NLRP基因家族在哺乳动物基因组中分布广泛,在免疫应答和生殖相关过程中发挥重要作用.本研究根据已报道的人类NLRP基因,利用PFAM、CDD、UniProt等数据库,采用ClustalW、MEGA X、ExPASy、MEME、GSDS和STRING等软件对牦牛NLRP家族成员进行全基因组鉴定和分析,并且基于高通量测序数据分析NLRP基因在牦牛不同组织中的表达情况.研究共鉴定出8个不同的NLRP基因家族成员,包括NLRP2、NLRP3、NLRP5、NLRP6、NLRP8、NLRP9、NLRP12、NLRP14,其中NLRP9有2个可变剪切.牦牛NLRP蛋白均有多个磷酸化位点,GRAVY值均小于0,属于亲水性蛋白;不稳定系数均大于40,均为不稳定蛋白,无跨膜结构.系统进化树显示同一家族成员氨基酸序列相似性高,在6个物种中都聚在一起,免疫相关基因与生殖发育相关基因在进化中已经分开.蛋白互作网络显示牦牛NLRP蛋白与其他物种已经研究的功能相似,不仅与参与免疫相关的蛋白互作,也与生殖标记蛋白互作.如NLRP3与多种参与炎症反应的蛋白PYCARD、MLKL、GSDMD互作,NLRP5也与生殖细胞标记蛋白GDF9、BMP15、ZAR1等互作.转录组分析结果显示,牦牛NLRP基因在6种组织中的表达较为微弱或者不表达,具有组织特异性.牦牛NLRP家族在结构及理化性质上与其他物种同源基因具有相同的特点,部分试验和预测证实其同样参与了免疫和生殖相关过程.本研究结论不仅对牦牛NLRP基因家族进行了系统分析,同时为进一步研究牦牛先天免疫和生殖相关过程提供理论依据.

Background High spontaneous miscarriage rate in yak, especially during late pregnancy, have caused a great economic loss to herdsmen living in the Qinghai-Tibet plateau. However, the mechanism underlying spontaneous miscarriage is still poorly understood. In the present study, placenta protein markers were identified to elucidate the pathological reasons for yak spontaneous miscarriage through isobaric tags for relative and absolute quantification (iTRAQ) proteomic technology and bioinformatic approaches. Results Subsequently, a total of 415 differentially expressed proteins (DEPs) were identified between aborted and normal placentas. The up-regulated DEPs in the aborted placentas were significantly associated with “spinocerebellar ataxia”, “sphingolipid signalling”, “relaxin signalling”, “protein export”, “protein digestion and absorption” and “aldosterone synthesis and secretion” pathway. While the down-regulated DEPs in the aborted placentas mainly participated in “valine, leucine and isoleucine degradation”, “PPAR signalling”, “peroxisome”, “oxidative phosphorylation”, “galactose metabolism”, “fatty acid degradation”, “cysteine and methionine metabolism” and “citrate cycle” pathway. Conclusions The results implied that the identified DEPs could be considered as placental protein markers for yak miscarriage during late pregnancy, and biomacromolecule metabolic abnormality and oxidative damage might be responsible for the high spontaneous miscarriage rate in yak. These findings provide an important theoretical basis for deciphering the pathologic mechanism of late spontaneous miscarriage in yak.

Additional file 3. Upregulated DEPs in immature FF.

驯化、自然选择和人工选择都会在基因组上留下选择信号,研究这些选择信号是筛选功能基因的重要策略之一.本研究基于野牦牛(Bos mutus)和天祝白牦牛(Bos grunniens)的重测序数据,利用GATK(genome analysis toolkit)获得 SNP(single nucleotide polymorphisms)变异位点,然后利用群体分化指数 FST和Tajima's D法分析两个牦牛群体的选择信号分布情况.选择FST值的top 5％和Tajima's D值两端前2.5％(top2.5％和bot2.5％)作为选择阈值,将阈值以内的位点定义为强选择信号位点.经SnpEff注释后,利用FST选择信号方法筛选得到868个候选基因,GO分析富集到细胞增殖的正向调节、肌动蛋白细胞骨架组织、镁离子结合等肌肉相关条目.KEGG富集到肌肉发育相关通路与低氧适应相关的通路,包括MAPK信号通路、肌动蛋白细胞骨架的调节、PI3K-Akt信号通路、ECM受体相互作用、细胞周期、Rapl信号通路.Tajima's D法分析结果显示,在天祝白牦牛基因组中筛选出377 214个(Tajima's D＞1.974 72)和539 869个(Tajima's D＜-1.780 03)个位点,分别注释得到38个和22个候选基因.在野牦牛基因组中筛选出413 240 个(Tajima's D＞1.854 31)和 332 871 个(Tajima's D＜-1.512 31)位点,分别注释得到 30 个和 24 个候选基因.对两种方法筛选出的强选择信号重叠位点进行注释,分别在野牦牛和天祝白牦牛基因组上中得到90和83个候选基因,从中鉴定出2个驯化相关基因(SCRIB1、SNCA)、1个低氧适应相关基因(THADA)、2个毛色相关基因(MAPK3、NNT).本研究通过FST和Tajima's D法筛选到一系列与野牦牛和天祝白牦牛遗传分化和表型差异相关的基因和通路,为进一步研究牦牛基因组与表型之间的关系以及确定控制牦牛重要经济性状的基因提供理论基础.

Additional file 5. GO of upregulated DEPs in immature FF.

Luteinizing hormone receptor (LHR), prolactin receptor (PRLR), growth hormone (GH) and insulin-like growth factor 1 (IGF1) have been shown to be key regulators of germ cell development. However, the role of LHR, PRLR, GH and IGF1 in the development of yak testis remains unclear. In this study, we aimed to describe and compare gene expression and protein localization of LHR, PRLR, GH and IGF1 in the development of yak testes. Testes were collected from 6, 24, 36 and 72 months yak, and the kidney, liver, testicular, lung, skeletal muscle, heart and spleen tissues were collected from 36 months yak. The quantitative real-time PCR (qRT-PCR) results showed that the expression of these four genes was widely expressed in kidney, liver, testicular, lung, skeletal muscle, heart and spleen, while the LHR and PRLR were highly expressed in the kidney, skeletal muscle and testis, and higher levels of GH and IGF were expressed in spleen and testis. Moreover, the mRNA expression of these genes in adults was higher than in pre-pubertal yak. In the testis, the LHR-, PRLR-, GH- and IGF1-positive signals were detected in the Leydig cells of the 6 months, while the intense positive signals were discovered in Leydig cells, spermatogonia and spermatocytes of the 36 and 72 months. Thus, LHR, PRLR, GH and IGF1 may be involved in the development of spermatids and spermatocytes, and in the regulation of spermatogonia proliferation and Leydig cell function.

BACKGROUND:Ovarian follicle fluid (FF) as a microenvironment surrounding oocyte plays critical roles in physio-biochemical processes of follicle development and oocyte maturation. It is hypothesized that proteins in yak FF participate in the physio-biochemical pathways. The primary aims of this study were to find differentially expressed proteins (DEPs) between mature and immature FF, and to elucidating functions of the mature and immature FF in yak.RESULTS:The mature and immature FF samples were obtained from three healthy yaks that were nonpregnant, aged from four to five years, and free from any anatomical reproductive disorders. The FF samples were subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ). The FF samples went through correlation analysis, principle component analysis, and expression pattern analysis based on quantification of the identified proteins. Four hundred sixty-three DEPs between mature and immature FF were identified. The DEPs between the mature and immature FF samples underwent gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) analysis. The DEPs highly expressed in the mature FF mainly took parts in the complement and coagulation cascades, defense response, acute-phase response, response to other organism pathways to avoid invasion of exogenous microorganisms. The complement activation pathway contains eight DEPs, namely C2, C5, C6, C7, C9, C4BPA, CFH, and MBL2. The three DEPs, CATHL4, CHGA, and PGLYRP1, take parts in defense response pathway to prevent invasion of exogenetic microorganism. The coagulation cascades pathway involves many coagulation factors, such as F7, F13A1, FGA, FGB, FGG, KLKB1, KNG1, MASP1, SERPINA1, and SERPIND1. While the DEPs highly expressed in the immature FF participated in protein translation, peptide biosynthetic process, DNA conformation change, and DNA geometric change pathways to facilitate follicle development. The translation pathway contains many ribosomal proteins, such as RPL3, RPL5, RPS3, RPS6, and other translation factors, such as EIF3J, EIF4G2, ETF1, MOV10, and NARS. The DNA conformation change and DNA geometric change involve nine DEPs, DDX1, G3BP1, HMGB1, HMGB2, HMGB3, MCM3, MCM5, MCM6, and RUVBL2. Furthermore, the expressed levels of the main DEPs, C2 and SERPIND1, were confirmed by western blot.CONCLUSIONS:The differential proteomics revealed the up-regulated DEPs in mature FF take parts in immunoreaction to prevent invasion of microorganisms and the up-regulated DEPs in immature FF participate in protein synthesis, which may improve our knowledge of the follicular microenvironment and its biological roles for reproductive processes in yak. The DEPs, C2 and SERPIND1, can be considered as protein markers for mature yak follicle.

为了解牦牛与犏牛睾丸组织转录组、蛋白组比较研究进展,本文通过检索2012～2020年的相关文献,主要从转录、翻译水平对牦牛与犏牛睾丸组织差异分析归纳.研究发现,转录组学找到了牦牛与犏牛睾丸组织存在的差异及与犏牛雄性不育相关的基因和非编码RNA,发掘了一大批与犏牛精子发生阻滞的相关的差异表达基因;蛋白组学获得了一批表达差异明显的蛋白质,这些差异蛋白在细胞生长、细胞周期、精子发生等方面发挥重要作用.通过牦牛与犏牛睾丸组织转录组、蛋白组比较研究分析,旨在为研究牦牛与犏牛睾丸组织转录组、蛋白组比较及犏牛雄性不育提供基础资料.

2020年8月份,青海省大通种牛场1.5岁"阿什旦"育成牦种公牛发生腹泻,为了确诊其病因,无菌采取18头腹泻犊牛粪便进行细菌分离鉴定、生化试验、PCR鉴定、致病性实验、虫卵检测和药敏试验等.实验结果(病料编号为10#)的样品:生化结果表明:符合大肠杆菌生化特性;PCR结果表明:引物能有效扩增大肠杆菌16SrRNA基因;致病性实验表明:将纯化后的细菌在营养肉汤培养24 h,将其腹腔注射昆明系小白鼠,每只0.2 mL,注射两只,白鼠24 h死亡,且小鼠腹腔呈现肠道炎症状样,该菌具有致病性,为致病性大肠杆菌.病毒性腹泻(BVDV)RT-PCR阴性,沙门氏菌PCR阴性.虫卵检测14/18线虫阳性.药敏结果表明:分离纯化的大肠杆菌对恩诺沙星、氨苄青霉素、呋喃妥因、阿米卡星、先锋霉素、庆大霉素、畜痢灵高度敏感;采自(编号为10#)1头犊牛为大肠杆菌感染,本研究为该牛场犊牛腹泻的病因分析和防治提供了指导.

牦牛是青藏高原地区的重要畜种,为牧民提供生产和生活资料.但是牦牛的繁殖力较低,通常为2年1胎或3年2胎,选育改良进展缓慢.卵母细胞体外成熟培养作为体外受精和胚胎发育必不可少的步骤,已被应用于牦牛科学研究和繁殖生产上.由于牦牛卵母细胞体外成熟能力远远低于体内,因此完善体外培养条件,提高牦牛卵母细胞体外培养的成熟率,对牦牛繁育技术研发和育种生产都有重要的意义.本文从卵巢保存、卵母细胞采集、卵母细胞培养、培养基添加剂等方面对卵母细胞体外成熟的影响因素进行了分析,旨在提高卵母细胞体外成熟发育质量,为牦牛体外受精技术提供技术参考.

Domestication is a key factor of genetic variation; however, the mechanism by which domestication alters gut microbiota is poorly understood. Here, to explore the variation in the structure, function, rapidly evolved genes (REGs), and enzyme profiles of cellulase and hemicellulose in fecal microbiota, we studied the fecal microbiota in wild, half-blood, and domestic yaks based on 16S rDNA sequencing, shotgun-metagenomic sequencing, and the measurement of short-chain-fatty-acids (SCFAs) concentration. Results indicated that wild and half-blood yaks harbored an increased abundance of the phylum Firmicutes and reduced abundance of the genus Akkermansia, which are both associated with efficient energy harvesting. The gut microbial diversity decreased in domestic yaks. The results of the shotgun-metagenomic sequencing showed that the wild yak harbored an increased abundance of microbial pathways that play crucial roles in digestion and growth of the host, whereas the domestic yak harbored an increased abundance of methane-metabolism-related pathways. Wild yaks had enriched amounts of REGs in energy and carbohydrate metabolism pathways, and possessed a significantly increased abundance of cellulases and endohemicellulases in the glycoside hydrolase family compared to domestic yaks. The concentrations of acetic, propionic, n-butyric, i-butyric, n-valeric, and i-valeric acid were highest in wild yaks. Our study displayed the domestic effect on the phenotype of composition, function in gut microbiota, and SCFAs associated with gut microbiota, which had a closely association with the growth performance of the livestock. These findings may enlighten the researchers to construct more links between economic characteristics and gut microbiota, and develop new commercial strains in livestock based on the biotechnology of gut microbiota.

本研究通过对青海省牦牛种畜经营主体及其良种繁育、自身建设和市场供需等情况进行调研,全面分析青海省牦牛种业发展现状,针对牦牛种业发展面临供种能力不足、种牛生产不平衡、经营主体发展水平低等问题,提出完善良种繁育体系建设、强化优良品种保护利用、坚持实施良种工程项目、加强人才队伍建设和优化产业布局等应对策略来促进青海牦牛种业发展.

In this study, we assembled the complete mitochondrial genome of Yanglong yak (Bos grunniens) from Illumina sequencing reads. The mitochondrial genome is 16,323 bp long with an A + T-biased nucleotide composition, and encodes 13 protein-coding, 22 tRNA, and two rRNA genes along with a noncoding control region. In addition, its gene order is identical to those of the previously published mitochondrial genomes of its congeners. Phylogenetic analysis indicates that this breed is closely related to Datong yak, Pamir yak, Tianjun yak, polled yak, Seron yak, Sunnan yak, a series of Domestic Yak and wild yak, followed by Jinchuan yak and Gannan yak, and slightly far away from Bison and Bos taurus.

This study was to investigate the effect of calcium iodine content in dietary on rumen fermentation in vitro of yaks. Rumen fermentation in vitro was carried out with calcium iodine as additive and yak diet as substrate.. The iodine content of the substrate was 0.1, 0.3, 0.5, 0.7, 0.9 mg/kg respectively, the gas production volume, rumen fermentation index and digestive enzyme activity were determined after 48 h. The results showed that while the content of iodine in substrate was 0.3 mg/kg, the concentrations of NH3-N, content of microbial protein (MCP), activity of lipase (LPS), trypsin (TYS), cellulose (CLS) reached the highest, which were14.267 mg/100 mL, 4.619 g/L, 0.462 U/mL, 65.463 U/mL, 69.527 U/mL respectively, acetic acid, propionic acid and total volatile fatty acid (TVFA) reached the highest, which were 56.142 mmol/L, 30.877 mmol/L, 87.460 mmol/L respectively, there was no significant difference between the groups (P>0.05); The ratio of acetic acid to propionic acid reached the minimum value of 1.819, which was significantly lower than that of all other groups (P<0.05).When the iodine content was 0.5 mg/kg, the activity of amylase (AMS) and cellulose (CLS) reached the highest, which were 1.832 U/mL, 69.527 U/mL respectively; When the iodine content was 0.7 mg/kg, the total gas production and methane production reached the highest, which were 80.8 mL、11.88 mL respectively. It was concluded that when the iodine content of Yak fodder is between 0.3 mg/kg and 0.7 mg/kg under in vitro condition, which is help to rumen fermentation and forage degradation.

短链脂肪酸(SCFA)是反刍动物吸收饲草、饲料中营养物质的重要形式.肠道菌群能够降解食物生成SCFA并影响其比例.本文通过16S rDNA测序和气相色谱质谱联用仪,分别测定了野牦牛(Bos mutus)和家牦牛(Bos grunniens)粪便菌群组成及SCFA含量,通过比较分析两种牦牛肠道菌群与SCFA的关系,筛选出野牦牛肠道中与SCFA高浓度有正相关关系的菌群.结果 显示,野牦牛粪便菌群主要有厚壁菌门(Firmicutes)(66.47％)、拟杆菌门(Bacteroidetes)(26.00％)和变形菌门(Proteobacteria) (3.48％),主要的科有瘤胃球菌科(Ruminococcaceae) (55.18％)、拟杆菌科(Bacteroidaceae) (8.75％)和毛螺菌科(Lachnospiraceae)(7.57％),家牦牛的菌群结构和组成与野牦牛相似.野牦牛粪便中SCFA以乙酸和丙酸为主,乙酸、丙酸、异丁酸、正丁酸和正戊酸的含量均显著高于家牦牛(P＜0.01).Spearman相关分析显示,野牦牛粪便菌群中紫单胞菌科(Porphyromonadaceae)、拟杆菌科(Bacteroidaceae)、普雷沃氏菌科(Paraprevotellaceae)、理研菌科(Rikenellaceae)和韦荣球菌科(Erysipelotrichaceae)与SCFA具有较强相关性(r＞0.4),而家牦牛仅有弱相关性(r＜0.3).说明牦牛后肠道具有丰富的能够促进SCFA生成的益生菌群,进而提高食物的转化效率.

旨在研发半舍饲条件下淘汰母牦牛育肥及犊牦牛培育技术.选取带犊母牦牛和公犊牛各66头,分别随机分为试验组和对照组.在犊牛培育期间(2017年12月4日—2018年3月5日),对试验组母牦牛进行补饲,试验组犊牦牛断乳前(3月5日断乳)随母牦牛哺乳,并进行补饲;对照组母牦牛不进行补饲,按常规放牧方式饲养,对照组犊牦牛进行断乳并补饲.育肥期间(2018年3月5日至出栏)对试验组母牦牛进行补饲,于6月错峰出栏;对照组母牦牛不进行补饲,于集中出栏时间出售(9月或10月).在犊牦牛培育期间,观察试验组母牦牛的体重变化情况;每月4日测定2组犊牦牛的体重和体尺指标,并进行统计学分析.计算试验组母牦牛错峰出栏收益.结果表明,在犊牦牛培育阶段,由于试验组犊牦牛随母牦牛哺乳,试验组母牦牛体重平均下降1.17 kg/头;2018年1月4日,试验组犊牦牛的体重极显著(P<0.01)高于对照组,体斜长和胸围显著(P<0.05)高于对照组;2018年2月4日和3月4日,试验组犊牦牛的体重、体高、体斜长、胸围均极显著(P<0.01)高于对照组.试验组母牦牛错峰出栏的效益为1063.05元.研究结果为提高半舍饲条件下淘汰母牦牛养殖效益以及提升犊牛培育质量提供了技术支撑.

为了使牦牛能够提前发情,缩短牦牛的繁殖周期,减少冷季对犊牛的影响,试验采用冷季补饲的方式,提高母牦牛营养促进早期发情.结果表明,试验组母牦牛在6月下旬发情16头,发情率为32.00％,到7月上旬共发情37头,总发情率达74.00％.对照组母牛6月份未发情,到7月上旬发情67头,发情率为51.54％.2015年、2016年数据显示6月份母牦牛未发情,到7月上旬各发情5头,发情率分别为2.63％和3.07％.因此,适时补饲能有效调控母牦牛提前发情配种.

This experiment conducted to compare the differences of growing yak meat nutritional content in different regions, the nearly 4 years old grazing yak in the two regions were selected of Henan county and Gangcha county in Qinghai province.The contents of crude protein,crude fat,ash,dry matter, cholesterol, amino acids and fatty acids of the longest muscle samples were measured and analyzed by using the national standard.Their differences were compared by using principal component analysis(PCA).The results showed as follows:The two regions of conventional nutrients the crude protein,crude fat,dry matter and cholesterol in the yaks meat have no obvious difference(P>0.05).The ash differed significantly among yaks meat samples from different regions(P<0.01).In the content of amino acids,the difference of yak in the two regions was significant. For the contents of essential amino acid(EAA),non-essential amino acid(NEAA)and total amino acid(TAA), Henan county of yak meat were significantly higher than that of Gangcha county(P<0.01).There was no significant difference between the saturated fatty acid(SFA),unsaturated fatty acid(UFA),monounsaturated fatty acid(MUFA)and polyunsaturated fatty acid(PUFA)in two areas of yak meat(P>0.05).However,the contents of SFA and UFA in yak meat of Henan county were slightly higher than that of Gangcha county. In conclusion,The quality of yak meat in Henan county was better than that of yak meat in Gangcha county.Both of them have the characteristics of"high protein,low fat,rich nutrition",and amino acids and unsaturated fatty acids are abundant.

The experiment was conducted to study the effect of different additions of canola rapeseed on the activity of digestive enzymes in jejunum and cecal chyme and the relationship among the activities of each enzyme.A total of 18 healthy castrated yaks with the same age,gender and body condition were randomly divided into 3 groups and fed with rapeseed for 21.75%(H),10%(M)and 0%(L)of the concentrate feed,six treatments were repeated for each treatment group,and each repeat was fed alone.The results showed that:(1)There was no significant effect of different rapeseed additions on the activi-ties of amylase(AMS),lipase(LPS)and cellulase(CLS)in jejunum(P>0.05).The trypsin(TRS)activity in H group was significantly higher 51.30%,45.08% than that in M group and L group.(2)The activities of AMS and CLS in H group were significantly higher than those in M and L groups(P<0.05).The AMS activity in H group was higher 75.26% and 72.22% than that of M group and L group,CLS activity was higher 68.73% and 52.48% than that in M group and L group.(3)There was a significant positive correlation between AMS and CLS activities in the digestive enzyme activity of jejunum chyme (P <0.01);and there was a significant positive correlation between AMS,LPS,TRS and CLS in the digestive enzyme activity of cecal chyme(P<0.01).

In order to compensate for the problem of insufficient support for monitoring of the nutrient value of pasture in the Qinghai-Tibet Plateau,we examined the dynamic nutritional value of grassland pasture under grazing conditions in the Qinghai-Tibet Plateau.During the period September 2015 to August 2016,we selected typical Tibetan herdsmen and grazing pastures to track yak grazing,and simulated the collection of edible for-age for sampling.The nutrient composition and total energy of the pasture were analyzed,and the dry matter digestibility of pasture was determined using a two-step in vitro method.The following results were obtained. 1)The contents of crude protein,ether extract,acid detergent fiber,ash,calcium,and phosphorus were 3.68%~19.21%,0.86%~2.74%,9.65%~43.83%,44.70%~76.23%,5.40%~9.62%,2.13%~5.12%,and 0.027%~0.190%,respectively,and the contents of CP and ash in May were significantly higher than those in other months (P <0.01).2)The digestibility of pasture was 42.72%~72.47%,which was significantly higher than that of other months (P <0.01 ).3)Total energy (GE),digestive energy (DE),and metabolic energy (ME)were 17.76 ~ 19.67,7.81 ~ 13.26,and 6.40 ~ 10.87 MJ ·kg-1 ,respectively.There was no significant difference in the GE of forage in different months (P >0.05),although it was the highest in June,followed by November.DE and ME were significantly higher in May than in other months (P <0.05),and the lowest in December,followed by April and June.In conclusion,the natural forage of Haiyan County,Qinghai Province in May and June is of good quality,whereas in October to March,the nutritional value of forage is low.There-fore,in view of the different types of seasonal grassland grazing throughout the year,it is recommended that pastoralists and farmers use natural grassland to supplement feed for breeding yaks in October to March.In the future,we will conduct follow-up monitoring of forage grass,and determine the fine feeding of grazing yak.