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Kafe 바로가기주관연구기관 | 경남과학기술대학교 |
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연구책임자 | 장인석 |
참여연구자 | 문양수 , 손시환 , 강선영 , 고영현 , 윤서현 , 김세윤 , 안영숙 , 박정근 , 김재민 , 그외 다수 |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2015-03 |
과제시작연도 | 2012 |
주관부처 | 농촌진흥청 |
과제관리전문기관 | 농촌진흥청 Rural Development Administration |
등록번호 | TRKO201500010157 |
과제고유번호 | 1395027401 |
DB 구축일자 | 2015-07-11 |
Ⅳ. 연구개발결과
○ 닭에서 스트레스에 따른 면역, 대사 및 DNA 발현 조사로 biomarker 발굴
1. Microarray 및 qRT-PCR을 통한 biomarker 유전자 발굴: 친염증 사이토카인, 대사 및 ER 유전자, DNA 기능 유전자 및 DNA 손상율(DNA fragmentation) 등을 이용한 스트레스 유전자 발굴 및 확인
(1) 친염증 사이토카인 및 항산화 효소 유전자: IL-1β, IL-6, IL-18, IFN-γ, SOD, GPX, CAT 등 Biomarker 유전자 발굴
(2) 대사 및
Ⅳ. 연구개발결과
○ 닭에서 스트레스에 따른 면역, 대사 및 DNA 발현 조사로 biomarker 발굴
1. Microarray 및 qRT-PCR을 통한 biomarker 유전자 발굴: 친염증 사이토카인, 대사 및 ER 유전자, DNA 기능 유전자 및 DNA 손상율(DNA fragmentation) 등을 이용한 스트레스 유전자 발굴 및 확인
(1) 친염증 사이토카인 및 항산화 효소 유전자: IL-1β, IL-6, IL-18, IFN-γ, SOD, GPX, CAT 등 Biomarker 유전자 발굴
(2) 대사 및 ER 유전자: PPARγ, C/EBPα, C/EBPβ, FASN, FABP4, ACSL1, HSPs, ATFs, XBP1, REBP1 등
(3) DNA 기능 유전자: Telomere, HMGCR, DNA fragmentation assay 등
○ 닭에서 스트레스에 따른 품종별 면역, 대사 및 DNA 기능유전자 발현 양상
1. 닭의 품종별 스트레스 표지 유전자 발현 조사: 스트레스에 따른 닭의 품종[(한국재래계 vs. 화이트레그혼, 육계(Ross vs. Cobb), 산란계(Hyline vs. Lohmann Brown)별 스트레스 biomarker 유전자(면역, 항산화 효소, 대사 및 DNA 기능 유전자)의 mRNA 발현은 품종 특이적으로 영향을 나타냄
(1) 한국재래계가 화이트레그혼순종보다 스트레스에 대한 유전자 발현정도가 높아 국내 환경스트레스에 대한 적응도가 높을 것으로 예상 됨
(2) 실용육계에서 품종간 Cobb가 일부 스트레스 연관 유전자의 발현도가 상대적으로 높게 발현되어 스트레스에 대한 민감도가 높은 것으로 나타남
(3) 산란계에서는 Hyline Brown 품종이 Lohmann Brown 품종에 비해 일부 스트레스 반응 biomarker 유전자 지표에서 높게 나타나는 것으로 예측
○ 닭에서 스트레스 반응에 따른 면역, 대사 및 DNA 기능 유전자 발현 양상
1. 닭의 스트레스에 따른 표지 유전자 발현 변화: 한국재래계, 육계(Ross vs. Cobb), 산란계(Hyline vs. Lohmann Brown) 품종에서 밀사 및 감염 스트레스에 따른 면역, 항산화 효소, 대사 및 DNA 기능 유전자의 mRNA 발현은 스트레스에 따라 특이적으로 영향을 미침
(1) 밀사 및 감염 스트레스에 따른 친염증 사이토카인, ER 스트레스, HSPs 및 DNA 기능(telomere 및 DNA 손상율)의 발현정도가 현저히 증가 됨
(2) 실용육계 및 산란계에서 고밀도 밀사스트레스가 스트레스 반응 유전자(친염증 사이토카인, ER 스트레스, HSPs 및 DNA 기능) 지표의 발현정도에 영향을 미침
The objective of this project was to determine the effects of stress factors(stocking density, LPS, etc) and strain(Korean native chickens, White leghorn, Ross, Cobb, Hyline brown and Lohmann brown) on the performance and physiological adaptive gene responses including immunological, metabolic and D
The objective of this project was to determine the effects of stress factors(stocking density, LPS, etc) and strain(Korean native chickens, White leghorn, Ross, Cobb, Hyline brown and Lohmann brown) on the performance and physiological adaptive gene responses including immunological, metabolic and DNA functional biomarkers in chickens. We conducted three detailed parts of this project (Study I, II and III) according to yearly basis (4 years). The summaries of three detailed study were described as follows;
(STUDY I)
The objective of this study was to determine the effects of stress factors(stocking density, LPS, etc) and strain (Korean native chickens, White leghorn, Ross, Cobb, Hyline brown and Lohmann brown) on the performance and physiological adaptive responses including corticosterone and mRNA expression of pro-inflammatory cytokines and antioxidant enzymes in chickens. We conducted 4 series of experiments (EXP 1, 2, 3 and 4) according to yearly basis (4 years). In EXP 1, we tried to find and develop stress related biomarkers especially immunity and antioxidant defense system. In our study, micro-array assay and subsequent quantitative RT-PCR were applied to assess the mRNA expression of stress related biomarker genes. We identified several novel cytokines and antioxidant genes, which responded to stress factors as biomarkers. In EXP 2, two strains(Korean native chickens vs. White leghorn) were allotted into two stocking densities(Control vs. LPS injection) in battery cages by 2 x 2 factorial designs. As a result, there was no significant strain effect on body weight and feed intake and the relative spleen weight at 3 hr after LPS treatment. However, the relative liver weight was found to be significantly (P<0.05) affected by LPS injection. Plasma corticosterone level was affected by LPS but not strain effect. The hepatic mRNA expression of pro-inflammatory cytokines including IL-1β, IL-6, IL-18 and IFN-γ and antioxidant genes including SOD, GPX and CAT was significantly altered by LPS or strain effect. However, in the spleen, the most of biomarker genes (IL-1 β, IL-6, IL-18, GPX and CAT) were significantly(P<0.05) affected by LPS treatment. In EXP 3, two strain of broiler(Ross strain, Cobb strain) were allotted into two stocking densities(Standard stocking density vs. High stocking density) in battery cages by 2 x 2 factorial designs with 8 replicates. There was significant(P<0.05) strain and stress effects(stocking density) on body weight, feed intakes and feed: gain ratio and the relative organ weights. Plasma corticosterone level was not affected by both stocking density and strain effects. In the liver, the mRNA expression of pro-inflammatory cytokines including IL-1β, IL-6, IL-18 and IFN-γ was not significantly changed by the effects of strain and stocking density. However, the mRNA expression of GPX was affected by strain, showing that Ross strain resulted in a significantly(P<0.05) lower GPX expression. With respect to the effect of stocking density, there was a significant(P<0.05) increase in SOD and CAT and GPX mRNA expression in the liver from high stocking density group. Splenic pro-inflammatory cytokine expression also was not affected by stocking density and strain, except that IL-18 mRNA significantly(P<0.05) decreased in Cob strain under high stocking density. The mRNA expression of SOD and CAT was significantly (P<0.05) affected by the effects of stocking density and strain. In EXP 4, two strain of commercial laying hens(Hyline brown, Lohmann Brown) were allotted into two stocking densities (Standard stocking density vs. High stocking density) in battery cages by 2 x 2 factorial designs with 8 replicates. There was significant(P<0.05) strain and stocking density effects on body weight, feed intakes and feed: gain ratio, however the relative liver and spleen weights were not affected by strain or stocking density. Plasma corticosterone and total antioxidant capacity were not affected by both stocking density and strain effects. In the liver, the mRNA expression of IL-6 and IL-18 and CAT was affected(P<0.05) by strain, showing that Lohmann strain might resulted in more sensitively responded to stocking density stressor. With respect to the effect of stocking density, there was a significant(P<0.05) changes in pro-inflammatory cytokinnes and antioxidant enzymes in the liver in response to stocking density. Splenic pro-inflammatory cytokine expression was not affected by strain. However, the mRNA expression of IL-18, HSP70 and CAT was significantly(P<0.05) affected by the effects of stocking density. In conclusion, the performance was affected by stocking density(stressor). The mRNA expression of major pro-inflammatory cytokines was influenced by stocking density or less frequently by strain, but antioxidant enzyme was partially affected by stocking density or strain in birds under stress conditions. More detailed studies are still needed to explore the effects of environmental stressors and genetic background on immunological and antioxidant responses in chickens.
(STUDY II)
The objective of the present study was to explore the stress-related functional genes in chickens for the establishment of disease-resistance chicken strains. To achieve the goal of the project, four experiments were conducted as follows: 1) analysis of lipid and muscle related metabolic gene transcriptional profiling to the stress of high stocking density(HSD) in broilers, 2) comparison of the mRNA expressions of stress and lipid metabolism associated genes in the liver between Korean native chicken(KNC) and white leghorn(WL) raised in high stocking density, 3) investigation of the gene expressions associated with stress, ER-stress and lipid metabolism in two strains of chickens raised in high stocking density, and 4) investigation of the gene expressions associated with stress, ER-stress and lipid metabolism in two strains of laying hens raised in high stocking density. The results of first project were described as follows. The chickens assigned to the HSD group had a significantly lower body weight, weight gain, and feed intake compared with those of the control group (p<0.05). The mortality of chickens was higher in the HSD group than that of the control group. The stress associated genes such as HMGCR, HSP90α, HSPA5 (GRP78/Bip), DNAJC3 and ATF4 were up-regulated, and interferon-γ and PDCD4 genes were down-regulated in microarry analysis. The endoplasmic reticulumn stress associated genes, HSPA5 (GRP78/Bip), DNAJC3 and ATF4, were highly expressed in HSD group. The genes, ACSL5, TMEM195 and ELOVL6, involved in fatty acid synthesis were elevated in the HSD group. The genes, ACAA1, ACOX1, EHHADH, LOC423347 and CPT1A, related to fatty acid oxidation were also activated by HSD. These results suggest that HSD rearing system stimulates the genes associated with fatty acid synthesis as well as fatty acid oxidation in the liver of broiler chickens. The results of second project were described as follows. The expression levels of hsp70 and HMGCR were higher in WL subjected to stress with high stocking density compared with those genes in control (P<0.05), while the expressions of genes were not affected in KNC. ER stress marker genes including ATF4, ATF6 and XBP1 were also highly expressed in WL with stress (P<0.05), but the stress of high stocking density did not influence to ER stress marker genes in KNC. Lipid metabolism associated genes and glucose transporter genes were highly expressed in WL compared with KNC when subjected to high stocking density stress (P<0.05). The data indicate that WL is more sensitive to the stress of high stocking density compared with KNC. The results of third project were described as follows. The high stocking density altered the gene expression of stress marker genes in commercial broilers Cobb and Ross. The expressions of HSP90a, and HSP90b genes were 2-3 fold highly expressed in Cobb compared with Ross when subjected to the stress of high stocking density(P<0.05). ER-stress marker genes including HSPA5, ATF4, XBP1 were also highly expressed in Cobb(P<0.05). But lipid metabolism associated genes were not affected in both strains. The data indicate that Cobb is more sensitive to the stress of high stocking density compared with Ross. The results of fourth project were described as follows. The expressions of stress-marker genes, HSP70, HSP90a, HSP90b, HMGCR were highly expressed in Hyline Brown compared with Lohmann Brown(P<0.05). ER-stress marker genes including HSPA5, ATF4, ATF6 were also highly expressed in Hyline Brown(P<0.05) compared with Lohmann Brown. Lipid metabolism associated genes were highly expressed in Hyline Brown compared with Lohmann brown when subjected to high stocking density stress (P<0.05). The data indicate that Hyline Brown is more sensitive to the stress of high stocking density compared with Lohmann Brown.
(STUDY III)
Chicken are exposed to a various husbandry stressors such as stocking density, unsuitable feeding, over and under rear temperature, transportation, fear and disease. These stressors cause an upset in the homeostasis of the body. Therefore, they have adverse effects on chicken performances. Many studies have been conducted to identify biological markers for assessing the physiological and immunological response of birds under stress conditions. To explore the effects of stressors on physiological parameters, we applied a combination of two stressors, high stocking density with feed restriction, to chickens and then analyzed the quantity of telomeric DNA, quantified total DNA damage and determined the gene expression levels of HSPs and HMGCR in blood. We concluded that the telomere length, especially the telomere-shortening rates, the quantification of total DNA damage and the expression levels of the HMGCR and HSP90α genes can be used as sensitive physiological stress markers in chickens. We also compared the stress response with chicken breeds that were subjected to a high stocking density on Single Comb White Leghorn (WL) and Korean Native Chicken (KNC) breeds. There was no significant difference between KNC and WL in body weight, weight gain, telomere shortening rate and DNA damage rate. However, the growth rate significantly decreased in chickens raised under high stocking density conditions, as compared to the control group. The telomere-shortening rate, DNA damage and HSPs expression of the lymphocytes were significantly higher in the high stocking density group than the control. The stress condition and breeds had a significant effect on the expressions of HSP70, HSP90-α and HSP90-β in lymphocytes, except HMGCR. The stress response of WL was higher than that of KNC, as analyzed to the expression of HSP70 and HSP90-α. Therefore, we concluded that the chickens which were exposed to a high stocking density had increased the individual physiological stress response regardless of breeds, and White Leghorns are more susceptible to stress condition than Korean Native Chickens. In additions, we compared the production performance and the stress reponses with commercial layers (Hyline Brown and Lohmann Brown) and boilers (Ross and Cobb) that were subjected to a high stocking. Stress response was analyzed by the quantity of telomeric DNA, the rate of DNA damage and the expression levels of heat shock proteins (HSPs) and hydroxyl-3-methyl-glutaryl coenzyme A reductase (HMGCR) genes on tissues and blood. The telomere length and telomere shortening rates were analyzed by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes and tissues. The DNA damage rate of lymphocytes was analyzed by the comet assay. The expression levels of HSP70, HSP90-α, HSP90-β and HMGCR genes were measured by quantitative real-time polymerase chain reaction in lymphocytes. In results of commercial laying hens, there was no significant difference between Hyline Brown and Lohmann Brown in viability, feed intake, feed conversion ratio, age at first egg, egg production, hen-housed egg production, telomere shortening rate, DNA damage, HSPs expression and rectum temperature. The body weight, feed intake, feed conversion ratio, egg laying performance were significantly lower in the high stocking density group than the low stocking density. Telomere shortening rate, DNA damage, HSP90-α and HSP70 expression of the lymphocytes were significantly higher in the high stocking density group than the low stocking density. Stocking stress significantly increased HSP90-α and HSP70 in both breeds. Hyline Brown was more increase the expression values of HSP90-α and HSP70 genes than Lohmann Brown. In the results of commercial boilers, there was no significant difference between Ross and Cobb in viability, feed intake, feed conversion ratio, body weight, weight gain, telomere length, telomere shortening rate, DNA damage. However, the growth rate significantly decreased in chickens raised under high stocking density conditions, as compared to the low group. Telomere shortening rate, HSP90-α, HSP90-β and HSP70 expression of the lymphocytes were significantly higher in the high stocking density group than the low stocking density. The expression value of HSP70 genes was more increased in Ross than Cobb under high stockung density. Therefore, we concluded that the chickens which were expoused to a high stocking density had increased the individual physiological stress response regardless of breeds. In commercial layer, the Hyline Brown is more susceptible to stocking stress than the Lohmann Brown. In addition, the Ross is also more susceptible to stress condition than the Cobb with analyzing the expression of HSP genes.
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