天祝白牦牛肾组织成纤维细胞系的建立与生物学特性研究

采集天祝白牦牛胚胎肾组织,用胰蛋白酶热消化法制备原代细胞,通过差速消化和差速贴壁法继代培养和细胞纯化,扩增至F3代后冷冻保存,并对复苏细胞的形态、活力、生长曲线、荧光蛋白质粒转染表达、核型以及乳酸脱氢酶同工酶等生物学特性进行了分析。结果显示,原代和传代细胞生长形态良好,群体倍增时间为27.2 h;染色体2n=60;二倍体为74%,占主体;乳酸脱氢酶同工酶电泳图谱有明显特征,LDH5浓度较高,活性较强;外源质粒在该细胞中能进行复制和表达;细菌、真菌、病毒、支原体检测呈阴性。表明本研究已成功建立天祝白牦牛肾组织成纤维细胞系,该细胞系的建立,使天祝白牦牛这一国家重要种质资源在细胞水平上得以保存,也为基因组文库和体细胞克隆等研究提供了理想的生物材料。     

青海大通马耳缘组织成纤维细胞系的建立及生物学特性研究

为了建立青海大通马耳缘组织成纤维细胞系,使青海大通马这一重要种质资源在细胞水平上得以保存,试验采集青海大通马耳缘组织用组织块法制备原代细胞,通过差速消化法和差速贴壁法纯化细胞,扩大培养至第3代用液氮保存,细胞复苏后进行了活力、形态、生长曲线、微生物污染、核型及荧光蛋白质粒转染表达等特性研究。结果表明:大通马耳缘组织原代和传代细胞呈成纤维型,生长良好,最大增殖浓度为4.27×105/mL,倍增时间为31.02 h;细菌、真菌、病毒、支原体检测呈阴性;染色体2n=64,二倍体率为80%,占主体;红色荧光蛋白质粒在该细胞中能进行复制和表达。     

岷县黑裘皮羊裘皮品质研究

本文研究了岷县黑裘皮羊不同年龄的被毛特征和产毛性能。对岷县黑裘皮羊的裘皮类型、裘皮色泽和光泽、毛股结构、花弯及花型、裘皮面积进行了分析,对岷县黑裘皮羊裘皮品质鉴定和羊分级进行了探讨。岷县黑裘皮羊品质鉴定一般进行三次;岷县黑裘皮羊的分级根据岷县黑裘皮羊体型外貌、体尺大小、公母羊体重、毛股自然长度、被毛颜色等指标,将岷县黑裘皮羊分成一级、二级、三级和等外。分析了岷县黑裘皮羊裘皮产业存在的问题并提出了对策。     

岷县黑裘皮羊产肉性能及肉品质研究

本试验通过屠宰测定和对屠宰羊背部肌肉、腹部肌肉、腿部肌肉作为肉质常规指标测定和氨基酸测定样品。通过测定,生长期岷县黑裘皮羊平均宰前活重、胴体重、净肉重均低于贵德黑裘皮羊、托克逊黑羊等国内黑羊品种;岷县黑裘皮羊羊肉中含有18种氨基酸,显现出较高的营养价值。粗脂肪含量腹部17.42%、腿部为14.87%、背部13.92%;表明岷县黑裘皮羊肉是一种口感鲜美的滋补食品。逐步建立岷县黑裘皮羊本品种选育体系,综合提高黑裘皮羊的生产性能,充分发挥岷县黑裘皮羊在当地养羊业中的作用。     

银狐垂体细胞系构建研究

分别采用组织块法和消化法2种接种方法进行银狐垂体细胞系构建研究。在消化法中使用不同浓度的胰蛋白酶来检验效果,同时在原代培养期使用反复差速贴壁法纯化,传代培养期结合使用反复差速贴壁以及两步消化法进行处理。结果表明：组织块法接种后成纤维细胞生长明显,原代培养后期成纤维细胞已为主要细胞;降低胰蛋白酶浓度能降低对细胞损伤,但消化时间会增长;采用消化法接种在抑制成纤维细胞生长方面效果好于组织块法。纯化效果上,结合反复差速贴壁法与两步消化法处理后获得了较好的效果,在原代培养时期能较好去除成纤维细胞并通过持续使用该方法可在传二代后获得较高纯度的细胞,纯度能达到80%以上,能够建立细胞系。     

银狐垂体细胞系构建研究

分别采用组织块法和消化法2种接种方法进行银狐垂体细胞系构建研究。在消化法中使用不同浓度的胰蛋白酶来检验效果,同时在原代培养期使用反复差速贴壁法纯化,传代培养期结合使用反复差速贴壁以及两步消化法进行处理。结果表明:组织块法接种后成纤维细胞生长明显,原代培养后期成纤维细胞已为主要细胞;降低胰蛋白酶浓度能降低对细胞损伤,但消化时间会增长;采用消化法接种在抑制成纤维细胞生长方面效果好于组织块法。纯化效果上,结合反复差速贴壁法与两步消化法处理后获得了较好的效果,在原代培养时期能较好去除成纤维细胞并通过持续使用该方法可在传二代后获得较高纯度的细胞,纯度能达到80%以上,能够建立细胞系。     

贵德黑裘皮羊引种适应性观察及杂交效果试验

贵德黑裘皮羊引入到甘肃岷县,在放牧加补饲饲养条件下,各生理指标正常,各月龄体重、主要体尺等指标与引入地接近,其生长发育、抗病能力、繁殖性能、耐粗饲等方面表现良好。贵×岷F1和岷县黑裘皮羊相比较,羊的初生重、1月龄、6月龄、1岁龄平均体重,公羔分别比岷县黑裘皮羊公羔提高0.11kg、0.67kg、0.97kg和1.33kg;母羔分别比岷县黑裘皮羊母羔提高0.08kg、0.40kg、1.21kg和.1.65kg。4月龄、6月龄的贵×岷F1羔羊在体斜长、胸围、体高方面都有所提高,数据表明,贵×岷F1杂交一代性能好于岷县黑裘皮羊,在甘肃省岷县生态条件下可以生存、繁殖和发展,并可用于杂交改良岷县黑裘皮羊,提高岷县黑裘皮羊的生产性能。     

岷县黑裘皮羊遗传资源保护与开发利用对策

岷县黑裘皮羊是我国优良的地方绵羊品种。为有效保护和开发利用这一地方种群资源,本文阐述了岷县黑裘皮羊的形成历史以及在外貌特征、生长发育、繁殖性能、肉用性能方面的特征特性,并根据岷县黑裘皮羊品种现状及产区实际,提出了岷县黑裘皮羊保种及开发利用对策。     

三角城藏羊红细胞乳酸脱氢酶同工酶酶谱

采用聚丙烯酰胺凝胶电泳(PAGE)法对83只三角城藏羊的红细胞乳酸脱氢酶(LDH)同工酶酶谱进行分析。结果发现:①红细胞LDH同工酶总共有11条区带;②第1区带的电泳迁移率为54.3％,第11区带的电泳迁移率为25.0％;③红细胞LDH有两种电泳表型,78只羊为LDHⅠ型,占93.98％,5只羊为LDHⅡ型,占6.02％。     

放牧补饲条件下岷县黑裘皮羊饲养效果观察

岷县黑裘皮羊产区自然条件严酷,饲养方式落后,养殖效益偏低。为了改变传统的饲养方式,建立适合当地生态条件的饲养模式,提高黑裘皮羊生产水平和养殖效益,开展了黑裘皮羊放牧补饲试验。试验结果表明岷县黑裘皮羊经短期放牧补饲饲养,增重效果明显,但增收效益不是十分显著。     

岷县黑裘皮羊微卫星遗传多样性分析

试验旨在分析岷县黑裘皮羊群体内遗传多样性,筛选出理想的遗传标记,为岷县黑裘皮羊的选育保种提供理论依据。选取144只岷县黑裘皮羊,颈静脉采血并提取DNA,对24对微卫星引物进行PCR扩增,用毛细管电泳技术进行基因分型,计算其等位基因数、等位基因大小及频率、有效等位基因数、杂合度和多态信息含量。结果显示,24个位点共检测到210个等位基因,平均等位基因数为8.75;群体等位基因频率为0.0104~0.7396,等位基因片段大小为97~285 bp;有效等位基因数为1.7581~8.2433个;群体平均观测杂合度为0.4839;平均期望杂合度为0.6959;平均多态信息含量为0.6527。其中MAF70位点等位基因数、有效等位基因数、期望杂合度和多态信息含量最高;OarFCB128位点观测杂合度最高;OarAE129位点等位基因数最少,SRCRSP9位点期望杂合度、多态信息含量最低;OarFCB304位点观测杂合度最低。Hardy-Weinberg平衡分析结果表明,所选位点中有19个处于非平衡状态。结果表明,岷县黑裘皮羊的遗传背景复杂,群体内遗传多样性丰富,可为其遗传资源的评估和选育保种工作提供理论依据。     

Establishment and characterization of a fibroblast cell line derived from Mongolian sheep

The Mongolian sheep ear marginal tissue fibroblast cell line (MSF32) from 32 samples was successfully established by using primary explants technique and cell cryoconservation technology. MSF32 cells were adherent, with a population doubling time of 28.2 h. Chromosome analysis showed that >90.2% of cells were diploid (2n = 54) prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the MSF22 cells had no cross‐contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses and mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP‐N3, pEGFP‐C1, pECFP‐N1, pECFP‐mito, pDsRed1‐N1 and pEYFP‐N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 12.3% and 63.3%. Every index of the MSF32 cell line meets all the standard quality controls of American Type Culture Collection (ATCC). Not only has the genetic resources of the Mongolian sheep been preserved at the cell level, but also valuable materials had been provided for genome, postgenome and somacloning research.     

甘肃地方绵羊品种微卫星遗传多样性与系统发育分析

通过对甘肃省6个地方绵羊品种(240只个体)15个微卫星座位的等位基因的研究,结果共发现了179个等位基因。其中在藏羊中发现的等位基因数最多(136个),兰州大尾羊最少(94个)。多态信息含量、期望杂合度和观察杂合度的数据表明:在研究的6个绵羊品种中藏羊和甘肃高山细毛羊的遗传多样性较丰富,而兰州大尾羊和岷县黑裘皮羊的遗传多样性较低。DA遗传距离和D s遗传距离构建的NJ系统发育树均表明:兰州大尾羊、蒙古羊和滩羊具有较近的亲缘关系,预示这3个品种可能具有相同的原始祖先。而甘肃高山细毛羊与其他5个甘肃地方品种的遗传距离较远,另为一类。     

Activity of serum creatine kinase, lactate dehydrogenase and LD isoenzymes in piglets affected with congenital tremor: a case report.

The activities of serum creatine kinase (CK), lactate dehydrogenase (LD) and LD isoenzymes were studied in 14 Prestice black pied pigs from a herd affected with congenital tremor. Mean CK activity was 19.57 +/- 3.56 mu kat.l-1 for 6 adult pigs, and it was 21.03 +/- 1.33 mu kat.l-1 and 20.42 +/- 1.23 mu kat.l-1 for the affected (n = 5) and control (n = 3) piglets, respectively. No significant differences were demonstrable between the groups in CK activity. Total serum LD and LD-4 as well as LD-5 isoenzyme activities were higher in sows. Piglets affected with congenital tremor showed an increase in total LD enzyme and LD-5 isoenzyme activity. It is concluded that no relationship exists between congenital tremor and serum CK activity in piglets. At the same time, there is a positive relationship between congenital tremor and significantly (P < 0.01) elevated LD enzyme and LD-5 isoenzyme activity. The results allow us to suggest that total lactate dehydrogenase and LD-5 isoenzyme activities could be used as biological markers of congenital tremor in piglets.     

Variations in serum sorbitol dehydrogenase, aspartate transaminase, and isoenzyme 5 of lactate dehydrogenase activities in horses given carbon tetrachloride

Seven horses were given 0.5 mg of carbon tetrachloride/kg of body weight via a nasogastric tube. Subsequent hepatocellular damage was monitored by serum enzyme determinations of sorbitol dehydrogenase, isoenzyme 5 of lactate dehydrogenase, and aspartate transaminase activities. Creatinine kinase activity was evaluated as an indicator of muscle cell damage. Sorbitol dehydrogenase, isoenzyme 5 of lactate dehydrogenase, and aspartate transaminase activities were significantly (P less than 0.05) increased by 24 hours after carbon tetrachloride administration. Isoenzyme 5 of lactate dehydrogenase and sorbitol dehydrogenase activities returned to baseline several days before aspartate transaminase activity returned to baseline. Creatine kinase activity remained unchanged.     

Heat stability of serum lactate dehydrogenase and its isoenzymes in young and adult cattle and sheep. Evaluation of a relative heat stability test and serum determination of alpha-hydroxybutyrate dehydrogenase in diagnostic work

The heat stability of lactate dehydrogenase (LDH) has been investigated in serum from young and adult cattle and sheep. The thermoresistance of the isoenzymes was determined by electrophoresis of serum samples preincubated at different temperatures. Marked differences were found in the percentage distribution of isoenzymes in serum from the two species as well as in the heat stability. LDH in serum from sheep was inactivated at a lower temperature than that in serum from cattle, and inactivation occurred at a lower temperature in young than in adult animals. The enzyme was in both species less tolerant to elevated temperatures than what is reported for human serum. Procedures worked out for a so-called relative heat stability test of LDH in human clinical diagnosis may therefore give misleading results if they were applied uncritically to sera from these animals.The LDH isoenzyme pattern of some main organs in calves and sheep indicates that a serum heat stability test may be useful in the diagnosis of skeletal muscle injuries in sheep. In cattle the tissue isoenzyme distribution is assumed to be too uniform to give information about specific organ lesions either by serum electrophoresis or by a heating technique.In contrast to what has been reported in man, serum levels of α-hydroxybutyrate dehydrogenase (HBD) in cattle and sheep, as earlier reported in swine, are found to be far better correlated to total LDH than to the most thermostable isoenzyme, LDH₁.     

中国地方绵羊群体TYRP1基因遗传变异研究

本研究旨在了解酪氨酸酶相关蛋白1（tyrosinase related protein 1，TYRP1）基因在中国地方绵羊群体内的遗传变异，以及TYRP1基因突变与不同毛色表型绵羊群体的相关性。通过直接测序法和PCR-RFLP技术对10个中国地方绵羊群体进行单核苷酸多态性（SNP）检测，利用Beagle、PLINK和POPGENE等软件对突变位点数据进行单倍型构建、连锁不平衡分析和遗传变异研究。突变位点检测结果表明，在绵羊TYRP1基因内识别了13个SNPs，其中位于TYRP1基因外显子上的10个SNPs位点，除个别位点在大尾寒羊、中国美利奴羊和岷县黑裘皮羊中没有发生突变外，其他突变位点在所有绵羊品种中均出现不同程度变异，说明中国地方绵羊群体具有较高的遗传多样性。单倍型分析结果表明，所有样本中共有42个单倍型，优势单倍型0000000000（245/918）、0100000001（91/918）在所有绵羊群体中均存在，除单倍型0101100000（93/918）在中国美利奴羊中没有出现，单倍型0001000001（69/918）在岷县黑裘皮羊、哈萨克羊群体中没有出现外，在其他群体中均存在。连锁分析结果表明，10个SNPs在所有样本中均存在2个连锁模块。群体遗传变异分析表明，中国地方绵羊群体具有较高水平的群体内遗传变异，各绵羊品种间存在明显的遗传分化模式，且各品种遗传关系与其品种传统分类结果基本一致。本研究为进一步研究TYRP1基因对绵羊毛色遗传性状的影响提供了参考依据。     

Lactate dehydrogenase and creatine kinase isoenzyme levels in the tissues and serum of normal lambs

The normal activities of lactate dehydrogenase and creatine kinase isoenzymes in the tissues and serum of clinically normal five-and-a-half-month-old lambs are presented. The percentage distribution of the lactate dehydrogenase isoenzymes in serum, liver, heart, lung and skeletal muscle were in agreement with previous studies but the distribution in ovine abomasum, small intestine, large intestine and red blood cells has not been previously described. Up to five creatine kinase isoenzymes were detected in ovine tissues and four in serum. One creatine kinase isoenzyme present in serum and tissues was thought to represent a mitochondrial isoenzyme which is absent from the normal serum of other species. Estimations of both lactate dehydrogenase and creatine kinase isoenzymes allowed all the tissues examined to be distinguished and were therefore more likely to allow tissue-specific isoenzyme patterns to be detected in serum than the estimation of the isoenzymes of either enzyme alone.     

Lactate dehydrogenase and its isoenzymes in the tissues and sera of clinically normal dogs

The total activity of lactate dehydrogenase (LDH) and the percentage distribution of its isoenzymes in the tissues and sera of clinically normal adult dogs are presented. Total LDH activity was greatest in skeletal muscle followed by heart muscle, kidney, small intestinal mucosa, liver, lung, pancreas and bone. Each tissue had a unique isoenzyme pattern and the proportions of the isoenzymes in serum suggested that liver is the source of normal serum LDH. The tissue isoenzyme patterns were similar to those obtained by other authors in human beings, horses, cattle, sheep and cats although in liver, differences between ruminants and monogastric animals including dogs were evident. The data presented provide a basis for the interpretation of serum LDH isoenzyme patterns in canine disease.     

Activities of some serum enzymes in halothane reacted and non-reacted pigs

Eight- and nine-week-old Hungarian Landrace pigs were tested with halothane as described by Laky et al. (1985). Immediately after the test blood samples were taken for determination of the activity of serum creatine kinase (CK), creatine kinase MB (CK-MB) isoenzyme, aldolase (ALD), lactate dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH). Elevated creatine kinase, creatine kinase MB isoenzyme and aldolase activities indicating enhanced susceptibility to stressors were found in 92% of the halothane reacted and 16% of the halothane non-reacted animals. In these individuals the activities of lactate dehydrogenase and alpha-hydroxybutyrate dehydrogenase were also high. Data of the literature show a close relationship between enhanced susceptibility to stressors and halothane reaction in pigs. It was suggested, therefore, that determination of the activity of appropriate serum enzymes might be used for detecting this enhanced susceptibility.