CB·氨基酸在猪卵母细胞孤雌激活及体外培养中的作用

[目的]研究CB、氨基酸在猪卵母细胞孤雌激活及体外培养体系中的作用,为优化相关技术体系提供依据。[方法]卵母细胞经体外成熟培养,采用不同孤雌激活方法(Ele.+CB组、CB组、Ele.组、对照组),研究CB在激活中的作用。激活后采用PZM3培养液,并去除氨基酸成分,探索氨基酸对体外培养胚胎的作用。[结果]CB+Ele.组的卵裂率显著地高于其他3组(P<0.05),囊胚率为32.7%,囊胚细胞数为61.4,而其他3组均未出现囊胚。电激活(脉冲电压100 V/mm,脉冲时程100μs,脉冲次数1次)联合CB处理,可激活卵母细胞体外发育至囊胚,CB有助于提高卵裂率、囊胚率。添加氨基酸的培养液中的卵母细胞的卵裂率、囊胚率、囊胚细胞数显著提高(P<0.05),表明氨基酸能提高猪孤雌激活胚胎培养效果。[结论]添加CB、氨基酸在猪卵母细胞孤雌激活及体外培养体系中能提高胚胎的培养效果。     

猪卵母细胞不同孤雌激活方法的研究

通过对不同电激活参数的摸索,电激活和化学激活联合的研究,以确定适合于猪卵母细胞孤雌激活的最佳方案。结果表明,体外成熟猪卵母细胞在电场时程60μs,1次直流脉冲的条件下,电场强度1.6kV/cm时其卵裂率(88.68%)和桑葚胚率(81.13%)较其他各组高。在电场强度为1.6 kV/cm,1次直流脉冲的条件下,脉冲时程20μs时,卵母细胞卵裂率仅为75.38%,桑葚胚率为64.62%;40和80μs时,卵裂率分别是84.62%和77.17%;100μs时,卵母细胞的卵裂率和桑葚胚率都明显下降。在电场强度为1.6kV/cm,电场时程为60μs的条件下,2次电脉冲激活卵母细胞的卵裂率(87.50%)、桑葚胚率(81.25%)和1次电脉冲的卵裂率(88.68%)、桑葚胚率(80.13%)之间无显著性差异(P﹥0.05),但两者均显著高于3次电脉冲的卵裂率(72.50%)和桑葚胚率(70.27%)。成熟卵母细胞经电刺激(ES)后,分别用CB(7.5μg/mL)、CHX(10μg/mL)、6-DMAP(2 mmol/L)、CB(7.5μg/mL)+CHX(10μg/mL)、CB(7.5μg/mL)+6-DMAP(2 mmol/L)各自处理4 h,ES+CB+CHX和ES+CB+6-DMAP组卵裂率显著高于其他3组,但其桑葚胚率差异不显著。结果显示,电场强度为1.6 kV/cm,电场时程60μs,1次脉冲的电刺激对体外成熟的猪卵母细胞(IVM)孤雌激活效果最好;1次或2次电脉冲就足以激活猪卵母细胞,过高脉冲对细胞反而有伤害作用;电激活和化学激活联合应用可提高猪卵母细胞的卵裂率。     

猪体细胞核移植电融合参数的研究

试验旨在研究不同电融合参数(场强、脉冲时程)对猪核移植胚融合率及发育能力的影响。采用44～48h成熟培养的去核猪卵母细胞作为受体细胞,颗粒细胞为供体。结果表明,在20μs脉冲时程条件下,0.8kV·cm-1组融合率显著高于0.4、1.2、1.6kV·cm-1组(P0.05)。0.8、1.2kV·cm-1组卵裂率显著高于0.4、1.6kV·cm-1组(P0.05)。场强为0.8kV·cm-1条件下,20μs组融合率显著高于30、40、50μs组(P0.05)。卵裂率20、30μs组显著高于40、50μs组。根据试验数据,本试验室最佳融合电参数为:0.8kV·cm-1、20μs,1次脉冲。     

利用电极盘进行猪卵母细胞电激活

[目的]研究不同电激活参数以及电脉冲联合6-DMAP对猪卵母细胞孤雌激活效果的影响。[方法]使用电极盘,测定不同场强（1．1、1．3、1．5、1．7、1．9kV／cm）,不同脉冲时程（30、50、70、90、110ps）,1次脉冲下猪卵母细胞囊胚率的变化。[结果]结果表明,脉冲强度以1．5和1．7kV／cm效果最好,在80胂时其猪卵母细胞囊胚率分别达到（22．35±5．16）％和（20．59±9．41）％;脉冲强度1．5kWcm,1次电脉冲后4h联合6-DMAP激活卵母细胞,当脉冲时程达到70和90μs时,囊胚率分别达到（21．65±9．95）％和（23．10±16．27）％。[结论]在试验条件下,使用电极盘的最适电激活参数为脉冲强度1．5-1．7kV／cm,脉冲时长80μs,1次脉冲电激活。     

不同激活方法对绵羊体外成熟卵母细胞孤雌生殖的影响

为确定绵羊体细胞核移植胚胎的激活条件,本实验探讨了不同激活方法对绵羊体外成熟卵母细胞的孤雌激活及其后的发育影响.选择体外成熟绵羊卵母细胞,随机分为3组:①电激活 6-DMAP 4h;②I(5μm)5min 6-DMAP 4h;③7%乙醇7min 6-DMAP 4h,培养48h后,各组卵母细胞的卵裂率分别为60.93%,79.96%,78.28%;培养到7d时,各组的桑椹/囊胚发育率分别为17.03%,42.84%,39.39%.化学激活的卵裂率、桑椹/囊胚率显著高于电激活(P<0.05);乙醇与Ionomycin相比,其卵裂率、桑椹/囊胚率差异不显著,但Ionomycin处理组效果稍好于乙醇组.结果表明,电激活、Ionomycin、7%乙醇都可以有效激活绵羊卵母细胞,并孤雌发育到囊胚,Ionomycin的激活效果最佳;同时也比较了不同电压对绵羊体外成熟卵母细胞激活的影响,以1.300kV/cm-1.350kV/cm,脉冲时间10us,脉冲次数为2次,间隔0.5s效果较好.     

有无透明带猪卵母细胞电激活参数的优化及体外培养方式的建立

[目的]摸索无透明带猪体外成熟卵母细胞(ZFOs)电激活的条件和无透明带孤雌激活胚胎(ZFEs)的体外培养方式,为手工克隆法生产广西巴马小型猪体细胞核移植奠定基础。[方法]用不同场强、脉冲时程和脉冲次数激活ZIOs、ZFOs,用不同培养方式(微滴法和微穴法)培养ZIEs和ZFEs,检查第7天的囊胚率及囊胚的总细胞数。[结果]有带猪体外成熟的卵母细胞电激活最佳场强和脉冲时程为125 V/(mm.80μs),微滴法培养的囊胚发育率为(30.54±11.06)%;无带猪体外成熟的卵母细胞电激活最佳场强和脉冲时程为85V/(mm.80μs),微穴法培养,其囊胚发育率为(12.77±6.98)%;单次脉冲的激活效果好于多次脉冲。[结论]对有无透明带胚胎的培养方式,微穴法培养无透明带胚胎,微滴法培养有带胚胎较合适,一次脉冲可以有效激活卵母细胞。     

SrCl2联合CB及DMSO对小鼠卵母细胞孤雌激活试验

[目的]研究SrCl2联合CB及DMSO对小鼠卵母细胞孤雌激活及激活后胚胎发育的影响。[方法]选择不同浓度的SrCl2分别联合一定浓度的CB和DMSO对小鼠卵母细胞作用不同的时间,检测其卵母细胞的激活率及激活后卵母细胞的体外发育率。[结果]SrCl2联合DMSO对小鼠卵母细胞的孤雌激活率显著高于SrCl2联合CB（P〈0．05）,但激活后胚胎的囊胚发育率极低;SrCl2联合CB对小鼠卵母细胞的激活率〉80％,2mmolSrCl2,5μg／mlCB激活1h的囊胚发育率达45．5％,与其他组差异显著（P〈0．05）。[结论]2mmolSrCl2,5μg／mlCB作用1h对小鼠卵母细胞孤雌激活及激活后胚胎发育效果较好。     

电激活参数和培养基对猪卯母细胞孤雌发育的影响

为优化电激活和胚胎培养的相关技术体系,研究电激活参数和培养液对猪卵母细胞孤雌胚胎发育的影响.结果表明:在脉冲电压为150v(以每lmm电激槽宽计,下同)、脉冲持续时间为100 μs、脉冲1次的电激活条件下,猪卵母细胞孤雌激活的效果较好;在脉冲持续时间和脉冲次数恒定的条件下,脉冲电压150V组的囊胚率显著高于100 V组...     

猪卵母细胞激活方法的研究

【目的】探讨猪卵母细胞体外胚胎生产的最佳激活条件。【方法】以不同电场强度(100,150,200,250V/mm)、脉冲时间(2,30,40,60,80μs)、电脉冲次数(1,2,3次)的电激活方法及电激活(电场强度为200 V/mm,脉冲时间为60μs,激活次数为1次)联合化学处理(NCSU-23+CHX、NCSU-23+6-DMAP和NCSU-23+CHX+6-DMAP)方法对猪卵母细胞进行激活,研究不同激活方法对猪卵母细胞孤雌活化的影响。【结果】电激活猪卵母细胞时,在200 V/mm、60μs、1次激活的条件下其卵裂率和囊胚率均最高。电激活联合NCSU-23+CHX、NCSU-23+6-DMAP、NCSU-23+CHX+6-DMAP化学处理时,在孤雌激活后期的卵裂率分别为69.17%,82.31%和79.67%,囊胚率分别为24.17%,39.46%和39.84%,在统计学上各处理间无显著差异。【结论】猪卵母细胞孤雌激活的最佳条件,是在电场强度为200 V/mm,脉冲时间为60μs,激活次数为1次的电激活后,联合NCSU-23+6-DMAP处理4 h,再移入胚胎培养液NCSU-23+4 g/L BSA中继续孵育(40±4)h。     

不同因素对小鼠卵母细胞电激活及孤雌发育的影响

研究了卵龄、电脉冲参数、氯化锶、亚胺环己酮(CHX)对小鼠卵母细胞电激活及孤雌发育效果的影响。结果表明,卵龄为17h的小鼠卵母细胞在场强1.0kV/cm,脉冲宽度30μs,2次电脉冲,激活液中含Ca2+、Mg2+和Sr2+的条件下激活后,再于含CHX的培养液中培养,可较好地激活卵母细胞,提高体外孤雌胚的发育率,激活率及桑囊胚率分别可达83.3%和57.5%。     

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 μs, with a blastocyst development rate of (20.12 ± 8.18)% (P ＞ 0.05). No significant difference was found between treatments of multiple pulses and a single pulse ( P ＞ 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35)% ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) %, P ＞ 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 ± 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79,P ＜ 0.01 ). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2 (5 % CO2:7%O2:88%N2) also showed no significant difference from those in high O2 (5% CO2 in air) [ (20.78 ± 8.80) % and 17.00 ± 6.12 vs. (25.30 ± 7.55) % and 18.01 ± 6.79, P ＞ 0.05 ]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.     

猪体细胞克隆胚胎的体外生产试验

 【目的】通过优化猪体细胞核移植程序,建立获得克隆囊胚的有效方法。【方法】比较不同电激活参数、不同体外培养条件对猪体细胞克隆胚发育能力的影响。【结果】选用1.5 kV?cm-1、80 μs 和1 DC的参数组合,对猪核移植重构胚进行电融合和电激活,可获得较高的卵裂率和最高的囊胚发育率（分别为71.4%和14.3%）,且囊胚发育率显著高于其它组（P＜0.05）;0.4% BSA NCSU 23 培养液培养克隆重构胚72 h,半量换液并未改善克隆胚的发育,但添加10% FBS 可显著提高囊胚发育率（15.1%对10.3%,P＜0.05）和DNA 完整率(56.8%对46.6%,P＜0.05) ;采用猪卵泡颗粒细胞（pGC）共培养体系未能显著提高克隆胚发育率（P＞0.05）,但卵丘细胞（pCC）单层共培养时,囊胚发育率显著高于对照组（16.7%对9.8%,P＜0.05）,培养5 d 的克隆胚凋亡率也显著低于对照组（39.5%对54.2%,P＜0.05）。【结论】采用1.5 kV?cm-1、80 μs 和1 DC 的参数组合对猪克隆重构胚进行电融合和电激活,以0.4% BSA NCSU 23培养液作 pCC 单层共培养72 h,然后换用10% FBS NCSU 23 培养液,可明显提高囊胚发育率。     

绵羊卵母细胞孤雌激活及其随后体外发育的初步研究

对绵羊卵母细胞体外成熟培养和孤雌激活的影响因素作了初步研究.卵母细胞在TCM199培养液中培养24 h后,采用1.3 kV/cm、60 μs、0.1 mmol Ca2+和1.4 kV/cm、40 μs、0.1 mmol Ca2+2种处理对成熟卵母细胞进行电激活,卵裂率分别为76.1%和77.0%,2种处理差异不显著(P＞0.05).发生卵裂的卵母细胞在SOFaaBSA培养液中培养(分换液和不换液2种处理)至7～8 d统计囊胚率,换液的囊胚率为12.2%,不换液的囊胚率为22.8%,二者差异显著(P＜0.05).     

乙二胺四乙酸钠、能量基质、氨基酸对猪卵母细胞体外成熟和早期孤雌发育的影响

猪的卵巢卵母细胞在含不同剂量葡萄糖+丙酮酸钠的成熟培养基中体外成熟培养44-48 h,检查核成熟率(试验一),进行化学法孤雌激活后,在含不同浓度的乙二胺四乙酸钠(EDTA-Na)和不同剂量的葡萄糖+谷氨酰胺+牛磺酸+亚牛磺酸的培养基中进行体外培养,检查孤雌激活胚第48小时的卵裂率和第168小时的囊胚发育率(试验二),研究成熟培养基中葡萄糖+丙酮酸钠剂量对卵母细胞体外成熟核成熟率的影响及培养基中不同浓度EDTA-Na和葡萄糖+谷氨酰胺+牛磺酸+亚牛磺酸的剂量对孤雌激活胚胎体外发育的影响.结果表明:(1)葡萄糖+丙酮酸钠在1倍剂量时,猪卵母细胞体外成熟的核成熟率为(59.3±5.0)%;当剂量加倍时,核成熟率极显著下降(P<0.01),为(51.0±4.4)%.(2)当EDTA-Na浓度增高,囊胚发育率上升,当浓度达50μmol.L-1时囊胚发育率最高,为(7.2±4.1)%,此后随浓度升高囊胚发育率下降;添加适当浓度(25-100μmol.L-1)EDTA-Na对猪孤雌激活胚的囊胚发育有利(P<0.05),当浓度达200μmol.L-1时其有利作用消失.(3)培养基中葡萄糖+谷氨酰胺+牛磺酸+亚牛磺酸的剂量过高对卵裂率无显著影响(P>0.05),但对囊胚发育不利(P<0.05).可见:(1)适当浓度(25-100μmol.L-1)EDTA-Na对猪早期胚胎体外发育具有促进作用;(2)成熟培养基中能量基质剂量过高会抑制猪卵母细胞体外成熟;(3)胚胎培养基中葡萄糖+谷氨酰胺+牛磺酸+亚牛磺酸的剂量过高会抑制猪早期胚胎的体外发育.     

Effects of Ages of Donors and Conditions of Preserving Ovaries on Porcine Oocytes Maturation in vitro and Efficiency of Parthenogenetic Activation

Effects of different ages of donors and different conditions of preserving ovaries on porcine oocytes maturation in vitro and efficiency of parthenogenetic activation were studied. The experiments included: 1) effects of different temperatures (22, 30, 37, 38.5and 40℃) of preserving ovaries on porcine oocytes maturation in vitro and developmental potential; 2) effects of periods of preserving ovaries on porcine oocytes maturation in vitro and development in vitro; 3) effects of different ages of donors on porcine oocytes maturation in vitro and developmental potential. The results of the experiment showed:1) There were no statistical differences (p＞0.05) of the parthenogenetic cleavage rate (79.64% vs 76.18%) and blastocyst rate (18.11% vs 33.82%) between oocytes from ovaries preserved at 38.5℃ and those preserved at 37℃. When the preserving temperature was increased to 40℃, the cleavage rate (21.68%) and the blastocyst rate (0) were great significantly lower than those at 37℃(p＜0.01). The cleavage rate (80.79% vs 76.18%) and blastocyst rate (29.61% vs 33.82%) were not different between 30 and 37℃(p＞ 0.05). When the preserving temperature was decreased to 22℃, the rate of cleavage was not different,but the rate of blastocyst was significantly lower, compared with that at 37℃; 2) The cleavage and blastocyst rates of the porcine oocytes collected after slaughter 2 or 6h were not different (p＞0.05); 3) The cleavage rate of oocytes from gilts and sows after maturation was not different, but the blastocyst rate of the sow group was significantly higher than that of gilt group (p＜ 0.05). The blastocyst cell number of sows and gilt showed no difference (p＞0.05).     

Effects of Chemical Activation on the Parthenogenetic Development of Porcine in vitro Maturation Oocytes

The objective of this study was to determine the effects of ionomycin combined with cytochalasin B (CB), cycloheximide (CHX), or 6-dimethylaminopurine (6-DMAP) on the activation of porcine oocytes. In Experiment 1, in vitro matured oocytes were activated with 15,20,25 or 30 mmol L-1 ionomycin separately. Activation rates of 20,25 mmol L-1 and 30 mmol L-1 treatments were higher (P<0.05) than that of 15 mmol L-1 treatment. In Experiment 2, in vitro matured oocytes were activated with 20 mmol L-1 ionomycin for 10,20,30,40 or 50 min and then incubated with 2 mmol L-1 6-DMAP for 6 h.Cleavage and blastocyst rates [(72.40±13.02)％, (25.37±11.43)％] after treatments for 40 min were higher (P>0.05) thanthose of the other treatments. In Experiment 3, matured oocytes were activated with ionomycin and then incubated with 7.5 mgmL-1 CB, 10 mg mL-1 CHX, 2 mmol L-16-DMAP, 7.5 mg mL-1 CB + 10 mg mL-1 CHX or 7.5 mg mL-1 CB + 2 mmol L-1 6-DMAP for6 h. The rates of activation, cleavage and blastocyst of 2 mmol L-1 6-DMAP treatment [(86.05±4.29)％, (61.77±8.10)％ and(21.62±3.31)％] were higher (P<0.05) than those of 7.5 mg mL-1 CB treatment. In Experiment 4, matured oocytes wereactivated with ionomycin and then incubated with 2 mmol L-1 6-DMAP for 3.5, 5.5 or 7.5 h. Cleavage rates and blastocyst rates of 5.5 h treatment [(66.59±14.36)％ and (25.40±10.16)％] were higher (P>0.05) than those of other treatments. In conclusion, activation of porcine oocytes appears to be most successful using the combination of ionomycin (20 mmol L-1,40 min) followed by 6-DMAP (2 mmol L-1, 5.5 h).     

培养液体积对猪孤雌胚早期发育的影响

为了探讨培养液体积对猪早期孤雌胚发育的影响,旨在优化猪早期胚胎培养体系。试验一、二、三、四分别把30、20、15、10个枚孤雌激活胚放入60,45,30,15,10,5μL(10个胚胎)的培养滴,第48小时观察分裂率,第168小时观察囊胚形成率。结果表明:试验一:30~60μL组囊胚率略高于10~15μL组;试验二:60μL组的囊胚率略高;试验三:各组分裂率和囊胚率差异不显著(P>0.05),但45~60μL组分裂率和囊胚率比其他组略高;试验四:15μL组分裂率高于45μL组(P<0.05),15μL组囊胚率显著高于5μL组(P<0.05),其余各组差异不显著。由此表明:30枚猪孤雌胚时,胚胎数∶培养滴体积=1∶(1~2)较好;15~20枚猪孤雌胚时,胚胎数∶培养滴体积=1∶3较好;10枚猪孤雌胚时,胚胎数∶培养滴体积=1∶1.5较好。