【作者】 张春梅；

【导师】 侯生珍； 桂林生；

【作者基本信息】 青海大学 ， 畜牧学， 2022， 硕士

【摘要】 本试验研究不同NFC/NDF水平日粮对青海黑藏羊瘤胃发育及瘤胃内环境的影响,旨在为青海黑藏羊合理的日粮NFC/NDF水平的配置提供理论依据。试验选取健康且体况相近的黑藏羊公羔60只（2月龄断奶）平均体重10.45±0.96kg。试验采用单因素随机区组设计,随机分为H组、M组和L组,分别饲喂NFC/NDF水平为1.82、1.16和0.71的日粮,每组20只,组内5个重复。试验期127 d,其中预饲期7 d,正试期120 d。试验结束后,从每组中随机选取5只试验羊屠宰,采集瘤胃组织制作冷冻切片,测量瘤胃角质层、瘤胃颗粒层、肌层厚度、乳头长度、乳头宽度;采集瘤胃组织提取总RNA,q RT-PCR测定瘤胃上皮组织发育、吸收代谢、屏障功能、炎症相关基因相对表达量;于同一部位分别取瘤胃内容物用于测定瘤胃抗氧化能力、消化酶活性、瘤胃发酵参数及细菌多样性。结果表明:1)M组和H组瘤胃的角质层厚度和肌层厚度显著小于L组（P<0.05）;M组瘤胃的颗粒层厚度显著大于H组（P<0.05）;M组瘤胃的乳头长度显著大于H组（P<0.05）;M组瘤胃的乳头宽度显著大于L组和H组（P<0.05）;各组间瘤胃的中央肌层厚度差异不显著（P>0.05）,但随着日粮NFC/NDF水平降低呈增大趋势。综上所述,日粮NFC/NDF水平为1.16时瘤胃组织形态发育状态最好。2)以M组为对照组,M组IGFBP-1、IGF-1 m RNA表达量与H组和L组相比差异均不显著（P>0.05）,但H组IGFBP-1、IGF-1 m RNA表达量显著高于L组（P<0.05）;H组MCT1、NHE1、PPARA、SLC14A1、HMGCS2 m RNA表达量呈上升趋势（P<0.10）,M组N+/K+-ATPase m RNA表达量显著低于H组（P<0.05）,且显著高于L组（P<0.05）;H组与L组ZO-1、claudin-1、claudin-4m RNA表达量显著低于M组（P<0.05）,各组desmoglein m RNA表达量差异不显著（P>0.05）;H组与M组瘤胃上皮炎症基因IL-6 m RNA表达量显著高于L组（P<0.05）。综上所述,当日粮NFC/NDF水平为1.16时,瘤胃发育程度、吸收代谢、屏障功能综合状态最佳。3)H组、M组、L组三组的GSH-Px活性差异不显著（P>0.05）,H组的SOD活性显著高于M组和L组（P<0.05）,H组的T-AOC、CAT活性均显著低于M组和L组（P<0.05）,M组的MDA含量显著低于H组和L组（P<0.05）;H组的α-淀粉酶活性显著高于L组（P<0.05）,L组的纤维素酶活性显著高于H组和M组（P<0.05）,M组的脂肪酶活性显著高于H组和L组（P<0.05）,L组的糜蛋白酶活性显著低于H组和M组（P<0.05）,H组、M组、L组三组的胰蛋白酶活性差异不显著（P>0.05）。综上所述,日粮NFC/NDF水平为1.16时青海黑藏羊瘤胃抗氧化能力最强且消化酶活性最高。4)M组瘤胃液p H显著高于H组且显著低于L组（P<0.05）;H组瘤胃液NH₃-N含量显著高于L组且显著低于M组（P<0.05）;M组瘤胃液MCP含量显著高于L组且显著低于H组（P<0.05）;M组瘤胃液乙酸含量显著高于H组且显著低于L组（P<0.05）;L组的VFA含量显著高于H组和M组（P<0.05）;3组瘤胃液乙酸/丙酸的比值差异显著（P<0.05）。97%的一致性共得到1463个OTUs;门水平下,Bacteroidetes、Firmicutes是3组的优势菌门,Proteobacteria在L组的丰度显著高于H组和M组（P<0.05）;属水平下,Prevotella＿1是3组的优势菌属,Ruminococcus＿2、Butyrivibrio＿2、Prevotellaceae＿NK3B31＿group在L组的丰度显著高于H组（P<0.05）,Lachnoclostridium＿1、Prevotellaceae＿Ga6A1＿group、probable＿genus＿10在L组的丰度显著低于H组（P<0.05）,Butyrivibrio＿2、Acetitomaculum、Moryella在L组的丰度显著高于M组（P<0.05）。瘤胃发酵参数和瘤胃细菌相关性分析:共性优势菌门Bacteroidetes与p H、Acetic Acid、VFA均呈正相关,与其他指标呈负相关;而Firmicutes与Bacteroidetes与发酵参数指标的相关性恰好相反。次优势菌门Proteobacteria与NH₃-N呈显著正相关,与Butyric Acid呈显著负相关;Patesicbacteria与Butyric Acid呈显著负相关,与VFA极显著负相关;Tenericates与VFA呈显著负相关;Synergistetes与Propanoic Acid呈显著正相关。综上所述,不同NFC/NDF水平日粮会改变黑藏羊瘤胃发酵类型,同时会影响瘤胃微生物组成及多样性。综上所述,不同NFC/NDF水平日粮会影响黑藏羊瘤胃组织形态的发育,会影响瘤胃发育程度、吸收代谢、屏障功能等相关基因的表达量,会影响瘤胃抗氧化能力及消化酶活性,同时会影响黑藏羊瘤胃发酵类型及瘤胃细菌的多样性。综合来看,当日粮NFC/NDF水平为1.16时,瘤胃发育状态及瘤胃内环境稳态最佳。更多还原

【Abstract】 This experiment was conducted to study the effects of different dietary NFC/NDF levels on rumen development and rumen internal environment of Qinghai Black Tibetan sheep,in order to provide theoretical basis for rational dietary NFC/NDF level configuration of Qinghai Black Tibetan sheep.In the experiment,60 black Tibetan lambs（2-month-old weaned）who were healthy and similar in body condition were selected with an average weight of 10.45±0.96 kg.Using a single-factor randomized block design,they were randomly divided into H,M and L groups,fed diets with NFC/NDF levels of 1.82,1.16,and 0.71,respectively,with 20 animals in each group,with 5 replicates within each group.The experimental period was 127 days,of which the pre-feeding period was 7 days and the positive test period was 120 days.After the experiment,5 experimental sheep were randomly selected from each group for slaughter,rumen tissue was collected to make frozen sections,and the rumen cuticle,rumen granular layer,muscle layer thickness,nipple length,and nipple width were measured;the rumen tissue was collected to extract total RNA,and qRT-PCR was used to determine the relative expression of rumen epithelial tissue development,absorption and metabolism,barrier function,and inflammation-related genes;the rumen contents were collected from the same site for determination of rumen antioxidant capacity,digestive enzyme activity,rumen fermentation parameters and bacterial diversity.The result shows::1)The thickness of cuticle and muscle layer of rumen in group M and H were significantly smaller than those in group L（P<0.05）;the thickness of the granular layer in the rumen of the M group was significantly greater than that of the H group（P<0.05）;the length of the rumen papilla in the M group was significantly greater than that in the H group（P<0.05）;the rumen papilla width of M group was significantly larger than that of L and H groups（P<0.05）;there was no significant difference in the thickness of the central myometrium of the rumen among the groups（P>0.05）,but it showed an increasing trend with the decrease of dietary NFC/NDF levels.In conclusion,the morphological development of the rumen was the best when the NFC/NDF level of the diet was 1.16.2)Taking the M group as the control group,There was no significant difference in IGFBP-1 and IGF-1 mRNA expression between group M and group H and group L（P>0.05）,but the expression levels of IGFBP-1 and IGF-1 mRNA in group H were significantly higher than those in group L（P<0.05）;the mRNA expressions of MCT1,NHE1,PPARA,SLC14A1 and HMGCS2 in group H showed an upward trend（P<0.10）,the expression of N+/K+-ATPase mRNA in group M was significantly lower than that in group H（P<0.05）,and significantly higher than that in group L（P<0.05）;the mRNA expressions of ZO-1,claudin-1,and claudin-4 in groups H and L were significantly lower than those in group M（P<0.05）,there was no significant difference in desmoglein mRNA expression among the groups（P>0.05）;the rumen epithelial inflammation gene IL-6 mRNA expression in H and M groups was significantly higher than that in L group（P<0.05）.In conclusion,when the dietary NFC/NDF level was 1.16,the comprehensive state of rumen development,absorption and metabolism,and barrier function was the best.3)There was no significant difference in GSH-Px activity among the three groups H,M and L（P>0.05）,the SOD activity of group H was significantly higher than that of group M and group L（P<0.05）,the activities of T-AOC and CAT in group H were significantly lower than those in group M and group L（P<0.05）,the MDA content of group M was significantly lower than that of group H and group L（P<0.05）;the α-amylase activity of group H was significantly higher than that of group L（P<0.05）,the cellulase activity of group L was significantly higher than that of group H and group M（P<0.05）,the lipase activity of group M was significantly higher than that of group H and group L（P<0.05）,the chymotrypsin activity of group L was significantly lower than that of group H and group M（P<0.05）,there was no significant difference in trypsin activity among the three groups H,M and L（P>0.05）.In conclusion,when the dietary NFC/NDF level was 1.16,the rumen of Qinghai Black Tibetan sheep had the strongest antioxidant capacity and the highest digestive enzyme activity.4)The pH of rumen juice in group M was significantly higher than that in group H and significantly lower than that in group L（P<0.05）;the content of NH3-N in the rumen fluid of group H was significantly higher than that of group L and significantly lower than that of group M（P<0.05）;the content of MCP in rumen fluid of group M was significantly higher than that of group L and significantly lower than that of group H（P<0.05）;the content of acetic acid in the rumen juice of the M group was significantly higher than that of the H group and significantly lower than that of the L group（P<0.05）;the content of VFA in group L was significantly higher than that in group H and group M（P<0.05）;the ratio of acetic acid/propionic acid in rumen fluid of the three groups was significantly different（P<0.05）.A total of 1463 OTUs were obtained with 97%agreement.At the phylum level,Bacteroidetes and Firmicutes were the dominant phyla of the three groups,the abundance of Proteobacteria in L group was significantly higher than that in H and M groups（P<0.05）.At the genus level,Prevotella₁ is the dominant genus of the 3 groups,the abundances of Ruminococcus₂,Butyrivibrio₂,and Prevotellaceae_NK3B31_group in group L were significantly higher than those in group H（P<0.05）,the abundances of Lachnoclostridium₁,Prevotellaceae_Ga6A1_group,and probable genus₁0 in group L were significantly lower than those in group H（P<0.05）,The abundances of Butyrivibrio₂,Acetitomaculum and Moryella in group L were significantly higher than those in group M（P<0.05）.Correlation analysis between rumen fermentation parameters and rumen bacteria:The common dominant bacterial phylum Bacteroidetes was positively correlated with pH,Acetic Acid and VFA,and negatively correlated with other indicators;Firmicutes and Bacteroidetes have opposite correlations with indicators of fermentation parameters.The subdominant phylum Proteobacteria was significantly positively correlated with NH3-N,and significantly negatively correlated with Butyric Acid;Patesicbacteria was significantly negatively correlated with Butyric Acid and extremely negatively correlated with VFA;Tenericates were significantly negatively correlated with VFA;Synergistetes showed a significant positive correlation with Propanoic Acid.In conclusion,different NFC/NDF levels of diets can change the type of rumen fermentation in black Tibetan sheep,and also affect the composition and diversity of rumen microbes.To sum up,diets with different NFC/NDF levels will affect the development of rumen tissue morphology in black Tibetan sheep,affect the expression of rumen development,absorption metabolism,barrier function and other related genes,and affect the rumen antioxidant capacity and digestive enzymes.It also affects the rumen fermentation type and the diversity of rumen bacteria in black Tibetan sheep.On the whole,when the NFC/NDF level of the diet was 1.16,the rumen development status and rumen homeostasis were the best.更多还原