Shouguang Chicken Research Articles

PGC-mediated conservation strategies for germplasm resources of Rugao Yellow Chicken and Shouguang Chicken in China1

PGC-mediated conservation strategies for germplasm resources of Rugao Yellow Chicken and Shouguang Chicken in China1

Effects of Breeds on the Content of Functional Nutrition in Eggs.

The purpose of this study was to compare the differences in the content of functional nutrients in eggs, performance parameters, and egg quality parameters of different chicken breeds. In Trial 1, 150 41-week-old hens of each breed, including the Dwarf Layer, White Leghorn, Silky fowl, Beijing-you chicken, and Shouguang chicken, were randomly assigned to the control (CON) and 2.5% flaxseed oil (FSO) groups to compare the difference in n-3 polyunsaturated fatty acid (PUFA) content in eggs. The contents of α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and total n-3 PUFA in eggs were increased (p < 0.05) in the FSO groups. The ALA (4.28%), DHA (2.03%), and total n-3 PUFA (6.46%) contents in eggs of Dwarf Layer were the highest among the five breeds (p < 0.05). To further verify if other functional nutrients also have such differences, 600 24-week-old White Leghorn and Dwarf Layer were allocated to four groups: CON, FSO, 0.02% selenium-enriched yeast (SEY), and 0.20% marigold flower extract (MFE), in Trial 2. The content of functional nutrients in eggs was significantly increased (p < 0.05) after feeding these additions. After feeding FSO, the eggs of the Dwarf Layer had a higher n-3 PUFA content than the White Leghorn (p < 0.05). However, no significant differences were found in selenium and lutein content in different breeds. Performance and egg quality were not negatively impacted by FSO, SEY, or MFE.

Regulation of mRNA and miRNA in the response to Salmonella enterica serovar Enteritidis infection in chicken cecum

BackgroundSalmonella enterica, serovar Enteritidis (SE) is a food-borne pathogen, which can cause great threat to human health through consumption of the contaminated poultry products. Chicken is the main host of SE. The mRNA and microRNA (miRNA) expression profiles were analyzed on cecum of Shouguang chicken via next-generation sequencing and bioinformatics approaches. The treated group was inoculated SE, and the control group was inoculated with phosphate buffer saline (PBS).ResultsThere were 1760 differentially expressed mRNAs in the SE-infected group, of which 1046 were up-regulated mRNA, and 714 were down-regulated mRNA. In addition, a total of 821 miRNAs were identified, and 174 miRNAs were differentially expressed, of which 100 were up-regulated and 74 were down-regulated. Functional enrichment of differentially expressed mRNAs was similar to miRNA target genes. The functional analysis results of differentially expressed mRNAs and miRNAs were performed. Immune-related processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were enriched by up-regulated mRNA. The down-regulated mRNAs were enriched in tissue development and metabolic-related KEGG pathways. The functional analysis of up-regulated miRNA target genes was similar to the down-regulated mRNAs. The down-regulated miRNA target genes were enriched in metabolic-related GO (Gene Ontology) -BP (Biological process) terms and KEGG pathways. The overlap of the up-regulated mRNA and the up-regulated miRNA target genes (class I) was 325, and the overlap of the down-regulated miRNA target genes (class II) was 169. The class I enriched in the immune-related GO-BP terms and KEGG pathways. The class II mainly enriched in metabolic-related GO-BP terms and KEGG pathways. Then we detected the expression of mRNA and miRNA through qRT-PCR. The results shown that the expression of HHIP, PGM1, HTR2B, ITGB5, RELN, SFRP1, TCF7L2, SCNN1A, NEK7, miR-20b-5p, miR-1662, miR-15a, miR-16-1-3p was significantly different between two groups. Dual-luciferase reporter assay was used to detect the relationship between miR-20b-5p and SCNN1A. The result indicated that miR-20b-5p regulate immune or metabolic responses after SE infection in Shouguang chickens by directly targeting SCNN1A.ConclusionsThe findings here contribute to the further analysis of the mechanism of mRNA and miRNA defense against SE infection, and provide a theoretical foundation for the molecular disease-resistant breeding of chickens.

Genome-Wide Association Study and Transcriptome Differential Expression Analysis of the Feather Rate in Shouguang Chickens.

The feather rate phenotype in chicks, including early-feathering and late-feathering phenotypes, are widely used as a sexing system in the poultry industry. The objective of this study was to obtain candidate genes associated with the feather rate in Shouguang chickens. In the present study, we collected 56 blood samples and 12 hair follicle samples of flight feathers from female Shouguang chickens. Then we identified the chromosome region associated with the feather rate by genome-wide association analysis (GWAS). We also performed RNA sequencing and analyzed differentially expressed genes between the early-feathering and late-feathering phenotypes using HISAT2, StringTie, and DESeq2. We identified a genomic region of 10.0–13.0 Mb of chromosome Z, which is statistically associated with the feather rate of Shouguang chickens at one-day old. After RNA sequencing analysis, 342 differentially expressed known genes between the early-feathering (EF) and late-feathering (LF) phenotypes were screened out, which were involved in epithelial cell differentiation, intermediate filament organization, protein serine kinase activity, peptidyl-serine phosphorylation, retinoic acid binding, and so on. The sperm flagellar 2 gene (SPEF2) and prolactin receptor (PRLR) gene were the only two overlapping genes between the results of GWAS and differential expression analysis, which implies that SPEF2 and PRLR are possible candidate genes for the formation of the chicken feathering phenotype in the present study. Our findings help to elucidate the molecular mechanism of the feather rate in chicks.

Mitochondrial DNA Haplotypes Influence Energy Metabolism across Chicken Transmitochondrial Cybrids.

The association between mitochondrial DNA haplotype and productive performances has been widely reported in chicken breeds. However, there has not been physiological evidence of this seen previously. In this study, chicken transmitochondrial cells were generated using the nucleus of the DF-1 cell line and mitochondria of primary cell lines derived from two native chicken breeds, Tibetan chicken and Shouguang chicken. Generally, Tibetan chicken primary cells showed a stronger metabolic capacity than Shouguang chicken primary cells. However, the Tibetan chicken cybrids had a dramatic drop in relative mtDNA copies and oxygen consumption. Higher rates of oxygen consumption (OCR) and expression levels of mitochondrial biogenesis and fusion genes were observed in Shouguang chicken cybrids, potentially reflecting that the mitochondrial DNA haplotype of Shouguang chicken had better coordination with the DF-1 nucleus than others. Meanwhile, mitonuclear incompatibility occurred in Tibetan chicken cybrids. The results demonstrate functional differences among mitochondrial DNA haplotypes and may shed light on the interaction between the mitochondria and nucleus in Gallus gallus domesticus.

Dynamic Transcriptomic Analysis of Breast Muscle Development From the Embryonic to Post-hatching Periods in Chickens.

Skeletal muscle development and growth are closely associated with efficiency of poultry meat production and its quality. We performed whole transcriptome profiling based on RNA sequencing of breast muscle tissue obtained from Shouguang chickens at embryonic days (E) 12 and 17 to post-hatching days (D) 1, 14, 56, and 98. A total of 9,447 differentially expressed genes (DEGs) were filtered (Q < 0.01, fold change > 2). Time series expression profile clustering analysis identified five significantly different expression profiles that were divided into three clusters. DEGs from cluster I with downregulated pattern were significantly enriched in cell proliferation processes such as cell cycle, mitotic nuclear division, and DNA replication. DEGs from cluster II with upregulated pattern were significantly enriched in metabolic processes such as glycolysis/gluconeogenesis, insulin signaling pathway, calcium signaling pathway, and biosynthesis of amino acids. DEGs from cluster III, with a pattern that increased from E17 to D1 and then decreased from D1 to D14, mainly contributed to lipid metabolism. Therefore, this study may help us explain the mechanisms underlying the phenotype that myofiber hyperplasia occurs predominantly during embryogenesis and hypertrophy occurs mainly after birth at the transcriptional level. Moreover, lipid metabolism may contribute to the early muscle development and growth. These findings add to our knowledge of muscle development in chickens.

Identification and Genetic Effect of Haplotypes in the Distal Promoter Region of Chicken &lt;i&gt;CCT6A &lt;/i&gt;Gene Associated with Egg Production Traits

The chaperonin containing TCP-1 complex protein 1 subunit zeta (CCT6A) is the only cytosolic chaperonin in eukaryotes assisting in the folding of cytoplasmic proteins. Previous study revealed that the mRNA expression of chicken CCT6A gene was remarkably elevated in the sexually mature ovaries. However, the mechanism underlying chicken CCT6A expression changes remains largely unknown. In this study, haplotypes caused by two single nucleotide polymorphisms (SNPs) of chicken CCT6A gene promoter (g.−2215 T>C and g.−1959 T>C) were identified and their associations with egg production traits as well as effects on gene expression were analyzed. Altogether four haplotypes including A (C−2215-T−1959), B (C−2215-C−1959), C (T−2215-T−1959) and D (T−2215-C−1959) were detected in all of the five chicken populations. Diplotypes AA, AD and DD were predominant in Xinyang brown hens, among which diplotype AD was associated with higher egg number at the age of 28 weeks old (E28) (P<0.05). In addition, diplotype AD was also predominant in Xinyang brown and Hy-line brown chicken populations with high egg production; whereas in Wenchang and Shouguang chicken populations which are Chinese indigenous chicken breeds and relatively lower in egg production, diplotype AA was predominant. Compared with diplotypes AA and DD, the mRNA expression of CCT6A in diplotype AD birds is the highest in F1, F5, and POF1 follicles of Hy-line brown hens (P<0.05). These results suggest that the two SNPs in chicken CCT6A promoter region are potential DNA marker for improving egg production trait.

Expression pattern of heme oxygenase 1 gene and hypoxic adaptation in chicken embryos.

Heme oxygenase 1 (HO-1), a rate-limiting enzyme of heme catabolism, has a crucial role of cytoprotective functions under hypoxia. The objective of the present study was to investigate potential differences in protective effect of HO-1 gene on chicken (Gallus gallus) embryo lung during late incubation. At embryonic day (D) D16, D18, D19, and D20 of incubation, the expression of HO-1 in the lungs of chicken embryos (Tibet and Shouguang chickens) incubated in normoxic (21% O2) and hypoxic (13% O2) conditions was measured. SNPs were screened within 5'-flanking region and coding regions with PCR-sequencing and the genotype of the SNPs was determined with PCR-RFLP in Tibet, Chahua and Shouguang chicken populations. In conclusion, the Tibet chicken had higher HO-1 expression on D19 under hypoxic incubation and had two SNPs with different frequency distributions from other chicken breeds, which might be a way that the Tibet chicken had hereditary adaptation to hypoxia during embryonic development.

English

Hypoxia is a major factor that affects the subsistence and development of multicellular organisms. Tibet chicken, as a unique native chicken breed in altiplano, shows genetic adaptation to hypoxia comparing with the breeds at the low altitude. In the present study, to explore effects of hypoxia on chicken fetal livers, eggs of Tibet chicken and Shouguang chicken were collected and the samples from each breed were divided into two groups, incubated in hypoxia and in normoxia respectively. The blood of embryos on the 16th day of incubation was collected and the serum chemistry parameters indicating liver metabolism were determined, which included glutamic-pyruvic transaminase (GPT), aspartate aminotransferase (GOT), total bilirubin (TB), direct bilirubin (DB), total bile acid (TBA), gamma glutamyltransferase (GGT), alkaline phosphatease (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), glucose and creatinine. The results show that biochemical indices varied significantly between hypoxia and normoxia except for GPT and glucose. Moreover, the concentration of ALP and LDH showed significant differences between the breeds and the incubations. The results suggest that the livers of both Shouguang chicken and Tibet chicken suffered damages in hypoxia, but the former was more serious. The results of this study support the opinion that Tibet chicken had better genetic adaptability on hypoxia, and made a good basis for further study of the genetic mechanism of adaptation to hypoxia. &nbsp; Key words: Hypoxia adaptation, liver metabolism, serum chemistry, Tibet chicken, chicken embryo.&nbsp;

Identification of chicken eNOS gene and differential expression in highland versus lowland chicken breeds

Nitric oxide (NO), an endothelium-derived relaxing factor, is synthesized from l-arginine by endothelial nitric oxide synthase (eNOS) in the endothelium. The objective of the present study was to preliminarily illuminate the expression of the eNOS gene in hypoxic adaptation of chicken embryonic development. The eNOS expression profiles between the Tibet and Shouguang chickens incubated under both normoxic and hypoxic conditions were detected by TaqMan real-time PCR. In this study, the chicken eNOS gene was found by both in silico cloning and RACE approaches. From the eNOS gene, we obtained a 3,310-bp mRNA sequence and a 10,666-bp DNA sequence and discovered that it was located on chicken chromosome 2 and had 7 unique transcripts. eNOS mRNA was detected in abundant amounts in some chick embryo organs (i.e., heart, liver, chorio-allantoic membrane, and lung), and expressed stably with the lowest levels in the brain. We observed that when exposed to hypoxia (13% O(2)) different embryo organ tissues had various sensitivities to hypoxia as determined by their eNOS expression profiles. Compared with the Shouguang chicken, the eNOS expression in the Tibet chicken was higher in the lung and liver, lower in the heart, and similar in the brain. In chorio-allantoic membranes, eNOS expression was higher in the Shouguang chicken than the Tibet chicken under hypoxic conditions, but not markedly different under normoxic conditions. The differences of eNOS expression between the 2 breeds may be relative to the hypoxic adaptation ability in Tibet chickens during embryonic development. This work will provide reference for future studies on the role of eNOS in hypoxic adaptation and response.

Differential Embryo Development among Tibetan Chicken, DRW and Shouguang Chicken Exposed to Chronic Hypoxia

: Avian embryos at high altitude are independent of maternal protection against hypoxia, which is contrary to mammals. It is well known that chronic hypoxic exposure at key points can significantly impact on avian development. Tibetan Chicken, a Chinese indigenous breed, living in Tibetan areas with an altitude of 2.2 to 4.1 thousand meters, has an adaptive mechanism to hypoxia. In the present study, fertilized eggs of Tibetan Chicken were incubated under 13% and 21% oxygen concentration. Two lowland chicken breeds, Shouguang Chicken, an indigenous chicken breed in Shandong Province of China, and Dwarf Recessive White Chicken, an imported breed in Beijing, were used as control groups. The embryo mass and some organs such as brain, heart, liver, stomach and eye weight in the three species were measured at Hamburger-Hamilton stage 39, 41, 43 and 45 under hypoxic and normal conditions. The results showed that in hypoxia Tibetan Chicken significantly differed from the two lowland chicken breeds in embryo mass at Hamburger-Hamilton stage 41, 43 and 45 (p 0.05). In addition, heart and the other organs showed different susceptibility to hypoxia at the studied stages. In conclusion, chronic hypoxia induced a change in the embryo development of the three different species and Tibetan Chicken showed adaptation to hypoxia. Of note, the embryo developmental physiology of Tibetan Chicken in response to hypoxia will shed light on the process of physiological acclimation or evolutionary adaptation as well as the study of clinical disease.

Study on Tibetan Chicken embryonic adaptability to chronic hypoxia by revealing differential gene expression in heart tissue

Oxygen concentration is essential for appropriate metabolism. Hypoxia can exert a significant impact on physiological alteration of the cell and organism. Tibetan Chicken (Gallus gallus) is a Chinese indigenous breed inhabiting in Tibetan areas, which is also a chicken breed living at high altitude for the longest time in the world. It has developed an adaptive mechanism to hypoxia, which is demonstrated by that Tibetan Chicken has much higher hatchability than low-land chicken breeds in high-altitude areas of Tibet. In the present study, Tibetan Chicken fertilized full sib eggs were incubated up to Hamburger-Hamilton stage 43 under 13% and 21% oxygen concentration, respectively. Shouguang Chicken and Dwarf Recessive White Chicken were used as control groups. The hearts in all of the 3 chicken breeds under hypoxic and normoxic conditions were isolated and hybridized to GeneChip(R) Chicken Genome Array to study molecular mechanisms underlying the adaptation to high altitude of Tibetan Chicken. As a result, 50 transcripts highly expressed in hypoxia are screened out. Among up-regulated genes, some are involved in the gene ontology (GO) such as cell growth, cell difference, muscle contraction and signal transduction. However, the expression levels of 21 transcripts are lower in hypoxia than those in normoxia. Some down-regulated genes take part in cell communication, ion transport, protein amino acid phosphorylation and signal transduction. Interestingly, gene enrichment analyses of these differential gene expressions are mainly associated with immune system response and ion channel activity in response to stimulus. Moreover, the transcriptional expression profiles analyzed by hierarchical clustering and CPP-SOM software in all of the 3 different chicken breeds revealed that Tibetan Chicken is much closely related to Shouguang Chicken rather than Dwarf Recessive White Chicken. In addition, 12 transcripts of Tibetan Chicken breed-specific expressed genes were identified, which seem to result in a more effective and efficient induction of energy demand and signal transduction of transcription and suppression of abnormal development in response to hypoxia. These findings will be beneficial in clarifying the adaptive molecular mechanism of Tibetan Chicken as well as providing new insight into cardiovascular disease at high altitude medicine.

Sequencing and alignment of mitochondrial genomes of Tibetan chicken and two lowland chicken breeds

Tibetan chicken lives in high-altitude area and has adapted well to hypoxia genetically. Shouguang chicken and Silky chicken are both lowland chicken breeds. In the present study, the complete mitochondrial genome sequences of the three chicken breeds were all sequenced. The results showed that the mitochondrial DNAs (mtDNAs) of Shouguang chicken and Silky chicken consist of 16784 bp and 16785 bp respectively, and Tibetan chicken mitochondrial genome varies from 16784 bp to 16786 bp. After sequence analysis, 120 mutations, including 4 single nucleotide polymorphisms (SNPs) in tRNA genes, 9 SNPs and 1 insertion in rRNA genes, 38 SNPs and 1 deletion in D-LOOP, 66 SNPs in protein-coding genes, were found. This work will provide clues for the future study on the association between mitochondrial genes and the adaptation to hypoxia.

Differential gene expression of phosphoglyceric kinase (PGK) and hypoxic adaptation in chicken

Four single-nucleotide polymorphisms (SNP) of the Phosphoglyceric Kinase (PGK) gene were discovered based on comparison of the sequences from an altiplano chicken breed (Tibetan chicken) and two lowland breeds (White Leghorn and Shouguang chicken). Gel-shift results indicate that one of these SNPs, an A-->G mutation at position 59 in exon10, is able to bind hypoxia-induced factor-l (HIF-1), functioning as a hypoxia response element (HRE). The mutant gene results in M-->T mutation at position 379 amino acid. The combined activity of this HRE and HIF-1 could increase correspondingly under a hypoxic stimulus. Hypoxia leads to increased death rates of chicken embryos; while the M-->T mutation described herein is prevalent in healthy embryos grown under hypoxic conditions, thus it may represent an adaptation to hypoxia. Fluorescence quantitative reverse transcription PCR results revealed that HIF-1 upregulates the transcript level of the glycolytic enzyme PGK in the brain and skeletal muscle of animals subjected to hypoxia. Thus, a large amount of ATP is produced by increased glycolysis, allowing the organism to meet energy metabolism demands. As such, we believe this SNP to be an adaptation to the external anoxic environment.

Differential gene expression of hypoxia inducible factor –1a and hypoxic adaptation in chicken

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor first discovered in cancer cells under hypoxic conditions, and was demonstrated to play a key role in the organism's adaptation to changing oxygen tensions. The Tibetan chicken, a breed indigenous to the Tibetan plateau is particularly adaptable to the low oxygen tension and low temperature conditions in the high altitude environment. By comparison, the White Leghorn and ShouGuang chicken are low-altitude chicken breeds. The eggs of these three chicken breeds were incubated in a fitted hatcher that simulated hypoxic condition. The results demonstrated that the hatching rate for Tibetan chicken was significantly higher than the two low altitude breeds, and Tibetan chicken displayed higher endurance in the hypoxia environment. To a certain degree, the hypoxic condition proved fatal to hatching for the low-altitude chickens. Gene expression of HIF-1alpha was detected in brain and skeletal muscle tissues for three chicken varieties using the TaqMan probe FQRT-PCR method. The results showed that HIF-1alpha mRNA displayed tissue specific differential expression, with the highest in the brain. In addition, the expression of HIF-1alpha mRNA in the brain of Tibetan chicken embryos was similar to that of the low-altitude chickens when they were hatched under normal oxygen tensions.

Identification of single‐nucleotide polymorphisms in 5′ end and exons of the PRKAG3 gene in Hubbard White broiler, Leghorn layer, and three Chinese indigenous chicken breeds

5'-AMP-activated protein kinase plays an important role in regulating the level of ATP in the presence of metabolic stress. Previous studies revealed that polymorphisms in 5'-AMP-activated protein kinase gamma 3 subunit (PRKAG3) gene are associated with meat quality in pigs. In the present study, single-nucleotide polymorphisms (SNPs) in the 5'-end and exons of chicken PRKAG3 gene were identified with the method of single-strand conformation polymorphism in Hubbard ISA White broiler, Leghorn layer, and three Chinese indigenous chicken breeds, Tibet Chicken, Shouguang Chicken and Beijing Yellow Chicken. Two SNPs in the 5'-end of the gene and 10 SNPs in exons 3, 4, 9 and 11, of which three caused amino acid substitutions, were identified in the PRKAG3 gene of the five chicken breeds. The results will facilitate further study on the association between the mutations of PRKAG3 and chicken meat quality.

The Investigation of Cell Culture Conditions to Maintain Chicken Embryonic Stem Cells as Totipotent Cells

The ES cell can provide a useful system for studying differentiation and development in vitro and a powerful tool for producing transgenic animalds. To investigate the culture condition of chicken embryonic stem (CES) cells which can retain their multipotentiality or totipotency, three kinds of feeder layer cells, SNL cells, primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells, were used as the feeder cells in media of DMEM supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and stem cell factor (SCF) for co-culture with blastoderm cells from stage X embryos of chicken. The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The results showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The typical CES cells clone shape revealed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong alkaline phosphatase (AKP) reactive cells were observed in the fourth passage cells. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro. The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8.2% (22/269) survived to hatching, 5 feather chimeras had been produced. This suggests that an effective culture system established in this study can promote the growth of CES cells and maintain them in the state of undifferentiated and development, which lays a solid foundation for the application of CES cells and may provide an alternative tool for genetic modification of chickens.