催乳素基因多态性及其与小尾寒羊高繁殖力关系的研究

设计5对引物，采用PCR-SSCP技术检测催乳素（prolactin，PRL）基因外显子1至外显子5在高繁殖力绵羊品种（小尾寒羊和湖羊）和低繁殖力绵羊品种（多赛特、特克塞尔和考力代）中的单核苷酸多态性，同时研究该基因对小尾寒羊高繁殖力的影响。通过克隆测序首次获得了绵羊PRL基因外显子全序列。结果表明：外显子2在5个绵羊品种中均存在单核苷酸多态性，而其它4个外显子只在湖羊中存在单核苷酸多态性。本研究初步表明小尾寒羊PRL基因外显子2的CC型平均产羔数分别比CD型和DD型多0.39只（P<0.05）和0.98只（P<0.05），CD型和DD型小尾寒羊平均产羔数差异不显著（P>0.05）。     

小尾寒羊BMP15基因多态性及其与高繁殖力关系的研究

以控制Belclare、Cambridge、Hanna、Inverdale和Lacaune绵羊高繁殖力的骨形态发生蛋白15基因(bonemorpho-geneticprotein15,BMP15)为候选基因,根据绵羊BMP15基因全序列设计6对引物覆盖全部的外显子,采用PCR-SSCP方法检测BMP15基因在高繁殖力绵羊品种(小尾寒羊)和低繁殖力绵羊品种(多赛特羊)中的多态性,同时研究该基因对小尾寒羊高繁殖力的影响。结果表明,只有外显子1的第1对引物扩增片段具有多态性,其余5对引物扩增片段都没有多态性。在127只小尾寒羊母羊和30只多赛特母羊中都检测到两种基因型(AA和AB)。统计结果表明,小尾寒羊和多赛特羊AA基因型频率分别为0.638和0.800,AB基因型频率分别为0.362和0.200。测序结果表明,AB基因型和AA基因型相比在cDNA第28bp处缺失了3个碱基(CTT),小尾寒羊和多赛特绵羊都发生了与Belclare、Cambridge、Hanna和Inverdale绵羊相同的CTT缺失突变。AB基因型小尾寒羊平均产羔数比AA基因型多0.20只,但差异不显著(P>0.05)。研究结果提示,BMP15基因的CTT缺失突变对小尾寒羊高繁殖力没有显著影响。     

GnRHR基因多态性及其与小尾寒羊高繁殖力关系的研究

设计8对引物,应用聚合酶链式反应-单链构象多态性（PCR-SSCP）技术检测促性腺激素释放激素受体（gonadotropin releasing hormone receptor, GnRHR）基因外显子1、外显子2和外显子3在高繁殖力品种（小尾寒羊和湖羊）和低繁殖力品种（南非肉用美利奴、考力代和中国美利奴绵羊）中的单核苷酸多态性,同时研究该基因对小尾寒羊高繁殖力的影响。结果发现引物P4和P7扩增片段具有多态性,其余6对引物的扩增片段都不存在多态性。对于P4扩增片段,在湖羊中检测到AA和BB基因型,在其余4个绵羊品种中只检测到AA基因型,BB型与AA型相比在外显子1有5个突变（+692G→A、+706T→A、+747T→C、+748A→T和+802T→A）,并引起氨基酸改变（Gly→Ser、Asp→Glu和Leu→Pro）。对于P7扩增片段,在小尾寒羊和湖羊中都检测到CC和DD基因型,在低繁殖力的3个绵羊品种中均只检测到CC基因型,DD型与CC型相比在外显子2有+50A→G和+101A→C两个突变,并引起氨基酸改变（Glu→Gly和Gln→Pro）;小尾寒羊CC和DD基因型频率分别为0.87和0.13,突变纯合型（DD）小尾寒羊平均产羔数比野生型（CC）多0.81只（P<0.01）。     

Kinetics of abamectin disposition in blood plasma and milk of lactating dairy sheep and suckling lambs

Abamectin (ABM) has been used worldwide as an anthelmintic drug in veterinary medicine and as an agricultural pesticide. Its pharmacokinetics and permeation into milk was evaluated in dairy sheep after subcutaneous administration. ABM elimination half-lives and mean residence times were 1.7 and 3.7 days for blood plasma and 1.9 and 3.8 days for milk, respectively. The ABM milk to plasma concentration ratio (0.89) primarily depends on milk fat content. Transfer of ABM residues to suckling lambs was evaluated by determination of ABM concentration time courses in lambs' plasma. Mean maximal concentration in lambs was 1.6 microg L(-1) at 3.3 days, and elimination half-life was 2.7 days. In ewes' plasma and milk, ABM was detected up to 23 days. Because of different pharmacokinetics, ABM exposure in lambs was almost 10% of the exposure in ewes, although the amount excreted in milk was only 1.0% of the dose.     

小尾寒羊BMP4基因多态性及其与高繁殖力关系的研究

采用PCR-SSCP技术检测骨形态发生蛋白4（bone morphogenetic protein 4,BMP4）基因外显子2、3和4在高繁殖力绵羊品种（小尾寒羊）和低繁殖力绵羊品种（中国美利奴绵羊、考力代绵羊、南非肉用美利奴绵羊）中的单核苷酸多态性,同时研究该基因对小尾寒羊高繁殖力的影响。结果表明：外显子2和4在4个绵羊品种中都不存在多态性,外显子3则都存在多态性。对于外显子3的引物,在4个绵羊品种中都检测到3种基因型(AA、AB、BB);统计结果表明：小尾寒羊、中国美利奴绵羊、考力代绵羊、南非肉用美利奴绵羊AA基因型频率分别为0.017、0.216、0.115、0.429,AB基因型频率分别为0.102、0.317、0.269、0.500,BB基因型频率分别为0.881、0.467、0.616、0.071;测序结果表明：BB型和AA型相比在BMP4基因外显子3第305位碱基处发生C→A的突变,该突变导致氨基酸由丙氨酸改变为天冬氨酸;BB型小尾寒羊平均产羔数分别比AB型和AA型多0.61只（P<0.05）和1.01只（P<0.05）。     

小尾寒羊MTNR1A基因外显子2的克隆与序列分析

对常年发情的绵羊品种小尾寒羊和季节性发情的绵羊品种多赛特羊共20只母羊的褪黑激素受体1A(melatonin receptor 1A，MTNR1A)基因外显子2的824 bp扩增产物进行了克隆测序及序列比较分析。结果表明, 小尾寒羊MTNR1A基因外显子2的核苷酸序列与已发表的绵羊序列(GenBank登录号U14109)完全相同。小尾寒羊与多赛特羊MTNR1A基因外显子2的差异由5个变化的核苷酸(分别为C329T、G355T、C566T、C580A和A675G)组成，核苷酸同源性为99.4%。小尾寒羊与山羊、母牛、猪、人、小鼠、挪威大鼠、西伯利亚仓鼠和鸡之间MTNR1A基因外显子2的核苷酸序列同源性为73.2%～98.5%，氨基酸序列同源性为78.5%～98.5%。     

绵羊多胎主效基因FecB分子检测方法的建立与应用

FecB（Fec,fecundity;B,Booroola）是在布鲁拉美利奴(Booroola Merino)绵羊（Ovis aries）中发现的能增加排卵数和产羔数的一个常染色体突变基因,是在绵羊中识别出的第1个高繁殖力主效基因。建立了绵羊多胎主效基因FecB的PCR-RFLP和PCR-SSCP检测方法,对小尾寒羊、湖羊、中国美利奴、东弗里生、杜泊、萨福克、特克塞尔、多赛特、考力代、德国肉用美利奴、南非肉用美利奴以及杂种羊共6 647只绵羊的检测结果表明,两种检测方法均能准确判型,稳定可靠性好,而且两种方法的检测结果完全一致。检测结果表明小尾寒羊和湖羊携带FecB突变（A746G）,中国美利奴、东弗里生、杜泊、萨福克、特克塞尔、多赛特、考力代、德国肉用美利奴和南非肉用美利奴绵羊都不携带该突变。     

The Boreray sheep of ST Kilda,outer hebrides,Scotland: The natural history of a feral population

The Boreray sheep have survived for 50 years without interference by man and maintain high density on rich maritime pastures heavily manured by seabirds. They are a blackface breed obtained from a cross between old Scottish shortwool and early blackface sheep. There is a population of about 400 on about 55 ha of pasture and the rams and ewes (with lamb and yearling rams) run in separate groups. The survival of rams is poor compared with ewes with an adult sex-ratio of about 10 ewes to 1 ram. Numbers of sheep fluctuate between 330 and 460 without causing sheet erosion, landslipping and disruption of the vegetation. The conservation plan for Boreray rests on continued non-interference with the sheep and no sheep should be introduced to the island.     

Composting as a Means of Disposal of Sheep Mortalities

Four studies explored the feasibility of year-round composting of lamb and mature sheep mortalities within the arid climate of the Canadian prairies. In all studies, a ratio of 2:1:1 (manure : mortalities : chopped straw) was maintained, although depth of the mortality layer within the bin, number of layers of mortalities per bin, age of animal (lamb or mature sheep) and time of year (summer or winter) were varied. Composting neonatal lambs in the spring/summer was successful whether a single layer (n=15 lambs, weight 99.7 kg) or two, separated layers of mortalities (n=41 lambs, weight 198 kg) were added to a 2.4 m³ open bin. Residual bone, wool and soft tissues were negligible after the lamb compost had completed one heating cycle. In contrast, composting mature sheep in the fall/winter was more difficult due to: (1) over wet manure (31% dry matter) resulting in continuous anaerobic decomposition of carcasses; (2) fat/grease accumulation when composting a layer of carcasses 71 cm in depth (weight of sheep 1020 kg). For mature sheep mortalities, 2 heating cycles were required to eliminate soft tissues and wool. As compost in all studies heated in excess of 60°C in the primary and/or secondary bin, bacterial isolates taken after the compost completed the secondary heating cycle were all innocuous species. Provided that compost is protected from excessive moisture and compost is aerated by turning into a secondary bin, a 2:1:1 (manure:mortalities:straw) ratio allowed for year-round composting of lamb and mature sheep mortalities.     

Diplonine, a neurotoxin isolated from cultures of the fungus Stenocarpella maydis (Berk.) Sacc. that induces diplodiosis

Diplodiosis is a neuromycotoxicosis of cattle and sheep caused by ingestion of maize infected with the ear-rot fungus Stenocarpella (= Diplodia ) maydis . Apart from ataxia, paresis, and paralysis, the toxin is responsible for stillbirths and neonatal losses characterized by the presence of spongiform degeneration in the white matter of the brain in the offspring of dams exposed to infected maize cobs. In the present study a toxin, named diplonine, which induced neurological signs in guinea pigs resembling some of those occurring in cattle and sheep, was isolated from S. maydis cultures. Purification of diplonine was achieved by methanol extraction followed by chromatographic separation on silica gel and RP-18 stationary phases. The structure and relative configuration of diplonine were defined by analysis of NMR and MS data as (S)-2-amino-2-[(1R,2S)-1-hydroxy-2-methylcyclopropyl]acetic acid or the (S)-2-amino-2-[(1S,2R)-diastereomer.     

绵羊同期发情及冻胚移植效果观察

羊胚胎移植技术是优秀种羊扩群和引种的有效手段。在绵羊繁殖季节采用两种同期发情处理方案对150只受体羊处理,同期发情率为88.67%(133/150)。采用手术法,对其中126只受体羊进行了纯种肉羊冻胚移植,受体羊可用率为94.74%(126/133)。用B超诊断妊娠率为44.00%(55/125),产羔59只,单羔存活率93.62%(44/47),双羔存活率66.67%(8/12)。试验结果表明,用冷冻胚胎移植繁殖种羊可行。     

Biomedical applications of poisonous plant research

Research designed to isolate and identify the bioactive compounds responsible for the toxicity of plants to livestock that graze them has been extremely successful. The knowledge gained has been used to design management techniques to prevent economic losses, predict potential outbreaks of poisoning, and treat affected animals. The availability of these compounds in pure form has now provided scientists with tools to develop animal models for human diseases, study modes of action at the molecular level, and apply such knowledge to the development of potential drug candidates for the treatment of a number of genetic and infectious conditions. These advances are illustrated by specific examples of biomedical applications of the toxins of Veratrum californicum (western false hellebore), Lupinus species (lupines), and Astragalus and Oxytropis species (locoweeds).     

Trace analysis of chloramphenicol residues in eggs, milk, and meat: comparison of gas chromatography and radioimmunoassay

A radioimmunological assay (RIA) to detect chloramphenicol (CAP) residues in eggs, milk, and meat is described. For tissues and other edible products of chloramphenicol-treated animals (chickens, cows, and pigs), the limit of detection is about 200 ng/kg. Residue levels above 1 microgram/kg can easily be quantitated. When highly specific antisera produced in sheep were used, cross-reactivity was insignificant except for metabolites deviating from the parent compound in the acyl side chain only. Thiamphenicol fails to bind to the antisera; hence, it does not interfere with the assay. In the procedure described, the role of cleanup is merely to remove lipids. Thus, skim milk can be analyzed following appropriate dilution without cleanup. The results obtained by RIA were confirmed by gas chromatography with electron capture detection. The new RIA allows rapid, sensitive, and specific screening of large numbers of samples.     

Glycoalkaloids and metabolites inhibit the growth of human colon (HT29) and liver (HepG2) cancer cells

As part of an effort to improve plant-derived foods such as potatoes, eggplants, and tomatoes, the antiproliferative activities against human colon (HT29) and liver (HepG2) cancer cells of a series of structurally related individual compounds were examined using a microculture tetrazolium (MTT) assay. The objective was to assess the roles of the carbohydrate side chain and aglycon part of Solanum glycosides in influencing inhibitory activities of these compounds. Evaluations were carried out with four concentrations each (0.1, 1, 10, and 100 microg/mL) of the the potato trisaccharide glycoalkaloids alpha-chaconine and alpha-solanine; the disaccharides beta(1)-chaconine, beta(2)-chaconine, and beta(2)-solanine; the monosaccharide gamma-chaconine and their common aglycon solanidine; the tetrasaccharide potato glycoalkaloid dehydrocommersonine; the potato aglycon demissidine; the tetrasaccharide tomato glycoalkaloid alpha-tomatine, the trisaccharide beta(1)-tomatine, the disaccharide gamma-tomatine, the monosaccharide delta-tomatine, and their common aglycon tomatidine; the eggplant glycoalkaloids solamargine and solasonine and their common aglycon solasodine; and the nonsteroidal alkaloid jervine. All compounds were active in the assay, with the glycoalkaloids being the most active and the hydrolysis products less so. The effectiveness against the liver cells was greater than against the colon cells. Potencies of alpha-tomatine and alpha-chaconine at a concentration of 1 microg/mL against the liver carcinoma cells were higher than those observed with the anticancer drugs doxorubicin and camptothecin. Because alpha-chaconine, alpha-solanine, and alpha-tomatine also inhibited normal human liver HeLa (Chang) cells, safety considerations should guide the use of these compounds as preventative or therapeutic treatments against carcinomas.     

Development of a fluorescent latex immunoassay for detection of a spectinomycin antibiotic

There is a need to develop a rapid and sensitive method to detect spectinomycin residues in animal tissues. A latex fluorescent immunoassay was designed using reagents developed for this assay. The spectinomycin antibody was produced in sheep, and the immunoglobulin (IgG) was purified through a Protein G affinity column and was immobilized onto latex particles. Spectinomycin was labeled with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF). The optimum assay conditions consisted of preincubating the latex-IgG with spectinomycin in buffer solutions or in bovine kidney extracts. DTAF-spectinomycin was added and was further incubated. The bound spectinomycin-DTAF/IgG-latex complex was separated by centrifugation at 4000 g for 10 min. The fluorescence signals of the unbound spectinomycin-DTAF in the supernatant were measured at 485/535 nm excitation/emission. The measured signals were directly proportional to the concentration of spectinomycin in the samples, and spectinomycin was detected at 0-100 ppb with minimum detectability of 5 ppb. The mean regression correlation of four trials in buffer was 0.936 when the % bound complex vs spectinomycin concentration was plotted. Analysis of the kidney extract spiked with 0-100 ppb spectinomycin had a regression correlation of 0.959. This assay provides a rapid screening method for low ppb detection of spectinomycin.     

Qualitative and quantitative polymerase chain reaction assays for an alfalfa (Medicago sativa)-specific reference gene to use in monitoring transgenic cultivars

Genetically modified (GM) alfalfa (Medicago sativa) was marketed for the first time in 2005. For countries with established thresholds for GM plants, methods to detect and quantify their adventitious presence are required. We selected acetyl CoA carboxylase as a reference gene for the detection and quantification of GM alfalfa. Two qualitative polymerase chain reaction (PCR) assays (Acc1 and Acc2) were designed to detect alfalfa. Both were specific to alfalfa, amplifying DNA from 12 separate cultivars and showing negative results for PCR of 15 nonalfalfa plants. The limits of detection for Acc1 and Acc2 were 0.2 and 0.01%, respectively. A quantitative real-time PCR assay was also designed, having high linearity (r > 0.99) over alfalfa standard concentrations ranging from 100 to 2.0 x 10(5) pg of alfalfa DNA per PCR. The real-time PCR assay was effective in quantifying alfalfa DNA from forage- and concentrate-based mixed diets containing different amounts of alfalfa meal.     

Detection of streptomycin residues in whole milk using an optical immunobiosensor.

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples ( approximately 3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).     

Symposium on microbiology update: old friends and new enemies. Staphylococcus aureus

The analytical methods for the detection of the staphylococcal enterotoxins can be divided into 2 categories: (1) methods for detection of enterotoxin-producing staphylococcal strains; (2) methods for detection of enterotoxin in foods. Gel diffusion methods (Ouchterlony, microslide), in which the enterotoxin produced by any given strain is compared to one of the identified enterotoxins, are used most frequently for strain testing. The sensitivity of these methods is from 0.1 to 0.5 micrograms enterotoxin/mL, which is normally adequate to determine the enterotoxigenicity of strains. The methods for the detection of enterotoxin in foods need to be much more sensitive to detect less than 1 ng of enterotoxin/g of food that may be present. The radioimmunoassay (RIA), the enzyme-linked immunosorbent assay (ELISA), and the reversed passive latex agglutination (RPLA) method have the necessary sensitivity to detect 1 ng/g of enterotoxin in foods without the use of complicated extraction-concentration procedures. Kits based on the ELISA and RPLA methods are now available commercially for the detection of enterotoxins in foods. Tests have shown that the ELISA methods are somewhat more sensitive than the RPLA method.     

Development of immunoassays for tyramine and tryptamine toxins of Phalaris aquatica L

The leaves of the perennial pasture grass Phalaris aquatica L. (phalaris) contain two groups of known toxins, indole alkaloids, primarily dimethyltryptamines and N-methyltyramines, which cause illnesses in grazing animals, especially sheep. Using amino-reactive and phenolic hydroxyl-reactive homobifunctional reagents, simple methods were devised for coupling toxins representative of those in phalaris to carrier proteins and enzymes for ELISA development. ELISAs were produced for both groups of toxins. Dimethyltryptamines were most sensitively detected [lower limit of detection (LLD) of 1 microg/L for bufotenine] using rabbit anti-bufotenine antibodies, coupled to ovalbumin using divinyl sulfone, with detection using a peroxidase conjugate prepared using the same hapten coupled with 1, 4-butanediol diglycidyl ether. The assay cross-reacted with other toxins of the same class (N,N-dimethyltryptamine and N, N-dimethyl-5-methoxytryptamine) but not with the structurally related amino acids histidine and tryptophan. The most sensitive N-methyltyramine assay (LLD of 1 microg/mL for N-methyltyramine) utilized antisera to tyramine with N-methyltyramine coupled to peroxidase. Significant cross-reaction was seen with the low-grade toxin hordenine, but detection of tyramine was poorer, whereas the amino acid tyrosine was not detected. These assays could be applied to the analysis of simple extracts of Phalaris leaves with minimal interference. A good correspondence was observed between toxin levels by ELISA and estimates from a more tedious thin-layer chromatography method. The method has now been incorporated in a Phalaris breeding program.     

60Co-γ辐照模式下玉米中赤霉烯酮和品质的变化及对绵羊定时人工授精的影响

为研究经赤霉烯酮(ZEN)自然污染与⁶⁰Co-γ辐照处理后的玉米对绵羊定时人工授精中澳洲白种公羊、湖羊母羊的生殖性能的影响,本试验选取含ZEN玉米样本,分别用0、3、5、8、10 kGy剂量的⁶⁰Co-γ辐照,并测定辐照后玉米主要营养成分变化。然后分别将未污染玉米(Ⅰ对照组)、经10 kGy ⁶⁰Co-γ辐照的污染玉米(试验Ⅱ组)、污染玉米(试验Ⅲ组)饲喂定时人工授精的绵羊,检测澳洲白种公羊的精液质量,测定湖羊母羊的繁殖性能指标。结果表明,未经⁶⁰Co-γ辐照的玉米含ZEN 2 153 μg·kg^-1,在10 kGy ⁶⁰Co-γ辐照剂量下,玉米中ZEN降解率为83.7%;当⁶⁰Co-γ辐照剂量低于10 kGy时,玉米淀粉表面结构无变化,主要营养成分无显著变化。试验Ⅱ组澳洲白种公羊的采精量、精子活力、精子密度与试验Ⅲ组相比显著升高(P<0.05),与Ⅰ对照组相比无显著差异(P>0.05);试验Ⅲ组澳洲白种公羊第30天精子畸形率与试验Ⅱ组、Ⅰ对照组相比均显著升高(P<0.05),显微镜下观察可见双尾精子、双头精子、无尾精子等现象,其中双尾精子头部、尾部不完整;试验Ⅱ组湖羊母羊同期发情率、受胎率、产羔率和羔羊始重等指标与试验Ⅲ组相比显著升高(P<0.05),与Ⅰ对照组无显著差异(P>0.05)。综上,使用经10 kGy ⁶⁰Co-γ辐照后ZEN含量低于2 153 μg·kg^-1的玉米饲喂繁育绵羊,对绵羊定时人工授精无不利影响。本研究结果为⁶⁰Co-γ辐照模式下玉米中赤霉烯酮和品质的变化对绵羊定时人工授精的影响研究提供了理论依据。