添加丙酸钙对奶公犊生长性能的影响和调控瘤胃发育机理的研究
发布时间：2018-08-14 14:54
【摘要】：本课题研究了丙酸钙添加水平对奶公犊生长性能的影响和调控瘤胃发育的机理。通过不同时间的饲养屠宰试验,研究了丙酸钙添加水平对犊牛生长性能、内脏器官以及胃肠道发育的影响；通过对丙酸钙影响瘤胃发酵、血液生化指标和瘤胃上皮相关mRNA基因表达量的研究,阐明了丙酸通过内分泌途径和G蛋白偶联受体途径促进瘤胃上皮发育的机理；应用基于转录组学技术筛选了丙酸对瘤胃上皮差异表达的基因,为调控犊牛瘤胃发育提供了更为全面的理论框架和研究基础。试验1：研究了丙酸钙添加水平对不同饲喂阶段奶公犊生长性能、内脏器官以及胃肠道发育的影响。选用娟姗公犊牛54头(日龄7±1d,体重23.1±1.2kg),随机分成3个处理：1)对照组不添加丙酸钙(CON)；2)添加5%丙酸钙(5%CaP,DM基础)；3)添加10%丙酸钙(10%CaP,DM基础)。试验期160 d,分为3阶段：第1阶段(饲喂0d-30d),第2阶段(饲喂30d-90d),第三阶段(饲喂90d～160d)。在每个阶段结束时,随机从每个处理组中选择6头犊牛屠宰并取样。添加丙酸钙能够提高犊牛饲喂90d(P=0.013)、120d(P=0.018)以及160d(P=0.006)的体重。饲喂90d的犊牛体重随着丙酸钙添加水平的提高而提高,饲喂120d和160d的犊牛体重在丙酸钙的添加水平之间没有显著性差异。10%CaP组显著提高了30d～60d(P=0.013)、60d～90d(P=0.013)和120d～160d(P=0.080)的平均日增重,而5%CaP组只提高了120d～160d的犊牛平均日增重(P=0.046)。10%CaP组脾脏重量在饲喂160天时显著高于CON组和5%CaP组(P=0.010)。5%CaP和10%CaP组肝脏重量在饲喂90d时都显著高于CON组(P=0.003),而在160d时,只有10%CaP组肝脏重量显著高于CON组(P=0.037)。添加丙酸钙可以提高犊牛饲喂90d和160d的瘤胃重量(P=0.006,0.010),提高十二指肠绒毛高度(P=0.007)和隐窝深度(P=0.0002),提高空肠绒毛高度(P=0.0037)、隐窝深度(P=0.0065)以及绒毛高度与隐窝深度的比例(P=0.0080),并提高回肠的绒毛高度(P0.001)和隐窝深度(P0.001)。小结：添加丙酸钙可以提高犊牛的生长性能、促进肝脏和脾脏等内脏器官以及胃肠道的发育。丙酸钙添加量在饲喂90d以前添加10%效果较好,而在饲喂90d～160d期间,添加5%的丙酸钙效果与10%效果相当。试验2：研究了丙酸钙添加水平对不同饲喂阶段犊牛瘤胃发酵以及瘤胃发育的影响。试验动物和处理同试验1。添加丙酸钙可以显著提高犊牛饲喂30d瘤胃内总挥发酸含量(P=0.0370),并有提高乙酸(P=0.0632)、丙酸(P=0.0903)和异丁酸(P=0.0591)浓度的趋势。10%CaP组降低了犊牛饲喂90d瘤胃内氨态氮的含量(P=0.0013)。添加5%丙酸钙可以提高饲喂160d犊牛瘤胃内丙酸(P=0.0029)和戊酸的含量(P=0.0133)。添加丙酸钙提高了犊牛饲喂30d(P=0.0085)和90d(P=0.0126)的瘤胃壁厚度,提高了饲喂30d犊牛瘤胃乳头的长度(P=0.0012),且随着丙酸钙添加量的增加,瘤胃乳头长度加大,但对乳头宽度没有影响(P=0.2754)。提高丙酸钙添加水平可以增加饲喂90d犊牛瘤胃上皮乳头的长度(P=0.0016)和上皮乳头的宽度(P=0.0293),其中添加5%丙酸钙可以提高饲喂160d犊牛瘤胃上皮乳头的长度(P0.001),而添加10%的丙酸钙可以提高瘤胃上皮乳头的宽度(P=0.0025)。小结：添加丙酸钙能够促进瘤胃发酵,促进瘤胃上皮乳头的发育。瘤胃上皮乳头的长度与瘤胃内的丙酸含量呈正相关关系。在饲喂90d以前添加10%丙酸钙对犊牛瘤胃发酵和瘤胃发育的效果较好,饲喂90d到160天期间添加5%的丙酸钙效果更好。试验3：研究了丙酸钙添加水平对犊牛血液生化指标和瘤胃上皮基因表达量的影响。试验动物和处理同试验1。在每个阶段结束时前一天,随机从每个处理组中选择6头犊牛颈静脉采血,并且从饲喂160d的CON和5%CaP组随机选择3头犊牛的瘤胃背囊部样品,用于荧光定量PCR测定。添加丙酸钙可以提高不同饲喂阶段犊牛血糖含量,血糖含量随着丙酸钙添加水平的提高而增加。10%CaP组显著提高了饲喂30d犊牛血糖含量(P0.05)。添加丙酸钙对犊牛血清胰岛素含量的影响和血糖一致。添加丙酸钙可以在数值上提高犊牛血清中1GF-1的浓度,其中在饲喂160d时,5%CaP组浓度最高。添加5%丙酸钙可以提高瘤胃上皮CCND1、CDK4、GPCR43、IGF-1R和INSR的基因表达量,其中CCND1和GPCR43都达到了显著性水平。小结：添加丙酸钙一方面可以提高血液中的葡萄糖、胰岛素和1GF-1的含量,进而提高瘤胃上皮INSR和IGF-1R基因表达量：另一方面丙酸可以直接作为信号分子通过G蛋白偶联受体途径提高GPCR43的基因表达量,来直接促进瘤胃上皮的发育。试验4：通过高通量转录组测序(RNA-Seq)方法筛选添加丙酸钙对犊牛瘤胃上皮差异表达的基因,探讨丙酸对瘤胃上皮发育影响的潜在分子机制。从试验一中CON和5%CaP处理组中随机选择3头饲喂160d的犊牛屠宰后取得的瘤胃上皮作为研究对象,经过样品总RNA提取、转录组文库的制备,然后在HiSeq2500测序平台上测序。样本匹配到牛基因组的基因共有18084个,其中只在5%CaP组表达的基因有718个,只在CON组表达的基因有696个。在两个处理组中均有表达的基因有16670个,显著差异表达的基因数量为369个,其中5%CaP组比CON组高表达的基因有216个,低表达的基因有153个。通过GO富集分析发现,添加丙酸钙可以上调与上皮细胞发育相关的TGM3、TGM1、OVOL1、DSP、IVL、SPINK5、CLDN4、CNFN、KRT36基因的表达量促进瘤胃上皮的发育。KEGG结果分析表明,差异基因主要通过代谢、信号转导以及内分泌系统影响瘤胃上皮的发育。小结：本试验成功筛选了丙酸钙调控犊牛瘤胃上皮发育的候选基因,为调控犊牛瘤胃上皮发育提供了理论基础以及更多的研究靶点和思路。
[Abstract]:The effects of calcium propionate on growth performance and rumen development of dairy male calves were studied. The effects of calcium propionate on growth performance, visceral organs and gastrointestinal development of dairy male calves were studied by feeding and slaughtering experiments at different times. The study on the expression of rumen epithelium-related mRNA genes clarified the mechanism of propionic acid promoting the development of rumen epithelium through endocrine pathway and G protein-coupled receptor pathway; the differentially expressed genes of rumen epithelium were screened by transcriptome-based technology, which provided a more comprehensive theoretical framework and research for the regulation of rumen development of calves. Experiment 1: The effects of calcium propionate on growth performance, visceral organs and gastrointestinal development of male calves at different feeding stages were studied. Fifty-four Juanshan male calves (7 6550 At the end of each stage, six calves were randomly selected from each treatment group for slaughter and sampling. Calcium propionate could increase the feeding time of calves by 90 days (P = 0.013) and 120 days (P = 0.018). The body weight of the calves fed 90 days increased with the increase of calcium propionate, and there was no significant difference in the body weight of the calves fed 120 days and 160 days. The body weight of the calves fed 10% CaP increased significantly by 30 days to 60 days (P = 0.013), 60 days to 90 days (P = 0.013) and 120 days to 160 days (P = 0.080). The average daily gain (P = 0.046) of calves increased only 120 days to 160 days. The spleen weight of 10% CaP group was significantly higher than that of CON group and 5% CaP group at 160 days (P = 0.010). The liver weight of 5% CaP and 10% CaP group was significantly higher than that of CON group at 90 days (P = 0.003) and only 10% CaP group was significantly higher than that of CON group at 160 days (P = 0.037). The rumen weight (P=0.006,0.010) and the height of duodenal villi (P=0.007) and the depth of crypt (P=0.0002) were increased, the height of jejunal villi (P=0.0037), the depth of crypt (P=0.0065) and the ratio of the height of villi to the depth of crypt (P=0.0080) were increased, and the height of ileal villi (P=0.001) and the depth of crypt (P=0.001) were also increased. Summary: Calcium propionate can improve the growth performance of calves, promote the development of liver, spleen and other visceral organs as well as gastrointestinal tract. Calcium propionate supplementation was better when 10% was added before 90 days, but the effect of adding 5% calcium propionate was similar to that of 10% during 90 days to 160 days. The effect of calcium propionate on rumen fermentation and rumen development of calves at the same feeding stage was the same as that of experimental animals and treatments. 1. Calcium propionate significantly increased the content of total volatile acids (P = 0.0370) in the rumen of calves fed 30 days, and increased the concentration of acetic acid (P = 0.0632), propionic acid (P = 0.0903) and isobutyric acid (P = 0.0591) in the rumen of calves. The contents of propionic acid (P = 0.0029) and valeric acid (P = 0.0133) in rumen were increased by adding 5% calcium propionate (P = 0.0013). Calcium propionate increased the thickness of rumen wall and the length of rumen papilla (P = 0.0012) of calves fed with calcium propionate for 30 days (P = 0.0085) and 90 days (P = 0.0126). The length of rumen papilla and the width of epithelial papilla increased with the increase of calcium propionate (P = 0.0016) and 5% calcium propionate (P = 0.0293), but the length of rumen papilla increased with the increase of calcium propionate (P = 0.2754). 10% calcium propionate could increase the width of rumen papilla (P = 0.0025). Summary: Calcium propionate could promote rumen fermentation and the development of rumen papilla. The length of rumen papilla was positively correlated with the content of propionic acid in rumen. Experiment 3: The effects of calcium propionate supplementation on blood biochemical indexes and rumen epithelial gene expression in calves were studied. The animals and treatments were the same as experiment 1. Six calves were randomly selected from each treatment group one day before the end of each stage. Blood samples from the rumen backpack of three calves were randomly selected from the CON and 5% CaP groups fed for 160 days and used for quantitative fluorescence PCR determination. Calcium propionate could increase the concentration of 1GF-1 in calf serum, and the concentration of 5% CaP was the highest at 160 days. Adding 5% calcium propionate could increase the expression of CCND1, CDK4, GPCR43, IGF-1R and INSR genes in rumen epithelium, including CCND1 and GPCR43. Conclusion: On the one hand, calcium propionate can increase the levels of glucose, insulin and 1GF-1 in the blood, and then increase the expression of INSR and IGF-1R genes in the rumen epithelium. On the other hand, propionic acid can be directly used as a signal molecule to increase the expression of GPCR43 gene through G protein-coupled receptor pathway, thus directly promoting the tumor. Gastric epithelial development. Experiment 4: Genes differentially expressed in rumen epithelium of calves were screened by high-throughput transcriptome sequencing (RNA-Seq) to explore the potential molecular mechanism of the effects of calcium propionate on rumen epithelial development. Three calves were randomly selected from the CON and 5% CaP treatment groups in trial 1 after slaughtering for 160 days. The total RNA was extracted from the samples, the transcriptome library was prepared, and then sequenced on the HiSeq2500 sequencing platform. The number of differentially expressed genes was 369, of which 216 were overexpressed in 5% CaP group and 153 were underexpressed in CON group. The results of GO enrichment analysis showed that calcium propionate could up-regulate the expression of TGM3, TGM1, OVOL1, DSP, IVL, SPINK5, CLDN4, CNFN and KRT36 genes related to epithelial cell development. KEGG analysis showed that the differentially expressed genes affected the development of rumen epithelium mainly through metabolism, signal transduction and endocrine system.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823.5
本文编号：2183214
[Abstract]:The effects of calcium propionate on growth performance and rumen development of dairy male calves were studied. The effects of calcium propionate on growth performance, visceral organs and gastrointestinal development of dairy male calves were studied by feeding and slaughtering experiments at different times. The study on the expression of rumen epithelium-related mRNA genes clarified the mechanism of propionic acid promoting the development of rumen epithelium through endocrine pathway and G protein-coupled receptor pathway; the differentially expressed genes of rumen epithelium were screened by transcriptome-based technology, which provided a more comprehensive theoretical framework and research for the regulation of rumen development of calves. Experiment 1: The effects of calcium propionate on growth performance, visceral organs and gastrointestinal development of male calves at different feeding stages were studied. Fifty-four Juanshan male calves (7 6550 At the end of each stage, six calves were randomly selected from each treatment group for slaughter and sampling. Calcium propionate could increase the feeding time of calves by 90 days (P = 0.013) and 120 days (P = 0.018). The body weight of the calves fed 90 days increased with the increase of calcium propionate, and there was no significant difference in the body weight of the calves fed 120 days and 160 days. The body weight of the calves fed 10% CaP increased significantly by 30 days to 60 days (P = 0.013), 60 days to 90 days (P = 0.013) and 120 days to 160 days (P = 0.080). The average daily gain (P = 0.046) of calves increased only 120 days to 160 days. The spleen weight of 10% CaP group was significantly higher than that of CON group and 5% CaP group at 160 days (P = 0.010). The liver weight of 5% CaP and 10% CaP group was significantly higher than that of CON group at 90 days (P = 0.003) and only 10% CaP group was significantly higher than that of CON group at 160 days (P = 0.037). The rumen weight (P=0.006,0.010) and the height of duodenal villi (P=0.007) and the depth of crypt (P=0.0002) were increased, the height of jejunal villi (P=0.0037), the depth of crypt (P=0.0065) and the ratio of the height of villi to the depth of crypt (P=0.0080) were increased, and the height of ileal villi (P=0.001) and the depth of crypt (P=0.001) were also increased. Summary: Calcium propionate can improve the growth performance of calves, promote the development of liver, spleen and other visceral organs as well as gastrointestinal tract. Calcium propionate supplementation was better when 10% was added before 90 days, but the effect of adding 5% calcium propionate was similar to that of 10% during 90 days to 160 days. The effect of calcium propionate on rumen fermentation and rumen development of calves at the same feeding stage was the same as that of experimental animals and treatments. 1. Calcium propionate significantly increased the content of total volatile acids (P = 0.0370) in the rumen of calves fed 30 days, and increased the concentration of acetic acid (P = 0.0632), propionic acid (P = 0.0903) and isobutyric acid (P = 0.0591) in the rumen of calves. The contents of propionic acid (P = 0.0029) and valeric acid (P = 0.0133) in rumen were increased by adding 5% calcium propionate (P = 0.0013). Calcium propionate increased the thickness of rumen wall and the length of rumen papilla (P = 0.0012) of calves fed with calcium propionate for 30 days (P = 0.0085) and 90 days (P = 0.0126). The length of rumen papilla and the width of epithelial papilla increased with the increase of calcium propionate (P = 0.0016) and 5% calcium propionate (P = 0.0293), but the length of rumen papilla increased with the increase of calcium propionate (P = 0.2754). 10% calcium propionate could increase the width of rumen papilla (P = 0.0025). Summary: Calcium propionate could promote rumen fermentation and the development of rumen papilla. The length of rumen papilla was positively correlated with the content of propionic acid in rumen. Experiment 3: The effects of calcium propionate supplementation on blood biochemical indexes and rumen epithelial gene expression in calves were studied. The animals and treatments were the same as experiment 1. Six calves were randomly selected from each treatment group one day before the end of each stage. Blood samples from the rumen backpack of three calves were randomly selected from the CON and 5% CaP groups fed for 160 days and used for quantitative fluorescence PCR determination. Calcium propionate could increase the concentration of 1GF-1 in calf serum, and the concentration of 5% CaP was the highest at 160 days. Adding 5% calcium propionate could increase the expression of CCND1, CDK4, GPCR43, IGF-1R and INSR genes in rumen epithelium, including CCND1 and GPCR43. Conclusion: On the one hand, calcium propionate can increase the levels of glucose, insulin and 1GF-1 in the blood, and then increase the expression of INSR and IGF-1R genes in the rumen epithelium. On the other hand, propionic acid can be directly used as a signal molecule to increase the expression of GPCR43 gene through G protein-coupled receptor pathway, thus directly promoting the tumor. Gastric epithelial development. Experiment 4: Genes differentially expressed in rumen epithelium of calves were screened by high-throughput transcriptome sequencing (RNA-Seq) to explore the potential molecular mechanism of the effects of calcium propionate on rumen epithelial development. Three calves were randomly selected from the CON and 5% CaP treatment groups in trial 1 after slaughtering for 160 days. The total RNA was extracted from the samples, the transcriptome library was prepared, and then sequenced on the HiSeq2500 sequencing platform. The number of differentially expressed genes was 369, of which 216 were overexpressed in 5% CaP group and 153 were underexpressed in CON group. The results of GO enrichment analysis showed that calcium propionate could up-regulate the expression of TGM3, TGM1, OVOL1, DSP, IVL, SPINK5, CLDN4, CNFN and KRT36 genes related to epithelial cell development. KEGG analysis showed that the differentially expressed genes affected the development of rumen epithelium mainly through metabolism, signal transduction and endocrine system.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823.5
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