# BRAIN VITAMIN D3 SIGNALING REGULATES CHICK ENERGY METABOLISM

J. Shiraishi*(1), T. Hayakawa(1), Y. Akasaka(1), T. Aoki(1), Y. Ohta(1)

(1) Nippon Veterinary and Life science University, Tokyo, Japan

* Corresponding author: jshira@nvlu.ac.jp

**Introduction:** Vitamin D3 (D3) is hydroxylated in the liver, circulates as 25-hydroxyvitamin D3 (25OH-D3), and then hydroxylated in the kidney is converted to an active vitamin D metabolite (1,25-dihydroxyvitamin D3). These vitamin D signals act on target organs expressing vitamin D receptor (VDR). VDR is also known to be expressed not only in peripheral organs such as liver, gastrointestinal tract, and kidney, but also in the brain.

In mammals, it has been revealed that the vitamin D signals in the brain act on the melanocortin system, which is the key regulator of energy metabolism, and control whole-body energy metabolism. However, little is known about the effect of brain vitamin D signaling on chick energy metabolism. Therefore, in this study, to clarify the function of the brain vitamin D signal in the neonatal feeding behavior and energy metabolism, we investigated 1) gene expression level of diencephalon VDR during growth and 2) effect of feeding D3 and 25OH-D3 on brain melanocortin system.

**Materials and Methods:** All experiments were demonstrated commercial broilers (Ross308). Experiment 1: Diencephalic VDR gene expression at 15, 18 and 21 days of embryonic stage and 3, 7, 15 and 42 days after hatching was relatively quantified by quantitative PCR (qPCR). Experiment 2: Four types of diets were mixed for D3 (3000 or 5000 IU) and 25OH-D3 (0 or 69mg), and fed for 11 days after hatching. The chick body weight, feed intake and feed efficiency were measured during the experimental period. At the end of experiment periods, the chicks were sacrificed, and diencephalon was collected. The expression level of VDR and melanocortin peptide (POMC, AgRP, NPY) genes were quantified by qPCR.

**Results and Discussion:** VDR gene expression in the diencephalon increased with embryonic development, and was higher in chicks immediately after hatching. Addition of D3 and 25OH-D3 to the starter diet for 11 days after hatching did not changed in the body weight, feed intake and feed efficiency. The POMC gene expression level in the D5000 treatment group was higher than that of D3000 treatment group, and these upregulation was diminished by the addition of 25OH-D3. Furthermore, positive correlation between diencephalic VDR and POMC gene expression level were observed. These results suggest that D3 and 25OH-D3 act on the melanocortin system in the chick brain and may be involved in regulating energy metabolism, particularly through the activity of POMC neurons.
THE EFFECT OF FREE FATTY ACIDS CONTENT AND SATURATION LEVEL ON DIETARY FAT PREFERENCE IN HENS. PRELIMINARY RESULTS

M. Palomar*(1), M.Soler(1), O.Piquer(1), A.Orozco(1), M.Calero(1), P.Úbeda(1), P.Rodríguez(1), C.Garcés-narro(1)

(1) Department of Animal Production and Health, Public Veterinary Health and Food Science and Technology, Faculty of Veterinary Medicine, Universidad CEU Cardenal Herrera, CEU Universities, Spain, Valencia, Spain

* Corresponding author: maria.palomarlloris@uchceu.es

Behavioural and genetic evidence show that avian taste is intimately related to nutrient sensing and, consequently, to poultry nutrition practices. Clarification of the mechanism of avian fat taste could provide meaningful information for improving new feedstuff in poultry nutrition. In this sense, acid oils could be economical and sustainable feedstuffs. Acid oils are fat by-products from the edible oil refining industry with similar fatty acids composition to their respective crude oils but richer in free fatty acids (FFA). A trial was conducted in order to study the effect of FFA content and ratio unsaturated:saturated (U:S) on dietary fat preference in hens. 48 Lohmann Brown laying hens were housed individually in cages. Three test diets containing Soybean acid oil (SA; high U:S, 50% FFA), PalmOil (PO; low U:S, 5% FFA) and Palm Fatty Acid Distillate (PFAD; low U:S, 50% FFA) were offered in a series of double choice tests (after a 7 d training period) against a reference diet with Soybean Oil (SO;high U:S, 5% FFA). In each diet, fat was added to a basal diet at inclusion rate of 6%. Two equally sized containers were placed in front of the cage: one of them holding the reference diet (SO) and the other one of the three experimental diets assigned following a complete block design. A control "reference vs. reference" two-way comparison was included to detect such possible effects of position preferences and feeders' position was changed after every measurement. Feed intake was measured during 2 h twice a day after 1 h of fasting. The consumption was analyzed as a standard preference index (% of test diet intake over total intake). Preference value were compared to the random choice value of 50%. For statistical analysis, a One Sample t-Test was performed. Significant differences were considered for P < 0.05. Preference measured for the double reference control did not differ from 50%(51.9%), indicating that the observed preference values may be attributed to dietary changes in fat ingredients. Hens showed feed preference for PO (60.9%) and PFAD (57.0%). However, preference values less than 50% were observed for SA (37.9%). These results suggest that with high U:S, hens prefer feed with less FFA. Furthermore, saturation level plays an important role on dietary fat preference: hens prefer low U:S fats even when they are rich in FFA. Acknowledgements: Financed by Generalitat Valenciana and the European Social Fund.
THE EFFECT OF DIFFERENT DIETARY RATIOS OF ARGININE AND METHIONINE TO LYSINE ON PROTEINS NITRATION, REDOX STATUS OF TURKEYS BLOOD AND DNA OXIDATION

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

(1)University of Life Science, Lublin, Poland

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

(3) University of Life Sciences, Lublin, Poland

* Corresponding author: kasiaognik@poczta.fm

The aim of this study was to determine the effect of different ratios of arginine (Arg) and methionine (Met) in diets with high lysine (Lys) content on performance, metabolism, and oxidation processes of tissues and DNA in turkeys. The Hybrid Converter turkeys were assigned to 6 groups with 8 replicates per group and 18 birds per replicate. Six feeding programs, with 3 dietary Arg levels (90, 100 and 110%) and 2 dietary Met levels (30 and 45%) relative to dietary Lys content were compared. During each of four feeding phases (4 weeks each), birds were fed ad libitum isocaloric diets containing high level of Lys; approximately 1.83%, 1.67%, 1.49% and 1.20%, respectively.

The applied dietary treatments had no effect on daily feed intake and body weight in each of feeding phases. Comparing to the lowest dietary Arg ratio (90% Lys), the both increased ratios (100 and 110% Lys) were associated with the increased levels of nitrotyrosine-3-NT (139.6 vs. 158.8 vs. 179.1 nmol/L, P<0.001), malondialdehyde-MDA (2.3 vs. 3.2 vs. 3.1 µmol/L, P=0.04), and decreased activity of superoxide dismutase-SOD (1616.7 vs. 1272.1 vs. 780.3 U/gHb, P<0.001) in the turkey blood. Birds fed diet of highest Arg ratio (110% Lys) had the highest level of PC (4.0 vs. 4.2 vs. 4.7 nmol/mg, P=0.002) in the blood comparing to birds fed other dietary Arg ratios (100 and 90% Lys). Irrespective to the dietary Arg ratio, a increased ratio of Met in the diet (45% Lys) resulted in lower blood levels of proteincarbonyl-PC (4.5 vs. 4.1 nmol/mg, P=0.008), 3-NT (162.7 vs. 155.7 nmol/L, P=0.05), and 8-hydroxydeoxyguanosine (8.5 vs. 7.9 ng/mL, P<0.001).

Our data indicated, that feeding turkeys diets containing 90% Arg and 45% Met in relation to Lys, could positively stimulate antioxidant defense system. The beneficial effect of such treatments was evidenced in down regulation of oxidation and nitration processes of key molecules in the biological system.

This work was supported by the National Science Centre, Grant No. 2017/27/B/NZ9/01007
STUDIES ON DOSE AND DURATION OF FEEDING COMBINATION OF NUCLEOSIDES IMPROVED PERFORMANCE, IMMUNITY AND NUTRIENT TRANSPORT IN BROILERS THAN INDIVIDUAL COUNTERPARTS

G. Marappan*(1), J.Rokade(1), S.Ts(2), G.Kolluri(1), M.Velusamy(3), J.Tyagi(1)

(1)ICAR-Central Avian Research Institute, Bareilly, India

(2)ICAR-Indian Veterinary Research Institute, Bareilly, India

(3)Veterinary College and Research Institute, Namakkal, India

* Corresponding author: gopsgopi72@gmail.com

Genetic improvement and environmental conditions have led to a shift in the concept of nutrients essentiality, especially in broiler chickens. Nucleotides were once regarded as non-essential but could be considered as conditionally essential at early rapid growth and stress conditions. The chicks during the early rapid growth periods become dependent on preformed nucleotides than its de novo synthesis. To assess the impact of nucleotides, a series of experiments were carried out. In the first experiment, the effect of nucleosides (adenosine, guanosine, cytosine and Uridine) were assessed individually and in combination. A total of six experimental groups as control; adenosine (0.1%); Guanosine (0.1%); cytosine (0.1); Uridine (0.1) and combination of four having equal proportions (0.1%). All the groups were administered with respective nucleosides orally daily morning before feeding for the first 14 days of age. The birds fed with the combination of nucleosides resulted in better growth performance and immune response than their individual counterparts and control group. Based on this results, the second experiment was carried out to determine the dose as well as the duration of supplementation. Chicks (N=400) were divided into eight groups of five replicates as Group N1: (Control no antibiotics/nucleosides); N2 (positive control - BMD@150ppm) for 42 days. Group N3, 4 and 5: combination of nucleosides @ 0.05, 0.1 and 0.15% for the initial 14 days; Group N 6, 7 and 8: combination of nucleosides @ 0.05, 0.10 and 0.15% for the initial 21 days of age. The production performance, immunity and relative expression of nutrient transporter genes were studied at various time intervals and the data were analyzed using single factor analysis of variance. Higher body weight (P0.05) difference among the treatment groups at 7 and 14 days. The results indicated that feeding combination of nucleosides @0.10% for initial 14 days of age improved the performance and immunity in broiler chickens.
MODULATING MICROBIOME BY NUTRITIONAL STRATEGY TO OPTIMISE PERFORMANCE OF BROILER
CHICKENS

X. Rousseau*(1), G.Gonzales-ortiz(1), T.Tedeschi dos santos(1), G.Gomes(1)

(1)AB VISTA, Marlborough, United Kingdom

* Corresponding author: xaviere.rousseau@abvista.com

Microbiome is often seen as a second brain as in fact he is involved in many physiological pathways. Intestinal microbiome has a crucial role and is part of new emerging nutritional strategy as fibre represents main of the substrate available to ferment by the bacteria. That's why it becomes relevant to stimulate specific populations to take more benefits from the fibre fraction. The objective of this study was to investigate the impact of 2 different nutritional strategies on growth performance and how hindgut fermentation and caecal microbiome could be affected. Two dietary treatments were tested where treatment 1 (XYL+XOS) was a diet supplemented with a combination of xylanase and a specific xylo-oligosaccharide to stimulate the microbiome to ferment fibre and treatment 2 (XYL+BUT) was the same diet supplemented by a xylanase and micro-encapsulated butyrate offering substrate directly to the microbiome. Treatments were fed to day old male Ross 308 placed in floor pens with 65 birds/pen in 4 feeding phase program (0-10d, 11 to 21d, 22 to 32d, >33d) with 9 replicates pens per treatment. Growth performance (body weight, feed intake per pen) were measured, FCR and EPEF calculated. Caecal samples were collected on 9 birds per treatment at 42 days and analysed for their VFA content and %G+C profile. Data were analysed by a one-way ANOVA. Differences in terms of growth performance appeared at the last growing phase with XYL+XOS, improving BW (p=0.032) and FCR (p=0.041) by 67 g/bird and 12 points respectively that is translated to an overall improvement of the bwcFCR by 5 points (p=0.087) and of the EPEF by 46 points (p=0.068). Along to the performance, a reduction of BCFAs production was also noticed (p=0.12) without any changes regarding all the others SCFA analysed. The %G+C profile show differences where XYL+XOS increases the butyrate producers' population (p<0.05). These data suggest that the product from xylanase hydrolysis in treatment 1 combined with the specific XOS had positive influence on caecal bacterial population that may have allowed to extract more energy from the fibre arriving in the hind gut that is translating in better growth performance. This beneficial effect is not only related to butyrate production or the concentration may have been too low. Caecal SCFA analysis may also not be the most relevant criteria as it's a point in time measurement of a dynamic flow of production/absorption so microbiome analysis could be more meaningful.
ID: 1155

URIC ACID IS SECRETED BY ENTEROCYTE AS AN ANTIOXIDANT BUT NOT A WASTE TO PROTECT AGAINST OXIDATIVE STRESS

M. Wang\*(1), H.Lin(1), X.Wang\*(1)

(1)Shandong Agricultural University, Taian, China

\* Corresponding author: wangminghui825@163.com

Abstract

Objective Uric acid has been found an important antioxidant in avian and human. Gut is considered as an organ to excrete uric acid (UA) except of kidney. The gastrointestinal (GI) tract has the most extensive surface to be constantly exposed to a wide variety of potentially harmful substances. This study re-evaluated the role of UA secretion in the intestines to test the hypothesis that enterocytes can synthesize and secret UA into intestinal lumen to protect from oxidative stress. Synthesis, secretion, and antioxidant effects of UA were investigated in the intestinal tract and enterocytes of chickens.

Design UA and allantoin concentrations in chyme were measured in various segments of the small intestine. mRNA expression of genes related to UA synthesis and transport was determined. Synthesis, secretion, and antioxidant effects of UA were evaluated in enterocytes and everted duodenum sacs in the presence of inosine, which is a precursor of UA. In order to test the hypothesis that enterocyte can synthesized UA to protect against oxidative damage, the in vitro cultured enterocytes was exposed to H2O2 challenge and the mRNA was extracted and reverted for transcriptome sequencing analysis. We further confirmed the effect of mild oxidative stress on synthesis and secretion of UA in enterocytes and cultured everted gut sacs.

Results Along the GI tract, UA and allantoin were present in the chyme and the duodenum had the highest ones, which was in line with the mRNA levels of the key enzymes in the purine nucleotide salvage-catabolism pathways. In enterocytes and everted gut sacs, inosine treatment increased UA synthesis and secretion. Low concentration of H2O2 stimulated UA synthesis and secretion by enterocytes. Transcriptomic study identified 206 differentially expressed genes (DEGs) with significantly higher expression level in H2O2 treatment, and 174 DEGs with low expression level, compared with control. In particular, the phosphodiesterase 1A (PDE1A) involved in guanine metabolism and XDH, ABCC4, ABCG2 mRNA levels increased significantly in H2O2 treatment. In enterocytes, UA attenuated H2O2-induced oxidative damage by Nrf2-ARE pathway.

Conclusions UA is synthesized by enterocytes and secreted into the intestinal lumen, where it elicits antioxidant effects on the intestinal epithelium. The result suggests that UA secretion into intestinal lumen is a protective mechanism of intestinal tract in facing to oxidative stress, rather than to excrete waste.
EFFECT OF IN OVO ADMINISTRATION OF SELENIUM ON EGG TYPE CHICKEN HATCHABILITY

K. Tona(1), K.Tona(1), A.Fafiolu(2), W.Pitala(3)

(1) Centre Régionale sur les Sciences Aviaires, Lomé, Togo

(2) Federal University of Agriculture Abeokuta (FUNAAB), Abeokuta, Nigeria

(3)Centre d'excellence Régional sur les Sciences Aviaires, Lomé, Togo

* Corresponding author: kwassitona@yahoo.fr

Health status and stress impact poultry production. To alleviate the effects of those factors, many approaches are developed like control housing and use of drugs. But substances like Selenium can be used to reduce the negative effect. Selenium is an antioxidant that protect the organism against oxidative stress and health status of animals. The aim of this study was to evaluate the effects of in ovo Selenium administration on layer type chicks' performance. A total of 600 eggs were numbered, weighed and incubated. At 18 days of incubation, the eggs were candled and those with evidence of living eggs were divided into five groups of 120 eggs each. These groups were eggs in T1 that were not injected, T2 received Nacl 90/00 injection as positive control, T3 received 10 µg (Se), T4 received 20 µg (Se) and T5 received 30 µg (Se). The eggs were injected at the air chamber site. A hole was created at the injection site with a syringe needle. The solution was administered using an automatic syringe. The dosage of injection is 0.1 ml per egg. After the injection the sites were well sealed with a scotch tape before transferring the eggs to the hatcher. Embryonic parameters such as pipping time, incubation durations, hatchability, chick's quality using tona score, organs and chicken weight at hatch were evaluated. Result showed that birds that were not injected had the shortest hatching duration compared to others. The control group, the negative and the positive controls groups showed the lowest hatchability while groups of eggs that were injected with 20 µg (Se) and 30 µg (Se) had the highest rate. At hatch, chicks of the highest selenium dosage (30 µg (Se)) injection were the highest in term of chick's quality compared to others and the quality increased as the concentration of Se increases in the solution injected. No significant (p>0.05) difference was observed for chicks and organs weight after hatch. In conclusion the injection of Selenium in the fertile eggs improve the performance of the chicks after hatch. But the Selenium administrated at high level have a negative effect on the birds because of it's proven toxicity.

Key words: Selenium, In ovo administration, Hatchability, Chick's quality.
CIRCADIAN RHYTHMS OF CALCIUM AND PHOSPHORUS DIGESTIBILITY IN COMMERCIAL LAYING HENS

M. Sinclair-black\*(1), P.Plumstead(2), R.Angel(3), W.Li(4)

(1)University of Pretoria, Johannesburg, South Africa

(2)Chemunique Pty Ltd, Johannesburg, South Africa

(3)University of Maryland, Maryland, United States

(4)DuPont Industrial Biosciences, Wilmington, United States

\* Corresponding author: micaela@chemunique.co.za

The objectives of this study were to investigate the circadian rhythms of digestible calcium (Ca) and phosphorus (P) in extended production cycle laying hens, with the intention to improve the efficacy of Ca and P utilization. The experimental design was a 2x2 factorial of 2 particle sizes of limestone (LM), grit (1.5mm geometric mean diameter (GMD)) or fine (0.2mm GMD) and two levels of added phytase from Buttiauxella sp. (0 or 600 FTU/kg). A corn-soy diet was mixed to contain 3.6% Ca from each LM and 0.11% Ca from the basal diet. Cr2O3 was included at 0.4% as a marker. The ileal digestibility of Ca and P were tested, at 3 hours post oviposition (POP) and at 11 hours POP. At 3 hours POP Ca and P are actively being added to the medullary bone matrix (Wilson & Duff, 1990) and at 11 hours POP the eggshell is undergoing calcification (Van De Velde et al., 1984). Amberlink hens at 33 wks of age were used and digestibility measurements were taken over four days at each time period (3;11 hours POP). Each hen served as a replicate (n = 1 hen) and 20 hens (5 per treatment) were sampled per time period per day. Distal ileal digesta was taken, lyophilized, ground and analysed for Ca and P and Cr. Data were analysed using SAS MIXED model with day of sampling as a random effect. Time POP affected Ca and P digestibility (P<0.01). Ca digestibility was higher at 11 hours POP than at 3 hours POP (P0.05). Phytase improved P digestibility with the greatest improvement noted at 3 hours POP (P<0.05). The results from this study suggests that hens are able to increase the capacity to absorb nutrients according to physiological demands for Ca and P differently during medullary bone mineralization vs eggshell calcification and, that phytase improves P digestibility in laying hens. These time-dependent changes in the efficacy of Ca and P utilization should be considered when formulating diets for laying hens using a split feeding approach.

References

Van De Velde, J. P., Loveridge, N., & Vermeiden, J. P. W. (1984). Parathyroid hormone responses to calcium stress during eggshell calcification. Endocrinology, 115(5), 1901-1904.

Wilson, S., & Duff, S. R. (1990). Morphology of medullary bone during the egg formation cycle. Research in Veterinary Science, 48(2), 216-220.
ID: 1254

BLOOD PH AND BLOOD GASSES OF BROILERS AT DAY 7 AND 21 AS AFFECTED BY THE ACID BINDING CAPACITY OF THE DIET, WATER ALKALINITY, AND WATER ACIDIFICATION.

K. Venter*(1), P.Plumstead(2), T.Nkukwana(3), M.Sinclaie-black(4)

(1)University of Pretoria, Johannesburg, South Africa

(2)Chemunique, Johannesburg, South Africa

(3)University of Pretoria, Pretoria, South Africa

(4)Chemunique, Lanseria, South Africa

* Corresponding author: kyle@chemunique.co.za

The objectives of this study were to determine if blood pH, bicarbonate and blood gasses [partial pressure of CO2 (PCO2), partial pressure of O2 (PO2), total CO2 (TCO2), O2 saturation percentage (sO2), bicarbonate (HCO3-), blood sodium (Na) and blood base excess (BEecf)] were affected by the acid-binding capacity (ABC) of the feed, alkalinity of water, and water acidification. The experimental design was a 2x2x2 factorial of 2 dietary treatments (High ABC and Low ABC), 2 water alkalinities (84 ppm CaCO3 and 416 ppm CaCO3) with or without the addition of a water acidifier that reduced water pH to 4.0. Male Ross 308 birds were placed in 96 cages with 12 replicates per treatment. Two starter (0-10d) and finisher (11-21d) diets were formulated with a low and high ABC. The water with high (416 ppm CaCO3) and low (84 ppm CaCO3) alkalinity values was provided ad lib. At 7 and 21d birds were fasted for 6 hours to ensure the gastrointestinal tract was empty and birds were then provided feed for 12 minutes prior to drawing blood from the brachial vein. All blood parameters were measured using an i-STAT point-of-care laboratory system (Abbott Point of Care, East Windsor, NJ). The effects of dietary ABC, water alkalinity, acidification and their interactions on the various blood parameters were tested by SAS MIXED model using repeated measures (SAS, 9.4). Age had a significant effect on all the measured parameters (P<0.01) apart from PO2 and sO2. Blood pH increased significantly (P<0.0001) from 7.32 to 7.39 at 7d and 21d, respectively. There was a significant (P<0.05) interaction between water alkalinity and acidification on blood pH. There was a significant (P<0.001) interaction water alkalinity, acidification and day of sampling for TCO2 and Base excess. Blood bicarbonate was significantly affected by day of sampling (P<0.0001) and a 3-way interaction between water alkalinity, acidification and day of age (P<0.005). The bicarbonate in the blood increased from day 7 (22.32) to day 21 (26.11). There was a correlation between blood pH and HCO3 (R=0.46) illustrating alkaline tide. The results of this study demonstrated that certain blood gasses varied significantly when the bird's stomach was buffered through feed or water. The consequences of this on nutrient digestion and live-performance will be evaluated in future studies.
A NOVEL APPROACH FOR THE MEASUREMENT OF BROILER CHICKEN ALBUMIN SYNTHESIS RATE

J. Peinado-izaguerri\*(1), F.Riva(2), M.Mclaughling(3), D.Mckeegan(3), M.Bain(3), M.Bhide(4), T.Preston(3)

(1) University of Glasgow, University of Veterinary Medicine and Pharmacy in Kosice, Glasgow, United Kingdom

(2) University of Glasgow, University of Zagreb, Glasgow, United Kingdom

(3) University of Glasgow, Glasgow, United Kingdom

(4) University of Veterinary Medicine and Pharmacy in Kosice, Kosice, Slovakia

* Corresponding author: Jorge.Peinado@glasgow.ac.uk

**INTRODUCTION:** Protein Fractional Synthesis Rate (FSR) can be used as an early biomarker in health and disease. This work aims to facilitate the introduction of proteomics platforms to estimate FSR of multiple proteins by measuring tracer incorporation at the peptide level. This novel approach for FSR measurement involves the use of heavy water (D2O), which labels free amino acids during de novo synthesis and transamination. The objective of the current study is to validate a 'dynamic proteomics' approach through isolation and conventional analysis of a single protein. Albumin is the ideal candidate due to its accessibility, high concentration in plasma and its importance as a marker of nutritional status. We applied a single D2O bolus to calculate the FSR of multiple proteins in broiler chickens by dynamic proteomics using a non-linear model of tracer incorporation.

**METHODOLOGY:** 21 day old male broiler chickens of around 1kg body weight were administered an oral bolus of 10 g D2O/kg body weight. Chickens were injected with 2mg/kg E.Coli lipopolysaccharide (LPS) or saline solution intravenously one hour after D2O administration. One chicken per pen was blood sampled and culled at 4h, 24h, 48h and 96h after D2O dosage. Blood was collected in 2ml tubes coated with EDTA and plasma was obtained after centrifugation. Deuterium enrichment in body water was quantified by FTIR analysis of plasma. Free amino acids were isolated by cation exchange columns and their deuterium enrichment was analysed by GC-MS. Albumin was isolated from plasma by differential solubility, acid hydrolysed and the deuterium enrichment of bound amino acids was analysed by GC-MS.

**RESULTS:** The elimination of a D2O bolus follows a single exponential. Elimination half time was 2.34 days for chickens injected with LPS and 2.10 days for control birds. Mean albumin FSR of chickens injected with LPS was 78.2% day-1 calculated from body water enrichment and 77.1% day-1 calculated from free alanine incorporation. Mean albumin FSR of control chickens was 100.6% day-1 calculated from body water enrichment and 97.7% day-1 calculated from free alanine incorporation.

**CONCLUSION:** New data on male broiler chicken albumin FSR has been obtained. This data will be compared with FSR measured at peptide level by proteomic platforms and will contribute to unravel the effect of E.Coli LPS on protein dynamics in broiler chicken.
DEVELOPMENTAL PROGRAMMING OF FAT ACCRETION IN BROILER CHICKS THROUGH LONG CHAIN
OMEGA-3 POLYUNSATURATED FATTY ACIDS IN THE HEN DIET

M. Kim(1), K.Piacquadio(1), U.Jung(1), A.Bettaieb(1), S.Das(1), J.Wilson(2), B.Voy*(1)

(1)University of Tennessee, Knoxville, United States

(2)University of Georgia, Athens, United States

* Corresponding author: bhvoy@utk.edu

Developmental programming can produce lasting effects on growth and metabolism in poultry (Lesuisse et al., Poult. Sci., 2018). Programming occurs in part through epigenetic mechanisms that persist across generations, yielding an efficient tool with which to improve performance. We previously demonstrated that developmental programming through long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) in the broiler-breeder hen diet reduced fat accretion in chicksafter hatch (Beckford et al., Sci. Rep, 2017). The objectives of this study are to determine if LC n-3 PUFAinfluence embryonic development of chick adipose tissue and to identify mechanisms for programmingof body composition. Broiler-breeder hens (Cobb 500) were fed corn-soybean diets supplemented withfat (2.3%) from fish oil (FO; LC n-3 PUFA) or soybean oil (SO; n-6 PUFA control) for4-7 weeks (n>20 hens/diet). Fertilized eggs were incubated to days E12, E14, E16, E18, and E20. Maternal diet effects at each age were compared using T-test (α=0.05). Embryo weights were significantly lower in FO vs. SO at E12 and E14, but not at later ages. Absolute and relative subcutaneous adipose weights were not affected by diet at any age. However, histological analyses revealed that maternal FO significantly reduced adipocyte size and increased adipocyte number, suggesting an effect of hen diet on adipogenesis. Breast muscle (p. major) weight was lower in FO embryos at E12 and E14, but by E18 was significantly greater than in SO controls. In mammals, maternal LC n-3 PUFA have been shown to reduce fat accretion and increase lean mass through effectson mitochondrial metabolism and fatty acidoxidation (Rudolph et al., Diabetes, 2018). To test this possibility, primary preadipocytes and satellite cells were isolated from SO and FO chicks two days after hatch and induced to adipogenic and myogenic differentiation. Oxygen consumption rates (Seahorse XF, Agilent.com) revealed that hen FOsignificantly altered mitochondrial respiration and enhanced fatty acid utilization in both cell types. Collectively, these results indicate that dietary programming by LC n-3 PUFA may reduce fat accretion after hatch through effects on adipogenesis that begin in the embryo. In addition, maternal FO enhances metabolic flexibility in both adipose and muscle, which may improve energy utilization by the developing chick. Efforts to identify the underlying molecular basis for metabolic programming byLC n-3 PUFA are underway.
SELECTED EXCRETA BIOMARKERS OF INTESTINAL BARRIER FUNCTION IN BROILER CHICKENS
SUBJECTED TO TWO GUT LEAKAGE MODELS WITH OR WITHOUT PROBIOTIC SUPPLEMENTATION

R. Barekatain\*(1), G.Howarth(2), N.Willson(2), D.Cadogan(3), S.Wilkinson(4)

(1)South Australian Research and Development Institute, Roseworthy, Australia

(2)University of Adelaide, Roseworthy, Australia

(3)Feedworks Pty Ltd, Romsey, vic, Australia

(4)Feedworks Pty Ltd., Romsey, vic, Australia

(5)Universidade Federal de Goiás, Goiânia, Brazil

(6)Universidade Federal de Jataí, Jataí, Brazil

* Corresponding author: Reza.Barekatain@sa.gov.au

Perturbation of the intestinal barrier can lead to increased intestinal permeability (IP), elevated inflammation, high risk of enteric diseases and compromised performance in poultry. Common assessments of intestinal barrier function have been mainly through invasive methods involving differentially sized sugar tests, tissue sampling, or blood collection. Such methods are often complex, time-consuming and less field relevant. The present study aimed to identify selected biomarkers in excreta of broilers to facilitate the non-invasive assessment of gut barrier function. A 3 × 2 factorial arrangement of treatments was used with the main factors being gut barrier dysfunction models (control, rye-based diet, and dexamethasone – DEX) with and without probiotic supplementation (a three-strain Bacillus). Seventy-two male Ross 308 day-old chickens were kept in two groups given the same diets with or without probiotic supplementation. From days 13 to 21 of age, birds were individually housed and subjected to experimental treatments. Each of the 6 experimental treatments was replicated 12 times. On d 14, 16, 18 and 20, birds in the DEX group (n=24) were injected with DEX (0.5 mg/kg BW). Fluorescein isothiocyanate dextran (FITC-d) uptake into serum was used to test IP on d 21. Fresh excreta samples were collected on d 20. The excreta concentrations of Alpha 1 Antitrypsin (A1AT), Intestinal Fatty acid Binding Protein (IFABP-2), Fibronectin (FN) and Intestinal Alkaline Phosphatase (IAP) were measured using chicken specific ELISA assays. Data were subjected to two-way ANOVA to assess main effects and interaction. Treatment means were separated by Fisher's LSD test (P<0.05). DEX and rye-based diet depressed feed intake, weight gain and increased feed conversion ratio compared with control birds. Only DEX increased FITC-d passage to the blood, indicating a greater IP. The excreta concentration of A1AT and IFABP-2 were unaltered by the experimental treatments. DEX increased (P<0.05) FN concentration in excreta compared with control birds. Conversely, inclusion of rye in the diet reduced (P<0.05) FN. Independently, DEX decreased IAP (P<0.05) in excreta compared with control and rye-fed birds. There was no demonstrable effect of probiotic addition on any of the studied parameters. Subject to further validation studies the results reveal that FN and IAP, determined by ELISA, show promise as excreta biomarkers for rapid assessment of gut barrier function in poultry.
SUN DRIED AZOLLA ALEAF MEAL AFFECTS GUT MICROBIOME AND INTESTINAL DEVELOPMENT THROUGH REGULATION OF P70S6 KINASE IN BROILER CHICKEN

A. Abdelatty*(1), M.Mandouh(2), A.Al-mokaddem(3), S.Busato(4), O.Badr(5), S.Mohamed(6), A.Elolimy(7), M.Moustafa(8)

(1) Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt

(2) Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

(3) Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt
(4) Oregon State University, Oregon, United States

(5) Department of Genetics and Genetic Engineering, Faculty of Agriculture, Benha University, Qalyubia, Egypt., Benha, Egypt

(6) Department of Genetics and Genetic Engineering, Faculty of Agriculture, Benha University, Qalyubia, Egypt., Giza, Egypt

(7) Department of Pediatrics, University of Arkansas for Medical Sciences, Little rock, ar, United States

(8) Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

(9) Department of Animal and Rangeland sciences, Oregon State University, Oregon, United States

* Corresponding author: raramehmet@gmail.com

The inclusion of soybean in other industrial branches like biodiesel will reduce its availability for poultry feeding. Therefore, other feed alternatives are crucial to sustain poultry farming. Azolla leaf meal (ALM) was used as a novel alternative protein source in broiler chicken diet (Basak et al., 2002). However, the effect of ALM on gut health, intestinal development, and major bacterial population in broiler cecum is not known.

We hypothesized that inclusion of ALM in broiler chicken diet will affect growth performance and gut health of broiler chicken.

A total of 135 male Ross 308 broiler chicks were randomly allocated into 3 dietary treatments (3 replicate/treatment; 15 birds /replicate). The groups were CON; fed on diet contain 0% ALM, AZ5; fed on diet contain 5% ALM, AZ10; Fed on diet contain 10% ALM for 37 days, diets were formulated to be iso-nitrogenous and iso-caloric.

Feed intake and body weight were recorded at the end of each feeding stage (starter, grower, and finisher). Nine birds from each group were euthanized and used for sampling of cecal contents for microbiota study, and a segment from each intestinal part was collected in formalin for pathological evaluation with routine H&E staining using microarray technique. The abundance of total and activated P70S6 Kinase protein, the main activator of mTOR pathway that regulates cell growth, was measured by immunohistochemistry using microarray technique. Gene expression of selected bacterial genera was performed using qPCR, through isolation of 16S rRNA.

Data were analyzed via one-way ANOVA using the General Linear Models (GLM) procedure of SAS 9.4.
Significance was set at P ≤ 0.05 and tendency when P > 0.05 but P ≤ 0.10.

Feed intake and body weight gain were increased in both ALM fed groups (P = 0.02), body weight tended to increase (P = 0.10), however FCR was similar in all groups (P = 0.15).

Class Bacilli expression was downregulated in AZ10% (L = 0.03). Family Enterobacteriaceae was markedly downregulated in both ALM fed groups (L = 0.01).

The noted effect of ALM on P70S6 Kinase was in duodenum and jejunum. The ratio of activated: total Kinase was increased in duodenum of ALM fed groups, while, it was only increased in jejunum of AZ5 group (P
References

Basak, B., Pramanik, Md.A.H., Siddiqur Rahman, M., Tarafdar, S.U., Roy, B.C., 2002. Azolla (azolla pinnata) as a feed ingredient in broiler ration. Int. J. Poult. Sci. 1, 29–34.
THE POTENTIAL OF CANNABIDIOL AND NANOSIZED SELENIUM IN MODULATING IMMUNE RESPONSE
IN CLOSTRIDIUM PERFRINGENS CHALLENGED CHICKENS

P. Konieczka*(1), D.Szkopek(2), M.Kinsner(2), M.Michalczuk(3), J.Banach(4)

(1)The Kielanowski Institute of Animal Physiology and Nutrition, Olsztyn, Poland

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

(3)Warsaw University of Life Sciences WULS-SGGW, Institute of Animal Sciences, Warszawa, Poland

(4)Institute of Natural Fibres and Medicinal Plants, Poznań, Poland

* Corresponding author: p.konieczka@ifzz.pl

The process of inflammation resulting from specific challenging conditions can be controlled via preventive veterinary. Nutrients with bioactive properties are the potential mediators that present biological activities in different levels. This study investigated actions of Cannabis sativa-derived cannabidiol (CBD) and nanosized selenium (nano-Se) on the expression of genes encodes cannabinoid receptors (CBD1 and CBD2) and peroxisome proliferator-activated receptor gamma (PPAR-γ) in brain and spleen of broilers not challenged or challenged with necrotic enteritis (NE).

A total of 216 male broiler Ross 308 were assigned to 3 groups with 8 replicates per group and 9 birds per replicate. From days 8 to 35 birds were feed: (i) control diet (C), (ii) diet supplemented with CBD and nano-Se, and (iii) diet as (ii) but birds were subjected to NE. At days 15, 16, 17 and 18 of life birds were challenged with C. perfringens in a subclinical dose. At day 23 of life, samples of brain and spleen were collected from 8 birds of each treatment. Differences between groups were determined using one-way ANOVA with a least significant difference test.

The body weight gain (BWG) for the period between days 9-23 was similar in C and diet with CBD and nano-Se, but was lower in challenged birds (P=0.018). BWG calculated for the whole period was similar in all groups (P>0.05). The feed conversion ratio (FCR) in the period of 9-23 days did not differ in C and treatment with CBD and nano-Se, but was worsened in challenged birds comparing to C (P=0.005). FCR for the whole period of the experiment did not differ between treatments (P>0.05). The expression level of CBD1 and CBD2 in birds brain and spleen was similar in all treatments (P>0.05). Expression level of PPAR-γ in brain did not differ between treatments however, in spleen it was significantly higher in challenged birds comparing to C and that fed CBD and nano-Se (P=0.003).

Our data indicated, that feeding chickens diets supplemented with CBD and nano-Se was not associated with the compromised performance, and it did not evoke higher expression of cannabinoid receptors genes. The results also suggest that feeding diets with CBD and nano-Se could positively stimulate immune system in challenged birds. The beneficial effect of such treatments was evidenced in up regulation of expression of gene determining immune function in birds.

This work was supported by the National Science Centre, Grant No. 2018/29/B/NZ9/01351
INGESTION AND DIGESTION
INFLUENCE OF CALCIUM LEVEL, CALCIUM SOURCE, AND PHYTASE ON PRECAECAL AMINO ACID DIGESTIBILITY AND INTESTINAL MICROBIOTA OF BROILER CHICKENS

J. Krieg(1), W.Siegert*(2), D.Borda-molina(2), D.Feuerstein(3), A.Camarinha-silva(2), M.Rodehutscord(4)

(1)University of Hohenheim, University of hohenheim, Germany

(2)University of Hohenheim, Stuttgart, Germany

(3)BASF SE, Lampertheim, Germany

(4)University of Hohenheim, Germany, Germany

* Corresponding author: w.siegert@uni-hohenheim.de

Reports on effects of phytase supplementation on precaecal digestibility (pcd) of amino acids (AA) are inconsistent. Phytase supplementation increased pcd of AA in some but not all studies. The Ca level of the diet previously has been shown to influence gastrointestinal phytate degradation. The Ca source might also be a relevant factor involved in phytate degradation. Therefore, we investigated whether pcd of AA is influenced by Ca level and Ca source and whether any effect is related to changes in microbiota composition in ileal digesta.

Six diets without mineral phosphorus were formulated with 5.8 or 8.2 g Ca/kg dry matter by inclusion of CaCO3, CaCO3+formic acid (FA), or Ca-formate (Ca-F). Each diet was used without or with phytase (1500 FTU Natuphos E/kg). Diets were tested in 6 pens with 15 broiler chickens each. Experimental diets were provided from day 16 post-hatch. Digesta from the terminal small intestine was collected and pooled on a pen-basis on day 21 and 22. Statistical models comprised Ca source, Ca level, phytase supplementation, and interactions between these factors as fixed effects, and a random block effect. Effects were considered to be significant if P<0.05.

The Ca level × phytase interaction was significant for pcd of all AA. Other interactions were not significant. Without supplemented phytase, pcd of all AA was lower at the high than at the low Ca level, with an average difference of 2.7 percentage points (pp) between Ca levels. No difference between Ca levels was determined when phytase was supplemented. The effect of Ca source was significant for all AA except for Cys. Adding FA to CaCO3 increased pcd of all AA except Cys by an average of 1.1 pp. Exchange of CaCO3+FA with Ca-F increased pcd of all AA except for Asx, Cys, and Hissignificantly, on average by 1.1 pp. Main effects, but not interactions were significant when microbiotaabundance in ileal digesta was analysed using permanova. Similar to pcd of AA, permanova showed asignificant difference in microbiota composition of CaCO3 compared to CaCO3+FA and Ca-F. Correlations of relative abundance and pcd of most AA were significantly positive for Lactobacillus johnsonii and negative for Gallibacterium sp. and Streptococcus alactolyticus.

The results suggest that dietary Ca levels, but not Ca sources contribute to inconsistency of phytase effects on pcd of AA found in the literature. Changes in pcd of AA may be connected to accompanying changes in microbiota composition.
EFFECTS OF NUTRITIONAL SUPPLEMENTATIONS ON GROWTH AND SELECTED BLOOD BIOMARKERS IN BROILER CHICKENS VACCINATED AGAINST COCCIDIOSIS

L. Bignon\*(1), C.Picart(2), O.Gestin(3), E.Crenn(3), B.Yang(2), C.Margetyal(4)

(1)WISIUM, Saint-gregoire, France

(2)ADM, Saint-nolff, France

(3)WISIUM, Saint-nolff, France

(4)ADM, Chierry, France

\* Corresponding author: laure.bignon@wisium.com

With the increasing concerns of drug resistance, vaccination against coccidiosis has become an important tool to control the disease. However, in practice vaccinated birds are usually observed with a deterioration of body weight gain. This loss could possibly due to the nutrients spared for immune systems, inflammatory and oxidative stress, as well as the damaged gut integrity. This study aimed to identify nutritional supplementations which compensate this loss. 960 Ross 308 males, including 240 non-vaccinated chicks (NV) and 720 hatchery-vaccinated (V) were weighted and divided into 64 pens with 8 treatments and 8 replications. Experimental treatments were as following: NVC: NV with coccidiostats, NVS: NV with standard diet, VS: V with standard diet, VArg: V with higher level of Arginine, VThr: V with higher level of threonine, Valgae: V and sulfated polysaccharides from algae, VEp: V and Echinacea purpurea, VPE: V with a blend of essential oils. The feed program included 3 phases: starter (0-10d), grower (11-20d) and finisher (21-35d). Chicken weight and feed consumption were measured at D10, D20 and D35. Blood samples were taken at D0, D20 and D35 to measure malondialdehyde (MDA) and glutathion peroxydase activity (GPx), as oxidative stress biomarkers. At D20, 2 chickens per pen were randomly selected to carry out the coccidiosis lesion score. At birth, V chicks were 1.5% lower than NV chicks (p<0.001). During starter period, no effect of treatment was observed on zootechnical performance. During grower period, NVC and NVS had significantly greater average daily gain (ADG; 63.7 g/d) than VPE (58.8 g/d) whereas the other treatments were intermediate (p=0.01). No effect was recorded on feed conversion ratio (FCR). During finisher period, no effect was seen on ADG but the FCR was improved for VArg and VThr (1.55) compared to NVS (1.59) (p=0.015). On the whole period, no significant difference was highlighted, but all nutritional strategies permitted a numerically improvement of growth (2 to 3%) in comparison with VS group. Concerning the total mean lesion score, it was low for all treatments, with the highest score in VPE (1.31) and the lowest in NVS (0.5) (p=0.019), the other treatments were intermediate. No significant effect of treatments was observed on GPx and MDA, but GPx of VArg and VThr tended to be higher than GPx of VS. To conclude, this work needs further investigations but these nutritional strategies seem to be promising.
INFLUENCE OF FEED FORM ON STANDARDISED ILEAL AMINO ACID DIGESTIBILITY OF PROTEIN SOURCES
IN BROILER CHICKENS

M. Barua(1), F.Zaefarian*(1), M.Abdollahi(1), T.Wester(1), C.Girish(2), R.Ravindran(1)

(1) School of Agriculture and Environment, Massey University, Palmerston north, New Zealand

(2) Evonik (SEA) Pte Ltd, Nordic european centre, Singapore

* Corresponding author: f.zaefarian@massey.ac.nz

Knowledge on the digestibility of amino acids (AA) in raw materials is critical for precise feed formulation and, for more efficient and sustainable use of feed resources. Published data on digestible AA values for broilers are based on mash feed, because of its simplicity. Broilers, however, are fed pelleted diets and the applicability of data generated with mash diets to pelleted diets could be questioned. Based on the above, it was hypothesised that broilers fed the same ingredient but in different feed form (FF) may show different AA digestibility values. Therefore, 336, 19-day-old male Ross 308 broilers were used to determine the influence of feed form (FF) on standardised ileal digestibility (SID) of nitrogen (N) and AA in different protein sources (PS). A completely randomised design was used to develop six experimental diets in a 3 × 2 factorial arrangement including three PS (i.e. meat and bone meal [MBM], soybean meal [SBM], and canola meal [CM]) in mash and pelleted forms. The inclusion rate of PS in three assay diets, were 383, 413 and 553 g/kg diet, respectively, and each assay diet contained about 18% protein. The basal endogenous N and AA losses were determined by offering a N-free diet (NFD) to broilers. The assay diets and NFD were fed to birds from day 19 to 23. The ileal digesta was collected from all the birds on day 23. The SID of N was higher (P < 0.05) in SBM followed by MBM and CM. The average SID values for indispensable AA (IAA) were similar in SBM and MBM and greater (P < 0.05) than CM. Average SID values for dispensable AA (DAA) was significantly (P < 0.05) higher in SBM than MBM and CM. Histidine was the only indispensable AA influenced by FF, and its SID was reduced (P < 0.05) by pelleting. Pelleting, however, resulted in significant (P < 0.001-0.05) reduction in the SID of all DAA and average of all AA. Pelleting decreased (P < 0.05) the standardised digestible protein and average of all AA contents. Overall, present data demonstrates that FF can influence N and AA digestibility of PS, in particular digestibility of DAA. Therefore, FF should be taken into account in AA evaluation assays.
IMPROVING FEED EFFICIENCY BY BOOSTING DIGESTIVE AND EXOGENOUS ENZYMES THANKS TO CLAY AND ALGAE

M. Gallissot\*(1), P. Aleixo(2), L. Albino(2), H. Rostagno(2), R. Pereira(1), M. Rodriguez(1)

(1) Olmix Group, Bréhan, France

(2) University of Viçosa, Minas gerais, Brazil

* Corresponding author: mgallissot@olmix.com

Several types of feed additives are available to improve feed digestibility, including exogenous enzymes which target specific nutrients that are not digested by endogenous enzymes. Recently, an innovative algo-clay complex (ACC) developed by Olmix Group (France) has shown interest in increasing the activity of endogenous digestive enzymes and feed efficiency. The present work aims at reviewing the effect of this ACC on broilers performance depending on the use of exogenous enzymes in the diet. Three different studies were implemented in the Poultry Research Center of the University of Viçosa (Brazil). All studies used Cobb 500 male broilers fed corn-soybean diets. Animals were placed in pens of 20 (Study 1) or 22 (Studies 2 and 3) chicks, with 10 replicates per treatment. In each study, the treatments were control (no ACC supplementation) and test (with 0.1% ACC in all feeding phases). The diets in the 3 studies differed by their supplementation in exogenous enzymes: no exogenous enzymes were used in Study 1, phytase was used in all groups in Study 2 and phytase and carbohydrates (NSP enzymes) were used in all groups in Study 3. Results of the Study 1 showed a decrease in feed conversion ratio (FCR) when using the ACC in grower phase (day 22-35; -4%, P<0.05) and total cycle (day 0-35, -3%, P<0.05). Growth rate tended to be higher in the test group than in control the grower phase (+4%, P<0.10) with a final weight (at 35 days) of 2.449 kg in the test group and 2.392 kg in the control group (P=0.127). In the Study 2, the ACC supplementation lead to a decreased FCR of 2% in the total period (day 0-42, P<0.05) with the highest effect in the grower phase (-4%, P<0.05). The final weight (42 days) was increased by 78g in the test group compared to control (respectively 3.337 kg vs 3.259 kg, P=0.163). In the Study 3, the FCR (day 0-42) was decreased by 3% in the test group compared to control (P<0.05) with the highest effect in the finisher phase (-8%, P<0.05). Growth rate followed the same trend with an increase in average daily gain (ADG) of 4% (P<0.01), resulting in a 134g higher final weight (42 days). The highest effect was observed in the finisher phase with a 12% increase in ADG (P<0.01). All in all, these studies highlight the capacity of the ACC to improve feed efficiency and growth of broilers independently of the use of exogenous enzymes. The demonstration of a synergistic effect between the ACC and exogenous enzymes requires further investigation.
ID: 387

EXOGENOUS XYLANASE IMPROVES BROILER GROWTH PERFORMANCE BY INCREASING THE RATE OF DIGESTION

C. Matthiesen\*(1), D.Pettersson(1), A.Smith(2), A.Storm(1)

(1)Novozymes A/S, Lyngby, Denmark

(2)DSM Nutritional Products (UK) Ltd, Derbyshire, United Kingdom

\* Corresponding author: cfrm@novozymes.com

The effect of dietary supplementation with the feed-enzyme RONOZYME® WX (DSM Nutritional Products Switzerland), a mono-component xylanase, on broiler performance and digestibility kinetics was investigated in Ross 308 chickens. We hypothesized that the xylanase would improve growth performance through an increase in the intestinal rate of digestion. The study included 2850 1-day old male broilers, divided into 5 treatments using 19 pens per treatment and 30 birds per pen. The positive control (PC) diet was formulated for optimal growth and based on wheat, soya bean meal and corn. Corn was substituted with increasing amounts of wheat-by-products and rye in the negative control and negative control high viscosity diets with and without RONOZYME® WX. The negative control diets were optimized using the Ross 308 Nutrition Specifications 2014 except for a reduction in apparent metabolizable energy (1 MJ/kg) and digestible amino acid content. Data were analyzed by a mixed model with age as repeated measures, showing a positive effect on final body weight at d35 and increased average feed intake compared to PC birds. The xylanase supplementation resulted in a 3% reduction in the feed conversion ratio (P<0.001) and the European feed efficiency of the enzyme treatments were no different from PC, but higher than unsupplemented diets (P=0.02). The enzymatic activity of the xylanase on cereal cell wall components was illustrated by microscopy in several parts of the digestive tract including crop, duodenum, jejunum and ileum. This was confirmed by improved digestibility of organic matter and starch (P<0.001) in the first half of the small intestine in xylanase supplemented birds, using TiO2 as a digestibility marker. The improvement was expressed less in the lower parts of the small intestine. This may indicate that the improved growth performance originated from a change in the intestinal site of digestion. It is speculated that the absorbed nutrients available for growth are greater with the dietary supplementation of xylanase, because of a more rapid digestion in the proximal part of the intestine. This may decrease the microbial uptake of nutrients in the distal part of the small intestine, thus ensuring an overall greater uptake of nutrients by the bird. In conclusion RONOZYME® WX improves the growth performance in broiler chickens and promotes the proximal digestion of organic matter and starch, giving a higher final bodyweight at d35 than PC fed birds.
# THE DIGESTIBILITY OF SOLUBLE NON-STARCH POLYSACCHARIDES AND FREE OLIGOSACCHARIDES IN BROILER CHICKENS OFFERED CORN- OR WHEAT-BASED DIET

E. Kim*(1), N.Morgan(1), A.Moss(1), L.Li(3), P.Ader(2), M.Choct(1)

(1)University of New England, Armidale, Australia

(2) BASF SE, Lampertheim, Germany

(3) BASF SEA, #35-01 Suntec Tower One, Singapore

* Corresponding author: ekim22@myune.edu.au

Poultry lack endogenous enzymes for efficient utilisation of non-starch polysaccharides (NSP), and therefore digestion of NSP is achieved by bacterial fermentation in the absence of exogenous enzymes. The efficiency of NSP digestion varies depending on animal age and the physical/chemical properties, feed processing and net concentration of NSP in the diet. This study aimed to investigate the digestibility of soluble NSP and free oligosaccharides in corn- or wheat-based diets. A total of 144 Cobb 500 mixed sex day-old broiler chickens were offered either wheat- or corn-based diets in pelleted form, with 12 replicates of 6 birds per treatment. Titanium dioxide was added at 5 g/kg to all diets as an inert marker and quantified by UV-spectroscopy to calculate the apparent digestibility. On 35 days of age, excreta was collected and pooled by pen and digesta was collected from the jejunum and ileum of four randomly selected birds per pen. Soluble NSP and free oligosaccharides in the diet, digesta and excreta samples were isolated by an enzymatic-chemical method and released constituent sugars were determined by gas chromatography. After testing for normality, an independent-sample t-test was used to compare the two treatments. The wheat-based diet was determined to contain greater soluble NSP content compared to the corn-based diet (15.9 vs. 6.2 g/kg DM), but free oligosaccharide concentrations were similar in both diets (36.9 vs. 37.2 g/kg DM). Apparent digestibility of soluble NSP in the jejunum and ileum was not affected by dietary treatment; but total tract digestibility was higher (P<0.001) in birds offered the wheat-based diet compared to the corn-based diet (44.8 vs. 23.2%). This suggests hindgut microbiota may prefer to utilise soluble NSP from wheat rather than corn. Apparent jejunal and ileal digestibility of free oligosaccharides was greater in birds offered the corn-based diet than those fed the wheat-based diet (P=0.006 and P<0.001, respectively). Total tract free oligosaccharide digestibility was higher in birds offered the corn-based diet compared to the wheat-based diet (82.3 vs. 75.83%, P=0.034). Thus, it appears free oligosaccharides from corn are more digestible throughout the small and large intestine compared to those derived from wheat. In conclusion, the digestive dynamics of soluble NSP and free oligosaccharides present in corn and wheat differ owing to the net quantities and source as well as intestinal environmental conditions.
ID: 517

COMPARING THE EFFECT OF TWO COMMERCIALLY AVAILABLE COCCIDIA VACCINE CHALLENGE ON APPARENT ILEAL NUTRIENT DIGESTIBILITY AND INTESTINAL MORPHOLOGY IN 21 DAY-OLD BROILER CHICKENS FED A CORN-SOYBEAN MEAL-PORCINE MEAT AND BONE MEAL-BASED DIET

S. Adedokun*(1), I.Adejumo(1), O.Olojede(2)

* Corresponding author: tayo.adedokun@uky.edu

The effect of two commercially available coccidia vaccine challenge (CVC) on the ileal amino acid, energy, calcium, and phosphorus digestibility and intestinal morphology was evaluated in 21 day-old broiler chickens. On day 14, one hundred and sixty-eight day-old broiler chicks were allotted to three treatments in a completely randomized design with seven replicate cages/treatment and eight birds/replicate cage. The treatments were the control, and control challenged with either Coccivac® B52 or D2 (Merck Animal Health). The Coccivac®-B2 contained live oocysts of Eimeria acervulina, E. maxima, E. mivati, and E. tenella while Coccivac®-D2 contained live oocysts of E. brunetti and E. necatrix in addition to those found in B52. All birds were fed a standard corn-soybean meal-porcine meat and bone meal-based diet from day 0 to 21. On day 14, the control birds were orally gavaged with 0.6 mL of distilled water, while the CVC birds were orally gavaged with 0.6 mL of distilled water containing 20x (approximately 2x of the recommended dose for day-old chick, by body weight) of B52 and D2 vaccine, respectively. Birds and feed were weighed on day 14 and 21. On day 21, birds were euthanized followed by ileal digesta collection. The mid-section of duodenum, jejunum, and ileum were removed for histology. Liver and spleen weights were also determined. Data were analyzed using the GLM procedure of SAS with mean separation using Tukey's test, where necessary. Specific contrast was used to compare the effect of B52 and D2. Bodyweight gain was highest (P < 0.05) in the control birds and lowest (P < 0.05) in the D2-challenged birds. Feed efficiency was lower (P < 0.05) in the D2-challenged birds compared to the control birds. The weight of the spleen relative to bodyweight for the D2-challenged birds was lower (P < 0.05) compared to the B52-challenged birds. Bone breaking strength (kgF) was lower (P < 0.05) in the B52-challenged birds compared to the control birds. Apparent ileal digestibility of dry matter, N, energy, Ca, amino acids, and digestible energy was lower (P < 0.05) in the CVC birds compared to the control birds. Duodenal and jejunal crypt depth, and villus height:crypt depth ratio was lower (P < 0.05) in the CVC birds compared to the control birds. However, ileum crypt depth was deeper (P < 0.05) in the CVC birds. Data from this study showed that both coccidia vaccine type reduced apparent ileal nutrient and energy digestibility compared to the control.

<footnote>(1)University of Kentucky, Lexington, United States</footnote>

<footnote>(2)University of Kentucly, Lexington, United States</footnote>
NEW DIETARY β-MANNANASE IMPROVES BROILER PERFORMANCE AND NUTRIENT DIGESTIBILITY

A. Fickler*(1), P.Ader(2), M.Francesch(3)

(1) BASF SE, Lampertheim, Germany

(2) BASF, Lampertheim, Germany

(3) IRTA, Constantí, Spain

* Corresponding author: anna.fickler@basf.com

Soybean meal is one of the main feed ingredients in poultry nutrition due to its high protein level and favourable amino acid composition. However, soybean meal contains non-starch polysaccharides such as β-mannans that are in general considered for being so called antinutritive factors. These factors can potentially decrease nutrient utilization and thereby impair performance of animals. Therefore, we evaluated the efficiency of a supplemented fungal β- mannanase in broiler on nutrient digestibility and growth. A total of 960 Ross-308 1 d-old male broilers were distributed to 24 pens with 40 animals each. The study was set up as a randomized complete block design, with two treatments replicated 12 times each. Dietary treatments were: a control diet based on corn-soybean meal without any additional enzyme supplementation, and the control diet supplemented with mannanase at 800 Thermostable Mannanase Units (TMU) per kg (BASF SE, Germany). Diets were fed in 2 phases (starter: 1-21d, finisher: 22-35 d). Performance from 1 to 35 d of age and apparent ileal digestibility (AID) of dry matter, energy, fat, nitrogen and amino acids at 36 d of age were measured. Mannanase increased daily gain and feed to gain ratio from 22-35 d (p<0.05), and daily gain from 1-35 d and final live weight at 35 d (p<0.05) (58.0 g/d vs. 60.4 g/d and 2071 g vs. 2156 g; respectively). Mannanase tended to improve overall feed to gain ration, from 1.474 to 1.459 (p=0.084). Moreover, the European Production Efficiency Factor was increased by mannanase from 384 to 411 (p<0.05). Mannanase increased the AID of dry matter, energy and fat (p<0.05), and nitrogen (p<0.001). In addition, mannanase increased the AID of all amino acids (p<0.05; p<0.01; p<0.001), on average by +2.4 percentage points, expect the AID of methionine. In conclusion, dietary mannanase increased the digestibility of the nutrients in the diet and thereby improved the performance of broiler chickens.
CHESTNUT TANNINS IN BROILER DIETS AND THEIR EFFECT ON NUTRIENT DIGESTIBILITY

K. Buyse* (1), L. Goethals (2), B. Wegge (3), E. Delezie (4), G. Janssens (5), M. Lourenço (6)

(1) ILVO, Melle, Belgium

(2) Sanluc International, Ghent, Belgium

(3) Sanluc International, Gent, Belgium

(4) ILVO, Animal Sciences Unit, Melle, Belgium

(5) Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

(6) 1 ILVO, Animal Sciences Unit, Melle, Belgium

* Corresponding author: Kobe.Buyse@ilvo.vlaanderen.be

The working mechanism of chestnut tannins and their effect on broiler metabolism are not yet fully elucidated. These tannins can be used as an additive to improve gut health and performance. The goal of this study was to determine the effect of chestnut tannins on nutrient digestibility of broilers fed maize-soy diets or wheat-rapeseed-palm oil diets. This trial is part of a larger project focused on unravelling the working mechanism of chestnut tannins.

Two contrasting basal diets, a corn-soy based and a wheat-rapeseed-palm oil based challenge diet were used to assess the interaction of chestnut tannins with dietary nutrients. These diets were formulated to similar nutritional value and chestnut tannins (Tanno-SAN®, Sanluc International, Gent, Belgium) were added to these diets (0 mg/kg, 500 mg/kg or 2000 mg/kg) resulting in 6 different treatments. 216 broiler chicks (Ross 308) of 21 days of age were allocated in 72 digestibility units (3 chicks per cage and 12 replicates per treatment) for a balance period of 5 consecutive days after which blood and faeces were collected to determine gross energy, crude protein, crude fat and uric acid. From these results apparent digestibility coefficients for gross energy, crude fat, metabolizable energy corrected for N, and crude protein corrected for uric acid were calculated. On blood samples non-esterified fatty acids (NEFA), triglycerides and glucose were determined. Results were analyzed with RStudio using linear models with dose of tannins and diet as factor.

Despite comparable nutritional values, there were significant differences between the two basal diets. Corn based diets resulted in better digestibility coefficients than wheat based diets. In contrast, addition of tannins showed no differences in all digestibility coefficients. There was no interaction between diet and tannin dose. Regarding blood values, corn based diets resulted in significantly higher NEFA and triglycerides values. Glucose levels were not different between diets. There was no effect of tannins on blood parameters nor significant interactions between diet and tannins.

From these results, it seems that chestnut tannins do not affect broiler digestibility. However, from other trials, an effect of chestnut tannins was observed on performance. We are currently looking in more detail to other parameters to help explain these apparently contradictory effects, but these results are not yet available at time of submission of this abstract.
ILEAL DIGESTA FLOW AND BASAL ENDOGENOUS LOSS OF PHOSPHORUS RESPONSE TO A PHOSPHORUS-FREE DIET WITH DIFFERENT FEEDING DURATION FOR BROILER CHICKENS

S. An(1), S.Hyeong(1), C.Kong*(1)

(1)Kyungpook National University, Sangju, South Korea

* Corresponding author: changsukong@gmail.com

The standardized ileal digestibility (SID) of phosphorus (P) is known to be more additive rather than apparent ileal digestibility for feed formulation and are calculated by correcting basal endogenous losses (BEL) which is commonly determined by feeding P-free diet (PFD). However, the impact of feeding length of PFD on BEL of P is not fully understood. Therefore, the objective of the present study was to determine the proper feeding duration of PFD to estimate the BEL of P for broiler chickens. A total of 384 male broilers (Ross 308) were fed a commercial starter diet for 14 days. On d 15, the birds were weighed and allocated to three treatment groups in a randomized complete block design with 8 replicate cages per treatment and 16 birds per cage. Birds in each of the treatment groups were fed the PFD for 2, 3, or 4 days, respectively. Chromic oxide was used as an index for determining the BEL of P. After feeding of PFD for 2, 3, or 4 days, the birds were asphyxiated with CO2 and ileal digesta and ileum samples were collected. Growth performance and weight of ileal digesta and ileum were measured for each sampling day. The birds fed the PFD for 2 days had lower (P < 0.05) final BW compared to the birds fed diet for 3 or 4 days. The relative dried weight of the ileum (mg/100 g of BW) was greater (P < 0.05) in birds fed the PFD for 2 days compared with the birds fed diet for 3 or 4 days. However, the amount of the dried ileal digesta (g/bird) was not affected by the duration of PFD feeding. The BEL of P was calculated to be 103.2, 102.1, and 110.6 mg/DMI in birds fed the PFD for 2, 3, and 4, respectively, but there was no significant difference among the treatments. The collection of ileal digesta from broilers fed PFD for 2, 3, or 4 days might not give different amount of ileal digesta as well as different values of BEL of P.

Keywords: feeding duration, phosphorus free diet, endogenous loss, phosphorus, broiler

References

Kong, C., and Adeola, O. 2013. Additivity of amino acid digestibility in corn and soybean meal for broiler chickens and White Pekin ducks. Poult. Sci. 92:2381-2388.

Kong, C., and O. Adeola. 2014. Invited review: Evaluation of aminoacid and energy utilization in feedstuff for swine and poultry di-ets. Asian-Australas. J. Anim. Sci. 27:917-925.
THE SUPPLEMENTATION OF BROILER FEEDS WITH CAPSICUM BASED ADDITIVE IMPROVES ANIMAL PERFORMANCE. THE RESULTS OF A META-ANALYSIS.

D. Menoyo(1), M.Blanch*(2), C.De blas(1), M.Ibáñez(1)

Capsaicin is the main active compound in capsicum oleoresin derived from red pepper (Capsicum spp.). Studies have demonstrated that the inclusion of plant extracts high in capsaicin can improve the productivity of broiler chickens. The meta-analysis is a useful tool to perform a joint analysis of different studies as it increases the statistical power of the test when comparing the mean of a control diet and a treatment (i.e. a feed additive). In this study, we used the technique of meta-analysis to address if the phytogenic additive based on capsaicin LUCTAROM® Convert (LOM) improves broiler chicken performance. A total of 8 in-vivo nutritional trials run by the company LUCTA S.A. between 2003 and 2019 were analysed. They were performed in different experimental facilities testing the effect of the additive LOM on average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio FCR and mortality in broilers fed from 1 to 21 and from 22 to 35-42 days of age. The data were analysed using a mixed model with LOM supplementation, sex, dietary fat type and their interactions as fixed effects and the trial as a random effect. Dietary fat level and age at the end of the growth period were included as linear covariates. The supplementation with LOM during the first period (1-21 days of age) significantly improved FCR 1.431 vs. 1.418 g/g for control and treatment respectively (P = 0.0062). Also, the additive inclusion tended to increase ADG (P = 0.052) with no effect on ADFI (P = 0.80) and mortality (P = 0.25). The positive effect of the additive on FCR tended to be higher (P = 0.060 vs. 0.198) in feeds with animal source as added fat rather than with vegetal. Similar findings were observed for the second period from 22 to 35-42 days of age. The supplementation with LOM in grower feeds improved ADG from 95.1 in control to 96.3 g/d in treatment groups (P = 0.028) and tended to improve FCR from 1,759 to 1,744 g/g (P = 0.065). Again the positive effect of the additive on FCR tended to be higher (P = 0.10) in feeds with animal source as added fat than with a vegetal fat source (P = 0.22). The meta-analysis of 8 trials with LOM shows a significant improvement of this additive based on capsaicin on broiler performance.

<footnote>(1)Universidad Politécnica de Madrid, Madrid, Spain</footnote>

<footnote>(2)Lucta S.A., Bellaterra, Spain</footnote>

<footnote>* Corresponding author: david.menoyo@upm.es</footnote>
THE IMPACT OF XYLANASE ON THE FATE OF NUTRIENTS AND NON-STARCH POLYSACCHARIDES ACROSS THE GASTROINTESTINAL TRACT OF BROILERS FED WHEAT DIETS

D. Kouzounis*(1), H.Schols(1), N.Soares(2), M.Jonathan(1)

(1)Laboratory of Food Chemistry, Wageningen University, Wageningen, Netherlands

(2)Huvepharma NV, Berchem, Belgium

* Corresponding author: dimitrios.kouzounis@wur.nl

Feed supplementation with enzymes active on non-starch polysaccharides (NSP) is widely practiced to improve animal performance. Enzyme inclusion has been reported to decrease the digesta viscosity facilitating feed digestion1. Xylanase addition could also lead to the formation of arabinoxylan oligosaccharides (AXOS), compounds exhibiting prebiotic potential2. The aim of this research is to determine the impact of xylanase on wheat NSP structure and passage through the gastrointestinal tract (GIT) and evaluate possible implications on animal performance.

During this research, 48 male broilers were assigned to two treatments following a randomized block design in 6 pens per treatment. The diets consisted of a wheat-soy control diet (WC) and a wheat-soy diet supplemented with commercial xylanase and cellulase (WE). Acid insoluble ash (AIA) was used as digestibility marker. After 28 days, the birds were euthanized and samples from gizzard, ileum and caeca were collected. Excreta samples were collected between day 24 and 28. Samples were freeze-dried and analyzed for dry matter, marker, starch, protein and total sugar composition.

Enzyme inclusion in the diet (WE) increased body weight and lowered feed conversion ratio compared to the control diet (WC). Additionally, enzyme supplementation resulted in increased apparent ileal digestibility of organic matter and total glucose, while the effect on individual feed components (protein, starch) is currently under investigation. The relative ratio of NSP xylose and arabinose to AIA was similar in the feed and ileum digesta for both WC and WE. These ratios decreased significantly in the excreta samples, suggesting carbohydrate fermentation in the hindgut. In excreta, the Xyl:AIA ratio did not differ between WC and WE but suggested a trend (0.1>p>0.05), indicating that xylanase improved xylose utilization in the hindgut. To further determine the impact of xylanase supplementation on NSP, ileal samples were subjected to Mass Spectrometry and the presence of AXOs was shown in WE, but not in WC. In both WE and WC, hexose oligomers (maltodextrins) were present as well.

Xylanase activity did not affect NSP passage in the GIT, but altered their structure, forming potentially prebiotic AXOs and improving glucose digestibility, leading to improved animal performance.

References

1. Kiarie et al. Nutr. Res. Rev. 26, 71–88 (2013)

2. Courtin, C. M. et al. J. Sci. Food Agric. 88, 2517–2522 (2008)
A NEW FUNGAL B-MANNANASE IMPROVES BROILER PERFORMANCE IN A XYLANASE AND GLUCANASE CONTAINING DIET

E. Van eerden\*(1), A. Fickler(2), P. Ader(2)

(1)Schothorst Feed Research, Lelystad, Netherlands

(2)BASF SE, Lampertheim, Germany

* Corresponding author: EvEerden@schothorst.nl

β-Mannans belong to the NSP fraction in feed. They are found in e.g. soybean meal, soybean hulls, and guar meal. They are considered anti-nutritional factors, potentially decreasing nutrient digestibility. Therefore, enzymes that break down β-mannans, i.e. β-mannanases, can possibly improve performance. Commercial broiler diets usually contain NSP enzymes such as xylanase and glucanase. Although its substrate is different, it is unclear whether β-mannanase adds value to diets already containing xylanase and glucanase. Thus, a broiler performance trial was carried out to study the effect of β-mannanase at two dose levels in the presence of xylanase and glucanase.

The trial had a randomized complete block design with 4 treatments and 12 replicates. Each replicate was a floor pen with 20 Ross 308 male broilers. Treatment 1 was a Control without NSP enzymes. Treatments 2-4 contained xylanase and glucanase (Natugrain® TS, BASF SE, Germany) added to the Control (= XG). Treatments 3 and 4 were additionally supplemented with β-mannanase (BASF SE, Germany) at 400 and 800 Thermostable Mannanase Units (TMU) per kg (= XGM400 and XGM800). All diets contained 500 FTU phytase (Natuphos E 5000G, BASF SE, Germany) per kg. Birds were fed with a 3-phase feeding regime: starter (0-14 d), grower (14-28 d) and finisher (28-35 d). The pelleted diets were based on wheat-maize-SBM and contained also barley, rye and soybean hulls. Drinking water and feed was available ad libitum during the whole trial. The data per phase and overall period were analyzed with one-way ANOVA.

Results: Broilers fed the XG diets showed a significantly improved FCR compared to broilers fed the Control diet in the starter phase, with no additional improvement due to mannanase. In the finisher phase, XG diets had a numerically lower FCR than the Control diet, which became significantly lower when β-mannanase was supplemented (XGM400 and XGM800). The overall experimental period showed a clear distinction between the different treatments. Adding xylanase and glucanase to the Control diet significantly improved FCR. Moreover, supplementation with β-mannanase resulted in an even further significantly improved FCR.

In conclusion, mannanase at 400 and 800 TMU/kg in NSP-rich diets had positive effects on FCR in broilers, even on top of xylanase and glucanase. The significant effect was found for both mannanase dose levels.
# THE EFFECT OF UNHEATED, LOW-TRYPsin INHIBITOR SOYBEANS ON THE DIGESTIBILITY AND GROWTH PERFORMANCE OF TOM TURKEYS REARED TO 14 AND 28 DAYS OF AGE

C. Evans*(1), J.Garlich(2), C.Stark(1), J.Grimes(2)

Two trials (T) were conducted to evaluate the effect of poult age on the optimal inclusion rate of a novel low trypsin inhibitor soybean in turkey starter diets. Poults were either raised in groups of 6 from hatch to 2 wk (T1) or groups of 5 from 2 to 4 wk (T 2). Male Hybrid Converter turkey poults (528) were randomly assigned to 48 battery cages. Poults were provided ad libitum access to water and feed. All finished feeds were iso-caloric and iso-nitrogenous with treatments defined by the inclusion of solvent extracted soybean meal (SBM), the unheated low trypsin inhibitor (LTI) soybean at 20, 30, or 40%, or the conventional unheated soybean (CB) at 20 or 40%. Poults and feeders were weighed weekly to determine body weight (BW) and feed intake (FI) to calculate feed conversion ratio (FCR). At d 14 and 28 (T1 & T2) excreta were collected and pooled by pen for the determination of apparent metabolizable energy (AMEn) and apparent lipid digestibility (ALD). All poults were euthanized via cervical dislocation and pancreas excised. Pancreas weights were expressed relative to poult BW (PRW). Overall, increased inclusion of the LTI bean from 0 to 40% linearly decreased BW (384, 350, 360, and 329 g, ± 14.2) and FI (415, 390, 375, and 370 g, ± 15.4) from 0 to 14 d, while increasing FCR (0.97, 1.11, 1.05, and 1.16, ± 0.05) and PRW (0.37, 0.45, 0.50, and 0.52%, ± 0.01) when compared to SBM. Based on performance and PRW of the 14 d poults, the TI was still present at levels sufficient to affect the protein digestibility. Performance was improved in T2 with older poults. At d 28, diets containing increasing LTI from 0 to 40% remained comparable to SBM in BW (1599, 1570, 1590, and 1545 g, ± 32.3), FI (1841, 1818, 1772, and 1731 g, ± 51.2) and FCR (1.75, 1.78, 1.70, and 1.74, ± 0.04), though PRW (0.28, 0.34, 0.37, and 0.41%, ± 0.01) remained greater in relation to increasing LTI inclusion. Feeding CB diets typically suppressed performance compared to SBM and LTI. At 14 d, feeding 20 or 40% CB reduced BW (312 and 240 g, ± 14.2) and FI (348 and 315 g, ± 15.4) and increased FCR (1.15 and 1.45, ± 0.05) and PRW (0.71 and 0.89%, ± 0.01). At 28 d, CB again reduced BW (1510 and 1409 g, ± 32.3) and FI (1694 and 1550 g, ± 51.2) with increased PRW (0.51 and 0.68%, ± 0.01), though FCR (1.77 and 1.80, ± 0.04) remained similar. Therefore, the more mature poult was better able to utilize the nutrients in the LTI diets.

<footnote>(1) Kansas State University, Manhattan, United States</footnote>

<footnote>(2) North Carolina State University, Raleigh, United States</footnote>

<footnote>* Corresponding author: caitlinevans@ksu.edu</footnote>
DIFFERENCES IN ORIGIN AND DOSE OF DIETARY ENDOXYLANASES RESULT IN DISTINCT ARABINOXYLAN DEGRADATION PATTERNS IN THE GASTROINTESTINAL TRACT OF YOUNG BROILERS FED WHEAT-BASED DIETS

A. Bautil\*(1), J.Buyse(1), M.Bedford(2), C.Courtin(1)

(1)KU Leuven, Leuven, Belgium

(2) AB Vista, Marlborough, United Kingdom

\* Corresponding author: an.bautil@kuleuven.be

Despite their general use in poultry feed, the predominant mode of action of endoxylanases in improving broiler performance is not fully understood. The abundance of different endoxylanases used in the field and the variable response to their application often complicates a clear understanding of the functionality of endoxylanase in vivo. In this study, we hypothesized that arabinoxylan (AX) hydrolysis and microbial fermentation in broilers fed a wheat-based diet is different for endoxylanases of different origin and at different dosages. To investigate this hypothesis, a digestibility trial was designed to provide further insights into the contribution of dose (10, 100, 1000 ppm) and origin (B. subtilis vs. N. flexuosa) of endoxylanases on the structure-function relationship of AX products formed in vivo in the hindgut of young (d 11) and old (d 36) broilers (Ross 308). The residual AX population, the AX digestibility coefficients and the metabolites produced during microbial AX fermentation were analyzed at the level of the ileum, caeca and feces. Data were statistically analyzed using a three-way ANOVA with enzyme, dose and age and their second order interactions as model effects. Endoxylanase supplementation resulted in a more extensive degradation of wheat AX and reduction in intestinal viscosity compared to the control (P < 0.05). In spite of their difference in substrate preference, solubilizing the wheat AX was clearly the main activity of both endoxylanases. Endoxylanases produced from B. subtilis stimulated precaecal digestion of dietary AX by creating a large pool of solubles, thereby increasing ileal viscosity compared to broilers fed an endoxylanase from N. flexuosa (P < 0.05). This latter endoxylanase mainly triggered caecal AX fermentation in the young broiler by delivering easily fermentable AX substrates having a low polymerization degree (P = 0.03). At least 100 ppm for either endoxylanase was required to obtain improved hydrolysis of the dietary AX along the GIT against the control (P < 0.05). The effects observed were particularly present in young broilers as they are more prone to anti-nutritional factors and dependent on the fiber degrading capacity of the young intestinal microbiome. From this study, it is clear that the origin and dose of endoxylanases added to a wheat-based diet determine the nature of the heterogeneous pool of AX substrates that is formed, which in turn affects intestinal viscosity and AX digestion at young ages.
EVOLUTION OF LIPID CLASSES ALONG THE GASTROINTESTINAL TRACT IN BROILER STARTER CHICKENS:
EFFECT OF SOYBEAN ACID OIL COMBINED WITH PALM OIL

B. Jimenez moya*(1), A.Barroeta(1), A.Tres(2), D.Solà-oriol(1), C.Garcés-narro(3), R.Sala(1)

(1) Animal Nutrition and Welfare Service, Department of Animal and Food Sciences, Universitat Autònoma de Barcelona, Barcelona, Spain

(2) Nutrition, Food Science and Gastronomy Department-XaRTA-INSA, University of Barcelona,
Barcelona, Spain

(3) Department of Animal Production and Health, Public Veterinary Health and Food Science and Technology, Faculty of Veterinary Medicine, Universidad CEU-UCH, Valencia, Spain

* Corresponding author: beatriz.jimenez@uab.cat

Acid oils are by-products from the refining process of edible oils, which have a similar fatty acids (FA) profile than their corresponding crude oils, but higher proportions of free FA (FFA: 40-90%). Their use in poultry nutrition could contribute to improving the sustainability of the food chain. It has been suggested that the absorption of fats is more important as a limiting step comparing with their hydrolysis. Therefore, it has been hypothesized that the study of the evolution of lipid classes (TAG: triacylglycerols; DAG: monoacylglycerols; MAG: monoacylglycerols, and FFA) during the absorption process will allow for a better understanding the differences observed in the digestibility of FA. The objective of the present study is to evaluate the effect of the combination of soybean acid oil (SA) with palm oil (P) on the lipid-class composition along the gastrointestinal tract (GIT) and excreta in starter broiler chickens. A total of 480 birds (16 per cage; 6 replicas per treatment) were assigned to 5 dietary treatments from 0 to 11d. A basal diet was supplemented at 6% with: P (TAG: 78%; FFA: 9%), S (TAG: 72%, FFA: 14%), SA (TAG: 28%, FFA: 56%), or the combination of 33%SA-66%P, and 66%SA-33%P. At 11d, content from upper and lower jejunum, upper and lower ileum, and excreta was collected for the determination of lipid-class content (mg/g) /Ti (mg/g). Data was statistically analysed by one-way ANOVA using the SAS statistical package. In general, TAG, DAG and FFA decreased along the GIT studied, but an increase of these lipid classes was observed in excreta. The highest disappearance of the main final lipolytic product (FFA) occurs from the upper to lower jejunum, which is associated with the highest FA digestibility values obtained in this segment. The replacement of SA by P caused lower levels of MAG and higher content of FFA in all GIT and excreta samples studied (P0.05), in agreement, these fats followed similar pattern in the digestibility of FA along the GIT. The results suggest that the study of the lipid classes can be used to understand the dynamics of fatty acids absorption. The inclusion of palm oil is not recommended in young broiler diets. Instead, soybean acid oil could be adequate in starter broiler chickens, obtaining similar FA absorption than soybean oil.
ID: 1190

# EFFECT OF FEEDING PROBIOTIC, ACIDIFIER OR NUCLEOTIDE ON THE PERFORMANCE OF BROILER CHICKENS

J. Doley(1), S.D*(1), J.Mahanta(1)

(1) College of Veterinary Science, Guwahati, India

* Corresponding author: debensapcota@yahoo.com

## EFFECT OF FEEDING PROBIOTIC, ACIDIFIER OR NUCLEOTIDE ON THE PERFORMANCE OF BROILER CHICKEN

Jyoti Doley, D. Sapcota and Jogadev Mahanta

Department of Poultry Science, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, India.

Substantial growth in the poultry industry has been achieved in the recent past mainly due to exploitation of various growth-promoting agents. Probiotics, acidifiers and nucleotide are the front runners among them. The present experiment has been planned to study the growth performance of broiler chickens by feeding probiotics (Bacillus subtilis, 1.6×10¹⁰ cfu/g), acidifier (a mixture of acetic, propionic and sorbic acids) or nucleotide in the ration. Day-old commercial Cobb-100 chicks (n=140) were randomly distributed into four equal groups, viz., T0, T1, T2 and T3 containing 35 chicks, which were further divided into 5 replicates of 7 each. The experiment was conducted under the randomized block design. Four dietary treatments were formulated as per BIS (2007) and were assigned to the above group as T0 (no growth promoters in feed, i.e., control), T1 (T0 with probiotic @ 50g/ton of feed), T2 (T0 with acidifier, @ 2 kg/ton of feed) and T3 (T0 with nucleotide, @1 kg/ton of feed). Feeding was accomplished for 6 weeks under the battery cage system of management. The experimental data were subjected to statistical analysis (Snedecor and Cochran, 1994).

From among all groups the probiotic fed showed significantly (Pth week this group achieved 1.62% higher body weight as compared to the control group. Parallelly, the retentions of dry matter, crude protein, ether extract, calcium and phosphorus were improved to the extent of 2.95%, 4.61%, 4.41%, 4.08% and 4.74%, respectively. The probiotics containing Bacillus subtilis organism might have helped in synthesizing more body proteins, lipids and minerals resulting in higher body growth. The broiler performance efficiency factor was highest in the probiotic fed group (98.30) followed by nucleotide (94.90), acidifier (93.33) and the control diet-fed group (91.82). Feeding of growth promoters reduced the intestinal pH whereas, significantly increased the carcass qualities. The additive fed groups showed significantly reduced blood cholesterol level, the result was more prominent in the probiotic fed group. The probiotic bacteria also improved the zootechnical parameters, gut health influencing overall performance. From among all, the probiotic fed group showed the lowest cost of feeding followed by the acidifier, nucleotide and control groups. It could be concluded that the use of probiotic fed group showed the best result followed by acidifier and nucleotide fed groups.

## Reference:

BIS (2007): Indian Standard: Poultry Feed Specifications, 5th Revision. Bureau of Indian Standards, New Delhi.

Snedecor, G. W. and Cochran, W. G. (1994). Statistical Methods. 18th Ed. Oxford and IBH Publishing Co., Calcutta, India.
ID: 1259

A SPECIFIC BLEND OF ESSENTIAL OILS AND OLEORESINS OF SPICES IMPROVES FEED EFFICIENCY AND REDUCE NITROGEN AND PHOSPHORUS LOSSES

J. Gabarrou*(1), G.Desrousseau(1), M.Sims(2), D.Hooge(3)

(1)Phode, Tersac, France

(2) Virginia Diversified Research Corp., Harrisonburg va, United States

(3)Consulting Poultry Nutritionist, Pleasant grove, United States

* Corresponding author: jfgabarrou@phode.fr

One new challenge for animal production is to reduce environmental impact like nitrogen (N) and phosphorus (P) losses. Considering the simple reduction in P and N recommendations might have a negative economic impact, search for alternative additives has been incentivised. The main goal of this experiment was to evaluate the effects of a blend of essential oils and oleoresins of spices on performance of broiler between 7 to 42 days compared to usual conditions of production.

A total of 1,200 newly hatched chicks (Ross-308) were randomly distributed (30/pen) into 3 groups: Negative Control (NC), Positive Control (PC) and Treatment (OLEO) respectively with 12, 14 and 14 replicates each. All broilers received from d 0 to d 7 the same non-medicated starter. At day 4 they all receive a previous dropping samples sprayed on the litter to apply a sanitary challenge. Thereafter, Negative Control group received only non-medicated feed (starter from day 0 to day 14, grower from day 15 to day 28 and finisher from 29 to 42). Birds from Positive Control group received 55 ppm of Bacitracin Methylene Disalicylate (BMD®) from day 8 to day 28 shuttled to 22 ppm of Stafac® from 29 to 42 days. In the tested group (OLEO), birds were received a phytogenic feed additive (Oleobiotec, Laboratoires PHODE - France) at 100 g/MT. Statistical analysis: A completely randomized design was used (P≤0.05), and means were separated by LSD (P=0.05). Statistical analysis was carried out using the General Linear Model procedure of SPSS®, Analytical Software, IBM USA.

The Control group presented significantly lower performances from 0-42 d compared to Positive Control or OLEO group (1.906 vs. 2.111 kg BW; 1.933 vs. 1.765 feed conversion ratio or FCR), confirming the negative effect of the challenge. Both groups (OLEO vs PC) presented statistically equivalent performance on mortality rate (5.48% vs. 6.19%), growth performance (2.061 vs. 2.111 kg live weight) and feed efficiency (1.775 vs. 1.765 FCR).

N and P losses are reduced in the OLEO group vs. NC (28% vs. 32% for N and 41% vs. 44% for P). These results could be explained by FCR and Live weight improvement in OLEO group.

Oleobiotec was an effective feed additive to improve feed efficiency and reduce Nitrogen and Phosphorus losses. Further studies need to be done to investigate whether this product improves digestibility of energy and nitrogen to help explain those results.
ID: 1287

EFFECT OF A SPECIFIC BLEND OF ESSENTIAL OILS AND OLEORESINS OF SPICES ON GROWTH PERFORMANCE OF BROILERS IS INFLUENCED BY CHALLENGE ACUITY

J. Gabarrou*(1), M.Létourneau-montminy(2), B.Medina(3)

(1)Phode, Tersac, France

(2)University of LAVAL, Quebec, Canada

(3)Probiotec, St hyacinthe, Canada

* Corresponding author: jfgabarrou@phode.fr

The consistence of plant extract efficacy is often discussed. A part of the explanation of variable result could be in the level of challenge animals have to face. The main goal of this study was to evaluate the effects of a blend of essential oils and oleoresins of spices, Oleo, with a specific focus on founding modulating factor of their response on growth performances of broilers from a meta-analysis approach. An exhaustive database including 25 trials performed from 2001 to 2018 with Oleo was built to quantify its impact on average daily gain (ADG) and feed conversion ratio (FCR) of broilers. Dietary treatments were either a positive control (C+) with antibiotic growth promoter, a negative control (C- ) without antibiotic or a treatment consisting in an addition of Oleo at 100 ppm. To test the hypothesis that Oleo response was influence by the challenge the bird experienced, when C+ and C- were both present in the same trial (n=9), the relative difference between them (C+/C-, %) was also calculated as Challenge Acuity Index and used as an X variable. Metabolizable energy and crude protein has been recalculated based on diet composition using table of feedstuff composition and expressed based on requirement of the genetic line studied. Regarding the range of trial durations and slaughter ages of broilers, the Y variables were converted as relative difference in FCR and ADG expressed in percent between Oleo and C+ (ADG_Oleo/C+, FCR_Oleo/C+) and Oleo and C- (ADG_Oleo/C-, FCR_Oleo/C-). The mixed procedure of Minitab (Version 18.0) was used with the effect of the trial as random effect and the dose of Oleo and C+/C- as X variables. When comparing Oleo and C- treatments, results showed 2.8±0.70% (P<0.001; R2=65%) reduction of FCR and 3.6±1.2% (P=0.006; R2=56%)

improvement of ADG with 100 ppm of Oleo. However, when comparing Oleo with C+ treatment there was no significant effect on FCR or ADG, indicating that Oleo and C+ are equal. The FCR_Oleo/C- was negatively correlated (Linear, P=0.03; Quadratic, P=0.02; R2=85%) with the Challenge Acuity Index (C+/C-) indicating that in challenge condition (when C+ performed better than C-), so in challenge condition, the reduction of FCR with Oleo addition was higher. This work help quantifying the average effect of Oleo and leads to better understand the variations observed between studies for the same alternative to antibiotics.
THE DOSE RESPONSE OF A PHYTASE ON THE DIGESTIBILITY AND ON THE BONE ASH CONTENT IN MALE BROILERS

S. Leleu*(1), E.Delezie(1), L.Nollet(2)

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

(2)Huvepharma NV, Berchem, Belgium

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

The objective of this trial was to investigate the effect of different doses of a phytase (OptiPhos) on the ileal digestibility and tibia quality of male Ross308 broilers. During the starter period, the diet wasthe same for all broiler chicks. From day 9 on, broilers were divided into eight treatment groups (pelleted grower): a positive control group (PC; corn-soybean meal based diet) and a negative controlgroup (NC, -1.5 g/kg available phosphorus (aP) and calcium), and six different doses of the phytase added to the NC: 250, 500, 750, 1000, 1250, and 1500 FTU/kg (T3 to T8). On day 17, broilers were transferred to digestibility units (six replicates of four or five broilers per treatment) and on day 27 theileal digesta and the right tibia were collected and pooled per digestibility unit. No significant differences were found in crude protein and crude ash digestibility. Adding the phytase (except for T5),significantly increased the total P digestibility (range 72.5-74.8%) compared to the PC (55.8%). Concerning the phytate P digestibility, all groups had a significantly higher digestibility (range 57.6- 81.1%) compared to the PC (41.1%). Compared to the NC (57.6%) only the two highest doses (T7 and T8; 74.3% and 81.1%, respectively) were significantly higher. The crude ash concentration of the tibia was significantly higher for all treatments (range 39.5-45.4%) compared to the NC (36.3%), where theaddition of the two highest phytase doses (T7 and T8; 43.6% and 43.9%, respectively) no longer significantly differed from the PC (45.4%). Based on these results, it can be concluded that adding thephytase at 1250 and 1500 FTU/kg could compensate for the decrease in available phosphorus.
FEEDING IN ALTERNATIVE PRODUCTION
SYSTEMS
GROWTH PERFORMANCE AND PHYSIOLOGICAL RESPONSES OF BROILER CHICKENS REARED ON DIFFERENT LEGUME PASTURES DURING DRY SEASON

O. Oke*(1), J.Akande(2), I.Sodipo(2), O.Smith(3), O.Onagbesan(2)

(1)Regional Centre for Poultry Science (CERSA), University of Lome, Togo, Lome, Togo

(2)Federal University of Agriculture, Abeokuta, Abeokuta, Nigeria

(3)Federal University of Agriculture, Abeokuta, Nigeria, Abeokuta, Nigeria

* Corresponding author: emaoke7@yahoo.co.uk

There has been a growing interest in pastured poultry production as a result of improved welfare and meat quality. The objective of this study was to evaluate the comparative benefits of different legumes pastures on the performance and physiological response of broiler chickens reared during the dry season in a tropical environment. The study involved the use of a total of 200 day-old unsexed Marshall Broiler chickens were used to investigate the growth performance and physiological responses of broiler chickens reared on different legume pastures. The chicks were weighed and assigned randomly into five experimental treatments namely; deep litter without access to run (DL), deep litter with access to Stylosanthes hamata (SH), deep litter with access to Stylosanthes guanensis (SG), and deep litter with access to Mucuna pruriens (MP) and deep litter with access to free run (FR) during dry season for 5 weeks. Each treatment had 4 replicates consisting of 10 birds each. Data were collected on growth performance, gait score, haematotological parameters, serum biochemical indices, tonic immobility and plasma triiodothyronine (T3) hormone. Data obtained were subjected to One-Way Analysis of Variance in a Completely Randomized Design using SAS (2008). Results showed that the weight gain and final weights of the birds reared on SH were higher (P < 0.05) than those of the other treatment groups. Generally, the feed conversion ratio of the birds on pastures was better than those without pasture. Throughout the experimental period, the different legume pastures had no significant (P > 0.05) effect on plasma triiodothyronine (T3) concentration. The gait score of the birds on different legume pasture were superior (P < 0.05) to those in the deep litter and free runs without pastures. The total protein of the birds in SH, SG, MP was similar but higher than those of DL and FR. Also, globulin of SH was comparable to those of SG and MP but higher (P < 0.05) than those of FR and DL while MP, SG, FR and DL were similar (P > 0.05). It was concluded that access to legume pastures; Stylosanthes hamata, Stylosanthes guianensis and Mucuna pruriens improved performance and gait score without adverse effect during dry season in the tropical environment. Access to Stylosanthes hamata appeared better than the other legumes.
ID: 141

EFFECTS OF ALTERNATIVE PROTEINS TO SOYBEANS AND LIPID SOURCES ON THE GROWING PERFORMANCE AND CARCASS AND MEAT QUALITY OF EXPORT CHICKEN

E. Baeza-Campone\*(1), M.Lessire(1), H.Juin(2), M.Guillevic(3),

G.Chesneau(3)(1)INRA, Nouzilly, France

(2) INRA, Surgères, France

(3) Valorex Company, Combourtillé, France

\* Corresponding author: elisabeth.baeza-campone@inra.fr

There is an increasing demand in the world for healthy food. In many countries, the place of chicken meat in diets is more and more important. Regarding nutritive value of chicken meat, the more variable component is lipid quantity and quality. The aim of the present study was to increase the amount of n-3 fatty acids (FA) in the edible part of broiler grillers dedicated to export market by testing different levels of extruded linseeds (LS) and rapeseed oil in growing and finishing diets. Another challenge was to replace a part of soybeans by faba beans. For that purpose, 3168 chickens from JV15 line were distributed into six groups. Each group comprised eight pens with 33 males and 33 females per pen. The groups were R1=control diet, R2=3 then 5% LS, R3=4 then 6% LS, R4=5 then 7% LS, R5=3 then 5% LS and low amount of soybean meal in the finishing diet, R6 = 3 then 5% LS and no soybean meal in the finishing diet. For each feeding period, all diets were isocaloric and isonitrogenous. The growing performance and mortality rate were recorded. At 31 days of age, all chickens were slaughtered in a commercial slaughter plant at an average weight of 1322 g. Thirty carcasses per group were cut to determine cut yields. The edible part of half carcass was removed to determine the chemical composition and fatty acid composition of meat with skin. The sensorial quality of fillet was evaluated on 20 carcasses per group after roasting. Data were tested with variance analysis. The effect of sex and group and their interaction were analysed by comparing means with a t-test and P value vs. 0.3 g in control group) without modifying growing performance, carcass composition and sensorial quality of meat. The environmental impact was favourable for the climate change but unfavourable for the land occupation. The extra cost of feed, in our experimental conditions, represented +3 to +8% for groups R2 to R6 compared to group control R1.
VARIETAL SELECTION AND TECHNOLOGICAL PROCESSES MAKE PULSES AN ALTERNATIVE OF SOYBEAN MEAL BY IMPROVING THEIR NUTRITIONAL VALUES

H. Juin*(1), M.Lessire(2), M.Guillevic(3), G.Chesneau(3)

(1)INRA EASM, Surgères, France

(2)INRA BOA, Tours, France

(3)Valorex, Combourtillé, France

* Corresponding author: m.guillevic@valorex.com

A major challenge for poultry nutrition is to greatly reduce the use of synthetic inputs and to rely on more ecological processes, and consequently reduce soya bean meal in the diet. Faba beans may represent an alternative to soya but they are still poorly valued by animals; thus, improve nutritional values by genetics and processing may increase their interest in poultry. In this project, specific varieties and adapted technological processes (dehulling, extrusion) were used. Balance study was conducted on broilers, Ross strain male, between 21 and 25 days of age, with total and individual excreta collection for 3 days. Seven raw materials were compared simultaneously: faba bean alone, dehulled faba bean, faba bean 80% + full-fat soybean 20% extruded, faba bean 80% + linseed 20% extruded, faba bean variety with low vicin and convicin 80% + linseed 20% extruded, dehulled faba bean 80% + linseed 20% extruded, dehulled faba bean 80% + dehulled linseed 20%. Diets consisted in a basal part for 60 % and the tested product for 40%. 8 groups of 10 birds were randomly allocated to one of the experimental diets, one group was used to determine the digestibility of basal diet. Dry matter, gross energy and nitrogen were analysed on tested products, diets and excreta, and uric acid on excreta. AMEn (apparent metabolisable energy) and apparent digestibility of nitrogen were calculated for each diet and tested product. For raw faba bean alone, dehulling resulted in an improvement of protein content (+10.6%), digestibility of energy (+12.7%) and nitrogen (+3.7%). When extruded and associated with faba bean, replacement of full-fat soybean by linseed, improved digestibility of energy (+25.3%) but reduce nitrogen digestibility (-3.1%). Compared to entire seed, dehulling of linseed improved protein content (+1.7%), digestibility of energy (+22.4%) and nitrogen (+8.5%). Selection of faba and linseed with low antinutritional factors improved digestibility of energy (+7.0%) and nitrogen (+4.7%). These results suggested that an appropriate selection of seeds, associated with an adapted technological treatment, such as dehulling and thermomechanical processing give opportunities for an improvement in nutritional value leading to a higher inclusion of local sources of protein for broilers.
ID: 483

USE OF ALTERNATIVE PROTEINS TO SOYBEANS IN BROILERS: EFFECT ON GROWTH PERFORMANCE AND ENVIRONMENT

M. Guillevic\*(1), H.Juin(2), M.Lessire(3), G.Chesneau(1)

(1)Valorex, Combourtillé, France

(2)INRA EASM, Surgères, France

(3)INRA BOA, Nouzilly, France

\* Corresponding author: m.guillevic@valorex.com

A major challenge for poultry nutrition is to greatly reduce the import of soybean meal and the use of synthetic inputs favouring the use of more ecological processes and the use of local alternative protein's sources. The objective of this trial was to evaluate the effect of different alternative strategies to soya bean in broiler on zootechnical, environmental and economic point of view. For this, 1080 broilers, Ross strain males, were randomly distributed into 24 floor pen at D1 (1st day of life, day 1), and all fed a standard starter diet till D12, and then experimental diets : grower from D12 to D22 and finisher from D22 to D33. From D12 to D33, each pen was allocated to one the four experimental groups: R1 control group with soybean meal as main protein source, R2, with sunflower, rapeseed and corn gluten as protein sources, R3 and R4 with soya bean drastically reduced, but 15% and 20 % of crude or thermomechanical processed faba bean with low content in antinutrients in grower and finisher, respectively. Diets were calculated to provide the same nutritional values for AME and digestible amino-acids. At D22, there were no significant differences between the groups for growth performance. At D33, there were no significant differences between groups for liveweight (1792 g). Feed to gain ratios from D12 to D33 were 1.563, 1.618, 1.552 and 1.542 for R1, R2, R3 and R4, respectively, and were only significantly different for R2 and R4. No differences were observed between the groups for podermatitis and breast muscle yield. Soya bean meal consumption per animal between D12 and D33 was 576 g for control group, 195 g for low soya group (R2), 40 and 37 g for R3 and R4, respectively. The environmental impact is favourable to local pulses, and in particular for processed seed, for fossil fuel consumption (-15%, -16%, -21%) and in particular for climate change (-20%, -28%, -41%) but is unfavourable on the land occupation (-14%, +19, +14%) for R2, R3 and R4, respectively. Finally, the economic impact is neutral since it represents a saving for the average French consumption of 0.13 € / consumers / year for chicken consumption for R4. To conclude, alternative protein's sources and particularly processed faba bean, can contribute to reduce imported and GM soya bean meal in poultry diets, to maintain growth and feed to gain ratio, to be benefit for the environmental point of view and neutral for the economic point of view.
INTERACTIVE EFFECTS OF DIETARY LEVELS OF FIBER, PROTEIN AND ENZYMES SUPPLEMENTATION ON GROWTH PERFORMANCE AND SHORT-CHAIN FATTY ACID PROFILE IN BROILERS

Y. Lin\*(1), O.Olukosi(1)

(1)University of Georgia, Athens, United States

* Corresponding author: yl91334@uga.edu

A total of 384 Cobb 500 male broiler chicks were used in a 21-day experiment to examine the influence of xylanase and protease supplemented individually or in combination on growth performance and short-chain fatty acid (SCFA) profile in broiler diets with different levels of dietary fiber and protein. Birds were allocated to 16 treatments in a randomized complete block design. Each treatment had 6 replicates with 4 birds per replicate cage. The 16 treatments were corn-soybean meal basal diets with different low (L) or high (H) levels of protein (P) and fiber (F) to give LFHP, LFLP, HFHP and HFLP. Wheat bran was used to increase the dietary fiber content. The HF diets also had lower ME content. All the diets were supplemented with phytase at 500 FTU/kg). Each of the basal diets were divided into four: non-enzyme supplemented or supplemented with xylanase, protease, xylanase plus protease. Birds and feed were weighed on days 0 and 21 and ceaca content of birds were collected on day 21. Correlation analysis was done to relate growth performance and caecal short-chain fatty acids (SCFA) data in addition to ANOVA. There was no significant 3-way interaction for any of the responses except for FCR. FCR was lower in birds fed with individual xylanase and protease in LP, but not HP diets. Birds fed HF diets had greater (P
FEEDING BEHAVIOR OF LOCAL CHICKENS FED WITH DRIED LARVAE FROM MUSCA DOMESTICA,
CRACKED MAIZE, AND A LAYER DIET, IN CAFETERIA FEEDING SYSTEM.

S. Pousga*(1), F.Sankara(1), M.Kenis(2), G.Mensah(3)

(1)Universite Nazi Boni, Bobo dioulasso, Burkina Faso

(2)CABI, Delemont, Switzerland

(3)INRAB, Cotonou, Benin

* Corresponding author: pousgasalimata@yahoo.fr

The aim of this study was to evaluate fly larvae as protein source in traditional poultry feeding.
Specifically, the objective was to assess the feeding behavior of 18-month-old local chickens, choice-
fed with three types of food: a balanced laying diet, crushed maize, and dried domestic fly larvae. Fifty-
four chickens (54) were divided into three (3) batches representing three (3) periods of the day during
which the cafeteria test was conducted: in the morning at 8am; noon at 12 pm and in the afternoon at
3 pm. In each batch, the chickens were randomly divided into 4 groups representing replications with
4 birds in two replications and 5 birds in the other 2. The cafeteria test was conducted for 6 weeks.
Observations on the behavior of the chickens from the recording of filmed sequences showed that, at
the beginning of the test, the chickens were attracted to the feeders containing the crushed maize.
From the fourth week, the chickens were distributed homogeneously around the 3 feeders. At the
beginning of the 5th to the 6th week all the chickens went to the feeders where the larvae were. The
results showed average consumption of 6.7 ± 2.6 g; 7.2 ± 3.3 g and 4.8 ± 2.2 g per chicken respectively
for dried larvae, crushed maize and the layer diet (P ≤ 0.05). According to the time of the day, the
morning cafeteria showed consumption of 5.0 ± 0.1 g; 5.5 ± 0.2 g and 3.7 ± 0.1 g respectively for larvae,
crushed maize and layer diet (P ≤ 0.05). The corresponding values for the noon test were 8.1 ± 0.2 g;
8.41 ± 0.2 g and 5.6 ± 0.2 g respectively for larvae, crushed maize and the balanced diet (P ≤ 0.05). The
afternoon test showed 6.9 ± 0.2 g; 7.8 ± 0.2 g and 4.94 ± 0.1 g respectively for larvae, maize and the
balanced feed (P ≤ 0.05). The noon larval consumption (8.1 ± 0.2 g) was significantly higher (P ≤ 0.05)
compared to the morning (5.0 ± 0.1 g) and the afternoon (6.9 ± 0, 2 g). The daily intake of the layer
diet after the 30 min test was also higher (P ≤ 0.05) in the noon group (55.0 ± 1.00 g) compared to the
morning (48, 7 ± 0.8 g), and the afternoon (53.9 ± 1 g). In addition, Total daily intake of dietary
supplement (maggot + maize + layer feed) tested was also higher (P ≤ 0.05) at noon (77.2 ± 1.5 g)
compared to the morning (62.9 ± 1.0 g) and the afternoon groups (73.6 ± 1.4 g). It was concluded that
maggots are appreciated by chickens and are more consumed at noon.
ID: 1114

TRIPHALA: JOURNEY FROM TRADITIONAL MEDICINE TO GROWTH PROMOTER AND IMMUNE-
MODULATOR IN BROILER CHICKENS

M. Velusamy*(1), P. Shamsudeen (2), G. Rajmanohar (2), G.Marappan(3), S. Senthilkumar(1)

(1)Veterinary College and Research Institute, Namakkal, India

(2)College of Poultry Production and Management, Hosur, India

(3)Central Avian Research Institute, Bareilly, India

* Corresponding author: vmadhupriyavet@gmail.com

Triphala is called in different names at various parts especially Asia and Africa is an agglomeration three
fruits with various biological activities. They are being used in the traditional human medicine for
treatment gastric disturbances and also as a digestive stimulant. Known for its digestive stimulation
and immune-modulation property, the present study was carried out in broiler chickens to explore its
suitability for antibiotic-free meat production. The experiment was carried out with 300 one-day-old
male broiler chicks grouped into six treatments having five replicates each. The chicks were reared
from day-old to thirty-five days of age in deep litter system in a gable-roofed, open-sided poultry house.
The birds were fed with either one of the following six diets: basal diet without supplementation(T1) or
with supplementation of oxytetracycline at 0.004 per cent (T2), or with supplementation of Triphala at
0.025 per cent (T3), 0.05 per cent (T4), 0.075 per cent (T5) or 0.10 per cent (T6). The data were
subjected to analysis of variance single factor to interpret the observations. The experiment revealed
that the birds fed with T2 had significantly (P<0.01) higher body weight and gain than control, which
recorded the lowest values. The Triphala fed groups (T3, T4, T5 and T6) not differ significantly from the
antibiotic fed group in growth performance. The group T6 has the highest body weight and body weight
gain among the Triphala fed groups. The feed conversion ratio of broilers was significantly (P<0.05)
improved in T2 and T6 followed by T5, T3 and T4 groups than the control group. There was no
difference in livability among treatment groups as there were non-specific mortality among the groups
were observed. The immune-modulatory property was assessed in birds against the Newcastle disease
virus vaccine. The titre values in the group T5 was significantly (P0.05) with T5. Based on results from the
experiment, it could be concluded that traditional medicine Triphala could be used as an additive @
0.10% for improving the production performance and immune response in broiler chickens for
antibiotic-free production.
ID: 1171

# INCLUSION OF SILKWORM PUPA AS PROTEIN SUPPLEMENT IN BROILERS RATION

T. Yirgu*(1), M.Urge(2), T.Dessie(3)

(1)Ethiopian Institute of Agricultural Research, Addis Ababa, Ethiopia

(2)Haramaya University, Dire-dawa, Ethiopia

(3)International Livestock Research Institute, Addis Ababa, Ethiopia

* Corresponding author: tekalegney@gmail.com

Inclusion levels of silkworm pupa (SWP) and noug seed cake for soybean meal (SBM) as a protein supplement in broiler starter and finisher rations were evaluated at Debre-Zeit Agricultural Research Centre. The objectives of the study were to assess the performance of broilers on the basis of dry matter and nutrient intake, growth, feed conversion efficiency, survivability, carcass characteristics, as well as the economics of production. Two hundred twenty-five day-old Hubbard Classic unsexed chicks with uniform average group weight (42.32±0.297g (±SEM)) were randomly allotted to five dietary treatment groups in a completely randomized design (CRD) each with three replicates of fifteen chicks per replicate. The treatments were T1 that contains no SWP (0% SWP+30% SBM (the recommended level of SBM)), T2 (1.25% SWP+22.5% SBM), T3 (2.5% SWP+15% SBM), T4 (3.75% SWP+7.5% SBM) and T5 (5% SWP (the recommended level of SWP) +0% SBM) of the total rations. The treatment is based on substitution of SBM by SWP and NSC hence, the formulation also requires NSC to vary, therefore NSC increases as SBM is replaced by SWP. So as the level of SWP inclusion increased the noug seed cake was also increased. Every 1.25% SWP and (5% and 4% NSC for starter and finisher ration, respectively) inclusion, replaced 7.5% SBM in the total ration. The crude protein content of SWP and noug seed cake was 61.18 and 36.79%, respectively. During the 49 days of the experiment, dry matter and nutrient intake, body weight change, dry matter, and nutrient conversion ratio and efficiency, mortality, carcass characteristics, organ weight, and percent, and economic responses were assessed. The results revealed that daily DM (59.2-63.9g/bird/day/ (SEM=±1.65)), CP (12.4-13.4g/bird/day (SEM=±0.37)) and ME (404.8-430.2Kcal/bird/day (SEM=±10.8)) intake, and body weight gain (19.4-23.1g/bird/day (SEM=±0.87)) during the entire experimental period were not significantly (P>0.05) affected by the dietary treatments. The amount of dry matter consumed per unit of weight gain or dry matter conversion ratio (DMCR) (g/g) was higher (P
METABOLISM
IN VIVO ASSESSMENT OF THE EFFECT OF STARCH DAMAGE LEVEL IN SOFT AND HARD WHEAT CULTIVARS CAUSED BY PROCESSING AND ITS IMPACT ON AMEN AND STARCH DIGESTION RATE IN BROILER CHICKENS

Y. Khanfas*(1), R. Newkirk(1)

(1)University of Saskatchewan, Saskatoon, Canada

* Corresponding author: yousef.khanfas@usask.ca

Hardness of wheat grains may affect starch digestibility due to physical starch damage during feed processing. The aim of this study was to evaluate the level of starch damage (SD) in different wheat cultivars as affected by feed processing and its impact on starch digestibility and apparent metabolizable energy corrected to nitrogen (AMEn) of broiler chickens. The experiment was a 2 × 4 × 2 factorial RCBD design with two types of wheat (soft and hard), four cultivars of each and two feed processing methods (mash vs pellet). A total of 540 one-day old broiler chicks (Ross 708) were randomly distributed to 16 treatment diets consisting of 6 replicates (cage) with 5 birds per cage and fed treatment diets from day 7 to 22. Statistical significance was considered at (P ≤ 0.05). SD as a % of total starch was significantly lower (P<0.05) in mash soft wheat (6.62%) compared to hard wheat (9.76%). Pelleting increased SD % in both wheat types, 10.53 vs 12.28%, in soft and hard wheat, respectively. Body weight gain and feed intake were increased by pelleting (P < 0.05). Feed conversion efficiency was improved by pelleting especially in hard wheat cultivars, 1.35, 1.28, 1.25 and 1.20 for soft wheat mash, soft wheat pellet, hard mash and hard wheat pellet, respectively (P = 0.001). There was no effect on empty proventriculus weight (g/kg body weight), but relative empty gizzard weight was greater in mash diets as compared to pelleted diets (P < 0.0001). In mash diets, AMEn of hard wheat cultivars were higher than soft wheat cultivars (3157 vs 3386 kcal/kg DM) soft and hard wheat, respectively except paramount (soft wheat cultivar) which had a similar AMEn (3352) to the hard wheat cultivars. Pelleting increased the AMEn of both soft and hard wheats, but hard wheats had higher AMEn (P = 0.0001; 3267 vs 3396 kcal/kg DM for soft and hard wheat, respectively). Starch digestion at proximal jejunum and distal ileum in mash diets were significantly higher in hard wheat cultivars (P < 0.0001) compared so soft wheat cultivars. Pelleting increased starch digestion at proximal jejunum and at distal ileum of soft wheat cultivars (P < 0.0001; 60.85 vs 64.34% proximal jejunum) in soft and hard wheats, respectively, and (96.42 vs 98.61% distal ileum) soft and hard wheats, respectively.). These results indicated that pelleting increases starch damage in both soft and hard wheat and improves starch digestibility and AMEn.

Keywords: AMEn, starch damage, wheat cultivars, broiler.
A SYNERGISTIC BLEND OF GUT HEALTH ENHANCING FEED ADDITIVES IMPROVES PERFORMANCE OF BROILERS RAISED WITHOUT ANTIBIOTICS

Y. Van der horst\*(1), Y.Han(1), G.Page(1), W.Mansilla(3)

(1)Trouw Nutrition, Amersfoort, Netherlands

(3)Trouw Nutrition, Casarrubios del monte, Spain

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

With the reduction of antibiotics in their production strategies, the main concern of broiler meat producers relate to potential decreases in animal productivity. The objective of this study was to test different dietary solutions on their efficacy to increase growth performance in broilers raised without antibiotics (RWA = no in-feed antibiotic or coccidiostat use during production phase; coccidiosis vaccination at hatchery).

A total of 2,420 one-day-old, male Ross 708 broiler chickens were allocated to each of 44 pens with 55 birds in a randomized complete block design. Dietary treatments included negative control (T1), synergistic blend of organic acids and medium chain fatty acids including target release butyrates and a phenolic compound (SBSMBP; T2), T2 + copper hydroxychloride (T3), T3 + dietary structure in the form of oat hulls (T4). Treatments were all-vegetable based diets and were fed based on a 3-phase feeding program. Treatments were all-vegetable based diets and were fed based on a 3-phase feeding program. The animals were monitored for body weight (BW), average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) was calculated. Differences in least square means were analysed through SAS.

Analysis of overall cumulative data (day 0 – 36) showed a significantly higher ADG (+1.9 g/d) in broilers fed T3 and T4 relative to those fed the control diet (p p p = 0.067) compared to T1. Cumulative FCR was analysed with the exclusion of mortality weights to reflect local commercial FCR calculations. Broilers fed T3 exhibited a significantly lower FCR relative to broilers in T1 (-2.11%) and T4 (-2.70%) (pp = 0.36) and within typical commercial practice levels.

The present study demonstrates the efficacy of SBSMBP to significantly improve FCR and numerically increase market weights under RWA production conditions. The addition of copper hydroxychloride to the above mentioned treatment further improves RWA program performance, resulting in a significant improvement in both, market weights and lower FCR. Data suggests no added benefit of inclusion of dietary structure in RWA programs.
KEY METABOLITES PRODUCED BY THE PROBIOTIC BACILLUS SUBTILIS 29784 STRENGTHEN INTESTINAL BARRIER AND DAMPEN INFLAMMATORY RESPONSE

P. Choi(1), E.Pinloche(2), P.Thiery*(3), K.Sidelmann brinch(4), E.Devillard(2), E.Goossens(1), R.Ducatelle(1), F.Van immerseel(1), L.Rhayat(2)

(1)Ghent University, Faculty of Veterinary Medecine, Merelbeke, Belgium

(2)ADISSEO France SAS, CERN, Malicorne, France

(3) ADISSEO France SAS, Antony, France

(4) Novozymes A/S, Animal Health & Nutrition, Lyngby, Denmark

The probiotic strain Bacillus subtilis 29784 (Bs29784) produces a large number of metabolites. Niacin (NA), hypoxanthine (HPX) and pantothenate (PTH), are produced in high quantity in in vitro cultures. These key metabolites have been also shown to be produced in vivo, in the digestive tract of poultry, mainly in the upper part. The purpose of the present study was to determine the effects of those key metabolites on the development of the intestinal epithelium, the enhancement of the intestinal barrier function, and the resilience to inflammation using Caco-2 cell models.

Caco-2 cells were subjected to different treatments through incubation with NA, HPX, or PTH at a concentration close to what is measured in the gastrointestinal tract of broilers and were compared to non-treated conditions. Differentiation was assessed by measuring alkaline phosphatase activity after 72h treatment once the cells had reached confluence (on day 5). Resilience to inflammation was evaluated by measuring TEER (transepithelial electrical resistance) and cytokine release after 2h incubation with the treatment followed by 4h incubation with Cytomix (TNF/INF/IL1). For each condition, two biological replicates, with at least 4 repeats each, were carried out and data were subjected to the ANOVA procedure of XLSTAT.

None of the metabolites tested had an impact on cell differentiation. The drop of TEER observed following Cytomix stimulation was less pronounced with the addition of both PTH and HPX. HPX treatment also led to a decrease of IL6 secretion by 47.3% compared to the control treatment group (p<0.0001). The results of this in vitro study suggest that the key metabolites produced by Bs29784 have a direct effect on the host and could directly support the metabolic activity of the host epithelial cells, and therefore could reinforce the intestinal barrier at the level of the small intestine. In a next step, those observations should be evaluated in vivo in order to correlate the mode of action of Bs29784 and its effect on animal response.

<footnote>* Corresponding author: lamya.rhayat@adisseo.com</footnote>
EFFECTS OF CHRONIC INTESTINAL INFLAMMATION CAUSED BY DEXTRAN SODIUM SULFATE ON FUNCTION OF EGG PRODUCTION IN LAYING HENS

T. Nii*(1), T. Bungo(1), N. Isobe(1), Y. Yoshimura(1)

(1) Hiroshima University, Higashi-hiroshima, Japan

* Corresponding author: tanii@hiroshima-u.ac.jp

Intestinal health is important for sustaining normal meat and egg production in chickens. It is considered that gut environment such as microbiota, mucosal barrier function and cytokine production affects egg production in laying hens. Intestinal inflammation is known to change gut environment. Previously, we reported that oral administration of dextran sodium sulfate (DSS: 0.9 g/kg BW) for 5 days caused sever intestinal inflammation and reduction of egg laying in the laying hens. However, there is no long-term investigation for mild inflammation on egg production in laying hens with DSS. Thus, the goal of the study was to determine the effects of long-term intestinal inflammation caused by low dose of DSS on egg production in laying hens. White Leghorn hens (330-day-old) were oral administrated with or without 0.225 g DSS/kg BW in drinking water for 28 days (n = 8, each). Laid eggs and blood plasma were collected once every week. Cecal contents, intestinal tissues and liver were collected 1 day after the final treatment. These samples were used for the analysis of egg quality, intestinal environment and egg yolk precursor production. Operational taxonomic unit was decreased and beta-diversity was changed in cecal contents in DSS group compared with control group. The ratio of villus height/crypt depth was lower in the cecum and colon in DSS group. Gene expression of IL-6 in cecum was higher, and of TGF-β4 in colon was lower in DSS than control group. These results suggested that the oral administration of DSS caused intestinal environment such as change of microbiota in cecal contents and enhancement of intestinal inflammation in lower segment. Egg yolk weight was 15% decreased during 1-3 weeks of experiment in DSS group. While the gene expression of yolk precursor production related molecules in the liver was not changed by DSS treatment in liver, and plasma VLDL concentration was higher in DSS group. In conclusion, disruption of intestinal environment caused by DSS could reduce egg yolk size for long term by disorder of normal follicular growth. The disfunction of follicular growth may be caused by disorder of egg yolk precursor uptake in ovarian follicles, but not disorder on supply of yolk precursor.
ID: 1058

# CLONING OF CHICKEN GLUT13 AND ITS EXPRESSION FEATURE

P. Du(1), Y.Huang*(1), W.Chen(1)

(1)Henan Agricultural University, Zhengzhou, China

* Corresponding author: hyanqun@aliyun.com

## INTRODUCTION

In mammals, facilitated transport of glucose into cells is mediated by a family of facilitative glucose transporter (GLUT) proteins[1]. As the member of GLUT family, GLUT13 (HMIT or SLC2A13) exhibits transport activity for myo-inositol, but not for glucose in mammals [2]. SLC2A13 can be a potential markers for CSC in various tumors[3]. The aim of this study was to clone the transcriptional sequence of chicken GLUT13, and to detect the tissue expression of GLUT13.

## MATERIALS AND METHODS

The coding region of chicken GLUT13 was cloned by RT-PCR technique, and the biological characteristics of its encoded protein were analyzed by bioinformatics technology. The expression of GLUT13 in different tissues and time after insulin treatment was detected by qPCR.

## RESULTS

Chicken GLUT13 was successfully cloned based on predicted sequence (XM_001232939.5). It is predicted to encode a 459 AA protein. The CDS is 561 bp short than that in XM_001232939.5. Nucleotide and amino acid sequence alignment showed that chicken GLUT13 was highly homologous among species, and collinear analysis showed that the region of chromosome 1 where GLUT13 was located was also highly conserved among species. The real-time fluorescence quantitative PCR showed that the expression of GLUT13 gene was the highest in chicken brain and kidney, which is similar to the expression pattern of human GLUT13. Exogenous insulin significantly inhibited the expression of GLUT13 gene at 240 min in brain and kidney.

## DISCUSSION AND CONCLUSION

The predicted chicken GLUT13 was highly similar to known human GLUT13, While we gained the CDS region of chicken GLUT13 561bp short than the predicted sequence in NCBI, Which results to chicken GLUT13 lack of an MFS_HMIT_like domain which may result to the function change in chicken. Preliminary analysis showed that chicken GLUT13 may be related with glucose intake under insulin stimulation.

Key words: GLUT13; gene cloning; bioinformatics analysis; tissue expression

## Reference

1. Byers, M.S., C. Howard, and X. Wang, Avian and Mammalian Facilitative Glucose Transporters. Microarrays 2017. 6(2): p. 1-15.

2. Di Daniel, E., et al., Evaluation of expression and function of the H+/myo-inositol transporter HMIT. BMC Cell Biol, 2009. 10: p. 54.

3. Lee, J.H., et al., H+-myo-inositol transporter SLC2A13 as a potential marker for cancer stem cells in an oral squamous cell carcinoma. Current Cancer Drug Targets, 2011. 11(8): p. 966-975.
ID: 1063

LOWER AND HIGHER CONCENTRATIONS OF BUTYRATE REGULATE FAT ACCUMULATION IN CHICKEN ADIPOCYTES VIA DIFFERENT MECHANISMS

H. Li*(1)

(1) Shandong Agricultural University, Tai'an, China

* Corresponding author: hfli1228@163.com

**Background:** Short-chain fatty acids (SCFAs), mainly include acetate, propionate and butyrate, are the fermentation products produced by microorganisms using dietary fibers in the cecum and colon. The beneficial role of butyrate in health, such as combating obesity and type 2 diabetes mellitus, has been widely studied in mammals and humans. The role of butyrate in fat metabolism has been demonstrated to be dose dependent. Besides, lipid metabolism in chickens is different from that in mammals, of which liver is the primary site for fat synthesis, and the adipose tissue is mainly for fat deposition. Therefore, we speculated that there might exist differences in the regulation of butyrate on fat metabolism between chickens and that in mammals. In the present study, we determined the effects of serial concentrations of sodium butyrate (SB) on fat accumulation in chicken adipocytes.

**Experimental design:** Firstly, in vitro experiments were performed to detect the effects of serial concentrations of SB (0.01 to 2 mM) on fat accumulation in chicken adipocytes, by using Oil-red-O staining, Bodipy-staining, TG content determination, qRT-PCR, and western blot analysis. Secondly, the role of SB in cell proliferation was examined via EdU and CCK-8 assays in preadipocytes. Subsequently, the involvement of free fatty acid receptors (FFARs), extracellular regulated protein kinase (ERK) signaling, AMP-activated protein kinase (AMPK) signaling, and inhibition of HDAC in the role of low and high concentrations of SB was elucidated, respectively. Lastly, animal experiment was carried out to determine the influence of low dose butyrate (basal diets supplementing with 0.1% SB coated with polyacrylic resin Π) on fat deposition of broiler chickens.

**Results:** Both Oil-red-O and Bodipy staining showed that the 0.01 and 0.1 mM butyrate groups had fewer stained lipid droplets than the control and that droplet sizes did not change significantly. However, SB at or higher than 0.5 mM resulted in larger lipid droplets and reduced cell numbers, especially at the 1 mM and 2 mM concentrations. SB activated FFAR2 and FFAR3 mRNA expression, with 2 mM showing the highest expression compared to other groups. The 0.01 mM SB treated group, had the PPARG (peroxisome proliferators-activated receptor γ), FABP4 (fatty acid binding protein 4), FAS (fatty acid synthase) and LPL (lipoprotein lipase) mRNA levels statistically lower than those in the controls. Contrarily, SB-treated groups higher than 0.5 mM showed marked elevated expressions of all adipogenic genes tested. Western blotting revealed that adipocytes treated with 1 mM or 2 mM butyrate had upregulated protein expression of PPARG and FABP4 compared with untreated cells. These findings indicate that although both lower and higher concentrations of SB reduced adipocytic fat accumulation, they exhibited different cell morphology and distinct lipogenic genes expression.

Since butyrate at all concentrations increased FFAR2 and FFAR3 mRNA expression, siRNAs specific to the two receptors were used to down-regulate their expression. We found siRNA-FFARs reversed the lessened fat droplets under 0.01 mM SB treatment. Results indicated that neither the p-ERK/ERK nor the p-AMPK/AMPK ratio was markedly altered by 0.1 or 1 mM SB. Strikingly, 1 mM SB significantly inhibited HDAC activity. Therefore, TSA, which is a highly specific inhibitor of HDACs, was used to mimic the effect of SB. The acetyl-histone H3 protein expression level was elevated by the 1 mM SB and 10 nM TSA treatments, respectively. Oil red O staining and TG content determination showed that both 1 mM SB and 10 nM TSA groups accumulated more lipids than the control group. Similar to the effects of 1 mM SB, 10 nM TSA also vastly elevated FFAR expression, and moderately increased the FAS and LPL mRNA levels. These findings demonstrate that inhibition of HDAC activity is at least partially involved in the role of 1 mM SB in fat accumulation.
Animal experiments indicated that dietary supplementation of low dose coated SB (0.1%) inhibited fat deposition in both livers and abdominal fat tissues of broilers. Liver index was reduced in SB-treated animals at d 21. H&E staining showed that fat deposition in the livers was decreased with SB supplementation. In accordance, the TG content in SB-treated livers was significantly lower than that in control livers. In contrast to the controls, SB supplementation substantially although not significantly reduced the abdominal fat ratio at both d 21 and d 42, which was decreased by 16.5% and 17.6%, respectively. The transcription of some adipogenic genes (Srebp-1c and ACC in liver; PPARG in adipose tissue) was inhibited in SB-treated livers and adipose tissues. The serum TG level from SB-treated broilers was significantly decreased compared to that from the control animals. However, the growth of the broilers and the feed to gain ratio were not changed by SB administration. These findings indicate that fat deposition in broilers was reduced by dietary supplementation of low dose SB.

**Conclusion:** This study showed a novel understanding on the differential between lower and higher concentrations of butyrate on fat accumulation in chicken adipocytes, and the suppression of low dose SB on fat deposition ensured a promising application of using SB as feed additive in chicken breeding.
ID: 1222

LEVEL AND SOURCE OF DIETARY METHIONINE: THEIR RESPECTIVE EFFECTS ON GROWTH AND PROTEIN METABOLISM IN GROWING BROILERS

S. Tesseraud\*(1), J.Conde-aguilera(2), M.Lessire(1), D.Batonon-alavo(3), Y.Mercier(3), J.Van milgen(2)

(1)INRAe, Nouzilly, France

(2) INRAe, Saint Gilles, France

(3) Adisseo, Antony, France

\* Corresponding author: sophie.tesseraud@inra.fr

Like other amino acids (AA), sulfur AA affect protein metabolism. Protein synthesis and deposition are reduced when an insufficient level of sulfur AA is provided. This response may originate from the dietary AA deficiency and/or excessive AA utilization for other purposes such as the synthesis of acute-phase proteins during catabolic stress. Although dietary sulfur AA have been extensively studied, little is known about how different supplies and sources can affect protein metabolism. The aim of this study was to investigate growth performance and protein turnover of different tissues (liver and 2 types of muscles) in broilers receiving a diet either deficient or sufficient in sulfur AA and supplemented with either DL-Methionine (DL-Met) or DL-OH-Met (methionine analog) during 3 weeks. For the two Met sources, the supply of Met and sulfur AA in the deficient diets was respectively 43% and 30% below those of the sufficient diets. The birds were pair-fed to ensure the same feed intake and housed individually in wire-floored cages. Forty birds (10 birds per treatment) were used to determine protein synthesis (using the flooding dose procedure, Tesseraud et al., 1996), proteolytic enzyme activities, and expression of genes related to proteolysis. The deficient Met supply significantly reduced performance. In Met-deficient compared to Met-sufficient birds, the weight and protein mass of pectoralis major (PM) muscles were also lower (P<0.001), but not those of the liver and thigh muscles. Similarly, for both Met sources, Met deficiency decreased the amount of proteins synthesized per day in PM, but not in the liver and thigh muscles. Irrespective of the tissue studied, the proteolytic activity of proteasome was greater (P<0.01) in the Met-deficient groups compared with the Met-sufficient groups. Results on proteolysis-related gene expression measured in PM agreed with these findings. There were no differences between dietary DL-Met and DL-OH-Met sources on all these parameters denoting in particular that DL-OH-Met supports as DL-Met protein synthesis in PM when adequately supplied. In conclusion, a Met deficiency greatly alters chicken growth, PM development, and tissue protein turnover, whereas different Met sources do not.

Tesseraud S, Peresson R, Lopes J and Chagneau AM (1996) Br J Nutr 75: 853-65.
ID: 1390

EFFECTS OF EXOGENOUS MULTI-ENZYME CARBOHYDRASE AND CORN DERIVED PHYTASE ON MALE BROILER PERFORMANCE FED A REDUCED ENERGY DIET

S. Llamas-moya*(1), R.Adhikari(2), B.Lumpkins(3), M.Lanahan(4)

(1)Kerry, Naas, Ireland

(2) Kerry Ingredients and Flavors, Beloit, United States

(3) Southern Poultry Feed and Research, Inc., Athens, United States

(4)Agrivida, Woburn, United States

* Corresponding author: sara.llamasmoya@kerry.com

The objective of the current study was to evaluate the effects of multi-carbohydrase enzyme (MCE) containing alpha galactosidase, xylanase, and B-mannanase enzyme compared to a single xylanase enzyme and the effect of corn derived phytase over microbial derived phytase. A total of 3250, one day old Ross 708 broilers were randomly assigned to 65 replicate pens with 50 birds per pen. Pens were randomly allocated in complete randomized block design to one of the five treatments; 1) a positive control (PC) with 1500 FTU/kg of microbial fermentation derived phytase, 2) negative control (NC) with a reduction of 99 kcal/kg ME compared to the PC and phytase similar to PC, 3) the NC + xylanase, 4) the NC MCE, 5) the NC + multi-carbohydrase enzyme with phytase replaced by 1500 FTU/kg of corn derived Phytase. 0.15% ca and 0.15% P credit was provided to the diet for both source of phytase used in the experiment. Feed and water was provided ad libitum in a 4 phase feeding program for up to 48 days. Feed intake, body weight gain, and FCR was measured at d 14, 28, 42, and 48. Data was analyzed by one-way ANOVA using SAS 9.2 and tukey HSD test was used as post hoc comparison with significant difference considered at P≤0.05. Reduction of dietary energy in NC diet reduced (P≤0.05) the weight gain and FCR of broilers compared to PC diet in all phase of the experiment. Inclusion of both xylanase and MCE increased (P≤0.05) the weight of birds compared to NC diet in all phase of the experiment. Broilers fed MCE in the diets had higher (P≤0.05) body weight gain compared to birds fed xylanase enzyme on day 48. Replacing fermentation based phytase with corn derived phytase increased (P≤0.05) body weight gain at all phase of the study. The result of the study indicates the use of exogenous enzyme can improve BWG and feed efficiency in broilers by sparing more energy to the diet. Use of multi carbohydrate enzyme had benefit over the single xylanase enzyme by acting on different substrates of the diet. Inclusion of corn derived phytase improved the yield in broilers compared to the fermentation derived phytase while used at similar FTU and Ca and P matrix.
INTERACTION BETWEEN NEUROPEPTIDE Y AND FEEDING REGIMENS IN AMINO ACID METABOLISM OF THE CHICK BRAIN

P. Tran*(1), V.Chowdhury(1), M.Elhussiny(1), M.Furuse(1)

(1)Kyushu University, Fukuoka, Japan

* Corresponding author: vc-sur@artsci.kyushu-u.ac.jp

Recently, we identified that feeding behavior of neonatal chicks was characterized by short resting periods between brief times spent on food intake and reported the involvement of free amino acids in the brain as innate signals (Tran et al., 2015, 2016). We hypothesized that appetite-related neuropeptides and amino acids collaborate to centrally regulate feeding behavior in neonatal chicks. This study aimed to investigate the relationship between orexigenic effect of neuropeptide Y (NPY) and brain amino acid metabolism in neonatal chicks under different feeding regimens. In Experiment 1, 5-days-old chicks (n=7-10) were intracerebroventricularly (ICV) injected with saline or NPY (375 pmol/10 µl/chick) and then subjected to fasting and ad libitum feeding condition for 30 min. Brain samples (diencephalon) and plasma were collected after experiment to analyze free amino acid concentrations. In Experiment 2, we further investigated the influence of central administration of NPY on L-tyrosine catabolism in the diencephalon due to its significant decline by NPY injection under feeding condition in Experiment 1. Chicks 5-days-old (n=9) were ICV injected with saline or NPY (375 pmol/10 µl/chick) and then subjected to ad libitum feeding condition for 30 min. The diencephalons were collected to examine the mRNA expression of tyrosine hydroxylase. Statistical analysis was conducted by the Stat View after the elimination of outliers by Thompson rejection. The stimulation of food intake by central NPY was confirmed. The results showed that central NPY significantly decreased diencephalic L-phenylalanine and L-isoleucine levels in both feeding and fasting condition (PPP
EF3

GENETICS

QUANTITATIVE GENETICS

MOLECULAR GENETICS

NEW TRAITS FOR POULTRY BREEDING
QUANTITATIVE GENETICS
GENOMIC CHARACTERIZATION OF EXTREME FEATHER PECKING IN LAYING HENS

H. Iffland\*(1), R.Wellmann(1), S.Preuss, J.Tetens(3), W.Bessei(1), J.Bennewitz(1)

(1)Institute of Animal Science, University of Hohenheim, Stuttgart, Germany

(3)Department of Animal Science, University of Göttingen, Göttingen, Germany

* Corresponding author: hanna.iffland@uni-hohenheim.de

Feather pecking is a behavior frequently occurring in commercial layer flocks. It often leads to skin injuries and cannibalism. Besides economic losses, severe animal welfare problems can also not be ignored. Breeding against feather pecking is possible, but phenotyping the hens in a commercial environment is at present economically not viable due to a lack of appropriate techniques. The distribution of feather pecking is not homogenous. Several studies reported the presence of a subgroup of extreme feather peckers within groups of laying hens. It is assumed that the extreme feather peckers play a leading role in the development of feather pecking in poultry flocks. The genomic characterization of extreme feather pecking might help to select against this damaging behavior. We developed a statistical method to detect extreme feather peckers. A mixture of two negative binomial distributions was fitted to two feather pecking datasets. The first dataset consisted of about 900 hens of a large F2 cross of the 11th generation of two laying hen lines selected divergently for severe feather pecking. The second dataset consisted of about 500 hens of the 15th generation of these two lines. For phenotyping, the hens were kept in groups of about 40 animals and observed for in total 420 min over several consecutive days. The hens were genotyped with the Illumina 60K chicken Infinium iSelect chip. After data filtering, about 29,000 SNPs remained in the datasets. In both studies, the proportion of extreme feather peckers was about 35%. Hens in this subgroup showed on average over both studies 2.8 series of severe feather pecking and pecked feathers on average 8.53 times within the series. In contrast, hens in the remaining subgroup showed on average 1.63 series of feather pecking and pecked feathers only 1.65 times within the series. In order to identify the extreme feather peckers individually, the posterior probability of each hen belonging to the extreme feather pecking subgroup was calculated and used as a novel trait for genetic analyzes. Heritabilities for this novel trait were 0.35 in the F2 cross and 0.26 in the two divergently selected lines. A genomewide association study revealed a QTL region on chromosome 1. The putative candidate genes found in this QTL region are part of the GABAergic system, which has already been linked to feather pecking.
AN EQTL IN THE CYSTATHIONINE BETA SYNTHASE GENE IS LINKED TO OSTEOPOROSIS IN LAYING HENS

H. McCormack*(1), D.De Koning(2), N.Dominguez-gasca(3), A.Law(1), D.Morrice(1), R.Fleming(4), A.Rodriguez-navarro(5), E.Sanchez-rodriguez(6), R.Preisinger(7), M.Schmutz(8), V.Šmídová(9), F.Turner(10), P.Wilson(11), R.Zhou(12), I.Dunn(11)

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

(2)Swedish University of Agricultural Sciences, Uppsala, Sweden

(3)Universidad de Granada, Granada, Spain

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

(5)Universidad de Granada, Spain., Granada, Spain

(6)Universidad de Granada Spain., Granada, Spain

(7)Lohmann Tierzucht GmBH, Cuxhaven, Germany

(8)Lohamnn Tierzucht GmBH, Cuxhaven, Germany

(9)Mendel University in Brno, Brno, Czech Republic

The Roslin Institute, University of Edinburgh, Roslin, United Kingdom

(11)The Roslin Insitute, University of Edinburgh, Roslin, United Kingdom

* Corresponding author: heather.mccormack@roslin.ed.ac.uk

High incidences of bone fractures and other forms of skeletal damage are being reported in egg-laying birds across a wide range of modern husbandry systems. The physiological adaptations for egg laying make laying hens susceptible to osteoporosis, resulting in increased bone fragility and susceptibility to fracture. We have previously shown that genetic factors underlie the variation in the susceptibility of individual birds to osteoporosis and bone fracture and have demonstrated that genetic factors explain approximately 40% of variation in end-of-lay bone quality (1). A quantitative trait locus (QTL) of large effect, located on Chromosome 1, was characterised in a F2 population of hens divergently selected for bone strength (2).

The aim of this study was to confirm and fine-map the QTL in progressive populations of the white leghorn strain used as the foundation of the selection lines (1). A number of SNP markers were identified which had highly significant associations with tibial breaking strength (BS), the most significant of which had BS values of 200.4 vs 218.1 Newton (p<0.002) for the two genotypes.

The transcriptome of the tibia for each genotype indicated four differentially expressed genes at the locus, with one gene, cystathionine beta synthase (CBS), having a nine-fold greater expression associated with the allele for low bone quality. CBS catalyses the conversion of homocysteine to cystathionine in the transsulphuration pathway. The genotype associated with higher expression of the CBS gene and poorer bone quality showed a small but significantly increased concentration of homocysteine (19.13 vs 17.18 mM/L; p=0.016).

We have defined and validated markers predicting bone strength that can be used in selective breeding. The results suggest that differences at the locus surrounding the CBS gene are involved in phenotypic effects of the QTL on bone quality. The pivotal role of CBS in mammalian sulphur metabolism, and in particular the methionine cycle, may give us additional routes to manage bone health in addition to genetic selection.

1. Bishop SC, Fleming RH, McCormack HA, Flock DK, Whitehead CC. Inheritance of bone characteristics affecting osteoporosis in laying hens, Br. Poult. Sci. 2000; 41:33-40.

2. Dunn IC, Fleming RH, McCormack HA, Morrice D, Burt DW, Preisinger R, Whitehead CC. A QTL for osteoporosis detected in an F-2 population derived from white leghorn chicken lines divergently selected for bone index, Anim. Genet. 2007; 38:45-9.
TOWARDS A CANALISATION OF EGG WEIGHT. AN ILLUSTRATION IN DUCKS.

H. Chapuis*(1), L.Bodin(1)

(1)INRAE, Castanet tolosan, France

* Corresponding author: herve.chapuis@inra.fr

Canalization can be defined as the action of selecting to maintain a constant phenotype in fluctuating environments or to decrease the sensitivity to environmental variations. Here, canalization parameters were addressed by a heteroscedastic model involving genetic values for the mean and the variability (log variance) associated with a single phenotypic value: the egg weight of Muscovy female ducks from an experimental population.

Two successive generations (68 females in generation 1 and 83 females in generation 2) laid 6859 individually weighed eggs (mean egg weight μ=80.3±8.3 grams). Across females, the number of weighed eggs ranged from 20 to 87 while the standard deviation of egg weight ranged from 2.8 to 8.2. The estimation of genetic parameters through a homoscedastic model provided additive variance for the egg weight: σ_u^2=18.77±5.64 and residual variance: σ_ε^2=20.38±0.41. Consequently, heritability of egg weight was h^2=0.42±0.10 and repeatability was r=0.54±0.03, in accordance with literature. A Double Hierarchical Generalized Linear Model (DHGLM) was used to estimate the genetic and non-genetic effects affecting the mean (location parameters) and the residual variance (dispersion parameters). For the dispersion, the fixed effects of generation and age of layer were then retained beside the random genetic effect v which led to an estimation of σ_v^2=0.19±0.03. The magnitude of σ_v^2, accounted for a ratio of 2.4 for egg weight standard deviation, between extreme females exhibiting a low or high sensibility to environmental variation for egg weight. A genetic correlation of p_uv=-0.11±0.11 was finally estimated between genetic effects for location and dispersion of egg weight. This nearly null estimated correlation is favorable to implement a selection to reduce individual variability of egg weight. However, an improvement of the confidence interval for this correlation is needed. That will be obtained with data from future generations.

Such methodology could profitably be implemented in laying breeding companies in order to improve
homogeneity of products, which could easily be attained without impairing the selection goal on mean
values when the correlation between location and dispersion parameters is negative or close to zero.
ESTIMATION OF VARIANCE COMPONENTS AND HERITABILITY OF BODY WEIGHT GAIN IN IRANIAN NATIVE CHICKENS

M. Gholizadeh\*(1), H.Hafezian(1)

(1) Department of Animal Science, Faculty of Animal and Aquatic Science, Sari Agricultural Sciences and Natural Resources University, Sari, Iran, Sari, Iran

* Corresponding author: mh_gholizadeh@yahoo.com

Recently, much attention has been paid to native chickens due to increasing consumption demand and its resistance to environmental conditions. Investigation of chicken growth over time is critical for the genetic improvement of meat production. Twenty one generations of Iranian (Mazandaran) native chickens were used to estimate the variance components and heritability of body weight gain in different interval of ages. Body weight gains were recorded from hatching to 8 weeks of age (bwg0-8), from hatching to 12 weeks of age (bwg0-12), from hatching to sex maturity (bwg0-SM), from 8 weeks of age to 12 weeks of age (bwg8-12), from 8 weeks of age to sex maturity (bwg8-SM) and from 12 weeks of age to sex maturity (bwg12-SM). Variance components were obtained by the AI-REML algorithm under six different animal models by implementing or ignoring maternal effects and the covariance between direct and maternal genetic effects. Significant fixed effects (generation, hatch and sex) were detected using the GLM procedure of the SAS software. Also, for sex maturation related traits, Age at sex maturation was considered as covariate. The most appropriate model for each trait was determined by the Akaike Information Criterion (AIC). For all traits measured, maternal permanent environmental effect was significant. Estimates of direct heritabilities (h2) were, respectively, 0.17, 0.21 and 0.26, 0.07, 0.31 and 0.26 for bwg0-8, bwg0-12, bwg0-SM, bwg8-12, bwg8-SM and bwg12-SM. Ratios of maternal permanent environmental effect were, respectively 0.06, 0.05, 0.05, 0.02, 0.04 and 0.02 for bwg0-8, bwg0-12, bwg0-SM, bwg8-12, bwg8-SM and bwg12-SM. Significant maternal permanent environmental effect on traits studied highlight the importance of considering this effect in genetic evaluation of birds for body weight gain. Also, Estimates of heritability show that improvement through selection is possible.
MULTIPLE ANCESTRAL HAPLOTYPES HARBORING REGULATORY CAUSATIVE MUTATIONS
CUMULATIVELY CONTRIBUTE TO A MAJOR QTL AFFECTING DIFFERENT CHICKEN GROWTH TRAITS

Y. Wang(1), X.Cao(1), C.Luo*(2), Ö.Carlborg*(3), X.Hu*(1)

(1)China Agricultural University, Beijing, China

(2) Guangdong Academy of Agricultural Sciences, Guangzhou, China

(3)Uppsala University, Uppsala, Sweden

* Corresponding author: yuzhe891@163.com

Introduction

In depth studies of quantitative trait loci (QTL) can provide insights to the genetic architectures of complex traits. A most investigated major QTL on chicken chromosome 1 has been associated with growth traits in multiple populations, but there is still no conclusive consensus of causal mechanisms in the last two decades.

Materials and Methods

Here we fine-mapped this QTL in a fifteen-generation chicken advanced intercross population including 1,119 birds and explored in further detail using 222 sequenced genomes from 10 high/low body weight chicken stocks. We developed a low-pass sequencing (0.5× coverage) method to obtain millions of SNP markers in thousands of individuals with extremely low cost. We combined linkage analysis, association analysis, haplotype association, identical-by-descent analysis, transcriptome sequencing, ATAC-seq and dual-luciferase reporter gene assay to explore this QTL interval.

Results

The strongest association in F2 and F9 generation was detected to a mosaic haplotype model inside the QTL. We traced the history of chromosomal transmission and demonstrated polymorphisms at multiple loci in the region cumulatively contributed 14.4% of the genetic variance for growth. Further, nine mosaic precise intervals (Kb level) which contain ancestral regulatory variants were fine-mapped in other 10 breeds/lines and gene expression analysis also indicated that multiple genes within these 9 haplotypes might be involved in the body growth.

We used ATAC-seq to identify chromatin accessibility near these intervals and chose one of them to demonstrate the key regulatory role in the duodenum. CAB39L gene was the candidate genes and two regulatory SNPs were the causative mutations. Besides, combining the QTL information and gene expression of gizzard, we discovered 2 hub genes, MLNR and HTR2A, and a core small effect gene list which are related to food intake.

Conclusions

This is the first study to break down the detail genetic architectures for the well-known QTL in chicken.
It demonstrates the interaction mode of major-minor genes in biological pathway and provides novel
insights into the likely role of minor genes in complex trait development. This is a good example of the
fine-mapping of various of complex quantitative traits in any species and illustrates the common
genomic complexity of complex traits.
GENETIC CHARACTERIZATION OF INDIGENOUS CHICKEN POPULATIONS IN CANARY ISLANDS (SPAIN)

A. Torres\*(1), M.Fresno(2), J.Capote(2), M.Gibaud(3), M.Macri(4), G.Pizarro(5), A.Canales(6), J.Delgado(6)(6)

(1)Instituto Canario de Investigaciones Agrarias, La laguna, Sri Lanka

(2)Instituto Canario de Investigaciones Agrarias, La laguna, Spain

(3)Systèmes d'Elevage Méditerranéens et Tropicaux, Montpellier SupAgro, Montpellier, France

(4)Laboratorio de Genética Molecular Aplicada, Animal Breeding Consulting S.L, Córdoba, Spain

(5)Laboratorio de Genética Molecular Aplicada, Animal Breeding Consulting S.L., Córdoba, Spain

(6)Departamento de Genética, Universidad de Córdoba, Córdoba, Spain

Since their conquest, the Canary Islands became an important maritime trading station through which different chicken breeds were introduced. Nevertheless, over the centuries, the Canarian chickens have evolved as a breed, including a set of variables that distinguishes them for their rusticity and good performance for egg and meat production, which makes them potentially suitable for dual-purpose production. However, there are no studies about their genetic characterization. This study aimed at investigating the genetic diversity, relationship and population structure of 161 Canarian chickens derived from five colour varieties: Black Barred (46), Red Barred (41), Black (38), Columbian (37) and Partridge (36), using 30 microsatellite markers and then compared to a reference set of 18 breeds taken from BIOCHICKEN project. Number of alleles, expected and observed heterozygosity values and polymorphic information content (PIC) were calculated using MS®Excel Microsatellite Toolkit. The Genetix software v4.04 was used to calculate the global inbreeding of individuals within breed (FIS), population differentiation index (FST) and global heterozygosity deficit (FIT). Deviations from Hardy-Weinberg equilibrium using Bonferroni correction on loci was determined by using CERVUS software v3.03. All microsatellites were polymorphic, with a mean of 7.2 alleles per locus. The observed and expected heterozygosity ranged from 0.101 to 0.802 and from 0.339 to 0.891 for the all colour varieties, respectively. Thus, the means of the observed (0.618) and expected (0.667) heterozygosity were higher than those found in other Spanish and European chicken breeds using the same set of studied markers. The PIC values were highly informative for 24 markers (>0.50), and for the rest were fairly informative (0.25-0.50). Moreover, only 3 markers (MCW014, MCW067 and MCW034) showed significant deviations from Hardy-Weinberg expectations. Across the five colour varieties, the FIS, FST and FIT were 0.039, 0.044 and 0.081, respectively. The low FST value indicated a scarce genetic differentiation among them. Finally, Canarian chickens did not show genetic proximity with other Spanish breeds or with the commercial strains included in the study. The results of the current study can be used as baseline genetic information for conservation programs, which will allow the recovery and official recognition of the Canarian chickens as an autochthonous breed.

<footnote>* Corresponding author: aletor80@yahoo.com</footnote>
MORPHO-BIOMETRIC CHARACTERIZATION OF INDIGENOUS GUINEA FOWL (NUMIDA MELEAGRIS) POPULATIONS IN NORTHERN TOGO

A. Soara\*(1), E.Talaki(2), G.Dayo(3), K.Tona(4)

(1)Regional Excellence Center in Poultry Science (CERSA)/University of Lome-Togo, Lome, Togo

(2)Regional Excellence Center in Poultry Sciences (CERSA)/University of Lome, Lome, Togo

(3)Institut of Sahel (INSAH/CILSS), Bamako, Mali

(4)Regional Excellence Center in Poultry Sciences (CERSA)/University of Lome-Togo, Lome, Togo

* Corresponding author: edith.soara@gmail.com

The study which had for objective the morphobiometric characterization of indigenous guinea fowls populations was conducted between March and July 2018 in two agroecological zones in northern Togo. The study was carried out on 738 adult guinea fowl. Each guinea fowl was described by direct observation, and weight and body measurements were recorded. The results showed twelve (12) colours of the plumage with a dominance of Bonaparte (39.0%) and Pearl grey (27.1%) colours and a rarity of Cinnamon pied (0.7%), Isabelle (0.3%) and Blue coral (0.1%) colours. The plumage was mostly smooth (90.5%). The eyes were mostly brown (57.0%), but Albino guinea fowls predominantly had white colour (85.2%) for the eye. Regardless of the phenotype, the helmet was curved (73.2%) and mumps white-bluish colour (85.5%). In Atakora, the beak was brown (81.3%), the wattles were red-white (49.8%), the shanks were black-red (32.6%) and the toes were red (47.6%) while in the Dry Savannah the beak was red (52.9%), the wattles were red-bluish (38.4%), the shanks were black-orange (12.3%) and the toes were grey (35.2%). For body measurements such as beak length, drumstick length, body length and live weight, Dry Savannah guinea fowl showed significantly (p<0.05) higher values than those of Atakora guinea fowl. Moreover, guinea fowl with Pearl grey phenotype (1.36 ± 0.28 kg) were heavier (p<0.05) than the other phenotypes and appeared to be better for improving growth performance. However, further studies are needed to establish positive relationship between phenotype and biometric characteristics.

Key words: Guinea fowl, characterization, morphobiometric, diversity, Togo
GENETIC ANALYSIS OF EGG PRODUCTION AND EGG QUALITY TRAITS IN THREE VARIETIES OF
DOMESTICATED JAPANESE QUAILS HAVING DIFFERENT PLUMAGE

M. Madheswaran*(1), J.Jaywant(2), R.Narayan(2), V.Saxena(3), G.Mariyappan(2)

(1)ICAR-CARI, Uttar pradesh, India

(2)ICAR-CARI, Bareilly, up, India

(3)ICAR-CARI, Bareilly, up, India

* Corresponding author: monikakanimozhi@gmail.com

The ultimate goal of a poultry breeder is to improve the overall genetic economic worth of the bird through multi-trait selection. So, study of heritability and correlations between productive traits is of great importance in the development of poultry breeding programs. In this perspective, the study had been planned to evaluate genetic parameters for developing white egg shell variety, for commercial exploitation. Three varieties of J.quail CARI- Ujjawal, CARI- Brown and CARI- Suneheri maintained as a closed flock at CARI were used. The single pair mating was carried out in individual pedigree laying cages by using 160 sires of each variety with an equal number of dams. Pedigreed and fertile eggs were collected, incubated and hatched out. Five female layers of 40 dam-sire families from each variety was transferred to individual laying cages after 5th week of age for recording of egg production traits. Part-time egg production of each bird was recorded biweekly at 8th, 10th, 12th, 14th, 16th and 18th week of age. The genetic analysis of the data sets were done with Wombat - (Full sib analysis) mixed model analysis by REML. There were significant differences observed in the ASM and egg production at 8th week was influenced by varieties. Egg quality traits also significantly influenced by varieties. Heritability estimates of biweekly egg production of all ranged from 0.041 to 0.583. The h² estimate for ASM ranged as low heritable with coefficients 0.041 to 0.166, for all the three varieties. Egg weight was moderately heritable. The heritability estimates of egg quality traits range from high to low. The ASM was negatively correlated with the numbers of egg produced for all. But the correlation components with biweekly egg production were positive up to 18th week in all the three varieties. High positive correlations were obtained between the Haugh unit and albumen height and between yolk weight and yolk diameter. Egg shell color observed in different varieties shows that CARI-Suneheri, produces more white-shelled eggs. The heritability estimates of Egg production traits in CARI Suneheri were higher. Moderate to high heritability estimates reported in the present study may leads to Mass selection for rapid improvement in growth and production traits in CARI-Suneheri because growth and production traits are positively correlated and hence whenever one goes for improvement in body weight there would be simultaneous improvement on the egg production also.
PHENOTYPIC RELATIONSHIPS BETWEEN HOST RESPONSE TO LENTOGENIC AND VELOGENIC NEWCASTLE DISEASE VIRUS IN LOCAL GHANAIAN CHICKENS

B. Kayang(1), P.Botchway*(1), E.Amuzu-aweh(1), A.Naazie(1), G.Aning(1), H.Otsyina(1), H.Zhou(2), M.Walugembe(3), J. Dekkers(3),T. Kelly(2), S.Lamont(3), R.Gallardo(2), D.Bunn(2)

(1)University of Ghana, Legon, accra, Ghana

(2)University of California, Davis, United States

(3)Iowa State University, Ames, United States

* Corresponding author: bbkayang@ug.edu.gh

Newcastle disease (ND) is a highly contagious viral disease that constantly threatens poultry production. The velogenic (highly virulent) form of ND inflicts the most damage and can lead to 100% mortality in unvaccinated village chicken flocks while the lentogenic (non-virulent) form causes mild infections and is used in vaccine development. This study sought to identify phenotypic relationships between the response of local chickens in Ghana to lentogenic and velogenic NDV. Blood samples were collected from 1440 half-sib chicks and genotyped on a 600K Affymetrix chip in order to assign dams. At four weeks old, chicks were challenged with lentogenic ND virus (NDV) and then blood and tear samples collected for antibody detection and viral load estimation, respectively. Body weights were recorded weekly from hatch. Traits measured were pre- and post-infection growth rates (GR),antibody levels at 10 days post infection (dpi), viral load at 2 and 6 dpi, and viral clearance. The birds were then transferred to another facility and naturally exposed to velogenic NDV after anti-NDV antibody titres had reduced to 1:1000. Body weights, blood and tear samples were collected for analysis. Finally, the birds were euthanised and lesion scores (LS) recorded. Post-exposure GR, antibody levels at 21 and 34 days post-exposure (dpe), LS for trachea, proventriculus, intestines, cecaltonsils and average LS across tissues, were measured. Variance components and heritabilities were estimated for all traits using univariate animal models in ASREML with ecotype as a covariate. Pre-andpost-infection GR means were 6.26 and 7.93, respectively, but mean post-exposure GR was -1.96. Heritability estimates for the traits in the lentogenic trial ranged from moderate (0.23) to high (0.55) while those for the traits in the velogenic trial were very low, ranging from 0.00 (average lesion scores)to 0.08 (antibody levels at 21 and 34 dpe). There were some correlations within traits measured in thevelogenic trial and between traits measured in both trials. Lesion score means ranged between 0.52 (trachea) and 1.33 (intestine), with males having significantly higher (p<0.05) lesion scores compared to females. These results suggest that selection for the traits measured in the lentogenic trial may be more feasible compared to the velogenic trial, although there was some evidence of phenotypic correlations between the two trials. Keywords: local chicken, Newcastle disease, correlations
DETECTION OF QTL WHICH AFFECT EGG QUALITY TRAITS IN PUREBRED AND CROSSBRED LAYERS

P. Le roy*(1), D.Picard-druet(1), F.Hérault(1), T.Burlot(3)

(1)INRAE, Saint-gilles, France

(3)Novogen, Plédran, France

* Corresponding author: pascale.leroy@rennes.inra.fr

Genomic evaluation of egg quality traits was firstly carried out using ssGBLUP methodology in a purebred Rhode Island line and SNP effects were post estimated to detect QTLs. Secondly, performances from crossbred individuals issued from the same purebred line were added and new QTL locations were obtained. Egg quality traits were measured once a week on 7 982 purebred hens, from 60 weeks of age to 80 weeks of age. In addition, the performances of 37 232 crossbred hens were available. Each purebred male had 45 crossbred daughters in expectation, with unknown dams of different genetic type. Egg quality traits were measured once they reached 70 weeks of age. Purebred birds, i.e. 1 214 males and 1 148 females, were genotyped for 302 102 SNPs. Five egg quality traits were studied: egg weight (EW), eggshell color (ESC), eggshell strength (ESS), albumen height (AH) and egg shell shape (ESshape). Among the 26 QTLs detected using purebred performances, 20 were confirmed when crossbred hens performances were added and 10 new QTL locations were detected. Four to 8 QTL were detected per trait. All QTL influencing EW and ESshape detected in purebred were confirmed during the second analysis and 2 new QTL were detected for EW. Conversely, 2 or 3 QTL were not confirmed during the second analysis for ESC, ESS and AH.
GENETIC ANALYSIS OF FEED EFFICIENCY TRAITS IN BROWN-EGG LAYER CHICKEN

Q. Zhou\*(1), S.Gu(1), F.Lan(1), G.Li(2), Y.Yan(2), H.Li(2), G.Wu(2), C.Sun(1), N.Yang(1)

(1) China Agricultural University, Beijing, China

(2) Beijing Engineering Research Center of Layer, Beijing, China

* Corresponding author: 996042700@qq.com

Feed consumption represents a large percentage of production costs in the poultry industry. Genetic selection plays a predominant role in improving feed efficiency. This study aimed to estimate genetic parameters of feed efficiency and relevant traits in late laying periods of chickens. Feed consumption, body weight and egg mass were measured from 69 to 72 weeks of age in a pure line of 725 Rhode Island Red hens from a commercial breeding company. Feed and water were provided ad libitum for all birds. Metabolic body weight (MBW), daily body weight gain (BWG), daily egg mass (EM), daily feed intake (FI), feed conversion ratio (FCR) and residual feed intake (RFI) were calculated to evaluate phenotypic and genetic parameters. The phenotypes that did not follow a normal distribution were transformed for following genetic analysis in DMU packages by using AI-REML with animal model. Mean values of FI, FCR, RFI, MBW, BWG and EM were 119.42g/d, 2.42g/g, 0, 312.86g, 1.11g/d and 52.79g/d, respectively. The coefficients of variation (CVs) of FI and MBW were less than 10%, while CVs of FCR, BWG and EM were greater than 15%. Heritabilities for FI, FCR, RFI, MBW, BWG and EM were 0.38 ± 0.10, 0.30 ± 0.09, 0.51 ± 0.10, 0.56 ± 0.10, 0.04 ± 0.05 and 0.32 ± 0.09, respectively.

Phenotypic and genetic correlations among FI, FCR and RFI were high and positive, ranging from 0.31 to 0.86. Genetically, FCR was highly correlated with MBW (0.47), whereas that with EM was negative (-0.58). RFI was correlated with EM (0.20), whereas that of RFI with MBW was low (0.04). Notably, the standard error of estimates among FCR, RFI and relevant traits ranged from 0.06 to 0.84, suggested that genetic correlation should be evaluated using larger population in breeding practice. In addition, the phenotypic correlations between RFI and its component traits were near 0. Phenotypic correlations between FCR and relevant traits ranged from 0.21 to 0.31, except that with EM (-0.68). In conclusion, the high heritability estimates for both RFI and FCR suggested that feed efficiency can be directly improved by proper selection programs. Selection for low RFI could reduce FI without significant changes in EM, while selection on FCR will increase EM. The findings would help better understand the genetic background of feed efficiency and contribute to chicken breeding and further genomic studies.

Keywords: feed efficiency, heritability, genetic parameters, chicken
## QUANTIFYING TEMPORAL GENOMIC EROSION IN LOCAL CHICKEN BREEDS

C. Bortoluzzi\*(1), G. Restoux(2), M. Tixier-boichard(2)

(1) Wageningen University & Research, Wageningen, Netherlands

(2) INRA, Jouy en josas, France

\* Corresponding author: chiara.bortoluzzi.2@gmail.com

Livestock biodiversity is declining globally at rates unprecedented in human history. Of the avian species, local chicken breeds are the most affected, as their small population size makes them more susceptible to demographic stochasticity and genetic drift [1, 2, 3]. The maintenance of genetic diversity and control over genetic drift by breeding programs is well understood, but often overlooked [2]. In this study, we used temporal whole-genome sequencing data to assess the consequences of starting a breeding program on the genetic diversity, deleterious variation, and inbreeding of two local French chicken breeds, the Gasconne and Barbezieux.

Although genetic diversity declined over time in the two populations, the reduction was more pronounced in the Gasconne (Δπ: -0.0005) than Barbezieux (Δπ: -0.0001). The reduction in diversity is the result of an increased number of medium and long ROHs, which cover up to 29% of the Gasconne genome (ΔFROH: 0.0776) and 13% of the Barbezieux genome (ΔFROH: 0.0051). In the Gasconne we also observed a higher number of damaging mutations (ΔL: 0.0029) than in the Barbezieux (ΔL: 0.00016). Damaging variants were mostly found in medium (0.83, p-value: <2.2-16) and long (0.95, p-value: 2.2-16) ROHs, suggesting that recent inbreeding enables rare damaging mutations to exist in homozygous state [4].

Our study shows that the size of the founding population is an important factor that can overrule genetic drift. We also show that the structure of the breeding program is key to preserve genetic diversity and reduce inbreeding. We recommend screening of individuals for damaging mutations to become common practice in near future.

## References

[1] Beate D Scherf, Dafydd Pilling, et al. The second report on the state of the world's animal genetic resources for food and agriculture. 2015.

[2] Brett A Melbourne and Alan Hastings. Extinction risk depends strongly on factors contributing to stochasticity. Nature, 454(7200):100, 2008

[3] Chiara Bortoluzzi, Mirte Bosse, Martijn FL Derks, Richard PMA Crooijmans, Martien AM Groenen, and Hendrik-Jan Megens. The type of bottleneck matters: Insights into the deleterious variation landscape of small managed populations. Evolutionary Applications, 2019.

[4] Zachary A Szpiech, Jishu Xu, Trevor J Pemberton, Weiping Peng, Sebastian Zöllner, Noah A Rosenberg, and Jun Z Li. Long runs of homozygosity are enriched for deleterious variation. The American Journal of Human Genetics, 93(1):90-102, 2013.
MOLECULAR GENETICS
PROFILING OF MICROSATELLITE LOCI AND THEIR ASSOCIATION WITH LAYER ECONOMIC TRAITS IN
RHODE ISLAND RED CHICKEN

A. Sahu*(1), S.Kumar(2), S.Jain(3), C.R.(3)

(1)ICAR-NRC on Pig, Guwahati, India

(2) ICAR-Central Avian Research Institute, Bareilly, India

(3)ICAR-Indian Veterinary Research Institute, Bareilly, India

* Corresponding author: dramiyavet@gmail.com

The research work was carried out in Rhode Island Red (RIR) chicken to analyze the effects of various genetic and non-genetic factors on layer traits along with determination of allelic polymorphism at egg production-associated microsatellite (MS) loci and the influence genotypes on economic traits. Hundred and eleven pullets belonging to five hatches were used in the investigation which was maintained at institute poultry farm. The location of studied 10 MS loci were chromosome Z (ADL0273 and MCW0258), 1 (MCW0145), 2 (ADL0176 and MCW0044), 3 (MCW0103), 5 (ADL0023), 26 (MCW0069), E29 (ADL0158) and E48 (MCW0110). Data on body weight at 20 weeks of age (BW20) and layer traits viz. age at sexual maturity (ASM), egg weight at 28 weeks of age (EW28), egg weight at 40 weeks of age (EW40) and egg production up to 40 weeks of age (EP40) were analyzed by least squares analysis of variance taking sire as random and hatch as fixed effects. Least squares means of ASM, BW20, EW28, EW40 and EP40 were 135.19 ± 1.15 days, 1347.13 ± 15.28 g, 42.49 ± 0.27 g, 48.19 ± 0.42 g and 124.55 ± 1.94 eggs, respectively. Hatch effect was significant (P
COMPARISON OF TRANSCRIPTOMIC PROFILE OF PRIMORDIAL GERM CELLS ISOLATED FROM WHITE LEGHORN AND GREEN-LEGGED PARTRIDGELIKE CHICKEN EMBRYOS

A. Dunisławska*(1), M.Bednarczyk(1), A.Szczerba(1), M.Siwek(1)

(1)UTP, University of Science and Technology, Bydgoszcz, Poland

* Corresponding author: aleksandra.dunislawska@utp.edu.pl

The Green-legged Partridgelike (GP), a native, dual-purpose Polish chicken, became recognized as a breed at the end of nineteenth century. Presently, the GP chicken is protected from extinction by means of in situ preservation and is kept in a flock without subjecting to any selection process for more than 60 generations. The White Leghorn chicken (WL) is intensively selected for several dozen years mainly to improve reproductive traits. This breed has been used to create highly productive egg-laying hybrids. The germline chimeras have been successfully produced by the transplantation fresh or frozen/thawed donor primordial germ cells (PGCs) collected from GP into recipient WL embryos. The aim of the current study was transcriptome comparison of the WL and GP PGCs at three developmental stages: 4, 8 and 12 day of embryo development. PGCs were collected from 6 embryos at each embryo development stage for each breed and RNA was isolated with commercial kit for single cells application (GeneElute Single Cell RNA Purification Kit, Sigma Aldrich). The quality and quantity of RNA was controlled by 2100 Bioanalyzer instrument (Agilent). High quality and purity RNA was intended for microarray analysis (One-color microarray-based gene expression analysis, Agilent). Microarray analysis was carried out according to the manufacturer's protocol. After scanning and feature extraction, obtained data were analyzed by GeneSpring GX software (Agilent).

Statistically significant differences (p2) was demonstrated in the case of PGCs originating from GP in comparison to WL. Up - regulated genes are mostly involved in the pathways related to regulation of biological processes (578 genes) and cellular processes (548 genes), developmental process (362 genes) and anatomical structure development (348 genes).

The obtained results might suggest a faster development of GP embryos comparing to WL embryos.

The research was supported by the National Science Centre, grant no. UMO-2017/27/B/NZ9/01510.
ANALYSIS OF THE REGULATORY MECHANISM OF THE CHICKEN VANIN1 GENE: ROLES OF PPARALPHA AND MIRNA-181A-5P

Z. Gu\*(1), Z.Wang(2), J.Yu(3), N.Hua(3), L.Xu(3), W.Yao(4)

(1) Changshu Institute of Technology, Changshu, China

(2) School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, China

(3) School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, China

(4) College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China

* Corresponding author: zhilianggu88@hotmail.com

Introduction: Vanin1 (VNN1) is a pantetheinase that can catalyze the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies showed that VNN1 is specifically expressed in chicken liver and negatively regulated by microRNA-122, a class of lipid metabolism-related noncoding RNAs. However, the upstream transcriptional regulation mechanisms of VNN1 gene have not been fully elucidated in the chicken liver. Here, we aimed to investigate the roles of PPARα and miRNA-181a-5p in regulating chicken VNN1 gene.

Methods: 5'-RACE was performed to identify the transcription start site of chicken VNN1. JASPAR, TFSEARCH and miRanda were used to analyze the potential transcription factor binding sites in the 5'-regulatory region of VNN1 gene and miRNA binding sites in chicken VNN1 3'UTR. We used a knock-down function strategy to manipulate PPARα (or miRNA-181a-5p) expression levels in vitro to further investigate its effect on VNN1 gene. Mechanistically, luciferase reporter assays were used to explore the specific mechanisms by which PPARα (or miRNA-181a-5p) regulates VNN1 gene. These experiments were performed in triplicate wells and repeated for at least three independent trials.

Results: Several transcription factor-binding sites were recognized via sequence analysis of the 5'-regulatory regions, including HNF1α, PPARα and CEBPα. GW7647 (a specific agonist of PPARα) increased the expression level of VNN1 mRNA in chicken primary hepatocytes, whereas the knockdown of PPARα with siRNA was the opposite. Moreover, the predicted PPARα-binding site is indeed functional and plays a crucial role in the regulation of the VNN1 gene. In addition, VNN1 3'UTR contains a sequence that is completely complementary to nucleotides 1 to 7 of miRNA-181a-5p. Overexpression of miR-181a-5p significantly decreased the expression of VNN1 mRNA, probably by directly combining with the predicted target site of the VNN1 3'UTR.

Conclusion: We provide experimental evidence that PPARα plays an important role in the transcriptional activation of the VNN1 gene and that miRNA-181a-5p acts as a negative regulator of VNN1 gene expression in chicken hepatocytes.
SUPPLEMENTARY DIETARY BETAINE MODULATES PERFORMANCE, OXIDATIVE STATUS AND MRNA EXPRESSION OF HEAT SHOCK FACTORS AND HEAT SHOCK PROTEINS IN MEAT-TYPE CHICKENS UNDER HEAT STRESS

F. Eichie\*(1), A.Salako(2), S.Aggrey(3)

(1)Nigerian Institute of Animal Science (NIAS), Abuja, Nigeria

(2)University of Ibadan, Ibadan, Nigeria

(3)The University of Georgia, Athens, United States

* Corresponding author: francisca_eichie@yahoo.com

Heat stress (HS) negatively impacts poultry performances and distorts molecular and cellular functions. Betaine supplementation is associated with the regulation biological processes associated with heat stress. To better understand the molecular mechanism of dietary betaine supplementation in mitigation HS, 3-week old Abor Acres chicks were assigned to four treatments: two diets (19%CP + 0%betaine and 19%CP + 1%betaine) and two temperature levels (24°C and 40°C) and subjected to either acute heat stress-AHS (40C for 6 hours) or chronic heat stress-CHS (40C for 7days). Five birds per treatment were euthanized and the Pectoralis(P.) major, ileum and liver were sampled for mRNA expression of heat shock factors (HSFs) and heat shock protein (HSPs) genes. Data were analyzed using ANOVA at α0.05, relative expression of genes was calculated using the 2-ΔΔCT method and beta-actin was used as endogenous control. Elevated temperature reduced growth and feed intake under acute and chronic HS compared to their respective control counterparts. Betaine supplementation decreased corticosterone from 2.20±0.67 to 1.96±0.60 and improved FCR from 1.55±0.15 to 1.20±0.08, and decreased GSH:GSSG from 3.17±0.58 to 2.81±0.22 in AHS birds. Betaine supplementation differentially affected mRNA expression and was tissue specific. mRNA expression of HSFs and HSPs in the ileum under AHS were upregulated but downregulated in the liver and P. major respectively compared to the control. However, across the tissues, HSF2, HSF3, HSP60, HSP70 and HSP90 genes were all downregulated in the P. major except for HSF2 in the ileum and HSP90 in the liver under AHS with betaine supplementation compared to the control, while under CHS, betaine supplementation improved feed conversion ratio 1.56±0.07 to 1.41±0.18, decreased corticosterone from 2.25±0.72 to 2.08±0.58 and GSH:GSSG from 3.08±1.12 to 2.53±0.5. The mRNA expressions of HSF3, HSP60 and HSP90 were upregulated in the P. major; HSF3 and HSP60 were upregulated in the ileum and only HSF3 was upregulated in the liver. Across treatments in the liver HSF2, HSP60, HSP70, HSP90 except HSF3 were all downregulated. Betaine supplementation enhanced performance through improved feed efficiency, corticosterone and oxidation status under AHS. The changes in the heat shock factor- and heat shock protein- genes show tissue-specific regulation and may explain the efficacy of dietary betaine supplementation in mitigating heat stress in meat-type chickens.
IDENTIFICATION OF CHICKEN MT3 GENE PROMOTER FUNCTIONAL FRAGMENTS

J. Yu\*(1), Y.Wu(1), Y.Hu(1), X.Wu(1), L.Xu(1), Z.Gu(1)

(1)School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, China

* Corresponding author: bioyjf@sina.com

**Abstract:** Metallothionein 3 (MT3), is one isoform of the MTs that are low molecular weight proteins with rich-cysteine, which have high affinity to heavy metals. Our previous studies showed that the mRNA expression level of Mt3 in the chicken liver was raised highly after fasting. However, much less is known about the regulations of Mt3 gene in the chicken. In current study, the results of the quantitative PCR showed that the mRNA expressions of Mt3 in kidney and adipose tissues was higher than that of other tissues. Its level of expression in the liver was the next highest. In order to identify the transcription initiation site, the 5'UTR sequence of Mt3 with 72 bp was obtained using the 5'RACE (rapid-amplification of cDNA ends) method from chicken liver. The promoter deletion experiment was performed to find the functional segments to regulate Mt3 transcription and the potential binding transcription factors. The sequence from -1000 to +72 of Mt3 gene was analyzed and seven segments from the +72 site to different upstream sites of -57, -122, -220, -427, -556, -727 and -928 were amplified by PCR from chicken genome and were inserted into the reporter vector psiCHECK2. The relative activities of Renilla luciferase were measured after the reporter vectors being transfected to LMH cells for 48 hours, respectively. The results showed that the segment from -122 to +72 raised the activity of Renilla luciferase higher than the segment from -57 to +72. It indicated there were some cis-elements could upregulated Mt3 transcriptional level in the segment from -122 to -57. The analysis showed there were 4 clusters (-115 to -103, -106 to -95, -95 to -79 and -78 to -66) in this segment, containing 10 different potential transcription factor binding sites. After some oligonucleotides mutated in the segment from -79 to -66, the relative activity of Renilla luciferase was significantly decreased by the mutation segment from -122 to +72 to the wild segment. The 3 potential transcription factors of E2F transcription factor 1(E2F1), transcription factor AP-2 alpha (TFAP 2α) and TFAP 2β might upregulate Mt3 transcription through binding to the segment from -79 to -66. And the inferences would be verified through the co-transfection experiment of the reporter vectors and the overexpression vectors of these transcription factors, and ChIP in the future works.
ID: 413

DEVELOPMENT OF PARENTAGE ASSIGNMENT SNP PANELS IN PHEASANT (PHASIANUS COLCHICUS) AND GOOSE (ANSER ANSER)

S. Brard-fudulea*(1), M.Teissier(1), D.Guémené(1), L.Jaffrelo(3), D.Bourasseau(2), S.Tricoire(2), T.Ruer(4), M.Blanchet(4), B.Alletru(5)(6)

(1)SYSAAF, Nouzilly, France

(2)Gen'Ethic, Chambretaud, France

(3)INRA, Clermont-ferrand, France

(4) Grimaud Frères Sélection, Sèvremoine, France

(5)Orvia Gourmaud Sélection, Saint andré treize-voies, France

(6)CNRS, Montpellier, France

* Corresponding author: sophie.brard-fudulea@inra.fr

In avian selection programs pedigree recording are generally dependent on the use of individual caging and individual artificial insemination. In other production such as aquaculture, parentage assignment methods based on the use of genomic markers are widely used to establish pedigree (Vandeputte and Haffray, 2014). In pheasant and goose, parentage assignment may contribute to welfare improvement of reproducers and allow selection on new traits. In goose, an existing microsatellite panel reaches poor assignment rates (70%), and no tool was available for pheasant. The objective of FaisSigne and SNPoie programs were to develop assignment panels for both species. RAD-seq was performed to identify SNP (Single Nucleotid Polymorphism) on blood samples from commercial lines. DNA was pooled, (one pool/line and 15 individuals/pool). Each pool was sequenced with the Illumina HiSeq 3000 technology. In pheasant, 991K RAD loci were obtained, and 1.787 K in goose. To ensure identification of high quality SNP, several filters were used on sequencing depth, number of lines where a loci was sequenced, number of lines were a SNP was observed, number of observation of minor alleles, missing data, and minimum distance between SNP. After quality controls, 4.7 K SNPs were available for pheasant, and 3 K for goose. For each species, 288 SNP were genotyped on sire-dam-descendant trios with Kaspar technology. Genotypes were filtered on missing data, mendelian compatibility and Hardy-Weinberg equilibrium. In goose, 231 SNPs were kept. Minimum Allele Frequency (MAF) was estimated within line and the 96 SNP with the highest mean MAF were kept for the panel design. Parentage assignment with this panel was performed in a routine context on commercial goose lines using APIS R package (Griot et al., 2019). The high assignment rate (94% in one line for example) was in accordance with previous work on ducks (Teissier et al., 2019). The same developments are currently being conducted in pheasant to design a specific assignment panel.

Griot, R., Allal, F., Brard-Fudulea, S., Morvezen, R., Haffray, P., Phocas, F., Vandeputte, M., 2019. Mol. Ecol. Resour.

Teissier, M., Thébault, N., Riquet, J., Diot, C., Brard-Fudulea, S., Guémené, D., Alletru, B., Cornil, M., Bouleau, P., Blanchet, M., Le Mignon, G., Demeure, O., Vignal, A., 2019. Presented at the 2èmes Journées Techniques Génomique du SYSAAF, Rennes.

Vandeputte, M., Haffray, P., 2014. Front. Genet. 5.
MICROEVOLUTIONARY DYNAMICS OF CHICKEN GENOMES UNDER DIVERGENT SELECTION FOR
ADIPOSITY

H. Zhang\*(1), N. Wang(1), L. Leng(1), Y. Wang(1), S. Wang(1), Y. Li(1), Z. Cao(1), P. Luan(1), H. Yuan(1), Z-Q. Du(1), H. Li(1)

(1) Northeast Agricultural University, Harbin, China

* Corresponding author: huizhang@neau.edu.cn

**Introduction:** Decades of artificial selection have remarkably improved the performance and efficiency of animal production systems. However, little is known about the dynamics of genome microevolution during the intensive breeding process. Tackling the genome dynamics within the microevolution framework could improve our understanding of the genetic basis of complex trait. Material and methods: In this study, we sequenced 60 pooled-DNA samples in a time-series manner for birds used as parents from 15 generations (G4-G18) of chicken lines divergently selected for abdominal fat content for 19 generations. We explored how the chicken genomes changed dynamically by examining the genome heterozygosity levels and identified genome regions under positive selection using identity score (IS) and fixation index (FST). Furthermore, important genes for abdominal fat content were identified from time-series sequencing data coupled with a sequencing-based GWAS and multi-omics analyses. **Results:** Between our two chicken lines, continuous and directional selection on adiposity has caused striking phenotypic differences. We explored how the chicken genomes changed dynamically by examining the genome heterozygosity levels, which decreased significantly from 0.6% of G4 to 0.5% of G18. Analysis of allele frequency difference showed that the number of SNPs subjected to positive selection increased sharply from G9, which correlated closely with the pattern of selection responses, indicating that selection could recruit genomic variants to drive the phenotypic improvement. Between lines, FST increased but IS decreased from G4 to G18, whereas the opposite trend was found for within-line comparison. Divergent selection thus increased between-line but reduced within-line genomic differences. We reveal that artificial selection tend to recruit the preexisting variations of genes that related to adipose tissue growth. In addition, novel mutations contribute to the divergence of phenotype under selection, but less significantly than preexisting genomic variants. Integrating time-series genome sequencing, GWAS and multi-omics data further identified that genes involved in signaling pathways important to adipogenesis, such as autophagy and lysosome (URI1, MBL2), neural system (CHAT), and endocrine (PCSK1) pathways, were under strong selection. **Conclusions:** Our study provides insights into the microevolutionary dynamics of domestic animal genomes under artificial selection.
EXPRESSION LEVEL OF W-LINKED GENES IS UNLIKELY TO BE ASSOCIATED TO THE REPRODUCTION
TRAITS IN DUCKS

H. Gu\*(1), Z. Ning(2), Y. Chen(3), Y. Jia(4), L. Wang(3), X. Lv(3), W. Yang(3), L. Qu(2)

(1)China Agriculture University, Beijing, China

(2)China Agricultural University, Beijing, China

(3) Beijing Municipal General Station of Animal Science, Beijing, China

(4) Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China

* Corresponding author: guhc@cau.edu.cn

Birds have a ZZ/ZW system and are characterized by female heterogamety (ZW), and this sex
determination mechanism different from mammals means that the W chromosome follows female-
limit inheritance pattern and therefore the expression of W-linked genes may be related to female
fitness traits. Recent reports in chickens have shown that the female-selected chicken breeds have
higher gene expression levels compared to Red Jungle Fowl and male-biased selected breeds like
fighting chickens. Reproduction traits as the important "female fitness traits" are generally measured
in poultry by egg production performance. To determine whether the expression of W-linked gene in
duck is related to reproduction traits, we have performed the following experiment. DNA samples were
extracted from 3 ducks (1 Pekin duck, 1 Shaoxing duck, and 1 Mallard) were used for SMRT sequencing.
Jinding duck (JD), Pekin duck (PK) and Mallard (MD) represent high, medium and low fertility
respectively according to their egg production levels. The left gonad of 30 one-day-old ducks (5 males
and 5 females per breed) was collected to perform transcriptome sequencing. SMRT reads data of
three ducks were used for genome assembly at contigs level, all contigs blast with 12 known W-linked
gametologs and reference genome to identify candidate W-linked genes in duck. Transcriptome data
and PCR validation experiments were used to filter true W-linked genes. Finally, we obtained 31 W-
linked genes (containing known W-linked genes), and some genes are present in multiple copy
numbers. Analysis of RNA-Seq normalized reads counts found that there was no significant difference
in the expression of W-linked gene among ducks (JD, PK, and MD) representing different reproductive
capacity (paired t-test, p>0.05). By comparing the single gene expression of JD (high reproductive
capacity) and MD (low reproductive capacity), there were only 4 W-linked genes have significantly high
expression in JD (independent-samples t-test, p<0.05), is inconsistent with similar research on
chickens. Taken together, our results indicate that W-linked gene expression level was not significantly
correlated with reproductive capacity in ducks, and there is no evidence that short-term positive
selection of reproductive traits on ducks leads to high expression of the W-linked genes.

Keywords: W-linked gene, reproduction traits, duck, RNA-Seq
# GENOME-WIDE ANALYSIS OF THE GENETIC ARCHITECTURE FOR COMMERCIAL, LOCAL, FIGHT AND WILD CHICKEN POPULATIONS BY 600K SNP MICROARRAY DATA

J. Zhang\*(1), C.Nie(1), X.Li(1), Z.Ning(1), Y.Chen(2), Y.Jia(3), L.Wang(2), X.Lv(2),W. Yang(2),L. Qu(1)

(1) China Agricultural University, Beijing, China

(2) Beijing Municipal General Station of Animal Science, Beijing, China

(3) Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China

* Corresponding author: 1599436449@qq.com

**Abstract:** After chickens were domesticated, diversified chicken populations were formed by both natural and artificial selection, which help chicken enrich abundant genetic and phenotypic variation and could be used as an ideal genetic research model. To better understand genetic architecture of chicken populations under different selection pressures, we genotyped various chicken populations with specific selection targets, including local, commercial, fighting and wild ancestral chicken breeds, by using the 600K SNP array. We analyzed the population structure, genetic relationship, inbreeding index and effective population number (Ne) and some other genetic parameters.

The wild ancestral population, red junglefowl (RJF), possessed the richest diversity with comparison to all other domesticated populations, indicated by LD, Ne and ROH results, etc. And the fight chicken breeds, which were subject to stronger male-biased selection on fighting related traits, also present comparatively higher variation than in commercial and local breeds. After being intensely selected for reproductive and productive traits, the commercial lines showed poorest diversity. We also found the European local chicken conferred lower genetic variation than in Chinese local breeds, which could be attribute to relative shorter history of the European breeds. We also estimated the effective sex-ratio of these breeds to show the change ratio of both sexes. We found commercial chickens made more sex imbalance between female and male and the commercial lines showed highest values for the ratios of female to male. Interestingly, the red jungle fowl population present more males than females.

Our results provide the population genetics information of chickens being undergone different selection pressures and could help for better conservation strategies of different chicken breeds.

**Keywords:** Chicken, Genetic architecture, Genetic diversity, Linkage disequilibrium, Effective population size