Deoxynivalenol induces oxidative stress,inflammatory response and apoptosis in bovine mammary epithelial cells

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium graminearum. It is one of the most common feed contaminants that poses a serious threat to the health and performance of dairy cows. This study investigated the in vitro cytotoxicity of DON on bovine mammary epithelial cells (MAC‐T). DON at different concentrations (0.25, 0.3, 0.5, 0.8, 1 or 2 μg/ml) inhibited the growth of MAC‐T cells after 24 hr of exposure (p < .001). DON at 0.25 μg/ml increased lactate dehydrogenase (LDH) leakage (p < .05); decreased glutathione (GSH) levels (p < .001), total superoxide dismutase (T‐SOD) activity and total antioxidant capacity (T‐AOC; p < .01); and increased malondialdehyde (MDA) concentration (p < .01) in MAC‐T cells after 24 hr of exposure. We also observed that DON increased reactive oxygen species (ROS) levels in cells incubated for 9, 15 and 24 hr (p < .001). DON at 0.25 μg/ml triggered oxidative damage in MAC‐T cells. Furthermore, it induced an inflammatory response in the cells incubated for 9, 15 and 24 hr (p < .05) by increasing the mRNA expression levels of nuclear factor kappa B, myeloid differentiation factor 88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, cyclooxygenase‐2 and IL‐8. We further examined the effect of DON on apoptosis. DON prevented normal proliferation of MAC‐T cells by blocked cell cycle progression in 24 hr (p < .001). In addition, the apoptosis rate measured using annexin V‐FITC significantly increased (p < .05) with increase in the mRNA expression level of Bax (p < .01) and increase in the Bax/Bcl‐2 ratio (p < .01) in cells incubated for 24 hr. In summary, DON exerts toxic effects in MAC‐T cells by causing oxidative stress, inducing an inflammatory response, affecting cell cycle and leading to apoptosis.     

An investigation of the suitability of three support matrices for the culture of cells derived from the secretory alveoli of the bovine mammary gland

The suitability of three support matrices, (thick collagen gels, aluminium oxide and cellulose ester membranes, the latter two both thinly coated with collagen) for the production of primary cultures of bovine mammary epithelial cells was investigated. Single secretory alveoli were isolated from mammary tissue of animals in early lactation by enzymatic digestion and differential filtration. Cell growth was monitored by light and scanning electron microscopy. The cellulose ester membrane was found to give the best results, allowing growth of monolayers with a morphology closely resembling that of the natural epithelium of the gland. There were low levels of fibroblast contamination, and the membranes could be easily manipulated for further studies.Abbreviations EM electron microscopy - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - LM light microscopy - PCM phase contrast microscopy - PBS phosphate-buffered saline - RPMI Roswell Park Memorial Institute - SEM scanning electron microscopy     

抗保幼激素与蜕皮激素对蚕生长发育及咽侧体和前胸腺超微形态的影响

4龄期使用抗保幼激素,当龄经过延长,全龄期缩短,并诱导出100％的三眠蚕;5龄后期使用抗保幼激素,全龄经过稍延长,茧质丝质优于对照,巨纤度细。5龄前期使用蜕皮激素全龄期延长,5龄后期使用蜕度激素,则龄期明显缩短,蚕的强健性、茧质、丝质均较差。抗保幼激素处理后咽侧体细胞内有大量的脑神经分泌物,但腺体细胞本身缺少内质网,而处理后的前胸腺则边缘绒毛层明显,具有大型细胞核,线粒体、内质网等细胞器发达。蜕皮激素的作用则相反,处理后前胸腺边缘不规则,内质网不甚发达,合成作用减弱。     

水牛原代体细胞慢病毒感染体系的建立

试验旨在建立水牛原代体细胞慢病毒感染体系。通过比较慢病毒（lentivirus）感染水牛颗粒细胞（buffalo granulosa cell,BuGC）、水牛乳腺上皮细胞（buffalo mammary epithelial cell,BuMEC）、水牛成纤维细胞（buffalo fibroblast cell,BuFC）的效率及荧光强度,筛选出3种水牛原代体细胞的最佳感染复数（MOI）。分离培养获得BuGC、BuMEC及BuFC。将慢病毒质粒pLVX-Puro-GFP和包装质粒pSPAX2、pMAD2.G脂质体转染HEK293T细胞,于转染后48和72 h收集病毒并通过稀释计数法测定慢病毒滴度。将慢病毒按不同的感染复数（100、200、400、600、800）感染BuGC、BuMEC和BuFC,感染72 h后于倒置荧光显微镜下观察拍照,统计感染效率及荧光强度。慢病毒感染3种水牛原代体细胞后,用嘌呤霉素筛选。结果显示,在MOI≤200时,慢病毒感染后细胞荧光强度BuMEC最强,而BuGC和BuFC间无显著差异;在MOI≥400时,慢病毒感染后细胞荧光强度为：BuMEC>BuGC>BuFC。在MOI=100时,慢病毒感染效率为：BuGC>BuMEC>BuFC;在MOI=200时,BuMEC和BuGC感染效率达到100%;在MOI=800时,BuFC感染效率达到100%。不同细胞类型对慢病毒的毒性耐受存在差异,在嘌呤霉素的筛选和维持下,获得了3种水牛原代体细胞绿色荧光蛋白（green fluorescent protein,GFP）过表达慢病毒稳转细胞株。慢病毒感染BuMEC、BuGC、BuFC的最佳MOI分别为200、400和800;3种水牛原代体细胞均能通过慢病毒获得过表达外源基因细胞株。本研究结果为水牛的乳腺组织发育分化、泌乳调控、生殖发育及转基因动物制备等提供了较好的细胞模型。     

In vitro study to assess the efficacy of CDK4/6 inhibitor Palbociclib (PD‐0332991) for treating canine mammary tumours

Therapy of canine mammary tumours (CMTs) with classical antitumour drugs is problematic, so better therapeutic options are needed. Palbociclib (PD‐0332991) is an innovative and effective anticancer drug for the treatment of breast cancer in women. Palbociclib is an inhibitor of cyclin‐dependent kinase 4 (CDK4) and CDK6, which are key regulators of the cell cycle machinery and thus cell proliferation. In the present in vitro study, we investigated whether Palbociclib also represents a candidate drug to combat CMT. For this purpose, the effect of Palbociclib was analysed in P114 and CF41 cells, two CMT cell lines with an endogenous CDK4/6 co‐expression. Incubation of P114 and CF41 cells with Palbociclib resulted in a dose‐ and time‐dependent loss of phosphorylated retinoblastoma protein (pRb), a classical CDK4/6 substrate within the cell cycle machinery. Moreover, treatment of CMT cells with Palbociclib‐induced cell cycle arrest affected cell viability, prevented colony formation and impaired cell migration activity. Palbociclib also inhibited the growth of P114 and CF41 cell spheroids. Immunohistochemical analysis of canine patient samples revealed a consistent expression of CDK6 in different canine mammary carcinoma types, but an individual and tumour‐specific expression pattern of phosphorylated pRb independent of the tumour grade. Together, our findings let us suggest that Palbociclib has antitumour effects on CMT cells and that canine patients may represent potential candidates for treatment with this CDK4/6 inhibitor.     

Effect of interaction between leucine and acetate on the milk protein synthesis in bovine mammary epithelial cells

The interaction between Leucine (Leu) and acetate affecting milk protein synthesis in the bovine mammary epithelial cells (BMECs), and underlying the molecular mechanisms are not well understood. The objectives of this study were to investigate the effect of Leu, acetate, and their interaction on the expression of genes involved in milk protein synthesis, and JACK2/STAT5, mTOR and AMP‐activated protein kinase (AMPK) signaling pathway. The study was a 2 × 6 factorial arrangement with treatments: Leu concentration (0.45 and 1.8 mM) and acetate concentration (0, 4, 6, 8, 10, and 12 mM). The results showed that 1.8 mM Leu or 8–10 mM acetate had positive effect on ATP content, the expression of casein genes, JACK2/STAT5 and phosphorylation of mTOR pathway, but reduced AMPK phosphorylation. Leu at 1.8mM had a positive effect on the up‐regulation of acetate on ATP content, the expression of CSN1S1, CSN2, CSN3, and JACK2, the expression and phosphorylation of eukaryotic initiation factor 4E, p70 ribosomal protein S6 kinase‐1, and mTOR, but reducing AMPK phosphorylation. The results suggest that acetate, Leu, and their interaction have effect on milk protein synthesis through the JACK2/STAT5, mTOR, and AMPK pathway. Acetate addition up‐regulated the effect of Leu on milk protein synthesis, and Leu facilitated the up‐regulation of acetate on milk protein synthesis through these pathways.     

Effects of long-term administration of recombinant bovine somatotropin on the concentration of metabolites in milk in different stages of lactation in crossbred Holstein cattle

Effects of long‐term treatment with recombinant bovine somatotropin (rbST) on concentrations of cellular metabolites in the milk of 87.5% crossbred Holstein cattle were performed. The peak milk yield of rbST‐treated animals was 22% higher (P < 0.05) than that of the control animals in early lactation. The mammary glucose uptake of rbST‐treated animals increased in early lactation, but decreased in mid and late lactation, while plasma glucose concentrations were not affected. Lactose and milk triacylglycerol secretion of rbST‐treated animals significantly increased (P < 0.05) when compared with those of control animals in early lactation. The concentrations of milk glucose of rbST‐treated animals significantly increased in early and mid‐lactation (P < 0.05). The concentrations of milk galactose markedly increased (P < 0.05) whereas the concentrations of milk uridine 5′‐diphosphoglucose (UDP‐glucose) and UDP‐galactose showed no significant changes as lactation advances in both groups. The concentrations of isocitrate, 2‐oxoglutarate and citrate in milk from both groups showed no significant changes throughout experiment. The concentration of glucose‐6‐phosphate (G6P), glucose‐1‐phosphate and cyclic adenosine 3,5′monophosphate in milk from both groups markedly decreased as lactation advances exception in early lactation of rbST‐treated animals, which G6P was not affected. These findings suggest that prolonged rbST treatment exerts its galactopoietic action at least in early lactation through both intramammary and extra‐mammary changes. Increases in the concentrations of glucose and G6P in milk maintained the level of pretreatment in early lactation associated with increases in milk yields during rbST administration, reflect their concentrations in the cytosol or Golgi vesicles of mammary cells, which would be one of the factors regulating intermediary metabolites in the lactose biosynthetic pathway.     

去势对白来航母鸡垂体和肾上腺的影响

垂体和肾上腺在摘除卵巢的雌性动物性激素合成过程中起到重要作用。为研究去势对白来航母鸡垂体和肾上腺的影响,实验对6只9周龄白来航母鸡进行卵巢摘除手术,饲养至97周龄时,检测去势白来航母鸡与同周龄未去势母鸡的血清性激素水平、冠高和距长,比较分析垂体重、肾上腺重差异,并检测垂体发育相关基因GREB1的表达量。结果显示:去势母鸡雌二醇浓度(P<0.05)、肾上腺重量(P<0.01)以及GREB1相对表达量(P<0.05)低于未去势母鸡;冠高、距长和垂体重量高于未去势母鸡(P<0.05)。可见,摘除母鸡卵巢可抑制肾上腺发育,促进垂体发育。     

大豆异黄酮在奶牛生产中的研究进展

大豆异黄酮是豆科植物中存在的一类黄酮类化合物,由于其类雌激素活性而被称为植物雌激素,具有抗肿瘤、抗氧化应激、抗炎以及促进乳腺上皮细胞增殖等能力。本文主要从大豆异黄酮的主要成分、结构功能及其对奶牛瘤胃发酵、泌乳性能、抗氧化能力以及增殖性能的影响和作用机制等方面进行阐述。     

甘氨酸对奶山羊乳腺炎性应答的调节及其作用机制研究

本试验在体内条件下研究了甘氨酸对奶山羊乳腺炎性应答的调节及其信号通路的变化。选用15只泌乳期经产健康萨能奶山羊,分为对照组、脂多糖(LPS)模型组和3个甘氨酸干预组,每组3只,饲养管理程序一致。对照组奶山羊不注射LPS和甘氨酸,以1 mL生理盐水取代;LPS模型组奶山羊于左右乳区通过乳头管分别注射1 mL LPS溶液(4μg/乳区);3个甘氨酸干预组奶山羊分别按照1、5、25 g/(只·d)的剂量,于左右乳区通过乳头管分别注射0.5 mL甘氨酸,连续注射5 d,第6天在左右乳区通过乳头管注射1 mL LPS(4μg/乳区)。各组奶山羊在注射LPS或生理盐水24 h后屠宰取乳腺组织样。制作乳腺组织切片,苏木精-伊红(HE)染色后用于观察病理变化;采用实时定量PCR法检测乳腺组织中白细胞介素(IL)-1β、IL-6、IL-8、肿瘤坏死因子-α(TNF-α)、环氧合酶-2(COX-2)、Toll样受体(TLR)2、TLR4、TLR9、髓样分化因子88(MyD88)、β-干扰素TIR结构域衔接蛋白(TRIF)基因的表达水平;采用Western blotting法检测乳腺组织中核转录因子-κB(NF-κB)p65蛋白的表达水平。结果显示:注射LPS 24 h后,3个甘氨酸干预组试验羊体温降至39.5℃以下。与LPS模型组相比,3个甘氨酸干预组炎性细胞侵润明显减少,乳腺细胞坏死程度明显减轻;高、中、低剂量甘氨酸干预均显著下调了IL-1β、IL-8和COX-2基因的表达水平(P<0.05),只有中剂量甘氨酸干预显著下调了IL-6、TNF-α基因的表达水平(P<0.05);高、中、低剂量甘氨酸干预均显著下调了TLR4基因的表达水平(P<0.05),只有中剂量甘氨酸干预显著下调了TLR2、TLR9基因的表达水平(P<0.05);高、中、低剂量甘氨酸干预均显著下调了MyD88基因的表达水平(P<0.05),同时低、高剂量甘氨酸干预还显著下调了TRIF基因的表达水平(P<0.05);高、中、低剂量甘氨酸干预均显著下调了核转录因子NF-κB p65蛋白的表达水平(P<0.05)。综上可知:1)甘氨酸通过下调TLR4及其下游关键信号分子MyD88和TRIF基因的表达,抑制转录因子NF-κB p65磷酸化,从而减少炎性因子IL-1β、IL-6、IL-8、TNF-α、COX-2的分泌。2)本研究初步阐明了甘氨酸通过抑制TLR-NF-κB信号通路从而抑制炎性细胞的激活或活性,阻止其炎症介质的释放,进而阻碍炎症反应过程实现。