Morphology and Transcriptome Analysis of Intestinal Organoids in Bovine fetus

Intestinal health is an important guarantee for the healthy development of mammals. Therefore, the research on intestinal development and function continues to be a hot spot. And intestinal organoids are a hopeful alternative for the intestine, especially in large animals. In this study, we selected intestine of fetus calves as a target to study the differences in intestinal development and the development and functions of organoids in vitro, which were less reported in the past. The fetus intestine tissue is considered a preferred source for obtaining organoid because of its sterility and excellent developmental potential.


Abstract Background
Intestinal health is an important guarantee for the healthy development of mammals. Therefore, the research on intestinal development and function continues to be a hot spot. And intestinal organoids are a hopeful alternative for the intestine, especially in large animals. In this study, we selected intestine of fetus calves as a target to study the differences in intestinal development and the development and functions of organoids in vitro, which were less reported in the past. The fetus intestine tissue is considered a preferred source for obtaining organoid because of its sterility and excellent developmental potential.

Methods
We compared the morphology of crypts and epithelial cells from duodenum, jejunum, ileum and colon at 4-6 month age fetus calves by HE staining, immunohistochemistry and transmission electron microscopy. And we cultured organoids from jejunum tissue and analyzed the structure by optical and electronic microscopy, immunological methods. Subsequently, through RNA sequencing, we con rmed the characteristics of gene expression between organoids and crypts.

Results
The results showed the villus of jejunum and ileum was signi cantly higher than others, but the depth of the duodenum crypt was signi cantly deeper than the other two (P<0.001). And a large number of ssions were observed in duodenum crypts. In addition, intestinal epithelial stem cell markers LGR 5 and CTNNB1 were highly expressed in the epithelium, but there was signi cant difference in villus and crypt of different intestinal segments. And By electron microscopy, we noticed that crypt stem cells were typically long and arranged in single and multiple layers closely, with a big nucleus and a large number of microvilli at the top of the cells. Each cell contained a large number of organelles, such as mitochondria and ribosomes. There are more free ribosomes in jejunum, ileum and colon crypt. Some Paneth cells and goblet cells were interspersed among crypt cells. There are typical tight junctions and adherent junctions ( ngers cross pattern) between crypt cells. In addition, we obtained jejunum organoids by tissue culture possessing similar traits, such as microvillus and cell tight junctions. CTNNB1, PCNA and Lyso were highly expressed in the crypt cells around the surface of the organoids which hinted at the prominent capacity of cell proliferation. Furthermore, according to RNA sequencing, we found metabolism-related genes were signi cantly higher expression in organoids than in crypt stem cells (Q<0.001). On the contrary, immune and disease related genes were signi cant higher in crypt stem cells (Q<0.001). Finally, we found metabolic genes existed signi cantly differential (Q<0.001): crypt stem cells were biased towards energy metabolism (fructose and mannose) and vitamin metabolism(retinol), and organoids were biased towards amino acid process(glutathione metabolic process) and cholesterol process. This also implied that enteral nutrition absorption needed to be further processed in myo broblasts except the crypt epithelium.

Introduction
Cattle are economically important domestic animals in modern stock farming. Digestive system diseases, especially calf diarrhea, often lead to huge economic losses every year. Therefore, it is very important to understand the mechanism of pathogenesis and the function of the healthy intestinal mucosa. Intestinal tissue carries out several physiologic functions, such as immunization, digestion, and absorption of nutrition, etc. And the epithelial cells renew one time every 3-5 days for maintaining the intestinal epithelium integrity and function responding to the challenges of the complex environment in the intestine. Even though all these differentiated epithelial cells were known to derive from intestinal epithelial crypt stem cells located at the base of epithelial crypts and its niche environment to play a key role in forming the homeostasis [1]. When intestinal homeostasis is destroyed by pathogen, toxin, or other abnormal physical and chemical factors, the crypt stem cells could be immediately activated for recovering its function. But many complex mechanisms were unclear. Therefore, it is necessary to establish an ideal model to study it, especially for large animals.
At present, most of the intestinal studies focused on the mouse and human, and Lgr 5+ intestinal stem cells (ISCs) have been isolated and successfully develop intestinal organoids with 3D Matrigel. Several other markers of intestinal crypt stem cells have been identi ed including Bmi1, lrig1 [2][3][4], which represented the special population of stem cells which differentiate into other cells. And some growth factors are optimized to be used in the cell culture medium containing the stem-cell-niche factors WNT, Rspondin, EGF, Noggin(BMP inhibitor) which can remain ISCs genetically and phenotypically stable [5]. But the systematic comparison of morphology and characteristics was absent on the different parts of the intestine. But above combination had poor e ciency in forming the organoids and in proliferating the ISCs. In addition, in the adult intestine, large numbers of microorganisms restrict to obtain organoids and ISCs. Therefore, it is necessary to study the fetal intestine development and to culture the ISCs or intestinal organoids in vitro easily. It will be of great signi cance in the study of intestinal pathology and physiology instead of in vivo.
Every part of the intestinal system performs different functions, such as duodenum performs the chemical digestion, the jejunum is the main site for nutrient absorption, while the ileum absorbs residual nutrients, vitamins, and the main functions of the colon are to absorb water, electrolytes and microbial fermentation products [6]. Therefore, the different morphology and function of every part of the bovine fetus intestine need to be clear, which is helpful for explaining the differentiated intestinal physiology and pathology in bovine. Meanwhile, the development of bovine intestinal organoids represents a great opportunity to substitute e ciently the number of animals used for in vivo experiments. Although some scientists had reported a reliable procedure to get "mini gut" with Matrigel and organoids growth medium(mouse) from bovine ileal crypts [7]. In order to optimize the cultivation system, more studies are needed to be perfected and be veri ed. Therefore, it needs to establish an improved method for achieving intestinal organoids in bovine and make it easily to do. and to explore the different functions of every section of the intestine. The authors considered the bovine fetal intestinal tissue is a perfect object for this study. In the article, we compared the morphological characters of the bovine fetal intestine and tried to establish an easy way to obtain organoids without Matrigel and with normal culture medium. And the organoids could be enough for analyzing and evaluating the relation of the bovine intestine with nutrition, microorganism, and drug metabolism, etc. In addition, RNA sequencing was used to determine the difference in gene expression between organoid and crypt stem cells, and to determine the precise application and superiority of organoids in the research eld for studying the intestinal digestive function in large animals.

Animals
All intestines in this study were obtained from about 5-6-month-old bovine fetuses at local slaughterhouse. The fetal crown-rump length(CR) was measured to estimate fetal age. The fetuses with the whole uterus and intact placenta were kept in the icebox and transported to the lab within 1 hour.

Isolation of different parts of intestinal tissue
In this study, four parts of the intestine were recovered from every fetus, including duodenum, jejunum, ileum and colon. The whole intestine was isolated from the fetal enterocoelia and was washed 5 times with ice-cooled PBS. The mesentery was removed and the enteric cavity was ushed cleanly. Then the intestinal segments were cut 1-cm pieces for the next study.

Organoids Culture and Crypt Isolation
The excised intestinal jejunum segments were washed in PBS containing 0.3 % BSA and penicillin/streptomycin and opened longitudinally. And the subserous layer of intestine was stripped with ophthalmic forceps from the intestinal mucosa layer (Figure 1 ) was cut into smaller squares measuring 0.2-0.8 mm in diameter. Tissue pieces were transferred into a 4˚C PBS solution and washed 3 times to remove single cells and other debris by static settlement for 1min. Following the nal wash, the mini intestinal mucosa pieces were placed into a culture medium for 72 h at 5% CO 2 and 38.5 ˚C. Every 24h, the culture dishes were shocked or pipetted 2 times to prevent pieces from gathering together or adhering to the bottom of the dish. Then the primary organoids with multiple nger-like were picked up under stereomicroscope(XTZ-D) and transferred to the fresh medium for another 72 h. The culture dish was 100 X 20 mm (Easy Dish). The volume of medium was 15 ml and the number of intestinal pieces was adjusted to approximately 200-300 s in 1ml medium. The desired organoids were collected for morphological study and RNA sequencing analysis. The basic medium was DMEM (Hyclone) containing 10% FBS (Gbico), 100 U/ml penicillin, 100 mg/ml streptomycin, 5 μg /ml transferrin, 10 μg /ml insulin, 0.15 mM non-essential amino acid, 1mM sodium pyruvate, 1 μg /ml hydrocortisone.
Crypt stem cells were isolated from developed organoids by pipetting with thin micropipette tip repeatedly until crypt stem cell mass(epithelial stem cell) was completely separated from organoids. Then the crypt stem cell masses were collected under stereomicroscope and washed 3 times with PBS.

H&E staining
For histological analysis, different samples (duodenum, jejunum, ileum and colon) of fetal intestinal pieces were xed separately in PBS containing 4% (W/V) paraformaldehyde for 24 h. After xation, the samples were dehydrated in increasing concentrations of ethanol (50%, 70%, 80%, 95% and 100%), cleared in xylene, and para n-embedded for 24 h and made 5 µm sections on a Leica microtome. After the sections were depara nated in xylene, ethanol and water orderly. The sections were stained using standard hematoxylin and eosin protocols. After staining, the slides were mounted with coverslips using Entelan as the mounting medium and were examined under a light microscope.

Immunohistochemical staining
The intestinal sections were processed following the above protocols. After successively incubating with citric acid antigen retrieval solution (PH6.0) and 3 % H 2 O 2 for 25 mins, the slides were rinsed with PBS (PH7.4) three times, each for 5 mins and nonimmune binding of antiserum was blocked by incubating sections in PBS supplemented with 3% BSA for 30 mins. After pre-treatment, the slides were incubated with the primary antibody (1:50) (LGR5/GPR49 Rabbit Monoclonal Antibody, AF1582, Beyotime. Anti-beta Catenin Rabbit pAb, GB11015, Servicebio) overnight at 4°C and were rinsed with PBS (PH7.4) 3 times, each for 5 mins. Sections were incubated with corresponding secondary antibodies (HRP conjugated, GB23303) for 50 min. The sections were visualized with DAB. Finally, sections were counterstained with hematoxylin for 3 min and dehydrated and mounted with Pertex. The slides were then examined and photographed, and the data was collected and analyzed with Image J software.
Immuno uorescence staining were carried out to detect Expression of target proteins in organoid following the above methods. The primary antibodies were selected Anti-beta Catenin Rabbit pAb(GB11015, Servicebio ), Anti-PCNA Rabbit pAb(GB11010), Anti-Lysozyme Rabbit pAb(GB11345), respectively. Secondary antibody was Cy3-labeled Goat Anti-Rabbit IgG (GB21303, Servicebio). Then DAPI was then used for nuclear staining. Spurr embedding media (Ladd Research,) followed by 3 changes in 100% Epon812 for 12 h each. Finally, tissue blocks were embedded overnight at 60°C in fresh 100% Epon812 using at embedding molds.
Tissues were trimmed with razor blades and sectioned at 50 to 70 nm on a diamond knife using an Ultramicrotome(Leica EM UC7). Sections were stained on a grid with saturated uranyl acetate for 20 min at room temperature in the dark, followed by 3 water rinses, and 5 min in lead citrate with 3 water rinses.
Grids were analyzed using a JEM-1400 transmission electron microscope (Jeol Ltd, Tokyo, Japan) at 100 kV accelerating voltage( TEM Center of Henan University of Chinese Medicine) [8].
RNA sequencing RNA was extracted from Organoids and crypts using TRIzol® reagent (Invitrogen; Thermo Fisher Scienti c, Inc.). The lysate was loaded into an RNase-free centrifuge tube and RNA-seq (DNBSEQ-500 analysis platform ) was performed by Huada Gene Technology Co., Ltd (Wu han). Three samples were used in each group for RNA-sequencing assay. Every sample contained approximately 30 organoids or 300 crypts. Reference genome (Bos_taurus) version is ARS-UCD1.2_GCF_002263795.1. Cytoscape 3.8.2 (https://cytoscape.org/) and ClueGO app were applied to data analysis and mapping.

Statistical analysis
The results were analyzed using Student's two-tailed t-test for paired data or one-way ANOVA complemented with a post-test for multiple values where appropriate. A con dence interval of 95% with p ≤ 0.05 was considered statistically signi cant.

Morphological construction of the intestinal epithelium
Villus and crypts of bovine fetus intestine had different heights and depths. Villus of the jejunum and ileal were higher than of duodenum and colon(P<0.05), villus of the colon were the shortest than others(P<0.01)( Figure 3). Crypts of duodenum were nearly two times the depth of others(P<0.01)( Figure  4). In addition, duodenum crypts contained numerous goblet cells and ssions. By comparison, a lot of goblet cells were developed on the villus of ileum and colon, and cell nucleus of these villi was distributed at the bottom of columnar epithelial cells which demonstrated these cells were differentiated cells. And there was a mass of metabolite product in the ileal and colon cavity, which suggested metabolic level of whole intestinal epithelial cells was prompt at fetus period. At the crypts, most the cells possessed a large nucleus and arranged multiple layers, especially in the jejunum. So these crypt cells are target cells for researching intestinal stem cells (Figure 2).

Immunohischemical assay of intestinal tissue
Given this Lgr5 is a typical mark intestinal crypt stem cell and CTNNB1 is the core of WNT pathway which is essential to the development and differentiation of intestinal crypt stem cells. Lgr5 and CTNNB1 were expressed in stem cells of the different intestines. In this study, we found villus and crypts expressed both of them signi cantly. Lgr5 and CTNNB1 at all crypts were expressed signi cantly stronger than at villus. And Lgr5 expressions gradually decreased from duodenum, jejunum, ileum to colon(P<0.05). But the expression of CTNNB1 was the highest at jejunum crypts(P<0.05). Therefore, different segments of intestine gene expression exists various.

Transmission electron microscope(TEM) imaging
From TEM intestinal crypt ultrastructure, several structure characteristics were discovered in different intestinal segments including cell junctions, organelles, crypt stem cells, Paneth cells and goblet cells, and so on. First, the ultrastructure of crypts exhibits signi cantly various. In the crypts of the duodenum, numerous protein-containing vesicles (black) were generated in Paneth cells, which secrete a variety of small proteins from vesicles against pathogens. Other types of cell were intestinal stem cells with small nucleus distributed among Paneth cells. In the crypts of jejunum, stem cells were arranged tightly and multi-layered in some places. The cells were squeezed to a long strip shape with microvillus close to intestinal crypt lumen and cell nucleus were moved lightly to the bottom of the cells. At the peripheral of crypts, there was a layer of stroma cells with a small nucleus and cell volume. Ileum crypts had similar features with the jejunum and secretory cells were inserted among intestinal stem cells. At the colon crypts, except for all the tightly arranged stem cells, many goblet cells were observed at the bottom of the colon, in which were occupied by mucous granules and had very small cell nucleus. At all of crypts, tight junctions of cells were formed by several desmosomes close to the lumen which effectively made the intestinal epithelium intact. In addition, numerous cross-ngers like membrane structure strengthened the cell adherent's junctions between crypt cells. At the same time, abundant organelles were scattered around the cytoplasm of the crypt cells. In addition, in duodenum crypt cells, there is more rough endoplasmic reticulum than others. A mass of free ribosomes was clustered in crypt cell cytoplasm of jejunum, ileum, and colon.

Comparison of CTNNB1 expression in crypts of fetus and adult bovine
Comparing the CTNNB1 expression in the crypt, we found adult and fetus crypts had signi cantly different. All the intestinal crypt cells were highly expressed CTNNB1, but fetal intestinal villus also had a higher level of CTNNB1 than adults, which showed the fetal intestinal epithelium powerfully dynamic.

Jejunum organoids and crypt stem cells imaging
In the study, we could culture and obtained Jejunum's organoids effectively, appearing in different shapes and villus-like structures. Organoids were packaged tightly by intestinal epithelial cells. The body cavity of organoids was lled with plenty of matrix cells (myo broblasts) and scaffolds supporting the crypt epithelial cell development. Crypts developed round-shaped cell clusters in a very short time and occurred visually vesicles (Figure 8).

Immuno uorescent staining of organoid
According to the immuno uorescent staining of organoid, we found the epithelial cells highly expressed PCNA, CTNNB1, and lysosome, displaying the epithelial cells proliferative potential and vitality. Particularly, CTNNB1 was speci c in the epithelial cells, meaning the trait of stem cells. And myo broblasts under the crypt cell layer were lled in the organoids, expressing PCNA and lysosome, which displayed these cells potential roles in support of the development of the organoids (Figure 9).

Ultrastructure of the organoid
From the ultrastructure of the organoid, microvillus were observed to arrange at the surface of the crypt epithelial cells, and desmosomes junctions between the cells were clear. Epithelial cell had a large nuclear, abundant mitochondria and ribosomes, the characteristic of the cells were similar with in vivo intestinal crypt epithelial cells. But the shape of epithelial cells changed from at to columnar gradually depending on its space of development. And the junction of cells was looser than in vivo crypt stem cells( Figure 10).

Information of RNA sequencing in organoids and crypts(stem cells)
Organoids and crypts showed signi cant difference in gene expression( 3744 vs 3738 Q<0.05; 1453 vs 1751, Q<0.0001). By KEGG pathways enrichment, there were totally different pathways. In organoids (Figure 11), most of the high expressed genes were related to almost all metabolism pathways, such as Glutathione metabolism, mineral absorption, and lysosome, etc. And most of them shared key genes and affected each other forming networks. These demonstrated the organoids carried out the complex function of digestion and transport as a mini digestion organ. And some genes (LRP2, WNT2B, WNT11) were bene cial to organ formation. In addition, HOX genes (such as HOXA5, HOXA6, HOXB3 HOXB4 HOXB5 and HOXD4, Q<0.001), which are key in organ development and regulation, were active in organoids. Further, mitochondria development-related genes (MRPL58, MRPS33, MRPS25, MRPS24, MRPS34, MRPL34, Q<0.05) were signi cantly expressed in organoids Implementing complex functions( Figure 11). Conversely, in crypts, there were numerous genes related to cell junctions, In ammation, stem cell development, immunity and cancers. And some classically key driver genes were involved, such as Jun, NFKB1, MAPK8,CTNNB1 and IL6 etc. Cell cycle-regulation genes including CCND1, CDK6, CDKN1A were signi cantly high in expression which implied active in the proliferation and senescence of the crypt cells. It also conformed to the demand of intestinal epithelial cell fast renewal. It was said that Wnt signaling pathway was critical to forming the homeostasis of the intestinal stem cells. But its relevant genes existed inconformity. In crypts, almost of genes including key CTNNB1 were positive to Wnt signaling pathway. In organoids, several high expressed genes such as SFRP2 and SFRP5 were prohibitive to these pathways. It suggested matrix cells and crypt cells are different. But Gene RSPO2 and ROR2 were positive to the pathway, explaining the RSPO2 and ROR2 protein secreted from matrix cells and supporting the crypt or epithelial stem cell development (Figure 12).
To explore the metabolism pattern, we compared the genes of KEGG pathways in the metabolism between organoids and crypts to sum up the difference. The results displayed its features. In organoids, GGT1(1.9 folds) and GGT5(3 folds) were expressed highly related with amino acid and peptide catabolic process. And some key genes in glutathione metabolic processes was highly, such as GSTM1(2.2folds), GSMT2 etc. Another high expressed gene clusters gathered in steroid biosynthesis and cholesterol metabolic process(Q<0.001). Crypts groups showed distinct traits in metabolism. Fatty acid degradation and fat-soluble vitamin metabolism(like retinol) related genes were involved as CYP26A1(6.3 folds), CYP1A1(2.4folds) etc. (Q<0.001). Another area in crypts were energy metabolism process genes(PFKP , PGD and HKDC1) concerned fructose, mannose, glucose and pentose etc. Nucleosidase activity( Loc534181 8.9 folds, NT5C2 1.0 folds) and inositol phosphate metabolism (PI4K2B, 1.4folds, PLCE1, 1.3 folds)were highly expressed in crypt cells (Figure 13).

Discussion
The digest system is one of the most complicated developmental systems to carry out at least four major functions: digestion of food, absorption of nutrients, excretion of hormones, and defense against pathogens [9]. There were a lot of research outputs in experimental animals and human intestinal epithelial cells, and it continues to be top issue on intestinal stem cells for Intestinal development and pathology, particularly in crypt stem cells and in vitro organoids. Few references are available in the larger animals, so it was worthy to exploit it. These studies described the characteristic of epithelium structure in the bovine fetuses and cultured intestinal organoids by a simple method without Matrigel and special supplements. And from RNA sequencing, we analyzed the intestinal organoids at gene expression patterns.
Intestinal function and its structure have a close relationship. We found the duodenum revealed a deeper crypt with a few ssions and a thicker intestinal layer in the bovine fetus. The villus height was greater in jejunum and ileum segment than other two. The enteric and absorptive capacity could be related to villus height, volume, and density of the mucosa surface. The ileum showed the greatest number of goblet cells. The mucins are synthesized and secreted by exocytosis by the goblet cells into the gut lumen compounding a mucus layer, lubricating and protecting the intestinal epithelium [10].
Intestinal epithelial cells metabolize very rapidly and numerous exfoliated cells and villi were observed in the bovine fetus intestine. Although a marked similarity in surface membrane antigens was revealed between fetal intestinal epithelial cells and adult crypt cells [11]. We found LGR 5 and CTNNB1, showing the cell's differentiation and stem ability, expressed in the villus less than in crypts. And the villus epithelial cells had very limited regenerative potential and the most of cells showed a cell nucleus close to the bottom of the columnar epithelial cell and the rate of nucleus and cytoplasm was lower. But in the crypt cells, the nucleus is located in the central region and the rate of nucleus and cytoplasm was utmost higher. These indicate that the cell ampli cation capacity is completely different between villus and crypt at the fetus stage of bovine. It implied the villus cells began to differentiate into functioning cells even in the womb. And the crypt cells exhibited excellent peculiarity of ampli cation in comparison to the villus cells in vitro. Other researchers also concluded that the growth ability of intestinal stem cells in vitro was closely related to tissue maturation in vivo [12]. Therefore, the crypt cells are our target cells achieving intestinal development and organoids. Jordi had reported that villus and intervillus (crypts) cells are equipotent and have the same regenerative potential, which were isolated from the small intestine of mice at E16.5 [13]. The con icting conclusion may be of different species or developmental stages. Hence, it suggests only the crypt cells are used to isolate the intestinal stem cells in bovine fetus.
Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations and provide in vitro model system for pathways, and mechanisms involved in epithelial damage and repair [14,15]. Here, we have established a simple method for isolating and developing intestinal organoids and crypt stem cells without supplement of special growth factors and Matrigel. Although the 3D culture system was popular for developing intestinal organoids in vitro, its e ciency is still extremely lower for develop more organoids.
In the human intestine, colon cancer is the most serious disease of the digestive system. So lots of efforts were devoted to colon-relevant studies about physiology and pathology. And using colon cancer stem cells and Matrigel, the 3D organoids were established and applied to research the cancer mechanism and anticancer drug screening. On the contrary, in livestock like bovine, scientists mainly concentrate on intestinal absorption and abnormal symptoms (Diarrhea, Hematochezia) which have a worse effect on their health, growth or milk production. From these results in bovine fetus, the structure of intestine appeared the signi cant distinction in different intestinal segments. And jejunum is the main part for absorbing nutrition and is the longest segment. Therefore, jejunum crypt cells in bovine are prime targets that may be used to research the absorption of nutrition and diarrheal pathology by virus, bacteria and toxins, etc. And in these studies, we succeed to obtain the crypt cells and organoids from bovine fetal jejunum, the morphology and development of its organoid displayed similar features as in vivo intestine. The epithelial cells arranged on the surface of organoids and there is numerous microvillus on the top of the cell. And every epithelial cell has a large nucleus and abundant organelles. Between the epithelial cells exist tight desmosomes junction and membrane crimp links. At the same time, epithelial cells expressed strongly positive CTNNB1 playing a key role in crypt stem cell of organoid development in WNT signal pathway. Except in the crypt stem cell, Gene PCNA and Lyso also were positive in the matrix cells, illustrating the strong capacity of the cells in proliferation and metabolism. It was consistent with organoids RNA sequencing. So the methods of isolating and establishing organoid can be dependable to get high-quality organoids as bovine jejunum in vitro models. Furthermore, it needs to be clear the inner of organoid which are full of collagen and matrix cells, which were often thought hollow in the published reports. Certainly, It is necessary to gure out the crypt and organoid roles in Intestinal function and development.
According to the results of RNA sequencing, Organoids and crypts had signi cant difference in gene expression patterns. Organoid's genes relative to the metabolism pathway were highly expressed. Jonathan reviewed that metabolic pathways are the core of life and numerous discoveries have shed light on how metabolic pathways determine cellular fate and function [16]. Such as genes Kegg enrichment in glutathione metabolism, mineral absorption, Lysosome pathways, etc., which are closely related to the intestine function. Glutathione plays an important cytoprotective role in the gut and is one of the main intracellular reducing compounds for optimizing the intestinal Ca 2+ absorption and depleting drugs such as menadione or vitamin K 3 , sodium deoxycholate or diets enriched in fructose, which induce several features of the metabolic syndrome, produce inhibition of the intestinal Ca 2+ absorption [17]. The lysosome pathway is the major route for clearance of aberrant components to maintain protein homeostasis and normal cellular function [18]. And Increasing lysosome enzyme activities help to clear pathological cellular waste. In the bovine intestinal organoids, numerous genes were enriched in the lysosome pathway indicating the organoid's complicated function. In addition, genes involved in the vascular smooth muscle contract pathway were highly expressed, which made the organoid have a similar character with bowel movements. Other metabolism pathways on purine, pyruvate, histidine, etc. were positive. mTOR and PPAR signaling pathways were also discovered superior in the bovine intestinal organoids. mTOR pathway is nutrient-sensing pathway playing a central role in the synthesis of proteins. PPAR pathway can regulate various metabolic processes such as fatty acid metabolism [19]. HOX genes and mitochondria development-related genes (MRPL58, MRPS33, etc.) were highly expressed in organoids. One is in charge of the development of organs, the other is bene cial to mitochondrial function. Both of them suggested that organoids possessed organ specialty and high metabolic levels. In brief, the development of the organoids from bovine jejunum can be used as a model for metabolism studies, in which few reports were published on intestinal organoids.
The intestinal epithelium is the most rapidly renewed tissue and its renewal is strictly controlled by intestinal stem cells, which forms a highly dynamic and selective barrier that controls the absorption of uid and solutes while restricting pathogen access to underlying tissues. Barrier properties are achieved by intercellular junctions [20]. And diverse localization of epithelial intercellular junction proteins along the intestinal tract may correlate with differences in paracellular permeability and adhesion along the intestinal tract [21]. These studies agreed with the conclusion. We compared the gene expression between crypt stem cells and organoids. Distinguished from the Kegg pathway enrichment on metabolism in organoid, crypt stem cells showed the gene enrichment characteristics in cell junction, immunization and disease pathways. And the enriching genes in TNF/FoxO/NF-kappaB/PI3K-AKT signaling pathways formed a network in the crypt stem cells closely related to immunity and in ammation. And most genes involved Cellular senescence/Apoptosis/cancer signaling pathways. Moreover, ample genes in Wnt signaling pathway and regulating pluripotent stem cells fully showed the various points of crypt stem cells. Such as CTNNB1 was the one of key drive genes in the crypts and It's consistent with our IHC results, distinguishing from Wnt signaling pathway in organoids. Because The intestinal epithelium has one of the highest turnover rates, with 10 11 epithelial cells (~200 g) being lost every day in humans. This indicates an immense proliferative process driven by the stem cell compartment located in the crypt [22].
To compare the genes in metabolism between organoid and stem cell, we found there was biases in signaling pathways. Such as crypt stem cells had priority in fat-soluble vitamin metabolic process, energy metabolism and fatty acid degradation. Ilya Lukonin also reported that retinoic acid metabolism of organoids in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium from the regenerative state and driving enterocyte differentiation [23]. We found the retinoic acid metabolism was restricted in crypt stem cells. Relatively, organoids possessed the capacities in amino acid (peptide) catabolic process and steroid (metabolic) process. It implied matrix cells also play a digestive cooperative role except for the work of epithelial (crypt) cells in nutrition metabolism.

Conclusion
In conclusion, studying the intestinal development of fetal cattle, we have learned the structure and morphology of different developmental segments of the intestine, and the development characteristics of duodenum, jejunum, ileum and colon are different. Moreover, due to its high developmental potential and without contamination by intestinal microbial, we can easily develop organoids from jejunum epithelial tissue. According to transcriptome analysis, using in vitro crypt stem cells is competent in detecting Immune in ammation, cancer, intestine development and even Interaction mechanism with gut microbiota. By comparison, intestinal organoids are more compatible and closer to in vivo intestines, particularly for evaluating metabolism in large animals. The datasets produced and/or analyzed during the current study are available from the corresponding author on reasonable request.     Signi cantly high expressed genes in KEGG pathways in Organoids (Left) and differentially expressed genes enrichment in KEGG pathways(Right) Figure 12 Signi cantly high expressed genes in KEGG pathways in Crypts (Left) and differentially expressed genes enrichment in KEGG pathways(Right)