山羊瘤胃中主要乳酸代謝菌代謝機制及其調(diào)控瘤胃酸中毒的應(yīng)用研究
發(fā)布時間：2018-07-31 06:13
【摘要】：有關(guān)瘤胃酸中毒發(fā)生機制研究表明,瘤胃乳酸的累積可能對酸中毒誘導(dǎo)起重要作用,而飼喂高精料日糧下誘發(fā)瘤胃乳酸累積主要取決于瘤胃乳酸產(chǎn)生菌和乳酸利用菌間的平衡程度。本論文即研究瘤胃中主要乳酸代謝菌代謝機制與調(diào)控方法,旨在為反芻動物瘤胃酸中毒的乳酸中毒機制深入解析提供參考。試驗一建立山羊體外、內(nèi)瘤胃酸中毒模型。將5種主要乳酸代謝菌(Streptococcus bovis,Lactobacilli fermentum,Butyrivibrio fibrisolvens,Megasphaera elsdenii 和Selenomonasruminantium)體外共培養(yǎng)并通過底物淀粉濃度(1,3和9g/L)誘導(dǎo)建立體外多菌共培養(yǎng)瘤胃酸中毒模型。結(jié)果表明:1)乳酸產(chǎn)生菌(S.bovis,L.fermentum和B.fibrisolvens)數(shù)量隨底物淀粉濃度提高而提高(0.001),乳酸利用菌(M.elsenii和S.ruminantium)數(shù)量在急性酸中毒(ARA)組中顯著低于亞急性酸中毒(SARA)組(P0.001)。2)ARA組中乳酸、乙酸、甲酸及丙酸濃度顯著高于SARA組(P0.001),但丁酸濃度顯著低于SARA組(P0.001)。3)乳酸脫氫酶(LDH)活性隨底物淀粉濃度提高而顯著提高(P0.001),ARA組中α-淀粉酶(α-AMY)活性顯著低于SARA組(P0.001)。4)ARA組中脂多糖(LPS)濃度顯著高于SARA組和對照組(P0.001),但對照組和SARA組無顯著差異(P=0.659);選擇8只干奶期薩能奶山羊,采用自身對照試驗設(shè)計,通過高淀粉飼糧(50%玉米粉)誘導(dǎo)建立山羊SARA模型。結(jié)果表明:1)SARA 組 pH顯著低于對照組(P0.001)。2)SARA 組 S.bovis,M.elsenii和S.ruminantium數(shù)量顯著高于對照組(P0.001),Lactobacill 數(shù)量顯著低于對照組(P0.001),B.fibrisolvens數(shù)量組間無顯著差異(P = 0.800)。3)SARA組乳酸、甲酸、丙酸及丁酸濃度顯著高于對照組(P0.01),但乙酸濃度無顯著差異(P = 0.209)。4)SARA組LDH和α-AMY酶活及LPS濃度顯著高于對照組(P0.001)。本試驗結(jié)果表明:可以通過主要乳酸代謝菌共培養(yǎng)建立體外酸中毒模型及通過高淀粉飼糧誘導(dǎo)山羊瘤胃酸中毒發(fā)生。與此同時,S.bovis和M.elsdenii是瘤胃酸中毒進程中主要的乳酸產(chǎn)生菌和分解菌。試驗二研究不同底物(淀粉和葡萄糖)、底物濃度(1,3和9g/L)和pH(6.5和5.5)條件下S.bovis有機酸產(chǎn)生通路及調(diào)控。結(jié)果表明:1)乳酸產(chǎn)量隨底物濃度的提高顯著提高(P0.001),且以淀粉為底物時pH為6.5下乳酸產(chǎn)量顯著高于pH為5.5(P0.001)。甲酸和乙酸產(chǎn)量隨底物濃度提高波動,但占總有機酸比例較小。2)LDH和α-AMY酶活隨底物淀粉濃度提高而顯著提高(P0.05),且pH為5.5時顯著高于pH為6.5(P0.001),但以葡萄糖為底物時各組無顯著差異(P0.05)。3)LDH、α-AMY、甲酸裂解酶(PFL)及分解控制蛋白A(CcpA)編碼基因表達與底物濃度總體上呈正相關(guān)。4)果糖-1,6-二磷酸(FDP)濃度隨底物濃度提高而顯著提高(P0.05),且pH為6.5下FDP濃度顯著高于pH為5.5(P = 0.032)。5)相較底物淀粉濃度的影響,S.bovis產(chǎn)乳酸對pH變化更敏感。本試驗結(jié)果表明:底物類型、濃度及pH可在轉(zhuǎn)錄水平調(diào)控S.bovis有機酸產(chǎn)生模式。試驗三研究底物乳酸濃度(15,30和90 mM)和pH(6.5和5.5)條件下M.elsdenii乳酸分解通路及調(diào)控。結(jié)果表明:1)M.elsdenii分解乳酸主要產(chǎn)生乙酸,其次丙酸和丁酸。2)pH為6.5時乙酸、丙酸和丁酸產(chǎn)量均顯著高于pH為5.5(P0.05)。pH為6.5時,產(chǎn)乙酸比例顯著低于pH為5.5(P0.001),而產(chǎn)丙酸和丁酸比例顯著高于pH為5.5(P0.001)。3)底物乳酸濃度為90 m時,乙酸產(chǎn)量和比例顯著高于其他兩組(P0.05)。底物乳酸濃度為30 m組,丙酸和丁酸產(chǎn)量和比例顯著高于底物乳酸濃度為90 mM組(P0.05),且90 mM組顯著高于15 mM組(P0.05)。4)涉及乳酸分解相關(guān)關(guān)鍵酶編碼基因隨底物乳酸濃度及pH變化出現(xiàn)不出程度的顯著差異表達(P0.05)。本試驗結(jié)果表明:底物濃度及pH可以在轉(zhuǎn)錄水平調(diào)控M.elsenii乳酸分解通路。試驗四研究山羊瘤胃灌注低營養(yǎng)富集誘導(dǎo)M.elsdenii對高淀粉飼糧誘導(dǎo)SARA緩解作用。結(jié)果表明:1)灌菌組pH顯著高于SARA組,同時顯著低于對照組(P0.05)。2)SARA組乳酸和丙酸濃度顯著高于灌菌組及對照組(P0.05),但對照組與灌菌組差異不顯著(P0.05)。SARA組和灌菌組丁酸濃度顯著高于對照組(P0.05)。對照組乙酸濃度顯著高于灌菌組(P0.05),但SARA組與對照組和灌菌組差異不顯著(P0.05)。3)SARA組S.bovi 數(shù)量顯著高于灌菌組和對照組(P0.05)。灌菌組Lactobacilli和B.fibrisolvens數(shù)量顯著高于SARA組和對照組(P0.05)。SARA組和灌菌組M.elsdenii和S.ruminantium數(shù)量顯著高于對照組(P0.05)。4)SARA組α-AMY和LDH酶活顯著高于灌菌組(P0.05),且灌菌組α-AMY酶活顯著高于對照組(P0.05)。5)SARA組LPS濃度顯著高于灌菌組,且灌菌組顯著高于對照組。本試驗結(jié)果表明:通過瘤胃灌注低營養(yǎng)富集誘導(dǎo)M.elsdenii菌液可以改善高淀粉飼糧誘導(dǎo)SARA山羊瘤胃發(fā)酵,在一定程度上緩解酸中毒的發(fā)生。
[Abstract]:The study of the pathogenesis of gastric acidosis shows that the accumulation of lactic acid in the rumen may play an important role in the induction of acidosis, while feeding the rumen lactic acid accumulation under the diet of high concentrate depends mainly on the balance between the lactic acid producing bacteria in the rumen and the lactic acid use bacteria. Control methods are designed to provide a reference for the mechanism of lactic acidosis in ruminant gastric acidosis. A model of goats in vitro, internal tumor gastric acidosis was established. 5 major lactic acid metabolites (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, Megasphaera elsdenii, and Selenomonasruminantium) were established in vitro. The results showed that the number of lactic acid producing bacteria (S.bovis, L.fermentum and B.fibrisolvens) increased with the increase of substrate starch concentration (0.001), and the number of lactic acid bacteria (M.elsenii and S.ruminantium) in acute acidosis (ARA) group was increased by 1,3 and 9g/L induced by substrate starch concentration (1,3 and B.fibrisolvens). The concentration of lactic acid, acetic acid, formic acid and propionic acid in group ARA was significantly higher than that in group SARA (P0.001), but the concentration of butyric acid was significantly lower than that of SARA group (P0.001).3), but the activity of lactate dehydrogenase (LDH) in ARA group was significantly higher than that of SARA group (P0.001), but the activity of alpha amylase (alpha.2) in the ARA group was significantly lower than that of the SARA group (P0.001). 001).4) the concentration of lipopolysaccharide (LPS) in the ARA group was significantly higher than that in the SARA group and the control group (P0.001), but there was no significant difference between the control group and the SARA group (P=0.659); the selected 8 dried milk goats were designed by self control experiment, and the goat SARA model was established through the high starch diet (50% corn flour). The results showed that 1) pH of the SARA group was significantly lower than the control. Group (P0.001).2) the number of S.bovis, M.elsenii and S.ruminantium in group SARA was significantly higher than that of the control group (P0.001), the number of Lactobacill was significantly lower than that of the control group (P0.001), there was no significant difference between the B.fibrisolvens quantity groups (P = 0.800) and the concentration of lactic acid, propionic acid and butyric acid was significantly higher than that of the control group, but there was no significant difference in the concentration of acetic acid. LDH and alpha -AMY enzyme activity and LPS concentration in group SARA were significantly higher than those of control group (P0.001). The results of this experiment showed that the model of stereotactic acid poisoning and goaltumor gastric acidosis could be induced by major lactic acid metabolites and induced by high starch diet. At the same time, S.bovis and M.elsdenii were the main milk in the process of tumor gastric acidosis. Acid producing bacteria and decomposing bacteria. Experiment two studied the pathway and regulation of S.bovis organic acids in different substrates (starch and glucose), substrate concentration (1,3 and 9g/L) and pH (6.5 and 5.5). The results showed that 1) the production of lactic acid increased significantly with the increase of substrate concentration (P0.001), and the yield of lactic acid under the pH of 6.5 was significantly higher than that of pH at 5.5 when starch was the substrate. (P0.001). The yield of formic acid and acetic acid fluctuated with the concentration of substrate, but the proportion of the total organic acids was smaller.2). The activity of LDH and alpha -AMY increased significantly with the increase of substrate starch concentration (P0.05), and the pH was 5.5 higher than pH was 6.5 (P0.001), but there was no significant difference in each group (P0.05).3) LDH, alpha -AMY, formate lyase and fractions when the glucose was the substrate. The expression of A (CcpA) encoding gene was positively correlated with substrate concentration (.4). The concentration of fructose -1,6- two phosphoric acid (FDP) increased significantly with the increase of substrate concentration (P0.05), and the concentration of pH to 6.5 FDP was significantly higher than pH (P = 0.032).5) compared with the concentration of substrate starch. The substrate type, concentration and pH can regulate the production mode of S.bovis organic acid at the transcriptional level. Experiment three studies the pathway and regulation of the M.elsdenii lactic acid decomposition pathway under the conditions of substrate lactic acid (15,30 and 90 mM) and pH (6.5 and 5.5). The results show that 1) M.elsdenii decomposition lactic acid mainly produces acetic acid, and then propionic acid and butyric acid.2) pH is 6.5, acetic acid, propionic acid And the yield of butyric acid was significantly higher than that of pH 5.5 (P0.05).PH 6.5, the proportion of acetic acid was significantly lower than that of pH 5.5 (P0.001), while the ratio of propionic acid and butyric acid was significantly higher than that of pH 5.5 (P0.001).3). When the substrate lactic acid concentration was 90 m, the yield and proportion of acetic acid were significantly higher than those of the other two groups (P0.05). The substrate lactic acid concentration was 30 m group, propionic acid and butyric acid yield and ratio. The sample was significantly higher than the substrate lactate concentration of 90 mM (P0.05), and the 90 mM group was significantly higher than the 15 mM group (P0.05).4) involved in the significant difference expression of the lactic acid decomposition related key enzyme encoding genes with the substrate lactic acid concentration and pH changes (P0.05). The results showed that the substrate concentration and pH could regulate the M.elsenii lactic acid at the transcriptional level. Decomposition pathway. Experiment four study the effect of M.elsdenii induced SARA on high starch diet induced by rumen perfusion in goats. The results showed that: 1) the pH of the group was significantly higher than that of the SARA group, and the concentration of lactic acid and propionic acid in the SARA group was significantly higher than that of the control group and the control group (P0.05), but the difference between the control group and the perfusion group was worse than the control group (P0.05). The concentration of butyric acid in group P0.05.SARA and perfusion group was significantly higher than that of control group (P0.05). The concentration of acetic acid in the control group was significantly higher than that of the perfusion group (P0.05), but there was no significant difference between the SARA group and the control group and the control group (P0.05).3). The S.bovi number of the SARA group was significantly higher than that of the perfusion group and the opposite group (P0.05). The number of M.elsdenii and S.ruminantium in the group.SARA and the control group (P0.05) and the control group was significantly higher than that of the control group (P0.05).4). The enzyme activity of alpha -AMY and LDH in the SARA group was significantly higher than that of the group (P0.05), and the alpha -AMY enzyme activity of the group was significantly higher than that of the control group (P0.05), and the concentration of SARA was significantly higher than that of the control group, and the perfusion group was significantly higher than the control group. The results showed that the induced M.elsdenii bacteria could improve the rumen fermentation of SARA goats induced by high starch diet, and alleviated the occurrence of acidosis to a certain extent.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.27
本文編號：2154680
[Abstract]:The study of the pathogenesis of gastric acidosis shows that the accumulation of lactic acid in the rumen may play an important role in the induction of acidosis, while feeding the rumen lactic acid accumulation under the diet of high concentrate depends mainly on the balance between the lactic acid producing bacteria in the rumen and the lactic acid use bacteria. Control methods are designed to provide a reference for the mechanism of lactic acidosis in ruminant gastric acidosis. A model of goats in vitro, internal tumor gastric acidosis was established. 5 major lactic acid metabolites (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, Megasphaera elsdenii, and Selenomonasruminantium) were established in vitro. The results showed that the number of lactic acid producing bacteria (S.bovis, L.fermentum and B.fibrisolvens) increased with the increase of substrate starch concentration (0.001), and the number of lactic acid bacteria (M.elsenii and S.ruminantium) in acute acidosis (ARA) group was increased by 1,3 and 9g/L induced by substrate starch concentration (1,3 and B.fibrisolvens). The concentration of lactic acid, acetic acid, formic acid and propionic acid in group ARA was significantly higher than that in group SARA (P0.001), but the concentration of butyric acid was significantly lower than that of SARA group (P0.001).3), but the activity of lactate dehydrogenase (LDH) in ARA group was significantly higher than that of SARA group (P0.001), but the activity of alpha amylase (alpha.2) in the ARA group was significantly lower than that of the SARA group (P0.001). 001).4) the concentration of lipopolysaccharide (LPS) in the ARA group was significantly higher than that in the SARA group and the control group (P0.001), but there was no significant difference between the control group and the SARA group (P=0.659); the selected 8 dried milk goats were designed by self control experiment, and the goat SARA model was established through the high starch diet (50% corn flour). The results showed that 1) pH of the SARA group was significantly lower than the control. Group (P0.001).2) the number of S.bovis, M.elsenii and S.ruminantium in group SARA was significantly higher than that of the control group (P0.001), the number of Lactobacill was significantly lower than that of the control group (P0.001), there was no significant difference between the B.fibrisolvens quantity groups (P = 0.800) and the concentration of lactic acid, propionic acid and butyric acid was significantly higher than that of the control group, but there was no significant difference in the concentration of acetic acid. LDH and alpha -AMY enzyme activity and LPS concentration in group SARA were significantly higher than those of control group (P0.001). The results of this experiment showed that the model of stereotactic acid poisoning and goaltumor gastric acidosis could be induced by major lactic acid metabolites and induced by high starch diet. At the same time, S.bovis and M.elsdenii were the main milk in the process of tumor gastric acidosis. Acid producing bacteria and decomposing bacteria. Experiment two studied the pathway and regulation of S.bovis organic acids in different substrates (starch and glucose), substrate concentration (1,3 and 9g/L) and pH (6.5 and 5.5). The results showed that 1) the production of lactic acid increased significantly with the increase of substrate concentration (P0.001), and the yield of lactic acid under the pH of 6.5 was significantly higher than that of pH at 5.5 when starch was the substrate. (P0.001). The yield of formic acid and acetic acid fluctuated with the concentration of substrate, but the proportion of the total organic acids was smaller.2). The activity of LDH and alpha -AMY increased significantly with the increase of substrate starch concentration (P0.05), and the pH was 5.5 higher than pH was 6.5 (P0.001), but there was no significant difference in each group (P0.05).3) LDH, alpha -AMY, formate lyase and fractions when the glucose was the substrate. The expression of A (CcpA) encoding gene was positively correlated with substrate concentration (.4). The concentration of fructose -1,6- two phosphoric acid (FDP) increased significantly with the increase of substrate concentration (P0.05), and the concentration of pH to 6.5 FDP was significantly higher than pH (P = 0.032).5) compared with the concentration of substrate starch. The substrate type, concentration and pH can regulate the production mode of S.bovis organic acid at the transcriptional level. Experiment three studies the pathway and regulation of the M.elsdenii lactic acid decomposition pathway under the conditions of substrate lactic acid (15,30 and 90 mM) and pH (6.5 and 5.5). The results show that 1) M.elsdenii decomposition lactic acid mainly produces acetic acid, and then propionic acid and butyric acid.2) pH is 6.5, acetic acid, propionic acid And the yield of butyric acid was significantly higher than that of pH 5.5 (P0.05).PH 6.5, the proportion of acetic acid was significantly lower than that of pH 5.5 (P0.001), while the ratio of propionic acid and butyric acid was significantly higher than that of pH 5.5 (P0.001).3). When the substrate lactic acid concentration was 90 m, the yield and proportion of acetic acid were significantly higher than those of the other two groups (P0.05). The substrate lactic acid concentration was 30 m group, propionic acid and butyric acid yield and ratio. The sample was significantly higher than the substrate lactate concentration of 90 mM (P0.05), and the 90 mM group was significantly higher than the 15 mM group (P0.05).4) involved in the significant difference expression of the lactic acid decomposition related key enzyme encoding genes with the substrate lactic acid concentration and pH changes (P0.05). The results showed that the substrate concentration and pH could regulate the M.elsenii lactic acid at the transcriptional level. Decomposition pathway. Experiment four study the effect of M.elsdenii induced SARA on high starch diet induced by rumen perfusion in goats. The results showed that: 1) the pH of the group was significantly higher than that of the SARA group, and the concentration of lactic acid and propionic acid in the SARA group was significantly higher than that of the control group and the control group (P0.05), but the difference between the control group and the perfusion group was worse than the control group (P0.05). The concentration of butyric acid in group P0.05.SARA and perfusion group was significantly higher than that of control group (P0.05). The concentration of acetic acid in the control group was significantly higher than that of the perfusion group (P0.05), but there was no significant difference between the SARA group and the control group and the control group (P0.05).3). The S.bovi number of the SARA group was significantly higher than that of the perfusion group and the opposite group (P0.05). The number of M.elsdenii and S.ruminantium in the group.SARA and the control group (P0.05) and the control group was significantly higher than that of the control group (P0.05).4). The enzyme activity of alpha -AMY and LDH in the SARA group was significantly higher than that of the group (P0.05), and the alpha -AMY enzyme activity of the group was significantly higher than that of the control group (P0.05), and the concentration of SARA was significantly higher than that of the control group, and the perfusion group was significantly higher than the control group. The results showed that the induced M.elsdenii bacteria could improve the rumen fermentation of SARA goats induced by high starch diet, and alleviated the occurrence of acidosis to a certain extent.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.27
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1 趙培廳;日糧不同NFC/NDF比對奶山羊瘤胃發(fā)酵功能和微生物區(qū)系變化的影響[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2011年
2 魏德泳;瘤胃酸中毒發(fā)生的微生物學(xué)機制及阿卡波糖調(diào)控作用的研究[D];南京農(nóng)業(yè)大學(xué);2010年
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