热应激对奶牛泌乳性能的影响及其机制

韩佳良，刘建新，刘红云



热应激对奶牛泌乳性能的影响及其机制

韩佳良，刘建新，刘红云

（浙江大学奶业科学研究所，杭州 310058）

随着温室效应加剧，奶牛热应激问题日益凸显，对乳业造成巨大经济损失。热应激是一复杂的生理应答过程，奶牛在温热环境下表现为呼吸和心率加快，直肠温度升高，采食量下降，对其内分泌系统和免疫系统造成负面影响，严重降低奶牛泌乳性能。目前关于奶牛热应激的报道多集中于生产试验，特别是饲喂功能性饲料添加剂对应激的缓解作用，但其具体作用机制尚不清晰。文章介绍了热应激对国内外不同地区奶牛产奶量和乳品质的广泛影响，并从能量代谢、内分泌、氧化应激、细胞凋亡和自噬等方面综述了热应激对奶牛泌乳性能的影响机制。能量代谢方面，从奶牛采食量减少、脂质分解和能量代谢紊乱等内在分子学机理角度解释了奶牛在热应激下处于能量负平衡状态的原因；内分泌方面，介绍了热应激对奶牛下丘脑-垂体-肾上腺轴/甲状腺轴/性腺轴/生长轴的调控，分析了激素变化对机体的影响及其作用机制；氧化应激方面，重点阐述了热应激通过影响机体内ROS水平从而产生氧化应激的分子机制及激活的相关防御信号通路；细胞凋亡和自噬方面，介绍了高温胁迫引起奶牛乳腺上皮细胞损伤，细胞凋亡相关基因表达引发的内源性和外源性细胞凋亡，而过度自噬引发的细胞损伤也对乳腺泌乳起负面调控作用。笔者指出，在可预见的未来，热应激将是奶牛养殖业面临的最大难题，应建立可控的奶牛热应激模型运用于生产实践研究，并加强奶牛乳腺上皮细胞水平的基础研究，结合高通量数据分析技术，系统揭示热应激的发病机制，为缓解热应激提供全面的理论依据。

奶牛；热应激；泌乳性能

近几年，随着温室效应加剧，热应激对奶牛生产性能的影响已成为奶牛养殖业亟需解决的难题。中国奶业发展呈现南北不均态势，奶牛主要养殖在北方地区，而南方却有着巨大的市场需求。许多养殖企业尝试在南方饲养奶牛，以满足其市场空缺。泌乳性能作为奶牛最重要的生产指标，受高温影响显著，所以南方地区普遍存在的高温环境对奶牛泌乳性能的影响是奶农焦虑的主要问题之一。本文从能量代谢、内分泌、氧化应激、细胞凋亡和自噬等方面综述了热应激对奶牛泌乳性能的影响及其机制，并结合组学技术揭示转录、蛋白和代谢水平物质变化，为热应激状态下改善奶牛乳腺机能进而采取保护措施以提高泌乳性能提供理论依据。

1 热应激

热应激是动物机体应对环境高温所产生的非特异性应答反应。国外学者采用热应激表示动物机体应对高温环境产生的散热需求[1]。国内学者则认为热应激是所有与高温有关的因素引起的有利于避免动物机体功能紊乱并使其更好地适应环境而发生在动物细胞水平的自我调节[2]。奶牛热应激的影响程度一般采用温湿指数（temperature-humidity index, THI）进行估计[3]，但由于不同奶牛年龄、品种、生理状态等因素不同，单纯依靠THI很难准确评估奶牛热应激状态。因此，还可结合热休克蛋白表达水平、生理生化、生产性能、内分泌等指标评定奶牛热应激状态。随着养殖自动化程度提高，可运用红外线热像仪和加速度传感器监测奶牛呼吸频率和往返踱步行为，实现远程监控奶牛健康状况[4]，也可通过监测奶牛活动和反刍时间对热应激进行早期预警[5]。

热应激可致使奶牛行为异常，生产和繁殖性能下降，发生疾病甚至导致奶牛死亡[6-7]。国内外研究人员尝试运用多种方法缓解奶牛热应激，例如在干奶期饲喂营养免疫调节剂可缓解奶牛热应激并提高其泌乳性能[8]；近期研究关注开发包含烟酸、维生素C、硫酸钾、γ-氨基丁酸等抗应激物质的瘤胃保护胶囊，从而在营养调控层面缓解奶牛热应激[9]。饲喂高能量混合日粮以保证热应激奶牛所需能量，但会增加奶牛瘤胃酸中毒风险；目前奶牛养殖场多采用较为经济实用的喷雾风扇系统进行降温，但造成环境湿度过大，使奶牛多发肢蹄病和乳房炎[10]。最新研究利用基因组育种值预测奶牛的热耐受性，选育牛产奶量下降较少，因此用基因组选育耐热性奶牛不失为一种抗热应激手段，在未来对动物福利具有深远影响[11]。

2 热应激对奶牛泌乳性能的影响

2.1 热应激对产奶量的影响

产奶量是奶牛泌乳性能最基本的指标，其高低直接影响奶牛养殖场的经济效益。不同地区的高热环境均会降低奶牛产奶量，但由于热应激程度、持续时间、奶牛品种、饲养条件等不同，产奶量降低的程度也不同。当30＜THI＜60时，德国荷斯坦奶牛处于正常状态，奶产量随THI的升高而升高；当THI＞60时，奶牛受到轻度热应激，奶产量下降，并随THI的升高而逐渐降低[12]。短期中度热应激下，加拿大和美国北部地区奶牛日均奶产量下降4.8%，并对恢复阶段存在长期的负面影响[13]。当THI＞83时，巴西南部荷斯坦奶牛受到强烈热应激，与对照组相比日均奶产量降低21%[14]。国内研究中，李朝明等[15]试验表明热应激条件下荷斯坦、娟珊和娟荷杂交奶牛产奶量均下降，其中娟荷杂交奶牛产奶量优于其他组，表现出较好的耐热性能。可见不同地区或品种的奶牛对热应激应答程度不同。根据我国区域气候条件，选育适应高温环境的奶牛品种并深入研究南方地区奶牛热应激阈值显得尤为必要。

2.2 热应激对乳品质的影响

乳蛋白率、乳脂率、乳糖率和非脂固体含量等是奶牛乳品质的重要指标。乳蛋白和乳脂肪是构成牛奶的重要营养物质，是市场竞争的核心因素。许多研究表明发生热应激时，各泌乳阶段奶牛的乳蛋白率、乳脂率、非脂固体含量均有下降趋势，且在泌乳前期降低幅度最大[12-13, 16-17]，但对牛奶中乳糖率没有显著影响[18- 19]。热应激对乳蛋白的影响主要表现为乳腺上皮细胞酪蛋白等主要乳蛋白基因表达下调，乳中总酪蛋白减少而尿素浓度增加，导致营养价值降低[20-21]。最新研究表明，热应激奶牛乳中短链和中链脂肪酸含量，磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰胆碱、溶血磷脂酰胆碱和葡萄糖神经酰胺等5种极性脂质显著降低[22]。极性脂质是构成脂肪球膜的主要成分，起到乳化剂的作用，可确保乳液体系的稳定性[23]。此外，体细胞数是衡量原料乳质量的一个重要指标，热应激条件下奶牛乳中体细胞数显著增加，严重影响乳品质[24]。但由于奶牛乳腺泌乳代谢受诸多因素调控，其泌乳参数不尽相同，大大增加了研究难度。只有深入了解热应激对奶牛泌乳性能的影响机制，才能合理调控泌乳进而达到改善乳品质的目的。

3 热应激影响奶牛泌乳性能的机制

3.1 热应激影响奶牛能量代谢

机体在生命活动过程中，始终保持能量的动态平衡。奶牛受到热应激时表现为干物质采食量减少以最大限度减少热总负荷，并加快激活散热机制（出汗、血液流向皮肤）引起连锁生理调节使维持能量增加，用于生产泌乳的能量减少[25-27]。下丘脑是动物体温和摄食调节中枢，热信号经下丘脑整合后通过神经和内分泌系统影响采食行为，其可通过合成载脂蛋白A-IV抑制食物摄取[28]。啮齿动物研究表明，热应激使小鼠体内载脂蛋白A-IV表达上调，这可能是动物采食量下降的原因之一[29]。并且，奶牛在热应激状态下血液葡萄糖含量显著上升，刺激胰岛素分泌增加[30-31]，与免疫应答和糖异生相关的氨基酸含量升高，并利用非酯化脂肪酸作为供能物质和乳合成的前体物质，提示机体受热应激胁迫需要更多的氨基酸参与免疫反应和糖异生竞争，最终导致机体处于能量负平衡状态[32-34]。

热应激直接影响奶牛脂质分解和能量代谢。HE等[35]运用代谢组学检测热应激奶牛乳样中代谢物变化，发现牛奶和血浆中存在乳酸盐、丙酮酸盐、肌酸等十几种差异代谢物，表明奶牛受到热应激后代谢紊乱，使机体处于能量负平衡状态，血乳屏障特异性功能下降。研究表明急性热应激通过激素敏感性脂肪酶（hormone-sensitive lipase，HSL）和脂滴包被蛋白（perilipin）的蛋白激酶A（protein kinase A，PKA）磷酸化增加细胞对脂肪分解信号的应答[36]。泌乳早期奶牛对热应激的代谢适应包括增加肌肉过氧化物酶体系中的长链脂肪酸降解，允许肌肉葡萄糖利用以及减少肝脏供能，保证产奶能量供应[37]。试验还表明，热应激奶牛血清中脂联素和AMPK活性增加[38]。腺苷酸活化蛋白激酶（adenosine 5′ monophosphate–activated kinase，AMPK）是细胞重要的能量感受器，调节细胞内代谢平衡。负能量刺激下AMPK使raptor蛋白Ser位点磷酸化受到抑制，导致哺乳动物雷帕霉素靶蛋白复合体1（mammalian target of rapamycin complex 1，mTORC1）活性降低，进而造成细胞基因转录及生物合成代谢紊乱[39- 40]，最终影响奶牛乳腺泌乳性能。研究发现，热应激期间奶牛干物质采食量的下降仅能解释产奶量下降的35%—50%[30, 41]，证明还有其他因素对奶牛泌乳性能产生重要影响。

3.2 热应激影响奶牛内分泌系统

乳腺是乳汁合成的场所，在多种神经内分泌激素和生长因子控制下发育、分化，合成并分泌乳汁[42]。热应激条件下奶牛合成代谢相关的激素水平改变，并影响相应信号转导通路和泌乳相关基因表达，最终影响其泌乳性能[43]。

3.2.1 下丘脑-垂体-肾上腺轴 下丘脑-垂体-肾上腺轴在奶牛受到热应激时发挥重要作用。高热环境刺激动物下丘脑分泌促肾上腺皮质激素释放因子（CRF），进而刺激垂体分泌促肾上腺皮质激素（ACTH），促进肾上腺糖皮质激素（主要是皮质醇）合成[44-45]。皮质醇是反刍动物主要应激激素，其分泌增加可有效帮助机体抵御热应激，是导致血液葡萄糖增加的主要因素[46]。此外，肾上腺髓质分泌肾上腺素（AMH）和去甲肾上腺素（INN）作用于中枢神经系统，引起奶牛兴奋性增强、呼吸加快、血压升高等全身适应性反应，进而使奶牛以牺牲产奶量为代价保证机体内环境稳定[47-48]。

3.2.2 下丘脑-垂体-甲状腺轴 甲状腺受热应激影响主要表现为甲状腺功能降低以减少机体产热，甲状腺激素（TH）合成减少[49-50]。TH介导的细胞信号转导在调节体温、能量摄入和代谢适应方面具有关键作用。最新研究发现，热应激状态下产后奶牛肝脏组织的TH依赖性基因（甲状腺激素受体α、碘甲状腺素脱碘酶1、共激活因子PPARGC1）表达降低，蛋白质组学数据显示肝脏氨基酸的分解代谢减少，转而用于β-氧化和糖异生[51]。

3.2.3 下丘脑-垂体-性腺轴 热应激同样可以影响下丘脑-垂体-性腺轴，调节促性腺激素释放激素（GnRH）和促性腺激素（Gn）的合成分泌，进而使促黄体激素（LH）、雌二醇（E2）、孕激素（P4）浓度减少，通过影响乳腺腺泡和导管系统的形成降低泌乳性能[52-55]。P4通过与膜结合型孕激素受体结合，调节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）通路调控泌乳[56]。催乳素（PRL）与催乳素受体结合并激活酪氨酸激酶2（janus kinase 2，JAK2），催化信号转导和转录活化蛋白5（signal transducer and activator of transcription 5，STAT5）磷酸化，活化的STAT5以二聚体的形式进入细胞核调控乳蛋白基因表达[56]。

3.2.4 下丘脑-垂体-生长轴 热应激也会影响下丘脑-垂体-生长轴。RHOADS等[57]研究发现热应激期间奶牛机体减少了胰岛素样生长因子-1（IGF-1）的产生，同时减弱乳腺内对生长激素（GH）敏感的生物能量代谢过程。但热应激对IGF-1的影响仍存在争议，李林等[58]研究显示热应激奶牛血液中GH、IGF-1含量显著上升，激活肝脏糖异生作用。GH可刺激IGF-1活化诱导的胰岛素受体底物I，经自磷酸化修饰后生成胰岛素受体蛋白停泊位点，通过磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）-Akt-哺乳动物雷帕霉素靶点（mammalian target of rapamycin，mTOR）信号通路，从转录、翻译水平调控奶牛泌乳过程[59]。

乳腺内分泌调控奶牛泌乳的详细机理尚不清楚。进一步研究热应激对奶牛内分泌的影响，可结合组学方法深入分析下丘脑-垂体-乳腺轴的激素及相关基因表达变化，揭示热应激影响内分泌进而调控泌乳的分子生物学机制。

3.3 热应激诱导产生氧化应激

机体氧化物质的存在量超过其抗氧化能力，便会产生氧化应激[60]。奶牛机体内的氧化物和抗氧化酶水平可作为衡量氧化应激程度的标志物。试验证明，高温环境下奶牛血浆中硫代巴比妥酸反应产物（TBARS）浓度升高，细胞内活性氧（ROS）水平、超氧化物歧化酶（SOD）活性、过氧化氢酶活性等指标显著增加，提示热应激可诱导产生氧化应激，并触发抗氧化酶防御系统[61-62]。

热应激通过影响机体内ROS水平，进而产生氧化应激。其主要过程是动物体温上升影响机体内代谢酶活性，加速细胞和组织中的代谢反应以增加ROS产生。过多的自由基攻击生物大分子反应性Cys残基使靶蛋白失活，并造成脂质过氧化，最终引起蛋白质和DNA损伤[63-65]。急性热应激可损伤线粒体膜电位，导致细胞线粒体功能障碍[66]。通过β-氧化或三羧酸循环等方式氧化线粒体底物，解偶联蛋白水平下调而电子传递链活性增加，使超氧化物生成增加，随后被SOD分解产生过氧化氢[67]。另一方面，热应激通过增加铁蛋白释放铁的速率导致过多的过渡金属离子（transition metal ions，TMI）生成，TMI可促进超氧阴离子或过氧化氢生成[68]。过氧化氢以其恒定产生和相对稳定的特性作为细胞信号转导的常见ROS信使，主要通过上调Kelch样环氧氯丙烷相关蛋白1（Kelch-like ECH-associated protein-1，Keap1）-核因子2相关因子2（NF-E2-related factor 2，Nrf2）-抗氧化反应元件（antioxidant response element，ARE）信号通路，进而激活细胞内防御信号通路[69]。同时，奶牛脂肪组织蛋白组学结果亦证明热应激可通过Nrf2介导的氧化应激、核受体FXR/RXR和LXR/RXR等应激相关途径影响组织蛋白组，并对奶牛的代谢应激有叠加作用[70]。氧化应激可造成细胞功能障碍并发生病理变化，乳腺组织损伤对奶牛的泌乳性能造成严重影响。根据热应激诱导产生氧化应激机制，温热环境下可通过营养调控的方法增加机体抗氧化水平以减少血浆脂质过氧化，从而改善其应激状况[71]。例如葛根素可通过抑制ROS产生和上调热休克蛋白72的表达，明显改善细胞由热应激引起的氧化应激损伤[72]。

3.4 热应激促使乳腺上皮细胞凋亡并诱导自噬

细胞凋亡是经一系列物理、化学或环境刺激，在分子层面进行调节而导致细胞自我破坏的过程。高温可使奶牛乳腺上皮细胞超微结构发生变化，表现为常染色体聚积边缘化、片段DNA被细胞膜包裹形成凋亡小体、线粒体肿胀破裂等，说明热应激诱导的细胞凋亡与线粒体凋亡途径有直接关联[73]。利用SW480细胞研究发现，热应激可激活细胞中溶酶体-线粒体凋亡途径，增加细胞内活性氧物质并使溶酶体膜通透性改变，组织蛋白酶B释放到细胞质中，线粒体去极化并使细胞色素C释放到胞质溶胶，从而引起细胞凋亡[74]。奶牛乳腺上皮细胞试验表明，高温可引起细胞凋亡相关基因、蛋白酶激活因子表达显著上升[75]。在线粒体凋亡途径中起重要作用，由细胞色素C与以及组成的凋亡复合体激活[76]。此外，肿瘤坏死因子受体（TNFR）、、抗癌基因细胞凋亡信号因子上调，表明热应激可通过外源性凋亡途径诱导细胞凋亡，干扰正常生物活性[75]。信号通路在热应激诱导的细胞凋亡中起关键作用，其涉及线粒体电子传递链、糖酵解、细胞衰老死亡等多种生物途径[77-78]。

正常情况下，自噬是保持细胞稳态的关键机制。在哺乳动物细胞中，自噬可通过溶酶体降解损伤的蛋白质和细胞器，并利用所得的氨基酸、脂肪酸和糖类，满足细胞的能量需求[79]。而在病理条件下，细胞饥饿、缺氧、氧化损伤和高热都会诱发细胞自噬，过度的自噬会导致细胞损伤[80]。在奶牛研究中，热应激诱导干奶期奶牛乳腺细胞发生自噬，干扰了细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，雌二醇和孕激素可与自噬基因beclin 1形成复合物，调节Bcl-2磷酸化，诱导牛乳腺上皮细胞发生自噬[82]，提示热应激造成的奶牛激素紊乱可能促使乳腺细胞发生自噬。mTOR信号通路的激活可有效抑制牛乳腺上皮细胞产生自噬，是控制自噬的中心环节[83]。自噬发生时，转化生长因子β1（TGFβ1）可通过Smad蛋白（drosophila mothers against decapentaplegic protein）介导信号转导降低mTOR上游信号Akt浓度，从而缓解mTOR对自噬的抑制作用[84]。奶牛的泌乳功能与乳腺细胞数量密切相关，热应激时乳腺细胞发生程序性死亡（凋亡、自噬）而造成泌乳能力下降。此外，细胞焦亡是一种新的程序性死亡方式，其依赖于并伴有大量促炎因子释放[85]。有研究表明热应激通过ROS依赖性高迁移率族蛋白1（high-mobility group box 1，HMGB1）的释放而诱导大鼠肝脏炎症小体激活，促使细胞焦亡[86]。热应激奶牛血浆蛋白质组检测显示，转甲状腺素蛋白减少而血浆肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）增加，证明热应激可诱导奶牛机体产生炎症反应[87]，但其是否涉及乳腺细胞焦亡还需进一步研究。

4 结语

热应激一直是奶牛养殖业面对的重大难题。目前，国内外针对奶牛热应激问题已有诸多报道，但多集中于饲喂功能性饲料添加剂对热应激的缓解作用。今后，生产实践中可通过建立奶牛热应激模型，明确热应激对奶牛整体的影响；基础研究可通过奶牛乳腺上皮细胞热应激模型结合高通量数据分析技术，系统研究热应激的发生机制。缓解热应激对奶牛泌乳性能的提升具有长远影响，可为奶牛养殖业创造巨大的经济效益。因此，充分研究热应激对奶牛泌乳的影响机制并积极寻求缓解方法，具有重要的理论和实践意义。

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（责任编辑 林鉴非）

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”国家重点研发计划（2016YFD0500503）、国家自然科学基金（31672447）

韩佳良，E-mail：ls-han@foxmail.com。

刘红云，E-mail：hyliu@zju.edu.cn

10.3864/j.issn.0578-1752.2018.16.012