GMP玻璃化冷冻未成熟牛卵母细胞核成熟及冷冻损伤试验

本试验通过荧光染色的方法建立了未成熟牛卵母细胞在体外培养过程中第1次减数分裂的各个阶段的参考判定图谱;根据这个标准来观察毛细玻璃管(GMP)玻璃化冷冻对卵母细胞核成熟和冷冻损伤的影响。结果表明,从屠宰场废弃的卵巢表面卵泡内抽取的COCs,70%处于生发泡期,12.5%生发泡开始破裂,7.5%已开始浓缩,这说明从屠宰场获得的COCs有较高的异质性;卵母细胞在成熟培养22h时收获排出第一极体的卵母细胞,可得到丰度较高的极体-胞质染色体对称、紧密相邻的成熟卵母细胞;GMP玻璃化冷冻损伤主要有2种表现形式,首先,直接影响膜结构的完整性,包括细胞膜和核膜,这可从退化的细胞比例看出(8～24h,有21.9%～27.2%的细胞处于该阶段),其次,影响CONDENSED向MⅠ期的过渡,这可从处于CONDENSED卵母细胞的比例看出(8～24h,有24.1%～34.3%的细胞处于该阶段)。     

牛病毒性腹泻病毒检测方法研究进展

引起牛病毒性腹泻/黏膜病(bovine viral diarrhea—mucosal disease,BVD—MD)的病原为牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV),牛感染后会出现与其他腹泻病相似的症状,仅通过临床表现和病理观察很难做出准确鉴别;其次持续感染(PI)牛是该病主要传染源,如何鉴别、净化牛场中PI牛十分重要。为了检测BVDV病原和净化PI牛,选择一种最佳的检测方法非常重要。文章对目前常用的不同BVDV检测方法进行了比较分析。不同检测方法在敏感性、特异性等方面均存在差异。实际应用时应根据检测目的综合考虑,选择适宜的检测方法。     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响。结果显示,添加颗粒细胞传代接种第2天收集的20%颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P<0.05);然而单层颗粒细胞却显著抑制水牛卵母细胞体外成熟和早期胚胎发育(P<0.05)。结果表明,与单层颗粒细胞共培养相比,与颗粒细胞条件液共培养更有利于水牛卵母细胞体外成熟和早期胚胎发育。本研究为水牛卵母细胞体外成熟培养体系的完善提供一定理论依据。     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

山羊卵泡卵母细胞的采集及体外成熟效果研究

为了获取高质量的卵泡卵母细胞，提高山羊体外胚胎生产的效率，采用切剖法与抽吸法采集卵泡卵母细胞，并对获得的可用卵母细胞进行了体外成熟培养。结果表明，切剖法能够获得较多的可用卵母细胞，可以明显增加用于体外成熟的卵母细胞数；卵母细胞的体外成熟率与卵泡直径密切相关，大卵泡与中卵泡的卵母细胞成熟率明显高于(P<0.05)小卵泡；山羊卵泡卵母细胞的体外成熟培养液中FCS的添加比例以10% ~ 15%为益。在一定范围内，提高FCS的浓度，并不能提高卵母细胞的成熟率。     

牛TLR4基因生物信息学分析

为了更加深入地了解牛TLR4基因的功能、结构及与该基因有关联的疾病识别及其致病机理,利用牛TLR4基因的氨基酸序列对其编码蛋白的基本理化性质、蛋白质译后的磷酸化位点和糖基化位点以及跨膜结构域和蛋白质结构进行预测。结果显示,牛TLR4基因一共编码841个氨基酸,负电荷残基总和(Asp+Glu)为84,正电荷残基总和(Arg+Lys)为76,不稳定指数小于40,表明该基因编码产物稳定性较好。在该基因整个编码产物中,亲水氨基酸占比较多,平均分值为-0.011,由此得知TLR4基因编码的蛋白质是一种易溶蛋白。有9个潜在的N-糖基化位点;78个磷酸化位点以及存在一个跨膜蛋白和存在信号肽,其切割位点在第25和第26个氨基酸之间。蛋白质的二、三级结构预测结果显示,二级结构和三级结构均由α-螺旋、β-折叠和无规则卷曲组成,其在生物合成、基因表达与调控等方面有重要作用。本研究结果可为牛TLR4基因深入研究提供理论基础。     

An oculo-cerebellar syndrome caused by congenital bovine viral diarrhoea virus-infection

An epizootic characterized by birth of calves severly ataxic and blind were encountered in 3 herds 7–8 months after outbreaks of bovine virus diarrhoea. Serological and virological investigations indicated introduction of bovine viral diarrhoea virus (BVDV) into previously virus-free herds, followed by transplacental virus infection of the fetuses of cows in the first trimester. Clinical, pathological, serological, and microbiological examinations were performed on 10 calves. Pathological findings included microcephaly and cerebellar hypoplasia, ocular malformations, and thymic hypoplasia. BVDV was isolated from tissue and blood of 7 calves, and 4 calves, 1 of which had not received colostrum, had virus-specific neutralizing antibodies.This is the first report on natural occurrence of congenital bovine infection with BVDV among Danish cattle herds resulting in abortion and birth of calves with severe debilitating congenital anomalies. It draws attention to the importance of this virus for bovines of all age groups.