Optimizing injection time of GFP plasmid into sheep zygote     

Mohammadreza Ebrahimi  Laura Mara  Bernardo Chessa  Fabrizio Chessa  Abbas Parham  Maria Dattena 《Reproduction in domestic animals》2021,56(3):467-475

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.  相似文献   

复合双歧杆菌制剂对肉羊生长性能、养分表观消化率及血液生化指标的影响     

曹健  李果  曹亮 《中国饲料》2021,(3):64-67

为研究复合双歧杆菌制剂对肉羊生长性能、养分表观消化率及血液生化指标的影响,试验选择健康、体重相近的杜寒杂交肉羊60只随机分成4组,每组15个重复,每个重复1只,1组为对照组饲喂基础日粮,试验2、3、4组分别在基础日粮中添加2.5%、5.0%、10%的复合双歧杆菌制剂,预试验7 d,试验期56 d。结果表明:(1)试验3、4组的平均日增重较1组比分别提高13.5%、9.3%(P<0.05),料重比较1组比分别降低10.3%、9.4%(P<0.05),试验2、3、4组的干物质采食量均高于1组(P>0.05);(2)试验2、3、4组的干物质、钙、磷、酸性洗涤纤维表观消化率均高于1组(P>0.05),试验3、4组的粗蛋白质、中性洗涤纤维消化率较1组比分别提高17.2%、15.3%、12.5%、9.1%(P<0.05);(3)试验3、4组血清中的总蛋白(TP)含量较1组比分别提高16.0%、12.9%(P<0.05),试验2、3、4组血清中的白蛋白(ALB)含量均高于1组(P>0.05),试验2、3、4组血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)含量均低于1组(P>0.05),试验3、4组血清中尿素氮(BUN)含量较1组比分别降低15.4%、13.8%(P<0.05)。综上,不同水平的复合双歧杆菌制剂可以提高肉羊生长性能、养分表观消化率,且对血液生化指标无明显影响,以5.0%添加为适宜。  相似文献   

计算机视觉技术在智慧羊场中的研究进展     

姚州  谭焓  田芳  周勇  章程 《中国饲料》2021,1(7):7-12

智慧羊场具有科学化、标准化管理的优点,是羊场转型发展的趋势。计算机视觉技术是人工智能的核心,可以实现非接触性、自动化、实时监测羊的个体信息。本文总结并分析了计算机视觉在羊场的个体识别方法、行为识别方法、体尺与体重估测、疾病监测的研究现状,并指出计算机视觉在智慧羊场的进一步发展趋势。 [关键词] 计算机视觉|智慧羊场|个体识别|行为识别|体尺与体重估测|疾病监测  相似文献   

妊娠后期营养限制对蒙古绵羊胎儿生物原胺类神经递质的影响     

秦宇龙  马驰  杨欢  訾阳  乔依娜  刘迎春  高峰 《动物营养学报》2021,33(1)

本试验旨在研究妊娠后期营养限制对蒙古绵羊胎儿生物原胺类神经递质的影响。选择健康、体况相近的蒙古绵羊18只,对其进行同期发情、配种后,从妊娠第90天开始,随机分为3个组:营养限制1组[NG1组,n=6,代谢能(ME)=0.175 MJ/(kg BW 0.75·d)]、营养限制2组[NG2组,n=6,ME=0.330 MJ/(kg BW 0.75·d)]和对照组[CG组,n=6,ME=0.670 MJ/(kg BW 0.75·d)],在妊娠第140天时屠宰,取各组胎儿及其脑组织和血液。结果显示:NG1组胎儿的脑重较CG组显著增高(P<0.05),并且NG1组胎儿脑中5-羟色胺(5-HT)(P<0.01)、肾上腺素(EPI)(P<0.05)含量显著或极显著高于CG组,去甲肾上腺素(NA)的含量极显著低于CG组(P<0.01)。NG2组胎儿脑中5-HT含量极显著高于CG组(P<0.01),而NA(P<0.01)、多巴胺(DA)(P<0.05)含量显著或极显著低于CG组。另外,NG1组胎儿血浆中5-HT(P<0.01)、EPI(P<0.01)、NA(P<0.05)、DA(P<0.01)含量显著或极显著低于CG组;NG2组胎儿血浆中5-HT(P<0.01)、NA(P<0.01)、DA(P<0.05)含量显著或极显著低于CG组。由此得出,妊娠后期营养限制使得蒙古绵羊胎儿脑中5-HT、EPI含量升高和NA含量降低,血浆中5-HT、EPI、NA、DA含量降低。  相似文献   

Toll-like receptor signaling is changed in ovine lymph node during early pregnancy     

Jiaxuan Wu  Qiongao Zhang  Leying Zhang  Pengfei Feng  Meihong Gao  Zhenyang Zhao  Ling Yang 《Animal Science Journal》2021,92(1):e13541

Toll-like receptors (TLRs) participate in regulation of adaptive immune responses, and lymph nodes play key roles in the initiation of immune responses. There is a tolerance to the allogenic fetus during pregnancy, but it is unclear that expression of TLR signaling is in ovine lymph node during early pregnancy. In this study, lymph nodes were sampled from day 16 of nonpregnant ewes and days 13, 16, and 25 of pregnant ewes, and the expressions of TLR family (TLR2, TLR3, TLR4, TLR5 and TLR9), adaptor proteins, including myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), and interleukin-1-receptor-associated kinase 1 (IRAK1), were analyzed through real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry analysis. The results showed that mRNA and protein levels of TLR2, TLR3, TLR4, TRAF6, and MyD88 were upregulated in the maternal lymph node, but TLR5, TLR9, and IRAK1 were downregulated during early pregnancy. In addition, MyD88 protein was located in the subcapsular sinus and lymph sinuses. Therefore, it is suggested that early pregnancy induces changes in TLR signaling in maternal lymph node, which may be involved in regulation of maternal immune responses in sheep.  相似文献   

AANAT基因在滩羊不同繁殖时期卵巢中的转录差异及DNA甲基化程度分析     

张辉  李晓雨  狄冉  刘玉芳  储明星 《中国畜牧兽医》2021,48(1):226-234

试验旨在检测5-羟色胺-N-乙酰基转移酶（AANAT）基因在绵羊休情季节和繁殖季节（卵泡期和黄体期）卵巢组织中的转录差异,并分析转录差异是否由DNA甲基化修饰程度改变所导致。试验采用自然环境条件和饲养管理一致,且体重差异在0.5 kg范围内的空怀母滩羊作为试验动物,采集其休情期、卵泡期和黄体期（每个时期3只）的卵巢组织,采用SYBR染料法进行实时荧光定量PCR检测AANAT基因在滩羊不同繁殖时期卵巢组织中的转录水平。随后针对转录水平有差异的两个时期（休情期和卵泡期）的样本,利用MethPrimer 2.0在线软件预测AANAT基因启动子区和第一外显子区的CpG岛;用重亚硫酸盐测序法（BSP法）检测AANAT基因启动子区及第一外显子区的甲基化程度。试验结果显示,滩羊休情期卵巢组织中AANAT基因转录水平显著低于卵泡期的AANAT基因转录水平（P<0.05）,休情期与黄体期滩羊卵巢组织中AANAT基因的转录水平差异不显著（P>0.05）。滩羊卵巢组织中AANAT基因启动子区上存在着一个长度为173 bp的CpG岛,第一外显子区存在着一个长度为118 bp的CG岛。然而,两个甲基化岛区内的单个CpG位点甲基化程度在滩羊休情期和卵泡期之间均不存在显著差异,暗示AANAT基因的表达受甲基化修饰外的因素调控。本研究结果可为进一步探讨AANAT基因在季节性发情和卵泡成熟中的功能提供参考资料。  相似文献   

The utility of a novel formulation of alfaxalone in a remote delivery system     

Molly Patterson  Nigel Caulkett  Peter Neuhaus  Kirby Pasloske  Kathreen Ruckstuhl 《Veterinary anaesthesia and analgesia》2021,48(4):537-540

ObjectiveTo quantify induction time, reliability, physiological effects, recovery quality and dart volume of a novel formulation of alfaxalone (40 mg mL^?1) used in combination with medetomidine and azaperone for the capture and handling of wild bighorn sheep.Study designProspective clinical study.AnimalsA total of 23 wild bighorn sheep (Ovis canadensis) in Sheep River Provincial Park, AB, Canada.MethodsFree-ranging bighorn sheep were immobilized using medetomidine, azaperone and alfaxalone delivered with a remote delivery system. Arterial blood was collected for measurement of blood gases, physiologic variables (temperature, heart and respiratory rates) were recorded and induction and recovery length and quality were scored.ResultsData from 20 animals were included. Administered dose rates were alfaxalone (0.99 ± 0.20 mg kg^?1; 40 mg mL^?1), azaperone (0.2 ± 0.04 mg kg^?1; 10 mg mL^?1) and medetomidine (0.16 ± 0.03 mg kg^?1; 30 mg mL^?1). The mean drug volume injected was 1.51 mL. The median (range) induction time was 7.7 (5.8–9.7) minutes, and recovery was qualitatively smooth.Conclusions and clinical relevanceAn increased concentration formulation of alfaxalone was administered in combination with medetomidine and azaperone, and resulted in appropriate anesthesia for the capture and handling of bighorn sheep. The dart volume was small, with potential for reducing capture-related morbidity.  相似文献   

miR-301a inhibits adipogenic differentiation of adipose-derived stromal vascular fractions by targeting HOXC8 in sheep     

Bishi Zhao  Yangyang Pan  Liying Qiao  Jianhua Liu  Kaijie Yang  Yu Liang  Wenzhong Liu 《Animal Science Journal》2021,92(1):e13661

  相似文献   

Cryopreservation of ram semen: Manual versus programmable freezing and different lengths of equilibration     

Jakub Vozaf  Alexander V. Makarevich  Andrej Balazi  Jaromir Vasicek  Andrea Svoradova  Lucia Olexikova  Peter Chrenek 《Animal Science Journal》2021,92(1):e13670

The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.  相似文献   

绵羊BMP15基因B2突变可视化检测技术的建立     

胡瑞瑞  刘莉  郭涛  李雅心  王小奎  胡圣伟 《中国畜牧兽医》2021,48(3):810-817

骨形态发生蛋白15（bone morphogenetic protein 15,BMP15）基因突变对绵羊排卵率、产仔数以及繁殖力有很大影响,本研究旨在建立快速可视化检测BMP15基因B2突变的技术。将改良的扩增阻滞突变系统（amplification refractory mutation system,ARMS）与核酸染料SYBR Green Ⅰ结合,针对BMP15基因B2突变设计ARMS特异性引物,使引物下游3'端的最后一个碱基为A,并在下游引物3'端的第3位碱基处设计额外的错配,以提高引物的特异性;经ARMS PCR扩增后,向产物中加入SYBR Green Ⅰ,通过肉眼观察PCR管内的颜色变化来检测BMP15基因的B2突变。经测序发现所采集的样本均为野生型,因此,利用重叠延伸PCR构建BMP15基因B2突变型模板,测序结果显示BMP15基因B2突变型模板构建成功。ARMS PCR结果显示,ARMS特异性引物能够只扩增突变型模板,而不会扩增野生型模板,且扩增效果良好。ARMS PCR扩增后加入SYBR Green Ⅰ发现已知野生型模板与阴性对照一致,呈SYBR Green Ⅰ原始颜色橙黄色,而已知突变型模板呈亮绿色,且色差明显,肉眼可辨。用建立的可视化检测BMP15基因B2突变的技术检测50个小尾寒羊基因组DNA （随机向其中20个样本中加入构建的BMP15基因B2突变型模板）,将可视化检测的突变型样本编号与混合样本时记录的编号对比,结果表明该技术能够准确检测绵羊BMP15基因的B2突变,且准确率高达100%。将构建的BMP15基因B2突变型的模板进行梯度稀释,对本试验可视化检测体系的灵敏度进行检测,发现ARMS可视化检测技术的灵敏度达到0.006 ng/μL。因此,本研究建立的技术能够可视化地检测绵羊BMP15基因B2突变,且准确率高,有望为高繁殖力绵羊的选育提供技术支持。  相似文献