不同年龄段牦牛瘤胃组织形态学与转录组研究

doi:10.11843/j.issn.0366-6964.2022.11.008

摘要/Abstract

摘要： 旨在比较不同年龄牦牛瘤胃组织形态、转录组表达谱的变化，挖掘影响牦牛瘤胃发育的关键基因和信号通路，为进一步探讨牦牛瘤胃发育机制提供理论基础。本研究选取非反刍阶段（1日龄与20日龄）、过渡阶段（60日龄）和反刍阶段（15月龄与3岁龄）的健康牦牛作为研究对象，共5个年龄组，每组3个样本。测量样本的体重与瘤胃重量；采集瘤胃组织样品，切片观察组织形态，并统计肌层厚度、乳头高度与宽度；提取瘤胃组织总RNA进行测序，并以1日龄为对照进行转录组分析。选取差异表达基因进行荧光定量试验，关联其与转录组数据的表达趋势。组织形态学分析结果显示，1日龄至3岁的牦牛瘤胃肌层与乳头形态有极大程度的发育与分化，15月龄和3岁牦牛瘤胃重量与指数显著增加（P<0.05）；从20日龄开始，瘤胃的肌层厚度随着日龄的增加显著增加（P<0.05）；20日龄后，瘤胃乳头高度和宽度随着日龄增加而显著增加（P<0.05）。转录组分析结果显示，20日龄组DEGs富集到胆固醇合成、丁酸代谢和PPAR信号通路等与VFA代谢相关的通路，富集到与免疫相关的免疫反应生物过程和Th17细胞分化通路，富集到与瘤胃上皮细胞异源物代谢相关的细胞色素P450外源性物质的代谢途径；60日龄组DEGs富集到脂肪酸代谢、丙酸代谢通路和丙酮酸代谢通路等与VFA代谢相关的通路，富集到与VFA吸收相关的矿物质吸收通路；15月龄与3岁组DEGs富集到维持瘤胃上皮的完整性和屏障功能的ECM受体相互作用途径。从富集通路中发现，HMGCS2、SLC26A3、PPARG、PPARD和CCL5等基因是与瘤胃营养吸收、代谢和免疫相关的候选基因。荧光定量结果显示，挑选的差异表达基因的表达与转录组测序结果一致。上述结果表明，牦牛瘤胃形态结构发育较早，20日龄是其发育的重要临界点；牦牛瘤胃肌层发育比瘤胃乳头更早，肌层厚度从20日龄时快速增加，瘤胃乳头从20日龄后快速发育；20日龄瘤胃开始VFA代谢，促进瘤胃快速健康的发育。

关键词: 不同年龄段, 牦牛, 瘤胃, 形态学, 转录组

Abstract: This study aimed to compare the changes in rumen tissues morphology and transcriptome profile of the yak at different ages to explore the key genes and signaling pathways affecting the rumen development, and lay a foundation for further in-depth exploration of the rumen development mechanism of the yak. In this study, the histomorphological and transcriptomic analysis of the healthy yak's rumen at 5 age groups was performed, including non-ruminant stage (1 day old and 20 days old), transitional stage (60 days old) and ruminant stage (15 months old and 3 years old), 3 samples per group. The body and rumen weights of the samples were measured. The rumen tissue samples were collected and carried out histomorphology observation, the muscle layer thickness, papillae height and width were measured and counted. Total RNA was extracted from rumen tissue for sequencing, and transcriptome analysis was performed with 1-day-old as control. The differentially expressed genes were selected for RT-qPCR, the results of RT-qPCR was compared with the expression trend of transcriptome data. The results of histomorphological analysis showed that the rumen muscle layer thickness and papillae morphology of yaks from 1 day to 3 years old developed and differentiated to a great extent at the histomorphological level. The rumen weight and index increased significantly at 15 months and 3 years old (P<0.05). From 20 days old, the muscle layer thickness increased significantly with the increase of age (P<0.05). After 20 days old, the height and width of rumen papillae increased significantly with the increase of age (P<0.05). In the 20 days old group, transcriptome results showed that the pathways related to VFA metabolism were enriched in cholesterol synthesis, butyrate metabolism and PPAR signaling pathway; The pathways related to immunity were enriched in the immune response biological process and Th17 cell differentiation pathway; The pathways related to the metabolism of rumen epithelial cells xenobiotics was enriched in the metabolic pathway of cytochrome P450 exogenous substance. In the 60 days old group, the pathways related to VFA metabolism were enriched in the fatty acid metabolism, propionic acid metabolism pathway and pyruvate metabolism pathway; The pathways related to VFA absorption was enriched in mineral absorption. The 15 months old and 3-year-old groups enriched in ECM receptor interaction pathways that maintain the integrity and barrier function of rumen epithelium. HMGCS2, SLC26A3, PPARG, PPARD and CCL5 genes were selected from the above pathways, which related to the nutrient absorption, metabolism and immunity of rumen. The expression pattern of differentially expressed genes detected by RT-qPCR was consistent with the results of the transcriptome sequencing. The above results indicate that the rumen morphology of the yak develops early, and 20 days old is an important critical point for ruminal development; The development of ruminal muscle layer thickness was earlier than that of ruminal papillae, muscular thickness increased rapidly from 20 days old; and the rumen papillae developed rapidly after 20 days old; The rumen started VFA metabolism at 20 days old, which promotes the rapid and healthy development of rumen.

Key words: different ages, yak, rumen, histomorphology, transcriptome

中图分类号:

S823.85

唐娇, 夏果, 刘益丽, 闵奇, 冉嫆, 谢书琼, 马士龙, 江明锋. 不同年龄段牦牛瘤胃组织形态学与转录组研究[J]. 畜牧兽医学报, 2022, 53(11): 3797-3810.

TANG Jiao, XIA Guo, LIU Yili, MIN Qi, RAN Rong, XIE Shuqiong, MA Shilong, JIANG Mingfeng. The Research of Histomorphological and Transcriptomic of Yak Rumen at Different Ages[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3797-3810.

参考文献 45

[1]	BALDWIN VI R L, MCLEOD K R, KLOTZ J L, et al.Rumen development, intestinal growth and hepatic metabolism in the pre- and postweaning ruminant[J].J Dairy Sci, 2004, 87 Suppl 1:E55-E65.
[2]	PARSONS S D, STEELE M A, LESLIE K E, et al.Effect of a milk byproduct-based calf starter feed on dairy calf nutrient consumption, rumen development, and performance when fed different milk levels[J].J Dairy Sci, 2022, 105(1):281-300.
[3]	田发益, 武俊喜.放牧与舍饲对彭波半细毛羊瘤胃细菌群落的生物学信息影响[J].畜牧兽医学报, 2019, 50(11):2252-2263.TIAN F Y, WU J X.Effects of grazing and barn feeding on biological information of rumen bacterial communities in Pengbo semi-fine wool sheep[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(11):2252-2263.(in Chinese)
[4]	SEHESTED J, DIERNÆS L, MØLLER P D, et al.Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro:effect of SCFA chain length and pH[J].Comp Biochem Physiol Part A:Mol Integr Physiol, 1999, 123(4):359-368.
[5]	孙玲伟, 包凯, 李影, 等.奶牛临床和亚临床酮病的血浆代谢组学研究[J].中国农业科学, 2014, 47(8):1588-1599.SUN L W, BAO K, LI Y, et al.Plasma metabolomics study of dairy cows with clinical and subclinical ketosis[J].Scientia Agricultura Sinica, 2014, 47(8):1588-1599.(in Chinese)
[6]	ONTSOUKA E C, ALBRECHT C.Cholesterol transport and regulation in the mammary gland[J].J Mammary Gland Biol Neoplasia, 2014, 19(1):43-58.
[7]	韩郭皓, 高晓莎, 段晋伟, 等.酵母对亚急性瘤胃酸中毒绵羊缓解效果及作用机制研究[J].畜牧兽医学报, 2022, 53(5):1475-1485.HAN G H, GAO X S, DUAN J W, et al.Study on relieving effect and mechanism of yeast on subacute ruminal acidosis in sheep[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(5):1475-1485.(in Chinese)
[8]	胡新旭, 卞巧, 张善鹏, 等.奶牛瘤胃健康、机体健康和繁殖性能的关系[J].湖南饲料, 2021(3):31-34.HU X X, BIAN Q, ZHANG S P, et al.The relationship between rumen health, body health and reproductive performance in dairy cows[J].Hunan Feed, 2021(3):4.(in Chinese)
[9]	张涛, 牟英玉, 亓王盼, 等.亚急性瘤胃酸中毒耐受性不同的奶牛血浆和乳中脂肪酸及代谢物组成分析[J].草业学报, 2021, 30(7):101-110.ZHANG T, MU Y Y, QI W P, et al.Analysis of plasma and milk fatty acid and metabolite composition in lactating dairy cows with differing tolerance to subacute ruminal acidosis[J].Acta Prataculturae Sinica, 2021, 30(7):101-110.(in Chinese)
[10]	施振旦, 李孝伟, 田允波, 等.奶牛繁殖杀手——内毒素[J].中国奶牛, 2007(3):29-32.SHI Z D, LI X W, TIAN Y B, et al.Slayer of dairy cow's reproductive performance:endotoxin[J].China Dairy Cattle, 2007(3):29-32.(in Chinese)
[11]	KONG R S G, LIANG G X, CHEN Y H, et al.Transcriptome profiling of the rumen epithelium of beef cattle differing in residual feed intake[J].BMC Genomics, 2016, 17:592.
[12]	BALDWIN VI R L, LIU M, CONNOR E E, et al.Transcriptional reprogramming in rumen epithelium during the developmental transition of pre-ruminant to the ruminant in cattle[J].Animals, 2021, 11(10):2870.
[13]	WANG B, WANG D M, WU X H, et al.Effects of dietary physical or nutritional factors on morphology of rumen papillae and transcriptome changes in lactating dairy cows based on three different forage-based diets[J].BMC Genomics, 2017, 18(1):353.
[14]	崔占鸿.牦牛犊牛培育方式对生长和消化道发育的影响[D].杨凌:西北农林科技大学, 2020.CUI Z H.Effects of rearing patterns on growth and digstive tract development in yak calves[D].Yangling:Northwest A&F University, 2020.(in Chinese)
[15]	LI W L, GELSINGER S, EDWARDS A, et al.Transcriptome analysis of rumen epithelium and meta-transcriptome analysis of rumen epimural microbial community in young calves with feed induced acidosis[J].Sci Rep, 2019, 9(1):4744.
[16]	MACKEY E.Effects of ruminal acidosis on rumen papillae transcriptome[D].Newark:University of Delaware, 2013.
[17]	吕小康, 王杰, 刁其玉, 等.瘤胃上皮细胞增殖和物质转运分子机制的研究进展[J].动物营养学报, 2017, 29(8):2657-2664.LYU X K, WANG J, DIAO Q Y, et al.Molecular mechanisms of ruminal epithelial cell proliferation and substance transportation[J].Chinese Journal of Animal Nutrition, 2017, 29(8):2657-2664.(in Chinese)
[18]	张剑搏, 丁考仁青, 梁泽毅, 等.早期营养干预对幼龄反刍动物瘤胃微生物区系发育的影响[J].草业学报, 2021, 30(2):199-211.ZHANG J B, DING K R Q, LIANG Z Y, et al.Effect of early nutrition intervention on rumen microflora development in young ruminants[J].Acta Prataculturae Sinica, 2021, 30(2):199-211.(in Chinese)
[19]	BOLGER A M, LOHSE M, USADEL B.Trimmomatic:a flexible trimmer for Illumina sequence data[J].Bioinformatics, 2014, 30(15):2114-2120.
[20]	KIM D, LANGMEAD B, SALZBERG S L.HISAT:a fast spliced aligner with low memory requirements[J].Nat Methods, 2015, 12(4):357-360.
[21]	ANDERS S, PYL P T, HUBER W.HTSeq——a Python framework to work with high-throughput sequencing data[J].Bioinformatics, 2015, 31(2):166-169.
[22]	TRAPNELL C, WILLIAMS B A, PERTEA G, et al.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nat Biotechnol, 2010, 28(5):511-555.
[23]	ANDERS S, HUBER W.Differential expression of RNA-Seq data at the gene level-the DESeq package[R].Heidelberg, Germany:European Molecular Biology Laboratory (EMBL), 2012.
[24]	KANEHISA M, ARAKI M, GOTO S, et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Res, 2008, 36:D480-D484.
[25]	GRAHAM C, SIMMONS N L.Functional organization of the bovine rumen epithelium[J].Am J Physiol Regul Integr Comp Physiol, 2005, 288(1):R173-R181.
[26]	王立斌.在饲喂开食料的基础上补饲苜蓿对犊牛胃肠道发育的影响[D].北京:中国农业大学, 2013.WANG L B.Influence of supplementary feeding of alfalfa on the gastrointestinal tract development of calves feeding on milk and starter[D].Beijing:China Agricultural University, 2013.(in Chinese)
[27]	张科.陕北白绒山羊0~56日龄羔羊胃肠道发育及瘤胃微生物区系研究[D].杨凌:西北农林科技大学, 2017.ZHANG K.Researchon Gi development and rumen microbiome from 0 to 56-day-old at cashmere goat[D].Yangling:Northwest A and F University, 2017.(in Chinese)
[28]	NOROUZIAN M A, VALIZADEH R.Effect of forage inclusion and particle size in diets of neonatal lambs on performance and rumen development[J].J Anim Physiol Anim Nutr, 2014, 98(6):1095-1101.
[29]	RAGIONIERI L, CACCHIOLI A, RAVANETTI F, et al.Effect of the supplementation with a blend containing short and medium chain fatty acid monoglycerides in milk replacer on rumen papillae development in weaning calves[J].Ann Anat-Anat Anz, 2016, 207:97-108.
[30]	STEELE M A, VANDERVOORT G, ALZAHAL O, et al.Rumen epithelial adaptation to high-grain diets involves the coordinated regulation of genes involved in cholesterol homeostasis[J].Physiol Genomics, 2011, 43(6):308-316.
[31]	MALHI M, GUI H B, YAO L, et al.Increased papillae growth and enhanced short-chain fatty acid absorption in the rumen of goats are associated with transient increases in cyclin D1 expression after ruminal butyrate infusion[J].J Dairy Sci, 2013, 96(12):7603-7616.
[32]	SUN D M, YIN Y Y, GUO C Z, et al.Transcriptomic analysis reveals the molecular mechanisms of rumen wall morphological and functional development induced by different solid diet introduction in a lamb model[J].J Anim Sci Biotechnol, 2021, 12(1):33.
[33]	LIN L M, XIE F, SUN D M, et al.Ruminal microbiome-host crosstalk stimulates the development of the ruminal epithelium in a lamb model[J].Microbiome, 2019, 7(1):83.
[34]	SCHORMANN N, HAYDEN K L, LEE P, et al.An overview of structure, function, and regulation of pyruvate kinases[J].Protein Sci, 2019, 28(10):1771-1784.
[35]	YANG C L, ZHU B N, YE S J, et al.Isomer-specific effects of cis-9, trans-11-and trans-10, cis-12-CLA on immune regulation in ruminal epithelial cells[J].Animals, 2021, 11(4):1169.
[36]	HU R, ZOU H W, WANG Z S, et al.Nutritional interventions improved rumen functions and promoted compensatory growth of growth-retarded yaks as revealed by integrated transcripts and microbiome analyses[J].Front Microbiol, 2019, 10:318.
[37]	MCBRIDE B W, ALZAHAL O, STEELE M A, et al.Adaptation to high grain diets proceeds through minimal immune system stimulation and differences in extracellular matrix protein expression in a model of subacute ruminal acidosis in non-lactating dairy cows[J].Am J Anim Vet Sci, 2012, 7(2):84-91.
[38]	高景, 齐智利.瘤胃上皮短链脂肪酸的吸收和代谢[J].动物营养学报, 2018, 30(4):1271-1278.GAO J, QI Z L.Absorption and metabolism of short chain fatty acids in ruminal epithelium[J].Chinese Journal of Animal Nutrition, 2018, 30(4):1271-1278.(in Chinese)
[39]	庄一民, 刁其玉, 张乃锋.幼龄反刍动物瘤胃上皮细胞β-羟基丁酸代谢与调控机制[J].畜牧兽医学报, 2020, 51(4):660-669.ZHUANG Y M, DIAO Q Y, ZHANG N F.Metabolism and regulation mechanism of beta-hydroxybutyric acid in ruminal epithelium cells of young ruminants[J].Acta Veterinaria et Zootechnica Sinica, 2020, 51(4):660-669.(in Chinese)
[40]	NAEEM A, DRACKLEY J K, STAMEY J, et al.Role of metabolic and cellular proliferation genes in ruminal development in response to enhanced plane of nutrition in neonatal Holstein calves[J].J Dairy Sci, 2012, 95(4):1807-1820.
[41]	GÁLFI P, KUTAS F, NEOGRÁDY S.Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme[J].Histochemistry, 1982, 74(4):577-584.
[42]	XUE M M, WANG K J, WANG A S, et al.MicroRNA sequencing reveals the effect of different levels of non-fibrous carbohydrate/neutral detergent fiber on rumen development in calves[J].Animals, 2019, 9(8):496.
[43]	CONNOR E E, BALDWIN VI R L, WALKER M P, et al.Transcriptional regulators transforming growth factor-β1 and estrogen-related receptor-α identified as putative mediators of calf rumen epithelial tissue development and function during weaning[J].J Dairy Sci, 2014, 97(7):4193-4207.
[44]	CHAN O, BURKE D J, GAO D F, et al.The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis[J].J Biol Chem, 2012, 287(35):29406-29416.
[45]	ROH S.The control of homeostasis in rumen epithelium of Japanese Black cattle[J].J Anim Sci, 2018, 97(S3):1.