扩增SRY基因鉴定牛、羊胚胎性别的初步研究

本研究利用本试验室设计的SRY基因3对常规PCR引物扩增牛SRY基因和羊的SRY基因,结果表明:第2对引物扩增出公牛和公羊基因组的DNA 750 bp左右清晰条带,而母牛、母羊基因组DNA无此扩增条带,说明第2对引物可以应用于牛和羊的早期胚胎性别鉴定;     

牛早期胚胎的性别鉴定PCR技术的研究

根据牛SRY(Y-染色体性别决定基因)基因序列自行设计合成3对常规PCR引物作为牛性别鉴定特异性引物,对15头公牛、20头母牛的样品进行检测.结果表明,第2对引物可清晰扩增出公牛基因组DNA750 bp左右条带,而母牛基因组DNA无此扩增条带,证明这条引物可以应用于牛的早期胚胎性别鉴定研究与应用.     

兔早期胚胎PCR性别鉴定体系的建立

根据兔SRY基因序列设计两对引物作为兔雄性特异性引物,根据兔APP基因序列设计1对引物作为内标引物,分别建立了兔早期胚胎性别鉴定的双重PCR和巢式PCR反应体系,在不同浓度的基因组DNA和兔早期胚胎上进行性别鉴定应用,同时,对兔SRY巢式PCR引物特异性进行了分析。结果表明,多重PCR扩增兔基因组DNA可以准确判定其性别,扩增灵敏度为100pg基因组DNA;多重PCR鉴定24枚兔32细胞桑椹胚性别,只能对整胚成功鉴定。巢式PCR,公兔基因组DNA扩增出282bp的SRY基因片段,母兔没有扩增产物,扩增灵敏度为10pg;对24枚兔32细胞桑椹胚性别鉴定结果表明,巢式PCR可以对少至4个胚胎细胞进行准确鉴定,同一胚胎结果符合率为100%(24/24)。SRY引物只对兔雄性基因组DNA特异,而其他动物(人、牛、绵羊、小鼠)雄性DNA及兔的冲卵液,均无PCR产物。     

哺乳动物胚胎性别鉴定试剂盒的研究

依据牛、山羊、兔、猪等哺乳动物SRY基因高度同源性设计一对22 bp的SRY引物,按照3×2×3因子组合,建立了PCR扩增胚胎DNA最适条件。采用此最适条件扩增了15个羊-兔异种克隆胚胎DNA,结果表明PCR法可以用来鉴别哺乳动物胚胎的性别。采用设计的性别鉴定引物,按照最优PCR扩增胚胎DNA条件配制了PCR性别鉴定试剂盒。     

利用PCR技术鉴定常见肉类来源

在提取送检的未知肌肉组织样品以及市购的猪肉、牛肉、羊肉样品基因组DNA的基础上,分别选用1对通用引物与3对特异性引物进行扩增,用来检测未知样品的畜种来源。结果显示:通用引物扩增4种样品均出现阳性条带;分别用羊、猪、牛的特异性引物扩增样品DNA,送检未知样品只在牛特异性引物扩增时出现了特异性目的条带。经PCR产物直接测序及同源性比对进行验证确认,该未知样品与牛的同源性为100%,从而验证了该方法的准确性。     

SRY基因和MCM158、MAF45鉴定哺乳动物性别

为了研究SRY基因和Y染色体上微卫星标记对性别鉴定的影响,试验以哺乳动物为研究对象,采用多重PCR技术扩增绵羊和牛基因组中Y染色体上的2个微卫星标记和SRY基因,根据其基因型进行性别鉴定,试图通过一次DNA扩增同时提供性别鉴定和基因分型的信息。结果表明:所设计的SRY基因引物具有高度特异性,是性别鉴定的主要依据;Y染色体上的MCM158、MAF45两标记由于特异性不好而不适用于性别鉴定。     

奶牛性别鉴定的研究与应用

选取牛雄性性别决定基因SRY (sex region of Y chromosome),根据基因序列设计特异引物,应用PCR技术对5头荷斯坦奶牛DNA样品进行扩增,鉴定其性别;并对设计的引物灵敏度进行检测;对已有的公、母各20头荷斯坦奶牛DNA样品进行PCR盲检,获取奶牛高灵敏度特异性引物,用于奶牛性别鉴定。结果表明,4头公牛DNA样品可以扩增出目标条带(66 bp),1头母牛DNA样品无法扩增出条带,阴性对照扩增无条带;最佳引物灵敏度为1.6 pg/μL,可以很好地满足性别鉴定需要。40头个体中,20头个体DNA样品可以扩增出条带,其余20头个体DNA样品无法扩增出条带,检测结果与实际性别对比准确率为100%。试验结果表明,设计的引物灵敏度比较好,能够满足奶牛性别鉴定的需要。     

云南黑山羊性别决定基因体外扩增研究

对云南黑山羊 (10♂ ,2♀ )的基因组DNA性别决定基因 (SRY)进行了体外PCR扩增的研究 ,结果表明适当引物可特异性扩增出雄性个体的性别决定基因片段 (大小约为 185bp) ,而对雌性个体则不能扩增出任何片段 ;研究还利用已筛选出的性别决定基因特异性引物对少量的胚胎细胞进行了特异性扩增 ,检测了 2 0枚云南黑山羊的胚胎 ,其中有 5枚胚胎可扩增出约为 185bp的特异性片段。以上结果为山羊胚胎早期性别鉴定奠定了技术基础。     

牙釉质基因巢式扩增鉴定牛早期胚胎性别的研究

根据牙釉质基因在牛X染色体和Y染色体上存在的差异设计巢式引物,对牛静脉血基因组DNA样本以及10枚牛早期胚胎DNA样本进行巢式扩增和电泳分析,以鉴定性别。结果表明:利用此方法能够对牛10 pg量血液基因组DNA进行扩增鉴定性别,雌性产生1条片段长度为311 bp的源于X染色体的条带,雄性产生1条源于X染色体的条带(311 bp)和1条片段长度为251 bp的源于Y染色体的条带,对10枚胚胎进行鉴定,6枚为雄性,4枚为雌性。说明牙釉质基因巢式PCR扩增鉴定牛早期胚胎性别方法可靠,准确性和敏感性较高。     

应用PCR技术检测柞蚕微孢子虫

采用斑迹抽提法提取柞蚕微孢子虫(Nosema pernyi)基因组DNA,基因组DNA的琼脂糖凝胶电泳图谱中有大小约15kb的清晰、完整条带。选用已报道的微粒子属16S rRNA基因的保守序列设计P1/P2和N1/N22对引物,对柞蚕微孢子虫基因组DNA进行PCR扩增,结果2对引物分别扩增出1条大小不同的特异谱条带,其中用引物N1/N2扩增可检测出0.47ng的DNA模板。应用同样的PCR引物、体系和反应条件,可有效扩增出受感染柞蚕幼虫、成虫中的微孢子虫基因组DNA条带。该项检测技术有望应用于柞蚕微粒子病的早期诊断。     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.