非人灵长类动物体外受精与胚胎移植的研究进展

简要介绍了非人灵长类动物体外受精与胚胎移植的内容、方法和步骤,影响其体外受精的主要因素,以及对未来非人灵长类动物的研究方向。     

转基因奶牛胚胎移植技术的试验研究

2006年6月至2010年3月以来,北京奶牛中心与中国农业大学合作,在北京奶牛中心良种场开展了转基因牛胚胎移植试验研究。本试验利用中国农业大学培育的转基因母牛作为供体,进行超数排卵生产体内胚胎,将获得的胚胎移植给15～15.5月龄荷斯坦母牛受体。截止2010年3月21日,累计移植转基因奶牛胚胎87枚,移植受体牛87头,妊娠48头,移植妊娠率为55.17%;产犊43头,成活34头,犊牛成活率为79%;经中国农业大学研究人员检测,有14头公牛携带转入目的基因,这14头公牛已全部进入北京市种公牛站饲养。目前,有10头公牛到了试采精月龄,精液经农业部牛冷冻精液质量监督检验测试中心(北京)检测,质量符合国家标准。     

湿垫内“水─气”间热质交换过程的理论分析

为完善湿垫风机系统在农业建筑夏季蒸发冷却中的应用，研究了湿垫冷却装置在喷淋水完全蒸发条件下的热质交换特征，经理论推导给出了确定喷淋水量及湿垫合理参数的计算式。     

英语学习中母语语音负迁移现象的分析

“母语语音负迁移”是中国学生在英语学习中普遍遇到的问题，其消极影响是显而易见的。本文从探讨汉语拼音的发音方法对英语语音的负迁移现象入手，揭示英汉语音的相似性与差异性。对英汉语音相似性与差异性的认知能促使中国学生在说英语时有意识地进行自我纠音，因此笔者认为在语音教学实践中引入英汉语音对比的相关知识及其研究成果将能有效地降低中国学生母语语音对目的语的干扰作用，从而提高语音教学质量。     

猪胎盘营养转运相关基因在不同妊娠期的表达

【目的】探究不同妊娠时期猪胎盘的氨基酸、葡萄糖、脂肪酸转运体的表达模式。【方法】选择15头遗传背景、产仔数接近的杜洛克2~4胎经产健康母猪平均分为3组,所有母猪发情后使用相同公猪精液进行人工授精,在妊娠第40天(D40)、65天(D65)和95天(D95)通过麻醉分别取出每组母猪子宫,快速打开子宫分离出每个胎儿的胎盘组织,提取胎盘组织总RNA并反转录合成cDNA,利用合成的引物进行普通PCR扩增,用2.0%琼脂糖凝胶检测扩增产物。采用实时荧光定量PCR检测并比较3个时期胎盘中氨基酸、葡萄糖、脂肪酸转运体相关基因mRNA相对表达水平。【结果】PCR检测结果显示,氨基酸转运体相关基因(SLC7A1、SLC7A2、SLC7A3、SLC7A4、SLC7A10、SLC1A3、SLC1A5、SLC38A10、SLC36A1)、葡萄糖转运体相关基因(SLC2A1、SLC2A2、SLC2A3、SLC2A10、SLC2A12、SLC2A13)及脂肪酸转运体相关基因(FATP1、FATP2、FATP3、FATP4、FABP3、FABP5、FABP7、CD36)的片段长度均与预期相符。实时荧光定量PCR结果显示,在氨基酸转运体中,D65胎盘中SLC7A4、SLC7A10、SLC38A10基因表达水平显著高于D40胎盘(P＜0.05),而SLC7A2基因表达水平显著低于D40胎盘(P＜0.05),且D65胎盘的SLC1A3和SLC7A4基因表达水平均显著低于D95胎盘(P＜0.05);在葡萄糖转运体中,D65和D95胎盘的SLC2A3和SLC2A13基因表达水平显著高于D40胎盘(P＜0.05),D95胎盘的SLC2A1、SLC2A2和SLC2A12基因表达水平显著低于D65胎盘(P＜0.05);在脂肪酸转运体中,D65胎盘的FATP2、FATP4、FABP3、FABP5、FABP7和CD36基因表达水平显著高于D40胎盘(P＜0.05),而FATP1、FATP4和CD36基因表达水平显著低于D95胎盘(P＜0.05)。【结论】在猪妊娠过程中,胎盘中SLC7A10、SLC38A10、SLC7A4、SLC2A3、FATP1、FATP4、FABP5、CD36等基因可能是影响胎儿生长发育的重要营养转运基因。     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.     

Use of Fourier‐Transform Infrared Spectroscopy to Quantify Immunoglobulin G Concentrations in Alpaca Serum

Background

Rapid, economical, and quantitative assays for measurement of camelid serum immunoglobulin G (IgG) are limited. In camelids, failure of transfer of maternal immunoglobulins has a reported prevalence of up to 20.5%. An accurate method for quantifying serum IgG concentrations is required.

Objective

To develop an infrared spectroscopy‐based assay for measurement of alpaca serum IgG and compare its performance to the reference standard radial immunodiffusion (RID) assay.

Animals

One hundred and seventy‐five privately owned, healthy alpacas.

Methods

Eighty‐two serum samples were collected as convenience samples during routine herd visits whereas 93 samples were recruited from a separate study. Serum IgG concentrations were determined by RID assays and midinfrared spectra were collected for each sample. Fifty samples were set aside as the test set and the remaining 125 training samples were employed to build a calibration model using partial least squares (PLS) regression with Monte Carlo cross validation to determine the optimum number of PLS factors. The predictive performance of the calibration model was evaluated by the test set.

Results

Correlation coefficients for the IR‐based assay were 0.93 and 0.87, respectively, for the entire data set and test set. Sensitivity in the diagnosis of failure of transfer of passive immunity (FTPI) ([IgG] <1,000 mg/dL) was 71.4% and specificity was 100% for the IR‐based method (test set) as gauged relative to the RID reference method assay.

Conclusions and Clinical Importance

This study indicated that infrared spectroscopy, in combination with chemometrics, is an effective method for measurement of IgG in alpaca serum.     

A Cathepsin B Inhibitor,E-64, Improves the Preimplantation Development of Bovine Somatic Cell Nuclear Transfer Embryos

Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.     

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.