發(fā)布時間：2018-05-29 03:51

  本文選題：熱應(yīng)激 + 錳與鋅��； 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：本研究通過種母雞高溫長期應(yīng)激、雞胚孵化以及子代高溫長期應(yīng)激共5個系列試驗,從繁殖性能、抗氧化性能及表觀遺傳等方面,系統(tǒng)、深入研究飼糧微量元素錳、鋅對熱應(yīng)激產(chǎn)蛋期肉種雞、種蛋孵化和出殼后子代肉雞生長發(fā)育不同階段的抗熱應(yīng)激效應(yīng)及其分子機(jī)制。試驗一采用2×3雙因子安排的完全隨機(jī)設(shè)計。試驗設(shè)2個種雞飼養(yǎng)溫度處理(21℃常溫與32℃C高溫)和3個種母雞飼糧錳處理(添加錳0 mg/kg、硫酸錳形態(tài)錳120 mg/kg與中等絡(luò)合強(qiáng)度有機(jī)形態(tài)錳120mg/kg),共6個處理組,每個處理6個重復(fù),每個重復(fù)4只�；�礎(chǔ)飼糧為玉米-豆粕型飼糧。正式試驗期持續(xù)14周。結(jié)果表明,熱應(yīng)激顯著降低(P0.05)肉種母雞蛋重、產(chǎn)蛋率、產(chǎn)蛋量、采食量、蛋料比、蛋殼厚度和強(qiáng)度及血漿ALP酶活和T3含量及提高(P0.02)其肛溫及血漿LDH和CK酶活,飼糧添加錳顯著提高(P0.05)蛋殼強(qiáng)度,降低(P=0.05)血漿T3,T4和PCC含量。環(huán)境溫度和飼糧錳處理互作對產(chǎn)蛋率、產(chǎn)蛋率、采食量和蛋料比有顯著影響(P0.05)。常溫條件下,飼糧錳處理對上述各指標(biāo)無影響(P0.22)；高溫條件下,和對照組相比,有機(jī)錳組顯著提高(P0.03)肉種母雞產(chǎn)蛋率、產(chǎn)蛋率、采食量和蛋料比,而錳源及無機(jī)錳組與對照組之間均無差異(P0.18)。以上結(jié)果提示,熱應(yīng)激降低了肉種母雞產(chǎn)蛋性能和蛋殼品質(zhì),并引起脂質(zhì)過氧化損傷及相應(yīng)組織損傷；飼糧添加錳提高了耐熱度及降低蛋白質(zhì)損傷,尤其是飼糧添加中等絡(luò)合強(qiáng)度有機(jī)錳能有效緩解熱應(yīng)激引起的肉種母雞產(chǎn)蛋性下降。試驗二在試驗一的基礎(chǔ)上進(jìn)一步研究環(huán)境溫度和飼糧錳對肉種母雞組織抗氧化性能及熱休克蛋白／因子表達(dá)的影響,結(jié)果表明,熱應(yīng)激顯著降低(P0.01)肉種母雞肝臟和心肌錳存留量,提高(P0.05)組織HSF1、HSF3和MnSOD mRNA及HSP70 mRNA和蛋白表達(dá)；其中,只有胸肌組織出現(xiàn)脂質(zhì)過氧化損傷和MnSOD舌性升高及HSP90 mRNA和蛋白表達(dá)顯著升高(P0.05)；飼糧添加無機(jī)錳和有機(jī)錳均提高(P0.01)肉種母雞心肌Mn含量、MnSOD和CuZnSOD酶活及胸肌MnSOD蛋白表達(dá),抑制(P0.05)胸肌HSP70 mRNA和蛋白表達(dá)。其中,有機(jī)錳組脛骨錳含量顯著高于無機(jī)錳組(P0.03)。環(huán)境溫度和飼糧錳處理互作對上述指標(biāo)無影響)P0.05)。以上結(jié)果提示,熱應(yīng)激降低組織錳存留量并引起氧化損傷,而胸肌對于熱應(yīng)激最為敏感;飼糧添加錳可提高機(jī)體抗氧化能力,降低熱應(yīng)激引起胸肌HSP70過表達(dá)；脛骨錳含量方面,中等絡(luò)合強(qiáng)度有機(jī)錳生物學(xué)利用性要優(yōu)于無機(jī)硫酸錳。試驗三進(jìn)一步研究母代溫度和母代飼糧錳對雞胚心肌抗氧化性能及表觀遺傳指標(biāo)的影響。肉種母雞飼養(yǎng)階段如試驗一,然后收集肉種母雞試驗結(jié)束前2天所有種蛋按相應(yīng)處理和重復(fù)進(jìn)行孵化試驗。結(jié)果表明,母代高溫顯著降低(P0.002)入孵種蛋受精率、孵化率和肉仔雞初生重及提高(P0.05)胚胎死亡率及雞胚心肌HSP、COX2、CDK6和BAX mRNA表達(dá)及引起總DNA低甲基化和總AcH3K9低乙酰化；母代飼糧添加無機(jī)錳和有機(jī)錳均提高(P0.05)蛋黃和組織錳含量及雞胚心肌SODs活性和MnSOD mRNA表達(dá),并發(fā)現(xiàn)MnSOD mRNA表達(dá)上調(diào)與其啟動子區(qū)低DNA甲基化和高AcH3K9乙�；�有關(guān)。母代溫度和糧錳處理交互作用顯著影響(P=0.05)入孵種蛋孵化率及心肌BCL2 mRNA和蛋白表達(dá)。母代常溫條件下,母代飼糧錳處理對入孵種蛋孵化率及心肌BCL2 mRNA和蛋白表達(dá)無影響(P0.28)；母代高溫條件下,和對照組相比,無機(jī)錳組和有機(jī)錳組顯著提高了入孵種蛋孵化率及心肌BCL2 mRNA和蛋白表達(dá)(P0.05)。以上結(jié)果提示,母代高溫?fù)p害孵化性能同時引起雞胚心肌氧化損傷和細(xì)胞凋亡及表觀遺傳異常；母代飼糧添加錳,特別是中等絡(luò)合強(qiáng)度有機(jī)錳,可緩解母代熱應(yīng)激引起的孵化率和胚胎存活率下降,可能與其提高機(jī)體抗氧化和抗細(xì)胞凋亡能力有關(guān)。試驗四進(jìn)一步研究母代溫度和母代飼糧錳對子代肉仔雞生產(chǎn)性能、血液生化及胚胎和子代miRNA表達(dá)影響。在試驗三基礎(chǔ)上獲得各處理組子代肉仔雞,并于29日齡按體重和性別隨機(jī)分為常溫、高溫2個處理。因此,子代肉仔雞試驗共有12個處理組,每個處理組6個重復(fù),每個重復(fù)4只雞,公母各半。結(jié)果表明,母代高溫顯著提高(P0.05)22-28日齡子代肉仔雞末重、日增重和目采食量；與母代添加錳組相比,母代對照組提高(P0.05)1-21日齡子代肉仔雞日采食量,21日齡血漿UA和T4含量及降低高溫下42日齡子代腿肉仔雞肌紅度,說明母代熱應(yīng)激和母代飼糧缺錳均引起子代肉仔雞早期補(bǔ)償生長,機(jī)體代謝水平增加；但母代飼糧添加有機(jī)錳能有效緩解子代熱應(yīng)激引起的肉品質(zhì)下降。通過對雞胚miRNA高通量測序、RT-PCR驗證及其靶基因預(yù)測和雙熒光素酶法驗證,發(fā)現(xiàn)母代溫度和母代飼糧錳交互作用顯著影響(P0.05)雞胚心肌gga-miR-34c-5p和gga-miR-1551-5p表達(dá)及其調(diào)控靶基因絲裂原活化蛋白激酶MAP3K14和BCL2表達(dá)。即和母代常溫相比,母代高溫顯著下調(diào)母代對照組雞胚心肌gga-miR-34c-5p和gga-miR-1551-5p表達(dá)以上調(diào)其心肌靶基因MAP3K14和NF-κB mRNA及BCL2 mRNA和蛋白表達(dá),但母代溫度對母代無機(jī)錳組和有機(jī)錳組無影響(P0.05)；且上述效應(yīng)可持續(xù)至高溫條件下42日齡子代肉仔雞階段。以上結(jié)果提示,母代飼糧添加錳通過下調(diào)雞胚和子代心肌gga-miR-34c-5p和gga-miR-1551-5p表達(dá)來上調(diào)其靶基因mRNA和蛋白表達(dá),激活NF-κB通路和抗凋亡能力,并在雞胚階段和子代肉仔雞2個階段表現(xiàn)抗良好的熱應(yīng)激效應(yīng)。試驗五是在飼糧錳對肉雞抗熱應(yīng)激效應(yīng)及其分子機(jī)制研究思路基礎(chǔ)上,耗竭期采用嚴(yán)重缺鋅純合飼糧及正試期采用半純合飼糧飼喂肉種母雞,進(jìn)一步研究了母代種雞飼養(yǎng)環(huán)境溫度及其飼糧鋅對孵化和子代肉仔雞2個階段的抗熱應(yīng)激效應(yīng)。種母雞試驗采用2×3雙因子安排的完全隨機(jī)設(shè)計。試驗設(shè)2個母代種雞飼養(yǎng)環(huán)境溫度處理(21℃C常溫與32℃C高溫)和3個種母雞飼糧鋅處理(添加鋅0 mg/kg.硫酸鋅形態(tài)鋅110mg/kg與中等絡(luò)合強(qiáng)度有機(jī)形態(tài)鋅110mg/kg),共6個試驗處理組,每個處理24只種母雞,分為6個重復(fù),每個重復(fù)4只種母雞。子代肉雞試驗在種蛋孵化試驗基礎(chǔ)上,于子代肉仔雞29日齡時,另設(shè)2個子代肉雞飼養(yǎng)環(huán)境溫度處理(常溫21℃與高溫33℃),共12個處理,每個處理設(shè)6個重復(fù),每個重復(fù)4只子代肉仔雞(公母各半),每個試驗處理共24只雞。結(jié)果表明,母代熱應(yīng)激不僅損害孵化性能(Jp=0.05),還顯著降低(P=0.05)子代肉仔雞1-21、22-28和29-42日齡三個階段末重和日增重及42日齡子代肉仔雞屠宰率；特別是子代熱應(yīng)激更易提高來自母代高溫42日齡子代肉仔雞胸肌滴水損失及破壞其胰腺氧化-抗氧化系統(tǒng)平衡。母代飼糧添加鋅可提高(P0.05)入孵種蛋孵化率和健雛率、生長前期子代肉仔雞生長性能和存活率及雞胚肝臟MT4和MT1 mRNA表達(dá)；母代飼糧添加鋅,特別是中等絡(luò)合強(qiáng)度有機(jī)鋅,可緩解母代熱應(yīng)激引起的1-21及22-28日齡子代肉仔雞日增重及其28日齡末重下降(P0.05)；改善子代熱應(yīng)激引起的肉品質(zhì)下降(P0.05)。以上結(jié)果提示,母代熱應(yīng)激對子代肉仔雞生長性能的影響可從出生持續(xù)至屠宰上市階段：母代飼糧缺鋅引起雞胚肝臟表觀遺傳異常,而母代飼糧添加鋅提高孵化及抗氧化性能；其中肝臟MT4 mRNA高表達(dá)與其啟動子區(qū)高AcH3K9乙�；�調(diào)控有關(guān)。綜上所述,發(fā)現(xiàn)種母雞飼糧添加錳,特別是中等絡(luò)合強(qiáng)度有機(jī)錳,可有效緩解熱應(yīng)激引起的種母雞產(chǎn)蛋性能、孵化率、胚胎存活率及子代肉品質(zhì)下降,與種母雞抗氧化能力及雞胚和子代抗氧化和抗細(xì)胞凋亡能力提高有關(guān)；同樣,母代飼糧添加鋅,特別是中等絡(luò)合強(qiáng)度有機(jī)鋅,可緩解母代熱應(yīng)激引起的1-21和22-28日齡子代肉仔雞日增重下降,改善子代熱應(yīng)激引起的肉品質(zhì)下降。此外,母代熱應(yīng)激和母代飼糧缺錳、鋅均引起雞胚組織總體及特定基因啟動區(qū)表觀遺傳異常；其中,雞胚心肌MnSOD和肝臟MT4 mRNA高表達(dá)受到其啟動子區(qū)DNA低甲基化和AcH3K9高乙�；�水平調(diào)控。以上研究新成果為全球氣溫逐年升高形勢下有效降低熱應(yīng)激對雞等家禽的負(fù)面影響提供系統(tǒng)、深入的科學(xué)試驗依據(jù)。
[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 .
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S831
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本文編號：1949377