牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用

牛星状病毒(BAstV)是我国新发的犊牛腹泻病原,本试验的目的是建立检测BAstV的Real-time PCR方法。根据BAstV流行株的ORF1a基因序列设计引物,通过优化反应条件和体系,成功建立基于EvaGreen检测BAstV的Real-time PCR方法。结果表明,该检测方法的Ct值与标准品模板在1.36×10¹~1.36×10⁸拷贝/μL线性关系良好,相关系数R²=0.999,扩增效率为93.79%;该方法可特异性检出BAstV,对犊牛腹泻其他相关病原呈阴性;最低检测下限为13.6拷贝/μL;批间和批内的变异系数均小于2%,重复性好。对2017年9月至2019年5月采自河南省的221份犊牛腹泻样本进行检测,BAstV的检出率为18.1%(40/221),采样场阳性率为100.0%(14/14)。本试验所建方法灵敏度高、特异性强、稳定性好,为BAstV的检测和流行病学调查提供了有力手段。     

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.     

甘露寡糖添加方式对哺乳期犊牛瘤胃细菌菌群结构的影响

为利用16S rDNA技术研究甘露寡糖不同添加方式对哺乳期犊牛瘤胃细菌菌群结构影响,本研究选用出生日龄一致、体重接近、健康状况良好的荷斯坦公犊牛20头,随机分为4组。CR组为对照组,饮用乳及开食料中均不添加甘露寡糖;ORa组在饮用乳中添加5 g甘露寡糖,开食料中不添加甘露寡糖;ORb组在开食料中添加5 g甘露寡糖,饮用乳中不添加甘露寡糖;ORc组在饮用乳及开食料中各添加2.5 g甘露寡糖(混合添加)。应用16S rDNA测序技术,研究了甘露寡糖添加方式对犊牛瘤胃细菌构建的影响。结果表明:甘露寡糖添加方式会影响哺乳期犊牛瘤胃细菌总数,但对哺乳期犊牛瘤胃细菌菌群多样性未产生显著影响(P>0.05)。在门水平上,与对照组相比,ORb组颗粒料中添加甘露寡糖显著降低了厚壁菌门与放线菌门的丰度(P<0.05),极显著提高了变形菌门的丰度(P<0.01),优势菌门增加为3种,分别为拟杆菌门、厚壁菌门与变形菌门。在属水平上,甘露寡糖不同添加方式导致哺乳期犊牛瘤胃细菌菌群结发生了改变,其中甘露寡糖不同添加方式对小杆菌属和脱硫弧菌属在瘤胃内丰度均产生了影响,但对ORa组与ORc组犊牛瘤胃细菌影响不及ORb组;且ORb组犊牛瘤胃细菌功能性菌属普雷沃氏-7属、氨基酸球菌属、优杆菌属和琥珀酸弧菌等均发生显著变化。基于以上结果,得出如下结论:本研究条件下,甘露寡糖添加方式对哺乳期犊牛瘤胃细菌菌群多样性未产生显著影响(P>0.05),但开食料中寡糖添加方式对菌群结构产生部分影响,其中发挥瘤胃蛋白降解作用和淀粉降解作用的琥珀酸弧菌科-UCG-001属、利用乳酸的新月形单胞菌属丰度极显著提高(P<0.01),而利用乳糖的优杆菌属丰度显著降低,降解半纤维素的小杆菌属丰度极显著降低(P<0.01)。     

Pharmacokinetics of metoclopramide in calves with renal dysfunction

To clarify the effect of renal dysfunction on pharmacokinetics of the prokinetic agent metoclopramide (MCP), we administered intravenously 0.4 mg/kg MCP to healthy calves and calves subjected to right kidney vessel ligation (ligation) without or with a subsequent left nephrectomy (ligation plus removal). Plasma MCP concentration, glomerular filtration rate (GFR) and plasma prolactin level were measured by liquid chromatography-tandem mass spectrometry, simplified equation using iodixanol and enzyme-linked immunosorbent assay, respectively. Only in calves with ligation plus removal, plasma MCP concentrations were increased significantly 6, 8 and 12 hr after injection, showing that a negative correlation was observed between the plasma MCP concentrations and GFR value. A tendency to increase in plasma PRL concentration was noted also in these calves. In conclusions, plasma MCP concentrations depend on the GFR mode in calves, and its critical GFR value was estimated.     

断奶应激对犊牛血液生化、免疫和内分泌的影响

选择体质量和出生日期接近的26头犊牛,其中6头母犊,20头公犊。10日龄开始补充苜蓿干草、精饲料自由采食,60日龄断奶。分别在断奶前（-1d）和断奶后1,3,5,7,14d采集血液,测定血清中代谢物、激素及免疫球蛋白含量的变化情况。结果表明,断奶影响犊牛的消化代谢,血清中葡萄糖、总蛋白、尿素氮、甘油三酯含量变化显著（P〈0.01）。断奶造成犊牛血清皮质醇、急性反应蛋白、T3、T4、乳酸脱氢酶、碱性磷酸酶、溶菌酶显著变化（P〈0.01）,使免疫球蛋白、γ-IFN、IL-1含量有不同程度下降（P〉0.05）。因此,断奶不仅降低了犊牛的免疫力,而且对犊牛产生了严重的应激反应。     

开食料中性洗涤纤维水平对犊牛生长性能、血清生化指标和抗氧化功能的影响

【目的】探究开食料中性洗涤纤维（NDF）含量对犊牛生长发育、血清生化指标及抗氧化性能的影响,为犊牛开食料的合理配制提供试验依据。【方法】选取平均体重为（42±2.5）kg 的60头（包括36头公犊牛,24头母犊牛）新生荷斯坦犊牛,随机分为4个试验组,每组15头（9头公犊牛+6头母犊牛）,15日龄起依次饲喂10%（10N）、15%（15N）、20%（20N）和25%（25N）NDF水平的开食料。试验犊牛单个饲养于犊牛岛,70日龄断奶,饲喂至112 日龄。每日记录开食料采食量,每两周称量犊牛的体重,于35和112日龄测量犊牛的体尺,35、70、90和112日龄空腹颈静脉抽血测定血液生化和抗氧化指标。【结果】（1）70—112日龄,犊牛开食料采食量10N组显著低于25N组（P＜0.5）;42—70日龄,20N和25N组NDF采食量显著高于10N和15N组（P＜0.05）;70—112日龄,NDF采食量随开食料NDF水平升高而显著增加（P＜0.05）; （2）70—112和15—112日龄,15N组犊牛体增重显著高于其他三组（P＜0.05）,分别高出12.67%、43.56%、30.17%和6.16%、18.09%、15.16%;15N组体高显著高于20N和25N组（P＜0.05）,体斜长和胸围显著高于25N组。（3）90日龄,15N组血清球蛋白含量显著高于其他三组,白球比显著低于其他三组（P＜0.05）;112日龄,血清谷丙转氨酶、谷草转氨酶和碱性磷酸酶含量随NDF水平的提高而显著降低（P＜0.05）。（4）35、70和112日龄,15N、20N和25N组血清超氧化物歧化酶浓度显著高于10N（P＜0.05）组,血清丙二醛浓度低于10N组。【结论】犊牛开食料中NDF水平直接影响进食量和体增重,15—112日龄荷斯坦犊牛开食料中NDF的适宜水平为15%。     

不同剂量25羟基维生素D3对断奶前犊牛血液生化指标、抗氧化能力和免疫功能的影响

为研究不同剂量25羟基维生素D₃[25(OH)VD₃]对断奶前犊牛血液水平、抗氧化能力和免疫功能的影响,选取18 头健康状况良好的新生荷斯坦犊牛,随机分成3 组,每组6 头进行为期56 天的饲养试验。分为对照组[不添加25(OH)VD₃]、低剂量组[25(OH)VD₃添加量6 000 IU/头·天]和高剂量组[25(OH)VD₃添加量12 000 IU/头·天]。结果表明,与对照组相比,随着25(OH)VD₃添加剂量增加,血浆生长激素、胰岛素样生长因子-1、过氧化氢酶、超氧化物歧化酶、总抗氧化能力以及免疫球蛋白G(IgG)含量显著增加(P<0.05),而丙二醛含量随剂量增加而存在降低趋势(P=0.079)。与对照组和低剂量组相比,高剂量组血浆免疫球蛋白A(IgA)含量有增加趋势(P=0.059)。综上所述,断奶前犊牛饲喂高剂量25(OH)VD₃有利于促进体内生长激素和胰岛素样生长因子-1分泌,可提高犊牛抗氧化能力,增强免疫功能。     

Inlfuence of FSH Treatment on Expression of CDC25A,TSSK3 and P53in Vitro Cultured Sertoli Cells of Calf

CDC25A, TSSK3 and P53 expressionsin vitro in cultured sertoli cells after FSH treatment were studied in order to provide some data for further researches of spermatogenesis. Different concentrations of...