ANTIMICROBIAL RESISTANCE TRANSMISSION IN ANTIBIOTIC-FREE POULTRY PRODUCTION PYRAMID

S. Baucheron(1), A.Travel*(2), I.Foubert(1), C.Souchet(2), Y.Baumard(1), M.Le bourhis(1), S.Leclercq(1), B Doublet(1)

(1)INRAE, Nouzilly, France

(2)ITAVI, Nouzilly, France

* Corresponding author: sylvie.baucheron@inra.fr

The transmission of antimicrobial resistant (AMR) bacteria from food-producing animals to humans is an important public health issue. The aim of this project is to characterize the vertical and horizontal transmission as well as the persistence of AMR in the broiler production pyramid without antibiotic selective pressure, considering all stages over three successive generations of animals and their associated environments, to identify new levels of risk control.

AMR monitoring was realized in an experimental poultry farm integrating all steps of the broiler production chain (from broiler breeder females to chicken at slaughterhouse). The commercial chicken line chosen was bred to study the quality of the meat. Fourteen representative parent layers and progeny during 3 generations were sampled including 2 sibling batches at each generation, one for reproduction purpose so reared in protected houses and one for meat production purpose so in standard conditions (group in floor pen). Fecal and caecal samples, incubated eggs, neck skin, and meat were collected as well as environmental samples (drinking water, feed, houses surfaces, water bath before plucking). AMR Gram-negative bacteria were selected on MacConkey agar supplemented with different antibiotics. Their antimicrobial susceptibility phenotype was determined by the disk diffusion method.

Fecal carriage of Escherichia coli and Proteus mirabilis with different multi-drug resistance (MDR) phenotypes was found in all flocks/generations of broilers. E. coli isolates with identical MDR phenotypes were found in successive generations suggesting vertical transmission while several others were associated to only 1 flock. More diverse MDR phenotypes were observed in broilers production flocks than in reproduction ones indicating an important role of the rearing conditions. Sequencing of representative MDR isolates and plasmids is ongoing to determine their genetic relationship. Environmental MDR bacteria identified in the experimental farm might contribute to transmission of resistance genes by mobile genetic elements. Moreover, metagenomic sequencing will be performed to analyze global caecal resistomes.

Our first results indicate that MDR bacteria are widespread and persist in the experimental poultry production pyramid, in spite of the non-use of antibiotics for a decade. Further investigations will be conducted to decipher the transmission of AMR bacteria and mobile genetic elements identified in this system.
THE EFFECTS OF D. VS. L-METHIONINE SUPPLEMENTATION ON GROWTH PERFORMANCE AND INTESTINAL HEALTH OF BROILER CHICKENS AFTER A CHALLENGE WITH EIMERIA SPP.

F. Lima de souza castro*(1), Y.Hou tompkins(1), W.Kim(1)

(1)University of Georgia, Athens, United States

* Corresponding author: fefelimasc@hotmail.com

The aim was to study the effects varying total sulfur amino acid (TSAA) levels supplemented with either D. vs. L-Methionine (Met) on the performance and intestinal health of broilers during the recovery phase after a single challenge with Eimeria spp. A total of 720 d-old-male Cobb 500 chicks were randomly distributed in a factorial arrangement 5 by 2 (6 replicates/12 birds). The factors were diets and challenge (CHA) or not (UCH) with Eimeria spp. The diets were negative control (NC – TSAA and Met deficient), 85% of TSAA supplemented with either D or L-Met (85D and 85L), and 100% of TSAA supplemented with either D or L-Met (100D and 100L). At d 14, the CHA group received orally 12,500 E. maxima and E. tenella, and 62,500 E. acervulina sporulated oocysts. At d 26 (14 dpi), BWG, FI, and FCR were measured, and intestinal samples were collected (1 bird/replicate) for morphology. Two-way ANOVA was performed, and means were compared by Tukey's test using SAS (P<0.05). Considering the overall period (14-26d), interactions between treatments were found for FI and FCR (P<0.001), whereas no interactions were found for BWG (P=0.456). The FI of UCH birds fed 100% of TSAA (D and L-Met) was lower than the intake of UCH birds fed 85% of TSAA, regardless Met isomer. Additionally, birds fed NC in the UCH group had higher intake than the birds fed the same diet in the CHA group. The FCR of UCH birds fed 100% of TSAA (D and L-Met) was lower than that of birds fed 85L and NC in the UCH group, and lower than birds fed NC, 85% of TSAA (D and L-Met) and 100D in the CHA group. The BWG was affected by both challenge and diet. The UCH birds showed the highest weight gain (P<0.001), and birds fed NC showed the lowest BWG (P<0.001). No interactions were found for intestinal morphology traits. The duodenum of UCH birds showed higher villus height (P=0.007) and crypt:villi ratio (P=0.011) than CHA birds. The jejunum of UCH birds showed higher villus height (P=0.002), lower crypt depth (P<0.001), and higher villi:crypt ratio (P<0.001) than CHA birds, whereas the ileum of UCH birds had lower crypt depth (P=0.006) than CHA birds. In conclusion, the Eimeria spp.challenge reduced the performance and intestinal health during the recovery (14 dpi) phase. Additionally, the use of 100% of TSAA, regardless Met isomer, led to better growth and intestinal development, whereas no differences were seen between D and L-Met, suggesting that both isomers are equivalent after the challenge.
ANTIMICROBIAL RESISTANCE IN FECAL E. COLI AND SALMONELLA ISOLATES OF SMALL POULTRY
FLOCKS IN ONTARIO, CANADA

C. Varga(1), M.Guerin*(2), M.Brash(3), D.Slavic(3), P.Boerlin(2), L.Susta(2)

(1)Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Canada

(2)University of Guelph, Guelph, Canada

(3)Animal Health Laboratory, Guelph, Canada

* Corresponding author: mguerin@uoguelph.ca

Small flocks have become increasingly popular in urban and rural areas of North America. Small flocks can pose a health risk to their owners by exposing them to antimicrobial resistant zoonotic pathogens. The objective of this study was to determine the AMR patterns of fecal E. coli and Salmonella enterica isolates of chickens, turkeys, waterfowl, and game birds from Ontario small flocks submitted for laboratory diagnostic testing between October 2015 and September 2017 because of production issues, morbidity, or mortality. From each submission, a pooled cecal sample was collected and tested for the presence of fecal E. coli and Salmonella. Three isolates from each positive E. coli sample and one isolate from each positive Salmonella sample were tested for susceptibility to 14 antimicrobials using a broth microdilution method. A total of 433 E. coli isolates (358 chicken, 27 turkey, 24 duck, and 24 game bird) and 5 Salmonella isolates (3 chicken, 1 turkey, 1 duck) were recovered.

For E. coli, a moderate to high proportion of isolates were resistant to tetracycline (43% chicken, 81% turkey, 42% duck, and 38% game bird isolates), streptomycin (29% chicken, 37% turkey, and 33% game bird isolates), sulfisoxazole (17% chicken, 37% turkey, and 21% duck isolates), and ampicillin (16% chicken and 41% turkey isolates). Multidrug resistance was found in 37% of turkey, 20% of chicken, 13% of duck, and 8% of game bird E. coli isolates. Streptomycin-tetracycline-sulfisoxazole-trimethoprim-sulfamethoxazole was the most commonly identified resistance pattern in the chicken E. coli isolates. The Salmonella isolates were most frequently resistant to streptomycin, tetracycline, and sulfonamides. For E. coli and Salmonella, there was a very low frequency of resistance to cephalosporins, carbapenems, macrolides, and quinolones — antimicrobials important in human medicine.

Escherichia coli isolates were frequently resistant to antimicrobials commonly used to treat poultry bacterial infections, which highlights the necessity of judicious antimicrobial use to limit the emergence of multidrug resistant bacteria. Results can be used to educate veterinarians and small flockowners about issues surrounding antimicrobial resistance to improve flock health and mitigate public health risks.
ID: 510

ANTIMICROBIAL RESISTANCE IN CAMPYLOBACTER ISOLATES OF SMALL POULTRY FLOCKS IN ONTARIO,
CANADA

C. Varga(1), M.Guerin*(2), M.Brash(3), D.Slavic(3), P.Boerlin(2), L.Susta(2)

(1)Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Canada

(2)University of Guelph, Guelph, Canada

(3)Animal Health Laboratory, Guelph, Canada

* Corresponding author: mguerin@uoguelph.ca

Small flock farming has become increasingly popular in North America. Small flock poultry are used as a source of food and are also kept as pets, which can pose a health risk to flock owners through exposure to antimicrobial resistant zoonotic pathogens. The objective of this study was to determine the AMR patterns of Campylobacter isolates of chickens, turkeys, waterfowl, and game birds from Ontario small flocks submitted for laboratory diagnostic testing between October 2015 and September 2017 because of production issues, morbidity, or mortality. From each submission, a pooled cecal sample was collected and cultured for Campylobacter. Three isolates from each positive sample were tested for susceptibility to nine antimicrobials using a broth microdilution method. Cluster and multiple correspondence analyses were used to explore relationships among antimicrobials in terms of their similarity in Campylobacter resistance in the isolates from chicken samples.

A total of 176 isolates were recovered (141 chicken, 21 turkey, 6 duck, and 8 game bird). A high proportion of the C. jejuni isolates from chickens (77%) and turkeys (100%), and the C. coli isolates from turkeys (50%) and game birds (40%), were resistant to tetracycline. The odds of resistance to tetracycline were higher in the C. jejuni isolates (OR = 3.54, P ≤ 0.01) than the C. coli isolates. There was a low frequency of resistance to quinolones and a very low frequency of resistance to macrolides. Antimicrobial susceptibility testing results for ciprofloxacin and nalidixic acid were clustered together. The high frequency of tetracycline resistance emphasizes the importance of prudent antimicrobial use in small flocks. Although resistance to macrolides and quinolones was uncommon, these are used to treat severe cases of campylobacteriosis in humans, highlighting the need for proper food safety and infection control practices by small flock owners to prevent exposure to antimicrobial resistant Campylobacter. Results can be used to educate veterinarians and small flock owners about issues surrounding antimicrobial resistance to mitigate public health risks.
ID: 548

DEVELOPMENT OF A METHOD FOR ASSESSING THE BIOACTIVITY OF PLANT EXTRACTS ON THE ANTIOXIDATIVE STATUS AND INNATE IMMUNITY IN CHICKEN

P. Barat(1), R.Guabiraba(1), S.Crochet(1), V.Guillory(1), E.Audouin-cailleau(2), P.Chartrin(1), J.Bernard(1), A.Collin(1), A.Travel*(3), L.Guilloteau(1)

(1) INRAE, Nouzilly, France

(2) BOA, INRAE, Université de Tours, Nouzilly, France

(3) ITAVI, Nouzilly, France

* Corresponding author: laurence.guilloteau@inra.fr

The potential of plant extracts to strengthen the antioxidative status and innate immunity in chickens could lead to a reduction in the use of chemical antimicrobial drugs in poultry farms. The assessment of plant extract (PE) efficacy is generally directly tested in chickens using various in vivo protocols with lipopolysaccharide (LPS) challenge. The objective of this study was to develop a relevant, robust and as minimally as possible method to reproduce the oxidative stress and inflammation induced by LPS. An ex vivo method was evaluated using chicken whole blood stimulated in vitro with LPS and compared with a method based on blood sampled after LPS injection in vivo. Blood sampled from 20 days-old broilers was cultured for six hours in contact with LPS or not. In vivo, the chickens were injected with LPS and two blood samples were taken, one before the injection and one six hours later. Biochemical and molecular markers of redox balance and innate immunity were tested by biochemical and qRT-PCR assays and analyzed by ANOVA or Mann-Whitney or Kruskal-Wallis non-parametric analyses. Glutathione peroxidase activity and the oxidized/reduced glutathione ratio were only modified in vivo after LPS injection indicating the induction of an oxidative stress. The other biochemical indicators tested (uric acid concentration, superoxide dismutase activity, total antioxidant status, thiobarbituric acid reactive substrate, haptoglobin-like activity) were not modified whatever the method. IL1-b, IL-6, IL8(L2) cytokine and iNOS gene expressions were significantly increased by LPS for both methods, the range of gene expression being higher ex vivo than in vivo. This ex vivo method can be used for assessing the capacity of plant extracts to modulate the antioxidative status and innate immunity of chickens. It will be used in a project aiming to define a methodology to select plant extracts from the literature, to screen their safety and biological activities in vitro and in vivo on chicken performance, health and welfare.

Funding: this study is a part of a CASDAR project (MEXAVI) funded by the French Ministry of Agriculture.
EFFECT OF AN ESSENTIAL OILS BASED FEED ADDITIVE ON IMMUNE RESPONSE GENES EXPRESSION IN LOHMANN WHITE LAYING HENS

L. Ilina*(1), A.Dubrovin(2), E.Yildirim(2), G.Laptev(2), V.Filippova(2), D.Tiurina(2)

(1)Biotrof Ltd., Saint-petersburg, Russia

(2)Biotrof Ltd, Saint-petersburg, Russia

* Corresponding author: ilina@biotrof.ru

The «BIOTROF+» Ltd and All-Russian Research Veterinary Institute of Poultry Science (ARRVIPS) teams, have carried out the experiment on 40 Lohmann White adult laying hens in vivarium of ARRVIPS. The Experimental group received the feed additive based on essential oils mix from the first day of the experiment, while the Control group received standard feeding diet. 3 weeks after the start. Half of the hens of each group were infected with an epizootic strain of S. enteritidis in the amount of 5×10⁸ CFU and afterwards further parting into groups was performed: Control (C), Infected Control (CI), Experimental (E), Infected Experiment (EI). A day and a week after infection, a planned slaughter of hens with sampling was executed.

The gene expression analysis was performed in molecular-genetic laboratory of Biotrof Ltd (Saint-Petersburg, Russia). Analysis of the level of relative expression of immunity genes was performed by qPCR that requires preliminary steps of RNA extraction and cDNA synthesis. Total RNA from the samples was isolated using the Aurum™ Total RNA Mini Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Using the iScript™ Reverse Transcription Supermix (Bio-Rad) kit, a reverse transcription reaction was performed to produce cDNA using RNA template. The amplification reaction with the gene primers was performed using the SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) kit according to the manufacturer's protocol. The relative quantification (RQ) of gene expression was calculated using the 2-ΔΔCT method. As a reference gene, the β-actin protein (ACTB) gene was chosen.

One day after infection, a decrease of IL-6 gene expression level was noted in the CI group compared to the C group by more than 2 times. At the same time, in the EI group the IL-6 gene expression significantly increased in comparison with the E group, and was also 1.78 times higher than the C group level. This trend continued even after 7 days after infection.

The AvBD-10 gene expression a day after infection in all groups lower, the C group level, which could be more likely due to an increase in the expression level of the C group. But the tendency of AvBD-10 expression ratio among the CI, E and EI groups correlated with IL-6 gene expression indices. A week after infection, an increase in AvBD-10 gene expression in all groups in comparison to the C group was noticed.

The IL8L1 gene expression a day after infection in the CI group was 7 times higher than in the C group. The IL8L1 expression in the E group was 14 times higher than in the C group and the expression in the EI group was 15 times higher than in the CI group. 7 days after infection, an equalization of IL8L1 gene expression level among CI, E, EI groups was noticed.

In the CI group, the level of bactericidal activity of blood serum a day after infection decreased, while in the EI group the level of bactericidal activity increased. In the CI group a week after infection the level decreased even more, while in the EI group it raised even more.

In conclusion, it was found that addition of an essential oil based feed additive to the feed ration of hens led to an increase in the immune activity.
# EFFICACY OF AN ATTENUATED COCCIDIOSIS VACCINE IN COMBINATION WITH DIFFERENT FEED ADDITIVES ON PRODUCTION PERFORMANCE AND INTESTINAL LESIONS IN BROILERS CHALLENGED WITH NECROTIC ENTERITIS

L. Pantoja millas*(1), M.Dardi(1), E.Van eerden(2)

(1)Laboratorios HIPRA, Amer, Spain

(2)Schothorst Feed Research Center, Lelystad, Netherlands

* Corresponding author: luis.pantoja@hipra.com

## INTRODUCTION

The combination of vaccination against coccidiosis and the supplementation of the diet with feed additives could be a composite approach to the control of necrotic enteritis problems triggered by Eimeria spp. infestation in broilers. Therefore, the objective of this study was to test the efficacy of an attenuated coccidiosis vaccine in combination with different feed additives in preventing a loss of production performance and intestinal lesions in broilers challenged with NE

## MATERIALS&METHODS

Healthy day-old Ross 308 male broiler chicks (960 chickens in 48 groups of 20 chicks) were reared in cages on wood shavings as the bedding material. These were then equally distributed between 6 experimental groups.

NE was triggered with a primary infestation with Eimeria maxima, a secondary infection with Clostridium perfringens and by feeding birds during the growing phase with a predisposing diet. Groups 3-6 were vaccinated at one day of life against coccidiosis with an attenuated coccidiosis vaccine for broilers. Groups 1 and 2 were then reared without feed additives in the diet, whereas the rest of groups were reared with Short Chain Fatty Acids (SCFA), Medium Chain Fatty Acids (MCFA) or Phytogenic Feed (PFA) additives respectively in the diet. NE was then experimentally induced in groups 2-6, whereas group 1 was sham-infected. Data were analysed by ANOVA using the Fisher's post-hoc Least Significant Difference (LSD) to test for treatment effects. Statistical analyses were based on two-sided tests implemented in GenStat®

## RESULTS

Vaccine administered alone or in combination with feed additives in the diet significantly decreased the intestinal lesion score associated with C. perfringens and slightly but not significantly the mortality compared to the untreated group. MCFA group was the only one among those fed with additives in the diet, which showed a synergistic effect with the vaccine.

Vaccinated birds converted feed better than the non-vaccinated group as shown by the higher BWG and lower FCR. MCFA group was the one among those with a diet supplemented with additives which converted the feed more efficiently.

## CONCLUSIONS

Vaccination with an attenuated coccidiosis vaccine for broilers, was shown to be able to reduce intestinal lesions associated C. perfringens compared to non-vaccinated birds and as a consequence benefits in productive performances were observed as well. MCFA were shown to be able to further decrease intestinal lesion scores associated with C. perfringens than vaccination alone
SUPPLEMENTATION WITH SAPONIN RICH PLANT EXTRACTS AS EFFICIENT AS MONENSIN SUPPLEMENTATION TO MAINTAIN BROILERS' ZOOTECHNICAL PERFORMANCES UNDER COCCIDIOSIS CHALLENGE.

M. Benarbia*(1), P.Caillis(1), M.Lebot(2), P.Chicoteau(1)

(1)Nor-Feed SAS, Beaucouzé, France

(2)Joint Lab ANR FeedInTech (FIT: SONAS/Nor-Feed), Beaucouzé, France

* Corresponding author: amine.benarbia@norfeed.net

Introduction:

For decades, ionophores such as monensin have been used successfully to prevent coccidiosis in broilers flocks. However, the emergence of resistant strains of Eimeria and issues linked to residues in meat1 led poultry producers to find efficient alternatives.

Saponin rich plants, mainly Yucca schidigera (YS), were shown to be effective to control coccidiosis in broilers2. However, few studies assessed the effect of combination of saponins from different plants and compared their effects to usual coccidiostats.

The objective of this study was to assess the effect of Norponin XO2 (NPXO2), a combination of saponins from YS and Trigonella foenum-graecum (TFG) on zootechnical performances and intestinal lesions of broilers artificially challenged with coccidiosis.

Materials and methods:

192 broilers (males, ROSS 308) were housed in cages and allocated to the following groups: 1. uninfested untreated, 2. infested untreated, 3. infested, monensin treated, 4. infested, NPXO2 treated. Infested birds received at D16 orally and individually 805000 sporulated oocysts (52000 E.acervulina, 17500 E.tenella, 10000 E.maxima and 1000 E.mitis). Zootechnical performances and intestinal lesions were assessed at D22. Data were analyzed using linear mixed regression models. In all models, treatment groups were compared to the group 2 as reference.

Results:

Group 1 birds had a higher body weight at the end of the experiment. Birds of group 3 have no difference, however group 4 animals tend to have higher body weight (P= 0.08). Even if FCR of birds of group 1, 3 and 4 was numerically lower, no significant difference was observed. Intestinal lesion of E.acervulina, and E.tenella were reduced in group 3 and 4 significantly. Lesion due to E.maxima were reduced only in group 3. No significant difference was observed between group 3 and 4 in zootechnical performances and intestinal lesion.

Conclusion:

According to these results, feed supplementation with saponin rich plants, YS and TFC (NPXO2), is as efficient as monensin supplementation to maintain zootechnical performances of broilers under artificial coccidiosis challenge.

References:

1. Clarke, L. et al. A review of coccidiostats and the analysis of their residues in meat and other food. Meat Sci. 97, 358-374 (2014)

2. Oelschlager, M. L., Rasheed, M. S. A., Smith, B. N., Rincker, M. J. & Dilger, R. N. Effects of Yucca schidigera-derived saponin supplementation during a mixed Eimeria challenge in broilers. Poult. Sci. 98, (2019).
# CHARACTERISATION, THE CORNER STONE OF A SUSTAINABLE USAGE OF BOTANICALS IN POULTRY: CASE STUDY OF CITRUS EXTRACT SUPPLEMENTATION

S. Cisse*(1), A. Boumezrag(2), R. Cornet(1), H. Bui(1), A. Manière(1), P. Engler(3), M. Benarbia(1), D. Guilet(1)

(1) Joint Lab ANR FeedInTech (FIT: SONAS/Nor-Feed), Beaucouzé, France

(2) University of Tiaret, Tiaret, Algeria

(3) Joint Lab ANR FeedInTech (FIT: SONAS/Nor-Feed), Beaucouze, France

* Corresponding author: sekhou.cisse@norfeed.net

## Introduction

For a long time, antibiotics have been used as growth promoters for preventing diseases and improving zootechnical performances. For the one health purpose, this application has been banned in Europe, giving way to alternatives such as botanicals. Among botanicals, citrus extract have already shown beneficial effect on broiler performances and health1. However, all citrus extracts are not the same and can vary a lot in terms of composition and concentration of active compounds2. This trial has been carried out in order to compare the effect of a Characterised Natural Citrus Extract (CNCE) with a Citrus Peel Extract (CPE), in terms of composition and effect on performances.

## Experimental design

A solution of each extract has been prepared in methanol and characterised by HPLC-UV-DAD-DEDL, in order to compare their composition. In the meantime, 540 Arbor Acres one day old birds were divided into 3 groups. Each group contained 180 birds divided into 6 replicates:

* Group 1: CTL group: a standard diet without supplementation;

* Group 2: CNCE group: a standard diet supplemented with 250 ppm of CNCE (Nor-Spice AB, Nor-Feed SAS)

* Group 3: CPE group: a standard diet supplemented with 250 ppm of CPE

Birds were reared until day 46. Heat stress (30°C/4h) was applied, from day 26 to day 30. Zootechnical performances were weekly recorded.

## Results

HPLC analysis show differences between the composition profile of CNCE compared to profile of CPE. Regarding zootechnical performances, birds from CNCE group were heavier (2312g ±196) than birds from CPE group (2097g ±92) and CTL group (2114g ±120). Moreover, birds from CNCE group had a better carcass yield (77.16 ± 15 %) than birds from CPE (72.57 ± 1.7%) and CTL groups (73.11 ± 3.1%) (p

## Conclusions

Citrus extract effect on animal's performances differs between CNCE and CPE. This difference may be linked to their composition. These results showed that botanicals may be efficient alternatives to AGPs as long as they are characterised. Indeed, characterisation gives the possibility to determine their composition and understand their mode of action, making it the corner stone of a sustainable usage of botanicals.

1. Akbarian, A. et al. Growth performance and gut health parameters of finishing broilers supplemented with plant extracts and exposed to daily increased temperature. SPANISH JOURNAL OF AGRICULTURAL RESEARCH 11, 109–119 (2013).

2. Moset, V. et al. Ensiled citrus pulp as a by-product feedstuff for finishing pigs: nutritional value and effects on intestinal microflora and carcass quality. Spanish Journal of Agricultural Research 13, 0607 (2015).
# ALKYL ESTERS OF MEDIUM CHAIN FATTY ACIDS ENHANCE BROILER GUT HEALTH AND PERFORMANCE

Y. Van der horst\*(1), M. Lingbeek(1)

(1)Trouw Nutrition, Amersfoort, Netherlands

* Corresponding author: yvonne.van der.horst@trouwnutrition.com

The level of intestinal microbial activity affects animal growth efficiency. The microbial community in the gut competes for nutrition with the host, accelerates enterocyte turnover rates resulting in lower efficiency in nutrient absorption and decreases fat digestibility by deconjugating bile acids (Yadav and Jha, 2019). Higher diversity of microbial genera has been associated with higher resistance and better production performance in broilers and other species (Stanley et al 2016).

An alkyl ester is a chemical compound derived from an acid in which at least one hydroxyl group is replaced by an alkoxy group. Hydrolysis of alkyl esters of medium chain fatty acids (MCFA) bypancreatic lipase is 10-50 times slower compared to triglycerides (McKay, 2007). Consequently, the level of 'free' MCFA is higher in the lower parts of the small intestine. Multiple R&D studies showed that alkyl esters positively affect total microbial counts and diversity and related growth performancecompared to the 'free' form of lauric acid.

An in vivo study with 776 female broilers fed a corn-soybean meal based diet with or without 1.5kg/t alkyl ester C12 and exposed to a dietary challenge compared microbiota counts. The total bacterial count in the jejunum of broilers fed the alkyl ester C12 diet was significantly (p < 0.05) reduced compared to the negative control at 21 days of age. The phylogenetic representation of jejunal samples showed that alkyl esters help to increase diversity in jejunum by 22%.

In an in vivo study performed with 936 female broilers fed a corn-wheat-soybean meal based diet, birds were exposed to a negative control (NC), C12 (1 kg/t) and alkyl ester of C12 (1 kg/t) in the diet. A tendency (p = 0.079) to reduce Clostridium perfringens in jejunum compared to NC and C12 was found for the C12 alkyl ester diet. A 2% lower FCR was found for the alkyl ester diet compared to NC in the first 3 weeks of age (p = 0.01).

In a re-used litter challenge study with 192 broilers, birds fed diets with alkyl esters of C12, had a tendency (p < 0.1) for higher body weight at 42 days compared to a standard diet.

Alkyl esters of MCFA's reach lower parts of the gastrointestinal tract compared to free MCFA and triglycerides. This results in improved microbial diversity and lower microbiota counts which is reflected in improvements in zootechnical broiler performance.
EFFECTS OF THE DIETARY SUPPLEMENTATION WITH GRAPE POMACE AND CHESTNUT EXTRACTS ON GROWTH PERFORMANCE AND INTESTINAL MUCOSA OF BROILER CHICKENS

A. Trocino*(1), A.Pascual(2), M.Birolo(3), G.Pillan(4), F.Bordignon(4), G.Radaelli(4), M.Bortoletti(4), C.Ballarin(4), G.Xiccato(5)

(1)University of Padova, Department of Comparative Biomedicine and Food science, Legnaro, Italy

(2)Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, padova, Italy

(3) Department of Agronomy, Food, Natural Resources, Animal, and Environment, University of Padova, Legnaro, Italy

(4) Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy

(5)Department of Agronomy, Food, Natural Resources, Animal, and Environment, Legnaro, Italy

* Corresponding author: angela.trocino@unipd.it

During the last years, poultry production has been moving towards antibiotic-free production systems in which tannins can be used as feed additives to improve animal immune response and health, due to their anti-microbial, anti-oxidant, and anti-inflammatory activities. Results widely vary according to tannins source and structure, however. Thus, the present study assessed the effects of the dietary supplementation in broiler chickens with two vegetal extracts containing tannins, from grape pomace (GP) or chestnut (CN), on growth performance and morphology and immune response of intestinal mucosa. A total of 864 chickens (both sexes) in collective pens (24 birds/pen, 12 pens/group) were assigned to three experimental groups fed a control diet (C) or the same diet added with 0.2% CN extract (diet CN) or 0.2% GP extract (diet GP). At 14 d and 34 d of age, 12 chickens per diet were slaughtered to sample jejunum. Serial sections of 4 µm were stained with hematoxylin/eosin for morphometric evaluation and with antibodies against intraepithelial CD3+ T-cells and CD45+ leukocytes (only samples of the first slaughtering) to evaluate the anti-inflammatory activity. Data were submitted to ANOVA using a mixed model with diet as the main effect and pen (growth data) or an animal (intestinal mucosa data) as a random effect. The diet GP significantly increased daily weight gain (DWG) in the whole trial (70.5 g/d vs. 69.4 g/d and 67.1 g/d; P<0.01) and final live weight (3148 gvs. 3099 g and 3084 g; P<0.01) compared to diets C and CN, with no effect on feed intake (on average111 g/d), feed conversion ratio (1.59), and mortality (3.4%). Indeed, the diet GP promoted DWG in thesecond (+1.7%, P<0.001; 14-28 d) and in the third period of growth (+2.1%, P<0.05; 28-45 d) comparedto diet C and, mostly, diet CN. The diets CN and GP decreased villi height compared to diet C (954 µm and 934 µm vs. 1033 µm; P<0.01), whereas crypt depth and villi-to-crypt ratio did not change. Regarding intestinal immune status, chickens fed diet GP showed higher densities of both CD3+ (2302vs. 2116 cells/mm2; P<0.001) and CD45+ (2198 vs. 2040 cells/mm2; P<0.05) compared to those fed diet CN, whereas chickens fed diet C exhibited intermediate results. In conclusion, GP dietary supplementation improved chicken performance and promoted immune response in intestinal mucosa. Further insights are required to define the action mechanisms at the intestinal level.
BACILLUS PROBIOTIC AND YEAST PARIETAL FRACTION TO REDUCE COLONIZATION OF SALMONELLA ENTERITIDIS IN LAYERS

J. Padgett*(1), P.Price(2), T.Gaydos(3), J.Baxter(4), C.Hofacre(4), M.Sims(5), R.Berghaus(6), K.Macklin(7)

(1)Phileo by Lesaffre, Bryan, United States

(2)Phileo by Lesaffre, Milwaukee, United States

(3)Gaydos Technical Services, Dallas, United States

(4)Southern Poultry Research Group, Watkinsville, United States

(5)Virginia Diversified Research, Harrisonburg, United States

(6)University of Georgia, Athens, United States

(7)Auburn University, Auburn, United States

Frequent high occurrence of Salmonella in poultry flocks can lead to major public health issues, hence be controlled. 2 in vivo trials were setup to investigate the potential, of a Bacillus probiotic and a parietal yeast fraction prebiotic to reduce Salmonella Enteritidis colonization in laying pullets and hens. In the first trial, sixty 9-week-old Hy-Line Brown pullets were randomly allocated to individual cages in 15 replicate blocks. Animals were either supplemented the basal control diet, or the basal diet containing 500g/ton yeast parietal fraction, 500g/ton Bacillus spp probiotic or 1kg/ton yeast culture. On day 3 of the trial all birds received 3x107 CFU of a nalidixic acid resistant Salmonella Enteritidis by oral gavage. Seven days after inoculation all birds were humanely euthanized, and the ceca aseptically removed for CFU determination. In the second trial sixty Hy-Line Hens aged 56 weeks were placed in individual cages and fed a mash diet. The treatments were negative control, 500g/ton Bacillus spp. probiotic, 500g/ton yeast parietal fraction and a combination of yeast parietal fraction and Bacillus spp. probiotic. At 60 weeks of age all hens were challenged orally with 7 x 107 CFU of Salmonella Enteritidis. One week later, birds were humanely euthanized, and the ceca aseptically removed and analyzed for Salmonella Enteritidis prevalence and enumeration by the Most Probable Number (MPN)method. Results of the first trial show that the mean quantity of Salmonella Enteritidis detected in thececa expressed in log10 MPN/g was 2.52 in the control, 2.49 in the yeast parietal fraction treatment, 1.73 in the probiotic treatment, and 1.66 in the yeast culture treatment. In the second study, the control birds had 3.35 log10 MPN/g of Salmonella. Enteritidis in the ceca. The Probiotic group had 1.90 MPN/g, a reduction of 1.44 (p<0.05) and the yeast cell wall group had 1.84MPN/g a reduction of 1.51 (p<0.05). The combination had 2.56 MPN/g a numerical reduction of 0.79 (p=0.14). These studies confirm that both, the Bacillus spp. probiotic, the yeast parietal fraction or yeast culture could reduce the Salmonella Enteritidis colonization in the ceca of laying pullets and hens.

<footnote>* Corresponding author: c.padgett@phileo.lesaffre.com</footnote>
PROBIOTIC STRATEGIES TO REDUCE SALMONELLA INFANTIS COLONIZATION IN BROILERS

J. Padgett*(1), P.Price(2), J.Byrd(3), C.Bailey(4)

(1)Phileo by Lesaffre, Bryan, United States

(2)Phileo by Lesaffre, Milwaukee, United States

(3)USDA ARS, College station, United States

(4)Texas A&M University, College station, United States

* Corresponding author: c.padgett@phileo.lesaffre.com

With a recent growing push from consumers for poultry administered no antibiotics, feed additives such as parietal yeast fractions have become a potential option of microbial intervention that has gained interest from producers. Although previous research tends to indicate a trend toward their influencing Salmonella populations in chickens, more in vivo studies are needed to effectively evaluate their effects on Salmonella. The objective of this study was to evaluate the efficacy of a commercially available parietal yeast fraction on the reduction of Salmonella Infantis in the ceca of broilers and its translocation into other organs. In this trial, 240 Cobb 500 off-sex male chicks were obtained from a commercial hatchery on day of hatch and randomly distributed into stainless steel battery pens in groups of 5 birds per pen with 8 pen replicates per treatment (48 pens total). Treatment groups of interest consisted of a control diet, a parietal yeast fraction at 500g/ton, and a Bacillus spp probiotic at 500g/ton. At 3 days of age, all birds were orally gavaged with 1.25x10<sup>7</sup> CFU's of Salmonella Infantis. On days 14 and 21, chicks were humanely euthanized, and organ samples (liver, spleen and ceca) weretaken for detection of translocation between organs by enumeration and prevalence. Organ samples were analyzed for Salmonella Infantis enumeration by direct plating on XLT-4 agar treated with Nalidixic acid and Novobiocin, and for prevalence by direct plating after 24 hours of incubation in Rappaport Vassiliadis broth. The parietal yeast fraction showed reductions in Salmonella prevalence inthe liver at day 14, and a reduction in prevalence in all three organs at day 21. Although not significant, at Day 14 the Challenged Control treatment showed 2.15x10<sup>5</sup> CFU's of Salmonella in the ceca compared to 8.08x10<sup>3</sup> CFU's in the parietal yeast fraction, & 1.44x10<sup>5</sup> in the bacillus treatment, providing a reduction of approximately 1.4 logs of Salmonella in the ceca of birds fed yeast fraction. Cecal Salmonella load enumeration for bacillus treatment was reduced at day 21 to 4.55x10<sup>4</sup> thoughnot significantly different. Further reductions were observed in birds fed parietal yeast fraction, as loads were significantly reduced from 1.04x10<sup>5</sup> CFU's in the control treatment to 3.50x10<sup>3</sup> CFU's inthe parietal yeast fraction, a reduction of over 1.5 logs (p
ID: 1100

# CHARACTERIZATION AND ASSESSMENT OF POSTBIOTIC METABOLITES ON BACTERIOLOGICAL QUALITY OF LAYER EGGS: AN INNOVATIVE APPROACH

B. Pearlin vincent*(1), G.Marappan(1), J.Tyagi(1), J.Rokade(1), A.Manoharan(1), T.Tamilselvan(1), E.Sivam(1), S.Mohan(1)

(1)CENTRAL AVIAN RESEARCH INSTITUTE, Bareilly, India

* Corresponding author: beulahpearlin@gmail.com

The microbiological quality of chicken eggs is of zoonotic importance for consumer's welfare. The intrinsic relationship between the bird and the gut microbiota has crucial implications on their health and products quality. Probiotics have been an attractive alternative to antibiotic growth promoters in food animals for maintenance of gut performance but recent studies indicated that these selected organisms could transfer resistance genes to inhabitant pathogens. Postbiotics, the active metabolites of probiotic organisms can perform a similar function without any concern on resistance transmission and its study on layer chicken are much limited. Laboratory isolated probiotic Lactobacillus plantarum (LGFCP4) was incubated in selective broth for 24 hours and its metabolites were collected and stored at 4°C. The postbiotics were in vitro characterized and there was a linear reduction (p<0.05) in media pH, with an increase in osmolarity in comparison to the uninoculated media. Assessment of its anti-inflammatory activity against hemolysis of erythrocytes demonstrated a significant (p<0.05) protective effect which was comparable to that of ibuprofen. A biological experiment was carried out with 48 laying hens (45 weeks of age) in six replicates to study the effect of postbiotics on gut microbes and inturn the bacteriological quality of eggs. One group of hens were fed with 0.2% of postbiotics for a period of 7 days and another group served as control under t-test distribution. The faecal samples from individual birds were collected at 0, 3 and 9 days of postbiotic feeding. Representative samples of egg contents and egg shell rinse were collected at the end of postbiotic feeding trial. The total coliform count showed significantly (p<0.01) reduction in postbiotic group (6.75x10⁵ CFU/g) vs. control (8.95x10⁵ CFU/g) and the period effect showed a non-significant decrease. However, lactobacillus count remained unaltered by the supplementation of postbiotics but exhibited period increase (p<0.01) with 8.10x10⁵ CFU/g at day 9. There was significant (p<0.05) increase in lactobacillus colonies of egg shell rinse due to postbiotic supplementation (4.27x10⁵ CFU/g) over control (3.57x10⁵ CFU/g) but the coliforms or lactobacillus in egg contents were non-detectable. The study indicated that postbiotics could work similarly to probiotics in manipulating the gut environment and improving product quality.
CHICKEN CONSUMPTION INCREASES PREVALENCE OF POLYCYSTIC OVARY SYNDROME: A MYTH IN PERSPECTIVES OF PAKISTAN

M. Un nisa*(1), A.Masroor(1), M.Kamran khan(1), N.Ahmad(1), M.Sajid arshad(1), H.Anwar(2), M.Imran(1), H.Umbreen(1)

(1) Institute of Home and food sciences, Faisalabad, Pakistan

(2) Department of Physiology, Faisalabad, Pakistan

* Corresponding author: linknisa@gcuf.edu.pk

Polycystic ovary syndrome (PCOS) is common endocrine disorder in women of reproductive age, across the world. In this modern era food intake is mainly progressing towards fast foods and soft drinks. These unhealthy eating patterns as well as lack of exercise lead to PCOS. It is a common perception in Pakistan that junk foods especially made of chicken are the main cause behind PCOS.

The objective of this study was to find out the percentage of prevalence of PCOS in Faisalabad region and relationship of dietary habits of females with PCOS.

To fulfill this purpose, 500 female PCOS patients were chosen from private clinics and public hospitals of Faisalabad, Punjab, Pakistan. A Performa was developed to record information regarding subject's socio demographic, clinical record (i.e. hirsutism, acne etc), dietary habits on weekly basis, physical activity and menstrual history.

The findings of present study showed that highest number of females that was about 59.20% suffered in PCOS at the age of 21-30 years. However, lowest number of females about 10.40% had PCOS at the age of 15-20 years. About 30.40% PCOS patients were working women while 69.60% were non-workers. Regarding the marital status, PCOS prevalence was high as 63.40% in married women. On the base of BMI, highest percentage of PCOS was recorded in obese women (43%). The current study showed that 32%, 18%, 10%, 55% and 12% PCOS females had thin, dry, lusterless, easily plucked out and dandruff loaded hairs, respectively. About 55% PCOS females were having facial hairs and 45% were having neck hairs. Regarding the skin problems, dry, purplish spots, itchy and acne skin types were found in 20%, 18%, 22% and 40% PCOS females, respectively. About 42% PCOS females were facing irregular menstrual cycle. About 70% PCOS females were not exercising in their routine habits. Dietary habits data revealed that there was more consumption of fast foods, soft drinks, cereals, sweets and chocolates by PCOS women. However reduced intake of poultry, fish, fruits and vegetables and dairy products was observed.

It can be concluded from the results that PCOS women have unhealthy life style pattern including unhealthy eating habits which is responsible for worsening this condition. There was no solid link found between the chicken/poultry consumption with the incidence of PCOS. Excess of everything is bad so it has been proved that people have wrong conception about chicken intake and hormonal imbalance in young females.
ID: 1115

EFFECT OF DIFFERENT ADDITIVES IN THE PROTECTION OF BROILERS CHALLENGED EXPERIMENTALLY WITH CAMPYLOBACTER

C. Sol\*(1), M.Puyalto(2), C.Marín(3), S.Vega(3), J.Mallo(4)

(1)NOREL SA, Lliça de vall, Spain

(2) NOREL SA, 8185, Spain

(3) Universidad Cardenal Herrera-CEU, Moncada, Spain

(4)NOREL SA, Madrid, Spain

* Corresponding author: csol@norel.net

The major source of human campyobacteriosis is considered undercooked and cross-contaminated broiler meat. The society is demanding to reduce antibiotic use and alternatives to fight against bacterial infections in farms. In this context, the aim of the trial was to assess the effect of different additives on caecal colonization of Campylobacter in broilers. A total of 288 day-old chicks (negative to Campylobacter) were distributed in 4 treatments among 24-floor pens (n=6). The treatments were:T1, Control feed without additives; T2, GUSTOR N'RGY (2.7Kg/t of feed), based on sodium butyrate (NaBut) protected with sodium salts of palm fatty acids distillates (Active ingredient, AI=56% C4); T3, DICOSAN (4Kg/t), based on sodium salts of medium-chain fatty acids distillates (NaMCFA, AI=38% C8+C10+C12); and T4, DICOSAN+ (2.3Kg/t), based on NaBut protected with NaMCFA (AI=65% C4+C8+C10+C12). All additives were supplied by NOREL and were dosed to achieve the same quantity of active ingredient in the final feed. At d11 of rearing, 20% of birds were orally challenged with Campylobacter jejuni (6 log10 CFU/mL). From each treatment 18 chicks were euthanized for caeca sampling at 1, 2, 3 and 4 weeks post-inoculation (WPI). Data were analysed by one-way ANOVA using the software package SPSS 24.0. The results showed that no treatment was able to prevent the contamination by Campylobacter 3 days post-inoculation. All additives got a significant reduction on Campylobacter counts along the rearing period.

By week, a significant logarithmic reduction of Campylobacter counts at 4th WPI were achieved by T2, T3 and T4 (1.63, 1.23 and 1.26 Log R, Pst and 2nd WPI. However, at 3rd WPI, T4 tended to have the lowest counts (6.75y, 6.59xy, 6.86y and 6.06x, for T1, T2, T3 and T4, respectively). At 4th WPI, T2 and T3 had significant lower counts of Campylobacter (6.66b, 5.91a, 6.03a and 6.24ab, for T1, T2, T3 and T4, respectively).

In conclusion, all treatments achieved a significant reduction of caecal colonization by Campylobacter. However, DICOSAN+ at 32d of rearing with 1.4 log10CFU/g reduction and GUSTOR N'RGY at 42d of rearing with 1.6 log10CFU/g reduction were the most effective additives. It has been reported that Campylobacter reductions around 2 log10 CFU/g is estimated to reduce human campylobacteriosis cases attributable to broiler meat by at least 76%.
ID: 1169

EFFICACY OF SYNERGISTIC MIXTURE OF ORGANIC ACIDS AS AN ALTERNATIVE TO ANTIBIOTIC GROWTH
PROMOTERS IN COMMERCIAL BROILERS RAISED UNDER OPEN-SIDED HOUSING SYSTEM IN MYANMAR

Y. Aung*(1), R.Nayak(2), S.Ayudhya(2), D.Patnaik(3), Z.Min(4), K.Zaw(4), K.Wint(4), H.Myint(5), J.Boekhorst(6)

(1)Department of Medicine, University of Veterinary Science, Nay pyi taw, Myanmar

(2)Trouw Nutrition Asia Pacific (Nutreco Asia Co. Ltd.), Bangkok, Thailand

(3)Trouw Nutrition Myanmar Co. Ltd., Yangon, Myanmar

(4)Poppa Feed Mill and Poultry Production, Yangon, Myanmar

(5)Department of Medicine, Nay pyi taw, Myanmar

(6)Trouw Nutrition Asia Pacific (Nutreco Asia Co. Ltd.), Bangkok, Thailand

* Corresponding author: yehtutaung78@gmail.com

The use of antibiotic growth promoters (AGPs) in commercial broiler production in Myanmar is
currently abundant and a common practice applied by virtually all feed millers. With the increasing
concern of antimicrobial resistance, alternatives to the use of AGP in livestock production are tried
worldwide. Therefore, two field trials were conducted to evaluate efficacy of replacement of in-feed
AGPs with synergistic mixture of modified organic acids (OAs) (Selko pH, Trouw Nutrition) by drinking
water in commercial broilers under open-sided housing system in Myanmar. One-day-old commercial
broilers, 6120 Ross308 and 7602 Arbor Acres Plus in the first and the second trial, respectively, were
equally divided into two groups, AGP group and OA group with the same housing system. The broilers
from AGP group, with two replicates, were provided the same commercial feeds with in-feed AGPs,
Chlortetracycline, Zinc Bacitracin and Tylosin. The broilers from OA group, with two replicates, were
provided the same commercial feeds without AGP but synergistic mixture of organic acids (Formic Acid,
Acetic Acid, Ammonium formate and Copper) in the drinking water. The pH of drinking water for OA
group were adjusted to get between 3.6-4.1 by adding the OA mixture. Mortality rates, feed
consumption, live weights at the market age are recorded in both experiments. Broiler performance
indices (BPI) and uniformity of live weight were evaluated at 41-day old and the data obtained were
analyzed by Student t test. The BPI of 344.017 and 351.332 in the first trial and 407.555 and 411.889
in the second trial were observed in AGP and OA group, respectively. The uniformity of live weight
observed were 86.56% and 87.41% in the first trial and 91.25% and 88.60% in the second trial in AGP
and OA group, respectively. However, there were no significant differences (p>0.05) in BPI and
uniformity of live weight between the two groups. The results of the two field trials clearly indicate
that the use of in-feed AGP can be replaced by the drinking water organic acids synergistic mixture in
commercial broilers raised under open-sided housing condition in Myanmar.
A DIETARY COMBINATION OF MICROENCAPSULATED ORGANIC ACIDS AND ESSENTIAL OILS REDUCES SALMONELLA ENTERICA SV. TYPHIMURIUM IN CHALLENGED BROILER CHICKENS

N. Abdelli*(1), S.Campoy sánchez(2), E.Frutos grilo(3), D.Reyes camacho(1), A.Forouzandeh(1), J.Pérez hernández(4), D.Sola oriol(4)

(1)Universitat Autònoma de Barcelona, Facultat de veterinària, Bellaterra, Spain

(2)Universitat Autònoma de Barcelona, Cerdanyola del vallès, Spain

(3) Universitat Autònoma de Barcelona, Departament de Genètica i de Microbiologia, Cerdanyola del vallès, Spain

(4) Universitat Autònoma de Barcelona, Facultat de veterinària, Bellaterra, Spain

* Corresponding author: nedra.abdelli@uab.cat

Contamination of poultry meat with foodborne pathogens, such as Salmonella, remains an important public health issue. Therefore, ensuring the microbial safety of poultry meat products is crucial for the poultry industry. We hypothesized that organic acids and essential oils (EO) could reduce Salmonella in chickens digestive tract because of their reported antimicrobial effects.

In this context, a trial of 21 days was performed to test the efficacy of two targeted-release blends of organic acids (Blend A: Calcium butyrate, Blend B: Calcium butyrate, fumaric acid and lauric acid), combined with EO, on growth performance and Salmonella colonization in experimentally infected broilers (d0, 107 S. Typhimurium). A total of 255 one-day-old male broiler chicken were distributed into 3 dietary treatments: negative control (NC) and NC supplemented with either 0.7g/kg of blend A or 2g/kg of blend B, with 5 replicas per treatment and 17 chicks per replica. Birds were weighed and feed disappearance was determined at d3, 7, 14 and 21 on a pen basis to evaluate growth performance. On the same days, 3 birds per replicate were euthanized to collect cecum and spleen samples for Salmonella quantification. Blood samples were collected from the 3 chickens euthanized per replicate, at the end of the experiment, to analyse acute phase proteins. One-way ANOVA followed by a Tukey's multiple comparison test was used to analyse the significance of the differences among treatment groups. For the count of positive spleens a chi-squared analysis was performed. The significance threshold was set on 0.05.

On d21, all chickens presented lowest performance than the ROSS standards. However, birds supplemented with the blend B showed higher body weight than those of the NC (P=0.04) as a result of higher average daily gain (P=0.030). These birds also presented reduced α1-acid glycoprotein concentrations compared to those on blend A and NC (P< 0.001). On d21, the supplementation of blend A reduced Salmonella concentration in the ceca (P=0.02) compared to the rest of dietary treatments. As for spleen, difference among treatments was observed only on d3 where blend B showed the lowest positive spleens (P=0.03) compared to blend A and NC.

In conclusion, a protected blend of calcium butyrate and EO did not affect growth performance of challenged birds. However, it decreased Salmonella concentration in the cecum, which may suggest that this blend exerts some antimicrobial effect on S. Typhimurium.
ID: 1252

THE EFFECT OF DIFFERENT DIETARY RATIOS OF LYSINE AND ARGININE IN DIET OF HIGH OR LOW METHIONINE LEVEL ON OXIDATIVE AND EPIGENETIC DNA DAMAGE, GENE EXPRESSION OF TIGHT JUNCTION PROTEINS AND SELECTED INDICES OF METABOLISM IN CLOSTRIDIUM PERFRINGENS CHALLENG

J. Jankowski(1), P.Konieczka(1), Z.Całyniuk*(2), K.Ognik(3), D.Mikulski(1)

(1)University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

(2)University of Lifesciences in Lublin, Lublin, Poland

(3)University of Life Sciences in Olsztyn, Lublin, Poland

* Corresponding author: p.konieczka@ifzz.pl

Essential amino acids are dietary components with immune-enhancing functions, therefore their proper dietary ratio may help birds to cope with the bacterial infections. Two experiments were run to investigate effect of different ratios of arginine (Arg) to lysine (Lys) in diets of low (30% Lys; experiment 1) and high (45% Lys; experiment 2) methionine levels on selected indices of metabolism, oxidative and epigenetic damage of DNA, and mechanisms determining gut integrity in turkeyssubjected to C. perfringens infection. In each experiment, 108 one-day-old Hybrid Converter female turkeys were placed in 6 pens (18 birds per pen) and reared for 42 days. At days 34, 36 and 37 of life, half of the birds was subjected to C. perfringens challenge. A 3 x 2 factorial design with three levels of Arg relativeto Lys (90, 100 and 110%; Arg90, Arg100 and Arg110, respectively) and C. perfringens infection (-, +) was employed. Challenging birds with C. perfringens increased lipids oxidation and oxidation and methylation of DNA of gut mucosa as well as it did downregulate the activities of DNA- repairing enzymes. Markers of liver function and metabolism did not differ neither of dietary treatments nor of challenge. Feeding diets of Arg110 and high methionine level evoked DNA oxidationand methylation while, in birds fed diets of Arg90 they were significantly downregulated. Results indicate that diets of high methionine level and Arg90 has a beneficial action on gut-associated indicesof integrity in turkeys subjected to necrotic enteritis. Although, the obtained results indicating interactive actions of the investigated amino acids ratios and host gut integrity-maintaining systems, such dietary intervention appear likely supported birds for coping with necrotic enteritis.

This work was supported by the National Science Centre, Grant No. 2017/27/B/NZ9/01007
ID: 1275

COMPARISON OF DIFFERENT IBD VACCINATION PROGRAMS UNDER FIELD CONDITIONS.

E. Russo\*(1), S.Boldini(1)

(1) MSD Animal Health, Segrate (mi), Italy

* Corresponding author: elisa.russo@merck.com

Infectious bursal diseases (IBD) is a viral poultry disease characterised by immunosuppression and huge economic losses. IBD control is a crucial point of antibiotic reduction in broiler, and the only effective control system is vaccination, with live attenuated vaccines or with turkey herpesvirus construct vaccines expressing IBDV VP2 protein (rHVT-IBD). The main advantages of this innovative vaccine are absence of vaccine reactions and absence of interference with maternal antibodies.

The objective of this field monitoring was to compare serology for IBD and performances of birds vaccinated with traditional vaccine (at 21 days, according to Deventer formula, group A) and with rHVT-ND-IBD (an rHVT-IBD construct expressing also F protein of Newcastle disease virus) half dose (group B) and full dose (group C).

In a broiler integration weekly serological monitoring program, 13 farms were selected, 3 for group A, 5 for B and 5 for C, all birds came from same breeders and the level of maternal antibodies was alike. Serological titers for IBD of the three groups were compared at each time point using one way ANOVA test, and t Student test, significance was set at p<0,05.

Production data of the same integration coming from 3 months of production for each vaccination plan were compared.

Group A had a drop in serological titers from week 1 to 3, from week 4 the serological titers began to raise. Group B had a similar trend for weeks 1 and 2 (no significant difference vs group A), but at week 3 the titers started to increase (significant difference vs group A). Group C never had a drop in serological titers (significantly higher titers vs groups A from 1 and to 2 weeks, and vs group B at 1 and 2). From 5 weeks of age all groups showed a strong seroconversion.

Performances were similar for all vaccination programs, but the use of antibiotics seemed to be lower with rHVT-ND-IBD vaccine.

The group vaccinated with live vaccine (A) had a period of susceptibility to IBD, with birds negative to serology, whereas groups vaccinated with rHVT-ND-IBD (B and C) never had negative samples. These findings confirm that rHVT-ND-IBD provides protection against IBD since the beginning of the cycle without showing periods of susceptibility that usually affect traditional vaccination schemes.

Among the rHVT-ND-IBD groups the onset of immunity was quicker in the group vaccinated full dose. This finding supports the use of full vaccine dosage in order to have an early and uniform immunity.
ID: 1310

THE INTERACTIVE ACTION OF TWO STRAINS OF PROBIOTICS ON PERFORMANCE AND EXPRESSION OF SELECTED GENES DETERMINING TIGHT JUNCTION PROTEIN IN CHICKENS SUBJECTED TO NECROTIC ENTERITIS

P. Konieczka*(1), D.Sandvang(2), M.Kinsner(3), D.Szkopek(3), J.Jankowski(1)

(1)University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

(2) Chr. Hansen A/S, Hørsholm, Denmark

(3) The Kielanowski Institute of Animal Physiology and Nutrition, Jabłonna, Poland

* Corresponding author: p.konieczka@ifzz.pl

Microbial feed supplements are applied into poultry feed to protect birds from pathogen infections and to fortify the gut barrier function. Spore-forming bacteria seem to be particularly well-suited for use in feeds, as they are metabolically dormant and resilient to environmental stresses, including feed processing. The objective of this study was to evaluate the actions of two strains of commercial probiotics (GalliPro®Tect and GalliPro®Fit containing a spore-forming strains of probiotic bacteria).

A total of 432 male day-old broilers Ross 308 were divided into group cages of 9 birds. The 6 dietary treatments were allocated to T1 - T6, so that each treatment was applied to 8 cages of 9 broilers; T1 standard commercial diet (C), T2 - C + GalliPro®Tect, T3 - C + GalliPro®Fit, and treatments T4-T6 were as T1-T3, respectively but assigned birds were challenged with

C. perfringens in a subclinical dose at days 15 and 16 of age. At day 29 of life, samples of jejunum were collected from 8 birds of each treatment. Differences between groups were compared by two-way ANOVA as a 2 × 3 factorial design: challenged vs. not challenged with C. perfringens, and dietary supplementation: (i) without additives, (ii) with GalliPro®Tect, (iii) with GalliPro®Fit.

The body weight (BW) at day 21 (P=0.018), BW gain (BWG) for the period 12-21 and 0-21 days (P<0.02), and FCR for the period 12-21 and 0-35 days (P<0.05) were compromised due to the challenge. The most efficient feed conversion ratio for the period 12-21 days was found in the control and GalliProFit groups while, for the same period but in challenged birds it was the best in birds fed GaliPro®tect. C. perfringens challenge and probiotic supplementation significantly increased expression levels of zonula occludens-1, occludin and claudin-3 genes in the ileal tissue, which was also associated with the significant interactions (P<0.001).

Our findings suggest that beneficial action of GalliPro®fit and GalliPro®tect depends on the physiological status of birds. In normal conditions (without challenge) GaliPro®fit was better than GaliPro®tect in maintaining birds performance whereas opposite was true in birds applied to challenge.

The results regarding expression level of genes determining tight junction of the gut indicating an interactive actions of the investigated additives and host gut integrity-maintaining system however, such dietary intervention appear likely supported birds for coping with necrotic enteritis.
PERCEPTION OF HUMAN HEALTH RISKS RELATED TO AIR QUALITY AND PREVENTIVE BEHAVIOURS IN
DUCK OVERFEEDING SYSTEMS

N. Rousset*(1), A.Battaglia(1)

(1)ITAVI, Ploufragan, France

* Corresponding author: rousset@itavi.asso.fr

Duck housing systems during overfeeding have evolved significantly over the past 4 years, in line with animal welfare regulation. This change has impacted working conditions of farmers who complain of greater respiratory discomfort due to 1. more aerial dust due to greater freedom of movement for animals and 2. drier ambient air. In this context, the project AGAtE is underway on dust exposure levels and preventive measures to prevent respiratory diseases induced by inhalation of particles. A part of the project addresses the risk perception of duck producers linked with their preventive behaviors, to improve arguments and communication methods on prevention.

The survey guidelines used to carry out a qualitative study (14 interviews), include perception of working conditions (motivations, constraints, health, general knowledge about risks), awareness about risk linked to air quality in duck houses (air quality perception, farmer-specific risk evaluation, sources of information), and preventive behaviors. A longitudinal analysis and a typology have been performed. Most of the duck producers feel very concerned about the respiratory risks linked with dust inhalation. Risks awareness is mainly ensue from visible indicators, odors, respiratory or ocular discomfort, but also from the social network (e.g. exchanges between peers). Nevertheless, half of them, older and more experienced, describe a “quite good” air quality in duck houses, mainly arguing about their technical results. Wearing a protective mask is a largely widespread behavior, and it seems to be a professional standard. Nevertheless, young and less experienced farmers, who have no occupational pathology, seem to wear protective masks less longer than other (e.g. only at the beginning of the rearing period when particles are most perceptible). Finally, we identified four different profiles based on their air quality perception and preventive behaviors. Experience, probably associated with a large social network rather than person’s age and occupational pathology appear as the mains motivations for wearing a protective mask when handling ducks.

Exchange meetings between peers or videos may be constitute a good way to inform on respiratory risk among duck producers. Moreover, it seems import to alert and train technicians about respiratory health risks, because they could be a good vector to inform young farmers at the beginning of their professional life.
POULTRY PRODUCTION FOR INSURING
FOOD SECURITY
FEED THE FUTURE INNOVATION LAB FOR GENOMICS TO IMPROVE POULTRY: INCREASING FOOD SECURITY IN AFRICA BY ENHANCING RESISTANCE TO NEWCASTLE DISEASE AND HEAT STRESS IN CHICKENS

H. Zhou*(1), T.Kelly(1), S.Lamont(2), J.Dekkers(2), R.Gallardo(1), A.Muhairwa(3), B.Kayang(4), P.Msoffe(5), A.Naazie(4), H.Otsyina(4)

(1)University of California, Davis, Davis, United States

(2)Iowa State University, Ames, United States

(3)Sokoine Agriculture University, Morogoro, Tanzania

(4)University of Ghana, Accra, Ghana

(5)Sokoine Agriculture University, Morogoro, Tanzania

Homestead and small-scale poultry production has tremendous potential for alleviation of malnutrition and poverty in climate-stressed rural communities in Africa. Poor animal health and husbandry practices limit village poultry production. Newcastle disease (ND) is the number one constraint of raising poultry in Africa, causing high mortality among village flocks. This research and development program seeks to improve poultry production by households and small farmers, and thereby improve food security, nutrition, and livelihoods in Africa -- key goals of the USAID Feed the Future Program. Genetics plays a significant role in resistance of poultry to NDV infection. Our program is applying advanced genetics and genomics to sustainably enhance innate resistance to ND and heat stress in chickens adapted to African environment. In the past six years, we identified the most robust genes and markers for genetic resistance through an integrated analysis of NDV challenge experiments and genomic analyses of well-characterized chicken lines under strictly controlled environmental conditions by RNA-seq and genome-wide association analysis (GWAS) using chicken 600K SNP panel, and of six African indigenous chicken ecotypes by GWAS. Our results confirmed the polygenic control of resistance to NDV and suggested that viral load and antibody titer following infection are important parameters for evaluating disease resistance. We estimated heritabilities for these traits in African chicken to be moderate to high (0.14-0.55), which indicates that selection to improve these ecotypes for resistance to ND is feasible. In addition, natural exposure trials of about 3,000 birds of African ecotypes with velogenic NDV strains were conducted to identify associations of markers with viral shedding, survival time, and anti-NDV titers. Based on our results, a low-density 5K SNP panel for genetic resistance to NDV was developed to select and breed local ecotypes with enhanced resistance to ND. We are currently assessing correlations of disease resistance traits with crucial production traits, such as egg production and growth rate. We are also conducting value chain assessment and developing a business plan to enable distribution of improved chicken lines to rural farmers with a focus on women who would benefit the most of an increase in resistance and productivity.

<footnote>* Corresponding author: hzhou@ucdavis.edu</footnote>
SUPPLEMENTARY FEEDING ENHANCES INCOME OF FARM WOMEN IN EXTENSIVE SYSTEM OF POULTRY PRODUCTION

A. Panda*(1), B.Sahoo(2), A.Kumar(3)

(1)I, Bhubaneswar, India

(2) ICAR-CENTRAL INSTITUTE FOR WOMEN IN AGRICULTURE, PLOT 50-51, MAUZA-JOKALANDI, NEAR KALINGA STUDIO, Bhubaneswar, India, Bhubaneswar, India

(3) ICAR-CENTRAL INSTITUTE FOR WOMEN IN AGRICULTURE, PLOT 50-51, MAUZA-JOKALANDI, NEAR KALINGA STUDIO, Bhubaneswar, Odisha, India, Bhubaneswar, India

* Corresponding author: akpanda59@gmail.com

Family poultry production has been recognized globally to alleviate poverty, hunger and malnutrition in developing countries. It offers advantages over other agricultural sectors and is an entry point for promoting gender balance in rural areas. Vanaraja, a dual purpose bird developed by ICAR-Directorate of Poultry Research, Hyderabad, India is gaining popularity across India for family poultry farming. The present study was conducted to study the effect of supplementary feeding on production performance of Vanaraja laying hens and income of farm women reared under extensive system of production. A total of 300, four weeks Vanaraja chicks were distributed to 20 selected farm women (15 female chicks) in Chanrapada and Denua basta, Nimapada block of Puri district, Odisha, India. The farm women reared the chicks by allowing them scavenge outside during day time and during night they were provided night shelter (low cost house), constructed using locally available bamboo and paddy straw with provision of drinking water. At 16 weeks of age, supplementary feeding @ 35g/bird/day was given to one group (birds reared by 10 farm women). Egg production was recorded daily. Egg weight was taken at 32 and 40 weeks of age. Supplementary feeding @ 35g/bird/day during 16-40 weeks of age resulted in significantly higher egg production compared to those of without supplementation. The birds of supplementary fed group produced more eggs with higher egg weight compared to nonsupplementary one. Calculating the feed cost at Rs.20/kg, the net return was higher by Rs. 1790 per beneficiary due to supplementary feeding (around Rs. 128/ bird). From the results of the present study, it is concluded that supplementary feeding @ 35g/bird/day to Vanaraja laying hens in extensive system of production resulted in higher egg production and economic return.
ID: 1085

VILLAGE CHICKENS: OPTIONS TO IMPROVE NUTRITION AND HEALTH OF HIV AND AIDS AFFECTED INDIVIDUALS IN MARRACUENE DISTRICT, MOZAMBIQUE

R. Da costa* (1)

(1) Kyeema Foundation, Mozambique, Maputo, Mozambique

* Corresponding author: rosac@kyeemafoundation.org

The prevalence of HIV is high in Mozambique. In young women, aged 15 to 24, the estimated HIV prevalence rate is 10.7%, compared to a prevalence rate of 3.6% among young men in the same age group. Nearly half of the people with HIV living in poor urban areas have a high prevalence of food insecurity. Village chickens can provide a scarce resource – high quality protein and micronutrients – in the form of meat and eggs, and can be sold or bartered to meet essential family needs such as medicine, clothes and school fees. Two hundred and forty vulnerable households comprising women, orphans and vulnerable children and HIV-affected individuals were selected in collaboration with local leaders and relevant government agencies in Maracuene District, trained in low-cost improvements to village chicken production, and each received a chicken 'starter pack' (three indigenous hens and one rooster) to improve their nutrient intake and household food and nutrition security. Over a period of 9 months, 98 hens had hatched 1210 chicks. With the increased availability of chickens, farmers started selling more, but home consumption remained low during the first year. The number of chickens consumed per family over a 3 months period, increased slightly from 1.02 to 1.51 and the average number of chickens sold per family in three months period, increased from 0.38 to 2.53 chickens. Consumption of eggs remained low with households preferring to use them for hatching.

In collaboration with the District Veterinary Services, thirty-six (21 male and 15 female) community vaccinators were trained and they vaccinated 18 056 chickens against Newcastle disease (ND) in 1 957 households of the District, including people affected by HIV and AIDS. The average flock size in the households that vaccinated regularly increased from 8.5 to 10.54 after two vaccination campaigns. No reports of mortality of chickens with clinical signs of ND were made in the areas where vaccination was taking place.

KEYWORDS

HIV - affected individuals, nutrition; Village chicken; Newcastle disease.
# FACTORS AFFECTING EMPOWERMENT OF SMALL HOLDER WOMEN POULTRY FARMERS IN SEMBABULE DISTRICT, UGANDA

D. Kusiimakwe*(1), A.Mugisha(2), K.Mugabi(3), E.Kyewalabye(2), L.Tukahirwa(1)

(1)Makerere University, Kampala, Uganda

(2) Makerere University Uganda, Kampala, Uganda

(3) Ministry of Agriculture Animal Industry And Fisheries, Uganda, Entebbe, Uganda

* Corresponding author: dkusiimakwe@gmail.com

## Introduction

This research was part of the bigger project "Hearing their voices: Action research to support women's agency and empowerment in livestock vaccine distribution, delivery and use in Rwanda, Uganda and Kenya"

Indigenous chicken production is a preserve for women in Sembabule District but traditionally a cock belongs to a man.

## Objectives

To determine the factors that affect women empowerment in small holder poultry production in Sembabule District, this research assessed;

i. Gender dynamics in small holder livestock keeping households.

ii. How gender dynamics affect poultry production in small holder households.

iii. The effect of gender dynamics on livestock vaccine adoption at household level.

iv. The effect of women empowerment level on livestock vaccine adoption at household level.

## Methodology

The five USAID domains of gender analysis and qualitative methodologies were used in data collection during the months of November and December.

Women keeping a maximum of 80 chickens were targeted as research participants. Field work comprised of 6 jar voices, 4 focus meals, 6 focus group discussions (FGDs) and 15 key informant interviews which explored local understanding of empowerment, the livestock vaccine value chain and how these relate to rearing of indigenous chicken.

## Results

Generally in Sembabule, all resources in a home belong to a man; including a woman. In remote areas, men usually take dominance over women in resource control and distribution. Whereas in trading centres are unmarried women control their own resources.

Women can rear chicken on family land but marketing of chicken remains a man's duty. Conflicts in households mainly arise when chicken multiply and a man realizes his wife is generating significant income. Such a man demands a part of the income claiming the wife is farming on his land.

Where women controlled some resources, those homes were happier and better than where men were dictators.

Women are more involved in group associations; through these groups, they easily move out of home, access credit and own livestock with little interference from their husbands.

There was little vaccine use due to lack of cold chain and a knowledge gap on vaccines.

## Conclusions

This research identified the following intervention areas that can help empower women in Sembabule;

i. Training women on commercial poultry production.

ii. Creating opportunities for women to participate in the vaccine value chain.

iii. Involving men in the empowerment process.
VALORIZATION OF CRICKET FLOUR AS A SUBSTITUTE OF FISH MEAL ON QUAIL PRODUCTION
PERFORMANCES IN THE SUDANO-GUINEAN ZONE OF CAMEROON, UC

F. Djitie kouatcho\*(1), B.Aoudou(2), D.Moussa(2), F.Ngoula(3), L.Ngamo tinkeu

(1)University of Ngaoundérré, Ngaoundéré, Cameroon

(2)University of Ngaoundéré, Ngaoundéré, Cameroon

(3)University of Dschang, Dschang, Cameroon

* Corresponding author: franckdjitie@gmail.com

Fish meal is one of the relatively expensive ingredients that contribute to making the food nearly 70% of the cost in poultry farming (NRC, 1994; INRA, 1989). About 27% of marine fishing products are processed into fishmeal or fish oil without meeting farming needs (Cashion et al., 2017). The use of local resources like cricket (Acheta domesticus) flour could help to remedy these shortcomings.

The objective of the study was to increase the profitability of poultry farming in general.

To assess the effect of fish meal substitution by cricket flour on quail production performances, a study has been conducted from July to September 2018 in Ngaoundéré, Cameroon. For this purpose, 192 four weeks old quails were divided into 12 comparable batches of 8 females and 8 males. Each of the four experimental diets (T00, T15, T30 and T45), formulated on the basis of the level (0, 15, 30 and 45% respectively) of substitution of basic diet (Crude Protein: 20,18% and ME: 3013,78Kcal/Kg) fish meal by cricket flour was randomly assigned to 03 batches in a completely randomized designcorresponding to 04 treatments repeated 03 times each. Data were collected on growth, carcass and some reproductive parameters. One way ANOVA and Duncan test at 5% significance level were used for data analysis.

Main results show that diet containing cricket flour has increased body weight in males (204.32±5.69g for T45) and regardless of sex (226.72±29.45g for T30) compared to respectively 184.17±3.11g and 214.55±32.77g recorded with the control group. In females, substitution at 30% increased the body weight (253.80±6.48g) and 45% induced a reduction in body weight (216.67±6.49g) compared to the control group value (244.92±6.07g). Carcass yield and proportion of different parts were not significantly affected by experimental diets. The significantly higher liver proportions were recorded at 15% and 35% incorporation of cricket flour compared to that of 45%. First songs and laid eggs were recorded at 07 weeks with T15 which also led to an improvement of laying performances in contrast to other treatments. In T45 females, ovaries were poorly developed compared to others.

It has been concluded that cricket flour could be used to replace fish meal in the diet of quail at the
finisher and reproduction phases at the level of 15 and 30%

Key words: storage time, external, internal, chemical characteristics, battery eggs.

References:

Cashion, T., Le Manach, F., Zeller, D. & Pauly, D., 2017. Most fish destined for fishmeal production are food-grade fish. Fish and Fisheries. 18, 837–844.

Institut National de la Recherche Agronomique (INRA), 1989. L'alimentation des animaux monogastriques : Porcs, lapins, volailles. 2ème Ed. INRA, Paris. 282p

National Research Council (NRC), 1994. Nutrient Requirements of Poultry, 9th Rev. Ed. National Academy Press, Washington, DC.
REDUCING THE ENVIRONMENTAL
IMPACTS OF POULTRY PRODUCTION
EFFECT OF INCLUSION OF BACILLUS SUBTILIS TO ENERGY AND PROTEIN REDUCED DIET ON GROWTH
PERFORMANCES, NUTRIENT DIGESTIBILITY AND GAS EMISSION IN BROILERS

J. Bodin\*(1), S. Upadhaya(2), I. Kim(3), D. Sangvang(4)

(1)Chr. Hansen, DK, Hoersholm, Denmark

(2)Department of Animal Resource and Science, Dankook, Korea., Choongnam, South Korea

(3)1Department of Animal Resource and Science, Dankook, Korea., Choongnam, South Korea

(4)Chr. Hansen A/S, Hoersholm, Denmark

* Corresponding author: frjebo@chr-hansen.com

816 day-old Ross 308 broiler chicks were used in a 6-wk feeding trial. Birds were randomly allotted to 1 of 6 dietary treatments (8 replicate pens/treatment with 17 birds/replication pen). The trial design is a 3×2 factorial arrangement with 3 levels of metabolizable energy (ME) and crude protein (CP), (100%ME and CP (1+2), 98.8% ME and CP (2+3), and 97.6% ME and CP (4+5) with constant amino acid levels. Treatments 2, 4, 6 were supplemented with 500 g/ton of a Bacillus subtilis-based probiotic. Performance results of this study showed a significant reduction (P < 0.05) in Body Weight (BW) and a degradation of Feed Conversion Ratio (FCR) in relationship with diet density reduction. Birds fed probiotic supplemented diets during the growing and finishing periods were heavier (P<0.05) than those fed diets without probiotic. FCR improved (P< 0.05) in Bacillus-based probiotic supplemented groups during these periods, but Feed Intake (FI) remained unaffected when comparing nutrient densities and probiotic inclusion. Concerning nutrient digestibility, Apparent Total Tract Digestibility (ATTD) of nitrogen (N) and energy were significantly lower (P<0.05) in ME- and CP-reduced diets than the control diet. Dry matter (DM) ATTD, ME- and CP-reduced diets tended (P = 0.051) to be lower compared with the control. Overall, DM, energy and nitrogen AATD improved in correlation with the supplementation of a Bacillus-based probiotic. The ATTD of nitrogen (N) and energy was significantly lower (P <0.05) in ME and CP-reduced diets than the control diet. A decreasing trend (P = 0.059) of NH3 gas emission from the excreta was observed for birds fed ME- and CP-reduced diets compared with control diets. Birds fed Bacillus-based probiotic supplemented diets showed reduced (P<0.05) NH3 emission compared to those fed a diet without probiotic. Inclusion of a Bacillus-based probiotic to ME- and CP- reduced diets improved zootechnical performances and reduced ammonia emission from the excreta, which could be due to improved intestinal health associated with microbial balanceand lower levels of protein in the diet. Thus, inclusion of B. subtilis (DSM 17299) in nutrient-reduced diets could contribute to a more efficient feed to meat transformation, with lower nutritional safety margins and reduced NH3 emissions. These combined effects could contribute to more sustainable poultry production.

Keywords: Bacillus subtilis, broilers, nutrient metabolism, digestibility, gas emission
REDUCTION IN PHOSPHORUS EXCRETION USING BUTTIAUXELLA SP. (META-ANALYSIS) AND POTENTIAL REDUCTION OF INORGANIC PHOSPHATES USE IN BROILER DIETS

S. Gilani*(1), R.Hardy(2), D.Velayudhan(3), Y.Dersjant-li(3)

(1)Danisco Animal Nutrition (IFF), Oegstgeest, Netherlands

(2)Danisco Animal Nutrition (IFF), Marlborough, United Kingdom

(3)Danisco Animal Nutrition (IFF), Leiden, Netherlands

* Corresponding author: Saad.Gilani@iff.com

Buttiauxella sp. phytase has high activity at low pH and a wide pH range, meaning faster and more complete breakdown of phytate in the early gastrointestinal tract. This can result in more efficient release of phytate bound P, reduced dependency on inorganic phosphates, and can lead to reduced P concentration in excreta. To calculate P excretion reduction using Buttiauxella sp., data from ten studies were analysed (through meta-analysis) with 510 data points. These studies were conducted in different locations including UK, The Netherlands, USA, Canada and New Zealand. Commercial broiler chickens were used in these studies till the age of 21 days. Birds were fed ad libitum. The diets were mainly wheat or corn based with soybean meal as a protein source. Nutrient requirements (except Ca and P) were met using breed recommendations. All trials had a positive control (PC) with inorganic phosphate, a negative control (NC, without phytase and reduction of available P and Ca vs. PC) and graded levels of Buttiauxella sp. phytase added on to the NC (250, 500, 750, 1000 and 2000 FTU/kg). At day 21, excreta samples were collected to determine apparent total tract digestibility of P. Increasing phytase dose led to decrease in total P excretion in exponential manner (P< 0.001 for scale and growth rate, JMP Pro statistical program). At phytase dose of 2000 FTU/kg, the P excretion reduced by 50.7% and 63.4% respectively vs NC and PC. Based on the total broiler feed produced in 2018, in the EU (55 million tons, MT) and globally (274.7 MT), P excretion with inorganic phosphate would be 173,000 T in EU and 864,000 T globally respectively. P excretion can be reduced to 96,600 T and 482,000 T by using phytase at 500 FTU/kg in the EU and globally. This can further be reduced to 63,400 T and 316,500 T in the EU and globally with the higher phytase dose of 2000 FTU/kg. Similarly based on the ileal digestible P contribution, usage of inorganic phosphates (Monocalcium phosphate used for these estimations) in broiler feed can be reduced. Without use of Buttiauxella sp. vs inclusion of 500 and 2000 FTU/kg respectively, MCP inclusion can potentially be reduced from 2.5 MT to 0.63 MT and to 0.22 MT globally. The reduction in MCP usage at the EU level is from 0.51 MT to 0.13 MT and 0.04 MT at 500 and 2000 FTU inclusion respectively. In conclusion, inorganic phosphates and P excretion can be immensely reduced with the use of Buttiauxella sp. phytase in meat producing chickens.
## ATTAPOULGITE AND OREGANO OIL IN FEED INFLUENCES ENVIRONMENTAL MICROBIAL COUNT IN POULTRY HOUSES

I. Skoufos*(1), E.Bonos(1), I.Giannenas(2), E.Gouva(1), A.Katrachouras(1), A.Patsavouras(1), S.Skaftouros(1), D.Papanastasiou(3), T.Bartzanas(4), A.Tzora(1)

(1) School of Agriculture, University of Ioannina, Arta, Greece

(2) School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece

(3) Institute for Bio-economy and Agri-technology (IBO), Centre for Research and Technology Hellas (CERTH), Volos, Greece

(4) Department of Natural Resources Management and Agricultural Engineering, Agricultural University of Athens, Athens, Greece

* Corresponding author: jskoufos@uoi.gr

It has been reported that increased density of birds in the poultry houses results in the accumulation of various pollutants, including bacteria and their toxins, toxic gases and other volatile compounds. Contamination levels in the houses, increase steadily in proportion to the birds age and especially towards the end of the rearing process. The increased microbial loaded the air is associated with microbial contamination of the birds. Pathogenic bacteria have been found within three kilometers of poultry farms.

The aims of this study were to examine the microbial load of air in broiler chicken houses and to examine the possible beneficial effects of dietary attapulgite and oregano essential oil on the air microbial load.

In this trial, three identical experimental houses were used (14m x 58m), the first as control, where broiler chickens were fed commercial diets. In the second house, attapulgite (4000 ppm) was supplemented in the diets throughout the rearing period. In the third house, both attapulgite (4000 ppm) and oregano essential oil (300ppm) were supplemented in the diets. The outside environment of the poultry houses was also evaluated. The experiment was conducted in the months of Septemberand October. Sampling was performed two days before slaughter to determine the total mesophilic flora (TMF). Samples were taken with the air sampler "Air Ideal" (bioMerieux S.A, France) at one location in each chamber, near its center and at a distance of 1.5m from the ground. After incubation in Plate Count Agar at 35°C for 48 hours, colonies were counted with the «Scan 100 automatic colony counter» (InterScience). In the control house, mean TMF was 3.40 x 10^5 cfu/m^3 of air. In the secondand third houses, mean TMF was 2.45 x10^5 cfu/m^3 and 2.23 x 10^5 cfu/m^3 of air, respectively. In the outside environment air samples TMF was 4.80 x 10^4 cfu/m^3. The predominant bacteria encountered indoors were Staphylococcus, Micrococcus and in lower counts Streptococcus, Bacillus and Enterobacteriaceae, whereas in the outdoor measurements Staphylococcus, Bacillus and Aerococcus were the most abundant.

The results of this trial could be used for the improvement of chicken housing conditions and microbial growth.

Acknowledgment: This research has been co-financed by Greece and the European Union (European Regional Development Fund) within the Operational Program "Competitiveness, Entrepreneurship and Innovation - NSRF 2014-2020". Project Code: T1EΔΚ-03856. Acronym: GREEN POULTRY MEAT ANTIFREE
THE EFFECT OF FEED FORM AND CRUDE PROTEIN CONTENT ON NITROGEN DIGESTIBILITY IN BROILERS

M. Brink\*(1), E.Delezie(1), P.Demeyer(2), Ö.Bagci(2), G.Janssens(3)

(1) ILVO (Research Institute for Agriculture, Fisheries and Food), Melle, Belgium

(2) ILVO (Research Institute for Agriculture, Fisheries and Food), Merelbeke, Belgium

(3) Ghent University, Merelbeke, Belgium

* Corresponding author: madri.brink@ilvo.vlaanderen.be

Nitrogen (N) excreted by poultry is converted to ammonia (NH3) which presents an environmental risk and a health risk to the farmer and animals. A reduction in dietary crude protein (CP) can effectively reduce N excretion in broilers provided that amino acid (AA) requirements are met. Feed form influences gastrointestinal tract development, feed and water intake and therefore the N and moisture level of the excreta. The aim of this study was to investigate the extent to which feed form and CP content influence N digestibility broilers. In total, 72 30-day-old Ross 308 male birds were assigned to 36 digestibility cages. Birds received diets with 19.5% CP (control), a 1.5 and 3.0% reduction in CP, while digestible AA:Lysine ratios were maintained. Each treatment was fed in mash and pellet form, which resulted in a 3×2 (CP level x feed form) experimental design with 6 treatments, each with 6 replicates. The study consisted of an adaptation and excreta collection period of 4 days each. Excreta was analysed for dry matter (DM), total N, total ammonium N (TAN) and uric acid (UA). Feed and waterintake were also recorded. After the collection period, treatment diets containing 0.4% titaniumoxide(TiO) as an indigestible marker were fed for 3 days. To calculate apparent ileal N digestibility (AID), ilealdigesta samples were analysed for total N and TiO. Data were analysed with linear mixed models usingR 3.5.1 for Windows. Results were expressed as Ismeans and SE of those means. Pellets led to a higherfeed (P<0.001) and water intake (P=0.018) compared to mash, while water to feed intake ratio was higher for control than for reduced CP treatments (P=0.003). Excreta moisture content did not differ between treatments, while CP (P<0.001) and UA (P<0.001) decreased with decreasing dietary CP. Faecal TAN was higher for pellets than for mash (P=0.002), while for UA the opposite was observed (P=0.001). Faecal CP digestibility (corrected for UA) was higher for mash than for pellets (P=0.006). Compared to the control, AID decreased with the 1.5 and 3.0% reduced CP treatments (P<0.001). N retention increased with a 3.0% CP reduction and was higher for mash than pellets (P=0.034). Althoughmash diets resulted in better N digestibility and retention compared to pellets, these results suggest that a dietary CP reduction within both mash and pelleted diets increased N retention and decreased excreta UA in broilers, resulting in lower levels of NH3 emitted from broiler litter.
SUSTAINABLE BROILER FARMING: INCREASING BUTTIAUXELLA SP. PHYTASE DOSE TO 2000 FTU/KG LINEARLY REDUCED NITROGEN EXCRETION BASED ON META-ANALYSIS OF 10 TRIALS

Y. Dersjant-li*(1), S.Gilani(2), R.Hardy(2), A.Debicki-garnier(2), A.Bello(2), L.Marchal(2)

(1)DuPont Nutrition and Biosciences, Leiden, Netherlands

(2) DuPont Nutrition & Biosciences, Leiden, Netherlands

* Corresponding author: Yueming.dersjant-Li@dupont.com

One of the strategies to increase sustainability of broiler production is to improve feed efficiency and reduce nutrients excretion such as nitrogen (N) to the manure. Among others, feed enzymes such as phytase and protease can improve crude protein (CP) and amino acids (AA) digestibility and can lead to reduced N excretion. Phytate is the major storage form of phosphorus (P) in plant-based feedstuffs and it has not only low P availability, but also can bind to AA and increase endogenous AA losses. Phytase breaks down phytate, increases availability of P and reduces the anti-nutritional effect of phytate and improves the digestibility of protein and AA. This study evaluated the impact of increasing a Butiauxella sp. phytase dose level to 2000 FTU/kg on N excretion based on meta-analysis of data from 10 in vivo trials. These studies were carried out at Universities or Research Institutes globally in the last 10 years. In these studies, the phytase was added in a range from 250 to 2000 FTU/kg to a negative control diet that was deficient in P and calcium, but without reduction in dietary CP and without other enzymes. In each study, a positive control (PC) diet formulated to meet the nutrient requirements of broilers was used as a reference. Total tract excreta samples were collected in these studies to evaluate N excretion at 21 days of age (after 4 days collection). Diets were mainly based on corn and soybean meal or wheat and soybean meal with analysed CP level in the range of 19.6 to 23.0% (mean value of 21.5%), in mash or pelleted form and fed ad lib. In total, 513 data points were used in the analysis. Dose response was analysed for linear, quadratic or exponential curve using JMP 14. A significant linear (P < 0.001) response was observed on dietary digestible CP and total CP excretion. At phytase dose level of 1000 and 2000 FTU/kg, CP excretion decreased by 6.3 and 12.6 g/kg diet, compared to the nutritional adequate PC. It was estimated that 274 million MT broiler feed was produced in 2018. Based on this analysis, supplementation of the Butiauxella sp. phytase at dose level of 1000 and 2000 FTU/kg could contribute to a great reduction in N in the manure (276,000 and 552,000 MT reduction in N excretion, respectively). In addition, these studies were carried out without application of CP and digestible AA matrix values, when applying these matrices, the N excretion could be further reduced
PHYSICOCHEMICAL AND MICROBIAL PROFILING OF POULTRY LITTER OVER TIME POST-HARVEST

M. Hussein\*(1), F.Khattak(2), L.Vervelde(3), S.Athanasiadou(4), J.Houdijk(4)

(1) SRUC - Scotland's Rural College and The Royal (Dick) School of Veterinary Studies, Edinburgh, United Kingdom

(2) Monogastric Science Research Centre, SRUC, Edinburgh, United Kingdom

(3) The Roslin Institute and R(D)SVS, University of Edinburgh, Edinburgh, United Kingdom

(4) Monogastric Science Research Centre, SRUC, Roslin Institute Building, Easter Bush, Edinburgh, United Kingdom

\* Corresponding author: Marwa.Hussein@sruc.ac.uk

Whilst experimental exposure to used litter often aims to induce reduced performance arising from anticipated litter-derived challenges (1,2), performance may be improved instead (3,4). Such variation may be associated with differences in litter quality between studies, e.g. arising from variation in pathogen load and duration between litter collection and usage. Here we investigated changes over time in microbial profiling and quality of litter, derived from a 35d broiler trial. Litter was kept at room temperature at trial end (d0), 200 g samples were collected weekly in sterile plastic bags at d0, d7, d14, d21 and d28, and stored at -80 °C pending analysis of pH, moisture and microbial profiling. The latter included quantification of 16S ribosomal DNA for total bacterial counts, 18S ribosomal DNA for total fungal counts and PCR screens for selected pathogens. DNA extracted from litter-derived bacterial pellets (5) using the DNeasy® PowerSoil® kit, was used to quantify total bacteria and fungi, Salmonella spp., avian pathogenic E. coli O1:K1:H7 (APEC) and Clostridium perfringens through qPCR, with outcomes converted into log gene copy number per g litter (cpg). Analysis of variance showed that pH gradually reduced from 8.42 at d0 to 8.09 at d28 (s.e.d. 0.066; P=0.002). Moisture levels were stable between d0 and d14 at ~33.7%, and then gradually reduced to 12.5% at d28 (s.e.d. 2.28%; P<0.001). Total bacteria increased from 8.06 log cpg at d0 to 8.25 log cpg at d7, then gradually reduced to 7.71 log cpg by d21 and then increased to 8.23 log cpg at d28 (s.e.d. 7.44 log cpg; P<0.001). Total fungi gradually reduced from 6.85 log cpg at d0 to 6.30 log cpg at d28 (s.e.d. 6.49 log cpg; P=0.013). PCR results revealed that this litter was negative for Salmonella spp and Clostridium perfringens. However, APEC gradually reduced from 7.00 log cpg at d0 to 6.30 log cpg at d28 (s.e.d. 5.67 log cpg; P<0.001). Thus, microbial profiles and quality of litter may vary over time post-harvest. Future work employing reused litter methods may benefit from detailed litter characterisation to optimise study design, to define what constitutes a sufficient pathogenic load, and to assist explaining subsequent outcomes on broiler performance and gut health.

1. Kidd et al 2003. J App Poult Res 12, 115-123

2. O'Reilly et al 2016. Proteome Sci 15, 10

3. Kennard et al 2017. Poult Sci 30, 47-54

4. Yamak et al 2015. Kafkas Univ Vet Fak Derg 22, 85-91

5. Lu et al 2003. App Env Microbiol 69, 901-908
ROBOTICS AND BIG DATA FOR PRECISION
FARMING
PRECISION NUTRITION STRATEGIES TO IMPROVE GROWTH PERFORMANCE AND ECONOMIC PERFORMANCE OF BROILER CHICKENS.

S. Van kuijk\*(1), F. Sanchez(2), Y. Han(1)

(1) Trouw Nutrition, R&D, Amersfoort, Netherlands

(2) Trouw Nutrition, Amersfoort, Netherlands

* Corresponding author: sandra.van.kuijk@trouwnutrition.com

Near infrared reflectance spectroscopy (NIRS) is a rapid technique to evaluate the nutrient profile of feed ingredients. A more advanced precision nutrition system such as NutriOpt has incorporated NIRS, extensive nutrient databases and broiler modelling programs to improve animal and economic performance. The objective of the study was to compare the various precision feeding solutions to optimize growth and economic performance of broiler chickens.

In total 1350 male Ross 308 broiler chickens were used. Five treatments were applied, with 18 replicate pens of 15 birds each per treatment. In treatment 1 (T1) birds received a standard European diet. The raw ingredients used in T1 were analyzed via NIRS. Diets optimizations were done using NIRS results only (T2), NIRS + Trouw Nutrition nutritional database (TNB) (T3), NIRS + TNB + NutriOpt Broiler model for margin (T4) and NIRS + TNB + NutriOpt Broiler model for feed conversion ratio (FCR) (T5). All diets were fed in 3 phases, a starter (D0-14), grower (D14-28) and finisher (D28-42). The growth performance was measured per pen at the start and end of each feeding phase. On D42 two birds per pen were sampled for carcass measurements. Economic performance was calculated based on the growth performance results. All results were statistically analyzed via ANOVA (SAS).

During the entire study, T1 and T2 showed the lowest body weight. Until D28, T5 (1.58 kg) outperformed (P<0.05) the other treatments, followed by T4 (1.51 kg) which was heavier (P<0.05) than T1 (1.23 kg), T2 (1.18 kg), and T3 (1.41 kg). The FCR was the lowest (P<0.05) in T4 and T5 in the starter phase, and in T5 in the grower phase. An unexpected heat wave during the finisher phase caused that the overall FCR was highest for T4, which was similar to T5 and T1, but higher than T2 and T3.

The margin calculations based on the overall growth performance were not significantly different. Until day 28, before the heat wave, the margin of the treatments were in the following order: T1&T2<T3<T4<T5 (0.68, 0.66, 0.80, 0.88, and 0.92 euro/bird, respectively). There was no effect of the treatments on carcass yield, thigh yield or wing yield. The breast meat yield was significantly highest in T5 (23.7%) and lowest in T2 (21.0%).

In conclusion, using NutriOpt precision feeding strategy comprising of NIRS + global database + broiler model results in an optimal growth and economic performance compared to the traditional formulation approach.
SUPPORTING NORMAL FUNCTION OF THE GASTRO-INTESTINAL TRACT WITH EFFECTIVE PROBIOTICS

C. Bostvironnois(1), J.Bodin*(2)

* Corresponding author: frcbos@chr-hansen.com

It is well-known and thoroughly documented that effective probiotics support many of the normal functions of the gastrointestinal tract, specifically, the digestive, absorptive, barrier and immune functions. For decades, those investigating the efficacy of probiotics in poultry have reported improvements in variables of economic importance, such as feed conversion, average daily gain, morbidity, and mortality. It is our hypothesis that these types of differences measured in well-controlled, challenge-type studies or in field observation trials are due to a greater percentage of the birds in the probiotic-fed group simply being normal. That is to say, as the percentage of normal, healthy birds increases in one group versus another, then all variables of economic importance should likewise improve. Concomitantly, variation among the birds in the probiotics-fed group should decrease. To test this hypothesis, we worked with a major global broiler integrator and fed 1.8M Ross birds from 72 flocks from the same complex, placed as hatched (males and females mixed); half receiving a daily feeding of GalliPro Fit (1.6x10^6 cfu/g of feed) and other half only on the standard diets. Average age at market was 35.7 and 35.2 days respectively. For feed conversion, livability, and condemnation, the distribution of observations from the probiotics-fed group compared to the control group was shifted in an economically-positive direction and variation was reduced. As a consequence, average performance for each variable was significantly improved (FCR: 1.617 ± 0.074 vs 1.651 ± 0.106; livability: 96.12% ± 1.70% vs 95.73 ± 2.33%; and condemnation: 0.64% ± 0.39% vs 0.86% ± 0.64%; GalliPro Fit-fed vs Control, respectively). Daily feeding of an effective probiotic enables commercial broilers to digest and absorb nutrients in a normal manner, to maintain a balanced microbiome, and to achieve as much of their genetic potential as their environment allows.

<footnote>(1)Chr Hansen, Arpajon, France</footnote>

<footnote>(2) CHR HANSEN, St germain les arpajon, France</footnote>
DEVELOPMENT OF AUTOMATED MEASURES OF INDICATORS OF WELL-BEING AND HEALTH OF POULTRY: SPECIAL CASE OF AVIAN INFECTIOUS BRONCHITIS

M. Riou*(1), N.Pajusco(2), A.Travel(3), M.Ducatez(4), C.Rossignol(5), S.Breton(1), A.Pleau(1), L.Warin(3), C.Souchet(3), L.Mérat(1), A.Jbenyeni(4), J.Guérin(4), L.Simon(2), B.Brouard(2), P.Creach(3)

(1) INRAE, Nouzilly, France

(2) Laboratoire d'Acoustique de l'Université du Mans, Le Mans, France

(3) ITAVI, Nouzilly, France

(4) IHAP, Université de Toulouse, INRAE, ENVT, Toulouse, France

(5) INRAE, Université de Tours, Nouzilly, French Southern and Antarctic Lands

* Corresponding author: mickael.riou@inra.fr

Guaranteeing consumers a production process that respects animal welfare is one of the civil societal demands, especially for fast-growing poultry production (1). EBENE® method, developed by ITAVI, coupled with the new image and sound technologies must allow for finer and additional analysis than those carried out by man, and even better to manage health of poultry. From the video-surveillance methods/tools are proposed to measure general variations in the activity of chickens but none of them can be used to measure the animal welfare principles proposed by the OIE. One objective of the project is to demonstrate the feasibility to associate group-wide sound measurements signing the state of well-being and health of poultry in the course of a respiratory pathologies. This project (E-broiler-track) received financial support from French ministry of agriculture (CAS DAR). The project will thus lead to technical specifications that will serve as a basis for the development of an infectious diseases control tool for poultry breeders. First part of this study was to collect sound data after infectious inducted disease under experimental controlled conditions. Different protocols have evaluated welfare with EBENE® method and clinical symptoms were measured during avian infectious bronchitis (IBV). After infection, all infected chickens (30/30) for each chicken line (EOPS PA12 and ROSS 308) developed respiratory clinical signs. 100 percent of tracheal swabs were positive IBV at D4 post-infection (p.i.). Tracheal lesions of IBV (7/7 chicken after necropsy at D7 p.i) confirmed the IBV infection, as compared to the control group. Chicken rales, coughs and sneezes have therefore been characterized and analysed using an algorithm developed here. The 2/3 of the registered sound were coughs and sneezes and 1/3 were rails in infected chickens. These initial results made possible to better characterize the pathophysiology of IBV from zootechnical, behavioural, acoustic and histological points of view. The acoustic approach is innovative and has made possible the development of new algorithms and sound analysis to differentiate healthy from diseased chickens. Those results gave us relevant data for early predictive information of a potential infection. In short, this approach will aim to develop a useful tool for poultry farmers to monitor their farms.

(1) Dawkins MS. Evolution and Animal Welfare. The Quarterly Review of Biology. 1998;73(3):305 28.
ID: 411

MEASURING CARBON DIOXIDE CONCENTRATIONS IN BROILER HOUSES

C. Pauline*(1), P.Robin(2), G.Amand(1), A.Keita(3), C.Nicolas(4), E.Pigache(5), J.Prigent(6), M.Lecomte(2), M.Tavares(3), P.Galliot(1), N.Rousset(1)

(1)ITAVI, Ploufragan, France

(2)INRA, Rennes, France

(3)ANSES, Ploufragan, France

(4)Chambre d'agriculture de Bretagne, Carhaix, France

(5)Chambre d'agriculture des Pays de la Loire, Angers, France

(6)Avipole Formation, Ploufragan, France

European directive 2007/43/CE establishing standards for the protection of broilers, sets a limit of 3000 ppm of carbon dioxide (CO2) concentration not to be exceeded at animal's level, over the entire duration of the flock. Since then, CO2 concentration sensors are developing in French poultry buildings. The purpose of this article is to provide methodological advices for continuous measurement of CO2 concentrations in a broiler house, by looking at CO2 commercial sensors' technical specifications and their optimal position in barn. This study, part of a project named GestCO2, received financial support from the French Agriculture Ministry (CAS DAR). Various CO2 sensors were compared in a commercial barn. Analysis of accuracy, response time and drift in time of each sensors showed that two sensors over five were not suitable to be used for CO2 monitoring and/or control in poultry houses. The position of the CO2 sensor at 80 cm +/- 20 cm of height, between pipets and feeders appeared to be the best compromise to measure, not only a representative CO2 concentration at animal's level but also of the whole house. However, at the end of the flock, this height can underestimate CO2 concentrations at animal's level in case of high CO2 productions by animals and litter. In the observed commercial barns, the horizontal heterogeneity of CO2 was higher at the beginning of the flock than at the end. These results suggest using more than one CO2 sensors for continuous measurements in poultry barns to characterize this heterogeneity. According to the sensors's tests, first level investment should be in high-performance sensor and in its maintenance than purchasing an additional sensor.

<footnote>* Corresponding author: creach@itavi.asso.fr</footnote>
FROM MEASURING AVERAGE BODY WEIGHT OF THE FLOCK TO PRECISION FEEDING IN BROILERS: A MODELLING APPROACH TO ADJUST DAILY FEED COMPOSITION

B. Méda*(1), E. Pampouille(2), M. Talineau(3), L. Dusart(2), A. Narcy(3), Y. Guyot(2)

(1) INRA UMR Biologie des Oiseaux et Aviculture, Nouzilly, France

(2) ITAVI, Nouzilly, France

(3) INRAE, Nouzilly, France

(9) University of New England, Armidale, Australia

* Corresponding author: bertrand.meda@inra.fr

Precision feeding in broiler production could reduce feed cost and nitrogen and phosphorus excretion. However, in order to adjust daily feed composition, daily requirements of birds according to their actual growth performance must be evaluated. In commercial farms, body weight (BW) is now frequently measured with automatic weighing scales. We propose here to use the data provided by these devices to 1) forecast BW gain to come, 2) estimate associated metabolizable energy (ME), digestible lysine (dLys) and available phosphorus (avP) requirements, and 3) recompose a full diet using two types of pellets with different nutritional characteristics. Firstly, BW data collected during the previous days are used to fit a quadratic function (BW=f(age)). Using this function, BW(d) and BW(d+1) are predicted, and "most probable" BWG for the next day (BWG(d+1)) is then calculated as BW(d+1) – BW(d). This first model was validated by comparing BW and BWG predictions (Y) to measured data (X, n = 814). BW is very well predicted (Y = 1.04 X – 0.03, R² > 99%), while BWG prediction quality is lower but still acceptable (Y = 0.76 X + 0.02, R² = 56%). Secondly, lipid and protein depositions according to actual BW(d) and predicted BWG(d+1) are estimated with allometric relationships developed using literature data. Requirements for ME and dLys are then estimated from these daily depositions. For avP, we considered an equation from the literature, directly using BW(d) and BWG(d+1) values. The requirements sub-model was validated by comparing predictions (Y) to measurements of ME, dLys and avP daily intake (X, n = 325). ME predictions are very good (Y = 1.04 X, R² = 96%) while the predictive quality for dLys and avP is slightly lower but still acceptable (Y = 0.93 X + 139, R² = 75% and Y = 0.99 X + 31, R² = 71% respectively). Thirdly, 1001 blend of A/B pellets are computed (increase of %A in the blend from 0 to 100% with a step of 0.1%), with A being poorer in ME and richer in dLys and avP than B, respectively. For each blend, assuming that feed intake is regulated on an energy basis, daily feed intake is estimated using ME requirement and blend content. dLys intake is then calculated and compared to dLys requirement. The best blend is the first one where dLys intake is above daily requirement. In practice, this blend could be easily prepared and distributed with a commercial weighing/mixing hopper.
# BIRD-E: A NEW DEVICE TO MEASURE THE FEED INTAKE, FEEDING BEHAVIOR AND GROWTH IN POULTRY

E. Guettier\*(1), S. Mignon-grasteau(1), I. Bouvarel(2), S. Urvoix(1), B. Quentin(1), A. Mika(2), P. Servant(1)

(1)INRA, Nouzilly, France

(2)ITAVI, Nouzilly, France

\* Corresponding author: Elodie.Guettier@inra.fr

In poultry production, feed represents more than 60% of the total production cost. So, it's important to find out strategies to improve feed efficiency through better nutrition and appropriate genetic selection. Apart from the economic impact, a better feed efficiency is also associated to a lower environmental impact.

Calculation of feed efficiency implies to record feed intake and growth. Until recently, to obtain these individual measures, animals had to be reared in cage, not allowing them to express their natural feeding and social behaviors. Electronic feeders are thus essential tools to record individual intake of animals raised in group and on floor, i.e. in conditions comparable to usual rearing conditions, especially for organic or label rouge systems. Several devices have been developed, but none include all the required characteristics, i.e. feed intake and body weight measures, from hatch to slaughter, without a corridor for the access to feed. INRAE, together with ITAVI and SYSAAF thus designed BIRD-e (Bird Individual Ration Dispenser-electronic), a feed station meeting all these needs.

BIRD-e is a circular feeder with 8 free accesses to the feed. It can feed 100 animals as soon as they hatch. One access is composed of a tray and a feed tube ending on a manger. Both are fixed on scales, giving respectively the weight of the animal and the feed weight. The presence of an animal is detected thanks to a microchip recognized by an antenna attached above each access. All data obtained with scales and antennas are synchronized and transmitted to an internal computer.

We used this feed station to test the adaptation of two genotypes of chickens (slow-growing chickens and rapid growing chickens) to sustainable diets. Apart from performances showing that animals were able to adapt to alternative diets without major effects on performances (detailed results on performances are presented in Berger et al. abstract), daily results showed that in some cases, transitions between starter, grower of finisher diets should be moved forward, as performances start to decrease several days before the transition.

As our new tool also give us access to information such as number, length or intensity of meals per day and intervals between meals, we can finely study transition phases and thus detect whether some behavioral profiles of birds are connected to differences in adaptation to transitions, even with similar final performances.

ITAVI: Institut Technique de l'Aviculture

SYSAAF: Syndicat des Sélectionneurs Avicoles et Aquacoles Français
TOWARDS LONGER CARRIERS IN LAYERS
HOW DO HOUSING STRUCTURES IN FREE-RANGE PRODUCTION AFFECT LAYING HEN WELFARE?

V. Sandilands*(1), L.Baker(2)

(1) SRUC, Penicuik, United Kingdom

(2) SRUC, Ayr, United Kingdom

* Corresponding author: vicky.sandilands@sruc.ac.uk

Free-range laying hens in the EU are housed with either single-tier ('flat deck', FD) or multi-tier (MT) housing structures in the shed. MT allows a greater number of hens per square meter of shed space, because each tier adds to the useable space. As a result, their use is growing in popularity, but with what welfare consequences? A total of 42 commercial free-range laying hen flocks were visited once near end-of-lay (FD: mean age 73 weeks, median age 72 weeks; MT mean age 75 weeks, median age 74 weeks) in the UK. Production data were collected from farm records, and welfare measures (e.g. feather condition, as an indicator of feather pecking, using a 0-5 scoring method of Savory and Mann 1999; keel bone fracture (KBF) and deviation (KBD), using a binary system devised by Casey-Trott et al. 2015; and foot condition, using a 0-2 scoring method from Welfare Quality 2009) were taken from up to 5 hens in 6 different zones of the shed and range (zones 1-4 inside the shed; zones 5-6 on the range, where zone 5 was within 5 m of the popholes, zone 6 was 5-10 m from the shed. Data were transformed where necessary for normality, and analysed by linear mixed models or generalised linearmixed models in Genstat. Mean values shown are original means. Average flock size was larger with MT (n=17 flocks, 14,671 hens) than FD (n=25 flocks, 8,896 hens). Mortality was not significantly different due to high variation (P=0.189, FD 7.5%, MT 4.8%). Mean feather condition was low (i.e. good) with both structures and did not differ (P=0.533, MT 0.90, FD 0.74) but was significantly lower in zone6 compared to any zone inside the shed (P
BETAINE AND ITS EFFECT ON BONE STRENGTH.

M. Maidin\*(1), H.Mccormack(2), P.Wilson(2), S.Caughey(2), N.Whenham(3), I.Dunn(2)

(1)Roslin Institute, Edinburgh, Uzbekistan

(2) Roslin Institute, Midlothian, United Kingdom

(3)AB Vista, Marlborough, United Kingdom

\* Corresponding author: maisarah.maidin@roslin.ed.ac.uk

Introduction: Laying hens can suffer from osteoporosis (OP). Genetics plays a role in susceptibility to OP and bone fracture1,2. Cystathionine Beta Synthase (CBS) was identified as a strong candidate gene at a QTL for bone strength (BS)3. Poor BS was associated with high homocysteine (HC), the substrate for CBS. HC can be methylated into methionine with betaine (BT), a common feed additive4. Phytase (PY) breaks down phytate from grains to increase the availability of phosphorus, an important factor in bone quality5. This study aims to evaluate the effects of BT and superdosing PY on bone quality, plasma HC and egg production.

Methods and materials: 1920 LSL-lite laying hens were reared following the Lohmann management guide, split into 4 treatment groups with 24 replicates. A 2x2 design, 1000mg/kg BT was provided both alone and with PY. The control was 300 PY units (FTU), and the superdosing level was 1000FTU. Plasma and bone (keel, humerus and tibia) were collected at 45 and 70 weeks of age (Wk) from 400 birds each time. Measurements of plasma HC level and bone radiodensity were taken. Humerus and tibia were subjected to 3-point bending and maximum load was recorded. Statistical analysis was carried out using linear mixed models with the main effects BT, PY and age as fixed effects and the replicate as a random effect.

Results: At 45Wk, HC levels was lower (p=0.0440) in birds fed BT (13.8±0.470µM/L) and those not fed BT (14.7±0.441µM/L); no difference at 70Wk. At 45Wk, tibia BS (p=0.0250) was higher in birds fed BT (288±3.25N) than in birds not fed BT (280±3.92N). At 70Wk, tibia BS (p=0.0360) was higher birds fed BT (281±3.82N) than in birds not fed BT (274±4.24N). There was no difference in tibia density (D) at 45Wk, but at 70Wk tibia D (p=0.0160) was higher in birds fed BT (2.04±0.0127) than in birds not fed BT (2.00±0.0127). There was no effect of PY. No difference was seen with keel and humerus. There was no effect of BT or PY on bird performance or on egg quality parameters.

Conclusion: The addition of dietary BT successfully reduced plasma HC level and improved tibia BS without compromising production. BT could reduce the effect of OP and improve laying hens' welfare. Ongoing investigation includes tibia histology. Future studies with different BT concentrations and relevant factors may be valuable.

References: 1 Bishop SC et al BPS 2000. 2 Fleming RH et al BPS 2006. 3 Dunn IC et al AG 2007. 4 Simon J WPSJ 1999. 5 Selle PH and V Ravindran AFST 2007.
EFFECT OF HERBAL CALCITRIOL - IN LAYING HENS ON PERFORMANCE AND EGG QUALITY IN THE LATE LAYING PERIOD

M. Devlikamov*(1), C.Wild(2), A.Pastor(3), K.Damme(4)

(1)Phytobiotics Futterzusatzstofe GmbH, Eltville, Germany

(2)LfL Kitzingen, Kitzingen, Germany

(3)Phytobiotics Futterzusatzstoffe GmbH, Eltville, Germany

(4)LfL Kitzingen, Eltville, Germany

* Corresponding author: m.devlikamov@phytobiotics.com

Decreased laying performance and lower egg quality of high-performing laying hens are often reducing egg producers profits. One of the reasons for this issue is the changing metabolization capacity of vitamin D3 in the birds, as a result of aging, physiological changes and production disorders. The objective of this study was to evaluate the effect of an additonal herbal active vitamin D3 on performance parameters and egg quality in the late production period in laying hens.

73-week-old Novogen Brown (NB) laying hens were randomly divided into two groups of 225 birds per group (with 3 replicates per group) for a trial duration of 8 weeks. The birds were kept in floor pens and had ad libitum access to water and feed. Both groups received a wheat and corn-based diet. 450mg/kg of herbal calcitriol product was added to the treatment group on top of regular vitamin D3 (3000 IU D3/kg in both groups), whereas the control group received no herbal-calcitriol product. The egg count and animal losses were recorded daily. Sorting and egg weight recording was carried out once a week. Feed consumption by back weighing in a 28-day rhythm. The egg quality (breaking strength, egg weight, haugh units etc.) was determined at the start, after 28 days and after 56 days on the basis of a sample of 50 eggs per compartment.

In the study, plant-based calcitriol numerically improved laying performance (+4.8%), total egg mass (4.8%) and reduced feed conversion rate (-3.3%). The herbal calcitriol product increased the albumen consistency significantly improved the eggshell breaking strength by 4.08 N (p ≤ 0,05 +/- 0,999). No significant effects were seen on egg weight and mortality. Furthermore, herbal calcitriol product showed no effect on number of cracked or dirty eggs.

In conclusion, the results of the study indicated that the herbal-calcitriol product tended to improve laying performance in older laying hens. Moreover, the product had a positive influence on the persistence of eggshell stability. A supplementation of the active form of vitamin D seems to be an efficient supplement to maintain performance in aged laying hens.
THE EFFECT OF GENOTYPE AND AGE ON LAYING PATTERN AND EGG WEIGHT OF LAYING HENS

M. Ketta*(1), E.Tůmová(2), D.Chodová(2)

(1)Czech University of Life Sciences Prague, Praha 6, Czech Republic

(2)Czech University of Life Sciences Prague, Praha, Czech Republic

* Corresponding author: kettam@af.czu.cz

The study evaluated laying pattern, egg weight and oviposition time of seven different laying hen genotypes at the beginning and end of laying cycle. In order to fulfil the study purpose, an experiment was conducted on Moravia BSL, Bovans Sperwer, ISA Sussex, ISA Dual, Moravia Barred, Dekalb White and Bovans Brown. The same birds were kept at the beginning (20-26 weeks of age) and at the end of laying cycle (64-70 weeks of age). Total number of 105 laying hens were individually housed in cages (550 cm2 per bird). Egg production, oviposition time and egg weight data were recorded daily. The mean rate of lay, mean sequence length and the mean internal cycle length were then calculated. The data were analysed using ANOVA two-way analysis of variance PROC GLM in SAS. The study results indicated an interaction between hen genotype and age for the rate of lay (P
HOW TO FEED NON-BEAK TRIMMED LAYING HENS?

L. Star*(1), A.Van de ven(2), D.Van vilsteren(3), W.Ten brinke(4), A.Hoogendoorn(5), A.Dijkslag(6), T.Lemmens(7), S.Lefebure(8)

(1)Schothorst Feed Research, Lelystad, Netherlands

(2)ABZ Diervoeding, Nijkerk, Netherlands

(3)AgruniekRijnvallei, Wageningen, Netherlands

(4)C.A.V. Den Ham, Den Ham, Netherlands

(5)E.J. Bos Mengvoeders, Ederveen, Netherlands

(6)ForFarmers N.V., Lochem, Netherlands

(7)Vitelia Voeders B.V., Oirlo, Netherlands

(8)Voergroep Zuid, Deurne, Netherlands

* Corresponding author: lstar@schothorst.nl

Since 2018, it is not allowed to beak trim laying hens in the Netherlands. It was expected that an intact beak would result in more feed selection and feather damage. The aim was to test which feed structure would be beneficial for performance and feather condition in non-beak trimmed (NBT) Dekalb White laying hens. The experiment was carried out as a randomised block design with 6 dietary treatments and 2 beak treatments (NBT vs. beak trimmed by laser (BT)). Diets differed in feed structure: coarse mash, fine mash, crumble, pellet, and pellet + wheat (similar in ingredient and nutrient levels; 2800 kcal/kg, 6.8 g dLYS/kg). A sixth dietary treatment was a coarse mash with an energy level of 2600 kcal/kg and dLYS level of 6.3 g/kg. These diets were fed to either BT or NBT birds. Each Diet x Beak treatment was tested with 3 aviary pens (= replicates) with 330 laying hens each. Production performance (feed intake, laying rate, egg weight, egg mass, feed conversion ratio (FRC), mortality rate) was measured from 25-72 weeks of age. Feather condition (5 birds per pen; scale 0-5; 0 = intact feathers, 5 = completely denuded) was determined every 8 weeks starting from 24 weeks of age onwards. Experimental data were analysed by ANOVA with Diet, Beak and interaction as factors.

Significant interactions were found for feed intake (P

To conclude, non-beak trimmed birds should be fed a fine mash diet to have best production performance and feather condition.
ID: 733

SKELETAL QUALITY IS RELATED TO KEEL BONE DAMAGE, BUT IS NOT EXPLAINED BY VARIATION IN EGG PRODUCTION

I. Dunn*(1), H.Mccormack(1), R.Fleming(1), P.Wilson(1), M.Maidin(1), J.Liang(1), B.Andersson(2), M.Schmutz(2)

(1)The Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom

(2)Lohmann Tierzucht, Cuxhaven, Germany

* Corresponding author: ian.dunn@roslin.ed.ac.uk

The physiological adaptations for egg laying make hens susceptible to osteoporosis. There is also a welfare paradox; cage free production is associated with better bone quality but also with greater incidence of bone damage, often the keel. The 'Long Life Layer' has great benefits for sustainability, but long production could increase the risk, it is often stated that poor bone quality is due to selection for increased egg production. However, we do not believe the evidence for a negative relationship between the numbers of eggs laid and bone quality is strong. In this study, we examine the hypothesis that there is negative genetic correlation between egg production and bone quality and demonstrate that keel bone damage is related to general skeletal quality.

Heritability and genetic correlations between egg production and bone quality in a White Leghorn (WL, 53 weeks of age n=956) and a Rhode Island Red (RIR, 68 weeks of age, n=973) pure line were calculated using ASREML. Early egg numbers, related to age at first egg, and the remaining periods (egg number) were analysed separately. Tibia and humerus breaking strength and the radiographic density of the tibia, humerus and keel bone were measured. In the WL the keel bone was given a numerical fracture grade (0-5), with higher graded keel bones having more damage.

Heritability estimates for bone quality traits are between 0.19±0.07 and 0.59±0.09. Heritability of keel bone density was near zero. Genetic correlations between egg number and bone quality were not significant. In the WL there was a negative genetic correlation between bone quality and early egg number (-0.50±0.11). When bone quality in grade 0 and grade 5 keels were compared, tibia breaking strain was 217.4±3.3 and 181.2±14.6 (p < 0.001) respectively.

There was no evidence for a relationship between egg production and bone quality. However, there was an effect of age at first egg (WL). Longer laying periods may not affect bone quality, but the onset puberty needs to be considered. The confounding effects of damage, as fracture callus is radio dense, may explain the lack of heritability for keel bone density. Results also show an inverse relationship between keel bone grade and tibia and humerus breaking force - higher graded fractures have lower breaking force. This demonstrates that keel bone grade is related to skeletal health and that bone quality overall can be improved by genetic selection.

ERANET grant BB/M028291/1; CA15224 Keel Bone Damage
ID: 781

# IS THERE A NEED TO REDUCE THE ENERGY AND/ OR PROTEIN LEVEL IN THE FEED OF AGED LAYING HENS?

S. Teerlynck\*(1), T. Van Limbergen, I. Kempen, E. Delezie, N. Everaert

(1)ILVO, Melle, Belgium

\* Corresponding author: sarah.teerlynck@ilvo.vlaanderen.be

Very little information is available on the nutritional requirements of aged laying hens (>70 wks). However, considering the tendency towards extended production cycles, gaining insight in these demands is of utmost importance. Feeding laying hens to meet their needs is essential to fully exploit the genetic potential on production and simultaneously maintaining good bird health. It can be assumed that the energy and protein demand of aged laying hens decreases. Nevertheless specific guidelines are lacking.

An experiment was conducted to determine whether there is a need to reduce the energy and/ or protein level in the feed of aged laying hens. A total of 288 ISA Brown laying hens (74-91 wks) were distributed over 4 dietary treatments with 8 replications and 9 birds per experimental unit (enriched cage). The hens of the control group (T1) were fed a standard laying hen diet containing 2759 kcal/kg ME and 15.5% CP. In dietary treatment 2 the CP-level and limiting amino acid levels (meth, cyst, lys, and thr) were reduced by 10% (ME 2759 kcal/kg; CP 13.95%). Treatment 3 contained a reduced energy level (1%) based on a reduction of starch (ME 2601 kcal/kg; CP 15.5%). Whereas for dietary treatment 4 both the energy and protein level (+limiting AA's) were reduced (ME 2613 kcal/kg; CP 13.95%). All four diets were corn- soy based with a stable 5.6% crude fat. Heterogeneity, common in older flocks, results in large within-groups variation (e.g. body weight). Therefore 32 ISA Brown laying hens were individually monitored from 97 to 101 weeks of age as well. Performance traits, egg production and egg quality (mean egg weight, dynamic stiffness, breaking strength, Haugh Units, yolk color and shell thickness) were measured for each 28-day period. Liver health associated parameters (lactate dehydrogenase, aspartate transaminase, liver weight, abdominal fat content, liver haemorrhagic and color scores) and plumage condition were determined at 74, 87 and 91 weeks (n=8/ treatment) of age. Performance traits for the individually kept laying hens were measured every 7-day period.

Both energy and protein level did not significantly influence feed intake, feed conversion ratio, or other performance traits. Egg production nor egg quality were affected by dietary energy or dietary protein level. No significant differences were found for liver health associated parameters, plumage condition was no different for hens fed a reduced energy and/ or protein level. It can be concluded that the energy and protein content in the feed of aged laying hens can be reduced by 1%, and 10% respectively without compromising performance traits or egg quality. Hopefully the results of the digestibility trial will explain the absence of effect on mean egg weight, hen weight and abdominal fat content. These results are currently being analyzed.
POSITIVE IMPACT OF PREBIOTICS AND ANTIOXIDANTS ON EGG QUALITY AT THE END OF THE LAYING HEN PRODUCTION CYCLE

K. Oueslati\*(1), B.Ribeiro(1), D.Chavatte(2), C.Alleno(3), R.Bouvet(3)

(1)NUWEN by Timab Produits Industriels, Dinard, France

(2)DLJConsultant, Saint brieuc, France

(3)Zootests, Ploufragan, France

* Corresponding author: christophe.alleno@zootests.fr

Laying hens are confronted with different stressors (heat stress, parasites, mycotoxins...) which leads to a higher production of free radicals that affect layer health and egg quality. Indeed, oxidation decreases the eggs quality. CalSEAGrow® contains pectic oligosaccharides, which have a prebiotic effect and chlorogenic acid which has an antioxidant effect. The current study aimed to evaluate the efficiency of this combination on egg quality and laying performance at the end of the production period (from 69 to 79 weeks-old). Indeed, when hens get older, egg producers face a decrease of egg quality and performance.

A total of 576 laying hens were assigned to 1 out of 2 treatments as follow: T1 – Control (Basal Diet), T2 – Basal Diet + tested product (1.0kg/ton of feed). Each treatment is divided into 12 cages of 24 hens during production period. Experiment is divided into 2 periods:

- 1st period (14 days) Hens receive the same feed during this period in order to test comparability of the initial groups,

- 2nd period (70 days) is the experimental phase.

Production data (feed consumption and egg production) were collected during the entire period of the trial. Egg quality was measured at 67 and 79 weeks old through Haugh Units and vitelline membrane strength. Along with the ageing of the hens, these criteria can be deteriorated. Kirunda et al. (2001) demonstrated a positive impact of vitamin E (antioxidant effect) on vitelline membrane strength.

Data were analyzed with Anova (with fixed effect: treatment and block) on SAS software.

First period results analysis showed no difference between the two groups concerning performance and egg quality. Thus, comparability of initial groups is admitted.

Second period analysis shows significant difference for T2 group that present higher Haugh Units: $80.0 \pm 9.6$ (T2) > $77.6 \pm 10.3$ (T1), p = 0.014; and better vitelline membrane rigidity (N/mm): $8.6 \pm 1.0$ (T2) > $8.4 \pm 1.2$ (T1), p = 0.036. Although statistical tests can't assure significant difference for feed conversion ratio, it also seems better for T2 group: $2.20 \pm 0.10$ (T1); $2.18 \pm 0.10$ (T2), p = 0.463; while maintaining egg mass (g/hen/week) T1 = T2 (390 ± 0.02), p = 0.427.

This study reported that the tested product enhances laying hens egg quality and performances, notably through its antioxidant properties that contribute to maintain the oxido-redox balance, thus reducing the negative consequences generated by oxidation like degradation of egg quality.
ID: 1258

EFFECTS OF A GLOBAL ENZYME SOLUTION ON PERFORMANCE, NUTRIENT DIGESTIBILITY AND BONE METABOLISM OF EGG LAYING HENS FROM 18 TO 84 WEEKS OF AGE

M. Jlali(1), A.Preynat(1), D.Korver*(2)

Exogenous enzymes allow for dietary nutrient density reductions. The objective was to determine the efficacy of a multicarbohydrase and phytase complex (MCPC) in laying hens. The hypothesis was that MCPC added to nutrient-reduced diets would support productivity equivalent to that of the positive control (PC; no nutrient reduction). Single-comb White Leghorn hens were housed in 56 cages of 4 birds for each of 5 dietary treatments from 18 to 84 weeks of age (woa). The PC diet met the primary breeder nutrient specifications; negative control 1 (NC1) was reduced in apparent metabolizable energy (ME) and digestible amino acids (dAA) by 5%, available P (avP) and Ca reduced by 0.18% of the diet; NC2 was reduced in ME and dAA by 7%, and avP and Ca reduced by 0.18% of the diet; and each of NC1 and NC2 supplemented with 1,250 xylanase units, 860 b-glucanase units and 1,000 FTU of phytase/kg of feed (NC1+MCPC and NC2+MCPC, respectively). Daily egg production was recorded, and on a 4-week basis, hen body weight (BW), % production, egg weight, specific gravity, shell breaking strength, and feed conversion ratio (FCR; kg feed:kg eggs) were determined. Apparent ileal digestibility (AID) of crude protein and P, apparent ileal digestible energy (AIDE) and bone ash were determined at 36, 56, and 84 woa. The cage was the experimental unit; data were analyzed by ANOVA for diet and age main effects and their interaction, with differences significant at P 0.05. From 20 to 84 woa, BW, egg weight and shell quality were not influenced by diet, but egg production and average daily egg mass were reduced by NC1 (P = 0.02 and 0.03, respectively) and NC2 (P = 0.03 and 0.008, respectively) relative to PC; MCPC restored performance of each NC diet (P = 0.24 and 0.26, respectively, for egg production and daily egg mass). FCR was lower for the PC hens than either NC diet (P

<footnote>(1)Adisseo France S.A.S, Commentry, France</footnote>

<footnote>(2)University of Alberta, Edmonton, Canada</footnote>

<footnote>* Corresponding author: doug.korver@ualberta.ca</footnote>
ID: 1461

TOWARDS THE IDENTIFICATION OF THE MOLECULAR PATHWAYS UNDERLYING THE ALTERATION OF CALCIUM METABOLISM IN HENS AT THE END OF LAY

A. Gloux(1), N.Le roy(1), N.Meme(1), M.Piketty(3), D.Prié(2), G.Benzoni(4), J.Gautron(1), Y.Nys(1), A.Narcy(1), M.Duclos*(1)

(1)BOA INRAE Université de Tours, Nouzilly, France

(2)INSERM, Paris, France

(3)GH Necker Enfants Malades, Paris, France

(4)ADM, Saint noffl, France

* Corresponding author: michel.duclos@inrae.fr

The current objective of the egg industry is to increase the hen's productive carrier up to 100 weeks. However, numerous studies have reported that egg quality and hen's bone strength deteriorate with age, because of altered calcium (Ca) homeostasis. This study was designed to make an integrative description of Ca homeostasis pathways in laying hens at peak and end of lay (23 and 90 weeks old). Brown laying hens aged 20 or 87 weeks were reared in individual cages to record oviposition and fed a commercial layer's diet containing 3.5 g/kg CaCO3 (1/3 fine, 2/3 coarse particles). At 23 or 90 weeks of age, hens were euthanized at 0-1h, 9-10h or 18-19h post ovulation (PO) prior to the collection of plasma and tissue samples (6 hens per stage PO). Plasma samples served for the assay of iP and vitamin D metabolites. RNA were prepared from parathyroid gland (PG), liver, jejunum, medullary bone (MB) and kidney for a quantitative study of candidate gene expression. All data were analyzed using a linear model by robust regression. The effects of the main factors PO stage, age and the interaction were tested. In case of significant effects in the model (P < 0.05), all pairwise comparisons were performed using the least-square means method and a Tukey adjustment. Parathyroid hormone (PTH) gene expression in the PG did not vary with age, while the expression of several Ca transporters and vitamin D receptor (VDR) in the jejunum, and that of the transient receptor potential channel subfamily V member 5 (TRPV5), mediating Ca reabsorption in the kidney, decreased with age (p<0,05). This could explain the previously reported reduced Ca retention in older hens. The higher expression of the Carbonic Anhydrase 2 (CA2) gene (p<0,05) and the lower expression of collagen type I alpha 1 chain (COL1A1) gene (p<0,05) in the MB also suggested exacerbated bone resorption and impaired bone accretion in the older hens. An increased expression of Fibroblast growth Factor 23 (FGF23) was also observed in the MB. This likely triggered the decreased plasma levels of 1.25(OH)2D3 and expression of target genes under its regulation. Our data highlights the molecular mechanisms underlying the osteoporotic syndrome previously documented in aged laying hens, thus providing new perspectives for future dietary interventions.
UNDERSTANDING NUTRITION X GENETICS
INTERACTION TO OPTIMIZE FEED
EFFICIENCY
EFFECT OF LEAF MEALS AND THEIR COMPOSITE ON THE PERFORMANCE, ANTI-OXIDANT, SERUM AND HAEMATOLOGY PROPERTIES OF TWO BROILER CHICKEN BREED

A. J. oluwasola*(1), A.Ayeni(1), M.Adegbenro(1), T.Oladayo(1), O.Dick(1), E.Oyedokun(1), I.Olaseinde(1), W.Ogundare(1)

(1) The Federal University of Technology, Akure, Nigeria

* Corresponding author: joagbede@futa.edu.ng

One of the reasons for the inclusion of leaf meals (LMs) in broiler diets is to take the advantages of their bioactive components to improve the growth performance (Sugiharto et al., 2019). Thus, the present study seeks to assess the efficacy of Bamboo Leaf Meal (BLM), Avocado Leaf Meal (ALM) and their Composite (mixed at 1:1) as phytogenic growth promoters in broiler chicken diets. A basal diet was formulated to contain 21% crude protein and 3000 kcal/kg metabolizable energy and used as straight diet from day-old to 42 days. The basal diet was divided into four portions such that the first portion was without LM (control) and the remaining 3 portions were supplemented with 5g/kg of BLM, ALM and their Composite (CLM), respectively. One hundred and twenty eight (128) each of Cobb and Arbor acre were purchased and randomly distributed into the four (4) treatments arranged in 2x4 factorial (2 breeds x 4 treatments) in Completely Randomised Design. Each treatment had four (4) replicates with eight (8) birds per replicate. Leaf Meal supplementation significant (p0.05) affected. The glutathione (GSH), superoxide dismutase (SOD) and catalase were not significant (p>0.05) for supplement effect, breed effects and their interaction. However, broilers fed supplemental LM diets recorded lower SOD and catalase but higher GSH than those fed control diet. Haematological indices measured were not significantly (p>0.05) affected by the supplemental LMs except for MCV, MCH, Lymphocytes and monocytes. Although, breed effect was not significantly (p>0.05) different for all haematological indices measured, the interactive effects of breed and supplemental LM were significant (p

Keywords: Leaf Meals, Anti-oxidants, Blood, Broiler Chicken, Phytogenic.

Reference

Sugiharto, S., Yudiarti, T., Isroli, I., Widiastuti, E., Wahyuni, H.I. and Sartono, T.A. (2019), Recent advances in the incorporation of leaf meals in broiler diets. Lrrd 31(109). http://www.lrrd.org/lrrd31/7/sgu_u31109.html
ID: 321

ADAPTATION OF SLOW- AND RAPID-GROWING BROILERS TO ALTERNATIVE DIETS

B. Quentin\*(1), É.Guettier(1), S.Urvoix(1), E.Le bihan-duval(1), S.Mignon-grasteau(1)

(1)INRA, Nouzilly, France

* Corresponding author: quentin.berger@inra.fr

To refine feed efficiency selection of broilers, we developed an electronic feed station allowing a continuous record of body weight and feed intake of animals reared in group on floor. Using this station, we compared slow-growing Label Rouge (LR) and fast-growing standard (S) chickens fed either with a corn-soybean diet (CD) or with an alternative diet (AD) including DDGS, fava beans, sunflower and rapeseed meals. Within a genotype, diets were isoproteic, isoenergetic and fulfilled the need of the animals during the three phase of their growth (S birds: 2850, 2900, 2950 kcal.kg⁻¹ and 21.5, 20, 18.5 % CP; LR birds: 2750, 2850, 2900 kcal.kg⁻¹ and 20, 18, 16.5 % CP).

For each genotype, the diet effect was estimated by an ANOVA on daily data of body weight, feed intake and feed efficiency, and on carcass composition and meat quality traits at slaughter (84 and 35 d for LR and S, respectively). The potential effect of the diet on performance homogeneity was checked through an ANOVA on the coefficient of variation including the effects of the period (starter, grower, finisher), the diet and their interaction. Correlations between slaughter traits and daily data were calculated by strain and diet.

LR chickens fed with AD were 4 to 9% heavier and had a 6 to 12% lower FCR during the starter period, but no effect was observed on feed intake nor meat characteristics. For S chickens, AD had no impact on the daily BW and feed efficiency but led to a slightly less acidic (+1.7% for pH), less yellow meat (9.7% for the b* index) and leaner carcass (-14% for abdominal fat yield). Unexpectedly, groups fed with AD were more homogeneous for all traits than with CD. For example, CV was respectively 20 and 22% lower for FCR in LR and S chickens.

Correlations between daily and slaughter traits differed between genotypes and diets. For example, for the group fed with the alternative diet, cumulated FCR was correlated with breast yield in LR chickens (-0.23), but not in S chickens (-0.13). Moreover, these correlations showed that some traits recorded during the starter period could be used as precocious indicators of slaughter traits. For example, bodyweight during the starter phase was positively correlated with fat yield in LR chickens fed with AD.

These first results indicate that animals can adapt to alternative feedstuffs, even those with a rapid growth rate. A complementary study is in progress on pedigree birds from a heavy line of broilers fed with the alternative diet, in order to suggest new criteria of selection related to feed efficiency and adaptability.
CONVENTIONAL VS. SLOW-GROWING BROILER CHICKENS: APPARENT RETENTION OF NUTRIENTS, CECA DIGESTA MICROBIAL METABOLITES, JEJUNAL HISTOMORPHOLOGY, AND TIBIA ASH WITH OR WITHOUT A DIETARY MULTI-ENZYME SUPPLEMENT

M. Mohammadigheisar\*(1), V Shouldice(1), S. Torrey(1), T. Widowski(1), E. Kiarie(2)

(1) University of Guelph, Guelph, Canada

(2) University of Copenhagen, Department of Veterinary and Animal Sciences, Roskilde, Denmark

* Corresponding author: mmohamm@uoguelph.ca

Emerging market differentiation for broiler meat production in terms of conventional (C) and slow-
growing (SG) strains is necessitating comparative research on various production aspects of these
strains. We investigated the effects of a dietary multi-enzyme supplement (MES) on apparent retention
(AR) of components, ceca digesta short chain fatty acids (SCFA), jejunal histomorphology, and tibia ash
in C and SG strains. A total of 640 d-old chicks of 4 strains (C and 3 SG: F6, J10, and N14) were housed
in metabolic cages (10 birds/cage). The strains differed by time they took to reach 2.2 kg BW
corresponding to 37, 43, 47, and 50 d for C, F6, J10, and N14, respectively. An antibiotic-free corn
soybean meal-basal diet was formulated for two-phase program (starter and grower). Half of the birds
per strain received basal diet with 0.04% MES containing phytase, protease, cellulase, and β-glucanase.
Diets were allocated by strain, creating a 2x4 factorial arrangement of treatments (8 treatments and
8 replicates/treatment). Birds were allocated equal amount of feed in each phase (starter, 476 g;
grower, 1,360 g) and had free access to water. Excreta samples were collected at the last 3 days of trial
and pooled within cages. Birds were euthanized at the end (upon the consumption of allocated feed)
for cecal digesta, jejunal tissue, and left tibia samples. There was no interaction (P>0.05) between strain
and diet on all parameters with exception of ceca digesta concentration of acetic acid (P<0.01). There
was strain effect (P<0.01) on AR of nitrogen (J10 and N14 had the lowest retention) and fat (N14had the
lowest retention). Dietary MES improved the AR of dry matter (DM), ash, nitrogen (N), fat, NDF, and
gross energy (GE; P<0.05). Inclusion of MES improved the AR of DM (3.4%), ash (22.0%), N (2.7%), fat
(4.1%), NDF (6.2%), and GE (2.4%) compared to the birds fed without MES. There was strain effect on
the concentration of lactic, propionic, and iso-butyric acid (P<0.05). We did not observe any effects on
jejunal histomorphology. Tibia ash (g/kg BW) weight was higher (P<0.01) for strain J10 compared with
strain C and F6 whilst strain N14 was intermediate. The data indicated that the MES improved AR of
components independent of strain, indicating effects of feed enzymes are not influenced by genetic
background of broiler chickens. The SG strains had higher tibia ash content, indicating higher bone
mineralization compared to conventional strains.
EFFECT OF REARING CONDITIONS AND NUTRITIONAL CONCENTRATION ON GROWTH PERFORMANCE AND MEAT QUALITY OF FAST- AND INTERMEDIATE-GROWING BROILERS BREEDS

L. Lorieau\*(1), B.Cheve(1), B.Renouf(1)

(1)Euronutrition, Saint-symphorien, France

* Corresponding author: lucie.lorieau@euronutrition.fr

There is a growing consumer demand for higher poultry welfare and meat quality in Europe. Such demand may be met by using slower growing broiler breeds. Nutritional concentration and rearing practices may vary across farms or countries. Hence, the aim of this study was to evaluate the effect of nutritional concentration (Reco ; recommended vs Dec; -3% metabolizable energy and -5% crude protein and amino acids) and rearing conditions on the performance and meat quality of fast (Fast; Ross 308) or intermediate (Int; JA 957) growing broiler breeds. Rearing conditions were either optimal (Opt) or suboptimal (SOpt). Birds in SOpt conditions were housed in higher density (18 vs 15/m²), on a less absorbent litter (straw vs sawdust) and were fed diets with higher byproducts inclusion rates and without xylanase compared to their Opt counterparts.

2088 day old chicks were randomly allocated into a 2x2x2 factorial design with breed (Int or Fast), nutritional concentration (Reco or Dec) and rearing conditions (Opt or SOpt) as experimental factors. There were 6 replicates per group. Feed intake and bodyweight gain were measured at 0, 10, 21, 35 and 40 days. Footpad dermatitis, myopathies and breast meat yields were measured at 41 day for 24 birds/group. A three-way ANOVA was performed to compare groups.

Compared to their Int counterparts, Fast birds showed a higher bodyweight gain (2.3 vs 1.8 kg, p<0.001) and tended to have a lower feed conversion ratio (1.64 vs 1.67, p<0.10) when fed recommended diets and reared under optimal conditions. Hence, Fast birds appeared more sensitive to dietary or environmental interventions than Int birds. In addition, Int birds had lower breast meat yields, foodpad dermatitis and myopathies scores than Fast birds (P<0.05) irrespective of the other experimental factors. Also, footpad dermatitis scores were significantly affected by rearing conditions (P<0.05).

Overall, this study suggests that Int birds may be able to demonstrate higher welfare outcomes than Fast birds under various experimental factors, as measured by lower footpad dermatitis and myopathies scores. Fast birds have, however, a higher economic profitability through superior bodyweight gain and breast meat yield. Hence, future improvements in broiler welfare are partly conditioned by the consumer's willingness to spend more money in supporting improved rearing conditions or by purchasing slower growing birds.
THE DISCOVERY OF HUNDREDS OF NOVEL MEMBERS OF THE CHICKEN CAECAL MICROBIOTA USING
METAGENOMIC SEQUENCING

L. Glendinning*(1), R.Stewart(2), M.Pallen(3), K.Watson(2), M.Watson(2)

(1)The University of Edinburgh, Edinburgh, United Kingdom

(2)University of Edinburgh, Edinburgh, United Kingdom

(3)Quadram Institute Bioscience, Norwich, United Kingdom

* Corresponding author: laura.glendinning@roslin.ed.ac.uk

The microbes which inhabit the chicken caeca play an important role in chicken nutrition, principally by converting dietary fibre into short chain fatty acids which can be used as an energy source by the host. They also play an important role in pathogen protection and the development of the immune system. By utilising sequencing data we are able to construct microbial genomes from these communities, allowing us to gain a greater understanding of their diversity and function. In this study we extracted DNA from the caecal contents of 24 chickens, 12 of which belonged to a slower-growing breed (Ranger Classic) and 12 of which belonged to a faster-growing breed (Ross 308). Birds were fed either a vegetable-only diet or a diet containing dietary fish meal. DNA was sequenced using shotgun sequencing on an Illumina NovaSeq. Reads were assembled then binned to construct metagenome assembled genomes.

Using 1.6T of sequencing data we constructed 469 high-quality draft bacterial genomes, which included 283 novel species (5 of which were Lactobacillus) and 42 novel genera. We then mapped our genomes to metagenomic data generated by a previous study of commercial chicken farms across nine EU countries, demonstrating that our genomes were abundant in chickens from all nine of the countries studied. We also compared the abundance of our genomes and their carbohydrate-active enzymes between our samples, finding significant differences between birds from different lines and on different diets, as well as a core microbiome shared between all groups. In conclusion, we have substantially increased the amount of chicken microbiota derived genomes present in public databases through the construction of metagenome assembled genomes. Our data can be used as a reference for future studies of the structure and function of the chicken microbiota.
THE EFFECT OF A MULTI-CARBOHYDRASE ENZYME AND YEAST-DERIVED PRODUCT ON INTESTINAL MICROBIOME STRUCTURE AND GUT FUNCTION OF TURKEYS

A. Rogiewicz*(1), A.Jozefiak(2), D.Mikulski(3), J.Juśkiewicz(4), Z.Zduńczyk(4), J.Jankowski(5), D.Józefiak(6), R.Patterson(7), B.Slominski(1)

(1) Department of Animal Science, University of Manitoba, Winnipeg, Canada

(2) Department of Preclinical Sciences and Infectious Diseases, Poznan University of Life Sciences, Poznan, Poland

(3) Department of Poultry Science, University of Warmia and Mazury, Olsztyn, Poland

(4) Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland

(5) Department of Poultry Science, University of Warmia and Mazury, Olsztyn, Portugal

(6) Department of Animal Nutrition and Feed Management, University of Life Sciences, Poznan, Poland

(7) Canadian Bio-Systems, Calgary, Canada

* Corresponding author: bogdan.slominski@umanitoba.ca

The aim of this study was to determine the effect of a multi-carbohydrase enzyme (E) and its combination with an enzymatically-modified yeast cell wall (Y) on the abundance of ileal and cecal microbiota and gut function of turkeys. The experimental diets, including the control (C), C+E, and C+E+Y, were fed to 7 replicate pens of 4 birds each from 22 to 56 days of age. In comparison to C, consecutive effects of E and E+Y on gut microbiome were observed. The addition of E+Y resulted in a decrease in relative abundance of the phylum Firmicutes (from 86.9 to 61.7; P=0.010), including the family Lactobacillaceae (from 57.8 to 23.2; P=0.074), and was followed by a significant increase in the number of Actinobacteria (from 2.5 to 14.5; P=0.036), including the family Bifidobacteriaceae (from 2.2 to 15.6; P=0.003), Cyanobacteria (from 2.9 to 15.1; P=0.065), and Proteobacteria (from 1.1 to 3.0; P=0.026) in the ileal digesta. A significant decrease in the abundance of Firmicutes was not followed by the order Clostridiales (26.1 vs. 30.3) with the family Ruminococcaceae, the butyrate-producing bacteria, showing somewhat, although not significant (P>0.05), increase in their relative abundance (2.8 vs. 6.9). This coincided with an increased concentration of butyric acid in the ileal digesta (P=0.023), as well as the proportion of butyric acid within the total SCFA content (P=0.022). As opposed to the ileum, the addition of E or E+Y did not affect the abundance of Firmicutes in the ceca. Within this phylum, however, the E+Y diet increased the relative abundance of Lachnospiraceae (from 11.2 to 17.5; P=0.049) and Blautia (from 5.9 to 11.1; P=0.027). Furthermore, a trend towards increased abundance of Proteobacteria (P=0.066) and Enterobacteriales (P=0.067) was observed. The addition of E+Y increased the activities of bacterial α-arabinopyranosidase, α-arabinofuranosidase, β-xylosidase and β-cellobiosidase. The SCFA content of the cecal digesta was much higher than that of ileum, which would reflect differences in microbial activities in both compartments as well as possible increased rate of absorption of SCFA, including butyric acid, from the ileum. In this context, butyrate is the preferred energy source for the epithelial intestinal cells and has been implicated in the prevention of intestinal inflammation. Overall, it appears evident that the positive effects of enzyme and yeast supplements on bacterial communities would be more pronounced in the small intestine.
GUT HEALTH OF BROILER CHICKENS REARED WITH INSECT LARVAE AS ENVIRONMENTAL ENRICHMENT:
FOCUS ON INTESTINAL MORPHOLOGY AND MICROBIOTA MODULATION

I. Biasato*(1), I.Ferrocino(2), E.Colombino(3), S.Bellezza oddon(2), A.Imarisio(3), M.Pipan(4), M.Meneguz(2), L.Gasco(2), M.Capucchio(3), A.Schiavone(3)

(1)Department of Agricultural, Forest and Food Sciences - University of Turin, Grugliasco (to), Italy

(2)Department of Agricultural, Forest and Food Sciences, University of Turin, Grugliasco (to), Italy

(3)Department of Veterinary Sciences, University of Turin, Grugliasco (to), Italy

(4)Entomics Biosystems, Cambridge, United Kingdom

* Corresponding author: ilaria.biasato@unito.it

Introduction. Insect larvae have recently been tested as feed additive for broiler chicks [1], but their use as environmental enrichment to improve animal health and welfare represents a novel approach. This study investigated the effects of Tenebrio molitor (TM) and Hermetia illucens (HI) larvae as environmental enrichment on gut morphology and microbiota of broiler chickens.

Material and methods. A total of 180 4-day-old male broiler chickens were randomly allotted to 3 experimental treatments (6 pens/treatment, 10 birds/pen): a) control group (C, commercial feed), b) HI group (commercial feed + HI live larvae) and c) TM group (commercial feed + TM live larvae). The insect larvae were daily provided as 5% of the expected daily feed intake and feces were collected at 0, 7, 14 and 38 days. The animals (3 birds/pen) were slaughtered at 38 days of age and gut samples and cecal contents were collected. Small intestine morphology was evaluated by morphometric analysis, while fecal and cecal microbiota were assessed by 16S rRNA amplicon based sequencing. Data were analyzed by R software (P<0.05). Fecal microbiota showed an increase in α- and β-diversity measures as a function of the sampling time (P<0.05). At genus level, HI- and TM-fed broilers showed a decrease in Butyricicoccus and Ruminococcus (short chain fatty acids [SCFAs]-producing bacteria [2]), an increase in Clostridiales (common fecal bacteria) and an increase in Lactobacillus (beneficial bacteria) and Staphylococcus (potential pathogenic bacteria, P<0.05). At genus level, Clostridium, TM7, Victivallaceae, Eubacterium (the latter two being SCFAs-producing bacteria [3]) and Collinsella (modulator of virulence and pathogenicity of potential enteric pathogens [4]) were characteristic of insect larvae-fed broilers (P<0.05).

Conclusion. The utilization of HI and TM larvae as environmental enrichment caused no negative effects on gut health of broiler chickens in terms of unaffected gut morphology and partial, positive cecal microbiota modulation. The controversial results of fecal microbiota analysis may be attributed to its temporal fluctuations, as it consists of microbial mixtures originating from different gastrointestinal segments.

References. [1] Lee et al. (2018) J Vet Med Sci. 80:736-740. [2] Wang et al. (2016) Front Microbiol. 7:593. [3] Bag et al. (2017) Genome Announc. 5:e01361-17. [4] Levine et al. (2013) Appl Env Microbiol. 79:3879-3881.
UNDERSTANDING THE MICROBIOME FOR
AN IMPROVED MANAGEMENT OF
HEALTH AND WELFARE
A PRECISION GLYCAN MICROBIOME MODULATOR REDIRECTS CORE METABOLIC PATHWAYS OF THE MICROBIOME LEADING TO IMPROVED GASTROINTESTINAL FUNCTIONALITY AND GROWTH PERFORMANCE

B. Blokker\*(1), G.Schyns(2), V.Jacquier(1), M.Walsh(3), J.Geremia(4)

(1)DSM Nutritional Products, Village-neuf, France

(2)DSM Nutritional Products, Lexington, United States

(3)DSM Nutritional Products, Kaiseraugst, Switzerland

(4)Midori USA Inc., Cambridge, United States

\* Corresponding author: britt.blokker@dsm.com

The strong motivation to eliminate antibiotic growth promoters (AGPs) from animal production provides an enormous opportunity to rethink the conventional approach to animal performance, health and welfare. Most current alternatives to AGPs focus on the different microbial species present in the gut. Rather, we reconsider the microbiome as an organ whose collective metagenome is responsible for thousands of metabolic functions that strongly influence the host. Although the microbiota themselves can vary significantly between farms and pens, our approach targets core metabolic pathways and functions that are highly conserved across microbiomes.

Here we present a precision glycan microbiome modulator (M2-1) and demonstrate a controlled shift in the functional metagenome of the gut microbiome with a consequential benefit on host biomarkers and performance.

Broiler caecal microbiomes sampled from various locations, with significant underlying variation in the phylogenetic composition, were exposed to M2-1 under anaerobic ex vivo conditions. Targeted metabolomic analysis demonstrated a consistent increase in the production of short chain fatty acids via the induction of the C3 and C4 microbial pathways and a decrease in ammonia.

A second study was performed with broiler chickens over 42 days to confirm the effects in vivo. Cobb 500 broilers were fed in two treatment groups with 21 replicates per treatment and 40 birds per replicate. Control birds received a standard corn-soy diet. Treated birds received the control diet supplemented with 500 ppm M2-1. Body weight gain, feed intake and weight-corrected feed conversion (cFCR) were measured. From 10 birds per treatment; caecal microbiome samples were analysed by functional metagenomics shallow shot gun sequencing and ileum tissue samples were analysed by gene expression microarrays.

Induction of C3 and C4 microbiome pathways was confirmed in vivo. Additionally, the nitrogen utilization pathway of the microbiome was downregulated in broilers fed M2-1 compared to control. In the ileal tissue KEGG pathways cytokine-cytokine interaction and nitrogen utilization were significantly altered by feeding M2-1, indicating a host response to the metabolites produced by the microbiome. Treated birds exhibited an improvement of 6.9 points in cFCR versus control.

In conclusion, M2-1 redistributes metabolic fluxes into core metabolic pathways of the microbiome, aiding gastrointestinal functionality and improving animal performance.
A PRECISION GLYCAN MICROBIOME MODULATOR CONSISTENTLY IMPROVES BROILER GROWTH PERFORMANCE

V. Jacquier*(1), B.Blokker(2), M.Walsh(3), J.Geremia(4)

(1)DSM, Saint-Louis Cedex, France

(2)DSM Nutritional Products, Village-neuf, France

(3)DSM Nutritional Products, Kaiseraugst, Switzerland

(4)Midori USA Inc., Cambridge, United States

* Corresponding author: vincent.jacquier@dsm.com

One of the largest global trends influencing how animals are produced is a decreased reliance on AGPs. This trend is motivated by the need to control the risk of developing antibiotic microbial resistance and by an ever-increasing consumer pressure. Most current alternatives to AGPs focus on the promotion of specific bacteria within the gastro-intestinal tract, underestimating the strength of the microbiome as a whole and its thousands of metabolic functions that strongly influence the host animal.

We present a precision glycan microbiome modulator (M2-1) that has been selected from over 200 possible glycan feed ingredients. M2-1 was selected because it targets core metabolic pathways and functions that are highly conserved across microbiomes, leading to improved gastrointestinal functionality. Here, we demonstrate the selection of M2-1 for consistent broiler performance across multiple growth conditions through a meta-analysis of 15 in vivo studies involving candidate glycans. We then highlight a subset of these studies focused on M2-1.

A total of 20,820, 1-day old male broilers were used, (Ross 308, Ross 708, and Cobb), and fed either a corn soybean-meal or a wheat soybean-meal based diet, from 0 to 35 or 42 days of age. The experimental treatments were: T1, control; T2, T1 + M2-1 included at 500 ppm. All diets contained 1,000 FYT/kg Ronozyme HiPhos. Feed intake (FI) and weight gain (BWG) were measured at the end of each study. Mortality was recorded daily. Corrected feed conversion ratio (cFCR) was calculated by considering the weight of dead birds and corrected with a common body weight. The data was analysed by means of a linear mixed model with treatment group as categorical fixed variable, control group as reference level, and study ID as random intercept. Trial-specific variances were allowed by the model, which also weighted the data proportionally to the inverse of the study variance. All analyses were performed in R (Version 3.4.3). At the end of the growing period, M2-1 had significantly improved cFCR by 4.9 points on average (P < 0.001) versus the control group, with improvements between 2.7 points and 6.9 points. This was driven by a significant increase in BWG, with an average improvement of +1.9%.

In conclusion, the M2-1 microbiome modulator, selected through a strong screening platform, consistently increased performance of broilers regardless of locations and diets. This has the potential to close the gap created by the AGP removal.
A PRECISION GLYCAN MICROBIOME MODULATOR REDIRECTS CORE METABOLIC PATHWAYS OF THE MICROBIOME LEADING TO IMPROVED RESILIENCE TO ENTERIC DYSBIOSIS AND INFLAMMATION

B. Blokker*(1), E.Perez-calvo(1), G.Schyns(2), V.Jacquier(1), M.Walsh(3), J.Geremia(4)

(1)DSM Nutritional Products, Village-neuf, France

(2)DSM Nutritional Products, Lexington, United States

(3)DSM Nutritional Products, Kaiseraugst, Switzerland

(4)Midori USA Inc., Cambridge, United States

* Corresponding author: britt.blokker@dsm.com

The removal of antibiotic growth promoters (AGPs) opens the potential for health challenges, formerly supressed by AGPs, to re-emerge in the broiler industry. For example, AGPs reduced intestinal inflammation by broadly supressing the gut microbiota. Our view is that AGP removal provides an opportunity to rethink how we achieve resilience to enteric dysbiosis and inflammation. We consider the microbiome as a beneficial organ, whose collective metagenome is responsible for thousands of metabolic functions that strongly influence host animal health.

Here, we present a novel precision glycan microbiome modulator (M2-1) that improves broiler resilience to enteric dysbiosis and inflammation by stimulating protective pathways in the gut microbiome, despite natural variations in phylogenetic composition between animals. The effects of M2-1 on health were assessed by an in vivo inflammation challenge study in broilers.

Ross 308 broilers were housed in battery cages for 28 days. Unchallenged control birds were fed a standard corn-soy diet. Challenged birds received a dysbiosis component (day 14 administration of Paracox-5 at 10x commercial dose) as well as a nutritional pro-inflammatory component (16% potato protein starting on day 7). Treated birds were subjected to the challenge model supplemented with 1000 ppm of M2-1. Results were analysed using one-way ANOVA (GraphPad 5.0).

Ileal histology showed reduced villi damage in M2-1 treated birds compared to challenged birds (p<0.01), as quantified by Alcian blue staining. M2-1 birds demonstrated increased gene expression associated with ileal nutrient absorption (FABP2, MGAM and SLC34A2) versus challenged birds (p<0.05) and increased expression of the SCFA transporter SLC5A8 (p=0.02). Proinflammatory genes NOS2, IL1B and IFNG tended towards decreased expression in the M2-1 group versus challenged birds.

In the Peyer's patches the percentage cytotoxic T cells was increased in the challenge group, while less T helper cells were observed; M2-1 supplementation attenuated this. Intestinal permeability was improved in M2-1 fed birds as shown by claudin 5 expression (p=0.05). Liver histology showed improved phenotypes in the M2-1 treated group, which also exhibited a decrease in plasma hepatic acute phase Alpha-1-acid glycoprotein levels.

In conclusion, M2-1 supports gastrointestinal functionality via its effects on core metabolic pathways of the microbiome, making birds more resilient to enteric dysbiosis and inflammation.
# EVALUATION OF PROBIOTICS EFFECT ON GUT MICROBIOTA OF CHICKENS BY ANALYZING FRACTAL STRUCTURES

N. Vorobyov\*(1), I.Kochish(2), I.Nikonov(2), E.Anchikov(2)

(1) Federal State Budget Scientific Institution All-Russia Research Institute for Agricultural Microbiology RAS, St. petersburg, Russia

(2) Moscow state Academy of Veterinary Medicine and Biotechnology - MVA by K.I. Skryabin, Moscow, Russia

* Corresponding author: danzim@mail.ru

## INTRODUCTION

Gut microbial communities in chickens are self-organized to implement joint biochemical processes and extract dietary components from substrates. As a result of adaptation to birds' natural environment the gut microbiota was able to self-organize into fractal structures that enable complex biochemical transformations of plant substrates with minimal energy costs. The microbial biosystem is regularly adjusted depending on the feed entering the gut evenly distributing the enzymatic load between the microbial components. Calculating the index of the fractal structures is proposed to assess the qualitative and quantitative parameters of these processes.

## MATERIALS AND METHODS

The experiment investigated the microbial communities in the intestines of chickens with different diets and two types of probiotics. The method is based on fractal analysis of the frequency spectrum of the operational taxonomic units (OTUs) in the microbial communities obtained by molecular genetic method. The initial OTU spectrum is converted into a reduced spectrum of OTU groups with each group combining OTUs that occur at a similar frequency. A two-dimensional fractal portrait of the microbial community is constructed where each OTU group is represented by a point with coordinates that depend on this group's frequency. A fractal portrait is necessary to conduct a topological analysis of the point location and isolate fractal structures in the microbial community. The analysis uses triangles with vertices at the OTU groups points as a fractal measure. The smallest triangle heights are chosen as the fractal measure scale. Their share in the microbial community determines the index of fractal structures (IFS) which also takes into account the uniform enzymatic load distribution between OTUs. The obtained frequency-taxonomic spectra were used to calculate the index of fractal structures.

## RESULTS

An analysis demonstrated that probiotics affect the self-organization of microbial communities (IFS up to 0.7–0.9) when grain (barley, wheat) is included in the feed. In other cases the probiotic effect on the self-organization of the microbial gut communities was low (IFS about 0.06). The chicken gut microbiota had not yet adapted to individual diet components and had formed an efficiently functioning microbial biosystem for digesting a new substrate.

## CONCLUSION

The experiment with chicken diets demonstrated that self-organization level of the gut microbial communities depends on the diet.
ORGANIC AND MEDIUM CHAIN FATTY ACIDS SUPPLEMENTS FOR BROILER CHICKENS - EFFICIENT POULTRY PRODUCTION AND MEAT QUALITY BASED ON MICROBIOTA MODIFICATION

A. Dauksiene*(1), M.Ruzauskas(1), R.Gruzauskas(2), V.Lele(1), V.Starkute(1), D.Klupsaite(1), P.Zavistanaviciute(1), E.Zokaityte(1), E.Mozuriene(1), E.Bartkiene(1)

(1) Lithuanian University of Health Sciences, Kaunas, Lithuania

(2) Kaunas University of Technology, Kaunas, Lithuania

* Corresponding author: agila.dauksiene@lsmuni.lt

The ban of antibiotic growth promoters in European Union (EU) has resulted in a need for new strategies to modulate the microflora metabolism in chickens (Buclaw M., 2017). Many different feed additives were developed, but effect of them depend on many factors including management conditions, stock density, litter quality, age and gender of broiler chickens, microbial ecology of digestive tract and etc. (Hajati H. 2018). In this study, the impact of organic acid (OA) and medium chain fatty acids (MCFA) on broilers caecal microbiota, productivity, and meat quality under industrial conditions were analyzed. The comparison study was conducted on 60 000 broiler chickens allocated to two groups (fed with OA and fed with MCFA supplements). Basal diet of the above mentioned two groups was supplemented with (I) MCFA (caproic, caprylic, capric and lauric acids) at the level of 15 g/kg and (II) with OA (formic, lactic, propionic, butyric, acetic acid, citric, sorbic acids) at the level of 20 g/kg. Chickens were slaughtered at the 42 day of age, and the caecum microbial profiles, short chain fatty acids (SCFA) and meat quality characteristics (carcass yield, physical, chemical and technological parameters) were evaluated. In statistical analysis differences were classified by Tukey test and results were considered statistically significant at p≤0.05. OA supplement showed higher effectiveness, in compare with MCFA, on increasing quantity and variety of beneficial bacteria strains (Lactobacillus and Bifidobacterium) in caecum of broilers. Also, MCFA showed significant influence on SCFA increasing, including acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids. As well MCFA increases breast meat yellowness and water holding capacity (WHC), however, higher carcass yield (leg and abdominal fat yield) and better growth performance (higher body weight and lower mortality) of the group fed with OA were established. Finally, both tested supplements showed desirable influence, however, the OA supplement was more efficient on beneficial bacterial profile, growth performance, and carcass traits, and the MCFA was more appropriate in increasing amount of SCFA and breast meat technological parameters.

References:

BUCLAW M. (2017) Inulin in poultry production. World's Poult Sci J. 73: 301-308.

HAJATI H. (2018) Application of organic acids in poultry nutrition. Int J Avian & Wildlife Biol. 3(4): 324-329.
CHICKEN GUT MICROBIOTA AS A POTENTIAL SOURCE OF THE OVIDUCT MICROBIOTA

N. Shterzer\*(1), N.Rothschild(1), Y.Sbehat(2), D.Eytan(1), J.Dayan(1), Z.Uni(1), E.Mills(1)

(1)Hebrew University of Jerusalem, Rehovot, Israel

(2) Hebrew University of Jerusalem, Reovot, Israel

\* Corresponding author: naama.shterzer@gmail.com

The mechanism in which the poultry gut microbiota is established in the newly hatched chick is unknown. It is well known that Salmonella, a poultry pathogen, can transfer from the chicken's intestines to the oviduct and be incorporated into the developing egg1. This mechanism might be employed by commensal bacteria and facilitate their transfer from hen to chicks. Recently, diverse bacterial DNA has been found in the oviduct, which suggests the existence of an oviduct microbiota and the possibility of bacterial vertical transmission2.

The objective of this study was to compare the oviduct microbiota and the gut microbiota, and to provide evidence of viable bacteria in the oviduct and the existence of a functioning oviduct microbiota.

We collected cecum, jejunum and oviduct samples from 10 broiler breeders (Cobb; 37 weeks). We analyzed and characterized the bacterial composition in each sample using 16S rDNA sequencing. Samples were also plated in anaerobic conditions to determine the viability of the bacteria.

We found that the similarity between the oviduct microbiota and the cecum microbiota (22.42 ± 17.23%) was significantly higher than the similarity between the oviduct microbiota and the jejunum microbiota (5.55 ± 3.07%). Overall, 353 OTUs were shared between ceca and oviducts, representing 52% of the cecum OTUs (making up 90% of total cecum abundance) and 62% of the oviduct OTUs (making up 60% of total oviduct abundance). Finally, preliminary trials of culturing bacteria from the oviduct demonstrate that there are viable bacteria in the oviduct, belonging to the families Lactobacillaceae and Enterobacteriaceae.

The high similarity between the gut microbiota and the oviduct microbiota, and the fact that the oviduct microbiota is similar throughout its length, indicate that one of the sources of the oviduct microbiota is the gut microbiota. It can be assumed that material from the gut reaches the top of the oviduct. Our ability to culture at least some of the oviduct bacteria establishes the existence of this microbiota and refutes the main claim against 16S data which only prove the existence of bacterial DNA and not necessarily of viable bacteria.

References:

1. Gantois, I. et al. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Reviews 33, 718-738 (2009)

2. Lee, S. et al. Characterization of microbial communities in the chicken oviduct and the origin of chicken embryo gut microbiota. Scientific Reports 9, 6838 (2019)
# THE EFFECT OF ESSENTIAL OILS BASED FEED ADDITIVE ON BACTERIAL PATHOGENS IN CAECUM OF LAYING HENS WHILE S. ENTERITIDIS INFECTION

L. Ilina*(1), A.Dubrovin(1), D.Tiurina(2), G.Laptev(2), E.Yildirim(2), S.Cherepanov(2)

(1)Biotrof Ltd., Saint-petersburg, Russia

(2)Biotrof Ltd, Saint-petersburg, Russia

* Corresponding author: ilina@biotrof.ru

The effect of a feed additive based on a mix of essential oils on caecum microbiome of laying hens was observed.

The experimental part of the study was carried out on 40 Lohmann White adult laying hens in All-Russian Research Veterinary Institute of Poultry Science vivarium.

The Experimental group received the feed supplement based on essential oils from the first day of the experiment, while the Control group received standard feeding diet. 3 weeks after the start of the experiment, half of each group hens was infected with an epizootic strain of S. enteritidis in the amount of 5×10⁸ CFU, afterwards further parting into groups was performed: Control, Infected Control, Experimental, Infected Experimental.

The metagenomics analysis was performed in molecular-genetic laboratory of Biotrof Ltd (Saint-Petersburg, Russia). Metagenomic analysis was performed on a MiSeq genomic sequencer (Illumina, Inc., USA) with a MiSeq Reagent Kit v3 (Illumina, Inc., USA) sequencing of the variable V3 and V4 regions of the 16S rRNA gene. The reference database was RefSeq Ribosomal Database Project (RDP) 16S v3 May 2018 DADA2 32bp.

It was noticed that the introduction of essential oils-based feed additive into the hens' diet resulted in significant decrease of total pathogenic bacteria proportion in the caecum content. The part of pathogenic microorganisms in caecum samples was 5% -16% lower than the group with the standard diet.

Infection of a hens by S. enteritidis did not lead to its detection in the caecum content with 16S metagenomics method, but already a day after infection it caused an increase in the content of other pathogenic microorganisms: in the Control group, the content of pathogenic bacteria after infection increased by 31.5%, and in the Experimental group by 65%. It should be noted that the number of pathogenic bacteria in the Control Infected group was the highest and higher than in the Experimental Infected group by 6.5%.

A week after infection, the content of pathogenic bacteria in the studied groups decreased among infected hens by 30%, which indicates a gradual normalization of the pathological process. Still in samples from an Experimental Infected group, the content of pathogenic bacteria was 5.5% lower than in Control Infected group.

Uninfected groups had a lower part of pathogenic bacteria in comparison with infected analogues: in the Control group it was 35% lower than in the Control Infected group, and in the Experimental group it was 32% lower than Experimental Infected group.

In conclusion, it should be noted that the content of pathogens was highest in samples from Control Infected group hens, and the lowest - in samples from Experimental group hens.
CHARACTERIZATION OF GUT MICROBIOTA IN CHICKENS WITH EMBRYONIC DEVELOPMENT

F. Akinyemi\*(1), J.Ding(2), H.Zhou(2), K.Xu(2), C.He(2), C.Han(2), Y.Zheng(2), H.Luo(2), K.Yang(3), C.Gu(4), Q.Huang(4), H.Meng\*(2)

(1)Shanghai Jiao Tong University, Shanghai, China, Shanghai, China

(2)Shanghai Jiao Tong University, Shanghai, China

(3) Shanghai Academy of Agricultural Sciences, Shanghai, China

(4) Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai, China

Greater attention has been given to gut microbiota in poultry industry, due to its important roles. However, there are limited reports on gut microbiota development of chicken embryo with time. To bridge this gap, we examined the gut microbiota composition in the chicken embryos across different stages of development.

Fifty-four samples, collected in Beijing Fatty, Shiqiza and Bobcork (Specific Pathogen Free) breeds including whole embryos that were incubated for three days, guts from chickens embryo that were incubated for 12th and 19th days for the study. DNA extraction was conducted immediately and 16S rRNA gene Sequencing was performed. Furthermore, QIIME, a general bioinformatics pipeline was used for microbiome analysis. KEGG helped in the alignment of the predicted genes and their functions. Microbiota compositions among different groups were further analyzed using LEfSe.

96 genera such as Ochrobactrum, Phylobacterium, and Amycolatopsis were core microbiota in three stages of development. Secondly, 94 genera of microbes were observed to change significantly between E3 and E12, 143 genera significantly differed between E12 and E19 of chicken embryo ($P < 0.05$). Ochrobactrum and Amycolatopsis reduced with growth changes of E3 (30.4%), E12 (25.1%), E19 (13.6%) and E3 (11.5%), E12 (7.4%) and E19 (5.6%) respectively. Contrarily, Phyllobacterium rate increased to 47.9% at E19, indicating the growing trend of microbial diversity among the embryo's development. Moreover, the Principal Component Analysis showed a high level of similarities between the E3 and E12 compared with E19 while the alpha analysis showed more diversity of gut microbiota at E19. Furthermore, the functional predictions showed that metabolic pathways such as energy metabolism, genetic information processing were significantly enriched on day 3 and day 12 in our study, suggesting their strong influence on growth, development and immunity of chicken embryo.

Our results indicated time as a cogent factor in gut microbiota composition, and a significant change in the gut microbiota population as they developed with time was observed. Our findings indicate the growing trend of microbial diversity among the embryo's development, also establish a background for understanding the distribution of gut microbiotas in the chicken embryo, across different developmental stages.

Keywords: Gut Microbiota, Embryo Development, 16S rRNA gene sequencing, Chicken

<footnote>* Corresponding author: fisayo@sjtu.edu.cn</footnote>
EFFECTS OF ORAL SUPPLEMENTATION WITH LIQUID WHEY AND ACIDAL IN DRINKING WATER ON GUT
PH AND MICROFLORA AND PRODUCTIVE PERFORMANCE IN LAYING HENS.

T. Bouassi\*(1), D.Libanio(2), Y.Ameyapoh(2), M.Mesa(3), A.Hil hernandez(3), K.Tona(2)

(1)CERSA/ Université de Lomé, Lomé, Togo

(2)Université de Lomé, Lomé, Togo

(3)Universidad de Granada, Granada, Spain

* Corresponding author: btchilo@ correo.ugr.es

Whey, a liquid remaining from cheese production, seems to act as a natural prebiotic in birds. The objective of this study was to evaluate the effect of liquid whey (LW) and an organic acid (ACIDAL) administered in the drinking water on improving production performance and gut health in hens. Seven hundred and fifty Isa Brown hens' chicks were randomly assigned to five treatments groups (n=150) and each group divided in five replicates. The animals were reared for 48 weeks. Treatments were administered in the drinking water at dosages: 250 mL/L of LW (Lacto 25) and 500 mL/L of LW (Lacto 50), and 1 mL/L of ACIDAL (Aci). A positive control group (T+) was treated with 500 mg/L of Tétracolivit (antibiotic). The negative control group (T-) did not receive any treatment in the drinking water. At weeks 8, 12, 24, 36 and 48, 10 birds from each treatment were sacrificed, and crop, proventriculus, ileum and caecum pH were measured. Fecal samples were taken from ileum and caecum contents and analyzed for Lactobacillus, Escherichia coli, total coliforms and totalstreptococcus. Age and weight of hens at first lay as well as weights of the first eggs were recorded. Egg production was collected daily. Egg weight, egg component ratio, Haugh units and yolk color intensity were recorded weekly. Administration of LW and ACIDAL in the drinking water reduced significantly (P<0.05) the gastrointestinal pH, the number of total coliforms and Escherichia coli, and improved Lactobacillus counts compared to the negative control group. Birds of Lacto 50 and Aci groups laid their first eggs earlier. Weight of birds at first lay and first egg weight in the four treated group was higher than those of control. Furthermore, egg production was increased by 10.44% in Lacto25 but the weight and quality traits were unaffected, while the egg shell ratio was higher in Aci group compare with the rest of the groups. In conclusion, LW and ACIDAL were as effective as antibiotic on improving gut health, without the inconveniences of antibiotic bacterial resistance.
# IMPACT OF STRUCTURAL POLYSACCHARIDE IN COHERENCE WITH A PROBIOTIC ON HENS CAECUM MICROBIAL COMMUNITY

D. Tiurina*(1), Z.Fedorova(2), G.Laptev(1), L.Ilina(1)

(1)BIOTROF Ltd, Saint-petersburg, Russia

(2)All-Russian Scientific Research Institute of Agricultural Animals Genetics and Breeding, Saint-petersburg, Russia

* Corresponding author: filippova@biotrof.ru

The paper presents the study of effect of a structural polysaccharide in coherence with a probiotic on hens caecum microbial community. The most effective ways to regulate energy metabolism in laying hens is limited feeding, but it can lead to stress. To prevent stress, it is recommended to use feeds with high fiber content. As a source of fiber there was used granular alfalfa grass meal.

To help the bird absorb hardly hydrolyzable ingredients of grass meal there was added Cello bacterin- T (biologically active additive that functions as feed enzyme and a probiotic). The studies were carried out on the basis of the Collective Use Center "Genetic Collection of Rare and Endangered Chicken Breeds" in All-Russian Research Institute of Agricultural Animals Genetics and Breeding. The hens of Amrock breed were kept on the floor in aviaries. At 112 days of age the hens were divided into 3 equal groups of 23 hens and 6 cocks in each group.

I Group - Control, II Group - Experimental 1, III Group - Experimental 2.

Starting from 126 days of age during the entire experiment (84 days) the hens of group I were fed by standard diet. In the Group II the diet was modified by addition of 10% alfalfa grass in the form of granules, and in the Group III in addition to alfalfa grass there was added "Cello bacterin-T" (Biotrof Ltd - St. Petersburg, Russia).

During the post-mortem examination the samples of caecum contents were taken to determine the different groups of bacteria inhabiting the digestive tract of hens using the T-RFLP analysis.

The metagenomics analysis was performed in molecular-genetic laboratory of Biotrof Ltd (Saint-Petersburg, Russia). An analysis has shown that in the caecum of hens a significant part of the microorganisms was represented by uncultivated bacteria. In birds of Group III, amount of uncultivated bacteria was 33.4% higher than in the Group I. The number of Lactobacillus sp. in birds of Group III increased by 2.8%, the number of class Actinobacteria decreased from 8.9% to 4.2%, and family Enterobacteriaceae from 4.7% to 2.7%. The number of family Fusobacteriaceae has decreased from 1.7% to 0.96%, Peptococcus sp. from 1.6% to 0.3%, Staphylococcus sp. from 0.12% to 0.07%.

The egg production in 60 days in Groups II and III was 4% and 9.8% higher, compared with the Group I.

Thus, the use of alfalfa grass with probiotic "Cello bacterin-T", contributed to increase the beneficial microflora and to a decrease in the undesirable bacteria in the caecum of hens.
# THE EFFECT OF THE BACILLIUS SUBTILIS ON POULTRY'S GASTROINTESTINAL TRACT MICROBIOME AND THE EGGS QUALITY

D. Tiurina*(1), S.Cherepanov(1), N.Tarlavin(1)

(1)BIOTROF Ltd, Saint-petersburg, Russia

* Corresponding author: filippova@biotrof.ru

The goal of the research was evaluation of the effect of the Bacillius subtilis strain (part of the "Liquipro" water-soluble probiotic) on poultry's gastrointestinal tract microbiome and the eggs quality. High incubation properties of eggs depend on the balance and ratio of the nutrients in parent stock diet. Improper feeding leads to a lack or absence of the vital components, proteins, vitamins, minerals necessary for the normal development of the embryo in the egg.

The studies were carried out in a poultry farm of Voronezh Region. The Hisex Brown hens of 343 days age were observed. The hens were divided into 2 groups, in the amount of 25558 hens in the Experimental group (Group I) and 57275 hens in the Control group (Group II).

Throughout the experiment (65 days), the hens of Group I were fed with a standard diet with the addition of water-soluble feed additive "Liquipro" (Biotrof Ltd - St. Petersburg, Russia), the hens of the Group II were fed with a standard diet without additions.

During the post-mortem examination of hens, samples of caecum contents were taken to determine the different groups of bacteria inhabiting the digestive tract of hens using the T-RFLP analysis. The analysis was performed in molecular-genetic laboratory of Biotrof Ltd (Saint-Petersburg, Russia). An analysis of the data showed that in the caecum of hens, a significant part of the microorganisms was represented by cellulolytic bacteria. In birds of the Group I, amount of cellulolytic bacteria was 4.3% higher than in the Group II. The number of family Lactobacillaceae in the Group I birds increased by 0.7%, the number of family Pasteurellaceae decreased from 2.6% to 1.3%, and Fusobacterium sp. from 3.1% to 0.6%.

Group I also showed an increase in egg protein mass of 1.4 g and an egg shell thickness of 0.03 cm as compared to the Group II.

In conclusion, the use of water-soluble feed additive "Liquipro" in the poultry diet led to the increase of beneficial microflora, decrease in the number of opportunistic and pathogenic bacteria in hens' gastrointestinal tract, and improved the quality of eggs.
EFFECT OF TWO NOVEL PLANT EXTRACTS (CITRUS AND CUCUMBER) ON BROILER CHICKEN PERFORMANCE AND GUT MICROBIOTA

F. Riva*(1), J.Peinado-izaguerri, D.Mcguinness(2), D.Eckersall(1), D.Mckeegan(1), M.Mclaughlin, G.Bruggeman(3), D.Hermans(3), M.Bain(1)

(1) University of Glasgow, Glasgow, United Kingdom

(2) Glasgow Polyomics, Glasgow, United Kingdom

(3) NuScience, Ghent, Belgium

* Corresponding author: Francesca.riva@glasgow.ac.uk

The need to find good alternatives to antibiotic growth promoters is a major challenge for the broiler industry. The presence of active molecules such as pectins, limonene, carotenoids and polyphenols in novel plant extracts makes them potential candidates due to their reported but unconfirmed modulation of the gut microbiota, metabolism and immune system of chickens (1)(2)(3). The aim of this study was to test the hypothesis that a baseline diet (diet 1) supplemented with citrus (diet 2) or cucumber extract (diet 3) can improve gut health of broiler chickens through the modulation of the gut microbiota and performance.

A dietary trial (12birds x 4replicates x 3dietary treatments) was performed in the experimental poultry facility at University of Glasgow. Feed intake, body weight gain (BWG) were recorded twice a week from day 0 until day 28. The cumulative feed intake and body weight of birds was used to estimate the feed conversion ratio (FCR). To study the microbiota 12 birds per treatment were culled at day 14 and 28 and the DNA of the ceca and jejunum was extracted and amplified by a 16S specific-PCR, sequenced using Illumina Sequencer and analysed with QIIME 2™ at Polyomics, Glasgow.

Birds performance data was analysed by two-way ANOVA. The birds fed the citrus diet showed a significantly higher Feed intake and FCR compared to those on the cucumber and basal control diets while those fed the cucumber diet were similar to the basal diet. However, no differences in treatments were revealed on the BWG basis. Alpha and beta diversity analysis showed that the citrus and cucumber diets did not significantly affect the entire bacterial taxonomy. However a significant difference in the microbial composition was found between the two different tissues (ceca versus jejunum). Bird age also had a small effect (day 14 versus day 28). Ongoing work using LEFSE (Linear discriminant analysis Effect Size) determined that citrus and cucumber were able to modulate the growth of some bacterial strains. The results from this study form part of the European Joint Doctorate in Molecular Animal Nutrition (MANNA) (www.phd4manna.eu) and will be used to search for novel biomarkers of health status and develop new dietary plans that would enhance bird growth, maximize feed efficiency and protection from diseases.

(1) D. Savoia., 2012, Fut Microbiol., 7(8):979-90

(2) U. M. Agatemor et al., 2015, Br J Pharm Res., 1-8

(3) S. Rafiq et al., 2018, Saudi J Biol Sci., 17(4):351-358
TEMPERATURE, HUMIDITY AND AMMONIA ARE IMPORTANT ENVIRONMENTAL PARAMETERS
MODULATING THE GUT MICROBIOME OF BROILERS UNDER "RAISED WITHOUT ANTIBIOTICS"
PRODUCTION

J. Johnson*(1), B.Zwirzitz(2), A.Oladeinde(3), M.Milfort(1), A.Fuller(1), T.Looft(4), A.Bosch(5), G.Zock(1), M.Sommers(5), S.Tu(6), J.Plumblee lawrence(3), D.Cudnik(3), S.Aggrey(1)

(1) University of Georgia Department of Poultry Science, Athens, United States

(2) Institute of Food Safety, Food Technology and Veterinary Public Health, Vienna, Austria

(3) United States National Poultry Research Center, Athens, United States

(4) National Animal Disease Center, Ames, United States

(5) University of Georgia Environmental Health Sciences Department, Athens, United States

(6) Khon Kaen University Department of Animal Science, Khon kaen, Thailand

* Corresponding author: jcj36120@uga.edu

The chicken gut microbiome consists of all microorganisms including bacteria, viruses, protozoa, and
fungi, and their collective genetic material present in the gastrointestinal tract (GIT). The gut
microbiome plays key roles in nutrition absorption, development of immunity, and disease resistance.
Gut microbiome may have positive or negative effects on feed efficiency, productivity, and health of
chickens. However, we do not know how the poultry house environmental parameters (temperature,
ammonia, humidity) modulate the bacterial community residing in the GIT. In this study, we raised six
hundred Cobb-500 broiler chicks in floor pens with clean pine shavings in two houses (300 birds/house;
25 per pen) for 49 days under environmental conditions reflecting those of commercial "Raised
Without Antibiotics (RWA)" broiler production. We performed 16S rRNA gene sequencing on the
meconium of day-old chicks, on the ceca when the birds were 14, 28, 42 and 49-days old and on weekly
litter samples. Furthermore, we tested the effect of an 8-hour feed withdrawal period on the
microbiome of 240 birds (120 birds/house). We also monitored the temperature, relative humidity and
ammonia levels in the house and the moisture, pH and culturable E. coli in the litter. The bacterial
community in the meconium, ceca and litter were significantly different throughout the study (p <
0.05), and the ceca had the highest alpha diversity. The top genera in the meconium and ceca were
Enterococcus and Bacteroides, respectively. For the litter, the top genera consistently changed from
day 7 to day 49. The bacterial community in the ceca and litter were significantly different between
houses (house 1 and 2) from day 28 – day 49 (p < 0.02). Temperature and morning (7 – 10 am) relative
humidity had a significant effect on the β-diversity of the ceca. In addition to house temperature,
relative humidity and ammonia, the pH and moisture of the litter were the important parameters
affecting the β-diversity of the litter. An 8-hour feed withdrawal before slaughter had no significant
effect (p> 0.05) on the bacterial community of the ceca. Together, our results indicate that the
environmental conditions in the farm house significantly affected the gut microbiome of broiler
chickens. As a consequence, broilers in house 1 had significantly different ceca/litter microbiome and
higher live weights than broilers in house 2 upon harvest.
HEPATOTOXIC EFFECT OF FUMONISIN - B1 IN BROILER CHICKEN

K. Satheesh\*(1)

(1)AH Polytechnic, Banavasi- 518360, Kurnool (Dt), Kurnool, India

\* Corresponding author: koksatvet@gmail.com

Fumonisin-B1 (FB1), a mycotoxin extracted from Fusarium moniliforme, has been implicated in a variety of clinical syndromes in several animal species including human beings. FB1 was fed experimentally to one hundred and twenty five healthy, unsexed, day old Vencob broiler chicks, randomly divided into five groups of twenty five each @ 0 (control), 50,100,200 and 400 ppm per kg feed. The effect was dose dependant and showed hepatotoxicity characterized grossly by severe enlargement with yellowish discoloration. Microscopically, mild to moderate congestion, cellular swelling, nodular infiltration of mononuclear cells, various degrees of necrosis, hepatitis, fatty changes, mild to moderate fibroplasia with formation of pseudolobules in an acinar pattern giving adenomatous appearance (prominent in high dose group), stenosis of bile duct, degenerative / necrotic changes and or hyperplasia and hypertrophy of bile ductular lining epithelium was noticed. The hepatic lesions described in the experimental birds are suggestive of precancerous stage and hepatotoxic nature of Fumonisin-B1.

Key words: Fumonisin-B1, Broiler chicks, hepatotoxicity, adenomatous appearance, precancerous stage
MICROBIAL ESTABLISHMENT AND DYNAMIC CHARACTERISTICS OF PIGEON MILK

J. Ding\*(1), N. Liao(2), Y. Zheng(2), L. Yang(2), K. Xu(2), C. He(2), C. Qin(2), C. Tang(3), L. Wei(4), H. Meng(2)

(1) Shanghai Jiaotong University, Shanghai, China

(2) Shanghai Jiao Tong University, Shanghai, China

(3) Shanghai Xinrong Big Emperor Pigeon Breeding Professional Cooperation, Shanghai, China

(4) Fengxian District Animal Disease Prevention and Control Center, Shanghai, China

\* Corresponding author: dingjinmei@sjtu.edu.cn

The breast milk of mammals is rich in microbes which can be transmitted to their offspring. Like mammals lactate for their offspring, the parent pigeons vomit the pigeon milk out of craw and feed the squabs mouth to mouth. However, there is little research on the microbiota of pigeon milk. In this study, high-throughput sequencing technology was adopted to detect and analyze the diversity and composition of microbiota. A total of 24 pigeon milk samples were collected, 12 parent pigeons and 12 squabs at different ages (4 each at 0, 5 and 10 days). Based on the results of operational taxonomic units, 8 phyla and 106 genera of microbiota in pigeon milk were annotated. Four major phyla dominated the pigeon milk Firmicutes (67%), Actinobacteria (20%), Proteobacteria (5%), and Bacteroidetes (5%). At the genus level, the microbiota of pigeon milk of parent pigeons were dominated by Lactobacillus, which was the most abundant genus with a proportion of 42% followed by Enterococcus (9%), Veillonella (9%), and Bifidobacterium (8%). While, the proportion of Lactobacillus (61%) and Bifidobacterium (15%) were significantly higher in squabs than parent pigeons. Lactobacillus exhibited an increasing trend with age which suggested that it may be an essential microbe in squabs. The Veillonella and Prevotella in squabs showed a gradual decline from day 0 to 10 days after hatch. In addition, we found that the predominant gut microbiota in pigeon were Lactobacillus and Turicibacter. The gut microbiota of squabs are more abundant than parents, but the parents have high gut microbial diversity. Our findings provide evidence to support the hypothesis that part of the microbial colonizers harbored in squabs were derived from parents and beneficial microbiota may be preferentially colonized in squabs.
MICROBIOME GWAS UNCOVER NOVEL GENETIC MUTATIONS OF THE HOST ASSOCIATED WITH
INFECTION OF SALMONELLA PULLORUM

H. Zhou\*(1), L.Xiao(2), L.Luo(2), C.He(2), J.Ding(2), L.Yang(3), K.Yang(3), K.Xu(2), Q.Huang(3), H.Meng(2)

(1) shanghai jiao tong university, Shanghai, China

(2) Department of Animal Science, School of Agriculture and Biology, Shanghai Jiao Tong University;
Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai, Shanghai, China

(3) Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Science,
Shanghai, China., Shanghai, China

* Corresponding author: haozhou69@gmail.com

The aim of this study was to explore the effect of relationship between host genome and the gut microbiota on salmonella pullorum disease. Here, we used composition of their gut microbiota as the informative intermediate trait of host. The tool of microbiome GWAS was employed to perform the association of genetic variation and beta diversity of gut microbial communities. Two sets of data are required for this approach: one is 366,189 high-quality host genomic variations obtained from reduced-representation genome sequencing (Personal Biotechnology Limited Company, Shanghai) in our previous study. The variations were from 135 positive and 140 negative pullorum infected individuals, for whom microbiome was profiled for the present study. The other is pairwise microbiomedistance matrix of weighted UniFrac calculated using their stool microbiome data. The mutations withadjusted P-value that was corrected by skewness and kurtosis less than 10-5 were considered as significantly associated with microbial communities. After that, we found that 425 significant mutations located on 141 annotated genes. Pathway analyses and GO keywords enrichments of thesegenes were conducted by DAVID. Interestingly, 26 genes of them, including GDF6, TH, SOX9, VPS33B, TGFB1, were significantly involved in the keywords of disease mutation (P=0.02). Additionally, several pathways related to immune function were remarkably enriched, such as inflammatory mediator regulation of TRP channels, TGF-beta signaling pathway, and Rap1 signaling pathway. In this study, using Unifrac, a widely used distance metric, as the intermediate trait of chicken with pullorum positively and negatively infected, we identified novel disease-related genetic variants that are associated with gut microbiome composition. They may affect gene transcription and activity to further influence the chicken immunity against pullorum infection. Overall, our results contribute to understanding the effect of host genetics and their gut microbiota on salmonella pullorum infected chicken.

KEYWORDS: microbiome GWAS; host genetics; microbiome; salmonella pullorum