【作者】 杨旭；

【导师】 史莹华；

【作者基本信息】 河南农业大学 ， 畜牧学， 2023， 硕士

【摘要】 国内养猪业规模的日渐扩大以及国际经济贸易形式的影响导致蛋白质饲料原料紧缺,“人畜争粮”矛盾日益严重。本课题组将苜蓿茎叶分离得到具有蛋白质含量高、氨基酸含量丰富的苜蓿叶粉,有望替代常规饲粮中的豆粕,但苜蓿叶粉作为一种蛋白质饲料原料对妊娠母猪繁殖性能、肠道微生物、机体代谢等的影响尚不清楚。本试验选取妊娠63日龄的60头初产母猪,按照健康状况良好、背膘厚基本一致的原则随机分为5组,分别为对照组(CTRL)、苜蓿叶粉25%替代豆粕组(ALM(25%))、苜蓿叶粉替代50%豆粕组(ALM(50%))、苜蓿叶粉替代75%豆粕组(ALM(75%))和苜蓿叶粉替代100%豆粕组(ALM(100%))。预饲期7天,妊娠70日龄试验正式开始,试验期间自由饮水。试验一:主要研究苜蓿叶粉替代不同比例饲粮豆粕对妊娠母猪繁殖性能及仔代哺乳期生长的影响。试验结果表明,1)苜蓿叶粉替代不同比例饲粮豆粕对妊娠期母猪背膘无显著影响(P>0.05),而ALM(100%)组母猪哺乳28天背膘厚显著降低(P<0.05)。2)ALM(50%)组、ALM(75%)组和ALM(100%)组母猪初生个体均重显著高于CTRL组和ALM(25%)组(P<0.05),ALM(100%)组母猪初生窝重极显著高于CTRL组(P<0.01);3)苜蓿叶粉替代不同比例饲粮豆粕能够改善初生仔猪体重分布,CTRL组、ALM(25%)组、ALM(50%)组、ALM(75%)组和ALM(100%)组仔猪体重在最佳生长发育能力范围即1.2～1.8 kg的比例分别为29.47%、58.49%、56.51%、60.00%和87.24%。4)各组仔猪根据初生个体重进行组间调整,确保五组调后个体均重、调后窝重无显著差异(P>0.05),而ALM(100%)组仔猪14d个体重、哺乳28d个体重及哺乳28d窝重高于CTRL组(P<0.05)。基于母猪繁殖性能及哺乳期仔猪生长性能的结果,初步得出结论,苜蓿叶粉替代妊娠母猪饲粮中豆粕的最佳比例为100%。试验二:主要研究苜蓿叶粉替代不同比例饲粮豆粕对母、仔猪机体抗氧化水平及炎症水平的影响。研究结果表明,1)妊娠85d时,ALM(100%)组母猪血清SOD水平极显著高于CTRL组(P<0.01),ALM(75%)组母猪血清GSH-Px酶活极显著高于其他各组(P<0.01);妊娠105d时,ALM(75%)组、ALM(100%)组母猪血清T-AOC以及SOD酶活极显著高于CTRL组和ALM(25%)组(P<0.01),ALM(100%)组母猪血清MDA水平显著低于CTRL组(P<0.05),CTRL组母猪GSH-Px酶活极显著低于各苜蓿叶粉替代组(P<0.01);哺乳28d时,CTRL组母猪血清T-AOC水平显著低于各苜蓿叶粉替代组(P<0.05),CTRL组和ALM(50%)组血清SOD酶活极显著低于ALM(75%)和ALM(100%)组(P<0.01),与CTRL组相比,ALM(75%)组母猪血清MDA水平显著降低(P<0.05),而ALM(25%)组、ALM(75%)组、ALM(100%)组母猪血清GSH-Px酶活极显著降低(P<0.01)。2)妊娠85d时,ALM(100%)组母猪血清IL-6水平极显著低于CTRL组(P<0.01),ALM(100%)组母猪血清IL-10水平极显著高于其他各组(P<0.01);各苜蓿叶粉替代组母猪血清ROS水平极显著低于CTRL组(P<0.01);ALM(75%)组、ALM(100%)母猪血清TNF-α水平极显著低于CTRL组和ALM(25%)组(P<0.01)。妊娠105d时,ALM(25%)组、ALM(75%)组、ALM(100%)组母猪血清IL-6水平极显著低于CTRL组(P<0.01),ALM(100%)组母猪血清IL-10水平极显著高于CTRL组、ALM(25%)组和ALM(50%)组(P<0.01);与CTRL组相比,各苜蓿叶粉替代组母猪血清ROS水平极显著降低(P<0.01);ALM(25%)组母猪血清TNF-α水平较CTRL组极显著提高(P<0.01)。哺乳28d时,与CTRL组相比,ALM(50%)组和ALM(100%)组母猪血清IL-6水平极显著降低(P<0.01);CTRL组和ALM(25%)组血清IL-10水平极显著低于ALM(75%)和ALM(100%)组(P<0.01);与CTRL组相比,ALM(25%)组和ALM(100%)组母猪血清ROS水平极显著提高(P<0.01),ALM(75%)组极显著降低(P<0.01)。3)仔猪哺乳28d时,与CTRL组仔猪相比,ALM(100%)组仔猪血清SOD酶活极显著提高(P<0.01);ALM(50%)组、ALM(75%)组、ALM(100%)组仔猪血清GSH-Px酶活极显著低于CTRL组和ALM(25%)组仔猪(P<0.01)。4)仔猪哺乳28d时,ALM(50%)组、ALM(75%)组、ALM(100%)组仔猪血清IL-6水平显著低于CTRL组(P<0.01);ALM(50%)组、ALM(75%)组、ALM(100%)组仔猪血清IL-10水平极显著高于CTRL组和ALM(25%)组(P<0.01)。妊娠期母猪及哺乳期母、仔猪机体抗氧化水平、炎症水平的结果表明,ALM(100%)对母、仔猪机体抗氧化水平、炎性水平具有更大的改善作用。试验三:主要研究苜蓿叶粉替代不同比例饲粮豆粕对母、仔猪粪便SCFAs和微生物区系的影响。经试验一、试验二结果分析得出苜蓿叶粉替代妊娠母猪饲粮豆粕的最佳比例为100%,因此使用CTRL组和ALM(100%)组母、仔猪粪便进行粪便SCFAs和微生物区系测定。试验结果表明,1)Alpha多样性分析得出,ALM(100%)组仔猪哺乳28d时Shannon指数显著高于CTRL组(P<0.05);由Beta多样性分析得到,妊娠后期,ALM(100%)组母猪粪便微生物和CTRL组母猪粪便微生物组成存在差异(P<0.01);在哺乳28d时,ALM(100%)组和CTRL组母、仔猪粪便微生物具有相似性(P>0.05)。2)粪便微生物门水平差异分析得到,妊娠105d时,ALM(100%)组母猪较CTRL组粪便Actinobacteriota的相对丰度极显著提高(P<0.01);哺乳28d时,ALM(100%)组仔猪粪便Desulfobacterota、Synergistota相对丰度较CTRL组仔猪显著降低(P<0.05)。3)粪便微生物属水平分析表明,母猪妊娠85d时,ALM(100%)组粪便微生物群中Clostridium＿sensu＿stricto＿1、Bifidobacterium细菌的的相对丰度显著高于CTRL组(P<0.05);母猪妊娠105d时,ALM(100%)组粪便Subdoligranulum的相对显著高于CTRL组(P<0.05),而Blautia的相对丰度极显著高于CTRL组(P<0.01);仔猪哺乳28d时,与CTRL相比,ALM(100%)组粪便Ruminococcus＿gauvreauii＿group的相对丰度显著升高(P<0.05),Desulfovibrio的相对丰度显著降低(P<0.05)。4)在母猪妊娠85d时,与CTRL组相比,ALM(100%)组母猪粪便中乙酸、丙酸、戊酸及异戊酸含量均极显著升高(P<0.01);母猪妊娠105d时,ALM(100%)组母猪粪便中乙酸、丙酸含量显著高于CTRL组(P<0.05),丁酸、戊酸含量极显著高于CTRL组(P<0.01);在母猪哺乳28d时,与CTRL组相比,ALM(100%)组粪便乙酸水平显著升高(P<0.05),丁酸、戊酸、异戊酸含量极显著提高(P<0.01),而丙酸含量极显著降低(P<0.01);在仔猪哺乳28d时,与CTRL组相比,ALM(100%)组粪便乙酸、丙酸、丁酸、戊酸含量极显著升高(P<0.01),异戊酸含量显著提高(P<0.05)。5)母、仔猪微生物垂直传递分析表明,苜蓿叶粉影响了norank＿f＿Selenomonadaceae、Ruminococcus＿gauvreauii＿group等菌属的垂直传递,改善了哺乳期仔猪的肠道健康。6)共享菌属同仔猪哺乳期生长的相关性分析表明,norank＿f＿Selenomonadaceae同仔猪粪便中乙酸、戊酸、异戊酸含量呈显著正相关关系(P<0.05),同仔猪哺乳14d个体重、哺乳28d个体重及哺乳28d窝重呈显著正相关关系(P<0.05),同仔猪血清MDA水平呈极显著负相关关系(P<0.01);Ruminococcus＿gauvreauii＿group同仔猪粪便中乙酸、丙酸、丁酸、戊酸、异戊酸等含量呈显著正相关关系(P<0.05),同仔猪血清中IL-6水平呈极显著负相关关系(P<0.01),同仔猪血清中MDA水平呈显著负相关关系(P<0.05)。母、仔猪粪便微生物及SCFAs含量的变化,证明了苜蓿叶粉能够改善母猪肠道健康,并通过母仔传递改善仔猪肠道健康,提高SCFAs含量,促进哺乳仔猪的生长发育。试验四:主要研究苜蓿叶粉替代饲粮豆粕对妊娠期、哺乳期母猪血浆代谢组的影响。经试验一、试验二结果分析得出苜蓿叶粉替代妊娠母猪饲粮豆粕的最佳比例为100%,因此使用CTRL组和ALM(100%)组母猪各阶段血浆进行测定。试验结果表明1)血浆代谢组PCA和OPLS-DA分析表明,妊娠期两组母猪CTRL组和ALM(100%)组在阴、阳离子模式下均表现出分离,而哺乳期两组母猪血浆代谢组在阴、阳离子模式下均有重合部分。2)妊娠85d时,母猪血浆中组氨酸代谢、β-丙氨酸代谢途径中的精胺、肌肽水平极显著升高(P<0.01),嘧啶代谢途径中胸腺嘧啶水平极显著升高(P<0.01);妊娠105d时,叶酸生物合成途径中的7-氨甲基-7-氨基鸟嘌呤、对羟基苯甲酸水平极显著升高(P<0.01),D-精氨酸和D-鸟氨酸代谢途径中的L-精氨酸水平极显著降低(P<0.01);母猪哺乳28d时,血浆中嘧啶代谢途径中胸腺嘧啶水平极显著升高(P<0.01),β-丙氨酸水平极显著降低(P<0.01),精氨酸生物合成途径中瓜氨酸水平极显著升高(P<0.01))。3)母猪繁殖性能-血浆代谢组学-母猪粪便微生物的相关性分析表明,妊娠105d时有益菌Blautia、Subdoligranulum同平均活仔重呈极显著正相关关系(P<0.01),同初生窝重呈显著正相关关系(P<0.05);同母猪血浆代谢物中的7-氨甲基-7-氨基鸟嘌呤呈极显著负相关关系(P<0.01),同对羟基苯甲酸呈显著负相关关系(P<0.05)。综上所述,使用苜蓿叶粉替代妊娠母猪饲粮中的豆粕能够改善母猪机体抗氧化能力,降低炎症水平,提高母猪分娩时仔猪初生重及初生窝重,改善初生仔猪体重分布范围,苜蓿叶粉100%替代豆粕效果最佳。同时粪便微生物和母猪血浆代谢组分析发现,苜蓿叶粉能够调控母猪微生物区系,增加短链脂肪酸的产生,能够改善母猪机体的诸多代谢通路,在提高母体抗氧化、降低母猪炎症以及促进胚胎生长及仔猪生长方面发挥有益作用。更多还原

【Abstract】 The increasing expansion of pig industry and the influence of strict international economic and trade forms had led to the shortage of domestic protein materials in feed,and the contradiction of"food competition between humans and livestock"had become increasingly serious.Alfalfa leaf meal with high protein content and rich amino acid content was obtained by seprating alfalfa stems and leaves in our research group,which is expected to replace soybean meal in conventional feed.However,the effects of alfalfa leaf meal as a protein feed source on productive performance,gut microbiota and metabolism of pregnant sows are still unclear.Sixty primiparous sows at 63 days of gestation were randomly divided into five groups according to the principle of good health and consistent backfat thickness.They were divided into control group(CTRL),ALM(25%)group,ALM(50%)group,ALM(75%)group and ALM(100%)group.The prefeeding period was 7 days,and the test was officially started at 70 days of gestation,during which water was consumed AD libitum.Experiment 1:The effects was investigated of different proportions with alfalfa leaf meal replacing soybean meal in pregnancy feedstuff on reproductive performance of sows and growth of their offspring during lactation.The results showed as follows:1)Replacement of dietary soybean meal with alfalfa leaf meal had no significant effect on the backfat thickness of sows during pregnancy(P<0.05),while the backfat thickness of sows in ALM(100%)group was significantly reduced on the 28th day of lactation(P<0.05).2)The average weights of newborns in ALM(50%),ALM(75%)and ALM(100%)groups were significantly higher than those in CTRL and ALM(25%)groups(P<0.05),and the litter weights at birth in ALM(100%)group were significantly higher than those in CTRL group(P<0.01).3)Replacement of dietary soybean meal with alfalfa leaf meal in different proportions can improve the body weight distribution of newborn piglets,and the proportions of piglets in the optimal growth and development ability range(1.2-1.8 kg)in CTRL,ALM(25%),ALM(50%),ALM(75%)and ALM(100%)groups were29.47%,58.49%,56.51%,60.00%and 87.24%,respectively.4)The piglets in each group were adjusted according to the birth weight to ensure that there was no significant difference in individual weight and litter weight after adjustment among the five groups(P<0.05),while the body weight in 14 days in lactation,body weight and litter weight in 28 days in lactation of ALM(100%)group were higher than those of CTRL group(P<0.05).Based on the analysis of reproductive performance of sows and growth performance of piglets during lactation,it was preliminarily concluded that the optimal ratio of alfalfa leaf meal to replace soybean meal in diet of pregnant sows was 100%.Experiment 2:The effects of of alfalfa leaf meal replacing different proportions dietary soybean meal on antioxidant levels and inflammation levels of sows and piglets were investigated.The results showed as following:1)On 85th day of pregnancy,the serum SOD level of ALM(100%)group was significantly higher than that of CTRL group(P<0.01).The serum GSH-Px enzyme activity in ALM(75%)group was significantly higher than that in other groups(P<0.01).The T-AOC and the activities of SOD in ALM(75%)and ALM(100%)groups were significantly higher than those in CTRL and ALM(25%)groups on 105th day of pregnancy(P<0.01).Serum MDA level in ALM(100%)group was significantly lower than that in CTRL group(P<0.05).The enzyme activity of GSH-Px in CTRL group was extremely significantly lower than that in all alfalfa leaf meal groups(P<0.01).At 28th day of lactation,the serum T-AOC level of sows in CTRL group was significantly lower than that in all alfalfa leaf meal groups(P<0.05),and the serum SOD activity of sows in CTRL group and ALM(50%)group was significantly lower than that in ALM(75%)and ALM(100%)group(P<0.01).Compared with CTRL group,the serum MDA level was significantly decreased in ALM(75%)group(P<0.05),and the activity of serum GSH-Px was significantly decreased in ALM(25%),ALM(75%)and ALM(100%)groups(P<0.01).2)On 85th day of pregnancy,the serum level of IL-6 in ALM(100%)group was extremely significantly lower than that in CTRL group(P<0.01),and the serum level of IL-10 in ALM(100%)group was extremely significantly higher than that in other groups(P<0.01).The serum ROS level of sows in all alfalfa leaf meal groups was significantly lower than that in CTRL group(P<0.01).The levels of serum TNF-αin ALM(75%)and ALM(100%)groups were extremely significantly lower than those in CTRL and ALM(25%)groups(P<0.01).On 105th day of gestation,serum IL-6 levels in ALM(25%),ALM(75%)and ALM(100%)groups were extremely significantly lower than those in CTRL group(P<0.01).The serum IL-10 content in ALM(100%)group was extremely significantly higher than that in CTRL group,ALM(25%)group and ALM(50%)group(P<0.01).Compared with the CTRL group,the serum ROS level of sows in all alfalfa leaf meal groups was significantly decreased(P<0.01).The serum level of TNF-αin ALM(25%)group was extremely significantly higher than that in CTRL group(P<0.01).On lactation day 28,compared with the CTRL group,the ALM(50%)and ALM(100%)groups had extremely significantly lower IL-6 levels(P<0.01),and the CTRL and ALM(25%)groups had extremely significantly lower IL-10 levels than ALM(75%)and ALM(100%)groups(P<0.01)in serum.Compared with the CTRL group,the level of ROS was significantly increased in ALM(25%)and ALM(100%)groups(P<0.01),and extremely significantly decreased in ALM(75%)group(P<0.01).3)Compared with the CTRL group,the activity of serum SOD in ALM(100%)group was extremely significantly increased on lactation day 28(P<0.01).The serum GSH-Px activity in ALM(50%),ALM(75%)and ALM(100%)groups was extremely significantly lower than that in CTRL and ALM(25%)groups(P<0.01).4)On lactation day 28,serum IL-6 levels in ALM(50%),ALM(75%)and ALM(100%)groups were extremely significantly lower than that in CTRL group(P<0.01).The serum IL-10 levels in ALM(50%),ALM(75%)and ALM(100%)groups were significantly higher than those in CTRL and ALM(25%)groups(P<0.01).The results of antioxidant levels and inflammation levels of sows and piglets showed that ALM(100%)had a greater improvement effect on the antioxidant levels and inflammation levels of sows and piglets.Experiment 3:The effects of replacing dietary soybean meal with alfalfa leaf meal at different proportions on fecal SCFAs and microflora of sows and piglets were investigated.The results of experiment1 and 2 showed that the optimal ratio of alfalfa leaf meal to replace dietary soybean meal for pregnant sows was 100%.Therefore,SCFAs and microbiota were determined using the feces of sows and piglets in CTRL group and ALM(100%)group.The results showed as following:1)According to the analysis of fecal microorganism,the Shannon index of ALM(100%)group was significantly higher than that of CTRL group on 28th day in lactation of piglets(P<0.05).And Beta diversity analysis showed that,there was a significant difference in fecal microbial composition between ALM(100%)group and CTRL group on gestation(P<0.05).On 28th day in lactation of sows and piglets,the fecal microbiota of ALM(100%)group and CTRL group were similar(P>0.05).2)The analysis of fecal microbial in Phylum level showed that,compared with the CTRL group,the ALM(100%)group had a significantly higher relative abundance of Actinobacteriota in the feces of sows on day 105 of pregnancy(P<0.01).On 28th day in lactation,the relative abundance of Desulfobacterota and Synergistota in feces of piglets in ALM group was significantly lower than that of CTRL group(P<0.05).3)The analysis of fecal microbial in Genus level showed that the relative abundance of Clostridium＿sensu＿stricto＿1 and Bifidobacterium in ALM(100%)group was significantly higher than that in CTRL group on day 85 of pregnant sows(P<0.05).On 105th day of gestation sows,the relative abundance of Subdoligranulum was significantly higher than that of the CTRL group(P<0.05),while the relative abundance of Blautia was significantly higher than that of the CTRL group(P<0.01).On lactation day 28,compared with CTRL group,the relative abundance of Ruminococcus＿gauvreauii＿group was significantly increased(P<0.05)and the relative abundance of Desulfovibrio was significantly decreased(P<0.05)in feces of piglets in ALM(100%)group.4)Compared with the CTRL group,the sows in ALM(100%)group had extremely significantly higher fecal contents of acetic acid,propionic acid,valeric acid and isovaleric acid in 85th day of pregnancy(P<0.01).In the sows of 105th day in pregancy,the contents of acetic acid and propionic acid in ALM(100%)group were significantly higher than those in CTRL group(P<0.05),and the contents of butyric acid and valaric acid in ALM(100%)group were extremely significantly higher than those in CTRL group(P<0.01).Compared with the CTRL group,the ALM(100%)group had a significantly higher fecal acetic acid content(P<0.05),extremely significantly higher fecal contents of butyric acid,valeric acid,and isovaleric acid(P<0.01),and a significantly lower fecal propionic acid content(P<0.01)at day 28 in lactation of sows.Compared with the CTRL group,the ALM(100%)group had extremely significantly higher contents of acetic acid,propionic acid,butyric acid,and valeric acid(P<0.01)and significantly higher content of isovaleric acid(P<0.05)in feces of piglets at 28 days in lactation.5)Analysis of vertical transmission of sows and piglets showed that alfalfa leaf meal affected vertical transmission of norank＿f＿Selenomonadaceae and Ruminococcus＿gauvreauii＿group and improved intestinal health of lactating piglets.6)The correlation analysis between the shared bacteria and the growth of piglets during lactation showed that norank＿f＿Selenomonadaceae was significantly positively correlated with the content of acetate,valerate and isovaleric acid in feces of piglets(P<0.05),and was the same as the body weight of piglets in 14 days of lactation There was a significant positive correlation between norank＿f＿Selenomonadaceae and body weight of 28d and litter weight of 28d(P<0.05),and a very significant negative correlation with serum MDA level of piglets(P<0.01);Ruminococcus＿gauvreauii＿group had a significant positive correlation with acetic,propionate,butyrate,valeric,and isovaleric acid in piglet feces There was a significant positive correlation between Ruminococcus＿gauvreauii＿group and serum IL-6 level of piglets(P<0.05),and the bacteria had a significant negative correlation with serum MDA level of piglets(P<0.05).The changes of fecal microorganisms and SCFAs contents of sows and piglets indicate that alfalfa leaf meal can improve intestinal health and SCFAs content of sows,improve intestinal health of piglets through mother-to-calf vertical transmission,and promote the growth and development of Suckling piglets.Experiment 4:To investigate the effects of alfalfa leaf meal replacing soybean meal on plasma metabolome of sows during pregnancy and lactation.The results of experiment 1 and 2 showed that the optimal ratio of alfalfa leaf meal to replace dietary soybean meal for pregnant sows was 100%,so the plasma of sows in CTRL group and ALM(100%)group were used for determination.The results showed as follows:1)PCA analysis and OPLS-DA analysis of plasma metabolome showed the separation in negative and positive modes between CTRL group and ALM(100%)group during pregnancy,while the plasma metabolome overlapped in negative and positive modes during lactation.2)Compared with CTRL group,the levels of spermine and carnosine in the histidine metabolism andβ-alanine metabolism pathway were extremely significantly increased(P<0.01),while the level of thymidine was extremely significantly increased(P<0.01)in the plasma of sows in ALM(100%)group on day 85 of pregnancy.The levels of 7-aminomethyl-7-aminoguanine and P-hydroxybenzoic acid in folate biosynthesis pathway were extremely significantly increased(P<0.01),while the level of L-arginine in D-arginine and D-ornithine metabolism pathway were extremely significantly decreased(P<0.01)on 105 days of gestation.At 28 days of lactation,the level of thymine in the pyrimidine metabolism pathway was significantly increased(P<0.05),the level ofβ-alanine was significantly decreased(P<0.01),the level of citrulline in the arginine biosynthesis pathway was extremely significantly increased(P<0.01).3)The correlation analysis of microorganisms,metabolites,sow reproductive performance showed that,on the 105th day of pregnancy,Blautia and Subdoligranulum were extremely significantly positively correlated with the average weight of newborns(P<0.01)and the litter weight at birth(P<0.05).There was an extremely significant negative correlation between Blautia,Subdoligranulum and 7-aminomethyl-7-aminoguanine(P<0.01)and P-hydroxybenzoic acid(P<0.05).In conclusion,using alfalfa leaf meal to replace soybean meal in the diet of pregnant sows can improve the antioxidant capacity of sows,reduce inflammation level,increase birth weight and litter weight of piglets at delivery,improve the weight distribution range of newborn piglets,and make more piglets within the optimal growth range.The effect of 100%replacement of soybean meal by alfalfa leaf meal is the best Meanwhile,fecal microbial and plasma metabolome analysis of sows showed that alfalfa leaf meal could regulate the microflora of sows,increase the production of short-chain fatty acids,improve many metabolic pathways of sows,and play a beneficial role in improving maternal antioxidant,reducing inflammation of sows,and promoting the growth of embryos and piglets.更多还原