THE COMMON PHEASANT GENOME PROVIDE UNIQUE INSIGHTS INTO ITS ADAPTATION AND EVOLUTION

C. He(1), L.Zhao(2), L.Xiao(1), K.Xu(1), J.Ding(1), H.Zhou(1), H.Yuan(3), X.Lu(2)(1)

(1)Shanghai Jiao Tong University, Shanghai, China

(2)Shanghai Animal Disease Control Center, Shanghai, China

(3)Shanghai Xinhao Rare Poultry Breeding Co. Ltd., Shanghai, China

* Corresponding author: mars-he@sjtu.edu.cn

The common pheasant (Phasianus colchicus), also called ring-necked pheasant, is a widely introduced game bird that originates from Asia. It has been the most consumed special poultry since it was reintroduced into Chinese market in 1980s. The pheasant is characterized by strong sexual dimorphism, thus an ideal model for sexual selection study. Here we present the de novo assembly of the common pheasant using single-molecule real-time sequencing, next-generation sequencing and high-throughput chromosome conformation capture approaches (Hi-C). Single-molecule sequencing polished with next-generation mapping generated an assembly with a contig N50 size of 1.33 Mb. Subsequent scaffolding by using Hi-C data generated 589 sequences with N50 size of 59.46 Mb, resolving 11 macro-chromosomes into single scaffolds. We annotated the genome using its transcript data, the method of ab initio gene prediction and homolog-based protein evidence, thus identified 23,058 genes. Quality assessment of the final assembly found 94% of the ring-necked pheasant canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies. Comparative analysis of 13 species identified 5,526 single copy orthologous genes of the common pheasant, based on which, a fully resolved phylogenetic tree was reconstructed with 100% bootstrap supports. The alignment between the common pheasant and the chicken chromosomes reveal high consistence. However, two inversions were found in the regions corresponding to chicken's MHC class I and class II genes, respectively. With the presence of reference, we performed whole genome re-sequencing to 15 individuals from 5 breeds. A total of 633 Gb of raw reads were mapped against our reference assembly, generating 12,696,645 variants across the whole genome. Selective sweep analysis was performed followed the work on Tibetan pigs. Some of the genes that showed artificial selection signatures regulate chicken's growth and productive traits, though further validation should be done for confirmation. Overall, we achieved a high-resolution reference genome assembly for the common pheasant by integrated 2nd and 3rd generation sequencing data. Having characterized the genome, we undertook detailed analyses of artificial selected genes and gene families to gain knowledge of this bird's macro- and micro-evolution.

Key words: common pheasant, de novo, MHC, comparative analysis, selective sweep
REFERENCE-GRADE GENOME ASSEMBLY OF WILD MALLARD (ANAS PLATYRHYNCHOS)

F. Zhu\*(1), J.Smith(2), R.Kuo(2), F.Martin(3), L.Eory(2), D.Ogeh(4), Z.Zhang(5), L.Lu(6)(7), S.Hou(7), N.Yang(1), Z.Hou(7)

(1) National Engineering Laboratory for Animal Breeding and MOA Key Laboratory of Animal Genetics and Breeding, Department of Animal Genetics and Breeding, China Agricultural University, Beijing, China

(2) The Roslin Institute & R(D)SVS, University of Edinburgh, Midlothian, United Kingdom

(3) European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, United Kingdom

(4) European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, United Kingdom

(5) College of Biological Sciences, China Agricultural University, Beijing, China

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

(7) State Key Laboratory of Animal Nutrition; Key Laboratory of Animal (Poultry) Genetics Breeding and Reproduction, Ministry of Agriculture; Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China

* Corresponding author: fengzhu@cau.edu.cn

Mallard duck has been long worldwide as a well model organism and considered ancestral to domestic ducks. The draft genome sequences are available but they are fragmented and incomplete, and built from domestic duck. In this study, we report high-quality genome assembly and annotation for wild mallard (CAU_wild1.0) by combining Pacbio single-molecular real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. We generated 112 Gb of genome sequences, with an estimated depth of coverage of 100x. After assembling and polishing, a total length of 1.2 Gb of sequences were produced with scaffold N50 of 77.62Mb. The final assembly includes 1665 scaffolds, of which the largest 41 super-scaffolds representing the chromosomes occupied 95.20% of all sequences. In this assembly, we predicted 17,953 genes with 28,562 annotated transcripts. Compared with previously published draft genome (CAU1.0), new genome assembly represents the significant improvement in contiguity (The scaffold N50 7-fold against CAU1.0). Another improvements of new assembly are achieved in genome gap-filing and accuracy assembly of highly complex regions. The number of gaps of new assembly is only 309 (about 1/37 of gap number of CAU1.0). Moreover, 502 avian "missing" genes are identified for the first time, which were considered in the complex regions. Our findings will accelerate evolutionary and functional genomics studies in birds and inform future breeding programs for economic traits of domesticats ducks.
A HIGH-QUALITY PEKIN DUCK GENOME ASSEMBLY PROVIDES NEW INSIGHTS INTO EVOLUTION DURING DUCK DOMESTICATION

Z. Yin\*(1), F.Zhu(2), F.Zhang(2), X.Li(2), J.Smith(3), R.Kuo(4), L.Eory(3), F.J. martin(5)(5)(2), L.Lu(6),
Z.Zhou(7), S.Hou(7), N.Yang(2), Z.Hou(7)

(1) China agriculture university, Beijing, China

(2) Department of Animal Genetics and Breeding, China Agricultural University, Beijing, China

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

(4)The Roslin Institute & R(D)SVS, University of Edinburgh,, Midlothian, United Kingdom

(5) European Molecular Biology Laboratory, European Bioinformatics Institute, Cambridge, United Kingdom

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

(7) Chinese Academy of Agricultural Sciences, Beijing, China

* Corresponding author: yinzhongtao@cau.edu.cn

Abstract

Pekin duck is one of the major global breeds of meat-type duck and account for an increasing market share. Although the previous version(CAU_1.0) of the genome is available, the short read length of the second-generation sequencing technology leads to poor assembly performance in the de novo assembly process for regions with high repeat structure and high heterozygosity in the genome, making it difficult to obtain longer fragment. In this study, we report high-quality genome assembly and annotation for pekin duck using Pacbio single-molecular real-time sequencing (depth coverage: 92.6x), BioNano optical mapping (depth coverage: 141.07x) and high-throughput chromosome conformation capture techniques (depth coverage: 101.92x). After assembling and polishing, a total length of 1.2 Gb of sequences were produced with contig N50 of 5.46Mb and scaffold N50 of 76.28Mb.Hic data was used to assemble scaffold into 40 chromosome-level sequences (39 autosomes and Z chromosomes), with an average chromosome loading rate of 95%. Compared with previously published Peking duck genome (CAU1.0), the N50 length of new assembled genome has increase of atleast 7 times at the scaffold level, and the number of GAPs is 1/37(559/20,679) of the latter. The resultsof the BUSCO showed an improvement of at least 3.3% in genomic completeness. In this assembly, 18507 protein coding genes were predicted using full-length transcripts. OC-17 plays an important role in the calcification of eggshells in poultry and our assembled pekin genome was the first poultry genome to obtain the complete genetic structure of OC17. Our study provides new insights into evolution during duck domestication and constitutes a promising resource on economically importantgenes in fowl.
# A NOVEL LONG NONCODING RNA LNCPRDM16 IDENTIFIED TO BE RELATED TO FAT DEPOSITION IN BROILERS

Y. Chen\*(1), R. Ding(1), S. Zhao(1), H. Li(1), Z. Du(1)

(1) Northeast Agricultural University (Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030,

* Corresponding author: 374989370@qq.com

## Introduction

For over half a century, commercial broiler has been selected intensively for growth rate and feed efficiency. However, intensive selection on fast growth rate brings along adverse outcomes, such as obesity and related metabolic syndromes. Currently, to reduce fat deposition is still a main objective of commercial broiler selection and breeding program. In recent years, noncoding RNAs, including microRNAs and long non-coding RNAs (lncRNAs), that regulate the proliferation and differentiation of adipocytes have been successively reported. A large number of lncRNAs have been identified to be of complicated functions in a wide variety of physiological and biological processes. However, research on the regulatory function of lncRNA on chicken adipose tissue development is still in its infancy.

## Materials and methods

The experimental animals were derived from the 23th generation of Northeast Agricultural University broiler lines selected divergently for abdominal fat content (NEAUHLF). Total RNAs were isolated from 19 tissues using Trizol reagent (Invitrogen). A cDNA synthesis kit from Takara (Dalian, China) was used for the synthesis of cDNA. The full lengths of the lncRNA were obtained by the FirstChoice RLM-RACE Kti (Thermo). The quantitative real-time PCR was performed to detect the expression patterns of LncPrdm16. Data were analyzed by the t-test.

## Results

We obtained the transcript sequences of LncPrdm16 from an unpublished RNA-seq experiments on abdominal fat tissues from NEAUHLF. The expression levels of LncPrdm16 were examined in various tissues from 7-wk-old fat and lean broiler lines. In fat and lean birds, LncPrdm16 was highly expressed in pancreas, moderately expressed in gizzard fat, crop fat, cerebrum, proventriculus, kidney and gizzard fat, and relatively low in heart, duodenum, pectoralis muscle and leg muscle. RACE analysis found that the full-length of LncPrdm16 was 996 bp using abdominal fat cDNA as template. In addition, LncPrdm16 was mainly expressed in mature adipocytes as compared to that of preadipocytes (p

Conclusion

Our results provide a foundation for future studies on the function of LncPrdm16 to adipose tissue growth and development in chickens.

§ Funding: The Chinese National Natural Science Foundation (grant no. 31472088).
A HIGH PERFORMING BLUE EGG LAYER LINE DERIVED THROUGH MARKER-ASSISTED INTROGRESSION INTO WHITE LEGHORN

A. Weigend(1), B.Andersson(2), C.Reimer(3), C.Dierks*(1), D.Cavero(4), H.Simianer(3), M.Schmutz(2), N.Ha(3), R.Preisinger(5), S.Weigend(1), T.Pook(3)

(1)Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics, Neustadt, Germany

(2)Lohmann Tierzucht GmbH, Cuxhaven, Germany

(3)University of Goettingen, Center for Integrated Breeding Research, Göttingen, Germany

(4)H&N International, Cuxhaven, Germany

(5)EW GROUP GmbH, Visbek, Germany

The objective of the present study is to demonstrate an efficient way to transfer a specific monogenic trait maintained in a gene bank, here blue egg shell color from the Araucana breed, into a contemporary high performing White Leghorn line (WL) by marker-assisted introgression. This project is part of the EU project IMAGE (Innovative Management of Genetic Resources). Blue eggshell color is inherited in a dominant way and is caused by a large insertion on chromosome 1 upstream of SLCO1B3. Since 2016, an initial F1 generation, two marker-assisted backcross generations (BC1 and BC2) and a final intercross generation (IC) were generated, aiming at a high performing homozygous blue layer line. Breed/line specific SNP markers surrounding the introgression locus were genotyped for the detection of recombinant animals using Merlin. Furthermore, genotypes from a 52K SNP array were used for the selection of animals with the highest proportion of the recipient genome and the highest degree of diversity. Selection was based on analyses with MoBPS. Through marker assisted selection, the WL genome content increased up to 91.9 % in the BC2 animals, which is 4.4 % more than the expected 87.5 % in a BC2 generation, and the proportion of the Araucana genome in the BC2 thus could be reduced by about 35% (8.1% instead of 12.5%). In 2019, an intercross population with a totalof 751 animals hatched (424 females, 327 males), of which 188 animals (116 females, 72 males) were homozygous carriers of the introgressed gene, 367 were heterozygous and 196 were non-carriers. Currently, analyses of genotyping results of the intercross generation are underway. Performance tests for the IC has not been finished yet, but will be reported. Preliminary analyses of performance data of the IC generation encompassing data until week of life 39 in comparison with commercial WL yielded promising results. The laying rate was quite similar between intercross and White Leghorn hens. The peak laying rate of IC animals reached 91.7 % in homozygous carriers, 91.1 % in heterozygous carriers, 94.0 % in non-carriers and 94.8 % in the WL control group. The eggshell strength increased from BC1 to BC2 up to 45 N on average, and was significantly lower than in WL control line (~50 N). The mean egg weight of 61.5 g in BC2 hens was about 2 g lower compared to the control group. Performance tests of the IC and genotyping results will enable to evaluate the success of the marker-assisted introgression.

<footnote>* Corresponding author: claudia.dierks@fli.de</footnote>
HOST TRANSCRIPTOME AND METABOLOMIC RESPONSES TO EIMERIA SPP INFECTION IN MEAT-TYPE CHICKENS

S. Aggrey*(1), M.Milfort(1), J.Foutz(1), A.Fuller(1), R.Rekaya(1)

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

* Corresponding author: saggrey@uga.edu

A study was conducted to identify transcriptome differences between two chicken genotypes infected with Eimeria (E.) acervulina and to understand the underlying gene expression changes and further delineate genotype-specific effects. Fourteen day old chicks of an unimproved (ACRB) and improved (COBB 500) genotype were orally infected with 2.5 x 10⁵ sporulated E. acervulina oocysts. At 6 day-post infection, 5 birds were bled for serum and the jejunum from each genotype and their controls were used for global transcriptome analysis. The serum was used to determine the global metabolomic profiles using ultra performance liquid chromatography/tandem mass spectrometry. There were 5,262 differentially expressed genes (DEGs) between the ACRB infected and controls, and 2,884 DEGs between COBB infected and controls. There were common pathways between ACRB and COBB genotypes in response to E. acervulina infection. Among the common pathways were actin cytoskeleton, MAPK signaling, focal adhesion and cell adhesion molecules. However, there were also genotype-specific pathways in response to the infection. Whereas Toll-like receptor and retinol pathways were specific to the COBB genotype, oxidative phosphorylation and calcium signaling pathways were specific to the ACRB genotype. Both ACRB and COB genotypes have shared pathways in responding to Eimeria spp infection, however, each genotype also show distinct transcriptome signature. From the metabolomic study, unique metabolic signatures were identified in fatty acid metabolism, beta-oxidation, oxidative stress and inflammation. There were also microbiome metabolites that were putatively associated with malabsorption during infection.
# COMBINED ATAC-SEQ AND TRANSCRIPTOME ANALYSIS TO IDENTIFY GENE REGULATION NETWORK AFFECTING CHICKEN GROWTH IN DUODENUM

X. Cao\*(1), Y. Wang(2), X. Hu(1)

(1) College of Biological Sciences, China Agricultural University, Beijing, China & State Key Laboratory of Agro-biotechnology, China Agricultural University, Beijing, China, Beijing, China

(2) College of Biological Sciences, China Agricultural University, Beijing, China & 2.State Key Laboratory of Agro-biotechnology, China Agricultural University, Beijing, China & College of Animal Science and Technology, China Agricultural University, Beijing,

\* Corresponding author: caoxuemin1991@163.com

## Background

Duodenum is important in food digestion and absorption and then provide nutrition for animal growth. The intestine length and body weight have the same QTL position[1], it's of great value to study chicken growth at the point of duodenum.

## Materials and Methods

Here we used Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) and RNA sequencing to profile the open chromatin regions and transcriptome in high and low body weight chicken duodenum.

## Results

According to the ATAC-seq analysis, peaks at open chromatin mapped to transcription start sites (TSS) regions, as well as intergenic and intron regions. The differentially expressed peaks are associated with signal transduction and transmembrane transport, which are the important parts of duodenum function. GO analysis showed that many of the differentially expressed genes were involved in the regulation of biological processes key to intestinal function, notably the neuroactive ligand-receptor interaction pathway has important role on chicken appetite and regulate growth[2]. We also found that 263 down-regulated genes with down-regulated peaks in high body weight chickens were significantly enriched in ion transport, amino acid transport, transmembrane transport and hormone metabolic process, we considered these genes as important candidates for further exploration to find genes affecting chicken growth.

Transcriptome and ATAC-seq data revealed that in most region (including TSS, exon, intron and transcriptional termination site), the chromatin accessibility increased when the gene expression increased, while in intergenic regions the chromatin accessibility decreased when the gene expression increased suggesting that there may be function elements inhibiting gene's expression in the intergenic regions.

## Conclusion

In conclusion, we found several pathway and biological processes regulating chicken growth together and the intergenic regions are associated with the gene expression inhibition.

## Reference

[1] S. Mignon-Grasteau, N. Rideau, I. Gabriel, C. Chantry-Darmon, M.Y. Boscher, N. Sellier, M. Chabault, E. Le Bihan-Duval, and A. Narcy, Detection of QTL controlling feed efficiency and excretion in chickens fed a wheat-based diet. *Genet Sel Evol* 47 (2015) 74.

[2] Z. Li, X. Liu, P. Zhang, R. Han, G. Sun, R. Jiang, Y. Wang, X. Liu, W. Li, X. Kang, and Y. Tian, Comparative transcriptome analysis of hypothalamus-regulated feed intake induced by exogenous visfatin in chicks. *BMC Genomics* 19 (2018) 249.
ID: 861

POPULATION HISTORY OF DOMESTIC JAPANESE QUAIL INFERRED FROM MITOCHONDRIAL GENOME SEQUENCES AND GENOME-WIDE SNP DATA

M. Nunome* (1), A. Hata (1), Y. Matsuda (1)

(1)Nagoya University, Nagoya, Japan

* Corresponding author: mtnunome@nuagr1.agr.nagoya-u.ac.jp

To understand the genetic diversity of domestic Japanese quail, we previously examined mitochondrial D-loop sequences and microsatellite (MS) markers for 19 Japanese quail populations (Nunome et al., 2017). The populations included nine laboratory lines established in Japan, six meat-type lines reimported from Western countries, one commercial population in Japan, and three wild quail populations collected from three Asian areas. The phylogenetic tree of D-loop sequences showed two distinct haplotype groups, MT-G1 and MT-G2. MT-G1 included a dominant haplotype, which represented most of the domestic populations and wild quail. MT-G2 was composed of minor haplotypes found for six laboratory lines, two meat-type lines, and a few individuals of commercial and wild quail. Genetic clustering analyses with microsatellite markers revealed two genetic groups, MS-G1 and MS-G2. MS-G1 was found for six laboratory lines. MS-G2 was seen in the other three laboratory lines, all meat-type lines, and commercial and wild quail populations. We have recently conducted a whole mitochondrial genome (mt-genome) sequencing and a genome-wide SNP genotyping: Mt-genome sequencing was performed using the Oxford Nanopore MinION platform. The mt-genome sequences of 22 individuals from six laboratories, two meat-type, and one wild quail populations have been determined. Genome-wide SNP data for one individual each from four laboratories and two meat-type lines were obtained with new genotyping technology, GRAS-Di (Enoki, 2019; Enoki & Takeuchi, 2018). Tentative phylogenetic study for each of mt-genome sequences and genome-wide SNP data supported the two genetically different groups of D-loop sequences and microsatellite markers, respectively. Thus, we assumed two genetic sources of domestic Japanese quail populations with different genetic backgrounds. One was represented by MT-G2 (D-loop) and MS-G1 (microsatellite markers), which may originate in the population that was exported to the United States from Japan before World War II. The other was represented by MT-G1 and MS-G2 and was probably derived from limited founder populations re-established in Japan after the war. Now we are carrying out detailed phylogenetic and population genetic analyses using mt-genome sequences and genome-wide SNP data for more individuals from various quail populations to infer the population history of quail populations from genetic data.
COMPARATIVE MICROSATELLITE ANALYSIS OF SIX HUNGARIAN NATIVE CHICKEN BREEDS

N. Sztán\*(1), N. Pálinkás-bodzsár(1), A. Hidas(1), T. Molnár(1)

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

* Corresponding author: nikoletta.sztan@gmail.com

Genetic diversity investigations of endangered breeds of farm animals have been going on for several
years. The aim of this study was to investigate the change of genetic diversity of six Hungarian
indigenous chicken breeds in 15 years.

In total 180 individuals were genotyped at 29 microsatellite loci, with 4-6 markers at the same time as a multiplex. Mean number of alleles, expected and observed heterozygosity, inbreeding coefficient and effective population sizes were calculated within populations, between populations Wright's fixation indices and pairwise FST, furthermore a cluster analysis was performed with Bayesian approach (STRUCTURE). Results obtained from populations sampled in 2002 (old populations) were compared to the stocks of the same breeds sampled in 2017 (young populations).

The range of basic diversity measures was generally more narrow and lower in the young stocks than
in the old populations, consequently the inbreeding coefficient was higher. While the old populations
were close to the Hardy-Weinberg equilibrium state, the expected and observed heterozygosity of the
young stocks differed significantly. The effective population size increased during 15 years for all
breeds, but the values of populations sampled in different periods showed quite low estimates
indicating lower genetic variability of the breeds. The pairwise FST estimates were considerable higher
in the young populations suggesting larger differentiation between them, likely due to genetic drift.
STRUCTURE results showed a clear clustering of the Hungarian populations. Based on Evanno's method,
the most likely clustering was K=6, assorting the old and young populations of the same breed as one
group. At higher level of K values the old and young flocks of the Black and Speckled Transylvanian
Naked Neck chickens split from each other respectively, suggesting larger distinction between the old
and young populations of those breeds.

There was no mentionable allele loss found in the Hungarian native chicken breeds investigated 15
years later; however one allele (frequency of 30%) seems to be missing from the Black Transylvanian
Naked Neck breed, may allude to a selection effect. The inbreeding level was generally higher in the
young populations which can be reduced using random sire rotation. With some improvements the
breed management of the Hungarian local chicken breeds can be an appropriate solution to conserve
the genetic variance.
# CLONING AND EXPRESSION ANALYSIS OF THE CHICKEN NEUREGULIN 4

Y. Guo\*(1), W. Wang(1), F. Mu(1), B. Ning(1), N. Wang(1)

(1) Northeast Agricultural University, Harbin, China

* Corresponding author: 1559247166@qq.com

## Introduction

Neuregulin 4 (NRG4), a member of the epidermal growth factor (EGF) family of extracellular ligands, is highly expressed in adipose tissues. The role of NRG4 in liver has been extensively studied in mammals and demonstrated that NRG4 can improve diet-induced metabolic disorders, including insulin resistance and hepatic steatosis. To date, the role of NRG4 in adipose tissue development and adipogenesis has not been fully explored. In the present study, we cloned chicken NRG4 gene, investigated its tissue expression profile and expression difference in abdominal adipose tissue between fat and lean chicken lines.

## Materials and methods

Twenty different tissues and organs were collected from the 7-week-old broiler chickens of the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). Total tissue RNA was extracted using Trizol reagent. Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser. Based on chicken NRG4 mRNA sequence, Primer 5.0 software was used to design the primers for gene expression analysis and the coding sequence cloning of chicken NRG4 gene. Quantitative real-time RT-PCR analysis was performed using the SYBR Premix Ex Taq and ABI Prism 7500 systems. Statistical differences were evaluated using two-tailed unpaired Student's t-test.

## Results

The full-length coding sequence (CDS) of chicken NRG4 gene was amplified by RT-PCR, cloned and sequenced. The sequence analysis showed that the full-length CDS of chicken NRG4 gene was 339 bp in length, encoding 112 amino acids. Nucleic acid sequence analysis showed that the NRG4 shared 24.9% and 40.65% sequence identity with human and mouse NRG4, respectively. Protein sequence analysis showed that chicken NRG4 shared 57.76% and 52.59% amino acid sequence identity with human and mouse NRG4, respectively. Real-time quantitative RT-PCR analysis showed that chicken NRG4 gene was expressed in all 20 tested tissues and organs, with the highest expression level in the kidney and the lowest expression level in the spleen. Unlike mammalian NRG4 gene, chicken NRG4 gene was moderately expressed in chicken adipose tissue. Further gene expression analysis showed that adipose NRG4 expression was significantly higher in the fat chicken line than in lean chicken line (p<0.05).

## Conclusion

Understanding the role and regulation of NRG4 in adipose tissue will provide insight into chicken adipose tissue growth and development.
# PREDICTING THE FUNCTIONAL CONSEQUENCES OF NON-SYNONYMOUS SINGLE NUCLEOTIDE POLYMORPHISMS IN LEPR GENE IN CHICKENS

Y. Li(1), C.Zhang(1), Z.Li(1), S.Wang(1), H.Li(1), N.Wang(1)

(1)Northeast Agricultural University(1 Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin 150030, China), Harbin, China

* Corresponding author: 710827104@qq.com

## Introduction

LEPR, a high-affinity receptor for leptin, is the key to the role and function of leptin. LEPR participates in many processes such as fat metabolism and appetite regulation in chickens. The objective of the current study is to identify the functional consequences of non-synonymous single nucleotide polymorphisms (nsSNPs) of LEPR gene in chickens based on bioinformatics tools and association analysis.

## Materials and methods

In this study, the 19th generation broilers from the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) were used as experimental materials. The SNPs on the exon of LEPR gene were screened by whole-genome re-sequencing. A variety of online bioinformatics tools (SIFT, Polyphen-2, PhD-SNP, SNPs & GO, SNAP) were used to predict the functional SNPs. The conservation of mutant amino acids was analyzed by the online multi-sequence alignment tool Clustal-Omega and ConSurf. The stability was analyzed by I-Mutant3.0 and Mupro. The secondary and tertiary structure changes of the LEPR protein were modeled by SOPMA and Modeller 9.23 software, respectively. Finally, we used a mixed linear model to analyze the association between SNPs and phenotypic traits (abdominal fat weight and abdominal fat percentage).

## Results

This study indicated that two possible functional nsSNPs (Exon19-N867I, Exon20-C1002R) may lead to significant changes in the structure and function of the LEPR protein. Association analysis showed that Exon20-C1002R was significantly correlated with abdominal fat weight and abdominal fat percentage. This significant mutation site is the first to be identified and further laboratory validation is required.

## Conclusions

Based on bioinformatic prediction and association analysis, it is concluded that Exon20-C1002R is a functional SNP that could change in the physical and chemical properties of LEPR protein. The findings will provide a new insight into lipid formation and help decrease abdominal fat in broiler using marker-assisted selection (MAS) in further broiler breeding program.
GENETIC DIVERSITY AND EVOLUTIONARY HISTORY OF THAI NATIVE CHICKENS AND WILD RED JUNGLEFOWL INFERRED FROM MITOCHONDRIAL SEQUENCES

A. Hata*(1), M.Nunome(2), P.Duengkae(3), T.Suwanasopee(4), S.Koonawootrittriron(5), K.Srikulnath(6), Y.Matsuda(7)

(1) Nagoya University, Nagoya, Japan

(2) Avian Bioscience research center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

(3) Forest Biology Department, Faculty of Forestry, Kasetsart University, Bangkok, Thailand

(4) Tropical Animal Genetic Unit (TAGU), Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand

(5) Tropical Animal Genetic Unit (TAGU), Department of Animal Science, Faculty of Agriculture, Bangkok, Thailand

(6) Laboratory of Animal Cytogenetics and Comparative Genomics (ACCG), Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, Thailand

(7) Laboratory of Avian Bioscience, Department of Animal Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

* Corresponding author: ayano.h@ku.th

Molecular phylogenetic studies of domestic chickens have been performed for a wide variety of chicken breeds throughout the world to understand their geographical origins and genetic diversities (Liu et al., 2006; Miao et al., 2013). To understand the evolutionary history of domestic chicken populations, genetic characterization of their wild progenitor, the red junglefowl (Gallus gallus, RJF), is prerequisite; however, their population genetic surveys are still limited (Akishinonomiya et al., 1994; 1996).

In this study, a part of the mitochondrial DNA D-loop region (780 bp) were sequenced for 298 RJFs (G. gallus and G. g. spadiceus) from 12 populations in Thailand and 138 chickens of 10 native chicken breeds from Thailand (Betong, Black meat chicken with white/black feather, Luarg Hang Khaw, Chee, Pradu Hang Dum, Kheaw Palee, Decoys, fighting-cock) and Vietnam (Dong Tao). Phylogenetic trees were constructed using D-loop sequences obtained in our study and more than 5,000 sequences retrieved from Genbank. Demographic histories of haplogroups of D-loop sequences (e.g. Haplogroup A-I) were inferred by Bayesian skyline plot analysis. Twenty-nine out of 44 haplotypes found in our study were newly discovered haplotypes, and 15 of them were only found in RJFs. In addition, a rare haplogroup-like lineage, which may have separated from other haplotypes around 2,000 years ago, was constructed by one new haplotype found in three RJFs. These results suggest that some haplotypes of RJFs were never involved during the process of chicken domestication. Bayesian skylineplot analyses indicated that four major haplogroups (A, B, CD and E) exponentially increased their lineages from 250 to 500 years ago, and particularly, two waves of lineage diversification occurred in Haplogroup A. Thai fighting-cook had the Haplogroup H, which is also found in Japanese fighting cooks (Shamo) in Okinawa (Komiyama et al., 2003), suggesting the possibility that Shamo chicken in Okinawa originated from Thailand.

It is well known that the distribution of domestic chickens spread throughout the world by human migration, culture contact, and trade after chicken domestication. Our present results reconfirmed the a long history of strong relationship between chickens and humans. We are now conducting whole mitochondrial genome sequencing for RJFs to perform highly resolved demographic analysis with about 150 mitochondrial genome sequences of chickens deposited on Genbank.
# INVESTIGATING ALLELE SPECIFIC EXPRESSION IN CHICKEN SPLEEN TRANSCRIPTOME RESPONSES TO AVIAN PATHOGENIC ESCHERICHIA COLI INFECTION

M. Monson\*(1), M.Kaiser(1), A.Wolc(1), S.Lamont(1)

(1)Iowa State University, Ames, United States

* Corresponding author: msmonson@iastate.edu

Colibacillosis, caused by extraintestinal infections with avian pathogenic Escherichia coli (APEC), negatively impacts poultry health, welfare, and performance. In chickens, exposure to APEC can reduce growth and egg production, and virulent strains, such as APEC O1:K1:H7, can even cause high mortality. Our long-term goal is to reduce the negative impacts of APEC infections through novel strategies for veterinary control or selective animal breeding. This study aims to improve our understanding of host functional responses to APEC by identifying allele specific expression (ASE), or allelic imbalance, in the chicken spleen transcriptome that occurs in the early stages of infection. APEC-associated ASE SNPs act as markers for cis-acting regulatory elements that affect the host response to APEC, which could be potential selection targets to improve resistance to APEC. For pathogen challenge, F1 progeny from reciprocal crosses between Fayoumi (disease-resistant) and broiler (disease-susceptible) chicken lines were inoculated at 14 days of age with APEC O1:K1:H7 or sterile PBS via intra-airsac injection. Samples of spleen tissue were collected 1 or 2 days post infection (DPI) and used for RNA-sequencing (RNA-seq) on the Illumina HiSeq 3000 (n = 5-6 samples/F1 cross/inoculation type/DPI). Resulting reads were mapped to the chicken genome (Galgal6a) and used to identify SNPs and to detect ASE at these loci (with the GATK4 pipeline). Significant ASE was detected within individual samples for 8,832 SNPs, of which 3,054 (almost 35%) were unique to APEC infected samples and were confirmed to be significant across biological replicates (at the group level). The Fayoumi sire x Broiler dam cross had a greater number of ASE SNPs at 1 DPI, while the Broiler sire x Fayoumi dam cross had more at 2 DPI. Many of these APEC-specific ASE loci were identified within genes that also had significant differential expression under infection, including SERPING1 and MGST1. These loci were also used for functional analysis in Ingenuity Pathway Analysis (IPA) to understand pathways where APEC induces cis-regulation of gene expression. Splenic ASE revealed the importance of cis-regulation in transcriptome responses to APEC and provided targets to investigate for markers of resistance to colibacillosis. Support: This work was funded by USDA-NIFA-AFRI #2015-67015-23093 as part of the joint NIFA-BBSRC Animal Health and Disease program and Hatch project #5424 and #5458.
THE FUNCTIONAL ROLE OF CELLULAR SENSOR IN THE RECOGNITION OF RNA LIGAND IN CHICKEN

K. Chungu\*(1), S.Lee(1), Y.Park(1), S.Woo(1), D.Rengaraj(1), J.Han(1)

(1)Seoul National University, Seoul, South Korea

* Corresponding author: frkellychungu@gmail.com

Sensing and recognition of pathogens is the first line of defense in host innate immunity. The host pattern recognition receptors (PPRs) recognizes the pathogen-associated molecular patterns (PAMPs) and interact with adapter proteins to form complexes that subsequently triggers an antiviral state via the interferon-mediated signaling pathway. In mammals, RIG-1 has been identified as a major PRRs for RNA viruses [1]. However, in avian species such as chicken, the RIG-1 is lacking implying that chicken could be more susceptible to RNA virus mediated infections. Since RIG-1 has been lacking in chicken, we investigated to identify which PPRs could compensate for the function of RIG-1.

In order to investigate which PPR is critical for recognition of RNA ligands, we first assessed mRNA expression of cellular sensors in DF-1 cells in response to poly I:C or influenza virus and then conducted knockdown studies to assess the functional effect on IFN-β activity. Thereafter, we established knockout cell lines to identify which PPR plays a pivotal role in the establishment of antiviral innate immune response of IFN-β activity. Significant differences between mock and poly I:C-treated or AIV groups were determined by one-way ANOVA with Turkey's multiple comparison, and differences of relative mRNA levels between groups with different treatments were conducted by the Student t test.
A value of P < 0.05 indicated statistical significance.

In our results, in response to poly I:C or influenza infection, the mRNA of PPRs related genes in DF-1 cells were significantly upregulated due to IFN-mediated positive feedback. Then our knockdown revealed that two of the PPRs are involved in the induction of interferon-mediated innate immune response in response to RNA ligand. By using CRISPR/Cas9 gene knockout, we found that, of the two identified cellular, one cellular receptor had the immune response impaired while the other could still stimulate immune response. In double knockout studies, we identified that of the two sensors, one could abolish the recognition of RNA ligands thus been the major receptor while the other sensor was secondary.

In conclusion, we demonstrated that even in the absence of RIG-1, chicken has a potent sensor of RNA ligands that regulate IFN-β pathway.

Reference

1. Kato, H., et al., Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature, 2006. 441(7089): p. 101.
INTEGRATED GENOME-WIDE MRNA AND PROTEIN EXPRESSION FOLLOWING SALMONELLA ENTERICA
SEROVAR ENTERITIDIS INOCULATION IN CHICKEN CECUM

Y. Wang(1), X.Miao(1), H.Li(1), Y.Dong(1), Y.Zhao(1), L.Liu(2), X.Li(1)

(1)Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention,
College of Animal Science and Technology, Shandong Agricultural University, Taian, China

(2)College of Life Science, Shandong Agricultural University, Taian, China

* Corresponding author: xyli@sdau.edu.cn

Salmonella enterica serovar Enteritidis (S. Enteritidis) can infect a wide range of hosts and cause food-
borne disease in the world. Poultry is considered as a primary source of Salmonella Enteritidis infection.
The correlation between mRNA and protein responding to S. Enteritidis infection remains unclear.
One hundred twenty two-day S. Enteritidis free Jiningbairi chickens were divided into two groups.
Chickens in inoculated group were orally inoculated with S. Enteritidis. Chickens in non-inoculated
group were mock inoculated with PBS. The cecum were collected at 3 days post inoculation for RNA
and protein isolation. The genome-wide transcriptome and proteome in cecum were analyzed through
bioinformatic tool.

There were 365 significantly differentially expressed genes including 200 up-regulated and 165 down-
regulated genes (P < 0.05, fold-change > 2.0). The up-regulated genes were mainly associated with
TLR6 signaling pathway, detection of diacyl bacterial lipopeptide, cellular response to interferon-
gamma, regulation of inflammatory response, cellular response to tumor necrosis factor, immune
response, inflammatory response, innate immune response, T cell receptor signaling pathway. Down-
regulated genes were related with multicellular organismal water homeostasis, sodium ion
homeostasis and glutathione metabolic process. There were 563 differentially expressed proteins
consisting of 225 up-regulated and 338 down-regulated ones. Enriched immune-related GO-BP terms
associated with differentially expressed proteins were defense response to bacterium, acute-phase
response and positive regulation of protein secretion. There were 74 differentially expressed mRNA
and 57 proteins matched. Related mRNA-proteins with same regulation direction were associated with
enriched in GO terms of oxidation-reduction process, JAK-STAT cascade, retina homeostasis,
endodermal cell differentiation and immune response. Related mRNA-proteines with reverse
regulation direction were associated with enriched GO terms of substrate adhesion-dependent cell
spreading, extracellular exosome, plasma membrane, adherens junction and brush border.

Transcriptome and proteome play different roles in the response to S. Enteritidis inoculation. The immune-related function was mainly associated with transcriptome, however, metabolism mainly associated with proteome.

This project was supported by the NSFC (31601980, 31872343), SDAIT (SDAIT-11-02), SDNSFC (ZR2018MC026) and SDASP(2017LZN007).
ID: 1103

# GENOME-WIDE VARIATION FACILITATES RESPONSE TO BIDIRECTIONAL SELECTION FOR SHEEP RED BLOOD CELL ANTIBODY RESPONSE IN CHICKENS

Q. Li\*(1)

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

* Corresponding author: liqinghe@caas.cn

## Objective:

Disease resistance is an important economic trait in the production of broilers. Long-term selection experiments provide a powerful approach to obtain empirical information on adaptation, allowing researchers to discover the objectives of the selection and infer their contributions to the mode and pace of adaptation. Here we implement a 55K SNP chip approach to investigate the consequences of 10 generations of bidirectional selection in White Leghorn chickens for a humoral immune trait: antibody response to sheep red blood cells.

## Material and methods :

Three groups were included in the study (up selection line, down selection line and the control group). At 56d of age, 30 birds from each group were intramuscularly injected with 1ml of 25% SRBC diluted in PBS. The peripheral blood samples were collected from all the chickens from the wing vein of each bird at 6d after challenged (on day 56). Antibody titer to sheep red blood cells (SRBC) was determined. Genomic DNA was isolated from peripheral blood and hybridized to a chicken 55k DNA chip.

## Results:

We observed the broad variation in the chicken genome in response to this selection regimen. Many genomic regions were highly differentiated as a result of this experimental selection regimen, although genetic drift has certainly contributed to this. Pathway enrichment analysis showed immunoglobulin, immune response, deubiquitination superoxide-generating NADPH oxidase activity, toll-like receptor signaling pathway have been significantly enriched in the candidate genes. We also identified five strong candidate genes associated with response to SRBC, including NFKB1, NOX1, TLR3, TLR15, and USP1.

## Conclusions:

Adaptation to bidirectional selection for antibody response in this experimental chicken population has been facilitated by revealing genetic variation across many regions of the genome, functional studies highlight four particularly interesting candidate sweeps and underlying candidate genes involved in immune response.
THE ANALYSIS OF SPLEEN TRANSCRIPTOME RESPOND TO INFECTIOUS BRONCHITIS VACCINATION IN
TAIWAN COUNTRY CHICKEN

S. Chuwatthanakhajorn(1), P.Tang(2), C.Chen*(2)

(1)Mahidol University, Nakhon pathom, Thailand

(2)National Chung Hsing University, Taichung, Taiwan

* Corresponding author: cfchen@dragon.nchu.edu.tw

Vaccination is the first line to protect chicken from severity of diseases, then rapid and high antibody
level responses to vaccine are achieving results after vaccination. Local chicken breeds usually have
better disease resistance. Our previous studies have shown that Taiwan Country chicken can induce
high antibody level response to Marek's and Newcastle disease vaccines in a short period. In addition,
Taiwan Country chicken has more resistance to coccidiosis and Leucocytozoonosis compared with
White Leghorn chicken. To find out the genetic effects on immune responses of Taiwan Country chicken
compared to White Leghorn chicken. B strain, Taiwan Country chicken which selected for body weight
at 8 weeks of age over 30 generations were used in this experiment. B strain and Single Comb White
Leghorn were vaccinated with live attenuated infectious bronchitis virus (IBV) vaccine
(2(3.6log10EID50)) via a nasal route at 3 weeks of age. Spleens from control and vaccinated groups
from both breeds were collected at day 7 post-vaccination then analyzed with RNA-sequencing. The
results indicated that after comparing between B strain and Single Comb White Leghorn, In vaccinated
group, B strain chicken show higher expression in C-type lectin domain family 2 member L-like and IgG
Fc-binding protein-like responses to vaccination. While in White Leghorn chicken, T cell-related genes
like T-cell surface glycoprotein CD8 alpha chain-like and T-cell surface glycoprotein CD8 alpha chain-
like were highly expressed. In unvaccinated group, B strain was greatly expressed in immune related-
genes including, interleukin 4 induced 1, interleukin 6, interleukin 22, glutamate decarboxylase2. In
another way, White Leghorn chicken highly expressed genes were interleukin 1 receptor-like 2,
mitogen-activated protein kinase 10, and T -cell-interacting, activating receptor on myeloid cells
protein 1-like responses to IBV vaccination. In conclusion, differences in gene expression will affect the
pattern of immune responses and this study provides the basis for the future study about immune-
related genes responses to IBV vaccination in Taiwan Country chicken.
THE INTEGRATION OF MULTI-OMIC DATA IN MULTI-TISSUES FROM LAYERS DIVERGENT FOR FEED EFFICIENCY PROVIDES SOME INSIGHTS INTO THE MOLECULAR MECHANISMS ASSOCIATED WITH THIS COMPLEX TRAIT.

F. Jehl(1), M.Boutin(2), C.Desert(2), D.Gourichon(3), C.Klopp(4), D.Laloë(5), Y.Ramayo-caldas(6), F.Pitel(7), T.Zerjal(5), S. Lagarrigue*(2)

(1)INRA - AGROCAMPUS OUEST, Saint-gilles, France

(2)AGROCAMPUS OUEST, Rennes, France

(3) INRA, Nouzilly, France

(4) INRA, Castanet-tolosan, France

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

(6)IRTA, Caldes de montbui, Spain

(7)INRA - INPT-ENSAT - INP-ENVT, Castanet-tolosan, France

* Corresponding author: sandrine.lagarrigue@agrocampus-ouest.fr

Improving feed efficiency remains a challenge to meet the needs of a growing human population and to contribute to the sustainability of the livestock sector. Improving feed efficiency also contributes to improve production efficiency considering that feed represents up to 60% of the production cost in monogastrics, and to reduce the competition between livestock feed and human food for the use of cereals.

In this work, we aimed at better understanding the pathways involved in feed efficiency in layer chickens. We used two experimental lines divergent in feed efficiency thanks to 40 years of selection that led to efficient birds, the R- and inefficient ones, the R+. First, we conjointly analyzed their transcriptomes in the adipose tissue, blood, hypothalamus and liver in order to determine the contribution of each tissue in the lines differentiation, and to identify genes and pathways shared between tissues. To this end, we realized a Multiple Factor Analysis (MFA) that provided a good separation between lines at the first factorial plan.

We identified approximately 7000 genes contributing to this axis with roughly 1000 shared by at least two tissues and less than 100 shared by the four tissues. Using regularized Canonical Correlation Analysis or co-Inertia Analysis, we produced correlation-based networks that we are currently studying to find potential hub- and regulator- genes. Moreover, using a recently produced (Jehl et al, Sci Rep., submitted) annotation of the chicken genome enriched in long non-coding RNA genes (LNC) known to be regulatory gene, we are analyzing the line difference in LNC. For the liver, transcriptomic data were enriched with lipidomic and metabolomic data and for the adipose tissue with lipidomic data. We conjointly analyzed these data within each tissue, selected between 1000 and 2000 variables found by MFA to separate the two lines, and observed interesting patterns of connectivity between genes and molecules in the resulting networks. In particular, we found genes highly correlated with different lipids and metabolites, some of which being known regulators of fatty acids metabolism. Overall, this work provides interesting insights in the mechanisms at work at the molecular level in different tissuesin relation with contrasted feed efficiencies in layer chickens. This work was supported by the French National Research Agency (Chickstress project - ANR-13-ADAP) and Europe (Feed-a-Gene project - grant agreement no. 633531).
GENOMIC SNP DETECTION BY RNA-SEQ: LESSONS FROM MULTI-TISSUE & MULTI-POPULATION DATA ANALYSIS IN CHICKENS

F.Jehl(1), M.Bernard(2), F.Degalez(1), L.Lagoutte(1), M.Coulee(1), S.Leroux (3), O.Bouchez(4), M.Tixier-Boichard(5), T.Burlot(6), D.Gourichon(7), B.Bed'hom(5), T.Zerjal(5), F.Pitel(3), C.Klopp(8), S.Lagarrigue*(1)

(1)PEGASE, INRA, AGROCAMPUS OUEST 35590, Saint Gilles, France

(2)SIGENAE, GABI, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France

(3)GenPhySE, INRA, Centre Occitanie-Toulouse, 31326 Castanet-Tolosan, France

(4)GetPlagGE, GenPhySE, INRA, Centre Occitanie-Toulouse, 31326 Castanet-Tolosan, France

(5)GABI, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France

(6)NOVOGEN, Mauguérand, 22800 Le Foeil, France

(7)PEAT UE, INRA, Nouzilly, France

(8)SIGENAE, GenPhySE, INRA, Centre Occitanie-Toulouse, 31326 Castanet-Tolosan, France

The RNA-seq, a technology that gives access to tissue transcriptomes, is mainly used for gene expression analysis to address a variety of scientific questions in a given species. However, since this technology involves a sequencing step, it can also be used to detect DNA variants in the expressed regions of the genome, thus allowing to take advantage of the accumulation of this type of data in multiple tissues and populations.

The identification of a large number of variants in the genome is crucial to unravel the relationship between genotype and phenotype, to analyze the genetic diversity between populations or to detect variants with deleterious effect in protein-coding regions.

However, the transcriptome is much more complex than the genome, thus requiring specific bioinformatic tools for sequence data analysis: it is characterized by mature transcripts corresponding to spliced genomic regions that can have highly variable expression levels, leading to depths of a few reads to tens of thousands of reads from one genomic position to another. Nevertheless, few pipelines have been proposed since 2013 for detecting reliable SNPs in RNA-seq data, but no benchmarking has been undertaken. Here we used the reference tools for RNA-seq sequence alignment (STAR-2-pass) and for variant calling (PicardTools and GATK pipeline). We analyzed 700 RNA-seq samples from more than 300 chickens belonging to 8 experimental and commercial layer and broiler populations with more than 16 individual per population.

We first compare SNPs detected from RNA-seq data with those detected from DNA-seq data in two populations for which both data types, on the same individuals and tissue, were available and show the reliability of the SNP detection procedure with RNA-seq data and commented the filters proposed by GATK. We then provide a description of the SNPs detected from RNA-seq data for the eight populations, obtaining an overview of the SNP distribution at the population scale, with several millions of reliably detected SNPs. We refine this analysis by analyzing these SNPs at the individual scale, selecting those with reliable genotypes resulting in hundreds of thousands of SNPs with a call rate > 95% for which we provide their frequencies in each population. Finally, we describe some interesting variations across populations.

This study was supported by INRA (Elastic project), the French Research National Agency (FatInteger and ChickStress projects) and Europe (Feed-a-Gene).

<footnote>* Corresponding author: sandrine.lagarrigue@agrocampus-ouest.fr</footnote>
COMPLETE TRANSCRIPTIONAL INSIGHTS INTO KEY GENES AND PATHWAYS INFLUENCING EGG YIELD IN NANDANYAO CHICKENS

T. Sun*(1), C.Xiao(1), Z.Yang(1), J.Deng(1), X.Yang(1)

(1)Guangxi university, Nanning, China

* Corresponding author: 1592972106@qq.com

**INTRODUCTION:** Nandan-yao chicken is an excellent local breed in Guangxi, which has been included in ANIMAL GENETIC RESOURCES IN CHINA POULTRY. Egg production is a complex economic trait controlled by multiple genes with low heritability. Therefore, this study intends to use RNA-seq to analyze the full transcriptional state of the ovary and uterine tissues of Nandan-yao chicken with high and low egg production, and to screen out important candidate genes and related regulatory pathways related to egg production.

**MATERIALS AND METHODS:** 300 Nandan-yao chickens, raised to 60 weeks old, and recorded their egg production. The three chickens with the highest egg production were selected as the high-yielding group (185 eggs), and the three with the lowest egg production were selected as the low-yielding group (82 eggs). Uterine and ovarian tissues from high- and low-yielding egg groups were collected for transcriptome sequencing. FASTQC, HISET2, htseq-count, DEseq2, cluster profiler and other software were used to process data. The differentially expressed genes were screened by the general criteria (log2|Fold Change|>1&FDR)

**RESULTS:** In the ovary, the numbers of differentially expressed mRNA, IncRNA and circRNA, respectively, were found to be 95, 558, 5, respectively. GO and KEGG analysis revealed their target genes were enriched in extracellular matrix regions for regulation, intracellular parts, protein binding, and immune system processes, etc. In addition, 16 differential miRNAs were screened (p, the numbers of differentially expressed mRNA, IncRNA, miRNAs, circRNA were found to be 402, 1421, 97, 45 respectively. GO and KEGG analysis revealed their target genes were enriched in ECM receptor interactions, oxidative phosphorylation, cell cycle, DNA metabolism, DNA methylation, cell cycle, oocyte meiotic division, cell metabolism, metabolic process of organic matter, etc. Among them, the ECM receptor interaction pathway is significantly enriched in both uterine and ovarian tissues, and the genes mainly enriched in this pathway include COL1A2, COL4A4, ITGB6, ITGA8, COL3A1 and VTN, which are most likely related to the regulation of egg-producing traits.

**CONCLUSION:** At present, we are still carrying out in-depth data mining and follow-up verification, which will be of great significance to the improvement and development of genetic resources of local chicken breeds in Guangxi.

**ACKNOWLEDGMENT:** The project is supported by the regional fund of the national natural science foundation of China (31660635) and the guangxi innovation driven development project (GK AA17204027).
ONE THOUSAND CHICKEN GENOMES

M. Tixier-boichard\*(1), F. Lecerf(2), P. Bardou(3), C. Klopp(4)

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

(2) ACO, Rennes, France

(3) INRAE, Castanet-tolosan, France

(4) INRA, Toulouse, France

\* Corresponding author: michele.tixier-boichard@inra.fr

The 1000 Gallus genomes project aims at gathering whole genome sequence data of chickens, produced by research teams as well as breeding companies, on the model of the 1000 Bull genomes project. The main interest is to increase the power of analyses by increasing the number of genomes. Applications are to improve the knowledge of the chicken genome, to characterize with a high accuracy the genetic diversity at the species level, to facilitate the identification of causal mutations and to provide support to genomic selection by facilitating imputation of sequence data from SNP data. A pilot project has started in France with the collection of 207 individual genome sequences produced by 9 publicly funded projects on a range of chicken populations (fast-growing and slow-growing broilers, layers, local breeds, wild ancestors). Technical metadata include sequencing method, depth and technology, fragment length, individual or pooled sequencing, date of data production. Animal metadata include population type, breed or line name, sex, pedigree information. No phenotypic information is required. All sequences have been aligned on GalGal5 reference genome and SNP calling was performed with a common pipeline. The organization of the project makes possible to identify the analysis of common interest to be performed on the whole data set, whereas the leader of a given project can use the entire set of SNPs to address a specific question, while recognizing the origin of the whole data set. The number of SNPs varied from 5 to 10 millions per project whereas the number of private alleles varied from 1000 to 10000 according to the project. A Structure analysis led to group the individuals in 7 genetic clusters. Among them, recently selected broilers were well separated from old broiler lines, showing the impact of selection within broilers, whereas a variable pattern of diversity was observed in local breeds with no real clustering between them. As expected, brown-egg layers were clearly separated from white-egg layers. Considering that these two types of lines exhibit very contrasted egg shell color, we developed a case study on 7 candidate genes involved in the metabolism of protoporphyrin IX which determines egg shell color. We search for SNPs exhibiting contrasted allele frequencies between White Leghorns and Rhode Island Red layer lines: 57 SNPs were identified in 5 of the 7 genes, most of them involved FECH and ABCB6 genes. Regarding ABCB6, 13 variants showing opposed frequencies (0/1) covered a segment of 6920 bases, including one non-synonymous variant, which suggest a potential selection signature. Functional consequences of this SNP are being explored.
IDENTIFYING SIGNATURES OF SELECTION RELATED TO COMB DEVELOPMENT

Y. Tu\*(1), J.Shu(1), Y.Liu(1), M.Zhang(1), Y.Shan(1), X.Ju(1), G.Ji(1), J.Zou(1)

(1)Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou, China

* Corresponding author: tyj3030@126.com

**Abstract:** The aim of this study was to identify the genes involved in the comb development and to provide insights into molecular mechanism of comb formation in chickens. Fixation index (FST) and average number of base differences ($\theta\pi$) of two groups of males with large and small combs were calculated based on whole-genome resequencing data. All males used in the present study were sourced from a Partridge Shank chicken breeding line with single comb. To obtain males with different comb sizes at 9 weeks of age for sequencing, comb areas and heights of 200 males determined from the descendants of 6 families of Partridge Shank chicken line were measured. Based on those results, 22 males with bigger combs (BC) and 19 males with smaller combs (SC) were selected for further analysis. DNAs in each population (BC, SC) were pooled in each tube. Two pooled samples (BC and SC) were separately sequenced using next-generation sequencing technique on the Illumina sequencing platform (HiSeq 2000). The chromosome regions with larger FST value and smaller $\theta\pi$ were considered as candidate regions of selection. Through further annotation of gene functions and pathways, we sought to screen possible selected genes associated with comb development. By screening whole genome resequencing data, the FST and $\theta\pi$ were calculated using a 40Kb sliding window strategy. Eight regions were identified. QTLs (FOX1 gene) related to comb length were found on chromosome 1. QTLs (GLP1R, BTBD9, MIR6633, MDGA1 genes) related to comb weight were found on chromosome 3. QTLs (ALDH1A1, TMC1, ANXA1 genes) associated with comb area were found on the Z chromosome. Nineteen genes, Wnt signaling pathway and neuroactive ligand-receptor interaction signaling pathway directly or indirectly related to comb growth and development were found through functional annotation and GO analysis. Among the selected genes LYN, GLP1R, FOX1, TBK1, STRAP, ST6GALNAC and Wnt signaling pathways were related to immunity. MDGA1, BTBD9, MTSS1, SrGAPs and neuroactive ligand receptor interaction signaling pathways related to neural function were screened. ALDH1A1, ANXA1, THBS, HIF-1$\alpha$, ACTN1 genes were related to heat dissipation. Among the selected genes FOX1, MDGA1 and ANXA1 associated with immunity, neurological function, and heat dissipation function coincided with genes affecting the length, weight, and area of the comb. Comprehensive analysis suggested that comb development was due to multiple genes and signaling pathways.

**Key words:** comb; whole-genome resequencing; selection signatures; candidate genes; genetic differentiation

**Acknowledgments:** The current research was funded in part by the China agriculture research system (CARS-41-G02), National Natural Science Foundation of China (31402032), Jiangsu Province major agricultural new varieties creation project (PZCZ201728), Jiangsu Provincial Agricultural three new Project (JATS [2018]249), Agricultural Science and Technology Independent Innovation Funds (CX (18)3003).
NEW TRAITS FOR POULTRY BREEDING
ASSOCIATIONS OF FEATHER PHENOTYPES WITH BODY AND FEATHER GROWTH PERFORMANCES IN
SLOW OR RAPID FEATHERED QINGYUAN PARTRIDGE CHICKENS

Z. Chen\*(1), Y. Sun(2), Z. Kuang(3)

(1) Quanzhou Institution of Agricultural Science, Quanzhou, China

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

(3) Guangdong Aijiankang Biotechnology Co., Ltd., Qingyuan, guangdong, China

* Corresponding author: chenzhixuancau@126.com

Introduction: In China, quality chickens are sold as whole carcass with skin. Immature plumage may result in black spots in the feather follicles of skin after feathers removal, which may therefore severely reduce the product quality and value, especially for breeds with dark plumage. This study was conducted to evaluate associations of feather phenotypes with body and feather growth performances.

Materials&Methods: A total of 320 day-old Qingyuan partridge chicks from the slow feathering strain (80 males and 80 females) and the rapid feathering strain (80 males and 80 females) were randomly selected. The two strains have been developed by phenotypic selection of five generations. Feathering parameters including primary feather length (PFL), primary-covert feather length (PCFL), average feather length (AFL), and feather length difference (FLD) were measured as hatched. Body weight (BW), comb height (CH), the percentage of matured feathers of dorsum (MFD), and the number of matured feathers of wing (MFW) were measured at 110-day-old, a typical marketing age of quality chickens in China. Effects of feather phenotypes and sex on growth traits, and correlations between feathering parameters and growth traits were estimated by GLM and CORR with SAS9.2.

Results: The MFW for rapid feathering strain was higher than slow feathering strain (PPr=-0.44, P=0.002) in rapid-feathering males, and between MFW and BW (r=-0.31, P=0.04) in slow-feathering males. For rapid-feathering males, there were positive correlations between PFL and MFW (r=0.33, P=0.02), PCFL and MFW (r=0.43, P=0.002), AFL and MFW (r=0.38, P=0.007); for slow-feathering males, there were negative correlations between PCFL and MFW (r=-0.36, P=0.02), AFL and MFW (r=-0.33, P=0.03); for rapid-feathering females, there was a positive correlation between FLD and MFD (r=0.33, P=0.03); for slow-feathering females, there was a negative correlation between FLD and CH (r=-0.32, P=0.02).

Conclusion: Influences of feathering parameters on growth traits of quality chicken breeds were proved first time by our results, providing scientific basis for the breeding of local chicken breeds. Further genetic correlation analysis is needed to confirm the possibility of improving feather maturity of Qingyuan partridge chickens by positive selection of PCFL and AFL for rapid-feathering males, and by negative selection of PCFL and AFL for slow-feathering males.
INDIVIDUAL FEED INTAKE AND FEEDING BEHAVIOUR COMPARISON BETWEEN PEKIN AND MUSCOVY
DUCKS BASED ON HIGH THROUGHPUT PHENOTYPING

J. Fablet*(1), B.Alletru(1)

(1)ORVIA - Gourmaud sélection, St andre 13 voies, France

* Corresponding author: julien.fablet@orvia.fr

The production of duck meat is covered by different species including the Pekin and the Muscovy
ducks. Generally, Pekin ducks are commonly used in the Asian market and Muscovy ducks are mainly
used in European market. Most of the studies published present result on feed efficiency on a small
number of animals (Bley et al, 2008, Howie et al, 2010). The aim of the study is to compare those two
genetic on the individual feed intake and feeding behaviour.

Animals of 2 Pekin lines (C, D) and 2 Muscovy lines (A, B) of Orvia hatched in 2016 were kept on floor to express their natural feeding behaviour as they do in normal production environment. Large group were studied and compared using high throughput phenotyping. Data have been collected using automatic feed station with ducks equipped of a radio frequency identification. A total of 12 068 ducks have been measured. Ducks were housed in large pen of around 1 500 animals. The ducks were feed ad libitum with a pelleted grower. Measurement have been done from week 3 to week 7 for the Pekin lines and from week 3 to week 12 for the Muscovy lines. The number of meals per duck has been record, meal size, duration of meal and the frequency of meals, feed intake, duration of feed intake per day. A total of 20 430 239 records have been studied.

R software has been used to remove abnormal data and to carry out the statistical treatments. Anova and Duncan tests have been performed. A focus from 3 to 7 weeks have been done. All differences between lines are significant. Muscovy meal duration per day is 1539s vs 826s for Pekin. Feed intake per meal is respectively 9.8g and 9.0g for Muscovy and Pekin. During the day Pekin are coming more often to the feeder 20.8 vs 13.9 for the Muscovy. Feed consumption per day is higher for the Pekin (186g) than Muscovy (136g). A clear variability within lines and between products have been identified. Although Pekin and Muscovy duck are on the meat market, these results highlight the fact that they have different feeding behaviour. This implies different managements. Future analyses will look the difference in term of genetic component of those traits within different Pekin lines and Muscovy lines.
References:

Bley, et al., 2008, Recording of individual feed intake and feeding behaviour of Pekin ducks kept in groups. Poultry Science 87:215-221

Howie, et al, 2010, Short-term feeding behaviour has a similar structure in broilers, turkeys and ducks.

British Poultry Science 51:714-724
ID: 653

# GENOME-WIDE ASSOCIATION STUDY FOR THE DYNAMIC CHANGE OF EGGSHELL GLOSS ACROSS THE WHOLE LAYING PERIOD IN CHICKEN

Q. Li(1), G.Li(1), G.Wu(1), Y.Yan(1), H.Li(1), J.Zheng(2), G.Xu(2), C.Sun(2), N.Yang(2)

(1) Beijing Huadu Yukou Poultry Industry Co. Ltd, Beijing, China

(2) China Agricultural University, Beijing, China

* Corresponding author: sweetquanlin@cau.edu.cn

The appearance of eggshell can influence the purchasing behavior of consumers. Eggs with a shiny appearance are more attractive than others. Previous studies found that the eggshell gloss was related to the texture of the surface. However, the genetic architecture underlying the dynamic change of eggshell gloss remain elusive. Herein, we measured the eggshell gloss at 6 age points (36, 56, 60, 72, 80 and 90 wk) and conducted comprehensive genome-wide association studies (GWAS) with 50 K density SNP array in a pure line of 706 Rhode Island Red hens. The results showed that the average value of gloss ranged from 2.21 GU to 3.15 GU with a variation of 15.09% to 23.73%. The eggshell gloss of 36 wk old hens was higher than that at other ages. Also, significant positive correlations were found between the eggshell gloss of hens with adjacent ages (R=0.51, 0.53, 0.51, 0.52 and 0.59). Subsequently, 6 genome-wide significant SNPs were identified at 60 wk. According to these candidate loci, six genes (ACADSB, IKZF5, GPR26, SLC2A11L3 and SLC2A11L2) were considered to play an important role in eggshell gloss. This study will greatly advance the understanding of the genetic basis and has the practical significance in breeding program for the improvement of eggshell gloss.

Keywords: chicken, eggshell gloss, GWAS, 50 K SNP array
THE DIFFERENCE IN SOME PHYSICAL EGG QUALITY AND PRODUCTIVE TRAITS BETWEEN PARENTAL LINES OF WL AND RIR AND THEIR RECIPROCAL HYBRIDS OF DOMINANT TINTED D 723 AND D 732

O. Krunt* (1), L.Zita(1), A.Kraus(2), H.Tyllerová landová(3), V.Anderle(3), R.Trott(3), M.Hruška(4)

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

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

(3)DOMINANT CZ, Lazne bohdanec, Czech Republic

(4)University of Hradec Kralove, Hradec kralove, Czech Republic

* Corresponding author: krausa@af.czu.cz

The creation of new hybrid combinations in poultry husbandry is connected with practical testation of selected and predicted lines and their crosses. The objective of the present work was to evaluate the effect of the particular genotype, age of layers and their interactions on egg quality traits in laying type hens Dominant. The following genotypes were examined in the experiment: as parental lines White Leghorn, slow- and fast-feathering (next WL FF, WL SF) and Rhode Island Red, slow- and fast-feathering (next RIR FF, RIR SF) and the crossbreeds of the above mentioned strains in the F1 generation (Dominant TINTED D 723 = LW FF x RIR SF and Dominant TINTED D 732 = RIR FF x WL SF). There was used feather sexing method. The layers were fed by feed mixture N1 (16.64% of CP) from the 20th week of age and by feed mixture N2 (15.02% of CP) from the 40th week. Husbandry conditions met the regular requirements for laying type hens. Egg quality traits were examined at the layers' age of 28, 36 and 59 weeks. For analysis, 300 eggs were collected from each genotype per time. The eggs were weighed using an electronic scale Ohaus. Eggshell strength was determined by the analyzer Instron. Eggshell thickness was measured by a digital micrometer. The proportions of yolk, albumen and eggshell were calculated in relation to egg weight and expressed as percentages. The egg shape, yolk and albumen index, yolk to albumen ratio and Haugh unit score were computed as well. The statistical analysis was processed by the computer application SAS. All monitored traits of physical eggquality were affected by genotype (P≤0.05) and age of hens (P≤0.05). There were detected significantly (P≤0.05) mutual interactions between genotype and age of hens in yolk weight, yolk and albumen proportion and their index, yolk to albumen ratio, Haugh unit score and eggshell thickness. Eggs from crossbreeds (D 723 and D 732) had better quality than those from parent lines (e.g. egg weight, eggshellthickness, albumen weight, yolk weight and proportion). The results of this study helped to select and start practical use of the new laying programme DOMINANT TINTED D 723, which produced egg production per hen day 338 eggs in age of 518 days in XIV. International performance test of commercial layers – alternative system (described in The final report 2015 – 2016) and started to be popular among programmes of DOMINANT CZ company. This research was funded by an “S” grant of the MEYS of the CR.
EF4

EGG QUALITY

CONSTRUCTION OF EGG QUALITY
QUALITY OF EGG PRODUCTS
CONSTRUCTION OF EGG QUALITY
AGING-ASSOCIATED CHANGES IN THE ISTHMIC AND UTERINE MUCOSAE LOWERING THE EGGSHELL QUALITY

M. Elhamouly\*(1), T.Nii(2), N.Isobe(2), Y.Yoshimura(2)

(1)Faculty of Veterinary Medicine, University of Sadat City, Sadat city, Egypt

(2)Hiroshima University, Hiroshima, Japan

\* Corresponding author: Mostafa.elhamoli@vet.usc.edu.eg

Understanding why aged hens produce inferior quality eggs may help to produce long-life layers. Thus, the aim of this study was to determine the aging-associated molecular changes in the isthmic and uterine mucosae causing an inferior quality eggs produced by aged hens. White Leghorn hens were used in this study to compare the expression of proteins related to eggshell membranes formation in the isthmus and eggshell biomineralization in the uterus of young (35 weeks old) and aged (130 weeks old) hens at 16 h following oviposition. The results showed that collagen V protein density was lower in the isthmic mucosa and eggshell membranes of aged hens than in young hens. The mRNA expression of calbindin, osteopontin, and ovocalyxin-36, and the densities of calbindin and carbonic anhydrase-2 were lower in the uterine mucosa of aged hens than in young hens. There was no observable histological difference in the isthmus or uterus between young and aged hens. We conclude that the eggshell quality is decreased in aged hens compared to young hens due to a molecular disorder to produce structural and functional proteins that share in the eggshell membranes formation and eggshell biomineralization process.
MICROSTRUCTURE OF THERMALLY INDUCED EGG YOLK GEL

R. Zhang\*(1)

(1)China agricultural university, Beijing, China

* Corresponding author: 1114695270@qq.com

Egg is a kind of animal food with balanced nutrition. Gel properties are one of the most important processing characteristics of eggs in food processing. The thermal gelation process of yolk is a complex process, which determines the rheological properties and gel properties of egg yolk gel products. Through a lot of research and breeding work on local chicken breeds in China, it is found that some local chicken breeds in China have the characteristics of producing yolk. Therefore, it is necessary to find out the causes of egg yolk non blocking at the molecular level

The experiment was divided into four parts: (1) pigeon eggs, common eggs and oil eggs were steamed for 10 minutes, 20 minutes and 30 minutes respectively. (2) cook the common eggs at the corresponding time, including duck eggs, eggs, goose eggs, quail eggs and pigeon eggs (3) cook 8 big black eggs and 8 Luodao red eggs at the same time, and send the above three groups of cooked eggs to the company for scanning electron microscopy observation. (4) lipoproteins were extracted from 20 large black eggs, 20 Luodao red eggs and 20 pigeon eggs, and the content was calculated.

The results showed that: (1) the yolk particles of egg yolk and pigeon egg were smaller than those of common eggs. (2) cooking time had no significant effect on the size of yolk particles. (3) the size of yolk granules of five kinds of bird eggs is very different, but they are basically granular. Under 500 nm scanning electron microscope, the structure of goose egg and duck egg is significantly different from that of other birds. (4) in the determination of lipoproteins, the content of lipoproteins in big black chicken was the highest, while that in Luodao red egg was the lowest.

The conclusions are as follows: (1) the reason for the good oil property of egg yolk and non choking may be that the egg yolk particles are small. (2) due to the existence of oil, duck eggs and goose eggs are closely arranged at 500 nm, and their oil shape is larger. (3) the content of lipoproteins may be related to the particle size, but it needs to be further explored.

[1] Xiao-le Xiang, Yuan-yuan Liu, Yang Liu, Xiao-yun Wang, Yong-guo Jin. Changes in structure and flavor of egg yolk gel induced by lipid migration under heating[J]. Food Hydrocolloids, 2020, 98.

[2] Uysal Reyhan Selin, Boyacı İsmail Hakkı, Soykut Esra Acar, Ertaş Nusret. Effects of heat treatment parameters on liquid whole egg proteins[J]. Food chemistry, 2017, 216.
# THE EFFECTS OF THE "SUBJECTIVE DAY" LENGTH ON DAILY OVIPOSITION RHYTHM AND PRODUCTIVE PERFORMANCE IN LAYING HENS

I. Saleeva*(1), A.Kavtarashvili(1), E.Novotorov(1), L.Prisyazhnaya(1)

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

* Corresponding author: saleeva@vnitip.ru

The effects of the "subjective day" length and intermittent lighting regimes on daily oviposition rhythm and productive performance in laying hens (cross SP-789). Four treatments of hens (100 birds per treatment) were kept from 124 to 290 days of age in cage batteries (5 birds per cage). The lighting regimes for treatments 1-4 were 1L:6D:4L:2D:3L:8D; 1L:5D:4L:2D:3L:9D; 1L:4D:4L:2D:3L:10D and 1L:4D:4L:2D:3L:10D; the "subjective day" length was 16, 15, 14 and 13 hours, respectively. First switch-on was at 2 am for all the regimes; light intensity was 10 Lx. The oviposition time was determined during 5-day periods in the beginning of the trial, in the middle and in the end of the trial using a camera recorder on a 24-hour basis. It was found that the decrease in the "subjective day" length resulted in earlier onset and earlier termination of the oviposition: in treatments 1-4 the oviposition started at 4, 3, 2, and 1 am and ended at 6, 4, 4, and 3 pm, respectively. The percentage of eggs laid between 2 and 8 am in treatments 1-4 was 52.3; 70.8; 88.0 and 89.9%; between 2 and 10 am 61.0; 76.9; 95.2 and 91.9%; between 2 am and 1 pm 92.9; 90.8; 98.8 and 98.0%, respectively. The average oviposition time in these treatments was 8 hrs 40 min; 7 hrs 22 min; 5 hrs 22 min and 5 hrs 18 min (time of the day), or 14 hrs 40 min; 14 hrs 22 min; 13 hrs 22 min and 14 hrs 16 min after the onset of the longest dark phase of the respective regimes (the difference between treatments 1 and 2 was significant at P<0.05; between treatments 3, 4 and 1, 2 at P<0.001). Average live bodyweight in treatments 1-4 at 290 days of age was 1664, 1660, 1714, 1681 g (the difference between treatments 2 and 3 was significant at P<0.05); mortality levels were 2.0; 3.0; 1.0 and 1.0%; egg production per initial hen 123.9; 124.1; 130.3 and 126.1 eggs (the difference between treatments 3 and 1, 2 was significant at P<0.001; between treatments 3 and 4 at P<0.05); average egg weight 60.0; 59.9; 60.4 and 59.7 g (the difference between treatments 3 and 4 was significant at P<0.05); feed conversion ratio per 10 eggs laid 1.37; 1.36; 1.32 and 1.36 kg, per 1 kg of eggs laid 2.28; 2.26; 2.18 and 2.26 kg, respectively. The conclusion was made that manipulations with the "subjective day" length can regulate daily oviposition time in correct direction; the best parameters of productivity and feed conversion were found at the 14-hr length (treatment 3).
GUINEA FOWL EGGSHELL STRUCTURAL ORGANIZATION AND PARTICULAR ORGANIC MATRIX PROTEIN PATTERNS TO DECIPHER ITS EXCEPTIONAL BIOMECHANICAL PROPERTIES

N. Le roy(1), L.Combes-soia(1), A.Brionne(2), V.Labas(2), A.Rodriguez-navarro(3), Y.Nys(2), M.Hincke(4), J.Gautron*(2)

(1)INRA, Nouzilly, France

(2)INRAE, Nouzilly, France

(3)University of Granada, Granada, Spain

(4)University of Ottawa, Ottawa, Canada

* Corresponding author: joel.gautron@inra.fr

The Guinea fowl (Numidameleagris) presents a highly resistant eggshell compared to the other birds. We will describe in this study the particular ultrastructure of the Guinea fowl shell that confers exceptional mechanical properties and how changes in organic matrix components controls the development of this structure. The inner part of the shell is similar to other birds, but an additional change in the size and orientation of crystals (switch) is observed at about 1/3 of the calcified layer. Large columnar calcite units break into smaller crystal units with varying crystallographic orientations forming a microstructure with an intricate interlacing of calcite crystals.

We recently reported the Guinea fowl shell structural organization from the micro to the Angstrom, which underlined that this particular shell is a bilayer structure. Organic matrix is suspected to firstly induce the initial microstructure shift and then the secondary nucleation events resulting in smaller crystals with increasing misorientations. Consequently, the change of the intra-crystalline organic matter level during the crystal switch was also investigated. A proteomic survey allowed us to identify and characterize 149 proteins in Guinea fowl shell. These proteins were quantified at five calcification stages corresponding to the first events of mineral deposition, the growth of calcite units just prior the shift of crystal orientation, then to the period of the deposition of newly formed crystalline shape and to later stage when the growth of the newly formed crystals is stabilized after the microstructure shift and the secondary nucleation events. We have observed 61 matrix proteins only present in the shift period and potentially responsible of the change of mineral in the shell. Amongst them are calcium binding proteins (NPNT-X1, CALBP1, Protein S100-A6, ANXA1 and 2, CDH2...), core proteins of proteoglycans (TSKU, GPC4...), and other proteins regulating the activity of proteins driving the mineralization (SSP1, OC-116GDF6...).

These proteins interact with mineral to produce changes in crystal size and orientation and consequently the new shell structure and its resulting mechanical properties. Data obtained will allow the determination of biological markers that will be used for the genomic selection of chicken layers with improved mechanical shell mechanical properties. Additionally, they provided a list of organic products that will be tested as additives for material and ceramics.
COMPARATIVE STUDY OF PERCENTAGE OF EGG LAYING AND EGG QUALITY TRAITS IN ITALIAN SLOW-GROWING CHICKEN BREEDS

S. Dabbou\*(1), V. Zambotto(2), M. Gariglio(2), S. Bellezza oddon(3), D. Soglia(4), S. Sartore(4), F. Gai(5), C. Garcés narro(6)(3)(4)

(1)Center Agriculture Food Environment (C3A), University of Trento, Italy, Trento, Italy

(2)Department of Veterinary Science, University of Turin, Turin, Italy

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

(4)Department of Veterinary Science University of Turin, Italy, Turin, Italy

(5)Institute of Science of Food Production, National Research Council, Turin, Italy, Turin, Italy

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

* Corresponding author: sihemdabbou@gmail.com

Introduction. Bionda Piemontese (BP) and Bianca di Saluzzo (BS) were the most widespread local chicken breeds of Piedmont region and currently are reared in small-scale farms for eggs and meat production. The aim of this study was to characterize the percentage of egg laying, as well as its physical and shell parameters of these two Italian chicken breeds.

Material and methods. A total of 180 laying hens of both BP and BS (23 wk of age) were homogenously distributed in 12 pens with outdoor access (15 hens/pen/breed); all the birds were fed the same commercial feed (ME 11.8 MJ/kg; CP 14.2%) for 36 wk. Percentage of egg laying and mean egg weight were recorded per pen throughout the laying hen cycle. From 37 to 61 wk of age, egg samples were collected every 6 wk, (5 eggs/pen/breed) to determine both egg and egg shell characteristics. Data were analysed by IBM SPSS Statistics V25.0.0 software. Percentage of egg laying and mean egg weight of the two breeds were analysed using Student's t-tests for independent samples. General Linear Model (GLM) procedures have been performed for all egg quality parameters, using breed and age as principal effects. Tukey multiple comparisons have been performed after the GLM procedure for slaughtering time. Significant differences are considered at P≤0.05.

Results. No significant difference was observed for the percentage of egg laying (BP 54.6 ± 2.7%; BS 53.3±3.0%; mean ± SEM) and for mean egg weight (BP 58.6±0.8g; BS 56.9±0.6g; mean ± SEM) among breeds. Physical egg parameters (egg weight (BP 62.4g, BS 59.8g), width (BP 43.3mm, BS 43.0mm); length (BP 59.7, BS 58.1 mm), yolk weight (BP 18.8g, BS 17.7g), albumen weight (BP 37.4g, BS 36.5g) yolk/egg percentage (BP 30.1%, BS 29.6%) and total egg surface (BP 73.6 cm2, BS 71.5 cm2) resulted higher in BP hens (P≤0.05). Egg shell parameters did not differ among breeds and age except for colour indices. Age influenced all the egg quality parameters with linear trend over the age (P≤0.05). Interaction between breed and age of hens were observed for yolk weight, yolk/egg ratio and for colour indices of yolk and egg shell (P≤0.05).

Conclusion. Promising results were obtained in both production and physical quality parameters of eggs between BP and BS hens. This yielded evidence for several valuable egg characteristics that might be exploited for the improvement of these Italian pure breeds.
DIETARY SUPPLEMENTATION OF LINOLEIC ACID FOR INCREASING THE PULLET EGG SIZE

B. Dey*(1), A.Roy(1), B.Ray(1), S.Sarker(1)

(1) Bangladesh Agricultural University, Mymensingh 2202, Mymensingh, Bangladesh

* Corresponding author: dey_bau@yahoo.com

Dietary supplementation of linoleic acid for increasing the pullet egg size

Linoleic acid (LN) is beneficial to human health for its multiple properties of antiobesity, antidiabetic, anticancer, antiatherogenic, and immunity improvement. The use of LN in laying hen diets to produce eggs enriched with essential nutrients has become a common practice in the table egg industry and linoleic acid levels in egg yolks increase in a dose-dependent manner. An experiment was conducted to investigate the effect of LN of the diet on egg weight, egg production and egg quality characteristics on NovoGen brown commercial layers of 20 to 40 weeks of age. There were three dietary treatments of four replications per treatment maintaining four birds per replication. All the diets were iso-caloric and iso-nitrogenous except for the doses of LN. The diets contained T1 (control diet with no LN), T2 (control diet + 1.5% LN) and T3 (control diet + 2.5% LN). In this experiment, we supplied soybean oil as the potential source of LN, as because soybean oil contain about 53% LN, soybean oil frequently use as the laying birds ration formulation and we had try to avoid the use of synthetic LN for the safer egg production. We had measured the boiled yolk weight, because the yolk weight directly reflects the egg weight. The results showed the egg weight, yolk weight, and egg quality parameters were better in the diet contained higher in linoleic acid. The yolk weight was significantly improved in the diet containing 2% soybean oil (2.5% LN) compared to 1% soybean oil (2.5% LN) and control (0% LN) groups. Additionally, both the internal and external egg quality parameters, especially shell thickness, Haugh Unit (HU), yolk index were improved in the dietary group treated with both 1% and 2% soybean oil (both 1.5 % and 2.5% LN). Taken together, it may be concluded that LN has a positive effect in not only the increase in egg size but also in the egg quality characteristics and thus improve the overall health benefit for human consumption.

Key words: Linoleic acid, pullet, egg size, diet, supplementation.

*Corresponding author: Dr. Bapon Dey, Associate Professor, Department of Poultry Science, Bangladesh Agricultural University, Mymensingh 2202. E-mail: dey_bau@yahoo.com
LAYING HENS PRODUCTION EFFECTIVENESS INCREASING AND QUALITY IMPROVING BY INCLUDING TO THEIR DIET SUSTAINABLE PLANTS (HELIANTHUS TUBEROSUS L.)

A. Dauksiene*(1), J.Klementaviciute(1), R.Gruzauskas(2), D.Klupsaite(1), E.Bartkiene(1)

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

(2) Kaunas University of Technology, Kaunas, Lithuania

* Corresponding author: agila.dauksiene@lsmuni.lt

Nutritionally enhanced eggs could play a larger role in promoting the nutritive value of people's diets (Shinn et al. 2018). The factors that affect egg shell quality and egg internal quality are housing systems, management, storage period and environmental temperature, diseases, age of hen and nutrition (Tamiru et al., 2019). To improve egg quality different feed additives are tested, as well as Helianthus tuberosus L. tubers, as an inulin source, can be very promising from this point of view. In the study was hypothesized that feeding with H. tuberosus L. tubers (HTT) powder, rich in inulin, might improve laying hens' egg quality characteristics. A total of forty 30-week-old laying hens we distributed into two groups and for 8 weeks were fed by diets supplemented with nothing (control) and HTT powder at the level of 2g/kg of feed. The fatty acids (FAs) profile in egg yolk by gas chromatography, yolk cholesterol by HPLC and egg, yolk, albumen and egg shell weight, eggshell breaking strength, albumen height and Haugh unit as well as body weight (BW), feed conversion ratio (FCR), laying rate were evaluated. In statistical analysis, data were subjected to univariate analysis of variance (ANOVA) and the Tukey HSD test was applied. Results were considered statistically significant at P≤0.05. In the present study, the treatment with HTT powder did not significantly influence the total sum of yolk SFAs, MUFAs, PUFAs and omega-6/omega-3 FAs ratio. The total cholesterol content in egg yolk was significantly lower in treatment group by 15.95% compared with the control. There were no significant effects of HTT on BW, FCR, but the laying rate of hens increased by 4%, compared with the control group (P≤0.05). A significant effect of HTT on shell breaking strength by 2% and Haugh unit by 5% was found, compared with the control (P≤0.05). The results of the study indicate that Helianthus tuberosus L. tubers powder has a positive effect on eggshell quality, Haugh unit, and it reduces yolk cholesterol level. Finally, laying hens productivity effectiveness and quality of egg can be improved by including to their diet sustainable plants.

References:

SHINN S., PROCTOR A. and BAUM J. (2018) Egg yolk as means for providing essential and beneficial fatty acids. J Am Oil Chem Soc. 95: 5-11.

TAMIRU H., DUGUMA M., FURGASA W. and YIEMER L. (2019) Review on chicken egg quality determination, grading and affecting factors. Asian J Med Sci Res & Rev. 1(1): 34-42.
IDENTIFICATION OF POLYMORPHISMS IN PROTEIN CODING GENES WHICH AFFECT EGGSHELL QUALITY TRAITS IN LAYERS

F. Herault\*(1), C.Diot(2), T.Burlot(3), J.Gautron(4), T.Moreau(4), P.Le roy(2)

(1)INRA, Saint Gilles, France

(2)INRA, Saint-Gilles, France

(3)Novogen, Pledran, France

(4)INRA, Nouzilly, France

\* Corresponding author: frederic.herault@inra.fr

Chicken egg is a high quality food at a low cost to the consumer but must be healthy and free from pathogens. Consumer demand for the development of alternative farming systems to cages is growing (barns, free-range, etc.). However, these systems present an increased risk of microbial contamination of eggs, which reinforces the importance given by breeders to the eggshell quality. A large number of QTLs influencing production and egg quality traits have been detected. However, the number of known causal polymorphisms remains low. In the context of genomic selection, knowledge of these polymorphisms is a key step to increase the evaluation accuracy. In the context of scientific knowledge, identifying genes and polymorphisms involved in the variability of traits is also important.

To address these two contexts, we have selected potential causal polymorphisms in QTL and candidate genes. Polymorphisms in DNA from laying hens were first selected in QTL regions according to a strong contribution to additive genetic variance of a trait. Then, they were selected according to their position in candidate genes in QTL regions. We have defined candidate genes as genes known to play a role in eggshell quality, differentially expressed in uterus or coding proteins with different levels in uterine fluid during eggshell formation. Finally, deleterious polymorphisms localized in coding regions (creation or loss of start or stop codon, reading frame shift or modification of splicing site) were identified. This strategy for identification of putative candidate genes and causal polymorphisms susceptible to affect protein abundance, structure and function is generic and can be applied to other species and traits. In our study, we have identified 4849 deleterious loci as putative causal mutations, localized in 418 candidate genes for eggshell formation.

Finally, we have selected 255 deleterious polymorphisms which have been genotyped on 480 laying hens from 8 laying breeds. Association studies analysis will be conducted to validate the effect of these polymorphisms on eggshell quality.

Polymorphisms in coding regions are a small proportion of all polymorphisms identified in QTL regions. We don't suggest that polymorphisms outside coding regions are not causal mutations. Conversely, when experimental validations are needed it's more obvious to analyze the effect of these polymorphisms on the function of proteins and define a link between polymorphism and protein function and trait.
EFFECTS OF ADDING THE FUNCTIONAL CO-EXTRUDATES AND NATURAL PIGMENTS IN THE DIET OF LAYING HENS ON EGG QUALITY

N. Spasevski*(1), T.Peulić(1), V.Banjac(1), S.Rakita(1), N.Puvača(2), B.Kokić(1), O.Đuragić(1)

(1)Institute of Food Technology, University of Novi Sad, Novi sad, Serbia

(2)Department of Engineering Management in Biotechnology, University Business Academy, Novi sad, Serbia

* Corresponding author: nedeljka.spasevski@fins.uns.ac.rs

Eggs are cheap and nutritionally valuable products of animal origin. Using specially designed mixtures for the production of table eggs, it is possible to produce eggs enriched in ω-3 fatty acids, as well as in natural pigments and vitamins. Thus, this study aimed to investigate the effects of inclusion of co-extrudate based on flaxseed and camelina seed in combination with natural pigments in hen's diet on egg quality parameters.

One hundred and eighty Lohmann Brown laying hens divided into six groups (two controls (C1 and C2) and four experimental (E1-E4)) were fed for four weeks. The laying hens were fed with corn-soybean meal based diet (C1 and C2). Flax-corn meal co-extrudate was added in amount of 13.5% (E1) and 22.5% (E2) while camelina-corn meal co-extrudate was added in amount of 16.6% (E3) and 27.6% (E4) in based diet. C1 contained up to 3% fat without added pigments, while C2 contained up to 5% fat and synthetic pigments. The same amount of natural pigments (1% carrot and 0.5% paprika) was added in all experimental treatments. Content of fat in E1 and E3 was 3%, while in E2 and E4 was 5%.

No significant changes (p>0.05) on the internal egg quality characteristics were observed for eggs from the experimental treatments when compared with eggs from control treatments. All experimental treatments achieved desirable color demanded by consumers, from 12.67 to 13.28 RYCF value (Roche Yolk Color Fan). The content of PUFAs in eggs from all experimental treatments was significantly higher (p<0.001) than in eggs originated from hens fed with control diets. The ω-6/ω-3 ratios of 1.43, 1.01, 1.74 and 1.73 in treatments E1, E2, E3 and E4, respectively, were significantly lower (p<0.001) than in treatments C1 and C2 (9.40 and 8.88, respectively). The addition of co-extruded flaxseed in treatments E1 and E2 had a negative effect (p0.05).

Based on obtained results it can be concluded that with the addition of natural pigment, as well as selected co-extrudates, it is possible to design functional eggs that will have good egg quality parameters, optimal yolk color, increased content of ω-3 fatty acids, better ω-6/ω-3 fatty acids ratio, as well as good sensory profile of the eggs.

Keywords: functional eggs, flaxseed, camelina seed, co-extrudates, natural pigment

Acknowledgement: This work was a part of the project III46012 financed by the Ministry of education, science, and technological development, Republic of Serbia.
GENETIC VARIATIONS FOR THE EGGSHELL CRYSTAL STRUCTURES REVEALED BY GENOME-WIDE
ASSOCIATION STUDY IN CHICKEN

Z. Duan\*(1), Q.Li(2), C.Sun(2), N.Yang(2), G.Xu(2)

(1)1China Agricultural University, 2National Animal Husbandry Service, Beijing, China

(2)China Agricultural University, Beijing, China

* Corresponding author: sweetquanlin@cau.edu.cn

Avian eggshell is a complex bio-ceramic characterized by layer structure. Eggshell forms an embryonic
chamber for the developing chick by providing a stable internal environmental. For the egg industry
worldwide, the integrity of eggshells is critical to the economic viability of the poultry production.
However, the genetic architecture underlying the eggshell crystal polymorphism remain to be
elucidated. Herein, we measured the eggshell crystal structures including integral intensity of the first
nine major diffraction peaks, total integral intensity (TA) and eggshell crystal orientation degree (OD),
then the genome-wide association studies in 927 F2 hens was conducted. The results showed that TA
had a positive correlation with the ultrastructure and common quality traits of eggshells. There was a
negative correlation between TA and OD, indicating that the larger the crystals, the lower the degree
of orientation, the more prone to random distribution. The SNP-based heritabilities of TA and OD were
0.23 and 0.06. Moreover, 621 SNPs located in 55.7-69.3 Mb in GGA1 were significantly associated with
TA, and the most significant SNP accounted for 4.87% of the phenotypic variance. Based on mRNA and
protein expression in uterine tissue, we suggested that DERA gene may play an important role in the
regulation of eggshell crystal growth. The findings greatly advance our understanding of the genetic
basis underlying the crystal ultrastructure of eggshell quality and has the practical significance in
breeding program for the improvement the eggshell quality.

Keywords: chicken, eggshell, crystal structure, X-ray diffraction, GWAS
EVALUATION OF EGG QUALITY TRAITS OF FOUR VARIETIES OF THE CANARIAN HEN IN FREE-RANGE
CONDITIONS

A. Torres\*(1), N. López(2), J. Pérez(2), C. Rodríguez(2), G. Lobo(3), M. Fresno(3)

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

(2)Universidad de La Laguna, La laguna, Spain

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

* Corresponding author: aletor80@yahoo.com

Despite the economic and cultural importance of poultry sector in the Canary Islands throughout their history, there is no information to date on the main productive performance and egg quality characteristics of Canarian hens. The objective of this study was to determine the quality of the egg produced by four colour varieties of Canarian hen (Black Barred, Red Barred, Black, and Partridge) reared under free-range conditions and comparing with commercial barn eggs from Isa Brown hens. All hens were fed with the same standard layer feed during the experimental period. A total of 180 eggs were collected at 32, 36 and 40 weeks of age, and were used to evaluate external and internal parameters. For the statistical analysis, the genotype effect was determined using an ANOVA followed by the post hoc Tukey's method. Statistical differences were considered significant at Pvs. 75.07 (0.30), respectively. There were not differences in shell and albumen weights, but yolk weight was higher in Canarian than in commercial eggs: 18.12g (0.17) vs. 16.19g (0.24), respectively. Moreover, eggshell colour parameters did not vary among Canarian varieties. However, significant differences were reported for the three coordinates defined by the International Commission on Illumination: lightness (81.48 vs. 62.34, SEM 0.687), redness index (5.64 vs. 19.72, SEM 0.459) and yellowness index (17.88 vs. 30.94, SEM 0.505) for local and commercial eggs, respectively, showing the Canarian eggs are less brown than the commercial ones. No differences were found for shell hardness, yolk index and pH of the yolk and the albumen, and ranged between 31.87-37.84N, 46.85-50.59%, 6.09-6.20, and 8.99-9.16, respectively. Finally, there were not differences for yolk colour parameters among Canarian varieties, but the yolk from local hens had a higher yellowness index than the yolk from commercial laying hens: 54.10 (0.43) vs. 48.53 (0.57), respectively. In conclusion, the results obtained confirm there is no division among the four Canarian varieties in terms of differences in egg quality traits. Nevertheless, the local hens presented some differentiating attributes, as shell and yolk colours, which may contribute to consumers' initial selection.
# STUDY OF MEASUREMENT METHODS ON PHENOTYPE OF TRANSLUCENT EGGS

D. Wang\*(1)

(1)Hebei Agricultural University, Baoding, China

\* Corresponding author: theconcertevent@cau.edu.cn

Scoring is a commonly used method to evaluate eggshell translucency, which mainly depends on the area size and density of translucent spots on eggshells. However, a lack of common scoring criteria and quantitative measurements of spots on eggshells does not allow for effective comparisons between research papers and greatly hinders the progress of research on translucent eggshell formation. To make measurement of translucent eggshells more objective, we optimized the scoring method and explored two new methods: the grayscale recognition and colorimeter methods, and further compared these three methods. Briefly, a total of 354 eggs from 600 395-d-old dwarf brown laying hens were collected and classified into 4 score levels subjectively according to their translucent degrees. This process was repeated five times. Then we captured profile side of each egg using a camera and measured spot characteristics by grayscale recognition, including quantities of spots (QS), diameter of each spot (DS), average area of each spot (AAES), sum of spot areas (SUSA), sum of shell area (SUSHA), and ratio of SUSA to SUSHA (RSS) on an eggshell. Furthermore, L, A, and B values of each egg on sharp, middle, and blunt ends were separately measured using a colorimeter. As results, average values of 31.31%, 29.78%, 29.81%, and 9.08% of all eggs were divided into score levels 1, 2, 3, and 4 (from opaque to translucent), which corresponds with RSS values of 1.34%, 3.23%, 6.21%, and 11.89%, respectively. By grayscale recognition, QS, DS, AAES, SUSA, SUSHA, and RSS all increased along with increased translucency scores (P < 0.05). Using scoring, an egg with specific RSS value was more easily divided into specific score level (50%) and adjacent score levels (50%). The L, A, and B values of eggshells that fall into score level one were significantly different from those of eggshells scored as levels 3 or 4; however, the adjacent score levels were not. In summary, the present study explored objective reference metrics for measurements of eggshell translucency.
MITOCHONDRIAL TRANSCRIPTION FACTOR A-INDUCED DECLINE IN MITOCHONDRIAL BIOGENESIS IN RELATION WITH THE DEPIGMENTATION OF BROWN EGGSHELL

M. Lu(1), J.Wang(1), S.Wu(1), G.Qi(1), H.Zhang*(1)

(1) Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China

* Corresponding author: zhanghaijun@caas.cn

Eggshell color is an important characteristic of poultry eggs and has a positive effect on consumer preference. It would be favorable for table eggs with dark brown color. However, poor shell color of aged hens results in unacceptable shell color. The objective of this study was to examine what factors were related with the occurrence of brown eggshell depigmentation in aged laying hens. There eggshell color was determined by brightness value (L*). We selected 24 chickens laying stable dark (46 ≤ L* ≤ 53) and light-eggshell color (59 ≤ L* ≤ 65) respectively from 300 62-wk-old Hy-Line brown-egg laying hens reared in individual cages. Oviposition time was recorded after collecting the successive 7 eggs per hen. A total of 8 hens per group were slaughtered at 15 h after the oviposition and were collected the eggshell gland region of the oviduct. All analyses were performed using Student's t-test with SAS9.1. Results showed that egg weight (P < 0.05), eggshell weight (P < 0.01), protoporphyrin IX (Pp IX) content of the eggshell and the shell gland (P < 0.001), and biliverdin content of the shell gland (P < 0.001) were significantly declined in the light-eggshell group. The mRNA relative expression of δ-aminolevulinic acid synthase 1 (P < 0.05), coproporphyrinogen oxidase (P < 0.01), ATP-binding cassette transporter ABCG2 (P < 0.01) and mitochondrial transcription factor A (TFAM) (P < 0.01) was downregulated in hens laying lighter brown eggs. There was significant decrease in mitochondrial DNA (mtDNA) copy numbers (P < 0.01), mitochondrial ND4 (P < 0.05), ATP8 (P < 0.05) and COX1 (P < 0.01) relative expression in the shell gland of the light-eggshell group. In addition, NAD+ contents of the shell glands were increased in the dark-shell group (P < 0.01). In conclusion, the remarkable triats of the depigmentation are showed to reduce Pp IX content in the eggshell and shell gland; the downregulation of ALAS1 mRNA relative expression make Pp IX biosynthesis declined; downregulated TFAM mRNA relative expression induce the decline in mitochondrial biosynthesis independent of peroxisome prolife-activated receptor-γ coactivators β pathway, and it is associated with the depigmentation by lessening the synthesis of ALAS.
QUALITY OF EGG PRODUCTS
EVALUATION OF PHYSIOCHEMICAL CHANGES IN BOILED EGGS STORED AT DIFFERENT TEMPERATURE

S. Eragama(1), H.Aruppala(1), A.Marcus(2), J.Pitawala(1), S.Abeyrathne*(1)

(1)Uva Wellassa University, Badulla, Sri Lanka

(2) Maxies & Company (Pvt) Ltd, Wennappuwa, Sri Lanka

Poultry Research Foundation, the University of Sydney, Sydney, Australia

Hard boiled eggs are used widely in the world but once these eggs are stored under refrigerated conditions lead to customers' rejections due to its rubbery texture. Therefore, objective was to evaluate the effect of storing temperature on structural changes in hard boiled eggs. Newly laid, medium sized eggs were purchased from a commercial farm and boiled at 95 0C for 15 minutes and stored at 27 0C, 4 0C and -18 0C for 0, 6, 12, 18, 24 and 48 hours as different time intervals. 18 eggs per each experimental group were studied and subjected to analyze the texture and Fourier Transform Infrared (FTIR) spectroscopy. The color was measured as lightness (L*), redness (a*) and yellowness (b*). Egg white surface was observed under gemological microscope for changes in structure. Sensory evaluation done by 7-point hedonic scale with 30 untrained panelists [Age 21-24 years, both male and females]. Salmonella was checked in boiled eggs stored at 27 0C. Texture and color data analysis were performed by one way ANOVA model and sensory data were analyzed with Freidman test in MINITAB 17.1. According to the results, frozen eggs showed the highest hardness due to formation of the ice crystals by freezing of water in the elastic gel of cooked egg white (denatured protein). The starting lightness (0 hours) of boiled egg white was 97.69± 1.15 and gradually decreased after 48 hours in 270C, 4 0C and -18 0C as 93.84± 1.27, 96.36± 1.60, and 95.10± 0.84 respectively while changing the color from white to greenish white due to microbial and chemical activities. Boiled eggs stored for 48 hours at 27 0C were positive for Salmonella may be due to removal of barriers in egg and destroying of proteins which act as anti-microbial agents. Frozen eggs showed a significantly lower acceptance than those kept refrigerated or at 27°C (p0C could not use for the sensory analysis due to spoilage. Texture hardness of the frozen eggs (0 hours) was 272.00±28.3 and it has increased up to 1060.75± 595 after 48 hours while chewiness (8.15±0.0707, to 14.966±12.80), gumminess (227.50±2.12 to 619.00±380) springiness (3.623±0.0802 to 3.40±0.157) and cohesiveness (0.825 ±0.0778 to 0.55 ± 0.1365) decreased. This texture variation has clearly explained by the reflection of light changes in microscopic images. FTIR data explained the textural changes in bonds of Amide A (3271 cm-1), Amide I (1626.2 cm-1), Amide II (1539.0 cm-1), C=O stretch of COO- (1397 cm-1), asymmetric PO2- stretch (1240 cm-1). Concluding, refrigeration condition (4 0C) for 24 hours was the more suitable way to store the boiled eggs to avoid consumer rejection when consider with texture profile and egg white lightness.

<footnote>* Corresponding author: sandun@uwu.ac.lk</footnote>
EVALUATION OF PHYSIOCHEMICAL CHANGES OF RAW CHICKEN EGGS STORED AT DIFFERENT TEMPERATURES

H. Pathirana(1), H.Aruppala(1), J.Pitawala(1), S.Abeyrathne*(1)

(1)Uva Wellassa University, Badulla, Sri Lanka

* Corresponding author: sandun@uwu.ac.lk

Eggs are great and cheap source of high-quality protein which are widely used in the food processing industry. However, eggs are undergone deterioration with the time due to its' high perishability and affects to economic losses. Refrigeration is very effective in preserving egg quality. The study was carried out to evaluate the physiochemical changes of raw chicken eggs (RCE) stored at room temperature (28-31°C) and refrigeration temperature (4°C). A total of 111 medium sized white shelled RCE obtained from 61-week old Hyline. 6 eggs per each experimental group were studied and subjected to analyze weight loss (%), yolk color, Haugh unit, USDA grade, yolk and white pH and Fourier transform infrared (FTIR) spectrums at both room temperature at 4°C for 0, 1, 4, 8, 12, 16, 20, 24, and 29 days. Salmonella examined was done to RCE in room temperature at day 0. The data were analyzed by one-way ANOVA model in MINITAB 17.1. According to the result the eggs were not contaminated by Salmonella. USDA grade has decreased in both temperature from AA to B as at room temperature after 8 days and after 16 days at (4°C). Yolk color in room storage RCE increased significantly than those kept at refrigeration temperature (4°C) (p<0.05) and symmetric CH2 stretching (3304 cm⁻¹) in room storage condition. No chemical changes observed in egg albumin of refrigerated RCE. Moreover, secondary structural changes were detected in protein in RCE albumin during the storage. In conclusion, RCE stored at refrigerated storage showed less physiochemical changes in weight loss, Haugh unit, albumin pH, yolk pH and chemical structures during the storage period up to 29 days than room temperature.
EFFECTS OF GENOTYPE ON THE EGG QUALITY CHARACTERISTICS IN BROWN-EGG LAYING HENS

M. Đukić stojčić*(1), L.Perić(1), S.Spiridonović(1)

(1)University of Novi Sad Faculty of Agriculture Department of Animal Science, Novi sad, Serbia

* Corresponding author: mirjana.djukicstojcic@stocarstvo.edu.rs

Egg quality characteristics are a very important factor both for the consumers, as well as for the egg producers. The main factors that affect egg quality characteristics are hens strain, hens age, storage temperature and time, humidity, laying season, and nutrition. On the market there is a number of different hybrids, and egg producers are often in doubt as to which hybrid to choose.

This study aimed to determine the influence of hybrid on egg quality parameters (external and internal) of commercial laying hens. 60 eggs of Hy line silver and Hy line brown were examined from the hens of the same age (50 weeks). All eggs were collected on the same day. The hens were reared on same farm, under same conditions, and fed with same diets.

Eggs were analysed in the laboratory for determining poultry meat and egg quality on the Faculty of Agriculture in Novi Sad, Department of Animal Science. The determinants of external and internal egg quality traits were: egg weight, shape index, shell cleanness, shell colour, shell breaking force, shell thickness, shell weight, yolk colour, albumen height, yolk height, Haugh unit (HU), yolk width, yolk index, USDA, blood / meat spots, and albumen and yolk pH.

Results obtained showed that both strains produced eggs which have good external and internal egg characteristics. Excluding egg shell breaking force, albumen and yolk height, yolk pH and index, all other egg characteristics studied were not significantly (p ≤ 0.05) affected by the strain.

The eggs of Hy line brown were superior in terms of internal parameters (albumen and yolk height, yolk index, and pH), while the hybrid Hy line silver was better in shell strength. The albumen and yolk height, yolk index, and pH significantly differed (p ≤ 0.05) between Hy line brown (7.37mm; 19.51mm; 48.82; 6.42), and Hy line silver (6.55mm; 18.82mm; 46.28; 6.25). There was statistically significant difference (p ≤ 0.01) between shell strength of observed hybrids. In Hy line silver eggs shell strength was 4.38, while in Hy line brown it was 3.45. A slight difference was found in HU, 83.92 for Brown vs. 80.48 HU for Silver.

The egg weight, shape index, shell cleanness, shell colour, shell thickness, shell weight, yolk colour, HU, yolk width, USDA, blood / meat spots, albumen pH were not significantly affected by the genotype.

The results obtained within this research suggest that different strains may possess different quality.

This has a large influence on the specific egg use. Concerning the egg breaking industry, the eggs from Brown hens showed superior egg quality, due to their higher internal quality. On the other hand, for the egg producers, the Silver hens are more suitable for rearing, owing to their strong shell.
EVALUATION OF CHEMICAL EGGS COMPONENTS IN LOCAL POULTRY BREEDS AND DEVELOPMENT OF NIRS SPECTROSCOPIC TECHNIQUE FOR THE PREDICTION OF THE BIOCHEMICAL COMPOSITION.

F. Cendron\*(1), S.Currò(1), C.De fassi negrelli rizzi(1), M.Cassandro(1)

(1)University of Padova, Legnaro, Italy

* Corresponding author: filippo.cendron@unipd.it

The present study aims to analyse quality eggs traits and develop infrared prediction models of Italian local chickens breeds.

The chemical analyses (proteins, moisture, lipids and ashes in yolk and white) were determined using reference methods on 25 eggs belonging to 8 genotypes: 4 breeds with white shell eggs (Padovana Camosciata, Padovana Dorata, Polverara Nera and Polverara Bianca) and 4 breeds with red shell eggs (Pépoi, Ermellinata di Rovigo, Robusta Maculata and Robusta Lionata), reared under the same farming conditions from a single farm. Spectra information were collected on visible and near-infrared part (from 400 to 2500 nm) using DS2500 (Foss, Hillerød, Denmark). Prediction models were carried out using a cross-validation approach and the accuracies of calibration equation were evaluated through the coefficient of determination (R2), error standard of prediction (SEcv), and the ratio of performance to deviation (RPD) in cross-validation. Red shell eggs show a lower yolk-to-egg white ratio compared to white shell eggs. The yolks of white shell eggs are richer in protein and moisture but lower in lipids compared to the red shell eggs. In detail, Padovana breed differs from the others for the greatest content of moisture and the lowest amount of lipids in the yolk; whereas in the albumen, Padovana differs just to Polverara showing a higher level of proteins and a lower amount of ashes. In the group of the red shell breeding hens, Pepoì differs significantly to the other breeds, both in yolk and albumen, for a higher amount of proteins and moisture, and lower quantities for the other considered chemical traits. The accuracy of the predictions were higher for the freeze-dried compared to the fresh egg yolk matrix, as expected. The fitting statistics of prediction models for eggs quality traits allowed a quite satisfactory prediction with a R2 range from 0.234 (ash) to 0.662 (moisture) in fresh eggs and from 0.486 (ash) to 0.951 (moisture) in the freeze-dried. Taken together, these results suggest an innovative approach to predict the chemical composition of eggs from local hens, that could be used as a discriminatory analysis.
NON-DESTRUCTIVE PREDICTION OF SHELL EGG QUALITY BY NEAR-INFRARED SPECTROSCOPY

E. Loffredi(1), S.Grassi(1), C.Alamprese*(1)

(1)DeFENS - University of Milan, Milan, Italy

* Corresponding author: cristina.alamprese@unimi.it

Freshness and quality characteristics play a pivotal role in defining the acceptability of eggs by the consumers. Different approaches for their assessment are available, but they are mostly destructive, while food industries call for rapid, easy and non-destructive methods, able to grade shell eggs along the entire production and distribution chain. In this context, the aim of this work was to evaluate the possibility of using near-infrared (NIR) spectroscopy as a non-targeted technique for non-destructive egg quality assessment.

Eighty-four shell eggs from organic and barn farming were analysed at different times and storage conditions for weight, albumen and yolk ratios, Haugh Unit (HU), air cell height, yolk coefficient, and shell strength and thickness. Diffuse reflectance spectra (12,500 to 4,500 cm⁻¹, FT-NIR, MPA, Bruker Optics, Milan, Italy) were collected by a fibre optic probe at four sampling points: two in the equatorial region and two at the poles. Before spectral acquisition, the effectiveness of beam penetration was assessed by comparing the spectra obtained by substituting the egg content with water and ethanol with those of pure water and ethanol.

The spectral information was reduced (7,300-4,200 cm⁻¹) and pre-treated with SNV, smoothing and first derivative, alone or in combination, to be used for the development of prediction models based on the Partial Least Squares regression algorithm. The calibrated models were evaluated for internal (cross-validation) and external prediction (test set with 29% of the total samples). The best models were obtained for HU (range = 5.93 – 87.16; standard deviation = 19.95) and air cell height (range = 4 - 13 mm; standard deviation = 2.4 mm). The calculated Root Mean Square Error (RMSE) values for the prediction were similar to those previously reported in studies on egg freshness (Giunchi et al., 2008; Kemps et al., 2006). However, the models here developed can be considered more robust as the reliability of beam penetration was assessed and they were built considering eggs from different rearing methods.

Thus, the presented results do the groundwork for a non-destructive tool to assess egg freshness and quality at industrial level.

Giunchi, A., Berardinelli, A., Ragni, L., Fabbri, A., & Silaghi, F. A. (2008). J. Food Eng., 89, 142-148.

Kemps, B. J., Bamelis, F. R., De Ketelaere, B., Mertens, K., Tona, K., Decuypere, E. M., & De Baerdemaeker, J. G. (2006). J. Sci. Food Agric., 86, 1399-1406.
EFFECT OF DIFFERENT COMBINATIONS OF NATURAL ZEAXANTHIN (YELLOW) PIGMENTS, AND NATURAL CAPSANTHIN AND SYNTHETIC CANTHAXANTHIN (RED) PIGMENTS ON COMMERCIAL LAYER HEN-EGG-YOLK COLOUR

J. Quinteros* (1), T. Wilson (2), C.Clark (3), A.Anwar (2), T.Scott (2), D.Holden (2), P.Scott (2)

(1) Research and Development division, Scolexia Pty Ltd, Melbourne, Australia

(2) Scolexia, Melbourne, Australia

(3) Premium Agri Products, Sydney, Austria

* Corresponding author: jquinteros@scolexia.com.au

Addition of pigments in the diet is a common practice in the laying hen industry, responding to the perception of the consumers about the correlation between yolk colour and egg quality, and their direct benefit to ocular and skin health. Recently, there has also been an increased preference for the use of natural pigments over their more communly used synthetic variants. Carotenoids are responsible for colouring egg yolks and chickens cannot synthetise them de novo so they need to be provided in the diet. In Australia, ration raw materials that contain natural pigments, such as maize, are not typical ingredients in layer diets because of limited availability. The objective of the trial was to compare the effect on the yolks colour of different combinations of natural yellow pigments derived from marigold flowers (Lutein; Zeaxanthin), with natural red pigments from paprika Capsicum annuum (Capsanthin) and synthetic red pigments (Canthaxanthin).

Hy-Line Brown laying hens (111) were divided into 6 groups (n = 24, 17, 18, 17, 18 and 17 from Groups 1 to 6), and fed without the addition of pigments. At 10 days before yolk colour scoring, designated groups received diets with different combinations of natural Zeaxanthin pigments (yellow), two concentrated (Leader Lutein Ester 10% and Colortek 10%) and one of typical source (Leader Yellow 2%). They also received red pigments, two from a natural origin derived from paprika (Leader Red Natural 1% and Xarocol 2%) and one synthetic (Leader red 10%, Canthaxanthin). Groups 1 and 2 received only concentrated yellow pigments from different sources, while groups 3 to 6 received different combinations of natural yellow and natural and synthetic red pigments. Yolk colours were measured every week for three weeks.

Median yolk colour scores did not differ between Groups receiving only concentrated yellow natural pigments, being 6 (4-7) for Leader Lutein Ester and 6 (4-8) for Colortek. Median scores in Groups 3 to 6, 12 (10-13), 12 (9-13), 12 (10-12) and 13 (11-14), were significantly higher. Inclusion rate for the non-concentrated Leader Yellow was 2.35 times higher than the concentrated alternatives and inclusion rate of natural reds was 3.50 and 3.52 times higher than that of synthetic reds.

Concentrated yellow pigments have proved to be as effective in adding colour to yolks as the typical non-concentrated pigments and with a lower inclusion rate. Natural red and synthetic red pigments bioequivalence was calculated as 3.5:1.
YOLK INDEX (YI) AS A FRESHNESS INDICATOR OF HEN EGGS ALSO RELATE TO SALMONELLA GROWTH INSIDE SHELL EGG.

H. Hatta*(1), M.Yamashita(2), N.Kubo(1), M.Osada-oka(3)

(1)Kyoto Women's University, Kyoto, Japan

(2)Ise Egg Research Center, Tokyo, Japan

(3)Kyoto Prefectural University, Kyoto, Japan

* Corresponding author: hatta@kyoto-wu.ac.jp

[Introduction] It has been known that hens infected with Salmonella enterica Enteritidis (SE) lay eggs with SE-contaminated on yolk membranes inside their shell eggs. Since Japanese eat raw eggs, it is necessary to ensure the freshness and safety of shell eggs. The expiry date for hen eggs is calculated by the relationship (Humphrey's equation) between the storage temperature and SE growth at the surface of yolk membrane inside shell eggs. D=86.939-4.109T+0.048T2 (D: days of SE rapid growth T: stored temperature)

Iron and yolk components are required for the growth of SE, and when the yolk membrane is weakened during egg storage, SE growth is accelerated due to the iron and yolk components leaked from the yolk. [Experimental design] Fresh eggs were stored at 30°C with 75% humidity for 0-15 days, and at 25°C for 0-4 weeks. Then, the egg freshness was measured during the storage time. For the HU and YI, digital egg tester (DET6000: Navel Co., Ltd.) was used. Yolk membrane strength was measured as a breaking load (gf/cm2). Egg yolk of shell eggs stored at 30°C with 75% humidity for 0, 3, 6, 9, 12 and 15 days and stored at 25 °C for 0.1, 2, 3, and 4 weeks were aseptically separated. Then, each separated yolk was individually immersed in 10-100 CFU/ml SE bacterial solution, and after 24 hours incubation, the amount of the bacteria was individually measured using an ATP luminescence measurement method (BackTiter-Glo: Promega). Statistical analysis was performed using ANOVA.

[Results] The strength of yolk membrane decreased in the eggs stored at 30 °C and 25 °C in correlation to decreases of both HU and YI. The SE bacterial count with being soaked egg yolk significantly increased when the yolk of the eggs whose expiration date have passed (P

[Conclusions] In this study, the validity of the expiration date, which indicates the shelf life of eggs in Japan, was confirmed. Furthermore, using "YI" as a new freshness index for hen eggs, we were able to propose, "YI = 0.3 or more" as a cut-off value for judging that raw eggs can be eaten even in egg SE-contaminated.
EF5

MEAT QUALITY

CONSTRUCTION OF MEAT QUALITY

MEAT PROCESSING
CONSTRUCTION OF MEAT QUALITY
POLYPHENOLS AS EFFECTIVE SOLUTION TO IMPROVE MEAT QUALITY AND ANTIOXIDANT STATUS OF BROILERS

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: astrid@impextraco.be

Intense farming contributes to increased oxidative stress, with harmful implications for health, productivity, carcass dressing and meat quality. The aim of this study was to determine the effect of different doses of a polyphenol blend (ELIFE®) on the antioxidant potential and meat quality of broiler chickens. The experimental design comprised 1440 Cobb® male day-old chicks (4T*9R*40) assigned to 4 dosages: T1 – negative control, T2 – supplementation of 250 mg kg⁻¹, T3- supplementation of 500 mg kg⁻¹, and T4 – supplementation of 1000 mg kg⁻¹ in finisher phase only. A 3-phase standard corn soybean meal diet (60 ppm vit E) was fed. At the end of the experiment, breast muscle and legs were removed from the carcass and weighed to calculate carcass yield (n=9). Meat quality was determined by the evaluation of thiobarbituric acid reactive substances (TBARS) on breast muscle and oxidative stress was determined by the analysis of glutathione (GSH), glutathione transferase (GST), and lipid peroxidation (LPO) on the liver samples from these same birds. Although not significant, a dose response was found for proportional breast muscle weight. A one percent increase was also found for proportional drumstick weight for the highest inclusion compared to the control. TBARS are formed as a byproduct of lipid peroxidation and indicate damage produced by oxidative stress. TBARS were significantly (p<0.0001) lower for all supplemented groups compared to control. LPO in the liver was also reduced with the inclusion of the polyphenol blend compared to the control, although not significant. GSH content in the liver significantly (p=0.0066) increased with supplementation, indicating less oxidation. GST is an enzyme that interferes in the conversion of reduced glutathione (GSH) into its oxidized form (GSSG). High intracellular concentrations indicate injury to those cells. Its concentration in the liver decreased significantly (p=0.019) with supplementation above 500 mg kg⁻¹ compared to control. In conclusion, supplying polyphenols to fast growing broilers protects them against oxidative stress, with decreased lipid peroxidation and improved meat quality as a consequence.
DIETARY ANTIOXIDANT SUPPLEMENTATION IMPROVES MEAT QUALITY OF FEMALE KORAT CHICKEN.

M. Magnin(1), C.Rozier*(2), C.Messant-monier(2), W.Wonganusorn(3), C.Suwanvichanee(4), S.Kubota(4), W.Molee(4), J.Yongsawatdigul(5)(4)

(1)ID4FEED, Annecy, France

(2)ID4FEED SAS, Annecy, France

(3)PS Nutrition Co, Bangkok, Thailand

(4)School of Animal Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon ratchasima 30000, Thailand

(5)School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon ratchasima 30000, Thailand

We investigated the effects of a dietary antioxidant supplementation (ID PHYT BIOLOX, ID4FEED SAS, France) composed of natural or nature-identical ingredients (containing 7% carvacrol, 6% thymol, 4% 1-8 cineol, 7% alpha-pinene, 0.03% carnosic acid, 3% Capsicum powder) when given to Korat slow - growing female chicken (KR). Chicken were fed practical corn-soybean meal diets with a single feed the first 3 weeks of age. After 3 weeks, female chickens were randomly allocated to 2 treatments, CT = control and ANT = control + 0.07% of antioxidant preparation. The performance, body weight, body weigh gain, feed intake and feed conversion ratio were measured at the starter period (3-6 week) and finishing period (6-10 weeks). At the 10 weeks of age, 5 chickens per replicate were slaughtered and breast meat were collected to measure the driploss, muscle color (L*, a*, b*), pH values at 48 min and 24 hour, cooking loss and TBARS values. The results showed that the growth performance, muscle color and pH values were not affected in ANT birds (P>0.05) but cooking loss, TBARS and drip loss were significantly decreased (respectively -4.8%, -25.1% and -13.0% ; P<0.05) in ANT treatment. These results suggest that plant antioxidants may reduce meat lipid oxidation and retain the properties of meat proteins during processing. The reducing of water loss is the parameter to indicate the meat juiciness in which has been affecting to consumer acceptance.

Key words: antioxidant, plant extract, chicken, meat quality

<footnote>* Corresponding author: camille.rozier@id4feed.com</footnote>
UNDERSTANDING THE ROLE OF HISTIDINE DIPEPTIDES ON POST-MORTEM PH DECLINE IN BROILER BREAST AND LEG MUSCLES

G. Baldi\*(1), F.Soglia(2), L.Laghi(1), M.Petracci(1)

(1) University of Bologna, Cesena, Italy

(2) University of Bologna, Cesena, Italy

* Corresponding author: giulia.baldi4@unibo.it

The rate and the extent of muscular post-mortem acidification influence several qualitative and technological properties of meat. Poultry muscles are highly abundant in histidine dipeptides (HD) such as anserine and carnosine, that possess a great buffering ability in vivo and thus might influence muscular acidification during early post-mortem, where muscle pH is still close to their action range. The aim of the study was exploring the role of HD on post-mortem metabolism in 3 chicken muscles having different energy metabolism and belonging to the main cut-ups (breast, thigh and drumstick), previously selected based on their ultimate pH and carnosine content as indicators of muscle glycolytic activity. For this purpose, 8 carcasses of broiler chickens (Ross 308 strain, females, 49 days of age, 2.8 kg body weight) were obtained from a commercial producer and samples from bone-in Pectoralis major (PM; breast), Extensor iliotibialis lateralis (EIL; thigh) and Gastrocnemious pars interna (GI; drumstick) muscles were collected at 15, 60, 120 and 1,440 min post-mortem, instantly frozen in liquid nitrogen and used to evaluate pH and glycolytic metabolites. At 1,440 min, buffering capacity and HD (anserine and carnosine) concentration were also assessed. Data were analyzed using One-Way ANOVA to test the effects of both sampling time and muscle type. According to their metabolic characteristics, selected muscles showed different acidification kinetics and carbohydrate metabolism. Indeed, if post-mortem glycolysis terminated at 120 min in both EIL and GI due to their lower glycolytic potential (P<0.05), it continued in PM allowing the achievement of a significantly lower ultimate pH (P<0.05) measured at 1,440 min. However, considering both its lactate production pattern and the existence of residual glycogen at 24 h post-mortem, PM should have shown a faster/greater pH decline. We suggest that the remarkably higher content of HD detected in breast muscle (P<0.001) exerted an effect on post-mortem metabolism by reducing the rate and the extent of muscle acidification. Indeed, if compared to leg muscles, PM showed significantly higher buffering capacity in the pH range 6.0-7.0 (P<0.001). In light of these outcomes, dietary strategies aimed at modulating themuscular content of histidine dipeptides could regulate post-mortem acidification kinetics, thus improving poultry meat quality traits (i.e. limit the incidence of PSE-like meats and improve microbial stability).
CAN ONE WEEK'S USE OF NETTLE IN BROILER FEED AFFECT THE CHEMICAL QUALITY OF MEAT?

M. Lukić*(1), N.Stanišić(1), Z.Škrbić(1), V.Petričević(1), N.Tolimir(2), V.Dosković(3), T.Stamenić(1)

(1)Institute for Animal Husbandry, Belgrade, Serbia

(2) Institute for Science Application in Agriculture, Belgrade, Serbia

(3)Faculty of Agronomy, University of Kragujevac, Čačak, Serbia

* Corresponding author: miloslukic.izs@gmail.com

This experiment aimed to evaluate the effects of adding fresh or dry nettle (Urtica dioica L.) to broiler diets during the last week of fattening on proximate composition, fatty acid and mineral content of meat. The trial was performed on a total of 600 ROSS-308 unsexed day-old chickens, divided into 3 groups (C - Control group; FN - Fresh Nettle group; DN - Dry Nettle group). All broilers were bred and fed equally, with standard diets from age 1-42 days, except for the last seven days of fattening when in FN group was added daily to the feeders (on top) 20g/chicken freshly collected, blanched and chopped nettle, or in case of DN group of broilers, 10g/kg finely ground dry nettle mixed into a finisher. On day 42, random sample of 6 male and 6 female birds were taken from each group and slaughtered. Meat samples (breasts and drumsticks) were collected and the proximate composition analysis was conducted on each portion of each chicken (total of 72 samples). The fatty acid composition (Shimadzu GC-2014 with FID detector) and mineral analysis (Na, Mg, K, Ca, Mn, Fe, Cu, Zn and Se content measured by iCap Q) was conducted on each portion (breast and drumstick) of 6 chicken per group (3 male and 3 female). An analysis of variance and t-test (IBM SPSS Statistics 20 software) was performed. Meat fat content was significantly lower (P=0.028) in broilers fed with the addition of dry nettle compared with other two groups, which could have an impact on quality characteristics of meat, such as flavor and tenderness. The addition of fresh or dry nettle influenced meat ash content as well, which was significantly larger compared with C group (P<0.001). Total MUFA was highest in DN group (31.20%), while there were no significant differences between other two groups (P<0.001), mainly due to higher share of palmitoleic and oleic acid found in meat from DN group. The lowest amount of total PUFA (35.08%) and n-6 fatty acids (32.19%) was found in DN group (P<0.001), specifically significantly lower content of linoleic acid. Also, DN group have better n-6/n-3 ratio (11.83) compared to control (13.46, P=0.036), while there was no effect when fresh nettle was used. Analysis of minerals in meat showed significantly higher level of Na and Se in DN group, and Fe and Zn in FN group. Generally, research shows that extra addition of 1% dry nettle in broiler feed during the last seven days of fattening can result in different chemical, fatty acid and mineral composition of meat.
INTERACTION BEWEEN CHICKEN GENOTYPE AND FEEDING REGIMEN FOR THE CONTROL OF MEAT QUALITY TRAITS

E. Tumova*(1), D.Chodova(2), M.Ketta(2), V.Skrivanova(3)

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

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

(3)Institute of Animal Science Prague, Prague, Czech Republic

* Corresponding author: tumova@af.czu.cz

In poultry production is great interest to provide consumers with high quality and safe meat. Meat quality has been affected by variable factors of which genotype and feeding regime play an important role. There is a question if both factors interact in meat quality traits, therefore, the aim of the study was to evaluate interaction between chickens genotypes of variable growth intensity and feeding regime in physical and chemical meat parameters. In the study, meat quality was analysed in 120 chickens; fast-growing Ross 308, medium-growing JA 575 and slow-growing ISA Dual chickens. In each genotype, a half of chickens were fed ad libitum (AL) and the second half was restricted (R) from 14 to 21 days of age. During restriction chickens received 70 % ad libitum feeding. The meat quality was assessed in breasts of chicken's slaughtered at live weight 2 kg, which Ross chickens reached at the age of 32 days, JA 757 45 days and ISA Dual 80 days. Results were processed by two-way analysis of variance with interaction of genotype and feeding regime. Meat pH, colour and cooking loss were significantly affected only by chicken genotype. Interaction of genotype and feeding regime was detected in texture (P≤0.05). The lowest texture was in restricted JA 757 (8.4 N), in Ross (AL 15.2 N vs. R 11.7 N) and JA 757 (AL 9.1 N vs. R 8.4 N) texture was significantly lower in the restricted groups, whereas, in ISA Dual in the ad libitum fed group (AL 9.8 N vs. R 11.3 N). In meat chemical composition, interactions were observed in dry matter (P≤0.05), ether extract (P≤0.001) and hydroxyproline (P≤0.001). On the other hand, crude protein was the highest (P≤0.001) in ISA Dual (AL 23.7 % and R 23.9 %). Based on the data of the present study we can assume that interactions of genotype and feeding regime are more important in meat nutritional value than in physical meat traits, and genotype more affected meat quality traits than feeding regime.
THE MEAT QUALITY OF MALES AND FEMALES OF FAST-, MEDIUM- AND SLOW-GROWING CHICKEN GENOTYPES

D. Chodova(1), E.Tumova*(2), M.Ketta(1)

(1)Czech University of Life Sciences Prague, Prague suchdol, Czech Republic

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

* Corresponding author: tumova@af.czu.cz

The aim of the study was to evaluate the meat quality parameters of males and females of fast- (Ross 308), medium- (JA 757) and slow-growing (ISA Dual) chickens slaughtered at 2 kg of weight. Males and females of each genotype were fattened under identical conditions. At 2 kg live weight, 20 males and 20 females were slaughtered. For meat quality analysis, the samples of Pectoralis major (PM) were taken 24 hours post mortem. The fast-growing chickens reached 2 kg slaughter weight at 32 days of age, medium- at 35 days of age and slow-growing at 74 days of age. No effect of sex or interactions of genotype and sex were detected in monitored traits except of meat tenderness. The dressing out percentage and breast percentage were lower (P≤0.001) in ISA Dual compared to medium and fast growing chickens. From physical meat characteristics, the pH value was affected by genotype (P≤0.001) with lower values in JA 757 and ISA Dual compared to the Ross. All of meat colour characteristics (L*, a* and b*) were influenced by genotype. The ISA Dual chickens had lighter (P≤0.001) an less intensive colour parameter a* (P≤0.001), but more intensive colour parameter b* (P=0.004) in PM than both genotypes Ross and JA 757. The drip loses were not affected by any of tested parameter. However, cooking losses were higher (P≤0.001) in JA 757 than in other genotypes. The toughest meat was observed in cockerels of ISA Dual (P≤0.001) and the most tender in Ross of both sexes. It can be concluded that slow-growing chickens had lower dressing out percentage and breast yield and less intensive meat colour than fast- or medium-growing chickens.
DIETARY INCLUSION OF HEMP (CANNABIS SATIVA L) AND DILL SEED (ANETHUM GRAVEOLENS)
IMPROVES SHELF LIFE AND QUALITY OF BROILER MEAT

M. Vispute*(1), D.Sharma(2), A.Rai(3), A.Biswas(4), A.Biswas(4), J.Rokade(4), G.M.(4), A.Chaple, A.Mandal(4)

(1) ICAR-INDIAN VETERINARY RESEARCH INSTITUTE, IZATNAGAR, BAREILLY, INDIA, Bareilly, India

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

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

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

* Corresponding author: mayurmv125@gmail.com

A study was conducted with dietary inclusion of hemp (Cannabis sativa L.) and dill (Anethum graveolens) seed in varying combinations against antibiotic growth promoter in broiler diet with an objective to study their possible synergistic effect on meat quality of broiler chickens. 192 CARIBRO-VISHAL broiler chickens were grouped under completely randomized design into six dietary treatments for 42 days comprising varying combinations of hemp and dill seed in addition to the basal diet. Each treatment had four replicates with eight birds per replicate, viz. Diet T1 (Basal Diet), T2 (BD+0.2% HS), T3 (BD+0.2% HS+0.3 DS), T4 (BD+0.3% HS), T5 (BD+0.3% HS+0.3 DS) and T6 (BD+0.025% Bacitracin Methylene Disalicylate). At 42 days post-hatch, eight birds per treatment group were sacrificed to collect breast and thigh meat samples to study the quality of fresh and stored meat (30 days). The muscle lipid, cholesterol content, DPPH and ABTS+ activity, TBARS value and Free fatty acid was determined by using methods of AOAC (1990); Wybenge et al. (1970); Kato et al. (1988); Shirwaikar et al. (2006); Witte et al. (1970) and Koniecko (1979) respectively. Statistical significance was declared at P<0.05 using the Tukey's multiple comparison test. Lipid content of thigh and cholesterol of both thigh and breast meat remained unchanged due to seed supplementation. Free fatty acid content in breast meat did not differ significantly, however, increased significantly at low inclusion levels (T3 & T2). TBARS value was significantly higher (P<0.001) in both pre and post storage breast and thigh meat of control birds. ABTS+ (P<0.001) radical scavenging activity was reduced significantly in fresh and stored meat. Lowest inhibition was witnessed in control (T1) under pre and post storage phases. DPPH activity increased significantly (P<0.05) in fresh meat, however, remained comparable in all groups in post-storage conditions except for antibiotic control (T6). It is concluded here that, the dietary hemp and dill seed, especially in combination improved shelf life of broiler meat under cold storage without compromising the meat quality.

Keywords: Hemp, Dill, meat quality, shelf life, Broiler

References:

Shirwaikar et al. (2006). In vitro antioxidant study of Sphaeranthus indicus (Linn). Ind. J. of Exp.Biol.
44:993-996

Witte et al. (1970). A new extraction method for determining 2-thiobarbituric acid values of pork and beef during storage. J. Food Sci. 35:582-585
SYNTHETIC VITAMIN E AND A NATURAL ALTERNATIVE : COMPARISON ON BROILER ZOOOTECHNICAL PERFORMANCE AND MEAT QUALITY

A. Atkinson*(1), J.Le roux(2), J.Gonzales foretic(3), C.Alleno(4)

(1)NUPROXA FRANCE, Ploufragan, France

(2)Nuproxa France SA, Ploufragan, France

(3)NUPROXA SWITZERLAND SA, Etoy, Switzerland

(4)ZOOTESTS, Ploufragan, France

* Corresponding author: jf.leroux@nuproxa.ch

Vitamin E (VE), found in most of raw materials used in poultry feed, is a biological antioxidant beneficial for farming under stressful environment such as high stocking densities, high temperature and immune suppression. It is also known for its interest in meat quality improvement. Synthetic VE is a mix of eight isomers of α-tocopheryl acetate with variable bioavailability. Natural VE is more complex and is composed of tocopherols, tocotrienols under 4 forms, α, β, γ and δ. Natural alternatives such as Herbal E, a polyherbal mixture composed of Ocimum Sanctum, Phyllantus emblica, Emblica officinalis are interesting because of their biological antioxidant activity. The purpose of this experiment was to compare the impact of synthetic VE and Herbal E on broiler zootechnical performance and meat quality.

The experiment was conducted with 400 broilers divided in 4 groups of 10 repeats of 10 Ross 308 males from day 21 to 42. The treatments were: T1: Negative Control; T2: 30 ppm of synthetic vitaminE; T3: 30 ppm of Herbal E; T4: 15pm of synthetic vitamin E and 15ppm of Herbal E. Mortality and individual weight were measured at days 21, 32, 42. Feed consumption and feed conversion ratio (FCR)were measured for periods 21-32 and 32-42 days. Meat quality, pH, colour and drip loss, were evaluated at day 42. Fixed effects were tested using ANOVA, SAS software.

There was no effect (p > 0.05) of treatment on average body weight at day 32 and 42. During period 21-32 days, FCR was higher (p < 0.001) for T1 and T2 (1.58 and 1.59, respectively) vs T3 and T4 (1.53 and 1.55, respectively). This difference was not significant (p > 0.05) for the period 32-42 days but remained significant (p < 0.001) during the whole period 21-42 days for T1 and T2 group (1.72 and 1.74, respectively) vs T3 and T4 groups (1.69 for both). There were not differences (p > 0.05) in carcass and breast yield, meat pH and colour between treatments. There was a reduction (p < 0.001) of drip loss in filets of groups T3 and T4 (1.07 and 1.12%, respectively) vs T1 and T2 (1.41 and 1.29%, respectively). Vitamin E had no effect on zootechnical performance and meat quality under the conditions of this experiment but broilers fed diets supplemented with the natural alternative improved both FCR and filet drip loss. Polyherbal mixture of plants known for their biological antioxidant activity can be an interesting alternative to VE for finishing broilers.
# MANIPULATION OF LIGHT INTENSITY IMPACT ON GROWTH, MEAT QUALITY, BLOOD PARAMETER AND BEHAVIOR OF BROILERS

K. Ryu\*(1), C.Lim(2), M.Rana(2), H.Choe(3), H.Chang(4)

(1)Department of Animal Science, Jeonbuk National University, Jeonju, South Korea

(2)Jeonbuk National University, Jeonju, South Korea

(3)Department of Animal Biotechnology, Jeonbuk National University, Jeonju, South Korea

(4)Department of Veterinary Infectious Diseases and Avian Diseases Control, College of Veterinary Medicine and Center for Poultry Disease Control, Chonbuk National University, Iksan, South Korea

* Corresponding author: seon@jbnu.ac.kr

The aim of this study was to investigate the influence of different light intensities on the growth performance, meat quality, blood parameters and behavior of broilers raised to 42 days of age. A total of 1280 one-day-old broiler chicks were randomly assigned to eight treatment groups based on light intensity in 32-floor pens of 2.86 m2 per pen. Each light intensity treatment had four replicates of forty chicks each. The light intensity treatments were as follows: 30-30-30; 30-30-20; 30-20-20; 30-20-10; 20-20-20; 20-20-10; 20-10-10; and 20-10-5 lx for the pre-starter (1 to 11 d), starter (12 to 25 d) and grower (26 to 42 d) period, respectively. Experimental birds were fed ad-libitum commercial mash diets during respective rearing period and given free access water. Data were subjected to statistical analysis using the GLM procedure of SAS software (SAS 9.1, 2009) in a completely randomized design. The results of the study showed that weight gain was differed by the light intensity treatments over the experimental period from 1 to 42 days of age and higher was in 20-10-10 lx intensity group (3050.71 g; p
SKIN COLOUR PREDICTION IN BROILER ACCORDING TO THE SEX AND CAROTENOID IN THE DIET

M. Campo*(1), J.Pérez(1), A.Guerrero(1), V.Resconi(1), M.Barahona(1), J.Olleta(1), R.Berrocal(2), F.Bacha(2), M.Mur(2)

(1)University of Zaragoza, Zaragoza, Spain

(2)UVESA, Tudela (navarra), Spain

* Corresponding author: marimar@unizar.es

A total of 1080 one-day-old males and 1080 one-day-old females, both ROSS 308, were randomly divided into 3 groups with 6 replicates (cages) of 60 animals each from the same sex. All were fed with 34 ppm of natural xanthophylls (mainly lutein + zeaxanthin) in the starter phase (1 to 14d). In the grower phase (15 to 41 d), all were fed with 5 ppm of canthaxanthine, but three different pigments were added: 68 ppm of lutein (natural pigment, group A), 34 ppm of apo-carotene-ester (synthetic pigment, group B) and 68 ppm of natural xanthophylls (group C). At days 14 and 28, both at the farm, and at day 41 at the abattoir, the colour of the skin was measured with a Minolta CM 600d in the CIEL*a*b* space in 5 chickens per cage, in two regions: underneath the wing and at the tibiotarsal joint in the thigh. Analyses of variance were used though a GLM model with sex and diet as fixed effects, and regression equations were calculated per each treatment. At 14 days, no differences were found due to the diet, but females showed higher redness in both measured areas. Since the amount of xanthophylls was identical for each gender, we can assume that differences in fatness in subcutaneous fat were responsible for colour differences at this early age. At 28 days of age, no differences were found at the tibiotarsal joint, but underneath the wing females showed higher yellowness (13.02 vs 11.29, P<0.01) with higher saturation (13.34 vs 11.62, P<0.01) than males. Also group A and C diets showed higher yellowness and saturation underneath the wing than group B diet (P<0.01). At the end of the trial, females had the skin underneath the wing more yellow (28.74 vs 25.31, P=0.001) and with higher saturation than males, whereas males showed lighter skin (69.20 vs 70.39, P=0.001) and slightly redder (P<0.05) than females at the tibiotarsal joint. The lower amount of carotenoid in group B showed lower colour indexes in both measuring areas. However, although with similar amount of carotenoid in the diet, group C showed similar colour indexes than group A underneath the wing, but higher redness (P<0.01), yellowness and saturation (P<0.60) than underneath the wing (R2<0.35), better in females than in males. Quadratic predictions of yellowness are slightly better than linear predictions, very similar in both sexes and measuring areas in diets A (R2=0.83) and C (R2=0.86), and more variable in diet B (R2=0.64-0.79).
BROILER BREAST COLOUR SHELF LIFE ACCORDING TO THE SEX AND CAROTENOID IN THE DIET

M. Campo*(1), J.Pérez(1), A.Guerrero(1), V.Resconi(1), M.Barahona(1), J.Olleta(1), R.Berrocal(2), F.Bacha(2), L. Mur(2)

(1)University of Zaragoza, Zaragoza, Spain

(2)UVESA, Tudela (navarra), Spain

* Corresponding author: marimar@unizar.es

A total of 1080 one-day-old males and 1080 one-day-old females, both ROSS 308, were randomly divided into 3 groups with 6 replicates (cages) of 60 animals each from the same sex. All were fed with 34 ppm of natural xanthophylls (lutein + zeaxanthin) in the starter phase (1 to 14d). In the grower phase (15 to 41 d) all were fed with 5 ppm of canthaxanthine but three different pigments were added: 68 ppm of lutein (natural pigment from Marigold, group A), 34 ppm of apo-carotene-ester (synthetic pigment, group B) and 68 ppm of natural xanthophylls (lutein + zeaxanthin, group C). At 24h after slaughter, and 2d and 6d after display with O2 permeable film at 4oC in the dark, colour was measured in the external surface and sliced breast, with a Minolta CM 600d in the CIEL*a*b* space in 15 chickens per treatment. Analyses of variance were used though a GLM model with diet and display as fixed effects within sex. Diet highly influenced colour variables in females, but did not affect lightness in males. In the sliced breast, group C showed higher yellowness (25.55, 25.90) and saturation (21.97, 21.79) in both males and females respectively, but lower in the intact breast in males (21.66, 19.44, respectively), than in groups A and B. Group B had the lowest values of yellowness, especially in females in the sliced breast (18.70). Display had a large effect in males, but did not affect redness in females. Throughout display, meat was darker, with less yellowness, Chroma and Hue. The decrease was especially evident in males, which started with higher values than females in yellowness (26.90 vs 23.40) and Chroma (27.21 vs 23.79), but after 2 days of display colour was already less intense, whereas females did not show a significant decrease between 24h after slaughter and 2 days of display in the sliced breast, keeping better the colour. After 6 days of display, the lost in yellowness and Chroma was very intense, especially in males (21.56, 10.62, respectively). The studied pigments at the used doses behave differently according to the sex, although group C with natural xanthophylls shows more intense colour than the rest of groups. Both intact and sliced breast lose colour intensity throughout display, more evident in males than in females.
# THE USE OF TURMERIC (CURCUMA DOMESTICA) AND PAPAYA LEAVES (CARICA PAPAYA L.) POWDER MEAL AS FEED ADDITIVE IN THE BASAL DIETS OF BROILER CHICKENS TO IMPROVE THEIR MEAT QUALITY

Y. Henuk\*(1)

(1) University of Sumatera Utara, Medan, Indonesia

\* Corresponding author: profesorhenuk@gmail.com

Sonya E. Silaen, Iskandar Sembiring and Yusuf L. Henuk\*

Department of Animal Science, Faculty of Agriculture, Universitas Sumatera Utara (USU), Medan, North Sumatera 20155 INDONESIA

\*Co-author/Corresponding/Presenting author: profesorhenuk@gmail.com

An experiment was conducted to determine the use of turmeric (Curcuma domestica) and papaya leaves (Carica papaya L.) powder meal as feed additive in the basal diets of broiler chickens to improve their meat quality. A total of 100 day old chicks were divided into four groups, then sub-divided into five replicates of five birds/treatment. The turmeric powder and papaya leaves powder were not added into the dietary treatment groups as T0 (only basal diet as control), T1(basal diet+0.5% turmeric powder), T2 (basal diet+2.5% papaya leaves powder), and T3 (basal diet+0.5% turmeric powder+2.5% papaya leaves powder). The meat quality traits of broiler chickens measured were water content, protein content, fat content, tenderness and cooking loss of broiler meat. Data on broiler chicken moisture content were 76 to 78% within the ranged of normal poultry meat of 60 to 80% water (Florkowski et al., 2002). The results of protein content within the ranged of normal poultry meat of 15 to 25% protein (Florkowski et al., 2002). The fat content is higher than 1.5 to 5.3% lipids reported by Florkowski et al. (2002). The tenderness of broiler meat were extremely tender of (<3.3 kg/cm-2; Suryati et al., 2008). The cooking loss of meat were 30.34 to 34.01% within the ranged of normal cooking loss of broiler meat of 15.55 to 36.0% (Rehman et al., 2012). In conclusions, the addition of turmeric and papaya leaf powder as feed additives in the basal diets of broiler chickens affected the content of protein, fat, and meat tenderness. The addition of papaya leaves powder alone can increase their protein content, water and tenderness while reduce their fat content and produced the lowest water content in broiler chicken meat.

## References

Florkowski T., M. Słowiński, K. Jasiewicz. 2002. Colour and technological quality of chicken meat. Mat. XXXIII Sesji Nauk. KTiChŻ PAN, 161 Abstract (in Polish).

Rehman, G., A. Basit, Rahman, I., M. Khan, and S. A. Kham. 2012. Comparative study on cooking loss in broiler and layer meat. Depart. of Veterinary. Medicine, Fac. of Animal Husbandry and Veterinary Science, Sindh Agriculture University Tandojam.

Suryati, Arief II, Polii. 2008. Korelasi dan kategori keempukan daging berdasarkan hasil pengujian menggunakan alat dan panelis. Anim. Prod., 10:188-193.
USE OF ALLYL-ISOTHIOCYANATE AND CARVACROL TO PRESERVE FRESH CHICKEN MEAT DURING CHILLING STORAGE

K. Hussein*(1), L.Friedrich(1), G.Kisko(1), E.Ayari(1), C.Nemeth(2), I.Dalmadi(1)

(1)Szent Istvan University, Budapest, Hungary

(2) Capriovus Ltd, Budapest, Hungary

* Corresponding author: khagri86@yahoo.com

The effect of active compounds (ACs); Allyl-isothiocyanate (AITC) and carvacrol (CARV), as natural additives on the quality of fresh chicken meat was evaluated. The meat was treated with 500, 1000-ppm of ACs vacuum packaged and stored at 4 °C up to 8 days. Samples were taken at different time intervals for different analyses on day 1, 3, 6 and 8. Physicochemical characteristics: pH, colour values (L*, a* and b*, Chroma and hue angle), lipid oxidation (thiobarbituric acid reactive substances-TBARS), microbiological evaluation: in vitro three Gram-positive (Listeria monocytogenes CCM, Staphylococcus aureus ATCC and Bacillus cereus) and three Gram-negative (Escherichia coli O157:H7, Salmonella Typhimurium and Pseudomonas lundensis) were used as target bacteria, in meat model aerobic mesophilic counts-AMCs were tested, sensorial properties (Electronic-nose based) were examined. AITC, particularly 1000-ppm, showed greater activity than CARV and resulted in cumulative colour changes higher and increasing L*, b* and h*, decreasing a* values, accumulative odour production. Besides AITC triggered reduction in the growth of Pseudomonas lundensis, Staphylococcus aureus, and Bacillus cereus and 3 log10 CFU/g reduction in AMCs. However, CARV was more active in increasing chroma properties and reducing the growth of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium. Concomitantly, 500-ppm CARV showed greater activity than AITC in controlling lipid oxidation, producing higher level of water holding capacity and protecting the colour changes. The knowledge of properties and mode of inhibition of AITC and CARV, may contribute the great potential of these natural preservatives to preserve the quality characteristics and extend the shelf life of meat and meat products.
INVESTIGATION OF THE RELATIONSHIP BETWEEN PH AND COLOUR OF CHICKEN BREAST MEAT AND LIPID PEROXIDATION PARAMETERS BY FEEDING COCONUT, PALM AND SUNFLOWER OIL

Á. Zimborán(1), M.Erdélyi(2), R.Szabó(2), M.Mézes(2), Z.Ancsin(2), K.Balogh(3), E.Zándoki(2), M.Kovács-weber*(2)

(1)Szent István Egyetem, Gödöllő, Hungary

(2)Szent István University, Gödöllő, Hungary

(3)Szebt István University, Gödöllő, Hungary

* Corresponding author: weber.maria@mkk.szie.hu

Our aim was to measure the effect of coconut, palm and sunflower oils on lipid peroxidation and meat
quality parameters and to investigate the correlation between these values.

Cobb500 cocks (n=245) were divided into five experimental groups: C (control), CO (5% coconut oil), PO (5% palm oil), COPO (2.5-2.5% coconut+palm oil), SO (5% sunflower oil). After the fattening period (42 days), the pH of the breast meat was measured 45 minutes (pH45) and 24 hours (pH24) after slaughter. The colour (CIELab L*a*b*) was detected on fresh surface (n=40). Lipid peroxidation parameters (reduced glutathione-GSH and malondialdehyde-MDA content, and glutathione peroxidase activity-GPx) were determined in blood plasma, red blood cell hemolysate and liver. Significant correlations were found in plasma: a*-MDA (r=0.3306; p=0.0372); b*-MDA (r=-0.4019; p=0.0102) and in liver: b*-GSH (r=0.3575; p=0.0235); pH45-GSH (r=0.3216; p=0.043); pH45-GPx (r=0.4043; p=0.0097); b*-MDA (r=0.3295; p=0.0379) according to the complete dataset. The correlations were also performed separately to analyse the correlation differences of the four experimental groups.

In the liver samples of SO group, pH45-GSH (r=-0.2443; p=0.5598) and pH45-GPx (r=-0.1891; p=0.6538) parameters were contradictory, suggesting that the addition of sunflower oil may have influenced the metabolism in the liver compared to other oils containing moderately lower level of long-chain unsaturated fatty acids.

In plasma, a* value was positively correlated with MDA in each group except the control, which is believed to be influenced by the additional oil. The b* value was also associated with MDA negatively in plasma and with GSH and MDA positively in liver which was reinforced by the addition of coconut oil.

Further studies are needed to clarify the knowledge about these relationships.
THE COMPONENTS OF VOLATILE FLAVOR COMPOUNDS IN CHICKEN BREAST MEAT AND THEIR
RELATED GENES

Y. Jin(1), H.Cui(2), L.Liu(3), X.Liu(3), Y.Wang, J.Ding, R.Liu(3), H.Li(4), J.Wen*(3), G. Zhao(3)

(1) College of Animal Science and Technology, China Agricultural University, Beijing 100193, China, Beijing, China

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

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

(4)School of Life Science and Engineering, Foshan University, Foshan, China, Foshan, China

Background

The quality of food flavor will affect the choice of consumers. In different foods, the source of flavor is also different. The phenotype of flavor character is composed of different volatile compounds. The formation of flavor substances in meat products mainly depends on Maillard reaction, lipid oxidation and degradation and their interaction. The volatile components degraded by esters include aliphatic hydrocarbons, aldehydes, ketones, alcohols, carboxylic acids and esters. Therefore, in order to determine the main volatile compounds that affect the flavor of chicken breast meat, the relative content of flavor compounds was determined by GC-MS. Through genome-wide association analysis (GWAS), the possible genes related to flavor were screened to provide molecular reference for the analysis of complex flavor characters.

Materials

Population: Jingxing (DW, n = 520) population, Qingyuan Ma (n = 400) population and Wenchang (n = 53) population

Determination of flavor substances: GC-MS

GWAS model:

α: PCA and batch effect (fixed effect)

β: marker effect (fixed effect)

ξ: polygene effect (random effect)

The likelihood ratio test (LRT) was used to test the significance of each point effect.

Results

Hexanal and 1-octen-3-ol are the main volatile components in chicken breasts through Principal component analysis (PCA). Correlation analysis results show that hexanal and 1-octen-3-ol have a significant negative correlation. The relative contents of hexanal and 1-octene-3-ol were used as phenotypic data, and the genotype of DW population was used for GWAS analysis. In the significant association region located by hexanal and 1-octen-3-ol, the predicted candidate genes are LPPand BCL6genes.

Discussion

Reports show that 1-octen-3-ol and hexanal can change the taste of dry-cured ham, which means that 1-octen-3-ol and hexanal are the main volatile components, and their contribution to the flavor cannot be ignored. As their candidate genes, LPPand BCL6 genes provide a possible range for the study of the genetic mechanism of flavor.

Keywords: Chicken; Volatile flavor compounds; GC-MS; Metabolomics

<footnote>* Corresponding author: zhaoguiping@caas.cn</footnote>
IDENTIFICATION OF MAJOR GENES FOR INTRAMUSCULAR FAT BY A GENOME-WIDE ASSOCIATION STUDY IN CHICKEN

J. Wen*(1), H.Cui(1), L.Liu(1), X.Liu(1), Y.Wang(1), S.Xing(1), R.Liu(1), Q.Li(1), M. Zheng(2), G.Zhao(1)

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

* Corresponding author: wenjie@caas.cn

Introduction: Chicken, known for the exquisite and delicious meat quality, is the important animal protein to the human as the second largest meat product after pork. Intramuscular fat is a quantitative trait collectively controlled by minor-major genes and has a great contribution to meat color, tenderness and flavor meat quality. To date, the genetic mechanism of IMF metabolism and many functional genes had been reported. However, the systematic screening of major gene on IMF deposition of chicken is yet not clear.

Experimental design and methods: In this study, the excellent Jingxing-yellow (JX-Y) chicken population with 16 generations selection of IMF content, and the unselected natural population as the control, were used to systematically elucidate the genetic basis of IMF metabolism by a genome-wide association study (n = 516).

Results: IMF was a complex of lipids, we found that triglycerides (TG) was the main component of IMF with the high positive genetic and phenotypic correlations, so TG could replace IMF for follow-up analysis. As expected, we excavated 131 SNPs associated with TG, and these 131 SNPs were located in the inside of LIN52 (Chr 5) and in the upstream of SLC16A7 (Chr 1). Excitingly, the results by XP-EHH analysis revealed that LIN52 was selected in used two population. And then, a big data of functional validation and the clarification of regulatory mechanism of SNP had supported that SLC16A7 was the major gene on TG biosynthesis in pectoralis of chicken. Meanwhile, our results by XP-EHH analysis revealed that the SNPs in the upstream of SLC16A7 (Chr 1) also were selected in used two population. The further structural analysis of variable region and functional verification of SLC16A7 confirmed that SLC16A7 was another major gene on TG biosynthesis in pectoralis of chicken.

Conclusions: In summary, we identify two major genes (LIN52 and SLC16A7) on TG biosynthesis in pectoralis of chicken, and our findings will help to understanding and perfect the underlying molecular regulatory mechanisms of IMF deposition in chickens.
EFFECT OF DIFFERENT SOURCES AND LEVELS OF SELENIUM ON SLAUGHTER ANALYSIS AND MEAT QUALITY OF BROILER CHICKENS

M. Michalczuk\*(1), P. Konieczka(2), A. Gajewska(3)

(1) University of Life Sciences WULS-SGGW, Warsaw, Poland

(1) The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jabłonna, Poland

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

* Corresponding author: monika_michalczuk@sggw.edu.pl

Selenium (Se) is an essential mineral that has antioxidant properties in poultry. The supplementation diets with Se is required because of the wide variation of Se concentration in feedstuff. The objective of this study was to evaluate the effects of dietary levels of inorganic and organic Se, salinized yeast and nano form of selenium (nano-Se) on slaughter analysis and meat physicochemical characteristics of broilers chickens.

A total of 300 d-old male broilers Ross 308 were divided into 4 experimental groups in 5 replication of 75 birds each. Birds were fed following treatments: (i) standard commercial diet containing inorganic Se at the level of 0.3 mg/kg diet (C), (ii) diet with the increased level of inorganic Se (0.5 mg/kg diet), (iii) as in treatment (ii) but with salinized yeast as Se source, and (iv) as (ii) but nano-Se was used as a Se source. At 42 day of life, 10 birds were selected from each group, and dissected to achieve breast muscle samples for analyses. The pH24 value and water holding capacity (WHC) where replicated three times, and the mean value was reported. Color parameters (a *, b *, L *) were analyzed in minced meat while, the drip loss was computed as the percentage ratio of meat weight after and before storage. The analysis of variance (ANOVA) was applied to determine the effect of diet supplementation with the selenium on slaughter analysis and broiler meat quality.

The dietary treatments significantly affected slaughter yield of birds. Both, the highest slaughter yield and the highest yield of breast muscle was found in birds fed diet with nano-Se while, the lower yields of these cuts was in birds fed diet with increased level of inorganic Se (P<0.01). Legs meat share was the highest in groups fed diet with salinized yeast whereas, the lower one was found in treatment with increased level of inorganic Se (P<0.01). This group was also characterized by the highest relative weight of heart, liver and gizzard but the lowest weight of adipose fat comparing to other groups (P<0.05). Feeding diets of increased level of inorganic selenium affected color of the breast meat (the highest L* value) and WHC of the breast meat (the lowest level) comparing to remaining groups (P<0.05).

Our data indicated that feeding birds diet containing nano-Se at the level of 0.5 mg/diet throughout the 42-days feeding period did not compromised quality indices of the meat.

This work was supported by the National Science Centre, Grant No. 2018/29/B/NZ9/01351
ID: 1280

EFFECT OF AFLATOXIN ON PERFORMANCE AND CERTAIN MEAT QUALITY PARAMETERS IN BROILER
CHICKEN

M. Erdélyi*(1), Z.Ancsin(1), M.Kovács-wéber(2), K.Balogh(1), E.Balogh-zándoki(1), M.Cserháti(3), V.Zollai(4), M.Mézes(1)

(1) Szent István University, Faculty of Agricultural and Environmental Sciences, Department of Animal Nutrition, Gödöllő, Hungary

(2) Szent István University, Faculty of Agricultural and Environmental Sciences, Institute of Animal Husbandry, Gödöllő, Hungary

(3) Szent István University, Faculty of Agricultural and Environmental Sciences, Department of Environmental Safety and Ecotoxicology, Gödöllő, Hungary

(4) Zollai Meat and Poultry Processing Ltd., Kóka, Hungary

* Corresponding author: erdelyi.marta@mkk.szie.hu

Mycotoxins can cause production losses and health risks both for livestock and human. Aflatoxin is considered to be the most hazardous one as it has carcinogenic property and it might be excreted in animal products (i.e. milk and egg). Due to climate change, nowadays, aflatoxin producing Aspergilli intriguing troubles for poultry farmers even in the temperate regions.

In our study effects of aflatoxin contaminated diet on performance and certain physico-chemical quality parameters of meat were investigated.

Three groups of Cobb 500 cockerels (n=600) were created (5 replicates each). Control birds (T1) were fed with three-phase commercial broiler diets. Birds in the treatment groups were fed with control diets contaminated with 0,1 mg AFB1/kg (quintuple of limit value 0.02 mg/kg (574/2011 EC)) between 1-21 or 21-42 days of age in Group T2 and T3, respectively. Live weight, feed consumption and feed efficiency were analysed in each group. At Day 42 birds were culled and meat productivity was evaluated and breast fillets were taken from 30 birds/group for further analysis. Colour, drip loss, texture, kitchen loss were analysed in breast samples.

Feed intake in Group T2 was significantly reduced on the first week of aflatoxin load, but from Day 21 birds strated to compensate the initial low consumption. As a result, live weight in Group T2 was significantly lower than in the control, but became similar by culling. Feed refusal caused by aflatoxin was confirmed also in Group T3 as from the start of aflatoxin load feed intake dopped markedly. However, weight of birds in Group 3 was notably different from the control only on Day 35. Altogether, feed efficiency was debased in both treatment groups.

Proportion of primary cuts was reduced mainly in Group T2. Considering meat quality the most remarkable changes were found in the colour as the breast samples in Group T3 were lighter and yellower, which resulted in visible difference, as well. However, statistically significant difference was revealed also in water holding capacity, as drip loss was notably higher of the breast samples in Group T2 than in the control (T1).

Altogether, aflatoxin has harmful effect both on performance and on certain quality traits of meat (water holding capacity), and these are more prevalent, when contamination occurs in the initial period of fattening.

The project was co-financed by the EU and the European Social Fund EFOP-3.6.3-VEKOP-16-2017-00008 and NVKP-16-1-2016-0009.
PHYSICOCHEMICAL TRAITS AND FATTY ACID PROFILE IN BREAST MUSCLES OF CHICKENS KEPT IN A SMALL-SCALE FARM, FED WITH FABA BEAN AND RAPESEED MEAL

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

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

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

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

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

The possibility of using other protein feeds than soybean meal raises the importance of conducting research and seeking solutions to produce good quality broiler meat in small farms in EU based on own feed. The aim of the study was to compare the fatty acid profile in the breast muscles of chickens fed with feed with faba bean and rapeseed meal as a high-protein replacement for soybean meal. ROSS 308 chickens were divided into 2 groups (170 birds/group). In the control group (I), nutrition was based on soybean meal. Experimental group (II) was fed with faba bean and rapeseed meal. After 6 weeks of rearing, 10 randomly selected chickens from each group were slaughtered. The breast muscles (BM) were collected, weighed, and the color of the meat was assessed with a colorimeter (Konica Minolta) in the CIE system (1986) (L*,a*,b*), water-holding capacity (Grau and Hamm, 1952) and the fatty acid profile was determined. Lipid extraction from BM was performed (Folch et al., 1957). Fatty acids (FA) were quantified as methyl esters (FAME) using a GC Trace 2000 gas chromatograph (ThermoQuest EC). Results are expressed as a percentage of total FAs identified. To assess the nutritional implications, the ratio of n-6 to n-3 FA (n-6 / n-3) and the ratio PUFA to SFA (P/S) were calculated. Atherosclerotic index (AI) and thrombogenic index (TI) were calculated (Ulbricht and Southgate, 1991). Numerical data were analyzed in the Statistica 10.0 (2011). Body weight before slaughter and carcass weight were significantly higher in group I (p0.05). A significantly higher proportion of C16:0 (palmitic), C22:4 n-6 (docosatetraenoic) acids was found in group II (P <0.05). In group II, a significant increase in the ratio of n-6 to n-3 acids (p<0.05) was found. The results indicate no negative impact of the use of feed with faba bean and rapeseed meal in chicken feed, except for an increase in C16:0 content and a positive increase in C22:4n6, which belongs to EFA, which allows to state that the use of replacement soybean meal may be recommended for rearing broiler chickens.

*The study was carried out under measure of the Multiannual Programme "Increased use of domestic feed protein to produce high-quality animal products under the conditions of sustainable development". This work has been supported by the Polish National Agency for Academic Exchange under Grant No. PPI/APM/2019/1/00003"
MEAT PROCESSING
SENSORY SCORES AND MICROBIAL COUNTS OF KILISHI-LIKE CHICKEN JERKY MADE WITH THREE SLURRY TYPES

N. Aladi\*(1), I.Okoli(2), N.Okeudo(2)

(1)Federal University of Technology. Owerri, Owerri, Nigeria

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

* Corresponding author: nnanyerealadi@gmail.com

Introduction

Production of kilishi-like product from chicken in this region increase demand for chicken and help to stabilize income for poultry farmers who are often compelled to sell their produce at competitively low prices during periods of glut. The quality acceptability of Kilishi is affected by the nature of slurry used (Ogbonna and Linus, 2009; Mgbemere et al., 2011). This study assessed the effect three slurry types; groundnut paste, melon paste and soya bean residue (Okara) on sensory qualities and microbial status of kilishi-like jerk from chicken after 12 weeks of storage.

Materials and Methods

Thinly sliced chicken meat from breast and thigh muscles were broiled for 2 hours at 60 °C, and then infused for 15 min in a slurry mixture containing either groundnut cake, melon cake or okara (280g) blended with water (1 L), onion (100g), garlic (25g), ginger (50g), ground pepper (20g), 2 Maggi® cubes, granulated sugar (20g), Yaji (kilishi pepper). The samples were then oven dried. The products were cooled to room temperature and hygienically packed in airtight polyethylene bags and stored on the shelf. After 12 weeks, they were assessed by a trained sensory panel using a 9 point category hedonic scale. 1 g of jerky from slurry type was dropped in a test tube containing 9 ml of distilled water, shaken vigorously and then allowed to settle for 5 min. The resulting solution was used to inoculate six media namely; Mackonkey, CLED, SSA, Sabourand desxtrose, chocolate blood and blood agar and the set up were incubated for 24 hours. The data were subjected to analysis of variance in a completely randomized design. Significantly different means were separated using LSD.

Results and Conclusion

No significant differences (p<0.05) were found for all the sensory parameters evaluated. The overall hedonic rating for the product types were very high (7.44 to 8.44) and no significant microbial growth was observed in all the cultures. It is therefore concluded it is feasible to produce Kilishi-like jerky from chicken using defatted groundnut paste, melon or okara as slurry materials and the product is safe to consume and will be stable after 12 weeks of storage under ambient conditions.

References

Mgbemere, V.N., Akpapunam, M.A. and Igene, J.O. (2011). Afr. J. of Food, Agric, Nutr and Dev, 11(2): 4718-4738.

Ogbonnaya, C. and Linus, I. I. (2009). World J. of Agric. Sci., 5 (1): 34-39
COMPARISON OF THREE DIFFERENT METHODS USED FOR ESTIMATING COOK LOSS IN BROILER BREAST
MEAT

J. Zhang(1), B.Pang(2), B.Bowker(3), Y.Yang(4), H.Zhuang*(3)

(1) Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forest Science, Beijing, China

(2) College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, China

(3)USDA, Agricultural Research Service, Russell Research Center, Athens, United States

(4)College of Engineering, China Agricultural University, Beijing, China

* Corresponding author: zjcau@126.com

The objective of this study was to evaluate the different methods used for estimating moisture loss in cooked broiler breast meat (pectoralis major). Two experiments were conducted. In the first experiment, both intact breast meat and cored meat samples were used to compare three different methods (5 min in room temperature, 5-min; cooling down to ambient temperature RM; or chilled down to room temperature in ice water, IW) for estimation of cook loss in broiler breast meat. In the second experiment, cut meat samples were used to compare the effect of the endpoint temperature (23, 40, 53, 57, 68, 75, or 90 °C) on the cool loss estimation using the 5-min and RT methods. Chicken breast meat were collected from local chicken processing plants and trimmed, cored, or cut into similar size/weight prior to cooking. Moisture loss after cooling, moisture retention, and total moisture loss after 24 h in cooked broiler breast meat were measured for comparison. Data showed that there were no significant differences (P > 0.05) between 5-min and RT methods in the cook loss (19-21%) and total loss (21-25%) regardless of coring. However, the cook loss (< 17%) and total loss (19.3%) of the IW method was significantly lower (P < 0.05) than those of the other two methods in whole breast meat samples but there were no significant difference in the cored samples (23.2%). When the endpoint temperature was ≤ 40°C or ≥ 75°C, no moisture accumulation was observed in packages between 5-min and RT method after 24 h. However, when the temperatures were ≥ 53 and < 75°C, cook loss estimations were significantly different (P < 0.05) between these two methods (more than 4%), with reducing the endpoint temperature resulting in increased differences (from less than 5% to more than 9%). These results demonstrate that the cook-loss estimating methods may affect moisture loss results in cooked broiler breast meat and the differences in cook loss results between estimating methods could be affected by the final endpoint temperature.
SENSORY CHARACTERISTICS, PROXIMATE AND MICROBIAL COUNTS OF KILISHI LIKE JERKY FROM CHICKEN

N. Aladi\*(1), V.Iyiola(2), E.Onwudufor(3), N.Okeudo(3)

(1)Federal University of Technology. Owerri, Owerri, Nigeria

(2)Federal University, Wukari, Wukari, Nigeria

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

* Corresponding author: nnanyerealadi@gmail.com

Introduction and Objective

In many sub-Saharan African countries, the demand for Kilishi – a shelf stable, intermediate moisture product that is prepared from thin flat beef slices infused in slurry made from a mixture of defatted groundnut paste and spices, is on the increase (Mgbemere et al., 2011). Successful production of kilishi-like product from chicken will help to stabilize farm income for poultry farmers who are often forced to sell at abysmally low prices due to poor marketing arrangements and in seasons of glut. This study evaluated the feasibility of producing a kilishi-like jerky (meat crackers) from chicken using ovendrying method.

Material and Methods

Meat crackers were produced from chicken and beef following the usual procedures for Kilishi production from beef (Ogbonna and Linus, 2009; Igene et al., 1990) except that charcoal oven was used in place of sun drying. The samples were subjected to sensory evaluation on day of production and after 7 days of storage. Proximate analysis and total plate count were also conducted on 7 day. The data generated was subjected to t-test at 0.05 and 0.01 significance levels.

Results

The panellists preferred meat jerky from chicken to beef on the day of production, but no significant differences (p<0.05) were found in the degree of likeness, flavour, texture and off-flavour intensity after 7 days of storage. Meat jerky from chicken had significantly higher moisture (34.21%) and Nitrogen Free Extract content (17.73%) but lower protein (42.65%) and fat (2.30) contents when compared to those from beef (21.05, 13.03, 51.02 and 10.19 respectively). The mean bacterial load of the chicken jerky was 3.501 x 10⁵ (E. coli/ml) was higher (p<0.05) than those jerky from for the beef jerky samples (1.159 x 10⁵ E. CFU/ml).

Conclusion

It is feasible to produce a kilishi-like product from broiler chicken meat using the oven drying method. The product was well accepted to consumers, had sensory scores similar to traditional kilishi from beef and the microbial load are within the recommended safety levels for human food.

References

Igene, J.O., Farouk, M.M. and Akanbi, C.T. (1990). J. Sci. Food Agric; 50: 89-98

Ogbonnaya, C. and Linus, I. I. (2009). World J. of Agric. Sci., 5 (1): 34-39.

Mgbemere, V.N., Akpapunam, M.A. and Igene, J.O. (2011). Afr. J. of Food, Agric, Nutr and Dev, 11(2): 4718-4738.
ASSESSMENT OF MICROBIOLOGICAL QUALITY OF GRILLED CHICKEN MEAT AND SPICES ACCORDING TO PROCESSING METHOD IN SOUTH OF BENIN REPUBLIC

K. Edikou*(1), A.Diantom(2), A.Akakpo(3), K.Ekpo(4), E.Osseyi(2), D.Karou(2), J.Dossou(5)

(1)Regional Centre of Excellence on Poultry Sciences (CERSA)/University of Lomé, Lomé, Benin

(2)College of Biological and Food Techniques (ESTBA), University of Lome, Lome, Togo

(3)Regional Centre of Excellence on Poultry Sciences (CERSA)/ University of Lome (Togo), Lome, Togo

(4)Regional Center of Excellence on Poultry Sciences (CERSA), University of Lome, Lome, Togo

(5)Faculty of Agronomic Sciences (FSA), University of Abomey-Calavi, Abomey-calavi, Benin

* Corresponding author: speral@yahoo.fr

Grilled chicken meat are well appreciated by consumers. However, grilling and sale conditions may affect quality grilled chicken meat (Djoulde et al., 2015; Alonge et al., 2017; Somda et al., 2018). This study aimed to assess microbiological quality of grilled chicken meat sold in Benin. A total of 65 samples of grilled chicken meat from different grilling processes at Cotonou and Abomey-Calavi (the two biggest cities) in Benin were collected respectively after grilling and 2h after exposure for selling. A total of 35 spices used for grilled chicken meat consumption were also collected. They were analyzed for microbiological quality according to ISO standards methods. Data were analyzed by student T test and ANOVA one-way followed by tukey post test using GraphPad 5. Results showed that total plate count of all chicken grilled kinds exceeded maximum limits of 3×10⁶ cfu/g fixed by EU regulation (n°2073/2005) except gaz-grilled chicken meat (1.47×10⁶ ± 6.26×10⁵ cfu/g). Total coliforms, E. coli, ASR, yeast and mold respected regulation's limits. As well, fecal coliforms exceeded limits in some grilled chicken meat. Microbial loads did not change significantly within 2 hours of exposure during selling. Spices collected from barrel and charcoal cabinet kiln grilling processors were higher in total plate count exceeding limits, contrary to gaz-cabinet and local chicken grilling processors. Spices collected for gaz-grilled chicken meat consumption contained lowest load of total (7.7× 10¹± 3× 10¹ cfu/g) and fecal (7.1×10¹ ±6.8×10¹ cfu/g) coliforms, and spices used for local chicken grilling recorded highest loads. E.coli, ASR, mold and yeast loads were under maximum limit. Staphylococcus aureus and salmonella were not found in all samples of grilled chicken meat and spices analyzed. In conclusion, this study suggested that efforts should be made by processors for grilling and selling chicken meat in hygienic conditions.
DIFFERENCES IN TEXTURE ANALYSES OF CHICKEN BREAST FILLETS AFFECTED BY SEVERE WOODEN BREAST AND SPAGHETTI MEAT MYOPATHIES

A. Pascual\*(1), A. Trocino(1), L. Susta(2), S. Barbut(2)

(1) University of Padova, Padova, Italy

(2) University of Guelph, Guelph, Canada

\* Corresponding author: anton.pascualguzman@phd.unipd.it

Texture of broiler meat is one of the most important attributes for consumers, and it can be affected by breast meat myopathies, such as wooden breast (WB) and spaghetti meat (SM). However, differences in textural measurements between samples can vary by the test applied, meat preparation (raw vs. cooked), and severity of myopathy. Thus, this study employed three tests: compression, Meullenet-Owens razor (MORS), and Allo-Kramer (AK) - to compare normal, WB, and SM fillets. A total of 209 fillets (68 normal, 71 WB, 70 SM) were selected from 3 flocks, at a large commercial processing plant, 3 h after slaughter. The fillets were analyzed for dry matter, crude protein, and fat contents (9 per meat type) as well as pH plus color, and then frozen for later texture analyses (181 fillets; 59 normal, 61 WB, 61 SM). Thawed fillets were submitted to: compression test (raw and cooked), and later cooked samples to the MORS test (blade: 0.5 mm thick, 8.9 mm wide, and the AK test (5 blades, 2 mm blade thickness). Data were analyzed by a mixed model with myopathy and flock as the main effects (SAS, 2013). Normal meat had lower fat content than WB meat (0.91% vs. 1.54%; P<0.05) with intermediate values for SM (1.25%), whereas protein content was higher in normal meat (23.9% vs. 22.3% and 22.7% in WB and SM; P<0.01); similar to previous results. Normal breasts were lighter (188 vs. 230 and 262 g in WB and SM; P<0.01), had lower L* and a* values than abnormal meat, and lower cooking losses (22.4% vs. 27.8% and 26.9% in WB and SM; P<0.001). Raw, normal and SM meat showed lower compression force (5.61 and 4.69 vs. 9.52 N), work (25 and 22 vs. 45 N x mm) and Young's modulus (2.71 and 2.11 vs. 4.29 N/sec) than WB (P<0.001); the same pattern was observed in cooked meat. For the MORS test, SM showed lower shear force (12.8 vs. 14.7 N), work (249 vs. 288 N x mm) and fewer peaks (5.39 vs. 7.57) than normal meat (P<0.01), whereas WB had intermediate values. For the AK test, SM showed lower force (10.5 vs. 14.5 N) and Young's modulus (31.0 vs. 46.0 N/sec) than WB (P<0.01), whereas normal meat had intermediate values. Overall, results revealed that texture tests show different sensitivity with respect to meat preparation and type. The compression test was useful to identify WB even in raw meat as well as in cooked meat. In cooked samples, MORS distinguished SM from normal fillets, whereas the AK test identified SM as different from WB.
BACTERIAL MICROFLORA IN POULTRY GROUND MEAT TREATED WITH CARNOSINE AND COLD PLASMA WERE ASSESSED BY SINGLE CARBON UTILIZATION

H. Yeh*(1), J-Line(1), A.Hinton(1), Y.Gao(3), H.Zhuang(1)

(1)USDA ARS USNPRC, Athens, United States

(3) National Center of Meat Quality and Safety Control, Nanjing Agricultural University, Nanjing, China

* Corresponding author: hungyueh.yeh@usda.gov

Introduction: Poultry meat contains abundant nutrients for human health and also harbors many potential spoilage and foodborne microorganisms. To provide safer food for human consumption, many reagents and technologies have been applied to reduce and/or eliminate these microorganisms in food matrices. However, bacterial communities in food before and after treatments with cold plasma, carnosine or both have not been extensive investigated. In this communication, the bacterial communities in poultry meat samples after various treatments were assessed with EcoPlates™.

Experiment Design: After treatment with cold plasma and carnosine, the ground meats were diluted in PBS, and the supernatants were dispensed into 96-well EcoPlates™. The plates were incubated at 25°C for seven days in an OmniLog® chamber. Bacterial growth in the plates was monitored on formazan production every 30 minutes at OD590 nm. The OD readings of the chemicals were adjusted by the subtraction from the average of the OD values of the water blank in the plates. Means of the resulting OD values for each chemical compound were calculated. The SAS software package (version9.4) was used to determine the bacterial growth curves based on three-parameter Gompertz growth algorithm. The PAST software package (version 3.22) was used to calculate principal coordinates analysis based on the variance-covariance matrix. GraphPad Prism® 8 (GraphPad Software) was used for plotting the bacterial growth curves.

Results: The kinetics of bacterial growth patterns were well fitted a sigmoidal Gompertz curve with the lag, log and stationary phases, but with different inflection times and asymptotes at the log phase and the stationary phase, respectively. The cold plasma technology and carnosine could inactivate bacterial growth in treated meat samples. Among 31 chemicals tested, seven chemicals could not be used by bacteria in meat samples as the carbon source: phenylethylamine, alpha-D-lactose, D,L-alpha-glycerol phosphate, 2-hydroxybenzoic acid, gamma-hydroxybutyric acid, alpha-ketobutyric acid and D-malic acid.

Conclusions: Whether the chemicals that inhibit growth of bacteria from the samples can be used as food preservatives is required for further investigations. Further, whether the profiling on bacterial communities be as an indicator for effectiveness of meat sample treatments is needed. To address what the bacterial compositions are in the communities of the meat samples is also needed.
IMPACT OF NATURAL PRESERVATIVE FORTIFICATION ON OXIDATIVE STABILITY OF CHICKEN SEEKH KEBAB PREPARED BY USING SPENT HEN MEAT

M. Nampalle*(1), A.Biswas(2), A.Biswas(2), J.Rokade(3), G.Marappan(4), M.Vispute(5), J.Saharia(4), M.Madeswaran(4)

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

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

(3) ICAR- Central Avian research Institute, Izatnagar, Bareilly, India

(4) ICAR-Central Avian Research Institute, Izatnagar, Bareilly, India

(5) ICAR-Central Avian Research Institute, Izatnagar, Bareilly, India

* Corresponding author: mk.nampalle333@gmail.com

Present study was carried out with in vitro inclusion of natural preservatives viz., curcumin, α-tocopherol acetate and eugenol @ 0.025, 0.04, and 0.02% respectively to observe their effect on oxidative stability of chicken seekh kebab (CSK). Spent hens were taken as meat source following standard slaughter technique at division of Post-Harvest Technology ICAR-CARI, lizatnagar. Formulation was selected on the basis of earlier studies carried out in the Post-Harvest Technology division, CARI, Izatnagar. Pre-standardized processing and cooking techniques were followed for CSK preparation. For comparison a control devoid of natural preservatives and treatment sample containing a mixture of curcumin 0.025%+α-tocopherol acetate 0.04%+eugenol 0.02% were prepared. These samples were then packed in self-sealing low density polyethylene bags aerobically and stored at 4±1 °C till insipient spoilage occurs. At an interval of 5 days different physicochemical and sensory quality parameters viz., TBARS value (Witte et al., 1970), free fatty acid, peroxide value (Koniecko, 1979), protein oxidation (Metmyoglobin formation %) (Krzywicki, 1979) and antioxidant activity (ABTS+ and DPPH activity) (Biswas et al., 2015) were evaluated. Duncan's multiple range test was used to test the statistical significance. During early storage period, differences amongst various physicochemical and sensory parameters varied numerically but as storage time progressed it varied significantly. TBARS value FFA, and PV lowered significantly (P

Keywords: Natural preservatives, oxidative stability, curcumin, eugenol.

Krzywicki, K. 1979. Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef. Meat Sci. 3(1): 1-10.

Biswas, A. K., Beura, C. K., Yadav, A. S., Pandey, N. K., Mendiratta, S. K. and Kataria, J. M. 2015. Influence of novel bioactive compounds from selected fruit by-products and plant materials on the quality and storability of microwave-assisted cooked poultry meat wafer during ambient temperature storage. LWT - Food Sci. Techn. 62: 727-733.
EF6

REPRODUCTION

REPRODUCTIVE PHYSIOLOGY

REPRODUCTIVE BIOTECHNOLOGIES
REPRODUCTIVE PHYSIOLOGY
ID: 62

# GENE EXPRESSION OF GONADAL GERM CELLS (GGCS) IN DOMESTIC CHICKEN

M. Kishi(1), M. Takehara(1), M. Onuma(2), N. Nakajima(2), A. Asano(3), A. Tajima*(4)

(1)Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan

(2)National Institute for Environmental Studies, Tsukuba, Japan

(3)Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan

(4)University of Tsukuba, Tsukuba, Japan

Study on the understanding of mechanism of sexual differentiation is crucial for the poultry industry, since the commercial use of poultry is heavily skewed in favor of either one of the sex. Accordingly, it is evident that the development of the technology to produce newly hatched chick of desired phenotypical sex regardless of the genetic sex benefit the poultry industry. At present, however, studies on the sexual dimorphism of chick embryos has been primarily focused on the gonadal sex differentiation, whereas limited information is available on the sexual differentiation of gonadal germ cells(GGCs). The present study was conducted, therefore, to examine the genetic factors that regulate the sexual differentiation of GGCS in chick embryonic gonads.

Fertilized eggs were incubated in an air forced incubator for 5 to 9 days and developing gonads were dissected carefully using a sharp edge of 30 gauge needle under a dissection microscope. GGCs were isolated from isolated gonads by using PBS(-) method as described by Nakajima et al. (2011). Briefly, embryonic gonads were incubated for 1 hour in the phosphate buffered saline without containing Ca and Mg ions [PBS(-)]. After incubation, GGCs dispersed in the PBS(-) from embryonic gonads were manually recovered using a fine glass pipette under dissection microscope. RNA were recovered from GGCs and subjected to gene expression analysis by RNAseq. Quantitative gene expression data were obtained by annotating the outcome of RNAseq data using chicken genome database.

Results showed that by 9th day of incubation, down regulation of genes characteristics to undifferentiated germ cells such Nanog and Prdm14 were observed, whereas upregulation of genes characteristics for sexual differentiated cells such as Dnd1 in male and Stra8 in female were observed. The results from present study strongly indicate that sexual differentiation of GGCs takes places in concert with gonadal sex differentiation. Future study should be directed toward identifying the initial factor that regulate the cascade of germ cell sexual differentiation in the poultry.

<footnote>* Corresponding author: tajima.atsushi.gb@u.tsukuba.ac.jp</footnote>
SPERM WITH A LONGER FLAGELLUM EXHIBIT BETTER FERTILIZATION SUCCESS BY FACILITATING SPERM ENTRANCE INTO THE SPERM STORAGE TUBULES IN JAPANESE QUAIL (COTURNIX JAPONICA)

T. Sasanami*(1), M. Matsuzaki(2), N. Hirohashi(3), S. Mizushima(4)

(1)Shizuoka University, Shizuoka, Japan

(2)Hiroshima University, Higashi-hiroshima, Japan

(3)Shimane University, Matsue, Japan

(4)Hokkaido University, Sapporo, Japan

During passage through the female reproductive tract, the ejaculated spermatozoa massively decrease in number, and a small number of sperm are able to reach the site of fertilization in animal reproduction to fuse with ova. In birds, vagina and sperm storage tubules (SSTs) located in the utero-vaginal junction are thought to be the site of sperm selection because less than 1% of sperm inseminated are successfully stored in the SSTs. However, the exact mechanism of sperm selection by which sperm enter the SSTs is poorly understood. Here, we investigated sperm entry into the SSTs and subsequent fertilization success under a competitive situation created by artificial insemination of a sperm mixture obtained from two males. We employed two quail strains, a wild type and a dominant black (DB) type, that allow easy assessment of any offspring's paternity by feather coloration. We found paternity of embryos was significantly biased towards DB males when mixed sperm were artificially inseminated into females, despite the two strains having similar sperm motility in normal medium assessed by computer-assisted sperm motility analysis. Our novel sperm staining method with two different fluorescent dyes, Hoechst33342 and pHrodo-red-AM clearly demonstrated that the DB-biased fertilization is due to the better ability of DB sperm to enter the SSTs. Moreover, we found that DB sperm had longer flagella solely coupled with a longer midpiece and they swam fast in with high viscosity medium created by the addition of methyl cellulose that may be a similar environment to the lumen of the female reproductive tract. Our results indicated that sperm competition occurs to win a place in the SSTs, and that filling the SSTs with their own spermatozoa is a critical step to achieve better fertilization success for the male Japanese quail. Thus, males produce the sperm with longer flagellum may have advantage for fertilization in birds.

<footnote>* Corresponding author: atsasan@shizuoka.ac.jp</footnote>