蒙古马全身主要骨骼肌表型谱的构建及比较研究

为探究蒙古马骨骼肌在运动和肉质方面的特点，本研究选取2匹5岁健康未调教的成年公马全身主要骨骼肌52块(2匹×26块)作为研究对象。试验采用石蜡切片、免疫组化、HE染色的方法得到26个部位骨骼肌形态、快慢肌纤维分布情况、肌纤维横截面积。因不同部位骨骼肌存在差异，本研究将肌肉组织分为5组(头颈部、前肢、躯干部、腹壁侧、后肢)分别进行统计。将其中对应的24个部位与前期得到的纯血马骨骼肌快慢肌纤维比例进行分组比对。结果得到蒙古马全身主要骨骼肌的表型谱，其中颈锯肌、冈上肌、臂二头肌、腕桡侧屈肌、腓肠肌、菱形肌、胸腹侧锯肌的慢肌纤维占比较低；腕桡侧伸肌、臂三头肌、背最长肌、臀中肌慢肌纤维占比达70%以上；且慢肌纤维占比与肌纤维横截面积呈负相关。蒙古马骨骼肌慢肌纤维占比基本高于纯血马，前肢、躯干部差异极显著(P<0.01),后肢、腹壁侧差异显著(P<0.05)。其中蒙古马与纯血马在臂三头肌、臀中肌、半膜肌和背最长肌慢肌纤维的差值大于50%。结果表明，蒙古马颈锯肌、冈上肌、臂二头肌、腕桡侧屈肌、腓肠肌、菱形肌、胸腹侧锯肌具有耐力训练的潜力；腕桡侧伸肌、臂三头肌、背最长肌、臀中肌品质好，可为...     

马eCG/LHβ亚基基因多态性研究

马绒毛膜促性腺激素(equine chorionic gonadotropin,eCG)是由妊娠期母马胎儿尿膜—绒毛膜细胞分泌的一种由α、β两个亚基组成的异源二聚体糖蛋白,具有FSH、LH的双重活性。采用PCR-SSCP和测序方法对28匹蒙古马和25匹纯血马的eCG/LHβ亚基基因的多态性进行研究,结果发现,在蒙古马eCG/LHβ亚基基因5′调控区存在一突变位点:G-415A分为AA、BB、AB 3种基因型,AA、BB、AB基因型频率分别为0.89、0.07、0.04;纯血马此基因中未检测到多态性。     

中国蒙古马mtDNA D-Loop高变区序列分析

对5匹中国蒙古马mtDNA D-Loop高变区400bp核苷酸序列的变异情况进行分析。结果发现5匹中国蒙古马mtDNA D-Loop高变区的平均核苷酸变异率为3．65％，其核苷酸变异类型包括转换、颠换和缺失3种形式，其中以转换最为常见。核苷酸变异位点较多，且个体之间差异大，说明该5匹中国蒙古马的mtDNA D-Loop高变区多态性丰富。     

蒙古马与锡林郭勒马的产肉性能及肉品质量的比较

通过对7匹2－4岁蒙古马和10匹2－4岁锡林郭勒马的产肉性能及肉品质量比较，结果表明：锡林郭勒马在活体重（307.45kg)、胴体重（166.85kg)、净肉重（138.80kg)等方面显著高于蒙古马（216.03kg、115.87kg、91.48kg)，屠宰率增加0.63%，净肉率增加2.8%；平均骨重（28.05kg)和蒙古马（24.39kg)差异不显著，骨肉比（1：4.95)高于蒙古马（1：4．38）。从这两个品种马肉的化学组成、脂肪品质、肌纤维的测定结果可看出，两品种马肉在理化性质上无显著的变化，具有马肉的水分大（＞70％）、蛋白质含量高（＞20％）、脂肪含量低（＜5％）、不饱和脂肪酸高（60％－65％）等一般特征。分析比较结果表明，锡林郭勒马的生产性能和肉品等级较高，且拥有蒙古马肉的营养特点。     

ZD制剂对马肌肉软组织损伤的超微结构观察

为了解ZD制剂对马损伤的肌肉软组织超微结构的影响,试验选择4匹健康蒙古马,将3匹造模后随机分为试验药物组、阳性药物组、阴性对照组,每组1匹,另1匹设为空白对照组。试验药物组马使用ZD制剂治疗,阳性药物组马使用按摩乳,阴性对照组马匹使用空白膏,药物用法用量同试验药物组,空白对照组不做任何处理。在用药后第7,14,21天用一次性取样器在各组马匹损伤处随机分3点采样进行电镜观察。结果表明:试验第7天,与正常马骨骼肌比,阴性对照组、试验药物组和阳性药物组马肌纤维断裂、排列紊乱,但与试验药物组比,阳性药物组肌纤维断裂、肿胀和排列紊乱更加明显。试验第14天,试验药物组和阳性药物组马肌纤维断裂、肿胀和排列紊乱情况得到修复,各组马线粒体数量均明显减少,但试验药物组改善明显。试验第21天,试验药物组马肌纤维形态、线粒体形态和数量基本恢复正常,与正常马骨骼肌电镜照片相似;阳性药物组马肌纤维断裂和明暗带不清晰的情况仍存在;阴性对照组马肌纤维断裂、肿胀和排列紊乱情况仍未明显改善,线粒体仍存在数量减少、肿胀、嵴消失等异常。正常马骨骼肌肌原纤维中暗带长约2.0μm,明带长约1.0μm,马骨骼肌细胞线粒体多为卵圆形颗粒状或长杆状,肌细胞核呈扁椭圆形。说明ZD制剂可促进马骨骼肌肌原纤维、线粒体、肌细胞核形态改善与组织修复,效果优于阳性药物组。     

斑点蒙古马TRPM1和RFWD3基因分型及分子选育方案制定

被毛颜色不仅是品种和个体鉴别的重要依据,而且也是制定选育方案时需要考虑的重要性状之一。TRPM1和RFWD3是决定豹点毛色表型的2个关键候选基因。本研究首次将60匹斑点蒙古马为研究对象,对2个基因进行分型,从分子水平对其毛色做出鉴定,验证斑点蒙古马的毛色基因型是否与已公布的马豹点毛色研究结果一致。结果:60匹斑点蒙古马TRPM1基因与对照组(非斑点毛色马)相比,均存在C/T(ECA1 108 249 293)杂合位点;通过测定RFWD3基因的3′调控区序列, ECA3 23 658 447上存在3种不同的多态性,分别为T/T、T/G和G/G。本研究为今后斑点蒙古马的分子育种奠定理论基础。     

蒙古马高负荷运动训练前后COX4I2、FABPpm和CAV-1基因表达量的研究

为研究蒙古马高负荷运动训练前后COX4I2、FABPpm和CAV-1基因表达量的影响,实验选择3匹5岁雌性蒙古马,采用qRT-PCR方法检测为期4个月的强度递增高负荷运动训练后蒙古马臀中肌3种基因在mRNA水平的表达变化。结果表明:经训练后蒙古马肌肉中COX4I2和FABPpm基因表达量均显著升高(P<0.05);CAV-1基因表达量也有升高(P>0.05)。可见,COX4I2、FABPpm和CAV-1基因与蒙古马的运动能力存在密切关系。     

基于高速双倍自助法对蒙古马系统发育树的研究

旨在利用高速双倍自助法研究蒙古马系统发育树的问题。本研究选用30匹蒙古马的mtDNA D-loop区碱基序列,其中包括5个蒙古马类群(巴尔虎马、三河马、乌审马、乌珠穆沁马、锡尼河马)各6匹,对其所有105个可能系统树(普氏野马作为外群,6匹)进行分析,利用生物信息学软件Mega6和PAMAL4.9,以最大似然法估计蒙古马的最大似然系统树。最后利用生物信息软件CONSEL、R3.0中的SDBP包等计算105个候补系统树的基于高速双倍自助法的可靠度(speedy double bootstrap P-value,SDBP)。结果表明,SDBP值最大的蒙古马系统树共有3个分支,巴尔虎马与乌审马合并为一个分支,具有较近的遗传关系;乌珠穆沁马与锡尼河马具有较近的遗传关系;三河马独立于其他4种蒙古马,构成一个系统发育分支。SDBP值最大的蒙古马系统树与三河马和其他4类蒙古马具有较远的预测血缘关系是一致的,同时此结果的系统树也具有最大似然值。本研究结果还表明,采用最大似然法结合SDBP值分析蒙古马的系统拓扑关系是较最大似然法结合渐近无偏(approximately unbiased,AU)值以及结合自助(bootstrap P-value,BP)值分析蒙古马系统拓扑关系更有效。同时也比非加权组平均法(unweighted pair-group method with arithmetic means,UPGMA)有效,而与邻接法(neighbor joining,NJ)得到了相近的结论。上述结果提示,我们的研究结果使蒙古马的进化理论奠定在更坚实的遗传学基础上,可推动蒙古马的进化理论进一步发展与完善。     

蒙古马马鼻狂蝇蛆病细胞免疫反应

为了研究马鼻狂蝇蝇蛆感染蒙古马的细胞免疫反应,2015年在内蒙古呼和浩特市郊区屠宰场收集感染马鼻狂蝇蝇蛆的蒙古马4匹,健康蒙古马2匹,采集马的咽部和咽后淋巴结进行HE染色和免疫组化试验,并利用graphpad prism5.0软件对感染组和对照组的CD3、CD20、CD68阳性细胞表达率进行分析。免疫组化结果显示,感染组与对照组相比,咽部组织内B淋巴细胞表达率差异显著(P0.05),T淋巴细胞、巨噬细胞表达率差异极显著(P0.001)。咽后淋巴结内的T淋巴细胞的表达率差异显著(P0.05),B细胞和巨噬细胞表达率差异极显著(P0.001)。在咽部和咽后淋巴结B淋巴细胞增多说明,获得性免疫反应发挥作用以后产生了针对马鼻狂蝇蝇蛆的特异性免疫球蛋白。T淋巴细胞数量增多说明,在抗原提成细胞中T细胞识别抗原产生,宿主已经产生了获得性免疫反应。巨噬细胞数量增多,说明巨噬细胞在感染组的抗原提成中发挥着巨大作用,能够提呈抗原而且发起细胞介导免疫反应。     

不同营养水平对羔羊肌肉组织学性状的影响

本试验旨在研究羔羊在营养限制期和补偿期背最长肌、半腱肌和股二头肌的肌纤维直径、面积和密度的变化规律.选用蒙古羔羊(羯羊)80只,随机分为4个组,即:对照组(C组)、中等受限1组(R1组)、中等受限2组(R2组)、严重受限组(R3组),进行营养限制期60 d和补偿期90 d的羔羊生长试验.限制期4个组饲粮能氮水平分别为代...     

Immunohistochemical identification and fiber type specific localization of protein kinase C isoforms in equine skeletal muscle

OBJECTIVE: To investigate whether protein kinase C (PKC) isoforms are expressed in equine skeletal muscle and determine their distribution in various types of fibers by use of immunofluorescence microscopy. ANIMALS: 5 healthy adult Dutch Warmblood horses. PROCEDURE: In each horse, 2 biopsy specimens were obtained from the vastus lateralis muscle. Cryosections of equine muscle were stained with PKC isoform (alpha, beta1, beta2, delta, epsilon, or zeta)-specific polyclonal antibodies and examined by use of a fluorescence microscope. Homogenized muscle samples were evaluated via western blot analysis. RESULTS: The PKC alpha, beta1, beta2, delta, epsilon, and zeta isoforms were localized within the fibers of equine skeletal muscle. In addition, PKC alpha and beta2 were detected near or in the plasma membrane of muscle cells. For some PKC isoforms, distribution was specific for fiber type. Staining of cell membranes for PKC alpha was observed predominantly in fibers that reacted positively with myosin heavy chain (MHC)-IIa; PKC delta and epsilon staining were more pronounced in MHC-I-positive fibers. In contrast, MHC-I negative fibers contained more PKC zeta than MHC-I-positive fibers. Distribution of PKC beta1 was equal among the different fiber types. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that PKC isoforms are expressed in equine skeletal muscle in a fiber type-specific manner. Therefore, the involvement of PKC isoforms in signal transduction in equine skeletal muscle might be dependent on fiber type.     

蒙古马高负荷运动训练前后MSTN基因、CKM基因表达量分析

为探讨蒙古马高负荷调教训练前后MSTN、CKM基因的表达情况,实验首先采用SYBR GreenⅠ荧光定量RT-PCR方法对4个常用内参基因(TTN、18s r RNA、GAPDH、β-actin)在臀中肌内的稳定性进行评估;确定内参基因后,对MSTN、CKM基因转录水平进行测定。结果表明:在蒙古马臀中肌中稳定度依次为GAPDH(1.087)β-actin(1.211)TTN(1.285)18s r RNA(1.460),选取稳定度最高的GAPDH基因作为内参基因;高负荷运动训练后MSTN基因m RNA表达量较训练前上调,而CKM基因则下调。本研究为蒙古马运动性能相关基因的研究奠定了基础。     

Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses

Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.     

蒙古斑点马黑、白毛色区域皮肤中相关基因的表达研究

本试验以蒙古斑点马为研究对象,分别对蒙古斑点马体躯白色区域和黑色区域皮肤的表型和差异表达基因进行分析并验证,试图解析蒙古斑点马毛色形成的分子机制,为今后保护和开发利用蒙古斑点马奠定基础.选择蒙古斑点马体躯白色和黑色区域皮肤制作组织切片,HE染色后在显微镜下观察其表型差异;对白色和黑色区域皮肤组织进行转录组测序,比较差异...     

Metabolic response to racing and fiber properties of skeletal muscle in standardbred and thoroughbred horses

Muscle biopsies were obtained from 17 Standardbred trotters (S) and 22 Thoroughbred racehorses (T) after races which varied in length from 1200 to 2840 m. Biopsies were taken between 2 and 14 minutes from the conclusion of the race. Histochemical and biochemical analyses were performed on all muscle samples. Standardbred horses possessed fewer Type II fibers, especially Type IIB fibers, and demonstrated a higher oxidative and lower glycolytic capacity in muscle than Thoroughbred horses. Intramuscular glycogen and lac-tate concentrations were lower in Standardbred horses after racing (mean 420 and 82 mmol/kg d.w. respectively) com-pared to Thoroughbred horses (mean 545 and 148 mmol/kg d.w. respectively). Low levels of intramuscular adenosine triphosphate (ATP) (mean S 17, T 15 mmol/kg d.w.) and creatine phosphate (mean S 28, T 42 mmol/kg d.w.) were recorded in both breeds after racing. Differences in muscle enzyme activities and metabolic response to exercise between breeds were related to the different muscle fiber compositions of Standardbred and Thoroughbred horses. Within both breeds, a high percentage of Type IIB fibers was associated with a low oxidative and high glycolytic capacity in muscle and a greater lactate accumulation after racing. In Standardbred horses low intramuscular ATP concentrations post-race were associated with a high percentage of Type IIB fibers and high lactate levels. No relationship was found between placing or speed of racing and intramuscular lactate and ATP concentrations post-race. This may have been due to many other factors which affected the outcome of a race and also due to the fact that it was not known when during a race lactate accumulation and ATP depletion occurred. Since the properties of fiber types appeared to be very different, the metabolic response within individual fiber types may be more closely related to fatigue than the metabolic response measured in whole muscle.     

Investigation of the expression and localization of glucose transporter 4 and fatty acid translocase/CD36 in equine skeletal muscle

OBJECTIVE: To investigate the expression and localization of glucose transporter 4 (GLUT4) and fatty acid translocase (FAT/CD36) in equine skeletal muscle. SAMPLE POPULATION: Muscle biopsy specimens obtained from 5 healthy Dutch Warmblood horses. PROCEDURES: Percutaneous biopsy specimens were obtained from the vastus lateralis, pectoralis descendens, and triceps brachii muscles. Cryosections were stained with combinations of GLUT4 and myosin heavy chain (MHC) specific antibodies or FAT/CD36 and MHC antibodies to assess the fiber specific expression of GLUT4 and FAT/CD36 in equine skeletal muscle via indirect immunofluorescent microscopy. RESULTS: Immunofluorescent staining revealed that GLUT4 was predominantly expressed in the cytosol of fast type 2B fibers of equine skeletal muscle, although several type 1 fibers in the vastus lateralis muscle were positive for GLUT4. In all muscle fibers examined microscopically, FAT/CD36 was strongly expressed in the sarcolemma and capillaries. Type 1 muscle fibers also expressed small intracellular amounts of FAT/CD36, but no intracellular FAT/CD36 expression was detected in type 2 fibers. CONCLUSIONS AND CLINICAL RELEVANCE: In equine skeletal muscle, GLUT4 and FAT/CD36 are expressed in a fiber type selective manner.     

Myosin heavy chain composition in normal and atrophic equine laryngeal muscle

The myosin heavy chain (MHC) composition of a given muscle determines the contractile properties and, therefore, the fiber type distribution of the muscle. MHC isoform expression in the laryngeal muscle is modulated by neural input and function, and it represents the cellular level changes that occur with denervation and reinnervation of skeletal muscle. The objective of this study was to evaluate the pattern of MHC isoform expression in laryngeal muscle harvested from normal cadavers and cadavers with naturally occurring left laryngeal hemiplegia secondary to recurrent laryngeal neuropathy. Left and right thyroarytenoideus (TA) and cricoarytenoideus dorsalis (CAD) were obtained from 7 horses affected with left-sided intrinsic laryngeal muscle atrophy and from 2 normal horses. Frozen sections were evaluated histologically for degree of atrophy and fiber type composition. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of muscle protein. Histologic atrophy was seen in all atrophic muscles and some right-sided muscles of 3 affected horses, as well as the left TA of 1 normal horse. Fiber type grouping or loss of type I muscle fibers was observed in the left-sided laryngeal muscles in all but 1 affected horse, as well as in the right muscles of 2 affected horses, and the left TA of 1 normal horse. SDS-PAGE showed 2 bands corresponding to the type I and type IIB myosin isoforms in the CAD and TA of the 2 normal horses. Affected horses demonstrated a trend toward increased expression of the type IIB isoform and decreased expression of the type I isoform in atrophic muscles. This study confirmed the presence of histologic abnormalities in grossly normal equine laryngeal muscle, and it demonstrated an increased expression of type IIB MHC with a concurrent decreased expression of type I MHC in affected muscles. Evaluation of muscle fiber changes at the cellular level under denervated and reinnervated conditions may aid in assessing future strategies for reinnervation or regeneration of atrophic laryngeal muscle.     

猪骨骼肌发育过程中免疫相关基因的表达分析

为探究猪骨骼肌发育过程中免疫相关基因的表达模式以及相关生物学功能,本研究根据蓝塘猪和长白猪骨骼肌10个发育时期的转录组数据,对各时期免疫相关基因的表达数目和表达量进行分析,鉴定了相邻发育时期之间以及同一时期品种间差异表达的免疫相关基因,并对后者进行功能注释分析。结果表明:在蓝塘猪与长白猪骨骼肌发育过程中存在1 129个表达的免疫相关基因;胚胎期35 d和49 d、胚胎期91 d和生后期2d这2组相邻时期之间的差异表达免疫相关基因数目最多;品种间差异表达免疫相关基因数目则在胚胎期49d以及生后期180d最多,且趋向于在蓝塘猪上调表达,并富集在NIK/NF-κB信号传导、细胞凋亡的负调控等生物学过程中。本研究表明免疫相关基因在猪骨骼肌发育过程中存在表达,长白猪与蓝塘猪骨骼肌生长发育差异可能与NF-κB介导的细胞凋亡有关。