黑猩猩发情周期观察和促黄体生成素测定

为准确掌握雌性黑猩猩排卵时间以利于人工授精,自2008年1月至2009年6月对长春市动植物公园1只成龄雌性黑猩猩发情表现进行观察,记录发情周期天数,并用不孕检测试纸测定促黄体生成素(LH)的分泌情况。结果显示:黑猩猩发情周期平均35.6 d。跟踪测试黑猩猩的3个发情周期尿液中的LH分泌水平,结果为LH峰值在40~60之间,且峰值出现在发情周期的第21天左右。     

Effect of Kisspeptin on Regulation of Growth Hormone and Luteinizing Hormone in Lactating Dairy Cows

Kisspeptin (KP),a neuroendocrine regulator of reproduction,is hypothesized to be an integrator of metabolism and hormones critical to the regulation of reproduction.Lactation is associated with enhanced growth hormone (GH) responsiveness and reduced fertility.Our study was designed to determine the effects of lactation on KP-stimulated GH and luteinizing hormone (LH) secretion.Five nonlactating and five lactating dairy cows were used in the study.Experiments were conducted with lactating cows at weeks 1,5 and 11 after parturition.The experimental treatments (saline and KP [ 100 and 400 pmol/kg body weight] ) were given intravenously and blood was collected and plasma was stored until later assay to determine concentrations of GH,LH,progesterone and non-esterified fatty acids.We found that neither dose of KP stimulated an increase in GH secretion.The low dose of KP increased (P ＜0.05)LH concentrations only in lactating cows.The higher dose of KP elicited an increase in circulating LH concentrations in both lactating and non-lactating cows.The lower dose of KP increased ( P ＜ 0.05 ) the area under the curve for LH only in cows during week 5 of lactation,and the area under the curve of LH following the highest dose of KP was greater ( P ＜ 0.05 ) in cows during week 5 of lactation than that for the other groups of cows.In summary,lactation status and stage of lactation did not change the sensitivity of the GH system to KP.However,an effect of stage of lactation on KP-stimulated LH secretion was detected in the dairy cows.Study of the KP system during lactation in dairy cows may provide critical insights into the mechanisms for lactation-associated changes in the reproductive axis.     

发情周期不同阶段牦牛子宫中黄体生成素受体的表达变化

为研究发情周期不同阶段牦牛子宫中黄体生成索受体(LHR)的定位及表达变化,笔者利用免疫组织化学SP法分别检测发情期、发情后期、间情期和发情前期牦牛子宫中LHR的表达,并进行光密度值分析.结果表明,LHR免疫阳性产物在牦牛子宫腺上皮细胞、基质细胞、血管内皮细胞、血管平滑肌细胞和肌层平滑肌细胞中均有表达;腺上皮细胞、基质细胞和肌层平滑肌细胞中LHR在发情前期和发情期表达最弱,发情后期表达增加,间情期表达最强(P＜0.05);子宫内膜血管平滑肌细胞中LHR的表达在发情期最强,间情期最弱(P＜0.05);血管内皮中LHR在发情期和发情前期表达很强,发情后期和间情期显著下降(P＜0.05).结果表明LHR参与了发情周期不同阶段牦牛子宫功能变化的调控.     

促黄体素受体在母畜生殖道的分布与表达

在多种动物包括灵长类动物、猪、牛、火鸡和人,子宫内都发现有促黄体素(LH)或人绒毛膜促性腺激素(hCG)及其mRNA的结合位点.牛、猪在整个发情周期子宫内都存在LH受体(LHR)蛋白及其mRNA,并在黄体期表达最高.LH通过激活腺苷酸环化酶和磷脂酶C信号途径,增加前列腺素合成酶(PGSH),也称环加氧酶(COX)的活性,从而促进前列腺素(PG)的合成.牛、猪、羊在发情周期的15 d左右PGF2α升高,这与子宫内LHR的活化有关.在不同种动物的输卵管、子宫内膜、子宫肌层和子宫颈,LHR的释放呈现出动态特征,表明LH在发情周期和胚胎附植的分子自分泌-旁分泌调节中可能具有重要的作用.     

促卵泡素受体和促黄体素受体在乏情期和怀孕期牦牛卵巢中的表达

应用免疫组织化学技术和图象分析方法对乏情期、怀孕期牦牛卵巢的促卵泡素受体(FSHR)、促黄体素受体(LHR)的表达特点进行了研究。结果表明,牦牛卵巢的皮质、髓质、颗粒层和膜内层、黄体中都分布有FSHR和LHR。且牦牛卵巢皮质、髓质中的FSHR光密度值在乏情期显著大于怀孕期(P0.05);怀孕期无黄体侧卵巢颗粒层和膜内层中FSHR光密度值均大于乏情期和怀孕期有黄体侧(P0.05)。怀孕期有黄体侧卵巢皮质处LHR光密度值最大,乏情期次之,怀孕期无黄体侧最小,各组间差异显著(P0.05);在怀孕期无黄体侧卵巢髓质处LHR光密度值显著小于怀孕期有黄体侧和乏情期(P0.05);在乏情期、怀孕期无黄体侧和怀孕期有黄体卵巢颗粒层和膜内层处LHR光密度值之间差异均不显著(P0.05)。表明牦牛卵巢中FSHR和LHR随着生殖阶段的不同而变化。     

Radioimmunoassay of Bovine,Ovine and Porcine Luteinizing Hormone with a Monoclonal Antibody and a Human Tracer

Forsberg, M., R. Tagle, A. Madej, J.R. Molina and M.-A. Carlsson. Radioimmunoassay of bovine, ovine and porcine luteinizing hormone with a monoclonal antibody and a human tracer. Acta vet. scand. 1993, 255-262.– A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human ¹²⁵ ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH and oLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 µg/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar.     

Recombinant Equine Luteinizing Hormone Stimulates Production of Progesterone from Murine Leydig, Ovine Small Luteal, and Equine Granulosal Cells

Recombinant equine luteinizing hormone (reLH) was evaluated for its ability to stimulate production of progesterone in cell lines from three species including murine Leydig tumor (MA-10), equine granulosal, and ovine small luteal cells (SLC). The response to reLH was compared with that obtained with human chorionic gonadotropin (hCG), equine chorionic gonadotropin, ovine luteinizing hormone, and equine luteinizing hormone (eLH). Media were collected hourly for 6 hours and assayed for progesterone content through radioimmunoassay. In MA-10 cells, production of progesterone was stimulated above baseline by reLH and hCG (P < .05). Ovine SLC responded to treatment with eLH, reLH, ovine luteinizing hormone, and hCG by increasing production of progesterone above that stimulated by vehicle control (P < .05). Production of progesterone in equine granulosal cells was maximally stimulated by treatment with hCG (P < .05), followed by reLH and eLH (P < .05). In conclusion, reLH elicited a progesterone response in MA-10, ovine SLC, and equine granulosal cells. Thus, reLH stimulates the production of progesterone in cell lines from three species.     

鹌鹑卵泡发育过程中颗粒细胞黄体生成素受体mRNA的表达

用Ｎorthern杂交的方法研究了鹌鹑排卵泡内颗粒细胞黄体生成素受体（ＬＨＲ）mＲＮＡ的表达。在预计排卵前２０h和３h分别取出最大的３个卵泡（Ｆ１、Ｆ３卵泡）以及小黄卵泡（ＳＹＦ）,剥离颗粒层提取出总ＲＮＡ,并经变性凝胶电泳后将ＲＮＡ转移到滤膜上。杂交所用的探针是用特异的引物经反转录一多聚栈链式反应（ＲＴ－ＰＣＲ）扩增出编码ＬＨＲcＤＮＡ的细胞外区（ＥＣ）和跨膜区（ＴＭ）cＤＮＡ。结果表明,颗粒细胞ＬＨＲmＲ     

黄体生成素和褪黑素受体在不同日龄雏鸵鸟睾丸内的分布研究

采用免疫组织化学SABC法对1 d、30 d和90 d雏鸵鸟睾丸内黄体生成素(1uteinizing hormone,LH)和褪黑素受体(melatonin receptor,MR)的分布进行定位,结果显示:LH阳性细胞胞质或胞膜出现棕黄色,胞核无阳性反应;MR阳性反应颗粒呈棕褐色或棕黄色,位于胞质、胞膜或胞核,呈散在...     

促黄体素对牛卵母细胞体外核成熟的影响

为了研究促黄体素(luteinizing hormone,LH)在牛卵母细胞成熟培养体系中的时间依从性,试验通过向牛卵母细胞体外成熟培养的不同时间段添加LH来观察其核成熟的情况。首先用无LH的基础成熟液I和添加LH的成熟液Ⅱ分别培养,在成熟的不同时间点(3、6、9h)用醋酸地衣红染色观察各自的生发泡破裂率。然后在体外成熟培养的不同时间段(0～3、0～6、0～9、0～24h)添加LH,24h后统计各自的第一极体率。结果表明,添加LH组在6h的生发泡破裂率显著低于对照1组(P0.05),24h添加LH组的第一极体率显著高于对照2组(P0.05)。因此,添加LH能延迟牛卵母细胞的核成熟,成熟培养中24h全程添加LH较不添加组能显著提高核成熟率。     

重度联合免疫缺陷（SCID）小鼠排卵,胚胎发育能力及妊娠母体激素动态

为探讨先天性Ｔ－Ｂ淋巴细胞联合缺陷对生殖功能的影响,用正常ＢＡＬＢ／ｃ小鼠作对照,对重度联合免疫缺陷（ＳＣＩＤ）小鼠的排卵,胚胎（胎儿）发育能力及妊娠体外周血浆促黄体素（ＬＨ）和孕酮（Ｐ）水平的变化进行了研究。首先,观察了妊娠期、窝产仔数,发现ＳＣＩＤ小鼠大多在妊娠第２０天（检栓当天为第１天）凌晨分娩,平均窝产卫数为３．８６±１．６７）只,ＢＡＬＢ／Ｃ小鼠多在妊娠第２０天较晚或第２１天凌晨分娩,平     

促黄体素受体研究进展

促黄体素 (L uteinizing hormone re-cepter,L HR)是哺乳动物生殖调控的重要激素 ,生理作用是通过其受体介导的。近几年来 ,L HR的研究已取得了重大的进展。文章对 L HR结构、受体的配体结合域、激素 -受体相互作用、信号转导与 G蛋白偶联、受体糖基化及脱敏、受体基因结构与调节、受体表达及受体突变对生殖机能的影响等方面进行了综述 ,以促进 L HR在生殖调控和疾病治疗中的进一步研究     

Enzyme Immunoassays for the Determination of Ovine LH and FSH

The development of competitive enzyme immunoassays for ovine plasma LH (oLH) and FSH (oFSH) is described. Standards and plasma samples were preincubated with diluted antiserum to oLH or oFSH and the reacted solution (100 μl per well) was transferred to plates previously coated with oLH or oFSH, respectively. The second antibody used was anti‐rabbit IgG horseradish peroxidase. The measuring range was 0.39–50 ng/ml for each hormone and the 50% relative binding sensitivity was 9 ng/ml for oLH. The respective value for oFSH was 3.5 or 34 ng/ml with different hormone and antibody preparations used for the assay. The enzyme immunoassays were used to determine oLH and oFSH levels in plasma from ewes of two breeds during the oestrous cycle. The assays detected the first FSH surge coincident with the LH surge, the second FSH surge about 24 h later and the periodic fluctuations of FSH concentrations during the luteal phase of the oestrous cycle. These enzyme immunoassays are an efficient and economic alternative to the established radioimmunoassays (RIA) for oLH and oFSH.     

黑猩猩月经周期与尿液促黄体生成素的监测

月经周期与尿液促黄体生成素（LH）的监测是预测黑猩猩排卵期的基本方法,本研究对1只雌性黑猩猩（呼名蔓丽）的月经周期和性皮肿胀情况进行了长达3 a的观察和记录,并采用人用的LH半定量检测试纸测定了黑猩猩部分月经周期尿液中促黄体生成素的含量。结果显示：蔓丽的月经周期平均为34.6±3.1 d,性皮肿胀最充分时的持续时间平均为5.8±2.3 d,尿液中LH水平与性皮肿胀开始消退的时间恰好吻合。本研究采用的人用LH半定量试纸测定黑猩猩尿液LH值,能够反映月经周期内尿液LH值的变化规律。     

激素处理产后母牛对产犊间隔及卵巢状态的影响研究

为了确定促黄体素释放激素A2(LRH-A2)和氯前列烯醇对缩短奶牛产犊间隔的影响,选择产后适宜的激素处理时间,选用51头年产奶量在5 000 kg~6 000 kg的经产荷斯坦奶牛,根据产后的时间分为对照组、试验Ⅰ组(产后2周)、试验Ⅱ组(产后20 d~40 d)、试验Ⅲ组(产后40 d~60 d),分别用LRH-A2、氯前列烯醇或LRH-A2 氯前列烯醇进行处理。并在每组随机选择3头,用B超观测处理前后奶牛的卵巢状态变化。结果表明,奶牛产后2周用LRH-A2处理,有利于缩短产犊间隔。     

褪黑激素ELISA方法的建立

将褪黑激素（MLT）与牛血清白蛋白（BSA）连结的复合物（MLT：BSA＝12：1）免疫5只雄性新西兰大耳兔，收集高效价血清，并提纯抗体。在96孔微量反应板上，以1：10000的抗体包被，将MLT与酶标MLT先后加入到板中竞争结合抗体，制定标准曲线，建立了MLT酶联免疫测定法（MLT－ELISA）。此方法灵敏度为17．31pg,板内变异系数8．1％，板间变异系数15．4％，用建立的此法测定夏至时考力代羊昼夜血样，测定结果跟国外报道的基本一致；用放射免疫法检测同一批血样，也获得类似结果，从而表明此方法可信。     

3个品种绵羊超数排卵处理后卵巢上促黄体素受体表达差异的研究

本研究旨在探讨在具有不同超数排卵效果的绵羊品种中,在进行超排处理后其卵巢上的促黄体素受体(LH receptor,LHR)分布情况,为进一步研究二者的相关性以及据此为改进超排效果奠定基础.根据超数排卵效果的优劣依次选择3个我国地方绵羊品种:小尾寒羊、白滩羊和乌珠穆沁羊.在常规超排最后一次注射FSH后的8、16和24 h分别各取3只绵羊的卵巢,采用免疫组织化学法观察3个品种绵羊卵巢上LHR的分布情况.结果表明:LHR存在于3个地方品种绵羊卵巢有腔卵泡的颗粒细胞和膜细胞上,但在早期卵泡上不表达;各品种绵羊的近成熟卵泡上LHR阳性细胞率都显著高于相应的次级卵泡(P＜0.05);3品种绵羊近成熟卵泡中LHR的阳性变化率存在显著差异,从高到低依次为小尾寒羊＞白滩羊＞乌珠穆沁羊(P＜0.05).籍此可以得出以下结论:LH对绵羊早期卵泡发育的诱导可能不需要通过其特异性受体起作用;3品种绵羊近成熟卵泡中LHR的阳性变化率与超排效果是一致的,表明超数排卵的效果可能与绵羊卵巢上的LH受体分布存在一定的联系.     

Validation of a Commercial Enzyme Immunoassay for Detection of Clostridium difficile Toxins in Feces of Horses with Acute Diarrhea

Background: Clostridium difficile infection (CDI) is a recognized cause of colitis in the horse. Identification of its toxins is important for management of individual cases and for prevention of transmission and zoonosis. In humans, CDI diagnosis is performed with enzyme immunoassays, none of which have been validated for horses. Hypothesis/Objectives: (1) Establish which test for CDI diagnosis was more frequently used by diagnostic laboratories, (2) determine the identified test's performance, sensitivity, and specificity, and (3) validate its use in diarrheic horses. Animals: Samples were obtained from 72 horses presented with acute diarrhea and hospitalized at the Ontario Veterinary College, University of Guelph. Methods: A survey was conducted to establish which of the tests for CDI diagnosis in horses is most commonly used throughout North America. A questionnaire was sent to all laboratories registered in the Veterinary Infection Control Society and the American Association of Veterinary Laboratory Diagnosticians. The performance of the test was evaluated by comparison to a cell cytotoxicity assay (CTA), the accepted Gold Standard for C. difficile toxin detection. Results: The Techlab C. difficile Tox A/B II ELISA was the most frequently used test. Compared with the CTA, no significant difference was observed, and a good level of agreement (93%) was obtained. The diagnostic performance of the ELISA test was adequate (84% sensitivity and 96% specificity). Conclusions and Clinical Importance: Results demonstrate that the Techlab C. difficile Tox A/B II ELISA is a reliable, adequate, and practical tool for identification of C. difficile toxins in horse feces.     

羊弓形虫病间接ELISA诊断方法的建立

为比较5种弓形虫抗原的诊断效果,建立实用的羊弓形虫病诊断方法,本研究制备了弓形虫速殖子全虫抗原和4个重组蛋白(GRA1、MIC3、MAG、BAG1),正交试验比较其免疫反应性,从中筛选出相对优势抗原并建立羊弓形虫病间接ELISA(iELISA)诊断方法,用于检测弓形虫特异的IgG抗体。研究结果表明：除BAG1外上述4种抗原均具有较好的免疫反应性,综合考虑选择致密颗粒蛋白1(GRA1)作为诊断抗原建立了GRA1-iELISA,其检测弓形虫抗体灵敏度大于1∶100(血清稀释度),且不与羊莫氏巴贝斯虫、吕氏泰勒虫、捻转血毛线虫阳性血清发生交叉反应,特异性良好;运用建立的方法和改良凝集试验分别对采自山羊屠宰场的80份血清样品进行检测,测得的弓形虫感染阳性率分别为28.75%(23/80)和31.25%(25/80),二者总符合率为82.5%。本研究建立的ELISA检测方法对羊弓形虫病的诊断以及商业化试剂盒的开发具有重要参考价值。     

酶免疫法测定磺胺喹恶啉残留的研究：磺胺喹恶啉抗体的制备

磺胺喹恶啉（ＳＱ）常用于鸡球虫病的防治，但它有一定的毒性，因此该化合物在肉类食品中的残留日益受到人们的关注。本试验用戊二醛法将ＳＱ与牛血清白蛋白（ＢＳＡ）联接形成完全抗原，用紫外扫描对其进行鉴定，并测定其ＳＱ与ＢＳＡ的分子比为１２．１∶１。随后将此抗原免疫家兔制备高效价的抗ＳＱ血清，并用ＥＬＩＳＡ法和琼脂扩散法对其进行鉴定。