猪孤雌胚胎体外培养影响因素

对NCSU-23和PZM-3等2种培养基体外培养猪孤雌激活(PA)胚胎的效果进行了比较,结果显示,PZM-3组与NCSU-23组PA胚胎囊胚孵化率差异显著(32.6%vs 18.9%,P<0.05);NCSU-23中添加2%必需氨基酸(EAA)显著降低猪PA胚胎的囊胚发育率(20.1%vs 25.1%,P<0.05);添加1%非必需氨基酸(NEAA)显著提高猪PA胚胎的囊胚率(24.7%vs 19.7%,P<0.05),但是联合添加NEAA和EAA对猪PA胚胎体外发育无显著影响(P>0.05).     

不同血清类型对水牛胚胎体外培养效果的影响

从屠宰场收集水牛卵巢上抽取的卵母细胞,经体外成熟培养后分别进行孤雌激活(PA)和体外受精(IVF),将获得的PA或IVF胚胎分别培养于添加浓度均为5%的牛血清白蛋白(BSA)、胎牛血清(FBS)、炭情水牛血清(OBS)、发情牛血清(OCS)和混合血清(OCS与FBS按1:1混合)的培养液中培养7～9 d,并观察胚胎的体外发育情况.结果,PA胚胎培养在OCS和混合血清组的卵裂率(73.52%,68.07%)和囊胚率(30.59%,27.73%)均显著高于BSA(38.37 %.6.40%)、OBS(44.97%,10.05%)和FBS(52.02%,17.04%)组(P＜0.05);IVF胚胎的卵裂率和囊胚率以OCS组最高(62.50%,22.62%),显著高于BSA(30.50%,6.38%)、OBS(35.48 %,7.10 %)和FBS组(40.11%,11.76%,P＜0.05),但与混合血清组(60.42%,21.35%;P＞0.05)差异不显著.结果表明,在水牛胚胎体外培养体系中添加5%的OCS或混合血清均能有效地促进水牛胚胎的体外发育.     

甘草酸单铵盐对猪卵母细胞体外成熟及胚胎发育能力的影响

将收集的猪COCs置于添加不同浓度(0.0,0.3,0.6,1.2μmol/L)甘草酸单铵盐(monoammonium glycyrrhizinate, MAG)的卵母细胞体外成熟培养液中培养46 h,统计成熟率，通过免疫荧光染色检测成熟卵母细胞ROS表达水平；对成熟卵母细胞体外受精，于胚胎培养液内体外培养48,120 h,分别统计体外受精胚胎卵裂率、囊胚率，并用Hochest荧光染色检测囊胚总细胞数；结合成熟率及IVF胚胎发育囊胚率选定最佳添加浓度，在体外成熟培养液中添加最适浓度MAG,以0μmol/L MAG为对照组，体外培养46 h后，利用免疫荧光染色检测猪成熟卵母细胞的线粒体膜电位水平和细胞凋亡水平。结果显示，与对照组相比，不同浓度MAG处理组猪卵母细胞体外成熟率均有所提高，但差异均不显著(P>0.05);不同浓度MAG添加均可降低猪卵母细胞ROS水平，与对照组相比，0.3,0.6μmol/L组差异显著(P<0.05),1.2μmol/L组差异极显著(P<0.01)。0.3μmol/L添加组显著提高了猪体外受精囊胚率(P<0.05),但不同MAG添加组对...     

柠檬苦素对小鼠卵母细胞体外成熟及其体外受精胚胎发育的影响

【目的】研究柠檬苦素(limonin,Lim)对小鼠卵母细胞体外成熟(IVM)及后续体外受精(IVF)胚胎发育潜能的影响,旨在为体外成熟培养系统的优化提供参考。【方法】在小鼠体外成熟培养液中添加不同浓度的Lim(0、10、20、50 μmol/L),成熟培养12 h后统计小鼠卵母细胞第一极体(PBI)排出率,筛选体外成熟培养液中添加Lim的最适浓度;在体外成熟培养液中添加最适浓度的Lim,以0 μmol/L Lim为对照组,成熟培养12 h,通过免疫荧光染色检测活性氧(ROS)、谷胱甘肽(GSH)以及线粒体膜电位(MMP)水平;通过实时荧光定量PCR检测卵母细胞抗氧化及凋亡相关基因的mRNA表达水平。将最适Lim组及对照组卵母细胞体外成熟24 h后进行体外受精,于体外受精24 h和3.5 d分别统计胚胎卵裂率和囊胚率,并用Fluorescein-dUTP和Hoechst 33342染色分别检测囊胚总细胞数及囊胚内凋亡细胞比率。【结果】与0 μmol/L Lim组相比,20 μmol/L Lim组小鼠卵母细胞PBI排出率显著升高(P＜0.05),后续试验均用20 μmol/L Lim进行处理。与对照组组相比,20 μmol/L Lim组小鼠卵母细胞内ROS水平显著降低(P＜0.05),GSH、MMP水平均显著增加(P＜0.05),抗氧化相关基因(GPx3、CAT和Prdx3)、抗凋亡相关基因(Bcl-2、Bcl-xl)表达水平均显著上调(P＜0.05),促凋亡相关基因(Caspase-3)表达水平显著下调(P＜0.05);体外受精胚胎的卵裂率、囊胚率、囊胚总细胞数均显著增加(P＜0.05),囊胚内细胞凋亡比率显著下降(P＜0.05)。【结论】在体外成熟培养液中添加20 μmol/L Lim可以通过抑制氧化应激和细胞凋亡、增加MMP水平提高小鼠卵母细胞质量,从而提高体外受精胚胎的发育潜力。     

胚胎培养液中能量底物对猪体细胞克隆胚胎体外培养的影响

如何根据猪胚胎发育的不同阶段合理使用不同成分的培养液,建立高效的体细胞克隆胚胎体外培养体系。本研究把所获得的猪体细胞克隆胚胎用不同的培养液培养,比较体外囊胚发育力和囊胚细胞数。结果表明:PZM-3比NCSU-23更适用于克隆胚胎的培养;培养前4d使用PZM-3,后2d更换含有葡萄糖的NCSU-23,可以提高胚胎的囊胚细胞数。说明胚胎在不同发育阶段对能量底物的需求不同,合理利用可以有效提高胚胎发育力。     

NGF和BDNF对猪孤雌激活胚胎体外发育的影响

在NCSU-23改良培养液中分别添加不同质量浓度的脑源性神经生长因子(不rain-derived neurotrophic factor,BDNF)和神经生长因子(nerve growth factor,NGF),观察其对猪孤雌激活胚胎体外生长的影响。结果显示:在培养液中添加40μg/L的BDNF,可促进猪孤雌激活胚胎的囊胚形成;在猪PA胚胎培养液中添加不同质量浓度的NGF,对猪孤雌激活胚胎的在卵裂率、囊胚率和囊胚孵化率影响不明显。结果表明,在NCSU-23改良培养液中添加一定质量浓度的BDNF可促进猪孤雌激活胚胎体外培养过程中囊胚的形成。     

氨基酸、维生素对水牛体外受精胚胎体外发育的影响

通过在培养液中添加不同浓度的氨基酸或维生素,探讨其对水牛体外受精(IVF)胚胎体外发育的影响.结果表明:(1)非必需氨基酸可显著提高水牛卵母细胞IVF后胚胎的分裂率,但对囊胚发育率无显著影响;(2)低浓度的必需氨基酸对水牛IVF胚胎的发育具有一定促进作用,但高浓度则有抑制作用;(3)维生素对水牛IVF胚胎发育则有促进作用.在培养液中添加维生素,水牛IVF胚胎的分裂率和第7天囊胚发育率显著提高.     

牛体外成熟卵母细胞的孤雌激活及其胚胎体外培养方法的研究

本试验比较了在SOFaa培养体系下化学激活(5 μmol/L离子霉素5 min,2mmol/L 6-DMAP 4h)与电激活(1.3 kv/cm,60 μ s,1次脉冲)方法对囊胚率的影响,结果表明化学激活囊胚率显著高于电激活(12.30% vs 2.4%,p＜0.01),而二者的卵裂率(90.61%vs 94.40%)和囊胚细胞数(48.24±13.12 vs 38±3.56)均没有显著性差异(p＞0.05);同时比较了在SOFaa培养体系下第4天更换添加10% FBS的SOFaa液与在SOFaa液中连续培养7天对囊胚率的影响,结果更换添加FBS的培养液使囊胚率显著降低(3.69% vs 11.64%,p＜0.01).     

表皮生长因子和胎牛血清对猪体细胞克隆胚胎发育的影响

为了研究胚胎培养液中添加表皮生长因子(EGF)和胎牛血清(FBS)对猪体细胞克隆胚胎发育的影响,试验将卵母细胞成熟培养42h后构建克隆胚,进行电融合激活,再使用6-DMAP(2mmol/L)进行化学辅助激活,然后移入NCSU-23培养基培养,激活后第2天添加0,10,20,30ng/mLEGF,7d后检查囊胚率、囊胚细胞数;激活后第0,2,4,6天添加10%FBS,7d后检查囊胚率、囊胚细胞数。结果表明:2～4细胞阶段添加EGF,添加20ng/mLEGF组的囊胚率显著高于对照组(P0.05)及其他处理,而囊胚细胞数与对照组无显著差异(P0.05);在第4天添加FBS组的囊胚率显著高于对照组(P0.05)和其他处理组,而囊胚细胞数与对照组无显著差异(P0.05)。结果说明添加20ng/mL的EGF能促进猪体细胞克隆胚胎的发育率,在第4天添加FBS更有利于胚胎的发育。     

SAHA处理对猪手工克隆胚胎体外发育潜能的影响

为了探讨组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸(SAHA)对德保猪手工克隆胚胎(HMC)发育潜能的影响,试验摸索SAHA的适宜处理浓度[0(对照),1.0,2.5,5.0,7.5,10.0μmol/L]和时间(0,6,12,24 h);之后分为4组,SAHA组、体外受精(IVF)组、孤雌激活(PA)组、对照(HMCC)组,分别在体外发育的1细胞期、2细胞期、4细胞期、囊胚期收集胚胎,在相同时期下比较各组胚胎组蛋白H4K8乙酰化(Ac H4K8)水平差异和相关基因(HDAC1、HAT1、ASF1A、OCT-4)相对表达量。结果表明:7.5μmol/L SAHA处理囊胚率显著高于对照(P0.05),12 h囊胚率显著高于0 h(P0.05);所以适宜处理浓度为7.5μmol/L,适宜处理时间为12 h。在1细胞期、2细胞期、囊胚期,SAHA组Ac H4K8水平接近IVF组水平(P0.05)。在囊胚期,SAHA组HDAC1基因相对表达量接近IVF组(P0.05);在囊胚期,SAHA组OCT-4基因相对表达量接近IVF组(P0.05)。说明SAHA可以使HMC胚胎Ac H4K8水平接近IVF水平,并纠正克隆胚胎乙酰化的异常,从而提高克隆胚胎发育潜能。     

Iloprost对猪早期胚胎体外发育的影响

本研究旨在探讨PGI2类似物iloprost对猪胚胎体外发育的影响。试验以IVF胚胎为研究对象,分别在不同时期(0、24、48、72h)将不同浓度(0、0.2、0.5、1.0、2.0、5.0、10.0μmol.L-1)iloprost加入到猪胚胎培养液中,于156h时记录囊胚发育率和囊胚细胞数,筛选获得最佳添加方案,检测胚胎脂肪代谢速度和代谢相关基因(cox2、creb、pparδ、pdk、cpt2)的表达水平,分析iloprost影响胚胎发育的机制。结果显示,最佳的添加方案为,在受精后48h加入2.0μmol.L-1 iloprost,胚胎囊胚发育率(28%)和囊胚细胞数(49.42)显著高于(P<0.05)对照组的囊胚发育率(16%)和囊胚细胞数(28.22);添加iloprost后,胚胎脂肪酸降解速度也显著加快(P<0.05),脂肪酸代谢相关基因cox2、creb、pparδ、cpt2的表达量上升,糖代谢相关基因pdk表达量无显著变化。结果表明,PGI2类似物iloprost可以促进胚胎降解脂肪酸,为胚胎发育提供能量,提高了胚胎体外发育能力。     

丙戊酸处理对猪手工克隆重构胚体外发育潜能的影响

为探讨组蛋白去乙酰化酶抑制剂丙戊酸（valproic acid, VPA)对猪手工克隆(HMC)胚胎发育潜能的影响,本研究比较了不同浓度（0、25、50、75和100 nmol/L）VPA处理猪HMC重构胚对后期胚胎发育率、内细胞团数和组蛋白乙酰化程度的影响。结果表明,50、75 nmol/L VPA处理组HMC重构胚的卵裂率和囊胚发育率均显著高于0、25和100 nmol/L VPA处理组（P<0.05）;与0、25、75和100 nmol/L相比,50 nmol/L VPA处理组能显著提高囊胚内细胞团细胞数（P<0.05）,囊胚阶段VPA处理组的组蛋白H3K14乙酰化（AcH3K14）水平高于空白组和孤雌激活囊胚。因此,应用VPA处理可提高猪HMC重构胚胎分裂率、囊胚率及增加内细胞团细胞数,提高了猪HMC胚胎的体外发育潜能。     

奶牛体外受精胚胎的性别控制研究

为加速母牛胚胎的生产,实现性控制胚胎产业化和体外扩繁优质品种的奶业工程发展,本研究在体外受精技术（in vitro fertilization,IVF）不断成熟和完善的基础上,通过对牛非性控IVF胚胎与常规人工受精体内胚胎移植的妊娠成功率及其两者出生后牛的性别比例进行比较分析,结果发现,两组妊娠成功率基本相同,且IVF胚胎出生的公牛比例高于体内胚;采用性控和非性控精液进行IVF试验,并对非性控IVF胚胎及性控IVF胚胎用牛Y染色体性别决定特异基因（SRY）核心序列进行分子生物学性别鉴定,结果显示,性控IVF胚胎的公牛比例显著低于普通IVF胚胎;此外,作者还比较了性控IVF与普通非性控IVF的囊胚发育率,结果显示两者并无显著差异。以上研究结果证明,体外扩繁优质良种母牛的奶业工程必须依靠性别控制体外授精技术来提高其效率和有效性。     

Treatment of porcine oocytes with MEM vitamins during in vitro maturation improves subsequent blastocyst development following nuclear transfer

This study was carried out to investigate the effects of minimum essential medium (MEM) vitamins during in vitro maturation (IVM)/in vitro culture (IVC) of porcine nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. Porcine cumulus-oocyte complexes (COCs) were divided into five groups, matured for 44 h in maturation medium with various concentrations of MEM vitamins (0, 0.05, 0.1, 0.2 and 0.4%), and observed for maturation rate. Also, COCs were matured in NUSU-23 media without MEM vitamins for 44 h and cultured in PZM-3 media with various concentrations of MEM vitamins (0, 0.05, 0.4 and 1.0%) for 6 days following nuclear transfer. Factorial (IVM/IVC) experiments were also performed in NCSU-23 medium with or without 0.05% MEM vitamins and PZM-3 medium with or without 0.4% MEM vitamins. They were then tested by examining in vitro development of the porcine reconstructed embryos. The maturation rates of the COCs treated with the MEM vitamins did not differ significantly among the MEM vitamin-treated groups. Addition of vitamins to culture medium did not affect development of porcine reconstructed embryos in vitro. However, addition of low concentrations of MEM vitamins only to maturation medium increased (P<0.05) the proportion of NT embryos developing into blastocysts compared with the control group. Addition of MEM vitamins to IVC medium did not enhance the developmental rate compared with the control group. Thus, addition of MEM vitamins to IVM medium could improve subsequent blastocyst development of porcine NT embryos.     

Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41–42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 ± 2% vs 48.1 ± 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.     

Stage‐specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium‐3 (mPZM‐3) with increased NaCl to 138 mmol/L (mPZM‐3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM‐3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM‐3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM‐3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM‐3+NaCl for the first 2 days and then cultured in mPZM‐3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM‐3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.     

甲基-β-环化糊精对猪体外受精及早期胚胎发育的影响

为了优化猪体外受精技术体系,本试验探索了甲基-β-环化糊精(methyl-beta-cyclic dextrin,MBCD)对猪体外受精以及早期胚胎发育的影响。在体外受精0和4 h向受精液(modified Tris-buffered medium,mTBM)中添加不同浓度(0,0.5,1,2,5,10,15,20μmol/mL)的MBCD,受精孵育结束后转至PZM-3培养液中进行胚胎培养。对各处理组卵母细胞的受精情况以及胚胎发育能力进行了系统的检测,并用金霉素(chlortetracycline,CTC)染色法评估了MBCD处理后精子获能状态。结果显示:1)体外受精0 h添加5μmol/mL MBCD组的卵裂率、囊胚率、囊胚细胞数显著高于(P<0.05)对照组和除10μmol/mL MBCD组之外的其他试验组。2)体外受精0 h添加5和10μmol/mL MBCD组、单精入卵率显著高于(P<0.05)对照组和其他试验组,而多精入卵率显著低于(P<0.05)对照组和其他试验组。3)添加5μmol/mL MBCD组,0～1 h,F型精子迅速减少(78.56～19.43),B型精子迅速增加(10.79～69.86);1～4 h,F型精子和B型精子基本保持不变(B型:69.86～78.78,F型:19.43～9.11)。上述结果表明在体外受精0 h向mTBM中加入5μmol/mL MBCD可以显著提高获能精子比例,减少多精受精发生,提高早期胚胎发育潜能。     

Development of in vitro produced porcine embryos according to serum types as macromolecule

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.     

Effects of ghrelin on in vitro development of porcine in vitro fertilized and parthenogenetic embryos

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.     

转GFP基因核移植牛胚胎的研究

试验采用脂质体转染法与电穿孔法,以携带绿色荧光蛋白（GFP）-新霉抗性（neo-）双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF（表皮生长因子）对转基因胚的影响,不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示,体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高,但对后期转基因重构胚的囊胚发育率的影响,以添加EGF 20 ng/mL组的最高;以胎牛耳成纤维细胞为供体细胞,不同转染方法转染供体细胞构建重构胚,其囊胚发育率差异不显著（P>〖JP2〗0.05）;mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好,该体系更适合体细胞核移植法生产转基因牛胚胎。