不同激活条件及半胱氨酸处理对猪ICSI胚胎发育影响研究

本试验探讨了不同辅助激活方法(Calciumionophore A23187激活、Calciumionophore A23187+6-DMAP联合激活和电激活)、不同精子预处理方法(液氮冻融处理和0.1%Triton X-100处理)和在添加半胱氨酸的胚胎培养液中培养不同时间(0 h、4 h、12 h和168 h)对猪卵母细胞内单精子注射(ICSI)胚胎体外发育的影响。结果显示:与无辅助激活相比,A23187+6-DMAP联合激活和电激活均能显著提高ICSI卵母细胞的激活率、卵裂率和囊胚率(P0.05),A23187+6-DMAP联合激活能显著提高ICSI卵母细胞的受精率(P0.05)。液氮冻融精子组ICSI卵母细胞的雄原核形成率显著高于活精子组(P0.05)。在添加半胱氨酸的胚胎培养液中培养4 h的ICSI卵母细胞受精率、雄原核形成率和囊胚率显著高于0 h组(P0.05)。以上结果表明,猪卵母细胞在ICSI后需要辅助激活来启动胚胎顺利发育,A23187+6-DMAP激活效果较好。液氮冻融精子可以促进ICSI后雄原核的形成。半胱氨酸处理4 h对猪ICSI卵母细胞受精和发育均有促进作用。     

不同因素对奶牛分离精子ICSI效率的影响

本研究旨在探讨不同的联合激活处理、精子预处理方法和单精子注射方式对奶牛分离精子ICSI效率的影响。借助显微操作仪将经不同预处理的奶牛分离X精子直接注入体外成熟22～24h的牛卵母细胞胞质内,注射过程采用回吸胞质和不回吸胞质2种处理,最后分别用3种激活方案对注射卵进行激活。结果表明:用CR1aa+Ionomycin+6-DMAP对ICSI注射卵进行激活后,卵裂率和囊胚率都高于A23187+CHX和7%ET+CHX两种联合激活组(63.74%vs61.09%、53.75%;28.54%vs17.68%、22.00%,P0.05);采用percoll密度梯度离心法处理精子,卵裂率(77.10%vs62.19%,P0.05)与囊胚率(26.95%vs22.82%,P0.05)均高于上游法处理组;ICSI时回吸胞质获得的注射卵,其卵裂率和囊胚率均显著高于不回吸胞质处理组(63.10%,25.35%vs41.56%,19.40%,P0.05)。结果表明,用percoll密度梯度离心法处理精子、注射时回吸胞质、用CR1aa+Iono-mycin+6-DMAP激活ICSI注射卵,可显著提高奶牛分离精子ICSI的效率。     

不同激活方法对绵羊胞质内显微授精的影响

为了研究激活方法对绵羊胞质内显微授精的影响,分别对卵子采用:①假注射,不激活;②注射精子,不激活;③假注射,Ionomysin+6DMAP激活;④注射精子,Ethanol+6DMAP激活;⑤注射精子,Ionomysin+6DMAP激活。结果表明:第1、2组无论注射精子与否,但都不激活,卵裂率(7.5%、5.3%)和囊胚发育率(0.94%、1.8%)都很低,差异均不显著(P>0.05)。第3组与第4、5组比较,卵裂率(65.7%、71.6%、75.6%;P>0.05)无明显差异,但囊胚发育率(5.7%、15.6%、26.0%;P<0.01)却明显低于4、5组。采用Ethanol+6DMAP和Ionomysin+6DMAP2种方法处理时,卵裂率差异不显著(71.6%、75.6%;P>0.05),但囊胚发育率前者要显著低于后者(15.6%、26.0%;P<0.05)     

山羊卵母细胞孤雌激活及孤雌胚的体外培养

为了比较不同的激活方法对卵母细胞孤雌激活的影响,以及培养液(CR1aa)中添加不同类型的血清对孤雌激活胚体外发育的影响。采用Eth 6-DMAP、A23187 6-DMAP和Ion 6-DMAP三种方法激活山羊卵母细胞,Eth 6-DMAP组孤雌激活胚的卵裂率和囊胚率(35.2%和4.8%)显著低于A23187 6-DMAP组(68.9%和24.1%)和Ion 6-DMAP组(88.1%和48.0%),表明以Ion 6-DMAP激活最为理想。在培养液(CR1aa)与颗粒细胞共同培养条件下,分别添加100 mL/L的发情牛血清(OCS)、胎牛血清(FBS)和新生牛血清(NCS)。添加OCS和FBS后,孤雌胚囊胚率分别为48.1%和45.0%,显著高于添加NCS组(26.0%)。表明OCS、FBS和NCS能有效的提高孤雌胚的体外发育能力,尤其是对提高孤雌胚的囊胚发育能力更佳。     

β-环糊精胆固醇包合物对牛冷冻精子品质的影响

为了研究添加胆固醇与β-环糊精的包合物(CLC)对荷斯坦牛冷冻精子品质(AR)的影响,试验分不同剂量CLC组和对照组,分别测定牛冻精的精子活率、精子顶体反应(AR)并进行比较。结果:添加1.5 mg CLC冷冻后活率最高[(60.4±7.38)%,P<0.05];孵育时间对冷冻后精子活率影响显著(P<0.05),添加1.5 mg CLC孵育15 min精子活率显著高于对照组(P<0.05);精子在BO液孵育15,30,45和60 min的酸性磷酸酶(ACP)吸光度值,1.5 mg CLC组与对照组比较差异都极显著(P<0.01)。试验证实,CLC可提高牛冷冻精子的活率,可用于牛精子的冷冻保存。     

不同化学激活方法对绵羊孤雌生殖胚体外发育的影响

本研究利用常规化学激活剂离子酶素(Ionomycin)和乙醇分别与6-二甲基氨基嘌呤(6-Dimethylaminoputine,6-DMAP)和细胞松弛素B(CB)的不同组合对绵羊卵母细胞的激活后体外发育效果进行了比较。结果表明:在桑囊胚率上,Ionomycin联合6-DMAP与联合6-DMAP CB差异不显著(40.4%vs46.0%),而与联合CB组差异显著(40.4%、46.0%vs 29.5%,p<0.05),与单独Ionomycin激活差异极显著(40.4%、46.0%vs 15%,p<0.01);乙醇联合6-DMAP CB与联合6-DMAP、CB差异显著(46.9%vs 34.2%、36.2%,p<0.05),而与单独乙醇激活差异极显著(46.9%vs 21%,p<0.01)。     

6-DMAP对延边黄牛末期卵母细胞核移植的影响

为探讨6-二甲基氨基嘌呤（6-DMAP）在延边黄牛末期去核的体细胞克隆中的作用,本试验主要研究了单独使用离子霉素处理与离子霉素联合添加6-DMAP处理对体外成熟的老化延边黄牛卵母细胞的激活率的不同影响;以及不同时期添加6-DMAP对重构胚后期发育能力的影响。试验结果显示,体外成熟的老化卵母细胞,使用离子霉素单独处理后91%被激活,而在离子霉素联合添加6-DMAP处理后却没有细胞被激活,也就是没有第二极体的排出。另外,融合后使用6-DMAP处理,所得重构胚的囊胚发育率最低,而激活后的卵母细胞立即用6-DMAP处理,所得重构胚的发育能力与无6-DMAP处理的相近。综上所述,离子霉素联合添加6-DMAP可抑制老化的延边黄牛卵母细胞的激活,阻止第二极体的排出,但对重构胚的后期发育没有影响。而融合使用添加6-DMAP的培养液,抑制了重构胚的后期发育。     

应用ICSI技术生产转基因水牛胚胎的初步研究

本研究旨在探讨水牛精子完整质膜和破损质膜的方法对水牛单精子注射(ICSI)技术转基因效果的影响。水牛精子经冻融、Triton×-100、超声波3种方法破损精子质膜后与外源质粒DNA混合,采用ICSI的方法将基因转染后的精子注射到水牛体外成熟卵内,观察水牛ICSI卵激活后的发育状态和外源基因的表达效果。结果表明:精子质膜完整性实验中,冻融破损精子质膜组早期胚胎基因表达率为21.8%,显著高于活精子组的5.1%(P<0.01);冻融组与活精子组的卵裂率、囊胚发育率均无显著差异(P>0.05)。以冻融、Triton×-100、超声波3种方法破损精子质膜,冻融组的囊胚发育率(16.7%)最高,显著高于Triton×-100组(P<0.01)。同时,冻融组的早期胚胎基因表达率亦显著高于Triton×-100组和超声断尾组(60.3%vs.31.7%vs.18.2%,P<0.01)。上述结果说明,使用ICSI技术转外源基因可获得表达EGFP基因的水牛早期胚胎;冻融精子质膜破损法能有效破损精子质膜,有利于外源DNA与精子的结合,且提高转基因效率。     

牛血清白蛋白对山羊精液冷冻效果的影响

用冷冻稀释液添加牛血清白蛋白(BSA)进行影响海门山羊冷冻精液品质的研究。选用3个处理组进行比较试验,结果表明:以添加6%BSA处理组效果最好,其冷冻解冻后精子活力高于对照组,但差异不显著(P>0.05),而SPA试验表明精子穿卵活力极显著高于对照组(P<0.01)。研究表明在冷冻稀释液中添加BSA进行海门山羊精液冷冻具有良好的效果。     

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).     

Consequences for the bovine embryo of being derived from a spermatozoon subjected to post-ejaculatory aging and heat shock: development to the blastocyst stage and sex ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.     

Full‐Term Development of Rabbit Embryos Produced by ICSI with Sperm Frozen in Liquid Nitrogen without Cryoprotectants

The aim of the present study was to establish the technology of intracytoplasmic sperm injection (ICSI) in rabbit by using the sperm frozen without cryoprotectants. Observation under an electron microscope revealed that the rabbit spermatozoa frozen without cryoprotectants had severe damage especially in the plasma membrane and junction between head and tail. However, after being injected into the oocytes, the sperm frozen without cryoprotectants retained the capability of supporting the cleavage and development of the ICSI oocytes, with no significant difference from that of fresh sperm, although the development of ICSI embryos derived from either frozen sperm or fresh sperm is much lower than that of in vivo‐fertilized zygotes. When additional artificial activation was applied following ICSI, the rates of cleavage and blastocyst formation of ICSI oocytes were significantly increased when compared with the oocytes without additional activation. Yet, the cell numbers in blastocysts were not significantly different between the activation and non‐activation group. After embryo transfer, four offspring were obtained from the oocytes microinjected with the sperm frozen without cryoprotectants. The technology established by this study may facilitate exploring the ICSI‐based transgenic method in rabbit and broaden the application of ICSI technique in related field.     

Effect of Pre‐Treatment of Ovine Sperm on Male Pronuclear Formation and Subsequent Embryo Development Following Intracytoplasmic Sperm Injection

The objective of this study was to determine the effects of various methods of sperm pre‐treatment on male pronuclear (MPN) formation and subsequent development of ovine embryos derived from in vitro‐matured oocytes and intracytoplasmic sperm injection (ICSI). The effect of treatment of injected oocytes with dithiothreitol (DTT) on embryo development was also assessed. In Exp. 1, the injected oocytes with non‐treated sperm were activated with three different procedures. The cleavage and blastocyst rates in those activated with DTT was lower (p < 0.05) than those activated with either ionomycin (Io) + 6‐dimethylaminopurine (6‐DMAP) or DTT + I + 6‐DMAP. In Exp. 2, the effects of sperm pre‐incubated with DTT, sodium dodecyl sulphate (SDS) or DTT + SDS as well as two‐time frozen/thawed sperm (without cryoprotectant) on MPN formation and oocyte activation were examined. The non‐treated sperm served as controls. The MPN formation in DTT + SDS group was higher (p < 0.05) than other groups except for freeze–thaw group. No difference in the rate of activated ICSI oocytes was observed among groups. In Exp. 3, the effect of pre‐treatment of sperm on subsequent development of ICSI embryos and blastocyst cell numbers were examined. The rates of cleavage and blastocyst formation as well as the blastocyst cell numbers were similar among the pre‐treated and control groups. In conclusion, pre‐treatment of sperm with DTT + SDS positively affected MPN formation, although the subsequent development capacity of the resulting embryos remained limited. Moreover, DTT was not effective on oocyte activation compared with Io + 6‐DMAP after ICSI.     

The Optimization of Preparating Conditions on Pig ICSI Embryos

In order to optimize the preparating conditions on pig intracytoplasmic sperm injection (ICSI) embryos,the Piezo operating system was used in this experiment and the embryonic survival rate,cleavage rate and cell numbers of blastocysts developed in various stages were measured as traditional and Piezo operating system,the concentration of CB,the operating temperature with Piezo operating system and different sperm processing method in preparating pig ICSI embryos.The results showed that Piezo operating system could significantly improve the survival rate and cleavage rate of injected oocytes (P<0.05),but there was no significant effect on blastocyst rate.There was a slight damage to oocyte added CB in the operating fluid,and adding 7.5 μg/mL CB was most concentration,30℃ was most appropriate operating temperature,but there was no significant effect of early development on pig ICSI embryos with breaking sperm tail with ultrasonic wave.The results suggested that Piezo operating system could significantly improve the survival rate.The results suggested that breaking sperm tail with ultrasonic wave,adding 7.5 μg/mL CB in operating fluid and 30℃ of operating temperature and using Piezo operating system was more suitable for preparating pig ICSI embryos.     

猪ICSI胚胎制备条件的优化

为了进一步优化猪胞浆内单精子注射（intracytoplasmic sperm injection,ICSI）胚胎制备条件,本试验将Piezo操作系统应用于猪ICSI胚胎制备过程中,并以胚胎各阶段存活率、分裂率和囊胚率作为衡量指标,对比Piezo操作系统与传统斜口针操作、Piezo操作条件时操作液中添加CB浓度、操作台温度及精子的不同处理方法对猪ICSI胚胎发育的影响。结果表明,Piezo操作系统可以显著提高注射卵的存活率和卵裂率（P<0.05）,但对囊胚率没有显著影响（P>0.05）;利用Piezo进行猪ICSI时,操作液中添加CB,可以减少显微操作时注射针卵母细胞的损伤,其中以7.5 μg/mL CB效果最佳;操作台温度30℃最适合制备猪ICSI胚胎;精子预先进行超声波断尾处理对猪ICSI胚胎的早期发育无显著影响。研究结果表明,精子预先进行超声波断尾处理后,操作液中添加7.5 μg/mL CB、操作台温度30℃,应用Piezo操作系统比较适合猪ICSI胚胎制备。     

单精注射法生产转GFP基因猪胚胎的研究

卵胞质内单精子注射(ICSI)的出现为治疗人类男性不育提供了新的途径。作为一种家畜胚胎工程新技术，ICSI可以解决猪的多精子受精问题。本研究用冷冻/解冻的猪精子与GFP基因孵育后对猪IVM卵母细胞进行ICSI，通过对猪ICSI卵母细胞发育能力和基因表达效率的分析，初步研究以精子为载体的转基因与卵细胞质内单精注射相结合的技术(SMGT-ICSI)生产转基因猪胚胎的技术路线的可行性。用GFP基因转染的精子注射后化学激活的ICSI卵母细胞基因表达率显著高于电激活处理的ICSI卵母细胞的基因表达率。用精子头部注射的猪卵母细胞和用完整精子注射的猪卵母细胞总基因表达效率无显著差异(P>0.05)。结果表明，用冷冻/解冻后的猪精子或精子头部与外源DNA孵育后通过ICSI方法生产转基因胚胎是可行的。     

ICSI受精卵胞质注射生产转基因猪胚胎的初步研究

试验旨在探讨单精子胞浆内注射（intracytoplasmic sperm injection,ICSI）法生产猪体外受精卵和应用猪ICSI受精卵进行胞质注射生产转基因胚胎的可行性。首先对比了猪体外受精（in vitro fertilization,IVF）受精卵与ICSI受精卵的胚胎发育效率;然后观察了猪ICSI受精卵的双原核形成时间及效率,对精子注射到胞质后6~18 h分6个时间段进行地衣红染色,对比精子进入卵胞质后的状态及原核形成;最后对猪IVF受精卵受精后8~10 h及ICSI受精卵受精后12~14 h进行EGFP-N1质粒（20 ng/μL）胞质注射,观察胚胎发育效率及转基因效率。结果表明,ICSI受精卵的胚胎发育率（卵裂率89.4%和67.9%、囊胚率36.5%和16.1%）显著优于IVF组（P<0.05）,适合用于猪的体外受精卵试验;猪ICSI受精卵双原核在精子注射到卵胞质后12~14 h形成,双原核形成率为54.90%,显著高于其余5个试验组（P<0.05）;ICSI受精卵胞质注射组胚胎卵裂率（86.2%和66.3%）、囊胚率（30.0%和13.6%）及转基因效率（18.5%和0）均显著高于IVF受精卵胞质注射试验组（P<0.05）。本试验结果为采用ICSI受精卵进行胞质注射生产转基因猪的研究奠定了基础。     

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF.     

卵丘细胞对卵母细胞成熟、受精和胚胎发育的影响试验

将从猪卵巢上获取的卵母细胞根据卵丘细胞层数的多少分为两类,并将这两类卵母细胞进行成熟培养、体外受精和孤雌激活,以验证两类卵母细胞的发育潜能.结果显示,将两类卵母细胞分别进行成熟培养时,优级卵的成熟率高于次级卵（67.42±177;1.52％vs 48.33±177;2.85％）,且差异极显著（P＜0.01）;将成熟的卵母细胞进行孤雌激活后,优级卵的囊胚率高于次级卵（58.00±177;2.88％ vs 41.00±177;6.40％）,且差异极显著（P＜0.01）,但囊胚总细胞教两者无显著差异（P＞0.05）;将成熟的卵母细胞进行体外受精后,优级卵的囊胚率、囊胚总细胞数均高于次级卵（18.00土5.70％ vs 4.25±177;2.31％,41.7±177;3.33 vs 36.2土2.10）,且差异极显著（P＜0.01）;进行受精卵孵育时,未去除卵丘细胞的胚胎卵裂率、囊胚率、囊胚总细胞数均高于去除卵丘细胞的（88.14土7.48％ vs 58.69±177;2.89％,51.2±177;7.33％ vs 11.92±177;2.29％,41.7±177;3.33 vs 36.8±177;3.15）,且差异极显著（P＜0.01）.以上结果说明：卵丘细胞层数较多的卵母细胞成熟率高,且其孤雌和体外受精胚胎的发育潜能也好;卵丘细胞存在时,有助于卵母细胞的受精及以后的胚胎发育.