The contents of AA as well as the concentration of TiO₂ as indigestible marker were determined in the digesta samples and in the feed. Ileal endogenous AA flow in birds fed the NFD was calculated as mg of amino acid (AA) flow per 1 kg of DM intake basis using the following formula reported by MOUGHAN et al. (1992):

$$ \text{IEAA (g/kg DM)} = [\text{AA in ileal digesta (mg/kg)}*(\text{diet TiO}_2, \text{mg/kg} / \text{ileal TiO}_2, \text{mg/kg})]. $$

The concentration of the marker in feed and digesta together with the content of AA in the feed and digesta were used to calculate the standardized ileal amino acid digestibility (SIAAD %) using the formula given by LEMME et al. (2004):

$$ \text{SIAAD (\%)} = \text{AID (\%)} + ((\text{IEAA flow, g/kg})/(\text{amino acid content of diet, g/kg}))*100 $$

where AID (%) apparent ileal digestibility= 100 - [(diet TiO₂, mg/kg /ileal digesta TiO₂, mg/kg) x (AAs in diet, g/kg/ AAs in ileal digesta, g/kg)] x 100

Data were subjected to one-factorial analysis of variance per tested sample of wheat (factor: protease supplementation), using the software ‘Stat Box Pro Agri’, version 7.1.9 (Grimmer soft, 1985-2011).

## Results

Quantification of endogenous intestinal AA losses indicated low losses (<3% of total AA) for Met, His and Cys whereas losses between 6.5% and 13.6% were recorded for Pro, Leu, Ser, Asp, Glu and Thr (Table 1). Although various methods are available to determine the endogenous losses, all indicate that the loss of Glu, Asp and Thr are high in comparison with the other essential AA (LEMME et al., 2004) and are due to mucin secretion.

Whereas several publications are available on AID of feed ingredients for poultry (GARCIA et al., 2007; ADEDOKUN et al., 2007), no data were reported on AID of feed ingredients supplemented with protease. SIAAD values of Lys (+ 2.0 %), Val (+ 3.7 %) and Thr (+ 4.8 %) from wheat DDGS were numerically increased with protease supplementation. Furthermore, SIAAD values of His and Gly from wheat middlings were significantly increased by 8.0 % and 5.5 %, respectively. Numerical improvements by more than 2% for SIAAD values of Ile & Phe (+2.8 %), Thr (+3.5 %), Ala & Tyr (+2.5 %) and Asp (+3.7 %) were also recorded. Improvement of SIAAD in the presence of protease is not the same for all AA and varies from one ingredient to another one. COWIESON and ROOS (2014) have shown that the response to the addition of the protease on AA digestibility may range from >5% for Thr and Cys to < 3% for Trp and Glu. The effects of added protease are dependent on feed composition and on intrinsic digestibility of AA. Indeed, the current results showed that SIAAD values of WM were much higher than those recorded for wheat DDGS which maybe influenced during the industrial processing (e.g. drying procedure).

Moreover, factors such as type and quality of the ingredients and the presence of anti-nutritional substances such as tannins, phytates, trypsin inhibitors in plant species also modulate the AID in poultry feed and thus the effectiveness of exogenous protease. Finally, the physiological state of animals (growth or maintenance), feed consumption or the nutritional feed quality could also influence the digestibility values. Characterization of raw materials is important to guide diet composition and to maximize the effects of protease supplementation.

## Conclusion

In this study, protease supplementation was effective in improving the standardized ileal digestibility of almost all the amino acids in wheat middlings.
<table><caption>**Table 1:** Ileal endogenous amino acid flow and standardized ileal digestibility coefficients (%) of amino acids (SIAAD) for broilers fed experimental diets</caption><thead><tr><th rowspan="3"></th><th colspan="4">IEAA (mg/kg DM)</th><th colspan="4">SIAAD (%)</th></tr><tr><th colspan="2">1</th><th colspan="2">4</th><th colspan="2">2</th><th colspan="2">3</th></tr><tr><th colspan="2">-</th><th colspan="2">+</th><th colspan="2">-</th><th colspan="2">+</th></tr><tr><th>Enzyme<sup>1</sup></th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>WM</th><th>p=</th><th>SEM</th><th>WDDGS</th><th>p=</th><th>SEM</th></tr></thead><tbody><tr><td><strong>EAAs<sup>2</sup></strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Arg</td><td>248</td><td>90.7</td><td>259</td><td>100.7</td><td>81.5</td><td>82.2</td><td>0.684</td><td>0.27</td><td>72.1</td><td>72.9</td><td>0.516</td><td>0.24</td></tr><tr><td>His</td><td>83</td><td>24.8</td><td>86</td><td>31.0</td><td>74.9</td><td>80.9</td><td>0.007</td><td>0.35</td><td>88.0</td><td>87.3</td><td>0.197</td><td>0.16</td></tr><tr><td>Ile</td><td>159</td><td>54.6</td><td>165</td><td>72.9</td><td>77.5</td><td>79.9</td><td>0.293</td><td>0.32</td><td>67.8</td><td>65.8</td><td>0.239</td><td>0.28</td></tr><tr><td>Leu</td><td>338</td><td>124.1</td><td>359</td><td>135.7</td><td>81.3</td><td>82.4</td><td>0.449</td><td>0.26</td><td>78.1</td><td>78.8</td><td>0.490</td><td>0.21</td></tr><tr><td>Lys</td><td>269</td><td>105.3</td><td>298</td><td>104.4</td><td>80.2</td><td>78.1</td><td>0.292</td><td>0.31</td><td>73.0</td><td>74.5</td><td>0.409</td><td>0.28</td></tr><tr><td>Met</td><td>68</td><td>16.6</td><td>67</td><td>30.9</td><td>83.4</td><td>83.4</td><td>0.945</td><td>0.24</td><td>74.2</td><td>75.2</td><td>0.490</td><td>0.25</td></tr><tr><td>Phe</td><td>203</td><td>74.7</td><td>214</td><td>68.9</td><td>81.4</td><td>83.5</td><td>0.142</td><td>0.26</td><td>83.3</td><td>83.4</td><td>0.990</td><td>0.19</td></tr><tr><td>Thr</td><td>651</td><td>175.1</td><td>701</td><td>166.3</td><td>68.9</td><td>71.3</td><td>0.272</td><td>0.31</td><td>65.4</td><td>68.6</td><td>0.115</td><td>0.31</td></tr><tr><td>Val</td><td>170</td><td>66.2</td><td>188</td><td>69.4</td><td>78.2</td><td>78.9</td><td>0.743</td><td>0.30</td><td>70.0</td><td>72.6</td><td>0.175</td><td>0.30</td></tr><tr><td><strong>NEAAs<sup>3</sup></strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ala</td><td>270</td><td>76.8</td><td>287</td><td>90.9</td><td>74.8</td><td>76.4</td><td>0.358</td><td>0.28</td><td>67.6</td><td>65.5</td><td>0.178</td><td>0.27</td></tr><tr><td>Asp</td><td>473</td><td>127.2</td><td>515</td><td>176.1</td><td>73.5</td><td>76.0</td><td>0.177</td><td>0.29</td><td>47.7</td><td>42.7</td><td>0.053</td><td>0.36</td></tr><tr><td>Cys</td><td>138</td><td>23.7</td><td>152</td><td>13.8</td><td>75.2</td><td>74.8</td><td>0.809</td><td>0.28</td><td>67.9</td><td>68.7</td><td>0.707</td><td>0.31</td></tr><tr><td>Glu</td><td>570</td><td>178.3</td><td>620</td><td>221.5</td><td>88.7</td><td>89.6</td><td>0.314</td><td>0.21</td><td>85.0</td><td>83.7</td><td>0.129</td><td>0.20</td></tr><tr><td>Gly</td><td>266</td><td>73.3</td><td>286</td><td>62.6</td><td>85.6</td><td>90.3</td><td>0.058</td><td>0.35</td><td>60.4</td><td>52.7</td><td>0.068</td><td>0.45</td></tr><tr><td>Pro</td><td>311</td><td>86.0</td><td>351</td><td>62.0</td><td>86.2</td><td>86.9</td><td>0.410</td><td>0.20</td><td>84.4</td><td>84.5</td><td>0.869</td><td>0.19</td></tr><tr><td>Ser</td><td>395</td><td>104.0</td><td>439</td><td>105.7</td><td>76.8</td><td>75.8</td><td>0.752</td><td>0.38</td><td>72.7</td><td>72.7</td><td>0.990</td><td>0.23</td></tr><tr><td>Tyr</td><td>168</td><td>56.8</td><td>184</td><td>53.4</td><td>79.2</td><td>80.7</td><td>0.278</td><td>0.31</td><td>79.6</td><td>79.8</td><td>0.898</td><td>0.26</td></tr></tbody></table>

## Literature

ADEDOKUN, S.A., O. ADEOLA, C. M. PARSONS, M. S. LILBURN, T. J. APPLEGATE, 2008: Standardized ileal amino acid digestibility of plant feedstuffs in broiler chickens and turkeys poults using a nitrogen-free or casein diet. Poult. Sci. **87**, 2535-2548

COWIESON A.J, ROOS F. F., 2014: Bioefficacy of a mono-component protease in the diets of pigs and poultry: a meta-analysis of effect on ileal amino acid digestibility. J. Appl Ani. Nut. **2**, 1-8

GARCIA, A. R., A. B. BATAL, N. M. DALE, 2007: A comparison of methods to determine amino acid digestibility of feed ingredients for chickens. Poult. Sci. **86**, 94-101

LEMME, A., V. RAVINDRAN, W. L. BRYDEN, 2004: Ileal digestibility of amino acid in feed ingredients for broilers. World's Poult. Sci. J. **60**, 423-437

MOUGHAN, P. J., G. SCHUTTERT, M. LEENAARS, 1992: Endogenous amino acid flow in the stomach and small intestine of the young growing rat. J. Sci. Food Agric. **60**, 437-442

<footnote n="1">¹- No added enzyme, + Added enzyme (Ronozyme® ProAct at 15000 PROT/kg); ²EAAs: essential amino acids; ³NEAAs: non-essential amino acids</footnote>
P-090 (ID 286)

# EFFECTS OF INCLUSION OF A MONO COMPONENT SERINE PROTEASE ON APPARENT ILEAL DIGESTIBILITY OF CRUDE PROTEIN AND AMINO ACIDS OF SOYBEAN MEALS OF DIFFERENT ORIGINS IN BROILERS

L. Cámara<fnref n="1" />,<fnref n="2" />, M. V. Kimiaeitalab<fnref n="1" />, B. Saldaña<fnref n="1" />, H. M. Mandalawi<fnref n="1" />, C. R. Angel<fnref n="3" />, A. Smith<fnref n="4" />, and G. G. Mateos<fnref n="1" />,<fnref n="2" />

**Corresponding author:** gonzalo.gmateos@upm.es

## Abstract

We studied the effects of including in the diet a mono component serine protease (PRO; RONOZYME® ProAct, DSM Nutritional Products, Basel, Switzerland) on apparent ileal digestibility (AID) of DM, CP and AA of soybean meals (SBM) of USA (n=3), Brazil (n=3) and Argentina (n=3) origin, in 22 d-old broilers. The 6 experimental treatments formed a 3 x 2 factorial with 3 SBM origins (USA, BRA and ARG) and 2 levels of PRO (0 and 200 mg/kg to give 0 and 15,000 PROT units/kg feed). Three different batches of each SBM were nested within each of the 3 SBM origins. Each of the 6 treatments were replicated 24 times (8 times per batch). The experimental unit was a cage with 8 broilers. Birds were fed a commercial corn-soybean meal diet from 1 to 18 d of age followed by the experimental diets that consisted of a mixture of 42.6% SBM and 57.4% of a nitrogen free diet for 4 d. The supplementation with PRO improved the AID of all the AA (ranging from 0.5 to 1.6 percentage units) but the differences were not significant. The AID of DM, CP and Lys was 1.5%, 1.1% and 1.0% higher in bird fed PRO than in birds fed the un-supplemented diets (P > 0.10). In general, the AID of DM, CP and AA was higher for the USA meals than for the South America meals but the differences were not significant. In addition, the AMEn of the diets increased by 1.1% as an average, with PRO inclusion (P < 0.001). In summary, the inclusion of RONOZYME® ProAct increased numerically the AID of all the AA of the SBM and the DM of the diets but the differences were not significant.

**Keywords:** amino acid digestibility, broiler, protease, soybean meal origin

## Introduction

Soybean meal (SBM) is the most common source of protein and amino acids (AA) in poultry diets worldwide. This is due to its high CP content, and the excellent AA profile that complements that of the cereals, and the high AA digestibility. SBM contributes up to 70% of dietary CP in a typical corn-soybean meal broiler diet. The United States (US), Brazil (BRA), and Argentina (ARG) are the main suppliers of meals in the global SBM market. (RAVINDRAN et al., 2014). The AA profile and quality of the CP of commercial SBM depend on factors such as processing conditions during oil extraction as well as soil, latitude and environmental variables during the growing and harvesting of the beans. Therefore, the nutritive value of SBM might vary depending on the crushing plant and the country of origin of the beans (GARCÍA-REBOLLAR et al., 2014). Research on the use of exogenous enzymes in broiler diets has been ongoing for decades. However, the commercial use of enzymes, especially of proteases, is more recent. The interpretation of results from studies conducted with proteases is sometimes difficult because of the confounding effects of the presence of more than one enzymatic activity in a single treatment (FREITAS et al., 2011). The objective of this study was to study the influence of a mono component serine protease on the AA digestibility of soybean from different origins in broilers of 22d of age.

## Material and Methods

In total 1,152 one-d-old (Ross 308) broiler chicks (divided into 2 blocks) were collected from a commercial hatchery (Avícola Moraleja, S. A., Madrid, Spain). After arrival to the experimental farm, chicks were weighed and distributed in groups of 8 in battery cages in an environmentally controlled room. All cages had wire flooring and were equipped with 2 nipple drinkers and an open trough feeder. Birds were fed a commercial corn-soybean meal diet from 1 to 18 d of age followed by the experimental diets that consisted of a mixture of 42.6% SBM and 57.4% of a nitrogen free diet, for 4 d. At d 22, birds were weighed and euthanized by CO₂ asphyxiation and fresh digesta was collected from the terminal ileum, defined as the segment of the gastrointestinal tract from 15 to 2 cm anterior to the ileocaecal junction. Ileal digesta was collected by gently flushing the contents with distilled water. The contents from the 8 birds within the cage

<footnote n="1">¹ Departamento de Producción Agraria, Universidad Politécnica de Madrid, 28040 Madrid, Spain</footnote>

<footnote n="2">² CEI Campus Moncloa, UCM-UPM, 28040 Madrid, Spain</footnote>

<footnote n="3">³ Department of Animal and Avian Sciences, University of Maryland, College Park, 20742, Maryland, USA</footnote>

<footnote n="4">⁴ DSM Nutritional Products (UK) Ltd. Heanor Gate Industrial Estate, DE 75 7SG Heanor, Derbyshire, UK</footnote>
were pooled and frozen (-20 °C) until the AID of the DM, CP and AA could be determined. The 6 experimental treatments formed a 3 x 2 factorial with 3 SBM origins (USA, BRA and ARG) and 2 levels of PRO (0 and 200 mg/kg to give 0 and 15,000 PROT units/kg feed). Three batches of SBM were nested within each of the 3 SBM origins. Each of the 6 treatments was replicated 24 times (8 times per batch) and the experimental unit was a cage with 8 broilers.

## Results

The interactions between origin and supplementation with protease was not significant and therefore only main effects are presented (Table 1). The supplementation with PRO improved the AID of all the AA (ranging from 0.5 to 1.6 percentage units) but the differences were not significant. The AID of DM (73.5 vs. 72.4%), CP (77.8 vs. 77.0%) and Lys (81.0 vs. 80.1%) was 1.5%, 1.1% and 1.0% higher in birds fed PRO than in birds fed the un-supplemented diets (P > 0.10). In general, the AID of DM (73.9 vs. 72.5 and 72.6%) and CP (78.2 vs. 76.8 and 77.4%) was higher for the USA meals than for BRA and ARG meals but the differences were not significant. Also for most AA the AID was higher for USA SBM than BRA and ARG SBM but the differences were not significant. The AMEn of the diets increased by 1.1% as an average with PRO inclusion (P < 0.001) (data not shown).

## Conclusions

The inclusion of RONOZYME® ProAct in the diet increased the AID of all the AA in the SBM and the DM of the diets, regardless of SBM origin, but the differences were not significant. The apparent ileal digestibility of DM, CP and almost all AA were numerally higher for the USA than for the ARG and BRA SBM.

<table><caption>**Table 1.** Apparent ileal digestibility of the amino acids of soybean meal of different origins with and without protease</caption><thead><tr><th></th><th>DM</th><th>Thr</th><th>Cys</th><th>Val</th><th>Met</th><th>Iso</th><th>Lys</th><th>Total</th><th>CP</th></tr></thead><tbody><tr><td><strong>Origin</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>USA</td><td>73.9</td><td>73.5</td><td>65.2</td><td>79.3</td><td>79.2</td><td>81.2</td><td>81.4</td><td>80.1</td><td>78.2</td></tr><tr><td>BRA</td><td>72.5</td><td>72.5</td><td>64.4</td><td>79.0</td><td>79.5</td><td>80.9</td><td>79.8</td><td>79.4</td><td>76.8</td></tr><tr><td>ARG</td><td>72.6</td><td>72.1</td><td>65.1</td><td>78.8</td><td>77.9</td><td>80.4</td><td>80.4</td><td>79.1</td><td>77.4</td></tr><tr><td>SEM<sup>1</sup></td><td>0.548</td><td>0.861</td><td>0.773</td><td>0.568</td><td>0.566</td><td>0.572</td><td>0.793</td><td>0.577</td><td>0.487</td></tr><tr><td><strong>Protease</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Without</td><td>72.4</td><td>72.9</td><td>64.2</td><td>78.4</td><td>78.0</td><td>80.4</td><td>80.1</td><td>79.1</td><td>77.0</td></tr><tr><td>With</td><td>73.5</td><td>72.6</td><td>65.5</td><td>79.6</td><td>79.6</td><td>81.3</td><td>81.0</td><td>79.9</td><td>77.8</td></tr><tr><td>SEM<sup>2</sup></td><td>0.470</td><td>0.688</td><td>0.756</td><td>0.622</td><td>0.825</td><td>0.582</td><td>0.616</td><td>0.571</td><td>0.571</td></tr><tr><td><strong>P-value</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Origin</td><td>0.200</td><td>0.551</td><td>0.658</td><td>0.869</td><td>0.178</td><td>0.599</td><td>0.423</td><td>0.460</td><td>0.371</td></tr><tr><td>Protease</td><td>0.101</td><td>0.729</td><td>0.231</td><td>0.194</td><td>0.174</td><td>0.297</td><td>0.274</td><td>0.327</td><td>0.325</td></tr></tbody></table>

The interaction was not significant

## Literature

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

GARCÍA-REBOLLAR, P., DE BLAS, C., LÁZARO, R., CÁMARA, L., MATEOS, G. G. 2014. Origin of the beans affects protein quality and nutritive value of commercial soybean meals. Poult. Sci. **93** (E-Suppl. 1), 145 (Abstr.).

MATEOS, G. G., SUEIRO, S., GONZÁLEZ, M., HERMIDA, M., FICKLER, J., REBOLLAR, P. G., SERRANO, M. P., LÁZARO, R. 2011: Differences among origins on nutritional and quality parameters of soybean meal.

RAVINDRAN, V., ABDOLLAHI, M. R., BOOTWALLA, S. M. 2014: Nutrient analysis, metabolizable energy, and digestible amino acids of soybean meals of different origins for broilers. Poult. Sci. **93**, 2567-2577.

<footnote n="1">¹ Standard error of the mean (16 replicates of 8 chicks each one).</footnote>

<footnote n="2">² Standard error of the mean (72 replicates of 8 chicks each one).</footnote>
P-091 (ID 284)

INFLUENCE OF PROTEASE ENZYME SUPPLEMENTATION ON APPARENT ILEAL DIGESTIBILITY OF AMINO ACIDS OF CORN, WHEAT AND BARLEY GRAINS

L. Cámara<fnref n="1" />,<fnref n="2" />, M. V. Kimiaeitalab<fnref n="1" />, J. D. Berrocoso<fnref n="1" />, C. E. Grewehr<fnref n="1" />,<fnref n="3" />, P. Guzmán<fnref n="1" />, C. R. Angel<fnref n="4" />, A. Smith<fnref n="5" />, and G. G. Mateos<fnref n="1" />,<fnref n="2" />

**Corresponding author:** gonzalo.gmateos@upm.es

**Abstract**

We studied the effects of a commercial multi-enzyme complex [with a minimum of 8,000 U/g of endo-1,4-β-glucanase, 18,000 U/g of endo-1,3(4)-β-glucanase and 26,000 U/g of endo-1,4-β-xylanase (XYL) (Roxazyme G2 G, DSM Nutritional Products)] and a mono component serine protease (PRO; Ronozyme ProAct, DSM Nutritional Products) on the apparent ileal digestibility (AID) of DM, CP and AA of 3 varieties of the 3 cereals in 22-d old broilers. The 12 experimental treatments formed a 3 x (2 x 2) factorial, with 3 main cereals (corn, wheat and barley), 2 levels of XYL (0 vs. 100 mg/kg), and 2 levels of PRO (0 vs. 200 mg/kg) with cereal variety nested within the corresponding cereal. Each treatment was replicated 24 times. Birds were fed the experimental diets from 18 to 22 d (92:8 mixture of the cereal and a N free diet). The AID of DM, Met and Leu, across variety and enzyme supplementation, was higher for corn than for barley with that of wheat being intermediate (P < 0.05). The inclusion of PRO increased (P < 0.05) Gly AID and tended to increase (P < 0.10) the AID of DM and of most AA, including Cys, Glu, Met, Thr and Val. The inclusion of XYL improved (P < 0.05) the AID of DM and of CP, and all AA, except Met and Lys. The combination of both enzymes did not improve AA digestibility further. Trends observed, when combining different enzyme activities in short term digestibility trials, need further study and may not reflect the response seen combining different enzyme combinations on animal performance studies or when fed as part of a complete diet from hatching to slaughter.

**Keywords:** amino acid digestibility, broiler, cereal, protease, xylanase

**Introduction**

Cereals are the most widely used ingredients as energy sources in poultry feeds. In addition to supplying starch, cereals also provide part of the CP and amino acids (AA) requirement of the bird. Starch utilization by poultry depends on the cereal used, because starches differ in their nature and structure. Corn and wheat are the two most commonly used cereals in poultry diets. In some countries, barley is also a commercial alternative. Corn has less protein and dietary fiber and more starch, fat and AMEn than wheat or barley (FEDNA, 2010). In addition, the chemical composition and nutritive value of corn is quite uniform compared to that of wheat and barley, but in all grains the energy and AA contents vary depending on factors such as cultivar, agronomic practices, weather conditions and length of storage (FRIKHA et al., 2011; PÉREZ BONILLA et al., 2011). Wheat contains relatively high levels of non-starch polysaccharides (NSP) in cell walls and these cannot be digested in the small intestine of non-ruminants because of the lack of adequate presence of enzymes in their digestive tract. The accumulation of NSP in the alimentary tract can result in water retention and an increase in the viscosity of the digestive contents. High digesta viscosity has a negative effect on nutrient digestion and absorption including that of the AA's. Consequently, exogenous enzymes (carbohydrases) are required to improve nutrient utilization (REBOLÉ et al., 2010). The objective of this work was to determine the effect of exogenous carbohydrates used with or without proteases on energy and amino acids (AA) digestibility of cereal grains.

**Material and Methods**

A commercial multi-enzyme complex with β-glucanase and β-xylanase activity (XYL; ROXAZYME® G2 G, DSM Nutritional Products) and a mono component serine protease (PRO; RONOZYME® ProAct, DSM Nutritional Products) were used to study the apparent ileal digestibility (AID) of DM, CP and AA in three different cereals. The experiment was carried out in 4 time blocks due to facility limitation. In each block, 576 one-d-old male broiler chicks (Ross 308) were used. On arrival at the experimental farm, chicks were weighed and distributed in groups of 8 into 72 battery cages in

<footnote n="1">¹ Departamento de Producción Agraria, Universidad Politécnica de Madrid, 28040 Madrid, Spain</footnote>

<footnote n="2">² CEI Campus Moncloa, UCM-UPM, 28040 Madrid, Spain</footnote>

<footnote n="3">³ Current address: Universidade do Estado de Santa Catarina, Lages, Brazil</footnote>

<footnote n="4">⁴ Department of Animal and Avian Sciences, University of Maryland, College Park, 20742, Maryland, USA</footnote>

<footnote n="5">⁵ DSM Nutritional Products (UK) Ltd., Heanor Gate Industrial Estate, DE75 7SG Heanor, Derbyshire, UK</footnote>
an environmentally controlled room. All cages had wire flooring and were equipped with 2 nipple drinkers and an open trough feeder. Birds were fed a commercial corn-soybean meal based diet from 1 to 18 d followed by the experimental diets (92:8 mixture of the cereal and a nitrogen free diet) to 22 d. On d 22, birds were weighed and euthanized by CO2 asphyxiation and fresh digesta was collected from the terminal ileum, defined as the segment of the gastrointestinal tract from 15 to 2 cm anterior to the ileocaecal junction. Ileal digesta was collected by gently flushing the contents with distilled water. The contents, from the 8 birds within the cage were pooled and frozen (-20 °C) until the AID of the DM, CP and AA could be determined. The 12 experimental treatments were formed by a 3 x (2 x 2) factorial arrangement of 3 main cereals (corn, wheat and barley), 2 levels of XYL (0 vs. 100 mg/kg), and 2 levels of PRO (0 vs. 200 mg/kg) with cereal variety (n= 3) of each of the 3 main cereals nested within the corresponding cereal. In total, each main treatment was replicated 24 times.

## Results

The AID of DM (81.8 vs. 71.3 and 72.1%, respectively), Met (85.6 vs. 78.4 and 82.2, respectively) and Leu (87.3 vs. 78.2 and 82.3%, respectively), across variety and enzyme supplementation, were higher for corn than for barley with that of wheat being intermediate (P < 0.05) (Table 1). The AID of Ala (85.9 vs. 74.6 and 72.0; P < 0.01, respectively) was higher for corn than for wheat and barley. However, the AID of Pro was higher for wheat than for corn and barley (90.9 vs. 84.5 and 84.3; P < 0.05, respectively) and the Glu was higher for wheat than for barley, with that of corn being intermediate (92.5 vs. 85.4 and 86.6; P < 0.05, respectively). The inclusion of xylanases improved the digestibility of DM (75.4 vs. 74.5; P < 0.01), CP (77.8 vs. 75.7; P < 0.01) and of all the AA (P < 0.01) except for Met and Lys that were not affected. The inclusion of PRO increased (P < 0.05) AID of Gly and tended to increase (P < 0.10) that of DM and of most AA, including Cys, Glu, Met, Thr and Val The combination of both enzymes did not improve AA digestibility further.

## Conclusions

The AID of most of the AA was higher for corn and wheat than for barley. The inclusion of a multi-enzyme complex with β-glucanase and β-xylanase activity increased the AID of DM, CP and of all the AA, except Met and Lys. The inclusion of the mono component serine protease increased the AID of Gly and tended to increase the digestibility of most of the other AA. The combination of both enzymes did not improve AA digestibility further. Trends observed when combining different enzyme activities in short term digestibility trials need further study and may not reflect the response seen combining different enzyme combinations on animal performance studies or digestibility studies when fed as part of a complete diet from hatch to slaughter.

## Literature

FUNDACIÓN ESPAÑOLA DESARROLLO NUTRICIÓN ANIMAL, 2010. In Normas FEDNA de composición y valor nutritivo de alimentos para la fabricación de piensos compuestos. 3rd ed. C. de Blas, G. G. Mateos and P. G. Rebollar. Eds. FEDNA, Madrid, Spain.

FRIKHA, M., SAFAA, H. M., SERRANO, M. P., JIMÉNEZ-MORENO, E., LÁZARO, R., MATEOS, G. G. 2011. Influence of the main cereal in the diet and particle size of the cereal on productive performance and digestive traits of brown-egg laying pullets. Anim. Feed Sci. Technol. **164**, 106-115.

PÉREZ-BONILLA, A., FRIKHA, M., MIRZAIE, S., GARCÍA, J., MATEOS, G. G. 2011. Effects of the main cereal and type of fat of the diet on productive performance and egg quality of brown-egg laying hens from 22 to 54 weeks age. Poult. Sci. **90**, 2801-2810.

REBOLÉ, A., ORTIZ, L. T., RODRÍGUEZ, M. L., ALZUETA, C., TREVIÑO, J., VELASCO, S. 2010. Effects of inulin and enzyme complex, individually or in combination, on growth performance, intestinal microflora, cecal fermentation characteristics, and jejunal histomorphology in broiler chickens fed a wheat-and barley based diet. Poult. Sci. **89**, 276-286.
<table><caption>**Table 1.** Apparent ileal amino acid digestibility (%) of the amino acid of the main cereal of the diet with and without enzymes</caption><thead><tr><th></th><th>DM</th><th>Thr</th><th>Glu</th><th>Pro</th><th>Gly</th><th>Ala</th><th>Cys</th><th>Val</th><th>Met</th><th>Leu</th><th>Lys</th><th>CP</th></tr></thead><tbody><tr><td><strong>Cereal</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Corn</td><td>81.3<sup>a</sup></td><td>66.6</td><td>86.6<sup>ab</sup></td><td>84.5<sup>b</sup></td><td>72.8</td><td>85.9<sup>a</sup></td><td>76.4</td><td>76.2</td><td>85.6<sup>a</sup></td><td>87.3<sup>a</sup></td><td>76.5</td><td>76.4</td></tr><tr><td>Wheat</td><td>72.1<sup>ab</sup></td><td>69.4</td><td>92.5<sup>a</sup></td><td>90.9<sup>a</sup></td><td>75.2</td><td>74.6<sup>b</sup></td><td>81.8</td><td>76.1</td><td>82.2<sup>ab</sup></td><td>82.3<sup>ab</sup></td><td>73.1</td><td>79.7</td></tr><tr><td>Barley</td><td>71.3<sup>b</sup></td><td>66.7</td><td>85.4<sup>b</sup></td><td>84.3<sup>b</sup></td><td>70.3</td><td>72.0<sup>b</sup></td><td>76.6</td><td>74.5</td><td>78.4<sup>b</sup></td><td>78.2<sup>b</sup></td><td>73.7</td><td>74.1</td></tr><tr><td>SEM<sup>1</sup> (n=32)</td><td>2.252</td><td>2.772</td><td>1.424</td><td>1.534</td><td>2.568</td><td>1.499</td><td>2.325</td><td>2.270</td><td>1.262</td><td>1.395</td><td>2.138</td><td>2.260</td></tr><tr><td><strong>Enzyme</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Xylanase Protease</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Without Without</td><td>71.8<sup>b</sup></td><td>63.3<sup>b</sup></td><td>86.2<sup>b</sup></td><td>84.2<sup>b</sup></td><td>68.9<sup>b</sup></td><td>74.2<sup>c</sup></td><td>74.6<sup>c</sup></td><td>71.8<sup>b</sup></td><td>79.6<sup>b</sup></td><td>80.1<sup>c</sup></td><td>71.3<sup>b</sup></td><td>73.5<sup>c</sup></td></tr><tr><td>With With</td><td>76.4<sup>a</sup></td><td>69.1<sup>a</sup></td><td>88.7<sup>a</sup></td><td>87.2<sup>a</sup></td><td>74.3<sup>a</sup></td><td>79.0<sup>ab</sup></td><td>79.7<sup>ab</sup></td><td>77.0<sup>a</sup></td><td>83.5<sup>a</sup></td><td>83.5<sup>ab</sup></td><td>75.9<sup>a</sup></td><td>78.0<sup>ab</sup></td></tr><tr><td>With Without</td><td>77.1<sup>a</sup></td><td>70.2<sup>a</sup></td><td>89.5<sup>a</sup></td><td>88.2<sup>a</sup></td><td>75.1<sup>a</sup></td><td>79.8<sup>a</sup></td><td>81.0<sup>a</sup></td><td>78.2<sup>a</sup></td><td>83.4<sup>a</sup></td><td>84.5<sup>a</sup></td><td>76.4<sup>a</sup></td><td>79.0<sup>a</sup></td></tr><tr><td>With With</td><td>74.5<sup>ab</sup></td><td>67.5<sup>a</sup></td><td>88.3<sup>a</sup></td><td>86.7<sup>a</sup></td><td>72.8<sup>a</sup></td><td>77.0<sup>b</sup></td><td>78.0<sup>b</sup></td><td>75.6<sup>a</sup></td><td>81.6<sup>ab</sup></td><td>82.2<sup>b</sup></td><td>74.2<sup>ab</sup></td><td>76.5<sup>b</sup></td></tr><tr><td>SEM<sup>1</sup> (n=72)</td><td>0.709</td><td>0.781</td><td>0.404</td><td>0.407</td><td>0.727</td><td>0.687</td><td>0.587</td><td>0.731</td><td>0.623</td><td>0.549</td><td>1.021</td><td>0.669</td></tr><tr><td><strong>Xylanase</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Without</td><td>74.1</td><td>66.2</td><td>87.5</td><td>85.7</td><td>71.6</td><td>76.6</td><td>77.1</td><td>74.4</td><td>81.6</td><td>81.8</td><td>73.6</td><td>75.7</td></tr><tr><td>With</td><td>75.8</td><td>68.9</td><td>88.9</td><td>87.5</td><td>74.0</td><td>78.4</td><td>79.5</td><td>76.9</td><td>82.5</td><td>83.4</td><td>75.3</td><td>77.8</td></tr><tr><td><strong>Protease</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Without</td><td>74.5</td><td>66.8</td><td>87.8</td><td>86.2</td><td>72.0</td><td>77.0</td><td>77.8</td><td>74.9</td><td>81.5</td><td>82.3</td><td>73.9</td><td>76.3</td></tr><tr><td>With</td><td>75.4</td><td>68.3</td><td>88.5</td><td>87.0</td><td>73.5</td><td>78.0</td><td>78.8</td><td>76.3</td><td>82.6</td><td>82.9</td><td>75.0</td><td>77.2</td></tr><tr><td>SEM<sup>1</sup> (n=144)</td><td>0.502</td><td>0.552</td><td>0.285</td><td>0.288</td><td>0.514</td><td>0.486</td><td>0.415</td><td>0.517</td><td>0.441</td><td>0.388</td><td>0.722</td><td>0.473</td></tr><tr><td><em>P-value</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cereal</td><td>0.029</td><td>0.673</td><td>0.027</td><td>0.038</td><td>0.458</td><td>0.001</td><td>0.254</td><td>0.837</td><td>0.019</td><td>0.011</td><td>0.538</td><td>0.288</td></tr><tr><td>Xylanase x protease</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.002</td><td>&lt;0.001</td></tr><tr><td>Xylanase</td><td>0.002</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.001</td><td>0.007</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.123</td><td>0.004</td><td>0.109</td><td>0.003</td></tr><tr><td>Protease</td><td>0.096</td><td>0.069</td><td>0.082</td><td>0.055</td><td>0.041</td><td>0.142</td><td>0.090</td><td>0.065</td><td>0.069</td><td>0.233</td><td>0.268</td><td>0.134</td></tr></tbody></table>

<footnote n="1">¹ The experimental unit was a cage with 8 chicks.</footnote>
P-093 (ID 202)

# THE EFFECT OF SUPPLEMENTING A PURIFIED BACTERIAL ENDO-XYLANASE IN THE DIET OF OLD LAYING HENS ON PRODUCTION AND EGG SHELL QUALITY

E. Delezie<fnref n="1" />, R. Mombaerts<fnref n="2" />, A. Goderis<fnref n="2" />, S. Leleu<fnref n="1" />, L. Maertens<fnref n="1" />

**Corresponding author:** evelyne.delezie@ilvo.vlaanderen.be

## Abstract

A trial was set up to investigate the effect of a bacterial endo-xylanase (B) in the diet of laying hens, 54 weeks old, on performance and egg shell quality. Two dietary treatments were studied: a standard wheat-corn based diet supplemented without (NC) or with B (100 mg/kg feed). Five replicates of 18 laying hens were allocated to each of these two treatments during five consecutive periods of 28 days. Supplementing the feed with B significantly improved the laying percentage (87.9 vs 83.7%) and egg mass (58.3 vs 55.3 g) resulting in a significant better feed conversion ratio (1.974 vs 2.085). The incidence of broken and dirty eggs was moderate and comparable between treatment groups, whereas the incidence of soft-shelled eggs was significantly lower (0.04 vs 0.47%) for the B supplemented group. Egg quality was not influenced by treatment, only for the Haugh units (HU) a significantly higher value for the laying hens fed the diets supplemented with B (90.14 vs 87.60) was noticed. To conclude, supplementing a bacterial endo-1,4-β-xylanase resulted in a more efficient egg production and increased HU when compared to the NC diet. Based on these results, we hypothesize that this bacterial xylanase not only reduces the anti-nutritional effects of the non-starch polysaccharides but also creates prebiotic arabinoxylan-oligosaccharides which are fermented in the ceca to produce high levels of butyrate.

**Keywords:** xylanase, bacterial, laying hens, performance, egg quality

## Introduction

The objective of the current trial is to provide efficacy data of non-starch polysaccharide (NSP) enzymes for use in laying hens. For this purpose a bacterial endo-xylanase (B) was supplemented in a standard wheat corn based diet. The bioefficacy of the NSP enzyme was determined in a zootechnical performance trial.

## Material and Methods

Laying hens (Lohmann brown), 54 weeks of age at the start of the experiment, were used in this trial. In each pen unit 18 laying hens were housed divided over 2 enriched cages (9 hens/cage) and reared under conventional conditions for lighting, heating and ventilation. Drinking water and feed (in finely ground meal form) was provided *ad libitum*. Two dietary treatments were studied: a standard wheat-corn based diet supplemented without (NC) or with a bacterial endo-xylanase (B) (Nutrase Xyla, 100 mg/kg feed). The trial was designed as a 2-factorial Complete Block Design (2 treatments x 5 replicates) with 18 medium weight laying hens per pen with a total number of 180 birds. Duration of the trial was 140 days as there were 5 periods of each 28 days.

Average pen body weight was recorded at the start and at the end of the experiment. Feed intake was recorded and feed conversion and daily feed intake per bird were calculated for each period per 18 laying hens. Daily number of eggs per pen unit of 18 laying hens were counted and every 2 weeks eggs were weighed. Furthermore, every day the incidence of cracked, soft-shelled and dirty eggs was recorded. Daily mortality and cullings were recorded for each pen. Corrections for mortality calculating zootechnical performances were done using the number of 'animal days' (number of animals x days alive). Egg quality was measured in terms of egg shell deformation (under a 500 g load, in micrometer), yolk colour, egg shell thickness and Haugh Units at the start, during period 3 and at the end of the trial. The thickness of the eggshell, after the removal of the egg content and the egg membranes, was measured (μm) with a micrometer at 3 different areas. The egg content was broken onto a glass-top table with a mirror, the thick albumen height was measured at the distance of approximately 1 cm from the yolk. The height of thick albumen in mm (H) and the egg weight in g (W) were used to calculate the Haugh Units (HU) from following formula (Monira et al., 2003): $HU = 100 \log (H - (1.7 * W^{0.37}) + 7.6))$. The yolk colour was determined with the 15-point scale of Roche.

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

<footnote n="2">²Nutrex nv, Achterstenhoek 5, 2275 Lille, Belgium</footnote>
All parameters were subjected to a factorial analysis of variance (“2 x 5 x 5 / 2 dietary treatments x 5 blocks x 5 periods”) and the corresponding LSD-multiple range test (with P ≤ 0.05 as the threshold value) (STATISTICA 64.0).

## Results

There was a significant effect of period on laying rate, egg weight and daily feed intake. Laying rate significantly decreased (P = 0.030) whereas egg weight increased with the age of the laying hens. In general, egg mass decreased with ongoing age of the laying hens but not at the 5% significance level. Supplementing the feed with B significantly affected the laying percentage (P = 0.020), daily egg mass (P = 0.016) and feed conversion (P = 0.016). Hens fed the diet supplemented with B had a significantly higher laying percentage compared to the other group of laying hens. Due to the differences in laying percentage, daily egg mass was significantly higher resulting in a significantly better feed conversion for the B supplemented broilers compared to their counterparts. The percentage of broken and dirty eggs was moderate and comparable between treatments but the incidence of soft-shelled eggs was significantly lower for the B supplemented group (P = 0.028 (Table 1)).

<table><caption>**Table 1:** Effect of dietary treatment on laying rate (%), egg mass (g), feed conversion, incidence of soft-shelled eggs (%) and haugh units (n = 5)</caption><thead><tr><th></th><th>Laying rate (%)</th><th>Egg mass (g)</th><th>Feed conversion</th><th>Soft-shelled eggs (%)</th><th>Haugh Units</th></tr></thead><tbody><tr><td>Control diet</td><td>83.7<sup>b</sup></td><td>55.3<sup>b</sup></td><td>2.085<sup>b</sup></td><td>0.47<sup>b</sup></td><td>87.60<sup>b</sup></td></tr><tr><td>B-supplemented diet</td><td>87.9<sup>a</sup></td><td>58.3<sup>a</sup></td><td>1.974<sup>a</sup></td><td>0.04<sup>a</sup></td><td>90.14<sup>a</sup></td></tr><tr><td>P-value</td><td>0.020</td><td>0.016</td><td>0.016</td><td>0.028</td><td>0.010</td></tr></tbody></table>

It is obvious from the results that egg quality changed with the age of the hen: with increasing age of the hens egg shell deformation was higher, egg shells became thinner and Haugh unit values decreased. A significantly higher percentage broken and dirty eggs were obtained during the last periods of the experiment this is in accordance with the thinner egg shell and higher egg shell deformation at this stage of the trial.

There was no treatment effect on egg quality only for the Haugh units a significant higher value was reached if laying hens were fed the B supplemented diet (Table 1). No interaction between period and treatment was present indicating that these effects were similar for each period.

At the start and the end of the trial, body weight of the hens was not significantly different between treatments. During the experiment laying hens lost some weight, but this was comparable between treatments. No significant effect of dietary treatments on body weight or body weight loss was observed.

Mortality percentage during the trial was rather high and main causes were cannibalism and age of the laying hens. Due to the low number of birds per treatment no statistical conclusions can be drawn.

## Conclusions

Feeding laying hens the diet supplemented with B significantly influenced their performance results. By supplementing the bacterial endo-xylanase a significantly higher laying percentage, daily egg mass and lower feed conversion was obtained. The incidence of broken and dirty eggs was comparable between groups whereas the incidence of soft-shelled eggs was significantly higher if laying hens were given the control diet. No dietary treatment effect could be detected for egg shell quality, but internal egg quality was significantly higher for the B supplemented laying hens. Based on these results, we hypothesize that this bacterial xylanase not only reduces the anti-nutritional effects of the non-starch polysaccharides but also creates prebiotic arabinoxylan-oligosaccharides which are fermented in the ceca to produce high levels of butyrate inducing an increased bioavailability of minerals (COURTIN et al., 2008).

## 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, AND J.A., DELCOUR, 2008: Dietary Inclusion of Wheat Bran Arabinoxylan-oligosaccharides Induces Beneficial Nutritional Effects in Chickens. Cereal Chem., **85**(5): 607-613.

MONIRA, K., N.M., SALAHUDDIN, AND G., MIAH, 2003: Effect of breed and holding period on egg quality characteristics of chicken. Int. J. of Poult. Sci. **4**: 261-263.

<footnote>a-b means with the same letter are not significantly different from each other at P ≤ .05.</footnote>
P-094 (ID 249)

REPLACEMENT VALUE OF RAW FULL-FAT SOYBEAN FOR COMMERCIAL SOYBEAN MEAL SUPPLEMENTED
WITH A NEW-GENERATION PROTEASE

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

Correspondence: piji@une.edu.au

**Abstract**

The aim of this study was to assess the potential of replacing commercial soybean meal with raw soybean meal (RSBM) in broiler diets supplemented with a protease product. Birds were housed in a cage rearing system in climate controlled rooms. The concentrations of antinutritive factors (ANF), particularly trypsin inhibitors, in the RSBM (13098.0 TIU/g) and diets (ranging from 1730.5 to 9913.2 TIU/g) were measured while bird response to the diets was assessed at 10, 24 and 35 days of age. Feed intake (P<0.01) and body weight gain (P<0.001) to 35 days were reduced by rising levels of RSBM in the diet. Feed conversion efficiency was also reduced (P<0.001) as a result of inclusion of RSBM. Body weight gain to 35 days was marginally (P<0.06) improved as a result of enzyme supplementation of the diets. There were no significant interactions between RSBM levels and enzyme supplement on the gross responses. Inclusion of RSBM in the diet tended to reduce carcass dressing percentage and parts yield but this effect was not significant. Breast meat yield and thigh weight were increased (not significantly) by enzyme supplementation. The weight of drumsticks and wings was also increased (P<0.05) as a result of enzyme supplementation. The relative weight of visceral organs was unaffected except for the weight of the small intestine at 10 d, which was increased (P<0.05) by rising levels of RSBM but reduced (P<0.01) by enzyme supplementation. Inclusion of RSBM in the diets increased the weight of the pancreas in entire study periods. It can be concluded that the enzyme supplement may enable the utilization of low levels of RSBM in diets for broiler chickens.

**Keywords:** Commercial SBM; proteases enzymes; raw full-fat soybean meal; trypsin inhibitors.

**Introduction**

The price of soybean meal (SBM) has consistently been on the increase over the years and this trend is likely to continue into the future (SHI et al., 2012; PETTERSSON & PONTOPPIDAN, 2013). POPESCU and CRISTE (2003) have suggested that full-fat SBM could be used as an alternative to both SBM and supplemental oils, which are included in various diets for poultry feeding. However, raw full-fat soybean meal (RSBM) contains antinutritional factors (ANF) such as protease inhibitors, lectins and phytate (CHEN et al., 2013). Exogenous feed enzymes have been routinely used in commercial poultry diets for several decades to improve dietary quality, through a reduction in or modification of the structure of ANF. The development of new enzyme products offers the incentive to the exploration of ingredients such as RSBM in poultry feeding. The objective of the current research was to assess the benefits of a protease enzyme product in diets containing graded levels of RSBM for broiler chickens.

**Materials and methods**

A 3 x 3 factorial study was designed to assess the potential of replacing commercial soybean meal with raw soybean meal (RSBM) at 0, 10 or 20 %. The diets were supplemented with a protease product (DSM Nutritional Products, Australia Pty. Ltd) at 0.1, 0.2 or 0.3 g/kg and were formulated to be iso-energetic and iso-nitrogenous. The recommended level of microbial phytase (0.1 g/kg DSM Nutritional Products, Australia Pty. Ltd) was uniformly added on each diet. Each of the nine treatments was replicated 6 times, with 9 broiler chicks per replicate. Birds were housed in cages, in climate controlled housing and fed on starter (1-10 d), grower (11-24 d) and finisher (25-35 d) diets.

**Results and discussion**

The analysed trypsin inhibitor (TI) concentrations ranged from 1730.5 to 9913.2 TIU/g diets). Some of these TI values are beyond the threshold levels for birds as set by FRIEDMAN & BRANDON (2001), who reported that diets containing more than 0.77 mg TI/g diets can affect the performance of non-ruminant animals. Increasing RSBM contents of the diets reduced (P<0.01) the feed intake of birds; however, their BWG and FCR over 1-35 days were unaffected due to

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

<footnote n="2">² DSM Nutritional Products, 2-Havelock Road #04-01, 059763, Singapore</footnote>

<footnote n="3">³ Ethiopian Institute of Agricultural Research, Debre-Zeit Centre, Ethiopia</footnote>
protease supplementation. This result agrees with those of other scholars (JACELA et al., 2013; BARLETTA, 2011), who reported that microbial proteases can break down both stored proteins and proteinaceous anti-nutrients and improve the nutritional values of feeds. The weight of drumsticks and wings of birds was improved as a result of protease enzyme supplementation of diets containing RSBM. However, the dressing percentage weights of other meat parts such as breast, thighs and neck were not affected. This result partly disagrees with those of RADA et al. (2014), who reported that the meat part of broilers is positively affected by addition of microbial enzymes.

<table><caption>Table: Feed intake (g/b), live weight gain (g/b) and feed conversion ratio (FCR) of broiler chickens between hatch and 10, 24 or 35 days of age</caption><thead><tr><th rowspan="2">RSBM¹ (%)</th><th rowspan="2">Enzyme level</th><th colspan="3">Feed intake (days)</th><th colspan="3">Live weight gain (days)</th><th colspan="3">FCR (days)</th></tr><tr><th>1-10</th><th>1-24</th><th>1-35</th><th>1-10</th><th>1-24</th><th>1-35</th><th>1-10</th><th>1-24</th><th>1-35</th></tr></thead><tbody><tr><td rowspan="3">0</td><td>L</td><td>253.6</td><td>1894.7</td><td>3897.9<sup>a</sup></td><td>238.1<sup>ab</sup></td><td>1411.2</td><td>2453.3</td><td>1.08<sup>b</sup></td><td>1.36</td><td>1.60</td></tr><tr><td>M</td><td>261.6</td><td>1753.8</td><td>3669.6<sup>ab</sup></td><td>242.8<sup>a</sup></td><td>1414.3</td><td>2474.6</td><td>1.07<sup>b</sup></td><td>1.29</td><td>1.48</td></tr><tr><td>H</td><td>267.0</td><td>1817.8</td><td>3776.0<sup>a</sup></td><td>245.1<sup>a</sup></td><td>1442.0</td><td>2562.4</td><td>1.07<sup>b</sup></td><td>1.25</td><td>1.47</td></tr><tr><td rowspan="3">10</td><td>L</td><td>264.4</td><td>1839.1</td><td>3581.4<sup>b</sup></td><td>231.7<sup>b</sup></td><td>1383.1</td><td>2348.7</td><td>1.09<sup>b</sup></td><td>1.33</td><td>1.53</td></tr><tr><td>M</td><td>267.6</td><td>1747.2</td><td>3418.6<sup>b</sup></td><td>241.8<sup>a</sup></td><td>1349.4</td><td>2350.7</td><td>1.10<sup>b</sup></td><td>1.30</td><td>1.46</td></tr><tr><td>H</td><td>263.3</td><td>1734.2</td><td>3502.4<sup>b</sup></td><td>242.8<sup>a</sup></td><td>1407.5</td><td>2475.2</td><td>1.07<sup>b</sup></td><td>1.24</td><td>1.42</td></tr><tr><td rowspan="3">20</td><td>L</td><td>259.9</td><td>1735.7</td><td>3458.2<sup>b</sup></td><td>219.7<sup>c</sup></td><td>1356.4</td><td>2348.4</td><td>1.16<sup>a</sup></td><td>1.32</td><td>1.48</td></tr><tr><td>M</td><td>245.7</td><td>1820.6</td><td>3586.0<sup>b</sup></td><td>220.1<sup>c</sup></td><td>1361.3</td><td>2437.1</td><td>1.14<sup>ab</sup></td><td>1.31</td><td>1.47</td></tr><tr><td>H</td><td>271.1</td><td>1786.1</td><td>3529.4<sup>b</sup></td><td>235.9<sup>ab</sup></td><td>1418.7</td><td>2484.6</td><td>1.14<sup>ab</sup></td><td>1.26</td><td>1.45</td></tr><tr><td colspan="2">Polled SEM</td><td>2.0</td><td>15.4</td><td>34.7</td><td>1.6</td><td>8.6</td><td>21.4</td><td>0.01</td><td>0.01</td><td>0.02</td></tr><tr><td colspan="11"><em>Sources of variation</em></td></tr><tr><td colspan="2">RSBM¹ level</td><td>NS</td><td>NS</td><td>**</td><td>***</td><td>NS</td><td>NS</td><td>***</td><td>NS</td><td>NS</td></tr><tr><td colspan="2">Enzyme level</td><td>NS</td><td>NS</td><td>NS</td><td>**</td><td>*</td><td>0.06</td><td>NS</td><td>**</td><td>NS</td></tr><tr><td colspan="2">RSBM¹ x enzyme</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr></tbody></table>

Regardless RSBM contents of diets, protease supplementation reduced the weight of the small intestines, but unaffected other visceral organs such as gizzard + proventriculus (G+P), liver, heart, bursa and spleen.

## Conclusion

It can be concluded that the inclusion of RSBM is detrimental to the physiological functions and growth of broiler chickens. However, some of these effects can be ameliorated by supplementation with the test protease enzyme.

## References

* BARLETTA, A. (2011). Introduction: Current Market and Expected Developments. In M. R. Bedford & G. G. Partridge (Eds.), *Enzymes in farm animal nutrition* (2nd ed., pp. 1-11): CABI, UK.

* CHEN, Y., DUAN, W., WANG, L., ZHANG, S., ZHOU, Y., ULUPI, N., AL-NASSER, A. (2013). Effects of Thermostable Phytase Supplementation on the Growth Performance and Nutrient Digestibility of Broilers. Int. J. Poult. Sci., 12(8), 441-444.

* FRIEDMAN, M., & BRANDON, D. L. (2001). Nutritional and health benefits of soy proteins. *Journal of Agricultural and Food Chemistry*, 49(3), 1069-1086.

* RADA, V., LICHOVNIKOVA, M., & FOLTYN, M. (2014). The effect of serine protease on broiler growth and carcass quality. *Acta Fytotechnica et Zootechnica*, 17(3), 87-89.

<footnote>a,b,c Means bearing uncommon superscript within a column are significantly different; ¹RSBM = Raw soybean meal; NS= not significant; * P<0.05; **P<0.01; ***P<0.001; L= low level of enzymes (0.1 g/kg); M= mid-level of enzymes (0.2 g/kg); H= high level of enzymes (0.3 g/kg).</footnote>
## AMYLASE IMPROVES RESISTANT STARCH DIGESTIBILITY OF DIETS

V.G. Schramm<fnref n="1" />, J.O.B. Sorbara<fnref n="2" />, R. Hermes<fnref n="2" />, L.N.E. Barrilli<fnref n="1" />, J.F. Durau<fnref n="1" />, L.P. Domingues<fnref n="1" />, A.P. Félix<fnref n="1" />, and A. Maiorka<fnref n="1" />

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

### Abstract

Enzymes are added to broiler diets in order to improve the utilization of nutrients. Amylase as an isolated enzyme is not commonly available on the market. The objective of this study was to investigate the effect of increasing levels of amylase on the starch digestibility of diets. For this, 315 male Cobb 500 chicks were used in a completely randomized design. Increasing levels of amylase (0, 66.7, 133.3, 200, 233.3 ppm) with 9 replicates of seven broilers were used. For ileal digestibility, 1% of acid insoluble ash was added to the diet. Data were analyzed by analysis of variance (P<0.05) and when significant it was presented as a linear and quadratic regression (P <0.05). Treatments, with or without amylase were compared and contrasted. No enzyme effect in available starch digestibility was observed. However, a linear improvement was found in the resistant starch digestibility associated with increasing levels of amylase (P <0.01). In addition, a quadratic effect on digestibility of the total starch (P = 0.03) was seen. It was concluded that amylase improves RS digestibility of corn-soy diets.

**Keywords:** enzymes, starch.

### Introduction

Feed enzymes are used in animal nutrition in order to improve the digestibility of the diet. Recent studies have shown that the addition of a mixture of amylase, protease, and xylanase brings benefits for birds fed diets based on corn and soybean meal (Zanella et al., 1999; Café et al., 2002). However, information on the influence of amylase as a single exogenous enzyme added to broiler diets is scarce. Starch is a major fraction of the diet and is the main energy source of broilers (Weurding et al., 2003). Thus the objective was to investigate the effect of increasing levels of amylase on the starch digestibility of diets.

### Materials and Methods

For this, 315 male Cobb 500 chicks were used in a completely randomized design. Increasing levels of amylase (0, 66.7, 133.3, 200, 233.3 ppm) with 9 replicates of seven broilers were used. The birds were housed in cages with water and feed *ad libitum*. At 25 days of age the birds were euthanized and the ileal content was collected. The material was frozen in liquid nitrogen and stored at -18 °C. Subsequently, the samples were lyophilized (Liofilizador ModulyoD, Thermo Electron Corporation, Waltham, MA, USA). The diets and ileal content samples were analyzed for available starch (AS), resistant starch (RS) and total starch (TS). For ileal digestibility, 1% of acid insoluble ash was added to the diet. Data were analyzed by analysis of variance (P<0.05) and when significant it was presented as a linear and quadratic regression (P <0.05). Treatments, with or without amylase were compared and contrasted.

### Results

No enzyme effect was observed on available starch digestibility, Table 1. However, a linear improvement was found in the resistant starch digestibility associated with increasing levels of amylase (P <0.01; y = 0.041x + 72.46; R<fnref n="2" /> = 0.594), Figure 1. In contrast the inclusion of the enzyme improved the resistant starch digestibility. In addition, a quadratic effect was seen on digestibility of total starch (P = 0.03; y = -0.001x<fnref n="2" /> + 0.024x + 93.33; R<fnref n="2" /> = 0.161), Figure 2.

<footnote n="1">¹ Dept. of Animal Science, Federal University of Paraná, Curitiba-PR, Brazil</footnote>

<footnote n="2">² DSM Nutritional Products, São Paulo-SP, Brazil</footnote>
<table><caption>**Table 1.** Available starch (AS), resistant starch (RS) and total starch (TS) apparent ileal digestibility coefficients of diets with different levels of amylase.</caption><thead><tr><th>Amylase (ppm)</th><th></th><th>AS</th><th>RS</th><th>TS</th></tr></thead><tbody><tr><td>0</td><td></td><td>94.81</td><td>70.77</td><td>93.13</td></tr><tr><td>66.7</td><td></td><td>95.70</td><td>78.01</td><td>95.11</td></tr><tr><td>133.3</td><td></td><td>94.73</td><td>77.61</td><td>93.97</td></tr><tr><td>200</td><td></td><td>95.59</td><td>78.66</td><td>94.12</td></tr><tr><td>266.7</td><td></td><td>93.48</td><td>84.31</td><td>91.97</td></tr></tbody></table><table><thead><tr><th><i>P</i>-value</th><th></th><th>Linear</th><th>Quadratic</th><th>Contrast(S)</th></tr></thead><tbody><tr><td></td><td>Linear</td><td>0.316</td><td>0.208</td><td>0.948</td></tr><tr><td></td><td>Quadratic</td><td></td><td>0.660</td><td></td></tr><tr><td></td><td>Contrast(S)</td><td></td><td>&lt;0.001</td><td></td></tr></tbody></table>

<figure><img src="image_38.png" /><figcaption>**Figure 1.** Resistant starch apparent ileal digestibility coefficient of diets with increasing levels of amylase (ppm).</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>**Figure 2.** Total starch apparent ileal digestibility coefficient with increasing levels of amylase (ppm). Resistant starch is divided into 3 fractions. but in starch analysis is stated that only the RS3 is included. which is retro-graded. because fractions RS1 and RS2 are solubilized by grinding and gelatinization during the analysis process (ASP, 1996; WOLF et al., 1999).</figcaption></figure>
Conclusions

It was concluded that amylase improves resistant starch digestibility of corn-soy diets.

Literature

ASP. N. G. 1996: Dietary carbohydrates: classification by chemistry and physiology. Food Chem. v.57. n.1. p.9-14.

CAFÉ. M. B., C. A. BORGES, C. A. FRITTS, and P. W. WALDROUP. 2002: Avizyme improves performance of broilers fed cornsoybean meal-based diets. J. Appl. Poult. Res. 11:29-33

WOLF. B.W., L.L. BAUER, G.C. FAHEY Jr. 1999: Effects of chemical modification on in vitro rate and extent of food starch digestion: an attempt to discover a slowly digested starch. J. Agr. Chem.. v.47. p. 4178-4183.

ZANELLA. I., N. K. SAKOMURA, F. G. SILVERSIDES, A. FIQUEIRDO, and M. PACK. 1999: Effect of enzyme supplementation of broiler diets based on corn and soybeans. Poult. Sci. 78:561-568.
P-098 (ID 73)

# MICROBIAL PHYTASE IMPROVES PERFORMANCE AND BONE TRAITS IN BROILERS FED DIETS BASED ON SOYBEAN MEAL AND CONTAINING LUPIN MEAL

S.A. Kaczmarek<fnref n="1" />, A.J. Cowieson<fnref n="2" /> M. Hejdysz<fnref n="1" />, and A. Rutkowski<fnref n="1" />

**Corresponding author:** Email: sebak1@up.poznan.pl

## Abstract

A completely randomized design study with a 3 x 2 factorial arrangement was conducted. A total of 480 male broiler chickens were used to determine the influence of phytase addition, plant protein source (soybean meal, soybean meal + 200 g/kg of blue lupin meal (*Lupinus angustifolius* L.) or soybean meal + 200 g/kg yellow lupin meal (*Lupinus luteus* L.)) on performance of broiler chickens, ileal Ca, P and protein digestibility, tibia bone characteristics and AMEn. All diets were deficient in Ca, available P as well as lysine and total sulphur amino acids. Birds fed different protein sources were characterized by similar BWG and FI during trial but FCR was higher when birds were fed containing yellow lupin meal (P < 0.05). Phytase improved BWG and FCR regardless of protein source (P < 0.05). Diets containing 200 g/kg blue or yellow lupin meal were found to be lower in AMEn than soybean meal diets (P < 0.05). Birds fed diets supplemented with phytase were characterized by higher utilization of Ca and P than birds whose feed was not supplemented with phytase (P < 0.05). Tibia bone ash as well as Ca content was similar across protein sources (P > 0.05), while tibia P content was lower when SBM or blue lupin meal was used (P < 0.05). Birds fed diets containing blue lupin meal were characterized by the highest tibia P content (P < 0.05). Birds fed phytase-supplemented diets were characterized by higher tibia Ca and P content irrespective of protein source.

**Keywords:** lupines, legumes, exogenous enzyme, tibia, performance

## Introduction

Whole lupin seeds contain meaningful concentrations of phytate P possibly limiting the use of lupin in poultry feeding. Phytate in plant feedstuffs can interact electrostatically with dietary (and possibly endogenous) proteins and amino acids which negatively influences protein utilization and increases endogenous protein loss (KONG and ADEOLA, 2011, GEHRING et al., 2013).

The research performed earlier by STEINER et al. (2007), showed that about two thirds of total P in legume seeds, are bound to phytate and lupin seeds may contain up to 4.4 g phytate-P/kg DM. The presence of phytate P in common maize/wheat SBM diets is no longer a problem because of widespread phytase use. However, the effectiveness of phytase in SBM-lupin diets has attracted less attention. As reported by BIEHL and BAKER (1997), supplemental phytase failed to elicit a gain or feed efficiency response in broiler chickens fed a maize-peanut meal diet, in contrast to SBM-based diets. (STEINER et al., 2007) found that the location of phytate in cereal grains and oilseeds varies among sources and in lupins strong binding of phytate to protein occurs. The objective of the research was to test the hypothesis that phytase efficacy could be affected by a diet protein source (representing phytate-protein matrix). In addition, ileal digestibility was performed to determine whether phytase improved protein, Ca and P utilization.

## Material and Methods

The study consisted of a completely randomized experimental design with a 3 x 2 factorial arrangement of treatments, three sources of protein (SBM, yellow or blue lupin meal) and with or without phytase (RONOZYME HiPhos, DSM Nutritional Products, Kaiseraugst, Switzerland, contained 5000 U of phytase activity/g). The experiment was conducted with 480 broilers (Ross 308). Chicks (initial individual weight: 40±2 g) were divided into six dietary treatments with 10 replications with 8 birds in each. On day 36, 10 chickens from each group were sacrificed by CO₂ gas, according to the recommendations for euthanasia of experimental animals and the ileum was removed. Ten digesta were flushed from the terminal ileum (15 cm, adjacent to the ileo-caecal junction) and pooled by cage (2 birds/sample) to provide sufficient material for chemical analysis (n=5). The left tibiae of experimental birds were taken from 5 randomly chosen birds per treatment.

<footnote n="1">¹Poznan University of Life Sciences, Department of Animal Nutrition and Feed Management, ul. Wołyńska 33, 60-637 Poznań, Poland</footnote>

<footnote n="2">²DSM Nutritional Products, Kaiseraugst, Switzerland</footnote>
All diets were deficient in protein (-13 g/kg), Ca (-3.9 g/kg), available P (-2.3 g/kg) as well as lysine (-3.4 g/kg) and total sulphur amino acids (-2.4 g/kg). Other nutrients met or exceeded Aviagen recommendations for 1-10d old broiler chickens (Aviagen 2009). One phase feeding model was used to keep phytate and vegetable protein in the diet at constant level throughout the whole trial.

## Results

From day 1 to day 35 there were no interactions between any of the main treatment terms. Birds fed different protein sources were characterized by similar BWG and FI during the whole trial. Feed conversion ratio was not affected by dietary treatments during the first two weeks of the experiment. However, considering the grower period (d14-35) of the trial or the whole experiment, birds fed diets containing YLM were characterized by the highest FCR (P < 0.05). There were no differences in FCR between birds fed SBM and blue lupin meal.

The use of phytase improved (P < 0.05) BWG and FCR regardless of the lupin type implemented during the grower period and the whole experiment. Diets containing 20% lupin meal were found to be lower in AMEn than SBM diets (P < 0.05). There was no effect of enzyme on AMEn and no significant treatment interaction. The ileal digestibility of protein of SBM and BLM diets was found to be lower than those based on YLM (P<0.05).

<table><caption>**Table 5.** Bird performance fed diets with soybean meal, blue lupin or yellow lupin, supplemented without (-) or with (+) phytase – main effects only.</caption><thead><tr><th rowspan="2">Protein source</th><th rowspan="2">Phytase addition</th><th colspan="4">1-35d</th><th colspan="4">At 35d</th></tr><tr><th>BWG</th><th>FI</th><th>FCR</th><th>AM En</th><th>CP</th><th>P</th><th>Ash</th><th>P</th></tr></thead><tbody><tr><td rowspan="2"></td><td>-</td><td>2039</td><td>32<br>49</td><td>1.60</td><td>11.<br>7</td><td>0.77<br>0</td><td>0.51<br>9</td><td>39.4</td><td>11.5</td></tr><tr><td>+</td><td>2122</td><td>32<br>67</td><td>1.54</td><td>11.<br>8</td><td>0.78<br>3</td><td>0.69<br>7</td><td>49.0</td><td>18.0</td></tr><tr><td rowspan="3">SBM</td><td>SBM</td><td>2106</td><td>32<br>35</td><td>1.54<sup>b</sup></td><td>12.<br>2<sup>a</sup></td><td>0.75<br>5<sup>b</sup></td><td>0.61<br>5</td><td>43.8<sup>b</sup></td><td>14.5<sup>b</sup></td></tr><tr><td>SBM+BL</td><td>2102</td><td>32<br>18</td><td>1.53<sup>b</sup></td><td>11.<br>5<sup>b</sup></td><td>0.76<br>8<sup>b</sup></td><td>0.61<br>7</td><td>43.1<sup>b</sup></td><td>15.0<sup>a</sup></td></tr><tr><td>SBM+YL</td><td>2035</td><td>33<br>21</td><td>1.63<sup>a</sup></td><td>11.<br>4<sup>b</sup></td><td>0.80<br>6<sup>a</sup></td><td>0.59<br>1</td><td>45.2<sup>a</sup></td><td>14.6<sup>b</sup></td></tr><tr><td rowspan="2">Phytase</td><td></td><td>&lt;0.0<br>01</td><td>n.<br>s.</td><td>&lt;0.0<br>01</td><td>n.s.<br>n.s.</td><td>n.s.<br>n.s.</td><td>&lt;0.0<br>01</td><td>&lt;0.0<br>01</td><td>&lt;0.0<br>01</td></tr><tr><td>Protein source</td><td>n.s.</td><td>n.<br>s.</td><td>&lt;0.0<br>01</td><td>&lt;0.<br>05</td><td>&lt;0.0<br>5</td><td>n.s.</td><td>n.s.</td><td>n.s.</td></tr></tbody></table>

+, phytase added; -, no enzyme added. n.s., Not significant (P > 0.05); abc – P < 0.05, SBM – soybean meal, BLM – blue lupine meal, YLM – yellow lupine meal.

Birds fed YLM diets retained less N than those fed SBM or BLM (P < 0.05). Phosphorus as well as Ca digestibility was affected by phytase addition (P < 0.05) but not by plant protein source. Birds fed diets supplemented with phytase were characterized by higher utilization of Ca and P than birds whose feed was not supplemented with phytase (P < 0.05). There was no interaction between protein source and phytase addition for apparent ileal digestibility. Tibia bone ash as well as Ca content was similar across protein sources (P > 0.05), while tibia P content was different (P < 0.05). Birds fed diets containing blue lupin were characterized by the highest tibia P content (P < 0.05). Tibia ash, Ca and P content was affected by phytase supplementation. Birds fed phytase-supplemented diets were characterized by higher tibia Ca and P content irrespective of protein source. There was no interaction between protein source and phytase addition for all tibia traits.
## Conclusions

It can be concluded that the use of YLM as a protein source in poultry diets may be limited due to antinutrients concentrations, when compared to other plant protein sources such as SBM. The benefits of phytase addition seem independent of the protein meal employed. It can be suggested that the use of phytase was an effective tool to improve phosphorous availability from maize-SBM and maize-SBM-YLM or maize-SBM-BLM diets. The extent to which the effect of phytase addition on nutrient digestion is associated with employed plant protein source, requires further research.

## Literature

* **BIEHL, R. and BAKER, D.** (1997) Microbial phytase improves amino acid utilization in young chicks fed diets based on soybean meal but not diets based on peanut meal. *Poultry Science* **76**: 355-360.

* **GEHRING, C. K., BEDFORD, M. R. and DOZIER, W. A.** (2013) Interactive effects of phytase and xylanase supplementation with extractable salt-soluble protein content of corn in diets with adequate calcium and nonphytate phosphorus fed to broilers. *Poultry Science* **92**: 1858-1869.

* **KONG, C. and ADEOLA, O.** (2011) Protein utilization and amino acid digestibility of canola meal in response to phytase in broiler chickens. *Poultry Science* **90**: 1508-1515.

* **STEINER, T., MOSENTHIN, R., ZIMMERMANN, B., GREINER, R. and ROTH, S.** (2007) Distribution of phytase activity, total phosphorus and phytate phosphorus in legume seeds, cereals and cereal by-products as influenced by harvest year and cultivar. *Animal Feed Science and Technology* **133**: 320-334.
P-099 (ID 326)

EFFECTS OF AN NSP-ENZYME SUPPLEMENTATION ON PRECAECAL NUTRIENT DIGESTIBILITY AND ARABI-
NOXYLAN DEGRADATION IN TURKEYS.

H. Kluge <fnref n="1" />, E. von Heimendahl <fnref n="2" />, S. L. Kinzinger <fnref n="2" />, G. I. Stangl <fnref n="1" />

**Corresponding author:** elke.von.heimendahl@lohmann-an.de

**Abstract**

The influence of NSP enzyme supplementation on arabinoxylan degradation in the ileal chyme of turkeys was investi-
gated in order to gain more insight in their effects on nutrient digestibility. 72 1-day old turkeys were randomly allocated
to 3 treatments: T1-control; T2-enzyme blend (ENZY CARBOPLUS; Lohmann Animal Nutrition GmbH), with 100 mg/kg
feed; T3-with 300 mg/kg feed. Birds were kept in pens from day 1 to 28. On day 28 birds were transferred to balance
cages and diets were supplemented with TiO2. Fecal samples were collected for ME calculation over 5 days. At study
end, birds were sacrificed and digesta collected for determination of prececal protein digestibility and enzymatic hy-
drolysis of arabinoxylan. Enzyme supplementation significantly improved weight gain (80.3 vs. 73.6 g/day, T3 vs. T1;
P=0.009) and FCR (1.44 vs. 1.58 g/day, T3 vs. T1; P=0.001). This is most likely the consequence of improved ME
which was numerically increased in T2 turkeys compared to T1 while ME and prececal protein digestibility of T3 was
significantly increased compared to T1. The analyses of free sugars in the ileal chyme showed a significant increase of
arabinose and xylose and a significant decrease of glucose in turkeys fed the enzyme blend. The significant decrease
of glucose in the ileal chyme of birds fed enzymes might be a result of increased resorption before the terminal ileum as
a result of higher starch degradation in the enzyme groups.

**Keywords:** turkeys, NSP enzymes, arabinoxylan, free sugars

**Introduction**

Cereal-based turkey diets are characterized by high amounts of NSP such as water-soluble arabinoxylan (AX) and β-glucans. However, the digestibility of diets containing high levels of NSP is limited and the apparent metabolizable energy (AME) and the amount of soluble NSP are negatively correlated (Choct & Annison, 1992). Both can be improved by the addition of enzymes which hydrolyze AX to low molecular compounds and monomer sugars such as xylose and arabinose. The current study investigates the prececal (pc) protein digestibility and enzymatic hydrolysis of AX in the ileal chyme in response to xylanase and β-glucanase in turkeys.

**Material and methods**

72 one-day old turkeys were randomly allocated to 3 treatments. The control group received a diet without enzymes (T1). T2 was fed a diet with 100 mg/kg diet of a xylanase/ β-glucanase blend (Enzy Carboplus; Lohmann Animal Nutrition GmbH, xylanase activity 7000 LXU/g and β-glucanase activity 600 LXU/g). T 3 received 300 mg of the enzyme blend. The turkeys were kept in 12 pens of 6 animals each and were fed a diet P1 (day 1-14) and a diet P2 (day 14-36). The diet composition is presented in Table 1.

At day 28, all animals were transferred to individual balance cages. Excreta were collected for ME calculation over 5
days. During the balance period feed was restricted.

At the end of the study, animals were sacrificed and digesta were collected from the proximal two-thirds of the intestine
between Meckel's diverticulum and 2 cm anterior to the ileo-ceco-colonic junction. Samples from 3 animals were pooled
for analyses. Samples were freeze dried and free sugars were analyzed after solution in water and after derivatization
to alditol acetates by gas chromatography. Individual body weight was measured at 0, 14, 28 and 36 days. Feed intake,
average daily gain (ADG) and FCR were calculated for all phases.

Experimental data were analysed statistically by means of analysis of variance (ANOVA) using the program STA-
TISTIKA FOR WINDOWS™. In case of significant F values (P<0.05), mean values of the treatment groups were tested
for significant differences by means of Tukey-HSD-test.

<footnote n="1">¹Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, Germany</footnote>

<footnote n="2">² Lohmann Animal Nutrition GmbH, Cuxhaven, Germany</footnote>
<table><caption>**Table 1:** Ingredient composition (%); AME and protein content of diets</caption><thead><tr><th></th><th>P1</th><th>P2</th></tr></thead><tbody><tr><td>Wheat</td><td>44.6</td><td>44.9</td></tr><tr><td>Rye</td><td>5.0</td><td>10.0</td></tr><tr><td>Soybean meal (48% CP)</td><td>40.0</td><td>36.0</td></tr><tr><td>Potato protein</td><td>2.0</td><td>0</td></tr><tr><td>Palm oil</td><td>3.0</td><td>3.2</td></tr><tr><td>Premix*</td><td>1.0</td><td>1.0</td></tr><tr><td>Monocalcium phosphate</td><td>2.5</td><td>2.5</td></tr><tr><td>Limestone</td><td>1.0</td><td>1.0</td></tr><tr><td>NaCl</td><td>0.3</td><td>0.3</td></tr><tr><td>Choline chloride</td><td>0.1</td><td>0.1</td></tr><tr><td>Lysine HCl</td><td>0.3</td><td>0.32</td></tr><tr><td>DL-Methionine</td><td>0.2</td><td>0.18</td></tr><tr><td>TiO<sub>2</sub></td><td>0</td><td>0.5</td></tr><tr><td>AME (MJ)</td><td>11.7</td><td>12.0</td></tr><tr><td>Protein (g)</td><td>269</td><td>241</td></tr></tbody></table>

**Results and discussion**

Turkeys receiving the enzyme blend showed significantly improved ADG and FCR during the performance period from day 1-28 days on trial compared to the control (Tab. 2).

<table><caption>**Table 2.** Zootechnical performance of turkeys (1 to 28 days on trial)</caption><thead><tr><th>Parameter</th><th>T1<br>Control</th><th>T2<br>(100 mg/kg)</th><th>T3<br>(300 mg/kg)</th><th>SEM</th><th>p-value</th></tr></thead><tbody><tr><td>BW, 1 day (g)</td><td>62.3</td><td>63.3</td><td>63.3</td><td>0.48</td><td>0.710</td></tr><tr><td>BW, 28 days (g)</td><td>840<sup>a</sup></td><td>1170<sup>b</sup></td><td>1090<sup>b</sup></td><td>5.0</td><td>&lt;0.001</td></tr><tr><td>ADFI (g/d)</td><td>49.3<sup>a</sup></td><td>60.8<sup>b</sup></td><td>57.2<sup>ab</sup></td><td>1.8</td><td>&lt;0.011</td></tr><tr><td>ADG (g/d)</td><td>27.9<sup>a</sup></td><td>39.4<sup>b</sup></td><td>36.8<sup>b</sup></td><td>1.6</td><td>&lt;0.001</td></tr><tr><td>FCR</td><td>1.77<sup>b</sup></td><td>1.54<sup>a</sup></td><td>1.55<sup>a</sup></td><td>0.04</td><td>0.006</td></tr></tbody></table>

During the balance period, birds fed the enzymes also had significantly higher body weights (P<0.001), improved ADG (P=0.006) and improved FCR (P=0.002) compared to control birds.

This is most likely the consequence of improved ME which was numerically increased in T2 and significantly increased in T3 turkeys compared to the control as well as the improved prececal protein digestibility which was numerically increased in T2 and significantly increased in T3 compared to control (Tab. 3).

The analyses of free sugars in the ileal chyme showed a significant increase of arabinose and xylose in treatment groups. A significant decrease in the concentration of glucose and mannose was observed in turkeys fed the enzyme blend (Tab. 3).

<footnote>*per kg premix: Ca 330 g, Vit. A 1200000 IU, Vit. D<sub>3</sub> 400000 IU, Vit. E 4000 mg, Vit. K<sub>3</sub> 240 mg, Vit. B<sub>1</sub> 210 mg, Vit. B<sub>2</sub> 720 mg, Vit. B<sub>6</sub> 400 mg, Vit. B<sub>12</sub> 2.3 mg, niacin 6,6 g, Ca-P- pantothenate 1.4 g, folic acid 150 mg, biotin 17,5 mg, iron 5.0 g, copper 1g, zinc 6.0 g, manganese 8.0 g, cobalt 25 mg, iodine 100 mg, selenium 35 mg</footnote>
<table><caption>**Table 3:** Free sugar concentrations, nutrient digestibility and energy utilization</caption><thead><tr><th>Parameter</th><th>T1<br>Control</th><th>T2<br>(100 mg/kg)</th><th>T3<br>(300 mg/kg)</th><th>SEM</th><th>p-value</th></tr></thead><tbody><tr><td>AX diet (% DM)</td><td>4.69</td><td>4.66</td><td>4.69</td><td></td><td></td></tr><tr><td colspan="6">Free sugars in the prececal digesta</td></tr><tr><td>Arabinose (% DM) <sup>1</sup></td><td>0.13 <sup>a</sup></td><td>0.17 <sup>ab</sup></td><td>0.21 <sup>b</sup></td><td>0.01</td><td>0.003</td></tr><tr><td>Xylose (% DM) <sup>1</sup></td><td>0.06 <sup>a</sup></td><td>0.10 <sup>b</sup></td><td>0.13 <sup>c</sup></td><td>0.01</td><td>&lt;0.001</td></tr><tr><td>Mannose (% DM) <sup>1</sup></td><td>2.01 <sup>b</sup></td><td>0.68 <sup>a</sup></td><td>1.14 <sup>ab</sup></td><td>0.21</td><td>0.026</td></tr><tr><td>Glucose (% DM) <sup>1</sup></td><td>16.5 <sup>b</sup></td><td>4.8 <sup>a</sup></td><td>8.5 <sup>ab</sup></td><td>1.44</td><td>&lt;0.001</td></tr><tr><td>Galactose (% DM) <sup>1</sup></td><td>0.19</td><td>0.19</td><td>0.14</td><td>0.03</td><td>0.655</td></tr><tr><td>Prececal protein digestibility (%) <sup>1</sup></td><td>81.2 <sup>a</sup></td><td>83.2 <sup>ab</sup></td><td>83.7 <sup>b</sup></td><td>0.43</td><td>0.029</td></tr><tr><td>ME (MJ/kg feed) <sup>2</sup></td><td>11.7 <sup>a</sup></td><td>11.8 <sup>ab</sup></td><td>12.0 <sup>b</sup></td><td>0.05</td><td>&lt;0.004</td></tr></tbody></table>

Data reveal that turkeys receiving the enzyme blend had lower amounts of glucose in the digesta than the control. We assume that the low glucose content of digesta is caused by a higher starch digestibility and an improved absorption of glucose as it was found in broiler chickens fed xylanase (Choct et al., 1999).

## Conclusion

Dietary inclusion of the enzyme blend significantly improved ADG and FCR of turkeys. This is the result of an improved ME content and precaecal digestibility of protein, improved NSP degradation and therefore increased availability of free sugars.

## Literature

CHOCT, M., R. J. HUGHES, M. R. BEDFORD, 1999: Effects of a xylanase on individual bird variation, starch digestion throughout the intestine, and ileal and caecal volatile fatty acid production in chickens fed wheat. Br. Poult. Sci., **40**, 419-422.

CHOCT, M., G. ANNISON, 1992. The inhibition of nutrient digestion by wheat pentosans. Br. J. Nutr. **67**, 123-132.

<footnote n="1">¹ 8 replicates/treatment of 3 pooled bird samples/replicate, ² 24 replicates/treatment</footnote>

<footnote>a,b Values in the same row with no common superscript are significantly different (P≤0.05).</footnote>
P-101 (ID 150)

THE EFFECT OF DIETARY FEED STRUCTURE, PHYTASE AND XYLANASE APPLICATION ON BROILER PERFORMANCE

I. Kühn¹, R. Ten Doeschate², M. Bedford² and K. Kozłowski³

¹ AB Vista, Darmstadt, Germany

² AB Vista, Marlborough, UK

³ University of Warmia and Mazury, Olsztyn, Poland

**Corresponding author:** imke.kuehn@abenzymes.com

**Abstract**

900 broilers were distributed randomly to one of 10 treatments (10 pens x 9 birds each). The birds were fed pelleted wheat-soybean-based diets, in 5 treatments with part of the wheat as whole grain. Starter, grower and finisher diets were adequate (PC, 4.5, 4.4 and 4.1 g/kg P<sub>av</sub> and 10, 8.8 and 8.2 g/kg Ca; respectively) or reduced by 1.5g/kg P<sub>av</sub> and 1.6g/kg Ca (CC<sub>500</sub>). All CC<sub>500</sub> diets contained 500 FTU/kg phytase or were supplemented with a further 1000 FTU/kg phytase, and were fed with or without 16,000 BXU/kg xylanase. Performance was measured at d10, d24, and d42, and data analysed as a 5 x 2 factorial ANOVA (5 treatments, with or without whole kernel inclusion). Differences at P<0.05 were declared as significant. No interactions were detected (P>0.05). Birds fed CC<sub>500</sub> diets achieved the same ADG and FCR as birds fed PC diets (P<0.05). Compared to CC<sub>500</sub>, the additional phytase improved ADG and FCR from the beginning of the trial and outperformed birds fed the PC diets. Addition of xylanase improved ADG and FCR in the last feeding period (d24-42) and this was significant over the whole trial period (d1-42). When added on top of the high phytase application, xylanase further improved FCR (from 1.52 to 1.49 P<0.05). The inclusion of whole wheat grains resulted in similar improvements in FCR from d25 onwards (P<0.05) as the xylanase. The data shows that addition of xylanase and/or whole wheat grains into diets containing a high dosage of phytase results in further improvements in broiler performance.

**Keywords:** phytase, xylanase, whole wheat grain, broiler

**Introduction**

Addition of whole wheat grains to broiler diets is common practice but different strategies in respect of the inclusion rate and application are applied commercially (fed along with or included in the pellet). Benefits of whole wheat grain feeding include improved growth and feed efficiency due to increased gastric retention time and endogenous enzyme secretion, resulting in better nutrient digestibility (Svihus et al., 2004). Because phytases and xylanases are typically added to wheat-based broiler diets and experiments following the effect of whole wheat grains often do not contain these enzymes, the objective of this trial was to examine the interaction between whole or ground wheat, xylanase and phytase dose on bird performance.

**Materials and Methods**

900 male broilers (Ross 308) were distributed to 10 treatments (10 pens, 9 birds each). The birds received starter (d1 to 10), grower (d11-24) and finisher diets (d25-42) based on wheat, soybean meal, corn and soya oil (for wheat, Na, Ca and P<sub>av</sub> contents, see Table 1). Adequate diets (PC) were not supplemented by enzymes whereas all mineral reduced diets (CC) contained phytase (Quantum Blue, 6-phytase produced by T. reesei) at 500 (CC<sub>500</sub>) or 1500 (CC<sub>1500</sub>) FTU/kg feed, without or with xylanase (Econase XT, endo-1,4-beta-xylanase produced by T. reesei, 16,000 BXU/kg feed; CC<sub>500</sub> + Xyl or CC<sub>1500</sub> + Xyl). All feeds were pelleted, with the wheat included either milled or partly added as whole grains (50, 150 and 250 g/kg in starter, grower and finisher feeds, respectively).
<table><caption>**Table 1.** Wheat, P, Ca and Na contents (g/kg) in the positive (PC) and commercial diets (CC)</caption><thead><tr><th></th><th>PC (starter, grower, finisher)</th><th>CC (starter, grower, finisher)</th></tr></thead><tbody><tr><td>Wheat</td><td>400, 464, 567</td><td>400, 481, 585</td></tr><tr><td>P available</td><td>4.8, 4.4, 4.1</td><td>3.3, 2.9, 2.6</td></tr><tr><td>Ca</td><td>10, 8.8, 8.2</td><td>8.4, 7.2, 6.6</td></tr><tr><td>Na</td><td>1.7, 1.7, 1.6</td><td>1.4, 1.4, 1.3</td></tr></tbody></table>

Birds were individually weighed at the start and end of each feeding phase and feed consumption measured accordingly. Nutritional contents of the diets were analysed following AOAC (2000) methods and enzyme contents analysed by the supplier using internal ELISA methods. Data was analysed as a 5 x 2 factorial ANOVA (5 treatments, with or without whole grains) with the pen as experimental unit, and differences at P<0.05 declared as significant.

## Results and Conclusion

The analytical composition of experimental feeds was in good agreement with the calculated values, with P and Ca contents being slightly above the estimated values, but the targeted differences between PC and CC diets were as expected. Analysed phytase activities were <50 (PC), 793-990 FTU/kg (CC₅₀₀ treatments), 2240-2930 FTU/kg (CC₁₅₀₀ treatments) and analysed xylanase activities were 14200-20700 BXU/kg in diets with and <2000 BXU/kg in those without added xylanase.

Performance results of birds achieved during the entire trial period are shown in Table 2. No interactions were analysed between wheat form and enzyme application.

The addition of whole wheat grains improved FCR (P<0.05) over the whole trial period without any effect on BW.

Birds fed diets including a standard phytase content (500 FTU/kg feed, CC₅₀₀), accompanied with the dietary P, Ca and Na reduction as recommended for the phytase tested, achieved breeder target BW and FCR in all test periods and gave the same performance as PC birds (P>0.05). Feeding an increased phytase level (1500 FTU/kg feed) further improved FCR (CC₁₅₀₀ compared to CC₅₀₀ and PC treatments, P<0.05).

These results confirmed recently published data showing that high dosages of the present phytase tested in diets reduced in P, Ca and Na as recommended for a standard phytase dose improves broiler performance above the level achieved by nutritionally well-balanced diets (Walk et al., 2013).

Xylanase, when added to diets which already contained a standard phytase level, further improved BW and FCR (CC₅₀₀ +Xyl compared to CC₅₀₀, P<0.05)

On top of the high phytase application, the xylanase effect on broilers BW was not significant but FCR was further improved (CC₁₅₀₀ +Xyl compared to CC₁₅₀₀, P<0.05). This additional effect mainly occurred in the final feeding phase (data not shown). The most beneficial FCR was in birds fed the higher phytase dose plus xylanase (CC₁₅₀₀+Xyl, P<0.05 compared to all other treatments).

Whether the additional effect of xylanase is due to an increased nutrient digestibility or has influenced phytate degradation, as shown for the tested enzymes by Zeller et al., 2015, needs further investigation. Additionally, xylanase also might increase digesta retention time through changes in the caecal microbiome inducing a hormonal effect on gastric retention time (Singh et al., 2012).

It can be concluded that dietary inclusion of whole wheat grain, a high phytase level and xylanase can improve FCR of broilers.
<table><caption>**Table 2.** Performance of broilers (day 1 to 42, n = 10)</caption><thead><tr><th>Treatment</th><th>BW at day 42 (kg)</th><th>ADFI (g)</th><th>FCR (kg/kg)</th></tr></thead><tbody><tr><td colspan="4"><strong>Wheat milled:</strong></td></tr><tr><td>PC</td><td>3.314 ± 0.039</td><td>125.2 ± 5.7</td><td>1.569 ± 0.056</td></tr><tr><td>CC<sub>500</sub></td><td>3.362 ± 0.096</td><td>122.2 ± 3.5</td><td>1.546 ± 0.029</td></tr><tr><td>CC<sub>500</sub> +Xyl</td><td>3.428 ± 0.092</td><td>126.2 ± 9.2</td><td>1.516 ± 0.016</td></tr><tr><td>CC<sub>1500</sub></td><td>3.388 ± 0.084</td><td>124.0 ± 2.7</td><td>1.532 ± 0.035</td></tr><tr><td>CC<sub>1500</sub> +Xyl</td><td>3.422 ± 0.093</td><td>123.3 ± 4.3</td><td>1.495 ± 0.021</td></tr><tr><td colspan="4"><strong>Whole wheat:</strong></td></tr><tr><td>PC</td><td>3.361 ± 0.107</td><td>124.1 ± 5.1</td><td>1.543 ± 0.033</td></tr><tr><td>CC<sub>500</sub></td><td>3.345 ± 0.127</td><td>123.5 ± 9.8</td><td>1.537 ± 0.030</td></tr><tr><td>CC<sub>500</sub> +Xyl</td><td>3.350 ± 0.109</td><td>123.7 ± 5.3</td><td>1.511 ± 0.031</td></tr><tr><td>CC<sub>1500</sub></td><td>3.414 ± 0.061</td><td>121.2 ± 2.7</td><td>1.506 ± 0.025</td></tr><tr><td>CC<sub>1500</sub> +Xyl</td><td>3.450 ± 0.089</td><td>120.5 ± 4.7</td><td>1.482 ± 0.022</td></tr><tr><td colspan="4"><strong>Wheat form</strong></td></tr><tr><td>Wheat milled</td><td>3.383 ± 0.106</td><td>124.2 ± 5.5</td><td>1.532<sup>b</sup> ± 0.042</td></tr><tr><td>Whole wheat</td><td>3.404 ± 0.106</td><td>122.6 ± 5.9</td><td>1.516<sup>a</sup> ± 0.035</td></tr><tr><td colspan="4"><strong>Enzyme</strong></td></tr><tr><td>PC</td><td>3.338<sup>b</sup> ± 0.121</td><td>124.7 ± 5.3</td><td>1.556<sup>c</sup> ± 0.047</td></tr><tr><td>CC<sub>500</sub></td><td>3.354<sup>b</sup> ± 0.110</td><td>122.9 ± 7.2</td><td>1.541<sup>c</sup> ± 0.029</td></tr><tr><td>CC<sub>500</sub> +Xyl</td><td>3.439<sup>a</sup> ± 0.099</td><td>125.0 ± 7.4</td><td>1.514<sup>b</sup> ± 0.024</td></tr><tr><td>CC<sub>1500</sub></td><td>3.401<sup>ab</sup> ± 0.073</td><td>122.6 ± 3.0</td><td>1.519<sup>b</sup> ± 0.032</td></tr><tr><td>CC<sub>1500</sub> +Xyl</td><td>3.436<sup>a</sup> ± 0.090</td><td>121.9 ± 4.6</td><td>1.489<sup>a</sup> ± 0.022</td></tr><tr><td>SEM</td><td>0.011</td><td>0.576</td><td>0.004</td></tr><tr><td>P (wheat form)</td><td>0.287</td><td>0.180</td><td><strong>0.013</strong></td></tr><tr><td>P (enzyme)</td><td><strong>0.004</strong></td><td>0.374</td><td><strong>0.001</strong></td></tr><tr><td>P (interaction)</td><td>0.900</td><td>0.771</td><td>0.737</td></tr></tbody></table>

## References

ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS, 2000: Official methods of analysis. 17th ed. Association of Official Analytical Chemist. Washington (DC).

SINGH, A., H.V. MASEY O'NEILL, T.K. GHOSH, M.R. BEDFORD and S. HALDAR, 2012: Effect of xylanase supplementation on performance, total volatile fatty acids and selected bacterial population in caeca, metabolic indices and peptide YY concentrations in serum of broiler chickens fed energy restricted maize-soybean based diets. Animal Feed Science and Technology 177, 194-203

SVIHUS, B., E. JUVIK, H. HETLAND and Á. KROGDAHL, 2004: Causes for improvement in nutritive value of broiler chicken diets with whole wheat instead of ground wheat. British Poultry Science 45(1), 55-60

WALK, C.L., M.R. BEDFORD, T.S. SANTOS, D. PAIVA, J.R. BRADLEY, H. WLADECKI, C. HONAKER and A.P. McELROY, 2013: Extra-phosphoric effects of superdoses of a novel microbial phytase. Poultry Science 92, 719-725

ZELLER, E., M. SCHOLLENBERGER, I. KÜHN and M. RODEHUTSCORD, 2015: Effect of diets containing enzyme supplements and microwave-treated or untreated wheat on inositol phosphates in the small intestine of broilers. Animal Feed Science and Technology 204, 42-51

<footnote>ab Values with different superscripts within column and parameter differ significantly (P<0.05)</footnote>
P-102 (ID 327)

EFFICACY OF PHYTASE SUPPLEMENTATION IN UNCOATED AND COATED FORM IN MALE BROILER CHICKENS

K. Männer<fnref n="1" />, E. von Heimendahl<fnref n="2" />, and S. L. Kinzinger<fnref n="2" />

**Corresponding author:** stefan.kinzinger@lohmann-an.de

**Abstract**

Heat stability of enzymes is important in pelleted feed. Coating is one option to increase the survival rate but might affect the enzyme release in the intestine. The aim of this study was to evaluate the effect of uncoated and coated phytase on performance and bone mineralization in broilers. 100 day-old chicks were housed in balance cages and allotted to 5 treatments. Starter and grower diets were formulated according to recommendations, except that P was reduced to allow a response to phytase addition. Treatments were control, 250 FTU/kg uncoated, 500 FTU/kg uncoated, 250 FTU/kg coated and 500 FTU/kg coated *E.coli* derived 6-phytase (Enzy Phostar, Lohmann Animal Nutrition). Due to inadequate P supply control birds reached 80% of the breed specific body weight (BW). Phytase addition led to significantly improved BW gain (13.4%) and FCR (10.4%) compared to control birds. Animal performance correlated with the dose level irrespective of the phytase form. Based on ileal P-digestibility the coated phytase was numerically (250 FTU/kg) or significantly (500 FTU/kg) better compared to the uncoated form. Both, ileal digestibility and tibia bone deposition of P and Ca also were dose-dependent (partly significant). In conclusion, for ileal digestibility and tibia mineralization the coated phytase at dose levels of 250 and 500 FTU/kg was similar or superior to the uncoated form. It was shown that the coating technology did not affect the biological efficacy of this new phytase.

**Keywords:** phytase, coating, tibia mineralization, broiler

**Introduction**

Enzymes like all proteins are heat sensitive. However, as the biggest proportion of feed is pelleted heat stability of enzymes is very important. Coating is one option in order to increase enzyme survival rate during the pelleting process. Nevertheless, coating might affect the release of the enzyme in the gastro-intestinal tract and therefore compromise its efficacy. The objective of the present study was to demonstrate the efficacy of a new phytase as uncoated or coated formulation when added to low phosphorus diets for broiler chickens.

**Material and methods**

100 one-day-old male broiler chickens (COBB) were housed in balance cages with 2 birds per cage and allotted to 5 treatments with 10 cages per treatment. The feeding period included starter (1-14 d) and grower diets (15-35 d) formulated according to the recommendations of GfE (1999), except that phosphorus was reduced in order to allow for a response to additional phytase supply as coated or uncoated product (*E. coli*-derived 6-phytase, Enzy Phostar, Lohmann Animal Nutrition) at dose levels of 250 or 500 FTU per kg feed. Diet composition is shown in table 1. Investigated parameters were body weight gain, feed intake, FCR as well as ileal digestibility (P, Ca), and left tibia bone mineralization at the end of the trial period.

Statistical analyses were performed with SPSS (IBM SPSS Version 21) and based on Oneway Anova. All treatment least squares means were compared with each other and the Tukey adjustment was used to control for experiment wise error.

<footnote n="1">¹ Institute of Animal Nutrition, Free University of Berlin, Germany</footnote>

<footnote n="2">² Lohmann Animal Nutrition GmbH, Cuxhaven, Germany</footnote>
<table><caption>**Table 1.** Ingredients and nutritional characteristics of the basal starter and grower diet</caption><thead><tr><th>Ingredients</th><th></th><th>Starter (1-14 d)</th><th>Grower (15-35 d)</th></tr></thead><tbody><tr><td>Wheat</td><td>%</td><td>46.33</td><td>49.46</td></tr><tr><td>Maize</td><td>%</td><td>10.00</td><td>10.00</td></tr><tr><td>Soybean meal</td><td>%</td><td>34.50</td><td>30.15</td></tr><tr><td>Soybean oil</td><td>%</td><td>5.60</td><td>6.80</td></tr><tr><td>Limestone</td><td>%</td><td>1.56</td><td>1.36</td></tr><tr><td>Monocalcium phosphate</td><td>%</td><td>0.50</td><td>0.13</td></tr><tr><td>DL-Methionine</td><td>%</td><td>0.23</td><td>0.24</td></tr><tr><td>L-Lysine HCI</td><td>%</td><td>0.08</td><td>0.16</td></tr><tr><td>Premix</td><td>%</td><td>1.20</td><td>1.20</td></tr><tr><td colspan="4"><strong>Nutritional characteristics</strong></td></tr><tr><td>ME<sub>N</sub>*</td><td>MJ/kg</td><td>12.20</td><td>12.60</td></tr><tr><td>Crude protein</td><td>g/kg</td><td>220.00</td><td>205.00</td></tr><tr><td>Lysine</td><td>g/kg</td><td>12.40</td><td>11.80</td></tr><tr><td>Methionine / cystine</td><td>g/kg</td><td>9.40</td><td>9.10</td></tr><tr><td>Calcium</td><td>g/kg</td><td>8.00</td><td>6.50</td></tr><tr><td>Total phosphorus</td><td>g/kg</td><td>5.00</td><td>4.00</td></tr><tr><td>Digestible phosphorus</td><td>g/kg</td><td>2.50</td><td>2.20</td></tr></tbody></table>

## Results and discussion

Due to the inadequate phosphorus supply, productive performance (table 2) of birds fed diets without phytase addition reached approximately 80% of the intended weight gain given by the breeding company. Phytase supplementation led to significantly increased body weight gain (+13.4%) and significantly reduced (10.4%) FCR compared to birds receiving no phytase. When feeding the new phytase at the dose level of 500 FTU the body weight gain was slightly (3.3%) higher than that recorded for the lower dose level (250 FTU). Feed intake showed no consistent preparation or dose dependent effects. FCR of the control was 1.69. Phytase supplementation significantly improved FCR in all groups by an average of 10.4% due to the enhanced body weight. Regarding body weight gain and FCR there were no preparation dependent effects detected.

As expected, the present study demonstrated that dietary inclusion of phytase significantly improved apparent ileal P-digestibility (table 2) in comparison to broilers fed a low phosphorus diet without phytase addition. Feeding phytase (uncoated, coated) at 500 FTU resulted in significantly better apparent ileal P-digestibility when compared to the lower dose level (250 FTU). Dose dependent effects on ileal digestibility were also found for crude ash and Ca. However, phytase effects in comparison to the control diet were lower than those recorded for digestibility of phosphorus. At each phytase dosage, coating did not influence ileal digestibilities of ash and Ca when compared to the uncoated formulation, while for P-digestibility it was found that the coated phytase gave numerically (250 FTU) or significantly (500 FTU) higher figures in comparison to the uncoated preparation.

Significant enzyme effects on tibia mineralization (table 3) were seen for crude ash, P and Ca contents in the fat-free dry matter in comparison to broilers fed the control diet (A). Deposition of crude ash, P and Ca showed no statistically firm effect of coating except for P-deposition at the 500 FTU dosage. In general, tibia parameters also exhibited a numerical, dose dependent effect.

For demonstrating the efficacy of the new phytase in uncoated or coated form, ileal digestibility and tibia mineralization were more sensitive than productive performance. Therefore, based on apparent ileal P-digestibility it could be shown that the coated form of the phytase was numerically (250 FTU) or significantly (500 FTU) better in comparison to the uncoated preparation. These findings were not expected and are in contrast to the report by Owusu-Asiedu et al. (2007) who could not find any significant difference in the efficacy of a coated and uncoated *E. coli* phytase.

<footnote>* Estimated according to equation of WPSA 1984</footnote>
<table><caption>**Table 2.** Effect of phytase on performance and digestibility during the total feeding period</caption><thead><tr><th>Treatment</th><th></th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th></tr></thead><tbody><tr><td>Phytase</td><td>FTU kg<sup>-1</sup></td><td>0</td><td>250 n-ct</td><td>500 n-ct</td><td>250 ct</td><td>500 ct</td></tr><tr><td>BW gain</td><td>g</td><td>1546.4<br/>± 68.5<sup>a</sup></td><td>1736.1<br/>± 79.6<sup>b</sup></td><td>1783.7<br/>± 58.5<sup>b</sup></td><td>1713.8<br/>± 63.0<sup>b</sup></td><td>1781.8<br/>± 34.8<sup>b</sup></td></tr><tr><td>FCR</td><td></td><td>1.691<br/>± 0.081<sup>a</sup></td><td>1.536<br/>± 0.063<sup>b</sup></td><td>1.485<br/>± 0.060<sup>b</sup></td><td>1.534<br/>± 0.044<sup>b</sup></td><td>1.504<br/>± 0.048<sup>b</sup></td></tr><tr><td colspan="7"><strong>Ileal digestibility</strong></td></tr><tr><td>Crude ash</td><td>%</td><td>46.89<br/>± 5.56<sup>a</sup></td><td>50.80<br/>± 4.86<sup>ab</sup></td><td>54.07<br/>± 3.73<sup>b</sup></td><td>50.10<br/>± 2.93<sup>ab</sup></td><td>53.64<br/>± 4.95<sup>b</sup></td></tr><tr><td>Phosphorus</td><td>%</td><td>42.95<br/>± 6.15<sup>a</sup></td><td>52.41<br/>± 3.30<sup>b</sup></td><td>60.29<br/>± 2.95<sup>c</sup></td><td>55.65<br/>± 3.98<sup>bc</sup></td><td>65.93<br/>± 3.29<sup>d</sup></td></tr><tr><td>Calcium</td><td>%</td><td>52.75<br/>± 6.03<sup>a</sup></td><td>58.38<br/>± 4.98<sup>abc</sup></td><td>62.02<br/>± 2.83<sup>bc</sup></td><td>57.04<br/>± 4.12<sup>ab</sup></td><td>63.84<br/>± 4.25<sup>c</sup></td></tr></tbody></table>

<table><caption>**Table 3.** Effect of phytase on tibia bone mineralization of broilers at 35 days</caption><thead><tr><th>Treatment</th><th></th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th></tr></thead><tbody><tr><td>Phytase</td><td>FTU kg<sup>-1</sup></td><td>0</td><td>250 n-ct</td><td>500n-ct</td><td>250 ct</td><td>500 ct</td></tr><tr><td>Tibia bone weight (as is)</td><td>g</td><td>10.1<br/>± 1.9</td><td>10.9<br/>± 1.6</td><td>11.6<br/>± 1.1</td><td>11.1<br/>± 1.2</td><td>10.7<br/>± 1.0</td></tr><tr><td>Tibia fat-free dry matter</td><td>%</td><td>36.88<br/>± 4.10</td><td>37.98<br/>± 2.54</td><td>37.10<br/>± 1.33</td><td>37.45<br/>± 2.99</td><td>37.07<br/>± 2.08</td></tr><tr><td colspan="7"><strong>Tibia bone mineralization</strong></td></tr><tr><td>Crude ash</td><td>g kg<sup>-1</sup> DM</td><td>262.0<br/>± 21.3<sup>a</sup></td><td>308.2<br/>± 23.8<sup>b</sup></td><td>313.1<br/>± 13.6<sup>b</sup></td><td>311.9<br/>± 32.1<sup>b</sup></td><td>320.3<br/>± 22.1<sup>b</sup></td></tr><tr><td>Phosphorus</td><td>g kg<sup>-1</sup> DM</td><td>34.6<br/>± 2.9<sup>a</sup></td><td>40.5<br/>± 2.9<sup>b</sup></td><td>43.3<br/>± 2.9<sup>b</sup></td><td>42.4<br/>± 4.9<sup>b</sup></td><td>49.6<br/>± 4.5<sup>c</sup></td></tr><tr><td>Calcium</td><td>g kg<sup>-1</sup> DM</td><td>92.8<br/>± 9.8<sup>a</sup></td><td>106.4<br/>± 7.1<sup>bc</sup></td><td>110.5<br/>± 6.6<sup>bc</sup></td><td>108.7<br/>± 6.6<sup>bc</sup></td><td>113.1<br/>± 5.5<sup>c</sup></td></tr></tbody></table>

## Conclusions

Based on apparent ileal digestibility and tibia bone mineralization it can be concluded that the coating had no negative influence on the biological efficacy of the tested phytase. The new phytase in coated form at the dose levels of 250 and 500 FTU per kg of diet was superior (partly significant) to the uncoated form. Moreover, the benefits of uncoated or coated new phytase correlated with the dose level.

## Literature

GfE, 1999: Empfehlungen zur Energie- und Nährstoffversorgung der Legehennen und Masthühner. Ausschuss für Bedarfsnormen der Gesellschaft für Ernährungsphysiologie. DLG-Verlags-GmbH.

WPSA, 1984: The prediction of apparent metabolisable energy values for poultry in compound feeds. World's Poult. Sci. 40, 181-182.

OWUSU-ASIEDU, A.; C. GILBERT, P. H. SIMMINS, C. M. NYACHOTI, 2007: Effect of a new coating on the bioefficacy of a phytase product in broilers fed corn-soybean meal-based diets. World Poultry Science Association, Proceedings of the 16th European Symposium on Poultry Nutrition, Strasbourg, France, 26.-30. August, 2007, pp. 591-594.

<footnote>ab Values with different superscripts within lines differ significantly (P<0.05) n-ct = uncoated, ct = coated</footnote>

<footnote>ab Values with different superscripts within lines differ significantly (P<0.05) n-ct = uncoated, ct = coated</footnote>
P-104 (ID 203)

ADDITIONAL EFFECT OF ENDOXYLANASES ON TOP OF SUPERDOSED PHYTASE ON BROILER PERFORMANCE

Ronny Mombaerts and Anne Goderis

Nutrex nv, Achterstenhoek 5, 2275 Lille, Belgium

E-mail corresponding author: ronny.mombaerts@nutrex.be

Abstract

To study the additional effect of xylanase on top of superdosed phytase (SD), a trial was organised at KU Leuven using 450 day-old Ross 308, floor pen housed, male broilers. Three dietary treatments were included: a standard wheat-corn based diet supplemented with 1500 U/kg feed of 6-phytase (= SD), SD with additional bacterial (Bacillus subtilis) endo-1,4-β-xylanase (100 mg/kg feed) (= BX), and SD with additional fungal (Trichoderma citrinoviride) endo-1,4-β-xylanase (100 mg/kg feed) (=FX). Ten replicates of 15 broilers were allocated to each of these 3 treatments during 42 days.

Supplementing SD with BX or FX significantly improved body weight (BW) (2925 vs 3085 and 3022 g) and corrected FC (FCcorr - 2 kg) (1.31 vs 1.21 and 1.26). With a FC of 1.65, BX was numerically better than FX (FC = 1.68) and SD (FC = 1.67). Comparing the results of both endo-1,4-β-xylanase products, the bacterial showed to be more efficient on FCcorr - 2 kg (p <0.05), BW (p< 0.1) and FC (numerically).

To conclude, endo-1,4-β-xylanases proved to be beneficial on top of superdosed phytase, and clear differences were
seen between fungal and bacterial endo-1,4-β-xylanase.

Keywords: xylanase, bacterial, broilers, performance

Introduction

Since superdosing of phytase is gaining interest in the broiler industry and speculations are made about the mode of
action of the “extra-phosphorous” effect, some doubts can arise whether there will still be a sufficient additional effect of
NSP enzymes. Phytases and xylanases act on complete different substrates and the mode of action, improving broiler
performance, is different as well. Maybe the effect of combining both classes of enzymes is not strictly additive, enough
arguments exist to believe in cost effectiveness of using both enzymes. The bio-efficacy of 2 different xylanases, on top
of superdosed phytase, was determined in a zootechnical performance trial.

Material & methods

The experiment was organised and carried out by the Catholic University of Leuven and consisted of a floor pen trial, using 30 pens (3 treatment * 10 replicates) of 15 male broiler chickens per pen. The experimental house is divided into pens of equal size, arranged along a central aisle. The pens had a surface of 0.8 m² each, with built up wood shavings as bedding. The temperature of the building was monitored and environmental conditions during the trial were optimum to the age of the animals. The lighting scheme was 24 hours light per day from day 1 to day 14 and a repeating cycle of 1 hour light and 3 hours dark from day 15 to day 42. The diets were provided ad libitum in one feeding trough per pen. Water was provided ad libitum with two drinking nipples per pen. Standard floor pen management practices were used throughout the experiment.

Male Ross 308 chickens were obtained from Belgabroed, Belgium. The breeder flock history and vaccination record at the hatchery were recorded. The broilers were vaccinated at the farm on day 14 against Gumboro and Newcastle Disease via drinking water. Broiler weights by pen by sex were recorded on days 1, 14 and 42. Feed intake was measured immediately after weighing on day 14 and day 42.

Three dietary treatments were included: a standard wheat-corn based diet supplemented with 1500 U/kg feed of 6-phytase (= SD), SD with additional bacterial (Bacillus subtilis) endo-1,4-β-xylanase (100 mg/kg feed) (= BX), and SD with additional fungal (Trichoderma citrinoviride) endo-1,4-β-xylanase (100 mg/kg feed) (= FX). Ten replicates of 15 broilers were allocated to each of these 3 treatments during 42 days.

All experimental diets (composition in table 1) were manufactured by Research Diet Services in the Netherlands. All feeds were fed in pellet form. Starter feed was fed from day 1 to 14. On day 14, non-consumed starter was weighed by
pen and discarded. Grower and finisher feeds were issued and fed until day 28 and 42 respectively. On day 42, non-consumed feed was weighed by pen and discarded.

<table><caption>TABLE 1: DIET COMPOSITION</caption><thead><tr><th>Ingredients (%)</th><th>Starter</th><th>Grower</th><th>Finisher</th></tr></thead><tbody><tr><td>Wheat</td><td>40,57</td><td>44,58</td><td>52,84</td></tr><tr><td>Corn</td><td>20,00</td><td>20,00</td><td>20,00</td></tr><tr><td>Soybean Meal 47</td><td>33,22</td><td>29,37</td><td>21,89</td></tr><tr><td>Fats/oils</td><td>2,23</td><td>3,09</td><td>2,75</td></tr><tr><td>Monocalciumphosphate</td><td>0,89</td><td>0,46</td><td>0,22</td></tr><tr><td>Limestone</td><td>1,23</td><td>0,87</td><td>0,64</td></tr></tbody></table>

<table><thead><tr><th>Nutrients (%)</th><th></th><th></th><th></th></tr></thead><tbody><tr><td>Crude protein</td><td>22,00</td><td>20,50</td><td>18,00</td></tr><tr><td>Crude fat</td><td>4,19</td><td>5,01</td><td>4,64</td></tr><tr><td>Crude fibre</td><td>2,66</td><td>2,61</td><td>2,53</td></tr><tr><td>Calcium</td><td>0,72</td><td>0,51</td><td>0,36</td></tr><tr><td>Phosphorous P</td><td>0,60</td><td>0,49</td><td>0,41</td></tr><tr><td>Sodium</td><td>0,16</td><td>0,15</td><td>0,14</td></tr><tr><td>Potassium</td><td>0,99</td><td>0,91</td><td>0,79</td></tr><tr><td>6-phytase (U/kg)</td><td>1500</td><td>1500</td><td>1500</td></tr></tbody></table>

Statistical analysis of average daily gain, feed intake and feed conversion was done with a linear model (SAS software). Model choice was based on the R²-value, on the root MSE and the distribution of the residuals.

## Results

In this trial significant treatment effects were observed for final body weight (P = 0.001) and corrected feed conversion (P = 0.001), while feed intake was not influenced by the treatments (P = 0.48).

The results in Figure 1 show that both NSP enzymes were able to increase significantly the final body weight of the animals. Adding xylanases increased final body weight by 5.5 % and 3.3 % for resp. BX and FX. Improving daily gain without changing feed intake means that feed utilisation became more efficient after xylanase supplementation. Corrected feed conversion improved by adding xylanase in a superdosed phytase diet. FCcor - 2 kg improved by 7.5 and 3.8 % for resp. the BX and FX supplemented group.

Although both xylanase sources improve the performance of the broilers, it's important to mention that they cannot be considered as equally effective. BX supplemented broilers were 63 g heavier (P < 0.1) at the end of the trial and their FCcor - 2 kg was 0.05 lower (P < 0.05) compared to the FX animals.

These data confirm the hypothesis that the mode of action of xylanase - which is a xylanase-specific combination of reducing viscosity, liberating entrapped nutrients and creating prebiotic substances - is not the same as the mode of action of phytase, even when superdosed.

## Conclusions

Based on this trial, there shouldn't be any doubts about the technical or economical benefits of adding xylanase in combination with superdosed phytase. A closer look however to the source and type of xylanase is important. Efficacy may vary considerably between different types of xylanases.

<figure><img src="image_40.png" /></figure>
P-105 (ID 307)

EFFECT OF A CARBOHYDRASES COMPLEX IN FEEDS WITH DIFFERENT METABOLIZABLE ENERGY AND AVAILABLE AMINO ACIDS LEVELS FOR BROILERS REARED UNDER A HOT CLIMATE CONDITION

R. Montanhini Neto, P. Cozannet, F. Rouffineau, and A. Preynat

Centre of Expertise and Research in Nutrition, Adisseo France SAS, Malicorne, France

Corresponding author: roberto.neto@adisseo.com

**Abstract**

The objective of this study was to evaluate the effect of a carbohydrate complex (CC) added to corn-soybean-based broiler feed formulated with different apparent metabolic energy (AME) and digestible amino acids (DAA) levels. Four diets were prepared as following: T1, Positive Control (PC) with regular commercial nutritional levels; T2, Negative Control (NC) 1, with less 80 kcal of AME kg⁻¹ and less 2% of DAA; T3, NC 2, -80 kcal kg⁻¹ and -4% of DAA; and T4, NC 3, -80 kcal kg⁻¹ and -6% of DAA. CC (Rovabio® ADVANCE L) was added or not in each of those four diets, totalling 8 treatments, with 12 repetitions and 40 male birds in each. Air temperature inside the facility was kept above the breeder recommendations (29 °C) and humidity was in constant rise during the experimental (0-36 d). DAA reduction in feed linearly reduced (P<0.01) all performance parameters. No interaction between feed nutritional levels and the addition of the CC was observed. For the overall period, the addition of CC improved (P<0.01) BWG (+3.3%) and FCR (-2.7%), regardless of nutritional reduction levels. The efficacy of energy and protein deposition (calculated as g of BWG for each cal of AME or mg of digestible Lysine ingested) were significantly improved by the CC addition (+2.7% and +2.8%, respectively). This study also allowed to estimate the ideal DAA reduction thanks to CC addition between -2% and -4%, when AME is reduced by 80 kcal.

**Keywords:** NSP-enzymes, broilers, amino acids, nutritional matrix, nutrient deposition

**Introduction**

The benefits of adding carbohydrate complex (CC) to the diets of non-ruminant animals, particularly poultry, are well established (Campbell and Bedford 1992). Enzymes have been shown to improve performance and nutrient digestibility when added to poultry diets containing cereals (Bedford and Schulze 1998). Therefore, data illustrating practical reformulation were scarce. Improved knowledge of CC effect on practical feed formulation utilization could result in development of CC directed towards far more specific targets. Improvement of diet AME content have already been evaluated previously. Data on amino acid deposition were less important. The aim of the present study was (1) to investigate the effect of a reduction of DAA on performance and (2) to measure the effect of enzyme supplementation on diets.

**Material and Methods**

Experiment were carried out in a commercial farm. The study was conducted as a 4 × 2 factorial blocked arrangement of treatments with 4 levels of apparent metabolizable energy adjusted for zero nitrogen retention (AMEn), and digestible essential amino acids (DAA), with or without CC supplementation. Diet nutrient constraints for positive control (PC) were fixed to meet the minimum requirements relative to energy content according to the Rhodimet Nutrition Guide (Adisseo 2013). Feed were based on corn soybean meal. Digestible amino acids and metabolizable energy for negative control (NC) were obtained by reduction of vegetable oil and synthetic amino acid/soybean meal reduction, respectively. The feeding program consisted of 3 phases: starter diet from 0 to 14 d, grower diet supplied from 15 to 28 d and finisher diet from 29 to 36 d. Reformulation resulted in 3 diets: negative control (NC) 1, with less 80 kcal of AME kg⁻¹ and less 2% of DAA; T3, NC 2, -80 kcal kg⁻¹ and -4% of DAA; and T4, NC 3, -80 kcal kg⁻¹ and -6% of DAA. Enzyme was included at 200ml/ton.

For this experiment, 3840 one-d old Ross PM3 male broilers (body weight 40±1.4g) were used and allocated into 96 pens, with 40 chickens per pen. Pens were used in factorial and completely blocked design with 12 pens per treatment. High temperature was maintained during the experiment at 29°C from day 4 to 36. Humidity was gradually increased from 45 to 78% from day 0 to 36. Feed and water were provided *ad libitum* throughout the experiment. Animal feed intake (FI) and body weight gain (BWG) were recorded 4h after the removal of feed at 21 and 35 d of age corresponding to the end of the starting, the growing and the finishing periods, respectively. Mortality was recorded daily. Carcass
analysis were performed at the end of the experiment. Carcass, breast and legs were weighted and mass relative to the live body weight were estimated.

## Results

All the animal remained healthy during the experiment. Performance of the positive control were in agreement with standard performances observed in the facility. Results are presented Table 1. During the overall period, DAA reduction resulted in a linear reduction BWG and increase of FCR of animal fed control diet (R² = 0.65 and 0.73, respectively; P < 0.001). Enzyme effect significantly increased the performances of broilers. Average increase of BWG and Feed efficiency were +3.2 and 2.6%, respectively. Effect were more pronounced for FCR during the starter period (-5.0%) and for BWG during the growing period (+3.9%).

<table><caption>**Table 1.** Combined effect of the reduction of digestible amino acids (DAA) content and the addition of a carbohydrate complex (CC) in feeds for broilers reared under hot climate condition in different periods (n=96).</caption><thead><tr><th>DAA Reduction</th><th colspan="2">0</th><th colspan="2">2%</th><th colspan="2">4%</th><th colspan="2">6%</th><th colspan="3">Statistic<fnref n="1" /></th></tr><tr><th>CC addition</th><th>wo</th><th>with</th><th>wo</th><th>with</th><th>wo</th><th>with</th><th>wo</th><th>with</th><th>CC</th><th>DAA Red</th><th>Inter</th></tr></thead><tbody><tr><td colspan="12" style="text-align: center; font-weight: bold;">Starter period</td></tr><tr><td>BWG</td><td>369<sup>ab</sup></td><td>372<sup>ab</sup></td><td>369<sup>ab</sup></td><td>383<sup>a</sup></td><td>359<sup>b</sup></td><td>368<sup>ab</sup></td><td>353<sup>b</sup></td><td>366<sup>ab</sup></td><td>***2</td><td>**</td><td>NS</td></tr><tr><td>FI</td><td>431<sup>bc</sup></td><td>414<sup>c</sup></td><td>442<sup>b</sup></td><td>447<sup>ab</sup></td><td>456<sup>ab</sup></td><td>445<sup>b</sup></td><td>472<sup>a</sup></td><td>453<sup>ab</sup></td><td>NS</td><td>*</td><td>NS</td></tr><tr><td>FCR</td><td>1.168<sup>cd</sup></td><td>1.112<sup>d</sup></td><td>1.199<sup>c</sup></td><td>1.168<sup>cd</sup></td><td>1.272<sup>ab</sup></td><td>1.211<sup>bc</sup></td><td>1.337<sup>a</sup></td><td>1.236<sup>bc</sup></td><td>*</td><td>***</td><td>NS</td></tr><tr><td colspan="12" style="text-align: center; font-weight: bold;">Grower period</td></tr><tr><td>BWG</td><td>856<sup>ab</sup></td><td>895<sup>a</sup></td><td>825<sup>bc</sup></td><td>857<sup>ab</sup></td><td>794<sup>bc</sup></td><td>813<sup>bc</sup></td><td>780<sup>c</sup></td><td>816<sup>bc</sup></td><td>*</td><td>**</td><td>NS</td></tr><tr><td>FI</td><td>1357</td><td>1376</td><td>1360</td><td>1378</td><td>1362</td><td>1382</td><td>1375</td><td>1400</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>FCR</td><td>1.585<sup>cd</sup></td><td>1.542<sup>d</sup></td><td>1.649<sup>bc</sup></td><td>1.615<sup>c</sup></td><td>1.717<sup>ab</sup></td><td>1.694<sup>b</sup></td><td>1.768<sup>a</sup></td><td>1.717<sup>ab</sup></td><td>*</td><td>***</td><td>NS</td></tr><tr><td colspan="12" style="text-align: center; font-weight: bold;">Finisher period</td></tr><tr><td>BWG</td><td>586</td><td>608</td><td>586</td><td>609</td><td>602</td><td>607</td><td>588</td><td>603</td><td>*</td><td>NS</td><td>NS</td></tr><tr><td>FI</td><td>1159</td><td>1150</td><td>1169</td><td>1193</td><td>1203</td><td>1197</td><td>1223</td><td>1238</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>FCR</td><td>1.981<sup>ab</sup></td><td>1.903<sup>b</sup></td><td>1.998<sup>ab</sup></td><td>1.966<sup>ab</sup></td><td>2.007<sup>ab</sup></td><td>1.977<sup>ab</sup></td><td>2.086<sup>a</sup></td><td>2.057<sup>a</sup></td><td>*</td><td>**</td><td>NS</td></tr><tr><td colspan="12" style="text-align: center; font-weight: bold;">Overall period</td></tr><tr><td>BWG</td><td>1811<sup>b</sup></td><td>1875<sup>a</sup></td><td>1780<sup>c</sup></td><td>1849<sup>a</sup></td><td>1755<sup>cd</sup></td><td>1788<sup>b</sup></td><td>1721<sup>d</sup></td><td>1785<sup>b c</sup></td><td>**</td><td>**</td><td>NS</td></tr><tr><td>FI</td><td>2947</td><td>2940</td><td>2971</td><td>3018</td><td>3021</td><td>3024</td><td>3070</td><td>3091</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>FCR</td><td>1.627<sup>d</sup></td><td>1.568<sup>e</sup></td><td>1.669<sup>cd</sup></td><td>1.632<sup>d</sup></td><td>1.721<sup>bc</sup></td><td>1.691<sup>c</sup></td><td>1.784<sup>a</sup></td><td>1.732<sup>b</sup></td><td>**</td><td>***</td><td>NS</td></tr></tbody></table>

<table><caption>**Table 2.** Equations performance parameters (BWG and FCR during overall period) based on reduction of digestible amino acids (DAA) content and the addition of carbohydrates in feeds for broilers reared under hot climate condition in different periods (n=96).</caption><thead><tr><th>Parameter</th><th>CC addition</th><th>Equation<fnref n="1" /></th><th>R²</th></tr></thead><tbody><tr><td rowspan="2">BWG</td><td>without</td><td>BWG = 1884 - 30 * red DAA</td><td>0.58</td></tr><tr><td>with</td><td>BWG = 1949 - 33 * red DAA</td><td>0.41</td></tr><tr><td rowspan="2">FCR</td><td>without</td><td>FCR = 1.532 + 0.051 * red DAA</td><td>0.77</td></tr><tr><td>with</td><td>FCR = 1.488 + 0.052 * red DAA</td><td>0.85</td></tr></tbody></table>

No interaction were observed among AA reduction and enzyme inclusion for all parameters. Linear equation were established between DAA reformulation and performance with and without CC. The equation were presented in Table 2. From this equation, BWG difference between supplemented and non-supplemented diets was estimated to 2.09% increase DAA whereas corresponding value for FCR was 0.85%. As a result, enzyme effect could be consider with a potential of 2.09 + 0.85 = 2.94% increase DAA. All effects were observed on FCR adj. For this parameter, difference between supplemented and non-supplemented diets was estimated to 2.09%. Carcass measurement performed at the end of the trial did not suggest difference among treatment and not alteration

<footnote n="1">¹ Data were subject to variance analysis with fixed effect CC (n=2) and two covariables DAA and AME content and interaction AA content*CC (n=8). 2 NS = P>0.05; * = P<0.05; ** = P<0.01; *** = P<0.001.</footnote>

<footnote n="1">¹ Data (n= 96) were analysed by regression using DAA red as predictive variable and CC as fixed effect.</footnote>
Improvement was observed regardless of the level of DAA of the diet. This results were in agreement with studies suggesting improvement of DAA using enzyme (Nian et al. 2011; Kalmendal et Tauson 2012). Reformulation of poultry diet taking into account metabolizable and DAA improvement might increase the CC value.

## Conclusions

Present study suggest important impact of DAA on animal BWG and FCR. Additive CC allowed an increase of animal performance. These improvement was estimated to 3% improvement of DAA.

## Literature

ADISSEO, 2013: Rhodimet Nutrition Guide. Adisseo France SAS, Antony.

BEDFORD M.R. and SCHULZE H., 1998: Exogenous enzymes for pig and poultry. Nut. Res. Rev. 11, 91-114.

CAMPBELL G.L. and BEDFORD M.R., 1992: Enzyme applications for monogastric feeds: a review. Can. J. Anim. Sci. 72, 449-466.

KALMENDAL R. and TAUSON R., 2012: Effect of a xylanase and protease, individually or in combination, and an ionophore coccidiostat on performance, nutrient utilization, and intestinal morphology in broiler chickens fed a wheat-soybean meal based diet. Poult. Sci. 91, 1387-1393

NIAN F., GUO Y.M., RU Y.J., PERON A. and LI F.D., 2011: Effect of xylanase supplementation on the net energy for production, performance and gut microflora of broilers fed corn/soy-based diet. Asian-Aust. J. Anim. Sci. 24, 1282-1287.
P-106 (ID 305)

# EFFECT OF THE ADDITION OF A CARBOHYDRASE COMPLEX ON PERFORMANCE AND CARCASS YIELD OF BROILER CHICKENS FED WHEAT-SOYBEAN-BASED DIETS

R. Montanhini Neto, Pierre Cozannet and A. Preynat

Centre of Expertise and Research in Nutrition, Adisseo France SAS, Malicorne, France

Corresponding author: roberto.neto@adisseo.com

## Abstract

The objective of this study was to evaluate the effect of the addition of a carbohydrate complex (CC) on the performance and nutrients' deposition of broiler chickens fed wheat-soybean-based diets. Six control diets were formulated with different digestible amino acids (dAA) content (-7.5%, -5.0%, -2.5%, 0.0%, +2.5%, and +5.0%) based on commercial recommendations, but with the same ideal protein profile by controlling essential dAA content (Met, Met+Cys, Lys, Thr, Val, Iso, Arg and Trp) in formulation of feeds. Six treatment diets were prepared by adding CC (Rovabio® ADVANCE L, 200 mL ton⁻¹) to the six control diets reduced in energy (85 kcal kg⁻¹) in order to isolate the energy effect. Each of the 12 treatments included eight repetitions of 15 male birds. Performance parameters (BWG, FI and FCR), efficacy of energy and protein deposition (calculated as g of BWG for each cal of AME or mg of dig. Lys ingested) and carcass and cuts yield were calculated for the period between 0-42 d. The reduction of DAA linearly degraded (P<0.05) all performance, nutrient deposition and carcass parameters. Carcass and cuts yield were not affected by CC addition or DAA reduction (P>0.05). No interaction was observed between DAA content and the addition of the CC. The addition of the CC significantly improved (P<0.001) BWG (+3.2%), FCR (-2.7%), and deposition of energy (+5.7%) and protein (+2.8%). In conclusion, the addition of CC can improve the performance and deposition of nutrients in order of 3%, regardless the level of tested DAA in diets.

**Keywords:** NSP-enzymes, broilers, amino acids, nutritional matrix, nutrient deposition.

## Introduction

The quality of feedstuffs for feed production can be highly variable, especially considering their nutritional composition (Maisonnier et al. 2001). Plant feed ingredients have different types of anti-nutritional factors and indigestible components. Monogastric animals lack enzymes to digest cellulose, arabinoxylan, beta-glucans, pectins, among other non-starch polysaccharides that make up the cell wall of some feeds (Ribeiro et al., 2011). To improve the availability of nutrients and minimize the anti-nutritional factors, the use of diets containing carbohydrate is becoming common (Avila et al. 2012). When added to poultry diets, carbohydrates usually result in numerous beneficial effects, such as increased utilization of nutrients, improved the utilization of apparent metabolizable energy (AME), increased growth rate, improved feed to gain ratio, decreased viscosity of intestinal digesta (Costa et al. 2008). The objective of this study was to evaluate the effect of the addition of a carbohydrate complex on the performance and nutrients' deposition of broiler chickens fed wheat-soybean meal-based diets.

## Material and Methods

This study was carried out in the Centre of Expertise and Research in Nutrition (CERN), Adisseo France SAS, in Malicorne, France (46°18'14" N, 2°44'07" E), between October and December 2014. The 1,440 male Ross PM3 broilers were divided in 12 treatments, with 8 repetitions of 15 birds each. Repetition consisted floor pens (2.5 m², disposed in bloc) between 1 and 42 days of age, in 3 phases: initial (d1-14), grower (d15-28) and finisher (d29-42). Six control wheat-soybean meal-based diets were formulated with different digestible amino acids (dAA) content (-7.5%, -5.0%, -2.5%, 0.0%, +2.5%, and +5.0%) relative to the recommendations of Rhodimet Nutrition Guide (Adisseo 2013). Essential dAA (Met, Met+Cys, Lys, Thr, Val, Iso, Arg and Trp) were controlled by the use of their industrial sources to ensure adequate inclusion of soybean meal (Table 2). Six treatment diets were prepared by adding of a carbohydrate complex (Rovabio® ADVANCE L, 200 mL ton⁻¹) to the diets reduced in energy (85 kcal kg⁻¹) (Table 1). Broilers and feed were weighted at d0, d14, d28 and d42 in order to calculate the performance parameters (BWG, FI and FCR) for each single phase and the overall period. Energy and protein efficiency were estimated dividing BWG by either metabolizable energy or digestible lysine intake (expressed as g of BWG AME or mg of dig. Lys ingested) for the overall period (d1-42). At d42, five birds per pen were killed and submitted to a controlled slaughtering where carcass and cuts were weighted, in order to determine their yield.
<table><caption>**Table 1.** Experimental design and information of treatments.</caption><thead><tr><th>Treatments</th><th>Additio of Carbohydrase Complex<sup>1</sup></th><th>Energy (AME) Reduction</th><th>Digestible Amino Acids Reduction<sup>2</sup></th></tr></thead><tbody><tr><td>T1</td><td>No</td><td>0</td><td>-7.5%</td></tr><tr><td>T2</td><td>No</td><td>0</td><td>-5.0%</td></tr><tr><td>T3</td><td>No</td><td>0</td><td>-2.5%</td></tr><tr><td>T4</td><td>No</td><td>0</td><td>0.0%</td></tr><tr><td>T5</td><td>No</td><td>0</td><td>+2.5%</td></tr><tr><td>T6</td><td>No</td><td>0</td><td>+5.0%</td></tr><tr><td>T7</td><td>With</td><td>85 kcal (2.8%)</td><td>-7.5%</td></tr><tr><td>T8</td><td>With</td><td>85 kcal (2.8%)</td><td>-5.0%</td></tr><tr><td>T9</td><td>With</td><td>85 kcal (2.8%)</td><td>-2.5%</td></tr><tr><td>T10</td><td>With</td><td>85 kcal (2.8%)</td><td>0.0%</td></tr><tr><td>T11</td><td>With</td><td>85 kcal (2.8%)</td><td>+2.5%</td></tr><tr><td>T12</td><td>With</td><td>85 kcal (2.8%)</td><td>+5.0%</td></tr></tbody></table>

<table><caption>**Table 2.** Composition of experimental diets.</caption><thead><tr><th rowspan="2">Composition (%)</th><th colspan="2">Starter (d 0-14)</th><th colspan="2">Grower (d 15-28)</th><th colspan="2">Finisher (d 29-42)</th></tr><tr><th>T1-T6</th><th>T7-T12</th><th>T1-T6</th><th>T7-T12</th><th>T1-T6</th><th>T7-T12</th></tr></thead><tbody><tr><td>Wheat</td><td>58.4</td><td>60.3</td><td>65.5</td><td>67.5</td><td>70.0</td><td>72.0</td></tr><tr><td>Soybean meal</td><td>30.5</td><td>30.0</td><td>23.5</td><td>23.0</td><td>19.0</td><td>18.5</td></tr><tr><td>Soybean oil</td><td>6.1</td><td>4.7</td><td>6.5</td><td>5.0</td><td>7.0</td><td>5.5</td></tr><tr><td>Nucleus<sup>1</sup></td><td>5.0</td><td>5.0</td><td>4.5</td><td>4.5</td><td>4.0</td><td>4.0</td></tr></tbody></table>

## Results

No effects from bloc or interaction between dAA reduction and the addition of the carbohydrate complex were observed for any evaluated parameter, and no differences of variances were observed among treatments. Treatments with the addition of carbohydrate complex, compared to those without enzyme addition, obtained better (P < 0.05) BWG and FCR in the initial and finisher phases as well as during the overall experimental period (+3.2% in BWG and +2.7% in FCR). The addition of the complex promoted significant improvement (P<0.01) of efficacy of energy (+5.7%) and protein (+2.8%) deposition. However, no difference (P>0.05) was observed for carcass and cuts yield with the addition (Tables 3 and 4).

<table><caption>**Table 3.** Performance results during the three production periods of broilers fed diets with or without the addition of a carbohydrate complex (CC).</caption><thead><tr><th rowspan="2">Production Periods</th><th colspan="3">Initial (d0-14)</th><th colspan="3">Grower (d15-28)</th><th colspan="3">Finisher (d29-42)</th></tr><tr><th>BWG<sup>1</sup></th><th>FI<sup>2</sup></th><th>FCR<sup>3</sup></th><th>BWG</th><th>FI</th><th>FCR</th><th>BWG</th><th>FI</th><th>FCR</th></tr></thead><tbody><tr><td>CC Addition</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Without</td><td>412</td><td>541</td><td>1.314</td><td>1010</td><td>1665</td><td>1.650</td><td>1495</td><td>2905</td><td>1.947</td></tr><tr><td>With</td><td>422</td><td>543</td><td>1.286</td><td>1028</td><td>1675</td><td>1.631</td><td>1559</td><td>2911</td><td>1.871</td></tr><tr><td>P-Values<sup>4</sup></td><td>&lt;0.01</td><td>0.55</td><td>&lt;0.01</td><td>0.08</td><td>0.51</td><td>0.14</td><td>&lt;0.01</td><td>0.88</td><td>&lt;0.01</td></tr></tbody></table>

<footnote n="1">¹ Rovabio ADVANCE L, 200 mL ton⁻¹, Adisseo France SAS, Antony, France. ² Reductions were based in the recommendation of Rhodimet Nutrition Guide (Adisseo 2013), as expressed in the level 0.0% of reduction.</footnote>

<footnote n="1">¹ Nucleus composition: Premix (vitamins, trace minerals and other additives), macro minerals (sodium sulphate, salt, dicalcium phosphate and limestone), and industrial amino acids (calcium salt of methionine hydroxy analogue, L-Lysine, L-Threonine, L-Tryptophan, L-Valine, L-Arginine, and L-Isoleucine).</footnote>

<footnote n="1">¹ Body weight gain (in g) for the respective period. ² Feed intake (in g) for the respective period. ³ Feed conversion ratio (in g g⁻¹) for the respective period. ⁴ All variables were tested for normality and homoscedasticity, allowing parametric statistics application. Treatments were compared by T-Test (P<0.05).</footnote>
<table><caption>**Table 4.** Performance during the overall experimental period (d1-42), efficacy of energy and protein deposition, and carcass yield of broilers fed diets with or without the addition of a carbohydrate complex (CC).</caption><thead><tr><th rowspan="2">Period</th><th colspan="3">Performance</th><th colspan="2">Efficacy of Deposition</th><th colspan="3">Carcass Yield</th></tr><tr><th>BWG</th><th>FI</th><th>FCR</th><th>Energy<sup>1</sup></th><th>Protein<sup>2</sup></th><th>Carc.<sup>3</sup></th><th>Brest<sup>4</sup></th><th>Legs<sup>5</sup></th></tr></thead><tbody><tr><td>CC Addition</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Without</td><td>2917</td><td>5111</td><td>1.754</td><td>0.185</td><td>56.69</td><td>72.13</td><td>30.05</td><td>13.51</td></tr><tr><td>With</td><td>3009</td><td>5129</td><td>1.706</td><td>0.196</td><td>58.27</td><td>72.69</td><td>30.72</td><td>13.46</td></tr><tr><td>P-Values<sup>4</sup></td><td>&lt;0.01</td><td>0.70</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>0.62</td><td>0.12</td><td>0.86</td></tr></tbody></table>

On the other hand, the reduction of dAA linearly degraded (P<0.05) all performance, nutrient deposition, and carcass parameters.

## Conclusions

Based in the analysis of the results obtained in this study, the addition of the evaluated carbohydrate complex can improve the performance, carcass yield and deposition of energy and protein in the order of 3% of broilers, regardless the level of dAA used in their diets.

## Literature

ADISSEO, 2013: Rhodimet Nutrition Guide. Adisseo France SAS, Antony, 10-13.

AVILA E., ARCE J., SOTO C. et al., 2012: Evaluation of an enzyme complex containing non-starch polysaccharide enzymes and phytase on the performance of broilers fed a sorghum and soybean meal diet. J. Appl. Poult. Res. 21, 279-286.

COSTA F., GOULART C.C., FIGUEIREDO D.F., et al. 2008: Economic and environmental impact of using exogenous enzymes on poultry feeding. Int. Poult. Sci. 88, 140-148.

MAISONNIER S., GOMEZ J., CHAGNEAU A.M. and CARRÉ B., 2001: Analysis of variability in nutrient digestibilities in broiler chickens. Brit. Poult. Sci.42, 70-76.

RIBEIRO T., LORDELO M.M.S., PONTE P.I.P. et al., 2011: Levels of endogenous beta-glucanase activity in barley affect the efficacy of exogenous enzymes used to supplement barley diets for poultry. Poult. Sci., 90, 1245-1256

<footnote n="1">¹ Calculated as g of BWG for each calorie of AME ingested in the same period. ² Calculated as g of BWG for each g of Dig. Lysine ingested in the same period. ³ Calculated as carcass weight compared to body weight (presented in %). ⁴ Calculated as entire breast weight (with muscle and bones) compared to carcass weight (in %). ⁵ Calculated as one entire leg weight (with muscle and bones) compared to carcass weight (in %).</footnote>
P-109 (ID 169)

EFFECT OF HOSTAZYM® X ON DIGESTIBILITY OF CEREALS AND PROTEINACEOUS BY-PRODUCTS IN BROILERS

L. Nollet, K. Bierman and N. Soares

Huvepharma NV, Antwerp, Belgium

Corresponding author: lode.nollet@huvepharma.com

Abstract

A trial was conducted to evaluate the effect of Hostazym® X on digestibility of wheat, corn, barley, soybean meal, rapeseed meal and DDGS (of wheat origin) in broilers. In total 56 cages, of 6 birds each, were fed a general starter diet until 13 days of age. From 13-22 days, birds were fed the different test feeds with or without Hostazym® X (1500 EPU/kg). Feeds contained either 65 % of wheat, corn or barley (supplemented with a 35 % soybean meal based protein concentrate), or 30 % of either DDGS or soybean meal (supplemented with a 70 % corn/wheat based carbohydrate concentrate). Furthermore, also 2 test diets containing 10 or 20 % rapeseed meal were incorporated in the trial. Digestibility was measured between 18 and 22 days, based on the method of total faecal collection. Firstly, results showed that Hostazym® X inclusion led to an increase in metabolisable energy of 45, 104, 94, 89, 120, 33 and 111 kCal/kg of feed. Secondly, the Hostazym® X treatment yielded an increment-in overall fat digestibility by 1.3, 5.0, 3.7, 1.2, 8.8, 2.0 and 3.0 g fat/kg of feed for wheat, corn, barley, soybean meal, DDGS, 10 and 20 % rapeseed meal based feeds respectively. Thirdly, faecal droppings were reduced by 4 – 14.5 % when using Hostazym® X, and water intake was reduced at a rate of 0.5 up to 6.1 %. From this trial it could be concluded that the inclusion of Hostazym® X in different types of feed, gave a clear improvement in digestibility while reducing faecal output and water consumption.

**Keywords:** xylanase, Hostazym® X, digestibility, wet litter

Introduction

Enzymes are used to degrade non-starch polysaccharides, mainly to reduce viscosity in the intestine and to liberate more nutrients which are entrapped by insoluble fibrous structures. This improves the digestibility of the nutrients present in the feed, while reducing excretion, optimizing performance and improving gut health status. Hostazym® X is a well-established enzymatic complex product used in many types of diets, and is known to exert a consistent and beneficial effect on the digestibility of nutrients in poultry diets. However, new data on this effect needs to be generated continuously, in order to confirm the existing matrix values.

Trial description

The digestibility study was composed out of 56 cages of 6 female broilers (ROSS 308) per cage. Broilers were fed a starter feed till 13 days of age. From 13 till 22 days of age, animals were fed with the treatment feeds. Day 13-18 was considered as the adaptation period, while the digestibility assay was conducted from day 18 till day 22. Different feeds were produced with or without Hostazym® X at 1500 EPU/kg of feed. The first composed diets contained a 65 % inclusion level of wheat, corn or barley (each 2 different batches), combined with a 35 % protein based (soybean meal) complementary feed. Secondly, feeds were composed containing either 30 % soybean meal or 30% wheat-based DDGS (of each 2 different batches), which were added to a complementary feed containing corn/wheat as cereal sources in a 50/50 ratio. Finally, feeds containing 10 or 20 % rapeseed meal (at the expense of soybean meal) were produced, in which corn/wheat was dosed in a 50/50 ratio. Eight cages per feedstuff were used, of which 4 cages received the control feed and 4 cages received the control feed + Hostazym® X at 1500 EPU/kg. During the period of digestibility (d18-22) daily feed intake, total faecal droppings and water intake were measured. Digestibility was determined by analyzing the feed (dry matter, gross energy, starch and fat) and quantifying the total amount of faecal material produced (dry). After homogenisation of the faecal material and subsampling, 250 g was freeze-dried to determine the dry matter content. The dry matter was then analyzed for energy, starch and fat. The digestibility was calculated as follows: digestibility (%) = (total nutrient intake by feed – total nutrient output by faeces)/(total nutrient intake by feed) x 100 %
## Results

First of all, the inclusion of Hostazym® X increased the metabolisable energy (ME) of all feeds, ranging from 33 kCal/kg (rapeseed meal 10%) up to 120 kCal/kg (DDGS 30%) (Fig. 1). Secondly, the inclusion of Hostazym® X increased the fat digestibility by 1.2% (soybean meal 30%) up to 7.7% (DDGS 30%) (Fig. 2).

<figure><img src="image_41.png" /><figcaption>Fig. 1: increase in ME (kCal/kg feed) when including Hostazym® X at 1500 EPU/kg feed</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>Fig. 2: fat digestibility (%) with or without Hostazym® X at 1500 EPU/kg feed</figcaption></figure>

The inclusion of Hostazym® X led to a reduction in faecal droppings, indicating a better digestion of the feed (Fig. 3). Additionally, the inclusion of Hostazym® X resulted in a reduced daily water consumption, which might be linked to a reduced viscosity in the gut when using Hostazym® X (Fig. 4). As a consequence of the effects shown in Fig. 3 and 4, a reduction in wet litter in field conditions can be expected when using Hostazym® X.
<figure><img src="image_43.png" /><figcaption>**Fig. 3:** daily faecal droppings (g/bird/d) with or without Hostazym® X at 1500 EPU/kg feed</figcaption></figure>

<figure><img src="image_44.png" /><figcaption>**Fig. 4:** daily water consumption with or without Hostazym® X at 1500 EPU/kg feed</figcaption></figure>

## Conclusions

The inclusion of Hostazym® X at 1500 EPU/kg led to an increase in ME which was highest in the DDGS-based diet, followed by the diets containing 20 % rapeseed meal, 65 % corn or 65 % barley. It also resulted in an increase in fat digestibility (highest in the corn diets followed by the 30 % DDGS diet), as well as reduction in daily faecal droppings, thereby showing an increased digestibility of the feed dry matter and also a reduced water consumption. Both of these key findings effectuate a lower risk of wet litter.
P-110 (ID 70)

OPTIPHOS® IMPROVES PERFORMANCE AND SHOWS A HIGH AP VALUE IN BROILERS

L. Nollet, K. Bierman and N. Soares

Huvepharma NV, Antwerp, Belgium

Corresponding author: lode.nollet@huvepharma.com

**Abstract**

A 42 d broiler pen trial was set-up at Valley View Research Farm (USA), consisting of 5 treatments having 8 replicates of 22 male birds each (Cobb x Cobb). Feeds were corn/soy based with low phosphorous level (starter 0.8 % Ca and 2.0 g/kg aP (available P); grower 0.7 % Ca and 1.4 g/kg aP; finisher (0.7 % Ca and 1.0 g/kg aP). To this feeds either no addition (negative control), or increasing levels of inorganic P as DCP (di-calcium phosphate) were added to yield an extra 1.0, 1.5 or 2.5 g/kg feed of aP (positive controls). To the remaining feed a dose of OptiPhos® at the rate of 550 OTU/kg feed was applied. Body weight, daily growth, feed intake, and mortality-corrected feed conversion (FCR) were measured for every phase. Also at 42 days of age, two tibias were collected per pen for ash analysis. Results showed that higher aP levels through OptiPhos® addition or through DCP addition, increased the weight gain while reducing FCR. The inclusion of OptiPhos® at 550 OTU brought the body weight performance to the same level/height as for the negative control + 1.5 g aP level. However 550 OTU OptiPhos® reduced FCR to come close to the FCR observed for the negative control + 2.5 g aP treatment. From the response curve between tibia ash and added P as DCP, an equivalence could be established between the OptiPhos® phytase unit (OTU) and aP in the ratio/ of 1.67 g aP for 550 OTU OptiPhos®.

**Keywords:** phytase, poultry, available P, matrix values, OptiPhos®

**Introduction**

Phytases are used in commercial broiler nutrition to reduce the need of and dependency on inorganic P sources by liberating P from phytate, present in vegetal feed materials. The correct estimation of the matrix value of the different phytases are is therefore essential to allow a nutritionist to formulate feeds with optimal P levels. This correct estimation of the matrix values requires multiple trials which have to be set-up in a scientifically correct manner.

**Material and methods**

A broiler trial was conducted at the trial site of Valley View Research Farm (USA) in the period of November/December 2013 (46 days). Male broilers (Cobb x Cobb) were split over 5 treatments, with 8 replicates of each 22 birds per treatment. Feeds were corn/soy based and were pelleted, except for the starter feed which was crumbled. There were 5 treatments including one negative control NC (starter: 0.8 % Ca and 2.0 g/kg aP; grower: 0.7 % Ca and 1.4 g/kg aP and finisher: 0.7 % Ca and 1.0 g/kg aP), and also three positive controls using increasing levels of extra inorganic P as DCP (dicalciumphosphate) added in the NC feed to yield an extra 1.0, 1.5 or 2.5 g/kg feed of aP. In the last treatment group, OptiPhos® was added at a rate of 550 OTU/kg in the NC feed. Body weight, daily growth, feed intake, and mortality-corrected feed conversion (FCR) were measured, while at day 42 two tibias were collected per pen for bone ash analysis.

**Results**

Higher aP levels by adding OptiPhos® or by adding DCP increased the weight gain while reducing FCR (Table 1). The inclusion of OptiPhos® at 550 OTU/kg feed brought the body weight performance to the same level as the one observed for the NC + 1.5 g aP treatment. However 550 OTU OptiPhos® reduced FCR to the extent that it came close to the FCR observed for the NC + 2.5 g aP treatment.

From the response curve between tibia ash and added P as DCP, an equivalence between the OptiPhos® phytase unit (OTU) and aP in the ratio of 1.67 aP for 550 OTU OptiPhos® could be established. This is very close to the commercially advised matrix value of 1.68 aP for 500 OTU OptiPhos®.
<table><caption>**Table 1:** effect on technical performance</caption><thead><tr><td></td><td>End weight (kg)</td><td>FCR</td><td>Tibia ash (%)</td></tr></thead><tbody><tr><td>NC*</td><td>2462<sup>a</sup></td><td>1.84<sup>a</sup></td><td>37.7<sup>a</sup></td></tr><tr><td>NC + 1.0 g aP</td><td>3115<sup>b</sup></td><td>1.76<sup>bcd</sup></td><td>42.7<sup>b</sup></td></tr><tr><td>NC + 1.5 g aP</td><td>3187<sup>b</sup></td><td>1.75<sup>c</sup></td><td>43.1<sup>b</sup></td></tr><tr><td>NC + 2.5 g aP</td><td>3268<sup>b</sup></td><td>1.71<sup>b</sup></td><td>44.4<sup>b</sup></td></tr><tr><td>NC + OptiPhos® 550 OTU</td><td>3189<sup>b</sup></td><td>1.72<sup>bcd</sup></td><td>43.1<sup>b</sup></td></tr></tbody></table>

**Conclusions**

OptiPhos® added at 550 OTU/kg feed gave similar to better technical results than the addition of 1.5 g aP as DCP. Equivalence calculations between OptiPhos® and P as DCP, revealed an aP value of 1.67 g aP for 550 OTU.

<footnote>*NC= negative control</footnote>

<footnote>a-d: values in a column followed by a different letter are significant different (P< 0.05)</footnote>
P-111 (ID 72)

OPTIPHOS® OUTPERFORMS QUANTUM® BLUE ON PH ACTIVITY RANGE AND SPEED OF PHYTIC ACID HYDROLYSIS

L. Nollet, K. Bierman and N. Soares

Huvepharma NV, Antwerp, Belgium

Corresponding author: lode.nollet@huvepharma.com

**Abstract**

OptiPhos® and Quantum® Blue were purified from commercial sources to obtain the pure phytase proteins of both enzyme products. Research was performed on the pure phytase protein *in vitro*, to determine the pH profile and Michaelis-Menten kinetics. The pH activity assay was conducted by measuring the phytase activity in Glycine-HCl buffers at pH 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 during a period of 10 min. Michaelis Menten kinetics were determined in a glycine-HCl buffer at pH 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5, after which the Vmax (maximum speed) and Kcat (turnover number) were both calculated. OptiPhos® showed a much higher activity at multiple pH levels than Quantum® Blue did, which indicates that OptiPhos® is performing better, especially under more acidic conditions. The Kcat value of OptiPhos® was also higher at all measured pH levels, in particular at lower pH. On average, the Kcat value of OptiPhos® was 893 vs. 645 per second for Quantum® Blue (i.e. +28 %). The Vmax value of OptiPhos® was also higher at most pH levels, in particular at lower pH. On an average basis covering all pH levels, the Vmax value of OptiPhos® was 1045 vs. 886 µmol P/min. per mg enzyme for Quantum® Blue (i.e. +15 %). It could be concluded that OptiPhos® has a higher activity at a broader pH range than Quantum® Blue, while OptiPhos® also works faster in breaking down phytic acid at *in vivo*-relevant pH levels.

**Keywords:** phytase, pH, kinetics, speed, OptiPhos®

**Introduction**

Phytase releases P from phytic acid present in vegetal feed materials, so decreasing the need for dietary supplementation of inorganic P sources and thereby reducing feed costs and P excretion. However, phytic acid can only be degraded by a phytase when it is in a solubilised state. As the solubility of phytic acid in feeds decreases with increasing pH due to formation of e.g. Ca salts, the phytase should especially work in a pH range below 4.5. With regard to the gastro-intestinal tract of poultry, this corresponds to the area of the proventriculus and the gizzard. Furthermore, considering that the transit time in this part of the gastro-intestinal tract is quite fast, a phytase should definitely be working at a high speed. The specific aim of this set-up was to evaluate purified OptiPhos® phytase on these intrinsic parameters, and to empirically compare it with Quantum® Blue.

**Material and methods**

The pure/naked phytase molecules of OptiPhos® and Quantum® Blue, were purified/extracted from the commercially available OptiPhos® and Quantum® Blue products. In brief, the phytases were solubilized overnight at 18 °C in 200 ml Tris-HCL buffer followed by a filtration on glass fibre pre-filter of 1 µm, on a membrane filter of 0.45µm and finally on a 0.22 µm GP Express PLUS Membrane. The protein solutions were concentrated by diafiltration. Proteins were precipitated by ammonia sulphate, and the phytase fraction was separated by chromatography.

The pH activity assay was conducted by measuring the phytase activity using phytate as a substrate in Glycine-HCl buffers at pH 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 for a period of 10 min (according to ISO 30024:2009).

Michaelis-Menten kinetics were determined for the phytases in a Glycine-HCl buffer at pH levels of 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5, after which Vmax and Kcat were calculated. Vmax represents the maximum rate of speed for P release from phytate, at maximum (see saturating) concentrations of the (phytate) substrate. Kcat is the turnover number, which is the maximum number of substrate (phytate) molecules converted per enzyme molecule per second.

**Results**

OptiPhos® showed a much higher activity at different pH levels than Quantum® Blue (Fig. 1), which indicates that OptiPhos® is also performing better under more acidic conditions.
<figure><img src="image_45.png" /><figcaption>**Fig. 1:** relative phytase activity at different pH levels after incubation during 10 minutes</figcaption></figure>

The Kcat value of OptiPhos® is also higher at all pH levels, in particular at lower pH. On an average basis, the Kcat value of OptiPhos® was 893 per second, compared to 645 per second for Quantum® Blue (+28 %, Fig. 2). The Vmax value of OptiPhos® was higher at most pH levels, in particular at lower pH. In this view the mean Vmax value of OptiPhos® was 1045 vs. 886 µmol P/min. per mg enzyme for Quantum® Blue (+15 %, Fig. 3).

<figure><img src="image_46.png" /><figcaption>**Fig. 2:** the Kcat value of OptiPhos® and Quantum® Blue at different pH levels</figcaption></figure>
<figure><img src="image_47.png" /><figcaption>Fig. 3: the Vmax value of OptiPhos® and Quantum® Blue at different pH levels</figcaption></figure>

Conclusions

OptiPhos® has a higher phytase activity at a broader pH working range compared with Quantum® Blue, and it also acts much faster in breaking down phytic acid at in vivo-relevant pH levels.
P-112 (ID 71)

OPTIPHOS® OUTPERFORMS RONOZYME® HIPHOS ON PH ACTIVITY RANGE, RESISTANCE TO PEPSIN AND SPEED OF PHYTIC ACID HYDROLYSIS

L. Nollet, K. Bierman and N. Soares

Huvepharma NV, Antwerp, Belgium

Corresponding author: lode.nollet@huvepharma.com

**Abstract**

OptiPhos® and Ronozyme® HiPhos were purified from commercial available sources, to obtain the pure phytase protein. On the pure phytase protein *in vitro* determination of the pH profile, pepsin resistance and Michaelis-Menten kinetics was done. The pH activity assay was conducted by measuring the phytase activity in Glycine-HCl buffers at pH 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 during 10 min. Pepsin resistance was measured by incubating pepsin and phytase at different pepsin/phytase ratios at 37°C. This was done/carried out for different time periods in 15 mM HCl (pH 2), after which the remaining phytase activity was measured in a 0.2 M Glycine-HCl buffer (pH 3.5) at 39°C. Michaelis Menten kinetics was determined in glycine-HCl buffer at pH 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 from which Vmax (maximum speed) and Kcat (turnover number) were calculated. Results showed that OptiPhos® had a much higher activity at a number of different pH levels than Ronozyme® HiPhos, indicating that OptiPhos® is a truly superior phytase at (mild) acidic conditions. OptiPhos® was by far more resistant to pepsin, both when incubated with relative low doses of pepsin for a long time (2 h), as well as when incubated with a higher pepsin/phytase ratio during a shorter period of time (40 min.). The Kcat value of OptiPhos® was also higher at all measured pH levels averaging at 893 per second, compared to 668 per second for Ronozyme HiPhos® (i.e. +35%). The Vmax value of OptiPhos® was higher at most pH levels averaging at 1045 vs. 806 µmol P/min per mg enzyme for Ronozyme® HiPhos (i.e. +23 %). It can be concluded that OptiPhos® has a more suitable pH profile, is more resistant to pepsin and will work faster than Ronozyme® HiPhos at *in vivo*-relevant pH levels.

**Keywords:** phytase, pH, pepsin resistance, kinetics, OptiPhos®

**Introduction**

Phytase releases P from phytic acid present in vegetal feed materials, so decreasing the need for dietary supplementation of inorganic P sources and thereby reducing feed costs and P excretion. However, phytic acid can only be degraded by a phytase when it is in a solubilised state. As the solubility of phytic acid in feeds decreases with increasing pH due to formation of e.g. Ca salts, the phytase should especially work in a pH range below 4.5. With regard to the gastro-intestinal tract of poultry, this corresponds to the area of the proventriculus and the gizzard. As phytases are in fact proteins and thus degradable by proteases, a good phytase should also be resistant to pepsin. Furthermore, considering that the transit time in this part of the gastro-intestinal tract is quite fast, a phytase should definitely be working at a high speed. The specific aim of this experimental set-up was to evaluate purified OptiPhos® phytase on these intrinsic parameters, and to empirically compare it with Ronozyme® HiPhos.

**Material and methods**

The pure/naked phytase molecules of OptiPhos® and Ronozyme® HiPhos were purified/extracted from the commercially available OptiPhos® and Ronozyme® HiPhos products. The phytases were solubilized overnight at 18 °C in 200 ml Tris-HCL buffer followed by a filtration on glass fibre pre-filter of 1 µm, on a membrane filter of 0.45µm and finally on a 0.22 µm GP Express PLUS Membrane. The protein solutions were concentrated by diafiltration. Proteins were precipitated by ammonia sulphate, and the phytase fraction was separated by chromatography. The purified enzymes were tested *in vitro* on their pH profile, pepsin resistance and Michaelis-Menten kinetics. The pH activity assay was conducted by measuring the phytase activity using phytate as a substrate in Glycine-HCl buffers at pH 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 for a period of 10 min (according to ISO 30024:2009). Pepsin resistance was measured by dissolving pepsin into 15 mM of HCl (final pH 2). In a final volume of 100 ml, 10 mg of purified phytase was incubated with pepsin at different pepsin/phytase (w/w) ratios at 37°C. This was carried out for different periods of time, after which the remaining phytase activity was measured in a 0.2 M Glycine-HCl buffer (pH 3.5) at 39°C. Michaelis-Menten kinetics was determined for the phytases in a Glycine-HCl buffer at pH levels of 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5, after which Vmax and Kcat were calculated. Vmax represents the maximum rate of speed for P release from phytate, at maximum (see saturating)
concentrations of the (phytate) substrate. Kcat is the turnover number, which is the maximum number of substrate (phytate) molecules converted per enzyme molecule per second.

## Results

OptiPhos® showed a much better activity at different pH ranges compared with Ronozyme® HiPhos, which indicates that OptiPhos® is also performing better at acidic conditions (Fig. 1).

<figure><img src="image_48.png" /><figcaption>**Fig. 1:** relative phytase activity at different pH levels after incubation during 10 minutes</figcaption></figure>

OptiPhos® was by far more resistant to pepsin, either when incubated with relative low doses of pepsin for a long time (2 h, Fig. 2) or when incubated with a higher pepsin/phytase ratio during a shorter period of time (data not shown).

The Kcat value of OptiPhos® was also higher at all pH levels. On an average basis, the Kcat value of OptiPhos® was 893 per second, compared to 668 per second for Ronozyme® HiPhos (+35 %, data not shown). The Vmax value of OptiPhos® was higher at most pH levels. In this view the mean Vmax value (in µmol P/min. per mg enzyme) of OptiPhos® was 1045 vs. 806 for Ronozyme® HiPhos (+23 %, Fig. 3).

<figure><img src="image_49.png" /><figcaption>**Fig. 2:** phytase recovery after prolonged exposure of OptiPhos® and Ronozyme® HiPhos to different low pepsin levels during a 2h period</figcaption></figure>
<figure><img src="image_50.png" /><figcaption>**Fig. 3: the Vmax values of OptiPhos® and Ronozyme® HiPhos at different pH levels**</figcaption></figure>

## Conclusions

OptiPhos® has a much broader pH working range with higher phytase activity than Ronozyme® HiPhos. OptiPhos® is far more resistant to pepsin breakdown, and works much faster in breaking down phytic acid at in vivo-relevant pH levels between 2.0 and 4.5.
P-113 (ID 69)

# THE EFFECT OF OPTIPHOS® AND RONOZYME® HIPHOS ON TECHNICAL PERFORMANCE AND AVAILABLE P IN BROILERS

L. Nollet, K. Bierman and N. Soares

Huvepharma NV, Antwerp, Belgium

**Corresponding author:** lode.nollet@huvepharma.com

## Abstract

A broiler trial was set up at Southern Poultry Research, Athens (USA) using male broilers (Cobb x Cobb). Birds were distributed over 8 treatments with 8 replicates of 8 broilers each and fed with a corn/soy mash feed from 0-14 d (start) and 15-28 d (grower). Treatments consisted of one deficient control (0.20 % aP (available P)), three positive controls with increasing levels of added inorganic P (dicalciumphosphate, DCP) to yield an overall 0.30, 0.35 and 0.45 % aP in the feed, and two phytase treatments (OptiPhos® and Ronozyme® HiPhos) at 2 levels: OptiPhos® at 550 and 750 OTU/kg and Ronozyme® HiPhos at 1000 and 1500 FYT/kg). The technical performance data showed that the increase in weight gain and the decrease in FCR corresponded linearly with increasing levels of aP from DCP or by the inclusion of phytase. The technical performance at day 14 and 28 of OptiPhos® treatment was generally better compared to Ronozyme® HiPhos treatment, in particular at the lowest inclusion level regarding BW gain, and at the highest inclusion rate regarding FCR. Increasing the dose of OptiPhos® reduced FCR significantly at 28 days, demonstrating that a higher dose of OptiPhos® has a benefit on technical performance. These results also indicated that the commercially used exchange rate of 2 FYT Ronozyme® HiPhos for 1 OTU OptiPhos® is in fact an underestimation and should rather be situated between 2.5 and 3.

**Keywords:** phytase, poultry, available P, matrix values, OptiPhos®

## Introduction

Phytases are used in commercial broiler nutrition to reduce the need of and dependency on inorganic P sources by liberating P from phytate, present in vegetal feed materials. The use of phytase thereby leads to a lower feed cost (optimizing technical performance) and to a reduced excretion of P, having a positive environmental effect. However, plural scientifically correct trials need to be set-up in order to verify if the claimed matrix value and inclusion level of different phytases are correct, and leading to a similar or even improved performance compared to a feed purely supplemented with inorganic P sources.

## Material and methods

A broiler trial was set up at Southern Poultry Research, Athens (USA) in the period January/February 2014. Male broilers (Cobb x Cobb) were distributed over on Petersime batteries (8 birds per cage, 8 repetitions per treatment) which were fed a corn/soy based diet (0-14 d: starter feed and 15-28 d: grower feed). There were 8 treatments: one deficient negative control NC (0.2 % aP), three positive controls with increasing levels of added inorganic P (DCP) on the NC to yield 0.30, 0.35 and 0.45 % aP in the feed, and two phytase treatments (OptiPhos® and Ronozyme® HiPhos) at 2 levels: OptiPhos® at 550 and 750 OTU/kg and Ronozyme® HiPhos at 1000 and 1500 FYT/kg added on the NC. Body weight, daily growth, feed intake, and feed conversion (FCR) were measured at day 14 and 28.

## Results

The weight gain increase and the FCR decrease corresponded linearly with increasing levels of aP (Table 1). The technical performance at day 14 and 28 of OptiPhos® was generally better than Ronozyme® HiPhos, in particular at the lowest level for BW gain, and at the highest inclusion rate for FCR (Table 1). This indicates that the used exchange rate of 2:1 of Ronozyme® HiPhos to OptiPhos®, is an underestimation of the aP value of OptiPhos® compared to Ronozyme® HiPhos. Increasing the dose of OptiPhos® reduced FCR significantly at 28 days, demonstrating that super-dosing OptiPhos® does have a benefit on technical performance.
<table><caption>**Table 1:** effect of the inclusion of OptiPhos® and Ronozyme® HiPhos at two different levels on body weight gain and mortality-adjusted FCR at 14 and 28 days of age</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="2">Day 14</th><th colspan="2">Day 28</th></tr><tr><th>Weight gain (g)</th><th>Adj. FCR</th><th>Weight gain (g)</th><th>Adj. FCR</th></tr></thead><tbody><tr><td>Deficient control (DC) (0.20% aP)</td><td>0.267<sup>a</sup></td><td>1.402<sup>a</sup></td><td>929<sup>ab</sup></td><td>1.65<sup>a</sup></td></tr><tr><td>DC + 0.10% aP</td><td>0.287<sup>b</sup></td><td>1.382<sup>ab</sup></td><td>939<sup>abc</sup></td><td>1.62<sup>ab</sup></td></tr><tr><td>DC + 0.15% aP</td><td>0.296<sup>bcd</sup></td><td>1.348<sup>cd</sup></td><td>948<sup>abcd</sup></td><td>1.58<sup>cd</sup></td></tr><tr><td>DC + 0.25% aP</td><td>0.316<sup>d</sup></td><td>1.333<sup>d</sup></td><td>993<sup>abd</sup></td><td>1.55<sup>de</sup></td></tr><tr><td>DC + OptiPhos® (550 OTU)</td><td>0.302<sup>cde</sup></td><td>1.36<sup>ce</sup></td><td>967<sup>abcd</sup></td><td>1.61<sup>b</sup></td></tr><tr><td>DC + Ronozyme® HiPhos (1000 FYT)</td><td>0.294<sup>bcd</sup></td><td>1.38<sup>be</sup></td><td>921<sup>b</sup></td><td>1.62<sup>ab</sup></td></tr><tr><td>DC + OptiPhos® (750 OTU)</td><td>0.303<sup>cde</sup></td><td>1.34<sup>cd</sup></td><td>986<sup>abcd</sup></td><td>1.57<sup>cde</sup></td></tr><tr><td>DC + Ronozyme® HiPhos (1500 FYT)</td><td>0.303<sup>cde</sup></td><td>1.35<sup>c</sup></td><td>976<sup>abcd</sup></td><td>1.60<sup>bc</sup></td></tr></tbody></table>

## Conclusions

OptiPhos® outperformed Ronozyme® HiPhos especially at lower inclusion levels, revealing OptiPhos® to be the more efficient phytase. OptiPhos® at 50 % of the Ronozyme® HiPhos dose (e.g. 750 OTU vs 1500 FYT) showed better performance, illustrating that the ratio OptiPhos®/ Ronozyme® HiPhos is, in fact, higher than 2.

<footnote>a-e: values in a column with different letter are significant different (P < 0.05).</footnote>
P-114 (ID 68)

# THE EFFICACY OF OPTIPHOS® AND RONOZYME® HIPHOS ON PERFORMANCE AND TIBIA ASH IN YOUNG BROILERS

L. Nollet, K. Bierman and N. Soares

Huvepharma NV, Antwerp, Belgium

Corresponding author: lode.nollet@huvepharma.com

## Abstract

A trial was conducted at Schothorst Feed Research (NL), to evaluate the effect of OptiPhos® and Ronozyme® HiPhos on growth performance and tibia ash content. Male Ross308 broilers in 6 replicates of 23 birds per treatment (reduced to 20 birds at day 5) were fed a starter feed which was equal for all birds from day 0 to 5. From day 5 to 21, trial feeds were fed to the different treatments (10 in total). Treatments used were one negative control (NC) with low Ca and low P, and three positive controls using the NC to which monocalciumphosphate (MCP) was added to increase the P level with 0.6, 1.2 and 2 g per kg of feed. Furthermore, there were also six NC + phytase treatments (OptiPhos® liquid dosed at 25, 50 and 100 mL/T providing 160, 320 and 640 OTU/kg, and Ronozyme® HiPhos dosed at 17.5, 35 and 70 mL?/T providing 490, 980 and 1960 FYT/kg). Feeds were pelleted (80°C, 3 mm) and enzymes were dosed after pelleting. Body weight, daily growth, feed intake, and feed conversion were measured during the trial period. At day 21, four tibias per pen were collected and pooled into one sample. The results showed that (a) increasing the dose of OptiPhos® above the recommended levels (250 OTU/kg) had a positive effect on body weight gain, (b) increasing the levels of OptiPhos® above the recommended dose (250 OTU/kg) had more effect on tibia ash than increasing levels of Ronozyme® HiPhos, and (c) the real potential of OptiPhos® in replacing P as MCP was 1.43 g aP per kg at 250 OTU.

**Keywords:** phytase, poultry, available P, matrix values, OptiPhos®

## Introduction

Phytases are used in commercial broiler nutrition to reduce the need of and dependency on inorganic P sources by liberating P from phytate, present in vegetal feed materials. The correct estimation of the matrix value of the different phytases is therefore essential to allow a nutritionist to formulate feeds with optimal P levels. This correct estimation of the matrix values requires multiple trials which have to be set-up in a scientifically correct manner.

## Trial description

A trial was set up at the Schothorst Feed Research, the Netherlands (January – February 2013) using male Ross308 broilers distributed over 10 treatments with 6 replicates of 23 birds per treatment (reduced to 20 birds at day 5). Broilers were fed a started diet till day 5 which was similar for all birds. From day 5 till day 21 the trials feeds were provided. There were 10 treatments including one negative control NC (corn/soy based: 57.6 and 30.3 %; 2900 kCal AMEn/kg, 21 % crude protein, 10.5 % dig. Lys, 0.65 % Ca, 0.44 % P and 0.25 % aP), and three positive controls adding P as monocalciumphosphate (P-MCP) to the NC at 0.6, 1.2 and 2 g per kg. There were also six phytase treatments: OptiPhos® liquid dosed at 160, 320 and 640 OTU/kg and Ronozyme® HiPhos dosed at 490, 980 and 1960 FYT/kg (analyzed values). Feeds were pelleted (80°C, 3 mm) and enzymes were added in liquid form–post-pelleting. Body weight, daily growth, feed intake, and feed conversion were measured. At day 21, four tibias were collected per pen (day 21) and pooled into one sample (so 6 samples per treatment) for bone ash analysis.

## Results

The negative and positive control (= not supplemented with phytase) were negative on phytase activity (Table 1). Increasing P availability by increasing phytase levels or adding MCP, increased the BW and tibia ash percentage while reducing FCR.
<table><caption>**Table 1:** Effect on technical performance and tibia ash</caption><thead><tr><th>Tr.</th><th>Phytase</th><th>Phytase (per kg)</th><th>BW (g)</th><th>FCR</th><th>Tibia ash (g/kg fat free DM)</th></tr></thead><tbody><tr><td>1</td><td>NC</td><td>&lt; 100 FTU</td><td>677<sup>a</sup></td><td>1.384<sup>e</sup></td><td>354<sup>a</sup></td></tr><tr><td>2</td><td>PC 1 (NC + 0.6 P-MCP)</td><td>&lt; 100 FTU</td><td>708<sup>b</sup></td><td>1.351<sup>d</sup></td><td>396<sup>b</sup></td></tr><tr><td>3</td><td>PC 2 (NC + 1.2 P-MCP)</td><td>&lt; 100 FTU</td><td>849<sup>c</sup></td><td>1.327<sup>c</sup></td><td>454<sup>de</sup></td></tr><tr><td>4</td><td>PC 3 (NC + 2.0 P-MCP)</td><td>&lt; 100 FTU</td><td>922<sup>fg</sup></td><td>1.305<sup>ab</sup></td><td>486<sup>g</sup></td></tr><tr><td>5</td><td>Optiphos®</td><td>160 OTU</td><td>851<sup>c</sup></td><td>1.331<sup>c</sup></td><td>440<sup>c</sup></td></tr><tr><td>6</td><td>Optiphos®</td><td>320 OTU</td><td>890<sup>de</sup></td><td>1.322<sup>bc</sup></td><td>465<sup>ef</sup></td></tr><tr><td>7</td><td>Optiphos®</td><td>640 OTU</td><td>905<sup>efg</sup></td><td>1.303<sup>a</sup></td><td>486<sup>g</sup></td></tr><tr><td>8</td><td>Ronozyme® HiPhos</td><td>490 FYT</td><td>865<sup>cd</sup></td><td>1.328<sup>c</sup></td><td>446<sup>cd</sup></td></tr><tr><td>9</td><td>Ronozyme® HiPhos</td><td>980 FYT</td><td>896</td><td>1.321<sup>bc</sup></td><td>472<sup>f</sup></td></tr><tr><td>10</td><td>Ronozyme® HiPhos</td><td>1960 FYT</td><td>934<sup>g</sup></td><td>1.3<sup>a</sup></td><td>487<sup>g</sup></td></tr></tbody></table>

From the results as indicated in Table 1, a response curve between tibia ash percentage and added P as MCP could be established. The obtained fitted line (linear equation) offers an opportunity to estimate a P value for a certain inclusion of phytase (OTU or FYT) and P as (Fig. 1).

<figure><img src="image_51.png" /><figcaption>**Fig. 1:** the effect of increasing inorganic P on bone ash concentration.</figcaption></figure>

From this regression curve, an equivalence between Ronozyme® HiPhos to OptiPhos® could be calculated for 125 to 750 OTU as shown in Table 2. It can be observed that the ratio between Ronozyme® HiPhos and OptiPhos® was on average 2.63, ranging from 2.4 at low dose to 2.9 at high dose. This indicates that levels of OptiPhos® above the standard recommended inclusion have more extra effect on tibia ash, than increasing levels of Ronozyme® HiPhos. In table 2 it is demonstrated that 250 OTU OptiPhos® is equal to 1.43 g P as MCP, which is close to the currently advised value of 1.45 g aP per kg of feed.

<table><caption>**Table 2:** equivalence between OptiPhos® and Ronozyme® HiPhos based on tibia ash and P regression</caption><thead><tr><th>OptiPhos®<br>(OTU/kg)</th><th>Ronozyme® HiPhos<br>(FYT/kg)</th><th>P-MCP (g/kg)</th><th>Ratio Ronozyme® Hi-<br>Phos/OptiPhos®</th></tr></thead><tbody><tr><td>125</td><td>295</td><td>1.10</td><td>2.4</td></tr><tr><td>250</td><td>630</td><td>1.43</td><td>2.5</td></tr><tr><td>500</td><td>1375</td><td>1.77</td><td>2.8</td></tr><tr><td>750</td><td>2175</td><td>1.97</td><td>2.9</td></tr></tbody></table>

## Conclusions

It can be concluded that increasing the dose of OptiPhos® above the normal recommended levels had an additional positive effect on body weight gain, and that the equivalence of 1 OTU OptiPhos® is on average 2.63 FYT of Ronozyme® HiPhos. It can also be observed that increasing levels of OptiPhos® above the recommended dose have more extra effect on tibia ash than increasing levels of Ronozyme® HiPhos, and that the real potential of OptiPhos® in replacing P as MCP is close to its claimed matrix value of 1.45 g aP/kg feed.

<footnote>a-g Mean values in a column with a different superscript letter are sign. different (P≤ 0.05).</footnote>
P-115 (ID 217)

ENERGY METABOLISABILITY AND ILEAL DIGESTA VISCOSITY OF GROWING TURKEYS FED DIETS CONTAIN-
ING MALTED SORGHUM SPROUTS SUPPLEMENTED WITH ENZYME OR YEAST

*Florence Oke*<fnref n="1" />, *Oluwaseyi Oduguwa*<fnref n="2" />, *Abimbola Oso*<fnref n="2" />, *Olajide Adeyemi*<fnref n="2" />, *Adebayo Jegede*<fnref n="2" />, *Adekoya Oso-
*fowara*<fnref n="2" />, *Adewale Adeoye*<fnref n="2" />, *Adeboye Fafiolu*<fnref n="2" />), *V. Pirgozliev*<fnref n="3" />

**Introduction**

Nigerian livestock enterprise has suffered more than any other industry as a result of rising costs of production as feed
alone accounts for up to 75% of total cost of broiler production (Ubosi, 2000) and short supply of essential raw materials
such as maize, ground nut cake and soybean meal. The resultant effects include low production level, narrow profit
margin and collapse of the once prosperous poultry industry. Moreover, the competition between man and livestock for
these feed ingredients necessitate the use of alternative sources of ingredient. Example of such is Malted Sorghum
Sprout.

Malted sorghum sprout (MSP) is a non - conventional feed stuff and a by-product of sorghum malting. MSP has been reported by Oduguwa et al. (2001) to have low nutritive value for monogastric animals and cannot be used as a main protein source. The low nutritive value may be due to its content of non-starch polysaccharide and fibre as well as tan- nin. Therefore, there is a need for the use of appropriate feed enzymes to improve the nutritive value of this cereal by- product. Bedford and Schulze (1998) documented that supplementation of diets with exogenous enzyme reduced the adverse effects of some of these compounds especially those produced by carbohydrates and proteins. Enzyme had been used to improve fibre digestion and reduced viscosity of digesta (Bedford et al., 1991 and Atteh, 2001). Enzyme treatments have been used to improve performance of broiler chickens, increase weight gain, dry matter digestibility and nutrient digestibility, respectively (Sunduet al., 2006).

Bakers' yeast has a relatively high lysine content and protein efficiency ratio. This yeast is very rich in essential miner-
als, crude protein (40 - 45 %) and vitamin B complex except B₁₂ (Moyad, 2008). Yeast extracts have been widely re-
ported as successful growth promoter in poultry industry. It helps in the metabolism of protein, lipids and carbohydrates
(Spring, 2002). Yeast promoted growth of intestinal microflora (Spring et al., 2000) and increased growth (Parks et al.,
2001). LutfulKabir (2009) noted that yeast improved digestion and stimulated the immune system.

The study reported below evaluated the response of turkeys fed diets containing MSP supplemented with enzyme and or yeast.

**Materials and Methods**

One hundred and twenty eight old British United Turkeys (BUT) were brooded for 28d pre-experimental period. At 28th day, the birds were randomly allocated to 6 dietary treatments of 20 poults per treatment replicated four times with 5 poults each such that each treatment contained either enzyme or yeast at 200ppm in diets containing MSP at 0,50, or 100g/kg respectively to investigate the energy metabolisability and ileal digesta viscosity of growing turkeys. The exogenous enzyme used is produced by (DSM Nutritional products Philippines, Inc.) is a commercial blend of xylanase (EC3.2.1.8), β glucanase (EC3.2.1.4) and protease (EC 3.4.2.1). The yeast used is the commercially available baker's yeast produced by LALLEMAND Canada Inc. At day 84, 6 groups of 6turkeys each were allocated to 6 experimental diets while an additional group of 6 turkeys were used for estimation of endogenous losses. All the turkeys were housed individually in a two tier steel metabolic cage. Three days of acclimatization were allowed before the commencement of the digestibility study. Known weights of feed (based on the daily requirement) were given to respective birds in the metabolic cage. Leftovers were measured and discarded on a daily basis. Excreta collection was done daily for a period of three days, unlike the former group of birds only water was administered to this group of birds so as to determine the endogenous losses. At the lapse of 24 hours, excreta was collected quantitatively on daily basis for two days. Apparent metabolisable energy (AME), AME corrected for nitrogen retention (AMEn), true metabolisable energy (TME) and TME corrected for nitrogen retention were determined according to the method of Sibbald (1979).

<footnote n="1">¹ Federal University Dutse, Nigeria</footnote>

<footnote n="2">² Federal University of Agriculture Abeokuta, Nigeria</footnote>

<footnote n="3">³ Harper Adams University College, UK</footnote>
At day 112, 2 birds per replicate, 48 turkeys in all were selected based on final live weight of 8175g to determine the ileal digesta viscosity. The turkeys were slaughtered and digesta content was collected from the Merckel's diverticulum to the ileo-caecal junction. The ileal content of each replicate were emptied into labeled sample bottles. Uniform weights of samples were taken with a sensitive scale and were diluted to a volume of 400ml. The ileal digesta viscosity was determined using a BROOKFILED DV-E viscometer. The readings were taken at 50, 60 and 100 revolution per minute. Data generated were analysed using ANOVA. Polynomial contrast (Linear and quadratic) was used to determine effects of enzyme or yeast inclusion using SPSS (1999).

## Results

Birds fed 50 and 100g/kg MSP had higher (P < 0.05) AME, AMEn and TMEn, while the least values for all the measurements were recorded for birds fed control diet. The TME values showed no effect (P > 0.05) following dietary inclusion of MSP. Dietary supplementation with enzyme or yeast showed no effect (P > 0.05) on apparent and true metabolisable energy values. MSP inclusion showed significant effect (P < 0.05) on ileal digesta viscosity of birds for all revolution per minutes used. At 50rpm, ileal viscosity quadratically reached its peak (P < 0.0001) and reduced when the MSP inclusion was increased to 100g/kg. At 60 revolutions per minute, ileal viscosity linearly increased (P < 0.01) in all birds fed diet containing MSP. Similar values were recorded for birds fed 50 and 100 g/kg MSP. At 100 rpm, ileal viscosity significantly (P < 0.01) increased for birds fed 100 g/kg MSP. Lower but similar values of viscosity were recorded for turkeys fed control diet and diet containing 50g/kg MSP.

<table><caption>**Table 1:** Effects of MSP inclusion and Enzyme or Yeast Supplementation on the Apparent and True Metabolizable energy of growing Turkeys.</caption><thead><tr><th>Measurements</th><th>0</th><th>50</th><th>100</th><th>SEM</th><th>L</th><th>Q</th><th>Enzyme</th><th>Yeast</th><th>SEM</th><th>P value</th></tr></thead><tbody><tr><td>AME (MJ/kg)</td><td>15.29<sup>b</sup></td><td>15.63<sup>a</sup></td><td>15.77<sup>a</sup></td><td>0.08</td><td>0.019</td><td>0.025</td><td>15.57</td><td>15.56</td><td>0.12</td><td>0.14</td></tr><tr><td>AMEn (MJ/kg)</td><td>15.23<sup>b</sup></td><td>15.57<sup>ab</sup></td><td>15.70<sup>a</sup></td><td>0.08</td><td>0.019</td><td>0.055</td><td>15.52</td><td>15.48</td><td>0.13</td><td>0.15</td></tr><tr><td>TME (MJ/kg)</td><td>16.43</td><td>16.75</td><td>16.78</td><td>0.07</td><td>0.057</td><td>0.112</td><td>16.66</td><td>16.65</td><td>0.13</td><td>0.17</td></tr><tr><td>TMEn (MJ/kg)</td><td>16.38<sup>b</sup></td><td>16.93<sup>a</sup></td><td>16.71<sup>ab</sup></td><td>0.08</td><td>0.086</td><td>0.010</td><td>16.78</td><td>16.57</td><td>0.13</td><td>0.16</td></tr></tbody></table>

<table><caption>**Table 2:** Effects of MSP inclusion and enzyme or yeast supplementation on Viscosity of growing Turkeys.</caption><thead><tr><th rowspan="2">Speed</th><th colspan="3">MSP Inclusion (g/kg)</th><th colspan="2">P value</th><th colspan="3">Additives (mg/kg)</th><th rowspan="2">SEM</th><th rowspan="2">P value</th></tr><tr><th>0</th><th>50</th><th>100</th><th>L</th><th>Q</th><th>Enzyme</th><th>Yeast</th><th>SEM</th></tr></thead><tbody><tr><td>50</td><td>297.8<sup>b</sup></td><td>329.9<sup>a</sup></td><td>308.8<sup>b</sup></td><td>4.56</td><td>0.232</td><td>0.000</td><td>313.1</td><td>311.2</td><td>3.72</td><td>4.12</td></tr><tr><td>60</td><td>250.7<sup>b</sup></td><td>277.4<sup>a</sup></td><td>274.1<sup>a</sup></td><td>4.38</td><td>0.008</td><td>0.003</td><td>267.7</td><td>267.1</td><td>3.74</td><td>4.23</td></tr><tr><td>100</td><td>216.2<sup>b</sup></td><td>222.6<sup>b</sup></td><td>230.5<sup>a</sup></td><td>2.25</td><td>0.004</td><td>0.019</td><td>218.3</td><td>227.8</td><td>1.83</td><td>2.10</td></tr></tbody></table>

## Conclusion

Turkeys were able to metabolise MSP at 50 g/kg and 100g/kg for improved AME, AMEn and TMEn irrespective of the additive used. Supplementation with 200mg/kg enzyme or yeast did not significantly affect ileal digesta viscosity.

<footnote><sup>abc</sup> Means on the same row having different superscript differ significantly (P < 0.05)</footnote>

<footnote><sup>abc</sup> Means on the same row having different superscript differ significantly (P < 0.05)</footnote>
References

Atteh, J. O. 2001. Versatility of nutrasexyla as a feed additive for monogastric animals. A paper presented at a two day seminar on starting the millennium with an array of tailor-made biotechnology improvement of flour milling and baking industry at Sheraton hotel, Lagos.

Bedford, M. R. and Schulze, H. 1998. Exogenous enzymes for pigs and poultry. *Nutrition Research Review*. 11:91-114.

Bedford, M. R., Classen, H. L. and Campbell, G. L. 1991. The effect of pentosans on the viscosity of the intestinal content and the performance of broilers fed rye. *Poultry Science* 70: 1571-1577.

LutfulKabir, S. M. 2009. The role of probiotics in the poult industry. *International Journal of Molecular Science*, 10: 3531-3546.

Moyad, M. A. 2008 Brewer's/baker's yeast (Saccharomyces cerevisiae) and preventive medicine: Part II. Urologic Nursing, 28: 73-75.

Oduguwa, O. O., Fanimo, A. O., Oduguwa, B. O. Iyayi, E. A. and Opadotun, A. I. 2001. Effects of enzyme supplementation on the nutritive value of MSP in the rat. *Tropical Journal of Animal Science* 4(1): 189-195

Parks, C. W., Grimes, J. L., Ferket, P. R. and Fairchild, A. S. 2001. The effect of mannan oligosaccharides, bambermycins, and virginiamycin on performance of large white male market turkeys. *Poultry Science*, 80: 718-723.

Sibbald, I.R. 1979.In: Recent advances in Animal Nutrition, Haresign, Wand Lewis, D (Eds) 1979.Pp 35-49

Spring, P. 2002. The role of yeast cell wall derived manna-oligosaccharide in nutrition and health. *Feed Compounder*, 22:14-18.

Spring, P., C. Wenk, H. A. Dawson and K. E. Newman. 2000. The effects of manna-oligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of salmonella- challenged broiler chicks. *Poultry Science*, 79: 205-211.

SPSS. 1999. Statistical Package for Social Sciences. SPSS for Microsoft Windows Release 6.0 User's manual.

Sundu, B., Kumar, A. and Dingle, J. 2006. Response of broiler chicks fed increasing levels of Copra meal and enzymes. *International Journal of Poultry Science* 5(1): 13-18

Ubosi, C. O. 2000. Improving current animal production the new millennium challenges and options. Book of proceedings of 25th annual conference of Nigeria Society of Animal Production (NSAP) Michael Okpara University of Agriculture, Umudike, Abia State, March 19-23, 2000 pp 4-7.
P-117 (ID 104)

# EFFECT OF MULTI-ENZYME SUPPLEMENTATION ON THE UTILIZATION OF MALTED SORGHUM (SORGHUM BICOLOR) SPROUTS BY BROILER CHICKENS

Onimisi, P.A; Lasisi, T.O and Bawa, G.S.

Department of Animal Science, Faculty of Agriculture, Ahmadu Bello University, Zaria, Nigeria

Corresponding author: onimisiphil@gmail.com

## Abstract

A feeding trial was conducted to evaluate the utilisation of Malted Sorghum Sprouts (MSSP) supplemented with Maxigrain® (an enzyme cocktail containing α-amylase, Xylanase, β-glucanase, Exo-Cellulase, Pectinase, Protease, Phytase and Lipase) by broiler chickens. Standard diets were formulated for 270 starter and finisher chickens with graded levels of MSSP. Birds were distributed in completely randomised design into 5 treatments with 3 replicates each. MSSP was included at 0%, 10% without maxigrain, 10 + maxigrain; 15% without maxigrain and 15 + maxigrain. Birds were supplied with treatment diets and water *ad libitum*. Data collected included the initial and final weight for each phase while weight gain and feed consumption were recorded weekly. Feed/gain ratio and feed cost/Kg gain were computed. Data were subjected to ANOVA and means were compared using DMRT. At the starter phase, birds fed 10% MSSP with Maxigrain® had significantly (P < 0.05) highest final body weight and daily weight gain, and also had the best feed/gain ratio and lowest feed cost per Kg gain. At the finisher phase, all the treatments had similar (P > 0.05) final weight and daily weight gain of birds. Feed conversion was however best for birds fed 15% MSSP with Maxigrain®, which also had the best and lowest feed cost per Kg gain. Broiler Chickens effectively utilised and obtained best performance with 10% and 15% MSSP in diet at starter and finisher phase respectively with Maxigrain® supplementation.

**Keywords:** Moulted Sorghum Sprouts, Broiler Chickens, Maxigrain® Enzyme, Growth Performance

## Introduction

*Sorghum bicolor* (L) Moench is widely grown in the semi-arid and arid savannah regions of Nigeria. MAUNDER (2002) reported that sorghum is a traditional crop of much of Africa and Asia and an introduced and hybridized crop in the western hemisphere. Sorghum is the world's fifth most important cereal and is grown in semi-arid regions of Africa being well adapted to the harsh climate and naturally resistant to many pests (BELTON *et al*., 2003).

It benefits from an ability to tolerate drought, soil toxicities and temperature extremes effectively than other cereals. In terms of the nutritive value, cost and availability, sorghum grain is the next alternative to maize in poultry feed (SUBRAMANIAN AND METTA, 2000).

Malted sorghum sprout (MSSP) is a by-product of sorghum malting. The separated roots and shoots which are left after malt extraction from the young germinating sorghum seedlings are collectively called sorghum sprout (ALETOR *et al*., 1998). MSSP has a lot of prospect as a feed stuff of the livestock industry. It is rich in organic nitrogen. It contains (g/kg); 226 crude protein, 48 crude fibre, 33 ether extract, 16 ash, 522 nitrogen free extract and 16.26 MJ/kg DM gross energy. Magnesium was reported to be the most abundant mineral while potassium was the least in MSSP. Among the trace minerals, Zinc is the most abundant while copper is the least. Sorghum sprout is reported to contain a considerable number of amino acids with low level of methionine, lysine and threonine (ANING *et al*., 1998; IKEDIOBI, 1989).

## Materials and Methods

**Experimental site:** The experiment was conducted at the Teaching and Research Livestock Farm of the Department of Animal Science, Ahmadu Bello University, Zaria, Nigeria. Zaria is located within the Northern guinea savannah zone on latitude 11° 9' 45"N and longitude 7° 38' 8"E, with an altitude of 610m above sea level (OVIMAPS, 2014).

**Malted sorghum sprouts:** MSSP was sourced from Nigerian Brewery Company (NBC) Kaduna Nigeria. It was dried in the sun and then incorporated into broiler diets.

**Experimental Design and management of birds:** Two hundred and seventy (270) day-old broiler chicks were used. The birds were assigned in a completely randomized design (CRD) to five dietary treatments with three replicates consisting of 18 birds each. Five diets were formulated; Diet 1, was maize based diet as the control, Diets 2 and 4 had 10% and 15% MSSP respectively; Diets 3 and 5 had 10% and 15% MSSP respectively, both supplemented Maxigrain® - an
enzyme cocktail - at 0.01%. Diets were adjusted to meet standard requirements of birds at the starter and finisher phases each of which lasted for 28 days. Feed and water were provided ad libitum. Records on weight gain, feed intake, feed conversion ratio, cost per kg weight gain and mortality were taken at both phases.

## Results

The effect of MSSP on growth performance of starter and finisher Broiler chickens is presented in table 1. For the starter broiler chickens, the final weights were significantly (P < 0.05) higher for enzyme supplemented diets both at 10 % and 15 % MSSP. Best performance was obtained for birds fed 10 % MSSP with Maxigrain® supplementation in terms of final weight, weight gain, feed to gain ratio and the cost of producing 1 Kg of meat.

At the finisher phase, all the treatments had similar (P > 0.05) final weight and daily weight gain of birds. Feed conversion was however best for birds fed 15 % MSSP with Maxigrain®, which also had the best and lowest feed cost per Kg gain.

Enzyme supplementation of diets containing MSSP resulted into better utilization of this feed resource.

## Conclusion

* Broiler starter chickens can efficiently utilise 10 % moulted sorghum sprouts in the diets with optimum performance when supplemented with Maxigrain enzyme cocktail.

* Broiler finisher chickens can efficiently utilise 15 % moulted sorghum sprouts in the diets with optimum performance when supplemented with Maxigrain enzyme cocktail.

<table><caption>Table 1: Effect of graded levels of MSP on the performance of broiler chickens</caption><thead><tr><th>Parameters</th><th>T1</th><th>T2</th><th>T3<br>% MSSP</th><th>T4</th><th>T5</th><th></th></tr><tr><td></td><td>0</td><td>10</td><td>10+Max</td><td>15</td><td>15+Max</td><td>SEM</td></tr></thead><tbody><tr><td colspan="7"><strong>Starter Phase</strong></td></tr><tr><td>Initial wgt (g/bird)</td><td>85.7</td><td>85.0</td><td>85.7</td><td>85.33</td><td>86.33</td><td>2.13</td></tr><tr><td>Final wgt (g/bird)</td><td>797.3<sup>a</sup></td><td>630.6<sup>b</sup></td><td>897.10<sup>a</sup></td><td>550.17<sup>b</sup></td><td>613.69<sup>b</sup></td><td>53.48</td></tr><tr><td>Wgt gain(g/bird/day)</td><td>25.4<sup>a</sup></td><td>19.5<sup>b</sup></td><td>28.98<sup>a</sup></td><td>16.60<sup>b</sup></td><td>18.83<sup>b</sup></td><td>1.89</td></tr><tr><td>Feed intake (g/bird/day)</td><td>54.4<sup>a</sup></td><td>42.3<sup>bc</sup></td><td>43.17<sup>b</sup></td><td>40.79<sup>bc</sup></td><td>39.43<sup>c</sup></td><td>1.58</td></tr><tr><td>Feed/gain ratio</td><td>2.14<sup>b</sup></td><td>2.18<sup>b</sup></td><td>1.53<sup>a</sup></td><td>2.47<sup>c</sup></td><td>2.13<sup>b</sup></td><td>0.13</td></tr><tr><td>Feed cost/kg gain (N)</td><td>209.4<sup>c</sup></td><td>196.26<sup>bc</sup></td><td>138.76<sup>a</sup></td><td>213.79<sup>c</sup></td><td>185.69<sup>b</sup></td><td>11.59</td></tr><tr><td>Mortality</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>--</td></tr><tr><td colspan="7"><strong>Finisher Phase</strong></td></tr><tr><td>Initial wgt (g/bird)</td><td>753.3</td><td>750.0</td><td>750.0</td><td>753.3</td><td>753.3</td><td>5.16</td></tr><tr><td>Final wgt (g/bird)</td><td>1833.3</td><td>1850.0</td><td>1916.7</td><td>1833.3</td><td>1923.3</td><td>45.19</td></tr><tr><td>Wgt gain(g/bird/day)</td><td>38.7</td><td>39.3</td><td>41.7</td><td>38.6</td><td>41.8</td><td>1.65</td></tr><tr><td>Feed intake (g/bird/day)</td><td>108.8<sup>a</sup></td><td>92.8<sup>b</sup></td><td>89.5<sup>b</sup></td><td>82.8<sup>c</sup></td><td>81.6<sup>c</sup></td><td>2.01</td></tr><tr><td>Feed/gain ratio</td><td>2.82<sup>c</sup></td><td>2.37<sup>b</sup></td><td>2.15<sup>a</sup></td><td>2.15<sup>a</sup></td><td>1.97<sup>a</sup></td><td>0.09</td></tr><tr><td>Feed cost/kg gain (N)</td><td>216.6<sup>d</sup></td><td>168.1<sup>c</sup></td><td>154.4<sup>b</sup></td><td>146.7<sup>ab</sup></td><td>135.7<sup>b</sup></td><td>6.09</td></tr><tr><td>Mortality</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>--</td></tr></tbody></table>

a, b, c: Means in the same row having different superscript are significantly different (P<0.05).

SEM: Standard error of means. Max: Maxigrain enzyme
## References

ALETOR, V. A., I. I. HAMID, E. NIEB, E. PFEFFER, 1998: Low-protein amino acids supplemented diets in Broilers. 1. Effects on growth performance, relative organ weight and carcass characteristics. In: *Proceedings of Silver Anniv. Conf. Nigerian Society of Animal Production*. Pp. 153-154.

ANING, K.G, A.G OLOGUN, A. ONIFADE, J.A. ALOKAN, V. A. ALETOR, A. I. ADEKOLA, 1998: Effect of replacing dried brewers' grain with sorghum rootlets on growth, nutrient utilization and some blood constituents in the rat. *Animal Feed Science and Technology* 71: 185-190.

BELTON, P, J. TAYLOR, G. DUODU, G. MILLS, C. GAO, P. SHEWRY, J. FORSYTH E.CHAMBA, 2003: Out of Africa: Sorghum, the sustainable cereal. Royal Society Summer Science Exhibition. https://royalsociety.org/summer-science/2003/out-of-africa

IKEDIOBI, C.O, 1989: Industrial utilization of sorghum. A Paper presented at the ICRSAT-WASP-LAR workshop held in Kano 4-6 Dec. 1989. In: Raw Material Research and Development Council (RMRDC) Bulletin No. 001, Oct. 1990.

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

OVIMAPS, 2012: Ovi location map; Ovi earth imagery date; May 20th, 2012.

SUBRAMANIAN, V, V.C. METTA, 2000: Sorghum grain for poultry feed. In: Technical and Institution Options for Sorghum Grain Mold Management. Proc. International Consultation.

Chandrasher A, Bandyopadhayi R and Hall AJ (eds.). International Crop Research for the Semi-Arid Tropics (ICRISAT). Patacheru 502 – 504, Andhra Pradesh, India, pp. 242 – 247.
P-118 (ID 22)

EFFECT OF RONOZYME® PHYTASE ON THE UTILIZATION OF TWO POPULAR VARIETIES OF SORGHUM
(SORGHUM BICOLOR) IN NIGERIA BY BROILER CHICKENS

Onimisi, P.A; Oladipo, M.F and Bawa, G.S.

Department of Animal Science, Faculty of Agriculture, Ahmadu Bello University, Zaria, Nigeria

Corresponding author: onimisiphil@gmail.com

Summary

The effect of feeding 300 broiler chickens with two common varieties of sorghum in Nigeria, *Jandawa* (Red sorghum) and *Kaura* (SAMSORG-17) with or without phytase was evaluated in a Completely Randomised Design of five dietary treatments, each replicated thrice with 20 chicks. Treatment 1 was maize- T2, *Jandawa*- T3, Kaura- T4, *Jandawa* with phytase- and T5, *Kaura* with phytase- based standard diets were formulated for starter and finisher chickens. Water and feed were offered to the birds *ad libitum* for the eight-week period. Collected data were initial and final weight while weight gain and feed consumption were recorded weekly. Feed/gain ratio and feed cost/Kg gain were computed. At 8 weeks, three birds per treatment were used for carcass and bone mineral analyses. Serum Ca and P were determined. Data were subjected to ANOVA and means were compared using DMRT. Birds fed *Kaura* with phytase had significantly (P < 0.05) highest final body weight and weight gain at the starter phase, non-significant (P > 0.05) differences at the finisher phase but had significantly (P < 0.05) lowest cost of production for the eight weeks period. Diets with phytase inclusion had significantly (P < 0.05) higher tibia Ca and P but non-significantly higher serum Ca and P than other treatments. Phytase in *Kaura* based diets significantly improved growth performance of broiler chicks, reduced cost of production and enhanced bone mineralization than *Jandawa* and maize based diets.

**Keywords:** Jandawa, Kaura, Ronozyme Phytase, Growth Performance, Bone Minerals

Introduction

Grain sorghum is the third most important cereal crop grown in the United States and the fifth most important cereal
crop grown in the world. In 2010, Nigeria was the world's largest producer of grain sorghum, followed by the United
States and India (FAO, 2012). MAUNDER (2002) reported that sorghum is a traditional crop of much of Africa and Asia
and an introduced and hybridized crop in the western hemisphere.

Enzyme inclusion in poultry feeds is one of the major nutritional advances in the last fifty years. Some of the enzymes that have been used over the past several years and have potential for use in the feed industry include cellulase (β-glucanases), xylanases and associated enzymes, phytases, proteases, lipases, and galactosidases (KHUSHEEBA AND SAJID, 2013). The addition of phytase to feed improves phosphorus utilization in both ruminant and monogastric animal; thus reducing the need for supplemental inorganic phosphate and helps in reducing the environmental problems that arise from organic phosphate excretion. Commercial phytases are available and are effective in improving phytate phosphorus utilization in poultry diets (WU et al., 2003; DILGER et al., 2004).

Amongst cereal grains, sorghum contains relatively high phytate concentrations (SELLE et al., 2003) and it follows that broilers on sorghum-based diets should be advantaged by phytate-degrading enzymes.

**Materials and Methods**

**Experimental site:** The experiment was carried out at the Poultry Unit of Animal Science Departmental Teaching and Research farm, Ahmadu Bello University, Zaria, Nigeria. Zaria, located in the Northern Guinea Savannah zone is on latitude 11° 9' 45"N and longitude 7° 38' 8"E, with an altitude of 610m above sea level. The climate is characterized by a well-defined dry and wet seasons with a mean annual rainfall of 700-1400mm (OVIMAPS, 2014)

**Experimental Design and management of birds:** Three hundred broiler chickens were allotted to five dietary treatments with 3 replicates of 20 birds each in a completely randomized design (CRD). The birds were housed in deep litter pens and managed with all necessary routine management practices with feed and water supplied *ad libitum*. Five treatment diets were formulated to meet standard requirements. Diet 1: (Control) Maize based diet. Diet 2: Jandawa based diet; Diet 3: Kaura based diet. Diet 4: Jandawa based diet with phytase. Diet 5: Kaura based diet with phytase
**Growth Study:** Initial and final weights of birds were taken at the beginning and at the end of both phases. Weight gain and feed intake were measured weekly while feed/gain ratio and cost per Kg gain were computed for both phases. Mortality was recorded as they occurred.

**Carcass Evaluation and Bone Mineral Analysis:** Three birds were selected from each treatment at the end of the feeding trial, fasted of feed overnight, weighed, slaughtered for carcass evaluation. Carcass weight, cut parts and organ weights were recorded. The prime cuts and organ weights were expressed as a percentage of dressed weight and live weight respectively. The tibia bones were removed carefully from the thigh. The fresh bones were oven dried at 100°C overnight to obtain a constant weight. The dry bones were ashed at 550°C for 6 hours. The bone ash was analysed for calcium content using Atomic Absorption Spectrophotometry (AAS) and the phosphorus content using flame photometry. Serum Calcium and Phosphorus were also determined using standard procedures.

**Data Analysis:** All data obtained were statistically analysed using the GLM Procedure of SAS and significant difference between treatment means were determined using Duncan's Multiple Range Test (SAS, 2002).

## Results

### Growth Performance of broiler chickens

Table 1 shows the performance of broiler chickens. Starter birds fed Kaura with phytase had significantly (P<0.05) higher final body weight and weight gain, which agrees with KHAN et al., (2013) who reported that phytase addition in broiler feed greatly enhanced the growth rate and weight gain of chicks. The maize and Jandawa diets (T1 and T2) had significantly lower feed consumption than all the other treatments which were at par. The Kaura diet with phytase increased feed consumption but with a significant increase in weight gain. Therefore, birds efficiently utilized Kaura diets.

Kaura diet with phytase had the least cost of production (N148.96) compared to other treatments with 158.44, 167.67, 159.01 and 176.33 for T1-T4 respectively. The use of phytase with Kaura significantly reduced the cost of producing broiler chickens than the traditional maize based diet.

Broiler finishers showed no significant (P > 0.05) difference in the final body weight, weight gain, feed intake and feed to gain ratio across all treatment diet.

The feed cost required to produce one kilogram of meat was lowest and similar for Kaura with enzyme and maize diet. This was followed by Kaura without enzyme and then Jandawa with enzyme. The highest cost of production was recorded for Jandawa without enzyme.

Phytase inclusion resulted into a general improvement in nutrient digestibility.

### Bone mineral contents of birds

Table 2 shows the result of mineral analysis of the tibia bones of broiler chickens fed Kaura and Jandawa with and without enzyme inclusion. There were significant (P<0.05) differences across treatments. Phytase inclusion increased tibia calcium of birds fed Jandawa diet with phytase (up to 6%) and tibia phosphorus (up to 3%) and Kaura diet with phytase (up to 7%) and tibia phosphorus (up to 3%). This is a good indication of increased availability of Phosphorus from phytate mineral complex by the action of phytase as also earlier reported by SABESTIAN et al., (1997) and MONDAL et al., (2007).

## Conclusion

* Complete replacement of maize with Kaura and Jandawa variety of sorghum did not impair body weight, feed intake, feed conversion ratio, nutrient digestibility, carcass and organ weights and bone mineral content in the diets of broiler chicken.

* Phytase inclusion in Sorghum based broiler diets significantly improved growth performance and mineral contents of bones.