chicks, whose maternal received HMSeBA attained body mass almost 6% higher than the birds of Se-Yeast group. The both groups had similar liveability and feed intake (Tables 3 and 4) but HMSeBA group attained 0.06 better feed conversion rate (Table 4) than Se-yeast group.

<table><caption>Table 1. Body weight gain of chicks obtained at the first incubation (g/chick).</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="3">HMSeBA (n=10)</th><th colspan="3">Se-Yeast (n=10)</th><th rowspan="2">P=</th></tr><tr><th>Mean</th><th>Stderr</th><th>CV</th><th>Mean</th><th>Stderr</th><th>CV</th></tr></thead><tbody><tr><td>Number of eggs placed hatchery</td><td>35</td><td>0.00</td><td>0.00</td><td>35</td><td>0.00</td><td>0.00</td><td>0.0000</td></tr><tr><td>No of total chicks at hatching</td><td>22.40</td><td>1.56</td><td>22.09</td><td>21.22</td><td>1.31</td><td>18.52</td><td>0.5762</td></tr><tr><td>Body weight at day 0</td><td>49.25</td><td>1.28</td><td>0.28</td><td>50.78</td><td>0.78</td><td>3.43</td><td>0.1021</td></tr><tr><td>B.W. gain at 7 days old</td><td>134.66</td><td>2.00</td><td>3.32</td><td>119.81</td><td>4.90</td><td>9.15</td><td><strong>0.0230</strong></td></tr><tr><td>B.W. gain at 14 d old</td><td>445.89</td><td>5.92</td><td>2.97</td><td>419.80</td><td>9.27</td><td>4.94</td><td><strong>0.0451</strong></td></tr><tr><td>B.W. gain at 21 days old</td><td>893.38</td><td>12.80</td><td>3.20</td><td>855.43</td><td>1.94</td><td>3.12</td><td>0.0620</td></tr><tr><td>B.W. gain at 28 days old</td><td>1756.64</td><td>21.87</td><td>2.78</td><td>1725.51</td><td>18.06</td><td>2.34</td><td>0.3042</td></tr><tr><td>B.W. gain at 35 days old</td><td>2620.76</td><td>34.77</td><td>2.97</td><td>2555.55</td><td>40.84</td><td>3.57</td><td>0.2587</td></tr><tr><td>B.W. at 35 days old</td><td>2670.01</td><td>21.99</td><td>2.72</td><td>2606.32</td><td>40.11</td><td>3.44</td><td>0.3201</td></tr><tr><td>Expected body weight at 35 days old</td><td colspan="3">2021</td><td colspan="3">Difference</td><td></td></tr><tr><td>No of birds at 35 days old</td><td colspan="3">209 (107+102)</td><td colspan="3">202 (103+99)</td><td>7 (3.5%)</td></tr><tr><td>Body Mass (kg/group)</td><td colspan="3">558 (2670x209)</td><td colspan="3">526 (2606x202)</td><td>32 (6%)</td></tr><tr><td>Liveability (%)</td><td colspan="3">93% (209/224)</td><td colspan="3">92% (202/220)</td><td>1%</td></tr></tbody></table>

<table><caption>Table 2. Cumulative feed intake and feed conversion efficiency of chicks obtained at the first incubation.</caption><thead><tr><th rowspan="2">Weeks of age</th><th colspan="4">Feed Intake (g/chick)</th><th colspan="4">Feed Conversion Rate (g feed/g gain).</th></tr><tr><th colspan="2">HMSeBA</th><th colspan="2">Se-Yeast</th><th rowspan="2">P=</th><th colspan="2">HMSeBA</th><th colspan="2">Se-Yeast</th><th rowspan="2">P=</th></tr><tr><th></th><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th><th></th><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th></tr></thead><tbody><tr><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2</td><td>159.68</td><td>7.34</td><td>161.28</td><td>8.01</td><td>0.8863</td><td>1.19</td><td>0.05</td><td>1.36</td><td>0.10</td><td>0.1624</td></tr><tr><td>3</td><td>605.79</td><td>19.95</td><td>618.85</td><td>31.97</td><td>0.7379</td><td>1.36</td><td>0.03</td><td>1.48</td><td>0.09</td><td>0.2229</td></tr><tr><td>4</td><td>1483.92</td><td>42.05</td><td>1503.51</td><td>71.12</td><td>0.8185</td><td>1.46</td><td>0.04</td><td>1.46</td><td>0.09</td><td>0.3654</td></tr><tr><td>5</td><td>2553.23</td><td>68.30</td><td>2530.78</td><td>97.68</td><td>0.3343</td><td>1.45</td><td>0.04</td><td>1.47</td><td>0.05</td><td>0.5004</td></tr><tr><td>6</td><td>3973.32</td><td>89.20</td><td>3859.62</td><td>108.3</td><td>0.1663</td><td>1.52</td><td>0.04</td><td>1.51</td><td>0.04</td><td>0.4107</td></tr><tr><td>Expected at 6 weeks of age</td><td colspan="3">3248</td><td colspan="3"></td><td colspan="3">1.61</td><td></td></tr></tbody></table>

<table><caption>Table 3. Growth performance (g/chicks) of chicks obtained at the second incubation.</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="3">HMSeBA (n=10)</th><th colspan="3">Se-Yeast (n=10)</th><th rowspan="2">P=</th></tr><tr><th>Mean</th><th>Stderr</th><th>CV</th><th>Mean</th><th>Stderr</th><th>CV</th></tr></thead><tbody><tr><td>Number of eggs placed hatchery</td><td>35.00</td><td>0</td><td>0</td><td>33.70</td><td>1.3</td><td>12.2</td><td>0.3306</td></tr><tr><td>No of total chicks at hatching</td><td>22.80</td><td>1.5</td><td>20.76</td><td>21.80</td><td>1.39</td><td>20.15</td><td>0.6302</td></tr><tr><td>Body weight (B.W.) at day 0</td><td>44.67</td><td>0.35</td><td>1.77</td><td>44.09</td><td>0.26</td><td>1.29</td><td>0.2230</td></tr><tr><td>B.W. gain at 7 d old</td><td>90.70</td><td>2.83</td><td>6.98</td><td>89.42</td><td>2.51</td><td>6.27</td><td>0.7429</td></tr><tr><td>B.W. gain at 14 d old</td><td>349.52</td><td>8.06</td><td>5.16</td><td>341.86</td><td>8.56</td><td>5.60</td><td>0.5332</td></tr><tr><td>B.W. gain at 21 d old</td><td>955.95</td><td>17.24</td><td>4.03</td><td>924.98</td><td>13.30</td><td>3.21</td><td>0.1927</td></tr><tr><td>B.W. gain at 28 d old</td><td>1708.89</td><td>37.84</td><td>4.95</td><td>1658.12</td><td>24.09</td><td>3.25</td><td>0.2906</td></tr><tr><td>B.W. gain at 35 d old</td><td>2486.39</td><td>42.62</td><td>3.83</td><td>2480.79</td><td>50.31</td><td>4.53</td><td>0.4724</td></tr><tr><td>B.W. at 35 d old</td><td>2531.06</td><td>42.46</td><td>3.75</td><td>2480.79</td><td>50.31</td><td>4.53</td><td>0.4671</td></tr><tr><td>Expected body weight at 35 days old</td><td colspan="3">2021</td><td colspan="3">Difference</td><td></td></tr><tr><td>No of birds at 35 days old (male+female)</td><td colspan="3">210 (99+111)</td><td colspan="3">202 (94+108)</td><td>8 (3.5%)</td></tr><tr><td>Body Mass (kg/group)</td><td colspan="3">531 (2531x210)</td><td colspan="3">501 (2480x202)</td><td>30 (6%)</td></tr><tr><td>Liveability (%)</td><td colspan="3">92% (210/228)</td><td colspan="3">92% (202/218)</td><td>-</td></tr></tbody></table>
<table><caption>**Table 4.** Cumulative feed intake and feed conversion efficiency of chicks obtained at the second incubation.</caption><thead><tr><th rowspan="2">Weeks of age</th><th colspan="4">Feed Intake (g/chick)</th><th colspan="4">Feed Conversion Rate (g feed/g gain).</th></tr><tr><th colspan="2">HMSeBA</th><th colspan="2">Se-Yeast</th><th rowspan="2">P=</th><th colspan="2">HMSeBA</th><th colspan="2">Se-Yeast</th><th rowspan="2">P=</th></tr><tr><th>1</th><th>Mean</th><th>sderr</th><th>Mean</th><th>Sderr</th><th>Mean</th><th>sderr</th><th>Mean</th><th>Sderr</th></tr></thead><tbody><tr><td>2</td><td>114.65</td><td>5.78</td><td>111.06</td><td>7.79</td><td>0.5276</td><td>1.25</td><td>0.04</td><td>1.24</td><td>0.08</td><td>0.3143</td></tr><tr><td>3</td><td>482.59</td><td>21.94</td><td>489.95</td><td>15.22</td><td>0.8743</td><td>1.38</td><td>0.06</td><td>1.43</td><td>0.06</td><td>0.5344</td></tr><tr><td>4</td><td>1295.50</td><td>40.34</td><td>1278.83</td><td>27.25</td><td>0.7558</td><td>1.35</td><td>0.04</td><td>1.38</td><td>0.03</td><td>0.6059</td></tr><tr><td>5</td><td>2336.88</td><td>42.47</td><td>2323.26</td><td>31.90</td><td>0.8042</td><td>1.37</td><td>0.03</td><td>1.40</td><td>0.02</td><td>0.4355</td></tr><tr><td>6</td><td>3702.46</td><td>59.95</td><td>3765.21</td><td>46.41</td><td>0.4318</td><td>1.49</td><td>0.01</td><td>1.55</td><td>0.04</td><td>0.2324</td></tr><tr><td>Expected at 6 weeks of age</td><td colspan="4">3248</td><td colspan="4">1.61</td></tr></tbody></table>

**Conclusion**

The results obtained in the experiment showed that chicks from the maternal receiving HMSeBA had numerically higher (P>0.05) slaughter weight and better (P>0.05) feed conversion rate than chicks of Se-Yeast group.

The performance values of broilers obtained from the both groups were found to be greatly higher than breeder's target in both trials.

The results of the present study reported here suggest that providing selenium in organic form, especillay HMSeBA could be of value increasing progeny performance in terms of growth performance.

**Literature**

1. Cheeke PR. Applied Animal Nutrition, Feeds and Feeding, 3. Edit. Pearson, Prentice Hall, Upper Saddle River, New Jersey 07458, 2005

2. Surai, P.F. Antioxidant Systems in the Animal Body. In Natural Antioxidants in Avian Nutrition and Reproduction; Nottingham Press: Nottingham, England, 2002, pp:1-25.

3. Ebeid TA. Organic selenium enhances the antioxidative status and quality of cockerel semen under high ambient temperature. Br Poult Sci. 2009; 50: 641-647.

4. Pappas AC, Acamovic T, Sparks NH, Surai PF, McDevitt RM. Effects of supplementing broiler breeder diets with organoselenium compounds and polyundaturated fatty acids on hatchability. Poult Sci. 2006; 85: 1584-1593.

5. Briens M, Mercier Y, Roffineau F, Vacchina V and Gereart PA. Comparative efficiency of a new organic selenium source vs. seleno-yeast and mineral selenium sources on muscle selenium enrichment and selenium digestibility in broiler chickens. British Journal of Nutrition, 2013; 110: 617-624.

6. McQuoid, D. Operation of multiple-stage and single-stage incubation systems. World Poultry, v.18, n.2, 2000.

7. Ross. Parent Stock Performance Objectives, June 2011. www.aviagen.com

8. SAS Institue; SAS User's Guide. Statistics. Version 5th Edition. 1996, SAS Institue Inc., Cary, NC
# EFFECT OF SELENIUM SOURCE ON EGG PRODUCTION AND EGG QUALITY OF BROILER BREEDERS

H.R.Kutlu<fnref n="1" />, L.Çelik<fnref n="1" />, N. Saber<fnref n="1" />, F. Yenilmez<fnref n="2" />, H.Cinli<fnref n="1" />, N. Toy<fnref n="1" />, F. Çakıroğlu<fnref n="1" />, M. Kutlu<fnref n="1" />, O. Yücelt<fnref n="1" />

## Abstract

The present study was conducted to evaluate two different sources (Se-hydoxy-4-methylselenobutanoic acid; HMSeBA vs. selenium yeast) of organic selenium in female broiler breeders in terms of egg production, fertility, egg quality, hatchability and chick growth performance. Two hundred and twenty, 54 weeks old Ross-308 male (20) and female (200) breeders were used in the experiment. Standard breeder (female) diet based on corn and soya were used. Birds were placed in a complete randomized design with 2 dietary treatments (HMSeBA vs. Selenium yeast) and 10 replications of 10 females and 1 males per pen sized 2x1.5m. Each pen had five nests with a female tubular feeder and one individual male feeder, and nipple drinkers. The experiment occurred while the broiler breeders were from 54 to 62 weeks of age and the lighting and feeds were supplied according to the Breeders' recommendations. Eggs were collected everyday and measured for exterior and interior quality measurements once a week. Throughout the experiment, feed intake, feed conversion efficiency, egg production, egg yield, hen-day egg production were recorded. The data obtained in the study were analysed statistically by t-test.

The results showed that birds attained almost 240 grams of weigh in both groups without any significant difference (P>0.05). Feeding the birds according to the breeder's recommendation maintained animal performance as breeders' target in terms of body weight, fertility and also hatchability and hatching yield. The results showed that providing selenium in the form of HMSeBA or Se-yeast did not affect egg production significantly (P>0.05), but the group receiving HMSeBA gave numerically higher number of eggs and attained higher egg weigh and egg mass with better feed conversion rate. The results with respect to egg quality showed that at the last week of the trial eggs obtained from the group receiving HMSeBA exhibited better values in terms of egg shell strength (P<0.07), shell thickness (P<0.09), yolk height (P<0.06) and yolk width (P<0.01). The results obtained in the trial suggest that providing dietary organic selenium in the form of HMSeBA could be of value in improving egg quality of broiler breeder hens.

**Keywords:** Organic Selenium, Breeder, Egg, Fertility, Hatching, Broiler, Performance

## Introduction

It is well documented that selenium with the conjunction of vitamin E is the vital nutrient source to maintain health, growth and product quality in mammals and birds (1). Vitamin E is a fat-soluble nutrient found in body fat depots, plasma lipoproteins, and cell membrane phospholipids, where it serves as an important antioxidant (2). Selenium (Se) fulfils an antioxidant role as a component of glutathione peroxidises (GSHPx). Se is widely distributed in the body, but the most labile reservoir is in the liver (2). It is found in body tissue principally as selenomethionine (SeMet) or as selenocysteine (SeCys), the latter found in GSHPx. Both nutrients are should be examine together because of their related functions and similar deficiency signs. However, Se and vitamin E each have unique metabolic roles, and the factors that alter the oxidative state of an animal may differentially affect their dietary needs. It is now well known that Se plays an important role in maintaining semen quality (3). An optimal Se status in male birds is considered to be an important factor in ensuring the fertility of breeding stocks, while an optimal Se status of the eggs of females needs for antioxidant system to maintain embryo development and also hatchability (4).

In poultry nutrition, selenium requirements meet through trace mineral premixes. Recent literature showed that besides the rational source of supplemental selenium in mineral forms mainly sodium selenite in animal diets, organic selenium sources have also been developed through selenize yeast with Se in the form of selenomethionine and also 2-hydoxy-4-methylselenobutanoic acid (HMSeBA) in recent years (5). Comparative studies showed that organic Se source provides some advantages with higher bioavailability and lower inclusion rate in the diet (5). However no comparative studies have been undertaken to evaluate different sources of organic selenium in female broiler breeders in terms of laying performance and egg quality.

<footnote n="1">¹ Department of Animal Science, Faculty of Agriculture, University of Çukurova, Turkey</footnote>

<footnote n="2">² Vacational School of Tufanbeyli, University of Çukurova, Turkey</footnote>
The present project was aimed to evaluate effect(s) of organic sources of Se-hydroxy-4-methylselenobutanoic acid (HMSeBA of Adisseo; Selisseo) in contrast to the other organic selenium source as selenium yeast on laying performance and egg quality of broiler breeders.

## Materials and Methods

The present study was carried out in the Broiler Breeder Unit of the Experimental Farm of the Department of Animal Science, Faculty of Agriculture, University of Çukurova, Adana-Turkey. Two hundred and twenty, 54 weeks of age Ross-308 male (20) and female (200) breeders were used in the experiment lasted 9 weeks. Experimental animals were weighed at the first day and last day of the trial to assess weigh changes throughout the experiment, in which standard breeder (female) diet based on corn and soya was used.

Two hundred female Ross-308 and 20 Ross-308 male broiler breeders were placed in the unit at 52 weeks of age. After 2 weeks pre-feeding period they were allocated according to body weight and also egg production in a completely randomized design into 2 treatment (0.2 ppm Se-Yeast or 0.2.ppm HMSeBA) groups with 10 replicate pens, 10 females and 1 males per pen sized 2.0 x 1.5 m in the breeder unit, where 20 subgroup pens were available for the trial. Extra 5 males were raised separately to replace sexually inactive or dead males.

Each pen of the breeder unit had five nests bedded wood shaving, sizeing 25x43x35 cm each, a female tubular feeder and one individual male feeder, and nipple drinkers. Animals were placed on wood shaving litter 7-8 cm height. The experiment lasted while the broiler breeders were from 54 to 62 weeks of age and the lighting and feeds (female: 156 gram/day, male: 130 gram/day) were supplied according to Ross Breeders recommendations (6) without drinking water restriction. The environmental temperature was maintained within the animal comfort zone using foggers and tunnel ventilation. Male performance was watched and the male with low mating performance was replaced with a spare one. Eggs were collected every day and measured for exterior quality (size, weigh, and crack incidents) measurements. Weekly egg production, hatching eggs, and eggs with defects were obtained through daily collections and were expressed in percentage. Defective eggs were considered those with physical deformities and those bearing cracks. Double yolk and floor eggs will be added up to total production, but were not incubated. In the experiment feed intake, feed conversion efficiency, egg production, egg yield, hen-day egg production, mortality rate were recorded. In the third week of the trial and thereafter (57-62th weeks of age) eggs obtained on the third day of every week analysed for interior quality (albumen, yolk measurements, Haugh Unit etc.) and also exterior quality measurements such as egg shape index, shell thickness, shell weigh, shell strength etc. The data obtained in the study were analysed using t-test procedure of SAS; the Statistical Analysis System (7). Result obtained in this study will be presented as means per bird with its standard error.

## Results and Discussion

In order to observe body weigh changes of breeders throughout the experiment, at the beginning of the experiment when the birds 54th week of age, and at the end of the experiment when the birds 62nd week of age, all the birds were weighed individually. The results with respect to body weight changes of the hen showed that birds attained almost 240 grams of weigh in both groups without significant difference (Table 1). Throughout the experiment, the birds were given feed according to Breeder company recommendation. Foods were given to female birds 156 gram/day, male birds 130 gram/day. Feeding the birds according to the breeder's recommendation keep animal performance as breeders' target in terms of body weight, fertility and also hatchability and hatching yield. The results with respect to body weigh changes and feed intake are given in Table 1.

<table><caption>**Table 1.** Live weigh changes and feed intake of birds throughout the experiment.</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="2">HMSeBA (n=10)</th><th colspan="2">Se-Yeast (n=10)</th><th rowspan="2">P=</th></tr><tr><th>Mean</th><th>Std Error</th><th>Mean</th><th>Std Error</th></tr></thead><tbody><tr><td>Live weigh at the first day of the trial (g/hen)</td><td>4696.7</td><td>27.25</td><td>4737.9</td><td>29.83</td><td>0.3213</td></tr><tr><td>Live weigh at the last day of the trial (g/hen)</td><td>4940.0</td><td>35.75</td><td>4976.2</td><td>36.02</td><td>0.4862</td></tr><tr><td>Weigh changes (g/hen)</td><td>243.3</td><td>39.84</td><td>238.33</td><td>22.28</td><td>0.9172</td></tr><tr><td>Feed Intake (g/hen/day)</td><td>156</td><td></td><td>156</td><td></td><td></td></tr><tr><td>Feed Intake (g/cock/day)</td><td>130</td><td></td><td>130</td><td></td><td></td></tr></tbody></table>
After 2 weeks pre-feeding period from 52-53 weeks of age, animals were grouped according to body weight and also egg production. At 54 weeks of age and thereafter laying performance parameters were analysed weekly. The results with respect to laying performance at 56 and 62 (two weeks after and eight weeks after the initiation of the experiment) weeks of age are given in Table 2. The results showed that providing selenium in the form of HMSeBA or Seyeast did not affect egg production significantly (P>0.05). However, mean production values at 62nd week of age seemed to be favour of HMSeBA groups (Table 2). Additionally, the performance values obtained at 56-62 weeks of age in the present trial were higher than the values set by the Breeder Company (6).

<table><caption>**Table 2.** Egg Production of the breeders at the second week and last week of the trial</caption><thead><tr><th rowspan="3">Parameters</th><th colspan="4">56th weeks of age</th><th colspan="4">62nd weeks of age</th></tr><tr><th colspan="2">HMSeBA (n=10)</th><th colspan="2">Se-Yeast (n=10)</th><th rowspan="2">P=</th><th colspan="2">HMSeBA (n=10)</th><th colspan="2">Se-Yeast (n=10)</th><th rowspan="2">P=</th></tr><tr><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th></tr></thead><tbody><tr><td>Egg production (number/week)</td><td>4.78</td><td>0.34</td><td>4.65</td><td>0.25</td><td>0.293</td><td>4.13</td><td>0.22</td><td>3.86</td><td>0.20</td><td>0.368</td></tr><tr><td>Egg weight (g/day)</td><td>46.79</td><td>3.03</td><td>45.59</td><td>2.22</td><td>0.215</td><td>41.79</td><td>2.23</td><td>38.85</td><td>2.02</td><td>0.342</td></tr><tr><td>Egg weight (g/egg)</td><td>68.67</td><td>0.25</td><td>68.76</td><td>0.38</td><td>0.850</td><td>70.76</td><td>0.22</td><td>70.52</td><td>0.50</td><td>0.657</td></tr><tr><td>Egg mass (g/week)</td><td>327.6</td><td>23.47</td><td>319.37</td><td>17.22</td><td>0.215</td><td>292.54</td><td>15.38</td><td>271.95</td><td>14.17</td><td>0.342</td></tr><tr><td>Feed Conversion Rate (g feed/g egg)</td><td>3.19</td><td>0.23</td><td>3.52</td><td>0.24</td><td>0.332</td><td>3.74</td><td>0.22</td><td>4.01</td><td>0.23</td><td>0.402</td></tr></tbody></table>

The results with respect to quality of eggs at the beginning (56th week of age) and at the last weeks (62nd week of age) of the experiment are given in Tables 3. The results showed at the beginning of the experiment all the quality measurements of the treatment groups were similar to each other. However at the last weeks of the trial eggs obtained from the group receiving HMSeBA exhibited better values in terms of egg shell strength (P<0.07), shell thickness (P<0.1), yolk height (P<0.06) and yolk width (P<0.01). The improvement in egg quality could be attributed to selenium and selenomethionine role in synthesis of egg shell matrix, mineralization and also its contribution to antioxidant capacity and membrane integrity.

<table><caption>**Table 3.** Quality measurements of eggs obtained at the beginning and end of the trial.</caption><thead><tr><th rowspan="3">Parameters</th><th colspan="4">56th weeks of age</th><th colspan="4">62nd weeks of age</th></tr><tr><th colspan="2">HMSeBA (n=10)</th><th colspan="2">Se-Yeast (n=10)</th><th rowspan="2">P=</th><th colspan="2">HMSeBA (n=10)</th><th colspan="2">Se-Yeast (n=10)</th><th rowspan="2">P=</th></tr><tr><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th><th>Mean</th><th>Stderr</th></tr></thead><tbody><tr><td>Egg weight (g)</td><td>69.25</td><td>0.774</td><td>68.40</td><td>0.772</td><td>0.436</td><td>72.66</td><td>0.88</td><td>71.68</td><td>0.65</td><td>0.3688</td></tr><tr><td>Albumen weight (g)</td><td>40.09</td><td>0.532</td><td>39.68</td><td>0.624</td><td>0.615</td><td>41.35</td><td>0.79</td><td>40.79</td><td>0.57</td><td>0.5676</td></tr><tr><td>Yolk weight (g)</td><td>22.54</td><td>0.241</td><td>21.95</td><td>0.308</td><td>0.134</td><td>24.02</td><td>0.43</td><td>23.52</td><td>0.31</td><td>0.1363</td></tr><tr><td>Egg shell weight (g)</td><td>6.63</td><td>0.099</td><td>6.72</td><td>0.090</td><td>0.485</td><td>7.43</td><td>0.14</td><td>7.53</td><td>0.14</td><td>0.6279</td></tr><tr><td>Shell strength (kg/cm²)</td><td>3542</td><td>181.0</td><td>3564</td><td>155.1</td><td>0.858</td><td>3793</td><td>173.0</td><td>3599</td><td>152.86</td><td><strong>0.0664</strong></td></tr><tr><td>Egg width (mm)</td><td>45.54</td><td>0.18</td><td>45.72</td><td>0.332</td><td>0.992</td><td>45.54</td><td>0.17</td><td>46.06</td><td>0.16</td><td>0.3908</td></tr><tr><td>Egg length (mm)</td><td>59.93</td><td>0.36</td><td>60.07</td><td>0.399</td><td>0.676</td><td>60.17</td><td>0.49</td><td>61.26</td><td>0.36</td><td>0.4717</td></tr><tr><td>Egg Shape index (%)</td><td>76.08</td><td>0.50</td><td>76.23</td><td>0.61</td><td>0.779</td><td>75.85</td><td>0.61</td><td>75.28</td><td>0.53</td><td>0.8537</td></tr><tr><td>Yolk height (mm)</td><td>18.96</td><td>0.134</td><td>19.03</td><td>0.182</td><td>0.788</td><td>19.94</td><td>0.26</td><td>19.37</td><td>0.14</td><td><strong>0.0521</strong></td></tr><tr><td>Albumen height (mm)</td><td>5.89</td><td>0.221</td><td>5.91</td><td>0.189</td><td>0.159</td><td>6.42</td><td>0.29</td><td>5.63</td><td>0.21</td><td>0.1669</td></tr><tr><td>Yolk width (mm)</td><td>47.16</td><td>0.637</td><td>46.69</td><td>0.512</td><td>0.626</td><td>46.17</td><td>0.50</td><td>50.27</td><td>1.08</td><td><strong>0.0012</strong></td></tr><tr><td>Albumen width (mm)</td><td>86.59</td><td>2.942</td><td>85.24</td><td>2.013</td><td>0.706</td><td>88.40</td><td>3.62</td><td>87.17</td><td>1.81</td><td>0.7586</td></tr><tr><td>Albumen length (mm)</td><td>106.48</td><td>4.014</td><td>100.9</td><td>1.861</td><td>0.214</td><td>112.37</td><td>3.79</td><td>110.20</td><td>1.75</td><td>0.59780</td></tr><tr><td>Shell thickness (μm)</td><td>324.72</td><td>3.703</td><td>333.1</td><td>4.316</td><td>0.142</td><td>358.47</td><td>5.65</td><td>339.78</td><td>5.11</td><td><strong>0.0981</strong></td></tr><tr><td>Haugh Unit</td><td>71.60</td><td>1.27</td><td>71.66</td><td>1.60</td><td>0.977</td><td>74.33</td><td>1.79</td><td>70.86</td><td>1.37</td><td>0.2123</td></tr></tbody></table>

It is well documented that selenium is an essential nutrient. Its role in metabolism is mainly related to the synthesis of Se-amino acid and Se-protein complexes that act as potent antioxidants. In addition to its antioxidant function, selenium has potential to affect egg quality. Wakebe (8) and Papas et al. (9) showed that Se addition to layer diets can
mitigate the reduction of Haugh units in stored eggs. Our results on increased shell strength are confirmed by previous studies (10), they reported that shell strength could be related to higher Se concentration in the shell and shell membrane. These two last factors are particularly increased when diet contains organic Se sources, suggesting that the high Se concentration could be the main factor for increased shell strength through its role in formation of organic matrix. It is well-known that eggshell membrane is mainly consisted of fibrous protein or collagen-like proteins (11), which derives organic matrix. It has been shown that fortifying poultry feeds with selenium, especially organic source, have a great potential to increase egg selenium contents (12). Our results suggest that selenium in the egg have a powerful agent with its antioxidant properties and also being a significant factor in synthesis of organic matrix of egg shell. It is also reported that the natural selenium-containing antioxidant enzymes like glutathione peroxidise and thioredoxin reductase have the potential to reduce the effect of free radicals on the aging process (13). Disulfide bonds (bonds between sulphur atoms) can cross-link proteins, decreasing enzyme function and increasing the sinew associated with aging collagen. Protein oxidation is known to be related to selenium deficiency (14). The present knowledge with respect to selenium and its protective effect on structural protein could be suggested that selenium is a key factor for the formation of organic matrix derived by shell membrane by maintaining integrity of fibrous protein or collagen-like proteins, leading to increase in shell strength.

## Conclusion

The results with respect to body weight changes of the hen during the trial showed that birds attained almost 240 grams of weigh in both groups without a significant difference (P<0.5).

Providing selenium in the form of HMSeBA or Se-yeast did not affect egg production significantly (P>0.05).

The performance values of breeders obtained from the both groups in the present study were higher than the values set by Breeder Company; Ross.

The breeder receiving dietary HMSeBA exhibited better values in terms of egg shell strength (P<0.07), shell thickness (P<0.1), yolk height (P<0.06) and yolk width (P<0.01).

## Literature

1. Cheeke PR. Applied Animal Nutrition, Fedds and Feeding, 3. Edit. Pearson, Prentice Hall, Upper Saddle River, New Jersey 07458. 2005;

2. Surai PF. Antioxidant Systems in the Animal Body. In Natural Antioxidants in Avian Nutrition and Reproduction; Nottingham Press: Nottingham, England. 2002; pp: 1-25.

3. Ebeid TA. Organic selenium enhances the antioxidative status and quality of cockerel semen under high ambient temperature. Br. Poult. Sci. 2009; 50: 641-647.

4. Pappas AC, Acamovic T, Sparks NH, Surai PF, McDevitt RM. Effects of supplementing broiler breeders diet with organoselenium compounds and polyunsaturated fatty acids on hatchability. Poult Sci. 2006; 85: 1584-1593.

5. Briens M, Mercier Y, Roffineau F, Vacchina V and Gereart PA. Comparative efficiency of a new organic selenium source vs. seleno-yeast and mineral selenium sources on muscle selenium enrichment and selenium digestibility in broiler chickens. British Journal of Nutrition, 2013; 110: 617-624.

6. Ross. Parent Stock Performance Objectives, June 2011; www.aviagen.com

7. SAS Institue; SAS User's Guide. Statistics. Version 5th Edition. 1996, SAS Institue Inc., Cary, NC

8. Wakebe M. Organic selenium and egg freshness. Japanese Patent Office, Application Heisei 8-179629. Poster Presentation 15th Annual Symp. on Biotechnology in the Feed Industry, Lexington, KI, USA, 1999.

9. Pappas AC, Acamovic T, Sparks NHC, Surai PF, McDevitt RM. Effects of supplementing broiler breeder diets with organic selenium and polyunsaturated fatty acids on egg quality during storage. Poult. Sci, 2005; 84: 865-874.

10. Invernizzi G, Agazzi A, Ferroni M, Rebucci R, Fanelli A, Baldi A, Dell'Orto V, Savoini G. Effects of inclusion of selenium-enriched yeast in the diet of laying hens on performance, eggshell quality, and selenium tissue deposition. Italian J. Anim. Sci., 2013; Vol.12:e1.
11. Tullett SG. Egg shell formation and quality. In: Wells, R.G., Belyavin, C.G. (Eds.), Egg Quality—Current Problems and Recent Advances. Butterworths, London, 1987; 123–146.

12. Jlali M, Biens M, Rouffineau F, Geraert PA and Mercier Y. Effect of 2-hydroxy-4-methylselenobutonoic acid as a dietary selenium supplement to improve the selenium concentration of table eggs. J.Anim. Sci., 2013; 91:1745-1752.

13. Best B. Selenium: AntiOxidant, Anti-Carcinogen and Immune System Booster. 2014; http://www.benbest.com/nutrceut/Selenium.html

14. Moskovitz J, Stadtman ER, Selenium-deficient diet enhances protein oxidation and affects methionine sulfoxide reductase (MsrB) protein level in certain mouse tissues. Proc Natl Acad Sci U S A. 2003, 100:7486-7490.
P-180 (ID 306)

PHOSPHORUS AND CALCIUM DIGESTIBILITY IS IMPROVED WITH THE ADDITION OF A CARBOHYDRASE
COMPLEX IN BROILER CHICKENS FED WHEAT-SOYBEAN-BASED DIETS

R. Montanhini Neto, Pierre Cozannet and A. Preynat

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

Corresponding author: roberto.neto@adisseo.com

**Abstract**

This study aimed to evaluate the efficacy of a carbohydrate complex (CC) in improving the digestibility of P and Ca in broilers' wheat-soybean-based diets, compared to that observed for a single 6-phytase. Four diets were formulated with different available phosphorus levels (2.0, 2.7, 3.4, and 4.1 g kg⁻¹), with a fixed Ca:P ratio. To each diet, either CC (Rovabio® ADVANCE L) at 200 mL ton⁻¹ or a 6-phytase (Phzyme® XP 5000L) at four different inclusion levels (0, 80, 160, and 240 FTU kg⁻¹) was added. For each diet, six birds were reared between d 10-28 in individual cages suitable for digestibility trials. Excreta collections were performed at d 15-18 and d 25-28 for the determination of the apparent digestibility of P and Ca. We estimated the potential of the CC in comparison with the phytase effect using the factorial regression of dietary P levels and the phytase inclusion. No significant interaction was observed between P levels and phytase inclusion. P levels promoted a linear reduction (P<0.001) of the digestibility of both minerals in the two periods of evaluation. The phytase inclusion promoted a linear improvement (P<0.05) of P and Ca digestibility. The addition of the CC increased (P<0.05) the P and Ca digestibility. For P apparent digestibility, the improvement rate was 7.9% and 3.8% respectively at d 18-21 and d 25-28; for Ca, it was 5.0% and 5.8%. These improvements were observed regardless the level of P in the diets.

**Keywords:** NSP-enzymes, phytase, calcium, phosphorus, digestibility.

**Introduction**

The model the action of the carbohydrates in improving the global digestibility of feeds is mediated by effects on digesta viscosity, on the physical accessibility to nutrients and on modifications in the intestinal microflora. All these effects are associated to the partial or complete hydrolysis of both soluble and insoluble non-starch polysaccharides (NSP) chains from plant raw material (Adeola and Cowieson 2010). Since these NSP, as main constituents of the cell wall, act to 'envelope' nutrients in the cell cytoplasm; this effect of NSP is frequently called "cage effect" (Smeets et al. 2014). Thus, by adding NSP-carbohydrases into feeds, not only nutrients directly released by the hydrolysis (such as monomeric sugars from fibres, for example) are made available to the normal digestion process of animal, but all other nutrients of interest present inside intact vegetable cells and other caged compartments can be acceded (Smeets et al 2013). Several studies have reported the effect of NSP-carbohydrases in promoting accessibility and indirectly improvement of digestibility of amino acids, starch and fat (Slominsky 2011). However, little is known about the effect on other compounds with nutritional and economic interest This study aimed to evaluate the efficacy of a carbohydrate complex (CC) in improving the digestibility of P and Ca in broilers' wheat-soybean-based diets, compared to that observed for a single 6-phytase.

**Material and Methods**

This study was carried out in the Centre of Expertise and Research in Nutrition (CERN), Adisseo France SAS, in Malicorne, France (46°18'14" N, 2°44'07" E), between October and November 2014. The 120 male Ross PM3 broilers were divided in 20 treatments, with six repetitions (individual cages suitable for digestibility trials) of 1 birds each between 1 and 28 days of age, in 2 feeding phases: initial (d1-7), grower (d8-28). Four diets were formulated with different available phosphorus levels (2.0, 2.7, 3.4, and 4.1 g kg⁻¹), with a fixed Ca:P ratio of 1.40. To each diet, either CC (Rovabio® ADVANCE L at 200 mL ton⁻¹) or a 6-phytase (Phzyme® XP 5000L) at four different inclusion levels (0, 80, 160, and 240 FTU kg⁻¹) was added. The CC and the 6-phytase were not evaluated in combination. Excreta collections were performed at d15-18 and d25-28 for the determination of the apparent digestibility of P and Ca. By considering the factorial regression of dietetic P level and phytase inclusion rate, were estimated the digestibility improvement potentials on P and Ca of the CC in comparison with the phytase effect, in FTU kg⁻¹.
## Results

No significant interaction was observed between dietary available P levels and phytase inclusion. P levels promoted a linear reduction (P<0.001) of the digestibility of both minerals in the two periods of evaluation. The phytase inclusion promoted a linear improvement (P<0.05) of P and Ca digestibility. The prediction equations obtained from the factorial regression of dietary available P levels and phytase levels on P and Ca apparent digestibility had R² between 0.35 and 0.60 (Table 1).

<table><caption>**Table 1.** Prediction equations obtained from the factorial regression of dietary available P levels (x, in %) and phytase levels (y, in FTU kg⁻¹) on apparent digestibility P and Ca at d18 and d28.</caption><thead><tr><th>Parameter</th><th>Prediction equation</th><th>R² (Prob.)</th></tr></thead><tbody><tr><td>Dig. P (d18)</td><td>69.193 + 53.308x + 0.102y - 210.713x² - 0.148xy - 6.063E10⁻⁵y²</td><td>0.349 (0.032)</td></tr><tr><td>Dig. P (d28)</td><td>72.763 + 57.365x + 0.063y - 220.908x² - 0.091xy - 5.373E10⁻⁵y²</td><td>0.506 (0.004)</td></tr><tr><td>Dig. Ca (d18)</td><td>69.357 + 102.603x + 0.082y - 262.085x² - 0.140xy - 1.416E10⁻⁶y²</td><td>0.436 (0.027)</td></tr><tr><td>Dig. Ca (d28)</td><td>67.847 + 22.160x + 0.006y - 195.413x² - 0.064xy - 1.182E10⁻⁵y²</td><td>0.591 (0.009)</td></tr></tbody></table>

The addition of the CC significantly increased (P<0.05) the P and Ca digestibility at d18 and d28 (Table 2). For P apparent digestibility, the improvement rate was 7.9% and 3.8% respectively at d 18-21 and d 25-28; for Ca, it was 5.0% and 5.8% (Table 3).

<table><caption>**Table 2.** Digestibility of P and Ca in different diets varying the available P levels with or without the addition of a carbohydrate complex (CC) at d18 and d28.</caption><thead><tr><th>Dietary P<sub>av</sub> levels (%)</th><th colspan="2">Dig. P (d18, %)</th><th colspan="2">Dig. Ca (d18, %)</th><th colspan="2">Dig. P (d28, %)</th><th colspan="2">Dig. Ca (d28, %)</th></tr><tr><th>CC Addition</th><th>without</th><th>with</th><th>without</th><th>with</th><th>without</th><th>with</th><th>without</th><th>with</th></tr></thead><tbody><tr><td>0.20</td><td>71.40</td><td>73.60</td><td>72.31<sup>b</sup></td><td>75.51<sup>a</sup></td><td>75.67<sup>b</sup></td><td>81.2<sup>a</sup></td><td>65.14</td><td>66.05</td></tr><tr><td>0.27</td><td>71.16</td><td>73.70</td><td>74.25</td><td>77.24</td><td>72.15</td><td>72.55</td><td>60.66</td><td>62.32</td></tr><tr><td>0.34</td><td>60.57<sup>b</sup></td><td>69.54<sup>a</sup></td><td>67.31<sup>b</sup></td><td>72.75<sup>a</sup></td><td>67.00</td><td>68.47</td><td>50.23<sup>b</sup></td><td>55.16<sup>a</sup></td></tr><tr><td>0.41</td><td>55.43<sup>b</sup></td><td>60.93<sup>a</sup></td><td>60.68</td><td>62.72</td><td>58.97<sup>b</sup></td><td>61.97<sup>a</sup></td><td>44.65<sup>b</sup></td><td>48.79<sup>a</sup></td></tr><tr><td>Average (all P levels)</td><td>64.64<sup>b</sup></td><td>69.44<sup>a</sup></td><td>68.64<sup>b</sup></td><td>72.06<sup>a</sup></td><td>68.45<sup>b</sup></td><td>71.05<sup>a</sup></td><td>55.17<sup>b</sup></td><td>58.08<sup>a</sup></td></tr></tbody></table>

<table><caption>**Table 3.** Improvement rate of digestibility of P and Ca in different diets varying the available P levels promoted by the addition of a carbohydrate complex (CC) at d18 and d28.</caption><thead><tr><th>Dietary P<sub>av</sub> levels (%)</th><th>Dig. P (d18)</th><th>Dig. Ca (d18)</th><th>Dig. P (d28)</th><th>Dig. Ca (d28)</th></tr></thead><tbody><tr><td>0.20</td><td>3.08%</td><td>4.42%*</td><td>7.32%*</td><td>1.40%</td></tr><tr><td>0.27</td><td>3.56%</td><td>4.03%</td><td>0.56%</td><td>2.74%</td></tr><tr><td>0.34</td><td>14.82%*</td><td>8.08%*</td><td>2.19%</td><td>9.82%*</td></tr><tr><td>0.41</td><td>9.93%*</td><td>3.37%</td><td>5.08%*</td><td>9.27%*</td></tr><tr><td>Average (all P levels)</td><td>7.85%*</td><td>4.98%*</td><td>3.79%*</td><td>5.81%*</td></tr></tbody></table>

By using the prediction equations with the improvements observed with the addition of CC, it was found that this complex had a potential compared to 99.6 FTU kg⁻¹ for P digestibility and 108.9 for Ca digestibility (Table 4).

<footnote>a Different letters in same line means significant difference (P<0.05) of digestibility between “without” and “with” addition of the CC by the Mann-Whitney non-parametric test.</footnote>

<footnote>* Significant (P<0.05) improvement rate of digestibility by the Mann-Whitney non-parametric test.</footnote>
<table><caption>**Table 4.** Improvement rate of digestibility of P and Ca in different diets varying the available P levels promoted by the addition of a carbohydrate complex (CC) at d18 and d28, compared with that observed for the effect of inclusion of a 6-phytase (presented in FTU kg⁻¹).</caption><thead><tr><th>Dietary P<sub>av</sub> levels (%)</th><th>Dig. P (d18)</th><th>Dig. Ca (d18)</th><th>Dig. P (d28)</th><th>Dig. Ca (d28)</th></tr></thead><tbody><tr><td>0.20</td><td>56.30</td><td>58.24*</td><td>193.44*</td><td>143.45</td></tr><tr><td>0.27</td><td>53.14</td><td>119.31</td><td>24.28</td><td>131.17</td></tr><tr><td>0.34</td><td>158.45*</td><td>140.82*</td><td>107.01</td><td>89.98*</td></tr><tr><td>0.41</td><td>131.77*</td><td>58.76</td><td>72.87*</td><td>127.90*</td></tr><tr><td>Average (all P levels)</td><td>99.92*</td><td>94.28*</td><td>99.40*</td><td>123.11*</td></tr></tbody></table>

These results confirm the hypothesis that NSP-carbohydrases are capable to release internal nutrients present caged by cell wall in plant compartments, even if those nutrients are not directly linked to enzyme substrates: the NSP. Once released, these internal nutrients become accessible for normal digestion process. Even if most part of phosphorus inside plant tissues are presented as phytate – normally indigestible for monogastrics – endogenous alkaline phosphatase can hydrolase the phosphate radicals from myo-inositol mono, di and triphosphate (Abudabos 2012). Besides, all sources of phosphorus (plant or mineral) in the experimental feed were taken into account in the measured digestibility. The expected reduction of digest viscosity by the CC can also played an important role in the indirect improvement of mineral digestibility, as observed in several studies with other nutrients (Smulikowska and Mieczkowska 1996; Langhout et al. 1997).

## Conclusions

The study demonstrated the efficacy of the evaluated carbohydrate complex in improving the digestibility of phosphorus and calcium, giving a strong scientific support to the concept of global digestibility improvement by this enzymatic complex.

## Literature

ABUDABOS A.M., 2012: Intestinal phytase activity in chickens (*Gallus domesticus*). Afric. J. Microb. Res., 6, 4932-4938.

ADEOLA O. and COWIESON J., 2001: Opportunities and challenges in using exogenous enzymes to improve non-ruminant animal production. J. Anim. Sci. 89, 3189-3218.

LANGHOUT D., SCHUTTE J.B., GEERSE C. et al., 1997: Effects on chick performance and nutrient digestibility of an exoglucanase added to a wheat- and rye based diet in relation to fat source. Brit. Poult. Sci. 38, 557-563.

SLOMINSKY B.A., 2011: Recent advances in research on enzymes for poultry diets. Poult. Sci., 90, 2013-2023.

SMEETS N., NUYENS F., CAMPENHOUT L. and NIEWOLD T., 2014: Effect of single or combined feed enzymes on the *in vitro* degradation of non-starch polysaccharides from wheat. In Proceedings of the 19th European Symposium on Poultry Nutrition.

SMEETS N., NUYENS F., CAMPENHOUT L. and NIEWOLD T., 2014: Variability in the *in vitro* degradation of non-starch polysaccharides from wheat by feed enzymes. Anim. Feed Sci. Tech., 187, 110-114.

SMULIKOWSKA S. and MIECZKOWSKA A., 1996: Effect of rye level, fat source and enzyme supplementation on fat utilization, diet metabolizable energy, intestinal viscosity and performance of broiler chickens. J. Anim. Feed Sci. 5, 379-393.

<footnote>* Significant (P<0.05) improvement rate of digestibility by the Mann-Whitney non-parametric test.</footnote>
P-185 (ID 287)

BONE ASH AS A CRITERION FOR ASSESSING MINERALISATION IN BROILERS

C. Sanni, D. Scholey, N. Morgan and E. Burton

School of Animal, Rural and Environmental Sciences, Nottingham Trent University, UK

Corresponding author: colin.sanni@ntu.ac.uk

**Abstract**

Two common divergences in the methodology for determining the degree of bone mineralisation were examined. The first objective was to compare the effect of varying fat extraction time on poultry tibia bones. The second objective was to evaluate the effect of autoclaving bones prior to fat extraction on bone ash. 264 day old Ross 308 male broilers were fed standard commercial diets and were allocated to 33 replicate pens; 8 birds per replicate. At 42d, 1 bird per replicate pen was culled. For objective 1, all adhering tissues were removed from the right tibia bones. The bones, including their cartilage caps, were then dried at 105°C for 24h prior to fat extraction by Soxhlet extraction for 1h using petroleum ether, and then dried at 105°C for 24h and dry bone weight recorded. This process was cumulatively repeated 8 times in total. For objective 2, the left tibia bones were autoclaved and then dried at 105°C for 24h prior to fat extraction in petroleum ether for 8h. All bones from right and left tibias were then ashed at 650°C for 24h. There were significant increases in fat extracted for each additional hour from 1-5h, P<0.05. After 5 hours of extraction no significant differ- ences were observed in percentage fat extracted. Autoclaving bones prior to fat extraction did not have any significant effect on bone ash.

**Keywords:** poultry, bone mineralisation, bone ash, methodology

**Introduction**

Proper bone mineralisation is important considering the fast rate of growth of modern broilers and the amount of weight put on relatively immature bones. Fast growing broiler chickens are known to be susceptible to bone abnormalities, causing major problems for broiler producers (Shim et al., 2008); the skeleton remains a weak link in physically support- ing heavy carcasses in young birds (Applegate and Lilburn, 2002).

Bone status is commonly used as an indicator of mineral adequacy in poultry diets (Onyango et al., 2003), and various bone measurements have already been identified as indicators of bone mineralisation (Hall et al., 2003, Shim et al., 2012). Tibia ash remains the most common bone used in evaluating bone mineralisation in poultry research (Shaw et al., 2010).

The Association of Official Analytical Chemists recommended method of determining tibia ash (AOAC, 2000) is widely cited in literature. The process involves determining ash on a fat-free, dry weight basis. However different divergences to this methodology have been cited in literature (Yan et. al., 2005) and it is thought that the variations in methodology may have an effect on the result obtained.

This study was conducted to evaluate the effect of two common divergences in the bone ash methodology: the effect of varying time on fat extraction using the Soxhlet method, and the effect of autoclaving prior to fat extraction on bone ash.

**Material and Methods**

264 day- old Ross 308 male broilers fed standard commercial diets were allocated to 33 replicate pens; 8 birds per replicate. Diets and water were provided ad libitum to all birds during the experimental procedure. At 42d, 1 bird per replicate pen was culled by cervical dislocation and tibia bones were manually removed and frozen at -20°C until further analysis.

For objective 1, all adhering tissues were removed from the right tibia bones. The right tibia bones, including their carti-lage caps, were dried at 105°C for 24h prior to fat extraction by Soxhlet extraction for 1h using petroleum ether. They were then dried at 105°C for 24h and dry bone weight recorded. This process was cumulatively repeated 8 times in total. For objective 2, the left tibia bones were autoclaved and dried at 105°C for 24h prior to fat extraction in petroleum ether for 8h. All bones from right and left tibias were then ashed at 650°C for 24h.
Data was analysed using the IBM SPSS statistical software, version 22. For objective 1, data was analysed as a one-way repeated measure ANOVA and post hoc test was determined using the Bonferroni procedure. For objective 2, one-way ANOVA was performed on data using the univariate model procedure. Post hoc test was determined using the Turkey's Honestly Significant Difference procedure.

## Results

### Objective 1: Effect of varying time on fat extraction

The total % mean fat extracted increased significantly for every additional hour of fat extraction upto 5 hours, and for every additional hour of fat extraction, there was a reduction in the additional % mean fat extracted.

<table><caption>**Table 1.** The effect of varying time on fat extraction</caption><thead><tr><th colspan="3">Mean % additional fat extracted per hour</th></tr><tr><th>Fat extraction time (h)</th><th>% additional mean fat extracted</th><th>SE*</th></tr></thead><tbody><tr><td>1</td><td>5.724<sup>a</sup></td><td>0.405</td></tr><tr><td>2</td><td>3.893<sup>b</sup></td><td>0.232</td></tr><tr><td>3</td><td>2.406<sup>c</sup></td><td>0.153</td></tr><tr><td>4</td><td>1.504<sup>d</sup></td><td>0.145</td></tr><tr><td>5</td><td>0.720<sup>e</sup></td><td>0.101</td></tr><tr><td>6</td><td>0.446<sup>f</sup></td><td>0.064</td></tr><tr><td>7</td><td>0.354<sup>f</sup></td><td>0.052</td></tr><tr><td>8</td><td>0.349<sup>f</sup></td><td>0.053</td></tr></tbody></table>

Means with different letters differ significantly (*P* < 0.05).

### Objective 2: Effect of autoclaving prior to fat extraction

Autoclaving bones prior to fat extraction did not have any significant effect on bone ash weight or percentage ash *P*>0.05

<table><caption>**Table 2**</caption><thead><tr><th>Processing method</th><th>Ash weight (g)</th><th>SE*</th><th>Ash %</th><th>SE*</th></tr></thead><tbody><tr><td>Fat extracted bones<sup>1</sup></td><td>2.3282</td><td>0.093</td><td>42.12<sup>a</sup></td><td>0.355</td></tr><tr><td>Autoclaved bones<sup>1</sup></td><td>2.3048</td><td>0.091</td><td>42.95<sup>a</sup></td><td>0.284</td></tr></tbody></table>

1 represents the mean of 33 observations per treatment

## Conclusions

The result from this study shows a minimum of 6 hours is required when using the Soxhlet extraction method to extract fat prior to ash determination. Autoclaving prior to fat extraction did not have any effect on bone ash parameters (ash weight and ash percentage). It is of paramount importance a common methodology is adopted by researchers for measuring ash content when evaluating tibia bone mineralisation in poultry.

<footnote>* Standard error</footnote>

<footnote>* Standard error</footnote>
## Literature

APPLEGATE, T. AND LILBURN, M., 2002. Growth of the femur and tibia of a commercial broiler line. Poultry Science, **81** (9), 1289-1294.

ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS, 2000. Pages 61-62 in Official Methods of Analysis of the Association of Official Analytical Chemists. Vol. 2. 17th ed. Assoc. Off. Anal. Chem., Washington, DC.

HALL, L.E., SHIRLEY, R.B., BAKALLI, R.I., AGGREY, S.E., PESTI, G.M. AND EDWARDS JR., H.M., 2003. Power of two methods for the estimation of bone ash of broilers. Poultry Science, **82** (3), 414-418.

ONYANGO, E., HESTER, P., STROSHINE, R. and ADEOLA, O., 2003. Bone densitometry as an indicator of percentage tibia ash in broiler chicks fed varying dietary calcium and phosphorus levels. *Poultry Science*, **82** (11), 1787-1791.

SHAW, A.L., BLAKE, J.P. AND MORAN, E.T., 2010. Effects of flesh attachment on bone breaking and of phosphorus concentration on performance of broilers hatched from young and old flocks. Poultry Science, **89** (2), 295-302.

SHIM, M.Y., ET AL., 2008. The Effect of Breeder Age and Egg Storage Time on Phosphorus Utilisation by Broiler Progeny Fed a Phosphorus Deficiency Diet with 1a-OH Vitamin D₃. Poultry Science, **87** (6), 1138-1145.

SHIM, M.Y., ET AL., 2012. The effects of growth rate on leg morphology and tibia breaking strength, mineral density, mineral content, and bone ash in broilers. Poultry Science, **91** (8), 1790-1795.

YAN, F., KEEN, C.A., ZHANG, K.Y AND WALGROUP, P.W., 2005. Comparison of methods to evaluate bone mineralization. Journal of Applied Poultry Research, **14** (3), 492-498.
# THE IMPACT OF SODIUM AND CHLORIDE LEVELS AND RATIOS ON THE PERFORMANCE OF BROILERS FED DIETS CONTAINING LASALOCID AT TWO DIETARY LEVELS

V. Stanev<fnref n="1" />, M. LaVorgna<fnref n="2" />, T. Barbosa<fnref n="3" />, M. Quiroz<fnref n="3" />, J. Dickson<fnref n="3" />, D. Vancraeynest<fnref n="1" />

## Abstract

The objective of the study was to evaluate the performance parameters of broilers fed with two different levels of a divalent ionophore (lasalocid) in combination with 12 different sodium:chloride (Na:Cl) ratios at moderate to low field coccidiosis infection pressure. A total of 4224 Cobb 500 male broilers were divided among 192 pens across 24 treatments. Results indicated that there was an interaction between electrolytes (Na & Cl), thus the ratios of these key nutrients can be as important as the actual levels. Best performance was achieved when the Cl level in the diet was 20% higher than the Na level. Water consumption was not statistically affected by the lasalocid dose, but it was in respect to dietary Na treatments. Also, broilers receiving a moderate (90 ppm) level of lasalocid during the latter stages of the growing cycle had higher body weights and lower feed conversions than those receiving a high (125 ppm) level. However, since there is a clear dose - anticoccidial efficacy response in ionophores, it could be that the situation would look differently under high coccidiosis infection pressure: there, the merits of a higher inclusion rate in preventing coccidiosis might become more important than a possible drawback. For this reason infection pressure should be taken into account in determining the actual anticoccidial dose in feed, when dose range is available.

## Introduction

In feed anticoccidials are largely used as a prevention tool in control of coccidiosis in poultry under modern production practices. In its commission report 233/2008, the European Commission recognizes their use as "essential". Ionophores are the most commonly used anticoccidial drugs in the modern poultry production. They are lipid-soluble molecules that bind to and transport ions - mainly mono- and divalent cations, across the bipolar lipid layer of biological membranes, which is normally non permeable for such ions. This leads to disruption of transmembrane ion concentration gradients of microorganisms which suppresses their proliferation. Due to this ion binding and transporting mode of action of ionophores, it has been speculated that different levels of electrolytes may affect performance of broilers fed with ionophores (Frigg & Broz, 1983; Cervantes and all., 1982; Christmas & Harms, 1988). It is known that levels of sodium (Na) and chloride (Cl) vary among commercial broiler diets, giving great variability to dietary electrolyte balance (DEB) levels. The study design evaluated the performance parameters of broilers fed with two different levels of a divalent ionophore (lasalocid) in combination with 12 different sodium:chloride (Na:Cl) ratios, under moderate to low field coccidiosis infection pressure.

## Materials and Methods

**Animals.** 4224 Cobb 500 males were divided into 192 pens at 1d of age. Birds were housed in an environmentally controlled building in 0.9m x 1.5m concrete floor pens at the stocking density of 16 birds per m<fnref n="2" />. Birds were vaccinated for Marek's disease in ovo and for Newcastle and infectious bronchitis by spray application at day of hatch.

**Experimental design.** Table 1 describes the group distribution for the study. The study had 24 different groups divided into two different levels of a divalent ionophore (lasalocid) in combination with 12 different Na:Cl ratios. No phytase has been used in order not to deviate the available levels from the analytical ones.

For each Na level, there were three relative percentage levels of Cl (approximately 80%, 100% and 120% of the Na levels, respectively). Each treatment had eight replicates distributed in a randomized complete block design, with the pen as the experimental unit. Birds were kept for 42 d with feed *ad libitum*. Feed schedule consisted of 3 different diets: Starter (0-18 d), Grower (19-28 d) and Finisher (29-42 d).

<footnote n="1">¹ Zoetis International Services, Paris, France</footnote>

<footnote n="2">² Devenish Nutrition, Fairmont, MN, USA</footnote>

<footnote n="3">³ Zoetis Global Poultry, Durham, NC, USA</footnote>
**Data collection.** At the beginning of each feed phase (d 0, 18, and 28), water consumption was measured for a period of four days. Feed consumption and body weight gain was measured at 18, 28, 35 and 42 d. Mortality was observed twice a day and all mortality was necropsied and weighted for feed conversion adjustment. On Study Day 42 all birds in every pen were scored for foot pad lesions. Each bird was scored according to the following: 0 = Normal (no burn, scab or lesion); 1 = Pad Burn (dermis only); 2 = Pad Scab (healing) on one or both feet; 3 = Pad Lesion (open sore) on one or both feet.

**Statistical analysis.** The model for this study was a randomized complete block with the pen as the experimental unit and repeated measures. All hypothesis tests were conducted at the 0.05 level of significance using two-sided tests. Primary variables were weight gain per bird, calculated by dividing pen weight by the number of animals and subtracting D0 pen weight divided by the number of animals at D0, and adjusted feed conversion, calculated on study d 18, 28, 35 and 42 using the total feed consumption for the pen divided by the total weight of surviving birds with D0 weight subtracted.

<table><thead><tr><th rowspan="3">Treatment</th><th rowspan="3">Lasalocid (ppm)</th><th colspan="2">Starter</th><th colspan="2">Grower</th><th colspan="2">Finisher</th></tr><tr><th colspan="2">(0-18 days)</th><th colspan="2">(19-28 days)</th><th colspan="2">(29-42 days)</th></tr><tr><th>%Na</th><th>%CI</th><th>%Na</th><th>%CI</th><th>%Na</th><th>%CI</th></tr></thead><tbody><tr><td>1</td><td rowspan="3">90</td><td rowspan="3">0.16</td><td>0.12</td><td rowspan="3">0.16</td><td>0.12</td><td rowspan="3">0.16</td><td>0.12</td></tr><tr><td>2</td><td>0.16</td><td>0.16</td><td>0.16</td><td>0.16</td></tr><tr><td>3</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.2</td></tr><tr><td>4</td><td rowspan="3">90</td><td rowspan="3">0.19</td><td>0.15</td><td rowspan="3">0.19</td><td>0.15</td><td rowspan="3">0.19</td><td>0.15</td></tr><tr><td>5</td><td>0.19</td><td>0.19</td><td>0.19</td><td>0.19</td></tr><tr><td>6</td><td>0.23</td><td>0.23</td><td>0.23</td><td>0.23</td></tr><tr><td>7</td><td rowspan="3">90</td><td rowspan="3">0.22</td><td>0.18</td><td rowspan="3">0.22</td><td>0.18</td><td rowspan="3">0.22</td><td>0.18</td></tr><tr><td>8</td><td>0.22</td><td>0.22</td><td>0.22</td><td>0.22</td></tr><tr><td>9</td><td>0.26</td><td>0.26</td><td>0.26</td><td>0.26</td></tr><tr><td>10</td><td rowspan="3">90</td><td rowspan="3">0.18</td><td>0.14</td><td rowspan="3">0.2</td><td>0.16</td><td rowspan="3">0.22</td><td>0.18</td></tr><tr><td>11</td><td>0.18</td><td>0.2</td><td>0.2</td><td>0.22</td></tr><tr><td>12</td><td>0.22</td><td>0.24</td><td>0.24</td><td>0.26</td></tr><tr><td>13</td><td rowspan="3">125</td><td rowspan="3">0.16</td><td>0.12</td><td rowspan="3">0.16</td><td>0.12</td><td rowspan="3">0.16</td><td>0.12</td></tr><tr><td>14</td><td>0.16</td><td>0.16</td><td>0.16</td><td>0.16</td></tr><tr><td>15</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.2</td></tr><tr><td>16</td><td rowspan="3">125</td><td rowspan="3">0.19</td><td>0.15</td><td rowspan="3">0.19</td><td>0.15</td><td rowspan="3">0.19</td><td>0.15</td></tr><tr><td>17</td><td>0.19</td><td>0.19</td><td>0.19</td><td>0.19</td></tr><tr><td>18</td><td>0.23</td><td>0.23</td><td>0.23</td><td>0.23</td></tr><tr><td>19</td><td rowspan="3">125</td><td rowspan="3">0.22</td><td>0.18</td><td rowspan="3">0.22</td><td>0.18</td><td rowspan="3">0.22</td><td>0.18</td></tr><tr><td>20</td><td>0.22</td><td>0.22</td><td>0.22</td><td>0.22</td></tr><tr><td>21</td><td>0.26</td><td>0.26</td><td>0.26</td><td>0.26</td></tr><tr><td>22</td><td rowspan="3">125</td><td rowspan="3">0.18</td><td>0.14</td><td rowspan="3">0.2</td><td>0.16</td><td rowspan="3">0.22</td><td>0.18</td></tr><tr><td>23</td><td>0.18</td><td>0.2</td><td>0.2</td><td>0.22</td></tr><tr><td>24</td><td>0.22</td><td>0.24</td><td>0.24</td><td>0.26</td></tr></tbody></table>

## Results

A number of interactions were identified as related to sodium and chloride levels. Evaluation of the effect of chloride at the different levels of sodium found that at 18 d of age, for birds receiving high levels of dietary sodium (0.22%), the high chloride (0.26%) treatments produced significantly lower feed conversions than did the medium chloride levels. Also, at d 28, 35, and 42, for those birds receiving high dietary sodium levels (0.22%), the high chloride (0.26%) treatments produced feed conversions that were lower than either the medium (0.22%) or low (0.18%) chloride levels.

<figure><img src="image_67.png" /><figcaption>**Fig 1.** Male broiler performance at different Cl level in relation to Na (high - Cl 20% more than Na; moderate - Cl equal to Na; and low - Cl 20% less than Na) at 42 days</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>**Fig 2.** Male broiler performance at 18, 28, 35 and 42 days with 90 or 125ppm of Avatec independently of the Na:Cl ratios</figcaption></figure>
<figure><img src="image_69.png" /><figcaption>**Fig.3** Percentage of Footpad lesions score 2 at 42 days.</figcaption></figure>

Moreover, for birds receiving low dietary levels of sodium (0.16%), those treatments that received low chloride (0.12%) levels produced feed conversion ratios at d 28, 35, and 42 that were lower than those receiving high (0.20%) chloride levels. Evaluation of the effect of sodium at different levels of chloride found that for birds receiving high levels of dietary chloride (T3/15, T6/18, T9/21 and T12/24), those receiving high (0.22%), medium (0.19%), or progressive (0.18 to 0.22%) sodium levels produced significantly lower feed conversions at 28, 35, and 42 d than birds receiving low (0.16%) levels of dietary sodium. Additionally, for birds receiving medium levels (100%) of dietary chloride, those that received medium sodium levels (0.19%) exhibited lower feed conversions at 28, 35, and 42 d than did those birds receiving high sodium levels (0.22%).

For the overall compound effect of the different Cl levels (120% of Na, 100% of Na and 80% of Na), no statistical significance was obtained as there were interactions between the different Na levels. Anyway numerical trend is clear that best performance (both BW and FCR) is obtained when Cl level exceeds Na level (fig 1).

Performance data for birds receiving 90 and 125 ppm of lasalocid are shown in Figure 2. BW and FCR were not significantly different between 90 and 125ppm treatments at d 18. Cumulative body weight gains for birds receiving 90 ppm of lasalocid (T1-T12) were superior to those receiving 125 ppm (T13-T24) at d 42. FCR followed the same pattern, with birds receiving diets with 90 ppm of lasalocid (T1-T12) exhibiting significantly lower feed conversions than did those receiving 125 ppm (T13-T24) at d 42.

Water consumption was not statistically affected by the lasalocid dose, but it was in respect to dietary Na treatments in the period between D28-D31, where birds with the high sodium level (0.22%) consumed significantly more water than the low levels (0.16%) of sodium. When foot pad lesion scores were analyzed, it was noticed that foot pad scores as measured by the % of those scoring #2, tracked with sodium levels. This is tracking with water consumption. Note that no treatments produced litter conditions that would be described as "wet"

**Discussion and Conclusions**

The trial data indicated that there was interaction between electrolytes (Na & Cl), thus the ratios of these key nutrients can be as important as the actual levels. For broilers receiving medium (0.19%) or progressive (0.18% / 0.20% / 0.22%) dietary sodium levels, there was a trend for increased weight gains and reduced feed conversions when dietary chloride levels were 120% of respective sodium levels. Body weight gain and feed consumption were also affected by different levels of ionophores inclusion in the diets. In the conditions present in this study, where coccidial challenge was at moderate to low field infection pressure, broilers receiving a high (125 ppm) level of lasalocid during the latter stages of the growing cycle had lower body weights and higher feed conversions than those receiving a moderate (90 ppm) level. This appears to be due, at least in part, to a reduction in feed intake. This is in agreement with findings of Frigg and Broz (1983), who described that high levels of monensin reduced feed intake in older birds. However, since there is a clear dose – anticoccidial efficacy response in ionophores, it could be that the situation would look differently under high coccidiosis infection pressure: there, the merits of a higher inclusion rate in preventing coccidiosis might become more important than a possible drawback. For this reason infection pressure should be taken into account in determining the actual anticoccidial dose in feed, when dose range is available. Water intake and FPD lesions increased significantly with the increase of Na level, but there was no interaction with lasalocid level
References

Cervantes.H.M., Jensen. L.M., Brenes. A., 1982. Moderation of monensin induced growth depression by dietary potassium. Poultry Science 61:1107-1112.

Christmas R.B. & Harms R.H, 1988. Effect of Energy on the Performance of Broiler Chicks Fed Various Levels of Monensin. Poultry Science 67 :452-454.

Commision of the European Union., 2008. REPORT FROM THE COMMISSION TO THE COUNCIL AND THE EURO-PEAN PARLIAMENT ON THE USE OF COCCIDIOSTATS AND HISTOMONOSTATS AS FEED ADDITIVES.
COM(2008)233 final.

Frigg M. & Broz J., 1983. Effect of various doses of lasalocid and monensin in combination with increasing potassium levels on performance and water consumption of broiler chicks. Arch. Gefügelk.,47, 153-158, ISSN 0003-9098. © Eugen Ulmer GmbH & Co., Stuttgart.
THE EFFECT OF HOUSING SYSTEM AND CALCIUM ON EGGSHELL QUALITY

J. Svobodová<fnref n="1" />, E. Tůmová<fnref n="1" />, M. Englmaierová<fnref n="2" />

The aim of study was to evaluate the effect of housing system and feed calcium content on quality of the eggshell. The laying hens (ISA Brown) were housed in enriched cages (750 cm<fnref n="2" /> per hen), on litter (7 birds per m<fnref n="2" />) and in aviary (15 birds per m<fnref n="2" />). There were two groups differing in calcium content (3 % and 3.5 %) in the diet. The significant interaction between housing system and calcium content was found in egg weight (P ≤ 0.002). The heaviest eggs (62.2 g) were detected in aviary with 3% calcium, whereas the lightest eggs (60.4 g) were laid in cage with 3% calcium in diet. Housing system had significant effect on egg weight (P ≤ 0.002), when heavier eggs were produced on litter with compared to other housing systems. The significant interaction of housing and Ca was also found in eggshell strength (P < 0.001), the highest values were in cage with 3.5% calcium and the lowest on litter with 3.5% calcium. This parameter was affected by housing system with higher values in enriched cage. Calcium content not significantly influenced eggshell thickness, however, eggshell strength was significantly higher in 3.5% Ca. These results regarding the quality of the shell probably related to the different needs of calcium in different systems of housing.

<table><thead><tr><th rowspan="2">characteristics</th><th rowspan="2">Ca (%)</th><th colspan="3">Housing system</th><th colspan="3">significance</th></tr><tr><th>cage</th><th>litter</th><th>aviary</th><th>Housing system</th><th>Ca</th><th>Housing x Ca</th></tr></thead><tbody><tr><td rowspan="2">Egg weight (g)</td><td>3,0</td><td>60.4<sup>d</sup></td><td>61.1<sup>bcd</sup></td><td>62.2<sup>ab</sup></td><td rowspan="2">0.002</td><td rowspan="2">NS</td><td rowspan="2">0.002</td></tr><tr><td>3,5</td><td>60.7<sup>cd</sup></td><td>62.1<sup>a</sup></td><td>60.6<sup>cd</sup></td></tr><tr><td rowspan="2">Eggshell weight (g)</td><td>3,0</td><td>6.0<sup>b</sup></td><td>6.2<sup>a</sup></td><td>6.2<sup>a</sup></td><td rowspan="2">NS</td><td rowspan="2">NS</td><td rowspan="2">0.002</td></tr><tr><td>3,5</td><td>6.2<sup>a</sup></td><td>6.1<sup>ab</sup></td><td>6.2<sup>a</sup></td></tr><tr><td rowspan="2">Eggshell thickness (mm)</td><td>3,0</td><td>0.431</td><td>0.363</td><td>0.365</td><td rowspan="2">NS</td><td rowspan="2">NS</td><td rowspan="2">NS</td></tr><tr><td>3,5</td><td>0.370</td><td>0.359</td><td>0.367</td></tr><tr><td rowspan="2">Eggshell strength (g/cm<fnref n="2" />)</td><td>3,0</td><td>4651.2<sup>b</sup></td><td>4637.6<sup>b</sup></td><td>4605.8<sup>b</sup></td><td rowspan="2">&lt; 0.001</td><td rowspan="2">0.001</td><td rowspan="2">&lt; 0.001</td></tr><tr><td>3,5</td><td>4893.1<sup>a</sup></td><td>4563.1<sup>b</sup></td><td>4798.5<sup>a</sup></td></tr><tr><td rowspan="2">Eggshell ratio (%)</td><td>3,0</td><td>12.2<sup>ab</sup></td><td>12.1<sup>b</sup></td><td>11.6<sup>d</sup></td><td rowspan="2">&lt; 0.001</td><td rowspan="2">0.022</td><td rowspan="2">&lt; 0.001</td></tr><tr><td>3,5</td><td>12.3<sup>a</sup></td><td>11.8<sup>c</sup></td><td>12.2<sup>ab</sup></td></tr><tr><td rowspan="2">Eggshell colour</td><td>3,0</td><td>31.3<sup>a</sup></td><td>30.5<sup>b</sup></td><td>30.9<sup>ab</sup></td><td rowspan="2">0.006</td><td rowspan="2">&lt; 0.001</td><td rowspan="2">&lt; 0.001</td></tr><tr><td>3,5</td><td>28.9<sup>c</sup></td><td>30.3<sup>b</sup></td><td>31.1<sup>ab</sup></td></tr></tbody></table>

<footnote n="1">¹ Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Czech Republic</footnote>

<footnote n="2">² Institute of Animal Science, Prague-Uhříněves, Czech Republic</footnote>
P-190 (ID 260)

# DETERMINATION OF PHOSPHORUS DIGESTIBILITY OF MONO-DICALCIUM PHOSPHATE AND MONOCALCIUM PHOSPHATE IN BROILER CHICKENS

T. Trairatapiwan, S. Attamangkune Y. Ruangpanit and O. Songserm

Dept. of Animal Science, Kasetsart University, Kamphaeng Sean, Nakhon-Pathom, 73140, Thailand

Corresponding author: tas_tt@yahoo.com

## Abstract

This study was conducted to determine the preceacal digestibility of phosphorus of mono-dicalcium phosphate (MDCP) and monocalcium phosphate (MCP) in broiler chickens using the regression method. Prior to formulating experimental diets, Ca and P concentation of MDCP and MCP were analyzed and found to be 16.1 and 21.3%, 14.5 and 22.4%, respectively. Basal diet was formulated to contain 0.24% total P and each of test ingredients was supplemented into the basal diet to produce 3 experimental diets. The MDCP and MCP test diets containing 0.31, 0.38, 0.45 and 0.30, 0.36, 0.42% total P, respectively. The total Ca to total P ratio in all diets was maintained at 1.2:1 to 1.4:1. Titanium dioxide at the level of 0.3% was used as indigestible marker. One-day-old male broiler chicks were fed with a commercial starter diet from 1 to 20 day of ages. At 21 day of ages, a total of 252 birds were assigned into 7 experimental diets with 6 replicate cages (6 birds per cage). Birds were fed with experimental diets for 8 days. At 29 day of ages, all birds were sacrificed and ileal digesta were collected from the posterior half of ileum. Coefficients of P digestibility of MDCP and MCP were calculated by linear regression analysis. The analyzed concentrations of P increased with raising inclusion levels of MDCP and MCP in the diets and were in agreed with the calculated values. Coefficients of prececal digestibility of P in MDCP and MCP were 0.67 and 0.63, respectively. The present study indicated that the availability of P in MDCP and MCP are lower from the values currently used.

**Keywords:** phosphorus, digestibility, feed ingredient, broiler

## Introduction

Phosphorus is one of major minerals for poultry. The database for accurate phosphorus availability of feed ingredients is considered a useful tool for improving the accuracy of feed formulation. However, there are several approaches used to determine P availability (Shastak and Rodehutscord, 2013). Prececal phosphorus digestibility is the measurement of phosphorus disappearance at the end of ileum. Prececal digestibility have been adopted for estimate the digestibility of several feedstuffs due to this method based on quantitative measurements. Therefore, the value will be unaffected by post-ileal microbial activity and the phosphorus excretion in the urine can be excluded.

MDCP and MCP are P sources that are commonly used in poultry diets. However, the data of digestible P of MDCP and MCP are limited. Therefore, this study was conducted to determine the preceacal digestibility of P from MDCP and MCP in broiler chickens using the regression method.

## Material and Methods

### Animals

One-day-old male broiler chicks of Ross 308 were raised in evaporative cooling system house with rice husk as litter material and fed with a commercial starter diet from 1 to 20 day of ages. At 21 day of ages, birds were individually weighed. A total of 252 birds that had body weight in the range of 10% from average body weight were introduced to the experiment, such that the average weight across experimental treatments was similar. Birds were assigned into 7 experimental diets with 6 replicate cages (6 birds per cage) and fed with experimental diets for ad libitum intake for 8 days.

### Experimental diets

Prior to formulating experimental diets, MDCP and MCP were analyzed for Ca and P content. Basal diet was formulated to contain 0.24% total P. Each of test ingredients was supplemented into the basal diet at the expense of a P-free ingredient to produce 3 experimental diets. The MDCP and MCP test diets contained 0.31, 0.38, 0.45 and 0.30, 0.36, 0.42% total P, respectively. The total Ca to total P ratio in all diets was maintained at 1.2:1 to 1.4:1 by the addition of
ground limestone. Titanium dioxide at the level of 0.3% was used as indigestible marker. Experimental diets were offered in mash form. Ingredient and nutrient composition of experimental diets are shown in Table 1.

## Sample Collection and Chemical Analysis

At 29 day of ages, all birds were sacrificed and ileal digesta were collected from the posterior half of ileum. The ileum defined as the section between Meckel's diverticulum and 2 cm prior to the ileo-caeco-colonic-junction. The content was flushed out with distilled water, pooled for all birds from one cage, immediately frozen and later freeze-dried and ground.

Diets and ileal digesta samples were analyzed for DM, Ca, total P, and Ti. Dry matter was determined by method as described by AOAC (2005). Ca and P concentrations were determined by the inductively coupled plasma atomic emission spectroscopy method (ICP-OES). Titanium dioxide was determined by the colorimetric method as described by Short et al. (1996).

## Calculations

The precaecal digestibility of P (pcdP) from the supplemented sources were calculated according to the procedure outlined by Rodehutscord (2013).

The prececal digestible P was calculated for each diet and replicated pen using the following equation:

$$ \text{pcdP (\%)} = 100 - \left[ \frac{100 \times (\text{MDiet} \times \text{PDigesta})}{(\text{MDigesta} \times \text{PDiet})} \right] \text{ [equation 1]} $$

where

MDiet and MDigesta = analysed marker concentration in the diet or digesta (g/kg DM);

PDiet and PDigesta = analysed P concentration in the diet or digesta (g/kg DM).

The content of pcdP in the diet was calculated as:

$$ \text{pcdP (g/kg of diet)} = \text{pcdP (\%)} \times \text{PDiet} / 100 \text{ [equation 2]} $$

The pcdP (g/kg of diet) was plotted against total P concentration (g/kg of diet) in a linear regression analysis. The slope of the regression line gives the coefficient of precaecal digestibility of P from the supplemented source.

## Results

The concentration of Ca and P in MDCP and MCP samples was 16.1 and 21.3%, 14.5 and 22.4%, respectively. The analyzed concentrations of P increased with an increase inclusion levels of MDCP and MCP in the diets and the values were in agreed with the calculated values (Table 1). Strong linear relationships (P<0.001) were observed between dietary total P concentration and content of pcdP in the diet for both MDCP and MCP. The digestibility coefficients of P in MDCP and MCP were 0.67 and 0.63, respectively (Table 2).

## Conclusions

Inorganic phosphate is assumed to be highly availability P sources. Under the condition of the present study, the primary conclusion could be made as the digestibility of P in MDCP and MCP are lower from the available values currently used to formuaited diet (INRA, 2002). The similar coefficient of P digestibility between of MDCP and MCP were observed.

## Literature

AOAC International, 2005: Official Methods of Analysis. 18th ed. AOAC Int., Washington, DC.

RODEHUTSCORD, M, 2013: Determination of phosphorus availability in poultry. World's Poultry Science Journal 69: 687-698.

INRA, 2002: Tables of composition and nutritional value of feed materials. Wageningen Academic Publishers, Wageningen, ISBN 2-7380-1046-6.

SHASTAK, Y., M. RODEHUTSCORD, 2013: Determination and estimation of phosphorus availability in growing poultry and their historical development. World's Poultry Science Journal 69: 569-586.

SHORT, F. J., P. GORTON, J. WISEMAN, and K. N. BOORMAN, 1996: Determination of titanium dioxide added as an inert marker in chicken digestibility studies. Animal Feed Science Technology 59:215-221.
<table><caption>**Table 1.** Ingredient and nutrient composition (%) of experimental diets</caption><thead><tr><th rowspan="2">Ingredient composition</th><th rowspan="2">Basal diet</th><th colspan="3">MDCP</th><th colspan="3">MCP</th></tr><tr><th>Level 1</th><th>Level 2</th><th>Level 3</th><th>Level 1</th><th>Level 2</th><th>Level 3</th></tr></thead><tbody><tr><td>Test ingredient</td><td>-</td><td>0.33</td><td>0.64</td><td>0.97</td><td>0.26</td><td>0.51</td><td>0.78</td></tr><tr><td>Corn</td><td>51.40</td><td>51.40</td><td>51.40</td><td>51.40</td><td>51.40</td><td>51.40</td><td>51.40</td></tr><tr><td>Soybean meal, 45% CP</td><td>23.60</td><td>23.60</td><td>23.60</td><td>23.60</td><td>23.60</td><td>23.60</td><td>23.60</td></tr><tr><td>Dried egg albumen</td><td>8.00</td><td>8.00</td><td>8.00</td><td>8.00</td><td>8.00</td><td>8.00</td><td>8.00</td></tr><tr><td>Dextrose</td><td>13.65</td><td>13.26</td><td>12.90</td><td>12.52</td><td>13.28</td><td>12.94</td><td>12.60</td></tr><tr><td>Soybean oil</td><td>2.00</td><td>2.00</td><td>2.00</td><td>2.00</td><td>2.00</td><td>2.00</td><td>2.00</td></tr><tr><td>Limestone</td><td>0.39</td><td>0.45</td><td>0.50</td><td>0.55</td><td>0.50</td><td>0.59</td><td>0.66</td></tr><tr><td>Salt</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td></tr><tr><td>Sodium bicarbonate</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td></tr><tr><td>Trace mineral-vitamin premix<fnref n="1" /></td><td>0.25</td><td>0.25</td><td>0.25</td><td>0.25</td><td>0.25</td><td>0.25</td><td>0.25</td></tr><tr><td>Choline chloride</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td></tr><tr><td>Titanium dioxide</td><td>0.30</td><td>0.30</td><td>0.30</td><td>0.30</td><td>0.30</td><td>0.30</td><td>0.30</td></tr><tr><td colspan="8">Calculated analysis</td></tr><tr><td>ME, kcal/kg</td><td>3,148</td><td>3,134</td><td>3,122</td><td>3,109</td><td>3,135</td><td>3,123</td><td>3,112</td></tr><tr><td>Crude protein</td><td>21.0</td><td>21.0</td><td>21.0</td><td>21.0</td><td>21.0</td><td>21.0</td><td>21.0</td></tr><tr><td>Calcium</td><td>0.33</td><td>0.41</td><td>0.48</td><td>0.55</td><td>0.41</td><td>0.48</td><td>0.55</td></tr><tr><td>Total phosphorus</td><td>0.24</td><td>0.31</td><td>0.38</td><td>0.45</td><td>0.30</td><td>0.36</td><td>0.42</td></tr><tr><td>Ca:total P ratio</td><td>1.4</td><td>1.3</td><td>1.3</td><td>1.2</td><td>1.4</td><td>1.3</td><td>1.3</td></tr><tr><td colspan="8">Analyzed composition</td></tr><tr><td>Calcium</td><td>0.34</td><td>0.40</td><td>0.48</td><td>0.53</td><td>0.42</td><td>0.48</td><td>0.52</td></tr><tr><td>Total phosphorus</td><td>0.26</td><td>0.32</td><td>0.41</td><td>0.44</td><td>0.31</td><td>0.35</td><td>0.41</td></tr></tbody></table>

<table><caption>**Table 2.** Linear relationship between pcdP (g/kg of diet) and total P concentration (g/kg of diet) of MDCP and MCP</caption><thead><tr><th>Item</th><th>Regression equation</th><th>SE of the slope</th><th>SE of the intercept</th><th>r<sup>2</sup></th><th>Digestibility coefficient</th></tr></thead><tbody><tr><td>MDCP</td><td>Y = 0.6747X - 0.1723</td><td>0.0372</td><td>0.1481</td><td>0.94</td><td>0.67</td></tr><tr><td>MCP</td><td>Y = 0.6316X - 0.0212</td><td>0.0469</td><td>0.1758</td><td>0.91</td><td>0.63</td></tr></tbody></table>

<footnote n="1">¹Supplied per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 3,000 IU; vitamin E, 15 IU; vitamin K3, 1.5 mg; thiamine, 1.5 mg; riboflavin, 5.5 mg; pyridoxine, 2 mg; vitamin B12, 0.01 mg; nicotinic acid, 25 mg; pantothenic acid, 12 mg; folic acid, 0.5 mg; biotin, 0.12 mg, Mn, 80 mg; Zn, 60 mg; Fe, 40 mg; Cu, 8 mg; I, 0.5 mg; Co, 0.1 mg; Se, 0.1 mg</footnote>
P-191 (ID 163)

# INHIBITORY ACTION OF ANALYTICAL GRADE ZINC OXIDE AND OF A NEW POTENTIATED ZINC OXIDE ON THE IN VITRO GROWTH OF ESCHERICHIA COLI AND OF SALMONELLA ENTERICA STRAINS

W. Vahjen<fnref n="1" />, A. Roméo<fnref n="2" />, S. Durosoy<fnref n="2" /> and J. Zentek<fnref n="2" />

**Corresponding author:** aromeo@animine.eu

## Abstract

Zinc oxide (ZnO) is a feed additive well known for its antimicrobial activity. A new source of potentiated ZnO and a regular ZnO product were compared; their inhibitory action on the *in vitro* growth of enteric bacterial strains was studied in liquid media.

Two pathogenic *E. coli* strains, *Salmonella enterica* biovar Enteritidis (DSM9898) and *S. enterica* biovar Typhimurium were inoculated in 96 well microtiter plates with Zn concentrations ranging from 0.00125 to 0.32 µg Zn/mL, for each Zn source (regular ZnO and potentiated ZnO HiZox®), using Brain-Heart-Infusion (BHI) medium at pH 4.6 and 6.5. Measurements were taken every 2 min for 8h for media at pH 6.5 and every 4 min for 16h at pH 4.6. Coefficients for lag time and maximum growth in the stationary phase were calculated.

Lag phase, exponential growth and stationary phase could be distinguished at sub-inhibitory concentrations. Compared to regular ZnO, lag times were significantly (p<0.05) higher in potentiated ZnO for all the strains except for S. Typhimurium, at all Zn concentrations and at both pH, but only below 0.01 µL/mL for *E. coli* PS7 at pH 6.5; results were higher for S. Typhimurium at pH 6.5 and similar at pH 4.6. All strains showed globally higher maximum growth for standard ZnO compared to potentiated ZnO at all Zn dosages at pH 4.6. Maximum growth tended to be lower for both S. *enterica* in potentiated ZnO at pH 6.5.

In conclusion, compared to the regular ZnO, the new source of potentiated ZnO improved growth inhibition against two pathogenic *E. coli* and two S. *enterica* strains.

**Keywords:** zinc, inhibitory action, enteric bacteria

## Introduction

The inhibitory action of a potentiated ZnO (HiZox®) and analytical grade ZnO on the *in vitro* growth of pathogenic enteric bacterial strains was studied in liquid media. Not only the typical minimal inhibitory concentration (MIC), which determines complete inhibition at certain concentrations, but also growth below these concentrations were investigated in order to assess the growth repressing effect of ZnO at different concentrations.

## Material and Methods

The following strains were compared:

- *Escherichia coli* PS 79 (O147:K89:K88)

- *Escherichia coli* PS 7 (O138:K81)

- *Salmonella enterica* subsp. *enterica* biovar Enteritidis DSM 9898

- S. *enterica* subsp. *enterica* biovar Typhimurium DSM 5569

Saturated suspensions of Zn-containing media were produced by adding 10g of ZnO (analytical grade ZnO or HiZox®) to a total of 100 mL BHI media at pH 4,6 and pH 6.5. After incubation for 1h, the suspension was autoclaved. After centrifugation the supernatants were analysed for total Zn concentrations. All media were diluted with BHI for final concentrations of 0.00125 to 0.32 µg Zn/mL medium.

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

<footnote n="2">² Animine, Sillingy, France</footnote>
Cells of each strains were inoculated in 96 well microtiter plates. A microplate reader with 650 nm filter was used to record turbidity as measure for bacterial growth. Measurements were taken every 2 min over a period of 8h for media at pH 6.5 and every 4 min for 16h for media at pH 4.6.

A nonlinear regression analysis (3-sigmoidal curve fit) was employed to calculate coefficients for lag time [min] and maximum growth [OD]. Three-dimensional plots of incubation time vs. ZnO concentration vs. growth were used to visualize the effects of ZnO concentrations below MIC values. Curve fits were deemed satisfactory, when r² values of >0.999 were achieved from the growth data, which were compared at each Zn-concentration using the t-test. The level of significance was set to up ≤0,05.

## Results

Compared to the analytical grade ZnO, potentiated ZnO showed increased solubility at both pH values: +30% at pH 4.6 and +410% at pH 6.5 in BHI medium.

Growth of all tested strains at pH 6.5 below the inhibitory Zn-concentration of 0.32 µg/ml yielded typical three phase, sigmoid growth curves only at 0.04 µg/ml and lower concentrations. Here, lag phase, exponential growth and stationary phase could be distinguished. Therefore, a 3-parameter sigmoid curve fit was applied to these growth curves to describe the influence of different Zn-sources on bacterial growth. Zn-concentrations of 0.08 and 0.16 µg/ml yielded growth curves that showed starting inhibition of growth.

The lag phase describes the time period of bacterial growth before the onset of exponential growth and is a measure of the “fitness” of a bacterial strain in the medium. At pH 6.5, compared to analytical grade ZnO, *E. coli* PS79 tended to show higher lag times for potentiated ZnO at all Zn-concentrations, while *E. coli* PS7 showed higher lag times only at 0.00125 – 0.005 µg/ml Zn. Both *Salmonella* strains displayed higher lag times for potentiated ZnO at all subinhibitory Zn concentrations than for analytical grade ZnO. The lag time for all strains was markedly higher at pH 4.6 than at pH 6.5. Compared to analytical grade ZnO, *E. coli* strains showed increased lag times at all subinhibitory Zn concentrations in potentiated ZnO supplemented medium. The growth of *S. enterica* strains was severely reduced at pH 4.6, even without Zn in the medium. As a consequence, sigmoidal growth curves were only observed for low Zn-concentrations (0 – 0.02 µg/ml for S. Enteritidis; 0 – 0.01 µg/µml for S. Typhimurium). The identical lag times for S. Typhimurium must therefore be attributed to extremely low growth at pH 4.6 for this strain.

The maximum growth coefficient describes the final cell density in the stationary phase. At pH 6.5 and at subinhibitory concentrations, maximum growth was influenced by Zn-source. *Salmonella* strains displayed higher growth in media supplemented with analytical grade ZnO. Growth at pH 4.6 for 8h was not sufficient to generate typical sigmoid curves that are usually seen in bacterial in vitro growth. Instead, 16 h incubation had to be employed to describe growth behaviour with a sigmoid curve fit. All tested strains showed higher maximum growth for ZnO than for potentiated ZnO. However, growth was severely reduced for *S. enterica* strains in acidic pH and thus, results should be interpreted with caution.

Although identical total Zn-concentrations were employed, one has to keep in mind that there may be a difference between total Zn and the “bioavailability” of the different Zn-sources at low Zn-concentrations. As potentiated ZnO displayed a 5-fold increase of solubility, the actual available Zn-concentration may be higher due to aggregation of analytical grade ZnO to components of the growth medium.

As the total Zn-concentrations were identical in tested media, the increased bactericidal effect of potentiated ZnO must be attributed to the “bioavailability” of Zn²⁺. Potentiated ZnO may show less aggregation to components of the medium and thus, the amount of available Zn²⁺ may be higher. This would be especially effective at lower pH, as ZnO exhibits increasing solubility as pH decreases.

## Conclusions

Compared to analytical grade ZnO, potentiated ZnO HiZox® led to increased growth reduction against two pathogenic *E. coli* and two *S. enterica* strains. Minor strain specific differences regarding the growth reducing efficiency were observed. The reduction of growth, particularly at low pH, may be attributed to the higher solubility of HiZox®, which may originate from its higher surface area.
P-192 (ID 255)

NON-PHYTATE PHOSPHORUS LEVELS AND PARTICLE SIZE OF CALCIUM SOURCE IN THE DIET OF COMMERCIAL LAYERS AT THE END OF THE FIRST PRODUCTION CYCLE

Reza Vakili

Department of animal science, Kashmir Branch, Islamic Azad University, Kashmir, Iran; rezavakili2010@yahoo.com

**Abstract**

This study evaluated the effects of particle size distribution of calcium source in different levels non-phytate phosphorus diets on first-cycle layer performance and egg quality. A completely randomized experimental design with 5 treatments include control: 100% medium limestone particle size distributions and 100 % Non-phytate phosphorus Level requirement, treatment 2: 50% fine and 50% coarse and 70 % Non-phytate phosphorus Level requirement, treatment 3: 50% fine and 50% coarse and 50 % Non-phytate phosphorus Level requirement, treatment 4: 30% fine and 70% coarse and 70 % Non-phytate phosphorus Level requirement, treatment 5 30% fine and 70% coarse and 50 % Non-phytate phosphorus Level requirement was applied, with four replicates of 12 birds each. The treatments did not influence the most of evaluated performance and internal and external egg quality parameters. However, limestone particle size distribution quadratically affected with percentage of defective eggs, with the lowest percentage obtained with the distribution 50 % fine and 50% coarse limestone and 50 % Non-phytate phosphorus Level requirement. Increasing dietary non-phytate phosphorus significantly increased eggshell weight per surface area. It was concluded that the combination of the dietary 70 % Non-phytate phosphorus Level with 50% replacement of fine-particle limestone by coarse limestone results in better eggshell and increases the number of marketable eggs.

**Keywords:** Commercial Layers, Non-phytate phosphorus Levels, Particle Size of calcium source, first-cycle layer performance, egg quality

**Introduction**

Calcium is one of the essential minerals in poultry nutrition. In addition to its vital functions as the main component of bone structure and participation in acid-base balance and enzymatic system, calcium is the also the main component of the eggshell. It is estimated that each egg contains 2.2g of calcium, present mainly in the eggshell. When limestone particle size is reduced, its solubility increases. Calcium has several functions in bird metabolism and it is the main component of the eggshell. Dietary calcium levels, particle size and solubility, according to source, influence eggshell quality. Therefore, the knowledge on the quality of feedstuffs used for bird feeding is essential to promote better performance. The present study aimed at determining the effects of particle size distribution of calcium source in different levels non-phytate phosphorus diets on first-cycle layer performance and egg shell quality.

**Material and Methods**

The experiment was carried out at the experimental facilities of the Poultry Sector of the Islamic Azad University, Kashmir branch, Kashmir. In this study, 240, 22-week-old Hy- line layers in their first production cycle were used. Birds were housed in a layer house equipped with 60 metal cages (48cm long x 42cm deep x 45cm high). A photoperiod of 15 hours of light per day was applied. Composition of basal diet shown in Table 1.

A completely randomized experimental design with 5 treatments include 100% medium limestone particle size distributions and 100 % Non-phytate phosphorus Level requirement, 50% fine and 50% coarse and 70 % Non-phytate phosphorus Level requirement, 50% fine and 50% coarse and 50 % Non-phytate phosphorus Level requirement, 30% fine and 70% coarse and 70 % Non-phytate phosphorus Level requirement, 30% fine and 70% coarse and 50 % Non-phytate phosphorus Level requirement was applied, with four replicates of 12 birds each. The following performance parameters were evaluated. Egg production data were collected daily, and egg weight and feed intake were determined weekly. Eggshell quality and internal egg quality were evaluated every 28 days during a total period of 56 days. The obtained results were recorded and presented as mean values for the 56-day period. Birds were offered feed and water ad libitum during the entire experimental period (22 to 30 weeks).
<table><caption>**Table 1.** Composition of experimental diets (as feed basis)</caption><thead><tr><th>Ingredients</th><th>%</th><th></th><th></th></tr></thead><tbody><tr><td>Yellow Corn</td><td>60.70</td><td>Calculated value:</td><td></td></tr><tr><td>Soybean meal, 445 g crude protein / kg</td><td>16.10</td><td>Crude protein (g/kg)</td><td>14.70</td></tr><tr><td>Flaxseed</td><td>0.0</td><td>ME (Mcal/kg)</td><td>2700</td></tr><tr><td>Wheat bran</td><td>3.40</td><td>Calcuim (g/kg)</td><td>3.20</td></tr><tr><td>Barley</td><td>10.00</td><td>AvaiableP ppppPPphosphorous (g/kg)</td><td>0.28</td></tr><tr><td>Dicalcium Phosphate</td><td>0.69</td><td></td><td></td></tr><tr><td>Oyster shells</td><td>7.90</td><td></td><td></td></tr><tr><td>NaCl</td><td>0.26</td><td></td><td></td></tr><tr><td>Vitamin premix<sup>a</sup></td><td>0.50</td><td></td><td></td></tr><tr><td>Mineral premix<sup>b</sup></td><td>0.40</td><td></td><td></td></tr><tr><td>DL-Methionine</td><td>0.05</td><td></td><td></td></tr><tr><td>Total</td><td>100</td><td></td><td></td></tr></tbody></table>

## Results

The treatments did not influence the most of evaluated performance and internal and external egg quality parameters. However, limestone particle size distribution quadratically affected with percentage of defective eggs, with the lowest percentage obtained with the distribution 50 % fine and 50% coarse limestone and 70 % Non-phytate phosphorus Level requirement. Increasing dietary non-phytate phosphorus significantly increased eggshell weight per surface area. Egg loss (defective and broken eggs) results are consistent with those obtained by Oliveira et al. (2002), who worked with 2.8 to 4.4% Ca levels.

There was a significant effect (p<0.05) of limestone particle size distribution on the percentage of defective eggs (Table 2), with the highest percentage (0.71%) when 30% fine limestone and 70% coarse limestone were fed, and the lowest percentage with 50% fine and 50% coarse limestone. The calcium derived from dietary limestone is influenced by limestone particle size. When particle size distribution contained more than 50% coarse limestone, calcium release decreased due to the lower solubility of Ca in coarser particles, explaining the higher percentage of broken and defective eggs caused by limestone particle size distribution of 30% fine and 70% coarse particles

<table><caption>**Table 2.** Effect of limestone particle size distribution on the percentage of defective eggs.</caption><thead><tr><th rowspan="2">Variables</th><th colspan="5">Treatments</th><th rowspan="2">SEM</th><th rowspan="2">P-values</th></tr><tr><th>Control</th><th>T2</th><th>T3</th><th>T4</th><th>T5</th></tr></thead><tbody><tr><td>Broken eggs (%)</td><td>2.09<sup>a</sup></td><td>2.01<sup>a</sup></td><td>2.43<sup>b</sup></td><td>2.61<sup>c</sup></td><td>2.73<sup>c</sup></td><td>0.17</td><td>0.042</td></tr><tr><td>Defective eggs(%)</td><td>0.39<sup>a</sup></td><td>0.37<sup>a</sup></td><td>0.41<sup>b</sup></td><td>0.59<sup>b</sup></td><td>0.65<sup>b</sup></td><td>0.04</td><td>0.039</td></tr><tr><td>Egg shell(%)</td><td>9.88<sup>b</sup></td><td>9.67<sup>a</sup></td><td>9.61<sup>a</sup></td><td>9.91<sup>b</sup></td><td>9.86<sup>b</sup></td><td>0.08</td><td>0.7761</td></tr></tbody></table>

<footnote><sup>a</sup>Vitamin premix supplied the following per kilogram of diet: Vitamin A, 440.000 IU; Vitamin D3, 80.000 IU; Vitamin E, 96. mg; Vitamin K3, 2000 mg; Vitamin B1, 6120 mg; Vitamin B2, 3000 mg; Vitamin B6, 612 mg; Calcium Pantothenate, 8800 mg; Niacin, 50 mg; Biotin, 2 gr; Folic acid, 1.25 mg; Vitamin B12,640 mg (Telavang co., Tehran, Iran).</footnote>

<footnote><sup>b</sup>Mineral premix supplied the following per kilogram of diet: Cu (CuSO4·5H2O, 25.45% Cu) 8 gr; Fe (FeSO4·7H2O, 20.29% Fe) 100 gr; Mn (MnSO4·H2O,32.49% Mn) 64.52 gr; Zn (ZnO, 80.35% Zn) 33.8 gr; Se (NaSeO3, 45.56% Se) 8 mg (Telavang co., Tehran, Iran).</footnote>

<footnote><sup>a, b</sup> Means within a row lacking a common superscript differ (P < 0.05).</footnote>
Conclusions

It was concluded that the combination of the dietary 70 % Non-phytate phosphorus Level with 50% replacement of fine-particle limestone by coarse limestone results in better eggshell and increases the number of marketable eggs.

Literature

Cheng TK, Coon CN. Effect on layer performance and shell quality of switching limestones with different solubilites. Poultry Science 1990b; 69(12):2199-2203.

Clunies M, Parks D, Leeson S. Calcium and phosphorus metabolism and eggshell thickness in laying hens producing thick or thin shells. Poultry Science 1992; 71(3):490-498.

Guinotte F, Nys Y. Effects of particle size and origin of calcium sources on eggshell quality and bone mineralization in egg laying hens. Poultry Science 1991; 70:583-592
# Protein and amino acids nutrition

P-197 (ID 271)

## FACTORIAL MODEL TO ESTIMATE DIGESTIBLE VALINE, ISOLEUCINE AND TRYPTOPHAN REQUIREMENTS OF BROILER BREEDER HENS

M. B. Lima<fnref n="1" />, N. K. Sakomura<fnref n="1" />, E. P. Silva<fnref n="1" />, J. C. P. Dorigam<fnref n="1" />, J. B. K Fernandes<fnref n="2" /> and C. A. Gonçalves<fnref n="1" />

**Corresponding author:** sakomura@fcav.unesp.br

### Abstract

The objective of this study was to determine amino acid utilization efficiency (k) and develop a factorial model to estimate valine (Val), isoleucine (Ile) and tryptophan (Trp) intakes. One hundred ninety-two hens were used in each amino acid (AA) assay. The experimental design consisted of eight treatments, eight replicates and one hen per cage. The diets were formulated by dilution technique using a summit diet and a nitrogen free diet, resulting in AA levels that ranged from 1.97 to 9.85g/kg for Val and Ile, and 5 to 2.5 g/kg for Trp. Each assay lasted nine weeks. Data obtained were AA intake (AAI), body weight (BW) and egg output (EO). Broken line with one inclination was used to evaluate the responses and with two inclinations to estimate k. The factorial model was $AAI=[AA_m×(BW×0.196)^{0.73}]+[(N_{egg}×EO×AA_{egg})/k]$, where $AA_m$ is AA for maintenance (247, 134 or 37 mg/BP<sub>m</sub><sup>0.73</sup> for Val, Ile and Trp respectively) determined in previous studies at Unesp; $BP_m^{0.73}$ is mature body protein; k is 0.66 for Val, 0.60 for Ile or 0.56 for Trp determined in this study; $N_{egg}$ is the N content in the egg (1.89 g/100g); $AA_{egg}$ is the AA content in the egg (413, 338 or 108 mg/g for Val, Ile and Trp respectively). The AAI estimated by the model at 30 weeks were 853, 692 and 228 mg/day for Val, Ile and Trp, respectively. Our model incorporates the most physiologically relevant metrics, units, and values, which provide accurate estimates of amino acid requirements for broiler breeder hens.

**Keywords:** amino acid, broiler breeder hens, efficiency of utilization, egg output, mathematical model

### Introduction

Mathematical models represent an important tool that incorporates variables of productivity to update nutritional requirements. These models also allow for greater flexibility than experimental methods in determining what variables might be more relevant to specific regions or even individual producers (Gous, 2014). Specifically regarding broiler breeder hens, the factorial models mostly rely on coefficients and data from commercial laying hens. This is clearly not an optimal situation. For example, laying hens have an amino acid utilization efficiency of 85% whereas for broiler breeder hens the amino acid utilization efficiency is 49% (Bowmaker and Gous, 1991; Silva et al. 2015). These differences arise from the egg-laying potential of the two types of hens, therefore, requiring specific coefficients for the model to predict nutritional requirements. Thus, the objective of this study was to determine amino acid utilization efficiency (k) and develop a factorial model to estimate valine (Val), isoleucine (Ile) and tryptophan (Trp) intakes.

### Material and Methods

The experimental design consisted of eight treatments, eight replicates and one hen per cage. The diets were formulated by dilution technique using a summit diet and a nitrogen free diet, resulting in AA levels that ranged from 1.97 to 9.85g/kg for Val and Ile, and 5 to 2.5 g/kg for Trp. Each assay lasted nine weeks. Data obtained were AA intake (AAI), body weight (BW) and egg output (EO). Broken line with one inclination was used to evaluate the responses and with two inclinations to estimate k. Two models were compared, one from the literature (M1, from Bornstein et al. (1979)) and other model developed in this study (M2). The factorial model was $AAI=[AA_m×(BW×0.196)^{0.73}]+[(N_{egg}×EO×AA_{egg})/k]$, where $AA_m$ is AA for maintenance (247, 134 or 37 mg/BP<sub>m</sub><sup>0.73</sup> for Val, Ile and Trp respectively) determined in previous studies at Unesp; $BP_m^{0.73}$ is mature body protein; k is 0.66 for Val, 0.60 for Ile or 0.56 for Trp determined in this study; $N_{egg}$ is the N content in the egg (1.89 g/100g); $AA_{egg}$ is the AA content in the egg (413, 338 or 108 mg/g for Val, Ile and Trp respectively). To predict valine, isoleucine, and tryptophan intake, the data of body weight and egg mass of 60 broiler breeder hens of Cobb (500) strain were used. These data were obtained from individual measurement of the hens during the period from 25 to 60 weeks of age. The statistical analyses were performed using SAS software (Statistical Analysis System, version 9.2).

<footnote n="1">¹Dept. of Animal Science, São Paulo State University, Jaboticabal, São Paulo, Brazil</footnote>

<footnote n="2">²Aquaculture Center of São Paulo State University, Jaboticabal, São Paulo, Brazil</footnote>
## Results

Fitted models for efficiency of utilization in function of amino acid intake were: Efficiency<sub>Val</sub> = 65.50-0.05×(X-654)+0.21×(654-X), Efficiency<sub>Ile</sub> = 59.65-0.05×(X-495)+0.27×(495-X), Efficiency<sub>Trp</sub> = 55.69-0.14×(X-171.2)+0.67×(171.2-X). Factorial models M1 and M2 were simulated with body weight and egg output from 60 individually monitored hens. The results of the simulation are shown in Table 1. The M1 model compared to M2 predicted 20% and 15% higher intakes for Val and Ile, respectively. The predicted intake for Trp was 50% lower in M1 than M2. The observed differences between the models remained during the whole period assessed.

<table><caption>**Table 1.** Prediction of valine, isoleucine, and tryptophan intake for broiler breeder hens from 25 to 60 weeks of age using two mathematic models with body weight (BW), body weight gain (BWG), and egg output (EO) data.</caption><thead><tr><th rowspan="2">Age weeks</th><th rowspan="2">BW kg</th><th rowspan="2">BWG g/day</th><th rowspan="2">EO g/day</th><th colspan="2">Valine intake</th><th colspan="2">Isoleucine intake</th><th colspan="2">Tryptophan intake</th></tr><tr><th>M1<sup>a</sup></th><th>M2<sup>b</sup></th><th>M1<sup>a</sup></th><th>M2<sup>b</sup></th><th>M1<sup>a</sup></th><th>M2<sup>b</sup></th></tr></thead><tbody><tr><td>25</td><td>3.89</td><td>6</td><td>37.1</td><td>790</td><td>645</td><td>601</td><td>507</td><td>111</td><td>166</td></tr><tr><td>30</td><td>4.10</td><td>6</td><td>53.9</td><td>1057</td><td>853</td><td>793</td><td>692</td><td>149</td><td>228</td></tr><tr><td>35</td><td>4.32</td><td>6</td><td>55.7</td><td>1098</td><td>883</td><td>824</td><td>716</td><td>155</td><td>236</td></tr><tr><td>40</td><td>4.54</td><td>4</td><td>55.6</td><td>1108</td><td>889</td><td>834</td><td>719</td><td>156</td><td>237</td></tr><tr><td>45</td><td>4.68</td><td>4</td><td>50.0</td><td>1032</td><td>828</td><td>781</td><td>662</td><td>145</td><td>217</td></tr><tr><td>50</td><td>4.82</td><td>0</td><td>48.7</td><td>1020</td><td>817</td><td>774</td><td>650</td><td>143</td><td>213</td></tr><tr><td>55</td><td>4.83</td><td>0</td><td>47.7</td><td>1005</td><td>805</td><td>764</td><td>640</td><td>141</td><td>209</td></tr><tr><td>60</td><td>4.83</td><td>0</td><td>45.6</td><td>974</td><td>780</td><td>741</td><td>617</td><td>137</td><td>202</td></tr></tbody></table>

The average egg output and body weight values for 60 hens at 25, 30, 35, 40, 45, 50, 55, and 60 weeks of age were applied in the M2 model to predict Val, Ile, and Trp intake (Table 2). For egg output, the coefficient of variation (CV) at 25 weeks was high (35%) because of the low rate of lay, but after this age the CV of egg output stabilized at the average value of 19%. For body weight, the CV ranged between 7 and 10% of the average value of 4.5 kg. The average egg output value and standard deviation accounted for 46.55% of the population, whereas maximum egg output values accounted for 90.07% of the population. For body weight, the average values corresponded to 54.55% of the population and maximum values for 94.66% of the population. Predicted values for Val, Ile, and Trp intake varied by 544, 487, and 165 mg/hen per day, respectively (Table 2). Differences between the maximum and average intakes were 223 (Val), 197 (Ile), and 66 (Trp) mg/hen per day, yielding maximum values approximately 30% higher than average intake values.

## Conclusions

The average efficiency of utilization for the three amino acids (Val, Ile, and Trp) was 66%. The AAI estimated by the model at 30 weeks were 853, 692 and 228 mg/day for Val, Ile and Trp, respectively. Our model incorporates the most physiologically relevant metrics, units, and values, which provide accurate estimates of amino acid requirements for broiler breeder hens.

<footnote><sup>a</sup>M1: AAI=[(AA<sub>m</sub>/0.85)×BW]+[BWG×(0.21×AA<sub>t</sub>)]+[EO×(63×AA<sub>y</sub>+158×AA<sub>t</sub>)] from Bornstein et al. (1979).</footnote>

<footnote><sup>b</sup>M2: AAI=[AA<sub>m</sub>×(BW×0.196)<sup>0.73</sup>]+[(N<sub>egg</sub>×EO)×AA<sub>egg</sub>)/k] developed in this study.</footnote>
<table><caption>**Table 2.** Data from a population of 60 broiler breeder hens and prediction of the minimum (min), average and standard deviation (μ±SD), and maximum (max) values for valine, isoleucine, and tryptophan intake based on the application of the factorial models.</caption><thead><tr><th rowspan="3">Age<br>weeks</th><th colspan="6">Population data</th></tr><tr><th colspan="3">Egg output (g)</th><th colspan="3">Body weight (kg)</th></tr><tr><th>Min</th><th>μ±SD</th><th>Max</th><th>min</th><th>μ±SD</th><th>max</th></tr></thead><tbody><tr><td>25</td><td>13</td><td>37±14</td><td>53</td><td>3.3</td><td>3.9±0.3</td><td>4.4</td></tr><tr><td>30</td><td>20</td><td>54±10</td><td>70</td><td>3.5</td><td>4.1±0.3</td><td>4.6</td></tr><tr><td>35</td><td>35</td><td>56±7</td><td>70</td><td>3.7</td><td>4.3±0.4</td><td>5.0</td></tr><tr><td>40</td><td>29</td><td>56±10</td><td>80</td><td>3.6</td><td>4.5±0.4</td><td>5.3</td></tr><tr><td>45</td><td>19</td><td>50±10</td><td>65</td><td>3.6</td><td>4.7±0.4</td><td>5.5</td></tr><tr><td>50</td><td>15</td><td>49±10</td><td>68</td><td>3.4</td><td>4.8±0.4</td><td>5.5</td></tr><tr><td>55</td><td>24</td><td>48±9</td><td>68</td><td>3.4</td><td>4.8±0.4</td><td>5.6</td></tr><tr><td>60</td><td>22</td><td>46±10</td><td>63</td><td>3.1</td><td>4.8±0.5</td><td>5.7</td></tr></tbody></table>

<table><thead><tr><th rowspan="3"></th><th colspan="8">Amino acid intakes predicted</th></tr><tr><th colspan="2">Valine</th><th colspan="2">Isoleucine</th><th colspan="2">Tryptophan</th><th colspan="2">Isoleucine</th></tr><tr><th>Min</th><th>μ±SD</th><th>max</th><th>min</th><th>μ±SD</th><th>max</th><th>min</th><th>μ±SD</th><th>max</th></tr><tr><th></th><th></th><th></th><th></th><th colspan="2">mg/bird per day</th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>25</td><td>349</td><td>645±171</td><td>833</td><td>244</td><td>507±153</td><td>676</td><td>76</td><td>166±52</td><td>223</td></tr><tr><td>30</td><td>454</td><td>853±121</td><td>1051</td><td>332</td><td>692±109</td><td>869</td><td>105</td><td>228±37</td><td>288</td></tr><tr><td>35</td><td>641</td><td>883±87</td><td>1054</td><td>498</td><td>716±78</td><td>870</td><td>162</td><td>236±26</td><td>288</td></tr><tr><td>40</td><td>582</td><td>889±117</td><td>1186</td><td>439</td><td>719±106</td><td>982</td><td>141</td><td>237±36</td><td>326</td></tr><tr><td>45</td><td>458</td><td>828±122</td><td>1022</td><td>328</td><td>662±109</td><td>832</td><td>103</td><td>217±37</td><td>274</td></tr><tr><td>50</td><td>423</td><td>817±127</td><td>1063</td><td>292</td><td>650±113</td><td>865</td><td>91</td><td>213±38</td><td>285</td></tr><tr><td>55</td><td>533</td><td>805±113</td><td>1061</td><td>392</td><td>640±100</td><td>866</td><td>125</td><td>209±34</td><td>286</td></tr><tr><td>60</td><td>485</td><td>780±127</td><td>1011</td><td>358</td><td>617±119</td><td>816</td><td>114</td><td>202±38</td><td>268</td></tr></tbody></table>

## Literature

BORNSTEIN, S., HURWITZ, S., LEV, Y. 1979: The amino acid and energy requirements of broiler breeder hens. Poult. Sci. **58**, 104-116.

BOWMAKER, J.E., GOUS, R.M. 1991: The response of broiler breeder hens to dietary lysine and methionine. Br. Poult. Sci. **32**, 1069-1088.

GOUS, R.M. 2014: Modeling as a research tool in poultry science. Poult. Sci. **93**, 1-7.

SILVA, E.P., MALHEIROS, E.B., SAKOMURA, N.K., VENTURINI, K.S., HAUSCHILD, L., DORIGAM, J.C.P., FERNANDES, J.B.K. 2015: Lysine requirements of laying hens. Livest. Sci. **173**, 69-77.

SAS, 2008: User's Guide: Statistics (version 9.2). SAS Institute Inc.
# MAINTENANCE REQUIREMENTS OF VALINE, ISOLEUCINE, AND TRYPTOPHAN FOR POULTRY

M. B. Lima, N. K. Sakomura, J. C. P. Dorigam, E. P. Silva and J. Gobi

Dept. of Animal Science, São Paulo State University, Jaboticabal, São Paulo, Brazil

Corresponding author: sakomura@fcav.unesp.br

## Abstract

The objective of this study was to determine maintenance requirements of valine (Val), isoleucine (Ile), and tryptophan (Trp) for poultry. Each nitrogen (N) balance trial lasted five days and was conducted for each amino acid (AA). After 48 hours of fasting (receiving water + sucrose), 40 g of each diet limiting in Val, Ile, or Trp were fed by tube, once a day, for three days to give a range of intakes from 0 to 101, 0 to 119, and 0 to 34 mg for Val, Ile, and Trp per kg of body weight (BW<sub>kg</sub>), respectively. A N-free diet was offered *ad libitum* during these three days. Excreta were collected during the last three days of the balance period and the N content of the excreta was analyzed. Nitrogen retention (NR) was obtained by the difference between N intake and N excretion. For each AA, a linear regression between NR and AA intake (mg/BW<sub>kg</sub> per day) was performed. The equations obtained were: NR= -98.6+2.4×Val (R<sup>2</sup> = 0.90), NR= -46.9+2.3×Ile (R<sup>2</sup> = 0.92), and NR= -39.5+7.3×Trp (R<sup>2</sup> = 0.92). The Val, Ile, and Trp required to maintain the body at zero NR were calculated to be 41, 20, and 5 mg/BW<sub>kg</sub> per day, respectively. Expressing mg per kg of metabolic weight, the intake of Val, Ile, and Trp was 59, 32, and 9, respectively. Considering the degree of maturity of the birds and body protein content (BP<sub>m</sub><sup>0.73×U</sup>), the amount of Val, Ile, and Trp required for maintenance was calculated to be 247, 134, and 37 mg/BP<sub>m</sub><sup>0.73×U</sup> per day. Maintenance requirement is more adequately expressed as body protein content.

**Keywords:** isoleucine, maintenance, nitrogen balance, tryptophan, valine

## Introduction

Many studies to determine maintenance requirement express the values as mg of amino acid per kg of body weight or mg of amino acid per kg of metabolic body weight (Leveille and Fisher 1960; Baker et al., 1996). However, there are differences in body composition of birds, especially regarding the fat content. Therefore, it is more adequate to express amino acid maintenance requirement based on protein weight of the bird, which would allow comparisons between studies (Burnham and Gous, 1992; Bonato et al., 2011). Thus, the aim of this study was to determine the requirements of digestible valine, isoleucine, and tryptophan for maintenance considering different unit systems and using Cobb 500 roosters.

## Material and Methods

One hundred forty four Cobb 500 roosters were used in three nitrogen balance studies. Forty two roosters were used in valine trial, 54 in isoleucine and 48 in tryptophan trials. The roosters were individually housed in metabolic cages with individual feeder and nipple type drinkers. After 48 hours of fasting (receiving water + sucrose), 40 g of each diet limiting in Val, Ile, or Trp were fed by tube, once a day, for three days to give a range of intakes from 0 to 101, 0 to 119, and 0 to 34 mg for Val, Ile, and Trp per kg of body weight (BW<sub>kg</sub>), respectively. A N-free diet was offered *ad libitum* during these three days. Excreta were collected during the last three days of the balance period and the N content of the excreta was analyzed. Nitrogen retention (NR) was obtained by the difference between N intake and N excretion. The nitrogen retention responses to the amino acid intakes were expressed in three unit systems: 1) mg per kg of body weight 2) mg per kg of metabolic weight and 3) mg per unit of metabolic protein weight at maturity. Statistical analyses were performed using the PROC REG from SAS 9.2 software.

## Results

The lowest feed intake was observed in valine trial (118 g/day), followed by isoleucine (143 g/day) and tryptophan (167 g/day) trials (Table 1). The experimental diets provided nitrogen retentions that ranged from negative to positive NR values. As the amino acid intake increased the NR values also increased. The responses to the control diets were limiting in the diet, because they provided retentions between levels 2 and 3, validating this study.
<table><caption>**Table 1.** Mean values for feed intake (FI), amino acid intake (AAI), and nitrogen retention (NR) in different systems of units.</caption><thead><tr><th rowspan="2">Levels (%)</th><th colspan="2">FI</th><th colspan="3">AAI, mg</th><th colspan="3">NR</th></tr><tr><th>g/d</th><th>kg BW d</th><th>kg BW<sup>0.75</sup></th><th>kg BP<sub>m</sub><sup>0.73</sup> d</th><th>kg BW d</th><th>kg BW<sup>0.75</sup></th><th>kg BP<sub>m</sub><sup>0.73</sup> d</th></tr></thead><tbody><tr><td><em>Valine</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0.000</td><td>111.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>-96.3</td><td>-137.5</td><td>-577.0</td></tr><tr><td>0.235</td><td>124.5</td><td>21.8</td><td>30.5</td><td>129.8</td><td>-37.8</td><td>-53.0</td><td>-222.5</td></tr><tr><td>0.470</td><td>108.6</td><td>43.4</td><td>61.2</td><td>256.8</td><td>-10.4</td><td>-15.8</td><td>-66.0</td></tr><tr><td>0.705</td><td>110.8</td><td>60.0</td><td>86.2</td><td>362.4</td><td>55.6</td><td>79.0</td><td>332.0</td></tr><tr><td>0.940</td><td>126.3</td><td>81.8</td><td>116.0</td><td>488.3</td><td>102.8</td><td>145.5</td><td>611.0</td></tr><tr><td>1.175</td><td>125.8</td><td>100.8</td><td>143.2</td><td>601.6</td><td>141.6</td><td>199.8</td><td>839.0</td></tr><tr><td>Root MSE</td><td>16.1</td><td>18.3</td><td>13.9</td><td>42.6</td><td>40.1</td><td>54.9</td><td>230.4</td></tr><tr><td><em>Isoleucine</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0.000</td><td>112.3</td><td>0.0</td><td>0.0</td><td>0.0</td><td>-58.2</td><td>-89.7</td><td>-379.5</td></tr><tr><td>0.404</td><td>142.8</td><td>27.0</td><td>41.5</td><td>175.0</td><td>20.5</td><td>31.2</td><td>131.3</td></tr><tr><td>0.606</td><td>148.6</td><td>39.6</td><td>60.6</td><td>256.6</td><td>67.2</td><td>103.6</td><td>438.0</td></tr><tr><td>0.808</td><td>133.5</td><td>50.8</td><td>79.17</td><td>335.8</td><td>68.8</td><td>106.7</td><td>451.5</td></tr><tr><td>1.010</td><td>153.8</td><td>64.7</td><td>100.5</td><td>424.7</td><td>94.2</td><td>145.5</td><td>615.8</td></tr><tr><td>1.212</td><td>144.8</td><td>78.2</td><td>120.6</td><td>510.6</td><td>140.6</td><td>216.8</td><td>916.6</td></tr><tr><td>1.414</td><td>155.5</td><td>94.5</td><td>144.0</td><td>608.3</td><td>189.0</td><td>286.8</td><td>1211.5</td></tr><tr><td>1.818</td><td>156.2</td><td>118.6</td><td>183.2</td><td>774.4</td><td>221.4</td><td>342.0</td><td>1448.2</td></tr><tr><td>Root MSE</td><td>29.86</td><td>6.04</td><td>6.96</td><td>28.74</td><td>27.39</td><td>40.54</td><td>30.83</td></tr><tr><td><em>Tryptophan</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0.000</td><td>158.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>-48.8</td><td>-76.2</td><td>-322.0</td></tr><tr><td>0.095</td><td>154.0</td><td>6.0</td><td>9.8</td><td>40.5</td><td>13.8</td><td>21.3</td><td>89.5</td></tr><tr><td>0.190</td><td>162.8</td><td>12.0</td><td>18.8</td><td>79.8</td><td>61.2</td><td>95.7</td><td>405.5</td></tr><tr><td>0.285</td><td>162.7</td><td>17.5</td><td>27.7</td><td>118.0</td><td>82.8</td><td>129.8</td><td>550.0</td></tr><tr><td>0.380</td><td>166.2</td><td>23.2</td><td>36.8</td><td>155.8</td><td>121.2</td><td>191.2</td><td>810.2</td></tr><tr><td>0.475</td><td>172.3</td><td>29.7</td><td>46.3</td><td>196.5</td><td>182.8</td><td>287.0</td><td>1215.2</td></tr><tr><td>0.570</td><td>194.7</td><td>34.0</td><td>53.7</td><td>227.2</td><td>207.0</td><td>327.5</td><td>1388.3</td></tr><tr><td>Root MSE</td><td>33.5</td><td>1.5</td><td>1.7</td><td>7.0</td><td>29.2</td><td>44.4</td><td>187.7</td></tr></tbody></table>

BW = Body weight; BP<sub>m</sub><sup>0.73</sup> = Body protein at maturity; Root MSE = root mean square error.

The regression equations obtained for all the different systems of units regarding the valine, isoleucine, and tryptophan trials are shown in Table 2. The maintenance requirements determined for valine, isoleucine and tryptophan were 41, 20 and 5 mg/BW, respectively. The requirements determined considering the mg of amino acid per unit of metabolic protein weight at maturity were 24, 134, and 37 mg/BP<sub>m</sub><sup>0.73</sup> per day for valine, isoleucine, and tryptophan, respectively. Many researchers recommend requirements for maintenance using the system mg/BW per day or mg/BW<sup>0.75</sup> per day (Leveille and Fisher, 1960; Leveille et al., 1960). However, these systems do not consider the differences in body composition. According to Taylor (1980) and Emmans (1989), the requirement for maintenance should be related to protein weight of the bird and maturity rate of body protein (u), which is the ratio of the current protein weight of the bird divided by the mature protein weight. Because the scale used by these authors does not consider the fat content, it is suitable for avoiding imprecision in the definition of amino acid requirements due to variation of fat content in the body of the birds, which can vary widely in birds of the same genotype and body weight (Burnham and Gous, 1992). These authors suggested that the best way to express the amino acid requirement for maintenance in growing and adult birds is according to protein weight, because there is no demand for amino acid to maintain the adipose tissue.
<table><caption>**Table 2.** Maintenance requirements for valine, isoleucine, and tryptophan estimated from linear equations considering three units.</caption><thead><tr><th>Amino acid</th><th>Regression equation<sup>1</sup></th><th>R<sup>2</sup></th><th>Requirement</th></tr></thead><tbody><tr><td><strong>Valine</strong></td><td></td><td></td><td></td></tr><tr><td>mg/kg BW d</td><td>NR = -98.6 (±10.1) + 2.4 (±0.2) × Val</td><td>0.90</td><td>41</td></tr><tr><td>mg/kg BW<sup>0.75</sup> d</td><td>NR = -140.6 (±14.7) + 2.4 (±0.2) × Val</td><td>0.90</td><td>59</td></tr><tr><td>mg/kg BP<sub>m</sub><sup>0.73</sup> d</td><td>NR = -591.7 (±62.2) + 2.4 (±0.2) × Val</td><td>0.90</td><td>247</td></tr><tr><td><strong>Isoleucine</strong></td><td></td><td></td><td></td></tr><tr><td>mg/kg BW d</td><td>NR = -46.9 (±7.1) + 2.3 (±0.1) × Ile</td><td>0.92</td><td>20</td></tr><tr><td>mg/kg BW<sup>0.75</sup> d</td><td>NR = -72.6 (±11.1) + 2.3 (±0.1) × Ile</td><td>0.92</td><td>32</td></tr><tr><td>mg/kg BP<sub>m</sub><sup>0.73</sup> d</td><td>NR = -307.9 (±46.7) + 2.3 (±0.1) × Ile</td><td>0.92</td><td>134</td></tr><tr><td><strong>Tryptophan</strong></td><td></td><td></td><td></td></tr><tr><td>mg/kg BW d</td><td>NR = -39.5 (±7.7) + 7.3 (±0.4) × Trp</td><td>0.92</td><td>5</td></tr><tr><td>mg/kg BW<sup>0.75</sup> d</td><td>NR = -62.9 (±12.5) + 7.3 (±0.4) × Trp</td><td>0.92</td><td>9</td></tr><tr><td>mg/kg BP<sub>m</sub><sup>0.73</sup> d</td><td>NR = -266.7 (±52.4) + 7.3 (±0.4) × Trp</td><td>0.92</td><td>37</td></tr></tbody></table>

## Conclusions

Based on the concepts presented in this study, the requirement of valine, isoleucine and tryptophan for maintenance are 247, 134, and 37 mg/kg BP<sub>m</sub><sup>0.73</sup> d, respectively.

## Literature

BAKER, D.H., FERNANDEZ, S.R., PARSONS, C.M., EDWARDS III, H.M., EMMERT, J.L., WEBEL, D.M. 1996: Maintenance requirement for valine and efficiency of its use above maintenance for accretion of whole body valine and protein in young chicks. J Nutr. **126**, 1844-1851.

BONATO, M.A., SAKOMURA, N.K., SIQUEIRA, J.C., FERNANDES, J.B.K., GOUS, R.M. 2011: Maintenance requirements for methionine and cysteine, and threonine for poultry. South Afr J Anim Sci. **41**, 209-222.

BURNHAM, D., GOUS, R.M. 1992: Isoleucine requirements of the chicken: requirement for maintenance. Br. Poult. Sci. **33**, 59-69.

EMMANS, G.C. 1989: The growth of turkeys. In: NIXEY, C., T.C. GREY: Recent Advances in Turkey Science 1989. Butterworths, London, 135-166.

LEVEILLE, G.A., FISHER, H. 1960: Amino acid requirements for maintenance in the adult rooster III. The requirements for leucine, isoleucine, valine and threonine, with reference also to the utilization of the d-isomers of valine, threonine and isoleucine. J Nutr. **70**, 135-140.

TAYLOR, St C. S. 1980: Genetic scaling rules in animal growth. Anim Prod. **30**, 161-165.

<footnote n="1">¹The data from control diet was not used in the statistical analyses. BW = Body weight; BP<sub>m</sub> = Body protein at maturity; NR = nitrogen retention; Val = valine; Ile = isoleucine; Trp = tryptophan.</footnote>
# PARTIAL REPLACEMENT OF COMMERCIAL PROTEIN CONCENTRATE BY FEATHER MEAL IN BROILER DIETS

A. A. Bhuiyan¹, Shijun Li¹*, A. A. Bhuyan² and M. N. Islam³

¹Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University; P.R. China

²Animal Biotechnology Division, National Institute of Biotechnology, Bangladesh

³Poultry Production Research Division, Bangladesh Livestock Research Institute, Bangladesh

*Corresponding author: lishijun@mail.hzau.edu.cn, aab76_blri@yahoo.com; Tel: +86-27-8738-7480, Fax: +86-27-8728-0408

## Abstract

Two hundred and sixty four straight run day old chicks were reared for a period of 35 days to know the effect of inclusion of RFM and TFM as a replacement of CPC in broiler diets. The inclusion levels of RFM and TFM were 0, 2, 4 and 6% of the diets. The RFM and TFM respectively replaced 0, 25, 50, and 75% of CPC in the diets where CPC content of control diet was 8%. A total of 24 healthy birds of either sex from non-feather meal dietary group were reared from 36 to 49 days for estimating the digestibility of CPC, RFM and TFM in metabolic trials. The crude protein contents of CPC, RFM and TFM were estimated as 53.7, 83.5 and 81.3%, respectively. Feed intake, live weight gain, feed efficiency and PER of the birds 2% RFM and TFM diet were considerably better than others. Feed intake and liveweight of broiler birds decreased linearly with the increase of RFM and TFM level beyond 2% level. Mortality of birds did not differ significantly among the tested diets. The percent digestibility was highest in CPC, intermediate in TFM and lowest in RFM diet. Inclusion of TFM in broiler diets may be suggested at 2% level by replacing similar amount of CPC.

**Keywords:** Commercial protein concentrate, feather meal, broiler diet

## Introduction

Rapid growth in poultry production demands increased amount of protein concentrate along with other feed ingredients. Cheap and locally available alternative sources of proteins should be identified to meet the growing demand. Dale (1992) reported that feather meal contained a level of high energy (3092-3996 kcal/kg ME). Heating feather meal results in cleavage of disulfide bonds of cystine, which allow better digestion of this product in growing chicks (Moran et al., 1966). Due to differences in processing conditions, commercial feather meals vary widely in their protein digestibilities (Papadopoulos et al., 1985; Han and Parsons, 1991). Burgos et al. (1974) reported that the amino acids in feather meal are almost completely digestible (95%). On the other hand, some other researchers (Parsons et al., 1982; Sibbald, 1986) found much lower digestibility (about 32%) of these amino acids. However, the digestibility of feather meal could be improved through treatment. The present study has therefore been designed to examine the effects of the replacement of protein concentrate by graded levels of feather meal in broiler diets.

## Materials and Methods

The study was conducted with 264 straight run Hubbard Classic broiler chicks and birds were reared on floor under almost similar management condition. Commercial protein concentrate (CPC) in diets were replaced by two different types of feather meals (raw and treated) with four different levels (0, 2, 4, and 6% of the diet). Twenty four separate pens having 3 replications in each dietary group were used for the trial. Seven iso-nitrogenous and iso-caloric diets were fed to the birds. Raw feather meal (RFM) was treated with alkali and autoclaved for 20 minutes under 15 PSI. Thereafter, the materials were oven dried and ground to produce treated feather meal (TFM).

## Protocol of metabolic trial

After 35-day trial, 24 selected healthy birds of either sex (mean liveweight of 810±42g) from control pens were transferred to individual cages and reared for 14 days for metabolic trial. Ten days acclimatization period was provided for all birds with clean drinking water and standard broiler diet containing 22.04% CP and 2881 kcal ME/kg. Six birds were randomly allocated to each of the test feeds (i.e. CPC, RFM and TFM) and rest six birds for feeding glucose to determine endogenous loss. After 10 days adjustment period, individual bird was fasted for 24h. Birds of all groups were then drenched 40g of each test feeds into the crop through a 60ml syringe fitted with 18cm plastic tube of 3.85mm internal and 6.60mm external diameter. During this period, excreta were collected for 24h from individual bird. Fresh excreta was then weighed and preserved at -20°C until they were used for chemical analysis.
## Chemical analysis, determination of endogenous losses and digestibility

All test materials and homogenized fresh excreta were subjected to chemical analysis and were determined by using proximate analysis procedure according to AOAC (1984). Proportion of nutrient found in excreta (abraded intestinal mucusa, bile, digestive fluid etc.) during glucose feeding period was considered as the endogenous loss and true digestibility was estimated according to Banerjee (1988).

## Statistical analysis

All recorded and calculated data were for a 4 (FM levels) × 2 (FM types) × 3 (replication) in completely randomized design. The analysis of variance was done using SPSS 11.5 software (SPSS, 1999) and the significant differences between means were identified by Duncan's New Multiple Range Test.

## Results and Discussion

Chemical analysis indicates that moisture, OM, CP and CF contents were much higher in feather meals (Table 1). Crude protein contents of CPC, RFM and TFM were 53, 83 and 81% respectively. Crude protein content of feather meals got support from earlier studies (Dale, 1992; Chubb, 1982 and Waldroup, 1982). On the other hand, just opposite scenario persisted for EE and Ash content.

<table><caption>**Table 1.** Chemical compositions of CPC, RFM and TFM</caption><thead><tr><th rowspan="2">INGREDIENTS</th><th colspan="7">Available nutrients (g / 100g)</th></tr><tr><th>Moisture</th><th>DM</th><th>OM</th><th>CP</th><th>CF</th><th>EE</th><th>Ash</th></tr></thead><tbody><tr><td>CPC</td><td>4.61</td><td>95.39</td><td>75.12</td><td>53.74</td><td>1.43</td><td>9.02</td><td>20.27</td></tr><tr><td>RFM</td><td>8.76</td><td>91.24</td><td>90.25</td><td>83.50</td><td>2.96</td><td>2.79</td><td>0.99</td></tr><tr><td>TFM</td><td>9.09</td><td>90.91</td><td>89.04</td><td>81.25</td><td>2.32</td><td>4.49</td><td>1.87</td></tr></tbody></table>

The effect of feather meal types, inclusion levels and their interaction on feed intake, body weight, feed conversion, mortality, protein intake and protein efficiency ratio of broilers were calculated. Except final liveweight, none of the performance characteristics was affected by feather meal types, or by its interaction with level of inclusion. On the contrary, all of the parameters studied, other than mortality of birds, were remarkably influenced by levels of feather meals inclusion in broiler diets. Results indicate that birds fed 2% TFM diet consumed highest amounts (2009.85g) of feed, which was more or less close (P>0.05) to that of CPC diet (2006.33g). Replacement beyond 2% of CPC in broiler diets adversely affected (P<0.01) the feed intake of birds, which in turn lowered the growth performance of birds. The FM types, replacement levels and their interaction on body weight were statistically significant (P<0.01). The highest weight gain was achieved in 2% level of TFM (991g) followed by the 0% (928g), 4% (849g) and 6% (781g). Feed intake and live weight of broiler birds decreased linearly with increased RFM and TFM levels. But at 2% level of RFM and TFM birds ate more and gained higher liveweight. This superior growth was simply due to higher nutrient intake and better nutrient utilization by the birds. As a result, protein intake was also higher for the birds fed 2% FM diet. The feed conversion showed significant differences among levels; birds fed on 2% FM diet converted feed more efficiently than others. The PER decreased with the increasing level of RFM and TFM, that could be due to the poor digestibility of protein of FM. Sibbald (1986) also reported similar findings. However, results indicate that the feed intake, liveweight and feed conversion were superior with TFM at 2% inclusion level in broiler diet. The true digestibility of the CPC, RFM and TFM are shown in Table 2, which indicates that the digestibility of nutrients of CPC is much higher (P<0.01) than that of RFM and TFM. Low digestibility of FM may explain its naturally occurring carotinoid compounds and disulfide bonds of cystine. The findings got support from some other researchers (Liener, 1980). Digestibility of TFM was found superior to that of RFM except EE and Ash. Crude protein digestibility of FM increased from 43% to 47% which might be as a result of alkali treatment.
<table><caption>**Table 2.** True digestibility of different nutrients of CPC, RFM and TFM</caption><thead><tr><th rowspan="2">Ingredients</th><th colspan="6">Digestibility</th></tr><tr><th>DM</th><th>OM</th><th>CP</th><th>CF</th><th>EE</th><th>Ash</th></tr></thead><tbody><tr><td>Protein concentrate</td><td>61±4.9<sup>b</sup></td><td>64±4.6<sup>b</sup></td><td>65±1.4<sup>c</sup></td><td>41±1.6<sup>c</sup></td><td>52±3.2<sup>c</sup></td><td>32±3.8<sup>c</sup></td></tr><tr><td>Raw feather meal</td><td>44±5.6<sup>a</sup></td><td>48±5.2<sup>a</sup></td><td>43±4.3<sup>a</sup></td><td>29±2.3<sup>a</sup></td><td>47±3.8<sup>b</sup></td><td>22±4.4<sup>b</sup></td></tr><tr><td>Treated feather meal</td><td>47±4.0<sup>a</sup></td><td>52±3.9<sup>a</sup></td><td>47±1.4<sup>b</sup></td><td>37±3.7<sup>b</sup></td><td>40±4.0<sup>a</sup></td><td>11±3.5<sup>a</sup></td></tr><tr><td>F value and sig. level</td><td>20.82**</td><td>19.75**</td><td>108.42**</td><td>31.11**</td><td>15.87**</td><td>39.19**</td></tr></tbody></table>

Values bearing different superscripts in the same row differ significantly; NS, Not significant; **, P<0.01

**Conclusions**

It may be concluded that TFM due to alkali treatment may lose some of its nutrient quantity but at the same time digestibility could be increased. Inclusion of FM in broiler diets may have a positive effect up to 2% level. Further study is necessary to overcome the factors that limiting the utilization of FM as feed ingredients for broiler.

**Acknowledgements**

The authors are pleased to acknowledge to Dr. Shijun Li, Associate Professor, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University; for funding to carry out this research work.

**Literature**

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

BANERJEE G.C, 1988: Feeds and principles of animal nutrition (Revised edition of Animal Nutrition). Oxford and IBM Publishing Co. Pvt. Ltd, New Delhi, India.

BURGOS, A.M., J. I. FLOYD and E.L. STEPHENSON, 1974: The amino acid content and availability of poultry by-product meal and feather meal. Poult. Sci., 53: 198-203.

CHUBB, L.G, 1982: Anti-nutritional factors in animal feed-stuffs, pages 21-37. In: Recent Advances in Animal Nutrition. Ed. William Haresign. Butterworth, London England

DALE, N, 1992: True metabolizable energy of feather meal. The J.Appl. Poult.Res., 1:331-334.

HAN, Y and C.M. PARSONS, 1991: Protein and amino acid quality of feather meals. Poult. Sci. 70: 812-822.

LIENER, I.E, 1980: Toxic Constituents of Plant Foodstuffs, 2nd ed. New York: Academic Press.

MORAN, E.T., JR., J.D. SUMMERS and S.J. SLINGER, 1966: Keratins as sources of proteins for the growing chick. I. Amino acid imbalance as the cause for inferior performance of feather meal and the implication of disulfide bonding in raw feathers as the reason for poor digestibility. Poult. Sci., 45: 1257-1266.

MORITZ, J.S. and J.D. LATSHAW, 2001: Indicators of nutritional value of hydrolyzed feather meal. Poult. Sci., 80: 79-86.

PARSONS, C.M., L.M. POTTER and R.D. BROWN, JR, 1982: Effect of dietary protein and intestinal microflora on ex-cretion of amino acids in poultry. Poultry Sci. 61:939-946.

SIBBALD, I.R, 1986: The TME system of feed evaluation: Methodology, feed composition data and bibliography. Tech. bulletin 1986-E. Agriculture Canada, Ottawa, ON, Canada.

SPSS, 1999: Statistical Package for Social Sci. SPSS Inc. South Wacker Drive, Chicago, USA.

WALDRUP, P.W, 1982: Whole soybeans for poultry feeds. Worlds Poult. Sci. J., 38: 28-35.
P-200 (ID 197)

# EFFECT OF REPLACING SOYBEAN MEAL WITH DIFFERENT PROTEIN SOURCES DURING STARTER PERIOD ON PERFORMANCE AND FOOT PAD DERMATITIS IN BROILERS

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

**Corresponding author:** sinisa.bjedov@stocarstvo.edu.rs

## Abstract

Focus on early nutrition in boiler feeding has lately gained more attention. Fishmeal (FM), potato protein (PP), corn gluten (CG) and HP AviStart is common use as partly replacements of SBM. FM, PP and CG have all in common low K content in comparison to SBM. Too high K levels may leads to the consumption of large amount of water, and as a consequence there is an increase risk in litter moisture. That creates a predisposition for the development of FPD (foot pad dermatitis). In this study, we examined the effect of different sources of protein in starter feed on production performance and occurrence of FPD in broiler chickens. The experiment was conducted with 1,110 Ross 308 broiler chickens in duration of 42 days. The experiment was set up in five treatments with six replicates (37 chicks per replicate). Treatments were as follows: T1 soybean meal, T2 potato protein, T3 fishmeal, T4 HP AviStart and T5 corn gluten. All used starter feeds had the same energy content and similar protein content. Feeding with starter diets lasted for 11 days, after which all groups consumed the same feed - grower (from 12-28 days) and finisher (29-42 days). There were no statistically significant differences among treatments at day 42 in live weight. Scoring of foot pads lesions showed that the lowest level of foot pad damage for T4 group (0.52), and highest for T5 (0.89) and T1 (0.92) (P<0.05). Lowest FCR for T4 (1.74) and highest for SBM (1.81) (P<0.05) were found.

**Keywords:** protein sources, performance, FPD, broilers

## Introduction

Factors such as minerals, crude protein level and quality, digestibility of raw materials, fat quality and feed form play a role in the consistency of the faeces (DE JONG et al., 2013) and thereby be a contributing cause for FPD. Prolonged contact of feet with fecal material and high moisture of the litter could contribute to the development of pododermatitis. Due to an all-vegetable diet typical will have higher content of K in the feed, the use of animal protein sources is widely seen as solution for dealing with the FPD, especially if only SBM is used a sole protein source. EICHNER et al. (2007) found that birds raised on an all-vegetable diet had a higher incidence of FPD than did birds rose on a mixed animal and plant based diet. EICHNER et al. (2007) found that addition of corn gluten meal to an all-vegetable diet reduced the incidence of FPD when compared with a vegetable - and animal-based diet. Studies on protein level and source have provided inconsistent results. The aim of the present study is to compare a new novel protein source HP AviStart to other protein source on its effect on performance and potential effect on FPD.

## Material and Methods

The experiment was conducted at the Experimental farm of Faculty of Agriculture in Novi Sad, Serbia. The experiment was conducted with 1,110 Ross 308 broiler chickens in duration of 42 days. The experiment had five treatments with six replicates (37 chicks per replicate). Treatments were as follows: T1 soybean meal, T2 potato protein, T3 fishmeal, T4 HP AviStart and T5 corn gluten. All used starter feeds had the same energy content and similar protein content. Feeding with starter diets lasted for 11 days, after which all groups consumed the same feed - grower (from 12-28 days) and finisher (29-42 days). Measurements of body weight were performed weekly. Mortality and feed intake were recorded on a daily basis. Feed consumption was measured weekly. Feed conversion ratio and average weekly weight gain were calculated at the end. Foot pad lesions were recorded on all birds at 42days of age using a 0-3 scoring system. Briefly, the scores were 0: no lesion; 1: lesion on <25% of the pads; 2: lesion on 25 to 50% of the pads; 3: lesion on >50% of the pads (EICHNER et al., 2007). European Production Efficiency Factor (EPEF) was calculated using following equation:

$$EPEF = \frac{\text{body weight (g) x survival rate (\%)}}{\text{feed conversion x duration of trial (days)}}$$

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

<footnote n="2">²Hamlet Protein, Horsens, Denmark</footnote>
Data were analysed by ANOVA using the GLM procedure and means separated by Duncans post hoc test using StatSoft computer package (STATISTICA 11).

<table><caption>**Table 1. Feed composition**</caption><thead><tr><th rowspan="2">Ingredients,%</th><th colspan="5">Starter (0-11)</th><th>Grover (12-28)</th><th>Finisher (29-42)</th></tr><tr><th>T-1</th><th>T-2</th><th>T-3</th><th>T-4</th><th>T-5</th><th>All groups</th><th>All groups</th></tr></thead><tbody><tr><td>Corn</td><td>52.00</td><td>59.23</td><td>60.10</td><td>54.99</td><td>54.89</td><td>57.55</td><td>59.77</td></tr><tr><td>Potato prot.</td><td>0.00</td><td>5.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>Corn gluten</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>5.00</td><td>0.00</td><td>0.00</td></tr><tr><td>HP AviStart</td><td>0.00</td><td>0.00</td><td>0.00</td><td>5.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>Fish meal</td><td>0.00</td><td>0.00</td><td>5.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>Full fat soya</td><td>5.00</td><td>5.00</td><td>5.00</td><td>5.00</td><td>5.00</td><td>5.00</td><td>5.00</td></tr><tr><td>Soya 44%</td><td>34.40</td><td>24.20</td><td>24.40</td><td>27.20</td><td>28.00</td><td>29.00</td><td>26.30</td></tr><tr><td>Soybean oil</td><td>4.34</td><td>2.25</td><td>1.83</td><td>3.52</td><td>2.73</td><td>4.54</td><td>5.43</td></tr><tr><td>Rest <sup>1</sup></td><td>4.25</td><td>4.42</td><td>3.78</td><td>4.29</td><td>4.42</td><td>3.89</td><td>3.53</td></tr><tr><td>Total</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td colspan="8">Calculated composition</td></tr><tr><td>Protein, %</td><td>21.4</td><td>21.1</td><td>21.2</td><td>21.3</td><td>21.9</td><td>19.5</td><td>18.5</td></tr><tr><td>ME, MJ/kg</td><td>12.85</td><td>12.85</td><td>12.85</td><td>12.85</td><td>12.85</td><td>13.20</td><td>13.60</td></tr><tr><td>Lys</td><td>1.30</td><td>1.30</td><td>1.30</td><td>1.30</td><td>1.30</td><td>1.15</td><td>1.09</td></tr><tr><td>Met+Cys</td><td>0.97</td><td>0.97</td><td>0.97</td><td>0.97</td><td>0.97</td><td>0.88</td><td>0.83</td></tr><tr><td>K</td><td>0.98</td><td>0.82</td><td>0.85</td><td>0.95</td><td>0.86</td><td>0.89</td><td>0.83</td></tr></tbody></table>

¹Lys, Met, Thr, MCP, NaHCO3, CaCO3, NaCl and premix

## Results

No significant difference among treatments for body weight at day 42 was found (Table 2). Lowest FCR for T4 (1.74) and highest for SBM (1.81) (P<0.05) were found. For neither mortality or EPEF no significant difference among treatments was found.

<table><caption>**Table 2. Performance of broiler chicken**</caption><thead><tr><th>Item</th><th>T1</th><th>T2</th><th>T3</th><th>T4</th><th>T5</th></tr></thead><tbody><tr><td>Average body weight at 42 day (g)</td><td>2,238</td><td>2,207</td><td>2,188</td><td>2,224</td><td>2,290</td></tr><tr><td>Feed conversion ratio</td><td>1.81<sup>b</sup></td><td>1.80<sup>b</sup></td><td>1.76<sup>ac</sup></td><td>1.74<sup>a</sup></td><td>1.80<sup>bc</sup></td></tr><tr><td>Mortality (%)</td><td>5.4</td><td>3.6</td><td>1.8</td><td>0.9</td><td>4.1</td></tr><tr><td>European Production Efficiency Factor</td><td>279</td><td>282</td><td>292</td><td>302</td><td>292</td></tr></tbody></table>

a-c Values among rows with no common superscript are significantly different (P<0.05)

Significant difference among treatments were found, with lowest level of foot pad damage for T4 group (0.52), and the highest for T5 (0.96) and T1 (0.92) (P<0.05) (Table 3). These results are similar with those noted by EICHNER et al. (2007) with the birds fed all CG showing the lowest incidence of foot pad dermatitis (P ≤ 0.05).

<table><caption>**Table 3. Footpad lesion score at day 42**</caption><thead><tr><th rowspan="2">Treatments</th><th rowspan="2">Total broilers</th><th colspan="4">Number of broilers with lesion score</th><th colspan="4">% of broilers with lesion score</th><th rowspan="2">Average score</th></tr><tr><th>0</th><th>1</th><th>2</th><th>3</th><th>0</th><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>T1</td><td>210</td><td>59</td><td>105</td><td>46</td><td>0</td><td>28.1</td><td>50.0</td><td>21.9</td><td>0.0</td><td>0.92<sup>c</sup></td></tr><tr><td>T2</td><td>214</td><td>94</td><td>98</td><td>22</td><td>0</td><td>43.9</td><td>45.8</td><td>10.3</td><td>0.0</td><td>0.66<sup>b</sup></td></tr><tr><td>T3</td><td>218</td><td>91</td><td>109</td><td>18</td><td>0</td><td>41.7</td><td>50.0</td><td>8.3</td><td>0.0</td><td>0.76<sup>b</sup></td></tr><tr><td>T4</td><td>220</td><td>114</td><td>97</td><td>9</td><td>0</td><td>51.8</td><td>44.1</td><td>4.1</td><td>0.0</td><td>0.52<sup>a</sup></td></tr><tr><td>T5</td><td>213</td><td>61</td><td>115</td><td>37</td><td>0</td><td>28.6</td><td>54.0</td><td>17.4</td><td>0.0</td><td>0.89<sup>c</sup></td></tr></tbody></table>

a-c Values between columns with no common superscript are significantly different (P<0.05)
VIERIA and LIMA (2005) found that feeding a corn-soybean meal diet formulated with animal feed component (3% pork by-product, 2.5% poultry by-product and 1.5% feather meal) and 0.14% less K produced litter with less moisture than diets formulated exclusively with corn and soybean meal.

## Conclusions

Usage of different alternative protein sources like FM and PP, together with HP AviStart offer solution for reduction in occurrence and intensity of damage to the foot pads in broilers.

## Literature

DE JONG, I. C., T. VELDKAMP, J. VAN HARN, 2013: Management tools to reduce footpad dermatitis in broiler chickens. In: Proceedings of the 19th European Symposium on Poultry Nutrition, Potsdam, Germany, 26-29 August 2013, pp. 78-83.

EICHNER, G., S. L. VIEIRA, C. A. TORRES, J. L. B. CONEGLIAN, D. M. FREITAS, O. A. OYARZABAI, 2007: Litter Moisture and Footpad Dermatitisas Affected by Diets Formulated on an All-Vegetable Basis or Having the Inclusion of Poultry By-Product. J. Appl. Poult. Res. **16**, 344-350.

VIEIRA, S.L., I. L. LIMA, 2005: Live Performance, Water Intake and Excreta Characteristics of Broilers Fed All Vegetable Diets Based on Corn and Soybean Meal. **Int. J. Poult. Sci. 4**(6), 365-368.
P-201 (ID 46)

ESTABLISHING AN AMINO ACID PATTERN AND IDEAL RATIO FOR MAINTENANCE OF POULTRY BY DELE-
TION METHOD

J. C. P. Dorigam, N. K. Sakomura, M. B. Lima, M. F. Sarcinelli, and R. M. Suzuki

Department of Animal Science, São Paulo State University "Julio de Mesquita Filho," College of Agrarian and Veteri-
nary sciences, Jaboticabal, São Paulo, Brazil

**Corresponding author:** sakomura@fcav.unesp.br

**Abstract**

This study aimed to estimate the essential amino acid (EAA) profile and the ideal ratio (IAAR) for maintenance of poultry by deletion method. A nitrogen balance (NB) trial was conducted using 138 adult roosters, housed individually in metabolic cages. The treatments were 23 purified diets being 11 diets with an amino acid mixture providing high protein intake of 500 mg N/BW<sub>kg</sub><sup>0.75</sup> per day and 11 diets providing low protein intake of 250 mg N/BW<sub>kg</sub><sup>0.75</sup> per day (in each diet one EAA was diluted 50%) and one nitrogen free diet. Each treatment had six replicates. After 48 h of fasting receiving water plus sucrose, the roosters were fed 40 g of the diets by tube once a day for three days. The excreta were collected within 72 h after the first feeding. The diets and excreta were analyzed for nitrogen content. For each EAA studied, a linear regression was fitted by NB and amino acid intake (AAI). The maintenance requirements were estimated as the AAI to maintain the NB equal to zero. The daily EAA requirements for maintenance were estimated to be: Lys 11, Met 40, Thr 30, Trp 7, Arg 54, Val 29, His 6, Gly 57, Phe 50, Leu 82 and Ile 25 mg/BW<sub>kg</sub><sup>0.75</sup> per day. Therefore, the IAAR for maintenance was concluded to be Lys 100, Met 372, Thr 274, Trp 66, Arg 498, Val 264, His 55, Gly 522, Phe 460, Leu 760 and Ile 235% independent of the scale. The EAA profile and the IAAR for maintenance of poultry estimated in this study contribute to improve factorial model for estimating EAA requirements for poultry.

**Keywords:** amino acid, maintenance, nitrogen balance, purified diet, rooster

**Introduction**

Maintenance requirement is an important component in factorial models because in young animals the maintenance
requirement is lower in relation to the total requirement and assumes greater importance as the poultry reaches matur-
ity due to the lower need for essential amino acids (EAA) for growth (OWENS and PETTIGREW, 1989). One alternative
approach used to determine the maintenance requirement for each EAA is the deletion method, based on monitoring
the nitrogen balance (NB) that arises as the EAA are reduced from the diets. This approach allows determining the
requirements for all EAA in one set of experiments and is generally accepted as an efficient and rapid tool to estimate
the ideal EAA profile. Thus, in the present study, the deletion method was used to update and obtain new coefficients
for maintenance requirements for poultry to derive an ideal EAA ratio for maintenance.

**Material and Methods**

A NB trial was conducted using 138 adult Cobb roosters. The treatments consisted of two protein levels (low and high) where 11 EAA were evaluated (lysine, methionine, threonine, tryptophan, arginine, valine, phenylalanine, histidine, glycine, isoleucine, and leucine) and a nitrogen-free (NF) diet, totalling 23 treatments with six replicates each. The purified diets were formulated to meet the requirements for energy and other nutrients for roosters (ROSTAGNO et al., 2011), except protein and EAA. The EAA composition of the diets with low (LP) and high protein concentration (HP) was based on the EAA profile for maintenance established by LEVEILLE et al. (1960). The series of HP diets were made, each providing one of the EAA tested in the quantity required for maintenance but with twice the other EAA required for maintenance, resulting in a daily nitrogen intake (NI) of 500 mg/BW<sub>kg</sub><sup>0.75</sup>. The series of LP diets were made, each providing half of the EAA tested in the quantity required for maintenance and the other EAA in adequate quantities to meet their requirements for maintenance, totalling a daily NI of 250 mg /BW<sub>kg</sub><sup>0.75</sup>. The birds underwent a fasting period of 48 h, receiving 60 ml of water with sucrose (50:50), once a day, by intubation. In the subsequent 72 hours the birds received 40 g of the diet by intubation. The NF was made available ad libitum during the trial. The excreta were collected daily and, at the end of the trial, leftover feed was quantified to determine feed intake. Diets and excreta were analysed for total nitrogen content to quantify NI, nitrogen excretion (NEX) and NB (NI-NEX). Data from NB trial were subjected to a linear regression, considering the amino acid intake (AAI) as the independent variable and the NB as dependent variable. The maintenance requirements were estimated as the AAI to maintain the NB equal to zero (in mg/BW<sub>kg</sub><sup>0.75</sup>)
## Results

The mean values and the standard deviations (±SD) from NB assay are presented in Table 1. The SD of the data was low considering the small amount of data obtained for each EAA. The roosters fed diets limiting in lysine and tryptophan in the LP diets presented higher NB than other EAA, which were above the maintenance requirement. However, roosters fed limiting diets in valine and histidine in LP diets had the NB close to zero, which were closer to nitrogen equilibrium in the LP diets. The linear equations obtained using the NB data, daily maintenance requirements expressed in mg/BW<sub>kg</sub><sup>0.75</sup> and the ideal EAA ratio are presented in Table 2. The difference between the EAA pattern required for growth and that required for maintenance concerns lysine (JANSEN, 1974). The same authors also explains that of all EAA, lysine turnover is quite low and this implies that lysine required for maintenance is also quite low (JANSEN, 1974). This low requirement was also observed. Consequently, the ideal ratio relative to lysine (Table 2) was quite different from those established for growth (ROSTAGNO et al. 2011), with higher values. However, the EAA ratio presented in Table 2 was similar to that presented by LI et al. (2003).

<table><caption>**Table 1.** Mean of daily values (±SD) resulting from the nitrogen-free diet (NF) and individual amino acid (EAA) deletions in the low (LP) and high protein (HP) diets, in mg/BW<sub>kg</sub><sup>0.75</sup></caption><thead><tr><th rowspan="2">EAA</th><th rowspan="2">Diets<sup>1</sup></th><th rowspan="2">AA<br>Intake</th><th colspan="3">Nitrogen</th></tr><tr><th>Intake</th><th>Excretion</th><th>Balance</th></tr></thead><tbody><tr><td rowspan="3">Lys</td><td>NF</td><td>0.0 ± 0.0</td><td>66.1 ± 2.7</td><td>258.7 ± 3.3</td><td>-192.6 ± 1.5</td></tr><tr><td>LP</td><td>17.7 ± 0.6</td><td>312.0 ± 9.9</td><td>234.2 ± 6.0</td><td>77.8 ± 6.1</td></tr><tr><td>HP</td><td>35.8 ± 0.8</td><td>557.4 ± 15.5</td><td>200.7 ± 6.9</td><td>356.7 ± 15.0</td></tr><tr><td rowspan="2">Met</td><td>LP</td><td>54.5 ± 1.2</td><td>231.1 ± 5.5</td><td>194.3 ± 6.8</td><td>36.9 ± 7.7</td></tr><tr><td>HP</td><td>109.1 ± 2.1</td><td>550.7 ± 11.6</td><td>219.3 ± 9.3</td><td>331.4 ± 9.0</td></tr><tr><td rowspan="2">Thr</td><td>LP</td><td>45.0 ± 0.7</td><td>298.9 ± 4.7</td><td>231.8 ± 7.9</td><td>67.1 ± 7.6</td></tr><tr><td>HP</td><td>89.6 ± 1.4</td><td>542.8 ± 8.9</td><td>181.7 ± 12.2</td><td>329.4 ± 15.3</td></tr><tr><td rowspan="2">Trp</td><td>LP</td><td>11.6 ± 0.1</td><td>286.6 ± 5.3</td><td>204.6 ± 10.1</td><td>82.0 ± 5.8</td></tr><tr><td>HP</td><td>23.1 ± 0.4</td><td>560.8 ± 9.7</td><td>191.8 ± 5.3</td><td>369.0 ± 9.3</td></tr><tr><td rowspan="2">Val</td><td>LP</td><td>34.5 ± 0.4</td><td>285.4 ± 5.3</td><td>290.9 ± 9.4</td><td>-5.5 ± 4.4</td></tr><tr><td>HP</td><td>67.6 ± 1.2</td><td>457.7 ± 7.1</td><td>239.2 ± 7.0</td><td>218.5 ± 5.4</td></tr><tr><td rowspan="2">Arg</td><td>LP</td><td>67.5 ± 1.1</td><td>273.7 ± 4.2</td><td>261.7 ± 7.1</td><td>12.0 ± 9.0</td></tr><tr><td>HP</td><td>139.9 ± 2.4</td><td>486.4 ± 9.8</td><td>210.9 ± 4.7</td><td>275.5 ± 11.0</td></tr><tr><td rowspan="2">Ile</td><td>LP</td><td>41.5 ± 0.4</td><td>265.0 ± 3.3</td><td>203.8 ± 6.4</td><td>61.3 ± 4.6</td></tr><tr><td>HP</td><td>80.1 ± 1.3</td><td>460.7 ± 6.9</td><td>185.8 ± 18.4</td><td>274.9 ± 7.1</td></tr><tr><td rowspan="2">Leu</td><td>LP</td><td>69.7 ± 0.6</td><td>263.6 ± 3.6</td><td>240.8 ± 5.8</td><td>22.8 ± 4.5</td></tr><tr><td>HP</td><td>138.4 ± 1.0</td><td>441.3 ± 3.5</td><td>200.9 ± 13.3</td><td>240.4 ± 1.8</td></tr><tr><td rowspan="2">His</td><td>LP</td><td>4.5 ± 0.0</td><td>308.3 ± 2.6</td><td>305.7 ± 5.4</td><td>2.6 ± 6.4</td></tr><tr><td>HP</td><td>8.9 ± 0.1</td><td>442.0 ± 5.6</td><td>253.6 ± 7.4</td><td>188.5 ± 5.1</td></tr><tr><td rowspan="2">Phe</td><td>LP</td><td>33.9 ± 0.3</td><td>243.9 ± 1.6</td><td>265.6 ± 6.9</td><td>-21.6 ± 6.0</td></tr><tr><td>HP</td><td>67.0 ± 0.5</td><td>436.0 ± 3.5</td><td>241.7 ± 5.2</td><td>194.3 ± 5.7</td></tr><tr><td rowspan="2">Gly</td><td>LP</td><td>47.5 ± 0.5</td><td>280.9 ± 2.8</td><td>255.7 ± 3.7</td><td>25.1 ± 4.3</td></tr><tr><td>HP</td><td>95.2 ± 1.2</td><td>499.5 ± 3.8</td><td>253.7 ± 6.6</td><td>245.8 ± 4.7</td></tr></tbody></table>
<table><caption>**Table 2.** Linear equations obtained from nitrogen balance (NB) trial with their coefficient of determination (R²), calculated requirements and the ideal ratio for maintenance (IAAR)</caption><thead><tr><th>Amino acid</th><th>Equations from linear regression</th><th>Requirement (mg/BW<sub>kg</sub><sup>0.75</sup>)</th><th>IAAR</th><th>R<sup>2</sup></th><th>Probability</th></tr></thead><tbody><tr><td>Lys</td><td>NB=-171.9(±4.16)+16.1(±0.32)×Lys</td><td>11</td><td>100</td><td>0.98</td><td>&lt;0.001</td></tr><tr><td>Met</td><td>NB=-198.4(±6.25)+5.0(±0.16)×Met</td><td>40</td><td>372</td><td>0.96</td><td>&lt;0.001</td></tr><tr><td>Trp</td><td>NB=-188.4(±5.17)+25.1(±0.61)×Trp</td><td>7</td><td>66</td><td>0.96</td><td>&lt;0.001</td></tr><tr><td>Thr</td><td>NB=-178.0(±5.33)+6.0(±0.17)×Thr</td><td>30</td><td>274</td><td>0.96</td><td>&lt;0.001</td></tr><tr><td>Arg</td><td>NB=-167.3(±5.15)+3.1(±0.10)×Arg</td><td>54</td><td>498</td><td>0.96</td><td>&lt;0.001</td></tr><tr><td>Val</td><td>NB=-156.8(±5.36)+5.4(±0.23)×Val</td><td>29</td><td>264</td><td>0.94</td><td>&lt;0.001</td></tr><tr><td>Ile</td><td>NB=-165.8(±3.64)+6.6(±0.13)×Ile</td><td>25</td><td>235</td><td>0.97</td><td>&lt;0.001</td></tr><tr><td>Leu</td><td>NB=-171.4(±2.60)+2.1(±0.05)×Leu</td><td>82</td><td>760</td><td>0.98</td><td>&lt;0.001</td></tr><tr><td>Phe</td><td>NB=-164.0(±4.63)+3.3(±0.20)×Phe</td><td>50</td><td>460</td><td>0.96</td><td>&lt;0.001</td></tr><tr><td>Gly</td><td>NB=-158.3(±3.13)+2.8(±0.09)×Gly</td><td>57</td><td>522</td><td>0.98</td><td>&lt;0.001</td></tr><tr><td>His</td><td>NB=-163.0(±4.57)+26.0 (±1.48)×His</td><td>6</td><td>55</td><td>0.93</td><td>&lt;0.001</td></tr></tbody></table>

**Conclusions**

The deletion method was valid to update the EAA requirements and establish an IAAR for maintenance of poultry, which can contribute to improve the factorial models.

**Acknowledgements**

The authors gratefully acknowledge Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP (Brazil) for financial support.

**Literature**

JANSEN, G.R., 1974: The amino acid fortification of cereals. In: ALTSCHUL, A.M.: New Protein Foods: Technology, Volume 2, Part 2. Academic Press, New York, ISBN 978-1-483-21595-2, 40-120.

LEVEILLE, G.A., R. SHAPIRO, H. FISHER, 1960: Amino acid requirements for maintenance in the adult rooster: IV. The requirements for methionine, cystine, phenylalanine, tyrosine and tryptophan; the adequacy of the determined requirements. J. Nutr. **72**, 8-15.

LI, G.; M. QU, N. Zhu, X. YAN, 2003: Determination of the Amino Acid Requirements and Optimum Dietary Amino Acid Pattern for Growing Chinese Taihe Silky Fowls in Early Stage. Asian Australas. J. Anim. Sci. **16**, 1782-1788

OWENS, F.N., J.E. PETTIGREW, 1989: Subdividing amino acid requirements into portions for maintenance and growth. In: FRIEDMAN M.: Absorption and utilization of amino acids. CRC Press, Boca Raton. ISBN 978-0-849-36007-7, 15-30.

ROSTAGNO, H.S., L.F.T. ALBINO, J.L. DONZELE, P.C. GOMES, R.F. OLIVEIRA, D.C. LOPES, A.S. FERREIRA, S.L.T. BARRETO, R.F. EUCLIDES, 2011: Brazilian Tables for Poultry and Swine: Composition of Feedstuffs and Nutritional Requirements, 3rd edn. UFV publishing, Minas gerais.
P-204 (ID 282)

STOCHASTIC MODEL TO PREDICT LYSINE REQUIREMENT AND EGG PRODUCTION OF BROILER BREEDER HENS

N.T. Ferreira<fnref n="1" />, N.K. Sakomura<fnref n="1" />, J.C.P. Dorigam<fnref n="1" />, J.B.K. Fernandes and R.M. Gous<fnref n="2" />

**Corresponding author:** sakomura@fcav.unesp.br

**Abstract**

The aim of this study was to develop a stochastic model to estimate lysine (Lys) requirements for broiler breeder hens, depending on body weight (BW) and egg production (EP). A population of broiler breeder hens was generated considering average BW of 2.34 (±0.23) kg. The age from first egg (AFE) adopted was 193d, which was estimated from the lighting program applied during the growing phase by Bristol-Reading Model. The model incorporated BW, AFE, internal cycle length (ICL=25.40+8.99×(0.75AFE)+0.07AFE) to determine the EP (ICL/24), yolk (YW=18.03×[1-e⁻⁰.⁰¹⁵×(AFE-103.4)]×e⁻⁰.⁰⁰¹AFE), albumen (AW=14.38×YW⁰.³⁷⁵) and shell (SW=0.358×(YW+AW)⁰.⁶⁸⁷) weights. These data were used to simulate EP and developing a model to calculate Lys requirements (Lysᵣ): Lysᵣ=((600*BP⁰.⁷³*BP)+((Lysᵣ*(0.03*YW)+ (Lysᵃ*(0.02*AW)/0.6)/EP) where BP is body protein content calculated from BW, Lysᵧ and Lysᵃ are the Lys contents of yolk and albumen, 0.03 and 0.02 are nitrogen content YW and AW, respectively, and 0.6 is efficiency of utilization. The maximum EP estimated was 87% at 28 weeks of age and declined to 57% at 60 weeks of age. The mean egg weight (EW=YW+AW+SW) increased from 63 to 77g, YW from 15 to 23g and AW from 40 to 47g. Considering the BW and EP, the Lys intake required by the flock increased to 838 mg/d at 28 weeks then declined to 753 mg/d at 60 weeks of age. The recommendation of Lys would be 5.3 g/kg of diet for broiler breeder hens at peak production for a daily feed intake of 160 g/d.

**Keywords:** broiler breeder, modelling, requirements

**Introduction**

Feed properly broiler breeder flocks has become essential to ensure the increasing production of hatching eggs. Thus, mathematical models have been used as a tool in poultry nutrition to adjust the nutritional levels. This approach has raised a number of issues that were previously ignored by researchers or were subjects to isolated studies that were previously unrelated to model development. Such models have been the basis of several trials, which the measurements considered the growth rates of ovary and oviduct prior to the onset of lay (Bowmaker and Gous, 1989; Ferreira et al., 2015), changes in internal cycle length over time (Johnston and Gous, 2003), and allometric relationships between yolk, albumen and shell weight (Johnston and Gous, 2007). However, issues still need to be studied in order to update and improve mechanistic models. Moreover, the modelling process could be used to identify gaps in the knowledge on physiological systems. Therefore, the aim of this study was to develop a stochastic model to predict the lysine requirements of broiler breeder flocks during the production of eggs, which can be calculated according to the requirements for different conditions such as BW and the lighting program used during growing period.

**Material and Methods**

The model described here simulates the potential for egg production and hence lysine requirements for a flock of broiler breeder hens. The information required for developing this simulation model was age and BW at the beginning of the laying period, the potential rate of EP, EW, changes in ICL, yolk, albumen and shell content, and body composition over the laying period. These variables were obtained from various sources, including a research reported by Ferreira et al. (2015). A population of broiler breeder hens was generated in Excel spreadsheet, considering the average BW of 2.34 (±0.23) kg. The age from first egg (AFE) adopted was 193d, which was estimated from the lighting program applied during the growing phase by Bristol-Reading Model. The model incorporated BW, AFE, internal cycle length (ICL=25.40+8.99×(0.75AFE)+0.07AFE) to determine the EP (ICL/24), yolk (YW=18.03×[1-e⁻⁰.⁰¹⁵×(AFE-103.4)]×e⁻⁰.⁰⁰¹AFE), albumen (AW=14.38×YW⁰.³⁷⁵) and shell (SW=0.358×(YW+AW)⁰.⁶⁸⁷) weights.

<footnote n="1">¹Department of Animal Sciences, São Paulo State University, UNESP, Jaboticabal, SP, Brazil</footnote>

<footnote n="2">²School of Agricultural, Earth and Environmental Sciences, University of KwaZulu-Natal, Pietermaritzburg, South Africa</footnote>
## Results and discussion

The mean reproductive performance (yolk, albumen and shell weight, EW, rate of laying and egg output), and the minimum, mean and maximum daily intakes of Lys required by the simulated population of broiler breeders hens are summarized at four-weekly intervals in Table 1. The maximum rate of lay of 87% was achieved at 28 weeks of age and declined up to 57% at 60 weeks of age. The laying performance simulated for a flock of broiler breeder hens in this exercise was similar to the Cobb-Vantress target breeding performance, validating the mechanistic method used to simulate rate of lay (Figure 1).

<figure><img src="image_70.png" /><figcaption>**Figure 1** Percentage of lay of a theoretical population of fifty broiler breeder hens over the 260 days after the first egg.</figcaption></figure>

Similarly, the increase in egg weight over time was similar to the changes that are expected in a commercial broiler breeder flock. It was observed that the requirements for the yolk and albumen increased over the weeks due to the increase in the size of the egg formed. However, the egg output decreases with the age and the requirements of daily amino acid also decreases (Gous and Nonis, 2010). The daily intake of Lys required by the flock increased to 838 mg/d and then declined to 753 mg/d by 60 weeks. Ekmay et al. (2013) estimated the amino acid requirements for broiler breeders at peak production, and recommend for Lys (916 mg/g), values above those found in this study compared to 28 weeks, probably due to BW difference.

The production of eggs, the body weight of the birds and the quality of feed, will directly influence the results of the requirement and consequently the concentration of nutrients. The concentration of nutrients estimated in this study is lower than that recommended by the breed manual. By considering the nutritional recommendations of the manual, the amount of feed offered (DFI) would be less than 160 g/d which was similar to the results of Gous and Nonis (2010). These authors reported that the DFI estimate is often considerably lower than the daily allowance, which is uncommon for broiler breeder hens. In this case, the model predicts a decrease in consumption as the supply of nutrients increases.

<table><caption>**Table 1.** The mean weight of yolk, albumen and shell (g), percentage of lay and egg output (g/d), and the minimum (Min), mean and maximum (Max) daily intakes of lysine (mg/d) required at four-weekly intervals of age for theoretical population of broiler breeders.</caption><thead><tr><th>Age<br>Week</th><th>Yolk<br>g</th><th>Albumen<br>g</th><th>Shell<br>g</th><th>Egg weight<br>g</th><th>Lay<br>%</th><th>Egg Output<br>g/d</th><th>Min</th><th>Lysine<br>Mean</th><th>Max</th></tr></thead><tbody><tr><td>24</td><td>15.0</td><td>39.7</td><td>5.6</td><td>60.3</td><td>84</td><td>50.4</td><td>360</td><td>773</td><td>979</td></tr><tr><td>28</td><td>17.1</td><td>41.7</td><td>5.9</td><td>64.6</td><td>87</td><td>56.0</td><td>767</td><td>838</td><td>939</td></tr><tr><td>32</td><td>18.6</td><td>43.0</td><td>6.1</td><td>67.7</td><td>81</td><td>55.1</td><td>748</td><td>827</td><td>895</td></tr><tr><td>36</td><td>19.7</td><td>44.0</td><td>6.2</td><td>69.9</td><td>77</td><td>53.8</td><td>717</td><td>819</td><td>884</td></tr><tr><td>40</td><td>20.6</td><td>44.7</td><td>6.3</td><td>71.6</td><td>73</td><td>52.2</td><td>694</td><td>765</td><td>854</td></tr><tr><td>44</td><td>21.3</td><td>45.3</td><td>6.4</td><td>73.0</td><td>69</td><td>50.2</td><td>668</td><td>730</td><td>777</td></tr><tr><td>48</td><td>21.9</td><td>45.8</td><td>6.5</td><td>74.1</td><td>66</td><td>48.6</td><td>712</td><td>734</td><td>751</td></tr><tr><td>52</td><td>22.4</td><td>46.2</td><td>6.5</td><td>75.1</td><td>62</td><td>46.8</td><td>738</td><td>739</td><td>740</td></tr><tr><td>56</td><td>22.9</td><td>46.5</td><td>6.6</td><td>76.0</td><td>60</td><td>45.3</td><td>745</td><td>746</td><td>747</td></tr><tr><td>60</td><td>23.4</td><td>46.9</td><td>6.6</td><td>76.9</td><td>57</td><td>43.9</td><td>752</td><td>753</td><td>754</td></tr></tbody></table>
It should be taken into consideration that in the nutrients partition, maintenance has priority, followed by the deposition of yolk and albumen. Besides, the egg output is dependent on the nutrient intake, since the requirements for maintenance and growth of the yolk are met. However, if the intake of the limiting nutrient for the day cannot sustain this minimum egg size, EP does not occur (Gous and Nonis, 2010). Therefore, to feed a flock of broiler breeders it is necessary to follow a daily pattern, even when no egg laying, the bird consumes the diet. This causes the energy consumed in excess to be deposited on the body.

**Conclusions**

The modelling approach presented here has the potential to help nutritionists to provide a more efficient feeding program for broiler breeder hen flocks. Moreover, the proposed model is dynamic and these parameters can be adapted to meet the requirement of the day. Improvements can be made in the model and also need to be re-evaluated regularly to keep pace with genetic advancements.

**Acknowledgements**

We wish to acknowledge the financial support for this research and the scholarship awarded from the São Paulo Research Foundation (FAPESP), grant 2015/06001-4.

**Literature**

Bowmaker JE and Gous RM 1989. A quantification of reproductive changes and nutrient requirements of broiler breeder pullets at sexual maturity. British Poultry Science 30, 663-675.

Ekmay RD, De Beer M, Mei SJ, Manangi M and Coon CN 2013. Amino acid requirements of broiler breeders at peak production for egg mass, body weight, and fertility. Poultry Science 92, 992-1006.

Ferreira NT, Sakomura NK, Dorigam JCP, Silva EP and Gous RM 2015. Modelling the egg components and laying patterns of broiler breeder hens. Animal Production Science 55, doi org/10.1071/AN14737.

Gous RM and Nonis MK 2010. Modelling egg production and nutrient responses in broiler breeder hens. Journal of Agricultural Science 1-15.

Johnston SA and Gous RM 2003. An improved mathematical model of the ovulatory cycle of the laying hen. British Poultry Science 44, 752-760.
P-205 (ID 318)

PROTEIN NUTRITION OF COMMERCIAL LAYING HENS

Araujo, L.F.<fnref n="1" />, D. V. Gambaro<fnref n="3" />, C. S. S. Araujo<fnref n="2" />, B. G. Leite<fnref n="1" />

Corresponding author: ifaraujo@usp.br

**Abstract**

Two experiments were conducted to investigate the reduction of the protein level in the diet of laying hens from 24 to 40 weeks of age. In each experiment, 240 laying hens were distributed in a completely randomized design with 3 treatments (3 levels of CP: 17.4%, 16.4% and 15.4%), 10 replications and 8 birds per pen each. It was used Bovans White and Bovans Brown laying hens in the first and second trial, respectively. The parameters evaluated were performance, egg weight and shell quality (specific gravity, shell thickness and breaking strength) and internal egg quality (albumen height, Haugh unit and yolk color). Birds were fed *ad libitum* and parameters were evaluated in 4 cycles of 28 days each. In experiment I, egg shell quality, albumen height and egg weight were not affected by dietary treatments. Hens fed 16.4% CP did not have their performance reduced. In experiment II, only yolk color was affected by treatments, with higher yolk color in eggs of hens fed diets with 15.4% of CP. Considering these results, it is concluded that laying hens fed diets with 16.4% CP showed better results for egg production and feed conversion. However, the reduction of protein to 15.4% reduced egg production. In addition, for semi-heavy hens it is possible to reduce protein to 15.41% without reduce egg production and egg quality.

**Keywords:** Amino acids, egg production, egg quality, protein

**Introduction**

The modern laying hen has high egg production and laying efficiency. Layer hens are able to digest protein that will supply the amino acids (AA) needed for maintenance or for producing new proteins for growth or egg production; however, only less than half of the nitrogen in AA can be utilize by chickens (Latshaw and Zhao, 2011). Because of that, it is convenient to decrease the level of crude protein (CP) in the diet, since synthetic AA are supplemented to meet the requirements of limiting AA. On the other hand, it is necessary to determine the extent to which the CP content of laying hen diets can be reduced without decrease hen performance. Thus, the aim of this study it was to investigate the reduction of the protein level in the diet of laying hens from 24 to 40 weeks of age.

**Material and Methods**

A total of 240 hens (Bovans White), in the first trial, and 240 hens (Bovans Brown), in the second trial, were randomly assigned to 1 of 3 dietary treatment groups varying in protein level (17.4%, 16.4% and 15.4%). Each of the 3 treatments was assigned to 10 replicate cages with 8 hens per cage. Hens were placed in an open side house with feed and water provided for *ad libitum* consumption. Diets were based on corn, wheat bran, and soybean meal. Calculated levels of nutrients of experimental diets are in Table 1. Evaluation of performance and egg quality parameters, started 4 wk after the trial began. The evaluation was carried out in 4 intervals of 28 d from 24 to 40 wk of age. The evaluated performance parameters were feed intake (g/bird-day), egg production (% hen-day), egg weight (g), feed conversion (kg of feed intake/number of eggs produced). The number of eggs per cage was recorded each day and summarized by 28 d periods. The mean production of 2 consecutive days, after each 28 d period, was used for estimating the other parameters. Egg and eggshell quality traits were evaluated by egg weigth (g), albumen height, Haugh units, yolk color, eggshell thickness (mm), egg breaking strength (kgf), and egg specific gravity (g/mL of H2O). The results were analyzed using the statistical program SAS (2012). The research was conducted under the approval of the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of the University of Sao Paulo.

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

<footnote n="2">² Dept. Nutrition and Animal Science, FMVZ, University of Sao Paulo, Pirassununga, Brazil</footnote>

<footnote n="3">³ Agroceres Multimix, Rio Claro, Brazil</footnote>
<table><caption>**Table 1.** Diets compositions</caption><thead><tr><th rowspan="2"></th><th colspan="3">Protein levels (%)</th></tr><tr><th>17,4</th><th>16,4</th><th>15,4</th></tr></thead><tbody><tr><td>Corn</td><td>62,450</td><td>63,380</td><td>64,320</td></tr><tr><td>Soybean meal</td><td>26,200</td><td>22,800</td><td>19,500</td></tr><tr><td>Wheat bran</td><td>0,000</td><td>2,400</td><td>4,500</td></tr><tr><td>Dicalcium phosphate</td><td>1,300</td><td>1,300</td><td>1,300</td></tr><tr><td>Fine limestone</td><td>5,210</td><td>5,166</td><td>5,188</td></tr><tr><td>Coarse limestone</td><td>4,000</td><td>4,000</td><td>4,000</td></tr><tr><td>Sodium chloride</td><td>0,350</td><td>0,350</td><td>0,350</td></tr><tr><td>DL - Methionine</td><td>0,190</td><td>0,220</td><td>0,250</td></tr><tr><td>L-Lysine</td><td>0,000</td><td>0,000</td><td>0,100</td></tr><tr><td>L-Threonine</td><td>0,000</td><td>0,058</td><td>0,103</td></tr><tr><td>L-Tryptophane</td><td>0,000</td><td>0,003</td><td>0,020</td></tr><tr><td>L-Valine</td><td>0,000</td><td>0,023</td><td>0,073</td></tr><tr><td>Mineral/vitamin mixture</td><td>0,300</td><td>0,300</td><td>0,300</td></tr><tr><td>Total</td><td>100,000</td><td>100,000</td><td>100,000</td></tr><tr><th colspan="4">Nutrient calculated</th></tr><tr><td>ME, kcal/kg</td><td>2.756</td><td>2.756</td><td>2.756</td></tr><tr><td>CP, %</td><td>17,4</td><td>16,4</td><td>15,4</td></tr><tr><td>Calcium, %</td><td>4,04</td><td>4,04</td><td>4,04</td></tr><tr><td>Available P, %</td><td>0,46</td><td>0,46</td><td>0,46</td></tr><tr><td>Sodium, %</td><td>0,16</td><td>0,16</td><td>0,16</td></tr><tr><td>Methionine + cystine (%)</td><td>0,77</td><td>0,77</td><td>0,77</td></tr><tr><td>Lysine, %</td><td>0,91</td><td>0,83</td><td>0,83</td></tr><tr><td>Threonine, %</td><td>0,67</td><td>0,67</td><td>0,67</td></tr><tr><td>Tryptophane, %</td><td>0,21</td><td>0,20</td><td>0,20</td></tr><tr><td>Valine, %</td><td>0,83</td><td>0,80</td><td>0,80</td></tr></tbody></table>

**Results**

In Table 2, are presented means of performance parameters and egg quality traits measured in Bovans White hens. Different levels of dietary protein did not affect (P > 0,05) egg shell quality, albumen height and egg weight. However, hens fed 16.4% CP did not have their performance reduced when compared with those fed 17,4 CP.

<table><caption>**Table 2.** Performance and egg quality parameters of Bovans White from 24-40 weeks of age.</caption><thead><tr><th rowspan="2"></th><th colspan="3">Protein levels (%)</th><th rowspan="2">SEM</th><th rowspan="2">P</th></tr><tr><th>17,4</th><th>16,4</th><th>15,4</th></tr></thead><tbody><tr><td>Egg production, %</td><td>93.05<sup>a</sup></td><td>93.45<sup>a</sup></td><td>90.05<sup>b</sup></td><td>1,17</td><td>0,048</td></tr><tr><td>Feed intake, g/bird/day</td><td>125<sup>b</sup></td><td>114<sup>a</sup></td><td>111<sup>a</sup></td><td>6</td><td>0,001</td></tr><tr><td>Feed conversion, kg/eggs</td><td>2,32<sup>c</sup></td><td>2,11<sup>a</sup></td><td>2,20<sup>b</sup></td><td>0,07</td><td>0,032</td></tr><tr><td>Specific gravity, g/mL of H2O</td><td>1,096</td><td>1,096</td><td>1,095</td><td>0,002</td><td>0,721</td></tr><tr><td>Egg weight, g</td><td>58</td><td>58</td><td>56</td><td>3</td><td>0,070</td></tr><tr><td>Albumen height, mm</td><td>6.1</td><td>6.3</td><td>6.5</td><td>0,2</td><td>0,139</td></tr><tr><td>Yolk color</td><td>5.8<sup>c</sup></td><td>6.0<sup>b</sup></td><td>6.1<sup>a</sup></td><td>0,05</td><td>0,034</td></tr><tr><td>Haugh units</td><td>78<sup>b</sup></td><td>79<sup>b</sup></td><td>82<sup>a</sup></td><td>2</td><td>0,038</td></tr><tr><td>Egg breaking strength, kgf</td><td>4.87</td><td>5.19</td><td>5.05</td><td>0,37</td><td>0,276</td></tr><tr><td>Eggshell thickness, mm</td><td>0.45</td><td>0.43</td><td>0.43</td><td>0,01</td><td>0,245</td></tr></tbody></table>

<footnote><sup>a,b</sup> Means within a row not sharing a common superscript differ significantly based on Tukey test (P<0,05)</footnote>
Performance and egg quality parameters measured in Bovans Brown hens are summarized in Table 3. The results show that different levels of dietary protein did not affect (P<0,05) performance, egg shell quality, and internal egg quality except yolk color with higher yolk color in eggs of hens fed diets with 15.4% of CP.

<table><caption>**Table 3.** Performance and egg quality parameters of Bovans Brown from 24-40 weeks of age.</caption><thead><tr><th>Protein levels (%)</th><th>17,4</th><th>16,4</th><th>15,4</th><th>SEM</th><th>P</th></tr></thead><tbody><tr><td>Egg production, %</td><td>94.4<br>8</td><td>94.23</td><td>93.26</td><td>2,24</td><td>0.690</td></tr><tr><td>Feed intake, g/bird/day</td><td>116</td><td>113</td><td>112</td><td>30</td><td>0,845</td></tr><tr><td>Feed conversion, kg/eggs</td><td>2,05</td><td>2,03</td><td>2,04</td><td>0,12</td><td>0,712</td></tr><tr><td>Specific gravity, g/mL of H2O</td><td>1,09<br>5</td><td>1,094</td><td>1,096</td><td>0,003</td><td>0,684</td></tr><tr><td>Egg weight, g</td><td>60</td><td>59</td><td>59</td><td>3</td><td>0,239</td></tr><tr><td>Albumen height, mm</td><td>5.5</td><td>5.0</td><td>5.2</td><td>0,4</td><td>0,182</td></tr><tr><td>Yolk color</td><td>4.9<sup>c</sup></td><td>5.3<sup>b</sup></td><td>5.5<sup>a</sup></td><td>0,1</td><td>0,001</td></tr><tr><td>Haugh units</td><td>71</td><td>69</td><td>70</td><td>3</td><td>0,393</td></tr><tr><td>Egg breaking strength, kgf</td><td>4.14</td><td>4.21</td><td>4.35</td><td>0,24</td><td>0,466</td></tr><tr><td>Eggshell thickness, mm</td><td>0.40</td><td>0.40</td><td>0.41</td><td>0,02</td><td>0,114</td></tr></tbody></table>

## Conclusions

Light laying hens fed diets with 16.4% CP improved feed conversion. For semi-heavy hens, it is possible to reduce protein to 15.41% without reduce egg production and egg quality.

## References

LATSHAW, J.D., L. ZHAO. 2011. Dietary protein effects on hen performance and nitrogen excretion. Poult. Scie. **90**:99-106.

SAS Institute. 2012. User's Guide. Version 9.3 4th Edition. SAS Inst. Inc., Cary, NC.

<footnote>a,b,c Means within a row not sharing a common superscript differ significantly based on Tukey test (P<0,05)</footnote>
P-207 (ID 224)

EFFECT OF DIFFERENT DIETARY ENERGY AND PROTEIN LEVELS ON PERFORMANCE AND CARCASS CHARACTERISTICS OF JAPANESE QUAIL

N. Ghavipanje, M. Afshin, and N. Afzali

Department of Animal Science, University of Birjand, Iran

Corresponding author: nafzali@birjand.ac.ir

Abstract

To investigate the effect of different levels of energy and protein on growth indices in Japanese quail, an experiment was carried out using 360 quail in a two way factorial arrangement on completely randomize design with two levels of energy (2800 and 2900 kcal/kg) and three levels of protein (22, 24 and 26 %). Four replicates with 15 quails were allocated to each experimental treatment and birds were reared for 45 days. Weekly measurements of feed intake, weight gain and feed conversion ratios were calculated. At the end of experiment, two birds from each experimental unit were slaughtered to determine carcass characteristics. The results showed that there were significant difference in weight gain, feed intake and feed conversion ratio between experimental treatments (p<0.05). Quails were fed rations containing 2900 ME and 26 % crude protein had highest body weight and lowest feed conversion ratio. The effects of interaction among energy and protein on carcass, breast, thigh and heart percentage were significant (p<0.05). The highest breast weight was observed in quails fed 2900 ME and 26 % crude protein. It can be concluded that using diets containing 2900 kcal ME/kg and 26% crude protein is recommendable to achieve a desirable performance in Japanese quails.

**Keywords:** Japanese quail, Energy, Protein, performance and Carcass characteristics

Introduction

Japanese quails (*Coturnix coturnix japonica*) are being raised for the purpose of producing eggs and meat. Due to the increasing demand for quail's meat and eggs, and economical production of these products, it is expected that quail farming will develop in the future. It is blessed with the unique characteristics of fast growth, early sexual maturity, high rate of egg production, that makes it suitable for diversified animal agriculture (KAUR *et al.*, 2008). The metabolizable energy and crude protein are the major components of poultry diet and on the other hand feeding cost is usually considered the most expensive item, but the energy and protein requirements as well as the efficiency of feed utilization are still poorly documented, especially for quails (SIYADATI *et al.*, 2011). In the number of studies on the effect of energy and protein levels in Japanese quail, different responses have been reported (SOARES *et al.*, 2003 and ATTIA *et al.*, 2012). Today, in addition to the quantity of poultry production, carcass quality is important. In studies on carcass characteristics of Japanese quail, different responses to different levels of energy and protein were observed (SIYADATI *et al.*, 2011, PARVIN *et al.*, 2010). Therefore, due to the genetic selection to achieve maximum production, the importance of diet energy and protein in poultry and lack of accurate data to estimate the nutritional requirements of Japanese quail, the present study was performed to investigate the effect of dietary levels of protein and energy on the performance and carcass characteristics of growing Japanese quail.

Material and Methods

The study included three hundred and sixty Japanese quails (5-day-old). A factorial design arrangement (2 x 3), using 6 treatments with four replicates of 15 chicks each was performed included two levels of metabolizable energy (2800, 2900 kcal ME/Kg) and three levels of crude protein (22, 24 and 26%), based on comletly randomize design on growth performance and carcass traits of Japanese quail. The dietary nutrients requirement (except energy and protein) was regulated based on the NRC (1994) tables and diets were balanced by UFFDA software. Quail chicks were grown on the floor and brooding temperature gradually decreased from 36 °C during the initial 7 days to 27°C by 21 days of age. The data of body weights and feed intake were recorded at weekly intervals and at the end of experiment (42 days), two birds from each experimental unit were slaughtered to determine carcass characteristics. The weight of the carcass, breast, thighs, back and neck of each quail (as a percentage of live weight) was measured separately. Data obtained from the experiment with GLM procedure of SAS software were analyzed; In addition differences among treatment means were tested using turkey's test.
## Results

The effect of treatments on the performance of Japanese quail during the growing period is provided in Table 1. A statistically significant difference was found in the productive performance on whole the growing period (5-42 days old) between the treatments (P <0.05). The highest weight gain, feed intake and also the highest feed conversion ratio was observed in quails were fed with diets containing 2900 kcal ME/kg and 26% crude protein. The growing quail metabolizable energy and crude protein requirements in some resources 2900 and 24% and 28% is mentioned, which is partly consistent with the results obtained in this study (LEESON and SUMMERS, 2008 and NRC, 1994). In the present study feed intake were increased with increasing levels of energy and protein, which is in contrast with the findings of SOARES et al. (2003). This researchers reported that the dietary protein level had no effect on feed intake (P >0.05). It is likely due to the higher level of oil which was used in our diet, therefore feed intake were increased in this group. Since the oil reduces dust and increased palatability of the diet, this were lead to a higher feed intake in this treat (KE-SHAVAREZ and JACKSON, 1992). In the study by SIYADATI et al. (2011), improved BWG and FCR in the quails were fed with diets containing higher metabolizable energy and crude protein was reported and also in another study (STERLING et al., 2005) the best feed conversion ratio was obtained with feeding diets containing 26% crude protein, which corresponded with the findings of the present investigation.

<table><caption>**Table 1.** Live weight gain, feed intake (g bird⁻¹) and feed conversion ratio of growing quails in 5-42</caption><thead><tr><th>Treatments</th><th>Live weight gain</th><th>Feed intake</th><th>FCR</th></tr></thead><tbody><tr><td>2800×22</td><td>193.807<sup>c</sup></td><td>649.768<sup>b</sup></td><td>3.356<sup>ab</sup></td></tr><tr><td>2800×24</td><td>178.824<sup>d</sup></td><td>641.206<sup>b</sup></td><td>3.586<sup>a</sup></td></tr><tr><td>2800×26</td><td>196.543<sup>bc</sup></td><td>693.592<sup>a</sup></td><td>3.530<sup>ab</sup></td></tr><tr><td>2900×22</td><td>192.395<sup>c</sup></td><td>659.972<sup>b</sup></td><td>3.430<sup>ab</sup></td></tr><tr><td>2900×24</td><td>206.258<sup>ab</sup></td><td>689.874<sup>a</sup></td><td>3.346<sup>ab</sup></td></tr><tr><td>2900×26</td><td>213.622<sup>a</sup></td><td>709.604<sup>a</sup></td><td>3.324<sup>b</sup></td></tr><tr><td>SEM</td><td>2.422</td><td>5.704</td><td>0.053</td></tr><tr><td>P_value</td><td>&lt;.0001</td><td>0.0071</td><td>0.0123</td></tr></tbody></table>

The effect of treatments on carcass, breast, thighs and back and neck as a percentage of live weight is reported in Table 2. Carcass weight in birds were fed diets containing 2800 kcal ME/kg and 24% crude protein was lower than the other treatments. The results of this study are in agreement with YAZERLOU et al. (2013). Those reported reduce carcass in quail fed diets containing lower energy (2750 kcal ME/kg) and 24% crude protein. The average weight of the thigh and breast were significantly different between experimental treatments (P <0.05). Better thigh and breast weight were observed in quails fed high levels of energy and protein. Which is in consistent with the result of SIYADATI et al. (2011) and CORREAL et al. (2008).

<table><caption>**Table 2.** Average carcass characteristics in different experimental treatments (as percentage of live weight)</caption><thead><tr><th>Treatments</th><th>Carcass</th><th>Thigh</th><th>Breast</th><th>Back and neck</th><th>Heart</th></tr></thead><tbody><tr><td>2800×22</td><td>69.598<sup>abc</sup></td><td>15.758<sup>ab</sup></td><td>26.573<sup>ab</sup></td><td>26.860<sup>a</sup></td><td>0.847<sup>a</sup></td></tr><tr><td>2800×24</td><td>66.856<sup>c</sup></td><td>14.801<sup>b</sup></td><td>25.743<sup>b</sup></td><td>26.401<sup>a</sup></td><td>0.748<sup>b</sup></td></tr><tr><td>2800×26</td><td>67.665<sup>bc</sup></td><td>15.049<sup>ab</sup></td><td>27.390<sup>a</sup></td><td>25.099<sup>b</sup></td><td>0.836<sup>a</sup></td></tr><tr><td>2900×22</td><td>70.878<sup>a</sup></td><td>15.925<sup>a</sup></td><td>27.363<sup>a</sup></td><td>26.246<sup>ab</sup></td><td>0.844<sup>a</sup></td></tr><tr><td>2900×24</td><td>69.379<sup>abc</sup></td><td>14.910<sup>b</sup></td><td>26.852<sup>ab</sup></td><td>26.207<sup>ab</sup></td><td>0.839<sup>a</sup></td></tr><tr><td>2900×26</td><td>70.494<sup>ab</sup></td><td>15.742<sup>ab</sup></td><td>27.417<sup>a</sup></td><td>27.335<sup>a</sup></td><td>0.730<sup>b</sup></td></tr><tr><td>SEM</td><td>0.643</td><td>0.224</td><td>0.262</td><td>0.288</td><td>0.007</td></tr><tr><td>P-value</td><td>0.0019</td><td>0.0051</td><td>0.0015</td><td>0.0010</td><td>&lt;0.0001</td></tr></tbody></table>

<footnote><sup>a,b,c</sup> Means within each column with uncommon superscript are significantly different (P <0.05)</footnote>

<footnote><sup>a,b,c</sup> Means within each column with uncommon superscript are significantly different (P <0.05)</footnote>
Conclusions

Due to higher weight gain, feed conversion ratio and good carcass, in general conclusion can be stated that a diet con-
taining 2900 kcal ME/kg and 26% CP leads to a desirable performance in Japanese quails.

Literature

ATTIA, A.I., KH.M. MAHROSE, I.E. ISMAIL, D.E. ABOU-KASEM, 2012: Responce of growing Japanese quail raised under two stocking densities to ditary protein and energy levels. **Egyptian journal of animal production**. **47**, 159-166.

CORREAL, G.S.S., M.A. SILVA, A.B. CORREAL, D.O. FONTES., G.G SANTOS., H.R, LIMANETO. Arq Bras Med Vet Zootec. 2008, **60**(1), 209-217.

GOLIAN, A., M. AMI AZGHADI and M. PILEVAR, 2010: Influence of various levels of energy and protein on perform-
ance and humoral immune responses in broiler chicks. Global Veterinaria. **4**(5), 434–440.

KAUR S., A.B, MANDAL., K.B, SINGH and M.M, KADAM, 2008: The response of Japanese quails (heavy body weight line) to dietary energy levels and graded essential amino acid levels on growth performance and immuno-cmpetence. Livestock Science. **117**,255-262.

KESHAVAREZ, K. and M.E. JACKSON, 1992: Performance of growing pullets and laying hens fed low protein, amino acid supplemented diets. Journal of poultry science, **71**, 905-918.

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PARVIN, N., T. K, MANDAL., V., SAXENA., S, SARKAR., and A, SAXENA, 2010: Effect of increasing protein
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9th revised edition pp: 155.
P-209 (ID 81)

EFFECT OF WHITE LUPINE SEEDS INCLUSION ON BROILER CHICKENS PERFORMANCE, DIGESTIBILITY AND VILLI STRUCTURE

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

Poznan University of Life Sciences, Department of Animal Nutrition and Feed Management, ul. Wołyńska 33, 60-637 Poznań, Poland

Corresponding author: sebak1@up.poznan.pl

Abstract

480 male broiler chickens were used in a 35-day study to determine the influence of white lupin inclusion (0, 100, 150, 200, 250 and 300 g/kg) on their performance and diet utilization as well as on ileal villi height and crypt depth. One-day-old male Ross 308 chicks were randomly assigned to six dietary treatments (ten replications per treatment). Birds fed diets with 150 g/kg white lupine were characterized by lower BWG, FI and higher FCR than the control birds. The use of 300 g/kg of white lupine decreased BWG by 400 g in comparison with the control (P<0.05). White lupine seeds were found to exert a negative effect on dietary AMEn and ileal crude fat digestibility as well as on crude protein (P<0.05). Ileal digesta viscosity increased together with the increase of white lupine seed content in experimental diets. The inclusion of 150 g/kg of white lupine seeds in experimental diet increased digesta viscosity from 1.76 cP (control treatment) to 2.41 cP (P<0.05). The highest digesta viscosity was determined when 300 g/kg of white lupine seeds was used – 3.74 cP. Villi height and villi surface area decreased as white lupine seeds content increased (P<0.05). The shallowest (P<0.05) crypts were determined in the control birds.

**Keywords:** broiler, villi, protein, viscosity

Introduction

Soybean meal (SBM) is the most common vegetable protein source in poultry diets but the inclusion of homegrown legume seeds in diets for non-ruminant animals as an alternative to soybean products has been receiving growing attention in countries that are not climatically suited to soybean production (NALLE et al., 2012). Good nutritional value of white lupine for broilers was reported previously by many authors. The AME of white lupine was determined to range from 8.0 to almost 15 MJ/kg. White lupine protein and amino acid (AA) ileal digestibility are similar to those obtained for soybean meal SBM (NALLE et al., 2012). NALLE et al. (2012) reported that average digestibility for white lupine AA ranged from 0.85 to 0.87. RAVINDRAN et al. (2005) showed that white lupine AA digestibility ranged from 0.75 to 0.88.

On the other hand, whole lupin seeds contain substantial concentrations of oligosaccharides, phytate or NSP limiting the use of white lupin in poultry feeding. Phytate in plant feedstuffs can interact electrostatically with dietary (and possibly endogenous) proteins and amino acids which negatively influences protein utilization and increases endogenous protein loss (SELLE et al., 2012). Additional, non-starch polysaccharides may increase viscosity of digesta affecting negatively nutrients absorption and consequently AME.

The aim of the study was to determine the effect of white lupine seeds dose on growth performance, nutrient utilization and villi structure in broiler chickens.

Material and Methods

The experiment was conducted with 480 broiler (Ross 308) chicks (initial individual weight: 40±2 g) divided into six dietary treatments, including a maize -SBM control diet (CON) and five experimental diets containing; 100, 150, 200, 250 and 300g/kg white lupine seeds. Each comprised 10 replications of 8 birds. Apparent total tract digestibility and AMEn value of the diets were calculated relative to the ratio of TiO₂ to the content of the nutrient in question in the feed, digesta or excreta. Data were analyzed by ANOVA using GLM procedure of R environment. The cage was the experimental unit and orthogonal contrasts were used to establish the difference between treatments. The contrasts used were the CON treatment versus the remaining 5 dietary treatments (100, 150, 200, 250 or 300 g of lupine seeds per kg of feed) as well as the contrast for linear (L) and quadratic (Q) effects. All data are presented as means.