In short, this SSF process is recommended to be easily installed into any feed production line. Significantly important improvements both in growth rate and in gut function of broiler chickens fed with the diets of fermented wheat and barley products promise a growth promoting effect in broilers. But this is not the case with oats, probably due to high level of crude fibre per se, limiting the regulation of feed intake to provide sufficiently enough amounts of nutrients available for intestinal uptake.

## Literature

ALBORES M., J.V. MEDINA, E.U. ÁLVAREZ, F.M. BUSTOS, E.M. MARTÍNEZ, 2009: Effect of citric acid on aflatoxin degradation and on functional and textural properties of extruded sorghum, Anim. Feed Scien. & Techn. **150**, 316-329.

CARLSON D., H.D. POULSEN, 1999: Phytate degradation in soaked and fermented barley and wheat (preliminary studies), Nor-dic Agric. Res. **81**, 278-283.

ENGBERG R.M., M. HAMMERSHØJ, N.F. JOHANSEN, M.S. ABOUSEKKEN, S. STEENFELDT, B.B. JENSEN, 2009: Fermented feed for laying hens: effects on egg production, egg quality, plumage condition and composition and activity of the intestinal micro-flora, Bri. Poult. Sci. **50**, 228-239.

FORBES J.M., F. SHARIATMADARI, 2005: Performance of broiler chickens given whey in the food and/or drinking water, Br. Poult. Sci. **46**, 498-505.

UCHEWA E.N., P.N. ONU, 2012: The effect of feed wetting and fermentation on the performance of broiler chick, Biotec. Anim. Husb. **28 (3)**, 433-439.

YASAR S., J.M. FORBES, 1999: Performance and gastro-intestinal response of broiler chickens fed on cereal grain-based foods soaked in water, Br. Poult. Sci. **40**, 65-76.

YASAR S., M.S. GOK, 2014: Fattening Performance of Japanese Quails (coturnix coturnix japonica) Fed on Diets with High Levels of Dry Fermented Wheat, Barley and Oats Grains in Whey with Citrus Pomace, Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Animal Science And Biotechnologies, **71(1)**, 51-62.
# INFLUENCE OF COMBINED EFFECT OF TEMPERATURE STIMULATION DURING THE LAST DAYS OF INCUBATION AND OF DIFFERENT PROTEIN AND ENERGY CONCENTRATIONS IN THE FEED ON GROWING PERFORMANCE OF COCKERELS OF DIFFERENT STRAINS

Ingrid Halle<fnref n="1" />, M. A. Lieboldt<fnref n="1" />, M. Henning<fnref n="2" />, B. Tzschentke<fnref n="3" />

**Corresponding author:** Ingrid.halle@fli.bund.de

## Abstract

Laying-type cockerels or spring chicken cannot be reared economically. Lohmann Tierzucht bred the dual-purpose chicken (producing eggs and meat) in response to growing criticism of conventional practices in modern egg production. Recent research with birds shows that incubation climate may have a long-lasting influence on poultry performance up to the age of slaughter. Therefore the following study was carried out, to investigate whether short-term variation in incubation temperature during the last days of incubation have a long-lasting effect on performance, also in laying-type cockerels. 2880 eggs (Lohmann Brown-LB/Lohmann Dual-LD) were incubated from days 1 to 17 under common incubation temperature (37°C). From day 18 until hatching the eggs were sorted in hatch incubators with different temperature programs. The male one-day-old chicks were randomly distributed in 8 treatment groups during a study time of 70 days: two origins of chicks (LB, LD), two hatch incubators and twofold protein/energy (200g crude protein/11 MJ AME<sub>N</sub>/kg; 215g/12 MJ) levels in the diet. Growing performance of LD cockerels was significantly better compared to LB males. Final body weight of LD birds was 1000 g higher and feed to gain ratio 10% lower. Short-term temperature stimulation during the end of incubation resulted in a 3.5% higher final body weight by LD cockerels.

**Keywords:** cockerels, incubation, temperature stimulation, growth

## Introduction

Laying-type cockerels or spring chicken cannot be reared economically (Damme and Ristic, 2003; Schäublin et al., 2005; König et al., 2010; Halle et al., 2012b). But routine culling of these day-old male chicks is more and more an ethical problem and to find alternative solutions is a great challenge. Lohmann Tierzucht bred the dual-purpose chicken (producing eggs and meat) in response to growing criticism of conventional practices in modern egg production. Recent research shows that incubation climate may have a long-lasting influence on poultry performance (Collin et al., 2007; Hulet et al., 2007; Pistun et al., 2008; Tzschentke and Halle, 2009; Halle and Tzschentke, 2011; Shinder et al., 2011; Halle et al., 2012a). The most important climatic incubation factor is the incubation temperature (Decuypere and Michels, 1992). In broiler chickens, for instance, short-term temperature training during the last 4 days of embryonic development, improved hatching results and later robustness and performance (Tzschentke and Halle, 2009). Therefore the following study was carried out, to investigate whether short-term variation in incubation temperature during the last days of incubation have a long-lasting effect on performance, also in laying-type cockerels.

## Material and Methods

In the trial 2880 eggs (Lohmann Brown-LB/Lohmann Dual-LD) were incubated from days 1 to 17 under common incubation temperature (37°C). From day 18 until hatching the eggs were sorted in hatch incubators with different temperature programs: 37°C (control) and 10°C over standard for 2 hours daily (38°C: short-term warm stimulation). The one-day-old chicks were sorted by sex and only the male laying-type cockerels were randomly distributed in 8 treatments (6 pens/group) resulted from the two origin of chicks (LB, LD), from two hatch incubators and twofold protein/energy (200 g crude protein/11 MJ AME<sub>N</sub>/kg - low; 215 g/12 MJ - high) levels in the diet (Table 1; 2). The duration of the trial was 70 days. Data from the trial were analyzed by a three-way ANOVA (SAS) ($P \le 0.05$).

<footnote n="1">¹ Institute of Animal Nutrition (FLI), Bundesallee 50, 38116 Braunschweig, Germany</footnote>

<footnote n="2">² Institute of Farm Animal Genetics (FLI), Höltystr. 10, 31535 Neustadt, Germany</footnote>

<footnote n="3">³ Humboldt-University of Berlin, Institute of Biology, Ziegelstrasse 30, 10117 Berlin, Germany</footnote>
<table><caption>**Table 1.** Study design</caption><thead><tr><th>Group</th><th>Cockerels origin</th><th>Temperature stimulation</th><th>Feed level</th></tr></thead><tbody><tr><td>1 / 5</td><td>LB / LD</td><td>Control</td><td>Low</td></tr><tr><td>2 / 6</td><td></td><td>Control</td><td>High</td></tr><tr><td>3 / 7</td><td></td><td>Short-term warm stimulation</td><td>Low</td></tr><tr><td>4 / 8</td><td></td><td>Short-term warm stimulation</td><td>High</td></tr></tbody></table>

<table><caption>**Table 2.** Ingredient composition and nutrients of the diets (g/kg) (Dry matter 885 g/kg)</caption><thead><tr><th rowspan="2">Ingredient</th><th colspan="2">Feed level</th></tr><tr><th>low</th><th>high</th></tr></thead><tbody><tr><td>Wheat/Corn/Bran</td><td>695</td><td>594</td></tr><tr><td>Soya bean meal/Oil</td><td>267</td><td>365</td></tr><tr><td>Vitamin and minerals</td><td>38</td><td>41</td></tr><tr><td>Crude protein</td><td>185</td><td>205</td></tr><tr><td>ME, MJ/kg</td><td>11.2</td><td>12.4</td></tr><tr><td>Lysine/Methionine+Cystine</td><td>10.0/8.5</td><td>11.5/9.8</td></tr></tbody></table>

## Results and conclusion

<table><caption>**Table 3.** Performance of laying type cockerels</caption><thead><tr><th>Genetic line</th><th>Temperature stimulation</th><th>Feed level</th><th>Feed intake g/bird/day</th><th>Final body weight, g/bird</th><th>kg feed/kg weight gain</th></tr></thead><tbody><tr><td rowspan="4">LB</td><td>no</td><td>Low</td><td>47.6</td><td>1336</td><td>2.568</td></tr><tr><td>no</td><td>High</td><td>43.3</td><td>1360</td><td>2.293</td></tr><tr><td>yes</td><td>Low</td><td>47.1</td><td>1336</td><td>2.544</td></tr><tr><td>yes</td><td>High</td><td>44.1</td><td>1374</td><td>2.309</td></tr><tr><td rowspan="4">LD</td><td>no</td><td>Low</td><td>78.6</td><td>2432</td><td>2.299</td></tr><tr><td>no</td><td>High</td><td>71.4</td><td>2482</td><td>2.049</td></tr><tr><td>yes</td><td>Low</td><td>81.9</td><td>2558</td><td>2.275</td></tr><tr><td>yes</td><td>High</td><td>73.9</td><td>2528</td><td>2.070</td></tr><tr><td colspan="6">ANOVA, P-value</td></tr><tr><td>Cockerels origin</td><td></td><td></td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>Temperature stimulation</td><td></td><td></td><td>0.14</td><td>0.09</td><td>0.96</td></tr><tr><td>Feed level</td><td></td><td></td><td>&lt;0.001</td><td>0.45</td><td>&lt;0.001</td></tr><tr><td>Temperature x Feed</td><td></td><td></td><td>0.9</td><td>0.5</td><td>0.2</td></tr><tr><td>Temperature x Origin</td><td></td><td></td><td>0.2</td><td>0.2</td><td>0.8</td></tr><tr><td>Origin x Feed</td><td></td><td></td><td>0.07</td><td>0.7</td><td>0.4</td></tr><tr><td>Origin x Temperature x Feed</td><td></td><td></td><td>0.6</td><td>0.4</td><td>0.9</td></tr></tbody></table>

Growing performance of LD cockerels was significantly better compared to LB males. Final body weight of LD birds was 1000 g higher and feed to gain ratio 10% lower. Short-term temperature stimulation during the end of incubation resulted in a 3.5% higher final body weight by LD cockerels. The daily feed intake and the feed to gain ratio was significantly improved through the increased protein/energy concentration of the "high" feed.

The results allow the conclusions that an incubation temperature profile, which includes short-term warm stimulation of +1°C for 2 h daily during final incubation, had a long-lasting effect on performance in male laying-type cockerels of origin Lohmann Dual. The most important factors on growing performance of the Lohmann Brown laying type cockerels were the protein-energy concentration in feed.
## Literature

COLLIN, A., C. BERRI, S. TESSERAUD, S. et al., 2007: Effects of Thermal Manipulation During Early and Late Embryogenesis on Thermo tolerance and Breast Muscle Characteristics in Broiler Chickens. Poultry Science 86, 795–800.

DAMME, K., M. RISTIC, 2003: Fattening performance, meat yield and economic aspects of meat and layer type hybrid. Poultry Science Journal 59: 50-53.

DECUYPERE, E., H. MICHELS, 1992: Incubation temperature as a management tool: a review. World's Poultry Science Journal 48: 28-38.

HALLE, I., B. TZSCHENTKE, 2011: Influence of Temperature Manipulation during the last 4 Days of Incubation on Hatching Results, Post-Hatching Performance and Adaptability to Warm Growing Conditions in Broiler Chickens. J. Poultry Sci. 48, 97-105.

HALLE, I., B. TZSCHENTKE, M. HENNING, P. KÖHLER, 2012a: Influence of temperature stimulation during the last 6 days of incubation on hatching results and later performance in Pekin ducks. Arch. Geflügelk. 76 (3), 176-183.

HALLE, I., H. KLUTH, S. DÄNICKE, 2012b: Effect of a graded dietary protein-energy-concentration on the growth performance of laying-type cockerels of different strains. Arch. Geflügelk. 76 (4) 223-229.

HULET, R., D. GLADYS, D. HILL, R. MEIJERHOF, T. EL-SHIEKH, 2007: Influence of egg shell embryonic incubation temperature and broiler breeder flock age on posthatch growth performance and carcass characteristics. Poultry Science 86: 408-412.

KOENIG, M., G. HAHN, K. DAMME, M. SCHMUTZ, 2010: Nutzung männlicher Legehybriden als Stubenküken. Fleischwirtschaft 12, 92-94.

PIESTUN, Y., D. SHINDER, M. RUZAL, O. HALEVY, J. BRAKE, S. YAHAV, 2008: Thermal Manipulations During Broiler Embryogenesis: Effect on the Acquisition of Thermotolerance. Poultry Science 87, 1516–1525.

SCHÄUBLIN, H., H. WIEDMER, R. ZWEIFEL, 2005: Vergleich der Mastleistungen und Fleischqualität von Hähnen ausgewählter Legelinien mit einem extensiven Masthybriden. Schlussbericht Versuchsprojekt M405, Aviforum, Schweiz.

SHINDER, D., M. RUZAL, M. GILOH, S. DRUYAN, Y. PIESTUN, YAHAV, 2011: Improvement of cold resistance and performance of broilers by acute cold exposure during late embryogenesis. Poultry Science 90, 633-641.

TZSCHENTKE, B., I. HALLE, 2009: Influence of temperature stimulation during the last 4 days of incubation on secondary sex ratio and later performance in male and female broiler chicks. Brit. Poult. Sci., 50, 634-640.
# EFFECT OF DIFFERENT DOSAGES OF COATED BUTYRIC ACID ON GROWTH PERFORMANCE AND ENERGY UTILIZATION IN BROILERS

Sebastian Kaczmarek<fnref n="1" />, Adriana Bari<fnref n="2" />, Marcin Hejdysz<fnref n="1" />, Andrzej Rutkowski<fnref n="1" />

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

## Abstract

Four dietary treatments were used in experiment I and II to investigate the effect of protected butyrate, on growth performance and nutrient digestibility in broiler chickens. One-day-old male Ross 308 chicks (total 960, 480 per trial) were randomly assigned to four dietary treatments. In experiment I, the basal diets were fed without or with protected calcium butyrate (B), inclusion (0.2, 0.3, or 0.4 g/kg). In experiment II, the basal diets were fed with or without B (0.3 g/kg), or with Avilamycin (A), or in combination of both. In experiment I, the use of B improved FCR (P<0.05) irrespective of the dose. Diets with B were characterized by higher AMEn value than the control treatment (P<0.05). Histomorphological evaluations showed that microencapsulated B had a positive influence (P<0.05) on villi height In experiment II, there was an effect (P<0.05) in improved BWG after B or A supplementation separately or in combination. The use of avilamycin improved (P<0.05) FCR in comparison with the control treatment. Ileal digestibility of crude protein and crude fat increased after B, A or the combination of both (P<0.05). The use (14, 35 and 42d) of B and A in combination improved AME content in comparison with the control treatment (P<0.05). The use of A, B or AB increased ileal digestibility of Thr, Ser and Pro (P<0.05). Valine, Ile, Leu, Tyr and His ileal digestibility was improved in comparison with the control after A addition (P<0.05).

**Keywords:** broiler, villi, amino acid, digestibility

## Introduction

It is certain that butyric acid is a very complex and dynamic molecule with different modes of action. After B supplementation, an increase in villi height and crypt depth has been observed in pigs and poultry (GALFI and BOKORI, 1990, LEESON et al., 2005). The growth in villi height increases the absorptive surface of the small intestine leading to better nutrient utilization, which is why recent studies have shown that when B is orally ingested it can have beneficial effects on animal performance (MAZZONI et al., 2008, CZERWIŃSKI et al., 2012). However, there is limited information on the effect of protected B on the AME of diets in broiler chickens. The limited literature data have shown that B may improve total tract digestibility, but not ileal nutrient digestibility in pigs (LE GALL et al., 2009). In contrast, MANZANILLA et al. (2006) reported that B reduced ileal digestibility of starch in weaned pigs.

The aim of this study was to investigate the effect that a protected butyrate can have on diet digestibility, AME, birds' performance and ileum histomorphology. This study consists of the results of two trials.

## Material and Methods

Ross 308 broiler chicks (total 960, 480 per trial) were obtained from a local hatchery. The birds were individually weighed and allocated to 10 weight classes. Groups of 10 birds were then randomly assigned to pens in such a way that the average initial bodyweight of birds was similar across pens. Ten replicate pens of 10 birds each were randomly assigned to four dietary treatments. All diets were fed in pelleted form throughout the 6-week experimental period.

The birds were held in pens and had *ad libitum* access to water and feed. On day 35 (Experiment I) and on days 14, 35 and 42 (Experiment II), excreta samples from each pen were collected over a 3-h period and subsequently frozen, freeze-dried, and finely ground. Collection trays were installed in floor pens to allow for excreta collection; ten excreta samples per treatment were collected. On day 35 (Experiments I and II), 10 birds from each treatment were randomly selected and killed via cervical dislocation. The contents of the terminal ileum (15 cm, adjacent to the ileo-cecal junction) were collected. Additionally, samples (n = 8) of the whole intestinal tract were removed, and segments of approximately 2 cm were collected. In experiment I, the basal diets were fed with or without protected calcium butyrate (B) (ButiPEARL™, Kemin) inclusion (0.2, 0.3, or 0.4 g/kg). In experiment II, the basal diets were fed with or without pro-

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

<footnote n="2">² Animal Nutrition and Health EMEA Toekomstlaan 42, Herentals, Belgium</footnote>
protected calcium butyrate (0.3 g/kg), (ButiPEARL, Kemin) or with avilamycin (6 mg/kg, active substance), (Maxus, Elanco) or with a combination of both.

## Results

In experiment I, considering the entire study period, the use of B improved FCR (P<0.05) irrespective of the dose. Apparent total tract crude fat digestibility was improved after B supplementation (P<0.05). Diets with B were characterized by higher AMEn values than the control treatment (P<0.05). Birds fed diets supplemented with B were characterized by higher VH than that of unsupplemented birds (P<0.05). At the lowest dose (0.2 g/kg), the difference was over 150 µm compared to the control treatment (P<0.05). Results from experiment II showed that avilamycin alone or in combination with B improved (P<0.05) BWG compared to the control treatment. A tendency (P<0.1) for improved BWG (14–42d) was observed after B or A supplementation, either separately or in combination. Similar differences (P<0.05) between A and B were observed through the whole trial period (0–42d). The use of avilamycin improved (P<0.05) FCR compared to the control treatment. To a similar extent, A, B and the combination of both improved (P<0.05) FCR compared to the control treatment. Birds from the control treatment were characterized by having the thickest mucosa (P<0.05). The thinnest mucosa was observed when birds were fed the control diet supplemented with the combination of B and A (P<0.05). Villous height was not affected when B or A were used separately; however, animals fed B showed numerically higher villi than the controls. Birds given the mixture of B and A consistently showed lower villi compared to the control treatment (P<0.05). Crypt depth decreased in the ileum for all treated animals compared to the control birds (P<0.05). The ileal digestibility of crude protein increased after B, A or the combination of both (P<0.05). Similar differences were observed in ileal crude fat digestibility, but significant differences (P<0.05) were noted only between the control and avilamycin treated animals. On day 14, the use of B and A in combination improved AME content compared to the control treatment (P<0.05) and the difference was over 150 kcal/kg. Considering days 35 and 42 of the experiment, determined AME increased after A, B or A and B combination supplementation compared to the control treatment (P<0.05). B, A or AB treated birds were characterized by higher total tract crude fat digestibility on days 14 and 35. On day 42, A and AB treated birds were different from the control (P<0.05). The most pronounced improvement was noted after A supplementation.

A or AB treated birds were characterized by higher Asp, Glu, Cys, Gly and Ala ileal digestibility than the control animals (P<0.05). The use of A, B or AB increased the ileal digestibility of Thr, Ser and Pro (P<0.05). Valine, Ile, Leu, Tyr and His ileal digestibility was improved compared to the control after A addition (P<0.05).

## Conclusions

The results of our studies indicate positive effects of feeding of up to 0.3 g/kg protected butyrate throughout the 42-d broiler growth period when the compound was used either alone or in combination with avilamycin. There is an indication that butyrate helped maintain intestinal integrity and, consequently, improve the digestion and absorptive processes. Future studies are warranted to investigate the dose response to protected butyric acid on enzyme secretion and starch, as well as other nutrient digestibility in birds.
<table><caption>**Table 3.** Broiler chicken performance (BWG, FI [g] and FCR [g:g]), ATTD, AID, AMEn and mucosa thickness – experiment I and II.</caption><thead><tr><th colspan="8">Experiment I</th></tr><tr><th rowspan="2">B dose [g/kg]</th><th colspan="2">BWG</th><th colspan="2">FI</th><th colspan="2">FCR</th><th rowspan="2">ATTD of CF</th><th rowspan="2">AMEn</th></tr><tr><th>0-14d</th><th>0-42d</th><th>0-14d</th><th>0-42d</th><th>0-14d</th><th>0-42d</th></tr></thead><tbody><tr><td>0</td><td>344</td><td>2616</td><td>512</td><td>4336</td><td>1.48</td><td>1.66</td><td>82.8</td><td>3,136</td></tr><tr><td>0.2</td><td>363</td><td>2696</td><td>511</td><td>4409</td><td>1.41</td><td>1.64*</td><td>86.9*</td><td>3,358*</td></tr><tr><td>0.3</td><td>354</td><td>2741</td><td>475</td><td>4348</td><td>1.31*</td><td>1.59*</td><td>85.5*</td><td>3,373*</td></tr><tr><td>0.4</td><td>357</td><td>2699</td><td>467*</td><td>4207</td><td>1.31*</td><td>1.58*</td><td>86.9*</td><td>3,263*</td></tr><tr><th colspan="9">Experiment II</th></tr><tr><th></th><th>BWG</th><th>FCR</th><th>Mucosa thickness</th><th>AID of CP (14d)</th><th>ATTD of CF (d14)</th><th colspan="3">AMEn at day:</th></tr><tr><td>C</td><td>2123</td><td>1.59</td><td>1210</td><td>69.6</td><td>92.5</td><td>14</td><td>35</td><td>42</td></tr><tr><td>C+B</td><td>2323*</td><td>1.49*</td><td>981*</td><td>78.1*</td><td>95.4*</td><td>3088</td><td>3105</td><td>3241</td></tr><tr><td>C+A</td><td>2356*</td><td>1.50*</td><td>895*</td><td>78.8*</td><td>95.6*</td><td>3154</td><td>3235*</td><td>3305</td></tr><tr><td>C+B+A</td><td>2361*</td><td>1.48*</td><td>837*</td><td>78.2*</td><td>94.4*</td><td>3148</td><td>3320*</td><td>3348*</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>3243*</td><td>3247*</td><td>3333*</td></tr></tbody></table>

CF – crude fat, AID – apparent ileal digestibility, CP – crude protein, ATTD – apparent total tract digestibility, C – Control treatment, C+B – control + ButiPEARL, C+A – control + avilamycin, C+B+A – control + ButiPEARL + avilamycin.

* – Means significantly different from control treatment (P < 0.05).

## Literature

CZERWIŃSKI, J., HØJBERG, O., SMULIKOWSKA, S., ENGBERG, R. M. and MIECZKOWSKA, A. (2012) Effects of sodium butyrate and salinomycin upon intestinal microbiota, mucosal morphology and performance of broiler chickens. *Arch Anim Nutr* 66: 102-116.

GALFI, P. and BOKORI, J. (1990) Feeding trial in pigs with a diet containing sodium n-butyrate. *Acta Vet Hung* 38: 3-17.

LE GALL, M., GALLOIS, M., SÈVE, B., LOUVEAU, I., HOLST, J. J., OSWALD, I. P., LALLÈS, J.-P. and GUILLOTEAU, P. (2009) Comparative effect of orally administered sodium butyrate before or after weaning on growth and several indices of gastrointestinal biology of piglets. *British Journal of Nutrition* 102: 1285-1296.

LEESON, S., NAMKUNG, H., ANTONGIOVANNI, M. and LEE, E. (2005) Effect of butyric acid on the performance and carcass yield of broiler chickens. *Poultry Science* 84: 1418-1422.

MANZANILLA, E. G., NOFRARÍAS, M., ANGUITA, M., CASTILLO, M., PEREZ, J. F., MARTÍN-ORÚE, S. M., KAMEL, C. and GASA, J. (2006) Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs. *Journal of Animal Science* 84: 2743-2751.

MAZZONI, M., LE GALL, M., DE FILIPPI, S., MINIERI, L., TREVISI, P., WOLINSKI, J., LALATTA-COSTERBOSA, G., LALLÈS, J.-P., GUILLOTEAU, P. and BOSI, P. (2008) Supplemental sodium butyrate stimulates different gastric cells in weaned pigs. *J Nutr* 138: 1426-1431.
# EFFECT OF ISO-ENERGETIC EXCHANGE OF DIETARY FAT AND STARCH ON THE GROWTH PERFORMANCE AND BODY COMPOSITION OF BROILERS

T. Veldkamp<fnref n="1" />, T. Schamp<fnref n="2" />, J. van Harn<fnref n="1" />, R. Dekker<fnref n="1" />, A.J.M. Jansman<fnref n="1" />

**Corresponding author:** teun.veldkamp@wur.nl

## Abstract

Dietary factors such as the concentrations of protein/amino acids, fat, and carbohydrates and their ratio, may affect the post-absorptive metabolism of energy and protein and energy deposition in the body. In a 2x2 factorial design, the effects of two dietary crude protein (CP; high protein (HP) vs. low protein (LP) concentrations; 200/190 vs. 170/160 g/kg in grower and finisher phase and two dietary fat/starch concentrations; high fat (HF; fat and starch 105 and 340 g/kg, respectively) and low fat (LF; fat and starch 65 and 420 g/kg, respectively) on growth performance and body composition of Ross 308 broilers were studied (9 to 35 d). Concentrations of apparent faecal digestible essential amino acids were similar in HP and LP diets. Feed intake (FI) of broilers fed HP diets tended (P=0.06) to be lower than in broilers fed LP diets. Body weight gain (BWG) of broilers fed HP diets was significantly higher than BWG of broilers fed LP diets (P<0.001) resulting in a significantly lower feed conversion ratio (FCR) of broilers fed HP diets (P<0.001). Exchange of dietary fat by starch resulted in a significantly lower FI and FCR, while BWG was not affected. The FCR was improved by substituting dietary fat by starch, the effect being more pronounced in LP than in HP diets (P=0.012). On d 35, the body fat content of birds fed LF diets was higher than in birds fed the HF diets (P=0.03). It can be concluded that dietary energy source and protein level in iso-energetic diets balanced for first limiting essential amino acids influence growth performance and body composition of broilers.

**Keywords:** broiler chicken, diet, energy, fat, starch, protein

## Introduction

Selection for improvements in body weight gain and breast meat yield affects protein and fat deposition in broilers. Energy deposition is the result of dietary energy intake and the efficiency of utilisation of digested energy for maintenance and deposition of protein and fat in the body. Energy distribution is not only affected by genetic factors but also factors such as climate and nutrition (quantitative feed intake and diet composition). In general, diets with high metabolisable energy or high energy to protein ratio result in a higher fat deposition (Swennen et al., 2007) and diets with amino acids content beyond requirements will result lower fat deposition but with poorer efficiency because elimination of excess nitrogen is an energy-consuming process. Reducing the dietary protein content to sub-optimal levels will result in a higher fat deposition (Buyse et al., 1992). Most studies in literature are focussed on the effect of dietary protein content (apparent faecal digestible amino acids) and less research is conducted on the effect of dietary energy source (carbohydrates and fat) on the post-absorptive metabolism of energy. Literature on effects of iso-energetic exchange of dietary fat and starch in broiler diets on growth performance and body composition is scarce and not consistent. In the present study, the effect of iso-energetic exchange of dietary fat and starch on the growth performance and body composition of broilers was studied.

## Material and Methods

Day-old male and female broiler chicks (Ross 308) were obtained from a commercial hatchery and divided over 72 floor pens with wood shavings (36 floor pens with males and 36 floor pens with females) in an environmentally controlled experimental broiler house. In each floor pen (1.00 x 0.75 m) 14 broilers were placed. Feed and water were provided ad libitum, and temperature was set initially at 34 °C and gradually reduced until 22 °C was reached at 25 days of age. From day 1 to 9, all broilers received a starter diet with 21% CP and 2,840 kcal ME/kg. The four experimental diets were randomly assigned to the floor pens (per gender 9 pens per diet). From 10 days of age, broilers in each pen received one of the four iso-energetic experimental diets. The diets contained the same ingredients, but different concentrations of particular ingredients to realise the experimental treatments. The levels of wheat, wheat middlings, cellulose, soybean meal, potato protein, corn gluten meal, corn starch and soybean oil were adjusted to create contrasts in macronutrient levels and to keep metabolisable energy content of all diets similar. Metabolisable energy content in the

<footnote n="1">¹ Wageningen UR Livestock Research, Wageningen, the Netherlands</footnote>

<footnote n="2">² ForFarmers, Lochem, the Netherlands</footnote>
grower (10-28 d) and finisher (29-35 d) diets was 12.24 MJ/kg. The high-protein (HP) and low-protein (LP) grower diets contained 200 and 170 g crude protein per kg diet, and the HP and LP finisher diets contained 190 and 160 g crude protein per kg diet, respectively. Free amino acids (lysine, methionine, threonine, valine, arginine, isoleucine and tryptophan) were included in the diets to meet the apparent faecal digestible amino acid requirements (CVB, 1996). The high-fat (HF) and low-fat (LF) grower and finisher diets contained per kg 105 and 65 g crude fat and 380 and 460 g starch + sugars, respectively. The same batches of ingredients were used to formulate the four experimental diets. All birds of each pen were weighed at 9, 18, 28 and 35 days of age and feed consumption was recorded per pen to determine feed intake (FI), body weight (BW), body weight gain (BWG) and feed conversion ratio (FCR). After weighing, two birds corresponding to the mean weight of each pen were selected and euthanized. Pooled in one sample, these birds were autoclaved, grinded, subsampled and analysed to determine dry matter (DM), protein, fat and ash concentration in the body.

## Results and Discussion

Results for the growth performance over the period of 0 to 35 days of age are presented in Table 1. Feed intake of the birds fed HP diets tended (P=0.06) to be lower than FI of broilers fed LP diets and FI of birds fed HF diets was significantly higher than FI of birds fed LF diets (101.6 vs. 99.2 g/d; P=0.01). Body weight gain of broilers fed HP diets was significantly higher than BWG of broilers fed LP diets (63.5 vs. 61.5 g; P=0.001). Body weight gain of broilers was not affected by the dietary energy source. Feed conversion ratio of birds fed HP diets was significantly lower than FCR of broilers fed LP diets (1.568 vs. 1.646; P<0.001). Feed conversion ratio of broilers fed HF diets was significantly higher than FCR of broilers fed LF diets (1.625 vs. 1.590; P<0.001). This effect was more pronounced in LP diets than in HP diets (Protein x Energy interaction; P=0.012). Despite inclusion of free essential amino acids in LP diets in order to meet (CVB, 1996) amino acid requirement values, BWG of birds fed LP diets was lower than on HP diets. The concentration of non-essential amino acids in LP diets may have been limiting BWG related to the 30 g/kg lower crude protein content in LP diets compared to HP diets. Body composition at 35 days of age of broilers fed HP diets showed a lower DM content, a higher protein content and a lower fat content compared to broilers fed LP diets. Body DM, ash and fat content in broilers fed HF diets was lower than in broilers fed LF diets. However, differences in fat content were rather small and the lower fat content in broilers fed HF diets was not expected. Body protein content was not affected by dietary energy source. No interaction effect with gender has been observed, neither for growth performance nor for body composition.

## Conclusions

Protein level and dietary energy source in iso-energetic diets balanced for most limiting essential amino acids influence growth performance and body composition of broilers. Lowering dietary crude protein content with 30 g/kg, however, in the grower and finisher period adversely affected growth performance of broilers. Partly substitution of fat by starch and sugars as dietary energy source improved growth performance. Low fat diets (higher in starch + sugars) resulted in a higher body fat content in birds at 35 days. The observed effects are related to differences in the post-absorptive utilisation of amino acids, carbohydrates (glucose and other sugars) and fatty acids and retention in the body.
<table><caption>**Table 1.** Effects of diet composition on the growth performance of broilers over the period of 0 to 35 days of age.</caption><thead><tr><th>Treatment</th><th></th><th>FI<br>(g/d)</th><th>GW<br>(g)</th><th>BWG<br>(g/d)</th><th>FCR</th></tr></thead><tbody><tr><td>HP</td><td></td><td>99.5</td><td>2264<sup>b</sup></td><td>63.5<sup>b</sup></td><td>1.568<sup>a</sup></td></tr><tr><td>LP</td><td></td><td>101.3</td><td>2196<sup>a</sup></td><td>61.5<sup>a</sup></td><td>1.646<sup>b</sup></td></tr><tr><td>HF</td><td></td><td>101.6<sup>b</sup></td><td>2233</td><td>62.6</td><td>1.625<sup>b</sup></td></tr><tr><td>LF</td><td></td><td>99.2<sup>a</sup></td><td>2227</td><td>62.4</td><td>1.590<sup>a</sup></td></tr><tr><td>HP</td><td>HF</td><td>100.6</td><td>2277</td><td>63.9</td><td>1.576<sup>a</sup></td></tr><tr><td>HP</td><td>LF</td><td>98.5</td><td>2251</td><td>63.1</td><td>1.559<sup>a</sup></td></tr><tr><td>LP</td><td>HF</td><td>102.6</td><td>2189</td><td>61.4</td><td>1.673<sup>c</sup></td></tr><tr><td>LP</td><td>LF</td><td>99.9</td><td>2202</td><td>61.7</td><td>1.620<sup>b</sup></td></tr><tr><td colspan="6"><strong>P-values</strong></td></tr><tr><td>Protein</td><td></td><td>0.060</td><td>0.001</td><td>0.001</td><td>&lt;0.001</td></tr><tr><td>Energy</td><td></td><td>0.010</td><td>0.760</td><td>0.760</td><td>&lt;0.001</td></tr><tr><td>Protein x Energy</td><td></td><td>0.757</td><td>0.342</td><td>0.342</td><td>0.012</td></tr></tbody></table>

<table><caption>**Table 2.** Effects of diet composition on the body composition of broilers at 35 days of age.</caption><thead><tr><th>Treatment</th><th></th><th>DM<br>(g/kg)</th><th>Ash<br>(g/kg)</th><th>Protein<br>(g/kg)</th><th>Fat<br>(g/kg)</th></tr></thead><tbody><tr><td>HP</td><td></td><td>323.2<sup>a</sup></td><td>24.2</td><td>180.3<sup>b</sup></td><td>122.3<sup>a</sup></td></tr><tr><td>LP</td><td></td><td>335.7<sup>b</sup></td><td>24.7</td><td>176.7<sup>a</sup></td><td>137.9<sup>b</sup></td></tr><tr><td>HF</td><td></td><td>325.4<sup>a</sup></td><td>24.0<sup>a</sup></td><td>178.8</td><td>127.0<sup>a</sup></td></tr><tr><td>LF</td><td></td><td>333.5<sup>b</sup></td><td>24.9<sup>b</sup></td><td>178.2</td><td>133.2<sup>b</sup></td></tr><tr><td>HP</td><td>HF</td><td>319.4</td><td>23.6</td><td>180.5</td><td>119.5</td></tr><tr><td>HP</td><td>LF</td><td>327.0</td><td>24.9</td><td>180.2</td><td>125.0</td></tr><tr><td>LP</td><td>HF</td><td>331.4</td><td>24.3</td><td>177.2</td><td>134.4</td></tr><tr><td>LP</td><td>LF</td><td>340.0</td><td>25.0</td><td>176.2</td><td>141.3</td></tr><tr><td colspan="6"><strong>P-values</strong></td></tr><tr><td>Protein</td><td></td><td>&lt;0.001</td><td>0.298</td><td>0.002</td><td>&lt;0.001</td></tr><tr><td>Energy</td><td></td><td>0.013</td><td>0.031</td><td>0.552</td><td>0.029</td></tr><tr><td>Protein x Energy</td><td></td><td>0.878</td><td>0.485</td><td>0.748</td><td>0.790</td></tr></tbody></table>

## Literature

BUYSE, J., E. DECUYPERE, L. BERGHMAN, E.R. KÜHN, F. VANDESANDE, 1992: Effect of dietary protein content on episodic growth hormone secretion and on heat production of male broiler chickens. Br. Poult. Sci. 33: 1101-1109.

CVB, 1996: Amino acid requirement of laying hens and broiler chicks. In: Schutte, J.B. (ed.) Dutch Bureau of Livestock Feeding. Report No. 18, Lelystad, the Netherlands.

SWENNEN, Q., E. DECUYPERE, J. BUYSE, 2007: Implications of dietary macronutrients for growth and metabolism in broiler chickens. World's Poult. Sci. J. 63, 541-556.
# STARCH DIGESTION IN XYLANASE SUPPLEMENTED MAIZE-SOY DIETS

S. A. Lee<fnref n="1" />, J. Wiseman<fnref n="2" />, H.V. Masey O'Neill<fnref n="3" />, S. Hill<fnref n="1" />

**Corresponding author:** stxsl11@nottingham.ac.uk

## Abstract

Emerging evidence suggests a beneficial effect of xylanase supplementation in maize-based diets through improved nutrient digestibility. Previous work has revealed that when xylanase is added to a maize diet and fed instantly there is no significant effect on starch digestion in broilers. This would suggest that the amount of time that xylanase is present in the digestive tract is a limiting factor for enzyme efficacy and would support the idea of an indirect mechanism of xylanase action. To test this hypothesis, the current study was designed to investigate the effect of adding xylanase to a maize-soy diet over different time scales. Ross 308 broilers (60) were divided between 6 treatment groups; a control group without xylanase, and groups supplemented with xylanase from 1, 2, 3, 4 or 5 weeks before slaughter. At day 35 and 36, digesta was collected from the gizzard, upper and lower small intestine, and both caeca. Scanning electron microscope (SEM) images revealed starch granules entering the caeca along with fractured plant material for all groups, signifying incomplete starch digestion. Greater cell wall degradation was not evident with xylanase supplementation. However, this may be explained by the *ad libitum* feeding of birds which would lead to the existence of feed particles at all stages of digestion in the tract. Understanding the effect that xylanase supplementation has on starch digestion is essential for improving bird performance while lowering feed costs.

**Keywords:** Xylanase, Maize, Starch, Digestion

## Introduction

For many countries, maize is the predominant source of starch and available energy in broiler diets. Maize-based diets are generally considered to be highly digestible, with a relatively high starch content and low concentrations of anti-nutritional factors, such as non-starch polysaccharides (NSP) when compared to other cereals such as wheat and rye (CHOCT, 1997). However, with increasing demand from the biofuels industry for cereals such as maize to be used for ethanol production, the cost of such feed in recent years has risen. Consequently, considerable attention has been given to improve nutrient utilisation of these diets through the use of exogenous enzymes, including xylanases, to release more energy from the diet. Traditionally, xylanases have been added to more viscosity-promoting diets, such as those based on wheat, to reduce digesta viscosity and improve nutrient digestion. However, there is also a beneficial effect of xylanase inclusion in maize-based diets through improved broiler performance (COWIESON et al., 2010), indicating that additional mechanisms other than reduction in viscosity are important. In previous work, adding xylanase to a maize diet and feeding on a single occasion did not significantly affect starch digestion in broilers. This suggested that the amount of time that xylanase was fed for may be a limiting factor for enzyme efficacy and would support the idea of an indirect mechanism of xylanase action. Therefore, the purpose of this study was to investigate the effect of feeding xylanase-supplemented maize-soy diets over different time periods before slaughter on starch digestion in broilers.

## Materials and methods

Day-old male Ross 308 broiler chicks were supplied from a commercial hatchery and raised on a maize-soy diet (Target Feeds, UK) with or without xylanase inclusion (Econase® XT 25; 100g/t; supplied by ABVista). A total of 60 birds were used in this study with 10 birds per treatment group; birds were group-housed in separate pens for a specific treatment group. Treatment groups (6) included a control group fed a diet without xylanase, and groups fed the diet supplemented with xylanase from 1, 2, 3, 4 or 5 weeks before slaughter. Diets and water were provided *ad libitum* throughout the trial. On days 35 and 36, birds were euthanised by intravenous injection of pentobarbital before dissection was carried out. Digesta was collected from the gizzard, upper and lower small intestine, and both caeca. The morphology of starch granules from the digesta was examined using a scanning electron microscope (SEM; Jeol JSM-6490LV). The total starch assay (Megazyme International Ireland Ltd) was performed for the determination of starch in digesta samples. Digesta samples were washed twice in 80 % ethanol and the precipitate digested with thermostable α-amylase (300 U)

<footnote n="1">¹ Division of Food Sciences, University of Nottingham, Sutton Bonington Campus, LE12 5RD, UK</footnote>

<footnote n="2">² Division of Animal Sciences, University of Nottingham, Sutton Bonington Campus, LE12 5RD, UK</footnote>

<footnote n="3">³ AB Vista Feed Ingredients, Marlborough, SN8 4AN, UK</footnote>
and amyloglucosidase (20 U). Glucose release was measured using a glucose analyser (Analox GM9 Analyser). Both ethanol washes, assumed to contain soluble glucose, were collected and the ethanol evaporated. Glucose concentration in this fraction was measured using the glucose analyser.

<figure><img src="image_20.png" /><figcaption>**Figure 1.** Starch granules in gizzard digesta taken from control birds (a) and birds fed diets supplemented with xylanase 1 (b), 2 (c), 3 (d), 4 (e) and 5 (f) weeks before slaughter.</figcaption></figure>

## Results

Birds were fed *ad libitum* before slaughter and therefore samples from the digestive tract will show feed particles that had been ingested for different periods of time. All gizzard samples showed cell wall fractionation (Fig.1), however greater cell wall destruction with xylanase treatment was not evident. Gizzard digesta from all treatment groups revealed a large number of trapped starch granules. The reasoning may not be solely down to cell wall materials, but also protein matrices that can form a continuous network that encapsulates starch granules, thereby preventing their release.

Total starch results demonstrate the progression of starch granule digestion as digesta passes through the digestive tract of birds (Fig.2). Starch content (Fig.2ia) was greatest in the gizzard, with decreased levels within the upper small intestine and then less again in the digesta of the lower small intestine, as starch becomes hydrolysed by endogenous amylases. As expected, the glucose content (Fig.2ib) of gizzard digesta was minimal as starch digestion does not occur in this tract section. Conversely, a higher glucose content was found in the upper small intestine, as this is the major site of starch digestion. By the lower small intestine, glucose levels fall again as monomers are absorbed into enterocytes. The content of starch and its digestion product, in any tract section, were not significantly affected by treatment (P > 0.05). Total starch results are also supported by SEM imaging (Fig.2ii) where starch granules appear to become more hydrolysed and are fewer in number as digesta enters more distal tract sections. Interestingly, a small number of starch granules escaped digestion and entered the caeca (Fig.2iid) for all treatment groups. Therefore, even when granules are released from their surrounding matrices, some are still resistant to digestion.

## Conclusions

In the current study there was no indication of greater cell wall destruction or starch digestion occurring in birds fed a xylanase supplemented diet. Starch fraction measurements and SEM imaging may not give a full picture of xylanase action due to the *ad libitum* feeding of birds and the complex environment of the digestive tract. Resistant starch granules that enter the caeca confirm the important issue of incomplete starch digestion in broilers. Further investigation is needed for a better understanding of the mechanisms of xylanase action on starch digestion in broilers fed a maize-based diet.

## Literature

* CHOCT, M., 1997: Feed Non-Starch Polysaccharides: Chemical Structures and Nutritional Significance. Feed Milling International. **7**, 13-26.

* COWIESON, A. J., M. R. BEDFORD, V. RAVINDRAN, 2010: Interactions between xylanase and glucanase in maize-soy based diets for broilers. Br. Poult. Sci. **51**, 246-257.
<figure><img src="image_21.png" /><figcaption>**Figure 2.** Progression of starch granule digestion through the tract of broilers. Graphs (i) show starch (a) and glucose (b) content of digesta taken from the gizzard, upper and lower small intestine of birds fed a xylanase-supplemented maize-soy diet (MS) for 0 (control), 1, 2, 3, 4 or 5 weeks before slaughter. SEM images (ii) show starch granule digestion as digesta moves from the gizzard (a) through to the upper (b) and lower (c) small intestine and into the caeca (d).</figcaption></figure>
# POSTER PRESENTATIONS

## Additives

P-001 (ID 94)

### THE EFFICACY OF CERTAIN FEED ADDITIVES FOR THE PREVENTION OF CAMPYLOBACTER JEJUNI INFECTION IN BROILER CHICKENS

Wafaa A. Abd El-Ghany*<fnref n="1" />, M. H. Awaad<fnref n="1" />, and Nagwa, S. R.<fnref n="2" />

### Summary

The efficacy of acidifiers (lactic and formic acids) and probiotics containing *Pediococcus acidilactici* (*P. acidilactici*) and *Saccharomyces boulardii* (*S. boulardii*) in reducing of *Campylobacter jejuni* (*C. jejuni*) infection in broiler chickens was investigated. Three birds out of 103 day old chick were euthanized for *C. jejuni* re-isolation. One hundred were assigned into 4 equal groups each; 25 birds. Groups 1, 2 and 3 were fed on ration containing acidifiers, *P. acidilactici* and *S. boulardii*, respectively; from day old till 5 weeks old. Group 4 was control. At 2 weeks old, each bird was orally challenged with *C. jejuni*. Groups were kept for 3 weeks recording signs and mortalities. Faecal swabs as well as liver and intestine were collected for *C. jejuni* shedding and re-isolation; respectively. Post-mortem lesions scoring of *C. jejuni* were calculated. Results cleared more severe signs of greenish diarrhoea in control than treated birds with no mortalities in all groups. Significant (p<0.05) reduction of *C. jejuni* shedding and re-isolation rates as well as lesions scoring was seen in groups treated with *S. boulardii* and *P. acidilactici* followed by acidifiers over control group. In conclusion, both acidifiers and probiotic preparations greatly reduced *C. jejuni* infection in broiler chickens.

**Keywords:** *C. jejuni*, Acidifiers, Probiotics, Chickens, Protection

### Introduction

*Campylobacter jejuni* (*C. jejuni*) infection is considered as one of the most important bacterial disease causing acute human enteric infection in developing countries (SHANE, 2000). Natural alternatives are widely used nowadays to control enteric infections in poultry with promising results (MOUNTZOURIS et al., 2010). Using of different organic acids to reduce *C. jejuni* intestinal colonization was studied successfully (SANTOS et al., 2008). Competitive exclusion compounds including probiotics are used effectively to protect chicks from intestinal pathogens colonization like *C. jejuni* (WILLIS and REID, 2008).

The aim of this study was to investigate the efficacy of acidifiers (lactic and formic acids) and probiotic preparations containing *Pediococcus acidilactici* (*P. acidilactici*) and *Saccharomyces boulardii* (*S. boulardii*) in reducing of *C. jejuni* infection in broiler chickens.

### Materials and Methods

Three out of 103 day old, meat type chicks were euthanized to prove their freedom of *C. jejuni* infection. One hundred chicks were assigned into 4 groups each; 25 birds. Chicks of groups 1, 2 and 3 were fed on ration containing lactic and formic acids (1 kg/ton), *P. acidilactici* (100 g/ton) and *S. boulardii* (100 g/ton); respectively; from day old till 5 weeks old. Chicks of group 4 were kept as control. All groups were orally challenged with 0.5 ml containing 5X10⁵ *C. jejuni* bird at 2 weeks old. Birds were kept for 3 weeks for signs and mortalities. Faecal swabs and liver and intestine were collected from all groups at different intervals for *C. jejuni* shedding and re-isolation; respectively. At 5 weeks old, a lesion scoring of *C. jejuni* after RABIE et al. (1998) was recorded. Microbiological examination of the organisms was done according to KONEMAN et al. (1995). Data were statistically analyzed and means were compared by one way ANOVA (p<0.05) using Post Hoc test according to SNEDECOR and COCHRAN (1980).

<footnote n="1">¹Faculty of Veterinary Medicine, Cairo University, Egypt</footnote>

<footnote n="2">²National Research Centre, Egypt</footnote>

<footnote>*Corresponding author: wafaa.ghany@yahoo.com</footnote>
## Results and Discussion

The results revealed no mortalities in all groups, but the severity of signs (depression and greenish diarrhae) was lower in treated than control birds. Figure (1) showed that the mean organ lesion score at 7, 14 and 21 days post infection was higher in acidifiers (2.04) than in *P. acidilactici* (1.40) and in *S. boulardii* (1.24) treated birds. Nevertheless, it was (3.04) in control group. Faecal swabs revealed presence of highly motile organisms. The rate of *C. jejuni* shedding decreased from 100 to 6.7% in acidifiers-treated group, from 100 to 0% in both *P. acidilactici* and *S. boulardii* treated birds, versus from 100 to 86.6% in control one (table 1). The rates of *C. jejuni* re-isolation showed descending patterns, viz. from 60 to 20% in acidifiers, from 80 to 20% in *P. acidilactici* treated groups, whereas it was 100% along 21 days post challenge in control group (figure 2).

Acidifiers and probiotics gave nearly similar degree of protection rate for *C. jejuni* infection in broilers (MOUNTZOURIS et al., 2010). PUCCI et al. (1988) mentioned that *P. acidilactici* can antagonize micoeaerophilic *C. jejuni* in their niches of intestine in which low dissolved oxygen tension exists. On the other hand, organic acids have a strong bactericidal effect on Campylobacter spp. (NASERI et al., 2012). EKLUND (1983) concluded that the un-dissociated acids diffused into the bacterial cell wall and released a proton that acidifies the cytoplasm. This result was supported by KROLL and PATCHETT (1991) and added that the cell death resulted from invisible denaturation of enzyme activity or DNA synthesis. Also, CHAVEERACH et al. (2004) showed that organic acids reduce the pH of crop to 3.8-4 and thus reduce the level of Campylobacter in the crop and the intestinal tract. However, treatments with viable probiotic bacterial cultures were effective in reducing *C. jejuni* in chickens due to bacteriocin (STERN et al., 2008). BLEHANT et al. (1989) explained the advantages of using yeast for combating enteropathogens as follows; yeast can grow at 37C, therefore be able to withstand the higher chickens body temperature, it is able to survive in low pH of the proventriculus and gizzard to reach the intestine and caeca where it can survive either aerobically and anaerobically and finally yeast is easily to be administered. Presence of mannose of *S. boulardii* on the intestinal cell wall may cause the yeast to act as a decoy for the attachment of pathogens (MILES, 1993), however RODRIGUES et al. (1996) attributed the protection to the ability of yeast to reduce the available amount of the produced toxins.

## Conclusion

Both acidifiers and probiotic preparations could greatly assist in reduction and elimination of *C. jejuni* infection risk in broiler chickens.

<figure><img src="image_22.png" /><figcaption>**Figure (1):** Post-mortem lesion scoring of *C. jejuni* in acidifiers and probiotic treated and untreated broiler chickens</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>**Figure (2):** Re-isolation of *C. jejuni* in acidifiers and probiotic treated and untreated broiler chickens</figcaption></figure>
<table><caption>**Table (1)** Shedding of *C. jejuni* in acidifiers and probiotic treated and untreated broiler chickens</caption><thead><tr><th rowspan="3">Day post<br>infection</th><th colspan="12">Treatment</th></tr><tr><th colspan="3">Acidifiers</th><th colspan="3">P. acidilactici</th><th colspan="3">S. boulardii</th><th colspan="3">Untreated control</th></tr><tr><th>No. of<br>birds</th><th>+ (%)</th><th>-</th><th>No. of<br>birds</th><th>+ (%)</th><th>-</th><th>No. of<br>birds</th><th>+ (%)</th><th>-</th><th>No. of<br>birds</th><th>+ (%)</th><th>-</th></tr></thead><tbody><tr><td>3</td><td>25</td><td>25<br>(100)</td><td>0</td><td>25</td><td>25<br>(100)</td><td>0</td><td>25</td><td>25<br>(100)</td><td>0</td><td>25</td><td>25<br>(100)</td><td>0</td></tr><tr><td>7</td><td>25</td><td>16*<br>(64)</td><td>9</td><td>25</td><td>11* (44)</td><td>14</td><td>25</td><td>13*<br>(52)</td><td>12</td><td>25</td><td>23 (92)</td><td>2</td></tr><tr><td>11</td><td>20</td><td>12*<br>(60)</td><td>8</td><td>20</td><td>13*<br>(13.3)</td><td>7</td><td>20</td><td>11*<br>(55)</td><td>9</td><td>20</td><td>18 (90)</td><td>2</td></tr><tr><td>15</td><td>15</td><td>3* (20)</td><td>12</td><td>15</td><td>2*<br>(13.3)</td><td>3</td><td>15</td><td>4*<br>(26.6)</td><td>11</td><td>15</td><td>14<br>(93.3)</td><td>1</td></tr><tr><td>18</td><td>15</td><td>3* (20)</td><td>12</td><td>15</td><td>1* (6.7)</td><td>14</td><td>15</td><td>0* (0.0)</td><td>15</td><td>15</td><td>12 (80)</td><td>3</td></tr><tr><td>21</td><td>15</td><td>1*<br>(6.7)</td><td>14</td><td>15</td><td>0* (0.0)</td><td>15</td><td>15</td><td>0* (0.0)</td><td>5</td><td>15</td><td>13<br>(86.6)</td><td>2</td></tr></tbody></table>

## Literature

BLEHANT, M.J., J. MASSOT, G.W. ELMER, R.H. LEVY, 1989: Disposition kinetics of *Saccharomyces boulardii* in man and rat. Biopharm. Drug Disposition, **10**, 353-364.

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

EKLUND, T. 1983: The antimicrobial effect of dissociated and undissociated sorbic acid at different pH levels. Appl. Bacteriol. **54**, 383-389.

KONEMAN, E.W, S.D. ALLEN, W.M. JANDA, W.C. WINN, 1995: Introduction to Diagnostic Microbiology. 5th ed., Lippincott Company, Philadelphia, (USA). PP. 365-398.

KROLL, R.G., R.A. PATCHETT, 1991: Biocide-induced perturbations of aspects of cells homeostasis: intracellular pH, membrane potential and solute transport. In: mechanisms of actions of chemical biocide. Pp: 189-202. S. P. Denyer and W. B. Hugal (eds). Blackwell, Oxford, England.

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<footnote>*Significant decrease over their untreated control group (p<0.05).</footnote>

<footnote>+= Positive</footnote>

<footnote>-= Negative</footnote>
# EFFECT OF SODIUM BENTONITE AND SEEDS OF SILYBUM MARIANUM ON LIVER ENZYMES, LYMPH ORGANS AND IMMUNE SYSTEM OF BROILER CHICKENS FED WITH AFLATOXIN

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

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

## Abstract

Aflatoxins (B1, B2, G1 and G2) are toxic groups of mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus. Hepatotoxicity, weakening the immune system and mutagenic are the major effects of Aflatoxins in different animal species. In this study the effect of different levels of dietary sodium bentonite (SB), *Silybum Marianum* seeds (SMS) and Aflatoxin B1 (AFB) on liver enzymes, lymph organs and immune system of broiler at the end of 42 days was studied. 224 Ross 308 broiler chicks in a completely randomized design based on factorial experiment (2 x 2 x 2) with 2 levels AFB (0and 500ppb), 2 levels SMS (0 and 0.5%) and 2 levels SB (0 and 0.5%) with 8 treatments, 4 replicates and 7 chickens per each. The experimental diets were fed from the first day. The results showed that compared with the control group, diets containing AFB significantly increased AST (Aspartate Aminotransferase) and ALT (Alanine Aminotransferase) however significantly decreased levels of immunoglobulin G (p≤0.05). SB with AFB causes a significant decrease in serum ALT levels and increase immunoglobulin G significantly (p≤0.05). Combination of SB, SMS and AFB decreased significantly serum AST compare to use each of them alone (p≤0.05). The results showed that the combination of SB and the SMS are more beneficial as separate application of them for deceased toxic effect of Aflatoxin B₁ in diets.

**Keywords:** Aflatoxin B1, Broiler, Immune system, Liver enzymes, Lymph

## Introduction

Mycotoxins are metabolites from moulds. Aflatoxins, a group of closely related biologically active mycotoxins, are produced by strains of *Aspergillus flavus* and *Aspergillus parasiticus* (sujatha et al., 2003). Fungi or mould growth in feedstuff is associated with the utilization of nutrients from the host. Consequently, alterations in the nutritional content of the feedstuff are expected. The extent of mould growth determines the degree of depletion in the nutrient content of the feedstuff. The germ of the grain is the main site for *Aspergillus* sp. development, which leads to a greater potential for the synthesis of AFB (brekke et al., 1975). Layered alumino silicates, such as sodium bentonite, have been found effective in counteracting mycotoxins (eraslan et al., 2005). The effects of *silymarin* or various products made from the seed of the plant *Silybum Marianum*. containing silymarin have also been tested on farm animals. The hepatoprotective activities were assessed on the basis of selected biochemical blood indicators in chicken broilers that had been exposed to the effects of aflatoxins (sujatha et al., 2003). The purpose of this study was to evaluate the changes of liver enzymes, lymph organ and immune system of broiler chickens when different levels of sodium bentonite and *Silybum Marianum* seeds were used as a feed additive in their diet.

## Material and Methods

Two hundred and twenty four of day old male Ross 308 broiler chickens obtained from a local hatchery were randomly distributed among eight treatments with four replicate groups per treatment and 7 chickens per replicate. the nutritional requirement of the chicks was based on Ross 308 company for starter (0-10 days) and grower (11-24 days) diets. Birds were distributed into the following eight treatments: 1) control 2)diet with (%) 500ppb aflatoxin B₁, 3) diet with 500ppb Aflatoxin B₁ and 0.5% *Silybummarinum* seeds, 4) diet with 500ppb aflatoxin B₁ and 0.5% Sodium Bentonite, 5)diet with 500ppb Aflatoxin B₁ and 0.5% *Silybummarinum* seeds and 0.5% Sodium Bentonite, 6)diet with 0.5% *Silybummarinum* seeds, 7) diet with 0.5% Sodium Bentonite, 8) diet with 0.5% *Silybummarinum* seeds and 0.5% Sodium Bentonite. At 42 day of age, two birds per pen were randomly selected and 2 mL of blood samples were obtained by wing puncture and centrifuged at 3000 rpm for 20 min to separate serum. Aspartate aminotransferase(AST), alanine aminotransferase

<footnote n="1">¹ Professor of Poultry Nutrition, Department of Animal Science, University of Birjand, Iran</footnote>

<footnote n="2">² MSc of management and poultry production, Department of Animal Science, university of Birjand, Iran</footnote>

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

<footnote n="4">⁴ Assistant professor of immunology, Department of Animal Science, University of Birjand, Iran</footnote>
(ALT), Imnoglubulin G and M and relative organ weight (bursa and spleen) were analyzed using the general linear model procedure of SAS.

## Results

The effect of experimental treatments on different parameters in Table 1 showed that compared with the control group, diets containing AFB significantly increased AST and ALT however significantly decreased levels of immunoglobulin G (p≤0.05). The increased serum AST and ALT activities observed by feeding naturally contaminated diets in the present study could be due to hepatic degeneration and subsequent leakage of enzymes into circulation (afzali and devegowda., 1999). SB with AFB causes a significant decrease in serum ALT levels and increase immunoglobulin G significantly (p≤0.05). Aflatoxin effects on humoral antibody levels compared to non-specific effects on the material seen below. However, interruption or reduction in antibody titer test has been reported against and reduce the amount of specific immunoglobulins (IgA and IgG) have been reported (campbell., 1983). These results are parallel with the findings reported (16), who observed a decrease in these parameters and an inhibition in protein synthesis during the aflatoxicosis in poultry. All treatment not significantly effects on relative weight of bursa and spleen. Combination of SB, SMS and AFB decreased significantly serum AST compare to use each of them alone (p≤0.05).

## Conclusions

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

## References

AFZALI,N. and DEVEGOWDA,G., 1999. Ability of modified manna oligosaccharide to counteract aflatoxicosis in broiler breeder hens. Poult. Sci, 78: 228.

BREKKE, O.L., PEPLISKI, A.J., Nelson, G.E.N., Griffin, E.L.: Pilotplant dry milling of corn containing aflatoxin. Cereal Chem., 1975; 52: 205-211.

3. CAMPBELL. M.L.Jr., J.D. May, W.E. Huff, and J.A. Doerr, 1983. Evaluation of immunity of young broilers chickens during simultaneous aflatoxicosis and ochratoxicosis. Poult. Sci. 62: 2183-2144.

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

5. SUJATHA, K., MATHURAM LN, SRIRAM P 2003: Hepatoprotective effect of silymarin in experimentally induced aflatoxicosis in broilers. Toxicology., 10: 55-59.
<table><caption>Table 1. effect of different treatment on liver enzymes (U/l), lymph organ (gr/kg live weight) and imnuglubolin G and M (mg/dl)</caption><thead><tr><th></th><th></th><th></th><th>AST</th><th>ALT</th><th>IGG</th><th>IGM</th><th>Bursa</th><th>Spleen</th></tr></thead><tbody><tr><td rowspan="8">Main effects</td><td>Aflatoxin (ppm)</td><td>0</td><td>179.31<sup>b</sup></td><td>6.68<sup>b</sup></td><td>1.06<sup>b</sup></td><td>2.18</td><td>0.15</td><td>0.13</td></tr><tr><td></td><td>500</td><td>208.90<sup>a</sup></td><td>8.87<sup>a</sup></td><td>1.75<sup>a</sup></td><td>1.56</td><td>0.14</td><td>0.13</td></tr><tr><td>p-value</td><td></td><td>0.00</td><td>0.00</td><td>0.02</td><td>0.05</td><td>0.09</td><td>0.53</td></tr><tr><td>Silybum (%)</td><td>0</td><td>196.18</td><td>8.25<sup>a</sup></td><td>1.56</td><td>2.18</td><td>0.15</td><td>0.13</td></tr><tr><td></td><td>0.5</td><td>192.03</td><td>7.31<sup>b</sup></td><td>1.25</td><td>1.56</td><td>0.15</td><td>0.12</td></tr><tr><td>p-value</td><td></td><td>0.25</td><td>0.03</td><td>0.28</td><td>0.05</td><td>0.77</td><td>0.42</td></tr><tr><td>Bentonite (%)</td><td>0</td><td>206.58<sup>a</sup></td><td>8</td><td>1.06<sup>b</sup></td><td>2</td><td>0.15</td><td>0.12</td></tr><tr><td></td><td>0.5</td><td>181.63<sup>b</sup></td><td>7.56</td><td>1.75<sup>b</sup></td><td>1.75</td><td>0.15</td><td>0.14</td></tr><tr><td></td><td>p-value</td><td></td><td>0.00</td><td>0.31</td><td>0.02</td><td>0.42</td><td>0.87</td><td>0.07</td></tr><tr><td></td><td>SEM</td><td></td><td>2.49</td><td>0.29</td><td>0.20</td><td>0.21</td><td>0.00</td><td>0.00</td></tr><tr><td rowspan="12">Dual interaction effects</td><td>Aflatoxin * silybum</td><td>0</td><td>178.50</td><td>6.62<sup>b</sup></td><td>1.37</td><td>2.75</td><td>0.15</td><td>0.13</td></tr><tr><td></td><td>0.5</td><td>180.13</td><td>6.75<sup>b</sup></td><td>0.75</td><td>1.62</td><td>0.16</td><td>0.13</td></tr><tr><td></td><td>500</td><td>213.86</td><td>9.87<sup>a</sup></td><td>1.75</td><td>1.62</td><td>0.14</td><td>0.13</td></tr><tr><td></td><td>500</td><td>203.93</td><td>8.87<sup>b</sup></td><td>1.75</td><td>1.50</td><td>0.14</td><td>0.12</td></tr><tr><td>p-value</td><td></td><td>0.11</td><td>0.01</td><td>0.28</td><td>0.12</td><td>0.77</td><td>0.23</td></tr><tr><td>Aflatoxin * ben</td><td>0</td><td>175.58<sup>b</sup></td><td>6.37<sup>c</sup></td><td>1.12<sup>b</sup></td><td>2.25</td><td>0.16</td><td>0.12</td></tr><tr><td></td><td>0.5</td><td>183.03<sup>b</sup></td><td>7<sup>bc</sup></td><td>1<sup>b</sup></td><td>2.12</td><td>0.15</td><td>0.14</td></tr><tr><td></td><td>500</td><td>237.58<sup>a</sup></td><td>9.62<sup>a</sup></td><td>1<sup>b</sup></td><td>1.75</td><td>0.14</td><td>0.12</td></tr><tr><td></td><td>500</td><td>180.21<sup>b</sup></td><td>8.12<sup>ab</sup></td><td>2.58<sup>a</sup></td><td>1.37</td><td>0.14</td><td>0.13</td></tr><tr><td>p-value</td><td></td><td>0.00</td><td>0.01</td><td>0.00</td><td>0.69</td><td>0.66</td><td>0.74</td></tr><tr><td>Silybum * ben</td><td>0</td><td>215.72<sup>a</sup></td><td>8.37</td><td>1.12</td><td>2.25</td><td>0.14</td><td>0.12</td></tr><tr><td></td><td>0.5</td><td>176.63<sup>c</sup></td><td>8.12</td><td>2</td><td>2.12</td><td>0.15</td><td>0.14</td></tr><tr><td></td><td>500</td><td>197.45<sup>b</sup></td><td>7.62</td><td>1<sup>b</sup></td><td>1.75</td><td>0.15</td><td>0.12</td></tr><tr><td></td><td>500</td><td>186.62<sup>bc</sup></td><td>7</td><td>1.50</td><td>1.37</td><td>0.14</td><td>0.13</td></tr><tr><td></td><td>p-value</td><td></td><td>0.00</td><td>0.66</td><td>0.51</td><td>0.69</td><td>0.11</td><td>0.89</td></tr><tr><td></td><td>SEM</td><td></td><td>3.52</td><td>0.42</td><td>0.28</td><td>0.31</td><td>0.00</td><td>0.00</td></tr><tr><td rowspan="12">Treatments</td><td>AFB * silybum * Ben</td><td>0</td><td>177.12<sup>c</sup></td><td>6.25</td><td>1.75<sup>ab</sup></td><td>2.50</td><td>0.14</td><td>0/12</td></tr><tr><td></td><td>0</td><td>179.87<sup>c</sup></td><td>7</td><td>1<sup>b</sup></td><td>3</td><td>0.17</td><td>0.14</td></tr><tr><td></td><td>0.5</td><td>174.5<sup>c</sup></td><td>6.5</td><td>0.50<sup>b</sup></td><td>2</td><td>0.17</td><td>0.12</td></tr><tr><td></td><td>0.5</td><td>186.22<sup>c</sup></td><td>7</td><td>1<sup>b</sup></td><td>1.25</td><td>0.14</td><td>0.14</td></tr><tr><td></td><td>500</td><td>254.32<sup>a</sup></td><td>10.50</td><td>0.50<sup>b</sup></td><td>2</td><td>0.15</td><td>0.13</td></tr><tr><td></td><td>500</td><td>173.40<sup>c</sup></td><td>9.25</td><td>3<sup>a</sup></td><td>1.25</td><td>0.14</td><td>0.14</td></tr><tr><td></td><td>500</td><td>220.85<sup>b</sup></td><td>8.75</td><td>1.5<sup>ab</sup></td><td>1.50</td><td>0.14</td><td>0.11</td></tr><tr><td></td><td>0.5</td><td>187.02<sup>c</sup></td><td>7</td><td>2<sup>ab</sup></td><td>1.50</td><td>0.15</td><td>0.12</td></tr><tr><td>p-value</td><td></td><td>0.01</td><td>0.88</td><td>0.00</td><td>0.12</td><td>0.05</td><td>0.99</td></tr><tr><td>SEM</td><td></td><td>4.98</td><td>0.59</td><td>0.40</td><td>0.43</td><td>0.00</td><td>0.01</td></tr></tbody></table>

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

SEM= Standard error of means. AFB= Aflatoxin B1. Ben= Bentonite.
P-008 (ID 146)

COMPARATIVE EFFICIENCY OF TWO MARIGOLD-BASED YELLOW PIGMENTS ON EGGS TARGETING PASTA INDUSTRY

M. Blanch¹, W. Kong², T. Hampton², J. Valle¹

¹ Novus Spain SA, Reus, Spain

² Novus International Inc., St. Charles, MO, USA

**Corresponding author:** marta.blanch@novusint.com

**Abstract**

This study compared the pigmenting efficiency of 2 liquid marigold-based pigments (XAMACOL® 15-X15 and XAMACOL® Max-XM obtained through a new technology developed by Novus Int. Inc.) on eggs intended for the pasta industry. A total of 194 Hyline W-36 laying hens aged 55 weeks and fed a white basal diet were used. Trial included 5 weeks of xanthophyll depletion (days -35 to -1) and 4 weeks of experimental diets (days 0 to 28). Two products were tested: X15 (15 mg total xanthophylls-TX/g product) and XM at 15 and 12 mg TX/g product (XM15 and XM12). There were 10 treatments: X15 and XM15 at 4 supplementation levels (20, 40, 60, and 80 mg TX/kg feed), XM12 at 48 mg TX/kg feed, and a negative control (no yellow pigment added). All treatments included 2 mg/kg feed of red xanthophylls from paprika. Measurements: egg yolk color (Minolta CR400 and Yolk Color Fan-YCF) for all eggs laid on days 21-24, and β-carotene equivalents in whole liquid eggs laid on days 25-28. XM15 showed significantly higher pigmentation (redness, ratio redness/yellowness, and YCF) in the egg yolk and higher β-carotene equivalents content in whole liquid egg compared to X15. No differences were observed between 400 g product/100 kg feed of X15 vs XM12 in any of the variables measured, although the concentration of xanthophylls was 20% lower when using XM12 than X15 (48 vs 60 mg TX/kg feed). XM technology showed higher pigmenting efficiency compared to X15; therefore lower levels of XM can be used to reach equivalent results.

**Keywords:** carotenoids, layers, pigments, yellow xanthophylls, egg yolk

**Introduction**

Highly colored egg yolks are required for the food processing industry in applications such as pasta production. Carotenoids are compounds responsible for pigmentation in poultry, but only the fat soluble carotenoids (xanthophylls) have this property (Hencken, 1992). Poultry cannot synthesize these compounds and therefore must obtain them from the diets. To achieve optimal yolk coloration, laying hen diets should be supplemented with yellow and red xanthophylls (Belyavin and Marangos, 1987). The main yellow pigments used in poultry feed are marigold extracts and β-apo-8'-carotenoic acid ethyl ester (apo-ester), which are derived from natural and synthetic origin, respectively (Sirri et al., 2007). In recent years, there has been a notable trend toward the use of natural, rather than synthetic, supplements in animal nutrition (Galobart et al., 2004). XAMACOL® 15 (X15) is a traditional, liquid marigold-based pigment (Novus Int. Inc.), and XAMACOL® Max (XM) is a new liquid marigold-based product obtained through a new technology. This study compared the pigmenting efficiency of 2 liquid marigold-based pigments (X15 and XM) on eggs for pasta production.

**Material and Methods**

This study was conducted at Green Acres Research facility (Novus Int Inc., MO, USA). A total of 194 Hyline W-36 laying hens aged 55 weeks were kept individually in battery cages in an environmentally controlled room. The trial lasted 9 weeks with 5 weeks of xanthophyll depletion (day -35 to day -1) during which the animals received a common basal diet low in xanthophylls (based on wheat and soybean meal), and 4 weeks of feeding the experimental diets (day 0 to day 28). All rations (provided in mash form) were designed to meet or exceed all dietary recommendations for W-36 (based on Hy-line guide 2009-11).

Two products were tested: X15 (15 mg total xanthophylls-TX/g product) and XM at 2 different concentrations of active ingredients (15 and 12 mg TX/g product, XM15 and XM12, respectively). Treatments arose from the addition of the test product to the basal diet at the corresponding dosages. The 10 treatments included: X15 and XM15 at 4 supplementation levels (133, 267, 400, and 533 g product/100 kg feed, corresponding to 20, 40, 60, and 80 mg TX/kg), XM12 at a single dose (400 g product/100 kg feed, corresponding to 48 mg TX/kg), and a negative control (no yellow pigment
added). Each treatment included 2 mg/kg of red xanthophylls from paprika. Each treatment had 19-21 replicates of 1
hen except the negative control, which had 14 replicates.

General performance data were recorded (BW d0 and d29, FI d14 and d29, egg weight d0 and d21-28, egg production daily). Egg yolk color was assessed by a Minolta CR-400 colorimeter (CIELab scale {illuminant D65, 2º standard observer}) and YCF (using Egg Analyzer®, ORKA Food Technology, Israel) for all eggs laid during days 21-24. β-carotene equivalents in whole liquid eggs were measured in eggs laid during days 25-28.

All data were statistically analyzed using the appropriate procedures of SAS software package (version 9.2, SAS Institute Inc., Cary, NC, USA). Data were previously explored to discard any possible outlier. Differences were declared at P<0.05.

## Results

No differences in performance were detected among treatments, which were considered normal for the conditions of this experiment (BWd0 = 1.53 ± 0.02 kg, BW d29 = 1.57 ± 0.02 kg, ADFI = 96.8 ± 0.56 g, egg weight = 65.1 ± 0.26 g/d, laying rate = 81 ± 0.7 %, FCR = 1.49 ± 0.01 g/g, mean ± SE, respectively). Because there were no differences in feed intake, differences in pigmentation are considered to be treatment related.

A treatment effect was observed in all egg yolk color variables and β-carotene equivalents when 10 treatments were compared. All treatments showed higher values compared to negative control, expect for luminosity (CIE L*) that was lower (data not shown).

Results from the factorial analyses (2 products: X15, XM15; 4 levels: 20, 40, 60, 80 ppm) showed higher pigmenting efficiency of XM15 compared to X15 (Figure 1). XM15 presented lower luminosity (CIE L*), and higher values in redness (CIE a*), redness/yellowness ratio (CIE a*/CIE b*), YCF and β-carotene equivalents compared to X15. No changes in yellowness (CIE b*) were observed among the two products at any dose.

An additional analysis was performed including only the three treatments with an inclusion level of 400 g product/100 kg feed (including X15, XM15, and XM12); these results are presented in Table 1. XM15 showed higher values in redness (CIE a*), redness/yellowness ratio (CIE a*/CIE b*), YCF and β-carotene equivalents compared to both X15 and XM12. XM12 demonstrated equivalent performance to X15 in the variables measured, despite having a 20% lower concentration of xanthophylls compared to X15 (48 vs 60 mg TX/kg feed).

## Conclusions

XM technology showed higher pigmenting efficiency compared to X15; therefore, lower levels of active ingredients from XM can be used to reach equivalent results.

## Literature

BELYAVIN, C. G., A. G. MARANGOS, 1987: Natural products for egg yolk pigmentation. In: W. HARESIGN D. J. A. COLE: Recent Advances in Animal Nutrition 1987. Butterworths, London, 47-68.

GALOBART, J., R. SALA, X. RICON-CARRUYO, E. G. MANZANILLA, B. VILA, J. GASA, 2004: Egg yolk color as affected by saponification of different natural pigmenting sources. J. Appl. Poult. Res. **13**, 328-334.

HENCKEN, H., 1992: Chemical and physiological behavior of feed carotenoids and their effects on pigmentation. Poult. Sci. **71**, 711-717.

SIRRI, F., IAFFALDANO, N., MINELLI, G., MELUZZI, A., ROSATO, M.P., A. FRANCHINI, 2007: Comparative pigmentation efficiency of high dietary levels of apo-ester and marigold extract on quality traits of whole liquid egg of two strains of laying hens. J. Appl. Poult. Res. **16**, 429-437.
<figure><img src="image_24.png" /><figcaption>**Figure 1.** Effect of X15 and XM15 on egg yolk color (CIE-Lab system and yolk color fan scores) and on β-carotene equivalents in whole liquid egg.</figcaption></figure>

<table><caption>**Table 1.** Effect of X15, XM15, and XM12 at 400 g product/100 kg feed on egg yolk color (CIE-Lab system and yolk color fan scores) and on β-carotene equivalents in whole liquid egg.</caption><thead><tr><th>Yellow xanthophylls</th><th>X15<br>(60 ppm)</th><th>XM15<br>(60 ppm)</th><th>XM12<br>(48 ppm)</th><th>SEM</th><th>P-value</th></tr></thead><tbody><tr><td>CIE L*</td><td>48.6</td><td>47.9</td><td>48.3</td><td>0.32</td><td>0.2793</td></tr><tr><td>CIE a*</td><td>4.37 b</td><td>5.46 a</td><td>4.64 b</td><td>0.145</td><td>0.0001</td></tr><tr><td>CIE b*</td><td>44.4</td><td>44.4</td><td>43.6</td><td>0.53</td><td>0.4907</td></tr><tr><td>CIE a*/CIE b*</td><td>0.099 b</td><td>0.124 a</td><td>0.108 b</td><td>0.0039</td><td>0.0001</td></tr><tr><td>YCF</td><td>9.62 b</td><td>10.19 a</td><td>9.63 b</td><td>0.087</td><td>0.0001</td></tr><tr><td>β-carotene (ppm)</td><td>34.0 b</td><td>37.5 a</td><td>34.6 b</td><td>0.49</td><td>0.0001</td></tr></tbody></table>
P-010 (ID 277)

# EFFECTS OF FEEDING ORIGINAL XPC ON SALMONELLA TYPHIMURIUM OR S. HEIDELBERG PREVALENCE AND LOAD IN BROILER CHICKENS

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

Diamond V, Cedar Rapids, Iowa, USA 52404

**Corresponding author:** jbroomhead@diamondv.com

## Abstract

Two *Salmonella* challenge studies were conducted in Cobb chicks and tested feeding XPC, a *Saccharomyces cerevisiae* fermentation product. Both were grown in floor pens; 11 pens with 25 chicks each (n=550; Study 1) or 8 pens with 50 chicks each (n=1200; Study 2). Two treatments were fed in Study 1: 1) Control or 2) 1.25 kg XPC/tonne from 0-21d and 0.62 kg XPC/tonne from 22-42d. In Study 2, the same two treatments were fed, in addition to a third: 1.25 kg XPC/tonne from 0-42d. All Birds in Study 1 were orally gavaged with a nalidixic acid resistant (NAR) strain of S. Typhimurium (10⁹ CFU) at 7d. In Study 2, one-half of birds in each pen (n=25) were tagged and orally gavaged with NAR S. Heidelberg (10⁹ CFU) at 0d. In Study 2, the 25 untagged birds in each pen were non-challenged and used to assess horizontal transmission (HT). In Study 1, fecal samples were collected at 14d and cecal samples collected at 35d from 3 birds per pen. In Study 2, cecal samples were collected from 10 birds per pen at 42d (5 tagged and 5 HT). Fecal and cecal samples were analyzed for *Salmonella*. FCR was statistically improved (*P*<0.05) in chickens fed XPC. *Salmonella* load was reduced (*P*<0.05) in feces of XPC fed birds in Study 1 (2.14 vs. 2.96 log₁₀ CFU/g in XPC vs. Control, respectively). Cecal *Salmonella* prevalence (% positive) was reduced in birds fed 1.25 kg XPC/tonne (0-42d; *P*<0.05) in Study 2. Based on these results, XPC-fed chickens had improved FCR and lower prevalence and numbers of *Salmonella*.

**Keywords:** Salmonella Typhimurium, Salmonella Heidelberg, Food Safety, Chickens

## Introduction

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

## Materials and Methods

Two independent studies were recently conducted, in which broilers were orally gavaged with approximately 10⁹ CFU of nalidixic acid resistant strains of *Salmonella* Typhimurium (Experiment 1) and *Salmonella* Heidelberg (Experiment 2). Experiments 1 and 2 were conducted by Colorado Quality Research in Wellington, CO and Southern Poultry Research Group near Nicholson, GA, respectively.

### Experiment 1: Salmonella Typhimurium Trial

Cobb chicks (n = 550) were assigned to one of 2 feed treatments (Control vs. XPC), with 11 replicate floor pens per treatment and 25 birds per pen. Control birds received industry standard diets with salinomycin but no antibiotic growth promoter. The XPC treatment received the same rations with the addition of Original XPC at 1.25 kg/tonne in the starter feed and 0.62 kg/tonne in the grower and finisher feeds. All birds and feed were weighed by pen at 0, 14, and 35 d and feed conversion calculated.

At 7 d, the number of birds per pen was adjusted to 23 and then all birds were challenged with S. Typhimurium. At 14 d, fecal samples were collected from three birds per pen. At 35 d, three birds per pen were euthanized and the ceca removed. Feces and cecal contents were diluted with 0.1% peptone and either direct plated or enriched in tetrathionate
broth with brilliant green and potassium iodide and plated on MacConkey Agar containing nalidixic acid to enumerate S. Typhimurium.

## Experiment 2: Salmonella Heidelberg Trial

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

At 0 d, 25-tagged birds per pen were challenged with S. Heidelberg. The remaining 25 birds per pen did not receive a tag or S. Heidelberg challenge, and were used to induce horizontal transmission (contact challenge between S. Heidelberg gavaged and non-gavaged birds). Ten birds per pen (5 tagged and 5 non-tagged) were sampled at 42 d and the ceca removed for S. Heidelberg colonization evaluation. Drag swabs (dragging sterile gauze swabs soaked in double strength skim milk) were taken from each pen (feces) at 42 d. Cecal and drag swab samples were diluted in buffered peptone water and spread on XLT-4 plates to determine S. Heidelberg positive samples.

## Results

Body weight gains were not significantly different (*P* > 0.05) between treatments in both the broiler studies. Feed conversion was significantly improved (*P* < 0.05) by the addition of XPC through 14 and 35 d in Experiment 1 (Table 1). Feed conversion was also significantly improved (*P* < 0.05) with 1.25 kg XPC/tonne (T3) through 35 d and with both XPC inclusion levels (T2 and T3) through 42 d (Table 1).

Fecal S. Typhimurium numbers (log10 CFU) were significantly reduced (*P* = 0.051) by 14 d when XPC was fed (Table 2). By 35 d, the S. Typhimurium numbers (CFU; Table 2) in the cecal contents were low and not significantly different (*P* > 0.05) between treatments. In Experiment 2, no statistical differences (*P* > 0.05) in S. Heidelberg positive drag swabs were observed between treatments (Table 3). However, prevalence of S. Heidelberg positive ceca was significantly reduced (*P* < 0.05) when 1.25 kg/tonne (T3) XPC was fed continuously, and intermediate when 0.62 kg XPC/tonne (T2) was fed during grower/finisher period (Table 3).

<table><caption>**Table 1.** Feed conversion ratio (FCR) of broilers in both experiments.</caption><thead><tr><th rowspan="2">Bird Age:</th><th colspan="2">Experiment 2 - FCR</th><th colspan="2">Experiment 1 - FCR</th></tr><tr><th>0-14 d</th><th>0-35 d</th><th>0-35 d</th><th>0-42 d</th></tr></thead><tbody><tr><td>T1; Control</td><td>1.38<sup>a</sup></td><td>1.62<sup>a</sup></td><td>1.68<sup>a</sup></td><td>1.79<sup>a</sup></td></tr><tr><td>T2; XPC (1.25 and 0.62 kg/tonne)<sup>1</sup></td><td>1.35<sup>b</sup></td><td>1.61<sup>b</sup></td><td>1.66<sup>ab</sup></td><td>1.77<sup>b</sup></td></tr><tr><td>T3; XPC (1.25 and 1.25 kg/tonne)<sup>1</sup></td><td>-</td><td>-</td><td>1.64<sup>b</sup></td><td>1.75<sup>b</sup></td></tr><tr><td>P value</td><td><i>P</i> < 0.05</td><td><i>P</i> < 0.05</td><td><i>P</i> < 0.05</td><td><i>P</i> < 0.05</td></tr></tbody></table>

<table><caption>**Table 2.** Broiler *Salmonella* Typhimurium (ST) in feces and ceca at 14 and 35 day of age, respectively, in Experiment 1.</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="2">ST fecal numbers at 14 d</th><th rowspan="2">ST cecal numbers<br>(CFU/g)</th></tr><tr><th>CFU/g</th><th>Log10 CFU/g</th></tr></thead><tbody><tr><td>Control</td><td>51,094</td><td>2.96<sup>a</sup></td><td>94</td></tr><tr><td>XPC (1.25 and 0.62 kg/tonne)<sup>1</sup></td><td>6,623</td><td>2.14<sup>b</sup></td><td>20</td></tr><tr><td>P value</td><td><i>P</i> > 0.05</td><td><i>P</i> = 0.051</td><td><i>P</i> = 0.12</td></tr></tbody></table>

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

<footnote n="1">¹ Starter and grower/finisher dietary inclusion rates, respectively.</footnote>

<footnote><sup>ab</sup> Values within a column with different superscripts are statistically different (*P* = 0.051).</footnote>

<footnote n="1">¹ Starter and grower/finisher dietary inclusion rates, respectively.</footnote>
<table><caption>**Table 3.** Broiler *Salmonella* Heidelberg (SH) prevalence in fecal swabs and ceca at 42 day of age in Experiment 2.</caption><thead><tr><th>Treatment</th><th>SH fecal<sup>2</sup> prevalence<br>(% Positive)</th><th>SH cecal prevalence<br>(% Positive)</th></tr></thead><tbody><tr><td>T1; Control</td><td>100</td><td>38.3<sup>a</sup></td></tr><tr><td>T2; XPC (1.25 and 0.62 kg/tonne)<sup>1</sup></td><td>100</td><td>33.3<sup>ab</sup></td></tr><tr><td>T3; XPC (1.25 and 1.25 kg/tonne)<sup>1</sup></td><td>88</td><td>17.5<sup>b</sup></td></tr><tr><td>P value</td><td><i>P</i> &gt; 0.05</td><td><i>P</i> &lt; 0.05</td></tr></tbody></table>

## Conclusions

A reduction in S. Typhimurium numbers was observed in broilers fed XPC. S. Heidelberg prevalence was reduced in the ceca of broilers fed XPC. Improvements in feed conversion were observed in broilers fed XPC in both experiments.

## Literature

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

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

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

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

<footnote n="1">¹ Starter and grower dietary inclusion rates, respectively.</footnote>

<footnote n="2">² Pen drag swabs collected from bedding material.</footnote>
P-011 (ID 220)

VITAMIN D ENRICHMENT OF EGGS FOR HUMAN HEALTH

Linda C. Browning¹²³ and Aaron Cowieson<fnref n="1" />

**Abstract**

Vitamin D is an essential component of vertebrate nutrition and epidemiological surveys confirm a chronic vitamin D insufficiency in the human population. Eggs are one of the few natural sources rich in vitamin D, containing both vitamin D₃ (D₃) and 25-hydroxyvitamin D₃ (25(OH)D₃). Can additional dietary vitamin D significantly increase the vitamin D content of table eggs? In order to establish the potential for enrichment of eggs with D₃ and 25(OH)D₃ a total of 162 hens were fed three levels of D₃, (2500 and 5000 IU/kg) in combination with three levels of 25(OH)D₃ (0, 34.5 and 69 µg/kg). Egg yolks were analysed for both their D₃ and 25(OH)D₃ content and egg production, egg weights and feed efficiencies recorded.

The concentration of D₃ and 25(OH)D₃ in egg yolk significantly increased with increasing dietary concentration. There was no significant difference in egg mass, egg weight or feed efficiency. Depending on the dietary concentrations used, it was possible to produce an egg with between 100-500 IU vitamin D, providing scope to help meet the recommended daily requirement of vitamin D for children or adults. The dietary addition of higher levels of D₃ and 25(OH)D₃ produced eggs with sufficient vitamin D to help meet the recommended daily requirements of adults and children without detrimental effect on production parameters.

**Keywords:** vitamin D, vitamin D₃, eggs, 25-hydroxyvitamin D₃, laying hens

**Introduction**

Vitamin D is an essential nutrient in vertebrate nutrition for the maintenance of good health and well being however epidemiological studies in most countries indicate a widespread vitamin D insufficiency in the population which potentially may have a detrimental effect on health. For example in Germany, a comprehensive survey was undertaken by Hintzpeter et al. (2008) which showed a widespread deficiency in vitamin D. This data is shown in Table 1.

<caption>**Table 1.** Prevalence of vitamin D-deficient and replete states by gender and seasonª</caption>

Extracted from: Vitamin D status and health correlates among German adults. (Hintzpeter et al., 2008)

<table><thead><tr><th></th><th>Severe deficiency<br>(&lt;12.5 nmol/l)</th><th>Moderate deficiency<br>(12.5–25 nmol/l)</th><th>Mild deficiency<br>(25–50 nmol/l)</th><th>Replete<br>(&gt;50 nmol/l)</th></tr></thead><tbody><tr><td><strong>Men</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Total (%)</td><td>2.2</td><td>13.4</td><td>41.2</td><td>43.2</td></tr><tr><td>May–October all ages (%)</td><td>1.1</td><td>8.7</td><td>35.4</td><td>54.8</td></tr><tr><td>Nov–April all ages (%)</td><td>3.3</td><td>18.1</td><td>46.8</td><td>31.8</td></tr><tr><td><strong>Women</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Total (%)</td><td>1.9</td><td>15.1</td><td>40.8</td><td>42.2</td></tr><tr><td>May–October all ages (%)</td><td>0.9</td><td>11.5</td><td>42.4</td><td>45.2</td></tr><tr><td>Nov–April all ages (%)</td><td>2.7</td><td>18.8</td><td>39.3</td><td>39.2</td></tr></tbody></table>

Egg yolks are rich in vitamin D (Parrish and Richter, 1979) because of an efficient transfer by the domestic fowl of vitamin D from the feed into the yolk and unlike other animal-based foods the egg yolk contains both D₃ and 25(OH)D₃, in fact eggs contain higher levels of 25(OH)D₃ than any other animal-based food such as fish and meat (Mattila et al., 1995). The fact that eggs contain 25(OH)D₃ is significant because in human nutrition 25(OH)D₃ has five times the relative biological vitamin D activity as compared to D₃ (Koshy and Van Der Slik, 1979, Reeve et al., 1982, Mattila et al.,

<footnote n="1">¹Poultry Research Foundation, University of Sydney, Camden, NSW 2570, Australia</footnote>

<footnote n="2">²Poultry CRC, PO Box U242, University of New England, Armidale, NSW 2351, Australia</footnote>

<footnote n="3">³To whom correspondence should be addressed: lbro6652@uni.sydney.edu.au</footnote>

<footnote>ª Categories of serum 25OHD levels as described by Lips (2004). The sum of each row is 100%.</footnote>
1993). The objective of this study was to evaluate the efficiency of deposition of vitamin D into egg yolks following the supplementation of both D₃ and 25(OH)D₃ in the feed of the laying hen.

## Materials and Methods

A total of 162 Isa-Brown laying hens of 58 weeks of age were divided into nine treatment groups of 18 birds each, being composed of six replicates of three birds per replicate. Each bird was housed separately in cages measuring 25 x 50 x 50 cm², with three adjacent cages forming the replicate unit. Prior to the commencement of the trial all birds were fed for 4 weeks on the control diet with 2,500 IU D₃/kg (acclimatisation period). During the subsequent trial period of 9 weeks, the birds were fed a mash diet *ad libitum* with the only difference between treatment groups being the inclusion of various levels of vitamin D₃ and 25(OH)D₃. The photoperiod regime was 16 hours of light and 8 hours of dark. Water was supplied *ad libitum*. An outline of the nine dietary treatments is shown in Table 2 and the inclusion rates of D₃ and 25(OH)D₃ are stated in terms of both international units and µg/kg of feed.

<table><caption>**Table 2.** Outline of dietary treatments</caption><thead><tr><th>Die<br>t</th><th>Diet Description</th><th>Added D<sub>3</sub> †IU/kg</th><th>Added 25(OH)D<sub>3</sub>††µg/kg</th></tr></thead><tbody><tr><td>1</td><td>Normal D<sub>3</sub></td><td>2,500</td><td>0</td></tr><tr><td>2</td><td>Normal D<sub>3</sub> + 34.5 µg 25(OH)D<sub>3</sub></td><td>2,500</td><td>34.5 µg/kg</td></tr><tr><td>3</td><td>Normal D<sub>3</sub> + 69.0 µg 25(OH)D<sub>3</sub></td><td>2,500</td><td>69.0 µg/kg</td></tr><tr><td>4</td><td>Medium-high D<sub>3</sub></td><td>5,000</td><td>0</td></tr><tr><td>5</td><td>Medium-high D<sub>3</sub> + 34.5 µg 25(OH)D<sub>3</sub></td><td>5,000</td><td>34.5 µg/kg</td></tr><tr><td>6</td><td>Medium-high D<sub>3</sub> + 69.0 µg 25(OH)D<sub>3</sub></td><td>5,000</td><td>69.0 µg/kg</td></tr><tr><td>7</td><td>High D<sub>3</sub></td><td>10,000</td><td>0</td></tr><tr><td>8</td><td>High D<sub>3</sub> + 34.5 µg 25(OH)D<sub>3</sub></td><td>10,000</td><td>34.5 µg/kg</td></tr><tr><td>9</td><td>High D<sub>3</sub> + 69.0 µg 25(OH)D<sub>3</sub></td><td>10,000</td><td>69.0 µg/kg</td></tr></tbody></table>

†DSM Nutrition I Products Pty Ltd Rovimix® D₃ 500 ††DSM Nutritional Products Rovimix®HY·D®

Throughout the 9 week trial all eggs were collected and weighed for each replicate group. The feed supplied to each replicate group was weighed out weekly and on completion of the experiment all remaining feed was weighed and feed conversion efficiency determined. At the end of the 9 week trial a total of five eggs per replicate (30 eggs per treatment) were collected and their yolks separated, weighed, blended and freeze dried prior to analysis for D₃ and 25(OH)D₃ content. The average weight of the egg yolks per replicate was used to calculate the D₃ and 25(OH)D₃ content of egg yolk per replicate (6 replicates per treatment).

## Results

After 9 weeks of feeding, the analytical results for the concentration of both D₃ and 25(OH)D₃ in egg yolk for each replicate group are shown in Table 3. The D₃ content per kg of egg yolk was increased (*P*<0.01) with the addition of higher concentration of D₃ (5,000 IU D₃/kg and 10,000 IU D₃/kg) compared with the low concentration of D₃ (2,500 IU D₃/kg) in layer feed. However, the higher dietary levels of D₃ did not significantly change the 25(OH)D₃ content of egg yolk. In contrast, the addition of 25(OH)D₃ (34.5 µg 25(OH)D₃/kg and 69.0 µg 25(OH)D₃/kg) in the diet of laying hens not only significantly increased the 25(OH)D₃ content of egg yolk (*P*<0.01) but also significantly increased the D₃ content of egg yolk (*P*<0.01). Consequently there was a significant positive interaction (*P*<0.05) between D₃ and 25(OH)D₃ when 25(OH)D₃ was included in the diet.

Not surprisingly the highest concentration of total vitamin D in egg yolk (in terms of IU per 100 g of egg yolk) was achieved at the highest level of D₃ (10,000 IU D₃/kg feed) in the feed in combination with the highest level of 25(OH)D₃ (69 µg 25(OH)D₃/kg feed) in the feed.
<table><caption>**Table 3.** Effect of various levels of D₃ and 25(OH)D₃ supplementation on D₃, 25(OH)D₃ and total vitamin D content of egg yolk</caption><thead><tr><th>D<sub>3</sub><br>(IU/kg)</th><th>25(OH)D<sub>3</sub><br>(µg/kg)</th><th>D<sub>3</sub> per 100g yolk<br>(µg)</th><th>25(OH)D<sub>3</sub> per 100<br>g yolk (µg)</th><th>Total vit D† per 100<br>g yolk (IU)</th><th>Total vit D<sup>1</sup><br>per egg yolk (IU)</th></tr></thead><tbody><tr><td>2500</td><td>0</td><td>6.48<sup>cd</sup> (±1.462)<sup>2</sup></td><td>1.61<sup>f</sup> (±0.757)</td><td>580<sup>e</sup> (±201.7)</td><td>96<sup>f</sup> (±33.1)</td></tr><tr><td>2500</td><td>34.5</td><td>5.96<sup>cd</sup> (±1.444)</td><td>3.30<sup>d</sup> (±1.448)</td><td>898<sup>d</sup> (±315.2)</td><td>152<sup>e</sup> (±50.2)</td></tr><tr><td>2500</td><td>69.0</td><td>4.90<sup>d</sup> (±0.532)</td><td>4.49<sup>c</sup> (±1.242)</td><td>1094<sup>cd</sup> (±246.2)</td><td>185<sup>de</sup> (±41.4)</td></tr><tr><td>5000</td><td>0</td><td>10.51<sup>c</sup> (±2.270)</td><td>2.06<sup>ef</sup> (±0.752)</td><td>832<sup>de</sup> (±186.1)</td><td>146<sup>e</sup> (±39.6)</td></tr><tr><td>5000</td><td>34.5</td><td>7.43<sup>cd</sup> (±1.673)</td><td>4.51<sup>c</sup> (±1.011)</td><td>1199<sup>c</sup> (±261.5)</td><td>213<sup>cd</sup> (±50.1)</td></tr><tr><td>5000</td><td>69.0</td><td>8.07<sup>cd</sup> (±3.022)</td><td>5.81<sup>b</sup> (±0.656)</td><td>1484<sup>b</sup> (±172.1)</td><td>256<sup>bc</sup> (±27.5)</td></tr><tr><td>10000</td><td>0</td><td>26.17<sup>ab</sup> (±8.280)</td><td>3.01<sup>de</sup> (±0.392)</td><td>1649<sup>b</sup> (±325.1)</td><td>270<sup>b</sup> (±56.4)</td></tr><tr><td>10000</td><td>34.5</td><td>23.60<sup>b</sup> (±5.208)</td><td>3.71<sup>cd</sup> (±0.481)</td><td>1686<sup>b</sup> (±173.0)</td><td>285<sup>b</sup> (±25.6)</td></tr><tr><td>10000</td><td>69.0</td><td>30.85<sup>a</sup> (±5.384)</td><td>8.05<sup>a</sup> (±1.117)</td><td>2845<sup>a</sup> (±251.3)</td><td>478<sup>a</sup> (±39.40)</td></tr><tr><td>Pooled SEM</td><td></td><td>1.648</td><td>0.381</td><td>99.3</td><td>16.98</td></tr><tr><td>Model P</td><td></td><td>&lt;0.01.</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td colspan="6"><strong>Main Effects</strong></td></tr><tr><td>2500</td><td></td><td>5.78<sup>c</sup></td><td>3.13<sup>c</sup></td><td>858<sup>c</sup></td><td>144<sup>c</sup></td></tr><tr><td>5000</td><td></td><td>8.67<sup>b</sup></td><td>4.12<sup>b</sup></td><td>1172<sup>b</sup></td><td>205<sup>b</sup></td></tr><tr><td>10000</td><td></td><td>26.87<sup>a</sup></td><td>4.93<sup>a</sup></td><td>2060<sup>a</sup></td><td>344<sup>a</sup></td></tr><tr><td>P</td><td></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>0</td><td></td><td>14.39<sup>a</sup></td><td>2.23<sup>c</sup></td><td>1021<sup>c</sup></td><td>171<sup>c</sup></td></tr><tr><td>34.5</td><td></td><td>12.33<sup>a</sup></td><td>3.84<sup>b</sup></td><td>1261<sup>b</sup></td><td>217<sup>b</sup></td></tr><tr><td>69.0</td><td></td><td>14.61<sup>a</sup></td><td>6.12<sup>a</sup></td><td>1808<sup>a</sup></td><td>307<sup>a</sup></td></tr><tr><td>P</td><td></td><td>N.S.</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td colspan="6"><strong>Interaction Terms</strong></td></tr><tr><td>D<sup>3</sup>*25(OH)D<sub>3</sub></td><td></td><td>N.S.</td><td>N.S.</td><td>P&lt;0.01</td><td>P&lt;0.01</td></tr></tbody></table>

Means in columns with no common superscript differ significantly

†Total vitamin D (IU) is calculated by the addition of D₃ (µg/kg egg yolk) and 25(OH)D₃ (µg/kg egg yolk) where 1.0 µg of D₃ equals 40 IU vitamin D and 1.0 µg of 25(OH)D₃ equals 200 IU vitamin D.

## Conclusion

Epidemiological surveys indicate a recognised chronic insufficiency of vitamin D within the human population. Vitamin D is essential for good skeletal health, healthy skin and a sound immune system and chronic insufficiencies may cause many diseases including osteoporosis. There are few naturally occurring foods which contain significant dietary levels of vitamin D, however the modern commercial laying hen has been shown to be unique in its ability to efficiently transfer vitamin D metabolites from its diet into egg yolk. Egg yolk represents one of the few foods which is rich in both D₃ and 25(OH)D₃. This experiment has demonstrated that a single egg from a laying hen supplemented with increased levels of D₃ and 25(OH)D₃ has the potential to contribute significantly to the recommended daily intake of vitamin D for both adults and children without detrimental effect on production parameters.

## Literature

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KOSHY, K. T., A. L. VAN DER SLIK, 1979: High-performance liquid chromatographic method for the determination of 25-hydroxycholecalciferol in chicken egg yolks. *J Agricult Food Chem*, 27, 180-183.

MATTILA, P., V. PIIRONEN, E. UUSI-RAUVA, P. KOIVISTOINEN, 1993: Determination of 25-hydroxycholecalciferol content in egg yolk by HPLC. *J Food Comp Analysis*, 6, 250-255.

MATTILA, P., V. PIIRONEN, E. UUSI-RAUVA, P. KOIVISTOINEN, 1995: Cholecalciferol and 25-hydroxycholecalciferol contents in fish and fish products. *J Food Comp Analysis*, 8, 232-243.

PARRISH, D. B., E. F. RICHTER, 1979: Determination of vitamin D in foods: A review. *Critical Reviews in Food Sci and Nutr*, 12, 29-57.

REEVE, L. E., N. A. JORGENSEN, H. F. DELUCA, 1982: Vit D compounds in cows milk. *J Nut*, 112, 667.
EFFECT OF NUCLEOTIDES ENRICHED YEAST EXTRACTS ON VILLUS LENGTH AND GOBLET CELL COUNT-
ING IN BROILER INTESTINE

M.T. Brufau<fnref n="1" />, N. González-Jurado<fnref n="1" />, A.M. Pérez-Vendrell<fnref n="2" />, R. D'Inca<fnref n="3" />, E. Auclair<fnref n="3" />, B. Vilà<fnref n="2" />, J. Brufau<fnref n="2" />, R. Ferrer<fnref n="1" />,
and R. Martín-Venegas<fnref n="1" />

Corresponding author: teresabrufau@gmail.com

**Abstract**

Dietary nucleotides have a key nutritional role in the development of tissues with a high renewal rate such as the intestine. In periods of fast growth, these nutrients can become conditionally essential so that exogenous supply may benefit animal development. Taking into account the dramatic changes that occur in the chicken intestine after hatching, our aim was to investigate two indicators of intestinal health (villus development and goblet cell counting) in animals fed four different diets supplemented with nucleotides. These diets were supplemented with yeast extracts containing different nucleotide concentrations (0-0.6 %). Cryostatic slices of the duodenum and ileum of 10-day-old male Ross chickens were prepared and treated with periodic acid-Schiff to stain mucus glycoproteins. Villus length and the number of goblet cells per epithelial length were measured in micrographs of known magnification. The results obtained indicate that beneficial effects of supplemented nucleotides are exerted in the duodenum by increasing the number of goblet cells from 12 to 22 % (p<0.05) and in the ileum by increasing villus length from 9 to 19 % (p<0.05). In conclusion, the inclusion of nucleotides-enriched yeast extracts in the starter diet for broilers may have beneficial effects by increasing absorptive surface area and improving the role of mucus in intestinal barrier function.

**Keywords:** poultry, nutrition, absorptive surface area, epithelial barrier function, mucus

**Introduction**

Nucleotides have significant effects on the development of tissues with a high replacement rates, such as the immune
system and gastrointestinal tract (Gardiner et al., 1995). During the early stage of development, a period of fast growth,
the availability of nucleotides can limit the maturation of these tissues and nucleotides can become conditionally essen-
tial so that exogenous supply may benefit animal development. In this sense, Jung and Batal (2012) have described in
the jejunum of 14-day-old chickens fed a diet supplemented with nucleotides an increase in villus length, an effect
which has been associated to an improvement of animal growth and conversion rate (Rutz et al., 2006; Esteve-García
et al., 2007; Liotta et al. 2010; Jung and Batal, 2012). Taking into account the dramatic changes that occur in the
chicken intestine after hatching (Geyra et al., 2001), our aim was to investigate in 10-day-old chickens, the effect of
increasing dietary nucleotide supplementation on two indicators of intestinal health, villus length as an indicator of the
absorptive available surface area and goblet cell counting as an indicator of epithelial barrier function.

**Material and Methods**

Male Ross 308 chickens (Granja Crusvi, Montblanc) were fed *ad libitum* from hatching until day 10 with balanced diets (Institut de Recerca i Tecnologia Agroalimentàries, IRTA; 3 replicates per treatment, 33 birds per replicate in floor pens) supplemented with yeast extracts containing increasing nucleotide concentrations (0, 0.2, 0.4 and 0.6 % in feed, Phileo-Lesaffre Animal Care, France, Control and treatments 0.2, 0.4 and 0.6, respectively). Villus length and goblet cell counting were determined in 6 animals per treatment (2 birds per replicate, randomly selected). After the animals were euthanized by cervical dislocation, the duodenum and the ileum were washed with ice-cold saline (NaCl 9 g/L, 4°C). Intestinal portions (5 mm in length) were taken and fixed in 3.0 % paraformaldehyde in 0.1 mol/L PBS (pH 7.4) during 2 h at room temperature. After fixation, the samples were cryoprotected in ice cold sucrose gradient in 0.1 mol/L PBS, mounted in optimal cutting temperature (Aname, Barcelona) and stored at -20 °C. Four groups of seven serial transverse cryostatic sections (10 µm, CM 3050 S, Leica, Wetzlar, Germany) were obtained from each intestinal portion at intervals of 300 µm to guarantee the observation of different villi. The samples were treated with periodic acid-Schiff to stain glycolproteins from mucus (Shao et al., 2013) and mounted with ProLongol (Life Technologies, Carlsbad, CA, E.U.A.).

<footnote n="1">¹Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Spain</footnote>

<footnote n="2">²Institut de Recerca i Tecnologia Agroalimentàries (IRTA- Centre Mas de Bover), Constantí, Spain</footnote>

<footnote n="3">³Phileo-Lesaffre Animal Care, Marcq-en-Barœul, France</footnote>
The images were captured with an Olympus BX41 microscope (Tokyo, Japan) at two magnification levels: 20 x for villus length and 100 x for goblet cell counting. Villus length was measured using the lamina propria as the base (Uni et al., 1995) in images processed by IMAT software (Centres Científics i Tecnològics of the Universitat de Barcelona). Goblet cell counting was performed in printed images of known magnification and refereed to epithelial length. The animal was used as the experimental unit. The statistical analysis was performed by ANOVA followed by a post-hoc test comparing the mean values of the treatments with the negative treatment (Control). P < 0.05 was considered to denote significance.

## Results

The results of villus length revealed (Figure 1A), as we have previously observed for Leghorn chickens (Mitjans et al., 1997), higher values in the duodenum than in the ileum (P < 0.05). Regarding the effect of the different treatments, the statistical analysis of the duodenal data indicated a progressive decrease for treatments 0.2 and 0.4 and no effect for the highest nucleotide concentration (treatment 0.6). In the ileum, the comparison of the supplemented diets with the Control revealed a progressive increase with nucleotide concentration.

<figure><img src="image_25.png" /><figcaption>**Figure 1.** Villus length (A) and number of goblet cells (B) in the duodenum and ileum of 10-day-old chickens feed the different treatments. Results were expressed as mean ± SEM (n = 6 animals). Different letters denote significant differences (P < 0.05) within the duodenum and the ileum.</figcaption></figure>

The values of goblet cell counting (Figure 1B) for the Control treatment were higher in the ileum than in the duodenum (P < 0.05). As for the comparison between treatments, the statistical analysis revealed in the duodenum, an increase for treatments 0.2 and 0.4 and no differences between treatments 0.4 and 0.6 and in the ileum, no effect for treatment 0.2 and a similar increase for treatments 0.4 and 0.6.

## Conclusions

The data obtained indicate that the best results were found for the highest nucleotide concentration (0.6%) which induced no changes in villus length and a 22% increase in the number of goblet cells in the duodenum, and in the ileum, an increase of 19% in villus length and 9% in goblet cell counting. In conclusion, the inclusion of nucleotides-enriched yeast extracts in the starter diet for broilers may have beneficial effects by increasing absorptive surface area and improving the role of mucus in intestinal barrier function.

## Acknowledgements

This work was supported by the Fundació Bosch i Gimpera and Phileo-Lesaffre Animal Care.
## Literature

ESTEVE-GARCIA, E., D. MARTINEZ-PUIG, E. BORDA, C. CHETRIT, 2007: Efficacy of a nucleotides preparation in broiler chickens. XVIIth European Symposium on Poultry Nutrition, 511-514.

GARDINER, K.R., S.J. KIRK, B.J. ROWLANDS, 1995: Novel substrates to maintain gut integrity. Nutr. Res. Rev. **8**, 43-66.

GEYRA, A., Z. UNI, D. SKLAN, 2001: Enterocyte dynamics and mucosal development in the posthatch chick. Poult. Sci. **80**, 776-782.

JUNG, B., A.B. BATAL, 2012: Effect of dietary nucleotide supplementation on performance and development of the gastrointestinal tract of broilers. Br. Poult. Sci. **53**, 98-105.

LIOTTA, I., F. MARINO, G. SAVIORINI, V. CHIOFALO, G. LANTERI, E. D'ALESSANDRO, B. CHIOFALO, 2010: Dietary nucleotides in broilers: Effects on productive performance and intestinal morphometry. In: CROVETTO, G.M.: Energy and protein metabolism and nutrition. Wageningen Academic Publishers, Wageningen, ISBN 978-90-8686-153-8, 201-202.

MITJANS, M., G. BARNIOL, R. FERRER, 1997: Mucosal surface area in chicken small intestine during development. Cell Tissue Res. **290**, 71-78.

RUTZ, F., M.A. ANGIUTI, J.L. RECH, F.M. GONSALVES, A.D. DELGADO, E.R. ROSA, N. ZAUK, C.L.G. RIBETRO, R.R. SILVA, P.R. GALLMANN, 2006: Performance and carcass traits of broilers fed diets containing yeast extract. Ci. Anim. Bras. **7**, 349-355.

SHAO, Y., Y. GUO, Z. WANG, 2013: β-1,3/1,6-Glucan alleviated intestinal mucosal barrier impairment of broiler chickens challenged with *Salmonella enterica* serovar Typhimurium. Poult. Sci. **92**, 1764-1773.

UNI, Z., Y. NOY, D. SKLAN, 1995: Posthatch changes in morphology and function of the small intestines in heavy- and light-strain chicks. Poult. Sci. **74**:1622-1629.
P-013 (ID 193)

EFFECT OF THE INCLUSION OF NA-BUTYRATE IN DIETS BASED ON CORN OR WHEAT, WITH DIFFERENT AMEN CONTENT, ON GROWTH PERFORMANCE OF BROILER REARED UNDER POOR HYGIENE CONDITIONS

L. Cámara<fnref n="1" />,<fnref n="2" />, M. V. Kimiaetalab<fnref n="1" />, I. Solis<fnref n="1" />, P. Guzmán<fnref n="1" />, A. Ortiz<fnref n="3" />, and G. G. Mateos<fnref n="1" />,<fnref n="2" />

**Corresponding author:** author@anyuniversity.co.uk

**Abstract**

In total, 800 one-d-old, straight run Ross 308 broilers were used to study the inclusion of 0.1% of a chemically protected Na-butyrate (Butirex C4) in diets based on corn or wheat with high (HE) or low (LE) AMEn content (60 kcal/kg difference) on growth performance of broilers. Chicks were placed in their respective pens immediately after the removal of the previous batch of birds, without any cleaning or disinfection. The experimental design was completely randomized with 8 treatments arranged as a 2 x 2 x 2 factorial with 2 cereals sources (corn vs. wheat), 2 AMEn concentration (HE vs. LE), and 2 levels of Na-butyrate (0 vs. 0.1%). Each treatment was replicated 4 times (a pen with 25 chicks). The trial lasted 40 d. From d 0 to 20, ADG (P < 0.05) and FCR (P < 0.01) were better in broilers fed corn than in broilers fed wheat. Also, FCR (P < 0.01) was improved in broilers fed the HE diets. Na-butyrate supplementation increased ADG (P < 0.001) and ADFI (P < 0.001) but did not affect FCR. Cumulatively (0 to 40 d of age), chicks fed corn tended to have better FCR than chicks fed wheat (P < 0.10) and chicks fed the HE diets had better FCR (P < 0.05) than chicks fed the LE diets. Na-butyrate increased ADG (P < 0.01) and ADFI (P < 0.05) of the birds. In summary, the supplementation with protected Na-butyrate improved ADG and ADFI from hatch to slaughter in broilers reared under poor hygiene conditions, independent of the main cereal and the energy content of the diet.

**Keywords:** broiler, cereal, growth performance, Na-butyrate

**Introduction**

The ban of use of antibiotics as "in feed" growth promoters in poultry diets has increased the interest on the utilization of other active feed additives (MATEOS et al., 2002). The inclusion in the diet of short chain fatty acids, such as butyric acid, might acidify the luminal contents and help in the control of the growth of the pathogen microbiota. Butyric acid, however, has a very unpleasant odor that reduces diet palatability (ANTONGIOVANNI et al., 2007). Consequently, this acid is frequently marketed as a salt, which is odourless and easier to handle. Several researches have compared diets based on corn or wheat in broilers (MASEY-O'NEILL et al., 2014). Broilers eat to satisfy their energy requirements and therefore, an increase in the AMEn content of the diet should result in a concomitant increase in feed intake and in an improvement in feed conversion (FCR) (SALEH et al., 2014). However, the effects of changes in the AMEn of the diet on average daily gain (ADG) are not well studied in broilers. The aim of this research was to study the effects of Na-butyrate supplementation on growth performance of broilers fed different type of diets when reared under dirty environmental conditions.

**Material and Methods**

In total, 800 one-d-old, straight run Ross 308 broilers were received from a commercial hatchery and randomly distributed in groups of 25 into 32 pens. Chicks were placed in the experimental barn immediately after the removal of the previous batch, without any cleaning or disinfection. The pens (1.1 m x 1.1 m) were bedded with wood shaving and provided with a hopper feeder and a bell drinker. The environmental conditions during the experiment were controlled automatically according to the age of the birds. The feeding program consisted in 3 diets supplied from 1 to 20 d (starter), 20 to 35 d (grower) and 35 to 40 d (finisher) of age. All diets met or exceeded the nutritional requirements for broilers (FUNDACIÓN ESPAÑOLA DESARROLLO NUTRICIÓN ANIMAL, 2008) and were presented as 3 mm Ø pellet. Within each of the 3 feeding periods, all diets were formulated to have the same AMEn: indispensable amino acid ratio. The diets were based on corn or wheat and contained 3,000 and 2,940 kcal/kg for the started diets, 3,140 and 3,077 kcal/kg for the grower diets and 3,170 and 3,107 kcal/kg for the finisher diets, for the high (HE) and low (LE) energy diets, respectively. Each of these 4 experimental diets by periods was supplemented or not with 0.1% Na-butyrate. Body weight of the chicks and feed consumption were determined by pen on d 1, 20, 35 and 40, and mortality was re-

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

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

<footnote n="3">³ NUTEGA, S. L., 28823, Coslada, Madrid, Spain</footnote>
corded and weighed as produced. From these data, ADG, average daily feed intake (ADFI) and FCR were determined. The experimental design was completely randomized with 8 dietary treatments arranged as a 2 x 2 x 2 factorial with 2 cereal sources (corn vs. wheat), 2 nutritional levels of the diet (differing in 60 kcal AMEn/kg) and 2 levels of inclusion of Na-butyrate (0 vs. 0.1%). Each treatment was replicated 4 times, and the experimental unit was a pen with 25 chicks. At d 20, after the growth performance control, the number of birds per pen was reduced to 15 to fit the European legislation on rearing density of broilers. Excess of birds were removed at random within each pen.

## Results

Mortality was 2.1% and was not related to treatment. From 0 to 40 d of age, FCR tended to be better in broilers fed corn than in broilers fed wheat (1.60 vs. 1.63; P = 0.094) and in broilers fed the HE diets than in broilers fed the LE diets (1.60 vs. 1.63; P < 0.05) (Table 1). Na-butyrate inclusion increased ADG (75.8 vs. 73.6 g; P < 0.01) and ADFI (122.1 vs. 119.2 g; P < 0.05). From 0 to 20 d of age, broilers fed the corn based diets had greater ADG (41.3 vs. 40.5 g; P < 0.05) and better FCR (1.34 vs. 1.39, P < 0.01) than broilers fed the wheat based diets. Broilers fed the LE diets had greater ADFI (56.8 vs. 55.0 g, P < 0.05) than chicks fed HE diets. Broilers fed Na-butyrate supplemented diets showed greater ADG (42.1 vs. 39.7 g; P < 0.001) and ADFI (57.8 vs. 54.0 g; P < 0.001) than broilers fed the control un-supplemented diet, but FCR was not affected.

## Conclusions

In summary, FCR was improved with corn or high energy diets. The addition of 0.1% of Na-butyrate in broilers feed is especially recommended at early ages (from 0 to 20 d of age) after this age, the effect is diluted.

<table><caption>**Table 1.** Effects of the main cereal, AMEn concentration, and inclusion of Na-butyrate (Butyrate) in the diet on growth performance from 0 to 40 days.</caption><thead><tr><th rowspan="2"></th><th colspan="2">0 - 20 d</th><th colspan="2">20 - 35 d</th><th colspan="2">35 - 40 d</th><th colspan="2">0 - 40 d</th></tr><tr><th>ADG<sup>1</sup><br>(g)</th><th>ADFI<sup>2</sup><br>(g)</th><th>FCR<sup>3</sup></th><th>ADG<br>(g)</th><th>ADFI<br>(g)</th><th>FCR</th><th>ADG<br>(g)</th><th>ADFI<br>(g)</th><th>FCR</th></tr></thead><tbody><tr><td>Cereal</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Corn</td><td>41.3</td><td>55.5</td><td>1.34</td><td>108.4</td><td>174.3</td><td>1.61</td><td>110.6</td><td>217.4</td><td>1.97</td></tr><tr><td>Wheat</td><td>40.5</td><td>56.4</td><td>1.39</td><td>107.5</td><td>175.5</td><td>1.63</td><td>110.5</td><td>216.0</td><td>1.95</td></tr><tr><td>AME<sub>n</sub>, kcal/kg</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2,940</td><td>40.8</td><td>56.8</td><td>1.39</td><td>108.5</td><td>177.0</td><td>1.63</td><td>110.3</td><td>217.2</td><td>1.97</td></tr><tr><td>3,000</td><td>41.0</td><td>55.0</td><td>1.34</td><td>107.3</td><td>172.8</td><td>1.61</td><td>110.8</td><td>216.1</td><td>1.95</td></tr><tr><td>Butyrate, %</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>-</td><td>39.7</td><td>54.0</td><td>1.36</td><td>106.4</td><td>173.6</td><td>1.63</td><td>111.1</td><td>216.6</td><td>1.96</td></tr><tr><td>0.1</td><td>42.1</td><td>57.8</td><td>1.37</td><td>109.4</td><td>176.3</td><td>1.61</td><td>110.1</td><td>216.7</td><td>1.97</td></tr><tr><td>SEM<sup>4</sup></td><td>0.265</td><td>0.482</td><td>0.0109</td><td>1.017</td><td>1.725</td><td>0.0124</td><td>1.846</td><td>2.205</td><td>0.0244</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="10" style="text-align: center;">P-value</td></tr><tr><td>Cereal</td><td>0.034</td><td>0.248</td><td>0.004</td><td>0.649</td><td>0.658</td><td>0.278</td><td>0.992</td><td>0.659</td><td>0.617</td></tr><tr><td>AME<sub>n</sub></td><td>0.704</td><td>0.021</td><td>0.005</td><td>0.427</td><td>0.115</td><td>0.223</td><td>0.716</td><td>0.723</td><td>0.393</td></tr><tr><td>Butyrate</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.472</td><td>0.064</td><td>0.305</td><td>0.344</td><td>0.437</td><td>0.973</td><td>0.356</td></tr></tbody></table>

<footnote n="1">¹ Average daily gain</footnote>

<footnote n="2">² Average daily feed intake</footnote>

<footnote n="3">³ Feed conversion ratio</footnote>

<footnote n="4">⁴ Standard error of the mean (n=16). The experimental unit was a pen with 25 or 15 birds per pen from 0 to 20 d and 20 to 40 d of age, respectively</footnote>

<footnote n="5">⁵ The interactions among main effects were not significant in all cases (P > 0.05)</footnote>
## Literature

ANTONGIOVANNI, M., BUCCIONI, A., PETACCHI, F., LEESON, S., MINIERI, S., MARTINI, A., and CECCHI R. 2007. Butyric acid glycerides in the diet of broiler chickens: effects on gut histology and carcass composition. Ital. J. Anim. Sci. **6**, 19-25.

FUNDACIÓN ESPAÑOLA DESARROLLO NUTRICIÓN ANIMAL, 2008. In Necesidades Nutricionales para Avicultura: Pollos de Carne y Aves de Puesta. R. Lázaro and G. G. Mateos, eds. FEDNA, Madrid, Spain.

MATEOS, G. G., LÁZARO, R., GRACIA, M. 2002. The feasibility of using nutritional modifications to replace drugs in poultry feeds. J. Appl. Poult. Res. **11**, 437-452.

SALEH, E. A., WATKINS, S. E., WALDROUP, A. L., and WALDROUP, P. W. 2004. Effects of dietary nutrient density on performance and carcass quality of male broilers grown for further processing. Int. J. Poult. Sci. **3**, 1-10.

MASEY-O’NEILL, H. V., SINGH, M., COWIESON, A. J. 2014. Effects of exogenous xylanase on performance, nutrient digestibility, volatile fatty acid production and digestive tract thermal profiles of broilers fed on wheat- or maize- based diet. Br. Polt. Sci. **55**, 351-359.
P-014 (ID 195)

EFFECT OF FEEDING DIFFERENT COMBINATIONS OF ORGANIC ACIDS, MONO-GLYCERIDES AND PROBIOTICS ON CAMPYLOBACTER COLONIZATION IN BROILERS

O. Casabuena<fnref n="1" />, M. Chemaly<fnref n="2" />, M. Den Hartog<fnref n="3" />, P. Vesseur<fnref n="3" />, and M.I. Gracia<fnref n="1" />

**Corresponding author:** mgracia@e-imasde.com

**Abstract**

An experiment was conducted within the EU-FP7 project CAMPYBRO in order to evaluate the effect of a combination of a blend of mono-glycerides and organic acids (MGOA) with a probiotic alone (B. subtilis C-3102, P) or with a multispecies probiotic (PS) and a plant extract based on garlic (G) added to the feed on *Campylobacter* counts in broilers. There were three treatments applied from 1 to 42 d of age, T1: Positive controls (*Campylobacter*, no additives), T2: T1+MGOA at 2.5%+P at 100 g/t and T3: T1+MGOA at 2.5%+PS at 1,000 g/t+G at 1,000 g/t. A total of 126 one-day-old Ross 308 broilers (half male and half female) were divided into the experimental treatments. At 14 d of age, all broilers were orally gavaged with 100 µl of a solution containing 1 x 10⁵ CFU/ml of ST-45 C. jejuni strain. On days 21, 35 and 42, ceca from 12 birds per treatment were collected and *Campylobacter* counts determined (ISO 10272). Data were analysed with a Kruskall-Wallis test (SPSS v.19.0). At 21 d, the two combinations used consistently decreased the *Campylobacter* counts compared with T1 (by 3-log; P < 0.05). At 35 and 42 d, only T2 (MGOA+P) decreased the colonization of *Campylobacter* compared with T1 (3.05 vs 7.15 log₁₀ CFU/g; P = 0.001 and 4.41 vs 8.39 log₁₀ CFU/g; P = 0.003; for T2 vs T1 at 35 and 42 d, respectively). The results suggest that the supplementation of a blend of mono-glycerides with organic acids together with a probiotic, especially *B. subtilis*, can effectively decrease *Campylobacter* in broilers.

**Keywords:** mono-glycerides, organic acids, probiotics, *Campylobacter jejuni*, broiler

**Introduction**

*Campylobacter* is the major cause of bacterial gastroenteritis in humans worldwide. The number of confirmed cases of campylobacteriosis in the European Union has followed a significant increasing trend in the last years, along with a clear seasonal trend. In 2013, it was the most commonly reported zoonosis with 214,779 confirmed human cases (EFSA, 2015). Few studies have evidenced a positive role of probiotics in preventing or reducing the shedding of C. jejuni in *in vivo* trials (MORISHITA et al., 1997; SANTINI et al., 2010), although *in vitro* studies reported a strong antimicrobial activity of several probiotic strains toward this pathogen (FOOKS and GIBSON, 2002; CHAVEERACH et al., 2004). A wide range of plant extracts and compounds have demonstrated strong bactericidal activity against *Campylobacter* spp. *in vitro* (FRIEDMAN et al., 2002; ROBYN et al., 2013). However, when evaluating *in vivo* results, data are not so conclusive. Also, *in vitro* studies have demonstrated that organic acids, medium chain fatty acids, or their monoglycerides have a strong bactericidal effect on *Campylobacter* spp. (CHAVEERACH et al., 2002; HERMANS et al., 2010). However, inconsistent results have been reported in *in vivo* trials (SOLIS DE LOS SANTOS et al., 2010; HERMANS et al. 2012). Therefore, it is clear that there exist differences in the results *in vitro* and *in vivo*. The objective of this work was to evaluate the effect of a combination of a blend of mono-glycerides and organic acids (MGOA) with a probiotic alone (*B. subtilis* C-3102, P) or with a multispecies probiotic (PS) and a plant extract based on garlic (G) added to the feed on *Campylobacter* counts in broilers.

**Material and Methods**

A total of 126 Ross 308 broilers (50% male and 50% female) from 1 to 42 days of age were used and allocated to the experimental treatments. There were three treatments applied from 1 to 42 d of age, T1: Positive controls (*Campylobacter*, no additives), T2: T1 + MGOA at 2.5% + P at 100 g/t and T3: T1 + MGOA at 2.5% + PS at 1,000 g/t + G at 1,000 g/t. The trial was carried out in an Animal Biosafety Level 2 Unit for broilers. Broilers were housed in cages in groups of three. The individual bird was the experimental unit. Mash (non-pelleted) feeds were fed *ad libitum* with no added coccidiostat, growth promoter or veterinary antibiotics. Diets were cereal based diets similar to those used commercially. Prior to the infection of the animals, caecal samples of 9 birds randomly selected were collected to verify that chicks were all *Campylobacter*-free. At 14 d of age, all broilers were orally gavaged with 100 µl of a solution containing

<footnote n="1">¹Imasde Agroalimentaria, S.L., Madrid, Spain</footnote>

<footnote n="2">²Anses, UHQPAP, Ploufragan, France</footnote>

<footnote n="3">³NEPLUVI, Houten, the Netherlands</footnote>
1 x 10⁵ CFU/ml of ST-45 C. jejuni strain. On days 21, 35 and 42, ceca were collected from 12 birds per treatment and *Campylobacter* counts determined (ISO 10272). Detection limit was 2 logs, and animals below limit of detection were assigned 2 logs for statistical analysis. Data were analysed with a Kruskall-Wallis test (SPSS v.19.0). Statistical significance is declared at P ≤ 0.05, with 0.05 < P ≤ 0.10 considered as a near-significant trend.

## Results

At 21 d, the two combinations used consistently decreased the *Campylobacter* counts compared with T1 (by 3.5-log P = 0.010 and by 3.0-log P = 0.032, for T2 vs T1 and T3 vs T1, respectively). At 35 and 42 d, only T2 (MGOA+P) decreased the colonization of *Campylobacter* compared with T1 (3.05 vs 7.15 log₁₀ CFU/g; P = 0.001 and 4.41 vs 8.39 log₁₀ CFU/g; P = 0.003; for T2 vs T1 at 35 and 42 d, respectively). This decrease was mainly due to the broilers below detection level, 75 and 8% and 58 and 17% in T2 and T3 at 35 and 42 d, respectively. Birds on T3 showed a non-significant reduction of 1.81-log compared to T1.

## Conclusions

Under our experimental conditions, it is concluded that: i) Both the supplementation of a combination of a blend of MGOA with a probiotic alone or with a multispecies probiotic and a garlic extract, significantly decrease *Campylobacter* counts in the ceca of infected birds at 21d, ii) Only the blend of MGOA together with the probiotic based on *B. subtilis* significantly decreased *Campylobacter* counts at 35 and 42d (a decrease by 4-log was reached at the end of the fattening period), and iii) the blend of MGOA with the multispecies probiotic and the garlic extract reached a non-significant decrease of 1.81-log in relation to controls.

The results suggest that the supplementation of a blend of MGOA together with a probiotic, especially *B. subtilis*, can effectively decrease *Campylobacter* in broilers.

## Acknowledgements

This work has been done within the project CAMPYBRO, which has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 605835.

<figure><img src="image_26.png" /><figcaption>**Figure 1.** Effect of dietary treatment (T1: Control, T2: T1 + MGOA at 2.5% + P at 100 g/t and T3: T1 + MGOA at 2.5% + PS at 1,000 g/t + G at 1,000 g/t) on *Campylobacter* counts (log₁₀ CFU/g) in the caeca of broilers at 21, 35 and 42 days of age</figcaption></figure>
## Literature

CHAVEERACH, P., D. A. KEUZENKAMP, H. A. URLINGS, L. J. LIPMAN, F. VAN KNAPEN, 2002: *In vitro* study on the effect of organic acids on *C. jejuni/coli* populations in mixtures of water and feed. Poult. Sci. **81**, 621-628.

CHAVEERACH, P., L. J. LIPMAN, F. VAN KNAPEN, 2004: Antagonistic activities of several bacteria on *in vitro* growth of 10 strains of *C. jejuni/coli*. Int. J. Food Microbiol. **90**, 43-50.

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

FOOKS, L. J., G. R. GIBSON, 2002: *In vitro* investigations of the effect of probiotics and prebiotics on selected human intestinal pathogens. FEMS Microbiol. Ecol. **39**, 67-75.

FRIEDMAN, M., P. R. HENIKA, R. E. MANDRELL, 2002: Bactericidal activities of plant essential oils and some of their isolated constituents against *C. jejuni*, *E. coli*, *L. monocytogenes*, and *S. enterica*. J. Food Prot. **65**, 1545-1560.

HERMANS, D., A. MARTEL, A. GARMYN, M. VERLINDEN, M. HEYNDRICKX, I. GANTOIS, F. HAESEBROUCK, F. PASMANS, 2012: Application of medium-chain fatty acids in drinking water increases *C. jejuni* colonization threshold in broiler chicks. Poult. Sci. **91**, 1733-1738.

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

MORISHITA, T. Y., P. P. AYE, B. S. HARR, C. W. COBB, J. R. CLIFFORD, 1997: Evaluation of an avian-specific probiotic to reduce the colonization and shedding of *C. jejuni* in broilers. Avian Dis. **41**, 850-855.

ROBYN, J., G. RASSCHAERT, D. HERMANS, F. PASMANS, M. HEYNDRICKX, 2013: Is allicin able to reduce *C. jejuni* colonization in broilers when added to drinking water? Poult. Sci. **92**, 1408-1418.

SANTINI, C., L. BAFFONI, F. GAGGIA, M. GRANATA, R. GASBARRI, D. DI GIOIA, B. BIAVATI, 2010: Characterization of probiotic strains: An application as feed additives in poultry against *C. jejuni*. Int. J. Food Microbiol. **141**, S98-S108.

SOLIS DE LOS SANTOS, F., M. HUME, K. VENKITANARAYANAN, A. M. DONOGHUE, I. HANNING, M. F. SLAVIK, V. F. AGUIAR, J. H. METCALF, I. REYES-HERRERA, P. J. BLORE, D. J. DONOGHUE, 2010: Caprylic acid reduces enteric *Campylobacter* colonization in market-aged broiler chickens but does not appear to alter cecal microbial populations. J. Food Prot. **73**, 251-257.
P-016 (ID 51)

# THE EFFECTS OF DIETARY PLANT ALKALOIDS SUPPLEMENTATION WITH VARIOUS LEVELS OF DIGESTIBLE LYSINE-METHIONINE AND CALCIUM-NON PHYTIC PHOSPHORUS RATIO ON PRODUCTIVE PERFORMANCES, CARCASS CHARACTERISTICS AND MEAT QUALITY OF BROILER

N. Chauychuwong, and K. Soisuwan

Dept. of Animal Science, Faculty of Agriculture, Rajamangala University of Technology, Srivijaya, Nakhon Si Thammarat, Thailand, 80110

**Corresponding author:** nantanachu@yahoo.com

## Abstract

This experiment was conducted to evaluate the effects of plant alkaloids (Sangrovit® Extra), digestible lysine-methionine (Lys-Met) and calcium-non phytic phosphorus (Ca-NPP) ratio on productive performances, carcass characteristics and meat quality of broiler. A 2x2x2 factorial arrangement of treatments comprising 2 dietary Lys-Met ratio (1.0:0.052% and 0.90:0.47%) and 2 levels of Ca-NPP ratio (1.09:0.49% and 0.98:0.44%) with and without Sangrovit® Extra (non and 60 mg/kg feed) was used. Cobb mixed sex broiler chicks (n = 360, 1-d old) were randomly divided into 8 dietary treatments (5 replicates of 9 birds per treatment). Birds were given 2 phase feeding regimen consisting of starter and grower periods. The results had shown that supplementation of plant alkaloids (Sangrovit® Extra) at the levels of 60 mg/kg feed tend to improve survival rate and significantly improve wing percentage (P<0.05). The dietary digestible Lys:Met ratio at the levels of 1.0:0.52% had better growth performance in term of weight gain (WG), average daily gain (ADG), feed efficiency and improve breast percentage when compare with the level of 0.90:0.47%. It also found that breast meat from 0.90:0.47% of digestible Lys:Met had significantly higher a* (redness) value than the control group (P<0.01). The interaction effect (P<0.01) between Sangrovit® Extra and digestible Lys:Met ratio were observed on WG, ADG and feed conversion rate (FCR). It was concluded, based on the data, that supplementation of Sangrovit® Extra at the levels of 60 mg/kg feed could improve survival rate and carcass yield in term of wing percentage. Digestible Lys:Met ratio at the levels of 1.0:0.52% had better growth performance while the level of 0.90:0.47% had higher significantly a* value of breast meat.

**Keywords:** digestible lysine-methionine ratio, calcium-non phytic phosphorus ratio, plant alkaloids, broiler

## Introduction

In recent years, the potential of the emphasis on alternative feed additive such as phytobiotics in reducing the extraction of N and P by farm animal to protect the environment, have received interest in the use of low protein, amino acid-supplemented diets for various kind of poultry (Keshavarz and Austic, 2004). The objective of this study was conducted to evaluate the effect of plant alkaloid (Sangrovit® Extra) in form of quaternary benzophenanthridine alkaloids (QBA) and protopine alkaloids (PA) from Phytobiotics (Thailand) Co; Ltd. supplementation with various levels of digestible lysine-methionine and calcium-non phytic phosphorus ratio on growth performance, carcass characteristics and meat quality of broilers.

## Material and Methods

Three hundred and sixty Cobb mixed sex broiler chicks of 1 day of age in five replications per treatment (9 birds/replication) were used. A 2x2x2 factorial arrangement of eight experimental diets comprising 2 levels of digestible lysine-methionine ratio (1.0:0.052%; control and 0.90:0.47%) with 2 levels of Ca-NPP ratio (1.09:0.49%; control and 0.98:0.44%) and supplementation with plant alkaloid (Sangrovit® Extra) in the levels of 0 and 60 mg/kg feed of isoenergetic diets. The reduced-digestible lysine-methionine and calcium-non phytic phosphorus ratio had approximately 10 percentage units lower than their control counterparts. Dietary treatments met the minimum requirements for another digestible essential amino acids recommended by NRC (1994). Birds were reared in open-side house and were given 2 phase feeding regimen consisting of starter and grower period. Feed and water were provided *ad libitum* throughout the experimental period (42 days of age). In each period, body weight, feed intake were recorded. FCR and ADG were calculated of each period and overall period. At the end of the experiment, 2 birds per replicate were killed by cervical dislocation. Carcass, abdominal fat and meat yields were collected and calculated relative to the final body weight. Breast samples were evaluated for drip loss, cooking loss, Warner-Bratzler Shear force (WSF) and meat color in term
of L*(lightness), a* (redness) and b* (yellowness). Data were analyzed by ANOVA and compared the difference between dietary treatment means by Duncan's New Multiple Range Test (Steel and Torrie, 1980) for a factorial completely randomized design. A significance level of P<0.05 was used.

## Results

Supplementation of plant alkaloids (Sangrovit® Extra) and various level of Ca-NPP ratio had no significant effect on growth performance of broilers. (Table 1). However, it was found that broiler fed with control group of digestible Lys:Met ratio had significantly more WG, ADG and feed efficiency than that the reduced-digestible Lys:Met ratio group. It was also found the interaction effect between Sangrovit® Extra and digestible Lys:Met ratio on WG, ADG and FCR (P<0.05). There has no differences (P>0.05) between treatments and interaction on eviscerated weight (EW) and abdominal fat. There were numerical trends for increasing wing yield as supplemented Sangrovit® Extra and increasing breast yield as due to the level of 1.0:0.52% digestible Lys-Met ratio in diet (P<0.05). The supplementation Sangrovit® Extra, level of digestible Lys:Met and Ca-NPP ratio in diets had no influence on drip loss and WSF of breast meat. However, Sangrovit® Extra supplementation had significantly higher (P<0.05) cooking loss than the control group (P<0.05). No differences in L*, a* and b* color exited between breast meat from broilers fed diets with various Sangrovit® Extra and level of Ca-NPP ratio. The a* value of meat from 0.9:0.47% digestible Lys:Met treatment was significantly higher (P<0.01) than the 1.0:0.52% digestible Lys:Met group (p<0.01). Results of this study were agreement with the report of Lilly et al. (2011) who reported that the dietary amino acid density in broiler feed had no effect on cooking loss and shear force of meat.

## Conclusions

The present study had shown that supplementation of plant alkaloids (Sangrovit® Extra) at the levels of 60 mg/kg feed could improve survival rate and carcass yield in term of wing percentage. It was also found that dietary digestible Lys:Met ratio at the levels of 1.0:0.52% had better growth performance while the level of 0.90:0.47% has significantly higher meat color in term of a* (redness) value.

## Acknowledgments

The authors would like to special thank for Phytobiotics Co Ltd. (Thailand) for the grant and financial, particularly for the material products (Sangrovit® Extra). The authors also thank you for Department of Animal Science, Faculty of Agriculture, Rajamangala University of Technology Srivijaya for the convenient equipment and experimental unit.

<table><caption>**Table 1.** Effects of dietary plant alkaloids (Sangrovit® Extra) supplementation with various Level of digestible Lys-Met and Ca-NPP ratio on growth performance and carcass composition of broilers</caption><thead><tr><th>Factors</th><th>WG<sup>2</sup><br>(g)</th><th>ADG<sup>2</sup><br>(g/d)</th><th>FCR<sup>1</sup></th><th>EW<br>(%)</th><th>Breast<sup>1</sup><br>(%)<sup>1</sup></th><th>Wing<sup>1</sup><br>(%)</th><th>Drumstick<br>(%)</th><th>Thigh<br>(%)</th></tr></thead><tbody><tr><td>Sangrovit®Extra (mg/kg feed)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0</td><td>2184.90</td><td>52.02</td><td>1.83</td><td>81.91</td><td>23.70</td><td>9.57<sup>b</sup></td><td>12.42</td><td>15.19</td></tr><tr><td>60</td><td>2176.70</td><td>51.83</td><td>1.82</td><td>78.18</td><td>24.91</td><td>10.45<sup>a</sup></td><td>12.18</td><td>15.96</td></tr><tr><td>Digestible Lys:Met (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1.0:0.52</td><td>2221.34<sup>a</sup></td><td>52.89<sup>a</sup></td><td>1.79<sup>b</sup></td><td>79.50</td><td>25.13<sup>a</sup></td><td>10.03</td><td>12.24</td><td>15.74</td></tr><tr><td>0.90:0.47</td><td>2140.26<sup>b</sup></td><td>50.96<sup>b</sup></td><td>1.86<sup>a</sup></td><td>80.59</td><td>23.48<sup>b</sup></td><td>9.99</td><td>12.36</td><td>15.41</td></tr><tr><td>Ca-NPP (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1.09:0.49</td><td>2176.72</td><td>51.83</td><td>1.83</td><td>80.05</td><td>24.57</td><td>9.82</td><td>12.24</td><td>15.58</td></tr><tr><td>0.98:0.44</td><td>2184.88</td><td>52.02</td><td>1.82</td><td>80.04</td><td>24.04</td><td>10.20</td><td>12.68</td><td>15.57</td></tr><tr><td>Pooled SEM</td><td>70.88</td><td>1.69</td><td>0.07</td><td>4.45</td><td>2.01</td><td>0.96</td><td>1.62</td><td>1.42</td></tr></tbody></table>

<footnote n="1">¹ a,b Means within a column with different superscripts differ significantly (P<0.05)</footnote>

<footnote n="2">² a,b Means within a column with different superscripts differ significantly (P<0.01)</footnote>
<table><caption>**Table 2.** Effects of dietary plant alkaloids (Sangrovit® Extra) supplementation with various Level of digestible Lys-Met and Ca-NPP ratio on meat quality of broilers</caption><thead><tr><th rowspan="2">Factors</th><th rowspan="2">Drip loss (%)</th><th rowspan="2">Cooking loss<fnref n="1" /> (%)</th><th rowspan="2">WSF (kg)</th><th colspan="3">Meat color</th></tr><tr><th>L*</th><th>a*<fnref n="2" /></th><th>b*</th></tr></thead><tbody><tr><td>Sangrovit®Extra (mg/kg feed)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0</td><td>9.43</td><td>29.30<sup>b</sup></td><td>2.65</td><td>53.71</td><td>4.99</td><td>15.65</td></tr><tr><td>60</td><td>10.64</td><td>31.92<sup>a</sup></td><td>2.70</td><td>54.79</td><td>4.66</td><td>15.48</td></tr><tr><td>Digestible Lys:Met (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1.0:0.52</td><td>10.23</td><td>30.04</td><td>2.67</td><td>54.22</td><td>4.44<sup>b</sup></td><td>15.52</td></tr><tr><td>0.90:0.47</td><td>9.84</td><td>31.19</td><td>2.68</td><td>54.27</td><td>5.21<sup>a</sup></td><td>15.61</td></tr><tr><td>Ca-NPP (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1.09:0.49</td><td>9.95</td><td>29.75</td><td>2.62</td><td>54.35</td><td>4.94</td><td>15.22</td></tr><tr><td>0.98:0.44</td><td>10.12</td><td>31.48</td><td>2.73</td><td>54.15</td><td>4.71</td><td>15.91</td></tr><tr><td>Pooled SEM</td><td>2.89</td><td>2.76</td><td>0.47</td><td>2.05</td><td>0.87</td><td>1.17</td></tr></tbody></table>

## Literature

KESHAVARZ, K., R.E. AUSTIC, 2004: The use of low-protein, low-phosphorus, amino acid and phytase supplemented diets on laying hen performance and nitrogen and phosphorus excretion. Poult. Sci. **83**, 75-83.

NRC, National Research Council, 1994: Nutrient Requirements of Poultry. 9 rev. ed. National Academy Press, Washington, DC. USA. 240p.

LILLY, R.A., M.W. SCHILLING, J.K. SILVA, J.M. MENTION, A. CORZO, 2011: The effects of dietary amino acid density in broiler feed on carcass Characteristics and meat quality. J. Appl. Poult. Res. **20**:56-67.

STEEL, R., J. H. TORRIE, 1980: Principle and procedures of statistics. A Biometrical Approach 2ed, Mc Grow Hill Book Co. New York.

<footnote n="1">¹a,b Means within a column with different superscripts differ significantly (P<0.05)</footnote>

<footnote n="2">²a,b Means within a column with different superscripts differ significantly (P<0.01)</footnote>
P-017 (ID 329)

EFFICACY OF COATED CALCIUM BUTYRATE ON PERFORMANCE, EGG QUALITY AND INCUBATION PARAMETERS IN BROILER BREEDERS

E. Delezie<fnref n="1" />, B. Wegge<fnref n="2" />, L. Goethals<fnref n="2" /> and L. Maertens<fnref n="1" />

Corresponding author: Beatrice@sanluc.be

**Abstract**

Organic acids salts are alternatives for antibiotic growth promoters, banned since 2006. Besides antibacterial and anti-inflammatory properties, it is known that butyric acid is an energy source for epithelial cells, improving gut morphology and absorption of nutrients in the intestinal tract. For an effective dietary application specific coating is required to bypass crop and/or stomach and to insure gradually release in the intestinal tract. Objective of this trial was to determine for the first time the efficacy of coated calciumbutyrate in broiler breeders, on performance and incubation parameters.

Two dietary treatments with 5 replicates of 31 broiler breeders and 3 roosters were used. Dietary treatments consisted of control diet and control diet supplemented with coated calciumbutyrate (*Globamax Enhanced*, Sanluc International nv). Productive performance of broiler breeders, egg quality, and incubation parameters were investigated.

Supplementing feed with coated calciumbutyrate resulted in significant increased laying rate and egg mass, resulting in 12.8% more eggs per hen. Also feed conversion was significantly lower in coated calciumbutyrate Although hens produced more eggs, egg shell and egg quality parameters remained equal. In coated calciumbutyrate a numerical higher haugh unit was seen. Fertility, hatchability and chick quality were each time numerically better after 1,2 and 3 months of treatment. Also late embryo mortality was lower. Therefore, a positive effect of coated calciumbutyrate was seen on performance, internal egg quality and some incubation parameters.

**Keywords:** broiler breeders, calcium butyrate, production, incubation

**Introduction**

Organic acids are considered potential alternatives to the banned antibiotic growth promoters. It is well recognized that butyric acid is an energy source for the epithelial cells of the villi, improving the gut morphology and absorption of nutrients in the small intestines. Moreover, the efficacy of butyrate likely will be improved if it is protected from immediate absorption in the upper tract by a coating. Several trials have been executed with calciumbutyrate (CB) to judge its effect on the zootechnical performances of broilers and diet's digestibility. Coated calciumbutyrate (CCB) showed a consistent positive effect on broilers' performances during different trials. Moreover, an amino acid sparing effect was demonstrated while it enhanced the metabolic energy value of wheat based diets.

This experiment was designed to determine the effect of supplementing the coated commercial CB (*Globamax Enhanced*, Sanluc International nv) at a dosage of 1000gram/tonne on 1) the productive performance of broiler breeders, 2) their egg quality, 3) fertility, embryonic mortality and hatchability and on 4) chick quality of their progeny.

**Materials and Methods**

**Birds and Housing.** Ross 308-broiler breeder hens (Belgabroed nv, Merksplas, Belgium) were housed in the poultry experimental facility at the Institute for Agricultural and Fisheries Research (ILVO) under standard conditions of lighting, heating and ventilation. The breeders were randomly placed in 10 pens, 31 hens per pen and 3 cockerels.

**Dietary Treatments.** The trial was designed as a randomized complete block design with 2 dietary treatments and 5 replicates each. The first treatment was a control treatment (Tr1) and treatment 2 consisted of the control treatment supplemented with Globamax Enhanced (1000gram/tonne). The feed was a wheat-corn based diet. During the trial period, feed was restricted based on body weight in order to obtain a body weight gain recommended by the Ross Management Guide (Aviagen EPI BV, Roermond, the Netherlands).

<footnote n="1">¹ ILVO-Belgium, Scheldeweg 68, B-9090 Melle, Belgium</footnote>

<footnote n="2">²Sanluc International nv., Schoolstraat 49, B-9860 Gijzenzele, Belgium</footnote>
**Performance Measurements.** 10 hens per pen were weighed individually on a 2-weekly basis and the average body weight was calculated per pen. Eggs were collected and counted daily. Laying rate (%), egg mass and feed conversion ratio (FCR) was calculated All pens were monitored daily for hen mortality and morbidity.

**Egg Quality Measurement and Incubation Parameters** After 1, 2 and 3 months, 120 eggs (60 eggs/treatment) were collected and analyzed for egg quality. Haugh unit was calculated using the following formula (MONIRA et al., 2003): $HU = 100 \log (H - (1.7 \times W^{0.37}) + 7.6))$. Egg shell deformation was assessed under a 500 mg load, in micrometers. Yolk color was determined using a 15-point scale color fan of DSM. The thickness of the eggshell, after the removal of the egg content and egg membranes, was measured with a micrometer at 3 different areas. An average value was calculated based on these 3 measurements. At each timepoint, 1000 eggs per treatment were collected and incubated to assess fertility, hatchability, embryonic mortality and chick quality. From 75 chicks of each treatment chick quality was scored as described by TONA et al. (2003).

**Statistical analysis** incubation parameters and broiler breeders performance were analysed using a factorial ANOVA to investigate the effect of dietary treatment and age and their interaction using Statistica 10 (StatSoft Inc., Tulsa, OK, USA). The egg quality measurements, with egg as a single replicate, were analyzed using a factorial ANOVA. Significant differences between treatments were tested with LSD test. Differences due to treatments or age were considered significant if the P-value was < 0.05.

## Results

The main results of the broiler breeders experiment are presented in table 1. No dietary effect was found on body weight and egg weight. Laying rate (P=0.018) and egg mass (P=0.002), however, were significantly higher if hens were fed the Globamax Enhanced supplemented diet. This effect was noticed weekly. As feed intake was equal for all diets, FCR was only influenced by laying rate, resulting in a significantly lower feed conversion if broiler breeders were given the Globamax Enhanced supplemented diet. No age or treatment effect was observed on the incidence of cracked, soft-shelled and floor eggs.

It is obvious from the results that egg quality did not change with increasing age of the hens, eggs became only heavier and more yellow. Probably, duration of the trial was too short to observe an age effect. There was also no significant effect of diet on egg quality. For the internal egg quality, a numerical increase was seen if breeders were fed the Globamax Enhanced supplemented diet compared to their control counterparts.

<table><caption>**Table 1:** Effect of age and treatment (T) on egg weight (g), laying percentage (%), egg mass (g) and feed conversion ratio (FCR) of broiler breeders.</caption><thead><tr><th>Age</th><th>T</th><th>Egg weight (g)</th><th>Laying percentage, %</th><th>Egg Mass, g</th><th>FCR</th></tr></thead><tbody><tr><td rowspan="6">Age effect</td><td>55</td><td>70.4</td><td>55.4<sup>ab</sup></td><td>39.2<sup>ab</sup></td><td>4.67</td></tr><tr><td>56</td><td>69.8</td><td>54.8<sup>ab</sup></td><td>38.2<sup>ab</sup></td><td>4.70</td></tr><tr><td>57</td><td>70.6</td><td>56.3<sup>a</sup></td><td>39.7<sup>a</sup></td><td>4.46</td></tr><tr><td>58</td><td>70.3</td><td>48.6<sup>ab</sup></td><td>34.2<sup>ab</sup></td><td>5.24</td></tr><tr><td>59</td><td>72.7</td><td>48.7<sup>ab</sup></td><td>35.4<sup>ab</sup></td><td>5.40</td></tr><tr><td>60</td><td>71.1</td><td>44.2<sup>b</sup></td><td>31.4<sup>b</sup></td><td>6.55</td></tr><tr><td>64</td><td>69.7</td><td>46.9<sup>ab</sup></td><td>32.6<sup>ab</sup></td><td>5.71</td></tr><tr><td rowspan="2">T effect</td><td>A</td><td>70.8</td><td>47.4<sup>b</sup></td><td>33.5<sup>b</sup></td><td>5.78<sup>b</sup></td></tr><tr><td>B</td><td>70.5</td><td>54.0<sup>a</sup></td><td>38.1<sup>a</sup></td><td>4.71<sup>a</sup></td></tr><tr><td rowspan="3">P-value</td><td>Age</td><td>NS</td><td>0.008</td><td>0.01</td><td>NS</td></tr><tr><td>T</td><td>NS</td><td>0.018</td><td>0.002</td><td>0.04</td></tr><tr><td>A x T</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr></tbody></table>

<footnote>a-b means with the same letter are not significantly different from each other at P ≤ .05.</footnote>
Egg weight at start of the incubation was comparable between dietary treatments. Chick weight was consequently also comparable between both groups of broiler breeders. For each incubation, at the age of 57, 62 and 66 weeks of age, numerical higher percentages of fertility and hatchability and lower embryonic mortality were obtained for the Globamax Enhanced supplemented fed breeders. The quality of chicks was very high although age of broiler breeders was high. As broiler breeders were already more than 50 weeks of age at the start of the trial, low fertility, hatchability percentage and high embryonic mortality percentages were reached. If broiler breeders were 45 weeks of age, cockerels were spiked, by replacing the worst cockerel out of the 3, by a younger new cockerel for the last time. The worse fertility and hatchability results in this trial, might thus be a consequence of the bad performance of the cockerels as the trial started about 10 weeks after spiking. It is known that both fertility and hatchability improve after cockerels are spiked.

## Conclusions

It can be concluded that a positive effect after supplementation of coated calcium butyrate was seen on performance of the broiler breeders, their internal egg quality and some incubation parameters.

## Literature

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

TONA, K., F. BAMELIS, B., DE KETELAERE, V., BRUGGEMAN, V.B.M., MORAES, J.BUYSE, O. ONAGBESAN, AND E. DECUYPERE, 2003: Effects of Egg Storage Time on Spread of Hatch, Chick Quality, and Chick Juvenile Growth. Poult. Sci. **82**: 736-741.
P-018 (ID 239)

HISTOLOGICAL STUDIES ON THE EFFECT OF PREBIOTICS ON BURSA OF FABRICIUS AND CAECAL TONSILS OF BROILERS

*¹El Habback, H. A., ¹El-Saba, A. A., ¹AbdRabou, M. I., ²EL-Werfaly, E. F.*

¹Cytology and Histology Department, Faculty of Veterinary Medicine, Cairo University, Egypt

²Cytology & Histology, Faculty of Veterinary Medicine, Alfateh University, Libya

*Corresponding author: hanyhaback@yahoo.com

**Summary**

Evaluation of effects of prebiotics on histological structure of bursa of Fabricius and cecal tonsils of broiler chickens was studied. A total of 4000 Arbor Acres broilers from local company (Ommat) were raised from 1-35 days old, and divided into two equal groups; control and treated one. The treated birds received water mixed with (INMUNAIR® 17.5) based prebiotic at inclusion rate 0.5-1 ml/ litter while the control ones received water free from a prebiotic. It was found that the used prebiotic enhanced the growth rate levels and immunity in treated birds as well as increased the cellular density of bursal lymph nodules with increasing in epithelial surface area. In addition, significant appearance of ileum and cecal tonsils with increase of musculosa thickness, villus and intestinal crypt depth and length were observed. It was noticed an increase in mucin dynamics in bursa of Fabricius and caecal tonsils of treated group. Conclusion, By adding prebiotics to poultry diets, Zootechnical performance body weight, feed conversion ratio and GIT health status were significantly improved.

**Keywords:** Prebiotic, Immunity, Broilers, Performance, Histology.

**Introduction**

A prebiotic substance has been defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of limited number of bacteria in the colon CHOUDHARI (2008). Several studies have shown that addition of prebiotics to the ration of broilers, layers and pigs improved performance through improving gut microflora (XU et al., 2003). Using of compounds that may have prebiotic effects is a possible way to improve intestinal health and animal performance in the absence of antibiotic growth promoters (KIM et al., 2011). From the available literature, the study of the effects of prebiotics on the Bursa of Fabricius and caecal tonsils of chickens is limited. So, the present study was conducted to evaluate the effect of prebiotics supplementation on bursa of Fabricius and gut performance as well as growth performance of broilers.

**Material and Methods**

The commercial corn soya bean starter and grower finisher diets were offered to the birds from one day old to 21 and from 22 to 35 days of age, respectively. The treated birds were received drinking water mixed with inactivated cells of *Propionibacterium acnes* and lipopolysaccharide from *E. coli* (INMUNAIR® 17.5) based prebiotic. Administration in drinking water was at a rate of 0.5-1 ml / Litter, while the control bird received water free from prebiotic.

**Administration of the product according to the recommendation of the manufacturer.**

<table><caption>Table (1): Administration guidelines of INMUNAIR® 17.5 (*)</caption><thead><tr><th>Animal</th><th>Age</th><th>Prophylactic use Breeding</th><th>Booster of antibiotic treatment</th><th>Dose</th></tr></thead><tbody><tr><td rowspan="3">Broiler</td><td>During the first 5 days, or days 3, 4 and 5</td><td>1 x 500 ml / 2.000 l. water</td><td></td><td>1 ml / 10 k bw</td></tr><tr><td>3<sup>rd</sup> week of life</td><td>1 x 500 ml / 2.500 l. water (during 2 days)</td><td></td><td>0.5 ml /10 k bw</td></tr><tr><td>Variable</td><td></td><td>0.5 ml / 10 kbw (during antibiotic treat.)</td><td></td></tr></tbody></table>

Immediately after sacrificing birds, specimens of bursa of Fabricius, ileum and caeci at the level of cecal tonsils were collected and fixed in 10% neutral buffered formalin solution. The trimmed sections were processed and stained by histological and histochemical stains according to BANCROFT and STEVENS (1996).
## Results

**Growth performance:** The result showed that dietary treatment of broiler chickens by (INMUNAIR® 17.5) at inclusion rate of 1 ml / litter showed significant higher body weight in the treated birds than control ones (Tables 2).

<table><caption>**Table (2):** Feed intake and growth performance</caption><thead><tr><th></th><th>Average body weight gain<br>(gm/bird/day)</th><th>Average feed intake<br>(gm/bird/day)</th><th>Feed conversion ratio</th></tr></thead><tbody><tr><td>days</td><td>1-21 22-35 1-35</td><td>1-21 22-35 1-35</td><td>1-21 22-35 1-35</td></tr><tr><td>Control</td><td>25 80 50.5</td><td>42.5 156.6 89.7</td><td>1.7 1.95 1.77</td></tr><tr><td>Prebiotic</td><td>25 98 55.5</td><td>42.6 166.12 92.1</td><td>1.7 1.69 1.65</td></tr></tbody></table>

Feed conversion ratio was calculated by the average body weight gain divided by corresponding average feed intake.

## Discussion

The effect of prebiotics on body weight gain showed some controversial results. The available information for different prebiotic used in poultry industry showed conflicting results. ORTIZ et al., (2009) and ALZUETA et al., (2010) found that by using inulin (Chicory extract) as dietary inclusion with dose up to 20g/kg did not significantly affect the body weight gain, feed intake or feed conversion ratio of broiler chickens. In contrast to these findings, REBOLE et al., (2010) showed that the inclusion of inulin in control diet improved the body weight gain of birds. In the mean time, using others prebiotic in several studies like Fermacto prebiotics (PIRAY et al., 2007 and KHAKSAR et al., 2008) has effect on growth performance. The current study showed that feeding INMUNAIR® 17.5 had no significant effect on body weight gain in starting period with improvement by bird getting older and significant effect gained in week 4th and 5th. In earlier study by ZELENIKA et al., (2007), it was found that using INMUNAIR® 17.5 in broiler induced significant improvement of zootechnical performance.

## Conclusion

By adding prebiotics to poultry diets, Zootechnical performance body weight, feed conversion ratio and GIT health status were significantly improved. Moreover, Prebiotics improve immune system to reduce colonization by pathogens.

## Literature

ALZUETA, C., M.L. RODRIGUEZ, L.T. ORTIZ, A. REBOLE, J. TREVINO, 2010: Effects of inulin on growth performance, nutrient digestibility and metabolisable energy in broiler chickens. Brit. Poult. Sci., 51, 393-398.

BANCROFT, O.D., A. STEVENS, 1996: Theory and practice of histological Technique. 4th Ed. Chirchil living stone, Edinburgh London and New York.

CHOUDHARI, A., S. SHINDE, B.N. RAMTEKE, 2008: Prebiotics and Probiotics as Health promoter. Vet. World, 1, 59-61.

KHAKSAR, V.G., A. KERMANSHAHI, H. MOVASSEGHIAN, A.R. JAMSHIDI. A., 2008: Effect of prebiotic Fermacto on Gut Development and performance of broiler chickens fed diet low in digestible amino acids. J. Anim. Vet. Adv., 3, 251-257.

KIM, G.B., Y.M. SEO, C.H. KIM, I.K. PAIK, 2011: Effect of dietary prebiotic supplementation on the performance, intestinal microflora, and immune response of broilers. Poult. Sci., 90, 75-82.
ORTIZ, L.T., OODRIGUEZ, M.L., ALZUETA, C., REBOLE, A., TREVINO, J., 2009: Effect of inulin on growth performance, intestinal tract sizes, mineral retention and tibial bone mineralization in broiler chickens. Brit. Poult. Sci., 50, 325-332.

PIRAY, A.H., H. KERMANSHahi, A.M. TAHMASBI, J. BAHRAMPOUR, 2007: Effects of cecal cultures and aspergillus meal prebiotic (Fermacto) on growth performance and organ weights of broiler chickens. Int. J. Poult. Sci., 6, 340-344.

REBOLÉ, A., L.T. ORTIZ, M.L. RODRÍGUEZ, C. ALZUETA, J. TREVIÑO, S. VELASCO, 2010: Effects of inulin and enzyme complex, individually or in combination, on growth performance, intestinal microflora, cecal fermentation characteristics, and jejuna histomorphology in broiler chickens fed a wheat and barley-based diet. Poult. Sci., 89, 276-278.

Xu, Z.R., C.H. Hu, M.S. Xia, X.A. Zhan, M.Q. Wang, 2003: Effects of dietary fructo oligosaccharide on digestive enzyme activities, intestinal microbiota and morphology of male broilers. Poult. Sci., 2, 1030-1036.

ZELENIKA, T.A., M. MIKEC, V. SAVIC, M. BALENOVIC, M. TIŠLJAR, M. DINARINA-SABLIC, 2007: Effect of "Inmunair® 17,5" to productivity and health of broilers. Proceedings of the 7th Symposium of Poultry Days 2007 with international participation, Poreč, Croatia, 7-10 May, 2007 pp. 51-55.
P-020 (ID 100)

EFFECT OF BEE POLLEN ON GROWTH PERFORMANCE AND IMMUNE STATUS OF YOUNG BROILER CHICKS

A.R. Fazayeli-Rad, N. Afzali, and M.R. Asghari

Dept. of Animal Science, University of Birjand, Birjand, Iran

Corresponding author: fazaelirad.alireza@gmail.com

**Abstract**

This study was carried out to evaluate the effect of supplementing a corn-soybean meal-based diet with bee pollen (BP) on growth performance and immune response. A total of 160 Ross 308 male broiler chicks were assigned to 4 dietary treatments (0, 10, 15 and 20 g kg⁻¹ of BP feed inclusion), consisting of 4 pens of 10 birds each, and were fed experimental diets from 1 to 21 d of age. Then, all birds received a common finisher diet from 22 to 35 d of age. The antibody response to sheep red blood cells (SRBC) was measured at 35 d. In general, average daily feed intake and relative weight of gizzard were not affected by the dietary groups compared to the control birds (*P*>0.05). Dietary supplementation increased (*P*<0.01) body weight, average daily gain (ADG), and improved feed conversion ratio (FCR). Relative pancreas, liver, bursa, intestine and spleen weights significantly increased (*P*<0.01) at 20 g kg⁻¹ of BP. Inclusion of 20 g kg⁻¹ of BP in the diet significantly increased (*P*<0.01) antibody response against SRBC at 35 d of age. In conclusion, these findings demonstrate that dietary supplementation with BP improved broiler growth performance and immunity. Thus, BP may be used as alternative feed additives in the poultry industry to improve immune response of young broiler chicks.

**Keywords:** bee pollen, broiler chick, immune response, performance

**Introduction**

The bee pollen (BP) is created in the male sexual organ of the flower – anthers with the purpose of fertilizing the stigma. It is the main source of proteins for bees (FAZAYELI-RAD et al., 2015). Honeybee collects pollen and mixes it with its own digestive enzymes. Although there is no specific chemical composition, BP is a rich source of protein (25%), essential amino acids, linolenic, palmitic and linoleic acids, flavonoids, carotenoids and phytosterols (XU et al., 2009). Bee pollen contains several antioxidant active compounds such as catalase, peroxidase and superoxide dismutase. Moreover, BP has antibacterial properties and increases the body's resistance to infection. Bee pollen makes the plumage of birds fuller and more vivid in color. OLIVEIRA et al. (2013) found that inclusion of BP increased number of humoral immune cells, phagocytes and red blood cells, and accelerated antibody production in broilers chickens. It seems that BP contains most of the essential nutritional elements necessary for growth and development in monogastric animals and humans (FAZAYELI-RAD et al., 2015). Therefore, the present study was conducted to evaluate the effects of BP on growth performance, relative weight of visceral organs, and immune response in broiler chicken.

**Material and Methods**

One hundred and sixty 1-d-old male broiler chicks of the Ross strain were obtained from a commercial hatchery and were used in this experiment, which lasted for 35 d. The chicks were individually weighed upon arrival and divided into 16 groups of 10 birds balanced for body weight. Birds had ad libitum access to feed and water during the experimental period and light was provided for 24 h throughout the experiment. Four corn-soybean meal-based diets were fed from 1 to 21 d of age. The diets included basal diet supplemented with 0, 10, 15 and 20 BP g kg⁻¹ of feed. The BP was mainly pollen collected by the honey bee from *Amygdalus scoparia* in the spring season. A common finisher diet (12.68 MJ metabolizable energy/kg and 190 g crude protein/kg) was fed from 22 to 35 d of age to all birds.

The BW and feed consumption for each cage were determined and were used to calculate data on average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR). At 21 d of age, three randomly chosen birds per replicate (12 birds per treatment) were killed by cutting the carotid artery and jugular vein, and processed manually and collections were made following a 4-h fast. The weights of the pancreas, liver, gizzard, intestine, bursa and spleen were recorded. The organs were cleaned with physiological saline solution, dried with filter paper and weighed. The weight of each organ was expressed relative to the total body weight (g/100 g BW). At 28 days of age, 3 birds from each pen were inoculated with a volume of 0.2 mL from a 8% suspension of sheep red blood cell (SRBC). Seven days after each injection, blood was collected in nonheparinized tubes by puncturing the brachial vein. Serum was obtained
by centrifuging at 2,500 × g for 15 min at 25°C, and stored at -30°C until assayed. Individual serum samples were analyzed for antibody responses against SRBC by ELISA technique using commercial kits, and the plates were read at 405 nm on an ELISA reader. The collected data were subjected to analysis of variance (ANOVA) using general linear model (GLM) of SAS (SAS Inst. Inc. Carry, NC) software and the corresponding means were compared by Tukey-Kramer's test. For the different statistical tests, significance was declared at *P* < 0.05.

## Results

The effect of dietary BP on growth performance is summarized in Table 1. The mean BW at 21 and 35 days of age, ADG and FCR were significantly improved by addition of BP in the diet (*P*<0.01), and it was maximized at 15 g kg⁻¹ supplemental BP. No change in relative gizzard weight was observed with dietary BP, while inclusion of 20 gkg⁻¹ of BP in the diet, significantly increased the relative weight of pancreas (*P*<0.05), liver (*P*<0.01), intestine (*P*<0.01), bursa (*P*<0.05), spleen (*P*<0.01), and antibody response against SRBC (*P*<0.01) at 35 d of age compared with birds fed the control diet (Table 2).

## Conclusions

The present study indicates that the addition of BP to diets improves the growing performance of broiler chickens compared with the un-supplemented, control treatment. The positive effect of BP on growth performance of broiler chickens could be related to the high contents of nutrients, among which essential amino acids, polyunsaturated fatty acids (PUFA), and some trace minerals (FAZAYELI-RAD et al., 2015) and these nutrients can be easily absorbed as BP components. It has been shown that nutrients content of BP pass from the stomach into the blood stream within 2 h after ingestion (XU et al., 2009).

During the experiment, we noticed that two important digestive organs, the liver and pancreas, were larger in the pollen group compared to the control group. The BP may promote effective contact between the digestive enzymes and their corresponding substrates, leading to significant modifications of the structure and function of intestine and organs (OLIVEIRA et al., 2013). To adapt to those changes, the activities of the intestinal secretory mechanisms may be enhanced. Thus, this may lead to increases in the size of the gastro-intestinal tract (GIT), pancreas, and liver. OLIVEIRA et al. (2013) indicated that this increased size of intestine and GIT could be an adaptive response to an increased need for digestive enzymes. It seems that BP raised digestive enzymes secretion in the gut for better digestion. OLIVEIRA et al. (2013) also implied that the reduction in relative organ weight had a direct economic benefit, as the dressing yield of broilers should increase proportionally. BP is rich in nutrients, such as proteins, amino acids, vitamins, and trace elements, among others, that promote faster cell proliferation and differentiation in the immune system of birds. According to WANG et al. (2007) dietary BP may stimulate the T lymphocytes formation, as well as the division, proliferation and activity of thymus cells. WANG et al. (2007) found that the thymus, spleen and bursa were heavier in broilers fed the diets with BP. To our knowledge, there is no published data on effect of the BP that was used in this study on antibody response to SRBC. The SRBC, a non-pathogenic antigen, is classified as a thymus-dependent antigen that obviously needs the help of T lymphocytes to produce antibodies. Bee pollen that is rich in flavonoids extends the activity of vitamin C, acts as antioxidant and may therefore enhance the immune function (WANG et al., 2007). Bee pollen is confirmed an interesting resource, able to improve productive performance and immune status of broiler chickens. Standing our results, BP could be used as a supplement in the young broiler chicks during the starter phase with positive effects on growth rate and feed conversion ratio, without increasing the final cost of feed. In addition, it could be a strategy to improve resistance to disease in broiler chickens and organic production.
<table><caption>**Table 1.** Effect of bee pollen (BP) supplementation on average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) of broiler chicks during 0 to 21d of age</caption><thead><tr><th></th><th>BW at 21 d (g)</th><th>ADG (g)</th><th>ADFI (g)</th><th>FCR (g/g)</th></tr></thead><tbody><tr><td>Basal diet (BD)</td><td>632<sup>b</sup></td><td>27.89<sup>b</sup></td><td>46.67</td><td>1.67<sup>a</sup></td></tr><tr><td>BD+ 10 BP g kg<sup>-1</sup></td><td>713<sup>a</sup></td><td>31.69<sup>a</sup></td><td>48.80</td><td>1.54<sup>b</sup></td></tr><tr><td>BD+ 15 BP g kg<sup>-1</sup></td><td>727<sup>a</sup></td><td>32.38<sup>a</sup></td><td>46.94</td><td>1.45<sup>c</sup></td></tr><tr><td>BD+ 20 BP g kg<sup>-1</sup></td><td>725<sup>a</sup></td><td>32.3<sup>a</sup></td><td>48.07</td><td>1.49<sup>bc</sup></td></tr><tr><td>Pooled SEM</td><td>19.30</td><td>0.83</td><td>0.89</td><td>0.03</td></tr><tr><td>P value</td><td>0.003</td><td>0.002</td><td>0.486</td><td>0.001</td></tr></tbody></table>

<table><caption>**Table 2** Effect of bee pollen (BP) supplementation on relative weight of organs (g/ 100 g BW) at 21 day of age, and SRBC responses at 35 day of age</caption><thead><tr><th></th><th>Pancreas</th><th>Liver</th><th>Gizzard</th><th>Intestine</th><th>Bursa</th><th>Spleen</th><th>SRBC (log<sup>2</sup>)</th></tr></thead><tbody><tr><td>Basal diet (BD)</td><td>0.23<sup>b</sup></td><td>1.97<sup>b</sup></td><td>1.59</td><td>2.43<sup>b</sup></td><td>0.18<sup>b</sup></td><td>0.15<sup>b</sup></td><td>6.25<sup>b</sup></td></tr><tr><td>BD+ 10 BP g kg<sup>-1</sup></td><td>0.23<sup>b</sup></td><td>1.99<sup>b</sup></td><td>1.61</td><td>2.45<sup>b</sup></td><td>0.20<sup>a</sup></td><td>0.15<sup>b</sup></td><td>6.50<sup>b</sup></td></tr><tr><td>BD+ 15 BP g kg<sup>-1</sup></td><td>0.24<sup>ab</sup></td><td>1.98<sup>b</sup></td><td>1.61</td><td>2.45<sup>b</sup></td><td>0.20<sup>a</sup></td><td>0.16<sup>b</sup></td><td>7.37<sup>ab</sup></td></tr><tr><td>BD+ 20 BP g kg<sup>-1</sup></td><td>0.25<sup>a</sup></td><td>2.05<sup>a</sup></td><td>1.63</td><td>2.62<sup>a</sup></td><td>0.21<sup>a</sup></td><td>0.20<sup>a</sup></td><td>8.13<sup>a</sup></td></tr><tr><td>Pooled SEM</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.03</td><td>0.01</td><td>0.01</td><td>0.41</td></tr><tr><td>P value</td><td>0.019</td><td>0.001</td><td>0.10</td><td>0.002</td><td>0.034</td><td>0.001</td><td>0.010</td></tr></tbody></table>

## Literature

FAZAYELI-RAD, A.R., N. AFZALI, H. FARHANGFAR, M.R. ASGHARI, 2015: Effect of bee pollen on growth performance, intestinal morphometry and immune status of broiler chicks. Europ. Poult. Sci. **79**, DOI: 10.1399/eps.2015.86.

OLIVEIRA, M.C., D.M. SILVA, F.C. LOCH, P.C. MARTINS, D.M.B. DIAS, G.A. SIMON, 2013: Effect of bee pollen on immunity and tibia characteristics in broilers. Braz. J. Poult. Sci. **15**, 323-328.

WANG, J., S. LI, Q. WANG, B. XIN, H. WANG, 2007: Trophic effect of bee pollen on small intestine in broiler chickens. J. Med. Food. **102**, 276-280.

XU, X., L. SUN, J. DONG, H. ZHANG, 2009: Breaking the cells of rape bee pollen and consecutive extraction of functional oil with supercritical carbon oxide. Innovative Food Sci. Emerg. Technol. **10**, 42-46.

<footnote><sup>a-c</sup>Mean values within a column with no common superscript differ significantly from each other (P<0.05).</footnote>

<footnote><sup>a-c</sup>Mean values within a column with no common superscript differ significantly from each other (P<0.05).</footnote>
P-021 (ID 101)

EFFECT OF BEE POLLEN ON INTESTINAL MORPHOMETRY AND SELECTED BLOOD PARAMETERS IN BROILER CHICKS

A.R. Fazayeli-Rad, N. Afzali, and M.R. Asghari

Dept. of Animal Science, University of Birjand, Birjand, Iran

Corresponding author: fazaelirad.alireza@gmail.com

Abstract

This study was conducted to determine the effect of bee pollen (BP) supplementation on intestinal morphometry and selected blood metabolites. A total of 160 Ross 308 male broiler chicks were assigned to 4 dietary treatments (0, 10, 15 and 20 g kg⁻¹ of BP feed inclusion), consisting of 4 pens of 10 birds each, and were fed experimental diets from 1 to 21 d of age. In general, phosphorus concentration in serum and morphometric indices of duodenum were not affected by the dietary groups compared to the control birds (*P*>0.05). Dietary supplementation decreased total serum cholesterol and triacylglycerides levels (*P*<0.01) at 20 g kg⁻¹ of BP. Addition of BP to the diets increased (*P*<0.01) villus length, crypt depth and villus length to crypt depth ratio in the jejunum compared to the control diet. In conclusion, these findings demonstrate that dietary supplementation with BP improved morphometric indices. Therefore, the present study suggests that a level of 15 g/kg BP supplementation in the diet is optimal for absorptive capacity in broilers.

**Keywords:** bee pollen, broiler chick, morphometric indices, blood parameters

Introduction

Pollen grains, a fine powder-like material, are the male seeds of a flower blossom which has been gathered by the bees and to which special elements (such as; Fe, Cu, Zn, Cr and Mn) from the bees has been added (FAZAYELI-RAD et al., 2015). Like propolis, other well-known honey bee products, the exact chemical composition depends on the plants the worker bees gather the pollen from, and can vary variable (OLIVEIRA et al., 2013). Addition of BP to the broiler chicken diet reduced cholesterol and triacylglycerides. Also, High-density lipoproteins (HDL) increased, whereas low-density lipoproteins (LDL) decreased (ATTIA et al., 2011). In addition, BP was able to improve gut health and normal gastrointestinal functions in poultry, protect kidney, and decrease triacylglycerides, cholesterol, creatinine, and blood urea N in rats (ATTIA et al., 2014). The present study was conducted to determine the effects of BP as dietary supplement on growth intestinal morphometry and blood biochemical parameters of broiler chickens.

Material and Methods

One hundred and sixty 1-d-old male broiler chicks of the Ross strain were obtained from a commercial hatchery and were used in this experiment, which lasted for 21 d. The chicks were individually weighed upon arrival and divided into 16 groups of 10 birds balanced for body weight. Birds had ad libitum access to feed and water during the experimental period and light was provided for 24 h throughout the experiment. Four corn-soybean meal-based diets were fed from 1 to 21 d of age. The diets included basal diet supplemented with 0, 10, 15 and 20 BP g kg⁻¹ of feed. The BP was mainly pollen collected by the honey bee from *Amygdalus scoparia* in the spring season.

At 21 days of age, 5 mL of blood was collected from the killed birds (the same birds were used for slaughtering) in non-heparinised tubes and kept on ice slashes until they were subjected to serum collection by centrifuging the whole blood sample at 2,500×g for 15 min. Before serum was collected, blood samples were held at room temperature for 4 h and were then refrigerated overnight. The concentrations of Ca, P, triacylglycerides and total cholesterol in serum samples were subsequently analyzed using colorimetric methods on an automatic biochemical analyzer, following manufacturer instructions for the corresponding reagent kit.

Fragments of approximately 5 cm were obtained from each of the following segments of the small intestine: the duodenum and the jejunum to evaluate the intestinal morphology. The fragments from each segment were then placed on polystyrene sheets, opened longitudinally, washed in saline solution, fixed in 10% formaldehyde solution, dehydrated and embedded in paraffin. The measurements of villus length and crypt depth were taken using a light microscope and a system that analyses computerised images, and the data were used to calculate the villus length to crypt depth ratio. The height of 30 villi and the depth of 30 crypts were measured from each segment and replicate, and the means were utilized for statistical analysis. The collected data were subjected to analysis of variance (ANOVA) using general linear
model (GLM) of SAS (SAS Inst. Inc. Carry, NC) software and the corresponding means were compared by Tukey-Kramer's test. For the different statistical tests, significance was declared at *P*< 0.05.

## Results

The effect of dietary treatment on cholesterol, triacylglycerides, Ca, and P concentrations in serum are presented in Table 1. At 21 d of age, none of the dietary treatments significantly altered the serum Ca and P concentrations. In this study, birds fed the dietary BP had lower cholesterol and triacylglycerides concentrations (*P*<0.01) compared with values for the control group. The effects of dietary BP on duodenal and jejunal criteria are presented in Table 2. Regarding the duodenal morphology, inclusion of BP in the diets did not have a significant effect on duodenum measured criteria. There were significant differences between treatments for jejunum morphometry. In this regard, feeding both 10 and 15 g kg⁻¹ of BP increased villus length and villus: crypt ratio as compared to those fed control and 20 g kg⁻¹ BP fed birds (*P*< 0.05). Jejunal crypt depth was higher in birds fed 15 g kg⁻¹ BP compared to control group (*P*< 0.05).

## Conclusions

Reduction of total cholesterol and triacylglycerides concentrations is the most beneficial effect on the lipid profile. It has been reported that bee pollen in a diet normalizes cholesterol and triacylglycerides levels in blood (FAZAYELI-RAD et al., 2015). After regular consumption of bee pollen, reports have been made of reduced cholesterol and triacylglycerides (ATTIA et al., 2014). The decrease in plasma lipids and cholesterol could be due to phospholipids and PUFA particularly linolenic acid (ATTIA et al., 2014). The small intestine is the main site for digestion and absorption of nutrients. The intestinal villi in the pollen group were longer and thicker than those of the control group. This enhanced intestinal villi development greatly increases the functional surface area of the mucosa for nutrient absorption in the small intestine. As a consequence, the absorption efficiency of the small intestine is accordingly increased. In addition, higher villus-to-crypt ratios suggest higher digestive and absorptive capacities (FAZAYELI-RAD et al., 2015). Thus, the increased ratios obtained here can be attributed to the beneficial effect of the BP in controlling proliferation of pathogenic bacteria and avoiding possible damage to the intestinal mucosa, which could result in reduced dimensions of the villi (ATTIA et al., 2011). Furthermore, the glands of the small intestine play an important role in the digestion of nutrients. The increased function of these glands also significantly contributes to the absorption of the nutrients in small intestines. Thus, addition of BP to the basic daily diet stimulates the digestive and absorptive functions of broilers and, in turn, promotes growth and development (ATTIA et al., 2014). Standing our results, morphometric indices of broilers were greater as the level of BP increased. Therefore, the present study suggests that a level of 15 g kg⁻¹ BP supplementation in the diet is optimal for maximizing absorptive capacity in broilers. In addition, it could be a strategy to decrease cholesterol in broilers meat.

<table><caption>**Table 1.** Effect of bee pollen (BP) supplementation on blood parameters (mg/dL) at 21 day of age</caption><thead><tr><th></th><th>Cholesterol</th><th>Triacylglycerides</th><th>Calcium</th><th>Phosphorus</th></tr></thead><tbody><tr><td>Basal diet (BD)</td><td>126.10<sup>a</sup></td><td>64.63<sup>a</sup></td><td>9.19</td><td>9.13</td></tr><tr><td>BD+ 10 BP g kg<sup>-1</sup></td><td>110.25<sup>b</sup></td><td>47.25<sup>b</sup></td><td>9.23</td><td>9.31</td></tr><tr><td>BD+ 15 BP g kg<sup>-1</sup></td><td>105.62<sup>b</sup></td><td>45.75<sup>b</sup></td><td>8.54</td><td>8.64</td></tr><tr><td>BD+ 20 BP g kg<sup>-1</sup></td><td>98.13<sup>b</sup></td><td>59.63<sup>ab</sup></td><td>8.38</td><td>8.39</td></tr><tr><td>Pooled SEM</td><td>3.66</td><td>4.03</td><td>0.33</td><td>0.26</td></tr><tr><td>P value</td><td>0.001</td><td>0.005</td><td>0.184</td><td>0.163</td></tr></tbody></table>

<footnote><sup>a-c</sup>Mean values within a column with no common superscript differ significantly from each other (*P*<0.05).</footnote>
<table><caption>**Table 2.** Effect of bee pollen (BP) supplementation on intestinal morphometry at 21 day of age</caption><thead><tr><th rowspan="2"></th><th colspan="3">Duodenum</th><th colspan="3">Jejunum</th></tr><tr><th>Villus length (µm)</th><th>Crypt depth (µm)</th><th>Ratio</th><th>Villus length (µm)</th><th>Crypt depth (µm)</th><th>Ratio</th></tr></thead><tbody><tr><td>Basal diet (BD)</td><td>1225</td><td>178</td><td>6.89</td><td>1148<sup>b</sup></td><td>158<sup>b</sup></td><td>7.26<sup>b</sup></td></tr><tr><td>BD+ 10 BP g kg<sup>-1</sup></td><td>1219</td><td>175</td><td>6.96</td><td>1187<sup>a</sup></td><td>160<sup>ab</sup></td><td>7.41<sup>a</sup></td></tr><tr><td>BD+ 15 BP g kg<sup>-1</sup></td><td>1248</td><td>181</td><td>6.88</td><td>1202<sup>a</sup></td><td>162<sup>a</sup></td><td>7.42<sup>a</sup></td></tr><tr><td>BD+ 20 BP g kg<sup>-1</sup></td><td>1235</td><td>175</td><td>7.06</td><td>1165<sup>ab</sup></td><td>159<sup>b</sup></td><td>7.33<sup>ab</sup></td></tr><tr><td>Pooled SEM</td><td>2.4</td><td>0.6</td><td>0.02</td><td>1.3</td><td>1.1</td><td>0.05</td></tr><tr><td>P value</td><td>0.087</td><td>0.092</td><td>0.102</td><td>0.021</td><td>0.014</td><td>0.035</td></tr></tbody></table>

## Literature

ATTIA, Y.A., A.E. AL-HAMID, M.S. IBRAHIM, M.A. AL-HARTHI, F. BOVERA, A.S. ELNAGGAR, 2014: Productive performance, biochemical and hematological traits of broiler chickens supplemented with propolis, bee pollen, and mannan oligosaccharides continuously or intermittently. *Lives. Sci.* **164**, 87-95.

ATTIA, Y.A., A.M. AL-HANOUN, A.E. TAG EI-DIN, F. BOVERA, E. SHEWIKA, 2011: Effect of bee pollen levels on productive, reproductive and blood traits of NZW rabbits. *J. Anim. Phys. Anim. Nutr.* **95**, 294–303.

FAZAYELI-RAD, A.R., N. AFZALI, H. FARHANGFAR, M.R. ASGHARI, 2015: Effect of bee pollen on growth performance, intestinal morphometry and immune status of broiler chicks. *Europ. Poult. Sci.* **79**, DOI: 10.1399/eps.2015.86.

OLIVEIRA, M.C., D.M. SILVA, F.C. LOCH, P.C. MARTINS, D.M.B. DIAS, G.A. SIMON, 2013: Effect of bee pollen on immunity and tibia characteristics in broilers. *Braz. J. Poult. Sci.* **15**, 323-328.

<footnote><sup>a-c</sup>Mean values within a column with no common superscript differ significantly from each other (P<0.05).</footnote>
P-023 (ID 332)

EXAMINATION OF BROILER CHICKEN VILLI EVOLUTION AFTER HERBAL EXTRACT FEEDING

Dr. János Gál PhD., Dipl. ECZM

Szent István University, Faculty of Veterinary Science, H-1078 Budapest, István u. 2., Hungary

e-mail: gal.janos@aotk.szie.hu

**Introduction**

Intestinal digestion of birds depends on the ingredients of the consumed feed (BARNES et al. 1972; ENGSTÖM 1992). The stability of the intestinal flora and the integrity of the intestinal epithelium are important for optimal digestion and absorption (COLEO et al. 1984, HORVÁTH-PAPP 2008). The absorption of the nutrition and the utilization of the feed depend on the condition of the villi and the epithelial cells covering them (YANG et al. 2009, COLEO et al 1984).

The aim of our study was to evaluate the differences in the individual morphological parameters of the villi in broiler chicken apropos of feeding with herbal extract supplemented feed. Changes in the villi significantly influence the digestive and the absorptive processes in the intestine, which affect the feed conversion and thus have an economic importance in broiler production.

**Material and methods**

For the study four groups of 115 broiler chicken were considered, out of the four three groups were infected with 2ml 10⁸CFU/ml Clostridium Perfringes bacteria three consecutive times (18, 21 and 24 days of age) using a crop probe. The negative control group was not infected and was fed with feed having the nutritive value specified in the technology instructions, ad libitum. The positive control group was fed the same feed, but was infected with Clostridium Perfringes. The feed of the other two groups were supplemented with Zn-bacitracin (1kg/T) and with complex plant extracts (Herbanoplex) (1kg/T) respectively.

The chickens were slaughtered according to animal rights laws and a small piece from the small intestine was fixed in 8% formaldehyde, and was segmented following paraffin embedding. After hematoxilin-eozin dying the length and the width of the villi as well as the depth of the crypts were investigated.

**Results and discussion**

Values of the measured villi parameters are summarized in table 1.

<table><caption>**Table 1.** The development of the parameters of the villi in case of the different experimental settings</caption><thead><tr><td></td><td>Length of villi</td><td>Width of villi measured in the middle</td><td>Depth of the crypts</td></tr></thead><tbody><tr><td>Negative control</td><td>2,21</td><td>0,196</td><td>0,330</td></tr><tr><td>Positive control</td><td>2,09</td><td>0,184</td><td>0,273</td></tr><tr><td>Treated with plant extracts</td><td>2,30</td><td>0,234</td><td>0,323</td></tr><tr><td>Treated with Zn bacitracin</td><td>2,20</td><td>0,189</td><td>0,285</td></tr></tbody></table>

Examining the results, we found that the villi parameters were more favorable in case of the group where the feed was supplemented with herbal extracts, then in case of the other groups. The villi were 5 % longer and 20 % wider as compared to the other groups. Moreover the crypts between the villi were deeper as well.

These results show that when the chicken consumed feed supplemented with herbal extract the absorptive surface of the intestine showed an improved development, which in turn results in a positive effect on feed conversion ratio and body weight improvement. We think that the additive containing complex plant extracts had a synergic positive effect on the villi and the epithelial cells when mixed into the feed. At the same time these results suggest the possibility to decrease antibiotics use with the application of herbal extracts.
Reference

BARNES, E. M. – MEAD, G. C. – BARNUML, D. A. – HARRY, E. G.: The intestinal flora of the chicken in the period 2 to 6 weeks of age, with particular reference to the anaerobic bacteria. British Poultry Science. 1972. 13. 311-326.

COLEO, P. J. – BAGUST, T. J. – IRELAND, L.: Development of the normal gastrointestinal microflora of specific pathogen-free chickens. Journal Hygenie. 1984. 92. 79-87.

ENGSTÖM, B.: Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Veterinaria Scandinavica. 1992, 33(4):369-378

HORVÁTH-PAPP, I.: Practical guide to broiler health management. Betűvet Ltd., Hungary, Budapest. 2008

YANG, Y. – IJL, P. A. – CHOCT, M.: Dietary modulation of gut microflora in broiler chickens: a review of the role of six kinds of alternatives to in-feed antibiotics. World's Poultry Science Journal. 2009. 65. 97-108.
P-027 (ID 200)

EFFECT OF DIETARY INCLUSION OF MEDIUM CHAIN FATTY ACIDS AND BUTYRATE ON PERFORMANCE OF LAYING HENS AND FATTY ACIDS PROFILE; SENSORY AND TEXTURAL PROPERTIES OF EGGS

R. Gruzauskas<fnref n="1" />, V. Sasyte<fnref n="1" />, V. Kliseviciute<fnref n="1" />, A. Raceviciute-Stupeliene<fnref n="1" />, V. Slausgalvis<fnref n="2" />, J. Al-Saifi<fnref n="2" />, A. Mieze-liene<fnref n="3" />, G. Alencikiene<fnref n="3" /> and G.J. Svirmickas<fnref n="4" />

**Corresponding author:** Romas.Gruzauskas@lsmuni.lt

**Abstract**

The aim of the present study was to investigate the effect of dietary supplementation of the mixture comprising medium chain fatty acids, butyrate, essential oils and *Saccharomyces cerevisiae* on laying hens' performance, fatty acids profile, sensory and textural properties of eggs. For the period of 8 weeks, 36 Hisex Brown laying hens were randomly assigned to 3 dietary treatments: 1) control diet, 2) diet supplemented with the mixture of medium chain fatty acids, butyrate, essential oils and *Saccharomyces cerevisiae* (*Lumance*®) at the level of 0.5 g/kg of feed, 3) diet supplemented with the same mixture as in treatment 2, but at a higher amount (1.5 g/kg of feed). All laying hens were kept under the same conditions. Sensory and textural attributes of the eggs (1 days and after storage for 28 days) were also evaluated. The results of the present study showed that neither 0.5 g/kg nor 1.5g/kg supplementation of feed additive affected egg production and feed consumption. No significant difference in saturated, unsaturated and polyunsaturated fatty acids between the treatments was observed. The diet supplemented with 0.5 g/kg of tested feed additive decreased toughness of egg yolk (1 days). After 28 days of storage, the hardness and solidity of egg white were higher in the 2nd and 3rd treatment groups (P<0.05). The inclusion of 1.5 g/kg feed additive had no significant effect on sensory properties of eggs, except the hardness – the egg white was decreased and egg yolk was increased compared with other treatments (P<0.05).

**Keywords:** laying hens, MCFA, butyrate, fatty acids, sensory and textural properties

**Introduction**

One of the most important quality criteria of table eggs are sensory characteristics. Sensory attributes and chemical composition of eggs can easily be influenced by feeding of hens (HUOPALAHTI et al., 2007), e.g. by the choice of feeding stuffs and feed additives.

Previous studies reported that organic acids such as fumaric, propionic and butyric acids and their salts have shown variable effects on egg production and egg quality parameters. These discrepancies would be related to the source, the amount of organic acids used, location, environmental condition and the composition of the diets (GRASHORN et al., 2013).

However, little information has been published on the synergetic effects of the *Lumance*® on fatty acids profile, sensory and textural properties of eggs as feed additive in laying hens diets. Therefore, the aim of this study was to investigate the effects of dietary supplementation of the *Lumance*® on laying hens performance, fatty acids profile, sensory and textural properties of eggs.

**Material and Methods**

Feeding trial was conducted on 36 laying hens of *Hisex Brown* strain at the age of 30 weeks divided into 3 groups (12 laying hens in each group) and fed a compound feed diets (Control group), supplemented with the mixture of medium chain fatty acids, butyrate, essential oils and *Saccharomyces cerevisiae* (Experimental I group, *Lumance*®, 0.5 g/kg of feed) and supplemented with the same mixture as in Experimental I group, but at a higher amount (Experimental II group, 1.5 g/kg of feed). During the feeding trial, the laying hens were held in the individual cages with stationary drinking-bowl and feed box under the same feeding and holding conditions. The birds were fed 8 weeks with wheat-soybean meal diet 125 g per day. Diets composition was adjusted to the respective requirements of the National Research Council (NRC, 1994).

<footnote n="1">¹ Institute of Animal Rearing Technology, Lithuanian University of Health Sciences Veterinary Academy, Kaunas, Lithuania</footnote>

<footnote n="2">² INNOV AD NV/SA, Essen, Belgium</footnote>

<footnote n="3">³ Laboratory of Sensory Science, Food institute of Kaunas University of Technology, Kaunas, Lithuania</footnote>

<footnote n="4">⁴ Institute of Animal Science, Lithuanian University of Health Sciences Veterinary Academy, Kaunas, Lithuania</footnote>