間歇溫度處理對育成蛋雞采食及腸道發育的影響

劉增民，潘亞麗，林海，焦洪超，趙景鵬，王曉鵑

間歇溫度處理對育成蛋雞采食及腸道發育的影響

劉增民，潘亞麗，林海，焦洪超，趙景鵬，王曉鵑

山東農業大學動物科技學院/山東省動物生物工程與疾病防治重點實驗室，山東泰安 271018

【】研究環境溫熱對家禽采食的影響，補充蛋雞飼養過程中的參數缺失，為蛋雞的正確飼養提供科學依據。【】選取11周齡的伊莎褐蛋雞360只，分為5個處理組，每組6個重復，每個重復12只雞。正式試驗前將試驗蛋雞分別轉入5間智能環控雞舍預飼1周。采用標準籠養，每籠3只雞。雞舍內相對濕度保持在60%，育成期每天光照8 h（9:00 — 17:00光照）。分為對照組和4個處理組，對照組保持基礎溫度22℃不變；4個處理組采用每日間歇溫度處理，即每天10:00 —18:00期間分別進行24℃、26℃、28℃、30℃的溫度處理，其余時間恢復到基礎溫度22℃（升溫與降溫時間均在1 h以內）。試驗期8周。試驗蛋雞自由采食和飲水，每周統計采食量，每兩周采集一次樣品，每組隨機挑選12只雞，稱重后斷頸處死，稱量腺胃重量，以及十二指腸、空腸和回腸的重量與長度。采集下丘腦、腺胃和十二指腸樣品于液氮中速凍，-80℃保存。試驗結束前連續3 d分別在熱處理期和非熱處理期統計采食量。【】在試驗的前4周，與對照組T22相比，T30組的采食量顯著降低（＜0.05）；在試驗后4周時，T24組的采食量顯著高于T28組和T30組（＜0.05）。在熱處理期，T30組的采食量顯著低于T22和T24；在非熱處理期，T30組的采食量顯著低于T22、T24和T26組（＜0.05）；各組熱處理期的采食量均顯著低于非熱處理期（＜0.05），并且保持T24組采食量最高，T30組采食量最低。通過統計器官指數，發現在16周時，與T22組相比，各處理組的腺胃指數極顯著升高（＜0.01）；18周時，與T22組相比，T30組的空腸指數顯著降低，且低于其他處理組（＜0.05）；20周時，與T22組相比，T24組的腺胃指數、空腸指數和回腸指數顯著升高（＜0.05），且空腸指數顯著高于T30組（＜0.05）。檢測食欲基因的表達，發現在14周時，與T22組相比，各處理組下丘腦（Neuropeptide Y）的表達量顯著升高（＜0.05），T30組下丘腦（Agouti-related protein）的表達量顯著降低（＜0.05），T30組十二指腸（Cholecystokinin）的表達量顯著升高（＜0.05）；20周時，與T22組相比，T24組下丘腦（Cocaine amphetamine-regulated transcript）的表達量顯著降低（＜0.05），T24組腺胃的表達量顯著降低（＜0.05）。【】育成期環境溫度保持在24℃可以促進蛋雞胃腸道的發育，提高下丘腦中促食因子的表達，抑制下丘腦抑食因子和腺胃的表達量，有利于蛋雞的生長發育。而30℃高溫處理會對蛋雞的腸道造成損傷，抑制下丘腦促食因子的表達，同時促進十二指腸抑食因子的表達，從而抑制蛋雞采食，降低采食量。

蛋雞；采食；溫熱；育成期；腸道；下丘腦

0 引言

【研究意義】溫度是影響畜禽生產的主要環境因素之一。家禽因其特殊的生理特點，特別容易受到高溫環境的影響。采食量與育成期蛋雞的生長發育密切相關，但目前的研究缺乏育成期蛋雞最佳采食量所需的溫度參數。本文旨在研究環境溫熱對育成期蛋雞采食的影響，為蛋雞適宜的溫熱參數提供一定的科學依據。【前人研究進展】采食是家禽消化吸收的首要環節，也是其維持生存、生長發育和生產的前提[1]。采食量與家禽生長發育和生產性能密切相關，是評估家禽能量代謝和營養需求的基礎。與哺乳動物一樣[2-3]，家禽的食欲調節系統是一個復雜的信號系統，涉及中樞和周邊調節。下丘腦是中樞神經系統調節采食量的重要器官，它在整合調控采食的各種信號并對采食行為做出調整中發揮著關鍵作用[4-5]。哺乳動物和家禽的下丘腦弓狀核都含有在食物攝入的中樞調節中起重要作用的前阿片黑素細胞皮質激素（POMC）和神經肽Y（NPY）/刺鼠相關蛋白（AgRP）[6-7]。中樞給藥NPY和AgRP刺激食物攝入，而中樞給藥α-黑素細胞刺激激素（α-MSH，一種源自POMC的神經肽）抑制哺乳動物和雛雞的食物攝入[8-11]。食物剝奪誘導哺乳動物和雛雞下丘腦中和的表達，并抑制的表達[12-13]。因此，下丘腦、和的mRNA水平可作為食欲的指標。可卡因-苯丙胺調節轉錄肽（CART）在體內多處區域都有表達，其中下丘腦表達量最高，其次為腸道。研究表明，CART能夠通過作用于下丘腦以及胃腸道來降低家禽食欲和飼料消化速率的途徑降低采食量[14]。膽囊收縮素（CCK）和肽YY（PYY）已被研究作為腸道激素，對肉雞和蛋雞的采食量調節非常重要[15]。這兩種基因在進食后將飽腹感信號傳遞給大腦，導致食欲減弱[16-18]。以上食欲調節因子在中樞神經系統和身體外周組織的交匯處起作用[19]，并受到包括環境溫度在內的多種因素的影響[20]。家禽屬于恒溫動物，當環境溫度處于一定變化范圍內時，家禽可以自我調節產熱和散熱來維持體溫的穩定[21]。由于羽毛厚實、汗腺缺乏和高代謝率，家禽特別容易受到熱應激的影響[22]。熱應激是影響家禽健康最為主要的環境影響因素之一[23]。研究發現，家禽暴露在高溫環境中會改變其生理內穩態，導致免疫紊亂、內分泌和電解質紊亂，導致體重減輕、產卵量減少，甚至死亡率增加[24]。在一定的溫度范圍內，蛋雞的采食量隨著環境溫度的升高而降低，在高溫下飼養的蛋雞的采食量和產蛋量較低[25]。下丘腦視前區的溫度調節中心在熱應激期間被激活，與采食調控相關的神經遞質或腦腸肽水平發生變化，包括NPY、AgRP、POMC和ghrelin等[25-27]。因此，高溫會通過調控中樞和外周的食欲相關基因影響蛋雞的采食量和生產性能。腸道是家禽主要的消化吸收場所，其功能好壞與家禽采食量息息相關。研究報道，急性熱應激影響肉鴨的小腸形態，降低空腸絨毛高度[28]。熱應激嚴重損傷腸道黏膜，降低消化吸收能力，影響體重的增加[29]。在熱應激期間，家禽降低采食量以降低消化熱。當機體處于輕微熱應激時，交感神經興奮，促使胃腸道蠕動減緩，增加食物在消化道內的滯留時間，降低采食量。而當機體處于嚴重熱應激時，血管膨脹充血，使得消化器官中循環血量降低，消化酶分泌減少，削弱消化功能，采食量大幅下降。嚴重時，還會造成消化器官損傷。熱應激會首先導致胃損傷和胰腺炎惡化，顯著升高血清中淀粉酶、脂肪酶、白介素、過氧化物酶的濃度[30]。熱應激還會導致血清皮質酮水平升高，法氏囊、胸腺、脾臟等免疫器官的比重顯著降低，引起腸道損傷[31]。綜上所述，家禽采食調控涉及到外源（環境溫度）與內源（食欲調控因子、消化器官發育）兩個方面，熱應激會通過調控食欲基因和消化器官發育來影響采食量。目前，國內外對家禽采食行為的研究已經有比較多的報道，但主要是以肉雞或產蛋期蛋雞為試驗對象，對蛋雞的育成期關注較少。此外，相關的長期熱應激試驗大多是全程高溫，這不符合溫度的日變化規律。【本研究切入點】因此，本文在蛋雞育成期設計了間歇溫度處理，即每天10：00— 18：00期間進行8 h的溫度處理，通過評估采食量、腸道發育及食欲調控因子的表達，為育成期蛋雞最佳采食量的適宜溫熱參數提供一定的科學依據。【擬解決的關鍵問題】本試驗從溫熱這一重要的環境因子入手，探究其對家禽采食行為的影響，旨在補充蛋雞飼養過程中的參數缺失，為蛋雞的科學飼養提供一定的理論基礎。

1 材料與方法

本試驗于2018年在山東農業大學動物生物工程與疾病防治山東省重點實驗室完成。試驗選用的海蘭褐蛋雞購自青島奧特種禽場。飼喂所用的飼料均為商品日糧，購自山東眾成飼料廠。

1.1 試驗動物與試驗設計

選取體重相近的11周齡伊莎褐蛋雞360只，分為5個處理組，每組6個重復，每個重復12只雞。試驗前將試驗蛋雞分別轉入5間智能環控雞舍預飼，環控舍定制于濟南科益試驗設備有限公司，每間3.75 m× 2.40 m，可獨立設定溫濕度（控溫范圍15—40℃，控溫精度± 0.5℃，控濕范圍50%—80%，控濕精度± 5%）。采用標準籠養，每籠3只雞。雞舍內相對濕度保持在60%，育成期每天8 h光照（9：00—17：00光照）。分為對照組（T22）和4個處理組（T24、T26、T28、T30），對照組保持基礎溫度22℃不變；4個處理組采用每日間歇溫度處理，即每天10：00—18：00期間分別進行24℃、26℃、28℃、30℃的溫度處理，其余時間恢復到基礎溫度22℃（升溫與降溫時間均在1 h以內）。預飼1周，試驗處理8周。試驗蛋雞自由采食和飲水，每周統計采食量，每兩周采集一次樣品，每組隨機挑選12只雞，稱重后斷頸處死，稱量腺胃重量，以及十二指腸、空腸和回腸的重量與長度。采集下丘腦、腺胃和十二指腸樣品于液氮中速凍，-80℃保存。試驗結束前連續3 d分別在熱處理期和非熱處理期統計采食量。

1.2 測定指標及方法

（1）采食量：每周統計采食量，計算平均日采食量。試驗結束前連續3 d分別在熱處理期和非熱處理期統計采食量，計算平均每小時采食量。

平均日采食量（g/d/hen）= 耗料量/試驗天數/試驗雞數；

平均每小時采食量（g/h/hen）= 耗料量/試驗時間/試驗雞數。

（2）器官指數

每組隨機挑選12只雞稱重后斷頸處死取樣，稱量腺胃、十二指腸、空腸和回腸的重量。

器官指數（%）=（器官的絕對重量/雞的體重）×100。

1.3 組織中相關基因mRNA表達水平的測定

用異硫氰二胍鹽法提取組織總RNA，提取RNA的濃度和質量分別采用核酸分光光度計和瓊脂糖凝膠電泳檢測。反轉錄參照Roche公司的反轉錄試劑盒說明書操作進行，根據Roche公司的Real-Time PCR試劑盒（04913914001）說明書進行熒光定量檢測。引物序列參見表1。以和的表達水平作為內參，采用“平均相對表達量=2-△△Ct”計算基因的相對表達量。

表1 基因特異性引物序列表

1.4 數據分析

試驗數據采用 SAS（Version 8e，SAS Institute，1998）統計軟件ANOVA程序進行單因子方差分析，Duncan 氏法進行多重比較。試驗數據用平均值±標準誤（Mean ± SE）表示，＜0.05表示處理間差異顯著，＜0.01 表示處理間差異極顯著。

2 結果

2.1 間歇溫度處理對育成蛋雞采食量的影響

如圖1所示，在試驗的前4周時，與對照組T22 相比，T30組的采食量顯著降低（＜0.05）；在試驗后四周時，T24組的采食量顯著高于T28組和T30組（＜0.05）。

試驗結束前連續3 d分別在熱處理期和非熱處理期統計采食量。如圖2-A所示，無論是在熱處理期還是非熱處理期，T30組的采食量均為最低。在熱處理期，T30組的采食量顯著低于T22和T24；在非熱處理期，T30組的采食量顯著低于T22、T24和T26組（＜0.05）。如圖2-B所示，各組熱處理期的采食量均顯著低于非熱處理期（＜0.05）。

同一圖中標不同字母者差異顯著，P＜0.05。下同

2.2 間歇溫度處理對育成蛋雞消化器官發育的影響

如圖3所示，14周時，各組的腺胃指數、十二指腸指數、空腸指數和回腸指數沒有顯著差異（＞0.05）。16周時，與對照組T22相比，各處理組的腺胃指數極顯著升高（＜0.01）；T30組的空腸指數和回腸指數顯著低于T28 組（＜0.05）。18周時，T26組和T30組的腺胃指數顯著低于T28 組（＜0.05）；T30組的十二指腸指數和回腸指數顯著低于T28 組（＜0.05）；與T22組相比，T30組的空腸指數顯著降低，且低于其他處理組（＜0.05）。20周時，與T22組相比，T24組的腺胃指數、十二指腸指數和空腸指數顯著升高（＜0.05），且空腸指數顯著高于T30組（＜0.05），T26組的回腸指數顯著升高，且高于T28 組（＜0.05）。其余指標無顯著差異（＞0.05）。

圖2 熱處理期與非熱處理期溫度對育成蛋雞采食量的影響

如表2所示，各組十二指腸、空腸和回腸的長度均無顯著差異（＞0.05）。

2.3 間歇溫度處理對育成蛋雞食欲基因表達水平的影響

如圖4所示，14周時，與對照組T22相比，各處理組下丘腦的表達量顯著升高（＜0.05），T26組、T28組和T30組下丘腦的表達量顯著降低（＜0.05），T30組十二指腸的表達量顯著升高（＜0.05）。16周時，與T22組相比，T30組下丘腦的表達量顯著升高（＜0.05）；T24組下丘腦的表達量顯著高于T28組（＜0.05）。18周時，T28組下丘腦的表達量顯著高于T24 組和T26組（＜0.05）；與T22組和其他熱處理組相比，T28組下丘腦的表達量顯著升高（＜0.05）。20周時，與T22組相比，T28組下丘腦的表達量顯著升高，且高于T26組和T30組（＜0.05），T24組、T28組和T30組下丘腦的表達量顯著降低（＜0.05），T24組和T26組腺胃的表達量顯著降低（＜0.05）；T28組下丘腦的表達量顯著高于T26 組和T30組（＜0.05）。其余指標無顯著差異（＞0.05）。

3 討論

3.1 間歇溫度處理對育成蛋雞生產性能的影響

家禽的適宜溫度為16—25℃，據統計，溫度在21—30℃之間每升高1℃，采食量下降1.5%，溫度在32— 38℃之間每升高1℃，采食量約下降4.6%。在較高的環境溫度下，家禽的產熱量隨著飼料消耗量的降低而降低[32]。當雞舍內溫度上升到一定程度時，家禽采食中樞會受到抑制，促使采食量下降。當外界溫度高于自身適宜溫度后，家禽會增加呼吸頻率，增加采食頻次，減少采食持續時間，降低總采食量[33]。目前的長期熱應激試驗大多是全程高溫，這不符合溫度的日變化規律。因此本試驗設計了間歇溫度處理，每天10：00—18：00進行8 h的溫度處理。升溫與降溫時間均在1 h內，符合溫度的日變化規律，且避免了因溫度控制不善對試驗結果產生的干擾影響。在間歇溫度處理前4周，T30組的采食量最低；在試驗后4周，T24 組采食量最高，T30組采食量仍然最低。主要是因為T24組的體重顯著高于其他處理組，而30℃高溫處理超出蛋雞適宜的生活溫度，損傷了蛋雞的消化系統，從而降低了蛋雞的采食量。而且，我們發現在非熱處理期，T30組的采食量仍然最低，表明經過長期高溫刺激，蛋雞的消化系統已經受到損傷。無論在哪個溫度處理中，非處理期間的平均每小時采食量均高于處理組，對高溫組來說，這是由于環境溫度下降促進了采食，而對低溫組來說，這可能是由于光照后的采食高峰引起的[34]。

圖3 間歇溫度處理對育成蛋雞消化器官指數的影響

表2 間歇溫度處理對育成蛋雞腸道長度發育的影響

同一行中標不同字母者差異顯著，＜0.05，n = 12 Means with different letters differ significantly,＜0.05, n = 12

圖4 間歇溫度處理對育成蛋雞食欲基因的影響

3.2 間歇溫度處理對育成蛋雞消化器官發育的影響

胃腸道是動物維持生長的消化系統的重要組成部分[35]。熱應激、氧化應激和缺氧條件對單胃動物胃腸功能和代謝的負面影響已得到充分證明[36]。研究表明胃腸道對應激源高度敏感，被認為是受熱應激影響的主要靶器官之一[37-38]。本試驗中，間歇溫度處理對腸道的長度沒有顯著影響。可能是因為常規的熱應激試驗設定的熱應激溫度在30—38℃之間，而本試驗是為了研究育成期蛋雞的最佳采食所需溫度，設定的溫度范圍是22—30℃，并未影響到蛋雞腸道的發育。Mashaly等研究表明，與間歇性熱應激相比，持續性熱應激對蛋雞的影響更嚴重[39]。間歇溫度處理4周后，各處理組的腺胃指數均升高，且T28 組的空腸指數和回腸指數顯著高于T30組，這表明育成期適當提高環境溫度有利于蛋雞的胃腸道發育。在試驗后期，T24組的腺胃指數、十二指腸指數和空腸指數均升高，而T30組的空腸指數顯著降低，這表明對育成期蛋雞進行24℃間歇溫度處理，會促進腺胃和腸道的發育，而30℃高溫處理會損傷蛋雞的腸道發育，從而影響采食，使采食量降低。

3.3 間歇溫度處理對育成蛋雞食欲基因表達的影響

食欲受到中樞和外周的調控，下丘腦是各種食欲調節信號的主要整合中心[40]。在家禽中，下丘腦在整合外部環境線索（尤其是應激源）方面起著關鍵作用，并作出適當的反應來影響采食量[19]。下丘腦神經元可以感知體溫的升高，并對負責控制攝食的細胞產生抑制作用。下丘腦既是體溫控制中樞，也是采食控制中樞。熱信號在傳入下丘腦后，不僅會開啟家禽的體溫平衡機制，而且還會傳遞到攝食中樞，從而改變家禽采食行為。家禽采食量一般與環境溫度呈負相關。一方面外界溫熱信號可能直接作用于家禽下丘腦食欲調控中樞，另一方面可以削弱消化道活動，導致消化道食物充盈從而抑制食欲。為期7 d的熱應激處理能夠顯著降低蛋雞采食量，顯著升高下丘腦中的和T的表達量，降低下丘腦的表達量[25]。研究表明，急性熱應激可以顯著增加腺胃、十二指腸和空腸中的表達水平，降低十二指腸的表達水平，而對中樞食欲基因表達無顯著影響[41]。從而證明了急性熱應激對食欲的調控位點主要在腺胃和腸道上。

本試驗屬于長期溫熱刺激，除了可以影響外周食欲基因外，還對中樞食欲基因作用明顯。整體來看，24℃溫度處理提高了下丘腦促食因子的表達，抑制了下丘腦抑食因子和腺胃的表達。而30℃高溫處理抑制了下丘腦促食因子的表達并提高了十二指腸抑食因子的表達。因此，高溫處理可能通過抑制中樞促食基因的表達，同時提高外周抑食基因的表達，來影響蛋雞的采食。環境的溫熱刺激可能是通過作用于蛋雞下丘腦和胃腸信號介導，使蛋雞在高溫環境下表現為厭食，適溫下采食增加。

4 結論

育成期環境溫度保持在24℃可以促進蛋雞胃腸道的發育，提高下丘腦中促食因子的表達，抑制下丘腦抑食因子和腺胃的表達量，有利于蛋雞的生長發育。而30℃高溫處理會對蛋雞的腸道造成損傷，抑制下丘腦促食因子的表達，同時促進十二指腸抑食因子的表達，從而抑制蛋雞采食，降低采食量。綜上所述，蛋雞育成期的飼養溫度保持在24℃對其生長發育最為有利。

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Effects of Intermittent Different Temperature on Feeding and Intestinal Development of Growing Laying Hens

LIU ZengMin, PAN YaLi, LIN Hai, JIAO HongChao, ZHAO JingPeng, WANG XiaoJuan*

College of Animal Science and Technology, Shandong Agricultural University/Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai’an 271018, Shandong

【】The objective of this study was to study the effects of ambient temperature on feeding and intestinal development of poultry, and to supplement the absent temperature parameters for laying hens rearing, so as to provide a certain scientific basis for the correct feeding of laying hens.【】A total of 360 Issa brown laying hens aged 11 weeks were selected and divided into 5 treatment groups with 6 replicates per group and 12 hens per replicate. The experimental laying hens were transferred to 5 intelligent environmental control chicken houses for 1 week of pre-trial and 8 weeks of formal experiment, with 3 chickens per cage. The relative humidity in the chicken house was kept at 60%, and the light was kept for 8 h (9:00-17:00) every day during the prelay period. The temperature of the control group was kept unchanged at 22℃, and the four treatment groups were carried out in a manner of daily intermittent, including 24℃, 26℃, 28℃, and 30℃ at 10:00-18:00 every day, respectively, and changed to the base temperature 22℃ for the rest of the time, the heating and cooling time were within 1 h. The experiment lasted for 8 weeks. The experimental laying hens were free to eat and drink, the feed intake was counted weekly, and the samples were collected once every two weeks. Twelve hens in each group were randomly selected and weighed, and then killed by neck cutting. The weight of glandular stomach, the weight and length of duodenum, jejunum and ileum were weighed. Hypothalamus, glandular stomach and duodenum samples were frozen in liquid nitrogen and stored at -80℃. Feed intake was calculated in heat treatment period and non-heat treatment period for 3 days before the end of experiment.【】Compared with T22 group, the feed intake in T30 group was significantly decreased at 13-16 week (＜0.05); the feed intake in T24 group was significantly higher than that in T28 and T30 groups at 17-20 week (＜0.05). During the heat treatment period, the feed intake of T30 group was significantly lower than that in T22 and T24 groups (＜0.05). The feed intake of T30 group was significantly lower than that in T22, T24 and T26 groups during the non-heat treatment period (＜0.05). The feed intake in the heat treatment period was significantly lower than that in the non-heat treatment period (＜0.05), and the highest feed intake was maintained in T24 group and the lowest in T30 group. Compared with T22 group, the glandular gastric index was significantly increased at 16 week (＜0.01). At 18 week, compared with T22 group, the jejunum index in T30 group was significantly lower than that in other groups (＜0.05). At 20 week, compared with T22 group, the glandular stomach index, jejunum index and ileum index in T24 group were significantly increased (＜0.05), and the jejunum index was significantly higher than that in T30 group (＜0.05). Compared with T22 group, the expression of(Neuropeptide Y) in hypothalamus of all treatment groups was significantly increased at 14 week (＜0.05). The expression of(Agouti-related protein) in hypothalamus of T30 group was significantly decreased (＜0.05), and the expression of(Cholecystokinin) in duodenum of T30 group was significantly increased (＜0.05). At 20 week, compared with T22 group, the expression of(amphetamine-regulated transcript) in hypothalamus of T24 group was significantly decreased (＜0.05), and the expression ofin glandular stomach of T24 group was significantly decreased (＜0.05).【】These results indicated that ambient temperature at 24℃ during the growing period could promote the development of gastrointestinal tract, increase the expression ofin hypothalamus, and inhibit the expression ofandin hypothalamus, which was beneficial to the growth and development of laying hens. However, the high temperature treatment at 30℃ damaged the intestinal tract of laying hens, inhibited the expression of hypothalamus feeding promoting factor, and promoted the expression of duodenal feeding inhibiting factor, thus inhibiting feeding intake and reducing feed intake of laying hens.

laying hens; feed intake; temperature; prelay period; intestine; hypothalamus

2021-12-12；

2022-05-27

國家重點研發計劃（2018YFE0128200）、山東省重點研發計劃（2019JZZY020602）、國家現代農業產業技術體系建設專項資金（CARS- 40-K09）、山東省“雙一流”獎補資金、泰山學者項目（201511023）

劉增民，E-mail：1325865598@qq.com。潘亞麗，E-mail：3229482556@qq.com。劉增民和潘亞麗為同等貢獻作者。通信作者王曉鵑，E-mail：wangxj@sdau.edu.cn

（責任編輯 林鑒非）