P-057 (ID 53)

EFFECTS OF ENHANCED ACIDIFIERS ON THE REDUCTION OF E. COLI IN CHALLENGED TURKEYS

S. Pruckner<fnref n="1" />, A. Kovács<fnref n="1" />, L. Vandi<fnref n="1" />, P. Massi<fnref n="2" /> and G. Tosi<fnref n="2" />

**Corresponding author:** sabine.pruckner@biomin.net

**Abstract**

A combination of synergistically acting organic acids and a permeabilizing substance in water application can reduce the microbial contamination in turkeys. An experiment was conducted to confirm these effects using water application with a blend of formic, acetic and propionic acid and a permeabilizing substance (Biotronic® Top liquid, BIOMIN, Austria). 60 day-old female turkey poults were randomly assigned to three groups with 20 turkeys each. A control group (NC) received a commercial diet with no antibiotic or NGP, trial group (NGP) received water supplemented with Biotronic® Top liquid 1.25 ml/l of water and positive control group (PC) received water supplemented with Enrofloxacin 0.5 ml/l of water. On day 10 the animals in all groups were challenged with E. coli O78. The trial lasted for 30 days. Bacterial counts of the intestinal tract were recorded on day 20 and 30. On day 20, E. coli counts of NGP group (5.42ª ± 1.07) were significantly (p<0.05) lower than NC (7.42ᵇ ± 0.78) and PC group (7.24ᵇ ± 1.18). On day 30, E. coli counts of NGP group (5.12ª ± 0.44) were as well significantly (p<0.05) lower compared to NC (8.09ᵇ ± 0.21) and PC group (7.89ᵇ ± 0.31). The results of the NGP group showed a significant (p<0.05) reduction of E. coli counts in the intestinal tract compared to NC and PC group.

**Introduction**

Antibiotic growth promoters (AGP) are antibiotics which are added to animal feed or water at subtherapeutic levels to improve the growth performance of the animals and to decrease the incidence of diseases (Khachatourians, 1998). AGP's have been used in animal production worldwide since 1946 (Moore et al., 1946). Nowadays antibiotic resistance has become a hot topic, as the use of antibiotics over time in animal health and growth promotion has resulted in bacterial resistance to antibiotics (Smith et al., 2010, Van de Bogaard and STOBBERRING, 1999). The problem of bacterial resistance has resulted in banning and/or regulating the use of AGP for growth promoting issues by a number of countries (Council regulation 98/2821/CEE, 1998; WHO, 2001). This leaded to the development of alternative products, which can be used instead of AGP's for growth promoting issues of the animals.

Organic acids (OA) as alternatvies to AGP have increasingly and successfully been supplemented in poultry feed. It is generally known that acidifiers improve poultry performance. Acids reduce the microbial load, aid the buffering capacity and reduce the pH in the feed and in the gastrointestinal tract, which enhances protein digestion (Eidelsburger and Kirchgessner, 1994, Overland et al., 2000). With the reduction of gastric pH, pepsinogen is activated into pepsine and besides that, the pH of the stomach is also brought closer to the optimum for pepsin activity (Mroz, 2000).

Despite the fact that natural replacements for AGPs are known, fighting Gram-negative bacteria is still a challenge. The outer membrane of Gram-negative bacteria acts as a barrier that prevents antimicrobial compounds from entering the cell and destroying vital functions (Canovas et al., 2005). This outer membrane can be destabilized by a permeabilizing substance (PS), which makes the bacterial cell more susceptible to antimicrobial compounds (Alakomi, 2007). The Biomin® Permabilizing Complex, is a PS, which boosts the antimicrobial effectivity of the organic acids.

**Material and Methods**

The trial was carried out at IZSLER, Italy, using 60 1-day old female turkeys (Big 6 Aviagen®), which were housed in 3 isolators. Turkeys were assigned to 3 treatments. The negative control group received a standard diet with no natural or antibiotic growth promoter, positive control group was fed a standard diet and water was supplemented with Enrofloxacin at 0.5 ml/l of water from day 11 to 20 and trial group was fed a standard diet and water supplemented with Biotronic® Top liquid at 1.25 ml/l of water (pH 4.5) from day 0 to 30.

The duration of the trial was 30 days. On day 10 the birds in all groups were orally challenged with E. coli O78 (1.38x10⁸ CFU/ml). 10 birds from each isolator were sacrificed 10 days post infection (dpi) and the rest of the birds

<footnote n="1">¹ Biomin Holding GmbH, Industriestraße 21, 3130 Herzogenburg, Austria</footnote>

<footnote n="2">² Istituto Zooprofilattico Sperimentale di Forlì, Forlì, Italy</footnote>
were sacrificed 20 dpi at the end of the trial. Bacterial counts (*E. coli*) of the intestinal tract posterior to duodenal loop were recorded on day 20 (10 dpi) and 30 (20 dpi) of the trial.

Bacteriological analysis: The samples were diluted 10-fold in Buffered Peptone Water (BPW). All samples were serially diluted to appropriate levels for plating. An aliquot from each sample (100 µL) was spiral plated on the following: Tryp-tone Bile X-Glucuronide (TBX) agar for the isolation and counting of *E. coli*. Any TBX plate containing isolated *E. coli* colonies were subcultured and sent to IZSLER headquarter laboratories for "O" serotyping to see if the isolated sero-type matched the *E. coli* serotype used in the challenge.

## Results

The results of the present study showed that water supplementation with Biotronic® Top liquid reduced *E. coli* counts in turkeys' intestinal tract. *E. coli* counts in the intestinal tract on day 20 and 30 of the experiment, in the trial group were significantly (p<0.05) reduced compared to NC and PC. On day 20, *E. coli* counts of the trial group were 27.0 % lower than NC and 25.1 % lower than PC. On day 30, *E. coli* counts of trial group were decreased by 36.7 % and 35.1 % in comparison to NC and PC.

<table><caption>**Table 1.** Bacterial counts intestinal tract of control and trial groups at day 20 and 30 of the experiment (log CFU/g)</caption><thead><tr><th>Parameter</th><th>NC</th><th>PC</th><th>NGP</th></tr></thead><tbody><tr><td>No of animals</td><td>20</td><td>20</td><td>20</td></tr><tr><td><i>E. coli</i> count day 20</td><td>7.42<sup>b</sup> ± 0.78</td><td>7.24<sup>b</sup> ± 1.18</td><td>5.42<sup>a</sup> ± 1.07</td></tr><tr><td><i>E. coli</i> count day 30</td><td>8.09<sup>b</sup> ± 0.21</td><td>7.89<sup>b</sup> ± 0.31</td><td>5.12<sup>a</sup> ± 0.44</td></tr></tbody></table>

## Conclusions

Supplementation of water with Biotronic® Top liquid reduced the number of *E. coli* in the intestinal tract of challenged turkeys. Based on this result it might be assumed that a healthy gut of the animals from first day of life is maintained, which protects against intestinal infections (Goren et al., 1988, Goren et al., 1984, Nurmi and Rantala, 1973). Potentially harmful bacteria, like *E. coli* can cause infections (Jeurissen et al., 2002), compete with the host for nutrients, stimulate the turnover of epithelial cells, secrete toxic compounds and increase the inflammatory processes in the animal. In this case the animals lose energy to the microflora and growth performance is reduced (Drew et al., 2003, Lan et al., 2005, Richards et al., 2005).

Based on the results of this experiment, the trial group outperformed the PC group, showing that Biotronic® Top liquid was more effective than Enrofloxacin in terms of antimicrobial efficacy, which confirms that NGP's can easily be replaced by AGP's.

## Literature

* ALAKOMI, H.-L. 2007. Weakening of the Gram-negative bacterial outer membrane-A tool for increasing microbiological safety. *VTT Publications*.

* CANOVAS, M., TORROGLOSA, T. & IBORRA, J. 2005. Permeabilization of Escherichia coli cells in the biotransformation of trimethylammonium compounds into L-carnitine. *Enzyme and Microbial Technology*, 37, 300-308.

* DREW, M. D., VAN KESSEL, A. G. & MAENZ, D. D. 2003. Absorption of methionine and 2-hydroxy-4-methylthiobutoanic acid in conventional and germ-free chickens. *Poultry science*, 82, 1149-1153.

* EIDELSBURGER, U. & KIRCHGESSNER, M. 1994. Zum Einfluß organischer Säuren und Salze im Futter auf die Masticierung von Broilern. *Archiv für Geflügelkunde*, 58, 268-277.

* GOREN, E., DE JONG, W. A., DOORNENBAL, P., BOLDER, N. M., MULDER, R. W. & JANSEN, A. 1988. Reduction of salmo-nella infection of broilers by spray application of intestinal microflora: a longitudinal study. *Veterinary Quarterly*, 10, 249-255.

* GOREN, E., DE JONG, W. A., DOORNENBAL, P., KOOPMAN, J. P. & KENNIS, H. M. 1984. Protection of chicks against salmo-nella infection induced by spray application of intestinal microflora in the hatchery. *Veterinary Quarterly*, 6, 73-79.
JEURISSEN, S. H. M., LEWIS, F., VAN DER KLIS, J. D., MROZ, Z., REBEL, J. M. J. & TER HUURNE, A. A. H. M. 2002. Parameters and techniques to determine intestinal health of poultry as constituted by immunity, integrity, and functionality. *Current Issues in Intestinal Microbiology*, 3, 1-14.

KHACHATOURIANS, G. G. 1998. Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria. *Canadian Medical Association Journal*, 159, 1129-1136.

LAN, Y., VERSTEGEN, M. W. A., TAMMINGA, S. & WILLIAMS, B. A. 2005. The role of the commensal gut microbial community in broiler chickens. *World's Poultry Science Journal*, 61, 95-104.

MOORE, P., EVENSON, A., LUCKEY, T., MCCOY, E., ELVEHJEM, C. & HART, E. 1946. Use of sulfasuxidine, streptothricin, and streptomycin in nutritional studies with the chick. *Journal of Biological Chemistry*, 165, 437-441.

MROZ, Z. Year. Supplementary organic acids and their interactive effects with microbial phytase in diets for pigs and poultry. In: Proc. Annu. Conf. Phytase in Animal Nutrition. Lublin, Poland, 2000.

NURMI, E. & RANTALA, M. 1973. New aspects of Salmonella infection in broiler production [20]. *Nature*, 241, 210-211.

OVERLAND, M., GRANLI, T., KJOS, N., FJETLAND, O., STEIEN, S. & STOKSTAD, M. 2000. Effect of dietary formates on growth performance, carcass traits, sensory quality, intestinal microflora, and stomach alterations in growing-finishing pigs. *JOURNAL OF ANIMAL SCIENCE-MENASHA THEN ALBANY THEN CHAMPAIGN ILLINOIS-, 78*, 1875-1884.

RICHARDS, J. D., GONG, J. & DE LANGE, C. F. M. 2005. The gastrointestinal microbiota and its role in monogastric nutrition and health with an emphasis on pigs: Current understanding, possible modulations, and new technologies for ecological studies. *Canadian Journal of Animal Science*, 85, 421-435.

SMITH, M., JORDAN, D., CHAPMAN, T., CHIN, J.-C., BARTON, M., DO, T., FAHY, V., FAIRBROTHER, J. & TROTT, D. 2010. Antimicrobial resistance and virulence gene profiles in multi-drug resistant enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhoea. *Veterinary Microbiology*, 145, 299-307.

VAN DE BOGAARD, A. & STOBBERRING, E. 1999. Antibiotic usage in animals: impact on bacterial resistance and public health. *Drugs*, 58, 589-607.
P-061 (ID 152)

SECONDARY PLANT COMPOUNDS AND THEIR ANTIMICROBIAL PROPERTIES TO INHIBIT THE GROWTH OF ANTIBIOTIC RESISTANT BACTERIA

T. Rothstein and I. Heinzl

EW Nutrition GmbH, Visbek, Germany

Corresponding author: timo.rothstein@ew-nutrition.com

**Abstract**

After the European ban of antibiotics as growth promoters in animal production, there is higher interest in naturally derived and generally recognised as safe substances as an appropriate replacement. The emergence of antibiotic resistant pathogens makes the demand for alternatives even more urgent.

In contrast to plant extracts, that can vary considerably in the content of bioactive compounds and thus also in their efficacy, purified secondary plant compounds allow the production of a standardised blend and thus a reproducible mode of action. Appropriate mixtures should be composed with a broad efficacy against major gram negative and positive pathogens.

In an attempt to determine the antimicrobial efficacy of Activo® Liquid, a water soluble mixture of standardized secondary plant compounds, we conducted in vitro experiments on major poultry pathogens by Agar Diffusion Tests. Here, the blend of secondary plant compounds showed an antimicrobial effect on all tested bacteria occurring in farm animals. The degree of growth inhibition positively correlated with the concentration of Activo® Liquid.

To further determine the antimicrobial efficacy in liquid medium, we tested Activo® Liquid against ESBL producing E.coli and Methicillin resistant S. aureus field isolates compared to their corresponding reference strains in a MIC assay.

The efficacy of Activo® Liquid against the tested strains could be demonstrated in a concentration dependent manner with antimicrobial impact at higher concentrations and bacteriostatic effects in dilutions of up to 0,1% (ESBL) and 0,2% (MRSA).

These encouraging results give reason for further investigations with Activo® Liquid in animal production.

**Keywords:** Antibiotic Resistance, Secondary Plant Compounds, ESBL, MRSA

**Introduction**

Antibiotics were and still remain the method of choice against bacterial diseases. Due to their additional positive effect on growth performance they increasingly have been used in animal husbandry. But every use of antibiotics is a selection of resistant bacteria and especially this prophylactic use at low dosage gives bacteria a better chance to adapt. For this very prophylactic and metaphylactic use secondary plant compounds could be an alternative: they are generally recognized as safe and a lot of them show antimicrobial characteristics, e.g. Carvacrol and Cinnamon aldehyde. They effectively act against Salmonella, E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Entero- and Staphylococcus and Candida albicans (Baiju and Modak, 2007; Khan et al., 2009; Yossa et al., 2011) but also against multi drug resistant strains of Escherichia coli, Klebsiella pneumoniae and Candida albicans (Khan et al., 2009). Some compounds influence digestion and others act as antioxidants. An optimal combination has both - positive influence on health and performance. As secondary plant compounds act via several modes, the question was if they can also have an effect against antibiotic resistant bacteria like ESBL producing E. coli or MRSA.

**Material and Methods**

Trial I: in so called "agar diffusion tests" (method available on request), the sensitivities of reference strains - representative for the different species of pathogens - were evaluated with different concentrations of a specific blend of secondary plant compounds. The effectiveness of the active substances was determined by the extent to which they prevent the development of bacterial overgrowth. The diameter of the bacteria free zone around an applied component can be translated into the antimicrobial efficacy against a densely grown bacterial population on a petri dish - the bigger the bacteria free zone, the higher the antimicrobial effect.
Trial II: Farm isolates of four ESBL producing *E. coli* and two Methicillin resistant *Staphylococcus aureus* strains were compared to nonresistant reference strains of the same species with respect to their sensitivity against Activo® Liquid. In a Minimal Inhibitory Concentration Assay (MIC) under approved experimental conditions (Vaxxinova Diagnostic, Muenster, Germany) the antimicrobial efficacy of Activo® Liquid in different concentrations was evaluated.

## Results

Trial I: In this trial, the blend of secondary plant compounds and organic acids (Activo® Liquid) showed an antimicrobial effect on all tested bacteria occurring in farm animals. The degree of growth inhibition positively correlated with the concentration of Activo® Liquid (see table 1).

<table><caption>**Table 1:** Inhibition of field isolated standard pathogens by different concentrations of Activo® Liquid</caption><thead><tr><th rowspan="2"></th><th colspan="3">Secondary Plant Compounds<br>(Activo® Liquid)</th></tr><tr><th>10%</th><th>2%</th><th>1%</th></tr></thead><tbody><tr><td><strong>Central Poultry Diagnostic Laboratory,<br>Kondapur, Hyderabad (India)</strong></td><td></td><td></td><td></td></tr><tr><td><em>E. coli</em> (reference strains)</td><td>++</td><td>+</td><td>+</td></tr><tr><td><em>Proteus vulgaris</em> (reference strains)</td><td>+</td><td>+</td><td>+</td></tr><tr><td><em>Pseudomonas fluorescens</em></td><td>++</td><td>+</td><td>-</td></tr><tr><td><em>Salmonella pulmorum</em></td><td>++</td><td>++</td><td>+</td></tr><tr><td><em>Salmonella gallinarum</em></td><td>++</td><td>++</td><td>+</td></tr><tr><td><em>Staphylococcus aureus</em> (reference strains)</td><td>+++</td><td>++</td><td>++</td></tr></tbody></table>

+++ 22 – 29 mm ZOI (zone of inhibition)

++ 15 – 21 mm ZOI

+ 10 – 14 mm ZOI

- < 10 mm ZOI.

Trial II: The efficacy of Activo® Liquid against the tested strains could be demonstrated in a concentration dependent manner with antimicrobial impact at higher concentrations and bacteriostatic efficacy in dilutions up to 0,1% (ESBL) and 0,2% (MRSA)(see table 2).

<table><caption>**Table 2:** Efficacy of secondary plant compounds (Activo® Liquid) against ESBL producing *E. coli* and MRSA</caption><thead><tr><th rowspan="2"></th><th colspan="4">Secondary Plant Compounds<br>(Activo® Liquid)</th></tr><tr><th>0.1%</th><th>0.2%</th><th>0.4%</th><th>1%</th></tr></thead><tbody><tr><td><strong>Laboratory: Vaxxinova, Münster, Germany</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><em>E. coli</em> Reference ATCC25922</td><td>+</td><td>++</td><td>++</td><td>++</td></tr><tr><td>ESBL 1 (Pig)</td><td>-</td><td>++</td><td>++</td><td>++</td></tr><tr><td>ESBL 2 (Pig)</td><td>+</td><td>++</td><td>++</td><td>++</td></tr><tr><td>ESBL 3 (Poultry)</td><td>+</td><td>++</td><td>++</td><td>++</td></tr><tr><td>ESBL 4 (Poultry)</td><td>-</td><td>++</td><td>++</td><td>++</td></tr><tr><td><em>S. aureus</em> Reference ATCC29213</td><td>-</td><td>+</td><td>++</td><td>++</td></tr><tr><td>MRSA 1 (Pig)</td><td>-</td><td>+</td><td>++</td><td>++</td></tr><tr><td>MRSA 2 (Pig)</td><td>-</td><td>+</td><td>+</td><td>++</td></tr><tr><td>- no effect</td><td colspan="4">+ growth inhibiting<br>++ bactericide</td></tr></tbody></table>
## Conclusions

In order to contain the emergence and spread of newly formed resistance mechanisms it is of vital importance to reduce the use of antibiotics. A general rethinking is necessary to rise to the challenge and give new approaches a chance. These approaches however, will only be successful in combination with good management practices. Antibiotics must not be used for growth promotion or metaphylactic treatment, but only as a pure curative instrument. In in vitro-trials the liquid blend of secondary plant compounds and organic acids (Activo® Liquid) showed very antimicrobial effects against prevalent livestock pathogens. This gives reason for further investigations with secondary plant compounds in animal production in order to use them for pro- and metaphylaxis and to reduce the use of antibiotics with their help in the future. The positive influence on performance parameters, as shown in many other trials, is an additional incentive for farmers to use these flavoring substances in the feed.

The high efficacy of secondary plant compounds against ESBL Producing *Escherichia coli* and Methicillin resistant *Staphylococcus aureus* (MRSA) could be a further step towards the reduction of antibiotic use. Due to the specific mode of action of antibiotics, resistance mechanisms are more likely to emerge. To support antibiotic treatment, Activo® Liquid, a blend of natural compounds with broad spectrum efficacy probably can be a safe supplement for the control of pathogenic organisms.

## Literature

BAIJU, N., S.M. MODAK, 2007: Broad-spectrum disinfectant composition containing a synergistic combination of cinnamon oil and citric acid. International Journal of Essential Oil Therapeutics 1, pp. 117-121

KHAN, R., B. ISLAM, M. AKRAM, S. SHAKIL, A. AHMAD, S. MANAZIR ALI, M. SID-DIQUI, A.U. KHAN, 2009: Antimicrobial activity of five herbal extracts against multi drug resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules 14, pp. 586-597 www.mdpi.com/journal/molecules

YOSSA, N., J. PATEL, D. MACARISIN, P. MILLNER, C. MURPH, 2012: Antibacterial activity of Cinnamaldehyde and Sporan against Escherichia Coli O157:H7 and Salmonella. University of Nebraska, Publications from USDA-ARS/UNL Faculty. Paper 1136
P-062 (ID 92)

1, 25 (OH)₂D₃-GLYCOSIDE ENHANCES GROWTH PERFORMANCE AND BONE MINERALIZATION OF BROILERS FED DIETS CONTAINING LOW AVAILABLE PHOSPHORUS IN THE PRESENCE OF PHYTASE

Y. Ruangpanit, S. Attamangkune, S. Rattanatabtimtong and C. Junchang

Department of Animal Science, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, Nakhon-Pathom 73140 Thailand

**Corresponding author:** agryos@ku.ac.th

**Abstract**

Vitamin D and its metabolites have been reported to improve phosphorus utilization. In commercial broiler diets, phytase is practically included to release phosphorus. To study the effectiveness of 1, 25 (OH)₂ D₃-glycoside when supplement to diets containing low available phosphorus (avP) in the presence of phytase, 1,400 one-day-old male broiler chicks (Ross 308) were divided into 7 dietary treatments. The diets were a positive control (PC, 0.5 and 0.45% avP in starter and grower diets, respectively), moderate avP reduction (0.3 and 0.25% avP in starter and grower diets, respectively), and high avP reduction (0.2 and 0.15% avP in starter and grower diets, respectively) supplemented with 500 U/kg phytase in combination with 100 and 200 g/ton of 1, 25 (OH)₂ D₃-glycoside (Panbonis®). Ca:avP ratio in all diets was maintained at 2:1. Each treatment consisted of 10 replications with 20 birds each. Growth performances of broilers decreased with a reduction of avP (P<0.05). The supplementation of 100 g/ton 1, 25 (OH)₂ D₃-glycoside improved broiler performances at both levels of avP reduction to the similar level of PC group (P<0.05). The supplement of 1, 25 (OH)₂ D₃-glycoside at 200 g/ton showed a superior effect on growth performances of broiler fed moderate but not high avP reduction diets (P<0.05). Similarly, bone breaking strength and bone mineralization were improved with 1, 25 (OH)₂ D₃-glycoside supplementation (P<0.05). The supplementation of 1, 25 (OH)₂ D₃-glycoside at higher level in the presence of phytase, therefore, elicits better broiler performances.

**Keyword:** Broiler, 1, 25 (OH)₂ D₃-glycoside, bone mineralization, tibia ash, Blood Ca and P

**Introduction**

Fast growing broilers are susceptible to bone abnormalities. Therefore, a good management to ensure good development of structural bone is critical for their growth performances. Phosphorus (P) is an important element for bone development. Supplementation of commercial phytase in broiler diets has commonly known to improve P digestibility and growth performance. In practical, phytase normally used in broiler diet with low available P (avP) to minimize feed cost while maintaining growth performance of broilers. However, superior effect of improving P utilization was observed when supplemented vitamin D in the presence of phytase since vitamin D helps to improve P absorption. This study was conducted to evaluate the effect 1, 25 (OH)₂ D₃-glycoside in diet containing phytase with different levels of low avP phosphorus on growth performance and bone characteristics of broiler.

**Material and Methods**

One thousand and four hundred, one day old, male broiler chicks of Ross 308 were divided into 7 dietary treatments (Table 1). Each treatment consisted of 10 replications with 20 birds per replication. All diets were formulated to be isocaloric and isonitrogenous. Phytase was supplemented at 500 FTU/. Body weight and average feed intake of all the birds were recorded at 1, 17 and 35 days of age for the calculation of feed conversion ratio. At the end of the study (35 DOA): 2 tibias (left) from 2 chicks per pen were for collected for bone breaking strength and % tibia ash and minerals content (Ca and P) analysis.
<table><caption>**Table 1. Description of treatments**</caption><thead><tr><td>Treatment</td><td>Description</td></tr></thead><tbody><tr><td>T1</td><td>Positive control with 0.50% avP (starter) and 0.45% avP (grower)</td></tr><tr><td>T2</td><td>Negative control 1 with 0.30% avP (starter) and 0.25% avP (grower)</td></tr><tr><td>T3</td><td>T2 + Phytase + 1, 25 (OH)2 D3-glycoside 100 g/ton</td></tr><tr><td>T4</td><td>T2 + Phytase + 1, 25 (OH)2 D3-glycoside 200 g/ton</td></tr><tr><td>T5</td><td>Negative control 2 with 0.20% avP (starter) and 0.15% avP (grower)</td></tr><tr><td>T6</td><td>T5 + Phytase + 1, 25 (OH)2 D3-glycoside 100 g/ton</td></tr><tr><td>T7</td><td>T5 + Phytase + 1, 25 (OH)2 D3-glycoside 200 g/ton</td></tr></tbody></table>

## Results

### Growth performances

The reduction of avP at both levels showed negative effect on growth performance of broiler. The worst performance was observed in bird fed high avP reduction (P<0.01). The supplementation 1, 25 (OH)₂ D₃-glycoside both at 100 and 200 g/ton diet in the present of phytase improved broiler performance fed diet containing both moderate and high reduction avP. However, only growth performance of broiler fed moderate avP reduction could be recovered to the same level of the control group (Table 2). These results were in agreement with ABURTO et al. (1998) that 1, 25 (OH)₂ D₃ supplementation improved broiler performance.

<table><caption>**Table 2. Effect of 1, 25 (OH)₂ D₃-glycoside supplementation on growth performance of broilers (1-35 days of age)**</caption><thead><tr><th>Treatment</th><th>Body weight (g)</th><th>Weight gain (g)</th><th>Feed intake (g)</th><th>FCR</th><th>Mortality (%)</th></tr></thead><tbody><tr><td>T1</td><td>2243.16<sup>b,c</sup></td><td>2197.61<sup>b,c</sup></td><td>3368.68<sup>a</sup></td><td>1.533<sup>b</sup></td><td>1.00<sup>b</sup></td></tr><tr><td>T2</td><td>1749.67<sup>e</sup></td><td>1704.51<sup>e</sup></td><td>2694.16<sup>c</sup></td><td>1.581<sup>b</sup></td><td>0.50<sup>b</sup></td></tr><tr><td>T3</td><td>2292.90<sup>a,b</sup></td><td>2248.16<sup>a,b</sup></td><td>3434.54<sup>a</sup></td><td>1.530<sup>b</sup></td><td>1.50<sup>b</sup></td></tr><tr><td>T4</td><td>2426.77<sup>a</sup></td><td>2381.76<sup>a</sup></td><td>3454.86<sup>a</sup></td><td>1.471<sup>b</sup></td><td>1.50<sup>b</sup></td></tr><tr><td>T5</td><td>811.07<sup>f</sup></td><td>765.72<sup>f</sup></td><td>1451.45<sup>d</sup></td><td>1.912<sup>a</sup></td><td>52.50<sup>a</sup></td></tr><tr><td>T6</td><td>2126.54<sup>c,d</sup></td><td>2081.18<sup>c,d</sup></td><td>3222.45<sup>b</sup></td><td>1.550<sup>b</sup></td><td>1.00<sup>b</sup></td></tr><tr><td>T7</td><td>2097.95<sup>d</sup></td><td>2052.80<sup>d</sup></td><td>3157.25<sup>b</sup></td><td>1.538<sup>b</sup></td><td>0.00<sup>b</sup></td></tr><tr><td>P-value</td><td>&lt;.0001</td><td>&lt;.0001</td><td>&lt;.0001</td><td>&lt;.0001</td><td>&lt;.001</td></tr><tr><td>SEM</td><td>18.2793</td><td>18.2851</td><td>14.5075</td><td>0.0181</td><td>0.554</td></tr></tbody></table>

### Bone breaking strength and mineralization

The reduction of avP at both levels caused a decrease in tibia breaking strength, tibia ash, Ca and P of broiler. The worst effect was observed in bird fed high avP reduction (P<0.01). In the presence of phytase, the supplementation 1, 25 (OH)₂ D₃-glycoside both at 100 and 200 g/ton diet improved tibia breaking strength tibia ash, Ca and P to be equivalent to that of the control group. Similarly, BACHMANN et al. (2013) report that the supplementation of 1, 25 (OH)₂ D₃ improved tibia breaking strength of broiler. In addition, tibia Ca and P of broiler fed high avP reduction were significant higher than that of the control group when fed with diet containing 200 g 1, 25 (OH)₂ D₃-glycoside (Table 3).

<footnote><sup>a,b,c,d,e,f</sup> Means in the same column with different superscripts are significantly different (P<0.05)</footnote>
<table><caption>**Table 3.** Effect of 1, 25 (OH)₂ D₃-glycoside supplementation on bone breaking strength and bone mineralization of broilers</caption><thead><tr><th>Treatment</th><th>Tibia breaking strength (N)</th><th>Tibia ash (%DM)</th><th>Tibia calcium (%DM)</th><th>Tibia phosphorus (%DM)</th></tr></thead><tbody><tr><td>T1</td><td>271.67<sup>a</sup></td><td>53.14<sup>a</sup></td><td>19.67<sup>b</sup></td><td>8.79<sup>b</sup></td></tr><tr><td>T2</td><td>188.38<sup>b</sup></td><td>47.99<sup>b</sup></td><td>18.30<sup>c</sup></td><td>7.51<sup>c</sup></td></tr><tr><td>T3</td><td>277.87<sup>a</sup></td><td>52.97<sup>a</sup></td><td>20.23<sup>ab</sup></td><td>8.47<sup>bc</sup></td></tr><tr><td>T4</td><td>298.46<sup>a</sup></td><td>53.39<sup>a</sup></td><td>20.34<sup>ab</sup></td><td>8.11<sup>b,c</sup></td></tr><tr><td>T5</td><td>87.48<sup>c</sup></td><td>39.83<sup>c</sup></td><td>15.70<sup>d</sup></td><td>6.47<sup>d</sup></td></tr><tr><td>T6</td><td>267.16<sup>a</sup></td><td>51.76<sup>a</sup></td><td>20.55<sup>a</sup></td><td>9.20<sup>ab</sup></td></tr><tr><td>T7</td><td>294.82<sup>a</sup></td><td>52.45<sup>a</sup></td><td>20.68<sup>a</sup></td><td>9.94<sup>a</sup></td></tr><tr><td>P-value</td><td>&lt;.0001</td><td>&lt;.0001</td><td>&lt;.0001</td><td>&lt;.0001</td></tr><tr><td>SEM</td><td>4.2888</td><td>0.2093</td><td>0.0956</td><td>0.1360</td></tr></tbody></table>

## Conclusions

The supplementation of 1, 25 (OH)₂ D₃-glycoside improved growth performances, bone breaking strength and bone mineralization of broiler fed low avP diets. Superior effect on growth performances was observed when supplemented 200g of 1, 25 (OH)₂ D₃-glycoside in broiler fed moderate avP reduction. The supplementation of 1, 25 (OH)₂ D₃-glycoside at higher level in the presence of phytase, therefore, elicits better broiler performances.

## Acknowledgements

The authors would like thank Herbonis, Basel, Switzerland for supporting the research funding and Poultry research and development center, department of Animal Science, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Thailand for assistance with the broiler study.

## Literature

ABURTO, A., H. M. EDWARDS, Jr., and W. M. BRITTON., 1998. The influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol, 25 hydroxycholecalciferol, and 1,25 dihydroxycholecalciferol in young broiler chickens. Poult. Sci. **77**:585–593.

BACHMANN, H., S. AUTZEN., U. FREY., W. RAMBECK., H. MCCORMACK, and C. C. WHITEHEAD., 2013. The efficacy of standardized product from dried leaves of Solanum glaucophyllum as source of 1, 25-dihydroxycholecalciferol for poultry. Br. Poult. Sci. **54**:642-652.

<footnote><sup>a,b,c,d</sup> Means in the same column with different superscripts are significantly different (P<0.05)</footnote>
P-063 (ID 93)

EFFECT OF QUATERNARY BENZOPHENANTRIDINE AND PROTOPINE ALKALOIDS ON GROWTH PERFORMANCE AND INTESTINAL MORPHOLOGY OF BROILERS

Y. Ruangpanit, S. Attamangkune and A. Reansoi

Department of Animal Science, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, Nakhon-Pathom 73140 Thailand

Corresponding author: agryos@ku.ac.th

**Abstract**

Current research has been focusing on the effects of plant extracts on poultry growth performance. It is hypothesised that potential improvements in performance parameters are related to changes in intestinal morphology. The aim of this experiment was to investigate the effects of a standardized formulation of quaternary benzophenanthridine and protopine alkaloids (QBPA and PA) from one selected plant of the *Papaveraceae* family on growth performance, villus height and crypt depth of broilers. 1600 one-day-old male broiler chicks (Ross 308) were assigned to 4 dietary treatments representing 0, 30, 60 and 100 mg/kg of QBA and PA (Sangrovit® Extra, Phytobiotics GmbH, Eltville, Germany). Each treatment consisted of 8 replicates with 50 birds each. Birds were vaccinated for coccidiosis at 4 days of age. Birds were fed with diets based on corn and soybean meal. Feed and water were provided *ad libitum*. Overall body weight gain did not differ between treatments (P>0.05). However, overall feed intake and feed conversion ratio were decreased (P<0.05) with increasing QBA and PA levels in the diet. Furthermore, QBA and PA reduced (P<0.05) mortality. Mortality was lowest when birds were fed 100 mg/kg QBA and PA. Supplementation of QBA and PA increased (P<0.05) villus height, villus width, villus height:crypt depth ratio in the jejunum and villus surface area in the jejunum, especially in the highest inclusion rate. In conclusion, feeding QBA and PA has potential to improve growth performance and gut morphology of broiler chickens.

**Keywords:** Broiler, quaternary benzophenanthridine, protopine alkaloids, intestinal morphology

**Introduction**

Plant extracts are increasingly being used in animal production industry as an alternative to antibiotic growth promotor. Sanguinarine and chelerythriene are the major alkaloids found in leaves of *Macleaya cordata*. These substances belong to quaternary benzophenanthridine and protopine alkaloids group. These alkaloids possess antimicrobial and anti-inflammatory effects (LENFELD et al., 1981). They also are known to improve endogenous digestive enzyme secretion, activate of the immune system and improve protein retention of farm animals (MELLOR, 2001). In the present study, benzophenanthridine and protopine alkaloids (QBPA and PA mixture) was supplemented in the diets to improve growth performance and intestinal morphology of broiler under hot climate management.

**Material and Methods**

One thousand six hundred, one day old, male broiler chicks of Ross 308 were divided into 4 dietary treatments including, corn-soy basal diet supplemented with 0, 30, 60 and 100 mg/kg of QBA and PA mixture (QBA 1.20 % and PA 0.23%). Each treatment consisted of 8 replications with 50 birds per replication. All birds were vaccinated for coccidiosis at 4 days of age. All diets were formulated to be isocaloric and isonitrogenous. Feed and water were provided *ad libitum*. The average environment temperature throughout the experimental period was 31.5°C (25.5-38.5°C) and the average relative humidity was 82% (50-99%). Body weight and average feed intake of all the birds were recorded at 1, 17 and 35 days of age for the calculation of feed conversion ratio. At 35 DOA, 2 birds from each replication were sacrificed and villus height, villus width, crypt depth, villus height/crypt depth and villus surface area of jejunum and ileum section were assayed (BRUNNSGAARD, 1998)

**Results**

**Growth performance**

There was no significant effect of QBA and PA mixture supplementation on body weight gain of broiler throughout the experimental period. However, feed intake and feed conversion ratio were significantly decreased with an increase of QBA and PA mixture levels in the diets (P<0.05). Similarly, LARISSA et al. (2013) indicated that chicks fed diet contain-
ing sanguinarine at 50 and 25 ppm had improved FCR. Broiler fed diet containing QBA and PA mixture had a significant reduction in mortality rate. The lowest mortality was observed in birds fed 100 ppm QBA and PA mixture (P<0.05). These results indicated that dietary QBA and PA mixture may exert growth-promoting effects on broiler chicks.

<table><caption>**Table 1.** Effects of QBA and PA mixture supplementation on growth performance of broilers</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="4">QBA and PA mixture level (ppm)</th><th rowspan="2">P-value</th><th rowspan="2">SEM</th></tr><tr><th>0</th><th>30</th><th>60</th><th>100</th></tr></thead><tbody><tr><td>Initial BW. (g)</td><td>42.08</td><td>41.85</td><td>41.94</td><td>42.03</td><td>0.6747</td><td>0.0690</td></tr><tr><td>BW gain (g)</td><td>512.81</td><td>505.76</td><td>511.30</td><td>517.89</td><td>0.3412</td><td>2.3131</td></tr><tr><td>FI (g)</td><td>723.10</td><td>729.55</td><td>710.08</td><td>703.45</td><td>0.1809</td><td>4.5093</td></tr><tr><td>FCR</td><td>1.410<sup>ab</sup></td><td>1.443<sup>a</sup></td><td>1.389<sup>ab</sup></td><td>1.359<sup>b</sup></td><td>0.0267</td><td>0.0094</td></tr><tr><td>Mortality (%)</td><td>0.75</td><td>0.25</td><td>0.50</td><td>0.50</td><td>0.8243</td><td>0.1860</td></tr><tr><td>Grower</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BW gain (g)</td><td>1,612.63</td><td>1,605.18</td><td>1,598.09</td><td>1,614.80</td><td>0.9001</td><td>8.6467</td></tr><tr><td>FI (g)</td><td>2,598.92<sup>a</sup></td><td>2,525.95<sup>b</sup></td><td>2,462.05<sup>c</sup></td><td>2,459.80<sup>c</sup></td><td>0.001</td><td>8.6687</td></tr><tr><td>FCR</td><td>1.613<sup>a</sup></td><td>1.575<sup>ab</sup></td><td>1.541<sup>b</sup></td><td>1.523<sup>b</sup></td><td>0.0158</td><td>0.0097</td></tr><tr><td>Mortality (%)</td><td>2.50<sup>a</sup></td><td>1.25<sup>ab</sup></td><td>1.00<sup>ab</sup></td><td>0.25<sup>b</sup></td><td>0.1582</td><td>0.3423</td></tr><tr><td>All peroid</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BW gain (g)</td><td>2,083.36</td><td>2,069.09</td><td>2,067.45</td><td>2,090.66</td><td>0.7339</td><td>8.5846</td></tr><tr><td>FI (g)</td><td>3,322.02<sup>a</sup></td><td>3,255.50<sup>b</sup></td><td>3,172.13<sup>c</sup></td><td>3,163.25<sup>c</sup></td><td>0.001</td><td>10.0187</td></tr><tr><td>FCR</td><td>1.595<sup>a</sup></td><td>1.574<sup>ab</sup></td><td>1.534<sup>bc</sup></td><td>1.514<sup>c</sup></td><td>0.0011</td><td>0.0069</td></tr><tr><td>Mortality (%)</td><td>3.25<sup>a</sup></td><td>1.50<sup>ab</sup></td><td>1.50<sup>ab</sup></td><td>0.75<sup>b</sup></td><td>0.1487</td><td>0.3824</td></tr></tbody></table>

a,b,c Mean values in a same row with no common superscripts are significantly different (P<0)

## Gut morphology

The supplementation of QBA and PA mixture significantly improved gut health by increasing villi height, villi width, villi height:crypt depth ratio and surface area of jejunum, especially, when supplement at 100 ppm in the diet (P<0.05) and significant increased villi height:crypt depth ratio of ileum (P<0.05). This result is partly in agreement with CHANG (2008) that the supplementation of QBA and PA mixture positively influenced villi height, villi:crypt depth ratio and villi surface area. This improvement could enhance rate of nutrient digestion, absorption and utilization which led to improve growth performance.

<table><caption>**Table 2.** Effects of QBA and PA mixture supplementation on jejunum and ileum morphology of broiler chicken at 35 days of age</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="4">QBA and PA mixture level (ppm)</th><th rowspan="2">P-value</th><th rowspan="2">SEM</th></tr><tr><th>0</th><th>30</th><th>60</th><th>100</th></tr></thead><tbody><tr><td><em>Jejunum</em></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Crypt depth (µm)</td><td>135.27</td><td>120.27</td><td>116.13</td><td>112.06</td><td>0.1894</td><td>3.9534</td></tr><tr><td>Villi height (µm)</td><td>1,182.97<sup>b</sup></td><td>1,376.30<sup>ab</sup></td><td>1,293.86<sup>ab</sup></td><td>1,385.70<sup>a</sup></td><td>0.1181</td><td>32.9307</td></tr><tr><td>Villi width (µm)</td><td>203.06<sup>b</sup></td><td>177.95<sup>b</sup></td><td>265.72<sup>a</sup></td><td>289.16<sup>a</sup></td><td>0.0002</td><td>9.3445</td></tr><tr><td>Villi height : Crypt depth ratio</td><td>9.55<sup>b</sup></td><td>11.56<sup>a</sup></td><td>10.70<sup>ab</sup></td><td>12.45<sup>a</sup></td><td>0.0219</td><td>0.3322</td></tr><tr><td>Villi surface area (mm<sup>2</sup>)</td><td>0.2407<sup>b</sup></td><td>0.2463<sup>b</sup></td><td>0.3686<sup>a</sup></td><td>0.4043<sup>a</sup></td><td>0.0001</td><td>0.0148</td></tr><tr><td><em>Ileum</em></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Crypt depth (µm)</td><td>121.39</td><td>116.44</td><td>102.83</td><td>105.47</td><td>0.2279</td><td>3.6227</td></tr><tr><td>Villi height (µm)</td><td>1,101.69</td><td>1,199.33</td><td>1,251.63</td><td>1,251.45</td><td>0.3545</td><td>33.6171</td></tr><tr><td>Villi width (µm)</td><td>248.88<sup>a</sup></td><td>252.59<sup>a</sup></td><td>195.19<sup>b</sup></td><td>201.59<sup>b</sup></td><td>0.0126</td><td>7.6612</td></tr><tr><td>Villi height : Crypt depth ratio</td><td>8.83<sup>b</sup></td><td>10.48<sup>ab</sup></td><td>10.64<sup>ab</sup></td><td>11.94<sup>a</sup></td><td>0.0312</td><td>0.3593</td></tr><tr><td>Villi surface area (mm<sup>2</sup>)</td><td>0.2936</td><td>0.3008</td><td>0.2937</td><td>0.2537</td><td>0.2933</td><td>0.0104</td></tr></tbody></table>

a,b Mean values in a same row with no common superscripts are significantly different (P<0.05).
**Conclusion**

Under hot climate management, the supplementation 60 ppm of QBA and PA mixture without the addition of growth promoter has a potential to improved growth performance of broiler. The superior effect on improving gut health was obtained with higher supplementation of QBA and PA mixture in the diet.

**Literature**

BRUNNSGAARD, G., 1998. Effects of cereal type and feed particle size on morphological characteristics epithelial cell proliferation and lectin binding patterns in the large intestine of pig. Animal Science. **76**: 2786-2798.

CHANG, W.K., 2008. Performance parameters and meat quality of broiler chickens fed Sangrovit as an alternative to antibiotics. Feeding trial report. Konkuk University.

LARISSA P., B. C. B. BEIRAO, R. M. HAYASHI, J. F. DURAU, M. C. LOURENCO, L. F. CARON, and E. SANTIN., 2013. Effect of sanguinarine in drinking water on Salmonella control and the expression of immune cells in peripheral blood and intestinal mucosa of broilers. J. Appl. Poult. **22**: 430-438.

LENFELD, J., M. KROUTIL, E. MARSALEK, J. SLAVIK, V. PREININGER and V. SIMANEK., 1981. Anti-inflammatory activity of quaternary benzophenanthridine alkaloids from Chelidonium majus. Planta Med. **4**: 161–165.

MELLOR, S., 2001. Natural appetisers from plants. Feed Mix **9**: 29–31.
P-064 (ID 91)

THE SUPPLEMENTATION OF 1, 25 (OH)₂ D₃-GLYCOSIDE IN COMBINATION WITH DIFFERENT PHOSPHORUS LEVELS ON GROWTH PERFORMANCES AND BLOOD CHEMICALS OF BROILERS

Y. Ruangpanit, S. Attamangkune, S. Rattanatabtimtong and T. Jeenyim

Department of Animal Science, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, Nakhon-Pathom 73140 Thailand

**Corresponding author:** agryos@ku.ac.th

**Abstract**

An improvement of Ca and P utilization could enhance bone quality during rapid growth of commercial broiler. 2100 one-day-old male broiler chicks (Ross 308) were used to study the effect of using 1, 25 (OH)₂ D₃-glycoside (Panbonis®) in combination with 3 patterns of dietary Ca and P on broiler performances and blood chemicals. During starter period, all birds were fed with diet containing equal level of Ca and available P (avP) and then divided into 6 dietary treatments including, normal avP, reduced avP 0.10% and 0.2% with and without 1, 25-(OH)₂ D₃-glycoside supplementation during grower and finisher periods. Ca:avP ratio in all diets was maintained at 2:1. Each treatment consisted of 7 replicates with 50 birds each (25 males and 25 females). All birds were fed with diets based on corn and soybean meal. Feed and water were provided *Ad-libitum*. There was no significant interaction and significant main effect on growth performances and carcass yields of broilers (P>0.05). However, an interaction effect was observed on serum Ca and P, bone breaking strength, tibia ash, tibia Ca and P (P<0.01). The supplementation of 1, 25 (OH)₂ D₃ glycoside increased serum Ca and P in all diets. The highest serum Ca and P was observed in birds fed normal diet (P<0.01). Bone breaking strength, tibia ash, tibia Ca and P of birds fed diets containing low avP were improved with 1, 25 (OH)₂ D₃-glycoside supplementation. Therefore, 1, 25 (OH)₂ D₃-glycoside could improve bone quality of broiler fed low avP diets.

**Keywords:** Broiler, tibia ash, bone breaking strength, 1, 25 (OH)₂ D₃-glycoside, Blood Ca and P

**Introduction**

Modern broiler chicken has a very rapid growth due to the extreme genetic improvement which could cause bone problem. Calcium and phosphorus are considered very important minerals for proper bone growth and development. It has been demonstrated that supplementation of vitamin D in the diet containing normal level of calcium appeared to be superior to calcium alone in maintaining optimal bone health and reducing bone fracture (NEWMAN and LEESON, 1999). A natural form of vitamin D metabolite, 1, 25 (OH)₂ D₃-glycoside, derived from *Solanum glaucophyllum* (SG) has been reported to improve bone breaking strength of broiler by improving Ca and P absorption (BACHMANN et al., 2013). In the present study, 1, 25 (OH)₂ D₃-glycoside was supplemented to broiler diet containing various levels of Ca and P to investigate the effect on growth performance and bone characteristics.

**Material and Methods**

The experiment was conducted using 2 x 3 Factorial in CRD. There were 2 levels of 1, 25 (OH)₂ D₃ supplementation and 3 levels of Ca: available P (avP) in the diet. A total of one thousand, on hundred and fifty two, one day old, broiler chicks of Ross 308 were fed with corn-soy basal diet for 39 days (Table 1). During starter period, all birds were fed with diet containing equal level of Ca:avP without and with 1, 25-(OH)₂ D₃-glycoside supplementation (100 ppm)and then divided into 6 dietary treatments including, including, normal avP, reduced avP 0.10% and 0.2% with and without 1, 25-(OH)₂ D₃-glycoside supplementation during grower and finisher periods (50 ppm). All experimental diets were corn-soy bean meal diet containing vitamin D₃ 3000 IU. Each treatment consisted of 7 replications with 50 birds per replication (25 males and 25 females). Body weight and average feed intake of all the birds were recorded at 1, 17 and 39 days of age for the calculation of feed conversion ratio. At the end of the study, 5 tibias (left) from 5 chicks per replication were collected for bone breaking strength, tibia ash and minerals content (Ca and P) analysis. Blood sample was collected from 2 birds per replication for serum Ca and P analysis.
<table><caption>**Table 1.** Dietary treatment</caption><thead><tr><th rowspan="2">Treatment<sup>1</sup></th><th rowspan="2">1, 25 (OH)<sub>2</sub>D<sub>3</sub><br>Supplementa-<br>tion</th><th colspan="3">Ca : avP level</th></tr><tr><th>Starter</th><th>Grower</th><th>Finisher</th></tr></thead><tbody><tr><td>T1</td><td>-</td><td>1.05:0.5</td><td>0.90:0.45</td><td>0.85:0.42</td></tr><tr><td>T2</td><td>+</td><td>1.05:0.5</td><td>0.90:0.45</td><td>0.85:0.42</td></tr><tr><td>T3</td><td>-</td><td>1.05:0.5</td><td>0.75:0.35</td><td>0.60:0.30</td></tr><tr><td>T4</td><td>+</td><td>1.05:0.5</td><td>0.75:0.35</td><td>0.60:0.30</td></tr><tr><td>T5</td><td>-</td><td>1.05:0.5</td><td>0.60:0.30</td><td>0.45:0.23</td></tr><tr><td>T6</td><td>+</td><td>1.05:0.5</td><td>0.60:0.30</td><td>0.45:0.23</td></tr></tbody></table>

## Results

### Growth performance

There was no significant interaction and significant main effect on growth performances and carcass yields of broilers (P>0.05). The reduction in Ca:avP level during grower and starter period had no effect on broiler performance (data not shown). The result clearly indicated that if the proper Ca:avP level was offered to broiler during early age, there will be no adverse effect when Ca:avP level was reduced in the later part of life. Moreover, all experimental diets had vitamin D3 3000 IU which, could help to maintain Ca and P utilization of bird fed low Ca:avP diets.

### Bone breaking strength and bone mineralization

The supplementation of 1, 25 (OH)<sub>2</sub> D<sub>3</sub>-glycoside improved bone breaking strength, tibia ash, tibia Ca and P of birds fed diets containing low Ca:avP level to be equivalent to that of the control group (Table 2). This possibly due to 1, 25 (OH)<sub>2</sub> D<sub>3</sub>-glycoside increased Ca and P absorption which led to higher deposition in the bone (VRIES et al., 2010)

### Serum Ca and P

There was a significant interaction effect of 1, 25 (OH)<sub>2</sub> D<sub>3</sub>-glycoside supplementation and Ca:avP level on serum Ca and P of broilers (Table 3, P<0.05). The supplementation of 1, 25 (OH)<sub>2</sub> D<sub>3</sub> glycoside increased serum Ca (P=0.0148) and P (P=0.0686) in all diets. The highest serum Ca and P was observed in birds fed normal diet with 1, 25 (OH)<sub>2</sub> D<sub>3</sub>-glycoside supplementation. Similar effect was reported by CHENG et al., 2004).

## Conclusion

Feeding low Ca:avP level to broiler received normal Ca:P during early age had no effect on growth performances. The supplementation of 1, 25 (OH)<sub>2</sub> D<sub>3</sub>-glycoside to broiler fed diet containing vitamin D3 showed no beneficial effect on growth performance. However, the supplementation of 1, 25 (OH)<sub>2</sub> D<sub>3</sub>-glycoside improved bone breaking strength, bone mineralization and serum Ca and P in broiler fed low Ca:avP level.

<table><caption>**Table 2.** Effects of 1, 25-(OH)2 D3-glycoside in combination with dietary calcium and phosphorus level on bone breaking strength and bone mineralization of broilers</caption><thead><tr><th>Treatment<sup>1</sup></th><th>1, 25-(OH)<sub>2</sub> D<sub>3</sub></th><th>Ca:avP ratio</th><th>Tibia breaking strength<br>(kg force)</th><th>Ash<br>(%)</th><th>Ca<br>(%)</th><th>P<br>(%)</th></tr></thead><tbody><tr><td>1</td><td>-</td><td>Normal</td><td>277.82<sup>c</sup></td><td>41.81<sup>a</sup></td><td>14.91<sup>a</sup></td><td>9.50<sup>a</sup></td></tr><tr><td>2</td><td>+</td><td>Normal</td><td>342.53<sup>a</sup></td><td>42.18<sup>a</sup></td><td>15.02<sup>a</sup></td><td>9.60<sup>a</sup></td></tr><tr><td>3</td><td>-</td><td>- avP 0.1%</td><td>287.60<sup>bc</sup></td><td>39.32<sup>b</sup></td><td>13.95<sup>b</sup></td><td>8.41<sup>b</sup></td></tr><tr><td>4</td><td>+</td><td>-avP 0.1%</td><td>327.85<sup>ab</sup></td><td>43.02<sup>a</sup></td><td>15.51<sup>a</sup></td><td>9.58<sup>a</sup></td></tr><tr><td>5</td><td>-</td><td>-avP 0.2%</td><td>271.00<sup>c</sup></td><td>38.96<sup>b</sup></td><td>13.41<sup>b</sup></td><td>8.38<sup>b</sup></td></tr><tr><td>6</td><td>+</td><td>-avP 0.2%</td><td>295.99<sup>bc</sup></td><td>43.05<sup>a</sup></td><td>15.38<sup>a</sup></td><td>9.20<sup>a</sup></td></tr><tr><td colspan="3">Source of variation</td><td colspan="3">P-value</td></tr><tr><td colspan="3">1, 25-(OH)<sub>2</sub> D<sub>3</sub> (A)</td><td>0.6529</td><td>0.9739</td><td>0.3752</td><td>0.3599</td></tr><tr><td colspan="3">Level Ca : avP (B)</td><td>0.0509</td><td>0.1517</td><td>0.1503</td><td>&lt;0.0001</td></tr><tr><td colspan="3">A*B</td><td>0.0034</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td></tr><tr><td colspan="3">SEM</td><td>48.6404</td><td>1.7095</td><td>0.8172</td><td>0.4255</td></tr></tbody></table>

<footnote>a,b,cMeans in the same column with different superscripts are significantly different (P<0.05)</footnote>
<table><caption>**Table 3.** Effects of 1, 25-(OH)2 D3-glycoside in combination with dietary calcium and phosphorus level on serum Ca and P of broilers at 39 days of age</caption><thead><tr><th>Treatment<sup>1</sup></th><th>1, 25-(OH)<sub>2</sub> D<sub>3</sub></th><th>Ca:avP Ratio</th><th>Ca (mg%)</th><th>P (mg%)</th></tr></thead><tbody><tr><td>1</td><td>-</td><td>Normal</td><td>4.33<sup>a</sup></td><td>6.22</td></tr><tr><td>2</td><td>+</td><td>Normal</td><td>4.56<sup>a</sup></td><td>6.79</td></tr><tr><td>3</td><td>-</td><td>- avP 0.1%</td><td>3.10<sup>b</sup></td><td>5.89</td></tr><tr><td>4</td><td>+</td><td>-avP 0.1%</td><td>4.44<sup>a</sup></td><td>6.30</td></tr><tr><td>5</td><td>-</td><td>-avP 0.2%</td><td>2.69<sup>b</sup></td><td>5.61</td></tr><tr><td>6</td><td>+</td><td>-avP 0.2%</td><td>3.07<sup>b</sup></td><td>6.37</td></tr><tr><th colspan="2">Source of variation</th><th colspan="2">P-value</th></tr><tr><td colspan="2">1, 25-(OH)<sub>2</sub> D<sub>3</sub> (A)</td><td>Normal</td><td>0.2550</td></tr><tr><td colspan="2">Level Ca : avP (B)</td><td>0.0003</td><td>0.1235</td></tr><tr><td colspan="2">A*B</td><td>0.0148</td><td>0.0686</td></tr><tr><td colspan="2">SEM</td><td>0.8123</td><td>0.7069</td></tr></tbody></table>

## Literature

CHENG, Y. H., J. P. GOFF, J. L. SELL, M. E. DALLORSO, S. GIL, S. E. PAWLAK and R. L. HORS., 2004. Utilizing *Solanum glaucophyllum* alone or with phytase to improve phosphorus utilization in broilers. Poult. Sci. **83**: 406–413.

NEWMAN S. and S. LEESON., 1999. The Effect of Dietary Supplementation with 1,25 Dihydroxycholecalciferol or Vitamin C on the Characteristics of the Tibia of Older Layin Hens. Poult. Sci. **78**:85-90.

VRIES, S.De., R.P. KWAKKEL and J. DIJKSTRA., 2010. Dynamics of calcium and phosphorus metabolism in lying hens, pp. 133-150. In D.M.S.S. VITTE and E. KEBREAB, eds. Phosphorus and calcium Utilization and Requirements in Fram Animal. AMA Dataset Ltd., Preton.

<footnote><sup>a,b,c</sup>Means in the same column with different superscripts are significantly different (P<0.05)</footnote>
P-065 (ID 194)

EFFECT OF SUPPLEMENTING VIA DRINKING WATER A BLEND OF MONO-GLYCERIDES, ALONE OR IN COMBINATION WITH ORGANIC ACIDS, ON CAMPYLOBACTER COLONIZATION IN BROILERS

J. Sánchez<fnref n="1" />, A. Parra<fnref n="2" />, Y. Carre<fnref n="3" />, A. Csorbai<fnref n="4" />, 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 blend of short and medium chain fatty acids (C3+C4+C6+C10+C12) mono-glycerides (MG) and a mixture (60:40 %) of the same mono-glycerides with organic acids (MGOA) added to the drinking water 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 + MG at 4ml/l from 0-21d and at 2ml/l at 21-42d and T3: T1 + MGOA at 9ml/l from 0-21d and at 4.5ml/l at 21-42d. 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 expressed as log₁₀ CFU/g caecal content were analysed by the nonparametric test of Kruskall-Wallis (SPSS v.19.0). Although treatments, especially T3, decreased the *Campylobacter* counts 0.9 to 2.3-log, no significant differences were detected between the two products tested and the control at any of the times evaluated (21, 35 and 42 d of age). It is concluded that the two mono-glyceride combinations tested did not significantly reduce *Campylobacter jejuni* colonization under the experimental model assayed.

**Keywords:** mono-glycerides, organic acids, *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). *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 (VAN DEUN et al., 2008; SOLIS DE LOS SANTOS et al., 2008, 2010; MOLATOVÁ et al., 2011; 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 blend of short and medium chain fatty acids (C3+C4+C6+C10+C12) mono-glycerides (MG) and a mixture (60:40 %) of the same mono-glycerides with organic acids (MGOA) added to the drinking water 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 to the drinking water from 1 to 42 d of age, T1: Positive controls (*Campylobacter*, no additives), T2: T1 + MG at 4ml/l from 0-21d and at 2ml/l from 21-42d and T3: T1 + MGOA at 9ml/l from 0-21d and at 4.5ml/l from 21-42d. 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 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, expressed as log₁₀

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

<footnote n="2">²CZ Veterinaria, Porriño, Spain</footnote>

<footnote n="3">³CIDEF, Rennes, France</footnote>

<footnote n="4">⁴BTT, Budapest, Hungary</footnote>
CFU/g caecal content, were analysed by the nonparametric test of Kruskall-Wallis (SPSS v.19.0). Statistical significance is declared at $P \le 0.05$, with $0.05 < P \le 0.10$ considered as a near-significant trend.

## Results

Both treatments showed numerically lower *Campylobacter* counts than control, with reductions of 0.60, 0.44 and 0.66-log for T2 and 0.90, 2.3 and 1.73-log for T3 at 21, 35 and 42d, respectively (Figure 1). However, the high variability observed mainly due to the broilers below detection level observed in T3 (8 and 42% and 17 and 33% in T2 and T3 at 35 and 42 d, respectively) caused that no significant differences in the *Campylobacter* counts were observed between the two products tested and the control treatment at any of the times evaluated (21, 35 and 42 d of age).

<figure><img src="image_32.png" /><figcaption>**Figure 1.** Effect of dietary treatment (T1: Control, T2: MG at 4ml/l from 0-21d and at 2ml/l from 21-42d and T3: MGOA at 9ml/l from 0-21d and at 4.5ml/l from 21-42d) on *Campylobacter* counts (log₁₀ CFU/g) in the caeca of broilers at 21, 35 and 42 days of age.</figcaption></figure>

## Conclusions

Under our experimental conditions, it is concluded that the two mono-glyceride combinations tested did not significantly reduce *Campylobacter jejuni* colonization under the experimental model assayed

## 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.

## 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 *Campylobacter jejuni/coli* populations in mixtures of water and feed. Poult. Sci. **81**, 621-628.

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.

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 *Campylobacter 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 *Campylobacter jejuni* in the ceca of colonized broiler chickens against the bactericidal effects of medium-chain fatty acids. Poult. Sci. **89**, 1144-1155.

MOLATOVÁ, Z., E. SKŘIVANOVÁ, J. BARÉ, K. HOUF, G. BRUGGEMAN, M. MAROUNEK, 2011: Effect of coated and non-coated fatty acid supplementation on broiler chickens experimentally infected with *Campylobacter jejuni*. J. Anim. Physiol. Anim. Nutr. **95**, 701-706.

SOLIS DE LOS SANTOS, F., A. M. DONOGHUE, K. VENKITANARAYANAN, M. L. DIRAIN, I. REYES-HERRERA, P. J. BLORE, D. J. DONOGHUE, 2008: Caprylic acid supplemented in feed reduces enteric *Campylobacter jejuni* colonization in ten-day-old broiler chickens. Poult. Sci. **87**, 800-804.

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.

VAN DEUN, K., F. HAESEBROUCK, F. VAN IMMERSEEL, R. DUCATELLE, F. PASMANS, 2008: Short-chain fatty acids and l-lactate as feed additives to control *Campylobacter jejuni* infections in broilers. Avian Pathol. **37**, 379-383.
P-069 (ID 32)

EFFECT OF TWO MANNANASES ON PERFORMANCE OF BROILERS FED GALACTOMANNAN ENRICHED DIETS

Y. Shastak¹, P. Ader¹, and C. Elwert²

¹BASF SE, E-ENE, Lampertheim, Germany

²feedtest, Wettin-Löbejün, Germany

**Corresponding author:** yauheni.shastak@basf.com

**Summary**

The objective of this study was to evaluate the effect of two mannanases on the performance of broilers fed galacto-
mannan-enriched diets. For the negative control (NC), a maize-soybean meal-based diet with 1.5% guar gum was for-
mulated to exceed dietary recommendations by about 5%. This NC diet was fed as is or supplemented with four levels
of a Trichoderma-β-mannanase (TMan) (40, 70, 120 or 220 U/kg feed, respectively) or a commercially available Bacil-
lus-β-mannanase product (BMan) at the highest recommended dosage (240 U/kg feed). A positive control diet (PC)
was formulated by using maize starch instead of guar gum in the NC. 672 one-day-old Cobb 500 male broiler chicks
were given pelleted experimental diets for 35 days. Inclusion of guar gum in the corn-soybean meal-based diet (NC)
significantly decreased (P < 0.05) daily BWG and increased feed:gain ratio compared with the PC. The supplementa-
tion of BMan and 40 U of TMan to the NC had a positive effect on broiler performance (P < 0.05), but birds were not
able to achieve the level of performance of the PC. Increasing TMan supplementation levels up to 70, 120 and 220 U/kg
feed resulted in performance values similar to the PC without a dose-response relationship. It was concluded that TMan
was more effective to hydrolyze water-soluble galactomannan as compared with BMan.

**Keywords:** mannan, mannanase, performance, broilers

**Introduction**

Mannan polysaccharides are anti-nutritional components present in the majority of poultry diets. In plant cells mannan act as a storage compound or as a building block of the plant cell wall. Protein meals and some other by-products used in poultry nutrition can contain substantial amounts of β-mannan.

In general, the presence of β-mannans in poultry diets may lead to reduced nutrient digestibility and growth performance. The reasons for the latter might be an increase in intestinal viscosity, adverse effects on innate immune response, and microbial proliferation in the gut (Shastak et al., 2015b). The negative effects of β-mannan can however be overcome by the addition of β-mannanases to the feed of domestic fowl.

Guar gum (GG) and guar meal predominantly contain water-soluble galactomannan with more than 5% by weight of D-galactose residues (Aspinall, 1959). GG, the endosperm of guar seeds, has often been used to mimic inclusion of feed ingredients high in galactomannan to practical diets (Daskiran et al., 2004).

The objective of this study was to evaluate the effect of two mannanases on the performance of broilers fed galacto-
mannan-enriched diets.

**Material and Methods**

In a previous study we evaluated a fungal β-mannanase at supplementation levels equal to or higher than 400 U/kg feed (Shastak et al., 2015a). The lowest supplementation level of the fungal β-mannanase (400 U/kg) to the negative control diet (containing guar gum) fully restored broiler performance to the level of birds fed a positive control diet (no guar gum). An increase of the β-mannanase supplementation level (up to 1600/U kg feed) led to no further improvement of broiler performance. Thus, the present study used lower dosages of the same β-mannanase source to determine the optimal supplementation level of the enzyme.

For the negative control (NC), a maize-soybean meal-based diet with 1.5% guar gum was formulated to exceed dietary recommendations (CVB, 2008) by about 5% (22% CP; 13.1 MJ ME/kg). This NC diet was fed as is or supplemented with four levels of a Trichoderma-β-mannanase (TMan) (40, 70, 120 or 220 U/kg feed, respectively) or a commercially available Bacillus-β-mannanase product (BMan) at the highest recommended dosage (240 U/kg feed). One mannanase unit (U) was defined as the amount of enzyme that produces reducing carbohydrates having a reducing power corresponding to one µmol mannose from mannan per minute under the assay conditions. A positive control diet (PC) was
formulated by using maize starch instead of guar gum in the NC. 672 one-day-old Cobb 500 male broiler chicks were given pelleted experimental diets for 35 days. Each of the seven diets was fed to eight replicate pens of 12 birds. Birds had continuous access to feed and water. Body weight for each pen was recorded on days 1 and 35 post hatch. Feed intake was measured on a pen basis. Total BW gain and mortality-corrected feed conversion ratio were calculated. Data were analyzed by one-factorial ANOVA, treatment differences were assessed by Tukey test.

## Results

Treatment effects were not significant for mortality (P > 0.05) (Table). Inclusion of guar gum in the maize-soybean meal-based diet (NC) significantly decreased (P < 0.01) daily BWG, FI and increased feed:gain ratio compared with the PC. The supplementation of BMan (240 U/kg) and 40 U/kg of TMan to the NC had a positive effect on broiler performance (P < 0.01), but birds were not able to achieve the level of performance of the PC and treatments NC+70, NC+120 or NC+220 U/kg feed of TMan (P < 0.05). Increasing TMan supplementation levels up to 70, 120 and 220 U/kg feed resulted in performance values similar to the PC without a dose-response relationship. Performance for all NC+70, NC+120 or NC+220 U/kg feed of TMan treatments was significantly increased (P < 0.05) as compared to the NC, NC+BMan or NC+40 U/kg TMan treatments (Table).

<table><caption>**Table 1:** BW Gain (BWG), Feed Intake (FI) and Mortality-Corrected Feed Conversion Ratio (FCR).</caption><thead><tr><th>Diet</th><th>BWG<br>g/d</th><th>FI<br>g/d</th><th>FCR<br>feed/gain</th><th>Mortality, %</th></tr></thead><tbody><tr><td>Positive control diet</td><td>67.4<sup>c</sup></td><td>104.5<sup>b</sup></td><td>1.55<sup>a</sup></td><td>8.3</td></tr><tr><td>Negative control diet (NC)</td><td>32.6<sup>a</sup></td><td>74.9<sup>a</sup></td><td>2.31<sup>c</sup></td><td>11.9</td></tr><tr><td>NC+240 U BMan</td><td>57.2<sup>b</sup></td><td>103.6<sup>b</sup></td><td>1.81<sup>b</sup></td><td>4.2</td></tr><tr><td>NC+40 U TMan</td><td>58.4<sup>b</sup></td><td>104.3<sup>b</sup></td><td>1.79<sup>b</sup></td><td>3.1</td></tr><tr><td>NC+70 U TMan</td><td>63.9<sup>c</sup></td><td>106.8<sup>bc</sup></td><td>1.67<sup>a</sup></td><td>13.5</td></tr><tr><td>NC+120 U TMan</td><td>67.6<sup>c</sup></td><td>110.3<sup>c</sup></td><td>1.63<sup>a</sup></td><td>10.4</td></tr><tr><td>NC+220 U TMan</td><td>67.8<sup>c</sup></td><td>108.6<sup>bc</sup></td><td>1.59<sup>a</sup></td><td>7.3</td></tr></tbody></table>

## Discussion

ß-Mannans from guar gum (galactomannans, water-soluble) in the NC diet had a negative effect on broiler performance (Table). Supplementation of both ß-mannanases to the NC improved daily BWG and feed:gain ratio compared to the NC. However, the results showed that two ß-mannanase sources had different abilities to hydrolyze water-soluble galactomannan, with higher inclusion levels necessary for BMan (240 U/kg feed) to degradate the same amount of galactomannan. Only 40 U/kg feed of TMan was required to achieve the same broiler performance level as compared with BMan. The reason for this might have been differences in resistance against proteolytic enzyme activity, pH optimum of enzyme, substrate specificity, etc. The three highest supplementation levels of TMan (70, 120 and 220 U/kg feed) fully restored broiler performance to the level of birds fed the PC diet.

## Conclusions

It was concluded that TMan was more effective in hydrolyzing water-soluble galactomannan as compared with BMan. 70 U/kg feed of TMan fully restored broiler performance to the level of birds fed the PC diet. Further studies are required to evaluate both enzymes on water-insoluble linear ß-mannan.

<footnote>*a-c*Means within columns without common superscript differ significantly (P < 0.05)</footnote>
References

ASPINALL, G.O., 1959: Structural chemistry of the hemicelluloses. Adv. Carbohydr. Chem. **14**, 429-468.

CVB, 2008: Livestock Feed Table (Veevoedertabel); Table Values Poultry Nutrition, 2007. Centraal Veevoederbureau, Lelystad, the Netherlands.

DASKIRAN, M., R.G. TEETER, D.W. FODGE, H.Y. HSIAO, 2004: An evaluation of endo-β-D-mannanase (Hemicell) effects on broiler performance and energy use in diets varying in β-mannan content. Poultr. Sci. **83**, 662-668.

SHASTAK Y., P. ADER, C. ELWERT, 2015a: Effect of supplementation of two different β-mannanase sources on broiler performance. Proc. Soc. Nutr. Physiol. **24**, 80.

SHASTAK Y., P. ADER, D. FEUERSTEIN, R. RUEHLE, M. MATUSCHEK, 2015b: β-Mannan and mannanase in poultry nutrition. World's Poult. Sci. J. **71**, 161-174.
P-072 (ID 19)

# THE EFFECTS OF DIETARY PLANT ALKALOIDS SUPPLEMENTATION WITH VARIOUS LEVELS OF DIGESTIBLE LYSINE-METHIONINE AND CALCIUM-NON PHYTIC PHOSPHORUS RATIO ON PRODUCTIVE PERFORMANCES AND EGG QUALITY OF LAYING HENS IN THE LATE PHASE OF PRODUCTION

K.Soisuwan and N.Chauychuwong

Department of Animal Science, Faculty of Agriculture, Rajamagala University of Technology Srivijaya, Nakhon Si Thamarat, Thailand, 80110

Corresponding author: ksoisuwan52@gmail.com

## Abstract

This study was conducted to evaluate the effect of plant alkaloids (Sangrovit® Extra), digestible lysine-methionine (Lys-Met) and calcium - non phytic phosphorus (Ca-NPP) ratio on productive performances and egg quality of laying hens in the late phase of production. A 2x 2 x 2 factorial arrangement of treatments comprising of 2 dietary Lys-Met ratio (0.81: 0.44 and 0.72:0.39%) and 2 levels of Ca-NPP ratio (3.75 : 0.45 and 3.38:0.41%) with and without Sangrovit® Extra (non and 60 mg/kg feed) was used. Isa brown hens (n=360,50 weeks of age) were randomly divided into 8 dietary treatments (5 replicates of 9 birds per treatment). The experiment was recorded for 12 weeks (50 to 62 weeks of age) and productive performances and egg quality traits were randomly every 4 week and shell quality traits were determined at 62 weeks of age. Dietary Lys-Met ratio and dietary Ca- NPP ratio had no effect (p>0.05) on egg production (EP), feed intake (FI), feed conversion ratio (FCR), egg weight (EW), egg mass (EM) and weight gain (WG). However, Sangrovit® Extra supplementation throughout the experimental period was significant lowered (P<0.05) mortality rate (MR) than without supplementation group. The supplementation with Sangrovit® Extra had no significant effect (P>0.05) on yolk color (YC), shell weight (SW), shell thickness (ST); however, feeding Sangrovit® Extra was significantly improved (P<0.05) % albumen (AP). It was also found that dietary Lys-Met and Ca- NPP ratio had no adverse effect (P>0.05) on egg quality. It was concluded, based on the data, that laying hens in the late phase of production required approximately 0.72 and 0.39% of digestible Lys-Met and required 3.38 and 0.41% of Ca- NPP, respectively. The supplementation of Sangrovit® Extra could improved survival rate and egg quality in term of albumen egg.

**Keywords:** digestible lysine-methionine, calcium- non phytic phosphorus, plant alkaloids and laying hens

## Introduction

In the recent years, the potential of the emphasis on alternative feed additive such as phytobiotics in reducing the excretion of N and P by farm animals to protect the environment, have received interest in the use of low-protein, amino acid-supplemented diets for various kinds of poultry (Keshavarz and Austic, 2004). The objective of this study was to evaluate the effect of plant alkaloid (Sangrovit® Extra) in form of quaternary benzophenanthridine alkaloid (QBA) and protopine alkaloid (PA) from Phytobiotics (Thailand) Co., Ltd. supplementation with various levels of digestible lysine-methionine and calcium- non phytic phosphorus ratio on productive performances and egg quality of laying hens in the late phase of production.

## Material and Methods

Three hundred and sixty Isa brown laying hens of 50 weeks of age in five replicates per treatment (9 birds/replication) were used. Eight experimental diets which contained of 2 levels of digestible lysine-methionine ratio (0.81:0.44, control and 0.72 : 0.39 %) with 2 levels of calcium- non phytic phosphorus ratio (3.75:0.45%, control and 3.38:0.41 %) and supplemental with plant alkaloid (Sangrovit® Extra) in the levels of 0 and 60 mg/kg feed of isoenergetic diets on laying hens performances and egg quality. The reduced-digestible lysine-methionine and calcium- non phytic phosphorus ratio had approximately 10 percentage units lower than their control counterparts while another nutrient contents met the minimum requirements by the recommendation of NRC (1994). Birds were raised in laying cage and received 16-h constant lighting regime throughout the experimental period from 50 to 62 weeks of age and received feed in mash form with feed and water were provided for ad librium consumption. The number of total dirty, broken, shell-less egg and mortality were recorded daily by replicate. Feed intake was recorded by replicate every 28d and in each period of experiment, egg production (%), feed intake (g/hen/day), FCR (kg feed / kg egg) were calculated of each 28-day period and overall (12 weeks). On the last 3 consecutive days of each period of experiment, egg quality was evaluated by the breaking method to measure egg weight, albumen weight, yolk weight, shell weight, yolk color and shell thickness. Data were
analyzed by ANOVA and compared the difference between dietary treatment means by Duncan's New Multiple Range Test (DMRT) by the recommendation of Steel and Torrie (1980) for a factorial completely randomized design. A significance level of P<0.05 was used.

## Results

There were no interaction between main treatments and between experimental periods, only main treatments are presented (Table 1). It was found that MR and AP of laying hens fed diets supplemented with Sangrovit® Extra throughout the experimental period significantly improved (P<0.05) when compared with control groups. These results were consistent with the literature of Keshavarz and Austic (2004) and Rama Rao et al., (2011) who reported that feeding diets formulated without crude protein minimum while supplemented with essential first limiting amino acid resulted in the similar of production performance and egg quality when compared with diets formulated base on crude protein minimum. However, MR was significantly improved (P<0.05) in birds fed diets supplemented with Sangrovit® Extra. It was supported by the report of Rangpanit (2014) who reported that diets supplemented with plant alkaloids can reduce serum cortisol hormone (stress hormone) when compared with the control group, so that it may be influenced birds to have high immunity to against the stress and disease, particularly when reared in high ambient temperature. It was also found that laying hens fed diets supplemented with Sangrovit® Extra had significantly improved (P<0.05) AP when compared with control group. These results were agreeded with the reported of Rangpanit (2014) who reported that laying hens when received diets supplemented with plant alkaloid will get an improvement in the gastrointestinal functions, particularly improved in villus height which could lead to a better absorption and utilization of nutrient such as essential amino acids and ultimately improved egg composition in term of % albumen.

<table><caption>**Table 1** Influence of dietary plant alkaloids (Sangrovit® Extra) supplementation with various levels of digestible lysine-methionine and calcium-non phytic phosphorus ratio on productive performances and egg quality of laying hens in the late phase of production.</caption><thead><tr><th rowspan="2">Factors</th><th colspan="8">Parameter</th></tr><tr><th>HD<br>(%)</th><th>EW<br>(g)</th><th>FCR<br>(kg/kg)</th><th>FI<br>(g/d)</th><th>MR<br>(%)<sup>1/</sup></th><th>AP<br>(%)<sup>1/</sup></th><th>YP<br>(%)</th><th>SP<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>84.17</td><td>58.49</td><td>2.19</td><td>115.70</td><td>7.94<sup>a</sup></td><td>61.97<sup>b</sup></td><td>23.20</td><td>12.68</td></tr><tr><td>60</td><td>83.76</td><td>58.17</td><td>2.17</td><td>116.50</td><td>6.07<sup>b</sup></td><td>63.28<sup>a</sup></td><td>23.70</td><td>12.75</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>0.81:0.44%</td><td>83.45</td><td>57.96</td><td>2.15</td><td>116.10</td><td>6.99<sup>ab</sup></td><td>62.47<sup>ab</sup></td><td>23.71</td><td>12.51</td></tr><tr><td>0.72:0.39%</td><td>83.67</td><td>57.24</td><td>2.13</td><td>116.30</td><td>6.81<sup>ab</sup></td><td>62.61<sup>ab</sup></td><td>23.52</td><td>12.52</td></tr><tr><td>Cal-NPP (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>3.75:0.45</td><td>83.12</td><td>58.13</td><td>2.17</td><td>115.90</td><td>6.77<sup>ab</sup></td><td>62.43<sup>ab</sup></td><td>23.51</td><td>12.40</td></tr><tr><td>3.38:0.41</td><td>83.24</td><td>58.26</td><td>2.14</td><td>115.70</td><td>6.67<sup>ab</sup></td><td>62.18<sup>ab</sup></td><td>23.47</td><td>12.42</td></tr><tr><td>Pooled SEM</td><td>1.98</td><td>0.46</td><td>0.07</td><td>0.87</td><td>0.12</td><td>0.32</td><td>0.21</td><td>0.09</td></tr></tbody></table>

## Conclusions

The present study has shown that it can be used diets composed of 0.72 and 0.39 % of digestible lysine-methionine and 3.38 and 0.41 % of calcium-non phytic phosphorus ratio for optimum egg production performances and egg quality of laying hens in the late phase of production. It was also found that the supplementation of Sangrovit® Extra in the levels of 60 mg/kg feed could improved survival rate and egg quality in term of albumen egg.

## Acknowledgments

The author would like to special thank for Phytobiotics Co.,Ltd. (Thailand) for the grant and financial support, particularly for the material products (Sangrovit® Extra) for this trial. We would like to thank you for Rajamagala University of Technology Srivijaya for supporting the convenient equipments and experimental unit.

<footnote>1/ a-b Mean within a column and under each main effect with no common superscripts differ significant (P<0.05)</footnote>
References

Keshavarz, K. and 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. 1994. National Research Council.1994.Nutrient Requiremets of Poultry. 9 rev. ed. National Academy Press, Washington, DC. USA. 240p.

Rama, Rao; S. V., V. Ravindran; T. Srilatha. A. K. Panda and M. V. L. N. Rayu, 2011. Effect of dietary concentration of energy, crude protion, lysine, and methionine on the performance of White Leghorn layers in the tropics. J. Appl. Res. 20:528-541

Rangpanit, Y. 2014. Effect of quarnenary benzophenanthridine alkaloid and protopine alkaloidsupplementation on laying hens performance and stress status. Kasetsart Uhiversity,Kamphangsaen, Nakhonpathom, Thailand. 12p.

Steel, R. and J. H. Torrie. 1980. Principle and procedures of statistics. A Biometrical Approach 2 ed., Mc Graw Hill Book Co. New York.
P-073 (ID 47)

EVALUATION OF THE EFFECT OF TWO COMMERCIAL BUTYRATE ADDITIVES ON THE PERFORMANCE OF BROILERS

R. Sygall, G. Wielsma

Perstorp Feed and Food, Waspik, the Netherlands

Corresponding author: richard.sygall@perstorp.com

Abstract

The objective of the study was to assess the effect of including tributyrin and a coated butyrate product at iso-butyric levels on the performance of broilers. A total of 714 male day old Ross broilers were used for the study. To better mimic commercial conditions the bedding was 'spiked' with dirty litter from a commercial unit. There were three treatment diets with 12 pens allocated to each treatment with 19/20 birds allocated to each pen at study start. The three treatment diets were control industry standard diet (T1), the control diet with tributyrin (T2) and the control diet with coated butyrate, (T3). The diets were fed for the first 9 days as phase 1 diets, from day 9 to 25 as phase 2 diets, and the final phase 3 diets were fed from day 25 to 35. Feed intake, body weight, FCR, FCE and % mortality were calculated for each phase and for the overall period (days 0 to 35). The results of the study indicate that the inclusion of tributyrin and coated butyric acid in standard broiler diets significantly improve bird live weight at days 25 and 35 as well as FCR and FCE corrected for mortality between days 9 to 25 and day 0 to 35 compared to birds offered standard industry diets. There were no overall significant differences in bird performance between the additions of tributyrin compared to coated butyrate at iso-butyric levels in this study. Both butyric acid products showed significant improvements in bird performance. This is thought to be through improved gut health

**Keywords:** Gut Health, Butyrate, Broilers, tributyrin, FCR improvement

Introduction

The objective of the study was to assess the effect of including Tributyrin, a glycerol ester of butyric acid, and a coated sodium-butyrate product on the performance of broilers fed wheat based diets at iso-butyric levels. Performance was assessed through measuring average daily feed intakes, live weight, feed conversion ratio (FCR - feed/gain), feed conversion efficiency (FCE - gain/feed) and mortality. To better mimic commercial conditions the bedding was 'spiked' with dirty litter from a commercial unit to disease challenge the birds.

Material and Methods

A total of 714 male, day old Ross broilers were used for the study. They were housed in three rooms in 36 pens. There were three treatment diets, with 12 pens allocated to each treatment, with 19/20 birds allocated to each pen at study start. The three treatment diets were

- control industry standard diet (T1);

- the control diet with Tributyrin (T2);

- the control diet with sodium butyrate, coated (T3)

The diets were fed for the first 9 days as phase 1 diets, from day 9 to 25 as phase 2 diets, and the final phase 3 diets were fed from day 25 to 35.

Food and water were provided *ad libitum* throughout the study. Birds were weighed on days 0, 9, 25 and 35 of the study. The amount of feed offered to each pen was weighed when added and refusals were weighed back on days 9, 25 and 35. Feed intake, body weight, FCR, FCE and % mortality were calculated for each feeding phase and for the overall period (days 0 to 35).
## Results

The results of the study indicate that the inclusion of Tributyrin in standard broiler diets significantly improve bird live weight at days 25 and 35 and FCR and FCE corrected for mortality between days 9 to 25 and day 0 to 35 compared to birds offered standard industry diets.

The inclusion of sodium butyrate coated product in standard broiler diets significantly improved bird live weights at day 25 and 35 and the FCR and FCE in phase 2 and the overall study compared to birds fed the control diet.

<table><caption>Table 1. Average live weight per bird (kg) for days 0, 9, 25 and 35 for all treatments</caption><thead><tr><th>Treatment</th><th>Day 0</th><th>Day 9</th><th>Day 25</th><th>Day 35</th></tr></thead><tbody><tr><td>T1 (Control)</td><td>0.039</td><td>0.262</td><td>1.511<sup>a</sup></td><td>2.511<sup>a</sup></td></tr><tr><td>T2 (Tributyrin)</td><td>0.039</td><td>0.268</td><td>1.569<sup>b</sup></td><td>2.627<sup>b</sup></td></tr><tr><td>T3 (Sodium Butyrate coated)</td><td>0.039</td><td>0.265</td><td>1.554<sup>b</sup></td><td>2.606<sup>b</sup></td></tr><tr><td>Significance Level</td><td>0.875</td><td>0.077</td><td><strong>0.017</strong></td><td><strong>0.033</strong></td></tr></tbody></table>

There were no overall significant differences in bird performance between the addition of Tributyrin compared to addition of sodium butyrate coated in a commercial broiler diet during phase 2, 3 and the overall study. There were significant differences in bird FCR and FCE corrected for mortality during phase 1 with birds offered the diet with the addition of Tributyrin performing better than the birds offered the diet containing the sodium-butyrate coated.

<table><caption>Table 2. Average FCR (corrected for mortality) for phase 1, 2, 3 and the overall study period for all treatments</caption><thead><tr><th>Treatment</th><th>Phase 1<br>(Day 0 – 9)</th><th>Phase 2<br>(Day 9 – 25)</th><th>Phase 3<br>(Day 25 – 35)</th><th>Overall<br>(Day 0 – 35)</th></tr></thead><tbody><tr><td>(Control)</td><td>1.093</td><td>1.403<sup>b</sup></td><td>1.756</td><td>1.515<sup>b</sup></td></tr><tr><td>(Tributyrin)</td><td>1.079</td><td>1.353<sup>a</sup></td><td>1.713</td><td>1.472<sup>a</sup></td></tr><tr><td>(Sodium Butyrate coated)</td><td>1.105</td><td>1.339<sup>a</sup></td><td>1.691</td><td>1.460<sup>a</sup></td></tr><tr><td>Significance Level</td><td>0.310</td><td><strong>&lt;0.001</strong></td><td>0.228</td><td><strong>&lt;0.001</strong></td></tr></tbody></table>

## Conclusions

Both butyric acid products (Tributyrin and sodium butyrate coated) included in the diets of broiler chickens produced significant improvements in bird performance thought to be due to their gut stimulation, growth promotion and / or anti-bacterial properties.

There were no detrimental effects of inclusion of Tributyrin and sodium-butyrate coated in broiler diets on bird health, welfare or performance.
P-075 (ID 235)

MILK SERUM: WASTE OR RECOURSE FOR BROILER DIET?

Tsiouris V.<fnref n="1" />,<fnref n="4" />, Georgopoulou I.<fnref n="1" />, Amvrosiadis I.², Batzios Chr.³, Sossidou E.<fnref n="4" />, Kontominas M.<fnref n="5" />

**Corresponding author:** biltsiou@yahoo.gr

**Abstract**

Whey is the milk by-product resulting from the production of cheese or curd cheese from milk. The nutritional value of whey as animal feed has been appreciated for a long time and it is no longer considered as a waste material. However, the absence of lactase in the digestive tract of poultry restricts the incorporation of high quantities in their diets. The objective of the present study was to investigate the effect of whey in various concentrations in poultry diets on the performance and the physico-chemical conditions of intestinal ecosystem of broiler chicks.

One hundred and twenty one-day-old broiler chicks were randomly allocated to four treatment groups: **group A**, negative control, **group B**, supplemented with 1% of whey in the diets of birds, **group C**, 2% of whey and **group D**, with 5% of whey, respectively. The following parameters were measured: body weight, pH and viscosity of intestinal digesta.

According to the results of this study, the supplementation of poultry diet with low concentration of whey (1-2%) increased significantly (P≤0.05) the body weight of birds and the pH of jejunum digesta, while reduced significantly (P≤0.05) the pH of caecum digesta and viscosity of jejunum and ileum digesta. Contrarily, the supplementation of poultry diet with high concentration of whey (5%) caused a significant reduction (P≤0.05) in the body weight, in the pH of caecum digesta and in the viscosity of jejunum digesta, while increased significantly (P≤0.05) the pH of duodenum and jejunum digesta as well as the viscosity of ileum digesta.

In conclusion, whey may constitute a valuable ingredient of poultry diet improving the performance and the gut health parameters of chicks. Contrarily, high concentrations of whey in poultry diets can cause growth retardation and impairment of gut health.

**Keywords:** whey, broiler chicks, performance, gut health

**Introduction**

Whey is the milk by-product resulting from the production of cheese or curd cheese from milk. More than 50% of its production is used by the feed industry on the basis of its excellent nutritional and functional properties. Moreover, the use of whey in animal feed is not only economically sensible, but it also solves the otherwise difficult problem of its disposal as waste. However, the absence of lactase in the digestive tract of poultry restricts the incorporation of high quantities in their diets (GULSEN et al., 2002; SHARIATMADARI et al., 2005). Therefore, few attempts have been made to evaluate the effect of whey usage on broiler performance. The objective of the present study was to investigate the effect of whey in various concentrations in poultry diets on the performance and on physico-chemical conditions of intestinal ecosystem of broiler chicks.

**Material and Methods**

One hundred and twenty one-day old broiler chicks were randomly allocated into four treatment groups of thirty (30) chicks each. The stocking density for each experimental group was 15 birds/m². Broilers in all groups fed a three phase ration (starter, grower and finisher), which was based on wheat, corn and soya. The addition of whey powder was done on grower and finisher rations. No antibiotic growth promoters, organic acids and phytobiotics were used. Feed and water were available ad libitum throughout the trial. The body weight was measured on the 13th, 23rd and 37th day of age, while mortality was recorded daily. After euthanasia on 37th day of age, the digesta of the duodenum, jejunum, ileum and cecum from each bird were immediately collected in separate tubes and vortexed. The pH of the duodenum and cecum from each bird was measured using a digital pH-meter. The content of the jejunum and the ileum, from each

<footnote n="1">¹Unit of Avian Medicine, ²Laboratory of Technology of Food of Animal Origin, ³Laboratory of Economics of Animal Production, School of Veterinary Medicine, Aristotle University of Thessaloniki, Greece</footnote>

<footnote n="4">⁴Institute of Animal Research, Hellenic Agricultural Organization-DEMETER, Thermi, Greece</footnote>

<footnote n="5">⁵Laboratory of Food Chemistry, School of Sciences, University of Ioannina, Greece</footnote>
bird, were centrifuged at 3,000 g for 45 minutes. Supernatants from each tube were taken and the pH as well as the viscosity were measured using a digital viscometer. Two readings were taken from each sample.

One-way analysis of variance was performed, to statistically evaluate possible effects of treatment on chicks' body weight, as well as on the pH and viscosity values of intestinal digesta. Shapiro-Wilk and Lilliefors tests were used to test for normality and the Levene's test for testing the homogeneity of variances. Differences between means of specific treatments were evaluated using Duncan's new multiple range test. Where assumptions about either variability or normality were seriously violated, the Kruskal-Wallis npar test was applied to evaluate treatment dependent differences, while the npar Wilcoxon rank sum test was used to determine whether the medians score values of specific treatments were different enough. All analyses were conducted using the statistical software program SPSS for Windows (v. 19.0).

## Results and Discussion

According to the results presented in Table 1, the supplementation of poultry diet with low concentrations of whey (1-2%) significantly increased ($P \le 0.05$) the body weight of birds and the pH of jejunum digesta, while significantly reduced ($P \le 0.05$) the pH of caecum digesta and viscosity of jejunum and ileum digesta. Thus, addition of whey to broiler diets by less than 2%, it may function as a natural growth promoter, since it contains lactose, proteins, minerals and yet unidentified growth factors (SHARIATMADARI et al., 2005). Moreover, whey increases the absorptive surface of intestinal mucosa and consequently, enhances effective utilisation of nutrients (GULSEN et al., 2002). In addition, lactose, the main sugar of whey, can be used as a readily available substrate for fermentation by intestinal lactic acid producing bacteria. Lactic acid influences the pH in the intestine, enhances the performance of birds and could suppress the growth of pathogenic bacteria (CORRIER et al., 1990; MCREYNOLDS et al., 2007).

However, the use of high concentrations of whey in poultry diets is compromised by its high lactose and sodium contents. The absence of lactase in the digestive tract of poultry indicates that incorporation of high concentrations of whey in the diet is likely to cause osmotic diarrhoea, with negative effects on broiler performance and gut health related problems (BILGILI and MORAN, 1990; MCREYNOLDS et al., 2007). As it is shown on the Table 1, the supplementation of poultry diet with high concentration of whey (5%) caused a significant reduction ($P \le 0.05$) in the body weight, in the pH of caecum digesta and in the viscosity of jejunum digesta, while it simultaneously increased significantly ($P \le 0.05$) the pH of duodenum and jejunum digesta as well as the viscosity of ileum digesta.

The mechanisms underlying on the effect of viscosity on nutrient digestion and absorption are rather complex. It is assumed that increased intestinal viscosity depresses nutrient digestibility by interference with the diffusion of digestive enzymes to their substrates and with the movement of digesta across the intestinal lumen. In addition, a higher intestinal viscosity increases the average retention time of the digesta and creates a favorable environment for bacterial activity, because the flow of digesta is reduced and the amount of undigested material in the intestinal tract is increased. This gives more time to the microbes to colonize the small intestine and results in greater competition with the host for nutrients (TSIOURIS et al., 2013).

<table><caption>**Table 1.** Effect of whey in various concentrations in poultry diets on the performance, pH and viscosity of intestinal digesta of broiler chicks ($x \pm SEM$).</caption><thead><tr><th rowspan="3"></th><th colspan="4">EXPERIMENTAL GROUPS</th></tr><tr><th>A</th><th>B</th><th>C</th><th>D</th></tr><tr><th>0 % whey</th><th>1 % whey</th><th>2 % whey</th><th>5 % whey</th></tr></thead><tbody><tr><td colspan="5"><strong>Body weight (Kg)</strong></td></tr><tr><td>13<sup>th</sup> day</td><td>0.435 ± 0.004<sup>a</sup></td><td>0.429 ± 0.004<sup>a</sup></td><td>0.431 ± 0.004<sup>a</sup></td><td>0.432 ± 0.004<sup>a</sup></td></tr><tr><td>23<sup>rd</sup> day</td><td>1.168 ± 0.014<sup>a</sup></td><td>1.165 ± 0.016<sup>a</sup></td><td>1.180 ± 0.014<sup>a</sup></td><td>1.137 ± 0.019<sup>a</sup></td></tr><tr><td>37<sup>th</sup> day</td><td>2.438 ± 0.065<sup>a</sup></td><td>2.586 ± 0.056<sup>b</sup></td><td>2.607 ± 0.037<sup>b</sup></td><td>2.239 ± 0.077<sup>c</sup></td></tr><tr><td colspan="5"><strong>pH</strong></td></tr><tr><td>Duodenum</td><td>5.96 ± 0.05<sup>a</sup></td><td>6.04 ± 0.04<sup>a</sup></td><td>6.03 ± 0.03<sup>a</sup></td><td>6.16 ± 0.04<sup>b</sup></td></tr><tr><td>Jejunum</td><td>5.19 ± 0.12<sup>a</sup></td><td>5.58 ± 0.10<sup>b</sup></td><td>5.62 ± 0.07<sup>b</sup></td><td>5.54 ± 0.11<sup>b</sup></td></tr><tr><td>Ileum</td><td>5.40 ± 0.18<sup>a</sup></td><td>5.54 ± 0.21<sup>a</sup></td><td>5.59 ± 0.19<sup>a</sup></td><td>5.58 ± 0.19<sup>a</sup></td></tr><tr><td>Caecum</td><td>6.53 ± 0.10<sup>a</sup></td><td>6.10 ± 0.08<sup>b</sup></td><td>6.05 ± 0.10<sup>b</sup></td><td>6.01 ± 0.09<sup>b</sup></td></tr><tr><td colspan="5"><strong>Viscosity (Centipoise)</strong></td></tr><tr><td>Jejunum</td><td>2.23 ± 0.09<sup>a</sup></td><td>1.51 ± 0.05<sup>b</sup></td><td>1.88 ± 0.05<sup>c</sup></td><td>1.82 ± 0.08<sup>c</sup></td></tr><tr><td>Ileum</td><td>2.42 ± 0.08<sup>a</sup></td><td>2.14 ± 0.09<sup>b</sup></td><td>2.62 ± 0.09<sup>ac</sup></td><td>2.85 ± 0.12<sup>c</sup></td></tr></tbody></table>

<footnote>a, b, c Mean values in the same row with a different superscript differ significantly ($P \le 0.05$).</footnote>
## Conclusions

The use of whey, in well tolerated concentrations, in poultry diets promotes the performance of birds and may suppress the growth of pathogenic bacteria, comprising an attractive feed additive in poultry diets. Further studies are required in order to elucidate the mechanisms underlying these actions as well as the effect of whey against intestinal pathogens.

## Acknowledgements

Authors acknowledge financial support under the Action "AgroETAK", MIS 453350, within the framework of the Opera-
tional Program 'Human Resources Development', co-funded by the European Social Fund through the National Strate-
gic Reference Framework (Research Funding Program 2007-2013) coordinated by the Hellenic Agricultural Organiza-
tion-DEMETER. The material presented is also based on MSc thesis data.

## Literature

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CORRIER D., A. HINTON, R. ZIPRIN, R.BEIER and J. DELOACH, 1990: Effect of dietary lactose on caecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks. Avian Diseases. **34**, 617-625.

GÜLŞEN, N., B. COŞKUN, H.D. UMUCALILAR, F. İNAL and M. BOYDAK, 2002: Effect of lactose and dried whey supplementation on growth performance and histology of the immune system in broilers. Archives of Animal Nutrition, 56, 131-139.

MCREYNOLDS J., J. BYRD, K. GENOVESE, T. POOLE, S. DUKE, M. FARNELL and D. NISBET, 2007: Dietary lactose and its effect on the disease condition of necrotic enteritis. Poultry Science. **86**, 1656-1661.

SHARIATMADARI F. and J. FORBES, 2005: Performance of broiler chickens given whey in the food and/or drinking water. British Poultry Science. **46**, 498-505.

TSIOURIS, V., I. GEORGOPOULOU, CHR. BATZIOS, N. PAPPAIOANNOU, A. DIAKOU, E. PETRIDOU, R. DUCATELLE and P. FORTOMARIS, 2013: The role of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chickens. Avian Pathology. **42**, 163-170
A 3-phase feeding scheme was applied with a starter period of 14 days followed by a grower diet between 15 and 28 days of age and a finisher diet between 29 and 38 days of age. All diets were prepared in the feed plant of Research Diet Services (Wijk-bij-Duurstede, the Netherlands) and delivered in bags to ZTC. Feed was provided *ad libitum* to the birds as a crumble (starter phase) or small pellets (grower and finisher phase). The trial was set up as a 3×2 factorial design with 3 diets differing in protein and energy content and 2 levels of NaDMG supplementation (0 or 1 g/kg NaDMG in the starter, 0 or 0.5 g/kg in the grower, and 0 or 0.3 g/kg in the finisher diet). Birds were fed one of the following treatments: (I) a wheat-soybean meal-corn-rapeseed meal based control diet (starter: 12.0 MJ/kg AME<sub>broiler</sub>, 20.5% CP and 1.10% dig. Lys; grower: 12.45 MJ/kg AME<sub>broiler</sub>, 19.5% CP and 1.03% dig. Lys; finisher: 12.66 MJ/kg AME<sub>broiler</sub>, 19.0% CP and 0.96% dig. Lys); (II) as (I) with 2% lower levels of CP and digestible amino acids; (III) as (I) with 2% lower AME<sub>broiler</sub> and 4% lower digestible amino acids and retainable P; (IV) as (I) with NaDMG; (V) as (II) with NaDMG; (VI) as (III) with NaDMG. All experimental diets contained NSP-enzymes, whereas no phytase and coccidiostats were included.

Average pen weight was recorded at 1, 14 and 38 days of age. Feed intake was recorded in the respective periods: 1-14 and 14-38 days. Feed conversion ratios (FCR), daily growth rate (ADG), bird-days and daily feed intake (ADFI) per bird were calculated for 1-14, and 14-38 days and also for the cumulative period 1-38 days. Litter quality was scored visually at day 21, 28 and 38 on a scale 0 to 3. A score 3 means very wet litter with a light (orange brown) colour; a score 0 implies very dry litter with a dark colour. On day 38 the foot pads of the present broilers per pen were scored (FPD scores). The scoring classes were on a scale of 0 (no or very mild lesions) to 3 (very severe lesions). All performance parameters were subjected to the analysis of variance (GLM procedure in SPSS).

## Results

The experiment was successfully conducted without any problems. Overall mortality was 4.0%. Both in the starter period (day 1-14) and the whole period (day 1-38) ADG was significantly affected by energy and protein level (P < 0.05), but not by NaDMG supplementation. FCR was in the starter period affected by both energy-protein level and NaDMG supplementation (P < 0.05). NaDMG supplementation significantly improved FCR (P < 0.05) in the whole period (-/0.04). Final body weight of the broilers fed NaDMG was numerically higher (+ 30 g). None of the treatments showed an effect on ADFI in the starter period and the whole period. The adjusted energy-protein levels and NaDMG supplementation did not improve litter scores and FPD scores. In this experiment, no interactions between energy-protein levels and NaDMG supplementation on the response parameters have been observed. Probably, it was not possible to prove significant interactions in this trial because of the very good experimental conditions and the limited amount of replicates. A summary of the main treatment effects on broiler performance and FPD score is represented in Table 1.

<table><caption>**Table 1:** Bird performance and FPD score (means ± SD) as affected by dietary treatment (day 0-38): main effects</caption><thead><tr><th rowspan="2"></th><th colspan="3">AME/CP level</th><th colspan="2">NaDMG</th><th colspan="3">Effect of:</th></tr><tr><th>control</th><th>low AA</th><th>low AME/ low AA</th><th>no</th><th>yes</th><th>AME/ CP</th><th>NaDMG</th><th>AME/ CP x NaDMG</th></tr></thead><tbody><tr><td>BW day 38 (g)</td><td>2765a<br>(± 97)</td><td>2701ab<br>(± 99)</td><td>2625b<br>(± 146)</td><td>2682<br>(± 138)</td><td>2712<br>(± 117)</td><td>P < 0.05</td><td>NS</td><td>NS</td></tr><tr><td>ADG (g/d)</td><td>71.6a<br>(± 2.6)</td><td>69.9ab<br>(± 2.6)</td><td>67.9b<br>(± 3.8)</td><td>69.4<br>(± 3.6)</td><td>70.2<br>(± 3.0)</td><td>P < 0.05</td><td>NS</td><td>NS</td></tr><tr><td>ADFI (g/d)</td><td>111.5<br>(± 4.8)</td><td>109.3<br>(± 5.3)</td><td>107.9<br>(± 4.6)</td><td>110.4<br>(± 4.8)</td><td>108.8<br>(± 5.1)</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>FCR</td><td>1.56<br>(± 0.03)</td><td>1.56<br>(± 0.04)</td><td>1.59<br>(± 0.06)</td><td>1.59x<br>(± 0.05)</td><td>1.55y<br>(± 0.03)</td><td>NS</td><td>P < 0.05</td><td>NS</td></tr><tr><td>FCR 1500 g</td><td>1.06b<br>(± 0.05)</td><td>1.10ab<br>(± 0.05)</td><td>1.16a<br>(± 0.11)</td><td>1.12x<br>(± 0.10)</td><td>1.07y<br>(± 0.06)</td><td>P < 0.05</td><td>P < 0.05</td><td>NS</td></tr><tr><td>Mortality (%)</td><td>4.7<br>(± 4.5)</td><td>5.3<br>(± 5.3)</td><td>2.0<br>(± 3.2)</td><td>4.9<br>(± 4.7)</td><td>3.1<br>(± 4.3)</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>FPD score</td><td>0.7<br>(± 0.8)</td><td>0.3<br>(± 0.6)</td><td>0.5<br>(± 1.1)</td><td>0.6<br>(± 1.0)</td><td>0.4<br>(± 0.8)</td><td>NS</td><td>NS</td><td>NS</td></tr></tbody></table>

ab, xy: significant differences (P < 0.05)
Conclusions

The results of this experiment suggest that NaDMG supplementation improves broiler performance parameters of which FCR was improved significantly. Although no significant interactions have been observed, the positive effect of NaDMG on FCR seems to be most pronounced in the diets with reduced energy and/or protein levels.

Literature

CLAPÉS, P., and M.R. INFANTE, 2002. Amino acid based surfactants: enzymatic synthesis, properties and potential applications. Biocatalysis Biotransform. 20: 215-233.

FRIESEN, R.W., E.M. NOVAK, D. HASMAN, and S.M. INNIS, 2007. Relationship of dimethylglycine, choline, and betaine with oxoproline in plasma of pregnant women and their newborn infants. Journal of Nutrition 137: 2641-2646.

KALMAR, I.D., A. COOLS, J. BUYSE, P. ROOSE and G.P.J. JANSSENS, 2010. Dietary *N*,*N*-dimethylglycine supplementation improves nutrient digestibility and attenuates pulmonary hypertension syndrome in broilers. Journal of Animal Nutrition and Animal Physiology 94: e339-e347.

PROLA, L., J. NERY, A. LAUWAERTS, C. BIANCHI, L. STERPONE, M. DE MARCO, L. POZZO, and A. SCHIAVONE, 2013. Effects of *N*,*N*-dimethylglycine sodium salt on apparent digestibility, vitamin E absorption and serum proteins in broiler chickens fed a high- or a low-fat diet. Poultry Science 92: 1221-1226.

VELDKAMP, T., 2010: Factors affecting foot pad dermatitis in broilers and turkeys. Proceedings II International Symposium. Highlights in nutrition and welfare in poultry production. Wageningen, the Netherlands.
P-079 (ID 229)

EFFECTS OF β-MANNANASE IN BROILERS ON LOW ENERGY DIETS

E. van Eerden<fnref n="1" />, C. Kwakernaak<fnref n="1" />, K. Poulsen<fnref n="2" />, and L. Suls<fnref n="2" />

**Corresponding author:** EvEerden@schothorst.nl

**Abstract**

β-Mannans are compounds present in e.g. soybean meal, with a structure that resembles pathogens. They may thus induce an immune response, which costs energy that cannot be used for production processes anymore. Therefore, enzymes breaking down β-mannans may have an energy sparing effect. The aim was to test if dietary supplementation with β-mannanase (Hemicell) could improve broiler performance in a practical-scale experiment. Four treatments were tested with eight replicate pens (820 Ross broilers per pen). For the starter, grower, and finisher phase a positive control (PC) with a standard energy level and a low energy control (LE = PC minus 120 kcal/kg AMEn) were formulated. One half of the LE diet was supplemented with β-mannanase. Also a medium energy diet (ME = PC minus 60 kcal/kg AMEn) with β-mannanase was formulated. The diets were maize/wheat/soybean meal based. The enzyme was dosed at 110 mL per tonne of feed. Diets were pelleted and freely available. Results from 0-38 days were analyzed with ANOVA. All treatments with reduced AMEn had a significantly higher feed intake than PC. Addition of β-mannanase to LE significantly improved body weight gain (81 g) and FCR (3.5 points). The supplemented LE treatment had a somewhat lower performance than PC, whereas the supplemented ME treatment performed similar to PC. Conclusion: dietary supplementation of β-mannanase improved broiler performance in low energy diets, suggesting an energy sparing effect.

**Keywords:** poultry, enzyme, β-mannanase, energy, production performance

**Introduction**

β-Mannans are fibers present in e.g. soybean meal, sunflower meal, palm kernel cake, guar meal, and rapeseed meal. Mannose sugar molecules, the main component of β-mannans are Pathogen-Associated Molecular Patterns (PAMPs). β-Mannans can thus be mistaken for invading pathogens and elicit an immune response. Energy needed for immune responses cannot be used for production processes. Therefore, enzymes breaking down β-mannans may have an energy-sparing effect. The aim of this experiment was to test if supplementation of β-mannanase (Hemicell®) to low energy diets could improve broiler performance under practical-scale conditions.

**Material and Methods**

The experiment had a randomized complete block design with four treatments and eight replicate pens per treatment. Each pen had a surface area of 47.5 m<fnref n="2" /> with fresh wood shavings, and contained 820 broilers (Ross 308, straight run). The trial had a starter phase from D0-10; a grower I phase from D10-21; a grower II phase from D21-31; and a finisher phase from D31-38.

The diets were maize/wheat/soybean meal based. For each feeding phase a positive control (PC) with a standard energy level and a low energy control (LE = PC minus 120 kcal/kg AMEn) were formulated. One half of the LE diet was supplemented with β-mannanase. Also a medium energy diet (ME = PC minus 60 kcal/kg AMEn) with β-mannanase was formulated. The treatments are summarized in Table 1.

<table><caption>**Table 1.** Energy content (kcal/kg) of the diets in each feeding phase</caption><thead><tr><th>Treatment</th><th>Description</th><th>Starter<br>D0-10</th><th>Grower I<br>D10-21</th><th>Grower II<br>D21-31</th><th>Finisher<br>D31-38</th></tr></thead><tbody><tr><td>1</td><td>Positive control (PC)</td><td>2820</td><td>2850</td><td>2900</td><td>2950</td></tr><tr><td>2</td><td>Low energy (LE)</td><td>2700</td><td>2730</td><td>2780</td><td>2830</td></tr><tr><td>3</td><td>LE + β-mannanase</td><td>2700</td><td>2730</td><td>2780</td><td>2830</td></tr><tr><td>4</td><td>ME + β-mannanase</td><td>2760</td><td>2790</td><td>2840</td><td>2890</td></tr></tbody></table>

<footnote n="1">¹ Schothorst Feed Research, Meerkoetenweg 26, 8218 NA Lelystad, the Netherlands</footnote>

<footnote n="2">² Elanco Animal Health, Plantin en Moretuslei 1A, 2018 Antwerp, Belgium</footnote>
ß-Mannanase was dosed at 110 mL per tonne of feed in a post-pelleting application. All diets contained xylanase and phytase, and were formulated to have the same amino acid content within a feeding phase. The starter diets were fed as crumbles, whereas the grower and finisher diets were fed as pellets.

Body weight, feed intake, and mortality were recorded per pen on a daily basis throughout the experimental period. Body weight gain and FCR were corrected for mortality in each feeding phase. The European Poultry Efficiency Factor (EPEF) was based on overall production figures and calculated as [live weight (kg) * survival (%)] / [age * FCR] * 100. The data were analyzed with ANOVA, using Genstat statistical software. Values with P≤0.05 were considered statistically significant. Statistics were based on two-sided tests. Treatment means were compared by least significant differences (LSD) after confirmation of significant treatment effects.

## Results

The birds performed well during the experiment, especially given the low dietary energy levels. Mean final body weight on day 38 was 2,356 g. The performance results are presented in Table 2. Reduction of AME by 120 kcal/kg and 60 kcal/kg significantly increased feed intake compared to PC. The addition of ß-mannanase to LE did not significantly affect feed intake compared to the non-supplemented LE treatment. Broilers eat to their nutritional requirements, and when dietary energy is reduced, feed intake will increase in order to meet energy requirements. If ß-mannanase would have a direct energy-releasing mode of action, then the inclusion of the enzyme in the low energy treatment would be expected to reduce feed intake compared to the low energy treatment without ß-mannanase. However, this experiment showed that this was not the case: feed intake in the low energy diet was numerically higher with ß-mannanase than without. This indicates that the mode of action of the ß-mannanase was not attributable to a direct energy-releasing effect, which supports the hypothesis of an energy-sparing effect due to lower costs spent on an immune response. However, actual effects on immune parameters were not measured.

The overall weight gain in LE was numerically lower than PC. The weight gain in the ß-mannanase supplemented treatments were numerically higher than PC without being statistically different. Adding ß-mannanase to the LE diet significantly increased body weight gain compared to the non-supplemented LE control.

The feed conversion ratio was significantly lower in the PC and the ß-mannanase supplemented ME diet than in both LE diets. However, adding ß-mannanase to the LE diet significantly improved FCR compared to the non-supplemented LE diet.

To account for differences in overall body weight gain, FCR was corrected to a standard body weight gain of 2,250 g, assuming 0.03 points FCR for each 100 g of body weight gain (FCR<sub>2250</sub> = FCR<sub>D0-38</sub> – 0.0003*(body weight gain<sub>D0-38</sub> – 250 g)). The results show that FCR<sub>2250</sub> was significantly higher in the non-supplemented LE treatment than in the other treatments, which were not significantly different from each other.

<table><caption>**Table 2.** Body weight gain (g), feed intake (g), feed conversion ratio (FCR; g/g), feed conversion ratio corrected to 2250 g body weight gain (FCR<sub>2250</sub>), and mortality (%) in broilers from 0 to 38 days of age, as affected by treatment. PC = positive control; LE = low energy; LEH = low energy + Hemicell; MEH = medium energy + Hemicell.</caption><thead><tr><th>Treatment</th><th>1</th><th>2</th><th>3</th><th>4</th><th></th><th></th></tr><tr><th>Description</th><th>PC</th><th>LE</th><th>LEH</th><th>MEH</th><th>LSD</th><th>P</th></tr></thead><tbody><tr><td>Body weight gain</td><td>2308 <sup>ab</sup></td><td>2278 <sup>a</sup></td><td>2359 <sup>b</sup></td><td>2344 <sup>b</sup></td><td>56.0</td><td>0.03</td></tr><tr><td>Feed intake</td><td>3690 <sup>a</sup></td><td>3822 <sup>bc</sup></td><td>3875 <sup>c</sup></td><td>3788 <sup>b</sup></td><td>60.9</td><td>&lt; 0.001</td></tr><tr><td>FCR</td><td>1.599 <sup>a</sup></td><td>1.678 <sup>c</sup></td><td>1.643 <sup>b</sup></td><td>1.616 <sup>a</sup></td><td>0.0216</td><td>&lt; 0.001</td></tr><tr><td>FCR<sub>2250</sub></td><td>1.581 <sup>a</sup></td><td>1.670 <sup>b</sup></td><td>1.610 <sup>a</sup></td><td>1.588 <sup>a</sup></td><td>0.0361</td><td>&lt; 0.001</td></tr><tr><td>Mortality</td><td>2.74</td><td>3.20</td><td>2.41</td><td>2.71</td><td>0.619</td><td>0.10</td></tr></tbody></table>

The EPEF results are presented in Figure 1. It shows that the non-supplemented LE treatment had a significantly lower EPEF than the other treatments. Supplementation of ß-mannanase in the LE diet significantly improved EPEF over the non-supplemented LE diet, resulting in similar EPEF as in PC.

<footnote>a,b,c Values without a common superscript within a row differ significantly (P≤ 0.05).</footnote>
<figure><img src="image_33.png" /><figcaption>**Figure 1.** European Poultry Efficiency Factor as affected by treatment. PC = positive control; LE = low energy; LEH = low energy + Hemicell; MEH = medium energy + Hemicell</figcaption></figure>

## Conclusion

It is concluded that dietary supplementation of β-mannanase in a low energy diet (120 kcal/kg below a standard energy level) significantly improved broiler performance over the same low energy diet without β-mannanase, which suggests an energy sparing effect of the enzyme. Supplementation of β-mannanase in a diet with 60 kcal/kg lower energy content resulted in similar performance as a non-supplemented positive control with a standard energy level.
P-080 (ID 1)

# THE EFFECT ON BROILER PERFORMANCE OF A CORN AND SOYBEAN MEAL DIET SUPPLEMENTATION WITH A SYNERGISTIC BUTYRATE BASED PRODUCT

Van Hamme V.¹ and Machado Junior P.C.²

¹ Impextraco nv, Heist-op-den-Berg, Belgium

² Impextraco Latin America, Curitiba, Brazil

**Corresponding author:** valentine@impextraco.be

## Abstract

The objective of this trial was to determine the effect on performance of a synergistic butyrate based supplementation of a corn and soybean meal broiler diet. A trial was carried out at the Experimental Broiler House of Impextraco Latin America, Brazil. Eight hundred day-old male Cobb® broilers were divided into 2 treatment groups, randomly distributed into 20 pens. Three high standard corn and soybean meal based diets were formulated according to bird's age. Treatments consisted of a negative control group (C) with standard feed and a group supplemented with a product based on coated calcium butyrate, calcium lactate and natural extracts (B) incorporated at 750g/ton in starter period and 500g/ton in grower and finisher period. Body weight (BW) and feed consumption were controlled weekly to provide data on feed conversion rate (FCR). A multivariate analysis of variance (MANOVA) was performed. At the end of the trial, the synergistic butyrate based supplementation of broiler feed improved FCR significantly and final body weight numerically. It may be concluded that, feeding a synergistic calcium butyrate based product on top of a gut health promoting corn based diet, still had beneficial effects on performance results of broilers.

**Keywords:** butyrate, broiler, performance

## Introduction

Butyrate is a natural substance present in mammals, with a biological role, as it is naturally produced by the gastrointestinal microbiota. Butyrate is the main energy source of colonocytes (Roediger, 1982). In physiological conditions, the positive effect on performance could be explained by the increased nutrient digestibility, the stimulation of the digestive enzyme secretions (Harada et al., 1983), the modification of intestinal microbiota (Van der Wielen et al., 2000), the improvement of the epithelial integrity (Sakata et al., 1983) and defense systems (Segain et. Al., 1999). Butyrate has also been implicated in down-regulation of bacterial virulence, both by direct effects on gene expression (Gantois et al., 2006) and by acting on cell proliferation of the host cells. In animal production, butyrate is a helpful feed additive, as it enhances performance (Hautekiet et al., 2011) and controls gut health disorders caused by bacterial pathogens.

## Material and Methods

The present trial was carried out at the Experimental Broiler House of Impextraco Latin America, located in Irati, Brazil. Eight hundred day-old male Cobb® broiler chicks, vaccinated against Infectious Bronchitis and Marek Disease at hatch, were individually weighed and divided into 2 groups, composed by 10 replicates of 40 animals each, and randomly distributed into pens (2 x 2m) covered with wood shavings. Two corn and soybean meal based diets were divided into three phases according to birds' age: starter (1 to 21 days of age), grower (22 to 35 days of age) and finisher feed (36 to 42 days of age). Treatments consisted of a negative control group (C) with standard feed and a group supplemented with a product based on coated calcium butyrate, calcium lactate and natural extracts (B) (BUTIFOUR® F) incorporated at 750g/ton in starter period and 500g/ton in grower and finisher period. Ad libitum access to water and feed was provided throughout the entire experiment, according to the CCAC guidelines (1993). Body weight (BW) and feed consumption were controlled weekly to provide data on feed conversion ratio (FCR). Weight of all dead animals was registered in order to correct FCR. The variables average FCR and average BW were analyzed using the software JPM® (SAS Institute Inc.). A multivariate analysis of variance (MANOVA) was performed, considering $p \le 0,05$.
<table><caption>**Table 1.** Composition of feeds</caption><thead><tr><th rowspan="2">INGREDIENTS (%)</th><th colspan="3">FEED</th></tr><tr><th>Starter</th><th>Grower</th><th>Finisher</th></tr></thead><tbody><tr><td>Corn</td><td>57,3383</td><td>59,4107</td><td>61,7346</td></tr><tr><td>Soybean meal</td><td>36,3133</td><td>33,7422</td><td>31,1283</td></tr><tr><td>Soybean oil</td><td>2,3821</td><td>3,0860</td><td>3,7061</td></tr><tr><td>Limestone</td><td>1,3824</td><td>1,3774</td><td>1,1974</td></tr><tr><td>Dicalciumphosphate</td><td>1,6541</td><td>1,4622</td><td>1,3216</td></tr><tr><td>Salt</td><td>0,4399</td><td>0,4421</td><td>0,4442</td></tr><tr><td>L-Lysine</td><td>0,0390</td><td>0,0493</td><td>0,0514</td></tr><tr><td>DL-Methionine</td><td>0,2352</td><td>0,2301</td><td>0,2096</td></tr><tr><td>Threonine</td><td>0,0157</td><td>0,000</td><td>0,0068</td></tr><tr><td>Mineral Premix</td><td>0,1000</td><td>0,1000</td><td>0,1000</td></tr><tr><td>Vitamin Premix</td><td>0,1000</td><td>0,1000</td><td>0,1000</td></tr></tbody></table>

**Results**

During this trial almost no significant effects of B on body weight could be detected. Due to an unknown reason, the product negatively influenced body weight on day 21. The impact on body weight was only temporary because at 42 days body weight was even numerically improved by B comparing to C.

<table><caption>**Table 2.** Body weight (mean ± standard deviation) (Prob>F: < 0,05 indicates significance)</caption><thead><tr><th rowspan="2"></th><th colspan="6">Body Weight (Kg)</th></tr><tr><th>7 days</th><th>14 days</th><th>21 days</th><th>28 days</th><th>35 days</th><th>42 days</th></tr></thead><tbody><tr><th>C</th><td>0,175±0,006</td><td>0,394±0,016</td><td>0,826±0,034</td><td>1,487±0,057</td><td>2,200±0,066</td><td>2,964±0,077</td></tr><tr><th>B</th><td>0,169±0,012</td><td>0,383±0,031</td><td>0,812±0,043</td><td>1,466±0,062</td><td>2,205±0,089</td><td>3,046±0,246</td></tr><tr><th>Prob>F</th><td>0,1284</td><td>0,0623</td><td>0,0303*</td><td>0,0747</td><td>0,3494</td><td>0,197</td></tr></tbody></table>

Feed conversion rate was significantly decreased by B starting from day 21 of evaluation, indicating that animals fed with B had improved intestinal digestive and absorptive capacities.

<table><caption>**Table 3.** Feed conversion ratio (mean±standard deviation) (Prob>F:<0,05 indicates significance)</caption><thead><tr><th rowspan="2"></th><th colspan="6">Feed Conversion Ratio</th></tr><tr><th>7 days</th><th>14 days</th><th>21 days</th><th>28 days</th><th>35 days</th><th>42 days</th></tr></thead><tbody><tr><th>C</th><td>0,872±0,039</td><td>1,359±0,080</td><td>1,461±0,085</td><td>1,499±0,055</td><td>1,585±0,041</td><td>1,690±0,028</td></tr><tr><th>B</th><td>0,889±0,049</td><td>1,341±0,122</td><td>1,420±0,063</td><td>1,471±0,04</td><td>1,557±0,04</td><td>1,628±0,084</td></tr><tr><th>Prob>F</th><td>0,5006</td><td>0,189</td><td>0,0465*</td><td>0,0298*</td><td>0,0498*</td><td>0,0045*</td></tr></tbody></table>

**Conclusions**

Findings from this trial reveal that the inclusion of a synergistic butyrate based product affects FCR significantly and body weight numerically. Animals that received a corn and soybean meal based diet supplemented with B, showed significant improvement in FCR starting from day 21 of the trial. Teirlinck et al. (2009) documented the protective effect of corn based diets on intestinal morphology and gut health comparing to wheat/rye based diets. One could expect it would be difficult to show any significant improvement by the addition of a gut health improving butyrate based product to this corn and soybean meal based diet. However this study has proven that a butyrate based product can significantly improve FCR. It may be concluded that, feeding a synergistic calcium butyrate based product on top of a gut health promoting corn based diet, still had beneficial effects on performance results of broilers.
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HARADA, E. & SATO, S. Effect of short-chain fatty acids on the secretory response of the ovine exocrine pancreas. American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 244no. G284-G290. 1983.

HAUTEKIET, V.; COLLET, P.; HAMME, V. van; GOETHALS, L.; SCHAUBLIN, H. Influence of an additive based on encapsulated calcium butyrate in two nutritional profiles on growth performance of broiler chickens. Actes des 9èmes Journées de la Recherche Avicole, Tours, France, p. 247-250. 2011.

ROEDIGER, W. E. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 83, p. 424-429. 1982.

SAKATA, T. & VON ENGELHARDT, W. Stimulatory effect of short chain fatty acids on the epithelial cell proliferation in rat large intestine. Comp Biochem Physiol A Comp Physiol 74, 459-462. 1983.

SEGAIN JP, RAINGEARD DE LA BLETIERE D., BOURREILLE A., (2000), Butyrate inhibits inflammatory responses through NFkB inhibition: implications for Crohn's disease. Gut 47, 397-403.

TEIRLYNCK E., BJERRUM L., EECKHAUT V., HUYGEBAERT G., PASMANS F., HAESEBROUCK F., DEWULF J., DUCATELLE R. AND VAN IMMERSEEL F., 2009, The cereal type in feed influences gut wall morphology and intestinal immune cell infiltration in broiler chickens, British Journal of Nutrition, 102, 1453-1461

VAN DER WIELEN, P. W.; BIESTERVELD, S.; NOTERMANS, S.; et al. Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth. Appl Environ Microbiol 66, p. 2536-2540. 2000
P-081 (ID 2)

# IMPACT OF AN AFLATOXIN CONTAMINATED FEED AND A MYCOTOXIN ELIMINATOR (ELITOX®) ON CIRCULATING IMMUNE CELLS AND SERUM ALBUMIN IN BROILERS

Koppenol A.<fnref n="1" />, Van Hamme V.<fnref n="1" />, Machado Junior P. C.<fnref n="2" /> and Caron L. F.<fnref n="3" />

**Corresponding author:** astrid@impextraco.com

## Abstract

The aim of the trial was to determine the impact of an early aflatoxicosis on serum albumin and circulating leucocytes and the protective effect of a mycotoxin eliminator. An in vivo trial was performed at UFPR, Brazil. Seventy two male day old Cobb® broilers were housed up to 21 days and divided into 3 treatments (T) (n=24): a negative control diet (C), a contaminated feed (500ppb of purified Aflatoxin B1 – AFB1) (AFLA), and contaminated feed supplemented with 0,2% of a mycotoxin eliminator (ME) (E-AFLA). At day 10 and 21, blood samples were collected (n=10/T) to quantify the amount of circulating activated cytotoxic T-cells, suppressor macrophages and B-cells through flow cytometry and to evaluate liver protein synthesis by quantifying serum albumin. Contamination with AFB1 led to a significant increase in activated cytotoxic T-cells at 10 days of age and a numerical increase in circulating suppressor macrophages comparing to C. The use of a ME reduced the amount of such cells down to the level of C. At d21, contamination with AFB1 resulted in numerically fewer B cells. The addition of ME again counteracted this effect. Serum albumin levels were significantly reduced in the AFLA group compared to the C group and brought back to control levels due to the inclusion of ME. The inclusion of a ME in broiler feed prevented early immunological impairment and liver damage induced by AFB1.

**Keywords:** Aflatoxin, Mycotoxin eliminator, Immune cells, Albumin, Liver

## Introduction

Aflatoxin is a secondary metabolite produced by mold species, predominantly *Aspergillus flavus* and *Aspergillus parasiticus* (Do, 2007), being reported by many authors as carcinogenic, mutagenic, teratogenic and growth inhibiting (Ortatatl, 2005). Animals intoxicated with aflatoxins present anorexia, decrease in growth rate, weight gain, egg production and weight, as well as increased mortality rate (Leeson et al., 1995). Macroscopic and microscopic alterations in liver (Espada et al., 1992), kidneys and spleen (Glahn et al., 1991) and humoral and cellular immunity impairment may also be observed (Oguz et al., 2003).

## Material and Methods

The trial was conducted at the Federal University of Paraná, Curitiba, Brazil. The experimental unit consisted of three 2m<fnref n="2" /> isolators covered with wood shavings, into a negative pressure ventilation system. Seventy two male day old Cobb® broiler chickens vaccinated against Infectious Bronchitis at hatch were used. Trial duration was 21 days. Ad libitum access to water and feed was provided throughout the entire experiment, according to the CCAC guidelines (1993). On day 1, all animals were individually weighed, identified with a wing tag, and equally distributed according to body weight into groups providing 3 treatments (T) (n=24): a control group (C), a contaminated feed (500ppb of purified Aflatoxin B1-AFB1) (AFLA), and a contaminated diet supplemented with 0,2% of mycotoxin eliminator (ME) (ELITOX®) (E-AFLA). On days 10 and 21, blood samples of 10 animals from each treatment were collected, to quantify different lymphoid cells (Imunova Biological Analysis Company) by flow cytometry and to evaluate the hepatic function (at the Veterinary Clinical Pathology Laboratory from the Federal University of Paraná). Serum was extracted by centrifugation to evaluate albumin content. Statistics were performed through ANOVA followed by the Tukey-Kramer Post Hoc Test, both considering $p \le 0,05$.

<footnote n="1">¹ Impextraco nv, Heist-op-den-berg, Belgium</footnote>

<footnote n="2">² Impextraco Latin America, Curitiba, Brazil</footnote>

<footnote n="3">³ Department of Basic Pathology, Sector de Ciências Biológicas, Centro Politécnico, Universidade Federal do Paraná (UFPR), Curitiba, Brazil</footnote>
## Results

Albumin, an important protein produced in liver, was decreased by both E-AFLA and AFLA on day 10. However, on day 21, ME returned albumin levels close to the control, which indicates some protective effect of ME on this variable.

<table><caption>**Table 1.** Mean albumin levels. Same letters within the same column indicate similar means.</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="2">Albumin</th></tr><tr><th>D10</th><th>D21</th></tr></thead><tbody><tr><td>C</td><td>1.35±0.14a</td><td>1.54±0.2a</td></tr><tr><td>AFLA</td><td>1.13±0.12b</td><td>1.35±0.12b</td></tr><tr><td>E-AFLA</td><td>1.1±0.14b</td><td>1.46±0.15ab</td></tr><tr><td>p>F</td><td>0.02*</td><td>0.0109*</td></tr></tbody></table>

Flow cytometry determinations showed significant differences for several immune cells. Activated cytotoxic T-lymphocytes were significantly impaired due to AFB1 contamination. AFB1 induced an initial increase and a further decrease, comparing to C and E-AFLA. Activated cytotoxic T-lymphocytes are a subset of T-cells that already migrated to the secondary lymphoid organs, where they get in contact with antigens. Such process occurs naturally as birds grow and are exposed to antigens. An initial higher number of these cells in the AFLA treatment may indicate an increased earlier need for cell activation and a later suppressive effect caused by the toxin, which may compromise birds' response against challenges. This negative effect was counteracted by ME.

<figure><img src="image_34.png" /></figure>

**Fig. 1.** Mean percentage of Activated Cytotoxic T-Lymphocytes related to Peripheral Blood Mononuclear Cells (PBMC) and mean percentage of B-lymphocytes. Same letters within the same points of evaluation indicate The amount of B-lymphocytes was numerically increased when comparing AFLA to C and significantly increased when comparing the former to E-AFLA. However, after 21 days of exposure, such pattern shifted to a significant reduction of B-lymphocytes in AFLA, when compared to E-AFLA and a numerical reduction when compared to C. B-lymphocytes are cells responsible for antibody production. Hence, later reductions in this cell subset may reflect an impairment in humoral immune response.

Exposure to AFB1 led to a numerical increase of circulating suppressor phagocytic monocytes (termed here as suppressor macrophages) in untreated birds after 10 and 21 days of exposure, while both C and E-AFLA showed numerical similar number of cells.
<figure><img src="image_35.png" /><figcaption>**Fig. 2** Mean percentage of Suppressor Phagocytic Monocytes (Macrophages).</figcaption></figure>

**Conclusions**

Findings of this trial could confirm Aflatoxin B1 at 500ppb significantly impaired liver function, as viewed through reduced albumin levels, and the immune system of animals. These negative effects could be counteracted by the use of a ME and thus prevent liver damage and early immunological impairment induced by AFB1.

**Literature**

DO, J. H., CHO, D. K., 2007, Aflatoxins: Detection, Toxicity, and Biosynthesis. Biotechnology and Bioprocess Engineering, 12:585-593

ESPADA, Y., DOMINGO, M., GOMEZ, J., CALVO, M. A., 1992. Pathological lesions following an experimental intoxication with aflatoxin B1 in broiler chickens. Research in Veterinary Science 53, 275-279

GLAHN, R. P., BEERS, K. W., BOTTJE, W. G., WIDEMAN, R. F., HUFF, W. E., THOMAS, W., 1991. Aflatoxicosis alters avian renal function, calcium, and vitamin D metabolism. Journal of Toxicology and Environmental Health 34, 309-321

LEESON, S., DIAZ, G., SUMMERS, J. D., 1995. Aflatoxins. In: Leeson, S., Diaz, G., Summers, J.D. (Eds.), Poultry Metabolic Disorders and Mycotoxins. University Books, Canada, Ont., pp. 248-279.

OGUZ, H., HADIMLI, H. H., KURTOGLU, V., ERGANIS, O., 2003. Evaluation of humoral immunity of broilers during chronic aflatoxin (50 and 100 ppb) and clinoptilolite exposure. Revue de Medicine Veterinaire 154, 483-486

ORTATATLI, M., OGUZ, H., HATIPOGLU, F., KARAMAN, M., 2005 Evaluation of pathological changes in broilers during chronic aflatoxin (50 and 100 ppb) and clinoptilolite exposure. Research in Veterinary Science, 78:61-68
P-082 (ID 211)

EFFECT OF FEEDING POLYPHENOLS ON CAMPYLOBACTER COUNTS IN BROILERS

P. Vazquez<fnref n="1" />, O. Casabuena<fnref n="1" />, S. Porras<fnref n="1" />, F.J. García-Peña<fnref n="2" />, and M.I. Gracia<fnref n="1" />

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

**Abstract**

An experiment was conducted in order to evaluate the effect of a blend of polyphenols (PPh; 1% hidroxibenzoic acids) added to the feed on cecal *Campylobacter* counts in broilers. There were two treatments applied from 1 to 42 d of age, T1: Positive controls (*Campylobacter*, no additives) and T2: T1 + PPh at 2 kg/t from 0-21 d and 4 kg/t from 21-42d. A total of 84 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 expressed as log₁₀ CFU/g ceca content were first tested for normality and then analysed by the non-parametric test of Kruskall-Wallis (SPSS v.19.0). No significant differences in the *Campylobacter* counts were observed between treatments at 21d (7.83 vs 7.43 log₁₀ CFU/g, for T1 vs T2; P = 0.85) or 42 d of age (6.77 vs 6.09 log₁₀ CFU/g, for T1 vs T2; P = 0.14). However, *Campylobacter* counts tended to be reduced by PPh supplementation at 35 d of age (5.99 vs 7.12 log₁₀ CFU/g; P = 0.07). It is concluded that supplementation of broiler diets with a blend of polyphenols might reduce *Campylobacter* infection in broilers.

**Keywords:** polyphenols, *Campylobacter jejuni*, broiler

**Introduction**

Campylobacteriosis is a foodborne disease associated with the infection of humans by thermophilic *Campylobacter* spp., mainly *C. jejuni* and *C. coli* (LASTOVICA AND SKIRROW, 2000). *Campylobacter* is the major cause of bacterial gastroenteritis in humans worldwide. In the EU, it is the most commonly reported gastrointestinal bacterial pathogen since 2005, with 214,779 human cases reported in 2013 (EFSA, 2015). *Campylobacter* preventive measures aim at reducing the probability of *Campylobacter* colonisation in birds (GHAREEB et al., 2013). For this purpose several approaches, as the use of bacteriophages (WAGENAAR et al., 2005), bacteriocins (LINE et al., 2008), organic acids (CHAVEERACH et al., 2004; VAN DEUN et al., 2008; SKÂNSENG et al., 2010) and their derivatives (HILMARSSON et al., 2006) or medium chain fatty acids (HERMANS et al., 2010) have been proposed over the past few years. Also phytochemicals could be considered as an alternative. These substances have low molecular weight and they are derived from the plant secondary metabolism and include glucosides, alkaloids, essential oils, saponins, mucilage, phenolic compounds, and flavonoids (MARTINS et al., 2000). Grape seeds and skins are good sources of phytochemicals such as gallic acid, catechin, and epicatechin and are suitable raw materials for the production of antioxidative dietary supplements. Flavonoids, as catechin and epicatechin, have been shown to be able to affect various biological functions: capillary permeability, cellular secretary processes involved in the inflammatory response and inhibition of enzymes, receptors and carriers (RHAMA and MADHAVAN, 2011). Thousands of phytochemicals, including phenols, have already been identified to be inhibitory towards microorganisms (COWAN, 1999). In fact, RHAMA and MADHAVAN (2011) proved that several flavonoids had even more *in vitro* antibacterial activity than tetracycline. Previous studies carried out by GANAN et al. (2009) also found that some phenolic compounds present in wine and grape residues significantly reduced *in vitro* the viability of *Campylobacter*. However, their ability to reduce *Campylobacter* has not been investigated *in vivo* and differences between *in vitro* and *in vivo* trials are not fully understood. Therefore, the objective of this study was to evaluate the effect of a blend of polyphenols (PPh; 1% hidroxibenzoic acids) added to the feed on cecal *Campylobacter* counts in broilers.

**Material and Methods**

A total of 84 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 two treatments applied from 1 to 42 d of age, T1: Positive controls (*Campylobacter*, no additives) and T2: T1 + PPh at 2 kg/t from 0-21 d and 4 kg/t from 21-42d. 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 experi-

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

<footnote n="2">²Laboratorio de Sanidad Animal de Algete, MAGRAMA, Spain</footnote>
mental 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, cae-
cal samples of 6 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 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 expressed as log₁₀ CFU/g ceca content were first tested for normality and then analysed by the nonparametric test
of Kruskall-Wallis (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

Although broiler fed PPh always showed less *Campylobacter* counts, no significant differences in the *Campylobacter* counts were observed between treatments at 21d (7.83 vs 7.43 log₁₀ CFU/g, for T1 vs T2; P = 0.85) or 42 d of age (6.77 vs 6.09 log₁₀ CFU/g, for T1 vs T2; P = 0.14). However, *Campylobacter* counts tended to be reduced by PPh supplementation at 35 d of age (5.99 vs 7.12 log₁₀ CFU/g; P = 0.07).

<figure><img src="image_36.png" /><figcaption>**Figure 1.** Effect of dietary treatment (T1: Negative Control and T2: Polyphenols) on *Campylobacter* counts (cfu/g of cecal content expressed as log₁₀) at 21, 35 and 42 days.</figcaption></figure>

## Conclusions

It is concluded that supplementation of broiler diets with a blend of polyphenols might reduce *Campylobacter* infection 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.
## Literature

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

COWAN M.M., 1999: Plant products as antimicrobial agents. Clin. Microbiol. Rev., **12**,564-582.

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.

GANAN, M., A.V. CARRASCOSA, A.J. MARTÍNEZ-RODRÍGUEZ, 2009: Antimicrobial activity of phenolic compounds of wine against *C. jejuni*. Food Control **20**, 749-752.

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

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

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

LASTOVICA, A.J., M.B. SKIRROW, 2000: Clinical significance of *Campylobacter* and related species other than *C. jejuni* and *C. coli*. In I. Nachamkin and M. J. Blaser (ed.), *Campylobacter*, 2nd ed. American Society for Microbiology, Washington, D.C, 89-120.

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

MARTINS E.R., D.M. CASTRO, D.C. CASTELLANI, J.E. DIAS, 2000: Buscando a saúde por medio das plantas medicinais. Plantas medicinais. CUFV ed. Viçosa pp. 17-55.

RHAMA S., S. MADHAVAN, 2011: Antibacterial Activity of the Flavonoid, patulitrin isolated from the flowers of *Tagetes erecta* L. Int. J. PharmTech Res., **3**, 1407-1409.

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

VAN DEUN K., F. HAESEBROUCK, F. VAN IMMERSEEL, R. DUCATELLE, F. PASMANS, 2008: Short-chain fatty acids and l-lactate as feed additives to control *C. jejuni* infections in broilers. Avian Pathol., **37**, 379-383.

WAGENAAR, J.A., M.A. VAN BERGEN, M.A. MUELLER, T.M. WASSENAAR, R.M. CARLTON, 2005: Phage therapy reduces *C. jejuni* colonization in broilers. Vet. Microbiol., **109**, 275-283.
# Feed enzymes

P-086 (ID 40)

## AMINO ACIDS CONCENTRATION OF INTERNAL CONTENTS AND ELEMENTAL CONTENTS OF EGGSHELL OF QUAIL EGGS DUE TO ENZYME-SUPPLEMENTED HIGH OR LOW FIBRE DIETS

*S.E. Alu<fnref n="1" />, S.N. Carew², F. G. Kaankuka³ and C.D. Tuleun³

*Corresponding Author: Tel. +2348033690937 & +2348091651998

E-mail: seafarms2000@yahoo.com or louderpraise@gmail.com

### Abstract

Three hundred and sixty 6 weeks old laying quails were utilized in an 8 week experiment to evaluate the effects of Maxigrain® enzyme supplementation of sugarcane scrapping meal-based diets on amino acids concentration of internal contents of quail eggs and the elemental contents of the eggshells. Six diets tagged T10₀, T10₅, T10₁₀, T15, T15₅ and T15₁₀ were formulated to be isonitrogenous (20%CP) and isocaloric (12.14 MJ/kg, ME). The enzyme was included at 0, 5 and 10mg/kg thus; treatments T10₅, T10₁₀, T15₅ and T15₁₀ contained 5 and 10mg/kg of the enzyme respectively. Treatments T10₀ functioned as the control for T10₅ and T10₁₀ while T15₀ was for T15₅ and T15₁₀, respectively. The birds were randomly allotted to the 6 dietary treatments at the rate of 60 birds per diet. Each treatment was replicated 4 times in a 3 x 2 factorial arrangement having 15 point-of-lay female birds per replicate. Three eggs of similar weights per replicate were randomly selected (n=72) for determination of amino acids concentration and their eggshells analyzed for mineral contents. Enzyme supplementation significantly (P<0.05) improved threonine (5.34 vs. 5.16 and 5.61mg/g), arginine (3.84 vs. 3.89 and 4.27 mg/g), methionine (2.66 vs. 2.72 and 2.86 mg/g), and phenylalanine (4.23 vs. 4.22 and 4.63 mg/g); all the acids evaluated were not influenced by the interactive effects of the enzyme and fibre. Enzyme or fibre did not influence (P>0.05) all the minerals analyzed; the interaction of the enzyme and fibre influenced all the minerals except for K which did not vary significantly (P>0.05). Birds fed the T10₀ produced better Ca values (11.92 and 12.75 %) compared to those fed the other diets. Same trend was recorded for Na (0.71 and 0.74 %) and Cl (0.69 and 0.73 %) concentration of the eggshells. Birds fed the T10₀ also produced better (P<0.05) result for P (8.75 %) than those fed the other diets. In view of the results gathered laying quails fed the high fibre-high enzyme supplemented diets will produce normal eggs.

**Keywords:** Layer quails, sugarcane scrapping meal, enzyme, amino acids, minerals and eggshell

### Introduction

Minerals are important for normal bone development, blood clot formation, muscle contraction and maintenance of good egg shell quality. They also take part in cellular membranes and are required for many metabolic functions. Some of them aid digestion as a component of hydrochloric acid (Olomu, 1979). The productivity, reproduction and health of flock greatly depend on the quality of feed they receive. Therefore, feeds should be properly formulated with appropriate requirement for particular group of animals (Adeola and Olukosi, 2008). The objective of this study is therefore; to evaluate the effect of replacing maize with sugarcane scrapping meal supplemented with exogenous enzyme on layer growth rate and layer parameters of laying quails.

### Materials and Methods

#### Study area

The experiment was carried out at the Teaching and Research Farm of the Faculty of Agriculture, Nasarawa State University, Keffi, Shabu - Lafia Campus, Nasarawa State, Nigeria.

#### Sugarcane scrapping

The proximate analysis of sample of sugarcane scraping meal (Table 1) were carried using the procedure outlined by AOAC (1984) while the fibre fractions were determined by methods of Vansoest and Robertson (1985) and the values reported on a dry matter basis.

<footnote n="1">¹Department of Animal Science, Faculty of Agriculture, Nasarawa State University, Keffi, P.M.B.135, Shabu-Lafia Campus, Nasarawa State, Nigeria; ²Department of Animal Production, ³Department of Animal Nutrition, College of Animal Science, University of Agriculture, P.M.B. 2373 Makurdi, Benue State, Nigeria</footnote>
<table><caption>**Table 1.** Proximate and energy composition of sugarcane scrapping</caption><thead><tr><th>Nutrient</th><th>CP</th><th>EE</th><th>CF</th><th>Ash</th><th>DM</th><th>NFE</th><th><sup>a</sup>Energy (MJ/kg ME)</th></tr></thead><tbody><tr><td>%</td><td>8.25</td><td>3.36</td><td>36.48</td><td>9.98</td><td>90.67</td><td>67.40</td><td>12.42</td></tr></tbody></table>

Six experimental diets tagged T10₀, T10₅, T10₁₀, T15, T15₅ and T15₁₀ were compounded to be isonitrogenous (23% crude protein) and isocaloric (11.67 MJ/kg, ME) with two levels of crude fibre, 10 and 15%. The birds were randomly allocated to the treatments at rate of 60 birds per diet in an 8 week experiment. Each treatment was replicated 4 times in a 3x2 factorial having 15 birds per replicate. Treatments T10₀, T10₅, and T10₁₀ contained 10% crude fibre and had 0, 5 and 10mg/kg of the enzyme while treatments T15₀, T15₅ and T15₁₀ contained 15% crude fibre level and had 0, 5 and 10mg/kg of the enzyme, respectively; such that treatments T10₀ and T15₀ served as the controls.

The birds were fed *ad-libitum* and allowed to have access to drinking water at all times. Other routine management practices were adopted as outlined by Musa *et al.* (2007). A total of 17 (2:15 male and female) sexually mature 9 weeks old quails per replicate were utilized for the experiment. Three eggs per replicate of similar weights were randomly selected (n=72) for determination of amino acids composition using the method of Speckman *et al.* (1958). The egg-shells so collected from each replicate were cleaned to be devoid of debris, oven-dried at 30°C for 18hrs to remove moisture, bulked, ground and thoroughly mixed to obtain a homogenous mixture. Samples of the mixture were analyzed for mineral according to standard methods outlined (AOAC, 1984).

### Statistical analysis

Data obtained were subjected to Two Way Analysis of Variance (ANOVA) and where significant differences (P>0.05) are observed, means were separated using Duncan's Multiple Range Test as described by Steel and Torrie (1980).

## Results and Discussion

Enzyme supplementation (Table 2) significantly (P<0.05) improved (5.34 vs. 5.16 and 5.61mg/g) threonine (3.84 vs. 3.89 and 4.27 mg/g), arginine (2.66 vs. 2.72 and 2.86 mg/g), methionine (2.66 vs. 2.72 and 2.86 mg/g) and phenylalanine (4.23 vs. 4.22 and 4.63 mg/g), but did not affect (P>0.05) all the minerals evaluated. This observation may be explained by the facts that availability of amino acids is affected by the presence of digestives endogenous enzyme, inhibitors, tannins and gossypol (Olomu, 1979); addition of exogenous enzyme in this study possibly increased the availability of these acids in the egg content. Dietary fibre (Table 2) reduced (P<0.05) the concentration of all amino acids except for cystine (0.42 mg/g) which remained uniform at both low and high fibre diets while mineral concentration was unaffected. The observations reported in this study agree with the earlier assertion of Olomu (1979).

All the amino acids evaluated were not influenced by the interactive effects of Maxigrain® enzyme and dietary fibre while all the minerals were reduced (P<0.05) except for K which did not vary significantly (P>0.05) (Table 3). Logically, the combination of high fibre diets with enzyme will reduce the negatives effects of fibre in the general performance of an animal (Adeola and Olukosi, 2008).

## Conclusions and Recommendations

In view of the results gathered laying quails fed the high fibre-high enzyme supplemented diets will produce eggs with normal chemical composition.

<footnote><sup>a</sup>Calculated from Pauzenga (1985)</footnote>
<table><caption>**Table 2.** Effect of Maxigrain® enzyme or dietary fibre on amino acids concentration of internal contents of quail eggs and mineral composition of eggshell</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">ENZYME TREATMENT MEANS</th><th colspan="4">FIBRE TREATMENT MEANS</th></tr><tr><th>No Enzyme</th><th>100ppm Enzyme</th><th>200ppm Enzyme</th><th>SEM</th><th>LOS</th><th>Low fibre</th><th>High fibre</th><th>SEM</th><th>LOS</th></tr></thead><tbody><tr><td><strong>Amino Acids (mg/g)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Lysine</td><td>4.90</td><td>4.87</td><td>5.11</td><td>0.13</td><td>NS</td><td>5.28<sup>a</sup></td><td>4.64<sup>b</sup></td><td>0.11</td><td>*</td></tr><tr><td>Histidine</td><td>2.22</td><td>2.21</td><td>2.25</td><td>0.03</td><td>NS</td><td>2.28<sup>a</sup></td><td>2.17<sup>b</sup></td><td>0.03</td><td>*</td></tr><tr><td>Arginine</td><td>5.34<sup>a</sup></td><td>5.16<sup>b</sup></td><td>5.61<sup>a</sup></td><td>0.08</td><td>*</td><td>5.55<sup>a</sup></td><td>5.19<sup>b</sup></td><td>0.07</td><td>*</td></tr><tr><td>Aspartic acid</td><td>8.21</td><td>8.02</td><td>8.49</td><td>0.13</td><td>NS</td><td>8.52<sup>a</sup></td><td>7.96<sup>b</sup></td><td>0.11</td><td>*</td></tr><tr><td>Threonine</td><td>3.84<sup>b</sup></td><td>3.89<sup>b</sup></td><td>4.27<sup>a</sup></td><td>0.12</td><td>*</td><td>4.33<sup>a</sup></td><td>3.66<sup>b</sup></td><td>0.10</td><td>*</td></tr><tr><td>Serine</td><td>3.55</td><td>3.64</td><td>3.74</td><td>0.08</td><td>NS</td><td>3.82<sup>a</sup></td><td>3.47<sup>b</sup></td><td>0.07</td><td>*</td></tr><tr><td>Glutamic acid</td><td>13.04</td><td>13.03</td><td>13.29</td><td>0.10</td><td>NS</td><td>13.31<sup>a</sup></td><td>12.92<sup>b</sup></td><td>0.08</td><td>*</td></tr><tr><td>Proline</td><td>3.29</td><td>3.17</td><td>3.52</td><td>0.10</td><td>NS</td><td>3.57<sup>a</sup></td><td>3.09<sup>b</sup></td><td>0.08</td><td>*</td></tr><tr><td>Glycine</td><td>6.30</td><td>6.23</td><td>6.69</td><td>0.18</td><td>NS</td><td>6.79<sup>a</sup></td><td>6.02<sup>b</sup></td><td>0.15</td><td>*</td></tr><tr><td>Alanine</td><td>5.21</td><td>5.30</td><td>5.46</td><td>0.11</td><td>NS</td><td>5.60<sup>a</sup></td><td>5.05<sup>b</sup></td><td>0.09</td><td>*</td></tr><tr><td>Cystine</td><td>0.41</td><td>0.42</td><td>0.43</td><td>0.01</td><td>NS</td><td>0.42</td><td>0.42</td><td>0.01</td><td>NS</td></tr><tr><td>Valine</td><td>5.07</td><td>5.05</td><td>5.45</td><td>0.15</td><td>NS</td><td>5.48<sup>a</sup></td><td>4.90<sup>b</sup></td><td>0.13</td><td>*</td></tr><tr><td>Methionine</td><td>2.66<sup>b</sup></td><td>2.72<sup>b</sup></td><td>2.86<sup>a</sup></td><td>0.05</td><td>*</td><td>2.85<sup>a</sup></td><td>2.64<sup>b</sup></td><td>0.04</td><td>*</td></tr><tr><td>Isoleucine</td><td>4.13</td><td>4.06</td><td>4.43</td><td>0.17</td><td>NS</td><td>4.47<sup>a</sup></td><td>3.94<sup>b</sup></td><td>0.14</td><td>*</td></tr><tr><td>Leucine</td><td>7.69</td><td>7.76</td><td>8.11</td><td>0.14</td><td>NS</td><td>8.26<sup>a</sup></td><td>7.45<sup>b</sup></td><td>0.11</td><td>*</td></tr><tr><td>Tyrosine</td><td>3.37</td><td>3.45</td><td>3.61</td><td>0.09</td><td>NS</td><td>3.69<sup>a</sup></td><td>3.27<sup>b</sup></td><td>0.07</td><td>*</td></tr><tr><td>Phenylalanine</td><td>4.23<sup>b</sup></td><td>4.22<sup>b</sup></td><td>4.63<sup>a</sup></td><td>0.12</td><td>*</td><td>4.69<sup>a</sup></td><td>4.03<sup>b</sup></td><td>0.10</td><td>*</td></tr><tr><td><strong>Minerals (%)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Calcium</td><td>12.33</td><td>4.94</td><td>5.14</td><td>0.25</td><td>NS</td><td>6.88</td><td>8.06</td><td>1.00</td><td>NS</td></tr><tr><td>Phosphorus</td><td>9.18</td><td>2.20</td><td>2.32</td><td>0.12</td><td>NS</td><td>4.26</td><td>4.87</td><td>1.01</td><td>NS</td></tr><tr><td>Potassium</td><td>1.64</td><td>0.72</td><td>0.91</td><td>0.34</td><td>NS</td><td>1.06</td><td>1.12</td><td>0.11</td><td>NS</td></tr><tr><td>Sodium</td><td>0.73</td><td>0.30</td><td>0.32</td><td>0.04</td><td>NS</td><td>0.44</td><td>0.46</td><td>0.06</td><td>NS</td></tr><tr><td>Chlorine</td><td>0.71</td><td>0.27</td><td>0.28</td><td>0.17</td><td>NS</td><td>0.40</td><td>0.44</td><td>0.06</td><td>NS</td></tr></tbody></table>

a,b- Means on the same row bearing different superscript differ significantly (P < 0.05), NS- No significant difference (P > 0.05),
LOS- Level of significant difference.
<table><caption>**Table 3.** Effects of Maxigrain® enzyme and dietary fibre on amino acids concentration of internal contents of quail eggs and mineral composition of eggshell</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="7">MAIN TREATMENT MEANS</th></tr><tr><th>T10<sub>0</sub></th><th>T10<sub>100</sub></th><th>T10<sub>200</sub></th><th>T15<sub>0</sub></th><th>T15<sub>100</sub></th><th>T15<sub>200</sub></th><th>SEM</th><th>LOS</th></tr></thead><tbody><tr><td colspan="9"><strong>Amino Acids (mg/g)</strong></td></tr><tr><td>Lysine</td><td>5.24</td><td>5.16</td><td>5.44</td><td>4.56</td><td>4.58</td><td>4.79</td><td>0.19</td><td>NS</td></tr><tr><td>Histidine</td><td>2.24</td><td>2.24</td><td>2.35</td><td>2.19</td><td>2.19</td><td>2.15</td><td>0.04</td><td>NS</td></tr><tr><td>Arginine</td><td>5.53</td><td>5.26</td><td>5.87</td><td>5.16</td><td>5.05</td><td>5.35</td><td>0.11</td><td>NS</td></tr><tr><td>Aspartic acid</td><td>8.39</td><td>8.27</td><td>8.88</td><td>8.02</td><td>7.77</td><td>8.10</td><td>0.19</td><td>NS</td></tr><tr><td>Threonine</td><td>4.22</td><td>4.11</td><td>4.67</td><td>3.45</td><td>3.67</td><td>3.87</td><td>0.17</td><td>NS</td></tr><tr><td>Serine</td><td>3.69</td><td>3.82</td><td>3.96</td><td>3.41</td><td>3.46</td><td>3.53</td><td>0.12</td><td>NS</td></tr><tr><td>Glutamic acid</td><td>13.20</td><td>13.13</td><td>13.62</td><td>12.88</td><td>12.94</td><td>12.96</td><td>0.14</td><td>NS</td></tr><tr><td>Proline</td><td>3.41</td><td>3.43</td><td>3.87</td><td>3.18</td><td>2.90</td><td>3.18</td><td>0.14</td><td>NS</td></tr><tr><td>Glycine</td><td>6.73</td><td>6.58</td><td>7.07</td><td>5.86</td><td>5.87</td><td>6.32</td><td>0.26</td><td>NS</td></tr><tr><td>Alanine</td><td>5.51</td><td>5.40</td><td>5.90</td><td>4.92</td><td>5.20</td><td>5.03</td><td>0.16</td><td>NS</td></tr><tr><td>Cystine</td><td>0.41</td><td>0.42</td><td>0.42</td><td>0.42</td><td>0.42</td><td>0.43</td><td>0.01</td><td>NS</td></tr><tr><td>Valine</td><td>5.41</td><td>5.18</td><td>5.87</td><td>4.74</td><td>4.93</td><td>5.03</td><td>0.22</td><td>NS</td></tr><tr><td>Methionine</td><td>2.78</td><td>2.82</td><td>2.97</td><td>2.55</td><td>2.61</td><td>2.76</td><td>0.07</td><td>NS</td></tr><tr><td>Isoleucine</td><td>4.51</td><td>4.12</td><td>4.78</td><td>3.75</td><td>3.99</td><td>4.08</td><td>0.25</td><td>NS</td></tr><tr><td>Leucine</td><td>8.19</td><td>8.04</td><td>8.56</td><td>7.20</td><td>7.48</td><td>7.68</td><td>0.20</td><td>NS</td></tr><tr><td>Tyrosine</td><td>3.53</td><td>3.61</td><td>3.93</td><td>3.22</td><td>3.30</td><td>3.30</td><td>0.12</td><td>NS</td></tr><tr><td>Phenylalanine</td><td>4.63</td><td>4.50</td><td>4.94</td><td>3.32</td><td>3.93</td><td>4.32</td><td>0.17</td><td>NS</td></tr><tr><td colspan="9"><strong>Minerals (%)</strong></td></tr><tr><td>Calcium</td><td>6.92<sup>a</sup></td><td>4.27<sup>b</sup></td><td>4.45<sup>b</sup></td><td>6.75<sup>a</sup></td><td>5.60<sup>b</sup></td><td>5.83<sup>b</sup></td><td>0.26</td><td>*</td></tr><tr><td>Phosphorus</td><td>3.75<sup>a</sup></td><td>2.20<sup>b</sup></td><td>2.32<sup>b</sup></td><td>2.03<sup>b</sup></td><td>2.40<sup>b</sup></td><td>2.60<sup>b</sup></td><td>0.13</td><td>*</td></tr><tr><td>Potassium</td><td>1.80</td><td>0.66</td><td>0.72</td><td>1.49</td><td>0.78</td><td>1.11</td><td>0.05</td><td>NS</td></tr><tr><td>Sodium</td><td>0.71<sup>a</sup></td><td>0.30<sup>b</sup></td><td>0.30<sup>b</sup></td><td>0.74<sup>a</sup></td><td>0.30<sup>b</sup></td><td>0.35<sup>b</sup></td><td>0.03</td><td>*</td></tr><tr><td>Chlorine</td><td>0.69<sup>a</sup></td><td>0.27<sup>b</sup></td><td>0.25<sup>b</sup></td><td>0.73<sup>a</sup></td><td>0.27<sup>b</sup></td><td>0.32<sup>b</sup></td><td>0.02</td><td>*</td></tr></tbody></table>

a,b- Means on the same row bearing different superscript differ significantly (P<0.05), **NS**- No significant difference (P>0.05), **LOS**- Level of significant difference.

References

Adeola, O. and O.A., Olukosi, 2008: Opportunities and challenges in the use of alternative feedstuffs in poultry production. *Nig. Poult. Sci.* J. 5(4) 147—155.

AOAC, 1984: Official methods of Analysis 14th ed. Arlington, V.A. Association of official Analytical chemists.

Musa, U., Haruna, E.S. and L.H. Lombin, 2008: Quail production in the tropics. Vom NVRI press. Pp 13, 24, 66–69.

Pauzenga, U. 1985: Feeding parent stock. Zootechnica International. December 1985 PP.22–24.

Olomu, J.M. (1979). Poultry production in Nigeria. Nutrient requirement of Poultry in Nigeria. Publ. NAPRI, Shika, Zaira, Nigeria pp 241—249.

Steel, R.G.D. and J H. Torrie, 1980: Principles and procedures of statistics 2nd Ed. McGraw, Hill New York, U.S. A. 633pp.

Speckman, D.H., Stein, E.H. and S. Moore, 1958: Automatic recording apparatus for use in the Chromatography of amino acids. Analytical Chemistry. 30: 1191.

Vansoest, P.J. and J.B. Robertson, 1985: Analysis of forages and fibrous foods. As 613 manual. Department of Animal Science, Cornell University, Ithaca. Pp105–106.
# DRESSING PERCENTAGE, CARCASS CUTS AND PERCENT ORGAN WEIGHTS OF MALE QUAILS FED LOW OR HIGH FIBRE DIETS SUPPLEMENTED WITH MAXIGRAIN® ENZYME

*S.E. Alu¹, C.D. Tuleun², F. G. Kaankuka², and S.N. Carew³

¹Department of Animal Science, Faculty of Agriculture, Nasarawa State University, Keffi, P.M.B.135, Shabu-Lafia Campus, Nasarawa State, Nigeria; ²Department of Animal Nutrition, ³Department of Animal Production, College of Animal Science, University of Agriculture, P.M.B. 2373 Makurdi, Benue State, Nigeria

*Corresponding Author: Tel. +2348033690937 & +2347085510322
E-mail: seafarms2000@yahoo.com or louderpraise@gmail.com

## Abstract

Three hundred and sixty 3 weeks old male quails were used in an 8-week experiment to evaluate the effects of Maxigrain® enzyme supplementation of sugarcane scrapping meal-based (SCSM) diets on carcass characteristics. Six diets, T10₀, T10₅, T10₁₀, T15, T15₅ and T15₁₀ were formulated to be isonitrogenous (20%CP) and isocaloric (11.67 MJ/kg, ME); diets T10₀, T10₅ and T10₁₀ contained 10% crude fibre while T15₀, T15₅ and T15₁₀ contained 15% crude fibre. The enzyme was included at 0, 5 and 10mg/kg. The birds were randomly allocated to the treatments at the rate of 60 birds per diet; each treatment was replicated 4 times in a 3 x 2 factorial setting with 15 birds per replicate. Four birds per treatment were selected, fasted for 12hrs, slaughtered and their carcasses evaluated. There were no variations (P>0.05) in all the parameters measured except for percent thigh (20.31 vs. 20.41 and 22.20%) which increased (P<0.05) due to enzyme supplementation while dietary fibre decreased (P<0.05) dressed weight (93.30 and 83.70%) and dressing percentage (81.60 and 74.10%) but increased (P<0.05) weight of gizzard (5.02 vs. 6.49%). Similarly, the interaction of enzyme and dietary fibre did not affect (P>0.05) all the parameters measured. It was therefore, concluded that SCSM supplemented with Maxigrain® at 10mg/kg can be included in the diets of growing male quails without reducing the carcass quality.

**Keywords:** male quails, sugarcane scrapping meal, enzyme, carcass cuts, dressing percentage and organ weights.

## Introduction

Maize, most often, constitutes the highest proportion of ingredient in diet formulation of any poultry ration (Agbede et al., 2002). This high inclusion rate translates into high cost of feed because of the seasonality of maize production and competition for its use by man (Agbede et al., 2002). This indicates the need to replace maize in poultry diets to reduce feed cost and overall production cost.

The objective of this study is therefore, to investigate effect Maxigrain® enzyme supplementation on the carcass characteristics of male quails fed sugarcane scrapping meal-based diets.

## Materials and Methods

### Study area

The experiment was carried out at the Teaching and Research Farm of the Faculty of Agriculture, Nasarawa State University, Keffi, Shabu - Lafia Campus, Nasarawa State, Nigeria.

### Sugarcane scrapping

Sugarcane scrapings were sourced from local sugarcane marketers within Lafia metropolis, sun-dried and milled to form the sugarcane scrapping meal.

The proximate analysis of sample of sugarcane scraping meal were carried using the procedure outlined by AOAC (2006) while the fibre fractions were determined by methods of Vansoest and Robertson (1985) and the values reported on a dry matter basis.

### Description of diets and experimental design

Six experimental diets tagged T10₀, T10₅, T10₁₀, T15, T15₅ and T15₁₀ were compounded to be isonitrogenous (23% crude protein) and isocaloric (11.67 MJ/kg, ME) with two levels of crude fibre, 10 and 15%. The birds were randomly allocated to the treatments at rate of 60 birds per diet in an 8 week experiment. Each treatment was replicated 4 times in a 3x2 factorial having 15 birds per replicate. Treatments T10₀, T10₅, and T10₁₀ contained 10% crude fibre and had 0,
5 and 10mg/kg of the enzyme while treatments T15₀, T15₅ and T15₁₀ contained 15% crude fibre level and had 0, 5 and 10mg/kg of the enzyme, respectively; such that treatments T10₀ and T15₀ served as the controls. Table 2 shows the gross composition of the experimental diets for finisher quails.

## Management of experimental birds

The birds were fed ad-libitum and allowed to have access to drinking water at all times. Other routine management practices were adopted as outlined by Musa *et al.* (2007).

## Data collection

The birds were slaughtered by severing the carotid arteries and jugular veins and blood drained under gravity. Carcass data were collected from four randomly selected birds per treatment group. The relative weights were determined using the formula (Abdulmojeed *et al.*, 2010):

$$ \text{Relative weight (cut part or organ)} = \frac{\text{Fresh weight of cut part or organ}}{\text{Final live body weight of bird}} \times 100 $$

## Statistical analysis

Data obtained were subjected to Two Ways Analysis of Variance (ANOVA). The separation of means was effected using Least Significant Difference (LSD) method and tested at probability level of 5% as described by Steel and Torrie (1980).

## Results and Discussion

The calculated metabolizable energy of SCSM was about 11.67 MJ/kg, ME (Table 1). It contains low (8.25%) crude protein, high crude fibre (36.48%) and low (3.36%) either extract. The dry matter was about 90.67% while ash and nitrogen free extract were about 9.98 and 67.40% respectively. This composition makes SCSM a fibrous material which will require some level of processing or pre-digestion such as milling and or enzyme supplementation if it must be fed to monogastric animals.

## Carcass characteristics of male quails

There were no variations (P>0.05) in the fasted live weight, dressing percentage, dressed weight, head, legs, visceral organs and wings except for thigh which was improved (P<0.05) by the supplementation of enzyme (Table 2). These observations were in contrast to that of Esuga *et al.* (2008) who observed a non-significant variation in percent thigh when they supplemented Maxigrain® enzyme in the diets of broiler chickens. There was significant reduction (P<0.05) in the percent dressed weight and dressing percentage due to dietary fibre. Similarly, all the carcass parameters measured (Table 3) did not vary (P>0.05) due to the interactive effects of enzyme supplementation and dietary fibre. The non-significant variation was in consonance with the earlier report (Alu *et al.*, 2010).

<table><caption>**Table 1.** Proximate and energy composition of sugarcane scrapping</caption><thead><tr><th>Nutrient</th><th>CP</th><th>EE</th><th>CF</th><th>Ash</th><th>DM</th><th>NFE</th><th><sup>a</sup>Energy (MJ/kg ME)</th></tr></thead><tbody><tr><td>%</td><td>8.25</td><td>3.36</td><td>36.48</td><td>9.98</td><td>90.67</td><td>67.40</td><td>12.17</td></tr></tbody></table>

Calculated from Pauzenga (1985).

## Conclusion and recommendation

Based on the findings of this experiment, supplementing high level (15%) of sugarcane scrapping meal-based diets with Maxigrain® enzyme at 5mg/kg is safe and economical and is recommended for inclusion in the diets of finisher quails.
## References

Agbede, S.O., Ajaja, K. and V.A. Aletor, 2002: Influence of Roxazyme G supplementation on the utilization of sorghum dust-based diets for broiler production. (NSAP). March 17th – 21st. Federal University of Technology, Akure,. Nigeria. 105 – 108.

AOAC, 1984: Official methods of Analysis 14th ed. Arlington, V.A. Association of official Analytical chemists.

Esuga, P.M., Sekoni, A.A., Omage, J.J. and G.S. Bawa, 2008: Evaluation of enzyme (Maxigrain®) treatment of graded levels of palm kernel meal (PKM) on performance of broiler chickens. *Pakistan Journal of Nutrition* 7(4):607-613.

Musa, U., Haruna, E.S. and L.H. Lombin, 2008: Quail production in the tropics. Vom NVRI press. Pp 13, 24, 66-69.

Pauzenga, U. 1985: Feeding parent stock. Zootechnica International. December 1985 PP.22 –24.

Steel, R.G.D. and J H. Torrie, 1980: Principles and procedures of statistics 2nd Ed. McGraw, Hill New York, U.S. A. 633pp.

Abdulmojeed, Y; Ayoade, J.A. and Y.A. Dairu, 2010: Effect of genotype and population density on growth performance, carcass characteristics and cost-benefits of broiler chickens in north central Nigeria. *Tropical Animal Health Production* (2010) 42:719-727

Alu S.E., Kaankuka, F.G. and O.I.A. Oluremi, 2010: The Interactive Effect of Nutrase Xyla(R) Enzyme Supplementation on the Carcass Characteristics of grower pigs Fed Low or High Fibre Diets. *International Journal of Food and Agricultural Research* 2010; 7 (1): 205-212.

<table><caption>**Table 2.** Effect of Maxigrain® enzyme supplementation or dietary fibre on carcass characteristics of male quails</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">ENZYME TMENT MEANS</th><th colspan="4">FIBRE TREATMENT MEANS</th></tr><tr><th>No Enzyme</th><th>100ppm Enzyme</th><th>200ppm Enzyme</th><th>SEM</th><th>LOS</th><th>Low fibre</th><th>High fibre</th><th>SEM</th><th>LOS</th></tr></thead><tbody><tr><td><em>Cut parts</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fasted weight (g/bird)</td><td>112.50</td><td>121.20</td><td>110.00</td><td>5.82</td><td>NS</td><td>115.00</td><td>114.20</td><td>4.75</td><td>NS</td></tr><tr><td>Dressed weight (g/bird)</td><td>85.00</td><td>91.00</td><td>89.50</td><td>2.60</td><td>NS</td><td>93.30<sup>a</sup></td><td>83.70<sup>b</sup></td><td>2.12</td><td>*</td></tr><tr><td>Dressing percentage (%)</td><td>75.70</td><td>76.20</td><td>81.60</td><td>2.89</td><td>NS</td><td>81.60<sup>a</sup></td><td>74.10<sup>b</sup></td><td>2.36</td><td>*</td></tr><tr><td>Thigh (%)</td><td>20.31<sup>b</sup></td><td>20.41<sup>b</sup></td><td>22.20<sup>a</sup></td><td>0.51</td><td>*</td><td>21.08</td><td>20.86</td><td>0.42</td><td>NS</td></tr><tr><td>Head (%)</td><td>7.13</td><td>6.71</td><td>6.94</td><td>0.20</td><td>NS</td><td>6.75</td><td>7.10</td><td>0.16</td><td>NS</td></tr><tr><td>Legs (%)</td><td>2.74</td><td>2.47</td><td>2.48</td><td>0.13</td><td>NS</td><td>2.52</td><td>2.61</td><td>0.10</td><td>NS</td></tr><tr><td>Wings (%)</td><td>10.15</td><td>9.15</td><td>9.73</td><td>0.40</td><td>NS</td><td>9.62</td><td>9.74</td><td>0.03</td><td>NS</td></tr><tr><td>Drumstick (%)</td><td>25.23</td><td>24.87</td><td>24.22</td><td>0.34</td><td>NS</td><td>25.34</td><td>25.67</td><td>0.04</td><td>NS</td></tr><tr><td>Breastplate (%)</td><td>38.55</td><td>38.90</td><td>37.23</td><td>0.44</td><td>NS</td><td>38.21</td><td>36.78</td><td>0.03</td><td>NS</td></tr><tr><td><em>Visceral organs (%)</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Heart (%)</td><td>1.13</td><td>1.06</td><td>1.13</td><td>0.06</td><td>NS</td><td>1.18</td><td>1.03</td><td>0.05</td><td>NS</td></tr><tr><td>Testes (%)</td><td>3.70</td><td>3.26</td><td>3.48</td><td>0.14</td><td>NS</td><td>3.30</td><td>3.65</td><td>0.12</td><td>NS</td></tr><tr><td>Gastrointestinal tract (%)</td><td>4.41</td><td>4.26</td><td>5.45</td><td>0.53</td><td>NS</td><td>4.65</td><td>4.76</td><td>0.43</td><td>NS</td></tr><tr><td>Liver (%)</td><td>2.06</td><td>1.84</td><td>2.04</td><td>0.16</td><td>NS</td><td>1.90</td><td>2.06</td><td>0.13</td><td>NS</td></tr><tr><td>Gizzard (%)</td><td>5.78</td><td>5.57</td><td>5.90</td><td>0.42</td><td>NS</td><td>5.02<sup>b</sup></td><td>6.49<sup>a</sup></td><td>0.35</td><td>*</td></tr></tbody></table>

a, b- Means on the same row bearing different superscript differ significantly (P < 0.05), **NS**- No significant difference (P > 0.05), **LOS**- Level of significance, **SEM**-Standard error of mean.
<table><caption>**Table 3.** Interactive effects of Maxigrain® enzyme supplementation and dietary fibre on carcass characteristics of male quails</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="3">10</th><th colspan="3">15</th><th rowspan="2">SEM</th><th rowspan="2">LOS</th></tr><tr><th>T<sub>0</sub></th><th>T<sub>100</sub></th><th>T<sub>200</sub></th><th>T<sub>0</sub></th><th>T<sub>100</sub></th><th>T<sub>200</sub></th></tr></thead><tbody><tr><td><strong>Cut parts</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fasted weight (g/bird)</td><td>115.00</td><td>120.00</td><td>110.00</td><td>110.00</td><td>122.50</td><td>110.00</td><td>8.23</td><td>NS</td></tr><tr><td>Dressed weight (g/bird)</td><td>90.00</td><td>96.00</td><td>94.00</td><td>80.00</td><td>86.00</td><td>85.00</td><td>3.69</td><td>NS</td></tr><tr><td>Dressing percentage (%)</td><td>78.40</td><td>80.20</td><td>86.10</td><td>73.00</td><td>72.10</td><td>77.20</td><td>4.09</td><td>NS</td></tr><tr><td>Thigh (%)</td><td>22.30</td><td>20.42</td><td>20.52</td><td>22.09</td><td>20.40</td><td>20.10</td><td>0.73</td><td>NS</td></tr><tr><td>Head (%)</td><td>6.91</td><td>6.48</td><td>6.86</td><td>7.36</td><td>6.95</td><td>7.01</td><td>0.28</td><td>NS</td></tr><tr><td>Legs (%)</td><td>2.56</td><td>2.29</td><td>2.70</td><td>2.93</td><td>2.64</td><td>2.26</td><td>0.18</td><td>NS</td></tr><tr><td>Wings (%)</td><td>9.92</td><td>9.25</td><td>9.69</td><td>10.39</td><td>9.04</td><td>9.78</td><td>0.57</td><td>NS</td></tr><tr><td>Drumstick (%)</td><td>25.01</td><td>25.11</td><td>24.82</td><td>25.22</td><td>25.00</td><td>25.32</td><td>0.05</td><td>NS</td></tr><tr><td>Breastplate (%)</td><td>40.11</td><td>39.22</td><td>39.09</td><td>38.45</td><td>39.90</td><td>39.51</td><td>0.09</td><td>NS</td></tr><tr><td><strong>Visceral organs (%)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Heart (%)</td><td>1.10</td><td>1.24</td><td>1.20</td><td>1.16</td><td>0.89</td><td>1.06</td><td>0.08</td><td>NS</td></tr><tr><td>Testes (%)</td><td>3.49</td><td>3.18</td><td>3.25</td><td>3.91</td><td>3.34</td><td>3.72</td><td>0.20</td><td>NS</td></tr><tr><td>Gastrointestinal tract (%)</td><td>4.92</td><td>3.38</td><td>5.65</td><td>3.91</td><td>5.14</td><td>5.24</td><td>0.75</td><td>NS</td></tr><tr><td>Liver (%)</td><td>1.65</td><td>1.96</td><td>2.08</td><td>2.47</td><td>1.73</td><td>1.99</td><td>0.23</td><td>NS</td></tr><tr><td>Gizzard (%)</td><td>4.78</td><td>5.09</td><td>5.17</td><td>6.78</td><td>6.05</td><td>6.63</td><td>0.60</td><td>NS</td></tr></tbody></table>

NS- No significant difference (P > 0.05), LOS- Level of significance, SEM-Standard error of mean.
P-088 (ID 108)

COMPARATIVE EFFICACY OF THREE PHYTASES ON GROWTH PERFORMANCE AND APPARENT ILEAL DIGESTIBILITY OF PHOSPHORUS OF BROILER CHICKENS

R. Aureli<fnref n="1" />, P. Guggenbuhl<fnref n="1" />, F. Fru<fnref n="1" /> and J. Broz<fnref n="2" />

**Corresponding author:** raffaella.aureli@dsm.com

**Summary**

In a five-week study, the efficacy of three solid thermostable phytase products from *C. braakii* (PHY-1), *E. coli* (PHY-2) and *Buttiauxella spp* (PHY-3) was compared in broilers. Growth performance, apparent ileal P digestibility (AIDP) and bone mineralization were measured. The birds were allotted to 5 treatments fed in 2 regimes (starter and grower). There was a positive control (PC) diet adequate in all nutrient requirements, and a negative control (NC) diet formulated to be deficient in nutrients and energy at equivalent levels as proposed for a standard recommended dose (RD) of PHY-1 by supplier. NC diets (1.75 g nPP /kg starter and 1.57 g nPP/kg grower) were fed as such and supplemented with phytases PHY-1, PHY-2 and PHY-3 at RD meaning 1000, 500 and 500 U/kg, respectively.

Phytases improved the weight gain (P<0.001) and FCR (P=0.047) compared to NC. Phytases performed equally or even outperformed the PC, irrespective of the measured parameters.

Over the whole trial period, all the phytases at RD were equally potent in terms of growth performance, tibia ash percentage and apparent ileal digestibility of P.

**Keywords:** phytase, phosphorus, ileal digestibility, bone mineralisation, broilers

**Introduction**

Phytate as P source is poorly utilized by monogastric animals as they have insufficient intestinal phytase to effectively digest phytate. The poor digestive utilization of phytate P by monogastric animals and its consequences in terms of diet costs and environmental pollution have led to extensive research efforts directed toward improving phytate digestion. Research has demonstrated definitively that phytase is the only enzyme that was able to initiate the release of P from the phytate molecule, making it available for absorption and utilization (AURELI et al., 2014a). The industrial demand for phytase with greater potency in intestinal phytate hydrolysis and better heat stability continues to stimulate the research for new enzyme sources. Enzyme preparations with phytases derived from *A. ficuum*, *Peniophora. lycii* and *E. coli* are available commercially. More recently, new microbial 6-phytase produced by synthetic genes, mimicking a gene from *C. braakii* or isolated from *Buttiauxella*, were introduced into the market. In the present study, the efficacy of phytase products from either *C. braakii*, *E. coli* or *Buttiauxella spp* was compared in terms of performance, ileal P digestibility and bone mineralization in broiler chickens.

**Material and Methods**

The study was carried out at the Research Center for Animal Nutrition and Health (DSM Nutritional Products France) using a total of 960 day-old male Ross PM3 broiler chickens randomly assigned into 5 treatments (6 replicates of 20 birds per treatment). Each group was placed in one floor pen littered with wood shavings and allocated to one of the different treatments. The chickens were housed in an environmentally controlled room and had *ad libitum* access to feed in pellets and tap water. The composition of the basal diets used in the trial is presented in Table 1. Diets I were fed as positive control diet (PC) and were adequate to meet all nutrient requirements of growing broilers in accordance to their age. Diet II was formulated to be deficient in nutrients and energy equivalent to the matrix values determined for PHY-1 standard recommended dose (RD). Diet II was fed as such (NC) and with the addition of PHY-1 (*C. braakii*, Ronozyme® HiPhos (GT)) at 1000 U/kg, PHY-2 (*E. coli* phytase produced by Schizosaccharomyces pombe) at 500 U/kg and PHY-3 (*Buttiauxella*-phytase) at 500 U/kg respectively.

<footnote n="1">¹Research Center for Animal Nutrition and Health, DSM Nutritional Products France, Saint Louis, France</footnote>

<footnote n="2">²DSM Nutritional Products Ltd, Basel, Switzerland</footnote>
<table><caption>**Table 1:** Composition and nutrient contents of the experimental diets</caption><thead><tr><th rowspan="2">Ingredients (%)</th><th colspan="2">STARTER (Day 1-21)</th><th colspan="2">GROWER (Day 22-36)</th></tr><tr><th>Diet I/PC</th><th>Diet II/NC</th><th>Diet I/PC</th><th>Diet II/NC</th></tr></thead><tbody><tr><td>Soybean meal</td><td>39.40</td><td>38.80</td><td>37.20</td><td>36.80</td></tr><tr><td>Maize</td><td>55.00</td><td>56.40</td><td>55.64</td><td>56.20</td></tr><tr><td>Soya oil</td><td>2.50</td><td>1.50</td><td>4.00</td><td>3.50</td></tr><tr><td>DL-Methionine</td><td>0.20</td><td>0.20</td><td>0.10</td><td>0.10</td></tr><tr><td>CaCO<sub>3</sub></td><td>0.49</td><td>0.69</td><td>0.55</td><td>0.65</td></tr><tr><td>DCP</td><td>1.20</td><td>0.20</td><td>1.20</td><td>0.20</td></tr><tr><td>Sodium chloride</td><td>0.15</td><td>0.15</td><td>0.15</td><td>0.15</td></tr><tr><td>Vitamin & mineral premix</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td></tr><tr><td>Coccidiostat (Lasalocid)</td><td>0.06</td><td>0.06</td><td>0.06</td><td>0.06</td></tr><tr><td>Sand</td><td>0.00</td><td>1.00</td><td>0.00</td><td>1.24</td></tr><tr><td>Titanium dioxide</td><td>-</td><td>-</td><td>0.10</td><td>0.10</td></tr><tr><td><u>Calculated content (g/kg)</u></td><td></td><td></td><td></td><td></td></tr><tr><td>Crude protein</td><td>224</td><td>221</td><td>214</td><td>212</td></tr><tr><td>P total</td><td>5.87</td><td>4.01</td><td>5.77</td><td>3.79</td></tr><tr><td>Avalaible P</td><td>2.61</td><td>1.13</td><td>2.59</td><td>1.10</td></tr><tr><td>Calcium</td><td>7.48</td><td>5.77</td><td>7.79</td><td>5.66</td></tr><tr><td><u>Analysed content (g/kg)</u></td><td></td><td></td><td></td><td></td></tr><tr><td>Crude protein</td><td>229</td><td>226</td><td>223</td><td>211</td></tr><tr><td>Metabolizable energy (MJ/kg)<sup>1</sup></td><td>13.3</td><td>12.9</td><td>13.5</td><td>13.3</td></tr><tr><td>P total</td><td>5.46</td><td>3.89</td><td>5.44</td><td>3.87</td></tr><tr><td>Calcium</td><td>6.57</td><td>5.31</td><td>6.62</td><td>5.17</td></tr></tbody></table>

On day 36, six chickens randomly chosen from each replicate were euthanized and the content of the terminal part of the ileum (17 cm and 2 cm proximal to the ileo-caecal junction), was sampled. The right tibiae were taken from the six chickens for bone strength determination and tibia ash analysis. The concentration of the marker in feed and digesta together with the concentration of P in the feed and digesta were used to calculate the apparent ileal digestibility coefficient (AID) of P according to the following formula:

$$AID (\%) = 100 - [(CM_f/CM_e) \times (CN_e/CN_f)] \times 100$$

CM<sub>f</sub> = concentration of marker in feed; CM<sub>e</sub> = concentration of marker in ileal digesta;

CN<sub>f</sub> = concentration of P in feed; CN<sub>e</sub> = concentration of P in ileal digesta

Performance data, ileal digestibility data, and tibia analyses were subjected to a one-factorial analysis of variance (ANOVA) with the treatment as the experimental factor. Where significant treatment effects (p < 0.05) were indicated, the differences among treatment means were subsequently analysed and compared using the Newman-Keuls test.

## Results

Over the whole period (day 1-36), the results on performance (Table 2) showed that inadequate dietary avP in the diet (NC) led to a significant decrease of WG whereas phytase supplementation was effective in improving significantly the WG and resulted in similar WG than that obtained with the adequate avP diet (PC). FCR was not significantly different among the supplemented phytase treatments. Dietary level of total P and phytase had significant effect on AIDP (Table 2). Indeed, addition of phytase to NC diet resulted in significant improvements of AIDP. Nevertheless, differences among the supplemented treatments were not significant, as already recorded by AURELI et al. (2014b). Whereas tibia-breaking strength was found to decrease with a reduction in dietary available P levels, the addition of phytase led to significant improvements of bone breaking strength and tibia ash percentages compared to NC diet. Although the dif-

<footnote n="1">¹Metabolizable Energy calculated with EC-equation based on analyzed crude nutrients according to the formula ME (MJ/kg) = ((15.51*crude protein+34.31*fat+16.69*starch+13.01*sugar)/1000)</footnote>
ference among the supplemented treatments was not significant, the effects on parameters of bone mineralization were similar when compared to PC (Table 2), showing that bone mineralization criteria are sensitive indicators of P status in the bird (RAVINDRAN et al., 1995).

<table><caption>**Table 2:** Effect of phytase supplementation on growth performance (day 1-36), apparent ileal digestibility (AID) of P and bone mineralization</caption><thead><tr><th rowspan="3">Product</th><th>Dose<br>expected</th><th>Dose<br>analysed<sup>1</sup></th><th>Weigth gain</th><th>Feed intake</th><th>FCR</th><th>AIDP</th><th colspan="2">Tibiae</th></tr><tr><th>U/kg</th><th>U/kg</th><th>g/b</th><th>g/b</th><th>%</th><th>%</th><th>strength<br>N</th><th>ash<br>%DM</th></tr></thead><tbody><tr><td>PC</td><td></td><td></td><td>2598<sup>a</sup></td><td>3771<sup>a</sup></td><td>1.452<sup>b</sup></td><td>40.6<sup>c</sup></td><td>298<sup>ab</sup></td><td>56.5<sup>a</sup></td></tr><tr><td>NC</td><td></td><td></td><td>1664<sup>b</sup></td><td>2466<sup>b</sup></td><td>1.482<sup>a</sup></td><td>53.7<sup>b</sup></td><td>212<sup>c</sup></td><td>55.0<sup>b</sup></td></tr><tr><td>NC+PHY-1</td><td>1000</td><td>1264</td><td>2590<sup>a</sup></td><td>3823<sup>a</sup></td><td>1.476<sup>ab</sup></td><td>64.6<sup>a</sup></td><td>290<sup>ab</sup></td><td>57.0<sup>a</sup></td></tr><tr><td>NC+PHY-2</td><td>500</td><td>485</td><td>2509<sup>a</sup></td><td>3689<sup>a</sup></td><td>1.470<sup>ab</sup></td><td>65.8<sup>a</sup></td><td>315<sup>a</sup></td><td>56.8<sup>a</sup></td></tr><tr><td>NC+PHY-3</td><td>500</td><td>529</td><td>2589<sup>a</sup></td><td>3820<sup>a</sup></td><td>1.476<sup>ab</sup></td><td>66.4<sup>a</sup></td><td>276<sup>b</sup></td><td>56.8<sup>a</sup></td></tr><tr><td>P values</td><td></td><td></td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.047</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.006</td></tr></tbody></table>

## Conclusions

The results obtained in this five-week efficacy study with broiler chicks indicated that phytases have a strong potency for improving P availability in maize/soybean meal based diets for broilers. Irrespective of the measured parameters, the phytase treatments performed equally to the PC. All the phytases at the recommended dose were equally potent in terms of growth performance, ileal digestibility of P and tibia ash percentage.

## Literature

AURELI, R., M. UMAR FARUK, J. BROZ, 2014a: Comparative efficacy of four phytase products on growth performance and apparent ileal digestibility of phosphorus in broiler chickens. Proc. XIVth European Poultry Conference, 23 – 27 June 2014, Stavanger, Norway, Abstract P123.

AURELI, R., M. UMAR FARUK, J. BROZ, 2014b: Effects of phytase supplementation on ileal digestibility of phytate phosphorus and total phosphorus of feedstuffs for broiler chickens Proc. XIVth European Poultry Conference, 23 – 27 June 2014, Stavanger, Norway, Abstract P122.

RAVINDRAN, V., E. T. KORNEGAY, L. M. POTTER, B. O. OGUNABAMERU, M. K. WELTEN, J. H.WILSON, M. POT-CHANAKORN, 1995: An evaluation of various response criteria in assessing biological availability of phosphorus for broilers. Poultry Science **74**,1820–1830.

<footnote><sup>a,b,c</sup> Means within a row, not sharing a common superscript, are significantly different (p<0.05)</footnote>

<footnote n="1">¹ phytase activity after pelleting in the grower diet</footnote>
P-089 (ID 109)

# EFFECTS OF PROTEASE SUPPLEMENTATION ON THE STANDARDIZED ILEAL AMINO ACID DIGESTIBILITY IN TWO WHEAT BY PRODUCTS IN BROILER CHICKENS

R. Aureli<fnref n="1" />, P.Guggenbuhl<fnref n="1" /> and J. Broz<fnref n="2" />

**Corresponding author:** raffaella.aureli@dsm.com

## Summary

A study was conducted to evaluate the effect of a serine protease on the standardized ileal digestibility of amino acids (SIAAD) in two wheat by-products (wheat DDGS and wheat middlings) in broiler chickens. A total of 288 20-day old male broiler chickens were used in a 4-day digestibility study. A nitrogen-free diet was used to determine basal endogenous amino acids losses of broiler chickens. The test feed ingredients served as the sole source of amino acids in experimental diets. The efficacy of the protease for improving the SIAAD in broiler chickens was investigated at the dietary inclusion level of 15 000 PROT/kg. The addition of the protease numerically improved the SIAAD of Val (+3.7 %) and Thr (+4.8 %) of wheat DDGS. Furthermore, SIAAD of Gly (+5.5 %) and His (+8.0 %) were significantly improved in wheat middlings whereas numerical improvements by more than 2% for SIAAD of Ile-Phe (+2.8 %), Thr (+3.5 %), Ala and Tyr (+2.5 %) and Asp (+3.7 %) were also recorded. In this study, protease supplementation was effective in improving the standardized ileal digestibility of almost all the amino acids in wheat middlings.

**Keywords:** protease, ingredients, amino acid, standardized ileal digestibility, broilers

## Introduction

Knowledge of amino acid (AA) digestibility of feed ingredients is necessary to feed broilers with properly balanced compound diets. For this reason, more attention has recently been given to the determination of AA digestibility of feed-stuffs, recognizing that it may vary greatly depending upon the feed ingredient. There is evidence demonstrating that the ileal digesta sampling is a more exact method of estimating the AA digestibility in chickens than the measurement of total AA flow via the excreta (LEMME et al., 2004; GARCIA et al., 2007). However, values of endogenous AA secretion have been shown to depend on the method of determination and the age of the bird (ADEDOKUN et al., 2007). A correction for the endogenous fraction by using a nitrogen-free diet allows determining the standardized ileal amino acid digestibility (SIAAD) which represents a better basis for comparison of dietary treatments and their effects on amino acid digestibility. By using this method, the efficacy of a protease (Ronozyme® ProAct) for improving the SIAAD in broiler chickens fed experimental diets containing wheat by-products (wheat DDGS and wheat middlings) was evaluated.

## Material and Methods

The study was performed at the Research Center for Animal Nutrition and Health (DSM Nutritional Products France, F-68305 Village-Neuf). Day-old male broiler chickens were housed in wire-floored battery cages, which were kept in an environmentally controlled room. The birds had ad libitum access to feed and tap water. They were given a standard broiler diet as mash from day old until day 20. On day 8, the chickens were divided by weight into groups of 8 birds, which were allocated to 6 treatments. 6 replicate groups were assigned to each treatment.

The 6 dietary treatments were as follows: Diets 1 and 4 were Nitrogen-free diet (NFD) to be used for estimating endogenous nitrogen and AA losses fed with or without protease supplementation. These values were used to standardize the apparent ileal digestibility (AID) and thus to determine SIAAD. Diets 2 and 3 were experimental diets containing corn starch and mineral sources in which wheat by-products such as wheat middlings (WM) and wheat DGGS (WDGGS) were the only source of dietary protein, respectively. Diets 5 to 6 were the same as diets 2 and 3, with the protease Ronozyme® ProAct added to supply 15000 PROT per kg feed. To enable the ileal digestibility determinations, titanium dioxide (TiO₂) was added to the feed as indigestible marker at a concentration of 1000 mg/kg.

The birds were provided the experimental diets in mash form from day 20 to day 24. On day 24 all the chickens per group were sacrificed and the content of the terminal part (17 cm proximal to a point 2 cm before the ileo-caecal junction) of the ileum was collected.

<footnote n="1">¹Research Center for Animal Nutrition and Health, DSM Nutritional Products France, Saint Louis, France</footnote>

<footnote n="2">²DSM Nutritional Products Ltd, Basel, Switzerland</footnote>
The contents of AA as well as the concentration of TiO₂ as indigestible marker were determined in the digesta samples and in the feed. Ileal endogenous AA flow in birds fed the NFD was calculated as mg of amino acid (AA) flow per 1 kg of DM intake basis using the following formula reported by MOUGHAN et al. (1992):

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

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

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

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

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

## Results

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

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

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

## Conclusion

In this study, protease supplementation was effective in improving the standardized ileal digestibility of almost all the amino acids in wheat middlings.