【作者】 张鑫；

【导师】 张莉莉；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2023， 硕士

【摘要】 宫内发育迟缓(intrauterine growth retardation,IUGR)指怀孕期间哺乳动物胚胎/胎儿或其器官生长发育受损,未能达到遗传学上生长潜能的现象。大量的研究表明,IUGR会对仔猪的肝脏形态造成损伤,并引起其线粒体功能障碍、能量代谢功能减弱和脂代谢功能紊乱等,从而对仔猪早期的生长发育造成不良影响。丁酸梭菌是一种广泛存在于动物粪便、土壤、干酪中的革兰氏阳性厌氧菌,已被证实可以提高动物的生长性能,改善脂代谢和增强机体的免疫功能。但丁酸梭菌的直接靶器官是肠道,它对肝脏代谢的具体调节机制仍有待进一步研究。因此,本试验试图从肠肝轴角度,研究丁酸梭菌对IUGR哺乳仔猪生长发育、肝脏线粒体功能和脂代谢的影响及相关机制,为IUGR猪早期的营养调控及丁酸梭菌在动物生产中的应用提供理论依据。1丁酸梭菌对IUGR哺乳仔猪生长性能、肝脏线粒体功能及能量代谢的影响试验一选取8头正常仔猪和16头IUGR仔猪,将它们分为3组:NBW-CON组、IUGR-CON组和IUGR-CB组,每组8个重复。NBW-CON组和IUGR-CON组仔猪每天灌服10 m L生理盐水,IUGR-CB组仔猪每天灌服10 m L含2×10~8 CFU/kg体重的丁酸梭菌菌液。试验从3日龄开始,试验期为21天。结果表明:(1)IUGR显著降低了24日龄仔猪的体重(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组仔猪24日龄的体重和ADG相较IUGR-CON组均显著提高(P<0.05)。(2)与NBW-CON组相比,IUGR-CON组仔猪的肝脏和肾脏重显著降低(P<0.05)。与IUGR-CON组相比,IUGR-CB组仔猪的肾脏重显著增加(P<0.05)、心脏指数显著降低(P<0.05)。(3)与NBW-CON组相比,IUGR-CON组仔猪肝脏出现了中央门静脉充血、肝索排列松散、肝血窦扩张的现象。而在灌服了丁酸梭菌后,仔猪肝脏的形态得到明显改善。(4)与NBW-CON组相比,IUGR-CON组仔猪肝脏的线粒体数量显著减少,且出现了线粒体嵴肿胀和囊泡化的现象。在灌服了丁酸梭菌后,IUGR-CB组仔猪肝脏线粒体的密度显著提高、IMJs的比例增加、线粒体嵴肿胀及囊泡化的现象消失。(5)IUGR显著降低了仔猪肝脏线粒体复合物Ⅱ和复合物Ⅴ的活性(P<0.05)。在补充了丁酸梭菌后,仔猪肝脏线粒体复合物Ⅰ和复合物Ⅱ的活性相较IUGR-CON组显著提高(P<0.05)(6)与NBW-CON组相比,IUGR-CON组仔猪血清中的葡萄糖含量显著降低(P<0.05),肝脏中ATP和NADH的含量显著降低(P<0.05),IDH、α-KGDH和MDH的活力显著升高(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组仔猪血清的葡萄糖水平极显著升高(P<0.001)、肝脏中NADH的活性显著提高(P<0.05),而IDH、α-KGDH和MDH的活力显著下降(P<0.05)。(7)与NBW-CON组相比,IUGR显著降低了仔猪肝脏AMPK、NRF1、TFAM、ERRα、m TOR和FOXO1的m RNA表达量(P<0.05)。在灌服丁酸梭菌后,IUGR-CB组仔猪肝脏AMPK、SIRT1、PGC-1α、NRF1、TFAM和ERRα的m RNA表达较IUGR-CON组显著提高(P<0.05)。由此可知,丁酸梭菌可以通过改善IUGR哺乳仔猪的肝脏形态及其线粒体功能,提高仔猪能量代谢的效率,从而促进其生长发育。2丁酸梭菌对IUGR哺乳仔猪肝脏脂肪酸代谢的影响试验二设计同试验一。结果表明:(1)与NBW-CON组相比,IUGR-CON组仔猪血清中NEFA的含量显著升高(P<0.05),肝脏HL和TL的活性显著下降(P<0.05)。在补充了丁酸梭菌后,IUGR-CB组仔猪血清中NEFA的水平显著下降(P<0.05),肝脏中TG、HL和TL的含量显著升高(P<0.05)。(2)与NBW-CON组仔猪相比,IUGR显著降低了仔猪肝脏CAV1的m RNA表达(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组仔猪肝脏CD36和CAV1的基因表达显著升高(P<0.05)。(3)IUGR-CON组仔猪肝脏DGAT1和DGAT2的m RNA表达较NBW-CON组显著降低(P<0.05),补充了丁酸梭菌后,IUGR-CB组仔猪肝脏ACC、DGAT1和DGAT2的表达量显著升高(P<0.05)。(4)与NBW-CON组相比,IUGR显著降低了仔猪肝脏LCAD的m RNA表达(P<0.05)。而在IUGR-CB组中,仔猪肝脏FXR、CPT1、PPARα、LCAD及ACOX的基因表达相较IUGR-CON组显著升高(P<0.05)。(5)与NBW-CON组相比,IUGR-CON组仔猪肝脏PPARα的蛋白表达显著降低(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组仔猪肝脏FXR和PPARα的蛋白表达相较IUGR-CON组显著提高(P<0.05)。由此可知,丁酸梭菌可以通过提高IUGR哺乳仔猪肝脏中与脂肪酸摄取、合成及氧化相关基因和蛋白的表达来改善仔猪的脂肪酸代谢紊乱,并降低其血清NEFA的水平。3丁酸梭菌对IUGR哺乳仔猪肝脏胆固醇和胆汁酸代谢的影响试验三设计同试验一。结果表明:(1)IUGR显著降低了仔猪血清HDL-C和肝脏TBA的水平(P<0.05),并提高了仔猪肝脏TC的水平(P<0.05)。在补充了丁酸梭菌后,IUGR-CB组仔猪血清中HDL-C、TBA和肝脏TBA的含量显著增加(P<0.05),且肝脏中TC的水平显著降低(P<0.05)。(2)与NBW-CON组相比,IUGR-CON组仔猪肝脏SREBF2的基因表达显著升高(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组仔猪肝脏SREBF2的表达显著下降(P<0.05),而LXRα、ABCA1、ABCG8和SCARB1的m RNA表达量显著升高(P<0.05)。(3)IUGR显著降低了仔猪肝脏中NTCP、OATP和OCT1的m RNA表达量(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组仔猪肝脏中NTCP、OATP和OCT1的m RNA表达量相较IUGR-CON组显著提高(P<0.05)。(4)IUGR显著降低了仔猪肝脏中CYP27A1和CYP7B1的m RNA表达量(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组仔猪肝脏CYP7A1、CYP27A1、CYP8B1和CYP7B1的m RNA表达量较IUGR-CON组显著升高(P<0.05)。(5)IUGR不会影响仔猪肝脏中与胆汁酸转运相关基因的表达。在添加了丁酸梭菌后,IUGR-CB组仔猪肝脏BSEP、MRP2和MDR3的m RNA表达量较IUGR-CON组显著提高(P<0.05)。(6)IUGR可以显著提高仔猪肝脏HMGCR和HMGCS的含量并降低CYP27A1的蛋白表达(P<0.05)。灌服丁酸梭菌后,IUGR-CB组仔猪肝脏HMGCR的含量显著下降,而LXRα、CYP7A1和CYP27A1的蛋白表达量显著提高(P<0.05)。由此可知,丁酸梭菌可以通过减少胆固醇的合成并促进胆固醇的外排及其向胆汁酸的转化来降低IUGR哺乳仔猪肝脏的胆固醇水平。4丁酸梭菌对IUGR哺乳仔猪回肠微生物组成及胆汁酸代谢谱的影响试验四设计同试验一。结果表明:(1)NBW-CON组与IUGR-CON组、IUGR-CON组与IUGR-CB组仔猪回肠微生物在α多样性上并无显著差异(P>0.05)。而IUGR-CB组回肠菌群的Simpson指数显著低于NBW-CON组(P<0.05)。(2)β多样性分析结果表明,NBW-CON组与IUGR-CON组仔猪回肠菌群并无显著差异(P>0.05),但IUGR-CON组与IUGR-CB组回肠菌群结构有显著差异(P<0.05)。(3)在门水平上,各组仔猪回肠食糜的优势菌门均为Firmicutes和Proteobacteria。在属水平上,NBW-CON组的优势菌属为Lactobacillus、Veillonella和Actinobacillus,IUGR-CON组的优势菌属为Lactobacillus、Streptococcus和Escherichia,IUGR-CB组的优势菌属为Lactobacillus、Actinobacillus和Escherichia。各组回肠菌群组成在门水平上并无显著差异(P<0.05)。在属水平上,IUGR-CON组仔猪回肠食糜中Streptococcus、Enterococcus和Moraxella的相对丰度较NBW-CON组显著升高(P<0.05)。在灌服了丁酸梭菌后,IUGR-CB组Streptococcus、Enterococcus、Rothia、Moraxella和Acinetobacter的相对丰度显著下降(P<0.05)。(4)THCA、THDCA、GHCA和GHDCA是仔猪回肠食糜中主要的结合型胆汁酸,而HCA、HDCA和CDCA是回肠食糜中主要的非结合型胆汁酸。仔猪肝脏中绝大多数是结合型胆汁酸,且GHDCA、GHCA、GCDCA、THCA和TUDCA为主要的胆汁酸。(5)IUGR-CON组仔猪回肠内容物中结合型胆汁酸的比例较NBW-CON组显著下降(P<0.05),而IUGR-CB组结合型胆汁酸和非结合型胆汁酸的比值较IUGR-CON组显著提高(P<0.05)。这具体表现为IUGR-CON组仔猪回肠中结合型胆汁酸THCA、GHCA、TCA和GCA的含量较NBW-CON组显著下降(P<0.05),而非结合型胆汁酸HDCA、6-keto LCA和DCA的浓度显著升高(P<0.05)。在补充了丁酸梭菌后,IUGR-CB组仔猪回肠内容物中THCA、GHCA、TCDCA)和TCA的含量显著提高(P<0.05)、6-keto LCA的含量显著降低(P<0.05)。(6)IUGR显著降低了仔猪肝脏中TUDCA的浓度(P<0.05),在灌服了丁酸梭菌后,IUGR-CB组仔猪肝脏中THCA、TUDCA、TCDCA的含量显著升高(P<0.05),GUDCA的浓度显著降低(P<0.05)。由此可知,丁酸梭菌灌服可以显著降低仔猪回肠微生物中部分条件致病菌的相对丰度。其中,Streptococcus和Enterococcus具有胆盐水解酶活性,它们丰度的降低可以增加回肠食糜和肝脏中THCA和TCDCA等结合型胆汁酸的含量。综上所述,IUGR会降低哺乳仔猪的生长性能,并对仔猪肝脏的肝脏形态结构和功能造成损伤。丁酸梭菌可以显著提高仔猪的生长性能,改善肝脏的线粒体功能,缓解仔猪的脂代谢紊乱。更多还原

【Abstract】 Intrauterine growth retardation(IUGR)can be defined as a phenomenon that the growth and development of the mammalian embryo/fetus or its organs are impaired and fail to reach their growth potential during pregnancy.A large number of studies have shown that IUGR can damage the liver morphology of neonatal piglets,and cause mitochondrial dysfunction,energy metabolism miopragia and lipid disorders.As a result,their growth and development can be adversely affected at the early stage.Clostridium butyricum is a kind of Gram-positive anaerobe that widely exists in animal feces,soil and cheese.It has been proved that it can improve the growth performance,lipid metabolism and immunity of animals.However,as a probiotic,the direct target of C.butyricum is the gut,and its regulatory mechanism on hepatic metabolism remains further study.Therefore,our study aimed to investigate the effects of C.butyricum on the growth and development,hepatic mitochondrial function and lipid metabolism of IUGR suckling piglets from the perspective of gut-liver axis,and to provide theoretical basis for the nutritional regulation of IUGR piglets and the application of C.butyricum in livestock production.1 Effects of supplemental C.butyricum on growth performance,mitochondrial function and energy metabolism of IUGR suckling pigletsEight normal piglets and sixteen IUGR piglets were selected,and then divided into 3groups:NBW-CON group,IUGR-CON group and IUGR-CB group(n=8).NBW-CON piglets and IUGR-CON piglets received 10 m L sterile saline per day,and IUGR-CB piglets received 10 m L bacterial fuid(containing 2×10~8 CFU/kg BW C.butyricum)per day.The trial started at 3 d of age and lasted for 21 d.The results showed that:(1)IUGR significantly decreased the body weight of piglets at 3 and 24 d of age(P<0.05).After C.butyricum supplementation,the body weight and ADG of piglets at 24 days of age were significantly increased(P<0.05).(2)Compared with the NBW-CON group,the weight of liver and kidney of piglets in IUGR-CON group were significantly decreased(P<0.05).Compared with the IUGR-CON group,kidney weight of piglets was increased and heart index was decreased in IUGR-CB group(P<0.05).(3)Compared with NBW-CON group,congestive central vein,loosely arranged cords and dilated sinusoids were observed in the liver of piglets in IUGR-CON group.However,C.butyricum supplementation effectively ameliorated these conditions.(4)Compared with the NBW-CON group,the number of mitochondria was obviously decreased,and vesiculated mitochondria and swollen mitochondrial cristae were observed in the liver of IUGR-CON piglets.After C.butyricum supplementation,mitochondrial density and the proportion of IMJs in the liver were significantly increased and the phenomenon of mitochondrial cristae swelling and vesiculation disappeared in the IUGR-CB piglets.IUGR significantly decreased the activities of hepatic mitochondrial complexⅡand complexⅤ(P<0.05).After C.butyricum supplementation,the activities of hepatic mitochondrial complexⅠand complexⅡof piglets were significantly increased compared with those of IUGR-CON group.Compared with the NBW-CON group,the content of GLU in the serum of IUGR-CON piglets was significantly decreased(P<0.05);the contents of ATP and NADH were significantly decreased(P<0.05),and the activities of IDH,α-KGDH and MDH were significantly increased in the liver(P<0.05).After C.butyricum supplementation,serum GLU level of IUGR-CB piglets was significantly increased(P<0.001);the activity of NADH was significantly increased(P<0.05),while the activity of IDH,α-KGDH and MDH were decreased(P<0.05).(7)IUGR significantly decreased the m RNA expressions of AMPK,NRF1,TFAM,ERRα,m TOR and FOXO1 in liver of piglets(P<0.05).C.butyricum supplementation significantly increased the hepatic m RNA expressions of AMPK,SIRT1,PGC-1α,NRF1,TFAM and ERRαof IUGR-CB piglets(P<0.05).Therefore,C.butyricum can accelerate the energy metabolism of IUGR Suckling piglets by improving their liver morphology and mitochondrial function,thus promoting their growth and development.2 Effects of supplemental C.butyricum on hepatic fatty acid metabolism of IUGR suckling pigletsThe design of experiment 2 was the same as that of experiment 1.The results showed that:(1)The content of serum NEFA was significantly increased(P<0.05),while the activities of hepatic HL and TL were significantly decreased in the IUGR-CON piglets,in comparison with the NBW-CON piglets(P<0.05).C.butyricum supplementation significantly decreased the IUGR-CB piglets’serum NEFA level(P<0.05),and increased their contents of hepatic TG,HL and TL(P<0.05).(2)IUGR significantly decreased the m RNA expression of CAV1 in liver of piglets(P<0.05).After C.butyricum supplementation,the hepatic expressions of CD36 and CAV1 were obviously improved in the IUGR-CB piglets(P<0.05).(3)The m RNA expressions of DGAT1 and DGAT2 in liver of IUGR-CON piglets were significantly decreased compared with those in NBW-CON group(P<0.05).C.butyricum supplementation significantly increased the m RNA expressions of ACC,DGAT1 and DGAT2 in the liver of IUGR-CB piglets(P<0.05).(4)IUGR significantly decreased the m RNA expression of LCAD in the liver of piglets(P<0.05).After C.butyricum supplementation,the gene expressions of FXR,CPT1,PPARα,LCAD and ACOX in the liver of IUGR-CB piglets were significantly increased compared with those in IUGR-CON group(P<0.05).(5)Compared with the NBW-CON group,the protein expression of hepatic PPARαwas lower in IUGR-CON piglets(P<0.05).C.butyricum supplementation significantly increased the protein expressions of FXR and PPARαin liver of IUGR-CB piglets compared with those in IUGR-CON group(P<0.05).Therefore,C.butyricum can improve the fatty acid metabolism and reduce the serum NEFA level of IUGR suckling piglets by increasing the expression of genes and proteins related to fatty acid uptake,synthesis and oxidation in liver.3 Effects of supplemental C.butyricum on hepatic cholesterol and bile acid metabolism of IUGR suckling pigletsThe design of experiment 3 was the same as that of experiment 1.The results showed that:(1)IUGR significantly decreased serum HDL-C and hepatic TBA levels,and increased hepatic TC content of piglets(P<0.05).After C.butyricum supplementation,the level of HDL-C in serum and the content of TBA in serum and liver were significantly increased,and the content of hepatic TC was significantly decreased in the IUGR-CB piglets(P<0.05).(2)Compared with the NBW-CON group,the m RNA expression of SREBF2 in liver of IUGR-CON piglets was significantly increased(P<0.05).After C.butyricum supplementation,SREBF2 expression was significantly decreased(P<0.05),while the m RNA expressions of LXRα,ABCA1,ABCG8 and SCARB1 were significantly increased in the liver of IUGR-CB piglets(P<0.05).(3)IUGR significantly decreased the m RNA expressions of NTCP,OATP and OCT1 in liver of piglets(P<0.05).C.butyricum supplementation significantly increased the piglets’m RNA expressions of NTCP,OATP and OCT1(P<0.05).(4)Piglets in the IUGR-CON group showed lower m RNA expression of CYP27A1 and CYP7B1 in liver of piglets(P<0.05).After C.butyricum supplementation,m RNA expressions of CYP7A1,CYP27A1,CYP8B1 and CYP7B1 in liver of IUGR-CB piglets were significantly increased(P<0.05).(5)There was no difference between NBW-CON group and IUGR-CON group in the expression of hepatic genes related to bile acid transport.C.butyricum supplementation significantly increased the m RNA expressions of BSEP,MRP2 and MDR3 in liver of IUGR-CB piglets(P<0.05).(6)In comparison with the NBW-CON group,IUGR-CON piglets showed significant higher contents of HMGCR and HMGCS(P<0.05)and lower protein expression of CYP27A1(P<0.05).After C.butyricum supplementation,the content of HMGCR was significantly decreased(P<0.05),while the protein expressions of LXRα,CYP7A1 and CYP27A1 were significantly increased in the liver of IUGR-CB piglets(P<0.05).Therefore,C.butyricum can lower cholesterol levels in the liver of IUGR suckling piglets by decreasing cholesterol synthesis,and promoting cholesterol efflux and its conversion to bile acids.4 Effects of supplemental C.butyricum on ileal microbial composition and bile acid profile of IUGR suckling pigletsThe design of experiment 4 was the same as that of experiment 1.(1)There was no signifcant diference noted in theα-diversity among the three groups.However,the IUGR-CB group’s Simpson index was lower than that of the NBW-CON group(P<0.05).(2)β-diversity analysis showed that there was no signifcant difference in ileal microbiota between NBW-CON and IUGR-CON group,and C.butyricum supplementation did make a diference(P<0.05).(3)At the phylum level,Firmicutes,Proteobacteria and Bacteroidetes predominantly constituted the ileal microbiota of the piglets,and no signifcant diference was found among the groups.At the genus level,Lactobacillus,Veillonella and Actinobacillus were the dominant genera in the NBW-CON group,while in the IUGR-CON group,Lactobacillus,Streptococcus and Escherichia accounted for the majority of the bacteria.In the IUGR-CB group,the Lactobacillus,Actinobacillus and Escherichia genera dominated.Compared to the NBW-CON group,the IUGR-CON group had a relatively higher abundance of Streptococcus,Enterococcus and Moraxella(P<0.05);however,piglets that received C.butyricum had clearly less(P<0.05)Streptococcus,Enterococcus,Rothia,Moraxella and Acinetobacter compared to piglets in the IUGR-CON group.(4)THCA,THDCA,GHCA and GHDCA accounted for the largest proportion of conjugated BAs in the ileum,while HCA,HDCA and CDCA represented the major unconjugated BAs in the ileum.The majority of the hepatic BAs were conjugated BAs,and GHDCA,GHCA,GCDCA,THCA and TUDCA were the primary conjugated BAs found.(5)Compared with the NBW-CON group,the proportion of conjugated BAs was significantly decreased in ileal chyme of IUGR-CON piglets(P<0.05),while the ratio of conjugated BAs to unconjugated BAs in IUGR-CB group was significantly increased(P<0.05).Thereinto,compared to the NBW-CON group,the contents of THCA,GHCA,TCA and GCA were decreased(P<0.05),and the levels of HDCA,6-keto LCA and DCA were increased(P<0.05)in the ileum of IUGR-CON piglets.C.butyricum supplementation significantly increased(P<0.05)the content of THCA,GHCA,TCDCA and TCA,and decreased(P<0.05)the concentration of 6-keto LCA in the liver of IUGR-CB piglets.(6)IUGR significantly decreased(P<0.05)the level of hepatic TUDCA compared to NBW-CON piglets.After C.butyricum supplementation,the contents of THCA,TUDCA and TCDCA were increased(P<0.05),and the level of GUDCA was decreased(P<0.05)in the liver of IUGR-CB piglets.Therefore,C.butyricum can significantly reduce the relative abundance of some opportunistic pathogens in the ileum of IUGR suckling piglets.Thereinto,Streptococcus and Enterococcus were BSH-producing microbes,and their decrease can increase the ileal and hepatic content of THCA and TCDCA of IUGR suckling piglets.In conclusion,IUGR decreased the growth performance of suckling piglets,and caused damage to their morphological structure and function of the liver.However,C.butyricum supplementation significantly improved the growth performance,hepatic mitochondrial function and lipid metabolism of the piglets.更多还原