;0.001</td></tr><tr><td>Asp</td><td>78.6 ± 2.5</td><td>81.0 ± 3.0</td><td>0.075</td></tr><tr><td>Thr</td><td>74.6 ± 3.3</td><td>77.4 ± 3.7</td><td>0.116</td></tr><tr><td>Ser</td><td>80.6 ± 2.5</td><td>82.8 ± 2.9</td><td>0.109</td></tr><tr><td>Glut</td><td>82.6 ± 2.2</td><td>84.9 ± 2.2</td><td>0.040</td></tr><tr><td>Gly</td><td>76.1 ± 2.6</td><td>79.3 ± 3.2</td><td>0.034</td></tr><tr><td>Ala</td><td>79.0 ± 2.5</td><td>81.8 ± 2.9</td><td>0.046</td></tr><tr><td>Val</td><td>76.7 ± 2.6</td><td>79.0 ± 3.5</td><td>0.143</td></tr><tr><td>Iso</td><td>80.1 ± 2.4</td><td>81.6 ± 3.6</td><td>0.332</td></tr><tr><td>Leu</td><td>82.6 ± 2.0</td><td>84.6 ± 2.6</td><td>0.088</td></tr><tr><td>Tyr</td><td>77.2 ± 4.0</td><td>75.4 ± 5.7</td><td>0.437</td></tr><tr><td>Phe</td><td>85.3 ± 1.9</td><td>86.3 ± 2.4</td><td>0.305</td></tr><tr><td>His</td><td>85.3 ± 1.6</td><td>87.1 ± 2.1</td><td>0.058</td></tr><tr><td>Lys</td><td>83.8 ± 1.9</td><td>85.9 ± 2.1</td><td>0.042</td></tr><tr><td>Arg</td><td>88.3 ± 1.5</td><td>89.1 ± 1.9</td><td>0.338</td></tr><tr><td>Pro</td><td>84.2 ± 2.0</td><td>85.5 ± 2.4</td><td>0.075</td></tr><tr><td>Met</td><td>94.1 ± 1.7</td><td>93.5 ± 1.5</td><td>0.116</td></tr><tr><td>Cys</td><td>71.6 ± 2.9</td><td>72.9 ± 3.7</td><td>0.109</td></tr><tr><td>TAA</td><td>81.0 ± 2.2</td><td>82.9 ± 2.8</td><td>0.040</td></tr></tbody></table>

Performance data for the whole trial period are shown in Table 2. Feed conversion ratio (FCR) tended to be improved (P<0.10) by 4.7% in birds fed the PFA. No significant differences were observed between the two groups in body weight gain (BWG) and feed intake (FI).

<table><caption>**Table 2.** Effects of a phytogenic feed additive (PFA) on performance in 21-day old broiler, when compared to an unsupplemeted control (CON) diet.</caption><thead><tr><th></th><th>Con</th><th>PFA</th><th>P-value</th></tr></thead><tbody><tr><td>BWG, g/d</td><td>750 ± 53</td><td>779 ± 53</td><td>0.277</td></tr><tr><td>FI, g/d</td><td>926 ± 87</td><td>916 ± 77</td><td>0.804</td></tr><tr><td>FCR, g/g</td><td>1.234 ± 0.074</td><td>1.176 ± 0.062</td><td>0.092</td></tr></tbody></table>

## Discussion and Conclusions

In the current study the tested PFA (Biostrong 510, Delacon, Austria) significantly improved ileal protein digestibility. This finding is in line with the results shown by Amad et al. (2011). In their publication a significant improvement of ileal protein digestibility was shown in broilers at 21 days, 35 days and 42 days of age when fed a phytogenic feed additive. Utterback at al. (2013) have reported a positive effect of a phytogenic feed additive on amino acid digestibility in laying hens. Similar to that publication, results of the current study revealed that feeding the phytogenic additive part of the amino acid digestibility values were significantly improved or tended to be. Improved protein and amino acid digestibility is most likely linked to an increased activity of pancreatic proteases through PFA feeding. Platel and Srinivasan (2004) have shown that the addition of phytogenic substances increases the activity of intestinal proteases in rats, whereas previous research has shown that the tested PFA increases activity of pancreatic enzymes in the lumen from 10 to 25 % (data not shown).
From the current study it is concluded, that protein and amino acid digestion was improved which resulted in an improved feed efficiency in broilers.

## Literature

AMAD, A. A., MÄNNER, K., WENDLER, K. R., NEUMANN, K., and ZENTEK, J. 2011: Effects of a phytogenic feed additive on growth performance and ileal nutrient digestibility in broiler chickens. Poultry Science. 12, 2811-2816.

AMAD, A. A., WENDLER, K. R., and ZENTEK, J. 2013: Effects of a phytogenic feed additive on growth performance, selected blood criteria and jejunal morphology in broiler chickens. Emir.J.Food Agric. 7, 549-554.

JAMROZ, D., ORDA, J., KAMEL, C., WILCZKIEWICZ, A., WERTELECKI, T., and SKORUPINSKA, J. 2003: The influence of phytogenic extracts on performance, nutrient digestibility, carcass characteristics, and gut microbial status in broiler chickens. Journal of Animal and Feed Sciences. 583-596.

PLATEL, K. and SRINIVASAN, K. 2004: Digestive stimulant action of spices: a myth or reality? Indian J.Med.Res. 5, 167-179.

UTTERBACK, P., SPANGLER, H., PARR, C., AARDSMA, M., MERRIMAN, L., PUGH, J., HILGENDORF, D., UTTERBACK, C., JUNGBAUER, L., and PARSONS, C. 2013: Effects of Biostrong 510 on Performance and Amino Acid Digestibility in Laying Hens. Poultry Science Annual Meeting 2013. Poultry Science 92 E-Suppl.1. 125.
P-036 (ID 331)

# EFFECTS OF COMBINING PHYTOGENIC SUBSTANCES WITH ESTERS OF SHORT AND MEDIUM CHAIN FATTY ACIDS ON PRODUCTION PERFORMANCE IN BROILERS

L. Jungbauer, Jan Dirk van der Klis

Delacon Biotechnik Gmbh, Steyregg, Austria

Corresponding author: leopold.jungbauer@delacon.com

## Abstract

An experiment was conducted to evaluate the effects of the combination of a phytogenic feed additive (PFA) and esters of short and medium chain fatty acids (SMFA) depending on duration of application on production performance in a subclinical necrotic enteritis infection in broiler chickens. A total 1'600 male Cobb x Cobb broilers were randomly allotted to four dietary treatments, each with 8 repetitions. Treatments were: infected non-treated (INT) and 3 groups infected treated (IT) with 750 mg/kg PFA+SMCFA till day 21 (T1), day 28 (T2) and day 35 (T3), all followed by PFA alone (150 mg/kg) till harvest at day 49. Birds were fed a corn/soy based diet for ad libitum intake. All birds were vaccinated against coccidiosis at day 1 (Coccivac-B) and challenged with a culture of C. perfringens on days 19, 20, and 21. On day 21 five birds per pen were necropsied for NE lesion scoring. Results on day 49 show that birds in all IT groups had significantly (p<0.05) better FCR compared to birds of the control. FCR between IT groups didn't differ significantly. However, with prolonged application FCR was numerically improved. Body weight on day 49 tended (p<0.10) to increase when birds fed combination of PFA and SMCFA followed by PFA alone. On day 21 NE lesions were significantly reduced (P<0.01) in birds fed PFA and SMCFA. It was concluded that the application of this combination of PFA and SMCFA followed by PFA alone till harvest significantly improved production performance and reduced NE related gut lesions.

**Keywords:** necrotic enteritis, health, performance, broiler

## Introduction

Currently the ban on the use of feed antibiotics is becoming common use in more and more countries. By the end of 2016 the USA will also ban in-feed antibiotics. Necrotic enteritis, caused by *Clostridium perfringens* is a disease in poultry that can have a high economic and animal welfare cost, and becomes more prevalent in birds fed AGP free diets (McDevitt et al., 2006). Thus, strategies to control and support intestinal health in broiler becomes more important. Some nutritional interventions, like modifications of feed composition and feed characteristics, that alleviate the consequences of intestinal disease challenges, decrease incidence of clostridial infections or simply improve nutrient availability in broilers, have been summarized (Van der Klis, 2014). Amad *et al*. (2013) have reported positive effects of a PFA containing thymol and anethol on gut morphology. Positive effects of thyme oil on intestinal integrity in broiler are also reported (PLACHA et al., 2014). In addition, SMFA exert direct antibacterial effects in the intestine. The objective of the present study was to investigate the effects of a combination of a PFA and SMFA on performance and gut lesions in broiler under a sub-clinical *C. perfringens* challenge.

## Material and Methods

A total of 1'600 male Cobb x Cobb broilers were randomly allocated to 4 dietary treatments. Each treatment was replicated 8 times with 50 broilers each. Treatments were: infected non-treated (INT) and 3 groups infected treated with 750 mg/kg PFA+SMFA till day 21 (T1), day 28 (T2) and day 35 (T3), all followed by PFA alone (150 mg/kg) till harvest at day 49. Birds were fed a starter (day 0-21), grower (day 21-35) and finisher (35-49) corn/soy-based diet with or without additives according to treatments. Feed and water were provided for ad libitum intake.

All birds were spray-vaccinated at the trial facility with the label recommended dosage of Coccivac-B on day of hatch. On day 19, 20 and 21 all were challenged with *C. perfringens*. Each day fresh inoculum was prepared and administered by mixing in the feed.

Production performance was measured per replicate per phase (data not shown) and over the total 49-day period. On Day 21, five birds from each pen were selected, sacrificed, group weighed, and examined for the degree of presence of Necrotic Enteritis lesions. The scoring was based on a 0 to 3 score, with 0 being normal and 3 being the most severe.Performance and lesion scores were statistically analyzed using SAS. After checking the homogeneity of the vari-
ances, analysis of variance was performed (Tukey test). Performance means were compared for all treatments whereas lesion scores were compared between non-treated and treated (T1, T2, T3). The significance level was set at P ≤ 0.05.

## Results

The effects of duration of supply of the PFA and SMFA are summarized in Table 1. Results show that with increased duration of supply BW and FCR were improved. Independent from duration of application, the combination of PFA and SMFA significantly (P<0.001) improved FCR. Body weight gain tended (P<0.10) to be improved with application of PFA and SMFA till day 35 (T3) compared to INT. In T1 and T2 was only numerically improved compared to INT.

<table><caption>**Table 1.** Effects of duration of supply of PFA in combination with SMFA on broiler performance on day 49</caption><thead><tr><th></th><th>Body weight (g)</th><th>Feed conversion ratio (feed/gain)</th></tr></thead><tbody><tr><td>INT</td><td>2'639<sup>A</sup></td><td>1.941<sup>a</sup></td></tr><tr><td>T1 (till day 21*)</td><td>2'682<sup>AB</sup></td><td>1.852<sup>b</sup></td></tr><tr><td>T2 (till day 28*)</td><td>2'729<sup>AB</sup></td><td>1.816<sup>b</sup></td></tr><tr><td>T3 (till day 35*)</td><td>2'788<sup>B</sup></td><td>1.803<sup>b</sup></td></tr><tr><td><i>p-value</i></td><td>0.087</td><td>&lt;0.001</td></tr></tbody></table>

Figure 1 shows the effects of duration of supply of the PFA and SMFA on necrotic enteritis (NE) gut lesions. Compared to the INT birds, treated birds had significant (P=0.008) lower NE related gut lesions on day 21.

<figure><img src="image_30.png" /></figure>

**Figure 1:** Effects of application of PFA and SMFA on necrotic enteritis related gut lesions in 21 day old broiler (P=0.008). Skrivanova et al. (2006) reported strong antibacterial effects of medium chain fatty acids against Gram-positive bacteria. Especially C. perfringens is very sensitive to these medium chain fatty acids. Such molecules prevent necrotic enteritis lesions due to growth inhibition or killing of C. perfringens in the gut. This supports the findings of the present study, which shows a significant reduction in NE related lesions. Performance results might be related to the positive effects of the PFA on performance as shown by Amad et al. (2011). From the current study it is concluded, that the combination of the tested PFA and esters of short and medium chain fatty acids reduces necrotic enteritis lesions and improves performance under a C. perfringens challenge.

## Conclusions

In the current study the combination of PFA and SCFA (Biostrong Forte, Delacon, Austria) significantly improved FCR and significantly reduced necrotic enteritis related gut lesions.

<footnote>* application of combination of PFA and SMCA followed by PFA at 150 mg/kg till slaughter</footnote>
## Literature

AMAD, A. A., MÄNNER, K., WENDLER, K. R., NEUMANN, K., and ZENTEK, J. 2011: Effects of a phytogenic feed additive on growth performance and ileal nutrient digestibility in broiler chickens. Poultry Science. 12, 2811-2816.

AMAD, A. A., WENDLER, K. R., and ZENTEK, J. 2013: Effects of a phytogenic feed additive on growth performance, selected blood criteria and jejunal morphology in broiler chickens. Emir.J.Food Agric. 7, 549-554.

MCDEVITT, R. M., BROOKER, J. D., ACAMOVIC, T., and SPARKS, N. H. C. 2006: Necrotic enteritis; a continuing challenge for the poultry industry. World's Poultry Science Journal. 2, 221-373, 377.

PLACHA, I., TAKACOVA, J., RYZNER, M., COBANOVA, K., LAUKOVA, A., STROMPFOVA, V., VENGLOVSKA, K., and FAIX, S. 2014: Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers. Br.Poult.Sci. 1, 105-114.

SKRIVANOVA, E., MAROUNEK, M., BENDA, V., and BREZINA, P. 2006: Susceptibility of Escherichia coli, Salmonella sp. and Clostridium perfringens to organic acids and monolaurin. Veterinarni Medicina. 3, 81-88.

VAN DER KLIS, J. D. 2014: Nutritional Strategies in feedin poultry under a highly challenged environment. Proc of the 27th annual convention of the Philippine Society of Animal Nutrisionsits.
P-037 (ID 162)

# A BACILLUS SUBTILIS BASED PROBIOTIC ASSIST IN OVERCOMING THE PERFORMANCE LOSS ASSOCIATED WITH DIETS REDUCED IN ENERGY

A. B. Kehlet<fnref n="1" /> and M. Sims<fnref n="2" />

**Corresponding author:** dkabk@chr-hansen.com

## Abstract

A study was carried out to evaluate the effect of a commercially available *Bacillus subtilis* based probiotic GalliPro® (GP) to corn/soybean based broiler diets with reduced energy levels. A total of 2160 Cobb 500 broilers were allocated to 6 treatments and reared over 42 days with ad libitum feeding. The positive control (100% ME) met NRC standards and GP was added with 8 x 10⁵ CFU/g of feed (500 ppm). Treatments were as follows: 1) 100% ME; 2) 98% ME; 3) 96% ME; 4) 100% ME + GP; 5) 98% ME + GP; 6) 96% ME + GP. By day 42, each pair of energy level showed significant improvement in bodyweight with GP addition. The 100% ME control showed similar weight gain as 98% ME + GP and 96% ME + GP. The FCR at day 42 was likewise improved by GP for 98% ME + GP being statistical similar to 100% ME control (1.814). Overall mortality was significantly lower in both 100% ME + GP (3.89%) and 98% ME + GP (2.50%) groups, compared to the same energy level without GP addition (both 6.11%). In conclusion, these results indicate that addition of a *Bacillus* based probiotic has the ability to reduce mortality and overcome performance loss associated with diets reduced up to 4% in energy.

**Keywords:** broilers, *Bacillus subtilis* probiotic, dietary energy

## Introduction

It is well known that the margins of the poultry industry and, therefore, its future prospects are largely dependent on feed costs, these being consequently one of the main concerns of poultry producers. Moreover, their goal today is to provide consumers with safe food, while promoting poultry health and welfare, in addition to carefully respect the environment. Given this challenging conjuncture, poultry producers really need tools to achieve all the referred objectives in the most competitive possible way.

The intestinal microflora has a great impact on avian performance, health and welfare. Furthermore, it is widely recognized that its profile can be modulated through dietary changes and by a variety of feed additives (Pedroso et al., 2013; Pan and Yu, 2014). In this sense, probiotics represent a valuable option to simultaneously improve production efficiency, avian health and welfare and poultry meat safety. GalliPro® (GP) is commercial heat stable *Bacillus* probiotic, which contains spores of *Bacillus subtilis* (>1.6 x 10⁹/gram) as the active ingredient. The use of GP as a growth promoter is not a new concept. While there have been several studies focusing on the effects of GP on broiler performance, intestinal microflora profile, pathogen bacteria counts in the gut and immune response (Opalinski et al., 2007; Knarreborg et al., 2008; Knap et al., 2011; Lourenco et al., 2012), few studies have approached the contribution of this probiotic to dietary energy efficiency in chickens. The objective of this study was to determine if broiler chickens fed reduced energy (ME) diets supplemented with GP at 8x10⁵ CFU's/gram of feed, perform similarly to broilers fed higher energy diets.

## Material and Methods

This experiment consisted of 72 pens with 30 straight run Cobb 500 broilers in each pen (2160 birds in total). The 76 pens were further grouped into 12 location blocks with 6 pens per block with each pen within a block randomly representing one of 6 treatment groups identified as 100% ME, 98% ME and 96% ME (without GP) or 100% ME+GP, 98% ME+GP and 96% ME+GP (with 500 ppm GP). On Day 7, each pen was top dressed with exactly 1,8 kg of previously used broiler litter from a flock having no disease issues or exposure to direct fed microbials. Continuous lighting was provided from Days 0 to 42. Feeds for this study were made up of corn, soybean and meat/bone meal. Diets were formulated to provide 3 different ME levels for each of the starter, grower and finisher dietary phases (9 basal diets in total). Pen body weights and feed weights (for feed conversions) were recorded at Days 0, 21 and 42. All deaths and removed birds (mortality) were documented. Data was analysed by ANOVA. Each response variable was evaluated by

<footnote n="1">¹Chr. Hansen, Boege Allé 10-12, DK-2970 Hørsholm, Denmark</footnote>

<footnote n="2">²Virginia Diversified Research, Corp. 1866 East Market Street, Suite 327 Harrisonburg, VA 22801, USA</footnote>
serial 2-tail t-tests comparing the means of each group to all others assuming unequal variances. The p-value of any
comparison found to be less than or equal to a "p" value 0.05 (p≤0.05) was considered as statistically different.

## Results

At day 21, birds were heaviest (p<0.05) for 100% ME group and lightest (p<0.05) for 96% ME group, with all other
groups being intermediate. Addition of GP in the diet did not improve live weight at 21 d. Live weight/bird of 100% ME +
GP and 98% ME + GP groups was heavier (p≤0.05) than all other groups with the exception of the 100% ME group,
which was not different (p>0.05). Final weights of each pair of 98%, and 96% ME treatment groups showed the GP-
supplemented group having the heaviest (p≤0.05). This observation also shows that the 42d live weights of 96% ME +
GP group was equal to that of the 98% ME group suggesting an energy uplift. Final live weights are shown in figure 1.

<figure><img src="image_31.png" /></figure>

Means followed by different letters are significantly different (p<0.05)

GP addition to the diet resulted in significantly improved FCR (p<0.05), with the exception of the lowest energy diet (96% ME). Mortality of the 3 groups receiving GP were either numerically lower (100% ME + GP and 96% ME + GP) or significantly (p≤0.05) lower (98% ME + GP) than their same ME level counterparts. In figure 2, mortality results are shown.

<figure><img src="image_32.png" /></figure>

Means followed by different letters are significantly different (p<0.05)

## Conclusions

The goal of this study was to determine if broiler chickens fed low energy diets supplemented with GP, perform similarly to broilers fed higher energy diets or do similarly to non-supplemented broilers on the same energy level. Under these experimental conditions, the results of this study demonstrated that diets with 2% lower energy and supplemented with GP have final feed conversions and live weights equal to non-supplemented diets with 2% higher energy. Furthermore, GP addition to the diet significantly reduced mortality in the group with 98% ME and numerically in 100% and 96% ME groups. In view of these results, it can be concluded that GP supplementation in broiler diets results in dietary energy utilization uplift and in mortality reductions.
## Literature

KNAP, I., A.B. KEHLET, M. BENNEDSEN, G.F. MATHIS, C.L. HOFACRE, B.S. LUMPKINS, M.M. JENSEN, M. RAM, and A. LAY, 2011: *Bacillus subtilis* (DSM17299) significantly reduces *Salmonella* in broilers. Poult. Sci. 90, 1690-1694

KNARREBORG, A., E. BROCKMANN, K. HØYBYE, I. KNAP, B. LUND, N. MILORA, T.D. LESER, 2008: *Bacillus subtilis* (DSM17299) modulates the ileal microbial communities and improves growth performance in broilers. Int. J. Probiotics Prebiotics 3, 83-88.

LOURENCO, M.C., L.N. KURITZA, P. WESTPHAL, E. MUNIZ, L. PICKLER, E. SANTIN, 2012: Effects of *Bacillus subtilis* in the dynamics of infiltration of immunological cells in the intestinal mucosa of chickens challenged with *Salmonella* Minnesota. Int. J. Poult. Sci. 11, 630-634.

PAN, D., Z. YU, 2014: Intestinal microbiome of poultry and its interaction with host and diet. Gut Microbes. 5, 108-119.

PEDROSO, A.A., A.L., HURLEY-BACON, A.S. ZEDEK, T.W. KWAN, A.P. O. JORDAN, G. AVELLANEDA, C. L. HOFACRE, B.B. OAKLEY, S.R. COLLETT, J.J. MAURER, M. D. LEE, 2013: Can probiotics improve the environmental microbiome and resistome of commercial poultry production? Int. J. Environ. Res. Public Health 10, 4534-4559.

OPALINSKI, M., A. MAIORKA, F. DAHLKE, F, CUHNA, F.S.C. VARGAS, E. CARDOZO, 2007: On the use of a probiotic (Bacillus subtilis - strain DSM 17299) as growth promoter in broiler diets. Braz. J. Poult. Sci. 9, 99-103.
P-040 (ID 250)

EVALUATION OF CHOLINE CHLORIDE REPLACEMENT BY A POLYHERBAL MIXTURE IN INTENSIVE BROILER PRODUCTION

Jean François LE ROUX¹*, Homero BORIN², Christophe ALLENO³

¹ Nutri Advance, Ploufragan, France

² Nutritec International, Etoy, Switzerland

³ Zootest, Ploufragan, France

*Contact: Jean François LE ROUX, jf.le.roux@nutri-advance.com

**Abstract**

**Material and methods:** In a 1200m² Colorado type house 26400 broilers ROSS PM3 were raised from which 820 males were divided into 20 cages inside the house; 2 batches of 10 repeats of 41 males were performed. Diet, based on corn, wheat and soybean meal was supplemented with choline chloride (CC group) or Natu B4® (B4 group) using a substitution ratio of 100g of Natu B4® for 430g of Choline chloride 70%. Feed consumption, body weight and FCR are measured at days 12, 24, 28 and 35. Blood analyses and liver weight are performed at day 35 on 43 animals per batch.

**Results:** Except for FCR which was significantly lower during the first 12 days for B4 group, final weights and FCR were similar at each age. Despite a low initial weight at day 1 (37.4 and 37.6 g), final weights at 35 days were heavy, 3255 and 3244g for CC and B4 group respectively. Final FCR were 1.54 for both groups. Most of blood parameters were similar in both groups (glucose, cholesterol, triglycerides, albuminemia, SGOT, etc.). Blood urea levels were significantly lower in B4 group. Liver weight were significantly lower in B4 group, 51.3 vs 55.4g.

**Conclusion:** Natu B4® is able to replace choline chloride supplementation allowing high level of performance. Natu B4® is a technical and economical substitute of choline chloride.

**Introduction**

Choline deficiency results in liver damage such as steatosis, hepatocarcinomas, inflammation, oxidative damage, abnormal phospholipid secretion, defects in lipoprotein secretion, Rinella et al, 2008. In animal production choline deficiency results also in reduction of zootechnical performance thus choline chloride is often supplemented in animal feed. The purpose of this trial is to compare the zootechnical performance of standard choline chloride supplementation and its substitution by Natu B4®, polyherbal product rich in phosphatidylcholine.

**Material and methods**

**Building and Animals**

In a 1200m² Colorado type house 26400 broilers ROSS PM3 were raised from which 820 males were divided into 20 cages inside the house. 2 batches divided into 10 repeats of 41 animals are performed. Feed consumption, body weight and FCR are measured at days 12, 24, 28 and 35. Blood analyses and liver weight are performed at day 35 on 43 animals per batch.

**Feeding treatments**

Two groups, CC & B4 are compared. CC group is supplemented in Choline chloride while Natu B4® is used in the B4 group, according the quantities reported in table 1

<table><caption>**Table 1:** Choline Chloride and Natu B4® content (g/T) in the different feed (age in days)</caption><thead><tr><th>Group</th><th>g/T</th><th>1-12 d</th><th>12-21 d</th><th>21-28 d</th><th>28-35 d</th><th>1-12 d</th><th>12-21 d</th><th>21-28 d</th><th>28-35d</th></tr></thead><tbody><tr><td>CC</td><td>Choline Cl</td><td>857</td><td>643</td><td>643</td><td>429</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>B4</td><td>Natu B4®</td><td>-</td><td>-</td><td>-</td><td>-</td><td>200</td><td>150</td><td>150</td><td>100</td></tr></tbody></table>
# Results

## Zootechnical performance

High performance levels were reached (table 2) with final weights at 35 days of 2355 and 2344 grams respectively for CC and B4 groups (1 day in advance versus genetic line standards). FCR, 1.54 for both groups at 35 days, is also better (-3.9%) compared to expected genetic line standard. A slight but significant improvement of FCR in the B4 group during the first 12 days was observed (1.02 vs 1.03).

<table><caption>**Table 2:** Zootechnical performance</caption><thead><tr><th rowspan="2"></th><th colspan="2">Day 12</th><th colspan="2">Day 35</th></tr><tr><th>CC</th><th>B4</th><th>CC</th><th>B4</th></tr></thead><tbody><tr><th>Weight g</th><td>421 ±10</td><td>425 ±5</td><td>2355±218</td><td>2344±227</td></tr><tr><th>Feed Conversion</th><td>1.03±0.01<sup>a</sup></td><td>1.02±0.01<sup>b</sup></td><td>1.54±0.02</td><td>1.54±0.03</td></tr></tbody></table>

## Blood parameters and liver weight

Glucose, total cholesterol, triglycerides, albumin, liver enzymes, were similar in the 2 groups. Urea and creatinine levels were significantly lower in B4 group. Liver weight is also significantly lower in the B4 group, 51.3g vs 55.4g, table 3.

<table><caption>**Table 3:** blood analysis (partial results) and liver weight at day 35</caption><thead><tr><th></th><th>CC</th><th>B4</th></tr></thead><tbody><tr><th>Glucose g/L</th><td>2.23</td><td>2.23</td></tr><tr><th>Urea g/L <sup>1</sup> < 0.05</th><td>27.3 %<sup>a</sup></td><td>50.0 %<sup>b</sup></td></tr><tr><th>Urea g/L <sup>1</sup> ≥ 0.07</th><td>20.5 %</td><td>15.9 %</td></tr><tr><th>Creatinine mg/L <sup>2</sup> < 0.6</th><td>15.9 %<sup>a</sup></td><td>34.1 %<sup>b</sup></td></tr><tr><th>Creatinine mg/L <sup>2</sup> ≥ 0.6</th><td>84.1 %</td><td>65.9 %</td></tr><tr><th>Liver weight at 35 days (g) <sup>3</sup></th><td>55.4±7.3<sup>a</sup></td><td>51.3±7.1<sup>b</sup></td></tr></tbody></table>

# Discussion

Same zootechnical performance and similar blood parameters show an equivalency on the mode of action between Choline chloride and Natu B4®.

Natu B4® is characterised by its high content in phosphatidylcholine and Choline chloride is a precursor of phosphatidylcholine. Lamaziere and Wolf, 2010, have given clear evidence that one important mode of action of phosphatidylcholine in the liver function is mediated through the peroxisome proliferator-activated receptor, PPARα nuclear receptors. Ip et al, 2004 have been able to correct a liver fibrosis and steatosis caused by a dietary deficiency in choline with a fibrate supplementation. Fibrates, a class of molecules used for the treatment of metabolic disorders (hypercholesterolemia) are also strong PPARα agonist, Staels et al, 1998. Among the different terpenoids present in Natu B4®, rutin has been also studied for its positive effect on liver cells. Wu and al, 2011, also found out the positive effect of rutin in the stimulation of PPARα and antioxidative enzymes

Nephroprotective activity of rutin through a reduction of blood creatinine and urea has been demonstrated by Sadeghnia et al, 2013. Demirbiliek et al, 2006, has also found a reduction of blood urea and creatinine with polyenylphosphatidylcholine, showing, on kidney protection, a similarity of action of this choline ester and rutin contained in Natu B4®.

# Conclusion

Possibly explained by a similar PPARα activation, Choline chloride and Natu B4® provide the same performance in broiler growth. Natu B4® is even able to do a little better through an improvement of feed conversion in the early age, a higher reduction of liver weight at slaughter, and a better kidney protection by reducing urea and creatinine level.

In industrial conditions, Choline chloride can be substituted by a polyherbal mixture rich in Phosphatidylcholine.

<footnote>a, b statistically different (P<0.05)</footnote>

<footnote n="1">¹ Urea a, b Khi 2 p = 0.036; ² Creatinine a, b Khi 2 p = 0.049; ³ Liver weight a, b Kruskal-Wallis Batch p = 0.042</footnote>
## Literature

RINELLA M, ELIAS MS, SMOLAK RR, FU T, BORENSZTAJN J, GREEN RM, 2008, Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet, J Lipid Res. 2008 May;49(5):1068-76

LAMAZIERE A, WOLF C., 2012, Phosphatidylcholine and PPARα: A relevant connection in liver disease?, Gastro-entérologie Clinique et Biologique, 34, 250-251

IP E, FARRELL G, HALL P, ROBERTSON G, LECLERCQ I, 2004, Administration of the Potent PPAR α Agonist, Wy-14,643, Reverses Nutritional Fibrosis and Steatohepatitis in Mice, Hepatology, 39:1286-1296

STAELS, B, DALLONGEVILLE, J, AUWERX J, SCHOONJANS K, LEITERSDORF, E, FRUCHART JC, 1998, Mechanism of Action of Fibrates on Lipid and Lipoprotein Metabolism, Circulation.,98:2088-2093

SADEGHNIA HR, YOUSEFSANI BS, RASHIDFAR M, BOROUSHAKI MT, ASADPOUR E, GHORBANI A., 2013, Protective effect of rutin on hexachlorobutadiene-induced nephrotoxicity, Ren Fail. 2013 Sep;35(8):1151-5.

WU CH, LIN MC, WANG HC, YANG MY, JOU MJ, WANG CJ., 2011, Rutin inhibits oleic acid induced lipid accumulation via reducing lipogenesis and oxidative stress in hepatocarcinoma cells. J Food Sci. 76(2):T65-72.

DEMIRBILEK S, KARAMAN A, BAYKARABULUT A, AKIN M, GÜRÜNLÜOGLU K, TÜRKMEN E, TAŞ E, AKSOY RT, EDALI MN., 2006, Polyenylphosphatidylcholine pre-treatment ameliorates ischemic acute renal injury in rats, Int J Urol. 2006 Jun;13(6):747-53.
P-041 (ID 222)

# EFFECT OF ARTICHOKE (CYNARA SCOLYMUS L.) EXTRACT SUPPLEMENTATION IN DRINKING WATER ON GROWTH PERFORMANCE OF BROILER CHICKENS

R. Lertpatarakomol<fnref n="1" />, P. Jaipeng<fnref n="2" />, S. Sirimongkolworakul<fnref n="2" />, K. Paditporn<fnref n="2" />, and J. Mitchaothai<fnref n="2" />

**Corresponding author:** rachakris@gmail.com

## Abstract

This study was conducted to determine the effect of artichoke (Cynara scolymus L.) extract (AE) supplementation in drinking water on growth performance of broiler chickens. A total of 272 day-old chicks were randomly distributed into four groups (control: without AE; AE-75: 75 ppm AE in drinking water; AE-150: 150 ppm AE in drinking water; AE-300: 300 ppm AE in drinking water). Each group was containing four replicates of 17 birds. All chicks were offered mash feed and drinking water *ab libitum*. Artichoke extract was provided on day 1-7 and 15-24 of age. Body weight and feed intake were recorded weekly in all replicates. Data were analyzed as a completely randomized design. The results showed that, 1 to 42 days of age, average daily gain (ADG) was significantly higher (P<0.05) in AE-75 (65.56 ± 3.97 g/d) and AE-300 (65.01 ± 0.52 g/d) compared with control (60.09 ± 3.18 g/d) and AE-150 (60.10 ± 2.04 g/d. Feed intake (FI) was significantly higher (P<0.05) in AE-300 (110.49 ± 11.12 g/d) compared with control (94.10 ± 2.54 g/d) and AE-150 (99.42 ± 5.63 g/d) but similar to AE-75 (102.77 ± 5.10 g/d). However, feed conversion ratio (FCR) did not difference between treatments (1.57 ± 0.09, 1.57 ± 0.04, 1.68 ± 0.10 and 1.63 ± 0.08, respectively). It could be concluded that artichoke extract supplementation in drinking water may improve growth performance of broiler chickens.

**Keywords:** Artichoke extract, *Cynara scolymus* L., Growth performance, Broiler chickens

## Introduction

Antimicrobial additives are often used to suppress or eliminate harmful microorganisms in the intestine and to develop growth and efficiency of feed. It has been shown that the continuous usage of antibiotics as growth promoters encourages the retention of residues in animal tissues and that the human consumption of such animal products would potentially increase processes of antibiotic resistance However, the use of antibiotics as feed additives has been banned in recently years because of the public concern over possible antibiotic residual effects and the development of drug resistant bacteria (JIN et al., 1997; LEESON, 2007; MOHAMMAD et al., 2011). One strategy is being developed to overcome these problems, supplementing the animal with bioactive components such as e.g. herbs, plant extracts and essential oil.

Artichoke (*Cynara scolymus* L.) is a member of the Compositae (daisy) family (NATEGHI et al. 2013). The major bioactive components of artichoke leaves are vitamin C, carotenoids, cynarin, flavonoids, phenolic acids, and caffeic acid (WANG et al., 2003; JOY and HABER, 2007; RADWAN, et al., 2007; FALLAH et al. 2013). Several research have shown that the artichoke extract (AE) had some beneficial effects such as protection the hepatotoxicity in broiler chickens (NATEGHI et al., 2013), reduction of blood urea, cholesterol and triglyceride in broiler chickens (FALLAH et al., 2013), improving the productive, reproductive performance and low cholesterol eggs of Mandarah hens (RADWAN, et al., 2007), modulating the oxidative stability and meat quality of Japanese quail (ABBASI and SAMADI, 2014) and enhancing the immune functions in broiler chickens (TAJODINI et al., 2014). Therefore, the objective of this study was conducted to determine the effect of AE supplementation in drinking water on growth performance of broiler chickens.

## Material and Methods

### Animals and Sample Collection

A total of 272 day-old chicks were randomly distributed in completely randomized experimental design (CRD) into 4 treatments with 4 replicates of 17 birds in each. The experimental treatments consisted of control (without AE), AE-75 (75 ppm AE in drinking water), AE-150 (150 ppm AE in drinking water) and AE-300 (300 ppm AE in drinking water). The dietary period was divided in two phases: starter (1 to 21 days) and grower (21 to 42 days). All chicks were offered

<footnote n="1">¹Department of Animal Science & Veterinary Basic Science, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand</footnote>

<footnote n="2">²Department of Clinical Veterinary Science, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand</footnote>
mash feed and drinking water *ab libitum*. AE was provided on day 1-7 and 15-24 of age. Routine medication, vaccination and husbandry practices were administered. Body weight and feed intake were recorded weekly in all replicates. Average daily gain (ADG), Feed Intake (FI) and feed conversion ratio (FCR) were calculated. The data were analyzed using the General Linear Models (GLM) procedure. The means were separated by Least Significant Difference (LSD) when a significant (P≤0.05) was observed.

## Results and Discussion

The result of this study was shown in Table 1. During 1 to 42 days of age, ADG was significantly higher (P<0.05) in AE-75 and AE-300 compared with control and AE-150. FI was significantly higher (P<0.05) in AE-300 compared with control and AE-150 but similar to AE-75. However, FCR did not difference between treatments. This results were in contrast with previous studies that indicated herbs, plant extracts, essential oil and the main components of essential oil did not affect body weight, feed intake or feed efficiency in broilers (BAMPIDIS et al., 2005; CROSS et al., 2007; MELO and HARKES, 2007; ABBASI and SAMADI, 2014). While, the finding of MELO and HARKES (2007) concluded that supplementing basal diet with Artichoke extract from 1-21 days of age improved feed efficiency of 21 and 42 day-old broilers. These authors suggested that improved feed conversion may be attributed to the lower feed intake reflected in those birds receiving Artichoke extract. In addition, this study has shown that supplementing AE in drinking water improved ADG of broilers. This could be explained by supplementation of Artichoke extract increased antibody levels, resulting in increased activity of the immune system. It has been proposed that immune-stimulation may have adverse effects on growth performance, because more nutrients are repartitioned to synthesize antibodies and develop immune organs, thereby decreasing the amount of nutrients available for growth (HEVENER et al., 1999; TAKAHASHI et al., 2000; TAJODINI et al., 2014).

<table><caption>**Table 1.** Effect of AE supplementation in drinking water on growth performance (1 to 42 days of age) of broiler chickens (Mean ± SD)</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">Groups</th></tr><tr><th>Control</th><th>AE-75</th><th>AE-150</th><th>AE-300</th></tr></thead><tbody><tr><td>ADG (g/d)</td><td>60.09 ± 3.18<sup>a</sup></td><td>65.56 ± 3.97<sup>b</sup></td><td>60.10 ± 2.04<sup>a</sup></td><td>65.01 ± 0.52<sup>b</sup></td></tr><tr><td>FI (g/d)</td><td>94.10 ± 2.54<sup>a</sup></td><td>102.77 ± 5.10<sup>ab</sup></td><td>99.42 ± 5.63<sup>a</sup></td><td>110.49 ± 11.12<sup>b</sup></td></tr><tr><td>FCR</td><td>1.57 ± 0.09</td><td>1.57 ± 0.04</td><td>1.68 ± 0.10</td><td>1.63 ± 0.08</td></tr></tbody></table>

## Conclusions

In conclusion, this finding determined by the results supplementation of 75 ppm artichoke extract in drinking water on day 1-7 and 15-24 of age may improve growth performance of broiler chickens.

## Literature

ABBASI, F., F. SAMADI. 2014. Effect of different levels of Artichoke (*Cynara scolymus* L.) leaf powder on the performance and meat quality of Japanese quail. Poult. Sci. J. **2**(2), 95-111.

BAMPIDIS, V.A., V. CHRISTODOULOU, P. FLOROU-PANERI, E. CHRISTAKI, P.S. CHATZOPOULOU, T. TSILIGIANNI, A.B. SPAIS. 2005. Effect of dietary dried oregano leaves on growth performance, carcase characteristics and serum cholesterol of female early maturing turkeys. British Poult. Sci. **46**, 595-601.

CROSS, D., R. MCDEVIT, K. HILMAN, T. ACAMOVIC. 2007. The effect of herbs and their associated essential oils on performance, dietary digestibility and gut microflora in chickens from 7 to 28 days of age. British Poult. Sci. **48**, 496-506.

FALLAH, R. A. KIANI, A. AZARFAR. 2013. Effect of Artichoke leaves meal and Mentha extract (*Mentha piperita*) on immune cells and blood biochemical parameters of broilers. Glob. Veterinaria **10**(1), 99-102.

HEVENER, W.P., A. ROUTH, G.W. ALMON. 1999. Effects of immune challenge on concentrations of serum insulin-like growth factor-I and growth performance in pigs. Canadian Vete. J. **40**, 782-786.

JIN, L.Z., Y.W. HO, N. ABDULLAH, S. JALALUDIN. 1997. Probiotics in poultry: modes of action. Worlds' Poult. Sci. J. **53**, 351-368.

<footnote><sup>a,b</sup>Means in the same row with different superscripts are significantly different (P≤0.05)</footnote>
JOY, J.F., S.L. HABER. 2007. Clinical uses of artichoke leaf extract. American J. Health Sys. Pharm. **64**, 1906-1909.

LEESON, S. 2007. Butyrate lancing science versus societal issues in poultry nutrition. Nutr. Abstr. Rev. (B): 71: 1-5.

MELO, J.E., R. HARKES. 2007. Productive evaluation of broiler chickens consuming Artichoke extract (Cynara Scolymus L.) during the first three weeks of life. XX Congreso Latinoamericano de aviculture, Brasil.

MOHAMMAD, A.A., M. HASHEMI, F. AHMADI. 2011. Effects of feed type with/without nanosil on cumulative performance, relative organ weight and some blood parameters of broilers. Glob. Vet. **7**(6), 605-609.

RADWAN, N.L. Z.M.A., ABDO, R.A. HASSAN.2007. Effect of feeding Artichoke leaves meal on productive and reproductive performance of Mandarah hens. Int. J. Poult. Sci. **6**(11), 826-834.

NATEGHI, R., F. SAMADI, F. GANJI, S. ZEREHDARAN. 2013. Hepatoprotective effects of *Cynara scolymus* L. extract on CCl4 induced liver injury in broiler chickens. Int. J. AgriScience. **3**(9), 678-688.

TAJODINI M., F. SAMADI, S.R. HASHEMI, S. HASSANI, M. SHAMS-SHARGH. 2014. Effect of different levels of Artichoke (Cynara scolymus L.) powder on the performance and immune response of broiler chickens. Int. J. AgriScience. **4**(1), 66-73.

TAKAHASHI, K.T., Y. MASHIKO, Y. AKIBA. 2000. Effect of dietary concentration of xylitol on growth in male broiler chicks during immunological stress. Poult. Sci. **79**:743-747.

WANG, M., J.E. SIMON, I.F. AVILES, K. HE, Q.Y. ZHENG, Y. TADMOR. 2003. Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.). J. Agri. Food Chem. **1**, 601-608.
P-042 (ID 234)

EFFECT OF DIETARY SODIUM DIFORMATE ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, GUT HEALTH AND PROFITABILITY IN BROILERS

Christian Lückstädt

ADDCON, Bonn, Germany

Corresponding author: christian.lueckstaedt@addcon.com

**Abstract**

Dietary formic acid and its salts act against pathogens, helping to decrease pressure on the immune system and improving nutrient digestibility. The use of the double sodium salt of formic acid, with the same antimicrobial properties as formic acid, but with improved handling properties, has become more prevalent in poultry diets. In a trial conducted at a research farm in Taiwan, sodium diformate (NaDF; 0.3% inclusion rate) was tested in a typical corn-soy diet, against a similar control diet containing no acidifier. 120 day-old broiler chicks (male Arbor Acres) were randomly selected and divided into 2 treatment groups with 3 replicates each. Feed and water were available *ad libitum*. The effects of NaDF on performance (final weight, FCR, broiler index), protein utilization and villi height were examined after 39 days. Data were analysed using the t-test and a confidence level of 95% was defined for these analyses.

Performance was boosted in the birds fed 0.3% NaDF. Treated birds were significantly heavier (2551 g vs. 2316 g; P<0.05), while the FCR improved only numerically (1.67 vs. 1.71). Protein utilization was also numerically better (63.3% vs. 61.6%), while the villi height in the jejunum and ileum was significantly increased by between 170 and 450 µm.

This study demonstrates that including NaDF in broiler diets is beneficial to performance, nutrient utilization and gut health and thereby increases the gross profit of broiler operations.

**Keywords:** broiler, sodium diformate, performance, digestibility

**Introduction**

Organic acids have a long history of use in animal nutrition, where they are usually added to stabilize compound feed, but also to enhance animal performance. While most early data on this class of additives were collected from studies in pig production (Cole et al., 1968; covering sows, piglets and fatteners), the available literature indicates a later focus on such additives in poultry production. One of the first reports of improved broiler performance when diets were supplemented with a single organic acid was for formic acid (Vogt et al., 1981).

Improvements in broiler performance or their hygienic production conditions with the aid of a variety of organic acids have been reported by many sources, as reviewed by Desai et al. (2007). An important limitation, however, is that organic acids are rapidly metabolised in the fore-gut (crop to gizzard) of birds, which can reduce their impact on growth performance. A newer salt of formic acid, sodium diformate (NaDF), has been demonstrated to be effective against pathogenic bacteria, including salmonella, throughout gastro-intestinal tract (Lückstädt and Theobald, 2009). Reducing the impact of pathogenic bacteria on the broiler, and improving the gut microflora, leads to a state of eubiosis in treated chickens and suggests that the inclusion of diformate in broiler diets can also improve bird performance. This hypothesis was confirmed statistically with a meta-analysis of data from trials carried out in recent years (Lückstädt, 2013). This study analyzed the average impact reported from all studies on the effect of the additive on the performance parameters weight gain, feed efficiency, mortality and productivity. However, these trials focused on weight gain and feed conversion outputs. This study aimed to investigate the effects of sodium diformate on growth performance parameters as well as nutrient digestibility and gut health in broilers.

**Material and Methods**

The trial was conducted at the research farm in Taiwan. One hundred and twenty-day-old male Arbor Acres broilers were randomly allotted into 2 treatment groups, with 3 replications and 20 birds in each pen. The commercially-available corn-soy-based feed in one group contained 0.3% NaDF; this group was compared against a negative control. Feed and water were provided *ad libitum*. Birds were weighed individually after 14 and 39 days; feed consumption was recorded per pen at the end of the trial. The effects of NaDF on performance (final weight, feed conversion ratio, European Broiler Index EBI), protein utilization and villi height were examined after 39 days.

Data were analysed using the t-test and a confidence level of 95% was defined for these analyses.
## Results

The inclusion of 0.3% NaDF in the diets of broilers for 39 days exerted significant effects on performance as well as protein digestibility in the birds.

The effect of the additive was already noticeable after only 14 days. At this stage, the body weight of the broilers receiving NaDF had increased significantly (P<0.05), by almost 21% (Table 1). At the end of the trial, treated birds were still significantly heavier (2551 g vs. 2316 g; P<0.05), while the FCR improved numerically (1.67 vs. 1.71). Furthermore, the mortality rate of birds was substantially reduced by 50%. As a result, the European Broiler Index, which includes the three most important productivity parameters in animal production – weight gain, survival and feed efficiency – improved markedly by almost 15%.

<table><caption>**Table 1:** Performance parameters of broilers fed with or without NaDF at 0.3% for 39 days</caption><thead><tr><td></td><td>Negative control</td><td>0.3% NaDF</td><td>Difference [%]</td></tr></thead><tbody><tr><td>BW-14 days [g]</td><td>320<sup>b</sup></td><td>386<sup>a</sup></td><td>+20.6</td></tr><tr><td>Final weight [kg]</td><td>2.316<sup>b</sup></td><td>2.551<sup>a</sup></td><td>+10.1</td></tr><tr><td>ADG [g]</td><td>58<sup>b</sup></td><td>64<sup>a</sup></td><td>+10.3</td></tr><tr><td>FCR</td><td>1.71</td><td>1.67</td><td>-2.3</td></tr><tr><td>Mortality [%]</td><td>3.33</td><td>1.67</td><td>-50</td></tr><tr><td>EBI*</td><td>329</td><td>378</td><td>+14.9</td></tr></tbody></table>

Means with a different superscript differ significantly at P<0.05

*EBI = Daily weight gain (g) x Survival (%) / 10 x FCR

Villus height in the jejunum and ileum of birds at 39 days showed a significant increase in birds fed 0.3% NaDF compared to controls (Table 2).

<table><caption>**Table 2:** Effect of sodium diformate (0.3%) on the morphology of the jejunum and ileum of broilers after 39 days.</caption><thead><tr><td></td><td>Negative control</td><td>0.3% NaDF</td><td>Difference [%]</td></tr></thead><tbody><tr><td>Jejunum - Villus height [µm]</td><td>1268<sup>b</sup></td><td>1721<sup>a</sup></td><td>+35.7</td></tr><tr><td>Ileum - Villus height [µm]</td><td>1011<sup>b</sup></td><td>1184<sup>a</sup></td><td>+17.1</td></tr></tbody></table>

Means with a different superscript differ significantly at P<0.05

Analysis of the protein utilization of the diets showed a numerical increase from 61.6% to 63.3% in the birds fed diets containing NaDF. This substantial improvement, of almost 3% in total, may be related to the larger intestinal surface area in NaDF fed birds due to the longer villi.

Similar trends (improved performance parameters as well as nutrient utilization) were reported as a result of a holanalysis of 59 published studies for potassium diformate in pig diets (Rosen, as reported by Lückstädt and Mellor, 2011). In broilers, improved zootechnical performance is thought to arise as a result of improved intestinal microflora, through suppressing pathogenic bacterial species; and improved protein digestion. This was supported by the present study as well as in a previous report under similar conditions (Lückstädt, 2014).

## Conclusions

As often seen with other additives, the performance improvements recorded are also influenced in part by the hygienic conditions. The current study was conducted at the research farm of a university. This study shows that even under these rather “clean” conditions, NaDF was able to significantly improve bird performance. Under practical conditions with a higher anticipated “bacterial load”, conditions might weigh even more in favour of the additive effects measured. This has been seen with other trials under commercial conditions. It can therefore be concluded that dietary sodium diformate can play an important role in improving broiler production.
## Literature

COLE, D.J.A., R.M. BEAL and J.R. LUSCOMBE (1968). The effect on performance and bacterial flora of lactic acid, propionic acid, calcium propionate and calcium acrylate in the drinking water of weaned pigs. Veterinary Record 83: 459-464.

DESAI, D., D. PATWARDHAN and A. RANADE (2007). Acidifiers in Poultry Diets and Poultry Production. In: LÜCKSTÄDT, C.: Acidifiers in Animal Nutrition – A Guide for Feed preservation and Acidification to Promote Animal Performance. Nottingham University Press, Nottingham, ISBN 978-1-904761-40-2, 63-69.

LÜCKSTÄDT, C. and P. THEOBALD (2009). Effect of a formic acid-sodium formate premixture on Salmonella, Campylobacter and further gut microbiota in broilers. Proceedings and Abstracts of the 17th European Symposium on Poultry Nutrition, 246.

LÜCKSTÄDT, C. and S. MELLOR (2011). The use of organic acids in animal nutrition, with special focus on dietary potassium diformate under European and Austral-Asian conditions. Recent Advances in Animal Nutrition – Australia 18: 123-130.

LÜCKSTÄDT, C. (2013). Effects of sodium diformate in broilers – a performance analysis. Proceedings and Abstracts of the 19th European Symposium on Poultry Nutrition, 191.

LÜCHSTÄDT, C. (2014). Effect of dietary sodium diformate on growth performance and nutrient utilization in broilers. Proceedings of the XIVth European Poultry Conference, 476.

VOGT, H., S. MATTHES and S. HARNISCH (1981). Der Einfluß organischer Säuren auf die Leistungen von Broilern und Legehennen. Archiv für Geflügelkunde 45: 221-232.
## Results

The results have shown that there were no differences (P>0.05) for average daily gain, average daily feed intake and % hen-day egg production of the hens among 3 experimental dietary treatments (Table 1.). However, at 18-22 week and 18-28 week of age, the laying hens offered the diet supplemented enzymes produced from *Trichoderma* sp. (T2) had better (P<0.05) feed efficiency per kg egg mass and higher values of % hen-day egg production of the laying hens when compared with those received enzymes produced from the *A. Niger* (T3), but there were no difference from the hens in the control group (T1), offered diet without enzyme addition. In the mean time, the laying hens obtained the dietary treatments in all studied groups had no difference (P>0.05) of feed efficiency per kg egg mass and % hen-day egg production at 23-28 week of age (Table 1.).

<table><caption>**Table 1.** Production performance of the experimental laying hens</caption><thead><tr><th rowspan="2">Item</th><th colspan="3">Treatment</th></tr><tr><th>T1</th><th>T2</th><th>T3</th></tr></thead><tbody><tr><td>Average daily gain (g/day)</td><td>0.82±0.72</td><td>0.36±0.55</td><td>0.26±0.21</td></tr><tr><td>Average daily feed intake (g/day)</td><td>103.17±1.88</td><td>103.20±1.42</td><td>101.33±2.03</td></tr><tr><td>Feed efficiency per kg egg mass</td><td></td><td></td><td></td></tr><tr><td>18-22 wk</td><td>4.03±0.58<sup>ab</sup></td><td>3.77±0.51<sup>a</sup></td><td>4.92±0.55<sup>b</sup></td></tr><tr><td>23-28 wk</td><td>2.40±0.05</td><td>2.34±0.14</td><td>2.50±0.16</td></tr><tr><td><strong>18-28 wk</strong></td><td>3.14±0.26<sup>ab</sup></td><td>2.99±0.30<sup>a</sup></td><td>3.60±0.30<sup>b</sup></td></tr><tr><td>% Hen-day egg production</td><td></td><td></td><td></td></tr><tr><td>18-22 wk</td><td>74.37±4.21</td><td>75.65±6.16</td><td>67.10±8.85</td></tr><tr><td>23-28 wk</td><td>89.15±1.07</td><td>89.41±3.39</td><td>83.87±6.51</td></tr><tr><td><strong>18-28 wk</strong></td><td>76.06±2.25</td><td>76.66±3.60</td><td>70.18±6.90</td></tr></tbody></table>

## Discussion

The results of the current study have shown that there was not different for growth rate and feed consumption of the laying hens among three experimental groups. The explanation for these results would be the fact that the studied lay hens already have passed growth phase, entering adult age with low growth rate. The experimental laying hens obtained diet supplemented with enzymes producing from *Trichoderma* sp. had better feed efficiency per kg egg mass than those received diet supplemented enzymes produced from the *A. niger*. This result is in accordance with earlier report (SATHYAVRATHAN and KRITHIKA, 2013), which showed that cellulases producing from *Trichoderma* sp. has higher efficient cellulases than that produced from *A. niger*. However, cellulases producing from both *Trichoderma* sp. and *A. niger* had no difference of feed efficiency per kg egg mass when compared with the laying hens in the control group. This would be influence of gastric condition affecting cellulase activities (WARD et al., 2003). For % hen-day egg production, there was no significant difference among 3 dietary treatments, but at 18-22 weeks of age the highest value of % hen-day egg production was found in the laying hens obtained the diet supplemented with enzymes producing from *Trichoderma* sp., which was in agreement with value of feed efficiency per kg egg mass. The same trend was found for the laying hens in the other groups. In addition, all laying hens were reared in a closed operating barn, resulting in less stress obtained from environment. At the period of 23-28 weeks of age, the laying hens in all experiment groups had rather closely values of feed efficiency per kg egg mass and % hen-day egg production. These would be explained by the more enzyme adaptation of the laying hens with age.

## Conclusions

Under the condition of the present study, the enzymes produced from *Trichoderma* sp. were more appropriate than those produced from *A. niger* for dietary supplementation for laying hens.

## Acknowledgements

The authors would like to thank Burapha University-Sakaeo Campus for facilities and financial support.

<footnote><sup>a,b</sup>means in the same row with different superscripts are significant difference (P<0.05)</footnote>
## Literature

KUHAD, R.C., R.GUPTA and A. SINGH, 2011. Microbial cellulases and their industrial applications, Enzyme Res., **Volume 2011**, Article ID 280696, 10 pages.

ILMÉN, M., A. SALOHEIMO, M.L. ONNELA and M.E. PENTTILA, 1997. Regulation of cellulase gene expression in the filamentous fungus *Trichoderma reesei*. *Appl. Environ. Microbiol.* **63(4)**, 1298-1306.

MAKI, M.L., M. BROERE, K.T. LEUNG and W. QIN, 2011. Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers. Int. J. Biochem. Mol. Biol. **2(2)**, 146–154.

SATHYAVRATHAN, P. and S. KRITHIKA, 2013. Comparison of cellulase production in *Trichoderma reesei* (NCIM – 1052) and *Aspergillus niger* (NRRL –322); Media Optimization and enzyme characterization of cellulase from *Trichoderma reesei* with lyophilization. Int. J. ChemTech Res. **5(1)**, 554-557.

Ward, M., L. Tierney, M. Gebert, M. Flynn and K. Clarkson, 2003. Feasibility assessment for the use of cellulase in biomass conversion for human applications. Interim Progress Report 08/19/2003 to 12/31/2003, ARO.
# THE ADDITION OF A NUTRITIONAL EMULSIFIER IMPROVES BROILER PERFORMANCE IN AN ENERGY DILUTED DIET

L. Maertens<fnref n="1" />, S. Leleu<fnref n="1" />, M. Rovers<fnref n="2" />, L. Segers<fnref n="2" />, E. Delezie<fnref n="1" />

**Corresponding author:** luc.maertens@ilvo.vlaanderen.be

## Abstract

Emulsifiers are used to enhance the fat digestibility and by consequence to improve the feed conversion ratio due to the enhanced dietary MEn content. In a three-phase production trial two emulsifiers, E484 (Excential Energy Plus, 350g/T) and E481 (1000 g/T), were added on top of a negative control (NC) diet. The decrease of energy (0.42 MJ MEn/kg) in the NC diet was induced by exchanging 2.14 % fat for 2.14 % wheat in the positive control (PC) diet. Meanwhile, the addition of the synthetic amino acids was adapted in order to obtain diets with the same content of digestible amino acids. Each treatment consisted of 6 replicates with 30 male Ross 308 broilers. Birds fed the emulsifier diets reached the same final bodyweight (between 2,550 – 2,569 g) as the PC birds but had a significant (P<0.01) higher bodyweight than NC birds (2,417g). Effects on both BWG as FCR were most pronounced in the first 4 weeks of the trial. FCR of both emulsifier supplemented diets was 4.7% better (P<0.05) than those fed the NC-diet. For the overall period (0-39 d), the FCR was significantly (P<0.05) different and amounted 1.553; 1.651; 1.620 and 1.612 for the PC, NC, E484 and E481 emulsifier supplemented diets, respectively. The results demonstrated that the addition of the tested emulsifiers enhanced the performances of broilers but could not fully compensate the energy dilution of 0.42 MJ MEn/kg.

**Keywords:** broiler, energy, fat, digestibility, emulsifier

## Introduction

Energy is a major cost component in broiler diets. Due to its high energy density, fats and oils are important energy sources in feed formulation. Improving the energy efficiency of these raw materials is of high interest from an economical point of view. Nutritional emulsifiers can be used to improve fat digestibility and thus improve the energy efficiency (Maertens et al, 2013). The objective of the trial was to evaluate the effect of 2 emulsifiers on the production performances of broilers. For this goal a negative control (NC) diet (with 0.42 lower energy content than the PC diet) was formulated and supplemented with one of the two test emulsifiers.

## Material and Methods

A total of 720 one day old, male broiler chicks (Ross 308) were used in this trial. The birds were housed in the poultry experimental facility of ILVO (Animal Sciences Unit, Section Small Stock Husbandry, Merelbeke, Belgium). A randomised block design was used, with in total 6 blocks of 4 pens and 30 birds per pen. The floor was covered with fresh wood shavings and no additional filling or cleaning of the floor was necessary during the trial.

The experiment consisted of 4 treatments. Treatment 1 was a positive control (PC) diet, formulated with a metabolizable energy content of 11.8, 12.2 and 12.5 MJ/kg for starter, grower and finisher diet, respectively. Treatment 2 was the negative control diet and formulated with 0.42 MJ/kg (100 kcal) lower energy content, compared to PC. In treatment 3, the NC diet was supplemented with emulsifier I (E484, Excential Energy Plus, 350g/T) and in treatment 4 the NC diet was supplemented with emulsifier II (E481, 1000g/T). The chosen inclusion levels were based on practical applied dosages and in accordance with the advice dosage of the supplier. Diets used were wheat-soybean meal based with animal fat as added fat source. Total fat in PC was 7.42%, 8.16% and 8.95% in starter, grower and finisher respectively. The reduction in energy in NC was induced by exchanging 2,14% fat for 2.14% wheat. Meanwhile, the addition of the synthetic amino acids was adapted in order to obtain diets with the same content of digestible amino acids. A three-phase feeding scheme was applied with a starter period of 12 days followed by a grower diet between 12 and 28 days of age and a finisher between 28 and 39 days of age. The diets were prepared at the Institute's feed milling facilities. Feed was provided *ad libitum* as mash and drinking water was provided *ad libitum* by drink belts.

Data were submitted to a two-factorial variance analysis with treatment as factor and block as random factor.

<footnote n="1">¹ ILVO (Institute for Agricultural and Fisheries Research), Animal Sciences Unit, Scheldeweg 68, 9090 Melle, Belgium</footnote>

<footnote n="2">² Orffa Additives B.V., Vierlingstraat 51, 4251 LC Werkendam, the Netherlands</footnote>
## Results

Bodyweight of the birds, body weight gain and feed conversion ratio's for period 0-28 and 0-39 days are presented in Table 2. Already at 12 days of age birds of both emulsifier added diets had a significant higher bodyweight than PC birds (data not shown). At day 28 and at the end of the trial, birds on the emulsifier diets had a significant higher weight than NC birds while at 39 days their weight was equal as birds fed the PC diet.

<table><caption>**Table 1:** Bodyweight (BW), Body weight gain (BWG) and Feed Conversion ratio (FCR) for period 0-28d and 0-39d</caption><thead><tr><th rowspan="2"></th><th colspan="2">BW</th><th colspan="2">BWG</th><th colspan="2">FCR</th></tr><tr><th>D28</th><th>D39</th><th>D0-28</th><th>D0-39</th><th>D0-28</th><th>D0-39</th></tr></thead><tbody><tr><td>Pos Control</td><td>1396<sup>ab</sup></td><td>2557<sup>a</sup></td><td>48,3<sup>ab</sup></td><td>64,4<sup>a</sup></td><td>1,456<sup>a</sup></td><td>1,553<sup>a</sup></td></tr><tr><td>Neg Control</td><td>1324<sup>b</sup></td><td>2417<sup>b</sup></td><td>45,7<sup>b</sup></td><td>60,8<sup>b</sup></td><td>1,574<sup>b</sup></td><td>1,651<sup>c</sup></td></tr><tr><td>Neg Cont + emulsifier I</td><td>1435<sup>a</sup></td><td>2550<sup>a</sup></td><td>49,6<sup>a</sup></td><td>64,2<sup>a</sup></td><td>1,500<sup>a</sup></td><td>1,620<sup>b</sup></td></tr><tr><td>Neg Cont + emulsifier II</td><td>1447<sup>a</sup></td><td>2569<sup>a</sup></td><td>50,1<sup>a</sup></td><td>64,7<sup>a</sup></td><td>1,500<sup>a</sup></td><td>1,612<sup>b</sup></td></tr></tbody></table>

a,b: means in the same column with a different letter are significantly different (P<0.05).

During the grower period as well as in the finisher period, birds fed PC diet had a significant lower FCR compared to NC. However, the addition of both emulsifiers improved the FCR significantly compared with the NC diet. For the grower period (0-28d) the FCR of both emulsifier diets was statistically not different from PC. The overall FCR (d 0-39) of the positive control group was 98 points (P<0.05) lower than the negative control group. The gap in energy between the PC and NC diet was not fully compensated by the addition of the emulsifiers. However, both emulsifiers had a significant positive effect on the overall FCR and induced an average improvement of 2.1%.

## Conclusions

The addition of both emulsifiers resulted in a comparable body weight gain (BWG) as observed in PC birds, while BWG was significantly improved (on average 6%) compared to the NC diet. The difference in energy content between PC and NC was clearly demonstrated by the significant different FCR. This gap was partly filled by the addition of the emulsifiers, both having a more favorable (P<0.05) FCR than the NC birds (2,1% improvement of FCR on average). However, the difference with the PC diet still remained significant. This indicates that a positive effect of 100 kcal (0,42 MJ)/Kg due to the addition of the emulsifier is overestimated. Both emulsifiers resulted in the same improvement in BWG and FCR, however emulsifier I (Excential Energy Plus) was more efficient in terms of dosing (350g/T) compared to emulsifier II (1000g/T).

The results indicate that the tested emulsifiers can improve energy efficiency in broiler diets and result in a significant better FCR, especially in the starter and grower period. The estimated energy upgrade under the tested diet composition is less than 100kcal. The practical application of this trial is that it shows a tool to improve feed efficiency that may lead to lower feed costs and more sustainable broiler production.

## Literature

MAERTENS, L., L. SEGERS, M. ROVERS, A van der AA, S. LELEU, 2013: The effect of different emulsifiers on fat and energy digestibility in broilers *Proc. Eur. Symp. Poult. Nutr. 19: PP V-86.*
DIFFERENT LEVELS OF SODIUM BENTONITE AND SILYBUM MARIANUM SEEDS IN REDUCING THE ADVERSE EFFECTS OF AFLATOXIN ON BLOOD LIPID LEVELS AND THE RELATIVE WEIGHT OF DIGESTIVE ORGANS OF BROILER CHICKENS

P. Malekinejad, ¹N. Afzali<fnref n="2" />, A. Mohammadi<fnref n="3" />, H. Sarir<fnref n="4" />

**Corresponding author:** pouyan.malekinejad@yahoo.com

**Abstract**

An experiment was conducted to evaluate the ability of different levels of sodium bentonite (SB), *Silybum Marianum* seeds (SMS) and their combination for reducing the adverse effects of Aflatoxin B₁ (AFB₁) in broiler chicks. A number of 224 broiler mail chicks (Ross) were used in 8 Treatments: 1) control, 2) diet with 0.5% SB, 3) diet with 0.5% SMS, 4) diet with 0.5% SMS and 0.5% SB, 5) diet with 500ppb AFB₁, 6) diet with 500ppb AFB₁ and 0.5% SMS, 7) diet with 500ppb AFB₁ and 0.5% SB, 8) diet with 500ppb AFB₁ and 0.5% SMS seeds and 0.5% SB. In a completely randomized design arranged with factorial experiment (2 × 2 × 2) with four replications was used from one to 42 days age. The results showed that compared with the control group, diets contaminated with AFB₁ increased the relative weight of gizzard and bile but decreased serum total protein and globulin significantly (p≤0.05). Broiler chickens fed diets containing SB, SMS and AFB₁ their biochemical indices (globulin and total protein) were improved significantly compare to treatment 5 (p≤0.05). Treatment 8 in comparison with treatments 6 and 7 to a greater extent reduced relative gizzard weight and increased serum concentrations of total protein (p≤0.05). The results showed that the combination of the seeds of SMS and SB can effectively eliminate the toxic effects of AFB₁.

**Keywords:** Aflatoxin B1, globulin, Silybum Marianum seeds, sodium bentonite, total protein

**Introduction**

Chicken meat is an important source of nutrients for human consumption. Broiler feed is exposed to various contaminants during the process of production, transportation and storage. One of the important injurious agents is mycotoxins as it has hepatotoxic, hepatocarcinogenic, mutagenic and teratogenicity effects in many animal species (IARC, 1987). Mycotoxins are metabolites from moulds. Aflatoxins, a group of closely related biologically active mycotoxins, are produced by strains of *Aspergillus flavus* and *Aspergillus parasiticus* (Tedesco et al., 2004). Numerous strategies for detoxification or inactivation of mycotoxin in contaminated feedstuff have been used. Most of these techniques are impractical or ineffective (Piva et al., 1995). One approach to the detoxification of mycotoxin is the use of nonnutritive absorptive materials in the diet to reduce the absorption of mycotoxins from the gastrointestinal tract (Thaxton et al., 1974). Layered alumino silicates, such as sodium bentonite, have been found effective in counteracting mycotoxins (Eraslan et al., 2005). The effects of *silymarin* or various products made from the seed of the plant *Silybum Marianum* containing silymarin have also been tested on farm animals. The hepatoprotective activities were assessed on the basis of selected biochemical blood indicators in chicken broilers that had been exposed to the effects of aflatoxins (Tedesco et al., 2004). The purpose of this study was to evaluate the changes of blood lipid levels and the relative weight of digestive organs of broiler chickens when different levels of sodium bentonite and *Silybum Marianum* seeds were used as a feed additive in their diet.

**Material and Methods**

Two hundred and twenty four of day old male Ross 308 broiler chickens obtained from a local hatchery were randomly distributed among eight treatments with four replicate groups per treatment and 7 chickens per replicate. The nutritional requirement of the chicks was based on Ross 308 company for starter (0-10 days) and grower (11-24 days) diets (Table I). Birds were distributed into the following eight treatments: 1) control 2)diet with (%) 500ppb aflatoxin B₁, 3) diet with 500ppb Aflatoxin B₁ and 0.5% *Silybummarinum* seeds, 4) diet with 500ppb aflatoxin B₁ and 0.5% Sodium Bentonite, 5)diet with 500ppb Aflatoxin B₁ and 0.5% *Silybummarinum* seeds and 0.5% Sodium Bentonite, 6)diet with 0.5% *Silybummarinum* seeds, 7) diet with 0.5% Sodium Bentonite, 8) diet with 0.5% *Silybummarinum* seeds and 0.5% So-

<footnote n="1">¹ MSc of management and poultry production, Department of Animal Science, university of Birjand, Iran</footnote>

<footnote n="2">² Professor of Poultry Nutrition, Department of Animal Science, University of Birjand, Iran</footnote>

<footnote n="3">³ Assistant professor of plant pathology, Department of plant pathology, University of Birjand, Iran</footnote>

<footnote n="4">⁴ Assistant professor of immunology, Department of Animal Science, University of Birjand, Iran</footnote>
dium Bentonite. At 24 day of age, two broilers per pen were randomly selected and 2 mL of blood samples were obtained by wing puncture and centrifuged at 3000 rpm for 20 min to separate serum. Serum total protein(TP), Glubulin and relative weight of digestive organs were analyzed using the general linear model procedure of SAS.

## Results

The effect of experimental treatments on different parameters in Table 1 show that compared with the control group, diets contaminated with AFB₁ increased the relative weight of gizzard but decreased serum total protein and globulin significantly (p≤0.05). AFB has been shown to cause inhibition of protein synthesis (16). Total protein levels in the serum proved to be sensitive indicators of aflatoxicosis in broilers. Use of absorbents in broiler diet will cause the subsequent absorption of toxin in their digestive tract and this prevents the reduction of blood serum albumin, globulin and protein in the case of using contaminating diet (7). However, in our case, the biochemical parameters for broilers fed diets containing SB + AF did not completely return to normal values, showing an inhibition of protein synthesis. Broiler chickens fed diets containing SB, SMS and AFB₁ their biochemical indices (globulin and total protein) were improved significantly compared to treatment 5 (p≤0.05). Treatment 8 in comparison with treatments 6 and 7 to a greater extent reduced relative gizzard weight and increased serum concentrations of total protein (p≤0.05).

## Conclusions

In conclusion Dietary inclusion of sodium bentonite had not any adverse effects on serum biochemical characteristics and it can also improve the values of TP in broiler chickens also results of the present study showed that consumption of level of SMS from 0.5 % in poultry diets can reduce the toxicity of AFB₁ in broiler chickens. But results showed that the combination of SB and the SMS are more beneficial as separate application of them for decreased toxic effect of Aflatoxin B₁ in diets.

## References

ERASLAN, G., Essz, D., AkDOGAN, M., SAHINDOKUYUCU, F. and general health of broilers. Supplementation of high Altrintas, L. (2005) The Effects of Aflatoxin and Sodium grade bentonite at 1.0 per cent level was found Bentonite and Alone on Some Blood Electrolyte Levels in Broiler Chickens. Turkish Veterinary. Hayvanclık Dergisi. 29: 601-605.

KUBENA, L.F., HARVEY, R.B., PHILIPS, T.D., CORRIER, D.E., HUFF, W.E.: Diminution of aflatoxicosis in growing chickens by the dietary addition of a hydrated sodium calcium aluminosilicate. Poultry Science., 1990; 69: 727-735.

PIVA, G., GALVANO, P., PIETRI, A., PIVA, A.: Detoxification methods of aflatoxins. A review. Nutrition Research., 1995; 15: 767-776.

SUJATHA, K., MATHURAM LN, SRIRAM P 2003: Hepatoprotective effect of silymarin in experimentally induced aflatoxicosis in broilers. Toxicology., 10: 55-59.

TEDESKO D, STEIDLER S, GALLETI, S, TAMENI M, SONZOGNI O, RAVAROTTO L. Efficacy of silymarin-phospholipid complex in reducing the toxicity of aflatoxin B1 in broiler chicks. Poultry Science. 2004; 83(11):1839-43.

THAXTON, J.P., TUNG, H.T., HAMILTON, P.B.: Immunosuppression in chickens by aflatoxin. Poultry Science., 1974; 53: 721-725.

TUNG, H.T., WYATT, R.D., THAXTON, P., HAMILTON, P.B.: Concentrations of serum proteins during aflatoxicosis. Toxicology Apply Pharmacology., 1975; 34: 320-326.
<table><caption>**Table 1.** effect of different treatment on TP and Glubulin (U/l) and digestive organ (gr/kg live weight).</caption><thead><tr><th></th><th></th><th></th><th>TP</th><th>Glubulin</th><th>Liver</th><th>Gizzard</th><th>Proventriculus</th></tr></thead><tbody><tr><td rowspan="8">Main effects</td><td>Aflatoxin (ppm)</td><td>0</td><td>2.44<sup>a</sup></td><td>1.44<sup>a</sup></td><td>1.80</td><td>1.46<sup>b</sup></td><td>0.45</td></tr><tr><td></td><td>500</td><td>2.01<sup>b</sup></td><td>1.25<sup>b</sup></td><td>1.84</td><td>1.69<sup>a</sup></td><td>0.44</td></tr><tr><td>p-value</td><td></td><td>0.00</td><td>0.00</td><td>0.41</td><td>0.00</td><td>0.95</td></tr><tr><td>Silybum (%)</td><td>0</td><td>1.98<sup>b</sup></td><td>1.19<sup>b</sup></td><td>1.82</td><td>1.69<sup>a</sup></td><td>0.45</td></tr><tr><td></td><td>0.5</td><td>2.47<sup>a</sup></td><td>1.50<sup>a</sup></td><td>1.82</td><td>1.45<sup>b</sup></td><td>0.44</td></tr><tr><td>p-value</td><td></td><td>0.00</td><td>0.00</td><td>0.98</td><td>0.00</td><td>0.39</td></tr><tr><td>Bentonite (%)</td><td>0</td><td>2.09<sup>b</sup></td><td>1.29</td><td>1.82</td><td>1.68<sup>a</sup></td><td>0.43<sup>b</sup></td></tr><tr><td></td><td>0.5</td><td>2.36<sup>a</sup></td><td>1.40</td><td>1.82</td><td>1.47<sup>b</sup></td><td>0.46<sup>a</sup></td></tr><tr><td colspan="2">p-value</td><td></td><td>0.00</td><td>0.09</td><td>0.97</td><td>0.00</td><td>0.04</td></tr><tr><td colspan="2">SEM</td><td></td><td>0.05</td><td>0.04</td><td>0.03</td><td>0.03</td><td>0.00</td></tr><tr><td rowspan="10">Dual interaction effects</td><td>Aflatoxin * silybum</td><td>0</td><td>2.21</td><td>1.28</td><td>1.79</td><td>1.50<sup>b</sup></td><td>0.46</td></tr><tr><td></td><td>0.5</td><td>2.66</td><td>1.61</td><td>1.80</td><td>1.42<sup>b</sup></td><td>0.43</td></tr><tr><td></td><td>500</td><td>1.75</td><td>1.11</td><td>1.85</td><td>1.89<sup>a</sup></td><td>0.44</td></tr><tr><td></td><td>500</td><td>2.27</td><td>1.39</td><td>1.83</td><td>1.49<sup>b</sup></td><td>0.45</td></tr><tr><td>p-value</td><td></td><td>0.65</td><td>0.68</td><td>0.72</td><td>0.00</td><td>0.13</td></tr><tr><td>Aflatoxin * ben</td><td>0</td><td>2.36</td><td>1.40</td><td>1.67</td><td>1.47<sup>b</sup></td><td>0.41<sup>b</sup></td></tr><tr><td></td><td>0.5</td><td>2.52</td><td>1.48</td><td>1.83</td><td>1.44<sup>b</sup></td><td>0.48<sup>a</sup></td></tr><tr><td></td><td>500</td><td>1.83</td><td>1.18</td><td>1.87</td><td>1.89<sup>a</sup></td><td>0.45<sup>ab</sup></td></tr><tr><td></td><td>500</td><td>2.19</td><td>1.32</td><td>1.81</td><td>1.49<sup>b</sup></td><td>0.44<sup>ab</sup></td></tr><tr><td>p-value</td><td></td><td>0.19</td><td>0.68</td><td>0.24</td><td>0.00</td><td>0.04</td></tr><tr><td>Silybum * ben</td><td>0</td><td>1.84</td><td>1.07</td><td>1.79</td><td>1.93<sup>a</sup></td><td>0.44</td></tr><tr><td></td><td>0.5</td><td>2.13</td><td>1.32</td><td>1.84</td><td>1.46<sup>b</sup></td><td>0.46</td></tr><tr><td></td><td>500</td><td>2.35</td><td>1.51</td><td>1.84</td><td>1.43<sup>b</sup></td><td>0.43</td></tr><tr><td></td><td>500</td><td>2.58</td><td>1.48</td><td>1.79</td><td>1.48<sup>b</sup></td><td>0.45</td></tr><tr><td colspan="2">p-value</td><td></td><td>0.70</td><td>0.05</td><td>0.38</td><td>0.00</td><td>0.39</td></tr><tr><td colspan="2">SEM</td><td></td><td>0.07</td><td>0.06</td><td>0.05</td><td>0.14</td><td>0.01</td></tr><tr><td rowspan="10">Treatments</td><td>AFB * silybum * Ben</td><td>0</td><td>2.03<sup>bcd</sup></td><td>1.07<sup>b</sup></td><td>1.70</td><td>1.58<sup>b</sup></td><td>0.43</td></tr><tr><td></td><td>0</td><td>2.40<sup>abc</sup></td><td>1.49<sup>ab</sup></td><td>1.87</td><td>1.42<sup>b</sup></td><td>0.50</td></tr><tr><td></td><td>0.5</td><td>2.70<sup>a</sup></td><td>1.73<sup>a</sup></td><td>1.82</td><td>1.36<sup>b</sup></td><td>0.40</td></tr><tr><td></td><td>0.5</td><td>2.63<sup>a</sup></td><td>1.48<sup>ab</sup></td><td>1.78</td><td>1.47<sup>b</sup></td><td>0.46</td></tr><tr><td></td><td>500</td><td>1.65<sup>d</sup></td><td>1.07<sup>b</sup></td><td>1.89</td><td>2.28<sup>a</sup></td><td>0.45</td></tr><tr><td></td><td>500</td><td>1.85<sup>d</sup></td><td>1.15<sup>b</sup></td><td>1.81</td><td>1.50<sup>b</sup></td><td>0.43</td></tr><tr><td></td><td>500</td><td>2.01<sup>cd</sup></td><td>1.29<sup>b</sup></td><td>1.85</td><td>1.49<sup>b</sup></td><td>0.46</td></tr><tr><td></td><td>0.5</td><td>2.54<sup>ab</sup></td><td>1.49<sup>ab</sup></td><td>1.80</td><td>1.48<sup>b</sup></td><td>0.44</td></tr><tr><td>p-value</td><td></td><td>0.02</td><td>0.00</td><td>0.27</td><td>0.01</td><td>0.95</td></tr><tr><td>SEM</td><td></td><td>0.11</td><td>0.09</td><td>0.07</td><td>0.06</td><td>0.01</td></tr></tbody></table>

Means within a column that do not have a common superscript are significantly different (P<0.05).

SEM= Standard error of means. AFB= Aflatoxin B1. Ben= Bentonite.
P-046 (ID 122)

EFFECT OF LIGNOCELLULOSE ON EGG PRODUCTION AND EGG QUALITY

N. Milošević<fnref n="1" />, M. Đukić Stojčić<fnref n="1" />, L. Perić<fnref n="1" />, S. Bjedov<fnref n="1" /> and M. Vukić-Vranješ<fnref n="2" />

**Corresponding author:** niko.milosevic@stocarstvo.edu.rs

**Abstract**

Fibre has no direct nutritious benefit in poultry nutrition and has until now been regarded as 'useles roughage'. However, increase of the content of raw fibers in the diets for poultry showed positive effects especially in broiler breeder production. In recent years, a lot of research results reported a positive effect on egg production in laying hens, as well as on the egg quality. Higher content of lignocellulose in diet have a positive impact on fecal consistency, litter quality and a reduced percentage of dirty eggs.

The experiment was conducted on 480 ISA Brown layers which were 37 weeks of age at the start of the experiment. Hens were divided in two groups: Control group with standard diet and Experimental group with standard diet+1% lignocellulose (Arbocel, JRS) "on top". The basic production parameters and egg quality characteristics were determined during the 12 consecutive weeks. The results showed that Arbocel had no significant effect on egg production or egg weight, but had a positive influence on feed consumption. A significant (P<0.05) positive effect of Arbocel on egg shell cleanness was established in the trial. Based on the results of this study it can be concluded that lignocellulose has a positive impact on feed consumption and egg shell cleanness.

**Keywords:** laying hens, lignocellulose, production, egg quality

**Introduction**

Fibre has no direct nutritious benefit in poultry nutrition and has until now been regarded as 'useles roughage'. However, increase of the content of raw fibers in the diets for poultry showed positive effects especially in broiler breeder production. In recent years, a lot of research results reported a positive effect on egg production in laying hens, as well as on the egg quality. Higher content of lignocellulose in diet have a positive impact on fecal consistency, litter quality and a reduced percentage of dirty eggs.

The term lignocellulose includes the mixture of cellulose, hemicellulose and lignin. The advantage of these products is standardized in its composition (the content of crude fiber content 50%-65%, depending on product), the absence of mycotoxins and the heat treatment which provides a microbiological quality (POTTGÜTER, 2008). Research on the effect of sodium in the diets of the laying hens showed that it has a positive effect on reducing the incidence of cannibalism, which is especially important in free range laying hens (VAN KRIMPEN et al, 2005; HARLANDER-MATAUSCHEK, 2007; VAN KRIMPEN 2008). ESMAIL (1997) found a direct relationship between the cellulose content in the diet and the occurrence of cannibalism and mortality in laying hens. In general, cannibalism is reduced with the increase of fiber content in the diet, especially the increase of insoluble fiber. Authors HARTINI et al. (2003) demonstrated that a higher concentration of sodium in the diet affects the performance of laying hens. The hens that were fed diets with a higher content of cellulose had improved production and had less mortality rates compared to that of hens fed diets with a low content of cellulose. It has been found that the portions with a high cellulose content improves the consistency of the feces, resulting in a dryer litter and decreasing the content of ammonia in the air (REZAEI et al., 2008).

**Material and Methods**

The trial was carried out on the experimental farm of the Department of Animal Science of the Faculty of Agriculture in Novi Sad. Investigation was conducted on 480 ISA Brown layers which were 27 weeks of age at the start of the experiment. Hens were divided in two groups (two treatments) with eight replicates each. One replicate consists 6 cages with 30 hens. The first group (Control) was fed a standard diet and the second (experimental) group was fed with a standard diet+1% lignocellulose (Arbocel, JRS) "on top". The standard diet containing 17% crude protein, 11,35 ME MJ/kg, 3.5% Ca, 0.35% available P and ad libitum water was supplied by nipple drinkers. Layers were beak trimmed. The basic production parameters were determined during the 12 consecutive weeks. The following parameters were used: Egg production parameter: laying percentage per housed hen, laying percentage per average hen and feed intake. Examination

<footnote n="1">¹ Department of Animal Science, University of Novi Sad, Faculty of Agriculture, Serbia</footnote>

<footnote n="2">² Institute for science application in agriculture, Belgrade</footnote>
of external and internal egg quality characteristics was carried out four time in four-week periods, on a random sample of 96 eggs per group (12 eggs from each replicate). External and internal egg quality characteristics: egg weight, shell color, shell cleanness, shell breaking force, yolk color and albumen height. Egg weight was measured by electronic digital balance. Shell cleanness was evaluated by a scoring system on a scale from 1 (very dirty shell) to 5 (completely clean). Shell color was visually evaluated with points 1 (weight) – 5 (dark). Egg breaking force was measured by an Egg Force Reader (Orka Food Technology Ltd, Israel). Egg yolk color was determined according to the Roche yolk colour fan. Albumen height was measured using a tripod micrometer. Data were analyzed by ANOVA and means were separated by Duncan's post hoc test using StatSoft computer package (STATISTICA 11, 2012).

## Results

The results showed that there were no statistically significant differences in egg production parameters between the control and arbocel group, although the arbocel group had a greater laying percentage than the control group. From the results in Table 2 it can be seen that the increase in cellulose content had a positive impact on increasing the weight of the eggs as well as the cleanliness of the shell. The difference in shell cleanliness was statistically significant. This is confirmed by the fact that higher cellulose content in food has an impact on the digestive tract and fecal consistency, which resulted in soiled eggs, which was lower in the arbocel group. A significant difference was observed in the yolk color in the group with increased cellulose content showing 0.17 points lower on the Roche scale.

<table><caption>**Table 1.** Egg production parameters</caption><thead><tr><th rowspan="2">Week</th><th colspan="2">Laying percentage, per housed hen (%)</th><th colspan="2">Laying percentage, per average hen (%)</th><th colspan="2">Feed intake (g)</th></tr><tr><th>Arbocel</th><th>Control</th><th>Arbocel</th><th>Control</th><th>Arbocel</th><th>Control</th></tr></thead><tbody><tr><td><strong>28</strong></td><td>94,55</td><td>93,87</td><td>94,55</td><td>93,87</td><td>126</td><td>124</td></tr><tr><td>29</td><td>94,64</td><td>93,94</td><td>94,64</td><td>93,94</td><td>138</td><td>135</td></tr><tr><td><strong>30</strong></td><td>95,49</td><td>94,48</td><td>95,49</td><td>94,7</td><td>131</td><td>126</td></tr><tr><td>31</td><td>94,93</td><td>94,61</td><td>94,93</td><td>94,61</td><td>131</td><td>126</td></tr><tr><td><strong>32</strong></td><td>93,93</td><td>94,18</td><td>94,33</td><td>94,39</td><td>125</td><td>115</td></tr><tr><td>33</td><td>94,88</td><td>94,9</td><td>94,88</td><td>94,9</td><td>125</td><td>126</td></tr><tr><td><strong>34</strong></td><td>94,32</td><td>94,96</td><td>94,32</td><td>94,96</td><td>134</td><td>132</td></tr><tr><td>35</td><td>94,21</td><td>93,94</td><td>94,21</td><td>93,94</td><td>129</td><td>127</td></tr><tr><td><strong>36</strong></td><td>93,71</td><td>93,35</td><td>93,71</td><td>93,35</td><td>144</td><td>144</td></tr><tr><td>37</td><td>93,21</td><td>93,35</td><td>93,21</td><td>93,43</td><td>125</td><td>123</td></tr><tr><td><strong>38</strong></td><td>94,08</td><td>94,28</td><td>94,08</td><td>94,28</td><td>128</td><td>125</td></tr><tr><td>Average</td><td><strong>94,36</strong></td><td><strong>94,18</strong></td><td><strong>94,4</strong></td><td><strong>94,21</strong></td><td><strong>131</strong></td><td><strong>128</strong></td></tr><tr><td colspan="7" style="text-align: center; font-weight: bold;">p-value</td></tr><tr><td>Treatments</td><td colspan="2"><strong>0,782</strong></td><td colspan="2"><strong>0,791</strong></td><td colspan="2"><strong>0,141</strong></td></tr><tr><td>Week</td><td colspan="2"><strong>0,051</strong></td><td colspan="2"><strong>0,033</strong></td><td colspan="2"><strong>0</strong></td></tr><tr><td>Interaction</td><td colspan="2"><strong>0,937</strong></td><td colspan="2"><strong>0,964</strong></td><td colspan="2"><strong>0,443</strong></td></tr></tbody></table>
<table><caption>**Table 2.** External and internal egg quality characteristics</caption><thead><tr><th rowspan="2">Week</th><th colspan="2">Egg weight (g)</th><th colspan="2">Shell color (points)</th><th colspan="2">Shell cleanliness (points)</th><th colspan="2">Albumen height (mm)</th><th colspan="2">Yolk color (Roche)</th><th colspan="2">Shell breaking force (kg)</th></tr><tr><th>A</th><th>C</th><th>A</th><th>C</th><th>A</th><th>C</th><th>A</th><th>C</th><th>A</th><th>C</th><th>A</th><th>C</th></tr></thead><tbody><tr><td>30</td><td>66</td><td>65</td><td>2,21</td><td>2,43</td><td>1,33</td><td>1,16</td><td>8,41</td><td>8,1</td><td>14,26</td><td>14,4</td><td>4,09</td><td>4,1</td></tr><tr><td>32</td><td>67,5</td><td>66,5</td><td>2,41</td><td>2,46</td><td>1,21</td><td>1,5</td><td>8,2</td><td>8,24</td><td>14,26</td><td>14,21</td><td>4,24</td><td>4,15</td></tr><tr><td>34</td><td>67</td><td>67,1</td><td>2,52</td><td>2,37</td><td>1,19</td><td>1,23</td><td>6,91</td><td>7,18</td><td>14,39</td><td>14,59</td><td>4,06</td><td>3,89</td></tr><tr><td>36</td><td>66</td><td>65,7</td><td>2,45</td><td>2,51</td><td>1,66</td><td>2,22</td><td>7,94</td><td>7,55</td><td>14,08</td><td>14,48</td><td>4,09</td><td>4,06</td></tr><tr><td>Average</td><td>67</td><td>66</td><td>2,4</td><td>2,44</td><td>1,17</td><td>1,28</td><td>7,86</td><td>7,77</td><td>14,25</td><td>14,42</td><td>4,12</td><td>4,05</td></tr><tr><td colspan="14" style="text-align: center;">p-value</td></tr><tr><td>Treatments</td><td>0,137</td><td>0,524</td><td>0,002*</td><td>0,161</td><td>0,023*</td><td>0,326</td><td>0,334</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Week</td><td>0,005*</td><td>0,162</td><td>0,013*</td><td>0,000*</td><td>0,027*</td><td>0,44</td><td>0,364</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Interaction</td><td>0,416</td><td>0,103</td><td>0,066</td><td>0,014*</td><td>0,074</td><td>0,447</td><td>0,403</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

A – Arbocel, C-Control,* p<0.05

**Conclusions**

It can be concluded that lignocellulose has a positive impact on feed consumption and egg shell cleanness and therefore it is necessary to continue the research on this subject, preferably during the whole production cycle.

*Supported by the Ministry of science of the Republic Serbia (Project No. TR 31033).*

**Literature**

ESMAIL, H.M., 1997: Fibre in Nutrition. Poultry International, July, 31-34.

HARLANDER-MATAUSCHEK, A., 2007: Untersuchungenüber die Motivation des Ederpickens bei Legehennen. Hohenheim, May 2007.

HARTINI, S., M. CHOCT, G. HINCH, J. NOLAN, 2003: Effect on diet composition, gut microbial status and fibre forms on „Cannibalism in Layers“; Australian Egg Corporation Limited, April 2003.

POTTGÜTER, R., 2008: Fibre in Layer Diets, Lohmann Information, **43**, 22-31.

REZAEI M., K. TORSHIZI, Y. ROUZBEHAN, 2008: Effect of processed and conventional fibre supplementation on performance, organ weight and immune response of broilers. XXIII World's Poultry Congress 2008, Brisbane, Australia.

VAN KRIMPEN, M., 2008: Impact of nutritional factors on eating behaviour and feather damage of laying hens. Dissertation, Wageningen, Sept. 2008.

VAN KRIMPEN, M.M., R.P. KWAKKEL, B.F.J. REUVEKAMP, C.M.C. VAN DER PEET-SCHWERING, L.A. DEN HARTOG, M.W.A VERSTEGEN, 2005: Impact of feeding management on feather pecking in laying hens. World Poultry Sci. Jou., **61**, 663–685.
P-047 (ID 226)

# EFFECT OF DIETARY SUPPLEMENTATION OF BOESENBERGIA PUNDURATA ON SMALL INTESTINAL MORPHOLOGY AND IMMUNOLOGICAL RESPONSE OF BROILER CHICKENS

J. Mitchaothai<fnref n="1" />*, P. Jaipeng<fnref n="1" />, R. Lertpatarakomol<fnref n="2" />, and K. Rojanamongkol<fnref n="1" />

## Abstract

The aim of the current study was to investigate the effect of dry powders of *Boesenbergia rotunda* (BR) on small intestinal morphology and immunological response of broiler chickens against Newcastle disease virus (NDV). Ninety-six chickens (1 day-old) were randomly allocated into four experimental groups equally and housed in 16 cages with 6 chickens each (4 cages per treatment). The chickens in group 1, 2, 3 and 4 were fed basal diet supplemented with 0.00% (T1), 0.05% (T2), 0.10% (T3) and 0.15% (T4) of dry powders of BR, respectively, lasting for 6 weeks. One chicken was randomly chosen from each cage at the age of 3 and 6 weeks in order to collect small intestines for measuring villous height (VH) and crypt depth (CD). All experimental chickens were vaccinated against NDV at 2 and 14 days old and the blood samples of the chickens were collected to measure the levels of heamagglutination inhibition (HI) titers against NDV at 2, 14 and 42 days old. The results have illustrated no significant difference (P>0.05) was seen for the VH, CD and VH:CD of small intestine. The HI titers against NDV at the day 14 of the chickens in T3 (3.00±1.07 log₂) was significant lower (P≤0.05) than those in T1 (4.25±0.71 log₂), while the HI titers against NDV of the chickens at day 2 and 42 was no significant differences among four experimental groups (P>0.05). Thus, the dietary supplementation of dry powders of BR for broiler chickens had no influence on small intestinal morphology, but the impact of the supplementation on immunological response to NDV was varied for broiler chickens.

**Keywords:** *Boesenbergia pundurata*, small intestinal morphology, immunological response, broiler chicken

## Introduction

There were tremendous interest for the use of local herbs for improving broiler chicken production and health, in order to alleviate residues from anti-microbial agents, especially in Asian countries. Several kinds of herbs were investigated for many aspects. One of a promising herb is *Boesenbergia rotunda* (BR) found many activities for control diseases. The main proposed active ingredient in BR was flavonoid compound containing important activities of anti-microbial and anti-inflammation (CHONG et al., 2012). There was very effective for inhibition of pathogenic *Escherichia coli* under *in vitro* condition (MIKSUSANTI et al., 2008). Both anti-microbial and anti-inflammation would be beneficial for health of broilers chickens, which have higher chance to get gastro-intestinal infection from bacteria, especially during nutrition stress from diet formula change. This would then lead to lower immunity responses to several and key infectious diseases. Therefore, the current study was aimed to investigate effect of dry powders of BR supplementation in diet with various doses on small intestinal morphology and immunological response of broiler chickens against Newcastle disease virus (NDV).

## Materials and Methods

A total of 96 male day-old broiler chickens were randomly allocated into four studied groups containing four replicates of 6 chickens in each. The chickens of group T1 (Control), T2, T3 and T4 were offered with the corn-soybean diet supplemented with dry *Boesenbergia pundurata* powders at 0.00%, 0.05%, 0.10% and 0.15%, respectively lasting for 6 weeks. All chicks were fed *ad libitum* and free to assess drinking water. One chicken was randomly chosen from each cage at the age of 3 and 6 weeks in order to collect small intestines at jejunum part for measuring villous height (VH), crypt depth (CD) and ratio of VH:CD. All experimental chickens were vaccinated against NDV at 2 and 14 days old and the blood samples of the chickens were collected to measure the levels of heamagglutination inhibition (HI) titers against NDV at 2, 14 and 42 days old. All data were expressed as mean ± standard deviation (SD) and analyzed as a

<footnote n="1">¹Department of Veterinary Clinical Science, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand</footnote>

<footnote n="2">²Department of Animal Science & Veterinary Basic Science, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand</footnote>

<footnote>*Corresponding author: jmitchaothai@yahoo.com</footnote>
completely randomized design (CRD) by ANOVA and then Duncan multiple test were used to detect significant difference between groups at $P<0.05$.

## Results

There were no significant difference ($P>0.05$) detected for VH, CD and VH:CD ratio of jejunum intestine at week 3 and 6 among four dietary treatments (Table 1.). The HI titers against NDV at the day 2 and 42 of the chickens were no significant difference ($P>0.05$) among four experimental groups. However, the HI titers against NDV at the day 14 of the chickens in T3 (3.00±1.07 $log_2$) was significant lower ($P<0.05$) than those in T1 (4.25±0.71 $log_2$) while the HI titers of the chickens at day 14 in the other groups were no significant difference ($P>0.05$) as illustrated in the Table 2.

<table><caption>**Table 1.** Small intestinal morphology of the chickens offered the dietary treatments</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Dietary treatment</th></tr><tr><th>0.00 %</th><th>0.05 %</th><th>0.10 %</th><th>0.15 %</th></tr></thead><tbody><tr><td><strong>Villous height (μm)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Wk3</td><td>1,020.56±97.40</td><td>1,009.45±108.31</td><td>917.50±126.92</td><td>942.46±50.86</td></tr><tr><td>Wk6</td><td>1,204.83±150.28</td><td>1,126.66±90.39</td><td>1,175.66±49.80</td><td>1,130.06±32.32</td></tr><tr><td><strong>Crypt depth (μm)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Wk3</td><td>81.61±9.00</td><td>88.34±17.36</td><td>90.75±21.21</td><td>79.06±26.28</td></tr><tr><td>Wk6</td><td>106.14±20.33</td><td>113.57±8.00</td><td>102.96±3.27</td><td>116.20±25.15</td></tr><tr><td><strong>Villous height : Crypt depth</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Wk3</td><td>12.57±1.24</td><td>11.76±2.59</td><td>10.85±4.30</td><td>12.72±3.27</td></tr><tr><td>Wk6</td><td>11.56±1.77</td><td>9.97±1.17</td><td>11.44±0.80</td><td>10.08±2.25</td></tr></tbody></table>

<table><caption>**Table 2.** Heamagglutination inhibition titer against the ND viruses of the chickens offered the dietary treatments</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Dietary treatment</th></tr><tr><th>0.00 %</th><th>0.05 %</th><th>0.10 %</th><th>0.15 %</th></tr></thead><tbody><tr><td><strong>Heamagglutination inhibition titer (log<sub>2</sub>)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Day2</td><td>6.38±1.06</td><td>7.25±0.87</td><td>7.00±1.41</td><td>6.75±0.71</td></tr><tr><td>Day14</td><td>4.25±0.71<sup>a</sup></td><td>3.25±1.04<sup>ab</sup></td><td>3.00±1.07<sup>b</sup></td><td>3.88±1.46<sup>ab</sup></td></tr><tr><td>Day42</td><td>1.75±1.04</td><td>0.75±0.89</td><td>1.13±1.73</td><td>1.13±1.00</td></tr></tbody></table>

## Discussion

From the results of the current study, there was not found effect of the dietary BR powders supplementation on VH, CD and ratio of VH:CD at jejunum of the broiler chickens. This would be the fact that no pathogenic *E. coli* challenge in the current study, resulting no severity of villous damage by *E. coli* in small intestine, mainly normal flora and less pathogenic bacteria. This results is in agreement with earlier study (DUARTE et al., 2014) reported that very small effect of flavonoid in crude propolis on small intestinal morphology. For immunological responses to NDV vaccination, in general all chickens had gradually decrease of the HI titers to NDV after vaccination, implying lower maternally derived antibodies (MDA) with chicken age. The pattern of immunological responses of the chickens in each studied group is rather similar each other. However, the chickens obtained the dry powders of BR at 0.10% had lower HI titer than those obtained on BR powders in diet at 14 day after vaccination, which is the day of building high level of immunity for protection. This result might be the variation of immunological responses and MDA of individual chickens (JALOB et al., 2011). In the clinical point of view, a HI titer of 3 $log_2$ is generally accepted as positive for specific immunity against ND (ALLAN and GOUGH, 1974). This would be interpreted that the HI titers of most studied chickens in the current study had protective immunities up to around 2 weeks of age, but had a risk of getting NDV infection somewhere between 4-6 weeks of age. Thus, there was hardly to make a conclusion for the influence of dietary BR supplementation on immunological responses. In general, the further study should test the effects of dietary BR supplementation with pathogenic *E. coli* challenge and measure the HI titer weekly to make more clear influence on immunological responses.

<footnote><sup>a,b</sup> means in the same row with different superscripts are significant difference ($P<0.05$)</footnote>
**Conclusions**

The dietary supplementation of dry powders of BR for broiler chickens had no influence on small intestinal morphology, but the impact of the supplementation on immunological response to NDV was varied for broiler chickens.

**Acknowledgements**

The authors would like to thank Mr.Chongpol Jaiwangdee, Ms.Watoo Todpetch, Ms.Prapatsorn Jaidee, Ms.Pensiri Thewvorksuwan and Ms.Narumol Ounyingcharean for their excellent assistant and Mahanakorn University of Technology for financial support.

**Literature**

ALLAN, W.H. and R.E. GOUGH, 1974. A standard haemagglutination inhibition test for Newcastle disease. (1). A comparison of macro and micro methods. Vet. Rec. **95**, 120-123.

CHONG, T.E., L.Y. KEE, C.C.FEI, H.C HAN, W.S. MING, C.T.L PING, F.G. TECK, N. KHALID, N.A. RAHMAN, S.A. KARSANI, S. OTHMAN, R. OTHMAN and R. YUSOF, 2012. *Boesenbergia rotunda*: From ethnomedicine to drug discovery. Evid.-Based Compl. Alt. Med. **Volume 2012**, Article ID 473637, 25 pages.

DUARTE, C.R.A., C. EYNG, A.E. MURAKAMI and T.C. SANTOS, 2014. Intestinal morphology and activity of digestive enzymes in broilers fed crude propolis. Can. J. Anim. Sci. **94**, 105-114.

JALOB, Z.KH., M.D. TAHA, M.M. JUMAA, D.A. NAJIM and A.A. SALMAN. 2011. Immune response against Newcastle disease virus (NDV) in broiler chickens. Al-Anbar J. Vet. Sci. **4**, 82-87.

MIKSUSANTI, B.S., B.S.L. JENIE, B.P. PRIOSOERYANTO, R. SYARIEF and G.T. REKSO, 2008. Mode of action Temu Kunci (*Kaempferia pandurata*) essential oil on *E. coli* K1.1 cell determined by leakage of material cell and salt tolerance assays. HAYATI J. Biosci. **15(2)**, 56-60.
# A NOVEL IN VITRO POULTRY MODEL TO COMPARE RELEASE KINETICS OF (UN)PROTECTED BUTYRATE

P.C.A. Moquet<fnref n="1" />, L. Onrust<fnref n="2" />, R. van Emous<fnref n="1" />, R.P. Kwakkel<fnref n="1" />

**Corresponding author:** pierre.moquet@wur.nl

## Abstract

The gastrointestinal (GIT) segment wherein dietary additives, such as butyrate, are released may influence their observed efficacy. Release kinetics are influenced by pH, gastric and enteric motility, retention time in the different GIT segments and enzymes concentration. Unfortunately, none of the available in vitro poultry model mimics accurately these conditions. As a consequence, a novel in vitro poultry GIT model was developed to compare the release kinetics of commercially available butyrate formulations. Four fat-embedded butyrate matrices originating from three suppliers as well as tributyrin, the triglyceride of butyrate, were evaluated in a three-step (pre-gastric, gastric and enteric) in vitro digestion procedure. Aliquots were drawn at 5 stages of digestion representing the end of the crop, proventriculus-gizzard, duodenum, jejunum and ileum. The percentage of the initial butyrate dose solubilized was calculated for each stage. Significant differences were found among formulations for each digestion stage (P < 0.001). Half of the fat-embedded butyrate matrices showed a sustained release profile, while the other released all their content in the pre-enteric regions. Tributyrin had a release profile targeting enteric segments. Results of this study indicate that despite almost identical visual appearance, release kinetics can vary significantly among butyrate derivatives. Such variations can possibly explain the inconsistency of the bird's growth response to dietary butyrate supplementation as shown in literature.

**Keywords:** *in vitro* model, release kinetics, feed additives, butyrate

## Introduction

In poultry nutrition, butyrate is used as a feed additive. This short chain fatty acid can be fed as an unprotected salt or in the form of protected derivatives such as butyrate glycerides or butyrate-loaded matrices. Butyrate elicits stimulatory, albeit inconsistent, effects on growth performance of broiler chickens. The GIT segment wherein butyrate is released may modulate its effects given the diversity of cell types and pH conditions encountered throughout the avian GIT, and the differences in microbiota composition in the different gut segments. Release properties of protected derivatives can therefore contribute to the understanding of the mode of action of butyrate. Release kinetics can be determined *in vivo* by measuring directly the butyrate content in the chyme or by measuring the oxidation of labelled butyrate in respiratory chambers. The first technique doesn't allow to discriminate between the dietary or fermentative origin of butyrate, while the prohibitive costs of the latter prevents its use when screening numerous formulations. *In vitro* models are interesting alternatives to the aforementioned techniques as they are quick, cost-effective, and the results are a not confounded by fermentation end-products. Release kinetics are being influenced by environmental factors e.g. pH, temperature, enzymatic activity or retention time. Specific conditions encountered in the avian GIT are described in the literature (Long, 1967; Philips and Fuller, 1983; Zyla et al., 1999; Dendow, 2015), but not in *in vitro* poultry models. The present work was undertaken to develop a novel *in vitro* poultry model, using realistic digestion parameters, to compare release kinetics of commercially available butyrate derivatives.

## Material and Methods

Tributyrin (T8626, Sigma-Aldrich, Saint-Louis, USA) and 4 commercially available fat-protected butyrate derivatives: Adimix 30 C (Nutri-ad International NV, Belgium), Ding Su (Xiamen Fujian Co., China), Sodium Butyrate 300 and 500 (Vega Pharma Limited, Hangzhou, China) were used in triplicate in the *in vitro* incubation procedure. The procedure consisted of pre-gastric, gastric and enteric steps. The enteric step had three different durations, resulting in a total of five incubation stages. Approximately 0.25 g of sample was incubated for 35 min in 20 ml of a 1 N tris-HCl buffer solution (pH 5.8) containing 1 g/l NaCl and 0.5 g/l of α-amylase from porcine pancreas (10 U/mg solid; A3176, Sigma-Aldrich). When only the pre-gastric digestion was considered, the procedure was stopped by transferring the tube to ice. Otherwise, incubation proceeded with addition of 2 ml of a 0.085 N HCl solution containing 11 g/l of pepsin from porcine

<footnote n="1">¹Animal Nutrition Group, Wageningen University, PO Box 338, 6700 AH Wageningen, the Netherlands</footnote>

<footnote n="2">²Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salis-burylaan 133, B-9820 Merelbeke, Belgium</footnote>
gastric mucosa (250 U/mg solid; P7000, Sigma-Aldrich). If necessary, pH was adjusted to 2.7 ± 0.05 with drops of 6 N HCl or 6 N NaOH. The second stage of incubation then proceed for 35 mn. The procedure was stopped when only the gastric digestion was considered by transferring the tubes to ice. Otherwise, 4 ml of a 1 N tris-buffer solution (pH 6.5) containing 1.875 g/l pancreatin from porcine pancreas (8 x USP specifications; P7545, Sigma-Aldrich) and 0.7 g/l bile salts (48305, Sigma-Aldrich) was added to the tube. If necessary, pH was adjusted to 6.5 ± 0.05 with drops of 6 N HCl or 6 N NaOH. The incubation was stopped by transferring the tubes to ice 10 mn, 80 mn or 170 mn after addition of pancreatin when duodenal, jejunal or ileal digestion were considered, respectively. A temperature of 40 ± 0.1°C and a stirring rate of 360 rpm were maintained throughout the different stages of incubation. Aliquot were drawn at the end of each digestion stage. Butyrate content was determined for each derivative by incubating ground samples in 20 ml of 1 M HCl solution during 24 hours. Aliquots were analysed for butyrate concentration by gas chromatography. Butyrate yield was expressed as the percentage of the total incubated butyrate dose that was solubilized at a certain time point. Butyrate yields were analysed for each incubation stage with the PROC GLM procedure of SAS® version 9.2 (SAS Institute Inc., Cary, NC, USA), using a significance level *P* of 0.05. LSD test was carried as a post-hoc test.

## Results

Table 1 presents the cumulative butyrate release per digestion stage of the assayed butyrate derivatives. For each digestion stage, significant differences were found among the derivatives (*P* < 0.001). Throughout the enteric phases of incubation, pH dropped gradually when tributyrin was incubated, resulting ultimately in a pH significantly lower than the one observed with fat-embedded butyrate matrices (*P* < 0.001).

<table><caption>**Table 4.** Cumulative butyrate release (%) per incubation stage for different butyrate formulations</caption><thead><tr><th rowspan="2">Butyrate derivative</th><th colspan="5">Incubation stage¹</th></tr><tr><th>Crop</th><th>Proventriculus -gizzard</th><th>Duodenum</th><th>Jejunum</th><th>Ileum</th></tr></thead><tbody><tr><td>Tributyrin</td><td>13.4<sup>d</sup> ± 1.5</td><td>13.9<sup>c</sup> ± 2.4</td><td>20.1<sup>d</sup> ± 2.2</td><td>34.0<sup>c</sup> ± 4.4</td><td>41.2<sup>c</sup> ± 2.8</td></tr><tr><td>Adimix 30</td><td>57.2<sup>c</sup> ± 3.3</td><td>77.8<sup>b</sup> ± 5.2</td><td>82.3<sup>c</sup> ± 3.3</td><td>89.7<sup>b</sup> ± 20</td><td>95.6<sup>b</sup> ± 6.1</td></tr><tr><td>Ding Su</td><td>88.3<sup>b</sup> ± 7.4</td><td>97.7<sup>a</sup> ± 4.1</td><td>105.2<sup>a</sup> ± 5.6</td><td>99.1<sup>ab</sup> ± 2.1</td><td>99.6<sup>b</sup> ± 0.2</td></tr><tr><td>Vega 30</td><td>66.8<sup>c</sup> ± 8.6</td><td>87.0<sup>b</sup> ± 9.1</td><td>100.0<sup>ab</sup> ± 5.9</td><td>107.7<sup>a</sup> ± 1.6</td><td>112.1<sup>a</sup> ± 9.9</td></tr><tr><td>Vega 50</td><td>99.2<sup>a</sup> ± 2.2</td><td>97.8<sup>a</sup> ± 1.7</td><td>96.5<sup>b</sup> ± 2.2</td><td>97.8<sup>ab</sup> ± 1.6</td><td>97.1<sup>b</sup> ± 0.4</td></tr><tr><td><i>P</i> value</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td></tr></tbody></table>

Values represent the mean ± standard deviation of three observations per time point. Means with different letters within a column differ significantly at *p* < 0.05. ¹Digestion stage 1: 35 min, 2: 70 min, 3: 80 min, 4: 160 min, 5: 250 min.

## Conclusion

Despite almost identical visual appearance, *in vitro* release profiles can differ significantly among fat-embedded butyrate matrices. In the present study two matrices showed a sustained release profile, with a gradual release of butyrate throughout all the digestion stages. Conversely, two other matrices showed no protection, with a complete release of their content in the first stage of digestion. Differences in release profiles may, in addition to the already described dose, health status and ages factors, be an underlying mechanism explaining the observed discrepancies in the literature concerning the use of fat-embedded butyrate matrices. Tributyrin showed a release profile targeting the enteric region, with a total cumulative yield being lower than the theoretical maximum. This could be due to an inhibitory effect of the butyric acid-induced drop of pH on pancreatic lipase during the enteric steps.

The present *in vitro* model allows to compare release profiles of different butyrate derivatives in quick and inexpensive way, thus meeting the goal of the study. There are, however, a few limitations related to the use of such a model. Avian-specific motility, e.g. gizzard contractions and anti-peristaltic movements of the chyme, are not included in the model. Yields greater than 100% indicates that the butyrate content determination needs to be refined. Also, pH stability can be compromised despite the use of a concentrated buffer when acidic compounds are assessed.
References

DENBOW, D.M., 2015: Chapter 14 - Gastrointestinal Anatomy and Physiology, in: SCANES, C. G.: Sturkie's Avian Physiology (Sixth Edition). Academic Press, San Diego, ISBN 9780124072435, 337-366.

LONG J.F., 1967. Gastric secretion in unanesthetized chickens. Am. J. Physiol. **212:6**, 1303-1307.

PHILIPS, S.M., R. FULLER, 1983. The activities of amylase and a trypsin-like protease in the gut contents of germ-free and conventional chickens. Br. Poult. Sci. **24:1**, 115-121.

ZYŁA K., D. GOGOL, J. KORELESKY, S. ŚWIATKIEWICZ, D.R. LEDOUX, 1999. Simultaneous application of phytase and xylanase to broiler feeds based on wheat: *in vitro* measurements of phosphorus and pentose release from wheats and wheat-based feeds. J. Sci. Food Agric. **79**, 1832-1840
P-053 (ID 98)

# EFFECTS OF ACIDIFIED AND ALKALINIZED DIETS ON GROWTH PERFORMANCE AND INTESTINAL MORPHOMETRY OF BROILER CHICKENS

R. Nourmohammadi<fnref n="1" />, H. Khosravinia<fnref n="1" />, and N. Afzali<fnref n="2" />

**Corresponding author:** Nourmohammadi.61@gmail.com

## Abstract

The purpose of this experiment was to study growth performance and intestinal morphology in broilers fed on acidified and alkalinized diets. Two hundred and fifty Ross 308 male broiler chicks were randomly allotted to 5 dietary treatments consisting a nutritionally balanced basal diet supplemented with 10, 20 and 30 g/kg citric acid (CA) or Ca (OH)₂ (ALK; 8.9 g/kg for growth period and 8.6 g/kg for finisher period) from day 7 up to day 42 of age. Inclusion of 30 g/kg CA significantly increased body weight, average daily weight gain, average daily feed intake and improved feed efficiency. Addition of 30 g/kg CA to the diets increased villus length in duodenum, jejunum and ileum, crypt depth and villus length to crypt depth ratio in the jejunum but reduced epithelial thickness in the duodenum and jejunum segments (P<0.05). Alkalinized diet significantly reduced average daily feed intake, whereas had no significantly effect on morphometrical parameters concerned. In conclusion, this study demonstrated that supplementation of broiler diets with 30 g/kg of CA improved performance and morphometric indices. Furthermore, change of acidity of gut toward alkaline by feeding an alkalinized diet resulted in a reduced growth performance.

**Keywords:** citric acid, calcium hydroxide, broiler chicken, intestinal morphology

## Introduction

Acidified diets further benefits to birds by activating the functionality of proteolytic enzymes, promoting feed intake, reducing ammonia and other growth-depressing microbial metabolites, favouring mineral absorption, and lowering the incidence of subclinical infections (CHAVEERACH et al. 2004). Citric acid (CA) appears to have a positive impact on the small intestine morphometry, thereby facilitating nutrient absorption and growth performance in broiler chicken (NOURMOHAMMADI and AFZALI, 2013). However, the effectiveness of acidified diets in broiler chicken may also depend upon composition of diet and its buffering capacity. The mode of action for dietary organic acids, including CA, is mainly believed to rely on their ability to acidify the diet and ultimately the contents of the digestive tract (NOURMOHAMMADI et al., 2012). These compounds are primarily used to prevent pathogen contamination such as Salmonella through sanitising the feed. However, experimental results showed that CA acts to improve nutrient digestion and absorption, mucosal immunity and exert topical effects on the intestinal brush border in broiler chicken (NOURMOHAMMADI et al., 2012). To the best of our knowledge, the supplemental effects of alkalinisation (ALK) diets in broilers are largely unrevealed. Therefore, the present study was conducted to compare the effects of acidified or alkalinized diets on growth performance and intestinal morphology in broiler chicken.

## Material and Methods

A total number of 250 male Ross 308 broiler chicks were obtained from a commercial hatchery and randomly distributed into 25 floor pens in an environmentally controlled house. Each of the five treatments was replicated five times, with 10 birds per pen. The experimental period was divided into grower (7 to 21 d) and finisher phases (22 to 42 d). The basal diet was supplemented with 10, 20 or 30 g/kg CA to provide three of the experimental diets. An experimental diet was also provided by addition of Ca (OH)₂ in the form of mile of lime (8.9 g/kg for growth period and 8.6 g/kg for finisher period). The pH value for the alkaline solution was set as 12.3. Considering pen as experimental unit, body weight (BW) and feed intake (FI) were recorded weekly and data were used to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed to gain ratio (FCR) for 7- to 42 d period of age. Fragments of approximately 5 cm were obtained from each of the following segments of the small intestine: the duodenum and the jejunum to evaluate the intestinal morphology. The fragments from each segment were then placed on polystyrene sheets, opened longitudinally, washed in saline solution, fixed in 10% formaldehyde solution, dehydrated and embedded in paraffin. The measurements of villus length and crypt depth were taken using a light microscope and a system that analyses computerised images, and the data were used to calculate the villus length to crypt depth ratio. The height of 30 villi and the depth of

<footnote n="1">¹Dept. of Animal Science, Lorestan University, Khorramabad, Iran</footnote>

<footnote n="2">²Dept. of Animal Science, University of Birjand, Birjand, Iran</footnote>
30 crypts were measured from each segment and replicate, and the means were utilized for statistical analysis. Data were analyzed using GLM procedure (SAS, Institute) in a completely randomized design. All of the treatment means were compared using Tukey-Kramer's test. For the different statistical tests, significance was declared at *P* < 0.05.

## Results

Inclusion of 30 g/kg CA increased BW, ADG, and ADFI, and decreased FCR compared to the control birds from 7 to 42 days of age (Table 1). Alkalinized diet (ALK) significantly decreased ADFI of the birds in comparison with the birds received control diet (*P* < 0.01). Dietary CA at 30 g/kg significantly increased villus length in duodenum, jejunum and ileum epithelium compared to the control birds (Table 2; *P* < 0.05). The acidified diets at 30 g/kg level also significantly increased the jejunal crypt depth and villi length: crypt depth ratio and reduced epithelial thickness in the duodenum and jejunum segments (*P* < 0.05).

## Conclusions

Results of the current study for increased ADFI and ADG due to dietary CA (30 g/kg) through day 42 of age are consistent with those reported by NOURMOHAMMADI et al. (2012). In the present study, all dietary treatments were isocaloric; therefore, the increased performance with 30 g/kg of CA at day 42 of age may be attributed in part to the positive effect of CA on improved gut health in favour of nutrients availability. It was shown that dietary acidification increases gastric proteolysis and protein/amino acid digestibility by enhancing digestive enzyme activities (LANGHOUT, 2000). The negative response or lack of response in most of the variables to alkalinized diet in the current research is unknown. However, ADFI was significantly reduced for ALK diet, a phenomenon which may be reasoned by impaired plasma acid-base balance or reduced palatability of the diet.

NOURMOHAMMADI and AFZALI (2013) reported that CA as natural growth promoters increased villus lengths in duodenum and jejunum through decreasing the intestinal colonization and infectious processes resulting in inflammatory reactions at the intestinal mucosa. The increased villus length represents an attempt to increase intestinal surface area to maximize absorption. Moreover, increased jejuna villus length could be hypothetically caused by reduced cell turnover and migration, resulting in the retention of epithelial cells on the villi (NOURMOHAMMADI and AFZALI, 2013). The positive influence of CA-added diets at 30 g/kg level on villus length and crypt depth in this study suggest that CA may enhance rate of nutrient absorption and a reduce rate of enterocyte cell migration from the crypt to the villus. The villus length to crypt depth ratio is an indicator of the likely digestive capacity of the small intestine. An increases ratio corresponds to an increased digestion and absorption. In conclusion, this study demonstrated that supplementation of broiler diets with 30 g/kg of CA improved performance and morphometric indices. Furthermore, change of acidity of gut toward alkaline by feeding an ALK diet resulted in a reduced growth performance in broiler chickens.

<table><caption>**Table 1.** Effects of citric acid (CA) and alkaline diet (ALK) on productive performance of broilers</caption><thead><tr><th rowspan="2">Parameter</th><th rowspan="2">Control</th><th colspan="3">Dietary acidification (g CA/kg)</th><th rowspan="2">ALK</th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>10</th><th>20</th><th>30</th></tr></thead><tbody><tr><td>BW at 42 d, g</td><td>1815<sup>b</sup></td><td>1856<sup>b</sup></td><td>1850<sup>b</sup></td><td>1993<sup>a</sup></td><td>1781<sup>b</sup></td><td>15.7</td><td>0.003</td></tr><tr><td>ADG, g/b/d</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7 to 42 d</td><td>48.2<sup>b</sup></td><td>49.6<sup>b</sup></td><td>49.3<sup>b</sup></td><td>53.8<sup>a</sup></td><td>47.6<sup>b</sup></td><td>1.35</td><td>0.007</td></tr><tr><td>ADFI, g/b/d</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7 to 42 d</td><td>91.8<sup>ab</sup></td><td>94.6<sup>a</sup></td><td>90.9<sup>b</sup></td><td>93.9<sup>a</sup></td><td>86.4<sup>c</sup></td><td>1.61</td><td>0.002</td></tr><tr><td>FCR, g/g</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7 to 42 d</td><td>1.91<sup>a</sup></td><td>1.90<sup>a</sup></td><td>1.84<sup>ab</sup></td><td>1.74<sup>b</sup></td><td>1.82<sup>ab</sup></td><td>0.05</td><td>0.041</td></tr></tbody></table>

<footnote><sup>a-c</sup> Means within a row with different superscripts are significantly different (*P* < 0.05).</footnote>

<footnote>SEM, standard error over general mean</footnote>
<table><caption>**Table 2.** Effects of citric acid (CA) and alkaline diet (ALK) on intestinal morphology of broilers</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th colspan="3">Dietary acidification (g CA/kg)</th><th rowspan="2">ALK</th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>10</th><th>20</th><th>30</th></tr></thead><tbody><tr><td colspan="9"><strong>Duodenum</strong></td></tr><tr><td>Villus length, µm</td><td>1726<sup>b</sup></td><td>1727<sup>b</sup></td><td>1733<sup>b</sup></td><td>1782<sup>a</sup></td><td>1738<sup>b</sup></td><td>6</td><td>0.001</td></tr><tr><td>Crypt depth, µm</td><td>148</td><td>149</td><td>148</td><td>151</td><td>150</td><td>1</td><td>0.147</td></tr><tr><td>Ratio</td><td>11.7</td><td>11.6</td><td>11.7</td><td>11.8</td><td>11.6</td><td>0.1</td><td>0.253</td></tr><tr><td>Epithelial thickness, µm</td><td>52.3<sup>a</sup></td><td>52.2<sup>a</sup></td><td>50.8<sup>ab</sup></td><td>50.0<sup>b</sup></td><td>51.3<sup>ab</sup></td><td>0.6</td><td>0.007</td></tr><tr><td colspan="9"><strong>Jejunum</strong></td></tr><tr><td>Villus length, µm</td><td>1224<sup>b</sup></td><td>1270<sup>b</sup></td><td>1260<sup>b</sup></td><td>1423<sup>a</sup></td><td>1261<sup>b</sup></td><td>4</td><td>0.001</td></tr><tr><td>Crypt depth, µm</td><td>132<sup>b</sup></td><td>132<sup>b</sup></td><td>133<sup>b</sup></td><td>136<sup>a</sup></td><td>133<sup>b</sup></td><td>1</td><td>0.004</td></tr><tr><td>Ratio</td><td>9.3<sup>b</sup></td><td>9.6<sup>b</sup></td><td>9.5<sup>b</sup></td><td>10.5<sup>a</sup></td><td>9.5<sup>b</sup></td><td>0.2</td><td>0.034</td></tr><tr><td>Epithelial thickness, µm</td><td>34.0<sup>a</sup></td><td>33.6<sup>ab</sup></td><td>33.5<sup>ab</sup></td><td>32.4<sup>b</sup></td><td>33.8<sup>ab</sup></td><td>0.4</td><td>0.041</td></tr><tr><td colspan="9"><strong>Ileum</strong></td></tr><tr><td>Villus length, µm</td><td>876<sup>b</sup></td><td>869<sup>b</sup></td><td>896<sup>ab</sup></td><td>934<sup>a</sup></td><td>873<sup>b</sup></td><td>3</td><td>0.028</td></tr><tr><td>Crypt depth, µm</td><td>114</td><td>116</td><td>115</td><td>118</td><td>116</td><td>1</td><td>0.373</td></tr><tr><td>Ratio</td><td>7.7</td><td>7.5</td><td>7.8</td><td>7.9</td><td>7.5</td><td>0.1</td><td>0.084</td></tr><tr><td>Epithelial thickness, µm</td><td>33.2</td><td>32.4</td><td>32.7</td><td>32.1</td><td>32.2</td><td>0.5</td><td>0.472</td></tr></tbody></table>

SEM, standard error over general mean

## Literature

CHAVEERACH, P., D.A. KEUZENKAMP, L.J. LIPMAN, F. VAN KNAPEN, 2004: Effect of organic acids in drinking water for young broilers on Campylobacter infection, volatile fatty acid production, gut microflora and histological cell changes. Poult Sci. **83**, 330-334.

LANGHOUT, P., 2000: New additives for broiler chickens. World Poultry-Elsevier. **16**, 22-27.

NOURMOHAMMADI, R., N. AFZALI, 2013: Effect of citric acid and microbial phytase on small intestinal morphology in broiler chicken. Ital. J. Anim. Sci. **12**, 44-47.

NOURMOHAMMADI, R., S.M. HOSSEINI, H. FARHANGFAR, M. BASHTANI, 2012: Effect of citric acid and microbial phytase enzyme on ileal digestibility of some nutrients in broiler chicks fed corn-soybean meal diets. Ital. J. Anim. Sci. **11**, 36-40.

<footnote><sup>a-c</sup> Means within a row with different superscripts are significantly different (P <0.05).</footnote>
P-054 (ID 99)

EFFECT OF DIETARY ACIDIFICATION AND ALKALINIZATION ON ILEAL DIGESTIBILITY AND TIBIA PARAMETERS OF BROILER CHICKENS

R. Nourmohammadi<fnref n="1" />, H. Khosravinia<fnref n="1" />, and N. Afzali<fnref n="2" />

**Corresponding author:** Nourmohammadi.61@gmail.com

**Abstract**

The objective of this study was to determine the effects of dietary acidification and alkalinization on ileal nutrients digestibility, serum enzyme activities and bone mineralization in broiler chickens. The birds were allotted to 5 dietary treatments for 35 d, each of which was replicated 5 times with 10 chicks per replicate. The dietary treatments consisting a nutritionally balanced basal diet supplemented with 10, 20 and 30 g/kg citric acid (CA) or Ca(OH)₂ (ALK; 8.9 g/kg for growth period and 8.6 g/kg for finisher period) from day 7 up to day 42 of age. Inclusion of 30 g/kg CA significantly increased tibia ash, tibia Ca content, ileal digestibility of crude protein and apparent metabolizable energy corrected to zero nitrogen retention at day 42 of age (P<0.05). Lactate dehydrogenase activity in serum was significantly elevated in the birds fed on the 30 g/kg CA-treated diets, whereas alkaline phosphatase activity was decreased (P<0.05). The results indicated that ALK diet significantly reduced tibia ash, tibial P and Ca contents. In conclusion, these findings demonstrate that inclusion of 30 g/kg CA bring about great economical benefits as nutrients digestibility and bone mineralization improvements. Also, the prominent impact of alkalinized diet was reduction of nutrients absorption.

**Keywords:** citric acid, calcium hydroxide, broiler chicken, serum enzyme activity, nutrient digestibility

**Introduction**

Inclusion of citric acid (CA) in poultry diet creates an acidic condition (pH 3.5 to 4.0) in gut favouring development of lactobacilli and other beneficial bacteria. Dietary acidification increases the activities of gastric enzymes, leading to improvements in digestion and absorption of protein and fibre (MARTIN et al., 2011). The CA appears to have a positive impact on histology of the small intestine, thereby facilitating nutrient absorption and growth performance in broiler chicken (NOURMOHAMMADI and AFZALI, 2013). On the other side, NOURMOHAMMADI et al. (2012) found that severe reduction pH in gut due to high dose dietary acidification brought about decreased digestion and absorption of nutrients, disrupted liver and kidney function in broiler chicks.

Despite of extensive body of research findings demonstrating favorite effects of acidified diets on performance, health and welfare of broiler chicken, effect of dietary alkalinization on poultry are not fully investigated. OWEN et al. (1994) reported that pH alteration of gut from the normal acid-base balance towards alkalinization affects health, welfare and production of broiler chicken. Therefore, the present study was conducted to evaluate the effects of acidified or alkalinized diets on ileal digestibility, intestinal histomorphology, bone mineralization, and serum enzyme activities in broiler chicken.

**Material and Methods**

A total number of 250 male Ross 308 broiler chicks were obtained from a commercial hatchery and randomly distributed into 25 floor pens in an environmentally controlled house. Each of the five treatments was replicated five times, with 10 birds per pen. The experimental period was divided into grower (7 to 21 d) and finisher phases (22 to 42 d). The basal diet was supplemented with 10, 20 or 30 g/kg CA to provide three of the experimental diets. An experimental diet was also provided by addition of Ca(OH)₂ in the form of mile of lime (8.9 g/kg for growth period and 8.6 g/kg for finisher period). The pH value for the alkaline solution was set as 12.3. At day 42 of age, blood were collected from two killed birds in non heparinised tubes and kept on ice slashes until they were subjected to serum collection by centrifuging the whole blood sample at 2,500 g for 10 min. The serum samples were analyzed for the activity of alkaline phosphatase (ALP) alanine aminotransferase (ALT) aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). At day 30 of age, titanium oxide (1 g/kg of diet) was added to all the diets for five days and was used as an analytical marker to determine the effect of treatments on digestibility of crude protein (CP), apparent metabolizable energy corrected to zero nitrogen retention (AMEn), calcium (Ca) and phosphorous (P). Tibia ash was determined by removing

<footnote n="1">¹Dept. of Animal Science, Lorestan University, Khorramabad, Iran</footnote>

<footnote n="2">²Dept. of Animal Science, University of Birjand, Birjand, Iran</footnote>
the adhering tissue, drying the bone at 110 °C for 12 h and extraction of fat with ether. The dry fat-free bones were ashed in a muffle furnace at 550°C for 3 h. Ash weight was calculated as a percentage of dry fat-free bone weight. The tibia contents of Ca and P were measured using dry-ashed bone samples following the same procedure of minerals estimation in feed and excreta. Data were analyzed using GLM procedure (SAS, Institute) in a completely randomized design. All of the treatment means were compared using Tukey-Kramer's test. For the different statistical tests, significance was declared at *P* < 0.05.

## Results

The effect of CA-containing and ALK diets on ileal digestibility and bone mineralization are summarized in Table 1. Inclusion of 30 g/kg CA in diet significantly increased ileal digestibility of CP (*P* < 0.05) and AMEn (*P* < 0.01). Tibia ash and Ca content were greater in the birds fed 30 g/kg CA than those received ALK and CON diets (*P* < 0.05). Alkalinized diet decreased tibia ash, Ca and P contents (*P*<0.05) compared to the acidified and CON diets. Inclusion of 30 g/kg CA in diet significantly reduced ALP activity (*P* < 0.01) and increased LDH activity (*P* < 0.01) compared to the control birds at day 42 of age (Table 2).

## Conclusions

In this study, all of the chicks fed on alkalinized diet showed severe leg paralysis at 24 d of age. It seems that the same birds were suffered from calcium tetany characterized by muscle weakness or paralysis and as a result of hypocalcemia (MARTIN et al., 2011). The risk factors associated in the development of calcium tetany in broilers are reported as poor flock uniformity and high calcium feed (> 1.2% Ca) (MARTIN et al., 2011). Calcium tetany may also develop when the normal response to a decline in blood Ca level becomes disrupted, for example by metabolic disturbances that increase blood pH and consequently cause blood Ca becoming unavailable. Metabolic disturbances that increase blood pH, called respiratory or metabolic alkalosis depending on the underlying cause, shift the plasma calcium flux from the ionized (active) form to the protein bound (inactive) form (MARTIN et al. 2011). It has been recognized that CA improve protein and energy digestibility by reducing the microbial competition with the host for nutrients, endogenous nitrogen losses and ammonia production in addition to their other beneficial effects for broilers (NOURMOHAMMADI et al., 2012).

The organic acids have shown positive impact on histology of the small intestine, thereby facilitating nutrient absorption and growth performance in broiler chicken (NOURMOHAMMADI and AFZALI, 2013). The decrease in serum ALP activity associated with the diets supplemented with 30 g/kg of CA might reflect the down regulation of this enzyme caused by increased availability of phosphorus (Huff et al. 1998). The increase in LDH activity that we observed in the birds fed on 30 g/kg CA may be related to normal function of liver, because this enzyme decreases sharply as the disease progresses.

<table><caption>**Table 1.** Effects of citric acid (CA) and alkaline diet (ALK) on ileal nutrients digestibility and bone mineralization of broilers</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th colspan="3">Dietary acidification (g CA/kg)</th><th rowspan="2">ALK</th><th rowspan="2">SEM</th><th rowspan="2">p-value</th></tr><tr><th>10</th><th>20</th><th>30</th></tr></thead><tbody><tr><td>CP, %</td><td>80.7<sup>c</sup></td><td>81.6<sup>bc</sup></td><td>83.1<sup>ab</sup></td><td>83.8<sup>a</sup></td><td>81.7<sup>bc</sup></td><td>0.4</td><td>0.038</td></tr><tr><td>AMEn, kcal kg<sup>-1</sup></td><td>2713<sup>b</sup></td><td>2709<sup>b</sup></td><td>2722<sup>b</sup></td><td>2775<sup>a</sup></td><td>2708<sup>b</sup></td><td>4</td><td>0.004</td></tr><tr><td>Ca, %</td><td>58.5</td><td>58.5</td><td>58.9</td><td>59.0</td><td>58.6</td><td>0.3</td><td>0.129</td></tr><tr><td>P, %</td><td>38.7</td><td>38.4</td><td>38.6</td><td>38.9</td><td>38.0</td><td>0.3</td><td>0.146</td></tr><tr><td>Tibia ash, %</td><td>46.0<sup>b</sup></td><td>46.5<sup>b</sup></td><td>46.1<sup>b</sup></td><td>48.6<sup>a</sup></td><td>43.1<sup>c</sup></td><td>0.3</td><td>&lt;0.001</td></tr><tr><td>Ca, %</td><td>37.0<sup>b</sup></td><td>38.0<sup>b</sup></td><td>37.8<sup>b</sup></td><td>38.9<sup>a</sup></td><td>31.6<sup>c</sup></td><td>0.3</td><td>&lt;0.001</td></tr><tr><td>P, %</td><td>19.17<sup>a</sup></td><td>19.21<sup>a</sup></td><td>19.20<sup>a</sup></td><td>19.45<sup>a</sup></td><td>18.63<sup>b</sup></td><td>0.08</td><td>0.037</td></tr></tbody></table>

<footnote><sup>a-c</sup> Means within a row with different superscripts are significantly different (*P* < 0.05).</footnote>

<footnote>SEM, standard error over general mean</footnote>
<table><caption>**Table 2.** Effects of citric acid (CA) and alkaline diet (ALK) on serum enzyme activities (U/L) of broilers</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th colspan="3">Dietary acidification (g CA/kg)</th><th rowspan="2">ALK</th><th rowspan="2">SEM</th><th rowspan="2">p-value</th></tr><tr><th>10</th><th>20</th><th>30</th></tr></thead><tbody><tr><td>ALP</td><td>14302<sup>a</sup></td><td>14315<sup>a</sup></td><td>14321<sup>a</sup></td><td>14107<sup>b</sup></td><td>14394<sup>a</sup></td><td>165</td><td>0.002</td></tr><tr><td>ALT</td><td>1.5</td><td>1.8</td><td>1.7</td><td>1.6</td><td>1.7</td><td>0.1</td><td>0.086</td></tr><tr><td>AST</td><td>239</td><td>238</td><td>237</td><td>238</td><td>241</td><td>3</td><td>0.073</td></tr><tr><td>LDH</td><td>714<sup>b</sup></td><td>726<sup>b</sup></td><td>717<sup>b</sup></td><td>749<sup>a</sup></td><td>718<sup>b</sup></td><td>7</td><td>0.007</td></tr></tbody></table>

SEM, standard error over general mean

## Literature

HUFF, W.E., P.A. MOORE, P.W. WALDROUP, A.L. WALDROUP, J.M. BALOG, G.R. HUFF, N.C. RATH, T.C. DANIEL, V. RABOYS, 1998: Effect of dietary phytase and high non-phytate phosphorus corn on broiler chicken performance. Poult Sci. **77**, 1899–1904.

MARTIN, M.P., M. WINELAND, O.J. FLETCHER, H.J. BARNES, 2011: Selected blood chemistry values in mobility-impaired broiler breeder hens with suspected calcium tetany using the i-STAT handheld clinical analyzer. Avian Dis. **55**, 340–345.

NOURMOHAMMADI, R., N. AFZALI, 2013: Effect of citric acid and microbial phytase on small intestinal morphology in broiler chicken. Ital. J. Anim. Sci. **12**, 44–47.

NOURMOHAMMADI, R., S.M. HOSSEINI, H. FARHANGFAR, M. BASHTANI, 2012: Effect of citric acid and microbial phytase enzyme on ileal digestibility of some nutrients in broiler chicks fed corn-soybean meal diets. Ital. J. Anim. Sci. **11**, 36–40.

OWEN, R.L., R.F. WIDEMAN, R.M. LEACH, B.S. COWEN, P.A. DUNN, B.C. FORD, 1994: Effect of age of exposure and dietary acidification or alkalinization on broiler pulmonary hypertension syndrome. J. Appl. Poult. Res. **3**, 244–252.

<footnote><sup>a-b</sup> Means within a row with different superscripts are significantly different (P <0.05).</footnote>
P-055 (ID 221)

# EFFECT OF ARTICHOKE (CYNARASCOLYMUS L.) EXTRACT SUPPLEMENTATION ON BLOOD CHEMISTRY OF BROILER CHICKENS

K. Paditporn<fnref n="1" />*, P. Jaipeng<fnref n="1" />, J. Mitchaothai<fnref n="1" />, R. Lertpatarakomol²and S. Sirimongkolworakul<fnref n="3" />

*Corresponding author: kornkamo@mut.ac.th

## Abstract

This study was carried out to investigate the effects of different levels of artichoke (Cynarascolymus L.) extract (AE) supplementation in drinking water on liver enzyme and blood chemistry of broiler chickens. A total of 272 one-day-old broiler chicks (Ross, 308) were randomly allotted to 4 treatment groups, each with 4 replicate cages containing 17 chicks for a period of 42 days. The treatment groups consisted of: control (C) group (without AE added in drinking water) and AE added in drinking water at the level of 75 ppm (T1), 150 ppm (T2) and 300 ppm (T3). Blood samples were collected at 0, 14, 28 and 42 days of age and then analyzed for the liver enzyme; Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), serum plasma protein (TP) and serum albumin (AL). The results have illustrated that there was significantly (P<0.05) higher of the AST at 42 days of the chicks in T1, T2 and T3 than that in the C. The ALT at 42 days of T2 was significantly higher (P<0.05) than that in the C groups, but no difference from the T1 and T3. The TP at 42 days of the chicks in the T3 were significantly lower (P<0.05) than the other groups and gradually increased (P<0.05) with lower level AE. The AL of the chicks in the T3 had lowest value, whereas the AL levels in other groups were varied without systematic pattern. It was concluded that AE supplementation in drinking water would improve liver function, while unclear effects on AL level in broiler chickens.

**Keywords:** artichoke extract, blood chemistry, liver, broiler chickens

## Introduction

Poultry industry produces animal proteins for human consumption most effectively and economically within the shortest possible time (JAVED et al., 2013). The current difficulties facing poultry industry is the occurrence of hepatotoxicity and oxidative stress condition which directly effects and damages the chicken liver and the health and quality of the meat of the chicken. Artichoke extract has been used since ancient times as a remedy for indigestion and liver problem. The major bioactive component of artichoke leave are cynarin, flavonoids, phenolic acid and caffeic acid (WANG et al., 2003; JOY and HABER, 2007). Obtaining the cynarin substance from artichoke extract and its usage in the medical science as hepatoprotective triggered the area of utilization particularly in the poultry farming. The motivation of keeping the liver function within the intended level in the broiler, where intensive farming is dealt has on the other hand increased the importance of such supplements (UZATICI and CELIK, 2014). Therefore, the objective of this study was to determine the effects of different levels of artichoke (Cynarascolymus L.) extract (AE) supplementation in drinking water on blood chemistry of broiler chickens.

## Material and Methods

### Animal and Sample collection

A total of 272 one-day-old broiler chicks (Ross, 308) mixed sex, day-old broiler chickens were housed in 6 x 10 m curtain-sided, open house system with rice husk as litter material. There were randomly allotted to 4 treatment groups, each with 4 replicate cages containing 17 chicks for a period of 42 days. The treatment groups consisted of: control (C) group (without AE added in drinking water) and AE added in drinking water at the level of 75 ppm (T1), 150 ppm (T2) and 300 ppm (T3). All chicks were offered mash feed and drinking water *ab libitum*. AE was provided on day 1-7 and 15-24 of age. Blood samples were collected at 0, 14, 28 and 42 days of age and then analyzed for the liver enzyme; Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), serum plasma protein (TP) and serum albumin (AL).

<footnote n="1">¹Department of Veterinary Clinical Science, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand</footnote>

<footnote n="2">²Department of Animal Science & Veterinary Basic Science, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand</footnote>

<footnote n="3">³Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand</footnote>
## Data analysis

Data from all experiments were analysed using the ANOVA procedure of SAS® software (SAS Institute, 2005) for completely randomized designs. A probability level of P<0.05 was used to determine statistical significance of differences among the treatments.

## Results

The results of the current study was shown in Table1. The results have illustrated that there was significantly (P<0.05) higher of the AST at 42 days of the chicks in T1, T2 and T3 than that in the C group. The ALT at 42 days of T2 was significantly higher (P<0.05) than that in the C groups, but no difference from the T1 and T3. The TP at 42 days of the chicks in the T3 were significantly lower (P<0.05) than the other groups and gradually increased (P<0.05) with lower level AE. The AL of the chicks in the T3 had lowest value, whereas the AL levels in other groups were varied without systematic pattern.

<table><caption>**Table1:** Effect of artichoke (*Cynarascolymus* L.) extract supplementation on blood chemistry of broiler chickens (Mean ± SD)</caption><thead><tr><th rowspan="2">Variable</th><th colspan="4">Group</th></tr><tr><th>Control</th><th>T1 (AE 75 ppm)</th><th>T2 (AE 150 ppm)</th><th>T3 (AE 300 ppm)</th></tr></thead><tbody><tr><td><strong>AST(IU/L)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>0 day</td><td>147.2 ± 77.73</td><td>147.71 ± 65.4</td><td>156.5 ± 86.43</td><td>107.14 ± 91.28</td></tr><tr><td>14 day</td><td>157.38 ± 38.77</td><td>168.25 ± 26.27</td><td>167 ± 35.64</td><td>190.75 ± 14.95</td></tr><tr><td>28 day</td><td>163.75 ± 54.14</td><td>188.13 ± 34.95</td><td>161.75 ± 62.49</td><td>174.13 ± 34.15</td></tr><tr><td>42 day</td><td>57.5 ± 39.96<sup>a</sup></td><td>254.75 ± 131.45<sup>b</sup></td><td>238.75 ± 119.43<sup>b</sup></td><td>171 ± 60.64<sup>b</sup></td></tr><tr><td><strong>ALT (IU/L)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>0 day</td><td>7 ± 4.9</td><td>5.88 ± 4.09</td><td>7.5 ± 4.04</td><td>8.43 ± 2.3</td></tr><tr><td>14 day</td><td>0.13 ± 0.35</td><td>2.12 ± 3.94</td><td>2.38 ± 2.2</td><td>1.25 ± 1.16</td></tr><tr><td>28 day</td><td>4.33 ± 2.66</td><td>4 ± 2.5</td><td>2.75 ± 2.49</td><td>2.25 ± 0.5</td></tr><tr><td>42 day</td><td>1.8 ± 0.84<sup>a</sup></td><td>5 ± 1.77<sup>ab</sup></td><td>6.13 ± 4.29<sup>b</sup></td><td>4.67 ± 2.88<sup>ab</sup></td></tr><tr><td><strong>TP (g/dL)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>0 day</td><td>3.27 ± 0.69</td><td>3.7 ± 0.97</td><td>3.82 ± 0.8</td><td>3.26 ± 0.49</td></tr><tr><td>14 day</td><td>2.74 ± 0.84<sup>a</sup></td><td>3.28 ± 0.34<sup>b</sup></td><td>3.08 ± 0.24<sup>ab</sup></td><td>3.28 ± 0.24<sup>b</sup></td></tr><tr><td>28 day</td><td>3.45 ± 0.54<sup>ab</sup></td><td>3.9 ± 0.73<sup>b</sup></td><td>3.75 ± 0.54<sup>ab</sup></td><td>3.15 ± 0.77<sup>a</sup></td></tr><tr><td>42 day</td><td>4.29 ± 0.83<sup>c</sup></td><td>3.85 ± 0.48<sup>bc</sup></td><td>3.55 ± 0.3<sup>b</sup></td><td>2.83 ± 0.53<sup>a</sup></td></tr><tr><td><strong>AL (g/dL)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>0 day</td><td>0.7 ± 0.18<sup>a</sup></td><td>0.93 ± 0.19<sup>ab</sup></td><td>0.95 ± 0.28<sup>b</sup></td><td>0.87 ± 0.16<sup>ab</sup></td></tr><tr><td>14 day</td><td>1.49 ± 0.78</td><td>1.31 ± 0.18</td><td>1.24 ± 0.2</td><td>1.24 ± 0.17</td></tr><tr><td>28 day</td><td>1.23 ± 0.16<sup>ab</sup></td><td>1.20 ± 0.19<sup>a</sup></td><td>1.40 ± 0.16<sup>b</sup></td><td>1.13 ± 0.21<sup>a</sup></td></tr><tr><td>42 day</td><td>1.53 ± 0.3<sup>ab</sup></td><td>1.88 ± 1.17<sup>b</sup></td><td>1.31 ± 0.12<sup>ab</sup></td><td>1.09 ± 0.33<sup>a</sup></td></tr></tbody></table>

All variations of AST, ALT, TP and AL were in the normal range of healthy chicks, indicating no hepatotoxic substances, such as $CCl_4$ toxin, which can damage the liver. In addition, the AST, ALT, TP and AL in this study agree with earlier report (ABDO et al., 2007). This would imply for beneficial influence of the AE supplementation to broiler chickens.

## Conclusions

In conclusion, the artichoke (*Cynarascolymus* L.) extract supplementation in drinking water would improve liver function, while unclear effects on albumin level in broiler chickens.

<footnote><sup>a,b</sup>means in the same row with different superscripts are significant difference (P<0.05)</footnote>
## Literature

ABDO, Z.M.A., N.L. RADWAN and N.A. SELIM, 2007: The effect of artichoke leaves meal on the utilization of dietary energy for broiler chicks. Int. J. Poult. Sci. **6**(12), 973-982.

JAVED, U., M.Z. KHAN, M.K. SALEEMI, A. KHAN, I. JAVED and S. RAFIQUE, 2013: Toxico-pathological effects of parenteral administration of gentamicin in growing broilers. Int. J. Agric. Biol. **15**, 529-534.

JOY, J.F. and S.L. HABER, 2007: Clinical use of artichoke leaf extract. Am. J. Health-Syst. Pharm. **64**, 1906-1909.

UZATICI, A. and K. CELIK, 2014: Analysis of the effects of Cynarion and Choline Chorite on the metabolic processes of broilers. Asian J. Anim. Sci. **8**(3), 86-92.

WANG, M., J.E. SIMON, I.F. AVILES, K. HE, Q.Y. ZHENG and Y. TADMOR, 2003: Analysis of antioxidative phenolic compounds of artichoke (Cynarascolymus L.). *J. Agric. Food Chem.* **51**(3), 601–608.