Yak Lactate Dehydgogenase A4: Purification,Properties, and cDNA Cloning

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.     

牦牛和黄牛的睾丸形态及其血管构筑特征

本实验旨在研究牦牛（Bos grunniens）和黄牛（B. taurus）的睾丸形态及其血管构筑和动脉管径特征,为牦牛睾丸在高原环境的生理适应性提供依据。从14头屠宰后的成年牦牛体内采集睾丸28枚,从9头成年黄牛体内采集睾丸18枚,测定其形态指标,利用血管铸型技术制作动静脉构筑标本,研究睾丸血管解剖学及主要动脉管径的特征。结果显示,牦牛和黄牛的睾丸、附睾的形态特征及动脉管径有差异,牦牛大部分睾丸动脉及其分支的管径与睾丸重的相对值极显著地高于黄牛（P < 0.01）,牦牛睾丸的主要动脉构筑特征与黄牛的相同,但其大的集合静脉数量较少,小静脉呈“编织袋”状紧密排布。研究认为,牦牛睾丸的血管解剖特征可为睾丸提供更为充足的血液,其相对发达的动脉血管和睾丸静脉“编织袋”状分布特点有利于睾丸的温度调节及精子成熟,可能是牦牛生殖器官适应高海拔环境的生理特征之一。     

Mitochondrial DNA sequence diversity and origin of Chinese domestic yak

In order to clarify the origin and genetic diversity of yak in China, we analysed mitochondrial DNA (mtDNA) control region sequences (approximately 891 bp) in 52 individuals from four domestic yak (Poephagus grunniens) breeds, as well as from a hybrid between yak and cattle (Pianniu). Twenty-five samples were further selected for partial (420 bp) cytochrome b sequencing based on control region sequence information. Two yak samples shared sequences with Chinese cattle (Bos taurus); the remaining yak mtDNAs converged into two major clades in the phylogenetic analysis. Genetic diversity varied substantially among the breeds, with the hybrid Pianniu yak demonstrating the highest diversity. Our results suggest that the Chinese yak was domesticated from two distinct matrilineal sources or from a heterogeneous pool containing both divergent lineages, with occasional gene introgression from cattle.     

Differences in mitochondrial gene expression profiles, enzyme activities and myosin heavy chain types in yak versus bovine skeletal muscles

Hypoxia can affect energy metabolism. We examined gene expression and enzyme activity related to mitochondrial energy metabolism, as well as myosin heavy chain (MyHC) types in yaks (Bos grunniens) living at high altitudes. Real-time quantitative PCR assays indicated that the yak has significantly lower levels of carnitine palmitoyltransferase (CPT) mRNA in the biceps femoris and lower levels of uncoupling protein 3 (UCP3) mRNA in both biceps femoris and longissimus dorsi than in Yellow cattle. No significant differences between yak and Yellow cattle were observed in the activities of mitochondrial β-hydroxyacyl-CoA dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase in the same muscles. Semi-quantitative RT-PCR analysis showed that the MyHC 1 mRNA levels in yak biceps femoris was lower than in Yellow cattle. We conclude that the yak has significantly lower mRNA levels of CPT, UCP3, and MyHC 1 in biceps femoris than in Yellow cattle, suggesting that the yak biceps femoris has lower fatty acid oxidation capacity and greater glycolytic metabolic potential.     

牦牛CAPN1基因的克隆与序列分析

CAPN1是影响肌肉嫩度的数量性状位点 (QTL)的候选基因。根据GenBank发表的普通牛CAPN1基因序列设计特异性引物,以天祝白牦牛cDNA为模板,分段进行PCR扩增,克隆,测序。应用生物软件BioEdit对各测序结果进行序列拼接共获得牦牛CAPN1 cDNA 片段2267bp,其中包含一个2151bp的完整的开放阅读框(ORF),以及3’和5’末端非编码区的部分序列(77bp和166bp) 。分析表明：牦牛CAPN1基因编码区全长2151bp,共编码716个氨基酸。与已报道的牛,猪,人小鼠的序列进行比较,核苷酸同源性分别为99.3%,93.9%,90.0% ,85.5% 。预测氨基酸的同源性分别为99.4%,96.1%,94.6%,89.0%,并且对牦牛CAPN1四个结构域分别进行NCBI BLAST发现四个结构域在以上四个物种中都显示出很好的保守性,最为保守的在结构域Ⅳ(>96%)。牦牛与牛产生的 14个核苷酸突变中,有3个产生了氨基酸突变,均发生在结构域Ⅲ。构建分子系统进化树表明：聚类结果与传统分类学相符。     

黄牛、牦牛和犏牛睾丸组织中Cdc2、Cdc25A基因mRNA表达水平

黄牛和牦牛远缘杂交后代犏牛雄性不育是牦牛杂交改良中的一大难题。Cdc2和Cdc25A是减数分裂的两个关键基因, 其表达水平的下降将使精子发生不能正常进行, 导致雄性不育。为了探讨Cdc2、Cdc25A基因mRNA表达水平与犏牛雄性不育的关系, 文章采用荧光定量PCR技术对Cdc2和Cdc25A基因的组织表达特征以及在黄牛、牦牛和犏牛睾丸组织中的表达水平进行了分析。结果表明: Cdc2和Cdc25A基因在牦牛各种组织中广泛表达, 说明Cdc2和Cdc25A基因在各种组织细胞分裂和细胞周期运行中均发挥作用; 黄牛和牦牛睾丸组织中Cdc2、Cdc25A基因表达水平均显著高于犏牛(P<0.05), 说明睾丸组织中Cdc2和Cdc25A基因的低表达可能与犏牛雄性不育相关。     

Species authentication of commercial beef jerky based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene

In this study,we determined species-specific variations by analyzing the mitochondrial 12S rRNA gene sequence variation(～440 bp) in 17 newly obtained sequences and 90 published cattle,yak,buffalo,goat,and pig sequences,which represent 62 breeds and 17 geographic regions.Based on the defined species-specific variations,two endonucleases,Alu I and Bfa I,were selected for species authentication using raw meat/tissue samples and the PCR-RFLP method.Goat and pig were identified using the Alu I enzyme,while cattle,yak,and buffalo were identified by digestion with Bfa I.Our approach had relatively high detection sensitivity of cattle DNA in mixed cattle and yak products,with the lowest detectable threshold equaling 20% of cattle DNA in a mixed cattle/yak sample.This method was successfully used to type commercial beef jerky products,which were produced by different companies utilizing various processing technologies.Our results show that several yak jerky products might be implicated in commercial fraud by using cattle meat instead of yak meat.     

The pulmonary circulation of some domestic animals at high altitude

Pulmonary haemodynamics and the histology of the pulmonary vasculature have been studied at high altitude in the yak, in interbreeds between yaks and cattle, and in domestic goats and sheep indigenous to high altitudes together with crosses between them and low-altitude strains. Cattle at high altitude had a higher pulmonary arterial pressure than cattle at low altitude. The yak and two interbreeds with cattle (dzos and stols) had a low pulmonary arterial pressure compared with cattle, while the medial thickness of the small pulmonary arteries was less than would be expected in cattle, suggesting that the yak has a low capacity for hypoxic pulmonary vasoconstriction and that this characteristic is transmitted genetically. Goats and sheep showed haemodynamic evidence of a limited response of the pulmonary circulation to high altitude, but no evidence that the high altitude breeds had lost this response. There were no measurable differences in the thickness of the media of the small pulmonary arteries between high- and low-altitude breeds of goats and sheep. All these species showed prominent intimal protrusions of muscle into the pulmonary veins but no specific effect of high altitude in this respect.     

Assessment of cattle genetic introgression into domestic yak populations using mitochondrial and microsatellite DNA markers

Hybridization between yak Poephagus grunniens and taurine Bos taurus or indicine B. indicus cattle has been widely practiced throughout the yak geographical range, and gene flow is expected to have occurred between these species. To assess the impact of cattle admixture on domestic yak, we examined 1076 domestic yak from 29 populations collected in China, Bhutan, Nepal, India, Pakistan, Kyrgyzstan, Mongolia and Russia using mitochondrial DNA and 17 autosomal microsatellite loci. A cattle diagnostic marker‐based analysis reveals cattle‐specific mtDNA and/or autosomal microsatellite allele introgression in 127 yak individuals from 22 populations. The mean level of cattle admixture across the populations, calculated using allelic information at 17 autosomal microsatellite loci, remains relatively low (mY_cattle = 2.66 ± 0.53% and Q_cattle = 0.69 ± 2.58%), although it varies a lot across populations as well as among individuals within population. Although the level of cattle admixture shows a clear geographical structure, with higher levels of admixture in the Qinghai‐Tibetan Plateau and Mongolian and Russian regions, and lower levels in the Himalayan and Pamir Plateau region, our results indicate that the level of cattle admixture is not significantly correlated with the altitude across geographical regions as well as within geographical region. Although yak‐cattle hybridization is primarily driven to produce F₁ hybrids, our results show that the subsequent gene flow between yak and cattle took place and has affected contemporary genetic make‐up of domestic yak. To protect yak genetic integrity, hybridization between yak and cattle should be tightly controlled.     

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) was estimated by 16S rDNA homology analysis. Two rumen 16S rDNA libraries were constructed. Of the 194 clones in the library of yak rumen, the sequences were mainly clustered to two phyla, low G+C Gram-positive bacteria (LGCGPB, 54.12% total clones) and Bacteroidetes (30.93%), respectively. While in the 197 clone-library of the cattle rumen, the sequences were mainly related to three phyla, Bacteroidetes (39.59%), gamma-Proteobacteria (26.9%) and LGCGPB (22.34%), respectively. The sequence analysis indicated that more than half of the species harbored in yak rumen belonged to the not-yet-cultured groups at <90% 16S rDNA similarity levels with cultured species, while 36% 16S rDNA sequences amplified from the rumen of Jinnan cattle fell in these catalogues. By comparing the uncultured sequences in yak rumen with those in Jinnan cattle and cow, the former formed distinct clusters loosely related to the later, implying that yak rumen could harbor some special prokaryote phyla. 10.8% sequences retrieved in yak rumen were related to the known rumen fibrolytic bacterial species; however none was related to the known amylolysis species. While 4% and 17.8% sequences retrieved from Jinnan cattle rumen were related to cultured fibrolytic and amylolysis species, respectively. The bacterial structures seemed to be in accordance with the feed of the two kinds of animals. In both rumens, retrieved methanogenic Archaea-related 16S rDNA sequences were at an unreasonable low level; in addition, none sequence was related to Ruminococcus albus, a classical rumen fibrolytic species. The reason can be due to the experimental biases.     

Genetic Structure and Differentiation of Three Chinese Indigenous Cattle Populations

Levels of genetic differentiation, gene flow, and genetic structure of three indigenous cattle populations (Luxi, Bohai, and Minnan) and two reference cattle populations (Chinese Holstein and Qinhai yak) in China were estimated using the information from 12 microsatellites, and 141 microsatellite alleles were identified. The mean number of alleles per locus ranged from 2.9005 in yak to 4.9722 in Holstein. The observed heterozygosity ranged from 0.5325 (yak) to 0.7719 (Holstein); 29 private alleles were detected. The global heterozygote deficit across all populations amounted to 58.5% (p < 0.001). The overall significant (p < 0.001) deficit of heterozygotes because of inbreeding within breeds amounted to 43.2%. The five cattle populations were highly differentiated (F _st = 26.9%, p < 0.001) at all loci. The heterozygote deficit within the population was highest in Luxi cattle and lowest in yak. The average number of effective migrants exchanged per generation was highest (1.149) between Luxi and Holstein, and lowest (0.509) between Luxi and yak. With the application of prior population information, cluster analysis achieved posterior probabilities from 91% to 98% of correctly assigning individuals to populations. Combining the information of cluster analysis, gene flow, and Structure analysis, the five cattle populations belong to three genetic clusters, a taurine (Luxi and Chinese Holstein), a zebu (Bohai and Minnan), and a yak cluster. This indicates that Bohai black is closer to Bos indicus than Luxi cattle. The evolution and development of three indigenous cattle populations are discussed.     

Identification and characteristics analysis of toll-like receptors family genes in yak

Toll-like receptors (TLRs) are innate pattern recognition receptors that play an important role in host resistance to pathogenic microbes. In this study, we cloned the coding region of the yak TLR family (1–10) genes and used bioinformatics to analyze gene characteristics. Real-time fluorescence quantitative polymerase chain reaction was used to detect TLR expression levels in different tissues. Yak TLR genes exhibited high homologies with other species. At the nucleotide level, yak shared more than 96 % homology with cattle and sheep and 75–87 % homology with human and mouse. At the amino acid level, yak shared 90–99 % homology with cattle and sheep and 64–86 % homology with human and mouse. Yak showed close evolutionary relationship with cattle and sheep, which formed a branch of mammals together with TLRs from human, horse, and mouse, among others, and formed a branch with a longer genetic distance with chicken. TLR1, 2, 6, and 10 and TLR7, 8, and 9 were clustered in 2 individual branches, respectively. Fluorescent quantitation results showed that TLRs were expressed in all yak tissues, but different members showed different expression patterns. TLR2, 4, and 6 showed the highest expression in the spleen, followed by ovary, small intestine, kidney, and liver. TLR1, 5, 7, 8, 9, and 10 were most highly expressed in the kidney and showed higher expression in the liver, kidney, spleen, and other tissues. Our results will be useful for studies on immune molecular mechanisms and disease resistance breeding of yak and other plateau animals.     

Complete sequence of the yak (Bos grunniens) mitochondrial genome and its evolutionary relationship with other ruminants

The yak (Bos grunniens) is the most important domesticated species in the Qinhai-Tibetan Plateau. In present study, the complete sequence of the yak mitochondrial genome was determined. Sequence analysis revealed that there are no differences with cattle in the yak mitochondrial genome organization. Interestingly, within the D-loop, the conserved sequence blocks are less conserved than surrounding regions. Neighbor-Joining (NJ) trees based on single genes, gene sets and concatenated genes of mitochondrial genome were constructed. The analysis identified the yak as a sister group of a cattle/zebu clade. Based on substitutions in 22 tRNA genes, 12S rRNA gene and 16S rRNA gene, the dating of divergence between yak and cattle/zebu, and yak and water buffalo, was proposed to have occurred 4.38-5.32 and 10.54-13.85 million years before present, respectively. This is consistent with the paleontologyical data. Yak and sheep/goat divergent dating predicts that their divergence occurred at 13.14-27.99 million years before the present day.     

藏黄牛β-乳球蛋白基因启动子部分序列特征

目的:阐明藏黄牛、牦牛、中国荷斯坦牛β-乳球蛋白(β-Lg)遗传变异体在乳中表达差异的分子基础。方法:采用聚丙烯酰胺凝胶电泳确定乳中β-Lg遗传变异体及其相对表达量,然后利用PCR方法扩增β-Lg启动子部分序列(375bp)并直接测序。结果:在杂合型β-Lg个体中,中国荷斯坦牛乳中β-Lg A的相对表达量(63.7±2.9%)均一致性地高于β-Lg B,而藏黄牛乳中二者比例十分接近,但个体差异差异较大。16个测序样品中共检测到13处碱基突变,其中有5处为牦牛特异的。另外,在牦牛β-Lg启动子-452与-453之间,还存在一个插入碱基。结论:β-Lg启动子-430碱基在中国荷斯坦牛中表现为等位基因特异的,而在藏黄牛中无等位基因特异性。     

高原慢性低氧对土生牦牛与迁饲黄牛肾功能影响的比较

目的：探讨长期慢性高原暴露对高原土生牦牛（yak）、高山迁饲黄牛（migratedcattle）和低海拔黄牛（lowland cattle）肾功能的影响,探究牦牛和高山迁饲黄牛适应和习服高原的不同特征及差异。方法：采集青海不同地区的牦牛血样,按海拔高度分为3 000 m,3 500 m,4 000 m和4 300 m四个组（n=84）,同时采集高山迁饲黄牛（n=22）及低海拔黄牛（n=39）血液,利用全自动血液生化分析仪测定尿素氮（BUN）、肌酐（Cr）、尿素氮/肌酐（BUN/Cr）、尿酸（UA）、二氧化碳结合率（CO₂cp）、葡萄糖（GLU）水平,分析不同海拔高度的牦牛之间及牦牛、高山迁饲黄牛和低海拔黄牛之间的差异。结果：随着海拔的升高,土生牦牛UA、CO₂cp两项升高较显著,与黄牛相比,BUN、BUN/Cr显著高于高山迁饲黄牛和低海拔黄牛, CO₂cp、GLU显著低于低海拔黄牛。低海拔黄牛和高山迁饲黄牛相比,BUN、BUN/Cr显著低于高山迁饲黄牛,UA、CO₂cp显著高于高山迁饲黄牛。结论：土生牦牛对于高原低氧环境有很强的适应能力;高山迁饲黄牛表现出对低氧的不适应性,处于对低氧的应激状态。     

Interferon-alpha genes from Bos and Bubalus bubalus

Interferon-a genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-a genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-a subtypes, the IFN-a gene of Holstein cow had only one point mutation with the BoIFN-aA subtype. The IFN-a gene of yellow cattle was similar to the BoIFN-aD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-aD1. The other four IFN-a genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-aI, BoIFN-aJ, BuIFN-a1, and BuIFN-a2, respectively. Each of the six clones was expressed in E. coli with molecular weight of approximately 20 kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-a (rIFN-a) all exhibited 1,000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-as could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-as a potential agent for clinical application against virus diseases in cattle industry.     

Comparative analysis of ISSR marker polymorphism in populations of yak (Bos mutus) and in F1 hybrids between yak and cattle in the Sayan-Altai region

The genetic variability in seven yak populations from the Sayan-Altai region and in F₁ hybrids between yak and cattle (khainags) was investigated with the help of a technique that involves the use of inter simple sequence repeat (ISSR) markers generated with PCR primers (AG)⁹C and (GA)⁹C. Samples for the analysis were collected in Mongolia, Tuva, and Altai from 2008 through 2012. The examined yak populations differed in in the presence/absence of ISSR fragments, as well as in their frequency. In total, 46 ISSR fragments were identified using two marker systems; the proportion of polymorphic loci constituted 76% and 90% for the AG-ISSR and GA-ISSR markers, respectively. For the total sample of yaks, total genetic diversity (H _t), within-population diversity (H _s), and interpopulation diversity (G _st) constituted 0.081, 0.044, and 0.459 for the AG-ISSR and 0.137, 0.057, and 0.582 for the GA-ISSR markers, respectively. Based on ISSR finger-printing, species- and breed-specific DNA patterns were described for the three groups of animals (yaks, cattle, khainags). For the domestic yak, the species-specific profile was represented by eight ISSR fragments. Genetic relationships between the yak populations, cattle breeds, and khainags were examined with the help of four different approaches used in the analysis of population structure: estimation of phylogenetic similarity, multidimensional scaling, principal component analysis, and cluster analysis. Clear evidence on the differentiation of the populations examined at the interspecific, as well as at intraspecific, level were obtained. Similar (relative), as well as remote (isolated), yak populations were identified. Khainags occupy an intermediate position between yak and cattle. However, the data on the ISSR-PCR marker polymorphism (genome polymorphism, population structure) indicate that part of the analyzed khainag genome was more similar to the yak genome than to the cattle genome.     

Profiling of the yak skeletal muscle tissue gene expression and comparison with the domestic cattle by genome array

Of all the mammals of the world, the yak lives at the highest altitude area of more than 3000 m. Comparison between yak and cattle of the low-altitude areas will be informative in studying animal adaptation to higher altitudes. To investigate the molecular mechanism involved in meat quality differences between the two Chinese special varieties Qinghai yak and Qinchuan cattle, 12 chemical–physical characteristics of the longissimus dorsi muscle related to meat quality were compared at the age of 36 months, and the gene expression profiles were constructed by utilizing the bovine genome array. Significant analysis of microarrays was used to identify the differentially expressed genes. Gene ontology and pathway analysis were performed by a free Web-based Molecular Annotation System 2.0. The results reveal ~11 000 probes representing about 10 000 genes that were detected in both the Qinghai yak and Qinchuan cattle. A total of 1922 genes were shown to be differentially expressed, 633 probes were upregulated and 1259 probes were downregulated in the muscle tissue of Qinghai yak that were mainly involved in ubiquitin-mediated proteolysis, muscle growth regulation, glucose metabolism, immune response and so on. Quantitative real-time PCR (qRT-PCR) was performed to validate some differentially expressed genes identified by microarray. Further analysis implied that animals living at a high altitude may supply energy by more active protein catabolism and glycolysis compared with those living in the plain areas. Our results establish the groundwork for further studies on yaks’ meat quality and will be beneficial in improving the yaks’ breeding by molecular biotechnology.