反芻動(dòng)物亞急性瘤胃酸中毒變異性的機(jī)制解析

閆佰鵬 蘭貴生 李發(fā)弟,2 李 飛*

(1.蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院，草地農(nóng)業(yè)生態(tài)系統(tǒng)國(guó)家重點(diǎn)實(shí)驗(yàn)室，蘭州 730030；2.甘肅省肉羊繁育生物技術(shù)工程實(shí)驗(yàn)室，民勤 733300)

目前，高能、高淀粉飼糧已成為奶牛泌乳階段或肉用反芻動(dòng)物育肥階段典型的營(yíng)養(yǎng)特征[1-3]，這一營(yíng)養(yǎng)策略在提高動(dòng)物生產(chǎn)性能的同時(shí)，也易引起瘤胃穩(wěn)態(tài)失衡，導(dǎo)致以亞急性瘤胃酸中毒(SARA)為主的一系列瘤胃健康問題。Malekkhahi等[4]研究表明，奶牛采食高谷物飼糧所誘發(fā)的SARA對(duì)干物質(zhì)采食量、產(chǎn)奶量、瘤胃微生物區(qū)系、血液代謝物等具有嚴(yán)重負(fù)面影響，其對(duì)動(dòng)物健康及生產(chǎn)性能的危害是現(xiàn)代反芻動(dòng)物生產(chǎn)面臨的突出問題[5-6]。諸多研究發(fā)現(xiàn)，反芻動(dòng)物過量采食高能、高淀粉飼糧時(shí)[7]，可導(dǎo)致瘤胃液pH長(zhǎng)時(shí)間低于正常生理范圍，這是引起SARA的主要發(fā)病途徑[8]和營(yíng)養(yǎng)因素[9-10]。研究表明，盡管飼養(yǎng)管理?xiàng)l件一致，飼喂相同飼糧的奶牛[11]、肉牛[12]及綿羊[13]，其個(gè)體間瘤胃液pH、發(fā)生SARA的風(fēng)險(xiǎn)存在顯著差異，這種差異使得反芻動(dòng)物生產(chǎn)管理和SARA的預(yù)防工作存在一定難度[11]，其主要影響反芻動(dòng)物營(yíng)養(yǎng)供應(yīng)策略、精準(zhǔn)飼養(yǎng)實(shí)現(xiàn)、分群飼養(yǎng)依據(jù)和群體瘤胃健康程度的評(píng)價(jià)等。個(gè)體間SARA的變異，尤其是反芻動(dòng)物本身的生理原因卻鮮有報(bào)道，包括營(yíng)養(yǎng)和生理途徑導(dǎo)致個(gè)體SARA易感性的變異、瘤胃微生物多樣性的改變等差異尚不清楚，因此，研究“反芻動(dòng)物個(gè)體間SARA變異性來源及機(jī)制”是重要的科學(xué)問題，本文從采食行為、瘤胃上皮吸收功能以及微生物區(qū)系3個(gè)方面對(duì)影響反芻動(dòng)物SARA易感性潛在營(yíng)養(yǎng)生理因素進(jìn)行綜述，旨在為相關(guān)研究提供科學(xué)依據(jù)。

1 SARA易感性識(shí)別的必要性與變異性

1.1 必要性

分階段、分群飼養(yǎng)是反芻動(dòng)物生產(chǎn)中實(shí)現(xiàn)精準(zhǔn)飼養(yǎng)的主要營(yíng)養(yǎng)管理手段[14]，該方式基于動(dòng)物群體在特定預(yù)期生產(chǎn)潛力或生產(chǎn)階段的營(yíng)養(yǎng)需要具有相似性，但實(shí)質(zhì)上此營(yíng)養(yǎng)供給方式并未充分考慮高產(chǎn)群體中個(gè)體間SARA耐受性的差異?？茖W(xué)研究中，由于試驗(yàn)動(dòng)物個(gè)體在遺傳及生活史等方面存在不同程度的異質(zhì)性，這種異質(zhì)性是導(dǎo)致試驗(yàn)結(jié)果產(chǎn)生變異的重要來源[15]，生物統(tǒng)計(jì)角度認(rèn)為這種變異稱為隨機(jī)誤差，盡管增加動(dòng)物數(shù)量、嚴(yán)格篩選試驗(yàn)動(dòng)物等方法可減小該誤差，但反芻動(dòng)物實(shí)際生產(chǎn)過程中個(gè)體間SARA易感性變異卻普遍存在，因此合理的飼養(yǎng)管理在集約化、規(guī)模化生產(chǎn)中至關(guān)重要。目前，基于隨機(jī)抽樣的瘤胃穿刺技術(shù)已應(yīng)用于群體SARA風(fēng)險(xiǎn)的評(píng)估[5,16-17]，但該方法并不能有效檢測(cè)出個(gè)體SARA易感性的差異，這意味著即使對(duì)高SARA風(fēng)險(xiǎn)的牛群改變飼糧策略(降低易發(fā)酵碳水化合物含量或增加粗飼料比例)以此降低群體SARA的風(fēng)險(xiǎn)，但同樣會(huì)損失部分SARA耐受性個(gè)體發(fā)揮其最大生產(chǎn)性能，因此降低或消除個(gè)體SARA易感性差異是保證群體高效和健康生產(chǎn)的關(guān)鍵，未來反芻動(dòng)物生產(chǎn)中可以考慮根據(jù)個(gè)體SARA耐受性的差異作為分群飼養(yǎng)的依據(jù)之一，結(jié)合特定生產(chǎn)階段和預(yù)期生產(chǎn)潛力施加針對(duì)性的營(yíng)養(yǎng)策略，保障動(dòng)物瘤胃健康及高效生產(chǎn)，完善精準(zhǔn)飼養(yǎng)手段。

1.2 變異性

生產(chǎn)實(shí)踐中，多種營(yíng)養(yǎng)調(diào)控與飼養(yǎng)管理手段被用于預(yù)防SARA，如保證飼糧物理有效中性洗滌纖維(peNDF)與淀粉比例、分群飼養(yǎng)[18-19]、降低熱應(yīng)激[20]、增加飼喂頻率[21]等。研究發(fā)現(xiàn)，牛羊采食相同飼糧時(shí)，其個(gè)體間發(fā)生SARA風(fēng)險(xiǎn)存在較大差異[11,13]。Macmillan等[21]研究發(fā)現(xiàn)，泌乳期荷斯坦奶牛(n=16)飼喂精粗比為70∶30飼糧后，其中7頭奶牛出現(xiàn)SARA癥狀(瘤胃液pH<5.8為535 min/d vs. 18.4 min/d)。Castillo-Lopez等[22]以肉牛為研究對(duì)象，表明育肥后期飼喂高谷物飼糧(含大麥谷物81.2%)肉牛(n=28)中平均有37.8%的個(gè)體發(fā)生SARA(瘤胃液pH<5.5超過180 min/d)，且個(gè)體間SARA發(fā)生概率在0～96.9%。Nasrollahi等[11]同樣研究發(fā)現(xiàn)，泌乳期奶牛(n=78)飼喂精粗比為65∶35飼糧后，31頭奶牛出現(xiàn)SARA癥狀(瘤胃液pH<5.8超過330 min/d)；類似結(jié)果在奶山羊上也有報(bào)道[23-24]。此外，泌乳奶牛分娩后個(gè)體間瘤胃酸中毒嚴(yán)重程度存在極大變異，且這種變異與采食量、飼糧組成、瘤胃揮發(fā)性脂肪酸(VFA)濃度及比例無關(guān)[25]。以上研究證明，相同飼糧條件下，動(dòng)物個(gè)體間SARA風(fēng)險(xiǎn)存在明顯的差異，且這種差異可能與營(yíng)養(yǎng)素的供給無關(guān)，受動(dòng)物自身生理因素的調(diào)控。

2 影響SARA易感性的生理因素

2.1 采食行為

瘤胃緩沖鹽主要源于動(dòng)物咀嚼產(chǎn)生的唾液、瘤胃上皮分泌的碳酸氫鹽及飼糧在瘤胃降解產(chǎn)生的氨[26]，其對(duì)穩(wěn)定瘤胃液pH、微生物生長(zhǎng)與養(yǎng)分消化有重要意義。其中唾液是瘤胃緩沖鹽的主要來源，對(duì)于高產(chǎn)奶牛唾液中的磷酸鹽和碳酸氫鹽緩沖系統(tǒng)可中和瘤胃中約37%的氫離子[27]，而反芻動(dòng)物唾液分泌量主要取決于其咀嚼時(shí)間(采食時(shí)間與反芻時(shí)間之和)。研究表明，反芻動(dòng)物的采食行為包括采食速度、反芻活動(dòng)、挑食程度等方面存在較大的個(gè)體間變異[28-29]。Giger-Reverdin等[24]報(bào)道表明，奶山羊采食精粗比為60∶40的飼糧，其平均咀嚼指數(shù)(咀嚼時(shí)間/干物質(zhì)采食量)為4.05 h/kg，但個(gè)體間咀嚼指數(shù)在1.83～6.30 h/kg變化。同時(shí)對(duì)奶山羊(n=12)飼喂精粗比為65∶35的飼糧，發(fā)現(xiàn)采食速度快的個(gè)體飼喂13 h后其瘤胃液pH在5.25～6.25波動(dòng)，采食速度慢的個(gè)體瘤胃液pH在6.25～6.50變化，表明盡管飼糧組成相同，由于個(gè)體采食速度過快，導(dǎo)致其SARA風(fēng)險(xiǎn)的提高[30]。挑食行為是反芻動(dòng)物飼養(yǎng)過程中不可避免的問題之一，長(zhǎng)期的挑食行為使得反芻動(dòng)物營(yíng)養(yǎng)攝入不均衡、消化代謝紊亂，導(dǎo)致動(dòng)物發(fā)生腹瀉、蹄葉炎和肝膿腫等營(yíng)養(yǎng)代謝疾病[31]，對(duì)畜牧業(yè)發(fā)展造成巨大損失。因此，一些研究通過改變飼糧結(jié)構(gòu)、飼喂策略等措施降低或消除其挑食行為[32]。Muhammad等[33]研究發(fā)現(xiàn)，斷奶犢牛(n=28)飼喂長(zhǎng)、短粒徑苜蓿干草后，犢牛對(duì)其長(zhǎng)粒徑飼糧的挑食行為顯著高于短粒徑飼糧；Macmillan等[21]以哺乳期荷斯坦奶牛(n=8)為研究對(duì)象發(fā)現(xiàn)，飼喂3次/d相對(duì)于飼喂1次/d，在降低挑食行為方面前者優(yōu)于后者，并且降低了高產(chǎn)奶牛患SARA的風(fēng)險(xiǎn)。上述研究表明，動(dòng)物采食相同飼糧其個(gè)體間采食行為存在較大變異，由于采食行為影響唾液分泌量(咀嚼時(shí)間)、進(jìn)入瘤胃營(yíng)養(yǎng)素的速度(采食速度)及組成(挑食行為)，這些行為差異均會(huì)影響瘤胃液pH(圖1)[34]，采食行為可能是導(dǎo)致動(dòng)物個(gè)體間SARA易感性差異的原因之一，但準(zhǔn)確結(jié)論還有待進(jìn)一步研究。

2.2 瘤胃上皮轉(zhuǎn)運(yùn)功能

瘤胃液pH波動(dòng)是瘤胃內(nèi)有機(jī)酸(VFA和乳酸)的產(chǎn)生、吸收、外流及緩沖鹽中和等因素共同作用的結(jié)果(圖1)，其中VFA移除的主要途徑是經(jīng)瘤胃上皮吸收，可占總VFA產(chǎn)量的50%～85%[26]。反芻動(dòng)物在適應(yīng)高谷物飼糧過程中其瘤胃上皮形態(tài)、瘤胃上皮生理功能(細(xì)胞增殖分化、吸收轉(zhuǎn)運(yùn)功能、代謝功能等[35])及電生理特性均發(fā)生改變，表現(xiàn)為基底層、棘層、顆粒層厚度降低[36-37]，上皮通透性提高[38]，內(nèi)毒性物質(zhì)增加等現(xiàn)象[39]。Penner等[40]發(fā)現(xiàn)，隨飼糧精料水平提高，奶牛瘤胃上皮對(duì)丙酸和丁酸的吸收速率分別提高65.7%(0.35 mol/h vs. 0.58 mol/h)和77.8%(0.18 mol/h vs. 0.32 mol/h)；同時(shí)，體外和體內(nèi)研究也證明，VFA濃度提高及pH下降會(huì)影響瘤胃上皮細(xì)胞VFA吸收[41-42]、細(xì)胞內(nèi)pH調(diào)節(jié)與質(zhì)子轉(zhuǎn)運(yùn)相關(guān)基因表達(dá)水平[43-44]，如Yan等[42]報(bào)道，適當(dāng)增加飼糧精料(10% vs. 35%)可提高山羊瘤胃上皮細(xì)胞VFA轉(zhuǎn)運(yùn)與胞內(nèi)pH調(diào)節(jié)相關(guān)基因表達(dá)，包括一元羧酸轉(zhuǎn)運(yùn)載體(MCT)(MCT1和MCT4)、陰離子轉(zhuǎn)運(yùn)載體[DRA、PAT1及陰離子交換蛋白2(AE2)]及Na+/H+交換載體(NHE)(NHE1、NHE2及NHE3)，促進(jìn)VFA吸收；與該結(jié)果類似，劉軍花等[45]發(fā)現(xiàn)高谷物飼糧(65∶35)顯著升高了MCT1、Na+/K+-ATP酶的表達(dá)量，同時(shí)顯著降低了MCT4的表達(dá)量。以上結(jié)果表明，瘤胃VFA吸收能力通過瘤胃上皮轉(zhuǎn)運(yùn)載體調(diào)控，隨著飼糧精料比例增加，瘤胃上皮基因NHE1、NHE2、NHE3和Na+/K+-ATP酶等的表達(dá)量增加，有可能導(dǎo)致VFA的吸收能力增強(qiáng)[46]。與之結(jié)果類似，Penner等[13]通過一次性灌注葡萄糖誘導(dǎo)綿羊SARA發(fā)現(xiàn)，相對(duì)于SARA易感(acidosis-susceptible,AS)組，SARA耐受(acidosis-resistant,AR)組綿羊瘤胃上皮對(duì)乙酸和丁酸的吸收更迅速，加速酸從瘤胃中的移除，提高瘤胃液pH；同時(shí)，Schlau等[12]針對(duì)肉牛的研究表明，AR組pH與AS組瘤胃液pH(6.05 vs. 5.59)的差異主要與瘤胃液VFA濃度有關(guān)(122 mmol/L vs. 164 mmol/L)，但該研究發(fā)現(xiàn)AR組瘤胃上皮細(xì)胞NHE3表達(dá)量顯著提高，而其他VFA吸收相關(guān)基因(MCT1和DRA)表達(dá)量無顯著差異。上述結(jié)果提示，反芻動(dòng)物應(yīng)對(duì)高精料飼糧過程中個(gè)體間瘤胃上皮轉(zhuǎn)運(yùn)載體基因的表達(dá)量存在差異，而瘤胃上皮轉(zhuǎn)運(yùn)載體基因的表達(dá)量同時(shí)受瘤胃液pH、VFA組成與濃度調(diào)控，瘤胃上皮轉(zhuǎn)運(yùn)基因表達(dá)量差異使得相同處理不同個(gè)體瘤胃上皮對(duì)VFA吸收能力存在較大變異，這種差異可能是導(dǎo)致動(dòng)物SARA易感性不同的潛在生理因素。未來應(yīng)針對(duì)個(gè)體瘤胃液pH、有機(jī)酸組成與瘤胃上皮轉(zhuǎn)運(yùn)載體三者結(jié)合，系統(tǒng)評(píng)估SARA變異的潛在機(jī)制。

圖1 采食行為和瘤胃上皮功能作用瘤胃液pH的主要途徑

3 瘤胃微生物及代謝物與SARA易感性

眾所周知，高精料飼糧誘導(dǎo)的SARA可導(dǎo)致瘤胃微生物區(qū)系的改變，對(duì)其進(jìn)行焦磷酸測(cè)序結(jié)果發(fā)現(xiàn)，SARA易感組瘤胃菌群豐度指數(shù)(Chao1和Ace指數(shù))和多樣性指數(shù)(Shannon指數(shù))顯著低于耐受組[47]，其在細(xì)菌水平上表現(xiàn)為淀粉利用菌、產(chǎn)乳酸菌和乳酸利用菌數(shù)量升高，而纖維分解菌數(shù)量下降[48-50]，瘤胃原蟲水平主要表現(xiàn)為原蟲數(shù)量的降低[51]。由于不同SARA易感性個(gè)體其瘤胃液pH環(huán)境存在差異，而pH直接影響瘤胃微生物的生長(zhǎng)與增殖，故推測(cè)反芻動(dòng)物SARA易感性變異會(huì)引起瘤胃微生物區(qū)系及發(fā)酵產(chǎn)物的差異?；赑CR-變性梯度凝膠電泳(DGGE)技術(shù)的研究證明，肉牛中AR(瘤胃液平均pH=6.02)與AS(瘤胃液平均pH=5.55)個(gè)體在瘤胃微生物多樣性方面存在明顯差異，且AS個(gè)體瘤胃更傾向于丙酸發(fā)酵型[52]；據(jù)張瑞陽(yáng)[47]研究發(fā)現(xiàn)高谷物誘導(dǎo)的SARA中，在門水平上，SARA組瘤胃擬桿菌門(Bacteroidetes)和蛋白菌門(Proteobacteria)的相對(duì)豐度顯著降低，硬壁菌門(Firmicutes)和放線菌門(Actinobacteria)的相對(duì)豐度顯著增加。在屬水平上，SARA組奶牛瘤胃中普氏菌(Prevotella)、密螺旋體屬(Treponema)、厭氧支原體屬(Anaeroplasma)、不動(dòng)桿菌屬(Acinetobacter)、帕匹桿菌屬(Papillibacter)的相對(duì)豐度顯著降低，而瘤胃球菌屬(Ruminococcus)、奇異菌屬(Atopobium)、雙歧桿菌(Bifidobacteria)、未分類的梭菌屬(unclassifiedClostridiales)的相對(duì)豐度顯著升高。關(guān)于瘤胃代謝產(chǎn)物與SARA易感性的關(guān)系尚不明確，但基于代謝組學(xué)技術(shù)研究發(fā)現(xiàn)，奶牛飼喂同一種高精料飼糧(精粗比45∶55)個(gè)體間部分瘤胃代謝產(chǎn)物乙酰乙酸、麥芽糖、甲酸等濃度差異在2～4倍[53]，表明采食相同飼糧條件下奶牛瘤胃代謝產(chǎn)物也存在差異。目前，宏基因組學(xué)(metagenomics)和代謝組學(xué)(metabolomics)技術(shù)已被廣泛應(yīng)用于挖掘瘤胃微生物信息，包括物種多樣性、種群結(jié)構(gòu)、代謝規(guī)律及功能活性等方面。Mao等[50]以山羊?yàn)檠芯磕Ｐ?，采?54焦磷酸測(cè)序技術(shù)及代謝組學(xué)方法揭示了SARA瘤胃微生物多樣性與瘤胃代謝組的關(guān)系，但不同SARA易感性是否引起瘤胃微生物在屬、種水平的變化尚不清楚，有必要從微生物基因組和代謝組角度揭示其變異性機(jī)制。瘤胃微生物系統(tǒng)的建立與穩(wěn)定取決于影響瘤胃環(huán)境的營(yíng)養(yǎng)與生理要素，采食行為和瘤胃上皮功能分別決定瘤胃的緩沖能力與VFA的移除速度，二者共同影響瘤胃液pH、滲透壓、氧化還原電勢(shì)等瘤胃內(nèi)環(huán)境參數(shù)，而上述參數(shù)即是影響瘤胃微生物區(qū)系的重要因素，也是判斷個(gè)體SARA是否發(fā)生與嚴(yán)重程度的主要依據(jù)。因此，闡明采食行為-瘤胃上皮功能-微生物區(qū)系三者之間的關(guān)系對(duì)揭示個(gè)體間SARA易感性的變異機(jī)制有重要意義。

4 小 結(jié)

反芻動(dòng)物個(gè)體間SARA的易感性存在差異，且這種差異主要受自身生理因素的調(diào)控，其中采食行為和瘤胃上皮的吸收能力的變異可能是導(dǎo)致個(gè)體SARA易感性差異的主要營(yíng)養(yǎng)生理因素，在未來的生產(chǎn)與科研中需明確動(dòng)物個(gè)體間SARA易感性變異的來源及機(jī)制、并針對(duì)不同個(gè)體采取相應(yīng)的營(yíng)養(yǎng)干預(yù)措施，保障動(dòng)物的高效生產(chǎn)與瘤胃健康。

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