Study on Polymorphisms in the Blood Protein of Tibetan Mastiff

[ Objective] The aim was to study the protein polymorphism in the blood of Tibetan Mastiff, and provide some theoretical basis for resource protection and reasonable development and utilization of Tibetan Mastiff varieties. [ Method] A total of 103 blood samples were taken from four populations of Hequ Tibetan Mastiff, Qinhai Tibetan Mastiff, Tibetan Spaniel and native dogs of Qinghai. Seven blood protein Iocus（Tf, Po, Sα2, Hb, AIb, Pr and Amy）were investigated by using vertical polyacrylamide gel electrophoresis with discontinuous buffer system. Then the genetic variation during different populations was analyzed. [ Result] Genetic variations were observed in Tf, Sα2 and Po in four populations, others were not polymorphic. There were three alleles at the locus of Tf and Po, two alleles at the loci of Sα2. Effective number of alleles and Nei＇s average expected heterozygosity were 1. 532 4 and 0.230 3 relatively, all higher in Tibetan Mastiff than other populations. [ Conclusion] Protein locus in blood of Tibetan Mastiff existed in genetic variation.

Effects of Cryopreservation on the Quality and Ultrastructure of Tibetan Mastiff Sperm

[Objective] This study aimed to investigate the effects of cryopreservation on the quality and ultrastructure of Tibetan Mastiff sperm. [Method] The effects of cryopreservation on the quality of Tibetan Mastiff sperm were evaluated via motility, membrane integrity rate and acrosome intact rate. On that basis, the effects of cryopreservation on ultrastructure of sperm were observed under SEM and TEM. [Result] In Experiment 1, EG gave the best results not only in post-thaw motility rate （36.3%）, but also in low membrane integrity rate （38.0%） and acrosome intact rate （42.0% ）, but there was no significant difference between EG group and Glycerol group （P0.05）. In Experiment 2, the 5 cm freezing height obtained the best freezing-thawing results, but there was no significant difference between 2 and 5 cm height （P 0.05）, besides in acrosome intact rate. In Experiment 3, SDS and Vc added separately or together into extenders could improve freezing-thawing results, but there was not obvious difference between SDS group and Vc group （P0.05）, besides the lower motility of Vc group （P0.05）. Addition of SDS and Vc obtained the best results in post-thaw motility rate （44.1%）, and also in membrane integrity rate （48.0%） and acrosome intact rate （48.2%）. The ultrastructure of frozen-thawed sperm was also evaluated under SEM and TEM, results showed that cryopreservation caused various degrees of damage to Tibetan Mastiff sperm, more serious damages were observed in the acrosome such as swelling, vesiculation and even disappearance. [Conclusion] This study confirms that EG, horizontal height of 0.25 ml straw above LN 2 surface and additives SDS and Vc together can improve freezing effect. However, cryopreservation has certain damage to ultrastructure of sperm.

Origin and phylogenetic analysis of Tibetan Mastiff based on the mitochondrial DNA sequence

At present, the Tibetan Mastiff is the oldest and most ferocious dog in the world. However, the origin of the Tibetan Mastiff and its phylogenetic relationship with other large breed dogs such as Saint Bernard are unclear. In this study, the primers were designed accord- ing to the mitochondrial genome sequence of the domestic dog, and the 2,525 bp mitochondrial sequence, containing the whole sequence of Cytochrome b, tRNA-Thr, tRNA-Pro, and control region of the Tibetan Mastiff, was obtained. Using grey wolves and coyotes as out- groups, the Tibetan Mastiff and 12 breeds of domestic dogs were analyzed in phylogenesis. Tibetan Mastiff, domestic dog breeds, and grey wolves were clustered into a group and coyotes were clustered in a group separately. This indicated that the Tibetan Mastiff and the other domestic dogs originated from the grey wolf, and the Tibetan Mastiff belonged to Carnivora, Canidae, Canis, Canis lupus, Canis lupus familiaris on the animal taxonomy. In domestic dogs, the middle and small breed dogs were clustered at first; German Sheepdog, Swedish Elkhound, and Black Russian Terrier were clustered into one group, and the Tibetan Mastiff, Old English Sheepdog, Leonberger, and Saint Bernard were clustered in another group. This confirmed the viewpoint that many of the famous large breed dogs worldwide such as Saint Bernard possibly had the blood lineage of the Tibetan Mastiff, based on the molecular data. According to the substitution rate, we concluded that the approximate divergence time between Tibetan Mastiff and grey wolf was 58,000 years before the present （YBP）, and the approximate divergence time between other domestic dogs and grey wolf was 42,000 YBP, demonstrating that the time of origin of the Tibetan Mastiff was earlier than that of the other domestic dogs.

Influence of Different Sources and Levels of Fat on Palatability of Tibetan Mastiff

In order to compare the palatability of Tibetan mastiff to different fat sources and fat levels, four kinds of raw lipid materials （ soybean oil, sunflower seed oil, chicken fat and butter） were selected to prepare five candidate diets with different fat source combinations and three different fat level combinations （6%, 10% and 14% ）. Ten healthy Tibetan mastiff adults were randomly divided into two groups. The intematianally useful double-pot cultivation design was employed to record the daily feed intake and preferred diet of each Tibetan mastiff, and further to calculate the intake rate. The butter group assumed higher feed intake and intake rate than the chicken fat group （P 〈0.01）, and the preferred diet as well （83%）. The feed intake and intake rate of chicken fat group was significantly higher than chicken fat and butter （ 1：1 ） combined group （ P 〈 0.01 ）. No significant difference was observed in the diet palatability of butter diet to Tibetan mastiff to different plant oil sources （ P 〉 0.05）. For various fat level combinations, the diet with highest fat content diet H23 assumed higher feed intake and intake rate than low fat diet H22 and H21 （P 〈0.05）. It is concluded that the palatability of butter diet to Tibetan mastiff is higher than chicken fat and its combined diet （ butter：chicken fat = 1：1 ） ; with fat level of 6% - 15%, high fat diet provides Tibetan mastiff a better palatability in comparison with low fat diet.

Genetic Structure and Genetic Differentiation of Tibetan Mastiff Populations

[ Objective] To analyze the genetic structure and genetic differentiation of Tibetan Mastiff populations and provide a theoretical basis for the protection and rational development and utilization of the Tibetan Mastiff breed resources. [ Method] A total of 86 blood samples were collected from two populations of Hequ Tibetan Mastiff （HTM） and Qinghai Tibetan Mastiff （QTM）. The genetic structure and genetic differentiation of nine blood protein loci of ＂If, Po, Es-1, Es-2, S（alpha）2, Hb, AIb, Pr and Amy between these two populations were examined by vertical polyacrylam- ide gel electrophoresis with discontinuous buffer system. [ Result] The effective number of alleles （Ne）, Shannon genetic diversity index （I） and Nei average expected gene heterozygosity （H） of the HTM were respectively 1.690 7, 0.485 5 and 0.295 9, and these were higher than those of QTM. The Nei＇s standard genetic distance （D） between HTM and QTM was 0.012 3, and the degree of genetic similarity was 0.987 8. The gene flow （Nm） of these two Tibetan Mastiff populations was 14.543 7. [ Conclusion] The genetic variability of intra-population of HTM was higher than that of QTM, and the genetic differentiation of inter-population was very low （ GsT = 0.018 7）. Thereby, the great gene flow （ Nm = 14.543 7） is the main factor leading to the low genetic differentiation of the inter-population in different geographical groups.

Tibetan Mastiffs in Yushu

A Tibetan Mastiff-Tanggya When I was a teenager,my family had a baby mastiff.Because a lock natural of white hair decorated the mastiff's chest,he was given the name"Tanggya".I remember Tanggya was a big dog when I was still a little girl. Whenever he stood on his hind legs,he seemed as tall as my father.When guests came to visit,he always jumped on them and barked constantly. Only my father could briefly calm him down,and nobody amongst our siblings dared to get close to him.Tanggya was never released u...

藏獒(TIBETAN MASTIFF)的核型研究

本文采用外周血淋巴细胞培养技术，制备藏獒染色体标本并对其核型进行了研究。藏类杂色体数目2n=78个，除x染色体为亚中着丝粒染色体外，其余均为端着丝粒染色体。参照家大染色体核型（《医用生物学实验指导》，杨抚华主编，1986年版图版XI），按染色体的特征，大小剪排出藏獒的核型。

High genetic diversity of Tibetan Mastiffs revealed by mtDNA sequences

Many studies have been reported the origin and evolution of domesticated animals, particularly dogs, however, few have system-atically examined the Tibetan Mastiff (TM), a dog that lives primarily in the Qing-Tibet Plateau. Here, we study the origin and evolution of TM based on the population comparison of mtDNA D-loop sequences from TM and other dogs across the world. Intriguingly, non-simultaneous arrivals of Tibetan Mastiffs ancestors to Tibet occurred, which may result from continuous Tibetans' settlement on Qing-Tibet Plateau supported by archaeological and genetic evidence. High genetic and haplotype diversity and robust haplotypes sharing with other dogs unique to East Asia are observed in Tibetan Mastiff, supporting that it is a very archaic breed derived probably from East Asia.

Conservation and breeding for Hequ Tibet Mastiff in China

This paper gave the standards to evaluate Hequ Tibetan Mastiff on body type, appearance, body measures and temperament. A nucleus group of breeding consisted of high-grade individual according this standards, and it was for enhancing the proceeding of selecting and breeding of Hequ Tibetan Mastiff. By this way, after working on three generations it had been gained that effective population size of the nucleus group of breeding was 96, increasing numbers of inbreeding coefficient was 0.005, male and female body height were 72.1 cm and 70.2 cm, their cannon circumference were 16.3 cm and 15.2 cm, respectively.

藏獒IFN-γ基因在毕赤酵母中的表达与抗病毒活性分析

采用毕赤酵母表达系统表达藏獒γ-干扰素(Tibetan mastiff interferon gamma,TmIFN-γ)并分析其生物活性,为抗病毒生物制品的研发与临床应用奠定基础。将构建的TmIFN-γ的真核表达质粒pPIC9k-TmIFN-γ转化至毕赤酵母GS115细胞,用不同浓度的G418筛选得到多拷贝重组菌株;利用甲醇进行诱导表达,以镍层析柱纯化目的蛋白,对表达产物进行SDS-PAGE和Western blotting鉴定;通过CCK-8试验检测重组TmIFN-γ蛋白的细胞毒性作用,利用VSV-MDBK系统检测其抗病毒生物活性。结果显示,成功筛选得到高拷贝pPIC9k-TmIFN-γ重组转化毕赤酵母菌株,利用甲醇诱导可获得TmIFN-γ重组蛋白,该蛋白对细胞无毒性,具有显著抗病毒活性,其抗病毒生物效价为1.727×10~5IU/mL。本研究获得了具有抗病毒活性的TmIFN-γ,有助于进一步探索TmIFN-γ的功能和开发新型基因工程抗病毒药物。

藏獒肺组织对高原低氧环境的适应特性

通过光镜观察了青海省果洛藏族自治州达日县8只藏獒肺脏的组织结构,并对其高原低氧环境的适应性进行了研究.结果表明:藏獒肺胸膜较厚,其中富含大量胶原纤维和弹性纤维;支气管、细支气管管壁较厚,固有层与外膜有较多弹性纤维;肺呼吸部肺泡管的数量多,每支呼吸性细支气管分出多条宽大的肺泡管;许多肺泡常呈现半充满状态的小囊,分布在肺泡管周围;肺泡隔内毛细血管丰富,并呈扩张状态,管腔内红细胞数量较多.

藏獒线粒体DNA D-loop区序列测定及其分类地位研究

根据家犬线粒体基因组DNA序列设计引物,获得了藏獒(Tibetan mastiff)线粒体DNA D-loop区全序列,并以灰狼、郊狼作为外类群对藏獒、家犬亚种内6个大型家犬品种进行了系统发育分析。试验结果发现:藏獒线粒体DNA D-loop区序列全长1250 bp,与家犬源序列的同源性为96.06%,其中有20个碱基缺失;系统发育分析发现藏獒、家犬亚种内6个大型家犬品种与灰狼聚为一大类,而郊狼自成一类,说明藏獒与其他家犬品种一样起源于灰狼,是家犬亚种内的一个品种,在分类地位上隶属于食肉目、犬科、犬属、狼种、家犬亚种;在家犬亚种内,藏獒与英国牧羊犬、兰伯格犬聚为一类,而德国牧羊犬与瑞典猎鹿犬、黑俄罗斯更犬聚在一起,再与圣伯纳犬聚为一类,从分子水平上验证了英国牧羊犬等大型犬可能含有藏獒血统的观点。

藏獒犬11个STR基因座遗传多态性

目的建立犬STR复合扩增体系，为犬个体识别和亲权鉴定提供一种检测方法。方法用自行建立的11个犬STR基因座复合扩增体系，使用AB13130XL遗传分析仪，对107份藏獒唾液DNA样本的扩增产物进行检测及统计学分析。结果除1个基因座杂合度低于60％，其他10个基因座杂合度均高于70％；11个基因座PIC值均在0．6以上；11个基因座中有8个基因座的个体识别率在0．938以上，其余3个基因座均在0．826以上；父权排除率除TETRA为0．267，其余在0．401—0．749之间，偶合机率1．08×10^-18。结论此11个犬STR基因座在藏獒犬中具有较高的个体识别能力，可用于犬类个体识别和亲权鉴定。

藏獒精液冷冻过程中pH值及甘油对其活力的影响

为探讨pH值和甘油对藏獒精液冷冻保存前后精子活力的影响，筛选6条种公藏獒在其繁殖季节（9—12月份）以不同pH值梯度（5．5，6．0，6．5，7．0，7．5）稀释液和不同甘油平衡时间（10，20，30，40，50，60，70，80，90min）对藏獒精液进行冷冻，检查精子活力变化。结果表明，在藏獒精液冷冻过程中，pH值为6．5的稀释液中精子在冷冻前后有较高的活力；甘油平衡时间对藏獒精液活力有较大影响，冻前精子活力随甘油作用时间而下降，在甘油平衡时间为20min所得藏獒冻后精子活力效果较好。

河曲藏獒生长发育的研究

对河曲藏獒初生、断奶、3月龄、6月龄、12月龄、18月龄、24月龄体质量和体尺进行了测定和分析。结果表明:河曲藏獒公犬体质量从3月龄开始极显著地大于母犬(P<0.01),体高从12月龄开始极显著地大于母犬(P<0.01),胸围只在6月龄极显著大于母犬(P<0.01)。在其它生长阶段中,公母犬间无明显差异(P>0.05)。河曲藏獒有较高的平均日增重,公、母犬最大日增重分别为220.66 g,193.66 g,12月龄体质量分别为48.80 kg,44.65 kg。在断奶到6月龄期间加强对藏獒幼犬培育十分重要。

藏獒的研究概况

本文介绍藏獒的培育与繁殖,包括藏獒的管理、饲喂、繁殖性能的研究等;藏獒生长发育的研究;从外形和外貌、毛色、适应性与气质等方面提供了藏獒综合鉴定标准及品种选育研究;对藏獒食欲低下、细小病毒病、犬瘟热、佝偻病、肠套叠、子宫脱出症等病症的治疗与防治做了概述;从藏獒血液核型、血液蛋白质、血液生化指标和参数等方面论述了藏獒的血液学参数研究概况。

基于Cytb基因多态性对藏獒群体遗传结构及系统进化的研究

本试验通过对甘肃藏獒和西藏藏獒共70个个体的线粒体Cytb基因进行扩增测序,应用生物信息学软件进行遗传结构和系统进化分析,为藏獒品种资源保护与合理开发利用提供参考依据。结果表明:在所分析的70条藏獒序列中,共检测到6种单倍型,8个多态位点,仅占分析总位点数的0.7%。甘肃藏獒与西藏藏獒的单倍型多样度(Hd)、核苷酸多样度(Pi)、平均核苷酸差异数(K)分别为0.595、0.002 37、2.703和0.674、0.002 51、2.857,即2个藏獒群体的遗传多样性水平相近。2个群体之间存在很强的基因流,未发生遗传分化,变异主要来自群体内部。根据Cytb基因单倍型网络图和系统发育树推断甘肃和西藏地区的藏獒为单一母系起源,逐步形成3个进化分支。

不同海拔高度的藏獒肺动脉血管特性及结构

[目的]探讨藏獒肺动脉血管的结构及对高海拔低氧的适应特性。[方法]通过光镜观察及形态学测量对来自青海和甘肃两地藏獒的肺动脉血管的特性及结构进行研究。[结果]藏獒肺动脉分为弹性动脉、肌性动脉和微动脉。肌性动脉又分为中动脉和小动脉。研究表明藏獒肺动脉平滑肌含量并没有随海拔而明显变化。2组藏獒在肺血管平滑肌含量方面差异不显著(P>0.05),但均高于牦牛和驼马。藏獒肺胸膜较厚,胸膜内有大量的胶原纤维和弹性纤维,肺脏表面有许多高低不平的皱襞,而且藏獒相邻肺泡间的肺泡隔内有丰富的毛细血管和大量红细胞。[结论]藏獒肺组织结构是其对高原低氧环境的一种遗传适应,与海拔高度无关。

2个中国藏獒群体遗传多样性及遗传分化的研究

本研究以青海土种犬和藏狮犬为对照,采用RAPD技术对河曲藏獒和青海藏獒群体的遗传多样性及遗传分化进行了分析。研究结果表明,河曲藏獒和青海藏獒群体的多态性位点百分率分别为85.53%和98.16%,平均多态性位点百分率为91.85%。藏獒群体遗传变异分析显示两个藏獒群体的有效等位基因数(Ne)、Nei氏平均预期基因杂合度(H)和Shannon遗传信息指数(I)的平均值分别为1.5354、0.3191和0.4807;且两个藏獒群体93.09%的变异来自群体内,仅6.91%变异来自群体间;两个群体间的基因流为3.3679。研究还发现两个藏獒群体之间的Nei氏标准遗传距离(D)为0.0505。本研究结果说明藏獒群体内遗传变异丰富,不同地域的藏獒群体之间存在广泛的基因交流,群体之间遗传分化程度很低,这为藏獒品种资源保护与合理开发利用提供了参考。

不同海拔地区藏獒肺组织结构及肺动脉血管壁的比较观察

通过光镜观察及形态学测量方法对青海省果洛藏族自治州达日县(海拔4 000 m左右)及甘肃省兰州地区各5只藏獒肺的组织结构和肺动脉血管壁的特性进行了研究。结果表明,藏獒肺胸膜较厚,其中富含大量胶原纤维和弹性纤维;支气管、细支气管管壁较厚,固有层与外膜有较多弹性纤维;肺呼吸部肺泡管的数量多,每支呼吸性细支气管分出多条肺泡管,肺泡管宽大;许多肺泡常呈现萎陷的小囊,分布在肺泡管周围;肺泡隔内毛细血管丰富,并呈扩张状态,管腔内红细胞数量较多。动脉血管壁测量结果显示,不同海拔地区藏獒肺小动脉中膜肌层厚度与血管外径的百分率差异不显著(P>0.05),但高于牦牛和驼马。