INFLUENCES OF DIETARY AMINO ACIDS SUPPLEMENTATION ON L CELLS IN THE CHICKEN SMALL INTESTINE.

K. Hiramatsu\*(1), K.Nishimura(1), K.Kita(2)

(1) Laboratory of Animal Functional Anatomy (LAFA), Faculty of Agriculture, Shinshu University, Kami-ina, nagano, Japan

(2) Laboratory of Animal Nutrition, Faculty of Agriculture, Iwate University, Morioka, iwate, Japan

\* Corresponding author: seitaik@shinshu-u.ac.jp

Glucagon-like peptide (GLP)-2 derived from proglucagon (PG) is secreted from intestinal L cells and affects the growth of intestinal epithelium. Dietary supplementation of amino acids, methionine (Met) and lysine (Lys), had influences on the secretion of GLP-1 coexisting with GLP-2 in the same cells (Nishimura et al., 2015). This finding indicates the possibility that GLP-2 secretion is controlled by the dietary amino acids. The present study aims at clarifying the influences of the dietary supplementation of Met and Lys on PG mRNA-expressing and GLP-2-immunoreactive cells.

Male White Leghorn chicks of 7-days-old (average BW 80 g) were divided into one control and three experimental groups; crude protein (CP) 0%, CP 0%+Met and CP 0%+Lys groups. Chicks were forcedly fed with the control or experimental diet three times a day for two days at 8 hour intervals. And then they were sacrificed by decapitation under anesthesia. The middle part of ileum was dissected out from each bird as tissue sample. Tissue samples were fixed with Bouin's fluid and embedded in paraffinwax. Paraffin sections were used for in situ hybridization (ISH) and immunohistochemistry (IHC). Oligonucleotide probe based on the PG sequence was synthesized for ISH. Rabbit anti-GLP-2 serum was used as primary antibody for IHC. Frequencies of the occurrence of PG mRNA- expressing and GLP-2-immunoreactive cells were calculated. Crypt depth (CD) and the number of cells in mitosis per a crypt (NoMC) were detected in each group. All parameters were compared by Scheffe's F test for multiple comparisons.

The frequency of PG mRNA-expressing cells was significantly higher in the control group than three experimental groups (p

It is concluded that dietary amino acids supplementation doesn't influence PG mRNA expression and GLP-2 secretion of the chicken ileal L cells.

Nishimura K et al., J. Poult. Sci., 2015, 52(3):221-226.
EFFECT OF FEED PROTEASE SUPPLEMENTATION ON PERFORMANCE AND DUODENDAL ENZYMES ACTIVITY OF BROILERS FED DIFFERENT TYPES OF WHEAT-SOY AND PEAS-CONTAINED DIETS

V. Vertiprakhov\*(1), K.Borisenko(1)

(1)VNITIP, Sergiev posad, Russia

* Corresponding author: Vertiprakhov63@mail.ru

The aim of the study was to evaluate effect of two doses of the feed protease added to wheat-soy, 5 and 10% peas inclusion broiler diets. Two hundred twenty-five 1-day old Cobb 500 broilers fed commercial diets for 35 days. Birds were randomly allocated by 25 birds each group: C1—wheat-soy diet, C2—wheat-soy diet, 5% of peas, C3—wheat-soy diet, 10% of peas. C2 and C3 fed with calculated amino acids reduction. Body weight (BW) and FCR recorded at 10,21,35 d. Six experimental groups with feed protease: E1—C1+50 mg/kg, E2—C1+100 mg/kg, E3—C2+50 mg/kg, E4—C2+100 mg/kg, E5—C3+50 mg/kg, E6—C3+100 mg/kg. For physiological study, totally 27 broilers age of Cobb 500 fed C1 diets, on 21d surgically fitted with a duodenum T-cannula, divided according experimental design above. Duodenum chyme collected for 6 days, analyzed amylase, proteolytic, lipase activity. BW was significantly decreased by 10% of peas inclusion on 3% (p<0.05). Protease inclusion significantly improved BW on 10,21,35 day in all types of diets (p<0.05). Observed no statistical differences between BW and doses of 50,100 mg/kg of feed protease. Dose of 100 mg/kg proteas with soy-wheatdiet significantly inhibited proteolytic activity to 18,44±1,27 (p<0.05). Whereas 5%, 10% of peas diets and 100 mg/kg of protease significantly (p<0.05) increased proteolytic activity to 52,56±2,66 and 46,5±1,89 respectively. Broilers fed 5%, 10% peas and 50 mg/kg of feed protease had significantly higher proteolytic activity than control in both diets (p<0.05). Only 5% of peas inclusion significantly decreased amylase activity in E2 diet (p<0.05). Diet with 10% of peas and 100 mg/kg protease significantly reduced amylase activity (p<0.05), comparing to 5% peas. Lipase activity was significantly affected by 5 and 10% of peas without protease. We suggest that feed protease significantly affected proteolytic activity in wheat-soy and peas-contained diets, improve BW, FCR. However, dose of 100 mg/kg protease and soy-wheat diet inhibited proteolytic, amylase activity we observed improvement in BW and FCR. Physiological adaptive and compensative response can have a place with high doses of feed protease.
ID: 417

# AGE AND ROUTE OF INFECTION WITH STAPHYLOCOCCUS AUREUS ELICIT DIFFERENTIAL RESPONSES IN LAYING HEN IMMUNE CELL PROFILES MEASURED BY FLOW CYTOMETRY

K. Fries-craft\*(1), M. Meyer(1), Y. Sato(2), M. El-gazzar(2), E. Bobeck(1)

(1) Iowa State University Department of Animal Science, Ames, United States

(2) Iowa State University Veterinary Diagnostic Laboratory, Ames, United States

* Corresponding author: kfcraft@iastate.edu

In 2019, instances of increased mortality in older caged laying hens in the United States were linked to Staphylococcus aureus infection. While this pathogen is commonly isolated from Staphylococcus infections in poultry, specific host responses and outcomes from varying infection routes remain unclear. The study objective was to examine systemic immune responses to an emerging disease in young and old laying hens infected with S. aureus by varying administration methods. 22-week and 96-week-old laying hens (100 birds/age) were randomly assigned to 1 of 4 treatment groups consisting of uninfected controls and birds inoculated with S. aureus isolated from affected laying colonies by oral gavage, subcutaneous (SQ), or intravenous (IV) injection. Prior to inoculation, 6 birds (3/age) were euthanized for baseline tissue sampling, with subsequent samples taken at 3 and 8d post-inoculation (pi). Body temperatures from 10 hens/ treatment were taken daily and blood was sampled at 6h, 1d, 3d, and 7d post-infection (dpi). Innate immune cell profiles in the spleen and peripheral blood were measured using multicolor flow cytometry. Results were analyzed by PROC MIXED (SAS 9.4) as a 2x4 factorial with fixed effects of age, infection route, and age×infection route (significant: P≤0.05). Prior to inoculation, 96-week-old hens had 34.9% more splenic CD1.1+ lipid antigen-presenting cells (P0.05). At 3dpi, 96-week-old hens infected with S. aureus by SQ and IV injection showed significant reductions in splenic CD1.1+ cells compared to the corresponding control group (P < 0.0001). At 3dpi, 22-week-old birds infected by IV injection displayed 24.2% reductions in T-cells compared to the corresponding control (P < 0.0001), indicating recruitment to peripheral infection sites; whereas older birds infected in the same manner did not display reductions in T-cells until 8dpi (P < 0.0001). T-cell subpopulations indicated that 22-week-old birds recruited CD3+CD8α+ cytotoxic T-cells at 3dpi (P = 0.0002), while older birds did not mobilize any of the examined T-cell populations. These results give insight into variations in the age-based immune response to S. aureus that may be underlying the increased mortality seen in older bids.
ID: 470

THE EFFECT OF SEX REVERSAL ON SEXUALLY DIMORPHIC OREXIGENIC AGRP EXPRESSION.

P. Wilson\*(1), S.Caughey(1), N.Homer(2), M.Clinton(1), Z.Debiao(1), I.Dunn(1)

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

(2)Queen's Medical Research Institute, Edinburgh, United Kingdom

\* Corresponding author: peter.wilson@roslin.ed.ac.uk

Sexual dimorphism for growth exists across the animal kingdom and it is well known that male chicks grow faster than females. In chickens, as in mammals, genes expressed in orexigenic neurones in the hypothalamus control feed intake and these appear to have been affected through selection for growth [1]. In previous studies we have demonstrated that there is a highly significant sexual dimorphism in hypothalamic gene expression of the orexigenic agouti related protein (AGRP) with a higher level in the faster growing male than the female [2]. The relationship between genetic loci that determine growth and body weight with the expression of AGRP have led us to the hypothesis that orexigenic AGRP expression may be important in determining body weight.

To specifically test the hypothesis in the case of sex, a sex reversal experiment was carried out to find
out if AGRP expression sexual dimorphism is related to the genetic sex of the chicken or alternatively
its steroid milieu.

Broiler breeder (BB) eggs were incubated at 37°C for 72h. At 72h 100ul of PBS (pH 7.2) or Fadrozole (10mg/ml PBS), an aromatase inhibitor which prevents ovary differentiation, was injected into the albumen and the eggs incubated until hatch. Chicks were raised in a single room on a standard BB programme in a 2x2 factorial design [male v female, fadrozole (FAD) v PBS (C)], repeated 3 times. Birds were sacrificed at 10 and 26 weeks, and samples taken including hypothalamus for expression analysis by qPCR.

Fadrozole treatment resulted in a clear sex reversal of females, which had bilateral testes-like gonads and plasma oestradiol (ng/ml) at 26 weeks of age of 0.01±0.002 v 0.44±0.13 in C females. AGRP expression (normalized units) in the hypothalamus was significantly different (p<0.001) between sexes, but not between treatments in either sex: Female C 0.166±0.043; Female FAD 0.141±0.044; Male C 0.479±0.079; Male FAD 0.346±0.152.

The clear effect of Fadrozole on the sexual phenotype of the hens and the lack of effect of Fadrozole on AGRP expression suggests that the sex differences in AGRP expression may be genetically determined. This supports the role of AGRP expression in determining genetic differences in growth and body weight and is further evidence of the effects of genetic sex [3] rather than steroid dependent phenotypes in chickens.

[1] Am. J. Physiol. Endocrinol. Metabol. 2013 304 E909-E921.

[2] Biology of Sex Differences. 2018; 9.

[3] Nature. 2010; 464:237-U115.
VARIATION IN MUSCLE ENERGY STORES: IMPACT ON REPRODUCTION AND EGG CHARACTERISTICS IN
MEAT-TYPE CHICKEN STRAINS

S. Métayer-coustard\*(1), A.Franzoni(2), S.Réhault-godbert(1), A.Collin(1), A.Travel(3), E.Raynaud(1), Y.Baumard(1), J.Delaveau(1), C.Rat(1), E. Cailleau-Audouin (1), A.Brionne(1), C.Berri(1), E.Lebihan-duval(1)

(1)INRAE, Nouzilly, France

(2)Pisa University, Pisa, Italy

(3)ITAVI, Nouzilly, France

* Corresponding author: sonia.metayer-coustard@inra.fr

The selection of meat-type chicken lines for increased growth and muscle development was paralleled with physiological changes. While considerable gains have been made in production performance, limits have appeared in terms of product quality and reproduction, questioning the sustainability of meat production. The genetic antagonism between production and reproduction remains a major challenge in maintaining the sustainability of selection on meat-type lines. In heavy strains, there is a decrease in muscle energy reserves, as assessed through the Glycolytic Potential. Divergent selection on breast meat ultimate pH from a commercial grandparental female broiler line has originated in two experimental lines: pHu+, presenting the lowest muscle energy status, and pHu-, exhibiting the highest energy status. These two lines constitute valuable genetic models to address the link between the energy status of the animal, reproduction, egg content, embryo development and ultimately chick quality at hatch.

The present study focused on the eggs of the breeding hens from the 11th generation of selection, where a difference of 0.6 pH units was observed for the selection criterion (pHu measured in chicken Pectoralis major muscle at 6 weeks of age). Data collected between 23 and 39 weeks of age showed a delayed first egg laid, lower egg laying rate, as well as a higher percentage of broken eggs in the pHu+ line compared to the pHu- line. Characterization of the resulting eggs revealed differences between the two lines, with heavier and more rounded eggs in the pHu+ line. Divergent selection also impacted animal fertility (lower in pHu+), embryonic mortality (higher in pHu-), and post-hatching mortality (higher between D0 and D7 in pHu+).

In conclusion, these results suggest an alteration in the reproductive performance and changes in egg characteristics when selecting for decreased muscle energy reserves. These preliminary observations pave the way for future genetic and physiological studies to assess the contribution of muscle energy status in reproductive and chick robustness traits, whose impairment penalizes meat-type broiler production. In an original way, they also suggest the possibility of identifying new indicators or biomarkers of energy status by measuring reproduction performance and egg characteristics.
This project (CHICK'TIP) received financial support from French Agriculture Ministry (CASDAR)
# EFFECTS OF DIETARY CARBOHYDRATE LEVEL ON GLUCAGON-LIKE PEPTIDE-IMMUNOREACTIVE CELLS IN THE CHICKEN SMALL INTESTINE.

M. Salahuddin\*(1), K. Hiramatsu(2), K. Kita(3)

(1) Shinshu University, Department of Science and Technology, Ina-shi, Japan

(2) Shinshu University, Faculty of Agriculture, Ina-shi, Japan

(3) Iwate University, Faculty of Agriculture, Morioka, Japan

\* Corresponding author: bau.salahuddin@gmail.com

Glucagon-like peptide (GLP)-1 and GLP-2 derived from proglucagon (PG) are secreted from intestinal L cells in response to nutrient ingestion and accomplish different physiological functions such as the stimulation of insulin secretion and the growth of intestinal epithelium. Dietary protein level affects the frequency of GLP-1-immunoreactive cells in the chicken ileum (Monir et al., 2014). But the effect of dietary carbohydrate (CHO) level on GLP-immunoreactive cells is not clear. The purpose of the study was to investigate effects of dietary CHO levels on GLP-1 and GLP-2-immunoreactive cells in the chicken small intestine.

White Leghorn male chicks of 6-week-old were divided into one control and two experimental groups; low CHO (12.5% of the control content) and CHO-free groups. After habituation for three days with the control feed chicks were fed with the control or experimental diets for 7 days. Proximal and distal parts of jejunum and ileum were removed from each chick as tissue samples under anesthesia. Tissue samples were fixed with Bouin's fluid and embedded in paraffin wax. Paraffin sections at 5 µm thickness were made from each sample. GLP-1 and GLP-2 were detected by streptavidin-biotinmethod. Frequencies of the occurrence of GLP-1- and GLP-2-immunoreactive cells were calculated with image analyzer and were statistically analyzed by one way ANOVA manner.

Endocrine cells showing immunoreactivity for GLP-1 and GLP-2 were observed in all intestinal parts of each chick. They were located in crypts and villous epithelium. No obvious differences were found in the distributional pattern of immunoreactive cells among three groups. Frequencies of GLP-1-immunoreactive cells of the control group were significantly higher than those of all intestinal parts in the CHO-free group and those of ileum and proximal jejunum in the low CHO group. No significant differences were recognized in frequencies of GLP-1-immunoreactive cells in ileum and distal jejunum between low CHO and CHO-free groups. Frequencies of GLP-2-immunoreactive cells were also significantly higher in all intestinal parts of the control group than the CHO-free group. No significant differences were recognized in frequencies of GLP-2-immunoreactive cells in ileum and distal jejunum between low CHO and CHO-free groups. It is concluded that the dietary CHO level affects GLP-1- and GLP-2-immunoreactive cells in the chicken small intestine.

Monir et al., Anim. Sci. J., 2014, 85: 581-587
ID: 677

# PROTEOMIC ANALYSIS OF TWO WEIGHT CLASSES OF MULE DUCK "FOIE GRAS" AT THE END OF OVERFEEDING PERIOD

B. Lo\*(1), H.Remignon(2), N.Marty-Gasset(1), C.Pichereaux(3)

(1)INRA, Auzeville-tolosane, France

(2)GenPhYSE, Toulouse, France

(3)IPBS, Toulouse, France

\* Corresponding author: bara.lo@toulouse-inp.fr

## INTRODUCTION:

Foie gras is the result of the accumulation of lipids in hepatocytes following a short period of gavage. Despite identical rearing conditions, different liver weights are always observed at the end of the overfeeding period. Thus, the aim of this study is to try to better understand what explain the difference in liver weights by comparing proteomic profiles of two groups of fatty livers differing in final weights: LWL =550-599g and HWL =700g.

## MATERIAL ET METHODS:

A flock of male mule ducks was normally reared (11weeks) and finally overfed twice a day (10 days) according to a standard overfeeding program. After slauthering, 24 livers (2x12) were randomly sampled to create 2 experimental groups (LWL and HWL). In addition to the gross biochemical contents of those samples, a proteomic analysis was performed according to a bottom-up approach (Chait, 2006) using NanoLC MS/MS. The raw data were obtained after searching in the local bank (Canard-Anas_Cairina 190524) and a label free protein quantification was also performed. To facilitate comparisons, statistical analyses were carried out on abundance values (peptide signal intensity in MS). Filters were applied to identify protein variants according to 3 conditions:

- The proteins must be detected in all the samples of one of the two conditions. If missing values are present in the other condition, they are replaced by an intensity value corresponding at 5% of the noise.

- The significance threshold of the Student test is <0.01.

- The expression ratio is 1,5 < < 0.66.

## RESULTS:

In accordance to previous observations (Rémignon et al., 2018), proteins content decreases significantly (-18%) in HWL (p< 0.001). However, no significant differences (p > 0, 05) were observed for dry matter and total lipid contents between the two studied groups.

Finally, 1444 quantified proteins were identified among which 78 respect the validation parameters described above. When comparing the LWL to the HWL group, 42 and 36 proteins were under- and overexpressed respectively.

## CONCLUSION:

The results showed that LWL are characterized by greater exports and beta-oxidation of lipids which indicate a more efficient storage of triglycerides in HWL. In HWL, this more massive storage of lipids reduced the amount of proteins accompanying cellular responses to hypoxia and apoptosis.

## REFERENCES :

Chait, B. T. 2006. Science 314:65-66.

Rémignon, H., R. Ben Haj Yahia, N. Marty-Gasset, and J. Wilkesman 2018. Poultry Science 97:2211-2217.
MSTN GENE KNOCKOUT CHANGES THE TRANSCRIPTOME PROFILE IN CHICK LEG MUSCLE

K. Xu\*(1), C.Han(1), H.Zhou(1), J.Ding(1), Z.Xu\*(1), C.He(1), F.Akinyemi(1), Y.Zheng(1), H. Luo(1), H. Meng(1)

(1)Shanghai Jiao Tong University, Shanghai, China

* Corresponding author: keristina@sjtu.edu.cn

Postnatal muscle growth is an important aspect of animal production. However, limited knowledge is available for molecular mechanisms underlying muscle growth. CRISPR has been used for postnatal gene editing to study gene function in mice, but less has been done in chicks. To verify whether postnatal gene editing could be achieved in chicks, we knocked out Myostatin (MSTN), an important negative regulator of muscle growth, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles to study the molecular mechanism of muscle growth. A pair of sgRNAs targeting MSTN exons 1 and 3 were packaged with Cas9 and adenoviruses (AdV) and delivered to the leg muscles of six newborn chicks. We collected muscles and detected the large fragment deletion by RT-PCR in 3-day-old (3d) and 14-day-old (14d). Transcriptome sequencing was used to determine the transcriptome profiles of 3d KO muscles, 3d WT muscles, 14d KO muscles and 14d WT muscles. We analyzed differentially expressed genes (DEGs) using the DESeq2 package and used the DAVID along with IPA software for enrichment analysis. RT-PCR and sequencing results indicated that large fragment deletions were achieved in the genome of all 3d KO muscles and 14d KO muscles. Compared to the WT group, there were 1339 DEGs at 3 days and 597 DEGs at 14 days. Several DEGs were associated with lipid metabolism (ARL4C), immune response (CCL19) and skeletal muscle growth (Ex-FABP). Gene Ontology analysis and KEGG pathway analysis results showed that many DEGs were involved in muscle growth, immune response, signal transduction and metabolism processes. IPA analysis revealed that two important classical pathways (PDGF signaling pathway and STAT3 signaling pathway) were associated with muscle growth. Our results confirm the feasibility of postnatal gene editing in chicks and provide valuable resources for studying the molecular mechanisms of muscle growth in chicks.
ID: 754

EFFECT OF EGG STORAGE TIME ON HEAT PRODUCTION AND HAEMATOLOGICAL PARAMETERS OF EMBRYONIC AND POST-HATCH DEVELOPMENT OF GUINEA FOWL BROILERS

Y. Kouame\*(1), D. Libanio(2), K. Tona(3)

(1)CERSA (Centre d'Excellence Regional sur les Sciences Aviaires, Lomé, Togo

(2)Centre d'Excellence Regional sur les Sciences Avaires, Lomé, Togo

(3)Centre d'Excellence Regional sur les Sciences Aviaires de l'Université de Lomé, Lomé, Togo

* Corresponding author: emmanuelle.kouame@gmail.com

It is well known that egg storage affects egg quality (Kirk et al., 1991) embryo survival and post hatch growth (Tona et al., 2003), gas exchanges and the change in embryonic CO2 metabolism (Fasenko, 2007). There is however dearth of information on the mechanisms involved. This study evaluated the effect of egg storage duration on heat production and haematological parameters on embryonic and post-hatch growth of guinea fowl broilers. A total of 800 guinea fowl hatching eggs were weighed and assigned to two treatment groups of storage duration: 5d and 10d having 4 replicates of 100 eggs each. Prior to setting, egg Haugh units were determined (Tona et al. 2002) then the eggs were incubated at 37.7°C of temperature; 55% of relative humidity. At 23 d of incubation and at 12 weeks post-hatch, 60 eggs with evidence of living embryo and 2 birds respectively per storage treatment were transferred to respiratory chambers to determine CO2 production and O2 consumption. CO2 and O2 were recorded and used to calculate heat production by the formula of Brouwer (1965) at internal piping, hatch and at 12 weeks posthatch. They were reared for 12 weeks including starter, grower and finisher phases. Feed intake and body weight were recorded and used to determine feed conversion ratio. Also Blood samples were collected at hatch and 12 week post-hatch from 24 guinea fowls per treatment for determination of haematological parameters. The guinea fowl were later slaughtered and carcass characteristics, organs and intestinal morphometric were determined. The data were analyzed with the Graph pad Prism 5 statistical software package. T test was used for analyse. The results showed that average Haugh unit of 5d stored eggs was higher (P<0.05) than of 10d stored eggs. Similarly heat production of embryo and 12 wk-old guinea fowl from eggs stored for 5d was higher (p< 0.05) compared to those from 10d. Also, basophil, eosinophil and lymphocyte concentration in guinea fowl from egg stored for 10d was higher (p< 0.05) than 5d. Body weights in guinea fowl for 5d were higher (p<0.05) than those 10d. Feed conversion ratio for 5d was higher (p< 0.05) than those 10d. Also, weight and length duodenum were higher (p< 0.05) for 5d than those 10d. It was concluded that egg storage affects the metabolism of embryos through heat production, immune system during embryonic and post-hatch growth.

Key words: Egg storage, egg quality, guinea fowls, heat production, haematological parameters.
RESPONSES OF EXOTIC TURKEYS FED SPIRULINA (ARTHROSPIRA PLATENSIS) WITH OR WITHOUT VITAMIN B2 AND B6 DURING DRY SEASON

O. Oso*(1), F.Bankole(2), E.Oke(3)

(1)Centre d'Excellence en Science Aviaires (CERSA), Université de Lomé, Lome, Togo

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

(3)Centre d'Excellence Régional sur les Sciences Aviaires (CERSA), Université de Lomé, Lome, Nigeria

* Corresponding author: osoheritagemoyin@gmail.com

Thermal stress is a challenging environmental stressor in poultry production especially in the tropics, resulting in poor growth rate, reduced productivity and immunosuppression. There is a dearth of information on nutritional intervention to ameliorate the effect of thermal stress on exotic turkeys during hot season in the tropics. This experiment was carried out to investigate the responses of exotic turkeys fed spirulina (Arthrospira platensis) with vitamin B6 or vitamin B2 during hot-dry season. Ninety unsexed exotic poults were allotted to 5 dietary treatments in a completely randomized design. The treatments were: corn-soybean meal basal diet (CT); basal diet supplement with 100mg/kg of spirulina (SP), 100mg/kg spirulina + 7mg/kg vitamin B6 (SP6); 100mg/kg of spirulina + 8mg/kg of vitamin B2 (SP2); 100mg/kg of spirulina +7mg/kg of vitamin B6 + 8mg/kg of vitamin B2 (SPC). The experimental diets were fed at the pre-starter phase, starter, grower and finisher phase. The birds were raised using a conventional turkey management method. Blood samples were collected from two birds in each replicate at each phase of growth for the determination of plasma corticosterone, Superoxide dismutase (SOD) and malondaldehyde. The results showed that at the grower phase, birds in SP2 and SPC had similarly low plasma corticosterone (P
RNA EXPRESSION DIFFERENCES IN THERMAL CONDITIONING AND THERMONEUTRAL BROILER CHICKENS FED DIETARY METHIONINE FROM DIFFERENT SOURCES AND LEVELS AND SUBJECTED TO HEAT STRESS

J. Foutz*(1), M.Milfort(1), W.Habashy(2), G.Schneiders(1), A.Fuller(1), R.Whelan(3), R.Rekaya(1), S.Aggrey(1)

(1)University of Georgia, Athens, United States

(2)Damanhour University, Damanhour, Egypt

(3)Evonik Nutrition and Care GmbH, Hanau-wolfgang, Germany

* Corresponding author: jamesfoutz@uga.edu

Heat stress (HS) negatively affects the performance and metabolism of chickens. Several cellular mechanisms are elicited to mitigate the negative effects of heat stress. Methionine is a precursor to glutathione, a major antioxidant in the body. It is therefore thought that methionine could mitigate some of the negative effects of HS. Thermal conditioning (TC) has also been a strategy used to mitigate HS. Our goal was to examine differential gene expression of heat stressed broiler chickens versus thermally conditioned broiler chickens as well as the effect of methionine source. For this study, Cobb500 broiler chickens were subjected to thermo-neutral temperature (TN) (25oC) or HS (35oC) from day 7 to 20. The two groups were all subjected to HS from day 21 to 35. Additionally, birds in both treatments were fed recommended diets supplemented with either DL-Methionine (DL-Met) or Methionine Hydroxy Analogue (MHA) at either 100% or 140% of requirement. RNA sequencing analysis was done on Pectoralis major samples (5 birds per treatment group) at day 10, 21 and 35. Only differentially expressed genes with a p<0.05 and log2 fold change either greater than 1 or less than -1 were considered significant. KEGG analysis revealed oxidative phosphorylation and phagosome pathways, among others, to be significantly downregulated in birds TC birds and fed either 100% DL-Met or MHA in relation to the TN birds at were heat stressed from day 21 to 35. Analysis shows a reduction in significantly differentially expressed genes in TC birds when supplemented with either source of methionine at 140% of the requirement. Interestingly, FOXO signaling pathway and the cell cycle was upregulated in TC birds fed 140% DL-Met and downregulated in TC birds fed 100% DL-Met, with similar genes being either up- or downregulated in those pathways. The regulation of significant genes of pathways in relation to heat stress, thermal conditioning, and dietary methionine elucidate the biological mechanisms that underlie thermal conditioning.
POSSIBLE EFFECTS OF LIMINAL MICROBIOTA AND PROBIOTICS ON THE INNATE IMMUNO-DEFENSE
SYSTEM IN THE CHICK INTESTINE

T. Terada(1), T.Nii(2), N.Isobe(2), Y.Yoshimura*(3)

(1)Hiroshima University, Higashi-hiroshima, Italy

(2)Hiroshima University, Higashi-hiroshima, Japan

(3)Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-hiroshima, Japan

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

[Introduction] Innate immunity plays a crucial role in preventing infection in the chick intestinal mucosa before the maturation of the lymphoid system. In the process of innate immune response, Toll-like receptors (TLRs) recognize microbe-associated molecular patterns, leading to the synthesis of cytokines and anti-microbial peptides (AMPs) including avian β-defensins (AvBDs) and cathelicidins (CATHs). The mechanism by which the expression of innate immune factors is regulated in the chick intestine remains to be established. We recently published the reports on the effects of microbiota regulation by antibiotics and probiotics treatments on the chick innate immune system. The present paper reviews those findings to discuss the effects microbiota and probiotics on the chick intestinal innate immune functions. [Experiment 1] We examined whether the expression of innate immune molecules, including that of TLRs, AMPs, and cytokines in the intestine was altered by antibiotics treatment. Broiler chicks were administered antibiotics (penicillin and streptomycin) daily for 7 or 14 days. It caused an increase in the relative frequency of the Enterobacteriaceae family and a decrease in somegram-negative and -positive bacteria families. The gene expression levels of some TLRs, cytokines, and AMPs showed a tendency to decrease by antibiotic treatment at day 7. [Experiment 2] We examined the effects of live probiotic Clostridium butyricum (CB) on the expression of innate immune molecules. Broiler chicks were administered 500 µl water with or without CB daily for 6 days. The expression of TLR2 and TLR5 as well as IL-1β and TGFβ2 in the ileum was upregulated by CB. The gene expressions of AvBD1 and CATH 3 were upregulated by CB in the cecum. [Conclusions] Our results revealed that the expression of cytokines and AMPs might be affected by luminal microbiota in the cecum because their expression was altered in association with the changes in microbiota frequency that were regulated by antibiotics. It was also found that probiotic live CB treatment affected the expression of innate immune molecules, which may be due to the recognition of CB molecules by TLRs and/or the stimuli by organic acid released from CB. Thus, we suggest that regulation of intestinal luminal microbiota by probiotics is an effective strategy to enhance a part of the innate immune defense system in the intestine of chicks.
ALKYLATION REPAIR HOMOLOG 5(ALKBH5) KNOCKOUT PROMOTES THE PROLIFERATION OF CHICKEN PREADIPOCYTES

Q. Zhang\*(1), C.Liu, H.Chen, B.Cheng, H.Li

(1)Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin, Heilongjiang, China, Harbin, China

\* Corresponding author: zhangxiaoqi0405@163.com

Introduction

M6A RNA modification plays an important role in the differentiation of mammalian preadipocytes and hepatic lipid metabolism. ALKBH5, the m6A demethylase, is involved in the regulation of a variety of biological processes, such as spermatogenesis, carcinogenesis and development. So far, the relationship between ALKBH5 and the growth and development of adipose has not been reported. The objective of the current study was to determine the role of ALKBH5 in preadipocyte proliferation.

Material and methods

Immortalized chicken preadipocyte (ICP2) was used as the cell model. ALKBH5 knockout cell line was established by CRISPR/Cas9 system. The expression levels of genes were detected by RT-qPCR and western blot. Cell proliferation was measured by CCK-8. Cell cycle was determined by flow cytometry.

Results

Three gRNAs were ligated to the Cas9 expression vector, respectively (named by ALKBH5-g1 to ALKBH5-g3). Then the Cas9/gRNA expression plasmid was transfected into ICP2 cells. 48 hours after transfection, the gRNA targeting efficiency was detected. The results of T7E1 enzyme digestion showed that only ALKBH5-g2 had genome cutting activity, so the ALKBH5-g2 vector was selected for the following experiment. The ALKBH5-g2 vector was transfected into ICP2 cells. 24 hours after transfection, monoclonal of the GFP positive cell was selected by flow cytometry. A total of 20 cell lines were obtained (named by ALKBH5-g2-1 to ALKBH5-g2-20). The results of sequencing and western blot showed that ALKBH5-g2-11 was the ALKBH5 knockout cell line (ALKBH5-/-). The results of CCK-8 showed that the proliferation of chicken preadipocytes was enhanced after the knockout of ALKBH5. The results of cell cycle showed that the proportions of cells in S-phase and G2-phase were increased significantly after the knockout of ALKBH5. The results of RT-qPCR showed that the expression levels of proliferation-related genes (Ki67 and PCNA) were increased after ALKBH5 knockout.

Conclusion

ALKBH5 knockout promotes the proliferation of immortalized chicken preadipocytes.
AGE AND SEX-SPECIFIC LONG-LASTING EFFECTS OF PERINATAL TEMPERATURE MANIPULATIONS ON
NEURONAL HYPOTHALAMIC MECHANISMS

B. Tzschentke* (1)

(1)Humboldt-Universität zu Berlin, Berlin, Germany

* Corresponding author: barbara.tzschentke@rz.hu-berlin.de

In precocial birds the hatching phase is a critical period in which perinatal temperature manipulations (PTM) induces long-lasting changes in physiological peripheral and central nervous mechanisms. This review focuses on own collaborative research projects on the influence of chronic and short-term PTM on the hypothalamic microstructure controlling body temperature, feed intake, body weight and metabolism in terms of age and sex dependence. All experiments were carried out in brain slices containing the preoptic area of the anterior hypothalamus (PO/AH) or the Nucleus infundibuli hypothalami of the ventrobasal hypothalamus (NI/VH). In Muscovy ducks aged between embryonic day (E) 28 and day (D) 10 post-hatching, the neuronal thermosensitivity of the PO/AH and its modification by chronic PTM (35°C; 38.5°C; control 37.5°C) were investigated using extracellular recording. During early ontogeny (E28-D5) the PO/AH is characterized by a high cold and low warm sensitivity (up to 30% and 5%, respectively). Between D5 and D10 a strong qualitative change occurs towards the “adult” neuronal thermosensitivity, which is characterized by high warm and low cold sensitivity (D10 15% warm and 14% cold sensitivity). PTM related modifications show also a clear age-dependent pattern starting with proximate non-adaptive changes up to a clear incubation temperature dependent and proximate adaptive modification at D10 (cold-incubation increased warm sensitivity and decreased cold sensitivity and warm-incubation induced opposite modifications, p<0.05. The NI/VH contains orexigenic neurons expressing neuropeptide Y (NPY) and anorexigenic neurons expressing proopiomelanocortin (POMC), which are also key player in stress response control. In female and male broiler chickens effects of short-term PTM (+ 1°C, 2 hrs/day) on neuronal NPY expression were investigated on D35 using immunohistochemistry. Analysis of 365 slices results in a significant (p<0.05) decrease in NPY expression exclusively in males. It is corresponding to changes in thyroid hormones (T3/T4) and performance. Females show no or only tendential changes in the same parameters. Studies on POMC gene expression in three weeks old normal incubated laying type chickens, showing correlation of methylation pattern and gene expression along with sex-specific differences (p=0.03). In conclusion, PTM induced lasting changes in the microstructure of the hypothalamic neuronal network are sex-specific and follow ontogenetic rules.
ID: 1135

THE EFFECT OF PHOTOPERIOD AND WAVELENGTH ON BROILER PERFORMANCE

N. Tyler\*(1), S.Mthembu(1), M.Ciacciariello(1)

(1)University of KwaZulu-Natal, Durban, South Africa

* Corresponding author: tyler@ukzn.ac.za

Previous studies have found that broilers reared on short (12h) photoperiods had reduced breast meat yield (BMY) and in some cases reduced body weight (Liu et al., 2010; Mlaba et al., 2015). Previous studies have also shown that broilers reared on green wavelengths have improved muscle growth and development (Rozenboim et al., 1999; Cao et al., 2008). Thus, this study aimed to determine if the loss in BMY in broilers reared on short photoperiods could be counteracted using green light. 3200 Ross 308 day old chicks were randomly placed in one of 8 light-tight rooms (400 chicks per room). A 2x2 factorial design was used with two wavelengths (green and white) and two photoperiods (short; 12L: 12D and long; the current commercial recommendation of 23L: 1D for the first week then down to 16L: 8D). The performance parameters body weight (BW), body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR) and BMY as a percent of BW (BMY%) from a sample of 8 birds/room were measured weekly until 35d of age. All results were analysed using ANOVA. Broilers that were reared under the short photoperiod had better FCR with a significantly higher BW at d 35 and had a significantly lower cumulative feed intake compared to broilers that were reared under the long photoperiod regardless of wavelength. Broilers on the white wavelength had a significantly higher BW at 21d than those on the green wavelength, but not at any other age. At 14d there was a significant interaction with BW of broilers on the long white treatment showing the highest BW. At 7d BMY% from broilers that were reared under a short green photoperiod and long white photoperiod was significantly higher compared to BMY % from the other groups. However, this interaction was not found at any other age. Mortality was not affected by photoperiod or wavelength. This study didn't find a reduced BMY from birds on short photoperiods, and regardless of wavelength there was an increase in BW on birds on short photoperiods, as well as an improved FCR, perhaps because of lower activity levels. However, the BW of all broilers was under target and therefore the results should be interpreted cautiously.
ID: 1269

# TRANSCRIPTIONAL INSIGHTS INTO KEY GENES AND PATHWAYS CONTROLLING ABDOMINAL FAT METABOLISM IN BROILER CHICKENS

C. Xiao\*(1)

(1)Guangxi University, Nanning guangxi, China

\* Corresponding author: 1101573274@qq.com

**INTRODUCTION:** With the increase in growth rate and feed conversion efficiency, the abdominal fat rate of Chinese domestic breeding broilers has been increasing. The increase in belly fat greatly reduces the slaughter rate and disease resistance of broilers. To explore genes and regulatory pathways related to abdominal fat deposition in Chinese domestic broiler breeds, transcriptome sequencing technology was used to detect the expression levels of genes in the high abdominal fat group and the low abdominal fat group of Guangxi Sanhuang chicken.

**MATERIALS AND METHODS:** San Huang chicken from Guangxi province, China, Under the same feeding and management conditions, the animals were raised to the day of listing. After slaughter, the abdominal fat of each chicken was measured. The abdominal fat rate was divided into high and low abdominal fat groups, and three samples of each group were taken Liver, back fat, pectoral muscle, and leg muscle tissues. RNA was extracted and sequenced. Sequence adapters and low-quality reads were removed by Trimmomatic, and quality control was performed with FastQC. Sequencing reads were mapped to the chicken reference genome using the HISAT2. To quantify the expression of the HTseq-count. Analysis of differential expression of transcripts with DESeq2. Genes with P-value RESULTS: A total of 2233 differentially expressed genes were identified between four groups. Among them, there were 474,459,1134,166 genes Differentially expressed in the liver, dorsal fat, pectoral muscle, and leg muscle. We conducted enrichment analysis on these differentially expressed genes, which found that these differentially expressed genes were mainly enriched in the ATP binding(MYH15, TOP2A, SYK, ASS1), extracellular region part(LPL, HBEGF, TSHB). PPAR signaling pathway(ACSL1, ACOX1, PLIN1).

**CONCLUSION:** This study showed that Genes related to abdominal fat deposition are mainly involved in ATP binding, extracellular region part, biological functions and PPAR signaling pathway. Additionally, key pathways related to lipid storage and metabolism (PPAR signaling pathway) may be the key pathways regulating differential lipid deposition.
THE INFLUENCE OF DIFFERENT FRACTION OF ALUMINOSILICATE IN SLAUGHTER CHICKEN DIET ON
JEJUNUM MORPHOLOGY

J. Bogucka*(1), M.Banaszak(2), A.Dankowiakowska(1), J.Biesek(2), B.Bigorowski(2), A.Zmudzińska(2)

(1) Department of Physiology, Zoophysiotherapy and Animal Nutrition, Faculty of Animal Breeding and Biology, UTP - University of Science and Technology, Bydgoszcz, Poland

(2) Department of Animal Breeding, Faculty of Animal Breeding and Biology, UTP - University of Science and Technology, Bydgoszcz, Poland

* Corresponding author: bogucka@utp.edu.pl

In slaughter chickens production more often is payed attention on high emission of toxic gases as the ammonia, hydrogen sulfate and carbon dioxide to the atmosphere. Searching of solutions a. e.: feed supplements for decreasing toxic gases emission level by the higher absorption capacity in digestive tract. The aim of this study was compare feed supplementation by different fractions of aluminosilicate (zeolite, halloysite) and evaluation of impact on slaughter chickens Ross 308 digestive tract morphology. In this study 3 groups of chickens Ross 308 (100 bird per group) were evaluated. In all groups birds were fed with standard diet. In group I (control) fed without aluminosilicate was used. The group II was fed with 1% halloysite additive. In the group III fed with 1% zeolite addictive was used. After 6 weeks slaughtered 30 chickens (10 bird per trial group). After slaughtering small intestine samples were collected (3-4 mm long) for histological analysis. In samples detected: villus height and widgt, which were used to villus surface area and crypt depth calculation. Statistical significance difference were found in villus height between group with 1% halloysite additive (II) and group with 1% zeolite additive (III) in fed. Similar differences were found between group I and III. No significant differences were found in the villus height between group I and II. The statistical significant differenceof villus widgt was found between groups I and III, II and III as well. High significance differences (P
IMPACT OF PHOTOPERIOD ON PRODUCTIVITY OF BROILERS RAISED WITHOUT ANTIBIOTICS

T. Shynkaruk\*(1), K.Schwean-lardner(2)

(1)University of Saskatchewan, Saskatoon, Canada

(2)Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Canada

* Corresponding author: tory.shynkaruk@usask.ca

An increasing percentage of the global broiler industry is shifting towards antibiotic free production. Moving from conventional rearing to raised without antibiotics (RWA) can have detrimental effects on productivity. To minimize the negative impacts associated with RWA flocks, producers must adapt management practices. While it is known that providing a dark period improves productivity (growth rate and feed efficiency and reduced flock mortality) in conventionally reared birds, it is unclear what benefits will be realized in RWA flocks. Therefore, the objective of this research was to assess the impact of photoperiod on the productivity of RWA broilers. Two identical blocked trials were conducted, each using 4,032 Ross 308 broilers (N=8,064) reared to 36 d. Birds were housed in 8 identical environmentally controlled rooms with 12 pens per room at a stocking density of 21 kg/m2 for each block. From 6 to 36 d, lighting treatments were 14L:10D, 17L:7D, 20L:4D, and 23L:1D. Each treatment was replicated in two rooms per trial resulting in 4 room replications overall. Body weight (BW), feed intake and feed efficiency were calculated at 7, 21, and 36 d. Flock uniformity was assessed at 34 d (n=336 birds per treatment per trial). Mortality was monitored daily and birds were necropsied by an independent laboratory to determine cause of mortality. Data were analyzed using regression analyses (Proc Reg and Rsreg of SAS 9.4). Differences were considered significant when P≤0.05. A quadratic relationship between BW and treatment existed at 7 (P<0.01), 21 (P<0.01), and 36 d (P<0.01) with the highest final BW at 36 d under 20L. Feed consumption responded quadratically at all periods with birds on 20L consuming the most feed. Feed efficiency with mortality correction responded in a linear fashion from 21-36 d and 0-36 d, with longer dark periods resulting in improved feed conversion. There was no effect of photoperiod on mortality. A quadratic response was observed for the percentage of birds within 5% of the mean BW for uniformity (P=0.05) with the highest percentage under 23L. A linear increase with increasing photoperiod was observed for the percentage of birds within 15% of the mean (P=0.04) with higher uniformity under 23L. In conclusion, the results indicate that including a minimum 4 h of continuous darkness in the lighting programs of RWA broilers improves performance.
ID: 1395

ONTOGENY OF HORMONAL SYSTEMS REGULATING CALCIUM AND PHOSPHORUS METABOLISM IN COMMERCIAL BROILER CHICKENS

L. Ellestad*(1), G.Boothby(1), J.Payne(1), M.Arango(1), H.Gil arenas(1)

(1)University of Georgia, Athens, United States

* Corresponding author: llelestad@uga.edu

Lameness due to pain or leg weakness is a major welfare concern in the broiler industry, with approximately 2-6% of birds manifesting skeletal problems even in the absence of nutritional deficiency, poor management, or injury. Though it is known that vitamin D3, parathyroid hormone (PTH), and calcitonin (CALC) play important roles in Ca and P homeostasis and are essential for bone strength, there remains a lack of data relating to physiological control of D3 conversion into its biologically active forms [25(OH)D3 and 1,25(OH)2D3] and hormonal regulation of Ca and P metabolism in broilers. The objective of this study was to investigate ontogenic expression of enzymes responsible for conversion of exogenous D3 to its metabolites and receptors for hormones critical in regulating Ca and P metabolism. Liver and kidney were collected from eight Cobb 500 broilers on day (D) of hatch (D0) and post-hatch D10, D20, D30, and D40. Levels of mRNA for enzymes involved in conversion of D3 and hormone receptors associated with Ca and P homeostasis were measured by RT-qPCR. Data were analyzed with one way ANOVA followed by Tukey's post hoc comparison and considered significant at P≤0.05. Levels of mRNA for CYP2R1, the enzyme responsible for converting D3 to 25(OH)D3, were highest in liver on D0 and decreased to the lowest levels on D20. In kidney, levels of CYP24A1, the enzyme responsible for deactivating 1,25(OH)2D3, were lowest on D0 and peaked by D10. Both RXRA and RXRG, transcriptional partners of vitamin D receptor, were highest onD0 and reduced to their lowest expression on D20 or D10, respectively, in liver. Levels of mRNA for both these genes remained relatively constant in kidney. In liver, mRNA for the two PTH receptors exhibited reciprocal expression patterns: PTH1R was highest at D0 and reduced steadily through D40,while PTHRL was lowest at D0 and increased steadily through D40. While PTH1R did not change in kidney, PTHRL was highest in this tissue on D0 and remained low after D20. Expression of CALC receptor was only detectable on D0 in liver; in kidney, CALC receptor levels were also highest on D0, though its expression remained detectable at all ages. These results indicate that systems involved in enzymatic conversion of vitamin D3 are not established until well after hatch and suggest that differential expression of receptors involved in hormonal control of Ca and P metabolism allows for tissue-specific regulation of this process.
INCUBATION PHYSIOLOGY
THE ASSIMILATION OF CARBOHYDRATES BY EMBRYOS OF BROILER AND LAYER CHICKEN

A. Dolgorukova*(1), V.Titov(2), A.Zotov(2), I.Gupalo(2), M.Tishenkova(2)

(1) Federal State Budget Scientific Institution Federal Scientific Center "All-Russian Research and Technological Poultry Institute" of Russian Academy of Sciences;; Sergiev posad, Russia

(2) Federal State Budget Scientific Institution Federal Scientific Center "All-Russian Research and Technological Poultry Institute" of Russian Academy of Sciences, Sergiev posad, Russia

* Corresponding author: anna.dolg@mail.ru

The difference in growth rate between broiler chicken and layers can be found since the embryonic stage of development. Certain differences in the metabolic rate can also be found: broiler embryos more intensively assimilate the lipids of yolk and produce more heat since day 12 of incubation in compare to embryos of layers. The study presented was performed on the embryos of Cornish broiler chicken and "Mini-myasnye" (universal breed). At day 18 of incubation eggs with live embryos were injected with the solutions of different carbohydrates (0.5 mL per egg). Four hundred eggs for each breed were allotted to four treatments: treatment 1 (negative control) was intact; treatment 2 (positive control) was injected with normal saline; treatments 3 and 4 were injected with 10% solutionsof glucose and dextrin, respectively; then the eggs were set for further incubation. Biochemical analyses of the embryos were performed in 1 hour and 1 day after the injection. It was found that concentration of glucose in embryonic blood in 1 hr after the injection was higher in the treatments injected with glucose (difference with control was significant at P<0.001) and dextrin (P<0.05). The assimilation of carbohydrates during days 18-19 of incubation was faster in Cornish embryos. The significant difference with control was found in the lactate concentration in embryonic blood in the glucose-injected Cornish eggs in 1 hr after the injection (2.16 vs. 4.28 mM/L in treatments 1 and 3, respectively, P<0.001); this effect was not found in the dextrin-injected eggs. Similar trend was found in the glucose-injected "Mini" eggs (1.71 vs. 2.34 mM/L, P<0.05). Since the increase in the lactate concentration is related to the anaerobic decomposition of glucose it can be assumed that the injectionof glucose prior to hatch stimulates the glycolysis. The concentration of lactate in embryonic blood from dextrin-injected eggs in 1 hr after the injection was at the level of control evidencing slow decomposition of the starch to glucose and gradual transfer of the latter to embryonic cells. In a day after the injection the concentrations of lactate were similar in all treatments for each breed. The conclusion was made that the injections of exogenous carbohydrates into the chicken embryos at day 18 of incubation improved the assimilation of glucose and enhanced glycolysis as a source of energy inthe hypoxic pre-hatch period; Cornish embryos were found to assimilate glucose faster.
# THE BIOCHEMICAL MARKER OF GENETICALLY PRECONDITIONED MEAT PRODUCTIVITY IN POULTRY AT THE EMBRYONIC STAGE OF DEVELOPMENT

A. Dolgorukova\*(1), V.Titov(2), I.Kochish(3)

(1) Federal State Budget Scientific Institution Federal Scientific Center "All-Russian Research and Technological Poultry Institute" of Russian Academy of Sciences;, Sergiev posad, Russia

(2) Federal State Budget Scientific Institution Federal Scientific Center "All-Russian Research and Technological Poultry Institute" of Russian Academy of Sciences, Sergiev posad, Russia

(3) Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia

\* Corresponding author: anna.dolg@mail.ru

For successful breeding to increase meat productivity it is necessary to know certain biochemical or morphological markers of the genetic predisposition to intense growth of muscle tissue. The difference in phenotypic parameters between meat and other breeds is manifested only at the postembryonic stage. But there are some biochemical factors acting at the embryonic stage and affecting the postnatal growth of muscle tissue. The synthesis of these factors, as the presence of their targets, may be predetermined genetically. Some researchers suggested that the activity of these factors is particularly related to the nitric oxide (NO) – universal cell mediator synthesized in living tissues from arginine. Using a highly sensitive and highly specific enzyme sensor that allows to assess the concentration of all nitro- and nitroso compounds in living tissues, the following patterns were established. The intensity of NO synthesis in the poultry embryos of one species is roughly the same while the rate of NO oxidation to nitrate is different in embryos of different breeds. In embryos of meat-type breeds this rate is high and about 90% of all synthesized NO undergoes oxidation to nitrate. In egg-type breeds this rate is low, and only several per cent of the synthesized NO oxidized and other NO accumulates within an embryo in content of co-called NO donor compounds – nitrosothiols, different nitrosyl-iron complexes. The difference in NO oxidation rate between the embryos of meat- and egg-type breeds is several orders of magnitude. But this difference varies by no more than 10% within a breed, line or cross. The intensity of NO oxidation in bird embryo is not depended on sex, age of the layer and feeding regime. These patterns were found in more than 50 breeds of 5 poultry species.

It was found that selection to increase meat productivity is always associated with the intensification of NO oxidation in embryo. In chickens after hatching the content of nitro- and nitroso compounds in the tissues sharply decreases, and becomes equal for all breeds of the species. Only embryonic NO oxidation is correlated with meat productivity.

We suppose that this rate of NO oxidation is predetermined by the presence or state of the NO targets. Thus, selection to increase meat productivity leads to a qualitative change in embryonic NO metabolism. Further research should reveal the mechanism of these changes.
COMPUTED TOMOGRAPHY (CT) AND MAGNETIC RESONANCE IMAGING (MRI) TO PHENOTYPE
FERTILIZED EGGS DURING INCUBATION

H. Adriaensen(1), F.Lecompte(1), V.Parasote(1), N.Bernardet(3), S.Métayer-coustard(1), N.Guyot(1), M.Halgrain(1), S.Réhault-godbert*(1)

(1)Institut national de recherche pour l'agriculture, l'alimentation et l'environnement, Nouzilly, France
(3)Institut national de recherche pour l'agriculture, l'alimentation et l'environnement, 37380, France

* Corresponding author: sophie.rehault-godbert@inrae.fr

Studies of the bird embryo has led to major advances in developmental biology in the 1950s, and the Hamburger and Hamilton table related to the different stages of chicken embryonic development is still very widely used as a reference by embryologists [1]. However, because of decades of intensive selection on meat and egg production criteria (fast-growing broilers versus slow-growing broilers, laying hens), we expect that some developmental/growth features may have changed since the publication of the reference table.

To further explore this hypothesis, we decided to focus on a commercial fast-growing broiler strain. We used Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), 1) to assess the evolution of egg quality during storage prior to incubation; 2) to analyze the growth and development of the embryo at various developmental stages.

Eggs from Ross 308 broiler strain (Aviagen) were stored for either 3 or 10 days (16°C, 75-85% relative humidity) and incubated for 11, 13 or 15 days prior to analysis (37.8°C, 55% relative humidity). CT on 10 eggs was performed on the day of lay, after 3 or 10 days of storage, and their global quality (egg weight, eggshell strength and thickness, internal quality of egg white and yolk) was assessed in parallel. As expected, most egg quality parameters decreased with duration of storage while the volume of the air chamber and the localization of egg yolk within the egg was also affected (CT). After 11,13 and 15 days of incubation, 10 eggs were analyzed by both CT and MRI. Embryos were removed from the egg, weighted and analyzed once again by CT followed by staining with Alcian blue and Alizarin Red for further analyses of their skeletal mineralization. The acquisition of all images is now achieved and the analyses of the results are in progress.

The final data integration will help to better appreciate internal movements of egg components during storage (which affects egg quality and potentially embryo viability) and during incubation, and to gain new insights into the development of the chicken embryo (anatomical characterization). In addition, by comparing these data with the Hamburger and Hamilton table, we will be able to determine whether some developmental features have been specifically affected by selection in this fast-growing broiler strain.

[1]. Hamburger V1, Hamilton HL. A series of normal stages in the development of the chick embryo. 1951. Dev Dyn. 1992 Dec;195(4):231-72.
INCUBATION TEMPERATURE AFFECT YOLK SAC DYNAMICS AND FUNCTIONALITY

J. Dayan\*(1), N.Reicher(2), T.Melkman-zehavi(2), Z.Uni(2)

(1) Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
(2) Department of Animal Sciences, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

\* Corresponding author: jonatan.dayan@mail.huji.ac.il

During incubation, the yolk, a main source of nutrients for the embryo, is enclosed by the yolk sac tissue (YST), a multifunctional organ responsible for nutrient absorption & digestion, for glycogen synthesis, for gluconeogenesis and for thyroid hormones (THs) regulation. Current work examined whether altered incubation temperatures (ITs) affect YST functions. Hence, 300 eggs were assigned to 3 temperature groups and incubated during E0-E18 in Control-37.8°C; Hot-39.3°C; Cold-36.3°C treatments. At E18 eggs were assigned to hatching trays and incubated in 37.8oc until hatch. At each sampling day along incubation (E5,E7,E9,E11,E15,E18,E19,E21), embryo and residual yolk (RSY) weights were recorded and YST was sampled. YST functionality was examined by regression analysis to RT-PCR expression patterns of genes encoding for lipid uptake & metabolism (LRP2,ApoA1), for oligopeptides uptake (PepT1), for gluconeogenesis (FBP1), for glycogen synthesis (GYS2) and for THs regulation (TTR,DIO1,DIO2).

Our results show that ITs significantly affect the expression of genes indicating YST functionality. In the Hot group almost all functionalities were affected while in the Cold group only few. Genes representing nutrient uptake showed a decrease in expression towards hatch, while the Control and Cold groups exhibited a linear increase. The expression of ApoA1 (indicating lipid metabolism) and DIO2 (THs activating enzyme) in Hot and Cold groups showed moderate linear expression pattern compared to Control. YST Glycogen synthesis was affected as expression of GYS2 in Hot and Cold groups had no significant change along incubation while in Control exhibited polynomial fit. The expression patterns of TTR (THs carrier) and FBP1 (gluconeogenic regulator) were not affected by ITs and showed similar expression patterns in all groups.

The effects on YST functionality probably led to the changes observed in yolk utilization, a parameter of hatchling quality. While the Control group showed optimal yolk consumption, as RSY decreased by 71.7% (E15-E21), the Cold and Hot groups exhibited poor consumption as RSY decreased by 63.8% and 37.2% respectively.

In conclusion, our findings show for the first time that IT's change the function of the YST and explains the reason for variations in RSY weight. It also highlights the importance of the YST in supporting the embryo and exhibit its functionality by fulfilling the roles of organs (intestine, liver) prior to their maturation in the embryo.
IN-OVO ELECTROLYTE FEEDING IMPROVES THE HATCHABILITY IN BROILER BREEDERS AND DOESN'T ALTER THE GROWTH TREND IN BROILERS

N. Mukhtar*(1), K.Imtiaz(2), T.Ahmad(3), A.Yousaf(2), F.Farooq(4), G.Ahmad(3)

(1)PMAS Arid Agriculture University Rawalpindi Pakistan, Rawalpindi, Pakistan

(2) Department of Clinical Studies -PMAS Arid Agriculture University Rawalpindi-Pakistan, Rawalpindi, Pakistan

(3) SB Feed Mill Mandara - SB Poultry, Rawalpindi, Pakistan

(4) Department of Poultry Sciences - PMAS Arid Agriculture University Rawalpindi-Pakistan, Rawalpindi, Pakistan

In-ovo electrolyte feeding improves the hatchability in broiler breeders and doesn't alter the growth trend in broilers

Perinatal period is the time when post hatch chick undergoes body functioning activities shift from egg nutrients to exogenous feed. In ovo feeding via amnion fluids also enhances or facilitates healthy gut development of embryo and chicks by facilitating production of healthy microbiome. Objective of current study was to check the effect of in ovo inoculation of different electrolytes to broiler breeder eggs and their subsequent effect on hatchability and growth performance of broiler chicks. Present experiment was conducted by injecting different concentration of electrolytes solution via in-ovo technique into the amniotic sac of two hundred and forty broiler breeder hatching eggs at 18th day of incubation. The aim of the present study was to check the effect of in-ovo feeding of different concentration of electrolytes solution on hatchability in broiler breeder eggs and its subsequent impact on growth response in broiler. Eggs were arranged into four experimental groups with sixty eggs in each experimental group. Electrolytes used in study were combination of sodium chloride (3.5g), potassium chloride (1.5g), sodium citrate (2.9g) and dextrose (20g), respectively. Treatment group 1 was labelled as control group while treatment group 2, 3 and 4 were inoculated with combination of electrolytes i.e. sodium chloride, potassium chloride, sodium citrate and dextrose in 100ml, 500ml and 1000ml volume of distilled water. All experimental eggs were kept in an aseptic environment. Eggs were kept in incubator for 18 days in setter and then experimental and non-experimental eggs trays were shifted to an isolated room where fumigation is done and in-ovo inoculation was performed and afterwards, transferred into hatcher. After successful hatch out analysis, it was observed that 52 eggs were hatched in control group while 56 eggs were hatched in all other three in-ovo electrolytes fed groups. All hatched out chicks were transported to farm for evaluation of growth performance parameters. Data regarding hatchability and growth parameters i.e. feed intake, weight gain, feed conversion ratio (FCR) and mortalities was analyzed by using Completely Randomized Design (CRD). The results indicated significantly higher (P 0.05) effect of in-ovo electrolytes feeding was noted on growth performance parameters viz., daily feed intake, weight gain, mortality and feed conversion ratio.

Key Words: Incubation, Concentration, FCR and Hatchability

<footnote>* Corresponding author: nmukhtar@uaar.edu.pk</footnote>
EFFECT OF SUPPLEMENTATION OF FISH OIL ON HATCHABILITY PERFORMANCE IN CHICKEN

E. Subbiah*(1), P.Chinnadurai(1), S.T. Selvan(1), A.Sundaresan(1), A.V.Omprakash(1)

(1)Tamil Nadu Veterinary and Animal Sciences University, Chennai, India

* Corresponding author: vet_ezhil2001@yahoo.co.in

The main energy source for the developing chick embryo is supplied by fats in the egg yolk. The alteration of the egg yolk fatty acid profile could improve the growth of the embryo during the incubation period. Hence, the present research was carried out to study the effect of fish oil on hatchability performance of native chicken namely TANUVAS Aseel and Kadaknath at Poultry Research Station, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India. The biological experiment was started using two hundred and seventy layers each of TANUVAS Aseel and Kadaknath (25 weeks of age) of single hatch were obtained and maintained under deep litter system of management with sex ratio of 1:6.These birds were randomly allotted into three treatment groups. All the treatment had three replicates and each consisted of 30 layers. The experimental birds fed from 25 weeks to 36 weeks with diets containing fish oil 0 % (T1-control),1.5 % (T2) and 3 % (T3). All the diets were made iso-nitrogenous and iso-caloric by adjusting the other ingredients. A total of 1450 hatching egg were collected from 30 weeks to 36 weeks. The per cent total hatchability, fertile hatchability and total embryonic mortality were recorded and analyzed.The data collected on various parameters were grouped and subjected to statistical analysis of one way ANOVA as per the procedure of statistical analysis system (SPSS, version 20.0 for windows). TANUVAS Aseel received fish oil had numerically lesser egg weight when compared to control group, whereas numerically higher egg weight recorded in birds received fish oil. Similar trend was recorded in chick weight. Fish oil treatment groups showed significantly (P<0.01) lower early and late embryonic mortalities during incubation. The statistical analysis did not show any significant variation between treatment groups on weak chicks in TANUVAS Aseel, whereas a significant (P<0.05) difference was noticed on weak chicks among treatment groups in Kadaknath. The hatchability (fertile and total egg set) was significantly (P<0.05) higher in birds fed with 3 per cent fish oil in TANUVAS Aseel, whereas 1.5 per cent fish oil fed group showed significantly (P<0.05) higher hatchability in Kadaknath. The cock maintained on fish oil diet recorded significantly higher (P<0.05)sperm motility and showed relationship between sperm motility and hatchability. This study concluded that incorporation fish oil in poultry feed will improve the hatchability in chicken.
USE OF MORINGA OLEIFERA LEAVES IN BROILERS PRODUCTION CHAIN: EFFECT ON HATCHABILITY OF SASSO BREEDER EGGS AND DAY-OLD CHICK SERUM LIPIDS

O. N'nanle*(1), D.Nideou(2), K.Voemesse(3), E.Decuypere(4), K.Tona(5), A.Tete-benissan(6)

(1)Centre d'Excellence Régional sur les Sciences Aviaires, Lomé, Togo

(2)Institut National Supérieur des Sciences et Techniques, Abéché, Chad

(3)Centre d'Excellence Régional en Sciences Aviaires, Lome, Togo

(4)K.U. Leuven, Leuven, Belgium

(5)Centre d'Excellence Régional en Sciences Aviaires, Lomé, Togo

(6)Université de Lomé, Lomé, Togo

* Corresponding author: nnanleo1@gmail.com

This study was designed to investigate the in ovo and or dietary effects of Moringa oleifera leaves or a combination of both on hatchability and serum lipids of day-old chicks. Hatching eggs (n=2400) from Sasso broiler breeders fed with or without Moringa oleifera leaves were incubated at standard incubation conditions. At day 18, both eggs were candled and those with evidence of living embryos were divided into 2 new groups of 450 eggs each. Moringa oleifera extract was injected into the air chamber of one group. After extract inoculation, eggs were transferred from the turning trays to hatching baskets. Between 456 and 510 h of incubation, hatching events such as external pipping and hatch were monitored every 3 h. Hatched chicks were recorded and weighed. Eighty newly chicks from each group were used for liver, heart and yolk sac weight; and for serum lipids determination. General Linear Models procedure was used to analyze data. When means were statistically different, then they were further compared using Tukey's test based on P<0.05. Results indicated that in ovo inoculation of extract reduced external pipping duration. Chick weights increased with Moringa oleifera leaves used only in broiler breeders diet but hatchability was greater in all groups treated with this material. Liver relative weights of day-old chick showed no remarkable changes while in ovo injection of extract reduced heart weights. Yolk sac utilization was better with Moringa oleifera leaves, regardless of the stage where this material was used. Serum concentration of total cholesterol and LDL cholesterol decreased with in ovo treatment while HDL cholesterol increased. These results suggest that Moringa oleifera leaves had positive effects on hatchability.

Keywords: Moringa oleifera leaves, broiler breeders, in ovo, hatchability, serum lipids.
INCREASING INCUBATOR CO2 LEVEL BY NON-VENTILATION DURING THE FIRST 10 D OF INCUBATION:
EFFECT ON THE EMBRYONIC DEVELOPMENT, HATCHABILITY AND POST-HATCH GROWTH OF 2
COMMERCIAL LAYERS STRAINS

A. Bilalissi*(1), H.Meteyake(2), K.Tona(3), K.Voemesse(3), O.N'nanle(3), D.Nideou(3), E.Decuypere(4), K.Tona(3)

(1) CERSA, Lome, Togo

(2) CERSA, University of Lome, (Togo), Lome, Togo

(3) CERSA, University of Lome, Lome, Togo

(4) Laboratory for Physiology, Immunology and Genetics of Domestic Animals, Department of Biosystems, K.U. Leuven, Belgium, Leuven, Belgium

* Corresponding author: abilalissi18@gmail.com

During embryonic development, Lowman Brown (LB) embryos differs from that of Lowman White (LW) in terms of embryonic growth and hatching time (Tona et al., 2010). Therefore, these strains might adapt differently on environmental factors such as exposure to high CO2. The aim of this study was to investigate the effect of increasing incubator CO2 concentration by non-ventilation during the first 10 d of incubation on the embryonic development and post-hatch growth of LB and LW strains. Prior to setting for incubation, a total of 1,200 hatching eggs produced by LB and LW breeders were weighed, individually numbered and randomly assigned to 4 replicates of 75 eggs each/strain. The eggs were then divided equally into 2 incubators that were either ventilated (control group) for the entire incubation or non-ventilated for the first 10 d and subsequently ventilated. At d 18, the eggs were weighed, candled and fertile eggs were transferred from the turning trays to hatching baskets. Between 457 and 510h, hatching eggs were checked individually every 3h for hatching events. After pull out at 21.5 d, chicks were raised until d 56. Data were processed with the statistical software package graph pad prism Version 5.0. Anova 2 way and Chi square test were used for data analysis. The results showed that CO2 level in non-ventilated group was higher (0.15%) compare to control group (0.03%). Hatchability was higher in non-ventilation incubator compare to control group in LB strain but the result was the reverse in LW strain. In both strains, non-ventilation treatment did not impact internal pipping duration, but external pipping duration was higher in LW strain compare to LB strain in both ventilated and non-ventilated group. The hatching window and the average time of hatching was shorter in non-ventilation group compare to ventilated group for both strains. Embryo weight was higher in non-ventilation treatment at ED12, ED14 and at IP compare to control group but there was no significant difference on day-old chick body weight. During post hatch, non-ventilation did not affect chick's body weight but chicks from LB strains had higher body weight compare to LW strains in both ventilated and non-ventilated group. It can be concluded that LB strain was more sensitive to incubator CO2 level than LW strain and the two strains do not have the same developmental trajectory.

Key words: Layer strain, embryo development, non-ventilation, post-hatch growth.
ID: 1267

INTERMITTENT THERMAL MANIPULATION DURING INCUBATION: EFFECT ON EMBRYO DEVELOPMENT, HATCHING PROCESS AND POST HATCH GROWTH OF TWO BROILERS STRAIN IN TROPICAL CLIMATE.+

H. Meteyake\*(1), A.Bilalissi(2), K.Tona(2)

(1)CERSA-UL, Lomé, Togo

(2)CERSA, Lome, Togo

\* Corresponding author: hezouwem0123@gmail.com

Several studies have shown that thermal manipulations have effects on embryo development and post-hatch growth. It is also known that genetic background of broiler strains influences embryonic development. It was hypothesised that effect of thermal manipulation might depend of the genetic background of broilers. Therefore, the aim of this study was to investigate the effects of thermal manipulation during incubation (TMI) on embryo development and post-hatch growth of Cobb and Ross broilers under tropical climate. A total of 1500 hatching eggs obtained from Ross and Cobb broiler breeders were incubated at standard incubation conditions (37.8°C and 60% relative humidity) until day 7 and then randomly divided into four groups of five replicates: Rosscont and Cobbcont groups incubated at standard conditions and Rosstemp and Cobbtemp group subjected to TMI (39.5 °C at 65% for 12 h/d from d7 to d16). Between 456 and 504h of incubation, hatching eggs were checked for hatching events. From day15 to day42 post hatch, chicks from the four groups were reared at a natural ambient temperature (ranging from 22 to 34°C). Embryo weight, Hatchability, hatching time, chick's temperature, day-old chick's weight and 6 week-old body weight were determined. Data were analysed using Graph pad prism 5.0 in two-way ANOVA and chi square test. Results indicated high hatchability in both control group (p<0.05) compared to treated groups. Hatching time was shorter in Cobb strain compared to Ross strain. The body temperature was lower in TMI groups compared to control group and higher in Cobb strain compared to Ross strain. There was an interaction between incubation temperature and the strain on hatching time and chicks body temperature (p<0.05). Chick weight was comparable in all groups but the 42day-old body weight was higher (p<0.05) in Cobb strain compared to Ross strain. In conclusion, the effects of thermal manipulation are influenced by the genetic background. Further research should be carried out for the optimization of thermal manipulation during incubation for each genetic line.

Key words: Thermal manipulation, Broilers strain, Embryo development, Post-hatch growth, hot climate.
CELLULAR AND MOLECULAR MECHANISMS WHICH ACCOUNT FOR EMBRYONIC SURVIVAL FOLLOWING
PROLONGED STORAGE

N. Pokhrel(1), O.Beyioku(1), Y.Cinnamon*(2)

(1) Agricultural Research Organization, Volcani Center, Department of Poultry and Aquaculture Science, & The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel., Rishon Letsiyon, Israel

(2) Agricultural Research Organization, Volcani Center, Rishon Letsiyon, Israel

* Corresponding author: yuvalcinnamon@gmail.com

The chicken embryo, known as the blastoderm, consists of ~100,000 cells by the time the egg is laid. Following oviposition, avian embryos can suspend their developmental progress, at low temperature in a process known as diapause. The diapause phenomenon is exploited in the poultry industry to store freshly-laid eggs prior to incubation. In our previous studies, we defined millstone criteria to challenge and predict the effects of different storage conditions on embryonic survival, following prolonged storage. These criteria consist of accurate analysis of developmental stage, total cell count, cell viability and mitotic index.

Comparing prolonged egg storage, for up to 28 days, at 12oC and 18oC, we found embryos stored at the lower temperature exhibit better cytoarchitectural characteristics, lower apoptotic-mediated cellular death, higher mitotic index and far better embryonic survival and hatchability. Moreover, we found that in freshly laid eggs in modern commercial broiler breeds, the predominant embryonic developmental stages, are XI EG&K and XII EG&K in young and old flocks, respectively. This reflects an advanced stage compared with similar pioneering studies, decades ago, which found that the predominant stage is around X EG&K. This variance can be accounted to differences between breeds or to changes in the reproductive system and/or developmental rate, which occurred during years of broiler genetic selection.

To disclose the roots of these variances, we compared the reproductive system, developmental stages and embryonic growth rate between modern commercial broilers and commercial legacy breed which was not subjected to genetic selection since 1986. Our results show a marked increase in growth rate in modern breeds. Furthermore, using RNA seq we identified signaling pathways which are involved in entering diapause state and promoting embryonic survival during prolonged storage. Notably, we found a marked down-regulation in genes associated with metabolism, while up-regulation was found in pathways associated with maintaining pluripotency, at lower temperatures. These results were further validated by real-time PCR and RNA in-situ hybridization techniques. Our study shows that maintaining the pluripotency during diapause, even when the metabolism is drastically decreasing, can be one of the mechanisms involved in embryonic survival during diapause.
ID: 454

SCREENING OF KEY PROTEINS IN EGG YOLK AND WHITE THAT INFLUENCING CHICKEN HATCHABILITY

C. Sun*, X.Wang(1), G.Xu(1), N.Yang(1)

(1)China Agricultural University, Beijing, China

* Corresponding author: cjsun@cau.edu.cn

During avian embryo development, the egg yolk and white provide almost all the nutritional and functional components. At present, the roles of these components in embryo development remain unknown. In this study, hens with divergent high/low hatchability (HH, LH; 100% vs. <65%) were selected from a population consisting of 88 males and 704 female Rhode Island Red chickens (♂: ♀ = 1: 8) to analyze the nutrients, minerals, and functional proteins in their egg yolks and whites. A tandem mass tag labeling quantitation approach was used to innovatively identify the differential proteins in (1) the egg whites/yolks of fresh eggs produced by hens with divergent high/low hatchability and (2) the egg whites of embryonated eggs with healthy and dead embryos. In egg yolks, a total of 401 proteins and 31 differential proteins were quantified. These differential proteins participate in some biological processes such as antimicrobial activity, protein processing, blood coagulation as well as differentiation and metabolism. The protein DNAJB11, which were upregulated significantly in high hatchability group and whose abundance were 270.65 times higher than that in the low hatchability group (P < 0.05), binds to misfolded proteins and regulates the correct folding and degradation proteins. Other proteins including WFDC2 and UMOD related to host defense contribute to embryo development when over-represented. In egg whites, a total of 378 proteins were quantified in egg white, and up to 102 differential proteins were identified. GO enrichment, pathway, and hierarchical clustering analysis revealed some of the differential proteins that are the main participants in several biological processes, including blood coagulation, intermediate filament, antibacterial activity, and neurodevelopment. A list of 11 putative protein biomarkers, such as keratin (KRT19, KRT12, KRT15, and KRT6A) and fibrinogen (fibrinogen alpha chain, fibrinogen beta chain, and fibrinogen gamma chain) were ultimately screened. The current study screened egg yolk and white proteins that can predict low hatchability and embryonic death and deciphered the role of these proteins in embryonic development, which is meaningful for the comprehensive understanding of embryonic growth.

Key words: chicken, hatchability, proteomics, egg white and yolk protein
AP1

SMALL SCALE FAMILY POULTRY FARMING
CLIMATE RESILIENT TANUVAS ASEEL X NANDANAM CHICKEN 4 CROSSBRED STRAIN (ANC4)

E. Subbiah*(1), P. Chinnadurai(1), S.T. Selvan(1), A. Sundaresan(1), A.V. Omprakash(1)

(1)Tamil Nadu Veterinary and Animal Sciences University, Chennai, India

* Corresponding author: vet_ezhil2001@yahoo.co.in

TANUVAS Aseel is a multicoloured native chicken variety which has got attractive plumage, unique juice flavor of the meat, good disease and heat tolerance, produce 160 eggs per annum and best suited for backyard rearing. Nandanam Chicken 4 developed for backyard rearing. It has got multi colour plumage, produces brown shelled eggs and produce 220 eggs per annum. Hence, the present research work was undertaken to evaluate the production and reproduction performance of TANUVAS Aseel x Nandanam chicken 4 crossbred strain (ANC4) and to develop a synthetic strain with Aseel characters for table purpose with native falvour of meat, to get more chicks per dam, owing to more egg production per dam at the Poultry Research Station, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India. For development of ANC4 Chicken, individual selection for male and family selection was carried out. In each generation 1200 day-old chick were hatched out as a replacement population.

A selection pressure of 1 in 2 for females and 1 in 16 for males were applied for each generation to achieve a target. Day old fourth generation ANC4 chicken were obtained from the Poultry Research Station. The collected data were subjected to statistical analysis of one way ANOVA as per the procedure of statistical analysis system (SPSS, version 20.0 for windows). ANC4 chicken production and reproduction parameters showed significantly (P<0.05) better than TANUVAS Aseel. The hatch weight of ANC4 chicken was 35.80 ± 0.93g. Body weight was recorded to select best individual for next generation. Eight week body weight of male was 488.12 ± 5.18 g, female 425.15 ± 4.26 g and twelfth week body weight of male was 1320.22 ± 17.53 g and female 1023.45.11.01g. Age at sexual maturity was 150 days. The per cent hen day (HDEP) and hen housed egg production (HHEP) was 50.05 ± 1.23 and 50.27 ± 1.32 respectively. The per cent total and fertile hatchability was 72.60 ± 1.39 and 87.54 ± 1.31 respectively. The annual egg number, day old chick per dam and per cent livability 183.13, 133.95.5 were recorded from 20 to 72 weeks. Liver samples was taken from ANC4 for transcriptome analysis. A total of 25,840,415 raw reads were observed and 94.75 per cent aligned against Red Jungle fowl. Differential gene expression analysis showed 133 genes were unregulated. The results revealed that production and reproduction performance of ANC4 was better than TANUVAS Aseel in terms of egg number and chick per dam.
FAMILY POULTRY AND PUBLIC POLICY: LESSONS, LINKAGES AND LOOKING FORWARD

R. Alders* (1), J. Wong(2), D. Anene(3), S. Moreland(2), H. Reed(4), B. Richardson(4), D. Heymann(2)

(1) Centre on Global Health Security, Chatham House and Kyeema Foundation, London, United Kingdom

(2) Centre on Global Health Security, Chatham House, London, United Kingdom

(3) University of Sydney, Camden, Australia

(4) Bill & Melinda Gates Foundation, Seattle, United States

* Corresponding author: RAlders@chathamhouse.org

INTRODUCTION

The number of people living with chronic hunger has risen for the third consecutive year to over 820 million in 2018 while incomes for men and women smallholder farmers are not increasing in line with SDG2 targets. Animal-source food and income from family poultry production can play a significant role in enhancing diet quality and quantity in resource-poor settings where monotonous cereal-based diets are common. However, family poultry does not always receive adequate or appropriate public policy attention which undermines farmers ability to achieve financially, environmentally and socially sustainable family poultry systems.

METHODOLOGY

As part of a wider livestock and aquaculture policy project, and with a focus on Bangladesh and Nigeria, we employed mixed methods - including stakeholder mapping, literature review, key informant interviews, online public consultation and national roundtables - to assess public policy successes, challenges and opportunities.

RESULTS

Globally a lack of inclusive policies, stakeholder consultation and implementation plans for existing smallholder livestock policies were highlighted as major challenges. Specifically, regarding family poultry, the key issues identified were: policy support for vaccination of family poultry without appropriate focus on adequate supply, cold chain and distribution contributes to ineffective disease prevention; policies banning the routine use of antibiotics as growth promotants without identification of and support for feasible transition strategies can cause additional antimicrobial resistance; inclusive policies that address adequate feed quantity and quality in relation to efficiently meeting the nutrient requirements of poultry without impacting negatively on supply of food fit for human consumption are largely lacking; introducing 'high producing' breeds without sound national breeding and indigenous breed conservation policies can lead to poor performance and impact negatively on farming household income; and focusing on increasing production without simultaneously addressing issues concerning food safety, efficient marketing and poultry product preservation can also impact negatively on farming households and consumers.

CONCLUSION

A multisectoral, inclusive approach is required to ensure that policies impacting on smallholder producers align optimally to facilitate efficient, safe, sustainable family poultry production that is available to consumers at affordable prices.
# CAN VILLAGE POULTRY IMPROVEMENT EMPOWER WOMEN?

B. Bagnol* (1)

(1)Witwatersrand University, Avignon, France

* Corresponding author: bagnolbrigitte@gmail.com

## Introduction

Gender analysis and gender sensitive instruments developed and used in development and research related to livestock and specifically to village poultry has evolved in the last three decades. Because poultry is the most widespread animal species in the world and because its potential for demand growth in the next decades is high it constitutes a very important animal husbandry to promote and improve. In addition, as poultry constitute one of the most common assets under the responsibility of women in the world, the motivations to use it as a leverage to increase rural women's visibility, economic benefit and empowerment is frequent.

## Methodology

A dozen female key players who participated in projects and research related to village poultry in Asia (HPAI) and in Southern Africa (Newcastle disease) in the last three decades speak about women empowerment and village poultry work and comment on interventions carried out and results achieved individually and collectively. The paper also put in perspective, revises, compiles and analyses quantitative and qualitative data collected in different contexts through the prism of current knowledges and practices toward gender equality and women's empowerment.

## Results

Women from different backgrounds and ages, in distinct period of their carrier and in diverse positions and roles in the several interventions under analysis experienced, exercised and valued different forms of power. By example, female students or female national researchers were given central role in the project empowering them both individually (power within), as professional (power to), as advocate for gender issues (power with). Approaches shared, methodologies applied, and experiences discussed, both in relation to research and development processes, in the search for gender equality and women's empowerment highlight the importance of placing women at the centre of the process.

## Conclusion

The diverse forms of women's empowerments experienced and achieved at different nodes of the interventions processes illustrate the multiplicity of ways by which gender sensitive interventions can transform individuals, processes as well as institutions.
COOCIDIOSIS IN KADAKNATH CHICKEN REARED IN MIXED FLOCKS UNDER SEMI-INTENSIVE HOUSING SYSTEM

G. Kolluri*(1), S.Rafiqui(2), A.Mariappan(2), P.Munuswamy(2), A.Yadav(1)

(1)ICAR-CENTRAL AVIAN RESEARCH INSTITUTE, Izatnagar, Bareilly, India

(2)ICAR-INDIAN VETERINARY RESEARCH INSTITUTE, Bareilly, India

* Corresponding author: drgau_vet@yahoo.com

An investigation was taken up during our routine field visit to backyard poultry farms wherein, few Kadaknath chicken flocks were reported to have mortality and morbidity issues for 6 consecutive days. The affected backyard farm comprised about 450 birds of 8 weeks age group which included Kadaknath and Aseel ecotypes, and were being maintained under semi-intensive system. These flocks were supplied by Central Avian Research Institute which is instrumental in enhancing rural poultry production in the country. Affected birds were witnessed to show anorexia, dullness with closed eyes and bloody droppings. Necropsy examination indicated dilation of the intestinal segments through distal end of jejunum in an increasing manner and its lumen contained necro-hemorrhagic contents amidst necrotic foci on the mucosal surface. Ceca showed varying intensities of mucosal hemorrhages and contained pasty to thick fluidic contents. Microscopically, H & E stained sections of intestinal segments including cecum revealed mucosal hemorrhages, marked epithelial necrosis, denudation of superficial lining cells of intestinal villi, and presence of numerous oocysts and schizonts in the epithelial cells of villus as well as crypt glands. The stromal tissues adjacent to the infected epithelium showed mild to moderate degrees of heterophilic as well as mononuclear cell infiltration. Molecular based diagnosis (PCR) revealed positive for Eimeria acervulina (321 bp), Eimeria mitis S (193 bp) and Eimeria tenella (278 bp). Administration of Diaverdin and sulphaquinoxaline combination in drinking water witnessed no further mortality. Indigenous breeds are considered to be adaptable to tropical conditions and resistant to many diseases despite their frequent exposure to contaminated places where the incidence of potential pathogens is relatively high, which might be due to strong innate immune response that they elicit. Scientific evidence confirming these assumptions are lacking. Kadaknath and Aseel are most popular breeds reflect that reflects India's heritage and are mostly preferred on various festive occasions, and therefore been used extensively for village chicken production. From this study, we understood that coccidiosis can affect diversified breeds of chicken with high disease incidence typically at the age of 8 weeks, especially in intensive housing conditions particularly during autumn and multiple strains of Eimeria are involved in exacerbating the intestinal pathology.
RELATIONSHIPS BETWEEN RESPONSES OF TANZANIAN FREE-RANGE LOCAL CHICKEN ECOTYPES TO
INFECTION WITH LENTOGENIC AND VELOGENIC STRAINS OF NEWCASTLE DISEASE VIRUS

J. Mushi\*(1), E.Amuzu-aweh(2), M.Walugembe(3), G.Chiwanga(4), E.Mollel(4), R.Max(5), R.Gallardo(6), T.Kelly(6), S.J. Lamont,(3), J.Dekkers(7), P.Msoffe(4), A.Muhairwa(8)

(1) Sokoine University of Agriculture, Morogoro, Tanzania

(2) Department of Animal Science, University of Ghana, Accra, Ghana, Morogoro, Tanzania

(3) Department of Animal Science, Iowa State University, Ames, IA 50011, USA, Iowa, United States

(4) Department of Veterinary Medicine and Public Health, Sokoine University of Agriculture, Morogoro, Tanzania, Morogoro, Tanzania

(5) Department of Veterinary Physiology, Sokoine University of Agriculture, Morogoro, Tanzania, Morogoro, Tanzania

(6) School of Veterinary Medicine, University of California, Davis, 95616, USA, Davis, United States

(7) Department of Animal Science, Iowa State University, Ames, IA 50011, USA, Morogoro, Tanzania

(8) muhairwa@yahoo.co.uk, Morogoro, Tanzania

Village poultry in Tanzania is dominated by free-range local chickens (FRLC), they provide about 100% of meat and eggs to households. They are resilient to harsh conditions of tropical hot climate, poor nutrition, diseases, and lack of veterinary care. These conditions, affect productivity in these chickens. The increasing human population in Tanzania is driving the need to increase production of FRLCs to supply essential animal protein in human diets as well as improvement of family income. Newcastle disease is still a major cause of massive losses to the FRLCs. This study aimed to improve production of the FRLCs through control of Newcastle disease. A breeding flock of three FRLC ecotypes was prepared from randomly sampled chickens, consisting of Kuchi, Morogoro-medium, and Ching'wekwe from the Lake, Central and Coastal climatic zones of Tanzania. The chickens are not pure ecotypes, genetically they belonged to two populations; (population 1 dominated by Ching'wekwe and Morogoro-medium and population 2 dominated by Kuchi) In a two-stage study, progenies of the breeders were infected with LaSota strain of Newcastle disease virus (NDV) and later exposed to virulent field strains of NDV (vNDV). In the first stage, chicks were infected with LaSota NDV at 28 days of age. Tear samples were collected at 2 and 6 days post infection (dpi) to determine viral load by PCR and blood samples at 10 dpi to determine antibody titers by ELISA. Body weights were measured weekly from hatch to six weeks. In stage two, the chicks were kept for four additional months to wane acquired immunity and then exposed to vNDV by contact with sick chickens. Body weights were measured at two-dayintervals. At mortalities, post-mortem examination and lesions scores of dead chickens were recordedfor three weeks, every morning and evening. Correlation analyses were used to determine relationships between the response traits. Survival analysis was also done. Pre- and post-infection growth rates were significantly positive but moderately correlated. There were no strong correlations between traits during infection with LaSota strain and exposure to vNDV in the FRLCs, apart from positive but weak (0.12) correlation of post-infection GR with average lesion scores during exposure to vNDV. Population 2 chickens with hazard ratio of 0.52 were more likely to survive vNDV infection than population1 chickens. Males of all populations were 1.4 times more likely to die of vNDV than females.

<footnote>* Corresponding author: jamessalakana_1979@sua.ac.tz</footnote>
GENDER DYNAMICS AND POULTRY HEALTH ISSUES IN SEMBABULE DISTRICT, UGANDA

L. Tukahirwa*(1), E.Kyewalabye(2), R.Nsibirano(3), W.Bikaako(4), A.Mugisha(2), H.Amuguni(5)

(1)Makerere University, Kampala Uganda, Kampala, Uganda

(2)Makerere University, Kampala, Uganda

(3)Makerere University, Kampala, Uganda

(4)OHCEA, Kampala, Uganda

(5)Tufts University, Kampala, United States

* Corresponding author: lilietuk@gmail.com

Introduction

This paper presents findings of the study that was part of a broader research project entitled "Hearing their voices: Action research to support women's agency and empowerment in livestock vaccine distribution, delivery and use in Uganda".

Poultry in Uganda is a source of income for many communities and offer some form of employment for a sizable number of households especially women and children. In Sembabule almost all rural households keep poultry. The objectives of the research study were:

1. To assess women's and men's access and control over poultry resources in Sembabule District

2. To assess gender roles, responsibilities and time use in poultry production in Sembabule District.

3. Prioritization of problems related to poultry vaccines in Sembabule District.

Research Methodology

The research was carried out in the two counties of Sembabule District, namely. Mawogola County which is predominantly agro-pastoralist and Lwemiyaga County, predominantly pastoralist. It was a cross sectional study using qualitative/participatory research methodologies, involving secondary data collection, stakeholder engagement, a rapid appraisal to the district for a scoping study and field data collection for gender analysis involving both traditional participatory methods, namely Focus Group Discussions, Key Informant Interviews and Participants observations. In addition, novel methods of collecting qualitative data, namely jar voices and focus meals were used. Domains 2 & 3 of The USAID Five Domains of Gender Analysis Framework were used to collect data. The data collected was analyzed using qualitative data analysis tools.

Results

Many women were found to have access and control over poultry resources. Men have control over land and cattle. Men are slowly encroaching on the goat resources which were previously a domain of women. It was found that more of the roles, responsibilities and time use in poultry production are done by women. The prioritized poultry health related challenges were: diseases especially New Castle Disease, lack of authentic drugs and vaccines, and lack of training.

Conclusions

In Sembabule District, Poultry is the only animal resource still accessed and controlled by women. If managed properly, poultry could be a starting point for enhancing livelihoods of women in this community. New Castle Disease is still a big challenge to poultry farming in the area, and more efforts are needed in combating this disease preferably through vaccination.
ID: 1291

# THE FAO CONTRIBUTIONS TO THE SUSTAINABLE DEVELOPMENT GOALS THROUGH POULTRY PRODUCTION IN DEVELOPING COUNTRIES

E. Guèye\*(1)

(1)Food and Agriculture Organization of the United Nations (FAO), Djibouti, Djibouti

\* Corresponding author: Fallou.Gueye@fao.org

Commercial poultry (CP) production has made substantial progress during the last seventy years in developing countries of Africa, Asia, Latin America and Oceania. This type of production depends on costly imported inputs such as day-old chicks from high-performance hybrid stocks, balanced feeds, drugs, vaccines, etc. and on the presence of consumers who can afford to buy commercial poultry products. Family poultry (FP), which make up more than 80% of poultry stocks in most developing countries, are still important. Analysing of grey literature and mid-term reports of targets and indicators towards achieving 17 Sustainable Development Goals (SDGs) by 2030 showed that FP are central to contribute to achieving directly 12 of the SDGs. The growing demand for poultry products in developing countries, driven by population growth, higher incomes, scarcity of land and urbanization, represents a huge opportunity for hundreds of millions of poor smallholder poultry farmers, processors and marketers, many of whom are women, to meet that market demand and rise out of poverty. Improving the efficiency of poultry production in developing countries, especially the productivity per poultry bird, can triple poultry productivity while reducing by more than a third its adverse environmental impacts, including reducing emissions of greenhouse gases (GHG), in those developing countries that are less GHG emitters but highly negatively impacted by them. However, FP are facing many constraints, including high mortality (mainly due to Newcastle and other avian diseases). Significant improvements in FP production systems can be achieved through well-designed and implemented research and development programmes that endow FP actors with necessary knowledge and skills. In addition to the need for substantial improvement in human and institutional capacity building, planners and policy makers must be sensitized about the potential of poultry as a tool in poverty reduction, food security and gender equity strategies.

The Food and Agriculture Organization of the United Nations (FAO) is committed to FP production and, through the International Network for Family Poultry Development (INFPD) and other initiatives such as the Hand-in-Hand. This paper highlights the importance of poultry for poverty reduction, food security, and the promotion of gender equality in developing countries and discusses the FAO contributions towards the SDGs adopted by the world leaders of 195 nations in 2015.
DIVERSIFIED POULTRY PRODUCTION IN INDIA

P. Rajamanickam\*(1)

(1)Former Vice-Chancellor, Tamil Nadu Veterinary and Animal Sciences University, Chennai, INDIA, Chennai, India

\* Corresponding author: profrppsc@gmail.com

Diversified poultry includes ducks, turkeys, Japanese quail, Guinea fowl, Goose, pigeons and commercial rearing of native chicken. Diversified poultry systems are now considered as viable livelihood options for small and marginal farmers in India. Ducks accounts for seven per cent of poultry population and are mostly found in coastal states of the county and the duck eggs contribute about 1.15 per cent of total egg production in the country and is preferred over chicken eggs by some consumers. Commercial Japanese quail farming in India can be a great source of handsome income and employment opportunity to small and marginal farmers. At present Japanese quail have become the third largest avian specie grown in the country. About 45 million meat type broiler quail are produced in the southern state of Tamil Nadu alone and about 30 % in other states; 30 million Japanesequail eggs produced annually. Turkey, guinea fowl and goose farming are still in their infant stage. Turkey farming is planned on a small scale and seasonal sales. Guinea fowl are reared in backyard of farm houses and a few Guinea fowl breeder farms do a lucrative business in Odisha and Tamil Nadu. Native chicken farming provides subsidiary income to the rural families. Native chicken meat and eggs attract premium price in local market. Hence, commercial intensive farming of native chicken has become popular and about 12 million native chicken are under intensive production. Whichever species is preferred based on local market preference, farmers mostly go for same species rearing and avoid multi species in the same farm. Commercial duck production for eggs and meat with improved breeds has not been attempted on a larger scale and its scope depends on change in demand for specific duck eggs and meat as chicken eggs and meat production costs are cheaper and management including health cover is easier. Change in consumption pattern with wider acceptance of frozen meat will usher in growth of turkey production in future. As the egg production performance of availability of turkey germplasm does not support viable commercial production, impart of quality small variety turkey breeds as parent stock is required. Guinea fowl meat is preferred only by a few. To diversify and substitute commercial chicken production in India especially to support small and marginal farmers, Japanese quail farming and intensive rearing of native chicken offer much scope. With proper inputs, turkey farming also may do so.
MONITORING OF RESPIRATORY PATHOGENS IN COMMERCIAL SPENT LAYING HENS AFTER
INTRODUCTION IN BACKYARD FLOCKS

M. Souvestre*(1), X. Nouvel(2), M. Hygonenq(2), G. Leloc'h(2), P. Belloir(3), L. Robertet(2), L. Manis(2), C. Citti(2)

(1) Veterinary School Toulouse, Toulouse, France

(2) ENVT, Toulouse, France

(3) Ecole Ingénieurs Purpan, Toulouse, France

* Corresponding author: marie.souvestre@envt.fr

Small scale family flocks (SSF) usually found in rural areas are getting more common in urban areas
worldwide. Some health surveys showed that SSF could be infected with Avibacterium paragallinarum
(AvP), Mycoplasma synoviae (MS), Mycoplasma gallisepticum (MG), Ornithobacterium Rhinotracheale
(ORT), Infectious bronchitis (IB), Chlamydia spp. (Chl.spp), Chlamydia gallinacea (Chl.g), Avian
metapneumovirus (aMPV) and Pasteurella Multocida (PM) which are pathogens of economic
importance for commercial poultry industry (Madsen et al., 2013, Donati et al., 2018). Studying how
SSF could be reservoirs for pathogens and understanding their transmission patterns are important
issues to prevent clinical outbreaks in small or large-scale poultry farms, especially in a recent context
of growing urban agriculture (Blecha et al., 2013). In France, a common practice for family flocks'
owners is to get spent hens from commercial farms. In order to assess the transmission of pathogens
from a "family flock" environment to commercial layers, a focus was made on AvP, MS, MG, ORT, IB,
Chl.spp, Chl.g, aMPV and PM status of commercial layers. Layers from a negative free-range
commercial farm for the previously cited pathogens were monitored after being introduced into 28
existing family flocks. Biosecurity practices were studied and sanitary status was assessed by qPCR at
introduction and six months after. Commercial layers became respectively positive for AvP (32%), MS
(39%), MG (11%), ORT (21%) after their introduction in SSF. All layers were negative for Chl.spp, Chl.g,
aMPV and PM. Median flock size was of 7 hens and 25% of SSF were flocks having less than 2 years old.
More than 80% of owners claimed to have layers for egg quality and recycling organic waste. They
observed no specific biosecurity practices. The study showed new agricultural practices in (sub)-urban
areas as well as flocks hosting pathogens. In this case, contamination from SSF to commercial farms
seemed limited since direct or indirect contact between commercial farms and urban family poultry
were rare (Alexander et al., 2007); (sub)-urbans family flocks could more be considered as "dead-end
hosts". Nevertheless, monitoring respiratory pathogens could be of interest to limit disease spread in
the SSF "compartment". Considering other pathogens such as Salmonella spp. and Campylobacter spp.
would help preventing eventual food-borne outbreaks and assess public health issues.
ID: 1331

ACTION RESEARCH TO IDENTIFY CONSTRAINTS AND OPPORTUNITIES FOR INCREASED NEW CASTLE DISEASE VACCINE ADOPTION AMONG WOMEN SMALL HOLDER POULTRY FARMERS IN KENYA, RWANDA AND UGANDA.

J. Amuguni*(1), F.Niyitanga(2), J.Oduma(3), J.Schurer(4), M.Rosenbaum(4), W.Bikaako(5), A.Yawe(5), M.Stanley(4), B.Miller(6), B.Bagnol(7), A.Mugisha(8)

(1)Tufts University Cummings school of Veterinary Medicine, North grafton, United States

(2)University of Rwanda, Kigali, Rwanda

(3)University of Nairobi, Kenya, Nairobi, Kenya

(4)Tufts University, North grafton, United States

(5)One Health Central and Eastern Africa, Kampala, Uganda

(6)International Goat Association, Little rock, arkansas, United States

(7)KYEMA Foundation, Marselles, France

(8)Makerere University, Kampala, Uganda

**Objectives:** In Rwanda, Kenya and Uganda, 75 to 90% of small-scale poultry farmers are women. Diseases are a major issue preventing them from optimizing production. Their flocks are frequently decimated by NCD despite availability of an effective vaccine. Packaging and reliable structures for vaccine delivery remain an obstacle to vaccine uptake and use. Women are disproportionately affected because of their reliance on poultry income. Our objectives were to: 1) identify barriers and opportunities for women's participation in NCD vaccine value chain (VVC) 2) formulate strategies that position women to effectively contribute to and benefit from poultry vaccines, 3) enhance women's participation in vaccine distribution, delivery and use, 4) provide data that impact programmatic and policy interventions.

**Methodology:** We used three methods for gender analysis and NCD-VVC analysis: 1) The USAID Five Domains of Gender Analysis Framework administered through focus groups captured barriers, opportunities and strategies for improving women's participation in poultry ownership and VVC. 2) The Women Empowerment in Livestock Index (WELI) a standardized household quantitative survey tool captured the empowerment index of women farmers and vaccine usage. 3) Outcome Mapping systematically mapped and tracked critical stakeholders in the VVC focusing on changes in gender norms and cultural practices, organizational systems, institutional and governance policies that lead to increased vaccine adoption. We also introduced novel tools such as Vaxer Calendars, focus meals, jar voices, VacZines and PhotoVax to collect data and identify entry points for interventions.

**Results:** Preliminary results highlight limited vaccine availability and accessibility, as well as infrastructure challenges, lack of knowledge and information to guide poultry farmers on vaccinable diseases, women's limited decision-making agency in their households and communities, gender related technical, social, cultural, and economic barriers, lack of partnership/networking with public and private sectors as key reasons for low adoption of NCD vaccines.

**Conclusion:** The project will pilot interventions that target women's individual and collective decision-making control within the households and communities, resulting in a recognized productive role in poultry management, strengthened skills and knowledge on vaccines, opportunities to influence governance structures leading to increased poultry vaccine adoption.

<footnote>* Corresponding author: Janetrix.amuguni@tufts.edu</footnote>
DEVELOPING SUSTAINABLE VALUE CHAINS FOR SMALL-SCALE POULTRY PRODUCERS

G. Leroy* (1), C.Ingabire(2), B.Besbes(2)

(1) FAO/INRA/AgroParisTech, Roma, Italy

(2)FAO, Roma, Italy

* Corresponding author: gregoire.leroy@fao.org

In developing countries, small-scale poultry production has an important role for food security and nutrition, income generation and women's empowerment. Small-scale poultry producers face however many challenges, including growing environmental constraints, poor access to quality input and services, market linkages and limited capacities and decision making in the value chains governance, which often hamper their productivity and competitiveness vis-à-vis their larger counterparts.

The sustainable food value chain development (SFVCD) framework developed by FAO is a market led approach that integrates the multifaceted concepts of value-addition and sustainability to address the challenges small-scale producers are facing. The value-chain development is a dynamic process, which begins with the characterisation of stakeholders and their activities, the enabling environment, the behavioural change and the governance of the value chain. This allows to identify root problems, leverage opportunities to upgrade the targeted value chain, and develop a vision and a strategy supported by appropriate action plans, in accordance with the value chain stakeholders.

The interest of the SFVCD for small-scale poultry production is illustrated through the results of various projects in Asia, Africa and Europe. The specific characteristics of small-scale poultry production systems (i.e. multi-functionality, poultry as a liquid asset, input and output provision, health and quality, social equity, etc.) underline the need to analyse value chains in a holistic manner, considering not only the supply chain itself, but also its broad environment, its dynamics and its connections to other systems. When considering value chain development involving/or/focusing on small-scale poultry producers, a particular challenge relates to the interest of these producers, especially in a scavenging system, to move from a subsistence to a market oriented system.
AP2

WATERFOWL
ID: 974

SUPERIOR EGG PRODUCTION REGISTERED IN PEKIN DUCKS REARED ON AFLATOXIN-SPIKED GROWER DIETS: POSSIBILITY OF EPIGENETIC ADAPTATION FROM PERINATAL CONDITIONING IN SURVIVORS

S. K. Mishra*(1), C.R. Jena(2), D. Kumar(2), B.K. Swain(2), P.K. Naik(2), G.R. Jena(3), S.K. Panda(3), N.C. Nath(4), P. K. Subudhl(5)

(1) ICAR Central Avian Research Institute, Baramunda, India

(2)ICAR-CARI Regional Center, Baramunda, Bhubaneswar, India

(3) Dept of Veterinary Pathology, CVSc & A.H,Ouat, Bhubaneswar, India

(4)Dept of Veterinary Physiology, CVSc, Assam Agricultural University, Guwahati, India

(5)Department of Veterinary Microbiology, C V Sc and AH, Central Agricultural University, Aizawl, India

* Corresponding author: suryakmishra@gmail.com

The negative impact of Aflatoxin-B1 (AFB1) on survival & productivity in ducks (Anas Platyrhynchos) is higher (>100 fold) than chickens; White Pekin (WP) ducks being more vulnerable than others. To improve ducks for better tolerance to AFB1 through epigenetic approach; a flock of WP were experimented, where 220 day-old ducklings (2 hours post hatch) were exposed to standard duck-chick crumbs spiked with 24ppb AFB1, with ad lib. feed & water; and standard management till 6 weeks; followed by withdrawal of AFB1 and growing of all survivors with standard grower and layer crumbs (Nil AFB1) till 18 and 70 weeks, respectively. In parallel, another trial was conducted with 200 ducklings [from same parents] on diets spiked with 150ppb AFB1 from day-old till 6 weeks, following withdrawal of AFB1 and survivors grown further till 40 weeks. Duck-day Egg production was measured on all AFB1 sensitized ducks, of both batches with respective controls till 70 weeks age (in former) and till 40 weeks age in latter batch. The AFB1 survivor ducks yielded significantly higher egg production than their counterparts across both batches. While survivors (24ppb sensitized) produced 169.7 eggs/duck versus controls who laid only 160.2 eggs; the latter batch survivors yielded 35.32 eggs/duck upto 40 weeks, versus their control which yielded 24.13 eggs/duck. While AFB1-exposed ducks consistently laid significantly higher number eggs (p < 0.05), in both batches, average egg weight (~64.5g) during laying in both groups remained non-significantly different between exposed vs unexposed lots. Mortality wise, while the former batch did not evince more than 5% mortality (0 to 6wks), the 150ppb-AFB1 ones experienced upto 12% mortality, till 6 weeks; with control showing no mortality in both batches consistently. While candidate gene analyses for 2 important genes: IGF1 and CYP-450 evinced significantly variant gene-expression between AFB1 survivors and control; the DNA methyl Transferase-1 (DNMT1) gene, having roles in methylation of DNA, also showed a differential expression in liver tissue (sacrificial samples) of AFB1-survived ducks, of both batches at 6 weeks age. It was concluded that: epigenetic adaptation to AFB1 through perinatal ages (till 6 weeks) have possibly conditioned survivor ducks into superior egg producers; when such sensitization is better achieved at lower dietary AFB1-doses (24ppb) than higher (150ppb), where toxic impacts could be more overwhelming than gains, in latter.
THE EFFECT OF MULTI STRAINS BACILLUS AMYLOLIQUEFACIENS ON GROWTH IN MUSCOVY DUCKS

S. Gilani(1), A.Debicki garnier*(2), J.Villatoro-hernandez(2), K.Waller(3), S.Roulleau(4), C.Henriet(5)

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

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

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

(4) MG2Mix, Zone à la Basse Haie, 35220, Châteaubourg, France

(5) Altilis, 23 Avenue Henri Brulle, 33500, Libourne, France

Probiotics are known to improve gut health in poultry. Bacillus amyloliquefaciens have shown to decrease pathogenic Escherichia coli, reduce intestinal permeability, increase nutrient digestibility and performance in broilers in recent paper (a). For these reasons, Bacillus amyloliquefaciens was applied in the growing ducks to monitor the performance. The trial was conducted in France at a semi-commercial setup, in MG2MIX trial farm facility. Male Muscovy ducks R71M, (n = 1312) were randomly distributed in two treatments i.e. with Bacillus amyloliquefaciens, serving as treated or without serving as control. The treatment groups received feed with 300,000 cfu/g for starter and 150,000 cfu/g for grower. Birds were kept on a raised slotted floor with free access to feed and water with 41 ducklings per pen and 16 replicates. Ducks were fed with a starter crumbles (day 1-21) and grower pellets (21-49 days). For starter and grower, diets mainly consisted of wheat and corn (respectively 72% and 83% of total cereals plus cereals by products) and soybean meal. Finished feeds were analyzed for crude protein (CP 19.3%), crude fat (CF 2.1%) and crude fiber (CFb 2.9%) for starter and for grower CP 15.1%, CF 3.0% and C Fb 2.65%. Body weight gain (BWG as gram/bird/day), feed intake (FI, g/b/d) were measured and feed conversion ratio (FCR) calculated at the end of starter and grower phases (day 21 and 49). The results were analyzed using JMP pro statistical analysis program and one-way ANOVA. Average BWG in control vs treated (23.6 vs 23.0), FI (36.5 vs 36.1 g/b/d) and FCR (1.55 vs 1.57) were not statistically different in the starter phase (d 1-21). These parameters improved significantly during grower phase (day 21-49) i.e BWG (65.7 vs 70.0) and FCR (2.13 vs 2.07). However, FI was not statistically different versus control (140 vs 145). Combining overall performance from day 1-49, FCR was significantly (P<0.05) improved by 2.0%. Improved gut health status and reduced immune challenge could be one explanation of increased performance, however, unfortunately these parameters were not measured in this study. In conclusion, addition of multi-strain Bacillus amyloliquefaciens improved BWG and reduced FCR in Muscovy ducks at 49 days of age.

a. K. Gibbs, E. White, D.J. Cadogan and S.J. Wilkinson. The effects of a multi-enzyme and bacillus probiotic combinations on calcium and phosphorus digestibility and broiler performance. 2018. Aust Poult Science Symposium 29.

<footnote>* Corresponding author: Saad.Gilani@iff.com</footnote>
FATTY ACIDS PROFILE OF BREAST MUSCLES FROM DUCKS FED WITH YELLOW LUPIN

J. Biesek*(1), M.Adamski(2), M.Hejdysz(3), M.Banaszak(2), A.Zmudzińska(2), G.Maiorano(4), B.Bigorowski(2)

(1) Department of Animal Breeding, Faculty of Animal Breeding and Biology, UTP - University of Science and Technology in Bydgoszcz, Bydgoszcz, Poland

(2) Department of Animal Breeding, Faculty of Animal Breeding and Biology, UTP - University of Science and Technology, Bydgoszcz, Poland

(3) Department of Animal Nutrition and Feed Management, UP Poznań University of Life Sciences, Poznań, Poland

(4) Department of Agricultural, Environmental and Food Sciences, Università degli Studi del Molise, Campobasso, Italy

* Corresponding author: jakub.biesek@utp.edu.pl

In duck rearing in small farms in EU more often is searched of alternative vegetable protein source for soybean meal. The possibility of using the high protein content plants produced in small farms is a reason for conducting studies about quality evaluation of duck meat produced in this conditions. The aim of the study was to comparison of fatty acids profiles in breast muscles of duck fed with diets based on yellow lupine (Lupinus luteus L.), brewery yeast and potatoes protein as high-proteinalternative for soybean meal. Chery Valley ducks were kept in small farm in pens on litter in two groups,100 birds in each. Both groups divided into 4 replications with 25 birds per each. The control group (I)was fed with balanced feed containing saybean meal. The treatment group (II) received balanced feedcontaining yellow lupine. Ducks were kept for 8 weeks. After 8 weeks 10 randomly chosen ducks fromeach group were slaughtered. From each carcass breast muscles were collected and weighted. In all collected samples the acids profile was determinate. Lipids extraction from breast muscles was madeby Folch at al. (1957) method. Fatty acids (FA) quantity level was determinate as fatty acids methyl esters (FAME) with using of gas chromatography GC Trace 2000 (ThermoQuest EC Instruments). Presented results embodied as percent complete determinate FA. Evaluation of feed implication was made by calculation of ratio between n-6 to n-3 FA (n-6/ n-3) and ratio of PUFA to SFA (P/S). Atherogenic index (AI) and thrombogenic index (TI) were calculated by Ulbricht and Southgate (1991)method. Numeral data were processed with Statistica 10.0 PL software (2011) ANOVA was processedand the significant differences was verified with Scheffe test when P<0.05. In group I the % of breast muscles in carcass was bigger for 0.26. Percent of linoleic acid (LA; C18:2 n-6) was significant bigger ingroup II and C18:1 n-9 (oleic acid) as well (P<0.05). It could be a positive effect of using the yellow lupine for profits from LA. In group II shown significant smaller quantity of C18:0, C:20:4 n-6, C22:5 n-3 (P<0,05). Shown bigger quantity in total of SFA in group I. Ratio P/S was significant bigger in group II(P<0.05). Significant smallerfactors of AI and TI were found in group II (P0.05). Results of this study shows positive influence of using feed with yellow lupin in ducks feeding, because it's improve quantityof linoleic acid and lower factorsAI and TI compared soybean meal diet. [Resolution No. 222/2015; Grant No. PPI/APM/2019/1/00003].
AP3
RATITES
GENETIC EVALUATION OF PRODUCTION TRAITS OF OSTRICHES REARED UNDER SUB-TROPICAL FARMING CONDITION

S. Tamil shanmugam*(1), A.Arunachallam(1), D.Palaniappan(1), K.Periyasamy(1), V.Raghuthoman(1)

(1)Tamilnadu Veterinary And Animal Sciences University, Chennai, India

* Corresponding author: drstselvan@gmail.com

In India, ostrich is being promoted as an alternative poultry farming. It is not a traditional farm of India. However, it is in existence since 2000. Our experience revealed that rearing, feeding, breeding and hatching is quite different from regular chicken rearing. Experiences also revealed that phenotypic / performance selection is not sufficient to maintain a good breeding stock. With this background, a study was undertaken to estimate the genetic parameters for production traits. Data pertaining to the chick weight, periodical body weights and pedigree information were collected (period from 2001 to 2018) from the ostrich breeding unit located at Post Graduate Research Institute in Animal Science, TANUVAS, Kattupakkam. The description of production traits studied were chick weight, body weight at different ages, and average daily weight gain. The effect of non-genetic fixed factors on the production traits were studied during the period 2000 – 03, 2004 – 07, 2008 - 11, 2012 – 15 and 2016 – 18) and seasons of hatch Winter (Jan– Feb), Summer (Mar – May), SWM (June– Sep) and NEM (Oct – Dec). All the analyses were carried out using WOMBAT software (Meyer, 2007). The overall mean chick weight was 766.54 + 8.15 g. The least square mean for body weight at 2, 4, 6, 8, 10 and 12 m were 10.35 + 0.31, 37.46 + 1.35, 52.22 + 0.74, 66.49 + 0.73, 85.04 + 1.28 and 99.78 + 1.33 kgs respectively. The least square mean ADG of 0-2, 2-4, 4-6, 6-8, 8-10 and 10-12 m were 166.07 + 5.97, 335.55 + 9.22, 346.07 + 10.64, 231.88 + 8.10, 321.3 + 11.48 and 245.28 + 12.74 g respectively. The effect of seasonal period of hatch was found significant (P<0.05) for chick weight, body weight traits [except 12 m (period) and 10 m (season)], ADG [except above 6 m (period) and 6-8 m (season)], mortality [except 3-6 m (period) and 6-18 m (season)]. The heritability estimates for chick weight, 2 months body weight and ADG (0-2 m) were high (above 0.3) and for body weight at 4 and 6 m and ADG (2-4, 4-6 and 8-10 m) were moderate (0.1 to 0.3). The estimates for body weight at 8, 10 and 12 m were low (0.0 to 0.1), the heritability estimate for chick weight was 0.551 + 0.143. Higher values of genetic correlation between early and late body weight traits provided better scope for improvement through indirect selection. Negative genetic trend was observed for early body weight traits and positive genetic trend was found for body weight traits at later ages.
ID: 736

OPTIMAL SPERM DOSE AND FREQUENCY OF INSEMINATION FOR MAXIMAL FERTILISATION RATE IN
OSTRICHES

P. Muvhali*(1), M.Bonato(1), I.Malecki(2), S.Cloete(1)

(1)Stellenbosch University, Stellenbosch, South Africa

(2)The University of Western Australia, Crawley, Australia

* Corresponding author: 20501609@sun.ac.za

Artificial insemination (AI) may help reducing infertility, mating incompatibility while improving genetic gain in ostriches. We investigated the minimum sperm dose (Trial 1) and optimum frequency of insemination (Trial 2) that would also maintain high fertility rates over extended periods. Twenty-two South African Black ostriches (7 males and 15 females) aged between 2-10 years old were used. Semen was collected using the dummy female method and diluted 1:4 (semen:diluent) with an ostrich specific semen diluent. In Trial 1, females were inseminated three times a week on every second day with various sperm doses of fresh semen from the same male, resulting in a total sperm dose of A=7.5 billion sperm/week. Fertilization status of the germinal disc of eggs produced after the last AI was determined using the egg breakout technique. The fertile period was calculated as the number of days fertilized eggs were laid after the last AI. The rate of sperm loss on eggs produced after AI was also estimated. In Trial 2, the minimum sperm dose along with the longest fertile period obtained in Trial 1 were used for inseminations on two consecutive days, followed by a single weekly dose for 4 weeks. Fertilization status, fertile period and rate of sperm loss were also evaluated. Generalized linear mixed models in SAS were applied on the data and P0.05). Rate of sperm loss between doses also did not differ (P>0.05). Preliminary results for Trial 2 revealed an overall fertilization rate of 47%. Hence, two consecutive AI events with sperm doses between 2.5-5 billion sperm/week followed by a single weekly dose appeared to maintain relatively good fertility rates of ostrich eggs. Additional data to expand on Trial 2 is underway to further refine the ostrich AI protocol and demonstrate its utility as a feasible reproduction option for ostriches.
BRISTLE HAIRS, FILOPLUMES AND PINHOLES IN OSTRICHES

A. Engelbrecht\*(1), S.Cloete(2)

(1)Western Cape Department of Agriculture, Oudtshoorn, South Africa

(2)Stellenbosch University, Matieland, South Africa

* Corresponding author: anele@elsenburg.com

Ostrich leather is deemed a luxury leather, with quality being of utmost importance. The widespread presence of small pinholes on ostrich skins has become increasingly problematic. Filoplumes and bristle hairs are implicated in the formation of the pinholes (Lunam & Weir, 2006; Weir & Lunam, 2011), and while both are deemed a natural phenomenon on avian skins, it is nonetheless regarded as a defect that retracts from leather quality. The presence of hair and hair follicles (pinholes) were consequently scored at different ages to determine environmental and genetic factors. It was found that the hair follicle scores on processed skins were heritable (±SE) at 0.40±0.06. The presence of hairson live birds was accordingly heritable at 0.51±11 in juvenile birds and 0.23±0.09 in adult birds assessed repeatedly. The animal permanent environmental variance ratio was also significant at 0.22±0.09 for adult birds. Hair follicle score was genetically correlated to hair score on live slaughter birds (0.49±0.23) while the phenotypic correlation between these traits amounted to 0.36±0.08. The genetic correlation was lower than expected but it should be considered that only 476 hair scores were available on SA Blacks, of which only about 20% (n=97) had hair follicle scores on their skins as well. Genetic correlations of hair scores on live adult birds with other traits of economic importance were 0.64±0.30 with egg production, -0.65±0.35 with chick production and 0.27±0.21 with adult live weight. Corresponding animal PE correlations were 0.93±0.38 with egg production, 0.78±0.32 with chick production and -0.10±0.21 with adult live weight. Phenotypic correlations amounted to respectively 0.14±0.07, 0.15±0.07 and 0.05±0.05. Hair scores on adult and juvenile birds essentially represented the same trait, as indicated by a genetic correlation of 0.93±0.11. Adult hair scores were also positively related to hair follicle scores in their slaughtered offspring, with the genetic correlation amounting to 0.61±0.27 between these traits.

References

Lunam C.A., Weir K.A. (2006) Storage of ostrich skin: effects of preservation methods on skin structure, physical properties and microbial flora. Rural Industries Research and Development Corporation Publication No. 06/054

Weir K.A., Lunam C.A. (2011) The Structure and Sensory Innervation of the Integument of Ratites. In: The Welfare of Farmed Ratites. Animal Welfare, vol 11. Springer, Berlin, Heidelberg
WVPA

HYGIENE AND PATHOLOGY
ID: 22

THE IMPACT OF CLEANING AND DISINFECTION OF POULTRY HOUSE ON BROILER PERFORMANCE AND MICROBIAL STATUS

J. Hamidu*(1), A.Patu(1), M.Okai(1)

(1) Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

(2)Institute for Animal Husbandry, Belgrade, Serbia

* Corresponding author: jahamidu.canr@knust.edu.knust

The study evaluated the impact of broiler house degree of cleaning and disinfection on growth performance and microbial status. The degrees of cleaning included (dry cleaning only (T1), dry and wet cleaning with ordinary water (T2), dry and wet cleaning with detergent (T3), dry and wet cleaning with detergent and disinfection (T4), and dry and wet cleaning with detergent, disinfection and whitewashing (T5). A total of 375 Ross 308 broiler chicks were randomly allocated to each of the treatment and reared for 8 weeks. Each treatment was replicated 3 times with 25 birds each. Data recorded included feed intake, weight gain, feed conversion ratio (FCR), liveability and microbial load. Data were analyzed as one-way ANOVA using the Proc. GLM SAS® at P ≤ 0.05. The bacterial counts were analyzed as log10 CFU and ANOVA obtained my GLM. Birds from T5 had higher feed intake than birds from T1 and T2 for the period of 1-28 days but none was different from T3 and T4 (P = 0.04). The broiler weight gain per bird tended to be different at 49 days (P = 0.07) and increased with the degree of cleaning. The FCR up to 49 was lower in T4 compared to T1 (P = 0.022). The weekly mortality reduced with increasing degree of cleaning (y = 1.24x2 – 13.70x + 46.95; R2 = 0.97). Similarly, bacterial load in the litter also reduced (y = -0.99x + 8.39, R2 = 0.82 for total viable count, and y = -0.92x + 7.64, R2 = 0.82 for E. coli count). It can be concluded that complete cleaning of the pens up to the application of disinfection increased broiler performance and there may not be the need to include whitewashing of the pen walls before broiler chick placement.
EFFECT A NOVEL TRIPLE STRAIN PROBIOTIC BACILLUS ON THE DIGESTIVE IMMUNE SYSTEM UNDER
SUB-CLINICAL NECROTIC ENTERITIS CONDITIONS

C. Bostvironnois*(1), D.Sandvang*(2), J.Schleifer(3)

(1)Chr Hansen, Arpajon, France

(2) Chr Hansen, Hoersholm, Denmark

(3)Chr Hansen, Milwaukee, United States

* Corresponding author: frcbos@chr-hansen.com

A commonly used immune-functionality technique is the examination of levels of cytokines. Cytokines are the communication substances secreted and received by cells in the body, particularly regarding immune function regulation. Research was conducted to measure the effect of a new 3-strain Bacillus-based probiotic (GALLIPRO® Fit) on the immune system, particularly investigating the impact on cytokines. In this trial, broilers were the study animal and a mild NE (Necrotic enteritis) challenge was included to better investigate cytokine levels and influence of the probiotic in the face of a pathogenic event.

36 pens were populated with 50 day-old birds each. There were 9 repetitions for each of the 4 treatment groups: negative control T1, negative control + probiotic T2, positive control, T3, positive control + probiotic T4. The NE challenge consisted of the addition of Clostridium perfringens at a dose of 1x10^8 CFU/ml drinking water on days 18, 19, and 20. On day 21, three randomly-obtained birds were sampled for intestinal cytokine gene expression. The pro-inflammatory cytokines IL-1b, IL-18, IL-6, and IL-12b and the regulatory cytokine IL-10 were assayed. The quantification was determined for each sample by comparing the number of cycles required to cross a certain threshold of fluorescence (Ct). The NE challenge group receiving the probiotic (T4) showed significant upregulation of IL-1b, IL-6, and IL-18. These cytokines are pro-inflammatory and anti-inflammatory. Of the three other treatment groups, only T3 (NE challenged, but not fed probiotic) showed a significant upregulation of IL-6. This is an expected result and indicates a normal, initial inflammatory response effect produced by the NE challenge. The two groups not receiving the NE challenge showed no upregulation of the cytokines. There was no expression of IL-12b and no significant upregulation of IL-10 for any of the treatment groups. These data indicate the new 3-strain Bacillus-based probiotic 'primes' the immune response. The probiotic treatment group and no NE challenge showed no up-regulation of the pro-inflammatory cytokines, thus maintaining normalcy but not stimulating the immune system. In the presence of the NE challenge, the probiotic-treated group showed significant enhancement of the intercellular cytokines.

This demonstrates the immune modulator activity of the novel probiotic to enable the triggering of
these defense mechanisms in the face of a disease challenge.
BACILLUS AMYLOLIQUEFACIENS CECT 5940 IMPROVES THE EXPRESSION OF IMMUNITY GENES IN BROILERS UNDER A SUBCLINICAL NECROTIC ENTERITIS CHALLENGE

K. Gharib-naseri*(1), J.De paula dorigam(2), S.Kheravii(1), K.Doranalli(2), R.Swick(1), M.Choct(1), N.Morgan(1), S.Wu(1)

(1) University of New England, Armidale, Australia

(2) Evonik Nutrition & Care GmbH, Hanau-Wolfgang, Germany

* Corresponding author: kgharib2@une.edu.au

The ban of in-feed antibiotics has negatively affected the poultry industry by causing necrotic enteritis (NE) to emerge in the flocks and has caused a significant economical concern for poultry farmers. Bacillus spp. namely, Bacillus amyloliquefaciens (BA), has shown to have substantial antimicrobial activities and the ability to produce several extracellular enzymes that enhance digestibility and absorption of nutrients. This strain can also produce bacteriocins, which can be effective in controlling C. perfringens (Bindiya et al. 2019). The study consisted of two 2 x 2 factorial arrangements where birds were subjected to NE challenge (yes or no) and probiotic treatment (yes or no). The dietary concentration of probiotic was 1x106 CFU of BA CECT 5940 per gram of feed. Oral administration of Eimeria oocysts (d 9) followed by inoculation with 1 mL 108 CFU/bird of Clostridium perfringens (d 14 and 15) was used to induce NE in the challenged groups. On day 24, NE challenged birds showed lower weight gain and increased feed conversion ratio (FCR) (P < 0.001), however the supplementation of BA improved the weight gain and FCR in all groups (P < 0.001 and P < 0.05, respectively). At d 16, jejunum tissue samples were collected for gene expression analysis. Results showed a NE challenge x BA interaction for interform-γ (IFN-γ) where IFN-γ expression was highest in the NE challenged group with no supplementation and BA supplementation significantly reduced INF-γ of challenged birds compared to the non-supplemented group (P < 0.001). Downregulation of IgA (P < 0.001) and IL-18 (P < 0.001) genes were observed in the NE challenged birds compared to the non-challenged groups. Furthermore, NE challenge x BA interaction was observed for IgG (P < 0.01) and IgM (P < 0.05), where the NE challenge with no supplementation of BA shows the lowest values, however, the supplementation of BA gave similar results as the unchallenged birds with or without supplementation. These findings suggest that supplementation of BA in broiler diets can help improve gut health by possessing positive effects on the expression of important genes related to the immunity functions in the gut.

Bindiya E, Tina K, Sasidharan RS, Bhat SG. Bacf3: Highly thermostable bacteriocin from Bacillus amyloliquefaciens btss3 antagonistic on food-borne pathogens. 3 Biotech 2019; 9(4): 1-7.
DIETARY SUPPLEMENTATION OF PLANT EXTRACT SHOWED IMPROVED FEED EFFICIENCY AND ENHANCED GUT INTEGRITY IN BROILERS CHALLENGED WITH NECROTIC ENTERITIS

A. Kumar*(1), S.K kheravii(2), M.Toghyani(2), C.Ionescu(3), A.Blanchard(3), C.Keerqin(2), N.K. sharma(2), R.A. Swick(2)

(1)University of New England, Australia, Armidale, Australia

(2)University of New England, Armidale, Australia

(3)Pancosma SA, Rolle, Switzerland

* Corresponding author: akumar26@myune.edu.au

Plants extracts are being increasingly researched for their potential to improve gut health as in-feed
antibiotics are phased out of the poultry industry. A feeding study was conducted to examine the
efficacy of plant extract (PE), a micro-encapsulated product composed of eugenol and garlic tincture
on growth performance, gut integrity and caecal microflora in broilers challenged with necrotic
enteritis (NE). A total of 960 d-old Cobb 500 broiler chicks were assigned to 48-floor pens each stocked
with 20 birds. A randomised complete block design was used with 6 treatments replicated 8 times and
the treatments were: T1 - unchallenged group, without additives; T2 - challenged group, without
additives; T3 - challenged group plus PE (100 g/t feed, Pancosma SA); T4 - challenged group plus
antimicrobial (AM, 625 g/t) (Maxiban, Elanco US Inc.); T5 - challenged group plus AM with PE; T6 -
challenged group plus half dose of AM (312.5 g/t) with PE in starter, grower and finisher phases,
respectively. Diets were based on wheat, sorghum, soybean meal, and meat and bone meal and were
supplemented with phytase. Challenged birds were given 1 mL per os field strains of Eimeria spp.
oocysts consisting of E. acervulina (5000), E. maxima (5000) and E. brunetti (2500) at d 9. On d 14,
challenged birds were gavaged with 1 mL per os 108 CFU/mL of Clostridium perfringens (NE-18). NE
caused mortality was determined by necropsy and mortality data were used to correct the FCR. Bird
performance was measured from d 0 to 35. A leaky gut marker, serum fluorescein isothiocyanate
dextran (FITC-d) and caecal microflora were measured at d 16. All data were analysed using JMP
software (v.14). The treatments T1, T4, T5, and T6 had higher body weight gain (BWG), higher livability
and lower FCR compared to T2 (P < 0.05). Birds supplemented with PE (T3) had lower FCR and feed
intake compared to T2 (P < 0.05). A higher concentration of serum FITC-d was observed in T2 compared
to unchallenged and challenged groups with additives (P < 0.05). Birds treated with PE (T3) had lower
counts of Lactobacillus spp. (P < 0.05) in the caecal contents than T2. These findings suggest that
different combinations of a PE with antimicrobials were effective in alleviating the impact of NE as
indicated by improved BWG, FCR, higher livability, and enhanced gut integrity. These results also
demonstrated that diet supplemented with plant extract helped to improve feed efficiency and
enhance gut integrity.
HAEMOPROTEUS INFESTATION IN A FLOCK OF COMMERCIAL BROILERS IN BELGIUM

P. De herdt\*(1), G.Roovers(2)

(1)MSD Animal Health Belgium, Brussels, Belgium

(2)Galluvet Veterinary Practice, Lummen, Belgium

\* Corresponding author: peter.de.herdt@merck.com

Haemoproteus constitutes a genus of parasites that infest the red blood cells of amphibians, reptiles and birds. More than 120 Haemoproteus species have been reported in birds, predominantly wild waterfowl, raptors and passerines (1). To the best of our knowledge, infestations of chickens have only been described in Bangladesh, Malaysia, Kenya, Mosambique and Iraq.

Since Haemoproteus is spread through blood-sucking insects, the geographic distribution might be explained by the presence or absence of appropriate vectors. In birds, these often include Culicoides spp. and Hippoboscid flies. The vectors for Haemoproteus in chickens have not been identified.

Haemoproteus can easily be recognized microscopically, based on morphological characteristics and the localization in the cytoplasm of erythrocytes.

In June 2018, a stagnation of feed and water intake was observed in a Belgian flock of 25-day-old commercial broilers. This resulted in reduced growth. The chicks did not give the impression of being sick and mortality was not increased. At 28 days of age, 10 broilers of the flock were euthanized and necropsied. From 4 of these, trachea, lung, heart, liver, kidney and bursa were examined cytologically. For this purpose, impression smears were stained with the Diff Quik staining reagents (Medion Diagnostics) and evaluated microscopically at a magnification of 1000 times.

In all necropsied birds, the intestinal tract was poorly filled with feed. Besides paleness of the heart and a few petechiae in the bursa of one broiler, gross pathological organ lesions were not observed.

Cytologically, moderate infiltrations of lymphocytes were detected in multiple organs of all birds.

Examination of the heart and lungs of one specific chick revealed gametocytes of Haemoproteus in the cytoplasm of multiple erythrocytes. Mononuclear phagocytic cells were often observed in close relationship to the infested cells, indicating onset of erythrophagocytosis.

To the best of our knowledge, this is the first report on Haemoproteus in chickens in Western Europe. Since this parasite usually spreads through vectors, the present observation implies the presence of an appropriate vector or any other, unknown mode of transmission.

It is not clear to which extent the lower performance of the flock can be ascribed to the infestation with Haemoproteus.

(1) Bermudez, A.J. 2014. Haemoproteus infections. In: Diseases of Poultry, 13th ed. D.E. Swayne et al., eds.Wiley-Blackwell, Ames, IA, pp. 1189-1190.
ANTIMICROBIAL RESISTANCE PROFILES OF KLEBSIELLA ISOLATES FROM DIFFERENT TYPES OF CHICKEN IN IMO STATE, SOUTHEAST NIGERIA

C. Okoli(1), V. Ajayi(2), C. Achonwa(2), N. Aladi(2), G. Okoli(1)

(1)National Open University of Nigeria, Abuja, Nigeria

(2)Federal University of Technology Owerri, Owerri, Nigeria

* Corresponding author: drokolicharles@gmail.com

Extensive use of antibiotics in poultry in Nigeria has resulted in the emergence of resistant bacteria in poultry pathogens, highlighting the need for frequent monitoring of antimicrobial resistance profiles in important pathogens. This study reports the antimicrobial resistance profiles of Klebsiella species isolated from the faecal droppings of different types of chicken raised by small-scale farmer in Imo state, in the rainforest south-eastern Nigeria. Six replicate cloacal samples each, from broilers starter (BS), broiler finisher (BF), grower birds (GB), layer hens (LH) and local chicken (LC) were collected with sterile swabs, at five different locations and cultured for Klebsiella species. Serial dilutions of each sample was streaked on MacConkey agar and incubated at 370C for 24 hours, and pink coloured colonies showing lactose fermentation sub-cultured further on eosin methylene blue agar to differentiate between Klebsiella and E. coli species. The isolates were subjected to antimicrobial resistance profiling on MacConkey agar using the disc diffusion method. Data generated were subjected to descriptive statistics such as means, percentages and histograms. An overall 43.00% Klebsiella prevalence was recorded; 60% in LC, 50.00% in GB, 40.00% in LH, 30% in BS and 35.00% in BF. Overall, the organisms exhibited very high resistance to trimethoprim (septrin), penicillin and nalidixic acid (77.42-87.1%), average resistance to streptomycin and gentamycin (51.61% each), moderate resistance to perfloxacin, ciprofloxacin and augumentin (29.00-35.48%) and low resistance to cephalexin and ofloxacin (16.13-19.35%). Isolates from GB were highly susceptible to ofloxacin, perfloxacin, ciprofloxacin, cephalexin and gentamycin, while those from LM were highly susceptibility to ciprofloxacin and cephalexin. BS isolates were also highly susceptible to the ofloxacin, perfloxacin and augumentin, while the BF isolates were susceptible to ofloxacin, augumentin, cephalexin and streptomycin. The isolates from LC were equally susceptible to ciprofloxacin, cephalexin and streptomycin. In conclusion, the floquinolones, augumentin and cephalexin could be used in the treatment of Klebsiella infection in the broiler and pullet flocks, while for the local chicken, ciprofloxacin, cephalexin, streptomycin and septrin should be used. Effective treatment of poultry infections in the study area should be based on the results of antimicrobial resistance profiling of causative pathogens.
# THE APPLICATION OF AMALGAM BACTERICIDAL LAMPS IN POULTRY PRODUCTION

I. Saleeva*(1), E.Zhuravchuk(1), A.Zaremskaya(1), E.Maksimova(1), A.Kavtarashvili(1)

(1) Federal Scientific Center “All-Russian Research and Technological Institute of Poultry” of Russian Academy of Sciences, Sergiev posad, Russia

* Corresponding author: saleeva@vnitip.ru

The trial was performed to evaluate the efficiency of ultraviolet (UV) irradiation for air disinfection in populated poultry house for floor-housed broilers kept in two boxes (15 m2 each) with sawdust litter. The air in the control box was not treated with UV. In the experimental box an amalgam bactericide lamp (87 Wt of UV at 253.7 nm) was set (2 m above the floor). UV irradiation of the air was performed by upper-room method in an intermittent regime: since 1 to 7 days of age – for 1 hour 6 times a day; from 8 to 28 days – for 10 min 12 times a day (each 2 hours); from 29 days of age to slaughter – for 15 min each 2 hours. The exposure to UV at the level of chicks was 11.4 mWt/m2; average exposure at the box 287.8 mWt/m2.

It was found that total microbial loads in the air of the experimental box were lower in compare to the control box: at 7 days of broilers' age by 76.4%; at 14 days by 37.5%; at 21 days by 66.3%; at 28 days by 47.5%; at 35 days by 50.9%. The elimination efficacy against E. coli at 7, 14, and 21 days of age was 100; 90.1; and 93.1%, respectively. Since the UV doses lethal for E. coli are also lethal for certain viruses it could be suggested that the studied regime of air disinfection with amalgam UV lamp can substantially decrease the risks of circulation of airborne diseases.

The decrease in total microbial loads in the air of experimental box resulted in the significant increase in average live bodyweight at slaughter (35 days of age) by 4.76% (P

No detrimental effects of UV were found on the sense of vision in chicks. No erhythemae were found on the skin of UV-treated chicks; color of the skin in open and feathered areas was similar in control and UV-treated chicks.

UV treatment also stimulated the chicks to attend feeders and drinkers more actively. As a result the bodyweight uniformity in the treated chicks (the percentage of chicks with bodyweight falling within the range of average value ±10%) was higher by 6.2% in compare to control treatments.

The conclusion was made that UV irradiation with new-generation ozone-free amalgam lamps in the studied intermittent regime significantly decreases the microbial load in the air of broiler poultry house and improve growth efficiency and immune status in broilers. The use of this modern UV equipment for air disinfection in populated poultry houses can substantially improve the efficiency of the prevention of hazardous infectious diseases.
SUCCESSFUL VACCINATION AGAINST GUMBORO DISEASE IN LABEL ROUGE SLOW GROWTH CHICKENS USING AN IMMUNE-COMPLEX VACCINE

L. Corrand(1), S.Mouchel*(2), E.Huchin(1), T.Langlade(2), C.Cazaban(2)

(1)ABIPOLE Veterinary Practice, Arzacq-arraziguet, France

(2) Ceva Santé Animale, Libourne, France

* Corresponding author: simon.mouchel@ceva.com

Label Rouge [Red Label] chicken is highly appreciated by the French consumers because of its traditional, free-range production methods using slow growing birds slaughtered at 81+ days of age. Its specifications ensure a meat with superior organoleptic properties, with a positive image in terms of animal welfare and consumers' health. Disease prevention is critical on such high-value birds with long lifetime. To preserve a good health status of their flocks, producers need an efficient, safe and convenient vaccination plan. Infectious bursal disease (IBD, Gumboro disease) is of significant economic importance, because it can induce immunosuppression, leading to morbidity, sometimes mortality, and frequent bacterial opportunistic superinfections which require using antibiotics. IBD immune-complex vaccines enable a timely immunization according to the maternal immunity status and a full protection against field viruses through bursa colonization as soon as the vaccine virus is freely released. Its technology allows the combination with vector HVT vaccines against other diseases (eg, rHVT-ND) and with Rispens vaccines. The aim of this study was to use a novel IBD immune-complex vaccine (Novamune) applied at the hatchery, in different conditions, in a large scale commercial operation of Label Rouge chicks. A total of 89 commercial flocks, representing almost 500,000 Label Rouge chickens were vaccinated with Novamune over a period of 3 months, including 11 flocks via subcutaneous route and 78 flocks via in ovo route (out of label). On the same period, a control group of 48 flocks was in-ovo vaccinated with a vector HVT-IBD vaccine. Other vaccines (including systematic Rispens vaccination) were applied as per the usual standards of the commercial operation. Post-vaccination monitoring criteria included production performances (mortality, ADG, FCR), IBD ELISA serology (D0, D28, D42 and D81), and IBDV RT-PCR (D28 and D42). The serology results confirmed the expected onset of immunity against IBD after immune-complex vaccination. This finding is confirmed by the detection of the vaccine strain (SYZA26) in the bursas. In terms of performance, no statistical difference between the two groups (immune-complex and vector rHVT IBD vaccination) was shown. The obtained results showed that this novel immune-complex vaccine is an efficient, safe and convenient option for preventing Gumboro disease in slow growing Label Rouge chickens via subcutaneous or in-ovo route.
APPLICATION OF INERT OILS IN THE CONTROL OF RED CHICKEN MITES DERMANYSSUS GALLINAE

A. Pavlicevic(1), I.Pavlović*(2), N.Zdravkovic(2), J.Yoon(3), B.Pan(4)

(1)Aves Mit doo, Subotica, Serbia

(2)Scientific Veterinary Institute of Serbia, Belgrade, Serbia

(3)Biogenoci Co Ltd, Seoul, South Korea

(4)College of Veterinary Medicine, China Agricultural University, Beijing, China

* Corresponding author: dripavlovic58@gmail.com

The use of inert oils in control of Dermanyssus gallinae (De Geer, 1778) is recommended due to: non-toxicity; effective, complex acaricidal action, which excludes the possibility of generating resistance; and multipurpose application (in prevention, curative and biosafety). The first dedicated formula of inert oils in control of red chicken mite is P 547/17. The new formula P 2020/0033, aims to increase economic accessibility to farmers and ease the conditions for more detailed application. The aim of this study is to consider the current arguments for the use of assigned inert oil formulations in the control of D. gallinae.

The study of 10% aqueous emulsion of formula P 2019/0033 was performed in a laboratory test, by the petri dish. By direct exposure of the adult D. gallinae to 1 min exposure, all of a 100% effect was found. With subsequent exposure on tin boxes with examination of prolonged action, after 1 month, we found the efficiency at an oblique position of 38%, inverted 81% and flat 98%.

Optimization of the clinical application of the formula P 547/17 was achieved by two treatments of detailed application using the spray method in an empty facility, at intervals of 5 or more days. Single application on cages and equipment with 20%, in repeated treatment at an absorbent surfaces (floor, walls) with 10% water emulsion and it proved the possibility of eradication of D. gallinae in parent breeding facilities (2 facilities with a capacity of 9,000 parents); in cage-laying for laying hens (8 facilities, capacity 18,000 chickens each); small farms for the exploitation of litters in a conventional cage system (capacity 2,500 and 4,000 stretchers). Project ID 1115 improves the application for large industrial cage-type facilities (conventional and enriched). In the first treatment, 20% excess water emulsion was applied. In the second treatment, a small dose of 50% water emulsion was used, which was applied only to the most important places of the cage, while for an absorbent surfaces 10% excess water emulsion was used. So far, eradication has been proved in two facilities with a capacity of 14,000 and 30,000 carriers. Eradication was observed after detailed and systematic examinations (early dust detection method, visual and tactile examinations, staff observations and surveillance of the flock's health status) for 12 months or more, and the established negative finding of D. gallinae.

It can be concluded that by the conditions and proper application of non-toxic formulations P 547/17 and P 2020/0033, we are able to effectively control D. gallinae and at the same time exclude the toxicological risk of acaricides.
# THE CAPABILITY OF A DETOXIFYING AGENT TO PREVENT FUNCTIONAL AND MORPHOLOGICAL LESIONS CAUSED BY T-2 TOXIN IN BROILER CHICKENS

M. Hinrichs(1), A.Schlagheck(1), C.Becker*(2)

(1)Biochem Zusatzstoffe Handels- und Produktionsgesellschaft mbH, Lohne, Germany

(2)Biochem Zusatzstoffe Produktions- und Handelsgesellschaft mbH, Lohne, Germany

* Corresponding author: cbecker@biochem.net

## Objective

Mycotoxins such as T-2 toxin (T-2) are responsible for severe pathological lesions and performance losses in poultry. Impaired gut morphology and liver function are corresponding consequences. The objective of this trial was to evaluate the capability of the detoxifying agent B.I.O.Tox® Farm (BTF; Biochem GmbH, Germany) to prevent the cytotoxic effects and performance losses due to T-2 toxin.

## Material and methods

360 one-day-old male broiler chicks (Cobb 500) with an average initial-weight of 39.1 g were randomly divided into 6 treatment groups (n = 6 pen, 10 chicks per replicate): negative control group (NCG) with no BTF or T-2 added, a positive control group (PCG) with 2 ppm T-2 in the diet, a BTF-0 group with 0.4% BTF, a BTF-1 group with 0.1% BTF plus 2 ppm T-2, a BTF-2 group with 0.2% BTF plus 2 ppm T-2 and a BTF-4 group with 0.4% BTF plus 2 ppm T-2. All birds were given ad-libitum access to feed and water during the whole trial period (d1-21). Diets were based on corn and soybean meal meeting the nutritional recommendations according to NRC 1994. Following parameters were observed: zootechnical performance, blood serum biochemistry, and jejunal morphology.

## Results

On d 21, body weight (BW), body weight gain (BWG), and feed intake (FI) were significantly decreased in the PCG compared to the NCG (9.7%, 12.0% and 6.3% respectively; P<0.05), whereby all BTF-groups showed significantly higher BW, BWG and FI compared to PCG and were comparable to NCG (P0.1). Total plasma protein (TPP) and albumin (ALB), both mycotoxin-specific parameters, were significantly lower in the PCG in comparison to the NCG and to BTF-groups (P<0.01). The villus:crypt ratio was significantly decreased in PCG compared to the NCG and BTF-groups (P<0.05). The relative weight of liver and the feed conversion ratio did not differ between treatments.

## Conclusions

This study confirms the capability of BTF to prevent the manifestation of T-2 associated cytotoxic effects. This was proven by maintained performance data, consistent serological values like TPP and ALB serum levels as well as a stabilized intestinal morphological parameter in the presence of 2 ppm T-2.
BACTERIOSTATIC AND BACTERICIDAL EFFICACY OF COMMERCIAL DISINFECTANTS AGAINST SALMONELLA INFANTIS VARIES CONSIDERABLY

V. Drauch*(1), C.Ibesich(1), C.Vogl(2), M.Hess(1), C.Hess(1)

(1)Clinic for Poultry and Fish Medicine, University of Veterinary Medicine Vienna, Vienna, Austria

(2)Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria

* Corresponding author: victoria.drauch@gmail.com

Salmonella (S.) Infantis is currently the most common serovar in broilers and in poultry meat in the European Union. In the field, eradication of S. Infantis in affected poultry flocks is considered extremely difficult. Despite stringent cleaning and disinfection measures between the placement of flocks, recurrent infections are often reported. Consequently, it could be hypothesized that S. Infantis has a higher tenacity compared to other Salmonella species, coincidencing with a higher resistance to disinfectants. So far, no studies are available which focus on the bacteriostatic and bactericidal efficacy of disinfectants against S. Infantis. Therefore, in the present in-vitro study the efficacy of 10 disinfectants (5x Glutaraldehyde + Quaternary ammonium (QUATS), 3x Peroxygen, 1x Chlorocresol, 1x Diamidine) was tested on 7 S. Infantis field isolates. The testings were performed according to the guidelines of the German Association of Veterinary Medicine [1]. The following variables were included in the tests: 1) different concentrations of disinfectants, 2) different temperatures (10°C, 20°C) and 3) low (3 g bovine serum albumin(BSA)/l) and high protein exposure (10 g BSA/l and 10 g yeast extract/l). Also, an additional incubation step after neutralizing the disinfectants was introduced to investigate the revitalisation potential of S. Infantis. The bacteriostatic efficacy could not be assessed for 3 disinfectants due to turbidity right after compounding. For 4 disinfectants a bacteriostatic effect could only be noticed by raising concentrations above recommendation. Testing disinfectants at different temperatures had no influence. In contrary, the bactericidal effect of 9 disinfectants decreased significantly in the presence of a high concentration of proteins. Additional incubation of the product-bacteria mixture after neutralizing the active disinfectant resulted for 9 disinfectants in a revitalisation of S. Infantis.

Overall, products based on QUATS were most efficient in the present test settings followed by Peroxygen, Chlorocresol and Diamidine. The present in-vitro investigations on the bacteriostatic and bactericidal efficacy of disinfectants against several S. Infantis isolates provide important data to improve performances of cleaning and disinfection measures of contaminated premises.

[1] DVG-Prüfrichtlinie, 2017. V. Tierhaltung - Anhang: Methoden der Prüfung von chemischen Desinfektionsmitteln für die Tierhaltung
STABILITY OF ANTIBIOTIC DRUGS IN DRINKING WATER - IMPACT OF TREATMENT WITH BIOCIDAL PRODUCTS

A. Dufay-lefort\*(1), L.Baduel(2), S.Mompelat(2), B.Le normand(3), A.Hemonic(4), A.Travel(5), T.Moreac(2), M.Liber(3), P. Guichard(6), A. Chevance(2)

(1)Institut Technique de l'Aviculture (ITAVI), Paris, France

(2)ANSES - ANMV, Fougeres, France

(3)SNGTV, Paris, France

(4)IFIP, Le rheu, France

(5)ITAVI, Paris, France

(6)IFIP, Paris, France

(7)ANSES, Fougeres, France

Biocidal products are often used to treat drinking water of animals, in order to improve its bacteriological quality. Antibiotic treatments are often administered to animals in drinking water, and there are no compatibility studies between medicines and biocidal products, in pharmaceuticals registration dossiers. This study aims to evaluate the impact of drinking water disinfectants on antibiotics stability in drinking water, in poultry, pig and rabbit farming.

Ten antibiotics formulations including doxycyclin, amoxicillin, sulphonamides, tiamulin and colistin were tested with two biocides (H2O2 and sodium hypochlorite) in standard, soft and hard waters, and compared to same formulations and waters without any biocidal treatment.

First part of the study consisted in testing different situations that reproduce field conditions : the stock solution storage (with concentrated antibiotics) during 24 hours, and then, injection of the stock solution in the drinking system during 6 hours, to simulate a dosing pump administration.

The second part of the study consisted in testing amoxicillin, tiamulin and doxycyclin with H2O2 in a field well water rich in iron and manganese.

The different antibiotic molecules were dosed by liquid chromatography coupled with UV detection. Same methodology was used for the two parts of the study (stability, dosage, data analysis). For each test (doubled), stability was stated as decreased if average concentration of the active compound was more than 10% lower to the control sample with significant difference (t-test, p<0,05).

In the first part of the study, water treatment with H2O2 at 50 ppm decreased concentration of only one antibiotic : amoxicillin.

Water treatment with sodium hypochlorite (0,5 ppm of active chlorine) had a negative influence on antibiotic stability of 7 antibiotic products, especially in hard water.

In the second part of the study, similar results compared to the first part were obtained for amoxicillin and tiamulin. Contradictory results were found for doxycyclin, demonstrating the multifactorial and complex nature of antibiotic stability in drinking water in the presence of biocidal products.

In conclusion, practical recommendations regarding the treatment with antibiotics in drinking water when using biocidal products are provided. Some proposals could have been sent to EU to improve recommendations in guideline EMEA/CVMP/540/03-Rev1 about the evaluation of stability for antibiotics in drinking water.

<footnote>* Corresponding author: lefort@itavi.asso.fr</footnote>
POULTRY HELMINTHS: HOW TO SEARCH FOR AND IDENTIFY THEM MORE EFFICIENTLY?

A. Dufay-lefort\*(1), A. Travel(2), J. Reperant(3)

(1)Institut Technique de l'Aviculture (ITAVI), Paris, France

(2)ITAVI, Paris, France

(3)ANSES, Ploufragan, France

* Corresponding author: lefort@itavi.asso.fr

To meet societal expectations, poultry sectors are moving towards more outdoor area farming (meat, egg laying), with a greater risk of exposure to outdoor parasites, such as helminths. At the same time, the desire to reduce the number of veterinary treatments in livestock and to use them in a more targeted way requires a good knowledge of parasites present in field. Nematodes, also known as roundworms, are usually the most frequent and pathogenic parasites encountered, especially Ascarids, Heterakis and Capillaria. Other worms can also infest poultry, such as trematodes and cestodes (tapeworms).

The most popular method for routine poultry helminths diagnosis is coproscopic analysis, detecting parasite eggs in the animal feces. But the immatures worms do not lay eggs, and the female worms can pause laying during several days, so this method is not fully reliable to detect helminths infestation. Furthermore, cestode eggs are often layed independently from feces emission.

Direct examination of the intestine and ceca allows evidence of large roundworms like Ascaridia and large tapeworms; but smaller worms, like Heterakis, are only visible in case of heavy infestation, and tiny worms located deep into the mucosa, like Capillaria and Davainea, are not visible although they are highly pathogenic.

Subsequently, current helminth screening methods used routinely in veterinary practice laboratories on the field are not satisfactory nor reliable, especially concerning Capillaria.

Therefore a specific method has been developed to improve helminths diagnosis, using successive sievings of the digestive content and superficial mucosa.

The content and superficial mucosa from intestines of the necropsied animals are collected and suspended in water, then sieved with a 1mm mesh strainer (that retains for example cestode heads and small Ascaridia), then with a 250 µm mesh strainer, and finally with a 100 µm mesh strainer (to withhold young Ascaridia and Capillaria detectable using a black background).

The same method is used for cecal content (using only 1mm and 250 µm mesh screen) in order to observe Heterakis worms when they are infrequent.

This method for easily identifying helminths will be deployed in poultry vet practices laboratories, in order to build an field observatory of helminths in France, as no inventory of parasitic helminth infestation is available. This field observatory will allow to use the anthelmintic treatments more efficiently, in a more targeted way.
ID: 414

A STAPHYLOCOCCUS AUREUS INFECTION HINDERED SPARE RESPIRATORY CAPACITY OF COMMERCIAL LAYING HEN IMMUNE CELLS MEASURED USING A METABOLIC ASSAY

M. Meyer\*(1), M.El-gazzar(1), Y.Sato(1), E.Bobeck(1)

(1)Iowa State University, Ames, United States

* Corresponding author: mmmeyer@iastate.edu

Staphylococcus aureus isolated from a commercial layer operation following unexplained mortality was used to inoculate 200 commercial laying hens to determine the possible route of infection and to study manifestation. Hens of 2 age groups, 22 and 96 wk, were randomly assigned to one of 4 treatments: CON (no Staph. aureus challenge), oral gavage (OG, intended CFU=108), subcutaneous injection (SQ), or intravenous injection (IV); n=25 hens per age group/treatment. Hens were transported from a commercial facility and allowed 48 hours of acclimation pre-inoculation. Ten randomly selected hens per age group/treatment were weighed daily. Blood was drawn from 4 hens per age group/treatment pre-inoculation, 6h post-inoculation, 1-day post-inoculation (dpi), 3dpi, and 7dpi. Peripheral blood mononuclear cells isolated from whole blood collections were cultured for use in the Seahorse Cell Energy Phenotype (Agilent) metabolic assay, which measures extracellular acidification rate (ECAR, glycolytic activity) and oxygen consumption rate (OCR, mitochondrial respiration) before and after a metabolic pathway inhibitor challenge. Spare respiratory capacity was calculated by subtracting baseline OCR and ECAR from the stressed/peak values following the assay's inhibitor challenge, and data were analyzed using Proc Mixed of SAS Version 9.4. There were no differences across treatments at the baseline or 6h post inoculation. At 1dpi, a dampened immune cell ECAR spare respiratory capacity was observed in the SQ treatment group compared to the control (P=0.0293), indicating a diminished glycolytic response. At 3dpi, the SQ hens' capacity recovered to the level of the control, but the IV treatment group had an ECAR response that was significantly lower than all other groups (P=0.0023). Interestingly, by 7dpi all inoculated treatments showed an ECAR spare respiratory capacity lower than the control, (P<0.05), indicating that Staph. aureus may not have attained its full impact until a week post-inoculation. Age did not have an impact on ECAR response (P=0.9847). OCR spare respiratory capacity was not affected by age (P=0.5449) nor treatment (P=0.7682). Treatment and timepoint did have an impact on body weight (interaction P=0.0001), with IV hens weighing less at 3dpi (0.34 kg lower) and 7dpi (0.39 kg lower). Hence, an intravenous route of Staph. aureus had the greatest impact on laying hen immune cell glycolytic capacity, an outcome that was corroborated by diminished body weight.
EVALUATION OF PROTECTION BY ND VACCINATION PROTOCOLS AGAINST EARLY CHALLENGE WITH VELOGENIC NEWCASTLE VIRUS-VII.1

W. Elfeil\*(1), M.Rady(2), W.Kilany(3), M.Elkady(4), M.Elsayed(5)

(1)Suez Canal University, Kilo 4.5 campus, Egypt

(2)Animal Health Research Institute, Agriculture Research Center, Giza, Egypt, Cairo, Egypt

(3)Animal Health Research Institute, Agriculture Research Center, Giza, Egypt

(4) Department of Poultry Diseases, Faculty of Veterinary Medicine, Beni-Suef University, Beni-suef, Egypt

(5) Middle East for Veterinary Vaccine Company (ME VAC), El-salhiya el-gededa, el-sharkia, Egypt

* Corresponding author: elfeil@vet.suez.edu.eg

Newcastle disease virus (NDV) is endemic in Egypt since 1960s and vaccination strategy is the main tool to control it in Egypt. Recently, NDV-VII.1 became endemic in Egypt associated with mortalities in vaccinated flocks, so the farmers moved to a combined live and inactivated vaccine strategy. The aim of this work was to evaluate effectiveness of three different vaccination protocols (G-1 double inactivated ND vaccine at 1/10 day of age, G-2 single inactivated ND vaccine at 7-day of age and G-3 single inactivated ND at one-day of age) in-combination with three-dose from live ND vaccine against challenge with velogenic NDV (vNDV). 180,000 one-day old chicks obtained from commercial hatchery were placed in three commercial broiler station "60,000 birds/station" and 40 one-day old chicks were moved to Biosafety level-3 isolators (BSL-3) at MEVAC facility to serve as control groups (non-vaccinated group challenged "G-4" and Non-vaccinated non-challenge-G-5). On a weekly basis, blood samples, cloacal swabs and oropharyngeal swabs were collected to check the development of humoral immune response and exposure to any life threatening Respiratory virus (Avian influenza" AIV", NDV and infectious Bronchitis "IB" virus). At 24-day of age, 25 birds from each station (G1-3) were moved to BSL-3 and kept under observation for 36 hours; cloacal and oropharyngeal swabs were collected and three birds from each group were euthanized and internal organs examined for three repeated times with 12 hours interval, to ensure that birds were free from any live threatening viral respiratory pathogen (AIV, NDV, IB). Birds in G1-4 were challenged with vNDV VII.1 (106 EID50) in 0.5 ml/ bird PBS via intranasal route, and birds in G-5 received 0.5 ml PBS via intranasal route. Birds in G1-5 were kept in BSL-3 for 10 days under observation and oropharyngeal swabs were collected on 3,6,9 days post challenge (dpc). Protection against mortalities after challenge with vNDV-VII.1 was 100% (12/12), 100%% (12/12), 91.7% (11/12), 0% (0/12) and 100% (12/12) in groups 1-5 respectively. Virus shedding of birds in G-1 was significantlu lower (amount of virus shedding and number of shedding) in comparison to G2/3. In conclusion, using NDV inactivated vaccine in two-dose protocol with live vaccines can provide protection to commercial broiler chicken against early challenge with vNDV in endemic areas as early as 26 day of age with significant lower shedding rate. The application of such protocol can be an effective tool to control NDV via a vaccination strategy.
EVALUATION PROTECTION OF H5 VACCINATION REGIMES AGAINST EARLY CHALLENGE WITH HPAI-
H5N1

W. Elfeil*(1), M.Rady(2), W.Kilany(2), A.Sedeik(2), M.Elkady(3), M.Elsayed(4)

(1)Suez Canal University, Kilo 4.5 campus, Egypt

(2) Animal Health Research Institute, Agriculture Research Center, Giza, Egypt

(3) Department of Poultry Diseases, Faculty of Veterinary Medicine, Beni-Suef University, Beni-suef, Egypt

(4) Middle East for Veterinary Vaccine Company (MEVAC), El-salhiya el-gededa, el-sharkia, Egypt

* Corresponding author: elfeil@vet.suez.edu.eg

The aim of this work was to evaluate effectiveness of inactivated H5 vaccine in different vaccination regime either as single vaccine dose at 7-day of age, two-dose regime (1/10-day of age) or using as one and half manufacture recommended dose at 7-day of age against early challenge at 26-day of age with HPAI-H5N1 clade 2.2.1.2 virus. 180,000 one-day old chicks obtained from commercial hatchery placed in three commercial broiler station "60,000 birds/station" (G-1 two-dose regime, G-2 one and half dose regime and G-3 single-dose regime) and 40 one-day old chicks moved to Biosafety level-3 isolators (BSL-3) at MEVAC facility to serve as control groups (non-vaccinated group challenged "G-4" and Non-vaccinated non challenge group "G-5"). On weekly basis blood samples, cloacal swabs and oropharyngeal swabs collected check develop of Humeral immune response and exposure to any life threatening Respiratory virus (Avian influenza" AIV", Newcastle disease "NDV" and infectious Bronchitis "IB" virus). At 24-day of age 25 birds from each station (G1-3) moved to BSL-3 and kept under observation for 36 hours; cloacal and oropharyngeal swabs collected and three birds from each group euthanized and internal organs examined for three repeated times with 12 hours interval to ensure that birds free from any live threatening viral respiratory pathogen (AIV, NDV, IB). Birds in G1-4 challenged with HPAI-H5N1 clade 2.2.1.2 (106 EID50) in 0.5 ml/ bird PBS via intranasal route and birds in G-5 received 0.5 ml PBS via intranasal route. Birds in G1-5 kept in BSL-3 for 10 days under observation and oropharyngeal swabs collected on 3,6,9 days post challenge (dpc). Regarding protection virus against mortalities following challenge with HPAI-H5; were 100% (8/8), 87.5% (7/8), 87.5% (7/8), 0% (0/8) and 100% (8/8) in groups 1-5 respectively. Regarding virus shedding birds in G-1 showed significant lower shedding virus (amount of virus shedding and number of shedding in comparison to G2/3. In conclusion, using homologous H5 inactivated vaccine in two-dose regime can provide protection to commercial broiler chicken against early challenge with HPAI-H5 virus as early as 26 day of age, with significant lower shedding rate: the application of such regime can be an effective tool to control HPAI under a vaccination strategy.
ID: 473

CONTRARY EFFECT OF DEOXYNIVALENOL (DON) AND DEEPOXY-DEOXYNIVALENOL (DOM-1) ON CAMPYLOBACTER JEJUNI RESOLVED IN VITRO

D. Ruhnau*(1), C.Hess(1), B.Grenier(2), B.Doupovec(2), D.Schatzmayr(2), M.Hess(1), W.Awad(1)

(1)Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria, Vienna, Austria

(2)BIOMIN Research Center, Technopark 1, 3430 Tulln, Austria, Tulln, Austria

* Corresponding author: daniel.ruhnau@vetmeduni.ac.at

Deoxynivalenol (DON) is the most common trichothecene mycotoxin detected in feedstuffs globally. Similarly, Campylobacter jejuni is very widespread in broilers and the most common zoonotic pathogen. In a recent study, we were able to demonstrate that DON not only increased the load of C. jejuni in the jejunum and cecum of infected birds but also raised the counts of these bacteria in the inner organs. In contrast, the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) reduced the Campylobacter load in vivo. Therefore, it can be hypothesized that DON and DOM-1 might have a direct or indirect effect on the growth of C. jejuni. The aim of the present study was to investigate the interaction between C. jejuni and DON or DOM-1 in vitro.

For this purpose, two C. jejuni strains were tested, one reference strain NCTC 12744 and one field strain 1303 derived from a broiler flock. Both strains (105 CFU/ml) were inoculated in 96-well plates and incubated without and with either DON or DOM-1 (5 and 20 µg/mL) under microaerophilic conditions at 41.5 °C. Colony forming units (CFU) of C. jejuni were determined in a time interval of 6 h (24 h – 30 h – 36 h – 42 h – 48 h). It was found that the presence of DON resulted in significantly higher growth rates of C. jejuni from 30 h incubation onwards confirming the in vivo data. On the contrary, the presence of DOM-1 lead to a significant decrease in the C. jejuni CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds.

Furthermore, RNA sequencing of Campylobacter jejuni incubated with either DON or DOM-1 was performed and revealed specific effects on bacterial metabolic mechanisms.
PERSISTENCE OF ANTIBIOTIC RESISTANT PATHOGENS AND COMMENSAL BACTERIA IN "NO ANTIBIOTIC EVER" PRODUCTION BROILER HOUSE LITTER

A. Oladeinde*(1), K.Cook(2), T.Strickland(3), D.Endale(3), Z.Abdo(4), J.Lawrence*(2), D.Cudnik(2), S.House(2)(4)

(1)US Department of Agriculture, Athens, United States

(2)USDA-ARS, Athens, United States

(3) USDA-ARS Southeast Watershed Research, Tifton, United States

(4) Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, United States

* Corresponding author: ade.oladeinde@usda.gov

We conducted an on-farm longitudinal study to evaluate the occurrence and persistence of pathogens (Salmonella and Campylobacter) and commensal bacteria (E. coli, Staphylococcus and Enterococcus) with antibiotic resistance under a "No Antibiotic Ever (NAE)" production system. The broiler farm was comprised of four broiler houses (> 22,000 birds/house) and used peanut hulls for bedding material. Broiler litter samples (n=288) were collected at the beginning (7 to 10 days after bird placement) and at the end (4-6 weeks after broiler placement) of each grow-out cycle (49 days), for three consecutive flocks. Pathogens and commensals were quantified, identified and characterized with VITEK® (bioMerieux), whole genome sequencing and antimicrobial susceptibility testing. Additionally, broiler house and litter physico-chemical parameters such as temperature, pH and moisture were monitored throughout the study. Overall, pathogen and E. coli populations in litter declined significantly (p< 0.05) from flock 1 to flock 3. Salmonella isolates (n = 47) were grouped into six O serogroups (C2, C3, D1, D2, B and E) and S. Kentucky (~ 56%) and S. Enteritidis (~ 30%) were the top serovars found in litter at the end of the 3rd flock. None of the Salmonella isolates tested carried antibiotic resistance. While Campylobacter coli isolates (~ 80%) were resistant to tetracycline, C. jejuni isolates (~ 90%) were susceptible to all drugs tested. The majority of E. coli isolates (~ 62.5%) were resistant to at least one class of antibiotics and multidrug resistant (resistance to two or more classes of antibiotics) E. coli persisted from flock to flock. The major Enterococcus spp identified were E. hirae (~ 65%) and E. faecalis (~ 15%), and these isolates harbored resistance to at least one class of antibiotics. Staphylococcus isolates (~ 45%) displayed resistance to lincomycin, and S. cohnii subsp. urealyticus (~ 27%) and S. xylosus (~15%) were the representative species present in litter from flock to flock. The temperature of the broiler house was the most important parameter that could explain the variability in our data. Our results suggest that the reuse of the same litter over the grow-out of multiple flocks under "NAE" production resulted in a reduction of Salmonella and Campylobacter population in litter, however, multidrug resistant commensal bacteria persisted from flock to flock.
A COMPARISON OF SALMONELLA ENTERITIDIS COLONIZATION IN BROILER CHICKENS CHALLENGED ON DAY 14 WITH EITHER AN OCULAR OR ORAL CHALLENGE

L. Brooks(1), J.Krehling(1), K.Chasteen(1), K.Macklin*(1)

(1)Auburn University, Auburn, United States

* Corresponding author: macklks@auburn.edu

Salmonella Enteritidis (SE) is an important food borne pathogen that is typically associated with poultry. This study was conducted to determine which organs are colonized by this bacterium when challenged with these different routes. For this study, 250 straight run broiler chicks were obtained from a commercial hatchery and were randomly placed into 10 pens (25 birds/pen). Standard management and feed practices were followed for the duration of the trial. On day 14, half of the pens were challenged orally by administering 500ul of 10x4 cfu of SE via oral gavage while the other 4 pens were challenged with 50ul of 10x4 cfu of SE into each eye.

The SE utilized in this study was nalidixic acid and novobiocin resistant. On days 35-38 a total of twenty birds were removed from each pen and necropsied. Samples collected include bursa and thymus (pooled), breast, ceca, crop, kidney, liver and spleen (pooled), skin, spinal cord, thigh, and trachea. A sterile swab was utilized to take samples from the abdominal cavity, bone marrow, cloaca and lung. After harvesting, samples were immediately placed into BPW and stored at 4C until all the birds were necropsied after which, samples were incubated at 37C for 24 hours. From each BPW sample a subsample was taken and used to inoculate TTB tubes. The TTB tubes were incubated for 24 hours at 41C. After that time samples were plated onto XLT4 agar that contained nalidixic acid and novobiocin. These plates were incubated at 37C for 48 hours after which the plates were removed, and Salmonella colonies noted. Data was analyzed using a t test at a p> 0.05. Results of this trial had shown at least one sample was positive for SE in all the birds. There were significant differences between the ocular (60.6%) and oral (18.4%) routes of challenge for the cloaca swab, lung swab (ocular=8.3%, oral=1.0%), pooled bursa and thymus (ocular=39.0%, oral=21.0%), ceca (ocular=61.0%, oral=28.0%), crop (ocular=59.6%, oral=20.0%), skin (ocular=21.0%, oral=6.1%). Differences in colonization may be due to the ability of this bacteria to more readily go systemic when administered via the eye compared to an oral gavage. This study shows that the route of inoculation may influence where the bacteria may colonize.
# PROTECTION AGAINST AL-2 AND 9109-LIKE VARIANT INFECTIOUS BURSAL DISEASE VIRUSES USING DUAL RECOMBINANT HVT-ND-IBD VACCINE ALONE AND IN COMBINATION WITH TWO LIVE IBDV VACCINE

A. Montoya\*(1), H.Sellers(2)

(1)MSD, Roswell, United States

(2)Poultry Diagnostic Research Center University of Georgia, Athens, United States

\* Corresponding author: andres.montoya@merck.com

Infectious bursal disease (IBD) is a highly contagious disease of young chickens caused by infectious bursal disease virus, characterized by immunosuppression and mortality generally at 3 to 6 weeks of age. IBDV is ubiquitous in commercial chicken operations. IBDV causes a prolonged B-lymphocyte immunodeficiency and increased susceptibility to various viruses and parasites. Both classic and variant strains of IBDV had been isolated in the southeastern United States. IBDV variant AL-2 (AL for Allen Laboratory) originated from Delaware and in recent years has affected broilers between the ages of 20 to 30 days or older with moderate to severe bursal atrophy. 9109 is a variant strain of IBD that has been a problem in the southern states of United States. 9109 has been characterized as a classical, very virulent, and variant strain. IBDV variant AL-2 and 9109 isolation in commercial broilers has increased in recent years in the southeastern United States, associated with broiler performance issues. A new first dual construct rHVT-ND-IBD vaccine (Innovax®-ND-IBD) has been launched in the United States. An evaluation of the protection against AL-2 and 9109 variants, alone or in combination with a live intermediate standard strain and 89/03 strain of IBDV vaccine, was performed. A subcutaneous injection was used for injection of the chicks for each treatment group at hatch. A minimum of 500 mls of each vaccine at 1x concentration was needed for vaccination. 300 SPF embryos were used in the study. The eggs were purchased from Charles River SPAFAS and set in a hatchery. The challenge viruses used in this study were the AL-2-like IBDV field isolate 124024 and 9109-like IBDV field isolate 127823 case submission to PDRC. The viruses were expanded in 3-week-old SPF chickens prior to the start of the study, then titrated in chicken embryos. The viruses were diluted to the target challenge dose in tryptose phosphate broth (TPB) to a target dose. Each bird in challenged treatment groups received 0.03 mls by the intraocular route of inoculation. Birds were housed in negative pressure isolation units and had unrestricted access to feed and water. Birds were fed unmedicated Southern States All Grain Start-N-Grow diet. Protection was evaluated by challenged birds at day 18. This early age was intended to mimic the age in the field when maternal antibody is expected to decline and when variant IBD challenge is commonly observed. The study was terminated on day 24 and birds had bursae weighed data for Bursa /Body Weight (Bu/BW) ratio were collected and bursas also were collected for histopathology to determine mean bursal lesion scores to determine atrophy. Protection against challenge was determined by Bu/BW ratios greater than or equal to the Bu/BW ratio of the Unvaccinated Challenge Group plus two standard deviations. The histopathology bursal lesion scoring system is based on scores of 0-4 where 0 is an absence of lesions and 4 is marked lymphoid atrophy. Statistics were performed on the Bu/BW ratios using the unpaired t-test (GraphPad Prism, v. 6). Statistics were performed on the bursal lesion scores using the unpaired t-test (Graph Pad Prism). Results showed protection in all the vaccinated groups when they were compared with the Non-vaccinated/Challenged group. The Bu/BW ratios for all vaccinated/ AL2 challenged groups were significantly higher as compared to NoVAXAL2 group, providing evidence that all vaccine combinations and the rHVT-ND-IBDV prevented significant bursal atrophy following AL-2 challenge. In addition, the Bu/BW ratios for all vaccinated/AL2 challenged groups were greater than 2 standard deviations above the mean Bu/BW of the NoVAXAL2 challenged group providing additional evidence for protection against challenge. While the Innovax vaccinated/AL2 challenged birds were protected, significant
bursal atrophy was observed in this group based on Bu/BW ratios when compared to the NoVAX and all other vaccinated/AL2 challenged groups. However, significantly less bursal atrophy was observed, based on Bu/BW, in the Innovax vaccinated/AL2 challenged group when compared to the NoVAXAL2 group. The extent of lymphocytic depletion in the bursa was most significant in the NoVAXAL2 group with a mean bursal lesion score of 4 compared to all other groups in this study. This confirms that the challenge virus and dose were valid. The bursal lesions scores for all vaccinated/AL2-challenged groups were significantly lower compared to the NoVAXAL2 group suggesting that all vaccine combinations provided protection against significant lymphocytic depletion in the bursa. In addition, the bursal lesion scores for the Innovax + 8902 vaccinated/AL2 challenged group, and Innovax+ Univax vaccinated/AL2 challenged group, were not significantly different from the NoVAX group. The bursal lesion scores for the Innovax vaccinated/AL2 challenged group were significantly higher than the NoVAX group and significantly lower than the NoVAXAL2 group. The mean lesion score for the Innovax vaccinated/AL2 challenged group was 2.4 and considered normal for broilers in the field. In fact, the mean bursal lesion score for the NoVAX group (1.1) is lower than normal birds in the field and likely due to the fact that birds were SPF and housed in HEPA filtered isolation units. Similar results were observed with the 9109 strain. The Bu/BW ratios for all vaccinated/9109 challenged groups were significantly higher, compared to the NoVAX9109 group, providing evidence that all vaccine combinations and the rHVT-ND-IBDV prevented significant bursal atrophy following 9109 challenge. In addition, the Bu/BW ratios for all vaccinated/9109 challenged groups were greater than 2 standard deviations above the mean Bu/BW of the NoVAX9109 challenged group providing additional evidence for protection against challenge. The bursal lesions scores for the rHVT-ND-IBDV group and the vaccine combinations were significant difference compared to the NoVAX9109 challenge group which provided evidence for protection against the 9109 challenge at 18 days of age. The rHVT-ND-IBD alone or in combination with a live IBD vaccine protected against AL-2 and 9109 like variant IBD virus when challenged at the early age of 18 days.
ID: 590

COMPARATIVE FIELD STUDY BETWEEN RHVT-IBD-ND VS RHVT-IBD VACCINE IN COMMERCIAL BROILERS

M. Fawzy*(1)

(1)MSD Animal Health, Sharkia, Egypt

* Corresponding author: mohmed.fawzy@merck.com

In this study, we determined the effects on the field performance parameters and serological responses in commercial broilers induced by rHVT-ND-IBD in comparison with a recombinant rHVT-IBD vaccine. For this purpose, a total 197,000 commercial Cobb broilers were monitored from a day of old till slaughter age for 3 sequential production cycles. In each production cycle, more than 60,000 birds were obtained from the same hatchery and reared in the same farm under the same management. These birds were separated in 2 houses with the same vaccination program except one house was vaccinated subcutaneously at a day of old with rHVT-ND-IBD and second house with a recombinant rHVT-IBD vaccine. Blood samples were randomly collected at slaughter age and the serum samples were tested for specific detection of IBDV antibodies by the IBD ELISA test using two different commercial IBD ELISA kits. Field performance parameters were evaluated for the two houses for the three sequential production cycles. these parameters were included; Mortality%, Survival rate%, Average slaughter age, Average Body Weight, FCR & EPE. The results demonstrated that both rHVT-ND-IBD and rHVT-IBD vaccinated birds showed high antibodies titer against IBDV at slaughter age. The field performance parameters did not show significant differences except a significant increase in mean body weight in rHVT-ND-IBD vs rHVT-IBD vaccinated birds with an average 100 gm per bird difference in rHVT-ND-IBD vaccinated birds. This comparison revealed that rHVT-ND-IBD® vaccinated birds have higher return of investment.
EFFECT OF ARTEMISIA ANNUA ON CHICKEN LEUCOCYTOZOONOSIS

Y. Chiang\*(1), C.Chen(2)

* Corresponding author: fs3291940@hotmail.com

Poultry leucocytozoonosis is an endemic disease in East Asia. It is caused by protozoan parasites Leucocytozoon caulleryi (L. caulleryi) and decreases the laying rate of layers. Unfortunately, laying hens should not be given antibiotics during the laying period. It is important to find the natural herbs that can replace antibiotics. In this study, we used the herb which has the effect of antimalaria, Artemisia annua (A. annua), as an additive. After artificial infection, 39 of three week-old Nogoya chickens were divided into three groups, control group (CON), antibiotic group (ANT) with 40 mg/kg Sulfadimethoxine, and A. annua powder group (TRT) with 0.5 g/day per chick. From Day 0 to 42 post of infection, body weight (BW), body weight gain (BWG), rectal temperature (RT), rectal temperature change (RTC), mortality rate, pale cockscomb rate, green feces rate, blood smears, polymerase chain reaction (PCR) and necropsy of the sudden death chicks investigated every week. The results show that success rate of infection was 23%. TRT had lower BW and BWG than ANT (P <0.05), but similar with CON. The rate of mortality, pale cockscomb and green feces of TRT and ANT were significantly lower than CON (P <0.05). Some important tissues of sick chicks were scattered with multifocal petechiae and white mottling which caused by second-generation schizonts. There is no significantly difference between TRT and CON on PCR, but the percentage of L. caulleryi in blood of TRT was significantly lower than CON by blood smears (P <0.05). In conclusion, feeding with 0.5 g of A. annua powder daily can effectively reduce the proportion of L. caulleryi and thereby reduce the mortality, pale cockscomb and green feces.

<footnote>(1) Department of Animal Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan</footnote>

<footnote>(2) National Chung-Hsing University Dept. of Animal Science, Taichung, Taiwan</footnote>
EFFICACY OF BIVALENT RHVT- ND-IBD VECTOR VACCINE AGAINST CHALLENGE WITH VERY VIRULENT
INFECTIOUS BURSAL DISEASE VIRUS IN COMMERCIAL BROILERS

H. Sultan*(1), S. Talaat(2), L. Tantawi(3), O. Yakout(2)

(1) Department of Birds and Rabbit Medicine, Faculty of Veterinary Medicine, University of Sadat City, Menoufiya, Egypt, Cairo, Egypt

(2) Department of Birds and Rabbit Medicine, Faculty of Veterinary Medicine, University of Sadat City, Menoufiya, Egypt, Menoufiya, Egypt

(3) Department of Pathology, Animal Health Research Institute. Dokki, Giza, Egypt, Giza, Egypt

* Corresponding author: sukkara@gmail.com

Infectious bursal disease (IBD) control has focused mainly on vaccinations, which provide protection against mortality and immunosuppression. The efficacy of commercial bivalent herpes virus of turkey -Newcastle disease and infectious bursal disease (rHVT-ND-IBD) vector vaccine against very virulent infectious bursal disease viruses (vvIBDV) Egyptian isolate (GenBank accession no.KX646373) was evaluated in commercial broiler chicks vaccinated at day-old by subcutaneous (sc) administration. Birds were challenged on 24 or 28-days of age, and the protection assessment were based on clinical signs, mortality, bursal/body weight ratios (B/BWR), gross lesions, mean severity index (MSI) of histopathological lesion scores of bursae and serological response at 7 days post-challenge (dpc). As result, the complete protection against mortality (100% and 100%) vs (100% and 96%) was adopted after challenge on days 24 and 28 in vaccinated and non-vaccinated challenged birds, respectively. In addition to partial and complete protection against bursal atrophy (B/BWR) were 0.57 and 0.42 vs 0.75 and 0.33 in vaccinated and non-vaccinated challenged birds at 24 and 28-days of age, respectively. A significant difference in MSI was shown (1.7 and 3 vs 1.5 and 3) in vaccinated and non-vaccinated birds when challenged on days 24 and 28, respectively. Our results revealed that the day-old vaccination of bivalent rHVT-ND-IBD vector vaccine is providing early onset of protection against the Egyptian vvIBDV in commercial broilers. In conclusion the pressure of vvIBDV in poultry industry, especially in IBDV-endemic countries, requires the early protection against vvIBDV. Indeed, the earliest development of immunity can be induced in chickens by day-old vaccination, moreover, day-old vaccination with rHVT-ND-IBD has been proven to be an efficient way of vaccine delivery in large scale poultry production.
INHIBITION OF SALMONELLA TYPHIMURIUM FECAL SHEDDING IN COBB 500 BROILERS WHEN FED ZN AND MN AMINO ACID COMPLEXES

M. Rebollo\*(1)

(1) ZINPRO, Eden prairie, United States

* Corresponding author: mrebollo@zinpro.com

Food borne diseases of humans, caused by Salmonella infected products, are an insidious problem that cause significant damage to consumers, food brands, and poultry companies. Zinc (Zn) and manganese (Mn) are essential nutrients, important for immune response and intestinal health. Feeding highly available Zn and Mn sources, such as those from amino acid complexed minerals (AACM), has resulted in improved epithelial tissue structure and function, as well as immunity. A challenge-study was designed to evaluate the effect of feeding Zn or Zn and Mn AACM (Availa®Zn and Availa®Z/M, Zinpro Corporation, Eden Prairie, Minnesota, USA) on fecal and cecal recovery of Salmonella Typhimurium. A total of 100 Cobb 500-day-old chicks were tested for being Salmonella--free and assigned to 4 treatments in a randomized complete block design. Treatments were: T1) Control, with no added Zn or Mn; T2) Availa-Zn60 with 60 ppm Zn and 0 Mn; T3) Sulfates with 40 ppm Zn and 40 ppm Mn; T4) Availa-ZM, 40 ppm Zn and 40 ppm Mn. Birds were placed in plastic containers and received an oral challenge on d 3, 6, 10, 13, and 16 with Salmonella Typhimurium DT104 at 1X108 CFU/bird. Dietary treatments were offered from d 22 to 42. Fecal samples were individually collected using rectal swabs on d 23, 30, 37, and 40. On d 42, birds were euthanized and cecal contents cultured. Fecal and cecal samples were diluted in Lennox L broth and cultured, then quantitated using Xylose Lysine Deoxycholate (XLD) agar (18 hr at 37°C). Bacterial counts and body weight were analyzed using Analysis of Variance with Tukey's ad hoc test for multiple comparison, and prevalence with Chi Square test (GraphPad Prism, v8). For all variables, significant differences were defined at P ≤ 0.05.

Compared to Sulfate or Control groups, birds fed Availa-Zn60 and Availa-ZM had: lower Salmonella excretion in feces (CFUs/g) at d 37 and 40 (P ≤ 0.05), lower Salmonella prevalence in feces at d 30 (P ≤ 0.01) and in ceca at d 42 (P ≤ 0.01), higher BW and lower FCR at d 42 (P ≤ 0.01). Supplementation of poultry feeds with amino acid complexed minerals, specifically Availa-Zn or Availa-Z/M, could be a useful tool in the prophylaxis of Salmonella infections.