猪2-细胞胚胎细胞的电融合

为了探索猪胚胎细胞核移植技术中细胞融合参数，进行了猪２－细胞胚胎细胞的电融合。电融合时，直流电场强度为２ｋＶ／ｃｍ，脉冲时程选４０μｓ时，可获得７０．４％的融合率和６８．４％的发育率。非电解质融合液（甘露醇）对于融合率和融合胚的发育均优于电解质融合液（ＤＰＢＳ）。在直流电脉冲前的交流脉冲是必要的，可以显著提高融合率（Ｐ＜０．０５）。     

体细胞核移植生产转基因牛胚胎的研究

为了优化利用体细胞核移植生产转基因牛早期胚胎的体系,以携带绿色荧光蛋白-新霉抗性双标记基因的pMSCV质粒转染的胎牛耳成纤维细胞为供体,以体外成熟的牛卵母细胞为受体构建克隆胚,研究供体细胞的传代次数对转基因胚的影响和重构胚在不同电场条件下（电场强度、直流电脉冲次数）融合效率及其在不同体外培养系统中的发育效果。结果表明：传代5次的细胞做核供体较有利于转基因胚的发育;在电场条件为电场强度1.6 kV/cm、直流脉冲1次的融合率最高;负压单层细胞共培养体系中的转基因囊胚发育率较好,与未转基因体细胞核移植胚的发育相比无显著差异（P〉0.05）。     

小鼠卵母细胞的电激活

采用宁波新科CRY 3 细胞融合仪及其配备的镀银电融合槽(电极宽度为0. 5mm)对影响小鼠卵母细胞电激活效率的诸多因素,如直流脉冲的强度(1. 0 kV/cm、1. 5kV/cm、2.0 kV/cm、2.5 kV/cm)、脉冲宽度(40μs、80 μs、100 μs、150 μs)和脉冲次数(1次、2次、3次)进行了比较研究,结果表明,卵母细胞激活率随电场强度的增加而升高,场强为2.0 kV/cm时,获得了较高的卵母细胞激活率和卵裂率(74.47%,77.14%),场强继续增加,卵母细胞的激活率和卵裂率反而开始下降。场强为2.0 kV/cm,脉宽为80μs时,卵母细胞的激活率最高(77.78%)。多次连续脉冲(3次,每次间隔10 min)处理卵子,可明显升高卵母细胞的激活率和囊胚发育率(89.58%,20.59%)。     

电融合条件对盘羊、北山羊异种核移植胚胎发育的影响

本试验通过对异种核移植后电融合的参数进行比较,发现盘羊的皮肤纤维细胞可以和绵羊的卵母细胞融合,北山羊的皮肤纤维细胞可以和山羊的卵母细胞融合;对盘羊细胞电融合强度为1.4 kV/cm、20 μs/次的融合率要较1.3 kV/cm、20μs/次高,并且降低融合后重构胚死亡率,显著提高重构胚的分裂率;对北山羊的细胞电融合强度为1.5 kV/cm、20 μs/次的融合率要较1.4 kV/cm、30 μs/次和1.6 kV/cm、20 μs/次高;融合后的重构胚胎能够正常分裂,并在子宫中着床发育.     

牛卵母细胞电激活研究

采用高场强单次脉冲（1600V/cm,80μs）及低场强3次脉冲（400V/cm,20μs,每次间隔20min）刺激体外成熟牛卵母细胞（成熟26h）,激活率分别为86.5%（167/193）和92.7%（101/109）,差异不显著（P>0.05）,其中多原核率分别为8.3%（16/193）和59.6%（65/109）,差异极显著（P<0.01）,但发育率差异不显著（20.6％和23.3％）。实验表明,电脉冲能显著提高牛成熟卵母细胞的激活率;多次电激会造成很大比例的多原核,但对卵母细胞的发育影响不大。     

电激活参数与渗透压阶段培养法对孤雌胚胎发育能力的影响

试验旨在摸索猪卵母细胞孤雌激活的电场强度和脉冲时间,并探索渗透压阶段培养法对孤雌胚胎后期发育的影响。猪卵母细胞成熟培养42~44 h后,分别在电场强度2.1、2.3、2.5 kV/mm和脉冲时间30、60、90 μs的9组电激活参数下进行孤雌激活试验;卵母细胞在2.1 kV/mm和30 μs的参数下进行孤雌激活后,分别培养于渗透压为271、280、290、302 mOsm的PZM-3中,48 h后移入渗透压280 mOsm的PZM-3中继续培养96 h;孤雌胚胎于电激活后先在含2 mmol/L 6-DMAP的PZM-3中培养4~6 h,然后移入不含6-DMAP的PZM-3中继续培养。试验结果表明,电场强度和脉冲时间两个参数间无显著的交互作用(P>0.05),脉冲时间相同条件下,卵裂率在不同电场强度条件下均无显著差异(P>0.05),2.1和2.5 kV/mm的电场强度条件下,脉冲时间为30 μs时的卵裂率显著高于60和90 μs(P<0.05),而2.3 kV/mm电场强度下3个脉冲时间试验组的卵裂率无显著差异(P>0.05),各试验组的囊胚率无显著差异(P>0.05);孤雌胚胎在渗透压为290~310 mOsm的PZM-3中培养48 h,卵裂率得到显著提高(P<0.05),渗透压对囊胚率无显著影响(P>0.05);6-DMAP对孤雌胚胎卵裂率无显著影响(P>0.05),但可以显著提高囊胚率(P<0.05)。结果提示,猪卵母细胞孤雌激活需要较高的电场强度(2.1~2.3 kV/mm)而脉冲时间不宜过长(30 SymbolmA@s);48 h的高渗培养和6-DMAP的辅助激活有助于孤雌胚胎的后期发育。     

融合液浓度梯度融合法不同融合条件对延边黄牛体细胞克隆效率的影响

采用延边黄牛耳皮肤成纤维细胞作供核细胞,0.28 mol/L蔗糖融合液,融合液浓度梯度融合法对重组卵母细胞进行融合操作,探讨不同融合电场强度、脉冲次数、脉冲时程对延边黄牛体细胞克隆胚胎融合率及重组胚体外发育率的影响。结果表明,适宜延边黄牛体细胞克隆、融合液浓度梯度融合法的融合电场强度为1200 v/cm;融合脉冲次数为2次;融合脉冲时程为40μs。     

兔卵母细胞的孤雌激活

从电脉冲次数、脉冲强度、脉冲间隔时间三方面对兔卵母细胞的孤雌激活进行了研究。结果表明，注射HCG后16h的MⅡ期卵，用1．4kV／cm、60μs的直流脉冲电激，脉冲次数分为1、2、4、8次4组，每次间隔30min，随着电激活次数的增加，活化率从46．5％增加到95．8％(P＜0．01)，但是电激活8次后，桑椹胚率却从63．6％下降列21．2％，囊胚率从23．6％下降列2．8％(P＜0．01)；用60μs的直流脉冲电激3次，每次间隔30min，分为0．4、1．2、2．4kV／cm3组，以1．2kV／cm脉冲强度的兔卵母细胞激活较为理想，脉冲强度增加到2．4kV／cm时，卵细胞死亡率升为67．1％(P＜0．01)。用1．4kV／cm、60μs的直流脉冲电激3次，电脉冲间隔时间分为30、120、240min3组，卵细胞活化率分别为68．6％、91．5％、91．9％（p＜0．01)。     

水牛体细胞核移植方法的研究

探讨电融合参数对水牛体细胞核移植效果的影响。体外成熟培养 22~24 h的水牛卵母细胞,在含有 5μg/mL细胞松弛素的操作液中进行去核,然后将经0 .1 mg/L Aphidicolin (APD)处理1 d, 再用0. 5% FBS培养2~9 d的水牛耳皮成纤维细胞或颗粒细胞注射到去核的卵母细胞卵周隙中,再经电融合形成重构胚。重构胚经 5μmol/L离子霉素激活处理5 min并在2 mmol/L的 6 DMAP中培养 3 h后,在含有颗粒细胞单层细胞的微滴中(30μL)培养7~9 d,观察其卵裂和胚胎发育情况。当电融合的电场强度为1 500 V·μs/mm(电压100 V/mm×脉时15μs)时,电脉冲3次,颗粒细胞核移植的融合率为 74.18%,分裂率为 71.82%,囊胚发育率为 10%,融合率显著高于2次电脉冲(52.03%,P<0. 05),卵裂率显著高于4次电脉冲(53.95%,P<0. 05);当电脉冲次数为 3 次时,电场强度为2 000 V·μs/mm的分裂率(53.54%)显著低于1 500 V·μs/mm,2 500 V·μs/mm的融合率(62.0%)和分裂率(53.23%)均显著低于1 500 V·μs/mm(P<0 .05)。采用耳皮成纤维细胞作供核的融合率(59.75%)和分裂率(57.45%)均显著低于颗粒细胞。染色体组型分析显示,66.7%的核移植胚胎具有正常的供体细胞二倍体核型。将来自1头22岁的摩拉公牛耳皮成纤维细胞的2枚冻胚移植给受体母牛,妊娠到215 d时发生流     

不同激活方法对猪卵胞质内单精子注射（ICSI）效果的影响

采用不同的注射方式结合人工激活方法处理猪卵母细胞,旨在探讨各处理方法对猪ICSI效果的影响。结果表明：假性注射＋未激活处理组卵母细胞的激活率虽然高于无机械刺激＋无激活处理对照组,但明显低于假性注射＋人工激活处理组;精子注射卵母细胞经CaCl2（1.8pL,30mmol/L）、离子霉素（15μmol/L,40min）和电脉冲（场强0.4kV/cm、脉冲时程90μs、1次脉冲）激活处理后,其激活率无显著性差异（P〉0.05）,CaCl2处理组的受精率显著高于离子霉素处理组（P〈0.05）,并与电激活处理组无显著性差异（P〉0.05）;但是,在假性注射组和对照组中,CaCl2处理组的孤雌发育率极显著地低于离子霉素和电激活处理组（P〈0.01）;CaCl2处理组的卵裂率（P〈0.01）和囊胚总细胞数（P〈0.05）显著高于对照组。因此,单纯注射性机械刺激对猪卵母细胞的激活效果较差,有必要进行人工激活;ICSI卵母细胞经CaCl2溶液激活处理后,能够取得较好的ICSI效果,而不显著增加其孤雌发育率。     

没食子酸对黄牛卵母细胞体外成熟的影响

试验旨在研究没食子酸（gallic acid，GA）对黄牛卵母细胞体外成熟及早期胚胎发育的影响，进一步优化黄牛卵母细胞体外成熟体系。在卵母细胞体外成熟液（M液）中添加不同浓度没食子酸（0、10、30、50、100 μmol/L），成熟22~24 h后，统计卵丘扩展情况及卵母细胞成熟率；同时，对成熟的卵母细胞进行正常体外受精（IVF），统计早期胚胎的分裂率、囊胚率、囊胚卵裂球数及卵裂球细胞凋亡率。根据试验结果，选择最优浓度，使卵母细胞在含该浓度没食子酸的成熟液中成熟24 h后，检测其细胞内的活性氧水平（ROS）和总谷胱甘肽（TGSH）含量。结果显示，M液中添加30 μmol/L没食子酸组卵丘扩展分值和成熟率显著高于对照组（0 μmol/L）（P<0.05），其他处理组与对照组无显著差异（P>0.05）；成熟的卵母细胞体外受精后进行后续胚胎培养，其中10和30 μmol/L组的分裂率均显著高于对照组和100 μmol/L组（P<0.05），50和100 μmol/L组分裂率较对照组也有所提高，但差异不显著（P>0.05）；早期囊胚率统计发现，与对照组相比，30和100 μmol/L能够显著提高囊胚发育率（P<0.05），10和50 μmol/L浓度组则无显著差异（P>0.05）；与对照组相比，30、100 μmol/L没食子酸均能显著提高IVF胚胎的早期囊胚卵裂球数（P<0.05）；但囊胚卵裂球凋亡率与对照组无显著差异（P>0.05）；对卵母细胞内活性氧和总谷胱甘肽含量检测时，发现30 μmol/L没食子酸可显著降低细胞内活性氧水平（P<0.05），且显著提高总谷胱甘肽含量（P<0.05）。综上所述，在黄牛卵母细胞体外成熟液中添加适量的没食子酸能有效降低卵母细胞内活性氧水平，提高总谷胱甘肽含量，进而提高卵母细胞成熟的质量及其后续IVF胚胎发育能力。     

Effects of Gonadotropins on In Vitro Maturation and of Electrical Stimulation on Parthenogenesis of Canine Oocytes

The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 μs with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 μs without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.     

Parthenogenetic Induction of Canine Oocytes by Electrical Stimulation and Ca-EDTA

In this study, we investigated parthenogenetic induction of canine oocytes by electrical stimulation following Ca-EDTA treatment. Oocyte maturation, parthenogenetic development, and cleavage rate in canine after various electrical stimulations (1.5, 1.8, 2.1 kV/cm) for 50 μs with single DC pulse following 1 mM Ca-EDTA treatment were investigated. In oocyte activated electrically at the voltage of 1.5 kV/cm after 1 mM Ca-EDTA treatment, the rate of pronucleus and two-cell was 4.1% and 2.7%, respectively. Although electrical stimulation could parthenogenetically induce immature oocyte to cleavage stage, degeneration rate in all experimental groups was more than 60%. This means that electrical stimulation after Ca-EDTA treatment could cause canine oocytes to be degenerated. However, two-cell in canine oocyte by parthenogenesis was for the first time induced. Therefore, we suggested that electrical stimulation for canine oocytes could induce parthenogenetically early embryonic cleavage. This result can be used as a basic data for parthenogenesis study in canine. Also, to perform more developed embryonic development, further study to parthenogenesis in canine need to be developed.     

绵羊去透明带卵母细胞体细胞核移植研究

为探索简化的核移植程序,本研究分析不同成熟培养时间、去核过程中紫外光照时间、融合电压强度、激活剂种类、不同培养方法对绵羊去透明带卵母细胞核移植的影响。结果表明:卵母细胞成熟培养18～19 h后去除透明带,其极体排出和附着效果最佳。采用1.9 kV/cm直流电压融合去核卵母细胞与颗粒细胞,融合率为88.2%,效果最好。离子霉素对重构胚具有较好的激活效果,卵裂率为82.1%,囊胚率为10.4%;压制WOW(s孔中孔)发育组卵裂率和囊胚发育率与四孔板培养组相比无显著差异,但卵裂率和囊胚发育率并不高;去除透明带的绵羊卵母细胞采用衰减1/4的UV照射10 s辅助去核后,卵裂率为35.6%,但重构胚未能发育至囊胚。结果显示,通过去透明带辅助显微操作去核的方法进行的绵羊体细胞克隆程序较易掌握。     

不同收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及发育的影响

试验旨在研究不同卵母细胞收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及体外受精的影响。以黄牛为研究对象,采用两种方法（抽卵法和割卵法）抽取卵泡中的卵母细胞,比较两种方法获取的卵母细胞成熟率,结果发现,抽卵法获得的卵母细胞成熟率显著高于割卵法（P<0.05）。将获取的卵母细胞分为4组：A组（对照组,只添加基础成熟培养液）、B组（基础成熟培养液+200 μmol/L半胱氨酸）、C组（基础成熟培养液+20 μg/mL肝素钠）、D组（基础成熟培养液+200 μmol/L半胱氨酸+20 μg/mL肝素钠）,结果发现,D组的卵母细胞成熟率显著高于A、B、C组（P<0.05）,B、C组间卵母细胞成熟率无显著差异（P>0.05）,但两组卵母细胞成熟率均显著高于A组（P<0.05）;A组卵母细胞卵裂率均显著低于B、C、D组（P<0.05）,B、C、D组间卵母细胞卵裂率无显著差异（P>0.05）;D组囊胚率显著高于其他3组（P<0.05）。结果表明,抽卵法获得卵母细胞效率显著高于割卵法,肝素钠及半胱氨酸对黄牛卵母细胞体外成熟和体外受精都有促进作用,且同时添加两种物质对体外成熟的效果更佳。     

The Construction of Cloned Sika Deer Embryos (Cervus nippon hortulorum) by Demecolcine Auxiliary Enucleation

The objective of our study was to establish the feasibility of experimental protocols for cloning sika deer. We performed auxiliary enucleation to improve the efficiency of nuclear transfer operation by optimizing the demecolcine concentration to induce cytoplasmic protrusions in the sika deer oocytes. In the present study,we had studied the impact of different demecolcine concentrations on cytoplasmic protrusions and enucleation rates. We determined that 95.9% of the sika deer oocytes formed cytoplasmic protrusions when treated for 1 h with 0.8 μg/ml demecolcine. The lowest observed rate of protrusion was 19.3% after overnight treatment with demecolcine. When the oocytes aged or had a poor cumulus expansion, they exhibited a significant decrease in the ability to form cytoplasmic protrusions. The rates of enucleation (94.9% vs 85.8%, p < 0.05), cell fusion (84.6% vs 70.1%, p < 0.05) and blastocyst formation (15.4% vs 10.9%, p < 0.05) using demecolcine auxiliary enucleation were significantly higher than those after blind enucleation. These results demonstrated that sika deer oocytes could be enucleated quickly and effectively using demecolcine auxiliary enucleation, which could enhance the enucleation rate, cell fusion rate and blastocyst rate of cloned embryos in vitro.     

钙离子及山梨醇对猪卵母细胞孤雌激活的影响

探讨电激液中不同Ca2+浓度与不同电脉冲强度相互关系对猪体外成熟卵母细胞孤雌激活的影响;以及山梨醇电激活液在猪孤雌激活中的应用。结果表明,当Ca2+浓度不高时,同时增加电激液Ca2+浓度和电脉冲强度能协同促进孤雌胚胎的卵裂率和囊胚率的提高,Ca2+浓度为0.05和0.1mmol/L时,分别以1.6kV/cm和1.2kV/cm激活,得到的卵裂率为73.75%、74.70%;囊胚率为37.50%、36.83%,显著高于其余各组(P0.05);当Ca2+过高,增加脉冲强度卵母细胞孤雌发育能力反而下降,退化率升高;以山梨醇液进行电激活,在1.6kV/cm时,卵裂率和囊胚率最高,分别为77.23%和34.15%;与甘露醇电激液不同,施加交流电,山梨醇液不能对孤雌胚胎发育起到促进作用;将山梨醇、甘露醇电激液等体积混合,交流脉冲后进行电激活,1.2、1.6kV/cm组卵裂率分别为72.33%和70.03%,囊胚率分别为31.03%和29.60%,虽然略低于甘露醇组(77.07%、36.03%),但优于山梨醇组(69.63%、26.93%),差异不显著(P0.05)。以上结果说明,猪卵母细胞激活所需内流Ca2+浓度存在临界值,临界值内,提高Ca2+浓度或电参数,都能提高卵母细胞的激活效果;超过临界值,效果相反;山梨醇液能够取代甘露醇液用于猪卵细胞的电激活;同时以山梨醇和甘露醇作为电激液的基本成份,完全可以用于猪卵母细胞的电激活或融合。     

Effect of activation treatments on actin filament distribution and in vitro development of miniature pig somatic cell nuclear transfer embryos

In the present study, we investigated the effect of activation treatments on the actin filament distribution and in vitro development of somatic cell nuclear transfer (SCNT) embryos in miniature pigs. We combined three activation methods, ionomycin (ION), electrical stimulation (ES), and cycloheximide treatment (CH), to prepare seven activation treatments (ION, ES, CH, ION + CH, ION + ES, ES + CH and ION + ES + CH). First, we investigated the activation rate of oocytes and in vitro development of parthenotes. The activation rates of the oocytes in the ION, ES, CH, ION + CH, ION + ES, ES + CH, and ION + ES + CH groups were 42.9, 51.3, 0.0, 82.1, 80.6, 78.1 and 78.6%, respectively, showing that the rates of the combined treatment groups were significantly higher (P<0.05) than those of the single treatment groups. Although there were no significant differences in the activation rates of the combined treatment groups, the developmental rate to blastocysts in the ION + CH treatment group (36.1%) was significantly higher (P<0.05) than the other combined treatment groups (14.6-24.7%). Subsequently, we investigated the in vitro development and distribution of microfilaments in SCNT embryos. The developmental rate to blastcysts of the SCNT embryos in the ION + CH treatment group (11.3%) was significantly higher (P<0.05) than in the ES and ION + ES + CH treatment groups (4.5 and 5.2%, respectively). The rate of normal actin filament distribution in the SCNT embryos activated with ION + CH was significantly higher (P<0.05) than those activated with ES or ION + ES + CH treatment (63.3 vs. 46.8 or 46.4%). In addition, the fragmentation rate of the SCNT embryos activated with ION + CH was significantly lower (P<0.05) than those activated with ION + ES + CH (14.9 vs. 26.1%). The present results suggest that an activation treatment of ionomycin combined with cycloheximide may avoid physical damage to microfilaments and result in improved subsequent development of miniature pig SCNT embryos.