Voluntary wheel running ameliorated the deleterious effects of high-fat diet on glucose metabolism,gut microbiota and microbial-associated metabolites

Exercise training is critical for the early prevention and treatment of obesity and diabetes mellitus.However,the mechanism with gut microbiota and fecal metabolites underlying the effects of voluntary wheel running on high-fat diet induced abnormal glucose metabolism has not been fully elaborated.C57BL/6 male mice were randomly assigned to 4 groups according to diets(fed with normal chow diet or high-fat diet)and running paradigm(housed in static cage or with voluntary running wheel).An integrative 16S rDNA sequencing and metabolites profiling was synchronously performed to characterize the effects of voluntary wheel running on gut microbiota and metabolites.It showed that voluntary wheel running prevented the detrimental effects of high-fat feeding on glucose metabolism 16S rDNA sequencing showed remarkable changes in Rikenella and Marvinbryantia genera.Metabolic profiling indicated multiple altered metabolites,which were enriched in secondary bile acid biosynthesis signaling.In conclusion,our study indicated that voluntary wheel running significantly improved glucose metabolism and counteracted the deleterious effects of high-fat feeding on body weight and glucose intolerance.We further found that voluntary wheel running could integratively program gut microbiota composition and fecal metabolites changes,and may regulate muricholic acid metabolism and secondary bile acid biosynthesis in high-fat fed mice.

Spatially resolved transcriptomic profiling of placental development in dairy cow

The placenta plays a crucial role in successful mammalian reproduction.Ruminant animals possess a semi-invasive placenta characterized by a highly vascularized structure formed by maternal endometrial caruncles and fetal placental cotyledons,essential for full-term fetal development.The cow placenta harbors at least two trophoblast cell populations:uninucleate(UNC)and binucleate(BNC)cells.However,the limited capacity to elucidate the transcriptomic dynamics of the placental natural environment has resulted in a poor understanding of both the molecular and cellular interactions between trophoblast cells and niches,and the molecular mechanisms governing trophoblast differentiation and functionalization.To fill this knowledge gap,we employed Stereo-seq to map spatial gene expression patterns at near single-cell resolution in the cow placenta at 90 and 130 days of gestation,attaining high-resolution,spatially resolved gene expression profiles.Based on clustering and cell marker gene expression analyses,key transcription factors,including YBX1 and NPAS2,were shown to regulate the heterogeneity of trophoblast cell subpopulations.Cell communication and trajectory analysis provided a framework for understanding cell-cell interactions and the differentiation of trophoblasts into BNCs in the placental microenvironment.Differential analysis of cell trajectories identified a set of genes involved in regulation of trophoblast differentiation.Additionally,spatial modules and co-variant genes that help shape specific tissue structures were identified.Together,these findings provide foundational insights into important biological pathways critical to the placental development and function in cows.

Repeated inoculation with rumen fluid accelerates the rumen bacterial transition with no benefit on production performance in postpartum Holstein dairy cows

Background The dairy cow’s postpartum period is characterized by dramatic physiological changes,therefore imposing severe challenges on the animal for maintaining health and milk output.The dynamics of the ruminal microbiota are also tremendous and may play a crucial role in lactation launch.We aim to investigate the potential benefits of early microbial intervention by fresh rumen microbiota transplantation(RMT)and sterile RMT in postpartum dairy cows.Twelve fistulated peak-lactation dairy cows were selected to be the donors for rumen fluid collection.Thirty postpartum cows were divided into 3 groups as the transplantation receptors respectively receiving 10 L fresh rumen fluid(FR),10 L sterile rumen fluid(SR),or 10 L saline(CON)during 3 d after calving.Results Production performance,plasma indices,plasma lipidome,ruminal microbiome,and liver transcriptome were recorded.After fresh and sterile RMT,we found that the molar proportion of propionic acid was increased on d 7 in the FR and SR groups and the bacterial composition was also significantly changed when compared with the CON group.A similarity analysis showed that the similarities between the CON group and FR or SR group on d 7 were 48.40%or 47.85%,whereas the similarities between microbiota on d 7 and 21 in the FR and SR groups were 68.34%or 66.85%.Dry matter intake and feed efficiency were not affected by treatments.Plasmaβ-hydroxybutyrate concentration in the FR group was decreased and significantly different lipids mainly included phosphatidylcholine and lysophosphatidylcholine containing polyunsaturated fatty acids.Hepatic transcriptomics analysis indicated acutephase response pathways were upregulated in the SR group.Conclusions Our study suggests that RMT can shorten the transition process of the ruminal microbiota of postpartum dairy cows with no benefit on dry matter intake or feed efficiency.Inoculation with rumen fluid may not be a useful approach to promote the recovery of postpartum dairy cows.

Enhancing milk quality and modulating rectal microbiota of dairy goats in starch‑rich diet:the role of bile acid supplementation

Background Diets rich in starch have been shown to increase a risk of reducing milk fat content in dairy goats.While bile acids(BAs)have been used as a lipid emulsifier in monogastric and aquatic animals,their effect on ruminants is not well understood.This study aimed to investigate the impact of BAs supplementation on various aspects of dairy goat physiology,including milk composition,rumen fermentation,gut microbiota,and BA metabolism.Results We randomly divided eighteen healthy primiparity lactating dairy goats(days in milk=100±6 d)into two groups and supplemented them with 0 or 4 g/d of BAs undergoing 5 weeks of feeding on a starch-rich diet.The results showed that BAs supplementation positively influenced milk yield and improved the quality of fatty acids in goat milk.BAs supplementation led to a reduction in saturated fatty acids(C16:0)and an increase in monounsaturated fatty acids(cis-9 C18:1),resulting in a healthier milk fatty acid profile.We observed a significant increase in plasma total bile acid concentration while the proportion of rumen short-chain fatty acids was not affected.Furthermore,BAs supplementation induced significant changes in the composition of the gut microbiota,favoring the enrichment of specific bacterial groups and altering the balance of microbial populations.Correlation analysis revealed associations between specific bacterial groups(Bacillus and Christensenellaceae R-7 group)and BA types,suggesting a role for the gut microbiota in BA metabolism.Functional prediction analysis revealed notable changes in pathways associated with lipid metabolism,suggesting that BAs supplementation has the potential to modulate lipid-related processes.Conclusion These findings highlight the potential benefits of BAs supplementation in enhancing milk production,improving milk quality,and influencing metabolic pathways in dairy goats.Further research is warranted to elucidate the underlying mechanisms and explore the broader implications of these findings.

Effects of forsythin extract in Forsythia leaves on intestinal microbiota and short-chain fatty acids in rats fed a high-fat diet

Forsythia suspensa,belonging to the deciduous shrubs of the Luteaceae family,a traditional Chinese medicine,has effects of alleviating swelling,clearing heat,detoxification and promoting blood circulation.The leaves of F.suspensa contain multiple chemical components and have a long history of use in folk medicines and health foods.The purpose of this study was to explore the effects of forsythin extract from F.suspensa leaves on intestinal microbiota and short-chain fatty acid(SCFA)content in rats with obesity induced by a high-fat diet.Forsythin extract in F.suspensa leaves increased the abundance of the intestinal microbiota,ameliorated intestinal microbiota disorders and inhibited the increase in total SCFA content in the intestinal tract in rats with obesity induced by a high-fat diet.These results suggested that forsythin extract in F.suspensa leaves may slow the development of obesity induced by a high-fat diet;thus,its active components and efficacy are worthy of further study.

Maternal supplementation with n-3 fatty acids affects placental lipid metabolism, inflammation, oxidative stress, the endocannabinoid system, and the neonate cytokine concentrations in dairy cows

Background The placenta plays a crucial role in supporting and influencing fetal development.We compared the effects of prepartum supplementation with omega-3(n-3)fatty acid(FA)sources,flaxseed oil(FLX)and fish oil(FO),on the expression of genes and proteins related to lipid metabolism,inflammation,oxidative stress,and the endocannabinoid system(ECS)in the expelled placenta,as well as on FA profile and inflammatory response of neonates.Late-pregnant Holstein dairy cows were supplemented with saturated fat(CTL),FLX,or FO.Placental cotyledons(n=5)were collected immediately after expulsion,and extracted RNA and proteins were analyzed by RTPCR and proteomic analysis.Neonatal blood was assessed for FA composition and concentrations of inflammatory markers.Results FO increased the gene expression of fatty acid binding protein 4(FABP4),interleukin 10(IL-10),catalase(CAT),cannabinoid receptor 1(CNR1),and cannabinoid receptor 2(CNR2)compared with CTL placenta.Gene expression of ECS-enzyme FA-amide hydrolase(FAAH)was lower in FLX and FO than in CTL.Proteomic analysis identified 3,974 proteins;of these,51–59 were differentially abundant between treatments(P≤0.05,|fold change|≥1.5).Top canonical pathways enriched in FLX vs.CTL and in FO vs.CTL were triglyceride metabolism and inflammatory processes.Both n-3 FA increased the placental abundance of FA binding proteins(FABPs)3 and 7.The abundance of CNR1 cannabinoid-receptor-interacting-protein-1(CNRIP1)was reduced in FO vs.FLX.In silico modeling affirmed that bovine FABPs bind to endocannabinoids.The FLX increased the abundance of inflammatory CD44-antigen and secreted-phosphoprotein-1,whereas prostaglandin-endoperoxide synthase 2 was decreased in FO vs.CTL placenta.Maternal FO enriched neonatal plasma with n-3 FAs,and both FLX and FO reduced interleukin-6 concentrations compared with CTL.Conclusion Maternal n-3 FA from FLX and FO differentially affected the bovine placenta;both enhanced lipid metabolism and modulated oxidative stress,however,FO increased some transcriptional ECS components,possibly related to the increased FABPs.Maternal FO induced a unique balance of pro-and anti-inflammatory components in the placenta.Taken together,different sources of n-3 FA during late pregnancy enhanced placental immune and metabolic processes,which may affect the neonatal immune system.

Feeding Calcium Salts of Linseed Oil on Metabolic Status and Reproductive Traits in Grazing Dairy Cows

This experiment aimed to evaluate the effect of calcium salts of linseed oil (rich in alpha-linolenic acid (ALA, C18:3n-3) on metabolic and reproductive traits in high-producing dairy cows under grazing. Thirty-six Holstein dairy cows were randomly assigned, in a complete block design, to receive ALA supplementation (0.85 kg∙day−1 of calcium salts of linseed oil) or to remain as untreated control (CON). The concentrate was formulated to offer the same amount of energy across treatments (CON cows received an extra kg of corn to compensate for the higher energy density of ALA treatment). A PMR + Alfalfa pasture was offered to all cows at the same time. A fixed time artificial insemination (FTAI) at 80 DIM, preceded by a Presynch plus Ovsynch protocol was implemented for the first service and later, on return to estrus, heat detection and artificial insemination (AI) were performed. Pregnancy diagnosis was checked at 30, 42, 60, and 90 d after AI. Blood and milk samples were taken biweekly. Treatment affected plasma cholesterol concentration (160.36 vs. 186.70 mg∙dl−1, p = 0.03, for ALA and CON, respectively) and on size of corpus luteum (CL, 17.6 vs. 13.7 mm, p = 0.02, for ALA and CON, respectively). Supplementation tended (p = 0.136) to increase conception rate by 200 DIM (81.69% vs. 55.43% in ALA and CON cows, respectively). However, treatment had no effect (p > 0.05) on body weight (BW), body condition score (BCS), and circulating levels of beta-hydroxybutyrate (BHBA), glucose, insulin, growth hormone (GH) and insulin-like growth factor (IGF-I). Our results suggest that supplementation with calcium salts of linseed oil could enhance ovarian function without affecting energy metabolism in early lactation dairy cows.

Effect of Extending Voluntary Waiting Period on Health of Holstein Friesian Dairy Cows

The aim of this of this experimental study to investigate the effect of 3 voluntary waiting periods (VWP) on health of dairy cows. A total of 100 lactations of 100 Holstein Friesian cows with high milk production (average 9.442 ± 620 kg) were randomly (based on the ear tags digits of cows) distributed to 1 to 3 experimental groups with VWP of 50 (VWPG50;n = 32), 100 (VWPG100;n = 34) or 150 days (VWPG150;n = 34). Observations relating to diseases of the cows were made by the farm veterinarian and research personnel. Were defined eight disease: puerperal fever, retained fetal membranes, displaced abomasum, metritis, clinical ketosis, clinical mastitis, ovarian cysts, and claw diseases. The experimental days (ED) were from 4 days in milk (DIM) to 100 days after the next calving. The experiment was managed at Dairy Research Farm ?imnic-Craiova January 2018 to December 2022. A clear set of clinical signs were used to define a case at the diseases without the need for laboratory confirmation. Values of Lactational incidence risk (LIR) median postpartum days at diagnosis and pairwise comparison of proportion between VWPs were calculated. LIR for puerperal fiver, retained fetal membranes, displaced abomasum, clinical ketosis, clinical mastitis, ovarian cysts and claw disease were 4;5;2;8;8;and 4% respectively. Numerically, cows with VWP of 100 and150 days had more disease cases compared with VWP of 50 days. The differences between experimental groups of cows regarding proportions of cows with disease cases were not statistically significant. Any of the eight disorders reported in this study was analyzed independently to other health problems. For all enrolled cows (n = 100) VWP was extended until 100 or 150 days postpartum with no effect on the lactational incidence risk for the eight disorders analyzed.

Therapeutic effects of Lingguizhugan decoction in a rat model of high-fat diet-induced insulin resistance

BACKGROUND Lingguizhugan(LGZG)decoction is a widely used classic Chinese medicine formula that was recently shown to improve high-fat diet(HFD)-induced insulin resistance(IR)in animal studies.AIM To assess the therapeutic effect of LGZG decoction on HFD-induced IR and explore the potential underlying mechanism.METHODS To establish an IR rat model,a 12-wk HFD was administered,followed by a 4-wk treatment with LGZG.The determination of IR status was achieved through the use of biochemical tests and oral glucose tolerance tests.Using a targeted metabolomics platform to analyze changes in serum metabolites,quantitative real-time PCR(qRT-PCR)was used to assess the gene expression of the ribosomal protein S6 kinase beta 1(S6K1).RESULTS In IR rats,LGZG decreased body weight and indices of hepatic steatosis.It effectively controlled blood glucose and food intake while protecting islet cells.Metabolite analysis revealed significant differences between the HFD and HFDLGZG groups.LGZG intervention reduced branched-chain amino acid levels.Levels of IR-related metabolites such as tryptophan,alanine,taurine,and asparagine decreased significantly.IR may be linked to amino acids due to the contemporaneous increase in S6K1 expression,as shown by qRT-PCR.CONCLUSIONS Our study strongly suggests that LGZG decoction reduces HFD-induced IR.LGZG may activate S6K1 via metabolic pathways.These findings lay the groundwork for the potential of LGZG as an IR treatment.

Time-dependent impact of a high-fat diet on the intestinal barrier of male mice

BACKGROUND Excessive saturated fat intake compromises the integrity of the intestinal mucosa,leading to low-grade inflammation,impaired mucosal integrity,and increased intestinal permeability,resulting in the migration of lipopolysaccharide(LPS)to other tissues.AIM To evaluate the chronic effects(at 10 and 16 wk)of a high-fat diet(HFD)(with 50%energy as fat)on the phylogenetic gut microbiota distribution and intestinal barrier structure and protection in C57BL/6 mice.METHODS Forty adult male mice were divided into four nutritional groups,where the letters refer to the type of diet(control and HFD or HF)and the numbers refer to the period(in weeks)of diet administration:Control diet for 10 wk,HFD for 10 wk,control diet for 16 wk,and HFD for 16 wk.After sacrifice,biochemical,molecular,and stereological analyses were performed.RESULTS The HF groups were overweight,had gut dysbiosis,had a progressive decrease in occludin immunostaining,and had increased LPS concentrations.Dietary progression reduced the number of goblet cells per large intestine area and Mucin2 expression in the HF16 group,consistent with a completely disarranged intestinal ultrastructure after 16 wk of HFD intake.CONCLUSION Chronic HFD intake causes overweight,gut dysbiosis,and morphological and functional alterations of the intestinal barrier after 10 or 16 wk.Time-dependent reductions in goblet cell numerical density and mucus production have emerged as targets for countering obesity-driven intestinal damage.

Effect of Yizhikang Powder on Hemorheological Indexes in Dairy Cattle with Retained Placenta

[ Objective ] The aim of the research was to reveal the mechanism of Yizhikang powder treatment on dairy cattle with retained placenta from the hemorheological perspective. [ Method] Dairy cattle with retained placenta were treated with oral administration of Yizhikang powder. And their hemorheological indexes were measured and compared with the corresponding indicators of pre-administration group, healthy group, and control group （sick but untreated）.[ Result] There was large decrease amplitude in the indexes （whole blood viscosity, plasma viscosity, whole blood viscosity reduction viscosity, ESR, ESR equation K value, fibrinogen content, platelet aggregation rate, RBC deformability IF value） of treated dairy cattle with retained placenta. By t test, the indexes, except hematocrit, decreased significantly after treatment （P〈0.05） and reached the status of post partum healthy cows. The hemorheological indexes didn＇t change significantly in the control group before and after treatment. [ Conclusion] Yizhikang powder could significantly improve blood flow state and reduced the occurrence of qi stagnation and blood stasis in perinatal period.

基于CPM-Dairy的几种我国常用奶牛饲料原料聚类分析

本试验依据康奈尔大学净碳水化合物净蛋白质体系(CNCPS)和CPM-Dairy配方软件的技术思想,对几种我国常用奶牛饲料原料进行聚类分析。采用近红外分析和实验室分析对4大类[苜蓿干草、干酒糟及其可溶物(DDGS)、玉米粉和玉米青贮],共1 169个我国常用奶牛饲料样品的16类指标进行测定,用聚类分析的方法对样品科学分类。结果表明,用聚类分析的方法,根据不同饲料的特点,分别将苜蓿干草分为12类、DDGS分为10类、玉米粉分为5类、玉米青贮分为6类。综合得出,聚类分析的方法得出结果符合CPM-Dairy饲料数据库要求,且可信度高。

Effects of Oral Administration of Propylene Glycol and Ca-P-Mg Mixture on Milk Quality and SCC of Dairy Cows after Birth

In order to further study the postpartum care technology of dairy cows,the effects of oral administration of propylene glycol or Ca-P-Mg mixture and compound oral rehydration salts on milk quality and SCC of dairy cows after birth were investigated. The results showed that the milk fat and dry matter contents in the dairy cows administered with Ca-P-Mg mixture and compound oral rehydration salts were higher than those in the dairy cows administered with propylene glycol（P 〈0.05）. However, there were no significant differences in milk sugar and protein contents and SCC between the two administration groups.

河南部分地区奶牛肠道纤毛虫感染情况及分子鉴定

为了明确奶牛肠道纤毛虫的感染情况及其人兽共患风险,从河南省不同地区8个集约化奶牛场中采集462份新鲜粪样,采用粪便直接涂片法镜检和基于18S rDNA和ITS1-5.8S rDNA-ITS2序列对样品中纤毛虫感染情况和分子特性进行了鉴定。结果显示,奶牛粪便中存在有槽巴克斯顿纤毛虫和结肠小袋纤毛虫,其中有槽巴克斯顿纤毛虫感染率为29.9%,成年牛感染率显著高于其他年龄段奶牛(P<0.01);结肠小袋纤毛虫感染率为3.9%,不同年龄段感染率差异不显著(P>0.05),8份样品为有槽巴克斯顿纤毛虫和结肠小袋纤毛虫混合感染。未从奶牛粪便中检出瘤胃内纤毛虫,说明奶牛瘤胃内的共生纤毛虫不随粪便排出体外。研究结果为集约化奶牛场纤毛虫病的防控提供了重要的基础数据。

新疆部分地区奶牛源大肠杆菌的流行性与耐药性分析

[目的]探明新疆部分地区奶牛养殖场患病奶牛乳和环境中大肠杆菌流行情况及耐药性,为该地区大肠杆菌的临床合理用药提供依据。[方法]试验采用平板划线法对新疆部分地区的4个集约化养殖场171份样本(55份患病奶牛乳样本、116份环境样本)进行细菌分离纯化,采用形态学观察、革兰染色镜检、生化鉴定及分子生物学方法对大肠杆菌分离株进行鉴定,并利用微量肉汤稀释法及PCR法对大肠杆菌分离株进行16种抗菌药物的耐药表型及耐药基因分析。[结果]从171份样本中共分离出45株大肠杆菌,其中8株来自患病奶牛乳样本,分离率为14.55%(8/55),37株来自环境样本,分离率为31.90%(37/116)。所有分离株均呈粉红色圆形菌落,革兰染色镜检呈红色、短杆的革兰阴性菌。生化鉴定结果显示,分离菌乳糖、甘露醇、靛基质与MR检测呈阳性,氧化酶、H_2S、VP和西蒙氏柠檬酸盐检测呈阴性,符合大肠杆菌生化特点。药敏试验结果显示,大肠杆菌分离株对头孢噻吩、氨苄西林、链霉素和四环素表现不同程度的耐药性,耐药率分别为82.22%、48.89%、33.33%和33.33%,而对美罗培南、卡那霉素、庆大霉素、环丙沙星和复方新诺明表现较高的敏感性,敏感率均在85.00%以上,其中有16株大肠杆菌为多重耐药菌,多重耐药率为35.56%。耐药基因检测结果显示,大肠杆菌分离株β-内酰胺类耐药基因bla_(TEM)的阳性率为13.33%,氨基糖苷类耐药基因addA1和strA的阳性率分别为6.66%和11.11%,四环素类耐药基因tetA和tetC的阳性率分别为13.33%和33.33%。[结论]新疆部分地区奶牛养殖场分离的大肠杆菌主要对β-内酰胺类抗生素高度耐药,且多重耐药情况严重。

乳制品中美拉德反应有害产物的生成途径、检测方法及其控制策略研究进展

美拉德反应能够赋予面包、咖啡等食品诱人的香气、颜色和质地,然而,对于牛奶和乳制品,美拉德反应会降低其营养价值,产生糠氨酸、糠醛和晚期糖化终末产物(advanced glycation end-products,AGEs)等多种美拉德反应有害产物,这些有害产物会导致人类各种慢性疾病。充分了解乳制品中美拉德反应有害产物的形成机制并建立控制方法对提高乳制品品质具有重要意义。本文详细阐述乳制品美拉德反应有害物的生物危害性,重点综述糠氨酸、5-羟甲基糠醛和AGEs的生成途径。此外,还综述美拉德反应有害产物检测方法的优点、缺点和最新进展,重点介绍当前乳制品中美拉德反应有害产物的控制策略。旨在为乳制品中美拉德反应有害产物的监测和控制提供科学指导。

奶牛肺炎克雷伯菌性乳房炎的研究进展

乳房炎是制约世界乳业发展的主要奶牛疾病之一,通常由传染性或环境性病原菌感染所致。在引起奶牛乳房炎的所有细菌性病原体中,肺炎克雷伯菌(Klebsiella pneumoniae)感染造成的牛奶损失比较大,患牛临床症状也比较严重。本文通过对奶牛肺炎克雷伯菌性乳房炎的临床症状、病原学、流行病学、致病机制、预防措施和治疗方法等进行综述,为现代奶牛场肺炎克雷伯菌性乳房炎的防控提供理论依据和实践经验,促进乳业健康发展。

Effects of Dairy Cattle Manure and Chemical Fertilizers on Fertility of Soils Grown with Cichorium intybus

[Objective] The aim was to explore recycling utilization of manure of dairy cattle through returning of manures into fields. [Method] Effects of dairy cattle ma- nure and chemical fertilizer on fertility of soils grown with Cichorium intybus were in- vestigated through a pot experiment. [Result] After manure of dairy cattle was ap- plied, it can be concluded that organic matter, total N, total P, alkali-hydrolyzable ni- trogen, available P, activities of urease and invertase in soils increased by 0.14-1.28 times, 43.8%-79.7%, 17.4%-30.8%, 147%-188%, 7 times, 17.2%-38.5%, and 1.36%- 3.34%, respectively. Furthermore, organic matter, total N. urease and invertase activi- ties in group of M7F3 increased most; total P and available P achieved the best in group of M3F7. These indicated that the applied manures of dairy cattle would maintain and improve soil fertility, providing better soils for Cichorium intybus. [Conclusion] The research provides reference for recycling of cattle manures and construction of ecological cyclical pattern of ＂grass planting-cattle breeding-methane fermentation-returning of manures into fields＂.

竹叶黄酮对H_(2)O_(2)诱导奶牛乳腺上皮细胞焦亡的保护作用

旨在以竹叶黄酮(BLF)为添加物,探究BLF对过氧化氢(H_(2)O_(2))诱导奶牛乳腺上皮细胞(BMECs)焦亡的保护作用。本研究以BMECs为研究对象,分为对照组、80μg·mL^(-1) BLF处理组、800μmol·L^(-1) H_(2)O_(2)处理组和80μg·mL^(-1) BLF+800μmol·L^(-1) H_(2)O_(2)处理组。利用RT-qPCR、Western blot、免疫荧光、流式细胞术等技术,检测细胞焦亡相关基因和蛋白表达以及凋亡相关微粒样蛋白(ASC)荧光强度。结果表明,与对照组相比,800μmol·L^(-1) H_(2)O_(2)处理BMECs 8 h显著增加了奶牛乳腺上皮细胞的焦亡(P<0.05),H_(2)O_(2)处理后,NLRP3、ASC、GSDMD、IL-18、IL-1β等焦亡相关基因的相对表达量均显著升高(P<0.05),尤其是NLRP3,说明H_(2)O_(2)显著增加BMECs焦亡。与H_(2)O_(2)处理组相比,BLF可以降低正常细胞焦亡水平,显著降低NLRP3、Nek7、ASC、GSDMD、IL-18、IL-1β基因mRNA上调的趋势,显著缓解了H_(2)O_(2)导致的NLRP3、ASC、Nek7、pro-caspase1、caspase1 p20、GSDMD-N、IL-18和IL-1β胞内焦亡相关蛋白表达的升高(P<0.05),ASC的荧光强度显著降低(P<0.05)。综上所述,H_(2)O_(2)显著上调NLRP3、ASC、Caspase-1、GSDMD、IL-1β、IL-18焦亡基因和蛋白表达水平,诱导奶牛乳腺上皮细胞焦亡。BLF通过降低NLRP3炎症小体通路蛋白和基因表达,降低了炎症因子水平,从而缓解奶牛乳腺上皮细胞焦亡。

基于不同ST型肺炎克雷伯菌感染的小鼠乳腺炎模型的差异分析

[目的]肺炎克雷伯菌感染是奶牛乳腺炎的常见病因,其诱导的临床型乳腺炎发病急、临床症状重,牛奶产量和质量降低,给奶牛场造成巨大的经济损失。本研究旨在探究奶牛乳腺炎源性不同序列型(sequence type, ST)肺炎克雷伯菌的毒力作用,筛选肺炎克雷伯菌发挥感染作用的潜在关键毒力基因。[方法]采用多位点序列分型(multi-locus sequence typing, MLST)分析山东、湖北2个大型奶牛场的129株临床乳腺炎源性肺炎克雷伯菌的ST型分布,并选择3种检出率较高的ST型肺炎克雷伯菌感染小鼠建立乳腺炎模型,探究小鼠乳腺的形态学损伤及炎性因子表达变化。通过分析肺炎克雷伯菌的感染特性与毒力基因分布,筛选与肺炎克雷伯菌感染相关的关键毒力基因。[结果]129株肺炎克雷伯菌共分为79种ST型,其中ST107占比最高,为19.37%;其次为ST2324(7.75%)和ST13(3.87%)。与ST107和ST13型相比,感染后24 h, ST2324型肺炎克雷伯菌诱导小鼠临床评分和乳腺载菌量均显著升高(P<0.05);乳腺内腺泡轮廓消失和大量炎性细胞浸润,乳腺中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和细胞色素C(Cyt C)mRNA表达量均显著升高(P<0.05)。毒力基因检测结果显示,3种不同ST型分离株共检测到74个毒力基因,其中ST2324型分离株中检出了ST13和ST107型分离株没有检出的毒力基因manB、pla和pilW。[结论]本研究从129株肺炎克雷伯菌中共检出79种ST型,其中ST2324、ST107和ST13检出率较高,ST2324型肺炎克雷伯菌对小鼠致病性最强,且携带的毒力基因manB、pla和pilW与肺炎克雷伯菌的高致病性密切相关。本研究结果为进一步解析肺炎克雷伯菌感染奶牛乳腺炎的机制提供了理论参考。