## Results

It was evident that feeding diets containing 200 g/kg WL seeds had a negative effect on the BWG and FCR of birds (0-35d), (BWG: CON – 2100 g, 200 – 1917 g, P<0.05). These could have been caused by a quadratic decrease in feed intake when WL seeds were introduced into the diet (FI: CON – 3053 g/bird, 200 – 2934 g/bird). Similarly, VIVEROS et al. (2007) reported that WL seeds had deleterious effects on the performance of broiler chickens. On the other hand, NALLE et al. (2012) found that broiler diets could contain up to 200g/kg of WL without a negative effect on growth. These discrepancies between above studies may reflect differences in cultivars or feed formulation strategies (NALLE et al., 2012). In our study, the observed decrease in the feed intake after WL addition could be partially explained by probable high insoluble NSP content. JØRGENSEN et al. (1996) reported that digesta retention time and nutrient digestibility are adversely affected by insoluble NSP content. The depression in birds' performance observed in our study may be attributed to determined increase in ileal viscosity. (Table 1). It is generally accepted that the high viscosity of digesta is associated with soluble NSP content in the diet. Adverse effects of soluble NSP are primarily associated with the viscous nature of these compounds. The modes of action include altered intestinal transit time, and changes in microflora composition and nutrient absorption (CHOCT and ANNISON, 1992).

<table><caption>**Table 6.** Apparent metabolisable energy of diet, ileal digestibility (CF - crude fat, crude protein and starch), viscosity (Vi) of ileal digesta, excreted NANA, villi height (VH) and area (VA) of broiler chickens fed diets with different doseges of WL.</caption><thead><tr><th rowspan="2">WL dose g/kg</th><th rowspan="2">AME<sub>N</sub> [MJ/kg]</th><th colspan="3">Apparent ileal digestibility:</th><th rowspan="2">Ileal Vi [cP]</th><th rowspan="2">Total NANA [µmol/gTiO<sub>2</sub>]</th><th colspan="2">Ileum</th></tr><tr><th>CF</th><th>CP</th><th>S</th><th>VH [µm]</th><th>VA [µm<sup>2</sup>]</th></tr></thead><tbody><tr><td>0 [CON]</td><td>12.8</td><td>0.74</td><td>0.74</td><td>0.99</td><td>1.76</td><td>219</td><td>861</td><td>415 800</td></tr><tr><td>100</td><td>11.5*</td><td>0.68</td><td>0.74</td><td>0.94</td><td>2.18'</td><td>282*</td><td>935</td><td>350 966'</td></tr><tr><td>150</td><td>11.6*</td><td>0.68</td><td>0.74</td><td>0.96</td><td>2.41*</td><td>314*</td><td>703*</td><td>301 265*</td></tr><tr><td>200</td><td>11.2*</td><td>0.68</td><td>0.73</td><td>0.96</td><td>3.06*</td><td>308*</td><td>698*</td><td>265 664*</td></tr><tr><td>250</td><td>10.9*</td><td>0.56*</td><td>0.70*</td><td>0.90*</td><td>3.08*</td><td>310*</td><td>721*</td><td>286 118*</td></tr><tr><td>300</td><td>10.4*</td><td>0.56*</td><td>0.68*</td><td>0.90*</td><td>3.74*</td><td>316*</td><td>704*</td><td>280 905*</td></tr><tr><td>Contrast</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>L</td><td>&lt;0.05</td><td>&lt;0.05</td><td>&lt;0.05</td><td>&lt;0.05</td><td>&lt;0.05</td><td>&lt;0.05</td><td>0.0006</td><td>&lt;0.05</td></tr><tr><td>Q</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>&lt;0.05</td><td>0.187</td><td>&lt;0.05</td></tr></tbody></table>

* - different from control treatment at P<0.05, ′ - different from control treatment at P<0.1

L - linear contrast, Q - quadratic contrast, NS - P>0.05

The increase in ileal viscosity following WL addition determined in our study is in agreement with the findings of OLKOWSKI et al. (2005) who reported that 400g/kg addition of WL to the diet caused an increase in digesta viscosity (from 1.8 to 4.2 cP). The diet utilization in our study was negatively affected by relatively low (100g/kg) WL inclusion. The increase in ileal digesta viscosity and high insoluble NSP content were probably the main factors affecting nutrient AID. NSP content was not determined in this study but the observed increase in ileal digesta viscosity after WL inclusion indicate that soluble and insoluble NSP content was increasing together with the increasing content of WL seeds in experimental diets. Ether extract, crude protein and starch AID (Table 1) were affected only by 250 and 300g/kg WL inclusion. The EE and CP AID could have been depressed by increasing ileal viscosity as well as insoluble NSP content. The use of over 150 g of WL depressed VH and VA (Table 1) with may explain the depression in nutrient utilization and, consequently, AME<sub>N</sub> reduction. Additionally, we observed a quadratic increase in excreted NANA together with WL inclusion. These increase in NANA excretion may have been caused by leveed mucin secretion, resulting in higher endogenous losses. The increased mucin production associated with diets containing WL meal may be due to an increase in bacterial numbers in the distal gut linked to an increase in substrate availability. As suggested by COWIESON et al. (2003), diets that increase mucin secretion are nutritionally expensive for birds with may explain the depression in birds' performance determined in our experiment.
Conclusions

It could concluded that the inherent variability of seeds may partially explain why birds did responded differently across trials (including this experiment) after similar dose of WL seeds. The NSP present in WL seeds caused high intestinal viscosity affecting nutrient absorption and, probably, endogenous losses.

Literature

CHOCT, M. and ANNISON, G. (1992) Anti-nutritive effect of wheat pentosans in broiler chickens: Roles of viscosity and gut microflora. Br Poult Sci 33: 821-834.

COWIESON, A. J., ACAMOVIC, T. and BEDFORD, M. R. (2003) Supplementation of diets containing pea meal with exogenous enzymes: Effects on weight gain, feed conversion, nutrient digestibility and gross morphology of the gastrointestinal tract of growing broiler chicks. British Poultry Science 44: 427-437.

JØRGENSEN, H., ZHAO, X.-Q. and EGGUM, B. O. (1996) The influence of dietary fibre and environmental temopera-
ture on the development of the gastrointestinal tract, digestibility, degree of fermentation in the hind-gut and
energy metabolism in pigs. British Journal of Nutrition 75: 365-378.

NALLE, C. L., RAVINDRAN, V. and RAVINDRAN, G. (2012) Nutritional value of white lupins (lupinus albus) for broilers: Apparent metabolisable energy, apparent ileal amino acid digestibility and production performance. animal 6: 579-585.

OLKOWSKI, B. I., CLASSEN, H. L., WOJNAROWICZ, C. and OLKOWSKI, A. A. (2005) Feeding high levels of lupine seeds to broiler chickens: Plasma micronutrient status in the context of digesta viscosity and morphometric and ultrastructural changes in the gastrointestinal tract. Poultry Science 84: 1707-1715.

RAVINDRAN, V., HEW, L. I., RAVINDRAN, G. and BRYDEN, W. L. (2005) Apparent ileal digestibility of amino acids in dietary ingredients for broiler chickens. Animal Science 81: 85-97.

SELLE, P. H., COWIESON, A. J., COWIESON, N. P. and RAVINDRAN, V. (2012) Protein-phytate interactions in pig and poultry nutrition: A reappraisal. Nutr Res Rev 25: 1-17.

VIVEROS, A., CENTENO, C., ARIJA, I. and BRENES, A. (2007) Cholesterol-lowering effects of dietary lupin (lupinus albus var multolupa) in chicken diets. Poultry Science 86: 2631-2638.
# INTERACTION BETWEEN THREONINE AND GLYCINE AT LOW DIETARY CRUDE PROTEIN AND THE EFFECT ON PRODUCTION PERFORMANCE, MEAT QUALITY AND PLASMA METABOLITES IN BROILERS

W. Lambert¹, M. Rovers³, J. Ensink³, S. Tesseraud², E. Corrent¹, L. de Lange⁴, L. Star⁴

¹ Ajinomoto Eurolysine s.a.s., 153 rue de Courcelles, 75817 Paris, France

² INRA, UR83 Recherches avicoles, 37380, Nouzilly, France

³ Orffa Additives B.V., Vierlinghstraat 51, 4251 LC Werkendam, the Netherlands

⁴ Schothorst Feed Research, Meerkoetenweg 26, 8218 NA Lelystad, the Netherlands

**Corresponding author:** Lambert_William@eli.ajinomoto.com

## Abstract

This trial was designed to study the interaction between Thr and Gly in 7-28d broilers. A total of 2,040 broilers were allocated to 17 treatments. A positive control (PC) diet (20.5% CP) was formulated to be adequate in essential AA. A negative control (NC) diet (18.5% CP, deficient in Thr and Gly) was supplemented with L-Thr and Gly to obtain a 4 Thr x 4 Gly design. The PC was compared to the other treatments by Student's t-test and the Thr x Gly interaction was tested by interactive regression analysis. Reducing CP increased feed intake and BWG (P<0.05) except for the NC diet. The four most deficient diets in Thr showed higher FCR than the PC diet irrespective of the Gly level. A Thr x Gly interaction was found for FCR for the 7-17d. Linear response of FCR to Thr was significant only at 2 levels of Gly and was stronger with the lowest Gly level. Plasma uric acid concentration was higher in the PC than in all the other diets and plasma triglyceride concentration was linearly decreased by Thr supplementation, but not by Gly. White striping and wooden breast occurrence at 28d were not affected. In conclusion, there is an interaction between Thr and Gly, and Thr is able to spare Gly in low CP diets.

**Keywords:** threonine, glycine, protein, performance, plasma metabolites

## Introduction

Lowering the dietary crude protein (CP) content in broiler feeds is an efficient tool to improve intestinal health, and to reduce foot pad lesions and the excretion of nitrogen to the environment (MEDA et al., 2011; WIDYARATNE, 2012). When CP content is reduced, the animal performance will be limited by the indispensable amino acids (IAA) but dispensable AA (DAA) and especially Gly might also become limiting (DEAN et al., 2006). However, Thr, the third limiting IAA in broilers, is a known precursor of Gly via the Thr dehydrogenase and aldolase pathways (DAVIS AND AUSTIC., 1982) and it was earlier demonstrated that Thr could spare the Gly requirement (OSPINA-ROJAS et al., 2013; SIEGERT AND RODEHUTSCORD, 2013). In this trial, the effects of Gly and Thr on production performance, meat quality and plasma metabolites were investigated in low CP diets.

## Material and Methods

A total of 2,040 male Ross 708 broilers were housed on litter in small pens at the experimental broiler facilities of Schothorst Feed Research, Lelystad, the Netherlands. Birds were fed a commercial starter diet from day 0 to 7, an experimental diet from day 7 to 28 and a finisher commercial diet from day 28 to 35. The experiment was performed with 17 treatments and 6 replicates, with 20 broilers per replicate. Treatment 1 was the positive control (PC) diet with a CP level of 20%, an apparent faecal digestible (AFD) Thr:Lys of 64% and an AFD Gly+Ser:Lys of 156 %. Treatment 2 was the negative control (NC) diet with a CP level of 18.5%, an AFD Thr:Lys ratio of 55% and an AFD Gly+Ser:Lys ratio of 135%. On top of the NC diet, the addition of 3 supplemental levels of L-Thr and Gly gave the treatments 2 to 17 (55, 58, 61, and 64% AFD Thr:Lys x 135, 142, 149, and 156% AFD Gly+Ser:Lys). The animals were fed an AFD Lys of 1.0% and AMEn of 3,000 kcal/kg. Performance parameters were measured at day 0, 7, 17, 28, and 35. Plasma sampling was performed at day 28 on 10 birds per treatment and analysed for uric acid, glucose, triglycerides and total AA levels. White striping and wooden breast incidence was evaluated in 5 treatments (PC + the 4 highest Thr treatments). The PC diet was compared to the other treatments by a 2-by-2 Student's t-test (PROC GLM, SAS 9.2). Quadratic and linear regressions were performed to test the effect of Thr and Gly at each level of Gly and Thr. Interactive regression analysis tested the interaction between Thr and Gly (PROC REG, SAS 9.2).
## Results

Reducing dietary CP resulted in higher feed intake (FI) and body weight gain (BWG) in most treatments (data not shown, P<0.05). FCR was degraded when dietary CP was low but only when dietary Thr level was 55% AFD Thr:Lys. Broilers fed the PC diet showed higher plasma uric acid concentration than any of the low CP treatments (data not shown, P<0.05).

<table><caption>**Table 1:** Effect of AFD Gly+Ser:Lys (%) and AFD Thr:Lys (%) on body weight gain (BWG, g/d), feed intake (FI, g/bird), feed conversion ratio (FCR), plasma uric acid (UA, mg/L), tryglycerides (g/L), glucose (Glu, g/L) and total amino acids (AA, mg/mL) circulating levels<fnref n="1" /></caption><thead><tr><th rowspan="2">AFD Gly+Ser: Lys (%)</th><th rowspan="2">AFD Thr:Lys (%)</th><th colspan="2">BWG (g/bird)</th><th colspan="2">FI (g/bird)</th><th colspan="2">FCR</th><th colspan="2">UA, mg/L</th><th colspan="3">TG, g/L Glu, g/L AA, mg/mL</th></tr><tr><th>D7-17</th><th>D7-28</th><th>D7-17</th><th>D7-28</th><th>D7-17</th><th>D7-28</th><th>D28</th><th>D28</th><th>D28</th><th>D28</th></tr></thead><tbody><tr><td rowspan="5">135</td><td>55</td><td>486</td><td>1511</td><td>671</td><td>2165</td><td>1.377</td><td>1.430</td><td>48.6</td><td>1.12</td><td>2.23</td><td>0.53</td></tr><tr><td>58</td><td>499</td><td>1575</td><td>684</td><td>2220</td><td>1.373</td><td>1.410</td><td>50.3</td><td>1.10</td><td>2.19</td><td>0.51</td></tr><tr><td>61</td><td>489</td><td>1548</td><td>664</td><td>2181</td><td>1.359</td><td>1.402</td><td>50.9</td><td>1.11</td><td>2.28</td><td>0.50</td></tr><tr><td>64</td><td>489</td><td>1541</td><td>658</td><td>2158</td><td>1.351</td><td>1.400</td><td>52.0</td><td>0.95</td><td>2.37</td><td>0.48</td></tr><tr><td>490</td><td>1545</td><td>672</td><td>2179</td><td>1.372</td><td>1.411</td><td>49.9</td><td>1.08</td><td>2.28</td><td>0.48</td></tr><tr><td>142</td><td>484</td><td>1539</td><td>663</td><td>2177</td><td>1.366</td><td>1.409</td><td>49.1</td><td>1.08</td><td>2.27</td><td>0.48</td></tr><tr><td>149</td><td>497</td><td>1552</td><td>673</td><td>2200</td><td>1.354</td><td>1.413</td><td>50.9</td><td>1.07</td><td>2.28</td><td>0.54</td></tr><tr><td>156</td><td>491</td><td>1538</td><td>669</td><td>2167</td><td>1.367</td><td>1.409</td><td>51.8</td><td>1.05</td><td>2.24</td><td>0.52</td></tr><tr><th colspan="13" style="text-align: left;">P-values</th></tr><tr><td rowspan="6">Linear effect of Thr</td><td>ALL<sup>2</sup></td><td>NS</td><td><strong>0.089</strong></td><td>NS</td><td>NS</td><td><strong>0.001</strong></td><td><strong>&lt;0.001</strong></td><td>NS</td><td><strong>0.004</strong></td><td><strong>0.03</strong></td><td><strong>0.003</strong></td></tr><tr><td>135<sup>3</sup></td><td>NS</td><td><strong>0.037</strong></td><td>NS</td><td>NS</td><td><strong>&lt;0.001</strong></td><td><strong>0.001</strong></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>142<sup>3</sup></td><td>NS</td><td>NS</td><td><strong>0.089</strong></td><td>NS</td><td>NS</td><td><strong>&lt;0.001</strong></td><td>NS</td><td><strong>0.059</strong></td><td>NS</td><td>NS</td></tr><tr><td>149<sup>3</sup></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td><strong>0.016</strong></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td><strong>&lt;0.001</strong></td></tr><tr><td>156<sup>3</sup></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td><strong>0.020</strong></td><td>NS</td><td>NS</td><td><strong>0.083</strong></td><td>NS</td></tr><tr><td>ALL<sup>4</sup></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><td><strong>&lt;0.001</strong></td></tr><tr><td rowspan="4">Linear effect of Gly</td><td>55<sup>5</sup></td><td><strong>0.063</strong></td><td><strong>0.017</strong></td><td><strong>0.038</strong></td><td>NS</td><td><strong>0.058</strong></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td><strong>&lt;0.001</strong></td></tr><tr><td>58<sup>5</sup></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td><strong>0.057</strong></td><td>NS</td><td>NS</td></tr><tr><td>61<sup>5</sup></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><td><strong>0.047</strong></td></tr><tr><td>64<sup>5</sup></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><td>NS</td></tr><tr><td>Interaction</td><td></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td><strong>0.088</strong></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr></tbody></table>

Supplementation of Thr linearly affected BWG but only when Gly was low (135 AFD Gly+Ser:Lys; Table 1). FCR was linearly decreased with increasing dietary levels of Thr in both periods of age (P<0.05). Addition of Thr also linearly decreased TG and total AA plasma levels and increased glucose plasma levels (P<0.05). On the contrary, Gly supplementation did not affect any of the parameters studied except when Thr level was the lowest where it resulted in an

<footnote n="1">¹ Data presented as means (n = 6 pens/treatment for performance criteria, n = 10 birds/treatment for plasma metabolites).</footnote>

<footnote n="2">² Linear regressions to threonine irrespective of glycine plus serine level.</footnote>

<footnote n="3">³ Linear regressions to threonine at each glycine plus serine level (135, 142, 149, or 156 % AFD Gly+Ser:Lys).</footnote>

<footnote n="4">⁴ Linear regressions to glycine plus serine irrespective of threonine level.</footnote>

<footnote n="5">⁵ Linear regressions to glycine plus serine at each threonine level (55, 58, 61, or 64 % AFD Thr:Lys).</footnote>

<footnote>NS (P-value > 0.1).</footnote>
increase of BWG, FI, and total AA plasma level (P<0.05). Quadratic regressions confirmed the linear effects (data not shown). White striping or wooden breast incidence were not affected by dietary CP or Gly level (data not shown).

Statistically, a tendency was detected for the interaction between Thr and Gly for FCR from day 7 to 17 but not for any other parameter or later in age (P=0.088). A response contour was performed using SAS 9.2 (PROC RSREG) to graphically represent this interaction (Figure 1). At low AFD Thr:Lys levels, the animals needed more Gly to show similar performance. At high Thr level, FCR was the lowest indicating that at least 64% AFD Thr:Lys is required for optimal feed efficiency. When dietary Thr was adequate, Gly addition had no effect on FCR.

<figure><img src="image_70.png" /><figcaption>**Figure 1:** Response contour with design points for feed conversion ratio from 7 to 17d (FCR_717). Gly = AFD Gly+Ser:Lys (%), Thr = AFD Thr:Lys (%).</figcaption></figure>

## Conclusions

Lowering dietary CP contributes to more sustainable broiler production. In this experimental trial, Gly was not limiting for growth at low CP levels unless Thr was deficient. In conclusion, the interaction between Thr and Gly is confirmed by this trial and Thr is able to spare the Gly requirement in low CP diets.

## Literature

* DAVIS, A. T., and R. E. AUSTIC, 1982: Threonine-degrading enzymes in the chicken. Poult. Sci. 61(10):2107-2111.

* DEAN, D. W., T. D. BIDNER, and L. L. SOUTHERN, 2006: Glycine supplementation to low protein, amino acid-supplemented diets supports optimal performance of broiler chicks. Poult. Sci. 85(2):288-296.

* MEDA, B., M. HASSOUNA, C. AUBERT, P. ROBIN, and J. Y. DOURMAD, 2011: Influence of rearing conditions and manure management practices on ammonia and greenhouse gas emissions from poultry houses. World's Poult. Sci. Journ. 67:441-456.

* OSPINA-ROJAS, I. C., A. E. MURAKAMI, C. A. OLIVEIRA, and A. F. GUERRA, 2013: Supplemental glycine and threonine effects on performance, intestinal mucosa development, and nutrient utilization of growing broiler chickens. Poult. Sci. 92(10):2724-2731.

* SIEGERT, W., and M. RODEHUTSCORD, 2013: Influence of dietary choline and threonine on responses of broilers to dietary glycine. Page 135 in Proceedings of the 19th European Symposium on Poultry Nutrition, Potsdam, Germany.

* WIDYARATNE, G. P., 2012: The role of protein and amino acid nutrition in controlling clostridium perfringens in the gastrointestinal tract of broiler chickens. Thesis dissertation, University of Saskatchewan.
P-211 (ID 188)

# IS META-ANALYSIS AN EFFECTIVE TOOL TO ESTIMATE THE GLYCINE PLUS SERINE REQUIREMENT OF BROILER CHICKENS?

W. Lambert<fnref n="1" />, A. Simongiovanni<fnref n="1" />, S. Tesseraud<fnref n="2" />, E. Corrent<fnref n="1" />

**Corresponding author:** Lambert_William@eli.ajinomoto.com

## Abstract

A meta-analysis was conducted to predict the Gly+Ser requirement of broilers. Dose-response trials from literature were entered in our database if they 1) were published after 1980, 2) provided specific information such as age or sex and 3) tested a basal diet supplemented with at least 3 levels of Gly. The database consisted of 14 trials and 76 data lines from 7 publications. The dietary energy, CP and AA levels were recalculated based on INRA (2004) tables. To be able to express the Gly+Ser requirement in % of the diet, 2 dose-responses (21 to 35d broilers) were excluded to obtain an homogeneous set of studies over the period 0 to 22d (n=12). A curvilinear-plateau model was then used to model the response to Gly+Ser. According to this model, the standardized digestible (SD) Gly+Ser requirement of 0-21d broilers was 1.78 % and 2.13 % for ADG and G:F, respectively. These results agree with previously published meta-analyses. However, in literature, low levels of Thr or Cys have been shown to give rise to metabolic interactions with Gly or Ser, thereby causing a reduced endogenous synthesis or an increased need of Gly or Ser. In the dose-response trials used in this meta-analysis, dietary Thr and Cys levels were often lower than suggested requirements. Due to the low number of dose-responses available and the influence of AA deficiencies, it is up to now difficult to give a reliable Gly+Ser requirement for broiler chickens.

**Keywords:** meta-analysis, glycine, requirement, interactions

## Introduction

Even though Gly is often considered as a dispensable AA (DAA), it is becoming a much studied AA in broiler research. Glycine requirement is always expressed as the sum of the Gly and Ser requirements as Ser to Gly conversion is equimolar and reversible. According to ROSTAGNO (2011), the digestible Gly+Ser requirement in ratio to Lys is higher for young broilers (147%, 0-21d) than for older broilers (134%, 21-42d). WU (2014) even suggested levels of 245% digestible Gly+Ser:Lys. Considering the conflicting evidence in literature, a meta-analysis was conducted to estimate the SD Gly+Ser:Lys requirement of broiler chickens.

## Material and Methods

Dose-response trials from literature were entered in the database if they 1) were published after 1980, 2) provided specific information such as age or sex and 3) tested a basal diet supplemented with at least 3 levels of Gly. The database consisted of 14 trials and 76 data lines from 7 publications. Respectively, 36, 36 and 28% of the trials were carried out in European Union, the United States and Brazil. The published diets were recalculated using INRA (2004) tables (SAUVANT et al., 2004). Trials were eliminated of the database if the diets were not sublimiting in digestible Lys (based on the average of two breeder recommendations) or deficient in one or more other indispensable AA (according to ROSTAGNO (2011) recommendations). From the 14 trials of the database, only 2 were matching the aforementioned criteria. However, 12 trials shared the same studied period of age (0 to 22 days) and could be used to model the response to Gly. Considering that the dietary Lys was not sublimiting in all trials, only the absolute SD Gly+Ser (% of the diet) requirement was expressed and not the ratio to Lys. In order to describe the response of ADG, ADFI and G:F to supplemental Gly, a curvilinear-plateau (CLP) model was used (PROC NLIN, SAS 9.2). The model based on SIMONGIOVANNI et al. (2012) was the following: $Y_{ij} = A_i (1 + U(R - X_{ij})^2) + \varepsilon_{ij}$ for $X_{ij} < R$; $Y_{ij} = A_i + \varepsilon_{ij}$ for $X_{ij} \ge R$ where $Y_{ij}$ is the response criterion (i.e. ADG, ADFI, G:F) for observation j of experiment i, $X_{ij}$ is the SD Gly+Ser supply, $A_i$ is the maximum response for experiment i, R is the minimum SD Gly+Ser supply required to reach the plateau (requirement) and U is the parameter describing the response to the SD Gly+Ser supply before the plateau value. The residual error term $\varepsilon_{ij}$ is assumed to be independent and normally distributed.

<footnote n="1">¹ Ajinomoto Eurolysine s.a.s., 153 rue de Courcelles, 75817 Paris, France</footnote>

<footnote n="2">² INRA, UR83 Recherches avicoles, 37380, Nouzilly, France</footnote>
## Results

Based on the 12 studies with young broilers, ADFI did not respond to Gly supplementation and is therefore not presented in this paper. ADG and G:F positively responded to SD Gly+Ser level (Figure 1a,b).

<figure><img src="image_71.png" /><figcaption>**Figure 1:** a) Average daily gain (ADG, g/d) and b) gain to feed (G:F; % of the highest treatment) according to the dietary SD Gly+Ser (%) level for the selected trials (n=12).</figcaption></figure>

The CLP model estimated that the Gly+Ser requirement was 1.78% SD Gly+Ser for ADG and 2.13% for G:F in 0 to 22 days old broilers (Table 1). However, the increase from 1.78 to 2.13% SD Gly+Ser only further improved G:F by 1.2% suggesting a weak response above 1.78% SD Gly+Ser. The requirement found in this meta-analysis is close to the recommendation based on other meta-analyses (AKINDE, 2014; SIEGERT et al., 2013). Similarly to AKINDE (2014), the present meta-analysis predicted a higher requirement for G:F than for ADG and the estimated requirements were also quite comparable between the 2 studies (in total Gly equimolar Ser). In contrast, SIEGERT et al. (2013) estimated a total Gly equimolar Ser requirement of 2.06% for ADG, higher than in AKINDE (2014; 1.93%) and the present meta-analysis (Table 1).

<table><caption>**Table 1:** Parameter estimates and error of estimates of the CLP model for average daily gain (ADG, g/d) and gain to feed (G:F) as response criteria (n=12).</caption><thead><tr><th></th><th>ADG</th><th>G:F</th></tr></thead><tbody><tr><td>Plateau (A)</td><td>35.1</td><td>0.758</td></tr><tr><td>Slope (U)</td><td>-0.2393</td><td>-0.1004</td></tr><tr><td>SD Gly+Ser requirement (R, %)</td><td>1.78 (0.09)</td><td>2.13 (0.09)</td></tr><tr><td>Total Gly equimolar Ser requirement (R, %)</td><td>1.88 (0.12)</td><td>2.08 (0.10)</td></tr></tbody></table>

Even though predicting the Gly+Ser requirement and response is theoretically possible, several factors in the protocols of the dose-response studies might have affected the estimation. First, the present requirement is only valid for young broilers (0-22d) and broilers are known to have a higher requirement in younger ages (ROSTAGNO, 2011; [1] in Table 2). Second, low dietary CP levels were used and convincing evidence suggested a higher Gly requirement at low CP levels (DEAN et al., 2006; [2]). Third, Gly is rich in N and increasing dietary levels of Gly logically increased dietary CP levels. Diets were therefore not isonitrogenous and the response to Gly might have been confounded with the response to protein [3]. Four, dietary Cys level was low on average and response to Gly may be increased at low dietary Cys (POWELL et al., 2011; [4]). Five, dietary Thr levels were low compared to recently published Thr requirement and response to Gly may be increased when Thr is low (OSPINA-ROJAS et al., 2013; [5]). To finish, the majority of the studies in the meta-analysis (9 out of 12) were deficient in one or more AA [6].

<table><caption>**Table 2:** Key features of the trials used to estimate the Gly+Ser requirement (n=12).</caption><thead><tr><th></th><th>Key features of the trials<br>(n=12)</th></tr></thead><tbody><tr><td>Age (days) [1]</td><td>0 to 22</td></tr><tr><td>CP of the lowest Gly treatment (average, %) [2,3]</td><td>18.7</td></tr><tr><td>CP of the highest Gly treatment (average, %) [2,3]</td><td>19.4</td></tr><tr><td>SD Cys:Lys (%) [4]</td><td>21</td></tr><tr><td>Deficient in SD Thr:Lys (&lt;67%; number of studies) [5]</td><td>7</td></tr><tr><td>At least one IAA limiting (Number of studies) [6]</td><td>9</td></tr></tbody></table>
## Conclusions

Theoretically, modelling the response of production performance to Gly supplementation and estimating a Gly+Ser requirement is possible. Experimental factors (dietary CP, AA levels) of the dose-response studies favored the estimation of a high Gly requirement as predicted in this paper. Until more knowledge on Gly is released, it is up to now difficult to give a reliable Gly requirement for broilers chickens.

## Literature

AKINDE, D. O, 2014: Amino acid efficiency with dietary glycine supplementation: Part 2. World's Poult. Sci. J. 70:575-584.

DEAN, D. W., T. D. BIDNER, and L. L. SOUTHERN, 2006: Glycine supplementation to low protein, amino acid-supplemented diets supports optimal performance of broiler chicks. Poult. Sci. 85(2):288-296.

OSPINA-ROJAS, I. C., A. E. MURAKAMI, C. EYNG, R. V. NUNES, C. R. DUARTE, and M. D. VARGAS, 2012: Commercially available amino acid supplementation of low-protein diets for broiler chickens with different ratios of digestible glycine+serine:lysine. Poult. Sci. 91(12):3148-3155.

POWELL, S., T. D. BIDNER, and L. SOUTHERN, 2011: Effects of glycine supplementation at varying levels of methionine and cystine on the growth performance of broilers fed reduced crude protein diets. Poult. Sci 90:1023-1027.

ROSTAGNO, H., 2011: Brazilian Tables for poultry and swine: composition of feedstuffs and nutritional requirements. 3rd edition.

SAUVANT, D., J. M. PEREZ, and G. TRAN. 2004. Tables of composition and nutritional value of feed materials. Second revised and corrected edition ed. Wageningen Academic Publishers, INRA Editions and AFZ, Paris.

SIEGERT, W., H. AHMADI, and M. RODEHUTSCORD, 2013: A meta-analysis on the influence of glycine and serine on growth and feed conversion of broilers using mixed model methodology. Proceedings of the Society of Nutrition Physiology.

SIMONGIOVANNI, A., E. CORRENT, N. LE FLOC'H, and J. VAN MILGEN, 2012: Estimation of the tryptophan requirement in piglets by meta-analysis. Animal 6(4):594-602.

WU, G., 2014: Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition. J. Anim. Sci. Biotech. 5(34):1-12.
P-212 (ID 186)

THREONINE REQUIREMENT OF BROILER CHICKENS: INTEGRATED PREDICTION BY META-ANALYSIS

W. Lambert<fnref n="1" />, A. Simongiovanni<fnref n="1" />, S. Tesseraud<fnref n="2" />, E. Corrent<fnref n="1" />

**Corresponding author:** Lambert_William@eli.ajinomoto.com

**Abstract**

Due to its incorporation into mucus glycoproteins and immunoglobulins and its role as glycine precursor, Thr is an effective nutrient in enhancing gut health, immune response and growth. However, the use of different strains, sexes, ages, AA systems or regression models to estimate Thr requirement, has contributed to the discrepancy in the published Thr recommendations. A meta-analysis was therefore conducted to estimate the standardized digestible (SD) Thr:Lys requirement. Dose-response trials from literature were entered in our database if they 1) were published after 1980, 2) provided specific information such as age or sex and 3) tested a basal diet supplemented with at least 3 levels of L-Thr. The database consisted of 81 trials and 469 data lines from 43 publications. The dietary energy, CP and AA levels were recalculated based on INRA (2004) tables. The diets had to provide sub-limiting Lys and adequate essential AA:Lys levels. Twenty-one trials were finally selected and a curvilinear-plateau model was used to describe the response of ADG and G:F to Thr. According to this model, the SD Thr:Lys requirement of broilers was 67.3% for ADG and 67.6% for G:F. The response between 60% SD Thr:Lys and the predicted requirement was estimated to result in an increase by 4.8 and 4.1 % for ADG and G:F, respectively. In conclusion, a dietary level of 67% SD Thr:Lys seems a reasonable compromise.

**Keywords:** meta-analysis, threonine, requirement, response

**Introduction**

Thr is the third limiting AA in broiler diets and has been widely studied since feed grade L-Thr has become available in the animal feed market. Due to its incorporation into mucus glycoproteins and immunoglobulins and its role as glycine precursor, Thr is an effective nutrient in enhancing gut health, immune response and growth. However, the use of different strains, sexes, ages, AA systems or regression models to estimate Thr requirement, has contributed to the discrepancy in the published Thr recommendations. For instance, MACK et al. (1999), ROSTAGNO (2011) and WU (2014) recommended levels of 63, 65 and 67% SD Thr:Lys, respectively, for maximizing performance. A meta-analysis was therefore conducted to estimate the standardized digestible (SD) Thr:Lys requirement of broiler chickens.

**Material and Methods**

Extensive review of the available literature was performed to collect trials studying the effect of Thr supplementation on growth performance. Dose-response trials from literature were entered in our database if they 1) were published after 1980, 2) provided specific information such as age or sex and 3) tested a basal diet supplemented with at least 3 levels of L-Thr. In total, 81 trials and 469 data lines from 43 publications were entered in the database. A majority (61.7 %) of the trials was performed in the United States, 66.7 % used male broilers and 67.9 % fed broilers with corn-based diets. The published diets were recalculated using INRA (2004) tables (SAUVANT et al., 2004). Trials were eliminated of the database if the diets were not sublimiting in digestible Lys (based on the average of two breeder recommendations) or deficient in one or more other indispensable AA (according to ROSTAGNO (2011) recommendations). In order to describe the response of ADG, ADFI and G:F to supplemental Thr, a curvilinear plateau (CLP) model was used based on SIMONGIOVANNI et al. (2012): $Y_{ij} = A_i (1 + U(R - X_{ij})^2) + \varepsilon_{ij}$ for $X_{ij} < R$; $Y_{ij} = A_i + \varepsilon_{ij}$ for $X_{ij} \ge R$ where $Y_{ij}$ is the response criterion (i.e. ADG, ADFI, G:F) for observation j of experiment i, $X_{ij}$ is the SD Thr: Lys supply, $A_i$ is the maximum response for experiment i (i.e. plateau), R is the minimum SD Thr:Lys supply required to reach the plateau and U is the parameter describing the response to the SD Thr:Lys supply before the plateau value. The residual error term $\varepsilon_{ij}$ is assumed to be independent and normally distributed. The modelling of the response trials was performed with the PROC NLIN of SAS 9.2.

<footnote n="1">¹ Ajinomoto Eurolysine s.a.s., 153 rue de Courcelles, 75817 Paris, France</footnote>

<footnote n="2">² INRA, UR83 Recherches avicoles, 37380, Nouzilly, France</footnote>
## Results

From the 81 trials of the database, 35 had non-sub-limiting levels of Lys and 24 presented deficiencies in AA (Met, Val, Trp). After the modelling of the 22 selected trials, the analysis of the residuals revealed that 2 points out of 4 of one dose-response were outliers and this dose-response was therefore eliminated, resulting in a final selection of 21 trials.

Based on these 21 dose-response studies, the response of ADFI, ADG and G:F to supplemental Thr was modelled again. Average daily feed intake did not respond to Thr supplementation and is therefore not presented in this paper. Average daily gain and G:F positively responded to SD Thr:Lys level (Figure 1a,b).

<figure><img src="image_72.png" /><figcaption>**Figure 1:** a) Average daily gain (ADG) and b) gain to feed (G:F) according to the dietary SD Thr:Lys level for the selected trials (n=21).</figcaption></figure>

According to the CLP model, the SD Thr:Lys requirement of broilers is 67.3% for ADG and 67.6% for G:F (Table 1). Recent studies (JIANG et al., 2014; MELOCHE et al., 2014) also support a SD Thr:Lys ratio of at least 68% depending on the growth criteria, close to the conclusion of this meta-analysis.

<table><caption>**Table 1:** Parameter estimates and error of estimates of the CLP model for average daily gain (ADG, g/d) and gain to feed (G:F) as response criteria (n=21 trials).</caption><thead><tr><th></th><th>ADG</th><th>G:F</th></tr></thead><tbody><tr><td>Plateau (A)</td><td>51.3</td><td>0.624</td></tr><tr><td>Slope (U)</td><td>-0.00086</td><td>-0.00067</td></tr><tr><td>SD Thr:Lys requirement (R, %)</td><td>67.3 (2.9)</td><td>67.6 (8.0)</td></tr></tbody></table>

WECKE and LIEBERT (2013) recently proposed an optimal Thr:Lys of 60 % for starter and 62 % for grower broilers based on nitrogen balance studies. However, recent studies have shown that challenged broilers require higher dietary Thr levels as Thr is the first AA in mucins and immunoglobulins (STAR et al., 2012). In the dose-response studies used in our meta-analysis, broilers are raised in good environmental conditions and results are based only on production performance. It is suspected that in practical conditions, in contrast to experimental facilities, broilers might require even more Thr for optimal health and growth.

Based on our estimation with the CLP model, ADG and G:F were represented from a deficiency of 10% (60% SD Thr:Lys) corresponding to the requirement proposed by WECKE and LIEBERT (2013). The CLP model predicts that an increase from 60% to the predicted SD Thr:Lys requirement improves ADG by 4.8% and G:F by 4.1%.

## Conclusions

According to the present meta-analysis, growing broilers require at least 67% SD Thr:Lys for optimal growth and feed efficiency. When SD Thr:Lys is increased from 60% to the predicted requirement, ADG is improved by 4.8% and G:F by 4.1%. In practice, Thr requirement might be even higher in case of challenging environmental conditions.
## Literature

JIANG, Z., W. DOZIER, and P. TILLMAN, 2014: Digestible threonine to lysine ratio of male broilers from 21 to 35 days of age. Poultry Science Association, Corpus Christi, Texas.

MACK, S., D. BERCOVICI, G. de GROOTE, B. LECLERCQ, M. LIPPENS, M. PACK, J. B. SCHUTTE, and S. VAN CAUWENBERGHE, 1999: Ideal amino acid profile and dietary lysine specification for broiler chickens of 20 to 40 days of age. Br. Poult. Sci. 40(2):257-265.

MELOCHE, K., P. TILLMAN, and W. DOZIER, 2014: Growth performance of male broilers fed diets varying in digestible threonine from 1 to 14 days of age. Poultry Science Association, Corpus Christi, Texas.

ROSTAGNO, H., 2011: Brazilian Tables for poultry and swine: composition of feedstuffs and nutritional requirements. 3rd edition.

SAUVANT, D., J. M. PEREZ, and G. TRAN. 2004. Tables of composition and nutritional value of feed materials. Second revised and corrected edition ed. Wageningen Academic Publishers, INRA Editions and AFZ, Paris.

SIMONGIOVANNI, A., E. CORRENT, N. LE FLOC'H, and J. VAN MILGEN, 2012: Estimation of the tryptophan requirement in piglets by meta-analysis. Animal. 6(4):594-602.

STAR, L., M. ROVERS, E. CORRENT, and J. D. VAN DER KLIS, 2012: Threonine requirement of broiler chickens during subclinical intestinal Clostridium infection. Poult. Sci(91):643-652.

WECKE, C., and F. LIEBERT, 2013: Improving the reliability of optimal in-feed amino acid ratios based on individual amino acid efficiency data from nitrogen balance studies in growing chicken. Animals. 3:358-373.

WU, G., 2014: Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition. J. Anim. Sci. Biotech. 5(34):1-12.
P-214 (ID 25)

# IMPACT OF DIFFERENT DIETARY L-ARGININE CONCENTRATIONS ON GROWTH OF PUREBRED LAYING HENS OF FOUR GENOTYPES DIFFERING IN PHYLOGENY AND PERFORMANCE LEVEL

M.A. Lieboldt<fnref n="1" />, I. Halle<fnref n="1" />, J. Frahm<fnref n="1" />, L. Schrader<fnref n="2" />, S. Weigend<fnref n="3" />, R. Preisinger<fnref n="4" />, and S. Dänicke<fnref n="1" />

**Corresponding author:** marc.lieboldt@fli.bund.de

## Abstract

In order to examine the impact of graded dietary L-arginine supply on growth of four purebred layer lines, two high performing genotypes (WLA - White Leghorn; BLA - Rhode Island Red) from LTZ were contrasted to two low performing genotypes (R11 - White Leghorn; L68 - New Hampshire) from FLI. Three diets containing 70 %, 100 % and 200 % of recommended L-arginine (NRC, 1994) were fed *ad libitum* to each genotype (n = 32 chicks per genotype and diet) from hatch to the end of 41st week of age. Growth data were fitted to Gompertz function by non-linear regression. Estimated parameters were compared by mixed procedure and Tukey-Kramer test. Asymptotic body weight and age at maximum body weight gain (tmax) were significantly affected by genotype and diet, while no interaction occurred. Brown hens achieved higher asymptotic body weight than white hens. The diet supposed to supply 70 % of recommended Arg induced significantly lower asymptotic body weight and delayed tmax in all genotypes. Although asymptotic body weight did not differ between diets of 100 and 200 % arginine, 100 % arginine caused a lower tmax in genotypes. WLA even showed lowest tmax independent of diet, while the other genotypes did not differ from each other. In conclusion, insufficient dietary arginine caused growth restriction and retardation, but sufficient Arg induced fastest growth in all genotypes. Brown hens grew stronger than white hens, whereas white ones grew faster.

**Keywords:** amino acids, L-arginine, genotypes, Gompertz function, growth

## Introduction

Due to bird's uricotelism, L-arginine (Arg) is considered as essential amino acid in poultry. It plays a decisive role in physiological processes such as growth and feathering and serves as precursor of proteins, creatine, polyamines, L-proline, various hormones and nitric oxides. The magnitude of required Arg depends on a large variety of environmental and genetic factors (reviewed in: KHAJALI and WIDEMAN, 2010). The objective of the present study was to examine the impact of graded dietary L-arginine supply on growth of purebred layer lines differing in performance and phylogenetic origin in order to calculate genotype specific growth curves from hatch to the end of the 41st week of age.

## Material and Methods

Two high performing, commercial genotypes from LTZ (WLA and BLA) were contrasted to two low performing ones from FLI (R11 and L68). White layer lines (WLA and R11) were of White Leghorn origin and phylogenetically closely related, but distant from the Rhode Island Red line BLA and its counterpart L68 (New Hampshire). Three diets were calculated for chicks (hatch to week 7), pullets (week 8 to 16) and laying hens (week 17 to 41) each. Diets of each age-group comprised of a basal diet with no Arg supplementation (low Arg, **LA**), a low Arg (adequate Arg, **AA**) and a high Arg (high Arg, **HA**) supplemented diet equivalent to 70, 100 and 200 % Arg of recommended level (NRC, 1994). Each group (genotype x diet) comprised 32 one-day-old female chicks equipped with individual wing-tags housed in floor-range pens. After rearing pullets were moved to floor-range systems of a layer facility. During the entire trial diets and water were provided *ad libitum* and chickens' body weight was recorded as follows: from hatch to week 4 once a week, from week 6 to 16 every second week and from week 16 to 41 every 28 days. Based on recorded body weights daily weight gain was calculated. The growth function of GOMPERTZ (1825) was fitted regressively to time-dependent individually recorded growth data from hatch to the end of 41st week of age using procedure "nonlinear regression" of STA-

<footnote n="1">¹ Institute of Animal Nutrition, Federal Research Institute for Animal Health, Friedrich-Loeffler-Institut (FLI), Bundesallee 50, 38116 Braunschweig, Germany</footnote>

<footnote n="2">² Institute of Animal Welfare and Animal Husbandry, Federal Research Institute for Animal Health, FLI, Dörnbergstraße 25-27, 29223 Celle, Germany</footnote>

<footnote n="3">³ Institute of Farm Animal Genetics, Federal Research Institute for Animal Health, FLI, Höltystraße 10, Neustadt-Mariensee 31535, Germany</footnote>

<footnote n="4">⁴ Lohmann Tierzucht GmbH (LTZ), Am Seedeich 9-11, 27472 Cuxhaven, Germany</footnote>
TISTICA 12.0 (STATSOFT INC., 2014). Parameters of growth function were estimated using iterative Quasi-Newton method.

$$y(t) = a \cdot e^{-b \cdot e^{-c \cdot t}}$$

Where y(t) = hen's body weight (g) at time t, expressed as function of a; a = hen's adult body weight (g; asymptotic limit); b,c = regression coefficients of the function; and t = time (weeks). The age at maximum daily weight gain (tmax), equivalent to the point of inflection of the cumulative, sigmoid growth curve, was calculated by the second derivative of the cumulative growth function:

$$t_{\max} = \frac{\ln b}{c}$$

Maximum daily weight gain was computed by inserting group-specific calculated tmax in the derivative of the corresponding cumulative growth function.

Statistical analysis of calculated growth function parameters was carried out by means of a two factorial ANOVA with genotype, diet and their interaction as fixed effects. The Tukey-Kramer test was applied for multiple comparisons of means. Data were reported as least square mean values and pooled standard error of mean. Differences were considered to be statistically significant for P < 0.05. ANOVA of growth curve parameters were performed using procedure MIXED of the software package SAS 9.4 (SAS INSTITUTE INC., 2012).

## Results

Table 1 summarized results of nonlinear regression. Genotype and diet significantly affected calculated parameters of growth function, tmax and corresponding maximum daily weight gain. The asymptotic limit of growth curves, equal to estimated adult body weight, received higher values in brown hens than in white ones (p < 0.001) and HA induced higher adult body weight in brown hens than LA (p < 0.01). WLA fed with AA reached tmax firstly and BLA fed with LA grew slowest (p < 0.05). Independent of diet, tmax of WLA differed significantly from that of the other genotypes. Latter ones showed no statistically differences between each other. In all genotypes AA caused fastest growth and differed significantly from HA and LA.

The derivatives of growth curves, identical to daily weight gain course showed a significant increase until group-specific tmax with its corresponding estimated maximum daily weight gain (graphs not shown). In the following, daily weight gain strongly decreased and approached a nearly zero-gain asymptotically after 40th week of age. Brown genotypes achieved higher maximum daily weight gain than white ones (p < 0.001). Yet, LA induced lower maximum daily weight gain at tmax and led to flatter curves than supplemented Arg diets (p < 0.001).

## Conclusions:

The present study confirmed the essential role of Arg in growing poultry. Insufficient dietary Arg led to a significant growth reduction and retardation compared to an adequate or surplus Arg supply. Negative effects caused by insufficient dietary Arg, such as reduced feed intake and secondary growth depression, might be the result of adverse effects of dietary imbalances between Arg and lysine as well as Arg and methionine (reviewed in: KHAJALI and WIDEMAN, 2010). A lack of metabolisable Arg might cause a deficit of endogenous L-ornithine, the precursor of growth-promoting polyamines. Genotype specific interactions with Arg were only found in WLA and L68 that led to the assumption that genetically determined differences in Arg utilization and metabolism occur. HEGSTEDT et al., 1941 reported on different rapidity in feathering that induced a higher Arg requirement in fast feathering genotypes than in slow feathering ones. The fast and well growing of WLA with additional Arg indicated that WLA belonged to fast feathering breeds, while L68 has been selected for slow feathering (K 30%, k 70%) consciously and should require less Arg during feathering.
<table><caption>**Table 1.** Effect of genotype and Arg supply on results of time-dependent individual growth data fitted to Gompertz function (LSMeans, PSEM, n = 32 chicks/experimental groups)</caption><thead><tr><th rowspan="2"></th><th colspan="3">WLA</th><th colspan="3">BLA</th><th colspan="3">R11</th><th colspan="3">L68</th><th colspan="3">ANOVA (p values)</th></tr><tr><th>LA</th><th>AA</th><th>HA</th><th>LA</th><th>AA</th><th>HA</th><th>LA</th><th>AA</th><th>HA</th><th>LA</th><th>AA</th><th>HA</th><th>PSEM</th><th>GT</th><th>DIET</th><th>GTxDIET</th></tr></thead><tbody><tr><td>a (g/chick)</td><td>1503<sup>d</sup></td><td>1492<sup>d</sup></td><td>1537<sup>d</sup></td><td>1756<sup>c</sup></td><td>1770<sup>bc</sup></td><td>1819<sup>b</sup></td><td>1342<sup>e</sup></td><td>1422<sup>e</sup></td><td>1404<sup>e</sup></td><td>1887<sup>b</sup></td><td>1964<sup>ab</sup></td><td>1977<sup>a</sup></td><td>29</td><td>&lt; 0.001</td><td>&lt; 0.05</td><td>n.s.</td></tr><tr><td>b</td><td>3.67<sup>bc</sup></td><td>3.66<sup>bc</sup></td><td>3.61<sup>c</sup></td><td>3.85<sup>a</sup></td><td>3.58<sup>c</sup></td><td>3.57<sup>c</sup></td><td>3.74<sup>b</sup></td><td>3.65<sup>bc</sup></td><td>3.63<sup>bc</sup></td><td>3.78<sup>ab</sup></td><td>3.74<sup>b</sup></td><td>3.91<sup>a</sup></td><td>0.04</td><td>&lt; 0.001</td><td>&lt; 0.01</td><td>&lt; 0.001</td></tr><tr><td>c</td><td>0.140<sup>c</sup></td><td>0.157<sup>a</sup></td><td>0.147<sup>b</sup></td><td>0.133<sup>d</sup></td><td>0.136<sup>cd</sup></td><td>0.133<sup>d</sup></td><td>0.135<sup>cd</sup></td><td>0.142<sup>bc</sup></td><td>0.138<sup>c</sup></td><td>0.135</td><td>0.143<sup>bc</sup></td><td>0.142<sup>bc</sup></td><td>0.002</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>n.s.</td></tr><tr><td>t<sub>max</sub> (weeks)</td><td>9.36<sup>bc</sup></td><td>8.33<sup>d</sup></td><td>8.76<sup>cd</sup></td><td>10.22<sup>a</sup></td><td>9.44<sup>bc</sup></td><td>9.65<sup>b</sup></td><td>9.82<sup>ab</sup></td><td>9.17<sup>c</sup></td><td>9.35<sup>bc</sup></td><td>9.94<sup>ab</sup></td><td>9.30<sup>bc</sup></td><td>9.60<sup>bc</sup></td><td>0.16</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>n.s.</td></tr><tr><td>R<sup>2</sup></td><td>0.998</td><td>0.998</td><td>0.998</td><td>0.997</td><td>0.998</td><td>0.997</td><td>0.998</td><td>0.998</td><td>0.998</td><td>0.998</td><td>0.998</td><td>0.998</td><td></td><td></td><td></td><td></td></tr><tr><td>RSD</td><td>24</td><td>22</td><td>20</td><td>25</td><td>26</td><td>22</td><td>22</td><td>20</td><td>19</td><td>26</td><td>24</td><td>30</td><td></td><td></td><td></td><td></td></tr><tr><td>Estimated MDWG (g/chick/d)</td><td>11.1<sup>e</sup></td><td>12.3<sup>cd</sup></td><td>11.9<sup>d</sup></td><td>12.3<sup>cd</sup></td><td>12.6<sup>c</sup></td><td>12.7<sup>c</sup></td><td>9.5<sup>g</sup></td><td>10.6<sup>ef</sup></td><td>10.2<sup>f</sup></td><td>13.4<sup>b</sup></td><td>14.8<sup>a</sup></td><td>14.7<sup>a</sup></td><td>0.2</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>&lt; 0.01</td></tr><tr><td>Achieved MDWG (g/chick/d)</td><td>11.0<sup>e</sup></td><td>12.1<sup>d</sup></td><td>12.1<sup>d</sup></td><td>12.9<sup>c</sup></td><td>12.7<sup>cd</sup></td><td>13.2<sup>c</sup></td><td>9.5<sup>f</sup></td><td>11.0<sup>e</sup></td><td>10.0<sup>f</sup></td><td>14.6<sup>b</sup></td><td>15.5<sup>a</sup></td><td>15.0<sup>ab</sup></td><td>0.3</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>n.s.</td></tr></tbody></table>

## Literature

GOMPERTZ, B, 1825: On the nature of the function expressive of the law of human mortality, and on a new mode of determining the value of life contingencies. Philosophical Transactions of the Royal Society. **2**, 513-585.

HEGSTEDT, D.M., G.M. BRIGGS, C.A. ELVEHJEM and E.B. HART, 1941: The role of arginine and glycine in chick nutrition. Journal of Biological Chemistry, **140**, 191-200.

KHAJALI, F., R.F. WIDEMAN, 2010: Dietary arginine: metabolic, environmental, immunological and physiological inter-relationships. World's Poultry Science Journal. **66**, 751-766.

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

SAS INSTITUTE INC, 2012. SAS 9.4. Cary, NC, USA.

STATSOFT INC, 2014. Statistica for the Windows operating system, version 12.0. Tulsa, Oklahoma.

<footnote>ªg LSMeans values with PSEM in the same row with different superscripts are significantly different (p < 0.05)</footnote>

<footnote>R² = coefficient of determination; RSD = residual standard deviation; PSEM = pooled standard error of mean; MDWG = maximum daily weight gain; n.s. = not significant</footnote>
# INTERACTION OF GRADED DIETARY L-LYSINE CONCENTRATIONS AND GENOTYPE ON PERFORMANCE AND EGG QUALITY OF PUREBRED LAYING HENS DIFFERING IN PHYLOGENY AND PERFORMANCE LEVEL

M.-A. Lieboldt<fnref n="1" />, Ingrid Halle<fnref n="1" />, S. Weigend<fnref n="2" />, R. Preisinger<fnref n="3" /> and S. Dänicke<fnref n="1" />

**Corresponding author:** marc.lieboldt@fli.bund.de

## Abstract

In order to examine the impact of graded L-lysine supply on performance and egg quality of five purebred layer lines, two high performing genotypes (WLA - White Leghorn; BLA - Rhode Island Red) from LTZ were contrasted to three low performing genotypes (R11 and G11 - White Leghorn; L68 - New Hampshire) from FLI. Three diets containing 80 %, 100 % and 120 % of recommended L-lysine (NRC, 1994) were fed *ad libitum* to each genotype (n = 8 hens per genotype and diet). From the 22nd to 61st week of age body weight, daily feed intake and laying performance were monthly recorded and in the 30th, 45th and 60th week of age egg quality was examined. Analysis of variance was performed using mixed procedure and Tukey-Kramer test. Body weight, daily feed intake and laying performance were significantly affected by genotype, age and their interaction, but not by dietary lysine. Depending on their genetic background WLA and BLA showed significantly higher daily feed intake, laying performance and albumen proportion as well as significantly lower feed to egg mass ratio than low performing genotypes. Eggs of the latter ones had higher yolk proportion, yolk to albumen ratio and more intensive coloured yolks. In contrast to the absent influence of dietary lysine on performance, relative weights of egg components varied significantly due to dietary lysine. In conclusion, dietary lysine level failed to alter performance, but modified egg proportions in genotypes equally. The differences in performance related traits traced back to the determined performance potential of genotypes.

**Keywords:** amino acids, egg quality, genotypes, L-lysine, performance

## Introduction

L-lysine (Lys) is an essential amino acid in poultry and associated with protein synthesis. In order to achieve their genetically determined performance potential, chickens depend on sufficient dietary supply with amino acids such as Lys. As most dietary plant proteins show relatively low amounts of Lys, a dietary supplementation with free L-lysine is frequently necessary in poultry nutrition. To approach the hypothesis that high performing genotypes are less capable to respond adequately to environmental changes such as insufficient nutrient supply compared to low performing ones, we conducted a performance trial with five lines of purebred laying hens differing in performance and phylogenetic origin. In order to examine interacting effects of genotype and Lys on laying performance and egg quality, hens were fed graded levels of dietary Lys.

## Material and Methods

Two high performing, commercial genotypes from LTZ (WLA and BLA) were contrasted to three low performing ones from FLI (G11, R11 and L68). White layer lines (WLA, G11 and R11) were of White Leghorn origin and phylogenetically closely related, but distant from Rhode Island Red line BLA and its counterpart L68 (New Hampshire). To enable individual records of performance and feed intake, 24 hens of each genotype were kept in single cages of a three-floor cage-system randomly. Each cage was equipped with a feeding trough, nipple drinker and perch. Feed and water were provided *ad libitum*. Hens were equally distributed to diets containing 80 %, 100 % (7.2 g Lys/kg diet) and 120 % of recommended Lys (NRC, 1994). The trial lasted from the 22nd to the 61st week of age and was subdivided into ten 28-day laying periods.

Hens were weighed at the end of each 28-day period. Eggs were recorded daily and egg weight was monitored by collecting laid eggs of each hen on four consecutive days in a two-week interval. Feed not consumed was recorded weekly. Based on recorded feed intake and egg mass the feed to egg mass ratio was calculated. Additionally, in the

<footnote n="1">¹ Institute of Animal Nutrition, Federal Research Institute for Animal Health, Friedrich-Loeffler-Institut (FLI), Bundesallee 50, 38116 Braunschweig, Germany</footnote>

<footnote n="2">² Institute of Farm Animal Genetics, Federal Research Institute for Animal Health, FLI, Höltystraße 10, Neustadt-Mariensee 31535, Germany</footnote>

<footnote n="3">³ Lohmann Tierzucht GmbH (LTZ), Am Seedeich 9-11, 27472 Cuxhaven, Germany</footnote>
30th, 45th and 60th week of age eggs of each hen were collected on three consecutive days, weighed and egg yolk and albumen were separated. Weight of shell and yolk were recorded and that of albumen was determined by difference. Egg components are presented as relative weights of total egg weight and the yolk to albumen ratio was calculated. Yolk colour was estimated by using a Roche-fan (15 fans, F. Hoffmann-La Roche Ltd., Basel, Switzerland).

Statistical analysis of examined traits was carried out by means of a three factorial analysis of variance (ANOVA) with genotype, diet, age and their interactions as fixed effects using procedure MIXED of SAS 9.4 (SAS INSTITUTE INC., 2012). As traits were measured repeatedly on the same animal a "repeated" statement was considered in the statistical model to account for similarities within subjects. The Tukey-Kramer test was applied for a multiple comparison of means and data were reported as least square mean values and pooled standard error of the mean. Differences were considered to be statistically significant for p < 0.05.

## Results

Table 1 summarizes average body weight and laying performance of genotypes during the entire trial. All examined traits were affected by genotype, age and their interaction (p < 0.001), while dietary lysine level failed to alter body weight and laying performance.

<table><caption>**Table 1.** Effects of genotype and lysine on the average performance of ten 28-day periods (LSMeans, PSEM, n = 8).</caption><thead><tr><th>Genotype</th><th>L-Lysine<br>(%)</th><th>Body weight<br>at week 22 (g)</th><th>Body weight<br>at week 61 (g)</th><th>Daily feed<br>intake (g/hen/d)</th><th>Laying<br>intensity (%)</th><th>Egg weight<br>(g/egg)</th><th>Daily egg mass<br>(g/hen/d)</th><th>Feed to egg mass<br>ratio (kg/kg)</th></tr></thead><tbody><tr><td rowspan="3">WLA</td><td>80</td><td>1470<sup>b</sup></td><td>1448<sup>c</sup></td><td>96<sup>b</sup></td><td>92.0<sup>a</sup></td><td>56.1<sup>b</sup></td><td>51.5<sup>b</sup></td><td>1.87<sup>a</sup></td></tr><tr><td>100</td><td>1499<sup>b</sup></td><td>1485<sup>c</sup></td><td>99<sup>b</sup></td><td>90.9<sup>a</sup></td><td>56.0<sup>b</sup></td><td>50.7<sup>b</sup></td><td>1.96<sup>a</sup></td></tr><tr><td>120</td><td>1425<sup>b</sup></td><td>1518<sup>c</sup></td><td>96<sup>b</sup></td><td>94.9<sup>a</sup></td><td>55.7<sup>b</sup></td><td>52.8<sup>b</sup></td><td>1.82<sup>a</sup></td></tr><tr><td rowspan="3">BLA</td><td>80</td><td>1624<sup>a</sup></td><td>1846<sup>b</sup></td><td>109<sup>a</sup></td><td>89.9<sup>a</sup></td><td>61.9<sup>a</sup></td><td>55.6<sup>a</sup></td><td>1.96<sup>a</sup></td></tr><tr><td>100</td><td>1647<sup>a</sup></td><td>1856<sup>b</sup></td><td>111<sup>a</sup></td><td>95.5<sup>a</sup></td><td>61.2<sup>a</sup></td><td>58.4<sup>a</sup></td><td>1.91<sup>a</sup></td></tr><tr><td>120</td><td>1641<sup>a</sup></td><td>1815<sup>b</sup></td><td>109<sup>a</sup></td><td>93.0<sup>a</sup></td><td>61.9<sup>a</sup></td><td>57.7<sup>a</sup></td><td>1.89<sup>a</sup></td></tr><tr><td rowspan="3">R11</td><td>80</td><td>1239<sup>c</sup></td><td>1415<sup>d</sup></td><td>80<sup>c</sup></td><td>67.5<sup>b</sup></td><td>51.3<sup>c</sup></td><td>34.8<sup>c</sup></td><td>2.30<sup>b</sup></td></tr><tr><td>100</td><td>1232<sup>c</sup></td><td>1381<sup>d</sup></td><td>76<sup>c</sup></td><td>63.5<sup>b</sup></td><td>49.3<sup>c</sup></td><td>31.3<sup>c</sup></td><td>2.43<sup>b</sup></td></tr><tr><td>120</td><td>1205<sup>c</sup></td><td>1279<sup>d</sup></td><td>75<sup>c</sup></td><td>62.2<sup>b</sup></td><td>49.7<sup>c</sup></td><td>30.9<sup>c</sup></td><td>2.43<sup>b</sup></td></tr><tr><td rowspan="3">L68</td><td>80</td><td>1735<sup>a</sup></td><td>2014<sup>a</sup></td><td>94<sup>b</sup></td><td>63.0<sup>b</sup></td><td>54.4<sup>b</sup></td><td>34.3<sup>c</sup></td><td>2.74<sup>c</sup></td></tr><tr><td>100</td><td>1694<sup>a</sup></td><td>1915<sup>a</sup></td><td>95<sup>b</sup></td><td>62.6<sup>b</sup></td><td>55.2<sup>b</sup></td><td>34.6<sup>c</sup></td><td>2.75<sup>c</sup></td></tr><tr><td>120</td><td>1752<sup>a</sup></td><td>2151<sup>a</sup></td><td>96<sup>b</sup></td><td>65.1<sup>b</sup></td><td>53.2<sup>b</sup></td><td>34.6<sup>c</sup></td><td>2.77<sup>c</sup></td></tr><tr><td rowspan="3">G11</td><td>80</td><td>1224<sup>c</sup></td><td>1364<sup>d</sup></td><td>76<sup>c</sup></td><td>67.4<sup>b</sup></td><td>45.9<sup>d</sup></td><td>30.9<sup>c</sup></td><td>2.46<sup>b</sup></td></tr><tr><td>100</td><td>1172<sup>c</sup></td><td>1344<sup>d</sup></td><td>78<sup>c</sup></td><td>68.5<sup>b</sup></td><td>45.9<sup>d</sup></td><td>31.4<sup>c</sup></td><td>2.48<sup>b</sup></td></tr><tr><td>120</td><td>1191<sup>c</sup></td><td>1371<sup>d</sup></td><td>74<sup>c</sup></td><td>64.5<sup>b</sup></td><td>45.5<sup>d</sup></td><td>29.3<sup>c</sup></td><td>2.53<sup>b</sup></td></tr><tr><td>PSEM</td><td></td><td>60</td><td>60</td><td>3</td><td>2.7</td><td>1.0</td><td>1.4</td><td>0.38</td></tr></tbody></table>

a-d: LSMeans within columns with no common superscripts are significantly different (p < 0.05)

Table 2 summarizes the average results of egg quality examinations in 30th, 45th and 60th week of age. The proportions of shell, yolk and albumen as well as the calculated yolk to albumen ratio were influenced by genotype, diet and age, but yolk colour was only affected by genotype and age (p < 0.001).
<table><caption>Table 2. Effect of genotype and lysine on the average egg quality (LSMeans, PSEM)<fnref n="1" /></caption><thead><tr><th rowspan="2">Genotype</th><th rowspan="2">L-Lysine<br>(%)</th><th colspan="3">Proportion (%) of</th><th rowspan="2">Yolk to<br>albumen ratio</th><th rowspan="2">Yolk colour (Roche<br>fan scale)</th></tr><tr><th>Egg shell</th><th>Egg yolk</th><th>Egg albumen</th></tr></thead><tbody><tr><td rowspan="3">WLA</td><td>80</td><td>12.7<sup>a</sup></td><td>29.7<sup>d</sup></td><td>57.6<sup>b</sup></td><td>0.518<sup>d</sup></td><td>12.4<sup>c</sup></td></tr><tr><td>100</td><td>12.7<sup>a</sup></td><td>29.4<sup>d</sup></td><td>57.9<sup>b</sup></td><td>0.511<sup>d</sup></td><td>12.5<sup>bc</sup></td></tr><tr><td>120</td><td>12.3<sup>ab</sup></td><td>30.0<sup>d</sup></td><td>57.7<sup>b</sup></td><td>0.522<sup>d</sup></td><td>12.4<sup>c</sup></td></tr><tr><td rowspan="3">BLA</td><td>80</td><td>12.0<sup>b</sup></td><td>26.0<sup>e</sup></td><td>62.0<sup>a</sup></td><td>0.422<sup>e</sup></td><td>12.2<sup>c</sup></td></tr><tr><td>100</td><td>12.3<sup>ab</sup></td><td>25.4<sup>e</sup></td><td>62.3<sup>a</sup></td><td>0.409<sup>e</sup></td><td>12.4<sup>c</sup></td></tr><tr><td>120</td><td>11.8<sup>b</sup></td><td>25.8<sup>e</sup></td><td>62.4<sup>a</sup></td><td>0.414<sup>e</sup></td><td>12.2<sup>c</sup></td></tr><tr><td rowspan="3">R11</td><td>80</td><td>12.5<sup>a</sup></td><td>31.4<sup>c</sup></td><td>56.1<sup>cd</sup></td><td>0.562<sup>c</sup></td><td>12.8<sup>b</sup></td></tr><tr><td>100</td><td>13.2<sup>a</sup></td><td>30.0<sup>cd</sup></td><td>56.8<sup>bc</sup></td><td>0.432<sup>e</sup></td><td>12.5<sup>bc</sup></td></tr><tr><td>120</td><td>11.6<sup>bc</sup></td><td>31.3<sup>c</sup></td><td>57.1<sup>bc</sup></td><td>0.551<sup>c</sup></td><td>13.0<sup>b</sup></td></tr><tr><td rowspan="3">L68</td><td>80</td><td>11.2<sup>c</sup></td><td>33.1<sup>ab</sup></td><td>55.7<sup>d</sup></td><td>0.598<sup>b</sup></td><td>13.7<sup>a</sup></td></tr><tr><td>100</td><td>11.3<sup>c</sup></td><td>32.2<sup>bc</sup></td><td>56.5<sup>bc</sup></td><td>0.573<sup>c</sup></td><td>13.6<sup>a</sup></td></tr><tr><td>120</td><td>11.6<sup>bc</sup></td><td>33.3<sup>ab</sup></td><td>55.1<sup>d</sup></td><td>0.609<sup>b</sup></td><td>13.7<sup>a</sup></td></tr><tr><td rowspan="3">G11</td><td>80</td><td>12.0<sup>b</sup></td><td>34.4<sup>a</sup></td><td>53.6<sup>e</sup></td><td>0.647<sup>a</sup></td><td>12.3<sup>c</sup></td></tr><tr><td>100</td><td>11.6<sup>bc</sup></td><td>33.5<sup>ab</sup></td><td>54.9<sup>de</sup></td><td>0.613<sup>b</sup></td><td>12.6<sup>bc</sup></td></tr><tr><td>120</td><td>11.9<sup>b</sup></td><td>33.8<sup>a</sup></td><td>54.9<sup>de</sup></td><td>0.627<sup>ab</sup></td><td>12.4<sup>c</sup></td></tr><tr><td>PSEM</td><td></td><td>0.1</td><td>0.3</td><td>0.3</td><td>0.008</td><td>0.1</td></tr></tbody></table>

## Conclusions

Due to their genetically determined performance potential, high performing layer lines achieved a significantly higher performance than their low performing counterparts. In the present experiment graded levels of dietary Lys failed to alter body weight, feed intake and laying performance independent of genetic background. Therefore, it can be assumed that the requirement for Lys is equal to or less than the lowest level used in this study, which was 80 % recommended Lys (NRC, 1994). The oversupply of dietary Lys (120 % Lys) showed neither enhancing nor depressing effects on hens' performance. This result indicates that high and low performing hens tolerated surplus Lys equally. However, lysine supply caused shifts in yolk to albumen ratio, while total egg weight remained constant. As amino acid pattern of egg proteins is genetically determined, they are hardly to influence by diet (LEESON, 1993; HUSSEIN and HARMS, 1994). That leads to the assumption that laying hens compensate dietary limitation and excess of Lys with a shift of egg proportions. In order to find an appropriate nutritional stressor for our animal model, further research with other amino acids or greater dietary imbalances seems to be necessary.

## Literature

HUSSEIN, S.M., R.H. HARMS, 1994: Effect of amino acid deficiencies on yolk:albumen ratio in hen eggs. J. Appl. Poultry Res. 3, 362-366.

LEESON, S., 1993: Potential of modifying poultry products. J. Appl. Poultry Res. 2, 380-385.

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

SAS INSTITUTE INC, 2012. SAS 9.4. Cary, NC, USA.

<footnote n="1">¹ eggs collected on three consecutive days of n = 8 hens per group in 30th, 45th and 60th week each a-e: LSMeans within columns with no common superscripts are significantly different (p < 0.05)</footnote>
P-216 (ID 290)

# EFFECT OF DIGESTIBLE ARGININE TO DIGESTIBLE LYSINE (DARG:DLYS) RATIOS ON PERFORMANCE OF MALE ROSS 308 BROILERS

L. Linares<fnref n="1" />, H.Willemsen<fnref n="1" />, S. Cerrate<fnref n="2" />, M.Silva<fnref n="2" />, J.McAdam<fnref n="1" />

**Corresponding author:** llinares@aviagen.com

## Abstract

Arginine (Arg) plays an important role on protein deposition in broilers. A review of the ratio of digestible Arg to digestible Lysine (dArg:dLys) is needed considering the genetic improvement on feed conversion rate (FCR). Ross 308 male broilers were fed crumbled starter (S; 0-10d), pelleted grower (G; 11-24d) and pelleted finisher (F; 25-46d) diets. Three dArg:dLys ratios were provided to birds by phase, (94, 107, 120), in a 3x3x3 factorial design (5 reps/trt - 20 birds/pen). Diets were formulated to meet Aviagen 2007 recommendations, except for dArg. At 10d, birds fed 94 dArg:dLys diets were lighter, and had lower feed intake and high FCR than those fed higher dArg:dLys (p<0.001). At 25d, no interaction between the S and G phase was observed. During this phase, birds fed both the 107 and 120 ratios of dArg:dLys had the highest liveweight (LWT) and the lowest FCR (p<0.001). At 46d, no interaction between feeding phases was observed. During this phase, birds fed 107 and 120 ratios of dArg:dLys resulted in the heaviest birds with the lowest FCR (p<0.001). However, the 120 dArg:dLys trt was comparable to the 94 dArg:dLys trt on LWT. Regression analysis of cumulative intake of dArg showed positive correlation (r<fnref n="2" /> =0.83 for FCR and 0.58 for LWT) suggesting that a dArg:dLys feeding strategy of 120, 107 and 120 during the S, G and F phases, respectively, was optimal for FCR. At the same time, maintaining a ratio of 107 across all feeding phases was optimal for growth, in line with the factorial data.

**Keywords:** broilers, nutrition, amino acid, digestible arginine, ratio

## Introduction

Arginine (Arg) is an essential amino acids for broilers, playing an important role in protein deposition rate, and key metabolic pathways that are necessary for maintenance, growth, reproduction, and immune status. Calculations from the more recent optimum amino acid balances studies suggested the optimum ratio of digestible Arg to digestible Lysine (dArg:dLys) to be in the range from 90 to 118% (Balanave and Brake, 2002). The following study aimed to review the ratio of dArg:dLys in light of the constant genetic improvement in growth rate and feed conversion rate (FCR).

## Material and Methods

A total of 2700 male Ross 308 broilers were studied over starter (0-10 days), grower (11-24 days) and finisher (25-46d days) feeding phases. Three different dArg:dLys levels were studied per phase (94, 107 and 120 dArg:dLys) in a 3 x 3 x 3 factorial design to give a total of 27 treatments, with 5 reps per treatment. Diets were wheat and soybean meal based, crumble form at starter, and pellet in grower and finisher phases. All diets were formulated according to 2007 Aviagen Nutrient Recommendations, except dArg.

## Results and Discussion

At day 10, birds fed 94 dArg:dLys diets were lighter and had lower feed intake with increased FCR compared to those fed higher dArg:dLys (p<0.001). The low feed intake of birds fed 94 dArg:dLys during first 10 days negatively affected their performance throughout the trial. The lack of difference in body weight (BW) and FCR between birds receiving 107 or 120 dArg suggests that despite receiving less Arg, birds on 107 dArg:dLys trt were not compromised. Therefore in the first 10 days the optimum for dArg is likely to be between 107 - 120.

At 25 days, no interaction between the starter and grower phase was observed for bird performance parameters. The heaviest birds were those that had been fed to one of the higher ratios of dArg:dLys in the starter (p=0.02) and grower phase (p<0.001). In terms of FCR, exposure to the highest ratio in the starter resulted in birds with lower FCR at 25d compared to birds fed to a ratio of 97 (p=0.03). Considering the grower phase independently, the most feed efficient birds were those fed both the 107 and 120 ratios of dArg:dLys (p<0.001). The improved performance of birds from the

<footnote n="1">¹ Aviagen Ltd, Newbridge, Scotland, UK</footnote>

<footnote n="2">² Aviagen Inc, Huntsville, Alabama, USA</footnote>
107 and 120 treatment ratios was due to an increased Arg intake, suggesting a ratio of 107 is optimal for growth and feed efficiency.

At 32 and 46 days, no interactions between feeding phases were observed for FCR or BW. At 32 days, the heaviest (p=0.01) and most feed efficient (p<0.01) birds were those that had been fed a ratio of 107 dArg:dLys over the starter and grower feeding phases, comparable to birds fed higher ratios of dArg:dLys. At 46 days, the influence of the starter period on BW and FCR had diminished, with only a tendency (p=0.09) for improved BW in birds exposed to the two higher ratios of dArg:dLys in starter phase, suggesting that for grow outs over 32 days, dArg does not have to be at a ratio of 107 to dLys in the starter feed to maximise growth. However, the two higher ratios in the grower phase did positively influence FCR (p<0.001) and ratios of 107 dArg:dLys in the grower phase yielded heavier birds (p=0.049) at 46 days than birds from the lowest treatment. Considering the influence of dArg:dLys in the finisher independent of the preceding phases, the 107 and 120 dArg:dLys treatments resulted in the heaviest birds with the lowest FCR (p<0.001), but the 120 dArg:dLys treatment was comparable to the 94 dArg:dLys treatment in terms of BW. Therefore the performance results at 46 days suggest, in line with the previous phases, that a ratio of 107 dArg:dLys is optimal.

Results of the regressions (Figures 1 and 2) of accumulative dArg intake on FCR and BW at 46 days significantly showed a strong positive correlation (r² =0.83 and 0.58 FCR and BW, respectively). The models run suggested that a dArg:dLys strategy of 120, 107 and 120 starter, grower, and finisher, respectively, was optimal for FCR while maintaining a ratio of 107 across feeding phases was optimal for growth, in line with the factorial data.

Since the interaction effects between phase and levels were not significant for the most processing carcass variables at 46 days, independent effects of arginine ratios in each phase are presented in the Table 1. The highest two ratios of dArg:dLys in the grower and finisher rations resulted in an increase in % eviscerated carcass, and this increase was linear in the finisher phase (p<0.001). Abdominal fat was lower in birds exposed to either the 107 or 120 treatments in the finisher period (p=0.012). However birds exposed to a ratio of 94 or 120 dArg:dLys in the starter had similar abdominal fat % (p<0.029). Birds fed the highest ratio in the finisher period displayed a significant increase in % breast meat, with the 107 dArg:dLys treatment yielding birds with a lower % breast but higher than those birds fed the 94 dArg:dLys treatment (p<0.001). The impact of the grower phase on breast was less dramatic, with both of the highest ratio treatments resulting in an increase in breast yield (p<0.001). The starter phase had no influence on breast meat yield at 46 days. Breast yield % was also measured at 32 days and the results were in line with the 46 day processed birds.

<table><caption>**Table 1.** Carcass traits at 46 d</caption><thead><tr><th rowspan="2"></th><th colspan="4">Eviscerated Carcass %</th><th colspan="4">Abdominal Fat %</th><th colspan="4">Breast Yield %</th></tr><tr><th>94</th><th>107</th><th>120</th><th>p</th><th>94</th><th>107</th><th>120</th><th>p</th><th>94</th><th>107</th><th>120</th><th>p</th></tr></thead><tbody><tr><th>S</th><td>73.2</td><td>73.97</td><td>73.95</td><td>0.088</td><td>1.58<sup>b</sup></td><td>1.49<sup>a</sup></td><td>1.55<sup>ab</sup></td><td>0.029</td><td>21.76</td><td>21.8</td><td>21.85</td><td>-</td></tr><tr><th>G</th><td>73.52<sup>b</sup></td><td>74.10<sup>a</sup></td><td>74.02<sup>a</sup></td><td>&lt;0.001</td><td>1.59</td><td>1.51</td><td>1.53</td><td>-</td><td>21.38<sup>b</sup></td><td>22.1<sup>a</sup></td><td>21.97<sup>a</sup></td><td>&lt;0.001</td></tr><tr><th>F</th><td>73.43<sup>c</sup></td><td>73.97<sup>b</sup></td><td>74.24<sup>a</sup></td><td>&lt;0.001</td><td>1.59<sup>b</sup></td><td>1.45<sup>a</sup></td><td>1.49<sup>a</sup></td><td>0.012</td><td>21.31<sup>c</sup></td><td>21.87<sup>b</sup></td><td>22.23<sup>a</sup></td><td>&lt;0.001</td></tr></tbody></table>

Results of the regression suggest that the ideal strategies for breast yield are ratios of 94, 120 and 107 for starter-grower-finisher, respectively; the lower ratio treatment in the starter is in line with the lack of effect of ratio observed from the Anova results. However the grower and finisher ratio treatments are slightly conflicting with the factorial results.

## Conclusions

Based on accumulative dArg intake a dArg:dLys strategy of 120, 107 and 120 in the starter, grower, and finisher, respectively, is optimal for FCR while maintaining a ratio of 107 across feeding phases was optimal for growth.

A dArg:dLys ratio of 107 or 120 in the grower and finisher rations increased the % of eviscerated carcass and reduced the % of abdominal fat at 46d days.

A dArg:dLys ratio of 120 in the finisher increased the % of breast meat.

To maximize performance traits, a minimum ratio of 107 dArg:dLys should be applied, in line with 2014 Aviagen Nutrient Recommendations
<figure><img src="image_73.png" /><figcaption>**Figure 1:** Response of 46 day BW to accumulative intake of digestible Arginine.</figcaption></figure>

<figure><img src="image_74.png" /><figcaption>**Figure 2:** Response of 46d FCR to accumulative intake of digestible Arginine.</figcaption></figure>

## Literature

AVIAGEN, 2007: Ross 308 Broiler Nutrition Specification, June 2007.

AVIAGEN, 2014: Ross 308 Broiler Nutrition Specification, 2014.

BALNAVE, D., J. BRAKE, 2002: Re-evaluation of the classical dietary arginine:lysine interaction for modern poultry diets: a review. World's Poult. Sci. J. **58**, 275-289.
# THE USE OF HERBAL METHIONINE AS AN ALTERNATIVE TO DL-METHIONINE IN BROILER CHICKENS

A. Moradi, S. Moradi, and Z. Zanganeh

Department of Animal Science, Faculty of Agriculture and Natural Resources, Razi University, Kermanshah, Iran

Corresponding Author: s.moradi@razi.ac.ir

## Abstract:

An experiment was conducted to evaluate the effects of graded DL-methionine replacement by herbal methionine on performance and carcass characteristics in broiler chickens. A total of 280 day-old male Ross 308 broiler chickens were used in 7 treatments, each treatment has 4 replicates, and 10 birds per replicate. Experimental treatments were as follows: Basal diet, in which total methionine requirement was supplied from DL-methionine (control), and in treatments 2 to 7, DL-methionine in basal diet was replaced by 20, 40, 50, 60, 80 and 100% of Herbal methionine (Meta-herb), respectively. Results indicated that BW in birds fed diets in which 20, 40 and 50% of DL-methionine were replaced by herbal methionine did not significantly differ than control group. No significant treatment impact were observed in feed intake, carcass percent and relative weights of breast, legs, liver, heart, thymus and bursa. In conclusion, it could be feasible to use herbal methionine in broiler diets as a replacement to supply part of the methionine requirement without affecting performance or carcass traits, while reducing production cost.

**Keywords:** Herbal methionine, Synthetic methionine, Broiler, Performance

## Introduction

Methionine is an essential amino acid for healthy and productivity of poultry. It is considered to be the first limiting amino acid in corn soybean based diets, followed by lysine and arginine. Synthetic methionine sources commonly used in the poultry industry are DL-methionine and methionine hydroxy analogue (MHA-FA). Today, the greatest resource that used is DL-methionine, that produced by chemical synthesis from acrolein, methyl mercaptan, and hydrogen cyanide. The increase in demand for meat has given rise to the use of these synthetic compounds in feed. Therefore, the prices of this amino acid changes with changes in the prices of the precursors. Recently, commercial sources of herbal methionine in some countries, including Sudan, India and recently in Iran be used, which is an additives plant and mixture of several plants rich in methionine, containing herbal ingredients that mimic the activity of methionine. Some researchers reported that broiler chickens fed on herbal methionine had similar performance in terms of body weight gain and feed conversion efficiency in comparison with birds that received DL-methionine (KALBANDE et al. 2009; HALDER and ROY. 2007). In contrast, some other researchers found that herbal methionine is not effective substitute for DL-methionine (YUAN et al. 2012; ITOE et al. 2010). The aim of this experiment was to evaluate the effects of graded DL-methionine replacement by herbal methionine on performance and carcass characteristics in broiler chickens.

## Materials and methods

The research protocol was approved by the Razi University animal care and use committee. A total of 280 one-day old male Ross 308 broiler chickens randomly assigned to 7 dietary treatments with 4 replicates per treatment (10 birds per replicate). The Experimental treatments were as follows: basal diet, in which 100% methionine requirement was supplied from DL-methionine (control), and DL-methionine in basal diet was replaced by 20 (HM 20), 40 (HM 40), 50 (HM 50), 60 (HM 60), 80 (HM 80) and 100% (HM 100) Herbal methionine (Metaherb), respectively. Herbal methionine (Metaherb) was supplied from Arosol Indian company. All diets were the same, only difference was the methionine source (Synthetic or herbal). Feed intake (FI), feed conversion ratio (FCR) body weight (BW) were weekly measured. Mortality was determined on a daily basis. At 42 days of age, one bird from each pen was randomly selected and slaughtered by cervical dislocation, and live BW, carcass percentage, abdominal fat pad, liver, breast, legs, heart, bursa, thymus and lien weight were determined. All data were analyzed with SAS software (version 9.2), with GLM procedure. Output data are given as means ± SD. Duncan's multiple range test was used to compare means.
## Results

The effects of experimental treatments on performance of broiler chickens are shown in Table 1. Results indicated that at 21 and 35 days of age diets supplemented with herbal methionine until 60% replacement, did not significantly affect BW, whereas 80 and 100% replacement significantly reduced BW in compare to control group (P≤0.05). At 42 days of age, until 50% replacement synthetic methionine with herbal methionine showed same BW with control group, while levels of 60, 80, 100% herbal methionine significantly decreased BW. Experimental treatments had significant effect on BWG of broiler throughout the experiment. Feeding diets supplemented until 60% replacement herbal methionine did not influence BWG until 21 days of age, but in treatments 80 and 100% replacement, it was significantly decreased than control group (P≤0.05). Whereas data of BWG during 22-42 days of ages indicated that there was no significant effect, until 40% replacement, but other treatments significantly reduced BWG (P≤0.05). Overall, Statistical analysis of body weight gain during 8-42 days of ages showed that no negative effects were observed from feeding herbal methionine supplemented diets up to 40% replacement on BWG, whereas replacement level higher than 40%, reduced BWG. Experimental treatments did not affect FI throughout the trial. During 8-21 days, treatments including 20 and 60% herbal methionine did not change FCR, whereas other treatments (40, 50, 80 and 100% replacement), significantly increased FCR (p≤0.05). During 22-42 and whole experimental period (8-42 days), FCR in all replacement levels were increased, except for the 20% replacement level that did not change FCR. Effect of herbal methionine supplementation on carcass characteristics at 42 day of age are shown in Table 2. No differences observed in carcass percent and relative weights of breast, legs, liver, heart, thymus and bursa in the control and experimental groups.

## Conclusions

Results showed that herbal methionine replacement with DL-methionine had no significant effect in terms of BW, BWG and FCR until levels of 50, 40 and 20%, respectively. But levels of 80 and 100% herbal methionine replacement significantly reduced the performance of broiler chickens. The results of the present study indicated that it could be feasible to use herbal methionine in broiler diets as a replacement to supply part of the methionine requirement without affecting performance or carcass traits, while reducing production cost.

<table><caption>Table 1. Effect of herbal methionine supplementation on growth performance in broiler chickens</caption><thead><tr><th>Treats</th><th></th><th>Control</th><th>20 HM</th><th>40 HM</th><th>50 HM</th><th>60 HM</th><th>80 HM</th><th>100 HM</th><th>P-value</th></tr></thead><tbody><tr><td rowspan="3">BW(g)</td><td>21 day</td><td>901.1±41.1<sup>a</sup></td><td>906.3±33<sup>a</sup></td><td>914.3±25.9<sup>a</sup></td><td>906.8±31.3<sup>a</sup></td><td>887.6±5.4<sup>a</sup></td><td>835.9±70.2<sup>b</sup></td><td>808.2±15<sup>b</sup></td><td>0.005</td></tr><tr><td>35 day</td><td>2189±56.2<sup>a</sup></td><td>2186.6±61.4<sup>a</sup></td><td>2139±75.8<sup>ab</sup></td><td>2154.7±39.3<sup>ab</sup></td><td>2114.3±96.8<sup>ab</sup></td><td>2010.5±139.9<sup>bc</sup></td><td>1944.6±105.8<sup>c</sup></td><td>0.02</td></tr><tr><td>42 day</td><td>2767.7±127.5<sup>a</sup></td><td>2732±92.4<sup>a</sup></td><td>2699±49.8<sup>a</sup></td><td>2665.4±69.4<sup>ab</sup></td><td>2573.3±54.5<sup>bc</sup></td><td>2577.9±13.6<sup>bc</sup></td><td>2472±116.8<sup>c</sup></td><td>0.001</td></tr><tr><td rowspan="3">BWG(g)</td><td>8 - 21</td><td>732.4±35.4<sup>a</sup></td><td>730.1±29.3<sup>a</sup></td><td>739.3±22.6<sup>a</sup></td><td>732.5±21.8<sup>a</sup></td><td>708.1±46.7<sup>ab</sup></td><td>677.3±76.1<sup>bc</sup></td><td>630.8±15.8<sup>c</sup></td><td>0.005</td></tr><tr><td>22 - 42</td><td>1852.9±119.9<sup>a</sup></td><td>1821±110.4<sup>ab</sup></td><td>1758.7±42.9<sup>abc</sup></td><td>1732.2±30.1<sup>bcd</sup></td><td>1680.7±22.4<sup>cd</sup></td><td>1762.3±62.7<sup>abc</sup></td><td>1638±94.6<sup>d</sup></td><td>0.001</td></tr><tr><td>8 - 42</td><td>2585.3±111.8<sup>a</sup></td><td>2551.1±103.7<sup>ab</sup></td><td>2498±35.1<sup>abc</sup></td><td>2455.7±34.2<sup>bc</sup></td><td>2388.8±46.9<sup>c</sup></td><td>2429.6±63.1<sup>bc</sup></td><td>2268.8±95<sup>d</sup></td><td>0.001</td></tr><tr><td rowspan="3">Fl(g)</td><td>8 - 21</td><td>1050.1±40.7<sup>ab</sup></td><td>1075±34.1<sup>b</sup></td><td>1096±34.8<sup>a</sup></td><td>1081.7±35.5<sup>ab</sup></td><td>1046.5±49.9<sup>ab</sup></td><td>1029.6±79.2<sup>b</sup></td><td>1025.5±27.4<sup>b</sup></td><td>0.02</td></tr><tr><td>22 - 42</td><td>3481.2±95.2<sup>ab</sup></td><td>3504.7±29.7<sup>ab</sup></td><td>3599.1±118.6<sup>a</sup></td><td>3503.8±39.9<sup>ab</sup></td><td>3512.2±71.8<sup>ab</sup></td><td>3530.2±119.5<sup>ab</sup></td><td>3405.1±76.6<sup>b</sup></td><td>0.001</td></tr><tr><td>8 - 42</td><td>4531.4±87.7<sup>bcd</sup></td><td>4579.7±45.5<sup>abc</sup></td><td>4695.2±140.8<sup>a</sup></td><td>4585.6±53.3<sup>ab</sup></td><td>4558.7±76.6<sup>abcd</sup></td><td>4438±87.7<sup>cd</sup></td><td>4430.6±102.9<sup>d</sup></td><td>0.001</td></tr><tr><td rowspan="3">FCR</td><td>8 - 21</td><td>1.42±0.05<sup>d</sup></td><td>1.47±0.05<sup>cd</sup></td><td>1.5<sup>bc</sup></td><td>1.5<sup>bc</sup></td><td>1.47±0.05<sup>cd</sup></td><td>1.55±0.05<sup>ab</sup></td><td>1.6<sup>a</sup></td><td>0.0004</td></tr><tr><td>22 - 42</td><td>1.87±0.09<sup>c</sup></td><td>1.9±0.14<sup>c</sup></td><td>2.05±0.1<sup>a</sup></td><td>2<sup>ab</sup></td><td>2.1<sup>a</sup></td><td>2±0.11<sup>ab</sup></td><td>2.07±0.09<sup>a</sup></td><td>0.0007</td></tr><tr><td>8 - 42</td><td>1.75±0.05<sup>c</sup></td><td>1.8±0.08<sup>bc</sup></td><td>1.9±0.08<sup>ab</sup></td><td>1.9<sup>ab</sup></td><td>1.9<sup>ab</sup></td><td>1.87±0.05<sup>ab</sup></td><td>1.97±0.05<sup>a</sup></td><td>0.001</td></tr></tbody></table>

All values represent the mean±SD of 4 replicates

Means within columns with different superscripts were significantly different (P≤0.05)
<table><caption>**Table 2.** Effect of herbal methionine supplementation on carcass characteristics (relative weight) in broiler chickens.</caption><thead><tr><th>Treats</th><th>Carcass, %</th><th>Breast, %</th><th>Legs, %</th><th>Liver, %</th><th>Heart, %</th><th>Thymus, %</th><th>Bursa, %</th></tr></thead><tbody><tr><td>Control</td><td>63.5±2.02</td><td>22.2±2.9</td><td>20.1±1.3</td><td>1.9±0.14</td><td>0.52±0.08</td><td>0.34±0.1</td><td>0.25±0.03</td></tr><tr><td>20 HM</td><td>63.06±2.9</td><td>21.2±2.6</td><td>20.9±0.82</td><td>2.1±0.29</td><td>0.51±0.08</td><td>0.52±0.3</td><td>0.22±0.08</td></tr><tr><td>40 HM</td><td>63.2±1.9</td><td>23.1±0.72</td><td>20.5±1.2</td><td>2.18±0.34</td><td>0.5±0.03</td><td>0.41±0.2</td><td>0.29±0.03</td></tr><tr><td>50 HM</td><td>63.26±1.4</td><td>22.6±2.9</td><td>21±0.46</td><td>1.9±0.2</td><td>0.5±0.04</td><td>0.51±0.2</td><td>0.27±0.11</td></tr><tr><td>60 HM</td><td>63.2±3.3</td><td>23.8±0.76</td><td>19.5±0.78</td><td>2.02±0.08</td><td>0.47±0.02</td><td>0.46±0.07</td><td>0.27±0.09</td></tr><tr><td>80 HM</td><td>63.06±2.9</td><td>21.6±1.1</td><td>20.4±1.1</td><td>2.09±0.2</td><td>0.53±0.02</td><td>0.56±0.2</td><td>0.180±0.1</td></tr><tr><td>100 HM</td><td>63.06±2.9</td><td>21.3±1.9</td><td>21.2±1.5</td><td>2.1±0.17</td><td>0.52±0.07</td><td>0.62±0.2</td><td>0.28±0.04</td></tr><tr><td>P-value</td><td>0.99</td><td>0.2</td><td>0.56</td><td>0.02</td><td>0.3</td><td>0.74</td><td>0.82</td></tr></tbody></table>

All values represent the mean±SD of 4 replicates

Means within columns with different superscripts were significantly different (P≤0.05)

## Literature

HALDER, G & B. ROY, 2007: Effect of herbal or synthetic methionine on performance cost benefit ratio, meat and feather quality of broiler chicken. Int. J. Poult. Sci. **12**, 987-996.

ITOE, S., II, DAFWANG & GS, Bawa, 2010: Evaluation of Methiorep as a Substitute for Methionine in broiler diets. Int. J. Poult. Sci. **9**:809-812.

KALBANDE, V.H., K. S. RAVIKANTH, MAINI, & D.S, REKHE, 2009: Methionine supplementation options in poultry. Int. J. Poult. Sci. **8**: 588-591.

YUAN, J., A.J. KARIMI., S.D. GOODGAME., C. LU, F.J. MUSSININ, & P.W. WALDROU, 2012: Evaluation of Herbal Methionine Source in broiler diets. Int. J. Poult. Sci. **11**(4): 247-250.
P-223 (ID 63)

VARIATIONS IN SOYBEAN MEAL AND EFFICIENCY OF PROTEIN HYDROLYSIS BY PANCREATIC PROTEASE AND MICROBIAL PROTEASE

K. Pontoppidan<fnref n="1" />, G. G. Mateos<fnref n="2" />, R. Angel<fnref n="3" /> and A. Smith<fnref n="4" />

**Corresponding author:** kpon@novozymes.com

**Abstract**

Variation in trypsin inhibitor activity (TIA) and protein solubility within commercial soybean meals (SBM) is believed to affect animal performance. The objective of this research was to investigate the proteolytic effect of purified pancreatic trypsin/chymotrypsin and a purified protease from *Nocardiopsis prasina* on 9 commercial SBM samples varying in chemical composition, TIA and protein solubility characteristics. SBM was incubated with or without protease (pH 7, 3 hours, 40°C) and the supernatants were analyzed by SDS-page and for level of free soluble amino ends using the o-phthalal-dialdehyde method. SDS-page analysis showed differences in the amount and composition of the soluble protein fraction of the SBM. The proteolytic efficiency of the lower dosages of pancreatic protease varied extensively between SBM batches whereas at the highest dose the efficiency was comparable for all SBM. The difference could not be explained by differences in protein solubility and TIA values as isolated variables, but the data strongly suggest that the hydrolysis of soya protein by pancreatic proteases depends on the commercial batch of SBM used. Incubation with *N. prasina* protease showed similar response for the different SBM, the main difference being a somewhat lower effect at high protease dose for one of the SBM batches, which might be in part explained by its distinct lower protein solubility.

**Keywords:** protease, soybean meal, trypsin inhibitor

**Introduction**

Soybean meal (SBM) is used in diets for non-ruminants because of its high protein content, superior amino acid profile, availability and cost. The crude protein fraction of SBM consists roughly of 80% storage proteins, 5% anti nutritional proteins including trypsin inhibitors (TI) and 15% of other proteins. Although it has been demonstrated, that commercial SBM varies significantly in chemical composition and nutritive value (de Coca-Sinova et al., 2008), due to the lack of simple reliable analytics the feed compound industry often accepts that the digestible amino acid content per unit of protein is constant irrespective of variables such as origin, genotype, processing, and storage conditions. TI inhibits the activity of the pancreatic digestive enzyme trypsin and high levels of TI activity in SBM have been associated with impaired animal performance (e.g. Perilla et al., 1997). The commercial availability of microbial proteases for inclusion in diets for non-ruminants offers an opportunity to complement the animals own digestive proteases. If these commercial proteases are not affected by TI it might be of particular interest to use such proteases in diets with moderately elevated TI levels to ensure sufficient amount of active protease in the intestinal tract to digest dietary protein.

**Material and Methods**

The 9 SBMs were obtained at the port at the arrival of the vessels by qualified personnel. All analysis was conducted at the University of Madrid. TI activity was assayed according to Hammerstrand et al. (1981). Two different enzyme preparations were used for the experiment: A purified preparation of pancreatic trypsin and chymotrypsin with approximately 90% trypsin and 10% chymotrypsin on a protein:protein basis and a purified preparation of an S1 protease from *Nocardiopsis prasina* (both Novozymes A/S, Denmark).

Each SBM was incubated (0.06 g DM/mL) in a pH 7 buffer (100mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CAPS, 12.5 mM CaCl₂·2H₂O, 150 mM KCl, 0.01% Triton X-100) either without or with the addition of protease diluted in an acetate buffer at pH 6 (100 mM Acetate, 5 mM CaCl₂, 0.01% BSA, 0.01% Tween 20). Samples were incubated with magnetic stirring (350 rpm) for 3 hours at 40°C. After incubation a stop reagent (220 mM TCA, 440 mM NaAc·3H₂O, 570 mM glacial acetic acid) was added at a 2:1 volume and samples were centrifuged (10 min, 4,000 rpm, 5°C). Supernatants were recovered and stored on ice bath until analysis. Protein composition of the non-protease

<footnote n="1">¹ Feed Applications, Novozymes A/S, Bagsvaerd, Denmark</footnote>

<footnote n="2">² Animal Science Department, Universidad Politécnica de Madrid, 28040 Madrid, Spain</footnote>

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

<footnote n="4">⁴ DSM Nutritional Products, Heanor, Derbyshire, UK</footnote>
treated supernatants was analyzed by SDS-page. Supernatants were diluted 5 times, mixed 1:1 with SDS-page loading buffer and incubated 5 minutes at 95°C and then cooled on ice bath before SDS-page analysis according to standard procedure. Protein bands were visualized by staining with Coomassie Blue. The effect of protease on release of soluble free amino groups was determined using a colorimetric assay based on the o-phthal-dialdehyde (OPA) method according to Nielsen et al. (2001). Supernatants were diluted 3-5 times in de-ionized water. To each 25 µL diluted sample, 200 µL of OPA reagent was added and samples were mixed by shaking (2 min, 700 rpm) before absorbance was read at 340 nm.

## Results

Analysis of the SBMs (Table 1) showed similar dry matter and crude protein content but considerable variation in solubility characteristics. SBM #7 from Brazil stood out as having lower solubility in water (PDI) and KOH as compared to all other samples. TI content ranged from 2.39 to 3.75 mg/g DM with the samples from the United States generally having higher TI activity than those from Brazil and Argentina. Urease activity was low in all samples indicating that urease activity is not suitable to predict TI activity when operating in the low end. Low protein solubility in KOH and water was correlated with high Heat Damage Index (R²=0.52 and 0.58 for KOH and PDI). Likewise high protein solubility in KOH and water was correlated with high TI activity (R²=0.50 and 0.76 for KOH and PDI).

<table><caption>**Table 1.** Analysed values for dry matter (DM, %), crude protein (CP, %), protein dispersibility index (PDI, %), protein solubility in KOH (KOH, %), urease activity (mg N/g DM), TI activity (TIA, mg/g DM) and Heat Damage Index (Ami-nored-Evonik)</caption><thead><tr><th>SBM batch</th><th>#1</th><th>#2</th><th>#3</th><th>#4</th><th>#5</th><th>#6</th><th>#7</th><th>#8</th><th>#9</th></tr><tr><th>Origin</th><th>USA</th><th>USA</th><th>USA</th><th>Arg</th><th>Arg</th><th>Arg</th><th>Bra</th><th>Bra</th><th>Bra</th></tr></thead><tbody><tr><th>DM, %</th><td>87.9</td><td>88.7</td><td>89.0</td><td>88.6</td><td>89.8</td><td>88.4</td><td>87.7</td><td>87.1</td><td>88.5</td></tr><tr><th>CP, %</th><td>53.8</td><td>52.4</td><td>52.9</td><td>53.6</td><td>52.9</td><td>52.8</td><td>53.7</td><td>54.2</td><td>53.2</td></tr><tr><th>PDI, %</th><td>25.6</td><td>24.8</td><td>9.7</td><td>10.4</td><td>11.7</td><td>10.1</td><td>7.0</td><td>15.2</td><td>12.5</td></tr><tr><th>KOH, %</th><td>80.2</td><td>79.7</td><td>78.6</td><td>75.4</td><td>77.3</td><td>71.4</td><td>67.9</td><td>77.2</td><td>81.4</td></tr><tr><th>Urease act., mg N/g DM</th><td>0.02</td><td>0.00</td><td>0.02</td><td>0.01</td><td>0.00</td><td>0.00</td><td>0.05</td><td>0.05</td><td>0.01</td></tr><tr><th>TIA, mg/g DM</th><td>3.75</td><td>3.49</td><td>3.15</td><td>2.71</td><td>2.67</td><td>2.49</td><td>2.39</td><td>2.76</td><td>2.71</td></tr><tr><th>Heat Damage Index</th><td>5</td><td>2</td><td>8</td><td>11</td><td>12</td><td>15</td><td>25</td><td>14</td><td>16</td></tr></tbody></table>

Evaluation of the non-protease treated SBM incubations by SDS-page showed that the samples had different amounts of solubilized protein. Protein band intensities were generally higher for those SBM batches with higher protein solubility and low Heat Damage Index.

The efficiency of pancreatic trypsin/chymotrypsin to hydrolyze protein varied markedly among SBMs. Protein in SBM 2, 4, 5 and 6 was hydrolyzed effectively at lower pancreatic protease levels compared to protein in SBM 1, 3, 7, 8 and 9. However, with sufficiently high inclusion level of protease, all SBMs were hydrolyzed to approximately the same extent. The difference in response to pancreatic protease could not be explained by differences in protein solubility and TI activity as isolated parameters. The efficacy of the protease from *N. prasina* was very similar on all SBMs except for SBM#7, which was hydrolyzed to a lower degree. The lower efficiency on SBM#7 might be explained, at least in part by its lower protein solubility as compared to the other batches, meaning that less protein was readily available for hydrolysis.

## Conclusions

The results clearly showed that the efficiency of pancreatic trypsin/chymotrypsin varied among SBMs. The differences observed could not be explained by differences in protein solubility and TI activity as isolated parameters. Incubation with the *N. prasina* protease resulted in effective and very similar responses for the different SBMs and did not appear to be affected by TI activity. Hence, the *N. prasina* protease seems to represent a promising tool to ensure a sufficient amount of active protease in the intestinal tract of animals fed diets with moderately elevated levels of TI.
<figure><img src="image_75.png" /><figcaption>**Figure 2.** Protease effect measured as release of soluble free amino groups after incubation of the different SBMs at pH 7, 3 h, 40°C with different proteases.</figcaption></figure>

## Literature

DE COCA-SINOVA, A., VALENCIA, D. G., JIMÉNEZ-MORENO, E., LÁZARO, R., MATEOS, G. G., 2008: Apparent ileal digestibility of energy, nitrogen and amino acids of soybean meals of different origin in broilers. Poult. Sci. **87**, 2613.

HAMMERSTRAND, G. E., BLACK, L. T., GLOVER, J., 1981: Trypsin inhibitor in soy products: modification of standard analytical procedures. Cereal Chem. **15**, 215-218.

KAKADE, M L., RACKIS, J. J., McGHEE, J. E., PUSKI, G., 1974: Determination of trypsin inhibitor activity of soy products: A collaborative analysis of an improved procedure. Cereal Chem. **51**, 376-382.

NIELSEN, P. M., PETERSEN, D., DAMBMANN, C., 2001: Improved method for determining food protein degree of hydrolysis. J. Food. Sci. **66**, 642-646.

PERILLA, N. S., CRUZ, M. P., DE BELALCAZAR, F., DIAZ, G. J., 1997: Effect of temperature of wet extrusion on the nutritional value of full-fat soya beans for broiler chickens. Br. Poult. Sci. **38**, 412-416.

WELHAM, T., DOMONEY, C., 2000: Temporal and spatial activity of a promotor from a pea enzyme inhibitor gene and its exploitation for seed quality improvement. Plant. Sci. **159**, 289-299.
# Other topics

## P-232 (ID 297)

### STABILITY OF SECONDARY PLANT COMPOUNDS BASED PRODUCTS IN FEED PROCESSING

R. Aschenbroich

EW Nutrition, Visbek, Germany

Corresponding author: rainer.aschenbroich@ew-nutrition.com

### Abstract

Supplementing feed with plant compound based products is gaining more and more attention since livestock industry demands alternatives for growth promoting antibiotics to increase performance and animal health. Most feed supplements based on secondary plant compounds (SPCs) are offered as free flowing, granulated products. But many secondary plant compounds are highly volatile and thus do not reach the animal's digestive tract to unfold their positive effects.

Encapsulation can protect SPCs from evaporation, but the products on the market differ greatly in design, technology, and performance.

To determine the stability during feed processing, we compared the recovery rate of the leader substance of 4 competitor products to Activo®, a matrix encapsulated blend of SPCs. For each product the recommended dosage was added to a standard pig or poultry diet, respectively. After mixing, pellets were produced in a feed press using different temperature conditions (70°C and 90°C; 3 min. heating). After the pelletizing process, 10 samples for each temperature were collected and analyzed by a certified laboratory in Germany. "Leader substances" were determined through GC/MS-analysis and recovery rate in the finished feed was calculated.

Our data show that the content of SPCs in feed can be considerably influenced by the processing temperature. Activo® maintained the highest content of active compounds in the feed after processing (94,7% compared to 53,3 – 85,7% in the competitor products at 70°C and 82,1 % vs. 50,5-79,9% at 90°C).

Micro droplet encapsulation with the appropriate matrix offers the most effective and economic solution for optimal use of SPCs in animal nutrition.

**Keywords:** secondary plant compounds, stability, micro droplet encapsulation, feed processing

### Introduction

Secondary plant compounds are incorporated into diets to improve productivity of livestock by ameliorating feed properties, promoting the animals' production performance and improving the quality of food derived from these animals. The commonly used SPC products are offered as free flowing and granulated products. On farm, this standard use of SPCs has a pleasant side effect namely the smell. However, the economic sense has to be questioned, since substances that reach our nose are no longer available in the product to find their way into the digestive tract of the animal.

To prevent these losses many products containing SPCs are encapsulated and have entered the market over the last few years. These products and the processes that generate them differ greatly in design, technology, and unfortunately, in terms of performance. The question is if the product is stable enough to overcome production steps like grinding, pelletizing or extrusion.

Encapsulation is a method of enclosing the active components within a carrier material in order to protect them from harmful environmental influences. With this innovation developed more than 50 years ago the delivery of veterinary and medical drugs has largely improved (Avgenaki, 2002). As the structure of the carrier material determines the degree of protection against volatilization, aspects like good film formation, emulsion stability, low viscosity, hygroscopicity, lack of an own taste or smell, and the probability of release have to be considered (Salzer and Siewek, 2014; Avgenaki, 2002). The inclusion in an amorphous carbohydrate-based matrix for example, provides protection against oxidative changes. However, the limiting factor of this method is the insufficient water resistance releasing the encapsulated substances upon contact with it.
For many applications utilizing the physical methods of spray drying, spray granulation or extrusion, the commonly used techniques are involving core-shell encapsulation (Coating) or matrix / micro droplet encapsulation (Figure 1).

## Core shell encapsulation (Coating)

Core shell encapsulation applies a layered coating around the outside of the active ingredient e.g. essential oils to form a protective barrier. The most commonly used substances to form such a barrier or shell are ethyl cellulose, polyvinyl alcohol, gelatin or sodium alginate.

One of the major disadvantages of core shell encapsulation is the limited stability it provides during feed processing like pelleting, grinding, extruding. The high temperature and pressure applied through such processing technologies very often leads to the damage of the outer shell and thus the release of liquid and easily volatile secondary plant compounds into the environment. This can significantly reduce the content of active compounds in final feed but can also change odor and taste.

## Matrix / Micro Droplet Encapsulation

Matrix encapsulation technology allows enclosing minute droplets of a complete mixture in different matrices like hydrocolloids, proteins or fat.

Micro droplet encapsulation with fat emerged as an effective and economical method to protect secondary plant compounds. One of its major advantages compared to core shell encapsulation is the increased protective capacity during feed processing. In the event of damage, only smallest amounts of substance leak out and higher quantities of the active substance remain in the feed. Thus, losses in active compounds can be reduced and a negative influence on odor and taste is minimized. The use of a combination of different sources of fats can optimize enzymatic degradation of the matrix through lipases. This allows the slow release of secondary plant compounds to exert their full potential in promoting digestion, reducing pathogenic load and inflammatory processes, and acting as antioxidants along the gastrointestinal tract.

<figure><img src="image_76.png" /><figcaption>**Figure 1:** Coating and micro droplet encapsulation and the results after processing</figcaption></figure>

## Material and Methods

The recovery rate of the leader substance in Activo® was compared to 4 competitor products after mixing and feed processing. The amount of recovered leader substance was analyzed through GC/MS analysis by a certified laboratory in Germany.

For each product the recommended dosage was added to a standardized feed (the ingredients of this feed formulation are characteristic for pig feed as well as for poultry feed). After mixing, pellets were produced in a feed press using different temperature conditions (70°C and 90°C). The heating time for each temperature was approx. 3 minutes. In the process, steam was added into the conditioner before the feed entered the shaping die. Subsequently, the pellets were cooled down with a belt cooler and for each temperature 10 samples were collected and analyzed. The amount of leader substance after mixing, but before heat processing was set as 100%.

## Results

As shown in figure 1, the critical point of the process is heating. Feed processing at 70°C can lead to losses in the leader substance of 5,3% in Activo® with 94,7% of the leader substance still remaining in the pelleted feed to 46,7% in the non-encapsulated product of competitor C. An increase in the processing temperature to 90°C decreases the resid-
ual content in leader substance from 82,1% (Activo®) to 50,5% in competitor C. In general, the encapsulated products provide a better protection against volatilization than the non-encapsulated products. An exception is competitor D that is offered as dried herbs, providing a better protection against heat related losses than the non-encapsulated extracts.

<figure><img src="image_77.png" /><figcaption>**Figure 2:** Recovery rate of the respective marker substances</figcaption></figure>

## Conclusions

Encapsulation is a good tool to protect the often volatile secondary plant compounds from volatilizing. The kind of encapsulation, however, is very important. Core-shell encapsulation can only protect, if the product is not undergoing extensive processing and is thus not damaged by milling. The more sophisticated method of micro-droplet encapsulation shields the mixtures in case of heating but also of milling. If damaged, only smallest amounts of the active components can lack, but the main part is ingested. If a fat matrix composed of different fats is used, release in the gastrointestinal tract can be regulated up to a certain degree. So, if secondary plant compounds are used in animal nutrition, an adequate method of encapsulation should be implemented to tap the full potential of these useful substances.

## Literature

AVGENAKI, G. 2002: Molekulare Verkapselung von Aromastoffen mit Amylose und Bestimmung ihrer Freisetzungsnetik. Inaugural Dissertation. University of Düsseldorf.

SALZER, U.-J., SIEWEK, F. 2011-2013: Handbuch Aromen und Gewürze. Behr's Verlag, Hamburg, ISBN 978-3-86022-558-5.
P-234 (ID 134)

EFFECT OF YEAST AS A SOURCE OF NUCLEOTIDE SUPPLEMENTED IN BROILER BREEDERS DIETS AND ITS CARRYOVER EFFECT ON PROGENY

M. A. Bonato<fnref n="1" />, L. F. Araújo<fnref n="2" />, R. L. C. Barbalho<fnref n="1" />, C. S. S. Araújo<fnref n="3" />

**Corresponding author:** melina.bonato@icc brazil.com.br

**Abstract**

In order to study the effects of dietary supplementation of nucleotides on broiler breeders' and progenies' performance, 4 trials were conducted. The 1st and 2nd trials with 80 broiler breeder females (Cobb) in each, distributed in a completely randomized design (CRD) with 2 treatments; 0 and 5kg/MT of nucleotide source (NU) from 25-45 weeks of age. Each treatment consisted of 10 reps, 4 breeders each. Breeders were inseminated at 35 and 45 weeks of age and the eggs incubated. Egg production, fertility and hatchability were evaluated. On 3rd trial, 300 male chicks were distributed in CRD in 2 groups (progeny from breeders supplemented or not) and fed with same feed, with 10 reps, 15 chicks each. On 4th trial, 240 male chicks were distributed in CRD in a factorial arrangement 2x2 (progeny from broiler breeders supplemented or not, and progeny supplemented or not, at 5kg/MT of NU), resulting in 4 treatments with 5 reps, 12 birds each. Study criteria included BWG, FI and, F/G at 21 and 42 days for both trials. The data were analyzed using the GLM (SAS) and means were compared by Tukey (P=0.05). The 1st and 2nd trials demonstrated that supplementation of NU to broiler breeders increases the number of live chicks by 6 and 13%, respectively. The 3rd and 4th trials showed a positive carryover effect on progeny performance for BWG (+5.6 and 4.3%) and F/G (+4.5% and 9.5), however, the NU supplemented broilers from the 4th trial had improved F/G (6 and 3.5%) in both breeders age.

**Keywords:** egg production, broilers, feed conversion

**Introduction**

Although the nucleotides are not considered as essential nutrients, they play an important role in several metabolic processes, in particular, in some body tissues and stages of animal life. The free nucleotides and nucleosides can be readily absorbed by the enterocytes in the intestine, and are especially important in tissues of rapid cell proliferation and limited capacity for de novo synthesis (major route of nucleotide production), such as intestinal epithelial cells, blood cells, hepatocytes and cells of the immune system. Then, the free nucleotides can be used by the salvage pathway, where the body can synthesize nucleotides with less energy consumption, as result of recycling bases and nucleotides from metabolic degradation of nucleic acids from dead cells or from the diet. However, when the endogenous supply is insufficient, exogenous (dietary) nucleotide sources become semi-essential or "conditionally essential" nutrients (CARVER and WALKER, 1995). This especially occurs in animals in rapid growth phases (early stages), reproduction, stress, and disease challenges.

The nutrition of broiler breeders is directly linked to egg production and quality of hatched chickens. Based on this, two studies were conducted with broiler breeders in order to evaluate the effects of supplementation of *Saccharomyces cerevisiae* yeast as a source of nucleotides on the production performance and over their progeny.

**Material and Methods**

Two trials were conducted with 80 broiler breeder females (Cobb) in each, distributed in a completely randomized design (CRD) with 2 treatments; 0 and 5kg/MT of nucleotide source (NU - *Saccharomyces cerevisiae* yeast, equivalent to 0.3kg/MT of free nucleotides) from 25-45 wks of age. Each treatment consisted of 10 reps of 4 breeders each. Breeders were inseminated at 35 and 45 wks of age and the eggs incubated. Egg production (*EP%*), egg fertility (*EF%*) and hatchability of incubated eggs (*HIE%*) and fertile eggs (*HFE%*) were evaluated in both trials; and total embryonic mortality (*TEM%*) and chick weight (CW,g) were evaluated only in 2nd trial. On 3rd trial, 2 groups of 150 male chicks; one group originated from breeders fed nucleotide supplemented diets, and other group originated from the breeders not given dietary nucleotides, were distributed in CRD, with 10 reps of 15 birds in each. All broiler groups received the

<footnote n="1">¹ ICC Industrial Comércio Exportação e Importação Ltda., Av. Brigadeiro Faria Lima, 1768 4C, CEP: 01451-909. São Paulo/SP, Brazil</footnote>

<footnote n="2">² Dept. of Animal Science, FZEA, University of São Paulo, Pirassununga/SP, Brazil</footnote>

<footnote n="3">³ Dept. of Animal Nutrition and Production, FMVZ, University of São Paulo, Pirassununga/SP, Brazil</footnote>
same growout diets over a 45 day grow out period (1 to 21, 22 to 35 and 36 to 45d). On 4th trial, 240 male chicks were distributed in CRD in a factorial arrangement 2x2 (being, progeny derived from broiler breeders which were supplemented or not, and progeny supplemented or not, at 5kg/MT of NU, up to 10 d), resulting in 4 treatments, 5 reps of 12 birds each. Study criteria included FI (g), BWG (g) and, F/G (g/g) at 45 d for 3th trial and 42 d for 4th trial. The data were analyzed using the GLM (SAS) and means were compared by Tukey at 5% of significance.

## Results

Nucleotide supplementation in the diet of broiler breeders improved (P<0.05) EP (1.6%), EF (1.7%), HIE (4.1%) and HFE (2.3%), in the 1st trial (Table 1). On the 2nd trial, nucleotide supplementation of the breeder diet improved (P<0.05) EP (2.6 and 2.9%), EF (2.5 and 2%), HIE (6.7 and 2.6%), HFE (9 and 6.9%), TEM (36.5 and 34.5%) and CW (2.2 and 4.4%), respectively, for hens from 25-35 and 36-45 wks (Table 2).

<table><caption>**Table 1.** Broiler breeder's performance from 25 to 45 weeks (1st trial)</caption><thead><tr><th>Treatments</th><th>Egg production (%)</th><th>Fertility (%)</th><th>Hatchability incubated eggs (%)</th><th>Hatchability fertile eggs (%)</th></tr></thead><tbody><tr><td>Control</td><td>74.3<sup>b</sup></td><td>95.1<sup>b</sup></td><td>85.2<sup>b</sup></td><td>89.2<sup>b</sup></td></tr><tr><td>Nucleotide Source</td><td>75.8<sup>a</sup></td><td>96.8<sup>a</sup></td><td>89.3<sup>a</sup></td><td>91.4<sup>a</sup></td></tr><tr><td>Probabilities</td><td>0.018</td><td>0.023</td><td>0.011</td><td>0.006</td></tr></tbody></table>

Means followed by different letters in the same column statistically differ by the Tukey test (P<0.05).

<table><caption>**Table 2.** Broiler breeder's performance from 25 to 45 weeks (2nd trial)</caption><thead><tr><th rowspan="2">Parameters / Treatments</th><th colspan="3">Breeders - 25 to 35 weeks</th><th colspan="3">Breeders - 36 to 45 weeks</th></tr><tr><th>Control</th><th>Nucleotide</th><th>P</th><th>Controle</th><th>Nucleotide</th><th>P</th></tr></thead><tbody><tr><td>Egg production (%)</td><td>81.14<sup>b</sup></td><td>83.27<sup>a</sup></td><td>0.041</td><td>76.43<sup>b</sup></td><td>78.66<sup>a</sup></td><td>0.011</td></tr><tr><td>Fertility (%)</td><td>94.49<sup>b</sup></td><td>96.88<sup>a</sup></td><td>0.037</td><td>95.81<sup>b</sup></td><td>97.74<sup>a</sup></td><td>0.012</td></tr><tr><td>Hatchability incubated eggs (%)</td><td>85.07<sup>b</sup></td><td>90.74<sup>a</sup></td><td>0.035</td><td>87.90<sup>b</sup></td><td>90.22<sup>a</sup></td><td>0.022</td></tr><tr><td>Hatchability fertile eggs (%)</td><td>86.02<sup>b</sup></td><td>93.78<sup>a</sup></td><td>0.044</td><td>89.28<sup>b</sup></td><td>95.45<sup>a</sup></td><td>0.019</td></tr><tr><td>Total embryonic mortality (%)</td><td>2.82</td><td>1.03</td><td>0.281</td><td>2.18</td><td>0.76</td><td>0.344</td></tr><tr><td>Chick weight (g)</td><td>45<sup>b</sup></td><td>46<sup>a</sup></td><td>0.006</td><td>45<sup>b</sup></td><td>47<sup>a</sup></td><td>0.003</td></tr></tbody></table>

Means followed by different letters in the same row statistically differ by the Tukey test (P<0.05).

Offspring from 35 and 45 wks old breeders (3th trial) fed diets supplemented with 5% of nucleotides had improved (P<0.05) BWG and F/G versus chicks from breeders not supplemented dietary nucleotides (Table 3). On the 4th trial, there was no interaction (P>0.05) between breeders and offspring supplementation, for either breeder age. Offspring from 35 and 45 wks old breeders fed nucleotide-added diets had better (P<0.05) BWG and F/G versus chicks from unsupplemented hens. The supplemented offspring groups had better (P<0.05) F/G compared to non-supplemented broilers from 35 and 45 wks breeders (Table 4 and 5).

<table><caption>**Table 3.** Progeny performance from breeders with 35 and 45 weeks (3th trial) at 45 days</caption><thead><tr><th rowspan="2">Treatments</th><th>FI (g)</th><th>BWG (g)</th><th>F/G (g/g)</th><th>FI (g)</th><th>BWG (g)</th><th>F/G (g/g)</th></tr><tr><th colspan="3">Breeders - 35 weeks</th><th colspan="3">Breeders - 45 weeks</th></tr></thead><tbody><tr><td>Control</td><td>5.48</td><td>3.30<sup>b</sup></td><td>1.66<sup>b</sup></td><td>5505</td><td>3244<sup>b</sup></td><td>1.70<sup>b</sup></td></tr><tr><td>Nucleotide source</td><td>5.58</td><td>3.45<sup>a</sup></td><td>1.61<sup>a</sup></td><td>5514</td><td>3457<sup>a</sup></td><td>1.60<sup>a</sup></td></tr><tr><td>Probabilities</td><td>0.327</td><td>0.042</td><td>0.04</td><td>0.417</td><td>0.027</td><td>0.035</td></tr></tbody></table>

Means followed by different letters in the same column statistically differ by the Tukey test (P<0.05).
<table><caption>**Table 4.** Progeny performance from breeders with 35 weeks (4th trial) at 42 days</caption><thead><tr><th rowspan="2">Progeny</th><th colspan="3">Breeders</th><th colspan="3">Probabilities</th></tr><tr><th>Control</th><th>Nucleotide</th><th>Average</th><th>Breeder</th><th>Progeny</th><th>Breeders x Progeny</th></tr></thead><tbody><tr><td rowspan="3">FI (g)</td><td>Control</td><td>5056</td><td>4769</td><td>4911</td><td rowspan="3">0.008</td><td rowspan="3">0.111</td><td rowspan="3">0.773</td></tr><tr><td>Nucleotide</td><td>4900</td><td>4581</td><td>4703</td></tr><tr><td>Average</td><td>4978<sup>A</sup></td><td>4675<sup>B</sup></td><td></td></tr><tr><td rowspan="3">BWG (g)</td><td>Control</td><td>2554</td><td>2757</td><td>2646</td><td rowspan="3">0.029</td><td rowspan="3">0.259</td><td rowspan="3">0.466</td></tr><tr><td>Nucleotide</td><td>2663</td><td>2711</td><td>2688</td></tr><tr><td>Average</td><td>2604<sup>B</sup></td><td>2703<sup>A</sup></td><td></td></tr><tr><td rowspan="3">F/G (g/g)</td><td>Control</td><td>1.98</td><td>1.73</td><td>1.86<sup>b</sup></td><td rowspan="3">0.012</td><td rowspan="3">0.048</td><td rowspan="3">0.92</td></tr><tr><td>Nucleotide</td><td>1.84</td><td>1.69</td><td>1.75<sup>a</sup></td></tr><tr><td>Average</td><td>1.91<sup>B</sup></td><td>1.71<sup>A</sup></td><td></td></tr></tbody></table>

Means followed by different letters in the same row (uppercase) and column (lowercase), statistically differ by the Tukey test (P<0.05).

<table><caption>**Table 5.** Progeny performance from breeders with 45 weeks (4th trial) at 42 days</caption><thead><tr><th rowspan="2">Progeny</th><th colspan="3">Breeders</th><th colspan="3">Probabilities</th></tr><tr><th>Control</th><th>Nucleotide</th><th>Average</th><th>Breeder</th><th>Progeny</th><th>Breeders x Progeny</th></tr></thead><tbody><tr><td rowspan="3">FI (g)</td><td>Control</td><td>4755</td><td>4734</td><td>4745</td><td rowspan="3">0.109</td><td rowspan="3">0.320</td><td rowspan="3">0.624</td></tr><tr><td>Nucleotide</td><td>4733</td><td>4712</td><td>4723</td></tr><tr><td>Average</td><td>4744</td><td>4723</td><td></td></tr><tr><td rowspan="3">BWG (g)</td><td>Control</td><td>2668</td><td>2835</td><td>2752</td><td rowspan="3">0.008</td><td rowspan="3">0.063</td><td rowspan="3">0.715</td></tr><tr><td>Nucleotide</td><td>2784</td><td>2856</td><td>2820</td></tr><tr><td>Average</td><td>2726<sup>B</sup></td><td>2856<sup>A</sup></td><td></td></tr><tr><td rowspan="3">F/G (g/g)</td><td>Control</td><td>1.78</td><td>1.67</td><td>1.73<sup>b</sup></td><td rowspan="3">0.036</td><td rowspan="3">0.049</td><td rowspan="3">0.414</td></tr><tr><td>Nucleotide</td><td>1.70</td><td>1.65</td><td>1.67<sup>a</sup></td></tr><tr><td>Average</td><td>1.74<sup>B</sup></td><td>1.66<sup>A</sup></td><td></td></tr></tbody></table>

Means followed by different letters in the same row (uppercase) and column (lowercase), statistically differ by the Tukey test (P<0.05).

## Conclusions

This study demonstrated that supplementation of nucleotides to broiler breeders increases the number of live chicks by 6 and 13% (1st and 2nd trials) and appears to have a positive carryover effect on their progeny performance for both BWG and F/G (3rd and 4th trials). In addition, the nucleotide-supplemented broilers from the 4th trial had improved F/G (6 and 3.5%) in both breeders age.

## Literature

CARVER, J.D, W.A. WALKER, 1995: The role of nucleotides in human nutrition. Nutritional Biochemistry 6, 58-72.
P-235 (ID 274)

EFFECTS OF FEEDING ORIGINAL XPC DURING A CAMPYLOBACTER CHALLENGE IN BROILERS AND TURKEYS

J.N. Broomhead, D.P. Smith, and D.R. McIntyre

Diamond V, Cedar Rapids, Iowa, USA 52404

Corresponding author: jbroomhead@diamondv.com

**Abstract**

Two studies were conducted to test the effects of feeding XPC during a *Campylobacter* challenge: 1) Cobb broiler chicks (n=1200) in Georgia, USA or 2) Hybrid hen poults (n=288) in North Carolina, USA. Both were placed in barns with 24 floor pens (50 chicks or 12 poults per pen). Three treatments were fed in the chicken study: 1) Control, 2) 1.25 kg XPC/tonne from 0-21d and 0.62 kg XPC/tonne from 22-42d, and 3) 1.25 kg XPC/tonne from 0-42d. Turkeys were fed one of 2 dietary treatments: Control or XPC (1.25 kg/tonne from 0-42d and 0.62 kg/tonne from 43-84d). At 14d (chickens) or 70d (turkeys), 25 or 5-tagged birds/pen (respectively) were orally gavaged with a gentamicin-resistant strain of *C. coli* (log 10⁶ CFU/ml). The remaining birds per pen were not tagged or challenged and were used to assess horizontal transmission (HT). Fifteen chickens at 42d or 10 turkeys at 84d were sampled per pen (5 tagged birds) and the ceca removed for *C. coli* analysis. FCR was statistically improved (*P* < 0.05) in chickens and turkeys from the addition of XPC. *C. coli* prevalence (% positive) in HT turkeys and chickens was reduced by feeding XPC (*P* < 0.05). HT chickens fed XPC had lower *C. coli* colonization (*P* = 0.09). In the turkeys, *C. coli* numbers were reduced (*P* < 0.05) from 4.5 to 3.5 Log₁₀ CFU/g in Control vs. XPC fed birds, respectively. Based on these results, XPC-fed chickens and turkeys had improved FCR and lower prevalence and numbers of *Campylobacter*.

**Keywords:** Campylobacter, Food Safety, Turkeys, Chickens

**Introduction**

*Campylobacter* is one of the leading causes of foodborne illness (Batz et al, 2012). Traditional methods for controlling *Campylobacter* contamination have been focused within the processing plant, trying to prevent cross contamination through washing and evisceration (Berghaus et al, 2013). It has been suggested that if preharvest levels of *Campylobacter* could be controlled in the birds' intestinal tract, then contamination of processed birds will also be reduced. Research has shown that biosecurity and pest management measures may improve preharvest control of pathogens, but additional measures (i.e. dietary ingredients, vaccination) may be needed to have the optimal preharvest pathogen control (Dale et al, 2015). The objective of this research is to evaluate the effects of feeding Original XPC™, a *Saccharomyces cerevisiae* fermentation product, on cecal *Campylobacter coli* colonization and transmission following an oral challenge.

**Materials and Methods**

Two independent studies were recently conducted, in which broilers or turkeys were orally gavaged with approximately 10⁶ CFU of gentamicin-resistant strains of *Campylobacter coli*. The broiler trial (Experiment 1) was conducted by Southern Poultry Research Group near Nicholson, GA and turkey trial (Experiment 2) was conducted at North Carolina State University near Raleigh, NC.

**Experiment 1: Broiler Trial**

Cobb chicks (n = 1200) were housed in 24 floor pens (50 birds/pen) and assigned to one of 3 feed treatments (8 replicates each). Control birds (T1) received industry standard diets with no antibiotics or coccidiostat. Treatments T2 and T3 received the control starter diet with the addition of XPC at 1.25 kg/tonne from 0-21 d. After 21 d, XPC was reduced to 0.62 kg/tonne for T2 birds, while T3 birds continued to receive feed with 1.25 kg/tonne of XPC. All birds and feed were weighed by pen at 0, 21, 35 and 42 d. All birds were vaccinated with Coccivac-B at 0 d.

At 14 d, 25-tagged birds per pen were challenged with *C. coli*. The remaining 25 birds per pen did not receive a tag or *C. coli* challenge, and were used to determine horizontal transmission (contact challenge between *C. coli* gavaged and non-gavaged birds). Fifteen birds per pen (5 tagged and 10 non-tagged) were sampled at 42 d and the ceca removed
for *C. coli* colonization evaluation. The MPN (most probable number) method was used to enumerate *C. coli* (Berghaus et al, 2013).

## Experiment 2: Turkey Trial

Hybrid hen poults (n = 288) were assigned to one of 2 feed treatments (Control vs. XPC), with 12 replicate floor pens per treatment and 12 birds per pen. Four diet phases were fed, as either pelleted and crumbled or pelleted, during the 12-week study; 2 starter and 2 grower ratios. Original XPC was added to the XPC rations at 1.25 kg/tonne in the two starter feeds and 0.62 kg/tonne in the two grower feeds. Diets did not contain any antibiotic or coccidiostat. All birds and feed were weighed at 10 weeks and at the end of the study (12 weeks of age) and feed conversion ratio calculated.

At 10 weeks of age, 5 birds from each pen were wing banded and challenged with *C. coli*. The remaining 7 birds per pen were not banded nor challenged, and were used to determine horizontal transmission of *Campylobacter* from challenged birds. At 12 weeks of age, 10 birds per pen (5 banded and 5 non-banded) were euthanized and ceca were aseptically removed. Cecal contents were either directly plated onto CCDA+gentamicin to determine presence of *C. coli* or serially diluted in buffered peptone water, plated on CCDA+gentamicin, and incubated in a microaerophilic environment for 48 h at 42 C to determine numbers of *C. coli*.

## Results

Body weight gains were not significantly different (*P* > 0.05) between treatments in both the broiler and turkey studies. Feed conversion was significantly improved (*P* < 0.05) in broilers by the addition of XPC at both inclusion levels on 35 d and 42 d, respectively (Table 1). Feed conversion was also significantly improved (*P* < 0.05) in turkeys, during the challenge (10-12 week) and overall (0-12 week; Table 1).

Cecal *C. coli* prevalence (percent positive) in contact challenged broilers was significantly reduced (*P* < 0.05) when 1.25 kg/tonne (T3) XPC was fed continuously and intermediate when 0.62 kg XPC/tonne (T2) was fed during grower/finisher period (Table 2). A similar response trend was observed in *C. coli* numbers (MPN; *P* = 0.09) in broilers when XPC was fed. In turkeys, cecal *C. coli* prevalence was significantly reduced (*P* < 0.05) in contact challenged and a trend for reduction (*P* = 0.09) in gavage (direct) challenged birds (Table 3). Cecal *C. coli* numbers were significantly (*P* < 0.05) reduced by one (1) log across all challenged turkeys (Table 3).

<table><caption>**Table 1.** Feed conversion ratio (FCR) in broilers and turkeys.</caption><thead><tr><th rowspan="2">Bird Age:</th><th colspan="2">Broilers FCR</th><th colspan="2">Turkeys FCR</th></tr><tr><th>0-35 d</th><th>0-42 d</th><th>10-12 wk</th><th>0-12 wk</th></tr></thead><tbody><tr><td>T1; Control</td><td>1.75<sup>a</sup></td><td>1.93<sup>a</sup></td><td>2.56<sup>a</sup></td><td>1.95<sup>a</sup></td></tr><tr><td>T2; XPC (1.25 and 0.62 kg/tonne)<sup>1</sup></td><td>1.73<sup>b</sup></td><td>1.90<sup>b</sup></td><td>2.38<sup>b</sup></td><td>1.90<sup>b</sup></td></tr><tr><td>T3; XPC (1.25 and 1.25 kg/tonne)<sup>1</sup></td><td>1.72<sup>b</sup></td><td>1.90<sup>b</sup></td><td>-</td><td>-</td></tr><tr><td>P value</td><td><i>P</i> < 0.05</td><td><i>P</i> < 0.05</td><td><i>P</i> < 0.05</td><td><i>P</i> < 0.05</td></tr></tbody></table>

<table><caption>**Table 2.** Broiler cecal *Campylobacter coli* prevalence and numbers in contact challenged birds at 42 day of age.</caption><thead><tr><th>Treatment</th><th>C. coli prevalence<br>(% positive)</th><th>C. coli numbers<br>(Most probable number)</th></tr></thead><tbody><tr><td>T1; Control</td><td>17.5<sup>a</sup></td><td>37</td></tr><tr><td>T2; XPC (1.25 and 0.62 kg/tonne)<sup>1</sup></td><td>10.0<sup>ab</sup></td><td>14</td></tr><tr><td>T3; XPC (1.25 and 1.25 kg/tonne)<sup>1</sup></td><td>1.3<sup>b</sup></td><td>0.9</td></tr><tr><td>P value</td><td><i>P</i> < 0.05</td><td><i>P</i> = 0.09</td></tr></tbody></table>

<footnote>ab Values within a column with different superscripts are statistically different (*P* < 0.05).</footnote>

<footnote n="1">¹ Starter and grower (turkey) or grower/finisher (broiler) dietary inclusion rates, respectively.</footnote>

<footnote>ab Values within a column with different superscripts are statistically different (*P* < 0.05).</footnote>

<footnote n="1">¹ Starter and grower/finisher dietary inclusion rates, respectively.</footnote>
<table><caption>**Table 3.** Turkey cecal *Campylobacter coli* prevalence in gavage and contact challenged birds and numbers in all birds at 12 week of age.</caption><thead><tr><th rowspan="2">Challenge method:</th><th colspan="2">C. coli prevalence<br>(% positive)</th><th>C. coli numbers<br>(log<sub>10</sub> CFU)</th></tr><tr><th>Gavaged</th><th>Contact</th><th>All birds</th></tr></thead><tbody><tr><td>T1; Control</td><td>87</td><td>93<sup>a</sup></td><td>4.51<sup>a</sup></td></tr><tr><td>T2; XPC (1.25 and 0.62 kg/tonne)<sup>1</sup></td><td>78</td><td>75<sup>b</sup></td><td>3.49<sup>b</sup></td></tr><tr><td>P value</td><td><i>P</i> = 0.09</td><td><i>P</i> &lt; 0.05</td><td><i>P</i> &lt; 0.05</td></tr></tbody></table>

## Conclusions

*Campylobacter* prevalence was reduced in contact challenged broilers and turkeys when XPC was fed. *Campylobacter* numbers were also reduced in broilers and turkeys fed XPC. Improvements in feed conversion were observed in broilers and turkeys fed XPC.

## Literature

BATZ, M. B., S. HOFFMANN, J. G. MORRIS, JR, 2012: Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation. J. Food Prot. **75**:1278–1291.

BERGHAUS, R. D., S. G. THAYER, B. F. LAW, R. M. MILD, C. L. HOFACRE, R. S. SINGER, 2013: Enumeration of Salmonella and Campylobacter in environmental farm samples and processing plant carcass rinses from commercial broiler chicken flocks. Appl. Environ. Microbiol. **79**, 4106-4114.

DALE, E. L., S. P. NOLAN, R. D. BERGHAUS, C. L. HOFACRE, 2015: On farm prevention of *Campylobacter* and *Salmonella*: lessons learned from basic biosecurity interventions. J. Appl. Poult. Res. **24**:222-232.

<footnote>ab Values within a column with different superscripts are statistically different (P < 0.05).</footnote>

<footnote n="1">¹ Starter and grower dietary inclusion rates, respectively.</footnote>
P-239 (ID 133)

# THE INFLUENCE OF OZONE TREATMENT ON THE DEGRADATION OF DIFFERENT MYCOTOXINS IN CONTAMINATED MAIZE

K. Dublecz<fnref n="1" />, L. Pál<fnref n="1" />, L. Wágner<fnref n="1" />, F. Husvéth<fnref n="1" />, F. Dublecz<fnref n="2" />, O. Hegyi<fnref n="1" />

**Corresponding author:** dublecz@georgikon.hu

## Abstract

Although commercial usage of ozone for grain treatments is not common, several results prove its efficiency against grain insects, moulds and different microbes. The scientific results of ozone on the different mycotoxin levels are contradictory.

In the present study the grinded maize samples were contaminated with different purified mycotoxins, aflatoxin B1 (AFB1); ochratoxin A (OTA); T2-toxin, Deoxynivalenol (DON); fumonisin B1 (FB1) and zearalenon (F2). The mycotoxin levels were adjusted according to the concentrations that can be found under field conditions. Maize samples were treated with 50 ppm ozone for 48 hours. Mycotoxin concentrations were measured after 24 and 48 hours treatments.

After 48 hours the following reduction in the mycotoxin concentrations were detected: AFB1: 26.1%; DON: 24.3%; F2: 44.5%; FB1: 53.7%; OTA: 12.6%; T2: 0,3%. Significant reduction was found after 24 hours in the concentrations of AFB1 (14.7%), DON (21.5%) and FB1 (44.7%). On the other hand the concentration of F2 has changed only during the second 24 hours of ozone treatment. Ozone seems to be less effective in breaking down T2 toxin.

**Keywords:** ozone, mycotoxins, maize

## Introduction

Mycotoxins have several negative effects and decrease the production rate of farm animals. Beside that animals can excrete mycotoxins with the animal products (milk, eggs, liver) that mean also food safety concerns. It is the reason, that several tools are used to decrease the incidence of toxicosis. Among them a new direction is the ozone treatment of the contaminated products. Ozone is well known as a disinfectant, but its effect on the mycotoxin contaminated grains is not fully understood. The efficiency of ozone treatment is influenced by several factors, like its concentration, the air flow, the exposure time and the humidity (ISIKBER et al., 2014). According to the results of MENDEZ et al. (2003), the ozone degrades rapidly in the first few hours while it flowing through a grain column, but remains stable afterwards. Even 30 day long treatment failed to cause any difference in the fatty acid and amino acid profile of soybean, wheat or maize. The aim of the present study was to determine the effect of ozone on the degradation of different mycotoxins.

## Materials and methods

Six mycotoxins, aflatoxin B1 (AFB1), zearalenon (F2), T-2 toxin (T2), fumonisin B1 (FB1), deoxynivalenol (DON) and ochratoxin A (OTA) have been purchased in purified form (Sigma Aldrich Fluka Analytical), solved in methanol and mixed with grinded maize samples. The toxin concentrations were adjusted close to the natural depressive levels (Table 1.). After evaporating the methanol, the samples were mixed and homogenized. The mycotoxin concentration of the samples was determined prior and after 24 and 48 hours of the ozone treatment. The grinded maize samples were analysed by HPLC after cleaning and separating the mycotoxins with toxin specific immune-affinity columns according to the validated methods of R-Biopharm Hungary Ltd.

Ozone treatments were carried out by an ExOzone (expanded capacity with a limited flashover high frequency cold charge emission with changed auxiliary electrode) type laboratory ozone generator. The actual treatment conditions (ozone concentration, temperature, humidity) were monitored with a TGM-100 type detector. The ozone concentration was adjusted according to the potential practical level.

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

<footnote n="2">² UBM Feed Ltd. Pilisvörösvár, Hungary</footnote>
<table><caption>Table 1. Adjusted mycotoxin concentrations of the maize samples</caption><thead><tr><th></th><th>Concentration (µg/kg)</th></tr></thead><tbody><tr><td>AFB1</td><td>100</td></tr><tr><td>F2</td><td>2500</td></tr><tr><td>T2</td><td>2500</td></tr><tr><td>FB1</td><td>2,5</td></tr><tr><td>DON</td><td>3,0</td></tr><tr><td>OTA</td><td>500</td></tr></tbody></table>

## Results

The average ozone concentration during the treatments was 50.3 ppm, the temperature 25.7°C and the relative humidity 54.9% (Figure 1. and 2.). After 24 hours no changes in the concentration of F2, T2 and OTA was measured. The following 24 hour long ozone treatment reduced the F2 concentration by about 44.5%. The decrease in OTA content was moderate, 12.6%. T2 concentration was not influenced by the ozone treatment.

<figure><img src="image_78.png" /><figcaption>Figure 1. The effect of ozone treatment on the F2, T2 concentrations (ppb)</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>Figure 2. The effect of ozone treatment on the OTA concentrations (ppb)</figcaption></figure>

Similarly to the T2 toxin the other trichothecene mycotoxin, DON showed also higher resistance against ozone, compared with FB1 or AFB1. In the case of FB1, its concentration decreased already after the first 24 hours by and only limited further decrease happened (Figure 3.). On the other hand gradual decrease was found in the AFB1 level (Figure 4.).
<figure><img src="image_80.png" /><figcaption>Figure 3. The effect of ozone treatment on the DON, FB1 concentrations (ppm)</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>Figure 4. The effect of ozone treatment on the AFB1 concentrations (ppb)</figcaption></figure>

## Conclusions

From the results it can be concluded, that ozone treatment seems to be effective to decrease the mycotoxin content of the cereal grains. Its efficiency is however, depends on the type mycotoxins and the exposure time. Trichothecene mycotoxins (T2 and DON) seem to be more resistant against ozone than the other toxins, investigated in this trial. The reason for this could be the chemical structure of the different toxins. The trichothecene skeleton contains less ligand that can interact with ozone. Further research needed to understand the interactions of ozone with the mycotoxin molecules, the biological activity of the modified substances and the optimal treating conditions for the different mycotoxins.

## Literature

1. ISIKBER, A. A., C. G. ATHANASSIOU. 2014: The use of ozone gas for the control of insects and micro-organisms in stored products. Journal of Stored Products Research. 1-7.

2. MENDEZ, F., D. E. MAIER, L. J. MASON, C. P. WOLOSHUK, 2003: Penetration of ozone into columns of stored grains and effects on chemical composition and processing perfomance. Journal of Stored Products Research. 39, 33-44.
P-242 (ID 192)

EFFECT OF PARTICLE SIZE AND FEED PRESENTATION (MASH VS PELLETS) ON CECAL MORPHOLOGY AND
CAMPYLOBACTER JEJUNI COLONIZATION OF BROILERS ORALLY INFECTED

M.I. Gracia¹, F.J. García-Peña², M. Guyard³, Gilles Le Pottier⁴, and M. Tenk⁵

¹Imasde Agroalimentaria, S.L., Madrid, Spain

²Laboratorio de Sanidad Animal de Algete, MAGRAMA, Madrid, Spain

³Anses, UHQPAP, Ploufragan, France

⁴CIDEF, Rennes, France

⁵MIKROLAB, Dabas, Hungary

Corresponding author: mgracia@e-imasde.com

Abstract

An experiment was conducted within the EU-FP7 project CAMPYBRO for evaluating the effect of particle size (PS) of wheat (W) and feed presentation (FP) in morphology and *Campylobacter jejuni* colonization in the caeca of broilers orally infected. There were four treatments factorially arranged with two W screen sizes (2mm vs 5mm) and two FP (mash vs pellets). A total of 144 one-day-old Ross 308 broilers were divided into cages (3 birds/cage) and experimental treatments (12 cages/treatment). At 14 days of age, all broilers were orally gavaged with 100 µl of a solution containing 1 x 10⁵ cfu/ml of ST-45 *C. jejuni* strain. On days 21, 35 and 42, caeca from 12 birds per treatment were collected and *Campylobacter* counts determined (ISO 10272). At 42d, the weight and the pH at caeca were taken. Data were analysed by GLM procedure of SPSS. Pelleting the diet decreased the relative importance of the caeca expressed as percentage of Gastrointestinal tract, body weight or empty body weight (P<0.05). There was an interaction between FP and PS for caeca weight: pelleting of fine size diets decreased the caeca weight and the opposite was observed for 5mm-diets (P=0.048). Neither the PS nor the FP caused a significant variation on *C. jejuni* counts or pH at the caeca of the birds. No interactions were observed between PS and FP. It is concluded that pelleting the diet decreased the relative weight of caeca but did not affect the pH or *C. jejuni* counts.

**Keywords:** *Campylobacter jejuni*, pelleting, particle size, broiler, gastrointestinal tract

Introduction

*Campylobacter* is the major cause of bacterial gastroenteritis in humans worldwide. In the EU, it is the most commonly reported gastrointestinal (GI) bacterial pathogen since 2005, with 214,779 human cases reported in 2013 (EFSA, 2015). *Campylobacter* preventive measures aim at reducing the probability of *Campylobacter* colonisation in birds (GHAREEB et al., 2013). For this purpose several approaches, as the use of bacteriophages (WAGENAAR et al., 2005), bacteriocins (LINE et al., 2008), organic acids (CHAVEERACH et al., 2004; SKÂNSENG et al., 2010) and their derivatives (HILMARSSON et al., 2006) or medium chain fatty acids (HERMANS et al., 2010) have been proposed over the past few years. However, few efforts have been done on modifying the upper GI tract in order to combat *C. jejuni* colonization in lower GI tract. HUANG et al. (2006) proposed that the gizzard may be a critical control point for reducing *Salmonella* contamination in growing broilers, and MOEN et al. (2012) have recently shown that a stimulated gizzard delays the horizontal spread of *C. jejuni* in broiler flocks. The aim of this work was to investigate the effect of particle size of wheat and feed presentation on *C. jejuni* colonization in broilers.

Material and Methods

A total of 144 Ross 308 broilers (50% male and 50% female) from 1 to 42 days of age were used and allocated to the experimental treatments. There were four treatments factorially arranged with two W screen sizes (2mm vs 5mm) and two FP (mash vs pellets). The trial was carried out in an Animal Biosafety Level 2 Unit for broilers. Broilers were housed in cages in groups of three. Mash (non-pelleted) feeds were fed *ad libitum* with no added coccidiostat, growth promoter or veterinary antibiotics. At 14 d of age, all broilers were orally gavaged with 100 µl of a solution containing 1 x 10⁵ CFU/ml of ST-45 *C. jejuni* strain. On days 21, 35 and 42, ceca were collected from 12 birds per treatment and *Campylobacter* counts determined (ISO 10272). Detection limit was 2 logs, and animals below limit of detection were assigned 2 logs for statistical analysis. At 42d, the weight of the gastrointestinal tract (GIT), empty BW (EBW), the weight of caeca and its pH were taken. Data were analysed by GLM procedure as a factorial design with PS and FP as main
effects (SPSS v.19.0). Statistical significance is declared at $P \le 0.05$, with $0.05<P\le0.10$ considered as a near-significant trend.

## Results

Neither the PS of the cereal nor the FP caused a significant variation on *C. jejuni* counts in the caeca of broilers at any of the periods studied (Figure 1). Also, no interactions were detected between PS and FP ($P < 0.05$)

<figure><img src="image_82.png" /><figcaption>**Figure 1.** Effect of particle size of cereal (2 vs 5 mm) and feed presentation (mash vs pellet) on *Campylobacter* counts ($\log_{10}$ CFU/g) in the caeca of broilers at 21, 35 and 42 days of age.</figcaption></figure>

Particle size did not affect any of the morphological parameters evaluated (Table 1). Pelleting the diet increased GIT weight (214.3 vs 178.4 g, for pellets vs mash; $P = 0.001$) and empty BW (2557 vs 1986 g, for pellets vs mash; $P = 0.001$). Pelleting also decreased the relative importance of the caeca expressed as percentage of GIT, BW or EBW ($P<0.05$). There was an interaction between PS and FP for caeca weight: pelleting 2mm diets decreased the caeca weight but the opposite was observed for 5mm-diets (1.17, 1.08, 1.08 and 1.23 g for 2mm mash, 2mm pellet, 5mm mash and 5mm pellet, respectively; $P = 0.048$). Dietary treatment did not affect pH at the caeca of the birds ($P > 0.05$).

## Conclusions

It is concluded that pelleting the diet increased BW, GIT weight and empty BW of broilers and decreased the relative weight of caeca without affecting the pH or *C. jejuni* counts.

## Acknowledgements

This work has been done within the project CAMPYBRO, which has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 605835.
<table><caption>**Table 1.** Effect of particle size of cereal (2 vs 5 mm) and feed presentation (mash vs pellet) on on caeca morphology and pH</caption><thead><tr><th rowspan="2"></th><th rowspan="2">BW, g</th><th colspan="2">GIT</th><th colspan="2">Empty BW</th><th colspan="4">Caeca</th><th rowspan="2">pH</th></tr><tr><th>g</th><th>%BW</th><th>g</th><th>%BW</th><th>g</th><th>%GIT</th><th>%BW</th><th>%EBW</th></tr></thead><tbody><tr><td colspan="11"><strong>Particle size</strong></td></tr><tr><td>2 mm</td><td>2440</td><td>191.7</td><td>7.91</td><td>2249</td><td>92.09</td><td>1.12</td><td>0.60</td><td>0.047</td><td>0.051</td><td>6.67</td></tr><tr><td>5 mm</td><td>2496</td><td>200.9</td><td>8.09</td><td>2295</td><td>91.91</td><td>1.15</td><td>0.59</td><td>0.047</td><td>0.051</td><td>6.75</td></tr><tr><td colspan="11"><strong>Feed presentation</strong></td></tr><tr><td>Mash</td><td>2165</td><td>178.4</td><td>8.27</td><td>1986</td><td>91.73</td><td>1.12</td><td>0.64</td><td>0.052</td><td>0.057</td><td>6.78</td></tr><tr><td>Pellet</td><td>2772</td><td>214.3</td><td>7.74</td><td>2557</td><td>92.27</td><td>1.15</td><td>0.55</td><td>0.042</td><td>0.045</td><td>6.64</td></tr><tr><td>SEM (n=24)</td><td>59.9</td><td>7.13</td><td>0.237</td><td>56.3</td><td>0.237</td><td>0.043</td><td>0.027</td><td>0.002</td><td>0.002</td><td>0.070</td></tr><tr><td colspan="11"><strong>Probability</strong></td></tr><tr><td>Particle size</td><td>0.512</td><td>0.367</td><td>0.607</td><td>0.559</td><td>0.607</td><td>0.591</td><td>0.906</td><td>0.897</td><td>0.884</td><td>0.400</td></tr><tr><td>Feed presentation</td><td><strong>0.001</strong></td><td><strong>0.001</strong></td><td>0.121</td><td><strong>0.001</strong></td><td>0.121</td><td>0.591</td><td><strong>0.017</strong></td><td><strong>0.001</strong></td><td><strong>0.001</strong></td><td>0.188</td></tr><tr><td>PS x FP</td><td>0.523</td><td>0.266</td><td>0.329</td><td>0.590</td><td>0.329</td><td><strong>0.048</strong></td><td>0.409</td><td>0.190</td><td>0.174</td><td>0.205</td></tr></tbody></table>

## Literature

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

EFSA, 2015: The European Union Summary Report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2013. EFSA Journal 2015; **13** (1), 3991, 162 pp.

GHAREEB, K., W.A. AWAD, M. MOHNL, G. SCHATZMAYR, J. BÖHM, 2013: Control strategies for *Campylobacter* infection in poultry production. World's Poult. Sci. J., **69**, 57-76.

HERMANS D, A. MARTEL, K. VAN DEUN, M. VERLINDEN, F. VAN IMMERSEEL, A. GARMYN, W. MESSENS, M. HEYNDRICKX, F. HAESEBROUCK, F. PASMANS, 2010: Intestinal mucus protects *Campylobacter jejuni* in the ceca of colonized broiler chickens against the bactericidal effects of medium-chain fatty acids. Poult. Sci., **89**, 1144-1155.

HILMARSSON H., H. THORMAR, J.H. THRAINSSON, E. GUNNARSSON, S. DADADOTTIR, 2006: Effect of glycerol monocaprate (monocaprin) on broiler chickens: An attempt at reducing intestinal *Campylobacter* infection. Poult. Sci., **85**, 588-592.

HUANG, D.S., D.F. LI, J.J XING, Y.X. MA, Z.J. LI, S.Q. LV, 2006: Effects of feed particle size and feed form on survival of *Salmonella typhimurium* in the alimentary tract and cecal *S. typhimurium* reduction in growing broilers. Poult. Sci., **85**, 831-836.

LINE J.E., E.A. SVETOCH, B.V. ERUSLANOV, V.V. PERELYGIN, E.V. MITSEVICH, I.P. MITSEVICH, V.P. LEVCHUK, O.E. SVETOCH, B.S. SEAL, G.R. SIRAGUSA, N.J. STERN, 2008: Isolation and purification of enterocin e-760 with broad antimicrobial activity against gram+ and gram- bacteria. Antimicrob. Agents Chemother., **52**, 1094-1100.

MOEN, B., K. RUDI, B. SVIHUS, B. SKÅNSENG, 2012: Reduced spread of *C. jejuni* in broiler chickens by stimulating the bird's natural barriers. J. Appl. Microbiol., **113**, 1176-1183.

SKÅNSENG B., M. KALDHUSDAL, B. MOEN, A.G. GJEVRE, G.S. JOHANNESSEN, M. SEKELJA, P. TROSVIK, K. RUDI, 2010: Prevention of intestinal *C. jejuni* colonization in broilers by combinations of infeed organic acids. J. App. Microbiol., **109**, 1265-1273.

WAGENAAR, J.A., M.A. VAN BERGEN, M.A. MUELLER, T.M. WASSENAAR, R.M. CARLTON, 2005: Phage therapy reduces *C. jejuni* colonization in broilers. Vet. Microbiol., **109**, 275-283.
# EFFECT OF PHYSICAL FEED QUALITY AND LOW ENERGY MASH DIET ON THE PERFORMANCE OF BROILER BREEDERS

E.Helander¹, H.Willemsen¹, C.Kemp², O. van Tuijl²

¹Aviagen Ltd. Midlothian, UK

²Aviagen EPI, the Netherlands

**Corresponding author:** ehelander@aviagen.com

## Abstract

Feed physical quality, fibre and energy content have been shown to influence eating-up time, gizzard function, feather pecking and plumage condition in laying hens. This experiment was conducted to study the effect of feed physical quality and of a low energy, high fibre diet on the performance and hatchability results of Ross 308 grandparent broiler breeders. Four treatments were given from 22 to 60 weeks of age in 4 replicates (147 females in each). Physical qualities of the diets for the first 3 treatments were crumble (C), coarse mash (CM) and coarse mash with 10% whole oats plus 5% whole wheat (CM15OW). The diets were formulated according to Aviagen recommendations (2013) (11.7 M/kg) The fourth diet was a mash with 20% whole oats and 10% whole wheat (M30OW), contained less energy (10.9 MJ/kg) and more digestible lysine (0.66%) than the other diets. All males (16 per replicate) were fed with CM15OW. Total hen week egg productions were 157, 165, 165.5 and 164.6; cumulative hatch of fertile eggs 80.89, 81.97, 83.17 and 82.43%, and 94, 100, 105 and 102 chicks per hen housed were obtained for C, CM, CM15OW and M30OW, respectively. Feeding breeders a coarse mash diet formulated according to recommendations gave better performance than a diet in crumble form. Whole cereal inclusion might have further improved hatchability and chick output. M30OW did not give additional benefits over CM15OW. Gizzard size was bigger on diets containing whole cereals.

**Keywords:** broiler breeder, nutrition, feed, physical quality

## Introduction

Lifetime uniformity and bodyweight control support expression of the full genetic potential of the modern broiler breeder. In addition to fast feed distribution, slow feed clean-up time may help maintaining flock uniformity. In the trial of Tanaka et al. 1983 (ref. van Krimpen et al. 2005), hens spent more time on feed intake as the particle size of the diet decreased: 100 min per day for pellets, 154 min for crumbles and 234 min for mash. According to Röhe et al. (2014), feeding of coarsely ground mash diets have positive effects on the gastrointestinal function and may lead to improved bird health and welfare. If included at the correct particle size, fibre rich raw materials will provide sufficient structure to stimulate gizzard development which will regulate the flow of nutrients into the small intestine, optimising digestive processes and nutrient utilization (Hetland et al. 2005). This experiment was conducted to study the effect of feed physical quality and of a low energy, high fibre diet on the performance and hatchability results of modern broiler breeders.

## Material and Methods

A total of 2352 grandparent Ross 308 female broiler breeders and 256 males were randomly allocated to 4 different experimental groups. Four dietary treatments were given from 22 to 60 weeks of age in 4 replicates (147 females and 16 males in each). Physical quality of the diets for the first 3 treatments were crumble (C), coarse mash (CM) and coarse mash with 10% whole oats plus 5% whole wheat (CM15OW). The diets were formulated according to Aviagen recommendations (2013) with energy content of 11.7 M/kg (WPSA). Digestible amino acids for raw materials were calculated using AminoDat4. The fourth diet was a mash with 20% whole oats and 10% whole wheat (M30OW), and contained less energy (10.9 MJ/kg) and more digestible lysine (0.66%) than the other diets. All males were fed CM15OW. Hammer mill (3000 rpm – 8 mm screen) was used for grinding the raw materials for treatments C and M30OW (excluding whole cereals). For CM corn, wheat and sunflower were roller milled (2.9 and 2.1 mm screen) and all other raw materials hammer milled (1050 rpm – 12 mm). Raw materials for CM15OW were grinded equally to treatment CM (excluding whole cereals). After peak feed the females were fed to equal pre-calculated ME intake and, thus, feed levels (g/d) were predefined according to the estimated ME level of the feed. Because of *Heterakis gallinarum* infection at 39 and 46 weeks of age the birds were treated with anthelminthic. After the second infection the birds lost weight and the feed levels had to be increased for all birds. Body weight (BW) and feed intake levels were recorded weekly until 35
weeks of age, fortnightly in weeks 35 – 45 and weekly again from 45 weeks onwards. Feathering was recorded at week 28, 38 and 48 and feed clean-up time at week 30, 40, 50 and 60 for all pens. Litter moisture and capping were measured at 30, 40, 50 and 60 weeks of age. Every other week eggs collected during one production week were set for hatching. At the end of the trial gizzard weight and thickness were measured of 16 birds per treatment.

## Results

Body weight gain (BWG) was higher than expected for all treatments with differences in BWG between the treatments: hens were lightest on the low-energy diet and heaviest on the crumble diet. Egg production was close to the target during the first part of production but reduced as *Heterakis* problems appeared at weeks 39 and 46 (Figure 1). From 48 weeks onwards the production started to recover and outreached the target at 50 – 56 weeks of age. CM15OW and M30OW recovered better than C and CM. Total hen week egg productions were 157, 165, 165.5 and 164.6; cumulative hatch of fertile eggs 80.89, 81.97, 83.17 and 82.43%, and 94, 100, 105 and 102 chicks per hen housed were obtained for C, CM, CM15OW and M30OW, respectively. Gizzard size and thickness increased as the amount of coarse particles or whole cereals increased in the diets. Litter capping and moisture % were significantly lower for CM15OW and M30OW until 45 weeks but later on the differences between the treatments mostly disappeared. Feed clean-up time varied between 195 – 307 minutes. with a lot of variation and inconsistency between the treatments and weeks measured. The highest mortality rate was found on the crumble diet.

Higher than expected BWG indicates the energy content of the diet was underestimated when pre-calculating the feed allocations. The heaviest hens and the poorest egg laying rate were found on the crumbled diet. According to Leeson and Summers (2005), oversupply of nutrients results in increased growth and obesity in broiler breeders and leads to reduced egg production. Increasing gizzard size and thickness as the amount of coarse particles or whole cereals increased in the diets, is in accordance with the findings of Hetland et al. (2005). The egg production of the hens fed coarse particles and/or whole cereals recovered better after the *Heterakis*-problem than the birds fed the crumbled diet. Whether the well-developed gizzard plays a role is unclear. The differences in plumage condition were negligible and in practise no feather pecking appeared. Better cumulative hatchability with whole cereal treatments and lower mortality rates on diets containing coarse particles might be linked to better gut health and bird welfare but more research is needed on this field.

<figure><img src="image_83.png" /><figcaption>**Figure 1.** Hen week egg production %.</figcaption></figure>

## Conclusions

Feeding breeders a coarse mash diet formulated to current recommendations gives better breeder performance than a diet in crumbled form. Whole cereal inclusion may further improve the hatchability results and chick output.
## Literature

HETLAND, H., B. SVIHUS, M. CHOCT, 2005: Role of Insoluble Fiber on Gizzard Activity in Layers. J. Appl. Poult. Res. **14**:38-46.

LEESON, S., J. D. SUMMERS, 2005. Feeding Programs for broiler breeders. In: LEESON, S., J. D. SUMMERS: Commercial Poultry Nutrition 2005, Guelph, Ontario, ISBN 0-9695600-5-2.

RÖHE, I., I. RUHNKE, F. KNORR, A. MADER, F. GOODARZI BOROOJENI, R. LÖWE, J. ZENTEK, 2014: Effects of grinding method, particle size, and physical form of the diet on gastrointestinal morphology and jejunal glucose transport in laying hens. Poult. Sci. **93**:2060-2068.

Van KRIMPEN, M.M., R.P.KWAKKEL, B.F.J. REUVEKAMP, C.M.C. Van Der PEET-SCHWERING, L.A. Den HARTOG, M.W.A. VERSTEGEN, 2005. Impact of feeding management on feather pecking in laying hens. World's Poult. Sci. J. **61**:663-686.
P-247 (ID 210)

DOES CAGE EFFECT REDUCTION THROUGH XYLANASE HAVE AN IMPORTANT EFFECT ON INTESTINAL MICROBIOTA?

Alireza Khadem<fnref n="1" />, Marta Lourenço<fnref n="1" />, Evelyne Delezie<fnref n="2" />, Luc Maertens<fnref n="2" />, Anne Goderis<fnref n="3" />, Ronny Mombaerts<fnref n="3" />, Monica Höfte<fnref n="4" />, Venessa Eeckhaut<fnref n="5" />, Filip Van Immerseel<fnref n="5" />, Geert P.J. Janssens<fnref n="1" />

**Corresponding author:** Alireza.Khadem@UGent.be

**Abstract**

Reduction of the cage effect (release of encapsulated nutrients) is one of the working mechanisms of xylanase, resulting in altered substrate for gut microbial fermentation. However, the true mechanism by which gut microbiota nutritionally are affected is not fully understood. Therefore, 720 1-d-old broiler chicks were randomly allocated to 24 pens. A corn-based diet was chosen to rule out the enzyme's effect on digesta viscosity. The corn was first frozen (5 days at -18°C) or not to separate the cage effect. Both diets were mixed with or without bacterial endo-1,4-β-xylanase. At day 34, caecum contents were collected for microbiota analysis (qPCR) and fresh digesta were taken for viscosity measurements. As expected, viscosity was not affected by enzyme or freezing. The bacterial xylanase significantly increased the numbers of Clostridium cluster IV (butyrate producers), total bacteria and the number of butyryl CoA-acetate CoA-transferase gene copies. Freezing corn resulted in increased total bacteria numbers and a tendency to increased Clostridium cluster IV. The results suggest the bacterial xylanase tested likely affected the gut microbial profile in 2 ways: 1) supply of fermentable arabinoxylan fragments and 2) changing digesta composition as substrate for the gut microbiota.

**Keywords:** cage effect, xylanase, frozen corn, gut microbiota.

**Introduction**

The use of xylanase is almost ubiquitous in poultry feeds. Nevertheless, the majority of research conducted with these enzymes has focused on determining their effects on animal performance and nutrient digestibility, and less on their effects on the intestinal microbiota.

Two major mechanisms have been proposed to explain the beneficial effects of xylanase on intestinal microbiota: 1) altering nutrient delivery to the host and gut microbiota 2) provision of fermentable oligosaccharides (Bedford and Cowieson, 2012). It is not clear, however the extent to which these mechanisms contribute to the beneficial effect of xylanase on gut microbiota. The aim of the present study was to assess how xylanase could affect intestinal microbiota in broilers.

**Material and Methods**

The experimental design was approved by the local Ethics Committee for Animal Experiments at the ILVO. A total of 720 male broiler chickens (Ross 308 Commercial strain) were purchased from a commercial hatchery (Belgabroed, Merksplas, Belgium) and were allocated at random in equal numbers of 24 floor pens (thirty chickens per pen) in an environmentally-controlled poultry house. Chickens received corn-soy based diets.

Half of the corn was frozen at -18 °C for 5 days, and milled after thawing. The experiment consisted of 4 dietary treatments 1) normal corn without bacterial endo-1,4-β-xylanase; 2) frozen corn without bacterial endo-1,4-β-xylanase; 3) normal corn with bacterial endo-1,4-β-xylanase (Nutrase Xyla®. Nutrex, Lille, Belgium) and 4) frozen corn with bacterial endo-1,4-β-xylanase. At day 34, caecum contents were collected for microbiota analysis (qPCR) and fresh digesta were taken for viscosity. Caecum contents of the chickens were placed into a plastic jar and frozen at -80°C until analysis. To extract the DNA from the caecum content (100 mg), the CTAB method was used (Griffiths et al., 2000, Kowalchuk et al., 1998). The concentration of the DNA was measured using the Nano drop ND 1000 spectrophotometer (Nanodrop

<footnote n="1">¹ Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium</footnote>

<footnote n="2">²Animal Sciences Unit, Institute for Agricultural and Fisheries Research, Melle, Belgium</footnote>

<footnote n="3">³Nutrex NV, Lille, Belgium</footnote>

<footnote n="4">⁴Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium</footnote>

<footnote n="5">⁵Laboratory of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium</footnote>
Technologies, Wilmington, DE, USA) and diluted to a concentration of 50ng/µl to test the quality of the DNA. Primers used for quantification of the different bacterial groups and genes are mentioned in Table 1.

<table><caption>**Table 1.** Primer information for quantitative RT-PCR assay</caption><thead><tr><th>Bacterial groups/ Functional gene</th><th>Primers (5'-3')</th><th>Reference</th></tr></thead><tbody><tr><td>Enterobacteriaceae</td><td>F: CATTGACGTTACCCGCAGAAGAAGC<br>R: CTCTACGAGACTCAAGCTTGC</td><td>Bartosch et al 2004</td></tr><tr><td>Clostridium Cluster I</td><td>F: TACCHRAGGAGGAAGCCA<br>R: GTTCTTCCTAATCTCTACGCAT</td><td>Song et al 2004</td></tr><tr><td>Clostridium Cluster IV</td><td>F: GCACAAGCAGTGGAGT<br>R: CTTCCTCCGTTTTGTCAA</td><td>Matsuki et al 2004</td></tr><tr><td>Clostridium Cluster XIV</td><td>F: GAWGAAGTATYTCGGTATGT<br>R: CTACGCWCCCTTTACAC</td><td>Song et al 2004</td></tr><tr><td>Lactobacillus</td><td>F: GGAATCTTCCACAATGGACG<br>R: CGCTTTACGCCCAATAAATCCGG</td><td>Abdulamir et al 2010</td></tr><tr><td>Total Bacteria</td><td>F: CGGYCCAGACTCCTACGGG<br>R: TTACCGCGGCTGCTGGCA</td><td>Hopkins et al 2005</td></tr><tr><td>Butyryl-CoA:Acetate-CoA transferase</td><td>F: GCIGAICATTTCACITGGAAYWSITGGCAYATG<br>R: CCTGCCTTTGCAATRTCIACRAANGC</td><td>Louis and Flint 2007</td></tr></tbody></table>

**Amplifications and detections were performed according to** Eeckhaut et al. (2011). Viscosity measurements were done as described by (Bedford and classen 1992) The main effects of freezing, enzyme and their interaction were statistically analyzed by 2x2 factorial model.

**Results and Discussion**

Viscosity was not affected by xylanase addition or freezing. Xylanase addition significantly increased the number of gene copies of butyryl CoA:acetate CoA-transferase, the numbers of Clostridium cluster IV and of total bacteria in the caecum (Table 2). Freezing increased numbers of total bacteria and of Clostridium cluster IV, although for the latter it was only a trend. Numbers of Enterobacteriaceae, Clostridium cluster XIV and Lactobacillus were not affected by freezing or by xylanase addition. No interactions of freezing with xylanase were observed for any of the bacterial groups assessed or for the number of gene copies of butyryl CoA:acetate CoA-transferase. Releasing of dietary encapsulated nutrients will affect the substrate available for gut microbial fermentation. Xylanase tested affected the gut microbial profile most likely by the combination of 1) provision of fermentable arabinoxylan fragments (originating from the broken cell walls) and of 2) a change in digesta composition for gut microbiota fermentation (from the release of the entrapped nutrients).

<footnote n="1">¹F: Forward; ²R: Reverse</footnote>
<table><caption>**Table 2.** Effect of freezing of corn (F), xylanase addition (X) and their interaction (F\*X) on colony forming units of bacterial species and enzyme (log CFU·g⁻<fnref n="1" />) per 100 mg of caecal contents in broiler chickens</caption><thead><tr><th rowspan="3"></th><th colspan="4">Dietary treatment(n=6)</th><th rowspan="3">SEM<sup>1</sup></th><th colspan="3">P-value</th></tr><tr><th colspan="2">Without xylanase</th><th colspan="2">With xylanase</th><th rowspan="2">F</th><th rowspan="2">X</th><th rowspan="2">F\*X</th></tr><tr><th>Normal corn</th><th>Frozen corn</th><th>Normal corn</th><th>Frozen corn</th></tr></thead><tbody><tr><td>Butyryl CoA:acetate CoA-transferase</td><td>8.01</td><td>8.25</td><td>8.32</td><td>8.34</td><td>0.04</td><td>0.275</td><td>0.016</td><td>0.155</td></tr><tr><td>Clostridium cluster I</td><td>8.29</td><td>8.38</td><td>8.44</td><td>8.55</td><td>0.04</td><td>0.184</td><td>0.072</td><td>0.727</td></tr><tr><td>Clostridium cluster IV</td><td>9.39</td><td>9.57</td><td>9.74</td><td>10.03</td><td>0.06</td><td>0.056</td><td>0.001</td><td>0.742</td></tr><tr><td>Clostridium cluster XIV</td><td>8.42</td><td>8.36</td><td>8.38</td><td>8.54</td><td>0.03</td><td>0.381</td><td>0.326</td><td>0.105</td></tr><tr><td>Lactobacillus</td><td>8.03</td><td>8.29</td><td>8.09</td><td>8.10</td><td>0.06</td><td>0.266</td><td>0.541</td><td>0.282</td></tr><tr><td>Enterobactericeae</td><td>6.83</td><td>6.42</td><td>6.83</td><td>6.85</td><td>0.09</td><td>0.410</td><td>0.400</td><td>0.247</td></tr><tr><td>Total bacteria</td><td>10.55</td><td>10.74</td><td>10.75</td><td>11.04</td><td>0.04</td><td>0.012</td><td>0.007</td><td>0.299</td></tr></tbody></table>

## Conclusions

The changes observed for the numbers of Clostridium cluster IV (where butyrate producers are present), total bacteria and butyryl CoA-acetate CoA-transferase gene copies indicate that xylanase supplementation was able to improve the intestinal microbiota and gut health in broilers fed with a corn–soy based diet. This could partly be attributed to cage effect reduction.

## Literature

* BEDFORD, M.R., A.J. COWIESON. 2012: Exogenous enzymes and their effects on intestinal microbiology. Anim. Feed Sci. Tech. **173**, 76–85

* BEDFORD, M.R., H.L. CLASSEN. 1992. Reduction of intestinal viscosity through manipulation of dietary rye and pentosanase concentration is effected through changes in the carbohydrate composition of the intestinal aqueous phase and results in improved growth rate and food conversion efficiency. The Journal of nutrition **122**:560–569.

* EECKHAUT, V., F. VAN IMMERSEEL, S. CROUBELS, S.D BAERE, F. HAESEBROUCK, R. DUCATELLE, P. LOUIS, P. VANDAMME. 2011. Butyrate production in phylogenetically diverse Firmicutes isolated from the chicken caecum. Microb. Biotechnol. **4**:503–512.

* GRIFFITHS, R.I., A.S. WHITELEY, A.G. O'DONNELL, M.J. BAILEY. 2000: Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl. Environ. Microb. **66**, 5488-5491.

* KOWALCHUK, G.A., P.L.E. BODELIER, G.H.J. HEILIG, J.R. STEPHEN, H.J. LAANBROEK. 1998: Community analysis of ammonia-oxidising bacteria, in relation to oxygen availability in soils and root-oxygenated sediments, using PCR, DGGE and oligonucleotide probe hybridisation. FEMS Microbiol. Ecol., **27**, 339–350.

<footnote n="1">¹Standard Error of Mean.</footnote>
P-248 (ID 155)

# PERFORMANCE AND INCIDENCE OF TIBIAL DISCHONDROPLASIA IN BROILERS SUPPLEMENTED WITH 25-HYDROXY-CHOLECALCIFEROL IN DIET

M.Laboissiere¹, R.M.Jardim-Filho ², M.B.Cafe ⁴,⁵, M.A.Andrade ¹, E.Gonzales ¹, J.H.Stringhini¹

¹Universidade Estadual de Goias, Sao Luis de Montes Belos, Brazil

² Sao Salvador Alimentos, Itaberai, Goias, Brazil

³ Medicina Veterinaria, Universidade Federal de Goias, Goiânia, Brazil

⁴ Producao Animal, Universidade Federal de Goias, Goiânia, Brazil

⁵ CNPq researcher, Goiania, Brazil

**Corresponding author:** michele.ufg@hotmail.com and jhstring@uol.com.br

## Abstract

Vitamin D3 and its metabolites could reduce the occurrence of Tibial Dyschondroplasia (TD) in broilers. Then, we evaluated the incidence of TD in broilers supplemented with vitamin D3 or 25-hydroxy-cholecalciferol (25OHD3) in diets. The experiment was conducted in the Federal University of Goias, Goiania, Brazil, with 405 Cobb 500 broilers. Treatments were: T1 – diets with 4.000 UI/kg vitamin D3; T2 – diets with 25OHD3; T3 – diets with 50% of each vitamin D3 source; with five replicates of 27 birds each. Birds and diets were weighed at 1st, 7th, 14th, 21st, 35th and 42nd days of age to measure performance variables. Incidence of TD was measured weekly as the growing area of the bone with a digital caliper. The scores used were: 0 = normal growing area; 1 = +/- 3mm; 2 = +/- 6mm; 3 = above 6mm. At the same ages, a histopathological study was done and the scores used were: 0 = normal; 1 = pre-hypertrophic chondrocytes; 2 = changes in chondrocytes cytoplasm and nucleus and divided in three levels (discrete, moderate and severe). These data were submitted to Kruskall Wallis test and performance was analyzed as ANOVA with "t" test (5%). No effect was observed in performance. No effect was observed in macroscopic evaluation of lesion score, that was characterized as score 0 in all treatments. In microscopic evaluation, 20% of tibias presented TD lesions at 28 days of age in all treatments. No effects of dietetic vitamin D3 sources alone or combined were observed in performance and TD lesions.

**Keywords:** feed conversion, feed intake, leg problems, 25OHD3, weight gain

## Introduction

Tibial dyschondroplasia (TD) is one of the most worrisome skeletal diseases, and a significant indicator in terms of animal welfare. To assist TD measurement, lesions can be monitored using various techniques. However, there is little data on the damage caused by TD, due to a scarcity of identification and quantification of these skeletal diseases in the industries. The high percentage of bone tissue deformities represents one of the major problems facing the poultry industry, especially on account of the intense genetic selection performed to increase the growth rate of animals.

In the animal production field, poultry breeding has seen the biggest genetic and technological advances. The rate of TD has increased significantly and is related to the pace of growth and the maximization of muscle mass in a short period of time. In recent years, vitamin D3 and its metabolites 1,25(OH)2D3 and 25(OH)D3 have been studied in the attempt to minimize problems caused by skeletal abnormalities. The purpose of this study was to evaluate the rate of tibial dyschondroplasia in chickens supplemented with 25-hydroxycholecalciferol in feed.

## Material and Methods

The experiment was conducted in the experimental breeding house at the Animal Production Department of the Veterinary and Animal Science School (Escola de Veterinária e Zootecnia - EVZ) of the Federal University of Goiás – UFG in Goiânia, Goiás, Brazil. The experimental protocol used in this study was approved by the Research Ethics Committee of UFG, under protocol no. 014/12.

To conduct the experiment, 405 one-day-old male chicks of the Cobb 500® breed were housed, from a single matrix lot (45 weeks old). The eggs were incubated under the same conditions, in a multiple stage machine. After hatching, the one-day-old chicks were standardized by weight and housed in three different groups of 135 birds, with five repetitions of 27 birds per box, reaching 15 experimental units, in which an entirely random outline was adopted. The experiment consisted of three treatments, from two sources of vitamin D supplementation (vitamin D3 and 25(OH)D3) described below: Treatments: T1: Control = the control group received the experimental diet containing vitamin D3 at a dose of
4,000,000 U.I./kg; T2: 25(OH)D3 = received no vitamin D3 and its equivalence was replaced by including the metabolite 25(OH)D3 in the experimental diet; T3: D3 + 25(OH)D3 = consisted of the combination of two sources of vitamin D, where the proportion in feed was 50% from each source used.

The birds and diets were weight on the 1st, 7th, 14th, 21st, 35th and 42nd days of age, and these data were used to calculate weight gain, feed consumption, feed conversion and viability for study of the performance variables during breeding phases. Each week the number of birds was reduced due to weekly collections of one bird per box for necropsies and laboratory analyses. One bird with identification per box was removed weekly, weighed and sacrificed by cervical dislocation, proceeding to remove the legs (leg + thigh + foot). The macroscopic evaluations of the birds' tibia were performed at 7, 14, 21, 28 and 35 days of age.

The rate of TD was assessed weekly by macroscopic viewing of the growth plate of the proximal portion of the right tibia, cutting the bone lengthwise. A digital caliper was used to measure the thickness of the growth plate. The methodology adopted was that described by TAKITA (1998) and presented by GONZALES & MACARI (2000), which stipulates the following scores: Score 0 = normal growth plate; Score 1 = growth plate with +/- 3mm thickening; Score 2 = growth plate with +/- 6mm thickening; Score 3 = growth plate with thickening above 6mm. To verify possible TD alterations during macroscopic evaluations of the right tibia, a histopathological study of the growth plates of the proximal portion of the marked birds' tibia was performed at 1, 7, 14, 21, 28 and 35 days of age. The methodology adopted was that described by TAKITA (1998), which histologically characterizes TD severity according to the following scores: Score 0 = normal growth plate; Score 1 = growth plate with a wide and distinct band of pre-hypertrophic chondrocytes; Score 2 = growth plates presenting degenerative changes in the cytoplasm and nucleus of chondrocytes, which in this study was subdivided into three levels (discrete, moderate and severe).

The data from the macroscopic and histological evaluation of the tibia growth plate were subjected to non-parametric statistical analysis, using the Kruskall Wallis test of the R (2.14.2) software statistical program. For statistical evaluation of performance, the data were subjected to analysis of variance, ANOVA, using the 5% significance level.

## Results and Discussion

There was no effect (p>0.05) for initial weight, final weight, average weight gain, average feed consumption, mortality and feed conversion (Table 1).

The macroscopic lesion scores from the proximal portion of the tibia bone, assessed at 7, 14, 21, 28 and 35 days old, presented no effect (p>0.05) from the different sources of vitamin D3 (Table 2). Upon macroscopically evaluating the growth plate thickening of the proximal portion of the tibia, it was observed that both treatments rated in the score (0) classification = normal TD growth plate.

<table><caption>**Table 1** Performance of chickens at 42 days of age, supplemented with different sources and combinations of vitamin D in feed</caption><thead><tr><th></th><th>AIW, g<sup>1</sup></th><th>AFW, g</th><th>AWG, g</th><th>AFC, g</th><th>FC</th><th>Mort, %</th></tr></thead><tbody><tr><td>D<sub>3</sub></td><td>45.92</td><td>3005</td><td>3003</td><td>5517</td><td>1.8358</td><td>4.5</td></tr><tr><td>25(OH)D<sub>3</sub></td><td>46.14</td><td>2997</td><td>2994</td><td>5439</td><td>1.8148</td><td>6.3</td></tr><tr><td>D<sub>3</sub> + 25(OH)D<sub>3</sub></td><td>46.17</td><td>3050</td><td>3048</td><td>5626</td><td>1.8448</td><td>4.5</td></tr><tr><td>P</td><td>****</td><td>0.14</td><td>0.14</td><td>0.37</td><td>0.74</td><td></td></tr><tr><td>C.V%</td><td>1.58</td><td>1.40</td><td>1.39</td><td>3.65</td><td>3.42</td><td>-</td></tr></tbody></table>

<table><caption>**Table 2** Average of macroscopic lesion scores from the proximal portion of the tibia bone, at 7, 14, 21, 28 and 35 days old, fed with different sources and combinations of vitamin D3</caption><thead><tr><th rowspan="2">Macroscopic lesion score</th><th colspan="3">Treatments</th><th rowspan="2">P *</th></tr><tr><th>D<sub>3</sub></th><th>25(OH)D<sub>3</sub></th><th>D<sub>3</sub> + 25(OH)D<sub>3</sub></th></tr></thead><tbody><tr><td>7 days</td><td>0.2</td><td>0.0</td><td>0.6</td><td>0.1078</td></tr><tr><td>14 days</td><td>0.4</td><td>0.6</td><td>0.2</td><td>0.4594</td></tr><tr><td>21 days</td><td>0.6</td><td>0.6</td><td>0.4</td><td>0.7788</td></tr><tr><td>28 days</td><td>0.8</td><td>0.6</td><td>0.2</td><td>0.1737</td></tr><tr><td>35 days</td><td>0.2</td><td>0.0</td><td>0.2</td><td>0.5836</td></tr></tbody></table>

<footnote n="1">¹ Upon housing, average initial weight AIW; average final weight AFW; average weight gain AWG; average feed consumption AFC; feed conversion FC; mortality Mort. NS effect = not significant (p>0.05).</footnote>

<footnote>* Not significant difference for Kruskal-Wallis test (p>0.05).</footnote>
When comparing the macroscopic results of lesion scores with the histopathological analyses, the non-existence of TD was confirmed. It was confirmed that there was no effect (p>0.05) from the different sources and combinations of vitamin D3 used in this experiment (Table 3). In the results, higher rates for TD were not found in either of the treatments (Table 4). Slight TD rates were noted, regardless of the treatment used, but without significant effect.

ATENCIO et al. (2005) concluded, in experiments conducted with reproducers, that the effect of the metabolite 25(OH)D3 in relation to vitamin D3 depends on the levels tested, and when compared to the sources of vitamin D3, the effect of the metabolite 25(OH)D3 was only higher when supplemented at low levels. According to BRITO (2008), the higher effectiveness of the metabolite 25(OH)D3 in reducing bone problems (reduction in rate) may be one of the explanations for improvement in the uniformity of lots reported in the field, and in the reduction in condemnations in slaughter. The results demonstrated that the combined use of the two sources, at the lower level of 25(OH)D3 supplementation in feed, generated similar values in rate of TD, when compared to the growing levels of 25(OH)D3 along, confirming the benefits of the more active metabolite as a source of vitamin D.

<table><caption>**Table 3** Histological lesions score for tibial dyschondroplasia (TD) in the proximal portion of the growth plate, at 21, 28 and 35 days old, fed with different sources and combinations of vitamin D in feed</caption><thead><tr><th rowspan="2">TD microscopic lesion score</th><th colspan="3">Treatments</th><th rowspan="2">P *</th></tr><tr><th>D<sub>3</sub></th><th>25(OH)D<sub>3</sub></th><th>D<sub>3</sub> + 25(OH)D<sub>3</sub></th></tr></thead><tbody><tr><td>21 days</td><td>0.0</td><td>0.2</td><td>0.2</td><td>0.5836</td></tr><tr><td>28 days</td><td>0.0</td><td>0.0</td><td>0.2</td><td>0.3678</td></tr><tr><td>35 days</td><td>0.2</td><td>0.0</td><td>0.0</td><td>0.3678</td></tr></tbody></table>

<table><caption>**Table 4** Frequency and severity of tibial dyschondroplasia (TD) in the proximal portion growth plate, at 21, 28 and 35 days old, fed with different sources and combinations of vitamin D in feed</caption><thead><tr><th rowspan="2">Age (days)</th><th rowspan="2">Treatments</th><th rowspan="2">TD (%)</th><th colspan="5">Degree of lesion (Score)</th></tr><tr><th>0</th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td rowspan="3">21</td><td>D<sub>3</sub></td><td>0</td><td>5/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>25(OH)D<sub>3</sub></td><td>20</td><td>4/5</td><td>1/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>D<sub>3</sub> + 25(OH)D<sub>3</sub></td><td>20</td><td>4/5</td><td>1/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td rowspan="3">28</td><td>D<sub>3</sub></td><td>0</td><td>5/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>25(OH)D<sub>3</sub></td><td>0</td><td>5/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>D<sub>3</sub> + 25(OH)D<sub>3</sub></td><td>20</td><td>4/5</td><td>1/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td rowspan="3">35</td><td>D<sub>3</sub></td><td>20</td><td>4/5</td><td>1/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>25(OH)D<sub>3</sub></td><td>0</td><td>5/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr><tr><td>D<sub>3</sub> + 25(OH)D<sub>3</sub></td><td>0</td><td>5/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr></tbody></table>

## Conclusions

The combined use of two sources of vitamin D supplementation (vitamin D3 and 25(OH)D3) did not alter the performance of chickens and did not aggravate tibial dyschondroplasia problems. It can thus contribute as a supplement source to minimize the effects of bone problem rates and condemnations in slaughter.

## Literature

ATENCIO, A.; PESTI, G.M; EDWARDS, JR. H.M. Twenty-five hydroxy-cholecalciferol as a cholecalciferol substitute in broiler breeder hen diets and its effect on the performance and general health of the progeny. Poultry Science, v.84, p.1277-1285, 2005.

BRITO, J. A. G. Vitamina D3 (Colecalciferol) e 25-Hidroxi-Colecalciferol (25-OHD3) em Rações de Frangos de Corte. Lavras (MG): UFLA, 2008. 120p. Tese (Doutorado) Universidade Federal de Lavras, 2008.

GONZALES, E.; MACARI, M. Doenças das Aves. Editora Facta. Campinas, SP, 2000.

SBCAL/COBEA: Sociedade Brasileira de Ciência em Animais de Laboratório (SBCAL) / Colégio Brasileiro de Experimentação Animal (COBEA) Proteção e Bem Estar de Animais de Laboratório. http://www.cobea.org.br/.

TAKITA, T. S. Efeito do genótipo, do ambiente e da interação genótipo x ambiente na incidência de dischondroplasia tibial em frangos de corte machos. Dissertação (Mestrado) Instituto de Biociências de Botucatu, Universidade Estadual Paulista, 43f, 1998.

<footnote>* Not significant difference for Kruskal-Wallis test (p<0.05).</footnote>
P-249 (ID 189)

EMULSIFIER INFLUENCING FAT AND GROSS ENERGY DIGESTIBILITY OF BROILERS FED DIETS CONTAINING
FAT POWDER

S. Leleu <fnref n="1" />, D. Hermans<fnref n="2" />, M. De Laet<fnref n="2" />, E. Delezie<fnref n="1" />, and L. Maertens<fnref n="1" />

**Corresponding author:** saskia.leleu@ilvo.vlaanderen.be

**Abstract**

Dietary emulsifiers can be used to enhance fat digestibility, and thus increase the energy efficiency. This can be profitable for young birds having only a limited natural fat digestibility, and for lowering the energy content of the diets resulting in a lower feed cost. The objective of the current digestibility trial was to evaluate the effect of an emulsifier (0.01%) – with a hydrophilic-lipophilic balance (HLB) of around 17-18 – on the fat digestibility coefficient (DC, via total fecal collection method) of fast growing male broilers (age of 18-22 days) fed two types of diets. Diet A had a high free fatty acid (FFA) content (20.0%) and an unsaturated/saturated FA ratio (U/S) of 1.60; while diet B had a FFA of 10.0% and a U/S of 2.00. However, both diets had the same ME (12.27 MJ/kg) and crude fat (8.5%) content. Soy oil and two types of fat powder were used as lipid sources in both diets. The emulsifier was added on top of half of each diet. As expected, the dietary fat and gross energy (GE) DC were significantly lower when diet A (with and without emulsifier) was fed (67.0% vs 73.9%, and 71.9% vs 73.8%, resp.). The emulsifier improved dietary fat digestibility with 2.8% (trend) and 4.2% (significant) for diet A and B, resp. The energy efficiency (MEn) was improved with 214 kJ (trend) and 292 kJ (significant) for diet A and B, resp. It can be concluded that the energy content and thus the feed cost can be lowered when the emulsifier is added, even when a fat powder is used.

**Keywords:** emulsifier, fat powder, U/S ratio, free fatty acid, broiler

**Introduction**

Mixing an emulsifier into the diet reduces the size of the fat globules and increases the total surface available for enzymatic digestion (Jones et al., 1992). As a result, dietary emulsifiers can be used to enhance fat digestibility, and thus increase the energy efficiency. This can be profitable for young birds having only a limited natural fat digestibility (Al-Marzooqi and Leeson, 1999), and for lowering the energy content of the diets resulting in a lower feed cost.

The objective of the trial was to evaluate the effect of the emulsifier tested on the digestibility of feed containing fat powder.

**Material and Methods**

Day-old male broilers (Ross 308) were housed on deep litter under conventional conditions for lighting, heating and ventilation. They were fed *ad libitum* the same standard starter with animal fat as main fat source.

From day 13 to 22, the birds were brought to the digestibility unit and placed in the balance cages. The balance cages consist of a wire bottom (with below the bottom a plastic tray for excreta collection), a feed trough in front, and a drinking cup at the back side. Groups of 5 birds (at 13 days of age) were assigned randomly to each of the 24 digestibility cages and received from than off their experimental diet. At day 18 an additional selection was done in order to have 4 comparable and healthy birds. During the main period (18-22 days of age) the birds were fed at a nearly *ad libitum* level, while water was freely available. The balance trial was further carried out according to the EU-reference method (Bourdillon *et al.*, 1990).

The experiment consisted of four treatments, each with six repetitions:

* T1: grower diet A

* T2: T-1 + Emulsifier (0.01% ; on top)

* T3: grower diet B

* T4: T-3 + Emulsifier (0.01% ; on top)

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

<footnote n="2">² Nuscience, Booiebos 5, 9031 Drongen, Belgium</footnote>
The grower diets used for the balance trial were wheat-soy-corn based. The two grower diets differed in FFA content and U/S ratio (Table 1). This was achieved by using different concentrations of soy oil and two types of fat powder as lipid sources in both diets.

<table><caption>**Table 1.** Calculated nutrient content of the grower diets</caption><thead><tr><th></th><th>FFA (%)</th><th>U/S ratio</th><th>Crude fat (%)</th><th>ME (MJ/kg)</th></tr></thead><tbody><tr><td>Grower diet A</td><td>20.0</td><td>1.60</td><td>8.5</td><td>12.27</td></tr><tr><td>Grower diet B</td><td>10.0</td><td>2.00</td><td>8.5</td><td>12.27</td></tr></tbody></table>

The two grower diets were prepared and divided in two equal parts. Using a small mixer, the emulsifier was added to produce the four experimental diets. The dietary levels of all nutrients were in agreement with the requirements of broilers following the AVIAGEN requirements.

Samples of feeds and freeze-dried excreta were analysed in the ILVO accredited laboratory for gross energy (GE; adiabatic calorimetry), nitrogen (macro-Kjeldahl; protein = N x 6.25) and fat (ether extract). The MEn-contents of the experimental diets were calculated from their respective "freeze-dried" excreta/feed (E/F) ratios as well as their corresponding GE-contents, followed by a correction for N-retention to zero, by using an energy equivalent of 34.36 kJ/g N-retained. The retention of fat was calculated from the E/F-ratio as well as the corresponding fat contents in the feed and the excreta.

All digestibility parameters were analysed by a linear mixed model with diet type and addition of the emulsifier as fixed factors (Statistica 12.5). Data that differ significantly have a P-value below 0.05.

## Results

The analysed dietary crude fat of the grower diets A and B was somewhat higher than the calculated content, and amounted to 9.24% and 8.92%, respectively.

No significant effect on feed intake was found. However, the birds treated with the emulsifier produced a significantly higher (17%) amount of excreta (fresh).

The E/F ratio was significantly influenced by the diet and the addition of the emulsifier: diet B had lower ratios compared to diet A and the addition of the emulsifier lowered the ratio in comparison to when no emulsifier was added (Table 2). As expected, diet B had a significantly higher fat and GE digestibility compared to diet A (with and without emulsifier) (67.0% vs 73.9%, and 71.9% vs 73.8%, resp.), because diets with a low U/S ratio and a high FFA content result in lower fat digestibility. Adding the emulsifier also significantly improved the fat and GE digestibility. Roy *et al.* (2010) also found that the use of a dietary emulsifier proved to be a tool to improve the utilization of fats in birds. Dietary fat digestibility was improved by the emulsifier with 2.8% (trend) and 4.2% (significant) for diet A and B, resp. The energy efficiency (MEn) was improved with 214 kJ (trend) and 292 kJ (significant) for diet A and B, resp.

<table><caption>**Table 2.** Excreta/feed (E/F) ratio, digestibility data and energy content of the diets.</caption><thead><tr><th rowspan="3">Grower diet</th><th colspan="4">Treatment (mean ± SD*)</th><th rowspan="3">Diet effect</th><th rowspan="3">Emulsifier effect</th></tr><tr><th>T1</th><th>T2</th><th>T3</th><th>T4</th></tr><tr><th>A (FFA:20.0%; US:1.60)</th><th>A (FFA:20.0%; US:1.60)</th><th>B (FFA:10.0%; US:2.00)</th><th>B (FFA:10.0%; US:2.00)</th></tr></thead><tbody><tr><td>Emulsifier</td><td>-</td><td>+</td><td>-</td><td>+</td><td></td><td></td></tr><tr><td>E/F ratio</td><td>0.301 ±0.011</td><td>0.292 ±0.005</td><td>0.288 ±0.010</td><td>0.273 ±0.009</td><td>&lt;0.001</td><td>&lt;0.01</td></tr><tr><td>Fat digestibility (%)</td><td>65.5 ±3.0</td><td>68.3 ±1.6</td><td>71.9 ±1.4</td><td>76.0 ±1.3</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>GE digestibility (%)</td><td>71.3 ±1.2</td><td>72.4 ±0.6</td><td>73.1 ±1.1</td><td>74.6 ±0.8</td><td>&lt;0.001</td><td>&lt;0.01</td></tr><tr><td>ME (kJ/kg DM)</td><td>14,166 ±239</td><td>14,381 ±127</td><td>14,375 ±215</td><td>14,672 ±167</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>N retention (kJ/kg)</td><td>830 ±14</td><td>830 ±10</td><td>803 ±19</td><td>808 ±17</td><td>&lt;0.001</td><td>NS<sup>§</sup></td></tr><tr><td>MEn (kJ/kg DM)</td><td>13,326 ±226</td><td>13,540 ±118</td><td>13,562 ±198</td><td>13,853 ±154</td><td>&lt;0.01</td><td>&lt;0.01</td></tr></tbody></table>

<footnote>*SD: Standard Deviation, §NS: not significant</footnote>
Conclusions

It can be concluded that the energy content and thus the feed cost can be lowered when the emulsifier is added, even when a fat powder is used and at different U/S ratios and FFA contents.

Literature

AL-MARZOOQI, W. and S. LEESON, 1999: Evaluation of dietary supplements of lipase, detergent, and crude porcine pancreas on fat utilization by young broiler chicks. Poultry Science, **78**, 1561-1566.

BOURDILLON, A., B. CARRÉ, L. CONAN, J. DUPERRAY, G. HUYGHEBAERT et al., 1990: European reference method for the determination of metabolisable energy with adult cockerels: reproducibility, effect of food intake and comparison with individual laboratory methods. British Poultry Science, **31**, 557-565.

JONES, D.B., J.D. HANCOCK, D.L. HARMON and C.E. WALKER, 1992: Effects of exogenous emulsifiers and fat sources on nutrient digestibility, serum lipids, and growth performance in weanling pigs. J. Anim. Sci. **70**, 3473-3482.

ROY, A., S., HALDAR, S. MONDAL and T. KUMAR GHOSH, 2010. Effects of supplemental exogenous emulsifier on performance, nutrient metabolism, and serum lipid profile in broiler chickens. Veterinary Medicine International, 2010, Article ID 262604. doi:10.4061/2010/262604
P-254 (ID 322)

# THE EFFECT OF INCREASING METABOLIZABLE ENERGY, LEVEL AND DURATION OF FEED RESTRICTION ON PERFORMANCE AND BLOOD PARAMETERS IN BROILER CHICKENS

Z. Zanganeh, S. Moradi, and A. Abdolmohammadi

Department of Animal Science, Faculty of Agriculture and Natural Resources, Razi University, Kermanshah, Iran

**Corresponding Author:** s.moradi@razi.ac.ir

## Abstract

The effect of increasing metabolizable energy (ME), level and duration of feed restriction on performance and serum metabolites was studied in broiler chickens fed pellet diets. A total of 480 Ross 308 male broiler chickens were randomly allocated to 12 treatments, each one consisted of 4 replicates, and 10 birds per replicate in 3×2×2 factorial arrangement. Experimental treatments consisted two levels of metabolizable energy (normal, and +100kcal/kg), two levels of feed restriction (90% and 85% restricted feed than recommended of Ross 308 management guide), and three periods of feed restriction duration including 14, 21 and 28 days. Results indicated that BW significantly increased in broilers fed high-ME diets and 90% feed restriction (P<0.01), except that, high-ME diets decreased BW at 7 days of age. FCR in High-ME diets significantly increased at 7 days of age, but decreased at 14 and 21 days of age (P<0.05). Broilers with 90% feed restriction and high-ME diets had lowest glucose concentration at 21 days, but interestingly, had highest glucose concentration at 35 days of age (P<0.01). Increasing ME level, elevated serum TG concentration at 42d of age (P<0.01). But increasing level and duration of feed restriction decreased TG level (P<0.05). In conclusion, energy level, duration and level of feed restriction in pellet diets affected performance and economical outcome of broiler farms, and need more researches to prepare relevant and practical program.

**Keywords:** Metabolizable energy, Pellet, Feed restriction, Broiler

## Introduction

Feeding strategies including pelleting and increasing metabolizable energy in broiler chickens resulted in higher growth performance and improving feed conversion ratio, while, increasing occurrence of metabolic disorders, fat deposition and mortality. One possible nutritional approach to reduce feed cost, improvement FCR and decreasing the occurrence of metabolic disorders and mortality is to restrict feed intake of the birds. Improvement in FCR and decrease in metabolic disorders noted with the use of feed restriction programs is due to reduced overall maintenance requirements and basal metabolic rate in feed-restricted birds linked with a smaller body weight during early growth (Rincon and Lesson, 2002). Therefore, the main objective of this study was to determine the suitable levels and timing of feed restriction regimes and level of metabolizable energy in pellet diet on productivity and blood parameters in broiler chickens.

## Material and Methods

The research protocol was approved by the Razi university animal care and use committee. A total of 480 one-d-old Ross 308 male broilers were placed in 48 individual cages and 12 treatments, arranged factorially including: 2 levels of ME (normal, and normal+100 kcal.kg⁻¹), 2 levels of restriction intensity (90% and 85% restricted feed than recommended of Ross 308 management guide), and 3 periods of restriction duration (8-21, 8-28 and 8-35d of age). Each treatment replicated 4 times. Body weight and feed intake were recorded weekly. At 21, 28, 35 and 42d of age one chick per each replicate were randomly selected and bled for GLU, TG and T₃ serum analysis. Data analyzed using GLM procedure of SAS software (2011).

## Results

No interaction between ME content of the diet and level and duration of feed restriction (FR) were observed for BW and FCR (Table 1). Increasing ME level of diet significantly improved BW and FCR from 8 to 21 and 1 to 42d of age (P<0.01), but interestingly, except for first week that decreased BW and elevated FCR (P<0.05). level of FR had no significant effect on BW and FCR at 8 to 21 and 22 to 42d of age but from 1 to 42d of age, high-restriction level (85%) decreased BW (P<0.01). The present study indicates that chickens subjected to 85% FR for complete recovery of BW needed more time rather than 90% FR (Lippenz et al., 2000). Applying feed restriction during ages 8-35 days significantly affected performance and decreased BW (P<0.01), also increased FCR (P<0.05) at 22-42 and 1-42 days of age.