# EXPANDING DUPLICATION OF A TESTIS GENE FAMILY IN THE CHICKEN GENOME

S. Fouchécourt\*(1), P. Monget(1)

(1)INRA, Nouzilly, France

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

**Background:** At the cellular and genetic levels, spermatogenesis represents a relatively well-conserved process event among phylogenetically distant animal species. In a previous study [1], we pointed out a substantial list of uncharacterised genes highly conserved in non-vertebrate and vertebrate species and exhibiting a high expression in chicken testis. Among these genes, we have studied two families of genes that exhibit numerous duplications in the chicken genome.

**Material&Methods:** Gene copy numbers investigated by PCR (genomic DNA from Leghorn adult roosters), gene conversion, and genomic localization by FISH were previously described in the lab [2][3]. Bioinformatics data were retrieved from Ensembl database (http://www.ensembl.org/index.html).

**Results:** We investigated the copy number of genes in two gene families, named "novel genes" in Ensembl database: 1) the chicken co-orthologs of the mouse PHD finger protein (7) gene and 2) the chicken co-orthologs of the Sun3-5/Spag4 genes. These genes are involved in the determination and/or differentiation of germ cells in vertebrates and we showed that they are highly expressed in the rooster testis. The gene dosage allows to confirm the high duplication of these genes in the chicken genome (around 60 copies for the first family and 20 for the second). The gene clusters of chicken PHD finger protein were localized by FISH on 8 loci on chromosomes 1, 2, 12, 19 and Z, as predicted by Ensembl.

**Conclusion:** We showed that two gene families of testis genes conversed across evolution and highly expressed in the gonad, are highly duplicated in the chicken genome. The identification of reliable fertility markers in farm chickens is critical in the context of the assessment of decline of reproductive performance (reduction in the longevity of male reproduction and deterioration in sperm quality). The characterization of such testis-specific (enriched) genes in the chicken might be of agronomic interest regarding the need for fertility markers in avian species.

[1] Fouchécourt et al. An evolutionary approach to recover genes predominantly expressed in the testes of the zebrafish, chicken and mouse. BMC Evol Biol, 2019. 19(1): p. 137

[2] Meslin, C., et al., Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome. Genome Biol Evol, 2015. 7(5): p. 1332-48.

[3] Solinhac, Romain, et al. "Integrative mapping analysis of chicken microchromosome 16 organization." BMC genomics 11.1 (2010): 616.
PROTEOME AND BIOLOGICAL FUNCTIONS OF INNER AND OUTER PERIVITELLINE LAYERS IN THE
CHICKEN EGG

M. Brégeon\*(1), D.Tomas\*(2), B.Bernay\*(3), M.Chessé\*(1), C.Zatylny-gaudin\*(4), V.Labas\*(2), S.Réhault-godbert\*(1), N.Guyot\*(1)

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

(2) INRAE, CNRS, Université de Tours, IFCE, PRC, CIRE-PAIB, Nouzilly, France

(3) Université de Normandie, SFR 4206 ICORE, Proteogen platform, Caen, France

(4) Université de Normandie, Alliance Université de Sorbonne, MNHN, UA, CNRS, IRD, BOREA, Caen, France

* Corresponding author: megane.bregeon@inra.fr

The chicken egg perivitelline layer (PL), also known as the vitelline membrane, is a proteinaceous structure surrounding the egg yolk, in contact with the albumen. In avian reproduction, PL is involved in the acrosome reaction, in the early embryonic development and in the physical and antimicrobial protection of the embryo. It thus contributes to the quality of both hatching and table eggs. It consists of two sublayers: the inner PL (IPL) and the outer PL (OPL), that are in contact with the yolk content and the egg white, respectively. About 140 different proteins were identified in the PL to date; however, the distribution of most of these proteins between IPL and OPL remains to be elucidated. The specific sites of synthesis, namely the ovary for the IPL and the oviduct for the OPL, suggest that these two sublayers possess different biological functions. The present study aims at characterizing the proteome composing IPL and OPL, and identifying the intrinsic putative functions of each perivitelline sublayer. For this purpose, PLs were harvested from ISA-Brown eggs collected on the day of lay and carefully washed to remove traces of yolk and egg albumen. IPL and OPL were carefully manually separated from isolated PLs and proteins composing these two layers were independently solubilized, fractionated by SDS-PAGE and analyzed by tandem mass spectrometry coupled to nanoliquid chromatography (GeLC-MS/MS). The proteomic analysis revealed more than 400 proteins in the whole PL including around 310 and 230 proteins that were identified in IPL and OPL, respectively. Among these, 141 were common to both sublayers and encompass the most abundant proteins of the PL. The tissue origin of some of these shared proteins was investigated by the analysis of gene expression (RT-qPCR) in different reproductive tissues assumed to be involved in the formation of IPL (liver, theca, granulosa) and OPL (oviduct segments). The functional annotation (gene ontology/bibliography), carried out on the entire set of identified proteins, revealed the presence of numerous proteins associated with fertilization and antimicrobial defense, but also with many other biological functions such as cell adhesion and migration, metabolism, morphogenesis, etc. Altogether, the data generated from this study give new insights into the structure and composition of the chickenegg PL and provide an integrative overview of the respective physiological functions of IPL and OPL.
# MIR-26A-5P REGULATES THE EXPRESSION OF VEGFA BY TARGETING NLK IN CHICKEN FOLLICULAR GRANULOSA CELLS

L. Kang\*(1), L. Hou(1)

(1) Shandong Agricultural University, Taian, China

* Corresponding author: likang916@126.com

## Introduction

The miRNAs are a class of small non-coding RNA molecules of about 22 nucleotides in length, which are involved in ovarian follicle function. Our previous study have found that miR-26a-5p facilitates theca cell proliferation in chicken ovarian follicles. However, the regulatory role of miR-26a-5p in chicken hierarchal follicular granulosa cells remains unclear. In this study, we found that miR-26a-5p regulates the expression of VEGF-A by targeting NLK in chicken follicular granulosa cells.

## Materials and methods

The follicular granulosa cells were obtained from Hy-line brown hens. NLK 3' UTR target site wild and mutant type luciferase reporter gene vectors as well as siNLK vector were constructed. The dual-luciferase reporter assay was used to detect the regulation relationship between miR-26a-5p and NLK. After overexpression, inhibition of miR-26a-5p and interference with NLK in chicken granulosa cells, RNA and protein were extracted for qRT-PCR and Western Blot, and the cell culture medium was used for ELISA test of VEGF-A. DNAMAN 6.0 and SAS 9.2 were used to design primers and analyze data. Differences were considered significant at p<0.05.

## Results

### (1) miR-26a-5p targets NLK to down-regulate its expression

The luciferase results showed that the relative luciferase activity with miR-26a-5p mimic and the mutant-NLK luciferase reporter vector showed significantly higher than that of the wild type, indicating that miR-26a-5p could target and negatively regulate the expression of NLK gene.

### (2) miR-26a-5p targets NLK to down-regulate the expression of VEGFA mRNA

After overexpression of miR-26a-5p in follicular granulosa cells, qRT-PCR showed that the levels of NLK and VEGF-A mRNA were significantly decreased; after inhibition of miR-26a-5p, the levels of NLK and VEGF-A mRNA were significantly increased. Furthermore, after interference with siNLK, the levels of VEGFA mRNA were also significantly decreased.

### (3) miR-26a-5p targets NLK to down-regulate the expression of VEGFA protein

After overexpression of miR-26a-5p, as well as interference with siNLK in granulosa cells, The Western Blot and ELISA results showed that the protein levels of NLK and VEGFA were significantly downregulated. After inhibiting miR-26a-5p, NLK and VEGF-A protein levels were significantly upregulated.

## Conclusions

(1) NLK is a direct target gene of gga-miR-26a-5p. (2) gga-miR-26a-5p regulates the expression of VEGFA by targeting NLK gene in chicken granulosa cells.
ID: 610

ROLE OF TTR, PRRP AND GHRELIN AND CRH SYSTEMS ON APPETITE REGULATION IN INCUBATING SILKIE.

M. Takeda\*(1), T.Ohkubo(1)

(1)United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchū, Japan

\* Corresponding author: s192619q@st.go.tuat.ac.jp

We previously identified that mRNA expression of the transthyretin (TTR) and prolactin-releasing peptide (PrRP) are changed significantly in the hypothalamus of incubating Silkie hens. However, it is unclear whether the observed changes are specifically associated with broodiness induced anorexia or not. In addition, Ghrelin and CRH suppress food intake in chicken, and these expressions are known to be regulated by thyroid hormone and PrRP, respectively. From these points, we investigated mRNA expression of TTR, PrRP, and ghrelin and CRH systems among different feeding states in order to clarify the regulatory mechanism of food intake in incubating hens.

Egg laying and behavior of matured female Silkies (36-week-old) were monitored daily, and divided into three groups (n=5); 1) incubating hens that sat in the nest with characteristic behaviors for at least 1 week, 2) laying hens that lay eggs regularly for at least 1 week, and 3) pair-feeding hens that were given feed equivalent to the level of incubating birds for 2 weeks. Finally, hens were weighed, sacrificed by decapitation, and the hypothalamus was collected. Hypothalamic TTR, PrRP, Ghrelin, Ghrelin receptor (GHS-R1alpha), CRH and CRH receptors (CRH1R, CRH2R) were quantitatively analyzed by RT-qPCR. The Smirnov-Grubbs test was applied to exclude outliers, and difference in mRNA expression was determined by the Tukey-Kramer test. P-values less than 0.05 were considered significant. All statistical analyses were performed by R statistical package (www.r-project.org).

TTR mRNA expression was significantly increased in pair-feeding compared to the other two groups. PrRP mRNA expression was significantly decreased in incubating and pair-feeding than in laying. Ghrelin mRNA expression tended to decrease in pair-feeding compared to laying and incubating. GHS-R1alpha mRNA expression in incubating showed an increasing trend compared to laying and pair-feeding. CRH mRNA expression was significantly reduced in incubating and pair-feeding compared to laying, and that was similar to the PrRP. CRH1R mRNA expression was not different among the groups, and CRH2R mRNA expression in pair-feeding was significantly reduced than in laying and incubating. In conclusion, TTR, PrRP and CRH systems are possible to respond generally to chronic under nutrition. Contrary, the ghrelin system may involve specifically regulating natural anorexia during incubation behavior in Silkies.
EFFECTS OF SEMINAL PLASMA ON THE SPERM REPLACEMENT IN CHICKEN SPERM-STORAGE TUBULES

M. Matsuzaki\*(1), S. Mizushima(2), T. Sasanami(3)

(1) Hiroshima University, Higashi-hiroshima, Japan

(2) Hokkaido University, Sapporo, Japan

(3) Shizuoka University, Shizuoka, Japan

\* Corresponding author: meimatsu@hiroshima-u.ac.jp

In birds, the ejaculated sperm does not directly travel to the site of fertilization, but they initially entered and stored in the sperm storage tubules (SSTs) located in the utero vaginal junction (UVJ) of the oviduct. After storage, sperm are gradually released from the SSTs and achieve fertilization in successive days (several days to months depending on the species). In domestic chicken, approximately 14 eggs are fertilized after single insemination by natural mating or artificial insemination (AI). Previously, in the chicken, it is reported that when consecutive AI of semen obtained from different males were performed, paternity of the chick significantly biased to the male which inseminated in the last. This phenomenon is referred to as last male sperm precedence (LMSP) and is also known to operate in the insect fertilization such as fruit fly. In this study, we investigated whether LMSP in the chicken is related to the replacement of the resident sperm in the SST after additional insemination. The ejaculated sperm of White Leghorn (WL) were fluorescently stained with pHrodo-Red and inseminated into WL female vagina, sperm filling rate which calculated as sperm filled SSTs/total SSTs decreased in a time-related manner, but approximately 50% of SSTs still contained the sperm after 5 days. When we performed another insemination of the sperm which fluorescently labelled with Hoechst33342 after 3 days of initial AI, we observed most of the resident sperm were labelled with H33342 dye under fluorescent microscope. This result suggests that LMSP in the chicken is occurred by the replacement of resident sperm with recently inseminated sperm. To examine the effects of seminal plasma on the sperm release from the SST, we injected seminal plasma of WL intravaginally for three consecutive days after AI, and the sperm filling rate was compared with the control birds (PBSinjection). As the results, sperm filling rate significantly decreased in the seminal plasma-injected birdscompared to the control. Moreover, when the seminal plasma of the second AI was removed, sperm replacement decreased, and this decrement recovered by the addition of seminal plasma. These results suggested that LMSP in the chicken operates at the stage of the sperm entrance into the SSTs, and seminal plasma components may play important roles in the process of sperm replacement.
TWO ISOFORMS OF DDX4 AND THEIR DIFFERENTIAL EXPRESSION DURING EARLY EMBRYO DEVELOPMENT OF JAPANESE QUAIL

S. Mizushima*(1), T. Sasanami(2), T. Ono(3), A. Kuroiwa(1)

(1) Hokkaido University, Hokkaido, Japan

(2) Shizuoka University, Shizuoka, Japan

(3) Shinshu University, Nagano, Japan

* Corresponding author: smizus@sci.hokudai.ac.jp

DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) encoding RNA helicase plays essential roles for proper germ cell formation in several animal species. In mice, it is reported that DDX4 is essential for spermatogenesis, whereas in chicken, DDX4 is necessary for maintenance of germ cells in both sexes, and subsequent oogenesis, not spermatogenesis. The difference in the role of DDX4 between mice and chicken is suggested to be due to the different mechanisms in germ cell specification in each species, however, authentic RNA targets of DDX4 remain unknown. In the present study, we investigated the expression profile of DDX4 during embryogenesis of Japanese quail. In addition, we attempted the screening of target RNA of DDX4 capable of interacting with DDX4 protein by in vitro binding assay. Western blot analysis using specific antibody revealed the presence of two DDX4 isoforms in the gonad of quail embryo. cDNA cloning and the sequencing analyses demonstrated the presence of short isoform which is the truncated version of DDX4 that lacks a part of the N-terminal region (DDX4-S), in addition to full length DDX4 (DDX4-L). Immunohistochemical and western blot analyses showed that DDX4-S isoform was expressed in male blastoderm and the gonad throughout the embryogenesis after the migration of primordial germ cells (PGCs) into their gonads. Interestingly, DDX4-S signals did not detect in the female blastoderm. On the other hand, DDX4-L was expressed in ovulated egg and was detectable through blastoderm and embryonic development, irrespective of genotypic sex. In addition, the number of genotypic male and female PGCs was apparently decreased in homozygous mutant DDX4 blastoderms generated by microinjecting CRISPR/Cas9 sgRNA into the egg at pronuclear stage, suggesting that DDX4 is an important germ cell determinant and/or regulates initial proliferation of male and female PGCs in Japanese quail. Furthermore, in vitro binding assays of DDX4 and blastodermal and gonadal mRNAs revealed that DDX4-S strongly binds to several male-specific mRNAs. Our results suggested that DDX4-L is essential for germ cell survival and proliferation in both sexes and DDX4-S may participate in male germ cell differentiation in Japanese quail.
# TRANSCRIPTOME SEQUENCING REVEALS CANDIDATE GENES FOR EGG RATE DURING PEAK LAYING PERIOD AND END-OF-LAY HENS

E. Hao\*(1), H.Chen(1), R.Huang(1)

(1)Hebei Agricultural University, Baoding, China

* Corresponding author: 277179119@qq.com

## Introduction

Laying hens have a sharp drop in laying rate when ovarian follicles are exhausted. However, low laying rate in aging laying hen is a result of a dynamic interaction between neuroendocrine changes that take place in the brain with the reproductive endocrine axis governing the function of ovaries.

## Material and Methods

To elucidate the key regulatory genes involved in Dawu Jinfen chicken with high and low rates of egg production, RNA from hypothalamus tissue during 35-week-old high laying rate 100%, 100%, 100% (Group YH:YH1, YH2 and YH3), 70-week-old high laying rate 100%, 98%, 100% (Group OH: OH1, OH2 and OH3) and 70-week-old low laying rate 55%, 45%, 30% (Group OL:OL1, OL2 and OL3) were sequenced using the Illumina HiSeq 2500 sequencing platform.

## Results

A total of 391.59 million clean reads with an average of 43.51 million reads per sample (range: 40.04 to 45.71 million) were acquired after removing the low-quality and adaptor sequences. A comparison between Groups OH and OL revealed 32 differentially expressed genes, with 15 displaying up, and 17 displaying down. A comparison between Groups OH and YH revealed 56 differentially expressed genes, with 30 displaying up, and 26 displaying down. A comparison between Groups OL and YH revealed 127 differentially expressed genes, with 30 displaying up, and 97 displaying down. Ten genes that are mainly involved in pathways for reproduction regulation, such as PRLH, ESR2, CGA, CRH, CYP19A1, CYP26B1, PGR, OXT. All of the predicted genes of them were mapped to terms in the GO and KEGG databases. In total, in OH-VS-OL groups, 2 GO terms and 55 KEGG pathways in hypothalamic tissues were significantly enriched.

## Discussion and conclusion

The hypothalamus is a key component of the HPG system. As the tip of the HPG axis, it plays an important role in hormone secretion and regulation of reproductive performance in laying hens. As we expected, our results aggregated many reproductive-related signaling pathways, such as the GnRH signaling pathway, the estrogen signaling pathway, and the prolactin signaling pathway. The obtained RNA sequencing data from the hypothalamus could be a valuable resource for genomic studies and provide new insights into the biological processes that regulate chicken reproduction. These data provide comprehensive gene expression information at the transcriptional level that might increase our understanding of the laying hens' reproductive biology.
EFFECT OF LEPTIN ON THE OVARIAN DEVELOPMENT AND FUNCTION IN JUVENILE CHICKS

A. Shaikat(1), M.Takeda(1), T.Ohkubo*(2)

(1) United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchū, Japan

(2) Ibaraki University, Ami, Japan

* Corresponding author: takeshi.ohkubo.0533@vc.ibaraki.ac.jp

Leptin advances the onset of laying and suggested to be involved in the regulation of reproduction. Leptin receptor (LEPR) appears in early embryonic days and continuously expresses in chicken ovary. However, the role of leptin in juvenile chicks' ovary is still not fully understood. Furthermore, formation of primordial follicles and its transition to primary follicles occurs around day 7 and day 28 after hatching in chicken. Therefore, the mRNA expression of LEPR, FSHR and CYP19A1 were measured in the ovary from day 1 to day 28 at 7 days interval. In addition, changes of the same gene expression after leptin administration was measured in the ovary in 7-and 28-days-old chicks.

Newly hatched White Leghorn female chicks were used in the present experiments. All birds were maintained at 24h light at 30°C for the first two weeks and 23h light at 29°C thereafter. All birds freely accessed to feed and water. In the first experiment, at the age of days 1, 7, 14, 21, and 28 (n = 5) were killed by decapitating and ovary was collected. In the second experiment, chicks received intraperitoneally recombinant mouse leptin at a dose of 25 and 250 µg/kg body weight (BW) at 7- and 28-days old. After 24h of administration, all birds were sacrificed by decapitation after taking blood and ovary was collected. mRNA expression of LEPR, FSHR and CYP19A1 was analyzed by RT-qPCR and serum estrogen was measured by ELISA in the second experiment.

In the first experiment, ovarian LEPR, FSHR, and CYP19A1 expressions showed an increasing trend by age progress and peaked at day 28. In the second experiment, leptin showed specific effects on ovarian gene expression, but the effect was differed by age. In 7-days-old chicks, both doses of leptin significantly increased LEPR and CYP19A1 mRNA expressions, and FSHR mRNA expression was dose-dependently increased by leptin. In the ovary of 28-days-old chicks, leptin dose dependently decreased LEPR mRNA. FSHR mRNA expression was elevated by lower and declined by higher doses of leptin. CYP19A1 mRNA remained unchanged by leptin treatment. Leptin increased significantly serum estradiol in the 7 days old chicks. Besides, high dose of leptin administration significantly decreased the serum estradiol in 28 days old birds.

In conclusion, leptin may have age specific effect on ovarian development in early juvenile chicks and that supports previous findings that leptin administration at early age advances puberty in birds.
ID: 1223

THE MOLECULAR MECHANISM STUDY OF SPERM STORAGE ABILITY BASED ON RNA-SEQ

Y. Sun*(1), J.Fan(1), L.Shi(1), P.Wang(1), L.Jiang(1), P.Ge(1), Y.Li(1), A.Ni(1), A. M. Ias(1), H.Ma(1), J.Chen(1)

(1)Institute of Animal Science, Beijing, China

* Corresponding author: yanyansun2014@163.com

Sperm storage tubules is a special tubular gland that exists in the hens' utero-vaginal junction, allowing sperm to survive in the fallopian tube and maintain fertility for a long time. The objective of this study was to identify the molecular regulation of sperm storage ability based on RNA-seq. A total of 158 White Leghorn laying hens and 28 rosters of 27 weeks of age were used in this study. After the artificial insemination with mixed semen for two consecutive days, the eggs were collected for each individual laying hen starting from the third day for 21 days and incubated. The fertility was calculated for each laying hens, based on which, the top four chickens with the highest fertility and the four with the lowest fertility were selected and named high and low sperm storage ability group, respectively. The uterus and vaginal junction area of these eight laying hens was dissected and divided into two parts in the longitudinal direction. One was made into paraffin sections and HE stained for the morphology evaluation including the number and cross section area of the sperm storage tubules, and the other one was used for transcriptome sequencing. There was no difference in sperm storage tubules numbers between high and low sperm storage ability groups (P > 0.05). The average cross section area of sperm storage tubules of high sperm storage ability group was higher than that of the low group (P < 0.05). Analysis of transcriptome data shows that the gene of high abundance in the utero-vaginal junction mainly includes the biological process of protein synthesis, genes related to sperm plasma membrane protein interaction and sperm state. A total of 555 mRNA were differentially expressed between groups, of which, 118 mRNA were up-regulated and 437 mRNA were down-regulated in the low sperm storage ability group. GO annotation analysis revealed that these genes were involved in muscle contraction, membrane receptors, signal transduction, and lipid synthesis and so on. The present study indicates that the cross section area of sperm storage tubules may be related to the sperm storage ability of laying hens. Based on the RNA-seq data, we get important clue that smooth muscle contraction and mucous membrane secretion-related lipids may also be involved in sperm release and ascending fertilization. The molecular mechanism involved in the sperm storage and release regulation needs further digging of the RNA-seq data.
ID: 586

EFFECTS OF PHOTOPERIOD UPON THE REPRODUCTIVE SYSTEM AND REPRODUCTIVE CYCLE IN THE
FEMALE NATIVE THAI CHICKEN: ROLES OF STEROID HORMONES

B. Kamkrathok(1), N.Sartsoongnoen(2), Y.Chaiseha*(1)

(1)Institute of Science, Suranaree University of Technology, Nakhon ratchasima, Thailand

(2)Faculty of Science and Technology, Nakhon ratchasima, Thailand

* Corresponding author: yupaporn@sut.ac.th

In birds, besides the neuroendocrine factors, the environmental factors may influence the reproductive cycle. This study was aimed to elucidate the roles of steroid hormones in the regulation of the reproductive cycle and its relationship with photoperiod. Female native Thai chickens, Pradoohangdum breed, were used. Blood samples were collected according to their reproductive stages (n=6); non-laying, laying, and incubating eggs. Plasma samples were analyzed for estrogen, progesterone, and testosterone by enzyme-linked immunosorbent assay (ELISA). The results showed that plasma estrogen and testosterone levels were significantly (p<0.05) higher in laying than that of non-laying hens, however; plasma levels were not different when compared with incubation hens. In contrast with estrogen and testosterone, plasma progesterone levels were not changed during the reproductive cycle. To investigate the role of photoperiod in relationship with steroid hormones, chickens were divided into 4 groups (n=6). They were reared in different photoperiodic treatments as long day (16 hours of light and 8 hours of darkness; 16L:8D), short day (8L:16D), normal day (12L:12D), and control group (giving natural light photoperiod). Blood samples were collected from each chicken 2 times a week for 4 weeks and then analyzed for estrogen, progesterone, and testosterone levels by ELISA. The results indicated that plasma estrogen and progesterone of chickens reared in long day, short day, and normal day were not different. In contrast, plasma estrogen and progesterone levels were higher in the control group when compared with other groups. Plasma testosterone levels were the same among 4 groups. These results support the pivotal role of steroid hormones in associated with the reproductive cycle of this equatorial gallinaceous species. In addition, photoperiod might have some effects on plasma estrogen and progesterone levels, but not on testosterone levels. These data presume to suggest that photoperiod may, in parts, have an effect on the reproduction of the native Thai chicken.
EFFECT OF NANOSELENIUM ON THE IMPROVEMENT OF CRYOPRESERVATION OF DOMESTIC FOWL SEMEN

B. Vegi*(1), Á.Drobnyák(1), Z.Szabó(1), É.Török(1), J.Prokisch(2), J.Barna(1)

(1)National Centre for Biodiversity and Gene Conservation, Gödöllő, Hungary

(2)University of Debrecen, Debrecen, Hungary

* Corresponding author: vegi.barbara@hagk.hu

During semen cryopreservation, the spermatozoa undergo notable stress effect. The avian
spermatozoa are extremely sensitive to the harmful effects caused by the formation of free radicals.
Under natural conditions various types of antioxidant systems in spermatozoa and seminal fluid are
able to bind to free radicals. Selenium with vitamin E is essential component of the antioxidant system
in a multi-step biochemical process. In several animal species, antioxidants added to the semen diluent
have been shown to improve the efficiency of semen freezing. Recent researches have shown that
nanoparticles could be more efficiently utilized due to their increased surface area and reactivity. In
the present work we investigated the effect of vitamin E and nanoselenium in various concentrations
in semen diluent on the changes of qualities of rooster spermatozoa after freezing/thawing.

Semen of 20 individually placed Hungarian yellow males was collected twice weekly. The pooled semen was divided into four equal parts and diluted with Lake's diluent in 1:2 ratios. 5 µg/ml α-tocopherol was added to the diluted semen samples with the exception of control group, as well as 50, 100 and 200 µg/ml of nanoselenium solution were put to the three experimental samples, respectively. After 20 min of equilibration at 5 °C, all samples were further diluted in 1:0.5 ratios with DMA containing diluent to give a final concentration of 6% DMA. The samples were frozen 1 cm above the surface of liquid nitrogen in 0.25 ml French straws for 20 minutes and then placed in liquid nitrogen. The samples were thawed at 5°C. From the spermatological parameters motility was examined using SCA® CASA. The live/dead cell ratio and the ratio of morphological abnormalities were determined by aniline blue-eosin staining.

After thawing, the proportion of motile cells in the samples with nanoselenium at different
concentrations did not differ significantly from the control group ((34.6%; 26.4%; 34.5% and 29.2%,
respectively). There were no differences in the survival rates of the cells (20.2%; 19.2%; 17.2%; 22.4%),
and the morphological abnormalities, either. Based on in vitro studies, the use of nanoselenium and α-
tocopherol in the given concentrations did not improve spermatological parameters after
freezing/thawing. Despite the in vitro results further studies are planned with artificial insemination to
investigate the effect of the above additives on the fertilization capacity of frozen/thawed sperm.
ID: 1330

EXTRACELLULAR VESICLES ISOLATED FROM SEMINAL PLASMA OF CHICKENS WITH CONTRASTED FERTILITY SHOW DIFFERENTIAL CHARACTERISTICS

L. Cordeiro\*(1), A.Carvalho(2), I.Grasseau(2), H.Lin(2), R.Uzbekov(3), E.Blesbois(2)

(1)INRAE, CNRS, Université de Tours, IFCE, UMR PRC, F-37380, Nouzilly, France, Nouzilly, France

(2)INRAE, CNRS, Université de Tours, IFCE, UMR PRC, Nouzilly, France

(3)Laboratoire Biologie Cellulaire et Microscopie Electronique, Fac. Médecine, Université de Tours, Tours, France

* Corresponding author: lavcordeiro@gmail.com

Extracellullar vesicles (EVs) are present in seminal plasma of different mammalian species but their
existence in chickens remains controversial. In addition, the relationship of these structures with male
fertility is still unclear. The aim of the present work was first to isolate the EVs from chicken seminal
plasma, then to characterize their morphology and protein content and finally to investigate their
interactions with sperm in both fertile and subfertile roosters. For this purpose, in vivo fertility was
determined by artificial insemination for 40 French SASSO T44 roosters and animals were classified as
fertile (F+, >90%) or subfertile (F-,
REPRODUCTIVE BIOTECHNOLOGIES
HOW SINGLE LAYER COLLOID CENTRIFUGATION CONTRIBUTES TO CHICKEN SPERM CONSERVATION IN VITRO

H. Lin*(1), I.Grasseau(1), L.Chen(2), A.Carvalho(1), E.Blesbois(1)

(1)INRA, Nouzilly, France

(2)TLRI, Tainan, Taiwan

* Corresponding author: linherbie@gmail.com

Conservation of animal genetic resources is a crucial issue for the future. In chicken, sperm cryopreservation is an important method of reproductive cell conservation to restore genetic diversity (Blesbois et al 2008). Glycerol is a highly efficient cryoprotectant for chicken sperm freezing but it must be removed before artificial insemination due to vaginal contraceptive effect. Stepwise dilutions followed by centrifugation is a general method used to remove glycerol from frozen-thawed semen. However, it is time-consuming, must be processed at 4°C, and could decrease fertilizing potential due to added sperm damages (Purdy et al 2009). Percoll™ is a colloid used for sperm preparation. This technique has recently been successful in chicken sperm selection (Lin et al 2019). Therefore, the purpose of this present study was to determine whether Percoll™ centrifugation could be a workable method to remove glycerol from cryopreserved semen. Twenty slow-growing, broiler type of T44 line roosters were used in this study. Semen was collected by dorso-abdominal massage, pooled, diluted 1:1 in Lake pre-cryopreservation (LPC) diluent and kept 10 min at 4°C under gentle shaking. LPC containing 22% glycerol was then added 1:1 (11% glycerol final concentration) for 10 min at 4°C. Percoll treatment: 1 ml of glycerolized semen was then gently placed on top of 2 ml Percoll™ 60%, 70% or 80% colloid or PBS in a 15 ml conical plastic tube and centrifuged at 800g for 20 min. Sperm pellets were resuspended in 100 µl Lake 7.1 (L7.1). Sperm motility was analysed by CASA over 3.5 hr. The results indicated that motile sperm (%) and progressive sperm (%) of 11% glycerolized chicken sperm treated with Percoll™ centrifugation were significantly higher than without. Furthermore, levels of motile sperm (%) and progressive sperm (%) stayed almost stable after Percoll™ centrifugation until 3.5 hr by opposite to 11% glycerolized sperm untreated with Percoll that lost 2/3 motile sperm in the first hour. Thus, Percoll™ centrifugation reduce the sperm motility alterations due to glycerol. This is an encouraging alternative method to remove glycerol in order to optimize the fertility obtained with cryopreserved semen in the chicken.

References

Blesbois E, Grasseau I, Seigneurin F, Mignon-Grasteau S, Saint Jalme M, Mialon-Richard MM (2008) Theriogenol. 69:252-261.

Lin HL, Chen YH, Lin, DY, Lai YY, Wu, MC, Chen LR (2019) Br. Poult. Sci. 61:86-91.

Purdy PH, Song Y, Silversides FG, Blackburn HD (2009) Poult. Sci. 88:2184-2191.
IMPROVED SERUM- AND FEEDER-FREE CULTIVATION OF CHICKEN PRIMORDIAL GERM CELLS

C. Koide\*(1), N.Hamai(2), S.Nomura(2), A.Takenouchi(2), M.Tsudzuki(2), Y.Nakamura\*(2)

(1)Hiroshima University, Higashi-hiroshima, Italy

(2)Hiroshima University, Higashi-hiroshima, Japan

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

Primordial germ cells (PGCs) are the embryonic progenitor cells for gametes. In chicken, a technique was developed in which intravascularly transplanted PGCs undergo germline transmission (Tajima et al. 1993). Recently, a serum- and feeder-free culture system for chicken PGCs was established (Whyte et al. 2015). However, the growth of PGCs was significantly slower than that of PGCs in serum-containing medium. Therefore, a need existed to improve a serum- and feeder-free culture for chicken PGCs. KnockOut Serum Replacement (KSR), a defined, serum-free formulation, could improve the growth of various types of stem cells in serum- and feeder-free culture (Garcia-Gonzalo et al. 2008; Kanatsu-Shinohara et al. 2014). Here, we demonstrate the impact of KSR on derivation and growth of chicken PGCs in serum- and feeder-free cultures. FAot medium (Whyte et al. 2015) was used as a basal medium for chicken PGC culture. We used male PGC-line (F5) of White Leghorn MK line that was derived under serum-free conditions. To investigate the effect of KSR on growth of PGCs, 1,000 PGCs were placed and cultured in FAot medium with different concentrations of KSR for 10 days. As a result, the addition of KSR to FAot medium could promote PGC propagation in vitro. FAot medium supplemented with 1% KSR was optimal for proliferation of PGCs. To investigate whether KSR could improve the derivation of PGCs in primary culture, blood sample (1µL) containing PGCs obtained from 2-day-old embryos of MK line were cultured in FAot medium with 1% KSR for 4 weeks. Resulted cultures that contained more than 50,000 PGCs were determined as derivation. In all, we demonstrated the addition of KSR accelerated the derivation of chicken PGCs in primary culture. In the presence of KSR, PGCs could propagate for more than 6 months and maintained normal morphology and karyotype. Immunocytochemical analysis showed that all PGC lines derived in a medium containing KSR were positive for Vasa and Dazl, germ cell markers, as well as SSEA-1, an undifferentiated cell marker. Germ cell transplantation showed that PGCs derived in KSR-containing medium could migrate toward and settle to the gonads of recipient embryos. In conclusion, the present study showed that serum- and feeder-free medium supplemented with KSR improves the derivation and propagation of chicken PGCs in culture.
DISTINCT BASE EXCISION REPAIR CHARACTERISTICS IN CHICKEN PRIMORDIAL GERM CELLS INDUCES
VARIOUS GENOTYPES BY BASE EDITING

K. Lee(1), H.Lee(1), S.Han(2), K.Lee(1), J.Park(2), J.Han(2)

(1)Seoul National University, Seoul, South Ossetia

(2)Seoul National University, Seoul, South Korea

* Corresponding author: leektmn@naver.com

Recently, base editing technology provides the opportunity to generate precisely genome-modified animal model (1). In addition, genome-modified chicken can be produced through genome-modified primordial germ cells (PGCs), but germ cells have a sophisticated DNA repair system. Therefore, we want to understand the influence of base editing in repair pathways of PGCs and to identify relationship between germ cell specific characteristics and the consequences of base editing.

First, we constructed all-in-one base editing plasmids. Then we applied the base editing plasmids into chicken DF-1 cells and PGCs, and genome modification was confirmed by T7E1 assay and sequencing analysis. Furthermore, qRT-PCR for BER pathway related genes and functional studies for BER gene on the effect of base editing purity was conducted. As a result, significant differences of gene expression and editing purity between cell types were evaluated by student t-test. Then, base edited WL PGCs were microinjected into the Ogye recipient embryo to generated genome-modified chickens.

First, we introduced base editing plasmids into two chicken cell lines. Specifically, in vitro genome modification showed that the base editing purity in DF-1 cells and PGCs were different as base editing purity was lower in PGCs in two economic related genes. Then, we examined the expression levels of several BER pathway related genes by qRT-PCR. As a result, most of the DNA glycosylases and BER complex genes are upregulated in PGCs. Subsequently, functional studies for upstream gene of the BER pathway demonstrated that regulation of BER gene affects base editing purity. By transplanting the base edited PGCs into recipient embryos, we produced more than 100 genome-modified chickens, which had nucleotide substitutions as well as indel mutation in the targeted locus.

Our results suggest that the distinct repair property of chicken PGCs causes low editing purity during genome-modification, but modulation of BER functions could enhance the production of genome-modified organisms harboring desired genotypes

Reference

1. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016;353(6305).
ID: 1153

SUCCESSFUL CRYOBANKING OF SEVEN INDIGENOUS CHICKEN BREEDS WITH PRIMORDIAL GERM CELL LINES

B. Lazar\*(1), R.Tóth(2), N.Tokodyné szabadi(2), N.Sztán(3), M.Molnár(3), B.Végi(3), Á.Drobnyák(3), K.Liptói(3), E. Patakiné Várkonyi(3), E. Gócza(1)

(1)NARIC ABC / NBGK HGI, Gödöllő, Hungary

(2)NARIC ABC, Gödöllő, Hungary

(3)NBGK HGI, Gödöllő, Hungary

* Corresponding author: lazbence@gmail.com

The economically important or indigenous chicken breeds are held in in situ populations worldwide, thus they are exposed to various risks. Therefore, it is increasingly important to protect the genetic information these breeds represent. From the available methods, cryopreservation of primordial germ cells (PGCs) is the most promising one.

The goal of the present study was to establish a cryobank based on PGCs for seven indigenous Hungarian chicken breeds and to test the viability of the cryopreserved cells by creating germline chimaeras with one of them; the Partridge colour Hungarian. The germline chimaeras were then backcrossed to recover the donor breed.

We collected blood from each embryo individually, then the isolated blood samples, containing the PGCs, were cultured in a medium which was selective for the PGCs. Later, samples from the cell lines were collected for DNA, RNA isolation and immunohistochemistry to characterize the quality of the cells and to perform microsatellite analysis to evaluate the representation of the genetic variability of the original population. As a next step, parallel vials were frozen from each PGC line. To evaluate the freezing process and to prove the functional integrity and migrating ability of PGCs, some of the vials were thawed and the cells were injected into recipient embryos. First, the cells were labelled with an in vivo fluorescent dye, thus the migration of the injected cells was followed toward the developing gonads, and the ratio of the colonization was analyzed. As a second experiment, after injection with the frozen/thawed PG cells, the eggs were left to incubate until hatching. The hatched chicks were then kept until maturation and are going to be cross-tested with animals from the donor genotype to examine the germline transmission.

During the study, 21 PGC lines from Partridge colour Hungarian chicken were established with a derivation rate of 31.1%. Based on the general diversification indexes which were used for the microsatellite analysis the cell lines represent the genetic variability of the original population. The PGC lines were frozen and then successfully thawed with a cell viability of 50%. The preserved cells were capable of colonizing the gonads of the recipient embryos; furthermore, we have 24 adults (13 roosters and 11 hens) which presumably contain the donor PGCs.

The research was funded by Horizon 2020 (n°677353 IMAGE) and by VEKOP-2.3.2-16-2016-00012.
FRENCH AVIAN CRYOBANK: DEVELOPMENT OF REPRODUCTIVE BIOTECHNOLOGIES BASED ON PRIMORDIAL GERM CELLS (PGCS) AND INVESTIGATION OF THE IMPACT OF IN VITRO STEPS ON PGCS INTEGRITY AND REPRODUCTIVE CAPACITY.

M. Govoroun*(1), S.Alves(1), H.Acloque(2), M.Bernard(3), C.Klopp(4), A.Jacques(1), F.Pitel(5), C.Hennequet-antier(1),A. Brionne(1), V. Coustham(1),E. Blesbois(1)

(1) INRA, Nouzilly, France

(2) GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-josas, France

(3) INRA, Jouy-en-josas, France

(4) INRA, Castanet-tolosan, France

(5) INRA, Castanet-tolosan, France

* Corresponding author: marina.govoroun@inra.fr

Introduction. Cryopreserved primordial germ cells (PGCs) derived in vitro present a tool complementary to frozen sperm for the conservation of avian genetic resources. Recently, we have developed in France reproductive biotechnologies based on PGCs and enriched the national avian cryobank by PGCs collections of a local breed La Poule Noire du Berry (NB), which have benefited from a national program of conservation for endangered breeds. However, little is known on the impact of in vitro steps on molecular integrity and reproductive capacity of PGCs. In this study we investigated the effect of culture duration and cryopreservation on DNA methylation, gene expression and germline transmission rate of NB PGCs.

Materials and methods. Male and female PGCs isolated from the embryos were derived in in vitro culture according to (Whyte et al., 2015). PGCs transcriptome and methylome were evaluated using RNAseq and RRBS analyses respectively after short term and long term cultures and after cryopreservation. To evaluate germ line transmission rate of in vitro derived cryopreserved NB PGCs, germline chimeras were created by transplanting PGCs after thawing and reamplification in vitro in Rhode Island embryos, and then crossing the obtained chimeras with Rhode Island chickens and obtaining their progeny.

Results and discussion. RNAseq study revealed important differences in transcript abundance between male and female PGCs that suggests their early sexual identity. Moreover, RRBS analyses showed that culture duration and cryopreservation significantly affected the epigenetic landscape of PGCs, and this effect was stronger in male PGCs than in those of females. The gender differential effect of culture duration was also observed on the PGCs transcriptome. In contrast, cryopreservation had little effect on gene expression in both sexes. The germline transmission rate for female and male NB PGCs was up to 60.6% and up to 42.4% respectively, and tended to vary according to the culture duration in a gender-specific manner.

Conclusions. This study shows that in vitro environment may affect PGCs at different molecular levels with consequences for their reproductive potential in gender dependent manner. This knowledge will be useful to better adapt in vitro steps to PGCs physiology and improve PGCs quality.

References:

Whyte J, Glover JD, Woodcock M, Brzeszczynska J, Taylor L, Sherman A, Kaiser P, McGrew MJ. Stem Cell Reports. 2015;5(6):1171-1182
ID: 1378

ANALYSIS OF REPRODUCTIVE QUALITY OF DUCK GERMLINE CHIMERAS

M. Doroshenko*(1), S.Kostenko(2), O.Konoval(3), L.Lu(4), A.Chepiha(1), P.Korol(5), X.Bu(4), L.Li(6)(2), P.Philipova(2)

(1)National University of Life and Environmental Sciences of Ukraine, Kiev, Ukraine

(2)National University of Life and Environmental Science of Ukraine, Kiev, Ukraine

(3) Institute of Animal Husbandry and Veterinary Science Zhejiang Academy of Agricultural Science, Kiev, Ukraine

(4) Institute of Animal Husbandry and Veterinary Science Zhejiang Academy of Agricultural Science, Hangzhou, China

(5) Institute of Animal Breeding and Genetics nd. a. M.V. Zubets NAAS of Ukraine, Chubynske, Ukraine

(6)Zhuji Guowei Poltri Development Company, Hangzhou, China

* Corresponding author: mariyamgart@i.ua

Avian species play a vital role in livelihood of human beings throughout the world, especially in developing countries. Duck is an under-researched scientific (breeding) object, but one of the most economically promising species of farm poultry. In Ukraine and in the world there is a problem of reduction and irreversible loss of genetic resources of birds, including local breeds of ducks. During the past several years, the production of transgenic animals has been a prominent theme in animal biology. The study was carried out to estimate the quality characteristics (external and internal) of germline chimeras, which was created by blastodermal cells method, Shan partridge and Shaoxing duck eggs through analyzing different physical properties of eggs. The research was conducted on a duck farm of the company Zhejiang Generation Biological Science and Technology Co., Ltd and at the Zhejiang Academy of Agricultural Sciences (Zhejiang Province, China) bird genetics laboratory. Recipients were sterilized using ultraviolet light irradiation. The isolated blastodermal donor cells were transfected with the DNA-construction (CRISPR/Cas9-mediated gene insertion into the duck genome) with Lipofectamine (liposome) which were inoculated under embryo of the (blastodisc) of the recipient eggs.

After hatching, eleven female and eight male alive birds (3 % of the manipulated embryos) were obtained. We formed 3 groups (n=6), according to the results of their sperm productivity of group of chimeras was higher compared to Shan partridge group (P > 0,01) and group of Shaoxing (P > 0,05) by collection sperm 6 times a week and higher when sperm collection was performed every other day compared to group 1 (P 0,05) in reproductive season. In total, we used 35 drakes to check the quality of the daughters. We conducted a comparative analysis and showed that for all productive traits, the chimera group had average indicators between the Shan partridge group and Shaoxing groups. We checked 10 loci of 5 genes and found that only a few of them are correlated with productive traits such as PRL, GH, DRD1, OIH. We found both positive correlations and negative correlations. It turned out that according to some signs, these mutational events are undesirable, such offspring should be discarded, and on the contrary, on the contrary, populations with such mutations should be maintained that give a good correlation with egg production and body weight. The prospect of further research is to evaluate the breeding qualities of the chimeric bird of the Shaoxing-Shan partridge breeds and to create new high-performance duck lines to produce products with optimal technological properties.
IMPLEMENTATION OF NEW METHODS IN THE HUNGARIAN IN VITRO POULTRY GENE BANK

K. Liptoi*(1), B.Vegi(2), A.Drobnyak(1), E.Kissne varadi(1), K.Buda(1), B.Lazar(1), M.Molnar(1), E.Patakine varkonyi(1), J.Barna(1)

(1) National Centre for Biodiversity and Gene Conservation - Institute for Farm Animal Gene Conservation (NBGK-HGI), Godollo, Hungary

(2) National Centre for Biodiversity and Gene Conservation - Institute for Farm Animal Gene Conservation, Godollo, Hungary

* Corresponding author: liptoi.krisztina@hagk.hu

The part of protection of biodiversity is the preservation of those farm animal species and breeds, which were excluded from public farming due to the use of intensive, high yielding lines and hybrids. The advantage of frozen storage of the reproductive material over in vivo preservation is that it has no animal health risk and does not involve cost of the housing and feeding. While the cryopreservation of gametes and embryos is a routine procedure in many species of mammals, in birds only the sperm freezing is still the most reliable preservation practice. The establishment of in vitro gene bank of the Hungarian poultry was started in 2012 - among the first ones in Europe - by NBGK-HGI. The work started with the frozen storage of spermatozoa according to the FAO protocol which gives recommendations only for domestic fowl, therefore the sperm cryopreservation of other poultry species is still under continuous development in our institute. In avian species, the females are heterogametic (ZW) and the males are homogametic (ZZ); the ovum carrying the W sex chromosome and the embryo per se cannot be frozen. Therefore, there is a need to develop methods, which allow the female genome to be involved in the in vitro preservation. Transplantation of the ovarian tissue of day-old chicks on one hand and the primordial germ cells (PGC) on the other may be the solution. Both materials can be frozen, by which gonadal chimeras are formed and after sexual maturation donor-derived ovum can be obtained. In usage of PGCs the challenge is to establish stable cell lines moreover improve hatchability after injection of donor cells into recipient embryos. According to our previous studies on tissue grafting not all breeds are suitable as recipients, thus one of the most difficult tasks is to form appropriate donor/recipient combinations. Although the optimization of these methods is still ongoing, reconstruction of the donor genotype has been already successful, so storage of PGCs and gonadal tissues from 7 native hens has begun. Currently, there are 1034 PGC samples stored from which 517 are core, while 517 working collections. In case of ovarian tissue of the day old chicks, 239 working collections are placed in the bank. The sperm bank currently has 250-300 straws/breed - altogether 4000 straws as core collection and 200 samples as working collection. This work was supported by Horizon 2020 "IMAGE" n°677353 and GÉNNET_21 - VEKOP-2.3.2-16-2016-00012.
A NEW MODEL FOR PGCS REINTRODUCTION INTO HOST ANIMAL USING INFERTILE INTERSPECIFIC HYBRIDS

M. Molnár*(1), B.Lázár(1), N.Sztán(1), B.Végi(1), Á.Drobnyák(1), K.Liptói(1), E.Gócza(2), S.Nandi(3), M. J. McGrew(3), E. Patakiné Várkonyi(1)

(1) National Centre for Biodiversity and Gene Conservation, Institute for Farm Animal Gene Conservation, Gödöllő, Hungary

(2) NARIC Agricultural Biotechnology Institute, Department of Animal Biotechnology, Applied Embryology and Stem Cell Research Group, Gödöllő, Hungary

(3) University of Edinburgh, The Roslin Institute and Royal Dick School of Veterinary Studies, Midlothian, United Kingdom

* Corresponding author: mariann092@gmail.com

Related to the avian gene preservation activities, the aim of our research was to produce interspecific hybrids, to investigate their ability hosting the primordial germ cells from native poultry breed (donor) and according to our expectations, this donor genotype may appear in the offspring of hybrids.

To achieve our goals, Hungarian guinea fowl x Hungarian yellow chicken hybrids were produced, the crossing was repeated inversely and the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described.

Crossing of Guinea fowl hens with domestic fowl males resulted 6.65% viable offspring but the reverse crossing was unsuccessful due to the high rate of infertile eggs (0.14% viable offspring). During the investigation of chromosomes 2.7% of the samples showed abnormalities (mosaicism) or aneuploidy, but this ratio is average compared to previous literary data. The hybrids hatched between the 21st and 27th day of incubation and raised until maturity. Three kinds of phenotype (light brown, dark brown and white spotted) were observed on the hatched hybrids (6.65%) and none of the hybrids had helmet, crest or facial wattles.

Between the 16th and 30th week of growth the gonads of hybrids weren't symmetrically positioned, their shape were bent and their size didn't increase really with the age. Histological analysis shows that the germinal epithelium is not active, but the normal tubular structure suggests that the gonads of the hybrids may be suitable for hosting of donor PG cells and to produce gametes.

Based on the investigations, the hybrids from the successful crossing were sterile male hybrids. 3 days old hybrid embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labelled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 13.6% of the injected hybrid embryos survived and 85% of the examined gonads contained fluorescent labelled donor PGCs.

This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

The research leading to these results was founded by the European Union's Horizon 2020 Research and Innovation Programme n°677353 IMAGE, by the VEKOP-2.3.2-16-2016-00012 and by the EFOP-3.6.3-VEKOP-16-2017-00008 grant, co-financed by the EU and the European Social Fund.
EFFECTS OF CALCIUM LACTATE ON VIABILITY AND BLOOD COMPONENTS OF CHICK EMBRYOS UNDER
THE SHELL LESS CULTURE SYSTEM.

K. Obara*(1), C.Hemmi-obara(2), M.Naito(3), I.Mitsui(2), Y.Une(4), A.Tajima*(5)

(1)Takanedai Animal Hospital., Funabashi, Japan

(2)Faculty of Veterinary Medicine, Okayama University of Science, Imabari, Japan

(3)National Institute for AgroBiological Science, Tsukuba, Japan

(4)Faculty of Veterinary Medicine, Okayama University of Science, Imabari, Japan

(5)University of Tsukuba, Tsukuba, Japan

* Corresponding author: tajima.atsushi gb@u.tsukuba.ac.jp

The complete shell-less culture system in chicken (cSLC) was developed by Tahara and Obara (2014). During the course of developing cSLC system, however, a supply of calcium lactate together with distilled water into the culture vessel were obligatory for the embryos to hatch. The aim of the present study, therefore, was to clarify effects of calcium lactate on the development of chick embryos under cSLC system.

Fertilized White Leghorn eggs (MB line, NLBS, Okazaki, Japan) were allocated to one of the following four treatments: Treatment 1. cSLC system supplemented with both calcium lactate together with distilled water; Treatment 2. cSLC system without calcium lactate but only with distilled water; Treatment 3. cSLC system without both calcium lactate and distilled water, Treatment 4. Intact fertilized eggs for control. At 17th day of embryo culture, blood was collected from the embryos and plasma lactate dehydrogenase (LDH), calcium (Ca), inorganic phosphorus (iP) were measured. The tibia length was also measured.

The viability of embryo in Treatment 1, 2, 3 and 4 were 24.1%, 75.0%, 100% and 100%, respectively. In Treatment 1, a significant bimodal decreases of the embryo viability were observed between 5th and 8th day and between 15th and 17th day of the culture (P<0.05). Plasma LDH concentration inTreatment 1 was significantly higher than in Treatment 3 (P<0.05). Blood Ca concentrations were significantly higher in Treatment 1 compared with other 3 treatments (P<0.05). On the other hand, nosignificant differences in iP concentration were observed among treatments. The tibia length was significantly shorter under cSLC systems (Treatment 1, 2, and 3) compared with Treatment 4 (P<0.05).In conclusion, even though calcium lactate supplementation is necessary for hatching of chick embryosunder cSLC system, adverse effects on the viability of embryos was observed. Future study should be directed toward decreasing the embryo mortality while supplying Ca source under cSLC system.
# WELFARE

## WELFARE AND BEHAVIOUR

## MANAGING POULTRY WELFARE
WELFARE AND BEHAVIOUR
ID: 432

THE IMPACT OF LIGHT INTENSITY ON KEEL BONE QUALITY AND TIBIA BONE STRENGTH OF EGG-STRAIN PULLETS HOUSED IN PERCHERY SYSTEMS FROM 0 TO 16 WEEKS OF AGE.

J.A. Chew(1), T. Shynkaruk(1), E.Herwig(2), T. Widowski(2), K.Schwean-Lardner(1)

(1)University of Saskatchewan, Saskatoon, Canada

(2) University of Guelph, Guelph, Canada

* Corresponding author: joann.chew@usask.ca

Light intensity can impact bird activity, which can affect musculoskeletal development. The objective of this study was to determine the effect of varying light intensity (L) on keel bone health and tibia bone strength of Lohmann Brown-Lite (Brown) and Lohmann LSL-Lite (White) pullets. A 3 x 2 factorial arrangement of L and strain (S) was performed in a randomized complete block design. Three L intensities (10, 30, or 50 lux, provided by white LED lights) and two S were used. Brown and White pullets (n=1,800 per S) were randomly assigned to floor pens (50 pullets per pen; 4 replications per L x S) from 0 to 16 weeks of age. Each floor pen contained a system of four parallel perches (0.3 m apart, 0.6 m high), ramp, two tube feeders, and a drinker line. All pullets were fed ad libitum on a commercial phase feeding program. Pullet body weight was collected on a pen basis at 0, 8, and 16 wk. At 16 wk, 10 pullets per pen (n=720) were palpated for keel bone deviations and fractures. An additional 9 pullets per pen (n=648) were euthanized and the keel bones and breast muscle were removed to assess keel bone damage and breast muscle weight. Right tibiae were collected, and bone strength was assessed using a 3-point-bending test. Mortality was recorded daily. The effect of L, S, and their interactions were analyzed using Proc Mixed (SAS 9.4) with Tukey's range test to separate means. Differences were significant when P<0.05. There was no effect of L, and no interactions between L x S, for any of the measured parameters. Brown pullets were heavier than White pullets at 8 and 16 wk. There was no effect of S on keel bone damage from palpated and dissected keels. No fractures were found from dissected keel bones. Brown pullets had a higher breast muscle weight than White pullets (86.3 vs 77.0 g; P<0.05), however relative to body weight, White pullets had a higher breast muscle mass (6.59 vs 5.97%, P<0.05). Brown pullets' tibiae were 1.31% heavier, 1.02% longer, and 1.18% wider than White pullets (P<0.05), however White pullets had a higher bone strength relative to bone size than Brown pullets (1,816 vs 1,277 kg/cm2; P<0.05). Mortality was higher in White pullets compared to Brown pullets (4.00 vs 1.22%; P<0.05). Overall, the results indicate that L, between a range of 10-50 lux, does not affect pullet keel bone health or musculoskeletal development, however S may play a role in these parameters.
CARCASS WEIGHT UNIFORMITY IN BROILER FLOCKS IS ASSOCIATED WITH PRODUCTION MEASURES ON
FARM AND AT SLAUGHTER

G. Vasdal\*(1), E.Granquist(2), E.Skjerve(2), I.Dejong(3), C.Berg(4), V.Michel(5), R.Moe(2)

(1)Animalia AS - Norwegian Meat and Poultry Research Centre, Oslo, Norway

(2) NMBU, Oslo, Norway

(3) Wageningen, Wagneingen, Netherlands

(4)SLU, Skara, Sweden

(5)Anses, Niort cédex, France

* Corresponding author: guro.vasdal@animalia.no

The flock weight uniformity in a poultry flock can be defined as the percent individuals within 10 % of the mean body weight (BW) and the variability of this uniformity can be expressed as the CV(coefficient of variance) of BW. Flock weight uniformity is a standardized and objective measure, and could potentially be used as a welfare indicator. Poor flock uniformity (i.e. a high CV) may indicate reduced animal welfare, due to either general housing or management problems, or bird health problems. However, little is known about the relationship between flock uniformity and other production measures on-farm or at slaughter. Therefore, the aim of this study was to investigate associations between carcass weight uniformity (CV of BW) and production measures on-farm and at slaughter in Norwegian commercial broiler flocks. A total of 45 randomly selected mixed-sex Ross 308broiler flocks were visited prior to slaughter at 28 – 30 days of age (average slaughter age 30.6 days). All flocks were raised under similar farm management systems. The Welfare Quality® protocol for broilers was used to assess different animal welfare indicators in each flock. For each flock, productiondata from the slaughterhouse were collected, including carcass weight uniformity (%), mortality (%), growth rate (g), feed conversion ratio (FCR), and rejected birds (%) in different rejection categories. Flock uniformity varied from 11 % to 18 % between flocks within the same hybrid, similar managementstandards and similar slaughter age (day 29 – 32). Poorer uniformity (i.e. higher CV) was associated with increased first week mortality (P < 0.004, r = 1.48), increased total mortality (P < 0.013, r = 0.01),increased FCR (i.e. less efficient growth) (P < 0.024, r = 0.06), reduced growth rate (P < 0.0012, r = - 0.01), and a reduced rejection rate at slaughter (P < 0.006, r = -0.01). The results show that flock uniformity varies across broiler flocks, and is associated with several production measures.
PILING BEHAVIOUR AND STIMULUS ATTRACTION IN LAYING HENS

J. Winter\*(1), M. Toscano(2), A. Stratmann(2)

(1) Centre for Proper Housing: Poultry and Rabbits, Animal Welfare Division, Veterinary Public Health Institute, Vetsuisse-Faculty, University of Bern, Switzerland, Zollikofen, Switzerland

(2) Centre for Proper Housing: Poultry and Rabbits, Animal Welfare Division, Veteterinary Public Health Institute, Vetsuisse-Faculty, University of Bern, Zollikofen, Switzerland

* Corresponding author: Jakob.Winter@vetsuisse.unibe.ch

Piling behaviour (PB), the tight agglomeration of several laying hens, often leads to suffocation and is therefore considered a major welfare problem in the loosed-house egg industry. Results of an observational study showed that PB is mostly triggered by specific stimuli (e.g. light spots and novel objects) and occurred when hens were attracted towards these stimuli (Winter et al. 2019). Since these observations lack experimental validity, we experimentally tested two hypotheses: one, that specific stimuli attract laying hens and, two that this attraction leads to PB. We conducted this study within 8 identical experimental pens, each holding 55 Lohmann Selected Leghorn hens between 21 - 27 weeks of age. Each pen consisted of two locations where stimuli were applied with opposite locations serving as controls, i.e., four locations/pen. We observed the animal responses towards three stimuli: a light spot (flashlight, presented for 10min), a glittering tape (for 35min), and increased local temperature (heating plate, mean: T ~44°C, ~20-24°C above environmental temperature, for 35 min) at the stimulus locations. Each pen received each stimulus once as well as a control with no presented stimulus stratified over a 7-week period. To test hypothesis one, we compared the number of hens at the stimulus and the control location at specific times where stimuli were presented: light spots: at 0, 5, 10min, glittering tape and increased local temperature at 0, 5, 10, 17.5, 25, 30, 35min. We assessed the influence of the interaction between stimuli and time on the number of attracted laying hens by applying generalised linear mixed-effects models. To test hypothesis two, we compared the number of PB events at the stimulus and the control locations within each pen. Our results showed that only the light spot affected the number of attracted hens (p< 0.0017) with more hens being present in the stimulus location when this stimulus was presented compared to the other stimuli. In addition, the attraction to light spots occasionally led to PB (3 PB at the stimulus vs. 0 PB at the control side, n=16). Our findings suggest that specific stimuli attract laying hens, but that stimulus attraction does not always lead to PB. Further studies should investigate which other factors (e.g. social mechanisms) lead to PB.

Winter J, Stratmann A, Toscano M. 2019. Piling Behaviour of Laying Hens in Switzerland: Origin and Contributing Factors. 53. ISAE Congress Proceedings, Bergen, Norway
UNDERSTANDING BEAK BLUNTING AND ITS POTENTIAL TO BE USED AS AN ALTERNATIVE TO CURRENT BEAK TREATMENT METHODS

S. Struthers*(1), T.Shynkaruk(2), K.Beaulac(2), S.Gomis(3), E.Herwig(2), K.Schwean-lardner(2)

(1)University of Edinburgh, Easter bush, United Kingdom

(2) University of Saskatchewan, Saskatoon, Canada

(3) Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada

* Corresponding author: sarah.struthers@ed.ac.uk

There is increasing pressure to ban the practice of beak treatment in the commercial egg industry and with this comes the need to find viable alternatives. Natural beak blunting by the inclusion of an abrasive material in the feeder has been suggested as an alternative but its effectiveness or impacts on pullets are not yet fully understood. The objective of this study was to examine the effects of beak blunting on the beak characteristics, production, and welfare of Lohmann Brown-Lite (LB) and Lohmann LSL-Lite (LW) pullets. Newly hatched chicks (n=900 per strain) were sorted into 3 beak treatments: sham untreated control (C), infrared beak treated (IR), or provided with a specialized beak blunting feeder (B), which had an abrasive inner feed pan. Pullets were housed in 18 floor pens (n=100 per pen; 900 cm2 per pullet) from 0 to 16 wk of age. Feed and water were provided ad libitum. Body weight, beak length, beak histology, and heterophil/lymphocyte (H/L) ratios were measured at 0, 2, 4, 10, and 16 wk. Mortality was recorded throughout the 16 wk. The study, a 3x2 factorial arrangement of beak treatment and strain, was performed in a completely randomized design. Data were analysed using PROC MIXED (SAS 9.4) with Tukey's test to separate means. Differences were significant when P≤0.05. Body weight did not differ between treatments except at 4 wk when C pullets were heavier than B pullets (290.8 vs. 284.3 g, respectively). Over the 16 wk, IR pullets had shorter top and bottom beaks compared to C and B pullets. No differences in beak length were found between C and B pullets. At all ages, B beaks showed no histological differences from C beaks. At 4 wk, all IR beaks except 1 had healed. H/L ratios did not differ between treatments at any age. Mortality was highest in the C pullets compared to B pullets over the 16 wk (7.7 vs. 3.6%, respectively), although the primary cause of mortality was yolk sac infection and unrelated to beak treatment. LB pullets were lighter than LW from 0 to 4 wk but heavier at 10 and 16 wk. LB pullets also had higher H/L ratios compared to LW at 2, 4, and 10 wk. Mortality was higher in the LW compared to LB (8.0 vs. 3.0%, respectively). Overall, the results suggest that while the specialized feeder did not negatively impact pullet body weight or welfare, it was not effective at blunting the beak, which poses welfare risks for outbreaks of severe feather pecking as the pullets age and enter lay.
TOE PECKING – AN EMERGING BEHAVIOR PROBLEM IN SWISS LAYING HENS

S. Gebhardt-henrich*(1), L.Zanini(2), S.Mueller(3), M.Toscano(1)

(1)ZTHZ, Division of Animal Welfare, University of Bern, Zollikofen, Switzerland

(2)ETH Zürich, Zollikofen, Switzerland

(3)Aviforum, Zollikofen, Switzerland

* Corresponding author: sabine.gebhardt@vetsuisse.unibe.ch

During the past 3 years, an increasing number of Swiss egg producers have reported mortality due to toe pecking. Studies on toe pecking in laying hens are rare. Therefore, we conducted a survey among Swiss egg producers, examined histological changes in affected toes, and scored toe damage after an experimentally altered feeding schedule. An online questionnaire was sent out to 490 Swiss egg producers. We included 96 complete questionnaires from 48 farms with and 48 farms without toe pecking. Results were analyzed using Fisher's Exact Tests and generalized linear models in univariate and multivariate analyses. Tissues of affected and intact toes of six laying hens were stained with HE and PAS for histology. Additionally, in an experimental barn, 4 of 8 pens missed the last feeding of the day. Toes were scored before and after 7 weeks of this change of feeding schedule at 39 weeks of age. Toe pecking at the own and other birds' toes and pecks to the comb were scored from videos. Data were analyzed with generalized mixed models. Pen was taken as a random variable and for the number of pecking events a Poisson distribution was chosen. Toe pecking was almost only reported in white hybrids (Fisher's Exact Test P = 0.01, N = 79). Infections with E. coli during the last 5 years (F1,50 = 5.69, P < 0.02), metal instead of plastic slats (F1,50 = 5.61, P < 0.02), and direct sunlight in the barn (F1,50 = 7.52, P < 0.008) increased the probability for toe pecking (N = 54). The presence of high frequency lights also increased the risk of toe pecking (χ21 = 4.73, P = 0.03, N = 45). Tissues affected by toe pecking showed ulcerating dermatitis in the deeper skin layers and the adjacent bone tissue. Treatment affected the frequency of toe pecking directed at other birds and was less frequent in the pens that missed the last feeding (treatment: F1,14 = 7.58, P = 0.02). The severity of toe lesions was positively associated with the frequency of toe pecking (F1,14 = 5.17, P = 0.04) and pecking at the comb (treatment: F1,14 = 10.59, P = 0.006; lesions: F1,14 = 12.27, P = 0.004; interaction: F1,14 = 4.09, P = 0.06). The severity of lesions of the toes and comb were correlated within hen (rs = 0.54, P = 0.007, N = 23). Although this study suggested that toe pecking, like feather pecking, was a multifactorial problem, genetics seemed to be a major factor as only white hybrids were affected. The histological findings indicate that toe pecking is of high relevance to bird welfare.
A PRACTICAL TOOL TO IMPROVE BROILER WELFARE AND TO DISCUSS ANIMAL WELFARE

L. Warin(1), P.Creach(2), A.Cotten(3), P.Galliot(2), C.Souchet(1), L.Vinco(4), I.Bouvarel*(1)

(1) ITAVI, Nouzilly, France

(2) ITAVI, Ploufragan, France

(3) Le Gouessant, Lamballe, France

(4) IZSLER, Brescia, Italy

* Corresponding author: warin@itavi.asso.fr

Animal welfare is a shared responsibility of all the actors involved in raising animals and farmers have to meet the European citizens' demand for high broiler welfare levels. The observation of footpad dermatitis (FPD) is a common measure used in Europe to assess broiler welfare. It provides information on the welfare status of broilers but it does not cover all dimensions of animal welfare. The self-welfare assessment method EBENE® was developed in France to provide a complete overview on the welfare status of broilers through 12 scores. Mainly animal-based indicators (behaviour and health) are observed with no need to handle animals and a mobile app is available. The aim of this article is to present farmer's perception of welfare and on the EBENE® method.

The welfare perception of broiler farmers in Italy (n=15) and in France (n=11) was assessed between April and November, 2019. Open questions, multiple-choice questions and Likert-scale questions from 0 to 5 were asked. French farmers also gave their opinion on the EBENE® app.

Among 6 proposals, to improve animal welfare is the 3rd priority for farmers. To assess animal welfare (open question), only half of them rely on animal-based indicators (eg behaviours) while the other half use only resource-based indicators (eg litter quality). When asked to choose among several proposals on how they assess animal welfare, almost 60% of the farmers answered behaviours. Not all the farmers consider behaviours as part of animal welfare assessment. Half of them declare that it is not easy to observe behaviours according to the EBENE® method because they are not used to it, which may explain why only few farmers observe behaviours. However, the behavioural observation is interesting for all of them, probably because it is different from their usual observations and that it gives them positive indicators to communicate on. Farmers are more used to negative animal-based indicators to assess welfare such as lameness, injuries or FPD, probably because these measures are easier to observe. Indeed, for around 80% of the farmers questioned, health indicators are easy to observe according to the EBENE® method.

There is a need for practical tools to help farmers talk about animal welfare. Welfare assessment tools such as EBENE® might help farmers to assess all the dimensions of animal welfare, and to be able to talk about positive animal-based indicators. It is also a practical tool to improve broiler welfare.
DISTURBANCE OF RESTING BEHAVIOUR OF BROILERS UNDER DIFFERENT ENVIRONMENTAL CONDITIONS

S. Forslind\*(1), H.Blokhuis(2), A.Brinch riber(3)

(1) Department of Animal Environment and Health, Swedish University of Agricultural Sciences, Uppsala, Sweden

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

(3) Department of Animal Science, Aarhus University, Tjele, Denmark

* Corresponding author: sara.forslind@slu.se

Sleep is essential for several important functions in the body and sleep deprivation can lead to health problems and stress. In broiler production, the chickens are generally kept in large flocks in a barren environment. In this production situation, chickens are exposed to physical disturbances by other individuals, forcing individuals to become active when they are highly motivated to rest.

Therefore, this study investigated separate resting places in the form of elevated platforms. By providing such platforms we hypothesized that the broilers would get more undisturbed rest compared to groups without elevated platforms. Three treatments were applied; treatment EP providing elevated platforms 30 cm above the litter (stocking density at 40 kg/m2), treatment SD with a lower stocking density at 34 kg/m2 and a control group C (no platforms and a stocking density at 40kg/m2). Behaviour of the chickens was video recorded in the pen, during both night and day, and recordings at days 20 and 34 of age were used for further analysis. Analysis was made in R (version 3.6.1) using an ANOVA test to compare the duration of resting bouts between treatment groups and post hoc comparisons of significant factors were made using Tukey's HSD test. Results presented as mean ± SE.

The results show that a separate resting place increased the duration of resting bouts and reduced the number of physical disturbances between chicks. A treatment effect of the duration of resting bouts (df=2, F=8.499, p

Our results show that elevated platforms provide an area where broiler chickens can rest with longer resting bouts and with less risk of being physically disturbed by other individuals. Also, reducing the stocking density increases the duration of resting bouts. In addition, the study shows that even with elevated platforms, disturbance of sleep occurs frequently.
ID: 393

ANIMAL WELFARE INDICATORS IN BROILER FARMS ARE NOT IMPAIRED BY COCCIDIOSIS VACCINATION
COMPARED TO COCCIDIOSTATS - FIELD STUDY.

N. Cariou*(1), S.Giraud(2), F.Quéré(3), G.Le bras(4), F.Mayette(3)

(1)MSD Santé Animale, Beaucouzé, France

(2)Nutrea, Carhaix-plouguer, France

(3)Nutrea, Languidic, France

(4)Nutrea, Carhaix plouguer, France

* Corresponding author: nadine.cariou@msd.com

Coccidia vaccination is often associated with the reduction of antibiotic use in broiler production.
Societal and regulatory expectations also include animal welfare compliance. Tarsal and breast burns,
scratches and infected wounds, and rejects are indicators measured at the slaughterhouse. Mortality
is the fourth criterion studied.

From March 2018 to May 2019 all chickens fed by one feed plant (plant B) were vaccinated at the hatchery with Paracox®-5 (approximately 16 million chickens). Results are compared with chicken batches fed a coccidiostat feed supplied by the same plant B over the previous period (February 2017 to March 2018) and with chicken batches fed a coccidiostat feed manufactured by another plant (plant A) from February 2017 to March 2019.

In order to take grower effect into account, only farms for which results from more than 6 batches
were available were retained for the statistical analysis with the software R. The analysis of 379 batches
from 32 farms is presented here.

For the mortality criterion, there was a (non-significant) trend towards a slight increase in total mortality (+0.28%) and mortality at 10 days (+0.18%) for the vaccinated batches. Since the use of TMP sulfa on vaccinated flocks is limited due to its negative effect on vaccinal coccidia recycling, it may have resulted in a few colibacillosis outbreaks with higher mortality.

It is interesting to note that while the rate of tarsal and breast burns evolves seasonally, with an increase on winter flocks, vaccination has no negative or positive impact on this criterion.

Similarly, the switch to vaccination has no impact on the rate of infected scratches and wounds.

Finally, the rate of rejects evolves in the same way over time for batches of broilers fed by plant A or plant B, before or after the introduction of vaccination.

This study therefore shows that under real field conditions, Paracox®-5 vaccination of broilers is compatible not only with the antibiotic reduction approach, but also with animal welfare regulations, the indicators on vaccinated batches being at the same level as on batches with coccidiostat.
INFLUENCE OF CATCHING METHOD ON ANIMAL HEALTH AND BEHAVIOUR OF BROILERS AT MANUAL LOADING

H. Louton*(1), E.Rauch(1), S.Klein(1), J.Frisch(3), J.Bachmeier(2), P.Schmidt(4), M.Erhard(1)

(1)LMU Munich, Munich, Germany

(2)Veterinary Practice, Straubing, Germany

(3)LMU Munich, 80539, Germany

(4)Statistical Consulting for Science and Research, Berlin, Germany

* Corresponding author: helen.louton@uni-rostock.de

Two manual loading techniques (1LCM = one leg catching method; 2LCM = two leg catching method) of broilers were compared concerning animal health and animal behavior. For this purpose broilers of one container of each method were evaluated within 12 loadings. Assessments of animal health prior and after loading and detailed analyses of the behavior were performed. A logistic regression model was used for statistical analysis.

By the use of the 2LCM (8048 broilers/hr) the time of loading was doubled compared to the 1LCM (3408 broilers/hr) and deviations from the target stocking density were observed with a higher variance. The risk for wing fractures was raised by the use of the 1LCM (1.12% in 1LCM, 0.42% in 2LCM). An increase of the catching duration and the height of the drawer in the container influenced the risk for wing fractures. Animals of low drawers were at higher risk for fractures, than broilers of middle or high drawers. Wing flapping was observed in 74.5% of the broilers of the 2LCM and in 83.6% of the 1LCM. A lower risk of wing flapping was achieved if the broilers were carried with a neighboring animal or grasped proximal to the body in the area of the tarsal joint. Weather the broilers are settled, dropped or thrown in the drawer significantly influences the behavior of the broilers in the drawer. The catching duration, the height of the drawer and the weight of the broilers influenced the method of settlement.

The design of the study needs to be considered as a limitation and accounted for when interpreting the results. The assessment of the broilers during loading was assessed on the basis of one container of each method within 12 loadings. In a previously performed study, we compared manual (2LCM) with mechanical loading of entire barns and observed wing fractures in 1.02% of the broilers of the 2LCM. Thus, it seems unlikely that the advantage of the 1LCM method observed in this study concerning wing fractures could be transferred to the loading of entire barns. An exhaustion of the loading staff when loading an entire barn with the 2LCM is expected and would lead to a higher risk for lack of concentration and careless or hectic performance. A deliberate and respectful handling of the broilers is essential for an animal friendly loading. By the consideration and implementation of the stated risk factors the risk for wing flapping and wing fractures can be lowered, irrespective of the number of grasped legs.
ID: 484

GENERAL PERFORMANCE AND EGG QUALITY OF LAYING HENS UNDER DIFFERENT REARING SYSTEMS

M. Adegbenro\*(1), D.Okedere(1), G.Onibi(1)

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

* Corresponding author: madegbenro@futa.edu.ng

Considering the issue of animal welfare which European Union (EU) and other nations are clamouring for, there is a need to consider such in sub-Saharan Africa. This study seeks to evaluate the general performance, production performance and egg quality of Isa-Brown laying hens reared under different systems. Ninety (90) Isa-Brown hens were used for this study and the study lasted for eighty-four days (84). The hens were randomly distributed to three (3) treatments (battery cage, deep litter and outdoor systems) of three (3) replicates and ten (10) hens per replicate in a Completely Randomized Design. Each hen was given 115g of feed per day, while fresh clean water was provided throughout the experimental period. Results show that highest final weight (1586.7g), highest percentage hen-day production (74%), highest egg weight (63.2g) and best feed conversion ratio (2.45) were observed in hens reared on battery cage system, while lowest percentage hen-day production (68.3%), lowest egg weight (56.7g) and worst feed conversion ratio (2.70) were observed in hens reared on the outdoor system. Meanwhile, highest total feed intake (9660g) was observed in hen reared on battery cage and deep litter systems, while the lowest total feed intake (8710g) was observed in hen reared on the outdoor system. Highest dressed weight (1446.6±112.6g) was observed in bird reared under the deep litter system, while lowest dressed weight (1383.3±63.6g) was observed in bird reared under the outdoor system. Highest gizzard weight (29.7±1.68g/kg body weight) was recorded in bird reared under the conventional battery cage system, while lowest gizzard weight (26.9±0.46g/kg body weight) was recorded in bird reared under the deep litter system. Hen on battery cage had the highest gross returns on the egg (N1555), while hen on the outdoor system had the lowest gross returns on the egg (N1475). In all, the highest profit (N309) was recorded in hen on the outdoor system, while the lowest profit (N284) was recorded in hen on deep litter system. In conclusion, outdoor could be considered as an alternative to battery cage system for hens considering the highest profit recorded and going by the European (EU) campaign on animal welfare.
ID: 494

LIVE INSECT LARVAE FOR LAYING HENS: EFFECTS ON PLUMAGE STATUS AND ANIMAL BEHAVIOR

S. Bellezza oddon*(1), I.Biasato(2), A.Imarisio(3), M.Pipan(4), D.Dekleva(4), S.Dabbou(3), M.Renna(3), G.Chemello(2), M. Gariglio(3), C.Caimi(2), L.Gasco(2), A.Schiavone(3)

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

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

(3)Department of Veterinary Science, Turin, Italy

(4)Entomics Biosystems, Cambridge, United Kingdom

* Corresponding author: sara.bellezzaoddon@unito.it

Introduction. Feather pecking and cannibalism are two phenomena widespread in intensive farming of laying hens that have negative impact on egg production. The aim of the study is to evaluate the effects of black soldier fly (Hermetia illucens) live larvae as environmental enrichment for laying hens on plumage status and animal behavior.

Material and methods. A total of 169 adult laying hens and 24 males of Bionda Piemontese (BP) and Bianca di Saluzzo (BS) breeds (308 days of age) were allotted to 12 pens. Each pen was assigned to one of two dietary treatments (6 replicates/treatment, from 12 to 19 birds/replicate) as follows: i) control diet (C): commercial feed and ii) HI: C + H. illucens live larvae. The commercial diet and water were distributed ad libitum and the amount of live larvae was distributed based on 6% of the expected daily feed intake (120 g/bird). To evaluate the plumage status, all the birds were given feathering scores for neck, breast, cloaca/vent, back, wings, tail and bumble foot lesions. The behavioral observations were carried out using video recordings. Each pen was filmed for 5 minutes before larvae administration in 4 different periods. The analysed behaviors were divided into 2 different categories: duration (walking, ground pecking, standing still, resting) and frequencies (scratching, grooming, allogrooming, trotting, severe feather pecking, stretching, chasing, sand bath, feather shake, wing flapping). Data were analysed by General Linear Mixed Model (IBM SPSS Statistics V20.0.0, P value < 0.05).

Results. No significant differences were observed for both the mean and the total feathering scores between the C and the HI hens independently of the breed (P<0.05). On the contrary, the HI BP animals showed higher (better) mean and total feathering scores when compared to the C group (P<0.05). The HI birds spent more time walking than the C group (P<0.05). Differently, the other duration behaviors were not affected by the treatment (P<0.05). Considering the frequencies, the HI hens displayed more grooming, allogrooming and trotting than the C birds. On the contrary, the C group performed more severe feather pecking than the HI hens.

Conclusions. The use of live larvae as environmental enrichment positively affects the plumage status in the BP breed. Furthermore, the addition of live larvae to the commercial diet leads to a decrease in severe feather pecking and an increase in some comfort behaviors.
ID: 498

IMPACT OF PHOTOPERIOD ON THE WELFARE OF BROILERS RAISED WITHOUT ANTIBIOTICS

T. Shynkaruk\*(1), K.Beaulac(2), 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

The North American broiler industry is facing increasing pressure to eliminate use of antibiotics which may impact bird welfare. Therefore, it is important to assess how management factors can ensure optimal welfare in flocks raised without antibiotics (RWA). The objective of this study was to investigate the impact of photoperiod on stress, gait and footpad scores, and behaviour of RWA broilers reared to 36d. Two repeated trials were conducted with a total of 8,064 mixed run Ross 308 broilers over both trials. Birds were housed in 8 environmentally controlled rooms (12 pens of 42 broilers per room). Lighting treatments were initiated at 6d and included 14L:10D, 17L:7D, 20L:4D, and23L:1D. Light was provided by white LEDs with an intensity of 10 lux (d6-36). At 25d, blood was collected from the brachial vein of 96 birds per treatment to assess H/L ratios. At 29d, gait (trial 2 only) and footpad scores were assessed on 40 and 80 birds per treatment, respectively. At 30d video cameras recorded behaviour in 4 pens per treatment for 24h; data were analyzed with scan samples every 15 minutes. Data were analyzed with regression analyses using Proc Reg and RSReg of SAS 9.4. Prior to analyses behaviour data were log +1 transformed. Significance was declared when P≤0.05 and a trend noted when P≤0.10. A quadratic response was observed for H/L ratio (P<0.01), with highest values, indicating increased stress, found in birds reared on 23L. Mobility, assessed via gait score, showed a linear tendency for poorer mobility under longer photoperiods (P<0.08). A linear increase in more severe footpad scores with increasing photoperiod (P<0.01) was observed. A linear increase in time performing nutritive behaviours was observed under longer photoperiods (P<0.01). Time spent standing responded quadratically (P<0.01) with the 14 and 23L treatments being highest. Preening responded in a linear fashion (P<0.01) with birds reared on 17 and 20L spending numerically more time preening. Birds raised on 20L spent more time environmental pecking (quadratic response; P=0.01). Object pecking occurred more under 14L (linear response; P=0.01). These results indicate that rearing RWA broilers on longer photoperiods has negative effects on bird welfare, as indicated by poorer mobility, chronic stress, more severe footpad lesions, and changes in behaviour.
# THE EFFECT OF A BACTERIAL LITTER PRODUCT APPLICATION ON LITTER QUALITY AND GROWTH PERFORMANCES OF BROILERS

B. Frayssinet(1), J.Plateau(1), A.Sacy(1), V.Demey*(1)

(1)Lallemand SAS, Blagnac, France

* Corresponding author: vdemey@lallemand.com

The objective of this trial was to examine the effect of the application of a dry litter product (formula with a complex of microorganisms and enzymes) on the physicochemical characteristics of the litter as well as the performances of broilers. For this 194 male broilers (Ross 308) were equally divided in 2 groups of 8 pens. Bedding material was sawdust. The litter of the treatment pens (T) was seeded with the dry litter product at the rate of 30g/m² (2 applications) at trial set up (below and above the sawdust). For the remainder of the trial application of 30g/m² was repeated weekly. The control pens (C) did not receive any litter treatment. Litter quality (humidity, temperature, litter scoring and pH) was monitored regularly from start (day 0) until end of the trial (day 35). Individual body weight (BW) was registered at day 0 and day 35. Footpad dermatitis (FPD) has been assessed at day 35. Litter crust height was measured as a visible indicator of litter quality at the end of the trial. Data were analyzed according to General Linear Model with Treatment as fixed effect (SPSS v24). Results indicate a significant difference between the control and the treatment group for humidity (C=64.6% vs T=56.8%; P<0.05), temperature (C=26.4°C vs 23.4°C; P<0.05) and litter score (C= 4.7 vs T=3.4; P<0.05) indicating an improved litter quality for the treatment group. Moreover, at the end of the trial, a strong tendency of improved growth was observed for the treatment group compared to the control (BW at day 35: C: 2432.3g vs. T: 2348.6g, P=0.06). There have been a significant reduction of number of broilers suffering from FPD (C=83% vs T=31%; P<0.05) and in the severity of these FPD (Average FPD-score: C=1.41 vs T=0.54; P<0.01). A lower height of the litter crust was also recorded (C=5.7 cm vs T=4.2 cm), indicating this could be a good visible sign of litter quality. These results indicate that the application of a bacterial dry litter product can improve litter quality and therefore help improve the environment of livestock barns. A better litter quality also reduces the occurrence of FPD, which could positively impact animal welfare.
OPTIMIZING SPACE AND ITS IMPACT ON WELFARE BEHAVIOUR AND PRODUCTION OF COMMERCIAL LAYING HENS IN CONVENTIONAL CALIFORNIA CAGE SYSTEM

P. Roy*(1), M.Kadam(2), S.Bhanja(3), J.Rokade(4), D.Bhaisare(2), S.Pawar(2)

(1) MAHARASHTRA ANIMAL AND FISHERY SCIENCES UNIVERSITY, Nagpur, India

(2) MAFSU, Nagpur, India

(3) CARI, IVRI, Izzatnagar, bareily, India

(4) CARI, IVRI, Nagpur, India

* Corresponding author: ingewarpin@gmail.com

In Asian countries commercial laying hens are mostly kept in confined conventional cages with automatic feeding, controlled lighting and ventilation to optimize production performance. Recently, in India cage rearing system was argued by animal welfare activists complaining that the welfare of the laying hens is not maintained ensuring reasonable space for standing up, lying down, flapping wings, turning around and access to feed and water. Scanty information are available in context to Asian condition to understand the welfare, behavior and stress-free production performance in conventional California cage rearing system. A 20-weeks experiment was conducted in 28 weeks old commercial white leghorn layers to investigate the required conventional cage floor space allowance per bird to optimize the welfare, behaviour and egg production. The four different experimental groups were assigned with different space allowance of 387, 484, 548 and 645cm2 per bird with 18 replicates in each. Laying birds were fed commercial diet (110 g/bird) and maintained under standard management practice. Data were analyzed using SPSS software package .Higher hen day egg production (89.71 and 90.92 %, P<0.001), best feed conversion ratio per dozen eggs (1.47 and 1.46, P <0.001) and best welfare parameter (better feather score, P<0.05) were recorded in the birds reared at 548 and 645 cm2 per bird (which were non-significant among themselves), respectively, compared to the 387 and 484 cm2 space provided birds. For H/L ratio and serum corticosterone measured at the start and end of the experimental period no significant difference was detected between treatments. Behaviors of the laying birds were observed through installed video recording cameras and different activities were calculated by distributing the state and event activities of 24 hour period into time frame of one minute. Significant different behavioral activities were observed for standing, sitting, walking, investigating, drinking and feather pecking among the different treatment groups (P<0.05). The birds reared at 548 cm2 per bird had highest activity of sleeping, feather-pecking (P<0.001), walking, sitting (P<0.05) as compared to others space allocated birds, indicating a more comfort zone. Hence it could be summarized that cage floor allowance of 548 cm2 per bird provides optimum space without compromising welfare behaviour and production of commercial laying hens in conventional California cage system.
EFFECT OF INCLUSION OF SUN-DRIED MANGO KERNEL MEAL ON HEMATOLOGICAL PARAMETERS OF JAPANESE QUAILS

J. Dzungwe*(1), A. Favour(2)

(1)Regional Center of Excellence for Avian Sciences, University of Lome, Togo, Lome, Togo

(2)University of Agriculture, Makurdi, Nigeria, Makurdi, Nigeria

* Corresponding author: torsagain@gmail.com

The Increasing cost of conventional feed ingredients has compelled most poultry farmers to compound their feed. Alternate use of non-conventional feed stuff can reduce production cost. This study was to determine the effect of inclusion of sun-dried mango kernel meal (SMKM) on hematological parameters of quails.

The study was conducted at the University of Agriculture, Makurdi, Nigeria. Experimental design used was complete randomized design. 144 unsexed Japanese quails of 2 weeks old were purchased from Veterinary research center, Vom, Nigeria. The Quails were fed the control diet (Maize= 55.2%, SMKM 0%, Full-fat soybean=26.67%, fish meal=5.2%, wheat offal=6.93%, bone ash=7%, salt=0.5%, vitamins/ mineral premix= 0.5%) for one week prior to the commencement of the experiment. They were randomly allotted to three dietary treatments where SMKM replaced maize at 0%, 25% and 50%. Each diet was replicated thrice with 16 quails per replicate. 3 wooden cages with wire mash floors, roofings and side covers were used for the experiment. Each cage had 3 layers to represent the 3 treatment replicates. Feed and water were given adlibitum for 28 days and no vaccines administered. Cocidiostat and antibiotic were administered at 6 weeks of age to control coccidiosis and bacterial infections. At the end of the trials, blood samples from 10 quails were collected from each replicate by cutting through the jugular vein using a sharp knife into bottles containing ethylene diamine tetracetic acid as anti-coagulant for hematological analysis. The hematological values were compared with standard reference values.

Results showed that, White blood cells (11.0-13.1 x 109/L) and Lymphocytes (63-74%) values increased above normal reference ranges across treatments. The values for Red blood cells (4.2-4.7 x 1012/L), hemoglobin (10.3-11.3 g/dL), packed cell volume (31-34%), Mean Corpuscular Volume (78-79 fL), Mean Corpuscular Hemoglobin (22-26 pg), Mean corpuscular Hemoglobin Concentration (33.1-33.2g/dL), basophil (0) and eosinophil (0) values were within the normal reference ranges. Monocytes (0-1%) and Neutrophils (25-37%) values were slightly below the reference ranges across treatments. Abnormalities observed in the hematological parameters could have been influenced by the antibiotics given to the quails. Since the abnormalities were not only limited to quails fed SMKM, it confirms that SMKM was not responsible for the abnormalities. Therefore, SMKM can replace maize for up to 50% in quail diets without deleterious effects on hematological parameters.
AN ADAPTABILITY INDEX FOR POULTRY GENOTYPES ORGANICALLY REARED

A. Cartoni mancinelli*(1), S.Mattioli(1), L.Menchetti(1), A.Dal bosco(1), M.Guarino amato(2), C.Castellini(1)

(1) Department of Agricultural, Environmental and Food Science University of Perugia, Italy, Perugia, Italy

(2) 3Council for Agricultural Research and Economics - Livestock Production and Aquaculture, Via Salaria, 31,00015 Roma, Italy, Roma, Italy

* Corresponding author: acartonimancinelli@gmail.com

In recent years, the interest towards friendly-farm systems increased. Especially in poultry, alternative rearing systems (free-range, organic, low input) spread. The EU reg. 889/2008, establishes that: "...the choice of breeds should take into account of their capacity to adapt to local conditions...". Moreover, the EU Reg. states that each Member State has to determine criteria for the definition of Slow-Growing (SG) genotypes evaluation of their adaptability to the organic system. In most EU countries, the definition of SG genotypes is based on the Daily Weight Gain (DWG); however, the relationship between genotype, growth rate and adaptability seems more complex. The present study develops an index to evaluate the adaptability of different poultry strains taking into account: explorative aptitude (calculated by time need for get out from the shelter), kinetic activity, foraging behaviour, plumage and body lesions. Accordingly, the adaptability of one hundred chicks males of six genotypes (Ranger Classic (R1), Ranger Gold (R2), Rowan Ranger (R3), Hubbard RedJA (A), CYgen5xJA87 (CY), M22xJA87(M)), were compared in organic condition (4 m2 outdoor run, organic diets, slaughtering at 81 days of age). Principal Component Analysis extracted a component including behavioural and body condition variables called Adaptability index (AI). Regression models were used to evaluate its relationships with genotype and DWG. ROC analysis was also performed to find the cut-off of DWG which discriminates high and low AI scores. The AI was positively related with grass intake, walking, drinking, and plumage score while negative loadings were found for explorative aptitude, static behaviours, and body lesions. The genotype strongly influenced the AI (P<0.001): R3 and A exhibited the highest adaptability score, R1 and R2 showed an intermediate value, whereas M and CY had the lowest scores. The optimal cut-off for DWG predicts the low and high adaptability of animals with a sensitivity of 87% and a specificity of 73%. Animals with DWG ≥38.6 g/d showed a low adaptability whereas for values < 38.6 the DWG did not affect the AI. In conclusion, even if all the genotypes tested are "theoretically" SG, they did not show the same adaptation. Moreover, a cut off for DWG within these strains can be identified. In particular, M and CY seemed not adapted to organic system and a DWG ≥38.6 g/d is associated with poor adaptability.
COMPARATIVE DIFFERENCES OF 6-WEEK OLD BROILERS AND 65-WEEK OLD BROILER BREEDERS
DURING VENTILATION SHUT DOWN WITH HEAT

D. Hodgson*(1), K.Livingston(1), R.Malheiros(1), K.Eberle(1), M.Martin(2), S.Shah(3), S.Nolan(1), K.Anderson(1)

(1) North Carolina State University, Raleigh, United States

(2) North Carolina Department of Agriculture and Consumer Services, Raleigh, United States

(3) Department of Biological and Agricultural Engineering, Raleigh, United States

* Corresponding author: ddhodgso@ncsu.edu

The US Poultry Industry weathered one of the worst highly pathogenic avian influenza (HPAI) disease outbreaks in history, with 48 million turkeys, ducks and chickens depopulated in 2015. This HPAI outbreak made its mark by illuminating a need for control measures pertaining to timely depopulation methods during an epidemic of pathogenic disease. Studies have been conducted to determine the effectiveness and physiological response of the procedure on different birds using Ventilation Shut Down Plus (VSD+). The addition of heat has been found to decrease the time to death and lower stress physiology markers of broilers. In 2019, the American Veterinary Medical Association included VSD+, in the depopulation decision tree as a method dependent upon resources and timeliness. Using data from the VSD+ studies with the addition of heat (VSDH), physiology parameters of 6-week old broilers versus 69-week old broiler breeders was examined. The simulation chambers allowed for 3.4 ft3 that represents the space volume/hen under industry depopulation conditions. Brain activity was monitored by affixing electroencephalogram electrodes to non-anesthetized birds and used as a component to determine time of death (TOD). VSDH was repeated four times with each bird type. Cloaca temperatures were also taken before and after the procedure. Blood was drawn pre- and post-VSDH to determine corticosterone (CORT), and blood chemistry parameters. Upon death, brain and liver tissue were collected and preserved in RNALater® to determine heat shock protein 70 mRNA expression. The analysis of the treatment differences was accomplished using one-way ANOVA through JMP Pro 14, alpha was 0.05. Despite a significant differences in body weight between the broiler breeders and broilers (P=0.0086), no differences in CORT levels or HSP70 in the brain were observed. However, HSP70 in the liver has a trend towards significance (P=0.051) with broiler breeders having 0.611 pg/ml and broilers having 0.492 pg/ml. TOD tended to be quicker for the broilers at 75 minutes and broiler breeders at 113.5 minutes (P=0.091). Broiler breeders have a greater expression of HSP70 in the liver compared to broilers while also having less post-CORT. Bird type/age appears to play a more important role in CORT values and HSP70 relationships and thereby heat tolerance more so than body weight. Further studies are needed to observe this relationship in successive time points to verify points of fluctuations.
FORMATION OF DOMINANCE HIERARCHY BY FIGHTING AMONG GUINEA FOWL (NUMIDA MELEAGRIS)

M. Shimoda\*(1), D.Waku(2), H.Ogawa(3)

(1)Tokyo Gakugei University Graduate School of Teacher Education, Tokyo, Japan

(2)Tokyo University of Agriculture, Tokyo, Japan

(3)Tokyo University of Agriculture, Kanagawa, Japan

* Corresponding author: m193408f@st.u-gakugei.ac.jp

Guinea fowl live in flocks over a wide area in Africa. But they are bred by artificial insemination in commercial production since they have a monogamous mating system. In order to efficiently breed naturally, it is necessary to know the process of forming relationships between individuals within a group. However, there are no studies on guinea fowl that have investigated the formation of hierarchical relationships between individuals in the flock by fighting. In this study, we examined the relationship between individuals in a group by focusing on the fighting behavior and squeal patterns, and aimed to enrich our knowledge base for better floor housing of guinea fowls. We reared guinea fowl in floor pens and observed their behavior via video footage, in particular, their fighting behavior and squeal patterns. In addition, squeals were analyzed by sound spectrogram analysis. We designed three experimental groups as follows: 1st group) both male and female (two individuals each), 2nd group) only male (four individuals), and 3rd group) only female (four individuals). All three groups were built three teams (total nine teams) and observed every two weeks each. We randomly picked the birds who make up all the groups and exchanged them for new individuals. As a result, in the mixed and only-male groups, male-to-male pecking and chasing were observed. In contrast, there was almost no female-to-female fighting. Peck order was determined in approximately 1-7 hours based on the result of the fighting between male-to-male fowls. The dominant individual ate a major portion of the food, whereas the subordinate individual ate little food and crouched in the corner of the pen. All females ate sufficient food. The lighter-built male tended to be pecked by dominant individuals. Further, the pecking behavior tended to occur between near-weight individuals. We found two new squeal patterns that are associated with fighting. These new squeals were produced by subordinate males when they crouched in the corner of the pen. These results suggest that attention must be paid while group rearing male individuals, but female-to-female and female and male groupings did not need such attention as there was no or little fighting in these groups. If there is more than one male being housed in the same area, preparing a larger area with sufficient number of feeders creates a better environment for subordinate male individuals and provides a better chance for their welfare.
# ABOLISHMENT OF BEAK TRIMMING IN LAYING HENS: FIRST RESULTS FROM NON-CAGE HOUSING SYSTEMS UNDER FIELD CONDITIONS IN GERMANY

L. Klambeck\*(1), K. Häffelin(2), B.Spindler(3), R.Andersson(2)

(1) Department of Animal Husbandry and Poultry Sciences, Osnabrueck, Germany

(2) Animal Husbandry and Poultry Sciences, University of Applied Sciences Osnabrück, Osnabrück, Germany

(3) Institute for Animal Hygiene, Animal Welfare and Farm Animal Behaviour, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

\* Corresponding author: L.Klambeck@hs-osnabrueck.de

In order to reduce the impacts of feather pecking and cannibalism e.g. on mortality, hens kept for egg production are routinely beak trimmed in most countries. In Germany, beak trimming in hens was abolished in 2017, which is in accordance with the German Animal Welfare Act. From 2017 to 2019 a total of 92 flocks (34,972 ± 18,676 hens per flock) of commercial layer breeds with untrimmed beaks were monitored under commercial housing conditions in floor, free-range and organic production systems. Mortality and laying performance were recorded daily. Additionally, the integument of the birds was visually scored in irregular intervals to derive indirect information about behavioural disorders. Deviating from former production conditions in Germany (production period until 65 – 75 weeks of age), the flocks in the current study were kept until 85.3 ± 4.5 weeks of age in white and 79.8 ± 8.4 weeks of age in brown genotypes, respectively. The cumulative mortality rate per flock and laying period ranged between 1.4 % and 29.8 %. Throughout the entire laying periods, mortality rate increased to ≥ 0.2% per week at an age of around 62 weeks in brown and 87 weeks of age in white genotypes. It could be carried out, that refraining from beak trimming requires specific management strategies. Influencing factors seem to be lighting (no flicker, no light-spots), feed quality (amount of aminoacids, sodium, crude fibre), feeding frequency (more than 3 times a day), litter management (manipulable and dry litter, usable for dust bathing) and environmental enrichment by offering additional materials such as alfalfa hay, straw or pecking blocks. Flocks using one or more of these strategies had less problems with behavioural disorders. Feather pecking occured in all flocks over the laying period, but at different weeks of age. The occurence of feather pecking did not differ between the different housing conditions. In case of the presence of severe behavioural disorders, reducing the light intensity below 20 lux was observed to be the first countermeasure in 76 of 92 flocks. Considering animal welfare aspects, early warning systems need to be established on-farm, accompanied by indicator based management strategies in order to reduce the risk for behavioural disorders corresponding with high mortality.

Keywords:

Abolishment of beak trimming, Layers, Hens, Non-cage-housing, Mortality, Behavioural disorder
EXPLORATION AND NEOPHOBIA IN DOMESTIC FOWL

V. Meuser*(1), I.Tiemann(1)

(1)Institute of Animal Science, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany, Bonn, Germany

* Corresponding author: verena.meuser@uni-bonn.de

Fear leads to distress which affects animal welfare and productivity in layers and broilers. Adaptation and coping strategies are favoured that enable the animal to deal with a changing environment. In local breeds, not only genetic resources might have preserved but also favourable behaviours. These behaviours include exploration behaviour, e.g. of unknown objects, which is negatively correlated with fear behaviour, e.g. neophobia. The objective of our study was to observe local breeds in different standardized tests concerning exploratory and fear behaviour directed towards objects and humans. For this reason, the dual-purpose breeds Bresse Gauloise (n = 24) and Malines (n = 25) were tested in the Novel Object test (NOT), Touch test (TT) and Avoidance Distance test (ADT) according to the Welfare Quality® assessment protocol for poultry (broilers, laying hens). For the NOT, the number of animals which undercut the distance of one animal's length to a novel object is recorded. For the TT, the number of animals which can be touched by a stationary experimenter is counted. For the ADT, the number of animals which undercut a one arm length distance to the stationary experimenter is counted. Every test was carried out at four different points in the pen on different days and a weekly repetition. Differences were found between breeds in each test, the NOT, TT and ADT (all P < 0.001). In each test, Bresse Gauloise have been found to show stronger fear reactions than Malines. According to the negative correlation between fear and exploration, Malines were found to exhibit a stronger exploration behaviour than Bresse Gauloise. The results of this study show that exploration and fear can be quantitatively measured in standardized tests. To favour better adapted animals, it is necessary to find a balanced relationship between these two behaviours. Local breeds like Malines and Bresse Gauloise show different adaptive values and might, therefore, be appropriate models to understand behavioural diversity and to unlock opportunities in modern breeding towards animal welfare.
PECKING BEHAVIOUR OF NON-BEAKTRIMMED LAYING HENS IN AVIARIES ON COMMERCIAL FARMS IN GERMANY

A. Schwarzer*(1), H.Louton(2), M.Elger(3), M.Erhard(3)

(1)Chair for Animal Welfare, Ethology, Animal Hygiene and Animal Husbandry, Munich, Germany

(2)Chair for Animal Welfare, Ethology, Animal Hygiene and Animal Husbandry, LMU Munich, Munich, Germany

(3)chair for Animal Welfare, Ethology, Animal Hygiene and Animal Husbandry, LMU Munich, Germany, Munich, Germany

* Corresponding author: angela.schwarzer@lmu.de

Severe feather pecking (SFP) impairs animal welfare due to pain and injuries of affected hens. The aim of the project was to investigate both SFP in different functional areas of commercially used aviaries and identify influencing factors.

Behavioural observations were conducted in commercial laying hen farms in Germany. 4 out of 8 farms used Lohmann Brown (LB)-, 3 mixed LB- and Lohmann Selected Leghorn (LSL)- and 1 farm Bovans Brown- and Dekalb White-flocks. Additionally to these non-beaktrimmed flocks, 4 farms kept beaktrimmed flocks under equal Management conditions (Multi-tier-aviaries, feeding and light regime according to the breeding companys management guidelines). Video cameras were installed at different functional areas (litter, aviary platforms, perches). Pecking behaviour was analysed using "continuous recording" for 5 minutes/hour during light day. For the distribution of the flocks in different functional areas "scan sampling" was used.

To analyse effects (partial Eta-Square) and significant effects of single independent factors an univariate multifactorial analysis of variance (UNIANOVA) with the area under the curve (AUC) derived from data of the functional area usage, was calculated. The calculated average of the AUC-data for AP and SFP were tested on normal distribution using a Shapiro-Wilk-Test. As data were not normally distributed, a Mann-Whitney-U-Test was used.

The functional areas were used in a circadian rhythm. While perches were not frequently visited during day, usage at night was as high as expected. However, in some cases aviary platforms were used for roosting instead. Usage of the litter area (25%) correlated with data found in literature. A consistently high number of hens were seen on aviary platforms. The functional area had a significant effect on SFP (p one laying hybrid only) revealed a significant effect on SFP (p flocks with white and brown layers) flocks. But in these cases the factor "management" had an influence on the result. LSL-hens were responsible for the majority of SFP (59%). Beaktrimming neither had an effect on the occurrence of aggressive pecking nor on SFP.

We found a high usage of aviary platforms and the highest rates of SFP in this area. Therefore trying to increase the space of this area and/or the use of other areas (eg. litter) could help to prevent SFP. But as SFP is a multifactorial problem, an individual risk analysis is recommended.
CORTICOSTERONE IN FEATHERS (CORTF) AS AN APPROACH TO QUANTIFY ANIMAL WELFARE: A
PROMISING TOOL FOR LAYERS?

K. Häffelin*(1), R.Lindenwald(2), B.Spindler(3), S.Döhring(1), F.Kaufmann(1), R.Preisinger(4), S.Rautenschlein(2), N.Kemper(3)(1)

(1) University of Applied Sciences Osnabrück, Osnabrück, Germany

(2) Clinic for Poultry, University of Veterinary Medicine Hannover (Foundation), Hannover, Germany

(3)Institute for Animal Hygiene, Animal Welfare and Farm Animal Behavior, University of Veterinary Medicine Hannover (Foundation), Hannover, Germany

(4)EW Group GmbH, Visbek, Germany

* Corresponding author: F.Kaufmann@hs-osnabrueck.de

CORTf has become a promising tool to monitor wild birds coping with environmental situations over a longterm period. The stress-related hormone is deposited into the feathers during growth, when they are supplied with blood. In layers, CORTf may be used to assess birds' ability to cope with rearing conditions and thus, to evaluate their susceptibility of developing behavioral disorders, such as feather pecking. It may also be suitable to perform selection decision to provide stress-resilient animals. The objective of the current study was to adapt and validate a protocol to measure CORTf in layers.

Ten tail and 80 interscapular feathers of three adult laying hens, Lohman Brown, were pooled and crushed. Tail and interscapular feathers were processed and analyzed separately. Three aliquots of 50mg each were created to examine precision and specificity of the assay. 40 aliquots of 10mg each were created and five aliquots were assigned to one assessment group. CORTf extraction was done using a modified protocol of Bortolotti et al. (2008), followed by an ELISA with four repetitions each sample (n=188). For precision, intra-assay CV within repetitions and inter-assay CV amongst two assays was calculated. Specificity was tested by examining the linearity of a serial dilution via linear regression. Aliquots of one assessment group went through the same treatment to examine the extraction efficiency using five different methanol volumes, two different methods of crushing and the effect of feather types. Group effects were calculated using Kruskal-Wallis or Mann-Whitney-U test.

Intra-assay CV was 7.5% (median over all repetitions), inter-assay CV 6.4%. Linearity (R²) was 99.7%.
No significant effect was detected between the five different methanol groups (p>0.05) and the two
crushing methods (p>0.05). CORTf values of interscapular feathers were significantly higher than values
of tail feathers (p<0.05).

It can be concluded that the validated protocol is suitable to analyze CORTf in layers. To choose feathers carefully is important, as our results indicate different values in different feather types. Subsequent studies should focus on individual feathers of single hens and identifying stressful conditions during feather growth and the related responses of individuals in terms of CORTf deposition.

Bortolotti, G.R., T.A. Marchant, J. Blas, T. German. 2008. Corticosterone in feathers is a long-term, integrated measure of avian stress physiology. Funct. Ecol. 22:494-500
A PARTIALLY PERFORATED FLOORING SYSTEM AND ITS EFFECT ON ANIMAL WELFARE IN BROILER HOUSING

C. Adler*(1), S.Hillemacher(2), A.Schmithausen(3), I.Tiemann(2), W.Büscher(4)

(1)Institute of Agricultural Engineering, University of Bonn, Bonn, Germany

(2) Institute of Agricultural Engineering, University of Bonn and Institute of Animal Science, University of Bonn, Bonn, Germany

(3) Corteva Agriscience, Munich, Germany

(4) Institute of Agricultural Engineering, University of Bonn, Bonn, Germany

* Corresponding author: c.adler@uni-bonn.de

In practice, broilers often spend the fattening period in contact with litter with an increasing moisture and excrement content over time. Contact to those litter conditions is known to reduce animal welfare. Totally (100%) perforated flooring systems separate the animals from the litter and showed positive effects on animal health. However, this system leads to a restriction in the natural behavior of the animals due to the missing bedding material. Furthermore, totally perforated flooring systems resulted in an increased ammonia concentration, if manure underneath the perforated floor was not removed between the fattening periods. The objective of our case-control study was to evaluate the effect of a partially (50%) perforated flooring system on health indicators and ammonia concentrations compared to a conventional deep-litter system. The experimental barn was partially (50%) equipped with a perforated floor directly underneath the feeders and water lines accessible by perforated ramps. Wood shavings were used in the remaining area (50%) and in the control barn (100%). Two fattening periods (repetitions) with 31 to 32 days were carried out with 500 Ross 308 broilers per barn (39 kg/m²). Foot pad dermatitis, hock burn, and plumage cleanliness were assessed weekly for 50 randomized selected birds per barn by Welfare Quality® (2009). Ammonia concentrations were measured continuously via photoacoustic infrared spectroscopy (LumaSense Technologies SA, Ballerup, Denmark). Because there was no homogeneity of variance, a Mann-Whitney-U-test was used to analyze the health indicators and Welch's t-test for the ammonia concentrations. Results for the whole fattening period showed a positive effect of the partially perforated flooring system on footpad dermatitis (U(999) = 93,420.0, Z = -7.47, P (999) = 111,189.0, Z = -3.89, P (999) = 116,878.5, Z = -1.88, P = 0.061). The ammonia concentration was higher in the barn with the partially perforated flooring system compared to litter flooring (T(2620.35) = -9.02, P
ID: 1142

# QUANTIFICATION OF THE BENEFITS OF LOW PROTEIN DIETS ON FOOT PAD DERMATITIS INCIDENCE AND SEVERITY IN BROILER CHICKENS

I. Minussi*(1), W.Lambert(1), M.Létourneau-montminy(2)

(1)Ajinomoto Animal Nutrition Europe, Paris, France

(2)Université Laval, Québec city, Canada

* Corresponding author: lambert_william@eli.ajinomoto.com

Foot pad dermatitis (FPD) is recognized as one of the main indicators of welfare in broilers. Specific nutritional strategies, especially low protein diets, are recognized as effective tools to reduce this condition. The effect of dietary protein content on dietary potassium, water intake, litter moisture and litter nitrogen content has already been quantified, allowing to better understand the mode of action of low protein diets on FPD. However, the quantification of the effects of low protein diets on FPD incidence and severity still needs further investigation.

All published and internal trials from 2007 which measured and reported both FPD incidence and severity were collected, leading to a database of 10 trials from 8 publications and 27 treatments. As birds were fed various levels of protein in different grow-out segments, weighted average crude protein content (WACP) was calculated to standardize protein supply. The different FPD scoring systems were harmonized: 0, 1 and 2 for birds with respectively no, moderate and severe FPD. The final score (foot pad score, FPS) was then recalculated according to the official method as $100 \times ((0.5 \times \text{birds scored 1}) + (2 \times \text{birds scored 2})) / \text{scored birds}$, resulting in a number from 0 and 200, 200 being the worst score. Mixed effects model of Minitab 19 was used to quantify the impact of dietary WACP on FPS and foot pad incidence (FPI). Experiment was taken as a random factor, and the level of FPS (cFPS) or FPI (cFPI) of the control treatment was also included to test for the interaction between cFPS or cFPI and WACP.

In the database, WACP, FPS and FPI averaged $19.0 \pm 0.17$, $44.8 \pm 6.5$ and $51.8 \pm 4.5$, respectively. Experiment effect explained only 6% of the variation (P=0.06; R2=97 vs 91%), making the model able to predict many situations. Dietary WACP content significantly and linearly reduced FPS and FPI (P<0.05). In conclusion, this work shows that low protein diets are an efficient strategy to control FPD by quantifying their beneficial effects on the FPD incidence and severity.
IMPACT OF HOUSING SYSTEMS ON BONE HEALTH AND WELFARE ISSUES IN JAPANESE QUAIL

F. Farooq\*(1), N.Mukhtar(2), W.Ali(2), T.Ahmad(3), A.Yousaf(4)

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

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

(3) Department of Livestock Production and Management - PMAS Arid Agriculture University Rawalpindi-Pakistan, Rawalpindi, Pakistan

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

* Corresponding author: dr_farhanf@hotmail.com

Impact of housing systems on bone health and welfare issues in Japanese quail

Quails can be used to obtain meat within a short period of 4-5 weeks. The high performance in quails depends on special care and management. Bone abnormalities like low bone mass, bone deformities, and bone fractures increased the risk of osteoporosis in quails. This leads to high mortality and reduce feed consumption, weight gain and feed efficiency. The present study was conducted to address leg problems and welfare concerns under two quail housing systems used in the country. A total of 500 quail one-day old chicks were maintained in into two experimental housing systems viz., battery cage system and deep litter system. Each housing system had five replicates and each replicate consisted of 50 quails. The experimental design consisted of Completely Randomized Design and analyzed by using General Linear Model techniques with the assistance of Statistical Analysis System. The results indicated non-significant (p>0.05) effect of two housing systems on feed intake, mean body weight gain and feed conversion ratio up to 6 week of quail age. The mortality rate was significantly higher (p0.05) in both treatment groups. Likewise, the carcass weight and the European broiler index as growth indicator was also non-significant (p>0.05) in both housing systems. Bone health parameters like fumer length, tibia weight, tibia length and tibia seeder index were significantly (p

Key words: welfare, quail, feed intake, weight gain, cage system, deep litter system
ID: 1300

COST ACTION 15224 'KEELBONEDAMAGE': BOOSTING RESEARCH, INNOVATION AND CAREERS IN GREECE AND SERBIA

E. Sossidou*(1), A. Dedousi(1), M. Đukić stojčić(2)

(1) Veterinary Research Institute, HELLENIC AGRICULTURAL ORGANISATION-DEMETER, Thessaloniki, Greece

(2) Faculty of Agriculture, University of Novi Sad Serbia, Novi sad, Serbia

* Corresponding author: sossidou.arig@nagref.gr

The aim of this paper is to present and discuss the networking opportunities and scientific outcomes of Short-Term Scientific Missions (STSMs) with exchange visits between researchers involved in CA15224 'Keel Bone Damage' in Greece and Serbia. Within the 1st STSM entitled 'KBD occurrence in different production systems in Greece', Prof Đukić Stojčić from the University of Novi Sad was hosted by the Veterinary Research Institute of the Hellenic Agricultural Organization in Greece and visited three laying hen farms with different production systems and made welfare measurements according to the Welfare Quality® Assessment Protocol for Poultry. Palpation of laying hens' keel bone and breast was made to assess both the fractures and deviations and feather plumage was scored. Egg samples were taken from each farm, in order to investigate their internal and external quality. Within the 2nd STSM entitled 'Effect of perch arrangements in furnished cages on welfare parameters, behavior of laying hens, egg quality and KBD', Dr Dedousi from the Veterinary Research Institute in Thessaloniki was hosted by the University of Novi Sad. and has visited three laying hen farms producing in different housing systems to measure welfare and behavior parameters and palpated one hundred (100) hens from each housing system. 1st STSM (Greece): The first investigation showed that KBD were present in all three observed production systems in Greece. The average range of keel damage observed in laying hens kept in enriched cages was 23.8%. Similar results were found in layers kept in free range system in which plumage score was on average 3.23. However, the incidence of KBD was higher in this system. In floor system, laying hens were 5.5 months old, with perfect feather coverage, and without other welfare problems. KBD was not recorded. Furthermore, the results showed a significant (P≤0.05) effect of housing system on the internal and external egg quality. 2nd STSM (Serbia): KBD was present only in two of the observed production systems, enriched cages and aviary system. In the aviary system, 56.4% of the birds had completely straight and flat keel, without deformation or fracture. However, KBD was recorded in 43.6% of the layers. In this production system 27.3% of the hens had deformation, 10.9% had fractures and 5.5% had both deformation and fractures.
EFFECTS OF ADDITIONAL LIGHT SOURCES ON TRAINING PERIOD OF LAYING HENS AND LAYING PERFORMANCE IN AN AVIARY HOUSING SYSTEM

S. Hillemacher\*(1), K.Geurts(2), W.Büscher(3), I.Tiemann(1)

(1)University of Bonn, Institute of Animal Science, Bonn, Germany

(2)HATO Agricultural lighting, Sittard, Netherlands

(3)University of Bonn, Institute of Agricultural Engineering, Bonn, Germany

* Corresponding author: sonja.hillemacher@uni-bonn.de

Recent studies provided important insights into the requirements of light sources for poultry housing systems and their impact on performance, behavior and welfare of laying hens. This study investigated the effect of additional light sources in an aviary housing system on the percentage of mislaid eggs in the bedding area. In addition, the effect of a training light above the perch area on the perching behavior of the hens in the evening was investigated. Hens were kept in a two-story aviary housing facility, matching conventional husbandry requirements and equipped with modern lighting solutions designed for poultry on the ceiling above the litter area and below the aviary (light period 10:14 after moving, extended by 1h/week to 16:8, 20 min. dim phase). The barn was subdivided into six compartments, each with 183 either brown (Lohmann Brown Classic, LB) or white (Lohmann Selected Leghorn Classic, LSL) layers. Compartments 1 (LB) and 2 (LSL) had a tubular LED light (2.3 W, BUBO, HATO, Netherlands) above the perching area, which was switched off with a 30 min. delay to the other lights in the evening to attract the hens to the perches. The number of hens remaining on the floor was counted 15 min. after lights off on the first 14 days. Compartments 3 (LB) and 4 (LSL) served as controls with no modifications. Compartments 5 (LB) and 6 (LSL) had additional tubular LED lightings below the aviary to investigate the effect on mislaid floor eggs (22 – 45 weeks of life). Statistical analysis was run using a Welch-ANOVA on all data sets, followed by Games-Howell Post-Hoc tests for the analysis of mislaid eggs and contrasts for the effect of the training light. The additional lights below the aviary reduced the percentage of mislaid eggs in the bedding area significantly in LB ($M \pm SE$; C3control 1.10% $\pm 0.08$; C5mod 0.25% $\pm 0.04$; Welch's F(5, 427.887) = 32.695, P < 0.001), in LSL number of floor eggs was constantly low (all $M \le 0.09$%). Furthermore, lighting above the perching area reduced the numbers of LB hens sleeping on the floor significantly (Welch's F(5, 38.010) = 2.859, P = 0.027). These modifications might reduce the workload for farmers and increase sales of shell eggs. The study emphasizes that functional lighting has a positive effect on the hens' behavior and performance by guiding the hens to the perches in the evening and decreasing the numbers of mislaid eggs on the floor. In conclusion, functional lighting supports animal welfare in layers.
INFLUENCE OF DIFFERENT FLOOR TYPES ON THE BEHAVIOR OF CHICKS

O. Müsseler*(1), C.Adler(2), W.Büscher(2), I.Tiemann(1)

(1) Institute of Animal Science, Rheinische-Friedrich-Wilhelms University Bonn, Germany, Bonn, Germany

(2) Institute of Agricultural Engineering, Rheinische-Friedrich-Wilhelms University Bonn, Germany, Bonn, Germany

* Corresponding author: o.muesseler@gmail.com

Although broilers have special demands on their housing system, they are often kept on litter with access to feed and water. In addition to produce meat food, the housing system should support the natural behavior of the chicks. Perforated plastic floors are discussed to improve animal welfare by separating the animals from their excrements. The disadvantage of those floor systems is the lack of bedding material to show natural behavior. In this study, perforated and plane littered areas were used to observe possible behavioral preferences. Furthermore, wood shavings, lignocellulose, maize spindle granulate, maize silage, straw pellets, spelt pellets, wood pellets, loose Miscanthus chaff, and different Miscanthus briquettes (shredded or 5 mm long fragments pressed at different pressure levels) were tested. A sieve tower analysis was used to determine which bedding materials are suitable for the two floor types. Miscanthus briquettes were most suitable for the perforated plastic floor and the other materials for the plane littered area. Chicks of the dual-purpose breed Bresse Gauloise were kept in rearing boxes and observed from 2nd to 6th day of life. For the observation, chicks were filmed continuously, and videos were analyzed using the scan sampling method with a 15-minute interval. All behaviors shown were analyzed based on an ethogram and compared to a baseline trial without bedding material. Data revealed that the chicks preened significantly more frequently (all p < .001) and showed more locomotion behavior (all p < .001) in the littered area. Compared to all other bedding materials in the plane littered area, chicks showed active behavior more often on maize silage (all p < .001), whereby comfort behavior was carried out most frequently on Miscanthus chaff (all p < .001). In the area of the perforated floor, active behavior was observed more frequently (all p < .01) in contact with the Miscanthus briquettes (shredded; 120 and 150 bar) in comparison to finer briquettes (5 mm; 80 bar). Furthermore, the latter was used more often to show comfort behavior (all p < .05). Quantitative analysis revealed that behavioral categories were exhibit on the littered area more often, although resting and comfort behavior was also observed on the perforated floor. In general, different floor types and litter materials could support the functional adaptation of the housing system towards the chick's behavioral needs, especially in terms of animal welfare.
HOW TO DEAL WITH INTACT-BEAK LAYING HENS IN PRACTICE?

N. Sleeckx*(1), E.Decru(2), K.De baere(1), I.Kempen(1)

(1) Experimental Poultry Centre, Geel, Belgium

(2) Experimetal Poultry Centre, Geel, Belgium

* Corresponding author: nathalie.sleeckx@provincieantwerpen.be

Feather pecking is an important welfare and economic problem in laying hens. Because of national legislation or market demands, more and more hens in Europe are non-beak trimmed. It has been shown that the consequences of injurious pecking in non-beak trimmed hens (NBT) can be much larger compared with beak trimmed hens (BT) resulting in a serious decrease of animal welfare, health and flock performance. Field studies show different outcomes of NBT flocks and conclusions are often contradictory. The first experiences of the Experimental Poultry Centre (EPC) with NBT hens showed a very high mortality (25%) and a significant lower laying % (-6% per hen present) at 77 weeks of age. A second flock was started where 5300 BT hens were compared with 5300 NBT hens in a controlled set-up. All hens (Isa Brown) were from the same breeder flock, were born on the same day, were reared in identical conditions in the same rearing house, were transported in the same way to the layer house and were placed in 4 identical aviaries (2 with NBT hens, 2 with BT hens). Compared to the previous flock, adaptations to feed and light management were implemented. All groups received pecking blocks and roughage as distraction. Production data were recorded, a flock behavior checklist was performed and plumage condition was assessed every 4 weeks. Differences between the groups were ascertained with MANOVA's. The aim of this study was to evaluate the onset and evolution of injurious pecking in the groups.

Until 71 weeks of age, all hens performed above breed standards. No differences were seen in mortality and laying percentage. However NBT hens showed higher feed intake and higher feed conversion ratio. In addition, plumage condition started to deteriorate at 60 weeks of age, especially at the lower back region, and more pecking behavior was observed in the NBT hens. Further follow-up of the flock until 85 weeks of age will give more information on the progression of the parameters assessed of the current flock.

In conclusion, the current groups of NBT hens show remarkable better results compared to the first flock. However, at 60 weeks of age a turning point seems to be reached and close observation until the end of lay is mandatory.
ID: 1145

EFFECT OF A DYNAMIC LIGHT SPECTRUM ON WELFARE AND PRODUCTION IN LAYING HENS

I. Kempen*(1), E.Decru(1), K.De baere(1), N.Sleeckx(1)

(1)Experimental Poultry Centre, Geel, Belgium

* Corresponding author: ine.kempen@provincieantwerpen.be

Prolonging the production cycle in laying hens can only be profitable when a flock shows optimal production, good welfare and health. Farmers are looking for strategies and tools to meet this ambition. Over the last few years, the importance of light in managing laying hens has been reconsidered. The evolution in lighting technology makes it possible to adapt light spectra and light intensity during the day or the production cycle.

At the Experimental Poultry Centre, a trial was conducted with 8 groups of 960 brown laying hens in commercial conditions. Four groups had a standard LED light program with a daily fixed spectrum. The other four groups received a dynamic light spectrum with longer wavelengths in the morning and evening and shorter wavelengths during the day. All the hens received the same management, feed and distraction materials. Production and welfare parameters were evaluated from 20 to 60 weeks of age. Light intensity (in lux) was measured at week 20, 40 and 60. Feather scoring and keel bone evaluation was conducted every 4 weeks.

At 60 weeks of age, there were no significant differences between the two treatments in mortality, laying percentage, daily feed consumption, feed conversion, percentage of second choice eggs and the amount of produced eggs per hen. Feather and keel bone evaluation showed no significant effect of a changing light spectrum. Although the trial could not demonstrate an effect on selected parameters, we can still conclude an important difference. Light intensity was changed during the production cycle based on flock behavior, farmer experience and group evaluation. The groups who received a dynamic light schedule could still be kept at an average of 20 lux at week 60 while the groups with a fixed program had to be kept at an average of 14.5 lux. We conclude that these dynamic light techniques can ensure longer a higher light intensity when keeping brown layers.
MANAGING POULTRY WELFARE
ID: 1422

DIFFERENCES IN PRODUCTIVITY, WELFARE AND CARCASS QUALITY FOR THREE SLOW GROWING BROILER GENOTYPES UNDER COMMERCIAL CONDITIONS

J.S. Petersen\*(1), S. Kabell

(1) SEGES, Aarhus N, Denmark

(2)HKScan Denmark, Vinderup, Denmark

\*Corresponding author: jtp@seges.dk

In Europe, due to animal welfare issues, the demand for slow growing broilers is increasing. Although slow growing broiler breeds have a lower potential for growth and feed intake than fast growing broiler breeds, the farmer must control that the growth of the broilers stays within the regulation for average daily gain and slaughter age. Likewise, in order to fulfill the consumer's demand for lean meat in a competitive and climate friendly way, the slaughter company must use a slow growing broiler breed that is optimal with regard to animal welfare, productivity and carcass quality. The purpose of this study was to compare growth, feed conversion rate, foot pad score, carcass and breast meat yield for three types of slow growing broiler breeds (Ranger Gold, Hubbard JA87CY and Rowan Ranger).

For each genotype 400 female and 360 male birds were placed as day old in 24 different pens (8 pens/genotype) on a commercial broiler farm. All the birds had ad libitum access to commercial compound feed for fast growing broilers. The birds were weighed pen-wise, and feed consumption was calculated, On day 45 the occurrence of foot pad dermatitis was determined and at day 49, ten birds/pen were randomly selected for manual slaughtering and carcass evaluation. Data was analysed statistically by JMP 14.

The results showed that Rowan Ranger had a lower daily gain (48 g/day) and a lower final body weight at day 45 than the other two genotypes (Hubbard JA87CY: 53 g/day and Ranger Gold:54 g/day). The feed efficiency was better for the Ranger Gold birds when compared to Hubbard JA87CY and Rowan Ranger birds. No foot pad dermatitis was observed in the genotype Rowan Ranger, and in Ranger Gold birds only minor levels of foot pad dermatitis was found. On contrary, a higher level of foot pad dermatitis was found in the Hubbard JA87CY birds. Hubbard JA87CY and Ranger Gold birds had the highest carcass weight. However, the ratio of breast meat to carcass weight was lower in Hubbard JA87CY birds (24 %) when compared to Rowan Ranger (27 %) and Ranger Gold (27 %).

It was concluded that under the current management system, Ranger Gold was superior in productivity, animal welfare and carcass quality when compared to Rowan Ranger and Hubbard JA87CY birds. For all three genotypes, the daily gain was higher than allowed for organic broiler production in DK, this may be explained by the fact that conventional broiler feed was used for the three genotypes in the present study.
ID: 292

PLANT EXTRACTS TO STRENGTHEN THE NATURAL DEFENSES OF POULTRY: DEVELOPMENT OF A SELECTION TOOL

T. Angélique*(1), F. Alleman(2), A. Roinsard(3), O. Tavares(3), D. Bellenot(4), B. Lemaire(4)

(1) ITAVI, Nouzilly, France

(2) UniLaSalle, Beauvais, France

(3) ITAB, Paris, France

(4) Iteipmai, Chemillé-en-anjou, France

* Corresponding author: travel@itavi.asso.fr

In order to reduce antibiotics use, plant extracts use to strengthen the animal natural defences is becoming increasingly important in poultry production. However, their development as ingredients is hampered, in part, by the lack of robust and related repeatable data. Our aim was to design and test a method to assist in selection, from literature, plant extracts potentially interesting to strengthen natural defences of poultry. We were inspired by Anses methodology that aims to evaluate scientific publications and results (Saisine 2013-SA-0122 – Fév. 2018). First step consisted in establishing, with help of scientists and practitioners, two reading grids. The first allows level of reliability of bibliographic resources to be noted by verifying 1. that studied extract is correctly characterized and 2. that experimental design and results analysis are relevant to conclude about effect of tested extract. The second grid evaluates effects of studied extract on indicators of immune, inflammatory and antioxidant status of poultry. For each article, reliability score of resource is cross-referenced with effect score of extract, thus highlighting the most effective extracts. Our method has been proven. A fairly extensive bibliographic research phase on plant extracts affecting poultry natural defences, led to the selection of 917 articles, representing 48 plants. 8 of them were selected because 1. mentioned in book Bruneton "Pharmacognosie-Phytochimie-Plantes médicinales " as having effects on immunity, inflammation and oxidative stress, 2. mentioned in at least 5 articles 3. cultivable in France. Evaluation grid and methodological considerations were allowed to select astragalus, echinacea, ginseng, nigella as interesting plant extracts to strengthen poultry natural defences.
CONSEQUENCES OF A LOW INFESTATION BY DERMANYSSUS GALLINAE ON LAYER HEN PERFORMANCE

N. Cariou\*(1), L.Daluzeau(1)

(1)MSD Santé Animale, Beaucouzé, France

* Corresponding author: nadine.cariou@msd.com

The consequences of a high infestation of chickens by Dermanyssus gallinae are well documented. They include drop in lay, increase in B-grade eggs, anemia, pecking and mortality. The overall rate of flock contamination with poultry mites in Europe is estimated as being greater than 80%. However, low poultry mite contaminations were so far not considered as detrimental to the egg production industry. This study provides documentation of the consequences of low level poultry mite infestation. A study done on six layer farms, with a history of repeated, low level infestation by Dermanyssus gallinae investigated specifically the economic effect of low poultry mite infestations. The flocks encompassed several chicken breeds in various housing systems such as enriched cages, closed aviary or free-range aviary. The number of hens ranged from 21,500 to 190,000 birds per farm, with a total of 700,000 layer hens.

Mite infestation in these flocks for the 3 previous production cycles were controlled by spray treatment when deemed necessary, and mite infestation was considered to be low or reasonably under control. These three production cycles were evaluated as the controls in this study.

Chickens in the study production cycle were treated with Fluralaner (0.5mg/kg BW twice at a 7-day interval, via drinking water) as soon as an active mite population was found, and before flocks reached 35 weeks of age. The average observation period following treatment was 36 weeks. The aim of the study was to treat at the very early stage of mite infestation, based on visual observation during a visit, or presence of blood-fed mites in traps. The visual score (Cox et al) was below 2 in average before treatment, then the mite load was reduced to undetectable.

The study showed that even low poultry mite infestation of layer hens induces a drop in performance when not treated. Based on deviation from the breed performance objectives, the results show an average improvement of 0,6% in egg production for fluralaner treated flocks, compared to the average deviation from the breed performance objectives of the control flocks for each farm. This improvement can be explained by less disturbance of hens by mites in the nests, leading to less floor eggs in aviaries, or by better persistency in lay. The average mortality rate was equal to that of the previous production cycles, as expected with low red mite infestation.

For 50% of the farms, A and B-grade egg number and feed intake were also reported. As no standard is available for these two criteria, we compared the trial flock to the control flocks. The average improvement in A-grade egg number was 2%, and the average decrease in feed intake was 3 grams per hen per day. It has already been described that anemia and stress due to red mites may result in higher FCR.

This study demonstrates that even when Dermanyssus gallinae infestation is low and kept under reasonable control by spray, economic losses due to reductions in production performance, A-grade eggs and feed efficiency can be eroding profits. More effective and earlier treatment, further to animal welfare improvement and the beneficial effect on farmer's health and comfort, can improve the economic performance of layer flocks.
ASYMMETRIC LENGTH OF BILATERAL MANDIBLE RAMUS CAUSES CROSSED BEAKS IN BEIJING-YOU CHICKENS

L. Shi(1), Y.Sun*(1), L.Jiang(1), J.Fan(1), P.Ge(1), S.Bian(1), Y.Zong(1), Y.Li(1), P. Wang(1), Y. Wang(1), A.Ni(1), A.Ias(1), H.Tesfay(1), H.Ma(1), J.Chen(1)

(1)Institute of Animal Science, Beijing, China

* Corresponding author: yanyansun2014@163.com

The crossed beak is a deformity featured by the misalignment of upper and lower beaks. The occurrence of the crossed beaks is from 0.8% to 7.0% in different chicken breeds and severely impaired welfare and production. The objective of this study was to identify the occurrence regularity and morphology characteristics of the crossed beaks based on Beijing-You chickens.

A total of 9,900 female one-day-old Beijing-You chicks without beak-trimming were observed until 70 d of age for the occurrences of beak deformity. According to the bent direction of the lower beak, the crossed beaks were classified into two categories: 1) the lower beak is left-bent (LEFT) and 2) the lower beak is right-bent (RIGHT). Four chicks of each category were randomly selected at 14 and 70 d respectively to sacrifice for the characterization of the lower beak, including the mandible weight, and length, thickness, and width of bilateral mandible ramus (MR). The normal chicks (NORMAL) were used as controls. Paraffin section and HE staining was made for the bilateral mandible of a crossed and a normal beak, respectively at 7 d for histomorphology analysis.

A total of 71 out of the 9900 chickens (0.72%) showed crossed beaks. This trait occurred between 0 to 56 d of age. The frequency of LEFT did not differ from RIGHT (χ² = 0.03, P > 0.05). The LEFT had shorter and thicker left MR length than those of NORMAL and RIGHT (P < 0.05). The RIGHT had shorter and thicker right MR length than those of NORMAL and LEFT (P < 0.05). The left MR length was shorter and thicker than that of the right-side in LEFT, and the right MR length was shorter and thicker than that of the left-side in RIGHT (P < 0.05). There was no difference in mandible weight among LEFE, RIGHT, and NORMAL groups (P > 0.05). No obvious histomorphological difference was observed between the bilateral mandible of normal and crossed beaks. The chicks with crossed beaks also have their upper beak often bent horizontally at the base along, and skull asymmetry (mainly nasals and orbits).

The present study indicates that the asymmetric length of the bilateral MR causing the lower beak deformity and leads to crossed beak in the Beijing-You chickens. The branches of the mandible on the short side crossed with the zygomatic arch and prevented normal occlusion, causing the curve of the zygomatic arch. The curved zygomatic arch and the muscle attached to it may have tension on the maxilla, lead the upper beak bend horizontally.
# RISK FACTOR IDENTIFICATION OF KEEL BONE FRACTURES IN DANISH LAYING HENS.

I. Thoefner*(1), H.Hougen(2), C.Villa(2), J.Dahl(3), J.Christensen(4)

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

(2)University of Copenhagen, Department of Forensic Medicine, Copenhagen, Denmark

(3)Danish Agriculture & Food Council, Copenhagen, Denmark

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

* Corresponding author: icnt@sund.ku.dk

Keel bone fractures (KBF) are considered the most important welfare problem for layer hens, with potential economic losses to the industry. The reported prevalences are alarmingly high in both caged and non-caged production systems, with up to 97% in non-caged systems. Identification of risk factors is necessary to understand KBF. Some efforts to identify risk factors have been made, however there has been some uncertainties in the definition of KBF, thus risk factor identification has shown to be not as conclusive as intended. The objective of the presented work was to identify risk factors of importance on the occurrence of KBF.

The current study presents the results from a prevalence study in Denmark including all production systems, including enriched cages, indoor non-caged flocks, outdoor non-caged flocks, and parent stock (60 weeks old). Forty flocks (120 birds/flock) were investigated at end-of-lay (>75 weeks old). Following euthanasia, the birds underwent inspection, palpation and necropsy. All observations and production data were analyzed (SAS statistical software).

The overall prevalence of KBF was 86%. In non-caged flocks, the KBF prevalence were 53-100%, whereas flocks from enriched cages ranged 50-98%. Furthermore, multiple KBF (≥4) in individual birds were often observed. Around 50% of the birds from non-caged flocks had ≥4 KBF, whereas 96% of KBF involved the distal end of the keel bone). Macroscopically KBF ranged from almost absence of callus, most frequent in caged birds, to large callus formations in and around the fracture lines, a frequent and typical finding in non-caged birds and birds with ≥4 KBF.

Parent birds had fewer KBF per bird, which is in contrast to the other birds from cage-free systems (P=0.01). Heavy birds from outdoor non-caged range flocks or parents stock had significantly lower KBF prevalence (P=0.009). Heavy birds had also fewer fractures than lightweight birds, regardless of production system. The odds ratio of age at onset of lay (+1 week) is 0.87; for every week onset of lay is increased, the risk of having fractures at the end-of-lay is reduced by 12%. Similarly, a 100 g increase in body weight at slaughter reduces the prevalence of KBF in end-of-lay hens by 3%. Other identified factors like housing, hen hybrid, and egg production are discussed.
EF10

TURKEYS
EFFECT OF BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS SUPPLEMENTATION IN TURKEY DIETS ON GROWTH PERFORMANCES

J. Bodin\*(1), M. Hanuszewska(2), K. Kozłowski(2), D. Sandvang(3)

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

(2) Uniwersytet Warminsko-Mazurski, Olsztyn, Poland

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

* Corresponding author: frjebo@chr-hansen.com

The objective of the study was to evaluate the influence of a probiotic preparation containing spores of Bacillus subtilis (DSM 5750) and Bacillus licheniformis (DSM 5749), added to feed on growth performances of female turkeys reared until 84 days of age. A total of 300 day old Hybrid converter female turkeys were randomly assigned to 20 pens (15 birds/pen) allocated to 2 treatments groups T1 (basal diet without probiotics supplementation) and T2 (basal diet -T1 with probiotic preparation supplemented at 1,28.106 CFU/g of feed). All diets were offered in crumbled/pelleted form, drinking water was supplied ad libitum. Body weight (BW) and body weight gain (BWG) were determined after individual weighing in each pen and recorded on a pen basis at 0, 28, 56 and 84 days of age, feed intake (FI), feed conversion ratio (FCR) for all experimental periods and European Productivity Index (EPI) for the entire experiment. Statistical analysis was performed by a one-way analysis of variance (ANOVA) and significant differences between treatments were determined by Duncan's multiple range test, P ≤ 0.05 were recognized as statistically significant. Mean concentrations of Bacillus subtilis and Bacillus licheniformis in different phases were satisfactory T2 diet samples and corresponded to target values. The probiotic strains were not detectable in control diets. During the second experimental period (days 29-56), BW and BWG were significantly increased for turkeys fed with probiotic preparation (T2) in comparison to the control group (T1) (+4.9% on BWG, P=0,003). At 84 days of age, over the entire period, turkeys fed with the probiotic diet (T2) had a significantly higher BW than the control group (T1) (+4.02%, 7.659 vs 7.363 kg). In this study, no significant difference on FCR in between T1 and T2 was obtained on all the feeding phases but an overall trend of -1.8% in favor of turkeys fed with probiotic preparation (T2) for the entire experimental period. In terms of productivity performances, female turkeys from group T2, fed-supplemented diets, achieved higher significant EPI values (481 vs 453, P=0,028). For birds which received the probiotic preparation, significantly higher final BW, BWG and EPI were achieved. The results of this study indicate that a preparation of Bacillus subtilis and Bacillus licheniformis positively impacted the zootechnical performances of female turkeys.

Keywords: Bacillus subtilis, Bacillus licheniformis, turkeys, zootechnical performances
EFFECT OF DIETARY SUPPLEMENTATION WITH VITAMIN E AND ORGANIC SELENIUM ON TURKEY MEAT QUALITY DURING STORAGE

F. Soglia\*(1), G.Baldi(1), F.Sirri(2), C.Torres(3), M.Petracci(1)

(1) Department of Agricultural and Food Sciences, Alma Mater Studiorum - University of Bologna, Cesena, Italy

(2) Department of Agricultural and Food Sciences, Alma Mater Studiorum - University of Bologna, Ozzano dell'emilia, Italy

(3) Zinpro Corporation, Eden prairie, United States

* Corresponding author: francesca.soglia2@unibo.it

Turkey meats have relatively high PUFA and heme pigment contents, which make them prone to oxidative reactions that can affect their storage stability. This study evaluated the effect of vitamin E level (vitE; dl-tocopheryl acetate at 70 [Basal-B] vs. 300 ppm [High-H]) and selenium (Se) sources (inorganic [I] vs. organic zinc-L-selenomethionine [O], both at 0.3 ppm) on quality traits and oxidative stability of turkey breast and desinewed minced thigh meat. A total of 1,064 one-day-old female turkey poults were randomly divided into 4 experimental groups: BI, BO, HI, and HO. At processing (100 d, avg live weight 9.7 kg), 200 Pectoralis major (PM; 50/group) muscles were randomly collected to evaluate quality and technological properties of fresh meat, as well as oxidative stability after 3, 7, 10, and 12 days of storage at 4°C in modified atmosphere packaging (30% CO2, 70% O2). The same parameters were assessed on desinewed minced thigh meat after 3 and 90 days of frozen storage at -20°C. Data were analyzed as a 2×2 factorial design (ANOVA) to investigate the main effects of vitE and Se source, as well as their interaction at P < 0.05. Supplementation with O-Se resulted in remarkably higher muscle deposition of this nutrient, as well as lower cooking losses at 3 d. In detail, HO exhibited the highest Se content (0.035 mg/100 g meat), suggesting that adding H-vitE may enhance muscular deposition of Se. Regardless of Se source, supplementing turkeys with H-vitE resulted in darker meat at 3 d and increased oxidative stability of the lipid fraction (up to 10 d). At this time point, a significant (P = 0.001) interaction was found for TBARS: BO and HO exhibited the greatest and lowest values, respectively, whereas BI and HI did not differ from each other. On the other hand, aside from the vitE level, a significant (P < 0.01) antioxidant effect (reduced TBARS) of O-Se was observed when performing the analyses at 12 d. With regard to desinewed minced thigh meat, H-vitE significantly (P < 0.05) increased the lipid fraction stability after 3 d of frozen storage. This can be ascribed to the ability of vitE to act as a scavenger against lipid oxidation. However, a time-dependent effect can be hypothesized, as this effect was not observed at 90 d. In conclusion, because of their time-dependent antioxidant effect, the supplementation with H-vitE and O-Se may be considered as a promising feeding strategy to control oxidative reaction in turkey meat during storage.
ID: 418

EVALUATION OF ALTERNATIVE ON-FARM EUTHANASIA METHODS FOR BROAD BREASTED WHITE TURKEYS

D. Bourassa*(1), R. Boyal(1), L. Jacobs(2), B. Mcrea(3), N. Bartenfeld-josselson(4), A. Campbell(2), G. Anderson(2), C. Harris(5), J. Buhr(4)

(1) Auburn University, Auburn, al, United States

(2) Virginia Tech, Blacksburg, va, United States

(3) Alabama Cooperative Extension Service, Auburn, al, United States

(4) USDA-ARS, US National Poultry Research Center, Athens, ga, United States

(5) University of Georgia, Athens, ga, United States

* Corresponding author: dvb0006@auburn.edu

Euthanasia of market weight turkeys using manual cervical dislocation is not feasible due to their size and the strength required by the operator, which has led to the use of blunt force head trauma. The objective of this study was to evaluate euthanasia application efficacy of four veterinary approved alternative methods: carbon dioxide (CO2), Turkey Euthanasia Device (TED), Koechner Euthanizing Device (KED), and electrical euthanasia in achieving rapid unconsciousness and death in broad breasted white turkeys. Euthanasia methods were applied to males and female turkeys (18.8, 12.8 kg)prior to exsanguination for processing. To improve safety, ease of use for a single operator, and permit timed observations, CO2, TED, and KED methods were performed with birds placed in a mobile bird euthanasia apparatus. For CO2 euthanasia the head was enclosed in a single-bird portable device for 4 min (n=13). TED was applied at the crest of the head (n=13). KED was placed at the base of the skull, perpendicular to the neck, and the handles pressed together (n=12). Electrocution was applied at 110 V AC for 15 s to induce cardiac fibrillation (n=16). Effectiveness was assessed based on kill success after one attempt, broken skin (presence/absence), external blood loss (presence/absence), and durations of persisting nictitating membrane reflex, gaping (jaws opening/closing), and musculoskeletal movements. Nonparametric data were analyzed using Chi-square and duration data using the GLM procedure of SAS with means separated by Tukey's HSD test. There were no differences between methods for kill success; CO2 (13/13), KED (12/12), TED (12/13), and electrical (15/16). Broken skin (BS) and blood loss (BL) were more frequent in TED (13/13 BS, BL) and KED (10/12 BS, BL) than CO2 (0/13 BS, BL) and electrocution (0/16 BS, BL, P<0.0001). The induced nictitating membrane reflex duration for KED (174 s) was longer than TED, CO2, and electrocution (0, 0, 0 s, P<0.0001). Gaping duration after KED application persisted longer (145 s) than TED, CO2, and electrocution (0, 0, 0 s, P<0.0001). KED resulted in significantly longer duration of muscle movement (184 s) than TED (144 s) which was longer than both CO2 and electrocution (0, 0 s, P<0.0001). All methods were determined to be successful with CO2 and electrical euthanasia resulting in minimal skin damage or blood loss. Electrical euthanasia and CO2 resulted in the shortest duration of reflex and muscle movement, followed by TED and KED.
ID: 489

# EFFICACY OF A NICARBAZIN/MONENSIN, A NOVEL COCCIDIOSTAT COMBINATION PRODUCT, FOR THE CONTROL OF COCCIDIOSIS IN TURKEYS

M. Vereecken*(1), M. Marien(1), B. Dehaeck(1), M. Geerinckx(1), S. De vries(1), K. De gussem(1)

(1)Huvepharma NV, Antwerp, Belgium

* Corresponding author: monita.vereecken@huvepharma.com

The efficacy of Monimax® (nicarbazin/monensin) at a concentration of 40mg/kg (40ppm) nicarbazin and 40mg/kg (40ppm) monensin, administered in feed was assessed in turkeys after an experimental coccidiosis challenge under floor pen conditions.

Birds were reared without coccidiostats till the age of 13 days when they were allocated to the different treatment groups. Monimax® treated birds were compared to an infected untreated control (IUC) and an uninfected untreated control (UUC) group. Each group consisted of 6 replicate pens (6 BIG 9 males/pen). Daily weight gain (DWG), feed conversion ratio (FCR), intestinal lesion scoring (ILS) for the pathogenic E. meleagrimitis and E. adenoeides species and oocyst shedding were compared after an experimental challenge with coccidiosis. Statistical analysis methodology was in accordance with those outlined in the WAAVP guidelines for evaluating anticoccidial drugs in chickens and turkeys (Holdsworth et al., 2004). All tests were 2-sided and the level of significance was set at 5%.

At 16 days of age all birds in the IUC and Monimax® groups were inoculated with a mixture of Eimeria meleagrimitis and E. adenoeides (European origin). Birds from the UUC were sham-inoculated.

The challenge was successful as proven by the significantly lower DWG, increased FCR, higher average lesion scores and oocyst production in the IUC compared to the UUC.

Administration of Monimax® was able to significantly improve performance (DWG and FCR). In the acute (D16-23) and subacute phase (D23-D28) after challenge no significant differences were noted between the Monimax® supplemented birds and the UUC. ILS at D23 for E. meleagrimitis and E. adenoeides as well oocyst counts at D23 and D28 were significantly suppressed by Monimax® supplementation in comparison to the IUC.

The results from this trial demonstrate the efficacy of Monimax® (nicarbazin/monensin) for prevention of coccidiosis in turkeys. Under present study conditions, we can conclude, based on significant lower oocyst shedding, lower coccidiosis lesions and significant better performance, that the administration of Monimax® proved efficacious against coccidiosis infection in turkeys.

Holdsworth PA, Conway DP, McKenzie ME, Dayton AD, Chapman HD, Mathis GF, Skinner JT, Mundt HC, Williams RB; World Association for the Advancement of Veterinary Parasitology. WAAVP guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys. (2004), Vet. Parasit. 121: 189-212.
# DIETARY NITROGEN REDUCTION STRATEGIES IN TURKEY FEEDS

M. Kenny*(1), J.Ralph(1)

(1)Aviagen Turkeys Ltd, Chester, United Kingdom

* Corresponding author: mkenny@aviagen.com

## Introduction and Objective

Minimising nitrogen emissions from turkey production systems is a major priority for both the industry and turkey breeding sector. An evaluation was conducted applying various strategies to reduce dietary Nitrogen in commercial turkey diets.

## Materials and Method

A total of 1,968 male Premium male turkey poults were allotted in groups of 80 to 24 pens, stocking density was 3.2 birds/m2. Treatment 1 (TRT1), was formulated to typical commercial diet crude protein (CP) levels. TRT2 CP was reduced by 1% in absolute terms while in TRT3 CP was reduced by 2%, in both these treatments Lys, Met+Cys and Thr specifications were the same as in TRT1. TRT4 CP was reduced by 1% with amino acids L-Ile, L-Arg and L-Val added to balance these amino acids from P1 to P7 as per TRT1. TRT5 CP was reduced by 2% with amino acids L-Ile, L-Arg and L-Val added to balance these amino acids as per TRT1. TRT6 CP was the same as TRT1 for P1 to P4 diets while P5 to P7 diets were the same as per TRT3 P5 to P7 diets.

## Results and Discussion

There was no significant difference in bodyweight between TRT1 (control) and the other treatments however bodyweight was significantly higher (P<0.05) in TRT4 and TRT5 relative to TRT2 suggesting a bodyweight response to correcting amino acid balance of lower crude protein density diets. TRT4 and TRT5 showed a numeric improvement in FCR relative to all other treatments. There was no difference in breast meat yield (BMY) between TRT 1 and the other treatments, however BMY in TRT4 and TRT5 was significantly higher than in TRT2 and TRT3 (P<0.05) suggesting that readdressing the amino acid balance of the lower crude protein density diets recovered BMY to the level of the control. Estimated nitrogen intake was significantly lower (P<0.05) in TRT 3 relative to the control while all other treatments trended toward lower intake relative to the control. Litter nitrogen was analysed in pens fed treatments TRT 1, 3, 5 and 6 and followed a similar trend to nitrogen intake, lower levels in TRT 3, 5 and 6 relative to the control.

## Conclusion

The results of the trial demonstrate that significant reduction of dietary protein, through all phases of the growing period, is possible without compromising performance. However the entire profile of essential amino acids also needs to be considered. Reduction in dietary CP levels reduced nitrogen intake which had a positive impact on litter nitrogen levels.
LED LIGHTING COMBINATION EFFECTS THYROID HORMONES IN TURKEYS

B. Bartz\*(1), J.Grimes(1)

(1) North Carolina State University, Raleigh, United States

* Corresponding author: bmbartz@ncsu.edu

Lighting consists of three main components: daylength, intensity, and chromaticity with each potentially impacting bird well-being, including plumage. Triiodothyronine (T3) is the bioactive form of thyroid hormone facilitating feather growth and linked to photorefractoriness in turkeys. To quantify the effects of lighting regiments on turkey hen feather coverage, the objective of these two studies were to evaluate the effects of lighting combinations on T3 hormone levels in turkey hens. The first study consisted of long and short (18h & 12h) daylengths (DL) in combination with 5000K (5K) LED and 5K LED + RED LED in environmental controlled rooms (ECR); natural decreasing DL and 5K LED blocked for 18h in natural, curtain sided facility for a total of 6 treatments. The second study consisted of high and low intensity (10 footcandles (FC) & 2FC) in combination with 5K LED and 2700K LED in ECR; natural lighting conditions supplemented with 14h of 75 Watt incandescent bulbs to match DL but naturally fluctuating intensity for a total of 5 treatments. In both cases, blood was collected from the brachial vein and serum was analyzed using a commercially available RIA kit for T3. Data were analyzed using JMP14 as a one-way ANOVA and significance obtained using Tukeys HSD procedure at P0.05). Based on the first study, there was a significant difference in feather coverage recorded by infrared imaging at 9 WOA (P<0.0001). However, this same effect was not recorded in the second study which focused on light intensity (P=0.56). At 9 WOA, birds exposed to an 18h DL with 5K LED in an ECR had significantly higher T3 (P=0.001) than bird exposed to natural conditions, natural conditions + 18h DL 5K LED, and 12h DL of 5K LED in ECR (5.29, 3.98, 3.90, and 3.86 ng/mL, respectfully). Birds exposed to RED LED, both 18h and 12h DL in ECR, had an intermediate response (4.65 and 4.37 ng/mL, respectfully). Based on previous results using infrared imaging, these results in hormone levels do not directly correlate with the recorded feather coverage (R2=0.688). Therefore, actual feather coverage may be further affected by behavioral responses of birds under various lighting regiments which will have to be quantified further to analyze feather pecking and other detrimental behaviors to feather coverage.
EFFECTS OF DIETARY SUPPLEMENTATION OF ORGANIC, INORGANIC AND NANO PARTICLES OF CHROMIUM ON IMMUNITY, ANTIOXIDANT PARAMETERS AND NUTRIENT COMPOSITION OF BREAST AND THIGH MUSCLES OF TURKEY POULTS.

A. Deshmukh*(1), A.Bhattacharya(2), P.Shukla(2), V.Kumar(3), A.Prakash(4), B.Yadav

(1)Department of Poultry Science, DUVASU, Mathura, Parbhani, India

(2) Department of Poultry Science, DUVASU, Mathura, Mathura, India

(3) Department of Animal Nutrition, DUVASU, Mathura, Mathura, India

(4) Department of Veterinary Pharmacology and Toxicology, DUVASU, Mathura, Mathura, India

* Corresponding author: abhidesh249037@gmail.com

Trace minerals are routinely added to diets, involved in numerous biochemical functions, making them
essential for optimal bird growth and health. Chromium (Cr) is essential for poultry. Chromium in
poultry feed is basically supplemented in three forms i.e. organic chromium, inorganic chromium and
nano particles of chromium picolinate. There are very few research findings showing comparative
effects of dietary supplementation of different forms of chromium as feed additive in poultry including
turkeys. Hence, the present study was designed to study the effect of dietary supplementation of
organic, inorganic and nano chromium particles on the immunity, antioxidant parameters and nutrient
composition of breast and thigh muscles of turkey poults. One-hundred-and-twenty-day old straight
run turkey poults were divided into four treatment groups comprising of three replicates and ten birds
in each replicate. T1 birds were fed basal turkey diet, T2 were fed basal diet + inorganic chromium@
0.5 mg/ kg diet, T3 were fed basal diet + organic chromium@ 0.5 mg/ kg diet, T4 were fed basal diet +
nano chromium@ 0.5 mg/ kg diet. The general innate immune-competence status of turkey poults
were assayed by measuring two important immunocompetence traits, as antibody response to SRBC
and cell mediated immune response to PHA-P after 8 weeks of age (Corrier and De Loach, 1990).
Concentration of IgG, IgM and Cortisol was determined in the serum of experimental birds by ELISA
kits. Superoxide dismutase (SOD) activity in serum was measured using the method as described by
Madesh and Balasubramanian (1998). The MDA assay was done by the TBARS (Thio Barbituric Acid
Reactive Substance) method of Ohkawa et al. (1979). Samples of the experimental feed, breast
(pectoralis major) and thigh (ilio tibialis) muscles were processed and analyzed for dry matter (DM),
crude protein (CP), ether extract (EE) and total ash (AOAC, 1990). Chromium content of the
experimental feed, breast and thigh meat was determined by Atomic Absorption Spectroscopy. The
data were analyzed statistically as per the standard procedure (Snedecor and Cochran, 1989) and
difference between the treatment means were obtained by using Duncan multiple range test (Duncan,
1955). There was no significance difference observed in humoral immune response to 1% SRBC and
cell mediated immune response to PHA-P among the treatment groups. There was no significant
difference observed in serum concentration of cortisol, IgG and IgM. Serum SOD values were
significantly higher (Ppectoralis major) and thigh (ilio tibialis) muscle among the treatment groups.
Chromium content was significantly higher (P
VALUE OF AN AUTOGENOUS VACCINE AGAINST ORNITHOBACTERIUM RHINOTRACHEALE IN TURKEY BROILERS ON TECHNICAL PERFORMANCES AND ANTIBIOTIC REDUCTION

M. Mellal\*(1), J.Durand(2), C.Babin(3), C.Delanghe(4), A.Ballot(5), S.Castagnos(6)

(1)VET&SPHERE - RESEAU CRISTAL, Malestroit, France

(2)LABOVET- RESEAU CRISTAL, Beaupréau-en-mauges, France

(3)FILIAVET- RESEAU CRISTAL, Bressuire, France

(4)RIVIEREVET - RESEAU CRISTAL, Redon, France

(5) MCVET - RESEAU CRISTAL, Sable, France

(6) CEVA - Biovac, Beaucouze, France

Nowadays, more than ever, commercial turkey producers need more alternatives to antibiotics and medication.

In the EcoAntibio 2012, a French national plan aiming to reduce the risks of antimicrobial resistance in veterinary medicine, it was stipulated that autogenous vaccines are an attractive alternative for minor species including turkeys.

Statistical results of RESALAB laboratories of the « Cristal Group », shown that Ornithobacterium rhinotracheale (ORT) is the second cause of pathology diagnosed in commercial turkey production after colibacillosis requiring antibiotic treatments.

According to the French network of animal diseases, 92% of ORT isolates are from commercial turkey production in 2018. This bacteria is mostly isolated of pneumonia and arthritis (49,5% and 29,8% of isolates respectively). In case of septicaemia, ORT infections are third after E. coli and Enterococcus.

With this in mind and in the logic to promote ORT autogenous vaccines in commercial turkey production, a group of veterinarians RESEAU CRISTAL conducted a comparative study based on economic and antibiotic exposure indicators of two groups of turkey flocks, without autogenous vaccines (N=20) and with autogenous vaccines (N=20).

Currently in France, there are not a commercial vaccines for ORT and the only effective solution offered to turkey producers are the autogenous vaccines. But this solution is still for the moment not systematically used for various reasons and obstacles that we want to overcome through this study.
We collected the antibiotic prescription data for each flock in order to calculate indicators of exposure to antibiotics, farm and slaughter data for one economic approach.

The results of this study and the market expectations tip the balance of the comparison in favor of the group of flocks having received the autogenous vaccines. For example, for the number of treatments, we reduce from 1,3 for flocks without autogenous vaccines to 0,9 for flocks with autogenous vaccines, and the ALEA from 2,05 to 0,8.

This allowed us to build relevant communication tools in favor of this alternative solution as a means of essential medication for the meat turkey sector.

For further, we carried out a serological follow-up study after autogenous vaccines to assess the success of the vaccination. The results demonstrate the benefit of serology to evaluate good practices of autogenous vaccines activity.

<footnote>* Corresponding author: mokrane.mellal@orange.fr</footnote>
ID: 1116

ASSOCIATIONS BETWEEN WELFARE MEASURES ON FARM AND SLAUGHTERHOUSE DATA IN FLOCKS OF TURKEY HENS (MELEAGRIS GALLOPAVO)

J. Marchewka\*(1), G. Vasdal(2), R. Moe(3)

(1)Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Magdalenka, Poland

(2)Norwegian Meat and Poultry Research Centre, Oslo, Norway

(3)Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Oslo, Norway

\* Corresponding author: j.marchewka85@gmail.com

There is a demand for documentation of animal welfare status in meat production industry. Work on turkey welfare has focused on toms at the end of production cycle, since this period is considered most challenging. Information on the relationship between welfare on farm and slaughter house recordings is currently lacking in flocks of meat turkey hens. The aim of this study is to investigate the relationship between routinely collected registrations at the slaughter house from turkey hens and their welfare measures on-farm by means of the transect walks. The current study was conducted between November 2017 and March 2018 in 20 commercial turkey flocks on farms in Norway. On each farm, one barn, divided into tom and hen area, was evaluated using the transect walk method when the birds were 11 weeks old. An observer walked the transects in random order and recorded the total number of birds per transect that were: immobile, lame, with visible head-, tail- or wing- wounds, small, featherless, dirty, sick, terminal, or dead. Routinely collected data from the slaughter house was provided for each flock. We calculated Spearman correlations using the PROC CORR in SAS 9.4. The prevalence of birds identified at the slaughterplant with old wing fractures was positively correlated (r=0.52) with the percentage of birds with wing wounds on-farm. This suggest that old wing fractures noted at the slaugherplant can be a potential indicator of the birds' condition during the production cycle. The results also showed that a higher prevalence of wing wounds on the farms was associated with more airsacculitis identified at the slaughterplant (r=0.47). The wing fractures observed at the slaughterplant may result from the final period of birds' life on-farm, when the disease could be intensifying and when birds are suspected to flap their wings to support breathing. We found that featherless birds had a higher rejection rate due to odor at slaughter (r=0.45). Featherless areas may reflect feather pecking occurring on farm, which is a well-known issue in turkeys, since olfaction may play role in regulating bird behavior, and has been suggested to be involved in feather-pecking. On farm welfare of the hen turkeys can be retrospectively suspected based on parameters obtained from the slaughterplant, mainly related to wing fractures. Some like rejections based on the odour may be promising on-farm welfare indicators collected at the slaughter, however need further investigations.
TURKEY PRODUCTION AND HEALTH: CURRENT CHALLENGES

A. Koppenol*(1), A.Soares(2), M.Ingberman(3)

(1) Impextraco NV, R&D Department, Heist op den berg, Belgium

(2) Universidade Esadual de Londrina, Departamento de Ciencia e Tecnologia de Alimentos, Londrina, Brazil

(3) Universidade Federal do Parana, Imunova, Curitiba, Brazil

* Corresponding author: Hafez.Mohamed@fu-berlin.de

In the future, several challenges and problems, in addition to already existing, will face turkey production worldwide. These include strong global competition between countries and other food producing animals. Furthermore, the continuous changes of consumer perceptions with regard to food safety, animal welfare and environmental protection.

The loss of consumer confidence and trust in the quality and safety of poultry meat and poultry products will remain a continuous major challenge. Several foodborne bacterial infections such as Salmonella and Campylobacter are linked to poultry and poultry products. Control and/or elimination of these organisms present a great challenge. Furthermore, the spread of antibiotic resistant bacteria such as Vancomycin-resistant Enterococci (VRE) and Methicillin-resistant Staphylococcus aureus (MRSA) will also be a continuous public health hazard.

The future concept of animal health will cover not only the absence of disease in birds, but also the relationship between the health of animals and their welfare. It will also take into account social, economic and ethical considerations, as well as support the achievement of a high level of environmental protection.

Emergence and re-emergence of infectious turkey diseases will remain an important never ending challenge. Currently and in the future, only a few authorised pharmaceutical veterinary products will be available for the treatment of turkeys as food producing animals. The development of efficient vaccines against bacterial infections will lead to a reduction of the use of antibiotics and subsequently will reduce the development of resistant bacteria.

Continuous improvement of rearing technology, management and nutrition to maintain bird comfort and health are essential.

Finally, farmers, veterinarians, stockholders and all other partners involved in the production chain need to share more responsibilities.
THE PREDICTION POWER OF BODY WEIGHT WITH MACHINE LEARNING AND DATA MINING OF MICROBIOME AND PERFORMANCE DATA OF MALE TURKEYS FED FORMIC ACID.

K. Flores\*(1), A.Fahrenholz(1), J.Grimes(1)

(1)North Carolina State University, Raleigh, United States

* Corresponding author: kflores2@ncsu.edu

Formic acid has been used in poultry feed to reduce the microorganism load and for acidification purposes. It affects the microbiome and ultimately the body weight (BW). The objective of this effort was to apply Machine Learning (ML) and Data Mining (DM) algorithms to predict with high confidence the BW at 19 weeks of age and to data-mine the most important attributes (variables) for the model. Nicolas Select male poults (1056) were randomly assigned to 48 concrete-floor pens. The experimental design was a completely randomized block design with a one-factor arrangement of 4 levels of formic acid at 0 lb/ton, 4lb/ton, 8lb/ton, and 12lb/ton (Amasil Na®, BASF SE, Ludwigshafen, Germany). BW at 19 weeks of age was analyzed in SAS 9.4 in a mixed model. Birds' BW, weekly body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR), gut pH, and microbiome data were used as variables for the ML and DM analysis. A total of 352 variables were used in ML and DM in both continuous (regression) and categorical (classification) analyses. BW at 19 weeks of age was classified as low and objective based on a 10% for BW margin of the Aviagen male objectives at 19 weeks of age. Support vector machine, neural networks, linear models, decision trees, Bayes, and k-nearest neighbors algorithm were used for ML with a 10-fold cross-validation system using WEKA 3.8. As a categorical analysis, the Support vector machine (SMO) yields an average of 91 % correctly classified BW at 19 weeks of age with an optimal of 7 variables. In comparison, decision trees (M5P) created a model with an average correlation coefficient of 0.93 to predict BW at 19 weeks with 3 variables. Different levels of formic acid were not statistically significant or were part of both classification and regression models to predict BW at 19 weeks of age. Both models decrease in efficiency when the attributes for modeling used are closer to the placement. In conclusion, Formic acid and its potential change in the microbiome were not deemed as important by ML and DM algorithms in both regression and a classification models. The predictive and attributes model is different depending on the question asked and the age of the attributes when the prediction is done. For this effort predicting with a regression model would serve as the most practical for application in the field. These ML and DM tools could be applied to turkey research and production systems for prediction and attribute selection.
ID: 1290

EFFECT OF THE DIETARY ADMINISTRATION OF A YEAST FRACTION RICH IN MANNAN-
OLIGOSACCHARIDES AND B-GLUCANS (1,3 AND 1,6) ON FECAL MICROBIOTA AND PRODUCTIVE
PERFORMANCE OF TURKEYS

M. Zampiga*(1), F.Sirri(1), E.Rondel(2), G.Manfreda(1), A.Lucchi(1), A.De cesare(3)

(1) Department of Agricultural and Food Sciences, Alma Mater Studiorum - University of Bologna, Bologna, Italy

(2) Phileo by Lesaffre, Marcq-en-baroeul, France

(3) Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Bologna, Italy

* Corresponding author: marco.zampiga2@unibo.it

The dietary administration of prebiotic compounds is thought to positively affect productive efficiency and health of poultry mainly by modulating the gut microbiota composition. The aim of this study was to investigate the effects of the dietary administration of a yeast fraction rich in mannan-oligosaccharides and β-glucans (1,3 and 1,6) on fecal microbiota and productive performance of turkeys. A total of 1,620 one-d-old female poults (BUT Big 6) were randomly divided in 2 experimental groups (9 replicates of 90 birds/each): CON, fed a commercial basal diet, and TRT receiving the CON diet supplemented with the feed additive (500 g/ton). Productive traits were determined on a pen basis at the end of each feeding phase (16, 37, 56, 77, 91, and 106 d). Fecal samples were collected at 21 and 85 d (n=18 samples/group/sampling time) and microbiota analyzed using a 16S rRNA-seq-based approach. Turkey-Kramer t-test was applied to compare the means of the relative frequency of abundance of specific taxonomic groups identified in CON and TRT whereas productive performance data were analyzed through Student-T test. Microbiota analysis revealed interesting effects ascribed to the treatment. Feces from TRT turkeys were characterized by a lower abundance of Gammaproteobacteria (mainly Enterobacteriaceae including Klebsiella, Shigella, Escherichia, Enterobacter, Erwinia and Serratia) at 21 d (0.45 vs. 20.6%, for TRT and CON group, respectively; P<0.001) associated with a significantly greater amount of health promoting microorganisms at 85 d (i.e. Lactobacillus spp., Clostridium bifermentas and Pediococcus acidilactici). Growth performances of CON and TRT turkeys were similar till 91 d. However, TRT group reached a lower body weight at slaughter (9,508 vs. 9,658 g; P<0.05) due to worsen productive performance during the last feeding phase (92-106 d) which might be possibly ascribed to an overdosage of the feed additive for this particular period of growth. Taken together, these results indicate that the dietary supplementation of the yeast fraction determines positive effects on fecal microbiota composition of turkeys promoting healthy gut conditions; however, the dietary inclusion level requires additional insights and fine tuning for the last period of life.
EF11

EDUCATION
VICKY THE VIRTUAL CHICKEN

T. Crowley\*(1)

(1)Poultry Hub Australia, Armidale, Australia

\* Corresponding author: tamsyn.crowley@deakin.edu.au

As part of Poultry Hub Australia's training initiative, we have developed a virtual chicken experience. 'Vicky' the virtual chicken allows users to experience all parts of the chicken from inside out. This project aims to provide not only an interactive learning tool but also an engagement tool that will facilitate a greater understanding and interest in the Poultry Industry.

The experience is run through virtual headsets and responds directly to the user ensuring each experience is unique. Both meat and egg chickens can be explored separately allowing the user to experience what each bird looks like inside. It is possible to explore different layers of the chicken such as organs, skeletal, muscle, separately just as you would if doing a live dissection. The meat chicken focuses on the different cuts of meat and the laying hen highlights the reproductive system allowing users to explore where an egg is made. There is also a testing function that asks the user to select a specified part of the chicken with tweezers and place on the tray, providing direct feedback to the user on a screen that indicates if they selected the correct part. The testing function can be played in a challenge mode providing an output score and printable certificate. The testing function collects metrics that may be used by an assessor to evaluate a user's competency.

This interactive tool has applications across all levels of education. We have already received fantastic feedback from users within Australia and we look forward to bringing this resource to the international poultry industry and public.
QUALIFICATION AND CONTINUING EDUCATION FOR PEOPLE WORKING IN THE POULTRY SECTOR

F. Kaufmann*(1), L.Klambeck(2), I.Goy(1), H.Grygo(1), R.Andersson(1)

(1)Osnabrück University of Applied Science, Osnabrück, Germany

(2)Osnabrueck University of Applied Science, Osnabrueck, Germany

* Corresponding author: f.kaufmann@hs-osnabrueck.de

The framework of the poultry production is constantly changing. New production systems and techniques, an increasing importance of animal welfare as well as management tools and the current level of scientific knowledge are challenging the competences of people who work with poultry at different stages of production. Furthermore qualification and continuing education in order to approve, expand or renew knowledge becomes more and more important in quality assuranceschemes (e.g. supplier contracts) and is even required e.g. in the German Animal Welfare Act.Therefore, on the one hand people are ‘required’ to participate in further training and on the other hand people participate voluntarily in order to improve their competences. Thus, continuing education – and the certificates to validate that it has happened – is therefore mandatory for those involved in the poultry industry. This emerging demand opens the discussion for a closer integration of vocational training, continuing education and advanced further education. Presently, the “in” catchwords are “Dual Course Studies” and “Open Universities”. Within a project, funded by the German Federal Ministry for Education and Research (BMBF), the University of Applied Sciences Osnabrück founded the “Osnabrücker Poultry Academy” (OPA) which worked out a concept that may meet all demands and requirements and may be best explained using the example of the certificate course “Poultry Professional” (PP). The PP is an on-the-job certificate course targeting people working in the poultry sector who want or need to approve, renew or expand their knowledge and skills. The course, which comprises 300 hours workload and covers level 5-6 of European Qualification Framework (EQF), is designed as a blended learning education program combining online digital media with traditional classroom methods. The gained certification “Poultry Professional” and acquired knowledge must be maintained by collecting 20 continuing education credits (CEPS) within 2 years. Therefore the “Poultry Professionals” have state-of-the-art knowledge. However, the same program can also be completed on EQF level 6-7 which includes further examinations and workload (600 hours). During the project, which started 2017 and ended in 2020, we were able to train 50 persons who now hold a PP certificate and are now obligated to maintain their certificate through continous training. The PP course is offered twice a year in two different forms: block course (11 days) or 11 single courses (one course per month). The different types of offerings adress the heterogenic target group and their respective possibility to take the course part-time. Generally, the demand for the PP course is high and cannot sufficiently be matched at the moment. Thus, the OPA will expand their offerings in width and depth as continuing education offers quality assurance in the whole production chain and addresses inter alia the sensitive issues of food safety and animal welfare and thus, may promote the public image of the poultry industry.
INVESTIGATION OF RISK TOLERANCE AND RISK ATTRIBUTES OF POULTRY FARMERS IN TAIWAN

I. Chang\*(1), H.Kuo(2)

(1) Taiwan Livestock Research Institute, Tainan, Taiwan

(2)Taiwan Livestock Research Institute, Tainan, China

* Corresponding author: sandychang75@gmail.com

The perceptions of risk and risk management of farmers have considerable influence on their management and implementation of preventive strategy. The study investigated the agricultural background, finical background, and risk self-assessment of poultry farmers in Taiwan, to provide the references of management strategy and self-risk checking for poultry industry. There were 81.3% male respondents in 107 valid questionnaires (missing value under 30%), 81.7% were full-time farmers and 82.2% was the breadwinner of a family. 35.5% loaned from the farmers' association and 46.7% were own funds. There was a significant relationship between the source of funding and the full-time / part-time (P<0.01) and working capital (P<0.05). Most poultry farmers did not have a relevant academic background but hold actual experiences. 57.0% of respondents were second generation of farmer, 74.0% had more than 5-year farming experiences, and 84.9% had at least one agriculture-related job before thought only 9.3% had agricultural academic background. The average of self-assessment risk tolerance level of poultry farmers were 6.3, high risk tolerance group seemed higher than other groups divided by age-adjusted risk interval. We investigated the selection of various risk sources of poultry farmers including human, production, financial, sale and organizational risks, each question in each risk section were given a certain related risk tolerance score. All respondents were divided into three group, which were conservative group, steady group and active group, according to the total risk scores. The results showed that the risk tolerance scores in agricultural related experiences, breeding methods, expenditure sustain, acceptance of price fluctuations, acceptance of investment loss ratio, level of financial loss impact, stop loss disposal, increasing investment, joining the organization and information sharing were different among groups by ANOVA test. Conservative group had lowest risk tolerance scores in agricultural related experiences, breeding methods, acceptance of price fluctuations, level of financial loss impact, stop loss disposal, increasing investment, joining the organization and information sharing. The scores of expenditure sustain and acceptance of investment loss ratio were highest in active group. In general, poultry industry farmers in Taiwan were steady on the risk attributes of investment.
ID: 1350

# GAUGING USER ENGAGEMENT OF BACKYARD POULTRY VIDEOS ON YOUTUBE FOR TARGETED OUTREACH

M. Cadena\*(1), M.Pitesky(2)

(1)UC Davis, Davis, United States

(2) University of California Davis, School of Veterinary Medicine, Davis, United States

\* Corresponding author: mmcadena@ucdavis.edu

**Introduction.** Backyard poultry (BYP) ownership is increasing in the United States (NAHMS, 2013). However, there are limited resources available to educate BYP owners. On top of this, survey and anecdotal observations indicate that BYP owners have poor biosecurity practices (USDA, 2005). This combination of poor biosecurity practices (e.g. exposure to wild birds) and unregulated bird movement can facilitate the spread of disease among poultry flocks. Consequently, the need for a robust outreach program is essential with respect to public health, food safety, animal health and the environment.
**Objective.** Gauge user engagement of BYP videos on YouTube to develop online outreach programs and to characterize online information using network analysis.

**Methods.** YouTube videos from searching “biosecurity for backyard chickens” were webscraped using Google Chrome’s extension, web scraper io, and called “featured videos.” Videos that were suggested as videos to watch next were also scraped and called “recommended videos.” Web scraped videos were classified as “excellent”, “adequate” or “poor” based on their biosecurity recommendations. Number of views as reported by YouTube was used to gauge user consumption. Number of likes and dislikes were used to gauge user participation. An edge list was uploaded to Gephi, a network analysis software.

**Results and Conclusion.** The resulting network consisted of 282 nodes representing featured and recommended videos and 249 directed links. Overall, the network seems decentralized with a network density of 0.004. In terms of social media engagement, it seems that users are not consuming content related to backyard poultry as much as they could. The number of views of featured videos with excellent content ranged from 8 to 3,345 views. Furthermore, it seems that participation or interaction with videos is as very low with only a few people liking (less than 30) or disliking (1) a video. From an outreach perspective, it seems that public engagement with YouTube videos can be improved and studying videos with the most views and likes could help understand user preference.

## Literature Cited

NAHMS. (2013). Urban Chicken Ownership in Four U.S. Cities.

USDA. (2005). Part I: Reference of Health and Management of Backyard/Small Production Flocks in the United States, 2004.

https://www.aphis.usda.gov/animal_health/nahms/poultry/downloads/poultry04/Poultry04_dr_Par-tl.pdf.
EF12

PHYSIOLOGY

GENERAL PHYSIOLOGY

INCUBATION PHYSIOLOGY
GENERAL PHYSIOLOGY