熱應激對奶牛泌乳性能的影響及其機制

韓佳良，劉建新，劉紅云

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熱應激對奶牛泌乳性能的影響及其機制

韓佳良，劉建新，劉紅云

（浙江大學奶業(yè)科學研究所，杭州 310058）

隨著溫室效應加劇，奶牛熱應激問題日益凸顯，對乳業(yè)造成巨大經濟損失。熱應激是一復雜的生理應答過程，奶牛在溫熱環(huán)境下表現為呼吸和心率加快，直腸溫度升高，采食量下降，對其內分泌系統(tǒng)和免疫系統(tǒng)造成負面影響，嚴重降低奶牛泌乳性能。目前關于奶牛熱應激的報道多集中于生產試驗，特別是飼喂功能性飼料添加劑對應激的緩解作用，但其具體作用機制尚不清晰。文章介紹了熱應激對國內外不同地區(qū)奶牛產奶量和乳品質的廣泛影響，并從能量代謝、內分泌、氧化應激、細胞凋亡和自噬等方面綜述了熱應激對奶牛泌乳性能的影響機制。能量代謝方面，從奶牛采食量減少、脂質分解和能量代謝紊亂等內在分子學機理角度解釋了奶牛在熱應激下處于能量負平衡狀態(tài)的原因；內分泌方面，介紹了熱應激對奶牛下丘腦-垂體-腎上腺軸/甲狀腺軸/性腺軸/生長軸的調控，分析了激素變化對機體的影響及其作用機制；氧化應激方面，重點闡述了熱應激通過影響機體內ROS水平從而產生氧化應激的分子機制及激活的相關防御信號通路；細胞凋亡和自噬方面，介紹了高溫脅迫引起奶牛乳腺上皮細胞損傷，細胞凋亡相關基因表達引發(fā)的內源性和外源性細胞凋亡，而過度自噬引發(fā)的細胞損傷也對乳腺泌乳起負面調控作用。筆者指出，在可預見的未來，熱應激將是奶牛養(yǎng)殖業(yè)面臨的最大難題，應建立可控的奶牛熱應激模型運用于生產實踐研究，并加強奶牛乳腺上皮細胞水平的基礎研究，結合高通量數據分析技術，系統(tǒng)揭示熱應激的發(fā)病機制，為緩解熱應激提供全面的理論依據。

奶牛；熱應激；泌乳性能

近幾年，隨著溫室效應加劇，熱應激對奶牛生產性能的影響已成為奶牛養(yǎng)殖業(yè)亟需解決的難題。中國奶業(yè)發(fā)展呈現南北不均態(tài)勢，奶牛主要養(yǎng)殖在北方地區(qū)，而南方卻有著巨大的市場需求。許多養(yǎng)殖企業(yè)嘗試在南方飼養(yǎng)奶牛，以滿足其市場空缺。泌乳性能作為奶牛最重要的生產指標，受高溫影響顯著，所以南方地區(qū)普遍存在的高溫環(huán)境對奶牛泌乳性能的影響是奶農焦慮的主要問題之一。本文從能量代謝、內分泌、氧化應激、細胞凋亡和自噬等方面綜述了熱應激對奶牛泌乳性能的影響及其機制，并結合組學技術揭示轉錄、蛋白和代謝水平物質變化，為熱應激狀態(tài)下改善奶牛乳腺機能進而采取保護措施以提高泌乳性能提供理論依據。

1 熱應激

熱應激是動物機體應對環(huán)境高溫所產生的非特異性應答反應。國外學者采用熱應激表示動物機體應對高溫環(huán)境產生的散熱需求[1]。國內學者則認為熱應激是所有與高溫有關的因素引起的有利于避免動物機體功能紊亂并使其更好地適應環(huán)境而發(fā)生在動物細胞水平的自我調節(jié)[2]。奶牛熱應激的影響程度一般采用溫濕指數（temperature-humidity index, THI）進行估計[3]，但由于不同奶牛年齡、品種、生理狀態(tài)等因素不同，單純依靠THI很難準確評估奶牛熱應激狀態(tài)。因此，還可結合熱休克蛋白表達水平、生理生化、生產性能、內分泌等指標評定奶牛熱應激狀態(tài)。隨著養(yǎng)殖自動化程度提高，可運用紅外線熱像儀和加速度傳感器監(jiān)測奶牛呼吸頻率和往返踱步行為，實現遠程監(jiān)控奶牛健康狀況[4]，也可通過監(jiān)測奶?；顒雍头雌c時間對熱應激進行早期預警[5]。

熱應激可致使奶牛行為異常，生產和繁殖性能下降，發(fā)生疾病甚至導致奶牛死亡[6-7]。國內外研究人員嘗試運用多種方法緩解奶牛熱應激，例如在干奶期飼喂營養(yǎng)免疫調節(jié)劑可緩解奶牛熱應激并提高其泌乳性能[8]；近期研究關注開發(fā)包含煙酸、維生素C、硫酸鉀、γ-氨基丁酸等抗應激物質的瘤胃保護膠囊，從而在營養(yǎng)調控層面緩解奶牛熱應激[9]。飼喂高能量混合日糧以保證熱應激奶牛所需能量，但會增加奶牛瘤胃酸中毒風險；目前奶牛養(yǎng)殖場多采用較為經濟實用的噴霧風扇系統(tǒng)進行降溫，但造成環(huán)境濕度過大，使奶牛多發(fā)肢蹄病和乳房炎[10]。最新研究利用基因組育種值預測奶牛的熱耐受性，選育牛產奶量下降較少，因此用基因組選育耐熱性奶牛不失為一種抗熱應激手段，在未來對動物福利具有深遠影響[11]。

2 熱應激對奶牛泌乳性能的影響

2.1 熱應激對產奶量的影響

產奶量是奶牛泌乳性能最基本的指標，其高低直接影響奶牛養(yǎng)殖場的經濟效益。不同地區(qū)的高熱環(huán)境均會降低奶牛產奶量，但由于熱應激程度、持續(xù)時間、奶牛品種、飼養(yǎng)條件等不同，產奶量降低的程度也不同。當30＜THI＜60時，德國荷斯坦奶牛處于正常狀態(tài)，奶產量隨THI的升高而升高；當THI＞60時，奶牛受到輕度熱應激，奶產量下降，并隨THI的升高而逐漸降低[12]。短期中度熱應激下，加拿大和美國北部地區(qū)奶牛日均奶產量下降4.8%，并對恢復階段存在長期的負面影響[13]。當THI＞83時，巴西南部荷斯坦奶牛受到強烈熱應激，與對照組相比日均奶產量降低21%[14]。國內研究中，李朝明等[15]試驗表明熱應激條件下荷斯坦、娟珊和娟荷雜交奶牛產奶量均下降，其中娟荷雜交奶牛產奶量優(yōu)于其他組，表現出較好的耐熱性能。可見不同地區(qū)或品種的奶牛對熱應激應答程度不同。根據我國區(qū)域氣候條件，選育適應高溫環(huán)境的奶牛品種并深入研究南方地區(qū)奶牛熱應激閾值顯得尤為必要。

2.2 熱應激對乳品質的影響

乳蛋白率、乳脂率、乳糖率和非脂固體含量等是奶牛乳品質的重要指標。乳蛋白和乳脂肪是構成牛奶的重要營養(yǎng)物質，是市場競爭的核心因素。許多研究表明發(fā)生熱應激時，各泌乳階段奶牛的乳蛋白率、乳脂率、非脂固體含量均有下降趨勢，且在泌乳前期降低幅度最大[12-13, 16-17]，但對牛奶中乳糖率沒有顯著影響[18- 19]。熱應激對乳蛋白的影響主要表現為乳腺上皮細胞酪蛋白等主要乳蛋白基因表達下調，乳中總酪蛋白減少而尿素濃度增加，導致營養(yǎng)價值降低[20-21]。最新研究表明，熱應激奶牛乳中短鏈和中鏈脂肪酸含量，磷脂酰乙醇胺、磷脂酰絲氨酸、磷脂酰膽堿、溶血磷脂酰膽堿和葡萄糖神經酰胺等5種極性脂質顯著降低[22]。極性脂質是構成脂肪球膜的主要成分，起到乳化劑的作用，可確保乳液體系的穩(wěn)定性[23]。此外，體細胞數是衡量原料乳質量的一個重要指標，熱應激條件下奶牛乳中體細胞數顯著增加，嚴重影響乳品質[24]。但由于奶牛乳腺泌乳代謝受諸多因素調控，其泌乳參數不盡相同，大大增加了研究難度。只有深入了解熱應激對奶牛泌乳性能的影響機制，才能合理調控泌乳進而達到改善乳品質的目的。

3 熱應激影響奶牛泌乳性能的機制

3.1 熱應激影響奶牛能量代謝

機體在生命活動過程中，始終保持能量的動態(tài)平衡。奶牛受到熱應激時表現為干物質采食量減少以最大限度減少熱總負荷，并加快激活散熱機制（出汗、血液流向皮膚）引起連鎖生理調節(jié)使維持能量增加，用于生產泌乳的能量減少[25-27]。下丘腦是動物體溫和攝食調節(jié)中樞，熱信號經下丘腦整合后通過神經和內分泌系統(tǒng)影響采食行為，其可通過合成載脂蛋白A-IV抑制食物攝取[28]。嚙齒動物研究表明，熱應激使小鼠體內載脂蛋白A-IV表達上調，這可能是動物采食量下降的原因之一[29]。并且，奶牛在熱應激狀態(tài)下血液葡萄糖含量顯著上升，刺激胰島素分泌增加[30-31]，與免疫應答和糖異生相關的氨基酸含量升高，并利用非酯化脂肪酸作為供能物質和乳合成的前體物質，提示機體受熱應激脅迫需要更多的氨基酸參與免疫反應和糖異生競爭，最終導致機體處于能量負平衡狀態(tài)[32-34]。

熱應激直接影響奶牛脂質分解和能量代謝。HE等[35]運用代謝組學檢測熱應激奶牛乳樣中代謝物變化，發(fā)現牛奶和血漿中存在乳酸鹽、丙酮酸鹽、肌酸等十幾種差異代謝物，表明奶牛受到熱應激后代謝紊亂，使機體處于能量負平衡狀態(tài)，血乳屏障特異性功能下降。研究表明急性熱應激通過激素敏感性脂肪酶（hormone-sensitive lipase，HSL）和脂滴包被蛋白（perilipin）的蛋白激酶A（protein kinase A，PKA）磷酸化增加細胞對脂肪分解信號的應答[36]。泌乳早期奶牛對熱應激的代謝適應包括增加肌肉過氧化物酶體系中的長鏈脂肪酸降解，允許肌肉葡萄糖利用以及減少肝臟供能，保證產奶能量供應[37]。試驗還表明，熱應激奶牛血清中脂聯素和AMPK活性增加[38]。腺苷酸活化蛋白激酶（adenosine 5′ monophosphate–activated kinase，AMPK）是細胞重要的能量感受器，調節(jié)細胞內代謝平衡。負能量刺激下AMPK使raptor蛋白Ser位點磷酸化受到抑制，導致哺乳動物雷帕霉素靶蛋白復合體1（mammalian target of rapamycin complex 1，mTORC1）活性降低，進而造成細胞基因轉錄及生物合成代謝紊亂[39- 40]，最終影響奶牛乳腺泌乳性能。研究發(fā)現，熱應激期間奶牛干物質采食量的下降僅能解釋產奶量下降的35%—50%[30, 41]，證明還有其他因素對奶牛泌乳性能產生重要影響。

3.2 熱應激影響奶牛內分泌系統(tǒng)

乳腺是乳汁合成的場所，在多種神經內分泌激素和生長因子控制下發(fā)育、分化，合成并分泌乳汁[42]。熱應激條件下奶牛合成代謝相關的激素水平改變，并影響相應信號轉導通路和泌乳相關基因表達，最終影響其泌乳性能[43]。

3.2.1 下丘腦-垂體-腎上腺軸 下丘腦-垂體-腎上腺軸在奶牛受到熱應激時發(fā)揮重要作用。高熱環(huán)境刺激動物下丘腦分泌促腎上腺皮質激素釋放因子（CRF），進而刺激垂體分泌促腎上腺皮質激素（ACTH），促進腎上腺糖皮質激素（主要是皮質醇）合成[44-45]。皮質醇是反芻動物主要應激激素，其分泌增加可有效幫助機體抵御熱應激，是導致血液葡萄糖增加的主要因素[46]。此外，腎上腺髓質分泌腎上腺素（AMH）和去甲腎上腺素（INN）作用于中樞神經系統(tǒng)，引起奶牛興奮性增強、呼吸加快、血壓升高等全身適應性反應，進而使奶牛以犧牲產奶量為代價保證機體內環(huán)境穩(wěn)定[47-48]。

3.2.2 下丘腦-垂體-甲狀腺軸 甲狀腺受熱應激影響主要表現為甲狀腺功能降低以減少機體產熱，甲狀腺激素（TH）合成減少[49-50]。TH介導的細胞信號轉導在調節(jié)體溫、能量攝入和代謝適應方面具有關鍵作用。最新研究發(fā)現，熱應激狀態(tài)下產后奶牛肝臟組織的TH依賴性基因（甲狀腺激素受體α、碘甲狀腺素脫碘酶1、共激活因子PPARGC1）表達降低，蛋白質組學數據顯示肝臟氨基酸的分解代謝減少，轉而用于β-氧化和糖異生[51]。

3.2.3 下丘腦-垂體-性腺軸 熱應激同樣可以影響下丘腦-垂體-性腺軸，調節(jié)促性腺激素釋放激素（GnRH）和促性腺激素（Gn）的合成分泌，進而使促黃體激素（LH）、雌二醇（E2）、孕激素（P4）濃度減少，通過影響乳腺腺泡和導管系統(tǒng)的形成降低泌乳性能[52-55]。P4通過與膜結合型孕激素受體結合，調節(jié)促分裂素原活化蛋白激酶（mitogen-activated protein kinase，MAPK）和蛋白激酶B（protein kinase B，Akt）通路調控泌乳[56]。催乳素（PRL）與催乳素受體結合并激活酪氨酸激酶2（janus kinase 2，JAK2），催化信號轉導和轉錄活化蛋白5（signal transducer and activator of transcription 5，STAT5）磷酸化，活化的STAT5以二聚體的形式進入細胞核調控乳蛋白基因表達[56]。

3.2.4 下丘腦-垂體-生長軸 熱應激也會影響下丘腦-垂體-生長軸。RHOADS等[57]研究發(fā)現熱應激期間奶牛機體減少了胰島素樣生長因子-1（IGF-1）的產生，同時減弱乳腺內對生長激素（GH）敏感的生物能量代謝過程。但熱應激對IGF-1的影響仍存在爭議，李林等[58]研究顯示熱應激奶牛血液中GH、IGF-1含量顯著上升，激活肝臟糖異生作用。GH可刺激IGF-1活化誘導的胰島素受體底物I，經自磷酸化修飾后生成胰島素受體蛋白停泊位點，通過磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）-Akt-哺乳動物雷帕霉素靶點（mammalian target of rapamycin，mTOR）信號通路，從轉錄、翻譯水平調控奶牛泌乳過程[59]。

乳腺內分泌調控奶牛泌乳的詳細機理尚不清楚。進一步研究熱應激對奶牛內分泌的影響，可結合組學方法深入分析下丘腦-垂體-乳腺軸的激素及相關基因表達變化，揭示熱應激影響內分泌進而調控泌乳的分子生物學機制。

3.3 熱應激誘導產生氧化應激

機體氧化物質的存在量超過其抗氧化能力，便會產生氧化應激[60]。奶牛機體內的氧化物和抗氧化酶水平可作為衡量氧化應激程度的標志物。試驗證明，高溫環(huán)境下奶牛血漿中硫代巴比妥酸反應產物（TBARS）濃度升高，細胞內活性氧（ROS）水平、超氧化物歧化酶（SOD）活性、過氧化氫酶活性等指標顯著增加，提示熱應激可誘導產生氧化應激，并觸發(fā)抗氧化酶防御系統(tǒng)[61-62]。

熱應激通過影響機體內ROS水平，進而產生氧化應激。其主要過程是動物體溫上升影響機體內代謝酶活性，加速細胞和組織中的代謝反應以增加ROS產生。過多的自由基攻擊生物大分子反應性Cys殘基使靶蛋白失活，并造成脂質過氧化，最終引起蛋白質和DNA損傷[63-65]。急性熱應激可損傷線粒體膜電位，導致細胞線粒體功能障礙[66]。通過β-氧化或三羧酸循環(huán)等方式氧化線粒體底物，解偶聯蛋白水平下調而電子傳遞鏈活性增加，使超氧化物生成增加，隨后被SOD分解產生過氧化氫[67]。另一方面，熱應激通過增加鐵蛋白釋放鐵的速率導致過多的過渡金屬離子（transition metal ions，TMI）生成，TMI可促進超氧陰離子或過氧化氫生成[68]。過氧化氫以其恒定產生和相對穩(wěn)定的特性作為細胞信號轉導的常見ROS信使，主要通過上調Kelch樣環(huán)氧氯丙烷相關蛋白1（Kelch-like ECH-associated protein-1，Keap1）-核因子2相關因子2（NF-E2-related factor 2，Nrf2）-抗氧化反應元件（antioxidant response element，ARE）信號通路，進而激活細胞內防御信號通路[69]。同時，奶牛脂肪組織蛋白組學結果亦證明熱應激可通過Nrf2介導的氧化應激、核受體FXR/RXR和LXR/RXR等應激相關途徑影響組織蛋白組，并對奶牛的代謝應激有疊加作用[70]。氧化應激可造成細胞功能障礙并發(fā)生病理變化，乳腺組織損傷對奶牛的泌乳性能造成嚴重影響。根據熱應激誘導產生氧化應激機制，溫熱環(huán)境下可通過營養(yǎng)調控的方法增加機體抗氧化水平以減少血漿脂質過氧化，從而改善其應激狀況[71]。例如葛根素可通過抑制ROS產生和上調熱休克蛋白72的表達，明顯改善細胞由熱應激引起的氧化應激損傷[72]。

3.4 熱應激促使乳腺上皮細胞凋亡并誘導自噬

細胞凋亡是經一系列物理、化學或環(huán)境刺激，在分子層面進行調節(jié)而導致細胞自我破壞的過程。高溫可使奶牛乳腺上皮細胞超微結構發(fā)生變化，表現為常染色體聚積邊緣化、片段DNA被細胞膜包裹形成凋亡小體、線粒體腫脹破裂等，說明熱應激誘導的細胞凋亡與線粒體凋亡途徑有直接關聯[73]。利用SW480細胞研究發(fā)現，熱應激可激活細胞中溶酶體-線粒體凋亡途徑，增加細胞內活性氧物質并使溶酶體膜通透性改變，組織蛋白酶B釋放到細胞質中，線粒體去極化并使細胞色素C釋放到胞質溶膠，從而引起細胞凋亡[74]。奶牛乳腺上皮細胞試驗表明，高溫可引起細胞凋亡相關基因、蛋白酶激活因子表達顯著上升[75]。在線粒體凋亡途徑中起重要作用，由細胞色素C與以及組成的凋亡復合體激活[76]。此外，腫瘤壞死因子受體（TNFR）、、抗癌基因細胞凋亡信號因子上調，表明熱應激可通過外源性凋亡途徑誘導細胞凋亡，干擾正常生物活性[75]。信號通路在熱應激誘導的細胞凋亡中起關鍵作用，其涉及線粒體電子傳遞鏈、糖酵解、細胞衰老死亡等多種生物途徑[77-78]。

正常情況下，自噬是保持細胞穩(wěn)態(tài)的關鍵機制。在哺乳動物細胞中，自噬可通過溶酶體降解損傷的蛋白質和細胞器，并利用所得的氨基酸、脂肪酸和糖類，滿足細胞的能量需求[79]。而在病理條件下，細胞饑餓、缺氧、氧化損傷和高熱都會誘發(fā)細胞自噬，過度的自噬會導致細胞損傷[80]。在奶牛研究中，熱應激誘導干奶期奶牛乳腺細胞發(fā)生自噬，干擾了細胞最佳增殖期的乳腺再生，引起泌乳性能降低[81]。最新研究表明，雌二醇和孕激素可與自噬基因beclin 1形成復合物，調節(jié)Bcl-2磷酸化，誘導牛乳腺上皮細胞發(fā)生自噬[82]，提示熱應激造成的奶牛激素紊亂可能促使乳腺細胞發(fā)生自噬。mTOR信號通路的激活可有效抑制牛乳腺上皮細胞產生自噬，是控制自噬的中心環(huán)節(jié)[83]。自噬發(fā)生時，轉化生長因子β1（TGFβ1）可通過Smad蛋白（drosophila mothers against decapentaplegic protein）介導信號轉導降低mTOR上游信號Akt濃度，從而緩解mTOR對自噬的抑制作用[84]。奶牛的泌乳功能與乳腺細胞數量密切相關，熱應激時乳腺細胞發(fā)生程序性死亡（凋亡、自噬）而造成泌乳能力下降。此外，細胞焦亡是一種新的程序性死亡方式，其依賴于并伴有大量促炎因子釋放[85]。有研究表明熱應激通過ROS依賴性高遷移率族蛋白1（high-mobility group box 1，HMGB1）的釋放而誘導大鼠肝臟炎癥小體激活，促使細胞焦亡[86]。熱應激奶牛血漿蛋白質組檢測顯示，轉甲狀腺素蛋白減少而血漿腫瘤壞死因子-α（TNF-α）和白細胞介素-6（IL-6）增加，證明熱應激可誘導奶牛機體產生炎癥反應[87]，但其是否涉及乳腺細胞焦亡還需進一步研究。

4 結語

熱應激一直是奶牛養(yǎng)殖業(yè)面對的重大難題。目前，國內外針對奶牛熱應激問題已有諸多報道，但多集中于飼喂功能性飼料添加劑對熱應激的緩解作用。今后，生產實踐中可通過建立奶牛熱應激模型，明確熱應激對奶牛整體的影響；基礎研究可通過奶牛乳腺上皮細胞熱應激模型結合高通量數據分析技術，系統(tǒng)研究熱應激的發(fā)生機制。緩解熱應激對奶牛泌乳性能的提升具有長遠影響，可為奶牛養(yǎng)殖業(yè)創(chuàng)造巨大的經濟效益。因此，充分研究熱應激對奶牛泌乳的影響機制并積極尋求緩解方法，具有重要的理論和實踐意義。

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（責任編輯 林鑒非）

Effect of Heat Stress on Lactation Performance in Dairy Cows

HAN JiaLiang, LIU JianXin,LIU HongYun

(Institute of Dairy Science, Zhejiang University, Hangzhou 310058)

With the steady aggravation of global warming, dairy cows face increasing heat stress, which results increasing economic loss to the dairy industry. Heat stress occurs when cows are exposed to extreme heat and cannot maintain their core temperature. Under hot temperature environment, dairy cattle is characterized by increased respiration, heart rate, and rectal temperature and decreased feed intake, which have a negative impact on endocrine system and immune system and seriously reduce milk performance of dairy cows. Previous studies of heat stress in dairy cows focused on its effects on productive performance, especially to alleviate the effect of heat stress by functional feed additives, but the specific mechanism of heat stress is not clear. This paper summarizes the widespread impact of heat stress on milk production and quality of dairy cows in different regions, and introduces the known mechanisms of heat stress from aspects of energy metabolism, endocrine, oxidative stress, apoptosis and autophagy. In the aspect of energy metabolism, heat stress induces a negative energy balance in cows by reducing food intake and inducing disorder in lipid breakdown and energy metabolism; In terms of endocrine system, heat stress affects the hypothalamic-pituitary-adrenal axis / thyroid axis / gonadal axis / growth axis of dairy cows and results in hormonal changes. In oxidative stress, heat stress affects the ROS levels in vivo and activates related defense signal pathways. In the aspect of apoptosis and autophagy, heat stress contributes to the damage of mammary epithelial cells by the expression of apoptosis-related genes and by excessive autophagy. The paper put forward that in the foreseeable future, heat stress will be a major challenge for the dairy industry. It is necessary to establish cows heat stress models to comprehensively investigate the basic mechanisms of heat stress in dairy cows using advanced cellular and molecular techniques. In addition, efforts should be placed to develop new interventions to reduce the damage of hear stress to dairy cows.

dairy cow; heat stress; lactation performance

2018-04-10；

2018-07-17

“十三五”國家重點研發(fā)計劃（2016YFD0500503）、國家自然科學基金（31672447）

韓佳良，E-mail：ls-han@foxmail.com。

劉紅云，E-mail：hyliu@zju.edu.cn