山羊TNNT3基因可变剪切及其对骨骼肌细胞分化的作用

【目的】快速骨骼肌肌钙蛋白T（fast skeletal troponin T3,TNNT3）作为肌钙蛋白(troponin, Tn)家族成员,调节横纹肌收缩、参与骨骼肌的生长发育并影响家畜肉质性状。通过获得山羊TNNT3基因的可变剪切体,分析山羊TNNT3基因可变剪切的表达模式及其在肌细胞分化中的作用,深入解析TNNT3基因在山羊骨骼肌生长发育过程中的作用机制。【方法】基于NCBI已公布山羊TNNT3基因（NM_001314210.1）和牛TNNT3基因（XM_010821200）mRNA序列,使用软件Primer Premier 6.0设计引物,以简州大耳羊胚胎期和出生后7个阶段骨骼肌为试验材料,克隆测序获得山羊TNNT3基因的CDS区可变剪切体,利用软件ORF Finder、EditSeq、DNAMAN、ClustalW和MEGA_X_10.1.8等对序列进行生物信息学分析;进一步设计实时荧光定量（real-time PCR,RT-qPCR）及半定量引物,研究TNNT3基因剪切体在7个不同组织（背最长肌（longissimus dorsi muscle,LD）、半膜肌（semimembranosus muscle,SM）、心、肝、脾、肺、肾）和7个发育阶段（胚胎期E75、E90、E105和出生后B3、B45、B150、B300）肌肉组织（背最长肌和半膜肌）中表达模式;此外,对转录本TNNT3_3进行体外编码能力检测确定其具有编码蛋白的能力,并在山羊骨骼肌卫星细胞（skeletal muscle satellite cells,MuSCs）中过表达,观察细胞形态变化以及检测标志基因的表达变化,研究其对山羊MuSCs分化的作用。【结果】①TNNT3（NM_001314210.1）CDS区全序列主要含有18个外显子,其中外显子16/17相互排斥,转录后单一表达。克隆发现山羊TNNT3基因 5个新转录本（TNNT3_1—5）,其外显子数分别是15、15、20、16、14。②生物信息学分析结果显示山羊TNNT3基因核苷酸序列和氨基酸序列与绵羊、牛、猪等哺乳动物具有很高的一致性,而与鱼类和爬行类动物的一致性较低,说明TNNT3基因序列在哺乳动物高度保守。③TNNT3 mRNA在背最长肌、半膜肌、心、肝、脾、肺、肾7个组织中都有表达,其中在骨骼肌中高度富集(P < 0.01),心脏及肺次之,其余组织中较低;TNNT3 mRNA在背最长肌和半膜肌中的表达始终处于一个动态变化中,胚胎期TNNT3在半膜肌的表达量高于背最长肌（P<0.05）;出生后则背最长肌中高于半膜肌（P<0.05）。④山羊TNNT3基因转录本TNNT3_3重复出现保守的外显子9—11（138bp）,体外翻译实验显示其可编码蛋白且蛋白大小与预期基本相符（37 kD）;相较于对照组,在山羊MuSCs中过表达该转录本使肌分化标志基因Myomaker、MyoG和MyH4 mRNA极显著升高(P < 0.01)。【结论】获得了山羊TNNT3基因具有完整CDS区5个新可变剪切体,TNNT3主要在肌肉组织（背最长肌和半膜肌）中高表达,在哺乳动物中高度保守且促进成肌分化。初步表明TNNT3基因在动物肌肉生长发育中具有重要的生物学功能。     

论山区生态环境保护与山羊养殖对策

因为缺乏统一而科学的长远规划,山羊养殖业在山区发展面临了许多新的问题,笔者尝试对此进行简单的讨论.     

Comparison of DNA probe test and cultivation methods for detection of Mycobacterium avium subsp paratuberculosis in caprine and ovine faeces

OBJECTIVE: To compare a DNA probe test with two cultivation methods for the detection of Mycobacterium avium subsp paratuberculosis in goat and sheep faeces. DESIGN: Comparison of the results of the three methods with histological examination as the reference standard. PROCEDURE: Faecal specimens were obtained from goats and sheep originating from flocks known to be affected with paratuberculosis and tested for Mycobacterium avium subsp paratuberculosis with a DNA probe test and two cultivation methods (old conventional culture and new double incubation method in Herrold's and Lowenstein-Jensen medium). RESULTS: In goats, the sensitivity of the various tests were for the DNA probe test 17.2%, for the double incubation culture method 25.4% and for the old conventional culture method 22.8% using the histopathological results as reference. In sheep the sensitivity of the various tests were for the DNA probe test 13.2%, for the double incubation culture method 8.8% and for the old conventional culture method 5.9% using the histopathological results as reference. The specificity of the above tests was 100% in goats and sheep and the specificity of the double incubation culture method in goats was 91%. CONCLUSIONS: The DNA probe test is a rapid and specific test that could be used in a control program if the sensitivity of the test were improved and possibly in combination with another test.     

影响波尔山羊超排效果的因素

对204只(次)波尔山羊采用C IDR+FSH+PG法超数排卵(以下简称超排),发情配种后第6天采用手术法从子宫角采胚,采胚成功率为92.16%(188/204),头均获胚(16.68±7.89)枚,其中头均获可用胚(14.11±8.37)枚,可用胚率84.66%(2 654/3 135)。对不同国家产FSH、不同剂量FSH、注射LRH+P4、不同季节、重复超排、左右侧卵巢等因素对波尔山羊超排效果的影响进行了研究。结果表明,以日本和国产(动物所)FSH超排效果为佳,头均获可用胚分别为(15.66±8.26)枚(n=89)和(15.39±6.91)枚(n=23);日本产FSH 14~16 mL组效果最佳,头均获可用胚(19.63±8.96)枚(n=19);注射LRH+P4组头均获可用胚(16.04±8.84)枚(n=80),极显著高于对照组(12.16±7.68)枚(n=62);春季、秋季、冬季超排头均获可用胚分别为(10.86±5.10)枚(n=7)、(11.30±8.29)枚(n=23)、(11.00±6.17)枚(n=10),差异不显著;重复超排(间隔12月)第1次头均获可用胚(15.08±5.12)枚(n=26),第2次头均获可且胚(16.92±8.32)枚(n=26)。     

日粮维生素E水平对山羊精液品质的影响

采用4×4拉丁方设计,给金堂黑山羊饲喂维生素E浓度不同(常量:30 mg/kg维生素E,10,50,100倍常量的添加量)而其他营养成分均一致的4种日粮,探索日粮中添加维生素E对山羊精液品质的影响。结果表明:日粮中添加维生素E有助于提高山羊采精量、精子密度和鲜精活率,改善其精液品质(P<0.05);随着日粮中维生素E浓度的增加,山羊采精量、精子密度和鲜精活率也随之改善,添加量为常量的50倍(1500 mg/kg)时,精子密度和鲜精活率最佳(P<0.01)。当添加量超过常量50倍时(100),精子密度和鲜精活率有所降低,但与添加量为50倍的试验组差异不显著(P>0.05)。     

Traders' preference for goat characteristics in selected markets of Pangasinan, Philippines

Preference for goat characteristics was determined from 42 traders in six markets in Pangasinan, an important trading center for goats in Luzon, the Philippines, and analyzed using a hedonic price model to determine whether these characteristics are important price determinants of goats. This information is important to goat raisers to respond to the needs of the local market, and thus to achieve a better price and higher income in goat raising. Meatiness, size, breed, sex and age (in descending order) were the dominant goat characteristics preferred by traders. Meatiness had the largest positive influence on price, with a price premium of US$6.24 (US$1.00 = PhP 53), whereas the age of the animal had the smallest influence at US$3.72. However, size and meatiness are manifestations of good breeds. Therefore, continued efforts to improve breeds in order to produce good quality goats are imperative to cater to market needs. Different characteristics implied different price premiums. Traders offered a higher price premium for better goat characteristics; thus, the improvement of these characteristics could result in higher returns for goat raisers.     

The Distribution of Soluble Copper- and Zinc-Binding Proteins in Liver and Kidney of Chronic Copper-Poisoned Goats

The distribution of copper and zinc among the soluble proteins in the liver and kidney from chronic copper-poisoned goats was examined after gel filtration of the proteins. The concentrations of copper in the liver and kidney cortex from five experimentally copper-poisoned goats were: 550–810 µg/g liver and 190–420 µg/g kidney cortex (wet weight). In general the copper-binding proteins from, both the liver and kidney samples were separated into two different fractions with approximate molecular weights (m.w.) of > 65,000 and 10,000, respectively. From the liver samples, varying amounts of copper were eluted in a fourth fraction with m.w. < 2,000. In the majority of kidney samples the dominating copper-binding protein fraction was the high molecular weight fraction. Absolute amounts of copper recovered in the metallothionein-like protein fraction were nearly the samt for all samples investigated. The distribution of zinc-binding proteins in both liver and kidney samples was nearly the same. The high molecular weight fraction dominated, and no zinc was bound to metallothionein-like proteins.     

波尔山羊精液高倍稀释试验研究

为充分发挥波尔山羊种公羊的作用,对精液高倍稀释各个技术环节进行了多方面的试验研究。采用新鲜羊奶稀释液进行了7个不同稀释倍数的试验,结果表明5倍与8倍稀释组情期受胎率分别为91.4%和91.5%,差异不显著(P>0.05);其它各组之间差异均显著或极显著(P<0.05或P<0.01);各组的情期受胎率随着稀释倍数的增大逐渐下降,15倍稀释组为84.0%,20倍为76.0%,25倍为68.8%,30倍为53.3%。25倍以下的稀释倍数,输入有效精子数在1000万以上,生产上是可以应用的。稀释液的筛选试验表明鲜奶稀释液受胎率较高;二次稀释法比一次稀释提高受精率9%(P<0.05);子宫颈深部输精比子宫颈口输精提高受胎率95.5%(P<0.01);10倍以上稀释有利于精液常温保存。