VEGF在绵羊生殖管道中的表达规律

以不同发情周期雌性绵羊子宫、输卵管为研究对象,采用免疫组织化学技术,针对血管内皮生长因子（VEGF）在绵羊子宫、输卵管的表达、定位和变化规律进行了检测,同时应用相关图像分析软件对抗原染色强度进行了定量分析。结果表明：输卵管在发情0～15d,VEGF表达量在第9天达到峰值后经历波动逐渐下降过程,输卵管内膜上皮细胞是VEGF抗原的主要靶细胞;而子宫角在发情0～15d,VEGF表达量在第5天达到峰值后经历波动逐渐下降过程,子宫内膜固有层及腺体周围细胞为VEGF抗原的主要靶细胞。该研究结果为绵羊生产中进一步提高受胎率和妊娠率及频密产羔等技术的应用提供了科学依据。     

Effect of L-lysine Added in Diluent on the Fresh Semen Quality of Dorper Sheep

In order to explore the effect of different L-lysine levels added in semen dilution on the fresh Dorper sheep quality stored at liquid state,3 health Dorper ram were used to collect semen which were equal-packaged and added to solution containing different levels (0,0.1,0.2,0.3,0.4 and 0.5 g) L-lysine in 120 mL diluent and at 0,6,24,30,48,54,72 and 78 h,the sperm motility,membrane integrity and acrosome integrity were detected.The results showed that the sperm motility in groups added 0,0.1 and 0.2 g L-lysine were higher than other groups,and that in 0.4 and 0.5 g L-lysine groups decreased faster than other groups;The sperm motility in 0.5 g L-lysine group reduced to 0 at 30 h,and it reduced to 0 at 48 h in the group added 0.4 g L-lysine;The effective survival time and survival index in the group added 0.1 g L-lysine was higher than other groups (P< 0.05);The membrane integrity of 0.1 g group was the highest,that had no significant difference with control group from 0 to 54 h (P >0.05),while after 72 h the two groups had significant differences (P< 0.05).The membrane integrity in 0.4 and 0.5 g groups were the worst,and that was significant different with other groups (P< 0.05);The acrosome integrity of 0.1 g group was the highest which had no significant difference with control group (P >0.05),0.4 and 0.5 g groups were the worst,the difference between them was not significant (P >0.05),while was extremely significant difference with other groups except for 0 and 6 h (P< 0.01).The results suggested that dilution added a high concentration of L-lysine could inhibit sperm quality,when the count of L-lysine was more than 0.2 g,sperm quality was significantly decreased,adding 0.1 g L-lysine could improve Dorper sheep fresh sperm quality stored at liquid state.     

白藜芦醇和褪黑素对绵羊胎儿成纤维细胞转染及卵母细胞体外成熟的影响

试验旨在利用白藜芦醇(Re)和褪黑素(MT)提高绵羊胎儿成纤维细胞电穿孔转染(电转)效率、优化卵母细胞体外成熟体系,以提高转基因动物生产效率。将绵羊胎儿成纤维细胞分为6组:对照组、添加白藜芦醇高、中、低(R^-5、R^-6、R^-7)3种浓度组及白藜芦醇和褪黑素联合添加组(R^-5M^-5、R^-6M^-7),通过电转效率和细胞凋亡情况确定电转体系优化方案;通过对细胞周期、线粒体膜电位及活性氧(ROS)的检测研究其作用机制。将绵羊卵母细胞分为对照组及白藜芦醇和褪黑素(R^-5M^-5、R^-6M^-7)联合添加组,通过绵羊卵母细胞体外成熟后卵丘细胞扩展、第一极体排出及体外受精胚胎的卵裂率检测其对卵母细胞体外成熟的作用。研究结果显示,分离的绵羊胎儿成纤维细胞P3代增殖旺盛,细胞生长曲线呈典型的S型;与对照组相比,所有试验组胎儿成纤维细胞的电转效率均显著提高(P<0.05),R^-5M^-5组电转效率最高;R^-6M^-7组显著降低细胞凋亡率(P<0.05),R^-5、R^-6、R^-5M^-5和R^-6M^-7组均显著促进细胞增殖(P<0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组G1期细胞增加,S期细胞相对减少,但差异均不显著(P>0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组成纤维细胞内ROS水平均显著降低(P<0.05),R^-6、R^-5M^-5和R^-6M^-7组线粒体膜电位均显著增加(P<0.05);R^-6M^-7组显著促进绵羊卵丘细胞扩展、提高卵母细胞第一极体排出率和体外受精胚胎发育能力(P<0.05)。综上,白藜芦醇和褪黑素可以优化绵羊胎儿成纤维细胞电转体系,提高卵母细胞体外成熟效率,为白藜芦醇和褪黑素在转基因动物生产中的应用提供参考依据。     

不同饲料效率与绵羊瘤胃组织形态学关系

【目的】利用单栏系统测定个体的饲料效率相关性状与瘤胃组织形态学指标,探讨绵羊饲料效率与瘤胃组织形态的关系,为解析绵羊饲料效率性状的影响因素研究提供基础数据。【方法】随机选取出生日龄相近、系谱信息详细、健康状况良好的187湖羊公羔,56 d断奶后转入单栏饲养,过渡期14 d,预饲期10 d,正试期100 d。正试期内所有羊只仅饲喂颗粒饲料,自由采食及饮水,并在80 d和180 d晨饲前空腹测定其体重（body weight,BW）和80—180 d间的采食量（feed intake,FI）,计算平均日增重（average daily gain,ADG）、中期代谢体重（metabolic body weight, MBW）、饲料转化率（feed conversion rate,FCR）和剩余采食量（residual feed intake,RFI）等饲料效率相关性状并对其进行描述性统计,于180 d饲养结束后屠宰采集瘤胃腹囊组织1 cm²保存于4%甲醛溶液中,用于制作组织切片并观测其瘤胃乳头长度、宽度和肌层厚度。最后将其与饲料效率相关性状进行相关分析和方差分析。【结果】饲料效率相关性状的变异系数均大于10%,且剩余采食量最大与最小的个体每天的剩余采食量之差达0.57 kg。饲料效率相关性状间的表型相关分析表明剩余采食量与饲料转化率（r= 0.68）和采食量（r= 0.48）呈极显著正相关（P<0.01）,与初始体重（r=0）、末期体重（r= -0.01）和平均日增重（r= -0.02）无显著相关（P>0.05）。饲料效率相关性状与瘤胃组织形态相关性分析发现,瘤胃乳头长度与平均日增重、采食量、初始体重和末期体重呈显著或极显著正相关（P<0.05或P<0.01）,肌层厚度与平均日增重、采食量和末期体重呈显著或极显著正相关（P<0.05或P<0.01）,而剩余采食量和饲料转化率与瘤胃组织形态无显著相关。不同RFI组羔羊采食量、饲料转化率和瘤胃肌层厚度存在显著或极显著差异（P<0.05或P<0.01）,瘤胃乳头长、宽无显著差异（P>0.05）,其中High-RFI组羔羊采食量和饲料转化率极显著高于Low-RFI组（P<0.01）,肌层厚度显著高于Medium-RFI组（P<0.05）;不同FCR组羔羊的剩余采食量、采食量、ADG、初始体重、末期体重和乳头长度存在显著或极显著差异（P<0.05或P<0.01）,肌层厚度和乳头宽度差异不显著（P>0.05）,其中High-FCR组羔羊剩余采食量、采食量、ADG、初始体重和末期体重均显著或极显著高于Low-FCR组（P<0.05或P<0.01）,Medium-FCR组羔羊乳头长度显著长于Low-FCR组（P<0.05）;除瘤胃乳头宽度外,不同FI组羔羊的上述指标均存在显著或极显著差异（P<0.05或P<0.01）,且High-FI组羔羊的剩余采食量、饲料转化率、ADG、初始体重、末期体重、肌层厚度和乳头长度均显著或极显著高于Low-FI组（P<0.05或P<0.01）;不同ADG组羔羊采食量、饲料转化率、初始体重、末期体重和肌层厚度均存在显著或极显著差异（P<0.05或P<0.01）,乳头长度和乳头宽度无显著差异（P>0.05）,其中High-ADG组羔羊采食量、剩余采食量、初始体重、末期体重和肌层厚度均显著或极显著高于Low-ADG组,饲料转化率则极显著低于Low-ADG组。【结论】剩余采食量与采食量和饲料转化率等饲料效率性状呈极显著正相关,表明其可作为衡量饲料效率的潜在指标。剩余采食量和饲料转化率与瘤胃组织形态学指标无显著相关,采食量和平均日增重与瘤胃乳头长度和肌层厚度呈显著正相关,表明羔羊瘤胃组织形态对采食量和增重有显著影响,但其进一步的作用机制有待深入研究。     

Effect of Lysine Supplement on the Digestion and Metabolism of the Rumen in Small-tail Han Sheep

5 male Small-tail Han sheep with the body weight of 45 kg at 1.5 years old,fitted with permanent rumen fistula,to study of the effect of the supplement of the untreated commercial lysine on the rumen microbiocoenosis,rumen metabolism and whole body metabolism by 5×5 Latin square design (5 groups:control,control with low urea (equal nitrogen),low lysine,control with high urea (equal nitrogen),high lysine).The amounts of lysine-HCl supplement were 0,4.0 and 8.0 g/kg diet (as DM basis) in each group respectively,with two urea supplement control groups of equal amount of nitrogen.The results showed that by supplement of 4.0 and 8.0 g/kg of lysine-HCl diet (as DM basis) respectively,the voluntary intake of dry matter of sheep were increased by 5.5%(P>0.05) and 11.8%(P<0.05) respectively.The total amount of rumen bacteria were increased by 18.9%(P<0.01) and 23.9%(P<0.01) respectively,as the amount of rumen Coccus was increased by 21.2%(P<0.01) and 30.1%(P<0.01) respectively.The amount of rumen big Bacillus was decreased by 16.7%(P<0.05) and 33.3%(P<0.01) respectively.The total VFA of was increased by 9.6%(P<0.05) and 12.2%(P<0.01) respectively,as the butyric acid was increased by 12.8%(P>0.05) and 20.2%(P<0.01) respectively.The apparent digestibility of organic matter was increased by 6.4%(P>0.05) and 10.0%(P<0.05) respectively,the apparent digestibility of crude protein was increased by 7.0%(P>0.05) and 18.3%(P<0.01) respectively;And nitrogen retention was increased by 46.4%(P>0.05) and 110.7%(P<0.01) respectively.However,there was no effect of lysine supplement on the amounts of the rumen protozoa and fungi,and the concentration of rumen ammonia nitrogen.It was concluded that the supplement of untreated commercial lysine to adult sheep would increase the voluntary intake,digestibility,nitrogen retention the rumen microbiocoenosis and the total amount of rumen bacteria,especially the rumen Coccus of adult sheep.The effect of lysine supplement could not be replaced by urea supplement completely.     

胚胎冻融技术及其在我国肉羊产业中的应用

胚胎冻融技术可使胚胎移植不受时间、地点的限制，从而达到提高胚胎移植效率的目的，研究和优化胚胎冻融技术对胚胎移植工程的发展具有重要的意义。主要对胚胎冻融技术的原理、特点及其在肉羊产业中的应用情况进行了综述，旨在阐明胚胎冻融技术对我国肉羊产业发展的重要作用。     

绵羊FGF10基因克隆、序列分析及其在毛囊发育中的表达分析

本试验旨在获得中国美利奴羊成纤维细胞生长因子10（fibroblast growth factor 10，FGF10）基因的编码区（CDS）全长序列并进行生物信息学分析，随后对FGF10基因在中国美利奴羊毛囊发育过程中的表达特征进行分析，明确其在中国美利奴羊毛囊发育过程中的表达模式，为进一步研究FGF10 mRNA表达水平与中国美利奴羊毛囊生长发育的表达调控机制奠定理论基础。采用PCR扩增获得中国美利奴羊FGF10基因CDS，并克隆到zero PCR@^TM-Blunt进行测序验证；利用实时荧光定量PCR技术检测FGF10在中国美利奴羊毛囊发育过程中的表达差异。结果表明，绵羊FGF10基因CDS长度为696 bp（序列上传GenBank，获得登录号：MT872422），编码231个氨基酸，与牛和山羊的氨基酸序列同源性达100%，存在1个信号肽和1个跨膜结构域，其为分泌通路信号蛋白；实时荧光定量PCR分析表明，FGF10基因在中国美利奴羊毛囊发育过程中均表达，在毛囊发育第85天表达最高，显著高于其他毛囊发育时期（P<0.05）。本研究获得中国美利奴羊FGF10基因完整的编码区序列和毛囊发育过程中的表达特征，生物信息学分析发现，FGF10基因编码区序列具有物种间的保守性，同时FGF10在绵羊毛囊不同发育阶段的皮肤组织中表达，由此表明，FGF10基因可能在绵羊毛囊的生长发育过程中发挥重要的生物学作用。     

Bioinformatics Analysis of Bone Morphogenetic Protein 4 (BMP4) of Sheep

The assay was aimed to explore the biological characteristics of bone morphogenetic protein 4 (BMP4) of sheep,NCBI,DNAMAN DNAStar,TMHMM Server v.2.0,PsortⅡ,SignalP various bioinformatical softwares were used to speculate the physical and chemical properties,hydrophobic property,phosphorylation site,conservative structure domain,protein secondary structure of BMP4 protein.Also,the three-dimensional structure was forecasted with the SWISS-MODEL Workspace software.The results indicated that the BMP4 of sheep had high homologies with the BMP4 of various species.The encoded protein was a hydrophilic protein which was unstable.There was no transmembrane regions and it was likely to be located in the nucleus.What was more,there was signal peptide and eighteen phosphorylation sites.Through the forecast of functional domains,the protein had two functional domains,including the transforming growth factor-beta (TGF-beta) superfamily and TGF-beta propeptide superfamily.The result was consistent with the function of BMP4 gene family,it also demonstrated that BMP4 was a growth factor and it had the function of signal transduction.The amino acid homology between the predicted 3D structure of protein and template 3bmp.1.A was 88.29%.The bioinformatics analysis of BMP4 gene could provide reference for the further study in practice.     

日粮钙水平对空怀期云南半细毛羊生长性能及血清生化指标和繁殖相关激素的影响

试验旨在探讨日粮钙水平对空怀期云南半细毛羊生长性能、配种前血清生化指标和繁殖相关激素的影响,为开展空怀期云南半细毛羊营养需要量研究提供参考。选用50只体况良好、体重相近、38月龄的经产2胎空怀云南半细毛羊,随机均分为5组,分别饲喂0.40%(无外源钙)、0.45%、0.53%、0.66%、0.85%5个钙水平的日粮。预试期14d,正试期30d。结果表明:0.53%钙水平组云南半细毛羊的总增重、平均日增重、平均日采食量、耗料增重比等指标相对优于其他组;0.40%钙水平日粮组的血清钙最高,血清碱性磷酸酶、甲状旁腺素、钙、磷组间差异不显著(P>0.05);繁殖相关激素各组间表现不一,0.40%钙水平组雌二醇水平最高(P>0.05)。在日粮磷水平为0.20%水平条件下,0.53%钙水平组的空怀期云南半细毛羊表现出较好的生长性能,可为临配种前生理生化和激素水平提供较好准备;增加日粮钙水平,未见对以上指标有明显的正向增强作用,可能与日粮钙磷比失衡有关。     

颗粒日粮中添加柠条对滩羊生长性能、血液生化指标、瘤胃发酵及羊肉品质的影响

[目的]研究颗粒日粮中添加柠条对滩羊生长性能、血液生化指标、瘤胃发酵及羊肉品质的影响。[方法]选择体重相近的健康去势滩羊20只,随机分成两组,每组10只;对照组饲喂含有10%菊花粕的颗粒日粮,试验组饲喂含有6%柠条和4%菊花粕的颗粒日粮;开展周期为50天的育肥试验,测定滩羊的生长性能指标;育肥试验结束后,测定滩羊血液生化指标;屠宰后测定滩羊的屠宰性能指标,并取瘤胃液测定瘤胃发酵指标;取背腰最长肌,测定粗脂肪、粗蛋白、氨基酸含量以及脂肪酸组成。[结果]试验组和对照组滩羊的初始体重和终末体重无显著（P>0.05）差异;试验组平均日增重较对照组降低10.84%,差异不显著（P>0.05）;对照组平均日采食量显著（P<0.05）高于试验组;两组试验羊料肉比相同。试验组滩羊血清中的总蛋白含量显著（P<0.05）高于对照组,其他血液生化指标无显著（P>0.05）差异。对照组滩羊屠宰率比试验组高1.59%,差异不显著（P>0.05）;两组试验羊肝脏重、肝脏指数、心脏和肺脏重、心脏和肺脏指数差异不显著（P>0.05）。试验组滩羊瘤胃液中丁酸浓度显著（P<0.05）高于对照组,异戊酸浓度显著（P<0.05）低于对照组。对照组和试验组滩羊背腰最长肌中分别检测出18种和20种脂肪酸,粗蛋白含量分别为17.51%和20.92%,粗脂肪含量分别为3.22%和3.89%,总氨基酸含量分别为13.25%和14.09%。[结论]颗粒日粮中加入柠条对滩羊生长性能、血液生化指标和屠宰性能影响不明显,但可改善瘤胃发酵,丰富羊肉中脂肪酸组成及含量,增加肉中粗蛋白、粗脂肪和氨基酸含量。