<table><caption>Table 1: Performance of broiler birds fed sorghum based with or without phytase enzyme</caption><thead><tr><th>Parameters</th><th>T1<br>Maize</th><th>T2<br>Jandawa</th><th>T3<br>Kaura</th><th>T4<br>Jandawa+<br>phytase</th><th>T5<br>Kaura+<br>phytase</th><th>SEM</th><th>LOS</th></tr></thead><tbody><tr><td colspan="8"><strong>Starter Phase (0-4 weeks)</strong></td></tr><tr><td>Initial wgt (g/bird)</td><td>43.53</td><td>43.40</td><td>43.53</td><td>43.40</td><td>43.30</td><td>0.16</td><td>NS</td></tr><tr><td>Final wgt (g/bird)</td><td>722.10<sup>b</sup></td><td>755.50<sup>b</sup></td><td>755.60<sup>b</sup></td><td>755.53<sup>b</sup></td><td>820.57<sup>a</sup></td><td>21.38</td><td>*</td></tr><tr><td>Wgt gain(g/bird)</td><td>678.57<sup>b</sup></td><td>712.10<sup>b</sup></td><td>712.07<sup>b</sup></td><td>712.13<sup>b</sup></td><td>777.27<sup>a</sup></td><td>21.46</td><td>*</td></tr><tr><td>Feed intake (g/bird)</td><td>1347.63<sup>b</sup></td><td>131.23<sup>b</sup></td><td>1412.4<sup>a</sup></td><td>1369.97<sup>a</sup></td><td>1440.87<sup>a</sup></td><td>42.63</td><td>*</td></tr><tr><td>Feed/gain ratio</td><td>1.98</td><td>1.84</td><td>1.99</td><td>1.92</td><td>1.85</td><td>0.05</td><td>NS</td></tr><tr><td>Feed cost (N/Kg)</td><td>80.02<sup>b</sup></td><td>91.18<sup>a</sup></td><td>80.02<sup>b</sup></td><td>91.68<sup>a</sup></td><td>80.52<sup>b</sup></td><td>2.89</td><td>*</td></tr><tr><td>Feed cost/kg gain (N)</td><td>158.44<sup>b</sup></td><td>167.67<sup>c</sup></td><td>159.01<sup>b</sup></td><td>176.33<sup>d</sup></td><td>148.96<sup>a</sup></td><td>4.27</td><td>*</td></tr><tr><td colspan="8"><strong>Finisher Phase (5-8 weeks)</strong></td></tr><tr><td>Initial wt (g/b)</td><td>722.10</td><td>755.50</td><td>755.60</td><td>755.53</td><td>820.57</td><td>21.38</td><td>NS</td></tr><tr><td>Final wt (g/b)</td><td>2272.2</td><td>2339.8</td><td>2257.9</td><td>2282.5</td><td>2395.2</td><td>83.86</td><td>NS</td></tr><tr><td>Wt gain (g/b)</td><td>1550.12</td><td>1584.28</td><td>1502.33</td><td>1526.99</td><td>1574.67</td><td>70.17</td><td>NS</td></tr><tr><td>Feed intake (g/b)</td><td>3902.50</td><td>4040.00</td><td>3883.20</td><td>4036.10</td><td>3999.50</td><td>143.58</td><td>NS</td></tr><tr><td>Feed/gain ratio</td><td>2.52</td><td>2.53</td><td>2.59</td><td>2.64</td><td>2.54</td><td>0.40</td><td>NS</td></tr><tr><td>Feed cost (N/Kg)</td><td>74.05<sup>a</sup></td><td>84.85<sup>c</sup></td><td>74.05<sup>a</sup></td><td>85.35<sup>d</sup></td><td>74.55<sup>b</sup></td><td>0.01</td><td>*</td></tr><tr><td>Feed cost/kg gain</td><td>186.60<sup>a</sup></td><td>216.65<sup>c</sup></td><td>191.54<sup>b</sup></td><td>225.61<sup>d</sup></td><td>189.36<sup>a</sup></td><td>3.19</td><td>*</td></tr></tbody></table>

a, b,c Means with different superscripts on the same row are significantly different (P<0.05)

LOS: Level of significance. NS: Non-significant difference (P>0.05) *: Significant difference (P<0.05)

SEM: Standard error of means

<table><caption>Table 2: Calcium and phosphorus contents of tibia bone and blood serum</caption><thead><tr><th>Parameters</th><th>T1<br>Maize</th><th>T2<br>Jandawa</th><th>T3<br>Kaura</th><th>T4<br>Jandawa+<br>phytase</th><th>T5<br>Kaura+<br>phytase</th><th>SEM</th><th>LOS</th></tr></thead><tbody><tr><td><strong>Bone</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Calcium (%)</td><td>29.11<sup>a</sup></td><td>23.39<sup>b</sup></td><td>24.69<sup>ab</sup></td><td>29.13<sup>a</sup></td><td>31.33<sup>a</sup></td><td>3.42</td><td>*</td></tr><tr><td>Phosphorus (%)</td><td>13.27<sup>a</sup></td><td>10.52<sup>b</sup></td><td>11.11<sup>ab</sup></td><td>13.11<sup>a</sup></td><td>14.10<sup>a</sup></td><td>1.55</td><td>*</td></tr><tr><td><strong>Serum</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Calcium (mmol/l)</td><td>1.81</td><td>1.78</td><td>1.76</td><td>1.84</td><td>1.88</td><td>0.09</td><td>NS</td></tr><tr><td>Phosphorus (mmol/l)</td><td>2.02</td><td>2.14</td><td>2.27</td><td>2.61</td><td>2.50</td><td>0.24</td><td>NS</td></tr></tbody></table>

a,b,c, Means with different superscripts on the same row are significantly different (P<0.05)

LOS: Level of significance. NS: Non-significant difference (P>0.05) *: Significant difference (P<0.05)

SEM: Standard error of means
References

DILGER, R.N., E.M. ONYANGO, J.S. SANDS, O. ADEOLA, 2004: Evaluation of microbial phytase in broiler diets. *Poultry Science*, 83: 962-970.

FAO, 2012: Food and Agricultural Organization Statistical data base. Rome, Italy.

KHAN, S.A, H.R. CHAUDHRY, Y.S. BUTT, T. JAMEEL, F. AHMAD, 2013: The effect of phytase enzyme on the performance of broiler Flock. *Poultry Science Journal*, 1 (2): 117-125

KHUSHEEBA., M, S. AJID, 2013: A review on role of exogenous enzyme supplementation in poultry Production. *Emirates Journal of Food and Agriculture*, 25 (1): 66-80.

MAUNDER, B, 2002: Sorghum-The global grain of the future. Retrieved October 1, 2014 from
http://www.sorghumgrowers.com/maunder.htm.

MONDAL, M. K., S. PANDA, P. BISWAS, 2007: Effect of microbial phytase in soybean meal based broiler diets containing low phosphorous. *International Journal of Poultry Science*, 6 (3): 201-206.

OVIMAPS, 2014: Ovi location map; Ovi earth imgery. December 3rd, 2014.

S.A.S, 2002: Statistical Analysis System Institute, User's Guide. Version 9 for Windows. North Carolina, U.S.A.

SEBASTIAN, S., S. P TOUCHBURN, R CHAVEZ E, P. C. LAGUE, 1997: Apparent digestibility of protein and amino acids in broiler chickens fed a corn-soybean diet supplemented with microbial phytase. *Poultry Science*. 76:1760–1769.

SELLE, P.H., A.R WALKER, W.L BRYDEN, 2003: Total and phytate-phosphorus content and phytase activity of Australian-sourced feed ingredients for pigs and poultry. *Australian Journal of Experimental Agriculture*, 43(5) 475-479.

WU, Y.B., V. RAVINDRAN, W.H HENDRIKS, 2003: Effects of microbial phytase, produced by solid-state fermentation, on the performance and nutrient utilization of broilers fed maize– and wheat–based diets. *British Poultry Science*. 44:710-718.
P-119 (ID 121)

SOLUBILISATION AND VISCOSITY REDUCTION EFFECTS ON CEREAL CELL WALLS MEDIATED BY XY-
LANASE AND β-GLUCANASE

J.Z.Ravn<fnref n="2" />, H.J. Martens<fnref n="1" />, D. Pettersson<fnref n="2" /> and N.R. Pedersen<fnref n="2" />*

Corresponding author: nrp@novozymes.com

**Abstract**

Viscosity reduction of three feed enzymes was measured in a high throughput pressure sensing system using commer-
cial dosages on purified polysaccharides (arabinoxylan and β-glucan). Fluorescence and antibody microscopy tech-
niques were also used to visualize effects on the solubilisation of endosperm cell walls of wheat and barley.

Viscosity data clearly demonstrated depolymerisation of mixed-linked β-glucans by the two multicomponent enzymes.
The two multicomponent enzymes also reduced the viscosity of the arabinoxylan solution, while the monocomponent
xylanase most effectively depolymerised the arabinoxylan. Microscopy data revealed that the multicomponent enzyme
chosen for the studies could solubilize both wheat and barley cell walls by targeting both the β-glucan strutures as well
as the arabinoxylans. The xylanase could effectively solubilise wheat and barley cell walls by merely attacking the ara-
binoxylans.

It's concluded that viscosity measurements provides information on differences in hydrolytic capacity of feed enzymes.
Additionally the study has shown that microscopy techniques that visualise cell wall solubilising effects of feed enzymes
can be useful for understanding enzyme mode of action and efficacy on different feed ingredients.

**Keywords:** xylanase, β-glucanase, solubilisation, viscosity

**Introduction**

Even after milling of feed ingredients some nutrients will remain within intact cell walls built up by indigestible fibre com-
ponents. Commercial exogenous feed enzymes, such as mixed-linked β-glucanases or arabinoxylanases, are used to
degrade fibre components in cereals by de-polymerising soluble fibres. This may reduce digesta viscosity and thereby
facilitate nutrient absorption. Exogenous microbial enzymes may also mediate solubilisation of the insoluble cell wall
matrix, thereby releasing captured nutrients.

**Materials and Methods**

Three commercial enzymes; RONOZYME WX (monocomponent xylanase), RONOZYME MultiGrain (a multienzyme product containing mainly xylanase and β-glucanase) and RONOZYME VP a commercial multicomponent enzyme containing over 40 different activities mainly targeting other components besides arabinoxylan and mixed-linked β-glucan.

The substrates used for the viscosity study, arabinoxylan and β-glucan, were obtained from Megazymes, Ireland.

Viscosity studies were conducted using a pipetting workstation, Hamilton, with eight microprocessor-controlled pipetting channels that are analogous to manual pipettes. It is capable of pressure sensing during aspiration and dispensing of solutions. The viscosity pressure (ViPr) assay displays a linear correlation between pressure measured (Pa) and kinematic viscosity (ABEL & PETTERSSON, 2011). During the complete aspiration and dispensing process the air displacement is continuously monitored in intervals of 10 ms by a pressure sensor to verify an uninterrupted substrate flow. Pressure measured in this work is defined as viscosity determined in relation to the ViPr assay.

Sections of barley and wheat (10 µm) were treated for 3h with RONOZYME MultiGrain or RONOZYME WX. Enzyme or buffer droplets were applied directly on the glass slide carrying the sample. Approximately 600 µL was applied to each glass slide.

Autofluorescence, as well as histochemical staining, was used to visualize the endosperm structures. Fluorescence microscopy was performed using a Nikon Eclipse 80i Microscope (Nikon Corporation, Japan) to visualize structures in both wheat and barley using Calcofluor white.

<footnote n="1">¹ Department of Plant and Environmental Sciences, University of Copenhagen, Denmark</footnote>

<footnote n="2">² Feed Applications, Novozymes A/S, Denmark</footnote>
Confocal images of immunolabeled samples were acquired from sections of barley and wheat by detecting the Alexa-555 fluorescein-signal from the LM11 immuno-label using a Leica CLSM SP2 microscop.

## Results

Within 30 minutes a distinct viscosity reduction was observed when adding the commercial products to a viscous arabinoxylan solution (Fig. 1). There was no noticeable viscosity reduction observed for the β-glucan solution when adding the monocomponent xylanase, as expected, while the two multicomponent products (RONOZYME Multigrain and VP) caused a marked viscosity reduction (Fig. 1) due to their β-glucanase activity.

<figure><img src="image_52.png" /><figcaption>**Figure 1.** Viscosity reduction of an arabinoxylan solution 1g/100 mL (left figure) and a β-glucan solution 0.5 g/100 mL (right picture) when adding commercially related dosages of the respective enzymes.</figcaption></figure>

Staining with calcoflour white revealed the mixed linked β-glucan structures present in the cell walls of barley (HES-SELMAN & ÅMAN, 1986) and on enzyme addition with the Ronozyme Multigrain the cell wall structures were disrupted and no longer visible (Fig. 2). A similar effect was not observed for the monocomponent xylanase (data not shown). Both the multicomponent and the monocomponent product efficiently degraded the arabinoxylan present in the cell walls (Fig. 3), as indicated by immuno-labeling with the LM 11 label targeting arabinoxylans.

<figure><img src="image_53.png" /><figcaption>**Figure 2.** Barley stained with calcoflour white, left picture (control), and the same sample after 3 h of enzyme incubation with RONOZYME Multigrain.</figcaption></figure>

<caption>**Figure 3.** Barley labelled with the LM 11 immuno label (left picture) and after 3 h when treated with RONOZYME Multigrain (middle picture) or RONOZYME WX (right picture).</caption>

## Conclusions

The results obtained in this study demonstrates the strong hydrolytic capacity of xylanases and β-glucanases that results in efficient viscosity reduction of soluble xylans and β-glucans. By using microscopy techniques it was also possible to demonstrate the cell wall degrading efficacy of commercial feed enzymes commonly used in animal production. A notable effect demonstrated was the capacity of a monocomponent xylanase to degrade the cell wall constituents of barley. This finding indicates that a β-glucanase is not necessarily essential for releasing nutrients encapsulated by insoluble cell walls partly made up by mixed-linked β-glucans. However, in situations where high concentrations of soluble β-glucans increase digesta viscosity to levels detrimental for nutrient absorption a dedicated β-glucanase may provide an efficient solution.

In the present study the use of microscopy techniques to visualize cell wall solubilisation of cereal cell wall components in vitro by feed enzymes agrees with the viscosity reductions obtained by these enzyme products.
The methods employed in this study may be used as a valuable *in vitro* methodology to demonstrate the complexity of feed ingredient degradation and solubilisation by commercial feed enzymes. Coupling of such results with actual animal performance data may provide an important tool for better understanding the relationship between cell wall degradation and viscosity reduction as predictors of the productive value of feed enzymes.

## Literature

ABEL, G., PETTERSSON, D. 2011. Viscosity pressure assay. Patent WO 2011107472 A1.

HESSELMAN, K., ÅMAN, P. 1986. The effect of β-glucanase on the utilization of starch and nitrogen by broiler chickens fed barley of low- or high viscosity. Anim. Feed Sci. Technol. **15**, 83-93.
P-120 (ID 107)

EFFECT OF A MICROBIAL 6-PHYTASE SUPPLEMENTATION ON PLASMATIC MYO-INOSITOL AND ON THE PECTORALIS MUSCLE TRANSCRIPTOME IN BROILERS

Jérôme Schmeisser¹, Aurélia-Anne Séon¹, Raffaella Aureli¹, Angelika Friedel², Patrick Guggenbuhl¹, Aaron J Cowieson², Fidelis Fru-Nji¹

¹DSM Nutritional Products France SA, Saint-Louis, France

²DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

Corresponding author: jerome.schmeisser@dsm.com

Summary

A floor pen trial of five weeks was conducted to evaluate the effects of a 6-microbial phytase supplementation in broilers in terms of growth performance, *myo*-inositol plasma concentration, bone mineralization, breast meat weight, and *Pec- toralis major* muscle transcriptome. Two diets were given *ad libitum*: A positive control diet (PC) and a negative control diet (NC) formulated with a moderate deficiency of phosphorus compared to PC. The NC diet was distributed with or without phytase supplementation at 1000 U / kg. Phytase supplementation led to a significant improvement of WG, FCR, *myo*-inositol plasma concentration, tibia ash percentage, and muscle weight compared to NC. A transcriptomic study showed that several genes involved in muscle development were up-regulated in chickens fed phytase compared to NC, involving transcription factors MEF2 activation via calcineurin A, and muscle differentiation through IGF with PI3 kinase. This study suggests that the addition of this phytase increases the concentration of *myo*-inositol, the final prod- uct of phytate hydrolysis, and influences muscle development in broilers.

**Keywords:** poultry, nutrition, muscle, *myo*-inositol, gene expression.

Introduction

Phytate is considered an anti-nutritional compound due to its ability to bind to cations, proteins and starch generating insoluble aggregates that lead to reduced bioavailability in poultry (Olukosi, 2012). Supplementation with microbial phy- tase has been shown to improve phytate-P digestibility in farm animals and to reduce environmental P pollution. In- creased retention of phytate-P and the associated beneficial effects on Ca solubility has a range of beneficial effects on feed intake and bone mineralisation (Aureli *et al.*, 2011). In addition, phytase improves growth performance, energy and amino-acid digestibility (Guggenbuhl *et al.*, 2012). Additionally, the involvement of *myo*-inositol in the improved growth efficiency of poultry has recently been implied (Cowieson *et al.*, 2014). The aim of the present work was to investigate physiological effects of phytase supplementation on plasma *myo*-inositol concentration and breast muscle transcrip- tome.

Material and Methods

The experiment was done at facilities of the Research Centre of Animal Nutrition (CRNA) of DSM Nutritional Products, France. On the day of arrival, broilers were divided by weight into 2 groups of 20 birds per treatment. Body weight gain (WG) and feed consumption for the whole period (d1-36) were determined for each cage.

The basal diets contained maize and soybean meal as main ingredients. The respective negative control diet was for- mulated with moderate deficiency of phosphorus (P total = 5.1 g/kg ; Av.P (available P) = 2.01 g/kg for the starter and and P total = 5.2 g/kg ; Av.P = 2.14 g/kg for the grower) ; and calcium (Ca) (Ca = 6.0 g/kg for the starter and Ca = 6.2 g/kg for the grower) and fed as such (negative control, NC) or supplemented with 2.0 g/kg non phytate-P from dicalcium phosphate dihydrate (DCP) as a positive control (PC). NC was supplemented with bacterial 6-phytase (DSM Nutritional Products Ltd, Switzerland) at 1000 FYT/kg (Phy1000). Biological samples were collected on d36 for plasma *myo*-inositol determination, tibia ash percentage, and breast muscle weight. Muscle RNA extraction was done by the phenol- chloroform method and microarray analysis was performed using the WT PLUS reagent kit and CHIGENE-1.0-ST ar- rays by Affymetrix (Affymetrix UK Ltd., High Wycombe, UK). Validation of microarray results was done by quantitative PCR (qPCR) on target genes.
## Results

The analysed CP content in all diets were slightly higher than formulated, while ME, P, Ca concentrations were on or close to target. Phytase activity recovered in the feed after pelleting was in line with expectations and in accordance with the design of the experiment.

The addition of either dicalcium phosphate or phytase to the NC group significantly improved weight gain (WG) and bone mineralization. A significant increase in plasma myo-inositol concentration on d36 was recorded with phytase supplementation compared to NC diet.

From the comparison of the both groups PC and Phy1000 versus NC, a total of respectively 300 and 625 genes were modulated. Significant influenced pathways (FDR<0.01) were observed for animals fed phytase compared to NC and were mainly associated to development (Table 1).

<table><caption>**Table 1:** Functional analysis from microarray data compared to NC.</caption><thead><tr><td>Treatment</td><td>Metabolic pathways</td><td>FDR</td></tr></thead><tbody><tr><td>PC vs NC</td><td>Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling</td><td>0.034</td></tr><tr><td>PC vs NC</td><td>Neurophysiological process_Netrin-1 in regulation of axone guidance</td><td>0.034</td></tr><tr><td>Phy1000 vs NC</td><td>Immune response_Function of MEF2 in T lymphocytes</td><td>0.002</td></tr><tr><td>Phy1000 vs NC</td><td>Development_Role of HDAC and calcium/calmodulin-dependent kinase (CaMK) in control of skeletal myogenesis</td><td>0.002</td></tr><tr><td>Phy1000 vs NC</td><td>Development_Non-genomic action of Retinoic acid in cell differentiation</td><td>0.002</td></tr><tr><td>Phy1000 vs NC</td><td>Development_Notch Signaling Pathway</td><td>0.004</td></tr></tbody></table>

A deeper analysis of the pathway "Role of Histone Deacetylase (HDAC) and calcium/calmodulin-dependent kinase (CaMK) in control of skeletal myogenesis" was performed. Differentially expressed genes associated with this network were up-regulated compared to negative control: Calmodulin (CALM1), calcineurin A (PPP3CB), Myocyte Enhancer Factors 2 (MEF2A and MEF2C), and P300/CBP associated factor (PCAF). The phospholipase C beta (PLCB1) expression was also up-regulated by qPCR. This enzyme enhances the effect of calmodulin/calcineurin A on muscle growth (Tokumitsu et al., 1999) but also the interactions between calcineurin and MEF2 (Li et al., 2013). The IGF/PI3K way seems to be affected since the phosphatidyl-inositide-3-phosphate kinase, regulatory subunit 1 (PI3KR1) was also significantly up-regulated, as the catalytic subunit (PIK3CA). These enzymes are involved in muscular development and differentiation (Wullschleger et al., 2006) via the binding of insulin or IGF on its receptor. Other studies showed insulin-mimetic effect of *myo*-inositol (Cowieson et al., 2014 and Józefiak et al., 2010) which was at a higher level in animals fed phytase compared to NC. The effect of *myo*-inositol on growth performance during grower period was also described by Cowieson et al. (2013). Results obtained could suggest a *myo*-inositol effect on IGF receptors, sensitive to insulin, and leading to modulation of gene expression linked to muscular development. Microarray data and confirmation of fold changes by qPCR are presented in table 2.

<table><caption>**Table 2.** Fold change in gene expression. (*: p<0.05, **: p<0.01.)</caption><thead><tr><td>Gene</td><td>Treatment</td><td>Fold change Microarray (n=4)</td><td>Fold change qPCR (n=16)</td><td>SEM (qPCR)</td></tr></thead><tbody><tr><td rowspan="2">PI3KR1</td><td>PC vs NC</td><td>1.32</td><td>1.51</td><td>-0.65 / +0.46</td></tr><tr><td>Phy1000 vs NC</td><td>2.02**</td><td>3.04*</td><td>-0.80 / +0.63</td></tr><tr><td rowspan="2">PI3KCA</td><td>PC vs NC</td><td>1.01</td><td>1.69</td><td>-0.66 / +0.47</td></tr><tr><td>Phy1000 vs NC</td><td>1.37</td><td>2.84*</td><td>-0.93 / +0.70</td></tr><tr><td rowspan="2">PPP3CB</td><td>PC vs NC</td><td>1.18</td><td>1.88</td><td>-0.73 / +0.53</td></tr><tr><td>Phy1000 vs NC</td><td>1.67*</td><td>2.67*</td><td>-0.59 / +0.48</td></tr><tr><td rowspan="2">MEF2A</td><td>PC vs NC</td><td>1.08</td><td>1.42</td><td>-0.21 / +0.19</td></tr><tr><td>Phy1000 vs NC</td><td>1.44*</td><td>1.68*</td><td>-0.30 / +0.26</td></tr><tr><td rowspan="2">MEF2C</td><td>PC vs NC</td><td>1.17</td><td>1.73</td><td>-0.50 / +0.39</td></tr><tr><td>Phy1000 vs NC</td><td>1.43*</td><td>2.14*</td><td>-0.50 / +0.41</td></tr><tr><td rowspan="2">PLCB1</td><td>PC vs NC</td><td>1.02</td><td>1.48</td><td>-0.50 / +0.37</td></tr><tr><td>Phy1000 vs NC</td><td>1.43</td><td>2.66*</td><td>-0.79 / +0.61</td></tr></tbody></table>
Conclusions

Exogenous phytase has a range of effects that extend beyond P and Ca and that these effects exhibit profound changes at a molecular level in broiler chickens. Addition of phytase significantly increases plasma *myo*-inositol concentrations and up-regulates key metabolic pathways involved in skeletal muscle development. These results explain why the addition of phytase to poultry diets often results in performance responses that cannot be readily explained by effects on nutrient digestibility alone. As phytase dosing continues to increase more attention should be given to dietary phytate concentrations, the antinutritional effects of phytate and the anabolic advantages of *myo*-inositol.

Literature

OLUKOSI OA, 2012: Biochemistry of phytate and phytases: Applications in monogastric nutrition. Biokemistri, **24**:58-63.

AURELI R., UMAR FARUK M., CECHOVA I., PEDERSEN P.B., ELVIG-JOERGENSEN S.G., FRU F. AND BROZ J., 2011: The Efficacy of a Novel Microbial 6-Phytase Expressed in Aspergillus oryzae on the Performance and Phosphorus Utilization in Broiler Chickens. Int. J. Poult. Sci. **10**(2), 160-168.

GUGGENBUHL P, WACHE Y, SIMOES NUNES C, FRU F, 2012: Effects of a 6-phytase on the apparent ileal digestibility of minerals and amino acids in ileorectal anastomosed pigs fed on a corn-soybean meal-barley diet. J. Anim. Sci., **90** **Suppl 4**:182-184.

COWIESON AJ, AURELI R, GUGGENBUHL P, FRU-NJI F, 2014: Possible involvement of myo-inositol in the physiological response of broilers to high doses of microbial phytase. Anim. Prod. Sci.:-.

TOKUMITSU H, TAKAHASHI N, ETO K, YANO S, SODERLING TR, MURAMATSU M, 1999: Substrate recognition by Ca²⁺/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain. J. Biol. Chem., **274**:15803-15810.

LI J, VARGAS MA, KAPILOFF MS, DODGE-KAFKA KL, 2013: Regulation of MEF2 transcriptional activity by calcineurin/mAKAP complexes. Exp. Cell. Res, **319**:447-454.

WULLSCHLEGER S, LOEWITH R, HALL MN, 2006: TOR signaling in growth and metabolism. Cell, **124**:471-484

JOZEFIAK D, PTAK A, KACZMAREK S, MACKOWIAK P, SASSEK M, SLOMINSKI BA, 2010: Multi-carbohydrase and phytase supplementation improves growth performance and liver insulin receptor sensitivity in broiler chickens fed diets containing full-fat rapeseed. Poult. Sci., **89**:1939-1946.
P-123 (ID 300)

EXOGENOUS PROTEASE IN MASHED AND PELLETED DIETS FOR BROILERS

C.S.S. Araujo<fnref n="1" />, R. F. Oliveira<fnref n="1" />, L.F. Araujo<fnref n="2" />, P. H. Mota<fnref n="1" />, N. B. P. Ultimi<fnref n="2" />, and C.A. Grangrelli<fnref n="1" />

**Corresponding author:** crsilva@usp.br

**Abstract**

The aim of this study was to evaluate the performance and carcass yield and cuts of broilers fed with mashed and pelleted diets with or without addition of protease. Seven hundred sixty eight birds were reared from 1 to 42 days old, distributed in a completely random design in a 2x2x2 > factorial arrangement (physical forms of feed, nutritional levels and with or without addition of protease) with eight treatments and eight replicates. For diets with low levels of nutrients, reduction of 4% was performed in crude protein (CP) and amino acids. The pelleted diets provided improvement in performance parameters of broilers at 42 days of age. The reduced levels of CP and amino acids increased feed conversion ratio. The addition of exogenous protease did not have main effect on the performance and carcass yield and cuts. However, there was interaction between the addition of protease and nutritional levels on carcass yield, and interaction between the physical forms of feed and addition of protease on breast yield.

**Keywords:** carcass yield, enzyme, pelleting, performance

**Introduction**

It is well known that the addition of exogenous enzymes in diets, as a nutritional strategy, has a practical goal of reduction of production costs and increase performance in poultry industry. There are many commercial enzyme products available, and research about use of phytase is well established in broiler nutrition. However, it is necessary research focused more on the other enzymes such as protease. Even though corn and soybean meal are widely used in diets for broilers because of their high nutritional value, it has been reported that considerable amount of protein pass through the digestive tract without complete digestion (Leme, et al. 2004). Because of this portion of protein no digested, the addition of exogenous proteases is a good opportunity to improve nutrient utilization (Freitas, et al. 2011). Ghazi et al, 2002 observed positive effects, in diets deficient or marginal in amino acids, when a single-component protease was used. Given the above, the aim of this study was to evaluate the performance and carcass yield and cuts of broilers fed with mashed and pelleted diets with or without addition of protease.

**Material and Methods**

All the experimental procedures used in this research were approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of the University of Sao Paulo. A total of 768 Cobb 500 male broiler chicks vaccinated for Marek's disease were obtained from a commercial hatchery. Broilers were reared from 1 to 42 days old, distributed in a completely random design in a 2x2x2 factorial arrangement (physical forms of feed, nutritional levels and with or without addition of protease) with eight treatments and eight replicates. Each pen was provided with nipple drinkers, one tube feeder and rice husk as litter. Feed and water were provided *ad libitum*. Live performance measurements (body weight gain, feed intake, and feed conversion) were determined at 21 and 42 days of age. At 43 days of age, two birds from each pen were randomly selected and processed for carcass yield and commercial cuts. Carcass weight was expressed as proportion of body weight, whereas carcass parts were expressed relative to the whole carcass. Corn and soybean meal were used as the major ingredients to formulate the feeds for a 4-phase program: 1 to 7 d, 8 to 21 d, 22 to 35 d, and 36 to 42 d of age. Dietary treatments were: T1 – mashed diet with reduced nutritional levels and without addition of protease; T2 – mashed diet with reduced nutritional levels and with addition of protease; T3 – mashed diet with recommended nutritional levels and without addition of protease; T4 – mashed diet with recommended nutritional levels and with addition of protease; T5 – pelleted diet with reduced nutritional levels and without addition of protease; T6 – pelleted diet with reduced nutritional levels and with addition of protease; T7 – pelleted diet with recommended nutritional levels and without addition of protease; T8 – pelleted diet with recommended nutritional levels and with addition of protease. Diets without reducing nutrient levels were formulated according to the requirements of Rostagno et al. (2011). For diets with low levels of nutrients, reduction of 4% was performed as suggested by the average of the manufacturer of the reduction in crude protein (CP) and amino acids (lysine, methionine

<footnote n="1">¹ Dept. Nutrition and Animal Science, FMVZ, University of Sao Paulo, Pirassununga, Brazil</footnote>

<footnote n="2">² Dept. Animal Science, FZEA, University of Sao Paulo, Pirassununga, Brazil</footnote>
and threonine). The commercial protease used in this study is produced by fermentation of *Bacillus licheniformis* containing transcribed genes *Nocardiopsis prasina*. The results were analyzed using the statistical program SAS (2012).

## Results

Live performance and carcass yield results are summarized in Table 1. Birds fed pelleted diets had improved all parameters performance when compared with those fed mashed diets from 1 to 42 days of age. In addition, the reduced levels of crude protein and amino acids resulted in impairment in feed conversion ratio. However, protease supplementation did not have main effect in bird performance. For the factorial part of the experiment there were no interactions between the effects evaluated for performance. Concerning to carcass yield traits, the addition of exogenous protease did not have main effect on carcass yield and cuts. However, there was interaction between the addition of protease and nutritional levels on carcass yield (Table 2), and interaction between the physical forms of feed and addition of protease on breast yield (Table 3).

<table><caption>**Table 1.** Live performance and carcass yield of broilers fed diets with different physical forms, reduced or recommended nutritional levels and with or without protease.</caption><thead><tr><th>Treatments</th><th></th><th>BWG (kg)</th><th>FI (kg)</th><th>FCR (kg:kg)</th><th>Carcass (%)</th><th>Breast (%)</th><th>Legs (%)</th></tr></thead><tbody><tr><td rowspan="2">Form</td><td>Mashed</td><td>2,70<sup>b</sup></td><td>4,880<sup>b</sup></td><td>1,87<sup>a</sup></td><td>72,91</td><td>31,35</td><td>32,93</td></tr><tr><td>Pelleted</td><td>2,82<sup>a</sup></td><td>5,350<sup>a</sup></td><td>1,75<sup>b</sup></td><td>73,16</td><td>30,98</td><td>33,37</td></tr><tr><td rowspan="2">Protease</td><td>Without</td><td>2,75</td><td>5,104</td><td>1,81</td><td>73,15</td><td>31,00</td><td>33,19</td></tr><tr><td>With</td><td>2,77</td><td>5,106</td><td>1,80</td><td>72,91</td><td>31,31</td><td>33,12</td></tr><tr><td rowspan="2">Nutritional Level</td><td>Reduced</td><td>2,73</td><td>5,220</td><td>1,86<sup>a</sup></td><td>72,92</td><td>30,85</td><td>33,46</td></tr><tr><td>Recommended</td><td>2,80</td><td>5,012</td><td>1,76<sup>b</sup></td><td>73,14</td><td>31,46</td><td>32,85</td></tr><tr><td colspan="2">Mains</td><td>2,6</td><td>5,105</td><td>1,81</td><td>73,03</td><td>31,15</td><td>33,16</td></tr><tr><td colspan="2">SEM</td><td>0,022</td><td>0,057</td><td>0,022</td><td>0,105</td><td>0,191</td><td>0,161</td></tr><tr><th colspan="2">Effects</th><th colspan="6">P-value</th></tr><tr><td colspan="2">Physical form</td><td>0,0068*</td><td>&lt;0,0001*</td><td>0,0047*</td><td>0,2503</td><td>0,3739</td><td>0,1844</td></tr><tr><td colspan="2">Protease</td><td>0,6893</td><td>0,9870</td><td>0,5783</td><td>0,2475</td><td>0,4168</td><td>0,7835</td></tr><tr><td colspan="2">Nutritional level</td><td>0,1002</td><td>0,0698</td><td>0,0156*</td><td>0,2714</td><td>0,1535</td><td>0,0589</td></tr><tr><td colspan="2">Form*Protease</td><td>0,6326</td><td>0,2791</td><td>0,1952</td><td>09021</td><td>0,0108*</td><td>0,1521</td></tr><tr><td colspan="2">Form*Nutritional</td><td>0,3493</td><td>0,1225</td><td>0,4922</td><td>0,1976</td><td>0,6230</td><td>0,5730</td></tr><tr><td colspan="2">Protease*Nutritional</td><td>0,4142</td><td>0,5922</td><td>0,5783</td><td>0,0172*</td><td>0,5301</td><td>0,4844</td></tr><tr><td colspan="2">Form*Protease*Nutritional</td><td>0,8375</td><td>0,1498</td><td>0,1156</td><td>0,3969</td><td>0,7595</td><td>0,2078</td></tr></tbody></table>

<table><caption>**Table 2.** Carcass yield of broiler fed diets with different nutritional levels and supplemented or not with protease.</caption><thead><tr><th rowspan="2">Carcass yield (%)</th><th rowspan="2"></th><th colspan="2">Nutritional level</th></tr><tr><th>Reduced</th><th>Recommended</th></tr></thead><tbody><tr><td rowspan="2">Protease</td><td>Without</td><td>73,09</td><td>73,11</td></tr><tr><td>With</td><td>72,74<sup>b</sup></td><td>73,17<sup>a</sup></td></tr><tr><td colspan="2">Mean</td><td>72,92</td><td>73,14</td></tr><tr><td colspan="2">SEM</td><td>0,148</td><td>0,149</td></tr></tbody></table>

<footnote><sup>a,b</sup> Means within a column not sharing a common superscript differ significantly based on test F (0,05)</footnote>

<footnote><sup>a,b</sup> Means within a row not sharing a common superscript differ significantly based on test F (0,05)</footnote>
<table><caption>**Table 3.** Breast yield of broiler fed diets with different physical forms and supplemented or not with protease.</caption><thead><tr><th rowspan="2">Breast yield (%)</th><th rowspan="2"></th><th colspan="2">Physical form</th></tr><tr><th>Mashed</th><th>Pelleted</th></tr></thead><tbody><tr><td rowspan="2">Protease</td><td>Without</td><td>31,67</td><td>30,32</td></tr><tr><td>With</td><td>30,96</td><td>31,52</td></tr><tr><td colspan="2">Mean</td><td>31,35</td><td>30,98</td></tr><tr><td colspan="2">SEM</td><td>0,266</td><td>0,273</td></tr></tbody></table>

**Conclusions**

Results demonstrated that pelleted diets improved broilers performance and the addition of protease in diets with recommended levels increased carcass yield.

**References**

FREITAS, D.M, S.L., VIEIRA, C.R. ANGEL, A. FAVERO, A. MAIORKA. 2011. Performance and nutrient utilization of broilers fed diets supplemented with a novel mono-component protease. Poult. Sci. **20**, 322-334.

GHAZI, S., J.A. ROOKE, H. GALBRAITH, M. R. BEDFORD. 2002. The potential for the improvement of the nutritive value of soya-bean meal by different proteases in broiler chicks in broiler cockrers. Brit. Poult. Sci. **43**, 70-77.

LEME, A., V. RAVIDRAN, W. L. BRYDEN. 2004. Ileal digestibility of amino acids in fed ingredients for broilers. World's Poult. Sci. **60**, 423-437.

ROSTAGNO, H. S., L. F. T. ALBINO, J. L. DONZELE. 2011. Tabelas Brasileiras para Aves e Suínos. Composição de Alimentos e Exigências Nutricionais. 3rd ed. UFV, Viçosa, Minas Gerais, Brazil.

SAS Institute. 2012. User's Guide. Version 9.3 4th Edition. SAS Inst. Inc., Cary, NC.
# ENERGY AND NUTRIENT UTILIZATION RESPONSES OF BROILERS FED DIETS SUPPLEMENTED WITH AN α-AMYLASE

J. O.B. Sorbara<fnref n="1" />, S. L. Vieira<fnref n="2" />, C. Stefanello<fnref n="2" />, G. O. Santiago<fnref n="2" />, C. T. Simões<fnref n="2" />, N. C. Serafini<fnref n="2" />, and R. G. Hermes<fnref n="1" />

**Corresponding author:** slvieira@ufrgs.br

## Abstract

A study was conducted to evaluate the effects of an α-amylase (RONOZYME® A, DSM Nutritional Products) supplemented in corn-soybean meal diets fed to a total of 480 slow-feathering Cobb 500 male broilers. Birds were randomly distributed to 10 treatments (8 replicates of 6 birds each). Birds were fed with a basal diet from 14 to 25 d (21% crude protein (CP), 1.15% dig. Lys, 0.86% dig. TSAA, 0.75% dig. Thr, 3,100 kcal apparent metabolizable energy (AME)/kg and 1% Celite), and to a basal diet replaced with corn at 40% (Test). basal and Test diets were supplemented with 40, 80, 120 and 160 kilo-Novo α-amylase units (KNU)/kg of feed. Total excreta were collected from 21 to 24 d whereas all birds were slaughtered at 25 d for ileum content collection. Feed, excreta and ileum samples were submitted to analysis for total tract retention and apparent ileal digestibility determinations. Amylase added to the basal diet resulted in quadratic increases (P < 0.05) in dry matter (DM), AMEn (Y = - 0.020x<fnref n="2" /> + 3.096x + 2,943; max = 3,063 kcal/kg; r<fnref n="2" /> = 0.20), and ileal digestible energy (IDE; Y = - 0.023x<fnref n="2" /> + 4.870x + 3,044; max = 3,302 kcal/kg; r<fnref n="2" /> = 0.28). Amylase added to the Test diet led to quadratic increases (P < 0.05) in AMEn (Y = - 0.026x<fnref n="2" /> + 3.662x + 3,475; max = 3,601 kcal/kg; r<fnref n="2" /> = 0.20), DM and CP digestibility of corn. In conclusion, when the amylase was tested in a complete diet 78 and 105 KNU/kg maximized AMEn and IDE release effect, respectively, whereas a maximum energy release of corn was estimated with 70 KNU/kg.

**Keywords:** amylase, broiler, metabolizable energy

## Introduction

Broiler diets have been formulated with corn and soybean meal, which supply the majority content of dietary energy and protein. The nutritional value of these ingredients can be variable depending on the content of protein, oil, starch, non-starch polysaccharides and antinutritional factors such as phytate, resistant starches and enzyme inhibitors (Cowieson, 2005). Although corn and soybean meal are considered highly digestible ingredients, there are possibilities for improvement of their nutritional value because some nutrients are not completely digested in the small intestine (Cowieson et al., 2011). In this context, studies with supplemental enzymes targeting the degradation of substrates that release energy for poultry have been increasing. Exogenous carbohydrates such as xylanases, amylases and glucanases, have been reported to improve energy utilization and the performance of broilers (Olukosi et al., 2008; Vieira et al., 2015). These enzymes may improve the access of endogenous enzymes to cell contents due to hydrolysis of cell wall arabinoxylans (Kocher et al., 2003) as well as to augment endogenous amylase in young animals (Gracia et al., 2003). Decreases in endogenous amino acids losses may also contribute to the beneficial effects of supplemental enzymes, possibly associated with a reduction in the antinutritional effect of some polysaccharides and/or through feedback mechanisms that reduce the need for endogenous enzyme synthesis and secretion (Jiang et al., 2008). Thus, the objective of the present study was to evaluate the effects of adding an exogenous α-amylase-β-glucanase complex on energy utilization, nutrient digestibility and total tract retention of corn or corn-soybean meal-based diets in broiler chickens.

## Material and Methods

A total of 480 one-day-old, slow-feathering Cobb × Cobb 500 male broiler chicks, vaccinated for Marek's disease at the hatchery, were randomly placed in 80 wire cages (0.9 × 0.4 m<fnref n="2" />). Each cage was equipped with one feeder and one drinker. Birds had ad libitum access to water and mash feeds. Birds were allocated to 10 experimental diets with 8 replications in a completely randomized design. A standard corn-soybean meal-based broiler starter was fed from 1 to 14 d (3,050 kcal/kg AMEn, 21.7% CP, 1.05% Ca, and 0.53% non-phytate P). Chicks were individually weighed into groups of 6 birds per cage before placement and the experimental diets were provided from 14 to 25 d. Birds were fed with a

<footnote n="1">¹DSM Nutritional Products, Av. Engº Billings, 1729, São Paulo, SP, Brazil, 05321-010</footnote>

<footnote n="2">²Dept. of Animal Science, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 7712, Porto Alegre, RS, Brazil, 91540-000</footnote>
basal diet (Basal) and a basal diet replaced with corn at 40% (Test). Basal and Test diets were supplemented with 40, 80, 120 and 160 kilo-Novo alpha-amylase units (KNU)/kg [Ronozyme A (CT)] of feed. Each gram of the α-amylase + β-glucanase complex product corresponds to 200 kilo-Novo α-amylase units and 350 fungal β-glucanase units (No-vozymes A/S, Bagsvaerd, Denmark). All experimental diets had 1% Celite as indigestible marker.

Excreta were collected twice daily on wax paper at 21, 22, 23 and 24 d being immediately mixed and pooled by cage and stored at -20°C until analysis. Previous to calorimetry, excreta were dried in a forced air oven at 55°C and ground to pass through a 0.5-mm screen. Intestinal contents were collected from all birds at 25 d after euthanasia by electrical stunning. Jejunal and ileal contents from all birds were flushed with distilled water into plastic containers, pooled by cage, immediately frozen in liquid nitrogen, and stored in a freezer at -20°C until lyophilized. Data were submitted to a 2-way ANOVA using the GLM procedure of SAS Institute (2009). Significance was accepted at P < 0.05 and mean differences were separated using Tukey's HSD test.

## Results

Amylase added to the basal diet resulted in quadratic increases (P < 0.05) in dry matter (DM), AME<sub>n</sub> (Y = - 0.020x<sup>2</sup> + 3.096x + 2,943; max = 3,063 kcal/kg at 78 KNU/kg; r<sup>2</sup> = 0.20), and ileal digestible energy (IDE; Y = - 0.023x<sup>2</sup> + 4.870x + 3,044; max = 3,302 kcal/kg at 105 KNU/kg; r<sup>2</sup> = 0.28) as shown in Table 1.

<table><caption>**Table 1.** Energy and nutrient utilization responses of broilers fed corn-soybean meal diets supplemented with an α-amylase + β-glucanase complex</caption><thead><tr><th rowspan="2">Item</th><th colspan="5">Total tract collection</th><th colspan="2">Ileal collection</th></tr><tr><th>DM, %</th><th>AME, kcal/kg</th><th>AME<sub>n</sub>, kcal/kg</th><th>Crude protein, %</th><th>Ether extract, %</th><th>DM, %</th><th>IDE, kcal/kg</th></tr></thead><tbody><tr><td colspan="8"><strong>Amylase, KNU/kg</strong></td></tr><tr><td>0</td><td>70.4</td><td>3,421</td><td>2,934</td><td>65.7</td><td>83.0</td><td>61.7</td><td>3,016</td></tr><tr><td>40</td><td>75.4</td><td>3,563</td><td>3,036</td><td>68.8</td><td>85.6</td><td>64.8</td><td>3,266</td></tr><tr><td>80</td><td>75.9</td><td>3,638</td><td>3,107</td><td>69.2</td><td>85.4</td><td>65.1</td><td>3,271</td></tr><tr><td>120</td><td>72.9</td><td>3,521</td><td>2,955</td><td>68.1</td><td>85.1</td><td>65.4</td><td>3,278</td></tr><tr><td>160</td><td>71.8</td><td>3,505</td><td>2,939</td><td>68.0</td><td>84.6</td><td>66.0</td><td>3,281</td></tr><tr><td>SEM</td><td>0.535</td><td>24.364</td><td>20.573</td><td>0.592</td><td>0.755</td><td>0.469</td><td>29.365</td></tr><tr><td colspan="8"><strong>P-value<sup>2</sup></strong></td></tr><tr><td>L</td><td>0.9571</td><td>0.4726</td><td>0.6317</td><td>0.3631</td><td>0.6424</td><td>0.0042</td><td>0.0074</td></tr><tr><td>Q</td><td>0.0002</td><td>0.0115</td><td>0.0066</td><td>0.1032</td><td>0.3353</td><td>0.1552</td><td>0.0274</td></tr></tbody></table>

The effects of an α-amylase + β-glucanase complex supplementation on total tract retention and ileal digestibility of corn in broiler chickens are shown in Table 2. Amylase added to the Test diet led to quadratic increases (P < 0.05) in AME<sub>n</sub> (Y = - 0.026x<sup>2</sup> + 3.662x + 3,475; max = 3,601 kcal/kg at 70 KNU/kg; r<sup>2</sup> = 0.20), DM (Y = - 0.0004x<sup>2</sup> + 0.087x + 77.1; max = 82% at 109 KNU/kg; r<sup>2</sup> = 0.16), and CP digestibility (Y = - 0.0005x<sup>2</sup> + 0.117x + 52.4; max = 59% at 117 KNU/kg; r<sup>2</sup> = 0.20) of corn.

<footnote n="2">²Linear or quadratic regression effects (P < 0.05).</footnote>
<table><caption>**Table 2.** Ileal digestibility and total tract utilization of nutrients and energy of corn supplemented with an α-amylase + β-glucanase complex</caption><thead><tr><th rowspan="2">Item</th><th colspan="5">Total tract collection</th><th colspan="2">Ileal collection</th></tr><tr><th>DM, %</th><th>AME, kcal/kg</th><th>AME<sub>n</sub>, kcal/kg</th><th>Crude protein, %</th><th>Ether extract, %</th><th>DM, %</th><th>IDE, kcal/kg</th></tr></thead><tbody><tr><td colspan="8"><strong>Amylase, KNU/kg</strong></td></tr><tr><td>0</td><td>77.4</td><td>3,561</td><td>3,493</td><td>52.0</td><td>73.1</td><td>72.3</td><td>3,512</td></tr><tr><td>40</td><td>79.9</td><td>3,607</td><td>3,545</td><td>57.0</td><td>75.5</td><td>77.2</td><td>3,525</td></tr><tr><td>80</td><td>80.8</td><td>3,681</td><td>3,597</td><td>57.9</td><td>74.9</td><td>79.1</td><td>3,772</td></tr><tr><td>120</td><td>81.5</td><td>3,653</td><td>3,466</td><td>58.9</td><td>73.4</td><td>78.8</td><td>3,586</td></tr><tr><td>160</td><td>79.2</td><td>3,474</td><td>3,560</td><td>58.0</td><td>70.6</td><td>73.8</td><td>3,540</td></tr><tr><td>SEM</td><td>0.587</td><td>32.111</td><td>29.539</td><td>0.882</td><td>0.749</td><td>0.993</td><td>42.137</td></tr><tr><td colspan="8"><strong>P-value<sup>2</sup></strong></td></tr><tr><td>L</td><td>0.1811</td><td>0.5800</td><td>0.2473</td><td>0.0977</td><td>0.1878</td><td>0.5217</td><td>0.6759</td></tr><tr><td>Q</td><td>0.0321</td><td>0.0397</td><td>0.0129</td><td>0.0254</td><td>0.0653</td><td>0.0076</td><td>0.1158</td></tr></tbody></table>

## Conclusions

Corn-soybean meal-based diets supplemented with α-amylase had a beneficial impact on energy utilization for broilers. When the amylase was tested in a complete diet 78 and 105 KNU/kg maximized AME<sub>n</sub> and IDE release effect, respectively, whereas a maximum energy release of corn was estimated with 70 KNU/kg.

## Literature

COWIESON, A. J., 2005: Factors that affect the nutritional value of maize for broilers. Anim. Feed Sci. Technol. **119**, 293–305.

COWIESON, A. J., P. WILCOCOCK, M. R. BEDFORD, 2011: Super-dosing effects of phytase in poultry and other monogastrics. World's Poult. Sci. **67**, 225–235.

GRACIA, M. I., M. J. ARANÍBAR, R. LÁZARO, P. MEDEL, G. G. MATEOS, 2003: Alpha-amylase supplementation of broiler diets based on corn. Poult. Sci. **82**, 436–442.

JIANG, Z., Y. ZHOU, F. LU, Z. HAN, T. WANG, 2008: Effects of different levels of supplementary alpha-amylase on digestive enzyme activities and pancreatic amylase mRNA expression of young broilers. Asian-australas. J. Anim. Sci. **21**, 97–102.

KOCHER, A., M. CHOCT, G. ROSS, J. BROZ, T. K. CHUNG, 2003: Effects of enzyme combinations on apparent metabolizable energy of corn-soybean meal based diets in broilers. J. Appl. Poult. Res. **12**, 275–283.

OLUKOSI, O. A., A. J. COWIESON, O. ADEOLA, 2008: Energy utilization and growth performance of broilers receiving diets supplemented with enzymes containing carbohydrate or phytase activity individually or in combination. Br. Poult. Sci. **99**, 682–690.

VIEIRA, S. L., C. STEFANELLO, H. V. RIOS, N. C. SERAFINI, R. G. HERMES, J. O. B. SORBARA, 2015: Efficacy and metabolizable energy equivalence of an α-amylase-β-glucanase complex for broilers. Braz. J. Poult. Sci. **17**, 227–236.

<footnote n="2">²Linear or quadratic regression effects (P < 0.05).</footnote>
# EFFECT OF PHYTASE SUPPLEMENTATION UP TO 4000 U/KG IN BROILERS FROM 22 TO 42 DAYS POST-HATCH GIVEN DIETS SEVERELY LIMITED IN AVAILABLE PHOSPHORUS

H.R. Taheri, A. Heidari, and M.H. Shahir

Dept. of Animal Science, University of Zanjan, Zanjan, 45371-38791, Iran

Corresponding author: taherihr@gmail.com

## Abstract

An experiment was conducted from 22 to 42 days post-hatch to evaluate the effectiveness of high concentrations of supplemental phytase in maize-soya bean meal-based diets severely limited in available phosphorus (P). Growth performance, plasma P and tibia ash (TA) were measured. The experiment used 220 21-d-old male broilers in 20 pens with 11 birds per pen. Dietary treatments included a positive control [PC, 4.3 g/kg nonphytate P (NPP)], negative control (NC, 1.4 g/kg NPP) and NC plus 1000, 2000 or 4000 phytase U/kg of the diet. Birds fed on the PC diet had higher average daily gain (ADG), gain to feed ratio (G:F), plasma P and TA than those fed on the NC. ADG and G:F values of the NC plus 4000 phytase U/kg reached those of the PC; nevertheless, plasma P values of the NC diets did not come up to that of the PC. While TA values of the NC, NC + 1000 or NC + 2000 did not reach that of the PC, TA of the NC + 4000 was greater than that of the PC. Overall mortality was approximately less than 3%, and not related to dietary treatments. Results of this study showed that, in the diets with 1.4 g/kg NPP, 4000 phytase U/kg can be sufficient to obtain a comparable performance in broilers to those given diets adequate in available P.

**Keywords:** broiler, performance, phosphorus, phytase

## Introduction

Microbial phytase has been used as a feed additive in the poultry industry since 1990. While the standard use of exogenous phytase as a source of dietary P often limits the inclusion in broiler diets to approximately 500 U/kg, some research has been conducted on the effects of high phytase supplementation. Shirley and Edwards (2003) showed improvements in broiler performance, bone ash and phytate P utilization with each additional dose of phytase supplied to a diet severely deficient in available P. Although most studies have examined dietary inclusion of phytase from 1000 to 2500 U/kg, there have been a few trials that have focused on the effect of large and graded concentrations of supplemental phytase (U/kg) up to 4000 (Han et al., 2009) or 12000 (Shirley and Edwards, 2003) on broiler performance.

There are three main reasons that justified the present study: (1) with the exception of the works of Shirley and Edwards (2003) and Han et al. (2009), nearly all of the previous studies have not examined the effect of phytase doses with the absence of the inorganic P source in the diets. Even, Shirley and Edwards (2003) have not eliminated the whole amount of inorganic P source from the negative control (NC) diets; (2) Shirley and Edwards (2003) have evaluated the effect of phytase superdoses on broiler chicks at 1 to 16 d of age, while the broilers have the highest feed intake and P excretion in the finisher phase and may respond to phytase doses differently; (3) Han et al. (2009) have evaluated the effect of graded inclusions of phytase up to 4000 U/kg, but they have not shown consistent trends and responses to phytase doses in terms of feed efficiency and plasma P. In addition, the PC has had 3.5 g/kg NPP in their study, but this concentration of NPP is lower than that (4.2 g/kg) recommended to Ross 308 (ROSS, 2009). We concluded that a research with higher NPP concentration in the PC must be compared with the NC diets supplemented with phytase superdoses. Therefore, in the present study, phytase was included at 0, 1000, 2000 or 4000 U/kg at minimum concentrations of NPP (1.4 g/kg) in the diets from 22 to 42 d post-hatch.

## Material and Methods

All diets were maize-soya bean meal-based and formulated to meet Ross 308 broiler nutrient requirements of finisher phase (Ross, 2009) with the exception of NPP in the NC diets. Experimental diets consisted of PC (with 4.3 g/kg NPP), NC (1.4 g/kg NPP), and NC plus 1000, 2000 or 4000 phytase U/kg of the diet. The Natuphos phytase (BASF, Mt. Olive, NJ) contained an enzyme activity of 1000 phytase U/g were used in the corresponding treatments.

The trial used 220 21-d-old Ross 308 male broilers in 20 pens from 22 to 42 d of age. All the birds had received a regular maize-soya bean meal-based diet with 5.0 and 4.5 g/kg NPP, respectively, from 1–10 and 11–21 d of age. Each treatment had 4 replicates and each replicate was assigned to a pen with 11 broilers (1.5 × 1.5 m). Birds were reared in
floor pens and in an environmentally controlled house; they were exposed to a 23:1 light:dark cycle. The experimental birds were given ad libitum access to water and mash diets.

Birds were weighed before placement (d 21) and on d 42 to calculate ADG. Feed intake was also measured from d 22 to 42 and used to calculate ADFI and G:F. On d 42, two birds/pen (8 birds/treatment) were randomly selected to obtain blood samples via wing vein. The concentrations of inorganic P in the plasma samples were analysed in duplicate using a spectrophotometer following the instructions of corresponding reagent kits (Pars Azmoon, Iran). On d 42, one bird/pen (4 birds/treatment) was randomly slaughtered to obtain tibia samples. TA percent was calculated as [TA weight/dry defatted tibia weight] × 100.

Data were analysed using the GLM procedure of SAS (2003). The pen was the experimental unit, except for TA content in which the individual bird was the experimental unit. Ash results presented as percentages were transformed to their arcsine square root before statistical analysis, and the nontransformed data are presented in the table. Contrast statement was performed to compare the mean of treatments. Orthogonal polynomial contrasts were used to determine significant linear and quadratic responses to phytase supplementation.

## Results

The objective of the trial presented was to evaluate the efficacy of a microbial phytase on growth performance, plasma P and TA of broiler chicks fed on a maize–soya bean meal-based diet severely limited in available P from 22 to 42 d post-hatch. Results showing the effect of phytase superdoses on growth performance are presented in table 1. Overall (d 22–42), mortality was less than 3%, and not related to dietary treatments (P > 0.05; data not shown). Birds fed on the PC had higher ADG, G:F, plasma P and TA than those given the NC. Supplementation of phytase to the NC diets significantly improved ADG and G:F. ADG exhibited linear and quadratic responses to phytase supplementation of the NC diets. G:F showed a linear response to phytase supplementation to the NC diets. ADG and G:F values of the NC plus 4000 phytase U/kg reached that of the PC. Improvements in ADG and G:F were, respectively, 20 and 19% with phytase supplementation of 4000 U/kg. The lack of differences in ADFI between treatments may indicate that young birds may be more susceptible to a NPP deficiency than the older birds which were used in the trial.

<table><caption>**Table 1.** Efficacy of graded level of phytase on growth performance<sup>a</sup>, plasma P (on d 42) and tibia ash (TA, on d 42) of male broiler chicks.</caption><thead><tr><th>Diets<sup>b</sup></th><th>ADG (g)</th><th>ADFI (g)</th><th>G:F (g/g)</th><th>P (mg/dl)</th><th>TA (%)</th></tr></thead><tbody><tr><td>PC</td><td>101.5</td><td>231.8</td><td>0.438</td><td>9.3</td><td>39.5</td></tr><tr><td>NC</td><td>81.4</td><td>219.4</td><td>0.371</td><td>2.8</td><td>33.0</td></tr><tr><td>NC + 1000 U/kg</td><td>88.6</td><td>221.7</td><td>0.400</td><td>4.9</td><td>36.3</td></tr><tr><td>NC + 2000 U/kg</td><td>91.3</td><td>230.3</td><td>0.396</td><td>4.3</td><td>37.6</td></tr><tr><td>NC + 4000 U/kg</td><td>97.5</td><td>221.1</td><td>0.441</td><td>5.6</td><td>41.1</td></tr><tr><td>SEM</td><td>2.42</td><td>5.21</td><td>0.010</td><td>0.56</td><td>0.85</td></tr><tr><td colspan="6">Source of variations (P > F)</td></tr><tr><td>Treatments</td><td>&lt;0.001</td><td>0.34</td><td>0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>Linear response</td><td>&lt;0.001</td><td>0.52</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>Quadratic response</td><td>0.01</td><td>0.20</td><td>0.08</td><td>0.02</td><td>0.003</td></tr><tr><td>PC vs. NC</td><td>&lt;0.001</td><td>0.11</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>PC vs. NC + 1000 U/kg</td><td>0.001</td><td>0.19</td><td>0.02</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>PC vs. NC + 2000 U/kg</td><td>0.01</td><td>0.84</td><td>0.01</td><td>&lt;0.001</td><td>0.03</td></tr><tr><td>PC vs. NC + 4000 U/kg</td><td>0.27</td><td>0.17</td><td>0.80</td><td>&lt;0.001</td><td>0.05</td></tr></tbody></table>

<footnote><sup>a</sup>Broiler chicks were on trial from 22-to 42-d-post-hatch. ADG = average daily gain; ADFI = average daily feed intake; G:F = gain to feed ratio.</footnote>

<footnote><sup>b</sup>PC = positive control (4.3 g/kg nonphytate phosphorus); NC = negative control (1.4 g/kg nonphytate phosphorus).</footnote>
Plasma P exhibited linear and quadratic responses to phytase addition to the NC diets (table 1). Plasma P values of the NC diets did not come up to that of the PC, indicating the amount of P release from phytate was not sufficient to boost plasma P in the absence of inorganic P source from the diet. TA exhibited a quadratic response to phytase supplementation to the NC diets (table 1). TA values of the NC, NC + 1000 or NC + 2000 did not reach that of the PC, however, TA was greater in broilers given 4000 phytase U/kg compared with those fed on the PC. There was approximately 4% increases in TA of the NC + 4000 compared with that of the PC.

## Conclusions

The results of this study showed that in diets with 1.4 g/kg NPP, 4000 phytase U/kg can be sufficient to obtain a comparable performance in broilers to those fed on diets adequate in available P.

## Literature

HAN, J. C., X. D. YANG, H. X., QU, M., XU, T., ZHANG, W.L., LI, J. H., YAO, Y. R., LIU, B. J., SHI, Z. F., ZHOU, X. Y. FENG, 2009: Evaluation of equivalency values of microbial phytase to inorganic phosphorus in 22- to 42-day-old broilers. J. Appl. Poult. Res. **18**, 707-715.

ROSS, 2009: Ross 308 Broiler Nutrition Specification. Ross Breeders Ltd: Newbridge, Midlothian, Scotland.

SHIRLEY, R. B., H. M. EDWARDS JR, 2003: Graded levels of phytase past industry standards improves broiler performance. Poult. Sci. **82**, 671-680.
P-126 (ID 227)

EVALUATION OF A NEW, HIGHLY EFFICIENT PHYTASE IN BROILERS

J. Tossenberger<fnref n="1" />, V. Halas<fnref n="1" />, K. Németh<fnref n="1" />, P. Ader<fnref n="2" /> and D. Feuerstein<fnref n="2" />

**Corresponding author:** halas.veronika@ke.hu

**Abstract**

For economic and environmental reasons use of phytase (500 FTU/kg) is common in monogastrics' feeding. Our aim was to evaluate a new, bacterial 6-phytase in corn-soybean meal diets with a maximum of 250 FTU/kg.

A total of 960 ROSS-308 broilers were allocated into 4 treatments (trt). Positive control feed was formulated without added phytase, its P and Ca content was in accordance with the recommendations (PC; P: 7.1 and 6.3, Ca/P: 1.3 and 1.2, in starter and grower phase, resp). Negative control feeds were formulated with reduced P and Ca (NC; P: 4.7 and 4.4, Ca/P: 1.3 and 1.2, in starter and grower phase, resp.) and supplemented with 0, 125, and 250 FTU/kg phytase.

Supplementation of 250 FTU/kg of phytase sufficiently compensated the negative effect of P deprivation on BW, FI, and ADG. Tibias were the weakest in NC group (187.2 N) significantly differed from PC and 250 FTU/kg phytase supplemented trts (mean of 238.4 N). The contrast in dietary P level resulted in consistent differences in bone ash, Ca, and P content (PC vs. NC; P<0.05) and the phytase supplementation increased the concentration of chemical components in tibia bone. However, added 250 FTU/kg to NC feed was not enough to achieve the same chemical composition as in PC group likely due to narrow Ca/P ratio and thus absolute lack of Ca.

In conclusion, P content of a corn-soybean broiler diet can be reduced by 30% without detrimental effect on performance and bone characteristics, if 250 FTU/kg new phytase is applied.

**Keywords:** new-phytase, broiler, performance, bone mineralization

**Introduction**

The bioavailability of P in grains is limited for monogastric farm animals. It is known that digestibility of dietary P can be improved by releasing phytate bound P with phytase. In broilers the recommended dose of commercial enzyme products in corn-soybean diets is 500 FTU/kg or more. Recently a high efficient, microbial 6-phytase produced by *Aspergillus niger* has been developed, and our aim was to evaluate this new phytase with maximum dose of 250 FTU/kg based on growth rate and bone characteristics of broilers.

**Material and Methods**

A performance and a bone mineralization study were conducted with identical 4 dietary treatments. The performance study was carried out with 960, 1-day-old ROSS-308 broiler cocks. A 2-phase feeding was used (starter phase: d 1-21, grower phase: d 22-38) and experimental diets were formulated on corn-soybean basis. Diet A was the positive control without added phytase, its P and Ca content was in accordance with NRC (1994) recommendations (PC; total P: 7.1 and 6.3, Ca/P: 1.3 and 1.2, non phytate P (NPP): 4.3 and 3.8 g/kg, in starter and grower phase, respectively). Diets in treatment B were formulated with reduced P and Ca (negative control: NC; total P: 4.7 and 4.4, Ca/P: 1.3 and 1.2, NPP: 2.0 and 1.8 g/kg, in starter and grower phase, resp.) and without added phytase. In treatments C and D the diet composition was the same as in diet B, but was supplemented with 125 FTU and 250 FTU new phytase (treatment C and D, respectively) per kg of feed. The birds were fed ad libitum from self-feeders during the trial. Drinking water was freely available from round drinkers. In the performance trial feed intake (FI), average daily gain (ADG), and the feed conversion (FCR) as well as mortality rate were studied.

The effect of the dietary P and phytase level on bone characteristics was evaluated with 10 birds taken from each dietary treatment at the end of the performance study (at 38 days of age). The assigned birds were slaughtered and the left tibia was dissected. The bone mineralization was determined considering the length, diameter, cross section area, and maximum breaking strength, as well as weight, ash, Ca, and P content of left tibia. The nutrient (dry matter, crude protein, crude fat, crude fiber, crude ash, Ca, P) content of the diets, as well as dry matter, Ca and P content of the tibia were determined in accordance with the AOAC (1989). The activity of the phytase products and feed samples were

<footnote n="1">¹Kaposvár University, Faculty of Agricultural and Environmental Sciences, Department of Animal Nutrition, Hungary</footnote>

<footnote n="2">²BASF SE, Lampertheim, Germany</footnote>
determined according to the method of Engelen et al. (2001). The effect of dietary treatments was analyzed with one way ANOVA (SAS, 2004).

## Results

The effect of dietary treatments on the growth performance of 38 days old broilers are presented in Table 1. Reduction of dietary P without enzyme supplementation resulted in 394 g (16.3%) lower final body weight due to the lower feed consumption (14,2 g/d identical with 16,2%; treatment A vs B; P<0.05). With the addition of phytase the body weight, the daily gain as well as the feed intake increased. Supplementation of 250 FTU per kg of the new phytase to the negative control diet was sufficient to compensate the detrimental effect of P deprivation on BW and ADG (treatment A vs D, P>0.05). Feed conversion, however, was independent on P supply or enzyme supplementation. The mortality rate was low regardless of dietary treatments in the course of the performance trial ranged from 2.1 to 4.6%.

<table><caption>**Table 1.** Effect of different dietary phytase supplementation on growth performance of broilers determined at 38 days of age</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">TREATMENTS*</th><th rowspan="2">RMSE</th><th rowspan="2">P value</th></tr><tr><th>A</th><th>B</th><th>C</th><th>D</th></tr></thead><tbody><tr><td>Body weight (g)</td><td>2412.9<sup>a</sup></td><td>2018.5<sup>c</sup></td><td>2148.6<sup>b</sup></td><td>2317.3<sup>a</sup></td><td>105.88</td><td>&lt;0.0001</td></tr><tr><td>Body weight gain (g/d)</td><td>61.4<sup>a</sup></td><td>51.9<sup>c</sup></td><td>55.5<sup>b</sup></td><td>59.5<sup>a</sup></td><td>3.09</td><td>&lt;0.0001</td></tr><tr><td>Feed intake (g/d)</td><td>87.9<sup>a</sup></td><td>73.7<sup>d</sup></td><td>77.4<sup>c</sup></td><td>81.5<sup>b</sup></td><td>3.34</td><td>&lt;0.0001</td></tr><tr><td>Feed conversion (kg/kg)</td><td>1.48</td><td>1.48</td><td>1.47</td><td>1.44</td><td>0.043</td><td>0.1696</td></tr></tbody></table>

*A: Positive control (without phytase supplementation); B: Negative control (with reduced Ca and P content, without phytase supplementation); C: Negative control + 125 FTU/kg; D: Negative control + 250 FTU/kg

a,b: Means with different superscripts are significantly different (P ≤0.05)

The effect of dietary P supply and phytase supplementation on the bone characteristics are shown in Table 2. Bone strength was the weakest in the NC group (187.2 N) and significantly differed from the PC and 250 FTU/kg phytase supplemented treatments (mean of 238.4 N). The contrast in dietary P level resulted in consistent differences in bone ash, Ca, and P content (treatment A vs. B; P<0.05) and the phytase supplementation increased the concentration of studied chemical components in tibia bone. However, addition of 250 FTU/kg to NC diet was not sufficient to achieve the same chemical composition as in PC group (treatment A vs D, P<0.05) likely due to small Ca/P ratio and thus absolute lack of Ca.

<table><caption>**Table 2.** Effect of different dietary phytase levels on bone characteristics and breaking strength as well as chemical composition (weight, ash, Ca and P content) of tibia</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">TREATMENTS*</th><th rowspan="2">RMSE</th><th rowspan="2">P value</th></tr><tr><th>A</th><th>B</th><th>C</th><th>D</th></tr></thead><tbody><tr><td>Length (mm)</td><td>85.22</td><td>81.09</td><td>85.53</td><td>83.96</td><td>3.431</td><td>0.0776</td></tr><tr><td>Diameter (mm)</td><td>6.84</td><td>6.78</td><td>7.05</td><td>6.82</td><td>0.578</td><td>0.7393</td></tr><tr><td>Max. breaking strength (N)</td><td>248.2<sup>a</sup></td><td>187.2<sup>b</sup></td><td>216.8<sup>ab</sup></td><td>228.5<sup>a</sup></td><td>40.78</td><td>0.0195</td></tr><tr><td>Weight (g)</td><td>10.2</td><td>9.7</td><td>10.1</td><td>9.8</td><td>1.06</td><td>0.6744</td></tr><tr><td>Ash (g/kg bone)</td><td>213.8<sup>a</sup></td><td>171.6<sup>c</sup></td><td>185.1<sup>b</sup></td><td>193.6<sup>b</sup></td><td>14.48</td><td>&lt;0.0001</td></tr><tr><td>Ca (g/kg bone)</td><td>67.4<sup>a</sup></td><td>53.4<sup>c</sup></td><td>59.4<sup>b</sup></td><td>62.3<sup>b</sup></td><td>4.89</td><td>&lt;0.0001</td></tr><tr><td>P (g/kg bone)</td><td>36.4<sup>a</sup></td><td>28.3<sup>c</sup></td><td>30.3<sup>bc</sup></td><td>32.2<sup>b</sup></td><td>2.76</td><td>&lt;0.0001</td></tr></tbody></table>

*A: Positive control (without phytase supplementation); B: Negative control (with reduced Ca and P content, without phytase supplementation); C: Negative control + 125 FTU/kg; D: Negative control + 250 FTU/kg

a,b: Means with different superscripts are significantly different (P ≤0.05)
**Conclusion**

In conclusion the P content of a corn-soybean broiler diet can be reduced by 30% without detrimental effect on performance and bone characteristics if 250 FTU/kg of a new phytase is applied.

**References**

AOAC, 1989: Official methods of analysis. Association of Official Analytical Chemist 13th ed. Washington, D.C.

ENGELEN AJ, VAN DER HEEFT FC, RANDSDORP PH, SOMERS WA, SCHAEFER J, VAN DER VAT BJ, 2001: Determination of Phytase Activity in Feed by a Colorimetric Enzymatic Method: Collaborative Interlaboratory Study, Journal of AOAC International, 84 (3) 629-33.

NRC, 1994: Nutrient Requirements of Poultry. National Academy Press, Washington, D. C.

SAS, 2004. SAS® ODBC Driver 9.1: User's Guide and Programmer's Reference. Cary, NC: SAS Institute Inc.
# STARCH DIGESTIBILITY AND ENERGY UTILIZATION OF BROILERS FED CORN-SOYBEAN BASAL DIETS SUPPLEMENTED WITH AMYLASE AND XYLANASE

S. L. Vieira<fnref n="1" />, C. Stefanello<fnref n="1" />, G.O. Santiago<fnref n="1" />, N.C. Serafini<fnref n="1" />, C.T. Simões<fnref n="1" />, J. O. B. Sorbara<fnref n="2" />, and R. G. Hermes<fnref n="2" />

**Corresponding author:** slvieira@ufrgs.br

## Abstract

A study was conducted to evaluate the effects of dietary α-amylase and β-xylanase supplementation of corn-soy diets formulated with or without supplemental phytase on energy utilization and starch digestibility in broiler chickens. A total of 336 slow feathering, Cobb × Cobb 500 male broilers were randomly distributed to 6 treatments having 8 replicates of 7 birds each. Birds were fed a common starter diet to 14 d. The experimental diets were provided afterwards until 25 d. A 2 × 3 factorial arrangement of 2 control diets (Basal = corn-soy diet without added phytase or PHY = corn-soy diet formulated with 1,000 phytase units/kg) and 3 carbohydrate supplementations (0, 80 kilo-Novo α-amylase units/kg, or 80 kilo-Novo α-amylase units/kg + 100 fungal β-xylanase units/kg) was used from 14 to 25 d. Excreta were collected from 21 to 24 d and all birds were euthanized at 25 d for jejunum and ileum content collection. Samples of feed, excreta, jejunal and ileal digesta were analyzed for determination of total tract retention and ileal apparent digestibility. No interactions between diet and carbohydrate were observed. Relative to the basal diet, AME<sub>n</sub> was increased (P < 0.01) by 70 kcal/kg and 99 kcal/kg when birds were fed the diet supplemented with amylase and amylase + xylanase, respectively. Starch digestibility in jejunum and ileum was increased (P < 0.05) by 3.5% and 2.4% when birds were fed the diet supplemented with amylase + xylanase. Results from this experiment show that corn-soy diets having phytase and supplemented with amylase and xylanase led to increased AME<sub>n</sub>, and starch digestibility in broilers. Furthermore, the efficacy of exogenous amylase and xylanase was independent of the presence of microbial phytase.

**Keywords:** amylase, broiler, phytase, starch, xylanase

## Introduction

Nutrient and energy digestibility of plant feedstuffs by poultry is limited by the proportion of their components for which there are no corresponding endogenous digestive secretions. This is the case of the non-starch polysaccharides that are present in and within the cell walls of soybean meal and corn (Bach Knudsen, 1997; Caffall and Mohnen, 2009). They are either indigestible or of very low digestibility when fed to poultry. Therefore, the energy derived from them is restricted by a lower organic combustion of these components as well as by the physical barrier represented by the cell wall itself that prevents enzyme access to substrates (Slominski et al., 1993). Additionally, phytate (myo-inositol hexaphosphate) is the main form of stored phosphorus in plant seeds. Besides the limitation in the use of phosphorus by poultry, phytate has also been reported to reduce energy utilization (Ravindran et al., 2006). Phytate and lower inositol phosphates can be hydrolyzed by the enzyme phytase (myo-inositol hexaphosphate phosphohydrolase), which has become a standard enzymatic additive in poultry diets (Cowieson et al., 2011).

As increasing numbers of enzyme products become available, the assembly of strategically optimized admixtures becomes a challenge in terms of targeting the complex mixture of components that have low digestibility. Whilst different enzyme classes hydrolyze different substrates and generate different products, a net effect on the increase in the digestibility of starch, protein/AA and fat with a cumulative energetic consequence seems to be an expected outcome of their action. While enzymes that target different substrates do not compete in terms of substrate degradation itself, they tend to overlap in nutrient digestion and performance, delivering sub-additive outcomes (Cowieson and Adeola, 2005). Increases in energy utilization from plant feedstuffs by broilers may derive from a large variety of components; however, since starch is quantitatively the largest energy supplier in broiler diets, any improvement in its degradation would lead to a significant benefit.

The objective of the present study was to evaluate the effects of an α-amylase alone or in combination with a β-xylanase on energy utilization and starch digestibility of broiler chickens fed corn-soy-based diets. These effects were

<footnote n="1">¹ Dept. of Animal Science, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 7712, Porto Alegre, RS, Brazil, 91540-000</footnote>

<footnote n="2">² DSM Nutritional Products, Av. Engº Billings, 1729, São Paulo, SP, Brazil, 05321-010</footnote>
assessed both in a phytase-free diet and in a diet that included phytase in order to explore the possibility that carbohydrate efficacy may be muted by the presence of phytase.

## Material and Methods

A total of 336 one-day-old, slow-feathering Cobb × Cobb 500 male broiler chicks, vaccinated for Marek's disease at the hatchery, were randomly placed in 48 wire cages (0.9 × 0.4 m2). Each cage was equipped with one feeder and one drinker. Birds had ad libitum access to water and mash feeds. Birds were allocated to 6 experimental diets with 8 replacements of 7 birds each in a completely randomized design. A standard corn-soybean meal-based broiler starter was fed from 1 to 14 d (3,050 kcal/kg AME<sub>n</sub>, 21.7% CP, 1.05% Ca, and 0.53% non-phytate P). A 2 × 3 factorial arrangement of 2 control diets (Basal = corn-soy basal diet without added phytase or PHY = corn-soy diet formulated with phytase) and 3 carbohydrate supplements (0, α-amylase, or α-amylase + β-xylanase) were provided from 14 to 25 d. The supplemental enzymes used in the present study are commercially available (Novozymes A/S, Bagsvaerd, Denmark). Their inclusion per kg of diet were: phytase [Ronozyme HiPhos (GT)] 1,000 phytase units (FYT), alpha-amylase [Ronozyme HiStarch (CT)] 80 kilo-Novo alpha-amylase units (KNU), and beta-xylanase [Ronozyme WX (CT)] 100 fungal xylanase units (FXU). All experimental diets had 1% Celite as indigestible marker.

Excreta were collected twice daily on wax paper at 21, 22, 23 and 24 d being immediately mixed and pooled by cage and stored at -20°C until analysis. Previous to calorimetry, excreta were dried in a forced air oven at 55°C and ground to pass through a 0.5-mm screen. Intestinal contents were collected from all birds at 25 d after euthanasia by electrical stunning. Jejunal and ileal contents from all birds were flushed with distilled water into plastic containers, pooled by cage, immediately frozen in liquid nitrogen, and stored in a freezer at -20°C until lyophilized. Data were submitted to a 2-way ANOVA using the GLM procedure of SAS Institute (2009). Significance was accepted at P < 0.05 and mean differences were separated using Tukey's HSD test.

## Results

There were no interactions between diet and carbohydrate for ileal digestibility, total tract retention and starch digestibility in 25-d-old broilers (Table 1). Differences in AME<sub>n</sub> and ileal digestibility of DM were found between PHY and basal diets (P < 0.01) with values, respectively of 3,483 kcal/kg and 65.2% for PHY and 3,412 kcal/kg and 63.4% for basal. In the present study, AME<sub>n</sub> was improved (P < 0.01) by 70 and 99 kcal/kg when amylase and amylase + xylanase were supplemented, respectively. Amylase and xylanase supplementation provided an increase (P < 0.01) in AME<sub>n</sub> of 2.8% when compared to the diet without carbohydrates. The digestibility of total starch in the jejunum and ileum was higher (P < 0.01) for broilers fed PHY or diets supplemented with amylase + xylanase. The digestibility of total starch in the jejunum and ileum was increased (P < 0.05) by 3.5% and 2.4%, respectively in broilers fed amylase + xylanase diet compared to broilers fed the diet without carbohydrates.

<table><caption>**Table 1.** Energy and nutrient utilization response, and apparent digestibility of starch by chickens fed experimental diets¹</caption><thead><tr><th rowspan="2">Item</th><th colspan="2">Ileal collection</th><th colspan="2">Total tract collection</th><th colspan="2">Jejunal starch, %</th><th colspan="2">Ileal starch, %</th></tr><tr><th>DM, %</th><th>IDE, kcal/kg</th><th>DM, %</th><th>AME<sub>n</sub>, kcal/kg</th><th>Digestible</th><th>Total</th><th>Digestible</th><th>Total</th></tr></thead><tbody><tr><td>Diet</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Basal</td><td>63.4</td><td>3,187</td><td>70.2</td><td>3,412</td><td>91.1</td><td>89.8</td><td>91.1</td><td>89.8</td></tr><tr><td>PHY</td><td>65.2</td><td>3,267</td><td>71.2</td><td>3,483</td><td>92.5</td><td>91.3</td><td>92.5</td><td>91.3</td></tr><tr><td>Carbohydrase</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0</td><td>62.9<sup>b</sup></td><td>3,150</td><td>69.6<sup>b</sup></td><td>3,391<sup>b</sup></td><td>91.1<sup>b</sup></td><td>89.6<sup>b</sup></td><td>91.1<sup>b</sup></td><td>89.6<sup>b</sup></td></tr><tr><td>Amylase</td><td>64.4<sup>ab</sup></td><td>3,234</td><td>70.9<sup>ab</sup></td><td>3,461<sup>a</sup></td><td>91.6<sup>b</sup></td><td>90.2<sup>ab</sup></td><td>91.6<sup>b</sup></td><td>90.2<sup>ab</sup></td></tr><tr><td>Amylase + xylanase</td><td>65.6<sup>a</sup></td><td>3,297</td><td>71.5<sup>a</sup></td><td>3,490<sup>a</sup></td><td>92.8<sup>a</sup></td><td>92.0<sup>a</sup></td><td>92.8<sup>a</sup></td><td>92.0<sup>a</sup></td></tr><tr><td>Mean</td><td>64.3</td><td>3,227</td><td>70.7</td><td>3,448</td><td>91.8</td><td>90.6</td><td>91.8</td><td>90.6</td></tr><tr><td>SEM</td><td>0.576</td><td>18.798</td><td>0.266</td><td>12.392</td><td>0.297</td><td>0.347</td><td>0.297</td><td>0.347</td></tr><tr><td>Main effect P-value</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Diet (D)</td><td>0.0192</td><td>0.1213</td><td>0.0458</td><td>0.0010</td><td>0.0188</td><td>0.0138</td><td>0.0188</td><td>0.0138</td></tr><tr><td>Carbohydrase (C)</td><td>0.0260</td><td>0.1683</td><td>0.0093</td><td>0.0007</td><td>0.0567</td><td>0.0129</td><td>0.0567</td><td>0.0129</td></tr><tr><td>D × C</td><td>0.6747</td><td>0.9702</td><td>0.4889</td><td>0.9714</td><td>0.8159</td><td>0.2075</td><td>0.8159</td><td>0.2075</td></tr></tbody></table>

<footnote>a-bMeans with different superscript letter differ (P < 0.05) based on Tukey's honestly significant difference test.</footnote>
Conclusions

Corn-soybean meal-based diets formulated with 1,000 phytase units/kg or supplemented with 80 kilo-Novo α-amylase units/kg and 100 fungal β-xylanase units/kg had a beneficial impact on energy utilization for broilers. AME<sub>n</sub> was also improved when birds were fed amylase + xylanase diet compared to the diet without carbohydrates. Finally, the digestibility of starch in the jejunum was lower than in the ileum, and corn-soy diets formulated with phytase or supplemented with amylase + xylanase improved starch utilization.

Literature

BACH KNUDSEN, K. E, 1997: Carbohydrate and lignin contents of plant materials used in animal feeding. Anim. Feed Sci. Technol. **67**, 319–338.

CAFFALL, K. H., D. MOHNEN, 2009: The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydr. Res. **344**, 1879–1900.

COWIESON, A. J., O. ADEOLA, 2005: Carbohydrases, protease, and phytase have an additive beneficial effect in nutritionally marginal diets for broiler chicks. Poult. Sci. **84**, 1860–1867.

COWIESON, A. J., P. WILCOCOCK, M. R. BEDFORD, 2011: Super-dosing effects of phytase in poultry and other monogastrics. World's Poult. Sci. **67**, 225–235.

RAVINDRAN, V., P. C. MOREL, G. G. PARTRIDGE, M. HRUBY, J. S. SANDS, 2006: Influence of an *Escherichia coli*-derived phytase on nutrient utilization in broiler starters fed diets containing varying concentrations of phytic acid. Poult. Sci. **85**, 82–89.

SLOMINSKI, B. A., W. GUENTER, L. D. CAMPBELL, 1993: New approach to water-soluble carbohydrate determination as a tool for evaluation of plant cell wall degrading enzymes. J. Agric. Food Chem. **41**, 2304–2308.
P-128 (ID 89)

COMPOSITE ANALYSIS OF TEN BROILER TRIALS EVALUATING STANDARD AND SUPERDOSES OF A MICROBIAL PHYTASE IN THE PRESENCE OR ABSENCE OF A XYLANASE

C.L. Walk, I. Kuehn, and M.R. Bedford

AB Vista, Marlborough, Wiltshire, UK

**Corresponding author:** carrie.walk@abvista.com

**Abstract**

In broiler diets the use of phytase to reduce inorganic phosphorus supplementation and of xylanase to improve nutrient utilisation is common practice. The objective of this analysis was to evaluate the impact of standard or superdoses of phytase, without or with a xylanase. A total of 10 broiler trials were conducted at research sites worldwide from 2012 to 2015 using diet, nutrient, husbandry and environmental conditions typical to the region. The dietary treatments included a diet with 500 FTU/kg of phytase and a reduction in available P and Ca by 0.15 and 0.16%, respectively (CON), the CON diet with superdoses (1000 to 1500 FTU/kg) of phytase (SD), the CON + xylanase (XYL), and SD + XYL. This resulted in a 2 x 2 factorial design and a total of 76 data points used in the model. In 4 of the trials, in addition to available P and Ca, the CON diet had a reduction in ME by 50 to 100 kcal/kg. The dosage of xylanase ranged from 8000 to 32000 BXU/kg. Body weight corrected FCR was calculated from broilers >35 days of age, and means were analysed using the fit model platform in JMP v. 11.2.1. Supplementation of broiler diets with phytase superdoses (1.552 vs 1.580) or xylanase (1.551 vs 1.581) improved (P < 0.0001) BW corrected FCR compared with broilers fed standard phytase or diets without xylanase. There was no phytase x xylanase interaction (P = 0.89), indicating phytase superdoses and xylanase work independently to improve the feed conversion ratio of broilers.

**Keywords:** poultry, phytase, xylanase, feed conversion ratio

**Introduction**

It has been estimated that approximately 95% of poultry diets contain an exogenous xylanase and approximately 90% an exogenous phytase. Supplementation of poultry diets with xylanase is reported to improve growth performance and nutrient digestibility through reduction of viscosity or improvements in energy availability through the breakdown of non-starch polysaccharides (NSP). While the content of NSPs in maize is not as high as that of wheat or rye, Masey O'Neill et al. (2012) reported beneficial responses on growth performance and nutrient digestibility when xylanase was supplemented in maize-based broiler diets. This may be associated with cell wall degradation (Meng and Slominski, 2005) or the production of oligosaccharides which exert a pre-biotic effect (Courtin et al., 2008) and thereby improve broiler performance and feed conversion ratio.

Phytase can hydrolyse phytate, thereby releasing phytate-phosphorus (P) and improving P digestibility. The use of exogenous phytase allows for reductions of inorganic phosphate in the diet while maintaining growth performance and bone ash (Zeller et al., 2015). Phytate is considered a source of P but also can be described as an anti-nutrient, and significantly reduces growth performance and nutrient digestibility in broilers. Recently, the use of superdoses of phytase has resulted in near complete phytate destruction and significant improvements in broiler feed conversion (Walk et al., 2014).

Both phytase and xylanase enzymes have different substrates and modes of action in broilers. However, broiler diets are commonly supplemented with both a xylanase and phytase. Therefore this composite analysis was conducted to evaluate the impact of standard or superdoses of phytase, without or with a xylanase, on broiler feed conversion ratio using 10 broiler trials conducted globally.

**Material and Methods**

Ten broiler trials were conducted from 2012 to 2015 in Brazil (n = 2), Mexico (n = 1), EU (n = 5), Thailand (n = 1) and India (n = 1). Trial diets were formulated to contain predominantly maize and soybean meal (Brazil, India, EU n = 1, and Thailand), sorghum and soybean meal (Mexico), or wheat and soybean meal (EU n = 4). The experimental diets were formulated to be nutrient adequate, with the exception of calcium and available P (avP), which was decreased by 0.16 and 0.15%, respectively, from requirement at each feeding phase. In addition, in four trials ME was reduced by 50 (India and Thailand), 80 (Brazil n = 1) or 100 (EU n = 1) kcal/kg from the requirement at each feeding phase. Phytase
(Quantum Blue, AB Vista, Marlborough UK) was formulated in the experimental diets at the standard dose (CON) of 500 FTU/kg or superdose (SD) of 1000 or 1500 FTU/kg. Xylanase (Econase XT, AB Vista, Marlborough UK) was added at 0 (- XYL) or variable doses which ranged from 8000 to 32000 BXU/kg (+ XYL; Table 1). Therefore, the experimental design included the CON - XYL, SD - XYL, CON + XYL or SD + XYL arranged as a 2 x 2 factorial of treatments. Only experimental means were used in the composite analysis (n = 84).

<table><caption>**Table 1.** Descriptive factors of each trial included in the composite analysis evaluating standard or superdoses of phytase, without or with a xylanase</caption><thead><tr><th rowspan="2">Location</th><th rowspan="2">n</th><th rowspan="2">Diet composition</th><th>Phase 1</th><th>Phase 2</th><th>Phase 3</th><th>Xylanase,</th></tr><tr><th colspan="3">ME, kcal/kg</th><th>BXU/kg</th></tr></thead><tbody><tr><td>India<fnref n="1" /></td><td>8</td><td>Corn and soybean meal</td><td>2950</td><td>3050</td><td></td><td>0 or 16000</td></tr><tr><td>Brazil</td><td>16</td><td>Corn and soybean meal</td><td>3050</td><td>3154</td><td></td><td>0 or 8000-16000</td></tr><tr><td>Mexico</td><td>4</td><td>Sorghum and soybean meal</td><td>3000</td><td>3100</td><td>3200</td><td>0 or 16000</td></tr><tr><td>Germany<fnref n="2" /></td><td>4</td><td>Wheat and soybean meal</td><td>2916</td><td>3100</td><td></td><td>0 or 16000</td></tr><tr><td>UK</td><td>4</td><td>Wheat and soybean meal</td><td>3000</td><td>3100</td><td></td><td>0 or 16000</td></tr><tr><td>UK</td><td>12</td><td>Corn and soybean meal</td><td>3000</td><td>3050</td><td>3100</td><td>0 or 8000-32000</td></tr><tr><td>UK</td><td>4</td><td>Wheat and soybean meal</td><td>3060</td><td>3150</td><td></td><td>0 or 16000</td></tr><tr><td>Brazil<fnref n="3" /></td><td>16</td><td>Corn and soybean meal</td><td>3050</td><td>3120</td><td>3170</td><td>0 or 8000-24000</td></tr><tr><td>Thailand<fnref n="1" /></td><td>8</td><td>Corn and soybean meal</td><td>2950</td><td>3000</td><td>3050</td><td>0 or 16000</td></tr><tr><td>Poland</td><td>8</td><td>Wheat and soybean meal</td><td>2919</td><td>3101</td><td>3149</td><td>0 or 16000</td></tr></tbody></table>

Body weight corrected feed conversion ratio was calculated from body weight and feed conversion ratio of broilers greater than 35 days of age. Means were analysed using the fit model platform in JMP v. 11.2.1. The model included phytase dose, xylanase and the interaction. Trial site was included in the model as a covariate. Significance was accepted at P < 0.05. Significant means were adjusted using t-tests.

## Results

The final model included 76 data points and had an R<fnref n="2" /> = 0.97. The mean BW corrected FCR was 1.576 with an RMSE = 0.023. Phytase dose × xylanase interaction was not significant (P = 0.89; Figure 1). Superdoses of phytase significantly (P < 0.0001) improved body weight corrected FCR from 1.580 to 1.552, regardless of the presence or absence of xylanase. Similarly, supplementing broiler diets with xylanase significantly (P < 0.0001) improved body weight corrected FCR of broilers from 1.581 to 1.551, regardless of the level of phytase supplied in the diet.

<footnote n="1">¹ Reflects a reduction in formulated ME by 50 kcal/kg.</footnote>

<footnote n="2">² Reflects a reduction in formulated ME by 100 kcal/kg.</footnote>

<footnote n="3">³ Reflects a reduction in formulated ME by 80 kcal/kg.</footnote>
<figure><img src="image_54.png" /><figcaption>**Figure 1.** Body weight corrected feed conversion ratio (FCR) of broilers fed standard (CON) or superdoses of phytase (SD) in the absence (- XYL) or presence (+ XYL) of xylanase</figcaption></figure>

## Conclusions

A composite analysis of 10 broiler trials conducted globally, using dietary and husbandry factors typical to the region, various ME matrices and grain-types indicated superdoses of phytase or the inclusion of xylanase into broiler diets results in significant improvements in body weight corrected feed conversion ratio. The lack of significant interaction would indicate phytase and xylanase work independently to improve FCR.

## Literature

COURTIN, C. M., W. F. BROEKAERT, K. SWENNEN, O. LESCROART, O. ONAGBESAN, J. BUYSE, E. DECUYPERE, T. VAN DE WIELE, M. MARZORATI, W. VERSTRAETE, G. HUYGHEBAERT, J. DELCOUR 2008. Dietary inclusion of wheat bran arabinoxylan oligosaccharides indicates beneficial nutritional effects in chickens. Cereal Chem. **85**, 607-613.

MASEY O'NEILL, H.V., N. LIU, J. P. WANG, A. DIALLO, S. HILL 2012. Effect of xylanase on performance and apparent metabolisable energy in starter broilers fed diets containing one maize variety harvested in different regions of China. Asian-Aust. J. Anim. Sci. **4**, 515-523.

MENG, X., B. A. SLOMINSKI 2005. Nutritive values of corn, soybean meal, canola meal and peas for broiler chickens as affected by a multicarbohydrase preparation of cell wall degrading enzymes. Poult. Sci. **84**, 1242-1251.

WALK, C. L., T. T. SANTOS, M. R. BEDFORD 2014. Influence of superdoses of a novel microbial phytase on growth performance, tibia ash, and gizzard phytate and inositol in young broilers. Poult. Sci. **93**, 1172-1177.

ZELLER, E., M. SCHOLLENGERGER, M. WITZIG, Y. SHASTAK, I. KUHN, L. E. HOELZLE, M. RODEHUTSCORD 2015. Interactions between supplemental mineral phosphorus and phytase on phytate hydrolysis and inositol phosphates in the small intestine of broilers. Poult. Sci. http://dx.doi.org/10.3382/ps/pev087.
# Feed raw materials

## P-130 (ID 110)

### THE EFFECT OF DIFFERENT LEVELS OF EXTRUDED HEMPSEED (CANNABIS SATIVA L.) ON PERFORMANCE, PLASMA LIPID PROFILE AND IMMUNE RESPONSE OF BROILER CHICKS

N. Afzali<fnref n="1" />, M. Barani<fnref n="2" /> and S.J. Hosseini Vashan<fnref n="1" />

**Corresponding author:** nafzali@birjand.ac.ir

### Abstract

The aim of this study was to evaluate the effect of extruded hempseed (EHS) on the performance, plasma lipid profile and immune response in broiler chicks during 1 to 42 days of age. A total of 250 one-day-old male broiler chick (Ross 308) were randomly assigned to 5 treatments. Each treatment was comprised of 5 replicates of 10 broiler chicks in each. The treatments were of: 1) Control diet (EHS0, diet contained no EHS); 2) diet contained %5 EHS (EHS5); 3) diet contained %10 EHS (EHS10); 4) diet contained %15 EHS (EHS15); 5) diet contained %20 EHS (EHS20). Diets provided similar metabolizable energy (ME) and crude protein (CP). Data were analyzed as a completely randomized design using of the PROC GLM procedure of SAS. The results of this study showed that inclusion of EHS into diet had no significant effect on performance parameters. The plasma Cholesterol (CHO), Triglyceride (TG), High density lipoprotein (HDL) and low density lipoprotein (LDL) did not significantly altered. Groups supplemented with EHS had higher IgG titres than control group. In conclusion, use of EHS into diet of broiler chicks could improve bird immune system and health.

**Keywords:** Extruded hempseed, Performance, Immune system, Broiler chick

### Introduction

Hempseed (Cannabis sativa L.) have been used as an important source of food, fiber and medicine for thousands of years (Pringle, 1997). Whole hempseeds contain approximately 25% proteins, 30% fat (Callaway, 2004) and high percentage of ash and cellulose (House et al, 2010). This ratio is considerably high when compared to some traditional feeds such as corn, wheat and soybean meal. High crude ash and cellulose levels in diets may cause lower BW due to decreasing of energy and digestibility of feeds. High levels of the fiber in diet has negative effects on nutrients digestibility and then bird performance (Krogdahl, 1986). A method for decreasing harmful effects of anti-nutritional factors is extruding process that improve nutritional value of feed (Allan et al., 2004). Hempseed oil contains over 80% polyunsaturated fatty acids (PUFA) and also it is a rich source of two essential fatty acids Linoleic acid (LA) and alpha-Linolenic acid (ALA) which is considered to be optimum for human health (Callaway, 2004). The oil of hempseed is comparable to black currant oil which improves immunity (Barre, 2001). The researchers reported that hempseed significantly reduced serum LDL, but had no significant effect on TG, CHO and HDL concentration (Konca et al., 2014b). The aim of this study was to evaluate the effect of extruded hempseed (EHS) on the performance, plasma lipid profile and immune response in broiler chicks.

### Material and Methods

A total of 250 one-day-old male broiler chick (Ross-308) were used in the study. All of the

birds were weighed and distributed into 5 treatment groups with 5 replicates and 10 birds per cage as replicate. The dietary treatments were: 1) Control diet (EHS0, diet contained no EHS); 2) diet contained %5 EHS (EHS5); 3) diet contained %10 EHS (EHS10); 4) diet contained %15 EHS (EHS15); 5) diet contained %20 EHS (EHS20). The diets were isocoloric and isonitrogenous. Performance parameters were measured in 10, 24 and 42 day of age. In 42 day of age, two birds were randomly selected from each cage and weighed individually and eviscerated. For determining serum lipid profile and (sheep red blood cell) SRBC test, 2 blood samples per selected bird were taken and were kept in ice and were sent to laboratory. SRBC test was carried out according to Nelson et al. (1995) method. Data were analyzed as a completely randomized design using General Linear Models procedure (PROCC GLM) of SAS (2002) and means were compared by Tukey test.

<footnote n="1">¹Professor and Assistant Professor respectively, Dept. of Animal Science, University of Birjand, Birjand, Iran</footnote>

<footnote n="2">²Ph.D. Student of Animal nutrition, Dept. of Animal Science, University of Birjand, Birjand, Iran</footnote>
## Results

The results of this study showed that inclusion of EHS into diet had no significant effect on performance parameters (Table 1).

<table><caption>**Table 1.** The effect of different levels of extruded hempseed on performance of broiler chicks</caption><thead><tr><th rowspan="2">Item</th><th colspan="7">Treatment</th></tr><tr><th>EH0</th><th>EHS5</th><th>EHS10</th><th>EHS15</th><th>EHS20</th><th><sup>1</sup>SEM</th><th>P-value</th></tr></thead><tbody><tr><td>BW (g/bird)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>10 old-day</td><td>172.98</td><td>172.48</td><td>179.84</td><td>188.52</td><td>177.86</td><td>5.03</td><td><sup>2</sup>NS</td></tr><tr><td>24 old day</td><td>771.71</td><td>767.62</td><td>763.01</td><td>752.20</td><td>740.24</td><td>22.65</td><td>NS</td></tr><tr><td>42 old-day</td><td>2379.92</td><td>2348.57</td><td>2248.94</td><td>2256.07</td><td>2304.53</td><td>60.56</td><td>NS</td></tr><tr><td>FI (g/bird/day)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>(1-10) day period</td><td>220.80</td><td>210.60</td><td>228</td><td>226.80</td><td>214.47</td><td>4.49</td><td>NS</td></tr><tr><td>(11-24) day period</td><td>1087.50</td><td>1128.48</td><td>1114.81</td><td>1141.60</td><td>1135.63</td><td>17</td><td>NS</td></tr><tr><td>(25-42) day period</td><td>2704.7</td><td>2683.1</td><td>2523.3</td><td>2487.4</td><td>2546.9</td><td>144.31</td><td>NS</td></tr><tr><td>(1-42) day period</td><td>4013</td><td>4020.2</td><td>3866.1</td><td>3855.8</td><td>3897</td><td>154.71</td><td>NS</td></tr><tr><td>FCR</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>(1-10) day period</td><td>1.28</td><td>1.27</td><td>1.22</td><td>1.20</td><td>1.21</td><td>0.02</td><td>NS</td></tr><tr><td>(11-24) day period</td><td>1.41</td><td>1.48</td><td>1.46</td><td>1.52</td><td>1.54</td><td>0.05</td><td>NS</td></tr><tr><td>(25-42) day period</td><td>1.84</td><td>1.81</td><td>1.86</td><td>1.88</td><td>1.77</td><td>0.1</td><td>NS</td></tr><tr><td>(1-42) day period</td><td>1.65</td><td>1.65</td><td>1.67</td><td>1.70</td><td>1.66</td><td>0.06</td><td>NS</td></tr></tbody></table>

EHS supplemented diets did not significantly affect the plasma Cholesterol (CHO), Triglyceride (TG), High density lipoprotein (HDL) and low density lipoprotein (LDL) concentration (Table 2).

<table><caption>**Table 2.** The effect of different levels of hempseed on plasma lipid profile in broiler chicks</caption><thead><tr><th rowspan="2">Parameter (mg/dl)</th><th colspan="5">Treatment</th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>EH0</th><th>EHS5</th><th>EHS10</th><th>EHS15</th><th>EHS20</th></tr></thead><tbody><tr><td>CHO</td><td>152.50</td><td>134.45</td><td>145.20</td><td>126.20</td><td>139.66</td><td>11.12</td><td>NS</td></tr><tr><td>HDL</td><td>51.10</td><td>63.35</td><td>68.40</td><td>60.65</td><td>66.25</td><td>5.90</td><td>NS</td></tr><tr><td>LDL</td><td>92.19</td><td>74.24</td><td>71.11</td><td>57.52</td><td>66.25</td><td>6.72</td><td>NS</td></tr><tr><td>TG</td><td>46.05</td><td>29.30</td><td>28.45</td><td>40.10</td><td>35.80</td><td>3.85</td><td>NS</td></tr></tbody></table>

The results showed that although the broilers fed with EHS supplemented diets hadn't significant difference in terms of humeral immune response, but they had higher IgG content compared to control group (EHS0) (Table3).

<footnote n="1"><sup>1</sup>SEM: Standard error of means</footnote>

<footnote n="2"><sup>2</sup>NS: Non-significance</footnote>
<table><caption>**Table 3.** The effect of extruded hempseed on anti body titre against SRBC in broiler chicks</caption><thead><tr><th rowspan="2">parameter</th><th colspan="5">Treatment</th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>EH0</th><th>EHS5</th><th>EHS10</th><th>EHS15</th><th>EHS20</th></tr></thead><tbody><tr><td>SRBC</td><td>8.66</td><td>9.33</td><td>9.00</td><td>9.33</td><td>8.00</td><td>0.63</td><td>NS</td></tr><tr><td>IgG</td><td>2.00</td><td>2.66</td><td>4.00</td><td>3.66</td><td>2.33</td><td>0.69</td><td>NS</td></tr><tr><td>IgM</td><td>6.66</td><td>6.66</td><td>5.00</td><td>5.66</td><td>5.67</td><td>0.01</td><td>NS</td></tr></tbody></table>

**Conclusions**

The obtained results of the study showed that adding extruded hempseed into broiler chicks diet hadn't significance effects on performance, serum lipid profile and immune response, but overall there is good potential in EHS to improve bird health and performance.

**Acknowledgements**

The authors express their gratitude to Hussein Razeghi, administrator of Razeghi commercial Co., for his cooperative in the study.

**Literature**

ALLAN, G.L., M.A. BOOTH, 2004. Effects of extrusion processing on digestibility of peas, lupins, canola meal and soybean meal in silver perch Bidyanus bidyanus (Mitchell) diets. Aquaculture Research, 35,981-991.

BARRE, D.E., 2001. Potential of evening primrose, borage, black currant and fungal oils in human health. Anal.Nutr. Metabolism, 45, 47-57.

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P-131 (ID 54)

CRUDE FIBRE IN LAYER FEED INFLUENCES PERFORMANCE AND PLUMAGE OF LSL HYBRID

Danielle Albiker*<fnref n="1" />, Rebecca Bieler<fnref n="2" /> und Ruedi Zweifel<fnref n="1" />

**Corresponding author:** danielle.albiker@aviforum.ch

**Abstract**

The influence of a high crude fibre content (7% CF) in a layer feed was compared to a control feed (3.5% CF) under Swiss growing conditions on the research farm of Aviforum Foundation regarding the production performance and plumage quality of 2860 non-beak-trimmed Lohmann Selected Leghorn hybrids. The 17 week old hens were evenly distributed to 8 compartments and randomly allocated to the two diets described above, allowing 4 compartment replications. Both groups showed an equally high laying performance of 94% at the age of 64 weeks. Hens fed the high CF (HCF) diet ate 2.6g per day and animal more and their FCR (g feed/g egg) was 0.03 units higher during the first 6 laying periods (LP) than compared to the control group (LCF), and they ate less during the last 5 LPs, thus lowering their FCR towards that of the control group. High dietary CF led to lighter birds (1804g for HCF; 1863g for LCF) which produced heavier eggs with less strong shells (HCF 64g, 41.7N; LCF 63.7g, 43.7N). The plumage covered the hens well and no significant difference was found in feather scoring. Hens eating the high CF diet suffered significantly less from cannibalism (HCF 0.4%; LCF 1.2%) which indicates a positive influence of high CF against pecking. This was also reflected by a slightly better feather score of the neck, back and cloaca area of hens fed a higher CF diet (HCF 3.05, 3.98, 2.45; LCF 2.90, 3.85, 2.13). In conclusion, a CF content of 7% in a layer diet had a positive influence on the welfare of non-beak-trimmed hens lowering cannibalism without jeopardizing laying performance.

**Keywords:** layers, crude fibre, plumage, performance

**Introduction**

An intact plumage is important for the hen to regulate its body temperature without losing much energy. A badly covered hen will eat more and is thus less economical. Fibres in the feed of layer hens have an influence on the quality of the plumage (Van Krimpen et al., 2005). A high fibre and a standard diet were tested under Swiss growing conditions on the research farm of Aviforum Foundation focussing on plumage quality and production performance.

**Material and Methods**

2860 LSL hybrids were evenly distributed into 8 compartments. These were randomly allocated to a diet containing 7% crude fibre (HCF) or a standard diet with 3.5% fibre (LCF), allowing 4 compartment replications with 1430 hens per diet. Both diets were isoenergetic and isonitrogenic. All animals were kept under the same conditions and management in an aviary system using wood shavings as litter material. The hens had daily access to a winter garden. They received a prelayer and two layer diets produced by Provimi Kliba SA, Kaiseraugst, until the age of 64 weeks. Laying performance, feed and water consumption as well as mortality were measured on a daily basis at the farm. Egg weights of all laid eggs were evaluated on one day of each laying period (LP). Egg shell strength and thickness as well as the yolk colour were measured during the peak performance, after 6 LPs and at the age of 64 weeks. Hen weight and plumage was evaluated regularly once per LP, scoring 10 hens per compartment on the neck, breast, stomach, back, wings and tail areas based on a scale of 4 (feathers intact) to 1 (no feathers) described by Tauson et al. (2005). To monitor excreta for abnormalities, the humidity of faeces was evaluated visually in the barn every 2 weeks and dry matter content was measured in the lab towards the end of the trial. Data were analyzed by ANOVA and means compared with the test of Tukey-Kramer.

**Results**

Laying performance (94% at the age of 64 weeks) and number of floor eggs (laid outside the nests) were not affected by treatments. HCF led to a higher feed consumption during the first 6 LPs. Neither the feed conversion ratio (FCR) nor the humidity of the faeces showed any significant differences between treatments (Table 1).

<footnote n="1">¹Aviforum Foundation, Burgerweg 22, 3052 Zollikofen, Switzerland</footnote>

<footnote n="2">²ETH Zurich, Institute of Agricultural Sciences, Universitätstr. 2, 8092 Zurich, Switzerland</footnote>
<table><caption>**Table 1.** Laying performance, floor eggs, feed consumption, FCR and humidity of faeces</caption><thead><tr><th></th><th>LCF<br>LP 1-6</th><th>HCF<br>LP 1-6</th><th>P-Level<br>LP 1-6</th><th>LCF<br>LP 1-11</th><th>HCF<br>LP 1-11</th><th>P-Level<br>LP 1-11</th></tr></thead><tbody><tr><td>Laying performance, %</td><td>94.0</td><td>93.8</td><td>0.798</td><td>94.0</td><td>94.0</td><td>0.908</td></tr><tr><td>Floor eggs, %</td><td>0.7</td><td>1.0</td><td>0.247</td><td>0.6</td><td>0.9</td><td>0.258</td></tr><tr><td>Feed per day and hen, g</td><td>118.7</td><td>121.3</td><td>0.076</td><td>122.6</td><td>123.7</td><td>0.449</td></tr><tr><td>FCR, kg feed/kg egg mass</td><td>2.056</td><td>2.082</td><td>0.324</td><td>2.047</td><td>2.056</td><td>0.768</td></tr><tr><td>Humidity of faeces,<br>1=dry to 3=very humid</td><td>1.383</td><td>1.520</td><td>0.431</td><td>1.459</td><td>1.534</td><td>0.692</td></tr></tbody></table>

HCF led to heavier eggs than the standard diet (61.4g LCF, 62.1g HCF over 6 LPs with P-Level 0.007 and 63.7g LCF, 64.0g HCF over 11 LPs with P-Level 0.058). During the whole trial, significantly more big sized eggs were laid with HCF compared to LCF. No significant differences could be shown between the treatments regarding egg shell quality. Due to the heavier eggs, egg shell strength was slightly lower with HCF at the age of 64 weeks. Yolk colour was different after 6 LPs only (Table 2). Hen weights showed no significant differences between treatments (1863g LCF and 1804g HCF).

<table><caption>**Table 2.** Egg weight and egg weight distribution</caption><thead><tr><th></th><th>LCF<br>LP 6</th><th>HCF<br>LP 6</th><th>P-Level<br>LP 6</th><th>LCF<br>LP 11</th><th>HCF<br>LP 11</th><th>P-Level<br>LP 11</th></tr></thead><tbody><tr><td>Egg weight. g</td><td>64.2</td><td>64.8</td><td>0.203</td><td>67.2</td><td>67.5</td><td>0.192</td></tr><tr><td>Small eggs <53g, %</td><td>0.3</td><td>0.2</td><td>0.875</td><td>0.3</td><td>0.0</td><td>0.159</td></tr><tr><td>Medium sized eggs 53-70g, %</td><td>89.5</td><td>84.7</td><td>0.006</td><td>80.1</td><td>76.7</td><td>0.038</td></tr><tr><td>Big sized eggs >70g, %</td><td>10.1</td><td>15.1</td><td>0.001</td><td>19.6</td><td>23.3</td><td>0.029</td></tr><tr><td>Egg shell strength, N</td><td>49.6</td><td>50.8</td><td>0.552</td><td>43.7</td><td>41.7</td><td>0.318</td></tr><tr><td>Egg shell thickness, µm</td><td>408</td><td>405</td><td>0.524</td><td>387</td><td>392</td><td>0.370</td></tr><tr><td>Yolk colour</td><td>12.1</td><td>11.9</td><td>0.013</td><td>12.2</td><td>12.0</td><td>0.168</td></tr></tbody></table>

Mortality reached an average of 5,5% (HCF) and 7,8% (LCF) after 11 LPs. Incidence of cannibalism in the control group (1.2%) was three times higher than with HCF (0.4%) and thus significantly different between treatments (P=0.043). The plumage covered the hens well and no differences could be found in overall feather scoring (3.68 LCF and 3.69 HCF). Kalmendal and Wall (2012) confirm this finding using sun flower cake containing soluble fibres in the feed. In this trial feeding lignocellulose (unsoluble fibres), the neck, back and cloaca areas were in a better state with HCF than with LCF (Figure 1).
<figure><img src="image_55.png" /><figcaption>**Figure 1.** Feather scoring of neck, back and cloaca area in LP 11</figcaption></figure>

## Conclusions

HCF in the diet with oat, lucerne meal, sun flower cake and 2.5% lignocellulose as components reduced cannibalism, indicating a positive influence of crude fibre in the diet preventing feather pecking, without diminishing laying performance. The lignocellulose helps to stabilize the water content of the faeces as it contains unsoluble fibres and has a high water holding capacity, leading to dry faeces and a good litter quality (Neufeld et al., 2014). Egg mass and percentage of big sized eggs were increased with HCF, favouring their commercialisation from the farm directly to the consumer. As Switzerland will abandon the trimming of the beak in the future, high crude fibre content in the feed should be tested on brown non-beak-trimmed hybrids, which are more likely to peck and suffer from cannibalism than LSL.

## Literature

KALMENDAL, R., WALL, H. (2012). Effects of a high oil and fibre diet and supplementary roughage on performance, injurious pecking and foraging activities in two layer hybrids. Br. Poult. Sci Vol. **53**(2), 153-161. Doi: 10.1080/0007 1668.2012.682146

NEUFELD, N., KROISMAYR, A. and NEUFELD, K. (2014). Neue Wege in der Rohfaserversorgung. Access: http://www.proteinmarkt.de/uploads/media/wpm49d5.pdf

TAUSON, R., KAJER, J., MARIA, G.A., CEPERO, R., and HOLM, K.E. (2005). Applied scoring of integument and health in laying hens. LAYWEL EU-project 502315, Access (28.05.13): http://www.laywel.eu/

VAN KRIMPEN, M.M., KWAKKEL, R.P., REUVEKAMP, B.F.J., VAN DER PEET-SCHWERING, C.M.C., DEN HAR- TOG, L.A., VERSTEGEN, M.W.A. (2005). Impact of feeding management on feather pecking on laying hens. World's Poult. Sci. Vol. **61**, 663-685
P-132 (ID 154)

EFFECT OF TWO SOURCES OF ZINC ON BROILER PERFORMANCE

C. Alleno<fnref n="1" />, A. Romeo<fnref n="2" />, and S. Durosoy<fnref n="2" />

Corresponding author: aromeo@animine.eu

Abstract

Various zinc sources are supplemented in poultry feeds, in order to fulfill animal requirements; zinc sulfate (ZnSO₄) and zinc oxide (ZnO) are among the most common. The aim of the study was to compare the effects of zinc from a new source of potentiated ZnO or from ZnSO₄ on broiler performance.

738 male broilers were divided into cages placed in a Colorado type house; 26 500 birds were raised in this building. The basal diet was based on wheat, corn and soybean meal. Experimental diets were supplemented with 60 ppm of zinc from ZnSO₄ or 60 ppm of zinc from potentiated ZnO (HiZox®). Zootechnical criteria (bodyweight, feed intake) were recorded at feed transition (12 days, 21 days, 28 days) and at the end of the trial (35 days). Litter score was evaluated at 28 days.

Broilers fed with potentiated ZnO showed a better start, with a significantly higher bodyweight at 12 days: +2.2% (p < 0.05). This difference could still be seen at 35 days (2251 g vs. 2231 g) but was not significant. Consumptions were equal between the 2 groups during the entire trial; the feed conversion ratio of the group fed with potentiated ZnO was better for the 0-12-days period after bodyweight standardization (p < 0.05). Litter quality was improved at 28 days for the group fed with potentiated ZnO, the difference between the 2 regimes is close to be significant (p = 0.08).

In conclusion, the new source of potentiated ZnO showed a positive impact on broiler growth performance and on litter quality, compared with ZnSO₄.

**Keywords:** zinc, broiler, performance

Introduction

Zinc (Zn) is an essential trace mineral that exists naturally in all plants, but its concentration is generally insufficient to meet animal requirements. In poultry, Zn deficiency leads to leg abnormalities, frizzled feathers and impaired growth performance. Consequently, Zn is usually supplemented in broilers diets, commonly in the form of zinc sulfate or the form of zinc oxide. Zn sources have different physicochemical characteristics and different effects on animal performance; in this study, zinc sulfate and a new source of potentiated ZnO (HiZox®) were compared.

Material and Methods

The trial took place in a Colorado type house, with dynamic transverse ventilation: 738 male broilers (Ross PM3) were divided into cages and raised among 26 500 birds. For each treatment, 9 cages of 41 broilers were used, with a density of 17 chicks/m<fnref n="2" />. Animals were given *ad libitum* access to water and feed; there were one nipple line and one feeder per pen. Wheat straw was chosen for litter.

Basal diets, based on wheat, corn and soybean meal, were supplemented with 60 ppm of zinc from ZnSO₄ or 60 ppm of zinc from potentiated ZnO. Analysed values for total zinc in the two feeding programs were around 90 ppm.

Zootechnical criteria (bodyweight, feed intake) were recorded at feed transition (12 days, 21 days, 28 days) and at the end of the trial (35 days). Experimental unit was the pen of 41 birds. Litter score was evaluated at 28 days (feeder area, water system area and rest area), according to the Table 1.

<footnote n="1">¹ Zootests SAS, Ploufragan, France</footnote>

<footnote n="2">² Animine, Sillingy, France</footnote>
<table><caption>**Table 1. Litter score**</caption><thead><tr><td>Score</td><td>Description</td></tr></thead><tbody><tr><td>1</td><td>Dry and crumbly</td></tr><tr><td>2</td><td>Crumbly but lightly wet</td></tr><tr><td>3</td><td>Crumbly but capping in some areas</td></tr><tr><td>4</td><td>Capping but we can find crumbly litter below</td></tr><tr><td>5</td><td>100% capping or completely wet</td></tr></tbody></table>

**Results**

The purpose of the test was to evaluate efficiency of two different feed programs on broiler performance. Experiment was conducted with two groups of 369 male broilers that received different diets:

Control: basal diet + 60 ppm of Zn from zinc sulfate

Treatment: basal diet + 60 ppm of Zn from a source of potentiated ZnO.

In this trial, average cumulative mortality and culling rate was 4.2 %. There were no statistically differences between treatments on mortality or culling rate.

At day 12, after the starter period, birds fed with potentiated ZnO showed statistically higher bodyweight than the control group: 416 g vs. 407 g (p = 0.037) [Fig 1]. At any other age, there was no statistically significant difference between groups. However, at day 35, the broilers of the treatment group had a numerically higher bodyweight (2251 g vs 2231 g).

<figure><img src="image_56.png" /><figcaption>**Figure 1. Average daily gain (0-12 days)**</figcaption></figure>

Feed consumptions were equal with both sources of zinc, for any period.

There were no statistically significant feed conversion ratio (FCR) differences according to group for any period. However, using Aviagen method to standardize bodyweight, we obtained a significant difference between groups for 0-12-d-old period: 1.05 for ZnSO4 > 1.03 for ZnO [Fig 2].
<figure><img src="image_57.png" /><figcaption>**Figure 2. Feed conversion ratio (0-12 days)**</figcaption></figure>

In addition, the source of potentiated ZnO seemed to show a positive impact on litter quality. Thus, p-value was closed to be significant (p = 0.081), when the number of observation is limited (only 9 observations per treatment) [Fig 3].

<figure><img src="image_58.png" /><figcaption>**Figure 3. Litter score**</figcaption></figure>

**Conclusions**

The potentiated ZnO (HiZox®) improved broiler performance (BW, FCR) during the starter period and showed a positive effect on litter quality at 28 days.
P-134 (ID 175)

EFFECT OF DIETARY SUPPLEMENTATION WITH FERMENTED RICE BRAN ON GROWTH PERFORMANCE, BLOOD PARAMETERS, AND INTESTINAL MICROFLORA IN BROILER CHICKENS

Han-Tae Bang, Hwan-Ku Kang, Ji-Hyuk Kim, Jong Hwangbo and Chan-Ho Kim

Poultry Science Division, Livestock Resource Development, National Institute of Animal Science, RDA, Senonghwan
330-801, Korea

Corresponding author: banght80@korea.kr

Abstract

The objective of this study was to investigate the effect of dietary supplementation with microbially-fermented rice bran on growth performance in broiler chickens. A total of one hundred and twenty 1-day-old broiler chickens, were randomly assigned to 5 dietary treatments in cages of 4 chicks each; each treatment had 6 replicate cages. A basal diet, formulated to be adequate in energy and nutrients, as well as four experimental diets, prepared by adding one of four ingredients 0.025% avilamycin, 1% Lactobacillus fermented rice bran, 1% Saccharomyces fermented rice bran, and 1% Bacillus fermented rice bran to the basal diet, were given to the birds over a 35-d period. The results indicated that BW gain during the first 21 d (starter period), growing periods (22 to 35d), and over the entire experimental period (0 to 35 d) was (P<0.01) different among treatments. Feed intake and feed conversion ratio varied significantly (P<0.05) across treatments in the starter period but not the growth period, or the overall experiment. The dietary supplements had no effect on any serum parameters except for total cholesterol, which was significantly lower for the supplements than the basal diet. Finally, intestinal flora Lactobacillus, Escherichia coli, and Salmonella did not vary across treatments. Our results suggest that the supplementation of poultry diet with fermented rice bran may be used to improve growth performance in broiler chickens.

**Keywords:** rice bran, fermentation, Bacillus, Saccharomyces, growth performance

Introduction

Antibiotics have been used as feed additives to improve growth performance and to control disease in animals. However, antibiotic use tends to produce antibiotic resistance, and leave residues in animal products. Since 2006, antibiotics have been banned from use as feed additives in the European Union. More recently, in July 2011, a full ban on the use of antibiotic growth promoters (AGPs) in animal feed came into effect in Korea in order to ensure the safety of livestock products for consumers. Therefore, it is necessary to find effective alternatives to antibiotic feed ingredients, such as prophylactic antibacterial substances and growth promoters. Rice bran, a by-product of rice milling, has high levels of important nutrients such as proteins, vitamins, minerals, complex carbohydrates, phytonutrients, phospholipids, and essential fatty acids (Ryan et al., 2011). However, the nutritional content of rice bran may vary widely because of differences in hull content, degree of polishing during milling, and differences in the variety of rice. It is abundantly available around the world, particularly in Asia, and Korea alone produces about 40 million tons of rice bran each year; this comprises 10% of the total weight of harvested paddy. Therefore, it a common feed supplement in animal production, including in poultry (Mu et al., 2011). Previous research on the supplementation of broiler chicken feed with rice bran has shown that it improves growth performance, however, studies on the usefulness of fermented rice bran for improving growth are scarce. The fermentation of materials with a microbial inoculum has been widely adopted in the development of novel functional ingredients because it may promote anti-inflammatory activity (Kim et al., 2012). However, response of the growth performance, blood parameters, and intestinal microflora of animals to feeds with fermented rice bran is yet to be described. The objective of this study was to determine whether the dietary supplementation of broiler chicken feed with microbially fermented rice bran affects growth performance, blood parameters, and intestinal microflora.

Material and Methods

A total of one hundred and twenty 1-day-old male chickens (initial BW 36 ± 0.74 g) of the commercial broiler strain ROSS × ROSS 308, were raised for an experimental period of 35 d. All chicks were housed in a battery cage (86 cm × 57 cm × 35 cm, width×length×height) in an environmentally controlled room maintained at 30°C during the first week of the experiment and then gradually decreased to 24°C at the end of the experiment as recommended by the Ross manual; the room was lit throughout the experiment. Based on a completely randomized design, 6 replicate cages, each
containing 4 chicks, were assigned to 1 of 5 experimental treatments: (1) a basal diet formulated to meet or exceed the energy and nutrient requirements of broiler chickens (NRC, 1994), (2) basal diet plus 0.025% Avilamycin (AGPS), (3) basal diet plus 1% Lactobacillus-fermented rice bran (LFR), (4) basal diet plus 1% Saccharomyces-fermented rice bran (SFR), and (5) basal diet plus 1% Bacillus-fermented rice bran (BFR). A 2-phase feeding program with a starter diet from 0 to 21 d and a grower diet from 22 to 35 d was followed. Feed and water were provided ad libitum for 35 d. Body weight gain (g; BWG) and feed intake (g; FI) were measured weekly. A feed conversion ratio (FCR) was calculated as the ratio of BWG to FI; BWG, FI, and FCR were corrected for dead birds. All data were analyzed by ANOVA as a completely randomized design using the PROC MIXED procedure.

## Results

Body weight gains during the starter period (0 to 21 d), growing period (22 to 35 d), and over the entire 35-day experimental period (0 to 35 d) were significantly higher for the experimental diets when compared to the basal diet (P<0.01). An orthogonal contrast revealed that birds fed with the antibiotic diet (AGPs) and fermented rice bran treatments (LFR, SFR, and BFR) displayed enhanced body weight gains of 14.1%, 9.5%, 8.9%, and 8.5% respectively, compared to the basal diet. Initial feed intake (during the 0 to 21-day starter period) was also significantly different across treatments (P<0.05) with SFR and BFR diets resulting in higher feed intake than the basal diet, AGPS, or LFR. However, feed intakes during the growth period and across the 35-day experimental period were similar across treatments. Similarly, initial feed conversion ratio (during the starter period), varied significantly across treatments (P<0.01) with higher initial FCR in birds that were fed SFR and BFR than in birds fed the basal diet, AGPS, and LFR; FCR did not vary across treatments during the growth period, or overall.

The positive effects of unfermented rice bran on the growth performance of broiler chicken may result from its high concentrations of protein, vitamin, minerals, complex carbohydrates, phytonutrients, phospholipids, essential fatty acids, and more than 120 antioxidants (Ryan et al., 2011). However, data explaining the positive effects of fermented rice bran products on growth performance in birds is unavailable, making it difficult to compare the present results with those of other studies. It is likely that the fermentation of plant materials increases the efficacy of their antioxidant and anti-inflammatory properties, when compared to those of unfermented materials (Cao et al., 2012; Kim et al., 2012). Therefore, the improved BWG and FCR reported here may be associated with increased antioxidants in microbially fermented rice-bran (MFR). This “increased antioxidants hypothesis” may also explain why supplementation of diets with relatively small amounts (1%) of MFR resulted in large positive effects on the growth performance of birds in this experiment.

<table><caption>**Table 1.** Growth performance of broiler chickens fed the diet containing fermented rice bran<fnref n="1" /></caption><thead><tr><th rowspan="2">Items</th><th colspan="5">Dietary treatments<sup>2</sup></th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>Basal</th><th>AGPs</th><th>LFR</th><th>SFR</th><th>BFR</th></tr></thead><tbody><tr><td>BW gain, g/bird</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0 to 21 d</td><td>686.1<sup>b</sup></td><td>763.7<sup>ab</sup></td><td>773.4<sup>a</sup></td><td>750.4<sup>ab</sup></td><td>736.4<sup>ab</sup></td><td>25.24</td><td>&lt;0.01</td></tr><tr><td>22 to 35 d</td><td>858.1<sup>c</sup></td><td>958.3<sup>a</sup></td><td>950.9<sup>ab</sup></td><td>945.8<sup>ab</sup></td><td>913.5<sup>b</sup></td><td>14.13</td><td>&lt;0.01</td></tr><tr><td>0 to 35 d</td><td>1,544.3<sup>b</sup></td><td>1,722.0<sup>a</sup></td><td>1,724.2<sup>a</sup></td><td>1,696.3<sup>a</sup></td><td>1,649.9<sup>a</sup></td><td>30.67</td><td>&lt;0.01</td></tr><tr><td>Feed intake, g/bird</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0 to 21 d</td><td>975.0<sup>b</sup></td><td>1,000.0<sup>b</sup></td><td>980.6<sup>b</sup></td><td>1,025.0<sup>ab</sup></td><td>1,150.0<sup>a</sup></td><td>44.37</td><td>0.02</td></tr><tr><td>22 to 35 d</td><td>1,686.1</td><td>1,713.4</td><td>1,645.6</td><td>1,671.1</td><td>1,606.5</td><td>164.94</td><td>0.99</td></tr><tr><td>0 to 35 d</td><td>2,661.1</td><td>2,713.4</td><td>2,626.2</td><td>2,695.1</td><td>2,756.5</td><td>170.31</td><td>0.93</td></tr><tr><td>FCR, feed/gain</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0 to 21 d</td><td>1.42<sup>ab</sup></td><td>1.31<sup>b</sup></td><td>1.28<sup>b</sup></td><td>1.36<sup>b</sup></td><td>1.56<sup>a</sup></td><td>0.05</td><td>&lt;0.01</td></tr><tr><td>22 to 35 d</td><td>1.97</td><td>1.79</td><td>1.74</td><td>1.77</td><td>1.75</td><td>0.18</td><td>0.88</td></tr><tr><td>0 to 35 d</td><td>1.72</td><td>1.58</td><td>1.53</td><td>1.59</td><td>1.67</td><td>0.10</td><td>0.70</td></tr></tbody></table>

<footnote>a-cValues with different superscripts in the same row are significantly different (P<0.05).</footnote>

<footnote n="1">¹Data are least squares means of 6 observations per treatment.</footnote>

<footnote n="2">²Basal = basal diet; AGPs = basal diet + 0.025% Avilamycin; LFR = basal diet + 0.1% Lactobacillus-fermented rice bran; SFR = basal diet + Saccharomyces-fermented rice bran; BFR = basal diet + Bacillus-fermented rice bran.</footnote>
The dietary supplements had no effect on any serum parameters except for total cholesterol, which was significantly lower for the supplements than the basal diet. Finally, intestinal flora Lactobacillus, Escherichia coli, and Salmonella did not vary across treatments.

## Conclusions

Our results clearly show that the supplementation of the diets of broiler chickens with fermented rice bran increases body weight gain, and reduces total cholesterol. Consequently, dietary fermented rice bran may be a valuable functional ingredient in improving overall growth performance in broiler chickens, and important in poultry.

## Literature

Cao, F. L., X. H. Zhang, W. W. Yu, L. G. Zhao, and T. Wang. 2012. Effect of feeding fermented Ginko biloba leaves on growth performance, meat quality, and lipid metabolism in broilers. Poultry Sci. 91:1210-1221.

Kim, C. H., G.-B. Kim, M. B. Chang, G. S. Bae, I. K. Paik, and D. Y. Kil. 2012. Effect of dietary supplementation of Lactobacillus-fermented Artemisia princeps on growth performance, meat lipid peroxidation, and intestinal microflora in Hy-Line Brown male chickens. Poultry Sci. 91:2845-2851.

Mu, K. S., A. B. Kasim, A. Ideris, and C. R. Saad. 2011. Effect of fermented rice bran, bio-converted byproduct on performance of broiler chickens. J. Anim. & Vet. Adv. 10:2990-2995.

Ryan, E. P., A. L. Heuberger, T. L. Weir, B. Barnett, C. D. Broeckling, and J. E. Prenni. 2011. Rice bran fermented with Saccharomyces boulardii generates novel metabolite profiles with bioactivity. J. Agric. Food Chem. 59:1862-1870.
P-135 (ID 111)

# THE EFFECT OF HEMPSEED (CANNABIS SATIVA L.) ON PERFORMANCE, SOME BLOOD BIOCHEMICAL PARAMETERS AND IMMUNE RESPONSE IN BROILER CHICKENS

M. Barani<fnref n="1" />, N. Afzali<fnref n="2" />, and S.J. Hosseini Vashan<fnref n="2" />

**Corresponding author:** barani1360@birjand.ac.ir

## Abstract

This study was conducted to investigate the effects of hempseed (HS) on performance, blood serum lipid profile and immune response in broiler chickens in during three growing periods (1-10; 11-24 and 25-42 day of age). A total of 250 one-day old male broiler chicks (Ross 308) were randomly distributed within 5 treatments with 5 replicates and 10 birds in each. The treatments were as follows: 1) Control diet (C, no HS in the diet); 2) 5% HS in the diet (HS5); 3) 10% HS in the diet (HS10); 4) 15% HS in the diet (HS15); 5) 20% HS in the diet (HS20). Data were analyzed as a completely randomized design using of the PROC GLM procedure of SAS. Overally, The results indicated that inclusion of HS over 10% to diet significantly reduced feed intake (FI), body weight (BW)(P<0.05) but had no significant effect on feed conversion ratio (FCR). The serum cholesterol (CHO), triglyceride (TG) and high density lipoprotein (HDL) were not significantly altered; but low density lipoprotein (LDL) concentration HS supplemented diets were lower than that C diet. HS supplemented groups had higher IgG titres than C group. In conclusion, HS could be a potential feed in reducing plasma LDL and maitaing health of broiler chicks.

**Keywords:** Hempseed, Broiler, Performance, Blood, Immune system

## Introduction

Hempseed (Cannabis sativa L.) is an annual, normally dioecious plant (Fortenbery and Bennet, 2004) known to have played a historically important role in food, fibre and medicine production (Callaway, 2004). It has been shown that whole hempseed contain over 30% oil, 25% protein and considerable amounts of fiber, vitamins and minerals. Hemp-seed oil contains over 80% polyunsaturated fatty acids (PUFA) and also it is a rich source of two essential fatty acids Linoleic acid (LA) and alpha-Linolenic acid (ALA) which is considered to be optimum for human health (Callaway, 2004). Consumers prefer foods rich in essential fatty acids, e.g. LA and ALA, due to their benefits for health. Linoleic acid and ALA are essential fatty acids for the human health and these should be taken with food (Goldberg et al., 2012). Some studies have shown that different levels of PUFA in the experimental diets of broiler had no effects on performance (Friedman and Sklan, 1995; Al-Khalifa et al., 2012; Konca et al., 2014a). The oil of hempseed is comparable to black currant oil which improves immunity (Barre, 2001). Konca et al. (2014b) reported that triglyceride, cholesterol and high density lipoprotein (HDL) were not significantly altered, however, low density lipoprotein (LDL) concentration in hempseed supplemented groups were lower than that of the control group. Also, hempseed may reduce LDL cholesterol and high blood pressure (Callaway, 2005). The objective of the present study was investigation of hempseed effects on performance, blood serum lipid profile and immune response in broiler chickens.

## Material and Methods

A total of 250 one-day-old male broiler chicks (Ross-308) were used in the study. All of the birds were weighed and distributed into 5 treatment groups with 5 replicates and 10 birds per cage as replicate. The dietary treatments were: 1) Control diet (C, no HS in the diet); 2) 5% HS in the diet (HS5); 3) 10% HS in the diet (HS10); 4) 15% HS in the diet (HS15); 5) 20% HS in the diet (HS20). The diets were isocoloric and isonitrogenous. Performance parameters were measured in 10, 24 and 42 day of age. In 42 day of age, two birds were randomly selected from each cage and weighed individually and eviscerated. For determinating serum lipid profile and (sheep red blood cell) SRBC test, 2 blood samples per selected bird were taken and were kept in ice and were sent to laboratory. SRBC test was carried out according to Nelson et al. (1995) method. Data were analyzed as a completely randomized design using General Linear Models procedure (PROCC GLM) of SAS (2002) and means were compared by tukey test.

<footnote n="1">¹ Ph.D. Student of Animal nutrition, Dept. Of Animal Science, University of Birjand, Birjand, Iran</footnote>

<footnote n="2">² Professor and Assistant Professor respectively, Dept. of Animal Science, University of Birjand, Birjand, Iran</footnote>
## Results

The results indicated that inclusion of HS up to 10% into diet significantly decreased feed intake (FI), body weight (BW)(P<0.05) but had no significant effect on feed conversion ratio(FCR) (Table1).

<table><caption>Table1. The effect of different levels of hempseed on performance of broiler chicks</caption><thead><tr><th rowspan="2">Item</th><th colspan="6">Treatment</th><th rowspan="2">P-value</th></tr><tr><th>C</th><th>HS5</th><th>HS10</th><th>HS15</th><th>HS20</th><th><sup>1</sup>SEM</th></tr></thead><tbody><tr><td>BW (g/bird)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>10 old-day</td><td>190.35</td><td>186.22</td><td>179.68</td><td>181.64</td><td>181.07</td><td>2.94</td><td><sup>2</sup>NS</td></tr><tr><td>24 old day</td><td>791.51a</td><td>782.89ab</td><td>698.50bc</td><td>659.30c</td><td>665.91c</td><td>20.07</td><td>*</td></tr><tr><td>42 old-day</td><td>2554.90a</td><td>2580.10a</td><td>2409.33ab</td><td>2247.04b</td><td>2282.08b</td><td>46.77</td><td>*</td></tr><tr><td>FI (g/bird/day)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>(1-10) day period</td><td>23.68a</td><td>23.22ab</td><td>22.90ab</td><td>22.44b</td><td>22.31b</td><td>0.25</td><td>*</td></tr><tr><td>(11-24) day period</td><td>82.54a</td><td>84.45a</td><td>80.83ab</td><td>75.21b</td><td>74.80b</td><td>1.50</td><td>*</td></tr><tr><td>(25-42) day period</td><td>172.83a</td><td>169.45a</td><td>158.42ab</td><td>147.98b</td><td>149.98b</td><td>4.59</td><td>*</td></tr><tr><td>(1-42) day period</td><td>103.27a</td><td>104.67a</td><td>100.02ab</td><td>92.67b</td><td>93.42b</td><td>103.27a</td><td>*</td></tr><tr><td>FCR</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>(1-10) day period</td><td>1.24</td><td>1.25</td><td>1.28</td><td>1.23</td><td>1.23</td><td>0.02</td><td>NS</td></tr><tr><td>(11-24) day period</td><td>1.46</td><td>1.52</td><td>1.63</td><td>1.60</td><td>1.57</td><td>0.05</td><td>NS</td></tr><tr><td>(25-42) day period</td><td>1.89</td><td>1.86</td><td>1.85</td><td>1.87</td><td>1.86</td><td>0.04</td><td>NS</td></tr><tr><td>(1-42) day period</td><td>1.70</td><td>1.71</td><td>1.74</td><td>1.73</td><td>1.72</td><td>0.03</td><td>NS</td></tr></tbody></table>

The results of the study showed that there were no statistical differences among the treatment groups in terms of plasma TG, CHO, LDL and HDL., but LDL concentration in HS supplemented diets significantly were lower than C diet (P<0.05) (Table2).

<table><caption>Table2. The effect of different levels of hempseed on plasma lipid profile in broiler chicks</caption><thead><tr><th rowspan="2">Parameter (mg/dl)</th><th colspan="5">Treatment</th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>C</th><th>HS5</th><th>HS10</th><th>HS15</th><th>HS20</th></tr></thead><tbody><tr><td>CHO</td><td>165.59</td><td>144.40</td><td>148.40</td><td>143.63</td><td>142.95</td><td>10.49</td><td>NS</td></tr><tr><td>HDL</td><td>72.45</td><td>79.50</td><td>82.40</td><td>80.10</td><td>74.90</td><td>6.62</td><td>NS</td></tr><tr><td>LDL</td><td>83.58a</td><td>57.69b</td><td>55.69b</td><td>56.77b</td><td>57.50b</td><td>4.55</td><td>*</td></tr><tr><td>TG</td><td>47.80</td><td>36.05</td><td>51.55</td><td>33.80</td><td>52.75</td><td>4.22</td><td>NS</td></tr></tbody></table>

The effect of feeding hempseed on immune response is shown in Table3. There were no significant difference among treatments, but HS supplemented groups had higher IgG titres than C group. The table 3 shows the results.

<footnote n="1">¹SEM: Standard error of means</footnote>

<footnote n="2">²NS: Non-significance</footnote>

<footnote>a-c: Values with different superscript in a row differ significantly *p<0.05</footnote>

<footnote>a-b: Values with different superscript in a row differ significantly *p<0.05</footnote>
<table><thead><tr><th rowspan="2">parameter</th><th rowspan="2">C</th><th colspan="4">treatment</th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>HS5</th><th>HS10</th><th>HS15</th><th>HS20</th></tr></thead><tbody><tr><td>SRBC</td><td>8.67</td><td>9.33</td><td>9.00</td><td>9.33</td><td>8.00</td><td>0.63</td><td>NS</td></tr><tr><td>IgM</td><td>6.67</td><td>6.67</td><td>5.00</td><td>5.67</td><td>5.67</td><td>0.69</td><td>NS</td></tr><tr><td>IgG</td><td>2.00</td><td>2.67</td><td>4.00</td><td>3.67</td><td>2.33</td><td>0.77</td><td>NS</td></tr></tbody></table>

## Conclusions

The results of the study indicated that using hempseed into broiler chicks diet over 10% significantly reduced FI and BW, but it hadn't significant effect on FCR. Also the study showed that adding hempseed into diet significantly reduced LDL concentration in compared to C treatment. In conclusion, HS could be a potential feed in reducing plasma LDL and maintaining health of broiler chicks.

## Literature

AL-KHALIFA, H., D. I., GIVENS, C., RYMER, P., YAQOOB, 2012. Effect of n-3 fatty acids on immune function in broiler chickens. Poult. Sci., 91,74–88.

BARRE, D.E., 2001. Potential of evening primrose, borage, black currant and fungal oils in human health. Anal.Nutr. Metabolism, 45, 47-57.

CALLAWAY, J.C., 2004. Hempseed as a nutritional resource: An overview. Euphytica 140,65-72.

CALLAWAY, J., U. SCHWAB, I. HARWIMA, P. HALONEN, O. MYKKANEN, P. HYWONEN, T. JARVINEN, 2005. Efficiency of dietary hempseed oil in patients with atopic dermatitis. The Journal of dermatological treatment, 16, 87-94.

FORTENBERY, T.R., M. BENNETT, 2004: Opportunities for commercial hemp production. Review of Agricultural Economics. 26(1), 97-117.

FRIEDMAN, A., D. SKLAN, 1995. Effect of Dietary Fatty Acids on Antibody Production and Fatty Acid Composition of Lymphoid Organs in Broiler Chicks. Poult. Sci. 74,1463-1469.

GOLDBERG, E.M., N. GAKHAR, D. RYLAND, M. ALIANI, R.A. GIBSON, J.D. HOUSE, 2012. Fatty acid profile and sensory characteristics of table eggs from laying hens fed hempseed and hempseed oil. Journal of feed science, 77, 153-159.

KONCA, Y., B. CIMEN, H. YALCIN, M. KALIBER, S. BUYUKKILIC, 2014. Effect of Hempseed (Cannabis sativa sp.) Inclusion to the Diet on Performance, Carcass and Antioxidative Activity in Japanese Quail (Coturnix coturnix japonica). Korean Journal of Food Science, 34(2), 141-150.

KONCA, Y., H. YALCIN, M. KARABACAK, M. KALIBER, F. DURMUSCELEBI, 2014. Effect of hempseed (Cannabis sativa L.) on performance, egg traits and blood biochemical parameters and antioxidant activity in laying Japanese Quail (Coturnix coturnix japonica) Br. Poult. Sci., 55(6),785-94.

NELSON, N.A., N. LAKSHMANAN, S.J. LAMONT, 1995.Sheep red blood cell and Brucella abortus antibody response in chickens selected for multi trait immunocompetence. Poult. Sci., 74, 1603-1609.

SAS Institute. 2002.SAA Users Guide: Statistics. SAS Institute Inc., Cary, NC.
# EFFECT OF DIFFERENT MAIZE BASED DIETS ON THE PRODUCTION PERFORMANCE AND CARCASS CHARACTERISTICS OF BROILER

A. A. Bhuyan<fnref n="1" />, Qi-Gai He<fnref n="1" />, A. A. Bhuiyan<fnref n="2" /> and N. R. Sarker<fnref n="3" />

**Corresponding author:** aab_bau@yahoo.com; Tel: +86-27-87286974; Fax: +86-27-87282608

## Abstract

A feeding trial was conducted for a period of 5 weeks with 160 day-old Starbro broiler chicks with a Randomized Block Design with 4 dietary treatments. Each treatment was replicated in 4 blocks with 10 chicks in each block. The dietary treatments were normal maize plus lysine and methionine, normal maize without lysine and methionine, Pacific 11 maize without lysine and methionine and Quality Protein Maize (QPM) without lysine and methionine. All diets were isocaloric and isonitrogenous that contained around 2975 Kcal ME/kg DM and 21.5% CP. At 5 weeks of age, 2 broilers from each block was randomly selected considering average body weight and slaughtered to analyze the meat yield traits. The QPM contained only slightly higher protein and lysine than Pacific 11. Other amino acid profiles of QPM were similar to that of Pacific 11. There were no differences in feed intake, water intake, total gain, feed conversion efficiency and mortality of broilers among the dietary treatments. Liveweight at slaughter (p<0.01), dressing percent (p<0.05) and breast meat (p<0.001) were highest, but heart (p<0.05) and digestive tract weight (p<0.05) were lowest in diet fed maize plus lysine and methionine. However, liveweight at slaughter and dressing percent did not differ (p>0.05) among the treatments without lysine and methionine. Morphological characteristics such as relationship between length of drumstick and carcass characteristics was promising.

**Keywords:** maize based diet, production performance, carcass characteristics, broiler

## Introduction

Feed is a major cost of poultry production that accounts for 60-70% of the total cost of poultry production. Maize is a major feed ingredients in poultry diet which constitutes 50-60% of the total diet and can contribute up to 30, 60 and 90% of the diets protein, energy and starch respectively (Eshetie, et al. 2011). Although normal maize contains about 8-9% protein, the quality of two essential amino acids, lysine and tryptophan, is below nutritional requirements for poultry. However, utilization of quality protein maize (QPM) which can correct this deficiency because QPM has approximately twice the lysine and tryptophan contents of normal maize (DMR, 2001). Therefore, a study was conducted to investigate the effect of replacement of QPM by hybrid maize and normal maize with or without lysine and methionine on production performance and carcass characteristics of broiler.

## Materials and Methods

A feeding trial was conducted for a period of 5 weeks with 160 day-old Starbro broiler chicks with a Randomized Block Design with 4 dietary treatments. Each treatment was replicated in 4 blocks with 10 chicks in each block. The dietary treatments were normal maize plus lysine and methionine (control diet), normal maize without lysine and methionine, Pacific 11 maize without lysine and methionine and Quality Protein Maize (QPM) without lysine and methionine. All diets were isocaloric and isonitrogenous that contained around 2975 Kcal ME/kg DM and 21.5% crude protein. Initial body weight of day old chicks was recorded immediately before the start of the trial and then weekly thereafter. FCR was recorded for whole of the period as total feed intake (Kg) per kg weight gain. Health status of birds was monitored every day and mortality of birds was recorded. At 5 weeks of age, 2 broilers from each block (total 32) was randomly selected considering average body weight and slaughtered to analyze the meat yield traits. Birds were slaughtered following “halal” method (Singh et al., 2003) and allowed to bleed completely and then plucked and weight to determine blood and feather losses (Kotula et al., 1960) and all data were recorded. Samples of feed ingredients and diets were analyzed for CP, CF and EE following AOAC, (1984). Amino acids of two maize hybrids, Pacific 11 and QPM were determined by using HPLC. Data was analyzed by RBD with 4 blocks (Snedecor and Cochran, 1989) using the procedure

<footnote n="1">¹The State Key Laboratory of Agricultural Microbiology, Ministry of Education, Huazhong Agricultural University; P.R. China</footnote>

<footnote n="2">²Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University; P.R. China</footnote>

<footnote n="3">³Animal Production Research Division, Bangladesh Livestock Research Institute, Bangladesh</footnote>
of SPSS 10 for windows (SPSS, 1998). Means compared for significant difference using Duncan's Multiple Range Test (Duncan, 1955).

## Results and Discussion

The QPM contained relatively higher ash, CP and CF than pacific11. QPM contained slightly higher Lysine, Threonine, Tryptophan, and Methionine than pacific 11(table 1). The CP content of QPM used in this study (9.6%) was within the range (7.4-9.5%) reported by DMR, 2001.

<table><caption>Table 1. Chemical composition and amino acid profiles (g/100g) of QPM and Pacific 11.</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="5">Chemical composition</th><th colspan="10">Amino acid</th></tr><tr><th>DM</th><th>Ash</th><th>CP</th><th>CF</th><th>EE</th><th>Gly</th><th>Thr</th><th>Arg</th><th>Ala</th><th>Trp</th><th>Met</th><th>Val</th><th>Phe</th><th>Ile</th><th>Leu</th><th>Lys</th></tr></thead><tbody><tr><td>QPM</td><td>90.2</td><td>1.7</td><td>9.6</td><td>1.8</td><td>6.1</td><td>0.3</td><td>0.2</td><td>0.3</td><td>0.5</td><td>0.1</td><td>0.1</td><td>0.3</td><td>0.3</td><td>0.2</td><td>0.7</td><td>0.2</td></tr><tr><td>Pacific 11</td><td>88.4</td><td>1.5</td><td>9.0</td><td>1.6</td><td>-</td><td>0.3</td><td>0.1</td><td>0.3</td><td>0.6</td><td>-</td><td>-</td><td>0.3</td><td>0.4</td><td>0.2</td><td>0.9</td><td>0.1</td></tr></tbody></table>

The data on final body weight, total gain, feed intake, FCR and mortality of broiler fed on different maize based diets are presented in Table 2. None of these characteristics differed (p<0.05) among diets. However, final weight (936g) and per day gain (31g) of broiler from 28 to 38 days reported by Miyasaka *et al.* (1997) was similar to the data of present study.

<table><caption>Table 2. Performances of broiler fed on different maize based diet</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">Treatments</th><th rowspan="2">F value and significance</th></tr><tr><th>Normal maize + LM</th><th>Normal maize</th><th>Pacific 11</th><th>QPM</th></tr></thead><tbody><tr><td>Initial weight (g)</td><td>38.7</td><td>39.1</td><td>40.8</td><td>40.7</td><td>0.32, NS</td></tr><tr><td>Final weight (kg)</td><td>1.1</td><td>1.2</td><td>1.2</td><td>1.1</td><td>0.32, NS</td></tr><tr><td>Total gain (kg)</td><td>1.1</td><td>1.1</td><td>1.2</td><td>1.1</td><td>0.55, NS</td></tr><tr><td>Feed intake (kg)</td><td>2.1</td><td>2.2</td><td>2.2</td><td>2.0</td><td>0.54, NS</td></tr><tr><td>FCR</td><td>2.0</td><td>1.9</td><td>2.0</td><td>2.0</td><td>0.15, NS</td></tr><tr><td>Mortality (%)</td><td>0.5</td><td>0.3</td><td>0.3</td><td>0.0</td><td>0.44, NS</td></tr></tbody></table>

NS= Non- significant (p>0.05).

Although lysine and methionine in normal maize based diet had no effect on production performance, it attached on the length of thigh (p<0.01) and drumstick (p<0.05). However lysine and methionine in normal maize-based diet did not affect (p<0.05) the length of neck, shank and wing of broiler (Table 3). Ishibashi *et al.* (2001) reported that when dietary lysine is low, bodyweight gain increased with increased lysine level.

<table><caption>Table 3. Morphological characteristics of broiler fed on different maize based diet</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">Treatments</th><th rowspan="2">F value and significance</th></tr><tr><th>Normal maize + LM</th><th>Normal maize</th><th>Pacific 11</th><th>QPM</th></tr></thead><tbody><tr><td>Neck (cm)</td><td>11.2</td><td>10.2</td><td>10.56</td><td>11.69</td><td>1.07, NS</td></tr><tr><td>Thigh (cm)</td><td>7.5<sup>b</sup></td><td>6.4<sup>a</sup></td><td>6.31<sup>a</sup></td><td>6.25<sup>a</sup></td><td>6.53, p>0.01</td></tr><tr><td>Drumstick (cm)</td><td>9.5<sup>b</sup></td><td>8.1<sup>a</sup></td><td>7.98<sup>a</sup></td><td>8.44<sup>a</sup></td><td>4.48, p>0.05</td></tr><tr><td>Shank (cm)</td><td>6.8</td><td>6.6</td><td>6.08</td><td>8.54</td><td>0.64, NS</td></tr><tr><td>Wing (cm)</td><td>17.3</td><td>16.1</td><td>16.56</td><td>16.25</td><td>1.54, NS</td></tr></tbody></table>

<footnote>ab, with different letters in the same raw differ (P<0.05); NS= Non- significant (p>0.05).</footnote>
Carcass characteristics of broiler fed of different diets based on maize are presented in Table 4. Dressing percent (p<0.05; 81%) and breast meat (p<0.05:15%) were highest, but heart (p<0.05; 0.58%) and digestive track weight (p<0.05; 11%) were lowest in birds fed maize plus lysine and methionine. Live weight of slaughter and dressing percent did not differ (p<0.05) between the treatment without lysine and methionine.

<table><caption>Table 4. Carcass characteristics of broiler fed on different maize based diet</caption><thead><tr><th rowspan="2">Parameters<br>(% of live wt.)</th><th colspan="4">Treatments</th><th rowspan="2">F value and significance</th></tr><tr><th>Normal maize + LM</th><th>Normal maize</th><th>Pacific 11</th><th>QPM</th></tr></thead><tbody><tr><td>Dressing percent</td><td>81.2<sup>b</sup></td><td>80.8b</td><td>77.0a</td><td>77.0a</td><td>5.39, p&lt;0.05</td></tr><tr><td>Heart weight</td><td>0.6<sup>b</sup></td><td>0.7ab</td><td>0.7ab</td><td>0.8a</td><td>1.89, p&lt;0.05</td></tr><tr><td>Breast meat weight</td><td>14.7<sup>c</sup></td><td>13.0b</td><td>10.4a</td><td>10.7a</td><td>13.84, p&lt;0.001</td></tr><tr><td>Shank weight</td><td>81.2<sup>b</sup></td><td>80.8b</td><td>77.0a</td><td>77.0a</td><td>5.39, p&lt;0.05</td></tr></tbody></table>

## Conclusions

There was no clear advantage to using QPM over normal maize or another hybrid Pacific 11 was found in terms of feed intake, total gain, feed conversion and mortality except for dressing percent. It is almost impossible to conclude from only one trial as the final weight of the broiler was only around 1.2kg, which should have been around 1.5kg in 35 days. Therefore, several batches of trials are needed to reach to a conclusion.

## Literature

* AOAC, 1984: Official Methods of Analysis. Centennial Edition. Association of Official Analytical Chemist. Washington DC. USA.

* DRM, 2001: Directorate of Maize Research In: Production technology of Quality Protein Maize, India Council of Agricultural Research, Pusa Campus, New Delhi, India.

* DUNCAN, D. B. 1955: Multiple ranges and multiple F-test. Biometrics 11:1-42.

* ESHETIE, T., F. MOGES and G. ZELEKE, 2011: Effect of Quality Protein Maize on Growth and Body Weight Gain Performance of Rhode Island Red (RIR) Breed Chickens. The IUP Journal of Life Sciences 5, 46-53.

* ISHIBASHI, T., K. KOIDA and C. YONEMOCHI, 2001: Possibility and limitation in amino acid nutrition in poultry. In: New challenges for animal science in a new century (Ed. E. Sato and J.K. Ha). Asian-Aust. J. Anim. Sci.14 (special Issue):164-175.

* ISLAM, M. R., M. P. MOSTARY, S. FARUQUE and Q. M. E. HUQUE, 2004: Effect of strains and age at slaughter on the performance and carcass characteristics of cockerels and estimation of carcass characteristics. A Report, Bangladesh Livestock Research Institute, Saver, Dhaka, Bangladesh.

* ISLAM, M. R., W. PERVIN and D. C. PAUL, 2002: Effect of levels of cassava leaf and root meal on intake, digestibility and performance of broiler. A Report, Bangladesh Livestock Research Institute, Saver, Dhaka, Bangladesh.

* KOTULA, A. W., J. E. THOMSON and J. A. KINNER, 1960: Weight increase during chilling of broilers as influence by method of opening the abnormal cavity during evisceration. Poult. Sci. 39: 26-27.

* MIYASAKA, N., K. TAKIZAWA, J. HIRATA, K. UENO, M. KADOWAKI and T. ISHIBASHI, 1997: Effects of age of amino acid requirements for maintenance of broilers. Hokuriku J. Zootech. Sci. 74:56-59

* SING, P. K., V. K. KHATTA, R. S. THAKUR, S. DEY and M. L. SANGWAN, 2003. Effects of phytase supplementation on the performance of broiler chickens fed maize and wheat based diets with different levels of non-phytate phosphorus. Asian-Aust. J. Anim. Sci. 16(1):1642-1649.

* SNEDECOR, G. W. and W. G. COCHRAN, 1989: Statistical methods, 8th edn. The Iowa State University Press, Ames, 1A, USA.

* SPSS, 1998. SPSS Base 8.0 Application Guide, SPSS Inc., USA.

<footnote>abc, with different letters in the same raw differ (P<0.05 to 0.001); NS= Non- significant (p>0.05).</footnote>
P-138 (ID 117)

THE EFFECT OF FEED RESTRICTION ON CHANGES OF MUSCLE FIBRE CHARACTERISTICS OF MUSCULUS PECTORALIS MAJOR IN BROILER CHICKEN

D. Chodová, and E. Tůmová

Department of Animal Husbandry, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague, Czech Republic

Corresponding author: chodova@af.czu.cz

**Abstract**

The aim of study was to evaluate the effect of one week feed restriction on muscle fibre characteristics in broiler chickens. The effect of quantitative feed restriction (80% ADL and 65% ADL) in the second week of age on histochemical characteristics of *musculus pectoralis major* (PM) was studied. Cockerels of Ross 308 (1215 chicken) were divided into 3 groups: *ad libitum* fed (ADL; 3×135), restricted 80 % of ADL (R80; 3×135) and with limited feed intake 65 % of ADL (R65; 3×135). Eight cockerels per group and age were slaughtered from 14 days of age till the end of the experiment at 35 days in a week interval to determine changes in muscle fibre characteristics. The number of muscle fibres in PM decreased (P≤0.001) with increasing age. Fibre cross sectional area (CSA) was significantly affected (0.027) by interaction of group and age. After the end of feed restriction (14 d) all groups had similar CSA (519.4 – 538.6 µm²), the differences between groups were observed at 35 days of age with the largest area in R65 chickens (2295.7 µm²), while R80 did not differ from ADL (1727.5 µm² and 1666.8 µm²). There was no effect of feeding regime on giant muscle fibre incidence.

**Keywords:** feed restriction, broiler chicken, muscle fibre, m. pectoralis major, giant fibre

**Introduction**

Growth performance has been increased in broiler chickens due to genetic progress, improvement of nutrition and controlled environment. This growth is associated with higher body fat deposition, mortality and increasing metabolic and skeletal disorders (BARBUT et al., 2008). The feed restriction is applied as a prevention of these problems (MENCH, 2002). Early feed restriction in poultry stimulates compensatory growth in the realimentation period (PLAVNIK and HURWITZ, 1990).

Meat quality depends on muscle fibre characteristics, from which number and fibre cross sectional area are the most important. In poultry muscle fibre formation and number of muscle fibres are complete before hatching (SMITH, 1963). Fast-growing chickens have larger fibre cross sectional area than the slow-growing ones. This increase can be associated with higher number of giant fibres, which have fibre cross-sectional area three to five times larger than normal muscle fibres (DRANSFIELD and SOSNICKI, 1999). The presence of giant muscle fibres is an indicator of interrupted metabolism and abnormal contraction of muscle fibres during *rigor mortis*. DALLE ZOTTE et al. (2001) reported that every normal muscle fibre can be converted into giant one. Increased occurrence of giant is indicator for the development of insufficient meat quality (DRANSFIELD and SOSNICKI, 1999; REHFELDT et al., 2004).

The aim of this study was to evaluate the effect of one week feed restriction applied from 8 days of age on basic muscle fibre characteristics and presence of giant muscle fibres.

**Material and methods**

In the experiment, male broiler chickens of genotype Ross 308 (n=1215) were divided into three groups with three replicates (3×135 per each treatment): the control group fed *ad libitum* (ADL), the second group (R80) received 80 % of ADL and the group R65 had feeding reduced on 65 % of ADL. The feed restriction was applied from 8 to 14 days of age. Before and following feed restriction chickens were fed *ad libitum*. Water was available free throughout the whole experiment. Birds were housed in 9 littered pens with environmental conditions corresponding with requirements for broiler chickens. Eight chickens from each group were slaughtered at 14, 21, 28 and 35 days of age. Immediately after slaughter samples of *musculus pectoralis major* (MPM) were taken for detection of histological characteristics of muscle fibres. The samples were frozen in 2-methylbutan cooled by liquid nitrogen and then stored at –80°C until analysis. Cross-sections (12 µm) of samples were cut using a cryostat Leica (Leica Microsystems, Nussloch GmbH, Nussloch, Germany) at –20°C. Subsequently, it was performed staining by haematoxylin and eosin for basic histological characteristics.
teristics of muscle fibres. Image analysis NIS Elements AR 3.1 (Laboratory Imaging, Nikon, Tokio, Japan) was used to detection number of muscle fibres per 1 mm² and fibre cross sectional area (CSA). The giant fibre percentage was calculated.

Data were processed by two-way analysis of variance, group and age using ANOVA procedure (SAS Institute Inc, 2003). P-value P≤0.05 was considered significant for all traits.

## Results and discussion

The results of one week feed restriction affecting muscle fibre characteristics are shown in Table 1. There was no interaction of group and age in number of muscle fibres per 1 mm². Together with increasing age, number of muscle fibres significantly (P≤0.001) decrease, which corresponds with the data of RYU and KIM (2005).

<table><caption>**Table 1.** The effect of feed restriction on *pectoralis major* muscle fibre characteristics</caption><thead><tr><th>Group</th><th>Age (days)</th><th>Number of muscle fibres per 1 mm²</th><th>Fibre cross sectional area (μm²)</th><th>Giant fibres percentage (%)</th></tr></thead><tbody><tr><td rowspan="4">ADL</td><td>14</td><td>1629.1</td><td>519.4g</td><td>0.09</td></tr><tr><td>21</td><td>1018.5</td><td>777.2fg</td><td>0.10</td></tr><tr><td>28</td><td>593.7</td><td>1360.7cd</td><td>0.05</td></tr><tr><td>35</td><td>464.6</td><td>1666.8bc</td><td>0.02</td></tr><tr><td rowspan="4">R80</td><td>14</td><td>1574.0</td><td>538.6g</td><td>0.04</td></tr><tr><td>21</td><td>944.0</td><td>852.9ef</td><td>0.06</td></tr><tr><td>28</td><td>650.5</td><td>1143.9de</td><td>—</td></tr><tr><td>35</td><td>441.0</td><td>1727.5b</td><td>0.03</td></tr><tr><td rowspan="4">R65</td><td>14</td><td>1626.0</td><td>520.5g</td><td>0.05</td></tr><tr><td>21</td><td>981.7</td><td>815.0fg</td><td>—</td></tr><tr><td>28</td><td>576.6</td><td>1418.7bcd</td><td>0.08</td></tr><tr><td>35</td><td>392.0</td><td>2295.7a</td><td>0.04</td></tr><tr><td>RMSE</td><td></td><td>209.7</td><td>303.3</td><td>0.09</td></tr><tr><td colspan="5"><strong>Significance</strong></td></tr><tr><td>Group</td><td></td><td>0.830</td><td>0.026</td><td>0.334</td></tr><tr><td>Age</td><td></td><td>≤0.001</td><td>≤0.001</td><td>0.711</td></tr><tr><td>Group * age</td><td></td><td>0.966</td><td>0.027</td><td>0.460</td></tr></tbody></table>

RMSE: root mean square error

a,b,c,d,e,f,g P≤0.05; Group: ADL – *ad libitum*, R80 – 80% feed of ADL, R65 – 65% feed of ADL

The CSA of muscle fibres was significantly (P≤0.027) influenced by interaction of group and age. All groups had similar CSA at 14 days of age, but at the end of experiment group R65 with the most intensive feed restriction had largest CSA, while R80 group with moderate feed restriction did not differ from ADL group. In contrast with our results REHFELDT et al. (2004) stated that qualitative and quantitative feed restriction reduced CSA of muscle fibres. Also LI et al. (2007) determined that restricted chickens revealed smaller CSA. In our experiment, the CSA significantly (P≤0.001) increased with age, which corresponds with DRANSFIELD and SOSNICKI (1999) and CHOI and KIM (2009).

Some fibres from *pectoralis major* exhibited histological features of giant fibres and also CSA of these fibres was typical for giant fibres. However, there was not detected the excessive incidence of giant muscle fibres in restricted groups and also age did not have an effect on giant fibre percentage. The percentage of giant muscle fibres was very low and according to REHFELDT et al. (2004) giant fibre percentage less than 0.5% is not associated with impairment in meat quality.

## Conclusion

The results of the current study indicated that early feed restriction in broiler chickens did not affect number of muscle fibres per 1 mm², but the fibre cross sectional area enlarged in restricted chickens. The CSA was affected by intensity of feed restriction. The excessive incidence of giant fibres was not detected in any group.
Acknowledgement

The study was supported by the Ministry of Agriculture of the Czech Republic (Project NAAR No. QJ1510192).

Literature

BARBUT, S., A. A. SOSNICKI, S. M. LONERGAN, T. KNAPP, D. C. CIOBANU, L. J. GATCLIFFE, E. HUFF-LONERGAN, AND E. W. WILSON, 2008: Progress in reducing the pale, soft and exudative (PSE) problem in pork and poultry meat. Meat Sci. **79**, 46–63.

CHOI, Y. M., B. C. KIM, 2009: Muscle fibres characteristics, myofibrillar protein isoforms, and meat quality. Livest. Sci. **122**, 105–118.

DRANSFIELD, E., A.A. SOSNICKI, 1999: Relationship between muscle growth and poultry quality. Poult. Sci. **78**, 743–746.

LI, Y., L. YUAN, X. YANG, Y. NI, D. XIA, S. BARTH, R. GROSSMANN, R.Q. ZHAO, 2007: Brit. J. Nutr. **98**, 310–319.

MENCH, J. A., 2002: Broiler breeders: Feed restriction and welfare. World Poultry Sci. J. **58**, 20–29.

PLAVNIK, I., S. HURWITZ, 1990: Performance of broiler chickens and turkeys poults subjected to feed restriction or to feeding of low-protein or low-sodium diets at an early age. Poult. Sci. **69**, 945–952.

REHFELDT, C., I. FIEDLER, N.C. STICKLAND, 2004: Number and size of muscle fibers in relation to meat production. In: TE PAS, M.F.W., M.E. HAAGSMAN, H.P. EVERTS: Muscle Development of Livestock Animals: Physiology, Genetics, and Meat Quality. CAB Int., Wallingford, UK, ISBN 0-85199-811-9, 1–30.

RYU, Y.C., B.C. KIM, 2005: The relationship between muscle fiber characteristics, postmortem metabolic rate, and meat quality of pig longissimus dorsi muscle. Meat Sci. **71**, 351–357.

SMITH, J.H., 1963: Relation of body size to muscle cell size and number in the chicken. Poult. Sci. **42**, 283–290.
P-140 (ID 178)

VARIABILITY OF STARCH DIGESTIBILITY IN BROILERS FED WITH WHEAT-BASED DIETS: EFFECTS OF AGE AND FEEDING BEHAVIOUR

Justine Danel¹, Jean-Paul Métayer², Maria Vilariño¹

ARVALIS - Institut du végétal, Poultry and swine nutrition research unit: ¹ Pouline, 41100 Villerable, France; ² Station expérimentale, 91720 Boigneville, France

Corresponding author: j.danel@arvalisinstitutduvegetal.fr

**Abstract**

The starch digestibility of wheat may be more or less variable. Chemical parameters of wheat or its fineness of grinding can only explain a little part of the variability of starch digestibility. Beyond results of nutrients digestibility trials in broilers, two extreme wheat profiles have been highlighted as for the starch digestibility (good: WheatSD+ or low: WheatSD-) and the individual variability. Total tract digestibility of starch, nitrogen and energy of diets with both wheat and one maize, with or without enzymes, were quantified in five ages (8, 13, 18, 23 and 28 days) on 4 pools of 3 animals by treatment. The feed intake was measured daily and even closer (all the 2 hours), the days matching 3 of the 5 collections (7, 17 and 27 days). With age, the starch digestibility decreased significantly (P<0.001), but only for WheatSD- and especially from 18 days of age. Whatever the diet, the digestibility of energy and nitrogen varied significantly and similarly (P<0.01 and P<0.001) with the highest values between 13 and 18 days. The starch excreta content, measured individually, was positively correlated with daily intake only for WheatSD- from 18 days (0.30<R²<0.52). The speed of ingestion changed during the day (P<0.001) with maxima before lights out (two to three times faster). For the last two hours, the value was positively correlated (R²=0.56) with starch excreta content but only for WheatSD-.

**Keywords:** wheat, starch, digestibility, age, broiler

**Introduction**

Wheat is an important raw material in the feeding of poultry and may lead variability in nutrients digestibility and in the available energy content, which can reduce the performances (Gutierrez *et al.*, 2008). Different factors (viscosity, hardness) had been highlighted without explaining all the variability (Carré *et al.*, 2005). Métayer *et al.* (2007) found contradictory results on the role of those two criteria and claimed a positive relation between starch digestibility (SD) and nitrogen corrected apparent metabolisable energy (AMEn). The results of a meta-analysis (Nugues *et al.*, 2013) allowed us to quantify the importance of the starch quantity in the wheat energetic value and remove many physical and chemical criteria as possible predictors of energy and starch digestibilities. Most of all, the important role of the animal makes difficult the prediction of the digestibility only from wheat characteristics. Other studies have been led to determine if technological parameters (wheat) or physiological mechanism (animal) could explain the variability. In this article, the role of ingestion rhythm and enzymes will be studied on the nutrients digestibility of two wheat models.

**Material and Methods**

Two wheat models, defined from a previous study, with extreme values concerning the SD (WheatSD+, Starch well Digested; WheatSD-, Starch not well Digested) were studied and compared to a corn. The effect of enzymes with xylanasic and β-glucanasic activities was also measured (E-/E+). The treatments were untitled : WheatSD-E-, WheatSD-E+, WheatSD+E-, WheatSD+E+, CornE- and CornE+, according to the raw material (RM) maximized in the feed and the addition (E+) or not (E-) of enzymes. A growing-phase feed were formulated (AMEn=2750 kcal/kg; Gross protein=19.5%) with an indigestible marker (TiO₂). The wheat-based diets were compounded of wheat (55.5%), corn (10%) and soybean meal (28.5%) and the corn-based diets of corn (60%), WheatSD+ (5.5%) and soybean meal (30%).

Every diet was tested on 12 male broilers (ROSS PM3). Excreta were collected individually in order to measure the starch content. 4 pools of 3 broilers were made by treatment to determine the starch, nitrogen and energy digestibilities at five ages (D8, D13, D18, D23 and D28). The growing performances of broilers (daily feed intake, total weight gain and FCR) were followed from D6 to D27. Individual feed intakes were measured every day and even all the two hours (between 8:30am and 8:30pm – 12 hours of light) to determine the speed of ingestion (D7, D17, D27). Statistics were performed with the software R v.2.15.0. Data were analysed by ANOVA with 3 factors (Age, RM, E), except for total performances (RM, E).
## Results

For the five ages, the results of ANOVA on the different digestibilities (nitrogen, starch and energy) got on the pools (4 pools per treatment of 3 chickens) are reported on the table 1.

For SD, the ANOVA showed effects of RM and Age (P<0.001) and an interaction of RM*Age (P<0.01) due to a decrease with age only for WheatSD- (-2.2 points for E-; -3.2 points for E+). This result attested the hypothesis of the degradation of SD with age but only for certain wheat profiles.

For each age, average and variability of individual starch content in excreta, which is a good predictor of SD (Métayer *et al.*, 2015), are shown on figure 1. Responses were similar to the ones got on the pools of excreta but the variability brought additional information. An interaction RM*E was observed (P<0.01) due to a negative effect of enzymes on WheatSD- (+2.2 points at D28; contradictory to the expected). The SD of corn-based diets was better and less variable than the wheat-based diets one. WheatSD- was significantly less valuable than Corn on this criteria, WheatSD+ is intermediary. The variability increased with age and was from 1.5 to 3 times more important for wheat than for corn-based diets. The purpose of these individual measures was to compare animal performances to the capacity for starch digestion. No relation could be established between feed intake and starch content in excreta all the diets considered. But, significant correlations were observed for WheatSD-E- at D18, D23 and D28 (R²=0.30, 0.45, 0.52), for WheatSD-E+ at D18 (R²=0.42) and for WheatSD+E+ at D18 and D23 (R²=0.31, 0.43). In this study, enzymes did not improve SD of diets.

The apparent nitrogen digestibility varied only with age (P<0.01). All diets considered, the highest values were observed at D18, whereas Métayer *et al.* (2015) reported a linear decrease with the age (from D6 to D20). The range of variation was of 2 points, except for WheatSD-E+ (4.6 points), but the mean deviation between the different ages was of only 1.2 point.

The energy and nitrogen digestibilities variations were similar but the difference between energy digestibility values of two treatments at the same age is bigger. Both criteria were well correlated (R²=0.58; P<0.001), in agreement with Métayer *et al.* (2015). The interactions RM*Age (P<0.01) and Age*E (P=0.05) did not allow a simple analyse of the results. Enzymes would tend to improve the energy digestibility in young chicks (from 8 to 18 days of age).

The study of performances did not show any effect of enzymes from D6 to D27. The FCR of corn-based diets are better (P<0.01) than those of wheat-based diets (respectively 1.44, vs. 1.50 and 1.50) except for WheatSD+E+, which is similar to corn.

The speeds of ingestion (SI) were calculated from the two-hour feed intakes at D7, D17 and D27. Whatever the age, the chickens eat faster (P<0.001) in the two hours before the lights out (respectively 5.0 g/h, 16.6 g/h and 30.5 g/h at D7, D17 and D27); twice or three times faster than the daily average of other periods, even at the lights on. No effect of the RM was highlighted, except to D17 (P<0.05). The correlation between SI at the lights out and starch content in excreta is significantly and positive only for WheatSD-E- (R²=0.56; figure 2), which attested our suggestion. However, the relation is the opposite for WheatSD-E+ (R²=0.47) with no explanatory hypothesis. It is in agreement with Svihus (2014) who reviewed a negative correlation between feed intake and SD.

<table><caption>**Table 1.** Results of ANOVA on Nutrients digestibility of WheatSD-, WheatSD+ and Corn-based diets without or with enzymes (E- or E+)</caption><thead><tr><th>Variable / Digestibility</th><th>Starch</th><th>Apparent Nitrogen</th><th>Energy</th></tr></thead><tbody><tr><td>RM</td><td>P&lt;0.001</td><td>NS</td><td>P&lt;0.001</td></tr><tr><td>Age</td><td>P&lt;0.001</td><td>P&lt;0.01</td><td>P&lt;0.001</td></tr><tr><td>Enzymes</td><td>NS</td><td>NS</td><td>P&lt;0.01</td></tr><tr><td>RM*Age</td><td>P&lt;0.01</td><td>NS</td><td>P&lt;0.01</td></tr><tr><td>RM*E</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>Age*E</td><td>NS</td><td>NS</td><td>0.05</td></tr><tr><td>RM*Age*E</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>RSD / RCV (%)</td><td>0.9 / 0.9</td><td>1.4 / 1.7</td><td>1.2 / 1.6</td></tr></tbody></table>
<figure><img src="image_59.png" /><figcaption>**Figure 1.** Starch content in excreta between D8 and D28 (averages and standard errors).</figcaption></figure>

<figure><img src="image_60.png" /><figcaption>**Figure 2.** Relationship between starch content in excreta and SI (2 hours before lights out).</figcaption></figure>

## Conclusions

Whatever the cereal, wheat or corn, nitrogen as energy digestibilities vary with the age, with maxima around 13 or 18 days of age. Starch digestibility is variable from wheat to another, as high and homogenous as corn or lower and more variable. For this special kind of wheat, chickens have a good starch digestion before 18 days of age, and a higher intake at the end of the day increases the starch content measured in the excreta. In this context, starch digestibility is not improved by the tested enzymes.

## Literature

CARRE, B., N. MULEY, J. GOMEZ, F.X. OURY, E. LAFFITTE, D. GUILLOU, C. SIGNORET, 2005: Soft wheat instead of hard wheat in pelleted diets results in high starch digestibility in broiler chickens. Brit. Poultry Sci. **46**(1), 66-74.

GUTIERREZ DEL ALAMO A., M.W.A. VERSTEGEN, L.A. DEN HARTOG, P. PEREZ DE AYALA, M.J. VILLAMIDE, 2008: Effect of wheat cultivar and enzyme addition to broiler chicken diets on nutrient digestibility, performance, and apparent metabolizable energy content. Poult. Sci. **87**, 759-767.

MÉTAYER J.P., M. VILARIÑO, J.M. GAÜZÈRE, F. SKIBA, 2007: Effets de la dureté et de la viscosité spécifique sur la valeur alimentaire du blé chez le poulet de chair. Proceedings of the 7ème Journées de la Recherche Avicole, Tours, France, 154-158.

METAYER J.P., P. LESCOAT, D. BASTIANELLI, I. BOUVAREL, Y. FOURNIS, M. VILARIÑO, 2015 : Facteurs de variation de la digestibilité des nutriments chez le poulet : synthèse des résultats du projet DigSPIR. Proceedings of the 11ème Journées de la Recherche Avicole, Tours, France, 504-508.

NUGUES M., J.P. MÉTAYER, F. DUYME, F. SKIBA, M. VILARIÑO, 2013: Effect of wheat characteristics on starch digestibility and apparent metabolisable energy in broiler chickens. Proceedings of the 19th ESPN, Potsdam, OP-15.

SVIHUS B., Starch digestion capacity of poultry, 2014: Poult. Sci. **93**, 2394–2399.
P-141 (ID 272)

# THE EFFECTS OF FEEDING MAIZE OR WHEAT BASED DIETS WITH OR WITHOUT XYLANASE SUPPLEMENTATION ON THE GUT MORPHOLOGY AND MICROFLORA OF BROILER CHICKS

F. Dublecz<fnref n="1" />, L. Pál<fnref n="1" />, L.Wágner<fnref n="1" />, F. Husvéth<fnref n="1" />, K. Dublecz<fnref n="1" /> and A. Molnár<fnref n="2" />

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

## Abstract

The aim of this trial was to get more understanding how maize and wheat based diets and using xylanase supplementation can modify the gut structure and the microbial fermentation in the caecum.

A total of 160 Ross 308 broiler cockerels were divided into four treatment groups and fed maize, wheat soybean meal based diets. Maize based diets contained only maize as the sole cereal. Wheat based diets contained beside maize 30, 40 and 50% wheat in the starter, grower and finisher phase respectively. Both maize and wheat based diets were fed with or without xylanase (0,15g/kg Aextra XB 201) supplementation (M+, M-, W+, W-). The most important difference in the morphology of jejunum was the significantly higher crypt depth and significantly lower villus:crypt ratio of treatment W+, compared with treatment W. Both W+ and W- treatments resulted higher viscosity in the ileum and higher Coliform bacteria concentration in the caecum compared with the M+ and M- groups. No significant changes in the number of Lactobacilli were found. Wheat based diets resulted higher concentration of short chain fatty acids (SCFA) and lower pH values in the caecum. Feeding wheat based diets decreased the caecal concentration of propionic acid and significantly increased the production of butyric acid.

From the results it can be concluded, that xylanase enzyme supplementation of wheat based diets results not only changes in the viscosity of the digesta, but modifies also gut morphology and the microbial fermentation in the caecum. Increasing the SCFA concentration and lowering the pH in the caecum can decrease the number of several pathogenic microbes and improve gut health.

**Keywords:** maize, wheat, xylanase, gut morphology, gut health

## Introduction

One of the most important differences between maize and wheat is, that wheat contains higher amounts of water soluble none starch polysaccharides (NSP-s), mostly arabinoxylans. Soluble NSP-s increase the viscosity of the digesta, causing lower digestibility of nutrients and worse litter quality (CHOCT et al., 2004). In order to get around these negative effects, the exogenous NSP degrading enzyme, xylanase is widely used in the practice. Beside the effects of viscosity and digestibility there are not consistent information how the different NSP-s of the main cereals can affect gut morphology and microflora (FERKET, 2011). Therefore the aim of this trial was to get more understanding how maize and wheat based diets, with or without exogenous xylanase can modify the morphology of the small intestine and the microbial fermentation in the caecum.

## Materials and methods

160 Ross 308 broiler cockerels were divided into four treatment groups. Maize based diets contained only maize as the sole cereal. Wheat based diets contained beside maize 30, 40 and 50% wheat in the starter (0-10d), grower (11-24d) and finisher (25-35d) phase respectively. Both maize and wheat based diets were fed with or without xylanase (0,15g/kg Aextra XB 201) supplementation (M+, M-, W+, W-). The soluble arabinoxylan contents of maize and wheat were 0.88 and 9.37 mg/kg respectively. Diets were isocaloric and isonitrogenous and were prepared to meet the nutrient requirements of Ross 308 hybrids. For the histomorphological analysis of the jejunum, the villus height, crypt depth, villus width, villus height:crypt depth ratio and thickness of muscle layer were measured. The viscosity of the ileal digesta was measured with a Brookfield DV II+ type viscometer. The number of Lactobacilli were counted on MRS agar after anaerobic incubation at 39°C for 48 h. Coliform bacteria were counted as red colonies on MacConkey agar, incubated aerobically at 39 °C for 24 h. For SCFA determination of caecal samples were measured by Gas Chromatograph, the pH with a portable pH meter. All data were analysed by two way analysis of variance by using SPSS 16.0 software.

<footnote n="1">¹ Department of Animal Science, University of Pannonia, Keszthely, Hungary</footnote>

<footnote n="2">² Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria</footnote>
## Results

The most important difference in the morphology of jejunum was the significantly higher crypt depth and significantly lower villus:crypt ratio of the W+ group, compared with the treatment W. The villus height and the other parameters were not affected by the treatments (Table 1.).

<table><caption>Table 1. Histomorphological parameters of the jejunum (µm)</caption><thead><tr><th></th><th>Maize</th><th>Maize+enzyme</th><th>Wheat</th><th>Wheat+enzyme</th></tr></thead><tbody><tr><td>villus height</td><td>825.0±22.0</td><td>791.5±35.3</td><td>812.5±31.3</td><td>823.4±21.1</td></tr><tr><td>crypt depth</td><td>177.7±6.3 ab</td><td>157.5±7.4 bc</td><td>150.8±5.1 c</td><td>183.4±7.3 a</td></tr><tr><td>apical transverse</td><td>121.0±6.5 b</td><td>143.3±6.1 a</td><td>129.7±5.1 ab</td><td>138.5±5.4 ab</td></tr><tr><td>villus/crypt ratio</td><td>5.0±0.2 ab</td><td>5.7±0.4 ab</td><td>5.6±0.2 a</td><td>4.8±0.2 b</td></tr><tr><td>muscle layer</td><td>119.3±3.8</td><td>131.9±23.8</td><td>115.0±4.0</td><td>122.7±3.9</td></tr><tr><td>basal transverse</td><td>175.0±9.5</td><td>191.6±6.9</td><td>185.5±8.8</td><td>199.0±8.8</td></tr></tbody></table>

Values of the table are means ±SEM; averages with different letter marks differ significantly (p<0.05)

The higher crypt depth in case of W+ treatment could cause increased mucus production which can affect the microflora of the small intestine content and the colonisation of the different bacteria. Due to the higher amount of soluble arabinoxylan content of wheat, significantly higher viscosity values were found in case of wheat based groups (W: 3.04; W+: 2.84 mPa·s) compared with the maize based diets (M: 2.31; M+: 2.56 mPa·s).

In the caeca significantly higher numbers of Coliform bacteria were observed in chickens fed the wheat based diets with or without enzyme supplementation (W: 5.78; W+: 5.31 CFU/g) compared with the maize based treatments (M: 4.47; M+: 5.01 CFU/g). No significant changes in the number of Lactobacilli were found (W: 8.16; W+: 7.86; M:7.97; M+: 7.93 CFU/g).

Wheat based diets resulted higher concentration of short chain fatty acids (SCFA) and lower pH values in the caecum compared with the maize based treatments. In the case of wheat based diets, the enzyme supplementation resulted further decrease in pH (Figure 1.).

Comparing the concentration of the different volatile fatty acids, feeding wheat based diets decreased the caecal concentration of propionic acid and increased the fermentation of butyric acid. The butyric acid content of caeca in W+ was significantly higher than those in the M and M+ groups (Figure 2.).

<figure><img src="image_61.png" /><figcaption>Figure 1. Caecal pH values</figcaption></figure>
<figure><img src="image_62.png" /><figcaption>Figure 2. Caecal SCFA contents (µmol/g)</figcaption></figure>

## Conclusions

From the results it can be concluded, that xylanase enzyme supplementation of wheat based diets results not only changes in the viscosity of the digesta, but modifies also gut morphology and the microbial fermentation in the caecum. Increasing the SCFA concentration and lowering the pH in the caecum can decrease the number of several pathogenic microbes and improve gut health.

## Literature

1. CHOCT. M., A. KOCHER, D.L. WATERS, D. PETTERSSON, G. ROSS, 2004: A comparison of three xylanases on the nutritive value of two wheats for broiler chickens. British Journal of Nutrition. 92. 1, 53-61.

2. FERKET. P. R., 2011: Nutritional-disease interactions regarding gut health in chickens. Proceedings of the 18th European Symposium on Poultry Nutrition-Izmir. Turkey. p.: 180-193
P-144 (ID 246)

RESPONSE OF BROILER CHICKS FED ON STEAM- OR COLD-PELLED DIETS CONTAINING RAW FULL-FAT SOYBEAN MEAL

Mammo M. Erdaw<fnref n="1" />,<fnref n="3" />, Rider A. Perez-Maldonado<fnref n="2" />, M.M. Bhuiyan<fnref n="1" /> and Paul A. Iji<fnref n="1" />

**Correspondence:** piji@une.edu.au

**Abstract**

A 2 x 4 factorial study was conducted to assess the response of broiler chickens fed steam- or cold-pelleted diets containing different levels (0, 10, 20 or 30 %) of raw full-fat soybean meal (RSBM). One half of each diet was steam-pelleted and the other half was cold-pelleted. Trypsin inhibitor contents of diets ranged from 1730.5 to 10484.4 TIU/g. Each of the eight treatments was replicated six times, with 8 birds per replicate. Chicks were housed under an environmentally controlled cage system and fed to 14 days of age. Feed intake (P < 0.05) and body weight gain (BWG) (P < 0.001) declined with increase in RSBM in the diets. However, BWG was reduced (P<0.001) in chicks on the steam-pelleted diets. Feed conversion ratio was negatively affected (P<0.05) by increase in RSBM levels. There were no interactions between RSBM level and pelleting condition. An increase in RSBM content and feeding on steam-pelleted diets resulted in an increase in the weight of gizzard + proventriculus (P<0.05), pancreas (P<0.001), duodenum (P<0.01) and jejunum + ileum. The relative weight of the spleen, bursa, heart and liver was not affected by the two factors. There is a need to assess the response of chicks over a longer production cycle. However, it would appear that steam-pelleting was not adequate at reducing the antinutritional effects of RSBM.

**Keywords:** Cold-pelleting; commercial SBM; steam-pelleting; raw full-fat

**Introduction**

Soybean meal is generally expensive due to its position as the premier vegetable protein ingredient for poultry and its supply fluctuates due to events in the major producing areas of the world in North and South America (SHI et al., 2012). The meal can be fed as full-fat or after oil extraction. Full-fat meals are higher in energy, but lower in protein, and require heat treatment, in a bid to reduce the levels of protease inhibitors that are responsible for poor growth of birds on diets containing raw soybean meals (RSBM). It is not well known if changing the pelleting methods reduces effects of trypsin inhibitors. The objective of this study was to evaluate the response of chicks fed on steam- or cold-pelleted diets containing RSBM.

**Materials and Methods**

Four diets were formulated, in which 0, 10, 20 or 30 % of RSBM replaced commercial SBM. One half of each diet was steam-pelleted and the other half was cold-pelleted. A 2 x 4 factorial arrangement (4 RSBM levels x 2 methods of pelleting) was used to assess the response of broiler chicks to diets containing RSBM. Each diet was replicated 6 times, with 8 birds per replicate. The diets were fed from hatch to 14 days of age.

**Results and Discussion**

Feed intake (FI) declined with increase in level of RSBM but this was significant (P<0.05) only for the steam-pelleted diets (Table). The interaction between RSBM level and pelleting method tended (P<0.07) to be significant. Body weight gain (BWG) was also reduced (P<0.001) by increase in RSBM level while birds on the cold-pelleted diets gained (P<0.001) more than those on the steam-pelleted diets, especially at the higher RSBM levels. On both pellet types, FCR was reduced (P<0.05) as a result of increase in RSBM. The interaction between RSBM level and pelleting method was not significant in the case of BWG and FCR. The weight of proventriculus + gizzard (G+P) (P<0.05), duodenum (P<0.01) and pancreas (P<0.001), in relation to the body weight, increased with rising level of RSBM (data not shown). Feeding the steam-pelleted diets also resulted in an increase in weight of G+P (P<0.01) and jejunum + ileum (P<0.001).

<footnote n="1">¹School of Environmental and Rural Sciences, University of New England, Australia</footnote>

<footnote n="2">²DSM Nutritional Products, Animal Nutrition and Health, 30 Pasir Panjang Road #13-31 Mapletree Business City Singapore 117440, Singapore</footnote>

<footnote n="3">³Ethiopian Institute of Agricultural Research, Debre-Zeit Centre, Ethiopia</footnote>
The reduction in FI with increase in level of RSBM may be due to the presence of antinutrients in raw soybean. Weight gain may have also been adversely affected through the negative effects of protease inhibitors, illustrated by the noticeably large weight of the pancreas (MOGRIDGE *et al.*, 1996). Contrary to expectations, steam-pelleting resulted in poorer growth than cold-pelleting. It is not known if the high conditioning temperature reduced the nutritive value of the meal.

<table><caption>Table 1 Feed intake (g/b), live weight gain (g/b) and feed conversion ratio (FCR) of broiler chickens fed on different diets between hatch and 14 days of age</caption><thead><tr><th>Pelleting method</th><th>RSBM¹ (%)</th><th>Feed intake</th><th>Body weight gain</th><th>FCR</th></tr></thead><tbody><tr><td rowspan="4">Steam</td><td>0</td><td>582.4<sup>a</sup></td><td>514.9<sup>a</sup></td><td>1.13</td></tr><tr><td>10</td><td>526.0<sup>bc</sup></td><td>487.6<sup>b</sup></td><td>1.15</td></tr><tr><td>20</td><td>554.7<sup>ab</sup></td><td>471.6<sup>b</sup></td><td>1.17</td></tr><tr><td>30</td><td>496.9<sup>c</sup></td><td>426.1<sup>c</sup></td><td>1.21</td></tr><tr><td rowspan="4">Cold</td><td>0</td><td>556.2<sup>ab</sup></td><td>517.1<sup>a</sup></td><td>1.11</td></tr><tr><td>10</td><td>562.2<sup>ab</sup></td><td>516.1<sup>a</sup></td><td>1.14</td></tr><tr><td>20</td><td>554.3<sup>ab</sup></td><td>503.6<sup>ab</sup></td><td>1.13</td></tr><tr><td>30</td><td>544.0<sup>ab</sup></td><td>479.8<sup>b</sup></td><td>1.17</td></tr><tr><td>Pooled SEM</td><td></td><td>6.05</td><td>4.90</td><td>0.01</td></tr><tr><td colspan="5"><strong>Sources of variation</strong></td></tr><tr><td>RSBM level</td><td></td><td>*</td><td>***</td><td>*</td></tr><tr><td>Pelleting method</td><td></td><td>NS</td><td>***</td><td>NS</td></tr><tr><td>RSBM x pelleting method</td><td></td><td>0.07</td><td>NS</td><td>NS</td></tr></tbody></table>

## Conclusion

Further testing, including grower and finisher diets, containing such a levelled RSBM with a partial economic analysis might be required.

## References

* MOGRIDGE, J., SMITH, T., & SOUSADIAS, M. (1996). Effect of feeding raw soybeans on polyamine metabolism in chicks and the therapeutic effect of exogenous putrescine. *Journal of Animal Science*, 74(8), 1897-1904.

* SHI, S., LU, J., TONG, H., ZOU, J., & WANG, K. (2012). Effects of graded replacement of soybean meal by sunflower seed meal in laying hen diets on hen performance, egg quality, egg fatty acid composition, and cholesterol content. *The Journal of Applied Poultry Research*, 21(2), 367-374.
# EFFECT OF EXTRUSION COOKING ON THE NUTRITIONAL VALUE OF FABA BEAN FOR BROILER CHICKENS

M. Hejdysz*, S.A. Kaczmarek and A. Rutkowski

Department of Animal Nutrition and Feed Management, Poznan University of Life Sciences, Wolynska 33, 60-637 Poznan, Poland

*Corresponding author email: marhej@up.poznan.pl

## Abstract

The aim of the investigations was to expand our knowledge regarding the effect of extrusion cooking on the nutrient value of currently cultivated Polish faba bean varieties. The experiment was conducted with 264 broiler male chickens, divided into eleven dietary treatments, 12 replications with 2 birds in each. The birds were kept (1-18d) individually in balance cages and received basal diets *ad libidum*. From day 19 to 23 birds were fed the basal diets which were mixed with faba beans meal in proportion 60:40. Five different cultivars of faba bean (Albus, Amulet, Kasztelan, Olga, Merlin) were used in the experiment. The birds from group one were fed only the basal diet. Diets of the remaining groups differed with regard to faba bean cultivars and form (raw or extruded). The following parameters were determined in the trial: ilea crude protein and amino acids digestibility and AME<sub>N</sub> value. For this purpose the difference method of digestibility determination and ME was used. TiO<sub>2</sub> was added to diets in the amount of 0.3% to carry out necessary calculations. Comparing the groups without and with extrusion, higher values for ileal digestibility were determined when extrusion was applied (crude protein - 85.9 % vs. 89.5%; Lys - 89.7% vs. 94.7%; Thr - 81.6% vs. 85.0%). The same differences were determined in the case of AME<sub>N</sub> (10.83 MJ/kg vs. 14.97 MJ/kg). The present trial demonstrated that extrusion exerted a beneficial effect on nutrient value of faba bean.

**Keywords:** faba bean, extrusion cooking, anti-nutritional factors

## Introduction

Faba bean seeds can be used as a valuable source of protein, energy and other nutrients in broiler chicken nutrition. However, faba bean seeds also contain variable amounts of anti-nutritional factors such as tannins, protease inhibitors, oligosaccharides (raffinose, stachyose and verbascose) and non-starch polysaccharides (NSP), which can negatively affect nutrient utilization and animal performance. Current low-tannin faba bean cultivars are characterized by low concentrations of tannins, but they still contain other anti-nutritional factors, which can reduce nutrient digestibility. Results have so far shown that low-tannin faba beans are characterized by better amino acid digestibility than conventional faba beans. However, the digestibility coefficients of amino acids contained in low-tannin faba beans are still lower than those contained in soybean meal (Woyengo and Nyachoti 2012).

According to some researchers, extrusion cooking of faba bean seeds is a process which may improve the performance of broiler chickens. An appropriate processing temperature is critical for the elimination of heat-labile anti-nutritional factors found in legume seeds (Diaz et al., 2006). The effect of extrusion cooking on the nutritional value of faba beans is not well understood. Moreover, there is no comparative information on the influence of extrusion cooking on the nutritive value of low-tannin faba beans.

The aim of the investigations was to expand our knowledge regarding the effect of extrusion on the nutrient value of currently cultivated Polish faba bean varieties.

## Material and methods

Raw and extruded faba bean seeds were analyzed in duplicate for crude protein (CP), starch (S), resistant starch (RS), phytin phosphorus, raffinose family oligosaccharides (RFO), tannin and trypsin inhibitor content. The experiment was conducted with 264 broiler male chickens, divided into eleven dietary treatments, 12 replications with two birds in each. The birds were kept (1-18d) individually in balance cages and received basal diets *ad libidum*. From day 19 to 23 birds were fed the basal diets which were mixed with faba bean meal in proportion 60:40. Five different cultivars of faba bean (Merlin, Olga, Albus, Amulet, Kasztelan) were used in the experiment. Birds belong to the first group were fed only the basal diet. Diets of the remaining groups differed with regard to faba bean cultivars and form (raw or extruded). The concentration of CP, S and amino-acids in diets and digesta were determined. The apparent metabolizable energy (AME<sub>N</sub>) of faba bean and apparent ileal digestibility (AID) were determined using the difference method and TiO<sub>2</sub> was
used as an indigestibility marker. The obtained results were subjected to two-factorial analysis of variance (ANOVA). Means from experiments were compared using Duncan test and the significance of differences was assumed to be at the level of p<0.05.

## Results and discussion

The analyzed composition of feed ingredients of raw and extruded low-tannin faba bean is presented in Table 1. The content of CP and S in DM was similar in raw and extruded faba beans and did not differ statistically significantly (Table 1). Faba beans in extruded form were characterized by lower concentrations of RS than faba beans in raw form (p ≤ 0.05). The content of RFOs and tannins in DM was similar in raw and extruded faba beans and did not differ statistically significantly (Table 1). Extrusion cooking had a positive impact on the decrease in trypsin inhibitors and phytic P content in seeds and this decrease was statistically significant (p ≤ 0.05). A similar relationship was confirmed by Alonso et al. (2000), who explained that during extrusion some molecules of inositol hexaphosphate were hydrolyzed to penta-, tetra- and triphosphates; therefore, the level of phytic P in seeds after extrusion was lower.

<table><caption>**Table 1** Chemical composition of five faba bean cultivars in raw and extruded form</caption><thead><tr><th rowspan="2">Item, % of DM</th><th colspan="2">Merlin</th><th colspan="2">Olga</th><th colspan="2">Albus</th><th colspan="2">Amulet</th><th colspan="2">Kasztelan</th></tr><tr><th>-1</th><th>+</th><th>-</th><th>+</th><th>-</th><th>+</th><th>-</th><th>+</th><th>-</th><th>+</th></tr></thead><tbody><tr><td>Crude protein</td><td colspan="2">29.9<sup>2</sup></td><td colspan="2">31.8</td><td colspan="2">32.6</td><td colspan="2">32.0</td><td colspan="2">28.5</td></tr><tr><td>Starch</td><td colspan="2">43.6</td><td colspan="2">39.6</td><td colspan="2">41.4</td><td colspan="2">40.8</td><td colspan="2">42.4</td></tr><tr><td>Resistant starch</td><td>17.3</td><td>1.43</td><td>17.3</td><td>0.79</td><td>18.4</td><td>0.93</td><td>20.2</td><td>0.79</td><td>17.8</td><td>1.16</td></tr><tr><td>Oligosaccharides</td><td colspan="2">2.55<sup>2</sup></td><td colspan="2">2.57</td><td colspan="2">3.00</td><td colspan="2">2.36</td><td colspan="2">2.68</td></tr><tr><td>Raffinose</td><td colspan="2">0.13</td><td colspan="2">0.18</td><td colspan="2">0.24</td><td colspan="2">0.18</td><td colspan="2">0.22</td></tr><tr><td>Stachyose</td><td colspan="2">0.62</td><td colspan="2">0.62</td><td colspan="2">0.86</td><td colspan="2">0.85</td><td colspan="2">0.82</td></tr><tr><td>Verbascose</td><td colspan="2">1.80</td><td colspan="2">1.77</td><td colspan="2">1.90</td><td colspan="2">1.33</td><td colspan="2">1.64</td></tr><tr><td>Tannins (mg/g)</td><td colspan="2">0.060</td><td colspan="2">0.062</td><td colspan="2">0.064</td><td colspan="2">0.060</td><td colspan="2">0.068</td></tr><tr><td>Trypsin inhibitors (mg/g)</td><td>0.60</td><td>0.30</td><td>0.50</td><td>0.50</td><td>0.60</td><td>0.20</td><td>0.50</td><td>0.02</td><td>0.60</td><td>0.30</td></tr><tr><td>Phytic P</td><td>0.38</td><td>0.12</td><td>0.32</td><td>0.35</td><td>0.50</td><td>0.19</td><td>0.34</td><td>0.29</td><td>0.40</td><td>0.21</td></tr></tbody></table>

The AID of CP, S, amino acids and AME<sub>N</sub> value is shown in Table 2. The Extrusion cooking of faba bean seeds had a statistically significantly positive effect on all determined parameters. There was no interaction between extrusion cooking and cultivars for CP. Moreover, in the trial carried out on broiler chickens, interactions were established between the faba bean cultivars and the form of seed with respect to AME<sub>N</sub> values, AID of starch and amino acids (Table 2). The obtained interactions showed that the effect of extrusion cooking depended on low-tannin faba bean cultivars. Following the analysis of the examined cultivars, very high Pearson's correlations between phytic P contents in raw and extruded seeds and AME<sub>N</sub> as well as starch and amino acid digestibility were found. These results confirmed that the effect of extrusion cooking depended on phytic P and RS concentrations in seeds (Table 3).

<footnote n="1">¹“-” faba bean in raw form; “+” faba bean in extruded form</footnote>

<footnote n="2">²Two means are shown if the value of raw and extruded forms was statistically significantly different. If not – only one mean is presented</footnote>