超排山羊卵巢卵母细胞体外成熟和体外受精的研究

以ＴＣＭ１９９＋１０ｍｍｏｌ／ＬＨＥＰＥＳ＋青霉素（６ｍｇ／Ｌ）＋链霉素（５ｍｇ／Ｌ）为基础培养液（ＢＭ），再分别加入不同成分，配成５种卵母细胞成熟液：（Ａ）ＢＭ＋１０％ＥＧＳ；（Ｂ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ（２．５ｍｇ／Ｌ）；（Ｃ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２（１ｍｇ／Ｌ）；（Ｄ）ＢＭ＋１０％ＦＣＳ＋ＨＣＧ＋Ｅ２；（Ｅ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２＋颗粒细胞（１．５×１０６～３．０×１０６个／ｍＬ）。在３８．５℃、５％ＣＯ２下培养２６ｈ，排卵后第１天卵巢卵母细胞体外成熟率分别为６７．６７％（２０／３０），６０．４２％（２９／４８），８３．６７％（４１／４９）和８０．８２％（５９／７３），排卵后第５天卵巢卵母细胞，在培液Ｄ中体外成熟率为７１．４３％（４５／６３）。排卵后第１天的９８枚卵巢卵母细胞，体外成熟体外受精卵裂率为２１．４３％，２１枚２细胞胚移植５头受体获２头羔羊。研究表明，超排山羊卵巢卵母细胞经体外成熟可获得大量廉价的成熟卵母细胞，并可通过体外受精获得试管山羊     

影响玻璃化冷冻兔胚胎效果的一些因素

试验对影响玻璃化冷冻兔胚胎效果的一些因素进行探讨，以找出理想的玻璃化冷冻方法。在测试的５种玻璃化溶液中，含３５％乙二醇（ＥＧ）和１．０ｍｏｌ／Ｌ蔗糖的溶液（ＶＳ１）对胚胎的毒性最小。用ＶＳ１冷冻桑椹胚和囊胚的理想程序是：在室温下使胚胎分别在２０％ＥＧ和３５％ＥＧ中平衡２、３分钟后，移入ＶＳ１中，０．５分钟内（囊胚也可在２分钟后）投入液氮中冷冻。桑椹胚的存活率为９１．７％（３３／３６），囊胚的存活率为９７．１％（３３／３４）～９７．３％（３６／３７）。８～１６细胞胚胎的理想冷冻程序为：在室温下使胚胎在２０％ＥＧ、３５％ＥＧ中平衡２、３分钟，移入４℃的３７％ＥＧ＋１．０ｍｏｌ／Ｌ蔗糖溶液中平衡２分或１０分钟后冷冻，胚胎存活率分别为１００％（３７／３７）、８６．１％（３１／３６）。     

不同冷保保护剂在鸡精液冷冻中的作用效果分析

本实验采用一定浓度的甘油，乙二醇，二甲基亚砜（ＤＭＳＯ），二甲基乙酰胺（ＤＭＡ）作为冷冻保护剂，用含冷冻保护剂的称释法液精稀释后常温保存，观察精子活率，比较精子生存指数，并进行输精实验，发现在常温下对精子毒害作用最大的是甘油，其次是ＤＭＳＯ，而ＤＭＡ及乙二醇对精子的毒害作用最小，以一定浓度的４种冷冻保护剂将精液冷冻后观察冻活率，发现以ＤＭＡ作为冷冻保护剂，解冻后精子活率最高。在输精实验中，以ＤＭＡ     

奶牛半胚裸露冷冻保存试验

本试验研究了不同冷冻方法，半胚冷冻前不同培养时间及不同冷冻保护液对奶牛７日龄半胚裸露冷冻效果的影响，结果以１０％（Ｖ／Ｖ）甘油＋１０％（Ｗ＋Ｖ）葡聚糖（Ｔ－５００）＋０．１Ｍ蔗糖＋２０％ＦＣＳ－ＰＢＳ溶液为冷冻保护液，裸露冷冻前培养１．０小时的半胚，获得了８２．４％（１４／１７）的较高存活率。试验结果表明，半胚冷冻前培养时间及不同保护液对奶牛半胚裸露冷冻有显著影响。     

猪核移植重组胚胎的发育能力

以ＭｃＧｒａｔｈ－Ｓｏｌｔｅｒ（１９８３）提供的去（注）核方法，将猪胚胎的单个卵裂球细胞注入去核的次级成熟卵母细胞，借助电融合的方法（ＡＣ；５Ｖ，１．５ＭＨｚ，１２－１５ｓ；ＤＣ：２ｋＶ／ｃｍ，４０μｓ。电激活／融合液：０．３ｍｏｌ／Ｌ甘露醇＋－．１ｍｍｏｌ／ＬＭｇＳＯ４＋０．１ｍｍｏｌＣａｃＬ２＋７．５ｍｇ／ｍｌＣＣＢ）融入受体胞质构成重级胚，体外培养（培养液为改衣ＴＣＭ－１９９；培养条件为５％     

扩张囊胚玻璃化超低温冷冻保存及移植技术的研究

本试验探讨了对鼠扩张囊胚玻璃化冷冻保存后，提高发育率的最佳条件。玻璃化溶液是用ＰＢＳ液稀释成的３０％聚蔗糖＋０．５Ｍ蔗糖混合法，再将乙二醇（ＥＧ）调制成２０、３０及４０％（ｖ／ｖ）的溶液，即ＥＦＳ２０、３０及４０。玻璃化冷冻保存是１０、２０及２５℃温度下，将扩张囊胚直接移入ＥＰＳ溶液后投入液氮中一步法令冻保存，其中２５℃下保存后的发育率最高（８７％）。继之将扩张囊胚用１０％ＥＧ溶液经５分钟处理后，再移入ＥＦＳ４０中二步法冷冻保存，发育率高达９４％。利用二步法冷冻保存的扩张囊胚移植后，妊娠受体的产仔率为６６％（１６８／２５５），同时照组产仔率６１％（８０／１３２）相比无显著性差异（Ｐ＞０．０５）。     

种鸡精液冷冻保护剂的确定

以农大褐父母代鸡为材料，以甘油、乙二醇、ＤＭＡ和ＤＭＳＯ为主要冷冻保护剂的试验表明，乙二醇和ＤＭＡ的毒性较小，以Ａ液为稀释液，加抗冷冻剂ＤＭＡ的解冻精子活力为０．５５，且以１０％的ＤＭＡ浓度最佳，精子活力最高为０．６０，显著高于其它组（Ｐ〈０．０５）。以Ｂ液为稀释液的解冻结果表明，应用混合冷冻保护剂的保护效果较好，可获得０．４１￣０．４５的精子活力，差异显著（Ｐ〈０．０５）。     

种鸡精液冷冻保护剂的确定

本实验以农大褐父母代种鸡为材料，以甘油、乙二醇、ＤＭＡ和ＤＭＳＯ为主要冷冻保护剂的试验表明，乙二醇和ＤＭＡ的毒性较小；以Ａ液为稀释液，加抗冷冻剂ＤＭＡ的解冻精子活力为０．５５，且以１０％的ＤＭＡ浓度最佳，精子活力最高为０．６０，显著高于其它组（Ｐ＜０．０５）。以日液为稀释液的解冻结果表明，应用混合冷冻保护剂的保护效果较好，可获得０．４１～０．４５的精子活力，差异显著（Ｐ＜０．０５）。     

鸡减蛋综合征病毒DNA结合蛋白的基因结构及同源分析

从中国发病鸡群中分离的鸡减蛋综合征病毒（ＥＤＳＶ）ＡＡ－２株，经常规方法提取其病毒核酸后，构建了限制性内切酶ＰｓｔⅠ及ＨｉｎｄⅢ水解片段的基因文库。对其中ＨｉｎｄⅢ－Ｆ片段（５２．９～５８．９ｍｕ）的正反２条链的序列测定发现，其反链存在１个编码容量为３８７个氨基酸（ａａ）的开放读码框架（ｏｐｅｎｒｅａｄｉｎｇｆｒａｍｅ，ＯＲＦ），经Ｇｅｎｅｂａｎｋ／ＥＭＢＬ同源搜寻后证实其编码产物为ＥＤＳＶ的ＤＮＡ结合蛋白（ＤＢＰ）。与其他腺病毒ＤＢＰ的氨基酸序列进行同源比较，其Ｎ端同源性在１９．０％～４６．２％之间，Ｃ端同源性在２７．７％～６０．４％之间。腺病毒ＤＢＰ的３个保守序列ＣＲ１，ＣＲ２，ＣＲ３在ＥＤＳＶＤＢＰ中有较大变化，但ＥＤＳＶＤＢＰ的锌指框架（Ｚｎ２＋ｆｉｎｇｅｒｍｏｔｉｆ）却保留了对其功能必需的残基。     

白枕鹤精液冷冻技术的研究

白枕鹤（ＧＲＵＳＶ　ＩＰＩＯ）羽毛青灰，红面，体型健美优雅，具有很高的观赏价值，已被国家列为二级保护动物。对白枕鹤类的人工授精、孵化、育雏以及冷冻精液技术的研究，可促进人工饲养条件下鹤类的增殖和种群的繁衍。本研究采用Ｂｅｌｔｓｖｉｌｌｅ稀释液做为基础液，４—６％ＤＭＳＯ做为冷冻保护剂，按常规冷冻法冻精配种受胎率达４０％，此项研究填补了我国在鹤类精液冷冻技术方面的空白。     

从猝死症黄牛分离出冠状病毒

应用电镜观察，在吉林省猝死黄牛的心，肝，脾和肾中发现典型的冠状病毒颗粒，其中心，肝样品中的冠状病毒较多。用牛胎肾传代细胞（ＭＤＢＫ）分离培养该病毒获得成功，并进行了电镜负染跟踪观察和电镜超薄切片观察。该病毒呈圆形和椭圆形为主的多形性，直径７０～１６０ｎｍ有囊膜，囊膜表面有突起，突起呈间隙较宽的顶端膨大成球形的２０ｎｍ长的柄状结构，即呈鼓锤样结构。     

山羊卵母细胞体外成熟的研究

在38℃和39℃不同温度条件下培养山羊卵母细胞,结果表明:在38℃和39℃下成熟率分别为63.6%和57.3%,二者之间差异不显著,说明只要培养温度在山羊正常体温的变动范围内,对其卵母细胞的成熟率没有影响;在38℃温度条件下用10?S的M199培养液成熟率为62.5%,而10 mg/ml BSA的M199培养液成熟率为58.7%,二者之间差异不显著,说明无血清培养可以代替血清培养.     

Characterization of an intravenous lipopolysaccharide inflammation model in calves with respect to the acute-phase response

Our objective was to develop a lipopolysaccharide (LPS) inflammation model in calves to evaluate the acute-phase response with respect to the release of pro-inflammatory cytokines and acute-phase proteins, fever development and sickness behaviour. Fourteen 4-week-old male Holstein Friesian calves were included and randomly assigned to a negative control group (n = 3) and an LPS-challenged group (n = 11). The latter received an intravenous bolus injection of 0.5 μg of LPS/kg body weight. Blood collection and clinical scoring were performed at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 18, 24, 28, 32, 48, 54 and 72 h post LPS administration (p.a.). In the LPS group, the following clinical signs were observed successively: tachypnoea (on average 18 min p.a.), decubitus (29 min p.a.), general depression (1.75 h p.a.), fever (5 h p.a.) and tachycardia (5 h p.a.). Subsequent to the recovery from respiratory distress, general depression was prominent, which deteriorated when fever increased. One animal did not survive LPS administration, whereas the other animals recovered on average within 6.1 h p.a. Moreover, the challenge significantly increased plasma concentrations of tumour necrosis factor-α, interleukin 6, serum amyloid A and haptoglobin, with peaking levels at 1, 3.5, 24 and 18 h p.a., respectively. The present LPS model was practical and reproducible, caused obvious clinical signs related to endotoxemia and a marked change in the studied inflammatory mediators, making it a suitable model to study the immunomodulatory properties of drugs in future research.     

Cellular composition of granulomatous lesions in gut-associated lymphoid tissues of goats during the first year after experimental infection with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) causes lesions in naturally and experimentally infected ruminants which greatly differ in severity, cellular composition and number of mycobacteria. Morphologically distinct lesions are already found during the clinically inapparent phase of infection. The complex local host response and number of MAP were characterized at the initial sites of lesions, organized gut-associated lymphoid tissue, in experimentally infected goats. Tissues were collected at 3, 6, 9 and 12 month post-inoculation (mpi) from goat kids that had orally received 10 times 10 mg of bacterial wet mass of MAP (JII-1961). The cellular composition of lesions in Peyer's patches in the jejunum and next to the ileocecal valve was evaluated in 21 MAP-inoculated goats, where lesions were compared with unaltered tissue of six control goats. CD68⁺, CD4⁺, CD8⁺, γδ T lymphocytes, B lymphocytes and plasma cells, MHC class II⁺ and CD25⁺ cells were demonstrated by immunohistochemistry in serial cryostat sections.At 3 mpi, extensive granulomatous infiltrates predominated, consisting of numerous epitheloid cells admixed with many CD4 and γδ T lymphocytes. Only single MAP were detected. This indicates a strong cellular immune reaction able to control MAP infection. γδ T lymphocytes were markedly increased in this type of lesion which may reflect their important role early in the pathogenesis of paratuberculosis. At 9 and 12 mpi, divergent lesions were observed which may reflect different outcomes of host–pathogen interactions. In five goats, minimal granulomatous lesions were surrounded by extensive lymphoplasmacytic infiltrates and no MAP were detected by immunohistochemistry. This was interpreted as effective host response that was able to eliminate MAP locally. In three goats, decreased numbers of lymphocytes, but extensive granulomatous infiltrates with numerous epitheloid cells containing increased numbers of mycobacteria were seen. This shift of the immune response resulted in uncontrolled mycobacterial multiplication. Focal and multifocal circumscribed granulomatous infiltrates of mainly epitheloid cells may represent sites of new infection, since they were observed in goats at all times after inoculation. Their presence in goats with minimal granulomatous lesions surrounded by extensive lymphoplasmacytic infiltrates may indicate that despite the local clearance, the infection may be perpetuated.The complex cellular immune reactions postulated for the pathogenesis of paratuberculosis were demonstrated at the local sites of infection. These early host–pathogen interactions are most likely essential for the eventual outcome of the MAP infection.     

Detection of circulating autoantibodies using living keratinocyte staining on MCA-B1 method in dogs with pemphigus foliaceus

In this study, we compared the sensitivity and specificity of three immunofluorescence techniques used to detect circulating autoantibodies in dogs with pemphigus foliaceus (PF); living keratinocyte staining on a canine keratinocyte cell line, MCA-B1, indirect immunofluorescence (IIF) on canine lip and IIF on bovine esophagus. Sera from canine PF cases were positive in four out of 27 dogs (14.8%) using living keratinocyte staining on MCA-B1 cells method, and five (18.5%) and eight sera (29.6%) using IIF on canine lip and bovine esophagus methods, respectively. By contrast, none of the 31 sera from dogs with non-pemphigus dermatoses reacted with MCA-B1 cells, whereas two (6.5%) as well as five sera (16.1%) obtained from those dogs showed positive reactivity with IIF on canine lip and bovine esophagus, respectively. Our results suggest that, although it exhibits the least sensitivity, the positive reactivity obtained by living keratinocyte staining on MCA-B1 cells can support the diagnosis of canine PF.     

鸡端脑和下丘脑向面神经腹侧核的传出性纤维联系—HRP逆行追踪法

将２０％ＨＲＰ水溶液注射于鸡的面神经腹侧核，逆行追踪支配该部的有关主同位神经核团。结果于端，下丘脑和脑干内出现标记细胞，端脑内的标记细胞较多地出现于古纹状体，少量出现于旧纹状体腹侧与枕中脑束之间的区域，两群标记细胞形态上明显不同。下丘脑的标记细胞较多地出现于下丘脑后区，室旁核出现少量，个别散在于下丘脑其他区域。     

Use of Fourier-transform infrared spectroscopy to quantify immunoglobulin G concentration and an analysis of the effect of signalment on levels in canine serum

Deficiency in immunoglobulin G (IgG) is associated with an increased susceptibility to infections in humans and animals, and changes in IgG levels occur in many disease states. In companion animals, failure of transfer of passive immunity is uncommonly diagnosed but mortality rates in puppies are high and more than 30% of these deaths are secondary to septicemia. Currently, radial immunodiffusion (RID) and enzyme-linked immunosorbent assays are the most commonly used methods for quantitative measurement of IgG in dogs. In this study, a Fourier-transform infrared spectroscopy (FTIR) assay for canine serum IgG was developed and compared to the RID assay as the reference standard. Basic signalment data and health status of the dogs were also analyzed to determine if they correlated with serum IgG concentrations based on RID results.Serum samples were collected from 207 dogs during routine hematological evaluation, and IgG concentrations determined by RID. The FTIR assay was developed using partial least squares regression analysis and its performance evaluated using RID assay as the reference test. The concordance correlation coefficient was 0.91 for the calibration model data set and 0.85 for the prediction set. A Bland–Altman plot showed a mean difference of −89 mg/dL and no systematic bias. The modified mean coefficient of variation (CV) for RID was 6.67%, and for FTIR was 18.76%.The mean serum IgG concentration using RID was 1943 ± 880 mg/dL based on the 193 dogs with complete signalment and health data. When age class, gender, breed size and disease status were analyzed by multivariable ANOVA, dogs <2 years of age (p = 0.0004) and those classified as diseased (p = 0.03) were found to have significantly lower IgG concentrations than older and healthy dogs, respectively.     

食用菌菌糠营养价值及利用途径的研究

菌糠作为饲料或饲料添加剂,可以替代一部分饲料用粮及其他营养元素。通过对菌糠饲料的营养价值、制作工艺、在养殖业的应用以及发展前景的论述。指出菌糠的营养相当于常规麦麸类饲料,可作为新型饲料资源应用于养殖业中,以降低饲料成本。     

奶牛不同品质的粗饲料日粮中添加不同浓度的异戊酸对其培养液VFA和BCP产量的影晌研究

本试验以荷斯坦奶牛为试验动物.用体外批次培养试验法.选择瘤胃调控剂异戊酸(1.25、2.50、5.00、10.00mg/g).以奶牛3种不同品质的粗饲料日粮(分别以苜蓿干草、羊草、玉米秸秆为主的粗饲料日粮)为底物进行了体外培养试验.主要研究了奶牛3种不同品质的粗饲料日粮中分别添加不同浓度的异戊酸对其培养液VFA和BCP浓度的影响.其结果为:(1)试验组培养液总VFA和BCP各时间点平均值显著高于对照组(P<0.05).试验组培养液乙酸、丙酸、丁酸各时间点平均值均高于对照组.(2)在3种不同品质的粗料日粮中,异戊酸对以玉米秸杆为主的粗饲料日粮体外发酵效果作用更明显.(3)在以苜蓿为主的粗饲料日粮中.异戊酸最适添加量为5.00mg/g;在以羊草为主的粗饲料日粮中.异戊酸最适添加量为5.00mg/g;在以玉米秸杆为主的粗饲料日粮中,异戊酸最适添加量为10.00mg/g.     

舒安对犬的麻醉效果观察

以新近研制的中小动物全身麻醉剂－舒安为实验药品，对１０只实验犬进行麻醉效果观察，并试用于９７例犬的手术麻醉，实验结果表明，舒安对犬具有良好的麻醉作用，毒副作用小，有较强的镇静、镇痛和肌松作用，对体温、脉搏、呼吸等生理功能影响小，恢复快。麻醉诱导平衡而迅速，苏醒期无明显兴奋现象。腺体分泌减少，呕吐机率低。