Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.     

Generation of RUNX3 knockout pigs using CRISPR/Cas9‐mediated gene targeting

Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies.     

Effects of voltage strength during electroporation on the development and quality of in vitro‐produced porcine embryos

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro‐produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm–40 V/mm with five 1‐msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single‐guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.     

Efficient generation of CLPG1‐edited rabbits using the CRISPR/Cas9 system

The sheep callipyge (CLPG) phenotype, a well‐known muscular hypertrophy syndrome, is caused by an A‐to‐G transition in the CLPG1 locus. The mechanisms of CLPG phenotype are very complicated and remain to be further studied. Lacking suitable animal models containing CLPG mutations may partially contribute to these unanswered mechanisms. In this study, we confirmed that the CLPG1 locus, especially the 12‐bp CLPG1 motif, is conserved in mammalian animals including rabbit. Then, we generated seven CLPG1‐edited rabbits with 100% efficiency using CRISPR/Cas9 system combined with cytoplasm injection technology. All the newborn rabbits were mosaicism with numerous kinds of mutations around the target sites. Among the nine screened potential off‐target sites (POTs) for the two sgRNAs used in this study, none off‐target effect was detected. This indicated that we efficiently and precisely generated CLPG1‐edited rabbits, and we believe that these newly generated rabbits will do help to unravel the mechanisms of the CLPG phenotype in the future.     

CRISPR/Cas9-mediated MSTN disruption accelerates the growth of Chinese Bama pigs

CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

CRISPR/Cas9介导的绵羊胎儿成纤维细胞肌肉生长抑制素基因敲除研究

肌肉生长抑制素(myostatin,MSTN)基因突变可引起动物出现"双肌"性状,提高产肉性能。利用CRISPR/Cas9技术制备MSTN基因敲除的绵羊胎儿成纤维细胞,为制备MSTN基因敲除羊提供材料。设计构建4个靶向MSTN基因的CRISPR/Cas9载体,脂质体转染细胞后,通过SURVEYOR分析和测序等方法对敲除效率进行检测,采用极限稀释法挑选稳定敲除的细胞系。试验成功构建了4个靶向MSTN基因的CRISPR/Cas9载体,细胞转染后,测序结果显示pX330-target 1和pX330-target 4载体作用的靶位点处出现突变,SURVEYOR分析检测其在靶位点产生切割的效率分别为24.20%和10.18%。通过极限稀释法,获得12个MSTN基因突变的细胞克隆,其中1个为纯合突变。序列比对发现靶位点处有小片段碱基插入或缺失突变,部分会出现移码突变。成功利用CRISPR/Cas9系统实现了绵羊MSTN基因敲除,证明该系统可有效应用于绵羊基因编辑,产生的突变细胞系为制备MSTN基因敲除羊提供了材料。     

Knockout of Myostatin Gene by CRISPR/Cas9 System in Ovine Fetal Fibroblasts

Mutation in myostatin (MSTN) gene resulted in double muscle effect,generating more mutton.To knock out MSTN gene in sheep fetal fibroblast by CRISPR/Cas9 system and obtain MSTN gene knockout cell lines,four plasmids were designed and constructed to target MSTN gene,and confirmed correctly by sequencing.The correct plasmids were delivered into the fetal fibroblast cells.The targeting efficiency was detected using SURVEYOR assay Kit.The stable transfected cell colonies were obtained via limiting dilution procedure.The sequence results demonstrated that the pX330-target 1 and pX330-target 4 plasmids could successfully knockout MSTN gene,and the targeting efficiency were 24.20% and 10.18%,respectively.Twelve MSTN gene knockout cell colonies were obtained via limiting dilution,and one of them was homozygous mutation.Several indel mutations were discovered at specific site,and some of them were frame-shift mutation.Therefore,we concluded that the CRISPR/Cas9 system could apply to the gene editing of sheep efficiently,and the gene knockout cell clones had potential application in generating MSTN gene knockout sheep.     

Low Occurrence of Extended‐Spectrum β‐lactamase‐Producing Escherichia coli in Finnish Food‐Producing Animals

ESBL/AmpC‐producing Escherichia coli is increasingly isolated from humans and animals worldwide. The occurrence of ESBL/AmpC‐producing E. coli was studied in food‐producing animals in Finland, a country with a low and controlled use of antimicrobials in meat production chain. A total of 648 cattle, 531 pig, 495 broiler and 35 turkey faecal samples were collected from four Finnish slaughterhouses to determine the presence of extended‐spectrum β‐lactamase (ESBL/AmpC)‐producing E. coli. In addition, 260 broiler and 15 turkey samples were screened for carbapenemase‐producing E. coli. Susceptibility to different class of cephalosporins and meropenem was determined with disc diffusion tests according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Determination of ESBL/AmpC production was performed with a combination disc diffusion test according to the recommendations of the European Food Safety Authority (EFSA). Plasmidic bla_ESBL/AmpC genes were characterized by polymerase chain reaction and sequencing. A collection of isolates producing AmpC enzyme but not carrying plasmidic bla_AmpC was analysed by PCR and sequencing for possible chromosomal ampC promoter area mutations. Altogether ESBL/AmpC‐producing E. coli was recovered from five cattle (0.8%), eight pig (1.5%) and 40 broiler samples (8.1%). No ESBL/AmpC‐producing E. coli was found in turkey samples. Carbapenem resistance was not detected. Altogether ESBL/AmpC‐producing E. coli was found on 4 (2.0%), 3 (4.5%) and 14 (25%) cattle, pig and broiler farms, respectively. From cattle samples 3 (27%) bla_CTX‐M‐1 and from broiler samples 13 (33%) bla_CTX‐M‐1 and 22 (55%) bla_CMY‐2 gene‐carrying isolates were detected. In pigs, no plasmidic bla_ESBL/AmpC gene‐carrying isolates were found. In all analysed isolates, the same mutations in the promoter region of chromosomal ampC were detected. The results showed low occurrence of ESBL/AmpC‐producing E. coli in Finnish food‐producing animals. In pigs, plasmidic bla_ESBL/AmpC‐carrying E. coli was not detected at all.     

CRISPR/Cas9技术及其在猪基因编辑中的应用

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)是一种新型基因编辑系统,是由细菌和古细菌对抗入侵病毒及外源DNA的适应性免疫防御系统演变而来的。该系统具有简易、精准、经济和高效的优点,目前已广泛应用于基因编辑及其相关研究中。本文总结了近几年CRISPR/Cas9技术在猪基因组编辑中的研究进展,并展望了其应用前景。     

CRISPR/Cas9技术在猪常见病毒性疾病防控中的应用

CRISPR/Cas系统是一种存在于古生菌和细菌中重要的适应性免疫系统,CRISPR/Cas9技术可以实现对特定基因序列进行编辑,目前已经成为应用最广泛的基因编辑工具,利用其可以实现对病毒进行基因改造或解析宿主与病毒相互作用,优化疫苗生产和抗病毒动物分子育种等.猪作为我国最重要的食品动物,在国民经济和社会稳定中发挥着重...     

Olive leaves (Olea europea L.) and α‐tocopheryl acetate as feed antioxidants for improving the oxidative stability of α‐linolenic acid‐enriched eggs

Ninety‐six brown Lohmann laying hens were equally assigned into four groups with six replicates. Hens within the control group were fed a corn–soybean‐based diet supplemented with 4% linseed oil. Two other groups were given the same diet further supplemented with 5 or 10 g ground olive leaves/kg feed, while the diet of the fourth group was further supplemented with 200 mg α‐tocopheryl acetate/kg. Supplementing diets with olive leaves had no effect on egg production, feed intake and egg traits. Eggs collected 28 days after feeding the experimental diets were analysed for lipid hydroperoxides and malondialdehyde (MDA) content, fatty acid profile, α‐tocopherol concentrations and susceptibility to iron‐induced lipid oxidation. Olive leaves were also analysed for total and individual phenolics, and total flavonoids, whereas their antioxidant capacity was determined using both the DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) and ABTS (2,2‐azinobis3‐ethylbenzothiazoline‐6‐sulphonic acid) radical scavenging activity assays. Results showed that neither α‐tocopheryl acetate nor olive leaves supplementation exerted (p > 0.05) any effect on the fatty acid composition of n‐3 eggs. Supplementing the diet with 5 g olive leaves/kg had no (p > 0.05) effect on the hydroperoxide levels of n‐3 eggs, while supplementing with 10 g olive leaves/kg or 200 mg α‐tocopheryl acetate/kg, the lipid hydroperoxide levels were reduced (p ≤ 0.05) compared to control. However, although hydroperoxides were reduced, MDA, a secondary lipid oxidation product, was not affected (p > 0.05). Iron‐induced lipid oxidation increased MDA values in eggs from all groups, the increase being higher (p ≤ 0.05) in the control group and the group supplemented with 5 g olive leaves/kg. The group supplemented with 10 g olive leaves/kg presented MDA values lower (p ≤ 0.05) than the control but higher (p ≤ 0.05) than the α‐tocopheryl acetate group, which presented MDA concentrations lower (p ≤ 0.05) than all other experimental diets at all incubation time points.     

Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A,B, and J

Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus(ALV)subgroups using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A(TVA) and generate chicken cells resistant to infection by this virus.Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely,overexpression of the wild-type TVA receptor(wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na~+/H~+ exchange 1(chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.     

The effect of M‐phase stage‐dependent kinase inhibitors on inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) expression and localization in pig oocytes

At fertilization, inositol 1,4,5‐trisphosphate receptor type 1 (IP₃R1) has a crucial role in Ca²⁺ release in mammals. Expression levels, localization and phosphorylation of IP₃R1 are important for its function, but it still remains unclear which molecule(s) regulates IP₃R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP₃R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP₃R1 were formed in the cortex of the oocyte except in a ring‐shaped band of cortex adjacent to the spindle. However, no such clusters of IP₃R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP₃R1 localization, phosphorylation and expression using M‐phase stage‐dependent kinase inhibitors. Our results show that treatments with roscovitine (p34^cdc2 kinase inhibitor) or U0126 (mitogen‐activated protein kinase inhibitor) did not affect IP₃R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI‐2536, an inhibitor of polo‐like kinase 1 (Plk1), dramatically decreased the expression level of IP₃R1 in pig oocytes in a dose‐dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP₃R1 expression in pig oocytes.     

CRISPR/Cas9系统在寄生原虫基因编辑中的应用

原虫生活史复杂，大部分难以通过体外培养完成整个生活史，缺乏高效的基因编辑系统。CRISPR/Cas9系统可以通过人工设计的方式，实现基因的精确、高效、快速编辑，被广泛应用于基因工程各个领域。CRISPR/Cas9系统为原虫的未知功能基因研究开辟了新途径。本文介绍CRISPR/Cas9系统的原理和目前在寄生原虫中的基因定位、基因功能研究、高通量基因家族筛选等方面的应用。     

家禽疱疹病毒CRISPR/Cas9基因编辑最新研究进展

基于CRISPR/Cas9系统的基因编辑是最新一代的基因组编辑技术，在向导RNA （gRNA）的介导下几乎可以靶向编辑任何一种基因，实现基因组的定点突变、敲除或插入。近年来将CRISPR/Cas9基因编辑技术应用于大基因组DNA病毒的研究，尤其是用于疱疹病毒的基因编辑已成为病毒学研究领域的最新国际热点。自2016年首次报道利用CRISPR/Cas9系统改造家禽疱疹病毒如马立克病病毒（MDV）基因组以来，短短5年时间已全面应用于家禽疱疹病毒的蛋白编码基因和非编码RNA基因的编辑、基因缺失疫苗和重组疫苗研发、抗病毒治疗以及抗病育种等领域。本文详细综述了当前CRISPR/Cas9基因编辑技术在家禽疱疹病毒中的应用进展和最新成果，并对其面临的问题和前景进行了展望，以期为后续研究提供重要参考。     

Fascin‐1 expression in canine cutaneous and oral melanocytic tumours

Fascin‐1 expression was examined in 9 cutaneous melanocytomas and 47 oral melanomas. The cases were scored on the basis of extent and intensity of staining, and combined scores were calculated. Fascin‐1 expression was observed in 5/9 (56%) melanocytomas and 46/47 (98%) melanomas. The combined score for fascin‐1 was significantly greater in stage III/IV melanomas than in stage I/II melanomas (P < 0.05). In addition, strong fascin‐1 staining was associated with a significantly shortened survival time (P < 0.05). The results of this study suggest that fascin‐1 overexpression correlates with the malignancy of canine melanoma and has the potential to be a new immunohistochemical marker to predict the clinical course of canine melanoma. In addition, targeted therapy for fascin‐1 may represent a new strategy for the treatment of canine melanoma.     

CRISPR/Cas9基因编辑技术在重要猪病毒病防控中的研究与应用

规律成簇的间隔短回文重复序列系统（clustered regularly interspaced short palindromic repeat，CRISPR）是一种广泛存在于古细菌和细菌中，由RNA介导在Cas蛋白协助下发挥作用的获得性免疫系统，目前，已发现的CRISPR系统中以CRISPR/Cas9应用最为广泛，本文主要对CRISPR/Cas9系统的基本原理和研究进展进行概述，着重介绍其在重要猪病毒病防控中的应用，包括改造宿主和改造病毒两方面，该技术为研究病毒致病机制、新型疫苗研发以及抗病育种研究等提供了强有力的工具，对疫病的控制有着深远的影响。     

IFN‐τ Acts in a Dose‐Dependent Manner on Prostaglandin Production by Buffalo Endometrial Stromal Cells Cultured in vitro

Interferon‐τ (IFN‐τ) has been recognized as the primary embryonic signal responsible for maternal recognition of pregnancy. Uterine endometrium produces both prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂). PGF_2α is responsible for the luteolysis; however, PGE₂ favours establishment of pregnancy by its luteoprotective action. In this study, the dose‐response effect of recombinant bovine IFN‐τ (rbIFN‐τ) on prostaglandin (PG) production by buffalo endometrial stromal cells cultured in vitro was studied. Buffalo endometrial stromal cells were isolated by double enzymatic digestion, initially with trypsin III followed by a cocktail of trypsin III, collagenase type II and DNase I and subsequently cultured till confluence. Further, cells were treated with different doses of rbIFN‐τ (0.001, 0.01, 0.1, 1.0 and 10 μg/ml) and keeping a separate set of control. Culture supernatant was collected after 6, 12 and 24 h of treatment. PG levels in the culture supernatant were measured by enzyme immune assay (EIA) and total cellular protein estimated by Bradford method. Results indicated that buffalo endometrial stromal cells following rbIFN‐τ treatment enhanced the secretion of both PGE₂ and PGF_2α, and also its ratio in a strict dose‐dependent manner with a significant increase (p < 0.01) in PGE₂ production at 1 μg/ml dose of rbIFN‐τ and maximal stimulation for both PG was observed at 10 μg/ml. Further, both PG production and its ratio were increased significantly (p < 0.01) in a time‐dependent fashion in all the groups at 6, 12 and 24 h post‐treatment with highest level achieved at 24 h as compared with control. Absolute levels of PGE₂ remained higher than PGF_2α indicating PGE₂ as the major PG produced by endometrial stromal cells. The dose‐dependent response of rbIFN‐τ signifies the importance of optimum concentration of IFN‐τ for the embryonic development especially during the critical period to establish successful pregnancy.     

Involvement of PKA signalling in anti‐inflammatory effects of chitosan oligosaccharides in IPEC‐J2 porcine epithelial cells

Weaning is characterized by intestinal inflammation, which is a big challenge in pig industry. Control of intestinal inflammation is important for improvement of growth performance and health. Therefore, the study was focused on the anti‐inflammatory activity of low‐molecular‐weight chitosan oligosaccharide (LCOS) in a porcine small intestinal epithelial cell line (IPEC‐J2). The results showed that TNF‐α, as inflammation inducer, significantly upregulated the mRNA expression of IL‐8 and MCP‐1. Afterwards, LCOS significantly attenuated mRNA expression of IL‐8 and MCP‐1 induced by TNF‐α in the cells. Mannose (MAN), as ligand of mannose receptor, had no effect on the anti‐inflammatory activity of LCOS, which suggested that mannose receptor may not involve in the anti‐inflammatory activity of LCOS in IPEC‐J2 cells. Interestingly, N‐[2‐(p‐bromocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide 2HCl hydrate (H89), as PKA (protein kinase A)‐specific inhibitor, reversed the mRNA expression of IL‐8 when co‐cultured with LCOS. Furthermore, LCOS concentration dependent downregulated the mRNA expression of claudin‐1 compared with TNF‐α treatment. However, the trans‐epithelial electric resistance (TEER) was not affected by LCOS when co‐cultured with TNF‐α in 3 hr. In conclusion, LCOS have a potent anti‐inflammatory activity, and as a feed additives, may be useful for the inhibition of inflammatory process in weaning period of pigs with intestinal inflammation occurring.