饲粮锰、锌对肉鸡的抗热应激效应及其分子机制的研究

发布时间：2018-05-29 03:51

  本文选题：热应激 + 锰与锌　； 参考：《中国农业大学》2016年博士论文

【摘要】：本研究通过种母鸡高温长期应激、鸡胚孵化以及子代高温长期应激共5个系列试验,从繁殖性能、抗氧化性能及表观遗传等方面,系统、深入研究饲粮微量元素锰、锌对热应激产蛋期肉种鸡、种蛋孵化和出壳后子代肉鸡生长发育不同阶段的抗热应激效应及其分子机制。试验一采用2×3双因子安排的完全随机设计。试验设2个种鸡饲养温度处理(21℃常温与32℃C高温)和3个种母鸡饲粮锰处理(添加锰0 mg/kg、硫酸锰形态锰120 mg/kg与中等络合强度有机形态锰120mg/kg),共6个处理组,每个处理6个重复,每个重复4只。基础饲粮为玉米-豆粕型饲粮。正式试验期持续14周。结果表明,热应激显著降低(P0.05)肉种母鸡蛋重、产蛋率、产蛋量、采食量、蛋料比、蛋壳厚度和强度及血浆ALP酶活和T3含量及提高(P0.02)其肛温及血浆LDH和CK酶活,饲粮添加锰显著提高(P0.05)蛋壳强度,降低(P=0.05)血浆T3,T4和PCC含量。环境温度和饲粮锰处理互作对产蛋率、产蛋率、采食量和蛋料比有显著影响(P0.05)。常温条件下,饲粮锰处理对上述各指标无影响(P0.22)；高温条件下,和对照组相比,有机锰组显著提高(P0.03)肉种母鸡产蛋率、产蛋率、采食量和蛋料比,而锰源及无机锰组与对照组之间均无差异(P0.18)。以上结果提示,热应激降低了肉种母鸡产蛋性能和蛋壳品质,并引起脂质过氧化损伤及相应组织损伤；饲粮添加锰提高了耐热度及降低蛋白质损伤,尤其是饲粮添加中等络合强度有机锰能有效缓解热应激引起的肉种母鸡产蛋性下降。试验二在试验一的基础上进一步研究环境温度和饲粮锰对肉种母鸡组织抗氧化性能及热休克蛋白／因子表达的影响,结果表明,热应激显著降低(P0.01)肉种母鸡肝脏和心肌锰存留量,提高(P0.05)组织HSF1、HSF3和MnSOD mRNA及HSP70 mRNA和蛋白表达；其中,只有胸肌组织出现脂质过氧化损伤和MnSOD舌性升高及HSP90 mRNA和蛋白表达显著升高(P0.05)；饲粮添加无机锰和有机锰均提高(P0.01)肉种母鸡心肌Mn含量、MnSOD和CuZnSOD酶活及胸肌MnSOD蛋白表达,抑制(P0.05)胸肌HSP70 mRNA和蛋白表达。其中,有机锰组胫骨锰含量显著高于无机锰组(P0.03)。环境温度和饲粮锰处理互作对上述指标无影响)P0.05)。以上结果提示,热应激降低组织锰存留量并引起氧化损伤,而胸肌对于热应激最为敏感;饲粮添加锰可提高机体抗氧化能力,降低热应激引起胸肌HSP70过表达；胫骨锰含量方面,中等络合强度有机锰生物学利用性要优于无机硫酸锰。试验三进一步研究母代温度和母代饲粮锰对鸡胚心肌抗氧化性能及表观遗传指标的影响。肉种母鸡饲养阶段如试验一,然后收集肉种母鸡试验结束前2天所有种蛋按相应处理和重复进行孵化试验。结果表明,母代高温显著降低(P0.002)入孵种蛋受精率、孵化率和肉仔鸡初生重及提高(P0.05)胚胎死亡率及鸡胚心肌HSP、COX2、CDK6和BAX mRNA表达及引起总DNA低甲基化和总AcH3K9低乙酰化；母代饲粮添加无机锰和有机锰均提高(P0.05)蛋黄和组织锰含量及鸡胚心肌SODs活性和MnSOD mRNA表达,并发现MnSOD mRNA表达上调与其启动子区低DNA甲基化和高AcH3K9乙酰化有关。母代温度和粮锰处理交互作用显著影响(P=0.05)入孵种蛋孵化率及心肌BCL2 mRNA和蛋白表达。母代常温条件下,母代饲粮锰处理对入孵种蛋孵化率及心肌BCL2 mRNA和蛋白表达无影响(P0.28)；母代高温条件下,和对照组相比,无机锰组和有机锰组显著提高了入孵种蛋孵化率及心肌BCL2 mRNA和蛋白表达(P0.05)。以上结果提示,母代高温损害孵化性能同时引起鸡胚心肌氧化损伤和细胞凋亡及表观遗传异常；母代饲粮添加锰,特别是中等络合强度有机锰,可缓解母代热应激引起的孵化率和胚胎存活率下降,可能与其提高机体抗氧化和抗细胞凋亡能力有关。试验四进一步研究母代温度和母代饲粮锰对子代肉仔鸡生产性能、血液生化及胚胎和子代miRNA表达影响。在试验三基础上获得各处理组子代肉仔鸡,并于29日龄按体重和性别随机分为常温、高温2个处理。因此,子代肉仔鸡试验共有12个处理组,每个处理组6个重复,每个重复4只鸡,公母各半。结果表明,母代高温显著提高(P0.05)22-28日龄子代肉仔鸡末重、日增重和目采食量；与母代添加锰组相比,母代对照组提高(P0.05)1-21日龄子代肉仔鸡日采食量,21日龄血浆UA和T4含量及降低高温下42日龄子代腿肉仔鸡肌红度,说明母代热应激和母代饲粮缺锰均引起子代肉仔鸡早期补偿生长,机体代谢水平增加；但母代饲粮添加有机锰能有效缓解子代热应激引起的肉品质下降。通过对鸡胚miRNA高通量测序、RT-PCR验证及其靶基因预测和双荧光素酶法验证,发现母代温度和母代饲粮锰交互作用显著影响(P0.05)鸡胚心肌gga-miR-34c-5p和gga-miR-1551-5p表达及其调控靶基因丝裂原活化蛋白激酶MAP3K14和BCL2表达。即和母代常温相比,母代高温显著下调母代对照组鸡胚心肌gga-miR-34c-5p和gga-miR-1551-5p表达以上调其心肌靶基因MAP3K14和NF-κB mRNA及BCL2 mRNA和蛋白表达,但母代温度对母代无机锰组和有机锰组无影响(P0.05)；且上述效应可持续至高温条件下42日龄子代肉仔鸡阶段。以上结果提示,母代饲粮添加锰通过下调鸡胚和子代心肌gga-miR-34c-5p和gga-miR-1551-5p表达来上调其靶基因mRNA和蛋白表达,激活NF-κB通路和抗凋亡能力,并在鸡胚阶段和子代肉仔鸡2个阶段表现抗良好的热应激效应。试验五是在饲粮锰对肉鸡抗热应激效应及其分子机制研究思路基础上,耗竭期采用严重缺锌纯合饲粮及正试期采用半纯合饲粮饲喂肉种母鸡,进一步研究了母代种鸡饲养环境温度及其饲粮锌对孵化和子代肉仔鸡2个阶段的抗热应激效应。种母鸡试验采用2×3双因子安排的完全随机设计。试验设2个母代种鸡饲养环境温度处理(21℃C常温与32℃C高温)和3个种母鸡饲粮锌处理(添加锌0 mg/kg.硫酸锌形态锌110mg/kg与中等络合强度有机形态锌110mg/kg),共6个试验处理组,每个处理24只种母鸡,分为6个重复,每个重复4只种母鸡。子代肉鸡试验在种蛋孵化试验基础上,于子代肉仔鸡29日龄时,另设2个子代肉鸡饲养环境温度处理(常温21℃与高温33℃),共12个处理,每个处理设6个重复,每个重复4只子代肉仔鸡(公母各半),每个试验处理共24只鸡。结果表明,母代热应激不仅损害孵化性能(Jp=0.05),还显著降低(P=0.05)子代肉仔鸡1-21、22-28和29-42日龄三个阶段末重和日增重及42日龄子代肉仔鸡屠宰率；特别是子代热应激更易提高来自母代高温42日龄子代肉仔鸡胸肌滴水损失及破坏其胰腺氧化-抗氧化系统平衡。母代饲粮添加锌可提高(P0.05)入孵种蛋孵化率和健雏率、生长前期子代肉仔鸡生长性能和存活率及鸡胚肝脏MT4和MT1 mRNA表达；母代饲粮添加锌,特别是中等络合强度有机锌,可缓解母代热应激引起的1-21及22-28日龄子代肉仔鸡日增重及其28日龄末重下降(P0.05)；改善子代热应激引起的肉品质下降(P0.05)。以上结果提示,母代热应激对子代肉仔鸡生长性能的影响可从出生持续至屠宰上市阶段：母代饲粮缺锌引起鸡胚肝脏表观遗传异常,而母代饲粮添加锌提高孵化及抗氧化性能；其中肝脏MT4 mRNA高表达与其启动子区高AcH3K9乙酰化调控有关。综上所述,发现种母鸡饲粮添加锰,特别是中等络合强度有机锰,可有效缓解热应激引起的种母鸡产蛋性能、孵化率、胚胎存活率及子代肉品质下降,与种母鸡抗氧化能力及鸡胚和子代抗氧化和抗细胞凋亡能力提高有关；同样,母代饲粮添加锌,特别是中等络合强度有机锌,可缓解母代热应激引起的1-21和22-28日龄子代肉仔鸡日增重下降,改善子代热应激引起的肉品质下降。此外,母代热应激和母代饲粮缺锰、锌均引起鸡胚组织总体及特定基因启动区表观遗传异常；其中,鸡胚心肌MnSOD和肝脏MT4 mRNA高表达受到其启动子区DNA低甲基化和AcH3K9高乙酰化水平调控。以上研究新成果为全球气温逐年升高形势下有效降低热应激对鸡等家禽的负面影响提供系统、深入的科学试验依据。
[Abstract]:The effects of dietary Mn and Zn on reproductive performance , oxidation resistance and apparent inheritance were studied in this study . The effects of Mn and Zn on reproductive performance , oxidation resistance and apparent inheritance were studied .
Compared with the control group , there was no difference between the egg laying rate , the laying rate , the feed intake and the egg - feed ratio , but the manganese source and the inorganic manganese group had no difference compared with the control group ( P 0 . 18 ) . The results suggested that the heat stress reduced the egg - laying performance and eggshell quality of the meat - type hens , and caused the injury of lipid peroxidation and the damage of the corresponding tissues ;
The effects of ambient temperature and dietary Mn on the antioxidant properties and heat shock protein / factor expression of broiler chicken were studied . The results showed that the heat stress significantly decreased ( P0.01 ) the effects of ambient temperature and dietary Mn on the antioxidant properties and the expression of heat shock protein / factor . The results showed that the heat stress significantly decreased ( P0.01 ) the liver and myocardial manganese retention of the broiler , increased ( P0.05 ) the expression of HSF3 and MnSOD mRNA and HSP70 mRNA and protein .
Among them , only breast muscle group had lipid peroxidation injury and MnSOD tongue increase and HSP90 mRNA and protein expression increased significantly ( P0.05 ) ;
The content of Mn , MnSOD , CuZnSOD and MnSOD in the breast muscle increased significantly ( P0.01 ) . The content of Mn SOD and CuZnSOD were significantly higher than those in the inorganic manganese group ( P0.05 ) .
The results showed that the parent ' s temperature was significantly decreased ( P0.05 ) .
The expression of SODs and MnSOD mRNA were increased ( P0.05 ) . The expression of SODs and MnSOD mRNA in chicken embryo were increased . The interaction of MnSOD mRNA was significantly affected by the interaction of parent temperature and manganese treatment ( P = 0 . 05 ) .
Compared with the control group , the hatching rate of the eggs and the expression of BCL2 mRNA and protein were significantly increased in the inorganic manganese group and the organic manganese group compared with the control group ( P0.05 ) .
The effects of Mn , especially moderate complex strength organic manganese , on the production performance , blood biochemistry and embryo and progeny miRNA expression of offspring were studied . The results showed that the parent temperature and the parent diet were divided into 12 treatment groups at room temperature and high temperature at random .
The serum UA and T 4 contents of the offspring were increased ( P0.05 ) from 1 to 21 days , and the levels of serum UA and T4 at 21 days were higher than those in the parent control group ( P0.05 ) .
The expression of gga - miR - 34c - 5p and gga - miR - 1551 - 5p in chicken embryo myocardium was significantly affected by high - throughput sequencing , RT - PCR and target gene prediction .
The experimental results suggested that the effect of adding manganese on the expression of target gene mRNA and protein , the activation of NF - 魏B pathway and anti - apoptotic ability by regulating the expression of gga - miR - 34c - 5p and gga - miR - 1551 - 5p in chicken embryo were studied .
in particular , that heat stress of the filial generation is more likely to improve the drop loss of the breast muscle of the offspring broiler from the parent parent high - temperature 42 - day - age children and damage the balance of the pancreatic oxidation - oxidation system thereof .
Zinc , especially organic zinc with moderate complexing strength , could be added to the female parent diet , which could alleviate the daily gain of 1 - 21 and 22 - 28 day - old offspring of female parent and its 28 - day - age weight decrease ( P0.05 ) .
The results suggested that the effect of heat stress on the growth performance of filial broiler could be continued from birth to slaughter marketing stage .
In conclusion , it is found that the high expression of MT4 mRNA in liver is related to the acetylation regulation of high AcH3 K9 in promoter region . In conclusion , it is found that the addition of Mn , especially the medium complex - strength organic manganese , can effectively alleviate the egg laying performance , hatchability , embryo survival rate and the decrease of the quality of offspring meat caused by heat stress , which is related to the anti - oxidation ability of the hen and the increase of anti - oxidation and anti - apoptotic ability of chicken embryo and filial generation .
In the same way , the addition of zinc , especially the medium - complex - strength organic zinc , can alleviate the daily gain and decrease of 1 - 21 and 22 - 28 - day - old offspring of the parent - generation heat stress , improve the meat quality caused by the heat stress of the offspring .
In this study , the high expression of MnSOD and MT4 mRNA in chicken embryo myocardium was regulated by low methylation of DNA in promoter region and high acetylation level of Ac3K9 .
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S831
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本文编号：1949377