瘤胃微生物总DNA快速提取法研究

本文介绍了一种简便快速地提取瘤胃微生物细胞总DNA的方法,该法是对提取植物的CTAB法进行改进而成。与经典反复冻融 SDS/蛋白酶K裂解法相比,该法可在数小时内高效地提取瘤胃内容物总DNA,经琼脂糖凝胶电泳对所提取瘤胃微生物总DNA进行检测,证明所获得的瘤胃总DNA符合分子生物学操作要求。     

瘤胃微生物总DNA快速提取法研究

本文介绍了一种简便快速地提取瘤胃微生物细胞总DNA的方法,该法是对提取植物的CTAB法进行改进而成.与经典反复冻融＋SDS/蛋白酶K裂解法相比,该法可在数小时内高效地提取瘤胃内容物总DNA,经琼脂糖凝胶电泳对所提取瘤胃微生物总DNA进行检测,证明所获得的瘤胃总DNA符合分子生物学操作要求.     

荧光光度法在瘤胃原虫对细菌吞噬作用研究中的应用

本试验旨在探讨荧光光度法研究瘤胃原虫对细菌吞噬作用的可行性.试验选用4只装有永久性瘤胃瘘管的徐淮山羊采集瘤胃液以获取瘤胃原虫和细菌,以4,6-二脒基-2-苯基吲哚(DAPI)标记细菌的荧光强度为主要测定指标,研究山羊瘤胃中原虫对细菌的吞噬作用.结果表明,DAPI标记的瘤胃细菌浓度与其荧光强度之间存在线性相关(R2=0.995 2).在日粮粗精比为7:3的条件下,徐淮山羊瘤胃原虫对细菌的吞噬速率为370.89 cells/(cell·h),瘤胃内原虫对细菌的吞噬速率存在一定的波动性,吞噬量在60 min达到饱和,其最大吞噬量为370,89 ceIls/cell.结果提示,荧光光度法可应用于瘤胃原虫对细菌吞噬作用的研究.     

单细胞凝胶电泳技术在临床兽医学中的应用

单细胞凝胶电泳技术是一种简便、快速、灵敏的检测真核细胞DNA损伤与修复的方法.其基本原理是将单个细胞包理于琼脂糖凝胶中进行裂解、电泳、荧光染色和显微镜观察,细胞DNA损伤产生的断链或碎片在电泳中迁移形成典型的"慧星"图像,根据迁移长度和荧光强度即可定量分析DNA损伤的程度.该文总结了该实验技术的基本原理、检测方法及其在临床兽医学中的应用前景.     

云南半细毛羊毛囊干细胞微生物污染检测

为检测云南半细毛羊毛囊干细胞（hair follicle stem cells,HFSCs）是否受到微生物污染,为后续的诱导分化等研究奠定基础.用培养法检测细菌、真菌污染;利用荧光染色剂Hoechst33342检测支原体污染.结果表明：实验组细胞和阴性对照中均没有发现细菌污染,实验组中有25％的细胞受到霉菌污染;实验组细胞经Hoechst33342荧光染色后,仅细胞核显色,细胞表面及细胞周围干净、清晰,无荧光小点.说明云南半细毛羊HFSCs没有细菌、支原体污染,仅有25％细胞受到霉菌污染.霉菌孢子对环境的耐受力很强,普通的消毒液很难清除,所以对操作环境带来严重危害.     

用细胞分类器进行精子性别分离

Johnson最近报道了他们用细胞分类器(flow cytometer或cell sorter)进行家畜性别分离的研究进展。他们根据X精子的DNA含量略多于Y精子的原理,用无毒的活体荧光染料将精子染色。将染色的活精子逐个通过细胞分流器的微柱。此处用一束激光激发荧光染料,使通过微柱的精子发光。X精子的发光量略高于Y精子。这种发光量的微小差别由光学测定仪记录并把信号送给电脑。精液通过激光束后,便被分成微滴。电脑把电子信息反馈到产生微滴的部位,使每个微滴荷电:X精子荷     

3种瘤胃纤维分解菌实时定量PCR方法的建立

为探索以减毒胞内侵袭菌介导的黏膜免疫对宿主动物胃肠道微生态的影响,本试验以白色瘤胃球菌、黄化瘤胃球菌和产琥珀酸丝状杆菌3种主要瘤胃纤维分解菌16S rRNA分别设计引物,以山羊瘤胃液提取细菌总DNA,分别扩增3种纤维分解菌目的DNA片段,并连接至pMD-18 T Vector上,经PCR和测序鉴定后,以不同稀释度的重组质粒为模板进行荧光定量PCR反应。结果显示,扩增得到的3种瘤胃纤维分解菌目的片段与已知菌种相应片段的同源性大于99%;以不同稀释度重组pMD-18 T为模板建立的荧光定量PCR扩增曲线差异明显,绘制标准曲线的相关系数均接近1,熔解曲线均呈单一峰值。因此,本试验成功建立了3种瘤胃主要纤维分解菌的实时定量PCR方法,为减毒胞内侵袭菌介导的黏膜免疫研究奠定了基础。瘤胃纤维分解菌;Real-time PCR;标准曲线;     

分子生物学技术在瘤胃细菌多样性研究中的应用

反刍动物的瘤胃是一个复杂的厌氧微生态发酵系统,研究其瘤胃微生物区系组成与功能已成为反刍动物营养学的重要内容,分子生物学技术是开展瘤胃微生物研究的重要技术手段。本文就变性梯度凝胶电泳(DGGE)技术、16S r DNA序列分析、宏基因组技术、DNA芯片技术等在瘤胃细菌多样性研究中的应用情况进行了总结与归纳,为今后开展此方面研究工作提供技术参考。     

采用PCR-DGGE技术对放牧羊补饲糖蜜尿素舔砖后细菌种群结构的分析

为了研究补饲糖蜜尿素舔砖对放牧羊瘤胃中细菌种群结构随时间变化的规律,试验采用基于16S DNA的PCR变性梯度凝胶电泳(DGGE)技术,在放牧羊舔食舔砖的不同时间点取样,进行瘤胃液的DGGE分析。结果表明:舔食糖蜜尿素舔砖后放牧羊瘤胃细菌主要以拟杆菌门、硬壁菌门为主。在舔食15 d后瘤胃细菌多样性显著增加,舔食30 d后瘤胃细菌多样性达到最高。糖蜜-尿素对瘤胃微生物种群具有明显的筛选作用,舔食前以Capnocytophaga canimorsus为优势菌群,舔食一段时间后各菌群或出现或消失,呈动态变化和定植过程;30 d后瘤胃种群结构基本保持稳定,Prevotella ruminicola Bryant处于优势地位。说明舔食舔砖前后细菌种群结构存在明显差异,舔砖可明显增加瘤胃微生物多样性。     

白色瘤胃球菌对秸秆细胞壁和微晶纤维素的附着比较

本文采用比浊法测定了白色瘤胃球菌 (R .albus 7)对球磨、未球磨玉米秸秆细胞壁和微晶纤维素的附着率。结果表明 ,白色瘤胃球菌对球磨玉米秸细胞壁的附着能力与微晶纤维素相当 ,且二者显著高于未球磨玉米秸细胞壁的附着率 (P <0 .0 1) ,说明增加细胞壁颗粒表面积可以提高纤维分解菌的附着程度。白色瘤胃球菌经酶 (胰蛋白酶、蛋白酶 )和高碘酸钠修饰后 ,可显著降低其对玉米秸细胞壁和微晶纤维素的附着能力 (P <0 .0 1) ,而用甲醛或戊二醛固定细菌蛋白质对细菌的附着能力没有显著影响 ,推测细胞表面的蛋白质和碳水化合物同时参与了对植物细胞壁的附着。细菌经氯化锂修饰后附着能力下降最为明显 ,说明S层蛋白质 (单一蛋白质或糖蛋白 )与白色瘤胃球菌的附着能力有很大关系     

Does cortisol inhibit vasopressin secretion in sheep?

Glucocorticoids inhibit the plasma vasopressin responses to hemorrhage and hypoxia in dogs. Attempts to demonstrate glucocorticoid inhibition of vasopressin secretion in fetal sheep have been unsuccessful, suggesting the possibility that there is an influence of development on the expression of this interaction, or that the interaction cannot be demonstrated in all mammalian species. This study was designed to investigate these two possibilities. Adult ewes chronically prepared with carotid arterial loops, were subjected to 5 hr infusions of cortisol at a rate of 6 ug/kg min or vehicle (5% ethanol in saline). The infusion of cortisol increased plasma cortisol concentration from 26 +/- 3 to 46 +/- 8 ng/ml, while vehicle infusion was associated with a decrease in plasma cortisol concentration from 23 +/- 4 to 15 +/- 3 ng/ml. One hr after the end of the cortisol or vehicle infusions, vasopressin secretion was stimulated by arterial hypotension produced by 10 min infusions of sodium nitroprusside (20 ug/kg min). Nitroprusside decreased arterial blood pressure equally in both groups. Plasma vasopressin concentrations were increased to peak concentrations of 92 +/- 33 and 116 +/- 20 pg/ml in the vehicle- and cortisol-infused groups, responses which were not significantly different as tested by ANOVA. We conclude that increases in plasma cortisol concentration, equal to those observed during responses to stressors, do not inhibit vasopressin secretion in this species.     

Useful Technique for the Study of Intracellular Calcium Fluxes in Single Porcine Granulosa Cells in Culture

Contents A new experimental model was applied to study calcium involvement in the mechanisms of action of the pituitary hormones on porcine granulosa cells (GC) on single cells in culture and in a time-dependent manner. The model involved laser cytometry with calcium-sensitive dye fluo-3AM. This study describes pituitary hormone-mediated changes of calcium (Ca²⁺) concentrations in cultured porcine GC, at the single cell level. Cells were isolated from medium sized follicles of cycling mature gilts (day 16–19 post-oestrus) and were cultured at a concentration of 50–100 × 10³ cells/Petri dish in 2 ml of enriched M199 medium for 2–3 days. Image scans were performed on individual cells that had been loaded with calcium-sensitive dye, acetoxymethyl ester of fluo-3 (fluo-3 AM; 488_ex/526_em nm). In Experiment A (n_exp = 6), cell response was measured after treatment with porcine luteinizing hormone (pLH) or prolactin (pPrl) (doses of 0, 1, 10 and 100 ng/ml in pH indicator-free HBSS containing 2 m m of Ca²⁺). In cultures treated with 1, 10 and 100 ng of pLH (N_cells = 329), increases in relative fluorescence were observed in 37.1, 80.0 and 72.9% of cells scanned, respectively. In cultures treated with similar dosages of pPrl (N_cells = 259), responses were noted in 41.8, 57.3 and 47.2% of cells scanned, respectively. In control cultures treated with medium alone (N_cells = 85) the fluorescence increases were not observed. Fluorescence intensity was increased in the hormone-treated cells only (p < 0.05). Seven-fold and five-fold relative fluorescence increases were observed for cells exposed to 100 ng of pLH or 10 ng of pPrl, respectively. In Experiment B, the effects of the Ca²⁺ ionophore (A23187) and Ca²⁺ chelator (EGTA) on the changes of calcium concentrations in GC-treated with both pLH (100 ng/ml) and pPrl (10 ng/ml) were examined. In the presence of A23187 (10 μm ), fluorescence of the cells increased two-fold (p < 0.05) within 400 s in cultures treated with combination of both pLH and pPrl. However, EGTA (2.5 m m ) completely abolished the responses of GC to both pLH and pPrl (N_cells = 168, n_exp = 16) and the fluorescence decreased to levels similar to the control group (N_cells = 159, n_exp = 5). The studies clearly demonstrated that intracellular free calcium changes occur in porcine GC when they are exposed to pLH or pPrl. The present study has provided a useful technique for studying the role of calcium in regulation of porcine GC by different factors at the single porcine cell level in culture and in a time-dependent manner.     

Comparative plasma dispositions of ivermectin and doramectin following subcutaneous and oral administration in dogs

This study evaluates the comparative plasma dispositions of ivermectin (IVM) and doramectin (DRM) following oral and subcutaneous administration (200 microg/kg) over a 40-day period in dogs. Twenty bitches were allocated by weight in to four groups (Groups I-IV) of five animals each. Animals in the first two groups (Groups I and II) received orally the injectable solutions of IVM and DRM, respectively, at the dose of 200 microg/kg bodyweight. The other two groups (Groups III and IV) received subcutaneously injectable solutions at the same dose rate. Blood samples were collected between 1h and 40 days after treatment and the plasma samples were analysed by high performance liquid chromatography (HPLC) using fluorescence detection. The results indicated that IVM produced a significantly higher maximum plasma concentration (C(max): 116.80+/-10.79 ng/ml) with slower absorption (t(max): 0.23+/-0.09 day) and larger area under the concentration versus time curve (AUC: 236.79+/-41.45 ng day/ml) as compared with DRM (C(max): 86.47+/-19.80 ng/ml, t(max): 0.12+/-0.05 day, AUC: 183.48+/-13.17 ng day/ml) following oral administration of both drugs; whereas no significant differences were observed on the pharmacokinetic parameters between IVM and DRM after subcutaneous administrations. In addition, subcutaneously given IVM and DRM presented a significantly lower maximum plasma concentration (C(max): 66.80+/-9.67 ng/ml and 54.78+/-11.99 ng/ml, respectively) with slower absorption (t(max): 1.40+/-1.00 day and 1.70+/-0.76 day, respectively) and larger area under the concentration versus time curve (AUC: 349.18+/-47.79 ng day/ml and 292.10+/-78.76 ng day/ml, respectively) as compared with the oral administration of IVM and DRM, respectively. No difference was observed for the terminal half-lives ((t(1/2lambda(z)) and mean residence times (MRT) of both molecules. Considering the pharmacokinetic parameters, IVM and DRM could be used by the oral or subcutaneous route for the control of parasitic infection in dogs.     

Safe and efficacious dosage of flecainide acetate for treating equine atrial fibrillation

To determine a safe and efficacious dose of flecainide acetate for treating equine atrial fibrillation (Af), the safe dosage level was determined by injecting 1, 2, or 3 mg/kg i.v. of 1% flecainide acetate solution at a rate of 0.2 mg/kg/min to five clinically healthy horses. Clinical signs and the ECG were monitored (HR, PR, QRS, and QT intervals) and blood was taken to measure the plasma flecainide concentration pre- and post-administration. No abnormal signs were observed in the 1- or 2-mg/kg groups, while agitation was observed in three of five horses in the 3-mg/kg group. The QRS, and QT intervals for the 3-mg/kg group increased significantly. The peak plasma flecainide concentrations were 1.316 +/- 358 (SD) ng/ml, 1,904 +/- 314 ng/ml, and 2,251 +/- 387 ng/ml for the 1-, 2-, and 3-mg/kg groups, respectively. To evaluate the efficacy of flecainide, Af was induced by right atrial pacing in six clinically healthy horses, and 1% flecainide acetate solution was then administered until they converted to sinus rhythm. All horses with induced Af converted. For the conversion, a total dose of 1.40 +/- 0.63 mg/kg flecainide was required, the duration of administration was 7.00 +/- 3.15 min and plasma flecainide concentration at conversion was 1,303 +/- 566 ng/ml. In conclusion, flecainide acetate is a safe and effective antiarrhythmic agent for equine Af, and the clinically effective dosage is 1 to 2 mg/kg.     

荧光报告质粒构建及其在布鲁菌细胞侵袭试验中的应用

采取质粒shuffling方法,将含GFPmut1的pPS858和能在布鲁菌中复制的质粒pBBRlMCS-2经KpnI酶切后连接,转化DH5a,利用双抗性筛选重组质粒pBBR1-GFP。将荧光质粒转入布鲁菌,获得荧光标记布鲁菌,并用构建好的荧光标记布鲁菌侵染巨噬细胞J774A．1,探讨其用于细胞侵袭试验的可行性。结果显示,成功构建布鲁菌荧光报告质粒pBBR1-GFP,用构建好的荧光标记布鲁菌侵染巨噬细胞后,在细胞内观察到带荧光的布鲁菌,且荧光强度与胞内细菌数成正比。细胞侵袭动态试验结果表明,细菌与细胞共孵育45min后,胞内细菌达到饱和。结果表明,成功构建了布鲁菌的荧光质粒报告系统,可用于布鲁菌细胞侵袭试验。     

Bioavailability and pharmacokinetics of metoclopramide in cattle

The bioavailability of metoclopramide was investigated in three steers following administration of 8 mg/kg by the oral, abomasal (cannula), and intravenous routes, using a Latin square design. The mean (± SD) oral and abomasal bioavailabilitles were 51.3 ± 30.7% and 76.2 ± 15.5%, respectively. The mean value for clearance ( C1 ) was 20.1 ± 5.9 ml/min and the volume of distribution ( V _d) was 0.51 ± 0.19 1/kg. Additionalpharmacokmetic parameters for metoclopramide were determined following intravenous administration to seven cows. A predominate two-compartment model of distribution was found in six cows with a t _1/2α harmonic mean of 24.2 min and a range of 11.2–72.4 min, a t _1/2β harmonic mean of 53.1 min and a range of 31.1–134.1 min, a Cl of 42.2 ± 8.7 ml/min, and a V _d of 2.1 ± 0.8 1/kg. To better define the relationship between metoclopramide concentration and release of prolactin, a treatment-by-subjects infusion study was conducted in which four different loading doses followed by constant infusion were used. A steady-state metoclopramide concentration ( MCP _ss) of 8.8 ± 2.6 ng/ml was associated with a three-fold elevation of prolactin to a mean value of 12.1 ± 3.1 ng/ml in six yearling steers. Steady state serum prolactin concentrations ( PRL _ss) did not rise significantly above 23.3 ± 6.9 ng/ml, even when MCP _ss reached a concentration of 518.5 ±151.2 ng/ml. The short half-life, moderate V _d, low minimum pharmacologically effective concentration, and rapid C1 found for metoclopramide in cattle in this study, suggest that a continuous release device could potentially be useful in the application of this drug in the prevention and treatment of fescue toxicosis.     

Secretion of dopamine and norepinephrine in hypophyseal portal blood and prolactin in peripheral blood of Holstein cattle

The objective was to test the hypothesis that dopamine regulates prolactin (PRL) secretion by determining acute changes in catecholamine concentrations in hypophyseal portal blood of cattle, and their relation to peripheral blood concentration of PRL in hypophyseal stalk-transected (HST) and sham-operated controls (SOC). Holstein heifers (606 +/- 21 kg BW; mean +/- SE) were subjected to neurosurgery for 8 h to collect hypophyseal portal blood with a stainless steel cannula designed with a cuff placed under the pituitary stalk and peripheral blood via a jugular vein catheter. PRL plasma concentration was measured by radioimmunoassay, and dopamine and norepinephrine in portal plasma by radioenzymatic assay. During anesthesia before HST or SOC, PRL plasma concentration ranged from 20-40 ng/ml throughout 255 min. PRL abruptly increased and remained above 90 ng/ml after HST compared with a steady decrease to <20 ng/ml in SOC heifers throughout 440 min. Within 5 min after severing the hypophyseal stalk, dopamine in portal blood (>8 ng/ml) was significantly increased (P < 0.05) compared with peripheral blood (<2 ng/ml). Norepinephrine concentration in portal blood was significantly greater (P < 0.05) than in peripheral blood during the first 60 min. The sustained high PRL level in peripheral plasma after severing the hypophyseal stalk stimulated hypothalamic dopamine secretion from hypophyseal portal vessels during the prolonged period of blood collection. Norepinephrine concentration in these cattle was greater in hypophyseal portal than in peripheral blood, implicating both an important hypothalamic source of the catecholamine as well as an adrenal gland contribution during anesthesia.     

Diurnal and episodic variations of plasma hydrocortisone concentrations in horses

Using a specific high-performance liquid chromatographic technique, plasma hydrocortisone values were measured hourly in 6 horses and every 10 minutes in 4 horses over 24 hours. Both circadian and episodic variation was observed. The mean plasma hydrocortisone concentration was a maximum of 58.8 ± 9.54 ng/ml at 9.19 ± 0.59 hr and a minimum of 27.85 ± 6.85 g/ml at 21.19 ± 0.59 hr. The number of episodes of secretion was 10.0 ± 1.41; the mean amplitude and duration of a peak were 26.21 ± 3.71 ng/ml and 105.25 ± 21.24 min respectively.     

Endocrine patterns in two strains of Japanese black cattle with growth retardation

Endocrine patterns were compared in 2 strains of Japanese black cattle with growth retardation; MHO- and HSK-paternal strains (MHO and HSK cattle, respectively). MHO cattle (n=8) displayed lower serum concentrations of insulin-like growth factor-1 (IGF-1), triiodothyronine (T3), thyroxine (T4), and cortisol (31.1+/-20.7 ng/ml, 73.9+/-51.9 ng/dl, and 2.9+/-2.9 microg/dl, 1.3+/-0.7 microg/dl, respectively) than those in both HSK cattle (n=5) (64.9+/-47.6 ng/ml, 97.8+/-40.7 ng/dl, 4.1+/-2.1 microg/dl and 1.8+/-1.1 microg/dl, respectively), and the controls (n=6) (314.7+/-197.2 ng/ml, 140.2+/-21.3 ng/dl, 5.8+/-1.7 microg/dl, and 3.0+/-1.4 microg/dl, respectively). The area under the concentration curve of growth hormone (GH-AUC 0-600 min) in MHO cattle (22210+/-18951 ng.min/ml) tended to be greater than those in HSK (7887+/-6340 ng.min/ml) and the controls (2811+/-1275 ng.min/ml). MHO cattle showed a high GH-AUC0-600 min in contrast to a low serum IGF-1 concentration, as well as lower serum T3, T4, and cortisol concentrations. HSK cattle exhibited the same secretory patterns, but much more moderately. Growth retardation in Japanese black cattle exhibits some variations based on pedigree.     

Opioid modulation of the hypothalamo-pituitary-adrenal axis in dairy cows.

Plasma cortisol responses to an intravenous bolus treatment with 250 mg naloxone, 300 mg morphine or a combination, were studied in Holstein-Friesian cows; 4 in early lactation (29-43 d postpartum) and 7 in mid-lactation (90-155 d post-partum). Blood samples were collected every 15 min from 60 min before to 90 min after treatment. Naloxone induced an immediate increase in cortisol concentration, reaching a peak within 30 min. The cortisol response (area under the curve) was positively correlated with pre-naloxone cortisol concentrations (r = 0.7, p < 0.05). The mean increase in cortisol concentration after naloxone appeared to be lower in early lactation (1.8 ng/ml) than in mid-lactation (8.3 ng/ml). In contrast, morphine consistently suppressed mean tonic plasma cortisol concentration by 2.7 ng/ml below baseline for at least 90 min. When given with morphine, naloxone counteracted the suppressive effects; the cortisol response was similar to that after naloxone alone. A cow in mid-lactation, suffering from chronic lameness (joint infection), gave opposite results, i.e., treatment with morphine alone increased cortisol concentration, whereas morphine with naloxone did not result in the expected large increase in plasma cortisol concentration. In conclusion, the hypothalamo-pituitary-adrenal axis of dairy cows appears to be under suppressive opioidergic control. However, the opioidergic system involved in hypothalamo-pituitary-adrenal functions of an animal under chronic stress behaved in an opposite manner.